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[125I]iodopride: a specific high affinity radioligand for labelling striatal dopamine D-2 receptors
European Journal of Pharmacology, 150 (1988) 203-205 Elsevier
203
EJP 0125R
Rapid communication
l l2Slllodopride: a specific high affinity radioligand for labelling striatal dopamine D-2 receptors A a r o n J a n o w s k y 1,,, T o m a s De Paulis 1,2, Jeffrey A. C l a n t o n 3, H o w a r d E. Smith 2, Michael H. Ebert 1 a n d R o b e r t M. Kessler 1,3 Departments of I Psychiatry, 2 Chemistry, and 3 Radiology Vanderbilt University, Nashville, TN 37235, U.S.A. Received 25 April 1988, accepted 25 April 1988
Substituted benzamides are currently among the most selective antagonists at dopamine D-2 receptors, and high affinity ligands have been developed by substituting halogens into the aromatic ring of the benzamides (De Paulis et al., 1986). The binding of the radiolabelled analogues of a number of substituted benzamides, including sulpiride, raclopride, remoxipride and eticlopride suggest that this class of coupounds has a relatively low affinity for dopamine D-1 receptors, as well as for the receptors of other neurotransmitters (Hall et al., 1986). The respective affinities (KdS) for these compounds at dopamine D-2 receptors ranges from approximately 10 nM for sulpiride to 0.17 nM for eticlopride (Hall et al., 1986, and references therein), and the more potent radiolabelled substituted benzamides appear to be useful for both the in vivo and in vitro characterization of dopamine D-2 receptors in brain (Seeman et al., 1986). The selectivity for the dopamine D-2 receptor recognition site also suggests that these benzamides may be superior radioligands, as compared to radiolabelled haloperidol and spiperone, which appear to bind to the same site but also have a relatively high affinity for other neurotransmitter receptors (Hall et al., 1986; See-
* To whom all correspondenceshould be addressed: Research Service (151-P), Veterans Administration Medical Center, 3710 S.W.U.S. Veterans Hospital Rd., Portland, OR 97207, USA.
man et al., 1986). We now report the high affinity, stereoselective, reversible, and sodium dependent binding of [azsI](S)-N-[(1-ethyl-2pyrrolidinyl)methyl]-5-iodo-2-methoxybenzamide, here called [125I]iodopride, to a membrane preparation from rat corpus striatum. Iodopride is a new iodinesubstituted benzamide (fig. 1, inset), which has a closer structural similarity to sulpiride than has the previously reported [125I]iodosulpride (Martres et al., 1985) or [125I]IBZM (Kung et al., 1988). [125I]Iodopride has an iodine atom in place of the aminosulfonyl group in the aromatic 5-position of (S)-sulpiride, and was prepared from the tributyltin-substituted intermediate by an iododestannylation reaction in the presence of 125I. The specific activity was 2000 C i / m m o l and the radiochemical yield was 56%. Striatal membranes were prepared by homogenization of freshly dissected rat striatum in 40 volumes (w/v) of ice cold Tris-HC1 buffer (50 mM, pH 7.4) containing 120 mM NaC1, 5 mM KC1, 2 mM CaCI: and 1 mM MgC12, using a Brinkman Polytron (Brinkman Instruments, Westbury, NY)(setting 5, 15 s). Following centrifugation at 30000 × g for 10 min at 4 ° C, the pellet was resuspended in 40 volumes of fresh buffer. The suspension was incubated at 3 7 ° C for 10 min, and then centrifuged again at 30 000 × g for 10 min at 4 ° C. The final pellet was resuspended in 100 volumes of ice cold buffer. The binding of [125I]iodopride was assayed in tubes containing 400 /~1 of the membrane preparation (0.3 mg protein) in a final volume of 500/~1. Tubes
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (BiomedicalDivision)
