Automated radiosynthesis and preclinical evaluation of Al[18F]F-NOTA-P-GnRH for PET imaging of GnRH receptor-positive tumors

Journal Pre-proof Automated radiosynthesis and preclinical evaluation of Al[18F]FNOTA-P-GnRH for PET imaging of GnRH receptor-positive tumors

Shun Huang, Hubing Wu, Baoyuan Li, Lilan Fu, Penghui Sun, Meng Wang, Kongzhen Hu PII:

S0969-8051(19)30580-3

DOI:

https://doi.org/10.1016/j.nucmedbio.2020.02.004

Reference:

NMB 8120

To appear in:

Nuclear Medicine and Biology

Received date:

3 December 2019

Revised date:

16 January 2020

Accepted date:

11 February 2020

Please cite this article as: S. Huang, H. Wu, B. Li, et al., Automated radiosynthesis and preclinical evaluation of Al[18F]F-NOTA-P-GnRH for PET imaging of GnRH receptorpositive tumors, Nuclear Medicine and Biology(2020), https://doi.org/10.1016/ j.nucmedbio.2020.02.004

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

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Automated Radiosynthesis and Preclinical Evaluation of Al[18F]F-NOTA-P-GnRH for PET Imaging of GnRH Receptor-Positive Tumors Shun Huang

a,1

, Hubing Wu

a,1

, Baoyuan Li b, Lilan Fu a, Penghui Sun a, Meng Wang a,

Kongzhen Hu a,* a

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Nanfang PET Center, Department of Nuclear Medicine, Nanfang Hospital, Southern Medical

Department of Nuclear Medicine, the Second Affiliated Hospital of Guangzhou University of Chinese

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b

ro

University,1838 Guangzhou Avenue North, Guangzhou, Guangdong Province, 510515, China

re

Medicine, Guangzhou, Guangdong Province, 510120, China

F-labelled GnRH for Imaging of GnRH Receptor-Positive Tumors

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18

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Short title:

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Correspondence: Kongzhen Hu, MD. Nanfang PET Center, Department of Nuclear Medicine, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou, Guangdong Province, 510515, China. Tel. +86 20 62786625. E-Mail: [email protected] 1

These authors contributed equally.

Keywords: Al[18F]F-NOTA-P-GnRH, Gonadotropin releasing hormone receptor, GnRH Receptor-Positive Tumors, PET Imaging, Radiosynthesis

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Abstract: Introduction: Gonadotropin releasing hormone (GnRH) receptor is overexpressed in many human tumors. Previously we developed a

18

F-labelled GnRH peptide. Although the

GnRH-targeted PET probe can be clearly visualized by microPET imaging in a PC-3

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xenograft model, clinical applications of the probe have been limited by complex labeling

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procedures, poor radiochemical yield, and unwanted accumulation in GnRH receptor F-labelled GnRH peptide that is

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more amenable to clinical development.

18

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negative tissues. In this study, we have designed a new

Methods: GnRH peptide analogues NOTA-P-GnRH was synthesized and automated

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radiolabeled with 18F using a Al[18F]F complex on a modified PET-MF-2V-IT-I synthesis

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module. The GnRH receptor affinities of AlF-NOTA-P-GnRH and NOTA-P-GnRH were

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determined by in vitro competitive binding assay. For in vitro characterization determination of stability and partition coefficients were carried out, respectively. Dynamic microPET and biodistribution studies of Al[18F]F-NOTA-P-GnRH were evaluated in xenograft tumor mouse models.

Results: The total radiochemical synthesis and purification of Al[18F]F-NOTA-P-GnRH was completed within 35 min with a decay-corrected yield of 35 ± 10%. The logP value of Al[18F]F-NOTA-P-GnRH was -2.74  0.04 and the tracer was stable in phosphate-buffered saline, and bovine and human serum. The IC50 values of AlF-NOTA-P-GnRH and NOTA-P-GnRH were 116 nM and 56.2 nM, respectively. Dynamic PET imaging together

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with ex vivo biodistribution analyses revealed that Al[18F]F-NOTA-P-GnRH was clearly delineated in both PC-3 and MDA-MB-231 xenografted tumors. Conclusion: Al[18F]F-NOTA-P-GnRH can be efficiently produced on a commercially available automated synthesis module and has potential for use in clinical diagnosis of GnRH receptor-positive tumors.

F-labelled GnRH tracer and preclinical evaluation for future clinical application.

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Advances in knowledge: Our studies developed the automated radiosynthesis of a new

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Implications for patient care: Quantitative and noninvasive imaging of GnRH expression

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would provide information for diagnosis and treatment of cancer patients.

