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Technetium-99m p-iodophenethyldiaminodithiol (DADT-IPE): Potential brain perfusion imaging agent for SPECT
Nucl. Med. Bid. Vol. 19, No. 3, pp. 303-310, ht. J. Radiar. Appl. Instrum. Parr B
0883-2897/92 $5.00 + 0.00 Pergamon Press plc
1992
Printed in Great Britain
Technetium-99m p -1odophenethyldiaminodithiol (DADT-IPE): Potential Brain Perfusion Imaging Agent for SPECT K. ‘Radioisotope
SHIBA’,
H. MORI’,
H. MATSUDA’
and
K.
HISADA*
Center and 2Department of Nuclear Medicine, School of Medicine, Takara-machi, Kanazawa 920, Japan
Kanazawa
University,
(Received 2 Augusr 1991) A new ligand, an N-p-iodophenethyl diaminodithiol (DADT-IPE), an anlog of N-isopropyl-p-iodoamphetamine (IMP), was synthesized and subsequently complexed with 99mTc, using stannous chloride as a reducing agent. Two complexes (a and b) were separated from 99mTc-DADT-IPE by high performance liquid chromatography (HPLC). Competitive inhibition studies showed that the IC, value of DADT-IPE (70pM) was similar to that of IMP (49pM). Biodistribution studies of one of the complexes fss’“Tc-DADT-IPE(a)l in rats showed that 0.65% of the injected dose of the tracer remained in the brain at 5 min after intravenous injection, with 0.53% of the dose-remaining in the brain at 60 min post-injection, whereas the corresoondina values for the other complex IWmTc-DADT-IPE(b)l were 0.34% dose in the brain at 5 min and 0.28% dose in the brain at 60 min‘ post-injection. TIe’half-life for clearance of 99mTc-DADT-IPE(a) from rat brain was found to be more than 5 h. These results suggested that 99mTc-DADT-IPE(a) has characteristics which are suitable for cerebral perfusion imaging.
Introduction
the blood-brain barrier (Neirinckx et al., 1988; Walovitch et al., 1988a,b). In the present study, we synthesized a %Tccomplex of a diaminodithiol (DADT) ligand functionalized with a p-iodophenethyl group FTCDADT-IPE), whose chemical structure is analogous to that of IMP, and we evaluated the characteristics of this 99mTc-DADT-IPE complex in the rat brain.
Iodine-123~labeled N,N,N’-trimethyl-N-[2-hydroxy-3methyl-S-iodobenzyll-1.3-propanediamine (HIPDM) (Kung et al., 1983; Tramposch et al., 1983) and N-isopropyl-p-iodoamphetamine (IMP) (Winchell et al., 1980a,b) have been developed as radiopharmaceuticals for cerebral perfusion imaging (CPI) with single photon emission computed tomography (SPECT). Their utility has been demonstrated in the diagnosis of stroke, epilepsy and various other cerebrovascular disorders. However, a technetium99m (99mTc) tracer would be more convenient and desirable. Previously we showed that the IMP retention mechanism in the brain was probably associated with the saturable binding of IMP to extremely highdensity, but relatively low-affinity binding sites; and that, furthermore the particular chemical structure of IMP would have an important role in this binding (Mori et al., 1990). The IMP retention mechanism may therefore be different from those of 99mTc-hexamethylpropyleneamine oxime (HMPAO) and 99mTc-ethyl cysteinate dimer (ECD) which are retained within the brain by being rapidly converted to hydrophilic metabolites that do not recross
Experimental General
Melting points were obtained on a Yanagimoto micro melting point apparatus and reported uncorrected. Infrared spectra (i.r.) were determined on a Hitachi Model 230 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Hitachi R-24B (taken in deuterated chloroform with tetramethylsilane as the internal standard). Mass spectra (MS) were recorded on a Hitachi M-80 spectrometer. Spectral properties were consistent with the proposed structures. Elemental analyses were performed on a Yanagimoto CHN-Corder MT-3; all values were within +0.3% of theoretical values. High performance liquid chromatography (HPLC) was 303
K. SHIBA et
304
carried out on a chiralcel OD cellulose carbamate column (Daicel Chemical Industries Ltd) eluted with ethanol/O.01 M NH,OAc (85: 15, v/v). The radioactive eluent was detected with a radioisotope detector (Beckman Model 170). Chemistry 3,3,6,6,lO,lO-Hexamethyl-1,2-dithiu-5,8-diazacyclo4,8-diene(2). This cyclic Schiff’s base (1) was pre-
pared according to a previously reported (Kung et al., 1984; Lever et al., 1985).
method
3,3,6,6,10,IO-Hexamethyl-1,2-diaza-S,8-diazacyclodecane(2). A solution of compound (1) (2.58 g,
10 mmol) in dry diethyl ether (40 mL) was saturated with dry hydrogen chloride. To this solution was added a solution of sodium cyanoborohydride, NaBH, CN(650 mg, 10 mmol), in absolute ethanol (5 mL) at 0°C; this was added as a single portion with stirring. The resulting solution was stirred for 1 h at O”C, and was then made alkaline by the addition of 1 N NaOH. The aqueous suspension was extracted with diethyl ether. The extracts were dried over anydrous Na*SO, and then evaporated until a pale yellow oil was obtained; this was then purified by column chromatography on silica gel, with hexane-ethylacetate (7 : 3, v/v) as an eluent, to yield compound 2 (2.19 g, 83.5%). 2: colorless oil. NMR(CDC1,) 6: 1.04-1.41(18H), 1.92 (2H, brs; disappearance after D,O addition), 2.36(1H, d, J = 11.8 Hz), 2.40(2H, s), 2.60(1H, d, J = 12.0 Hz), 2.65(1H, d, J = 11.8 Hz), 3.15(1H, d, J = 12.0 Hz). Mass spectrum(C1): 263(M+ + H). 8N-p-Zodophenylacetyl-3,3,6,6,10, J,2-dithia-5,8-diazacyclodecane(3).
ltkhexamethyl-
p-Iodophenylacetyl chloride (2.10 g, 7.5 mmol) was added dropwise to a solution of 2 (1.3 10 g, 5 mmol) in dry diethyl ether (50mL). A white precipitate formed immediately, and after 1 h at room temperature, the reaction mixture was made alkaline by the addition of K2C0, solution. The aqueous suspension was extracted with diethyl ether. The extracts were dried over Na,SO, and evaporated until an oil was obtained; this oil was purified by column chromatography on silica gel with hexantithyl acetate (1: 1, v/v) as an eluent to yield compound 3 (2.140 g, 84.6%). 3: colorless oil. i.r. (CC&) 1640 cm-‘. NMR(CDC&) 6: 0.94-1.33(18H), 2.41(1H, d, J = 13.4Hz), 2.97(1H, d.d, J = 13.4Hz, J= 3.0Hz), 3.32(0.5H, d, J= 16.4 Hz), 3.47(0.5H, d, J = 15.1 Hz), 3.65(2H, d, J = 2.0 Hz), 4.03(2H, m), 4.96(0.5H, d, J = 15.1 Hz), 5.51(0.5H, d, J = 16.4Hz), 7.01(2H, d, J=8.3Hz), 7.64(2H, d, J = 8.3 Hz). Mass spectrum(C1): 507(M+ + H). 8N-p-Zodophenethyl-3,3,6,6,10,10-hexamethyl1,2dithia-5,8-diazacyclodecane(4). 10 mL (10 mmol) of
1.OM borane solution in tetrahydrofuran (THF) was introduced into a freshly-dried three-necked flask fitted with a reflux condenser, magnetic stirrer and dropping funnel; this flask was placed in an ice-water bath. Then compound 3 (2.024 g, 4 mmol) in dry
al.
