The role of aryl hydrocarbon receptor (AHR) in drug-drug interaction and the expression of AHR in Pichia Pastoris

Author

Yujuan Zheng, University of the PacificFollow

Date of Award

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Miki S. Park

First Committee Member

William K. Chan

Second Committee Member

Jae H. Chang

Third Committee Member

Melanie Felmlee

Fourth Committee Member

Qinliang Zhao

Abstract

The aryl hydrocarbon receptor is a ligand-activated transcription factor that is involved in many important functions in the body. To study the role and function of AHR, an abundant amount of in vitro expressed and purified protein is needed. A baculovirus insect expression system is commonly employed to express AHR, however, there are several drawbacks with this method, such as mutation potential and high cost. A better in overexpression system is needed and we hypothesize that Pichia pastoris, a yeast expression system, could stably express AHR and ARNT (aryl hydrocarbon receptor nuclear translocator) in sufficient amount with reasonable cost. Codon optimized human AHR and ARNT genes were separately transformed into the Pichia pastoris genome and expressed. Co-immunoprecipitation, gel-shift assay and western analysis indicate Pichia pastoris was able to stably overexpress functional AHR and ARNT proteins in comparable yield and lower cost compared to baculovirus insect expression system and the expressed proteins were used to develop a new in vitro method to study AHR and ARNT binding.

Pharmacokinetic studies were performed to investigate the role of AHR in rutacarpine-caffeine interactions. Oral RUT pretreatment was shown to reduce oral caffeine area under the curve (AUC) in rats, due to an increase in caffeine clearance (CL) and a decrease in oral bioavailability (F). RUT, an AHR ligand, increases caffeine CL by inducing Cyp1A2 enzyme, but the mechanism by which RUT reduces caffeine F is not understood. We hypothesize that it is also mediated via AHR pathway. To test the hypothesis, wild type (WT) and AHR knock out (KO) mice were administered caffeine IV and orally, with and without VEH or RUT pretreatment. As expected, PK data show higher caffeine CL and lower F values in WT, but similar CL and F values in AHR KO mice, upon RUT co-administration.

Rats study, in which with pretreatment of vehicle, AHR ligands: RUT, beta-naphthoflavone or indole-3-carbinol before caffeine was dosed orally is consistent with mice study, that all three AHR agonists tested were able to reduce oral caffeine AUCs in rats.

RUT reduces caffeine’s oral bioavailability is through AHR signaling pathway, however, However, the mechanism by which AHR mediates the reduced caffeine F is not known.

Pages

142

Recommended Citation

Zheng, Yujuan. (2019). The role of aryl hydrocarbon receptor (AHR) in drug-drug interaction and the expression of AHR in Pichia Pastoris. University of the Pacific, Dissertation. https://scholarlycommons.pacific.edu/uop_etds/3580