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Fig. 1. Effects of dopamine and dopamine receptor antagonists on [125I]iodopride and [3H]sulpiride binding in a membrane preparation from striatum. The experiments were carried out as described in the text. ICs0 values (defined as the concentration of drug necessary to inhibit 50% of specific [125I]iodopridebinding) for various drugs were as follows: haloperidol, 1.2 nM; raclopride, 1.3 nM; (-)iodopride, 1.5 nM; sulpiride, 32.5 nM; (+)iodopride, 112.7 nM; SCH 23390, 600 riM; dopamine, 1 860 nM. The ICs0 values for inhibition of [3H]sulpiride binding by the same drugs were as follows: (-)iodopride, 3.3 nM; haloperidol, 6.0 nM; raclopride, 14 nM; sulpiride, 36 nM; (+)iodopride, 145 nM; dopamine, 1700 nM; SCH 23390, 1900 nM. In calculating the ICs0 values, 6-8 concentrations of each drug was used in the presence of 0.6-0.9 nM [125I]iodopride, or 5 nM [3H]sulpiride. The ICs0values are averaged from three experiments that were conducted with triplicate determinations. The correlation coefficient for inhibition of [3H]sulpiride binding and [125I]iodopride binding was 0.81; P < 0.027, using Pearson's Product moment. were incubated at 2 5 ° C for 30 min, and the incubation was terminated by vacuum filtration over Whatman G F / B filters, presoaked in 0.3% polyethylenimine. The filters were rinsed 3 times with 4 ml of ice cold buffer, and the radioactivity remaining on the filters was measured by conventional gamma-ray spectrometry. Specific binding, defined as the difference in binding observed in the presence and absence of 10 uM ( - ) iodopride, was approximately 60% of the total binding at a radioligand concentration of 1 nM. The use of sulpiride (10 uM) to define nonspecific binding yielded similar results. The specific binding of [125I]iodopride to striatal membrane preparations is saturable and of high affinity (K d - 3 nM) (data not shown). As with the binding of other radiolabelled benzamides, the specific binding of [125I]iodopride is sodium dependent, and optimal binding is observed at sodium concentrations greater than 40 mM. The sodium dependent binding of [125I]iodopride is unevenly distributed throughout the rat brain and is highly localized to the striatum, where it reaches a peak accumulation 20 min after intravenous
a d m i n i s t r a t i o n . T h e inhibition of specific [125I]iodopride and [3H]sulpiride binding in vitro by dopamine and dopamine antagonists is shown in fig. 1. The rank order of potency for these agents in displacing specific [125I]iodopride binding is similar to their abilities to displace [3H]sulpiride and [3H]spiperone from dopamine D-2 receptors in rat corpus striatum (fig. 1; Hall et al., 1986), and suggests that the iodinated ligand binds to the dopamine D-2 receptor recognition site. In addition, the relatively high lipophilicity, selectivity and receptor affinity of iodopride suggests that its 122I or 123I labelled analogues may be valuable tools for the in vivo imaging, using positron emission tomography or single photon emission tomography, respectively, of striatal dopamine D-2 receptors.
References De Paulis, T., Y. Kumar, L. Johansson, S. R~imsby, H. Hall, M. SNlemark, K. ,~ngeby-Mrller and S.-O. 0gren, 1986, Potential neuroleptic agents. 4. Chemistry, behavioral
205 pharmacology, and inhibition of [3H]spiperone binding of 3,5-disubstituted N-[(1-ethyl-2-pyrrolidinyl)methyl]-6methoxysalicylamides, J. Med. Chem. 29, 61. Hall, H., M. S~Uemark and E. Jerning, 1986, Effects of remoxipride and some related new substituted salicylamides on rat brain receptors, Acta Pharmacol. Toxicol. 58, 61. Kung, H., Y.-Z. Guo, J. Billings, X. Xu, R. Mach, M. Blau and R. Ackerhalt, 1988, Preparation and biodistribution of [125I]IBZM: a potential CNS D-2 dopamine receptor imaging agent, Nucl. Med. Biol. 15, 195.
Martres, M.-P., N. Sales, M.-L. Bouthenet and J.-C. Schwartz, 1985, Localization and pharmacological characterization of D-2 dopamine receptors in rat cerebral neocortex and cerebellum using [125I]iodosulpride, European J. Pharmacol. 118, 211. Seeman, P., D.E. Grigoriadis and H.B. Niznik, 1986, Selectivity of agonists and antagonists and D-2 dopamine receptors compared to D-1 and S-1 receptors, Drug Develop. Res. 9, 63.
