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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 projects (on , and ). 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 (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 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 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 . 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 , 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 in humans [197] 55 3.23 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 [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 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] 76 Study 20. Preliminary evaluation of thiopentone PK enantioselectivity in sheep [224] 76 Study 21. Evaluation of thiopentone PK enantioselectivity in humans [232] 76 Study 22. Evaluation of thiopentone PK enantioselectivity in rats [259] 76 Study 23. PK-PD study of thiopentone enantioselectivity in rats [262] 77 Study 24. Enantioselectivity of GABA A receptor effects of thiopentone [261] 77 Study 25. PK study of thiopentone enantiomers from i.v. infusion in humans [264] 77 Study 26. Tissue distribution and effects from thiopentone enantiomers in rats [265] 77 Study 27. Blood-brain equilibration of thiopentone enantiomers in rats [266] 78 Study 28. Nicotinic ACh receptor effects of thiopentone enantiomers [284] 78 Study 29. PK studies of high dose thiopentone in humans [287] 78 Review 14. Thiopentone – a neurological and a neurosurgical analysis in patients [303] 78 Study 30. Acid-base changes on PD and PK of bupivacaine and thiopentone [306] 79 Study 31. Direct cardiac effects of thiopentone and its enantiomers in sheep [307] 79 Postscript 2 79 3.4 Nonsteroidal anti-inflammatory drug program 80 Review 15. A potential role of NSAIDs for postoperative pain management [200] 81 Study 32. Ketorolac enantiomers: a rapid, robust chiral assay [216] 82 Study 33. Study of the renal effects of ketorolac in rats [242] 83 Study 34. Ketorolac - multimodal analgesia study in sheep [269]. 83 Postscript 3 83 3.5 Halothane project 87 Study 35. Halothane enantioselectivity pharmacokinetics in rats [267] 89 Postscript 4 89 3.6 Ketamine project 90 Study 36. Ketamine pharmacokinetics and metabolism in rats [289] 92 Study 37. Pharmacokinetics of ketamine in rats –interaction with [296] 93 Study 38. Potentiation of ketamine anaesthetic potency in rats by alfentanil [300] 93 Study 39. Stereoselective potentiation of potency of ketamine in rats by alfentanil [320] 93 Postscript 5 94 3.7 Thalidomide project 95 Study 40. A robust chiral assay for thalidomide in biological samples [316] 96 Study 41. Enantioselectivity of thalidomide pharmacokinetics in rats [319] 96 Study 42. Interpretation of the interaction between thalidomide and cisplatin [321] 97 Postscript 6 97 3.8 Some additional reviews involving stereopharmacology 98 Reviews 16, 17 and 18. Stereopharmacological impact in anaesthesiology [248,305,313] 98 Postscript 7 99 4. Discussion – and some conclusions 100 Afterword 107

v

Foreword In January 2013, reflecting my interest in history, I participated in the 8 th International Symposium on the History of Anaesthesia held in Sydney. My paper titled, somewhat cryptically and with apologies to Charles Dickens, 1848, and a Pharmacologic Tale of Two Cities, sought to apply some precepts of chemistry, my primary discipline, to anaesthesia, my adopted discipline, and I focused on research of that year by John Snow (1813-1858) in London, and Louis Pasteur (1822-1895) in Paris. I concluded the paper by projecting some of their insights onto my own research on thiopentone, a agent introduced into clinical anaesthesia in the 1930s that became one of the most important substances in the history of anaesthesia. The preparation of that paper became the stimulus for this thesis.

Snow was a “man of ”, in the Victorian language of the day, who had trained as an apothecary on the way to gaining his doctorate in medicine in 1844. 1 This, he achieved mainly by training and study applied to clinical practice. Pasteur was a physical scientist who had written theses in both physics and chemistry for his doctorate in science in 1847, mainly from his own experimental observations on the nature of matter. 2

Snow introduced the principles and practice of dose regulation of volatile anaesthetic agents and thereby the basis of controllable anaesthesia, as well as the basis of what would become pharmacokinetics and pharmacodynamics a century later. Pasteur observed mirror image morphology in crystals of the ‘same’ pure chemical substance, and that solutions thereof rotated plane polarized light but oppositely and equally, thereby laying the basis for what we now refer to as chirality and stereochemistry. In my paper, I elaborated by describing how different stereoisomeric forms of drugs can produce qualitatively and/or quantitatively different pharmacological effects, and that this is not unexpected since the body is built of many different stereochemical molecules with which such substances interact. I have referred to this as ‘stereopharmacology’. Finally, I related that thiopentone is synthesized and used clinically as a racemate, i.e., an equal part mixture of two mirror image stereoisomers or enantiomers. I concluded by describing some relevant aspects of my research on the racemate and separate enantiomers of thiopentone, speculating on how its history might have been different had thiopentone been developed as an enantiopure substance rather than a racemate.

Pasteur’s biographer Patrice Debré wrote an interesting comment – that he was “a chemist among the doctors”. This set me thinking. I trained as a chemist and I also worked among the doctors, attempting to apply the quantitative skills of chemistry to the pharmacology of anaesthesia and acute pain management which, in this thesis, I term ‘anaesthesiology’. This thesis comes from a selection of previously published contributions of stereopharmacology applied mainly to anaesthesiology based on work that I initiated, supervised, and/or at least strongly influenced. Thus it is an account of one particular aspect of the history of pharmacology in anaesthesiology, and my very small contributions to it. Looking back, I think that some of the contributions have remained quite leading edge, others less-so; nonetheless, all are fixed by time in the notions, issues and techniques of the day. I have also included a personal narrative of these events, in acknowledgement of the improbability that anyone will ever be interested enough to ask me to write about such things.

1 Richardson BW. John Snow, M.D., a representative of medical science and art of the Victorian era. In, Richardson BW, ed, The Asclepiad Vol IV, 1887, pp 274-300: Longmans, Green, and Co: London. 2 Debré P. (1998). Louis Pasteur . (Translated by E. Forster). Johns Hopkins University Press: Baltimore Maryland,. ISBN 0-8018-5808-9. vi

Acknowledgements My research was always multidisciplinary, with many colleagues, fellows, postgraduate students, nursing and technical staff of various backgrounds, contributing their abundant skills to an egalitarian research environment. They are named in the publications of our multidisciplinary research groups in various places, and I am pleased to again acknowledge the contributions of each to our research. I learned much from all, and even more from some.

In particular, I acknowledge the Australian and New Zealand College of Anaesthetists for the support provided to me by the inaugural award of the Douglas Joseph Professorship of Anaesthetics in 1993. This encouraged me to further expand my ideas on the stereopharmacology of anaesthetic agents, which eventually led to the theme for this thesis.

Our ‘human lab’ research studies in consenting patients and/or healthy volunteers were performed collaboratively between medical, nursing and scientist colleagues. Our animal research studies required no less expertise, and many of the same colleagues participated in both human and animal studies. Our ‘sheep lab’ studies were particularly ‘high tech’ and labour intensive, involving exacting surgical preparation of the subjects, each with their ongoing care and maintenance for periods of up to several months. Experimental studies typically required many pairs of hands concurrently treating and/or monitoring the subject, handling the instrumentation, performing serial blood sampling, usually from multiple catheters, etc. Our ‘rat lab’ studies were less labour intensive, but they were no less exacting, and also involved surgical preparation and maintenance, along with the pharmacological studies. All of our studies generated many blood samples for subsequent laboratory analysis, and this, too, required the highest expertise in analytical chemistry.

I especially acknowledge my principal co-authors in regard to the theme of this thesis. In alphabetical order, and noting the graduation of my postgraduate candidates who were involved: anaesthetist Stephen Barratt (PhD Syd , 2000); oncologist Fran Boyle; pharmacologist Dennis Chang; pharmacologist Mary Collins/Chebib; veterinary anaesthetist Sue Copeland (PhD Syd , 2008); neurologist Dennis Cordato (PhD Syd , 2001); indefatigable Head of Department Michael Cousins; molecular biologist Ross Davey; organic chemist Colin Duke; neurochemist Steven Edwards (PhD Syd , 2007); analytical chemist Bronwyn Fryirs/Dawson; analytical chemist Xiao-Qing (Sonia) Gu; pharmaceutical chemist Li Huang; cardiac surgeon YiFei Huang (PhD Adel , 1991); animal house manager the Late Ray Kearns; veterinary anaesthetist and surgeon Leigh Ladd (PhD Syd , 2007); anaesthetist Peter McCall; analytical chemist Colin McLean; anaesthetist Larry McNichol; anaesthetist Charles Minto (PhD Syd , 2002); biologist Susan Murphy (PhD Syd , 2007); anaesthetist Craig Nancarrow (PhD Flind , 1987); toxicologist and biostatistician John Plummer; biologist Marie Pryor; anaesthetist and intensivist Bill Runciman (PhD Flind , 1983); biochemist Al Rutten (PhD Flind , 1996); anaesthetist Nigel Sharrock; my wise postgraduate supervisor and medicinal chemist Jack Thomas; my longest-time collaborator and pharmacokineticist Geoff Tucker; pharmacometrician Richard Upton (PhD Flind , 1990); and anaesthetist Bernadette Veering.

I also acknowledge the crucial support of my family in the genesis of this thesis. Sylvia, my partner of more than a half century supported me through the elations and disappointments of research for most of that time, as did our kids, Cary, Neryl and Fiona. At various stages and places, they tolerated yet another late night in the lab finishing a long experiment, or yet another weekend in the office finishing a grant application because, like me, they believed that it all was ‘worth it in the end’. My eternal love and respect to each and all. vii

Thesis structure The thesis is structured as a Foreword , a narrative of four Sections outlining and discussing, rather than re-reviewing, the context and content of the publications, an Afterword , an Appendix, as well as various figures and photographs added to personalise the account. Explanatory notes and references to the work of others are reported in footnotes, as well as the selected publications.

The Foreword presents a brief account of how this thesis was conceived. Section 1 is titled Concepts and tools and here I have attempted to draw together various notions that underpin the theme. Section 2 is titled Translating chemistry into anaesthesiology – in stages and sets out the context of the research chronologically, and acknowledges the institutions and colleagues supporting the research. Section 3 is titled Stereopharmacological research in anaesthesiology and presents the main theme based upon knowledge relevant at the time of the original publications. Section 4 presents a discussion and some conclusions arising from the research. Each subsection is therefore intended to present a ‘stand-alone’ document in its historical context with an accompanying commentary, rather than to present an up-to-date review of each topic. The Afterword presents some additional reflections, largely from an historical perspective. The Appendix gives a full numbered list of publications (cited in square brackets in the narrative), followed by reprints of the selected publications (cited in boldface in square brackets in the narrative). Some repetition within and between sections is hard to avoid, so as to present coherent accounts and to give recognition to those to whom it is due, but hopefully it is not excessive.

Declarations This thesis is submitted in accordance with the University of Sydney (Higher Degree by Research) Rules 2011 (as amended), Part 5 Higher Doctorates.

Rule 5.02 in part, states: (1) The Academic Board may, on the recommendation of the relevant Faculty, award a higher doctorate for published work that, in the opinion of the examiners: (a) constitutes a distinguished contribution to knowledge or creative achievement; and (b) is recognized by scholars in the relevant field as constituting a distinguished contribution to knowledge or creative achievement in that field. Accordingly, I state that the work presented in this thesis comprises a record of published research and scholarship in a relevant field, and is submitted for consideration for the degree of Doctor of Medical Science.

Rule 5.03 in part, states: (1) Subject to this clause 5.03, to be eligible for admission to candidature for a higher doctorate, an applicant must: (a) hold a degree from the University that was conferred five or more years prior to the application date. Accordingly, I state that I hold the degrees of Master of Science (conferred 1968) and Doctor of Philosophy (conferred 1972) of the Faculty of Science of the University of Sydney.

Rule 5.04 in part, states: (2) The application must be accompanied by: (a) a list of the published works that the candidate proposes to submit for examination; (b) a description of the themes of the published works; and viii

(c) where there are a large number of publications whose dates range over a period of time and cover a range of subjects, a statement of how these publications are related to one another and to the theme. Accordingly, I state that this thesis is based on a selection of my publications on a theme in a relevant field – in this case, that of the bearing of drug stereochemistry to some or other aspect of pharmacology, mainly in the field of anaesthesiology.

Selected publications I am conscious that, over time, my principal research role evolved into being the director of various concurrent research programs and projects. This brought the responsibilities of the initiation and preparation of research grant applications, supervision of postgraduate students, maintenance of day-to-day progress in each project, and responsibility for submission of reports, reviews, and other publications arising from the studies performed. My main functions thereby became those of mentoring and providing proper welfare and resources; accordingly, I participated less and less in ‘hands-on’ involvement in day-to-day experiments. The selected publications present a cohesive theme from my work performed at several institutions, with numerous colleagues including many postgraduate candidates. Whenever practicable, any co-authoring postgraduate candidates were placed first in authorship.

I declare that I have been the author or co-author of each of the selected publications, numbered according to the list below. None has been previously submitted by me for a degree of The University of Sydney or any other university.

(a) Publications for which I was solely responsible: Nos. 200, 214, 243, 313, 327, 328

(b) Publications for which I initiated the work, provided significant input to the experimental work performed with my co-author student(s), fellow(s), and/or collaborator(s), and was solely or substantially responsible for the writing: Nos. 110, 170, 198, 244, 245, 248, 267, 282, 325

(c) Publications for which I initiated the work, contributed to the experimental work performed by my co-author student(s), fellow(s), and/or collaborator(s), and was substantially responsible for the writing: Nos. 48, 49, 185, 190, 197, 215, 216, 221, 242, 259, 262, 265, 266, 307

(d) Publications for which I initiated the work, contributed to the experimental work performed by my co-author student(s), fellow(s), and/or collaborator(s), and was partly responsible for the writing: Nos. 177, 188, 216, 217, 218, 250

(e) Publications that are mainly collaborative, for which I contributed to the concept, experimental design and/or data analysis of work performed by my co-author student(s), fellow(s), and/or collaborator(s), and was partly responsible for the writing: Nos. 197, 224, 246, 273, 284, 287, 303, 305

(f) Publications that have contributed to theses submitted for the degree of Doctor of Philosophy of The University of Sydney by a candidate under my supervision who is the first named author: Nos. 232, 261, 264, 273, 285, 287, 289, 296, 299, 300, 316, 319, 320, 321, 323, 324

ix

(g) Publications that have contributed to theses submitted for the degree of Doctor of Philosophy of The University of Sydney by a candidate under my supervision who is another of the named authors: Nos. 269, 276, 280, 306, 314

(h) Publications that were additionally included within a thesis submitted for the degree of Doctor of Science of The University of Sydney by my colleague Michael Cousins: Nos. 48, 259, 266, 267

The publications listed below are grouped under somewhat sketchy subheadings and the supporting institution, essentially in chronological order. Each publication is designated as a “Study” (experimental observations and new knowledge), a “Review” (synthesis of ideas based on relevant knowledge), or a “Patent”, and assigned the identifying number (in square brackets) according to the Appendix. Each is finally designated (in parenthesis) by authorship according to the above Declaration.

Basic concepts and experimental tools (Flinders University) Review 01. [110]. Mather LE, Runciman WB. The physiological basis of pharmacokinetics: concepts and tools. In, Quantitation, Modelling and Control in Anaesthesia , Stoeckel HO, ed. Georg Thieme: Stuttgart, pp 12-40, 1985 (b) Introductory reviews of stereochemistry-pharmacolology (The University of Sydney) Review 02. [198]. Mather LE, Rutten AJ. Stereochemistry and its relevance in anaesthesiology. Current Opinions in Anaesthesiology 4: 473-479, 1991 (b) Review 03. [214]. Mather LE. "Chirality is in your hands" - the Australasian Visitor’s Lecture for 1994. ANZCA Bulletin 3: 26-32, 1994 (a) Review 04. [215]. Mather LE, Björkman S. Pitfalls in pharmacokinetics. Anaesthesia Pharmacology Reviews 2: 260-270, 1994 (c) Local anaesthetics - basic concepts and tools (Flinders University) Review 05. [48]. Mather LE, Cousins MJ. Local anaesthetics and their current clinical use. Drugs 18: 185-205, 1979 (c,h) Review 06. [49]. Tucker GT, Mather LE. Clinical pharmacokinetics of local anaesthetic agents. Clinical Pharmacokinetics 4: 241-278, 1979 (c) The start of the stereopharmacology research - prilocaine (The University of Sheffield) Study 01. [197]. Tucker GT, Mather LE, Lennard MS, Gregory A. Plasma concentrations of the stereoisomers of prilocaine after administration of the racemate - implications for toxicity? British Journal of Anaesthesia 65: 333-336, 1990 (c) Local anaesthetics program – focus on chiral agents (Flinders University and The University of Sydney) Study 02. [170]. Mather LE. Disposition of mepivacaine and bupivacaine enantiomers in the sheep. British Journal of Anaesthesia 67: 239-246, 1991 (b) Study 03. [177]. Rutten AJ, Mather LE, McLean CF. Cardiovascular effects and regional clearances of IV bupivacaine in sheep: enantiomeric analysis. British Journal of Anaesthesia 67: 247-256, 1991 (d) x

Study 04. [188]. Rutten AJ, Mather LE, Plummer JL, Henning EC. Postoperative course of plasma protein binding of lignocaine, ropivacaine and bupivacaine in sheep. Journal of and Pharmacology 44: 355-358, 1992 (d) Study 05. [190]. Rutten AJ, Mather LE., McLean CF. Tissue distribution of bupivacaine enantiomers in sheep. Chirality 5: 485-491, 1993 (c) Study 06. [217]. Mather LE, Rutten AJ, Plummer JL. Pharmacokinetics of bupivacaine enantiomers in sheep: influence of dosage regimen and study design. Journal of Pharmacokinetics and Biopharmaceutics 22: 481-498, 1994 (d) Study 07. [218]. Mather LE, McCall P, McNichol PL. Bupivacaine enantiomer pharmacokinetics after intercostal neural blockade in liver transplantation patients. and Analgesia 80: 328-35, 1995 (d) Study 08. [246]. Sharrock N, Mather LE, Go G, Sculco TP. Arterial and pulmonary arterial concentrations of the enantiomers of bupivacaine following epidural injection in elderly patients. Anesthesia and Analgesia 86: 812-817, 1998 (e) Study 09. [250]. Gu X-Q, Fryirs B, Mather LE. High-performance liquid chromatographic separation and nanogram quantitation of bupivacaine enantiomers in blood. Journal of Chromatography B 719: 135-140, 1998 (d) Study 10. [244]. Huang YF, Pryor ME, Veering BT, Mather LE. Cardiovascular and central nervous system effects of bupivacaine and levobupivacaine in sheep. Anesthesia and Analgesia 86:7 97-804, 1998 (b) Study 11. [245]. Mather LE, Huang YF, Pryor ME, Veering BT. Systemic and regional pharmacokinetics of bupivacaine and levobupivacaine in sheep. Anesthesia and Analgesia 86: 805-811, 1998 (b) Review 07. [243]. Mather LE. Levobupivacaine – a viewpoint. Drugs 56: 355-364, 1998 (a) Review 08. [273]. Gennery B, Mather LE, Strichartz G. Levobupivacaine: New preclinical and clinical data. Seminars in Anesthesia 19: 132-148, 2000 (e) Patent 01. Inventors: Mather LE, Richards AJM. Levobupivacaine and its use . Patent Number: US6,069,155. Publication date: 2000-05-30. Assignee: Darwin Discovery Ltd (e) Patent 02. Inventors: Mather LE; Bardsley HJ. Levobupivacaine useful for managing chronic pain . Patent Number: US5,955,47. Publication date: 1998-06-11. Assignee: Ltd (e) Study 12. [276]. Chang DH-T, Ladd, LA, Wilson KA, Gelgor L, Mather LE. Tolerability of large dose intravenous levobupivacaine in sheep. Anesthesia and Analgesia 91: 671- 679, 2000 (g) Study 13. [285]. Ladd LA, Mather LE. Central effects index – a semi-quantitative method for assessment of CNS toxicity of local anaesthetic agents in sheep. Journal of Pharmacological and Toxicological Methods 44: 467-476, 2000 (published 2001) (f) Study 14. [280]. Chang DH-T, Ladd LA, Copeland S, Iglesias M, Plummer JL, Mather LE. Direct cardiac effects of intracoronary bupivacaine, levobupivacaine and ropivacaine in the sheep. British Journal of Pharmacology 132: 649-658, 2001 (g) Review 09. [282]. Mather LE, Chang DHT. Cardiotoxicity of local anaesthetics. Drugs 61: 333-343, 2001 (b) xi

Study 15. [299]. Ladd LA, Chang DH-T, Wilson K, Copeland S, Plummer JL, Mather LE. Effects of CNS site-directed carotid arterial infusions of bupivacaine, levobupivacaine and ropivacaine in sheep. Anesthesiology 97: 418-428, 2002 (f) Study 16. [306]. Mather LE, Ladd LA, Chang DH-T, Copeland SE. The effects of imposed acid-base derangement on the cardio-activity and pharmacokinetics of bupivacaine and thiopental. Anesthesiology 100: 1447-1457, 2004 (g) Review 10. [314]. Mather LE, Copeland SE, Ladd LA. Acute toxicity of local anesthetics: underlying pharmacokinetic and pharmacodynamic concepts. Regional Anesthesia & Pain Medicine 30: 553-568, 2005 (g) Study 17. [323]. Copeland SE, Ladd LA, Gu X-Q, Mather LE. The effects of general anesthesia on the central nervous and cardiovascular system toxicity of local anesthetics. Anesthesia and Analgesia 106: 1429-1439, 2008 (f) Study 18. [324]. Copeland SE, Ladd LA, Gu X-Q, Mather LE. The effects of general anesthesia on whole body and regional pharmacokinetics of local anesthetics at toxic doses. Anesthesia and Analgesia 106: 1440-1449, 2008 (f) Review 11. [325]. Mather LE, Tucker GT. Properties, absorption and disposition of local anesthetics. In: Neural Blockade , 4th edition. Cousins MJ, Bridenbaugh PO, Horlocker T, Carr DB, eds Lippincott: Philadelphia, pp 45-91, 2008 (b) Review 12. [327]. Mather LE. Comparative clinical pharmacology of levobupivacaine with other local anesthetics (in Japanese, translated by T Yamada, T Mori and A Asada) In: Basic Science and Clinical Experience of Levobupivacaine and Other Local Anesthetics . Asada A, Nishikawa K, eds. Kokuseido Publishing Co Ltd. Tokyo, pp 3- 50, 2010 (a) Review 13. [328]. Mather LE. The acute toxicity of local anesthetics. Expert Opinions on Drug Metabolism and Toxicology 6: 1313-1332, 2010 (a) Intravenous anaesthetics program - focus on thiopentone (The University of Sydney) Patent 03. Mather LE, Duke CC. Synthesis and uses of thiopentone enantiomers . WO2000/024358, 04 May 2000. Assignee: The University of Sydney, with the Filing Date of October 22, 1999 and Priority Date of October 22, 1998 (e) Study 19. [221]. Huang JL, Mather LE, Duke CC. High-performance liquid chromatographic determination of thiopentone enantiomers in sheep plasma. Journal of Chromatography B 673: 245-250, 1995 (c) Study 20. [224]. Mather LE, Upton RN, Huang JL, Ludbrook GL, Gray E, Grant C. The systemic and cerebral kinetics of thiopental in sheep: enantiomeric analysis. Journal of Pharmacology and Experimental Therapeutics 279: 291-297, 1996 (e) Study 21. [232]. Cordato D, Gross AS, Herkes G, Mather LE. The pharmacokinetics and pharmacodynamics of the enantiomers of thiopentone following a single intravenous bolus injection and under conditions of prolonged intravenous infusion. British Journal of Clinical Pharmacology 43: 355-362, 1997 (f) Study 22. [259]. Mather LE, Edwards SR, Duke CC, Cousins MJ. Enantioselectivity of thiopental distribution into central neural tissue of rats: an interaction with halothane. Anesthesia and Analgesia 89: 230-235, 1999 (c,h) xii

Study 23. [262]. Mather LE, Edwards SR, Duke CC. Electroencephalographic effects of thiopentone enantiomers in the rat: Correlation with drug tissue distribution. British Journal of Pharmacology 128: 83-91, 1999 (c) Study 24. [261]. Cordato D, Collins M, Johnston GAR, Mather LE. Stereoselective interaction of R-(+)-thiopentone and S-(-)-thiopentone enantiomers at the GABA A receptors. British Journal of Pharmacology 128: 77-82, 1999 (f) Study 25. [264]. Cordato DJ, Mather LE, Gross AS, Herkes GK. Pharmacokinetics of thiopental enantiomers during and following prolonged high-dose therapy. Anesthesiology 91: 1693-1702, 1999 (f) Study 26. [265]. Mather LE, Edwards SR, Duke CC. Electroencephalographic effects of thiopentone and its enantiomers in the rat. Life Sciences 66: 105-114, 1999 (c) Study 27. [266]. Mather LE, Edwards SR, Duke CC, Cousins MJ. Microdialysis study of the blood-brain equilibration of thiopentone enantiomers. British Journal of Anaesthesia 84: 67-73, 2000 (c,h) Study 28. [284]. Coates KA, Mather LE, Johnson R, Flood P. Thiopental is a competitive inhibitor at the human a7 nAChR. Anesthesia and Analgesia 92: 930-933, 2001 (e) Study 29. [287]. Cordato DJ, Herkes GK, Mather LE, Gross AS, Finfer SR, Morgan MK. Prolonged high-dose thiopental for neurological and neurosurgical emergencies. Anaesthesia and Intensive Care 29: 339-348, 2001 (f) Review 14. [303].Cordato DJ, Mather LE, Herkes GK, Morgan. MK. for acute neurological and neurosurgical emergencies – do they still have a role? Journal of Clinical Neuroscience 10:283-288, 2003 (e) Study 30. [306]. Mather LE, Ladd LA, Chang DH-T, Copeland SE. The effects of imposed acid-base derangement on the cardio-activity and pharmacokinetics of bupivacaine and thiopental. Anesthesiology 100: 1447-1457, 2004 (g) Study 31. [307]. Mather LE, Duke CC, Ladd LA, Copeland SE, Gallagher G, Chang DH-T. Direct cardiac effects of coronary site-directed thiopental and its enantiomers: a comparison to propofol in conscious sheep. Anesthesiology 101: 354-364, 2004 (c) Nonsteroidal anti-inflammatory drug program – focus on ketorolac (The University of Sydney) Review 15. [200]. Mather LE. Do the pharmacodynamics of the non-steroidal anti- inflammatory drugs suggest a role in the management of postoperative pain. Drugs 44(Supplement 5): 1-13, 1992 (a) Study 32. [216]. Mills MH, Mather LE, Gu XQ, Huang JL. Determination of ketorolac enantiomers in plasma using enantioselective liquid chromatography on an alpha 1- acid glycoprotein chiral stationary phase and ultraviolet detection. Journal of Chromatography 658: 177-182, 1994 (d) Study 33. [242]. Ronnhedh C, Jaquenod M, Cousins MJ, Power I, Eckstein RP, Jordan V, Mather LE. Factors influencing ketorolac-associated perioperative renal dysfunction. Anesthesia and Analgesia 86: 1090-1097, 1998 (c) Study 34. [269]. Mather LE, Cousins MJ, Huang YF, Pryor ME, Barratt SMcG. Lack of secondary hyperalgesia and central sensitization in an acute sheep model. Regional Anesthesia & Pain Medicine 25: 174-180, 2000 (g) xiii

Halothane project (The University of Sydney) Study 35. [267]. Mather LE, Fryirs BL, Duke CC, Cousins MJ. Lack of whole body pharmacokinetic differences of halothane enantiomers in the rat. Anesthesiology , 92: 192-196, 2000 (b,h) Ketamine project (mainly The University of Sydney) Study 36. [289]. Edwards SR, Mather LE. Tissue uptake of ketamine and enantiomers in the rat: indirect evidence for metabolic inversion. Life Sciences 69: 2051-2066, 2001 (f) Study 37. [296]. Edwards SR, Minto CF, Mather LE. Concurrent ketamine and alfentanil administration: pharmacokinetic considerations. British Journal of Anaesthesia 88: 94-100, 2002 (f) Study 38. [300]. Edwards SR, Mather LE. Alfentanil potentiates anaesthetic and electroencephalographic responses to ketamine in the rat. European Journal of Pharmacology 460: 27-35, 2003 (f) Study 39. [320]. Edwards SR, Mather LE, Smith MT. Studies with ketamine and alfentanil following Freund’s complete adjuvant-induced inflammation in rats. Clinical and Experimental Pharmacology and Physiology 34: 414-420, 2007 (f) Thalidomide project (The University of Sydney) Study 40. [316.] Murphy-Poulton SF, Boyle F, Gu X-Q, Mather LE. Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP. Journal of Chromatography B 831:48-56, 2006 (f) Study 41. [319]. Murphy S, Boyle FM, Davey RA, Gu X-Q, Mather LE. Enantioselectivity of thalidomide serum and tissue concentrations in a rat glioma model and effects of combination treatment with cisplatin and BCNU. Journal of Pharmacy and Pharmacology 59: 105-114, 2007 (f) Study 42. [321]. Murphy S, Davey RA, Gu X-Q, Haywood MC, McCann LA, Mather LE, Boyle FM. Enhancement of cisplatin efficacy by thalidomide in a 9L rat glioma model. Journal of Neuro-Oncology 85: 181–189, 2007 (f) Other reviews of stereopharmacology (The University of Sydney) Review 16. [248]. Mather LE, Edwards SR. Chirality in anaesthesia: ropivacaine, ketamine and thiopentone. Current Opinions in Anaesthesiology 11:383-390, 1998 (b) Review 17. [305]. Cordato DJ, Mather LE, Herkes GK. Stereochemistry in clincal medicine: a neurological perspective. Journal of Clinical Neurosciences 10: 649-654, 2003 (e) Review 18. [313]. Mather LE. Stereochemistry in anaesthetic and analgetic drugs. Minerva Anestesiologica 71: 507-516, 2005 (a)

Laurence Edward Mather Emeritus Professor, Discipline of Anaesthesia July 29 2015 xiv

We tried very hard…and sometimes succeeded... 1

1. Concepts and tools

“When the only tool you own is a hammer, every problem begins to resemble a nail.” Abraham Maslow (1908-1970)

This thesis is based on a selection of previously published works on the theme of stereopharmacological research in anaesthesiology . In this Section, I have attempted to draw together various concepts and tools used to construct my theme.

Chemistry is my undergraduate discipline, with a transition via pharmaceutical sciences as a postgraduate student to applied pharmacology as a profession, and I have attempted to apply the concepts and tools of applied pharmacology to anaesthesiology, my adopted discipline.

I have chosen to include a brief discourse on the origins of modern anaesthesia, not just because of its truly fascinating pharmacological history, or its enormous practical impact in permitting the development of modern surgery, or even the exceptional biography of the founding father of anaesthesia science, John Snow, but to make the case, as did John Snow in 1847, that anaesthesia has quantitative chemistry as its basis.

This Section also includes elements of the development of modern analytical chemistry, and necessarily so, because quantitative analyses of drugs and related substances in biofluids are the essential tools of pharmacokinetics and pharmacodynamics, themselves key concepts of modern pharmacology, and the matter of much of this thesis. These pharmacological concepts and tools, however, are rather more complex when the resolution of stereoisomers necessarily precedes their quantitation. Thus the analysis of biofluids for stereoisomers, although routine nowadays, is mentioned in greater measure. However, before proceeding with issues of pharmacology and anaesthesiology, I have included some basic medicinal chemistry to introduce the subject drugs of this thesis.

1.1 Basic medicinal chemical concepts Drugs are the ‘superstars’ of the chemical substance world. Most are ingested voluntarily to either extend our life or improve the way that we feel, typically as part of the treatment plan for a medically recognised condition. They typically act by altering or regulating some or other normal or deranged physiological function. Others, including the subject drugs of this thesis, are used mainly by medical specialists to modify particular physiological processes as part of a discrete procedure performed on their patients.

Traditionally made from folkloric-selected natural products on an empirical basis, the vast majority of contemporary drugs are produced synthetically or semi-synthetically from natural product starting materials. The science of medicinal chemistry evolved to assist in making the selection of drugs more systematic and less empirical, although some empiricism still remains among various legacy drugs as well as others having a structure that, at least superficially, seems out of place with an established mechanism of action.

The science of medicinal chemistry, as the name suggests, is based on understanding the relationship between the chemical and physical properties of a substance and the physiological modifying properties that confer the status on that chemical of being a drug. The science began with empirical observations of a particular chemical having a particular 2 effect and evolved largely when homologues, derivatives and/or analogues were selected and/or modified to enhance that particular effect.

Physicochemical properties of drugs dominate as determinants of their pharmacology and these, of course, are the direct resultant of their chemical structure. In a pharmacological context, the properties of drug acidity or basicity dominate through the exent of ionization at the environmental pH, as notated by the pKa (the negative logarithm of the acid dissociation constant, or of the conjugate acid in the case of a base). The other dominant property is lipophilicity because it determines, to some exent, the distribution of every chemical substance between aqueous and lipoidal milieux, and thus distribution into and across every body membrane - a precursor for most drug-derived effects. Lipophilicity is mainly related to the extent of hydrocarbon substitution in a molecule, and is often notated by a distribution or partition coefficient. These factors combine to determine the polarity of a molecule and how a particular substance moves down a concentration gradient, and thus achieves receptor accessibility and binding, both being precursors to drug effect.

The drug subjects of this thesis come from different pharmacological classes and, except within the several local anaesthetic agents, have markedly different chemical structures and consequent physicochemical properties. Various aspects of their chemistry and pharmacology are discussed in the selected publications and in Section 3, and so comments here are intended only as a brief introduction to the selected drugs.

(i) Local anaesthetic agents. The local anaesthetic agents all come from the chemical subclass usually referred to as amino amides (bases). For water solubility for injection they are mainly prepared as HCl salts, and thus are presented in a slightly acidic solution (pH 3.5 – 5.5). The conjugate acids of the agents have a pKa in the range of 7.7 - 8.1. The base forms are lipophilic, enabling neural uptake from an injectable aqueous solution. Among the subject drugs, prilocaine contains a secondary amino group; mepivacaine, ropivacaine, and bupivacaine each contains a tertiary amino group, with the rank order of lipophilicity of the bases of these substances increasing in the order mentioned.

Structural formula of mepivacaine shown as the base form. (ii) Thiopentone. Thiopentone is weakly acidic with a pKa of 7.7, a result of ionization of the thioenol form existent through dynamic equilibrium of the thioketo and thioenol tautomeric forms. Thiopentone itself is a highly lipophilic molecule that is made water soluble for injection by ionization with a sodium carbonate-bicarbonate base mixture.

Structural formula of thiopentone shown as the sodium salt of the thioenolate form. 3

(iii) Ketorolac. Ketorolac is a pyrrolizine carboxylic acid derivative with a pKa of 3.84. It is lipophilic in the acid form, and is made water soluble for injection by combination with the conjugate acid of (2-amino-2-(hydroxymethyl)-1,3-propanediol), having a pKa of 8.07, and often referred to as tris, THAM, tromethamine or trometamol.

Structural formula of ketorolac shown as the acid form. (iv) Halothane. The volatile liquid has a specific gravity of 1.86 at 20º, a boiling point of 50.2º at 760 mm Hg and a vapour pressure of 243 mm Hg at 20º and is thus readily administered in inspired air. It is a non-ionizing highly lipophilic substance with a water solubility of 0.345:100 parts, a blood:gas partition coefficient of 2.3 and an oil:gas partition coefficient of 330.

Structural formula of halothane. (v) Ketamine. Ketamine is a secondary amine with a pKa of 7.5, and is prepared as the HCl salt and thus is presented for injection in a slightly acidic solution ( pH 3.5 –5.5). The base is highly lipophilic and is readily distributed into the central nervous system.

Structural formula of ketamine shown as the base. (vi) Thalidomide (α-phthalimidoglutarimide) has a pKa of ~11.6 and thus is essentially non- ionizing but reasonably polar substance that is sparingly soluble in most solvents including water, ethanol, , glacial acetic acid, ether and but is readily soluble in dioxane, pyridine and . It is mainly used as an oral preparation, rather than an injectable, but may also be made into a suspension for some dosage forms.

Structural formula of thalidomide.

1.2 A glossary of basic stereochemical concepts Stereochemistry (i.e., chemistry in three-dimensional space) is crucial in most physiological signalling systems and so the stereochemistry of the drug may, as mentioned in various of the 4 selected publications, be another significant qualitative and/or quantitative determinant of drug action. Accounts of the principles of stereochemistry are given in many places; a concise summary of the notation and rules of naming substances has been published under the auspices of the International Union of Pure and Applied Chemistry (IUPAC) as the 1974 Recommendations. 3 Chirality is one form of stereochemistry, and is identifiable by the presence of one or more asymmetric centres in a molecule, most usually a carbon atom, and this is the basis of much of the present awareness of drug stereochemistry. All of the subject drugs of this thesis have a single carbon atom as the centre of chirality generating a single pair of stereoisomers. Apart from the direction of optical rotation the physicochemical properties of the resultant pairs of stereoisomers are identical.

Isomers : molecules having the same atoms in different arrangements. Stereoisomers : isomers with different spatial arrangements that cannot be made to be superimposed without the breaking and remaking of covalent bonds. Chiral : from Greek χειρ = hand; chirality = “handedness”. This is a general scientific term that indicates a form or system distinguishable from its mirror image; the concept is encountered in physics, mathematics, biology and chemistry. In this thesis, the term is used only to apply to chemical molecular properties. Chiral centre : locus for plane of asymmetry. In molecular chemistry, this is a source of left (–)- or right (+)-handed rotation of plane polarized light. A molecule may have one or more asymmetric (or stereogenic or chiral) centres, i.e., having a chemical structure in such a way that the structure and its mirror image are not superimposable. The analogy of human hands (Figure 1.1) is pertinent. A molecule lacking a chiral centre may be designated “achiral” if a point is to be made.

Figure 1.1: The hands ‘mirror image’ conceptualization of chirality superimposed with a ball-and-stick depiction of an amino acid – the chiral building blocks of proteins, and thus the basis for stereopharmacology.

Enantiomers or optical isomers : mirror image stereoisomers. These may be specified by several widely-used systems. (i) The original (c. 1848, empirical) system is based on the direction of optical rotation of plane polarized light and the molecules are designated as dextro -, d or (+) if rotating plane polarized light to the right, or alternatively levo -, l or (-) if rotating plane polarized light to the left. (ii) An older (c. 1900, functional) system known as

3 Cross LC, Klyne W. Rules for the Nomenclature of Organic Chemistry. Pure Appl Chem 45: 11-30, 1976 5 the Fischer System, was originally applied by Emil Fischer to carbohydrates. This does not relate to optical rotation, but designates the molecules as “D”, or alternatively “L”, on the basis of their structural resemblance to the reference molecules, respectively (+)- glyceraldehyde, arbitrarily assigned a “D-configuration”, and (-)-serine, arbitrarily assigned an “L-configuration”. (iii) The preferred designation is by absolute configuration, as R- (rectus = right) or alternatively S- ( sinister = left) on the basis of a priority order of molecular arrangement of substituents about the chiral centre, according to the CIP (Cahn-Ingold- Prelog) Priority (or Sequence) Rules, articulated during the mid-1960s. Unfortunately, even contemporary literature contains a mixture of naming conventions and print typeface. In this thesis, CIP designation is used wherever possible and, for simplicity, the optical rotation of a substance has been omitted where it is not germane, and regular font has been used in place of italic font.

Figure 1.2: The ‘mirror image’ depiction of chirality in the halothane molecule shown in planar and ball-and- stick drawings. The chiral carbon is marked *.

Cahn-Ingold-Prelog Priority (or Sequence) Rules: Briefly, Rule 1: atoms of higher atomic number take precedence over those of lower atomic number. Lone pairs of electrons are assigned the lowest priority; Rule 2: isotopes of higher atomic weight take precedence; Rule 3: in molecules where two or more of the atoms directly attached to the stereogenic centre are the same e.g. in the compounds below in which three of the atoms attached to the stereogenic central carbon are carbon, then the order of priority is of the next atoms along the chain; Rule 4: relates to molecules bearing unsaturated groups attached to the stereogenic central atom; Rule 5: when the difference between substituents is in configuration then (R) takes precedence over (S). Having established the priorities, the molecule is viewed so that the atom/group with the lowest priority points away in space and with the face of the molecule pointing outward, the three other groups are arranged in order of decreasing priority. A clockwise decreasing order is assigned the (R)-configuration and an anti-clockwise decreasing order is assigned the (S)-configuration. 6

Figure 1.3: Application of the CIP Rules to bupivacaine. The chiral carbon is marked *. Spatiality is indicated by the heavy bond extending from the plane of the page. The direction of the sequence priority is shown by the arrow and dotted lines. [From Review 03, 215 } Racemate : an equimolar mixture of R- and S-enantiomers of a chiral substance: it does not rotate plane polarized light, and may be designated as RS-, rac -, DL-, (±)- or may remain without such designation unless a point is being made about stereochemistry. For example, the intravenous anaesthetic agent thiopentone (discussed in Sections 2 and 3 of this thesis) is synthesized and used clinically without specification as the racemate ( rac - or RS-thiopentone) but is the equimolar mixture of (+)-R-thiopentone and (-)-S-thiopentone. Racemization is the conversion of a chiral form into its enantiomeric counterpart by chemical or biological reaction. A is an equal part preparation of the two enantiomers. A non- racemic mixture is a preparation of the two enantiomers in unequal parts. Enantiopure is used to refer to a substance being one enantiomer free from the other, or a ‘single enantiomer ’ in common parlance. Stereoselective or stereospecific are sometimes ambiguous terms. Chiral natural products are frequently enantiopure as enzymic synthesis is typically stereospecific. Chemical synthesis is typically not stereospecific (i.e., there is an equal probability of both enantiomers being products) unless chiral synthesis is specifically undertaken to preserve or to introduce a chiral centre. Most pharmacological-biological reactions are more or less stereoselective, i.e., there is typically an enantiomeric preference rather than an enantiomeric exclusivity. The presence of small quantities of the other enantiomer as a ‘contaminant’ or as a ‘metabolite’ due to metabolic or chemical racemization, however, can be problematic in the interpretation of pharmacological research. Hence, there is a requirement that appropriate stereospecific assays be used to examine any dependent claims. Selected are briefly mentioned here using International Union of Pure and Applied Chemistry (IUPAC) nomenclature to indicate absolute configuration and optical rotation, and are described pharmacologically in other sections, typically using their trivial names. (i) Local anaesthetics. Many local anaesthetics, including lignocaine (lidocaine), procaine and tetracaine, are achiral, but those derived from natural products, such as cocaine, typically are chiral. The publications comprising this thesis involve the following chiral local anaesthetic agents: Prilocaine [(RS)-N-(2-methylphenyl)-N2-propylalaninamide] consisting of the (+)-R- and (-)- S-prilocaine enantiomer pair. 7

Mepivacaine [(RS)-N-(2,6-dimethylphenyl)- 1-methyl-piperidine-2-carboxamide] consisting of the (-)-R- and (+)-S-mepivacaine enantiomer pair: (racemic) mepivacaine is also designated as DL-, (±)- or dl -mepivacaine (or just mepivacaine). Bupivacaine [(RS)-1-butyl-N-(2,6-dimethylphenyl) piperidine-2-carboxamide] consisting of the (+)-R- and (-)-S-bupivacaine enantiomer pair: (racemic) bupivacaine is also designated as DL-, (±)- or dl -bupivacaine (or just bupivacaine); levobupivacaine is the internationally approved name for the enantiopure local anaesthetic (-)-S-bupivacaine, which is also designated as L(-), (-)- or l-bupivacaine; its enantiomer, dexbupivacaine (or dextrobupivacaine), is (+)-R-bupivacaine and has been used as an experimental local anaesthetic only: it is also designated as, D-, (+) or d-bupivacaine. Inevitably and regrettably, the various terms appear synonymously and interchangeably, in both this thesis and elsewhere in the literature, according to the historical context. Note also that the optical rotations of the mepivacaine enantiomers are the opposite of those of the same configurations of bupivacaine. Ropivacaine , the propyl homologue of levobupivacaine, is (S)-(-)-N-(2,6-dimethylphenyl)-1- propylpiperidine-2-carboxamide. (ii) Thiopentone [(RS)-[5-ethyl-4,6-dioxo-5-(pentan-2-yl)-1,4,5,6-tetrahydropyrimidin-2- yl]sulfanide sodium], consisting of the (+)-R- and (-)-S-thiopentone enantiomer pair. (iii) Ketorolac [(RS)-5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid], consisting of the (+)-R- and (-)-S-ketorolac enantiomer pair. (iv) Halothane [(RS)-2-bromo-2-chloro-1,1,1-trifluoroethane], consisting of the (+)-R- and (-)-S-halothane enantiomer pair. (v) Ketamine [(RS)-2-(o-chlorophenyl)-2-methylaminocyclohexanone)], consisting of the (-)- R- and (+)-S-ketamine enantiomer pair. (vi) Thalidomide [(RS)-2-(2,6-dioxopiperin-3-yl)isoindol-1,3-dione)], consisting of the (+)-R- and (-)-S-thalidomide enantiomer pair.