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1. Introduction

The gonadotropin releasing hormone (GnRH) receptor is a member of the subfamily of

ligand,

the

hypothalamic

decapeptide

GnRH

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endogenous

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glycoprotein hormone receptors within the G protein-coupled receptor superfamily[1]. Its

(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) regulates the reproductive system through binding to high-affinity GnRH receptors on pituitary gonadotroph cells, triggering release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH)[2][3]. Overexpression of GnRH receptor has been found in various types of cancers, such as cancers of the endometrium, ovary, urinary bladder, prostate, breast, pancreas, and glioblastoma [4,5]. Accordingly, the GnRH receptor is an important target for cancer treatment and diagnostics. Radiolabeled GnRH derivatives are used as tracers to determine expression levels in

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vivo and affinity of GnRH receptors in vitro[5–16]. Positron emission tomography (PET) is a non-invasive molecular imaging technique using radiotracers to study and visualize human physiology[17]. To date, only a few PET radiotracers for GnRH receptors have been reported. Zoghi et al. reported a

68

Ga-labelled DOTA-triptorelin with high binding

affinity for GnRH receptor imaging[15]. However, this study did not report use of that

of

probe for imaging or measurement of tumor uptake. A GnRH probe based on fluorine-18 18

F is the most

ro

(t1/2 = 109.8 min) would be expected to be very useful in that regard, as

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widely used radionuclide in PET. In fact, it has a short but manageable 109.8 min half-life,

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which allows sufficient time for multistep radiosynthesis, and a short positron linear range

emitters.

Previously

we

lP

in tissue (2.3 mm), which gives the highest resolution PET images of all available positron have

developed

a

18

F-labelled

GnRH

peptide

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([18F]FP-D-Lys6-GnRH), which could be clearly visualized by microPET imaging in a

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PC-3 xenograft model[8]. However, this tracer has some problematic disadvantages. The radiosynthesis of [18F]FP-D-Lys6-GnRH with high-performance liquid chromatography (HPLC) purification was a lengthy and laborious multistep procedure with limited yield. The biodistribution of [18F]FP-D-Lys6-GnRH resulted high accumulation in the gall bladder and abdomen. These issues restricted the application of this particular 18F-labelled GnRH probe in clinical study. In order to develop

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F-labelled GnRH probes suitable for clinical applications, we

designed a new GnRH derivative with the introduction of a hydrophilic pegylated linker between the NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid) and a D-Lys6-GnRH

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motif. This peptide can be easily prepared and labeled via an automated Al[18F]F chelation reaction. Herein, we reported the automated radiosynthesis of this new 18F-labelled GnRH peptide tracer (Al[18F]F-NOTA-P-GnRH) and further evaluated this tracer in PC-3 and MDA-MB-231 xenografted tumor mice. 2. Materials and Methods

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2.1. General

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All chemicals were purchased from commercially available sources and used without

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further purification. The semi-preparative reverse-phase HPLC using C18 column (10 ×

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250 mm) was performed using the PET-MF-2V-IT-I synthesizer module (PET Co. Ltd.,

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Beijing, China). Analytical HPLC was performed with a flow rate of 1 mL/min using a LC-20AD HPLC system (Shimadzu, Kyoto, Japan) equipped with a ZORBAX Eclipse

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XDB-C18 analytic column (4.6 × 150 mm, 5 μm; Agilent Technologies, Palo Alto, CA,

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USA) or Kromasil 100-5 C18 analytic column (4.6 × 150 mm, 5 μm; Akzo Nobel). Radioactivity was measured with a calibrated ion chamber (Capintec CRC-15R, Capintec, Inc., Ramsey, NJ, USA) or a gamma counter (γ-counter) (CAPRAC-R, Capintec, Inc., Ramsey, NJ, USA). High-resolution mass spectra were recorded with a ThermoFisher Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). No-carrier-added

18

F-fluoride was obtained by reacting

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O(p, n)18F in a GE

PETtrace (800 series) cyclotron. 2.2. Peptide synthesis Peptides were synthesized on a 12-channel semi-automatic peptide synthesizer (China

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Peptides. Co., Ltd., Shanghai, China) by standard Fmoc-solid phase synthetic protocols. Peptides were synthesized on Rink Amide MBHA Resin with a loading value of 0.45 mmol/g. The peptide chain was elongated by sequential coupling and Fmoc deprotection of

Fmoc-Gly-OH,

Fmoc-L-Pro-OH,

Fmoc-L-Arg(pbf)-OH,

Fmoc-L-Leu-OH,

Fmoc-D-Lys(Dde)-OH, Fmoc-L-Tyr-OH, Fmoc-Ser-OH, Fmoc-Trp-OH, Fmoc-L-His-OH,

of

and (S)-(-)-2-pyrrolidone-5-carboxylic acid. All coupling reactions were performed once

was

activated

in

situ

by

-p

derivative

ro

using the standard conditions. An excess of three equivalents (eq.) for each amino acid standard

