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Synthesis and biodistribution of [5-131I]iodotropapride: a potential D2 dopamine receptor imaging agent
Nucl. Med. Bid. Vol. 21, No. 2, pp. 255-262, 1994
Pergamon
0969-8051(93)EOO40-C
Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0969~8051/94 $6.00 + 0.00
Synthesis and Biodistribution of [5-131IlIodotropapride: a Potential D2 Dopamine Receptor Imaging Agent ROBERT CANTINEAU’, MARCEL GUILLAUME’*, PHILIPPE CHRISTIAN LEMAIRE’, ALAIN PLENEVAUX’, FRANCOISE BERNARD POURRIAS’ and LEON CHRISTIAENS’ ‘Cyclotron Research
DAMHAUT’, GAUTHIER’,
Liege University, 4000 Liege, Belgium and 2Centre de Recherche Delalande, 10, Rue des Carriires, 92500 Rueil-Malmaison, France
Center,
(Accepted 22 July 1993) [5-“‘IJIodotropapride is a benzamidic compound which displays high affinity and selectivity for dopaminergic receptors. It was prepared from the corresponding brominated compound by a nucleophilic substitution with [“‘IJiodine (t ,,Z= 8.02 days, E, = 364 keV) based on the use of Cu(1) as catalyst and high specific activity of [“‘I]NaI. After i.v. injection in rats the tracer crosses the blood-brain barrier (0.42 + 0.06% of injected dose in the total brain) and demonstrates a high affinity binding to the striatum. The striatum-to-cerebellum ratio increases with time and reaches values of 9 and 22 at 30 and 120 min after injection, respectively. This specific uptake in the striatum is saturable and can be blocked by pretreatment with different D, antagonists. When labeled with ‘23I (f,,2 = 13 h, E,i = 159 keV), the corresponding [‘231]iodotropapride may be useful for the investigation of the D, dopamine receptors in humans with single photon emission computer tomography (SPECT).
Introduction Over the past several years, different benzamidic compounds were labeled with iodine-125 in view of the mapping of the D, dopamine receptors in the living human brain with the iodine-123 analogs and single photon emission computed tomography (SPECT) (Kung et al., 1988a, b. 1989; Kessler et al., 1989; Murphy et al., 1990). Promising results were recently obtained with these compounds on various neurological pathologies in humans (Kung et al., 1990; Brucke et al., 1991, 1993; Oertel et al., 1992; Schwartz et al., 1992). Iodinated butyrophenones were also developed to evaluate either receptor concentration (Mertens et al., 1989; Landvatter, 1985; Saji et al., 1987; Lever et al., 1989; Chalon et al., 1990) or cerebral brain perfusion (Moerlein et al., 1987). In 1988, a new iodinated benzamidic derivative [‘251]PKl1195 was suggested as a promising tracer for the study of peripheral benzodiazepine binding sites in the human brain (Gildersleeve et al., 1989). More recently, a new chemical structure, [1231]iodolisuride-an ergolene derivative-was successfully used for SPECT imaging of dopaminergic D, receptors (Maziire et al., 1989).
Tropapride (l), (exe)-2,3-dimethoxy-N-8-(phenylmethyl)-8-azobicyclo (3.2.1 act-3-yl)-benzamide (Fig. l), is a compound which displays important affinity and selectivity to dopamine D, receptors with a high potency as an antagonist of apomorphine-induced in vivo hyperactivity (Jalfre et al., 1983; Rumigny et al., 1984). Preliminary investigations with (‘3’I]iodotropapride (Cantineau et al., 1991) showed an exceptional affinity of this new radioligand for dopaminergic sites in the rat brain. Subsequent investigations with [“Flfluorotropapride (Damhaut et al., 1992) and [76Br]bromotropapride (Loc’h et al., 1993a, b) demonstrated the potential use of this nortropane-substituted benzamide in the study of dopaminergic neurotransmission. These attractive biochemical properties suggested that the radioiodinated analog had to receive attention as a possible
1: Tropapride 2: 5-Iodotropapride 2: SBromotropapride
*Author to whom all correspondence and reprint requests should
R=N R=I R = Br
Fig. I. Chemical structure of tropapride and its derivatives.
be addressed.
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ROBERT CANTINEAU et al.
new specific radioligand for the study of central nervous system dopamine receptors in humans. The main goal of the present work was the synthesis of [5-“‘Iliodotropapride in order to investigate the in vivo binding properties of this new radiopharmaceutical in the rat brain and evaluate its potentialities as a receptor imaging agent. Preliminary results obtained in rats are described including regional biodistribution and blocking experiments. In vitro characteristics are also determined such as the octanol-water partition coefficient and the protein nonspecific binding in isolated receptor preparations.