1.3 Essentials of experimental concepts and tools Stereopharmacology is formed by the intersection of drug stereochemistry and pharmacology. It’s not a word in wide usage – but the meaning is plain enough, especially when pharmacology is interpreted as encompassing the quantitative concepts and tools of pharmacokinetics and pharmacodynamics, as described later. The research described in this thesis is based on some chiral drugs in which various studies were performed to investigate the bearing of their stereochemistry on some or other aspect of their pharmacology. In this thesis, I refer to this as ‘ stereopharmacology’ .

My adopted disciplines, into which I attempted to apply the precepts of chemistry and pharmacology, are anaesthesia and pain medicine. Whereas some authorities prefer to regard anaesthesia and pain medicine as separate disciplines, others combine them, essentially reflecting the origins and purpose of anaesthesia to ablate pain during surgery. It is also reasonable to keep them separate as the practice of pain medicine is truly multidisciplinary, involving those who specialise in treating the ‘psyche’, as well as the ‘physicke’. Some who prefer to combine them typically tend to think more about the management of acute pain, particularly, as the continuum of the ablation of perioperative pain, as I have learned to do. In this thesis, I refer to this as ‘ anaesthesiology’ .

As discussed in Section 2.1, I commenced my postgraduate research career in 1966 – at a stage when quantitative measurement of small quantities of organic compounds was set to be 8 revolutionized through gas-liquid chromatography (GLC) which had been evolving from the late 1950s. Gas-liquid chromatography permitted a substantial improvement in specificity and sensitivity over the century-old spectroscopic methods, as well as the more recent and widely-used ion pair dye colorimetric methods current at that time. 4 At the start of my MSc research project, I tried using the (methyl orange) dye methodology to quantitate the concentrations of lignocaine (aka lidocaine 5) in biological fluids, but promptly changed to GLC as soon as it became available in our lab. This, thereby, became the start of a program on local anaesthetic agents that would span my entire postgraduate career. It is no exaggeration to point out that GLC, more than any other tool, facilitated the growth of pharmacokinetics, itself a new concept in pharmacy and pharmacology that also had been evolving from the late 1950s. However, at that stage, I was unaware that many of the concepts and tools of what would become my main research theme had originated over a century earlier, with the seminal work of anaesthetist John Snow and chemist Louis Pasteur.

1.4 Quantitation in anaesthesia – John Snow It is to John Snow and his writings of the mid-19 th Century that we now ascribe a quantitative approach to anaesthesia. I first learned about John Snow in my medicinal chemistry readings about the physicochemical basis of anaesthesia.

As is well known, the first public demonstration of the inhalation of the vapour of ‘sulphuric ether’ (so-known because of its method of production, chemically named , but commonly referred to simply as ‘ether’) was by William TG Morton for producing surgical anaesthesia in Boston on October 16 1846.

The first uses of ether for anaesthesia in Great Britain occurred in both London and Dumfries on December 19 1846. Its use in London is well documented: this was at the home of American botanist Francis Boott, recipient of the ‘news of etherization’ directly from surgeon Henry Jacob Bigelow of Boston. The ether was administered by James Robinson, highly respected Surgeon-Dentist to the Metropolitan Free Hospital, for the painless extraction of a diseased lower molar tooth. However, its intended repetition in other patients a few days later had only mixed success, 6 a scenario that turned out to be not uncommon due to design deficiencies in the administration apparatus used. Its use in Dumfries by William Scott has been much harder to document, but the claim for priority there remains intriguing.7

On December 21 1846, the first use of ether for general surgery occurred in the operating theatre of University College Hospital when Robert Liston amputated the thigh of a man “previously narcotised by inhalation of ether vapour” using “an ingenious apparatus extemporaneously contrived by Mr. Squire, of Oxford Street”, and this was reported in one

4 Sung CY, Truant AP. The physiological disposition of lidocaine and its comparison in some respects with procaine. J Pharmacol Exp Ther 112(4): 432-443, 1954 5 Lidocaine is specified in the United States Pharmacopeia (USP) as the anhydrous HCl salt whereas the British specifies lignocaine as the monohydrate of the HCl salt. Thus, in preparing a solution, the amount of lignocaine would be increased by the water to produce the correct strength, i.e. if specified as say 2% L.HCl so it would be 87/81 times the weight of L.HCl.H2O added, etc. See: Wiedling S. Xylocaine: The pharmacological basis of its clinical use . 2nd edition. Almqvist & Wiksell, Stockholm 1964, 148p 6 Letter to the editor from Mr J. Robinson, Surgeon-Dentist to the Metropolitan Hospital. The Medical Times No. 379: 2 January 1847, 273-274; also see Ellis RH. The introduction of ether anaesthesia to Great Britain. Anaesthesia 31(6): 766-777 and 32(2): 197-208, 1977. 7 Sykes WS. The beginning of things. Ch 3 in, Essays on the First Hundred Years of Anaesthesia , Vol 1. Longman Group Ltd: London, 1982, pp 48-76. 9 perfunctory paragraph in The Medical Times, a week later. 8 The ether was administered by an apparatus built around a sponge soaked in ether, from which the ether dripped downwards, and the vapour concentrated at the bottom of a collecting vase from which it was inspired through a flexible tube. It was explained in a footnote to the account that “No heat is required under any circumstances”. 9

The Medical Times of Saturday January 9 1847 ran a leading article entitled “Painless Operations” with the opening (and wonderfully prescient) paragraph “We anticipate, and with reason, that the year 1846 will be characterized by one of the most important discoveries that we have ever had to record…”. 10 John Snow witnessed ‘etherization’ for surgery during the last week of 1846. Amid assorted reports about anaesthesia, including those of its failures, made to the Westminster Medical Society on January 16 1847, John Snow explained that temperature had a great effect “over the relations of atmospheric air with the vapour of ether” and that “this had apparently been overlooked in the construction and application of the instruments hitherto used [and that]…This circumstance would explain in some measure the variety of the results and account for some of the failures” 11 – indeed, a hardly surprising outcome given the likely, even indoors, temperatures in London, at that time, in mid winter!

Thomas Jones Barker’s portrait of John Snow, aged 34, was exhibited at the Royal Academy of Arts, London in 1847. [Zuck D. Snow, Empson and the Barkers of Bath. Anaesthesia 56(3): 227-230, 2001]

The following week, Snow demonstrated to the Westminster Medical Society a prototype of his ether vaporizer: this was distinct from other ether inhalers in that it provided temperature control. 12 During the ensuing weeks, further contributions to the London Medical Gazette

8 Medical Correspondence: Operations without pain. The Medical Times 278: 26 December 1846, 251 9 Performance of surgical operations during the state of narcotism from ether. Lond Med Gaz IV: 38- 39, 1847 10 Leading Article: “Painless Operations”. The Medical Times , Volume The Fifteenth (October 3 1846 to February 20 1847), No. 380: January 9 1847, 289-292; Leading Article: “Painless Surgical Operations.”; “Insensibility during surgical operations produced by inhalation” as Read before the Boston Society of Medical Improvement, November 9 1846, by Henry Jacob Bigelow. The Medical Times No. 379: January 2 1847, 271-273. 11 Inhalation of ether. Report of the Westminster Medical Society, Saturday, January 16 1847. Lond Med Gaz IV:156-157, 1847 12 Apparatus for inhaling the vapour of ether. Report of the Westminster Medical Society, Saturday, January 23 1847. Lond Med Gaz IV: 201-202 10 gave accounts of the successes and the failures of ether, but without advancing any real insights related to the different outcomes among the patients. Snow’s contribution of March 19 1847 got straight to the fundamental issue. In his opening sentence, he stated “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”. 13 Snow realised that quantitation of dose was the key to successful administration. He explained that the dose of ether being delivered was a function of vapour pressure, and this was a function of temperature, requiring further adjustments for the residual in the ether from its manufacture, and of residual water used to wash the alcohol from the ether. Snow devised tables to correct vapour pressure for temperature and use with his temperature regulated ether delivery apparatus, and used this apparatus to perform dose-response experiments on animals, birds, reptiles, patients, and on himself, observing the time-course of onset and regression of effects, and keeping meticulous records of the results.

Snow also devised a progressional semi-quantitative scale of five stages of ether anaesthesia, which he observed during the induction and regression of anaesthesia.14 He also advocated a quantitative mass balance basis of the anaesthetic agent being used for surgical anaesthesia. Indeed, Snow specifically acknowledged Andrew Buchanan’s contributions to these concepts,15 saying that “Dr Buchanan, by considering the quantity of ether expended in inhalation, and making allowance for what is expired, without being absorbed, considered the quantity in the blood of the adult in complete etherization to be not more than half a fluid ounce; and this is, I believe, a pretty correct estimate”. 16 Snow demonstrated that the physical variables of anaesthetic vapour inhalers and the rate of inhalation, as well as physiological variables, such as respiratory rate, were relevant to the induction of anaesthesia, and that attention to quantitation held the answer to avoiding ‘etherization failures’. He further applied the graded response ‘stages’ to chloroform anaesthesia, and he attempted to calculate what we would later pharmacokinetically refer to as clearance, apparent distribution volume, and even measured the urinary excretion of a metabolite (chloride from chloroform).17 It is now recognized that Snow’s work of the mid-19 th C laid foundations for what would become pharmacokinetics and pharmacodynamics a century later. 18

1.5 Stereochemistry in pharmacology – Louis Pasteur I learned the basics of stereochemistry as an undergraduate, and had often measured optical rotation as part of the process of identification of an ‘unknown’ organic compound in chemistry laboratory classes. Indeed, I later taught this, myself, as a chemistry demonstrator. However, I did not know much about Pasteur, apart from the hallmark experiment in which he picked apart mirror image-appearing crystals of the same substance, sodium ammonium paratartrate, and measured their optical rotations, finding them equal but opposite.

13 Snow J. On the Inhalation of the Vapour of Ether. Lond Med Gaz IV: 498-502, 1847 14 Snow J. On the Inhalation of the Vapour of Ether in Surgical Operations : Containing a Description of the Various Stages of Etherization, and a Statement of the Result of Nearly Eighty Operations in which Ether Has Been Employed in St. George's And University College Hospitals. John Churchill, 1847. 15 Buchanan A. Physiological effects of the inhalation of ether. Lond Med Gaz III: 669- 671, and 715- 717, 1847, and IV: 929-938, 1847 16 Snow J. On narcotism by the inhalation of vapours. Lond Med Gaz VII: 850-854, 1848 17 Snow J. On narcotism by the inhalation of vapours (part 14). Lond Med Gaz 46: 321-327, 1850 18 Wagner JG. History of pharmacokinetics. Pharmacol Ther 12(3): 537-562. 1981; Tucker GT. Pharmacokinetics and pharmacodynamics – evolution of current concepts. Anesth Pharmacol Rev 2: 177-187, 1994 11

It is to Louis Pasteur that we attribute the founding of stereochemistry from his observations, in 1848, of the optical rotation of forms of tartaric acid in relation to their enantiomorphic crystalline structures. 19 Less than a decade later, Pasteur noted different rates of metabolism of the stereoisomers of tartaric acid by Penicillium glaucum , a common mould thereby linking biochemistry and stereochemistry. 20

Louis Pasteur as a 25 year old graduate laboratory assistant at the Ecole normale supérieure (c. 1847). [The Historical Medical Library of the College of Physicians of Philadelphia]

On December 7 1854, as the incoming Dean of the new Faculty of Sciences at Lille, Pasteur gave the opening speech in which he said, “In the fields of observation…chance favours the prepared mind”. 21 Pasteur was then speaking of Danish physicist Oersted and the almost ‘accidental’ way in which he discovered the basic principles of electro-magnetism, 22 but it is such a wonderful generalization for scientists of all disciplines. Pasteur’s biographer, the eminent immunologist Professor Patrice Debré, indicates that Pasteur instilled this in his students. Long before I had read Debré’s biography, I had become aware of this maxim, but was unaware that it came from Pasteur. It is exactly what I tried to instil in my postgraduate students, because there were many times when we had unexpected results…and we all needed prepared minds!

19 Mason S. The origin of chirality in nature. Trends Pharmacol Sci 7: 20-23, 1984; Gal J. Louis Pasteur, language, and molecular chirality. I. Background and dissymmetry. Chirality 23: 1-16, 2011 20 Pasteur L. Note relative au Penicillium glaucum et a la dissymetrie moleculaire des produits organiques naturels. Mallet-Bachelier, Imprimeur-Libraire. 1860 (http://searchworks.stanford.edu/view/1275777 ; accessed June 29 2015) 21 Lecture, University of Lille (7 December 1854): http://en.wikiquote.org/wiki/Louis_Pasteur (accessed January 23 2015); reprinted in: Pasteur Vallery-Radot, ed., Oeuvres de Pasteur (Paris, France: Masson and Co., 1939), vol. 7, page 131: http://gallica.bnf.fr/ark:/12148/bpt6k7363q/f137.chemindefer (accessed January 23 2015) 22 http://www.pasteurbrewing.com/articles/life-of-pasteur/louis-pasteur-chance-favors-prepared- mind/173.html (accessed 7 November 2014) 12

1.6 Stereopharmacology and anaesthesiology As a postgraduate student reading medicinal chemistry, I had become aware of the pharmacological impact of stereochemistry in the evolution of receptor theory (such as shown hypothetically in Figure 1.4). This was, of course, during the era of examining structure- action relationships among homologues and analogues to define receptor morphology and thereby to select the optimal drug structure – and was long before any notion of combinatorial chemistry and receptor mapping.

Figure 1.4. The Beckett and Casy conceptual model of a receptor for a hypothetical ‘optically active’ analgesic agent (e.g. methadone or ) showed two mirror image receptor forms to accommodate both configurations of a chiral carbon. [Beckett AH, Casy AF. and biological action. J Pharm Pharmacol 7(1): 433-455, 1955]

In my PhD thesis of 1972, I discussed the then-sparse stereochemical data on the local anaesthetic bupivacaine, but my project did not involve any such experimental work. My first stereochemical experiment with bupivacaine was an (unsuccessful) attempt to resolve its enantiomers for a pharmacokinetic study whilst working with Geoffrey T (Geoff) Tucker at the University of Washington in in 1972 (Section 2.2). Geoff is a graduate of Chelsea College of Pharmacy, University of London; we shared many research concepts and tools, and had corresponded over several years. He had completed his PhD a few years earlier than me under the supervision of the renowned medicinal chemist Arnold Beckett (1920-2010). Professor Beckett, notable for many medicinal chemistry concepts and tools, had proposed the existence of an ‘opioid receptor’, decades before its formal acceptance after cloning, purely on the basis of his studies of the stereochemistry of molecules that had analgesic activity (e.g. Figure 1.5).

Figure 1.5 The Beckett and Casy conceptual model of a receptor for a hypothetical ‘optically active’ opioid analgesic agent (e.g. morphine shown), modified to account for knowledge as to which configuration of an enantiomeric pair was the primary agonist. [Beckett AH, Casy AF, Harper NJ, Phillips PM. Analgesics and their antagonists: Some steric and chemical considerations. J Pharm Pharmacol 8(1): 860-873, 1956] 13

In the mid 1960s, Professor Beckett’s postgraduate students in pharmaceutical sciences were at the forefront of the emerging subdiscipline of pharmacokinetics, supported by the analytical chemistry of drugs by GLC. As a contemporary postgraduate student using similar concepts and tools, I read their publications avidly. 23 After graduation, I became acquainted with several of them through our mutual interests, in local anaesthetic pharmacokinetics, as cited in a number of my publications. Malcolm Rowland published pharmacokinetic models for lignocaine. Grant Wilkinson (1942-2006) published on pharmacokinetic models of hepatic drug clearance. RN (Nick) Boyes published on local anaesthetic pharmacokinetics and metabolism, and worked for Astra Pharmaceutical Company in the United States where we often met during the etidocaine project (Section 2.2). Felicity Reynolds, a clinical anaesthetist from St Thomas’ Hospital London, published on the placental transmission of drugs at the same time as my research on this topic, and later on the vascular stereopharmacology of the local anaesthetics (Section 3.2). EJ (Ted) Triggs (1942-2007) subsequently joined the academic staff of the University of Sydney and published on the pharmacokinetics of local anaesthetic agents with my postgraduate supervisor, Associate Professor John (Jack) Thomas, and others.

A concurrent and similar scientific movement in drug sciences was also occurring in Australian universities, particularly in the capital cities. In Sydney, a highly influential Department of Clinical Pharmacology was founded at St Vincent’s Hospital-University of NSW under the inaugural chair of Professor Denis Wade (one of my PhD examiners). At the University of Sydney, Jack Thomas supervised a succession of postgraduate students on clinical anaesthesia-orientated projects.

For several years, I shared Jack Thomas’ lab with pharmaceutical scientist Peter Meffin (1943-1987), who had started his research the year after me and who studied the pharmacokinetics and metabolism of mepivacaine for his PhD thesis.24 Peter later went on to work at the University of California-San Francisco and at Stanford before returning to Australia where he studied the pharmacological and metabolic implications of stereochemistry in nonsteroidal anti-inflammatory drugs in the Department of Clinical Pharmacology at Flinders Medical Centre (the Flinders University of South Australia, School of Medicine, hereafter referred to as “Flinders”) in Adelaide. Another colleague in our lab, who had started his research a couple of years after me, was chemist RG (George) Moore, who also published on local anaesthetics jointly with Ted Triggs and Colin Shanks (1936- 1998), a research-dedicated clinical anaesthetist who asked me to teach him to measure lignocaine concentrations in blood. 25 George Moore later went on to work at Foundation 41,

23 Some examples: Beckett AH, Boyes RN, Parker JBR. Determination of lignocaine in blood and urine in human subjects undergoing local analgesic procedures. Anaesthesia 20(3): 294-298, 1965; Beckett AH, Tucker GT, Moffat AC. Routine detection and identification in urine of stimulants and other drugs, some of which may be used to modify performance in sport. J Pharm Pharmacol 19(5): 273-294, 1967; Beckett AH, Moffat AC, Rowland M, Tucker GT, Wilkinson GR. Observations on the gas chromatographic behaviour of some amines used in anorectic formulations. J Chromatog A 30: 199-202, 1967; Reynolds F, Beckett AH. The determination of bupivacaine, lignocaine and mepivacaine in human blood. J Pharm Pharmacol 20(9): 704-708, 1968 24 Thomas J, Meffin P. Aromatic hydroxylation of lidocaine and mepivacaine in rats and humans. J Med Chem 15(10): 1046-1049, 1972; Meffin P, Robertson AV, Thomas J, Winkler J. Neutral metabolites of mepivacaine in humans. Xenobiotica 3(3): 191-196, 1973.; Meffin PJ, Thomas J. The relative rates of formation of the phenolic metabolites of mepivacaine in man. Xenobiotica 3(10): 625-632, 1973 25 Notably: Mihaly GW, Moore RG, Thomas J, Triggs EJ, Thomas D, Shanks CA. The pharmacokinetics and metabolism of the anilide local anaesthetics in neonates. Eur J Clin 14 a neonatal research unit at The Women’s Hospital in Sydney founded by William McBride (of thalidomide fame, also see Section 3.7), and later became head of the Astra Pharmaceutical Company in Japan. Colin Shanks later made many significant contributions to anaesthesiology research, most notably at Northwestern University in Chicago with pharmacokinetic-pharmacodynamic studies of thiopentone. 26

Interestingly, a number of other significant scientific-anaesthesiology research connections were built during that period. Among them, pharmaceutical scientist Denis Morgan (1950- 2009), another of Jack Thomas’ postgraduate students of the 1970s, who wrote his PhD thesis on the local anaesthetic agent, etidocaine,27 on which I had worked at the University of Washington some years earlier (Section 2.2). Some of Denis’s etidocaine research also involved Michael Cousins, who was then working at the Royal North Shore Hospital (before subsequently moving to Flinders (Section 2.3)). Denis later went on to work at the Victorian College of Pharmacy-Monash University. He and I co-supervised pharmaceutical scientist Carmen La Rosa for her MPharm research on pethidine in my lab at Flinders (Section 2.3). Also, Denis had supervised the PhD thesis of pharmaceutical scientist JiLui Huang who came to work in my lab at Sydney as a post-doctoral research officer on the initial stages of the thiopentone stereopharmacology project (Section 2.7).

In 1988, after feeling deeply affected by Peter Meffin’s premature death some months earlier, I took sabbatical leave in the Department of Anaesthetics at the University of Sheffield then chaired by Professor Walter Nimmo, an anaesthetist with strong expertise in pharmacological and pharmacokinetic research - mutual interests that formed a pathway to our professional friendship.28 I had previously appeared with Walter at several international symposia on opioid analgesics and drug delivery techniques, and we were both consultants to the Alza Corporation in Palo Alto on a major research program on the transdermal delivery of (now marketed as Durogesic®, Janssen-Cilag Pty Ltd). My sabbatical in his department at the University of Sheffield led directly to my interest stereopharmacology, which began when I visited Geoff Tucker’s nearby lab, in the Department of Clinical Pharmacology, and saw for

Pharmaco l 13(2): 143-152 1978; Moore RG, Thomas J, Triggs EJ, Thomas DB., Burnard ED, Shanks CA. The pharmacokinetics and metabolism of the anilide local anaesthetics in neonates. Eur J Clin Pharmacol 14(3): 203-212, 1978 26 Avram MJ, Sanghvi R, Henthorn TK, Krejcie TC, Shanks CA, Fragen RJ, Howard KA, Kczynski DA. Determinants of thiopental induction dose requirements. Anesth Analg 76(1): 10-17, 1993; Gentry WB, Krejcie TC, Henthorn TK, Shanks CA, Howard KA, Gupta DK, Avram MJ. Effect of infusion rate on thiopental dose-response relationships. Assessment of a pharmacokinetic- pharmacodynamic model. Anesthesiology 81(2): 316-24, 1994; Krejcie TC, Henthorn TK, Shanks CA, Avram MJ.. A recirculatory pharmacokinetic model describing the circulatory mixing, tissue distribution and elimination of antipyrine in dogs. J Pharmacol Exp Ther 269(2): 609-616, 1994 27 Thomas J, Morgan D, Vine J. Metabolism of etidocaine in man. Xenobiotica 6(1): 39-48, 1976; Morgan D, McQuillan D, Thomas J.. Pharmacokinetics and metabolism of the anilide local anaesthetics in neonates. II etidocaine. Eur J Clin Pharmacol 13(5): 365-371, 1978; Morgan DJ, Cousins MJ, McQuillan D, Thomas J. Disposition and placental transfer of etidocaine in pregnancy. Eur J Clin Pharmacol 12(5), 359-365, 1977; Morgan, DJ, Smyth MP, Thomas J, Vine J. Cyclic metabolites of etidocaine in humans. Xenobiotica 7(6): 365-375, 1977; Vine J, Morgan D, Thomas J. The identification of eight hydroxylated metabolites of etidocaine by chemical ionization mass spectrometry. Xenobiotica 8(8): 509-513, 1978; Cousins MJ, Augustus JA, Gleason M, Morgan DJ, Thomas J. Epidural block for abdominal surgery: aspects of clinical pharmacology of etidocaine. Anaesth Intens Care 6(2): 105-115, 1978 28 As examples: Nimmo WS, Heading RC, Wilson J, Tothill P, Prescott LF. Inhibition of gastric emptying and drug absorption by narcotic analgesics. Brit J Clin Pharmacol 2(6): 509-513, 1975:. Clements JA, Heading RC, Nimmo WS, Prescott LF. Kinetics of acetaminophen absorption and gastric emptying in man. Clin Pharmacol Ther 24(4): 420-431, 1978 15 the first time the analytical resolution of various racemic drugs on newly developed chiral stationary phase HPLC columns. 29

On my return to Flinders in 1989, I purchased the first of what would be many chiral stationary phase HPLC columns for quantitative studies, in the first instance, to test the possibility of metabolic inversion of mepivacaine and bupivacaine as part of our study program on local anaesthetics. A vital tool had arrived – but I need to go back in history to review the concepts requiring its use.

1.7 Pharmacokinetics Pharmacokinetics was developing both a concept and a tool during my postgraduate years. Apart from curiosity about drugs in the body, it held the promise of underpinning rational pharmacotherapy, as mentioned above.

The theory of what was to become pharmacokinetics was elaborated during the 1920s, at first based on endogenous substances and, in 1937, based on exogenous substances. The term ‘pharmacokinetics’ was coined in the 1950s and its purpose is to determine the effect of a body on a drug, i.e., how a dose of drug, once administered, becomes affected by body processes over time. Until the mid 1960s, pharmacokinetic studies were rather crude because of rather low sensitivity, mainly spectroscopic, analytical methods for drugs and their metabolites.

From the mid-1960s, gas-liquid chromatography (GLC) was becoming more affordable and, being capable of reasonably high sensitivity and specificity measurements of substances that could be made sufficiently volatile to pass into the gas phase, it was rapidly adopted as the standard laboratory measurement tool for drugs in biofluids, including by laboratories involved in sports doping investigations, an area for which Professor Beckett later became well-known. During the 1970s, development of high performance (originally named high pressure) liquid chromatography (HPLC) was to become the next major tool. From the late- 1970s, HPLC was becoming a versatile alternative to GLC as it did not require substances to be volatile. A range of more sensitive and versatile detectors progressively became available: these included -selective detectors for GLC, diode array detectors for HPLC, and later, mass spectrometric detectors for both. Thus, the quantification of drugs in biofluids dramatically improved, allowing pharmacokinetics to develop concurrently, practically and conceptually.

As commonly interpreted these days, ‘pharmacokinetics’ refers to the ‘whole body’ disposition of a substance, and is normally based on quantitative measurements of the time- course of drugs (and/or their metabolites) in blood (or plasma or serum) serially sampled from the systemic circulation. The results are thereby the time-averaged outcome of the distribution and/or clearance of the substance in every individual region, organ and cell of the body. ‘Compartment’ pharmacokinetics, being easy to conceive and compute, quickly became the standard methodology. This method requires the assumption that a blood sample is representative of the ‘circulating concentrations’ of the substance, an assumption only superficially true, as many of our own experiments would later demonstrate. Variant methodologies were developed for more specific purposes, e.g. ‘non-compartment’ that precluded most assumptions, and ‘physiological pharmacokinetics’ that required many more assumptions.

29 Hermansson J, Eriksson M. Direct liquid chromatographic resolution of acidic drugs using a chiral α1-acid glycoprotein column (Enantiopac®). J Liquid Chrom 9(2-3): 621-639, 1986 16

Pharmacokinetics has also grown to depend on computational power through hardware and software development, and this occurred rapidly during the 1970s. The experimental techniques of the 1960s had only very limited ways in which to make inferences from the time-course of drug concentrations. In those times, mainly the ‘half-life’ of a substance was determined from a semi-log plot on graph paper. Even at the time of my own PhD studies, this was only just germinating. 30 The terminology and methodology evolved into ‘drug disposition’, a term used to describe the combined concerted processes of drug distribution and elimination, and methods for quantitating these processes had to catch up both theoretically and practically.

There can be many reasons for quantification of a drug blood concentration time course and, essentially, all require the serial drug concentration data to be reduced to pharmacokinetic summary data. The summary data then can be used for information, mainly to compare values for the variables, such as extent of distribution or rate of clearance between individuals, or between drugs, or to examine the effects of an imposed condition or specific pathology on a particular drug. Additionally, such data are useful to be able to predict and compare, say the consequence of multiple doses, or alternative modes of administration. But such pharmacokinetic effort is somewhat hollow without linkage to information about drug effects. Accordingly, much effort has been invested in clinical laboratories for describing a drug’s ‘therapeutic window’, that is, the drug blood concentration below which ‘therapeutic effects’ were unlikely and above which ‘toxic effects’ were likely. Many specific algorithms for drug dosing regimens were built around this concept, for example for the prescribing of gentamicin. Pharmacokinetics is still evolving, most recently into ‘population pharmacokinetics’ in which physical and other variables of the subjects are evaluated for their predictive capacity for pharmacodynamic response to drugs. 31 Population pharmacokinetics has been prominent in anaesthesia literature, especially for intravenous induction agents, from soon after its inception. 32

A large portion of my work over many years, including some of that described in this thesis, has been given to developing novel experimental approaches for pharmacokinetics. One particular outcome was what we called ‘regional pharmacokinetics’, and it developed out of observations that the total body clearance of some drugs could not be accounted for by the sum of measured clearances from visceral organs known or expected to be responsible for drug clearance, and this is described in more detail in Section 2.5. This was not meant to overthrow the traditional pharmacokinetic methods but to counter their shortcomings.

30 As examples: Riegelman S, Loo JCK, Rowland M. Shortcomings in pharmacokinetic analysis by conceiving the body to exhibit properties of a single compartment. J Pharm Sci 57(1): 117-123, 1968; Riegelman S, Loo J, Rowland M. Concept of a volume of distribution and possible errors in evaluation of this parameter. J Pharm Sci 57(1): 128-133, 1968; Loo JCK, Riegelman S. New method for calculating the intrinsic absorption rate of drugs. J Pharm Sci 57(6): 918-928, 1968 31 As examples: Sheiner LB, Ludden TM. Population pharmacokinetics/dynamics. Annu Rev Pharmacol Toxicol 32(1): 185-209, 1992; Whiting B, Kelman AW, Grevel J. Population pharmacokinetics theory and clinical application. Clin Pharmacokin 11(5): 387-401, 1986; Mould DR, Upton RN. Basic concepts in population modeling, simulation, and model-based drug development. CPT: Pharmacometr Syst Pharmacol 1(9): e6, 2012 32 As examples: Schüttler J, Ihmsen H. Population pharmacokinetics of propofol: a multicenter study. Anesthesiology 92(3): 727-738, 2000; Russo H, Simon N, Duboin MP, Urien S. Population pharmacokinetics of high-dose thiopental in patients with cerebral injuries. Clin Pharmacol Ther 62(1): 15-20, 1997 17

1.8 Pharmacodynamics The term ‘pharmacodynamics’ is often used rather loosely. It really means what the drug does to the body in a quantitative sense, whereas drug ‘response’ or ‘effect’ is generally used in a more qualitative sense.

Once in the systemic circulation, the balance between a drug’s desirable and undesirable (or ‘therapeutic’ and ‘toxic’) pharmacological effects derives from the rates and extents of its concurrent uptake into, distribution within, and elution from, all ‘responsive’ and ‘non- responsive’ organs and regions of the body (although, plainly, only ‘responsive’ organs or tissues respond). This applies even if drugs are administered locally for local effects because of inevitable systemic absorption unless, in a rare event, the drug is locally destroyed chemically or metabolically. Thus, the nett effects of the drug depend in a very complex manner upon its ‘whole body’ disposition, but it can be more complex when the effects of the drug in one part of the body modify its pharmacokinetics and/or its effects in another part of the body (e.g., as with the brain/heart ‘systemic toxicity’ of local anaesthetics, Section 3.23).

The concept of a drug’s ‘therapeutic window’ remains an important part of contemporary clinical pharmacology, albeit a simplistic one because it suggests only a dichotomous or quantal drug response. The responses to a drug are almost always graded, normally in proportion to the occupancy by the drug of its receptors, thereby warranting a combined pharmacokinetic-pharmacodynamic modelling approach. Nonetheless, a dichotomous response in relation to a measured biofluid drug concentration, when used with a logistic regression algorithm, can give useful information regarding the probability of a defined event occurring to a patient during treatment. In this thesis, both kinds of pharmacokinetic- pharmacodynamic models were used in different studies (i.e., quantitative responses , as measured by quantitative electroencephalographic (qEEG) changes; qualitative responses , as measured by a patient with head injuries making a pupillary or motor response) as functions of thiopentone plasma concentrations.

With the evolution of techniques and technology, pharmacodynamics has come to mean the quantitative response or effect of drug in relation to its concentration in some relevant site. This site may be the blood or plasma, often the unbound concentration of the drug in blood or plasma, and sometimes the calculated concentrations in an ‘effect compartment’, the hypothetical site that contains the drug receptors causing the particular effect. The latter became an empirical modelling concept initially proposed concurrently in the late 1970s by prototypic pharmacometrician Lewis Sheiner (1940-2004) of the University of California-San Francisco and by mathematician-anaesthetist Chris Hull from the University of Newcastle- upon-Tyne. It was developed for understanding the quantitative equilibrium between the time-course of drug concentrations measured in plasma and that of the measured drug effect. The concept was first advanced for neuromuscular blockade from muscle relaxants where a graded response could be measured readily on a continuous scale from nil to complete.33 It was computationally developed further with additional physiological measures, and applied to intravenous anaesthesia.34

33 Notably: Sheiner LB, Stanski DR, Vozeh S, Miller R, Ham J. Simultaneous modeling of pharmacokinetics and pharmacodynamics: application to d-tubocurarine. Clin Pharmacol Ther 25 (3): 358-371, 1979; Holford NH, Sheiner LB. Understanding the dose-effect relationship. Clin Pharmacokin 6(6): 429-453, 1981; Hull CJ, English MJM, Sibbald A. Fazadinium and pancuronium: a pharmacodynamic study. Br J Anaesth 52(12): 1209-1221, 1980 34 As examples: Stanski DR, Hudson RJ, Homer TD, Saidman LJ, Meathe E. Pharmacodynamic modeling of thiopental anesthesia. J Pharmacokin Biopharm 12(2): 223-240. 1984; Scott JC, Ponganis KV, Stanski DR. EEG quantitation of narcotic effect: the comparative pharmacodynamics 18

1.9 Applied pharmacokinetics and pharmacodynamics As mentioned elsewhere, the generation of pharmacokinetic summary data is facilitated by likening the body to one or more compartments, a model borrowed from early 20 th C physiological concepts, where compartments are uniform spaces or volumes throughout which the drug analyte is distributed evenly in concentration. 35 Such models are mathematical-statistical constructs, although various anatomical-physiological constructs are often projected onto them, sometimes quite inappropriately. Ordinarily, only the kidneys can be isolated as a ‘real compartment’ if concurrent serial urine drug concentrations are measured and assumed to represent peritubular fluid drug concentrations. The advantage is that the time course of measured drug concentrations can be summarized by reference to the parameters of the compartment model used to simulate the action of the body on the drug.

Figure 1.6 shows a hierarchical family of (one, two and three) compartment models that may be consistent with a set of drug blood concentration-time data – and thereby provide a range of possible statistical solutions to pharmacokinetic questions – and with different possible interpretations. It can be difficult to assign a model a priori to a set of data, and the principle of parsimony, along with common sense, is required in acceptance of a model.

The compartments comprise a sampled (blood) or ‘central’ compartment, and maybe one or more compartments that are added as ballast to correct for the time course of drug ingress into, and egress from, one or more ‘peripheral compartments’. These are often described, in terms of the rates of transfer or exchange, using physiological terms such as ‘well perfused’ and ‘poorly perfused’ tissues. However, additional insight regarding how or where a drug is eliminated is required in making a choice among competing models. Clearly, the body is composed of many more ‘real’ compartments that can be justified on residual sums-of- squares type statistical criteria. Indeed, there are as many ‘real’ compartments as there are of regions with identifiably different blood flows, regional distribution coefficients, and rates of elimination. When these are not identifiably different, then the real ‘compartments’ become ‘lumped’ together as mathematical or statistical compartments. The problem with compartment models is that of identifiability, 36 and that a ‘whole body’ or ‘black box’ approach is thus used, and little-to-no information can be gleaned as to the time course of drug concentrations in actual responsive and non-responsive organs and regions of the body.

of fentanyl and alfentanil. Anesthesiology 62(3): 234-241, 1985; Colburn WA. Simultaneous pharmacokinetic and pharmacodynamic modeling. J Pharmacokin Biopharm 9(3): 367-388, 1981; Verotta D, Sheiner LB. Simultaneous modeling of pharmacokinetics and pharmacodynamics: an improved algorithm. Comp Appl Biosci: CABIOS 3(4): 345-349, 1987; Schnider TW, Minto CF, Stanski DR. The effect compartment concept in pharmacodynamic modelling. Anaesth Pharmacol Rev 2: 204-213, 1994 35 The outstanding example is Riggs DS. The Mathematical Approach to Physiological Problems, a critical primer. Williams and Wilkins, Baltimore, 1963; also see Cutler DJ. Linear systems analysis in the kinetics of anaesthetic agents. Anaesth Pharmacol Rev 2: 243-249, 1994 36 Hull CJ. Compartmental models. Anaesth Pharmacol Rev 2: 188-203, 1994 19

Figure 1.6: What are the implications of the choice of a model? Compartment models – statistical solutions to pharmacokinetic questions. The data may be fitted by various models, but which of the models seems more appropriate, and why? [From one of Geoff Tucker’s lectures]

Such criticism does not invalidate compartment models for drugs – indeed, they have been, and will remain, very useful tools, as long as the underlying concepts are respected. Only too often, however, one might read in a paper statements like ‘the data were fitted to a two (or whatever) compartment model…’, thereby betraying the lack of understanding that the model is used to represent the data (for whatever purpose), and not the other way. Or one might hear at a lecture statements like ‘(so-and-so) drug is a two (or whatever) compartment drug’, thereby betraying the lack of understanding that the experimental design contributes to the data and thus the associated model by way of site of blood sampling, timing and duration of blood sampling, and sensitivity (and specificity) of the drug assay, at least. Studying a racemate without stereospecific assay simply adds another uncertainty to interpretation – a point made forcefully by pharmaceutical scientists Geoff Tucker and Martin Lennard. 37

37 Tucker GT, Lennard MS. Enantiomer specific pharmacokinetics. Pharmacol Ther 45: 309–29, 1990; Lennard MS. Clinical pharmacology through the looking glass: reflections on the racemate vs enantiomer debate. Brit J Clinical Pharmacol 31(6), 623-625, 1991 20

Indeed, my own earlier pharmacokinetic work lacked stereospecific drug assays, but this was because analytical resolution techniques were not then available.

A major portion of my research started in the late 1970s during my collaboration with clinical anaesthetist and intensivist William B (Bill) Runciman (who became my 2 nd PhD student) when we strove to understand how whole body drug disposition could be accounted for by the actual body organs and tissues (Section 2.3). Some researchers refer to this concept as ‘physiological pharmacokinetics’, 38 but we broadened the concept and coined the term ‘regional pharmacokinetics’, where a region could be defined anatomically or physiologically, as a whole organ or a collection of tissues, and be demarcated by blood sampling catheters so that its properties, for example, its blood flow or oxygen consumption, could be measured as driving or responding to drug effects, and this was based simply on the fundamental concept of mass balance (Figure 1.7). Performed in sheep, this methodology became the basic tool for our investigating conceptually simple but practically difficult questions (such as ‘where are (specific) drugs cleared?’, and ‘does the sum of the measured regional drug clearances actually equal its calculated total body clearance?’), along with clinically-pertinent investigations (such as the comparative effects of general and spinal anaesthesia on regional blood flow, oxygen consumption and drug disposition). The latter included regional sampling of afferent and efferent blood concentrations in concert with a selective drug-affected selective signal from the subject region. Some particular applications of our methodology that include concurrent quantitative measurements of drug effects on the heart and brain are described more fully in Section 3.

Figure 1.7: Conceptual diagrams of an anatomical-physiological view of the body that allowed our experimental development of regional pharmacokinetic-pharmacodynamic models in an anatomically and physiologically correct manner. Blue demonstrates the region(s) of interest; green demonstrates where representative afferent blood could be sampled, red where representative efferent blood could be sampled, and white demonstrates mixed blood. In the left example, the kidneys are the region of interest; in the right example, the whole body is the region of interest with drug infusion made into the left pulmonary artery. [From Review 01, 110 ]

38 Himmelstein KJ, Lutz RJ. A review of the applications of physiologically based pharmacokinetic modeling. J Pharmacokin Biopharm 7(2): 127-145, 1979; Jarvis DA. Physiological pharmacokinetic models – a review of their principles and development. Anaesth Pharmacol Rev 2: 214-230, 1994 21

Despite their obvious value, whole body pharmacological approaches have waned over the past decade or so, as fashion and funding bodies have become more attracted to advances in molecular and cellular biology. As these techniques were not a part of my repertoire, it was becoming clearer that my ability to attract adequate funding for my (experimental ‘whole body’ pharmacology) group would diminish, and that it was time for me to consider retirement. Notwithstanding, and regardless of their obvious attractiveness, I remain concerned that the hastening rush of reductionist approaches will fail to reproduce the clinical problems that they were instituted to solve, and that the loss of expertise in whole body pharmacology will hamper future research. This, then, leads directly to the ‘elephant in the laboratory’.

1.10 The ‘elephant in the laboratory’ Application of the relevant concepts and tools in the pursuit of knowledge inevitably requires personnel, equipment and supplies. Plainly, experiments can be expensive – and these require funding. Funding, or rather the perpetual quest for funding, is the ‘elephant in the laboratory’.

It is self-evident that the availability of funding influences both research directions and output, and these are determined, to some extent, by ‘where the money is’. When I was a postgraduate student, my supervisor provided the resources for my research: these were rather austere – many ‘disposable’ items had to be re-used for example - but the resources were sufficient, and were hard-won and well used. In succession, it became my turn to provide sufficient resources for my postgraduate students and research activities.

For many of my research years, I had a fully supportive head of department, Michael Cousins, to whom I pay full tribute for his tireless work. Michael was sometimes a co-principal investigator on my projects, and I on his, as we raised funds to establish new academic departments, twice – in Adelaide in 1976 and again in Sydney in 1991 (Sections 2.3 and 2.7). Sometimes we approached pharmaceutical companies where we believed that one or other of their products could be used better for patient care through further research, and sometimes they approached us for the same reasons. The infrastructure thereby gained allowed us to apply for funds from the National Health and Medical Research Council of Australia (NHMRC) on a more competitive basis, especially with longer-established departments and research groups. We also obtained various research grants-in-aid and benefactions from other bodies and generous individuals. The record given in Table 1 indicates the major sources of support for my research programs and projects.

Anaesthesia is not really a ‘high profile’ research discipline, unlike say, cell biology, genetics or immunology. One NHMRC reviewer, critical of a project that I had submitted on thiopentone, once wrote ‘…that anaesthesia doesn’t kill people…’ – and fortunately this is the case (most of the time), but this comment reveals the reviewer’s lack of insight. Perhaps unsurprisingly, I didn’t get the funding. Nevertheless, the Australian and New Zealand College of Anaesthetists (ANZCA) and the Australian Society of Anaesthetists (ASA) were prepared to support many of my projects, particularly at early stages.

