Genetic or chemical knock-out of P-glycoprotein (P-gp;MDR1) at the rodent blood-brain barrier (BBB) significantly increases (by 10 to 30-fold) the distribution of P-gp substrate drugs into the brain. Based on these data, it has been widely postulated that P-gp plays a vital role in limiting drug distribution at the human BBB and that P-gp based drug interactions at the human BBB are likely to be profound. Our hypothesis challenges this well-established paradigm and claims that such interactions will be modest because therapeutic plasma concentrations of potential P-gp inhibitor drugs will be insufficient to profoundly inhibit Pgp at the human BBB. Moreover, we hypothesize that such drug interactions can be quantitatively predicted by in vitro cell models and in vivo studies in the rat. The development by our laboratory of a novel and innovative non-invasive, Positron Emission Tomography (PET) imaging method to measure P-gp activity at the human BBB will allow us to test these hypotheses. Since P-gp can demonstrate allosteric activation and multiple binding sites, predictions of P-gp based drug interactions can be complicated by these phenomena. Therefore, our specific aims will be: 1. In vitro studies: We will determine the potency of a variety of drugs (EC50) to inhibit P-gp efflux of verapamil-bodipy by LLCPK1 cells expressing the MDR1 gene or an empty vector (control cells). 2. In vivo rodent studies: For the drugs studied (aim 1), we will determine the ratio of the therapeutic maximum plasma concentration (Cmax) and EC50 (Cmax/ECso) as well as the unbound maximum plasma concentration (Cmaxu) and EC5o(Cmaxu/EC5o). For those drugs (n=4) that are potent inhibitors of P-gp at their therapeutic concentrations (highest ratios), we will determine their in vivo EC50 at the rat BBB using [3H]-verapamil as the P-gp substrate. In vivo human studies: The two most potent inhibitors identified from the rat studies will be tested (at Cmax) for their ability to inhibit P-gp activity at the human BBB by measuring, using PET, their effect on the distribution of [11C]-verapamil into the brain. 4. In vitro-vivo correlation: We will determine (a) if the above in vitro EC50 and in vivo ECso values in the rat are correlated;and (b) whether the in vitro and in vivo rodent data are predictive of the magnitude of interaction observed at the human BBB at the Cmax of the inhibitor. 5. P-QD allosterism and multiple binding sites: We will determine if the interaction of P-gp with its drug substrates demonstrates allosterism and multiple binding sites. If it does, these phenomena will need to be taken into consideration when predicting in vivo P-gp based drug interactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM032165-30
Application #
8380469
Study Section
Special Emphasis Panel (ZGM1-PPBC-6)
Project Start
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
30
Fiscal Year
2012
Total Cost
$283,705
Indirect Cost
$101,884
Name
University of Washington
Department
Type
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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