045P Edinburgh
BPS Summer Meeting 2009

Determining PK/PD relationships using LCMS based GABAA receptor occupancy

Rebecca Fish1, Nisha Singh2, Alan Edgington1, Cerian Powell1, Sidath Katugampola1, David Tattersall1. 1Pfizer, Sandwich, United Kingdom, 2University of Oxford, Oxford, United Kingdom.

 

Receptor occupancy (RO) is classically determined using ex vivo and in vivo radio-labelled studies. In the absence of suitable radiolabels, RO may still be determined by High Performance Liquid Chromatography coupled with Mass Spectrometry (LCMS/MS) using unlabelled tracers (Barth et al., 2006; Chernet et al., 2005; Need et al., 2007 and Hopkins et al., 2009). The aim of this study was to compare traditional ex vivo and in vivo methods with LCMS to determine the whole brain central GABAA RO/plasma relationships of bretazenil and diazepam. Flumazenil was used as either the radiolabel or unlabelled tracer in male CD rats (200-250g). Plasma dependent RO was observed with bretazenil and diazepam. There was no statistical difference in the plasma logED50 values obtained using ex vivo, in vivo and LCMS methodologies for bretazenil or diazepam (Unequal variance t-tests, P<0.05, Table 1).

 

Method
Dose, route, pre-treatment time		Ex vivo
[3H] Flumazenil, 1nM		In vivo
[3H] Flumazenenil, 10μCi/kg, i.v.		LCMS
Flumazenil, 10μg/kg, i.v.
Bretazenil
(0.1-5mg/kg, i.p., t=30min)		3.3 (2.3-4.8)2.7 (0.8-8.9)5.0 (2.0-12.4)
Diazepam
(1-10mg/kg, i.p., t=30min)		158 (86-293)87 (38-201)152 (78-297)

Table 1 Plasma levels (nM) of bretazenil or diazepam required to give 50% GABAA RO, 95% confidence intervals in brackets. NSB was determined with 5mg/kg, i.p. bretazenil, n=4. Bretazenil and diazepam showed dose dependant occupancy, ED50 values of 0.06, 0.06 and 0.04mg/kg and 3, 6 and 9mg/kg, for in vivo, ex vivo and LCMS respectively.

Such data is in agreement with the in vitro binding profile of bretazenil and diazepam (Giusti et al., 1993) providing translatable plasma PK occupancy values. Additionally, for novel targets where a radiolabel is unavailable, such a LCMS RO approach may provide an alternative means to help establish PK/PD relationships.

 

Barth V et al., (2006). Life Sci. 78: 3007-3012.
Chernet E et al., (2005). Life Sci. 78: 340-346.
Giusti et al., (1993). J. Pharm. Exp. Ther. 266: 1019-1028.
Hopkins et al., (2009). Biopharm. Drug Dispos. 30: 9-20.
Need AB et al., (2007). Life Sci. 81: 1389-1396.