Radiofluorinated Rhenium Cyclized r-MSH Analogues for PET Imaging ofMelanocortin Receptor 1

Gang Ren, Shuanlong Liu, Hongguang Liu, Zheng Miao, and Zhen Cheng*

Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford forCancer Early Detection, Stanford University, Stanford, California, 94305-5344, United States. Received September 1, 2010;Revised Manuscript Received October 18, 2010

In order to accomplish in ViVo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R),several R-melanocyte-stimulating hormone (R-MSH) analogues have been labeled with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. Tofurther pursue a radiofluorinated R-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl2-18F-fluoropropionate (18F-NFP) to radiofluorinate the transition metal rhenium cyclized R-MSH metallopeptidesfor PET imaging of MC1R positive malignant melanoma. Metallopeptides Ac-d,Lys-ReCCMSH(Arg11) (twoisomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesischemistry and rhenium cyclization reaction. The two isomers were then conjugated with 19F-NFP or 18F-NFP.The resulting cold or radiofluorinated metallopeptides, 18/19F-FP-RMSH-1 and 18/19F-FP-RMSH-2, were furtherevaluated for their in Vitro receptor binding affinities, in ViVo biodistribution, and small-animal PET imagingproperties. The binding affinities of 19F-FP-RMSH-1 and 19F-FP-RMSH-2 were determined to be within lownanomolar range. In ViVo studies revealed that both 18F-labeled metallopeptides possessed good tumor uptake inthe B16F10 murine model with high MC1R expression, while possessing much lower uptake in A375M humanmelanoma xenografts. Moreover, 18F-FP-RMSH-1 displayed more favorable in ViVo performance in terms ofhigher tumor uptake and much lower accumulation in the kidney and liver, when compared to that of 18F-FP-RMSH-2 at 2 h postinjection (p.i.). 18F-FP-RMSH-1 also displayed lower liver and lung uptake when comparedwith that of the same peptide labeled with 18F-SFB (named as 18F-FB-RMSH-1). Small animal PET imaging of18F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected,much lower tumor uptake and poorer tumor/normal organ contrast were observed for A375M model comparedto those of the B16F10 model. 18F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retentionwhen compared with 18F-FB-RMSH-1. 18F-FP-RMSH-1 demonstrates significant advantages over 18F-FB-RMSH-1 and 18F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1Rexpression in ViVo.

INTRODUCTION

The incidence of malignant melanoma in western countrieshas kept increasing in the last two decades. According to theNational Cancer Institute, there will be an estimated 68,000 newcases and 8,650 deaths in the United States in 2010. Althoughmelanoma represents only 5% of all skin cancer subtypes, itcontributes to more than 50% of deaths related to skincancer (1, 2). Melanoma is an aggressive disease with highmetastatic potential and resistance to cytotoxic agents. Currently,early diagnosis and accurate staging of melanoma remain crucialfor the improvement of management of melanoma patients (3, 4).

Melanocortin type 1 receptor (MC1R1) is a G-protein coupledreceptor and has been found to be overexpressed in many typesof murine and human melanomas, making it an attractive target

for receptor based melanoma imaging and therapy (5, 6). Overthe last two decades, a wide variety of alpha-melanocyte-stimulating hormone (R-MSH) peptides have been developedand extensively studied for MC1R targeted melanoma imagingand treatment (7, 8). To date, two classes of R-MSH analogueshave shown the most promising results for in ViVo targeting ofMC1R positive melanoma. One class is the linear R-MSH, Ac-Nle-Asp-His-d,Phe-Arg-Trp-Gly-Lys-NH2 (referred to as NA-Pamide) and its analogues (9-12); the other is transition metalrhenium cyclized R-MSH, ReO[Cys3,4,10, d,Phe7, Arg11]R-MSH3-13 [referred to as ReCCMSH(Arg11)] based metallopep-tides (13-18).

