Pharmacologic characterization in vitro and in vivo of iodine 123-labeled derivatives of the β-adrenoceptor antagonist CGP12177, designed for the imaging of cardiac β-receptors

Pharmacologic characterization in vitro and in vivo of iodine 123-labeled derivatives of the 13-adrenoceptor antagonist CGP12177, designed for the imaging of cardiac 13-receptors Eric A. Dubois, MD, G. A e r n o u t S o m s e n , MD, Jan C. v a n d e n Bos, MSc, A n t o n G. M. J a n s s e n , MSc, G e r a r d J. Boer, PhD, Harry D. Batink, Eric A. v a n R o y e n , MD, PhD, M a r t i n Pfaffendorf, PhD, a n d Pieter A. v a n Z w i e t e n , MD, PhD Background. Potential new radioligands for the noninvasive imaging of cardiac [3-adrenoceptors with single-photon emission computed tomography were investigated. Methods and Results. Two iodinated derivatives of CGP12177 para (S-CYBL2B) and ortho (CYBL2A) substituted CGP12177 and an iodinated form of nadolol (CYBL1) were synthesized. Their affinity was tested in vitro (left ventricular homogenates). The biodistribution of [123I]S-CYBL2B was evaluated in rabbits. Specific binding was assessed by pretreatment of the animals with 0.1 ~tmol propranolol. The inhibition constant values (in nanomolars, means + SEM; n = 3 to 5) were determined at 1.17 + 0.42, 28800 + 9260, 11.1 + 2.1, 53.0 + 19.9, and 1790 + 700 for CGP12177, CYBL2A, S-CYBL2B, nadolol, and CYBL1. Myocardial uptake of [~23I]S-CYBL2B was not inhibited by pretreatment of the animals with propranolol, but uptake by lung tissue could be blocked by propranolol (0.63% _+0.09% vs 0.33% + 0.02% % injected dose/g × kg; p < 0.05). In isolated right atria, preincubation with S-CYBL2B induced a parallel rightward shift of the concentration-response curve with isoprenaline. Conclusions. S-CYBL2B shows high affinity for cardiac [3-adrenoceptors, but binding proved nonspecific in vivo, whereas binding in lung tissue was specific. These results suggest that S-CYBL2B is probably not a suitable radioligand for receptor imaging. (J Nucl Cardiol 1996;3:242-52.) Key Words: ~-adrenoceptors • CGP12177 ° heart failure

[3-Adrenoceptors play an important role in the regulation of heart rate and cardiac contractile force, Both physiologic parameters are enhanced when these receptors are stimulated by their natural agonists, the catecholamines noradrenaline and adrenaline, or by synthetic 13-adrenoceptor agonists. On the basis of their ligand specificity, myocardial [3-adrenoceptors have From the Departments of Nuclear Medicine, Pharmacotherapy, and Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, and Cygne B.V. Eindhoven University of Technology, Eindhoven, The Netherlands. Supported by grant D92015 of The Netherlands Heart Foundation. Submitted for publication Aug. 22, 1995; revision accepted Nov. 3, 1995. Reprint requests: Eric A. Dubois, MD, Department of Nuclear Medicine, Room F2-238, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Copyright © 1996 by American Society of Nuclear Cardiology. t071-3581/96/$5.00 + 0 43/1/70480 242

been subdivided into at least two subtypes: ~-adrenoceptors, which are present mainly on myocytes, and [32adrenoceptors, which are found mainly on smooth muscle cells in the coronary vasculature and on adrenergic nerve endings. ~ Myocardial [3-adrenergic receptor density is known to be altered significantly under different pathophysiologic circumstances such as myocardial infarction, 2 chronic heart failure, 3 myocardial ischemia, 4 abnormal thyroid status,~ and diabetes mellitus. 6'7 In failing human hearts, down-regulation of [~lreceptors is observed as a result of prolonged exposure to the endogenous agonists noradrenaline and adrenaline.8-13 This down-regulation of [3-adrenoceptors has been established mainly in vitro in homogenates of ventricular tissue obtained invasively during endomyocardial biopsy. In congestive heart failure, neuroendocrine changes concerning both the sympathetic system and the renin-angiotensin-aldosterone system have been shown to be of great importance for pathophysiologic

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understanding, therapy evaluation, and patient management. Because [3-adrenoceptor density is assayed mainly in biopsy samples, little is known about the time course of changes in receptor density during the process of disease, the spatial distribution of these particular receptors, and the possible changes in distribution during the disease. Because a clear relationship between the degree of changes in cardiac adrenergic activation and the severity of the disease and its prognosis has been established, it can be imagined that measurement of [3-adrenoceptor density may be used as a prognostic marker in a variety of pathologic circumstances. ~4-16 The development of a noninvasive method to assess [3-adrenoceptor density in volunteers arid subsequently in patients with congestive heart failure or other cardiac disorders would be of great clinical interest for the visualization and possible quantification of these receptors in humans under different pathologic circumstances. It can even be conceived that the evaluation of therapeutic measures may be judged on the basis of changes in cardiac [3-adrenoceptor density. Several attempts have been made to develop new radioligands for the imaging of [3-adrenoreceptors in vivo with either single-photon emission computed tomography (SPECT) or positron emission tomography. Numerous [3-blockers have been labeled with " C or ~SF as potential ligands for positron emission tomographic imaging, although with limited success due to insufficient affinity for the receptor or relatively high nonspecific binding. CGP12177 (4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazole-one) is a well-known non-subtype-selective potent [3-blocker that has been labeled successfully with 'IC for the imaging and quantification of [3-adrenoceptor density in vivo by positron emission tomography, although specific activity showed great variety (specific activity ranging from 400 to 1300 mCi/gmol at the time of injection), t719 Suitable radioligands for the imaging of these receptors with SPECT have not been developed so far. Iodocyanopindolol labeled with ~25I or ~23I has been studied by several investigators as a potential radioligand for the imaging of [3-receptors in vivo. 2°'2' Although this ligand shows high affinity for the receptor, it also displays affinity for serotoninergic receptors, thus hampering genuine quantification of [3-adrenoreceptor density in vivo. This study was undertaken to investigate whether three newly synthesized radioligands, in particular derivatives of CGP12177, would be suitable for the SPECT imaging of [3-receptors in vivo. The affinity of the newly synthesized compounds for the [3-adrenoceptor was assessed, as well as the biodistribution, binding characteristics, and pharmacodynamic activity of one of the new radioligands in rabbits.