203
EJP 0125R
Rapid communication
l l2Slllodopride: a specific high affinity radioligand for labelling striatal dopamine D-2 receptors A a r o n J a n o w s k y 1,,, T o m a s De Paulis 1,2, Jeffrey A. C l a n t o n 3, H o w a r d E. Smith 2, Michael H. Ebert 1 a n d R o b e r t M. Kessler 1,3 Departments of I Psychiatry, 2 Chemistry, and 3 Radiology Vanderbilt University, Nashville, TN 37235, U.S.A. Received 25 April 1988, accepted 25 April 1988
Substituted benzamides are currently among the most selective antagonists at dopamine D-2 receptors, and high affinity ligands have been developed by substituting halogens into the aromatic ring of the benzamides (De Paulis et al., 1986). The binding of the radiolabelled analogues of a number of substituted benzamides, including sulpiride, raclopride, remoxipride and eticlopride suggest that this class of coupounds has a relatively low affinity for dopamine D-1 receptors, as well as for the receptors of other neurotransmitters (Hall et al., 1986). The respective affinities (KdS) for these compounds at dopamine D-2 receptors ranges from approximately 10 nM for sulpiride to 0.17 nM for eticlopride (Hall et al., 1986, and references therein), and the more potent radiolabelled substituted benzamides appear to be useful for both the in vivo and in vitro characterization of dopamine D-2 receptors in brain (Seeman et al., 1986). The selectivity for the dopamine D-2 receptor recognition site also suggests that these benzamides may be superior radioligands, as compared to radiolabelled haloperidol and spiperone, which appear to bind to the same site but also have a relatively high affinity for other neurotransmitter receptors (Hall et al., 1986; See-
* To whom all correspondenceshould be addressed: Research Service (151-P), Veterans Administration Medical Center, 3710 S.W.U.S. Veterans Hospital Rd., Portland, OR 97207, USA.
man et al., 1986). We now report the high affinity, stereoselective, reversible, and sodium dependent binding of [azsI](S)-N-[(1-ethyl-2pyrrolidinyl)methyl]-5-iodo-2-methoxybenzamide, here called [125I]iodopride, to a membrane preparation from rat corpus striatum. Iodopride is a new iodinesubstituted benzamide (fig. 1, inset), which has a closer structural similarity to sulpiride than has the previously reported [125I]iodosulpride (Martres et al., 1985) or [125I]IBZM (Kung et al., 1988). [125I]Iodopride has an iodine atom in place of the aminosulfonyl group in the aromatic 5-position of (S)-sulpiride, and was prepared from the tributyltin-substituted intermediate by an iododestannylation reaction in the presence of 125I. The specific activity was 2000 C i / m m o l and the radiochemical yield was 56%. Striatal membranes were prepared by homogenization of freshly dissected rat striatum in 40 volumes (w/v) of ice cold Tris-HC1 buffer (50 mM, pH 7.4) containing 120 mM NaC1, 5 mM KC1, 2 mM CaCI: and 1 mM MgC12, using a Brinkman Polytron (Brinkman Instruments, Westbury, NY)(setting 5, 15 s). Following centrifugation at 30000 × g for 10 min at 4 ° C, the pellet was resuspended in 40 volumes of fresh buffer. The suspension was incubated at 3 7 ° C for 10 min, and then centrifuged again at 30 000 × g for 10 min at 4 ° C. The final pellet was resuspended in 100 volumes of ice cold buffer. The binding of [125I]iodopride was assayed in tubes containing 400 /~1 of the membrane preparation (0.3 mg protein) in a final volume of 500/~1. Tubes
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (BiomedicalDivision)
204
1000 ._c -o ._E 100 m
~
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H
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~/3
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0 a.
