Melanoma Imaging Using 18F-Labeled Î±-Melanocyte-Stimulating

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Melanoma imaging using F-labeled #-melanocyte-stimulating hormone derivatives with positron emission tomography Chengcheng Zhang, Zhengxing Zhang, Kuo-Shyan Lin, Joseph Lau, Jutta Zeisler, Nadine Colpo, David M. Perrin, and Francois Benard Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01113 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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Molecular Pharmaceutics

Melanoma imaging using 18F-labeled α-melanocyte-stimulating hormone derivatives with positron emission tomography

Chengcheng Zhang1, Zhengxing Zhang1, Kuo-Shyan Lin1,2, Joseph Lau1, Jutta Zeisler1, Nadine Colpo1, David M. Perrin3, François Bénard*1,2

1

Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada

2

Department of Radiology, University of British Columbia, Vancouver, BC, Canada

3

Department of Chemistry, University of British Columbia, Vancouver, BC, Canada

*Corresponding Author Dr. François Bénard. Address: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Rm 14-111, Vancouver, BC V5Z 1L3, Canada. Phone: 604-675-8206. Fax: 604-675-8218. E-mail: [email protected].

Conflict of interest: The authors declare no conflict of interest.

Running title: MC1R-targeted imaging using 18F-labeled αMSH with PET

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 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

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ABSTRACT Melanocortin 1 receptor (MC1R) is specifically expressed in the majority of melanomas, a leading cause of death related to skin cancers. Accurate staging and early detection is crucial in managing melanoma. Based on the α-melanocyte-stimulating hormone (αMSH) sequence, MC1R-targeted peptides have been studied for melanoma imaging, predominately for use with single-photon emission computed tomography (SPECT), with few attempts made for positron emission tomography (PET). 18F is a commonly used PET isotope due to readily available cyclotron production, pure positron emission and a favorable half-life (109.8 min). In this study, we aim to design and evaluate αMSH derivatives that enable radiolabeling with 18F for PET imaging of melanoma. We synthesized three imaging probes based on the structure of Nle4cyclo[Asp5-His-D-Phe7-Arg-Trp-Lys10]-NH2 (Nle-CycMSHhex), with a Pip linker (CCZ01064), an Acp linker (CCZ01070), or an Aoc linker (CCZ01071). 18F labeling was enabled by an ammoniomethyl-trifluoroborate (AmBF3) moiety. In vitro competition binding assays showed sub-nanomolar inhibition constant (Ki) values for all three peptides. The 18F radiolabeling was performed via a one-step 18F-19F isotope exchange reaction that resulted in high radiochemical purity (> 95%) and good molar activity (specific activity) ranging from 40.7 to 66.6 MBq/nmol. All three 18F-labeled peptides produced excellent tumor visualization with PET imaging in C57BL/6J mice bearing B16-F10 tumors. The tumor uptake was 7.80 ± 1.77, 5.27 ± 2.38 and 5.46 ± 2.64 percent injected dose per gram of tissue (%ID/g) for [18F]CCZ01064, [18F]CCZ01070 and [18F]CCZ01071 at 1 h post-injection (p.i.), respectively. Minimal background activity was observed except for kidneys at 4.99 ± 0.20, 4.42 ± 0.54 and 13.55 ± 2.84 %ID/g, respectively. The best candidate [18F]CCZ01064 was further evaluated at 2 h p.i., which showed increased tumor uptake at 11.96 ± 2.31 %ID/g and further reduced normal tissue



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uptake. Moreover, a blocking study was performed for CCZ01064 at 1 h p.i., where tumor uptake was significantly reduced to 1.97 ± 0.60 %ID/g, suggesting the tumor uptake was receptor-mediated. In conclusion, [18F]CCZ01064 showed high tumor uptake, low normal tissue uptake and fast clearance, and is therefore a suitable and promising candidate for PET imaging of melanoma.

Keywords: Melanocortin 1 receptor; Melanoma; α-melanocyte-stimulating hormone; PET imaging; F-18; Trifluoroborate.


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INTRODUCTION Melanoma is the main cause of death related to skin cancers, with an estimated 87,110 new cases and 9,730 deaths in the United States for 2017.1 The incidence of melanoma has been increasing. Melanoma is particularly deadly at the advanced metastatic stage with only 34% 5year survival rate even with new treatment approaches such as immune checkpoint inhibitors.2 Therefore, early detection and accurate staging is crucial in managing melanoma. Positron emission tomography (PET) has emerged as a powerful tool for diagnostic imaging due to high sensitivity and spatial resolution compared to single-photon emission computed tomography (SPECT). PET imaging using 2-[18F]Fluorodeoxyglucose ([18F]FDG) has gained popularity in detecting various types of tumors, thanks to simple chemical synthesis, readily available 18F production in large quantities from medical cyclotrons, pure positron emission and a favorable half-life at 109.8 min. Coupled with computed tomography (CT), this modality is highly accurate in identifying cancerous events, including cutaneous melanoma3 However, due to the non-specific uptake of [18F]FDG, which relies on high metabolic rate of cancer cells, this examinations has some limitations, for example to detect early nodal metastases,4 and liver metastases in uveal melanomas.5 Targeting tumor-specific receptors with PET imaging might provide high specificity and sensitivity, which would aid in the accurate early detection of metastatic melanomas. The melanocortin 1 receptor (MC1R) is specifically expressed in the majority of melanomas, including late stage uveal melanoma,6 and no significant expression levels in normal tissues, which makes it an important protein for targeted molecular imaging. Recently, we reviewed the development of radioligands targeting MC1R for melanoma imaging using PET and SPECT.7 These compounds were designed based on the endogenous ligand of MC1R, i.e. α-