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METHODS Synthesis Treatment of nadolol (Sigma Chemical Co., St. Louis, Mo.) with iodine and mercury (II) acetate in acetic acid22 resulted in the selective formation of CYBL1 (melting point = 84 ° to 85 ° C). Iodination of racemic CGP12177 with iodine in the presence of silver trifluoroacetate in chloroform23 resulted in a 75:25 mixture of ortho-substituted (CYBL2A) and para-substituted (CYBL2B) forms, which were separated by high-performance liquid chromatrography (gBondapak, C- 18; Waters Associates, Milford, Mass.), with MeOH: H20: CF3COOH as elution medium (50:50:0.3, vol/vol/vol; 5 ml/min). S-CYBL2B ([~]D =-10.9°) was formed by iodination of S-CGP12177. The S-enantiomer of CGP12177 was synthesized by the reaction of the tosylate of S-(-)-2-phenyl3-tert-butyl-5-hydroxymethyl oxazolidine ~425 with 2-amino-3nitrophenol, followed by reduction with hydrogen and reaction of the obtained diamino derivative with phosgene. 26'27 [~23I]SCYBL2B was synthesized from nonradioactive S-CYBL2B by conversion to the corresponding trimethyltin derivative. Treatment of S-CYBL2B with hexamethylditin and tetrakis(triphenylphosphine)palladium in toluene at 70 ° C gave S-7-trimethylstannyl-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one. '6 The oxidative radioiododestannylation reaction of this trimethyltin precursor with Na~Z3I (specific activity _>5000 Ci/mmol and radionuclidic purity ___99.9%, produced indirectly by a proton reaction on 99.8% enriched '~-4Xe)2S yielded ['23I]S-CYBL2B with a specific activity of greater than 5000 Ci/mmol at the time of use. Nuclear magnetic resonance (NMR) imaging and mass spectroscopy (MS) were performed to confirm the chemical structure of the newly synthesized compounds. ~H-NMR spectra were measured on a Bruker AM-400 spectrometer at 400.13 MHz (Bruker Instruments, Inc., Billerica, Mass.). ~3CNMR spectra were run on the same apparatus at 100.62 MHz. Ion-spray MS was performed on a PE SCIEX API 300 MS/MS system (Perkin-Elmer SIEX Instruments, Norwalk, Conn.). All newly synthesized compounds were made by Cygne B.V., Eindhoven University of Technology, Eindhoven, The Netherlands. The chemical structures of the various compounds involved are depicted in Figure 1. The newly synthesized compounds are listed in Table 1.

In Vitro Analysis Animals. Male New Zealand White rabbits, weighing 1.5 to 2.5 kg, were anesthetized with an intramuscular injection of 5 mg/kg xylazine and 30 mg/kg ketamine and subsequently killed by an intramuscular injection of 200 mg/kg pentobarbitat. The thorax was opened and the heart was removed rapidly. The left ventricle was dissected, rinsed in 10 ml ice-cold 1 mmol/L KHCO3, and freeze clamped. The lungs were rinsed in 10 ml 20 mmol/L NaHCO 3 and also freeze clamped in liquid nitrogen. Both were stored at -80 ° C until use for radioligand-binding experiments. Radioligand-Displacement Experiments. Homogenates of left ventricles and lungs were used for radioliganddisplacement experiments. With some modifications, the method described by Engel et al. 29 was used: 100 mg left

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Bisoprolol

Nadolol 1 7

H O ~ I ~ itO@1~

~2

13

1.

2 OH H ~ I

3

CYBLI

.

o==~~]~

OH H (S)-CGPI2177

H' CGP12177

H',

l

z

22¢~_..6 o, ;,

°=(.1~5 " I4

H

S-CYBL2B

CYBL2A

SnMe3 (S)-4-(3-t-buty amino-2-hydroxypropoxy)-7trimethylstannyl-benzimidazole-2-one

Figure 1. Chemical structures of (S)-CGP12177, CGP12177, nadolol, bisoprolol, CYBL1, CYBL2A, S-CYBL2B, and precursor of ['2~I]S-CYBL2B.

