Design, preparation and biological evaluation of a 177Lu-labeled somatostatin receptor antagonist for targeted therapy of neuroendocrine tumors

Bioorganic Chemistry xxx (xxxx) xxxx

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Design, preparation and biological evaluation of a 177Lu-labeled somatostatin receptor antagonist for targeted therapy of neuroendocrine tumors Hossein Behnammanesha, Safura Jokara, Mostafa Erfanib, , Parham Geramifarc, Omid Sabzevarid,e, Mohsen Aminif,g, Seyed Mohammad Mazidib, Maliheh Hajiramezanalia, ⁎ Davood Beikic, ⁎⁎

a

Department of Nuclear Pharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Radiation Application Research School, Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran Research Center for Nuclear Medicine, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran d Department of Toxicology and Pharmacology, Faculty of Pharmacy, Toxicology and Poisoning Research Centre, Tehran University of Medical Sciences, Tehran, Iran e Toxicology and Poisoning Research Centre, Tehran University of Medical Sciences, Tehran, Iran f Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran g Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran b c

ARTICLE INFO

ABSTRACT

Keywords: Somatostatin receptors Antagonistic peptide Radionuclide therapy Lutetium-177

Somatostatin receptor-targeted radionuclide therapy has become an effective treatment in patients with neuroendocrine tumors. Recently, investigations on the development of antagonistic peptides are increasing with possible superior biological properties as opposed to the agonists. Herein, we have reported the development of a new somatostatin receptor peptide ligand labeled with 177Lu to achieve a therapeutic ligand for tumor treatment. The interactions of selected and drown ligands using Avogadro software were docked on somatostatin receptor by Dink algorithm. The best docked peptide-chelator conjugate (DOTA-p-Cl-Phe-Cyclo(D-Cys-L-BzThi-D-Aph-LysThr-Cys)-D-Tyr-NH2) (DOTA-Peptide 2) was synthesized using the Fmoc solid-phase method. DOTA-Peptide 2 was radiolabeled with the 177Lu Trichloride (177LuCl3) solution at 95 °C for 30 min and radiochemical purity (RCP) of 177Lu-DOTA-Peptide 2 solution was monitored by radio-HPLC and radio-TLC procedures. The new radiolabeled peptide was evaluated for stability, receptor binding, internalization, biodistribution and singlephoton emission computed tomography (SPECT) imaging using C6 glioma cells and C6 tumor-bearing rats. DOTA-Peptide 2 was obtained with 98% purity and efficiently labeled with 177Lu (RCP > 99%). 177Lu-DOTAPeptide 2 showed a high value of stability in acetate buffer (91.4% at 312 h) and human plasma (> 97% at 24 h). Radioconjugate exhibited low internalization (< 5%) and high affinity for somatostatin receptors (Kd = 12.06 nM, Bmax = 0.20 pmol/106 cells) using saturation binding assay. Effective tumor uptake of 7.3% ID/ g (percentage of injected dose per gram of tumor) at 4 h post-injection and fast clearance of radiopeptide from blood and other organs led to a high tumor-to-normal organ ratios. SPECT/CT imaging clearly showed the activity localization in tumor. The favorable antagonistic properties of 177Lu-DOTA-Peptide 2 on the somatostatin receptors can make it a suitable candidate for peptide receptor radionuclide therapy (PRRT). In the future study, the therapeutic application of this radiopeptide will be evaluated.

1. Introduction Somatostatin (SST) is an inhibitory peptide which induce reduction of intracellular cAMP and calcium via five SST subtypes (SST1–SST5)

[1,2]. It has been demonstrated in numerous studies that desensitization and internalization of SST receptor subtypes will happen in response to agonist stimulation [3,4]. Neuroendocrine tumors (NETs) are a relatively rare family of heterogeneous tumors that are mostly found

Corresponding author at: Research Center for Nuclear Medicine, Tehran University of Medical Sciences, Shariati Hospital, North Kargar Ave., 1411713135 Tehran, Iran. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (M. Erfani), [email protected] (D. Beiki). ⁎

https://doi.org/10.1016/j.bioorg.2019.103381 Received 30 July 2019; Received in revised form 26 September 2019; Accepted 21 October 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Hossein Behnammanesh, et al., Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2019.103381

