[3H]β-CIT: a radioligand for dopamine transporters in rat brain tissue

doi:10.1016/S0014-2999(99)00695-0

European Journal of Pharmacology

Volume 385, Issues 2–3, 3 December 1999, Pages 291-294

https://doi.org/10.1016/S0014-2999(99)00695-0 Get rights and content

Abstract

[³H]2-β-carbomethoxy-3-β-[4′-iodophenyl]tropane (β-CIT) was prepared and evaluated. With rat forebrain tissue, [³H]β-CIT showed high affinity for dopamine transporters (DAT), with selectivity for DAT over norepinephrine transporters, but not serotonin transporters, as well as DAT-stereoselectivity with β-CIT, amphetamine and methylphenidate. Affinity and selectivity for 53 compounds assayed with [³H]β-CIT and standard DAT radioligand [³H]GBR-12935 were highly correlated (r>0.95). [³H]β-CIT is proposed as a useful, high-affinity DAT radioprobe.

Introduction

Radioligands with high affinity or selectivity for dopamine transporters (DAT) include tritiated mazindol (Javitch et al., 1984), GBR-12935 (Andersen, 1987), phencyclidine analog N-(1-[1-benzo[b]thien-2-ylcyclohexyl])piperidine (BTCP) (Vignon et al., 1988) and phenyltropane congeners, including radioligands suitable for clinical neuroimaging Milius et al., 1991, Chally et al., 1996, Neumeyer et al., 1994, La Garza et al., 1999. Phenyltropanes include [³H]CFT (Madras et al., 1989) and [¹²⁵I]R-2β-carbomethoxy-3β-(4-iodophenyl)tropane (β-CIT, RTI-55) Boja et al., 1991, Innis et al., 1991. Since [³H]β-CIT is not available, we prepared and characterized it, finding close pharmacological similarity to the standard DAT radioligand [³H]GBR-12935, and a very high signal-to-noise ratio in radiotransporter binding and autoradiographic experiments.

Section snippets

Materials

Ten phenyltropanes were prepared (Neumeyer et al., 1994) at Research Biochemicals International (RBI, Natick MA): β-CIT, its precursor (nor-β-CIT), enantiomer (1S)-β-CIT, 3,4-diiodophenyl (CIIT), 4-fluorophenyl-(CFT) and 4-chlorophenyl congeners (CClT), as well as N-2-fluoroethyl-CIT (FE-CIT), N-3-fluropropyl-CIT (FP-CIT), 2β-carboisopropoxy-CIT (CIT-IP), and N-3-fluoropropyl-2β-carboisopropoxy (FP-CIT-IP) derivatives. [N-³H-methyl]β-CIT (87 Ci/mmol) was prepared from nor-β-CIT at New England

Binding of [³H]β-CIT with rat striatal homogenates

Striatal binding of [³H]β-CIT was linear vs. time (1–30 min, saturating by 45 min at 20°C), and linearly dependent on tissue-protein (to ±5-times standard assay conditions equivalent to 1.5 mg fresh striatum). Unlabeled β-CIT inhibited [³H]β-CIT binding with striatal homogenates monophasically (slope function, 0.99). K_d of [³H]β-CIT by Scatchard and kinetic analyses averaged 230 pM. At standard assay C=300 pM [³H]β-CIT, specific binding defined with 1 μM GBR-12909 averaged 92%.

Pharmacology of binding of [³H]β-CIT compared with [³H]GBR-12935

Potency (K_i, nM)

Discussion

Other than [³H]β-CIT, the only other commercially available tritiated-phenyltropane for labeling DAT is [³H]CFT, studied with primate brain tissue (Madras et al., 1989). Unlabeled β-CIT showed 57- and 15-fold higher affinity than CFT vs. [³H]β-CIT and [³H]GBR-12935 (Table 1). [³H]β-CIT was selective for DAT sites in autoradiographs of corpus striatum: 90% of total binding was displaced by the dissimilar but very potent DAT ligand, GBR-12909 (Table 1). The little [³H]β-CIT bound in extrastriatal

Acknowledgements

Supported by grants from NIH MH-19905, MH-34006, MH-47370, MH-49533, Bruce J. Anderson Foundation, funds of the McLean Private Donors Neuropharmacology Research Fund; and Saal van Zwanenbergstichtung. Drug substances were generously donated by manufacturers listed above.

