Folate-PEG-NOTA-Al18F: A New Folate Based Radiotracer for PET

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Folate-PEG-NOTA-Al18F, A New Folate Based Radiotracer for PET imaging of Folate Receptor-Positive Tumors Qingshou Chen, Xiangjun Meng, Paul McQuade, Daniel Rubins, Shu-an Lin, Zhizhen Zeng, Hyking Haley, Patricia Miller, Dinko Gonzalez Trotter, and Philip S. Low Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00415 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Molecular Pharmaceutics

Folate-PEG-NOTA-Al18F, A New Folate Based Radiotracer for PET imaging of Folate Receptor-Positive Tumors Qingshou Chen,†♦ Xiangjun Meng,‡ ♦ Paul McQuade,‡ Daniel Rubins,‡ Shu-An Lin,‡ Zhizhen Zeng,‡ Hyking Haley,‡ Patricia Miller,‡ Dinko González Trotter‡ and Philip S. Low†* †

Department of Chemistry, Purdue University, 720 Clinic Drive, West Lafayette, IN 47907 Tel: (765) 494-5273. Fax: (765) 494-5272. E-mail: [email protected]. ‡ Imaging, Merck Research Laboratories, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States ♦ Both authors contributed equally to this work

Corresponding author Philip S. Low, Ph.D. Professor Department of Chemistry, Purdue University, 720 Clinic Drive, West Lafayette, IN 47907 Tel: (765) 494-5273. Fax: (765) 494-5272. E-mail: [email protected]. First author Qingshou Chen, Ph.D. Department of Chemistry, Purdue University, 720 Clinic Drive, West Lafayette, IN 47907 Tel: (765) 494-5273. Fax: (765) 494-5272. E-mail: [email protected].

Folate-PEG-NOTA-Al18F for PET Imaging of FR+ Tumor ACS Paragon Plus Environment


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ABSTRACT The folate receptor (FR) has been established as a promising target for imaging and therapy of cancer (FR-α), inflammation, and autoimmune diseases (FR-β). Several folate-based PET radiotracers have been reported in the literature, but an 18Flabeled folate-PET imaging agent with optimal properties for clinical translation is still lacking. In the present study, we report the design and preclinical evaluation of folate-PEG12-NOTA-Al18F (1), a new folate-PET agent with improved potential for clinical applications. Methods: Radiochemical synthesis of 1 was achieved via a onepot labeling process by heating folate-PEG12-NOTA in presence of in situ prepared Al18F for 15 min at 105 °C, followed by HPLC purification. Specific binding of 1 to FR was evaluated on homogenates of KB (FR-positive) and A549 (FR-deficient) tumor xenografts in the presence and absence of excess folate. In vivo tumor imaging with folate- PEG12-NOTA-Al18F was compared to imaging with

99m

Tc-EC20 using

nu/nu mice bearing either KB or A549 tumor xenografts. Specific accumulation of 1 in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution with in the presence and absence of excess folate. Results: Radiosynthesis of 1 was accomplished within ~35 min, affording pure radiotracer 1 in 8.4 ± 1.3% (decay corrected) radiochemical yield with ~ 100% radiochemical purity after HPLC purification and a specific activity of 35.8 ± 15.3 GBq/mmol . Further in vitro and in vivo examination of 1 demonstrated highly specific FR-mediated uptake in FR+ tumor, with Kd of ~0.4 nM (KB), and reduced accumulation in liver. Conclusions: Given its facile preparation and improved properties, the new

ACS Paragon Plus Environment

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radiotracer, folate-PEG12-NOTA-Al18F (1), constitutes a promising tool for identification and classification of patients with FR over-expressing cancers. Keywords: folate receptor, folate conjugate, Al18F-NOTA, 18F-PET imaging, cancer imaging

INTRODUCTION Folic acid performs an essential role in methylation and methyl group transfer reactions in vivo,1 and the uptake of folic acid is facilitated by folate receptors (FRs). Four FR isoforms (FR-α, FR-β, FR-γ, and FR-δ) have been identified and characterized, with FR-α being over-expressed and fully accessible on many epithelial-derived cancers26

but nearly absent in most healthy tissues.2,

5

Due to its highly disease-restricted

expression pattern, FR-α is now well accepted as a promising receptor for development of targeted diagnostics and therapeutics for cancers.7-9 As FR shows roughly equal binding affinity to folate and folate conjugates (Kd = 10-9 M),10,

