Sulfonation of Tyrosine as a Method To Improve Biodistribution of

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Sulfonation of Tyrosine as a Method to Improve Biodistribution of Peptide-Based Radiotracers: Novel F-Labelled Cyclic RGD Analogues 18

Mohammad Baqir Haskali, Delphine Denoyer, Wayne Noonan, Carleen Cullinane, Christine Rangger, Normand Pouliot, Roland Haubner, Peter D. Roselt, Rodney J Hicks, and Craig A Hutton Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01062 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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Molecular Pharmaceutics

Sulfonation of Tyrosine as a Method to Improve Biodistribution

of

Peptide-Based

Radiotracers:

Novel 18F-Labelled Cyclic RGD Analogues Mohammad B. Haskali,†,‡,# Delphine Denoyer,#,¶ Wayne Noonan,# Carleen Culinane,# Christine Rangger,ǁ Normand Pouliot,#,§ Roland Haubner,‖ Peter D. Roselt,# Rodney J. Hicks,# Craig A. Hutton †,‡,* †

School of Chemistry and ‡Bio21 Molecular Science and Biotechnology Institute, The

University of Melbourne, VIC 3010, Australia #

The Centre for Molecular Imaging and Translational Research Laboratory, The Peter

MacCallum Cancer Centre, Melbourne, Victoria, Australia ǁ

*

Department of Nuclear Medicine, Medical University of Innsbruck, Innsbruck, Austria

School of Chemistry, Bio21 Institute Building, 30 Flemington Rd, The University of

Melbourne, VIC 3010, Australia. Phone: (+61 3) 8344 2393, email: [email protected].

ACS Paragon Plus Environment

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ABSTRACT. Control of the biodistribution of radiolabeled peptides has proven to be a major challenge in their application as imaging agents for positron emission tomography (PET). Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[18F]fluoropropionate and the biodistribution of the radiolabeled peptides was compared with that of their non-sulfonated, clinically relevant counterparts, [18F]GalactoRGD and [18F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da towards the MW, compared with 189 Da for both the ‘Galacto’ and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabeled peptides.

KEYWORDS. RGD peptides, PET imaging, fluorine-18, biodistribution, sulfonation, tyrosine3-sulfonate, tyrosine

Introduction: ACS Paragon Plus Environment

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Angiogenesis, the growth of new blood vessels from existing vasculature, is a natural biological phenomenon that facilitates physiological processes such as wound healing and embryonic development.1 However, dysregulated angiogenesis plays critical roles in tumor growth and the metastasis of solid tumors.1,2 During angiogenesis, integrin αvβ3 receptors are preferentially over-expressed on the endothelial cell surface.3 Integrin αvβ3 is a cell-adhesion receptor that binds extracellular proteins such as vitronectin and fibronectin via the Arg-Gly-Asp (RGD) tripeptide motif.4 Increased expression of this receptor on tumor cells is a positive marker for metastasis and is required for spontaneous dissemination of bone-metastatic cancer.5-7 Moreover, antagonism of integrin αvβ3 has been shown to suppress the formation of new blood vessels and therefore such antagonists hold great potential for the treatment of metastatic tumors.1,2,8 Hence, radiotracers that bind specifically to integrin αvβ3 are of great interest as diagnostic and therapeutic agents for metastatic cancers. Cyclic RGD-containing pentapeptides bind with high affinity and specificity to αvβ3 integrin and have been the focus of intense investigations toward the development of radiotracers for positron emission tomography (PET) imaging applications in the diagnosis and treatment of cancer.3,9-11 While the Arg-Gly-Asp sequence is essential for binding to αvβ3 integrin, the remaining residues in the cyclic pentapeptide are variable.4,12-14 Lysine is typically included to provide a side chain amine group suitable for ligation of the radionuclide-containing moiety, and an aromatic amino acid residue of D-configuration (D-Phe or D-Tyr) is usually introduced at the remaining site. However, a common problem observed with such cyclic RGD peptides is that their hydrophobic nature generally leads to greater hepatobiliary clearance, resulting in high concentrations of the radiotracer in the liver and intestine and ultimately undesired background activity.12,15,16 Commonly employed strategies to improve the pharmacokinetics of radiolabeled peptides consist of conjugation with hydrophilic compounds, including carbohydrate or polyethylene glycol


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(PEG) moieties. Increased hydrophilicity of radiopharmaceuticals usually correlates with enhanced renal excretion and, in the case of peptides, improved biodistribution and an increased non-plasma protein bound fraction.17 The RGD peptides in current clinical use, [18F]GalactoRGD 1 and [18F]FPPRGD2 2 (Figure 1), have both been successfully employed for the visualization of a range of tumors, exemplifying the utility of such hydrophilic ‘linkers’ in modifying the pharmacokinetic properties of radiotracers.15,16,18 Nevertheless, conjugation of such groups is not without problems. The galactose-derived ‘sugar amino acid’ (SAA) incorporated into [18F]GalactoRGD 1 requires a challenging, multi-step synthesis, while conjugation of either carbohydrate or PEG groups adds significant mass to the peptide, which can adversely affect binding affinity and other biological properties. For example, while incorporation of the SAA group into [18F]GalactoRGD resulted in optimal pharmacokinetic properties, incorporation of SAA into a dimeric RGD peptide resulted in inferior biodistribution.19 Moreover, PEGylation of the monomeric RGD peptide reduced the affinity of the PEGylated RGD peptide 2.20 Ideally, the one-step introduction of a small, highly hydrophilic group that imparts optimal pharmacokinetic properties with little or no impact on the target affinity or specificity of the peptide radiotracer is desirable.


