Development of 68 Ga-SCN-DOTA-Capsaicin as an Imaging Agent Targeting Apoptosis and Cell Cycle Arrest in Breast Cancer

Abstract

⁶⁸Ga-labeled capsaicin using a DOTA (1,4,7,10-tetraazocyclododecane-N,N′,N″,N′″-tetraacetic acid) derivative [⁶⁸Ga-SCN-Benzyl(Bn)-DOTA-capsaicin] was studied for the diagnosis of breast cancers, such as MCF-7 and SK-BR-3. The standard compound, ⁶⁹Ga-SCN-Bn-DOTA-capsaicin, was also prepared and characterized by spectroscopic analysis. The binding affinity of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was evaluated by using breast cancer cell lines (MCF-7, SK-BR-3) and colon cancer cell (CT-26); the biodistribution was carried out by using MCF-7-bearing nude mice, after which the positron emission tomography (PET) images were obtained at different time intervals (15–120 minutes). ⁶⁸Ga-SCN-Bn-DOTA-capsaicin showed a cellular uptake of 0.93% Injected Dose (ID) after 30 minutes of incubation, whereas ⁶⁸Ga-SCN-Bn-DOTA showed a lower uptake of 0.25% ID. The tumor-to-blood ID/g% ratios increased and were found to be 0.49, 0.22, and 0.77 for 15, 30, and 60 minutes, respectively. The small-animal PET study showed that the uptake of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was higher in the tumor regions even at 30 minutes after injection. These results suggest that ⁶⁸Ga-SCN-Bn-DOTA-capsaicin is a potential targeting agent for PET imaging of MCF-7.

Introduction

Breast cancer is one of the most common among other cancers in women because it undergoes uncontrolled growth and metastasizes to other organs.¹ Diagnosis of cancer in the early stages is important because the survival rate decreases with time.² Epithelial cells in the breast begin to grow and divide under the stimulation of female hormones, such as estrogen.³ Positron emission tomography (PET) has become an increasingly important technique for an evaluation of the position and size of a variety of cancer with high sensitivity and high resolution.^4,5 In general, ¹¹C and ¹⁸F produced by a cyclotron are the radionuclides used most widely for PET with a half-life of 20.39 and 109.8 minutes, respectively.^6,7

Recent interest has been focused on radio metals, such as ⁶⁸Ga, ⁴⁴Sc, ⁶⁴Cu, ⁶⁷Cu, and ⁸⁶Y because of their convenient labeling procedures and the stability of the radio metal-labeled compounds.⁸ Among the radiometals, positron-emitting ⁶⁸Ga and ⁴⁴Sc can be produced by ⁶⁸Ge/⁶⁸Ga and ⁴⁴Ti/⁴⁴Sc generator systems. The main advantages of the generator systems are the independence from a cyclotron/reactor and multi elutions per day of the daughter radio nuclide, which allows flexibility in its uses and applications.⁶⁸Ga has a half-life of 68 minutes and is formed from the decay of its mother nuclide ⁶⁸Ge with a half-life of 270 days.⁹⁶⁸Ga-labeled compounds have been studied widely because ⁶⁸Ga has a well-established chemistry and stability after chelation with a range of chelating agents, such as 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid [DOTA; log K_f (Ga-DOTA) = 21.3], 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA; log K_f (Ga-NOTA) = 31.0], diethylenetriaminepentacetic acid [DTPA; log K_f (Ga-DTPA) = 25.5], and 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid [TETA; log K_f (Ga-TETA) = 19.7]. Among these chelating agents, extensive research on DOTA^10

–13 and NOTA¹⁴ has been done. Other ⁶⁸Ga-labeled bifunctional chelating agents have also been reported, such as ⁶⁸Ga-SCN-DOTA-Benzamide,¹⁵ ⁶⁸Ga-DOTA-peptide,¹⁶ ⁶⁸Ga-DOTA-polyamido-amine dendrimer,¹⁷ ⁶⁸Ga-DOTA-TATE (⁶⁸Ga-N-(4,7,10-(tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetyl-d-Phe-c[Cys-d-Tyr-Trp-Lys-Thr-Cys]-Thr)),¹⁸ and ⁶⁸Ga-DOTA-NOC (⁶⁸Ga-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-Nal3-Octreotide).