HBTU

hexafluorophosphate)/DIPEA

re

(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

the

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(N,N’-diisopropylethylamine) procedure and twice deprotected with 20% piperidine in dimethylformamide (DMF). The group of Dde in Fmoc-D-Lys(Dde)-OH was deprotected

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with hydrazine in DMF and elongated by sequential coupling and Fmoc deprotection of

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Fmoc-NH-PEG3-CH2COOH and NOTA. Peptide cleavage from the solid support and the simultaneous removal of all protecting groups was carried out by treating the resin-bound peptide with TFA/H2O/1,2-ethanedithiol/triisopropylsilane (95:1:2:2) for a minimum of two hours followed by filtration. The desired product was purified by semipreparative HPLC and lyophilized. The purity of the product was >95% by analytical HPLC (tR = 13.1 min). MS: [M+H]+ = 1741.9 (m/z), calc: 1740.9 (C80H120N22O22) (Supplementary Fig. S1). 2.3. Preparation of AlF-NOTA-P-GnRH Aluminum chloride (0.46 mg, 0.35 μmol) and KF (0.28 mg, 0.7 μmol) in 0.1 mL

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deionized water was mixed with AcOH (10 μL) and NOTA-P-GnRH (0.4 mg, 0.23 μmol) in 350 μL MeCN. The reaction mixture was sealed and heated at 100 °C for 30 min. After cooling to room temperature, the reaction mixture was purified with a semipreparative HPLC. The purity of the product was >95% by analytical HPLC (tR = 12.9 min). MS: [MH]+ = 1785.8 (m/z), calc: 1784.9 (C80H118AlFN22O22).

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2.4. Radiosynthesis of Al[18F]F-NOTA-P-GnRH

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Effectiveness of the NOTA chelator for complexation of Al[18F]F on a

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PET-MF-2V-IT-I synthesizer (PET Co. Ltd., Beijing, China) was tested (Fig.1). Prior to

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delivery of 18F-fluoride to the synthesizer, reagents were loaded as follows: vial B1, NaCl

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(0.9%, 0.4 mL); vial B2, MeCN (1.5 mL); vial B3, a mixture of aluminum chloride (AlCl3), glacial acetic acid, deionized water, NOTA-P-GnRH, and organic solvents; vial B4, 0.1%

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trifluoroacetic acid (TFA) in water (1.5 mL). The Sep-Pak light QMA cartridge was

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preconditioned with 5 mL aqueous NaHCO3 (8.4%) and 10 mL sterile water. 18F-Fluoride (5.5−37 GBq) in [18O]H2O was transferred to the module and trapped on a Sep-Pak light QMA anion exchange cartridge. Trapped fluoride ions (18F-) were eluted from the cartridge into the reactor using 0.4 mL of 0.9% NaCl from vial B1. MeCN (vial B2) was added to the reactor and the mixture was azeotropically evaporated under a stream of nitrogen (80 mL/min) at 116 °C. Subsequently, a mixture solution was added from vial B3 to the residue, and heated for 10 min at 100 °C. The reaction mixture was diluted with 1.5 mL of 0.1% TFA in water, prior to its injection into the HPLC system using trifluoroacetic acid solution from vial B4. Purification was performed using a preparative Agilent ZORBAX

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SB-C18 column (9.4 × 250 mm) equipped with a UV detector and a radioactivity detector (Supplementary Fig. S2). The product Al[18F]F-NOTA-P-GnRH was collected and passed through a 0.22 μm Millipore filter. The identity of the radiopharmaceutical was confirmed using AlF-NOTA-P-GnRH as a reference.

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2.5. Octanol/water partition coefficient

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Octanol/water partition coefficients were determined for Al[18F]F-NOTA-P-GnRH by

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measuring the distribution of radiolabeled compound in n-octanol and phosphate-buffered

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saline (PBS) (pH = 7.4). Approximately 740 KBq of Al[18F]F-NOTA-P-GnRH was added

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to a vial containing 5 mL each of n-octanol and PBS. The mixture was vigorously vortexed for 1 min and centrifuged for 10 min to ensure complete the separation of layers. Three

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calculated (n = 4).