THF(20 mL) was added slowly through the dropping funnel to the borane solution, keeping the temperature at just below 10°C. After the addition was complete, the reaction mixture was stirred for 1 h at below 20°C and was then refluxed for 30min. The flask was allowed to cool to room temperature and 2 mL of 6N HCl and 4 mL of water were added. The THF was removed by distillation at atmospheric pressure. The aqueous solution was made alkaline with 2N NaOH solution and was extracted with diethyl ether. The extracts were dried over Na,SO, and evaporated until a crystalline solid was obtained; this was purified by column chromatography on silica gel, with hexane-ethylacetate(9 : 1, v/v) as an eluent to yield 4 (1.419 g, 72.1%). 4: m.p. 97-99°C. NMR(CDC1,) 6 : 0.92-l .52(18H), 1.86(1H, brs), 2.30(1H, d, J = 14.6 Hz), 2.65(18, d, J = 11.2 Hz), 2.71(2H, t), 2.74(2H, t), 2.75(1H, d, J = 15.0 Hz), 2.87(1H, d, J = 14.6 Hz), 3.00(1H, d, J = 11.2 Hz), 3.06(1H, d, J = 15.0 Hz), 6.89(2H, d, J = 8.3 Hz), 7.60(28, d, J = 8.3 Hz). Mass spectrum(C1): 493(M+ + H). Anal. calcd for C,,H,,N,S21: C, 48.77; H, 6.75; N, 5.69. Found: C, 48.54; H, 6.91; N, 5.48. 2,2,5,5,9,PHexamethyl-4,7-diaza7N-p-iodophenethyl-l,lO-decanedithiol(DADT-ZPE)(S). Compound
4 (984 mg, 2 mmol) was dissolved in dry diethyl ether (15 mL) in a freshly-dried 50-mL round bottomed flask and lithium aluminum hydride (LiAlH,) (76 mg, 2 mmol) was added. After being refluxed for 20 min, the reaction solution was hydrolyzed with saturated citric acid solution and was stirred for 30 min at room temperature. The aqueous suspension was extracted with diethyl ether, and the extractors were dried over Na,SO, and evaporated until an oil was obtained; this was purified by column chromatography on silica gel, with hexane-ethylacetate (5: 1, v/v) as an eluent, to yield compound 5 (972 mg, 98.4%). 5: colorless oil. NMR(CDC1,) 6: l.O8(6H, s), 1.35(6H, s), 1.43(68, s), 2.15(3H, s), 2.55(2H, s), 2.63(2H, s), 2.68(2H, s), 2.84(4H, m), 6.88(2H, d, J = 8.0 Hz), 7.56(2H, d, J = 8.0 Hz). Mass spectrum(CI): 495(M+ + H). 5 was converted to the hydrochloride salt by the passing of HCl gas. Anal. calcd for C,H,,N, &I: C, 42.33; H, 6.57; N, 4.94. Found: C, 42.44; H, 6.82; N, 5.10. Competitive inhibition studies
Inhibition studies of [12SI]IMPbinding by DADTIPE, phenethyl-diaminedithiol(DADT-PE) and various other drugs were routinely run in quadruplicate, in disposable polypropylene tubes which contained 0.1 mL of 50mM Tris-HCl buffer, pH 7.4 (with various concentration of drugs); 0.1 mL of [‘2sI]IMP (200 mM); and 0.4mL of rat brain homogenate (crude synaptosomal membrane) containing 250-400 pg of protein. After incubation for 30 min at eQ”C, the samples were quickly diluted with 5 mL of ice-cold buffer and filtered through glass filters (Whatman GF/B). The filters were washed twice with
DADT-IPE: potential brain perfusion imaging agent
5 mL of ice-cold buffer and air-dried. The radioactivity retained by the filters was counted in an autogamma scintillation counter (Aloka, ARC-360). The results of 3-4 experiments were averaged.
305
Autoradiography
In vitro autoradiography. Following 30 min of preincubation at 4”C, frozen 20-pm sections of male Sprague-Dawley rat brain were incubated in standard glass slide trays with approx. 2 PM Radiolabeling 99mTc-DADT-IPE complexes in the presence or abLabeling of the ligand DADT-IPE(5) with g9mT~ sence of excess (500 PM) cold DADT-IPE. The prewas performed as follows: to a solution of 0.5incubation and incubation medium contained 50 mM 1.0 mg of 5 in 1.OmL of water was added Tris-HCl (PH 7.4). Following 30 min of incubation at 37-370 MBq (I-10 mCi) sodium P9”Tc]pertechnetate 4”C, the sections were washed three times, for 30 s ([99mTc]Tc0; ) in 0.1 mL saline, followed by a freshly each time, in ice-cold buffer and dried. prepared solution of stannous chloride dihydrate Autoradiograms were obtained by overnight (SnCl,.2H,O) in water (0.1 mL, lo-‘M). The mixexposure of these mounted sections for x-ray film. ture was vortexed at 70-80°C for several minutes. The In vivo autoradiography. Male Sprague-Dawley rats reaction mixture was analyzed by thin-layer chroma(150-250 g) were injected intravenously with 0.4 mL tography (TLC), using 5% methanol in methylene of saline solution containing 40% ethanol and chloride. The separation of the two complexes was 370-l 110 MBq (l&30 mCi) of 9”Tc-DADT-IPE(a). carried out by HPLC using ethanol-O.01 M NH,OAc The rats were killed under anesthesia at 5 or 15 min (85:15, v/v) as the solvent. after injection. The brains were removed and frozen in embedding medium at -25°C; thin (20~pm) coroPartition coeficients nal sections of the midbrain were then cut, using a These were determined by mixing the 99mTc-com- cryostat microtome. Each section was then mounted plexes with 3 mL each of I-octanol and buffer on a glass slide and air dried. Autoradiograms were (pH 7.0, 0.05 M phosphate) in a test tube. The tube obtained by overnight exposure of these mounted was vortexed for 10 min. Two samples (0.2 mL each) sections to x-ray film. from the octanol and aqueous layers were then counted in an autogamma scintillation counter. The Results and Discussion partition coefficient was obtained by calculating the Chemistry ratio of the net cpm/mL of octanol to the net cpm/mL of the buffer. The partition coefficient of [“51]IMP The preparation method for DADT-IPE(5) is was determined by the same procedure as that which shown in Fig. 1. The macrocyclic diimine(1) was was used to determine the partition coefficient of the prepared by condensation of the dialdehyde and 99mTc-DADT-IPE complexes. diamine. The reduction of 1 with sodium cyanoboro-
Biodistribution studies Male Sprague-Dawley rats (150-250 g) were injected intravenously, into the tail vein, with 0.4mL (74-148 kBq, 2-4 PCi) of each of the 99mTc-DADTIPE complexes (a, b). At various times after injection, blood samples were collected by cardiac puncture, and the rats were killed by decapitation immediately thereafter. The organs of interest were subsequently dissected, weighed and prepared for counting. The percent of radioactivity in the whole blood was estimated using a blood volume of 7% of body weight. To investigate the stability of the 99mTc-DADT-IPE complexes in the blood and in the brain tissue, male Sprague-Dawley rats (150-250 g) were administered 370 MBq (10 mCi) of these 99mTc-DADT-IPE complexes. At 30 min post-injection, blood samples were collected by cardiac puncture, and the rats were sacrificed by decapitation. The brains were quickly removed and homogenized with 5 mL of 0.32 M sucrose. These homogenized tissues were subsequently extracted using 5 mL of methanol. The blood samples, the brain extracts and the 99mTc-DADT-IPE control samples were analyzed by thin-layer chromatography (TLC), using 5% methanol in methylene chloride.
hydride(NaBH,CN) in dry diethyl ether under acidic conditions at 0°C provided the expected diaminodisulfide(2) selectively, without providing the bicyclic imidazolidine. p-Iodophenylacetyl chloride
Fig. I. Synthesis scheme for the preparation of DADT-IPE.