In our previous research, both NAPamide and ReCCMSH-(Arg11) analogues had been synthesized and radiolabeled witha well-established radiofluorination synthon, N-succinimidyl-4-18F-fluorobenzoate (18F-SFB), for positron emission tomog-raphy (PET) imaging of melanoma (12, 19). The resulting 18F-probes exhibited good tumor imaging contrast as early as 1 hpostinjection (p.i.) in B16F10 tumor mouse models. Compared with18F-SFB labeled NAPamide, 18F-SFB conjugated ReCCMSH(Arg11)peptides displayed higher tumor uptake and retention, suggesting theadvantages of rhenium cyclized CCMSH(Arg11) as a scaffold fordeveloping MC1R PET imaging agents (19). Overall, these pioneerstudies highlight that 18F-SFB labeled R-MSH peptides canefficiently target malignant melanoma with overexpression of

* To whom correspondence should be addressed. Molecular ImagingProgram at Stanford, Department of Radiology and Bio-X Program,Canary Center at Stanford for Cancer Early Detection, 1201 WelchRoad, Lucas Expansion, P095, Stanford University, Stanford, CA, USA.Phone: 94305-5484, 650-723-7866. Fax: 650-736-7925. E-mail:zcheng@stanford.edu.

1Abbreviations: R-MSH, alpha-melanocyte stimulating hormone;MC1R, melanocortin type 1 receptor; PET, positron emission tomo-graphy; SPECT, single photon emission spectroscopy; HPLC, high-performance liquid chromatography; 18F-NFP, 4-nitrophenyl 2-18F-fluoropropionate; 18F-SFB, N-succinimidyl-4-18F-fluorobenzoate; [18F]-FDG, 2-deoxy-2-[18F]fluoro-D-glucose; p.i., postinjection.

Bioconjugate Chem. 2010, 21, 2355–2360 2355

10.1021/bc100391a 2010 American Chemical SocietyPublished on Web 11/12/2010

MC1R in mice and that they can be promising candidates forMC1R targeted melanoma PET imaging.

However, 18F-SFB conjugated ReCCMSH (Arg11) peptidesalso show moderate liver and lung uptake and relatively highuptake in the gallbladder, which is unfavorable for clinicaltranslation (19). In this research, we aimed to further optimizethe in ViVo behavior of 18F labeled R-MSH metallopeptides formelanoma PET imaging. 4-Nitrophenyl-2-[18F]fluoropropionate(18F-NFP) is a small prosthetic group with less hydrophobicityand has been widely used for the radiofluorination of manypeptides (20). The 18F-NFP conjugated galacto-RGD peptidehas even been evaluated in patients of different solid tumorsfor tumor angiogenesis imaging (21-23). The promising resultssuggested NFP as an attractive labeling moiety. Herein, wereport the radiosynthesis of 18F-NFP labeled Ac-d,Lys-ReCCMSH(Arg11) peptides (two isomers, abbreviated asRMSH-1 and RMSH-2, respectively) (Figure 1). The resultingprobes 18F-FP-RMSH-1 and 18F-FP-RMSH-2 were then evaluatedin C57BL/6 mice bearing subcutaneous murine B16F10 melanomatumors with high levels of the MC1R and Foxn1 nude mice bearinghuman A375M melanoma with low levels of the MC1R.

MATERIALS AND METHODS

General. 125I-(Tyr2)-[Nle4,D-Phe7]-R-MSH [125I-(Tyr2)-NDP]was purchased from Perkin-Elmer (Waltham, MA). All N-R-Fmoc-protected amino acids were purchased from AdvancedChemtech (Louisville, KY). Dimethylformamide (DMF) andmethylene chloride were from Fisher Scientific (Fair Lawn, NJ).Piperidine (20%) in DMF and 0.4 M N-methylmorpholine inDMF were from Protein Technologies Inc. (Tucson, AZ).Trifluoroacetic acid (TFA), O-benzotriazole-N,N,N′,N′-tetra-methyluronium hexafluoro-phosphate (HBTU), and 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink amideresin LS, 100-200 mesh, 1% DVB, 0.2 mmol/g) were fromAdvanced Chemtech. Pyridine, acetic anhydride, acetic acid,and anhydrous ether were from J.T.Baker (Phillipsburg, NJ).Triisopropylsilane (TIPS), N,N′-diisopropylethylamine (DIPEA),and 1,2-ethanedithiol (EDT) were purchased from Sigma-Aldrich (Milwaukee, WI). High performance liquid chroma-tography (HPLC) grade acetonitrile (CH3CN) and Millipore 18mΩ water were used for peptide purifications. 4-Nitrophenyl2-fluoropropionate (19F-NFP) was synthesized as previouslyreported (24). All other standard synthesis reagents werepurchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).All the other general materials (cell lines, mice, etc.) andinstruments [reverse phase HPLC, radioactive dose calibrator,and electrospray ionization mass spectrometry (ESI-MS), or

matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF-MS)] are the same as previouslyreported (12). The A375M cell line was a generous gift fromDr. M. Kolodny, University of California, Los Angeles, CA,and B16/F10 murine melanoma cells were obtained fromAmerican Type Tissue Culture Collection (Manassas, VA).C57BL/6 and Fox Chase SCID mice were purchased fromCharles River Laboratories (Boston, MA).

Synthesis of RMSH and 19F-FP-RMSH. The rheniumcyclized metallopeptide, RMSH, was synthesized as previouslyreported using the Ac-d,Lys-CCMSH(Arg11) peptide (amino acidsequence: Ac-kCCEHdFRWCRPV-NH2) and rhenium-gluco-heptonate transchelation reaction (19). Two major products(isomers RMSH-1 and RMSH-2) with identical molecularweights (MW) but different HPLC retention times wereobtained. The reference standards 19F-FP-RMSH-1 and -2 werethen prepared by a reaction of the metallopeptides with 19F-NFP (24). Briefly, RMSH-1 or -2 (1 µmol) in DMSO (100 µL)was added to 19F-NFP (5 µmol) in DMSO (100 µL), followedby the addition of DIPEA (5 µL). The mixture was heated at60 °C for 2 h. After it cooled down to room temperature, thereaction solution was quenched by adding TFA (50 µL) andinjected into a semipreparative HPLC for purification. The flowrate was 3 mL/min, with the mobile phase starting at 95%solvent A and 5% solvent B (0-3 min), going to 35% solventA and 65% solvent B at 33 min, then going to 15% solvent Aand 85% solvent B and maintaining this solvent compositionfor another 3 min (36-39 min), and returning to initial solventcomposition after 42 min had elapsed. Fractions containing theproduct were collected, lyophilized, and characterized by ESI-MS or MALDI-TOF-MS.

Radiosynthesis of 18F-FP-RMSH-1 and -2. The radioflu-orination synthon, 18F-NFP, was prepared on the basis of theprocedure reported previously (23, 25). 18F-NFP (specificactivity of 0.4-1.0 Ci/µmol or 14.7-37 GBq/µmol) dissolvedin acetonitrile (100 µL) was then added to the peptide (RMSH-1or -2; 100 µg) dissolved in DMSO and reacted for 20 min at60 °C. After adding TFA (50 µL) to quench the reaction, thereaction solution was injected into a semipreparative HPLCusing the same elution gradient as the one used in the synthesisof cold 19F-FP-RMSH (1 or 2). The HPLC fractions containingthe radiolabeled product were subsequently collected, combined,and evaporated with a rotary evaporator to dryness. Theradiolabeled peptide was reconstituted in phosphate-bufferedsaline (PBS, 0.01 M, pH 7.4) and passed through a 0.22 µmMillipore filter into a sterile vial for in Vitro and animalexperiments.

Figure 1. Scheme of the radiosynthesis of 18F-FP-RMSH.

2356 Bioconjugate Chem., Vol. 21, No. 12, 2010 Ren et al.

In Vitro Cell Binding Assay. B16F10 murine melanoma cellswere cultured in Dulbecco’s modified Eagle’s high-glucosemedium (DMEM) supplemented with 10% fetal bovine serum(FBS) and penicillin and streptomycin. The cells were main-tained at 37 °C in a 5% CO2 humidified incubator. The receptorbinding affinity studies of 19F-FP-RMSH-1 and -2 for theMC1R were performed using B16F10 cells. Briefly, 0.5 × 106

cells were resuspended in Dulbecco’s modified Eagle’s mediumcontaining 25 mM HEPES, 0.2% BSA, and 0.3 mM 1,10-phenanthroline. The cells were then incubated at 37 °C for 90min with either 19F-FP-RMSH-1 or -2 (peptide concentrationvarying from 10-12-10-6 M) and approximately 40,000 countsper minute (cpm) of 125I-(Tyr2)-NDP. Cells were washed threetimes with ice-cold PBS, and the radioactivity of the cells wasmeasured. Data was analyzed by Graphpad Prism 5.0 (Northamp-ton, MA). The IC50 values, the concentration of competitorrequired to inhibit 50% of the radioligand binding, of thepeptides were calculated.

