Evaluation of the Human Melanoma Targeting Properties ofRadiolabeled r-Melanocyte Stimulating Hormone Peptide Analogues

Yubin Miao,†,⊥ Donna Whitener,† Weiwei Feng,†,| Nellie K. Owen,§ Jianqing Chen,†,| andThomas P. Quinn*,†, ‡

Departments of Biochemistry, Radiology, and Internal Medicine, University of MissourisColumbia,Columbia, Missouri 65211, and Harry S. Truman Memorial Veterans Hospital,Columbia, Missouri 65201. Received May 5, 2003; Revised Manuscript Received August 20, 2003

The purpose of this study was to evaluate the human MC1 receptor-mediated melanoma targetingproperties of two metal cyclized R-MSH peptide analogues, 188Re-(Arg11)CCMSH and 188Re-CCMSH.Initially, the presence and density of the MC1 receptor were determined on a bank of human melanomacell lines. All eight human melanoma cell lines tested in this study displayed the MC1 receptor ata density of 900 to 5700 receptors per cell. Receptor affinity and biodistribution properties of188Re-(Arg11)CCMSH and 188Re-CCMSH were evaluated in a cultured TXM13 human melanoma-xenografted Scid mouse model. Biodistribution results demonstrated that 3.06 ( 0.68 %ID/g of188Re-(Arg11)CCMSH accumulated in the tumors 1 h postinjection and greater than 65% of the activityat 1 h postinjection remained in the tumors at 4 h after dose administration. Whole body clearanceof 188Re-(Arg11)CCMSH was very rapid, with approximately 82% of injected dose cleared throughurinary system at 4 h postinjection. There was very little activity in blood and major organs such asliver, lung, and muscle except for the kidney. 188Re-CCMSH exhibited similar tumor uptake andretention in TXM13 human melanoma-xenografted Scid mice as 188Re-(Arg11)CCMSH. However, thekidney uptake value of 188Re-CCMSH was two times higher than that of 188Re-(Arg11)CCMSH. Theresults of this study indicate that the MC1 receptor is present on the surface of a large number ofhuman melanoma cells, which makes the MC1 receptor a good imaging or therapeutic target. Moreover,the biodistribution properties of 188Re-(Arg11)CCMSH and 188Re-CCMSH highlight their potential astherapeutic agents for human melanoma.

INTRODUCTION

Malignant melanoma has become a serious publichealth problem due to its increase in incidence (1). In2001, it was estimated that there were 51 400 cases ofmalignant melanoma newly reported and 7800 fatalitiesin the United States (1). It is estimated that more than1.3% of Americans will develop malignant melanomaduring their lifetimes (2). Moreover, metastatic mela-noma deposits are difficult to discover and are resistantto conventional chemotherapy and external beam radia-tion therapy (2). Therefore, there is a great need todevelop novel treatment approaches for metastatic mela-noma.

Radiolabeled antibodies and antibody fragments havebeen investigated extensively to target melanoma andits metastases (3-5). However, their success has beenlimited because of the intrinsic limitations of radiolabeledantibodies and antibody fragments, such as slow circula-tion clearance (6, 7) and reduced rates of tumor penetra-

tion (8, 9). Recently, 123I-labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([123I]BZA) and N-(2-diethylaminoethyl)-3-iodo-4-methoxybenzamide ([123I]IMBA) have exhibitedhigh tumor uptake value ranging from 5 to 20 %ID/g inB16 tumor model (10, 11). However, their clinical ap-plication may be impeded due to the disadvantages ofiodine-123, such as routine availability and high cost.More promising results were obtained with 131I-labeledN-(2-diethylaminoethyl)benzamide derivatives (12). Incomparison with [123I]BZA and [123I]IMBA, superiortumor uptake and retention of (4-acetamido-N-(2-diethy-laminoethyl)-5-[131I]iodo-2-methoxybenzamide) providedconsiderable potential for melanoma imaging and radio-nuclide therapy. However, the high liver uptake (9.86%ID/g at 6 h pi) and slow clearance (3.72 %ID/g at 24 hpi) might limit its therapeutic application.

