Synthesis and biodistribution of a new 99mTc nitrido complex for cerebral imaging

Nuclear Medicine and Biology 29 (2002) 665– 669

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Synthesis and biodistribution of a new 99mTc nitrido complex for cerebral imaging JunBo Zhang, XueBin Wang*, ChunYun Li Department of Chemistry, Beijing Normal University, Beijing, 100875, Peoples Republic of China Received 20 September 2001; received in revised form 29 October 2001; accepted 4 March 2002

Abstract The bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(IPEDTC)2] (IPEDTC: N-isopentyl dithiocarbamato) has been synthesized by the reduction of 99mTcO4- into [99mTcN]2⫹ with stannous chloride in the presence of succinic dihydrazide and propylenediamine tetraacetic acid, followed by the addition of the sodium salt of N-isopentyl dithiocarbamate. The radiochemical purity of the complex was over 90% as measured by thin layer chromatography. In vitro studies showed that the complex possessed good stability under physiological conditions. Its partition coefficient indicated that it was a lipophilic complex. The electrophoresis results showed the complex was neutral. Biodistribution in mice showed that the complex accumulated in brain with high uptake and good retention. The brain uptake (ID%/g) was 2.22, 2.06 and 2.45 and the brain/blood ratio was 1.01, 2.34 and 3.22 at 5, 30 and 60 min post-injection, respectively. These results for the complex suggested that it could be a potential brain perfusion imaging agent. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Technetium-99m; [99mTcN]2⫹ core; Neutral; Lipophilic; Biodistribution; Brain uptake

1. Introduction In recent years, a number of complexes of 99mTc containing the monoxo[Tc⫽O]3⫹ and dioxo[O⫽Tc⫽O]⫹ cores have proved useful in diagnostic nuclear medicine as perfusion imaging agents, clearly demonstrating the wide role of technetium(V) chemistry. The [Tc⬅N]2⫹ core constitutes a group isoelectronic with [Tc⫽O]3⫹ and [O⫽Tc⫽O]⫹. The nitrido N3- ligand is considered to be the strongest ␲ donor known, and may act as a terminal or bridging atom which stabilizes the metal in high oxidation states. The preparation of 99mTc nitrido complexes at the no-carrier-added level was first reported by Baldas and Bonnyman [3]. This procedure is based on the reaction of 99m TcO4- with excess NaN3, in the presence of concentrated HCl, at reflux temperature. This method suffers from many limitations for its application to nuclear medicine. Lately, the preparation of 99mTc nitrido complexes at the tracer level and in sterile and pyrogen-free conditions has been extensively investigated [8]. So far, a number of 99mTcN * Corresponding author. Tel.: ⫹8610-62208126; fax: ⫹861062205562. E-mail address: [email protected] (X.B. Wang).

tracer agents with promising biological characteristics have been developed [9]. One such tracer agent is the bis(Nethoxy, N-ethyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(NOEt)2], which exhibits high myocardial uptake and significant redistribution behavior in various animal models and in humans [5]. In order to extend the investigation of the biological properties of the class of technetium-99m nitrido complexes, we report here the synthesis and biodistribution of the bis(Nisopentyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(IPEDTC)2](IPEDTC:N-isopentyl dithiocarbamato) as a potential cerebral imaging agent. It was found that the complex exhibited significant brain localization and good retention in mice, suggesting it could be potentially useful as a cerebral imaging agent.