In my own case, as I am sure is not dissimilar to other researchers, a lack of funding also meant that various ‘interesting ideas’ needed to be modified, curtailed, and/or abandoned. It does not necessarily follow, of course, that un-funded or under-funded quests were scientifically flawed or ill-judged. It is just that, in our research grant review system, one’s ‘grand plan program’, or ‘interesting idea project’, may not be sufficiently compelling to one’s peers when placed for funding on a ranked scale with competing programs or projects. 22

Looking back, as Principal or Co-Principal Investigator, I raised many millions of dollars to support my research programs or projects, over and above those funds required for general departmental infrastructure, growth and support. A significant part of my working life was directly involved in seeking funds to advance science in my own area, and assisting colleagues to advance theirs. However, the serious impact of the ‘elephant in the laboratory’ is that, every year, so much time is spent by the most highly educated people in the country, competing for a small pool of research funds - with a greater than 80% chance of failure – and this is largely unknown and unnoticed. New investigators, like many professional scientists, can be left without support, to become dispirited. This is such an awful waste of time and talent – not to mention the anguish and the direct financial costs. In so many ways, it is outrageous!

The peer-review grant application system, no doubt, can be perceived as fickle or even unjust, and probably both could be true. Whilst it is hard to propose a better way, the peer-review system highlights the issues of ‘small ponds’ where it, firstly, can be difficult to find suitable peers to assess a research proposal because of the small pool of cognisant yet impartial reviewers and, secondly, where the applicant’s ‘small pond’ research proposal is not amongst the perceived ‘hot issues’ of the day. I hasten to add that I am not suggesting that I have been affected by the former, but probably have been by the latter. Nevertheless, as indicated in Table 1, I managed acceptably, and am grateful to the NHMRC, ANZCA, and the ASA, along with other bodies, commercial sources and private benefactors for their support. Such support enabled postgraduate research student stipends and salaries for professional researchers, as well as purchase of laboratory capital equipment and maintenance supplies, without which our research simply could not have been performed.

Table 1: Program, project, and main grant-in-aid research support (1977-2005) National Health and Medical Research Council of Australia Haemodynamic influences on drug disposition . LE Mather, WB Runciman: 1980-1981. $47,479 project grant Interaction of oxygen availability, blood flow and drug dispositio n. LE Mather, WB Runciman: 1982-1983. $86,070 project grant Interaction of oxygen availability, blood flow and drug disposition. LE Mather, WB Runciman: 1984-1986. $105,485 project grant In vivo flux-effect relationships for drugs, hormones, and other active substances . LE Mather, WB Runciman, AM Snoswell: 1986. $10,000 equipment grant: sheep metabolic equipment In vivo flux-effect relationships for drugs, hormones, and other active substances. LE Mather, WB Runciman, AM Snoswell: 1987-1989. $98,452 project grant kinetics and its physiological and toxicological effects . LE Mather, WB Runciman, AM Snoswell, AH Ilsley: 1987-1990. $115,961 project grant Disposition of morphine and its conjugates: effects of renal impairment. LE Mather, R Nation (University of South Australia), WB Runciman, A Somogyi (University of Adelaide): 1989-1991. $198,219 project grant Efficacy and safety of combined opioid and antiprostanoid analgesics. LE Mather, MJ Cousins: 1991-1993. $210,000 project grant Perioperative epidural analgesia and postoperative nutritional support. RC Smith, LE Mather, MJ Cousin, S McG Barratt: 1992-1994. $134,840 project grant Gas-liquid chromatograph . LE Mather, MJ Cousins, C Duke: 1994. $54,000 equipment grant Pharmacological consequences of stereoisomeric mixtures of anaesthetic agents. L Mather, M Cousins, C Duke: 1994-1996. $168,546 project grant 23

Etiological factors influencing NSAID-induced perioperative renal dysfunction . MJ Cousins, I Power, LE Mather, E Gallery: 1996-1998. $270,825 project grant Determination of the impact of acid-base and blood flow changes in the treatment of bupivacaine toxicity . YF Huang, LE Mather: 1996-1998. $154,800 project grant Persistent postsurgical morbidity prevalence and cost implications . MJ Cousins, RC Smith, I Power, LE Mather: 1996-1998. $282,427 project grant Non steroidal anti-inflammatory drugs - a pharmacokinetic-pharmacodynamic study of differential actions and activity at relevant anatomical sites . LE Mather, I Power, MJ Cousins: 1997-1999. $239,514 project grant Studies of the effects of gender and metabolism on morphine antinociception in rats. LE Mather, co-chief investigator with MT Smith (University of Queensland): 1997-1999. $142,346 project grant Pain relief for the living and for the dying. NHMRC Centre of Clinical Excellence in Hospital-based Research. LE Mather and others, in consortium: 1998-2000. $630,000 program grant Blood gas analyzer for large animal laboratory. LE Mather: 2000. $32,000 equipment grant Australian and New Zealand College of Anaesthetists Stereoisomers in anaesthesia. Inaugural Douglas Joseph Professorship: Australian & New Zealand College of Anaesthetists. LE Mather:1993. $65,000 program grant Intravenous nutrition and analgetic regimens in surgical patients. Australian & New Zealand College of Anaesthetists MJ Cousins, LE Mather, S Barratt: 1994. $65,000 project grant Kinetics of brain uptake of anaesthetic drugs. Australian and New Zealand College of Anaesthetists CF Minto, LE Mather: 1999. $36,000 Inotropic effects of thiopentone enantiomers and propofol. Australian and New Zealand College of Anaesthetists LE Mather: 2001. $35,000 project grant New analgetic and anti-inflammatory treatment strategies related to NMDA receptor antagonism. Australian and New Zealand College of Anaesthetists LE Mather: 2003. $46,000 project grant Australian Society of Anaesthetists Pharmacokinetic approach to pain control. Australian Society of Anaesthetists: Jackson-Rees Research Grant. LE Mather, JV Stapleton 1977. $4,000 grant-in-aid Cardiotoxicity of local anaesthetics agents. Australian Society of Anaesthetists: Jackson-Rees Research Grant. C Nancarrow, WB Runciman, LE Mather, AH Ilsley: 1983. $48,000 grant-in-aid The efficacy of patient controlled analgesia. Australian Society of Anaesthetists. LE Mather, A Hobbes, A Woodhouse: 1992. $20,000 grant-in-aid Studies of factors in postoperative PCA drug use. Australian Society of Anaesthetists: Janssen-Cilag/ASA Research Grant. LE Mather, A Hobbes, A Woodhouse: 1994. $20,000 grant-in-aid Other major competitive sources Equipment and general maintenance for drug kinetic and dynamic studies. Flinders University of South Australia Research Committee. LE Mather: 1977-1981. $11,500 Cardiotoxicity of local anaesthetic agents . Flinders Medical Centre Research Foundation. WB Runciman, C Nancarrow, LE Mather: 1984. $6,087 equipment grant Purchase of sheep crates . Potter Foundation. LE Mather, WB Runciman, AM Snoswell: 1988. $4,500 equipment grant Drug kinetic and dynamic studies. Flinders University of South Australia Research Committee. LE Mather: 1983-1990. $11,500 equipment and general maintenance grant-in- aid Analgesia studies . Ramaciotti Foundation. LE Mather, MJ Cousins: 1991. $30,000 grant-in- aid 24

Efficacy and safety of combined drug therapy for prevention and treatment of severe pain. Ramaciotti Foundation. LE Mather, MJ Cousins: 1992. $31,780 equipment grant: pain bioassay technology How hypersensitivity to pain is produced and how it is influenced by -like drugs. Government Employees Medical Research Fund. LE Mather: 2000-2001. $60,000 project grant The role of the tryptophan metabolite kynurenate, an endogenous NMDA receptor antagonist, and its biosynthetic precursor, L-kynurenine, in centrally mediated antinociceptive effects of nonsteroidal anti-inflammatory drugs NSAIDs. Northern Sydney Area Health Service. LE Mather, SR Edwards: 1999. $20,000 project grant Mass spectrometer detector equipment for high performance liquid chromatography . DETYA Research Infrastructure Block Grants LE Mather: 2001. $96,000 equipment grant Mass spectrometer detector equipment for high performance liquid chromatography. University of Sydney Sesqui Research and Development Grant. LE Mather: 2001. $66,000 equipment grant Pharmaceutical and biotechnology industry Studies on the pharmacokinetics and pharmacodynamics of local anaesthetic agents. Astra Pharmaceuticals Pty Ltd (Australia). MJ Cousins, LE Mather: 1976-1982. $25,000 program grant-in-aid Pharmacokinetics and pharmacodynamics of , a new steroidal intravenous anaesthetic agent. Glaxo Pharmaceuticals (UK). LE Mather, MJ Cousins: 1978-1980. $64,000 program grant-in-aid Studies on the pharmacokinetics of chlormethiazole. Astra Pharmaceuticals Pty Ltd (Australia). LE Mather, MJ Cousins, LT Seow: 1977-1981. $15,000 project grant-in-aid Interaction of haemodynamics and drug disposition. Astra Pharmaceuticals Pty Ltd (Australia). LE Mather, WB Runciman: 1981-1984. $15,000 program grant-in-aid CNS and cardiotoxicity of local anaesthetic agents. Astra ALab AB (Sweden). LE Mather, WB Runciman, C Nancarrow: 1983-1987. $110,000 program grant-in-aid Pharmacokinetics and efficacy of transdermal fentanyl. Alza Corporation LE Mather, MJ Cousins: 1985-1987. $77,500 project grant-in-aid Regional pharmacokinetic studies on . Janssen Pharmaceutica (Australia). LE Mather, H Owen: 1987. $20,000 project grant-in-aid Aetiological factors in NSAID-induced renal failure associated with post-operative pain management . Roche/Syntex Group. I Power, MJ Cousins, LE Mather: 1994-1996. $93,000 grant-in-aid Pre-emptive use of the NMDA blocker : controlled study of efficacy in reducing analgesia requirements. Algos Corporation, New York. MJ Cousins, C Brown, I Power, LE Mather: 1996. $12,500 project grant in aid Studies of chirality of local anaesthetics. Chiroscience Ltd (UK). LE Mather: 1994-2001. $743,005 program grant Studies of transpulmonary drug absorption. Aradigm Corp (USA). LE Mather, co-chief investigator with MJ Cousins: 1996-2003. $988,567 program grant Studies of transpulmonary insulin pharmacokinetics and pharmacodynamics. Novo Nordisk A/G Denmark. LE Mather, co-chief investigator with A McElduff: 2000-2003. $574,536 program grant Studies of local anaesthetic intoxication. Abbott Laboratories, Australia. LE Mather: 2001- 2002. $35,000 grant-in-aid Effect of a new intravenous anaesthetic, Alfaxan CD, on myocardial performance. Jurox Pty Ltd LE Mather: 2004-2005. $108,995 project grant 25

2. Translating chemistry into anaesthesiology – in stages

“The true worth of an experimenter consists in his pursuing not only what he seeks in his experiment, but also what he did not seek." Claude Bernard (1813-1878)

Ideas and work on the stereopharmacology theme accumulated at every institution in which I worked. Along the way, my research necessitated my learning the elements of a new language, that of anaesthesiology. I have included this Section to convey my appreciation for my principal teachers and collaborators at those institutions that supported my research, to elaborate on concurrent projects as mentioned by publication in the Appendix, and to provide a passably chronological and definitely personal accompaniment to the selected publications.

2.1 The University of Sydney (1966-1972) In February 1958, I started my first laboratory job – as a ‘student chemist’ in the Sydney laboratories of the multinational pharmaceutical company, Johnson and Johnson Pty Ltd (J&J), mainly performing chemical analyses for product process control, along with some new product development. Concurrently, from 1958 to 1964, I undertook part-time (mainly night classes) studies in Applied Chemistry at Sydney Technical College and later the University of NSW, graduating from both institutions in 1965. For my first four years at J&J, I worked in the ‘rubber and plastics’ lab, and for my last four years, I worked in the ‘pharmaceutical products’ lab. My work in pharmaceutical product development generated the desire to undertake a postgraduate degree to learn the ways of drug research. After my initial inquiries in 1965 about pharmacology postgraduate study at two universities in Sydney came to nothing, I made plans to travel to the UK in 1966 for this to happen but, oddly, this was made possible - via an unexpected route.

In August 1965, I was working on a J&J subsidiary company development project on norbormide, a non-anticoagulant rat-selective rodenticide, that involved some collaboration with SE (Syd) Wright (1914-1966), Professor of Pharmaceutical Chemistry in the Department (now Faculty) of Pharmacy at The University of Sydney. During discussions with him, I mentioned my research aspirations, and he suggested that I might become a postgraduate student in his department. He organized an interview with TR (Tom) Watson (1927-2012), then Senior Lecturer in Pharmaceutical Chemistry (and who became successor to the Chair following Professor Wright’s sudden death at the age of 52). Tom appointed me to a teaching fellowship, commencing in 1966, as a demonstrator and tutor in analytical, physical and organic chemistry classes for 2 nd and 3 rd year undergraduate pharmacy students – whilst allowing me to candidate concurrently as a postgraduate student. This Department was a place with an amazing wide-ranging research strength, and was especially known for its research in natural products chemistry, mainly under the direction of Tom Watson. In a recent obituary, fellow department academic Associate Professor RE (Dick) Thomas wrote “To his colleagues, Professor Watson's passing marks the end of an era that began in the 1950s when two outstanding men, Sydney Wright and Thomas Watson, started the process of transforming a worn-out Department of Pharmacy into an outstanding centre for research and education, recruiting over the next two decades an exceptional team of young people who laid the foundation for the world-class institution that is now the Faculty of Pharmacy within the University of Sydney.”39 Yes, it was like that.

39 Dignified man of molecules. Sydney Morning Herald, 2 November 2012 . http://www.smh.com.au/comment/obituaries/dignified-man-of-molecules-20121101-28ml5.html 26

Professor Tom Watson, May 16 2006. He gave me the opportunity to enter life as a postgraduate research student, and taught me stereochemistry.

In November 1966, and a couple of weeks after our son was born, I passed my MSc(Qual) exams, and Jack Thomas, then Senior Lecturer (later Associate Professor) in Medicinal Chemistry, agreed to supervise my MSc candidacy. As well as his other interests in classical drug structure-action relationships, Jack had developed a project on methaemoglobinaemia and had worked collaboratively with Colin Richmond (Dick) Climie (1923-2013), a clinical anaesthetist at the Royal Hospital for Women in Sydney on a project on drug-induced methaemoglobinaemia involving the local anaesthetic, prilocaine, being trialled for obstetric anaesthesia.40 Jack asked if I was interested in working on a project related to obstetric anaesthesia? Yes, indeed I was!

Professor Jack Thomas, May 16 2006. This Lancastrian, scientist, humanitarian, and raconteur was my esteemed postgraduate supervisor, and taught me medicinal chemistry.

Jack had successfully applied that year for a grant to purchase new GLC equipment for his laboratory, and this was put to good use in measuring drug concentrations in my MSc project. My first project with Dick Climie involved studies of lignocaine (lidocaine) concentrations

40 Notably: McLean S, Robinson J, Starmer GA, Thomas J. The influence of anaesthetic agents on the formation of methaemoglobin induced by aniline in cats. J Pharm Pharmacol 19(12): 803-809, 1967; McLean S, Starmer GA, Thomas J. Methaemoglobin formation by aromatic amines. J Pharm Pharmacol 21(7): 441-450, 1969; Climie CR, McLean S, Starmer GA, Thomas J. Methaemoglobinaemia in mother and foetus following continuous epidural analgesia with prilocaine Clinical and Experimental Data. Br J Anaesth 39(2): 155-160, 1967 27 when being used for epidural anaesthesia in parturients. I next met Geoffrey (Geoff) Long, a clinical anaesthetist at King George V Hospital, and worked with him on studies with topical lignocaine analgesia in parturients. I also studied alcuronium, a semisynthetic muscle relaxant newly introduced into anaesthetic regimens for Caesarean sections and intended as a replacement for curare, but I had to devise a bioassay for this as its measurement was not possible by the chemical techniques then available. These studies [1-3, 9] formed the basis of my MSc thesis, submitted in 1968, not long before our first daughter was born. I next worked on my PhD project, also with Dick and Geoff, under Jack’s supervision. Dick had read about bupivacaine, a new long-acting local anaesthetic, and obtained some from the UK: it formed the basis of my PhD project studies [4-6, 10]. I knew that bupivacaine is a racemate but, there being no technique for resolving the enantiomers analytically, I measured it achirally, just like others then involved in similar research. 41

During my PhD candidacy, and not long before our second daughter was born, the Australian anaesthesia community hosted an international symposium 42 in Canberra and one of the plenary lecturers was John J Bonica (1917-1994), Professor and Chairman of the Department of Anesthesiology, and principal investigator on the US National Institutes of Health (NIH) funded Anesthesia Research Center (ARC), at the University of Washington in Seattle – a world-leader on regional anaesthesia and pain management. I travelled to the symposium with Colin Shanks, especially with a view to meeting Professor Bonica and, hopefully, to seek an opportunity to work with him after I had finished my PhD. He said that he had read my papers and invited me to join his group. My PhD thesis, which focussed on bupivacaine, was submitted in January 1972, and I was appointed as a temporary Research Fellow in the Department of Pharmacy prior to our mid-year move to the United States.

2.2 The University of Washington (1972-1976) Our family of five arrived in Seattle on June 24 1972, and were greeted at the airport by Geoff and Irene Tucker – a memorable welcome to a new life. Soon at work, I was greeted by Professor Bonica, and by the equally remarkable B Raymond (Ray) Fink (1914-2000), Professor and Director of the ARC - a British born polymath anaesthesiologist with much wisdom and wit, and a veteran medical officer of the World War II Desert Campaign - who led his own local anaesthesia-neuroscience research group.

The ARC was my formal introduction to multidisciplinary research. The purpose of the ARC was to advance knowledge pertinent to clinical anaesthesia and thus involved numerous anaesthesiologists. Geoff Tucker and I brought our chemical pharmacology skills, and others added skills including psychology, bioengineering, neurophysiology, neuropharmacology, and biochemistry. Both the anaesthesia human studies lab and my analytical chemistry lab were situated at Harborview Medical Center (HMC), a downtown Seattle art deco county

41 Notably: Tucker GT. Determination of bupivacaine (Marcaine) and other anilide-type local anesthetics in human blood and plasma by gas chromatography. Anesthesiology 32(3): 255-260, 1970; Tucker GT, Boyes RN, Bridenbaugh PO, Moore DC Binding of anilide-type local anesthetics in human plasma: I. Relationships between binding, physicochemical properties, and activity. Anesthesiology 33(3): 287-302, 1970; Tucker GT, Boyes RN, Bridenbaugh, PO, Moore DC. Binding of anilide-type local anesthetics in human plasma: II. Implications in vivo, with special reference to transplacental distribution. Anesthesiology 33(3): 304-313, 1970; Reynolds F. Metabolism and excretion of bupivacaine in man: a comparison with mepivacaine. Br J Anaesth 43(1): 33-37, 1971; Reynolds F, Taylor G. Maternal and neonatal blood concentrations of bupivacaine. Anaesthesia 25(1): 14-23, 1970 42 The Third Asian and Australasian Congress of Anaesthesiology, 19-23 September 1970; convened by eminent anaesthetists Leonard Shea and Brian Dwyer. 28 hospital and UW teaching hospital. Geoff had his lab at the nearby Virginia Mason Research Center (VMRC), part of the Virginia Mason Hospital (VMH) clinical, teaching and research complex, where the head of the anaesthesia department was Daniel C (Dan) Moore (1918- 2015), a highly respected researcher and teacher of anaesthesiology who also had written textbooks on regional anaesthesia.

Professor John J Bonica, in 1986, at the University of Washington. He was known, affectionately, as “The Godfather” of anaesthesiology. He gave me a life-changing career opportunity in a new discipline.

The Anesthesia Research Center hallway plaque in the BB Wing of the University of Washington School of Medicine. The ARC was funded from 1968 to 1978.

Soon after my arrival in Seattle, Dan Moore invited me to participate in a research planning meeting along with some of the well-known identities of regional anaesthesia, among them anaesthesiologists Gale Thompson and brothers Phillip O (Phil) and L Donald (Don) Bridenbaugh from VMH, and Phillip R (Phil) Bromage (1920-2013) from McGill University in Montreal. The meeting was convened by Benjamin G (Ben) Covino (1930-1991), then Medical Director of the Astra Pharmaceutical Company in Worcester, Massachusetts (later Professor of Anesthesia at the University of Massachusetts Medical School (UMass) and subsequently Chair of the Department of Anesthesia at Harvard University-Brigham and Women’s Hospital). In one way or another, these people all influenced the content of this thesis. Our purpose was to design a pre-registration research program on a new long-acting local anaesthetic, etidocaine, 43 which had been recently synthesized by the Astra Pharmaceutical Company at their laboratories then in Worcester, Massachusetts (rather than

43 IUPAC name: [(RS)-N-(2,6-dimethylphenyl)-2-(ethyl(propyl)amino)butanamide] Adams HJF, Kronberg GH, Takman BH. (1975). U.S. Patent No. 3,862,321. “Acyl xylidide local anesthetics” Washington DC: USPTO. Assignee: Astra Pharmaceutical Products, Inc. (Worcester, MA). 29 at the main laboratories of the Swedish parent company in Södertalje, the origin of most of their products).

This was the period of the Vietnam War, and regional anaesthesia afforded a useful anaesthesia technique for ‘battlefield surgery’. This, no doubt, provided something of an influence in the funding priorities of the ARC by the National Institutes of Health. Previous studies by John Bonica’s group included studies of the cardiovascular sequelae of epidural anaesthesia in healthy young adult male volunteers under various conditions such as haemorrhage. 44 Whereas Dan Moore’s group had performed their studies in surgical patients,45 John Bonica’s group had previously performed their studies in prisoner volunteers. Due to a national scandal, although not involving the University of Washington, prisoners were no longer being allowed to provide a medical research volunteer population.46 With this project, I gained a rapid introduction into writing proposals for a newly-formed Institutional Review Board (‘ethics committee’), to preparing information and consent documents, and to recruiting healthy volunteers for (what were rather invasive) research studies. Not only were such activities new for me personally, they were also new for medical researchers and institutions in many parts of the world. Subsequently, I also worked with Dan Moore’s group on clinical studies on bupivacaine in surgical patients.

Besides Geoff Tucker, my newly acquired colleagues were anaesthetists Terence M (Terry) Murphy (1937-1996) originally from Liverpool UK, and Michael D’A (Mike) Stanton-Hicks originally from Adelaide, South Australia; all were to remain close friends and colleagues. Also, anaesthetist Peter Berges, a veteran of previous Bonica group studies, assisted with commencing the etidocaine project. Both Terry and Mike were experienced researchers on local anaesthetics and each was expert in placing a needle. The main studies were to be performed in the human studies lab at Harborview Medical Center, assisted by experienced technical staff. Although Professor Bonica was the group leader, he was, not surprisingly, heavily involved with departmental matters and left the organization and the lab work to us.

The research program commenced in late-1972 and involved studying the comparative neural blockade characteristics and cardiovascular sequelae of epidural anaesthesia with etidocaine

44 Some prominent examples include: Bonica JJ, Berges P, Morikawa K. Circulatory effects of peridural block. I. Effects of level of analgesia and dose of lidocaine. Anesthesiology 33(6): 619- 626, 1970; Bonica JJ, Akamatsu TJ, Berges PU, Morikawa K, Kennedy Jr, WF. Circulatory effects of peridural block. II. Effects of epinephrine. Anesthesiology 34(6): 514-522, 1971; Bonica JJ, Kennedy WF, Akamatsu TJ, Gerbershagen HU. Circulatory effects of peridural block: 3. Effects of acute blood loss. Anesthesiology 36(3): 219-227, 1972; Stanton-Hicks M, Berges PU, Bonica JJ. Circulatory effects of peridural block. IV. Comparison of the effects of epinephrine and phenylephrine. Anesthesiology 39(3): 308-314, 1973. Our first paper, that involving etidocaine [15], became Part V of this series, and added the pharmacokinetic dimension reflecting the contribution that Geoff Tucker and I introduced. Additionally, many of the volunteers were recruited from the (where Terry Murphy was the team doctor!). 45 Some prominent examples include: Moore DC, Bridenbaugh DL, Bridenbaugh PO, Thompson GE. Bupivacaine hydrochloride: a summary of investigational use in 3274 cases. Anesth Analg 50(5): 856-872, 1971; Moore DC, Bridenbaugh LD, Bridenbaugh PO, Tucker GT. Caudal and epidural blocks with bupivacaine for childbirth: report of 657 parturients. Obst Gynec 37(5): 667-676, 1971; Moore DC, Bridenbaugh LD, Bridenbaugh PO, Thompson GE, Tucker GT. Does compounding of agents increase their toxicity in humans? Anesth Analg 51(4): 579-585, 1972; Bridenbaugh PO, Tucker GT, Moore DC, Bridenbaugh LD, Thompson GE, Balfour RI. Role of epinephrine in regional block anesthesia with etidocaine: a double-blind study. Anesth Analg 53(3): 430-436, 1974 46 Hornblum AM. They were cheap and available: Prisoners as research subjects in twentieth century America. BMJ 315(7120): 1437-1441, 1997 30 and several local anaesthetic agents, along with the pharmacokinetics. A snapshot (below) taken during one of the studies shows the lab activities: sensory testing, cardiovascular monitoring and blood sampling for gas monitoring and subsequent pharmacokinetic analysis. The program also involved our spending significant time in planning with the senior drug discovery and development scientists, mainly pharmacologist HJ (Jack) Adams, organic chemist Bertil Takman, neurophysiologist HG (Helen) Vassallo, and pharmacokineticist Nick Boyes, at the Astra Pharmaceutical Company in Worcester, Massachusetts which, at that time, had the ambience of a highly inspirational university department and was directed by pharmacologist Murray R Blair, Jr (1929-2010), previously a Dean of Medicine from Tufts University. 47

John Bonica’s regional anaesthesia research group in early 1973 at the University of Washington anaesthesia research laboratory in the top floor operating suite of Harborview Medical Center. Personnel (from left): Charles ‘Chuck’ Pearcy (ARC chief technician) monitoring the physiological recorder, Gary Ledray (ARC technician) about to measure cardiac output by dye dilution, (the bearded) Laurie Mather about to measure blood gases, Geoff Tucker sampling blood, Terry Murphy and Mike Stanton-Hicks determining sensory and autonomic blockade, and an unknown healthy volunteer patiently undergoing the testing. A pharmacokinetic tutorial, of sorts, can even be seen on the blackboard in the background.

Like bupivacaine, etidocaine was synthesized and used as a racemate. Various earlier studies had shown some differential pharmacology of the separate enantiomers, and this had been discussed in my PhD thesis. This was of concern for interpretation of our anticipated

47 Their relevant publications include: Truant AP, Takman B. Differential physical-chemical and neuropharmacologic properties of local anesthetic agents. Anesth Analg 38(6): 478-484, 1959; Keenaghan JB, Boyes RN. The tissue distribution, metabolism and excretion of lidocaine in rats, guinea pigs, dogs and man. J Pharmacol Exp Ther 180(2): 454-463, 1972; Adams HJ, Kronberg GH, Takman BH. Primary local anesthetic testing and acute toxicity of N,N ′-disubstituted 2,3- diamino-2′,6 ′-propionoxylidides. J Pharm Sci 62: 1677–1679, 1973; Blair MR. Cardiovascular pharmacology of local anaesthetics. Br J Anaesth 47 Suppl 1: 247-252, 1975; Covino BG, Vassallo HG. Local anesthetics: mechanisms of action and clinical use. New York: Grune & Stratton (pp. 1- 148), 1976 31 pharmacokinetic and pharmacodynamic data. In preparation for the pharmacokinetic studies that were to be performed, Geoff Tucker and I made a substantial effort to resolve the enantiomers using various chromatographic techniques, the most promising of which was on thin layer chromatography plates impregnated with enantiopure mandelic acid which, we expected, would differentially retard the migration of the enantiomers. Although some analogous literature suggested that this approach could work, for etidocaine and bupivacaine, it did not! Our studies then still had to be based on achiral assays [15-17, 20-29].

I also developed another long-term research theme at that time. I would see surgical patients being administered intramuscular injections of (achiral) pethidine48 as a premedicant and/or postsurgical analgesic. Some patients appeared more affected than others, and I speculated that this could be due to differences in the rate of absorption of the intramuscularly injected pethidine. Other opioids such as morphine or fentanyl were also used, but their chemical assay was not adequate from the doses used. The less potent pethidine, however, was given in large enough doses for its measurement using a relatively simple GLC assay which we adapted for use in the etidocaine program [11]. We performed a series of studies on pethidine in healthy volunteer subjects (the investigators!) and surgical patients to relate the time course of pethidine blood concentrations to its effects, as this had never previously been done [13, 14, 18, 19]. These studies generated an elementary pharmacokinetic-pharmacodynamic approach to opioid analgesia that would lead into patient controlled analgesia (PCA) in later years.

In 1975, the year that the Vietnam War ended, I was promoted to Assistant Professor of Anesthesiology and Pharmaceutical Sciences, but things began to change, again somewhat unexpectedly, by our deciding to move back to Australia.

My time in John Bonica’s inspirational department at the University of Washington shaped the remainder of my career. I left there with many newly-acquired concepts and tools, not least among them, was the notion of the strength of multidisciplinary research, and with the pharmacology of anaesthesiology creating my main research themes.

2.3 Flinders Medical Centre (1976-1981) Sometime in late 1974, in Dan Moore’s office at Virginia Mason Hospital, I met Michael J Cousins, a visiting Australian anaesthetist and researcher from the Royal North Shore Hospital in Sydney. I took Michael to dinner that night, not really expecting to see him again. However, in mid 1975, I received a letter from him saying that he had been appointed as the foundation Professor of Anaesthesia and Intensive Care at Flinders Medical Centre, the new integrated state hospital and School of Medicine of the Flinders University of South Australia in Adelaide, and asking if I would consider applying for the inaugural lectureship in anaesthesia. After much family discussion, I applied, and was offered the position. We moved to Adelaide in January 1976, to find a house in competition with the countless other new staff recently recruited from elsewhere to Flinders, which had already inducted its first cohort of undergraduate students, and was due to open for patients in April 1976.

At Flinders, we started with bare labs and progressively built them up with personnel, equipment and techniques. I decided to study the management of postoperative pain, a

48 Synthesized in Germany during the 1930s originally as an anticholinergic agent and potential atropine substitute, it was soon recognized as a useful morphine-like analgesic: known as in the USA as meperidine and/or Demerol® it also has a local anaesthetic potency similar to that of procaine. 32 continuation of the pethidine work started in Seattle, and an area where many editorialists were pressing for urgent improvements. Whist awaiting delivery of lab equipment, I was invited by Peter McDonald, Head of the Department of Microbiology to collaborate on a biopharmaceutical project on erythromycin, and this proved a very satisfactory way of assembling pharmacokinetic and other techniques for subsequent projects [31, 32, 35, 42, 43, 51, 66, 79, 88]. The pethidine work started in 1977 and became a PhD thesis project for my first postgraduate student, biochemist Kevin Austin. It developed into an ideal model to study the relationships between pharmacokinetics, pharmacodynamics and patient satisfaction with their clinical pain management [33, 34, 36, 37, 44-47, 50, 52, 53, 55, 63, 64, 71, 77, 78, 84, 89]. Variability in pharmacokinetics led to variability in clinical response, and clinical response could differ between patients even at similar blood concentrations of opioid. Being able to use the analgesic agents in a way that accounted for such variability, or even allow patients to dose themselves, would become the key to better pain management – and better patient satisfaction! When analytical techniques improved over the ensuing years, allowing analogous studies with more potent opioids such as morphine, methadone and fentanyl, the patterns were shown to be similar. 49

In 1978, we learned from Peter Wilson, who had recently joined the department from working at the Mayo Clinic, of experiments being performed there with morphine injected intrathecally to reach opioid receptors described as being located in the spinal cord. 50 My clinical anaesthetist colleagues Michael Cousins, Chris Glynn and Peter Wilson were interested in trying to emulate this in patients with pain. I suggested the alternative of using epidural pethidine (instead of intrathecal morphine) because pethidine has local anaesthetic activity equivalent to procaine, and I rationalized that this, along with any central activity from systemically absorbed pethidine, might augment any spinal analgesic activity. We were astonished to observe that pain relief without local anaesthesia occurred, and that the subsequently measured systemic blood pethidine concentrations were much smaller than those that we knew from our other experiments were needed for parenteral pethidine analgesia. It was a crucial breakthrough. Our first report in the Lancet in 1979 [39] was the third-ever clinical report in this area, and other studies followed [40, 41, 54, 56, 65]. Spinal opioids soon became world-standard pain management practice.

In 1979, we were approached by the British pharmaceutical company, Glaxo Pty Ltd, to study their new intravenous anaesthetic, minaxolone. 51 It was intended as a water-soluble replacement for Althesin®, a mixture of alphaxolone and alfadolone in an organic polyethylated castor oil adjuvant base, but the base was considered to be problematic due to an unacceptable risk of anaphylactoid reactions. We performed preclinical studies in healthy volunteers to measure activity in the central nervous system (CNS) through mental arithmetic tasks, in concert with a pharmacokinetic approach, and determined that the rapid recovery from anaesthesia was due to high clearance rather than extensive redistribution [62, 73, 74]. We also extended the human studies to other intravenous anaesthetic agents [61, 70, 72, 75,

49 As examples: Plummer JL, Gourlay GK, Cherry DA, Cousins MJ. Estimation of methadone clearance: application in the management of cancer pain. Pain 33: 313-322, 1988; Gourlay GK, Kowalski SR, Plummer JL, Cousins MJ, Armstrong PJ. Fentanyl blood concentration ‐analgesic response relationship in the treatment of postoperative pain. Anesth Analg 67: 329-337, 1988 50 Yaksh TL, Rudy TA. Studies on the direct spinal action of narcotics in the production of analgesia in the rat. J Pharmacol Exp Ther 202(2): 411-428, 1977 51 Aveling W, Fitch W, Waters A, Simpson P, Prys-Roberts C, Sear JW, Chang H, Cooper GM, Savege TM, Campbell D. Early clinical evaluation of minaxolone: a new intravenous steroid anaesthetic agent. Lancet 314(8133): 71-73, 1979; McNeill H, Clarke RJ, Dundee J. Minaxolone: a new water-soluble steroid anaesthetic. Lancet 314(8133): 73-74, 1979 33

76]. In Seattle, Terry Murphy and I had done pilot studies with local anaesthetics in sheep and had realized their benefits as large animal experimental subjects. I determined that some additional preclinical studies needed to be performed on minaxolone in a large animal species, and so a pair of pharmacokinetic and pharmacodynamic (cardiovascular) studies was performed in sheep [58, 67]. Minaxolone never made it past the preclinical evaluation stage, mainly because it caused excitatory involuntary skeletal muscular movements in humans, and our work helped the manufacturers realise that it was not useful to proceed further. However, the significant benefit to us was that it provided the impetus for establishing the ‘sheep lab’.

Again digressing back to Seattle days, Geoff Tucker had conceived a set of ‘physiological’ pharmacokinetic experiments designed to account for drug concentration-time profiles in actual body organs, thereby differing to the standard ‘compartmental’ approaches that we and others were using. The experiments were proposed to study the consequences of intravenous lignocaine infusions in surgically pre-prepared Rhesus monkeys, as part of John Bonica’s renewal application for the Anesthesia Research Center (ARC) that had been submitted to the NIH. However, Geoff returned to the UK in 1973, before the outcome of the application was determined. When the ARC was re-funded, I inherited the studies and had to work up the practical methodology to perform the experiments.

At that time, such ‘physiological’ pharmacokinetics were based on several variables: putative regional blood flows (in the relevant species if known, scaled from another species if not known, or guessed), drug tissue:blood distribution coefficients (usually determined experimentally post mortem after drug infusion, or by equilibration experiments with isolated tissues), and drug clearance (usually determined as the total body clearance from intravenous administration studies, along with the portion known or guessed to be renal; the remainder was assumed to be hepatic). 52 The theory was simple enough. Blood flow provided the maximum rate of drug delivery, distribution coefficients provided the maximum body tissue concentrations and, with some assumptions about instantaneous mixing of the drug between blood and tissues, the time course of drug concentrations in various organs and tissues could be calculated using a set of differential equations. 53 These could be solved on a digital computer, and the resultant predicted drug concentration time course in the various identified regions for a particular rate of drug administration, could be compared to any experimental values for validation of the assumptions.

The practical problem was how to determine regional drug uptake and elimination (i.e., by various organs and tissues), and to separate the primary determinants (i.e., regional blood flow = drug supply to tissues, and drug clearance = drug elimination from tissues) in real experiments, preferably in conscious subjects. The methodology for measuring regional blood flow serially was a then-recently developed technique in non-human primates in the Cardiovascular Research Institute at the University of California–San Francisco, and I went there in 1974 to learn the technique. This involved bolus injection through a previously- placed left ventricular catheter of 50 µm diameter carbon microspheres, labelled with one of five gamma emitting radio-isotopes, and then repeating the process at other time points with differently labelled microspheres. Regional blood flow was determined by completely

52 Rowland, M. Physiologic pharmacokinetic models: relevance, experience, and future trends. Drug Metabol Rev 15(1-2): 55-74, 1984 53 Benowitz N, Forsyth FP, Melmon KL, Rowland M. Lidocaine disposition kinetics in monkey and man. I. Prediction by a perfusion model. Clin Pharmacol Ther 16(1): 87-98, 1974; Benowitz N, Forsyth RP, Melmon KL, Rowland M. Lidocaine disposition kinetics in monkey and man. II. Effects of hemorrhage and sympathomimetic drug administration. Clin Pharmacol Ther 16(1), 99-105, 1974 34 dissecting the animal post mortem and determining the microsphere radioactivity embolized in samples of each and every tissue. The activity of each label, i.e., at each time point, was determined in each sample by deconvolving the gamma emission spectrum of each isotope mathematically, and expressing this activity as a fraction of the total administered dose to estimate the tissue-by-tissue distribution of cardiac output at each time point. The concentration of the drug of interest, which was lignocaine in our studies, was also assayed in each sample to account for the fraction of dose.

Although state of the art at that time, the microsphere methodology was very time consuming, requiring complete dissection of the subject for radio-counting, with representative sampling of required tissues: small organs such as adrenals were counted whole and sundry tissues such as skin and bone were cremated before radio-counting. The methodology also was subject to errors, notably due to any haemodynamic and/or metabolic changes associated with anaesthesia and euthanasia of the subject, as well as post mortem migration of the microspheres: but these problems were not realized at that stage. Others in the Anesthesia Research Center were using the method for studying regional blood changes due to spinal anaesthesia,54 and so we shared the expensive facilities required. We could estimate regional blood flow at only 5 occasions (the number of different isotopic labels), and we could not distinguish rates of regional drug uptake from elimination. I produced only one study with the method, reported briefly in conference proceedings [23], before I returned to Australia, believing that there had to be a better way, if such studies were to be performed.

At Flinders during the late 1970s, clinical anaesthetist/intensive care specialist and PhD candidate in our department, Bill Runciman, was struggling with his studies of catecholamine pharmacotherapy in patients having septicaemia, mainly because the available methodology for measuring catecholamine concentrations in blood wasn’t reliable. He was seeking a change of direction. I had discussed with Bill my interests in ‘physiologically based’ pharmacokinetics with the minaxolone project, along with the limitations of the then-available methodologies and my vision of the gains being possible from developments beyond them. He decided to change his PhD project to this area. With Bill’s practical skills, and with the assistance of several others, notably research officers Anthony Ilsley and Ronda Carapetis, we developed methodology for the regional placement of catheters into various blood vessels of the sheep for the infusion of blood flow tracers using classical physiological techniques,55 and for the sampling of blood for pharmacokinetic purposes (Figures 2.1-2.4). This became the basis of the ‘multicannulated sheep preparation’ and is described more fully in Section 3.13 (note that among the examples given to illustrate the principles were etidocaine, bupivacaine and prilocaine: all were assayed achirally as there was then no way of resolving the enantiomers analytically [ 110 ]).

54 Sivarajan M, Amory DW, Lindbloom LE, Schwettmann RS. Systemic and regional blood-flow changes during spinal anesthesia in the rhesus monkey. Anesthesiology 43(1): 78-88, 1975 55 Lassen NA, Perl W. Tracer Kinetic Methods In Medical Physiology , Vol. 189. New York: Raven Press, 1979 35

Figure 2.1: The multicannulated sheep preparation. Insertion of catheters at the vessels of the neck. The exteriorized catheters were constantly perfused with high pressure-low flow heparinized saline to maintain patency over long periods, often several months. (From the PhD Thesis of Bill Runciman).

Figure 2.2: The multicannulated sheep preparation. Left: Before closing, after insertion of catheters, including a Swan-Ganz thermodilution catheter for measuring cardiac output, through the vessels of the neck. Right: Before awakening from anaesthesia, after closing and attachment of catheter flushing apparatus.

The main thrust of the development of the multicannulated sheep preparation occurred with our 1980s program to determine the effects of general and spinal anaesthesia on haemodynamic variables, including regional blood flow, oxygen consumption and impact on the regional clearances of co-administered drugs under conscious and perturbed conditions (described in Section 2.5). This necessitated the facility to sample regional blood for determining the affluent and effluent concentrations of flow tracers, oxygen and the drugs to enable calculation of their regional extraction ratios, fluxes, and clearances. The results of the program was published as a six-part series in the British Journal of Anaesthesia between 1984 and 1986 [100-102, 114, 120, 121]. Figure 2.3 shows a schematic of one of our models based on infusion of 125 I-iodohippuric acid sodium salt (125 IOH) into a mesenteric artery that allowed determination of concurrent gut, hepatic and renal blood flows from a single indicator using the classical physiological techniques of indicator dilution and the Fick Principle. Verification of the placement of catheters was performed using X-ray opaque dye angiography and typical catheter laboratory techniques. Some examples are shown in Figure 2.4. The physiological data gathering techniques, especially of digitally acquired signals, 36 evolved during the 1990s, and these were essential to the research outlined in Sections 3.3 and 3.4.

Figure 2.3: The multicannulated sheep preparation. A conceptual depiction of methodology used to measure concurrent blood flows to the gut, liver and kidneys using classical physiological techniques in association with the steady state regional pharmacokinetics of a single tracer, in this case 125 I–iodohippurate sodium.

Figure 2.4: The multicannulated sheep preparation. Radiologic verification of renal venous vasculature for placement of catheters. Left: Bilateral venograms illustrating the anatomy of the right (short) and left (long) renal veins. Right: Venogram illustrating the anatomy of the right hepatic vein and its tributaries. (From the PhD Thesis of Bill Runciman)

The practical strength of this program cannot be underestimated. Pharmacokinetics had evolved during the 1970s from the simple approach of plotting blood or urine concentrations on semi-log graph paper of the 1960s to a more mathematically-statistically complex computer-based subdiscipline, mainly based on compartment theory. But such time-averaged approaches still viewed the body as a ‘black box’ in which the drug behaved as an inert tracer – and clearly this could be fraught, for example when dealing with an anaesthetic agent that could exert profound acute decreases in cardiac output and changes to its blood-flow distribution, regional enzyme activity, drug affinity, and consequent pharmacokinetics. At last, we were able to perform studies designed to place pharmacokinetics on an anatomically- correct, physiologically-sound basis [68, 69, 80-82, 85-87]. These studies could answer previously inaccessible problems about drug disposition under various conditions [e.g. 100- 102, 114, 118, 120, 121, 138, 144, 148]. The sheep preparation would later be central in the stereopharmacology studies described in this thesis (Section 3). 37

2.4 The University of Massachusetts Medical Center (1981-1983) In 1981, Mike Stanton-Hicks, my former colleague from Seattle, was now Professor and Head of the Department of Anesthesia at UMass in Worcester, Massachusetts. He wrote asking me to consider applying for a newly-established research professorship there. After much family deliberation, we moved to Worcester in August 1981. At UMass, with a financial recession happening, it was proving very difficult to attract funds and to start new research projects, and so I gave priority to completing various papers from our research at Flinders [77-83, 88, 89]. I also initiated several new papers [90-93], including a highly cited review on fentanyl and its congeners [92]. In March 1982, I was invited to give a plenary lecture at the 7th Annual Meeting of the American Society of Regional Anesthesia (ASRA), held in Monterey California [91]. After the meeting I was invited by anaesthesiologist and pharmacologist Donald R (Don) Stanski to spend a week in his labs at the Department of Anesthesiology of Stanford University and the Palo Alto Veterans Administration Hospital. Don and I went on to share much camaraderie through research that was later to have a direct link to this thesis through thiopentone (Section 3.3). I also spent many productive hours with Ben Covino’s colleague Hal Feldman in the cardiovascular pharmacology lab at Harvard University- Brigham and Women’s Hospital, and with Helen Vassallo at the Astra Pharmaceutical Company discussing research issues of local anaesthetic pharmacology that, ultimately, were leading to the next long-acting local anaesthetic, ropivacaine, and my involvement with it.56 In mid-1982, Mike Stanton-Hicks left UMass, initially for a sabbatical at the University of Düsseldorf, but decided not to return. At the end of 1982, Michael Cousins unexpectedly visited Worcester, and invited me to return to Flinders, which I did in 1983.