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melanocyte-stimulating hormone (αMSH), a tridecapeptide with the sequence of Ac-Ser1-Tyr2Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2. Native αMSH is prone to degradation in vivo, and therefore not suitable for imaging purposes. Over the years, three classes of αMSH derivatives were developed and have seen successful usage for melanoma imaging: 1) truncated linear αMSH derivatives with unnatural amino acid substitutions at Nle4 and D-Phe7, e.g. [Ac-Nle4,Asp5,D-Phe7]-αMSH4–11 (NAPamide);8 2) metal coordination-based cyclized αMSH derivatives, e.g. Rhenium-cyclized [Cys3,4,10,-D-Phe7]-αMSH3–13 (ReCCMSH);9 and 3) lactam bridge-based cyclized αMSH derivatives, e.g. Nle4-cyclo[Asp5-His-D-Phe7-Arg-Trp-Lys10]-NH2 (Nle-CycMSHhex).10 Unnatural amino acid substitution and cyclization of αMSH improved both in vivo stability and binding affinity to MC1R. Consequently, a large number of αMSH-based radiotracers have been designed and showed promising preclinical results for SPECT imaging of melanoma,7, 10-18 including 38 and 21 αMSH derivatives radiolabeled with 99mTc and 111In, respectively.7 In contrast, only five radioligands in three studies reported 18F-labeled αMSH derivatives for PET imaging of melanoma. The compounds were developed based on the NAPamide19 or ReCCMSH20, 21 core structures, and low tumor uptake and moderate tumor-tobackground contrast was observed with PET imaging in preclinical melanoma models. Moreover, 18F-labeled Nle-CycMSHhex derivatives have not been reported in literature. The aim of this study was to design and evaluate αMSH derivatives based on the NleCycMSHhex that enable radiolabeling with 18F for PET imaging. We synthesized three imaging probes, with a cationic singly charged 4-amino-(1-carboxymethyl) piperidine (Pip) linker (CCZ01064), a cationic doubly charged 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp) linker (CCZ01070), and a neutral 8-aminooctanoic acid (Aoc) linker (CCZ01071). 18F labeling was enabled by the introduction of an ammoniomethyl-trifluoroborate (AmBF3) moiety to all


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three peptides. We evaluated their binding affinity to MC1R, radiolabeling properties with 18F, and in vivo biodistribution and potential for PET imaging in a preclinical melanoma model.



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MATERIALS AND METHODS Peptide synthesis Peptides were synthesized on solid phase via standard Fmoc chemistry as described previously.22 Briefly, a sequence of Fmoc-Asp(O-2-PhiPr)-His(Trt)-D-Phe-Arg(Pbf)-Trp(Boc)Lys(Mtt)-OH was synthesized on Rink-Amide-MBHA resin. The protecting groups O-2-PhiPr and Mtt were selectively removed by treatment with 2.5% trifluoroacetic acid (TFA), and side chains of Asp and Lys were cyclized in presence of benzotriazole-1-yl-oxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP, 4 equiv) and N,NDiisopropylethylamine (DIEA, 4 equiv). The Fmoc group was removed, and Fmoc-Nle-OH was coupled to Asp. Subsequently, a linker, Fmoc-Pip-OH (CCZ01064), Fmoc-Acp-OH (CCZ01070) or Fmoc-Aoc-OH (CCZ01071) was coupled to Nle. For AmBF3 coupling, 2-azidoacetic acid (5 equiv) was coupled to the linker in presence of N,N’-diisopropylcarbodiimide (5 equiv) and N-hydroxysuccinimide (6 equiv). The peptide was then deprotected and cleaved from the resin by treating with 90/2.5/2.5/2.5/2.5 TFA/Phenol/H2O/triisopropylsilane/1, 2-ethanedithiol for 3 h at room temperature. The solution was filtered and the peptide was precipitated in diethyl ether, and purified using highperformance liquid chromatography (HPLC, Agilent). The HPLC eluate containing the azide peptide was collected and lyophilized, and the mass was verified by mass spectrometry (AB/Sciex 4000 QTRAP). A mixture of the azide peptide, N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate (5 equiv), CuSO4 (5 equiv), and sodium ascorbate (12 equiv) in 80/20 H2O and acetonitrile (ACN) was incubated at 45°C for 2 h. The desired product was purified by HPLC with 32% ACN and 68% 40 mM ammonium formate (NH4HCO2, 40mM, pH 6.0) for CCZ01064, 28% ACN and 72% 40 mM NH4HCO2 (40mM, pH 6.0) for CCZ01070, and


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34% ACN and 66% 40 mM NH4HCO2 (40mM, pH 6.0) for CCZ01071 at a flow rate of 4.5 mL/min. The AmBF3-peptides were collected and lyophilized, and the masses were verified by mass spectrometry (AB/Sciex 4000 QTRAP).

