Radiometallic Complexes of DO3A-Benzothiazole Aniline for Nuclear

Jan 30, 2018 - Consequently, this molecular platform represents a useful approach in nuclear medicine toward tumor-theranostic radiopharmaceuticals wh...
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Article Cite This: Mol. Pharmaceutics 2018, 15, 1133−1141

Radiometallic Complexes of DO3A-Benzothiazole Aniline for Nuclear Medicine Theranostics Ji-Ae Park,†,∇ Ji Woong Lee,†,∇ Hee-Kyung Kim,‡,§ Un Chol Shin,† Kyo Chul Lee,† Tae-Jeong Kim,‡ Yongmin Chang,∥,⊥ Kyeong Min Kim,# Jung Young Kim,*,† and Yong Jin Lee*,† †

Division of RI-Convergence Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Korea Division of Biomedical Engineering Science and §BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University, Daegu 702-701, Korea ∥ Department of Molecular Medicine and ⊥Department of Radiology, Kyungpook National University, Daegu 702-701, Korea # Division of Medical Radiation Equipment, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Korea ‡

S Supporting Information *

ABSTRACT: To develop a radioactive metal complex platform for tumor theranostics, we introduced three radiopharmaceutical derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acidbenzothiazole aniline (DO3A-BTA, L1) labeled with medical radioisotopes for diagnosis (68Ga/64Cu) and therapy (177Lu). The tumor-targeting ability of these complexes was demonstrated in a cellular uptake experiment, in which 177Lu-L1 exhibited markedly higher uptake in HeLa cells than the 177Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex. According to in vivo positron emission tomography imaging, high accumulation of 68GaL1 and 64Cu-L1 was clearly visualized in the tumor site, while 177LuL1 showed therapeutic efficacy in therapy experiments. Consequently, this molecular platform represents a useful approach in nuclear medicine toward tumor-theranostic radiopharmaceuticals when 68GaL1 or 64Cu-L1 is used for diagnosis, 177Lu-L1 is used for therapy, or two of the compounds are used in conjunction with each other. KEYWORDS: benzothiazole, radiopharmaceuticals, 68Ga, 64Cu, 177Lu

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INTRODUCTION Heterocyclic benzothiazole aniline (BTA) compounds have various biological effects, such as antimicrobial, anticancer, anthelmintic, and antidiabetic activity.1−4 In the past two decades, BTA compounds have been developed as antitumor agents, and several attempts have been made to modify these heterocycles to improve their antitumor activity.5−9 Furthermore, the potential abilities for cancer treatment have been explored in the development of imaging probe and therapeutic agents in nuclear medicine. For instance, a fluorescent rhenium complex conjugated to 2-(3-aminophenyl)benzothiazoles has been introduced as a promising radiopharmaceutical candidate for breast cancer. The analogous tricarbonyl technetium or rhenium complexes (M = 99mTc(CO)3 or Re) have been proposed for single-photon emission computed tomography (SPECT) imaging and targeted radiotherapy (TRT), although their direct therapeutic effect in vivo has not yet been demonstrated.10−12 More recently, BTA conjugates with 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) have been employed as multimodal magnetic resonance image (MRI)/optical and MRI/SPECT probes.13,14 In our previous work, we investigated the use of gadolinium © 2018 American Chemical Society

complexes with DO3A-BTA conjugates as a single-molecule theranostic agent.15,16 The complexes provided tumor-specific and enhanced intracellular MR images of the cytosols and nuclei of tumor cells such as MCF-7, MDA-MB-231, and SKHEP-1. The antiproliferative activity of DO3A-BTA and Gd(DO3A-BTA) was demonstrated by determining their in vitro growth inhibition values (GI50 and TGI) and monitoring tumor volume regression in vivo. On the basis of these observations, we attempt to develop tumor-theranostic metal complexes of DO3A-BTA conjugates labeled with the therapeutic radioisotope 177Lu and the diagnostic radioisotopes 68 Ga/64Cu in the same molecular structure. Efforts toward the development of analogous complexes with 99m Tc/188Re, 68Ga/90Y, 64Cu/67Cu, and 64Cu/177Lu as “matched pairs” for tumor imaging and TRT have been reported in the literature.17,18 Specifically, the most widely used radiometals for positron emission tomography (PET) imaging are 68Ga (T1/2 = Received: Revised: Accepted: Published: 1133

November 8, 2017 January 22, 2018 January 30, 2018 January 30, 2018 DOI: 10.1021/acs.molpharmaceut.7b00996 Mol. Pharmaceutics 2018, 15, 1133−1141

Article

Molecular Pharmaceutics Chart 1. Benzothiazole-Based Antitumor Molecular Platforms

67.7 m) and 64Cu (T1/2 = 12.7 h).19,20 As a radiometal for tumor therapy, 177Lu (T1/2 = 6.7 days) has good physical properties in terms of its beta radiation emission (β−: Emax = 497 keV, 79%) and average penetrating depth of soft-tissue of 0.23 mm (max. 1.7 mm).21,22 Moreover, because of their ability to interchange various types of medical radioisotopes with parallel chemical properties, radiometal−chelator systems represent an easy and convenient approach to the preparation of radiopharmaceuticals in the clinic. In this study, analogous 68 Ga, 64Cu, and 177Lu complexes of DO3A-BTA aniline conjugates were investigated for their in vivo PET imaging ability and in vitro cell therapeutic efficacy in a mice model of human cervical cancer and for their radioactive accumulation in a biodistribution study.