2.5 Flinders Medical Centre (1983-1990) My work on return to Flinders focussed on intravenous anaesthesia [103, 104, 115] and opioid analgesia [98, 99, 119], the latter becoming part of the development and scientific basis for opioid-based patient controlled analgesia (PCA) [97, 107, 136, 137, 146, 147, 153, 161, 162]. I also prepared a review with Michael Cousins on spinal opioids [96] that has become amongst the most highly cited papers in the anaesthesia literature. In 1984, I was elected to Fellowship of the Faculty of Anaesthetists of the Royal Australasian College of Surgeons (FFARACS) for “services to anaesthesia”.

Our sheep lab had continued with pharmacokinetic and pharmacodynamic studies. The sheep is a sufficiently large animal to allow exploration of anatomical and physiological issues in pharmacokinetics and pharmacodynamics under differing circumstances. Applications of the ‘multicannulated sheep preparation’ to various studies often produced significant and unexpected results, and these helped us to explore anatomically-based pharmacokinetics [100- 102, 114, 118-121]. This work formed the centre-piece of Bill Runciman’s PhD thesis, and we thereafter continued a very successful partnership investigating various physiological- pharmacological effects of anaesthesia, in particular, with the not-yet-approved intravenous anaesthetic propofol [124, 129, 132, 133, 144, 159, 163, 172, 173]. Bill and I co-supervised PhD students anaesthetist Craig Nancarrow, biochemist Al Rutten, surgeon YiFei Huang,

56 The Covino lab used techniques that complemented the ones developed in my Flinders lab: e.g., Liu P, Feldman HS, Covino BM Giasi R, Covino BG. Acute cardiovascular toxicity of intravenous amide local anesthetics in anesthetized ventilated dogs. Anesth Analg 61(4): 317-322, 1982;; Feldman HS, Covino BM, Sage DJ.. Direct Chronotropic and Inotropic Effects of Local Anesthetic Agents in Isolated Guinea Pig Atria. Reg Anesth Pain Med 7(4), 149-156, 1982; Liu PL, Feldman HS, Giasi R, Patterson MK, Covino BG. Comparative CNS toxicity of lidocaine, etidocaine, bupivacaine, and tetracaine in awake dogs following rapid intravenous administration. Anesth Analg 62(4): 375-379, 1983 38 biochemist Richard Upton and physiologist Michael Reid. 57 We developed methodology and performed experiments on a variety of drugs and conditions that used the power of the experimental preparation [ 110 , 138, 163, 166, 168, 169, 173, 182, 183, 187, 191, 192, 194]. The underlying principles of all these experiments were the same – determination of mass balance. Put simply, the dose of a drug had to be accounted for by the amount of drug in the sampled blood (or central compartment) + the amount of drug not yet absorbed (if not placed intravascularly) + the amount in the peripheral (or tissue) compartment(s) + the amount in the effect compartment (miniscule, and typically not measurable per se ) + the amount of drug already eliminated [139, 140]. Such measurements, although requiring significant surgical invasion, were reasonably amenable to experimentation.

Busy days in the ‘sheep lab’ at Flinders. Top left: The sheep is wearing a mask to monitor its oxygen consumption and carbon dioxide production during Craig Nancarrow’s study of the effects of acid-base manipulation on the distribution of local anaesthetics into heart and brain [131]. Top righ t: Sampling blood as part of Al Rutten’s PhD project comparing the cardiovascular effects of local anaesthetics [149]. Bottom left: This picture was often used in the (bearded) author’s research presentations, usually accompanied by the comment that “the sheep is on your right”. Bottom right: An experiment during Bill Runciman’s research on the effects of general anaesthesia on regional blood flow and drug disposition [132].

In 1983, I had participated in an international conference on regional anaesthesia in Saltsjöbaden, Sweden. Immediately afterwards, I participated in a small closed workshop of experts in Stockholm, chaired by Ben Covino (then at Harvard Medical School), to discuss early data on LEA-103, later named ropivacaine, a new local anaesthetic agent synthesized at

57 Michael Reid was the first combined BM BS PhD candidate at Flinders Medical Centre. This was an innovative combined degree intended for suitably qualified medical students already having a higher degree. The candidate directed their planned course electives into supervised laboratory research and added an intercalated year 5 to finish their PhD project research to graduate with the combined medical undergraduate degrees of BM, BS and PhD at the completion of their year 6. I introduced this degree during my tenure (1986-1990) as Assistant Dean (Postgraduate Affairs). 39

Astra AB, Sweden, that was being proposed as a ‘safer’ alternative to bupivacaine. This came about because of clinical morbidity evidence, reported in the late 1970s, suggesting that bupivacaine could have a disproportionately high risk of causing cardiotoxicity, manifest as ventricular arrhythmias associated with difficult resuscitation, compared to the shorter acting agents. New local anaesthetic agents with a greater margin of safety were sought to replace bupivacaine. The meeting recommended to Astra that development of LEA-103 should proceed…and it did.

In 1984, I was asked to set up a program to investigate the relative toxicity of ropivacaine in comparison to the standard shorter-acting lignocaine and longer-acting bupivacaine in our multicannulated sheep preparation. This was to start an extensive program on drug uptake and distribution, in relation to effects on the heart and brain in relation to regional pharmacokinetics, and formed the beginning of the new era of the stereopharmacology of local anaesthetics [123, 131, 139, 140, 145, 149, 164, 169, 170, 174, 175, 185, 186]. These studies involved determination of the myocardial effects of the local anaesthetics in relation to their disposition in the myocardium. This required cannulation of the coronary sinus to collect venous effluent of the heart.

Figure 2.5: Verification of the placement of catheters in a sheep subject under image intensification at Flinders.

Figure 2.6. Angiographic appearance of the coronary sinus of the sheep prior to ligation of the hemiazygos vein (left), and after ligation of the hemiazygos vein at the left aspect of the pericardium (right). (From the PhD thesis of Craig Nancarrow.)

Detailed examination of the vasculature revealed that the left hemiazygos vein partly drains the mixed tissues of the chest wall, and also enters into the left aspect of the left coronary sinus. This finding is consistent with the persistence of the left vena cava in the adult sheep. 40

Hence, during preparation, the hemiazygos vein had to be ligated and verified that it was ligated (Figures 2.5 and 2.6). Other research groups having similar objectives had neglected this significant anatomical issue.

My last major project at Flinders focussed on morphine. During the late 1980s, there was much controversy about the role of the kidney in morphine-induced clinical toxicity. It was thought that the pharmacologically active morphine 6-glucuronide metabolite could accumulate, and this led to its adverse respiratory effects. In 1987, I convened a consortium of Adelaide researchers interested in morphine pharmacology to apply (successfully) for an NHMRC grant to study the morphine metabolite/kidney clearance problem based on the multicannulated sheep preparation [160, 181, 189, 193, 195]. I also continued my interest in the pharmacology of pain management [116, 117, 122, 125-128, 130, 141-143, 148, 152-156, 158, 167, 176, 178-179, 184].

March and April of 1990 were incredibly busy months. In March 1990, I was promoted to a personal chair at Flinders named the Abbott Professor of Anaesthesia and Analgesia Research. We had the pleasures and pains, so to speak, of being the hosts of an international conference of some 2000 delegates with the 6th World Congress on Pain organised by the IASP being held in Adelaide during April 2-6, and where I delivered the plenary lecture “Novel methods of analgesic drug delivery” on April 4 [176]. Sadly, in the week before the Congress, I attended the funeral of my colleague biochemist Alan Snoswell (1930-1990), with whom I shared interests in ovine research methodology, as well as the metabolic aspects of nitrous oxide [171, 182, 187, 194].

2.6 The University of Sheffield (1988) In 1988, I took sabbatical leave at the University of Sheffield-Royal Hallamshire Hospital to work with Walter Nimmo in the Department of Anaesthetics and with my former colleague Geoff Tucker in the Department of Clinical Pharmacology. Walter’s department had a particularly strong pharmacokinetic-pharmacodynamic research program oriented towards drugs used in the perioperative period. Geoff, with Martin Lennard (Senior Lecturer in the Department of Clinical Pharmacology), had developed analytical techniques using the newly developed chiral stationary phases (derivatized cyclodextrins, immobilized α1-acid glycoprotein (AGP or AAG), and human serum albumin (HSA)) that had been found to have different binding affinities for many pairs of enantiomers. Bringing these themes together, we performed a relatively simple study on prilocaine (Section 3.22), separating the enantiomers on a derivatized cellulose (Chiracel OD) stationary phase, that demonstrated significant enantioselectivity in its human pharmacokinetics [ 197 ]. On returning to Flinders in January 1989, I purchased an early version EnantioPac HPLC-AGP column from Sweden. With this, we determined the pharmacokinetics of the separate mepivacaine and bupivacaine enantiomers to determine whether metabolic inversion occurred [ 170 ], as well as to determine the tissue distribution of the bupivacaine enantiomers after administration of the racemate [ 177, 188, 190 ].

41

A day out from the lab at Sheffield - with Cedric Prys-Roberts, Professor of Anaesthesia at the University of Bristol (left), and Geoff Tucker (right) at an international symposium on intravenous anaesthesia held at Cambridge University (March 28 1988).

2.7 The University of Sydney (1991-2007) In 1990, Michael Cousins accepted the Chair of Anaesthesia at University of Sydney to form a new academic Department of Anaesthesia and Pain Management at the Royal North Shore Hospital (RNSH). He thereby became the University’s second Professor of Anaesthesia, after Douglas Joseph (1925-1990). I was invited to apply for a research chair created in the same academic department, and was appointed Professor of Anaesthesia and Analgesia (Research), effective January 2 1991.

From our beginnings at RNSH, we were accommodated in old (c. 1930s) and remote hospital space until our new (office and laboratory) departmental accommodation, which was to be built within the first year, was commissioned. Two rather cold and leaky abandoned outpatient treatment rooms were assigned to us as temporary analytical chemistry and neurophysiology labs, and we did the best we could with what we had to set up our research projects. Elsewhere, a verandah of an old clinical suite was made available as a ‘pain clinic’, and a disused operating room within the main surgical suite was made available as a ‘human studies lab’. More encouragingly, during1992, an old animal house building some 400m remote from the main hospital building was rebuilt into excellent operating, experimental and holding rooms, and part of this became our permanent ‘sheep lab’. Eventually, after several false starts, a full ward was refurbished as a fully functional out-patient pain clinic, and a suite of laboratories and offices was commissioned in the main hospital building on the floor above our renovated departmental area. Thus, the Pain Management and Research Centre was finally commissioned and officially opened on May 9 1994 by NSW Minister for Health, Ron Phillips, and University of Sydney Deputy Vice-Chancellor Professor Susan Dorsch. By then, we were becoming a full academic department of anaesthesia, as well as the largest multidisciplinary pain centre yet assembled in the southern hemisphere. The initial research priorities were mainly on the neurophysiology of pain transmission in Michael Cousins’ labs, and diverse whole-body pharmacological studies of analgetics and anaesthetics in my labs. This combined approach would continue the multidisciplinary application of laboratory research to clinical problems and postgraduate teaching [201-211, 213, 215, 219, 220, 222, 225, 228, 237, 255, 274, 278, 281, 283, 286, 290-292, 298, 301, 312, 315].

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Our other major projects at RNSH included pharmacokinetic-pharmacodynamic analysis of i.v. anaesthesia, patient controlled analgesia, modes of opioid delivery and perioperative multimodal analgesia [223, 229-231, 233-236, 238-240, 247, 249, 256, 257, 260, 269, 272, 277, 293], studies of nonsteroidal anti-inflammatory agents and their [226, 242, 263, 270, 271, 279, 297, 304], and studies of algogenic peptides in saliva as biomarkers for oro-facial pain [237, 247, 283]. Concurrently, an academic research partnership was built with Maree Smith (then senior lecturer, now Professor) at the University of Queensland, for studies on morphine and its congeners [255, 281, 286, 310, 320]. Other clinical research focused on improving drug delivery techniques, especially aerosolized transpulmonary drug delivery, for pain control and for diabetes control [227, 231, 238, 253, 254, 275, 294, 295, 308, 310, 326].

On April 9 1994, in Chicago, I was awarded the John J Bonica Distinguished Medal of the American Society of Regional Anesthesia. This was indeed a great honour. John Bonica had a significant influence on me and my career, and I had hoped that he would be present. He sent me a congratulatory message, but unfortunately he was too ill to attend the meeting. Sadly, on August 15 1994, John Bonica died, only weeks after the death of his wife, Emma.

The writer with Professor John Bonica, in his office at the University of Washington in 1986.

In 2000, I was also honoured to be elected to Fellowship of the Royal College of Anaesthetists (UK). My presentation citation was prepared by neuroscientist Patrick Wall (1925-2001), who was famous for the “gate theory” of pain. In 2002, the Faculty of Pain Medicine of the Australian and New Zealand College of Anaesthetists awarded me an honorary Fellowship.

How did the stereopharmacology theme of this thesis emerge? Clearly, it had been primed by my prior studies of the chiral local anaesthetic agents - but these did not constitute a theme. The theme came about as a result of a ‘curious exchange’ over thiopentone with Don Stanski, in 1992.

Don Stanski became Chairman of the Department of Anesthesiology at Stanford University in 1992. From 1982, I had been in frequent contact with him as we had many interests in common and had together published a pharmacokinetic review [151]. In 1992 when I was visiting Stanford, Don asked me to review a grant proposal on thiopentone that he was about to submit to the NIH. His group had already published numerous pharmacokinetic- pharmacodynamic papers on thiopentone based on state-of-the-art qEEG techniques, and he was seeking to extend his laboratory rat model to include the role of regional blood flow in 43 pharmacokinetics. Being somewhat attuned to the potential importance of stereopharmacology, I knew that thiopentone, like many barbiturates, is synthesized and used as a racemate, 58 and so I suggested that differential pharmacokinetics or pharmacodynamics of its enantiomers could complicate the interpretations of the data. Don dismissed my concerns, responding that the reviewers wouldn’t know about that, and that neither he nor others had enantiomeric methodology, anyway. Nonetheless, he got his grant. However, out of this somewhat ‘curious exchange’ over thiopentone, another reason to study the impact of stereopharmacology materialized.

Professor Don Stanski in his Stanford office, at the time of our ‘curious exchange’ over thiopentone that led, indirectly, to this thesis.

By mid-1992, we had embarked upon our first large animal (sheep) research program in our new department. This was an NHMRC-supported study in our multicannulated sheep preparation to test the principles of multimodal analgesia based on the combination of an opioid and a nonsteroidal anti-inflammatory drug (NSAID), an area of rapidly growing interest. Our study was, in the first instance, to use (achiral) fentanyl as the opioid - chosen because of the convincing evidence for a lack of pharmacological activity of its metabolites, and ketorolac as the NSAID - chosen because of the convincing evidence for the analgetic effectiveness of its parenteral formulation. The study plan involved various aspects of stereopharmacology because ketorolac is a racemate, and various studies on its enantiomers had been performed indicating a good degree of stereoselectivity in its analgetic and anti- inflammatory actions, in common with other chiral NSAIDs. Accordingly, development of a chiral assay of ketorolac in biofluids was designated as a primary laboratory requirement, preceding the research program. However, it was thiopentone and the ‘curious exchange’ with Don Stanski that really intrigued me. Thiopentone was then still the most important and widely used anaesthetic induction agent of all time and we had previously studied its cardiovascular and other pharmacological effects [159, 163, 165]. However, it was very clear that very few people were aware that it was synthesized and used as a racemate.

58 Thiopentone has one chiral carbon, the alpha-carbon of the 5-(2’-methylbutyl) side chain, giving the (+)-R- and (-)-S-thiopentone enantiomer pair. See: Andrews PR, Mark LC. Structural specificity of barbiturates and related drugs. Anesthesiology 57(4): 314-320, 1982 44

I decided that the thiopentone-chirality issue was worthy of further research that I would engage with this research if the opportunity arose. And it did, in 1993, starting with my research proposal (successfully) submitted for the inaugural Douglas Joseph Professorship of the Australian and New Zealand College of Anaesthetists. The associated grant funds allowed me to develop what essentially became the thiopentone enantiomer research project (Section 3.3), thereby forming part of a longer-running program on intravenous anaesthetic agents commenced at Flinders during the 1980s. These efforts were reinforced, later in 1993, by an invitation from Chiroscience plc, a small chemical company from Cambridge UK, to collaborate on a new chiral local anaesthetic research program that had roots in my PhD project on bupivacaine, which fitted into the long-running local anaesthetic agent program established several decades earlier (Section 3.2). These more major programs, along with the NSAID program, were later supplemented by several more discrete projects involving stereopharmacology of ketamine, halothane, and thalidomide, thereby forming the basis of this thesis.

Not just a pretty face. Ovis aries , you taught us a lot about how things work.

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3. Stereopharmacological research in anaesthesiology: publications comprising the main theme

“On October 28 in 1853, Henry David Thoreau received back from his publisher the 706 unsold copies (out of 1000 printed) of his first book, 'A Week on the Concord and Merrimack Rivers,' published four years earlier at his own expense. In his journal later that same day, the ever-resilient Thoreau described his 'purchases' as 'a library of nearly nine hundred volumes, over seven hundred of which I wrote myself.’” (Today in Literature http://www.todayinliterature.com/ )

This Section introduces the publications selected in support of this thesis, along with a narrative of how the selected publications relate to the theme. The narrative is not intended to present updated reviews on the subject drugs as the thesis is based upon the knowledge as relevant at the time of the original publications.

The selections are labelled as ‘programs’ and ‘projects’. The programs comprise local anaesthetic agents: this is the largest section, and the selected publications focus on bupivacaine, prilocaine, ropivacaine and levobupivacaine; intravenous anaesthetic agents: the agents also included minaxolone, propofol, alphaxolone and chlormethiazole, but the selected publications focus on the work on thiopentone; and nonsteroidal anti-inflammatory drugs : the agents studied also included diclofenac and but the selected publications focus on ketorolac. The projects focus on single drugs: halothane , ketamine and thalidomide. All came from my own initiatives, except for that on thalidomide which came from an invitation to collaborate with the oncology research group led by oncologist Frances M (Fran) Boyle and molecular biologist Ross A Davey, and involved my co-supervision of PhD candidate biologist Susan F (Sue) Murphy. Reasonable chronology has been observed within each subsection.

Before proceeding to the programs, projects and publications comprising the main theme, I have outlined a brief historical introduction to various landmarks of the science of anaesthesiology that have influenced this thesis.

3.1 Introduction One of the first modern landmarks of science in anaesthesiology is assembled in the proceedings of a conference sponsored by The New York Academy of Medicine and the National Research Council (U.S.), Committee on Anesthesia, Section on Anesthesiology and Resuscitation, in New York on April 23-24 1962. 59 I discovered this remarkable volume early in my postgraduate career, and it was highly influential in shaping my understanding of anaesthetic agents. I note, in retrospect, that it didn’t discuss stereochemical aspects of pharmacology even though some of the prominent anaesthetic subjects, notably halothane and thiopentone, 60 were used as racemates. However, that particular omission is not surprising.

59 Published as: Papper EM, Kitz RJ (Eds). Uptake and Distribution of Anesthetic Agents . New York: McGraw-Hill, 1963, 321p 60 The original pharmacology of halothane was reported without any mention of its chirality. See: Raventós J, Goodall RR. The action of Fluothane*—a new volatile anaesthetic. Br J Pharmacol Chemother 11(4), 394-410, 1956. [The asterisk in the title denoted the name to be a registered trade mark of Imperial Chemical (Pharmaceuticals) Ltd.] However, in a lecture to the Manchester Medical Society in 1957, Charles Suckling, the chemist who developed halothane and the colleague of the pharmacologist James Raventós, commented “In passing we might note that the second carbon atom of halothane has bonds with four different atoms and halothane should 46

Certainly, the importance of stereochemistry in the pharmacology of endogenous and exogenous natural products, including neurotransmitters and hormones, and main-stream drugs such as cocaine, opium and the belladonna alkaloids, had been appreciated for more than half a century. However, when it came to synthetic drugs, medicinal chemists were only then learning about the biological significance of isosterism, i.e., the equivalence of space- filling by various functional groups of drug molecules, thus ester and amide linking groups in procaine and procainamide, for example, were seen to be bio-isosteric, and drugs containing these groups could have equivalent actions. Moreover, the analytical chemistry of the day was rarely sufficiently sensitive or specific enough for incisive pharmacokinetic inferences, let alone differentiation between stereoisomers, unless separate stereoisomers or diastereoisomers were being compared.

The scientific successor to the 1962 New York meeting occurred in 1984 in Bonn, and I was privileged to be a participant [ 110 , 111]. 61 This was the International Workshop on Quantitation, Modelling and Control in Anaesthesia, May 31 – June 2 1984, and organized by anaesthetist Horst Stoeckel and his colleagues, Jürgen Schüttler and Helmut Schwilden, then of the Department of Anesthesiology at the University of Bonn. The renowned anaesthesiologist E (Manny) Papper (1915-2002), co-editor of the proceedings of the 1962 New York meeting, also participated in this conference, and I was delighted to be able to talk with him – he, along with chemical pharmacologist Bernard B (Steve) Brodie (1907-1989), had shaped my earliest postgraduate role models in their pioneering of collaborative multidisciplinary chemical-clinical-pharmacology at Columbia University, New York, on drugs used in anaesthesiology, including pethidine 62 and thiopentone,63 that were to engross me for many years. I had been privileged to meet both of them on their visits to Sydney during my PhD candidacy (and Manny Papper was later to become one of my PhD examiners!). Many papers of the Bonn program involved pharmacokinetics and newly emerging pharmacodynamic methods (mainly qEEG and its variants) for measuring and controlling anaesthetic drug effects. However, of the various data presented on many chiral anaesthetic drugs (including thiopentone, methohexitone, , etc), only one chiral substance was formally recognized – ketamine, with one set of data describing the qEEG effects of its separate enantiomers. By then, the GLC and HPLC analytical techniques were certainly satisfactory, but the techniques for enantiomeric resolution were not yet ready, apart from cumbersome resolutions involving derivatization to make diastereoisomers. Even so,

therefore exist in two optically active isomers. The commercial product should be the racemic mixture, but we do not know how to resolve it into the dextro- and laevorotatory forms.”. Reprinted as: Suckling CW. Some chemical and physical factors in the development of Fluothane. Br J Anaesth 29(10): 466-472, 1957. Similarly, chemical-pharmacological accounts of the chiral barbiturates failed to mention the fact that they were administered as racemates. See: Lloyd JB. The barbiturates: chemical constitution and nomenclature. Br J Anaesth 29(10): 473-478, 1957 61 Published as: Stoeckel HO (Ed). Quantitation, Modelling and Control in Anaesthesia . Stuttgart: Georg Thieme, 1985, 310p.. 62 Notably: Apgar V, Burns JJ, Brodie BB, Papper EM. The transmission of meperidine across the human placenta. Am J Obstet Gynecol 64(6): 1368-1370, 1952; Burns JJ, Berger BL, Lief PA, Wollack A, Papper EM, Brodie BB.. The physiological disposition and fate of meperidine (Demerol) in man and a method for its estimation in plasma. J Pharmacol Exp Ther 114(3): 289-298, 1955 63 Notably: Brodie BB, Mark LC, Papper EM, Lief PA, Bernstein E, Rovenstine EA. The fate of thiopental in man and a method for its estimation in biological material. J Pharmacol Exp Ther 98(1): 85-96, 1950; Brodie BB, Mark LC, Bernstein E, Papper EM, Lief PA.. Acute tolerance to thiopental. J Pharmacol Exp Ther 102(4): 215-218, 1951; Brodie BB, Burns JJ, Mark LC, Lief PA, Bernstein E, Papper EM. The fate of in man and dog and a method for its estimation in biological material. J Pharmacol Exp Ther 109(1): 26-34, 1953; Mark LC, Burns JJ, Campomanes CI, Ngai SH, Trousof N, Papper EM, Brodie BB. The passage of thiopental into brain. J Pharmacol Exp Ther 119(1): 35-38, 1957 47 the pharmacological significance of stereopharmacology wasn’t yet widely appreciated, but other strong voices were now being heard. A subsequent program, again organized by the Department of Anesthesiology at the University of Bonn, was held in May 1994. This symposium built upon the scientific advances of informatics during the previous decade, and was concerned more with applications of the concepts and tools to anaesthesiology. 64

Additionally, among various other collections, a volume of the short-lived journal Anaesthetic Pharmacology Review contained a set of papers (some of which have already been cited elsewhere in this thesis) that provided insightful methodology reviews related to anaesthesiology. 65 Papers included the abstract methodology of deconvolution to account for body transit times of various solutes, the functional methodology of compartmental analysis to account for the time-averaged course of mass balance, and the practical methodology of flux and mass balance analysis at defined times as described in this thesis, as well as applications to many problems. This particular issue was most gratifying in that the Guest Editors were my former PhD students Bill Runciman and Richard Upton.

3.11 Growing recognition of stereopharmacological issues The current impetus to recognize the potential stereopharmacological problems of racemic drugs began with the international symposium ‘Stereochemistry and biological activity of drugs’ held in Noordwijkerhout in the Netherlands on October 21-22 1982, organized by the Dutch Society of Clinical Pharmacology and Biopharmacy, the Dutch Society of Pharmacology, and the Royal Dutch Chemical Society, Medicinal Chemistry Section. 66 In particular, it was pharmaceutical scientist Everhardus Jacobus Ariëns (1918-2002) of the University of Nijmegen who, in a now famous quotation, referred to the lack of attention to the stereochemistry in pharmacological and medical investigation as being the basis for producing "sophisticated nonsense in pharmacokinetics and clinical pharmacology." 67 This was an inconvenient truth for many drug researchers, drug manufacturers and drug regulators, alike. Professor Ariëns’ case was ably supported by many other scientists, among them Geoff Tucker and Martin Lennard, in whose labs at the University of Sheffield I first observed routine chromatographic enantiomeric resolutions (Section 2.6). However, the message about stereopharmacology still had not permeated into the discipline of anaesthesiology, at least not in any systematic way.

It was Tucker and Lennard, in particular, who pointed out that the pharmacokinetics of a racemic drug determined achirally may have no resemblance to the pharmacokinetics of either enantiomer if their pharmacokinetics differed markedly, using the simulation shown in Figure 3.1 to make the point. 68 Clearly, pharmacokinetic-pharmacodynamic studies based on a

64 Published as Schwilden H, Stoeckel H (Eds). Control and Automation in Anaesthesia . Berlin and Heidelberg: Springer-Verlag, 1995, 281p 65 Pharmacokinetics. Anaesthetic Pharmacology Review Volume 2(3), 1994, pp 1-295: SA Feldman (Ed) Castle House Publications: Tunbridge Wells, Kent (UK). This journal was started in 1993 by anaesthetist Stanley Feldman, from Westminster Hospital London - a noted researcher of neuromuscular blocking agent pharmacology. 66 Published as: Ariens E, Soudijn W, Timmermans PBMWM. (Eds) Stereochemistry and biological activity of drugs. Blackwell Scientific publications: Oxford 1983. 194p 67 Ariëns EJ. Stereochemistry, a basis for sophisticated nonsense in pharmacokinetics and clinical pharmacology. Eur J Clin Pharmacol 1984; 26: 663-668 68 See, as examples: Tucker GT Lennard MS. Enantiomer specific pharmacokinetics. Pharmac Ther 45: 309-329, 1990; Lennard MS. Clinical pharmacology through the looking glass: reflections on the racemate vs enantiomer debate. Br J Clin Pharmacol 31(6): 623-625, 1991 48 racemate could be problematic, even more so when the kinetics of exchange with receptors, or intrinsic activity at receptors, differed between enantiomers.

Figure 3.1: Enantiomers with different pharmacokinetics, as depicted in the figure, could generate misleading information if assayed achirally as the sum of the enantiomers. (From Tucker GT Lennard MS. Enantiomer specific pharmacokinetics. Pharmac Ther 45: 309-329, 1990)

As briefly introduced in Section 1, optical isomers (enantiomorphs or enantiomers, in the 1856 terminology of crystallographer Carl Friedrich Naumann 69 ) are characterized by a centre of asymmetry or stereogenic centre (named a “chiral” centre by Lord Kelvin, in 1894). Some substances have more than one chiral centre (diastereoisomers), others have none (achiral). Differences in the pharmacology of drug enantiomers have been well known for over a century. Stereospecificity pertains to active vs. inactive forms, and such differences can be expected from a chiral drug produced from a natural product (e.g. atropine, cocaine, morphine), but may be overlooked when a chemical synthesis, storage, or in vivo metabolism, produces a racemate. As many biological molecules, e.g. amino acids, carbohydrates and hormones, are chiral substances, it is not surprising that the body responds to chiral drugs that mimic, or interact with, natural substances in a stereoselective, but not necessarily stereospecific, manner.

About 25% of all existing drugs (at least, until the mid-1990s) were being made and used as a racemate, either because the technology was not available to accomplish it, or the need to produce them as enantiopure substances was not perceived. Stereoselectivity, which is far more common than absolute stereospecificity, occurs where actions of enantiomers are qualitatively and/or quantitatively different. The time-course of effects from a racemate derive from the summation of effects of the individual enantiomers. Similar or different pharmacokinetics may additionally occur, and/or there may be qualitative and/or quantitative differences in metabolic pathways, involving active and inactive metabolites, and even metabolic inversion or interactions between enantiomers. Enantioselective pharmacology can occur at any site where a drug interacts with an endogenous chiral centre. This can be a receptor binding protein (affecting drug action), metabolizing enzymes (affecting drug elimination) and/or other macromolecules (affecting drug distribution). It is not usually possible to predict from first principles to what extent different drug enantiomers will interact

69 Gal J. Carl Friedrich Naumann and the introduction of enantio terminology: a review and analysis on the 150th anniversary. Chirality 19: 89-98, 2007 49 differently with proteins or enzymes: the differences in their kinetics and dynamics must be determined experimentally. Among the drugs used by anaesthesiologists, some are achiral, e.g., , propofol, fentanyl, lignocaine, diclofenac; some are racemates, e.g., , ketamine, methadone, tramadol, bupivacaine, prilocaine, ketorolac, thiopentone; and others are enantiopure, e.g., morphine, cocaine, , levobupivacaine, ropivacaine, .

3.12 The main theme: stereopharmacological programs and projects In most of the following papers, the signs of optical rotation of enantiomer have been omitted for clarity as, in the main, optical rotation is not relevant to the stereopharmacology. Where possible, the CIP nomenclature has been used (also see Section 1.2).

All of the experimental studies were approved by the relevant institutional ethics review committees, and the interventions on patients or healthy volunteers, as appropriate, were performed by registered medical practitioners. The objective of animal studies was to gain and explain a greater understanding of the pharmacology of the subject drugs, as far as laboratory animal studies can be viewed as models for extrapolation to humans. Such models are founded on the basis that the underlying principles and mechanisms are essentially conserved between mammalian species. In some studies, particularly with those on local anaesthetics, the dosage regimens were chosen to resemble the clinical pattern that might have occurred in patients with accidental intravascular injection. In some other studies, dosage regimens were performed with either a single intravenous dose followed by a period of drug ‘washout’ conditions, or by single or multiple stage intravenous infusions or ‘washin’ conditions to produce ‘steady state’ conditions. In those cases, the different dosage paradigms were chosen to provide complementary information about pharmacokinetic and pharmacodynamic behaviour.

3.13 Basic concepts and experimental tools Stereopharmacology, although recognized wherever appropriate, was not a part of my early experimental studies because the appropriate techniques were not then available. Before exposition of the experimental studies comprising the applied chemical-stereochemical theme, I have included several publications, referred to in Section 2, reviewing the basic concepts and experimental tools.

Review 01. The rationale of our experimental techniques [110] In Section 2, I referred to the ‘power’ of a large animal chronic preparation that could be used to determine the concurrent effects of drugs and/or drug treatments such as general anaesthesia on drug effect in selected organs, regional blood flow and drug disposition. In my lab, the ‘multicannulated sheep preparation’ became that standard preparation. This review describes some of the theory and the practice of this form of whole body pharmacology.

The adult sheep is a normally docile animal, females more so than males, and readily adapts to a laboratory environment; being a prey animal, it is remarkably resistant to infection. Sheep are regarded by society as a source of food or wool, but respond to human companionship in both domestic and laboratory environments, creating physiological stability. Being a large animal (typical body weight of around 50 Kg), they have a large blood volume, and their size facilitates the placement of probes and transducers for measurement of blood flow and pressure, cannulae to sample afferent and efferent blood of organs of interest, and electrodes for electrocardiographic (ECG) and electroencephalographic (EEG) recording. Moreover, apart from being a ruminant species, their physiological norms, such as blood 50 pressure and heart rate, are similar to those of humans, and this contrasts markedly to laboratory rodents. With due care, chronically instrumented animals can easily be studied in the conscious state or while anaesthetized, and can be studied repeatedly, with different drugs or dosage regimens. Therefore there is great opportunity for data collection, allowing both within subject and between group study designs. If there are disadvantages of the sheep as a laboratory subject, they would be that highly specialized skills and facilities are required for their preparation, study and maintenance.

Our development of such a preparation was in stages, according to both experimental objectives and availability of the required techniques, and these have been detailed in our papers based on this preparation. The most elaborate published description of our sheep preparation and associated concepts and tools, was delivered first as a conference paper in 1984, and published in a multiauthored book in 1985 [ 110 ]. It was written reasonably early in the ‘sheep lab’ series, whilst we were still discovering techniques, and prior to the ensuing papers that involve specific variations and developments for specific objectives, such as heart and brain site-directed drug administration.

Reviews 02, 03 and 04. Early reviews of stereopharmacology [198,214,215] In 1991, I was invited by the editor of Current Opinion in Anaesthesiology to submit a translational review on how stereochemical issues might be significant in anaesthesiology [198 ]. I invited my PhD student Al Rutten to co-author this article with me. Various reviews on the consequences of neglecting drug stereochemistry had been aired previously.70 We reviewed the origins and nomenclature of isomerism, and then used a pharmacodynamic and pharmacokinetic framework to discuss how the enantiomers of chiral drugs can differ pharmacologically because of differences in their interactions with chiral biomolecules, e.g., in receptors (governing drug actions), membranes (regulating distribution), and enzymes (controlling clearance). We mentioned a growing list of examples where one enantiomer has replaced a racemate originally introduced into clinical use, often as a result of combining pharmacodynamic and/or pharmacokinetic advantages (e.g., for , for ) in a “single enantiomer switch” that often generates considerable gains to the pharmaceutical companies through intellectual property benefits, apart from any pharmacological benefits. 71 This review led on to my (successful) application for the Australian and New Zealand College of Anaesthetists inaugural Douglas Joseph Professorship in 1993. My Australasian Visitor’s Lecture presented to the ANZCA Annual General Meeting in 1994 was couched to present to Fellows the concepts and my research plans for the award [ 214 ]. Neither review would be seen as profound if judged by a chemist but these were among the first to be oriented towards anaesthesiology. Also in 1994, I was able to reinforce the message about disregarding stereoisomerism in a review forming part of an anaesthetic pharmacology series [ 215 ]. I invited my Swedish colleague pharmaceutical scientist Sven Björkman from Malmö to co-author this review. I had first met Sven at Stanford, where he was collaborating with Don Stanski on qEEG pharmacokinetic-

70 As primary examples: Ariens EJ. Stereochemistry, a basis for sophisticated nonsense in pharmacokinetics and clinical pharmacology. Eur J Clin Pharmacol 26(6): 663-668, 1984; Williams K, Lee E. Importance of drug enantiomers in clinical pharmacology. Drugs 30(4): 333-354, 1985; Ariëns EJ. Stereochemistry: a source of problems in medicinal chemistry. Med Res Rev 6(4): 451- 466, 1986; Ariens EJ. Implications of the neglect of stereochemistry in pharmacokinetics and clinical pharmacology. Drug Int Clin Pharm 21(10): 827-829, 1987; Tucker GT, Lennard MS. Enantiomer specific pharmacokinetics. Pharmacol Ther 45(3): 309-329, 1990 71 Notably: Tucker GT. Chiral switches. Lancet 355(9209): 1085-1087, 2000; Agranat I, Caner H, Caldwell J. Putting chirality to work: the strategy of chiral switches. Nature Rev Drug Disco v 1(10): 753-768, 2002 51 pharmacodynamic modelling projects. Our contribution, titled “Pitfalls in pharmacokinetics”, among the many possible pitfalls mentioned the need for stereospecific assays for pharmacokinetic and pharmacodynamic studies to forestall the pitfall of undifferentiated drug concentrations being correlated with effects of racemic drugs and producing ‘nonsense data’. Since then, many other accounts and reviews of chirality in anaesthetic drug pharmacology have been published so that stereopharmacology is now a very definite part of anaesthesiology. 72

3.2 Local anaesthetic agent program This Section is presented to give my account of selected salient events covering several local anaesthetic agents, and to preserve reasonable chronology regarding these events.

3.21 Introduction In April 1968, at the commencement of my PhD project, I went to the office of HM Customs at Sydney Kingsford-Smith Airport to collect a box containing 100 x 20 ml 0.5% bupivacaine HCl (Marcaine®) ampoules, ordered from the UK by Dick Climie for clinical use in obstetric epidural anaesthesia and as part of my PhD project. The Customs Officer refused to release the box, as it was not on his list of ‘permitted’ drugs, and he had never heard of that particular local anaesthetic agent! It took many months for my supervisor to convince the Federal Minister responsible for HM Customs that it should be released to us for our research. Despite this shaky start, I continued to work on some or another aspect of bupivacaine, as well as etidocaine, ropivacaine, and finally levobupivacaine - each being touted to be the ‘new bupivacaine’ in the name of clinical efficacy and safety.

In 1974, I attended my first international symposium 73 where I was awed by meeting many anaesthesia research luminaries of my postgraduate research, particularly anaesthetist JS (Jeff) Crawford (1922-1988), one of the pioneers of obstetric anaesthesia and research on the placental transmission of drugs, and anaesthetist DB (Bruce) Scott (1925-1998), one of the pioneers of research on local anaesthetic pharmacokinetics and neural blockade. For that symposium, Geoff Tucker and I co-authored a major review of modern local anaesthetic agent pharmacokinetics, much of it based on our own research at the University of Washington [16]. We described the systemic pharmacokinetics of local anaesthetics as being dominated by the rate of absorption from the site of injection, rather than the rate of clearance from the body (a ‘flip-flop’ model), with biphasic absorption patterns being due to the agents partitioning into the fatty milieu at the site of deposition, and with prolonged slower phases of absorption of the more lipophilic agents. Since that time, I have been the author or co-author of numerous reviews of the pharmacology of local anaesthetic agents [ 48 , 49 , 57, 59, 95, 108, 141, 186, 251, 252, 309, 322, 325 , 327 -329 , 331].

Reviews 05 and 06. Achiral aspects of local anaesthetic pharmacology [48,49] These two reviews were published in 1979, not long before chirality was to become a significant issue in local anaesthetic pharmacology, and have been selected for this reason. The first, contributed with Michael Cousins in the journal Drugs , presented a wide-ranging

72 As primary examples: Egan TD. Stereochemistry and anesthetic pharmacology: Joining hands with the medicinal chemists. Anesth Analg 83(3): 447-450, 1996; Sidebotham DA, Schug SA. Stereochemistry in anaesthesia. Clin Exp Pharmacol Physiol 24(2): 126-130, 1997; Burke D, Henderson DJ. Chirality: a blueprint for the future. Br J Anaesth 88(4): 563-576, 2002; Weiskopf RB, Nau C, Strichartz GR. Drug chirality in anesthesia. Anesthesiology 97(2): 497-502, 2002 73 Symposium on Local Anaesthesia, Edinburgh, Scotland, 2-3 September, 1974; proceedings published as Br J Anaesth Volume 47, Supplementary Edition, 1975 (Editor DB Scott) 52 overview of local anaesthetic pharmacology as then known, before the revelations gained through modern patch clamping and other neurophysiological techniques [48 ]. The second, contributed with Geoff Tucker in the companion journal Clinical Pharmacokinetics, although out-of-date, is possibly still the most comprehensive review yet published of the pharmacokinetics of local anaesthetics [49 ].

Many diverse chemical substances can cause local anaesthesia, but relatively few are used clinically for this purpose. Apart from the overall structure of the molecule, the essence of the chemical pharmacology of local anaesthetics resides in two primary physicochemical properties, lipophilicity and ionization, that dominate local anaesthetic activity and toxicity. Within molecular structures containing the functional groups that generate the local ‘anaesthesiophore’, chirality is a second order consideration. Most of this was known empirically, before the ion channel receptors of local anaesthetic actions were identified some 30 years ago.

Overall, lipophilicity determines both local anaesthetic activity and toxicity, and this is not surprising because toxicity, like neural blockade, is now known to derive from the blockade of the same ion channels in excitable membranes. In the (increasingly lipophilic) homologous series of N-alkyl piperidine (mepivacaine-like) local anaesthetics, the N-alkyl 4- and 5-carbon substances are at the peak of (acute) toxicity because of the balance between molecular intrinsic toxicity and penetration of vital organs.74 Shorter N-alkyl carbon chains have lesser intrinsic local anaesthetic activity; longer chains have lesser penetration. It was also known that ionization was important to activity, and that the balance between the relative concentrations of ionized and unionized species was important to neural penetration so that the pKa of local anaesthetics was a highly relevant property. Similarly, stereochemistry was known to be a relevant factor in intrinsic local anaesthetic activity and toxicity. Despite this, the local anaesthetics chosen from the homologous N-alkyl piperidines, C1 = mepivacaine and n-C4 = bupivacaine, were developed as racemates. However it is not surprising that, with continued research and technique evolution, a preferred enantiomer would eventually emerge from the racemic local anaesthetics – and these appeared in the S-enantiomer forms of ropivacaine and levobupivacaine, with ropivacaine being somewhat less lipophilic, less potent and less toxic, than levobupivacaine.

Knowledge of the pharmacological impact of chirality in local anaesthetic agents, however, long predates that of the ‘local anaesthetic receptor’. 75 The pharmacological significance of chirality in modern local anaesthetics was studied by two Swedish pharmacologist PhD candidates during the period of my own PhD candidacy. Gunnar Åberg, working in the research laboratories of AB Bofors was awarded a PhD of Linköping University in 1972 for his studies on mepivacaine and bupivacaine. 76 Bengt Åkerman, working in the research laboratories of AB Astra, was awarded a PhD of Uppsala University in 1973 for his studies on prilocaine . 77 During the same period, Felix P Luduena, a senior pharmacologist at the

74 Most importantly: Åberg G, Dhunér KG, Sydnes G. Studies on the duration of local anaesthesia: structure/activity relationships in a series of homologous local anaesthetics. Acta Pharmacol Toxicol 41(5): 432-443, 1977 75 As examples: Copeland AJ. Psicaine: A new local anaesthetic. Brit Med J 1(3340): 9, 1925; Watson-Williams E. Psicaine: An artificial cocaine. Brit Med J 1(3340): 11, 1925 76 Åberg G. "Studies on mepivacaine and its optically active isomers with special reference to vasoactive properties." PhD diss., Linköping University, Sweden 1972 77 Åkerman B. “Studies on the relative pharmacological effects of enantiomers of local anaesthetics with special regard to block of nervous excitation.” PhD diss. Uppsala University, Sweden 1973 53

Sterling-Winthrop Research Institute in Rensselaer New York, 78 was also working on bupivacaine, as that company had obtained a licence for its marketing in the USA.