Capsaicin is one of the widely used ingredients in the food and drug industries,¹⁹ and it is involved in sense, pain, and a host of chronic neuro-inflammatory conditions.^20,21 The anticancer activity of capsaicin has been intensively investigated in various types of cancer, including colon, bladder, prostate, and breast cancer, and it was confirmed that capsaicin has strong anticancer activity in vitro and a chemotherapeutic effect in in vivo tumor models.^22,23 Although the exact mechanism of anticancer activity of capsaicin is still not clear, many studies have reported that capsaicin has an effect on apoptosis, cell cycle arrest, and angiogenesis.^{24

–31} In particular, capsaicin has been reported to induce apoptosis and cell cycle arrest in diverse cancer cells and its activity seems to be related to TRPV1 and TRPV6, which are known to be capsaicin receptors.^{25,26,29,32,33} In breast cancer cells, capsaicin is known to efficiently inhibit the cellular progression and cell proliferation by inducing cell cycle arrest and apoptosis by modulation of EGFR and HER2 signaling in ER-positive and -negative cells,²⁸ which means that capsaicin could be a strong ligand targeting apoptosis and cell cycle arrest for the development of therapeutics and diagnostics against breast cancer.

Thus, based on the reported results on the anticancer effect of capsaicin, we hypothesized that radio metal-labeled capsaicin can be localized to tumor tissues and effectively visualize cancer in vivo with PET. This study could show some clues for understanding the mode of action of capsaicin against breast cancer, and it could also show the possibility of capsaicin as a ligand targeting apoptosis and cell cycle arrest in tumors. For this purpose, ⁶⁸Ga-labeled SCN-benzyl(Bn)-DOTA-capsaicin was prepared as a breast cancer target compound. This article reports the synthesis and preliminary biological evaluation of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin.

Materials and Methods

General

p-SCN-Bn-DOTA (8-Methyl-n-vanillyl-trans-6-nonenamide-1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid) was obtained from Futurechem (C₂₁H₂₇N₅O₇S, Korea). All other reagents were obtained from Sigma-Aldrich and were used without further purification. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a JEOL ECA-500 NMR spectrometer (500 MHz for ¹H and ¹³C; Jeol, Tokyo, Japan). The NMR spectrum was recorded in D₂O, and the chemical shifts were recognized in ppm relative to tetramethylsilane as a standard material. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was performed by using a UV detector (Wavelength, 254 nm) and Bioscan γ detector (Young-Lin, Inc., Korea & Bioscan, Chicago, USA). Both semi-preparative (Phenomenex Luna, C18, 10 mm × 250 mm) and analytical (Phenomenex Gemini C18, 4.6 mm × 250 mm) HPLC columns were used for analysis and purification of radiopharmaceuticals used in the studies. The ⁶⁸Ge/⁶⁸Ga generator was used with 0.05 N-HCl of eluent (Eckert & Ziegler, Berlin, Germany). The thin-layer chromatography-silica gel (ITLC-SG) plates were purchased from Merck (Darmstadt, Germany). CRC-15R calibrator (Capintec Instruments, USA) was used for radioactivity measurements. The ⁶⁸Ga activity was assayed by using a 1480 WIZARD-3 γ counter (Perkin Elmer, Waltham, MA, USA), whereas an in vivo study was performed by using a PET/X-ray instrument (Genesis 4, Perkin Elmer, USA).

Preparation of p-SCN-Bn-DOTA-capsaicin

A mixture of 4-(aminomethyl)-2-methoxyphenol hydrochloride (0.18 g, 10 mmol) and 6-aminohexanoic acid (10 mmol) in 1 mL dimethylformamide (DMF) containing TBTU(O-(benzotriazol-1-yl)-N,N,N′,N′,-tetramethyluronium tertrafluoroborate) (10 mmol) and DIEA(N,N-diisopropylethylamine) (10 mmol) was stirred for 5 hours at room temperature. The reaction mixture of 6-amino-N-(4-hydroxy-3-methoxybenzylhexanmide (1 mmol) and p-SCN-Bn-DOTA (1 mmol) was stirred in 1 mL DMF for 2 hours at room temperature. The solvent was evaporated under reduced pressure, and the resulting mixture was purified by semi-preparative HPLC (gradient 80% of H₂O for 4 minutes and 0%–100% MeCN for 10 minutes; flow rate, 4 mL/min; 240 nm; t_R, 20 minutes) to give p-SCN-Bn-DOTA-capsaicin as a pale yellow solid (50 mg, 99%) (Fig. 1).

