THE GUINEA PIG MODEL FOR ORGANOPHOSPHATE TOXICOLOGY AND THERAPEUTIC DEVELOPMENT A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Christopher Ruark B.S., Miami University, 2007 M.S., Wright State University, 2010 ______________________________________ 2015 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL May 2, 2015 I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Christopher Ruark ENTITLED The Guinea Pig Model for Organophosphate Toxicology and Therapeutic Development BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy. __________________________ Jeffery M. Gearhart, Ph.D. Dissertation Director ___________________________ Mill W. Miller Ph.D. Director, Biomedical Sciences Ph.D. Program ___________________________ Robert E. W. Fyffe, Ph.D. Committee on Vice President for Research and Final Examination Dean of the Graduate School ___________________________ Jeffery M. Gearhart, Ph.D. ___________________________ Adrian M. Corbett, Ph.D. ___________________________ James B. Lucot, Ph.D. ___________________________ Mateen M. Rizki, Ph.D. ___________________________ Gerald M. Alter, Ph.D. ABSTRACT Ruark, Christopher Ph.D., Biomedical Sciences Ph.D. program, Wright State University, 2015. The Guinea Pig Model for Organophosphate Toxicology and Therapeutic Development. Organophosphates (OPs) are highly toxic insecticides and nerve agents that have been designed to inhibit the hydrolysis of acetylcholine by binding to the serine active site of acetylcholinesterase (AChE). They are one of the most common causes of human poisoning worldwide and are frequently intentionally used in suicides in agricultural areas. For this reason, there is a need for therapeutics to rescue those from intoxication. Obvious ethical concerns prevent humans from being subjected to OP exposure for therapeutic efficacy and safety testing. Therefore, animal surrogates for humans must be appropriately selected. A new paradigm, described herein, incorporating both in silico and in vitro techniques may be able to reduce the use of animals in biomedical research. Historically, the guinea pig (Cavia porcellus) has been believed to be the best non-primate model for OP toxicology and therapeutic development because, similarly to humans, guinea pigs have low amounts of OP metabolizing carboxylesterase (CaE) in blood and tissues. To explore the hypothesis that guinea pigs are the most appropriate human substitute for studying OP toxicology iii and therapeutic development, I cloned, purified and enzymatically compared a recombinant guinea pig acetylcholinesterase (gpAChE) with the human and mouse enzyme variants. The guinea pig, mouse and human apparent inhibition constants for diisopropyl fluorophosphate were found to be 8.4±0.6 µM, 4.9±0.6 µM and 0.42±0.01 µM, respectively, indicating that species differences exist for OP inhibition. Furthermore, I developed a mechanistic quantitative structure-property relationship (QSPR) to predict OP and therapeutic tissue: plasma partition coefficient (Kt:pl) parameters for each species. Differences in tissue lipid, water and protein content contributed to species specific Kt:pl. For example, guinea pig and human lung Kt:pl predictions for paraoxon were found to be 0.3 and 0.17, respectively. Biological and chemical specific parameters were then incorporated into a SimBiology guinea pig and human physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) OP- therapeutic interaction model. A 7 regional compartment model was needed to adequately simulate the pharmacodynamics of VX in the brain. The OP PBPK/PD model was validated against the small amount of available data published in the literature and was used to predict and compare guinea pig and human species differences in response to exposure and therapeutic efficacy. It was found that the human is 3.45 times more sensitive than guinea pigs to VX as shown by the area under the curve in the brain, 1.14 times more sensitive than guinea pigs as shown by the area under the curve in the diaphragm and 1.11 times more sensitive as shown by the time to minimum iv concentration in the diaphragm. The OP PBPK/PD model structure, along with chemical parameters, can be altered to make predictions for other OP chemicals of concern. It was also shown that a constant intravenous infusion of a novel allosteric modulator that increases AChE’s velocity may be an effective means of treating dermal exposure in both guinea pigs and humans. In conclusion, this dissertation carefully evaluated physiological and enzymatic differences between these two species and greatly assisted in evaluating the suitability of the guinea pig as a model for human OP toxicity testing and therapeutic development. It is recommended that the guinea pig continue to be used as an animal model for OP toxicity testing and therapeutic development as long as the in silico and in vitro techniques, developed herein, are properly utilized to extrapolate to human populations. v TABLE OF CONTENTS Page I. INTRODUCTION…………………………………………………..