Development of a New FR-Targeting Agent

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The development of a new FR-targeting agent Tc-HYNFA with improved imaging contrast and comparison of multimerization and/or PEGylation strategies for radio-folate modification Zhide Guo, Linyi You, Changrong Shi, Manli Song, Mengna Gao, Duo Xu, Chengyu Peng, Rongqiang Zhuang, Ting Liu, Xinhui Su, Jin Du, and Xianzhong Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00536 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Molecular Pharmaceutics

The development of a new FR-targeting agent

99m

Tc-HYNFA with

improved imaging contrast and comparison of multimerization and/or PEGylation strategies for radio-folate modification

Zhide Guo†,‡, Linyi You†, Changrong Shi†, Manli Song†,#, Mengna Gao†, Duo Xu†, Chenyu Peng†, Rongqiang Zhuang†, Ting Liu†, Xinhui Su§, Jin Du‡, Xianzhong Zhang*,†

†

State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular

Imaging and Translational Medicine, School of Public Health, Xiamen University, 4221-116 Xiang’An South Rd, Xiamen 361102, China ‡

Department of Isotope, China Institute of Atomic Energy, P. O. Box 2108, Beijing 102413, China

#

The First Affiliated Hospital of Zhengzhou University, ZhengZhou 450000, China

§

Zhongshan Hospital Affiliated of Xiamen University, Hubin South Road, Xiamen 361004, China

1

ACS Paragon Plus Environment


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ABSTRACT This study aims to develop a new FR-targeting agent for SPECT imaging with improved contrast and evaluate the modification strategies of multimerization and/or PEGylation in the development of new radio-folates. A series of novel folate derivatives have been synthesized and radiolabeled with 99mTc using tricine and TPPTS as coligands. To better investigate their pharmacokinetics, cell uptake, biodistribution, and microSPECT/CT imaging were evaluated. Four radio ligands displayed high KB cell uptake after incubation for 2 h and 4 h. Pre-saturated with excess FA resulted in a significant blocking effect in KB cells, indicating specificity of these radioligands toward FR. Biodistribution and microSPECT imaging studies in KB tumor bearing mice showed that the folate conjugate

99m

Tc-HYNFA with PEG and triazole linkage displayed the highest tumor uptake

(16.30±2.01%ID/g at 2 h p.i. and 14.9±0.62%ID/g at 4 h p.i. in mice biodistribution) and best imaging contrast, indicating promising application prospect. More interestingly, the in vivo performance of this monomeric

99m

Tc-HYNFA was much better than that of FA multimers and

non-PEGylated monomers, suggesting that multimerization may be not a feasible method for the design of radio-folates. In conclusion, PEG linkage rather than FA multimerization should be taken into consideration in the development of folate-based radiopharmaceuticals in the future.

Keywords: 99mTc-labeling, folate receptor, SPECT/CT imaging, PEGylation, multimerization

2


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INTRODUCTION Folate receptor (FR) has been found to be overexpressed on the cell membrane of a wide variety of human tumors such as ovarian, breast, endometrial, colorectal and renal carcinomas,1-4 and is an ideal target for folate-functionalized probes and drugs. Owing to the ability of the folate conjugates with low molecular weight in specifically binding to macromolecules and nanoparticles, FR has been successfully used in the diagnosis and therapy of various cancers and inflammatory diseases.5-11 In the last decade, folate-based radiopharmaceuticals have been developed for diagnosis of FR-positive malignant tumors, and a variety of radionuclides (99mTc,

111

In,

67/68

Ga,

18

F) have

emerged as folate-based imaging agents for single photon emission computed tomography (SPECT) and positron emission tomography (PET).12-16 Among them,

99m

Tc is the most attractive

radionuclide due to its superior nuclear properties including the short half-life of 6 h, principal γ emission of 140.5 keV, easy acquisition, and the favorable price.17,18 PEGylation and multimerization are proposed to be two approaches to increase the targeting efficiency.19-21 In the present study, we intended to synthesize folate-targeted probes based on small polyethylene glycol (PEG) molecule and dendritic skeleton, respectively. As we know, three components should be indispensable parts for probe design, which were designated as bifunctional chelator (BFC), linker and targeting group in the present study.22 The linkers between the BFC and targeting biomolecule played an important role in the properties of corresponding conjugates. PEGylated dendritic skeleton with small chains acted as the stable linkers for multimerization design in the present study. Besides, 2-hydrazinonicotinic acid (HYNIC) as an efficient BFC was attached to one end of the linker via click reaction and (N-tris-(hydroxymethyl)-methylglycine)

and

99m

Tc-labeled by using tricine

trisodiumtriphenylpho-sphine-3,30,3″-trisulfonate

(TPPTS) as coligands.23 Meanwhile, folic acid (FA) was conjugated to the other end as a targeting moiety for specifically binding to FR on tumor cell membranes. 3