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Figure 1.

18

F-labelled RGD peptides in clinical use, 1 and 2, and the sulfonated RGD peptides

prepared in this study, 3 and 4.

Sulfonation of aromatic compounds is well established as a method to confer water solubility on dyes, polymers and catalysts, and to improve biodistribution of drugs and molecular probes.21-23 With a pKa of ~1.5, sulfonic acids are fully ionised at physiological pH and have the potential to increase hydrophilicity significantly despite their relatively small size. However, very few investigations of sulfonated peptides have been reported.24 Cysteine sulfonic acid – an oxidized form of cysteine – has been incorporated into radiolabeled peptides but, remarkably, a general method for the introduction of sulfonate groups into radiolabeled peptides has not been


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developed. The sulfonation of tyrosine with chlorosulfonic acid has been shown to generate the tyrosine-3-sulfonate derivative in good yield, both on the free amino acid and on tyrosinecontaining peptides.25 Accordingly, we herein report the sulfonation of tyrosine-containing RGD peptides and investigation of the effect of the sulfonate group on the lipophilicity and biodistribution of radiolabeled peptides. We report that the site-specific sulfonation of tyrosine residues increases the hydrophilicity of radiolabeled peptides and leads to improved biodistribution.

Experimental: All chemicals obtained commercially (Sigma Aldrich) were of analytical grade and used without further purification. No-carrier-added [18F]fluoride was obtained using a PETtrace 16.5 MeV cyclotron (Cyclotek) incorporating a high pressure niobium target via the nuclear reaction (98% 18O isotopic enrichment).

18

18

O(p,n)18F

F-Separation cartridges (QMA, Waters) were

preconditioned with the requisite base. Reversed phase solid phase extraction (SPE) cartridges (33µ polymeric reversed phase (30mg/ml), Phenomenex) were pre-conditioned with ethanol and rinsed with water before use. Radioactivity measurements were carried out with a CRC-15PET dose calibrator (Capintec) that was calibrated daily using Cs-137 and Co-57 sources (Isotope Products Laboratories). Radiation was detected using a solid state photodiode scintillator crystal detector (Knauer). Semipreparative HPLC purification of cold materials was performed using an Agilent 1200 series HPLC system, whilst QC analysis was obtained on an Agilent 1100 series HPLC system. Preparative High Performance Liquid Radiochemical Chromatography (HPLRC) was performed using a Knauer 1050 pump, 2500 UV detector and 5050 manager. Radiation was detected using a Knauer solid state photodiode scintillation crystal detector in a TO-5 case. Analytical HPLRC was performed using a Shimadzu HPLC system consisting of a SCL-10AVP


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system controller, SIL-0ADVP auto injector, LC-10 ATVP solvent delivery unit, CV-10AL control valve, DGU-14A degasser and SPD-10AVPV detector. This was coupled to a radiation detector consisting of an Ortec model 276 photomultiplier base with a 925-SCINT ACE-mate preamplifier, amplifier, bias supply and SCA and a Bicron 1M11/2 photomultiplier tube. 1

H NMR spectra of peptides were obtained on a 600 or 800 MHz Bruker Avance spectrometer.

ESMS data was obtained using an Agilent6 510 Q-TOF LC/MS mass spectrometer equipped with an Agilent 1100 LC system.

Peptide Synthesis. c(RGDyK) 5. The cyclic peptide was synthesized as previously reported.17 The crude peptide was purified by HPLC (0.1% TFA in 0–85% MeCN:H2O over 60 min, ProteCol C18HQ305 300Å 5µ 250×4.6 mm column, flow rate 8 mL/min). The appropriate fractions with the cyclic peptide were freeze-dried to afford the cyclic peptide 5 as white foam (80 mg, 26% yield). 1H NMR (800 MHz, D2O) δ 8.39 (d, 1H, J = 7.6 Hz, NH), 7.84 (d, 1H, J = 8.6 Hz, NH), 7.07 (d, 2H, J = 8.4 Hz), 6.78 (d, 2H, J = 8.4 Hz), 4.47 (dd, 1H, J = 10.7, 5.7 Hz), 4.31 (dd, 1H, J = 8.4, 6.3 Hz), 4.14 (d, 1H, J = 14.8 Hz), 3.77 (dd, 1H, J = 11.1, 3.4 Hz), 3.41 (d, 1H, J = 14.8 Hz), 3.17–3.08 (m, 2H), 2.98 (dd, 1H, J = 13.2, 5.8 Hz), 2.88–2.74 (m, 4H), 2.66 (dd, 1H, J = 16.7, 6.7 Hz), 1.78 (m, 1H), 1.67–1.52 (m, 2H), 1.51–1.35 (m, 5H), 0.92–0.78 (m, 2H). ESI-MS: m/z 620 [M+H]+, 310 [M+2H]2+. c(RGDy(SO3)K) 6. Chlorosulfonic acid (125 µL) was added to a solution of c(RGDyK) 5 (30 mg, 0.05 mmol) in TFA (2.5 ml). A white precipitate formed immediately. The mixture was stirred for 30 min, then H2O (1 ml) was added and the mixture was concentrated by a stream of nitrogen to ca. 0.5 ml. The concentrate was diluted with H2O (3.5 ml) and directly purified by HPLC (0.1% TFA in 0–40% MeCN:H2O over 85 min, ProteCol C18HQ305 300Å 5µ 250×4.6