K. SHIBAet nl.
306
was synthesized by iodination of p-aminophenylacetic acid, followed by acyl chlorination. The diaminodisulfide(2) was reacted with the piodophenylacetyl chloride to give 3. Reduction of the amido group of 3 was accomplished by reaction with borane-tetrahydrofuran solution to yield N-piodophenethyl-diaminodisulfide(4). 4 was successfully reduced with an equimolar amount of lithium aluminium hydride(LiAlH,) for 20 min, to yield N-p-iodophenethyl-diaminodithiol(DADT-IPE)(5) without deiodination. The N-p-iodophenethyldiaminodithiol(DADT-IPE)(S) was converted to the hydrochloride salt by the passing of HCl gas. Competitive inhibition studies IC, values showed the following potencies in competition of the [‘251]IMPbinding, listed in order of Table
increasing potency: IMP > DADT-IPE > N-benzylp-iodophenethylamine (BIPA) > N-isopropylamphetamine > methoamphetamine > DADT-IPE > dopamine > phenylalanine (Table 1). Compared with their nonhalogenated parent amines, IMP, DADT-IPE and BIPA, with a p-iodophenethyl carbon skeleton, were effective in inhibiting [1251]IMP binding, suggesting that these DADT-IPE characteristics were similar to those of IMP. Radiolabeling The 99”Tc-DADT-IPE complexes were prepared by simply mixing the ligand with ~Tc]TcO; and &Cl,. 2H,O for several minutes at 70-80°C. The radiochemical yield of the 99mTc-complex ranged from 85 to 95% on TLC analysis (R, values were 0.85Wl.90), but analysis of the reaction solution by
I. Inhibition of [‘2’1]IMP binding by drugs Chemical structure
Drugs IMP
Gn (flM)* 49 + 6
I
7Ok 18
DADT-IPE
N-Benzyl-p-iodophenethyl
0
CH2CH2-NH CH2 1-i
amine
CH 13." CH2-CH-N
O,\
N-Isopropyl amphetamine
-
Methoamphetamine \/ o-
1240 f 160 'CH(C"&
CH I 3 ," CH2-CH-N,
1300 +_140
CH3
1540 + 195
DADT-PE
CH2-CH2-NH2 Dopamine
Phenylalanine
125 + 25
0,-,
-CH2-CH2-NH2 I COO"
7800 + 2600
> 20,000
*It& represents the concentration of drugs inducing a 50% inhibition of specific binding. Values given are the mean + SEM taken from 3 to 4 experiments. N-Benzyl-p-iodophenethyl amine was prepared by reacting p-iodophenylacetyl chloride with benzyl amine, followed by reduction with boranetetrahydrofuran. DADT-PE was prepared by the same procedure as that used to prepare DADT-IPE, except that a phenylacetyl chloride was used instead ofp-iodophenyl acetyl chloride. N-Isopropylamphetamine was prepared by hydrodehalogenation of IMP with LiAI,. These drugs were identified on NMR and by mass spectrometer. [“‘1]IMP and nonradioactive IMP were obtained from Nihon Medi-Physica Inc. Methamphetamine, dopamine and phenylalanine were commercially available or were supplied by pharmaceutical companies.
Fig. 4. In virro autoradiograms of rat brain sections incubated for 30 min with 99mTc-DADT-IPE complexes in the absence (a) or presence (b) of excess (5OOpM) cold DADT-IPE.
Fig. 5. In vivo autoradiograms
of *Tc-DADT-IPE (a) in rat brain at 5 min (a) and 15 min (b) post-injection.
307
DADT-IPE: potential brain perfusion imaging agent
309
-CH3
Fig.
3. Proposed
structure
o-
R’
R
-CH2CH2-
\
,
I
of the %Tc-DADT-IPE
complex.
__AL
were more than 92% pure and were injected less than 1 h after isolation. In addition, TLC analyses demonstrated that both *Tc-DADT-IPE complexes were stable in blood and brain at 30min post-injection. Biodistribution studies
h
;r u)
0
4
8
12
16
20
Fig. 2. Radiochromatograms obtained by HPLC analysis of the 99”Tc-DADT-IPE complex.
HPLC showed the presence of two major peaks of approximately equal magnitude in the reaction solution (Fig. 2). These peaks were separated and checked for purity by HPLC. The peak exhibiting the shorter retention time was referred to as peak A PTc-DADT-IPE(a)], and the other, as peak B [99mTc-DADT-IPE(b)]. Each peak was shown to be stable in 40% ethanol in saline for at least 24 h. Based on the structure reported by Davison et al. (1981), the proposed structure of the %Tc-DADT-IPE complex is shown in Fig. 3. The samples injected into the rats Table 2. Biodistribution
Table 2 shows the biodistribution of 99mTc-DADTIPE(a,b) in rats. The brain uptake values of *TcDADT-IPE(a) and *Tc-DADT-IPE(b), at 5min post-injection were 0.65 and 0.34%, respectively, of the injected dose. At 15, 30 and 60 min post-injection, the corresponding values for ““Tc-DADT-IPE(a) were 0.60, 0.56 and 0.53%, respectively, whereas were 0.30, 0.29 and those for %Tc-DADT-IPE(b) 0.28%, respectively. Although WmTc-DADT-IPE(a) had a 2-fold higher uptake in the rat brain than *Tc-DADT-IPE(b), both 99mTc-complexes(a,b) were retained in the rat brain for long periods (the T,,r of both of these complexes was more than 5 h). 99”Tc-DADT-IPE(T,,, = 5 h) had a much longer retention in the rat brain than WmT~-292 f67619 29-hexamethyl-4,7-diaza-l,10-decandithiol(99”Tc-DADT-HM) [85% with T,,* = 10 min and 15% with r,,, = 57 min; these data were reported by Kung et al. (1984)]; that
of 99mTc-DADT-IPE(a
and b) in rats
DADT-IPE(a) 5 min Organ Brain Blood7 Lung Heart Liver Spleen Stomach Kidney Brainibloodf
15min 30min (% dose/organ)*
60min
0.65f 0.03 3.56f 0.42 1.86+ 0.09 1.80+ 0.03 46.58f4.43 0.79* 0.12 0.77+ 0.07 3.56; 0.50 1.5f 0.2
0.60f 0.04 3.12f 0.37 1.28f 0.08 1.19+0.04 35.27f 2.48 0.64f 0.05 0.61+ 0.06 2.14+ 0.34 1.7+ 0.2
0.56 + 0.02 2.75 + 0.25 I .20 f 0.07 I .Ol + 0.04 30.66 + I .98 0.50 * 0.05 0.52 k 0.05 2.23 + 0.10 l.9IO.2
0.53 f 0.03 2.48 + 0.30 0.90+0.18 0.82 f 0.09 26.71 f 0.75 0.42 + 0.05 0.49 f 0.04 2.11 +O.l6 2.OIO.l
0.34 f 0.03 6.50 f 1.24 1.56+0.18 1.34 kO.13 33.16 + 2.65 0.53 + 0.04 0.71 f 0.10 2.77 + 0.42 0.4 IO.0
0.30 4.69 1.03 0.92 29.84 0.44 0.75 2.18 0.6
0.29 3.65 0.86 0.70 27.89 0.35 0.76 2.05 0.7
0.28 k 0.02 3.09kO.12 0.69 + 0.08 0.52 _+0.03 24.63 f 0.85 0.30 * 0.03 I .OO+ 0.08 I .95 f 0.06 0.8 + 0.0
DADT-IPEe) Brain Bloodt Lung Heart Liver Spleen Stomach Kidnev Brain;blood$
*Values represent mean f SD (n = 4). tNormalized to body weight of 200 g. t% Dose/g or8an ratio.
