Radiometallic Complexes of DO3A-benzothiazole Aniline (BTA) for

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Radiometallic Complexes of DO3A-benzothiazole Aniline (BTA) for Nuclear Medicine Theranostics Ji Ae Park, Ji Woong Lee, Hee-Kyung Kim, Un Chol Shin, Kyo Chul Lee, TaeJeong Kim, Yongmin Chang, Kyeong Min Kim, Yong Jin Lee, and Jung Young Kim Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00996 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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Molecular Pharmaceutics

Radiometallic Complexes of DO3A-benzothiazole Aniline (BTA) for Nuclear Medicine Theranostics Ji-Ae Park,1,† Ji Woong Lee, 1,† Hee-Kyung Kim,2,3 Un Chol Shin,1 Kyo Chul Lee,1 Tae-Jeong Kim,2 Yongmin Chang,4,5 Kyeong Min Kim,6 Jung Young Kim,1,* and Yong Jin Lee,1,*

1

Division of RI-Convergence Research, Korea Institute of Radiological and

Medical Sciences, Seoul 139-706, Korea, 2Institute of Biomedical Engineering Research and

3

BK21 Plus KNU Biomedical Convergence Program,

Kyungpook National University, Daegu 702-701, Korea, 4Department of Molecular Medicine and 5Department of Radiology, Kyungpook National University, Daegu 702-701, Korea, 6Division of Medical Radiation Equipment, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Korea

Keywords: Benzothiazole, Radiopharmaceuticals, 68Ga, 64Cu, 177Lu

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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 acid-benzothiazole 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 (DOTA) complex. According to in vivo positron emission tomography (PET) imaging, high accumulation of

visualized in the tumor site, while

68

Ga-L1 and

64

Cu-L1 was clearly

177

Lu-L1 showed therapeutic efficacy in

therapy experiments. Consequently, this molecular platform represents a useful approach in nuclear medicine toward tumor-theranostic radiopharmaceuticals when 68Ga-L1 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.

2


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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 made to modify these heterocycles to improve their antitumor activity (5-9). Furthermore, the potential abilities of cancer treatment have been tried to develop the 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 =

99m

Tc(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 complexes with DO3A-BTA conjugates as a single molecule theranostic agent (15, 16). The complexes provided tumorspecific and enhanced intracellular MR images of the cytosols and nuclei of 3



tumor

cells

such

antiproliferative

as

MCF-7,

activity

of

MDA-MB-231,

DO3A-BTA

and

Page 4 of 41

and

SK-HEP-1.

The

Gd(DO3A-BTA)

was

demonstrated by determining their in vitro growth inhibition values (GI50 and TGI) and monitoring tumor volume regression in vivo. Based on these observations, we attempt to develop tumor-theranostic metal complexes of DO3A-BTA conjugates labeled with the therapeutic radioisotope

177

Lu and the

diagnostic radioisotopes 68Ga/64Cu in the same molecular structure. Efforts toward the development of analogous complexes with 68

Ga/90Y,

64

Cu/67Cu and

99m

Tc/188Re,

64

Cu/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 (T1/2 = 67.7 m) and therapy,

64

68

Ga

Cu (T1/2 = 12.7 h) (19, 20). As a radiometal for tumor

177

Lu (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,

64

Cu and

177

Lu complexes of DO3A-BTA aniline

conjugates were investigated for their in vivo PET imaging ability and in vitro 4


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cell therapeutic efficacy in a mice model of human cervical cancer and for their radioactive accumulation in a biodistribution study. 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-yl-phenylcarbamoyl)methyl]-7,10-bis-carboxymethyl-1,4,7,10,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 (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). 5



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Synthesis of 68Ga-Ls (L1, L2 and L3). For the elution of the radiolabeling, an eluate of

68

68

Ge/68Ga generator, HCl (1.0 M) was used. For

Ga (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. L1-3 (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 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 64CuCl2 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 6


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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 additionally 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 radioTLC scanner via elution with citrate buffer (0.1 M) on instant thin layer chromatography (ITLC) paper, and the formation of

64

Cu-L1 was further

corroborated by radio-HPLC. For the purpose of chemical structure confirmation, a natural analogue of

64

Cu-L1,

nat.