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mm column, flow rate 8 mL/min) to afford the sulfonated peptide 6 (20 mg, 60% yield). 1H NMR (600 MHz, D2O) δ 8.62 (d, J = 7.6 Hz, NH), 8.32 (d, J = 7.6 Hz, NH), 7.47 (s, 1H), 7.23 (d, 2H, J = 8.4 Hz), 6.90 (d, 1H, J = 8.4 Hz, y), 4.47 (dd, 1H, J = 10.7, 5.7 Hz, y ), 4.32 (dd, 1H, J = 8.4, 6.3 Hz, R), 4.15 (d, 1H, J =14.8 Hz, G), 3.74 (dd, 1H, J = 11.1, 3.4 Hz, K), 3.40 (d, 1H, J = 14.8 Hz, G), 3.18–3.07 (m, 2H, R), 3.04 (dd, 1H, J = 13.2, 5.8 Hz, y), 2.90–2.77 (m, 4H, f, K, D), 2.65 (dd, 1H, J = 16.7, 6.7 Hz, D), 1.82–1.64 (m, 1H, R), 1.55–1.53 (m, 2H, R, K), 1.50–1.32 (m, 5H, R, K), 0.91–0.83 (m, 1H, K), 0.73–0.64 (m, 1H, K). ESI-MS: m/z 700 [M+H]+, 350 [M+2H]2+. FP-c(RGDy(SO3)K) 3. 4-Nitrophenyl 2-fluoropropionate (NFP 7, 4 mg, 0.02 mmol) was added to a solution of c(RGDy(SO3)K) 6 (12 mg, 0.2 mmol) in DMF (0.5 ml) and TEA (7 µL, 0.6 mmol). The mixture was concentrated under vacuum, then H2O (10 ml) was added and the mixture was purified by HPLC (0.1% TFA in 0–40% MeCN:H2O over 60 min, ProteCol C18HQ305 300Å 5µ 250×4.6 mm column, flow rate 8 mL/min) to afford the cold standard 3 (8 mg, 60% yield). 1H NMR (600 MHz, D2O) δ 8.37 (d, J = 8.0 NH), 8.35 (d, J = 6.1 NH), 7.94 (d, J = 8.6, NH), 7.47 (d, J = 2.1, y), 7.19 (dd, J = 8.7, 1.9 Hz, y), 6.88 (d, J = 8.8 Hz, y), 5.0 (dq, J = 49.1, 6.0 Hz, FP), 4.50 (dd, 1H, J = 10.6, 5.9 Hz, y), 4.30 (dd, 1H, J = 9.5, 5.6 Hz, R), 4.15(d, 1H, J = 15.5 Hz, G), 3.77 (dd, 1H, J = 11.0, 4.0 Hz, k), 3.40(d, 1H, J = 15.4 Hz, G), 3.10 (m, 4H, R, k), 2.99 (dd, 1H, J = 13.9, 6.0 Hz, y), 2.84 (m, 2H, y, D), 2.66 (dd, 1H, J = 17.4, 6.4 Hz, D), 1.78 (m, 1H, R), 1.58 (m, 2H, R, k), 1.45 (dd, 3H, J = 25.2, 7.0 Hz, FP) 1.43 (m, 3H, R, k), 0.91 (m, 1H, k), 0.84 (m, 1H, k). ESI-MS: m/z 774 [M+H]+, 387 [M+2H]2+. E(RGDy(SO3)K)2 8. To a solution of HATU (24.5 mg, 60.0 µmol), Boc-Glu-OH (7.9 mg, 30.1 µmol) and DIPEA (112 mL, 0.64 mmol) in DMF (0.3 ml) was added c(RGDy(SO3)K) 6 (40 mg, 60.0 µmol) in DMF (0.3 ml). The reaction was stirred for 15 min and then TFA (3 ml) was added and the mixture stirred for another 1 h. The TFA was removed under a stream of nitrogen and the ACS Paragon Plus Environment