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hundred milliliters of each layer were measured using a γ-counter and logP values were

2.6. In vitro stability and in vivo metabolism The in vitro stability of Al[18F]F-NOTA-P-GnRH was evaluated by incubation of 3.7 MBq (100 μCi) of the probe with PBS (200 μL), bovine or human serum (200 μL) at 37 °C for 2 h. For the PBS study, 100 μL of solution was directly injected into a radio-HPLC for analysis. The bovine or human serum sample was filtered through a 30 KDa Millipore filter and analyzed by HPLC. In vivo metabolic analysis of Al[18F]F-NOTA-P-GnRH in plasma was performed using mouse blood samples (0.4 mL) that were collected at 60 min after tracer injection. Two

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blood samples were collected from nude mice. Centrifugation and collection of plasma samples were performed as previously described[8]. Blood was centrifuged (12000 g, 10 min). The supernatant was filtered through a 30 KDa Millipore filter and analyzed by HPLC; samples were collected in 0.5-min fractions for 25 min. Samples were then analyzed in a γ-counter for 20 s. Counts were decay-corrected to the injection time and

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added together. The counts were plotted intensity (cpm) versus fractions to show the

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profile of the sample. Data were plotted to reconstruct the HPLC spectrum.

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2.7. In vitro receptor binding assay

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The GnRH receptor affinities of AlF-NOTA-P-GnRH and NOTA-P-GnRH were

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determined by in vitro competitive binding assay according to previously published procedure[8]. Detailed procedures are included in the supplemental information.

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2.8. Preparation of animal tumor models

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All animal studies were conducted in compliance with Nanfang Hospital Animal Ethics Committee at the Southern Medical University. The MDA-MB-231 human breast cancer cell and PC-3 human prostate cancer cell lines were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China) and were cultured in L15 and DMEM/F-12 medium at 37°C in a humidified 5% CO2 atmosphere, respectively. Both media were supplemented with 10% FBS and 1% penicillin/streptomycin. The MDA-MB-231 tumor model was developed in 4 to 5-week-old female athymic nude mice (Animal experiment center of Nanfang Hospital) by inoculation of 12 × 107 cells in their right shoulders. The PC-3 tumor model was generated by subcutaneous injection of PC-3

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cells (25 × 106) into the right shoulders of male athymic nude mice (Animal experiment center of Nanfang Hospital). Animals were used in experiments when the tumor diameter reached 5-8 mm (3−5 weeks after inoculation). 2.9. MicroPET/CT imaging Dynamic microPET imaging studies were conducted in tumor-bearing nude mice using

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the Inveon MicroPET/CT scanner (Siemens, Erlangen, Germany). Tumor-bearing mice

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were intravenously injected with 5.55−7.4 MBq of Al[18F]F-NOTA-P-GnRH. For the

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blocking experiment, the tumor-bearing mice were coinjected with 15 mg/kg mouse body

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weight of D-Lys6-GnRH and Al[18F]F-NOTA-P-GnRH (n = 3 per group). Dynamic scans

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were conducted over a period of 2 h. PET images were reconstructed using a three-dimensional ordered-subset expectation maximum (OSEM) algorithm (Siemens,

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Erlangen, Germany) and were converted to the percentage of injected dose per gram of

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tissue (% ID/g) images. For data analysis, the regions of interest (ROIs) were manually drawn over the tumor and major organs on decay-corrected whole-body coronal images using Inevon Research Workplace 4.1 software (Siemens, Erlangen, Germany). 2.10.

Ex vivo PET study in Wistar rats

Al[18F]F-NOTA-P-GnRH (37 MBq) was injected intravenously while rats was anesthetized with 10% chloral hydrate solution (4 mL/kg). Rats were sacrificed 1 h after injection, and the pituitary and muscle were removed and placed on cardboard. The cardboard containing the organs was then placed in the Inveon MicroPET/CT scanner. Imaging started with a low-dose CT scan, immediately followed by a 20 min PET scan.

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2.11. The

In vivo biodistribution biodistribution

of

Al[18F]F-NOTA-P-GnRH

was

determined

in

PC-3

xenograft-bearing nude mice (n = 4 for each group). Each PC-3 xenograft-bearing nude mouse was injected with approximately 1.0 MBq of Al[18F]F-NOTA-P-GnRH in 0.10 mL of PBS via the tail vein. Animals were sacrificed at 30, 60, and 120 min postinjection. The

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tumors and other tissue samples of interest were rapidly dissected and weighed, and their

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radioactivity was measured with a -counter. The results were calculated as the percentage

Immunohistochemistry

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2.12.

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of injected dose per gram of tissue or organ (% ID/g) (n = 4).

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The immunohistochemical staining of hypophysis, muscle, PC-3, and MDA-MB-231 cancer-xenografted tumor lesions are included in the supplemental information.

