Radiosynthesis and Preclinical Evaluation of 18F-Fluoroglycosylated

Article pubs.acs.org/bc

Radiosynthesis and Preclinical Evaluation of 18F‑Fluoroglycosylated Octreotate for Somatostatin Receptor Imaging Simone Maschauer,† Marcus Heilmann,† Carmen Wan̈ gler,‡ Ralf Schirrmacher,§ and Olaf Prante*,† †

Molecular Imaging and Radiochemistry, Department of Nuclear Medicine, Friedrich Alexander University Erlangen-Nürnberg (FAU), Schwabachanlage 6, 91054 Erlangen, Germany ‡ Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine Medical Faculty Mannheim of Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany § Medical Isotope Cyclotron Facility, Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada ABSTRACT: Short synthetic octapeptide analogs derived from the native somatostatin peptides SST-14 and SST-28, namely, octreotate (TATE) or octreotide (TOC), bind with high affinity to somatostatin receptors (sstr), mainly to subtypes 2 and 5, which are expressed in high density on neuroendocrine tumors (NET). Therefore, radiolabeled TATE or TOC derivatives represent highly valuable imaging probes for NET diagnosis by positron emission tomography (PET). The aim of our study was the development of an 18F-labeled octreotate analog as an alternative radiotracer for the clinically established 68Ga-DOTATOC and 68Ga-DOTATATE. We applied our previously developed method based on copper(I)catalyzed azide−alkyne cycloaddition (CuAAC) to the radiosynthesis of 18F-fluoroglycosylated TATE ([18F]FGlc-TATE). [18F]FGlc-TATE was obtained in high yields of 19−22% (non-decay-corrected, referred to [18F]fluoride) and in high specific activities of 32−106 GBq/μmol. [18F]FGlc-TATE showed high affinity to sstr expressed on AR42J cells (IC50 = 4.2 nM) with fast and high internalization, and a beneficial logD7.4 of −1.8. In AR42J tumor bearing nude mice, [18F]FGlc-TATE showed high and specific tumor uptake of 5.6%ID/g at 60 min post-injection, as determined by blocking experiments using octreotide, and fast clearance from other organs, resulting in excellent tumor-to-blood ratios increasing from 9 to 17 from 30 to 60 min postinjection. Small animal PET studies revealed high uptake of [18F]FGlc-TATE in the tumor which could be blocked with octreotide by >99%. Overall, [18F]FGlc-TATE revealed excellent in vitro and in vivo properties and is therefore a viable alternative 18F-labeled radiopeptide for imaging somatostatin receptor-positive tumors by PET.

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With a half-life of only 68 min, 68Ga-labeled tracers are limited to clinics which are able to produce the radiopharmaceutical on site by a GMP-compliant process. With a half-life of 109 min, a high abundance of its positron emissions (97%), and a maximum positron energy of 635 keV, resulting in a shorter positron range and therefore a much better image resolution, fluorine-18 can be regarded as one of the most favorable radionuclides for PET imaging.5,6 Therefore, a radioligand for sstr2 labeled with fluorine-18 would provide the advantage that it could be used in nuclear medicine facilities located apart from its production site. Until now, only a few radiotracers for sstr labeled with fluorine-18 are published.7−13 Among these, the most promising were bearing glycosyl residues to reduce lipophilicity in order to enhance the clearance properties of the tracer in vivo.7,8,11−13 A chemoselective method for 18F-labeling and concomitant glycosylation in one reaction step is the use of [18F]FDG for oxim

INTRODUCTION

The somatostatin (SST) receptors belong to the family of Gprotein-coupled receptors and comprise five distinct subtypes termed sstr1−sstr5. Neuroendocrine tumors (NET) express high amounts of somatostatin receptors, mainly sstr2, followed by equal amounts of sstr1 and sstr5, whereas subtypes 3 and 4 are hardly expressed.1,2 The natural SST peptides, SST-14 and SST-28, bind with low nanomolar affinity to the five receptor subtypes, whereas short synthetic octapeptide analogs, such as octreotate or octreotide, bind with high affinity to only subtypes 2, 3, and 5.3 Currently, the most frequently used radiotracers for NET diagnosis by positron emission tomography (PET) are gallium-68-labeled 68Ga-DOTATATE and 68 Ga-DOTATOC. 68Ga-DOTATOC has high affinity toward sstr2 and sstr5, whereas 68Ga-DOTATATE only has affinity to sstr2 and its affinity is 10-fold higher compared to 68GaDOTATOC. Nevertheless, in one of the few head-to-head studies, direct patient-based comparison of both radiotracers showed almost no differences regarding diagnostic accuracy for the detection of NET lesions.4 © XXXX American Chemical Society

Received: August 25, 2016 Revised: October 6, 2016

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DOI: 10.1021/acs.bioconjchem.6b00472 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 1. Synthesis of FGlc-TATEa

a

(a) CuSO4, sodium ascorbate, H2O, DMSO (H2O/DMSO 10/1), room temperature, 90 min.