References (16)

J.W. Boja et al.
[¹²⁵I]RTI-55, a potent ligand for dopamine transporters
Eur. J. Pharmacol.
(1991)
E. Habert et al.
Characterization of [³H]paroxetine binding to rat cortical membranes
Eur. J. Pharmacol.
(1985)
R.B. Innis et al.
Single photo emission computed tomography imaging of monoamine reuptake sites in primate brain with [¹²³I]β-CIT
Eur. J. Pharmacol.
(1991)
F.I. Tarazi et al.
Localization of dopamine receptor subtypes in caudate–putamen and nucleus accumbens septi of rat brain: comparison of D₁-, D₂-, and D₄-like receptors
Neuroscience
(1998)
J. Vignon et al.
[³H]N-(1-[2-benzo(b)-thiophenyl]cyclohexyl)-piperidine ([³H]BTCP): new phencyclidine analog selective for the dopamine uptake complex
Eur. J. Pharmacol.
(1988)
P.H. Andersen
Biochemical and pharmacological characterization of [³H]GBR-12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex
J. Neurochem.
(1987)
R.J. Baldessarini et al.
Prolonged D₂ antidopaminergic activity of alkylating and nonalkylating derivatives of spiperone in rat brain
Mol. Pharmacol.
(1992)
T. Chally et al.
Radio-synthesis of [¹⁸F]N-3-fluoropropyl-2-β-carbomethoxy-3-β-(4-iodophenyl)nortropane and the first human study with positron emission tomography
J. Nucl. Med. Biol.
(1996)

There are more references available in the full text version of this article.

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European Journal of Pharmacology

Abstract

Introduction

Section snippets

Materials

Binding of [³H]β-CIT with rat striatal homogenates

Pharmacology of binding of [³H]β-CIT compared with [³H]GBR-12935

Discussion

Acknowledgements

References (16)

[¹²⁵I]RTI-55, a potent ligand for dopamine transporters

Eur. J. Pharmacol.

Characterization of [³H]paroxetine binding to rat cortical membranes

Eur. J. Pharmacol.

Single photo emission computed tomography imaging of monoamine reuptake sites in primate brain with [¹²³I]β-CIT

Eur. J. Pharmacol.

Localization of dopamine receptor subtypes in caudate–putamen and nucleus accumbens septi of rat brain: comparison of D₁-, D₂-, and D₄-like receptors

Neuroscience

[³H]N-(1-[2-benzo(b)-thiophenyl]cyclohexyl)-piperidine ([³H]BTCP): new phencyclidine analog selective for the dopamine uptake complex

Eur. J. Pharmacol.

Biochemical and pharmacological characterization of [³H]GBR-12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex

J. Neurochem.

Prolonged D₂ antidopaminergic activity of alkylating and nonalkylating derivatives of spiperone in rat brain

Mol. Pharmacol.

Radio-synthesis of [¹⁸F]N-3-fluoropropyl-2-β-carbomethoxy-3-β-(4-iodophenyl)nortropane and the first human study with positron emission tomography

J. Nucl. Med. Biol.

Cited by (42)

Effects of monoamine reuptake inhibitors in assays of acute pain-stimulated and pain-depressed behavior in rats

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Effects of methylphenidate hydrochloride on the cardiovascular system in vivo and in vitro: A safety pharmacology study

Metabolism and autoradiographic evaluation of [<sup>18</sup>F]FE@CIT: a Comparison with [<sup>123</sup>I]β-CIT and [<sup>123</sup>I]FP-CIT

Synthesis, radiolabeling and baboon SPECT imaging of 2β-carbomethoxy-3β-(3′-[<sup>123</sup>I]iodophenyl)tropa ne ([<sup>123</sup>I]YP256) as a serotonin transporter radiotracer

<sup>18</sup>F fluoroethylations: different strategies for the rapid translation of <sup>11</sup>C-methylated radiotracers

Short communication[3H]β-CIT: a radioligand for dopamine transporters in rat brain tissue

Abstract

Introduction

Section snippets

Materials

Binding of [3H]β-CIT with rat striatal homogenates

Pharmacology of binding of [3H]β-CIT compared with [3H]GBR-12935

Discussion

Acknowledgements

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Neuroscience

Eur. J. Pharmacol.

Biochemical and pharmacological characterization of [3H]GBR-12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex

J. Neurochem.

Prolonged D2 antidopaminergic activity of alkylating and nonalkylating derivatives of spiperone in rat brain

Mol. Pharmacol.

Radio-synthesis of [18F]N-3-fluoropropyl-2-β-carbomethoxy-3-β-(4-iodophenyl)nortropane and the first human study with positron emission tomography

J. Nucl. Med. Biol.

Short communication
[³H]β-CIT: a radioligand for dopamine transporters in rat brain tissue

Binding of [³H]β-CIT with rat striatal homogenates

Pharmacology of binding of [³H]β-CIT compared with [³H]GBR-12935

Biochemical and pharmacological characterization of [³H]GBR-12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex

Prolonged D₂ antidopaminergic activity of alkylating and nonalkylating derivatives of spiperone in rat brain

Radio-synthesis of [¹⁸F]N-3-fluoropropyl-2-β-carbomethoxy-3-β-(4-iodophenyl)nortropane and the first human study with positron emission tomography