FIG. 1.

Synthesis of ^68&69Ga-SCN-Bn-DOTA-capsaicin.

Preparation of ⁶⁹Ga-SCN-Bn-DOTA-capsaicin

The stable isotope reference compound, ⁶⁹Ga-SCN-Bn-DOTA-capsaicin was synthesized by reacting p-SCN-Bn-DOTA-capsaicin with GaCl₃ ⁺ (1 equation) in deionized water. The pH was adjusted to 4 with a 0.5 M sodium acetate buffer solution. The reaction mixture was stirred for 30 minutes at 100°C, and the crude compound was purified by RP-HPLC (gradient, 80% H₂O for 4 minutes and 20%–100% MeCN for 10 minutes; flow rate, 5 mL/min; 240 nm).

Preparation of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin

⁶⁸Ga (∼80 MBq) was eluted from ⁶⁸Ge/⁶⁸Ga generator with 0.05 N HCl. One milligram of p-SCN-Bn-DOTA-capsaicin was added to the HCl solution containing ⁶⁸Ga, and the pH of the solution was adjusted by adding 1 M sodium acetate buffer (pH 5) to give pH 4. The mixture was stirred for 10 minutes at 100°C.³⁴ The reaction mixture containing ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was injected in the RP-HPLC system (gradient, 70% H₂O for 5 minutes and 0%–100% MeCN for 10 minutes; flow rate, 4 mL/min; 240 nm). ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was confirmed by a coinjection of the authentic compound, ⁶⁹Ga-SCN-Bn-DOTA-capsaicin. This purified product was dried and dissolved in saline for the biological experiments.

Binding constant of gallium conjugate

The binding constant of p-SCN-Bn-DOTA with ⁶⁹Ga was measured according to the standard calibration curve method. The reaction is determinated by the on-rate constant [k_on] and the off-rate constant [k_off]. The binding constant can be calculated as: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm K } _a = \frac { k_ { on } } { k_ { off } } = \frac { [ HG ] } { [ H ] [ G ] } \end{align*} \end{document}

where [H], [G], and [HG] are the concentrations of the ⁶⁹Ga, p-SCN-Bn-DOTA-capsaicin and ⁶⁹Ga-SCN-Bn-DOTA-capsaicin complex, respectively.

Cell culture and tumor model

Human breast cancer cell MCF-7 and SK-BR-3 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Mouse colorectal cancer cell CT-26 was cultured in Dulbecco's modified Eagle's medium (DMEM) and supplemented with 10% FBS. All cancer cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO₂.^35,36 All animal studies were progressed according to a protocol approved by the Orient (Gyeonggi-do, Korea) and Korea Atomic Energy Research Institute. The mouse model consisted of female Balb/c nude mice (4–6 weeks old, body weight of 20–25 g). Human breast cancer MCF-7 cells xenograft models were injected by 5 × 10⁶ tumor cells into the left thigh. The tumors were cultured and grown 2 to 3 weeks before the micro animal-PET imaging experiments.

Partition coefficient (Log P)

The Log P was measured by mixing 1.85 MBq of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin with 1 mL of 1-octanol and 1 mL of phosphate buffer saline (PBS, pH 7.4) in a test tube. The test tube was agitated for 10 minutes at 25°C and centrifuged at 10,000 rpm for 5 minutes. Each aliquot, 200 μL of PBS and 200 μL of 1-octanol, was taken and counted on a γ counter in triplicate.³⁷ The log p-value was calculated by using the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \hbox { Log P } = \frac { Radioactivity \ in \ octanol } { { \rm Radioactivity \ pbs } } \end{align*} \end{document}

In vitro stability

A total of 370 kBq of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was incubated with 1.0 mL of human serum in a water bath at 37°C for 15, 30, 60, and 120 minutes, and then measured by radio-TLC; 0.1 M Na₂CO₃ and MeOH (1:1) were used as the solvents. All the data were collected in triplicate.