2. Materials and methods Succinic dihydrazide (SDH), propylenediamine tetraacetic acid (PDTA) and stannous chloride dihydrate were purchased from Aldrich Chemical Company, USA. N-methyl, S-methyl dithiocarbazate (DTCZ) was synthesized as reported previously [1]. The sodium salt of N-isopentyl di-

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thiocarbamate was prepared by reacting the isopentylamine with an equivalent amount of carbon disulfide in NaOH solution according to the literature [9]. A 99Mo/99mTc generator was obtained from the China Institute of Atomic Energy (CIAE). All other chemicals were of reagent grade and were used without further purification. 2.1. Preparation of the bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(IPEDTC)2] The preparation of the complex was carried out by the following two methods: Method 1: 1 ml of saline containing [99mTcO4]- (15 MBq) was added to a kit containing 0.05 mg of stannous chloride dihydrate, 5.0 mg of succinic dihydrazide (SDH), and 5.0 mg of propylenediamine tetraacetic acid (PDTA). The mixture was kept at room temperature for 15 min. Successively, 4.0 mg of the sodium salt of N-isopentyl dithiocarbamate dissolved in 1.0 ml of water was added and the reaction was allowed to proceed for 10 min at room temperature (25°C). The pH of the final solution was approximately 7⬃8. Method 2: 1 ml of saline containing [99mTcO4]- (15 MBq) was added to a vial containing 0.10 mg of stannous chloride dihydrate, 10.0 mg of propylenediamine tetraacetic acid (PDTA) and 1.0 mg of N-methyl, S-methyl dithiocarbazate (DTCZ) in freeze-dried form. The resulting solution was heated at 100 °C for 15 min and then cooled to room temperature. 1 ml of a water solution containing 4.0 mg of the sodium salt of N-isopentyl dithiocarbamate was then added. At last, the reaction vial was left to stand for 10 min at room temperature (25°C). The pH of the final solution was about 7⬃8. The radiochemical purity(RCP) of the product was evaluated by thin layer chromatography(TLC) and ranged from 90%–99%. The TLC was performed on a polyamide strip and eluted with saline and CH2Cl2: CH3OH⫽9:1 (V/V). 2.2. Determination of the partition coefficient for the complex The partition coefficient of the complex was determined by mixing the complex with an equal volume of 1-octanol and phosphate buffer (0.025 M, pH 7.0 and pH 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and then centrifuged at 5000 r/min for 5 min. From each phase, a 0.1 ml aliquot was pipetted and counted in a well ␥-counter. The measurement was repeated three times. Care was taken to avoid cross contamination between the phases. The partition coefficient, P, was calculated using the following equation: P ⫽ (cpm in octanol ⫺ cpm in background)/ (cpm in buffer ⫺ cpm in background)

Usually the final partition coefficient value was expressed as logP. 2.3. Stability study The stability of the complex in the reaction mixture was assayed by measuring the RCP at different times after preparation at room temperature (25°C). For in vitro stability in phosphate buffered saline (pH 7.4) and human serum, 100 ␮l of the radiolabelled complex were mixed with 2 ml each of phosphate buffered saline (pH 7.4) and human serum (1 mg/ml), respectively. TLC was carried out to assess the RCP after incubating at 37°C for different time intervals. 2.4. Paper electrophoresis 1 ␮l samples were spotted on Whatman 1 chromatography paper, saturated with 0.05 M pH 7.4 phosphate buffer, in an electrophoresis bath. 150V were applied across 12 cm of the strip for 1.5 hr. The strips were dried and the distribution of radioactivity on the strip was determined. 2.5. Biodistribution study A solution of the bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(IPEDTC)2] (0.1 ml, 740 KBq) was administered via a tail vein to Kunming mice(18⬃20 g) and the injected radioactivity measured with a well-type NaI(Tl) detector. The mice were sacrificed at 5, 30 and 60 min post-injection. The organs of interest and the blood were collected, weighed and measured for the radioactivity. All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation.