ventricular tissue was minced with scissors and homogenized in 10 ml ice-cold 1 mmol/L KHCO3 by a Polytron homogenizer (full speed, three times for 5 seconds). The homogenate was centrifuged at 1000 g for 20 minutes. The supernatant was filtered through four layers of cloth gauze and centrifuged at 45,000 g for 20 minutes. The pellet was resuspended, homogenized, and diluted to a level of 100 ml/g wet weight in incubation buffer (10 mmol/L Tris/HC1 buffer, 154 mmol/L NaC1, and 0.01% ascorbic acid at pH 7.4). An aliquot of the membrane suspension (150 gl) was incubated with 50 gl [~25I]-iodocyanopindolol (specific activity equals 2200 Ci/mmol) in a final concentration of 4.5 x 10-" mol/L (total radioactivity of 2.5 x 10-~ Ci) and 50 lal of 13 final concentrations ranging from 10 ~o to maximal 10-~ mol/L of the

iodinated compounds. The incubation was terminated by adding 3 ml ice-cold incubation buffer to the entire mixture followed by rapid vacuum filtration through Whatman GF/C filters (Whatman Laboratory Products Inc., Clifton, N.J.). Filters were washed twice with incubation buffer (3 ml) and counted for radioactivity at 74% efficiency in a Beckman y-counter. Nonspecific binding of iodocyanopindolol to the membrane suspension was defined as the radioactivity bound in the presence of a high concentration of (+)-CGP12177 (10 5 mol/L). Specific binding was defined as the total minus the nonspecific binding. As a reference compound, we used bisoprolol, a 131-selective receptor antagonist. In addition to the newly synthesized iodinated compounds, we also tested the affinity of the noniodinated forms of these new agents, nadolol and CGP12177. To investigate whether the newly synthesized radioligand S-CYBL2B binds specifically to ~-adrenoceptors, muscarinic receptors, or a-adrenoceptors, 150 gl of the membrane suspension of the left ventricle was incubated with 50 gl [L23I]SCYBL2B (specific activity >5000 Ci/mmol, at a final concentration of 1.87x10 -9 mol/L) and 50 gl S-CYBL2B, CGP12177, dexetimide, or phentolamine (50 gl; final concentration 10-5 mol/L). Further specificity was tested, with homogenates of lung tissue incubated for 1 hour in the presence of 50 gl ['23I]S-CYBL2B (specific activity >5000 Ci/mmol, at a final concentration of 1.87 x 10-9 mol/L) and addition of the [~-adrenoceptor antagonists S-CYBL2B, ICIl18,551 (a 92selective antagonist), CGP12177, or propranolol (50 gl; final concentration 10-5 mol/L). Lung tissue homogenate was prepared according to the method described by Engel et al.29:100 mg lung tissue was minced with scissors and homogenized in 10 ml ice-cold 20 mmol/L NaHCO 3 by a Polytron homogenizer (full speed, three times for 5 seconds). The homogenate was centrifuged at 1000 g for 20 minutes. The supernatant was filtered through four layers of cloth gauze and centrifuged at 45,000 g for 20 minutes. The pellet was resuspended, homogenized, and diluted to a level of 400 ml/g wet weight in incubation buffer (10 mmol/L Tris, 154 mmol/L NaC1, and 0.01% ascorbic acid at pH 7.4). Functional Studies: Isolated Right Atria. Right atria of male Wistar rats, weighing 200 to 250 gm, were used for functional studies. Rats were killed by a blow on the skull and the heart was removed quickly and placed immediately in a Krebs solution (at room temperature) of the following composition: NaC1, 119 mmol/L; KCI, 4.8 mmol/L; CaC12. 2.5 mmol/L; MgSO4, 1.2 mmol/L; NaHCO3, 25 retool/L; KH2PO4, 1.2 mmol/L; and glucose, 11.1 mmom/L, gassed with 95% O2 + 5% CO 2. Whole right atrium was cut carefully and rapidly perpendicular to the axis of the heart. Each atrium was placed in a 10 ml organ bath filled with 8 ml Krebs solution at 37 ° C (pH 7.4) and connected by a silk thread to a force transducer (UFL Co., Los Angeles, Calif.). The isometric force was recorded by a MacLab/8 computer system (A.D. Instruments, Castle Hill, Australia) and the rate of spontaneous beating of the right atria (defined as heart rate) was derived and recorded from the isometric force by a rate meter (A.D. Instruments). The resting tension of each atrium was adjusted to 0.5 g and the tissues were equilibrated for at least 60 minutes before the experiments were started. At 30-minute

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Table 1. List o f c h e m i c a l n a m e s a n d a b b r e v i a t i o n s of t h e r e f e r e n c e c o m p o u n d s C G P 1 2 1 7 7 a n d n a d o l o l and the newly synthesized compounds Chemical name

Abbreviation

4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazole-2-one 4-(3-t-butylamino-2-hydroxypropoxy)-5-iodo-benzimidazole-2-one (S)-4-(3-t-butylamino-2-hydroxypropoxy)-7-iodo-benzimidazole-2-one (2R, 3S)-5-[3-(t-nutylamino-2-hydroxypropoxy]-1,2,3,4-tetrahydronaphtalene-2,3-diol (2R, 3S)-8-iodo-5--[3-(t-nutylamino-2-hydroxypropoxy]-1,2,3,4-tetrahydronaphtalene-2,3-diol

CGP12177 CYBL2A ~CYBL2B Nadolol CYBL!