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crude peptide was purified by HPLC (0.1 % TFA in 0–50% MeCN:H2O over 80 min, ProteCol C18HQ305 300Å 5µ 250×4.6 mm column, flow rate 8 mL/min) to afford the sulfonated RGD dimer 8 (30.0 mg, 75%, ≥99% purity) as a fluffy white solid. 1H NMR (600 MHz, D2O) δ 7.44 (d, 2H, J = 2.2, y), 7.23–7.17 (m, 2H, y), 6.89 (d, 2H, J = 8.9 Hz, y), 4.71 (suppressed, D), 4.49 (dd, 2H, J = 10.6, 4.5 Hz, y), 4.31 (ddd, 2H, J = 9.4, 5.6 Hz, R), 4.15 (d, 2H, J = 15.7 Hz, G), 3.97 (t, 1H, J = 6.9, E), 3.74 (dd, 2H, J = 11.7, 4.1 Hz, K), 3.40 (d, 1H, J = 15.7 Hz, G), 3.21– 3.14 (m, 1H, K), 3.13–3.03 (m, 6H, R, K), 3.02–2.91 (m, 4H, K, y), 2.85–2.77 (m, 4H, y, D), 2.63 (dd, 2H, J = 16.6, 6.6 Hz, D), 2.34 (t, 2H, J = 8.2 Hz, E), 2.10 (t, 2H, J = 7.1 Hz, E), 1.82– 1.73 (m, 2H, R), 1.62–1.53 (m, 4H, R, K), 1.47–1.35 (m, 6H, R), 1.35–1.23 (m, 4H, K), 0.93– 0.84 (m, 2H, K), 0.83–0.70 (m, 2H, K). ESI-MS: m/z 1351 [M+H]+, 675 [M+2H]2+. FP-E(RGDy(SO3)K)2 4. E(RGDy(SO3)K)2 8 (5.0 mg, 3.0 µmol) and NFP 7 (1.1 mg, 40.0 µmol) were dissolved in DMF (0.1 ml) and DIPEA (20 µL, 0.15 mmol) was added. After 10 min, the solution was diluted with water (3.5 ml) and purified by HPLC (0.1 % TFA in 0–50% MeCN:H2O over 80 min, ProteCol C18HQ305 300Å 5µ 250×4.6 mm column, flow rate 8 mL/min) to afford the cold standard 4 (4.2 mg, 86%, ≥99 % purity) as a fluffy white solid. 1H NMR (600 MHz, D2O) δ 7.47–7.44 (m, 2H, y), 7.20–7.15 (m, 2H, y), 6.90–6.85 (m, 2H, y), 5.08 (dqd, 1H, J = 48.1, 7.0, 2.0 Hz, FP), 4.71 (suppressed, D), 4.52 (m, 2H, y), 4.29 (m, 2H, R), 4.21 (m, 1H, E), 4.15 (d, 2H, J = 15.5 Hz, G), 3.77 (m, 2H, K), 3.42 (d, 1H, J = 15.5 Hz, G), 3.18– 2.90 (m, 11H, R, K, y) 2.88–2.78 (m, 4H, y, D), 2.66 (dd, 2H, J = 17.1, 6.7 Hz, D), 2.30 (m, 2H, E), 2.06 (m, 1H, E), 1.96 (m, 1H, E), 1.82–1.73 (m, 2H, R), 1.72–1.48 (m, 2H, R, K), 1.47–1.37 (m, 2H, R, K), 1.47–1.35 (m, 8H, R, FP), 1.35–1.23 (m, 4H, K), 0.94–0.77 (m, 4H, K). ESI-MS: m/z 1614 [M+H]+, 807 [M+2H]2+, 538 [M+3H]3+. Radiochemistry. [18F]NFP 7. The radiolabeled synthon was prepared according to the literature procedure using a radiosynthesis module.26 In brief, anhydrous K222.K+18F– complex was treated


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with p-nitrophenyl 2-bromopropionate (20.0 mg, 0.07 mmol) in tBuOH:MeCN (2 ml, 8:2, vial 3). After 5 min at 100 °C, the solvent was evaporated to about 0.5 ml at 65 °C and then diluted with 0.1% TFA in 60:40 MeCN:H2O (1.5 ml, vial 4). The resulting mixture was transferred into the first HPLC loop loading vial. The reaction vial was washed with additional 0.1% TFA in 60:40 MeCN:H2O (1.5 ml, vial 6) and again transferred to the same HPLC loop loading vial. The crude ester was then purified by preparative HPLC 1 (0.1% TFA in 60:40 MeCN:H2O, Macherey-Nagel EP250/16 Nucleosil 100-7 C18 column equipped with EP30/16 guard column, flow rate 6 mL/min) to afford [18F]-7 as a clear solution. [18F]FP-c(RGDy(SO3)K 3. [18F]NFP 7 (2231–5550 MBq), isolated on a C18 Cartridge, was eluted with DMSO (300 µL) into a vial charged with c(RGDy(SO3)K) 6 (0.2 mg, 0.28 µmol). TEA:DMSO (1:20, 10 µL) was added. After 5 min, a portion (555 MBq) of the material was purified by HPLC (0.1 % TFA in 0–80% MeCN:H2O over 40 min, Phenomenex Luna 5µ C18(2) 100 Å 250×10 mm column, flow rate 4 mL/min) to afford the title compound [18F]-3 in a clear solution (2072–2701 MBq, 47–51 % yield n.d.c., SA: 135–154 GBq/µmol). [18F]FP-E(RGDy(SO3)K)2 4. [18F]NFP 7 (3293–3996 MBq) was eluted with DCM (1.5 ml) via a sodium sulfate cartridge into a vial. The DCM was evaporated at room temperature with a gentle flow of nitrogen. E(RGDy(SO3)K)2 8 (1.0 mg, 0.7 µmol) and TEA (15 µL, 0.11 µmol) in DMSO (0.35 ml) was transferred into the vial containing the dried [18F]NFP. The reaction was heated to 70 °C for 10 min. The residue was diluted with sterile water (3.0 ml, 0.1 TFA). The material was then purified by HPLC (0.1 % TFA in 10–80% MeCN:H2O over 40 min, Phenomenex Luna 5µ C18(2) 100 Å 250×10 mm column, flow rate 4 mL/min). The HPLC fraction containing the product was diluted with sterile water (40 ml) and isolated on C18 cartridge. The C18 cartridge charged with [18F]FP-E(RGDy(SO3)K)2, was washed with PBS (5 ml) and eluted with ethanol (0.4 ml). The elution lines were rinsed with PBS (5 ml) and filtered ACS Paragon Plus Environment

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into the sterile vial to obtain [18F]FP-E(RGDy(SO3)K)2, [18F]-4 (560–849 MBq, 15–19% yield n.d.c., SA: 111–133 GBq/µmol). An analytical HPLC trace of the 18F-labelled material matched that of the 19F-material.