Another class of promising agents for melanoma imag-ing and therapy are R-melanocyte stimulating hormone(R-MSH) peptide analogues. Wild-type R-MSH is a smalltridecapeptide (Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2), which is involvedin the control of skin pigmentation. The biological activityof R-MSH is mediated through interactions with themelanocortin 1 (MC1) receptor (13). The melanocortinreceptors belong to the superfamily of G-protein-coupledreceptors (GPCRs). At the present time, five melanocortinreceptors, namely MC1 receptor to MC5 receptor, havebeen identified and cloned (37-43). The MC1 receptor ismainly expressed in melanocytes and leukocytes andinvolved in skin pigmentation and animal coat coloration(13, 37, 44). The MC2 receptor has been found in the

* Corresponding author: Thomas P. Quinn, 117 SchweitzerHall, Department of Biochemistry, University of MissourisColumbia, Columbia, MO 65211. Phone: (573) 882-6099; Fax:(573) 884-4812; E-mail: quinnt@missouri.edu.

† Department of Biochemistry, University of MissourisColumbia.

‡ Department of Radiology, University of MissourisColumbia.⊥ Department of Internal Medicine, University of Missouris

Columbia.§ Harry S Truman Memorial Veterans Hospital.| Current address: Bracco Research USA, Princeton, NJ

08540.

1177Bioconjugate Chem. 2003, 14, 1177−1184

10.1021/bc034069i CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 10/31/2003

adrenal gland and regulates glucocorticoneogenesis (37).The MC3 receptor and MC4 receptor have been identifiedin the brain for the control of feeding behavior and energyhomeostasis (45). The MC5 receptor is expressed in avariety of peripheral tissues and participated in mediat-ing exocrine gland function (46). Among the members ofmelanocortin receptors, the MC1 receptor has been themost widely studied (13-16, 44). The MC1 receptor hasbeen identified both on human and mouse melanomacells (14, 15). More than 80% of human metastaticmelanoma tumor samples have been found to bear MC1receptor (16). Nanomolar receptor affinity of R-MSH andmany of its analogues for the MC1 receptor make MC1receptor an attractive target for the development of newmelanoma targeting peptide pharmaceuticals.

Recently, several R-MSH analogues have been exam-ined for their abilities to target melanoma (17-20, 52-53). In our lab, a novel class of metal-cyclized R-MSHanalogues has been developed for melanoma imaging andtherapy (21-23). Three cysteine were introduced into theamino acid sequence of CCMSH (Ac-Cys3-Cys4-Glu5-His6-D-Phe7-Arg8-Trp9-Cys10-Lys11-Pro12-Val13-NH2) to site-specifically coordinated radiometals such as 99mTc and188Re. Peptide cyclization with 99mTc and 188Re made themolecules resistant to chemical and proteolytic degrada-tion in vivo while retaining high bioactivities (22, 23).Both 99mTc-CCMSH and 188Re-CCMSH exhibited excel-lent tumor uptake and retention and rapid whole bodyclearance in B16/F1 murine melanoma bearing C57 mice(22, 23). However, the presence of nonspecific kidneyactivity associated with 188Re-CCMSH administrationindicated that nephrotoxicity might be a problem withhigh doses used in melanoma therapy trials. Thereforetwo strategies, chemical modification of the peptide andamino acid coinfusion, were investigated to reduce renaluptake of 188Re-CCMSH in our previous report (23). Thesubstitution of Lys at 11th position of 188Re-CCMSH withArg yielded 188Re-(Arg11)CCMSH, which possessed supe-rior tumor uptake and lower renal activity accumulationin B16/F1 murine melanoma bearing C57 mice. The renaluptake value of 188Re-CCMSH was significantly de-creased by amino acid coinfusion as well (approximately50%); however, the tumor/kidney ratio of 188Re-(Arg11)-CCMSH was higher than that of 188Re-CCMSH withamino acid coinfusion (23). The superior tumor uptakeand lower kidney accumulation of 188Re-(Arg11)CCMSHgreatly enhanced its potential as a melanoma therapeuticagent.