Radiochemistry The 18F labeling of the AmBF3 peptides was performed according to previously published procedures.23 Briefly, 18F-fluoride was produced using a TR19 cyclotron (Advanced Cyclotron Systems Inc.) via the 18O(p,n)18F reaction. Approximately 37-74 GBq of 18F-fluoride in 70 µL saline was mixed with the 19F-AmBF3 peptides (100 nmol) in 1:1 ratio of pyridazine-HCl buffer (1 M, pH 2.0) and N,N-dimethylformamide, and heated at 85°C for 5 min and another 15 min after vacuum was applied. The mixture was then quenched with PBS and purified by HPLC using 30% ACN and 70% NH4HCO2 (40mM, pH 6.0) at a flow rate of 4.5 mL/min for [18F]CCZ01064, 26% ACN and 74% H2O (0.1% TFA) for [18F]CCZ01070, and 28% ACN and 72% H2O (0.1% TFA) for [18F]CCZ01071. The 18F-AmBF3 peptides were further purified using C18 Sep-Pak cartridge (Waters), eluted with ethanol, air dried and formulated in saline. Quality control was performed with the same buffer conditions, and peptides with radiochemical purity of > 95% were used for imaging and biodistribution studies.

Cell Culture The B16-F10 melanoma cell line (Mus musculus) was obtained from ATTC (CRL-6475). The cell line was confirmed pathogen-free by the IMPACT 1 mouse profile test (IDEXX BioResearch). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, StemCell



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Technologies) supplemented by 10 % FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified incubator containing 5% CO2.

Receptor binding assays MC1R binding affinity of the peptides was assessed using previously published procedures.22 Briefly, 500,000 B16-F10 cells/well were seeded overnight. Growth media was removed, and reaction buffer containing 4.8 mg/mL HEPES and 2 mg/mL BSA was added. Increasing concentrations of non-radioactive peptides of interest along with 0.1 nM of 125I-[Nle4, D-Phe7]-αMSH (125I-NPD- αMSH, Perkin Elmer) were also added. The reaction mixture was incubated with moderate agitation for 1 h at 25°C. Cells were washed with ice-cold PBS twice, harvested, and measured for radioactivity on a WIZARD 2480 gamma counter (Perkin Elmer).

Tumor implantation All animal experiments were conducted in accordance to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. For tumor implantation, C57BL/6J mice were briefly anesthetized by inhalation with 2% isoflurane, and 1 - 2 × 106 B16-F10 cells were implanted subcutaneously on the right dorsal flank. Mice were used for imaging or biodistribution studies when the tumors reached 8-10 mm in diameter.

Preclinical PET/CT imaging PET/CT imaging studies were carried out on a µPET/CT scanner (Inveon, Siemens) using previously published procedures.24 Briefly, for static PET scans, each tumor-bearing


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mouse was injected with 4-6 MBq of radiolabeled peptides via tail vein under anesthesia. After the injection, mice were allowed to recover and roam freely in their cages. After 1 or 2 h, the mice were sedated with 2% isoflurane inhalation and positioned in the scanner. A baseline CT scan was obtained for localization and attenuation correction. This was followed by a 10 - 15 min static PET scan. For dynamic PET scans, 60 min list-mode acquisition was started at the time of intravenous injection of the radiolabeled peptides following a baseline CT scan. The PET images were reconstructed using the ordered subset expectation maximization and maximum a posteriori algorithm (OSEM3D/MAP), using 2 OSEM3D iterations followed by 18 MAP iterations, with a requested resolution of 1.5 mm.

Biodistribution studies Mice were anesthetized by 2% isoflurane inhalation, and injected with 1 - 2 MBq of radiolabeled peptides. For blocking studies, 10 µg of non-radioactive peptides was co-injected with the radioactive compound. After the injection, the mice were allowed to recover and roam freely in their cages, and euthanized by CO2 inhalation 1 h or 2 h later. Blood was promptly withdrawn, and the organs of interest were harvested, rinsed with 1× PBS (pH 7.4), and blotted dry. Each organ was weighed and the radioactivity of the collected tissue was measured using a Wizard 2480 gamma counter (PerkinElmer), and normalized to the injected dose using a standard curve and expressed as the percentage of the injected dose per gram of tissue (%ID/g).

