BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS Neuroleptic Binding Sites: Specific Labeling in Mice with [18]Haloperidol, A Potential Tracer for Positron Emission Tomography P. B. Zanzonico, R. E. Bigler, and B. Schmall Memorial Sloan-Kettering Cancer Center, New York, New York Haloperidol labeled with fluorine-18 (Ti/a = 110 min, positron emission 97%), prepared yielding .04 Ci/mlllimole by the Balz-Schiemann reaction, was evaluated in a murine model as a potential radiotracer for noninvasive determination, by posi tron-emission tomography, of regional concentrations of brain dopamine receptors in patients. As the haloperldol dose In mice was increased from 0.01 to 1000 fig/kg, the relative concentration of [18F]haloperidol (/¿Ciper g spec¡men/¿tCiper g of body mass), at one hour after injection decreased from 30 to 1.0 in the strl- atum and from 8.0 to 1.0 in the cerebellum. The striata! radioactivity, plotted as rel ative concentration against log of dose, decreased sigmoidally, presumably re flecting competition between labeled and unlabeled haloperidol for a single class of accessible binding sites. Because the cerebellum is relatively deficient in dopa mine receptors, the observed decrease in cerebellar radioactivity may reflect a saturable component of haloperidol transport into brain. The high brain concentra tions and the unexpectedly high striâtum-to-cerebellumconcentration ratios (>4 at haloperidol doses <1 ¿ig/kg)suggest that [18F]haloperidol warrants further in vestigation as a potential radiotracer for dopamine receptors. J NucíMod 24: 408-416,1983 Aberrant dopaminergic neuronal transmission has conjunction with a suitable radiotracer for dopamine been implicated in the pathogenesis of various neurologic receptors, for the noninvasive determination of regional disorders, including Parkinsonism (1-3), Huntington's concentrations of brain dopamine receptors in vivo. chorea (1,4,5), and tardive dyskinesia (6-10), as well Specific dopamine receptors are present in brain in as of certain neuroses (11) and psychoses (12-15). nanomolar concentrations (22), and therefore will be Characterization of dopamine receptors has been ac saturated at comparably low brain concentrations of a complished largely using radiometrie binding assays of dopaminergic ligand. In contrast, nonspecific binding synaptic membrane preparations derived from animal sites are practically nonsaturable (23-26). A potential or postmortem brain specimens (16-19). The highly dopamine-receptor-binding radiotracer must have a invasive and nonphysiologic nature of these procedures sufficiently high specific activity to yield an adequate severely limits their applicability to man and makes in photon flux for statistically significant radiation counting terpretation of results somewhat problematic. Conse at ligand doses that do not saturate the specific binding quently there is great interest in the potential utilization sites. Because theoretical maximum specific activity is of positron-emission tomography (PET) (20,21), in inversely proportional to half-life, dopaminergic ligands labeled with short-lived radionuclides—most notably carbon-11 (Ti/2 = 20.4 min, positron emission 100%) Received Sept. 24, 1982; revision accepted Dec. 17, 1982. and fluorine-18 (T|/2 = 110 min, positron emission For reprints contact: Dr. Pat Zanzonico, Biophysics Laboratory, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New 97%)—have great potential as dopamine receptor- York, NY 10021. binding radiotracers. For compounds singly labeled with 408 THE JOURNAL OF NUCLEAR MEDICINE BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS carbon-11 or fluorine-18, the theoretical maximum (Schmall B, Tilbury RS, Nisselbaum, JS, unpublished specific activities are 9.2 X IO6and 1.7 X IO6Ci/mmole, data). Although the resulting specific activity 400 respectively. mCi/mmole appears to be somewhat low for receptor Haloperidol and spiroperidol—potent neuroleptics of studies in vivo, such studies are feasible in the "partial- the butyrophenone series, widely used in the manage body counting facility" a high-sensitivity, low-back ment of schizophrenia—have been shown to bind spe ground radiation counting facility designed and installed cifically, reversibly, and with high affinity to synaptic in our center's biophysics laboratory (45). membrane preparations in vitro (22). Demonstration in vivo of specific spiroperidol binding sites has been ac MATERIALS AND METHODS complished using specifically tritiated spiroperidols of high specific activity, namely l-phenyl-[3-3H]spirop- Chemicals.All commerciallyobtained chemicals and eridol (sp. ac. 8-15 Ci/mmole) (27-30) and 1-phenyl- solventswere of reagent grade and were used as supplied. [4-3H]spiroperidol (30 Ci/mmole) (31,32), and also Haloperidol in crystalline form was a gift.