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3. Results

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3.1. Chemistry and radiochemistry

The synthesis of peptides was performed on solid resin by standard Fmoc-solid phase synthetic protocols. The radiometal chelator NOTA was conjugated to D-Lys6-GnRH via hydrophilic pegylated linker to yield NOTA-P-GnRH with more than 95% chemical purity. The AlF-NOTA-P-GnRH was synthesized and used as a standard for characterization of its radioactive counterpart in HPLC and for receptor binding assays. The molecular weights of NOTA-P-GnRH and AlF-NOTA-P-GnRH were confirmed by mass spectrometry (Supplementary Fig. S3 and S4).

Al[18F]F-NOTA-P-GnRH was radiosynthesized via an automated one-step, one-pot 11

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procedure on the modified PET-MF-2V-IT-I synthesizer as shown in Fig. 1. The labeling procedure was optimized with regard to the amount of peptide and organic solvents (Table 1). The influence of solvents has a significant impact on the yield of the reaction, and the decay-corrected yield was 7.5%, 7%, 16%, and 20%, in MeCN, EtOH, DMSO, and DMF, respectively. Another critical parameter for efficient labeling was the amount of peptide.

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An increase in the of amount of peptide from 57 to 114 nmol led to an increase of labeling

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efficiency to 35%.

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For the final standard labeling protocol, a solution containing 9.0 μL of 10 mM AlCl3, 8

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µL of glacial acetic acid, 50 µL of deionized water, 200 µg of NOTA-P-GnRH, and 334 µL

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of DMF was added to the reactor (Fig. 2.). The labeling was completed within 35 min with a decay-corrected yield of 35 ± 10% (n = 6). The radiochemical purity of

(n

=

6).

The

octanol/water

partition

coefficient

(log

P)

of

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GBq/µmol

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Al[18F]F-NOTA-P-GnRH was more than 98% with a high molar activity of 20–80

Al[18F]F-NOTA-P-GnRH was -2.74  0.04 (n = 4), indicating that this tracer is hydrophilic.

3.2. In vitro stability and in vivo metabolism Al[18F]F-NOTA-P-GnRH displayed good stability in PBS, bovine and human serum for up to 120 min, as analyzed by a reverse-phase HPLC. The results showed that the percentage of intact probes remained greater than 95% after 2 h incubation in PBS, bovine and

human

serum

at

37

°C

(Fig.

3a).

Metabolite

analysis

revealed

that

Al[18F]F-NOTA-P-GnRH was slowly metabolized in vivo, with 50% of intact probe in

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plasma at 1 h after injection, and defluorination was not observed (Fig. 3b). 3.3. In vitro receptor binding assay The GnRH receptor affinities for AlF-NOTA-P-GnRH and NOTA-P-GnRH were determined using human GnRH membrane preparations and [131I](His5, D-Tyr6)GnRH as the radioligand. The results of these assays are summarized in Fig. 4. The IC50 values of

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AlF-NOTA-P-GnRH and NOTA-P-GnRH were 116 nM and 56.2 nM, respectively (n = 3).

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3.4. MicroPET imaging studies

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Dynamic microPET studies on mice bearing PC-3 or MDA-MB-231 tumor xenografts

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were performed with Al[18F]F-NOTA-P-GnRH. MicroPET images of summed 2-h coronal

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sections were selected by manually selecting ROIs for visualization of tissue distribution and the calculation of time-activity curves for assessing kinetics (Fig. 5a, c and d). As the

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images demonstrate, tumors could be visualized within each animal model. The kinetic

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data indeed confirmed that the tracer exhibited higher uptake in tumor than muscle (background). Rapid tumor uptake was visualized within the first 10 min in each test animal. Kidneys showed rapid uptake of the tracer, which was quickly excreted through the urinary bladder. Liver uptake was relatively low. As the tracer was cleared rapidly from normal nontargeted organs, the ratio of radioligand in tumor vs. muscle consistently increased throughout the 2-h scan time (Fig. 5d). Kinetic analysis showed that tumor-to-muscle radioligand ratio was slightly higher in the PC-3 tumor model when compared to the MDA-MB-231 tumor model. The blocking studies in nude mice bearing xenograft tumors demonstrated that the accumulation of radioactivity in most organs,

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including tumor, was higher than observed in the control group (Supplementary Fig. S5). However, the ratio of tumor-to-muscle in the control group was higher than in the blocking group throughout the 2-h scan time After dissection, ex vivo animal PET studies rats were performed. High uptake of Al[18F]F-NOTA-P-GnRH is visualized in the hypophysis while low uptake in muscle (Fig.