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Previously, we have reported three 99m

Tc-HP3FA24,

99m

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Tc-labeled folate conjugates including multimeric

monomeric99mTc-HYNIC-T-FA25,

and

dimeric

99m

Tc-HYNIC-D1-FA226 as

FR-targeted agents. In order to further understand the effectiveness of multimerization or PEGylation on the in vivo imaging performance, here we developed several new 99mTc-folates with multimerization and/or PEGylation and compared their tumor-targeting properties with those previously reported ones via biodistribution and SPECT imaging. The structures of radio-folates were

shown

in

Figure

1a

and

1b,

including

Tc-HYNIC-T-FA

Tc-HYNIC-T-PEG-FA

99m

Tc-HYNIC-D1-PEG-FA2 and

99m

Tc-HP3FA. The HP3FA was a PAMAM-based (polyamidoamine-based) folate multimer with 7

99m

as

Tc-HYNFA);

dimers:

Tc-FA-D1-HYNIC2; and multimers:

99m

99m

and

99m

(denoted

99m

99m

monomers:

Tc-HYNIC-D1-FA2,

Tc-HYNIC-D2-FA4 and

folate groups reported by Song et al..24 HYNIC-T-FA and HYNIC-D1-FA2 were prepared via click reaction.25,26 In particular, HYNIC-D1-FA2 was a dimer with two folate moieties, and HYNIC-D2-FA4 was a tetramer with four folate moieties. Based on

99m

Tc-HYNIC-T-FA, a

hydrophilic PEG linker was added to increase the distance between targeting moiety and chelating group to get a new probe 99mTc-HYNFA. While 99mTc-HYNIC-D1-PEG-FA2 was a folate dimer with two identical targeting elements modified with hydrophilic linkers. On the contrary, 99m

Tc-FA-D1-HYNIC2 was a HYNIC dimer with two identical chelating groups, which was also

based on a unique branching structure. In the present study, these folate-based radiopharmaceuticals on our platform were selected to in-depth investigate the influence of the linker modification and targeting or chelating groups on the pharmacokinetics of probes. Several researches previously showed that forming dimmers or modification with PEG were effective strategies to improve tumor target. Among the conjugates, multimeric RGD peptides (cyclic arginine-glycine-aspartic peptides) used for αvβ3-targeted radiotracers can be taken as representatives.20,27 Although many construction of folate-based conjugates reported in the literature benefited from these modifications and design strategies, little attention has been concentrated on which is a better strategy for developing folate-based 4


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radiopharmaceuticals.

EXPERIMENTAL SECTION Methods and Materials. The Na99mTcO4 (technetium-99m as sodium pertechnetate) was obtained from Zhongshan Hospital Affiliated of Xiamen University. 18F-FDG was obtained from the First Affiliated Hospital of Xiamen University. γ-counter (WIZARD 2480, Perkin-Elmer, USA) and CRC-25R Dose Calibrators (CAPIN-TEC Inc., USA) were used for the radioactivity count. SPECT imaging study was performed using a nanoScan-SPECT/CT preclinical scanner (Mediso, HUNGARY). PET/CT scan with

