552
B. Lord et al. / European Journal of Pharmacology 765 (2015) 551–559
et al., 2014).
concentration of the radioligand was either associated with in-
creasing time of incubation or dissociated with an excess of a
competitor (A-804598; 1 M). For the association study, two
concentrations of tracer (close to Kd and 2Kd) were incubated with
increasing time (1, 5, 10, 15, 20, 30, 40, 60 and 120 min) with
High affinity and selective radioligands are used in drug dis-
covery programs to define binding affinity of compounds under
true equilibrium where complete displacement is achieved, or in
cases of partial or no displacement, a radioligand can reveal al-
ternate binding pockets. In addition, radioligands are also used to
determine the ex vivo receptor occupancy of central P2X7 re-
ceptors after peripheral administration of drugs (Able et al., 2011;
Bhattacharya et al., 2013; Lord et al., 2014). [3H] A-804598 (2-Cy-
ano-1-(1-phenylethyl)-3-quinolin-5-ylguanidine) is the first and
only radioligand described to date for P2X7 (Donnelly-Roberts
et al., 2009). However, we observed higher than optimal non-
specific binding and lack of complete displacement in rat brains
but not in cells expressing the recombinant rat P2X7 channel. We
focused on identifying a second generation P2X7 radiotracer and
we describe [3H] JNJ-54232334 (7-[2-Chloro-3-(trifluoromethyl)
benzyl]-6-methyl-3-pyrimidin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-
a]pyrazin-8(5H)-one), as a novel P2X7 radioligand.
μ
membranes in volumes of 40
10 l (buffer). For the dissociation study, the total incubation vo-
lume was 290 l (40 l tracer; 50 l membrane; 200 l A-804598;
M); dissociation was initiated by adding an excess of (200 l)
A-804598 (1 M) at different time points (240, 210, 180, 150, 120,
μ
l (tracer), 50
μ
l (membrane) and
μ
μ
μ
μ
μ
1
μ
μ
μ
105, 90, 75, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 2 and 1 min)
after a 2 h incubation with tracer and membrane alone. For dis-
placement binding, increasing concentrations of the cold ligand
were added to the tracer and membrane in a 100
μ
l (10
μ
l cold
ligandþ40 l tracerþ50 l membrane suspension) total volume
μ
μ
for an incubation time of 1 h. In all cases, the experiment was
terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and
washed with washing buffer (Tris–HCl 50 mM) followed by drying
the plate for 30–40 min. Radioactivity was read in TopCount
(Perkin Elmer) by adding MicroScint 0 in each well. Data are from
a single experiment with 4 replicates per data point. Radioligand
binding parameters (Kd, Bmax, Kon, Koff) were calculated in Graph
Prism Software version 5.0. All data are presented as mean7S.E.
M. unless otherwise stated.
2. Materials and methods
All animal work described in this paper was done in accordance
with the Guide Care for and Use of Laboratory Animals adopted by
the US National Institutes of Health. Animals were allowed to ac-
climate for 7 days after receipt. They were group housed in ac-
cordance with institutional standards, received food and water ad
libitum and were maintained on a 12 h light/dark cycle.
