Development and application of analytical chemical methods for the simultaneous analysis of the behavioral and pharmacokinetic response to amphetamine in rats

Abstract

Amphetamine is a psychostimulant that is used clinically for the treatment of attention-deficit hyperactivity disorder and narcolepsy. Using a force-plate actometer, amphetamine has been determined to produce a predictable behavioral phenomenon known as 'focused stereotypy' in Sprague-Dawley and Fischer 344 (F344) rats. Chronic administration of amphetamine can result in behavioral sensitization, a phenomenon by which later doses result in a more intense behavioral response than the same, initial dose. In rats, the behavioral response to a determined dose of amphetamine varies between strains and between individual rats within a strain. These variations result in uncertainty as to whether the resulting behavioral differences elicited by the drug are pharmacokinetic or pharmacodynamic in nature. Microdialysis provides a means to collect extracellular fluid from a particular brain region which can then be measured to determine the pharmacokinetic response to amphetamine in this region. A high performance liquid chromatography method using tandem mass spectrometry detection was developed for the analysis of amphetamine, the amphetamine metabolites (p-hydroxyamphetamine, norephedrine and p-hydroxynorephedrine), dopamine and clozapine to attempt to measure these compounds in dialysate obtained from the striatum of rat brains. This method was combined with high resolution behavioral measures obtained from a force plate actometer to determine how closely associated drug pharmacokinetics are to the behaviors they elicit. Results indicate that amphetamine induced behaviors are not clearly linked to amphetamine pharmacokinetics and that the observed behavioral differences are likely pharmacodynamic in nature. The atypical antipsychotic drug clozapine has been shown to lengthen the duration of amphetamine induced focused stereotypy in rats. Amphetamine/clozapine interactions cannot be ruled out as a possible cause of this phenomenon. Because microdialysis was ineffective in determining clozapine levels in rat brain, a liquid/liquid extraction method was used to determine whether amphetamine had an effect on whole brain clozapine concentrations. Using this method, it was determined that clozapine levels were not affected by amphetamine at the time point analyzed.