Cellular uptake of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin

The cellular uptake was measured with MCF-7, SK-BR-3, and CT-26 cells by using ⁶⁸Ga-SCN-Bn-DOTA-capsaicin and ⁶⁸Ga-SCN-Bn-DOTA. The cells were cultured in DMEM (Gibco Life Sciences) supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were incubated at 37°C in a 5% CO₂. The cells were sub-cultured in 12-well plates (1 × 10⁶ of MCF-7, SK-BR-3, and CT-26 cancer cells); incubated at 37°C for 15, 30, 60, and 120 minutes with 185 kBq/well of either ⁶⁸Ga-SCN-Bn-DOTA-capsaicin or ⁶⁸Ga-SCN-Bn-DOTA. After incubation, the 20 μL of supernatant was collected and the remainder of the media was discarded, after which cells were washed with 500 μL of cold PBS to remove the unbounded compound. For solubilization of the cell membrane, 200 μL of 0.1% sodium dodecyl sulfate was added to cell pellets.¹⁵ The radioactivity of the 20 μL of supernatant and 100 μL of cell suspension was determined by using a γ counter. Cellular uptake value of the ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was calculated and recorded as percentage of inoculation dose (%ID).

Biodistribution studies

Biodistribution was studied by measuring the radioactivity after injecting ⁶⁸Ga-SCN-Bn-DOTA-capsaicin in the mice. ⁶⁸Ga-SCN-Bn-DOTA-capsaicin (3.7 MBq) was injected intravenously via the tail vein in MCF-7-bearing mice. The mice organs were measured at 15, 30, 60, and 120 minutes after the injection in triplicate. A mouse blood sample was collected by heart puncture and organs were extracted with heart, lung, liver, spleen, stomach, intestine, pancreas, kidney, muscle, fat, bone, skin, tail, tumor, and brain, after which the radioactivity in these organs were measured by using the γ counter.

Animal-PET study

The mouse was anesthetized by ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (2.5 mg/kg). The MCF-7 xenograft mice were imaged at 15, 30, 60, and 120 minutes after the iv injection of 3.7 MBq of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin. The tumor blocking test of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was also done with MCF-7-bearing mice (18–20 g) treated with capsaicin, which presumably binds to TRPV 1. Capsaicin compound (3 mg/kg) was injected via tail vein, followed by an injection of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin after 10 minutes. The PET images were acquired after 60 minutes.

Results and Discussion

Chemistry

⁶⁹Ga-SCN-Bn-DOTA-capsaicin was prepared, as shown in Figure 1. The resulting reaction mixture was purified by RP-HPLC to give p-SCN-Bn-DOTA-capsaicin with ∼70% yield (Supplementary data available at www.liebertpub.com/cbr; Figs. S1, S2, and S3). However, purification of ⁶⁹Ga-SCN-Bn-DOTA-capsaicin was carried out by semi-preparative HPLC with ∼62% yield. The integrated chromatogram area on binding to DOTA was used to determine the binding constant of gallium for p-SCN-Bn-DOTA-capsaicin. A binding constant K_a = 196 M⁻¹ was observed for ⁶⁹Ga-SCN-Bn-DOTA-capsaicin. Characterization of each compound was confirmed by ¹H-NMR and mass spectrometry (MS).

p-SCN-Bn-DOTA-capsaicin; ¹H NMR (500 MHz, D₂O) δ = 7.24 (d, 2 H, J = 7.6 Hz), 7.14 (brs, 2 H), 6.90 (d, 1 H, J = 1.6 Hz), 6.82 (d, 1 H, J = 8.0 Hz), 6.76 (d, 1 H, J = 8.4 Hz), 4.24 (s, 2 H), 3.91 (brs, 4 H), 3.78 (s, 3 H), 3.72–2.49 (m, 23 H), 2.27 (t, 2 H, J = 7.2 Hz), 1.69–1.49 (m, 4 H), and 1.35–1.24 (m, 2 H); ¹³C NMR (100 MHz, D₂O) δ = 178.9, 175.7, 174.5, 173.7, 168.2, 147.2, 143.9, 135.7, 134.5, 130.4, 130.2, 125.4, 119.7, 119.1, 115.2, 111.2, 105.0, 55.6, 54.4, 53.9, 52.9, 52.1, 51.6, 50.0, 49.6, 49.1, 46.6, 44.4, 42.4, 35.6, 31.1, 28.0, 25.6, 25.2, and 0.8; MS (electrospray ionization): [M+H]⁺ = 818.6 (m/z), calc (molecular weight): 818.0 (C₃₈H₅₅N₇O₁₁S) (Figs. S4, S5, S6).