Åberg worked with the series of N-alkyl homologous heterocyclic local anaesthetics synthesized in the 1950s at AB Bofors, 79 then Astra’s Swedish rival. The original publication describing these compounds (Figure 3.2) included some physicochemical properties (Figure 3.3), as well as brief preclinical laboratory acute toxicity experiments, with one finding no difference in acute toxicity between the mepivacaine enantiomers. 80 With no studies then yet suggesting a preference for enantiopure substances, and based on the balance between duration of action in laboratory experiments and acute toxicity in laboratory rodents, mepivacaine and bupivacaine were selected for further clinical, and subsequent commercial, development – each as a racemate.

Figure 3.2: Chemical structures of the mepivacaine and bupivacaine enantiomers, depicting mirror images.

Among his many experiments, Åberg determined the acute toxicity (LD 50 ) in laboratory rodents of the mepivacaine enantiomers to be equal but, significantly, that of D-bupivacaine (i.e., R-bupivacaine) was greater than that of L-bupivacaine (i.e., S-bupivacaine), the absolute configurations had not then been determined, but pharmacological reasons for the differences were not then known. 81 Luduena et al .82 also demonstrated that the toxicity of S-bupivacaine was, in various comparative tests, 24-36% less than racemic bupivacaine but that of R- bupivacaine was not significantly different to bupivacaine from studies in various laboratory rodents.

78 Luduena FP. Duration of local anesthesia. Annu Rev Pharmacol 9: 503, 1969 79 USP 2,955,111 Synthesis of N-alkyl-piperidine and N-alkyl-pyrrolidine-α-carboxylic acid amides. Inventors: Bo Thuresson af Ekenstam, Bofors, and Bror Giista Pettersson, Karlskoga, Sweden, assignors to Aktiebolaget Bofors, Bofors, Sweden, a corporation of Sweden. Filed Jan. 28, 1957, Patented Oct. 4, 1960 80 af Ekenstam B, Egnér B, Pettersson G. N-alkyl pyrrolidine and N-alkyl piperidine carboxylic acid amides. Acta Chem Scand 11: 1183–1190, 1957; af Ekenstam B. The effect of the structural variation on the local analgetic properties of the most commonly used groups of substances. Acta Anaesth Scand 25 Suppl:10-18, 1966 81 Åberg G, Toxicological and local anesthetic effects of optically active isomers of two local anesthetic compounds. Acta Pharmacol Toxicol 31: 273-286, 1972 82 Luduena FP, Bogado EF, Tullar BF. Optical isomers of mepivacaine and bupivacaine. Arch Int Pharm Thér 200(2): 359, 1972 54

Stereochemistry may have other pharmacological repercussions, for example, in selectivity of metabolic pathways of elimination. Unpublished work by Peter Duguid and Jack Thomas at the Sydney University Department of Pharmacy in the 1980s found that, after intravenous injection of the separate bupivacaine enantiomers into healthy human volunteers, the majority measured urinary metabolite was conjugated 4-OH bupivacaine after R-bupivacaine, but was conjugated 3-OH bupivacaine after S-bupivacaine.

Figure 3.3: Solubility of mepivacaine (left) and bupivacaine (right) racemates and separate enantiomers in buffers made to different pH. [From Friberger P, Åberg G. Some physicochemical properties of the racemates and the optically active isomers of two local anaesthetic compounds. Acta Pharm Suec 8: 361-364, 1971]

Another relevant pharmacological difference between the bupivacaine enantiomers with functional clinical consequences was in their intrinsic vasoactivity. 83 This has been demonstrated in a variety of ex vivo and in vivo models, including intradermal injection of bupivacaine in human volunteer studies. At high concentrations, both enantiomers were found to act as vasodilators; at low concentrations, S-bupivacaine became vasoconstrictive, whereas R-bupivacaine did not. It could be assumed that any resultant vasoconstriction would augment a prolongation of neural blockade, and possibly contribute to the lower toxicity of S-bupivacaine, found after extravascular injection.

By the 1970s, laboratory study of systemic toxicity from local anaesthetics was still mainly being restricted to observing gross effects, with dose-response relationships for CNS toxicity (convulsions) and/or death (cardio-respiratory failure) under conditions that did not necessarily relate well to clinical conditions involving patients undergoing neural blockade.84

83 Particularly: Åberg G, Wahlström B. Mechanical and electrophysiological effects of some local anaesthetic agents and their isomers on the rat portal vein. Acta Pharmacol Toxicol 31(4): 255-266 1972; Adler R, Aler G, Åberg G. Effects of optically active isomers and racemate of mepivacaine (Carbocaine) in dental anaesthesia. Svensk tand tids (Swedish dental journal) 62(8): 501, 1969; Åberg G, Adler R. Thermographic registrations of some vascular effects of a local anaesthetic compound. Svensk tand tids 63(10) 671-678, 1970; Dhunér KG. Vascular effects of the isomers of mepivacaine. Acta Anaesth Scand 16: 45-52, 1972; Blair MR. Cardiovascular pharmacology of local anaesthetics. Br J Anaesth 47(Suppl 1): 247-252, 1975; Aps C, Reynolds F. An intradermal study of the local anaesthetic and vascular effects of the isomers of bupivacaine. Br J Clin Pharmac 6(1): 63-68, 1978; Aps C, Reynolds F. The effect of concentration on vasoactivity of bupivacaine and lignocaine. Br J Anaesth 48(12): 1171-1174, 1976 84 Particularly: Munson E, Martucci RW, Wagman IH. Bupivacaine and lignocaine induced seizures in rhesus monkeys. Br J Anaesth 44(10): 1025-1029, 1972; de Jong RH, Heavner JE, de Oliveira L. Intravascular lidocaine compartment: kinetics of bolus injection. Anesthesiology 37(5): 493-496, 1972; Munson ES, Tucker WK, Ausinsch B, Malagodi MH. Etidocaine, bupivacaine, and lidocaine seizure thresholds in monkeys. Anesthesiology 42(4): 471-478, 1975 55

Otherwise, observations were made following unfortunate clinical accidents, but these were of rare events, often made under such chaotic clinical conditions (multiple interventions, concomitant drugs, etc.) that the value of the observations could, in many or most cases, be considered of limited scientific value. Nevertheless, electrophysiological techniques were emerging that could help to finally reveal the mode of action of local anaesthetics for producing neural blockade as well as their, sometimes fatal, side effects

Despite the (quite reasonable) claims that bupivacaine has a high margin of safety in the hands of an experienced anaesthetist [27], others were suggesting that etidocaine and bupivacaine were more likely to be involved in a clinical mischance fatality than their shorter- acting counterparts. This viewpoint gained widespread traction after a prominent editorial by Stanford obstetric anaesthetist, George Albright, in 1979. 85 An ensuing FDA hearing in 1983 recommended that 0.75% bupivacaine should be precluded from obstetric use, and that bupivacaine should be contraindicated for intravenous regional anaesthesia. 86 However, some authorities claimed that the whole process condemning these agents was deficient. 87 Nevertheless, in order to introduce ropivacaine (or any other agent) as a potential replacement for bupivacaine, its toxicity/safety would need to be demonstrated in a variety of preclinical paradigms, and be found superior, to satisfy regulatory bodies.

With a grant from the Astra Pharmaceutical Company in Sweden, my research group became involved in developing preclinical experimental paradigms for determining the systemic effects and toxicity of ropivacaine and other local anaesthetics in our sheep preparation. Craig Nancarrow became the first of our PhD candidates to work on this program, starting in 1984, followed later by Richard Upton, YiFei Huang and Al Rutten. Our studies described various aspects of the systemic pharmacology of the local anaesthetics [118, 131, 134, 140, 145, 149] and contributed to the registration package of ropivacaine, but were yet to involve direct aspects of chirality.

3.22 Prilocaine As discussed in Section 2.6, I worked at the University of Sheffield in 1988, on sabbatical leave from Flinders University. There, my former colleague Geoff Tucker introduced me to his analytical lab techniques being used to resolve the stereoisomers of beta-adrenoceptor antagonists.

Study 01. Pharmacokinetics of prilocaine enantiomers in humans [197] The pharmacology of prilocaine was first reported in 1960, where it was described simply as a ‘racemic local anaesthetic’.88 Prilocaine has been recognized as a relatively safe local anaesthetic agent on the basis of its intrinsic CNS toxicity being somewhat less than its main alternative, lignocaine, and is thus viewed as the preferred agent for many peripheral nerve blocks, such as brachial plexus block. 89 We decided to perform a simple study to determine

85 Albright GA. Cardiac arrest following regional anesthesia with etidocaine or bupivacaine. Anesthesiology 51(4): 285-287, 1979 86 United States Department of Health and Human Services, Public Health Service, Food and Drug Administration. Minutes of 5th meeting, Anesthetic and Life Support Drugs Advisory Committee, October 4, 1983. 87 Writer W, Davies JM, Strunin L. Trial by media: the bupivacaine story. Canad J Anesth 31(1): 1-4, 1984 88 Wiedling S. Studies on α-n-propylamino-2-methylpropionanilide — a new local anaesthetic. Acta Pharmacol Toxicol 17(3): 233-244, 1960 89 Wildsmith JAW. Prilocaine-an underutilized local anesthetic. Reg Anesth Pain Med 10(4): 155-159, 1985 56 the pharmacokinetics of prilocaine were stereoselective in humans by analysis of plasma extracts using the newly available HPLC chiral stationary phases.

Previous work on prilocaine at Astra by Bengt Åkerman et al. described the local anaesthetic pharmacology of the enantiomers in 1967 and the selectivity of their metabolism in relation to prilocaine-induced methaemoglobinaemia in 1970. 90 Prilocaine has a high total body clearance, and was believed, correctly, to undergo extrahepatic clearance in addition to substantial hepato-splanchnic clearance. 91 We compared the plasma concentrations of the prilocaine enantiomers after parenteral and enteral administration to demonstrate the role of hepato-splanchnic metabolism in the total body clearance of prilocaine, and any enantioselectivity involved. Geoff Tucker had previously participated in a collaborative pharmacokinetic study on prilocaine used for brachial plexus block. 92 For simplicity, the original deep-frozen samples from that study would be re-analysed with enantiomeric resolution; a number of the staff, including the investigators, volunteered to collect timed blood samples following the oral ingestion of capsules containing prilocaine. After brachial plexus administration, the plasma concentrations of both prilocaine enantiomers were similar. After oral ingestion, the plasma concentrations of R-prilocaine were hardly detectable, but those of S-prilocaine were substantial. The conclusions were plain: the marked route- dependence of -prilocaine enantiomer concentration ratio was the resultant of blood flow limited clearance of both enantiomers after peripheral administration, whereas a marked enantiomeric difference in intrinsic hepato-splanchnic clearance was demonstrated by oral administration. More importantly, the analytical separation technology was to form the basis for my future studies on local anaesthetic and other chiral drugs.

3.23 Bupivacaine Bupivacaine, or LAC 43 as it was then known, was first described in papers given at the 3rd World Congress of Anaesthesiologists held September 20-26 1964 in São Paulo. It was intended as a long-acting local anaesthetic replacement for tetracaine and cinchocaine, both of which had clinical reputations for unreliability. Ropivacaine was launched at the 11 th World Congress of Anaesthesiologists meeting held April 14-20 1996 in Sydney. It was intended as a safer replacement for the reliable but more ‘toxic’ bupivacaine, and was the first synthetic enantiopure local anaesthetic agent.93 The research program on ropivacaine introduced many new experimental paradigms to enable its meaningful comparison with the main existent standard local anaesthetics, the shorter-acting lignocaine and the longer-acting bupivacaine. As mentioned above, my group at Flinders played a major role in the acquisition of data through whole body studies in our multicannulated sheep preparation [145, 149, 164]. In 1989, after our ropivacaine project, we commenced a new series of pharmacology curiosity-

90 Åkerman B, Persson H, Tegner, C. Local anaesthetic properties of the optically active isomers of prilocaine (Citanest®). Acta Pharmacol Toxicol 25(2): 233-241, 1967; Åkerman B, Ross S. Stereospecificity of the enzymatic biotransformation of the enantiomers of prilocaine (Citanest®). Acta Pharmacol Toxicol 28(6): 445-453. 1970 91 Scott DB, Jebson PJR, Braid DP, Ortengren B, Frisch P. Factors affecting plasma levels of lignocaine and prilocaine. Br J Anaesth 44(10), 1040-1049(1972).. Arthur GR, Scott DHT, Boyes RN, Scott DB. Pharmacokinetic and clinical pharmacological studies with mepivacaine and prilocaine. Br J Anaesth 51(6): 481-485, 1979 92 Wildsmith JAW, Tucker GT, Cooper S, Scott DB, Covino BG. Plasma concentrations of local anaesthetics after interscalene brachial plexus block. Br J Anaesth 49(5): 461-466, 1977 93 USP 4,695,576 L-N-N-Propylpipecolic acid-2,6-xylidide. Inventors: Bo T. af Ekenstam, Christer Bovin, both of Sweden. Assignee: Astra Lake Medel Aktiebolag, Sodertalje, Sweden. Filed Oct. 9, 1985; Patented Sep. 22, 1987; Ruetsch YA, Boni T, Borgeat A. From cocaine to ropivacaine: the history of local anesthetic drugs. Curr Topics Med Chem 1(3): 175-182, 2001 57 driven studies with bupivacaine in sheep and used our newly-acquired HPLC-chiral stationary phase columns to resolve the enantiomers in biological samples [ 170 , 177 , 188 , 190 , 217 ].

The work was continued collaboratively after we moved to the University of Sydney in 1991. In 1992, I was invited to participate in a symposium convened by anaesthetist Hans Wüst (1940-1999) of the Heinrich-Heine University of Düsseldorf on “Neue Aspekte in der Regionalanaesthesie”. My paper was presented in a session on admixtures of local anaesthetics (such as lignocaine + bupivacaine [e.g., 83]) which were intended to attain the best properties from each agent. In my paper [185], I treated the racemic local anaesthetic bupivacaine as an unintentional mixture of agents and went on to review its emerging stereopharmacology from our own recent experiments: this presented something of a surprise to some of the audience who had not previously thought of (racemic) bupivacaine in that light.

The following publications were selected to describe various aspects of bupivacaine stereopharmacology.

Study 02. Pharmacokinetics of mepivacaine and bupivacaine enantiomers in sheep [170] This study, based on chiral-HPLC resolution (Figure 3.4), was required to precede any further studies using racemic bupivacaine (and mepivacaine) to determine whether chiral inversion occurred, i.e., whether the alternative enantiomer was also present in the respective biological samples that could influence the interpretation of pharmacokinetic or pharmacodynamic data. It was performed with intravenous bolus doses of the separate enantiomers (gifts from Gunnar Åberg) in our sheep preparation, and various biofluid samples were analysed chirally for both enantiomers to describe their systemic pharmacokinetics. No evidence of inversion was found, thus giving confidence that future studies with these substances could produce reliable pharmacokinetic data.

Figure 3.4: Analytical resolution of mepivacaine (left, R- and S-mepivacaine (RM and SM), with R-bupivacaine internal standard) and bupivacaine (right, R- and S-bupivacaine (RB and SB) with S-mepivacaine internal standard) in extracts of blood.

Study 03. Bupivacaine pharmacokinetics with steady state infusion in sheep [177] In a follow-up study, we used a continuous intravenous infusion of bupivacaine to reach steady state conditions using a similar experimental design that had been used previously for 58 ropivacaine [164]. This allowed better measures of the regional clearances of the respective enantiomers than after bolus administration, because regional extraction ratios measured under non-steady state conditions require the assumption of distribution pseudo-steady state conditions. At the same time, the study allowed observation of the cardiovascular system effects of bupivacaine at different stages.

Study 04. Post-surgical plasma binding of bupivacaine enantiomers in sheep [188] It had been argued over several decades by many people, including Geoff Tucker and me [16, 17], that quantitative perioperative changes to plasma proteins can alter drug binding capacity with possible toxicological consequences. We performed this study to determine whether such changes were significant for three local anaesthetics, and whether bupivacaine was altered enantioselectively. Decreased plasma binding of each of the local anaesthetics was observed on the 1 st postoperative day, suggesting a common mechanism, and was attributed to haemodilution and/or an elevation of free fatty acids displacing the local anaesthetics. However, the consistency in binding after the 8 th postoperative day was an important finding and served to bolster the experimental design of our pharmacokinetic studies that normally commenced after that allocated postsurgical recovery time.

Study 05. Enantioselectivity of bupivacaine tissue distribution in sheep [190] This study complemented previous work by determining the tissue:blood distribution coefficients of the bupivacaine enantiomers at steady state and after fatalities caused by bupivacaine intoxication. Interestingly, there was a greater total distribution of the more toxic R-bupivacaine than its enantiomer into vital tissues, although this was extinguished when corrected for plasma binding. These observations became significant in supporting the registration of levobupivacaine.

Study 06. Influence of i.v. infusion on bupivacaine pharmacokinetics in sheep [217] In this study, we took a step back from the previous studies to examine the notion that drug administration conditions may constitute a study design variable, and that this may become manifest with the enantiomers of a racemic drug. In most of our previous studies on local anaesthetics, the drug administration regimen had been chosen as a brief infusion to mimic the possible consequences of an accidental intravenous injection, but the drug administration regimen had not been previously considered as a variable in the pharmacokinetic outcome (as still happens). In these experiments, racemic bupivacaine was administered in one of three typical intravenous regimens: a ‘bolus’, a ‘brief infusion’, and a ‘prolonged infusion’, and these represent the variations used by other researchers. The most significant outcome, apart from the expected enantiomeric differences, was that the slow half-life of both enantiomers increased with the increased duration of administration of the regimen, indicating the influence of the dosage regimen on the pharmacokinetic outcome.

Study 07. Bupivacaine pharmacokinetics with intercostal block in humans [218] At the request of Melbourne anaesthetists Peter McCall and Larry McNichol in 1993, we collaborated to determine the pharmacokinetics of bupivacaine in patients undergoing orthotopic liver transplantation and having postoperative pain management provided by intercostal neural blockade with bupivacaine. The implications being examined were that intercostal administration leads to the highest concentrations of bupivacaine per unit dose, compared to other sites of neural blockade, and that disturbances to hepatic function might further expose the patients to exaggerated blood concentrations of bupivacaine. Enantiomeric differences in both distribution and clearance produced a blood concentration ratio of the enantiomers that was time-dependent, and this had not been previously appreciated. This 59 study added to the literature by measuring the bupivacaine enantiomers and by allowing insight into whether any disturbance due to liver transplantation surgery might be enantioselective.

Study 08. Bupivacaine pharmacokinetics with epidural block in humans [246] At the request of New York anaesthetist Nigel E Sharrock in 1995, we collaborated to determine the pharmacokinetics of bupivacaine in elderly patients undergoing orthopaedic surgery with perioperative pain management provided by epidural neural blockade with bupivacaine. The issue being examined was related to the uptake of bupivacaine into the lungs. Since the work of Geoff Tucker and anaesthetist RA (Bob) Boas on lignocaine at the University of Washington in the early 1970s, it has been accepted that the lungs act as a ‘buffer’ against high arterial local anaesthetic concentrations, potentially leading to toxicity. 94 This study found that it took some 20-30 minutes for the concentrations of both enantiomers to equilibrate across the lungs, thereby adding to the literature by measuring the bupivacaine enantiomers, and allowing insight into whether any disturbance to ‘bupivacaine lung uptake’ might be enantioselective.

3.24 Levobupivacaine Levobupivacaine came to my attention as a new drug entity in 1993 when I was approached by Brian Gennery, medical director of Chiroscience plc from Cambridge UK, 95 about studying their ‘new’ enantiopure local anaesthetic, intriguingly brand-named Chirocaine®. The business of Chiroscience was the synthesis of enantiopure intermediates, but the company had decided for business reasons to bring to market some finished drugs of their own. Levobupivacaine was seen as a product that could be brought to market reasonably quickly because of the large amount of existent knowledge about bupivacaine and, being a drug of acute use, it did not require extensive long-term toxicity studies. Foremost, Chiroscience needed definitive assessments of its acute toxicity in comparison to bupivacaine. Geoff Tucker, who was a consultant to Chiroscience on other matters of stereopharmacology, recommended to Brian Gennery that I should be approached as a specialist in this area, based on our research experience with ropivacaine.

I visited Chiroscience in August 1993, en route to the International Association for the Study of Pain (IASP) 7th World Congress in Paris. I met with Brian Gennery, Andy Richards (company co-founder), Robert Gristwood (scientific director), Hazel Bardsley (project manager), Jane Greaves and Deborah Harding (project pharmacologists) and outlined our expertise and previous studies of ropivacaine, along with a willingness to set up a collaborative program in Sydney. We agreed to set up an investigator-led research program with mutual goals of understanding local anaesthetic systemic pharmacology/acute toxicity, and with full publication rights. A program like this, I envisaged, would provide an infrastructure boost for our ‘new’ university department at the Royal North Shore Hospital, and it fitted nicely with the stereopharmacology theme that I wanted to make a major departmental focus. Chiroscience thereby became a major foundation sponsor of our department’s laboratory research program, providing much needed analytical and

94 Tucker GT, Boas RA. Pharmacokinetic aspects of intravenous regional anesthesia. Anesthesiology 34: 538-549, 1971; Bertler Å, Lewis DH, Löfström JB, Post C.. In vivo lung uptake of lidocaine in pigs. Acta Anaesth Scand 22(5): 530-536, 1978; Jorfeldt L, Lewis DH, Löfström JB, Post C.. Lung uptake of lidocaine in healthy volunteers. Acta Anaesth Scand 23(6): 567-574, 1979 95 Union Chimique Belge. Now part of UCB , a branch of UCB Pharma S.A. of Belgium. UCB was established by Emmanuel Janssen in Brussels in 1928, primarily focusing on industrial chemicals (and was one of the first companies to distil ammonia from coal). 60 physiological monitoring equipment to help establish our laboratories, as well as funding that enabled several PhD candidacies. Later on, Brian, Robert and Deborah came to inspect our labs and audit our procedures.

The experimental strength that my lab brought to the levobupivacaine program lay in the version of the multicannulated sheep preparation originally developed at Flinders. With a small group initially consisting of YiFei Huang, Marie Pryor, joined by Bernadette Veering (who was on sabbatical leave from Leiden University), we commenced a program to study the comparative acute toxicity of levobupivacaine and bupivacaine in sheep. Although we drew heavily on the ropivacaine paradigm, we had the opportunity to improve upon it through newer generation technology, e.g., high fidelity catheter tipped sensors, ultrasonic transducer blood flow measurements and, above all, a fully digitalized physiological data acquisition system shown in the snapshots below (Figures 3.5, 3.6). Our methods became among the most sophisticated large animal preparations for studying whole body pharmacology to that date.

Figure 3.5 The ‘sheep lab’ at University of Sydney-RNSH being set up for determination of the regional pharmacokinetics and pharmacodynamics of local anaesthetics in the late 1990s.

After completion of the initial levobupivacaine project [ 244 , 245 , 250 ], YiFei Huang was recruited to the Department of Cardiology at Royal North Shore Hospital, Bernadette Veering returned to the Netherlands, and Marie Pryor was recruited to Astra Pharmaceuticals. Our group later expanded when Leigh A Ladd, DHT (Dennis) Chang and Susan E (Sue) Copeland, along with several research assistants, joined the program in 1997. Our work contributed to the preclinical data for the registration of levobupivacaine. On January 12 1999, in Washington DC, I gave evidence to the 87 th meeting of the Anesthetic & Life Support Advisory Committee of the Food and Drug Administration (FDA), chaired by Dr Terese Horlocker and in the presence of Dr Cynthia McCormick, Director, Division of Anesthetics, Critical Care and Addiction Products, on the US approvability of levobupivacaine. Among the various presentations of preclinical and clinical data made to the FDA, mine described the comparative pharmacodynamic and pharmacokinetic data on levobupivacaine and bupivacaine from our studies in sheep, and was considered by the Committee as “quite compelling”. 96 Levobupivacaine was approved in the US and elsewhere, and was launched at the European Society for Regional Anaesthesia meeting in Göteborg in April 2001, but my work with it did not stop there.

96 FDA document 3488t1 61

Figure 3.6: A set of digitally acquired data showing the effects of the onset of CNS toxicity on the cardiovascular system of a conscious sheep. A brief panel on the far left shows before, during, and after intravenous drug infusion of 100 mg bupivacaine HCl that started at 600 sec and terminated at 780 sec.

Levobupivacaine was to become a useful probe in some more fundamental pharmacological- toxicological studies of local anaesthetics. I proposed to Brian Gennery that Chiroscience fund further studies in my lab to attempt to test the ‘CNS hypothesis’ of local anaesthetic- induced cardiotoxicity, i.e., that local anaesthetic action on the brain stem was the primary action, and that the cardiac arrhythmogenic response was secondary.97 To isolate local CNS and cardiac effects from systemic effects, our studies would need to include separate close arterial infusions of the agents (bupivacaine, levobupivacaine and ropivacaine), respectively directed towards the brain and heart, but in the whole body system whilst conscious and under full neural control. A way to do this, is to enrich the selective afferent blood supply of the respective organ with drug, so that its received concentration will be similar to that in an intravenous administration, but with re-circulating drug being small and unlikely to alter the principal action. Ideally, the whole organ should be uniformly enriched with drug through all relevant afferent vessels, as with intravenous administration, but this is experimentally challenging and has not yet been done.

Specifically, enrichment of the heart blood supply could only be performed via the left coronary arteries, owing to the difficulty of access to the right sided arteries during thoracotomy (Figure 3.7). Others researchers concurrently reported using this approach with similar aims, but performed their studies in anaesthetized animals, an approach that we subsequently showed to be problematic. 98

97 Heavner JE. Cardiac dysrhythmias induced by infusion of local anesthetics into the lateral cerebral ventricle of cats. Anesth Analg 65(2): 133-138, 1986; Thomas RD, Behbehani MM, Coyle DE, Denson DD. Cardiovascular toxicity of local anesthetics: an alternative hypothesis. Anesth Analg 65(5): 444-450, 1986 98 Morrison SG, Dominguez JJ, Frascarolo P, Reiz S. A comparison of the electrocardiographic 62

Similarly, the preference for enrichment of the whole brain is because the nucleus tractus solitarius (NTS) is found fairly caudal in the medulla oblongata and, as the NTS controls heart function to a great extent, it should be included as part of the brain. It was possible to administer the local anaesthetics directly to the CNS only through enrichment via the accessible carotid arteries. Enrichment by the arterial brain supply via the circle of Willis is from a minimum of five vessels but, anatomically and practically, this is too complicated for present experimental techniques. Nevertheless, our work [Studies 14 and 15] remains a landmark of pharmacological ingenuity, and was achieved through the surgical dexterity of Leigh Ladd. Some of the vascular anatomy is shown in the resin-cast anatomical models prepared by Leigh Ladd (Figures 3.7 and 3.8).

Figure 3.7: Left: The superficial vascular anatomy of the sheep heart showing the left anterior descending coronary artery used for drug infusion. Right: A resin cast of the vascular anatomy of the sheep heart with the left main coronary artery (LCA) and the left anterior descending coronary artery used for drug infusion (labelled LAD). (Prepared by Leigh Ladd)

Figure 3.8: A resin cast of the vascular anatomy of the sheep brain with the left and right carotid arteries (as labelled) used for drug infusion. (Prepared by Leigh Ladd)

cardiotoxic effects of racemic bupivacaine, levobupivacaine, and ropivacaine in anesthetized swine. Anesth Analg 90(6): 1308-1314, 2000 63

Previous work had shown that local anaesthetic CNS toxicity was exaggerated by acidosis and/or hypercapnia; 99 nevertheless, others reported differences between various in vivo models. 100 To help resolve the differences, we performed a study to determine the consequences of acid-base imbalance using bupivacaine and thiopentone as probes [ 306 ]. These two drugs, respectively, constitute a basic and an acidic substance, each with a pKa value close to blood pH. They are thus susceptible to changes in ionization, with changes in ambient pH, and consequent changes in their binding to plasma proteins, thereby potentially altering their systemic and regional pharmacokinetics, and thus their effects. The specific objective of this study was to determine the effects of induced acid-base disturbance without hypoxia on the pharmacokinetics and actions of bupivacaine. The study design provided an opportunity to compare the effects of acid-base disturbance on the actions and pharmacokinetics of both a weak base, bupivacaine, and a weak acid, thiopentone. The results were reported in the same paper, and are again referred to in Section 3.3 on thiopentone.

Finally, we returned to the issue of general anaesthesia as an experimental variable in local anaesthetic induced toxicity, having first studied this issue at Flinders, a decade or so earlier. By necessity, much physiological and pharmacological research in animal subjects is carried out under general anaesthesia but, as we had found at Flinders in the 1980s, the use of general anaesthesia causes profound physiological and pharmacological sequelae. In attempting to clarify the ‘toxicity of local anaesthetics’ issue, many whole body cardiotoxicity experimental paradigms were being performed by various investigators in various animals. In some paradigms, such as ours, the subjects were chronically prepared and conscious, in others, they were acutely prepared and anaesthetized.

Administration of a general anesthetic agent is first line clinical pharmacotherapy for acute CNS toxicity from local anaesthetics, and this has several implications. First, it rapidly and appropriately depresses the CNS response, and this is significant because CNS response modifies the cardiovascular system response by reducing the risk of malignant cardiac dysrhythmias. Second, general anaesthetic agents have multiple intrinsic cardiovascular system effects: they depress the myocardium and alter regional haemodynamics, and these effects will be additive to any effects of the local anaesthetics. Third, as has been repeatedly documented, general anaesthetics modify drug disposition – by both direct effects on enzyme function and by indirect effects on the cardiovascular system and regional blood flow. All of these effects can have implications for the interpretation of data obtained in research experiments where subjects are anaesthetized, compared to those where they are conscious, and for translation of experimental findings to the treatment of patients where concomitant general anaesthesia can modify the body’s response to drugs [129, 205].

It was clear that whole body research on local anaesthetic toxicity needed further clarification as to effects in conscious vs . anaesthetized subjects. The specific aims were to determine the

99 Notably: Englesson S, Matousek M. Central nervous system effects of local anaesthetic agents. Br J Anaesth 47 suppl: 241-246, 1975; Rosen MA, Thigpen JW, Shnider SM, Foutz, et al. Bupivacaine- induced cardiotoxicity in hypoxic and acidotic sheep. Anesth Analg 64(11): 1089-1096, 1985; Haasio J, Rosenberg PH, Pitkänen M, Kyttä J. Treatment of bupivacaine-induced cardiac arrhythmias in hypoxic and hypercarbic pigs with amiodarone or bretylium. Reg Anesth Pain Med 15(4): 174-179, 1990. 100 Notably: Heavner JE. Cardiac toxicity of local anesthetics in the intact isolated heart model: a review. Reg Anesth Pain Med 27: 545-555, 2002; Groban L. Central nervous system and cardiac effects from long ‐acting amide local anesthetic toxicity in the intact animal model. Reg Anesth Pain Med 28: 3-1, 2003 64 influences of general anaesthesia on the haemodynamic, inotropic and electrocardiographic responses to local anaesthetic infusion, to determine the influences of general anaesthesia on the CNS responses to local anesthetic infusion, and to determine the influences of general anaesthesia on the systemic and regional pharmacokinetics of local anesthetics [ 323, 324 ]. All of the local anaesthetic agents in common perioperative use were studied in this paradigm. In addition to the published studies and reviews, I prepared various research reports on the long-acting local anaesthetics, privately for Chiroscience, and for presentation to various national and international symposia.

Study 09. A new enantiospecific HPLC assay for the levobupivacaine program [250] Ongoing research on the systemic effects of bupivacaine and its analogues had indicated the need for studies of pharmacodynamics to be performed in concert with relevant regional pharmacokinetics in order to gain greater insight into the results. As this project was conceived with this viewpoint, it was decided to set up an assay to cater for the many thousands of blood and other samples that would be generated. We required a rapid turnover time as well as high precision and sensitivity, and, as before, enantiospecificity was essential to verify the absence of metabolic inversion. By the mid 1990s, newer generation commercially available α1-acidglycoprotein (AGP) and human serum albumin (HSA) columns offered greater efficiency of chiral separation and longer life. Funds from the Chiroscience grant were used to purchase a new high performance liquid chromatograph with photodiode array detection, and my Research Assistants, analytical chemists Xiao-Qing (Sonia) Gu and Bronwyn Fryirs/Dawson, devised the separations and performed the analyses used for the ensuing pharmacokinetic studies.

Study 10. Cardiovascular effects of i.v. bupivacaine and levobupivacaine [244] This was the first whole body study in sheep with complete digital acquisition of data (example shown in Figure 3.6). The aims were to determine the relevant cardiovascular and central nervous systems responses to graded doses of levobupivacaine and bupivacaine, along with the time-courses of drug concentrations in pulmonary arterial, aortic, and coronary sinus blood. From our previous studies, we knew that the mean i.v. bolus convulsant dose of bupivacaine was approx. 45mg in a 45Kg sheep, and we knew that doses inducing convulsions profoundly influenced the haemodynamic responses of the animals due to autonomic stimulation. Therefore, the experiments comprising this study were divided into intentionally subconvulsive and intentionally convulsive . With subconvulsive doses, dose- dependent depression of myocardial contractility was shown to be a primary manifestation of cardiac toxicity. With convulsive doses, the attendant tachycardia and hypertension, along with effects on cardiac output and coronary blood flow, provided indices of relative effects. Overall, the effects were qualitatively similar, but occurred at higher doses of levobupivacaine than bupivacaine, and those arrhythmias that occurred with levobupivacaine were of a less malignant nature than those that occurred with bupivacaine.

Study 11. PK-PD analysis of bupivacaine and levobupivacaine [245] Serial drug blood concentrations gathered during Study 10 were measured by enantiospecific HPLC. First, a multi-compartment open model was fitted to the arterial blood drug concentrations. Second, the myocardial and brain net fluxes at each time point sampled were calculated from the respective products of the arterial-venous sinus blood drug concentration differences and regional blood flows; these values were numerically integrated over time to give the nett regional content of drug. These ‘mass balance’ parameters are independent of pharmacokinetic models, and give a good indicant of drug residence in tissue for correlating the time courses of drug effect and tissue concentration. Third, a non-parametric ‘effect 65 compartment model’ was prepared from the time course of arterial drug concentrations and dP/dt max . The pharmacokinetics of both enantiomers were found to be independent of dose. The pharmacokinetics of levobupivacaine did not differ whether it was administered alone or as a component of bupivacaine. The studies did not reveal any evidence of an enantiomer- enantiomer pharmacokinetic interaction for bupivacaine.

Reviews 07 and 08. Levobupivacaine: critical data [243,273] The editor of the journal Drugs invited me to prepare a one-page commentary on the potential single enantiomer switch from bupivacaine to levobupivacaine [ 243 ]. Additionally, Brian Gennery (from Chiroscience) received an invitation from the editor of Seminars in Anesthesia to write this ‘preclinical review’ [ 273 ] when levobupivacaine was being considered by the FDA. He invited Gary Strichartz, a neuropharmacologist collaborator from Harvard Medical School and protégé of Ben Covino, and me, to co-author the review. Gary had overseen many of the ex vivo neuropharmacological studies of the local anaesthetics that were complementary to our in vivo studies. Our paper, which reads rather like a composite of three reviews, had the desired effect of bringing the new local anaesthetic, levobupivacaine, to the attention of a wide anaesthesiology readership.

Patents 01 and 02. Use patents on levobupivacaine Part of the research program grant agreement with Chiroscience was that, although we had full publishing rights, sufficient funding for the research and several PhD candidacies, and would retain all of the laboratory equipment purchased for the program, the intellectual property resided with the company. The company’s goodwill extended to naming me as an ‘inventor’ on several patent families alongside the Chiroscience principals, and my participation in the intellectual property process created a new and interesting experience for me personally.

Study 12. Analysis of fatal toxicity from levobupivacaine [276] Study 10 showed clearly that levobupivacaine has a greater margin of safety than bupivacaine. However, having not observed any deaths from levobupivacaine administration in that protocol, we were unable to judge either the relative lethal doses or whether the cause of death would be due to the same mechanisms as with bupivacaine. This study was designed to extend the range of doses of levobupivacaine in order to determine the mode of death from fatal doses in sheep. We found that two patterns of fatal cardiac effects predominated. The sudden onset of ventricular fibrillation (VF) occurred in a minority, and electro-mechanical dissociation, with or without ventricular tachycardia and associated failure to fill, occurred in the remainder, but over longer time courses. The mechanism of death induced by bupivacaine in sheep is mainly VF. Levobupivacaine infusions at this large-dose range, produced increased electrocardiographic QRS width, along with a risk of ventricular arrhythmias, but these data confirmed a greater safety profile for levobupivacaine than bupivacaine.

Study 13. Chaos model of the CNS toxicity of local anaesthetics [285] Overt CNS toxicity of local anaesthetics is typified by generalized tonic-clonic convulsions, possibly followed by fatal cardio-respiratory depression. Prodromal CNS effects of increasing severity are readily described by healthy human volunteers, but the occurrence of convulsions is often the first detected sign of serious or life-threatening toxicity in patients and research animals, alike. In previous studies, the CNS toxicity of the local anaesthetics had been classified as the absence or presence of overt convulsions, i.e., as a quantal response. This obscures the continuum of responses and what we believe to be, a proper quantitative approach to describing the CNS effects of these and other drugs. Since the brain is a non- 66 linear system, it is capable of producing chaotic dynamics under certain circumstances. We hypothesized that convulsions represent early stages of the onset of chaotic dynamics, that would either extinguish or amplify in the next stages. This could manifest as a continuous scale of behavioural effects, and these could be useful for modelling the CNS effects of bupivacaine and levobupivacaine. This method, while perhaps in need of further development, might be used as an index of action for any non-linear system, particularly in conductive tissues, namely central and peripheral nervous systems, heart and voluntary muscle. This study arose from a task set for Leigh Ladd as part of his PhD thesis. It appears to be among the first attempts to use chaotic modelling of pharmacological response.

Study 14. Direct cardiovascular toxicity of local anaesthetics [280] The whole body response to cardiovascular system (CVS) toxicity is complex, involving direct effects on cardiac conduction and contractility, effects on vascular smooth muscle, and indirect effects mediated via the CNS control of CVS function. To simplify and to isolate operant variables, many investigators have studied electrical and mechanical effects of local anaesthetics in isolated tissues (e.g. Langendorff heart), and in subsets of isolated tissues (e.g. Purkinje fibres or beating atria) but such models preclude complicated issues associated with whole body pharmacokinetics and interactive control mechanisms. I decided that a preferred model would be to dose the relevant organs directly, i.e., by close arterial administration (to the heart and to the brain), in a fully intact conscious large animal preparation – with the same local blood-borne drug concentrations as with an intravenous administration, but precluding significant systemic blood-borne drug concentrations that could cause secondary responses. This required detailed exploration of the vascular anatomy of the respective regions before proceeding to the studies. The results demonstrated remarkably similar quantitative effects on myocardial contractility for each of the local anaesthetics, even allowing for differences in anaesthetic potency (Figure 3.9). This study, including the development of the surgical- anatomical approaches, formed part of the PhD thesis of Leigh Ladd.

Figure 3.9: Conscious sheep: with a 3-min intracoronary arterial infusion of each of 3 local anaesthetic agents to determine the comparative direct myocardial effects measured by left ventricular dP/dt max with minimal/negligible recirculated doses to cause CNS effects.

Study 15. Direct CNS toxicity of local anaesthetics [299] This study was necessary to determine the influence of CNS toxicity apart from any direct effects on the cardiovascular system in intact conscious animals. It was possible to administer the local anaesthetics directly, but only with enrichment via the accessible carotid arteries. Enrichment by the arterial supply to brain via the circle of Willis is from a minimum of five vessels. The preference for enrichment of the whole brain is because the Nucleus Tractus Solitarius (NTS) is found fairly caudal in the medulla oblongata and, as the NTS controls 67 heart function to a great extent, it should be included as part of the brain but, anatomically and practically, this is too complicated for present experimental techniques. The significance of this is that the ‘CNS hypothesis’ of local anaesthetic cardiotoxicity can’t be fully tested in the sheep, or any other preparation. Nonetheless, this study formed an innovative contribution to the studies of the comparative toxicity of local anaesthetics and also formed part of the PhD thesis of Leigh Ladd.

Review 09. Cardiotoxicity of local anaesthetics – rationale [282] In 2000, I was invited by the editor of Drugs to contribute an up-to-date perspective on local anaesthetic cardiotoxicity. It was to be wide ranging in current theories and lines of investigation, and non-partisan, including chirality. I invited my post-doctoral research officer, Dennis Chang, to work with me on this review in which we attempted to analyse the various models applied, as well as factors revealed by the models as significant in the causation of toxicity. We concluded with the hopefully very strong message, that despite the improvements in the margin of safety of the newer agents ropivacaine and levobupivacaine over the standard bupivacaine, the newer agents should be regarded as “safer, not safe”. The paper has been widely cited.

Study 16. Acid-base changes on PD and PK of bupivacaine (and thiopentone) [306] This study involved administration of bupivacaine rather than levobupivacaine, but is included in this subsection because of the methodology used, and its chronological place in the study sequence, and is repeated in the thiopentone series.

One of the fundamental tenets of medicinal chemistry is that of the ‘pH partition hypothesis’ describing the equilibration of weakly ionizable drugs across a semipermeable membrane originally proposed by Bernard Brodie and colleagues in connection with the gastrointestinal absorption of drugs. It has been found applicable to the distribution of (most) other drugs and (most) body compartments or spaces, including equilibration across the placental and blood- brain barriers, although more recent findings of selective transporter proteins now need to be incorporated into the general theory. This hypothesis is founded on the notion that a weakly ionizable drug attains an equilibrium between unionized and ionized species, in concentrations determined by the pKa of the drug and the pH of the medium according to the Henderson-Hasselbalch equation; that (only) unionized drug diffuses across the (lipoidal) membrane, achieving equal concentrations on both sides; the total concentrations of the drug may differ on the opposing sides of the semipermeable membrane due to re-altered equilibrium between the unionized and ionized species also due to the prevailing pH; and that changes in these concentrations can be wrought by changing the pH on one or both sides of the membrane, potentially altering the effects of that drug. As acid-base changes within reasonably narrow ranges can occur from physiological derangements in a clinical milieu, this may significantly affect the relative unionized and ionized concentrations of weakly acidic or basic drugs that have a pKa value close to that of the pH of blood. Both thiopentone (a weak acid pKa=7.9) and bupivacaine (a weak base pKa=8.1) are cardiac . This study was conceived to determine whether intentionally imposed acid-base changes could affect the pharmacokinetics and cardiac effects of thiopentone and bupivacaine, and it made a suitable PhD thesis topic for veterinary anaesthetist Sue Copeland, who was then working as a research officer on the levobupivacaine program. At the doses used, the cardiovascular changes caused by these drugs were essentially unaffected by the acid-base derangements.

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Review 10. PK and PD concepts and the acute toxicity of local anesthetics [314] This research review was written as a follow-up to an earlier well-prepared article on the “maximum recommended doses of local anaesthetics” by Per H Rosenberg (Helsinki), Bernadette Veering (Leiden) and William F (Bill) Urmey (New York). 101 Their paper laid out clinical and relevant criteria for considering doses in patients. I sought to present the underlying and, where possible, unifying concepts affecting the margin between safe and effective and unsafe and toxic doses and to interpret them in pharmacokinetic and pharmacodynamic terms. I invited my PhD students, Sue Copeland and Leigh Ladd, to join me to mull over the issues from our own and other experimental evidence, and this rather broad viewpoint was the outcome. It has been well-received and widely cited.