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in the gastrointestinal (GI) tract, pancreas, and lungs [5–7]. The bestknown clinical property of NETs is overexpression of somatostatin receptors (SSTRs) especially SSTR2 subtype, applied for imaging and treatment using high-affinity analogs targeting SSTRs [8–10]. Somatostatin receptor targeted radionuclide therapy is one of the treatment methods of NETs using a small peptide coupled with a therapeutic radionuclide as Lutetium-177 (177Lu) or Yttrium-90 (90Y) [7,11,12]. 177Lu as a desirable theranostic radioisotope (low-energy electrons emission (Eβ− avg = 134 keV, 100%) and low-energy gammas (Eγ = 113 and 208 keV)) has potential in simultaneous imaging and therapy of tumors [13]. 177Lu with lower β− energy has shorter tissue range leading to less toxicity and more patient tolerance during clinical treatment [14,15]. For decades, therapeutic somatostatin receptor agonists as 177LuDOTA-TOC and 177Lu-DOTA-TATE have been developed and are now used to treat patients with NETs. Clinical assessments of 177Lu-DOTATATE in Gastro-Entero-Pancreatic Neuroendocrine Tumors (GEP-NETs) showed complete and partial response in 2% and 28% of patients, respectively. Moreover, it can be selected as a safe and effective treatment for patients with large-sized tumors, fast proliferation, and receptor overexpression [16–20]. In recent years, the development of SST peptides with receptor antagonistic properties have been increasing. Because of the lower receptor desensitization and internalization of antagonists, they can bind to a larger number of binding sites that lead to a higher tumor uptake than the corresponding agonists [4,8,21,22]. Cescato et al. confirmed that 177Lu-DOTA-BASS (SST antagonist) had higher binding capacity than 177Lu-DOTA-TATE in all human ileal carcinoids samples by in vitro receptor autoradiography [4]. Although the BASS antagonist showed a lower receptor affinity than the TATE agonist, several new somatostatin antagonists have been developed by replacing amino acids in the peptide sequence and adding a chelator to improve the binding affinity to SST receptors [23]. A comparison between a novel SSTR2 antagonist, 177Lu-DOTA-JR11, and 177Lu-DOTATATE in the clinical pilot study showed desirable pharmacokinetic, prolonged intra-tumoral residence time and a higher effective dose for 177 Lu-DOTA-JR11 [24]. Therefore, based on the positive features of radiolabeled antagonists, development and assessment of radiolabeled SSTR antagonists as a new class of peptides for imaging and treatment of neuroendocrine tumors is of utmost importance. The aim of the present study was design and synthesis of a therapeutic radiolabeled antagonist for possible treatment of the SSTR-positive tumors. The selected peptide sequence using docking results was synthesized and conjugated to DOTA chelator for 177Lu labeling. The new radiolabeled peptide was evaluated for stability, receptor binding affinity, biodistribution and finally single-photon emission computed tomography (SPECT) imaging.

chromatography (HPLC, Sykam S7131, Eresing, Germany) was equipped with both ultraviolet (UV) absorption detector and Gabi radioactivity detector obtained from Raytest-Gabi, Straubenhardt, Germany. 2.2. Docking studies Homology modeling was performed using the Modeller 9.18 software [https://salilab.org/modeller/manual/node8.html]. SSTR2 amino acids sequence was taken from UniPort (http://www.uniprot.org/ uniprot/P30874). According to the blast of the sequence of the SSTR2 against the Protein Data Bank (PDB) proteins, “Active Mu-opioid Receptor” was the best template with 42% similarity to the SSTR2. Therefore, the PDB code 5c1m was selected to perform homology modelling. Then, the 3D structure for SSTR2 was equilibrated with the aqueous environment for 100 ns using the GROMOS 53a5 force field by Gromacs 5.1.3 software [25,26]. The selective ligands were drown and minimized using Avogadro software1.2.0 [27] by steepest descent method and UFF force field. The interactions of selected analogs to the SSTR2 were determined by the Dinc 2.0 algorithm [28] in blind and bound docking. Analysis of results was carried out using Ligplot software [29] to determine hydrogen and hydrophobic bonds. To validate the docking study, DOTA-LM3 peptide as a known SSTR2 antagonist and DOTA-TATE peptide as a known SSTR2 agonist were docked under the same condition by Dinc server. Fortunately, all of peptides were docked to the receptor with a reasonable score. 2.3. Synthesis of the selected peptide The chemical structure of the conjugate DOTA-LM3, DOTATATE, DOTA-Peptide 1 and DOTA-Peptide 2 ([(1,4,7,10Tricarboxymethyl-1,4,7,10-tetrazacyclododec-1-yl)acetyl]-(L)p(L-BzThi)Chlorophenylalanyl-(D)Cysteinyl-(L)-3-Benzothienylalanyl (D)-4-Aminocarbamoylphenylalanyl(D-Aph)-(L)-Lysyl-(L)-Threoninyl(L)-Cysteinyl-(D)-Tyrosine-NH2-cyclic disulfide) are shown in Fig. 1A–D. The peptide was synthesized according to the Fmoc solid-phase method on Rink Amide MBHA resin. All Fmoc-protected amino acids were used in a 3-equivalent excess based on the original substitution of the resin. Briefly, Fmoc-Tyr(tBu)-OH (3 equivalents) was attached to the deprotected Rink resin (200 mg) using DIPEA (9 equivalents), HOBT (3 equivalents) and DIC (3 equivalents) in anhydrous DMF (3 mL) for 4 h and slow rotation at room temperature. Then the Rink resin was washed with DMF (6 × 5 mL) and DCM (1 × 5 mL) and filtered off. The Fmoc group was removed using 20% piperidine in DMF in two 10-min treatments. The deprotection, coupling and washing cycles were repeated until the assembly of all amino acids of peptide sequence was completed. Cyclization was performed on the Rink resin using 1.5 equivalents of Tl(CF3COO)3 for 80 min and slow rotation at 0 °C. The coupling of DOTA moiety (3 equivalents) was performed using 3 equivalents HATU and 5 equivalents DIPEA in 3 mL NMP as a coupling agent. The peptide was cleaved and deprotected using TFA/TIS/H2O (95/2.5/2.5) and precipitated with cold petroleum ether/diisopropyl ether (40/60). The final product, DOTA-Peptide 2 was purified by semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC) equipped with C-18 reversed-phase column (NUCLEOSIL C18, 5 µm, 250/10 mm). The peptide was eluted using different gradients of TFA 0.1% in H2O (solvent A) and Acetonitrile (solvent B). The UV detection was done using a variable wavelength UV detector operating at λ = 280 nm. The conjugate DOTA-Peptide 2 was monitored by electrospray ionization mass spectrometry (positive ESI using the Agilent G6410 Triple Quadrupole Mass spectrometer) and RP-HPLC (C18, Reprosil-pur ODS-3.5, 250 Å~4.6 mm, 5 µm, Mobile phase: A: 0.1% TFA in H2O, B: 0.1% TFA in acetonitrile, flow rate: 1 mL/min, volume of injection 20 μL, total run time: 40 min) [30].

