Iododerivative of pargyline: A potential tracer for the exploration of monoamine oxidase sites by SPECT

Nucl. Med. Eiol. Vol. 22, No. 6, pp. 721-736, 1995 Copyright 0 1995 Eis&ier Science Ltd Printed in GreatBritain.All rightsreserved 0969-8051195$9.50 + 0.00

Iododerivative of Pargyline: A Potential Tracer for the Exploration of Monoamine Oxidase Sites by SPECT ISABELLE LENA, JEAN-EDOUARD OMBETTA, SYLVIE CHALON, ANNE-MARIE DOGNON, JEAN-LOUIS BAULIEU, YVES FRANGIN, LUCETTE GARREAU, JEAN-CLAUDE BESNARD and DENIS GUILLOTEAU* Laboratoire de Biophysique MCdicale et Pharmaceutique,INSERM U3 16 3 1 avenue Monge, 37200Tours-France (Accepted 13 February 1995)

Monoamine oxidases are important in the regulation of monoaminergic neurotransmission.An increase in monoamine oxidasc B (MAO B) has been observed in some neurodegenerative diseases, and therefore quantification of cerebral MAO B activity by SPECT would be useful for the diagnosis and therapeutic follow-up of

thesedisorders.We have developedan iodinatedderivative of pargyline, a selective inhibitor of MAO B, in order to explore this enzyme by SPECT. Stable bromo and iodo derivatives of pargyline were synthesized and chemically characterized. The radioiodinated ligand [‘ZSI]-2-iodopargyline was obtained with high specific activity from the bromo precursor by nucleophilic exchange. Affinity and selectivity of 2-iodopargyline were testedin vitro. Biodistribution study of [‘Z51]-2-iodopargylinewas performedin rats. Radioiodinatedligand were obtained in a no-carrier-addedform. 2-iodopargyline has a higher in vitro affinity for MAO B than pargyline. However, the in vitro selectivity for MAO B was better for pargyline than for 2-iodopargyline. Ex vivo autoradiographicstudiesand in viva saturationstudieswith selective inhibitors of MAO showed that the cerebral biodistribution

of [‘*‘I]-2-iodopargyline

in the rat is consistent with high level binding to MAO B sites in the

pineal gland and in the thalamus.In conclusion,2-iodopargyline preferentially binds in viva to MAO B sites with high affinity. However, its selectivity for MAO B in rats is not very high, whereas this ligand binds to a lesser extent to MAO A. It will be then of great value to evaluate the specificity of 2-iodopargyline in humans. This new ligand labeled with lz31should thereforebe a suitable tool for SPECT exploration of MAO B in the human brain.

Introduction Monoamine oxidases (MAO, E.C 1.4.3.4) play an important role in the regulation of monoaminergic transmission in the central nervous system. Indeed, neurotransmitter amines are released in the synapse and there is about 90% reuptake by presynaptic neurons where part may be stored in granules and another part is inactivated by deamination by MAO. Two types of MAO are described, MAO A and MAO B, identified by their different substrate and inhibitor specificity (Johnston, 1968; Knoll and Magyar, 1972): MAO A preferentially deaminates 5-hydroxytryptamine (5-HT) and is irreversibly and selectively inhibited by clorgyline; MAO B selectively oxidizes p-phenylethylamine (PEA) and is irreversibly and selectively inhibited by L-deprenyl. In rat brain (Levitt et al., 1982) and human brain (Thorpe et al., 1987), MAO A is preferentially expressed by noradrenergic neurons while MAO B is specifically expressed by serotoninergic neurons and glial cells. The two forms of MAO are distributed differently in organs and some variations exist in different