Statistical analysis Data analysis was performed using GraphPad Prism 7.0a. Student’s t tests were performed for all organs in the biodistribution studies, and one-way ANOVA was performed for



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receptor binding affinities; multiple comparisons were corrected using the Holm-Sidak method. Outliers were removed using the ROUT method with Q = 1%. The difference was considered statistically significant when p value was < 0.05.


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RESULTS Chemical properties and in vitro binding affinity The chemical structures of the three peptides, CCZ01064, CCZ01070 and CCZ01071, are shown in Figure 1. All peptides were synthesized with high purity (> 97%) determined by HPLC, and their masses were verified by mass spectrometry (Table 1). In vitro competition binding assays were used to determine their inhibition constant (Ki), which were 0.59 ± 0.05, 0.81 ± 0.07 and 0.90 ± 0.11 nM, respectively (Table 1, Figure 2). The binding affinity of CCZ01064 was significantly higher (p < 0.05) than the other two peptides.

Radiochemical characteristics The 18F radiolabeling of the peptides was performed in high radiochemical purity (> 95%) with radiochemical yield ranging from 5 to 14% (Supplementary Figure 1). Good molar activities (specific activities) were achieved ranging from 40.7 to 66.6 MBq/nmol (Table 2).

PET imaging and biodistribution PET images with all three radiolabeled peptides, [18F]CCZ01064, [18F]CCZ01070 and [18F]CCZ01071, provided excellent tumor visualization with minimal activity accumulation in normal tissues, except for kidneys for all three tracers and intestines for [18F]CCZ01071, at 1 h p.i. in C57BL/6J mice bearing B16-F10 tumor (Figure 3). Biodistribution data are summarized in Table 3 and showed consistent observation, i.e. tumor uptake at 1 h p.i. was 7.80 ± 1.77, 5.27 ± 2.38 and 5.46 ± 2.64 %ID/g for [18F]CCZ01064, [18F]CCZ01070 and [18F]CCZ01071, respectively. Minimal background activity was observed except for kidneys for all three tracers at 4.99 ± 0.20, 4.42 ± 0.54 and 13.55 ± 2.84 %ID/g, respectively, and intestines for



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[18F]CCZ01071 at 9.57 ± 2.57 %ID/g. Dynamic PET scans were also acquired and showed sustained tumor uptake with all three radiolabeled peptides with fast radioactivity clearance from normal tissues (Figure 4). The best candidate [18F]CCZ01064 was further evaluated at 2 h p.i. (Figure 5, Table 3), PET imaging and biodistribution data showed increased tumor uptake at 11.96 ± 2.31 %ID/g and further reduced normal tissue uptake. Moreover, a blocking study was performed with co-injection of 10 µg of non-radioactive CCZ01064 at 1 h p.i. (Figure 5, Table 3), tumor uptake was significantly reduced to 1.97 ± 0.60 %ID/g, which is an approximately 75% decrease compared to the unblocked study, confirming the tumor uptake was receptormediated.


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DISCUSSION MC1R-targeted melanoma imaging has been studied extensively with metal- or lactam bridge-based cyclized αMSH over the last decade. These peptides were predominately radiolabeled with 99mTc or 111In for SPECT imaging. Only few attempts have been made to develop 18F-labeled PET imaging probes targeting MC1R.19-21 In this study, we designed and evaluated the first 18F-labeled αMSH peptides based on the Nle-CycMSHhex core structure, i.e. AmBF3-Pip-Nle-CycMSHhex (CCZ01064), AmBF3-Acp-Nle-CycMSHhex (CCZ01070) and AmBF3-Aoc-Nle-CycMSHhex (CCZ01071). AmBF3 was coupled to the peptides via a copper-catalyzed click reaction. This moiety allowed a simple one-step 18F-radiolabeling via 18F-19F isotope exchange reaction in mild aqueous condition.23 Using this strategy, we have successfully designed 18F-labeled compounds for bradykinin,25, 26 bombesin27 and carbonic anhydrase IX (CA-IX)28 imaging with PET. For radiolabeling, the AmBF3-conjugated αMSH peptides were easily amenable. We performed HPLC purification to ensure > 95% radiochemical purity for biodistribution and PET imaging studies, and achieved good molar activity > 40.7 MBq/nmol. Three linkers were selected, Pip (+1 charge), Acp (+2 charge) and Aoc (neutral). For the Nle-CycMSHhex peptide, with a negatively charged amino acid glutamate, tumor uptake was significantly reduced compared to a neutral glycine.10 In contrast, we observed favorable binding affinity and tumor uptake using the cationic Pip linker for targeting MC1R22 and other receptors, e.g. bradykinin B1 receptor29 and neuropeptide Y1 receptor.30 Therefore, positively charged Pip and Acp were incorporated in the tracer design. As a control, the neutral linker, Aoc, was selected as higher tumor uptake was achieved compared to the other neutral PEG2, GlyGlyGly, and GlySerGly linkers in 99mTc-labeled Nle-CycMSHhex compounds.14