Peptide HPLC QC Analysis: The following HPLC columns were used for QC analysis of NFP and peptides: QC

analysis

of

cold

standards

FP-c(RGDy(SO3)K)

3,

FP-E(c(RGDy(SO3)K)2)

4,

c(RGDy(SO3)K) 6 and E(c(RGDy(SO3)K)2) 8: SGE ProteCol C18HPH125 120Å 5µ 150×4.6 mm column. QC analysis and specific activity determination of [18F]NFP 7 and [18F]-labelled peptides ([18F]-1–4): Phenomenex Luna 100Å 5µ C18(2) 150×4.6 mm column.

αvβ3-Integrin Receptor Binding Studies. Isolated human αvβ3-integrin receptors were used to determine the in vitro binding affinities of FP-c(RGDy(SO3)K) 3 and FP-E(RGDy(SO3)K)2 4 following the method of Nachman et al.27 with some modifications.28 Binding affinities of FPc(RGDy(SO3)K) 3 and FP-E(RGDy(SO3)K)2 4 were determined using 125I-c(RGDyV), which was prepared using the Iodogen method reported previously.13 The day before the experiment, 96well plates were coated with αvβ3-integrin receptors (20 ng receptor/well) diluted in coating buffer (25 mM tris(hydroxymethyl)aminomethane (Tris), 150 mM sodium chloride (NaCl), 1 mM calcium chloride (CaCl2), 0.5 mM magnesium chloride (MgCl2), 10 µM manganese (II) chloride tetrahydrate (MnCl2⋅4H2O)). The receptors were left to incubate for 16 h at 4°C, followed by 2 h incubation with blocking buffer (coating buffer plus 1% bovine serum albumin (BSA)) to reduce non-specific binding of the peptides to the wells. Then to each well (in triplicate) was added 50 µL of competitor (RGD peptides) in increasing concentrations (0.001ACS Paragon Plus Environment

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100 nM RGD in binding buffer (coating buffer plus 0.1% BSA)) and 50 µL radioligand solution (~25,000 cpm

125

I-c(RGDyV) in binding buffer). As a control assay, the same procedure was

carried out using c(RGDyV) as the competitor. The assay was stopped after 2 h by removing the solutions. Each well was washed twice with 150 µL ice-cold binding buffer. The remaining receptor bound activity was removed with 100 µL hot (~60°C) 2 M sodium hydroxide solution and transferred into plastic vials which were then measured in a gamma-counter. The halfmaximal inhibitory concentration (IC50) values were calculated by fitting the data with nonlinear regression.

Cells and Tumor Implantation. The 66cl4β3 mouse mammary carcinoma cells genetically engineered to over-express integrin αvβ3 were obtained as a gift from Dr N. Pouliot (Peter MacCallum Cancer Centre, Melbourne, Australia), prepared as previously described29 from the mouse mammary epithelial cell line 66cl4 derived by Dr F. Miller (Michigan Cancer Foundation, Detroit, MI, USA). A431 human squamous cell carcinoma line was purchased from ATCC. Cells were maintained in a 37 °C and 5% CO2 incubator under humidified atmosphere and cultured in α-MEM medium supplemented with 5% fetal calf serum. All animal experiments received Peter MacCallum Cancer Centre animal research ethics approval and were conducted in compliance with strict guidelines. 66cl4β3 cells (1x105) suspended in 100 µL PBS:Matrigel (1:1) were subcutaneously administered in the shoulder of 6–8 week old Balb/c mice. Mice were imaged once tumor graft volumes reached approximately 200mm3.

Small Animal PET Imaging. Mice (3 mice per time point) were injected with 9–13 MBq of sterile filtered radiotracer in 5% ethanol in saline (100–200 µL). Just prior to imaging, mice were anaesthetized via inhalation of 2.5% isoflurane in 50% O2 in air and imaged for 10 min on a


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small animal PET scanner (resolution 2.7 mm at the center of the FOV) at five time intervals (30 min, 60 min, 90 min, 120 min and 240 min). The energy window was within 450–700 keV and a 6 ns coincidence-timing window was used. Acquired data was in a 3D mode and was decay corrected for isotope decay and random disintegrations. Image reconstruction was established using the 3D RAMLA algorithm and the tracer uptake was measured using the region of interest (ROI) software. Briefly, ROIs were circled around tumors and background regions to express the mediastinal blood pool. The regions of tracer accumulation such as the kidneys and the bladder were excluded. Tumor-to-background ratios were obtained by taking the ratio of the maximum pixel intensity within the tumor ROI in relation to the average pixel intensity within the background ROI. The maximum standardised uptake value (SUV) was determined to aid the image comparison of each tracer. For the blocking studies, five mice were co-injected with the tracer and 0.2 mg of c(RGDyK) peptide and imaged at 90 or 120 min, as described above.