……………………………………….1 1.1 Acetylcholinesterase Background..……………………………………………………..….1 1.2 Organophosphate Background…………………………………………………….…………3 1.3 Therapeutic Background……………………………………………………………………..…5 1.4 The Guinea Pig as an Organophosphate Animal Model………………………….6 1.5 Recombinant Acetylcholinesterase…………………………………………………………8 1.6 Physiologically Based Pharmacokinetic and Pharmacodynamic Modeling…………………………………………………………10 1.7 Tissue: Plasma Partition Coefficients..………………………………………………….11 1.8 Mechanistic Tissue Composition Partition Coefficient Models……………..12 1.9 Research Summary………………………………………………………………………………14 II. METHODS…………………………………………………………..…………………………….…….….16 2.1 Guinea Pig AChE Cloning and Enzymology………..……………………………….….16 2.1.1 Guinea Pig AChE Cloning into Gateway Entry Vector….…………………16 2.1.2 Guinea Pig AChE Cloning into Gateway Mammalian Destination Vector and Protein Expression……………………………………………17 2.1.3 Generation of a Stable Cell Line………….……………………………………..…19 2.1.4 Guinea Pig AChE Purification….…………..………………………………………..19 2.1.5 Enzyme Kinetics………….…………………………………………………………..……20 2.1.6 Homology Modeling….………………………………………………………………….23 2.2 Partition Coefficients……………….……………………………………………………………24 2.2.1 Partition Coefficient Model Development….…………………………………24 2.2.2 Unbound Chemical Fraction……….…………………………………………………26 2.2.3 Tissue: Plasma Partition Coefficient……………….………………..…………..26 2.2.4 Fraction Unbound……………….……………………………………………………....27 2.2.5 Influence of pH Gradients………………….……………………………………..….30 2.2.6 Tissue Composition Partition Coefficients……….…………………………...31 2.2.7 Conversion of Kt:pl to Kt:bl………………….………………………………………..…32 2.2.8. Tissue Composition Data Collection and Preprocessing………….……32 2.2.9 Monte Carlo Analysis………….……………………………………………………..…33 2.3 PBPK/PD Modeling………….…………………………………………………………………….34 2.3.1 SimBiology Model Development…………….…………………………………….34 2.3.2 VX PBPK/PD Model Development…………….…………………………………..35 2.3.2.1 Model Dosing Routes…….……………………………………………..……..36 2.3.2.2 Allometric Scaling…….………………………………………………………....37 vi 2.3.2.3 VX Model Parameterization and Validation….……………….…….38 2.3.3 Allosteric PBPK/PD Model…………………………………………………………….39 2.3.3.1 Evidence Supporting Acetylcholinesterase Allostery…….…..…39 2.3.3.2 PBPK/PD Model of Positive Drug Allostery……….…..……………..41 III. RESULTS…………………………………………………………..…………………………………………45 3.1 Guinea Pig AChE Cloning and Enzymology………………………………………….…45 3.1.1 Guinea Pig AChE Cloning……….……………………………………………………..45 3.1.2 Guinea Pig AChE Transfection and Purification…………………………….46 3.1.3 Organophosphate Dose Response………………………………………………..49 3.1.4 Inhibitor Ki Determinations………..……………….…………………………….….52 3.1.5 Zero-Time Plot Kinetics…………...……………………………………………………54 3.1.6 Homology Modeling……………………………………………………………………..56 3.2 Partition Coefficient Model Derivation………………….………………………………58 3.2.1 Tissue Composition Data Collection and Processing………….………….58 3.2.2 Monte Carlo Analysis: Passive Partitioning…….…………………..………..60 3.3 VX and Allosteric Modulator PBPK/PD Modeling……….………………………….65 3.3.1 VX PBPK/PD Modeling………………………………………………………………….65 3.3.2 Human PBPK/PD Model Extrapolation…………………….……………………79 3.3.3 Allosteric Modulator PBPK/PD Modeling………………………………………81 IV. DISCUSSION………………………………………………………………………………………………..83 4.1 Organophosphate Inhibition…………….……………………………………………………83 4.2 Partition Coefficients…………….………………………………………………………………87 4.3 VX and Allosteric Modulator PBPK/PD Modeling……….………………………….90 4.4 Summary…….………………..………………………………………………………..….…………96 V. REFERENCES….………………………………………………..………………………………………….98 VI. APPENDIX..………………………………………………………………………………….……………129 vii LIST OF FIGURES Figure Page 1. AChE’s mechanism for hydrolysis of acetylcholine………………………………………………2 2. AChE 3’ Splice Variants.………………………………………………………………………………………3 3. General Organophosphate Structure………………………………………………………………….4 4. Mechanism of organophosphate inhibition, reactivation, spontaneous hydrolysis and aging………………………………………………………………………5 5. Research Summary……………………………………………………………………………………………15 6. SignalP 4.1 Signal Peptide Cleavage Prediction…………………………………………………17 7. Gateway pENTR/D-TOPO and pT-Rex-DEST30 Plasmid Maps……………………………18 8. Ellman’s Method………………………………………………………………………………………………19 9. SimBiology Model Used to Predict Ellman’s Assay..…………………..……………………..23 10. Kt:pl Model Schematic…………………………………………..……………………………………………25 11. VX PBPK Model Schematics………………………………….…………………………………………..36 12. Allosteric