Cu-L1, 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

64

Cu-L1 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 (Fig. S1 in the Supporting Information) and Rt (nat.Cu-L1) = 8.2 min (Fig. S2);

7



Page 8 of 41

MALDI-TOF-MS: m/z = 672.3 (C29H34CuN6O7S, calculated MW = 673.15) (Fig. 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 down to RT. As mentioned above, it was performed with additionally purification of C18 cartridge. The radiochemical purity of the two complexes was confirmed by a radio-TLC scanner. whereas

177

177

Lu-DOTA was used here without further purification,

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.

8


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The radiochemical stability of 68Ga-L1, 64Cu-L1 and by an ITLC method (vide supra).

68

Ga-L1,

64

177

Lu-L1 was assessed

Cu-L1 and

177

Lu-L1 were

separately incubated at 37 °C in human serum and mouse serum for different intervals, with free

64

Cu and

177

Lu used as references. The mobile phases for

68

Ga-L1 and 64Cu-L1 were citrate acid (0.1 M) and for

177

Lu-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

9



Page 10 of 41

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, MDA-MB-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 68GaLs 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

177

Lu-DOTA and

177

Lu-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 MDA-MB-231 cells 10


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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 kBq/0.1 mL) or

177

68

Ga-L1 (740 kBq/0.1 mL),

64

Cu-L1 (340

Lu-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 with 1.5% isoflurane and then injected with

68

Ga-L1 (18.5

MBq/0.1 mL) or 64Cu-L1 (7.4 MBq/0.1 mL) via tail vein. Thirty-minute static scans were acquired at 1 h and 4 h after injection with 68Ga-L1 and at 1 h, 4 h, 18 h and 36 h after injection with 64Cu-L1. Fourier rebinning and 2-dimensional 11



Page 12 of 41

ordered-subset 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 interests (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 cell. 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

177

Lu-DOTA and

177

Lu-L1 and incubated at

37 °C for 60 min. After washing twice with 10 mL of PBS, the cells were detached and seeded into 6-well 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 means ± SD (n = 4).

12


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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 177Lu-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:

The extrapolated TACs were used to calculate the effective dose (ED, mSv·Bq-1) 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, USA).

Statistical analysis

13



Page 14 of 41

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 considered significantly different.

RESULTS Radiochemistry 68

Ga-Ls (L1, L2 and L3), 64Cu-L1,

177

Lu-DOTA and

177

Lu-L1 were labeled

with a radiochemical purity of 98% or more. In particular, L1 could be quickly labeled with any of 68Ga, 64Cu or

177

Lu at 100 °C within 5 min and then further

purified to remove radioactive impurities using a C18 Sep-Pak cartridge, which afforded

64

Cu- (or

177

Lu)-L1 with a radiochemical purity that was normally

greater than 98%. Furthermore, the chemical structure of established by characterizing its natural analogue,

nat.

64

Cu-L1 was

Cu-L1, via analytical

HPLC (Fig. S4). The retention times (Rt) of nat.Cu-L1 and 64Cu-L1 were 9.0 min and 9.5 min, respectively, thereby demonstrating that the difference between the retention times of

nat.

Cu-L1 and

64

Cu-L1 was slight (0.5 min), particularly

considering the distance gap of 30 cm between the RI and UV detectors of 14


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HPLC system. In addition, after purification with the Sep-Pak cartridge, the specific activities of

68

Ga-L1,

64

Cu-L1,

177

Lu-L1 and

177

Lu-DOTA (without

cartridge) in all biological studies were 342.1 ± 4.9, 219.5 ± 11.9, 44.4 ± 1.3 and 39.2 ± 4.0 GBq/µmol, 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-MB231 and U87MG cells revealed that all three complexes (68Ga-L1, L2 and L3) exhibited high uptake in the HeLa cells (Figure 1 (A)). In all the cells, 68Ga-L1 showed particularly higher uptake than 68Ga-L2 and level of

68

that of

68

68

Ga-L3. Specifically, the

Ga-L1 in HeLa cells (3.31 ± 0.03% ID) was ten times greater than

Ga-L3 (0.36 ± 0.08% ID) (P < 0.001). Consequently, the well-

validated L1 ligand was used as a targeting molecule for in vitro and in vivo evaluation. Next, we determined the cellular uptake of

177

Lu-DOTA and

177

Lu-

L1 in HeLa cells. As shown in Figure 1 (B), 177Lu-L1 exhibited very high uptake compared with indicating that