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5b). These data were consistent with the expression of GnRH receptors in these tissues.

validate

the

microPET imaging

experiments,

biodistribution

studies

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To

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3.5. Biodistribution studies

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Al[18F]F-NOTA-P-GnRH were also performed in nude mice bearing PC-3 tumor at 30 min,

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1 h, and 2 h after intravenous injection. The results are shown in Table 2. This radiolabeled peptide displayed rapid blood clearance with time (1.44 ± 0.31 %ID/g at 30 min after

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injection, and 0.12 ± 0.03 %ID/g at 2 h after injection). Kidney uptake was initially high

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(6.50 ± 2.27 %ID/g, 30 min after injection), but decreased to 5.40 ± 0.86% ID/g at the end of 2 h. Moderate liver and gall bladder activity accumulation were observed with a relatively slow washout rate. After injection, brain and muscle exhibited low uptake of less than 0.5% ID/g at 30 min and decreased to less 0.1% ID/g at 2 h. GnRH-positive PC-3 tumors uptake showed respectable values (1.23 ± 0.73% ID/g, 30 min after injection). The PC-3 tumor-to-muscle ratio increased rapid from 2.80 at 30 min to 6.08 at 1 h. Results of biodistribution studies also showed a moderate hypophysis uptake at 30 min, due to pituitary tissue contains the high endogenous concentration of GnRH receptors. These data are consistent with in vivo microPET image quantification in the PC-3 mice xenografted

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animal model. 3.6. Immunohistochemistry GnRH receptor expression was determined by immunohistochemical staining in hypophysis and muscle from rat, PC-3 and MDA-MB-231 tumor xenografts dissected from nude mice. As shown in Fig. 6, the hypophysis, PC-3 and MDA-MB-231 tumor

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tissues exhibited extensive GnRH receptor expression. In contrast, cytoplasmic staining of

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the GnRH receptor in muscle was weak.

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4. Discussion

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A variety of human tumors overexpress the GnRH receptor, and GnRH derivatives have

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proven to be clinically useful. For this reason, the development of radiolabeled GnRH analogues for targeting tumors in vivo has attracted considerable attention. Several

111

111

In-DOTA-Ahx-(D-Lys6-GnRH1)[11]

and

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[5,6,11,15,16].

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radiolabeled GnRH peptides have been reported for GnRH-positive tumor imaging

In-DOTA-Aoc-D-Phe-(D-Lys6-GnRH)[16] radiotracers were reported in mouse models

for SPECT imaging of breast cancer and prostate cancer, respectively. PET offers higher spatial resolution, greater sensitivity, and higher temporal resolution than SPECT[18]. We recently reported the radiosynthesis and evaluation of [18F]FP-D-Lys6-GnRH, which could be clearly visualized in GnRH-positive PC-3 tumor lesions with PET[8]. However, its application in the clinic is restricted due to the laborious multistep synthesis and high uptake in the gall bladder and abdomen. In this study, we reported on the development of the radiometal chelator NOAT

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conjugated to the GnRH agonist D-Lys6-GnRH via a PEG3 linker, which has been used to improve the in vivo kinetics of various pharmaceuticals[19,20]. The resulting derivatized peptide retained potent GnRH receptor binding affinity. This makes it possible to radiolabel the peptide via complexation of Al[18F]F by a NOTA chelator, a technique which has already been successfully applied to several peptides[16-31]. However, in these

optimized

the

labeling

Al[18F]F-NOTA-P-GnRH

on

the

conditions

modified

to

ro

further

automate

PET-MF-2V-IT-I

production

synthesis

of

module.

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we

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studies radiolabeling was performed manually, using small volumes. In the present study,

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Automation made it possible to easily obtain the tracer in large volumes for clinical application, enabling it to retain high molar activity. The octanol/water partition coefficient

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showed that Al[18F]F-NOTA-P-GnRH was more hydrophilic than our previous developed

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[18F]FP-D-Lys6-GnRH[8]. Furthermore, Al[18F]F-NOTA-P-GnRH was found to be more

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stable against metabolic degradation than [18F]FP-D-Lys6-GnRH in blood at 1h[8]. However, the results on binding affinity indicated that the moiety of AlF-NOTA-PEG3 attached to the D-Lys6-GnRH showed a dramatically reduction the receptor binding affinity of the peptide. Further refinement of the linker between the NOTA and D-Lys6-GnRH to improve the binding affinity of the peptide is underway in our laboratories. Pituitary gonadotrophs contain a high concentration of GnRH receptors while muscle does not contain significant amounts of GnRH receptors[1]. Ex vivo microPET imaging studies in a rat confirmed that Al[18F]F-NOTA-P-GnRH shown a high uptake in hypophysis, suggesting that the tracer specifically bound to GnRH receptors in vivo. In

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vivo microPET studies using Al[18F]F-NOTA-P-GnRH exhibited the expected high uptake and retention in PC-3 and MDA-MB-231 xenografted tumor models, which are well known for GnRH-positive tumor imaging [8,11,16]. These data are consistent with biodistribution studies and the expression of GnRH receptors in these tissues. The blocking studies of Al[18F]F-NOTA-P-GnRH was consistent with literatures reported for

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[125I]Triptorelin[5] and [18F]FP-D-Lys6-GnRH[8]. The reason might be occasioned by a

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slowed tracer excretion caused by excess of D-Lys6-GnRH.

