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[99mTc]4-Dithiocarbamato-N-benzylpiperidine

[[99mTc]N(PNP)Pip-DTC]+

, MSc., , PhD, , MVSc, , PhD, , PhD, , PhD, and , PhD.

Author Information

Created: ; Last Update: May 14, 2008.

Chemical name:[99mTc]4-Dithiocarbamato-N-benzylpiperidine
Image NPNP.jpg
Abbreviated name:[[99mTc]N(PNP)Pip-DTC]+
Synonym:
Agent Category:Compound
Target:Sigma receptors
Target Category:Receptor-ligand binding
Method of detection:Single-photon emission computed tomography (SPECT), planar gamma imaging
Source of Signal/Contrast:99mTc
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Proposed structure of [[99mTc]N(PNP)Pip-DTC]+.

Background

[PubMed]

Sigma receptors belong to a unique family of proteins that are different from opioid and N-methyl-D-aspartate (NMDA) phencyclidine receptors (1, 2). They are known to be involved in several disorders of the central nervous system such as schizophrenia, depression, and dementia (3-5). These receptors are classified into two subtypes, sigma-1 and sigma-2. Of the two subtypes, only the sigma-1 receptors are well studied and characterized. It has been demonstrated that [3H](+)-pentazocine selectively labels only the sigma-1 sites, whereas di-o-tolyl guanidine (DTG) labels both the sigma-1 and sigma-2 receptors (6, 7). Because sigma receptors are expressed in high density in different cancerous tumors such as breast tumors, melanomas, non-small cell lung carcinomas, prostate tumors, and tumors of neural origin, they are an attractive target for the development of radiotracers that can be used in nuclear medicine for imaging and cancer therapy (8).

Many ligands, including various derivatives of piperidine and piperazine (9, 10), benzamides (11), and alkylamines (12), have been reported to have a high affinity for the sigma receptors. The presence of a nitrogen atom was suggested to be an essential pharmacophoric element for the binding of phenylalkylpiperidines and phenylalkylpiperazines to the sigma receptors (13). Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have shown that, when a tumor is found in the brain, sigma receptors are also present. In this respect, a variety of ligands labeled with 11C and 18F (14, 15), 99mTc (16), or 123I (17) were reported to selectively bind the sigma receptors. It is important to use radiolabeled ligands with high specific activity to study receptors, and this can be achieved by incorporating novel 99mTc-based cores such as [99mTc(CO)3(H2O)3]+ and [[99mTc]N]2+. The donor array of the chelating moiety is favorable for complexation with the [[99mTc]N]2+ core. However, due to steric constraints, formation of a four-way coordinated [[99mTc]N]2+ complex of the [[99mTc]N(BM)2] type requires coordination of two molecules of a biologically avid substrate (BM), where each molecule acts as a bidentate chelator to the [[99mTc]N]2+ core. This drawback is overcome by using the metal synthon [[99mTc]N(PXP)]2+ (where X = N, S, O) for the preparation of the receptor binding complexes. In such molecules the PXP ligand acts as a bidentate ligand that requires only a single moiety of the biomolecule for coordination and leads to the formation of an asymmetrical ([[99mTc]N(DTC)PXP]+) type complex (18). As a consequence, the radiolabeled complex of the dithiocarbamate derivative of 4-amino-N-benzylpiperidine, [[99mTc]N(PNP)Pip-DTC]+, was developed for the imaging of sigma receptors in tumors, and its in vivo activity was investigated in tumor-bearing mice.

Synthesis

[PubMed]

4-Dithiocarbamato-N-benzylpiperidine (Pip-DTC)