11

and exhibits internalization via receptor-mediated endocytosis, folic acid has

been often employed to deliver diagnostic and therapeutic agents selectively to pathologic cells that over-express FR.7-9, 11 A series of folate receptor-targeted imaging agents have been developed for optical,12-18 magnetic resonance19-25 and nuclear imaging26-30 of cancers (ovarian cancer, non-small lung cancer, kidney cancer, and endometrial cancer, etc.) and sites of inflammation (e.g. rheumatoid arthritis, atherosclerosis, and pulmonary fibrosis etc.). Given the intrinsic decay characteristics of positron emission tomography (PET), i.e. higher sensitivity, better spatial resolution and greater signal to noise ratio,31, 32 folate based PET imaging agents have attracted growing



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interest for accurate determination of FR-expression levels in patients33 who could potentially benefit from FR-targeted therapy. Of the radionuclides (e.g. 18F,

89

Zr,

66

Ga,

68

Ga,

64

Cu, and

124

I) that are commonly

employed for PET imaging, 18F is often preferred due to its commercial availability34 and optimal physical decay characteristics (t1/2 = 110 min, 97% β+ decay with Eβ+,max = 0.63 MeV), which allow multistep syntheses and enable distribution to clinics with limited access to radiochemistry. A variety of folate based

18

F PET agents have already been

developed and shown to yield highly resolved images of FR positive tumors in preclinical studies.35-49 Several radiolabeling strategies have been explored in the literature either through direct radio-labeling of 18F- on the backbone of folic acid 40-43 or via conjugating to 18

18

F- labeled prosthetic groups.27, 35-39, 44-46, 50, 51 Given the relatively short half-life of

F- (~ 110min), it is preferred to introduce

18

F- at later stages of radioimaging agent

synthesis.52-54 Our group has previously developed a new folate-NOTA-Al18F radiotracer, in which a NOTA chelator was introduced to facilitate one-pot 18F- radiolabeling through chelating of NOTA to in situ prepared Al18F.55 The radiochemical synthesis of folateNOTA-Al18F proved to be efficient, and the following in vitro and in vivo studies of folate-NOTA-Al18F displayed a high and specific binding affinity to FR, demonstrating potential as a PET imaging agent for clinical application. However, the subsequent micro-PET imaging and ex vivo biodistribution studies also revealed moderate radiotracer uptake in the liver, indicating that an unfavorable hepatobiliary excretion might be caused by the relative high lipophicility of the conjugate. Our motivation for seeking a folatetargeted PET agent in clinical application prompted us to develop a new radiotracer with ideal radiopharmaceutical properties for high quality of PET imaging. Therefore, folate-


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PEG12-NOTA-Al18F was designed, in which a PEG12 unit was introduced to improve the hydrophilicity of the folate conjugate. Herein, we report the chemical synthesis and onestep radiochemical labeling of folate-PEG12-NOTA to prepare folate-PEG12-NOTA-Al18F PET imaging agent and describe its improved radiopharmaceutical properties both in vitro and in vivo.

MATERIALS AND METHODS General Distilled water was obtained by deionization (18 MΩ/cm2) on a Milli-Q water filtration system (Millipore Corp., Milford, MA, USA). All chemicals and solvents, unless otherwise specified, were purchased from Sigma (St. Louis, MO, USA) and were used without further purification. Trt-EDA resin was purchased from CHEM-IMPEX (Wood Dale, IL, USA). 2,2'-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7triazonane-1,4-diyl)diacetic acid (NOTA-NHS) was obtained from CheMatech (France). N10-TFA-pteroic acid was kindly provided by Endocyte, Inc. The diammonium salt of 3