Biodistribution Studies. All animal experiments were per-formed in compliance with a protocol approved by StanfordUniversity Institutional Animal Care and Use Committee. Five-to six-week old male C57BL/6 mice were implanted with 1 ×106 B16F10 murine melanoma cells, and Foxn1 nude mice wereinoculated with 3 × 106 A375M human melanoma cells in theright flank. When the diameters of the tumors reached around8 mm, approximately 110-130 µCi (4.07-4.81 MBq) of 18F-FP-RMSH-1 or -2 was injected into each mouse through thetail vein. After injection of the radiotracer, the B16F10 mice (n) 3) were sacrificed at 1, 2, and 4 h p.i., while A375M (n ) 3)mice were sacrificed at 2 h p.i. by carbon dioxide. Tumors,blood, and major organs of interest were harvested, weighed,and counted in a Wallac 1480 automated γ-counter (Perkin-Elmer, Waltham, MA). The radioactivity uptake in tumors andnormal tissues was expressed as a percentage of the injectedradioactive dose per gram of tissue (% ID/g).

Small-Animal PET Imaging Studies. PET imaging of tumorbearing mice was performed on a small-animal PET R4 rodentmodel scanner (Siemens Medical Solutions USA, Inc., Knox-ville, TN). The mice bearing B16F10 or A375M tumors wereinjected with 110-130 µCi (4.07-4.81 MBq) of 18F-FP-RMSH-1 or -2 through the tail vein. At 1 and 2 h p.i., the micewere anesthetized with 2% isofluorane and placed in the proneposition near the central field of view in the small-animal PET.Five-minute static scans were obtained, and images werereconstructed by a two-dimensional ordered subsets expectationmaximum (OSEM) algorithm. Regions of interest (ROIs) werethen drawn over the tumor or organ of interest on decay-corrected whole-body coronal images. The mean counts perpixel per minute were obtained from the ROI and converted tocounts per milliliter per minute by using a calibration constant.By assuming a tissue density of 1 g/mL, the ROIs wereconverted to counts ·g-1 ·min-1. An image ROI-derived % ID/gof tissue was then determined by dividing counts per gram perminute with injected dose (ID). No attenuation correction wasperformed.

Statistical Methods. Statistical analysis was performed usingthe Student’s t-test for unpaired data. A 95% confidence levelwas chosen to determine the significance between groups, withP < 0.05 being significantly different.

RESULTS

Chemistry, Radiochemistry, and IC50. The linear peptide,Ac-d,Lys-CCMSH(Arg11), could react with rhenium-glucohep-tonate to produce two major products (RMSH-1 and RMSH-2) with same molecular weight (MW) and similar bindingaffinities to MC1R, which was reported previously (19). Thenonradioactive fluorinated metallopeptides were then prepared

by conjugation with cold 19F-NFP to allow for characterizationof the 18F labeled counterparts. Under the HPLC gradient(5-65% over 30 min) used in the study, the retention times for19F-FP-RMSH-1 and -2 were found to be 16.6 and 18.1 min,respectively (Figure 2). The desired products were purified bysemipreparative HPLC (>90% yield and >95% purity) andcharacterized by MALDI-TOF-MS. For all of the metallopep-tides prepared above, the measured MW was consistent withthe expected MW: m/z ) 1878.1 for [M + H]+ (C73H104FN23-O17ReS3, calculated MW ) 1878.2). The receptor binding assayshowed that the IC50 values of 19F-FP-RMSH-1 and -2 were6.1 and 33.7 nM, respectively (Table 1).