18

F-FDG was performed using an Inveon micro PET/CT scanner

(Siemens Medical Solutions Inc., USA). 1HNMR spectra were recorded on a Bruker (400 MHz) spectrometer. FT-IR spectrum (AVATAR 360, Nicolet Company, USA) was used to analyze reaction processes. The UV spectroscopy was analyzed using microplate spectrophotometer (Multiskan GO, Thermo Fisher, USA). Mass spectra (MS) were performed using a Brucker Apex IV FTM instrument. Kit formulation was lyophilized using lyophilizer (Freezone Triad 2.5L, LABCONCO, USA). DionexUlti-Mate 3000 HPLC (Thermo Scientific, USA) with flow-counter radioactivity detector (BioScan, USA) was used to test the radiochemical purity. Animal Model. KB tumor-bearing mice models were established with nude mice (female, aged 4-5 weeks). Approximately 5×106 cultured KB cells were suspended in PBS and subcutaneously implanted in the shoulder of mice. Mice were maintained on a folate-free diet for 7 days before the biodistribution and imaging studies. All the mice were obtained from Laboratory Animal Center of Xiamen University. Animal studies were carried out in compliance with the national laws related to the conduct of animal experimentation. Synthesis of Folate Conjugates. The folate conjugates used in the present study were prepared and characterized on the basis of our previous studies.25,26 The details were described in the 5



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supplementary file. Radiolabeling. A “kit-like” method was developed for radiolabeling. The radio-folates were prepared by addition of the

99m

TcO4- (370 MBq, 1.5 mL) to the folate kits containing folate

conjugates and other auxiliaries (as shown in Figure 1b). The chemical purities and identities of the radio-folates were examined by analytical HPLC (High Performance Liquid Chromatography). Octanol/Water Partition Coefficient. The octanol/water partition coefficients (log P) were obtained by following a reported procedure using phosphate buffer (PB, 0.02 mol/L, pH=7.0) and octanol.25,26 In a centrifuge tube, the purified radiotracer solution (370 kBq/100 µL) was mixed with 2.9 mL of PB and 3 mL of octanol. After blending the solution, the mixture was separated by centrifuging and partitioning. The counts of 100 µL organic layer and 100 µL inorganic layer were determined by γ-counter. The partition coefficient (log P) was calculated using the following equation: P = (activity in octanol−background activity)/(activity in aqueous layer−background activity). All experiments were performed in triplicate. Stability Experiments. The radiochemical stabilities of radio-folates in saline and mouse plasma (at 37 °C for 6 h) were analyzed by ITLC SG strips/ACD (acid-citrate-dextrose buffer, 0.068 mol/L citrate, 0.074 mol/L glucose, pH 5.0). All the strips were detected with MiniScan (BioScan). In Vitro Experiments. Cell uptake of radio-folates were studied in vitro using KB tumor cells. KB cells in 24-well plates were incubated with radiolabeled folate for various lengths of time at 37 °C. At different time points, the cells were washed with 0.5 mL cold PBS to remove dissociative radioactivity, then washed with 500 µL stripping buffer (pH 4.0, including 0.2% bull serum albumin) two times. The stripping buffer was collected in a tube. Finally, the cells were eluted by adding 0.5 mL NaOH (1 mol/mL) for 5-10 min. Similarly, the cells eluted by NaOH were collected in a tube. For blocking study, 10 µL FA (1mg/mL) was added into the incubation medium before the radiolabeled folate treatment. After incubation, 0.5 mL NaOH was added to elute the cells. All the counts were determined by γ-counter. All cell uptake studies were performed in triplicate. 6


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Biodistribution. For biodistribution, about 370 kBq (about 0.1 µg folate compound, in 100 µL PBS 7.4) of the radiolabeled folate was injected via a lateral tail vein for each mouse (n=3). The mice were sacrificed by decapitation at 2 and 4 h after injection of radiolabeled folate. For FA blocking study, the biodistribution of radio-folates was also evaluated following pre-injection with excess FA (100 µg). The interested tissues and organs were dissected, weighed and measured in γ-counter. The results were showed as a percentage of the injected dose per gram of tissue (%ID/g). Imaging Study. To further investigate the FR binding properties of radio-folates, SPECT imaging was performed using athymic nude mice bearing KB tumor. Animals were injected with 18.5 MBq of the radiotracers (100 µL per injection) via a lateral tail vein. For PMX (pemetrexed) blocking study of 99mTc-HYNFA, each mouse received 400 µg of PMX (2 mg/mL PMX in PBS) via intravenous injection 10 min prior to the radiotracer injection.28,29 Mice were anesthetized with isoflurane/air mixture and scanned with a nanoScan-SPECT/CT preclinical scanner equipped with low energy high-resolution collimators. The acquiring parameters were as follows: energy peak of 140.5 keV for

99m

Tc, window width of 20%, matrix of 256×256, medium zoom, and frame: 30 s.