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The expression of GnRH receptors on the two xenografted tumors, hypophysis, and

PET

imaging

and

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muscle tissues were further confirmed by immunohistochemical analysis. Comparison of biodistribution

results

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Al[18F]F-NOTA-P-GnRH

and

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[18F]FP-D-Lys6-GnRH[8] revealed that Al[18F]F-NOTA-P-GnRH had a much lower

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background and was more rapidly taken up by kidneys, and mainly excreted via the renal

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route. Together, these results suggested that Al[18F]F-NOTA-P-GnRH may have clinical potential for imaging of GnRH receptor-positive tumors. 5. Conclusions

In this study, we successfully designed and developed the automated radiosynthesis of a new 18F-labelled GnRH tracer (Al[18F]F-NOTA-P-GnRH) using a commercially available synthesis module. The tracer can be produced with a high labeling yield and high molar activity. Al[18F]F-NOTA-P-GnRH exhibited GnRH receptors specific in rat and in xenograft tumor mouse models. In vivo microPET studies in xenograft tumor mouse models showed significant tumor uptake and trapping inside tumor tissue in two

17

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GnRH-positive tumor models. Thus, the new tracer is a promising candidate for future clinical applications. Abbreviations gonadotropin releasing hormone

FSH

follicle-stimulating hormone

LH

luteinizing hormone

PET

Positron emission tomography

HPLC

high-performance liquid chromatography

NOTA

1,4,7-triazacyclononane-N,N',N''-triacetic acid

HBTU

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

DIPEA

N,N’-diisopropylethylamine

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GnRH

dimethylformamide

TFA

Trifluoroacetic acid

% ID/g

percentage of injected dose per gram of tissue

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ROIs

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DMF

regions of interest

Acknowledgments

We would like to thank Dr. Yuhua Zhong for helpful discussion of the data. This study was supported by grants from the National Natural Science Foundation of China (81701729), Scientific Research Foundation of Southern Medical University (C1034414), Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2017J010), Medical Scientific Research Foundation of Guangdong Province of China

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(A2019389, A2016445), and the President Foundation Project of Nanfang Hospital, Southern Medical University (2018Z003). Declaration of competing interest The authors have no conflicts of interest to disclose. Data Availability

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The data used to support the findings of this study are included within the article and

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supplementary information file.

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Appendix A. Supplementary data

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Supplementary Fig. S1-5 and detailed descriptions of experimental methods are provided in the supporting information.

Bjelobaba I. Editorial : Gonadotropin-releasing Hormone receptor Signaling

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[1]

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2018;54:8120–3. https://doi.org/10.1039/C8CC01400K. [33] Laverman P, McBride WJ, Sharkey RM, Eek A, Joosten L, Oyen WJG, et al. A novel facile method of labeling octreotide with 18F-fluorine. J Nucl Med 2010;51:454–61. https://doi.org/10.2967/jnumed.109.066902. [34] McBride WJ, D’Souza CA, Sharkey RM, Karacay H, Rossi EA, Chang C-H, et al. Improved 18F Labeling of Peptides with a Fluoride-Aluminum-Chelate Complex. Bioconjug Chem 2010;21:1331–40. https://doi.org/10.1021/bc100137x. [35] Liu S, Liu H, Jiang H, Xu Y, Zhang H, Cheng Z. One-step radiosynthesis of 24

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18F-AlF-NOTA-RGD2 for tumor angiogenesis PET imaging. Eur J Nucl Med Mol Imaging 2011;38:1732–41. https://doi.org/10.1007/s00259-011-1847-4. [36] D’Souza CA, McBride WJ, Sharkey RM, Todaro LJ, Goldenberg DM. High-Yielding Aqueous 18F-Labeling of Peptides via Al18F Chelation. Bioconjug Chem 2011;22:1793–803. https://doi.org/10.1021/bc200175c.

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[37] Schottelius M, Berger S, Poethko T, Schwaiger M. Development of Novel 68 Ga- and GnRHR-Targeting Efficiency F-Labeled GnRH-I Analogues with

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na

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High 2008:1256–68.

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Figure Legends

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Fig. 1. Schematic diagram of modified PET-MF-2V-IT-1 synthesizer that is used for

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the production of Al[18F]F-NOTA-P-GnRH.