Biodistribution Studies. Following PET scanning, all mice were sacrificed and a sample of the tumor, blood, kidneys, liver, spleen and muscle were taken and weighted before analysis of retained radioactivity. The percentage of injected dose per gram of tissue (%ID/g) was obtained by measuring the disintegrations at 511 keV (15% were quantified in a well counter combined with a multi-channel analyzer).

Autoradiography and H&E Staining Studies. Balb/C mice bearing 66cl4β3 tumors were injected intravenously with 8–15 MBq of either [18F]FP-c(RGDy(SO3)K) 3 or [18F]FP-Ec(RGDy(SO3)K)2 4. After 60 min, mice were sacrificed by cervical dislocation. Tumor tissues were then removed, embedded in Optimal Cutting Temperature medium (OCT) and snap-frozen in cooled isopentane. Frozen sections of 10 µm were cut on a cryostat at –12˚C and thawACS Paragon Plus Environment

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mounted on glass microscope slides. After air-drying, the sections were covered with solid scintillation paper and scanned in a high resolution autoradiographic imager. Disintegrations were recorded during a period of 12 h. After autoradiography, the sections were fixed for 1 min with 10% neutral buffered formalin before being stained with hematoxylin and eosin. Sections were then mounted in mounting medium and cover-slipped. Digital images of whole sections were taken with a dissecting microscope equipped with a digital camera. Images at higher magnification were taken with a microscope at ×40 objective. Nuclei appeared dark-blue and cytoplasm and collagen appeared pink. Results: Chemistry and Radiochemistry. The cyclic-RGD peptide c(RGDyK) 5 was treated with chlorosulfonic acid in TFA to generate the sulfonated peptide c(RGDy(SO3)K) 6 in good yield in a single, simple step (Scheme 1). Analysis by 1H NMR spectroscopy indicated sulfonation had occurred at the 3-position of the tyrosine aromatic ring, confirming the site-specific sulfonation. No protection of the side chain groups was necessary.


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Scheme 1: Preparation of 18F-labelled sulfonated cyclic RGD peptide [18F]-3.

The sulfonated dimeric RGD peptide, E(RGDy(SO3)K)2 8 was prepared by coupling of 2 equiv. of the sulfonated RGD peptide, c(RGDy(SO3)K) 6, with Boc-Glu using HATU (Scheme 2). The Boc group was then removed by treatment with TFA to give dimeric peptide 8. The fluoropropionylated peptides, FP-c(RGDy(SO3)K) 3 and FP-E(RGDy(SO3)K)2 4, were prepared by treatment of c(RGDy(SO3)K) 6 and E(RGDy(SO3)K)2 8, respectively, with 4nitrophenyl 2-fluoropropionate 7 in DMF. The cold standards were characterized by 2D NMR and HRMS.


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Scheme 2: Preparation of 18F-labelled, sulfonated cyclic RGD peptide dimer [18F]-4.

Subsequently, the radiofluorination of these peptides was undertaken with [18F]NFP ([18F]-7). [18F]NFP was synthesized according to a one-step radiofluorination method utilizing a radiosynthesis module,15 and was utilized directly to prepare both [18F]FP-c(RGDy(SO3)K) 3 and [18F]FP-E-c(RGDy(SO3)K)2

4.

The

radiosynthesis

of

[18F]FP-c(RGDy(SO3)K)

3

was

accomplished in 99%. Radiosynthesis of [18F]FP-E(RGDy(SO3)K)2 4 required more forceful conditions than those used to prepare the monomeric RGD peptide 3 (70 °C cf. 35 °C). This was presumed due to a ACS Paragon Plus Environment

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greater steric hindrance at the central glutamate α-amino group in the RGD dimer 8 than the terminal lysine amino group in the monomeric RGD peptide 6. Furthermore, pre-drying [18F]NFP 7 using a sodium sulfate cartridge was essential to reduce the extent of competing hydrolysis of the activated ester. [18F]FP-E(RGDy(SO3)K)2 4 was prepared in approximately 90 min with an overall radiochemical yield of 3% n.d.c. from free fluoride, and a specific activity of 118 GBq/µmol. The peptide was isolated in a radiochemical purity >99%.

Determination of Lipophilicity. Determination of the distribution coefficient, logD, of 3 and 4 was attempted using the shake-flask method.30,31 In brief, radiolabeled peptides 3 and 4 were dissolved in a 1:1 mixture of phosphate buffer (pH 7.4) and octanol at room temperature. The solutions were equilibrated by shaking for 5 min and then centrifuged to separate the two layers. The phases were isolated and counts contained in each phase measured using a well counter. However, all attempts to calculate the distribution coefficient D (log D) of the sulfonated RGD peptides using this method were hampered by the detergent-like properties of these compounds, which resulted in poorly reproducible partitioning of the peptides between the aqueous and octanol layers. Accordingly, relative hydrophilicities of these peptides were estimated from their retention times by RP-HPLC.32-35 FP-c(RGDy(SO3)K) 3 has a significantly reduced retention time compared with both GalactoRGD 1 and FPPRGD2 2 (6.7 min for 3 cf. 8.8 min for GalactoRGD 1, Figure 2), despite lacking the SAA linker. Similarly, the sulfonated RGD dimer 4 has a greatly reduced retention time compared with FPPRGD2 2 (6.9 min for 4 cf. 8.8 min for FPPRGD2 2, Figure 2), despite lacking the mini-PEG linker. These reduced retention times on RP-HPLC suggest the introduction of the sulfonate group has a more pronounced effect on increasing the hydrophilicity than conjugation of either SAA or PEG groups.