Eppendorf tube with ethanol (1.0 mL), and then the radiochemical purity was confirmed by a radio-TLC scanner. Synthesis of 64Cu-L1 and nat.Cu-L1 (Cold Complex). The title compound was initially prepared in the form of 64 CuCl2 in aqueous HCl (10 mL, 3.7−7.4 GBq), as described in the literature.23 The solution was then dried in a cone-shaped glass vial by purging with nitrogen gas at 90 °C for 20 min, and then a solution of sodium acetate buffer (0.2 mL, 1.0 M, pH 5.5) was added. A solution of L1 (0.1 mg, 0.163 μmol) in EtOH (0.01 mL) was added to this radioactive solution, which was then incubated at 50 °C for 30 min in a heating bath. As mentioned above, it was performed with additional purification of C18 cartridge after cooling the mixture at RT. The radiochemical purity and yield of 64Cu-L1 were confirmed to be greater than 98% by a radio-TLC scanner via elution with citrate buffer (0.1 M) on instant thin-layer chromatography (ITLC) paper, and the formation of 64Cu-L1 was further corroborated by radio-HPLC. For the purpose of chemical structure confirmation, a natural analogue of 64Cu-L1, nat.CuL1, was prepared via a slightly modified procedure as follows:24 To a solution of Cu(ClO4)·6H2O (9.2 mg, 0.025 mmol) and L1 (30.6 mg, 50 μmol) was added methanol (5 mL). The resulting clear blue solution was refluxed at 70 °C for 4 h, after which any solid impurities were removed by filtration through Celite. Diethyl ether was added to the filtrate to precipitate the product as a green powder. The chemical identification of 64CuL1 with nat.Cu-L1 was confirmed by a Waters HPLC system equipped with a C18 analytical column (5 μm, 3.0 × 150 mm) using the following separation conditions: An aqueous mixture of solvent A (MeOH) and solvent B (0.1% TFA in water) was employed with a 30 min linear gradient (from 50% A to 100% A) at a flow rate of 0.5 mL/min. Rt(L1) = 4.8 min (Figure S1 in the Supporting Information) and Rt(nat.Cu-L1) = 8.2 min (Figure S2). MALDI-TOF-MS: m/z = 672.3 (C29H34CuN6O7S, calculated MW = 673.15) (Figure S3). Synthesis of 177Lu-DOTA and 177Lu-L1. To synthesize the compounds labeled with radioactive lutetium, 0.1 mL of 177 LuCl3 (ITG Isotope Technologies Garching GmbH, Germany; ca. 9.62 MBq) was transferred into a reactor vial. This radioactive fraction was dried by purging the vial with nitrogen gas at 100 °C for 10 min. Mixtures of 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, 0.1 mg) and L1 with sodium acetate buffer (1.5 mL, 1 mM, pH 5.5) were prepared and were added separately to the dried 177 LuCl3 described above. The two reaction mixtures were heated at 100 °C for 5 min and then were cooled to RT. As mentioned above, it was performed with additional purification of C18 cartridge. The radiochemical purity of the two complexes was confirmed by a radio-TLC scanner. 177LuDOTA was used here without further purification, whereas