*Author for correspondence. 727

species (Youdim and Finberg, 1991). Thus, MAO B represents about 80% of total MAO activity in the human brain, whereas MAO B represents only 40% of total MAO activity in the rat brain. An increase in MAO B activity, without change in MAO A activity, has been observed in some neurodegenerative disorders such as Alzheimer’s disease (Adolfsson et al., 1980; Oreland and Gottfries, 1986; Reinikainen et al., 1987; Jossan et al., 1991), Huntington’s chorea (Mann et al., 1980, 1986) and amyotrophic lateral sclerosis (Eckblom et al., 1992). In Parkinson’s disease the modifications of MAO B activity are unclear (Strolin Benedetti and Dostert, 1989; Oreland, 1991). It would be therefore of great interest to be able to explore MAO B activity by single photon emission computed tomography (SPECT) for the diagnosis and therapeutic follow-up of these neurodegenerative disorders. The suicide inhibitors of MAO, [“C]-clorgyline, [‘lC]-pargyline and [“Cl-L-deprenyl (Fowler et al., 1987; Ishiwata et al., 1985) and the substrate of MAO B [’ ‘Cl-N,Ndimethylphenylethylamine (Shinitoh et al., 1987; Inoue et al., 1985; Halldin et al., 1989), have been used as positron emitting ligands for PET studies. Only preliminary results have been reported with iodinated inhibitors of MAO B for SPECT exploration (Macwhorter and

728

ISABELLE

Baldwin, 1991;Beer et al., 1993;Ohmomo et al., 1992). We therefore developed a radioiodinated derivative of pargyline, a potent irreversible inhibitor of MAO B (Kalir et al., 1981; Neff and Fuentes, 1976). We describe here the synthesis and radiolabeling of N-(2-iodobenzyl)-N-methyl-2-propynylamine, named 2-iodopargyline. The in vitro affinity and specificity for MAO B of 2-iodopargyline were determined on rat brain preparations and biodistribution studies were performed with [1251]-2-iodopargyline.

LENA

C, d.

(UV 254 nm) on an Ultrasphere IP 5 pm (4.6 x 25 cm) C,, reverse phase column using CH,CN/H,O (60140 v/v) as eluent at a flow rate of 1 mL/min. Radiolabeling (Fig. 1B)

[ ‘251]-2-iodopargyline was obtained from 2-bromopargyline by radioiodide for bromide nucleophilic exchange. Sodium [“‘II-iodide (specific activity 2200 Ci/ mmol, Amersham, England) was added to a vial containing 1 mg substrate, 455 pL mixture of reducing agents (25 mg gentisic acid, 35 mg citric acid, 1 mg Materials and Methods SnSO, in 2.25 mL of H,O and 25 pL glacial acetic acid) Synthesis of N-(2-bromobenzyl)-N-methyland 30 pL CuSO, solution (32.5 mg in 10 mL H,O) in 2-propynylamine or 2-bromopargyline, and a N, atmosphere.After heating for 40 min at 144X and N-(2-iodobenzyl)-N-methyl-2-propynylamine or cooling down, the reaction mixture diluted with water 2-iodopargyline (Fig. IA). was passedthrough a reverse phase cartridge (Sep Pak C,,, Waters).The radioiodinated product and its bromo We synthesizeda bromoderivative of pargyline in orprecursor were retained on the cartridge and were then der to use it as a precursor in the radiolabeling proceeluted by ethyl ether (0.5 mL x 4) and evaporated to dure. We also synthesized a stable iododerivative, 2-iodryness.The residue was taken up with acetonitrile and dopargyline, for the determination of the HPLC purifipurified with a Beckman HPLC system including UV cation conditions and for in vitro assays.A solution of (254 nm) and gammadetectors.Elution was performed N-(2-iodobenzyl) chloride or N-(2-bromobenzyl) broon an Ultrasphere IP 5pm (4.6 x 25 cm) C,, reverse mide (10 mmol; Aldrich, France) was added slowly at phasecolumn with a CH,CN/H,O (50/50 v/v) mixture at room temperatureto a solution of N-methyl-Zpropynyla flow rate of lmL/min. The peak corresponding to amine (10 mmol; Aldrich, France) in ethanol (10 mL). [‘251]-2-iodopargylinewas collected and passedthrough The resulting solution was stirred at room temperature a Sep Pak C,, cartridge to eliminate the solvent. The for 30 min in the presenceof pyridine (10 mmol) and radioiodinated product was then eluted with ethylether stirred again at reflux overnight. After removing the solvent under vacuum, the residuewas taken up with NaOH and evaporatedto dryness. The final product, [‘251]-2iodopargyline was dissolved in a propyleneglycol/water 1N (20 mL), extracted with ethyl acetate(20 mL) and mixture (70/30 v/v) and passedthrough a 0.22 pm filter. washed with water (20 mL x 3). The organic layer was A coinjection of the radioiodinated product with stadried over sodium sulfate and removedunder vacuum to ble 2-iodopargyline was carried out on an UltrasphereIP give a clear oil. Proton nuclear magnetic resonance(‘H-NMR) spectra 5 pm C,, column with CH,CN/H,O mixture (50/50 v/v) were recorded in CDCl, on a BIIker AM 200 (200 at a flow rate of 1 mWmin. MHz) spectrometer and the chemical shifts were expressedin parts per million (ppm) in relation to an in- Lipophilicity measurement ternal tetramethylsilane standard. The chemical purity The lipophilic characterof 2-iodopargyline was deterwas estimated by HPLC analysis with a LKB system mined by C,, reversephaseHPLC according to El Tayar