2.2. In vitro receptor binding autoradiography in rat, wild-type and
P2X7 knockout brain sections
Three naïve male Sprague Dawley Rats (approximately 400 g in
body weight, Harlan Laboratories Livermore, California, USA),
3 naïve C57/bl6 wild-type (WT) mice and 3 P2X7 knockout (KO)
mice (Solle et al., 2001) (8–12 weeks, Jackson Labs, Bar Harbor,
Maine, USA) were euthanized by CO2 asphyxiation. Brains were
rapidly frozen on powdered dry ice and stored at ꢀ80 °C before
sectioning. Tissue sections at the level of the hippocampus of
2.1. In vitro P2X7 pharmacology
The calcium flux assay and the radioligand binding assay were
used to define the potency and affinity of JNJ-54232334 and JNJ-
54140515, respectively. Bz-ATP was used to stimulate P2X7 in
functional assays and [3H] A-804598 was used as the radioligand
of choice to measure the affinity of JNJ-54232334 at the re-
combinant rat P2X7 membranes whereas [3H] A-804598 and [3H]
JNJ-54232334 were both used in rat brain homogenates. For cal-
cium flux assay, 1321N1 cells expressing P2X7 orthologues were
dissociated 18–24 h prior to the assay using 0.05% trypsin/EDTA
(Invitrogen, Grand Island, NY, USA), and plated at density of 25,000
20 m thickness were prepared for autoradiography as previously
μ
described (Langlois et al., 2001). The sections were kept at ꢀ80 °C
until use. Briefly, sections were pre-incubated for 15 min at room
temperature in 50 mM Tris HCl with 0.1% BSA and then incubated
for 1 h in fresh 50 mM Tris HCl, 0.1% BSA supplemented with [3H]
A-804598 (30 nM) or [3H] JNJ-54232334 (10 nM). The non-specific
binding was determined using adjacent sections incubated in the
cells wellꢀ1 into poly-
D-lysine coated 96-well black-walled clear
bottom plates (Becton-Dickinson, Bedford, MA,USA). On the day of
the experiment, cells were washed with assay buffer, containing
(in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH
7.4 and dye loaded with a 2 ꢁ Calcium-4 (Molecular Devices,
Sunnyvale, CA, USA). Cells were stained with the Calcium-4 dye in
staining buffer for 30 min at room temperature in the dark. Test
compounds were prepared at 250 ꢁ the final test concentration in
neat dimethylsulfoxide. Intermediate 96-well compound plates
presence of 100
54232334) A-740003 (N-[1-[[(Cyanoamino)(5-quinolinylamino)
methylene]amino]-2,2-dimethylpropyl]-3,4-dimethox-
μ
M
([3H] A-804598) or 10 ([3H] JNJ-
M
μ
ybenzeneacetamide). At the end of the incubation, sections were
washed 4 times (5 min each) in ice cold buffer followed by 2 dips
in deionized water and rapidly dry under a stream of cold air.
Digitized images were acquired with the TRacer
lowing a 3 h acquisition with beta acquisition software version
9.4 and high resolution images were acquired with the DFine -
β-Imager fol-
were prepared by transferring 1.2
of assay buffer. A further 3 ꢁ dilution occurred when transferring
50
L wellꢀ1 of the compound plate to 100 L wellꢀ1 in the cell
μ
L of the compound into 300
μL
β
Imager following 96 h session with beta D acquisition software
(Biospacelab, Paris, France)
μ
μ
plate. Cells were incubated with test compounds and dye for
30 min. Calcium flux was monitored in Fluorescence Imaging Plate
2.3. Ex vivo receptor autoradiography in rat brain sections
reader (FLIPRTetra
)
as the cells were challenged by adding
L wellꢀ1 of BzATP. The final concentration of Bz-ATP was
M. Experiments were run in triplicate with antagonistic
50
250
μ
Male Sprague Dawley Rats approximately 400 g in body
weight, Harlan Laboratories were used. JNJ-54140515 was for-
μ
potency values calculated in GraphPad Prism Software version 5.0
(San Diego, CA) and expressed as pIC50.
mulated in 20% Hydroxypropyl- -Cyclodextrin and delivered in a
β
volume of 5 ml/kg for p.o. dosing and 1 ml/kg for s.c. dosing. For
the 10 mg/kg s.c. time course experiment, three animals per time
point were used with a time course of 0.25, 0.5, 1, 2, 4, 6, and 24 h
post dose. Three animals per dose were used for the p.o. dose
dependent receptor occupancy experiment with doses of 0.01,
0.03, 0.1, 0.3, 1, 3, 10, 30 mg/kg with tissue taken 0.5 h post dose.
Ex vivo receptor binding was performed as described in the in
For radioligand binding with Sprague Dawley rat cortex, tissues
were homogenized followed by high-speed centrifugation
(32,000 g) of the supernatants for 30 min. The membrane pellet
was then re-suspended in ice cold assay buffer (Tris–HClþ0.1%
BSA) so that the final concentration of membrane was 400
μ
g/well.
fixed
For association and dissociation experiments,
a
Lord, Brian