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EXPERIMENTAL METHODS General Remarks. All reagents were purchased from commercial sources and used as received unless stated otherwise. Solvents were purified and dried according to previously published procedures. {4-[(4-Benzothiazol-2-ylphenylcarbamoyl)-methyl] -7, 10-bis-carboxymethyl1,4,7,10,tetraaza-cyclododec-1-yl}-acetic acid (L1, MW = 612.2), DO3A-1-[2-(4′-nitrophenyl)-6-(3″-bromopropoxyl)benzothiazole] (L2, MW = 628.2), and DO3A-1-[2-(4′aminophenyl)-6-(3″-bromopropoxyl)-benzothiazole] (L3, MW = 658.2) were prepared as described previously (Chart 1).15,16 C18 Sep-Pak cartridges were obtained from Waters Corporation (Milford, MA). Analytical HPLC was performed on a Waters 515 ternary pump with UV detection. All preparative separation was performed on an HPLC system equipped with a Gilson 321 pump, a UV/vis-151 detector, and a Bioscan flow-count photomultiplier tube (PMT) radioactivity detector. All animal experiments in this study followed protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Radiological and Medical Sciences (KIRAMS). Synthesis of 68Ga-Ls (L1, L2, and L3). For the elution of the 68Ge/68Ga generator, HCl (1.0 M) was used. For radiolabeling, an eluate of 68Ga (1.0 mL, ca. 370 MBq) was transferred into a reactor vial. The fraction was dried by purging with nitrogen gas at 100 °C for 15 min. L1−L3 (0.1 mg; L1 = 0.163 μmol, L2 = 0.159 μmol, L3 = 0.151 μmol) was dissolved in sodium acetate buffer (pH 5.5) (0.1 mL, 1.0 M) and then was added into the reactor vial. The vial was heated to 100 °C for 5 min. Next, the radiolabeled mixture was passed through a C18 cartridge (Waters Sep-Pak C18 plus) that had been conditioned with ethanol (10 mL) and distilled water (10 mL). Impurities were washed off the Sep-Pak cartridge with distilled water (10 mL). The radiolabeled product was eluted into an 1134

DOI: 10.1021/acs.molpharmaceut.7b00996 Mol. Pharmaceutics 2018, 15, 1133−1141

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Molecular Pharmaceutics

with 1.5% isoflurane and then injected with 68Ga-L1 (18.5 MBq/0.1 mL) or 64Cu-L1 (7.4 MBq/0.1 mL) via the tail vein. Thirty-minute static scans were acquired at 1 and 4 h after injection with 68Ga-L1 and at 1, 4, 18, and 36 h after injection with 64Cu-L1. Fourier rebinning and two-dimensional orderedsubset expectation maximization (OSEM) algorithms with no corrections for attenuation or scatter were used.25,26 On the plane of reconstructed images showing tumor lesions, circular 3D regions of interest (ROIs) were drawn on the areas of the tumors using analysis software (Inveon Research Workplace) provided by the vendor of the PET scanner. The pixel values of the reconstructed images were converted to %ID/g via a cross calibration factor obtained previously. Clonogenic Assay for 177Lu-DOTA and 177Lu-L1 in HeLa Cells. HeLa cells were plated at a cell density of 3 × 105 cells/well in 10 cm cell culture dish plates (10 mL) with optimized medium and incubated for 24 h in a 37 °C incubator with 5% CO2. The cells were treated with 1.85, 3.7, and 9.25 MBq (0.04, 0.08, and 0.2 μM) of 177Lu-DOTA and 177Lu-L1 and incubated at 37 °C for 60 min. After being washed twice with 10 mL of PBS, the cells were detached and seeded into 6well plates at a density of 1000 and 2000 cell/well and then were cultured in a 37 °C incubator for 2 weeks. The cells were stained with crystal violet solution (0.5% w/v in 25% MeOH), and photographed colonies of greater than 50 cells were counted. All data points are displayed as the mean ± SD (n = 4). Radiation Dosimetry. To estimate the radiation-absorbed dose for 68Ga-L1, 64Cu-L1, and 177Lu-L1, mice biodistribution data were used. The mean activity in mice organs at 1, 2, and 4 h p.i. for 68Ga-L1 and 1, 4, 18, and 36 h p.i. for 64Cu-L1 and 177 Lu-L1 was extrapolated to generate time−activity curves (TACs) of a male reference subject (70 kg) human as follows,27 where the average body weight of the mice was used for each compound:mouse