Similarly, 18F-FP-RMSH-1 and -2 were prepared by theconjugation of RMSH-1 or -2 with the radioactive 18F-NFP.On the basis of the retention times acquired from the nonra-dioactive fluorinated peptides, the radiofluorinated products wereidentified and collected using the same HPLC gradient. The totalradiosynthesis time for making the radiopeptide took about 100min. The decay-corrected radiochemical yields of 18F-FP-RMSH-1 and -2 were based on 18F-NFP and were 12.0% (2.7% and 10.5% ( 1.8% (n ) 4), respectively, at the end ofsynthesis (EOS). The radiochemical purities of the labeledpeptides were over 95%, as verified by analytical radio-HPLC.Since the difference in retention times of radiolabeled andunlabeled peptide was greater than 1.5 min, the radiofluorinatedRMSH-1 and -2 were easily separated from their nonradiola-beled counterparts by HPLC. The specific activity of the two18F-FP-RMSH-1 and -2 were estimated to be ∼20 GBq/µmol(decay corrected) on the basis of the labeling agent 18F-NFP.

In ViWo Biodistribution of 18F-FP-RMSH-1 and -2. The inViVo biodistribution studies of both 18F-FP-RMSH-1 and -2were examined in B16F10 murine allograft and A375M humanxenograft melanoma bearing mice. As shown in Table 2, for18F-FP-RMSH-1, the uptake in the B16F10 tumor with highexpression of MC1R was 1.80 ( 0.18, 2.12 ( 1.08, and 1.09( 0.23% ID/g at 1, 2, and 4 h p.i., respectively, suggestinggood tumor uptake and retention of this PET probe. 18F-FP-RMSH-1 also displayed rapid blood clearance and low uptakein muscle, resulting in decent tumor/blood and tumor/muscleratios (3.80 ( 0.89 and 9.36 ( 3.98, respectively) at 2 h p.i. Itwas also noted that 18F-FP-RMSH-1 displayed a much lower

Figure 2. HPLC profiles of 19F-FP-RMSH-1 (A) and 2 (B).

Table 1. IC50 Values of the Fluorinated r-MSH Analogues andTheir Expected and Measured Molecular Weights (M.W.) for [M +H]+ by ESI-MS or MALDI-TOF-MS

peptides expected M.W. measured M.W. IC50 (nM)19F-FP-RMSH-1 1878.2 1878.1 6.1 ( 0.619F-FP-RMSH-2 1878.2 1878.1 33.7 ( 3.0

18F-Metallopeptides for MC1R Imaging Bioconjugate Chem., Vol. 21, No. 12, 2010 2357

uptake in the lung and liver (0.80 ( 0.03 and 0.78 ( 0.05,respectively) compared with 18F-FB-RMSH-1 (19). Also com-pared with 18F-FB-RMSH-1, 18F-FP-RMSH-1 showed lowerkidney uptake in B16F10 tumor bearing mice (P < 0.05). Thelevel of accumulation in the kidney was 8.82 ( 0.76 and 6.76( 0.82% ID/g at 1 and 2 h p.i., respectively, for 18F-FP-RMSH-1. For the A375M model, tumor uptake of 18F-FP-RMSH-1was only 0.84 ( 0.39% ID/g at 2 h p.i., which was significantlylower than that in B16F10 tumors (P < 0.01).

For 18F-FP-RMSH-2, the uptake in the B16F10 tumor modelwas 0.78 ( 0.10% ID/g at 2 h p.i., which was much lower thanthat of 18F-FP-RMSH-1 (P < 0.05). Meanwhile, 18F-FP-RMSH-2 showed much higher kidney uptake of 15.13 ( 0.88%ID/g at 2 h p.i. (P < 0.05). In addition, 18F-FP-RMSH-2demonstrated a higher uptake in the liver when compared tothat of 18F-FP-RMSH-1. For other major organs including thelung, spleen, pancreas, and stomach, 18F-FP-RMSH-2 exhibitedan uptake similar to that of 18F-FP-RMSH-1 (P > 0.05). Lastly,18F-FP-RMSH-2 revealed very low tumor uptake (0.63 (0.19% ID/g), but high uptake (13.45 ( 0.91% ID/g) wasobserved in the kidneys at 2 h p.i in the A375M tumor model.

