Download Splice variant - Journal of Pharmacology and Experimental

AZD3355, a novel GABAB receptor agonist, inhibits transient lower esophageal
sphincter relaxation through a peripheral mode of action
Anders Lehmann, Madeleine Antonsson, Ann Aurell Holmberg, L. Ashley
Blackshaw, Lena Branden, Hans Brauner-Osborne, Bolette Christiansen, John Dent,
Thomas Elebring, Britt-Marie Jacobson, Jorgen Jensen, Jan P. Mattsson, Karolina
Nilsson, Simo S. Oja, Amanda J. Page, Pirjo Saransaari, and Sverker von Unge
jpet.109.153593
Supplemental Information
Effect of GABAB receptor agonists on various GABAB(1) splice variants
The human GABAB(1) splice variants GABAB(1a), GABAB(1b), GABAB(1e), GABAB(1g),
GABAB(1m) and GABAB(1o), as well as GABAB(2), were cloned from a human brain
cDNA and subcloned into pCI-Neo expression vectors. Transient co-transfections of
GABAB(1) splice variants and GABAB(2) in CHO-Gqi5 cells were performed using
lipofectamin (Life Technologies, Paisley, Scotland) according to the manufacturer’s
protocol. In brief, for each transfection, CHO-Gqi5 cells (≈1.4 million cells) were
seeded in T75 flasks 24 hours prior to the transfection to obtain 50–70% confluency.
Cells were washed with 5 mL OptiMEM (Life Technologies) and thereafter incubated
in 5 mL OptiMem for approximately 1 hour. Plasmid DNA (4 g), 4 mL OptiMEM
and 24 L lipofectamine were mixed (transfection mixture) and incubated for 45 min
at room temperature. Cells were incubated with transfection mixture at 37C. After 5
hours, the transfection mixture was removed and 14 mL culture medium added
(Ham’s F12 with Glutamax-1, supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, and 100 µg/mL streptomycin; Life Technologies) After 24 hours, cells
were washed with 5 mL phosphate buffered saline (PBS) without calcium and
magnesium (Life Technologies) followed by 6 mL PBS containing 500 BAEE units
trypsin and 0.5 mM ethylenediaminetetraacetic acid (Trypsin-EDTA (T/E), Sigma-
Aldrich, St. Louis, MO) to detach the cells. Cells from the same transfection were
then used either for fluorescence imaging or saturation binding analysis. For the
purposes of fluorescence imaging, cells were seeded (≈45,000 cells/well in 200 µL
culture medium) in poly-D-lysine coated, black walled plates (Becton Dickson,
Bedford, UK) for 24 hours. Results for agonistic activity of AZD3355 and baclofen
for different GABAB receptor splice variants are summarized in Supplemental Table
1.
For saturation binding analysis on whole cells using [3H]CGP54626 (Tocris, Bristol,
UK), cells (200,000–300,000 cells/well) were seeded in a 24-well plate and incubated
for 24 hours. For cell counting, cells were washed with 0.5 mL PBS, detached with
0.5 mL PBS containing T/E. The cell suspension was then mixed with Trypan blue
(Life Technologies) and counted in a Bürker chamber (excluding cells stained with
Trypan blue). For saturation binding analysis, cells were washed 2 times with 0.8 mL
Hank’s buffered salt solution (HBSS, Life Technologies) followed by the addition of
0.5 mL HBSS containing [3H]CGP546262 (0.1–30 nM) and 0.1% ethanol. Nonspecific binding was determined by the inclusion of 3 mM GABA in the assay buffer.
After 45 min incubation at 37C, the cells were rapidly rinsed with 2  0.8 mL icecold HBSS. Cells were solubilized by addition of 250 µL 0.1 M NaOH and then
transferred to a new 24-well plate (Wallac 1450-408). The wells in the original plate
were washed with 180 µL H2O. The cells were mixed with 0.5 mL scintillation
cocktail for 20 min and radioactivity determined in a Micro- counter (Wallac, Turku,
Finland). KD and Bmax values were calculated by fitting binding data to the equation
B=Bmax/(1+KD/[L]), using Xlfit for Microsoft Excel. Findings for [3H]CGP54626 are
summarized in Supplemental Table 2.
Uptake of radiolabeled GABAB receptor agonists in rat cerebrocortical slices
Rat cerebrocortical slices were prepared and incubated with 0.1, 1.0 or 10.0 M of
labeled compounds for 5, 10, and 30 min. The intracellular uptake of [3H]GABA
(Amersham International, Bristol, UK, specific activity 3.40 TBq/mol, postitive
control), [14C]baclofen (3.2 TBq/mol, radiochemical purity 99%), and [14C]AZD3355
(5.8 TBq/mol, radiochemical purity 96%) was calculated after subtraction of the
amount of label in the extracellular spaces, estimated with the hydroxyl[14Cmethyl]inulin (Amersham) method (Laakso and Oja, 1976).
