In Vivo Evaluation of a Radiogallium-Labeled Bifunctional Radiopharmaceutical, Ga-DOTA-MN2, for Hypoxic Tumor Imaging

Abstract

On the basis of the findings obtained by X-ray crystallography of Ga-DOTA chelates and the drug design concept of bifunctional radiopharmaceuticals, we previously designed and synthesized a radiogallium-labeled DOTA chelate containing two metronidazole moieties, ⁶⁷Ga-DOTA-MN2, for hypoxic tumor imaging. As expected, ⁶⁷Ga-DOTA-MN2 exhibited high in vivo stability, although two carboxyl groups in the DOTA skeleton were conjugated with metronidazole moieties. In this study, we evaluated ^67/68Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors. ⁶⁷Ga-labeling of DOTA-MN2 with ⁶⁷GaCl₃ was achieved with high radiochemical yield (>85%) by 1-min of microwave irradiation (50 W). The pharmacokinetics of ⁶⁷Ga-DOTA-MN2 were examined in FM3A tumor-bearing mice, and compared with those of ⁶⁷Ga-DOTA-MN1 containing one metronidazole unit and ⁶⁷Ga-DOTA. Upon administration, ⁶⁷Ga-DOTA-MN2 exhibited higher accumulation in the implanted tumors than ⁶⁷Ga-DOTA. Tumor-to-blood ratios of ⁶⁷Ga-DOTA-MN2 were about two-fold higher than those of ⁶⁷Ga-DOTA-MN1. Autoradiographic analysis showed the heterogeneous localization of ⁶⁷Ga-DOTA-MN2 in the tumors, which corresponds to hypoxic regions suggested by well-established hypoxia marker drug, pimonidazole. Furthermore, in positron emission tomography (PET) study, the tumors of mice administered ⁶⁸Ga-labeled DOTA-MN2 were clearly imaged by small-animal PET at 1 h after administration. This study demonstrates the potential usefulness of ^67/68Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors and suggests that two functional moieties, such as metronidazole, can be conjugated to radiogallium-DOTA chelate without reducing the complex stability. The present findings provide useful information about the chemical design of radiogallium-labeled radiopharmaceuticals for PET and single photon emission computed tomography (SPECT) studies.

Tumor hypoxia results from an imbalance between oxygen supply and consumption, which is caused by abnormal structure and function of microvessels supplying the tumor, increased diffusion distances between the nutritive blood vessels and the tumor cells, and reduced O₂ transport capacity of the blood.^1,2) Tumor hypoxia has been associated with an aggressive tumor phenotype, poor response to radiotherapy and chemotherapy, and increased risk of invasion and metastasis of tumors.^3,4) Thus, non-invasive measurement of tumor hypoxia with positron emission tomography (PET) would have a distinct effect on characterizing tumor malignancy and determining a course of therapy.

Metronidazole, nitroimidazole antibiotic medication used for anaerobic bacteria and protozoa in particular, has a tendency to accumulate in the hypoxic regions and enhance the lethal effect of ionizing radiation for hypoxic tissues.⁵⁾ The reduction of the nitroimidazole (RNO₂) within cells proceeds in successive steps involving enzyme-mediated electron transfer via the free radical anion (RNO^•₂⁻), hydroxylamine (RNHOH) and terminating in the less-reactive amine (RNH₂). Unlike in normal cells, where rapid reoxidation of RNO₂⁻ to RNO₂ can occur, resulting in diffusion out of the tissue, further reduction in hypoxic tissue leads to preferential retention of the nitroimidazole within cells.^6,7)

We have recently reported a novel radiogallium-labeled metronidazole derivative, Ga-DOTA-MN2 (Fig. 1a), for hypoxia imaging, based on the concept of bifunctional radiopharmaceuticals.⁸⁾ Bifunctional radiopharmaceuticals have the recognition site of the molecular target and the chelation site for the radiometal independently in one molecule.^9–12) Ga-DOTA-MN2 containing two metronidazole moieties was designed in support of the previous crystallographic findings that the four nitrogens of the cyclen ring and two oxygens of the opposite carboxyl groups are coordinated to the gallium metal.¹³⁾ As expected, ⁶⁷Ga-DOTA-MN2 exhibited high stability in vitro and in vivo and significantly accumulated in the mouse fibrosarcomas (NFSa) implanted in mice with high target-to-nontarget ratios. These results warranted further evaluation of radiogallium-labeled DOTA-MN2 as a hypoxic tumor imaging agent.

Fig. 1. Chemical Structures of Bifunctional Radiopharmaceuticals, (a) DOTA-MN2 and (b) DOTA-MN1

In this study, we first attempted to improve the radiochemical yield of ⁶⁷Ga-DOTA-MN2 with microwave-assisted reaction. Next, to assess the effectiveness of introduction of two metronidazole moieties, the pharmacokinetics of ⁶⁷Ga-DOTA-MN2 in mice bearing mouse mammary carcinoma (FM3A) were compared with those of ⁶⁷Ga-DOTA-MN1 containing one metronidazole unit (Fig. 1b) and ⁶⁷Ga-DOTA. Moreover, we investigated the localization of ⁶⁷Ga-DOTA-MN2 in hypoxic regions of tumors by comparison of the ex vivo autoradiographic images with immunohistochemical images for a well-established hypoxia marker drug, pimonidazole. Finally, PET imaging studies were conducted using ⁶⁸Ga-labeled DOTA-MN2.