H-folic acid (37 MBq/mL, 1483.7 GBq/mmol) was purchased from Moravek

Biochemicals (Brea, CA, USA). The folate-PEG12-NOTA PET precursor and all other intermediates were synthesized as described below. High-performance liquid chromatography (HPLC) analysis and purification of the folate-NOTA precursor were performed on an Agilent 1200. Carrier-free

18

F-fluoride was secured from PETNET

(Malvern, PA, USA). The scintillation fluid, Ultima Gold high-flash-point liquid scintillation cocktail, was purchased from Packard Instrument Co. (Meridien, CT, USA). Folic acid deficient RPMI cell culture medium (lacking folic acid, vitamin B12, and



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phenol red) was obtained from Cell Culture Technologies GmbH. Raw data acquired in vitro were analyzed using GraphPad Prism 4 software, and PET imaging files were evaluated using MATLAB (MathWorks, Natick, MA, USA). Chemistry Compound 3 was synthesized through an efficient solid phase synthesis. Briefly, Fmoc-PEG12-OH was introduced to 1,2-diaminoethane trityl resin under treatment of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate

(HATU)

and

N,N-diisopropylethylamine

3-oxid (DIPEA)

in

dimethylformamide (DMF). The Fmoc- protecting group was then removed by incubating the resin in 20% piperidine in DMF for 30 min then followed by washings with DMF and 2-propanol (i-PrOH). The above procedure was repeated for two additional peptide coupling steps for linkage of Fmoc-Glu-(OtBu)-OH and N10-TFAPtc-OH.

The

final

product

was

cleaved

from

the

resin

using

a

TFA:H2O:triisopropylsilane cocktail (95:2.5:2.5) and concentrated under vacuum. The resulting residue was then incubated in sat. Na2CO3, followed by purification using RP-C18 HPLC to yield pure folate-PEG12-EDA-NH2 (3) as a yellow solid, which was characterized by LC-MS. The synthesis of folate-PEG12-NOTA (2) was accomplished by the coupling of folate-PEG12-EDA-NH2 (3) to NOTA-NHS ester in DMSO, followed by HPLC purification (Figure 1A).

Radiochemistry A cartridge containing 18F-fluoride was washed with 1.5 mL of pure water and then

18

F-fluoride was eluted using 1.0 mL of 0.4 M KHCO3. The eluted 18F-fluoride


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solution (100 µL, ~3.7 GBq) was added to a stem vial charged with 10 µL acetic acid, 50 µL AlCl3 (2 mM in 0.1 M NaOAc buffer, pH 4) and 125 µL 0.1 M NaOAc pH 4. The solution was incubated for 2 min at room temperature, mixed with 0.50 mg folate-PEG12NOTA precursor (2) in 125 µL of 0.1 M NaOAc, pH 4, and heated immediately to 100 °C for 15 min. It was then transferred to a vial containing 0.7 mL of 0.1% formic acid, mixed and subjected to HPLC purification [Waters Xselect CSH C18 (250×10 mm, 130 µm)] using a gradient of 10-15% MeCN for 15 min at a flow rate of 5 mL/min, the balance being 0.1% formic acid. The peak corresponding to folate-PEG12-NOTA-Al18F was collected, the MeCN was removed in vacuo to yield folate-PEG12-NOTA-Al18F (1), Figure 1B). Specific activity and radiochemical purity were determined via a Waters Acquity LC/MS system (Milford, MA, USA) and a β-RAM Model 4 Radio-HPLC detector (IN/US Systems, Brandon, FL, USA).

Cell Culture and Cell Binding Studies Analysis of 3H-folate binding to FR was conducted on KB cells cultured in folate deficient RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (HIFBS), 1% L-glutamine and 1% penicillin at 37°C in a humidified atmosphere containing 5% CO2. Spent medium in each well was replaced with 10 nM [3H]-folate in the presence of increasing concentrations of either folate-PEG12-NOTA precursor (2) or free folic acid. After incubating for 1 h at 37 °C, cells were rinsed with PBS (2 x 0.5 mL) and 1 M trichloroacetic acid (1 x 0.5 mL) to remove any unbound radioactive material. Cells were dissolved in 1% sodium dodecylsulfate (0.5 mL) and transferred to Ecolume scintillation cocktail for scintillation counting. Experiments were performed in triplicate for each concentration and relative binding affinities (IC50) were calculated using a plot