177

Lu-L1 was purified through a C18 cartridge (Waters Sep-Pak C18 plus) that had been conditioned with ethanol (10 mL) and distilled water (10 mL). In Vitro Serum Stability. The radiochemical stability of 68 Ga-L1, 64Cu-L1, and 177Lu-L1 was assessed by an ITLC method (vide supra). 68Ga-L1, 64Cu-L1, and 177Lu-L1 were separately incubated at 37 °C in human serum and mouse serum for different intervals, with free 64Cu and 177Lu used as references. The mobile phases for 68Ga-L1 and 64Cu-L1 were citrate acid (0.1 M) and for 177Lu-L1 was NaOH (1 M):EtOH:DW, 1:5:10 (v/v/v). Partition Coefficient Determination. The octanol/water partition coefficient (log P) of 64Cu-L1 and 177Lu-L1 was determined according to the following protocol. To neat octanol (0.5 mL) was added a solution of 64Cu-L1 or 177Lu-L1 (3.7 kBq) in PBS (0.5 mL, pH 7.4). The resulting mixture was vigorously stirred for 5 min and then was centrifuged (12 000 rpm) for an additional 5 min. The radioactivity of both the PBS and octanol phases were measured in a gamma counter, and the log P values were calculated (n = 3). Cell Culture. Human cervix adenocarcinoma cell line (HeLa), human embryonic kidney cell line (293), human breast cancer cell line (MDA-MA-231), and human glioblastoma cell line (U87MG) were maintained in Dulbeccos’s modified Eagle’s medium (DMEM, WelGENE Inc., Korea) containing 10% fetal bovine serum (FBS) and 1% antibiotics and were grown in a humidified incubator at 37 °C and 5% CO2. The medium was changed every 3 days. In Vitro Cellular Uptake. Each cell line (293, HeLa, MDAMB-231, and U87MG) was plated at a cell density of 3 × 105 cells/well in 24-well plates with optimized medium. After 24 h of incubation, the cells were assessed for cellular uptake by determining 68Ga-Ls uptake at 37 °C for 60 min. Briefly, the cells were collected and incubated in 0.5 mL of Hank’s balanced salt solution (HBSS) containing 74 kBq of 68Ga-L at 37 °C for 60 min. The cells were washed twice with 2 mL of cold PBS, and the radioactivity of the detached cells was counted with a gamma counter (480 Wizard 3ralt solution (HBSS)). All data points are displayed as the means ± SD (n = 4). The cellular uptake study for 177Lu-DOTA and 177Lu-L1 in HeLa cells was also performed using the same methods and observed at different incubation times (30, 60, and 90 min). Tumor Xenograft Model. Female BALB/c nude mice (SLC, Hamamatsu, Japan) at 4−6 weeks of age were subcutaneously injected with 5 × 106 HeLa cells or MDAMB-231 cells suspended in 100 μL of DMEM in the left arm or right thigh. The mice were subjected to biodistribution and PET studies when the tumor volume reached 0.7−0.9 cm in diameter (30−50 days after implantation). Biodistribution. The receptor-specific uptake was determined using nude mice bearing HeLa cells. Mice were injected with 68Ga-L1 (740 kBq/0.1 mL), 64Cu-L1 (340 kBq/0.1 mL), or 177Lu-L1 (740 kBq/0.1 mL). Mice (n = 4 per time point) were sacrificed by exsanguination at different time points post injection (p.i.). The organs of interest (i.e., blood, muscle, heart, lung, liver, spleen, stomach, intestine, kidney, bone, brain, and tumor) were harvested and measured for radioactivity in a gamma counter (1480 Wizard, Wallac). The organ uptake was calculated and expressed in percent injected dose per gram (% ID/g). In Vivo PET Imaging. Whole-body PET images of mice were obtained using a dedicated small animal PET scanner (Inveon, Siemens Medical Solutions). Mice were anesthetized

⎛ mass body ⎞ ⎟⎟ %ID(t )human = %ID(t )mouse · ⎜⎜ · ⎝ massorgan ⎠mouse ⎛ massorgan ⎞ ⎜⎜ ⎟⎟ ⎝ mass body ⎠human

The extrapolated TACs were used to calculate the effective dose (ED, mSv·Bq1−) for an adult male in the same manner as the mice data using Organ Level Internal Dose Assessment/ EXponential Modeling software version 1.0 (OLINDA/EXM, Vanderbilt University, Nashville, TN). Statistical Analysis. Data were analyzed with GraphPad Prism statistical software (GraphPad Software, Inc.). Student’s two-tailed t test was used to determine statistical significance at the 95% confidence level. Differences among groups were assessed using one-way analysis of variance with Tukey’s post hoc test. P < 0.05 was considered significantly different.

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RESULTS Radiochemistry. 68Ga-Ls (L1, L2, and L3), 64Cu-L1, 177LuDOTA, and 177Lu-L1 were labeled with a radiochemical purity of 98% or greater. In particular, L1 could be quickly labeled with any of 68Ga, 64Cu, or 177Lu at 100 °C within 5 min and then further purified to remove radioactive impurities using a C18 Sep-Pak cartridge, which afforded 64Cu- (or 177Lu)-L1 with a radiochemical purity that was normally greater than 98%. 1135

DOI: 10.1021/acs.molpharmaceut.7b00996 Mol. Pharmaceutics 2018, 15, 1133−1141

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Figure 1. (A) Cellular uptake studies of 68Ga-Ls using 293, HeLa, MDA-MB-231, and U87MG cells (n = 3, mean ± SD) after 60 min incubation time. (B) Comparison of 177Lu-DOTA and 177Lu-L1 uptake in HeLa cells at different incubation times (n = 3, mean ± SD). ***P < 0.001 vs 68GaL1 in HeLa.