Materials and Methods

Synthesis of DOTA-MN1 and DOTA-MN2

Under argon, to a solution of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate)-10-acetic acid (DOTA-tris(t-Bu) ester, 50.0 mg, 0.087 mmol) in dry N,N-dimethylformamide was added N,N′-diisopropylcarbodiimide (33.0 mg, 0.26 mmol), 1-hydroxybenzotriazole (HOBt) (35.5 mg, 0.26 mmol), and 2-(2-methyl-5-nitroimidazol-1-yl)-ethylamine dihydrochloride (21.2 mg, 0.087 mmol) in an ice bath. After stirring for 48 h at room temperature, chloroform (30 mL) was added. The mixture was washed with distilled water, and then dried and evaporated in vacuo. The residue was purified with a Cosmosil 5C₁₈-PAQ column (10×250 mm, Nacalai Tesque Inc., Kyoto, Japan) at a flow rate of 2.4 mL/min with a mixture of water and acetonitrile containing 0.1% trifluoroacetic acid (TFA) (10–30% acetonitrile in 0–10 min and 30–50% acetonitrile in 10–60 min), and MN1-DOTA-tris(t-Bu) ester was obtained as a white solid (25.4%). ¹H-NMR (400 MHz, CDCl₃) δ ppm: 7.87 (1H, s), 4.47 (2H, br), 3.64 (2H, m), 3.23 (4H, br), 3.06 (4H, br), 2.82 (8H, br), 2.55 (3H, s), 2.35–2.17 (8H, br), 1.45 (18H, s), 1.26 (9H, s); IR (KBr) cm⁻¹: 3418, 2940, 1728, 1682, 1466; electrospray ionization (ESI) MS (m/z): 725.7 (M+H)⁺.

A mixture of MN1-DOTA-tris(t-Bu) ester and TFA was stirred at room temperature for 90 min, and then concentrated in vacuo. Purification by HPLC was carried out with a Cosmosil 5C₁₈-PAQ column (10×250 mm) eluted with water containing 0.1% TFA at a flow rate of 2.4 mL/min. The solvent was removed by evaporation in vacuo to afford DOTA-MN1 as a white solid. ¹H-NMR (400 MHz, D₂O) δ ppm: 7.90 (1H, m), 3.66 (4H, br), 3.38–3.23 (16H, m), 2.92 (8H, m), 2.35 (3H, s); IR (KBr) cm⁻¹: 3418, 3117, 2854, 1682, 1427; ESI-MS (m/z): 557.5 (M+H)⁺.

DOTA-MN2 was prepared as reported previously.⁸⁾ In brief, to a solution of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,7-bis(tert-butyl) ester was added triethylamine, 1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride, HOBt, and 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride in an ice bath. After stirring for 48 h at room temperature, chloroform was added. The mixture was washed with distilled water, and then dried and evaporated in vacuo. The residue was recrystallized from methanol to obtain di-tert-butyl 2,2′-[4,10-bis(2-{[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]amino}-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl]diacetate, followed by the reaction with conc. HCl at room temperature for 2 h. After concentrated in vacuo, the residue was dissolved in methanol, and dry ether was added to precipitate DOTA-MN2.

Synthesis of Ga-DOTA-MN1 and Ga-DOTA-MN2

The coordination with Ga to DOTA-MN1 and DOTA-MN2 was performed according to our previous report.⁸⁾ Ga-DOTA-MN1; ESI-MS (m/z): 623.3 (M+H)⁺, Ga-DOTA-MN2; ESI-MS (m/z): 775.3 (M+H)⁺.

⁶⁷Ga-Labeling

DOTA-MN2 (50 µg) was mixed with ⁶⁷GaCl₃ (FUJIFILM RI Pharma Co., Ltd., Tokyo, Japan) in 0.2 m ammonium acetate buffer (pH 5.8, 200 µL) followed by heating in an oil bath at 95°C or 140°C, or by microwave-assisted reaction (50 W, Initiator, Biotage AB, Uppsala, Sweden). The HPLC purification was carried out with a Cosmosil 5C₁₈-PAQ column (4.6×250 mm) eluted with water and acetonitrile containing 0.1% TFA (95 : 5) at a flow rate of 0.6 mL/min. ⁶⁷Ga-DOTA-MN1 was obtained by reacting DOTA-MN1 with ⁶⁷GaCl₃ at 95°C for 1 h, followed by HPLC purification with a Cosmosil 5C₁₈-PAQ (4.6×250 mm) eluted with water containing 0.1% TFA. ⁶⁷Ga-DOTA was also synthesized by reacting DOTA with ⁶⁷GaCl₃ at 95°C for 1 h. The radiochemical purity was determined by HPLC and cellulose acetate electrophoresis (CAE) run in veronal buffer (pH 8.6, I=0.06) at a constant current of 0.8 mA for 30 min as reported previously.⁸⁾