intervals the medium was exchanged by fresh Krebs solution. After equilibration, cumulative concentration-response curves to isoprenaline were constructed. The concentration of isoprenaline was increased only after the previous addition of isoprenaline had caused the maximal response. Only a single complete cumulative concentration-response curve was obtained in each atrium. In separate experiments, cumulative concentration-response curves to isoprenaline were constructed after 30 minutes of incubation with S-CYBL2B (3 x 10 ~ or 10_6 mol/L) or propranolol (10-6 mol/L). In Vivo Studies Male New Zealand white rabbits weighing 2.5 to 3.5 kg were anesthetized with an intramuscular injection of 5 mg/kg xylazine and 30 mg/kg ketamine. Subsequently, the animals received 50 ~tCi [L23I]S-CYBL2B (specific activity >5000 Ci/mmol) intravenously through a lateral ear vein. Animals were killed 5, 15, 30, 50, 60, 120, and 240 minutes and 24 hours (three animals per time point) after injection by intramuscular injection of 200 mg/kg pentobarbital. Blood was removed by a vacuum pump, through an intracardial puncture. The organs were removed and weighed and radioactivity was measured with a 't--counter (Auto-Gamma 5000; Packard Instrument Co., Inc., Downers Grove, Ill.) at 51% efficiency and corrected for decay, weight, and body weight and expressed as percent injected dose per gram times kilograms. In a separate experiment, five animals received an intravenous injection of 0.1 gmol (+) propranolol intravenously 10 minutes before the administration of the radioligand, and five animals received only 50 gCi [~2~I]S-CYBL2B, to establish nonspecific binding. Animals were killed 30 minutes after the intravenous administration of the radioligand as described above. Again, the organs were removed and weighed and radioactivity was measured with the aforementioned y-counter at 51% efficiency and corrected for decay, weight, and body weight. Calculations Displacement curves were fitted to the individual binding data by a computer program based on a sigmoidal model for a single receptor subtype (GraphPad Software, San Diego, Calif.). The inhibition constant (Ki) was derived from the equation Ki = ECso/(1 + L/Kd), where L equals the concentration

of the radioligand iodocyanopindolol and Kd equals the affinity constant of iodocyanopindolol for the 13-adrenoceptor. To compare the affinity of different compounds, the relative binding affinity was calculated for each compound by dividing the Ki value of each new agent by the Ki of the most potent compound (x100%). For the functional experiments, EDso values were calculated from pooled dose-response curves, with the aforementioned computer program. Statistical Analysis Data are expressed as means + SEM. Statistical analysis of the results was performed with a nonparametric MannWhitney test. A p value < 0.05 was considered to indicate statistical significance of differences. For the in vivo analysis, analysis of variance with a Bonferroni test was performed. Drugs Used (+)-CGP12177 was obtained from Ciba-Geigy (Basel, Switzerland). Iodocyanopindolol (specific activity 2200 Ci/mmol) was provided by Du Pont de Nemours0 New England Nuclear (Boston, Mass.). Newly synthesized compounds were provided by Cygne B.V., Eindhoven University of Technology. All other chemical compounds were obtained from Sigma Chemical Co. In Table 1 the newly synthesized compounds are listed.

RESULTS C h e m i c a l S y n t h e s i s . The chemical syntheses were performed according to procedures described in the literature, as mentioned in the Methods section.. N M R spectroscopy .and MS revealed the following analysis for C Y B L I : 1H N M R (CDC13:CD3OD = 9: 1): 8 7.63 (d, J = 8.7 Hz, 1H), 6.52 (d, J = 8.6 Hz, 1H), 4.4 (m, 1H), 4.15 to 4.0 (m, 4H), 3.3 (m, 1H), 3.12 (m, 1H), 2.90 (m, 4H), 1.45 (s, 9H). 13C NMR: ~i 157.9 (C1), 138.4 (C5), 138.0 (C3), 126.3 (C6), 111.9 (C2), 91.8 (C4), 71.9 (Cll), 70.3 (Ca), 69.5 (C9), 69.4 (C12), 53.2 (C(CH3)), 46.2 (C2), 42.0 (C10), 30.2 (C7), 27.9 (C(C_H3)), and MS (Ion-spray): 436 (M + H)+. • Nadolol is iodinated predominantly in the para

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125 100 Q_ >.0 03 .

cm

75

•

CGP12177

•

Bisoprolol

•

Nadolol

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50-

{.-

•-Q

25-

0, I

t

-11 -10

I

-9

I

-8

I

-7

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-6

I

I

-5

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-3

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-2

Log molar[antagonist] Figure 2. Displacement curves of bisoprolol, nadolol, CYBL1, and CGP12177 with respect to binding of iodocyanopindolol (ICYP) in homogenates of left ventricular tissue of New Zealand White rabbits. Values are expressed as means + SEM (n = 4). 12510013.. >O

•

CGP12177

•

CYBL2A

•

S-CYBL2B

75-

03

.50"I3 t"-

"Q

25-

I

I

I

I

I

I

I

I

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I

-11

-10

-9

-8

-7

-6

-5

-4

-3

-2

Log molar [antagonist] Figure 3. Displacement curves of iodinated forms of CGP12177 with respect to binding of iodocyanopindolol (ICYP) and its native compound CGP12177 in homogenates of left ventricular

tissue of New Zealand White rabbits. Values are expressed as means + SEM (n = 4). position of the moderately activating alkoxy group due to the relatively low reaction temperature. NMR spectroscopy and MS showed the following peaks for C Y B L 2 A and CYBL2B, respectively: C Y B L 2 A t a = 6.0 minutes, 1H N M R (CDC13:CD3OD = 9 : 1): 8 7.37 (d, J = 8.2 Hz, 1H), 6.26 (d, J = 8.2 Hz, 1H), 4.27 (m, 1H), 4.10 (dd, J = 4.62 Hz and J = 10.2 Hz, 1H), 4.05 (dd, J = 4.26 Hz and J = 10.2 Hz, 1H), 3.35 (dd, J = 2.55 Hz and J = 12.5 Hz, 1H), 3.10 (dd, J = 10.3 Hz and J = 12.5 Hz, 1H), 1.34 (s, 9H). 13C N M R (CDC13:CD3OD= 9:1): fi 156 ( C = O), 141.4 (Cl), 131.4 (C3), 130.9 (C5),