o~ 0.1 0.1
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1'0 Specific
160
10'00
[nSl]lodopride Binding (nM)
Fig. 1. Effects of dopamine and dopamine receptor antagonists on [125I]iodopride and [3H]sulpiride binding in a membrane preparation from striatum. The experiments were carried out as described in the text. ICs0 values (defined as the concentration of drug necessary to inhibit 50% of specific [125I]iodopridebinding) for various drugs were as follows: haloperidol, 1.2 nM; raclopride, 1.3 nM; (-)iodopride, 1.5 nM; sulpiride, 32.5 nM; (+)iodopride, 112.7 nM; SCH 23390, 600 riM; dopamine, 1 860 nM. The ICs0 values for inhibition of [3H]sulpiride binding by the same drugs were as follows: (-)iodopride, 3.3 nM; haloperidol, 6.0 nM; raclopride, 14 nM; sulpiride, 36 nM; (+)iodopride, 145 nM; dopamine, 1700 nM; SCH 23390, 1900 nM. In calculating the ICs0 values, 6-8 concentrations of each drug was used in the presence of 0.6-0.9 nM [125I]iodopride, or 5 nM [3H]sulpiride. The ICs0values are averaged from three experiments that were conducted with triplicate determinations. The correlation coefficient for inhibition of [3H]sulpiride binding and [125I]iodopride binding was 0.81; P < 0.027, using Pearson's Product moment. were incubated at 2 5 ° C for 30 min, and the incubation was terminated by vacuum filtration over Whatman G F / B filters, presoaked in 0.3% polyethylenimine. The filters were rinsed 3 times with 4 ml of ice cold buffer, and the radioactivity remaining on the filters was measured by conventional gamma-ray spectrometry. Specific binding, defined as the difference in binding observed in the presence and absence of 10 uM ( - ) iodopride, was approximately 60% of the total binding at a radioligand concentration of 1 nM. The use of sulpiride (10 uM) to define nonspecific binding yielded similar results. The specific binding of [125I]iodopride to striatal membrane preparations is saturable and of high affinity (K d - 3 nM) (data not shown). As with the binding of other radiolabelled benzamides, the specific binding of [125I]iodopride is sodium dependent, and optimal binding is observed at sodium concentrations greater than 40 mM. The sodium dependent binding of [125I]iodopride is unevenly distributed throughout the rat brain and is highly localized to the striatum, where it reaches a peak accumulation 20 min after intravenous
a d m i n i s t r a t i o n . T h e inhibition of specific [125I]iodopride and [3H]sulpiride binding in vitro by dopamine and dopamine antagonists is shown in fig. 1. The rank order of potency for these agents in displacing specific [125I]iodopride binding is similar to their abilities to displace [3H]sulpiride and [3H]spiperone from dopamine D-2 receptors in rat corpus striatum (fig. 1; Hall et al., 1986), and suggests that the iodinated ligand binds to the dopamine D-2 receptor recognition site. In addition, the relatively high lipophilicity, selectivity and receptor affinity of iodopride suggests that its 122I or 123I labelled analogues may be valuable tools for the in vivo imaging, using positron emission tomography or single photon emission tomography, respectively, of striatal dopamine D-2 receptors.
References De Paulis, T., Y. Kumar, L. Johansson, S. R~imsby, H. Hall, M. SNlemark, K. ,~ngeby-Mrller and S.-O. 0gren, 1986, Potential neuroleptic agents. 4. Chemistry, behavioral
205 pharmacology, and inhibition of [3H]spiperone binding of 3,5-disubstituted N-[(1-ethyl-2-pyrrolidinyl)methyl]-6methoxysalicylamides, J. Med. Chem. 29, 61. Hall, H., M. S~Uemark and E. Jerning, 1986, Effects of remoxipride and some related new substituted salicylamides on rat brain receptors, Acta Pharmacol. Toxicol. 58, 61. Kung, H., Y.-Z. Guo, J. Billings, X. Xu, R. Mach, M. Blau and R. Ackerhalt, 1988, Preparation and biodistribution of [125I]IBZM: a potential CNS D-2 dopamine receptor imaging agent, Nucl. Med. Biol. 15, 195.
Martres, M.-P., N. Sales, M.-L. Bouthenet and J.-C. Schwartz, 1985, Localization and pharmacological characterization of D-2 dopamine receptors in rat cerebral neocortex and cerebellum using [125I]iodosulpride, European J. Pharmacol. 118, 211. Seeman, P., D.E. Grigoriadis and H.B. Niznik, 1986, Selectivity of agonists and antagonists and D-2 dopamine receptors compared to D-1 and S-1 receptors, Drug Develop. Res. 9, 63.