⁶⁸Ga-Labeling

⁶⁸Ga was eluted from a ⁶⁸Ge/⁶⁸Ga generator (Eckert & Ziegler, Berlin, German) to a vial with 6 mL of 0.1 n HCl. To the eluate was added 3.2 mL of conc. HCl and the solution was passed through an anion exchange column (Chromafix, Macherey-Nagel GmbH & Co., KG, Düren, Germany) at a flow rate of 1.5 mL/min at room temperature. The vial was washed with 2 mL of 4 n HCl, and the wash solution was also passed through the same column. The ⁶⁸Ga was then eluted from the column with deionized water (500 µL) and mixed with 1.2 m sodium acetate (250 µL). DOTA-MN2 (75 µg) in 0.2 m ammonium acetate buffer (pH 5.8, 100 µL) was added to 400 µL of ⁶⁸Ga solution (1.8 MBq). The mixture was heated in an oil bath at 120°C for 30 min, and purified in the same manner as in the case of ⁶⁷Ga-DOTA-MN2. ⁶⁸Ga-DOTA was prepared by reacting DOTA with ⁶⁸Ga-acetate at 95°C for 1 h. The radiochemical purity was determined by HPLC and CAE.

Animal Model

Animal experiments were conducted in accordance with our institutional guidelines and were approved by the Animal Care and Use Committee, Kyushu University, and the Kyoto University Animal Care Committee. NFSa mouse fibrosarcoma (1 mm×1 mm) or FM3A cells (5×10⁶ cells suspended in 100 µL of phosphate-buffered saline (PBS)) were inoculated subcutaneously into the right thigh or right flank of 5-week-old female C3H/He mice (Japan SLC, Inc., Hamamatsu, Japan). When tumors were approximately 0.7–1.0 cm in diameter, the animals were used for autoradiography, biodistribution study, and PET imaging.

Biodistribution Study

FM3A-implanted mice were divided into three groups (n=5–6) with approximately equal distributions of tumor sizes on the day before the study. Animals were fasted for 6 h before administration of radiolabeled compounds. ⁶⁷Ga-DOTA-MN2, ⁶⁷Ga-DOTA-MN1 or ⁶⁷Ga-DOTA (37 kBq/100 µL in PBS/mouse) was injected via a tail vein, and the biodistribution was monitored at 30 min and 1, 3, and 6 h postinjection. Organs of interest were excised, weighed, and the radioactivity counts were determined with a NaI well-type scintillation counter (ARC-370M, Hitachi Aloka Medical, Ltd., Tokyo, Japan) using the injected dose as a standard. Data were calculated as the percentage injected dose per gram of tissue (%ID/g).

Autoradiography and Immunostaining

FM3A-implanted mice were intraperitoneally injected with pimonidazole hydrochloride (60 mg/kg, 100 µL PBS) and 30 min later, ⁶⁷Ga-DOTA-MN2 or ⁶⁷Ga-DOTA (5.55 MBq/100 µL PBS) was injected intravenously. After another 1 h, the mice were sacrificed and the tumor tissues were excised. The excised tumors were frozen, and cut into 20-µm-thick sections with a cryomicrotome. The sections were thaw-mounted in silane-coated slides, which were then placed on a phosphor image plate (BAS-SR 2040, FUJIFILM, Tokyo, Japan) for 5–6 d to obtain ⁶⁷Ga autoradiograms. The autoradiographic images were analyzed using a computerized imaging analysis system (BAS2500, FUJIFILM). The adjacent slides used in the autoradiographic study (10 µm) were subjected to immunostaining for pimonidazole. The immunostaining was performed using the Hypoxyprobe-1 Plus Kit (Hypoxyprobe, Inc., Burlington, MA, U.S.A.), according to the manufacturer’s protocol. Similar examination was conducted on NFSa mouse fibrosarcoma-bearing mice.

PET Imaging

⁶⁸Ga-DOTA-MN2 (6.7 MBq) or ⁶⁸Ga-DOTA (7.4 MBq) was injected into FM3A-implanted mice (n=2) intravenously. The mice were anesthetized with 1.5% isoflurane and placed on a scanner bed in a prone position. For 45–75 min after injection, the mice were imaged using eXplore VISTA (GE Healthcare U.K. Ltd., Amersham, U.K.). An energy window of 250–700 keV was used. Before reconstruction, the raw data were corrected for random and scattered coincidences, and radioactive decay. PET images were reconstructed according to a standard filtered back-projection procedure (FBP) with a Ramp filter (alpha, 1.0; cut-off, 1.0) or by using a two-dimensional ordered-subset expectation maximization algorithm (2D-OSEM). The regions of interest (ROIs) were drawn on the tumor, corresponding areas (muscle) in the left flank, liver, and kidney of the FBP-reconstructed images.

After the PET scans, X-ray images of the mice were obtained for an anatomical comparison using an R-mCT system (Rigaku, Tokyo, Japan) equipped with a fixed microfocus X-ray tube and X-ray sensor. This instrument exposes a cone-shaped X-ray beam on the 2D detector through the animal set on a stage. The revolving arm rotates 360° around the mouse for 17 s to acquire a full 3D computerized tomography (CT) data set.