f 0.03 k 0.40 kO.12 + 0.07 k 2.23 * 0.02 * 0.03 + 0.16 + 0.0
+ f f f f f f f +
0.04 0.33 0.09 0.05 0.92 0.02 0.04 0.08 0.0
K. SHIBA et
310 Table 3. Partition coefficients of 99mTc-DADTIPE(a and b) complexes and [‘251]IMP Compound DADT-IPE IMP ‘Octanol-buffer
Partition coefficient* a b
254 + 25 306 + 34 12.5; 0.6
partition coefficient at pH 7.0.
is, the inclusion of the p-iodophenethyl group at the less sterically hindered nitrogen atom of the DADT backbone resulted in significant alterations in brain retention, in comparison with the simple unsubstituted 99mTcDADT. The partition coefficient of 99mTc-DADT-IPE(a) was low (254) compared to that of 99mTc-DADTIPE(b)(306) (Table 3). In addition, the partition coefficient of 99mTc-DADT-IPE(a) was much higher than that of IMP( 12.5) although the rat brain uptake of 99mTc-DADT-IPE(a) (0.65% dose/organ at 5 min post-injection) was lower than that of IMP, as reported by Winchell et al. (1980a) (1.57% dose/organ at 5min post-injection). These results suggest the compound lipophilicity is a necessary but not sufficient condition for the crossing of the blood-brain barrier, and that a high brain trapping may require not only suitable lipophilicity, but also suitable molecular weight, polarity and structural and stereochemical characteristics. Autoradiography
Figure 4 shows the in vitro autoradiograms of rat brain sections incubated for 30min with 99mT~DADT-IPE complexes in the presence or absence of excess (500 PM) cold DADT-IPE. 99mTc-DADT-IPE complexes bound homogeneously to brain tissues; cold DADT-IPE was effective in inhibiting 99mT~DADT-IPE complexes, whereas DADT-PE and other deiodinate amines were ineffective in inhibiting 99mTc-DADT-IPE complexes (autoradiograms not shown). These results suggest that the trapping mechanism of 99mTc-DADT-IPE complexes could be related to their chemical structure and could be similar to that of IMP. The in vivo autoradiograms of 99mTc-DADTIPE(a) in rat brain at both 5 and 15 min post-injection [Fig. 5(a) and (b)] showed a high gray-white matter uptake ratio. These images were in agreement with those described previously for IMP (Kuhl et al., 1982), and they suggest that 99mTc-DADT-IPE(a) exhibits features of fixed regional distribution, reflecting regional perfusion.
These experimental data suggest that 99mTcDADT-IPE complexes, whose chemical structures were analogous to that of IMP, had a retention mechanism similar to that of IMP. In fact, the 99mTc-
al.
DADT-IPE(a) complex showed several characteristics suitable for CPI; it had relatively high uptake and long retention in the brain. Thus, DADT-IPE derivatives may be a good prospect for developing 99mTc-radiopharmaceuticals with high affinity and long retention in the brain. At present, by investigating the relationship between the structure of DADE-IPE analogs and their trapping characteristis, we are attempting to prepare a derivative of DADTIPE which exhibits greater brain uptake. Acknowledgement-We thank Nihon Medi-Physics Inc. for the supply of I-125 IMP and nonradioactive IMP.
References Davidson A., Jones A. G., Orvig C. et nl. (1981) A new class of oxotechnetium(5 +) chelate complexes containing a TcON,S, core. Inorg. Chem. 20, 162991632. Kuhl D. E., Barrio J. R., Huang S. C. et al. (1982) Quantifying local cerebral blood flow with N-isopropylp-[I-123liodoamphetamine tomography. J. Nucl. Med. 23, 196203. Kung H. F., Molnar M. and Billings J. (1984) Synthesis and biodistribution of neutral lipid-soluble Tc-99m complexes that cross the blood-brain barrier. J. Nucl. Med. 25, 326-332. Kung H. F., Tramposch K. M. and Blau M. (1983) A new brain perfusion imaging agent: [I-123lHIPDM: N,N,N’trimethyl-N’-[2-hydroxy-3-methyl-5-iodobenzyll-I .3propane&amine. J. Nucl. Med. tli 66-12. Kune H. F.. Yu C. C. and Billinas J. (1985) Svnthesis of new Bii(aminoethanethiol)(BAT) derivatives: possible ligands for %Tc brain imaging agents. J. Nucl. Med. 28, 1280-1284. Lever S. Z., Burns H. D., Kervitsky T. M. et al. (1985) Design, preparation, and biodistribution of a technetium99mtriaminedithol complex to assess regional cerebral blood flow. J. Nucl. Med. 26. 1287-1294. Mori H., Shiba K., Matsuda H., Tsuji S. and Hisada K. (1990) Charcteristics of the binding of N-isopropyl-p-[I1251 iodoamphetamine in the rat brain synaptosomal membranes. Nucl. Med. Commun. 11, 327-331. Neirinckx R. D., Burke J. F., Harrison R. C., Foster A. M., Andersen A. R. and Lassen N. A. (1988) The retention mechanism of Tc-99m-n,L-HMPAO: intracellular reaction with glutathione. J. Cereb. Bid Flow Metab. 8, (Suppl. 1) s4-12. Tramposch K. M., Kung H. F. and Blau M. (1983) Radioiodine labeled N,N’-dimethyl-N’-(2-hydroxy-3alkyl-5-iodobenzyl)-1.3-propanediamines for brain perfusion imaging. J. Med. Chem. 26, 121-125. Walovitch R. C., Makuch G., Knapik G., Watson A. D. and Williams S. J. (1988a) Brain retention of Tc-99mECD is related to in uivo metabolism. J. Nucl. Med. 29, 741. Walovitch R. C., Hall K. M., O’Toole J. J. and Williams S. J. (1988b) Metabolism of Tc-99m-ECD in normal volunteers. J. Nucl. Med. 29, 741. Winchell H. S., Baldwin R. M. and Lin T. H. (1980a) Development of I-123 labeled amines for brain studies: localization of I-123 iodophenyl alkylamines in rat brain. J. Nucl. Med. 21, 940-946. Winchell H. S., Horst W. D., Brau L. et al. (1980b) N-isopropyl[I-123]-p-iodoamphetamine: single pass brain uptake and washout; binding to brain synaptosome and localization in dog and monkey brain. J. Nucl. Med. 21, 941-952.