122.2 (C2), 107.9 (C4), 80.5 ( C 6 ) , 74.6 (C7), 65.7 (Ca), 56.8 (C[CH313), 44.6 (C9), and 25.4 (C[CH313). MS (Ion-spray): 406 (M + H)+; CYBL2B tR = 4.4 minutes, 1H N M R (CDC13 :CD3OD = 9: 1): ~ 7.26 (d, J = 8.2 Hz, 1H), 6.44 (d, J = 8.2 Hz, 1H), 4.32 (m, 1H), 4.12 (dd, J = 4.55 Hz and J = 9 . 5 H z , 1H), 4.03 (dd, J = 5.21 Hz and J = 9 . 5 Hz, 1H), 3.18 (dd, J = 2.53 Hz and J = 12.2 Hz, 1H), 3.06 (dd, J = 10.2 Hz and J = 12.2 Hz, 1H), 1.39 (s, 9H). 13C NMR (CD3OD): 8 157 (C = O), 144.5 (C1), 134.8 (C3), 131.5 (C5), 120.0 (C2), 108.5 (C6), 71.6 (Cv), 63.8 (C4), 67.2 (C8), 58.4 (C[CH313), 45.5 (C9),

Dubois e t a ] . [3-Adrenoceptors in receptor imaging

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247

125rn

t"Xl .9,oj o-~rn

100-

- -

-T-

=>-

75-

-Q ,j.,,

50-

Total binding

v////~

(+) p r o p r a n o l o l

r,-.-,\-.~ S - C Y B L 2 B phentolamine ,,

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n--

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[--]

14

0 Figure 4. Percentage binding of ['23I]S-CYBL2B after 1 hour of incubation with excess (final concentration equals 5000 times ['23I]S-CYBL2B) of muscarinic antagonist dexetimide, [~-adrenoceptor antagonists propranolol, CGP12177, and S-CYBL2B, and o~-adrenoceptor antagonist phentolamine with homogenates of left ventricular tissue of New Zealand White rabbits. Total binding resulted in 16,000 counts/min. Values are expressed as means + SEM (n = 4). 25.8 ( C [ C H 3 1 3 ) . MS (Ion-spray): 406 (M + H) +. MS(EI): 405.0 (M+), 390.0, 275.9, 274.9, 246.9, 218.8, 126.9, 92.1, 86.1, and 71.1. NMR and MS of S-7-trimethylstannyl-4- (3-tert-butylamino-2- hydroxypropoxy)-benzimidazol-2-one (which is the precursor of [~2q]SCYBL2B) revealed the following peaks: 1H NMR (CDC13): 8 7.00 (d, J = 8.0 Hz [with satellite peaks at 22.0 Hz], 1H), 6.63 (d, J = 8.0 Hz [with satellite peaks at 2.5 Hz], 1H), 4.10 (dd, J = 3.39 Hz and J = 8.9 Hz, 1H), 4.05 (m, 1H), 3.97 (dd, J = 6.3 Hz and J = 8.9 Hz, 1H), 2.75 (m, 2H), 1.14 (m, 9H), 0.37 (s [with satellite peaks at 26.8 and 28.0 Hz], 9H). MS (Ion-spray): 440, 441, 442, 443, 444, 445, 446, and 448 (M + H) + (only the most abundant peaks are given distribution equal to that of Sn isotopes). In Figure 1 the structural formulas of the newly synthesized agents and their native compounds are depicted (Table 1). In Vitro Analysis. In Figure 2 the displacement curves of bisoprolol, nadolol, CYBL1, and CGP12177 with respect to iodocyanopindolol binding to cardiac ventricular tissue are depicted. CGP12177, known to be a very potent [3-adrenoceptor antagonist, was significantly more active than both nadolol (Ki = 1.17 + 0.42 nmol/L for CGP12177 vs 53.0+ 19.9 nmol/L for nadolol; n = 4) and bisoprolol (Ki = 79.5 + 41.6 nmol/L; n =4). CYBL1 displayed significantly less affinity for the receptor than did nadolol (Ki = 1787 + 700 nmol/L; n =4).

The iodination of CGP12177 yielded two isomers: (S)(-)-para-iodinated (S-CYBL2B) CGP12177 and ortho-iodinated CGP12177 (CYBL2A), respectively. In Figure 3 the displacement curves obtained with these iodinated compounds and the native compound CGP12177 are shown. Interestingly, displacement experiments with CYBL2A revealed that iodination at the

Table 2. K~values and relative binding affinity for CGP 12177, nadolol, bisoprolol, CYBL 1, S-CYBL2A, and S-CYBL2B obtained from displacement experiments in left ventricular tissue of New Zealand white rabbits with [IZSl]iodocyanopindolol as the radioligand Compound CGP12177 CYBL2A 5-CYBL2B Nadolol CYBL1 Bisoprolol

Ki (nmol/t) 1.17_+ 28800 + 11.1 + 53.0 + 1790 + 79.5 +

0.42 9260* 2.1 * 19.9~F 700* 41.6t

RBA (%) 100 0.004 10.6 2.21 0.07 1.47

Data are expressed as m e a n s _+SEM (n = 3 to 5).