In brief, superficial slices (0.4 mm thick) were manually cut from both cerebral
hemispheres of 3-month-old Sprague-Dawley rats (weight about 300 g; Orion Ltd,
Espoo, Finland) and immediately transferred to 4 mL of medium (composition in
mM): NaCl 127, KCl 5, CaCl2 2.5, NaH2PO4 1.3, MgSO4 1.2, HEPES 10 and Dglucose 10, pH 7.4) and incubated at 37oC under O2 with 0.1, 1.0 or 10.0 M of
labeled compounds for 5, 30 or 60 min. The incubations were terminated by rapid
filtration and rinsing of the slices with 5 mL of ice-cold medium. The slices were
frozen in liquid nitrogen, weighed, and extracted with 5% (w/v) trichloracetic acid
solution. The slice extracts and medium were subjected to scintillation counting. The
amount of compound in the intracellular space was calculated by relating the
radioactivity in the slice extracts to the specific radioactivity in medium after
subtraction of the amount of radioactivity retained in the extracellular spaces. The size
of the extracellular space was estimated with the hydroxy[14C-methyl]inulin
(Amersham) method (Laakso and Oja, 1976). The results are given as mol/kg wet
weight of incubated slices.
[3H]GABA uptake assay and FLIPR® membrane potential assay in mammalian
cells transiently expressing the human GATs
Cell culture and transfections – tsA201 cells (a transformed HEK293 cell line) were
cultured in GlutaMAX-I DMEM supplemented with 10% fetal bovine serum,
penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37°C in a humidified
atmosphere of 95% air and 5% CO2. The constructs encoding each of the human
GABA transporters (termed hGAT-1, hBGT-1, hGAT-2 and hGAT-3) (Christiansen
et al., 2007; Kvist et al., 2009; in press) inserted in the pcDNA5/FRT vector
(Invitrogen) were transiently transfected into cells using PolyFect according to the
protocol of the manufacturer (Qiagen, West Sussex, UK). Human EAAT3 was used as
negative control, as previously described (Christiansen et al., 2007). The functional
assays were performed 36–48 hours later.
[3H]GABA uptake assay – tsA201 cells transfected with each of the four human
GABA transporters were split into poly-D-lysine-coated white 96-well plates (PerkinElmer, Boston, MA, USA). The next day, the medium was removed and cells were
washed with 100 µL assay buffer (HBSS supplemented with 20 mM HEPES, 1 mM
CaCl2, and 1 mM MgCl2, pH 7.4). Then 75 µL assay buffer supplemented with 30 nM
[3H]GABA (or 30 nM D-[3H]Asp in the experiments with human EAAT3) and
various concentrations of the test compounds was added to each well and the plate
was incubated at 37°C for 3 min. Cells were then washed with 3  100 µL ice-cold
assay buffer and 150 µL MicroscintTM20 scintillation fluid (Perkin-Elmer, Boston,
MA, USA) was added to each well. The plate was shaken for at least 1 hour and
counted in a Packard TopCount microplate scintillation counter. The [3H]GABA
competition curves were constructed based on measurements obtained for eight
different concentrations of the test compounds. The experiments were performed in
triplicate in at least three independent experiments.
The FLIPR® membrane potential (FMP) assay – The test compounds were
characterized functionally in the FMP assay (Molecular Devices, Crawley, UK)
essentially as described previously (Jensen and Bräuner-Osborne, 2004). Briefly,
tsA201 cells transfected with each of the four human GABA transporters were split
into poly-D-lysine-coated black clear bottom 96-well plates (BD Biosciences,
Bedford, MA, USA). The next day, the culture medium was removed, and the cells
were washed with 100 µL assay buffer (same buffer as used in the [3H]GABA uptake
assay). Then 100 µL assay buffer supplemented with FMP assay dye was added to
each well, and the plate was incubated at 37°C for 30 min. The plate was assayed at
30°C in a NOVOstarTM plate reader (BMG Labtechnologies, Offenburg, Germany)
measuring emission at 560 nm caused by excitation at 530 nm before and up to 1 min
after addition of 25 µL substrate solution (the substrate was dissolved in assay buffer).
The concentration-response curves were constructed based on measurements obtained
for eight different concentrations of the test compounds. The experiments were
performed in triplicate in at least three independent experiments.