Materials and Methods General
Each organic substrate and solvent was of analytical grade from Merck and Aldrich Chemical Company. Iodotropapride and bromotropapride were a gift from Delalande. Ketanserin, spiperone and halopemide were supplied by Janssen Pharmaceutics. Bromolisuride was a gift from Dr Mazikre (Orsay). No-carrier-added [1311]NaIwith a specific activity of 300 Ci/mmol was obtained in a 0.05N NaOH solution from Cintichem. Wistar rats were purchased from IFFA Credo. Radio high performance liquid chromatography (HPLC) was carried out using a Rheodyne 7125 injector (5 mL loop), a pump (Model 6000 A from Waters) and a 254 nm LambdaMax 480 U.V. detector coupled to a NaI (2” x 2”) scintillator associated with a monochannel gamma spectrometer from Labtech. The column was a semi-preparative Select-B from Merck (250 x 10 mm, particule size: 10 pm). Quality control was both performed on a Phenomenex C-18 column (30 x 3.9 mm, particule size: 5 pm) using Silica Gel plates from Macherey-Nagel. The specific activity was determined by comparison of the area of the U.V. peak with a calibration curve. Labeling procedure
In a 1 mL screwcap vial, the following reagents were introduced: 0.5-l mg of bromotropapride, 500 PL of a solution containing 25 mg of gentisinic acid, 35 mg of citric acid, 1 mg of SnSO,, 250 PL of acetic acid in 2.25 mL of water, 3OpL of a CuSO, .5H,O solution (32.5 mg/lO mL) and 20 PL (f 5 mCi) of the radioactive material. Before tight sealing, the vial was flushed with a flow of nitrogen for 15 min and the reaction was allowed to proceed at 160°C in an aluminum heating block for 30 min. Free iodide was removed from the reaction mixture by passing through a C-l 8 Sep Pak cartridge from Waters (part No. 51910) conditioned with 5 mL of ethanol and 5 mL of water. The cartridge was washed with 5mL of water and the activity eluted with 1 mL of methanol. The [“‘Iliodotropapride was isolated from the unreacted bromoderivative by HPLC. The chromatographic separations were carried out with the following eluent: methanol/
acetonitile/water/triethylamine/acetic acid: 161161 66/l/l at a constant flow rate of 6 mL/min. The collected radioactive peak was diluted 2.5-fold with water and NaOH 2N was added to the solution until pH 9. [‘3’I]Iodotropapride was fixed on a C-18 Sep Pak cartridge, washed with 20 mL of water and eluted through a Millex GV (0.22 pm) with 0.5 mL of ethanol. The activity was then diluted with NaCl 0.9% for animal studies. The radiopharmaceutical was produced with an overall radiochemical yield of 40% and a specific activity ranging from 200 to 300 Ci/mmol. Quality control was performed by HPLC and TLC using the following conditions: -HPLC:
-TLC:
Phenomenex C-18 column (4 = 3.9 mm, length = 300 mm) eluted with methanol/acetonitrile/water/acetic acid/ TEA: 20/20/60/1/l. The retention times were 16 and 20.5min for bromotropapride and [‘311]iodotropapride, respectively. the solvent used was chloroform/ methanol/ethylamine: 90/10/0.1. The R, was of 0.85 for the [‘311]iodo-derivatives, respectively.
Biodistribution
The biodistribution study was investigated in female Wistar rats (180-250 g) which were injected in the femoral vein under light ether anesthesia with 10-20 PCi (370-740 kBq) [‘3’I]iodotropapride. During the experiments, animals had no access to food. Rats were sacrificed at various times post-injection (5 min to 1 h) by cardiac excision. The entire organs, except blood and muscles assumed to be 7 and 40% of total body weight, respectively, were removed and counted in a 2.5 keV resolution 16 cm3 Ge(Li) detector. The percentages of injected dose (ID) per g of organ were calculated by comparison with a reference solution consisting of suitably diluted samples of the injected compound. Brain distribution study
Regional brain distribution was determined by dissecting, weighing and counting samples from different brain regions (frontal cortex, striatum, cerebellum) after femoral injection of [‘3’I]iodotropapride. The countings were performed with an automatic gamma sample changer (Berthold BF 5300 model). The results were expressed as the percentage of injected dose per g of tissue. From these results, region-to-cerebellum ratios were easily calculated. Studies on the carrier effect
To check the saturability of the iodotropapride brain uptake, the striatum-to-cerebellum ratios were determined in rats after intravenous injection (femoral vein) under light anesthesia of 15 pCi (555 kBq) of [‘3’I]iodotropapride at different decreasing specific activities (240.03 Ci/mmol). Cold
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Synthesis and biodistribution of [5-“‘Iliodotropapride iodotropapride was dissolved in saline containing 5% of ethanol. The rats (4 for each specific activity) were killed by decapitation 2 h after injection of the tracer.
Table 2. Radiochemical yield with Cur catalyst at 160°C as a function of the reaction time
Blocking experiments
20 30 60 90
Rats were intravenously injected (femoral vein) under light ether anesthesia with D, antagonists, such as halopemide (20mg/kg) (Loonen and Soudijn, 1985; Leysen, 1984; Clanton ef al., 1991) and bromolisuride (0.4 mg/kg) (Maziere et al., 1986) and an S, antagonist, such as ketanserine (2.5 mg/kg) (Laduron et al., 1982). Spiperone, a D, and S, antagonist, was also used at 2 mg/kg (Leysen, 1989). The drugs were dissolved in saline containing 5% of alcohol and one equivalent of tartaric acid to increase the drug solubility. The animals were pretreated with the antagonists 1 h before injection of the tracer and sacrificed 2 h post-tracer injection. Partition coeficient (P)
The n-octanol-phosphate buffer partition coefficient was measured at 37°C at blood pH (i.e. 7.4). 5 PCi (185 kBq) of [‘3’I]iodotropapride were mixed with an exactly known amount of n-octanol (5 mL) and 0. I M phosphate buffer (5 mL) at various pHs (pH buffers adjusted at 37°C) for 4 h. The two phases were separated after centrifugation, weighed and counted. Partition coefficients were calculated from the following equation:
Reaction time (min)
so 60 60 60
and 0.8 mL of 0.05 N phosphate buffer pH 7.4 were separated by a dialysis membrane (dialysis tubing visking size l-8/32” Medicell Int. Ltd). The dialysis cells were gently shaken in a water bath at 37°C for 18 h. After incubation, aliquots were taken and counted as the membrane as well for protein binding evaluation. Unchanged species in plasma
After centrifugation (3000rpm) of 4-5 mL of blood sample, the plasma was separated from blood cells and the total radioactivity measured from 0.5 mL aliquots. The separation of proteins from plasma was easily achieved by ultrafiltration (Ultrafree-CL filters from Millipore, cutoff at 10,000 NMWL) at 5OOOg for 30 min. In such conditions, 400-500 PL of deproteinized plasma were collected, weighed and measured with a NaI-y spectrometer. Unchanged iodotropapride was determined by analytical HPLC using the same conditions as for the quality control procedure.