* [G-3H]- />-[77Br]bromospiroperidol (5-10 Ci/mmole) (33,34). Haloperidol (14 Ci/mmole) was obtained commercially. Radiochemical stability of the tritiated ligands in vivo The radiochemical purity (97%) was verified by TLC on has been demonstrated by thin-layer chromatography silica gel* jn chloroform/methanol/ammonium hy (TLC) (27,29,31). In the case of l-phenyl-[3H]spirop- droxide (98:2:1). eridol, the striatum-to-cerebellum radioactivity con Animals. All experiments used male and female B6 centration ratio at 1hr after injection decreased from 8.8 mice, maintained on a 12-hr light-dark cycle, with free to 1.0 as the spiroperidol dose was increased from 44 to access to food and water and housed 8-10 to a cage. The 2000 Mg/kg (27,29). Because dopamine receptors are mice were approximately six months old and weighed abundant in the striatum and relatively deficient in the 20-25 g each. cerebellum (22), this decrease in the striatum-to-cere Preparationandanalysis of "de-fluorinatedhaloper idol". This compound, 4-[4-(4-chlorophenyl)-4-hy- bellum ratio presumably reflects saturation of striatal dopamine receptors and the ensuing dose-dependent droxypiperidino] butyrophenone, appears to be a ligand decrease in the proportion of specifically bound spirop binding to dopamine receptors (46), and was therefore eridol in brain. In contrast, demonstration in vivo of prepared as an analytical standard for high-pressure specific haloperidol binding sites using tritiated halo- liquid chromatography. It was synthesized by the N- peridol has not yet been accomplished. The specific ac alkylation of 4-(4-chlorophenyl)-4-hydroxypiperidine tivities of randomly labeled tritiated haloperidol (0.042 with -y-chlorobutyrophenone, and was purified by re- Ci/mmole) and of 4'-fluorobutyrophenone-[2'-3H]- crystallization from 95% ethanol (mp:125-128°C;Ref. haloperidol (0.1 Ci/mmole), though adequate for 46: 128-130°C). Thin-layer chromatography of the pharmacokinetic studies (35-40), are apparently too low product yielded a single spot (Rf = 0.75). The identity for the discrimination of specifically localized from of the product was verified by chemical ionization mass nonspecifically localized haloperidol in brain (37,40). spectrometry. Though of adequately high specific activity, the unex Preparationandanalysisof [18F]haloperidol.This was pected absence of selective striatal localization of 4- prepared from the corresponding tetrafluoroborate di (4-chlorophenyl)-[3-3H]-haloperidol (8-18 Ci/mmole) azonium salt (chemical yield 20-30%, radiochemical at a haloperidol dose of only 5 Mg/kg (30) suggests, yield 0.5-1.0%) by the Balz-Schiemann reaction using perhaps, that it may undergo spontaneous de-tritiation aqueous [18F]fluoride, as developed by Kook et al. (43) in vivo. and adapted by Schmall and Bigler (44). [I8F]Fluoride We have prepared [l8F]haloperidol by the Balz- was produced in our compact medical cyclotron via the Schiemann reaction and have evaluated it in a murine nuclear reaction 16O(3//e,p)18F by the irradiation of model as a radioligand for the specific labeling in vivo water with 22-MeV helium-3 ions (47,48). Chemical of neuroleptic binding sites. Because of the technical and radiochemical purity was greater that 95%, as de constraints imposed by a short-lived, cyclotron-produced termined by reverse isotope dilution analysis, thin-layer radionuclide such as fluorine-18, we have concurrently chromatography, and high-pressure liquid chromatog evaluated [G-3H]haloperidol as a convenient model raphy. Reverse isotope dilution analysis was accom radioligand for the determination of the dose-dependent plished by chromatographing an admixture of [I8F]- regional concentrations of haloperidol in brain. [G- haloperidol and a large excess of unlabeled haloperidol 3H]Haloperidol is commercially available and has been on neutral alumina (Brockman activity 1, 80-200 mesh) used in receptor binding assays in vitro (41,42). [I8F]- and comparing its specific activity before and after Haloperidol prepared by the Balz-Schiemann reaction chromatography. Analysis of the product by thin-layer includes carrier because fluorine-18 is introduced by chromatography on silica gel in chloroform/ isotopie exchange with stable fluorine in the corre methanol/ammonium hydroxide (98:2:1) and in chlo sponding tetrafluoroborate diazonium salt (43-44). roform/ethyl acetate/formic acid (5:4:1) yielded single Volume 24, Number 5 409 ZANZONICO, BIGLER, AND SCHMALL radioactive spots (Rf = 0.75 and 0.36, respectively) 1 Mg/kg or less, each specimen was placed in a small identical to those of haloperidol. High-pressure liquid polyethylene tube and the radioactivity counted by an chromatography of