Data analysis – All data were analyzed using Prism 5.0a (GraphPad Software, San
Diego, CA, USA). The curves were fitted by nonlinear regression using the equation
Y = Bottom + (Top – Bottom)/(1+10^((log IC50 – X)  Hill slope), where X is the
logarithm of the concentration, Y is the response, Top is the response at the top
plateau, Bottom is the response at the bottom plateau, log IC50 is the concentration
when the response is half way between Top and Bottom. The Hill slope describes the
steepness of the curve.
Transient lower esophageal sphincter relaxations (TLESRs) in ferrets
Experiments were performed on adult female ferrets (weight range 0.6–0.8 kg)
obtained from the Institute for Medical and Veterinary Science, Adelaide, South
Australia. In brief, chronic lateral cervical mucosa-to-skin esophagostomies were
constructed under anesthesia. Manometric studies were begun at least a month after
surgery. Manometry was performed with a micromanometric assembly (2.0-mm OD;
Dentsleeve, Wayville, Australia) inserted through the esophagostomy. A pH electrode
(0.8-mm OD; FlexilogTM Oakfield Instruments, Oxford, UK) was attached 10 mm
above the proximal end of the sleeve sensor and referenced to an Ag-AgCl electrode.
The combined pH/manometric assembly was introduced via the esophagostomy and
held in place by a Neoprene harness around the animal’s upper thorax and neck.
Swallows were detected with a microphone that was attached to the collar and
positioned over the hyoid bone. After preamplification (Synectics Polygraf),
manometric and pH outputs were acquired to disk and analyzed off-line with
Labview-based software. Audio signals from the throat microphone were converted to
a software time marker and integrated with other analog inputs.
TLESR in dogs dosed once daily with AZD3355 for 14 days
The inhibitory effect of baclofen on TLESR in the dog has been shown to remain
stable after once daily administration for 14 days (Lehmann et al., 2000). However,
since tolerance to GABAB receptor stimulation may be agonist-specific (Enna et al.
1998), it was deemed necessary to determine the effects of AZD3355 after repeated
dosing. Six dogs were administered AZD3355 at 7 mol/kg p.o. once a day for 14
days, and the effects on TLESR were assessed on day 1, 3, 7 and 14 using the same
protocol as described for the other experiments. The effect on day 1 was lower than
that established in the dose-response experiments, but there was no difference
between the different experimental days (Supplemental Figure 2). This indicates that
the effect of AZD3355 is maintained after repeated administration, at least in the
short-term.
Hypothermia after s.c. administration of GABAB receptor agonists in mice
For each substance and time, an exponential model, given by
Tj,k  a  exp (b  dosek )   j,k ,
dosek  0,  j,k ~ N( 0,  2 )
was fitted to data. The response T j,k was defined as the j th subject’s temperature
change from baseline adjusted with the average change from baseline of all animals
that were given dose zero. As baseline value, the average of all pre-drug
administration measurements was used. In this model  denotes the change from
baseline for dose zero and e  denotes the change in T when the dose increases with
one unit, that is, Tk /Tk 1 when dosek 1  dosek  1 . In addition, this model was
fitted under the assumption of homogeneous normal errors,  j,k ~ N( 0,  2 ) . The
fitted model was then used to calculate ED2 , the dose producing a 2ºC drop in
temperature.
Whole body autoradiography in rats
A group of four male rats (Long Evans black hooded male rats, M&B A/S, Denmark)
were each given 5.0 mL/kg of the test formulation corresponding to 10 µmol/kg (20
MBq/kg) body weight of [14C]baclofen. Following i.v. injection, individual rats were
killed at varying times ranging from 5 min to 6 hours. [14C]AZD3355 (5.4 mL/kg)
was administered at a nominal level of 20 µmol/kg body weight and the radioactive
doses were 5.7 MBq/kg body weight. Time of sacrifice ranged from 5 min to 48
hours. Both male and female rats were used (n=23; male Lister Hooded strain rats
were from Charles River, Margate, Kent, UK, and pregnant female rats of the same
strain, 18 days of gestation, were from Harlan, Bicester, Oxon, UK), and no sexrelated differences in distribution of radioactivity was noted. Also, dosing was either
i.v. or p.o. in males and i.v. in females, and again, there were no differences
attributable to the route of administration in terms of distribution of radioactivity to
the central nervous system (CNS). At the time of the experiment, the animals weighed
about 200 g. The sectioning and visualization of distribution, with specific reference
to the CNS, was done according to standard methods.