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Figure 2: HPLC retention times of GalactoRGD 1 (8.8 min, blue trace), FPPRGD2 2 (8.8 min, red trace), FP-c(RGDy(SO3)K) 3 (6.7 min, green trace) and FP-E-c(RGDy(SO3)K)2 4 (6.9 min, magenta trace). Absorbance detected at 254 nm.

αvβ3-Integrin Receptor Binding Assay. The ability of sulfonated peptides FP-c(RGDy(SO3)K) 3 and FP-E-c(RGDy(SO3)K)2 4 to inhibit the binding of

125

I-c(RGDyV) to isolated immobilized

αvβ3 receptors was determined in a competitive binding assay (Figure 3). Both peptides displayed an IC50 value in the nanomolar range and compared favorably with the control competitor peptide c(RGDyV). The sulfonated monomeric peptide FP-c(RGDy(SO3)K) 3 had an IC50 of 2.4 nM while the sulfonated dimer FP-E-c(RGDy(SO3)K)2 4 displayed an IC50 value of 0.70 nM indicating a higher binding affinity, in line with previous observations of dimeric RGD peptides. Thus, the presence of the sulfonated tyrosine residues in these RGD peptides has no deleterious impact on receptor binding affinity, with the sulfonated RGD peptides 3 and 4 exhibiting significantly higher binding affinities than the gold standards FP-Galacto-RGD 1 and FPP(RGD)2 2.36,37


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100

normalized binding

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


4 dimer Y monomer X 3 c(RGDyV)

80 60 40 20 0 -12

-10

-8

-6

log(concentration)

Figure 3: Inhibition curves (IC50) of FP-c(RGDy(SO3)K) 3 (blue), FP-E-c(RGDy(SO3)K)2 4 (green) and control peptide c(RGDyK) (red) in a competitive binding assay using immobilized αvβ3-integrin receptors.

Small Animal PET Imaging. Imaging studies were conducted on female Balb/c mice bearing αvβ3-expressing mammary carcinoma (66cl4β3). PET images were acquired at various time points (30, 60, 90, 120 and 240 min) post-injection (p.i.). Representative PET maximum intensity projection (MIP) images of both [18F]FP-c(RGDy(SO3)K) 3 and [18F]FP-Ec(RGDy(SO3)K)2 4 are shown in Figure 4. Tumor was clearly visible at each time point with the most favourable timepoint being at 120 min p.i. for both tracers.


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Figure 4: Representative whole-body PET images of 66cl4β3 tumor bearing mice at 30, 60, 90, 120 and 240 minutes after intravenous injection of either [18F]FP-c(RGDy(SO3)K) 3 (upper panels) or [18F]FP-E-c(RGDy(SO3)K)2 4 (lower panels). Tumor-to-background ratios (TBR) are indicated below each image and represent the mean±SD of 3 mice. p.i. = post-injection.

Biodistribution studies. Mice were sacrificed following PET scanning and the biodistribution of radioactivity determined. Primary tumor, blood, muscle, kidneys, spleen and liver were recovered and the radioactivity in these tissues was measured by well counting (Tables 1 and 2). For both radiotracers, radioactivity rapidly decreased from the blood during the first hour post injection, then continued to clear but at a much slower rate. This suggests that decrease in tracer activity in blood with time followed a bi-exponential kinetic pattern. Muscle uptake decreased over the course of the study. Uptake in the liver was moderate at 30 min (2.6 %ID/g for [18F]FPc(RGDy(SO3)K) 3 and 2.7 %ID/g for [18F]FP-E-c(RGDy(SO3)K)2) 4 and cleared to reach 0.7 %ID/g for [18F]FP-c(RGDy(SO3)K) 3 and 1.3 %ID/g for [18F]FP-E-c(RGDy(SO3)K)2) 4 at 240 min. While kidney uptake was 5.96 %ID/g and 12.9 %ID/g 30 min after [18F]FP-c(RGDy(SO3)K)


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3 and [18F]FP-E-c(RGDy(SO3)K)2 4 injection, respectively, radioactivity rapidly cleared with only 1.6 %ID/g and 1.8 %ID/g remaining in this organ at 240 min for [18F]FP-c(RGDy(SO3)K) 3 and [18F]FP-E-c(RGDy(SO3)K)2 4, respectively. Relatively high initial kidney uptake of [18F]FPc(RGDy(SO3)K) 3 and [18F]FP-E-c(RGDy(SO3)K)2 4, followed by a continuous decrease in the %ID/g in the liver, indicate predominant renal clearance presumably as a result of the enhanced hydrophilicity. In the tumor, [18F]FP-c(RGDy(SO3)K) 3 and [18F]FP-E-c(RGDy(SO3)K)2 4 %ID/g was comparable at 30 min and only slightly decreased at 240 min. Tumor-to-blood ratios increased gradually over the course of the experiment for [18F]FP-c(RGDy(SO3)K) and is maximal at 240 min post tracer administration, consistent with rapid blood clearance and good tumor retention (Figure 5A). A similar pattern was observed for the other tissues analyzed (Figure 5A). Tumor-to-tissue ratios of mice injected with [18F]FP-E-c(RGDy(SO3)K)2 4 were the highest at the 60 min and 90 min time points, indicating that [18F]FP-E-c(RGDy(SO3)K)2 PET scans could be performed earlier than [18F]FP-c(RGDy(SO3)K) PET scans (Figure 5B). Indeed, the shorter time interval between [18F]FP-E-c(RGDy(SO3)K)2 injection and image acquisition is a highly desirable feature for clinical translation of PET-tracers. The substantial tumor uptake combined with low retention of both radiolabeled peptides in non-target organs reflects the results observed in the small animalPET imaging study.