Ki, Inhibition constant; RBA, relative binding affinity. *p < 0.05 versus noniodinated form of the c o m p o u n d . t P < 0.05 versus CGP12177.

ortho position yielded an antagonist that was able to displace the radioligand from the 13-adrenoceptor only at high concentrations (Ki = 28800 _+9260 nmol/L; n = 4). The para-iodinated compound, S-CYBL2B, however, proved able to displace iodocyanopindolol from the [3-adrenoceptor (Ki = 11.1 + 2.1 nmol/L; n = 4), although this compound was significantly less potent than the noniodinated form. The various Ki values are listed in Table 2. On the basis of the above results, S-CYBL2B was selected for further in vitro and in vivo studies and therefore radioactively labeled with 123I. In Figure 4 the results of the in vitro displacement experiments with left ventricular tissue are shown. The binding of [I2q]SCYBL2B to [31-receptors in the left ventricle could not

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125nn

i Total binding v////~ (+) p r o p r a n o l o l

100-

k..-.-.-.~ S-CYBL2B

o~ nn

c>:50

75-

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I

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I IC1118,551

250 Figure 5. Percentage binding of ['23I]S-CYBL2B after 1 hour of incubation with excess (final concentration equals 5000 times (['23I]S-CYBL2B) of 13-adrenoceptor antagonists propranolol, CGP12177, ICI 118,551, and S-CYBL2B with homogenates of lung tissue of New Zealand White rabbits. Total binding resulted in 42,000 counts/rain. Values are expressed as means + SEM (n = 3). *p < 0.05 versus total binding (in absence of aforementioned antagonists). 400-

-' 300c E t~ 200..Q 09

o ¢-

•

Control

•

S-CYBL2B (3"10-8 M)

•

S-CYBL2B (10 -6 M)

zx Propranolol (10 -6 M)

1000

2

1

Log molar [isoprenaline] Figure 6. Increase (expressed as beats per minute) in heart rate compared with baseline in right atria of male Wistar rats weighing 200 to 250 g exposed to isoprenaline. S-CYBL2B and propranolol caused rightward shifts of concentration-response curves of isoprenaline. Data are expressed as means _+SEM (n = 4). be antagonized significantly by an excess of the I]-adrenoceptor antagonists propranolol or CGP12177. Similarly, the binding of [~23I]S-CYBL2B could not be antagonized significantly by dexetimide or phentolamine, thus indicating that [Ia3I]S-CYBL2B does not significantly bind to muscarinic receptors or c~-adrenoceptors. The results of the displacement experiments with homogenates of lung tissue are shown in Figure 5. Binding to ~2-receptors in homogenate of lung tissue could be antagonized significantly by all of the antagonists used. Isolated Right Atria. After the equilibration period, the frequency of beating of the isolated right atria was 298 + 23 beats/rain (n = 4). When propranolol at a final concentration of 10-~ mol/L or S-CYBL2B at a

final concentration of 3 x 10-8 mol/L was added, no significant changes in basal heart rate were observed. When S-CYBL2B was given at a final concentration of 10-6 tool/L, a slight but nonsignificant reduction in basal heart rate was observed. Concentration-response curves with isoprenaline with isolated whole right atria of male Wistar rats caused a concentration-dependent increase in heart rate (EDs0=0.31 +0.05 nmol/L; n = 4 ) . Propranolol shifted the concentration-response curve to the right, with an EDso of 5 . 2 6 + 0 . 1 5 nmol/L. In the presence of 3 x 10-8 mol/L S-CYBL2B, the concentration:response curve showed a slight rightward shift, although the EDso did not change significantly (EDso 0.83 + 0.22 nmol/L; n = 4). The presence of 10-6 mot/L S-CYBL2B produced a significant rightward shift of the

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dose-response curve (EDso = 847 + 21 nmol/L; n = 4). Hill slopes were not significantly different between the four experimental groups (1.25 +0.05 in the control group vs 1.57 + 0.22, 1.40 + 0.10, and 1.17 + 0.07 in the presence 10-6 mol/L propranolol, 3 x 10-~ mol/L S-CYBL2B, and 10-~ mol/L S-CYBL2B). Maximal increase in heart rate (absolute numbers) increased significantly at increasing concentrations of S-CYBL2B (maximal increase by 153 + 2 beats/min in the control group vs 186+3 and 2 6 4 + 2 8 in the presence of 3 x 10.8 molYL and 10-6 mol/L, respectively). The presence of propranolol did not alter the maximal effect. Biodistribution Studies. The biodistribution of radioactivity after the intravenous injection of ['23I]SCYBL2B in anesthetized New Zealand White rabbits is shown in Table 3. Radioactivity in the left ventricle rapidly increased after intravenous administration to reach a maximal value 15 minutes after injection (0.45% + 0.04% injected dose/g tissue x kg). Radioactivity in the right ventricle increased rapidly after injection to reach a maximal value 5 minutes after injection (0.35% + 0.08% injected dose/g tissue x kg). Between 30 minutes and 2 hours after injection, radioactivity decreased substantially. Two hours after injection, radioactivity decreased in both the ventricles and atria. Radioactivity in the lungs reached maximal values 5 minutes after injection (1.49% + 0.63% injected dose/g tissue x kg). From this time point onward, radioactivity decreased rapidly until 24 hours after injection, although radioactivity levels in the lungs were significantly higher than those measured in the heart, at all time points. Significant uptake of radioactivity was observed in the liver and kidney of the animals. Radioactivity in the liver remained unaltered and showed no significant changes at different time points. The kidney showed a significant uptake 5 minutes after injection. After this time point, radioactivity decreased slowly over time. The blood showed only significant levels of radioactivity (0.82%+0.73% injected dose/g tissue x kg) 5 minutes after injection. At all later time points, radioactivity measured in the blood remained below 0.05% injected dose/g tissue x kg. Both fat and muscle showed both relatively low uptake of the radioligand (<0.1% injected dose/g tissuexkg at all time points). The results of the experiments in which the injection of [I~I]S-CYBL2B was preceded by an intravenous injection of 0.1 p_mol (+)propranolol intravenously are shown in Table 4. Uptake in the left ventricles of these animals could not be counteracted significantly by the preinjection of 0.1 ~tmol (+)propranolol intravenously (in control animals vs treated animals), whereas in the lungs a significant decrease in uptake of S-CYBL2B was

Dubois et al. [3-Adrenoceptors in receptor imaging

249

observed in the animals pretreated with 0.1 ~tmol (+) propranolol intravenously.