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of cell bound radioactivity versus concentration of test compound using GraphPad Prism 4. Specific binding affinity of folate-PEG12-NOTA-Al18F (1) to FR was evaluated on two tumor homogenates, KB and A549. Briefly, tumors were collected, weighed, added to 9 volumes of lysis buffer (50 mM Tris-HCL containing 2 mM EDTA, pH 7.4), and homogenized at 4 °C using a Potter-Elvehjem homogenizer attached to a variable-speed drill and a tissuemizer. The homogenized tissue was centrifuged for 5 min in a microfuge at 1000 rpm, and the cell pellet was resuspended in folate deficient 1640 RPMI medium, seeded in 24-well (100,000 cells/well) Falcon plates and allowed to form monolayers over a period of 24 h. To each well were added increasing concentrations of folate-PEG12-NOTA-Al18F (1) in fresh medium (0.5 mL). After incubating for 1 h at 37 °C, cells were rinsed with PBS (2 x 0.5 mL) and 1 M trichloroacetic acid (1 x 0.5 mL) to remove any unbound radioactive materials. 1% sodium dodecylsulfate in PBS (0.5 mL) was then added and solubilized cells were transferred into individual test tubes and bound folate-PEG12-NOTA-Al18F was measured in a γ-counter (Wizard; PerkinElmer) using an energy window of 300-700 keV. Experiments were performed in triplicate at each concentration. The Bmax and Kd values were calculated using GraphPad Prism 4.

Tumor Implantation in Mice All animal procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Merck, West Point, PA. KB cells were cultured in medium containing folate free RPMI 1640 with 5% fetal bovine serum, while


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A549 cells were cultured in F-12K growth medium containing 10% fetal bovine serum at 37°C with 5% CO2. The growth media was changed 2 or 3x/week and the cells were subcultured at a ratio of 1:10 when desired. Female nu/nu mice (6-8 week-old) were ordered from Charles River Laboratories (Stone Ridge, NY, USA) and housed in a temperature and humidity controlled room and maintained on a folate-deficient rodent diet. After 7 to 14 days of acclimatization, tumors were implanted at the right shoulder with subcutaneously injection of 1x106 KB cells in 100 µL folate free RPMI, or A549 cells in 100 µL PBS + Growth Factor Reduced Matrigel (1:1). Animal experiments were then performed 2-3 weeks after tumor cell injection, when the tumors had grown to a mass of 200 to 400 mg.

MicroPET Imaging Folate-PEG12-NOTA-Al18F (1) was evaluated by microPET imaging on nude mice bearing either KB or A549 tumor xenografts both in the presence and absence of excess folic acid to block all accessible FR. PET experiments were carried out with a dedicated small-animal PET system (Focus220, Siemens Medical Solution, Hoffman Estate, IL). Anesthesia was introduced using 4-5 % isoflurane and maintained with 1-3% isofluorane using an air/oxygen mixture through a nose cone throughout the imaging sessions. Mice bearing KB tumor xenografts on their right shoulders were injected via tail vein catheter with 4-11MBq of folate-PEG12-NOTA-Al18F (1). The blockade group (n = 3 per tumor type) received a similar intravenous injection of 50 µg folic acid (FA) immediately before radiotracer administration, and the baseline (n = 5 per tumor type) group was injected with a corresponding volume of isotonic saline.



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PET data were collected for 90 min following radiotracer administration, and reconstructed using maximum a posteriori reconstruction (MAP, β = 0.2, 256 x 256 x 95 voxels, 0.9 x 0.9 x0.80 mm3,

57

Co transmission-based photon attenuation and

scatter correction). PET images were converted to percent injected activity per gram tissue (%IA/g). Image analysis was performed using MATLAB (MathWorks, Natick, MA, USA).