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All three peptides evaluated in this study showed excellent binding affinity to MC1R, with Ki values in the sub-nanomolar range. With the +1 charge Pip linker, significantly improved binding was observed at 0.59 ± 0.05 nM compared to the neutral Aoc linker at 0.90 ± 0.11 nM. Interestingly, the +2 charge Acp linker did not further improve the binding affinity. All three 18F-labeled peptides produced clear tumor visualization with PET imaging, the tumor uptake was 7.80 ± 1.77, 5.27 ± 2.38 and 5.46 ± 2.64 %ID/g for [18F]CCZ01064, [18F]CCZ01070 and [18F]CCZ01071, respectively. Minimal background activity accumulation was observed for the former two peptides, except for kidneys at 4.99 ± 0.20 and 4.42 ± 0.54 %ID/g, respectively. This suggests that the two peptides were cleared primarily through the renal pathway. For [18F]CCZ01071, higher background activity was observed in kidneys (13.55 ± 2.84 %ID/g), and also intestines (9.57 ± 2.57 %ID/g). This is likely due to the lipophilicity of the Aoc linker compared to the cationic linkers, which resulted in clearance via biliary pathway in addition to renal pathways. Furthermore, dynamic PET imaging showed sustained tumor uptake for all three peptides, and fast normal organ clearance suggesting good in vivo stability. The tumor uptake for [18F]CCZ01064 rapidly reached 4.0 – 4.5 %ID/g within the first 2 min, and the uptake was sustained for the duration of the dynamic scan. The slightly lower tumor uptake value compared to the static PET imaging might be due to the fact that the mice were under anaesthesia from the beginning of the dynamic scan. In contrast, for static scans, mice were allowed to wake up after radiotracer injection and roam freely before the imaging session. The hypotensive effect of prolonged sedation for the dynamic scan probably explains the lower tumour uptake with the dynamic scan. In addition, low bone uptake in dynamic and static PET scans as well as biodistribution data suggests that the AmBF3 peptides were stable in vivo with minimal defluorination.


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Due to the high tumor uptake and tumor-to-normal organ uptake ratios, [18F]CCZ01064 was evaluated further at 2 h p.i., PET imaging and biodistribution data showed consistent results: a significant increase in tumor uptake was observed at 11.96 ± 2.31 %ID/g, and kidney uptake was significantly decreased to 3.29 ± 0.61 %ID/g. Tumor-to-blood and tumor-to-muscle uptake ratios significantly increased to 72.37 ± 12.50 and 91.27 ± 16.43 respectively, which provided excellent image contrast for tumor visualization. In addition, a blocking study was performed with co-injection of non-radioactive CCZ01064 (10 µg), in which tumor uptake decreased significantly (75% reduction), along with significantly reduced tumor-to-normal organ contrast. These results confirmed that the tumor uptake was MC1R-mediated. The tumor uptake of 7.80 ± 1.77 and 11.96 ± 2.31 %ID/g at 1 h and 2 h p.i. respectively makes [18F]CCZ01064 an excellent 18F-labeled compound to target MC1R. This represents a five-fold improvement over the previously developed 18F-labeled αMSH derivatives, i.e. [18F]FB-NAPamide (1.19 ± 0.11 %ID/g) at 1 h p.i.,19 and [18F]FP-RMSH-1 (2.12 ± 1.08 %ID/g), [18F]FP-RMSH-2 (0.78 ± 0.10 %ID/g),20 [18F]FB-RMSH-1 (2.11 ± 0.12 %ID/g) and [18F]FBRMSH-2 (1.36 ± 0.18 %ID/g) at 2 h p.i. in the same tumor model.21 This improvement is likely due to sub-nanomolar inhibition constant (0.59 ± 0.05 nM) of CCZ01064 (Table 1), which is based on the Nle-CycMSHhex core structure, compared to the binding affinity ranging from 5.4 to 33.7 nM for the reported NAPamide- and ReCCMSH-based compounds.19-21 The B16-F10 mouse melanoma used in this study is commonly employed for evaluating MC1R-targeting radioligands with SPECT and PET imaging as well as biodistribution in preclinical animal models.19-21, 31, 32 This allowed a direct comparison to previously published results. However, lower MC1R expression levels on various human melanomas have been



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observed.33 Further investigations on the potential of [18F]CCZ01064 for PET imaging of human melanoma are needed. Previously, we also reported 68Ga-labeled Nle-CycMSHhex peptides, which showed a tumor uptake of 21.9 ± 4.6 %ID/g at 2 h p.i. in mice bearing B16-F10 melanoma.22 The higher tumor uptake value is likely a result of the higher molar activity of > 236.8 MBq/nmol, as the 68

Ga-labeled compounds could be separated from the unlabeled chelate-peptide precursor. In a

small animal model, receptor binding sites are more easily saturated than in a large human, and even low masses of injected peptides can decrease tumor uptake. However, the longer half-life of 18