Scheme 2. Radiosynthesis of 18F-Fluoroglycosylated TATEa

(a) [18F]fluoride, K2CO3, KH2PO4, crypt-222, CH3CN, 85 °C, 5 min; (b) 60 mM NaOH, 60 °C, 5 min; (c) hexin-TATE (60 nmol), Cu(OAc)2, sodium ascorbate, THPTA, sodium phosphate buffer (pH 8.0), 60 °C, 10 min.

a

conjugation with a aminooxy precursor.14,15 Recently, we established an alternative methodology for 18F-fluoroglycosylation based on copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC).16,17 This methodology was applied by us and others to a variety of biomolecules, resulting in a large number of new 18 F-labeled ligands for various molecular targets which had favorable bioproperties and were suitable for in vivo use in PET imaging studies.18−20 Consequently, it is our intention here to use this 18F-fluoroglycosylation method to provide access to an alternative new 18F-fluoroglycosylated octreotate derivative. We present here the synthesis, radiosynthesis, and in vitro evaluation of the new octreotate derivative [18F]FGlc-TATE. Finally, the biodistribution of [18F]FGlc-TATE was studied in AR42J tumor bearing nude mice as well as its suitability as radioligand for sstr imaging by small animal PET.

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For the radiosynthesis of the 18F-fluoroglycosylated octreotate analog [18F]FGlc-TATE, we followed our previously published three-step two-pot methodology.17,22 After 18Ffluorination of the tosyl precursor and its deacetylation with NaOH, the free 6-deoxy-6-[18F]fluoroglucosyl azide was coupled to the alkyne-bearing octreotate by CuAAC (Scheme 2). This last reaction step was optimized regarding the amount of alkyne-bearing peptide (Figure 1). Using 10−40 nmol of hexin-TATE the radiochemical yield (RCY) reached 70% after a reaction time of 20 min. Increasing the amount to 60−80 nmol of hexin-TATE led to the maximum RCY of >90%

RESULTS AND DISCUSSION

Chemistry and Radiosyntheses. The fluoroglycosylation of TATE peptide was achieved by CuAAC, starting from hexinTATE21 and 6-deoxy-6-fluoroglucosyl azide22 applying standard conditions using CuSO4 and sodium ascorbate (Scheme 1). An excess of the glucosyl azide was used in the reaction, such that there was no remaining hexin-TATE at the end of the reaction time. This allowed a simple workup procedure by solid-phase extraction without the need for HPLC separation and yielded the product FGlc-TATE in high purity of 98%.

Figure 1. Optimization of the click chemistry reaction step. Reaction conditions: 6-deoxy-6-[18F]fluoroglucosyl azide in 270 μL NaOH (60 mM), hexin-TATE, 40 nmol Cu(OAc)2, 200 nmol THPTA, 1 μmol sodium ascorbate, sodium phosphate buffer (270 μL, pH 8.0), 60 °C, 10 min. The amount of hexin-TATE was varied from 10 to 80 nmol. B


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Bioconjugate Chemistry

finding as it was shown in a study comparing five [99mTc/ EDDA/HYNIC0]TOC derivatives that the internalization rate significantly correlated with the tumor uptake of the tracer in vivo.26 The lipophilicity of [18F]FGlc-TATE was determined by the n-octanol/PBS distribution method, resulting in a logD7.4 of −1.80 ± 0.07 (n = 3). Since this value was in the same range as reported for Glc-Lys([18F]FP)-TOCA (logD7.4 = −1.70),7 an 18 F-labeled somatostatin analog which was successfully used in patients with sstr-positive tumors,27 we assume that [18F]FGlcTATE could be a true alternative tracer with similar properties regarding tumor uptake, biodistribution, and clearance behavior in vivo. In order to complete the determination of the in vitro characteristics of [18F]FGlc-TATE, the metabolic stability was determined by incubation of the radiotracer in human serum at 37 °C. [18F]FGlc-TATE showed high stability for up to 120 min, as determined by radio-HPLC analysis, which did not show any degradation products, but only the intact tracer (Figure 4). To sum up, the in vitro results on [18F]FGlc-TATE were highly promising and suggested excellent in vivo properties of the tracer, pointing us toward the in vivo evaluation.

achieved after the short reaction time of 10 min. The product [18F]FGlc-TATE was isolated by semipreparative HPLC in an overall yield of 19−22% (non-decay-corrected, referred to [18F]fluoride) and in high specific activities of 32−106 GBq/ μmol (n = 6). In Vitro Assays. It is well-known that [Tyr3]octreotate analogs bind preferably to somatostatin receptor subtype 2;23 therefore, the affinity of FGlc-TATE to somatostatin receptors was determined by using the pancreatic cell line AR42J, which is known to express high levels of the four sstr subtypes 1, 2, 3, and 5,24 in a competition binding experiment applying 177LuDOTATOC as radioligand. FGlc-TATE showed high affinity to SST receptors with an IC50 value of 4.2 nM, whereas IC50 values for nonglycosylated hexin-TATE and Lu-DOTATOC, which were used for comparison, were significantly higher by a factor of 5 (Figure 2). To verify the receptor expression of the

Figure 2. Determination of IC50 values for different octreotide derivatives by displacement of 177Lu-DOTATOC in AR42J cells. Each data point represents the mean value ± standard deviation (SD) from 2 to 3 experiments each performed in triplicate.

AR42J cells used in the present study, we also performed a saturation binding assay by the use of carrier-added [18F]FGlcTATE in a concentration range of 0.1−10 nM (Figure 3A). The Bmax was determined to be 460 fmol/mg at 37 °C, which equals about 56 000 binding sites per cell. In a study by Viguerie et al. a value of 150 000 sites per cell was measured in a binding assay at 25 °C. Considering the fact that at 37 °C only about 40% of the maximal number of binding sites are occupied,25 the determined Bmax (56 000 sites/cell, 37 °C) is in good accordance with the reported value (150 000 sites/cell, 25 °C). In addition, high internalization rates were observed for [18F]FGlc-TATE in AR42J cells with values of about 80% internalized radiotracer after 5 min of incubation, which was constant for at least 2 h (Figure 3B). This is an important

Figure 4. Radio-HPLC analyses of blood samples from mice after i.v. injection of [18F]FGlc-TATE at 2 and 10 min p.i., in comparison to [18F]FGlc-TATE from the quality control (QC) and to a sample taken after a 2 h incubation in human serum in vitro. HPLC method: Chromolith RP-18e, 100 × 4.6 mm, 10−50% acetonitrile in water (0.1% TFA) in 5 min, 4 mL/min, tR ([18F]FGlc-TATE) = 2.9 min.