Biodistribution Study The tissue distribution profile of folate-PEG12-NOTA-Al18F (1) to FR was further evaluated through ex vivo biodistribution studies in mice in comparison with

99m

Tc-

EC20 and folate-NOTA-Al18F under both baseline and blockage conditions. Immediately after PET imaging, mice were euthanized via 10-30% CO2 inhalation. Tissues (blood, plasma, heart, lung, liver, spleen, kidney, muscle and tumor) were collected, cleaned and weighed, then counted in a gamma counter (Wizard 3, PerkinElmer). All mice bearing KB tumors that underwent PET imaging with either 1 or folate-NOTA-Al18F, and also mice with A549 tumors, were included in the biodistribution study. For

99m

Tc-EC20 studies, 0.4-0.7 MBq was administered, and

mice were sacrificed by inhalation of 10-30% CO2 and dissected 120 min post injection (p.i.) of radiotracer. FA blockade was achieved with the same methodology as in 1. Radiotracer accumulation for all biodistribution studies was calculated in %IA/g.

Hot Saturation Binding Assay


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KB and A549 xenografts were harvested and stored at -80°C freezer before use. Crude homogenates of the xenografts were prepared by homogenizing them separately in ice cold buffer (50 mM Tris pH 7.5, 120 mM NaCl, 2mM KCl, 1 mM MgCl2, 1 mM CaCl2) for 30 sec. at 4°C on setting 5 of Polytron. Crude homogenates of KB and A549 xenografts (1 mg/mL of wet tissue weight) were used in the assay. Tissue homogenates were first pre-incubated at room temperature for 2 h in assay buffer (50 mM Tris-HCl pH 7.5, 120 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2), 1:1000 Protease Inhibitor (P-8340) with 2% DMSO or 5 µM self-block using Skatron tube strips (SK15776) covered with aluminum foil. Non-displaceable binding of folate-PEG12-NOTA-Al18F (1) was defined using 5 µM self-block. Following pre-incubation, twelve concentrations of 1 from 0.1 nM to 30 nM were added to the assay tubes in duplicates with final assay volume 0.25 mL per tube.

The assay tubes were mixed by brief vortex, and then

incubated at room temperature for 60 min. After completion of incubation, each assay tube mixtures were transferred onto Skatron GF/C filters (SK11731), which were presoaked in 0.2% PEI for 30 min at room temperature before use, using a Skatron Combi cell 12-well harvester. The filters were promptly washed 3 times on setting 3-3-3 with ice cold wash buffer (50 mM Tris, pH 7.5, 120 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2). The filters were punched into Pico Pro vials, and then counted in Gamma counter (Wizard 3, PerkinElmer). The data were analyzed using nonlinear fit (one site binding) model with Prism software (GraphPad Prism 4, CA).

RESULTS Synthesis of Folate-PEG12-NOTA Conjugate (2)



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A detailed description of the chemical synthesis of the PET precursor, FA-PEG12-NOTA 2, is available in the Figure 1A and Supporting Information. Briefly, starting from 50 mg 1,2-diaminoethane trityl resin (0.06 mmol), pure folate-PEG12-EDA-NH2 (3) was prepared as a yellow solid (32.5 mg, 50 %). The following coupling of 3 with NOTANHS followed by preparative RP-C18 HPLC afforded the pure FA-PEG12-NOTA 2 (4.09 mg, 68 %).

Radiochemistry Radiochemical synthesis of folate-PEG12-NOTA-Al18F (1) was achieved by heating the folate-PEG12-NOTA precursor 2 with in situ prepared Al18F in 0.1 M NaOAc, pH 4 buffer to yield a mixture of radiolabeled components (Figure 2A). Following radioactive HPLC purification afforded the final product 1 in 8.4 ± 1.3% (decay corrected) radiochemical yield with ~ 100% radiochemical purity (Figure 2B) and a specific activity of 35.8 ± 15.3 GBq/mmol The total radiochemical synthesis including radioactive HPLC purification was accomplished within 35 min.

In Vitro Characterization Relative Binding Affinity Relative binding affinity assay was performed on KB cells by evaluating the concentration required to block binding of 3H-folic acid. As shown in figure 3, folatePEG12-NOTA conjugate (2) exhibited high affinity for FR with an average IC50 of 33.8 nM, suggesting that introduction of PEG12- unit had minor effect on receptor


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affinity when compared to that of folate-NOTA (18.7 nM) and native folic acid (4.6 nM).