0883-2897/92 $5.00 + 0.00 Pergamon Press plc
1992
Printed in Great Britain
Technetium-99m p -1odophenethyldiaminodithiol (DADT-IPE): Potential Brain Perfusion Imaging Agent for SPECT K. ‘Radioisotope
SHIBA’,
H. MORI’,
H. MATSUDA’
and
K.
HISADA*
Center and 2Department of Nuclear Medicine, School of Medicine, Takara-machi, Kanazawa 920, Japan
Kanazawa
University,
(Received 2 Augusr 1991) A new ligand, an N-p-iodophenethyl diaminodithiol (DADT-IPE), an anlog of N-isopropyl-p-iodoamphetamine (IMP), was synthesized and subsequently complexed with 99mTc, using stannous chloride as a reducing agent. Two complexes (a and b) were separated from 99mTc-DADT-IPE by high performance liquid chromatography (HPLC). Competitive inhibition studies showed that the IC, value of DADT-IPE (70pM) was similar to that of IMP (49pM). Biodistribution studies of one of the complexes fss’“Tc-DADT-IPE(a)l in rats showed that 0.65% of the injected dose of the tracer remained in the brain at 5 min after intravenous injection, with 0.53% of the dose-remaining in the brain at 60 min post-injection, whereas the corresoondina values for the other complex IWmTc-DADT-IPE(b)l were 0.34% dose in the brain at 5 min and 0.28% dose in the brain at 60 min‘ post-injection. TIe’half-life for clearance of 99mTc-DADT-IPE(a) from rat brain was found to be more than 5 h. These results suggested that 99mTc-DADT-IPE(a) has characteristics which are suitable for cerebral perfusion imaging.
Introduction
the blood-brain barrier (Neirinckx et al., 1988; Walovitch et al., 1988a,b). In the present study, we synthesized a %Tccomplex of a diaminodithiol (DADT) ligand functionalized with a p-iodophenethyl group FTCDADT-IPE), whose chemical structure is analogous to that of IMP, and we evaluated the characteristics of this 99mTc-DADT-IPE complex in the rat brain.
Iodine-123~labeled N,N,N’-trimethyl-N-[2-hydroxy-3methyl-S-iodobenzyll-1.3-propanediamine (HIPDM) (Kung et al., 1983; Tramposch et al., 1983) and N-isopropyl-p-iodoamphetamine (IMP) (Winchell et al., 1980a,b) have been developed as radiopharmaceuticals for cerebral perfusion imaging (CPI) with single photon emission computed tomography (SPECT). Their utility has been demonstrated in the diagnosis of stroke, epilepsy and various other cerebrovascular disorders. However, a technetium99m (99mTc) tracer would be more convenient and desirable. Previously we showed that the IMP retention mechanism in the brain was probably associated with the saturable binding of IMP to extremely highdensity, but relatively low-affinity binding sites; and that, furthermore the particular chemical structure of IMP would have an important role in this binding (Mori et al., 1990). The IMP retention mechanism may therefore be different from those of 99mTc-hexamethylpropyleneamine oxime (HMPAO) and 99mTc-ethyl cysteinate dimer (ECD) which are retained within the brain by being rapidly converted to hydrophilic metabolites that do not recross
Experimental General
Melting points were obtained on a Yanagimoto micro melting point apparatus and reported uncorrected. Infrared spectra (i.r.) were determined on a Hitachi Model 230 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Hitachi R-24B (taken in deuterated chloroform with tetramethylsilane as the internal standard). Mass spectra (MS) were recorded on a Hitachi M-80 spectrometer. Spectral properties were consistent with the proposed structures. Elemental analyses were performed on a Yanagimoto CHN-Corder MT-3; all values were within +0.3% of theoretical values. High performance liquid chromatography (HPLC) was 303
K. SHIBA et
304
carried out on a chiralcel OD cellulose carbamate column (Daicel Chemical Industries Ltd) eluted with ethanol/O.01 M NH,OAc (85: 15, v/v). The radioactive eluent was detected with a radioisotope detector (Beckman Model 170). Chemistry 3,3,6,6,lO,lO-Hexamethyl-1,2-dithiu-5,8-diazacyclo4,8-diene(2). This cyclic Schiff’s base (1) was pre-
pared according to a previously reported (Kung et al., 1984; Lever et al., 1985).
method
3,3,6,6,10,IO-Hexamethyl-1,2-diaza-S,8-diazacyclodecane(2). A solution of compound (1) (2.58 g,
10 mmol) in dry diethyl ether (40 mL) was saturated with dry hydrogen chloride. To this solution was added a solution of sodium cyanoborohydride, NaBH, CN(650 mg, 10 mmol), in absolute ethanol (5 mL) at 0°C; this was added as a single portion with stirring. The resulting solution was stirred for 1 h at O”C, and was then made alkaline by the addition of 1 N NaOH. The aqueous suspension was extracted with diethyl ether. The extracts were dried over anydrous Na*SO, and then evaporated until a pale yellow oil was obtained; this was then purified by column chromatography on silica gel, with hexane-ethylacetate (7 : 3, v/v) as an eluent, to yield compound 2 (2.19 g, 83.5%). 2: colorless oil. NMR(CDC1,) 6: 1.04-1.41(18H), 1.92 (2H, brs; disappearance after D,O addition), 2.36(1H, d, J = 11.8 Hz), 2.40(2H, s), 2.60(1H, d, J = 12.0 Hz), 2.65(1H, d, J = 11.8 Hz), 3.15(1H, d, J = 12.0 Hz). Mass spectrum(C1): 263(M+ + H). 8N-p-Zodophenylacetyl-3,3,6,6,10, J,2-dithia-5,8-diazacyclodecane(3).
ltkhexamethyl-
p-Iodophenylacetyl chloride (2.10 g, 7.5 mmol) was added dropwise to a solution of 2 (1.3 10 g, 5 mmol) in dry diethyl ether (50mL). A white precipitate formed immediately, and after 1 h at room temperature, the reaction mixture was made alkaline by the addition of K2C0, solution. The aqueous suspension was extracted with diethyl ether. The extracts were dried over Na,SO, and evaporated until an oil was obtained; this oil was purified by column chromatography on silica gel with hexantithyl acetate (1: 1, v/v) as an eluent to yield compound 3 (2.140 g, 84.6%). 3: colorless oil. i.r. (CC&) 1640 cm-‘. NMR(CDC&) 6: 0.94-1.33(18H), 2.41(1H, d, J = 13.4Hz), 2.97(1H, d.d, J = 13.4Hz, J= 3.0Hz), 3.32(0.5H, d, J= 16.4 Hz), 3.47(0.5H, d, J = 15.1 Hz), 3.65(2H, d, J = 2.0 Hz), 4.03(2H, m), 4.96(0.5H, d, J = 15.1 Hz), 5.51(0.5H, d, J = 16.4Hz), 7.01(2H, d, J=8.3Hz), 7.64(2H, d, J = 8.3 Hz). Mass spectrum(C1): 507(M+ + H). 8N-p-Zodophenethyl-3,3,6,6,10,10-hexamethyl1,2dithia-5,8-diazacyclodecane(4). 10 mL (10 mmol) of
1.OM borane solution in tetrahydrofuran (THF) was introduced into a freshly-dried three-necked flask fitted with a reflux condenser, magnetic stirrer and dropping funnel; this flask was placed in an ice-water bath. Then compound 3 (2.024 g, 4 mmol) in dry
al.