Fig.

3.

HPLC

analysis

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Fig. 2. Reaction scheme for the radiosynthesis of Al[18F]F-NOTA-P-GnRH. the

stability

of

Al[18F]F-NOTA-P-GnRH.

a)

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Al[18F]F-NOTA-P-GnRH incubation in PBS, bovine or human serum for 2 h

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compared to Al[18F]F-NOTA-P-GnRH from the quality control and its reference

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standard AlF-NOTA-P-GnRH. b) Representative HPLC profiles displaying unchanged Al[18F]F-NOTA-P-GnRH (peak 1) and its metabolite (peak 2) in blood at 1 h after

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intravenous injection. ret. = retention time in min. Fig. 4. Competitive binding curves of AlF-NOTA-P-GnRH and NOTA-P-GnRH. Values are represented as mean  SD, n = 4. Fig.

5.

a)

Decay-corrected

whole-body

coronal

microPET

images

of

Al[18F]F-NOTA-P-GnRH in nude mice bearing MDA-MB-231 and PC-3 tumors after intravenous injection. b) Ex vivo microPET images of rats were evaluated. Tomographic images obtained by PET were comparable to planar photographs of organs. c) MicroPET time-activity curves of tumors and major organs after intravenous injection of Al[18F]F-NOTA-P-GnRH. d) Comparison of tumor-to-muscle 26

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ratio in PC-3 and MDA-MB-231 tumor models. Fig. 6. Immunohistochemical staining of GnRH receptor expression in hypophysis, muscle, PC-3, and MDA-MB-231 xenografted tumor tissues. a) Hypophysis, PC-3, and MDA-MB-231 xenografted tumor lesions exhibited strong brown cytoplasmic staining, whereas muscle tissue exhibited weak brown cytoplasmic staining. b) As a

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comparison, hypophysis, muscle, PC-3, and MDA-MB-231 xenografted tumor tissues were incubated in buffer without the primary goat anti-human GnRH antibody (

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400).

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Table

1.

Automated

radiosynthesis

of

Al[18F]F-NOTA-P-GnRH

using

NOTA-P-GnRH and Al[18F]F on the modified PET-MF-2V-IT-I synthesizer. Organic solvents

Decay-corrected yield

100 µg (57 nmol)a

MeCN

5.7%, 9.2% (n = 2)

100 µg (57 nmol)a

EtOH

5.0%, 9.1% (n = 2)

100 µg (57 nmol)a

DMSO

12.9%, 19.3% (n = 2)

100 µg (57 nmol)a

DMF

14.5%, 25.6% (n = 2)

200 µg (114 nmol)b

DMF

35 ± 10% (n = 6)

Reaction condition: peptide, AlCl3 (4.7 μL, 10 mM), glacial acetic acid (5 µL),

Reaction condition: peptide, AlCl3 (9 μL, 10 mM), glacial acetic acid (8 µL),

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b

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deionized water (50 µL), and solvent (334 µL).

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a

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Amount of peptide

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deionized water (50 µL), and solvent (334 µL).

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Journal Pre-proof Table 2. In vivo biodistribution of Al[18F]F-NOTA-P-GnRH in nude mice bearing PC-3 tumor after intravenous injection. All data are average % ID/g, means ± SD (n = 4). 30 min

60 min

120 min

Blood

1.44 ± 0.31

1.17 ± 0.33

0.12 ± 0.03

Brain

0.08 ± 0.02

0.06 ± 0.01

0.01 ± 0.00

Heart

0.68 ± 0.40

0.35 ± 0.06

Lung

1.46 ± 0.47

Liver

1.36 ± 0.17

Spleen

0.75 ± 0.05

Gall bladder

Kidney

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Organ

0.07 ± 0.02 0.16 ± 0.02

1.83 ± 0.75

1.25 ± 0.45

0.57 ± 0.11

0.70 ± 0.23

1.54 ± 0.48

1.65 ± 0.25

1.32 ± 0.46

6.50 ± 2.27

4.57 ± 1.15

5.40 ± 0.86

0.71 ± 0.28

1.30 ± 1.66

0.96 ± 0.78

0.44 ± 0.13

0.24 ± 0.08

0.06 ± 0.02

Stomach

0.81 ± 0.27

3.03 ± 2.64

0.32 ± 0.03

Bone

0.58 ± 0.21

1.28 ± 0.50

0.17 ± 0.11

Hypophysis

1.84 ± 1.19

--

0.32 ± 0.22

Tumor

1.23 ± 0.73

1.46 ± 0.73

0.38 ± 0.10

Ratio of tumor to

2.80

6.08

6.33

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Muscle

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Intestine

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0.68 ± 0.48

muscle

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6