Figure 2. Biodistribution of 68Ga-L1 (A), 64Cu-L1 (B), and 177Lu-L1 (C) in nude mice bearing HeLa tumors at different times (n = 4, mean ± SD). (D) Comparison of selected tumor/muscle ratios of 68Ga-L1, 64Cu-L1, and 177Lu-L1.

and 39.2 ± 4.0 GBq/μmol, respectively, as determined by the HPLC method described above. In Vitro Cellular Uptake. The results of cellular uptake studies of 68Ga-Ls in 293, HeLa, MDA-MB-231, and U87MG cells revealed that all three complexes (68Ga-L1, L2, and L3) exhibited high uptake in the HeLa cells (Figure 1A). In all the cells, 68Ga-L1 showed particularly higher uptake than 68Ga-L2 and 68Ga-L3. Specifically, the level of 68Ga-L1 in HeLa cells (3.31 ± 0.03% ID) was 10 times greater than that of 68Ga-L3 (0.36 ± 0.08% ID) (P < 0.001). Consequently, the wellvalidated L1 ligand was used as a targeting molecule for in vitro and in vivo evaluation. Next, we determined the cellular uptake

Furthermore, the chemical structure of 64Cu-L1 was established by characterizing its natural analogue, nat.Cu-L1, via analytical HPLC (Figure S4). The retention times (Rt) of nat.Cu-L1 and 64 Cu-L1 were 9.0 and 9.5 min, respectively, thereby demonstrating that the difference between the retention times of nat.Cu-L1 and 64Cu-L1 was slight (0.5 min), particularly considering the distance gap of 30 cm between the RI and UV detectors of the HPLC system. In addition, after purification with the Sep-Pak cartridge, the specific activities of 68Ga-L1, 64 Cu-L1, 177Lu-L1, and 177Lu-DOTA (without cartridge) in all biological studies were 342.1 ± 4.9, 219.5 ± 11.9, 44.4 ± 1.3, 1136

DOI: 10.1021/acs.molpharmaceut.7b00996 Mol. Pharmaceutics 2018, 15, 1133−1141

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Molecular Pharmaceutics

Figure 3. (A) In vivo PET images of mice bearing HeLa cell tumors in the right thigh (yellow arrow) with 68Ga-L1. (B) In vivo PET axial (upper panels) and coronal (lower panels) images of mice bearing HeLa cell tumors in the left arm (yellow arrow) with 64Cu-L1. (C) In vivo PET/CT axial (upper panels) and coronal (lower panels) images with 64Cu-L1 focused on the liver, gallbladder, and intestine.

of 177Lu-DOTA and 177Lu-L1 in HeLa cells. As shown in Figure 1B, 177Lu-L1 exhibited very high uptake compared with 177LuDOTA and maintained its signal for up to 90 min, thus indicating that 177Lu-L1 shares the tumor-targeting ability that is characteristic of BTA analogues. In Vitro Characterization. The determination of the radiochemical stability of the complexes in human and mouse serum and PBS indicated that both 64Cu-L1 and 177Lu-L1 had high stability against proteinases (Table S1). Radio-TLC analysis over 36 h showed that 64Cu-L1 exhibited little radiochemical degradation (>97%) and that 177Lu-L1 was highly stable (>99%). The log P values of 177Lu-L1 and 64Cu-L1 were −0.12 ± 0.03 and −0.36 ± 0.02, respectively. Biodistribution Studies. The results of the biodistribution studies of 68Ga-L1, 64Cu-L1, and 177Lu-L1 are presented in panels A, B, and C of Figure 2, respectively. At 1 h p.i., 68Ga-L1 exhibited higher lung (7.52% ID/g), kidney (8.64% ID/g), and spleen (4.60% ID/g) uptake than 64Cu-L1 and 177Lu-L1. The tumor uptake of 68Ga-L1 at 1, 2, and 4 h p.i. was determined to be 3.58, 3.51, and 2.33% ID/g, respectively. The liver uptake at 1 h p.i. of 64Cu-L1 (21.01% ID/g) was greater than that of 68 Ga-L1 (7.13% ID/g) and 177Lu-L1 (3.70% ID/g), which may be explained by the in vivo transmetalation of 64Cu. The intestinal uptake of 64Cu-L1 (14.26% ID/g) was also higher than that of 68Ga-L1 (2.86% ID/g) and 177Lu-L1 (2.60% ID/g) at 1 h p.i. The tumor uptake of 64Cu-L1 at 1, 4, 18, and 36 h p.i.

