Publication Date

2015

Document Type

Dissertation

Committee Members

Jeffery Gearhart, Ph.D. (Advisor); Adrian Corbett, Ph.D. (Committee Member); James Lucot, Ph.D. (Committee Member); Mateen Rizki, Ph.D. (Committee Member); Gerald Alter, Ph.D. (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

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 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 uM, 4.9+/-0.6 uM and 0.42+/-0.01 uM, 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 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.

Page Count

176

Department or Program

Biomedical Sciences

Year Degree Awarded

2015

Download


Share

COinS