Results and Discussion
,_F Labeling
A,,& w,h where: A,, = total count of n-octanol after separation W,, = weight (g) of n-octanol after separation D,, = density (g/mL) of n-octanol
A,, = total count of phosphate solution after separation W,, = weight of phosphate solution D,, = density of phosphate solution. Protein binding
This parameter was determined by equilibrium dialysis according to the following method: 1 mL of 0.05 N phosphate buffer pH 7.4 and a solution consisting of 0.1 mL of fresh human serum, 0.1 mL of [‘3’I]iodotropapride (2 PCi, 74 kBq) in ethanol/water Table I. Radiochemical yield versus reaction temperature with Cu+ as catalyst Reaction temperature (“C) RT 65 80 100 125 IS0 160 180
Yield (%)
Yield (%) 0 0 0 0 IO so 60 60
13’1labeling of iodotropapride was based on a nucleophilic exchange in the presence of Cu(I) and with an excess of reducing agents in conditions similar to those described by Mertens et al. (1987). The radiochemical yield obtained in the best experimental conditions was relatively low (60% for a reaction time of 40 min and a temperature of 160°C). As shown in Tables 1 and 2, the labeling yield was dependent on the temperature and the reaction time. The exchange only took place from 125°C. Iodotropapride was formed for temperatures ranging from 125 to 16OC. [‘3’I]lodotropapride was purified on a semi-preparative HPLC column which isolated the radioactive compound. One percent of triethylamine in the eluting solvent was required in order to avoid tailing of the starting material and to achieve the maximal specific activity which essentially depends on the starting 1311mspecific activity. It has indeed to be taken into account that bromotropapride shows binding affinity for dopaminergic receptor sites (Loch et al., 1993a), its presence in the iodotropapride peak reduces the apparent specific activity of the radiopharmaceutical. [‘3’I]Iodotropapride identification was performed by HPLC by comparison with an authentic reference sample. The iodinated compound showed a retention time of 52 min and the
ROBERTCANTINEAUet
258
al.
-
Lung
--1-
Kidney
-
Liver
__01
Intestine
-=-
Heart
-
Muscle
-*-
Blood
-
0
10
20
Thyroid
Time Fig. 2.
50
40
30
post
injection
60 (min )
Total “‘I activity [“Ainjected dose (ID)/g of tissue] in selectedtissues as a function of time following [13’I]iodotiopaprideinjection (n = 4). In vivo studies
bromo derivative 40min. After purification, no U.V. peak of bromotropapride could still be observed in the HPLC chromatogram ensuring a final chemical purity better than 99.95%. In order to validate [13’I]iodotropapride as an in uivo dopaminergic tracer, animal studies were carried out.
General biodistribution. Distribution of radioactivity in selected rat tissues was measured at 5, 15, 30 and 60 min as shown in Fig. 2. High initial uptake was observed in lungs, liver and kidneys. The liver activity remained high throughout the first hour. The
P P I-
1
I
O
1
200
100 Time
post
injection
(min )
Fig. 3. Total “‘I activity [% injected dose (ID)/d in brain as a function of time following [13’I]iodotropaprideinjection (n = 4).
Synthesis and biodistribution
of [5-‘3’I]iodotropapride
259
0
0‘ 2.0 *0
1.5 -
1.0 -
0.5 :1 :I
Time
post
injection
(min )
Fig. 4. Total 13’1activity [% injected dose (ID)/g of tissue] in different brain regions as a function of time following [‘3’I]iodotropapride injection (n = 4).
thyroid uptake (less than 0.05% ID/g at 10min increasing up to 0.20 at 4 h) was low suggesting no in vivo deiodination of the compound for the first 4 h after injection. Figure 3 indicates that 0.42X~4_5% of the injected dose appears in less than 5min in the total brain, decreases quickly to 0.30% and remains constant during the first 2 h showing finally a signifi-
cant constant
retention
by brain tissue (0.24.3%
ID/d.
Biodistribution data at later times will be required before human investigation in order to estimate the radiation dosimetry of the “‘I analogue. More than 95% of the radioactivity found in the plasma at different times post-injection belong to the
P P
I
0
x
I
m
I
0
I
Time Fig. 5. Striatum-to-cerebellum
1
200
100
and frontal cortex-to-cerebellum
post
injection
(min )
activity ratios vs time (n = 4).
ROBERT CANTWEAU et al.