was determined to be 3.40, 2.60, 2.73, and 2.06% ID/g, respectively. 177Lu-L1 (5.56 and 3.08% ID/g at 1 and 4 h p.i., respectively) had significantly higher blood uptake than 68GaL1 (2.46 and 0.87% ID/g at 1 and 4 h p.i., respectively) and 64 Cu-L1 (0.98 and 1.93% ID/g at 1 and 4 h p.i., respectively). The uptake of 177Lu-L1 led to moderate concentrations of the compound in the lung, liver, spleen, intestine, and kidney. The tumor uptake of 177Lu-L1 was determined to be 2.74, 1.45, 0.35 and 0.19% ID/g at 1, 4, 18, and 36 h p.i., respectively. The tumor/muscle ratio of 64Cu-L1 was higher than those of 68GaL1 and 177Lu-L1, which showed similar concentrations (Figure 2D). The tumor/muscle ratios of all complexes increased or were maintained up to 18 h (except 68Ga-L1, which was maintained up to 4 h). All complexes showed low levels of radioactivity in the brain and bone. In Vivo PET Imaging. Panels A and B of Figure 3 show animal PET images of 68Ga-L1 and 64Cu-L1, respecitvely, in HeLa cell tumor-bearing mice. High accumulation of 68Ga-L1 and 64Cu-L1 was clearly visualized in the tumor site. The tumor uptakes of 68Ga-L1 were 3.4 and 2.4% ID/g at 1 and 4 h p.i., respectively, which were consistent with the biodistribution data. For 64Cu-L1, the radioactivity in the tumor area was maintained up to 42 h p.i. with concentrations of 2.3, 2.6, 3, and 2.6% ID/g at 1, 4, 18, and 42 h p.i., respectively. These data were fully consistent with the values observed in the biodistribution studies. 64Cu-L1 was mainly cleared through 1137

DOI: 10.1021/acs.molpharmaceut.7b00996 Mol. Pharmaceutics 2018, 15, 1133−1141

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Molecular Pharmaceutics

Table 1. Dosimetry of 68Ga-L1, 64Cu-L1, and 177Lu-L1 Extrapolated to an Adult Human Based on the Biodistribution Data (n = 4)a

the hepatic pathway. As shown in Figure 3C, the gallbladder and intestinal uptake of 64Cu-L1 was significantly higher than the liver uptake at 1 and 4 h p.i. In Vitro Therapy. 177Lu-L1 and 177Lu-DOTA induces dosedependent inhibition of tumor cell survival. Survival values are shown as percent survival compared to the control. Figure 4

ED (mSv/MBq) source organ adrenals brain breast gallbladder wall lower large intestine wall small intestine stomach wall upper large intestine wall heart wall kidneys liver lungs muscle ovaries pancreas red marrow osteogenic cells skin spleen testes thymus thyroid urinary bladder wall uterus total body effective dose

Figure 4. Effect of the radiation dose of 177Lu-L1 and 177Lu-DOTA on HeLa cell proliferation (n = 3, mean ± SD). *P < 0.05, ** P < 0.01 (177Lu-L1 vs 177Lu-DOTA).

shows that higher concentrations of the probe result in a higher percentages of tumor cell killing. When 177Lu-L1 was administered at 1.85, 3.7, and 9.25 MBq, the cell survival values were 103.4 ± 20.89%, 36.3 ± 13.63%, and 5.3 ± 2.62%, respectively. By contrast, the cell survival values with the corresponding amounts of 177Lu-DOTA were 86.9 ± 5.07%, 84.5 ± 13.43%, and 35.3 ± 1.72%. Therefore, 177Lu-L1 significantly inhibited the survival of HeLa cells compared with 177Lu-DOTA, which was attributed to the tumor-targeting nature of the BTA analogue. When the cells were incubated with 9.25 MBq, 177Lu-L1 had a 7 times greater cell-killing effect than 177Lu-DOTA (P < 0.01). Radiation Dosimetry. The expected radiation-absorbed dose for the administration of 68Ga-L1, 64Cu-L1, and 177Lu-L1 to humans, which was derived from the mice biodistribution data, is shown in Table 1. In general, the effective dose (ED) of 64 Cu-L1 was higher than those of 68Ga-L1 and 177Lu-L1. The highest radiation-absorbed doses were found in the liver for 68 Ga-L1 (0.0373 mSv/MBq) and 64Cu-L1 (0.0928 mSv/MBq), followed by those in the kidney for 68Ga-L1 (0.0311 mSv/ MBq) and 64Cu-L1 (0.0317 mSv/MBq) and those in the lungs for 68Ga-L1 (0.0244 mSv/MBq) and 64Cu-L1 (0.0245 mSv/ MBq). The highest radiation-absorbed dose of 177Lu-L1 was in the kidney (0.0612 mSv/MBq), followed by the liver (0.0371 mSv/MBq) and the lung (0.0120 mSv/MBq). The mean ED of 68 Ga-L1, 64Cu-L1, and 177Lu-L1 was 0.0062, 0.1020, and 0.0054 mSv/MBq, respectively.

a

68

64

Ga-L1

2.81 7.72 9.01 3.91 1.16 1.46 2.80 2.17 8.03 3.11 3.73 2.44 4.29 1.49 2.63 1.23 9.70 6.35 1.80 6.11 1.25 8.02 9.39 1.39 3.86 6.26

× × × × × × × × × × × × × × × × × × × × × × × × × ×

−3

10 10−4 10−4 10−3 10−3 10−2 10−3 10−3 10−3 10−2 10−2 10−2 10−3 10−3 10−3 10−3 10−4 10−4 10−2 10−4 10−3 10−4 10−4 10−3 10−3 10−3