In Vivo Evaluation of [18F]FGlc-TATE. The biodistribution of [18F]FGlc-TATE was evaluated in nude mice bearing AR42J tumors on the left shoulder. The radiotracer was intravenously (i.v.) injected in the tail vein of mice and selected organs were removed, counted, and weighted after 30, 60, and 120 min post-injection (p.i.). The results are given in Table 1

Figure 3. (A) Saturation binding curve of [18F]FGlc-TATE determined after an incubation time of 60 min at 37 °C and (B) internalization of [18F]FGlc-TATE in AR42J cells in vitro. Nonspecific binding and uptake was measured in the presence of octreotide (1 μM). Each data point represents the mean value ± SD (n = 4). C


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Bioconjugate Chemistry Table 1. Biodistribution Data of [18F]FGlc-TATE Using AR42J Tumor Bearing Nude at 30, 60, and 120 min p.i.a 30 min blood lung liver kidney heart spleen brain muscle femur AR42J intestine tumor/blood tumor/kidney tumor/liver

0.92 1.09 1.45 8.96 0.52 0.46 0.06 0.58 1.02 7.87 0.69 9 1 6

± ± ± ± ± ± ± ± ± ± ±

0.15 0.23 0.34 1.72 0.19 0.49 0.02 0.34 0.39 1.11 0.23

30 min blockingb n.d. 5.85 3.56 31.71 2.86 1.44 0.13 1.14 1.42 0.63 0.70

± ± ± ± ± ± ± ± ± ±

60 min 0.34 0.47 0.91 3.43 0.15 0.15 0.04 0.46 0.58 5.62 0.44 17 2 6

0.55 1.02 7.65 0.68 0.31 0.03 0.19 0.05 0.26 0.89

± ± ± ± ± ± ± ± ± ± ±

0.11 0.11 0.25 1.79 0.06 0.14 0.03 0.78 0.37 1.53 0.15

60 min blockingb 2.38 4.38 4.36 48.87 1.53 1.34 0.14 1.03 1.25 0.95 1.33

± ± ± ± ± ± ± ± ± ± ±

0.14 1.78 0.97 15.38 0.80 0.84 0.06 0.44 0.45 0.60 0.37

120 min 0.16 0.25 0.33 0.80 0.10 0.17 0.01 0.07 0.21 2.13 1.13 15 3 6

± ± ± ± ± ± ± ± ± ± ±

0.05 0.09 0.07 0.11 0.02 0.13 0.01 0.05 0.03 0.58 0.32

a Values are expressed as percent injected dose per gram tissue (%ID/g, mean ± SD, n = 3). bCoinjection of [18F]FGlc-TATE and octreotide acetate (30 μg/animal).

Figure 5. Biodistribution data of [18F]FGlc-TATE using AR42J tumor bearing nude mice. (A) Uptake of [18F]FGlc-TATE at 30, 60, and 120 min p.i. (B) Tumor-to-organ ratios calculated from biodistribution data. (C) Uptake of [18F]FGlc-TATE in the AR42J tumor under control and under blocking conditions (30 μg octreotide acetate/animal) determined 30 and 60 min after injection. All data are expressed as mean values ± SD (n = 3).

and Figure 5, showing highest uptake values of 5.6% and 2.1% ID/g in the AR42J tumors after 60 and 120 min post-injection, respectively, and, apart from kidneys, negligible uptake in all other organs. [18F]FGlc-TATE was rapidly cleared from blood and from kidneys, indicating the expected renal clearance pathway. Due to the fast clearance, the tumor-to-blood-ratio increased from 9 to 17 from 30 to 60 min p.i., and the tumorto-kidney-ratio increased from 0.9 after 30 min to 2.7 at 120 min p.i. (Table 1, Figure 5B). In comparison with previously reported [18F]fluoroethyl-azide TATE derivatives by Leyton et al. (constant kidney uptake value of 25−30%ID/mL between 30 and 60 min p.i.),9 SiFAlin-TATE derivatives by Wängler et al. (approximate SUV of 7.5 at 30 min p.i. and 6.5 at 60 min p.i.),12 and even clinically etablished 68Ga-DOTATATE (constant kidney uptake of approximately 3 (SUV) between 30 and 85 min p.i.)12 that all showed slow washout from kidneys, [18F]FGlc-TATE is characterized by a significantly faster kidney clearance of 62% (9.0%ID/g at 30 min p.i. decreasing to 3.4%ID/g at 60 min p.i. and 0.8 at 120 min p.i.; Table 1), which is probably due to the 6-deoxy-6-[18F]fluoroglucosyl moiety. The specific uptake of [18F]FGlc-TATE in the tumor in vivo was proven by coinjection of the radiotracer with an excess amount of octreotide acetate (30 μg/animal). This blocking experiment showed significantly reduced tumor uptake with only 1%ID/g at 60 min p.i., which equals 83% decreased tumor uptake in comparison with control animals (Table 1, Figure