2. Materials and methods 2.1. Reagents and chemicals All protected amino acids and Rink Amide methylbenzhydrylalanine (MBHA) resin were purchased from Novabiochem (Merck, Darmstadt, Germany). 177Lu Trichloride (177LuCl3) and DOTA-TATE were provided by Pars Isotope Co. (Tehran, Iran). Rat C6 glioma cell line was provided from the Pasteur Institute of Iran (Tehran, Iran). All chemicals and solvents used in our work like Dimethylformamide (DMF), Dichloromethane (DCM), N-Methyl-2-pyrrolidone (NMP), 1hydroxybenzotriazole (HOBT), N,N′-Diisopropylcarbodiimide (DIC), N,N-Diisopropylethylamine (DIPEA), Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU), Trifluoroacetic acid (TFA) and Triisopropylsilan (TIS) were of analytical reagent (AR) grade. TLC–silica gel sheets by Gelman Sciences (Washington, DC, USA) were used for instantthin-layer chromatography (ITLC). The distribution of radioactivity on TLC sheets were plotted using a MiniGITA TLC Scanner (Elysia-raytest GmbH, Germany). The high-performance liquid 2

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Fig. 1. Chemical structures of (A) DOTA-LM3, (B) DOTA-TATE, (C) DOTA-Peptide 1 and D) DOTA-Peptide 2.

2.4. Radiolabeling and in-vitro stability of

177

Lu-DOTA-Peptide 2

volume samples of both organic and aqueous phases were measured in a gamma counter (EG&G/ORTEC, Model 4001 M) and the log P was calculated [9].

25 µL of 177LuCl3 (185 MBq) solution was added into a vial containing 5 nmol of DOTA-Peptide 2 in 0.5 mL of ammonium acetate buffer (0.25 M, pH 5.0) and then the mixture was heated at 95 °C for 30 min. Radiochemical purity (RCP) of 177Lu-DOTA-Peptide 2 solution was determined by radio-HPLC and radio-TLC procedures. Radio-HPLC was done using 0.1% TFA in water as solvent A and acetonitrile as solvent B (gradient program: 0–8 min, 20–65% solvent B, flow rate: 1.0 mL/min) and ITLC was developed using citrate buffer 0.1 M (pH 5.0) as mobile phase [31]. The in vitro stability of 177Lu-DOTA-Peptide 2 was evaluated in ammonium acetate buffer solution (NH4OAc, 0.25 M, pH = 5) for a total of 312 h post-preparation. A solution of 177Lu-DOTA-Peptide 2 (20 µL, 5 MBq) was added into 500 μL of NH4OAc. The stability of the reaction mixture was determined by radio-TLC at different time points during 312 h incubation at room temperature (25 °C) [32]. All tests were conducted in triplicate.