177

Cu-L1

5.69 2.31 1.57 9.30 1.75 2.41 7.84 4.17 2.19 3.17 9.28 2.45 5.15 2.39 5.30 2.09 1.58 1.03 2.23 7.76 1.97 1.05 1.32 2.18 6.19 1.02

× × × × × × × × × × × × × × × × × × × × × × × × × ×

−3

10 10−3 10−3 10−3 10−3 10−2 10−3 10−3 10−2 10−2 10−2 10−2 10−3 10−3 10−3 10−3 10−3 10−3 10−2 10−4 10−3 10−3 10−3 10−3 10−3 10−2

7.26 8.52 1.62 1.04 3.24 1.94 2.69 6.09 6.94 6.12 3.71 1.20 4.60 4.20 6.87 2.87 4.15 1.35 1.19 1.50 2.59 1.90 2.53 3.99 3.52 5.44

Lu-L1 × × × × × × × × × × × × × × × × × × × × × × × × × ×

10−4 10−4 10−4 10−3 10−4 10−2 10−3 10−4 10−3 10−2 10−2 10−2 10−3 10−4 10−4 10−4 10−4 10−4 10−2 10−4 10−4 10−4 10−4 10−4 10−3 10−3

ED: effective dose.

Because of the potential antitumor properties of BTA aniline, three types of radiopharmaceuticals of the form [RM(L)] [RM = 68Ga, 64Cu, 177Lu; L1 = DOTA-BTA; L2 = DOTA-BTANH2; L3 = DOTA-BTA-NO2] were designed and successfully synthesized for tumor diagnosis imaging and therapy. The radiochemical yield and purity of the complexes was high, as DOTA analogues generally have good metal coordination ability. The tumor cells employed in the cellular uptake experiments were from the human cervix adenocarcinoma cell line (HeLa), human embryonic kidney cell line (293), human breast cancer cell line (MDA-MA-231), and human glioblastoma cell line (U87MG). The results of the uptake experiments with a series of BTA analogues showed that in all the cells, 68Ga-L1 exhibited remarkably higher uptake than 68Ga-L2 and 68Ga-L3. Among the different cell lines, 68Ga-L1 exhibited especially strong uptake in the HeLa cells. Because of this result, we specifically selected L1 from the L conjugate series as an antitumor radiopharmaceutical candidate for HeLa cell, which was then confirmed by in vivo PET imaging of xenograft-bearing nude mice. Moreover, the uptake of 68Ga-L1 in a HeLa cell tumor was twice as high as its uptake in a MDA-MB-231 tumor (Figure S5), thereby demonstrating that L1 exhibits high tumortargeting ability in HeLa cells. The cellular uptake studies further confirmed the tumor-targeting ability of BTA analogues by comparing 177Lu-L1 with 177Lu-DOTA. Here, 177Lu-L1 showed 20-fold higher affinity than 177Lu-DOTA, potentially

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DISCUSSION The use of PET probes, such as the previously reported radiolabeled BTA derivatives, has been focused on Alzheimer’s disease (AD).28−30 Some 99mTc (or nat.Re) labeled complexes for cancer (i.e., SPECT) imaging and therapy have been reported, but these compounds have suffered from low uptake and resolution.10,11 To image (68Ga/64Cu) and treat (177Lu) tumors in mice bearing human cervical cancer cells, we introduced the first PET probes containing the BTA moiety. 1138