5C). Interestingly, uptake in most other organs (e.g., lung, liver, kidneys) was dramatically increased under blocking conditions (Table 1), a phenomenon which was also observed by Schottelius et al. for a series of 18F-labeled carbohydrated TATE derivatives.13 To further evaluate [18F]FGlc-TATE in vivo, we proceeded with small animal PET scans using the same animal model as for the biodistribution study. The PET imaging analysis showed that AR42J tumors were clearly visualized at 40−60 min p.i. and reached high uptake values of 13 ± 4%ID/g (n = 3, Figure 6, left side). This uptake was highly specific as indicated by blocking experiments in which mice were coinjected with [18F]FGlc-TATE and octreotide acetate (30 μg/animal). The percentage of blocking was >99%, consequently only negligible tumor uptake could be seen in those animals (0.1 ± 0.1%ID/g, n = 3; Figure 6, right side). The animal experiments were complemented with analyses of blood taken from mice at 2 and 10 min p.i. Due to the fast blood clearance of [18F]FGlc-TATE, the analysis of blood from later time points after injection was not possible. Radio-HPLC confirmed high stability of [18F]FGlc-TATE in the blood: at 2 min p.i. the intact radiotracer was recovered to >99%, and at 10 min p.i. the amount of intact tracer was still 77% when an unknown more lipophilic metabolite occurred (Figure 4). Until now, only a few 18F-labeled octreotide/octreotate derivatives have been reported. Some of them (e.g., [18F]FPGluc-TOCA,7 Gluc-S-Dpr([18F]FP)TOCA13) were synthesized D


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affinity to sst receptors, high metabolic stability, and favorable biodistribution with excellent clearance properties. Small animal PET scans indicated that [18F]FGlc-TATE has high potential as an alternative 18F-labeled radioligand for PET imaging of sstr positive tumors, predominantly due to its excellent tumor-tobackground-ratio. [18F]FGlc-TATE was obtained by a reliable and easy click chemistry-based procedure and showed excellent in vitro and in vivo properties. Therefore, [18F]FGlc-TATE represents a viable alternative 18F-labeled radiopeptide for imaging somatostatin receptor-positive tumors by PET.

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EXPERIMENTAL PROCEDURES General. The labeling precursor 2,3,4-tri-O-acetyl-6-Otoluenesulfonyl-β-D-glucopyranosyl azide and the glycosyl azide 6-deoxy-6-fluoro-β-D-glucopyranosyl azide were prepared as published previously.16,22 Radio-HPLC was performed on an Agilent 1100 system with a quarternary pump and variable wavelength detector and radio-HPLC detector D505TR (Canberra Packard). Computer analysis of the HPLC data was performed using FLO-One software (Canberra Packard). Electron-spray ionization (ESI) mass spectrometry analysis was performed using a Bruker Esquire 2000 instrument. Synthesis of hexin-TATE. The synthesis of hexin-TATE (TATE-5-hexynoic acid) was performed as described before.21 The purity was determined by RP-HPLC and was >99%. ESIMS: m/z calcd: 1142.5, found: 1143.5 [M + H]+, 1141.5 [M − H]−. Synthesis of FGlc-TATE. To a solution of 6-deoxy-6fluoro-β-D-glucopyranosyl azide (1.8 mg, 8.7 μmol) in water (450 μL) and hexin-TATE (2.74 mg, 2.4 μmol) in DMSO (50 μL) was added copper(II)sulfate pentahydrate (0.2 M, 10 μL) and sodium ascorbate (0.6 M, 10 μL). The mixture was incubated at room temperature for 90 min, and then diluted with water (20 mL). The product was purified using a SPE cartridge (Sep-Pak C18 Environmental Cartridge, Waters), preconditioned with CH3CN and water. FGlc-TATE was eluted from the cartridge with EtOH (2 mL), dried under reduced pressure, diluted with water (500 μL), and lyophilized to yield FGlc-TATE as a white solid: 1.2 mg, 0.89 μmol, 37%. The purity was determined by RP-HPLC and was 98% (Method A: Kromasil 100 C8, 5 μm, 4.6 × 250 mm, 1.5 mL/ min; 10−50% acetonitrile in water (0.1% TFA) in 30 min: tR = 18.3 min; Method B: Chromolith RP 18e, 4.6 × 100 mm, 4.0 mL/min; 10−50% acetonitrile in water (0.1% TFA) in 5 min: tR = 2.9 min). ESI-MS: m/z calcd: 1349.52, found: 1350.80 [M + H]+, 676.24 [M+2H]2+. Production of [18F]fluoride. No-carrier-added (n.c.a.) 18 [ F]fluoride was produced by the 18O(p,n)18F reaction on 18 O-enriched water (>98%, Rotem Ind. LTD) using a proton beam of 11 MeV generated by a RDS 111 cyclotron (Siemens/ CTI) and fixed on an anion exchanger cartridge (Sep-Pak Accell Plus QMA, Waters). Radiosynthesis of [18F]FGlc-TATE. 2,3,4-Tri-O-acetyl-6deoxy-6-[18F]fluoroglucosyl azide was prepared, isolated by semipreparative HPLC and deacetylated with NaOH (250 μL, 60 mM, 10% ethanol) at 60 °C for 5 min as described before.22 The crude product 6-deoxy-6-[18F]fluoroglucosyl azide was used for the click chemistry reaction with hexin-TATE in a onepot-procedure as described before.29 In brief, to the solution containing 6-deoxy-6-[18F]fluoroglucosyl azide (in 270 μL of 60 mM NaOH) was given a mixture containing hexin-TATE (60 nmol in 60 μL water), THPTA (10 μL, 20 mM), Cu(OAc)2 (4 mM, 10 μL) and sodium ascorbate (0.1 M, 10