⁶⁹Ga-SCN-Bn-DOTA-capsaicin; ¹H-NMR (500MHz, D₂O) δ = 9.33 (d, 2 H, J = 8.1 Hz), 7.25 (brs, 2 H), 6.99 (d, 1 H, J = 1.6 Hz), 6.82–6.70 (d, 2 H, J = 8 Hz), 4.74–4.60 (m, 1 H), 4.13 (s, 2 H) 3.71 (brs, 4 H), 1.87–1.80 (m, 4 H), and 1.30–1.01 (m, 2 H); ¹³C (125 MHz, D₂O) δ = 205.9, 176.9, 147.4, 144.9, 143.9, 130.8, 120.1, 118.1, 115.5, 111.7, 55.9, 42.6, 35.6, 25.4, and 25.1; MS (liquid chromatography-MS): 887.2 of C₄₀H₆₆GaN₇O₁₁S (Figs. S7, S8).

Radiochemistry

To confirm the optimal labeling yield, the excess amount of p-SCN-Bn-DOTA-capsaicin was added in ⁶⁸GaCl₃ solution. ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was easily obtained by a one-step reaction.

The ⁶⁸Ga-SCN-Bn-DOTA-capsaicin obtained through the RP-HPLC with radiochemical yield was about 70% (Fig. 2), and radiochemical purity was measured by radio-TLC with 98% (Fig. S9).

FIG. 2.

HPLC chromatograms of ^68&69Ga-SCN-Bn-DOTA-capsaicin (Left: after synthesis, Right: after purification) RP-HPLC condition (gradient, 80% H₂O for 4 minutes and 20%–100% MeCN for 10 minutes; flow rate, 5 mL/min; 240 nm; tR, 13 minutes).

Partition coefficient and stability in human serum

The measured log p-value of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was −2.32 ± 0.18. The log p-value shows that this radiopharmaceutical is water soluble. As hydrophilic drugs are rapidly removed from the body and there is less concern about drug toxicity, ⁶⁸Ga-SCN-Bn-DOTA-capsaicin will also show rapid clearance from the body.

The stability of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin in human serum was indicated by radiochemical purity of the tracer. As a result, the stability of coordinated ⁶⁸Ga proved to be better than 93% until 120 minutes (Fig. 3). These results are expected to be stable from a pharmacokinetic perspective.

FIG. 3.

In vitro serum stability of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin with incubation time (15, 30, 60, and 120 minutes) at 37°C (n = 3).

In vitro cellular uptake

Cellular uptake of capsaicin was evaluated with ⁶⁸Ga-SCN-Bn-DOTA-capsaicin and ⁶⁸Ga-SCN-Bn-DOTA. Cellular uptake results have been shown that affinity of cancer cells is dependent on the level of TRPV 1 receptor by the various cells with MCF-7, SK-BR-3, and CT-26 cells.⁶⁸Ga-SCN-Bn-DOTA-capsaicin showed a relatively high cellular uptake of 0.93% ID as compared with that of 0.25% for ⁶⁸Ga-SCN-Bn-DOTA after 30 minutes of incubation for MCF-7. These results indicate a high affinity of capsaicin for breast cancer. However, cellular uptake showed a negligible value for both ⁶⁸Ga-SCN-Bn-DOTA-capsaicin and ⁶⁸Ga-SCN-Bn-DOTA (Fig. 4).

FIG. 4.

Cellular uptake of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin and ⁶⁸Ga-SCN-Bn-DOTA in MCF-7, SK-BR-3, and CT-26 cells. The photographs shown were of MCF-7 (positive control) and SK-BR-3, CT-26 (negative control).