F (109.8 min) compared to 68Ga (67.7 min), and the availability of medical cyclotron in

hospitals that produce 18F in large quantity (at TBq level) might make 18F-labeled αMSH derivatives more practical and applicable, especially for patients, where receptor saturation is expected to be less significant. In conclusion, we designed and evaluated three 18F-labeled αMSH derivatives based on the structure of Nle-CycMSHhex. All three peptides have showed high binding affinity to MC1R and the 18F-labeled peptides produced high contrast tumor visualization with PET in a preclinical model of melanoma. The best candidate [18F]CCZ01064 is therefore a suitable and promising candidate for PET imaging of melanoma, and warrants further evaluation in human xenografts and potentially clinical studies.


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ACKNOWLEDGEMENTS This work was supported in part by the Canadian Institutes of Health Research (FDN148465 and MOP-119361), the BC Cancer Foundation, and the BC Leading Edge Endowment Fund. We thank Wade English, Baljit Singh, and Milan Vuckovic for their technical assistance.



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Supporting information The supporting information is available free of charge on the ACS publication website. Quality control and molar activity determination for 18F-labeled CCZ01064, CCZ01070 and CCZ01071.


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16. Morais, M.; Oliveira, B. L.; Correia, J. o. D.; Oliveira, M. C.; Jimé nez, M. A.; Santos, I.; Raposinho, P. D. Influence of the bifunctional chelator on the pharmacokinetic properties of 99mTc (CO) 3-labeled cyclic α-melanocyte stimulating hormone analog. J Med Chem. 2013, 56, (5), 19611973. 17. Raposinho, P. D.; Correia, J. D.; Oliveira, M. C.; Santos, I. Melanocortin-1 receptor-targeting with radiolabeled cyclic alpha-melanocyte-stimulating hormone analogs for melanoma imaging. Biopolymers 2010, 94, (6), 820-9. 18. Raposinho, P. D.; Xavier, C.; Correia, J. D.; Falcao, S.; Gomes, P.; Santos, I. Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc (CO) 3]+: effect of cyclization on tumor-seeking properties. J Biol Inorg Chem. 2008, 13, (3), 449-459. 19. Cheng, Z.; Zhang, L.; Graves, E.; Xiong, Z.; Dandekar, M.; Chen, X.; Gambhir, S. S. Small-animal PET of melanocortin 1 receptor expression using a 18F-labeled α-melanocyte-stimulating hormone analog. Journal of Nuclear Medicine 2007, 48, (6), 987-994. 20. Ren, G.; Liu, S.; Liu, H.; Miao, Z.; Cheng, Z. Radiofluorinated rhenium cyclized α-MSH analogues for PET imaging of melanocortin receptor 1. Bioconjugate chemistry 2010, 21, (12), 2355-2360. 21. Ren, G.; Liu, Z.; Miao, Z.; Liu, H.; Subbarayan, M.; Chin, F. T.; Zhang, L.; Gambhir, S. S.; Cheng, Z. PET of malignant melanoma using 18F-labeled metallopeptides. Journal of Nuclear Medicine 2009, 50, (11), 1865-1872. 22. Zhang, C.; Zhang, Z.; Lin, K.-S.; Pan, J.; Dude, I.; Hundal-Jabal, N.; Colpo, N.; Bénard, F. Preclinical Melanoma Imaging with 68Ga-Labeled α-Melanocyte-Stimulating Hormone Derivatives Using PET. Theranostics 2017, 7, (4), 805-813. 23. Liu, Z.; Pourghiasian, M.; Radtke, M. A.; Lau, J.; Pan, J.; Dias, G. M.; Yapp, D.; Lin, K. S.; Bénard, F.; Perrin, D. M. An Organotrifluoroborate for Broadly Applicable One‐Step 18F‐Labeling. Angewandte Chemie International Edition 2014, 53, (44), 11876-11880. 24. Lin, K.-S.; Pan, J.; Amouroux, G.; Turashvili, G.; Mesak, F.; Hundal-Jabal, N.; Pourghiasian, M.; Lau, J.; Jenni, S.; Aparicio, S. In vivo radioimaging of bradykinin receptor B1, a widely overexpressed molecule in human cancer. Cancer Res. 2015, 75, (2), 387-393. 25. Liu, Z.; Amouroux, G.; Zhang, Z.; Pan, J.; Hundal-Jabal, N.; Colpo, N.; Lau, J.; Perrin, D. M.; Bénard, F.; Lin, K.-S. 18F-Trifluoroborate derivatives of [des-Arg10] kallidin for imaging bradykinin B1 receptor expression with positron emission tomography. Molecular pharmaceutics 2015, 12, (3), 974-982. 26. Kuo, H.-T.; Pan, J.; Lau, J.; Zhang, C.; Zeisler, J.; Colpo, N.; Benard, F.; Lin, K.-S. Radiolabeled R954 derivatives for imaging bradykinin B1 receptor expression with positron emission tomography. Molecular Pharmaceutics 2017. 27. Pourghiasian, M.; Liu, Z.; Pan, J.; Zhang, Z.; Colpo, N.; Lin, K.-S.; Perrin, D. M.; Bénard, F. 18 FAmBF 3-MJ9: A novel radiofluorinated bombesin derivative for prostate cancer imaging. Bioorganic & medicinal chemistry 2015, 23, (7), 1500-1506. 28. Lau, J.; Liu, Z.; Lin, K.-S.; Pan, J.; Zhang, Z.; Vullo, D.; Supuran, C. T.; Perrin, D. M.; Bénard, F. Trimeric radiofluorinated sulfonamide derivatives to achieve in vivo selectivity for carbonic anhydrase IX–targeted pet imaging. Journal of Nuclear Medicine 2015, 56, (9), 1434-1440. 29. Zhang, Z.; Amouroux, G.; Pan, J.; Jenni, S.; Zeisler, J.; Zhang, C.; Liu, Z.; Perrin, D. M.; Bénard, F.; Lin, K.-S. Radiolabeled B9958 Derivatives for Imaging Bradykinin B1 Receptor Expression with Positron Emission Tomography: Effect of the Radiolabel–Chelator Complex on Biodistribution and Tumor Uptake. Molecular Pharmaceutics 2016, 13, (8), 2823-2832. 30. Zhang, C.; Pan, J.; Lin, K.-S.; Dude, I.; Lau, J.; Zeisler, J.; Merkens, H.; Jenni, S.; Guérin, B.; Bénard, F. Targeting the neuropeptide Y1 receptor for cancer imaging by positron emission tomography using novel truncated peptides. Molecular Pharmaceutics 2016, 13, (11), 3657-3664. 31. Carta, D.; Salvarese, N.; Morellato, N.; Gao, F.; Sihver, W.; Pietzsch, H. J.; Biondi, B.; Ruzza, P.; Refosco, F.; Carpanese, D. Melanoma targeting with [99m Tc (N)(PNP3)]-labeled α-melanocyte 22 ACS Paragon Plus Environment