Figure 6. Representative coronal (top) and transaxial (bottom) small animal PET images at 40−60 min p.i. of a AR42J tumor bearing nude mouse injected with [18F]FGlc-TATE (left) and coinjected at the following day with radiotracer and octreotide acetate (30 μg/animal; right). Red arrows indicate the tumors.

by a multistep and highly laborious radiosynthesis with low overall yields, while others, such as 18F-SiFA-Glc-PEG1-TATE,8 showed limited hydrophilicity, resulting in unfavorable clearance properties. One of the first published 18F-labeled TATE-derivatives was Cel-S-Dpr([18F]FBOA)TOCA which displayed high tumor uptake (24%ID/g at 60 min p.i.) as well as high tumor-to-background ratios and a fast kidney clearance.13 Compared to [18F]FGlc-TATE, the tumor uptake of Cel-S-Dpr([18F]FBOA)TOCA is superior; however, the kidney uptake of Cel-S-Dpr([18F]FBOA)TOCA is higher for all time points post-injection. This radiotracer could be synthesized by a chemoselective high-yield two-step methodology using 4-[18F]fluorobenzaldehyde ([18F]FBA) as prosthetic group in isolated yields of 40−60% after 50 min for the second reaction step.13 Considering the radiosynthesis of [18F]FBA (50−55% isolated yield after a synthesis time of 55 min),28 Cel-S-Dpr([18F]FBOA)TOCA was obtained in overall yields of about 20−30% in a total synthesis time of 105 min, which is slightly higher regarding isolated yield, but the radiosynthesis time is 35 min longer than that for [18F]FGlcTATE. In another series of 18F-labeled octreotate analogs, which were synthesized by CuAAC of [18F]fluoroethyl azide with alkyne-bearing TATE-derivatives in a two-step reaction in a synthesis time of 90 min, the most promising two derivatives (18F-FET-G-TOCA and 18F-FET-βAG-TOCA) showed tumor uptake values of 8−17%ID/g for AR42J tumor bearing mice.9 This result was within the same range as the tumor uptake values for [18F]FGlc-TATE in the present study. Compared to 68 Ga-DOTATOC which showed standard uptake values in the AR42J tumor model of about 4 at 65 min p.i.,12 the tumor uptake of [18F]FGlc-TATE was similarly high (13 ± 4%ID/g that equals an SUV of 4.6 ± 1.4).

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CONCLUSIONS In conclusion, we successfully adapted the 18F-fluorglycosylation methodology to the synthesis of the 18F-fluoroglycosylated octreotate analog [18F]FGlc-TATE, which confirmed high E


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Bioconjugate Chemistry μL) in sodium phosphate buffer (NaH2PO4/Na2HPO4 1:6.31, 0.5 M, pH 8, 270 μL) and the reaction mixture was left for 10 min at 60 °C without stirring. The RCY was >90% for [18F]FGlc-TATE as determined by analytical HPLC from a sample withdrawn from the reaction mixture. Isolation of [18F]FGlc-TATE was realized by semipreparative HPLC (Kromasil C8, 125 × 8 mm, 20−50% acetonitrile in water (0.1% TFA) in a linear gradient over 30 min, 4 mL/min, tR = 8.9 min). The product fraction was diluted with water, passed through a RP-18 cartridge (LiChrolut, Merck, 100 mg), and the product eluted with ethanol (2 mL). For in vitro and in vivo experiments the solvent was evaporated in vacuo and [18F]FGlc-TATE was formulated with 0.9% saline. Starting from [18F]fluoride (500 MBq), [18F]FGlc-TATE could be obtained in an overall yield of 95−110 MBq (19−22%, nondecay-corrected yield, referred to [18F]fluoride) in a total synthesis time of 70 min in specific activities of 32−106 GBq/ μmol (n = 6). Stability in Serum In Vitro. An aliquot of [18F]FGlcTATE in saline (0.9%, 40 μL, 5 MBq) was added to human serum (400 μL) and incubated at 37 °C. Aliquots (20 μL) were taken at various time intervals (5−120 min) and quenched in acetonitrile/10% TFA (1:1, 100 μL). The samples were centrifuged, and the supernatants were analyzed by radioHPLC (Chromolith RP-18e, 100 × 4.6 mm, 10−50% acetonitrile in water (0.1% TFA) in a linear gradient over 5 min, 4 mL/min, tR = 2.9 min). Determination of the Partition Coefficient (logD7.4). The lipophilicity of [18F]FGlc-TATE was assessed by determination of the water−octanol partition coefficient as described before.17 Cell Culture. The rat pancreas cancer cell line AR42J is known to express sst2 receptors abundantly.30 The cells were obtained from CLS (Cell Line Service GmbH, Eppelheim, Germany) and grown in DMEM (Dulbecco’s Modified Eagle’s Medium) containing glutamine (2 mM) supplemented with fetal bovine serum (FBS, 10%) at 37 °C in a humidified atmosphere containing of 5% CO2. Cells were routinely subcultured every 3−4 days. Routine tests of the cells for contamination with mycoplasma were always negative. Receptor Binding Assay. For receptor binding assays, AR42J cells were seeded in 96-well plates at a concentration of 300 000 cells per well in binding buffer (DMEM, 0.5% bovine serum albumin (BSA), 10 mM HEPES). Cells were centrifuged (150 g, 2 min), the supernatant was removed, and FGlc-TATE, hexin-TATE, Lu-DOTATOC (ABX) or octreotide acetate (Sigma-Aldrich) was added in increasing concentrations (0.1− 100 nM) in binding buffer (90 μL). To each well a solution of 177 Lu-DOTATOC (7 kBq in 20 μL, diluted with binding buffer to a concentration of 3.5 nM of DOTATOC, 177LuDOTATOC was obtained from the GMP radiopharmacy of the Department of Nuclear Medicine, FAU) was added and plates were incubated for 1 h at room temperature on a shaker. Afterward, the plates were centrifuged, the supernatants were sucked off, and the wells were washed twice with binding buffer. Subsequently, cells were lysed using warm NaOH (2 N), transferred to counting tubes and the radioactivity was measured in a γ-counter (Wallac Wizard, 1470, PerkinElmer). IC50 values were calculated using GraphPad Prism. Each experiment was performed twice in triplicate. Internalization Assay. AR42J cells were cultured in 24 well plates at a confluence of 70−80% for 48−72 h before the assay was performed. Initially, the medium was removed, cells were