Hot Saturation Binding Assay Saturation binding assay of folate-PEG12-NOTA-Al18F (1) was performed on homogenates of KB and A549 xenografts (1 mg/mL wet weight), respectively. The Kd values calculated approximately for folate-PEG12-NOTA-Al18F were 0.4 nM (KB) and 1.1 nM (A549) (Figure 4). Non-displaceable binding (assessed in the presence of 5 µM self-block) was low, approximately < 1% (KB) of total binding at concentrations near the Kd value, consistent with a single class of binding sites. Bmax values of folate-PEG12NOTA-Al18F were found to vary 120-fold between KB (241 nM) and A549 (2 nM) xenografts. Folate-PEG12-NOTA-Al18F binding potentials (Bmax/Kd) in xenografts were 603 (KB) and 2 (A549), correspondingly, resulting in 300-fold greater binding potential of folate-PEG12-NOTA-Al18F in KB xenografts than in A549 xenografts. The lower expression level of FR in A549 xenografts could account for the reduced retention of folate-PEG12-NOTA-Al18F (1) in A549 samples.

In Vivo microPET Imaging As demonstrated in figure 5, tumors were promptly visible under baseline conditions, whereas the uptake of folate-PEG12-NOTA-Al18F (1) was completely blocked with folic acid, supporting a high specificity of folate-PEG12-NOTA-Al18F binding to FR in vivo. As expected, the accumulation in liver was considerably reduced due to the introduction of more hydrophilic PEG12- unit in the radiotracer, which was consistent with the following biodistribution studies (Figure 6 and Table 1). The high radioactivity found in



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kidneys was likely due to the uptake mediated by FR that expressed in the proximal tubule cells in kidneys and the potential accumulation via renal excretion.

Ex Vivo Biodistribution Studies Ex vivo biodistribution studies revealed an uptake of folate-PEG12-NOTA-Al18F (1) in KB tumors as high as 9.20 ± 0.62 %IA/g in baseline animals, which was substantially reduced to 1.64 ± 0.13 %IA/g under FA blockade experiments, indicating a highly specific uptake mediated by the FR (Figure 7 and Table 1). On the contrary, the uptake of folate-PEG12-NOTA-Al18F (1) in lower FR expressing A549 tumors was 2.10 ± 0.23 %IA/g, considerably lower than that in KB tumors (Figure 7 and Table 1). Identical to the observation in microPET imaging, the accumulation of radiotracer in liver was reduced to 0.42 ± 0.07, over two-fold lower than that of folate-NOTA-Al18F (Figure 6 and Table 1). In agreement with the PET imaging data, the highest accumulation of folate-PEG12-NOTA-Al18F was found in kidneys (Figure 6) because of the higher FR expression in the proximal tubule cells and potential renal excretion mechanism. Furthermore, a blocking effect was also seen in lung, heart, muscle and spleen due to the FR expression in these normal tissues.5 Overall, the biodistribution of folate-PEG12-NOTA-Al18F (1) is comparable to those of

99m

Tc-

EC20 (SPECT) and folate-NOTA-Al18F (PET), but demonstrates more preferred pharmacokinetic properties.

