P326 Evaluation of P-glycoprotein expression in Pain Relevant Tissues Across Species for Understanding Translation of Efflux from Preclinical Species to Human

Renu S. Dhanikula , Dmpk, AstraZeneca R&D Montreal, Montreal, QC, Canada
Cyrla Hoffert , Translational Science, AstraZeneca R&D Montreal, Montreal, QC, Canada
Rebecca Grant , Translational Science, AstraZeneca R&D Montreal, Montreal, QC, Canada
Denis Projean , Dmpk, AstraZeneca R&D Montreal, Montreal, QC, Canada
Dajan O'Donnell , Translational Science, AstraZeneca R&D Montreal, Montreal, QC, Canada
Marie Roumi , Dmpk, AstraZeneca R&D Montreal, Montreal, QC, Canada
Translation of drug exposure and efficacy from preclinical species to human is imperative for the success of the drug discovery and development process. However, it is often a challenging endeavour, in particular, when the entry of molecules to the site of action is very limited and when little is known about the species differences in the expression and functionality of proteins or transporters expressed in the target tissue. Various efflux transporters, such as P-glycoprotein (Pgp), are now widely accepted to have profound influence on the disposition of substrates1. Nevertheless, there is paucity of information about their expression and functionality in pain relevant tissues (brain, spinal cord (SC) and dorsal root ganglia (DRG)) across various species. Therefore, our attempts were directed at evaluating the expression of Pgp at these target regions and understanding its effect on CNS disposition. Expression of Pgp protein in the fresh frozen brain, SC and DRG of Sprague Dawley rats, Cynomolgus monkeys and Human was examined using indirect immunohistochemistry with C219 and H241 antibodies (Calbiochem and Santacruz Biotechnologies). Diaminobenzidine was used as a chromogen for the purpose. Subsequently, cellular localization of Pgp was assessed by performing immunofluorescence based co-localization with C219 antibody and endothelial cell marker (Von-Willebrand factor antibody, Abcam). Leica DM4000B microscope and Zeiss confocal laser scanning TCS SPE microscope were used for capturing brightfield and fluorescence images, respectively. Evident differences were observed in the expression of Pgp in rat, monkey and human tissues. In the brain cortex, the expression of Pgp was relatively higher in rat than in monkey and human, while it was comparable in later two species (human and monkey). A similar trend was observed in the spinal cord expression of Pgp. Interestingly, there were no species differences in the Pgp expression in the DRG. Regional differences were observed within the spinal cord in all species, with the grey matter exhibiting higher protein levels relative to the white matter. Co-localization studies in all the examined tissues indicated that Pgp was primarily localized in the endothelial cells. These findings suggest that the exposure of Pgp substrates in human brain and SC could be higher than in rats but comparable to that in monkeys. Quantification of Pgp by LC-MS/MS is in progress to substantiate these findings. To the best of our knowledge this is the first report in the literature, wherein expression of Pgp in the DRG of various species has been demonstrated.