3. Results and discussion 3.1. Synthesis of the bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(IPEDTC)2] The preparation route for the bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex is as follows: [99mTcO4]⫺⫹H2NNHCOCH2CH2CONHNH2⫹SnCl2.2H2O ⫹(HO2CCH2)2NCH(CH3)CH2N(CH2CO2H)2 99m 3[99mTc§N]2⫹ Tc§N]2⫹ int ; [ int ⫹i⫺C5H11NHCS2Na

共1兲

399mTcN(i⫺C5H11NHCS2)2 [99mTcO4]⫺⫹H2NN(CH3)C(¢S)SCH3⫹SnCl2.2H2O ⫹(HO2CCH2)2NCH(CH3)CH2N(CH2CO2H)2

99m 3[99mTc§N]2⫹ Tc§N]2⫹ int ; [ int ⫹i⫺C5H11NHCS2Na

399mTcN(i⫺C5H11NHCS2)2

共2兲

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Table 1 (Rf values for some selected complexes)

saline CH2Cl2:CH3OH ⫽ 9:1(V/V)

[99mTcO4]⫺

99m

0.1 0.1

0.1 0.1

TcO2䡠nH2O

2⫹ [99mTc⬅N]int

[99mTcN(IPEDTC)2]

0.7⬃1.0 0.1

0.1 0.9⬃1.0

The bis(N-isopentyl dithiocarbamato) nitrido technetium99m complex could be prepared by using two alternative freeze-dried kit formulations, which differ in the choice of the donor of nitride nitrogen atoms (N3-). Succinic dihydrazide (H2NNHCOCH2CH2CONHNH2) and N-methyl, S-methyl dithiocarbazate (H2NN(CH3)C(⫽S)SCH3) all could play the role of an efficient donor of nitride nitrogen atoms(N3-). N-methyl, S-methyl dithiocarbazate (H2NN(CH3)C(⫽S)SCH3) was nearly insoluble in water at room temperature and needed to be heated to 100 °C to yield nitride nitrogen atoms (N3-) and this preparation required heating, whereas succinic dihydrazide (H2NNHCOCH2CH2CONHNH2) was able to afford nitride nitrogen atoms (N3-) at room temperature. At present, Method 1 represents the best kit formulation for preparing technetium-99m nitrido radiopharmaceuticals suitable for use in nuclear medicine. In the reaction, SnCl2䡠2H2O behaves as a reducing agent. The presence of propylenediamine tetraacetic acid ((HO2CCH2)2NCH(CH3)CH2N(CH2CO2H)2) is required in order to prevent precipitation of Sn2⫹ in the form of insoluble tin salts. The method is based on the reaction of [99mTcO4]- with succinic dihydrazide (SDH) or N-methyl, S-methyl dithiocarbazate (DTCZ) in the presence of stannous chloride as reducing agent to form a technetium-99m nitrido intermediate. The [99mTc⬅N]2⫹ is a suitable substrate for the substitution reaction with the sodium salt of N-isopentyl dithiocarbamate at room temperature to give the final complex bis(N-isopentyl dithiocarbamato) nitrido technetium-99m [99mTcN(IPEDTC)2]. Because the sodium salt of N-isopentyl dithiocarbamate is a weak base, it is not stable under acidic conditions. Although [99mTc⬅N]2⫹ can be prepared both under acidic conditions and under neutral conditions [9], the pH of the [99mTc⬅N]2⫹ solution should be adjusted to approximately 7. It is also a suitable pH for clinical use. The RCP of the complex was routinely checked by TLC. Rf values for some selected complexes are shown in Table 1 (Polyamide strip). The mean RCP of the product was 96 ⫾ 3% immediately after the preparation.

Bis(diethyldithiocarbamato) nitrido technetium-99m complex [99mTcN(DDC)2] is neutral and has a square pyramidal geometry with an apical Tc⬅N bond and two monoanionic dithiocarbamate ligands spanning the four positions in the basal plane through the four sulfur atoms [2]. Because the sodium salt of N-isopentyl dithiocarbamate and the sodium salt of diethyldithiocarbamate all belong to the dithiocarbamate ligands, it seems reasonable to presume that the structure of the bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex[99mTcN(IPEDTC)2] is similar to that of the bis(diethyldithiocarbamato) nitrido technetium-99m complex[99mTcN(DDC)2]. The proposed structure of [99mTcN(IPEDTC)2] is shown in Fig. 1. Its structure needs to be further investigated.

Fig. 1. The proposed structure of the [99mTcN(IPEDTC)2] complex.