DISCUSSION Cardiac [3-adrenoceptors are assumed to play a pivotal role in several cardiac disorders, in particular congestive heart failure. [3-Adrenoceptors are downregulated in these patients due to an increase in cardiac sympathetic activation. On the long term this neuroendocrine compensation may contribute to the deterioration of cardiac function. Therefore pharmacologic reduction of cardiac sympathetic activation and a subsequent restoration of myocardial [3-adrenoceptor density may improve functional capacity, myocardial function, and probably prognosis. To monitor these changes in [3-adrenoceptor density, the noninvasive assessment of cardiac [3-adrenoceptor density with a suitable radioligand would be of clinical importance. Moreover, the effects of pharmacologic intervention, such as the use of low-dose [3-adrenoceptor antagonists, may be evaluated. 3°-34 Several criteria have to be fulfilled for a new compound to be potentially useful as a radioligand for in vivo imaging of cardiac receptors. It is generally accepted that a high affinity for the receptor is necessary, preferably with a Ki value less than 1 nmol/L. Furthermore, the radioligand should display high specific binding, it should be metabolically stable and, if possible, hydrophilic to avoid accumulation in lung tissue. 35 In addition, in the case of imaging of cardiac [~-adrenoceptors, a [31-selective radioligand is preferable because, in patients with congestive heart failure, a down-regulation of mainly ~31-adrenoceptors is observed. 36~4° At present, no optimal radioligands for the imaging of these receptors in vivo with SPECT are available. Nadolol and CGP12177 are known [3-adrenoceptor antagonists. CGP12177 has been labeled successfully with ~'C for receptor imaging 35 with positron emission tomography. In this study, three newly synthesized compounds are described: CYBL1, which is the iodinated form of the [3-adrenoceptor antagonist nadolol, and two iodinated derivatives of the ~3-adrenoceptor antagonist CGP12177 (i.e., CYBL2A and S-CYBL2B). In displacement studies with homogenates of cardiac left ventricular tissue, which is considered to be rich with [31adrenoceptors, the affinity of nadolol decreased significantly after the introduction of the iodine molecule. Because CYBL1 displayed a rather low affinity in vitro for the receptor, CYBL1 is probably not a suitable radioligand for in vivo receptor imaging. The iodination of CGP12177 yielded two new compounds: CYBL2A and S-CYBL2B. A marked difference in affinity between these two new compounds was observed in

250

Dubois et al. [3-Adrenoceptors in receptor imaging

JOURNALOF NUCLEARCARDIOLOGY May/June 1996

T a b l e 3. Biodistribution of [' 231]S-CYBL2B after intravenous injection of 50 pCi t h r o u g h a lateral ear vein of anesthetized male N e w Zealand white rabbits Organ

Left ventricle Right ventricle Left atri u m Right atrium Lung Liver Kidney Spleen Intestine Muscle Fat Blood

5 min

0.28 0.35 O.28 0.45 1.49 0.33 0.51 0.22 0.07 0.01 0.02 0.82

_+0.07 + 0.08 _+O.07 _+0.32 _+0.63 _+0.12 + 0.29 _+O. 11 _+0.04 _+0.005 + 0.01 + 0.73

15 min

0.45 0.38 0.29 0.27 0.89 0.73 1.09 0.48 0.25 0.06 0.04 0.05

_+0.04* _+0.03 _+0.04 _+0.03 + 0.03 _+0.05 + 0.50 _+0.08 _+0.02 + 0.008 + 0.003 + 0.002

30 min

50 min

1 hr

0.20 + 0.03* 0.24 _+0.03* O. i 8 + 0.03 O. 13 + 0.03 0.66 + 0.11 0.49 + 0.09 0.48 + 0.07 0.36 + 0.04 O. 13 + 0.03 0.03 + 0.006 0.04 + 0.008 0.03 + 0.005

0.21 _+0.01 O. 17 _+0.007 O.20 _+0.01 O. 13 _+0.01 0.50 +_0.04 0.74 + 0.09 0.41 + 0.01 0.23 _+0.09 0.33 + 0.03* 0.04 + 0.005 0.03 + 0.002 0.03 + 0.001

0.20 + 0.01 O. 16 + 0.005 O. 17 + 0.02 O. 16 + 0.03 0.46 _+0.03 0.55 + 0.06 0.35 + 0.08 0.28 _+0.04 0.32 _+0.01 0.06 + 0.007 0.03 + 0.01 0.03 + 0.0008

Data are e x p r e s s e d as m e a n percent injected d o s e per g r a m tissue times kilogram body weight _+SEM (n = 3). *p < 0.05 versus previous time point (analysis of variance, with Bonferroni test).