2.7. Cell culture and binding affinity test Binding affinity test for the radiolabeled peptide was performed using Rat C6 glioma cell line [34–36]. The cells were cultured under controlled condition in RPMI-1640 (Gibco, UK) complemented with 10% fetal bovine serum, 2% L-Glutamine solution 2 mM, penicillin (50 U/mL) and streptomycin (50 μg/mL) at 37 °C with 5% CO2 [37]. The C6 cells were seeded in 6-well plates (approximately 105 cells/ 2 mL RPMI per well). After 24 h, cell culture was removed and cells were washed twice with ice-cold phosphate-buffered saline (PBS) and gently agitated with different concentrations (1–100 nM) of 177LuDOTA-Peptide 2 for 1 h at 37 °C. Non-specific binding of radiopeptide was estimated in the presence of 1000-fold excess of octreotide for 30 min at 37 °C. Then, the cell culture was removed and cells were washed twice with PBS. Finally, the cells were harvested and the bound activity was measured using the gamma counter. Dissociation constant (Kd) and the total concentration of SSTR receptors expressed on C6 cells (Bmax) values were estimated from nonlinear curve fitting of bound peptide versus the concentrations of 177Lu-DOTA-Peptide 2 (nM) (Prism; GraphPad software) [30,38]. All tests were conducted in triplicate.

2.5. Human plasma stability assay In brief, 177Lu-DOTA-Peptide 2 (50 µL, 12 MBq) was incubated with human plasma (950 µL) at 37 °C for 1, 4 and 24 h. After each interval, 100 µL of the mixture was treated with 100 µL cold ethanol (96%) so as to protein precipitation. The supernatant was separated from the pellet by centrifugation (7000 rpm for 10 min) and its RCP was analyzed by radio-TLC as previously described [33].

2.8. Internalization studies The internalization rate of 177Lu-DOTA-Peptide 2 was studied in C6 cells. The cells were seeded in 6-well plates (105 cells per well, triplicate for each time) and incubated overnight at 37 °C with 5% CO2. The cells were treated with the radioligand (2.5 pmol/well) and incubated at 37 °C. After defined times (0.5, 1, 2, 4 and 6 h), cell culture was removed, cells were washed with PBS and incubated twice for 5 min with 1 mL glycine buffer (0.1 M, pH 2.8) for dissociation of surface-bound

2.6. Determination of partition coefficient (log P) To determine the log P value of 177Lu-DOTA-Peptide 2, three vials containing 50 µL (12 MBq) of radiolabeled peptide in 1 mL NH4OAc (0.25 M, pH 5.0) and 1 mL n-octanol were vigorously shaken for 10 min and then two phases were separated. The extraction step was repeated three times as described above. The activity concentrations of equal 3

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radioligands and then cells were rinsed with 1 mL PBS. In the end, the cells were lysed using 1 mL NaOH solution (1 N) three times and transferred to tubes for quantifying the internalized radioactivity in a gamma counter. Specific internalization was assessed in the presence of octreotide (5 nmol). Result was expressed as percentage of the applied radioactivity [30,39].

interactions of selected compounds with the SSTR2 active site. A summary of the protein-ligand interactions in the docking studies is described in Table 1. Docking results exhibited that the docked DOTAPeptides 1 and 2 were successfully accommodated in the active site with the range of binding affinity energy from −3.37 to −6.8 kcal/mol (Table 1). Superimposition of docked peptides on SSTR2 showed high similarity in overall pose and coordination pattern (Fig. 2A). Interactions of DOTA-TATE and DOTA-Peptide 2 with active site residues are shown in Fig. 2B and C, respectively. Several hydrogen bonds with important residues of SSTR2 active site led to high binding affinity energy of DOTA-Peptide 2 which was close to DOTA-TATE as a template.

2.9. In vivo stability and biodistribution studies All animal experiments were performed in accordance with national research council's guide and the investigation was approved by the ethical committee at Tehran University of Medical Sciences (Code no: IR.TUMS.VCR.REC.1397.894). For in vivo stability assessment, normal rats (male Wistar rats, age, 8–10 wk; weight, 200–250 g) were intravenously injected with 20 µL (5 MBq) of 177Lu-DOTA-Peptide 2 in 200 μL saline. At 1, 4 and 24 h post-injection, the urine of each rat was collected and evaluated directly by radio-HPLC. All assays were done in triplicate [32]. 8-week-old male rats were subcutaneously injected with the C6 cells (1 × 107) suspended in PBS into the right leg. Three weeks after cell implantation when the average tumor volume reached 800–1000 mm3, in vivo studies were carried out [36]. Normal and tumor rat models were injected with 177Lu-DOTA-Peptide 2 (12 MBq) in 0.1 mL sterile saline via the tail vein. At 1, 4, 24 and 48 h after injection, rats were sacrificed under anaesthesia, selected organs were removed, weighted and their radioactivity was determined using a gamma counter. Blocking studies were performed using octreotide (100 μg) in 0.5 mL saline in tumor rat models. The biodistribution of 177Lu-DOTATATE (15 MBq in 0.1 mL) was studied in tumor rats as well. The results were calculated as a percentage of injected dose per gram (%ID/g) of organ or tissue mass [32,37].