Statistical Analysis

To compare the tumor-to-blood (T/B)ratios and tumor-to-muscle (T/M) ratios between ⁶⁷Ga-DOTA-MN1 and ⁶⁷Ga-DOTA-MN2, two-way factorial ANOVA followed by a Tukey–Kramer test was performed. Differences at the 95% confidence level (p<0.05) were considered significant.

Results

Synthesis of ^67/68Ga-DOTA-MN2, ⁶⁷Ga-DOTA-MN1, and ^67/68Ga-DOTA

The radiochemical yields of ⁶⁷Ga-DOTA-MN2 in the condition of heating at 95°C and 140°C in an oil bath or microwave-assisted reaction are summarized in Fig. 2. Under heating at 95°C, the radiochemical yield reached 80% at 2 h, which was higher than that at 140°C heating until 30 min. On the other hand, microwave-assisted reaction achieved a high radiochemical yield (>85%) in a very short period of time (1 min).

Fig. 2. Radiochemical Yields of ⁶⁷Ga-DOTA-MN2

■: Microwave-assisted reaction (50 W), ○: 95°C in an oil bath, ▲: 140°C in an oil bath.

⁶⁷Ga-DOTA-MN1, ⁶⁷Ga-DOTA, ⁶⁸Ga-DOTA-MN2 and ⁶⁸Ga-DOTA were synthesized with radiochemical yields of 90, >99, 67 and >99%, respectively. All the radiogallium-labeled compounds were obtained with a radiochemical purity of >99%.

Biodistribution Study

Results of in vivo biodistribution studies of ⁶⁷Ga-DOTA-MN2, ⁶⁷Ga-DOTA-MN1, and ⁶⁷Ga-DOTA are summarized in Tables 1–3. ⁶⁷Ga-DOTA-MN2 significantly accumulated in the tumor at an early stage after injection (0.52%ID/g at 1 h), which was equal to the level of ⁶⁷Ga-DOTA-MN1 (0.50%ID/g at 1 h). Tumor accumulation of ⁶⁷Ga-DOTA was significantly lower for 6 h than those for ⁶⁷Ga-DOTA-MN2 and ⁶⁷Ga-DOTA-MN1 (p<0.0001). The blood clearance of ⁶⁷Ga-DOTA-MN2 was more rapid than that of ⁶⁷Ga-DOTA-MN1, leading to significantly higher T/B ratios in ⁶⁷Ga-DOTA-MN2 (Tables 1, 2). T/M ratios of ⁶⁷Ga-DOTA-MN2 were also higher than those for ⁶⁷Ga-DOTA-MN1, although the difference was not statistically significant, and only a trend (Tables 1, 2). ⁶⁷Ga-DOTA-MN2 mainly accumulated in the kidneys, followed by the tumor, lung, and liver at 1 h after injection, which was similar to ⁶⁷Ga-DOTA-MN1. ⁶⁷Ga-DOTA showed the fastest clearance from the blood among the three compounds.

Table 1. Biodistribution of Radioactivity after Injection of ⁶⁷Ga-DOTA-MN2 in C3H/He Mice Bearing FM3A Mouse Breast Tumors

		Time after injection
		30 min (n=5)	1 h (n=6)	3 h (n=5)	6 h (n=5)
%ID/g	Blood	1.29±0.18	0.16±0.03	0.05±0.01	0.04±0.01
	Spleen	0.47±0.11	0.17±0.07	0.12±0.03	0.09±0.04
	Pancreas	0.35±0.07	0.07±0.02	0.08±0.02	0.06±0.03
	Stomach	0.62±0.10	0.12±0.02	0.09±0.02	0.07±0.02
	Intestine	0.73±0.29	0.17±0.02	0.10±0.02	0.10±0.04
	Kidney	3.77±0.66	2.28±0.36	1.58±0.24	1.41±0.43
	Liver	0.56±0.18	0.21±0.02	0.18±0.01	0.16±0.06
	Heart	0.50±0.12	0.10±0.04	0.06±0.01	0.03±0.02
	Lung	1.40±0.23	0.29±0.03	0.08±0.01	0.07±0.03
	Muscle	0.30±0.27	0.08±0.05	0.02±0.01	0.03±0.02
	Tumor	1.37±0.19	0.52±0.05	0.36±0.21	0.25±0.07

Each value represents the mean±S.D. of five or six animals.

Table 2. Biodistribution of Radioactivity after Injection of ⁶⁷Ga-DOTA-MN1 in C3H/He Mice Bearing FM3A Mouse Breast Tumors

		Time after injection
		30 min (n=5)	1 h (n=6)	3 h (n=5)	6 h (n=5)
%ID/g	Blood	1.36±0.26	0.25±0.04	0.11±0.03	0.08±0.01
	Spleen	0.56±0.09	0.21±0.03	0.14±0.04	0.22±0.04
	Pancreas	0.39±0.05	0.09±0.03	0.06±0.01	0.04±0.05
	Stomach	0.67±0.21	0.15±0.04	0.16±0.12	0.11±0.05
	Intestine	0.54±0.13	0.18±0.05	0.13±0.06	0.09±0.03
	Kidney	4.33±0.55	1.94±0.42	1.75±0.22	1.48±0.15
	Liver	0.82±0.07	0.46±0.03	0.37±0.22	0.38±0.03
	Heart	0.57±0.17	0.10±0.03	0.06±0.01	0.05±0.02
	Lung	1.33±0.25	0.28±0.03	0.12±0.03	0.09±0.01
	Muscle	0.32±0.07	0.10±0.04	0.04±0.02	0.05±0.03
	Tumor	1.34±0.22	0.50±0.05	0.36±0.09	0.23±0.05

Each value represents the mean±S.D. of five or six animals.

