SUPPLEMENTARY DATA
MATERIALS & METHODS
Radioligand displacement experiments
RBA were determined by incubating NRCM with 5 nM [3H]-CGP-12177 and
0.1 nM to 0.1 mM of unlabeled compound (AMI9, AMI9S, alprenolol,
atenolol, metoprolol, propranolol, pindolol, timolol) for 30 min at 37°C (n = 46 for each point) prior to rinsing and detachment using sodium dodecyl sulfate
1% in 10 mM sodium borate. Aliquots were removed for radioactivity
counting and protein content assessment. [3H] activity was assessed by liquid
scintillation counting (Ready Safe Beckman) following cell & organ (see
below) dissolution in tissue solubilizer BTS 450 (Beckman) at 60°C and
bleaching using perhydrol (Merck). Specific binding of [3H]-CGP-12177 was
determined by subtracting nonspecific binding (propranolol 10-5M) from total
binding. Results were expressed as % of [3H]-CGP-12177 specific binding in
the absence of unlabeled molecules, allowing for the determination of the
inhibition constant KI from the equation KI = IC50 / (I + L/KD) [1], where L
and KD are the dose and dissociation constant of [3H]-CGP-12177 for -AR,
respectively. For [3H]-CGP-12177, a KD value of 0.95 nM was determined by
preliminary binding experiments (data not shown). The RBA of each
compound was calculated by dividing its KI value by that of propranolol.
Synthesis and radiolabeling of AMI9 and AMI9S
The chemical structure of AMI9 is presented in Figure 1. The syntheses of the
AM 9-SnBu3/AMI9 and AM 9S-SnBu3/AMI9S compounds were performed
as previously described [2]. Enantiomeric purity reached 97.5% [3]. AMI9 and
AMI9S radiolabeling was performed by Iodine/SnBu3 exchange on the AMI9
and AMI9S precursors AM9-SnBu3 and AM9S-SnBu3, respectively.
Typically, 100 µg AM9-SnBu3 or AM9S-SnBu3 in 100 µl ethanol reacted
with 2 nmol Na125I and 100 µg chloramine T at RT for 1 hr, before the
reaction was stopped by 100 µg sodium hydrosulfite in 100 µl of 0.1 M
phosphate buffer. Purification was performed on an anion exchanger AG® 1X8 (chloride form) resin (Bio-Rad). The radiochemical purity as determined
using HPLC on a LiChrospher® RP-18 column (Merck) using 20 mM
phosphate buffer (pH 4)/acetonitrile (65/35; V/V) exceeded 95%.
Binding assays
AMI9 and AMI9S specific activities were ~74 TBq/mmol following 125I
labeling and purification. Saturation binding studies on NRCM were
performed using [125I]-AMI9 and [125I]-AMI9S (0.2-50 nM) and two
reference -AR radioligands, [3H]-CGP-12177 (0.1-20 nM) and [125I]- ICYP
(5-500 pM). Incubations were performed for 30 min at 37°C and the samples
were then processed as described above. Specific binding was determined as
the mean of individual differences between total binding and nonspecific
binding values, the latter being defined as the radioactivity not displaced by
10-25 mM of (±)-propranolol (n = 3-6 for each point). Bmax (in fmoles/mg of
protein) and KD (in M) were determined using Scatchard linear regression.
KD values of [125I]-AMI9 and [125I]-AMI9S were determined by kinetic
binding experiments on NRCM. Cells were incubated at 37°C with 20 nM
[125I]-AMI9 or 13.3 nM [125I]-AMI9S for 30 sec-40 min (association study)
or for 40 min prior to 30 sec-40 min co-incubation with a 1000-fold excess of
cold AMI9 or AMI9S (dissociation study). Incubation was stopped and the
samples were processed as described above. Nonspecific binding was assessed
following the addition of a 500-750-fold excess of (±)-propranolol (relative to
the dose of radioligand; n = 3 for each point). Specificity and selectivity of
[125I]-AMI9 and [125I]-AMI9S bindings were estimated by competition
experiments using specific and/or selective inhibitors. Assays were performed
by incubating NRCM with 20 nM [125I]-AMI9 (or 14 nM [125I]-AMI9S) and
0.1 nM - 100 mM of CGP 20712A (1-selective), ICI 118551 (2-selective),
prazosin (1-selective), yohimbine (2-selective) or atropine (muscarinic
specific) for 30 min at 37°C (n = 3-6 for each time point). Phentolamine 100
mM or (±)-propranolol 10 mM were used to estimate AMI9 & AMI9S specific
binding on - and -AR, respectively. Incubations were stopped and the
samples were washed and counted as described above. Competition curves
indicated % of total radioligand binding.