Small-Animal PET Imaging. Small-animal PET imaging ofmice bearing B16F10 using 18F-FP-RMSH-1 and -2 wasperformed, and the imaging results were compared (Figure3A,B). Visual examination showed that 18F-FP-RMSH-1 uptakein the tumor was higher than that of 18F-FP-RMSH-2 at both1 and 2 h p.i. Both probes showed moderate to high uptake inthe liver and kidney. Further quantification analysis of small-

animal PET images demonstrated that 18F-FP-RMSH-2 had amuch higher uptake in the kidney than that of 18F-FP-RMSH-1(P < 0.01) at 1 and 2 h p.i. (Figure 4). Improved quality ofPET images was also observed at 2 h p.i. for 18F-FP-RMSH-1. On the basis of the above findings, 18F-FP-RMSH-1 wasselected as a PET probe for further evaluation.

18F-FP-RMSH-1 was then imaged in the nude mice bearingA375M melanoma with low MC1R expression at 1 and 2 h(Figure 3C). Compared with B16F10 tumor bearing mice, muchlower tumor uptake and poor tumor-to-background ratio wereobserved in A375M tumors at both time points. The A375Mtumor was barely discernible, while high activity accumulationin kidneys was still observed.

DISCUSSION

It has been reported that overexpression of MC1R maycorrelate with the prognosis of malignant melanoma (26, 27).Peptide based radioactive probes have been extensively studiedfor tumor receptor targeted imaging and therapy (28-30).Recently, a number of studies have also been performed todevelop molecular probes that specifically target MC1R inmelanoma (12, 19). However, 18F-SFB labeled RMSH has itsown limitations such as suboptimal tumor uptakes and relativelyhigh uptakes in the liver and lung. Thus, the major goal of thisresearch is to study whether we can further optimize the in ViVoprofile of 18F-labeled RMSH analogues by using a different

Table 2. Biodistribution Data for 18F-FP-MSH-1 and -2 in C57BL/6 Mice Bearing Subcutaneously Xenotransplanted B16F10 Murine Melanomaand Foxn1 Nude Mice Bearing A375M Human Melanomaa

organ(% ID/g)

18F-FP-RMSH-1 18F-FP-RMSH-2

B16F10 A 375M B16F10 A 375M

1h 2h 4h 2h 2h 2h

tumor 1.80 ( 0.18 2.12 ( 1.08 1.09 ( 0.23 *0.84 ( 0.39 *0.78 ( 0.10 *0.63 ( 0.19blood 0.90 ( 0.10 0.42 ( 0.06 0.15 ( 0.04 0.33 ( 0.22 0.31 ( 0.11 0.20 ( 0.09heart 0.55 ( 0.03 0.31 ( 0.06 0.14 ( 0.06 0.41 ( 0.19 0.21 ( 0.06 0.18 ( 0.07liver 1.36 ( 0.22 0.78 ( 0.05 0.44 ( 0.11 0.69 ( 0.21 *1.97 ( 0.17 1.66 ( 0.08lung 1.72 ( 0.16 0.80 ( 0.03 0.31 ( 0.07 0.68 ( 0.10 0.64 ( 0.15 0.47 ( 0.21muscle 0.33 ( 0.11 0.17 ( 0.03 0.06 ( 0.01 0.19 ( 0.08 0.20 ( 0.05 0.10 ( 0.02spleen 0.61 ( 0.05 0.34 ( 0.07 0.20 ( 0.04 0.34 ( 0.19 0.53 ( 0.03 0.27 ( 0.03brain 0.11 ( 0.04 0.11 ( 0.04 0.04 ( 0.01 0.05 ( 0.01 0.04 ( 0.01 0.04 ( 0.01intestine 0.99 ( 0.45 1.14 ( 0.80 0.29 ( 0.18 0.40 ( 0.19 0.91 ( 0.33 0.17 ( 0.00stomach 0.67 ( 0.31 0.47 ( 0.24 0.31 ( 0.07 0.44 ( 0.02 0.42 ( 0.14 0.52 ( 0.50pancreas 0.32 ( 0.08 0.18 ( 0.03 0.06 ( 0.01 0.78 ( 0.49 0.10 ( 0.03 0.09 ( 0.01bone 0.69 ( 0.18 0.68 ( 0.37 0.17 ( 0.09 0.23 ( 0.11 0.52 ( 0.08 0.25 ( 0.12kidney 8.82 ( 0.76 6.76 ( 0.82 4.41 ( 0.78 6.59 ( 1.53 *15.13 ( 0.88 *13.45 ( 0.91