3.2. Synthesis of DOTA-peptide 2 DOTA conjugated Peptide 2 (Fig. 1) was synthesized on a Rink amid resin and obtained with the yield of 40% and purity 98%. The conjugate was characterized by RP-HPLC and ESI-MS (Table 2). 3.3. Radiolabeling and in vitro stability DOTA-Peptide 2 was radiolabeled with 177Lu after incubation for 30 min at 95 °C. Radiochemical yield of the reaction was > 98% by radio-HPLC and radio-TLC procedures. Labeled peptide and free 177Lu migrated to Rf 0.3–0.4 and 0.9–1.0, respectively using citrate buffer as mobile phase. As shown in Fig. 3, tR values for 177Lu-labeled peptide and free 177Lu were observed at 17.41 and 5.20 min, respectively. The in vitro stability of 177Lu-DOTA-Peptide 2 in ammonium acetate buffer solution using ITLC showed high values of RCP (> 90%) during 312 h incubation time (Table 3). 3.4. Human plasma and in vivo stability of

2.10. SPECT/CT imaging

177

Lu-DOTA-peptide 2

RCP of 177Lu-DOTA-Peptide 2 incubated with human plasma for 24 h at 37 °C were analyzed by radio-TLC. The radiopeptide remained stable for 24 h with RCP 97%. The in vivo stability of 177Lu-DOTAPeptide 2 using radio-HPLC showed a high value of RCP (93%) in urine during 24 h after injection (Fig. 4).

The SPECT/CT imaging was performed using a SIEMENS Symbia T scanner (SIEMENS, Erlangen, Germany). Tumor-bearing rats were anaesthetized during imaging sessions with a mixture of ketamine and xylazine. Additional anaesthesia was given during the SPECT imaging if necessary. 15 MBq of 177Lu-DOTA-TATE and 177Lu-DOTA-Peptide 2 were injected through the lateral tail vein, separately. Blocking rat model was prepared as described above. SPECT/CT imaging was performed on supine position at 1 and 4 h after radiopeptide injection. The SPECT acquisition protocol was flash 3D with 256 × 256 matrix size and ninety 40-second views in a circular orbit. Reconstruction was performed using the flash 3D algorithm with 8 iterations and 6 subsets. The reconstructed images were filtered by 4 mm-FWHM Gaussian filter. CT scans performed for anatomical reference and attenuation correction (spatial resolution 1.25 mm, 80 kV, 20 mAs) with a total CT scanning time of 5 s. Transmission data were reconstructed into a matrix of equal size yielding a co-registered image set.

3.5. Determination of log P, Kd and Bmax Radioactivity distribution of 177Lu-DOTA-Peptide 2 in n-octanol and normal saline was measured to explain the hydrophobic-hydrophilic properties of the radiolabeled peptide. The log p was calculated −2.1 that showed the hydrophilic nature of the radiolabeled peptide. A saturation binding assay was performed with rat C6 glioma cells as previously described. The Kd value of 177Lu-DOTA-Peptide 2 was determined by means of representative saturation binding curve as presented in Fig. 5. The Kd and Bmax values were calculated 12.06 ± 1.85 nM and 0.20 ± 0.008 pmol/106 cells, respectively.

3. Results

3.6. Internalization studies

3.1. Selection of SSTR2 antagonist using docking studies

Internalization studies were performed using rat C6 glioma cell line at 0.5, 1, 2, 4 and 6 h post-treatment of 177Lu-DOTA-Peptide 2 (2.5 pmol). Results showed no significant change (p < 0.05) in

A molecular docking study was carried out to evaluate the Table 1 Molecular docking affinities of peptides. Peptide

Sequence

Affinity binding (kcal/mol)

DOTA-Peptide 1 DOTA-Peptide 2 DOTA-LM3 DOTA-TATE

DOTA-p-Cl-Phe-Cyclo(D-Cys-L-Nal-D-Aph-Lys-Thr-Cys)-D-Tyr-NH2 DOTA-p-Cl-Phe-Cyclo(D-Cys-L- BzThi -D-Aph-Lys-Thr-Cys)-D-Tyr-NH2 DOTA-p-Cl-Phe-Cyclo(D-Cys-Tyr-D-Aph-Lys-Thr-Cys)-D-Tyr-NH2 DOTA-D-Phe-Cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr

−3.37 −6.80 −4.49 −8.12

4

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Fig. 2. A) Superimposition of DOTA-LM3 (yellow), DOTA-TATE (red), DOTA-Peptide 1 (blue) and DOTA-Peptide 2 (green) on SSTR2. (B) and (C) are schematic representations of the TATE and DOTA-Peptide 2 interactions with SSTR2 active site residues, respectively.