DOI: 10.1021/acs.molpharmaceut.7b00996 Mol. Pharmaceutics 2018, 15, 1133−1141

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Molecular Pharmaceutics indicating the former compound as an agent for tumortargeting therapy. Similar to peptide receptor radionuclide therapy (PRRT), the evaluation of the absorbed dose of radiation (dosimetry) generated by 177Lu-L1 in vivo can provide valuable information for treatment planning, dose−response analysis, and therapeutic efficacy predictions.31−33 177Lu, a medical radioisotope and β− particle emitter (Emax = 498 keV, 78%) with a long half-life (6.7 days), is well-known as a good candidate for the treatment of small tumors and micrometastases. Because it emits 208 keV (11%) and 113 keV (6.4%) γ radiation, 177Lu can also be used for SPECT imaging in vivo.21,22 177 Lu-labeled complexes capable of targeting tumor cells thus have the potential to be very useful for enabling simultaneous diagnosis and therapy; however, compared with 68 Ga complexes used for PET-CT imaging, few tumor-therapeutic assessments of these compounds, which require a dynamic scanning technique with high quantifiability and resolution, have been reported. In this respect, 177Lu-DOTA-TATE ([177Lu-DOTA0, Tyr3, Thr8] octreotide), a recent example of a compound employed in nuclear medicine, is used for treating neuroendocrine tumors and is used along with 68Ga-DOTATATE for dosimetry in clinical oncology.21 In a comparative analysis of radioactivity accumulation, 177Lu-L1 was demonstrated to have similar pattern to 68Ga-L1 compared to 64Cu-L1 with respect to the biodistribution and dosimetry results. Furthermore, the mean EDs of 68Ga-L1, 64Cu-L1, and 177Lu-L1, which, as shown in Table 1, ranged from 5−10 μSv/MBq, were lower than the in vivo radiation exposure of clinically used RGD-PET radiotracers (10−40 μSv/MBq).34 68Ga-L1 thus could be helpful for establishing pretherapeutic planning and dosimetry for the clinical use of 177Lu-L1. Similarly, the radioisotopes 124I/131I, 86Y/90Y, and 64Cu/67Cu are frequently used as matched pairs for diagnostics/therapy. Based on the in vivo clearance, physical/biological half-life, and tissue penetration of radiation, several studies have been performed to systematically match two medical radioisotopes to obtain improved tumor-theranostic radiopharmaceuticals.17,18 In addition, 64Cu-L1 was not observed to exhibit radiochemical degradation (radiochemical purity >93%) according to radio-TLC analysis of its in vitro serum stability, and 177LuL1 was stable for at least 36 h without evidence of degradation. The Lu(III)-DOTA complex is well-known to be more stable than Cu(II)-DOTA (Lu(III)-DOTA, log KML = 25.4; Cu(II)DOTA, log KML = 22.3).35 As expected, the log P value of 64CuL1 (−0.36 ± 0.02) indicated that the compound was slightly more hydrophilic than 177Lu-L1 (−0.12 ± 0.03). Furthermore, the difference in hydrophilicity between 177Lu-L1 and 64Cu-L1 stemmed from the charge effect of the complexes: overall, 177Lu (or 68Ga)-L1 has zero charge, whereas 64Cu-L1 has a negative charge (−1). After all, although in vitro 64Cu-L1 was showed good stability, 64Cu-L1 in biological studies (such as in Figures 2 and 3) was observed to have high liver uptake and slow liver clearance.36 The high liver uptake observed here can be explained by the low kinetic stability in vivo of copper complexes in aqueous solution, which is due to the “Jahn− Teller effect”.37

and 177Lu. Both 68Ga-L1 and 64Cu-L1 exhibited favorable tumor-targeting efficacy in in vivo PET imaging, and 177Lu-L1 achieved significant antitumor effects. For further preclinical or clinical studies, the in vivo dosimetry of these complexes was calculated.

CONCLUSION We designed three molecular platforms labeled with medical radioisotopes for tumor-theranostic applications, one of which, L1, was shown to be a good PET probe and radiotherapeutic in a preclinical mouse model when complexed with 68Ga, 64Cu,

(1) Bradshaw, T. D.; Westwell, A. D. The development of the antitumour benzothiazole prodrug, Phortress, as a clinical candidate. Curr. Med. Chem. 2004, 11, 1009−1021. (2) Shi, D. F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J.; Lelieveld, P.; Fichtner, I.; Stevens, M. F. G. Antitumor benzothiazoles. 3. Synthesis of 2-(4-aminophenyl)benzothiazoles and evaluation of their

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00996. Molecular formula strings (CSV), chemical compound characterization (ESI mass, HPLC spectra), in vitro stability data, biodistribution data, and in vivo animal PET/CT images of 68Ga-L1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: (+)82-2-970-1624. E-mail: [email protected]. *Phone: (+)82-2-970-1364. E-mail: [email protected]. ORCID

Ji-Ae Park: 0000-0001-6109-802X Yongmin Chang: 0000-0002-0585-8714 Kyeong Min Kim: 0000-0001-6284-0644 Jung Young Kim: 0000-0002-0755-3893 Author Contributions ∇

J.-A.P. and J.W.L. contributed equally to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Nuclear Research and Development Program of the National Research Foundation of Korea (NRF) grant funded by the Korean government (No. 2017M2A2A6A02019904) and grants of the Korea Institute of Radiological and Medical Sciences (KIRAMS) funded by Ministry of Science and ICT(MSIT), Republic of Korea. (No. 50532-2018).

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ABBREVIATIONS DO3A, 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid; DTPA, diethylenetriaminepentaacetic acid; DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; BTA, benzothiazole; SPECT, single-photon emission computed tomography; TRT, targeted radiotherapy; PET, positron emission tomography; HPLC, high-performance liquid chromatography; MRI, magnetic resonance imaging; ITLC, instant thin-layer chromatography; RT, room temperature; DMEM, Dulbecco’s modified eagle’s medium; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; PBS, phosphate buffered saline; OSEM, ordered-subset expectation maximization; ROI, regions of interest; ED, effective dose; TACs, time−activity curves

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