DISCUSSION


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The main objective of this investigation was to develop a new folate-targeted PET tracer with better pharmacokinetic properties for imaging of FR-positive tumors. In contrast to our earlier reported folate-NOTA, a PEG12- unit was introduced to improve the hydrophilicity of folate-PEG12-NOTA, which proved effective to reduce radioactivity accumulation in liver under in vivo studies. Moreover, the employment of a convenient solid phase synthesis in the chemical synthesis guaranteed the sufficient supply of the precursor for in vitro and in vivo study. A fast and robust radiochemical synthesis of folate-PEG12-NOTA-Al18F (1) was realized through one-step radiolabeling with an Al18F intermediate prepared in situ. The production of folate-PEG12-NOTA-Al18F is efficient and reproducible with a radiochemical yield of 8.4 ± 1.3% (decay corrected), high radiochemical purity (~ 100%, after HPLC purification), and a total synthesis time of ~ 35 min, which are essential for eventual clinical translation of the radiotracer. Although the radiolabeling efficiency of folate-PEG12-NOTA was relatively low compared to previous folate-NOTA, indicating a block effect from PEG12- unit, the highly specific binding affinity of 1, proved that introducing the hydrophilic PEG12- unit to folic acid has little effect on the binding affinity of the resulting folate conjugate. Biodistribution data and PET imaging experiments demonstrated that the uptake of folate-PEG12-NOTA-Al18F (1) was concentrated primarily in FR+ KB tumors and kidneys. As displayed in table 1 and figure 7, uptake of radiotracer 1 in FR+ KB tumors was 9.20 ± 0.62 %IA/g 90 minutes after injection. Accumulation of this radiotracer was also shown to be FR-mediated as the administration of excess free folic acid completely blocked its detection in tumor. Compared to folate-NOTA-Al18F, over two-fold reduced liver accumulation was observed for 1, indicating that the hydrophilicity improvement



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could advance the pharmacokinetic properties of the radiotracer. The highest uptake of radioactivity was found in the kidneys (61.9 ± 6.17 %IA/g, 90 min p.i.) due to the naturally prolific expression of FR in proximal tubule cells. Renal excretion of this new radiotracer may also contribute to the high accumulation in kidneys. In contrast, the biodistribution study in A549 xenografts revealed that in tumors with lower FR expression, only very limited accumulation of level 1 was found, which further confirmed that the highly specific uptake of 1 is mediated by FR. Our motivation for this study was to seek another folate-targeted PET agent that could meet the financial requirements for translation of a PET agent into the clinic. Indeed, a variety of folate-targeted18F PET agents have been reported to yield highly defined images of FR positive tumors in animal tumor models.35-47 Although more studies needed to improve the radiolabeling efficiency of folate-PEG12-NOTA, the present folate-PEG12-NOTA-Al18F (1) demonstrated some preferable properties that differ for each previous conjugate, including its higher specific activity,35,

39, 41, 46

comparable radiochemical yield,35, 41, 43, 44 or faster and simpler synthesis.35, 36, 41, 43, 44 In side-by-side comparison to 99mTc-EC20, a clinically proved SPECT imaging agent, folate-PEG12-NOTA-Al18F also showed very similar distribution in mice, with comparable tumor uptake but lower blood and liver accumulation at 90 and 120 min post-injection, respectively. Given the intrinsic characters of PET/CT in a clinical setting, i.e. better sensitivity and spatial resolution, shorter image acquisition time, and quantitation of radiotracer accumulation in lesions, folate-PEG12-NOTA-Al18F possess superiority over

99m

Tc-EC20. Considering its improved characteristics, we


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propose that folate-PEG12-NOTA-Al18F (1) may constitute an improved diagnostic tool for identification and staging of patients with FR-expressing cancers.

DISCLOSURE The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. No potential conflict of interest relevant to this article was reported.

ACKNOWLEDGEMENTS This work was supported by a research grant from Endocyte, Inc.

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FIGURE 1A. Synthesis of folate-PEG12-NOTA precursor (2)


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FIGURE 1B. Radiochemcial synthesis of folate-PEG12-NOTA-Al18F (1)



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FIGURE 2A. Representative analytic HPLC chromatogram of crude folate-PEG12NOTA-Al18F obtained from the radiolabeling reaction.

FIGURE 2B. Representative analytical HPLC chromatogram of folate-PEG12-NOTAAl18F after semi-preparative HPLC purification.


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FIGURE 3. Relative binding affinity evaluation of folate-PEG12-NOTA (1) to cultured KB cells in comparison to folic acid and folate-NOTA.



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A

C

B

D

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18

FIGURE 4. Specific binding affinity assay of folate-PEG12-NOTA-Al F (1) to FR in KB (A, B) and A549 (C, D) tumor crude homogenate (1 mg/mL). Displaceable and saturable binding was observed and the tracer binds one site with high affinity. KB xenografts have significantly higher binding site density than A549 xenografts.