A CH2-Y

+ H-N-CH2-Csi

C-H

CH2-N-CH,-C-C-H

-

X = Br or I Y = Br or Cl

B Br

/

d-

125 I

CH3

\-C&-;-CH2-Cz&H 2-bromopargyiine

‘251Na

C,H3

CH,-N-CH2-C-C-H 1sI-2-iodopargyline

Fig. 1. (A) Synthesisof 2-bromopargyline and2-iodopargyline;(B) Radiochemical synthesisof [‘2511-2-iodopargyline.

2-Iodopargylinefor explorationof MAO B

729

et al. (1985). The dluting solvant was a mixture of 3-Nmorpholino propanesulfonic acid (MOPS) buffer (20 mM, pH 7.5) containing 2 mL per liter of n-decylamine and 50-70% methanol concentrations.The logarithm of the apparentcapacity factors, log K,, were plotted versus methanol concentrations and the log K,, apparent lipophilicity at pH 7.5, was determinedby extrapolation to 0% methanol concentration.

tissue or blood. Thyroid uptake,which representsin vivo deiodination, was also determined. This parameterwas expressedas the percentageof total injected dose. Determination of the radioactivity bound to proteins was performed in rat plasma and brains 120 min after I.V. injection of 80 uCi of [‘251]-2-iodopargyline. Intracardiac sampleswere taken and brains were removed. Plasmasampleswere treatedwith 0.1 mL of 0.5 M perchloric acid to precipitate proteins. After centrifugation Assay of inhibition of MAO activity the radioactivity of the supematantsand of the pellets The inhibitory activity of 2-iodopargyline was as- was countedin a gammascintillation counter. The brains sessedby measuring its potency to inhibit in vitro the were treated as described by Murakami et al. (1992). They were homogenized in 1 mL of 0.5 M perchloric MAO activity in rat brain homogenateswith 5hydroxy [side chain-2-14C]tryptamine creatinine sulphate([ i4C]- acid and 1 mL of methanol, and then centrifuged. The 5 HT, Amersham,England) at a final specific activity of pellets were suspendedin 1 mL of methanol and centri1 uCi/umol as selective substratefor MAO A and phe- fuged twice. Free and bound radioactivity was measured nylethylamine hydrochloride p [ethyl-l-‘4C] ([14C]- in the combined supematantsand pellets, respectively. PEA, New England Nuclear, USA) at a final specific These values were corrected by a recovery coefficient activity of 10 pCi/umol as selective substratefor MAO determined by addition of [izs]-2-iodopargyline to B (Moureau et al., 1992). The inhibitory potencies of brains from noninjected rats before homogenization. To assessspecific in vivo binding of [‘?I-2-iodoclorgyline, L-deprenyl and pargyline were determinedas pargyline, rats were pretreated60 min before IV. injecreferences. tion of 10 pCi [‘251]-2-iodopargylinewith 10 mg/kg I.V. The whole brains of male Wistar rats (200-300g) of clorgyline, a selective MAO A inhibitor, or with 10 were homogenized using a Ultra Turrax (T 25) homogmg/kg IV. of L-deprenyl, a selective MAO B inhibitor. enizer in 20 volumes of 0.1 N sodium phosphatebuffer Rats were sacrificed 180 min after injection of [‘251]-2(pH 7.4) and all the assays were performed with this iodopargyline and the radioactivity of brain regions was crude preparation.A reaction mixture containing 100 uL measured as described above. of crude homogenate,50 pL of inhibitor (or 50 l.tL of water for controls) and 250 pL of buffer was preincu- Ex vivo autoradiographic studies batedfor 20 min at 37°C. The reaction was startedby the Rats were injected with 100 l&i of [‘251]-2-iodoparaddition of the substrate (100 FL) and incubation was gyline IV. and sacrificed 120 min after this injection. continued at 37°C for 5 min with [14C]-5HT (625 pmoll Brains were rapidly removed after sacrifice, frozen on 1)or for 1 min with [i4C]-PEA (40 umol/l). The reaction dry ice, and cut into 20 pm transversal slices. After was stopped by addition of 200 uL 4 N HCl. The medrying, the slices were apposedto a p max sensitive film tabolites thus formed were extracted with 7 mL of tol(HyperBlm B max, Amersham,England). The films were uene/ethyl acetate mixture (50/50 v/v). After shaking exposed for 5 wk and then developed with Kodak L 24 and freezing, the organic phase was transferred to a revelator, fixed and dried. Imageswere analyzed using a counting vial containing a mixture of 10 mL of toluene/ 2.5 diphenoloxazole (4 g/L) for determination of the densitometric image analyzer (Autoradlmstar). After identification of brain regions of interest according to the radioactivity. Paxinos and Watson atlas (1982), the binding intensity The results were expressedas the percentageof residwas evaluated by a semi-quantitative method. ual MAO activity calculated from control values. IC 50 values were calculated graphically from the inhibition Results curves obtained with inhibitor concentrations ranging from 10e4to lo-” mol/L. Synthesis of 2-bromopargyline and 2-iodopargyline Biodistribution