Study 17. Effects of general anaesthesia on local anaesthetic toxicity [323] General anaesthesia is often used in laboratory animal studies, either as an obligatory part of the surgical preparation or as a means of immobilizing the subjects. It is used in most experimental studies of local anaesthetic toxicity, yet clinical local anaesthetic toxicity mainly occurs in conscious patients undergoing regional anaesthesia. As shown during our 1980s program at Flinders, general anaesthesia significantly affects normal physiology, haemodynamics, as well as drug pharmacokinetics and metabolism. However, the concomitant effects of general anaesthesia mostly go ignored in laboratory models because it is necessary in the preparation of the subject or in the way that acute studies are performed (typically with the subject ‘not being recovered’ from the experiment). Despite knowledge that general anaesthetics can modify local anaesthetic toxicity, its role as a study design factor has been largely ignored. This study determined the effects of standardized general anaesthesia on the behavioural, cardiovascular, and pharmacokinetic responses in previously instrumented ewes to the intravenous infusion of putatively equi-anaesthetic doses of bupivacaine, levobupivacaine, ropivacaine, lidocaine, mepivacaine and prilocaine, compared to saline (control). For these studies, it was necessary for the subject to be placed in a sling to support its weight (Figure 3.10). Not unexpectedly, at the doses chosen, the local anaesthetics all caused frank convulsions with the expected EEG changes in conscious sheep, but no overt CNS effects in anaesthetized sheep. Most significantly, fatal cardiac arrhythmias occurred in some sheep when conscious, but in none when anaesthetized. However, despite exacerbation of cardiovascular depression from the general anaesthesia by all local anaesthetics, all anesthetized animals survived. This study showed clearly that general anaesthesia affects the CNS and cardiac responses to local anaesthetics, and must be considered an important experimental variable in any study design. This work was part of Sue Copeland’s PhD project.

Study 18. Effects of general anaesthesia on local anaesthetic pharmacokinetics [324] This study, a second aspect of Study 17, documents the pharmacokinetic changes caused to local anaesthetics by general anaesthesia. It was the first time that I had introduced WinNonlin (Pharsight, version 5.0.1) as a pharmacokinetic analysis engine into our work, having previously used other pharmacokinetic programs. This was done for several reasons. WinNonlin has become the ‘industry standard’ tool, and I was aware that some of our findings could be controversial, so I wanted to use ‘reviewer-proof’ methodology. WinNonlin has excellent data handling routines and I wanted to introduce it to my PhD students. We found that blood concentrations of all of the local anaesthetic agents were doubled under general anaesthesia because of decreased whole body distribution and clearance, a finding consistent with earlier experiments at Flinders in the 1980s with achiral

101 Rosenberg PH, Veering BT, Urmey WF. Maximum recommended doses of local anesthetics: A multifactorial concept . Reg Anesth Pain Med 29: 564-575, 2004 69 analyses. The total body clearances of R-enantiomers of bupivacaine and prilocaine exceeded those of their respective S-enantiomers, but mepivacaine clearance was not enantioselective. This study confirms that not only does general anaesthesia in a model alter responses to drugs, but it alters their pharmacokinetics, as well.

Figure 3.10: A sheep subject being supported by a sling to maintain stable posture, a necessity for both safety and physiological monitoring during the studies of general anaesthesia and local anaesthetics.

3.25 Broader aspects of local anaesthetic pharmacology involving chirality Reviews 11, 12 and 13. Translational pharmacology of local anaesthetics [325,327,328] Geoff Tucker and I contributed a chapter, intended as a wider discourse on the translational pharmacology of local anaesthetic agents, to the 1 st edition (1980) of Cousins’ and Bridenbaugh’s “Neural Blockade” [59], but it scarcely mentioned stereoisomerism. We continued to contribute our chapter to “Neural Blockade”, updated, through the 2 nd (1988), 3 rd (1998) and most recent 4 th (2008) editions, with the latest edition containing much discussion of the enantiopure local anaesthetics now in clinical use, and reflecting the change of knowledge [ 325 ]. The other reviews were published after I had retired. In 2010, I received an invitation to present the plenary lecture on local anaesthetics to the 13th Asian- Australasian Congress of Anesthesiologists in Fukuoka, Japan on June 1-5 2010. Apart from the honour of the invitation, it held special meaning for me as it was to this same congress in Canberra in 1970, that John Bonica had presented the plenary lecture on local anaesthetics. Afterwards, I received a request by the meeting organizer, Akira Asada from the University of Osaka, to contribute a chapter based on my lecture for a Japanese language textbook that he was coordinating for anaesthetists, focussing on the background, rationale and clinical uses for the introduction of levobupivacaine [ 327 ]. Soon after, I received another invitation to submit an ‘expert review’ for a pharmacology-toxicology journal, aimed at compiling critical ideas with suggestions for future research. This review was to become my final major contribution to the literature about local anaesthetics, at the end of many decades of personal data creation [ 328 ].

Postscript 1 The need for safe and reliable local anaesthetic agents has been recognized since the era of cocaine – indeed, it was the rationale for procaine being introduced over a century ago. Longer-acting local anaesthetic agents such as tetracaine and dibucaine, used from the 1930s, were replaced in the early 1970s when bupivacaine became the de facto standard because of its chemical stability and good reputation for reliability. However, by the late 1970s, its widespread use was thought to be accompanied by a disproportionately greater incidence of a sometimes fatal cardiac toxicity syndrome than was occurring with shorter-acting agents such as lignocaine and mepivacaine. Such sequelae are of particular concern after accidental 70 injection or unexpectedly rapid absorption into the systemic circulation, thus producing a relative overdose that has proved impossible to manage in some cases. The responded by introducing the enantiopure bupivacaine congeners, ropivacaine and levobupivacaine, each having a lower intrinsic risk of cardiac toxicity - and adding to the safety of regional anaesthesia.

In the mid 1980s, at the request of Astra Pharmaceuticals, we developed a program of studies on ropivacaine (Naropin®) and other local anaesthetics. In the mid 1990s, at the request of Chiroscience, we developed the program further to study levobupivacaine (Chirocaine®) and other local anaesthetics by adding new generation physiological measurement technology with analogue-to-digital processing and techniques for performing site-directed close arterial drug administration. Our ‘model’ was accepted by the US FDA as a benchmark paradigm for studying the cardiovascular and central nervous system effects of local anaesthetics. The multicannulated sheep preparation was thus highly influential in the development and registration of ropivacaine and levobupivacaine. Both agents are interchangeable with bupivacaine for neural blockade procedures when adjusted for dose, and both can cause serious and life-threatening acute toxicity, but generally at higher doses than those of bupivacaine. Levobupivacaine is anaesthetically equi-potent with bupivacaine, and thus has a greater margin of safety. Ropivacaine also has a greater margin of safety than bupivacaine, but being anaesthetically less potent (it requires greater doses than bupivacaine for equivalent neural blockade), some of its increased margin of safety is offset.

During the 1980s and 1990s development of ropivacaine and levobupivacaine, much was discovered and/or re-discovered about the reasons for the S-enantiomers being relatively less cardiac toxic. 102 Foremost, the kinetics of cardiac ion channel blockade are moderately stereoselective, favouring faster drug-channel dissociations of the S-enantiomers, and it is probable that similar stereoselectivity occurs elsewhere also. 103 Moreover, the enantiomers differed in their myocardial uptake and intrinsic effects, 104 and plasma protein binding. 105 That such differences can influence their systemic toxicity is now taken for granted.

102 Åkerman B. Uptake and retention of the enantiomers of a local anaesthetic in isolated nerve in relation to different degrees of blocking of nervous conduction. Acta Pharmacol Toxicol 32 :225- 236, 1973; Lee-Son S. Stereoselective inhibition of neuronal sodium channels by local anaesthetics. Anaesthesiology 77:324-355, 1992 103 Nau C, Wang SY, Strichartz GR, Wang GK. Block of human heart hH 1 sodium channels by the enantiomers of bupivacaine. Anesthesiology 93(4):1022-1033, 2000; Valenzuela C, Delpon E, Tamkun MM, Tamargo J, Snyders DJ. Stereoselective block of a human cardiac potassium channel (kv1.5) by bupivacaine enantiomers. Biophys J 69(2): 418-427, 1995; Valenzuela C, Snyders DJ, Bennett PB, Tamargo J, Hondeghem LM. Stereoselective block of cardiac sodium channels by bupivacaine in guinea pig ventricular myocytes. Circulation 92(10):3014-3024, 1995 104 Mazoit JX, Orhant EE, Boïco O, Kantelip JP, Samii K. Myocardial uptake of bupivacaine: I. Pharmacokinetics and pharmacodynamics of lidocaine and bupivacaine in the isolated perfused rabbit heart. Anesth Analg 77(3), 469-476, 1993; Mazoit JX, Boico O, Samii K. Myocardial uptake of bupivacaine. 2. Pharmacokinetics and pharmacodynamics of bupivacaine enantiomers in the isolated perfused rabbit heart. Anesth Analg 77(3): 477-482, 1993; Mazoit JX, Decaux A, Bouaziz H, Edouard A. Comparative ventricular electrophysiologic effect of racemic bupivacaine, levobupivacaine, and ropivacaine on the isolated rabbit heart. Anesthesiology 93(3), 784-792, 2000; Aya AG, Jean E, Robert E, Ripart J, Cuvillon P, Mazoit JX, Jeannes P, Fabbro-Péray P, Eledjam JJ. Comparison of the effects of racemic bupivacaine, levobupivacaine, and ropivacaine on ventricular conduction, refractoriness, and wavelength: an epicardial mapping study. Anesthesiology 96(3): 641-650, 2002 105 Mazoit JX, Cao LS, Samii K. Binding of bupivacaine to human serum proteins, isolated albumin and isolated alpha-1-acid glycoprotein - differences between the two enantiomers are partly due to cooperativity. J Pharmacol Exp Ther 276(1): 109-115, 1996 71

During the period of this research, clinical practice was concurrently improved through the strict use of test doses to detect intravascular needle placement, dose fractionation so that the bulk of the dose is not injected as one, the use of ultrasonic techniques to visualise the anatomical environment, the development of lipid rescue, etc.. While the chirality issue is not particularly important for local anaesthetics used in small doses, such as for subarachnoid block, it becomes much more important when larger doses are used, through the ‘fail-safe’ principle.

The acute systemic toxicity of local anaesthetics has become a study area, and has even acquired an acronym, LAST. 106 However, further improvements through enantiopurity in drug dosage forms have been resisted, so that the various developments by way of heavily loaded (i.e., high dose) slowly dissolving liposomal, polylactide or other depot preparations, have been based on racemic bupivacaine. The reasons for this are not obvious, perhaps it is because of patent-law protection, but it is not based on the pharmacology published to date. Despite the obvious appeal of these slowly dissolving depot preparations, the consequences of their accidental injection into a blood vessel have not yet been described. As shown during the introduction of ropivacaine and levobupivacaine, both regulatory authorities and anaesthesiologists are attuned to questions of local anaesthetic toxicity. It is clear that both will want assurances that any new preparation containing a long-acting local anaesthetic will have adequate safety in the event of rapid systemic absorption or accidental intravascular injection. However, I am sure that new dosage forms will require new studies, and hopefully these will make use of the models that have taken us so long to develop, rather than just reinvent them.

3.3 Intravenous anaesthetic agent program From around the 1920s, the quest for an intravenous agent for the induction of anaesthesia was focussing on barbiturates, notably somnifene (or Somnifen®, an equal part mixture of the diethylamines of diethyl barbituric acid and diallyl barbituric acid), pernoston (sodium sec- 107 butyl β-bromallyl ) and, a decade later, hexobarbitone and thiopentone.

Barbiturates derive from a golden age of European synthetic organic chemistry with the synthesis of barbituric acid, formed by the condensation of urea and malonic acid by JFW Adolph von Baeyer in 1864. This was followed by barbitone (diethylbarbituric acid) synthesized by Emil Fischer and Joseph Friederich von Mering in 1903, and numerous others. Intravenous barbiturate administration became successful with the water soluble sodium salt of amylobarbitone in 1928. Pentobarbitone was synthesized by Ernest Henry Volwiler (1893- 1992) and Donalee Tabern (1900-1974) at Abbott Laboratories in North Chicago, and first used intravenously in 1930. John S Lundy (1894–1973), from the Mayo Clinic, called it Embutal®, derived from the initial letters of its chemical name: E (= ethyl), M (= methyl), BUT (= butyl) and AL (= American barbiturate ending) – its sodium salt for intravenous injection was given the trade name Nembutal® ( N = Na +). Thiopentone is the sulphur analogue of pentobarbitone: it was synthesized in 1932, also by Volwiler and Tabern.

106 Weinberg GL. Treatment of local anesthetic systemic toxicity (LAST). Reg Anesth Pain Med 35(2): 188-193, 2010 107 Thalheimer M. Anesthesia by intravenous injection of 1-methyl-5, 5-allyl-isopropyl barbituric acid. Anesth Analg 16(2): 61-65, 1937; DeAngelis AM. Basal anesthesia with Pernoston: analysis of 1000 administrations. Anesth Analg 20(1): 50-57, 1941; Dundee JW. Thiopentone and other barbiturates . E&S Livingstone, Edinburgh, 1956, p312 72

Thiopentone was first administered clinically on March 8 1934 by Ralph M Waters (1883- 1979) at the University of Wisconsin Hospital in Madison. It became the ‘gold standard’ intravenous anaesthetic induction agent, lasting well into the 1990s. During these six decades, it was subjected to innumerable investigations, including many studies of its pharmacokinetics and pharmacodynamics that increased in complexity as research techniques evolved. Importantly, it was found in the 1950s that the rate of metabolism did not account for the ‘ultra-short’ duration of action or govern the rate of recovery from thiopentone anaesthesia, and this information was determined from the barely adequately sensitive assay methods available at that time. 108 From the 1960s, several pharmacokinetic models for thiopentone were proposed, based on limited data and many assumptions, and these were very influential because they connected the relationship between physiological variables and pharmacokinetic outcomes. 109 Rapid progress in understanding the pharmacokinetics and pharmacodynamics of thiopentone and its congeners became available with the development and application of GLC and HPLC during the late 1970s and early 1980s by a number of research groups, most notably at the University of Bonn, the University of Melbourne, Northwestern University, and Stanford University. 110 Such studies also provided data for thiopentone to be used by multi-stage infusions as a primary anaesthetic agent during surgery. 111

Thiopentone, and certain other barbiturates and congeners such as methohexitone, are chiral compounds made and used as racemates, but this is neither well known nor normally mentioned in accounts of their pharmacological or clinical use. The broad fact of enantioselectivity in thiopentone pharmacology seems first to have been published in the 1970s, 112 but this was generally overlooked, either out of commercial expediency or the

108 Brodie BB, Mark LC, Papper EM, Lief PA, Bernstein E, Rovenstine EA. The fate of thiopental in man and a method for its estimation in biological material. J Pharmacol Exp Ther 98 (1): 85-96, 1950; Brodie BB, Bernstein E, Mark LC. The role of body fat in limiting the duration of action of thiopental. J Pharmacol Exp Ther 105 (4): 421-426, 1952 109 Price HL. A dynamic concept of the distribution of thiopental in the human body. Anesthesiology 21(1): 40-45, 1960; Saidman LJ, Eger EI. The effect of thiopental metabolism on duration of anesthesia. Anesthesiology 27(2): 118-126, 1966; Bischoff KB, Dedrick RL. Thiopental pharmacokinetics. J Pharm Sci 57(8): 1346-1351, 1968 110 Some notable examples include: Schwilden H, Schüttler J, Stoeckel H. Closed-loop feedback control of anesthesia by quantitative EEG analysis in humans. Anesthesiology 67(3): 341-347, 1987; Crankshaw DP, Rosler A, Ware M. The short term distribution of thiopentone in the dog. Anaesth Intens Care 7(2): 148-151, 1979; Bjorksten AR, Crankshaw DP, Morgan DJ, Prideaux PR. The effects of cardiopulmonary bypass on plasma concentrations and protein binding of methohexital and thiopental. J Cardiothorac Anesth 2(3): 281-289, 1988; Avram, MJ, Krejcie TC, Henthorn TK. The relationship of age to the pharmacokinetics of early drug distribution: the concurrent disposition of thiopental and indocyanine green. Anesthesiology 72(3): 403-411, 1990; Bührer M, Maitre PO, Hung OR, Ebling WF, Shafer SL, Stanski DR. Thiopental pharmacodynamics I. Defining the pseudo-steady-state serum concentration-EEG effect Relationship. Anesthesiology 77(2): 226-236, 1992.; Hung OR, Varvel JR, Shafer SL, Stanski DR. Thiopental pharmacodynamics II. Quantitation of clinical and electroencephalographic depth of anesthesia. Anesthesiology 77(2): 237-244, 1992 111 Crankshaw DP, Edwards NE, Blackman GL, Boyd MD, Chan HNJ, Morgan DJ. Evaluation of infusion regimens for thiopentone as a primary anaesthetic agent. Eur J Clin Pharmacol 28(5): 543- 552, 1985; Crankshaw DP, Boyd MD, Bjorksten AR. Plasma drug efflux--a new approach to optimization of drug infusion for constant blood concentration of thiopental and methohexital. Anesthesiology 67(1): 32-41, 1987 112 Christensen HD, Lee IS. Anesthetic potency and acute toxicity of optically active disubstituted barbituric acids. Toxicol Appl Pharmacol 26: 495-503, 1973; Haley TJ, Gidley JT. Pharmacological comparison of R-(+), S-(–) and racemic thiopentone in mice. Eur J Pharmacol 36: 211-4, 1976; Mark LC, Brand L, Perel JM, Carrol FI. Barbiturate stereoisomers: direction for the future. Excerpta Medica 399: 143-146, 1977 73 inability to make further developments. The authors of that report claimed that there was greater anaesthetic (and apnoeic) potency of S-thiopentone and suggested that it might be usefully substituted for rac-thiopentone. This may be true, but the paucity of the data and the methodology used rendered the claims overly optimistic. Accordingly, much of the even- recent pharmacokinetic-pharmacodynamic research literature on thiopentone 113 is compromised to the extent that enantiomeric differences are significant.

Advances in describing the relevant anaesthetic pharmacodynamic effects of thiopentone were facilitated with the development of neural recording techniques, particularly qEEG, assisted by developments in online digital recording of selected variables. Many researchers in this area had been using EEG ‘models’ for many years, but the ‘Stanford’ approach became a benchmark paradigm for experiments on the relationship between CNS effects and pharmacokinetics of thiopentone in humans and in rats. 114

Figure 3.11: Results with the original legend from one of the Stanford thiopentone pharmacokinetic- pharmacodynamic studies, showing the biphasic qEEG response along with thiopentone plasma concentrations during a brief intravenous infusion of thiopentone in the rat [from Ebling WF, Danhof M, Stanski DR. Pharmacodynamic characterization of the electroencephalographic effects of thiopental in rats. J Pharmacokin Biopharm 19(2): 123-143, 1991]

113 Some examples include: Ilkiw JE, Benthuysen JA, Ebling WF, McNeal D. A comparative study of the pharmacokinetics of thiopental in the rabbit, sheep and dog. J Vet Pharmacol Ther 14: 134- 140, 1991; Russo H, Bressolle F. Pharmacodynamics and pharmacokinetics of thiopental. Clin Pharmacokin 35(2): 95-134, 1998. Björkman S, Nilsson F, Åkeson J, Messeter K, Rosen I. The effect of thiopental on cerebral blood flow, and its relation to arterial plasma concentration, during simulated induction of anaesthesia in a porcine model. Acta Anaesth Scand 38: 473-478, 1994; Upton RN, Ludbrook GL. A model of the kinetics and dynamics of induction of anaesthesia in sheep: variable estimation for thiopental and comparison with propofol. Br J Anaesth 82(6): 890- 899, 1999 114 Stanski DR, Hudson RJ, Homer TD, Saidman LJ, Meathe E. Pharmacodynamic modeling of thiopental anesthesia. J Pharmacokin Biopharm ;12: 223-40, 1984; Ebling WF, Danhof M, Stanskl DR. Pharmacodynamic characterization of the electroencephalographic effects of thiopental in rats. J Pharmacokin Biopharm 19: 123-143,1991 74

Thiopentone generates a biphasic effect on the qEEG, i.e., initial activation is followed by deactivation (shown in Figure 3.11, from one of the Stanford qEEG studies). Although the pharmacokinetic-pharmacodynamic concept was generating an objective framework for studying temporal aspects of its effects on the brain, questions about the significance of the chirality of thiopentone, particularly in respect to the biphasic qEEG response, had not been considered until my project sought to address that issue in the early 1990s. Interestingly, in Melbourne, some of our colleagues also began studies of stereopharmacological aspects of thiopentone, at around the same time.115

To initiate the stereopharmacology program, I applied, successfully, for the inaugural Douglas Joseph Professorship. This award was established under Regulation 21 of the Faculty of Anaesthetists, Royal Australasian College of Surgeons in 1991 as “a prestigious award for Fellows making an outstanding contribution to the advancement of the specialty to pursue scholarship and research in human anaesthesia in Australia, New Zealand, Hong Kong, Malaysia and Singapore”. 116 It commemorates Australia’s first academic Professor of Anaesthesia and was adopted by the Australian and New Zealand College of Anaesthetists following its establishment, to be awarded on a quadrennial basis starting in 1993. My application was focussed on thiopentone, then still the most widely used anaesthetic induction agent, as well as halothane, then an obsolescent but a toxicologically intriguing agent.

For this program, I decided that it would be sensible to use the same techniques that were being used in the key thiopentone pharmacokinetic-pharmacodynamic modelling projects by Don Stanski’s group at Stanford University. I visited Don’s department in 1993 and, armed with enthusiasm and a video camera, I learned from Eileen Osaki, Don’s research assistant, how to set-up and perform studies in the rat using the qEEG (Figure 3.12), along with computer controlled infusions of drugs using anaesthesiologist Steve Shafer’s Stanpump software package (Figure 3.13).117 It made sense that if we were going to compare outcomes with the existent literature, then we should use these same techniques, which I then passed on to my research assistant, Steven R (Steve) Edwards.

The Douglas Joseph Professorship also allowed me to appoint a postdoctoral research officer JuiLi (Li) Huang from Monash University (see Section 1.6). The project also led to further collaboration with my former PhD student Richard Upton, then at the University of Adelaide, and with Colin C Duke, a stereochemist of the University of Sydney Faculty of Pharmacy, who was later to become a collaborator on the nonsteroidal anti-inflammatory drug and halothane projects. Subsequently, human studies on thiopentone grew into a PhD project for Dennis J Cordato, a clinical neurologist and research fellow co-supervised by Geoffrey K Herkes from the RNSH Department of Neurology, and various other studies were performed collaboratively with several colleagues from other departments, most notably in Clinical

115 Nguyen KT, Stephens DP, McLeish MJ, Crankshaw DP, Morgan DJ. Pharmacokinetics of thiopental and pentobarbital enantiomers after intravenous administration of racemic thiopental. Anesth Analg 83:552-558,1996; Nguyen KT, Morgan DJ. Myocardial uptake of thiopental enantiomers by the isolated perfused rat heart. Chirality 1996;8:477-80. 116 http://www.anzca.edu.au/resources/regulations/regulation-list/regulation-21.html (accessed December 2013 ) 117 Notably: Maitre PO, Buhrer M, Shafer SL, Stanski DR. Estimating the rate of thiopental blood-brain equilibration using pseudo steady state serum concentrations. J Pharmacokinet Biopharm 18(3): 175-87, 1990; Ebling WF, Danhof M, Stanski DR. Pharmacodynamic characterization of the electroencephalographic effects of thiopental in rats. J Pharmacokinet Biopharm 19(2): 123-143, 1991; Gustafsson LL, Ebling WF, Osaki E, Harapat S, Stanski DR, Shafer SL. Plasma concentration clamping in the rat using a computer-controlled infusion pump. Pharm Res 9(6): 800- 807,1992 75

Pharmacology at RNSH and Pharmacology at University of Sydney. These involved a variety of paradigms, and were carried out in stages to investigate particular issues, as described in the following publications.

Figure 3.12 Preparation of the Stanford PK-PD rat model for studying the qEEG effects of thiopentone. Top left: scalp dissection and placement of 2 of 8 electrodes in the brain. Top right : all 8 electrodes placed. Bottom left : placement of an electrode contact plug in the brain (to allow quick and easy connection for pharmacodynamic monitoring). Bottom righ t: Electrode contact plug in place before final scalp closure.

Figure 3.13: The Stanford PK-PD model being used for studying the qEEG effects of thiopentone in the rat. This “Stanford qEEG” model was adopted for our ‘rat lab’ studies described in this thesis.

An obligation of the Douglas Joseph Professorship was to present the Australasian Visitor’s Lecture to the General Scientific meeting of the Australian and New Zealand College of Anaesthetists. At the 1994 meeting in Launceston, the town where, on June 7 1847, William Russ Pugh famously administered ether for surgery, I had the pleasure of presenting a plenary lecture entitled “Chirality is in your hands” [ 214] to outline to the assembled Fellows and guests my rationale for wanting to perform this work on stereopharmacology.

76

Patent 03. Synthesis and uses of thiopentone enantiomers The studies to be performed under the Douglas Joseph Professorship research grant required significant quantities of the separate enantiomers of thiopentone. Colin Duke became a close colleague on this program and was responsible for devising the novel chiral syntheses of the thiopentone enantiomers for which he and I were subsequently listed as ‘inventors’ on Patent Application number PCT/AU1999/000919 and Patent WO2000/024358. The methodology produced supplies of the thiopentone enantiomers to set up the enantiospecific assay of thiopentone in biological samples after administration of rac -thiopentone, as well as perform comparative pharmacological studies with the clinically used rac -thiopentone in the sheep and rat preparations.

Study 19. Thiopentone: a chiral assay for future studies [221] Our studies required development of an enantiospecific assay for thiopentone in extracts of blood and tissues, with resolution from the probable metabolite pentobarbitone, as well as chemical identification specifications for the chiral barbiturates. The assay technique was developed by Li Huang originally described for processing the sheep blood samples, but the method was transferred by Sonia Gu for all subsequent studies without significant changes for human and rat blood samples.

Study 20. Preliminary evaluation of thiopentone PK enantioselectivity in sheep [224] The first pharmacological study was designed to examine enantioselectivity of brain uptake and elution, and to compare the systemic pharmacokinetics of the thiopentone enantiomers after administration of a single dose of the rac -thiopentone in a “washout” paradigm. The initial study in sheep was undertaken in Richard Upton’s lab at the University of Adelaide, as our sheep lab was unable to do this with the urgency required, due to commitments with the levobupivacaine program. The blood analysis and data processing were undertaken in my lab. Although we found minor enantioselective differences in whole body mean residence time and slow half-life, we concluded that an enantiomeric difference in the brain kinetics would not account for the qEEG biphasic response.

Study 21. Evaluation of thiopentone PK enantioselectivity in humans [232] This first pharmacokinetic study in Dennis Cordato’s PhD series consisted of a series in human patients, either having rac -thiopentone for surgical anaesthesia, or being infused with rac -thiopentone as part of their neurological management of brain injury, in which serial blood samples were assayed enantiospecifically. This study found weak enantioselectivity in whole body pharmacokinetics, much like Study 20. However, the qEEG data collected in this study could not identify any particular events with either enantiomer, and this is a problem for the experimental design in clinical patients having normal polypharmacy clinical management. We decided that further study of qEEG would require a design in which complete control over the subject is attainable, e.g. in an animal model, and this was achieved with subsequent studies in the rat.

Study 22. Evaluation of thiopentone PK enantioselectivity in rats [259] This study used a Stanpump-controlled intravenous infusion pump to produce a stepped infusion “washin” paradigm (as used in the Stanford research) to complement the single dose “washout” paradigm of Study 20, and to evaluate possible effects of the subjects being anaesthetized. The controlled infusion was ‘pharmacokinetically designed’, i.e., it was algorithm-based using previously determined pharmacokinetic values in that species or subject. The rat, being a small animal, facilitated a detailed analysis of thiopentone distribution between blood and the various tissues of the CNS. The two-group design was 77 incorporated to anticipate comparison with the data generated by other research groups that use anaesthetized subjects without questioning whether the general anaesthetic agents have any impact on the results. Significant differences were, again, found between conscious and anaesthetized subjects.

Study 23. PK-PD study of thiopentone enantioselectivity in rats [262] This was the first of several studies performed with enantiopure thiopentone to compare with racemate and used a constant rate infusion until lethal, whilst recording both behavioural end- points and a qEEG signal to relate to the distribution of the thiopentone enantiomers into the CNS and other tissues. The infusions clearly produced the biphasic EEG activation- deactivation sequences that were similar for both enantiomers and the racemate. However, the study found that there was a margin of safety in favour of R-thiopentone over rac - thiopentone or S-thiopentone in terms of the ratio of lethal to anaesthetic doses, and showed enantioselectivity of the distribution of the agent between CNS tissues and blood. I believe that this research makes a major contribution to understanding the stereopharmacology of thiopentone, and indicates an advantage of R-thiopentone over the more potent S-thiopentone or clinically-used rac -thiopentone.

Study 24. Enantioselectivity of GABA A receptor effects of thiopentone [261] This was the second study performed with enantiopure and racemic thiopentone, and was published back-to-back in the same number of the British Journal of Pharmacology as Study 23. In an attempt to rationalise the potency differences of the thiopentone enantiomers recorded in Study 23, I arranged for Dennis Cordato to learn the techniques and perform some in vitro studies of their affinity to GABA A receptors, the target receptor of the barbiturates and some other anaesthetic agents, using the techniques being practised by Mary Collins (Chebib), then in the University of Sydney Department of Pharmacology (now Professor of Pharmaceutical Neuroscience in the Faculty of Pharmacy). Mary, a former colleague of Graham A R Johnston, one of the world’s most prominent neuropharmacological experts on GAB A receptors, hosted Dennis in her laboratory for this set of studies performed on GABA A receptors transfected into Xenopus laevis oocytes. These studies found S-thiopentone to be ≈2x more potent than R-thiopentone in the potentiation of GABAA at that receptor, thereby presenting a pharmacodynamic factor, in addition to the pharmacokinetic factors, accounting for the stereopharmacology of thiopentone.

Study 25. PK study of thiopentone enantiomers from i.v. infusion in humans [264] This study contained a small subset of patients infused with high dose thiopentone for producing decreased intracranial pressure. Others had observed that thiopentone at high dose develops pharmacokinetic non-linearity, or Michaelis-Menten kinetics, whereby the rate of metabolism proceeds at a constant mass rate of the presenting concentration (described as ‘zero order’) until the concentration becomes low enough to proceed at a constant fractional rate (described as ‘first order’). This study was designed to investigate whether the zero order kinetics could be an artefact, resulting from the two enantiomers of thiopentone being combined. The results showed that both enantiomers enter non-linear kinetics with a small, but significant, degree of enantioselectivity.

Study 26. Tissue distribution and effects from thiopentone enantiomers in rats [265] This, the second series performed in the Stanford rat model with computer controlled stepwise infusions of enantiopure and racemic thiopentone, was used to complement the data found in Study 23. The paradigm recorded a qEEG signal to relate to the distribution of the 78 thiopentone enantiomers into the CNS during both the ‘washin’ and ‘washout’ periods. The study also confirmed a margin of safety in favour of R-thiopentone.

Study 27. Blood-brain equilibration of thiopentone enantiomers in rats [266] The third series was also performed with enantiopure thiopentone to compare to racemate and used stepwise infusions. The paradigm used the new technology of a microdialysis catheter placed into brain tissue to examine the time course of the (diffusible) unbound drug concentrations to relate to the rates of attaining distribution-equilibrium of the thiopentone enantiomers between plasma and the CNS and other tissues. The study confirmed the outcome of Study 26 but with more specific methodology, by finding that there was no difference in the time courses of the uptake and distribution of the thiopentone enantiomers into CNS tissues.

Study 28. Nicotinic ACh receptor effects of thiopentone enantiomers [284] I had visited the Department of Anesthesiology at Columbia University in New York as a guest of Margaret Wood, Professor and Chair, and presented a seminar on our thiopentone work. This was the department where Manny Papper had performed his work on thiopentone a half century earlier. Not long afterwards, I was contacted by Pamela Flood of that department about collaborating on another in vitro receptor binding study with enantiopure thiopentone to compare to the racemate, this time on nicotinic acetylcholine receptors (nAChRs), in a transfected xenopus model that she had developed. The nAChRs in the central nervous system had emerged as a potential target for the anaesthetic effects of thiopental, as Pamela had found that the α7 nAChR was inhibited by thiopentone in a clinically relevant concentration range. This work was performed in Pamela’s lab in New York and was intended to clarify the range of stereoselective activities of the intravenous anaesthetic agents. It turned out that the separate S- and R- enantiomers of thiopentone caused inhibition indistinguishable from that caused by the racemate.

Study 29. PK studies of high dose thiopentone in humans [287] At the time, high dose thiopentone intravenous infusion for the treatment of patients with head injuries was standard treatment. We decided that Dennis Cordato should examine more closely the relationship between the thiopentone plasma concentration and several indicators of neurological response, and this was done by studying a prospective cohort of patients and reviewing existent data for another cohort. The project also involved both the RNSH medical treating team (Geoff Herkes, Michael K Morgan and Simon Finfer) and ‘therapeutic monitoring’ drug assays previously provided by RNSH Department of Clinical Pharmacology (by Dr Annette S Gross). I devised a logistic regression model to predict the responsiveness of patients in relation to their measured thiopentone concentrations. It was pointed out that, however appealing, the use of a thiopentone blood concentration may be to predict an individual patient’s clinical response (and it is appealing compared to using other variables such as dose/weight/time, etc.), it is more complicated than can be simply modelled with the then-available techniques for a racemate, where the enantiomers can have different pharmacokinetics, pharmacodynamics and active metabolites, as occurs with thiopentone.

Review 14. Thiopentone – a neurological and a neurosurgical analysis in patients [303] This was a ‘past, present and future’ pharmacological review assembled from the data gathered during Dennis Cordato’s PhD studies and placed into the clinical context of the prevailing philosophy, mainly of the treatment of patients with closed cranial injuries. It raised the question as to whether enantiopure thiopentone should be developed (by that stage we had decided that the less potent R-thiopentone had a greater margin of safety over the 79 clinically-used racemate or the more potent S-thiopentone), and responded that the answer remains “unknown”. That question never was answered. Even by that stage, the several pharmaceutical companies approached had declined interest in developing single enantiomer thiopentone: propofol was fast becoming the induction agent of choice, and the indication of thiopentone for the treatment of head injuries was insufficiently prominent as to be profitable.

Study 30. Acid-base changes on PD and PK of bupivacaine and thiopentone [306] As described more fully for Study 16, acid-base changes within reasonably narrow ranges can occur from physiological derangements in a clinical milieu and this may significantly affect the relative unionized and ionized concentrations of weakly acidic or basic drugs that have a pKa value close to that of the pH of blood. Both thiopentone (a weak acid pKa=7.9) and bupivacaine (a weak base pKa=8.1) are direct cardiac depressants. This study was conceived to determine whether intentionally imposed acid-base changes could affect the pharmacokinetics and cardiac effects of thiopentone and bupivacaine. This made a suitable PhD thesis topic for veterinary anaesthetist Sue Copeland who was then working as a research officer on the levobupivacaine program. At the doses used, the cardiovascular changes caused by these drugs were essentially unaffected by the acid-base derangements.

Study 31. Direct cardiac effects of thiopentone and its enantiomers in sheep [307] By the early 2000s, our lab had acquired many useful techniques that could be used to tease apart some of the physiological mechanisms by which anaesthetic agents exerted primary (or direct) cardiac effects from the secondary (or neurally mediated) effects. As part of the possible commercial development of an enantiopure thiopentone, an important test of our claims for a preferred single enantiomer of thiopentone over the racemate would need to show that it had a preferred cardiac effect profile, or at least not a worse one. I decided that we should use the close coronary arterial infusion paradigm, developed for the local anaesthetic studies, with the separate enantiomers and racemate of thiopentone, and this presented an opportunity to compare all three substances to propofol, which was, by then, becoming accepted in clinical anaesthesia (and would soon replace thiopentone). No advantage was found for either thiopentone enantiomer over the racemate, perhaps removing the most reasonable proposition for adoption of enantiopure thiopentone and, interestingly, no advantage was found for propofol over thiopentone.

Postscript 2 By the time that our experiments were completed, propofol had become the ‘new thiopentone’ as an induction agent – a combination of good pharmacology and good marketing. Indeed, thiopentone had had a good life – some 60 years – although it almost didn’t make it past the infamous but unfair ‘Pearl Harbor euthanasia’ tag. 118 Nonetheless, as its pharmacology became better known, anaesthesiologists learned how to use it for surgical patients of all kinds. Much of its pharmacology became better characterized through specific research into the pharmacokinetics and pharmacodynamics. 119 This, as pointed out elsewhere, is due to biofluid drug concentration assays being increased in specificity and sensitivity. Concurrently, numerous pharmacokinetic-pharmacodynamic studies of CNS-acting drugs have used some aspect of the EEG signal (e.g. the spectral edge of 95% power) with state-of- the-art modelling methods, to derive temporal and comparative relationships for the ‘drug concentration in the effect compartment’. Most, if not all, of such studies have ignored enantiomeric consequences of racemic drug administration. Moreover, although sophisticated

118 Bennetts FE. Thiopentone anaesthesia at Pearl Harbor. Br J Anaesth 75(3): 366-368, 1995 119 Krejcie TC, Avram MJ. What determines anesthetic induction dose? It’s the front-end kinetics, doctor!. Anesth Analg 89(3): 541, 1999 80 methods of pharmacokinetic analysis have been used to describe thiopentone kinetics, in none has the issue of enantioselectivity of even global pharmacokinetic parameters been mentioned. This thereby became the starting point for this program.

Some studies, essentially concurrent with ours, found mixed evidence for the stereoselectivity of thiopentone in several in vitro electrophysiological models. For these, the investigators used thiopentone enantiomers separated by preparative HPLC and the fractions, as judged by the chromatograms published, appeared to have had some, albeit small, residual enantiomeric contamination. 120 Overall, the evidence confirmed a greater potency of S-thiopentone at the GABA type A receptors, no difference at the neuronal α7 or muscle αβγδ nACh receptors, but a greater potency of R-thiopentone at the neuronal α4β2 nACh receptors. Interestingly, as the authors noted, this finding is diametrically opposite to their stereoselectivity for general anaesthesia. 121

Somewhat surprisingly, we were awarded a provisional patent for the “Synthesis and uses of thiopentone enantiomers” based on chiral syntheses from inexpensive, natural product, starting materials. No-one had previously perceived the need, but it was too late for developing enantiopure R-thiopentone as a new drug entity. Yes, it has a greater margin of safety and that could be particularly useful for generating barbiturate coma, but the advantages were probably marginal if used as a single dose for the induction of anaesthesia, and it still lacks the much more desirable pharmacokinetic profile of propofol.

In 2003, I gave a seminar to the Department of Anaesthetics at the Queen’s University of Belfast, the last academic home of John W Dundee (1921-1991), the great British academic anaesthetist-clinical pharmacologist who had performed very many landmark investigations on thiopentone, and was author of the first monograph on thiopentone. 122 In tribute to him, my topic was the “Pharmacology of the thiopentone enantiomers: a personal tribute to The Late Professor John W Dundee”. I had met him on one occasion only, at the British Society for Clinical Pharmacology meeting in Bradford in 1988, during my year in Sheffield, and I felt honoured that he acknowledged my work.

3.4 Nonsteroidal anti-inflammatory drug program My specific interest in the pharmacology of nonsteroidal anti-inflammatory drugs (NSAIDs) began when I was asked to contribute chapters on their pharmacology to multi-disciplinary textbooks on pain medicine [127] and anaesthesia [150] during the mid 1980s.

As is well known, NSAIDs are a chemically diverse group of drugs (salicylates, propionic acids, oxicams, etc). However, apart from an acidic nature, their chemical structure gives no clues as to the pharmacological modes of actions that they have in common. As a group, NSAIDs exert analgesic, anti-inflammatory and antipyretic effects – comprising a potentially beneficial combination of effects for pain management. These effects result from inhibition of part of the cyclooxygenase (COX) enzyme system, and this leads to their more useful functional pharmacological classification as cyclooxygenase inhibitors. This action blocks

120 Tomlin SL, Jenkins A, Lieb WR, Franks NP. Preparation of barbiturate optical isomers and their effects on GABA(A) receptors. Anesthesiology 90(6), 1714-1722, 1999 121 Downie DL, Franks NP, Lieb WR. Effects of thiopental and its optical isomers on nicotinic acetylcholine receptors. Anesthesiology 93(3): 774-783, 2000; Dickinson R, de Sousa SL, Lieb WR, Franks NP. Selective synaptic actions of thiopental and its enantiomers. Anesthesiology 96(4): 884-892, 2002 122 Dundee JW. Thiopentone and other thiobarbiturates . E & S Livingstone. Edinburgh, 1956, p312 81 one of the main oxidative pathways for arachidonic acid that is liberated from the membranes of injured cells, leading to blockade of the synthesis of new prostaglandins (PGs) and thromboxanes (TxBs), but does not affect those already synthesized. Prostaglandins are a family of chemicals that have offspring substances (such as PGE2) that can stimulate peripheral nerve endings to produce pain, as well as others that beneficially dilate blood vessels (notably PGI 2 or prostacyclin). Hence this action on PGs leads to their more accurate pharmacological description as prostaglandin synthetase inhibiting drugs or, simply, antiprostanoids. Although this is a generally accepted link, it has been called into question as the basis for their relieving ‘clinical’ pain.

Although traditionally described as ‘peripheral analgesics’, there is convincing evidence that NSAIDs exert both peripheral and central actions, and inhibit injury-induced synthesis of prostaglandins and other enzymes (e.g. of , iNOS) in producing their clinical actions. I decided to develop a research project to investigate this issue when, in 1995 anaesthetist Associate Professor Ian Power, a protégé of Walter Nimmo, was recruited to our department and shared similar interests. We attracted NHMRC and other research funds that enabled this program to go ahead and for toxicologist Yiguang Lin to be appointed as a research fellow to work on this. We later co-supervised the MSc thesis of anaesthetist Robert Grace on the central and peripheral effects of the NSAID diclofenac using the rat tail ischaemia-reperfusion model developed by pharmacologist Linda Gelgor for her PhD thesis at the University of Witwatersrand. Linda had earlier joined my group as a research fellow and had taught us the model. 123

The main focus of this Section is ketorolac. Ketorolac is synthesized and used as the racemate of (+)-(R)- and (-)-S-ketorolac, but the cyclooxygenase inhibitory action predominates in the S-enantiomer,124 a property shared with various other chiral NSAIDs such as and naproxen. What was different and clinically relevant about ketorolac was that it was introduced as a water soluble (tromethamine) salt, suitable for injection , and that it was being claimed to have an analgetic potency similar to morphine. 125 This parenteral presentation, I rationalized, may be useful in managing post-operative pain. I developed my ideas on this when I was invited to co-chair, with my long-standing academic colleague from Denmark, surgeon Henrik Kehlet, a satellite symposium on this theme on June 15 1992 at the 10 th World Congress of Anaesthesiologists meeting in the Hague [199]. A set of supportive papers and discussions filled out the one-day symposium and this was published as a supplement in the journal, Drugs .