260
Table 3. Effect of different competing S, and D, receptor ligands on the regional distribution of [“‘Iliodotropapride. The rats were pretreated I h before injection of I”iIliodotronauride and sacrificed 2 h after (n = 4) s2
K, (nM) D2
ID (mg/kg)
FCjCb (ratio k SD)
StjCb (ratio f SD)
0.64 220 0.63
0.26 3.1 0.3* 240
2 20 0.4 2.5
2.3 + 0.1 1.5 kO.2 2.2 f 0.2 2.1 * 0.2 1.5 f 0.2
21.5+_2 1.6 k 0.2 3.8 f 0.4 2.8 i 0.3 21+2
K, Wf) Cold ligand Control Spiperone Halopemide Bromolisuride Ketanserin
*Kd = dissociation constant. K, = inhibition constant.
unchanged species indicating a negligible metabolization. Regional biodistribution. The experimental tissue activity data obtained in three typical rat brain regions, cerebellum (Cb), striatum (St) and frontal cortex (FC), are illustrated in Fig. 4 and showed a very high uptake in the striatum. The time-course of 13’1radioactivity in cerebellum (Cb), striatum (St) and frontal cortex (FC) was determined in rats (n = 4). As shown in Fig. 4, the radioactivity in the striatum which contains more D, than S2 sites increased with time from 0.85 f_ 0.09% of the injected dose (ID) per g of tissue at 5 min to a value of 1.95 + 0.2 at 4 h after injection. The frontal cortex rich in S2 receptor sites displayed lower uptakes decreasing with time (0.67 +_0.08 to 0.07 + 0.01% ID/g of tissue at 5 min and 4 h after injection, respectively). The activity in the cerebellum, which relates to the non-specific binding, was low and also decreased with time from 0.37 + 0.05 at 5 min to 0.055 f 0.007% ID/g of tissue at 240 min after injection. The time-course of the tissue-to-cerebellum ratio is shown in Fig. 5. The frontal cortex-to-cerebellum ratios were low (2.8 + 0.20 and 2.4 f 0.20, 1 and 2 h after injection, respectively). On the contrary, the striatum-to-cerebellum ratio displayed an important increase with time and values of 12 and 22 were, respectively, obtained at 60 and 120min after injection. No plateau was reached even 4 h after injection, as observed for some other dopaminergic antagonists (Chalon et al., 1990). Blocking experiments. The specificity of the in vivo [‘311]iodotropapride binding was evaluated by blocking experiments using S, and D, antagonists. The results are summarized in Table 3. Spiperone showing a mixed D,-S, affinity (Leysen, 1989) strongly blocked the [‘3’I]iodotropapride acTable 4. Saturability assessment of the striatum uptake by addition of carrier (constant injected activity per rat: 15 nCi. Animals were killed 1.5 h post-injection, a = 4) Specific activity (Ci/mmol) 240 15 1.5 1.5 0.75 0.3 0.15 0.03
Striatum-to-cerebellum 16 16 16 16 11 7 4 1.4
cumulation in the striatum and frontal cortex. Drugs with higher affinity for D, than for Sz receptor sites, such as halopemide (Loonen and Soudijn, 1985; Leysen, 1984; Clanton et al., 1991) and bromolisuride (Maziere et af., 1986), decreased the striatum-tocerebellum ratios. These antagonists did not modify the FCjCb ratio. On the contrary, ketanserin (Laduron et al., 1982) which has a higher affinity for serotonergic S2 sites, did not affect the St/Cb ratio but blocked the accumulation of the tracer in the frontal cortex. These results clearly demonstrate that the in vivo binding of [‘3’I]iodotropapride is related to the dopaminergic receptor sites and, to a much lesser extent, the S2 sites. Saturability. The striatum uptake was drastically influenced by the specific activity of the radiopharmaceutical. Ratios of 16 and 4 were measured for n.c.a. [‘3’I]iodotropapride and addition of 50 pg of carrier, respectively. These data, summarized in Table 4, proved that the binding to structures rich in D, receptor sites is saturable. In vitro studies Lipophilicity. Partition coefficient (P) data are provided in Table 5. At blood pH, P was 2.9. The protein binding value measured as described in the experimental section was 65%. Kd values. Extremely high affinity for the dopaminergic Dz receptors (Kd = 0.030 nM) was evaluated on rat brain tissue (Schotte et al., 1992) indicating that iodotropapride is about 50 times more potent than iodolisuride (Kd = 1.6 nM, Martres et al., 1985), the most commonly used iodinated ligand for the in vitro labeling of dopamine D, receptors.
Conclusions No carrier added [5-‘3LI]iodotropapride was prepared via nucleophilic substitution of the bromoderivative with [1311]iodinein the presence of Cu(1) as catalyst. After i.v. injection into rats, this new
ratio Table 5. Variation of P values of [“sI]iodotropapride versus pH PH 6.5 7.0 7.4
P
2.31 2.5 2.9
Synthesis iodinated
benzamide
penetrated
and biodistribui :ion of [5-‘3’l]iodotropapride
the blood-brain
bar-
and localized mainly on Dz dopamine receptor sites with a relative high specificity compared to those observed for other dopaminergic antagonists (Kung et al., 1989; Chalon et al., 1990). This high affinity binding was saturable. This preliminary study provided evidence of the potentialities of the corresponding [‘Z31]iodotropapride (“‘I: tl!2 = 13 h, ET = I59 keV) as a suitable ligand for imaging D, dopamine receptors in the human cerebral nervous system with SPECT. These investigations are presently under study and already show higher striatum-to-cerebellum ratios (ranging between 3.2 at 1 h and 5 at 4 h) than any other dopaminergic ligand so far investigated on humans. rier
Acknowledgements-The authors thank Mrs F. Mombaerts-Czichosz and Mr G. Salvador for their helpful technical assistance. This research was supported through a grant from the “Fonds National de la Recherche Scientifique” (FNRS) of Belgium (3.9007.89F).