in rats

Male Wistar rats weighting 200 g received 10 uCi of [‘251]-2-iodopargyline IV. and were sacrificed 30, 60, 120, 180, and 240 min after injection. Different peripheral tissues (kidney, liver, intestine, lung, heart, and spleen) were removed and weighed. Brains were removed, dissected and cerebral regions (pineal gland, thalamus, frontal cortex, cerebellum, brain stem, hypothalamus, hippocampus, striatum) were weighed. The radioactivity of all samples was then measuredwith a gamma scintillation counter (LKB 1260 Multigamma). Intra-cardiac blood sampleswere taken.The results were expressedas the percentageof injected doseper gram of

‘H NMR spectrashowed that 2-bromopargyline and 2-iodopargyline had an identical chemical structure with regard to the side chain but that the structure of the phenyl group was different in relation to the bromide or the iodide atom in position 2. 2-bromopargyline ‘H-NMR (CDCl,) d: 2.22 (t, lH, 4J 2.38 Hz, ] C-H), 2.30 (s, 3H, N-CH,), 3.30 (d, 2H, 4J 2.38 Hz, N - CH, - C I), 3.59 (s, 2H, Ar - CH, - N), 7.04 (td, 1Harom.,3J7.62 Hz, ‘J 1.89Hz), 7.20 (td, 1Harom., 3J7.45 Hz, 4J 1.30 Hz), 7.32 (d.d, 1H arom., ‘J 7.61 Hz, “J 1.89 Hz), 7.47 (dd, 1Harom., 3J7.89 Hz, 4J 1.30 Hz). 2-iodopargyline ‘H-NMR (CDCl,) d: 2.22 (t, lH, 4J 2.35 Hz, j C-H), 2.30 (s, 3H, N-CH,), 3.39 (d, 2H, 4J 2.35 Hz, N - CH, - C I), 3.53 (s, 2H, Ar - CH, - N), 6.87

730

ISABELLE LENA et ~1.

(td, IH arom., 3J7.51 Hz, “J 2.00 Hz), 7.23 (td, 1Harom., ‘J 7.38 Hz, 4J 1.10 Hz), 7.32 (dd, 1H arom., 3J 7.60 Hz, “J 2.00 Hz), 7.76 (dd, IH arom., 3J7.88 Hz, 4J 1.10 Hz). The HPLC analysis showed a higher than 99% chemical purity for 2-bromopargyline and 2-iodopargyline. Radiolabeling

[‘251]-2-iodopargyline was obtained by nucleophilic exchange with a radiochemical yield of about 50%. The radiochemical purity determined by the HPLC system described above was higher than 99%. The retention time (Fig. 2) of the bromo precursorwas 19 min and that of the radioiodinated product was 27 min, allowing a total separationof the two compoundsand a no-carrieradded preparation of the tracer. Therefore the specific activity was comparableto that of the [‘*‘I]-iodide used (2200 Wmmol). The coinjection of unlabeled 2-iodopargyline and the pure radioiodinated product showed identical retention times.