Review 15. A potential role of NSAIDs for postoperative pain management [200] My opening address at the 1992 Hague symposium reviewed the evidence for and against the use of this class of agent in post-operative pain management, and this included aspects of their

123 Gelgor L, Phillips S, Mitchell D. Hyperalgesia following ischaemia of the rat's tail. Pain 24(2): 251- 257, 1986; Gelgor L, Phillips S, Butkow N, Mitchell D. Injectable aspirin and mepyramine abolish post-ischaemic hyperalgesia in rats. Pain 26(3): 353-359, 1986: Gelgor L, Butkow N, Mitchell D.. Effects of systemic non-steroidal anti-inflammatory drugs on nociception during tail ischaemia and on reperfusion hyperalgesia in rats. Brit J Pharmacol 105(2): 412-416, 1992; Sher GD, Cartmell SM, Gelgor L, Mitchell D. Role of N-methyl-D-aspartate and opiate receptors in nociception during and after ischaemia in rats. Pain 49(2): 241-248, 1992 124 Guzman A,Yuste F, Toscano RA, Young JM, Van Horn AR, Muchowski JM. Absolute configuration of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2-alpha]pyrrole-1-carboxylic acid, the active enantiomer of ketorolac. J Med Chem 29(4): 589-91, 1986 125 Rooks WH. The pharmacologic activity of ketorolac tromethamine. Pharmacotherapy 10(6P2): 30S- 32S, 1990; Buckley MM-T, Brogden RN. Ketorolac: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential. Drugs 39: 86-109, 1990 82 pharmacokinetics, pharmacodynamics and chirality [ 200 ]. My rationale, based on pharmacological first principles, was that NSAIDs could provide a better postoperative course both in their own right by inhibiting the local injury response and by reducing the use of opioid analgesics along with their typical side effects, hopefully allowing a faster course of rehabilitation, briefer hospital stay, and greater patient satisfaction. The principal concerns regarding their perioperative use was the risk of decreased haemostasis, delayed wound healing and renal side effects, particularly where the effects of anaesthesia and surgery may increase such risks, and particularly in elderly patients. Some of the benefits of NSAIDs derive from opioid sparing (e.g. reduction in perioperative nausea and vomiting and improvement in ventilation), but some studies were alluding to an enhanced quality of analgesia from the combination, compared with either NSAID or opioid alone. The chemistry of certain chiral NSAIDs, however, is complicated by chiral inversion occurring as a metabolic pathway, and this becomes significant since the pharmacology of such agents is highly enantioselective.

My main interest in ketorolac was as adjunctive therapy for perioperative pain management, but it is not easy to gain insight in research concept studies with patients because the patient’s clinical needs come first. Laboratory studies in experimental animals allow greater flexibility in study designs, dosage regimens and invasion for the collection of samples, etc., and are especially important in the early days of concept testing. I decided that studies would be most tractable through regional pharmacokinetic studies in our sheep preparation using the nociceptive mechanical pressure and thermal threshold models developed for sheep by Avril Waterman-Pearson and her colleagues at the Veterinary School at the University of Bristol and I went there to learn the techniques. 126 With Michael Cousins as a co-principal investigator, we attracted NHMRC funds that helped us to set up the animal analgesia [ 269 ] and renal dysfunction [ 242 ] research in our new department at the University of Sydney- RNSH. This led to a PhD project for anaesthetist Stephen (Steve) McG Barratt on ‘pre- emptive’ and ‘multimodal’ analgesia. These were, in that period, emerging concepts for improving the practice of pain management. Based on analogous pharmacological problems of chirality with other ‘profen’ NSAIDs, I knew that any further pharmacological efforts to study ketorolac would have consider the enantioselectivity of pharmacokinetics, thus requiring analytical resolution [ 216 ].

Study 32. Ketorolac enantiomers: a rapid, robust chiral assay [216] By the early 1990s, a chiral assay had been reported for ketorolac: it was a slow assay, being based on derivatization to produce diastereoisomers and these were separated on a reverse phase HPLC column. 127 We required a rapid and simple stereospecific assay for large numbers of biological samples to be set up initially for performing pharmacokinetic analysis of ketorolac in our multicannulated sheep preparation. Our assay was based on pH partitioning and liquid-liquid extraction before separation on an AGP chiral stationary phase with photodiode array detection. This was much less time consuming than the derivatization

126 Nolan A, Livingston A, Morris R, Waterman A. Techniques for comparison of thermal and mechanical nociceptive stimuli in the sheep. J Pharmacol Meth 17(1): 39-49, 1987; Nolan A, Livingston A, Waterman AE. Investigation of the antinociceptive activity of buprenorphine in sheep. Br J Pharmacol 92(3): 527-533, 1987; Waterman AE, Livingston A, Amin A. The antinociceptive activity and respiratory effects of fentanyl in sheep. Vet Anaesth Analg 17(1): 20-23, 1990; Chambers JP, Waterman A E, Livingston A. Further development of equipment to measure nociceptive thresholds in large animals. Vet Anaesth Analg 21(2): 66-72, 1994 127 Hayball PJ, Tamblyn JG, Holden Y, Wrobel J. Stereoselective analysis of ketorolac in human plasma by high-performance liquid chromatography. Chirality 5(1): 31-35, 1993 83 technique, and was more sensitive. Similar assays using chiral stationary phase resolution have subsequently been reported. 128

Study 33. Study of the renal effects of ketorolac in rats [242] Despite the clinical successes of post-operative ketorolac, we knew that the case for caution remained because NSAIDs have known risks of potentially serious renal side effects, but these had not been studied systematically in a perioperative setting. The renal complications are a direct consequence of the prostaglandin inhibition and may be exacerbated by the perioperative conditions, in particular, general anaesthesia and fluid imbalance. With collaboration from the Department of Clinical Pathology, I decided to investigate any impact of possible co-factors using an established postoperative model in (Fischer 344) rats undergoing isoflurane anaesthesia and laparotomy and with co-administration of gentamicin, a potentially nephrotoxic antibiotic used intraoperatively by many surgeons. This model was previously established by Michael Cousins for his renal toxicity studies from volatile anaesthetic agents. 129 We studied the renal effects of ketorolac, alone in low and high doses, with dehydration to represent intraoperative fluid imbalance. We found consistent changes in renal function, accompanied by severe, wide-spread histological changes of acute tubular necrosis, but only in animals co-treated with gentamicin and ketorolac, and not with ketorolac alone or gentamicin alone. Later, we changed to studying diclofenac as it had, by then, been produced in a parenteral dosage form, and had fewer experimental complications by being achiral [263, 268, 270, 271, 279, 304, 318].

Study 34. Ketorolac - multimodal analgesia study in sheep [269]. This study was conceived to test hypotheses around the concepts of pre-emptive analgesia and opioid-NSAID combinations with nociceptive threshold testing using the methodology developed at the University of Bristol in our perioperative sheep model with the combined use of ketorolac preceding fentanyl. The results, however, did not show evidence of significant pre-emptive analgesia, secondary hyperalgesia or a pharmacological interaction. Although the various individual components of the model seemed appropriate, it may be that the combined aspects of the thoracotomy surgical model and the choice of the sheep were not ideal. Moreover, concurrent pharmacokinetic studies of the ketorolac enantiomers were inexplicably inconclusive, and the project was curtailed due to lack of funding. Steve Barratt pursued the remainder of his PhD project on studies in humans [256, 293].

Postscript 3 Our studies comprised a series of pharmacokinetic studies on ketorolac in our multicannulated sheep preparation with various intravenous drug administration paradigms, performed with the objective of studying whole body and regional enantioselectivity of its pharmacokinetics. Although we produced many sets of pharmacokinetic data sets under many experimental

128 Jones DJ, Bjorksten AR. Detection of ketorolac enantiomers in human plasma using enantioselective liquid chromatography. J Chromatogr B 661(1): 165-167, 1994; Diaz-Perez MJ, Chen JC. Aubry A-F, Wainer IW. The direct determination of the enantiomers of ketorolac and parahydroxyketorolac in plasma and urine using enantioselective liquid chromatography on a human serum albumin-based chiral stationary phase. Chirality 6(4): 283-289, 1994; Campanero MA, Lopez-Ocariz A, Garcia-Quetglas E, Sadaba B, Azanza JR. Determination of ketorolac enantiomers in plasma using enantioselective liquid chromatography. Application to pharmacokinetic studies. Chromatographia 48(3-4): 203-208, 1998; Carlucci G, Carlucci M, Locatelli M. Analysis of anti-inflammatory enantiomers by HPLC in human plasma and urine: A review. Anti-Inflam Anti-Allergy Agents in Med Chem 11(1): 96-112, 2012 129 Barr GA, Mazze RI, Cousins MJ, Kosek JC. An animal model for combined and gentamicin nephrotoxicity. Br J Anaesth 45(4): 306-312, 1973 84 circumstances, our studies were never published due to our inability to draw coherent conclusions; some examples are given below. These studies were followed by a series of studies of multimodal analgesia, in which ketorolac was combined with fentanyl, and antinociception and ventilation were used as measures of relevant drug effects and side effects [269 ].

In the intravenous ketorolac studies, the drug concentration-time data were found to be adequately described by classical 2-compartmental ‘whole body’ pharmacokinetic behaviour after intravenous bolus administration. However, there was only weak evidence of enantioselectivity in the disposition of ketorolac, and only in some subjects. Next, on the basis of the pharmacokinetic model devised for each subject, a computer controlled infusion pump was used (with the Stanford University ‘Stanpump’ technique 130 ) to drive the arterial blood drug concentrations rapidly to a steady state for a preselected period. In one series, a steady state total arterial ketorolac concentration of 10 mg/l (a high-end therapeutic concentration for severe pain in humans) for 2 hours was selected, and this was achieved reasonably as shown by the data in Figure 3.14, but the resultant ketorolac enantiomer blood concentration ratios again differed inexplicably between subjects.

Figure 3.14a: Arterial plasma concentrations of the ketorolac enantiomers with a 2 hour computer controlled intravenous infusion (steady state target R- + S-ketorolac = 10 mg/L) in each of 4 sheep. It is apparent from the arterial blood concentration measurements that enantioselective disposition occurred in Subjects1, 2, and 3 but not 4. These data are inexplicably inconsistent in their enantioselectivity. [Unpublished data]

130 Shafer SL. The pharmacokinetic and pharmacodynamic basis of target controlled infusion. Available at web.stanford.edu/~sshafer/LECTURES.DIR/Notes/CCIP.DOC 85

Figure 3.14b: The regional ratio of R- to S-ketorolac plasma concentrations with the 2 hour computer controlled intravenous infusion in each of the 4 sheep shown in Figure 3.13a. It is apparent that enantioselective disposition occurred in Subjects 1, 3and 4 but not 2. These data are inexplicably inconsistent in their enantioselectivity. [Unpublished data]

Figure 3.14c: Arterial and regional plasma concentrations of the ketorolac enantiomers with a 2 hour computer controlled intravenous infusion (steady state target R- + S-ketorolac = 10 mg/L) in Subject 1 of Figure 3.13a. There is significant extraction by the kidneys, and some by the liver. It is apparent from pulmonary arterial blood concentration measurements that ketorolac is washing out of the lungs, but the similarity for R- and S- ketorolac suggests that enantioselective disposition was unlikely to have occurred in the lungs. [Unpublished data]

In another series programmed to achieve higher concentrations and to determine the regional pharmacokinetics, the blood concentrations of both enantiomers continued to increase after cessation of the infusion, but showed no clear evidence of enantioselective extraction by liver, 86 kidneys or lungs, possibly due to non-linearity and/or nett metabolic inversion. The results were confusing and we decided that any future systematic studies would need administration of the separate enantiomers. As we did not have them in sufficient quantities and, given our limited resources, we decided to abandon the ketorolac part of the program. Nevertheless, it was an instructive series of studies to be performed whilst setting up our new academic department in the early 1990s. It pulled together the components of a fully functioning ‘sheep lab’ with surgical, maintenance and experimental facilities, as well as an analytical chemistry lab. However, we did not abandon work on NSAIDs, then rapidly emerging as the important perioperative that they are today. The ketorolac work led directly to more tractable studies on diclofenac in which we performed a range of studies on its mode(s) and sites of action, its renal side effects, and its biodisposition in relation to its actions and sites of action. Although forming part of the nonsteroidal anti-inflammatory drug program, the diclofenac studies [263, 270, 271, 279, 304], being achiral pharmacology, are not included in this thesis.

In 2002, Colin Duke and I took a new chemical-pharmacological approach when we submitted a grant application to examine the central actions of NSAIDs based on diclofenac as a model. We proposed preparing various lipophilic ester derivatives of diclofenac that would act as ‘pro-drugs’ and have facile access to the central nervous system, but would be hydrolysed by local and/or plasma esterases. We rationalized that this approach, supported by biofluid measurements, would allow study of central and peripheral effects contributing to their analgesic actions. Unfortunately, our proposal was not funded, and later that year, my colleagues in this program accepted positions elsewhere. Ian Power went to the Chair of Anaesthesia at the University of Edinburgh, Steve Edwards went to a research fellowship at the University of Queensland, and Yiguang Lin went to a senior lectureship at the University of Technology, Sydney. Our ‘critical mass’ of nonsteroidal anti-inflammatory drug expertise was thus lost, and I decided to not pursue further funding for this area.

However, ketorolac unexpectedly returned to my lab in 2004 when I was asked to collaborate on a clinical project with anaesthetist Dennis Kerr and orthopaedic surgeon Lawrence Kohan. They wanted to test the analgetic usefulness of operative site infiltration of admixed ketorolac, ropivacaine and adrenaline in patients undergoing a hip or total knee replacement, as well as measure the circulating concentrations to assess the systemic absorption. Sonia Gu made the analytical measurements and we found marked enantioselectivity in ketorolac blood concentrations, with the circulating blood concentrations to be less than those associated with pain relief (Figure 3.15). Dennis published the clinical aspects of the studies after I had retired, unfortunately, without my input and without any mention of the pharmacokinetics. 131 The ‘Kerr-Kohan’ technique is now widely used, and is recognized as providing a useful alternative to systemic analgesics for patients with moderate to severe pain. 132 These days, ketorolac is used in newer and non-invasive dosage forms, including controlled-release oral, intranasal and transdermal forms for systemic treatments, as well as newer forms such as intraocular for local treatments. 133

131 Kerr DR, Kohan L. Local infiltration analgesia: a technique for the control of acute postoperative pain following knee and hip surgery: a case study of 325 patients. Acta Orthopaedica 79 (2): 174- 183, 2008; Nizam I, Kohan L, Kerr D. Do non-steroidal anti-inflammatory drugs cause endo- prosthetic loosening?. J Bone Joint Surg 91(Supp III): 473-473, 2009 132 Gillis JC, Brogden RN. Ketorolac. A reappraisal of its pharmacodynamic and pharmacokinetic properties and therapeutic use in pain medicine. Drugs ; 53: 139-188, 1997; White PF, Raeder J, Kehlet H. Ketorolac: Its role as part of a multimodal analgesic regimen. Anesth Analg ; 114: 250- 254, 2012 133 Sinha VR, Kumar RV, Singh G. Ketorolac tromethamine formulations: an overview. Expert Opin. 87

Ketorolac: total knee replacement #2 Ketorolac: hip relacement #3 1.0 0.5 R-ketorolac 0.8 S-ketorolac 0.4

0.6 0.3

0.4 0.2 Conc. (mg/L) Conc. Conc. (mg/L) Conc. 0.2 0.1 R-ketorolac S-ketorolac 0.0 0.0 0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240 Time (min) Time (min)

Figure 3.15: Plasma concentrations of ketorolac enantiomers in two patients after operative site infiltration - one having a total knee replacement (left) and another having a Birmingham hip replacement (right). These cases have been chosen to represent the series of results. Both show marked enantioselectivity in ketorolac blood concentrations. (Unpublished data)

3.5 Halothane project Halothane, the prototypic ‘second generation’ volatile anesthetic, was welcomed into clinical anaesthesia during the 1950s as a non-flammable, reasonably potent, inhalational general anaesthetic agent, and it quickly became the most widely used volatile anaesthetic agent. Progressively from the mid 1980s, however, the use of halothane declined due to hepatotoxicity which derives, at least in part, from metabolic intoxication. Halothane was progressively replaced by and isoflurane which are much less metabolized, and later by and sevoflurane which are negligibly metabolized. By the early 1990s, when my application for the Douglas Joseph Professorship was being prepared, halothane already was largely obsolescent for human anaesthesia, although its relatively lower price compared to the newer volatile anaesthetics kept it in use for veterinary anaesthesia. Among others, Michael Cousins has done much to document the metabolism of halothane and its toxicological consequences. 134 It is estimated that up to 20% of patients exposed to halothane develop a mild form of hepatotoxicity: a small subset of these will develop hepatitis. 135

The physicochemical properties of volatile anaesthetic agents, especially the oil:water, oil:gas and blood:gas distribution coefficients, are their most prominent features and these determine both their anaesthetic potency and their pharmacokinetics. These properties show structure- action correlations among subsets, e.g. halocarbons and halogenated ethers, with potency, where potency is typically given as the MAC. 136 Although it is largely overlooked even in the most revered and innovative of pharmacokinetic models, 137 halothane is synthesized and used

Drug Deliv 6(9): 961-975, 2009 134 Plummer JL, Cousins MJ, Hall P. Volatile anaesthetic metabolism and acute toxicity. Drug Metab Drug Interact 4 (1): 49-98, 1982 135 Gut J, Christen U, Huwyler J. Mechanisms of halothane toxicity: novel insights. Pharmacol Ther 58: 133-155, 1993 136 MAC = Minimum Alveolar Concentration, measured at 1 atm pressure, at steady state thereby allowing for tissue: blood equilibration, preventing movement in 50% of the subjects to a supramaximal surgical incision (it is therefore actually a ‘Median’ concentration). Eger EI, Saidman LJ, Brandstater B. Minimum alveolar anesthetic concentration: a standard of anesthetic potency. Anesthesiology 26 (6): 756-763, 1965; Quasha AL, Eger EI, Tinker JH. Determination and applications of MAC. Anesthesiology 53 (4): 315-334, 1980 137 Some examples: Zwart A, Smith TN, Beneken JE. Multiple model approach to uptake and distribution of halothane: the use of an analog computer. Comp Biomed Res 5(3): 228-238, 1972; Allott PR, Steward A, Mapleson WW. Pharmacokinetics of halothane in the dog. Br J Anaesth 88 as a racemate of (+)-R and (-)-S-halothane enantiomers. Indeed, its successors, enflurane, isoflurane and desflurane (but not sevoflurane) are also racemates, and this too is generally overlooked. I am grateful to chemist Jerrold Meinwald of Cornell University, New York, for his insight and dialogue some 25 years ago that helped to shape my thinking about this issue. 138 The possibility of its metabolic enantioselectivity 139 being involved in its induction of hepatitis was an intriguing question and so halothane was included in the research plan, more as an intellectual exercise to gauge whether a knowledge of stereopharmacology might have helped to save halothane from an early retirement.

During the early 1990s, several papers reported that isoflurane potency was stereoselective in several models, 140 but both enantiomers were found to exert similarly depressant effects on the isolated guinea pig myocardium. 141 In one study of halothane, no difference in potency of the enantiomers was found in an in vitro model synaptic transmission. 142 In other models, differences in potency were found between the enantiomers of halothane. 143 The debate about traditional structural non-specificity vs . structural specificity of action of these agents thus moved in favour of a chiral interaction with neural ion channel receptor proteins. One pathway of the metabolism of halothane has been found to be structurally specific. 144 Halothane is metabolized by oxidation, reduction and some glutathione conjugation, and it is unclear whether oxidative or reductive metabolism of halothane is associated with hepatic injury as the end-product metabolites have not been found to reproduce the halothane hepatotoxicity. It is believed that the mild changes in liver function after halothane anaesthesia are due to toxic intermediates formed by the reductive pathway, while severe and often fatal liver injury results from an immune response to antigenically altered hepatocytes. It is thought possible that the hapten provoking the immune response is a product of the oxidative metabolism of halothane.

While an extensive bibliography exists on the pharmacodynamics (potency) and pharmacokinetics/metabolism (induction and emergence) of halothane, attention has never

48(4): 279-296, 1976; Carpenter RL, Eger EI, Johnson BH, Unadkat JD, Sheiner LB. Pharmacokinetics of inhaled anesthetics in humans: measurements during and after the simultaneous administration of enflurane, halothane, isoflurane, methoxyflurane, and nitrous oxide. Anesth Analg 65(6): 575-582, 1986 138 Meinwald J, Thompson WR, Pearson DL, Konig WA, Runge T, Francke W. Inhalational anesthetics stereochemistry: optical resolution of halothane, enflurane, and isoflurane. Science 251(4993): 560-561, 1991 139 Martin JL, Meinwald J, Radford P, Liu Z, Graf MLM, Pohl LR. Stereoselective metabolism of halothane enantiomers to trifluoroacetylated liver proteins. Drug Metab Rev 27 (1-2): 179-189, 1995 140 Moody EJ, Harris BD, Skolnick P. Stereospecific actions of the inhalation anaesthetic isoflurane at the GABA A receptor complex. Brain Res 1993; 615: 101-6; Franks NP, Lieb WR. Stereospecific of inhalational general anesthetic optical isomers on nerve ion channels. Science 254: 427-430, 1991; Moody EJ, Harris BD, Skolnick P. Stereospecific actions of the inhalation anaesthetic isoflurane at the GABA A receptor complex. Brain Res 615: 101-106, 1993; Lysco GS, Robinson JL, Castro R, Ferrone RA. The stereospecific effects of isoflurane isomers in vivo. Eur J Pharmacol 263: 25-29, 1994 141 Graf BM, Boban M, Stowe DF, Kampine JP, Bosnjak ZJ. Lack of stereospecific effects of isoflurane and desflurane isomers in isolated guinea pig hearts. Anesthesiology 81: 129-136, 1994 142 Kendig JJ, Trudell JR, Cohen EN: Halothane stereoisomers: Lack of stereospecificity in two model systems. Anesthesiology 39: 518-524, 1973 143 Sedensky MM, Cascorbi HF, Meinwald J, Radford P, Morgan PG. Genetic differences affecting the potency of stereoisomers of halothane. Proc Nat Acad Sci 91(21): 10054-10058, 1994; Harris BD, Moody JE, Skolnick, P. Stereoselective actions of halothane at GABA A receptors. Eur J Pharmacol 341(2): 349-352, 1998 144 Martin JL, Meinwald J, Radford P, Liu Z, Graf MLM, Pohl LR. Stereoselective metabolism of halothane enantiomers to trifluoroacetylated liver proteins. Drug Metab Rev 27(1-2): 179-189,1995 89 been directed towards putting the issues together and investigating the metabolic- toxicological consequences of its enantiomeric duality. Thus the long-term experimental aims of this project were to investigate whether halothane-induced liver injury could be related to differential metabolism of the halothane enantiomers.

Study 35. Halothane enantioselectivity pharmacokinetics in rats [267] The initial study consisted of development of methodology for quantitative exposure, along with a GC-MS method to separate and quantitate the enantiomers from administration of rac - halothane to rats. This was achieved using the rate of change of head-space halothane enantiomer consumption during halothane anaesthesia (Figure 3.16). This pharmacokinetic study found no significant enantiomeric difference in the rate of halothane consumption using this ‘finite dose’ model, thus strongly indicating that there was no significant enantioselectivity in the respective rates of distribution and elimination. It may be that the whole body approach was too insensitive to reveal enantioselectivity in the regional pharmacokinetics of the gut, liver and kidneys, where it particularly matters.

Figure 3.16: Experimental setup for exposing rats to monitored concentrations of halothane. Left: exposure chamber, Centre: gas scrubbing chambers, Right: gas monitoring equipment.

Postscript 4 Halothane, the prototypic non-flammable ‘second generation’ inhalation anaesthetic agent designed from first principles for an ideal anaesthetic agent, rapidly became the ‘gold standard’ in Western anaesthesia, but is no longer so because it is metabolized in part to potentially toxic substances - a finding that was not envisaged until many years after its introduction. The introduction of the newer, less metabolized, volatile anaesthetic agents was based on the toxicological lessons learned from halothane.

This project arose from a curiosity-driven idea – to investigate whether there were sufficient enantioselective pharmacokinetic differences to indicate a preference that might have saved an enantiopure halothane. These pharmacokinetic differences were not found from the first study using racemic halothane and any further progress would have required synthesis and administration of the separate halothane enantiomers, along with quantitation of the metabolites produced. It was intended that the relevant enantiomers of halothane would be synthesized using novel and reported methods; however, the budget could not support this part of the work, and so the more prominent thiopentone work was prioritized. Thus, the importance of chirality, to the question of the relationship of metabolic activation of halothane to a toxicological outcome, was not able to be determined. The issue of stereopharmacology 90 remains in isoflurane and desflurane, and any future pharmacological complications due to their enantiomeric duality remain to be determined.

3.6 Ketamine project The fascinating history and unusual pharmacology of ketamine were recently described in a retrospective written by anaesthesiologist Ed Domino, the researcher attributed with performing the first studies of its anaesthetic pharmacology in ‘prisoner volunteers’ in 1964. 145 Ketamine has been in widespread clinical use as an injectable anaesthetic agent since the 1960s. 146 It was soon adopted as an anaesthetic agent in many animal laboratories 147 and, indeed, I used it as the primary anaesthetic agent in my primate lab studies at the University of Washington in the mid-1970s but, back then, like most others, I never thought about its chirality.

Ketamine is formally classified as a ‘’ anaesthetic agent, i.e., it is characterized by analgesia and changes to vigilance and perception, rather than hypnosis, with a ‘trance-like’ state. It also has anticonvulsant and psychotomimetic properties, the latter reflecting its genesis in a quest to find a substitute, without the severe adverse hallucinogenic side effects upon re-awakening found in its progenitors. Ketamine is generally considered to have a wide margin of safety in clinical use. 148

The pharmacological effects of ketamine are primarily mediated by its noncompetitive antagonism of the NMDA receptor, but ketamine also interacts with opioid, cholinergic, and monoaminergic receptors. 149 Clinically relevant doses of ketamine are associated with only minimal respiratory depressant effects, but they may produce significant increases in blood pressure and heart rate. The cardiovascular effects of ketamine are due to sympathetic stimulation, although it produces a direct depressant effect on the myocardium, consistent with local anaesthetic-like sodium channel blockade. The general clinical use of ketamine has tended to be limited mainly to induction of anaesthesia in haemodynamically unstable patients, sedation in the intensive care setting, and in short painful procedures such as changes of dressings in patients with burn injuries. However, over the past decade or so, there has been increasing use of its analgetic properties in patients having neuropathic pain. Overall, the benefits of ketamine derive from its dual anaesthetic and analgetic actions, but its therapeutic efficacy is somewhat limited by psychotomimetic emergence reactions and cardiovascular stimulating effects. 150

Standard pharmaceutical preparations for general clinical use contain racemic ketamine composed of the enantiomer pair (-)-R- and (+)-S-ketamine. I first became aware of the differential pharmacokinetics and pharmacodynamics (qEEG effects) of the ketamine

145 Domino EF. Taming the ketamine tiger. Anesthesiology 113(3): 678-684, 2010 146 Brown TCK, Cole WHJ, Murray GH. Ketamine: A new anaesthetic agent. Aust NZ J Surg 39(3): 305-310, 1970; Dundee JW, Bovill J, Knox JWD, Clarke RSJ, Black GW, Love SHS, Coppel DL. Ketamine as an induction agent in anaesthetics. Lancet 295(7661): 1370-1371, 1970 147 Bree MM, Feller I, Corssen G. Safety and tolerance of repeated anesthesia with CI 581 (ketamine) in monkeys. Anesth Analg 46(5): 596-600, 1967; Youth RA, Simmerman SJ, Newell R, King RA. Ketamine anesthesia for rats. Physiol Behav 10(3): 633-636, 1973 148 Green SM, Clem KJ, Rothrock SG. Ketamine safety profile in the developing world: survey of practitioners. Acad Emergency Med 3(6): 598-604, 1996 149 White PF, Way WL, Trevor AJ. Ketamine - its pharmacology and therapeutic uses. Anesthesiology 1982;56:119-136. 150 Kohrs R, Durieux ME. Ketamine: teaching an old drug new tricks. Anesth Analg 87(5): 1186-1193, 1998; Hocking G, Cousins MJ. Ketamine in chronic pain management: an evidence-based review. Anesth Analg 97(6): 1730-1739, 2003 91 enantiomers in discussions with anaesthesiologists Paul F White and Steven L Shafer during various visits to Stanford during the 1980s. 151 I also became aware of the growing use of ketamine in the treatment of chronic pain, as well as the possibilities for the use of enantiopure S-ketamine for greater selectivity, whilst I was participating in conferences in Germany. 152 In various human and animal studies, the analgesic and potency of enantiopure S-ketamine has been found to be ~1.5- to 4-fold greater than that of R-ketamine. Additionally, in clinical studies the recovery phase following administration of S-ketamine is shorter in comparison to racemic ketamine. Based upon such evidence, enantiopure S- ketamine was developed as a product that has now been approved for clinical use in Europe. 153

Our stereopharmacological studies of ketamine were conceived as a curiosity-driven part-time PhD project for neurochemist Steve Edwards, whom I had appointed as a research officer for the thiopentone program. We planned to examine the pharmacokinetics of the ketamine enantiomers in rats in association with two effect models: a carrageenan-induced hindpaw inflammation with spinal and/or peripheral NMDA receptors mediating the anticipated antihyperalgesic and anti-inflammatory effects, and the Stanford qEEG rat model. In a different rat model, others had previously described significant differences in the neuropharmacological profiles of the individual enantiomers as showing a clear therapeutic index advantage for S-ketamine, for example, the ED50 values for hypnosis in rats produced by intravenous R- and S-ketamine were found to be 10.3 and 3.5 mg/Kg, respectively, and the LD 50 values 41.5 and 35 mg/Kg, respectively. However, plasma and brain ketamine concentrations were found to be equivalent following i.v. administration of 30 mg/Kg doses of racemic ketamine or the individual ketamine enantiomers, 154 suggesting a similarity of brain pharmacokinetics and that the differences in their behavioural effects may be more a function of pharmacodynamic factors and/or ketamine metabolite activity. In studies of NMDA receptor binding affinity, it had been shown that that of S-ketamine was several times greater than that of R-ketamine. 155

Norketamine is the principal metabolite of ketamine. Work with human liver microsomes indicated that the rate of N-demethylation was greater with S-ketamine than with rac - ketamine or R-ketamine 156 and a clinical investigation indicated that norketamine also had analgetic properties. 157 A study of NMDA receptor binding affinities in rat cortical membranes found the comparative K i values for S- and R-ketamine to be 0.3 µM and 1.4 µM

151 White PF, Ham J, Way WL, Trevor A. Pharmacology of ketamine isomers in surgical patients. Anesthesiology 52(3): 231-239, 1980; White PF, Schüttler J, Shafer A, Stanski DR, Horai Y, Trevor AJ. Comparative pharmacology of the ketamine isomers: Studies in volunteers. Br J Anaesth 57(2): 197-203, 1985; Schüttler J, Stanski DR, White PF, Trevor AJ, Horai Y, Verotta D, Sheiner LB. Pharmacodynamic modeling of the EEG effects of ketamine and its enantiomers in man. J Pharmacokin Biopharm 15(3): 241-253, 1987 152 Klepstad P, Maurset A, Moberg ER, Oye I. Evidence of a role for NMDA receptors in pain perception. Eur J Pharmacol 187(3): 513-518, 1990. 153 Way WL. Ketamine-its pharmacology and therapeutic uses. Anesthesiology 56(2): 119-136, 1982. 154 Marietta MP, Way WL, Castagnoli N, Trevor AJ. On the pharmacology of the ketamine enantiomorphs in the rat. J Pharmacol Exp Ther 202: 157-165, 1977 155 Ulrich Zeilhofer H, Swandulla D, Geisslinger G, Brune K. Differential effects of ketamine enantiomers on NMDA receptor currents in cultured neurons. Eur J Pharmacol 213(1): 155-158, 1992; Oye I, Paulsen O, Maurset A. Effects of ketamine on sensory perception: evidence for a role of N-methyl-D-aspartate receptors. J Pharmacol Exp Ther 260(3): 1209-1213, 1992 156 Karasch ED, Labroo R. Metabolism of ketamine stereoisomers by human liver microsomes. Anesthesiology 77: 1201–1207, 1992 157 Grant IS, Nimmo WS, and Clements JA. Pharmacokinetics and analgesic effects of i.m. and oral ketamine. Br J Anaesth 53: 805–810, 1981 92 respectively, and 1.7 µM and 13 µM respectively for S- and R-norketamine. 158 Thus, we rationalized that the activity of the N-desmethyl metabolites, R- and S-norketamine, would need also to be considered in any model. 159 We planned that future experiments using comparative target-controlled norketamine enantiomer infusions would also need to be performed.

Our studies on ketamine were planned to add methodology in stages and to build on prior results. Unfortunately, research funds dwindled before completion of the planned studies, and a grant application, to complete the ketamine research plan and to test the generality by including other NMDA antagonists, was unsuccessful. Steve Edwards then moved to the University of Queensland to work with my colleague pharmaceutical scientist Maree Smith, completing his remaining PhD project work there, with Maree acting as an associate supervisor. The four studies on ketamine are reported here as a set, although only two of them involve the ketamine enantiomers.

Study 36. Ketamine pharmacokinetics and metabolism in rats [289] This was conceived as an exploratory pharmacokinetic study of the disposition of the ketamine enantiomers after administration of the racemate in rats. It required development of an enantiospecific assay, and this was followed by studies with two complementary paradigms: (i) a constant rate ‘washin’ infusion until fatal, (ii) a brief infusion followed by ‘washout’. These, respectively, allowed examination of ketamine and norketamine enantiomer serial plasma concentrations and tissue distribution at maximal and minimal drug effects (Figure 3.17). Both paradigms found enantioselectivity in the plasma concentrations and tissue distribution coefficients. Comparison of distribution coefficients of ketamine and norketamine enantiomers for the two paradigms provided indirect evidence for metabolic inversion. Different profiles for the tissue uptake and metabolism of ketamine enantiomers were apparent with the ‘washin’ and ‘washout’ paradigms, and this pharmacokinetic complexity plainly could have pharmacodynamic implications, should the norketamine metabolites be found to have significant analgetic activity, as later they were. 160 1.25 2.5

2.0 1.2 1.5 1.15 1.0 1.1 0.5

Ratio of R- to S-ketamine of R- Ratio 1.05 0.0

0 5 10 15 20 25 30 to S-norketamine of R- Ratio 0 5 10 15 20 25 30 Time (min) Time (min)

Figure 3.17: Strong evidence of ketamine enantioselective disposition was found in rats (n=8 individual plasma concentration data sets shown) infused with racemic ketamine until fatal. Left: Ketamine; Right: norketamine.

158 Ebert B, Mikkelsen S, Thorkildsen C, Borgbjerg FM. Norketamine, the main metabolite of ketamine, is a non-competitive NMDA receptor antagonist in the rat cortex and spinal cord. Eur J Pharmacol 333: 99-104, 1997 159 Leung LY, Baillie TA. Comparative pharmacology in the rat of ketamine and its two principal metabolites, norketamine and (Z)-6-hydroxynorketamine. J Med Chem 29(11): 2396-2399, 1986, Leung LY, Baillie TA. Studies on the biotransformation of ketamine. II—quantitative significance of the N ‐demethylation pathway in rats in vivo determined by a novel stable isotope technique. Biomed Env Mass Spect 18(6): 401-404, 1989 160 Herd DW, Anderson BJ, Holford NH. Modeling the norketamine metabolite in children and the implications for analgesia. Pediatr Anesth 17(9): 831-840, 2007 93

Study 37. Pharmacokinetics of ketamine in rats –interaction with alfentanil [296] This study was conceived both as a further stage in the development of the analytical methodology, and to gain data about the biodisposition of ketamine and norketamine. Previous studies had found that the EEG spectral changes were qualitatively the same for racemic ketamine and S-ketamine, but that S-ketamine was twice as potent as racemic ketamine in patients co-medicated with . 161 This study attempted to examine the pharmacokinetic effects of adding alfentanil, an opioid agonist, to the system. The rationale for using alfentanil in place of midazolam lay in the possible future clinical application of the combination of these agents for total intravenous anaesthesia (TIVA). In the presence of alfentanil, the mean plasma total ketamine concentration-time area under the curve was significantly lower, while tissue:plasma distribution coefficients for ketamine in the presence of alfentanil were significantly higher in forebrain, hindbrain, gut, and fat. This relatively simple study indicated that the distribution of ketamine into the brain was increased in the presence of low plasma concentrations of alfentanil, and this could have important clinical applications for TIVA and/or pain management.

Study 38. Potentiation of ketamine anaesthetic potency in rats by alfentanil [300] The next study required addition of the Stanford qEEG methodology to the pharmacokinetic and tissue distribution studies of ketamine. It was designed into the present series to evaluate the possible significance of the altered distribution into the CNS of ketamine in the presence of alfentanil. It was found that, compared to ketamine alone, alfentanil significantly increased both the duration of anaesthesia and the processed EEG power. Correspondingly, the plasma ketamine concentration producing half-maximal EEG effect was significantly reduced in the presence of alfentanil. The results indicate that low plasma alfentanil concentrations potentiate the anaesthetic and EEG effects produced by ketamine, again having positive implications for the use of these agents for TIVA or pain management.

Study 39. Stereoselective potentiation of potency of ketamine in rats by alfentanil [320] In this study, a comparison of the effects of racemic and S-ketamine were performed in an inflammatory antinociceptive rat model used in Maree Smith’s lab. This is based on the intraplantar injection of Freund’s Complete Adjuvant (FCA) which induces a persistent localized inflammatory state associated with thermal hyperalgesia and increased excitability of spinal cord dorsal horn neurons. Antinociceptive effects are determined behaviourally, by the threshold pressure to paw withdrawal upon application of graduated von Frey hairs (PWT). This study investigated the effects of rac - and S-ketamine alone, and then after administration of alfentanil, on inflammatory hyperalgesia in rats, 5 days after the intraplantar injection into one hind paw. The PWTs were significantly increased for both inflamed and non-inflamed hind paws by both rac - and S-ketamine, as well as alfentanil, but neither rac - nor S-ketamine produced further increases in threshold when the agents were combined. The findings complemented previous studies in which non-competitive NMDA receptor antagonists suppressed behavioural hyperalgesia. The study adds to the body of information that factors such as time-course, frequency and the mode of administration, in addition to the type of antinociceptive model (i.e., inflammatory compared with acute) and the nociceptive testing procedure (i.e., noxious mechanical compared with low threshold stimuli), are significant variables in revealing the pharmacological properties of agents used for pain management.

161 Hering W, Geisslinger G, Kamp HD, Dinkel M, Tschaikowsky K, Rugheimer E, Brune K. Changes in the EEG power spectrum after midazolam anaesthesia combined with racemic or S-(+)- ketamine. Acta Anaesthesiol Scand 38: 719-723, 1994 94

Postscript 5 This project began with the development of a stereospecific assay for a study of the pharmacokinetics of ketamine in rats, as a preliminary to its subsequent studies with qEEG measurements in a model for TIVA. Subsequent studies were to introduce alfentanil as another component of TIVA. 162 However, the pharmacokinetics of ketamine turned out to be complex, possibly due to metabolic inversion of R- to S-ketamine but this was not able to be tested as we did not have sufficient amounts of the separate enantiomers. We rationalized that if this was demonstrated in accord with the time course of antinociception, it could account for some of the apparent pharmacological preference for S-ketamine over the racemate, i.e., concentration of the more active enantiomer. However, equivocal differences were obtained when this was tested in the FCA rat models; thus, the remaining work on administration of the enantiomeric desmethyl (norketamine) metabolites in this model would have been needed, for a further stage, to help resolve this intriguing pharmacological problem. Taken together, the studies provided additional support for the use of pharmaceutical preparations of enantiopure S-ketamine over racemic ketamine, and they provided evidence for a pharmacodynamic interaction between ketamine and the opioid agonist, alfentanil, presumably mediated via noncompetitive antagonism of the NMDA receptor by ketamine. Nevertheless, additional pharmacokinetic factors involving the local distribution of drugs into injured tissues may also possibly contribute to the effects of NMDA receptor antagonists on opioid-induced antinociception – and this remains to be studied.

After the conclusion of our project, an exposition of ‘modern anaesthetics’ reviewed both the experimental and clinical stereopharmacology of ketamine and its enantiomers.163 Where the chirality of ketamine had been considered in receptor experimental paradigms, several-fold differences in binding affinity were found for NMDA (S > R), µ-opioid (S > R), κ-opioid (S > R), σ-opioid (R > S), but nACh (S = R). These differences were generally reflected in the associated clinical effects.

Currently, at the clinical level, ketamine is accepted as well tolerated, and have been found useful to ameliorate any psychotropic side effects. 164 The original anaesthetically and analgetically preferred enantiomer S-ketamine (also referred to as esketamine) is now also used clinically mainly in Europe, 165 but not in Australia. After further behavioural pharmacological studies in laboratory animals, R-ketamine has been found a potent and safe antidepressant and, relative to S-ketamine, surprisingly appeared to be free of psychotomimetic side effects. 166 R-ketamine, as of July 2014, is in phase II clinical trials for treatment-resistant depression. 167 Others have additionally shown the desmethyl

162 Schüttler J, Kloos S, Schwilden H, Stoeckel H. Total intravenous anaesthesia with propofol and alfentanil by computer-assisted infusion. Anaesthesia 43(s1): 2-7, 1988 163 Sinner B, Graf BM. Ketamine. In: Schüttler J, Schwilden H.. Modern anesthetics (Vol. 182). Springer. pp 313-333, 2008 164 Niesters M, Martini C, Dahan A. Ketamine for chronic pain: risks and benefits. Br J Clin Pharmacol 77 (2): 357-367, 2014 165 Marland S, Ellerton J, Andolfatto G, Strapazzon G, Thomassen O, Brandner B, Paal P. Ketamine: use in anesthesia. CNS Neurosci Ther 19 (6): 381-389, 2013; Zhang JC, Li SX, Hashimoto K. R(−)- ketamine shows greater potency and longer lasting antidepressant effects than S(+)-ketamine. Pharmacol Biochem Behav 116 : 137-141, 2014 166 Zhang JC, Li SX, Hashimoto K. R(−)-ketamine shows greater potency and longer lasting antidepressant effects than S(+)-ketamine. Pharmacol Biochem Behav 116 : 137-141, 2014 167 Wijesinghe R. Emerging therapies for treatment resistant depression. Ment Health Clin 4(5):89, 2014 95

(norketamine) metabolites are active in rat pain models,168 but the evidence in human pain models appear contradictory. 169

3.7 Thalidomide project Thalidomide is a originally prepared in 1954 by Chemie Grünenthal, a small pharmaceutical company in the former West Germany. In clinical trials it was found to promote deep, all-night, sleep, and soon became known for its remarkably high therapeutic index – it was not toxic to adult humans in any reasonable dose. It thus provided an apparently suitable replacement for the barbiturate ‘sleeping pills’ widely used at the time, and it was promoted from about 1957 for use in pregnant women with morning sickness. By 1960, in Germany, a rare birth defect, phocomelia, was being observed far more frequently than ever. In 1961, Sydney obstetrician William G (Bill) McBride delivered three babies with phocomelia and he realised that the only common factor was that their mothers had taken thalidomide during early pregnancy. 170 McBride, among others, contacted Grünenthal with suspicions that thalidomide might be the cause of these birth defects, and this was accepted. Thalidomide was withdrawn by the end of 1961 - and became infamous!

The rehabilitation of thalidomide began in the 1990s, mainly through the work of Folkman and D’Amato in Boston. 171 They correctly advanced the notion that the teratogenic effects of thalidomide were due to antiangiogenesis, and that antiangiogenesis would be useful in cancer therapy. 172 Thalidomide has useful immunomodulating and anti-inflammatory actions as well, and is now being used in pharmacotherapy of multiple myeloma and acute cutaneous manifestations of leprosy, with possible uses in the treatment of neuropathic pain, 173 but it is its use as an ingredient of cancer pharmacotherapy 174 that is presented in this thesis.