Carbon disulphide (15 μl, 0.25 mmol) was added dropwise to a mixture of 4-amino-N-benzylpiperidine (50 mg, 0.26 mmol) and sodium hydroxide (4.8 mg, 0.12 mmol) dissolved in diethyl ether (19). A white precipitate was obtained immediately, and the reaction mixture was stirred at room temperature overnight. After 24 h, the solvent was removed under reduced pressure, and the product was purified on a silica gel column with ammonia:methanol (5:95) as the eluting solvent. 1H-NMR (sigma ppm, CDCl3):7.4 (s, 5H, phenyl) 4.18 (s, 2H, benzylic) 2.21 (m, 1H, piperidine) 2.09 (m, 4H, piperidine) 1.5 (m, 4H, piperidine); 13C-NMR (300 MHz, CD3OD) 31.78 (2C, piperidine) 35.58 (2C, piperidine) 53.43 (C, piperidine) 64.03 (C, benzylic) 128.47 (C, phenyl) 129.36 (2C, phenyl) 130.82 (2C, phenyl) 138.58 (C, phenyl) 213.6 (C, NHCS2); MS (ESI): mass calculated for C13H17N2S2Na; 288, found 289 (M+1).

Preparation of the complex with the proposed structure of [[99mTc]N(PNP)Pip-DTC]+

The metal synthon ([[99mTc]N(PNP)]2+), which is required for radiolabeling, was prepared by addition of PNP ligand solution (0.1 ml, 1 mg) in ethanol to the preformed [[99mTc]N]2+ core (19). A high-performance liquid chromatographic (HPLC) analysis of the synthon showed two radioactive peaks at 11 min and 18.6 min with yields of 6% and 94%, respectively (Fig. 1; see Supplemental Section below). The complex was prepared by addition of 50 μL of 4-dithiocarbamato-N-benzylpiperidine (50 μg, 0.2 mM) to the preformed [[99mTc]N(PNP)]2+ core and heating the solution at 80°C for 40 minutes. The HPLC retention time of the complex was 17 min (1,020 s) (Fig. 2; see Supplemental Section below), and the log P value was 1.49 ± 0.13. The specific activity of the complex was >425 GBq/mmol (11.5 Ci/mmol) with >95% radiochemical yield and purity. When the complex was incubated with histidine and cysteine, there was no change in the HPLC pattern, which indicated that the complex was stable in terms of transchelation by other challenging ligands present in vivo.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Cell uptake studies were performed with melanoma and fibrosarcoma cell lines (19). The percentage binding of the [[99mTc]N(PNP)Pip-DTC]+ complex in melanoma cells and in fibrosarcoma cells were observed to be similar at a high concentration of the ligand, although they differed at low and medium concentrations (Tables 1 and 2).

Table 1. In vitro cell binding studies of [[99mTc]N(PNP)Pip-DTC]+ complex in fibrosarcoma and melanoma cells. (Data are expressed as mean value ± SD; n = 3 determinations.)

Tracer Concentration
(nmol)
% Binding
FibrosarcomaMelanoma
0.345.9 ± 0.35.2 ± 0.4
0.174.5 ± 0.16 ± 0.6
0.0344 ± 0.17.2 ± 0.5

Table 2. Inhibition studies for [[99mTc]N(PNP)Pip-DTC]+ in fibrosarcoma and melanoma cells using (+)pentazocine as a sigma receptor-specific ligand. (Data are expressed as mean value ± SD; n = 3 determinations.)

Concentration of
(+)-Pentazocine
(μg)
% Inhibition
FibrosarcomaMelanoma
10078 ± 2.591 ± 3.2
5071 ± 1.945 ± 1.1
1060 ± 2.234 ± 0.8

When melanoma cells were incubated with 100 μg (+)-pentazocine, 91% inhibition was observed; inhibition decreased to 45% when the concentration of the drug was reduced to 50 μg. In fibrosarcoma cells, 100 μg (+)-pentazocine led to 78% inhibition; inhibition decreased to 71% at 50 μg concentration (Table 2).

Animal Studies

Rodents

[PubMed]

In vivo biodistribution and receptor blocking studies

Biodistribution studies were performed with Swiss mice bearing fibrosarcoma tumors and with C57BL6 mice bearing melanoma tumors (19). Cell lines were obtained from the National Center for Cell Studies in Pune, India. Tumors were developed by subcutaneous injection of cells (~106 cells) into the animals. Three animals per time point were injected with the radiolabeled complex for biodistribution studies. Receptor blocking studies were carried out by intraperitoneal injection of 25 μg (+)-pentazocine in mice 30 minutes before administration of radiolabeled complex. Animals were killed 3 h after injection, and % radioactivity associated with each organ was estimated.