Table I. In vitro inhibition of MAO A and MAO B activity by acetylenic derivatives q. Compound

MAO A (*W

13.9 f 655.0 f 1125.0 f 3.7f

Z-iodopargyline Pargyline L-deprenyl Clorgyline

4.2 97.9 106.0 1.9

,cIn MAO B (*M) 5.2 * 3.0 19.8 zk7.1 13.5 f 3.5 2650.0 zt 354.0

MAO A/MAO

B

2.1 33.1 83.3 0.0014

lcso values were determined on brain homogenates with [14C]-5HT for MAO A activity and [14C]-PEA for MAO B activity. Each value represen& the mean f S.D. of four independent determinations.

those obtained for L-deprenyl and pargyline, described as potent MAO B inhibitors. The IC,, MAO A/K 50 MAO B ratio was lower for 2-iodopargyline (2.7) than for pargyline (33.1) and for L-deprenyl (83.3). In contrast this ratio was higher for 2-iodopargyline than for the selective MAO A inhibitor clorgyline (0.0014). Biodistribution

in rats

Table 2 shows the time-dependantbiodistribution of [‘251]-2-iodopargyline in rat peripheral tissues. The Lipophilicity measurement higher radioactivity was obtained 30 min after injection The apparentlipophilicity at pH 7.5, log k,, for par- of the ligand, except for kidney where the maximal level gyline and 2-iodopargyline was 2.24 and 3.5, respec- was obtained 2 h after injection. Highest levels were tively, indicating that 2-iodopargyline is more lipophilic measuredin kidney and liver. than pargyline. Table 3 shows the time-dependantbiodistribution of [‘*‘I]-2-iodopargyline in rat brains and blood. The acAssay of inhibition of MAO activity cumulation of radioactivity in all brain regions studied The IC,, values of 2-iodopargyline were 13.9 nM to- and in blood was highest between 30 min and 60 min ward MAO A and 5.2 nM toward MAO B respectively and decreaseduntil 180 min postinjection, the time at (Table 1). The IC,, value toward MAO B was lower than which radioactivity reacheda constantlevel. The highest

Rt = 27 min.

3 hromopnrgyline Rt= IY min.

13. Injection

Fig. 2. HPLCpurificationof [‘Z51]-2-iodopargyline. Detailsare

described

in Materials

and Methods.

2-Iodopargyline

for exploration of MAO B

7.71

Table 2. Biodistribution of [‘251]-2-iodopargylineafter intravenous injection in the rat 0.5 h

lh 2h 3h 4h 0.846 0.790 1.048 0.742 0.787 * 0.177 f0.1 18 -to.149 io.195 f 0.094 Liver 0.646 0.497 0.447 0.418 0.371 f 0.067 f0.134 It 0.029 f 0.084 k 0.082 Intestine 0.445 0.402 0.227 0.161 0.183 f 0.290 kO.189 *0.131 f 0.027 _+ 0.059 Lung 0.353 0.215 0.135 0.148 0.124 IL0.008 f 0.029 f 0.012 k 0.024 f 0.027 HeaIl 0.267 0.172 0.106 0.090 0.087 f 0.034 f 0.042 f 0.02I f 0.017 f 0.012 Spleen 0.233 0.143 0.083 0.071 0.069 f 0.044 f 0.087 f 0.006 +0.015 i 0.01I Animals received 10 pCi of [12SI]-2-iodopargylineand were sacrificed at different times after injection. The radioactivity of each tissuewas measured. Results we expressedas meanpercentageof injected dose per gram of tissue f S.D. n = 5-7 for each value. Kidney

accumulation of radioactivity was observedin the pineal gland. High accumulation was also observedin the thalamus.The radioactivity measuredin the thyroid reached 0.3% of the total injected dose 240 mm after injection. The radioactivity bound to plasmaproteins 120min after injection was 46% of the total plasmaradioactivity. The fraction bound to proteins in the brain was 64% of the total brain radioactivity. The results of the saturation studies, performed 180 min after injection when in viva binding of [lz51]-2iodopargyline was stable, are shown on Fig. 3. Radioactivity in the different regions of the brain was greatly decreased with pretreatment with L-deprenyl (p c 0.0005, t test). Pretreatmentwith clorgyline induced a small decreasein the level of radioactivity in four of the eight brain regions studied (p < 0.05, t test).