177

177

Lu-DOTA and maintained its signal for up to 90 min, thus

Lu-L1 shares the tumor-targeting ability that is characteristic

of BTA analogues. In vitro characterization

15



Page 16 of 41

The determination of the radiochemical stability of the complexes in human and mouse serum and PBS indicated that both 64Cu-L1 and

177

Lu-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

177

Lu-

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

177

Lu-L1

are presented in Figure 2A, B and C, 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 h, 2 h 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 ID/g) and

177

64

Cu-L1 (21.01% ID/g) was higher than that of

68

Ga-L1 (7.13%

Lu-L1 (3.70% ID/g), which may be explained by the in vivo

transmetalation of

64

Cu. The intestinal uptake of

64

Cu-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 h, 4 h, 18 h and 36 h p.i. was determined to be 3.40, 2.60, 2.73 and 2.06% ID/g, respectively.

177

Lu-L1 (5.56 and 3.08%

ID/g at 1 h and 4 h p.i., respectively) had significantly higher blood uptake than 16


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68

Ga-L1 (2.46 and 0.87% ID/g at 1 h and 4 h p.i., respectively) and

64

(0.98 and 1.93% ID/g at 1 h and 4 h p.i., respectively). The uptake of

177

Cu-L1 Lu-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 h, 4 h, 18 h and 36 h p.i., respectively. The tumor/muscle ratio of 64Cu-L1 was higher than those of 68Ga-L1 and

177

Lu-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 Figure 3A and 3B show animal PET images of

68

Ga-L1 and

64

Cu-L1 in

HeLa cell tumor-bearing mice. High accumulation of 68Ga-L1 and 64Cu-L1 was clearly visualized in the tumor site. The tumor uptake of 68Ga-L1 was 3.4 and 2.4% ID/g at 1 h and 4 h p.i., respectively, which were consistent with the biodistribution data. For

64

Cu-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 h, 4 h, 18 h and 42 h p.i., respectively. These data were fully consistent with the values observed in the biodistribution studies.

64

Cu-L1 was mainly cleared

through the hepatic pathway. As shown in Figure 3C, the gallbladder and 17



Page 18 of 41

intestinal uptake of 64Cu-L1 was significantly higher than the liver uptake at 1 h and 4 h p.i. In vitro therapy 177

Lu-L1 and

177

Lu-DOTA induces dose-dependent inhibition of tumor cell

survival. Survival values are shown as percent survival compared to the control. Figure 4 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

177

Lu-DOTA were 86.9 ± 5.07%, 84.5 ± 13.43%

and 35.3 ± 1.72%. Therefore, HeLa cells compared with

177

Lu-L1 significantly inhibited the survival of

177

Lu-DOTA, which was attributed to the tumor-

targeting nature of the BTA analogue. When the cells were incubated with 9.25 MBq,

177

Lu-L1 had a 7 times greater cell killing effect than

177

Lu-DOTA (P
93%) according to radio-TLC analysis of its in vitro serum stability, and

177

Lu-L1 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 64Cu-L1 (-0.36 ± 0.02) indicated that the compound was slightly more hydrophilic than

177

Lu-L1 (-0.12 ± 0.03).

Furthermore, the difference in hydrophilicity between

177

Lu-L1 and

stemmed from the charge effect of the complexes: Overall,

64

Cu-L1

177

Lu (or 68Ga)-L1 22


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has zero charge, whereas 64Cu-L1 has a negative charge (-1). After all, although in vitro 64Cu-L1 was showed good stability,

64

Cu-L1 in biological studies as

Figure 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).

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

Ga,

64

Cu and

177

Lu. Both

68

Ga-L1 and

64

Cu-L1 exhibited favorable tumor-

targeting efficacy in in vivo PET imaging, and

177

Lu-L1 achieved significant

antitumor effects. For further preclinical or clinical studies, the in vivo dosimetry of these complexes was calculated.

TABLE and FIGURES 23



Table 1. Dosimetry of 68Ga-L1, 64Cu-L1 and

177

Page 24 of 41

Lu-L1 extrapolated to an adult

human based on the biodistribution data (n = 4). (ED: Effective Dose)

24


Page 25 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chart 1. Benzothiazole-based antitumor molecular platforms.

25



Page 26 of 41

Figure 1. (A) Cellular uptake studies of 68Ga-Ls using 293, HeLa, MDA-MB231 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

Radiometallic Complexes of DO3A-benzothiazole Aniline (BTA) for

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