Uptake Ratio

tumor/blood 2.03 ( 0.33 3.80 ( 0.89 7.45 ( 0.69 3.11 ( 1.30 2.74 ( 0.80 2.90 ( 0.04tumor/muscle 5.78 ( 1.35 9.36 ( 3.98 16.97 ( 1.73 4.49 ( 0.17 3.98 ( 1.09 6.40 ( 2.66

a Data were expressed as the mean ( SD, indicating the percentage administered activity (injected dose) per gram of tissue (%ID/g) after intravenousinjection of 110-130 µCi (4.01-4.87 MBq) tracers (n ) 3). P < 0.05 when compared with 18F-FP-RMSH-1 at 2 h.

Figure 3. Representative decay-corrected coronal small-animal PET images of mice bearing B16F10 tumors on the right shoulder at 1 and 2 h aftertail vein injections of 18F-FP-RMSH-1 (A) and -2 (B) (n ) 3 for each group). T stands for tumor. (C) Representative decay-corrected coronalsmall-animal PET images of A375M tumor bearing mice at indicated time points after a tail vein injection of 18F-FP-RMSH-1.

2358 Bioconjugate Chem., Vol. 21, No. 12, 2010 Ren et al.

radiofluorination synthon, 18F-NFP. The resulting PET probe,18F-FP-RMSH, was also studied for its ability to imagemelanoma and differential MC1R expression in tumor micemodels.

The two isomers of rhenium cyclized Ac-d,Lys-CCMSH-(Arg11), RMSH-1 and -2, were reproducibly formed in previousstudies and proved to possess high MC1R binding affinity inthe nanomolar (nM) range. Further modification of RMSH withthe small prosthetic group 19F-NFP only slightly reduced theiraffinities, as evidenced by in Vitro cell binding assays (Table1). The NFP conjugation to RMSH did not change the bindingcapability to B16F10 cells which overexpress MC1R. Inaddition, 19F-FP-RMSH-1 displays much higher MC1R bindingaffinity than 19F-FP-RMSH-2 (6.1 vs 33.7 nM), which isconsistent with the study of the 19F-SFB conjugated RMSHpeptides (19). Since the affinities of both compounds are in thelow nanomolar range and thus very encouraging, further in ViVoevaluation of the 18F-NFP labeled metallopeptides is warranted.

In biodistribution studies in the B16F10 tumor mice model,a higher tumor uptake of 18F-FP-RMSH-1 can be observedcompared to that of 18F-FP-RMSH-2 along with much lowerkidney and liver uptake for 18F-FP-RMSH-1 at 2 h (P < 0.05).The higher tumor uptake with lower kidney uptake was furtherevidenced by small-animal PET imaging as well as quantifica-tion analysis of PET images. For 18F-FP-RMSH-1, B16F10tumors are clearly delineated over the background at 1 and 2 hp.i., while 18F-FP-RMSH-2 shows a much less prominent tumoruptake (Figure 3A,B). The quantification analysis of small-animal PET images further reveals significant differences in thetumor, liver, and kidney uptake between the two probes (P <0.01) (Figure 4). 18F-FP-RMSH-2 exhibits a much lower tumoruptake but higher kidney uptake than those of 18F-FP-RMSH-1at both 1 and 2 h in B16F10 tumor bearing mice. In the A375Mmodel, the tumor uptake is low for both 18F-FP-RMSH-1 and

-2. Interestingly, the higher uptake in the liver and kidney for18F-FP-RMSH-2 compared with that of 18F-FP-RMSH-1 (P <0.05) may further suggest the different clearance time of thesetwo isomers. Overall, 18F-FP-RMSH-1 clearly displays morefavorable in ViVo properties, which suggests that it could be abetter candidate for imaging MC1R in ViVo.