Discussion

Fluctuations of MAO activity in the human brain are observedin psychiatric and neurological diseases.A decreasein both MAO A and MAO B activity has been shown in alcoholism (Oreland et al., 1983), suicidal behaviours (Gottfries et al., 1975) and cycloid psychosis (Eckert et al., 1980). An increase in MAO B activity, without modification of MAO A activity, has been observed in neurodegenerativedisorders such as Alzheimer’s disease(Adolfsson et al., 1980;Oreland and Gottfries, 1986;Reinikainen etal., 1987;Jossanet al., 1991). Huntington’s chorea (Mann et al., 1980, 1986) and amyotrophiclateral sclerosis(Eckblom et al., 1992).This increase in MAO B may be the consequenceof a proliferation of glial cells, rich in MAO B, secondaryto a neuronal degeneration (Strolin Benedetti and Dostert, Ex vivo autoradiographic studies 1989; Oreland, 1991). This phenomenonmay be an acEx vivo autoradiographic study was performed 120 celeratedprocessanalogousto that observedwith aging min after injection of [‘251]-2-iodopargyline.Autoradio- where an increase in MAO B concentration has been gramsin a rat brain are shown in Fig. 4. The binding was demonstratedin rat brains (Arai et al., 1985) as well as particulary intense in the pineal gland, the raphe nuclei in human brains (Fowler et al., 1980).The type of glial and in the interpeduncular nucleus. Moderate binding cells involved in this process has not yet been estabwas obtained in the hippocampus.The relative intensi- lished for certain but the current data are in favor of ties of the binding of [‘251]-2-iodopargyline in rat brains astrocytes since the presenceof MAO B activity has beenrevealed in astrocytesof senile plaques(Nakamura are summarized in Table 4. Table 3. Regional brain and blood uptake of [ ‘251]-2-iodopargylineafter intravenous injection in the rat Pineal Gland Thalamus Hippocampus Hypothalamus Striatwn Frontal cortex Cerebellum Brain stem Blood

0.5 h 0.479 f0.015 0.259 f 0.020 0.247 f 0.026 0.200 f 0.019 0.227 f 0.025 0.205 * 0.019 0.200 * 0.020 0.183 to.018 0.287 f 0.040

Ih 0.546 f 0.023 0.229 f 0.029 0.190 f 0.025 0.161 f 0.024 0.180 f0.019 0.164 3~0.029 0.157 f 0.020 0.149 f 0.023 0.177 f 0.021

2h 0.416 f0.013 0.200 f0.013 0.139 f 0.012 0.132 f0.013 0.131 f 0.016 0.129 f0.013 0.119 f 0.012 0.116 f0.012 0.094 f 0.014

3h 0.228 f0.011 0.118 It 0.022 0.091 kO.015 0.087 * 0.015 0.090 * 0.017 0.086 f0.015 0.08I to.015 0.078 * 0.015 0.063 f 0.005

4h 0.224 + 0.023 0.123 f- 0.024 0.096 f 0.034 0.082 k 0.026 0.084 f 0.023 0.079 k 0.023 0.077 + 0.024 0.069 f 0.029 0.055 f 0.024

Animals received 10 pCi of [‘*‘I]-2kdopargyline and were sacrificed at different times after injection. The radioactivity of each brain region and blood was measured.Results are expressedas meanpercentageof injected dose per gram of tissuef SD. n = 5-7 for each value.

732

ISABELLE LENA rl UJ.

W Controls

q L-Deprenyl E Clorgyline

0.0

P

Th

Hi

Hyp

Str

Fc

Cer Br st

Fig. 3. Cerebral biodistribution of [ ‘251]-2-iodopargyline:saturationstudy. Values are expressedas mean percentageof injected dose per gram of tissue k S.D. n = 4 to 6 for each value. *p < 0.05, **p c 0.0005, t testfor unpaired values. P = pineal gland; Th = tbalamus; Hi = Hippocampus; Hyp = hypothalamus;Str = striatum; FC = Frontal cortex; Cer = Cerebellum; Br st = Brain stem.