Dynamic semiquantitative SPECT/CT imaging was acquired from 0 to 60 min after the 99m

Tc-HYNFA injection, 20×120 s. Time-radioactivity curves (TACs) were calculated by drawing

regions of interests (ROIs) on the SPECT/CT images. To test the specific binding of 99mTc-HYNFA, a blocking study was performed by pre-injection of free FA (100 µg FA in 100 µL PBS, pH=7.4) as a blocking agent 1 h prior to the intravenous injection of

99m

Tc-HYNFA (18.5 MBq in 100 µL PBS 7.4). Then the microSPECT imaging data

were acquired at 2 h after radiotracer injection. For a comparison, mice were injected with

18

F-FDG (18F-fluorodeoxyglucose, 3.7 MBq) and

scanned by PET/CT at 60 min after injection.

7



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RESULTS Chemistry and Radiolabeling. The synthesis routes of folate conjugates were shown in Scheme S1-S5. In the present study, propargyl-PAMAM dendron D1, D2 and hydrophilic PEG were served as linkers between FA and BFC. Carboxyl groups of FA were activated by NHS and DCC to afford FA-NHS.30,31 There were two forms of carboxylic acid (γ and α) presented on folate molecules. Comparatively, the γ-carboxylic group had higher reactivity relative to the α-carboxylic one. In this case, the reaction occurred mainly at the γ-carboxylic group rather than the α-carboxylic group via controlled the additions of NHS and DCC.24-26 The PEG is a low-molecular-weight polyethylene glycol with terminal amino groups. In order to decrease the byproduct folate-PEG-folate, FA-NHS was slowly added to excess PEG. The D1 and D2 were synthesized using propargylamine as a propargyl focal point, which had controlled divergent arms. The 3-azidopropyl-1-amine and dendrons with small molecular weights were confirmed by 1H NMR (Figure S1). Click chemistry is an efficient strategy and can be identified by IR spectra (alkynes and azides forming 1,2,3-triazole linkages using the Cu(I)-catalyzed [3+2] cycloaddition).32,33 The chemical structure of folate conjugates was characterized by IR (Figure S2) and UV (Figure S3) spectroscopy. Alkynyl and azido groups had characteristic peaks around 2100 cm-1 in FT-IR spectrums. The disappearance of characteristic peak indicated the successful click reaction of the alkyne and azide groups. Comparing the UV absorption peaks of FA and final products, the shift of peak positions (286 and 350 nm) showed that the folate groups have been introduced into the target molecules successfully. The 1H NMR spectrums (Figure S4) demonstrated that the folate compounds had been successfully synthesized. The characteristics of radio-folates were summarized in Table S1. By forming a ternary ligand system (tricine and TPPTS as co-ligands), the radio-folates were obtained in high radiochemical yield of above 90% (TLC method, Table S1) and radiochemical purity of above 95% (analyzed by radio-HPLC, Figure S5-S6 and Table S1). The specific activities of radiolabeled folates were above 8


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4 MBq/nmol. And the log P of 99mTc-HYNFA, 99mTc-HYNIC-D1-PEG-FA2, 99mTc-HYNIC-D2-FA4 and

99m

Tc-FA-D1-HYNIC2 were -2.54 ± 0.10, -2.63 ± 0.08, -2.33 ± 0.12 and -2.32 ± 0.17,

respectively, suggesting their well hydrophilic properties. Compared to FA derivatives previously investigated on our radio-folate platform, radiotracers with PEG linker had the better solubility (the data of solubility were shown in Figure 1b). High stability of these radio-folates in saline and mice serum was observed at different time, which was determined by ITLC-SG/ACD system (Figure S6, S7 and Table S2). There were no obvious signs of decomposition of the radiotracers over a time period of 6 h. In Vitro Experiments. As shown in Figure S8 and Table S3, during 4 h, the observed cell uptake of radiolabeled folate was increased gradually.

99m

Tc-HYNIC-D2-FA4 had the lowest total bindings

(25±2.7% and 35±1.6% after 2 h and 4 h incubation, respectively). By comparison,

99m

Tc-HYNFA

showed good merits in the cell uptake (57±2.9% and 70±3.1% after 2 h and 4 h incubation respectively). The addition of free FA in KB cells resulted in a sharp drop of the cell uptake (

Development of a New FR-Targeting Agent

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