The animals were anesthetized and killed by complete immersion in hexane, cooled to
-70°C. After removal of the limbs and tail, each carcass was embedded in a 2.5%
aqueous solution of carboxymethyl cellulose and frozen for at least 10 minutes at 70°C. The animal blocks were stored at -20ºC until sectioning. Each block was
subsequently mounted in a Leica CM3600 Cryostat (Leica Microsystems, Germany)
maintained at approximately -20ºC. After initial trimming of the block, sagittal whole
body sections (20-30 µm) were obtained at different levels through the carcass. Each
section was attached to tape and numbered consecutively.
Five to six sections from each rat were chosen for phosphor imaging to best
represent the different areas in brain and other organs of interest. The sections
were talced and placed against phosphor imaging screens for 43 to 88 hours
(baclofen experiments) or 7 days (AZD3355 experiments). The imaging plates
were exposed in refrigerated light tight cassettes in a shielding box to protect
from environmental radiation.
Following exposure the imaging plates were unloaded in subdued light and
scanned using Fuji BAS 2500 or 1500 radioluminography. The latent
images were visualized and stored in an electronic data file for subsequent
analysis.
Chemicals
GABA and baclofen were purchased from Tocris, Bristol, UK. AZD3355, ARH061719, and AR-H040551 were synthesized at AstraZeneca R&D, Mölndal
Sweden or Albany Molecular Research, Albany, NY, USA. The radiolabeled
compounds were synthesized by Roger Simonsson, AstraZeneca R&D
Mölndal, Sweden. All other chemicals were of highest commercially available
grade.
Extrapolation of results across species
The results in the present study are based on studies performed on recombinant human
GABAB receptors, rat and dog brain membranes, as well as in vivo experiments in the
rat, mouse, ferret, and dog. In order to be able to make any cross-species comparisons,
the similarities of the GABAB receptor between species have to be considered. The
GABAB receptor is one of the most highly conserved G-protein coupled receptors
known, and the homology across the species used here is >98% on an amino acid
level. It is therefore unlikely that there are any important species differences in terms
of potency of isosteric agonists such as AZD3355 and baclofen. Further, the
displacement of [3H]GABA from dog and rat brain membranes with AZD3355 was
identical. Previous work has shown that baclofen has very similar potency in dogs
(Lehmann et al., 1999), ferrets (Blackshaw et al., 1999), cats (Liu et al., 2002), and
humans (Lidums et al., 2000; van Herwaarden et al., 2002; Zhang et al., 2002; Lee et
al., 2003) as an inhibitor of TLESRs. It therefore seems reasonable to extrapolate
results obtained in one species to the other species. Indeed, apart from expected
differences in pharmacokinetics related to body mass (as illustrated by the different
pharmacokinetic properties of AZD3355 in dogs and rats; see Supplemental Table 3
and Supplemental Figure 3), the only species differences that have been demonstrated
in this regard pertain to the expression of certain splice variants in the rat but not in
humans (Pfaff et al., 1999). However, these variants have a pharmacology similar to
that of the other splice variants (Pfaff et al., 1999).
REFERENCES
Blackshaw LA, Staunton E, Lehmann A, and Dent J (1999) Inhibition of transient
LES relaxations and reflux in ferrets by GABA receptor agonists. Am J
Physiol 277: G867-874.
Christiansen B, Meinild AK, Jensen AA, and Brauner-Osborne H (2007) Cloning and
characterization of a functional human gamma-aminobutyric acid (GABA)
transporter, human GAT-2. J Biol Chem 282: 19331-19341.
Jensen AA, and Bräuner-Osborne H (2004) Pharmacological characterization of
human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a
fluorescence-based membrane potential assay. Biochem Pharmacol 67: 21152127.
Kvist T, Christiansen B, Jensen AA, and Bräuner-Osborne H (2009; in press) The
four human gamma-aminobutyric acid (GABA) transporters: pharmacological
characterization and validation of a highly efficient screening assay Comb
Chem High Throughput Screen
Laakso ML, and Oja SS (1976) Factors influencing the insulin space in cerebral
cortex slices from adult and 7-day-old rats. Acta Physiol Scand 97: 486-494.
Lee KJ, Vos R, Janssens J, and Tack J (2003) Differential effects of baclofen on lower
oesophageal sphincter pressure and proximal gastric motility in humans.
Aliment. Pharmacol. Ther. 18: 199-207.
Lehmann A, Antonsson M, Bremner-Danielsen M, Flardh M, Hansson-Branden L,
and Karrberg L (1999) Activation of the GABAB receptor inhibits transient
lower esophageal sphincter relaxations in dogs. Gastroenterology 117: 11471154.