3.8. SPECT/CT imaging

Table 2 Analytical data for DOTA-Peptide 2. Compound name

DOTA-Peptide 2

Mass spectroscopy

SPECT/CT imaging was performed for rats bearing C6 tumor after injection of 177Lu-DOTA-Peptide 2 (15 MBq) via tail vein. Fig. 6 shows SPECT/CT fused images of aforementioned rats 1 and 4 h after injection. Besides significant accumulation of radiotracer in abdomen and kidneys, SPECT/CT imaging of C6 xenograft model clearly showed the activity accumulation in tumor inoculated in the right leg of the rats as expected at 1 and 4 h after injection (Fig. 6A and 6B). Following administration of octreotide as blocking agent, the tumor uptake was decreased in SPECT/CT image at 4 h post-injection, indicating the specific binding of radiotracer with SSTRs in the tumor (Fig. 6C). As expected, C6 tumor was recognized in SPECT/CT imaging of 177LuDOTA-TATE in tumor bearing rat (Fig. 6D).

HPLC

Calculated M (Da)

Observed M (Da)

tR (min)

Purity (%)

1588.5

1589.7 [M + H]+ 795.5 [M + H]+/2

15.45

98

radioanalog internalization in the period study and Peptide 2 specific internalization was 3.2% during 6 h.

177

Lu-DOTA-

3.7. Biodistribution studies The radioactivity distribution profile of 177Lu-DOTA-Peptide 2 in the tumor-bearing rats at 1, 4, 24 and 48 h after injection were represented in Table 4. The pharmacokinetic of the radiopeptide showed a fast blood clearance, a high pancreas uptake and no significant retention in the liver in tumor-bearing and normal rats. Biodistribution results at 4 h post-injection showed the similar pharmacokinetic parameters of 177Lu-DOTA-Peptide 2 with 177Lu-DOTA-TATE as SSTR2 ligand in tumor-bearing rats.

4. Discussion Nowadays, it is believed that somatostatin antagonists are superior to agonists as imaging and therapeutic agents due to desirable pharmacokinetics, higher tumor uptake and better visualization of tumors [40,41]. In this study, new peptide sequence with SST antagonistic properties was designed, synthesized, radiolabeled with 177Lu and evaluated for possible treatment of NETs. 5

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Fig. 3. HPLC profile of

177

To synthesize purposefully, molecular docking studies were performed on the designed sequences as well as DOTA-LM3 and DOTATATE as pattern sequences. Based on SSTR2 structural information obtained from the previous studies, the following points were into consideration in the design of peptide structures: the presence of the lysine at position 5 on peptide structure needed for electrostatic interaction with the receptor binding site, the presence of tryptophan or amino acids with similar lipophilic properties at position 4 required for interaction with lipophilic pocket of the receptor binding site, and the presence of phenylalanine or similar amino acid at position 3 to create a π-π interaction with the π-rich domain of receptor binding site [42]. Acceptable affinity binding energy and superimposition of peptides with modeled SSTR2 are used to select the best candidates for more experiments. Among the designed peptides, DOTA-Peptide 2 showed the most similarity to patterns and the highest affinity binding energy (−6.8 kcal/mol). Schematic representations of the DOTA-Peptide 2 interactions with SSTR2 active site residues showed electrostatic interaction between lysine 5 and serine 285 as well as the presence of lipophilic BzThi beside D-Aph in DOTA-Peptide 2 sequence which resulted in more lipophilic interaction with binding site (Fig. 2). To convert an agonistic structure to antagonistic one, the main parameter is the inversion of chirality in position 2 and 3 of somatostatin analog sequences [43]. In the present study for the first time, new peptide structure for SSTR2 was synthesized using solid-phase chemistry on a Rink amid resin. The incorporation of an unusual amino acid (BzThi) in antagonistic peptide structure is one of the innovative aspects of this study, because the replacement of Tyr3 with more lipophilic BzThi causes the increase in affinity of peptide to SSTR2. Moreover, it may also improve the affinity of peptide to SSTR3 and SSTR5 [44]. The radiochemical yield and in vivo stability of radiolabeled peptides was strongly dependent on the chelating agent so, synthesized peptide was conjugated to DOTA that is currently the gold standard

Lu-DOTA-Peptide 2.

Fig. 4. Human plasma and in vivo stability of

177

Lu-DOTA-Peptide 2.

Fig. 5. Saturation binding curve for 177Lu-DOTA-Peptide 2 performed in rat C6 glioma cells.