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Saji H., Nakatsuka I., Shiba K., Tokui T., Horiuchi K., Yoshitake A., Torizuka K. and Yokohama A. (1987) Radioiodinated 2’-iodospiperone: a new radioligand for in uiuo dopamine receptor study. Life Sci. 41, 1999-2006. Schotte A., Janssen P. F. M., Verwimp M., Guillaume M. and Leysen J. E. (1992) [‘251]iodotropapride, a new high affinity ligand for dopamine D2 receptors. Proc. “9rh Int. Cong. Histochemistry and Citochemistry”, Maastricht, Augustus 30. Schwartz J., Tatsch K., Arnold G., Gasser T., Trenkwalder C., Kirsch C. M. and Oertel W. H. (1992) I-123-iodobenzamide-SPECT predicts dopaminergic responsiveness in patients with de nouo parkinsonism. Neurology 42, 556-561.
Pergamon
0969-8051(93)EOO40-C
Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0969~8051/94 $6.00 + 0.00
Synthesis and Biodistribution of [5-131IlIodotropapride: a Potential D2 Dopamine Receptor Imaging Agent ROBERT CANTINEAU’, MARCEL GUILLAUME’*, PHILIPPE CHRISTIAN LEMAIRE’, ALAIN PLENEVAUX’, FRANCOISE BERNARD POURRIAS’ and LEON CHRISTIAENS’ ‘Cyclotron Research
DAMHAUT’, GAUTHIER’,
Liege University, 4000 Liege, Belgium and 2Centre de Recherche Delalande, 10, Rue des Carriires, 92500 Rueil-Malmaison, France
Center,
(Accepted 22 July 1993) [5-“‘IJIodotropapride is a benzamidic compound which displays high affinity and selectivity for dopaminergic receptors. It was prepared from the corresponding brominated compound by a nucleophilic substitution with [“‘IJiodine (t ,,Z= 8.02 days, E, = 364 keV) based on the use of Cu(1) as catalyst and high specific activity of [“‘I]NaI. After i.v. injection in rats the tracer crosses the blood-brain barrier (0.42 + 0.06% of injected dose in the total brain) and demonstrates a high affinity binding to the striatum. The striatum-to-cerebellum ratio increases with time and reaches values of 9 and 22 at 30 and 120 min after injection, respectively. This specific uptake in the striatum is saturable and can be blocked by pretreatment with different D, antagonists. When labeled with ‘23I (f,,2 = 13 h, E,i = 159 keV), the corresponding [‘231]iodotropapride may be useful for the investigation of the D, dopamine receptors in humans with single photon emission computer tomography (SPECT).
Introduction Over the past several years, different benzamidic compounds were labeled with iodine-125 in view of the mapping of the D, dopamine receptors in the living human brain with the iodine-123 analogs and single photon emission computed tomography (SPECT) (Kung et al., 1988a, b. 1989; Kessler et al., 1989; Murphy et al., 1990). Promising results were recently obtained with these compounds on various neurological pathologies in humans (Kung et al., 1990; Brucke et al., 1991, 1993; Oertel et al., 1992; Schwartz et al., 1992). Iodinated butyrophenones were also developed to evaluate either receptor concentration (Mertens et al., 1989; Landvatter, 1985; Saji et al., 1987; Lever et al., 1989; Chalon et al., 1990) or cerebral brain perfusion (Moerlein et al., 1987). In 1988, a new iodinated benzamidic derivative [‘251]PKl1195 was suggested as a promising tracer for the study of peripheral benzodiazepine binding sites in the human brain (Gildersleeve et al., 1989). More recently, a new chemical structure, [1231]iodolisuride-an ergolene derivative-was successfully used for SPECT imaging of dopaminergic D, receptors (Maziire et al., 1989).
Tropapride (l), (exe)-2,3-dimethoxy-N-8-(phenylmethyl)-8-azobicyclo (3.2.1 act-3-yl)-benzamide (Fig. l), is a compound which displays important affinity and selectivity to dopamine D, receptors with a high potency as an antagonist of apomorphine-induced in vivo hyperactivity (Jalfre et al., 1983; Rumigny et al., 1984). Preliminary investigations with (‘3’I]iodotropapride (Cantineau et al., 1991) showed an exceptional affinity of this new radioligand for dopaminergic sites in the rat brain. Subsequent investigations with [“Flfluorotropapride (Damhaut et al., 1992) and [76Br]bromotropapride (Loc’h et al., 1993a, b) demonstrated the potential use of this nortropane-substituted benzamide in the study of dopaminergic neurotransmission. These attractive biochemical properties suggested that the radioiodinated analog had to receive attention as a possible
1: Tropapride 2: 5-Iodotropapride 2: SBromotropapride
*Author to whom all correspondence and reprint requests should
R=N R=I R = Br
Fig. I. Chemical structure of tropapride and its derivatives.
be addressed.
255
256
ROBERT CANTINEAU et al.
new specific radioligand for the study of central nervous system dopamine receptors in humans. The main goal of the present work was the synthesis of [5-“‘Iliodotropapride in order to investigate the in vivo binding properties of this new radiopharmaceutical in the rat brain and evaluate its potentialities as a receptor imaging agent. Preliminary results obtained in rats are described including regional biodistribution and blocking experiments. In vitro characteristics are also determined such as the octanol-water partition coefficient and the protein nonspecific binding in isolated receptor preparations.