AMI9 & AMI9S pharmacological activity
Isolated hearts from adult female Wistar rats were perfused as previously
described [4]. A latex balloon inserted into the left ventricle and linked to a
pressure transducer (Statham P23 db) connected to a recorder (Mac Lab) was
used to monitor variations in cardiac contractility (dp/dt max, mmHg/sec).
After 25 min of pre-perfusion (baseline), the hearts were perfused with the -
AR agonist isoproterenol (10 nM) for 10 min prior to 10 min co-perfusion of
10 nM isoproterenol and 0.1 nM - 10 mM AMI9 or AMI9S. (±)-propranolol
was used as a reference. Isoproterenol (10 nM) alone for 20 min was used as a
control (n = 4-9 hearts for each dose).
Lipophilicity
The partition coefficient (P) of AMI9 (AMI9S) was determined in noctanol/20 mM phosphate buffer (pH 7.4; 23°C) and expressed as log(P). The
retention coefficient (k') was determined using HPLC (Waters Bondapak
C18 column eluted by 65% phosphate buffer (0.01 M)/35% acetonitrile (pH 4;
23°C)). k' = (t-t0)/t0, where t is the compound retention time detected at 280
nm, and t0 the column dead time assessed using thiourea. k' was also measured
for compounds with known lipophilicity (practolol, oxprenolol, propranolol,
CGP-12177, CGP-20712A).
Biodistribution in mice
[125I]-AMI9 (230 kBq/28.9 pmole per mouse; 7.9 TBq/mmol), [3H]-CGP12177 (25 kBq/14.4 pmole per mouse; 1.7 TBq/mmol) or [125I]-ICYP (22
kBq/0.27 pmole per mouse; 80.5 TBq/mmol) were injected intravenously to
conscious and restrained female Swiss mice (26.6 ± 2.6 g body weight) with
the syringe activity being assessed immediately prior to and after injection
(Capintec CRC-15R dose calibrator) in order to determine the injected dose.
Animals were euthanized by cervical dislocation at 0.5 – 120 min postinjection (p.i.). Blood sampling was performed from the ventricles and the
heart, lungs, liver, kidneys, brain, gastrocnemius muscle & abdominal fat were
quickly removed, rinsed in saline, weighted and gamma-well counted (n = 3
except for heart, n = 6; Cobra II, Packard Instruments). All activity data were
expressed in counts per minute (cpm) using internal references for the
correspondence between the dose calibrator (MBq) and the gamma-well
counter (cpm). Results were expressed as DUR (cpm /g tissue)/(cpm injected/g
body weight).
Statistical analysis
All fits were performed using KaleidaGraph™ version 2.1.3 (Abelbeck
Software). Other graphs were performed using Cricket Graph III™ version
1.5.3 (Computer Associates Int., Islandia, NY). Differences between groups
were assessed by 2-tailed Student's t test. Two-way ANOVA analysis with
repeated measurements was used for comparison of dP/dt max values in the
perfused rat heart study. P values <0.05 were considered significant.
Source of compounds
[3H]-CGP-12177 (1.6-1.9 TBq/mmol) and [125I]-ICYP (74-81 TBq/mmol)
were purchased from Amersham and DuPont NEN. Na125Iwere supplied by
IBA, France. CGP-12177 and CGP-20712A were donated by Ciba-Geigy,
Basel, Switzerland. ICI 118551 was purchased from Tocris Cookson Ltd,
Bristol, UK. Practolol was synthesized by DCM, UMR CNRS 5250, Grenoble,
France. Alprenolol, atenolol, atropine, isoproterenol, metoprolol, oxprenolol,
phentolamine, pindolol, prazosin, propranolol, timolol and yohimbine were
purchased from Sigma-Alrdich.
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