Table 3. Biodistribution of Radioactivity after Injection of ⁶⁷Ga-DOTA in C3H/He Mice Bearing FM3A Mouse Breast Tumors

		Time after injection
		30 min (n=5)	1 h (n=6)	3 h (n=5)	6 h (n=5)
%ID/g	Blood	0.53±0.16	0.04±0.01	0.01±0.00	0.01±0.00
	Spleen	0.17±0.07	0.07±0.02	0.06±0.02	0.06±0.04
	Pancreas	0.16±0.03	0.04±0.03	0.01±0.01	0.03±0.01
	Stomach	0.28±0.12	0.06±0.03	0.04±0.02	0.03±0.02
	Intestine	0.28±0.08	0.12±0.08	0.07±0.01	0.07±0.01
	Kidney	2.86±0.61	0.98±0.17	1.04±0.16	0.91±0.18
	Liver	0.22±0.08	0.07±0.01	0.07±0.02	0.07±0.01
	Heart	0.21±0.06	0.03±0.01	0.02±0.01	0.01±0.01
	Lung	0.51±0.17	0.07±0.03	0.03±0.02	0.03±0.01
	Muscle	0.17±0.06	0.03±0.01	0.01±0.01	0.02±0.02
	Tumor	0.92±0.28	0.30±0.11	0.16±0.05	0.16±0.03

Each value represents the mean±S.D. of five animals.

Autoradiography and Immunostaining

The results of autoradiography (⁶⁷Ga-DOTA-MN2 and ⁶⁷Ga-DOTA) and immunostaining for pimonidazole are shown in Fig. 3. The autoradiograms obtained from FM3A-implanted mice showed a heterogeneous distribution of ⁶⁷Ga-DOTA-MN2 in the tumor tissue, which mostly corresponds to the pimonidazole-positive areas (Figs. 3a, b). Meanwhile, the distribution of ⁶⁷Ga-DOTA was not consistent with those of pimonidazole (Figs. 3c, d). Clear localization of ⁶⁷Ga-DOTA-MN2 within the pimonidazole-positive areas was also observed in NFSa mouse fibrosarcoma (Figs. 3e, f).

Fig. 3. Representative Autoradiograms (a, c, e) and Immunohistochemical Distribution of Pimonidazole (b, d, f) of the Tumor Sections

The tumors were excised 1 h postinjection of ⁶⁷Ga-DOTA-MN2 (a, b) and ⁶⁷Ga-DOTA (c, d) into FM3A-bearing mice or 30 min postinjection of ⁶⁷Ga-DOTA-MN2 (e, f) into NFSa-bearing mice. The localization of ⁶⁷Ga-DOTA-MN2 is well correlated with the pimonidazole-positive regions in both tumors.

PET Study

Figure 4 shows merged images of PET and CT at 1 h after injection of ⁶⁸Ga-DOTA-MN2 and ⁶⁸Ga-DOTA in FM3A-implanted mice. The tumor in the right flank was clearly visualized 1 h after injection of ⁶⁸Ga-DOTA-MN2 with high contrast to normal tissues, except the kidneys, while ⁶⁸Ga-DOTA showed low accumulation in the tumor, as indicated by the biodistribution study. Signal ratios of ⁶⁸Ga-DOTA-MN2 in the tumor to those in the breast tissue, liver, kidney, and muscle were 1.36, 1.48, 0.41, and 2.91, respectively, at 1 h postinjection.

Fig. 4. PET/CT Images in the Coronal and Transaxial Planes at 1 h after Injection of ⁶⁸Ga-DOTA-MN2 or ⁶⁸Ga-DOTA in FM3A-Bearing Mice

Arrows and K indicate tumors and kidneys, respectively. PET imaging with ⁶⁸Ga-DOTA-MN2 clearly visualized the tumor in the right flank.

Discussion

In this study, we validated the effectiveness of ^67/68Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors. Similar to the results obtained with NFSa mouse fibrosarcomas,^{8) 67}Ga-DOTA-MN2 showed the significant accumulation in FM3A mouse mammary carcinomas where hypoxic regions have been confirmed.¹⁴⁾ In ex vivo autoradiography, heterogeneous distributions of ⁶⁷Ga-DOTA-MN2 within both FM3A and NFSa were observed. This intratumoral distribution was well consistent with that of a hypoxic marker, pimonidazole, suggesting that ⁶⁷Ga-DOTA-MN2 specifically recognized the hypoxic regions in the tumors. Although there were a few pimonidazole-positive regions in which ⁶⁷Ga-DOTA-MN2 did not accumulate, this is probably because of the difference in the molecular size, charge, and lipophilicity between Ga-DOTA-MN2 and pimonidazole.