18F-FP-RMSH-1 exhibits longer retention times in theB16F10 tumor than all other normal organs but the kidney(Table 2). The tumor-to-blood and tumor-to-muscle ratios reachthe highest levels at 4 h. More importantly, it is noticed that18F-FP-RMSH-1 displays high tumor-to-background ratios, forexample, tumor-to-blood and tumor-to-muscle ratios at 4 h are7.45 ( 0.69 and 16.97 ( 1.73, respectively. While for anothertwo radiofluorinated R-MSH peptides reported previously, 18F-FB-NAPamide and 18F-FB-RMSH-1, their tumor-to-bloodratios at 4 h are only 3.60 ( 0.37 and 1.65 ( 0.11, respectively,and their tumor-to-muscle ratios at 4 h are 7.46 ( 2.68 and5.24 ( 1.43, respectively (12, 19). These increased tumor-to-background ratios as well as tumor residence time would makelongitudinal observation of the malignant melanoma withoverexpression of MC1R more feasible when using NFPconjugated RMSH-1 as PET probes. For the A375M tumormodel, the tumors are barely noticeable and show backgrounduptake at 1 and 2 h p.i., when the mice were injected with 18F-FP-RMSH-1 (Figure 3C). This low uptake is likely caused bynonspecific uptake. The better PET imaging quality in theB16F10 tumor compared to that in the A375M suggests that18F-FP-RMSH-1 could differentiate melanoma with differentMC1R expression in living subjects.

More importantly, compared with previous 18F-SFB conju-gated RMSH-1 (18F-FB-RMSH-1) that possesses moderate lungand liver uptake in the B16F10 tumor model (19), 18F-FP-RMSH-1 shows similar tumor uptake at 1 and 2 h, but theuptake gets significantly higher than that of 18F-FB-RMSH-1at 4 h (P < 0.05). The background uptake, particularly in theliver and lung, is much lower for 18F-FP-RMSH-1. Much highertumor-to-blood and tumor-to-muscle ratios are also observedfor 18F-FP-RMSH-1 than that of 18F-FB-RMSH-1. Further-more, the liver and lung uptakes are lower for 18F-FP-RMSH-2when compared with that of 18F-FB-RMSH-2 in both models(P < 0.05). All of these observations highlight that differentradiofluorination methods have significant impact in this met-allopeptide’s in ViVo properties. Additionally, these observationsfurther suggest that the uptake in the liver and lung are due tononspecific accumulation.

Taken together, 18F-NFP shows advantages over 18F-SFB forlabeling of RMSH peptides. Compared to 18F-FB-RMSH-1,18F-FP-RMSH-1 is more promising for translation into mela-noma MC1R PET imaging in clinics. The relatively low liverand lung uptakes of 18F-FP-RMSH-1 also validate the pos-sibility of using it as a potential PET probe for the earlydetection of malignant melanoma metastasis in these regions.18F-FP-RMSH-1 seems by far the best radiofluorinated probereported for imaging MC1R expression in ViVo.

CONCLUSIONS

In summary, radiofluorinated rhenium cyclized R-MSHanalogue, 18F-FP-RMSH-1 and -2, were successfully synthe-sized. Their characteristics including binding affinities and inViVo profiles were investigated in this study. 18F-FP-RMSH-1demonstrates significant advantages over 18F-FB-RMSH-1 and18F-FP-RMSH-2. 18F-FP-RMSH-1 displays favorable in ViVopharmacokinetics in MC1R overexpressing tumor mice models.It is a promising PET probe for imaging MC1R positivemelanoma and MC1R expression in ViVo.

Figure 4. PET quantification of the tumor and other major organuptakes derived from 1 (A) and 2 h (B) after tail vein injections of18F-FP-RMSH-1 or -2. Data are shown as the mean ( SD % ID/g(n ) 3).

18F-Metallopeptides for MC1R Imaging Bioconjugate Chem., Vol. 21, No. 12, 2010 2359

ACKNOWLEDGMENT

This work was supported, in part, by National Cancer Institute(NCI) In ViVo Cellular Molecular Imaging Center (ICMIC) grantP50 CA114747 and Melanoma Research Alliance. We alsothank the Radiochemistry Facility at Stanford for 18F productionand Dr. Zhe Liu for his help on peptides synthesis.

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