Lidums I, Lehmann A, Checklin H, Dent J, and Holloway RH (2000) Control of
transient lower esophageal sphincter relaxations and reflux by the GABAB
agonist baclofen in normal subjects. Gastroenterology 118: 7-13.
Liu J, Pehlivanov N, and Mittal RK (2002) Baclofen blocks LES relaxation and crural
diaphragm inhibition by esophageal and gastric distension in cats. Am J
Physiol 283: G1276-1281.
Pfaff T, Malitschek B, Kaupmann K, Prezeau L, Pin JP, Bettler B, and Karschin A
(1999) Alternative splicing generates a novel isoform of the rat metabotropic
GABABR1 receptor. European Journal of Neuroscience 112: 874-882.
van Herwaarden MA, Samsom M, Rydholm H, and Smout AJ (2002) The effect of
baclofen on gastro-oesophageal reflux, lower oesophageal sphincter function
and reflux symptoms in patients with reflux disease. Aliment. Pharmacol.
Ther. 16: 1655-1662.
Zhang Q, Lehmann A, Rigda R, Dent J, and Holloway RH (2002) Control of transient
lower oesophageal sphincter relaxations and reflux by the GABAB agonist
baclofen in patients with gastro-oesophageal reflux disease. Gut 50: 19-24.
Supplemental Table 1: Agonistic activity of AZD3355 and baclofen for different
GABAB receptor splice variants
AZD3355
Baclofen
Splice variant
EC50 (nM)
Intrinsic activity
EC50 (nM)
Intrinsic activity
GABAB(1a)
8.0  1.3 (3)
1.2 ± 0.2 (3)
130  40 (3)
1.2 ± 0.2 (3)
GABAB(1b)
6.6  1.0 (3)
0.9 ± 0.2 (2)
81  29 (3)
0.8 ± 0.2 (2)
GABAB(1e)
8.5 ± 0.9 (3)
1.1 ± 0.1 (3)
140 ± 54 (3)
0.9 ± 0.1 (3)
GABAB(1g)
19 ± 8.0 (2)
1.0 ± 0.1 (3)
300 ± 200 (2)
1.0 ± 0.03 (2)
GABAB(1m)
17 ± 14 (2)
1.1 ± 0.03 (2)
213 ± 71 (2)
1.1 ± 0.1 (2)
GABAB(1o)
11 ± 2 (2)
1.1 ± 0.2 (2)
130 ± 21 (2)
1.0 ± 0.1 (2)
Data are the mean ± S.E.M. of (n) experiments.
Supplemental Table 2: Binding affinity (KD) and maximal binding (Bmax) for
[3H]CGP54626 to different GABAB(1) splice variants
Splice variant
KD (nM)
Bmax (fmol/100 cells)
GABAB(1a)
1.0  0.24
3.6 
GABAB(1b)
1.7  0.35
4.8  0.56
GABAB(1e)
1.4  0.35
3.5  0.50
GABAB(1g)
1.4  0.11
3.8  0.68
GABAB(1m)
1.6  0.34
4.0  0.51
GABAB(1o)
1.3  0.28
1.7  0.29
Data are the mean ± S.E.M. of 3 experiments.
Supplemental Table 3: Pharmacokinetic properties of AZD3355 (7 mol/kg) in the
dog and rat, after i.v. and oral administration
Dog
Rat
i.v. administration (n=4)
i.v. administration (n=2)
t½lz (h)
7.6 ± 0.5
2.8; 2.8
Clearance (L/h/kg)
0.18 ± 0.01
0.7; 0.8
Vss (L/kg)
1.1 ± 0.2
2.2; 2.3
Oral administration (n=4)
Oral administration (n=3)
Cmax (µmol/L)
8.5 ± 3.3
1.1 ± 0.3
Tmax (h)
0.7 ± 1.6
2.4 ± 1.1
t½lz (h)
6.8 ± 2.7
4.1 ± 1.1
Bioavailability (%)
88.3 ± 5.2
103 ± 15
Data are mean ± S.E.M. (except for rat i.v. data, which are individual results).
Legends for Supplemental Figures
Supplemental Figure 1. Structures of the GABAB receptor agonists studied.
Supplemental Figure 2. Effects of once daily administration of AZD3355 on
inhibition of transient lower esophageal sphincter relaxation (TLESR) in the dog.
Data represent mean ± SEM (n=6).
Supplemental Figure 3. Plasma concentration–time curves of AZD3355 in (a) dogs
and (b) rats after intravenous and oral administration. Data represent mean ± SEM.