Biodistribution studies

The time-dependent biodistribution studies carried out in MCF-7 xenografted balb/c mice exhibit the highest and rapid (∼15 minutes) uptake of ⁶⁸Ga-labeled molecule by tumor as compared with that with the other organs. The uptake also follows rapid clearance, except in the case of the intestine, which manifests a bit higher time for complete removal of ⁶⁸Ga (Fig. 5). Thirty minutes of the uptake kinetics after injection are compatible with the half-life of ⁶⁸Ga and the tumor-to-blood (T/B) ratio, in addition to the tumor-to-muscle ratio as seen from Table 1.

FIG. 5.

Biodistribution studies in MCF-7-bearing mice at 15, 30, 60, and 120 minutes after an iv injection of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin. The data are expressed as the percentage of administered activity (injection dose) per gram of tissue (%ID/g, n = 3).

Table 1.

Tumor-to-Blood and Tumor-to-Muscle Ratios (n = 3)

	T/B	T/M
15 minutes	0.49 ± 0.12	0.09 ± 0.03
30 minutes	0.22 ± 0.05	0.35 ± 0.16
60 minutes	0.77 ± 0.10	0.03 ± 0.01

Animal PET image

PET image intensities clearly predict the relatively highest uptake values of ⁶⁸Ga-DOTA-capsaicin in xenografted tumor as compared with other organs under study, and they were achieved in 30 minutes. In addition, tumor-specific uptake was reconfirmed by the blocking studies, as shown in Figure 6. The results determined by this study suggest the possible use of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin as a potential PET imaging probe for breast cancer.

FIG. 6.

Animal-positron emission tomography images of MCF-7 xenografted mice of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin.

Conclusions

This article describes the synthesis and in vitro and in vivo evaluation of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin for MCF-7 breast cancer cell imaging. ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was synthesized successfully with >70% radiochemical yield and >98% radiochemical purity. Selective uptake of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin was observed in ER-positive MCF-7 cells in vitro. The biodistribution results of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin showed higher tracer uptake in xenograft tumors, suggesting that capsaicin can be used as a potent ligand targeting tumor for the development of chemotherapeutics and diagnostics. However, tumor uptake seems to slow release. Advantages of DOTA-conjugated tracers are the increased T/B ratios, rapid renal excretion caused by higher hydrophilicity of DOTA and capsaicin, and high in vivo stability of ⁶⁸Ga-chelated SCN-Bn-DOTA-capsaicin. Micro-PET studies of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin with an MCF-7 model showed that this tracer was significantly accumulated in the tumor cells. However, blocking study results confirmed the low affinity for tumor. These biological results suggest that ⁶⁸Ga-SCN-Bn-DOTA-capsaicin could be a promising PET probe for MCF-7 through specific tumor uptake. As mentioned in the Introduction, the mode of action of capsaicin in targeting tumors has not been clearly determined but some reports said that pro-apopotic activity of capsaicin may be related to the signaling of TRPV1 and TRPV6, which are the receptors binding with capsaicin.^{25,26,29,32,33} Thus, we have a plan to develop a capsaicin derivative with a higher binding affinity to TRPV receptors to confirm whether the uptake of capsaicin in tumor is mediated via TRPV1 and TRPV6, and we will report about our results.

Footnotes

Acknowledgments

This research was supported by the Nuclear R&D Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (2015M2A2A4A02043265, 2015M2A2A4A02043268, and 2016M2C2A1937989) and Dongguk University Research Fund, Republic of Korea.

Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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Development of 68 Ga-SCN-DOTA-Capsaicin as an Imaging Agent Targeting Apoptosis and Cell Cycle Arrest in Breast Cancer

Abstract

Introduction

Materials and Methods

General

Preparation of p-SCN-Bn-DOTA-capsaicin

Preparation of 69Ga-SCN-Bn-DOTA-capsaicin

Preparation of 68Ga-SCN-Bn-DOTA-capsaicin

Binding constant of gallium conjugate

Cell culture and tumor model

Partition coefficient (Log P)

In vitro stability

Cellular uptake of 68Ga-SCN-Bn-DOTA-capsaicin

Biodistribution studies

Animal-PET study

Results and Discussion

Chemistry

Radiochemistry

Partition coefficient and stability in human serum

In vitro cellular uptake

Biodistribution studies

Animal PET image

Conclusions

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material

Preparation of ⁶⁹Ga-SCN-Bn-DOTA-capsaicin

Preparation of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin

Cellular uptake of ⁶⁸Ga-SCN-Bn-DOTA-capsaicin