et al., 1990).Therefore, a selective radioligand of MAO B might be useful as a marker of astrocyteproliferation. Clorgyline, L-deprenyl and pargyline belong to the class of irreversible acetylenic MAO inhibitors: these inhibitors bind irreversibly to MAO by forming a stable covalent complex betweentheir acetylenic group and the flavine group of MAO (Maycock et al., 1976). In a first step, we have chosen to develop an iodinated analog of pargyline which have a high affinity but a moderate specificity towards MAO B, in vitro (Kalir et al., 1981) aswell as in viva (Neff and Fuentes,1976).An iodinated derivative of L-deprenyl, which possessesa higher selectivity towards MAO B, is yet under development, in spite of the higher complexity of the chemical synthesis of such a compound. We performed iodination of pargyline in the ortho position of the aromatic ring since many authors have reported that the ortho position for the halogenatedderivatives of acetylenic inhibitors offers higher affinity to MAO in vitro than meta and para positions (Swett et al., 1963; Fujita, 1973; Williams and Walker, 1984). After synthesisof a bromoderivative of pargyline, the labeling procedure was based on a nucleophilic exchange between the bromine atom of this precursor and [‘251]iodide, resulting in a no-carrier-added radioiodinated compound with a 50% yield and a very high degreeof purity (99%). In our in vitro experiments,the lcso values obtained for clorgyline, L-deprenyl and pargyline were in agreementwith those previously determined(Kalir et al., 1981). The 1c50 value of 2-iodopargyline toward MAO B (5.2 nM) was lower than that of pargyline (19.8 nM). Thus, the introduction of an iodine atom on the

ortbo position of the aromatic ring of pargyline resulted in an increase in the affinity toward MAO B. This increasein affinity may be related to the higher lipophilic characterof 2-iodopargyline as comparedto that of pargyline, which we determinedby HPLC, since it has been reportedthat the affinity of irreversible acetylenic inhibitors to MAO increaseswith their lipophilicity (Williams and Lawson, 1975). However, the selectivity of 2-iodopargyline toward MAO B was lower than that of pargyline since the ratio IC,, MAO A/K,, MAO B was 12 times lower for 2-iodopargyline than for pargyline.

In the in viva rat brain, the kinetic biodistribution of [ 1251]-2-iodopargylinedisplayed the maximal uptake 30 min after injection, suggesting that [ ‘251]-2-iodopargyline passesrapidly through the blood-brain barrier, probably due to its high lipophilic character.The whole brain uptake 120 min after injection is about 0.2% of the total injected dose.This low uptake may in part be explained by the high binding of the tracer to plasmaproteins. The study of thyroid radioactivity showed a very low percentageof total injected dose even 240 min after injection of [‘251]-2-iodopargyline (0.3% of total injected dose), indicating a low in vivo deiodination. The time-dependantbiodistribution study in the whole rat showed that the highest accumulation was found in

the kidney, suggestinga renal elimination of the ligand. The regional distribution of [‘*‘I]-2-iodopargyline in rat brains showed the highest accumulation in the pineal gland which exclusively contains MAO B (Tipton e6al., 1976). The accumulation was also high in the thalamus which is rich in MAO B (Willoughby et al., 1988).In the other brain regions, the radioactivity was lower.

2-Iodopargyline for exploration of MAO B

Fig. 4. Autoradiograms of [‘251]-2-icdopargylinein rat brain. (A) Coronal brain section in interaural plane 0.7 mm according to Paxinos and Watson(1982). a very high accumulationis observed in the pineal gland (P) and the raphe nuclei (Rn). (B) Coronal brain section in interaural plane 3.2 mm according to Paxinos and Watson (1982). Interpeduncularnucleus (IP) and hippocampus(Hi) are clearly labeled. Autoradiographic studies in rat brains confirmed the high level of binding of [‘251]-2-iodopargyline found in the pineal gland and in the thalamus. Moreover, autoradiographic studies permitted the definition of other spe-

cific MAO B binding sites. Thus, a high intensity I of [‘251]-2-iodopargyline binding in rat brains was also observed in raphe nuclei, the interpeduncular nucleus, the subfomical organ, the arcuate and periventricular nuclei

734

ISABELLE LENA et o/.