In biodistribution study, ⁶⁷Ga-DOTA-MN2 and ⁶⁷Ga-DOTA-MN1 showed two-fold higher uptake in the tumor than ⁶⁷Ga-DOTA, clearly indicating that the incorporation of metronidazole moiety(ies) contributed to the increase of accumulation in the tumor. Although the tumor uptake of ⁶⁷Ga-DOTA-MN2 was equal to that of ⁶⁷Ga-DOTA-MN1, the T/B ratios for ⁶⁷Ga-DOTA-MN2 were significantly higher than those for ⁶⁷Ga-DOTA-MN1 owing to the rapid clearance of ⁶⁷Ga-DOTA-MN2 from blood circulation. T/B ratios of ⁶⁷Ga-DOTA were higher than those of ⁶⁷Ga-DOTA-MN2 because the blood clearance of ⁶⁷Ga-DOTA was much faster compared with ⁶⁷Ga-DOTA-MN2. However, ⁶⁷Ga-DOTA did not accumulate in the hypoxic regions as shown in Fig. 3. Recently, a ^99mTc-labeled probe containing two 2-nitroimidazole moieties was reported to display higher accumulation in hypoxic cells than one containing a 2-nitroimidazole unit.¹⁵⁾ These results demonstrated the advantage of the probe containing two nitroimidazole moieties such as Ga-DOTA-MN2 in the recognition of hypoxic regions in the tumors.

To date, various hypoxia imaging agents, such as ¹⁸F-fluoromisonidazole (¹⁸F-FMISO) and 1-α-d-(5-deoxy-5-¹⁸F-fluoroarabinofuranosyl)-2-nitroimidazole (¹⁸F-FAZA), have been developed.^16–18) These nitroimidazole derivatives can recognize the hypoxic regions by the same mechanism as metronidazole. ¹⁸F-FMISO is a commonly used PET probe for imaging tumor hypoxia and for predicting the efficacy of radiotherapy. However, ¹⁸F-FMISO has many limitations for routine clinical application, such as slow accumulation in tumors, slow clearance kinetics from normoxic tissues, low target-to-background contrast and a significant amount of radioactive metabolite products.^{19) 18}F-FAZA, a sugar-coupled 2-nitroimidazole derivative, showed rapid clearance through the kidneys and high T/B ratios, but the uptakes in the hepatobiliary system and soft tissues were observed, indicating the generation of radiometabolites.²⁰⁾

^67/68Ga-DOTA-MN2 exhibited a moderate tumor uptake and a rapid clearance from the body, leading to high tumor-to-nontarget ratios. The T/B ratios of ⁶⁷Ga-DOTA-MN2 at 1 h postinjection were 3.19, which was higher than that of ¹⁸F-FMISO (1.19)²¹⁾ and equal to that of ¹⁸F-FAZA (3.27),²²⁾ although tumor cells examined in each experiment were different. At the same time point, ⁶⁷Ga-DOTA-MN2 exhibited higher T/M ratios (8.99) than ¹⁸F-FMISO (1.65) and ¹⁸F-FAZA (1.69).^21,22) Moreover, ^67/68Ga-DOTA-MN2 exhibited higher tumor-to-nontarget ratios for liver, intestine, and lung than ¹⁸F-FAZA.²²⁾ Because of lower levels of abdominal background radioactivity, ⁶⁸Ga-DOTA-MN2 would be superior to ¹⁸F-FMISO and ¹⁸F-FAZA for hypoxic tumor imaging. ^99mTc- and ⁶⁸Ga-labeled thiol-containing chelators derivatized with metronidazole(s) were previously reported to accumulate in the hypoxic regions in the tumor.^23,24) However, T/B or T/M ratios of these compounds were much lower than those of ⁶⁷Ga-DOTA-MN2, probably because of their high lipophilicity.

⁶⁸Ga is of great interest as a positron emitter for PET because of its radiophysical properties (t_1/2=68 min; β⁺=89% and EC=11%; E_β+max=1.899 MeV).^{25–27) 68}Ga is available from a ⁶⁸Ge/⁶⁸Ga generator (⁶⁸Ge, t_1/2=270.8 d), which renders it independent of an on-site cyclotron. Therefore, ⁶⁸Ga-labeled PET probes possess significant commercial potential and serve as a convenient alternative to cyclotron-based PET radionuclides, such as ¹⁸F. For hypoxic imaging, ⁶⁸Ga-labeled nitroimidazole derivatives were recently reported.^28,29) To produce the short-lived ⁶⁸Ga-labeled probes, rapid radiolabeling techniques are needed. To date, we have spent two hours radiolabeling ⁶⁷Ga-DOTA-MN2 with high radiochemical yields,⁸⁾ while microwave-assisted reaction contributed to the high radiochemical yield in a short time (ca. 1 min). Microwave heating has shown its potential in accelerating ⁶⁸Ga-labeling of DOTA-conjugated oligonucleotides³⁰⁾ or peptides.³¹⁾ In the future, we will apply microwave-assisted reaction to the labeling of DOTA-MN2 with ⁶⁸Ga.