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Table 1: Ex vivo biodistribution of [18F]FP-c(RGDy(SO3)K) 3 in selected tissues of 66cl4β3 tumor bearing mice at different time points. Results are expressed as percentage of injected dose per gram of tissue (%ID/g) and represent the mean±SD of 3 mice. %ID/g at time post-injection (min) Tissue

30

60

90

120

240

Tumor

3.80 ± 0.52

3.44 ± 0.51

2.54 ± 1.35

2.53 ± 0.37

2.73 ± 0.82

Blood

0.80 ± 0.11

0.44 ± 0.09

0.18 ± 0.04

0.19 ± 0.02

0.12 ± 0.01

Muscle

0.89 ± 0.08

0.75 ± 0.12

0.71 ± 0.39

0.52 ± 0.08

0.46 ± 0.29

Kidneys

5.96 ± 0.69

4.49 ± 0.53

2.74 ± 0.15

2.59 ± 0.03

1.56 ± 0.27

Spleen

1.33 ± 0.65

1.43 ± 0.05

0.88 ± 0.27

0.85 ± 0.09

0.54 ± 0.12

Liver

2.64 ± 1.51

1.82 ± 0.39

0.93 ± 0.20

1.07 ± 0.15

0.67 ± 0.24

Table 2: Ex vivo biodistribution of [18F]FP-E-c(RGDy(SO3)K)2 4 in selected tissues of 66cl4β3 tumor bearing mice at different time points. Results are expressed as percentage of injected dose per gram of tissue (%ID/g) and represent the mean±SD of 3 mice. %ID/g at time post-injection (min) Tissue

30

60

90

120

240

Tumor

4.36 ± 0.29

5.48 ± 0.99

4.51 ± 0.52

2.98 ± 1.40

2.47 ± 0.25

Blood

1.45 ± 0.54

0.59 ± 0.07

0.46 ± 0.03

0.52 ± 0.03

0.44 ± 0.03

Muscle

1.07 ± 0.11

0.66 ± 0.06

0.50 ± 0.07

0.82 ± 0.50

0.38 ± 0.02

Kidneys

12.94 ± 7.25

6.03 ± 2.42

3.56 ± 0.80

4.51 ± 1.48

1.84 ± 0.07

Spleen

3.13 ± 0.51

2.08 ± 0.70

1.91 ± 0.20

2.31 ± 0.84

1.32 ± 0.08

Liver

2.74 ± 1.54

2.51 ± 0.40

2.38 ± 0.35

2.50 ± 0.29

1.28 ± 0.15


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Figure 5: Tumor-to-tissue ratio of (A) [18F]FP-c(RGDy(SO3)K) 3 and (B) [18F]FP-Ec(RGDy(SO3)K)2 4 determined by well counting at the indicated time point. Results represent the mean±SD of 3 mice.


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Blocking studies. Blocking experiments were performed by co-injection of 0.2 mg of unlabeled

c(RGDyK)

peptide

with

either

[18F]FP-c(RGDy(SO3)K)

3

or

[18F]FP-E-

c(RGDy(SO3)K)2 4 (Figure 6). A decrease of over 80% of radioactivity in the tumor was observed in presence of excess c(RGDyK) compared with the control, confirming the specific binding of [18F]FP-c(RGDy(SO3)K 3 and [18F]FP-E-c(RGDy(SO3)K)2 4. Radioactivity was cleared significantly faster, and organ uptake was lower, when mice were administered with blocking peptide. This phenomenon has been observed with other radiolabeled RGD peptides38,39 and some of this binding can be attributed to the expression of αVβ3 in normal vasculature and to multiple cell types expressing αVβ3 including osteoclasts, activated endothelial and smooth muscle cells, platelets (αIIbβ3), megakaryocytes and macrophages.40

Figure 6: Small animal PET maximum intensity projection images of 66cl4β3 tumor bearing mice at 120 min or 90 min p.i. of (A) [18F]FP-c(RGDy(SO3)K) 3 and (C) [18F]FP-EACS Paragon Plus Environment

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c(RGDy(SO3)K)2 4, respectively, with (blocked) or without (control) co-injection of 0.2 mg of c(RGDyK). Black arrows indicate the tumors. Ex vivo biodistribution of (B) [18F]FPc(RGDy(SO3)K) 3 and of (D) [18F]FP-E-c(RGDy(SO3)K)2 4 following PET scanning. Results are expressed as percentage of injected dose per gram of tissue (%ID/g) and represent the mean±SD of 3 mice. Tissue uptake in control mice was compared to tissue uptake in mice co-injected with c(RGDyk) and statistical significance was determined using Student’s t test; ** p

Sulfonation of Tyrosine as a Method To Improve Biodistribution of

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