Materials and Methods General
Each organic substrate and solvent was of analytical grade from Merck and Aldrich Chemical Company. Iodotropapride and bromotropapride were a gift from Delalande. Ketanserin, spiperone and halopemide were supplied by Janssen Pharmaceutics. Bromolisuride was a gift from Dr Mazikre (Orsay). No-carrier-added [1311]NaIwith a specific activity of 300 Ci/mmol was obtained in a 0.05N NaOH solution from Cintichem. Wistar rats were purchased from IFFA Credo. Radio high performance liquid chromatography (HPLC) was carried out using a Rheodyne 7125 injector (5 mL loop), a pump (Model 6000 A from Waters) and a 254 nm LambdaMax 480 U.V. detector coupled to a NaI (2” x 2”) scintillator associated with a monochannel gamma spectrometer from Labtech. The column was a semi-preparative Select-B from Merck (250 x 10 mm, particule size: 10 pm). Quality control was both performed on a Phenomenex C-18 column (30 x 3.9 mm, particule size: 5 pm) using Silica Gel plates from Macherey-Nagel. The specific activity was determined by comparison of the area of the U.V. peak with a calibration curve. Labeling procedure
In a 1 mL screwcap vial, the following reagents were introduced: 0.5-l mg of bromotropapride, 500 PL of a solution containing 25 mg of gentisinic acid, 35 mg of citric acid, 1 mg of SnSO,, 250 PL of acetic acid in 2.25 mL of water, 3OpL of a CuSO, .5H,O solution (32.5 mg/lO mL) and 20 PL (f 5 mCi) of the radioactive material. Before tight sealing, the vial was flushed with a flow of nitrogen for 15 min and the reaction was allowed to proceed at 160°C in an aluminum heating block for 30 min. Free iodide was removed from the reaction mixture by passing through a C-l 8 Sep Pak cartridge from Waters (part No. 51910) conditioned with 5 mL of ethanol and 5 mL of water. The cartridge was washed with 5mL of water and the activity eluted with 1 mL of methanol. The [“‘Iliodotropapride was isolated from the unreacted bromoderivative by HPLC. The chromatographic separations were carried out with the following eluent: methanol/
acetonitile/water/triethylamine/acetic acid: 161161 66/l/l at a constant flow rate of 6 mL/min. The collected radioactive peak was diluted 2.5-fold with water and NaOH 2N was added to the solution until pH 9. [‘3’I]Iodotropapride was fixed on a C-18 Sep Pak cartridge, washed with 20 mL of water and eluted through a Millex GV (0.22 pm) with 0.5 mL of ethanol. The activity was then diluted with NaCl 0.9% for animal studies. The radiopharmaceutical was produced with an overall radiochemical yield of 40% and a specific activity ranging from 200 to 300 Ci/mmol. Quality control was performed by HPLC and TLC using the following conditions: -HPLC:
-TLC:
Phenomenex C-18 column (4 = 3.9 mm, length = 300 mm) eluted with methanol/acetonitrile/water/acetic acid/ TEA: 20/20/60/1/l. The retention times were 16 and 20.5min for bromotropapride and [‘311]iodotropapride, respectively. the solvent used was chloroform/ methanol/ethylamine: 90/10/0.1. The R, was of 0.85 for the [‘311]iodo-derivatives, respectively.
Biodistribution
The biodistribution study was investigated in female Wistar rats (180-250 g) which were injected in the femoral vein under light ether anesthesia with 10-20 PCi (370-740 kBq) [‘3’I]iodotropapride. During the experiments, animals had no access to food. Rats were sacrificed at various times post-injection (5 min to 1 h) by cardiac excision. The entire organs, except blood and muscles assumed to be 7 and 40% of total body weight, respectively, were removed and counted in a 2.5 keV resolution 16 cm3 Ge(Li) detector. The percentages of injected dose (ID) per g of organ were calculated by comparison with a reference solution consisting of suitably diluted samples of the injected compound. Brain distribution study
Regional brain distribution was determined by dissecting, weighing and counting samples from different brain regions (frontal cortex, striatum, cerebellum) after femoral injection of [‘3’I]iodotropapride. The countings were performed with an automatic gamma sample changer (Berthold BF 5300 model). The results were expressed as the percentage of injected dose per g of tissue. From these results, region-to-cerebellum ratios were easily calculated. Studies on the carrier effect
To check the saturability of the iodotropapride brain uptake, the striatum-to-cerebellum ratios were determined in rats after intravenous injection (femoral vein) under light anesthesia of 15 pCi (555 kBq) of [‘3’I]iodotropapride at different decreasing specific activities (240.03 Ci/mmol). Cold
257
Synthesis and biodistribution of [5-“‘Iliodotropapride iodotropapride was dissolved in saline containing 5% of ethanol. The rats (4 for each specific activity) were killed by decapitation 2 h after injection of the tracer.
Table 2. Radiochemical yield with Cur catalyst at 160°C as a function of the reaction time
Blocking experiments
20 30 60 90
Rats were intravenously injected (femoral vein) under light ether anesthesia with D, antagonists, such as halopemide (20mg/kg) (Loonen and Soudijn, 1985; Leysen, 1984; Clanton ef al., 1991) and bromolisuride (0.4 mg/kg) (Maziere et al., 1986) and an S, antagonist, such as ketanserine (2.5 mg/kg) (Laduron et al., 1982). Spiperone, a D, and S, antagonist, was also used at 2 mg/kg (Leysen, 1989). The drugs were dissolved in saline containing 5% of alcohol and one equivalent of tartaric acid to increase the drug solubility. The animals were pretreated with the antagonists 1 h before injection of the tracer and sacrificed 2 h post-tracer injection. Partition coeficient (P)
The n-octanol-phosphate buffer partition coefficient was measured at 37°C at blood pH (i.e. 7.4). 5 PCi (185 kBq) of [‘3’I]iodotropapride were mixed with an exactly known amount of n-octanol (5 mL) and 0. I M phosphate buffer (5 mL) at various pHs (pH buffers adjusted at 37°C) for 4 h. The two phases were separated after centrifugation, weighed and counted. Partition coefficients were calculated from the following equation:
Reaction time (min)
so 60 60 60
and 0.8 mL of 0.05 N phosphate buffer pH 7.4 were separated by a dialysis membrane (dialysis tubing visking size l-8/32” Medicell Int. Ltd). The dialysis cells were gently shaken in a water bath at 37°C for 18 h. After incubation, aliquots were taken and counted as the membrane as well for protein binding evaluation. Unchanged species in plasma
After centrifugation (3000rpm) of 4-5 mL of blood sample, the plasma was separated from blood cells and the total radioactivity measured from 0.5 mL aliquots. The separation of proteins from plasma was easily achieved by ultrafiltration (Ultrafree-CL filters from Millipore, cutoff at 10,000 NMWL) at 5OOOg for 30 min. In such conditions, 400-500 PL of deproteinized plasma were collected, weighed and measured with a NaI-y spectrometer. Unchanged iodotropapride was determined by analytical HPLC using the same conditions as for the quality control procedure.