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FIGURE 5. PET images with folate-PEG12-NOTA-Al18F (1) in comparison of folateNOTA-Al18F areshown in representative mice bearing KB tumors on right shoulders (arrows) under baseline and blocking conditions. A blocking dose of 50-100 µg folic acid was administered i.v. immediately before the radiotracer. PET images are corresponding coronal slices through the tumor and kidneys, with PET data summed from 60-90 min post radiotracer administration.



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FIGURE 6. Ex Vivo biodistribution of folate-PEG12-NOTA-Al18F (1) at 90 min p.i. in nude mice in comparison to the folate-NOTA-Al18F at 90 min.


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FIGURE 7. Tumor accumulation of folate-PEG12-NOTA-Al18F (1) at 90 min p.i. in nude mice bearing KB or A549 tumor xenografts in comparison to the folate-NOTA-Al18F at 90 min and SPECT tracer 99mTc-EC20 at 120 min.



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18

TABLE 1. Ex vivo biodistribution of folate-PEG12-NOTA-Al F (1) at 90 min post injection in nude 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

18

mice bearing KB tumor xenografts in comparison to folate-NOTA-Al F at 90 min and at 120 min post injection. Values are in units of %IA/ g ± SEM (except ratios).

Organ or Tissue

folatefolateRatio of folatefolatePEG12PEG1218 18 Baseline to 18 18 NOTA-Al F NOTA-Al F NOTA-Al F NOTA-Al F blockade

99m

Tc-EC20

99m

Tc-EC20

99m

Tc-EC20

Baseline (N = 5)

FA Block (N = 3)

Blood

0.09 ± 0.01

0.26 ± 0.11

0.34

0.20 ± 0.04

0.47 ± 0.24

0.27 ± 0.02

0.33 ± 0.08

Plasma

0.14 ± 0.02

0.30 ± 0.15

0.46

0.28 ± 0.06

0.84 ± 0.44

0.50 ± 0.03

0.62 ± 0.16

Heart

0.79 ± 0.16

0.15 ± 0.06

5.19

0.87 ± 0.01

0.09 ± 0.05

1.77 ± 0.04

0.15 ± 0.02

Kidney

55.25 ± 7.38

10.80 ± 3.91

5.12

87.86 ± 4.42

30.93 ± 24.85

107.26 ± 3.70

8.30 ± 2.59

Liver

1.64 ± 0.30

0.41 ± 0.05

4.05

5.40 ± 0.75

3.70 ± 1.01

3.89 ± 0.35

2.35 ± 0.47

Lung

0.48 ± 0.06

0.32 ± 0.13

1.51

1.00 ± 0.1

0.25 ± 0.12

2.03 ± 0.12

1.22 ± 0.25

Muscle

0.57 ± 0.11

0.09 ± 0.02

6.69

1.05 ± 0.09

0.08 ± 0.04

1.18 ± 0.04

0.11 ± 0.02

Spleen

0.45 ± 0.07

0.24 ± 0.04

1.88

0.51 ± 0.08

0.15 ± 0.03

0.70 ± 0.08

0.25 ± 0.07

Tumor

9.20 ± 0.62

1.64 ± 0.13

5.60

10.91 ± 2.69

1.32 ± 0.08

10.91 ± 0.60

0.92 ± 0.11

Tumor/Liver

6.19 ± 0.90

4.22 ± 0.70

1.47

1.97 ± 0.42

0.44 ± 0.16

3.09 ± 0.26

0.41 ± 0.06

Tumor/Kidney

0.18 ± 0.02

0.26 ± 0.15

0.68

0.12 ± 0.03

0.40 ± 0.34

0.10 ± 0.01

0.12 ± 0.02

Tumor/Blood

116.18 ± 20.60

10.32 ± 5.39

11.25

55.83 ± 11.54

14.03 ± 12.09

41.46 ± 2.21

2.94 ± 0.39

Baseline (N=4) FA Block (N=3)

In blockade group, each animal received 50 -100 µg of folic acid (FA) immediately before radiotracer injection


Baseline (N = FA Block (N = 3) 20)

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248x296mm (96 x 96 DPI)


Folate-PEG-NOTA-Al18F: A New Folate Based Radiotracer for PET

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