Table 3 Stability study of radiolabeled DOTA-Peptide 2 in acetate buffer. Values represent mean ± SD, (n = 3). In vitro stability of

177

Lu-DOTA-Peptide 2

Time (h)

1

24

72

144

312

RCP%

99.5 ± 0.23

99 ± 0.47

98.5 ± 0.26

95.1 ± 1.20

91.4 ± 2.57

6

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Table 4 Biodistribution of

177

Lu-DOTA-Peptide 2 and

177

Lu-DOTA-TATE in C6 tumor-bearing rats. Values represent mean ± SD, (n = 3).

%ID/g organ 177

Lu-DOTA-Peptide 2

*Blocking Tumor

177

Lu-DOTA-TATE

Organ

1h

4h

24 h

48 h

4h

4h

Blood Liver Kidneys Stomach Brain Heart Bone Thyroid Spleen Pancreas Lung Intestine Adrenal Muscle Tumor Whole body

0.21 ± 0.10 1.53 ± 0.90 11.39 ± 2.20 18.41 ± 3.10 0.21 ± 0.05 0.43 ± 0.15 0.44 ± 0.10 0.63 ± 0.17 3.66 ± 1.30 24.84 ± 5.60 1.59 ± 0.37 1.50 ± 0.40 7.44 ± 4.40 0.19 ± 0.10 5.1 ± 1.30 0.18 ± 0.07

0.07 ± 0.05 1.32 ± 0.70 7.24 ± 1.30 15.60 ± 3.70 0.11 ± 0.02 0.29 ± 0.15 0.36 ± 0.10 0.56 ± 0.18 2.94 ± 1.10 18.16 ± 3.70 1.43 ± 0.45 1.22 ± 0.35 7.06 ± 3.10 0.12 ± 0.10 7.3 ± 1.45 0.11 ± 0.02

0.01 ± 0.01 0.73 ± 0.40 5.79 ± 0.80 7.54 ± 2.10 0.0.5 ± 0.01 0.18 ± 0.07 0.14 ± 0.04 0.32 ± 0.11 1.66 ± 0.40 9.63 ± 2.70 0.28 ± 0.07 0.66 ± 0.20 4.00 ± 1.30 0.11 ± 0.07 6.4 ± 1.17 0.03 ± 0.00

0.00 ± 0.00 0.46 ± 0.40 3.35 ± 0.80 4.78 ± 1.31 0.1 ± 0.00 0.16 ± 0.06 0.12 ± 0.03 0.22 ± 0.08 0.64 ± 0.40 5.24 ± 0.90 0.20 ± 0.03 0.25 ± 0.06 2.89 ± 0.90 0.06 ± 0.03 3.74 ± 1.23 0.02 ± 0.00

0.10 ± 0.04 0.90 ± 0.50 5.95 ± 1.20 3.72 ± 1.20 0.09 ± 0.01 0.29 ± 0.02 0.32 ± 0.10 0.50 ± 0.13 3.38 ± 0.90 6.53 ± 1.80 0.30 ± 0.10 1.11 ± 0.47 2.1.00 ± 5.10 0.18 ± 0.10 0.68 ± 0.70 0.10 ± 0.02

0.13 ± 0.06 0.79 ± 0.60 6.89 ± 1.10 8.21 ± 1.71 0.06 ± 0.01 0.12 ± 0.08 0.09 ± 0.02 0.21 ± 0.09 2.90 ± 1.60 12.05 ± 2.50 0.28 ± 0.10 2.67 ± 0.97 4.90 ± 1.10 0.15 ± 0.08 5.8 ± 1.65 0.08 ± 0.01

Tumor-to-organ ratio Tumor-to-blood Tumor-to-kindy Tumor-to-liver Tumor-to-muscle

24.28 0.45 3.33 26.84

104.28 1.01 5.53 60.83

640.00 1.10 8.77 58.18

– 1.11 8.13 62.33

* Octreotide (50 μg) in 0.5 mL saline as a co-injection.

Fig. 6. SPECT/CT imaging of rats bearing C6 tumors at 1 h (A), 4 h (B), blocking at 4 h (C) after injection of 177 Lu-DOTATATE. Tumors are indicated by arrows.

chelator for 177Lu labeling [45]. Radiolabeling of DOTA-peptide conjugate with 177Lu as a theranostic radioisotope with favorable characteristics was performed for concurrent treatment and imaging in tumor rat models [13]. In radiolabeling step, 177Lu-DOTA-Peptide 2 was prepared with high specific activity (37 MBq/nmol) and RCP above 99% with no need for a further purification. Negligible 177Lu release from 177Lu-DOTA-Peptide 2 in acetate buffer and human plasma during incubation time confirmed the high stability of 177Lu-DOTA complexes. The similarity of HPLC chromatograms of extracted radiopeptide from rat urine with synthesized and radiolabeled peptide using UV and radioactivity detectors revealed the high in vivo stability of radiolabeled peptide. Partition coefficient (log P) is a critical factor which affect the biodistribution behavior of radioligands that higher hydrophilic nature is observed for more negative values of log P. For this reason, the 177Lu-