T a b l e 4. Radioactivity in the left ventricles and

lungs of N e w Zealand white rabbits 30 minutes after intravenous injection of 50 pCi ['f31]S-CYBL2B after a preinjection of 0.1 pmol (+) propranolol intravenously

Left ventricle Lungs

Control animals

Pretreated animals

0.24 _+0.02 0.63 + 0.09

0.20 +_0.01 0.33 + 0.02*

Data are e x p r e s s e d as m e a n percent inside diameter per g r a m tissue times kilogram body weight _+SEM (n = 5). *p < 0.05 compared with the control animals.

displacement experiments in vitro. In this study S-CYBL2B (para substituted) proved to be significantly more potent than C Y B L 2 A (ortho substituted), although both compounds were less active than the native agent CGP12177. Although S-CYBL2B showed less affinity for the receptor than CGP12177, it was radioactively labeled to perform further biodistribution studies. Radioactive labeling of S-CYBL2B yielded [J23I]SCYBL2B, with a specific activity of 5000 Ci/mmol > and a radiochemical purity of 99.9%_>. In vitro displacement experiments with homogenates of lung tissue showed that ['23I]S-CYBL2B could be displaced from ~2-receptors, thus demonstrating specific binding of ['23I]S-CYBL2B to this receptor. With homogenates of left ventricular tissue, ['z~I]S-CYBL2B could hardly be displaced by any of the antagonists used. Only S-CYBL2B proved able to counteract the binding to the receptor. However, this effect is merely

the result of a lowering of specific activity. Studies with isolated right atria showed that incubation with S-CYBL2B at a concentration of 10-6 mmol/L produced a reduction in basal heart rate. Concentration-response curves with isoprenaline were shifted parallel rightward by S-CYBL2B, thus demonstrating that S-CYBL2B is a functional and competitive receptor antagonist. At this concentration, S-CYBL2B proved even more potent in shifting the concentration-response curve to the right than propranolol. These results appear to be in contrast to the in vitro experiments with homogenates of left ventricular tissue, which suggested that the binding of S-CYBL2B is nonspecific. This apparent discrepancy may be explained by a high nonspecific binding of this compound, because high nonspecific binding would prevent the detection of specific binding with in vitro displacement experiments in case receptor density is low, whereas S-CYBL2B at a high concentration may be able to shift the concentration-response curve with isoprenaline to the right. Lipophilicity is probably not the reason for the absence of displacement in left ventricular tissue because no displacement was observed with lipophilic antagonists. Moreover, we observed no displacement with homogenates of left ventricular tissue. In addition, in lung tissue we observed no difference in displacement with different antagonists. In vivo biodistribution studies with ['23I]S-CYBL2B showed significant uptake in the left ventricle and lung of the animal. A constant level of radioactivity in the left ventricle was observed between 30 minutes and 2 hours after injection, suggesting specific binding. However, pretreatment of the animals with 0.1 gmol (+) propranolol could not prevent the uptake of [123I]SCYBL2B by the left ventricle. In contrast, uptake in the

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2 hr

O. 13 O. 11 O. 12 0.09 0.33 0.61 0.20 O. 17 0.22 0.07 0.01 0.02

__+0.003 + 0.005 __+0.02 + 0.007 + 0.02 __+O. 13 __+0.02 +__0.006 __+0.10 __+0.004 _+0.0001 _+0.001

4 hr

0.07 0.05 0.05 0.05 0.21 0.38 O. 13 O. 12 0.05 0.06 0.009 0.02

__+0.009 __+0.004 __+0.004 + 0.001 + 0.01 __+0.07 + 0.004 __+0,03 +__0,01 __+0,005 _+0,002 _+0,0007

Dubois et al. ~3-Adrenoceptors in receptor imaging

2 4 hr

0.024 0.03 0.03 0.03 0.08 0.25 0.05 0.05 0.05 0,03 0.009 0,03

+ 0.002 __+0.003 __+0.005 + 0.005 + 0.01 __+0.03 __+0.02 __+0.02 __+0.009 + 0.002 _+0.003 _+0.006

lungs could be p r e v e n t e d significantly by 0.1 g m o l (+) propranolol. This difference b e t w e e n in v i v o displacement in the lung and that in the left ventricle matches the a f o r e m e n t i o n e d in vitro data. There was no significant difference in affinity for ~ l - s e l e c t i v e adrenoceptors and ~2-selective adrenoceptors (displacement curves with lung tissue not presented), indicating that this n e w c o m p o u n d is not selective for one of the receptor subtypes. Possible m e t a b o l i s m cannot explain the difference b e t w e e n in v i v o d i s p l a c e m e n t in the lungs versus left ventricular tissue because [ ' 2 q ] S - C Y B L 2 B could be displaced f r o m the lungs, whereas it could not be displaced f r o m the left ventricles. A l t h o u g h these results strongly point to a high nonspecific binding, uptake by m u s c l e and fat, both tissues that are assumed to represent nonspecific binding, was rather low. T h e s e data suggest that C Y B L 1 and C Y B L 2 A s h o w too low affinity for the receptor to be useful for i m a g i n g purposes. S - C Y B L 2 B p r o v e d to be a m o d e r a t e l y potent n o n s e l e c t i v e 13-adrenoceptor antagonist. Despite its affinity for the receptor, the data presented also d e m o n strate that S - C Y B L 2 B shows high nonspecific binding. T h e r e f o r e it is unlikely that this radioligand m a y be useful for the m e a s u r e m e n t of cardiac 13-adrenoceptor density in vivo.

We thank J. A. J. M. Vekemans for helpful discussions on the synthesis and interpretation of nuclear magnetic resonance spectra. J. L J. van Dongen is gratefully acknowledged for performing the mass spectroscopy. P. A. 3/1. P. van Doremalen is acknowledged for pelforming the radiolabeling.

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