In PET studies, ⁶⁸Ga-DOTA-MN2 clearly visualized the implanted tumors. The intratumoral distributions of radioactivity were heterogeneous as confirmed in the autoradiographic study, suggesting that the localization of ⁶⁸Ga-DOTA-MN2 in PET images might reflect the heterogeneous oxygen microenvironment of tumors.³²⁾ Thus, ⁶⁸Ga-DOTA-MN2 could characterize the tumors, which affords the selection of therapeutic strategies such as chemotherapy, radiotherapy, and operation. We also assume the possibility of ⁶⁷Ga-DOTA-MN2 in SPECT imaging of the hypoxic tumor because ⁶⁷Ga produced with a cyclotron is an attractive radioisotope for SPECT (⁶⁷Ga, t_1/2=3.3 d). SPECT imaging with ⁶⁷Ga-DOTA-MN2 at 3–6 h postinjection would achieve more specific imaging of hypoxic regions in the tumors because tumor-to-normal tissue ratios on ⁶⁷Ga-DOTA-MN2 were increasing with time, as shown in the biodistribution studies.

Conclusion

⁶⁸Ga-DOTA-MN2, developed on the basis of previous findings obtained by X-ray crystallography of Ga-DOTA chelates and the drug design concept of bifunctional radiopharmaceuticals, clearly visualized the hypoxic regions of tumor with high contrast to normal tissues with small-animal PET. Furthermore, we validated the effectiveness of conjugation of two metronidazole moieties to radiogallium-DOTA chelate for the recognition of hypoxic regions in the tumors. Although further studies are needed for progression toward clinical application, these results demonstrate the potential of ^67/68Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors.

Acknowledgment

We would like to thank FUJIFILM RI Pharma Co., Ltd., Tokyo, Japan, for donating the ⁶⁷GaCl₃. This work was supported in part by a Grant-in-Aid for Scientific Research (B) (21390347) from the Japan Society for the Promotion of Science and the Project to Develop ‘Innovative Seeds’ of the Japan Science and Technology Agency.