washed twice with phosphate buffered saline (PBS), and incubated at 37 °C in binding buffer (DMEM, 1% BSA, 10 mM HEPES) containing [18F]FGlc-TATE (0.5 MBq/mL). After different time intervals (1, 2, 5, 15, 30, 45, 60, 90, and 120 min) the supernatant was removed and the wells were washed on ice twice with ice cold PBS. Afterward, the cells were rinsed with the acid wash solution (20 mM NaOAc, pH 5) twice for 1 min and the washing solutions were collected in a tube. Cells were lysed with NaOH (0.1 M, 1 mL) and collected in tubes. Cells and liquids were counted in a γ-counter (Wallac Wizard, 1470, PerkinElmer) and internalization rate was calculated as the percentage of the counts per minute (cpm) of the cells from the sum of cells (cpm) plus respective acid wash solutions (cpm). Nonspecific binding was determined by performing the assay in the presence of nonradioactive octreotide acetate (1 μM). The experiment was performed twice each in quadruplicates. Saturation Binding Assay. AR42J cells were cultured in 24-well plates at a confluence of 70−80% for 48−72 h before the assay was performed. Cells were washed once with binding buffer (1 mL) and then incubated with binding buffer (500 μL) containing various amounts of [18F]FGlc-TATE (0.1−10 nM) for 60 min at 37 °C. Subsequently, 50 μL of each well was collected for determination of the total radioactivity, the residue of the medium was removed, and cells were washed twice with ice-cold PBS (1 mL). The cells were lysed with NaOH (0.1 M, 1 mL) and collected. All samples were counted in a γ-counter (Wallac Wizard, 1470, PerkinElmer). Afterward, the protein mass in each well was analyzed by the Bradford assay. Nonspecific binding was determined by performing the assay in the presence of nonradioactive octreotide acetate (1 μM). The Bmax value was calculated using GraphPad Prism from two independent experiments each performed in quadruplicate. Animal Model. All animal experiments were performed in compliance with the protocols approved by the local Animal Protection Authorities (Regierung Mittelfranken, Germany, No. 54−2532.1−22/10). Male NMRI nude mice (nu/nu) were obtained from Harlan Winkelmann GmbH (Borchen, Germany) at 4 weeks of age and were kept under standard conditions (12 h light/dark) with food and water available ad libitum for at least 4 weeks. AR42J cells were harvested and suspended in sterile PBS at a concentration of 5 × 107 cells/ mL, respectively. Viable cells (5 × 106) in PBS (100 μL) were injected subcutaneously in the back on the left side. Two weeks after inoculation the mice, now weighing about 35−40 g and bearing tumors of 1−2 g, were used for biodistribution and small animal PET studies. Biodistribution Studies. AR42J xenografted nude mice were anesthetized with isoflurane (4%) and injected with [18F]FGlc-TATE into a tail vein (4−8 MBq/mouse). The animals were killed by cervical dislocation 30, 60, and 120 min p.i. The tumors, other tissues (lung, liver, kidneys, heart, spleen, brain, muscle, femur and intestine), and blood were removed and weighed. Radioactivity of the samples was measured using a γ-counter (Wizard Wallac, PerkinElmer), and expressed as percentage of injected dose per gram of tissue (%ID/g), from which tumor-to-organ levels were calculated. For determination of nonspecific radiotracer uptake (blocking experiment), nude mice were injected with a mixture of [18F]FGlc-TATE and an excess of octreotide acetate (30 μg/animal), and killed at 30 and 60 min p.i., using three animals in each group. Tumors were removed and the uptake (%ID/g) was determined as described above. F