After the injection of [[99mTc]N(PNP)Pip-DTC]+ the radioactivity cleared rapidly from blood. High uptake of radioactivity was observed in the intestine (48 ± 1.4% injected dose/gram tissue (% ID/g) at 3 h post-injection (p.i.)) (Table 3).

Table 3. Biodistribution of radioactivity after the injection of [[99mTc]N(PNP)Pip-DTC]+ in mice bearing melanoma and fibrosarcoma tumors and blocking studies using (+)-pentazocine (% ID/g of organ (mean ± SD); n = 3 animals.)

Organs3 hPentazocine 3 h24 h
Liver9.7 ± 1.16.2 ± 1.26.5 ± 1.3
Blood0.5 ± 0.090.3 ± 0.010.13 ± 0.01
Int +GB48 ± 1.438 ± 2.639 ± 5.4
Kidney14 ± 28.3 ± 1.27.5 ± 1.7
Heart5.1 ± 0.32.9 ± 0.24.6 ± 0.8
Lungs1.7 ± 0.30.8 ± 0.021.4 ± 0.3
Muscles1.8 ± 0.41 ± 0.030.4 ± 0.02
Spleen1.3 ± 0.20.25 ± 0.021.1 ± 0.1
Melanoma Tumor1.0 ± 0.20.6 ± 0.10.8 ± 0.1
Fibrosarcoma Tumor1.9 ± 0.31.1 ± 0.21.4 ± 0.4

and a significant amount of radioactivity was excreted by the hepatobiliary system, which may be attributed to the high lipophilicity of [[99mTc]N(PNP)Pip-DTC]+. The uptake values in fibrosarcoma and melanoma tumors were 1.9 ± 0.3% ID/g and 1.0 ± 0.2% ID/g at 2 h p.i. and 1.4 ± 0.4% ID/g and 0.8 ± 0.1% ID/g at 24 h p.i., respectively. The tumor/muscle ratio in mice bearing the melanoma tumors increased from 0.55 at 3 h p.i. to 2 after 24 h p.i., whereas the tumor/blood ratio increased from 2 to 6.1 after 24 h p.i. In mice bearing the fibrosarcoma tumors, the ratios were more pronounced, and the tumor/muscle ratio increased from 1.05 at 3 h p.i. to 3.5 after 24 h p.i. The tumor/blood ratio increased in these animals from 3.8 at 3 h p.i. to 10.7 after 24 h p.i. (Table 4).

Table 4. Tumor/muscle and tumor/blood ratios of [[99mTc]N(PNP)Pip-DTC]+ complex at 3 h and 24 h p.i. and % inhibition using (+)-pentazocine in mice bearing melanoma and fibrosarcoma tumors.

TumorTumor/MuscleTumor/Blood% Inhibition (3 h p.i.)
3 h p.i.24 h p.i.3 h p.i.24 h p.i.
Melanoma0.55226.140
Fibrosarcoma1.053.53.810.742

The high tumor/blood ratio indicated that the tumor uptake was not a result of blood pool activity in the tumor. In vivo receptor specificity was determined by performing blocking studies in which (+)-pentazocine was used for the saturation of the receptor sites. The radioactive uptake of 1.0 ± 0.2% ID/g at 3 h p.i. in animals bearing melanoma tumors decreased to 0.6 ± 0.1% ID/g with pre-administration of (+)-pentazocine (Table 4). In animals bearing fibrosarcoma tumors, the uptake was 1.9 ± 0.3% ID/g at 3 h p.i., and this decreased to 1.1 ± 0.2% ID/g in blocking studies. The ~40% decrease in radioactivity observed in both tumor types indicated partial receptor specificity of the complexes under in vivo conditions.

Other Non-Primate Mammals

[PubMed]

No publications are currently available.

Non-Human Primates

[PubMed]

No publications are currently available.

Human Studies

[PubMed]

No publications are currently available.

Supplemental Information

[Disclaimers]

Figure 1

Figure 2

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    This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.

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