Fig. 2. The stability of the [99mTcN(IPEDTC)2] complex.

3.2. Partition coefficient (logP) of the complex The partition coefficient (logP) of the complex was 1.04 and 0.98 at pH 7.0 and pH 7.4, respectively, showing no great difference between pH 7.0 and pH 7.4. The logP of the complex is within the range of values quoted for lipophilicity (0.9 –2.5) that are suitable for crossing the blood brain barrier (BBB). Due to its high lipophilicity, the complex is easily adsorbed on the walls of the vials and syringes. In order to avoid this adsorption, ␥-cyclodextrin or Tween 80 were added to the final solution. As compared with 99mTcd,l-HMPAO [7], the logP of the [99mTcN(IPEDTC)2] complex is less than that of the 99mTc-d,l-HMPAO (logP⫽1.20).

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Table 2 Biodistribution in mice of [99mTcN(IPEDTC)2] (x ⫾ s, n ⫽ 3) (ID %/g)

Heart Liver Lung Kidney Brain Blood Brain/Blood

5 min

30 min

60 min

15.70 ⫾ 2.45 37.31 ⫾ 2.05 13.10 ⫾ 2.37 13.30 ⫾ 0.68 2.22 ⫾ 0.28 2.20 ⫾ 0.28 1.01

3.36 ⫾ 0.68 35.87 ⫾ 5.55 4.22 ⫾ 0.88 5.70 ⫾ 0.44 2.06 ⫾ 0.24 0.88 ⫾ 0.01 2.34

3.27 ⫾ 0.82 36.24 ⫾ 3.48 4.89 ⫾ 0.62 5.56 ⫾ 0.80 2.45 ⫾ 0.16 0.76 ⫾ 0.06 3.22

3.3. Stability of the complex In vitro experiments showed that the RCP of the complex did not appreciably change with time (Fig. 2), suggesting it possesses a great stability under physiological conditions. The stability of the [99mTcN(IPEDTC)2] complex is superior to that of the 99mTc-d,l-HMPAO [7] (It is decomposed at room temperture at 1 hr after preparation). 3.4. Paper electrophoresis The paper electrophoresis pattern of the [99mTcN (IPEDTC)2] complex shows that the [99mTcN(IPEDTC)2] complex remains at the point of spotting (percentage of the radioactivity: 99.11%), suggesting that it is a neutral complex. 3.5. Biodistribution of the complex in mice Biological distribution results in mice for the [99mTcN (IPEDTC)2] complex are shown in Table 2. Results of biodistribution of [99mTcN(IPEDTC)2], 99mTc-d,l-HMPAO [4] are shown in Table 3 for comparison. From Table 2, the complex is seen to have a significant brain uptake and good retention. The complex also exhibited high initial myocardial uptake, but the washout from the heart was rapid. The blood uptake was low and its clearance was fast so that the brain/blood ratio was high, 1.01, 2.34, 3.22 at 5, 30 and 60 min post-injection, respectively. The liver uptake was significant and remained high at 60 min post-injection, suggesting the hepatobiliary system is the major route of excretion of the administered radioactivity. From Table 3, although the brain uptake and retention of the complex [99mTcN(IPEDTC)2] is not as good as that of Table 3 Comparison of biodistribution of [99mTcN(IPEDTC)2] and