Only a few studies have been published on MAO B selective radioiodinated derivatives as potential tools for Binding intensity Brain area SPECT imaging (Mac Whorter and Baldwin, 1991; * Pineal gland Ohmomo et al., 1992; Beer et al., 1993) but, to our * Interpedunculw nucleus knowledge, SPECT images have not been yet reported. Hypothalamus * Further studies are in progressto document biodistribuArcuate nucleus * Periventricular nucleus tion in the primate and to evaluate the dosimetry. * Raphe nuclei In conclusion, we have developed a new radioiodi* Subfomical organ Thalamus nated ligand, 2-iodopargyline, which preferentially laHabenular nucleus bels MAO B sites in rat brains with high affinity but Paravenhicular nucleus : moderateselectivity. The results demonstratethat a raVentricular ependyma Hippocampus : dioiodinated tracer may be valuable to explore enzymatStriamm ic activity in vivo. It is now necessaryto evaluate the Accumbens nucleus i specificity of this ligand for MAO B in humans. If this Globus pallidus Substautia nigra : specificity is not enough high, we envisageto increaseit Cerebral cortex s by modifying the chemical structure of 2-iodopargyline Semiquantitative evaluation of binding intensity (average estimations (position of iodide), or by developing an iodinated deby two independent observers). *, very high intensity; t, highintenrivative of another compound from the same family, sity; f, moderate intensity; 8, slightly higher than background. such as L-deprenyl. 2-iodopargyline labeled with lz31 may be a suitable SPECT radiopharmaceuticalfor MAO of the hypothalamus,and the ependyma.Moderate bind- studies in the human brain, valuable for the diagnosis ing was found in the hippocampus.The lowest levels of and follow-up of neurodegenerative disorders, which binding sites were seenin the cortex, the substantianigra would also help in IMAO drug development,as already and the striatum. Autoradiogramsof [12sI]-2-icdopargy- described for ’ ‘C labeled ligands (Bench et al., 1991; line binding sites were similar to the MAO B sites dis- Fowler et al., 1993). tribution in rat brains determined by a sensitive peroxidatic histochemical method identifying the highest level Acknowledgements-This work was supported by INSERM of MAO B activity in the pineal gland and the raphe and by P&e GBM, R6gion Centre. We thank the Centre de nuclei (Willoughby et al., 1988). Our ex viva autoradio- RechercheDelalande for valuable advice on the in vitro assays. graphic results are also in accordancewith those ob- We also thank Sylvie Bodard for excellent technical assistance, tained by in vitro autoradiography with [3H]-Ro Francine Wosciekowsky for preparation of the typescript and DoreenRainefor editorialassistance. 16-6329, a selective reversible MAO B inhibitor @aura et al., 1992). The saturation study showed that pretreatment by References L-deprenyl, a selective irreversible MAO B inhibitor, Adolfsson R., Gottfries C.-G., Oreland L., Wiberg A. and Winblad B. (1980) Increasedactivity of brain and platelet monovery significantly decreasedthe radioactivity in all brain amine oxidase in dementia of Alzheimer type. Life Sci. 27, regions investigated. After pretreatmentwith clorgyline, 1029-1034. a selective MAO A inhibitor, the binding of [“‘I]-2Arai Y., StenstrhmA. and Oreland L. (1985) The effect of age iodopargyline was slightly but neverthelesssignificantly on intra- and extraneuronal monoamine oxidase-A and -B decreasedin four of the eight brain regions studied. A activities in the rat brain. Biogenic Amines 2, 67-71. pretreatmentwith both L-deprenyl and clorgyline lead to Beer H. F., Rossetti I., Frey L. D., Remy N., Maier A. and Schubiger P. A. (1993) [lUI]-Ro 43-0463, a potential SPET almost the same decreasein the binding of [‘251]-2-iotracer for MAO-B imaging. Proceedings of the Tenth Interdopargyline than with L-deprenyl alone (data not national Symposium on Radiopharmaceutical Chemistry, shown). This was in agreementwith the selectivity deKyoto, Japan,October 1993,239. termined in vitro. The fraction bound to proteins in the Bench C. J., Price G. W., Lammertsma A. A., Cremer J. C., brain 120 min after injection was about 60%, supporting Luthra S. K., Turton D., Dolan R. J., Kettler R., Dingemanse the fact that [‘251]-2-iodopargyline binds covalently to J., Da Prada M., Biziere K., MC Clelland G. R., Jamieson V. L., Wood N. D. and Frackowiak R. S. J. (1991) MeasureMAO, as could be expected with an irreversible inhibiment of human cerebral monoamineoxidase type B (MAOtor. 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2-Iodopargyline

for exploration of MAO B

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