177

Lu-DOTA-Peptide 2 and 4 h (D) after injection of

DOTA- Peptide 2 (log P ~ −2.1) like other SST radiopeptides showed fast blood clearance and renal elimination. Glioma C6 cells which natively express the SSTR1, 2, 3 and 5 without overexpression of SSTRs were utilized for in vitro and in vivo assays [34–36]. Since the dissociation constant (Kd) is a scale of a radioligand binding affinity to a receptor, the low Kd value for 177LuDOTA-Peptide 2 (12.06 nM) revealed a good binding affinity to SSTRs. Moreover, the type of radiometal and chelating agent in the structure of an antagonist can affect the receptor affinity and the biodistribution of radiopeptides [9,46]. Therefore, the calculated Kd for 177Lu-DOTABASS as a promising SSTR2 antagonist with similar radioisotope and chelator was 8.16 nM which is comparable with our sequence [10]. An important feature of SSTR radioantagonists is lack of internalization after binding to cell surface receptors. The result of DOTA-Peptide 2 labeled with 177Lu showed a little amount of internalization (< 5%) in 7

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C6 cells after 6 h of incubation as expected. The other well-known radiopeptides with antagonistic properties have also been reported with < 5% internalization [10,38]. While, internalization for DOTATATE was obtained about 50% [31]. So our result confirmed the antagonistic properties of the synthesized radiopeptide as reported in previous works [37,47]. Based on biodistribution study in normal and tumor-bearing rats, the radiopeptide was cleared from kidney due to its hydrophilic characteristics. Expression of SST receptors in pancreas, stomach and adrenal led to the accumulation of the most radioactivity in these organs8. Due to the longer residence time to receptor, radiopeptide was cleared slowly from non-target organs similar to clearance pattern of 177 Lu-DOTA-TATE. The uptake percentage of the radiopeptide into C6 tumor was more than 7% at 4 h after injection which was decreased to 0.68% in tumor blocked with octreotide and revealed the specific affinity of 177LuDOTA-Peptide 2 to SSTRs. The maximum tumor uptake of 177Lu-DOTAPeptide 2 at 4 h post-injection was shown to be more than 177Lu-DOTATATE at the same time. The lack of overexpression of SSTRs in C6 cells is an important factor in the lower C6 tumor uptake than tumor inoculated with SSTRs-overexpressed cells like HEK-HSST2 [8]. A good retention of radiopeptide was observed in tumor (3.74% ID/g) over 48 h after injection. Ascending trend in tumor-to-blood ratio indicates the increasing tumor uptake and fast clearance of radiopeptide from blood circulation. Tumor-to-kidney ratios was increased approximately twofold up to 4 h, and remained unchanged for 48 h. Increase in tumorto-muscle ratios as a non-target organ indicates selective uptake of radiopeptide in tumor. SPECT/CT imaging of C6 tumor-bearing rats was carried out to demonstrate the in vivo interaction capability of 177Lu-DOTA-Peptide 2 with SSTRs at the surface of C6 cells. Accumulation of high radioactivity in tumor inoculated in the right leg of the rats at 1 and 4 h postinjection (Fig. 6A and B) in comparison with no detectable radioactivity in the tumor blocked with octreotide (Fig. 6C) indicates the specific binding of 177Lu-DOTA-Peptide 2 with SSTRs. The most activity accumulation in pancreas and kidneys were observed due to selective uptake in pancreas and renal clearance of radiopeptide which were correlated with biodistribution results. 177Lu-DOTA-TATE revealed a significant accumulation in tumor xenograft as anticipated (Fig. 6D). In vitro and in vivo assessment displayed acceptable results for subsequent evaluation of therapeutic aspects of 177Lu-DOTA- Peptide 2 in the future study.

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5. Conclusion To detect and treat the neuroendocrine tumors, various SSTR-targeting radiotracers have been studied so far. The development and use of radiolabeled SSTR antagonists may improve PRRT method in neuroendocrine tumors. In the present study, a new SST antagonist radiopeptide, 177Lu-DOTA-Peptide 2, was designed and synthesized. This antagonist radiopeptide manifested high affinity to SSTRs, good in vivo and in vitro stability, fast blood clearance and desirable capability in visualization of tumor lesions after 4 h using SPECT/CT imaging and can be a promising therapeutic agent for neuroendocrine tumors. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research has been part of a PhD thesis at Tehran University of Medical Sciences [Code no. 97-03-58-39735] and supported by Iranian National Science Foundation [Grant no. 96005739], Tehran, Iran. The 8

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