THF(20 mL) was added slowly through the dropping funnel to the borane solution, keeping the temperature at just below 10°C. After the addition was complete, the reaction mixture was stirred for 1 h at below 20°C and was then refluxed for 30min. The flask was allowed to cool to room temperature and 2 mL of 6N HCl and 4 mL of water were added. The THF was removed by distillation at atmospheric pressure. The aqueous solution was made alkaline with 2N NaOH solution and was extracted with diethyl ether. The extracts were dried over Na,SO, and evaporated until a crystalline solid was obtained; this was purified by column chromatography on silica gel, with hexane-ethylacetate(9 : 1, v/v) as an eluent to yield 4 (1.419 g, 72.1%). 4: m.p. 97-99°C. NMR(CDC1,) 6 : 0.92-l .52(18H), 1.86(1H, brs), 2.30(1H, d, J = 14.6 Hz), 2.65(18, d, J = 11.2 Hz), 2.71(2H, t), 2.74(2H, t), 2.75(1H, d, J = 15.0 Hz), 2.87(1H, d, J = 14.6 Hz), 3.00(1H, d, J = 11.2 Hz), 3.06(1H, d, J = 15.0 Hz), 6.89(2H, d, J = 8.3 Hz), 7.60(28, d, J = 8.3 Hz). Mass spectrum(C1): 493(M+ + H). Anal. calcd for C,,H,,N,S21: C, 48.77; H, 6.75; N, 5.69. Found: C, 48.54; H, 6.91; N, 5.48. 2,2,5,5,9,PHexamethyl-4,7-diaza7N-p-iodophenethyl-l,lO-decanedithiol(DADT-ZPE)(S). Compound
4 (984 mg, 2 mmol) was dissolved in dry diethyl ether (15 mL) in a freshly-dried 50-mL round bottomed flask and lithium aluminum hydride (LiAlH,) (76 mg, 2 mmol) was added. After being refluxed for 20 min, the reaction solution was hydrolyzed with saturated citric acid solution and was stirred for 30 min at room temperature. The aqueous suspension was extracted with diethyl ether, and the extractors were dried over Na,SO, and evaporated until an oil was obtained; this was purified by column chromatography on silica gel, with hexane-ethylacetate (5: 1, v/v) as an eluent, to yield compound 5 (972 mg, 98.4%). 5: colorless oil. NMR(CDC1,) 6: l.O8(6H, s), 1.35(6H, s), 1.43(68, s), 2.15(3H, s), 2.55(2H, s), 2.63(2H, s), 2.68(2H, s), 2.84(4H, m), 6.88(2H, d, J = 8.0 Hz), 7.56(2H, d, J = 8.0 Hz). Mass spectrum(CI): 495(M+ + H). 5 was converted to the hydrochloride salt by the passing of HCl gas. Anal. calcd for C,H,,N, &I: C, 42.33; H, 6.57; N, 4.94. Found: C, 42.44; H, 6.82; N, 5.10. Competitive inhibition studies
Inhibition studies of [12SI]IMPbinding by DADTIPE, phenethyl-diaminedithiol(DADT-PE) and various other drugs were routinely run in quadruplicate, in disposable polypropylene tubes which contained 0.1 mL of 50mM Tris-HCl buffer, pH 7.4 (with various concentration of drugs); 0.1 mL of [‘2sI]IMP (200 mM); and 0.4mL of rat brain homogenate (crude synaptosomal membrane) containing 250-400 pg of protein. After incubation for 30 min at eQ”C, the samples were quickly diluted with 5 mL of ice-cold buffer and filtered through glass filters (Whatman GF/B). The filters were washed twice with
DADT-IPE: potential brain perfusion imaging agent
5 mL of ice-cold buffer and air-dried. The radioactivity retained by the filters was counted in an autogamma scintillation counter (Aloka, ARC-360). The results of 3-4 experiments were averaged.
305
Autoradiography
In vitro autoradiography. Following 30 min of preincubation at 4”C, frozen 20-pm sections of male Sprague-Dawley rat brain were incubated in standard glass slide trays with approx. 2 PM Radiolabeling 99mTc-DADT-IPE complexes in the presence or abLabeling of the ligand DADT-IPE(5) with g9mT~ sence of excess (500 PM) cold DADT-IPE. The prewas performed as follows: to a solution of 0.5incubation and incubation medium contained 50 mM 1.0 mg of 5 in 1.OmL of water was added Tris-HCl (PH 7.4). Following 30 min of incubation at 37-370 MBq (I-10 mCi) sodium P9”Tc]pertechnetate 4”C, the sections were washed three times, for 30 s ([99mTc]Tc0; ) in 0.1 mL saline, followed by a freshly each time, in ice-cold buffer and dried. prepared solution of stannous chloride dihydrate Autoradiograms were obtained by overnight (SnCl,.2H,O) in water (0.1 mL, lo-‘M). The mixexposure of these mounted sections for x-ray film. ture was vortexed at 70-80°C for several minutes. The In vivo autoradiography. Male Sprague-Dawley rats reaction mixture was analyzed by thin-layer chroma(150-250 g) were injected intravenously with 0.4 mL tography (TLC), using 5% methanol in methylene of saline solution containing 40% ethanol and chloride. The separation of the two complexes was 370-l 110 MBq (l&30 mCi) of 9”Tc-DADT-IPE(a). carried out by HPLC using ethanol-O.01 M NH,OAc The rats were killed under anesthesia at 5 or 15 min (85:15, v/v) as the solvent. after injection. The brains were removed and frozen in embedding medium at -25°C; thin (20~pm) coroPartition coeficients nal sections of the midbrain were then cut, using a These were determined by mixing the 99mTc-com- cryostat microtome. Each section was then mounted plexes with 3 mL each of I-octanol and buffer on a glass slide and air dried. Autoradiograms were (pH 7.0, 0.05 M phosphate) in a test tube. The tube obtained by overnight exposure of these mounted was vortexed for 10 min. Two samples (0.2 mL each) sections to x-ray film. from the octanol and aqueous layers were then counted in an autogamma scintillation counter. The Results and Discussion partition coefficient was obtained by calculating the Chemistry ratio of the net cpm/mL of octanol to the net cpm/mL of the buffer. The partition coefficient of [“51]IMP The preparation method for DADT-IPE(5) is was determined by the same procedure as that which shown in Fig. 1. The macrocyclic diimine(1) was was used to determine the partition coefficient of the prepared by condensation of the dialdehyde and 99mTc-DADT-IPE complexes. diamine. The reduction of 1 with sodium cyanoboro-
Biodistribution studies Male Sprague-Dawley rats (150-250 g) were injected intravenously, into the tail vein, with 0.4mL (74-148 kBq, 2-4 PCi) of each of the 99mTc-DADTIPE complexes (a, b). At various times after injection, blood samples were collected by cardiac puncture, and the rats were killed by decapitation immediately thereafter. The organs of interest were subsequently dissected, weighed and prepared for counting. The percent of radioactivity in the whole blood was estimated using a blood volume of 7% of body weight. To investigate the stability of the 99mTc-DADT-IPE complexes in the blood and in the brain tissue, male Sprague-Dawley rats (150-250 g) were administered 370 MBq (10 mCi) of these 99mTc-DADT-IPE complexes. At 30 min post-injection, blood samples were collected by cardiac puncture, and the rats were sacrificed by decapitation. The brains were quickly removed and homogenized with 5 mL of 0.32 M sucrose. These homogenized tissues were subsequently extracted using 5 mL of methanol. The blood samples, the brain extracts and the 99mTc-DADT-IPE control samples were analyzed by thin-layer chromatography (TLC), using 5% methanol in methylene chloride.
hydride(NaBH,CN) in dry diethyl ether under acidic conditions at 0°C provided the expected diaminodisulfide(2) selectively, without providing the bicyclic imidazolidine. p-Iodophenylacetyl chloride
Fig. I. Synthesis scheme for the preparation of DADT-IPE.