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Figure 1. Chemical structures of the Nle-CycMSHhex derivatives, A. CCZ01064, B. CCZ01070, and C. CCZ01071.


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Figure 2. Representative competitive binding curves using 125I-NPD- αMSH for CCZ01064, CCZ01070, and CCZ01071 on B16-F10 cells.



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Figure 3. PET images of [18F]CCZ01064, [18F]CCZ01070 and [18F]CCZ01071 at 1 h p.i. in C57BL/6J mice bearing B16-F10 tumor. t, tumor; k, kidney; i, intestines; b, bladder.


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Figure 4. Time activity curves of A. [18F]CCZ01064, B. [18F]CCZ01070 and C. [18F]CCZ01071 using regions-of-interest drawn around the tumor, muscle, heart (blood), bone, and kidney. %ID/g is in log scale.



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Figure 5. PET images of [18F]CCZ01064 at 1 h p.i. and 2 h p.i., and 1 h p.i. with co-injection of 10 µg of non-radioactive CCZ01064 (blocking) in C57BL/6J mice bearing B16-F10 tumor. t, tumor; k, kidney; b, bladder.


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Table 1. Analytical data for αMSH analogues and affinities for MC1R. (One way ANOVA was performed to compare receptor binding affinites, multiple comparisons corrected using HolmSidak method, * p < 0.05, n = 3).

Peptide CCZ01064 CCZ01070 CCZ01071

Mass calculated 1369.7 1398.8 1370.8

Mass found 1371.9 (M+2H) 1400.0 (M+1H) 1372.0 (M+1H)

Purity (%) > 97% > 99% > 99%

Ki (nM, n = 3) 0.59 ± 0.05* 0.81 ± 0.07 0.90 ± 0.11



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Table 2. Radiochemistry data for 18F-labeled CCZ01064, CCZ01070 and CCZ01071.

Peptide

Radiochemical yield (%, decay-corrected)

Radiochemical purity (%)

Molar activity (MBq/nmol)

[18F]CCZ01064 [18F]CCZ01070 [18F]CCZ01071

12 ± 6 (n = 5) 14 5

97.3 ± 1.1 95.3 96.6

66.6 ± 22.2 40.7 59.2


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Table 3. Biodistribution data of 18F-labeled CCZ01064, CCZ01070 and CCZ01071 in C57BL/6J tumor-bearing mice. Blocking study was performed with co-injection of 10 µg of non-radioactive CCZ01064. (Multiple t tests were performed compared to 18F-labeled CCZ01064 at 1 h p.i., multiple comparisons were corrected using Holm-Sidak method, ** p < 0.01, *** p < 0.001, n ≥ 5). Values are in percentage of injected dose per gram of tissue (mean ± standard deviation).