In 2002, I was invited by oncologist Fran Boyle to co-supervise a PhD project for biologist Sue Murphy involving thalidomide, a new adjunctive treatment that is now considered potentially useful as part of multimodal chemotherapy of brain tumours. At that time, the oncology group had previously used thalidomide empirically in the treatment of glioblastoma multiformae (GBM), a rare but devastating brain tumour with poor treatment prospects, and had found partial responses in some patients. 175 Sue was using models in rats with transplanted tumours for assessing the effectiveness of chemotherapeutic substances in

168 Shimoyama M, Shimoyama N, Gorman AL, Elliott KJ, Inturrisi CE. Oral ketamine is antinociceptive in the rat formalin test: role of the metabolite, norketamine. Pain 81 (1): 85-93, 1999; Holtman Jr JR, Crooks PA, Johnson-Hardy JK, Hojomat M, Kleven M, Wala EP. Effects of norketamine enantiomers in rodent models of persistent pain. Pharmacol Biochem Behav 90 (4): 676-685, 2008 169 Olofsen E, Noppers I, Niesters M, Kharasch E, Aarts L, Sarton E, Dahan A. Estimation of the contribution of norketamine to ketamine-induced acute pain relief and neurocognitive impairment in healthy volunteers . Anesthesiology 117 (2): 353, 2012; Herd DW, Anderson BJ, Holford NH. Modeling the norketamine metabolite in children and the implications for analgesia. Ped Anesth 17(9): 831-840, 2007 170 McBride WG. Thalidomide and congenital abnormalities. Lancet 278 (7216): 1358, 1961 171 http://www.chemsoc.org/chembytes/ezine/2001/stephens_nov01.htm 172 D'Amato RJ, Loughnan MS, Flynn E, Folkman J. Thalidomide is an inhibitor of angiogenesis. Proc Nat Acadf Sci 91 (9): 4082-4085, 1994 173 Andrade P, Visser-Vandewalle V, Del Rosario JS, Daemen MA, Buurman WA, Steinbusch HW, Hoogland G. The thalidomide analgesic effect is associated with differential TNF-α receptor expression in the dorsal horn of the spinal cord as studied in a rat model of neuropathic pain. Brain Res 1450: 24-32, 2012 174 Fanelli M, Sarmiento R, Gattuso D, Carillio G, Capaccetti B, Vacca A, Gasparini G. Thalidomide: a new anticancer drug?. Expert Opin Invest Drugs 12 (7): 1211-1225, 2003 175 Marx GM, Pavlakis N, McCowatt S, Boyle FM, Levi JA, Bell DR, Wheeler HR. Phase II study of thalidomide in the treatment of recurrent glioblastoma multiforme. J Neuro-oncol 54 (1): 31-38, 2001 96 inhibiting their growth. My role was to facilitate chemical methodology for the chiral analysis of thalidomide and to supervise any emergent pharmacokinetic and pharmacodynamic aspects.

Thalidomide is prepared as a racemate, consisting of the single pair of the (+)-(R)- and (−)- (S)-thalidomide enantiomers. The enantiomers have different pharmacological effects: R- thalidomide has sedative properties, and S-thalidomide has immunomodulatory and teratogenic properties. However, there is good evidence that both enantiomers will rapidly racemize at physiological pH in vivo , thereby negating any potential advantage of using enantiopure thalidomide for seeking greater pharmacological selectivity. 176 Because the enantiomers have the potential for producing qualitatively different effects, as well as having quantitatively different pharmacokinetics, it was considered important to quantify their relative concentrations in relevant tissues, and to determine whether interactions occur between thalidomide and cytotoxic agents in association with their effects on tumour growth.

Study 40. A robust chiral assay for thalidomide in biological samples [316] The project required a chiral assay of thalidomide for studies in experimental animals and in patients having polypharmacy. This assay (Figure 3.18) allowed determination of whether there was enantioselectivity in thalidomide clearance and in its distribution between serum, tumours and other tissues. The enantiomeric concentration ratios and distribution coefficients demonstrated that enantioselectivity in thalidomide pharmacokinetics occurred in the rat, in a similar manner to that found in humans, after administration of the separate thalidomide enantiomers. 177 Enantioselectivity and racemization in thalidomide pharmacokinetics in the rat model indicated that a therapeutic advantage of a single enantiomer would be minimal, but also reinforced the need for considering the relevance of racemization in interpreting any pharmacodynamic studies.

Figure 3.18: Complete separation of the thalidomide enantiomers on a Vancomycin HPLC column, and showing the internal standard, phenacetin.

Study 41. Enantioselectivity of thalidomide pharmacokinetics in rats [319] This study was performed in the oncology research rat model in the Department of Oncology in which adult female F344 rats were implanted with 9L gliosarcoma tumours intracranially, subcutaneously (flank), or both. It was designed as a pharmacokinetic-pharmacodynamic study to examine whether the empirical clinical evidence of anti-tumour synergy between

176 Reist M, Carrupt PA, Francotte E, Testa B. Chiral inversion and hydrolysis of thalidomide: mechanisms and catalysis by bases and serum albumin, and chiral stability of teratogenic metabolites. Chem Res Toxicol 11 (12): 1521-1528, 1998 177 Eriksson T, Björkman S, Roth B, Höglund P. Intravenous formulations of the enantiomers of thalidomide: pharmacokinetic and initial pharmacodynamic characterization in man. J Pharm Pharmac 52 (7): 807-817, 2000 97 thalidomide and the antitumour agents 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or cisplatin could have involved altered pharmacokinetics of the thalidomide enantiomers. The study tested the effectiveness of thalidomide alone, and with intraperitoneal BCNU or cisplatin in combination chemotherapy, and was assessed by steady-state serum, tumour and other tissue concentrations of R- and S-thalidomide after several weeks of treatment. Marked enantioselectivity of thalidomide pharmacokinetics was found, with both serum and tissue concentrations of R-thalidomide being 40-50% greater than those of S-thalidomide; co- administration of BCNU or cisplatin did not alter the thalidomide concentration enantioselectivity. However, poor correlation between concentration and subcutaneous anti- tumour effect was found for individual treatments. Indeed, the consistency of the enantiomer concentration ratios across treatments strongly suggests that the favourable antitumour outcomes found with interactions between thalidomide and the cytotoxic agents did not involve enantioselectivity of thalidomide pharmacokinetics.

Study 42. Interpretation of the interaction between thalidomide and cisplatin [321] The final study was based on the same implanted tumour rat model of GBM as used in previous studies with co-treatment with cisplatin; tumour volume used as the primary endpoint for assessing drug effect. Additionally, mechanistic factors were sought as a means of explaining the synergism between thalidomide and cisplatin found in the study. Two factors were notable. First, treatment with thalidomide was associated with a greater distribution of cisplatin into tumours. Second, the growth modulator, vascular endothelial growth factor (VEGF) was found to be inhibited in the combination treatment. These data suggest that the increased antitumour activity of cisplatin in the presence of thalidomide had both a pharmacokinetic and a pharmacodynamic basis. Based on the selective effects of thalidomide on tumour cisplatin concentrations and the resulting increase in efficacy, thalidomide may also increase the efficacy of other drugs, such as taxanes, that are presently considered ineffective against glioblastoma.

Postscript 6 The role of combination pharmacotherapy with angiogenesis inhibitors has been validated in various preclinical cancer models and in clinical trials. However, concern has been raised that angiogenesis inhibitors targeting the VEGF pathway may ultimately lead to tumour adaptation and progression to stages of greater malignancy, with heightened invasiveness and, in some cases, increased lymphatic and distant metastasis. This would warrant clinical investigation, as the prospect has important implications for the development of enduring antiangiogenic therapies. 178 This is because the new blood vessels formed in the presence of VEGF inhibition are structurally and functionally abnormal, and that this could select for a more malignant phenotype – possibly with increased morbidity and mortality. Nonetheless, emerging anti-VEGF therapies are potentially effective in glioblastoma and can transiently normalize tumour vessels, providing a window of opportunity for optimally combining chemotherapeutics and radiation. 179 In particular, new combination therapies have been advanced based on the accrued knowledge. 180

178 Pàez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Viñals F, Casanovas O. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Canc Cell 15 (3): 220-231, 2009 179 Jain RK, Di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT. Angiogenesis in brain tumours. Nature Rev Neurosci 8 (8): 610-622, 2007 180 Gilbert MR, Gonzalez J, Hunter K, Hess K, Giglio P, Chang E, Yung WA. A phase I factorial design study of dose-dense temozolomide alone and in combination with thalidomide, isotretinoin, and/or celecoxib as postchemoradiation adjuvant therapy for newly diagnosed glioblastoma. Neuro-oncol doi: 10.1093/neuonc/noq100, 2010 98

One negative finding with thalidomide has been a marked increase in the incidence of both venous and arterial thromboses associated with its use. 181 This has led to the recommendation that thalidomide be administered in conjunction with low-dose warfarin, although the protective impact of a low-dose anticoagulant has not been fully established. Perhaps more importantly, thalidomide is now being supplanted with other thalidomide derivatives, such as lenalidomide and pomalidomide, with immunomodulatory properties. 182 These also demonstrate enantioselective pharmacokinetics, and so it may be some time before the stereopharmacology of these agents becomes settled in relation to the mode of action and their usefulness in the pharmacotherapy of cancer.183

3.8 Some additional reviews involving stereopharmacology Some additional included publications review the stereochemical impact on the pharmacolology of several drugs of interest.

Reviews 16, 17 and 18. Stereopharmacological impact in anaesthesiology [248,305,313] Another invitation from the editor of Current Opinion in Anaesthesiology in 1998, requested a review specifically directed towards several anaesthetically important drugs – ropivacaine, ketamine, and thiopentone [ 248 ]. I invited Steve Edwards, who had by then become a part- time PhD student, to co-author this paper.

The next, is one that we instigated. In 2003, my PhD student, neurologist Dennis Cordato suggested preparing a review to introduce stereopharmacology to his neurological colleagues. It contains yet another elementary discussion of the molecular origins of stereochemistry, chemical-historical introduction, and then refers to various relevant drugs used for the pharmacotherapy of Parkinson’s disease, epilepsies, headache, and adjuctive therapies such as anticoagulants [ 305 ].

In 2004, I received an invitation from anaesthesiologist-pain specialist Costantino (Nino) Benedetti, a former colleague from the University of Washington, to take part in a symposium in Capo Calavà, Sicily, on the 10 th anniversary of John Bonica’s death, along with a commemoration on the island of Filicudi, Bonica’s birthplace. Nino had gathered a guest faculty of Bonica alumni to review their current research at the symposium, which was hosted by the Italian Society of Anaesthesiologists. I chose to present two reviews: one focussing on the stereopharmacology of thiopentone and bupivacaine [313 ], and another on contemporary aspects of cannabinoid pharmacotherapy about which, by then, I had become involved through parliamentary inquiries as to its potential medicinal uses [312]).

181 Armstrong TS, Wen PY, Gilbert MR, Schiff D. Management of treatment-associated toxicites of anti-angiogenic therapy in patients with brain tumors. Neuro-oncology 14(10): 1203-1214, 2012 182 Ruchelman AL, Man HW, Zhang W, Chen R, Capone L, Kang J, Muller GW. Isosteric analogs of lenalidomide and pomalidomide: synthesis and biological activity. Bioorg Med Chem Lett 23(1): 360-365, 2013 183 Chen N, Kasserra C, Reyes J, Liu L, Lau H. Single-dose pharmacokinetics of lenalidomide in healthy volunteers: dose proportionality, food effect, and racial sensitivity. Cancer Chemother Pharmacol 70(5): 717-725, 2012; Li Y, Zhou S, Hoffmann M, Kumar G, Palmisano M. Modeling and simulation to probe the pharmacokinetic disposition of pomalidomide R-and S-enantiomers. J Pharmacol Exp Ther 114: 2014 Published online before print May 15, 2014, doi: 10.1124/jpet.114.21525 99

Postscript 7 Finally, I observe that, rather disappointingly, a recent otherwise truly excellent multiauthored account of the history of anaesthesia mentions stereopharmacology on but a few occasions – and then only briefly in connection with local anaesthetics, ketamine and isoflurane. 184 Nevertheless, despite the scientific and commercial complexities, regulatory bodies have since paid attention and now demand evidence from sponsors of new racemic drugs that the racemate is not inferior to a single enantiomer form of the drug. 185 And the medicinal -pharmacotherapy issue is far from being solved politically or medically: to me, it represents a classical case where ideology trumps evidence!

However…this is where it all began…

WESTMINSTER MEDICAL SOCIETY . Saturday, January 23,1847. Dr. Sayer, President ______DR. SNOW placed on the table an APPARATUS FOR INHALING THE VAPOUR OF ETHER It consisted of a round tin box, two inches deep, and four or five inches in diameter, with a tube of flexible white metal, half an inch in diameter, and about a foot and half long, coiled round and soldered to it. … Dr. Snow said it had been applied, in one case, at the temperature of seventy degrees, and had produced the effects of ether, very powerfully, in half a minute…

(Lancet i, 120-121, 1847)

(Pasteur L. Comptes rendus hebdomadaires de l’Académie des Sciences, Paris, Seance of May 15, 1848, 26 (21), 535–538 (Published on May 22, 1848))

184 Eger II EI, Saidman LJ, Westhorpe RN (Eds.). The Wondrous Story of Anesthesia . Springer: New York. 2014, p944. 185 De Camp WH. The FDA perspective on the development of stereoisomers. Chirality 1(1): 2-6, 1989 100

4. Discussion – and some conclusions

“One special advantage of the skeptical attitude of mind is that a man is never vexed to find that after all he has been in the wrong.” (Sir William Osler, The treatment of disease. The Canada Lancet 42: 899-912, 1909)

In this thesis, I have developed a narrative around the concepts and tools relevant to a theme of stereopharmacology based on the quantitative methodologies of medicinal chemistry, pharmacokinetics and pharmacodynamics, and have applied these concepts and tools to some clinically relevant drugs, mainly selected from the discipline of anaesthesiology. The thesis, being based on my publication record, thus contains no new experiments or data. I have, however, attempted to place the selected publications into an historical context, relating to the academic departments and personnel concerned, and to my own involvement within that context. I found this both an interesting and a satisfying approach, and I hope that any reader may also find it so.

It is difficult to draw any more meaningful conclusion than the inescapable one – that stereochemistry can have relevant pharmacological consequences. After all, the body is made of stereochemical building blocks. It should come as no surprise that for some drugs, at least, stereopharmacological differences can be truly large, even to the point of a quantal effect. For some drugs, pharmacokinetic differences can be largely due to direct metabolic preferences, or to distribution and transport differences, or to indirect differences, such as effects on the vasculature thereby affecting pharmacokinetics. Although the work described in this thesis did not turn up any examples of true stereopharmacologic specificity, there were a number of instances of stereopharmacologic selectivity or stereopharmacologic preferences.

While there are many examples of good pharmacological reasons for making a ‘single enantiomer switch’ for an existing drug racemate, there are, no doubt, many more examples of marginal pharmacological reasons yet strong commercial reasons - often a cause of ‘patent ever-greening’. Therefore, either or both pharmacological or commercial interests may lead to the clinical introduction of an enantiopure drug substance where a racemic substance had been introduced in the first instance. Rarely, is that racemic drug then deleted but more often produced as a ‘’ substance. In the main examples - notably of bupivacaine with ropivacaine and levobupivacaine, and ketamine with esketamine – there is a reasonable case for the pharmacological rationale, although the commercial backgrounds also generate some interest.

Ropivacaine, although a single enantiomer intended to supplant bupivacaine, is a single enantiomer of a homologue rather than that of the racemate. It was owned, developed, and promoted commercially with the brand name of Naropin® by the multinational AstraZeneca Corporation as part of their long history of local anaesthetics. It has been very successful clinically, and I may be counted among its satisfied recipients! The true single enantiomer switch for bupivacaine is levobupivacaine, nicely name-branded Chirocaine®, owned and developed by Chiroscience, a drug synthesis company that did not ‘end-market’ drugs. Levobupivacaine therefore required a ‘big pharma’ licensee to promote and market it for clinical uses. This was originally intended to have occurred through a 1995 agreement with the Swedish company Pharmacia, but Pharmacia became merged shortly afterwards with the US company Upjohn and, in April 1996, Chiroscience pulled out of the agreement. 186 On

186 http://ir.ucb-group.com/phoenix.zhtml?c=137495&p=irol-newsArticle&ID=844904 101

March 31 1998, Chiroscience entered into a licensing agreement with the British company for world distribution, excluding Japan, of levobupivacaine. On September 7 1998, Chiroscience entered into an agreement with Maruishi Pharmaceutical Co Ltd for the development and marketing rights to Chirocaine for Japan. 187 Zeneca had become the principal company of ICI Pharmaceuticals that had created propofol, and Maruishi were the originators of sevoflurane. Thus, both of these companies already had important anaesthetic products, and thereby an established sales network. However, the plans became unstuck when Astra and Zeneca announced their plans to merge. On March 1 1999, Chiroscience regained the licence for levobupivacaine to re-seek a marketing partner. On June 15 1999, Chiroscience announced that the US marketing rights for Chirocaine® had been licensed to Purdue Pharma, developers of long-acting opioid preparations, and to Abbott Laboratories for countries other than Japan and the USA. 188 Eventually, I believe that Abbott Laboratories, the originators of thiopentone, or one of its subsidiary companies, became the licensee for the USA as well. Chiroscience subsequently underwent a merger into Celltech in 1999 which, itself was acquired by the Belgian company UCB in 2004, and the name Chiroscience disappeared. 189

These various business mergers and other deals were occurring whilst I was involved in our ongoing levobupivacaine research program. As mentioned previously, I gave expert evidence on levobupivacaine to the FDA hearings in Washington DC in March 1997 and January 1999, and participated in various investigator workshops, as well as open symposia, including the official launch of Chirocaine® in Göteborg in 2001. The local anaesthetic agent program thereby remained a major part of my research, and it is fitting that it is the largest section in this thesis. Having been closely involved with levobupivacaine, I have formed the opinion that it has been somewhat let down by insufficient promotion of its safety. Certainly clinical practice has improved to make local anaesthetic toxicity less frequent and the newer single enantiomer agents have improved and have a greater safety profile when compared to bupivacaine. But I reiterate the conclusions of our Review 09 [ 282 ] “Despite optimism about the enhanced safety of these drugs, anaesthesiologists should remember that a syringe full of ropivacaine or levobupivacaine still contains a potentially lethal dose. Compared with bupivacaine, the newer agents may be seen as ‘safer’, but they must not be regarded as ‘safe’.”.

The ropivacaine and levobupivacaine programs were productive collaborations with the pharmaceutical industry. We hoped that this might also pertain in the development of the single R-thiopentone enantiomer, with its greater margin of safety. Colin Duke, who had become a close colleague on this program, was responsible for devising the novel chiral syntheses of the thiopentone enantiomers from inexpensive, natural product, starting materials, and for which we became inventors on Patent Application number PCT/AU1999/000919 ( Patent 03). With the award of provisional patents for enantiopure thiopentone (both enantiomers), I approached a number of pharmaceutical companies, including Chiroscience, with a view to their sponsoring further development as a possible ‘single enantiomer switch’. However, none would commit, basically on the grounds that

187 http://www.prnewswire.com/news-releases/chiroscience-and-maruishi-to-develop-chirocaine-in- japan-76489102.html (accessed 26 December, 2014); http://www.independent.co.uk/news/business/has-zeneca-chosen-wisely-1190661.html ; http://www.pharmaceuticalonline.com/doc/chiroscience-to-regain-license-for-chirocaine-0001 ; http://ir.ucb-group.com/phoenix.zhtml?c=137495&p=irol-newsArticle&ID=844904 ; 188 http://ir.ucb-group.com/phoenix.zhtml?c=137495&p=irol-newsArticle&ID=844904 189 Hughes C. Celltech and Chiroscience in pounds 700m biotech merger. The Independent , Wednesday 16 June 1999 102 there was not sufficient commercial advantage now that propofol was attracting a larger proportion of the anaesthesia market. And we well knew, ourselves, that nothing could give either thiopentone enantiomer the pharmacokinetic advantages of propofol. No-one had previously perceived the need for a ‘safer thiopentone’ and we came up with it a decade too late. Its history might have been different if the enantiomers had been studied a generation earlier.

Thiopentone has also, more recently, come under threat of annihilation from another source – the anti-capital punishment lobby, or more specifically the anti-lethal injection lobby. 190 Thiopentone was adopted by many states of the USA as part of a triad of agents (with muscle relaxant pancuronium and electrolyte potassium) used for execution of prisoners by lethal injection. Certainly, this particular use had no regard for a single enantiomer of thiopentone with a greater margin of safety! On March 31 2010, Hospira (a 2007 spin-off company from Abbott Laboratories), the sole U.S. manufacturer of thiopental sodium, issued a statement disapproving of the use of their drugs for executions. On January 21 2011, Hospira issued a statement that the company had intended to produce thiopentone sodium from its Italian plant but that the government of Italy, as part of the European Union, decided that they could not be assured that the drug would not be used in lethal injections. As a consequence of this and their open opposition to the use of thiopentone in lethal injections, Hospira decided instead to exit the thiopentone market. 191

Certainly, the relevant historical hints about stereopharmacology and thiopentone should have been heeded. But does it really matter? Probably not, now. Although various relatively minor pharmacokinetic differences between enantiomers were found by us and by others, the more significant difference was in the margin-of-safety that favoured R-thiopentone, particularly for use in high doses such as for the production of ‘thiopentone coma’ in patients with head injuries – a relatively minor use of thiopentone. The differences between enantiomers in thiopentone as an anaesthetic induction agent were probably not large enough to make it a commercial proposition. Thus, after some half century of providing the leading anaesthetic induction agent, thiopentone anaesthesia has almost ceased. And, still, probably many past, present and future anaesthetists will not have known about its enantiomeric duality – just as with halothane.

With ketorolac, the results were disappointingly inconclusive. In reality, there seemed to be no penalty for using the racemate and no good reason for developing the S-enantiomer associated with cyclooxygenase inhibition. Concurrently, other water soluble and injectable forms of various nonsteroidal anti-inflammatory drugs (diclofenac, valdecoxib, as examples) have been brought to market and are proving clinically useful.

Halothane, now an ‘anaesthetic antique’, was included out of curiosity. It was not worthwhile following up, given that our initial study found no detectable difference in whole body pharmacokinetics, and given the (diminishing) funds that would have been required for synthesising the separate enantiomers to use in the next phase, as well as the reluctance of anaesthetists to reconsider its use. The volatile anaesthetics of the current generation are serving patients well.

190 Woolston C. Death row incurs drug penalty: Bid to use common anaesthetic for executions threatens to cut off supply to US hospitals. Nature 502: 417–418, 2013 191 http://www.deathpenaltyinfo.org/home (accessed January 2014 ) 103

The ‘market penetration’ of clinical practice with esketamine (enantiopure S-ketamine) has been yet rather slow and the racemate still remains in ever increasing use with newer dosage forms, such as intranasal. Although these are also being developed for esketamine, the racemate shows little sign of being replaced.

And last, thalidomide, where interconversion between the enantiomers occurs, an enantiopure form has nothing to commend it, at least as far as can be seen at present. A variety of thalidomide derivatives has been prepared and many have been introduced into trials over the past decade, and more may come from the selection or development of one of these.

Most drugs act at proteinaceous receptors made of stereochemical building blocks. In my postgraduate student days, drug receptors were little more than inferences derived from structure-activity experiments, typically in an isolated organ preparation, using drug homologues and analogues, with attempts to match an optimal chemical structure to a particular activity profile. There is no doubt that this approach worked. Take, for example, the stereopharmacology of opioids: a stereoselective opioid receptor was proposed in the mid- 1950s by Arnold Beckett and his group on the basis of structure-action relationships and stereochemistry - long before the receptor was identified and cloned. The morphinoids agonists and antagonists are highly stereoselective, but many opioids, including pethidine and fentanyl, are achiral. However, the structure-action approach was inefficient. These days, new chimeric receptors can be assembled from various combinations of exofacial, transmembrane, and cytoplasmic segments domains into new structures, and these can be used to map binding sites, and to design an optimal drug, including which pair of enantiomers will be preferred. This technology has left me a generation or more behind.

I briefly digress here to raise an issue - why does molecular asymmetry exist? I was never challenged to think about any such reasons by my teachers, mentors or students – I was taught that chirality was, but never, why? Why are some molecules, notably the natural amino acid building blocks, always oriented one way? After all, that is the basis of stereopharmacology.

One physical explanation suggests that the left-handed molecular bias may be due to ‘circular polarization’ whereby light from space may have preferentially destroyed molecules with one kind of handedness. The origins of circular polarization have been suggested as being due to magnetic fields from space bodies causing preferential alignment of space dust around stars. Amino acids synthesized by chemical reactions may then have been preferentially left handed, and subsequently delivered to earth in meteorite showers. 192 Others agree about the meteorites, where evidence indicated that those containing the highest left-hand enrichment had the longest exposure to water, which amplifies any inequality in amino acids. Up to 15% more left-handed amino acids were found in some meteorites. The amplification factor is suggested as necessary to account for the magnitude of the bias, whereas the polarized light may only account for some 2% of the bias. 193 Biochemists, on the other hand, have tended to favour an explanation based on a chance occurrence in prebiotic chemistry leading to an initial chiral excess in the biochemistry of ribozymes which is dominated by right handedness. At some stage the disequilibrium in synthesized products led to a permanent chiral dominance. 194 But now I am completely out of my depth, so I return to discuss some personal philosophy about data acquisition and data insight.

192 Choi CQ. Did Asteroid Impacts Spark Life's "Left-Handed" Molecules? http://www.scientificamerican.com/article/asteroid-impacts-spark-left-handed-molecules/ 193 Hsu J. Why Life on Earth is Left-Handed. http://www.space.com/6463-life-earth-left-handed.html 194 http://www.quantamagazine.org/20141126-why-rna-is-right-handed/ 104

In the Foreword to this thesis I noted the remark that Patrice Debré wrote about Pasteur - he was “a chemist among the doctors”. 195 Next, to paraphrase mathematician and physicist Lord Kelvin (1824-1907),196 ‘if you can measure something, then you can say something about it’. As a bench chemist, I became skilled at measuring things, such as drug concentrations in biofluids, and responses to drugs, and I attempted to apply these skills to research in anaesthesiology. When physiologist Claude Bernard wrote “The true worth of an experimenter consists in his pursuing not only what he seeks in his experiment, but also what he did not seek.", he was clearly referring to insight into data. This has been facilitated by the enormous technological changes that have occurred during my days in the laboratory. Again, I briefly digress.

I started in my lab with GLC, first with flame ionization detectors, then moved on to GLC with nitrogen selective detectors for enhanced sensitivity towards the various amino- containing drugs and metabolites. Next I acquired HPLC and this allowed analyses of non- volatile and thermally unstable substances, first with general purpose UV detectors, then with diode array detectors that allowed greater selectivity towards mixed analytes, and finally, both GLC and HPLC with mass spectrometry detectors allowed the greatest versatility of all. This technology evolution occurred from the mid 1960s to the end of the 1990s and, by that stage, my job had evolved into preparing the grant applications to acquire the equipment and selecting the personnel to operate it.

My first pharmacological bioassay (MSc research on alcuronium) used a smoked drum recorder apparatus very little changed from the late 19 th century. My studies at the University of Washington in Seattle and at Flinders used then-current analogue techniques on high fidelity strip chart recorders, both for chromatography traces and recording of continuous physiological variables such as blood pressure, and all required manual extraction of data. Automation came first to my chemistry lab in Seattle in 1974 when I purchased a GLC integrator-recorder that detected peaks automatically from the rate of change of baseline, quantitated them from a standard curve acquired during the experiment, and printed out the assay results. Next, at Flinders in the early 1980s, both the GLC and HPLC devices were additionally equipped to process samples in a predetermined sequence, but each still remained a stand-alone device. Eventually full computer process control and chromatographic data acquisition was introduced. However, equivalent progress came somewhat later to the acquisition of physiological data. For my last series of sheep lab studies at Flinders in 1989- 1990, some analogue-to-digital microprocessing had been introduced for Al Rutten’s PhD thesis. By 1993, full quantitative digital signal acquisition of physiological data was becoming available, and I introduced it into my sheep lab in Sydney for the levobupivacaine program. This, along with further improvements in hardware and software, enabled large scale experiments to be performed in which we could expand scales for visual inspection, extract data automatically, and dump the results into spreadsheets for statistical analyses and graphical presentations, etc..

Indeed, the whole ‘digital revolution’ has occurred during the span of my research. In 1966, I was invited to join a cohort of postgraduate students to test the feasibility of using the University of Sydney’s first supercomputer for searching the published research literature with MEDLARS (Medical Library Acquisition Reference System), a batch processing system

195 Debré P.. Louis Pasteur . (Translated by E. Forster) Baltimore, Maryland: Johns Hopkins University Press. ISBN 0-8018-5808-9. (1998) 196 Thomson W.. Popular Lectures and Addresses , Vol. I. London: MacMillan. p. 80. (1891) 105

(and the forerunner of the online MEDLINE system now in use). We performed our searches with key words and journal titles that we had recorded onto punch cards against a data base of references created from the Index Medicus. It was a triumph just to get a search to run – let alone retrieve any useful citations! I took a class in FORTRAN IV the following year, but calculations for most of my research were performed on a slide rule and, occasionally, on the department’s sole Monroe electric calculator. It was not until I began working in Seattle that I first saw an electronic digital calculator, a Hewlett Packard HP-35 running the Reverse Polish Notation operating system. Geoff Tucker and I used the University of Washington’s CDC6400 mainframe computer, still with batch processing and punch cards, to run non-linear least-squares regression programs, but then with our added-on user-written pharmacokinetic subroutines in FORTRAN to perform our anaesthetic pharmacokinetic modeling calculations. This was followed by the Anesthesia Research Center’s own PDP-8 mini-computer (it only half-filled the room) located in the neuropharmacology lab of our anaesthesiologist colleague, Rudy de Jong (1928-2011), a noted researcher of local anaesthetic pharmacology. This computer originally booted and ran from a paper-tape reader at its own terminal, and later, from a demountable hard disk for ‘real-time’, not batch, modelling The progress was staggering!

At Flinders, we had our own departmental terminal and printer attached to the university mainframe – operating at the spellbinding speed of 1200 baud and printing at 300 baud. This allowed us to work up various pharmacokinetic routines, initially as part of the erythromycin studies, during the late 1970s. Next, in 1980, at my suggestion, our department became the first at Flinders to purchase a stand-alone word processer – an NBI OASys 3000 – a dual 8” floppy disk computer running a proprietary operating system. It arrived in time for my first PhD student’s thesis preparation and impressed everyone who saw it – including Gustav J (Gus) Fraenkel (1919-1998), the astute founding Dean of Medicine – who pronounced that it was ‘expensive, but probably the way of the future’. At UMass in 1982, I used remote computing over the telephone network by way of a brief-case sized acoustic coupler. I purchased my first home computer in 1983, from a specialty computer shop in Mountain View, California, during a visit to Stanford, and proudly brought it home to Adelaide. It was an Osborne Executive, running the CP/M operating system, with a 5” CRT screen, dual floppy 5¼” disks, mains powered, ‘weighing 28 pounds’ and the size of a home sewing machine – but it ‘fitted under an airline seat’ and thus was labelled ‘portable’! My next several home computers also were purchased on visits to Stanford, with improvements in their operation, weight and battery powered portability, as the prices and types available there surpassed those in Australia. I am now able to sit at home and retrieve mid-19 th century volumes of the London Medical Gazette and read the original writings of John Snow, on my laptop computer, as I compose this thesis. The progress, from my days of punching cards, to retrieve the reference to a possibly interesting paper in a library somewhere –not the paper itself - to having the latest reference list – and the associated papers - in front of me, has been truly astonishing!

Finally, I write this paragraph at a time when ‘reform’ of Australian universities is a high- stake political issue. The message being pushed is the notion that it is ‘competition that drives academic excellence’ (and apparently the all-important university rankings). I disagree. The collegiate university may, these days, be mortally wounded but it is still cooperative collaboration that drives excellence in teaching and in research. It did in my day, and I believe that it always will. Drawing on my own experience, I think that I have made abundantly clear the case for collaborative and cooperative multidisciplinary research. Competition would never have come close to providing a sensible methodology for the 106 problems that I have encountered over many years. To draw on an aphorism from another place and time “…there’s glory enough for all”. 197

Since retirement in 2007, I have maintained a strong interest in the medicinal applications of cannabis [312]. I had published a paper, seemingly Australia’s first, on the chemical composition of cannabis from an invitation to collaborate with pharmacognosist Lorna Cartwright from the Department of Pharmacy in 1972 [7], but hadn’t subsequently thought much about it, and not a lot was known at that time.

In 2000, I was invited to participate in a working party convened by the Premier of New South Wales, Robert John (Bob) Carr, to examine the case for introducing legislation to permit the medicinal use of cannabis in this state. This is clearly another area where collaborative cooperation – between basic and applied scientists, , medical and nursing practitioners, law makers and enforcers, concerned citizens, patients, and probably many others - is vital. After joining the Premier’s working party with an open mind, I became convinced of the medicinal merits of cannabis, and sought to undertake further research by proposing multidisciplinary projects that literally went from the plant to the bedside by involving plant scientists, medicinal chemists, pharmaceutical chemists, pharmacologists and neurologists. My grant applications were unsuccessful and I am not entirely convinced that it was because the science was lacking. Anyway, I became confident that cannabis did have medical uses and have made formal submissions to Parliamentary inquiries, written for the professional and lay press, and given countless media interviews on cannabis and cannabinoids [332, 334-337]. Most cannabinoids and their congeners are chiral compounds - but I haven’t yet had an opportunity to make reference to their stereochemistry to anyone.

The politics of cannabis is complicated enough without evoking stereopharmacology!

Anaesthetized sheep. A symbol of much - presented to Sonia Gu at her last day in the lab on June 6 2006 - not long before my own last day in the lab on September 30 2006.

197 Whitford I, Qureshi S, Szulc AL. The discovery of insulin: is there glory enough for all? Einstein Journal of Biology and Medicine 28(1):12-17, 2012 107

Afterword The inspiration for this thesis began with my developing a paper for a symposium on the history of anaesthesia, based on a long-standing interest in the work of John Snow, and discovering, in a book sale, a biography of Louis Pasteur. So, I now return to the 1848 world of John Snow and Louis Pasteur and the paper which I presented to that symposium [340].

By 1848, most of the world had heard about anaesthesia. In London, it was another cholera year. In Paris, it was another year of revolution. Medicine was still considered an art rather than a science. The first golden age of physiology that began with William Harvey, strengthened by polymath scientists such as Robert Hooke and Isaac Newton, had passed into a “period of consolidation”. 198 The physical chemistry of gases had been developed by Robert Boyle, Humphry Davy, Joseph Priestley, Joseph Gay-Lussac and others; optical physics had been established by Abbé Haüy and Etienne Malus and incorporated by Jean- Baptiste Biot into mineralogy and chemistry via crystallography; and the great age of organic chemistry had commenced with Friedrich Wöhler, Justus von Liebig and Jean-Baptiste Dumas. Neither pharmacology nor biochemistry had yet been actualized. A second golden age of physiology was about to begin with Claude Bernard, who was born in the same year as John Snow. In 1848, Snow was a 35 year old Lecturer on Forensic Medicine at the Aldersgate School of Medicine. He was more advanced professionally than Pasteur, who was then a 25 year old graduate assistant in the laboratory of Jérôme Balard at the École normale supérieure and under the tutelage in crystallography of Auguste Laurent. .

In 1848, Snow began writing “On narcotism by the inhalation of vapours” a seminal series in which he developed a narrative around the importance of the vapour pressure of the anaesthetic agent, as well as its inspired and resultant blood concentrations. 199 He incorporated the ideas that gas moved along concentration gradients in the body (alveolar to arterial to tissue to venous gas tensions), and ideas around the comparative properties of numerous substances considered as potential anaesthetic agents. Snow went on to describe the effects of anaesthesia on physiological functions, including oxygen consumption. He recognized the significance of relative safety, initially contrasting the convenience and agreeability of chloroform with the greater safety of the ether, and later comparisons with other possible agents. Indeed, his grand search, according to his friend and biographer, Benjamin Ward Richardson, was “for a narcotic vapour which, having the physical properties and practicability of chloroform, should, in its physiological effects, resemble ether in not producing, by any accident of administration, paralysis of the heart…” Perhaps he was foreshadowing halothane.

According to Richardson, Snow also “paid considerable attention to the subject of local anaesthesia, and tried various methods for attaining to a knowledge of a perfect local anaesthetic.” He did this in 1854 by cryo-anaesthesia with chloroform mixtures and solid carbon dioxide, but was never satisfied with the results “and soon relinquished the inquiry”. 200

198 Westerhof N. A short history of physiology. Acta Physiol 202: 601-603, 2011 199 Snow J. On narcotism by the inhalation of vapours. London Med Gazette, 1848; 41, 850-854 (part 1); 1848; 41: 893-895 (part 2); 1848; 41: 1074-1078 (part 3); 1848; 42: 330-335 (part 4); 1848; 42: 412-416 (part 5); 1848; 42: 614-619 (part 6); 1848; 42: 840-844 (part 7); 1848; 42: 1021-1025 (part 8); 1849; 43: 228-235 (part 9); 1849; 43: 451-456 (part 10); 1849; 43: 983-985 (part 11); 1849; 44: 272-277 (part 12); 1850; 45: 622-627 (part 13); 1850; 46: 321-327 (part 14); 1850; 46: 749-754 (part 15) ; 47: 622-627 (part 16); 1851; 48: 1053-1057 (part 17); 1851: 48: 1090-1094 (part 18) 200 Richardson BW. Life of John Snow M.D . In: Snow J, On chloroform and other anaesthetics: their action and administration. Edited with a memoir of the author by Benjamin W Richardson, MD. 108

Isolation of the cocaine alkaloid by Friedrich Gaedcke was described in 1855 201 but Snow was unlikely to have known about this, and no such reference appeared in his subsequent writings. Snow’s friend Richardson records that, on June 10 1858, Snow completed the editing, except for the construction of the index, of his assembly of personal research and observations embodied in “On chloroform and other anaesthetics: their action and administration”.202 This was to be his last publication. He had an episode of a recurring giddiness a little earlier that morning, before returning to his editing, but what was to be a fatal stroke soon followed. His condition worsened over the successive days, and his death occurred at 3 P.M. on Wednesday, June 16 1858. Snow was buried in Brompton Cemetery, London, on the Monday following. Richardson, who oversaw the publication of the book in August of that year, added a moving Preface as a eulogy and biography. In the concluding paragraph, he no longer refers to Snow as a “medical man” but, significantly, as a “science man”. 203 In a second, shortened memoir written some 30 years later, Richardson wrote that the book “…will always remain as a memorable record in the history of medical literature”. 204 Indeed so. Snow’s works evolved to become the quantitative basis of anaesthesia.

French scientists of the late 18 th C had a strong interest in mineralogy as one of the principal earth sciences. Chemical crystal structure was believed to depend on chemical composition structure. It was known that some crystals, notably of quartz - a common model for study, could be asymmetrical, but it was thought that this was a random phenomenon. It was into this milieu that the optical physics of polarization of reflected or refracted light rays was added, around 1812, by Biot, who was later to become a mentor of Pasteur.

In 1848, Pasteur was studying paratartaric acid [an old name for (2RS,3RS)-tartaric acid, aka DL-tartaric acid, (±)-tartaric acid, and racemic acid); it is found in the ‘cream of tartar’ in wines, and the name racemic acid takes its name from racemus , Latin meaning a bunch of grapes 205 ]. Pasteur observed that dissymmetric, otherwise identical mirror images, crystals of the sodium ammonium ‘double salt’ of paratartaric acid, did not rotate plane polarized light, but that the natural tartaric acid, which had the same molecular structure, did. 206 Astonishingly, Pasteur observed, under magnification, that the crystals of paratartaric acid salt (and certain other salts) came equally in two shapes, both asymmetric and otherwise identical, but mirror images of each other; in solution, these crystals each rotated plane polarized light, but in opposite directions. 207 Paratartaric acid itself does not rotate plane polarized light because it is equi-part mixtures of the two forms, but the right faceted crystals were dextrorotatory, exactly like the natural crystals from wine. This was a seminal finding by any measure. His paper “On the relation that can exist between crystalline form and chemical

John Churchill, London 1858. 201 Gaedcke F. Ueber das Erythroxylin, dargestellt aus den Blättern des in Südamerika cultivirten Strauches Erythroxylon Coca. Archiv der Pharmazie 132(2): 141–150, 1855 202 Snow J. On Chloroform and Other Anaesthetics: their Action and Administration . Edited, with a memoir of the author by Benjamin W. Richardson, M.D. London: John Churchill, 1958. 203 Richardson BW. Life of John Snow M.D . In: Snow J, On chloroform and other anaesthetics: their action and administration. Edited with a memoir of the author by Benjamin W Richardson, MD. John Churchill, London 1858, p. xliv. 204 Richardson BW. John Snow, M.D., A representative of medical science and art of the Victorian Era. The Asclepia d Vol IV. London: Longmans, Green and Co 1887. p. 274-300 (reprinted in Br J Anaesth 24: 267-291, 1952) 205 Aronson J. Grapes. BMJ 317(7159): 688, 1998 206 Gal J. Louis Pasteur, language, and molecular chirality. I. background and dissymmetry. Chirality 2011; 23: 1-16. 207 Tobe Y. The reexamination of Pasteur’s experiment in Japan. Mendeleev Communications Electronic Version 2003; 3: 1-2 109 composition, and on the cause of rotational polarization” 208 was read on May 22 1848,209 and led to the molecular basis of stereochemistry.

By 1853, Pasteur still had not discovered in molecular terms why paratartaric acid had two forms. In experiments with various alkaloids (as bases), he found that they, too, could exhibit equivalent optical rotation-crystal morphology phenomena. Eventually, he devised a method for separating the mirror image forms of paratartaric acid salt by combining the forms of an optically active acid with an optically pure base, thereby making products – one with positive rotation, one with negative rotation, and selectively crystallizing them from the mother liquor. This became Pasteur’s classical resolution and is still used, although more efficient methods have become available. In 1857-8, Pasteur found that microorganisms could metabolize (ferment) chiral substrates at different rates 210 - a biochemical-stereochemical interaction – the foundation of stereopharmacology - and a new direction for Pasteur began. Although Pasteur founded stereochemistry, stereochemistry was not tractable until 1874-5, when Jacobus Henricus van’t Hoff and Joseph Le Bel explained the tetrahedral bonding of carbon, and the basis of stereoisomerism of organic compounds was established.

Pasteur introduced stereochemistry to science; Snow introduced science to anaesthesia. Stereochemistry came late to anaesthesiology as the first century of its favoured molecules were achiral ( viz. , ether, chloroform, , ) and, until relatively recently, it was commonly taught that anaesthetic actions were structurally non-specific, being mediated through cell membrane actions by agents with essentially common physicochemical properties. 211 Not surprisingly, this view is pharmacologically too simple – it depends on one’s methods. 212 The next century of anaesthetics began with halothane – a racemate.

Within 150 years, all manner of pharmacokinetic and pharmacodynamic models were being applied to racemic drugs, and characterized by rates, volumes, clearances, ED 50 s, etc. It is all rather nice and tidy but, when based on achiral logic for racemic drugs – as most models are, then these models are just plain wrong! However, the practical awareness of the importance of stereopharmacology was re-awakened in 1984 when Everardus Ariëns scathingly proclaimed that ignoring stereochemistry and regarding racemic drugs as single drug entities was a basis for producing pharmacological “nonsense data”, however attractively packaged. Until two decades ago, analytically separating the enantiomers of racemic drugs, for pharmacokinetic-pharmacodynamic research was near to impossible. Now, studying how the body handles the enantiomers of racemic drugs is relatively easy, and ‘single enantiomer switches’ have led to the development of numerous new products.

It is sometimes said of Snow that ‘he made the art of anaesthesia a science’, and it is sometimes said of Pasteur that ‘he was a chemist that revolutionized medicine’. This inquisitive spirit of 1848 and the pharmacological tale of those two cities, that inspired me to write this thesis, hopefully lives on.

208 English title as recorded in Debré P. Louis Pasteur (translated by E Forster). Johns Hopkins University Press, Baltimore. 1998; pp 502-503 209 Gal J. When did Louis Pasteur present his memoir on the discovery of molecular chirality to the Academie Des Sciences? analysis of a discrepancy. Chirality 2008; 20: 1072-1084. 210 Gal J. The discovery of biological enantioselectivity: Louis Pasteur and the fermentation of tartaric acid, 1857—A review and analysis 150 yr later. Chirality 2008; 20: 5-19 211 Eger EI. II Anesthetic uptake and action . Williams and Wilkins, Baltimore, 1974 212 Yamakura T, Bertaccini E, Trudell JR, Harris RA. Anesthetics and ion channels: molecular models and sites of action. Annu Rev Pharmacol Toxicol 41 (1): 23-51, 2001