Results and Discussion
,_F Labeling
A,,& w,h where: A,, = total count of n-octanol after separation W,, = weight (g) of n-octanol after separation D,, = density (g/mL) of n-octanol
A,, = total count of phosphate solution after separation W,, = weight of phosphate solution D,, = density of phosphate solution. Protein binding
This parameter was determined by equilibrium dialysis according to the following method: 1 mL of 0.05 N phosphate buffer pH 7.4 and a solution consisting of 0.1 mL of fresh human serum, 0.1 mL of [‘3’I]iodotropapride (2 PCi, 74 kBq) in ethanol/water Table I. Radiochemical yield versus reaction temperature with Cu+ as catalyst Reaction temperature (“C) RT 65 80 100 125 IS0 160 180
Yield (%)
Yield (%) 0 0 0 0 IO so 60 60
13’1labeling of iodotropapride was based on a nucleophilic exchange in the presence of Cu(I) and with an excess of reducing agents in conditions similar to those described by Mertens et al. (1987). The radiochemical yield obtained in the best experimental conditions was relatively low (60% for a reaction time of 40 min and a temperature of 160°C). As shown in Tables 1 and 2, the labeling yield was dependent on the temperature and the reaction time. The exchange only took place from 125°C. Iodotropapride was formed for temperatures ranging from 125 to 16OC. [‘3’I]lodotropapride was purified on a semi-preparative HPLC column which isolated the radioactive compound. One percent of triethylamine in the eluting solvent was required in order to avoid tailing of the starting material and to achieve the maximal specific activity which essentially depends on the starting 1311mspecific activity. It has indeed to be taken into account that bromotropapride shows binding affinity for dopaminergic receptor sites (Loch et al., 1993a), its presence in the iodotropapride peak reduces the apparent specific activity of the radiopharmaceutical. [‘3’I]Iodotropapride identification was performed by HPLC by comparison with an authentic reference sample. The iodinated compound showed a retention time of 52 min and the
ROBERTCANTINEAUet
258
al.
-
Lung
--1-
Kidney
-
Liver
__01
Intestine
-=-
Heart
-
Muscle
-*-
Blood
-
0
10
20
Thyroid
Time Fig. 2.
50
40
30
post
injection
60 (min )
Total “‘I activity [“Ainjected dose (ID)/g of tissue] in selectedtissues as a function of time following [13’I]iodotiopaprideinjection (n = 4). In vivo studies
bromo derivative 40min. After purification, no U.V. peak of bromotropapride could still be observed in the HPLC chromatogram ensuring a final chemical purity better than 99.95%. In order to validate [13’I]iodotropapride as an in uivo dopaminergic tracer, animal studies were carried out.
General biodistribution. Distribution of radioactivity in selected rat tissues was measured at 5, 15, 30 and 60 min as shown in Fig. 2. High initial uptake was observed in lungs, liver and kidneys. The liver activity remained high throughout the first hour. The
P P I-
1
I
O
1
200
100 Time
post
injection
(min )
Fig. 3. Total “‘I activity [% injected dose (ID)/d in brain as a function of time following [13’I]iodotropaprideinjection (n = 4).
Synthesis and biodistribution
of [5-‘3’I]iodotropapride
259
0
0‘ 2.0 *0
1.5 -
1.0 -
0.5 :1 :I
Time
post
injection
(min )
Fig. 4. Total 13’1activity [% injected dose (ID)/g of tissue] in different brain regions as a function of time following [‘3’I]iodotropapride injection (n = 4).
thyroid uptake (less than 0.05% ID/g at 10min increasing up to 0.20 at 4 h) was low suggesting no in vivo deiodination of the compound for the first 4 h after injection. Figure 3 indicates that 0.42X~4_5% of the injected dose appears in less than 5min in the total brain, decreases quickly to 0.30% and remains constant during the first 2 h showing finally a signifi-
cant constant
retention
by brain tissue (0.24.3%
ID/d.
Biodistribution data at later times will be required before human investigation in order to estimate the radiation dosimetry of the “‘I analogue. More than 95% of the radioactivity found in the plasma at different times post-injection belong to the
P P
I
0
x
I
m
I
0
I
Time Fig. 5. Striatum-to-cerebellum
1
200
100
and frontal cortex-to-cerebellum
post
injection
(min )
activity ratios vs time (n = 4).
ROBERT CANTWEAU et al.