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(4) Poeppel, T. D., Binse, I., Petersenn, S., Lahner, H., Schott, M., Antoch, G., Brandau, W., Bockisch, A., and Boy, C. (2011) 68GaDOTATOC versus 68Ga-DOTATATE PET/CT in functional imaging of neuroendocrine tumors. J. Nucl. Med. 52, 1864−1870. (5) Le Bars, D. (2006) Fluorine-18 and medical imaging: Radiopharmaceuticals for positron emission tomography. J. Fluorine Chem. 127, 1488−1493. (6) Coenen, H. H., Elsinga, P. H., Iwata, R., Kilbourn, M. R., Pillai, M. R. A., Rajan, M. G. R., Wagner, H. N., Jr, and Zaknun, J. J. (2010) Fluorine-18 radiopharmaceuticals beyond [18F]FDG for use in oncology and neurosciences. Nucl. Med. Biol. 37, 727−740. (7) Wester, H., Schottelius, M., Scheidhauer, K., Meisetschläger, G., Herz, M., Rau, F., Reubi, J., and Schwaiger, M. (2003) PET imaging of somatostatin receptors: design, synthesis and preclinical evaluation of a novel 18F-labelled, carbohydrated analogue of octreotide. Eur. J. Nucl. Med. Mol. Imaging 30, 117−122. (8) Wängler, C., Waser, B., Alke, A., Iovkova, L., Buchholz, H. G., Niedermoser, S., Jurkschat, K., Fottner, C., Bartenstein, P., Schirrmacher, R., et al. (2010) One-Step 18F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET). Bioconjugate Chem. 21, 2289−2296. (9) Leyton, J., Iddon, L., Perumal, M., Indrevoll, B., Glaser, M., Robins, E., George, A. J., Cuthbertson, A., Luthra, S. K., and Aboagye, E. O. (2011) Targeting somatostatin receptors: preclinical evaluation of novel 18F-fluoroethyltriazole-Tyr3-octreotate analogs for PET. J. Nucl. Med. 52, 1441−1448. (10) Liu, Z., Pourghiasian, M., Bénard, F., Pan, J., Lin, K.-S., and Perrin, D. M. (2014) Preclinical evaluation of a high-affinity 18Ftrifluoroborate octreotate derivative for somatostatin receptor imaging. J. Nucl. Med. 55, 1499−1505. (11) Niedermoser, S., Chin, J., Wängler, C., Kostikov, A., BernardGauthier, V., Vogler, N., Soucy, J. P., McEwan, A. J., Schirrmacher, R., and Wängler, B. (2015) In vivo evaluation of 18F-SiFAlin-modified TATE: a potential challenge for 68Ga-DOTATATE, the clinical gold standard for somatostatin receptor imaging with PET. J. Nucl. Med. 56, 1100−1105. (12) Litau, S., Niedermoser, S., Vogler, N., Roscher, M., Schirrmacher, R., Fricker, G., Wängler, B., and Wängler, C. (2015) Next generation of SiFAlin-based TATE derivatives for PET imaging of SSTR-positive tumors: influence of molecular design on in vitro SSTR binding and in vivo pharmacokinetics. Bioconjugate Chem. 26, 2350−2359. (13) Schottelius, M., Poethko, T., Herz, M., Reubi, J. C., Kessler, H., Schwaiger, M., and Wester, H. J. (2004) First 18F-labeled tracer suitable for routine clinical imaging of sst receptor-expressing tumors using positron emission tomography. Clin. Cancer Res. 10, 3593−3606. (14) Hultsch, C., Schottelius, M., Auernheimer, J., Alke, A., and Wester, H. J. (2009) 18F-Fluoroglucosylation of peptides, exemplified on cyclo(RGDfK). Eur. J. Nucl. Med. Mol. Imaging 36, 1469−1474. (15) Namavari, M., Cheng, Z., Zhang, R., De, A., Levi, J., Hoerner, J. K., Yaghoubi, S. S., Syud, F. A., and Gambhir, S. S. (2009) A novel method for direct site-specific radiolabeling of peptides using [18F]FDG. Bioconjugate Chem. 20, 432−436. (16) Maschauer, S., and Prante, O. (2009) A series of 2-Otrifluoromethylsulfonyl-D-mannopyranosides as precursors for concomitant 18F-labeling and glycosylation by click chemistry. Carbohydr. Res. 344, 753−761. (17) Maschauer, S., Einsiedel, J., Haubner, R., Hocke, C., Ocker, M., Hübner, H., Kuwert, T., Gmeiner, P., and Prante, O. (2010) Labeling and glycosylation of peptides using click chemistry: a general approach to 18F-glycopeptides as effective imaging probes for positron emission tomography. Angew. Chem., Int. Ed. 49, 976−979. (18) Boss, S. D., Betzel, T., Müller, C., Fischer, C. R., Haller, S., Reber, J., Groehn, V., Schibli, R., and Ametamey, S. M. (2016) Comparative Studies of Three Pairs of α- and γ-Conjugated Folic Acid Derivatives Labeled with Fluorine-18. Bioconjugate Chem. 27, 74−86.

Small-Animal PET Imaging. PET scans and image analysis were performed using a small-animal PET scanner (Inveon, Siemens Medical Solutions). About 1−3 MBq of [18F]FGlcTATE was injected into a tail vein of each nude mouse (n = 3) under isoflurane anesthesia (4%). Animals were subjected to a 20 min scan starting at 40 min p.i. After iterative maximum a posteriori (MAP) image reconstruction of the decay, regions of interest (ROIs) were drawn over the tumor using the software AMIDE. The radioactivity concentration within the tumor region was obtained from the mean value within the multiple ROIs and then converted to uptake values (%ID/g) relative to the injected dose. For receptor-blocking experiments tumorbearing mice were scanned for the period 40−60 min p.i. after coinjection of radiotracer containing octrotide acetate (30 μg/ animal, n = 3). Stability in Mouse Blood Ex Vivo. Two mice (CD-1, Charles River) were injected under isoflurane anesthesia (4%) with 3−5 MBq of [18F]FGlc-TATE in the tail vein. At 2 or 10 min post-injection, the mice were killed by cervical dislocation and blood was collected in Li-heparine coated vials. The vials were centrifuged immediately, the supernatant (50 μL) was added to TFA (10%, 50 μL) to precipitate plasma proteins, and this mixture was centrifuged again. The supernatant was analyzed by radio-HPLC using two different methods: Method A: Chromolith RP-18e, 100 × 4.6 mm, 10−50% acetonitrile in water (0.1% TFA) in a linear gradient over 5 min, 4 mL/min, tR = 2.9 min; Method B: Kromasil 100 C8−5 μm, 250 mm × 4.6 mm, 10−50% acetonitrile in water (0.1% TFA) in a linear gradient over 30 min, 1.5 mL/min, tR = 18.3 min.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +49-9131-8544440. Fax: +499131-8539288. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Manuel Geisthoff for expert technical support, Peter Hennig for the radiosynthesis of 177LuDOTATOC and Dr. Jü rgen Einsiedel (Department of Chemistry and Pharmacy, FAU) for ESI-MS analysis. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, grant MA 4295/1-3).