References

• 1)  Vaupel P, Harrison L. Tumor hypoxia: causative factors, compensatory mechanisms, and cellular response. Oncologist, 9 (Suppl. 5), 4–9 (2004).
• 2)  Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev., 26, 225–239 (2007).
• 3)  Brizel DM, Scully SP, Harrelson JM, Layfield LJ, Bean JM, Prosnitz LR, Dewhirst MW. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res., 56, 941–943 (1996).
• 4)  Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res., 56, 4509–4515 (1996).
• 5)  Brown JM. Keynote address: hypoxic cell radiosensitizers: where next? Int. J. Radiat. Oncol. Biol. Phys., 16, 987–993 (1989).
• 6)  Chapman JD, Baer K, Lee J. Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells. Cancer Res., 43, 1523–1528 (1983).
• 7)  Varghese AJ, Whitmore GF. Binding to cellular macromolecules as a possible mechanism for the cytotoxicity of misonidazole. Cancer Res., 40, 2165–2169 (1980).
• 8)  Mukai T, Suwada J, Sano K, Okada M, Yamamoto F, Maeda M. Design of Ga-DOTA-based bifunctional radiopharmaceuticals: two functional moieties can be conjugated to radiogallium-DOTA without reducing the complex stability. Bioorg. Med. Chem., 17, 4285–4289 (2009).
• 9)  Ogawa K, Mukai T, Arano Y, Hanaoka H, Hashimoto K, Nishimura H, Saji H. Design of a radiopharmaceutical for the palliation of painful bone metastases: rhenium-186-labeled bisphosphonate derivative. J. Labelled Comp. Radiopharm., 47, 753–761 (2004).
• 10)  Ogawa K, Mukai T, Arano Y, Ono M, Hanaoka H, Ishino S, Hashimoto K, Nishimura H, Saji H. Development of a rhenium-186-labeled MAG3-conjugated bisphosphonate for the palliation of metastatic bone pain based on the concept of bifunctional radiopharmaceuticals. Bioconjug. Chem., 16, 751–757 (2005).
• 11)  Ogawa K, Mukai T, Arano Y, Otaka A, Ueda M, Uehara T, Magata Y, Hashimoto K, Saji H. Rhemium-186-monoaminemonoamidedithiol-conjugated bisphosphonate derivatives for bone pain palliation. Nucl. Med. Biol., 33, 513–520 (2006).
• 12)  Ogawa K, Mukai T, Inoue Y, Ono M, Saji H. Development of a novel ^99mTc-chelate-conjugated bisphosphonate with high affinity for bone as a bone scintigraphic agent. J. Nucl. Med., 47, 2042–2047 (2006).
• 13)  Heppeler A, Froidevaux S, Mäcke HR, Jermann E, Béhé M, Powell P, Hennig M. Radiometal-labelled macrocyclic chelator-derivatised somatostatin analogue with superb tumour-targeting properties and potential for receptor-mediated internal radiotherapy. Chem. Eur. J., 5, 1974–1981 (1999).
• 14)  Kudo T, Ueda M, Kuge Y, Mukai T, Tanaka S, Masutani M, Kiyono Y, Kizaka-Kondoh S, Hiraoka M, Saji H. Imaging of HIF-1-active tumor hypoxia using a protein effectively delivered to and specifically stabilized in HIF-1-active tumor cells. J. Nucl. Med., 50, 942–949 (2009).
• 15)  Huang H, Zhou H, Li Z, Wang X, Chu T. Effect of a second nitroimidazole redox centre on the accumulation of a hypoxia marker: synthesis and in vitro evaluation of ^99mTc-labeled bisnitroimidazole propylene amine oxime complexes. Bioorg. Med. Chem. Lett., 22, 172–177 (2012).
• 16)  Eschmann SM, Paulsen F, Reimold M, Dittmann H, Welz S, Reischl G, Machulla HJ, Bares R. Prognostic impact of hypoxia imaging with ¹⁸F-misonidazole PET in non-small cell lung cancer and head and neck cancer before radiotherapy. J. Nucl. Med., 46, 253–260 (2005).
• 17)  Nunn A, Linder K, Strauss HW. Nitroimidazoles and imaging hypoxia. Eur. J. Nucl. Med., 22, 265–280 (1995).
• 18)  Sorger D, Patt M, Kumar P, Wiebe LI, Barthel H, Seese A, Dannenberg C, Tannapfel A, Kluge R, Sabri O. [¹⁸F]Fluoroazomycinarabinofuranoside (¹⁸FAZA) and [¹⁸F]fluoromisonidazole (¹⁸FMISO): a comparative study of their selective uptake in hypoxic cells and PET imaging in experimental rat tumors. Nucl. Med. Biol., 30, 317–326 (2003).
• 19)  Grönroos T, Minn H. Imaging of tumour hypoxia using PET and ¹⁸F-labelled tracers: biology meets technology. Eur. J. Nucl. Med. Mol. Imaging, 34, 1563–1565 (2007).
• 20)  Souvatzoglou M, Grosu AL, Röper B, Krause BJ, Beck R, Reischl G, Picchio M, Machulla HJ, Wester HJ, Piert M. Tumour hypoxia imaging with [¹⁸F]FAZA PET in head and neck cancer patients: a pilot study. Eur. J. Nucl. Med. Mol. Imaging, 34, 1566–1575 (2007).
• 21)  Liu RS, Chou TK, Chang CH, Wu CY, Chang CW, Chang TJ, Wang SJ, Lin WJ, Wang HE. Biodistribution, pharmacokinetics and PET imaging of [¹⁸F]FMISO, [¹⁸F]FDG and [¹⁸F]FAc in a sarcoma- and inflammation-bearing mouse model. Nucl. Med. Biol., 36, 305–312 (2009).
• 22)  Piert M, Machulla HJ, Picchio M, Reischl G, Ziegler S, Kumar P, Wester HJ, Beck R, McEwan AJ, Wiebe LI, Schwaiger M. Hypoxia-specific tumor imaging with ¹⁸F-fluoroazomycin arabinoside. J. Nucl. Med., 46, 106–113 (2005).
• 23)  Das T, Banerjee S, Samuel G, Sarma HD, Korde A, Venkatesh M, Pillai MR. ^99mTc-labeling studies of a modified metronidazole and its biodistribution in tumor bearing animal models. Nucl. Med. Biol., 30, 127–134 (2003).
• 24)  Ito M, Yang DJ, Mawlawi O, Mendez R, Oh CS, Azhdarinia A, Greenwell AC, Yu DF, Kim EE. PET and planar imaging of tumor hypoxia with labeled metronidazole. Acad. Radiol., 13, 598–609 (2006).
• 25)  Antunes P, Ginj M, Zhang H, Waser B, Baum RP, Reubi JC, Maecke H. Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals? Eur. J. Nucl. Med. Mol. Imaging, 34, 982–993 (2007).
• 26)  Fani M, André JP, Maecke HR. ⁶⁸Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals. Contrast Media Mol. Imaging, 3, 67–77 (2008).
• 27)  Zhernosekov KP, Filosofov DV, Baum RP, Aschoff P, Bihl H, Razbash AA, Jahn M, Jennewein M, Rösch F. Processing of generator-produced ⁶⁸Ga for medical application. J. Nucl. Med., 48, 1741–1748 (2007).
• 28)  Hoigebazar L, Jeong JM, Choi SY, Choi JY, Shetty D, Lee YS, Lee DS, Chung JK, Lee MC, Chung YK. Synthesis and characterization of nitroimidazole derivatives for ⁶⁸Ga-labeling and testing in tumor xenografted mice. J. Med. Chem., 53, 6378–6385 (2010).
• 29)  Hoigebazar L, Jeong JM, Hong MK, Kim YJ, Lee JY, Shetty D, Lee YS, Lee DS, Chung JK, Lee MC. Synthesis of ⁶⁸Ga-labeled DOTA-nitroimidazole derivatives and their feasibilities as hypoxia imaging PET tracers. Bioorg. Med. Chem., 19, 2176–2181 (2011).
• 30)  Velikyan I, Lendvai G, Välilä M, Roivainen A, Yngve U, Bergström M, Långström B. Microwave accelerated ⁶⁸Ga-labeling of oligonucleotides. J. Labelled Comp. Radiopharm., 47, 79–89 (2004).
• 31)  Velikyan I, Beyer GJ, Långström B. Microwave-supported preparation of ⁶⁸Ga bioconjugates with high specific radioactivity. Bioconjug. Chem., 15, 554–560 (2004).
• 32)  Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res., 49, 6449–6465 (1989).