K. SHIBAet nl.
306
was synthesized by iodination of p-aminophenylacetic acid, followed by acyl chlorination. The diaminodisulfide(2) was reacted with the piodophenylacetyl chloride to give 3. Reduction of the amido group of 3 was accomplished by reaction with borane-tetrahydrofuran solution to yield N-piodophenethyl-diaminodisulfide(4). 4 was successfully reduced with an equimolar amount of lithium aluminium hydride(LiAlH,) for 20 min, to yield N-p-iodophenethyl-diaminodithiol(DADT-IPE)(5) without deiodination. The N-p-iodophenethyldiaminodithiol(DADT-IPE)(S) was converted to the hydrochloride salt by the passing of HCl gas. Competitive inhibition studies IC, values showed the following potencies in competition of the [‘251]IMPbinding, listed in order of Table
increasing potency: IMP > DADT-IPE > N-benzylp-iodophenethylamine (BIPA) > N-isopropylamphetamine > methoamphetamine > DADT-IPE > dopamine > phenylalanine (Table 1). Compared with their nonhalogenated parent amines, IMP, DADT-IPE and BIPA, with a p-iodophenethyl carbon skeleton, were effective in inhibiting [1251]IMP binding, suggesting that these DADT-IPE characteristics were similar to those of IMP. Radiolabeling The 99”Tc-DADT-IPE complexes were prepared by simply mixing the ligand with ~Tc]TcO; and &Cl,. 2H,O for several minutes at 70-80°C. The radiochemical yield of the 99mTc-complex ranged from 85 to 95% on TLC analysis (R, values were 0.85Wl.90), but analysis of the reaction solution by
I. Inhibition of [‘2’1]IMP binding by drugs Chemical structure
Drugs IMP
Gn (flM)* 49 + 6
I
7Ok 18
DADT-IPE
N-Benzyl-p-iodophenethyl
0
CH2CH2-NH CH2 1-i
amine
CH 13." CH2-CH-N
O,\
N-Isopropyl amphetamine
-
Methoamphetamine \/ o-
1240 f 160 'CH(C"&
CH I 3 ," CH2-CH-N,
1300 +_140
CH3
1540 + 195
DADT-PE
CH2-CH2-NH2 Dopamine
Phenylalanine
125 + 25
0,-,
-CH2-CH2-NH2 I COO"
7800 + 2600
> 20,000
*It& represents the concentration of drugs inducing a 50% inhibition of specific binding. Values given are the mean + SEM taken from 3 to 4 experiments. N-Benzyl-p-iodophenethyl amine was prepared by reacting p-iodophenylacetyl chloride with benzyl amine, followed by reduction with boranetetrahydrofuran. DADT-PE was prepared by the same procedure as that used to prepare DADT-IPE, except that a phenylacetyl chloride was used instead ofp-iodophenyl acetyl chloride. N-Isopropylamphetamine was prepared by hydrodehalogenation of IMP with LiAI,. These drugs were identified on NMR and by mass spectrometer. [“‘1]IMP and nonradioactive IMP were obtained from Nihon Medi-Physica Inc. Methamphetamine, dopamine and phenylalanine were commercially available or were supplied by pharmaceutical companies.
Fig. 4. In virro autoradiograms of rat brain sections incubated for 30 min with 99mTc-DADT-IPE complexes in the absence (a) or presence (b) of excess (5OOpM) cold DADT-IPE.
Fig. 5. In vivo autoradiograms
of *Tc-DADT-IPE (a) in rat brain at 5 min (a) and 15 min (b) post-injection.
307
DADT-IPE: potential brain perfusion imaging agent
309
-CH3
Fig.
3. Proposed
structure
o-
R’
R
-CH2CH2-
\
,
I
of the %Tc-DADT-IPE
complex.
__AL
were more than 92% pure and were injected less than 1 h after isolation. In addition, TLC analyses demonstrated that both *Tc-DADT-IPE complexes were stable in blood and brain at 30min post-injection. Biodistribution studies
h
;r u)
0
4
8
12
16
20
Fig. 2. Radiochromatograms obtained by HPLC analysis of the 99”Tc-DADT-IPE complex.
HPLC showed the presence of two major peaks of approximately equal magnitude in the reaction solution (Fig. 2). These peaks were separated and checked for purity by HPLC. The peak exhibiting the shorter retention time was referred to as peak A PTc-DADT-IPE(a)], and the other, as peak B [99mTc-DADT-IPE(b)]. Each peak was shown to be stable in 40% ethanol in saline for at least 24 h. Based on the structure reported by Davison et al. (1981), the proposed structure of the %Tc-DADT-IPE complex is shown in Fig. 3. The samples injected into the rats Table 2. Biodistribution
Table 2 shows the biodistribution of 99mTc-DADTIPE(a,b) in rats. The brain uptake values of *TcDADT-IPE(a) and *Tc-DADT-IPE(b), at 5min post-injection were 0.65 and 0.34%, respectively, of the injected dose. At 15, 30 and 60 min post-injection, the corresponding values for ““Tc-DADT-IPE(a) were 0.60, 0.56 and 0.53%, respectively, whereas were 0.30, 0.29 and those for %Tc-DADT-IPE(b) 0.28%, respectively. Although WmTc-DADT-IPE(a) had a 2-fold higher uptake in the rat brain than *Tc-DADT-IPE(b), both 99mTc-complexes(a,b) were retained in the rat brain for long periods (the T,,r of both of these complexes was more than 5 h). 99”Tc-DADT-IPE(T,,, = 5 h) had a much longer retention in the rat brain than WmT~-292 f67619 29-hexamethyl-4,7-diaza-l,10-decandithiol(99”Tc-DADT-HM) [85% with T,,* = 10 min and 15% with r,,, = 57 min; these data were reported by Kung et al. (1984)]; that
of 99mTc-DADT-IPE(a
and b) in rats
DADT-IPE(a) 5 min Organ Brain Blood7 Lung Heart Liver Spleen Stomach Kidney Brainibloodf
15min 30min (% dose/organ)*
60min
0.65f 0.03 3.56f 0.42 1.86+ 0.09 1.80+ 0.03 46.58f4.43 0.79* 0.12 0.77+ 0.07 3.56; 0.50 1.5f 0.2
0.60f 0.04 3.12f 0.37 1.28f 0.08 1.19+0.04 35.27f 2.48 0.64f 0.05 0.61+ 0.06 2.14+ 0.34 1.7+ 0.2
0.56 + 0.02 2.75 + 0.25 I .20 f 0.07 I .Ol + 0.04 30.66 + I .98 0.50 * 0.05 0.52 k 0.05 2.23 + 0.10 l.9IO.2
0.53 f 0.03 2.48 + 0.30 0.90+0.18 0.82 f 0.09 26.71 f 0.75 0.42 + 0.05 0.49 f 0.04 2.11 +O.l6 2.OIO.l
0.34 f 0.03 6.50 f 1.24 1.56+0.18 1.34 kO.13 33.16 + 2.65 0.53 + 0.04 0.71 f 0.10 2.77 + 0.42 0.4 IO.0
0.30 4.69 1.03 0.92 29.84 0.44 0.75 2.18 0.6
0.29 3.65 0.86 0.70 27.89 0.35 0.76 2.05 0.7
0.28 k 0.02 3.09kO.12 0.69 + 0.08 0.52 _+0.03 24.63 f 0.85 0.30 * 0.03 I .OO+ 0.08 I .95 f 0.06 0.8 + 0.0
DADT-IPEe) Brain Bloodt Lung Heart Liver Spleen Stomach Kidnev Brain;blood$
*Values represent mean f SD (n = 4). tNormalized to body weight of 200 g. t% Dose/g or8an ratio.
