PK of Medcm Against Nerve Agents, Which Have Been Integrated with PK and PD Data for the Nerve Agents Sarin and VX
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UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE Institute of Developmental Sciences The Pharmacokinetics of Medical Countermeasures Against Nerve Agents by Stuart Jon Armstrong Thesis for the degree of Doctor of Philosophy November 2014 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE Institute of Developmental Sciences Thesis for the degree of Doctor of Philosophy THE PHARMACOKINETICS OF MEDICAL COUNTERMEASURES AGAINST NERVE AGENTS Stuart Jon Armstrong Nerve agents are organophosphorus compounds that irreversibly inhibit acetylcholinesterase, causing accumulation of the neurotransmitter acetylcholine and this excess leads to an overstimulation of acetylcholine receptors. Inhalation exposure to nerve agent can be lethal in minutes and conversely, skin exposure may be lethal over longer durations. Medical Countermeasures (MedCM) are fielded in response to the threat posed by nerve agents. MedCM with improved efficacy are being developed but the efficacy of these cannot be tested in humans, so their effectiveness is proven in animals. It is UK Government policy that all MedCM are licensed for human use. The aim of this study was to test the hypothesis that the efficacy of MedCM against nerve agent exposure by different routes could be better understood and rationalised through knowledge of the MedCM pharmacokinetics (PK). The PK of MedCM was determined in naïve and nerve agent poisoned guinea pigs. PK interactions between individual MedCM drugs when administered in combination were also investigated. In silico simulations to predict the concentration-time profiles of different administration regimens of the MedCM were completed using the PK parameters determined in vivo. These simulations were used to design subsequent in vivo PK studies and to explain or predict the efficacy or lack thereof for the MedCM. The PK data generated in this study are the first such data in conscious guinea pigs, the small animal species of choice for determination of MedCM efficacy. These data show how PK can improve the understanding of MedCM efficacy. PK studies such as those reported here should be integrated into the development programmes of future MedCM. Contents 1. INTRODUCTION ........................................................................ 1 1.1 Background ......................................................................................... 2 1.2 Normal cholinergic function ................................................................ 3 1.3 Cholinesterases .................................................................................. 6 1.4 Nerve agents ....................................................................................... 7 1.4.1 Routes of exposure .................................................................... 13 1.5 Medical countermeasures .................................................................. 15 1.5.1 Post-poisoning therapy ............................................................... 17 1.5.1.1 Atropine .............................................................................. 17 1.5.1.2 Avizafone............................................................................. 20 1.5.1.3 Oxime .................................................................................. 22 1.5.2 Pretreatment .............................................................................. 25 1.5.2.1 Pyridostigmine bromide ....................................................... 25 1.5.2.2 Hyoscine and physostigmine ................................................ 27 1.5.2.3 Other pretreatments ............................................................ 28 1.5.3 Bioscavengers ............................................................................ 28 1.6 Choice of animal species ................................................................... 30 1.7 Pharmacokinetics and pharmacodynamics......................................... 31 1.8 Microdialysis ..................................................................................... 35 1.9 Aims and hypotheses ........................................................................ 38 2. MATERIALS AND METHODS ..................................................... 41 2.1 Animals ............................................................................................ 42 2.1.1 Animal supply and background .................................................. 42 2.1.2 Animal training .......................................................................... 43 2.1.3 Pre-surgical preparation ............................................................. 45 2.2 Surgical procedures .......................................................................... 45 2.2.1 Anaesthesia ................................................................................ 46 2.2.2 Implantation of venous and arterial cannulae ............................. 47 2.2.3 Muscle microdialysis probe implantation .................................... 48 2.2.4 Brain microdialysis probe implantation ....................................... 50 2.2.5 Post-surgical recovery procedures .............................................. 52 2.3 Study protocol .................................................................................. 53 2.3.1 Administration of test compounds ............................................. 53 2.3.1.1 Intravenous administration of medical countermeasures ...... 53 2.3.1.2 Intramuscular administration of medical countermeasures ... 53 2.3.1.3 Administration of human butyrylcholinesterase .................... 53 i 2.3.1.4 Subcutaneous administration of sarin ................................... 54 2.3.1.5 Application of percutaneous VX ............................................ 54 2.3.2 Automated blood sampling ......................................................... 54 2.3.2.1 Sampling volumes ................................................................ 58 2.3.3 Blood sampling protocols ........................................................... 58 2.3.4 Microdialysis sampling ............................................................... 60 2.3.5 Sample preparation .................................................................... 62 2.4 Assay of cholinesterase activity ......................................................... 62 2.5 Quantitation of analytes .................................................................... 63 2.5.1 Quantitation of VX ...................................................................... 63 2.5.2 Quantitation of pharmacological medical countermeasures ........ 65 2.5.3 Quantitation of butyrylcholinesterase ......................................... 70 2.6 In vitro calibration of microdialysis probes ........................................ 70 2.6.1 Medical countermeasure calibration ............................................ 70 2.6.2 VX calibration ............................................................................. 71 2.7 Pharmacokinetic analyses .................................................................. 71 2.7.1 Noncompartmental analysis ........................................................ 71 2.7.2 Compartmental analysis ............................................................. 72 2.8 Pharmacodynamic analysis ................................................................ 76 2.8.1 Noncompartmental analysis ........................................................ 76 2.8.2 Pharmacodynamic model ............................................................ 76 2.9 Linked pharmacokinetic and pharmacodynamic analysis ................... 79 2.10 Bioavailability .................................................................................... 81 2.11 Statistical analysis ............................................................................. 82 2.12 Histopathological analysis ................................................................. 82 2.13 Electron microscopy .......................................................................... 83 3. PHARMACOLOGICAL MEDICAL COUNTERMEASURES AGAINST NERVE AGENTS ....................................................................... 85 3.1 Chapter specific introduction ............................................................ 86 3.2 Chapter specific aims ........................................................................ 88 3.3 Materials and methods specific to the study of pharmacological medical countermeasure pharmacokinetics ....................................... 90 3.4 Results .............................................................................................. 93 3.4.1 General results ........................................................................... 93 3.4.2 Characterisation of microdialysis probes .................................... 93 3.4.2.1 Histopathological assessment .............................................. 93 3.4.2.2 In vitro microdialysis probe efficiency .................................. 94 3.4.3 Medical countermeasure pharmacokinetics administered by the intravenous route .................................................................................. 103 ii 3.4.4 Plasma bioavailability of medical countermeasures following intramuscular administration ................................................................. 106 3.4.5 Muscle and brain pharmacokinetics of medical countermeasures following intramuscular administration.................................................. 109 3.4.6 Medical countermeasure pharmacokinetics following combined administration by the intramuscular