Abstract
The purpose of this study was to demonstrate experimentally that alterations of in vivo transporter function at the blood-brain barrier (BBB) in disease and during pharmacotherapy can be reconstructed from in vitro data based on our established pharmacoproteomic concept of reconstructing in vivo function by integrating intrinsic transport activity per transporter molecule and absolute protein expression level at the BBB. Pentylenetetrazole (PTZ)-kindled and spontaneous model of epilepsy (EL) mice were used as models of chemically induced and spontaneous epilepsy, respectively. A mouse model of antiepileptic drug treatment was prepared by consecutive 5-week administration of phenytoin (PHT). Quantitative targeted absolute proteomic analysis of 31 membrane proteins showed that P-glycoprotein (P-gp/mdr1a) protein expression levels were significantly increased in brain capillaries of PTZ (129%), EL (143%), and PHT mice (192%) compared with controls. The brain-to-plasma concentration ratios (Kp brain) of P-gp/mdr1a substrate verapamil were 0.563, 0.394, 0.432, and 0.234 in control, PTZ, EL, and PHT mice, respectively. In vivo P-gp/mdr1a function at the BBB was reconstructed from the measured P-gp/mdr1a protein expression levels and intrinsic transport activity for verapamil per P-gp/mdr1a previously reported by our group. Then, the reconstructed P-gp/mdr1a functional activities were integrated with unbound fractions of verapamil in plasma and brain to reconstruct Kp brain of verapamil. In all mice, reconstructed Kp brain values agreed well with the observed values within a 1.21-fold range. These results demonstrate that altered P-gp functions at the BBB in epilepsy and during pharmacotherapy can be reconstructed from in vitro data by means of our pharmacoproteomic approach.
Footnotes
- Received May 13, 2014.
- Accepted July 24, 2014.
This work was supported in part by four grants-in-aid from the Japanese Society for the Promotion of Science (JSPS) for Scientific Research (S) [KAKENHI: 18109002], Scientific Research (A) [KAKENHI: 24249011], Young Scientists (B) [KAKENHI: 23790170], and a JSPS fellowship [KAKENHI: 207291]. This work was also supported in part by two grants for the Development of Creative Technology Seeds Supporting Program for Creating University Ventures and the Revitalization Promotion Program (A-STEP) from the Japan Science and Technology Agency.
T.T. and S.O. are full professors at Tohoku University and Kumamoto University, respectively, and are also directors of Proteomedix Frontiers. This work was not supported by Proteomedix Frontiers, and their positions at Proteomedix Frontiers did not affect the design of the study, the collection of the data, the analysis or interpretation of the data, the decision to submit the manuscript for publication, or the writing of the manuscript, and did not present any financial conflicts. The other author declares no conflicts of interest.
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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