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ABBREVIATIONS CuAAC, copper(I)-catalyzed azide−alkyne cycloaddition; DMSO, dimethyl sulfoxide; [18F]FDG, 2-deoxy-2-[18F]fluoroglucose; i.v., intravenously; NET, neuroendocrine tumor; PET, positron emission tomography; p.i., post-injection; RCY, radiochemical yield; SST, somatostatin; sstr, somatostatin receptor; THPTA, tris(3-hydroxypropyltriazolyl methyl)amine

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REFERENCES

(1) Johnbeck, C. B., Knigge, U., and Kjær, A. (2014) PET tracers for somatostatin receptor imaging of neuroendocrine tumors: current status and review of the literature. Future Oncol. 10, 2259−2277. (2) Reubi, J., Waser, B., Schaer, J.-C., and Laissue, J. A. (2001) Somatostatin receptor sst1−sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands. Eur. J. Nucl. Med. 28, 836−846. (3) Patel, Y. C. (1999) Somatostatin and its receptor family. Front. Neuroendocrinol. 20, 157−198. G


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Bioconjugate Chemistry (19) Maschauer, S., and Prante, O. (2014) Sweetening pharmaceutical radiochemistry by 18F-fluoroglycosylation: a short review. BioMed Res. Int. 2014, 214748. (20) Maschauer, S., Einsiedel, J., Hübner, H., Gmeiner, P., and Prante, O. (2016) 18F- and 68Ga-Labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1. J. Med. Chem. 59, 6480− 6492. (21) Wängler, C., Schäfer, M., Schirrmacher, R., Bartenstein, P., and Wängler, B. (2011) DOTA derivatives for site-specific biomoleculemodification via click chemistry: Synthesis and comparison of reaction characteristics. Bioorg. Med. Chem. 19, 3864−3874. (22) Maschauer, S., Haubner, R., Kuwert, T., and Prante, O. (2014) 18 F-Glyco-RGD peptides for PET imaging of integrin expression: efficient radiosynthesis by click chemistry and modulation of biodistribution by glycosylation. Mol. Pharmaceutics 11, 505−515. (23) Reubi, J. C., Schär, J.-C., Waser, B., Wenger, S., Heppeler, A., Schmitt, J. S., and Mäcke, H. R. (2000) Affinity profiles for human somatostatin receptor subtypes SST1−SST5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use. Eur. J. Nucl. Med. Mol. Imaging 27, 273−282. (24) Hofsli, E., Thommesen, L., Nørsett, K., Falkmer, S., Syversen, U., Sandvik, A. K., and Lægreid, A. (2002) Expression of chromogranin A and somatostatin receptors in pancreatic AR42J cells. Mol. Cell. Endocrinol. 194, 165−173. (25) Viguerie, N., Tahiri-Jouti, N., Esteve, J.-P., Clerc, P., Logsdon, C., Svoboda, M., Susini, C., Vaysse, N., and Ribet, A. (1988) Functional somatostatin receptors on a rat pancreatic acinar cell line. Am. J. Physiol. Gastrointest. Liver Physiol. 255, G113−G120. (26) Storch, D., Behe, M., Walter, M. A., Chen, J. H., Powell, P., Mikolajczak, R., and Macke, H. R. (2005) Evaluation of [99mTc/ EDDA/HYNIC0]octreotide derivatives compared with [111In-DOTA0,Tyr3, Thr8]octreotide and [111In-DTPA0]octreotide: Does tumor or pancreas uptake correlate with the rate of internalization? J. Nucl. Med. 46, 1561−1569. (27) Meisetschläger, G., Poethko, T., Stahl, A., Wolf, I., Scheidhauer, K., Schottelius, M., Herz, M., Wester, H. J., and Schwaiger, M. (2006) Gluc-Lys ([18F]FP)-TOCA PET in patients with SSTR-positive tumors: biodistribution and diagnostic evaluation compared with [111In] DTPA-octreotide. J. Nucl. Med. 47, 566−573. (28) Poethko, T., Schottelius, M., Thumshirn, G., Hersel, U., Herz, M., Henriksen, G., Kessler, H., Schwaiger, M., and Wester, H. J. (2004) Two-step methodology for high-yield routine radiohalogenation of peptides: 18F-labeled RGD and octreotide analogs. J. Nucl. Med. 45, 892−902. (29) Maschauer, S., Greff, C., Einsiedel, J., Ott, J., Tripal, P., Hubner, H., Gmeiner, P., and Prante, O. (2015) Improved radiosynthesis and preliminary in vivo evaluation of a 18F-labeled glycopeptide-peptoid hybrid for PET imaging of neurotensin receptor 2. Bioorg. Med. Chem. 23, 4026−4033. (30) Elberg, G., Hipkin, R. W., and Schonbrunn, A. (2002) Homologous and heterologous regulation of somatostatin receptor 2. Mol. Endocrinol. 16, 2502−2514.

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Radiosynthesis and Preclinical Evaluation of 18F-Fluoroglycosylated

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