f 0.03 k 0.40 kO.12 + 0.07 k 2.23 * 0.02 * 0.03 + 0.16 + 0.0
+ f f f f f f f +
0.04 0.33 0.09 0.05 0.92 0.02 0.04 0.08 0.0
K. SHIBA et
310 Table 3. Partition coefficients of 99mTc-DADTIPE(a and b) complexes and [‘251]IMP Compound DADT-IPE IMP ‘Octanol-buffer
Partition coefficient* a b
254 + 25 306 + 34 12.5; 0.6
partition coefficient at pH 7.0.
is, the inclusion of the p-iodophenethyl group at the less sterically hindered nitrogen atom of the DADT backbone resulted in significant alterations in brain retention, in comparison with the simple unsubstituted 99mTcDADT. The partition coefficient of 99mTc-DADT-IPE(a) was low (254) compared to that of 99mTc-DADTIPE(b)(306) (Table 3). In addition, the partition coefficient of 99mTc-DADT-IPE(a) was much higher than that of IMP( 12.5) although the rat brain uptake of 99mTc-DADT-IPE(a) (0.65% dose/organ at 5 min post-injection) was lower than that of IMP, as reported by Winchell et al. (1980a) (1.57% dose/organ at 5min post-injection). These results suggest the compound lipophilicity is a necessary but not sufficient condition for the crossing of the blood-brain barrier, and that a high brain trapping may require not only suitable lipophilicity, but also suitable molecular weight, polarity and structural and stereochemical characteristics. Autoradiography
Figure 4 shows the in vitro autoradiograms of rat brain sections incubated for 30min with 99mT~DADT-IPE complexes in the presence or absence of excess (500 PM) cold DADT-IPE. 99mTc-DADT-IPE complexes bound homogeneously to brain tissues; cold DADT-IPE was effective in inhibiting 99mT~DADT-IPE complexes, whereas DADT-PE and other deiodinate amines were ineffective in inhibiting 99mTc-DADT-IPE complexes (autoradiograms not shown). These results suggest that the trapping mechanism of 99mTc-DADT-IPE complexes could be related to their chemical structure and could be similar to that of IMP. The in vivo autoradiograms of 99mTc-DADTIPE(a) in rat brain at both 5 and 15 min post-injection [Fig. 5(a) and (b)] showed a high gray-white matter uptake ratio. These images were in agreement with those described previously for IMP (Kuhl et al., 1982), and they suggest that 99mTc-DADT-IPE(a) exhibits features of fixed regional distribution, reflecting regional perfusion.
These experimental data suggest that 99mTcDADT-IPE complexes, whose chemical structures were analogous to that of IMP, had a retention mechanism similar to that of IMP. In fact, the 99mTc-
al.
DADT-IPE(a) complex showed several characteristics suitable for CPI; it had relatively high uptake and long retention in the brain. Thus, DADT-IPE derivatives may be a good prospect for developing 99mTc-radiopharmaceuticals with high affinity and long retention in the brain. At present, by investigating the relationship between the structure of DADE-IPE analogs and their trapping characteristis, we are attempting to prepare a derivative of DADTIPE which exhibits greater brain uptake. Acknowledgement-We thank Nihon Medi-Physics Inc. for the supply of I-125 IMP and nonradioactive IMP.
References Davidson A., Jones A. G., Orvig C. et nl. (1981) A new class of oxotechnetium(5 +) chelate complexes containing a TcON,S, core. Inorg. Chem. 20, 162991632. Kuhl D. E., Barrio J. R., Huang S. C. et al. (1982) Quantifying local cerebral blood flow with N-isopropylp-[I-123liodoamphetamine tomography. J. Nucl. Med. 23, 196203. Kung H. F., Molnar M. and Billings J. (1984) Synthesis and biodistribution of neutral lipid-soluble Tc-99m complexes that cross the blood-brain barrier. J. Nucl. Med. 25, 326-332. Kung H. F., Tramposch K. M. and Blau M. (1983) A new brain perfusion imaging agent: [I-123lHIPDM: N,N,N’trimethyl-N’-[2-hydroxy-3-methyl-5-iodobenzyll-I .3propane&amine. J. Nucl. Med. tli 66-12. Kune H. F.. Yu C. C. and Billinas J. (1985) Svnthesis of new Bii(aminoethanethiol)(BAT) derivatives: possible ligands for %Tc brain imaging agents. J. Nucl. Med. 28, 1280-1284. Lever S. Z., Burns H. D., Kervitsky T. M. et al. (1985) Design, preparation, and biodistribution of a technetium99mtriaminedithol complex to assess regional cerebral blood flow. J. Nucl. Med. 26. 1287-1294. Mori H., Shiba K., Matsuda H., Tsuji S. and Hisada K. (1990) Charcteristics of the binding of N-isopropyl-p-[I1251 iodoamphetamine in the rat brain synaptosomal membranes. Nucl. Med. Commun. 11, 327-331. Neirinckx R. D., Burke J. F., Harrison R. C., Foster A. M., Andersen A. R. and Lassen N. A. (1988) The retention mechanism of Tc-99m-n,L-HMPAO: intracellular reaction with glutathione. J. Cereb. Bid Flow Metab. 8, (Suppl. 1) s4-12. Tramposch K. M., Kung H. F. and Blau M. (1983) Radioiodine labeled N,N’-dimethyl-N’-(2-hydroxy-3alkyl-5-iodobenzyl)-1.3-propanediamines for brain perfusion imaging. J. Med. Chem. 26, 121-125. Walovitch R. C., Makuch G., Knapik G., Watson A. D. and Williams S. J. (1988a) Brain retention of Tc-99mECD is related to in uivo metabolism. J. Nucl. Med. 29, 741. Walovitch R. C., Hall K. M., O’Toole J. J. and Williams S. J. (1988b) Metabolism of Tc-99m-ECD in normal volunteers. J. Nucl. Med. 29, 741. Winchell H. S., Baldwin R. M. and Lin T. H. (1980a) Development of I-123 labeled amines for brain studies: localization of I-123 iodophenyl alkylamines in rat brain. J. Nucl. Med. 21, 940-946. Winchell H. S., Horst W. D., Brau L. et al. (1980b) N-isopropyl[I-123]-p-iodoamphetamine: single pass brain uptake and washout; binding to brain synaptosome and localization in dog and monkey brain. J. Nucl. Med. 21, 941-952.