Tumor Blood Fat Seminal glands Testes Intestines Spleen Pancreas Stomach Liver Adrenal Glands Kidneys Heart Lungs Thyroid Bone Muscle Brain

[18F]CCZ01070 1 h p.i. 5.27 ± 2.38 0.45 ± 0.06 0.11 ± 0.03 0.11 ± 0.04 0.23 ± 0.04 0.60 ± 0.11 0.33 ± 0.04 0.15 ± 0.03 0.35 ± 0.26 0.61 ± 0.09 0.29 ± 0.02 4.42 ± 0.54 0.21 ± 0.05 0.92 ± 0.08 0.55 ± 0.16 0.40 ± 0.10 0.19 ± 0.04 0.02 ± 0.00

[18F]CCZ01071 1 h p.i. 5.46 ± 2.64 0.92 ± 0.25 0.16 ± 0.07 0.16 ± 0.05 0.32 ± 0.10 9.57 ± 2.57 0.66 ± 0.18 0.22 ± 0.04 0.40 ± 0.16 1.34 ± 0.31 0.46 ± 0.13 13.55 ± 2.84 0.43 ± 0.12 2.24 ± 0.73 1.00 ± 0.45 0.45 ± 0.17 0.26 ± 0.07 0.03 ± 0.01

Tumor/Blood Tumor/Muscle Tumor/Bone Tumor/Kidney

12.18 ± 6.68 29.39 ± 17.39 13.8 ± 6.93 1.24 ± 0.68

6.03 ± 2.87 22.09 ± 12.87 12.70 ± 6.83 0.42 ± 0.21

Tissue

1 h p.i. 7.80 ± 1.77 0.45 ± 0.01 0.07 ± 0.01 0.10 ± 0.03 0.19 ± 0.03 0.93 ± 0.29 0.35 ± 0.01 0.12 ± 0.01 0.39 ± 0.15 0.68 ± 0.04 0.31 ± 0.14 4.99 ± 0.20 0.21 ± 0.01 0.80 ± 0.06 0.84 ± 0.36 0.71 ± 0.42 0.19 ± 0.03 0.03 ± 0.02

[18F]CCZ01064 2 h p.i. 11.96 ± 2.31*** 0.17 ± 0.05 0.05 ± 0.02 0.12 ± 0.06 0.19 ± 0.06 1.31 ± 0.57 0.36 ± 0.10 0.10 ± 0.05 0.89 ± 0.75 0.65 ± 0.25 0.77 ± 0.74 3.29 ± 0.61*** 0.16 ± 0.06 0.46 ± 0.12 1.26 ± 0.48 0.77 ± 0.20 0.13 ± 0.03 0.06 ± 0.05

1 h p.i. blocked 1.97 ± 0.60*** 1.96 ± 0.60 0.31 ± 0.08 0.27 ± 0.10 0.58 ± 0.12 1.55 ± 0.17 1.16 ± 0.37 0.57 ± 0.23 1.08 ± 0.58 1.72 ± 0.36 0.81 ± 0.31 27.33 ± 14.22*** 0.90 ± 0.31 3.48 ± 1.30 0.87 ± 0.30 1.14 ± 0.52 0.69 ± 0.30 0.06 ± 0.02

17.09 ± 4.57 42.92 ± 13.63 15.28 ± 9.52 1.57 ± 0.39

72.37 ± 12.50*** 91.27 ± 16.43*** 15.90 ± 2.76 3.64 ± 0.32

1.02 ± 0.20*** 2.96 ± 0.85*** 1.94 ± 0.56** 0.08 ± 0.03***



Figure 1. Chemical structures of the Nle-CycMSHhex derivatives, A. CCZ01064, B. CCZ01070, and C. CCZ01071. 89x98mm (300 x 300 DPI)

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Figure 2. Representative competitive binding curves using 125I-NPD- αMSH for CCZ01064, CCZ01070, and CCZ01071 on B16-F10 cells. 82x67mm (300 x 300 DPI)



Figure 3. PET images of [18F]CCZ01064, [18F]CCZ01070 and [18F]CCZ01071 at 1 h p.i. in C57BL/6J mice bearing B16-F10 tumor. t, tumor; k, kidney; i, intestines; b, bladder. 55x37mm (300 x 300 DPI)


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Figure 4. Time activity curves of A. [18F]CCZ01064, B. [18F]CCZ01070 and C. [18F]CCZ01071 using regions-of-interest drawn around the tumor, muscle, heart (blood), bone, and kidney. %ID/g is in log scale. 173x366mm (300 x 300 DPI)



Figure 5. PET images of 18F-CCZ01064 at 1 h p.i. and 2 h p.i., and 1 h p.i. with co-injection of 10 µg of nonradioactive CCZ01064 (blocking) in C57BL/6J mice bearing B16-F10 tumor. t, tumor; k, kidney; b, bladder. 82x60mm (300 x 300 DPI)


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Melanoma Imaging Using 18F-Labeled Î±-Melanocyte-Stimulating

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