99m

the complex 99mTc-d,l-HMPAO, the blood uptake of the former is lower than that of the latter. The brain/blood ratio of the complex [99mTcN(IPEDTC)2] is superior to that of the complex 99mTc-d,l-HMPAO. The biodistribution results of [99mTcN(IPEDTC)2] in mice demonstrate that the brain is one of the principal target organs for this compound. This finding is somewhat surprising when compared to the biological properties of [99mTcN(NOET)2]. As reported previously [12], it was found that the latter complex exhibits high myocardial uptake and less brain uptake. These two species have identical square pyramidal structures. In vitro experiments revealed that they are highly stable and their chemical identities were not altered after incubation with phosphate buffered saline (pH 7.4) and human serum at 37°C. Therefore, it seems difficult to try to explain the different biological behavior of these two species on the basis of their minor structural differences. We think that the hydrogen atom bonded to the nitrogen atom of [99mTcN(IPEDTC)2] perhaps plays an important role in brain uptake and retention. The view is partially supported by our recent rearch work [10,11]. We have synthesized two complexes—[99mTcN(CHDTC)2] (CHDTC: N-cyclohexyl dithiocarbamato) and [99mTcN (DCHDTC)2] (DCHDTC: dicyclohexyl dithiocarbamato), which are different in lateral group bound to the ⬎NCS2 moiety. Different biodistribution results in mice of these two complexes were found. The former has a high brain uptake and good brain/blood ratio (ID%/g: 5.91; brain/ blood ratio: 2.10 at 60 min post-injection), while the latter has much less brain uptake and a poor brain/blood ratio (ID%/g: 0.41, brain/blood ratio: 0.03 at 60 min post-injection). The only difference in these two complexes is that [99mTcN(CHDTC)2] has a hydrogen atom bound to the ⬎NCS2 moiety, suggesting the hydrogen atom bound to the nitrogen atom of the dithiocarbamate ligand possibly affects the brain uptake and retention. This presumption may explain why the analogous compounds reported by Pasqualini [9] show less brain uptake. As we know, 99mTc-d,l-HMPAO is retained in the brain because it undergoes reactions to generate more polar or charged species that cannot diffuse from the brain. What is the mechanism of brain retention for [99mTcN(IPEDTC)2]? One possibility is that [99mTcN(IPEDTC)2] may undergo some reaction inside the brain which alters its chemical identity. In order to test this possibility, we extracted the activity from the brain tissue, after injection of [99mTcN

Tc-d,1-HMPAO in mice

[99mTcN(IPEDTC)2] t/min brain blood brain/blood

5 2.22 ⫾ 0.28 2.20 ⫾ 0.28 1.01

99m

30 2.06 ⫾ 0.24 0.88 ⫾ 0.01 2.34

60 2.45 ⫾ 0.16 0.76 ⫾ 0.06 3.22

Tc-d,1-HMPAO

5 4.00 ⫾ 1.46 6.43 ⫾ 1.17 0.62

30 4.17 ⫾ 0.24 3.90 ⫾ 0.87 1.07

60 4.13 ⫾ 1.13 3.23 ⫾ 1.12 1.28

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(IPEDTC)2] in mice, using the following procedure: the brain tissue was homogenized in a CH2Cl2:CH3OH⫽1: 1(V/V) mixture and centrifuged to remove particulates. The supernatant was taken to dryness and redissolved in a small amount of saline. Then the TLC and paper electrophoresis were performed by using the same methods as previously described. The TLC showed no change in the Rf value. Paper electrophoresis indicated that this tracer remained neutral after localization into the brain cells. From these facts, we may conclude that [99mTcN(IPEDTC)2] does not undergo chemical modification in the brain after injection. This is similar to that of [99mTcN(NOET)2]. The uptake of [99mTcN(NOET)2] in the heart is not followed by chemical modifications of the tracer [6], suggesting [99mTc⬅N]2⫹ core in 99mTc nitrido dithiocarbamate complexes displays a remarkable chemical and biochemical inertness. Maybe [99mTcN(IPEDTC)2] has a high binding affinity to some components in the brain, thus retaining the complex in the brain. This needs to be further investigated.

[5]

4. Conclusion

[8]

The bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(IPEDTC)2] has been successfully prepared under sterile and apyrogen conditions through an efficient method, which can be easily used for the preparation of a radiopharmaceutical through a freezedried kit formulation. The significant brain localization, good retention and high brain/blood ratio of the complex in mice were favorable properties, justifying further investigations in animals and humans. References [1] M.A. Ali, S.E. Livingstone, D.J. Phillips, Metals chelates of dithiocarbazic acid and its derivatives. III. complexes of the tridentate Schiff

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