260
Table 3. Effect of different competing S, and D, receptor ligands on the regional distribution of [“‘Iliodotropapride. The rats were pretreated I h before injection of I”iIliodotronauride and sacrificed 2 h after (n = 4) s2
K, (nM) D2
ID (mg/kg)
FCjCb (ratio k SD)
StjCb (ratio f SD)
0.64 220 0.63
0.26 3.1 0.3* 240
2 20 0.4 2.5
2.3 + 0.1 1.5 kO.2 2.2 f 0.2 2.1 * 0.2 1.5 f 0.2
21.5+_2 1.6 k 0.2 3.8 f 0.4 2.8 i 0.3 21+2
K, Wf) Cold ligand Control Spiperone Halopemide Bromolisuride Ketanserin
*Kd = dissociation constant. K, = inhibition constant.
unchanged species indicating a negligible metabolization. Regional biodistribution. The experimental tissue activity data obtained in three typical rat brain regions, cerebellum (Cb), striatum (St) and frontal cortex (FC), are illustrated in Fig. 4 and showed a very high uptake in the striatum. The time-course of 13’1radioactivity in cerebellum (Cb), striatum (St) and frontal cortex (FC) was determined in rats (n = 4). As shown in Fig. 4, the radioactivity in the striatum which contains more D, than S2 sites increased with time from 0.85 f_ 0.09% of the injected dose (ID) per g of tissue at 5 min to a value of 1.95 + 0.2 at 4 h after injection. The frontal cortex rich in S2 receptor sites displayed lower uptakes decreasing with time (0.67 +_0.08 to 0.07 + 0.01% ID/g of tissue at 5 min and 4 h after injection, respectively). The activity in the cerebellum, which relates to the non-specific binding, was low and also decreased with time from 0.37 + 0.05 at 5 min to 0.055 f 0.007% ID/g of tissue at 240 min after injection. The time-course of the tissue-to-cerebellum ratio is shown in Fig. 5. The frontal cortex-to-cerebellum ratios were low (2.8 + 0.20 and 2.4 f 0.20, 1 and 2 h after injection, respectively). On the contrary, the striatum-to-cerebellum ratio displayed an important increase with time and values of 12 and 22 were, respectively, obtained at 60 and 120min after injection. No plateau was reached even 4 h after injection, as observed for some other dopaminergic antagonists (Chalon et al., 1990). Blocking experiments. The specificity of the in vivo [‘311]iodotropapride binding was evaluated by blocking experiments using S, and D, antagonists. The results are summarized in Table 3. Spiperone showing a mixed D,-S, affinity (Leysen, 1989) strongly blocked the [‘3’I]iodotropapride acTable 4. Saturability assessment of the striatum uptake by addition of carrier (constant injected activity per rat: 15 nCi. Animals were killed 1.5 h post-injection, a = 4) Specific activity (Ci/mmol) 240 15 1.5 1.5 0.75 0.3 0.15 0.03
Striatum-to-cerebellum 16 16 16 16 11 7 4 1.4
cumulation in the striatum and frontal cortex. Drugs with higher affinity for D, than for Sz receptor sites, such as halopemide (Loonen and Soudijn, 1985; Leysen, 1984; Clanton et al., 1991) and bromolisuride (Maziere et af., 1986), decreased the striatum-tocerebellum ratios. These antagonists did not modify the FCjCb ratio. On the contrary, ketanserin (Laduron et al., 1982) which has a higher affinity for serotonergic S2 sites, did not affect the St/Cb ratio but blocked the accumulation of the tracer in the frontal cortex. These results clearly demonstrate that the in vivo binding of [‘3’I]iodotropapride is related to the dopaminergic receptor sites and, to a much lesser extent, the S2 sites. Saturability. The striatum uptake was drastically influenced by the specific activity of the radiopharmaceutical. Ratios of 16 and 4 were measured for n.c.a. [‘3’I]iodotropapride and addition of 50 pg of carrier, respectively. These data, summarized in Table 4, proved that the binding to structures rich in D, receptor sites is saturable. In vitro studies Lipophilicity. Partition coefficient (P) data are provided in Table 5. At blood pH, P was 2.9. The protein binding value measured as described in the experimental section was 65%. Kd values. Extremely high affinity for the dopaminergic Dz receptors (Kd = 0.030 nM) was evaluated on rat brain tissue (Schotte et al., 1992) indicating that iodotropapride is about 50 times more potent than iodolisuride (Kd = 1.6 nM, Martres et al., 1985), the most commonly used iodinated ligand for the in vitro labeling of dopamine D, receptors.
Conclusions No carrier added [5-‘3LI]iodotropapride was prepared via nucleophilic substitution of the bromoderivative with [1311]iodinein the presence of Cu(1) as catalyst. After i.v. injection into rats, this new
ratio Table 5. Variation of P values of [“sI]iodotropapride versus pH PH 6.5 7.0 7.4
P
2.31 2.5 2.9
Synthesis iodinated
benzamide
penetrated
and biodistribui :ion of [5-‘3’l]iodotropapride
the blood-brain
bar-
and localized mainly on Dz dopamine receptor sites with a relative high specificity compared to those observed for other dopaminergic antagonists (Kung et al., 1989; Chalon et al., 1990). This high affinity binding was saturable. This preliminary study provided evidence of the potentialities of the corresponding [‘Z31]iodotropapride (“‘I: tl!2 = 13 h, ET = I59 keV) as a suitable ligand for imaging D, dopamine receptors in the human cerebral nervous system with SPECT. These investigations are presently under study and already show higher striatum-to-cerebellum ratios (ranging between 3.2 at 1 h and 5 at 4 h) than any other dopaminergic ligand so far investigated on humans. rier
Acknowledgements-The authors thank Mrs F. Mombaerts-Czichosz and Mr G. Salvador for their helpful technical assistance. This research was supported through a grant from the “Fonds National de la Recherche Scientifique” (FNRS) of Belgium (3.9007.89F).
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