The receptor binding characteristics and in vivo bio-distribution properties of radiolabeled R-MSH peptidesare most often analyzed in the murine melanoma B16series of cell lines and in the C57-B16/F1 syngenicmurine melanoma mouse model (48, 57). However, ifradiolabeled R-MSH peptides are to be effective inmelanoma imaging and therapy, they must be able tobind a wide variety of human melanoma cells and showfavorable biodistribution properties in human melanomaxenografts. Differences between the human and murineMC1 receptor, tumor morphology, and physiology couldhave a significant impact on the imaging and therapeuticpotential of radiolabeled MSH peptide analogues. In thisstudy, the prevalence of the MC1 receptor on humanmelanoma was determined in a bank of human mela-noma cell lines. The tumor targeting properties of two188Re cyclized R-MSH peptide analogues, namely 188Re-(Arg11)CCMSH and 188Re-CCMSH, were determined ina TXM13 human melanoma-xenografted, severely com-promised immunodeficient (Scid) mouse model. The invitro receptor binding affinities of (Arg11)CCMSH, CC-

MSH, and nonradioactive rhenium-conjugated Re-(Arg11)-CCMSH and Re-CCMSH in TXM13 human melanomacells were examined by a competitive displacement cellbinding assay using 125I-Tyr2-NDP ([Nle4, D-Phe7]R-MSH).Pharmacokinetic properties of 188Re-(Arg11)CCMSH and188Re-CCMSH were determined in TXM13 human mel-anoma-xenografted Scid mice to evaluate their potentialfor human melanoma therapy. Results presented in thisstudy demonstrated that both 188Re-(Arg11)CCMSH and188Re-CCMSH exhibited substantial tumor uptake andgood retention in TXM13 human melanoma-xenograftedScid mice, which highlighted their potential as thera-peutic agents for human melanoma.

MATERIALS AND METHODS

Chemicals and Reagents. Amino acids and resinwere purchased from Advanced ChemTech Inc (Louis-ville, KY). 188ReO4

- was obtained from a 188W/188Regenerator from Oak Ridge National Laboratory. Allchemicals used in this study were purchased from FischerScientific and used without further purification. Thehuman melanoma cell lines were obtained from NationalCancer Institute except for the TXM13 cell line wassupplied by Dr. Isaiah J. Fidler and Dr. Janet Price fromthe Cell Biology Department, University of Texas M. D.Anderson Cancer Center.

MC1 Receptor Quantitation Assay. The Bmax of thehuman melanoma cell lines were determined by a methodpreviously described (23). Briefly, 1 × 106 human mela-noma cells were incubated at 37 °C for 1.5 h in thepresence of an increasing concentration of 125I-(Tyr2)-NDP(1.56 nCi to 200 nCi) in 0.5 mL of binding media (MEMwith 25 mM HEPES, pH 7.4, 0.2% BSA, 0.3 mM 1,10-phenathroline). The reaction media was aspirated afterincubation. Cells were rinsed with 0.5 mL of ice-cold pH7.4, 0.2% BSA/0.01 M PBS three times. The activity incells was measured in a NaI well counter. Nonspecificbinding was determined by incubating cells and 125I-(Tyr2)-NDP with nonradioactive NDP at a final concen-tration of 10 µM. Scatchard plots were obtained byplotting the ratio of specific binding to free 125I-Tyr2-NDPvs concentration of specific binding (fmole/million cells).The Bmax was the X intercept of linear regression line.

Peptide Purification. High performance liquid chro-matography (HPLC) analysis was performed on an ISCOsystem (Lincoln, NE) equipped with absorption detectorand Packard radiometric detector (Meriden, CT). Watercontaining 5 mM hydrogen chloride and acetonitrile wereused as HPLC solvents A and B, respectively. A C-18reverse phase column (218TP54, Vydac, Hesperia, CA)was used to purify 188Re conjugates with a flow rate of1.0 mL/min. A 20-min gradient of 18-28% acetonitrilein H2O/5 mM HCl was used for radiolabeled peptidepurification.

Preparation of 188Re-Labeled R-MSH Peptides.The R-MSH peptide analogues, CCMSH and (Arg11)-CCMSH, were synthesized by using Fmoc chemistry onamide resin with a Synergy 432A desktop solid-phasepeptide synthesizer (Applied Biosystems, Foster City, CA)as previously described (23). The peptides were depro-tected, cleaved from the resin, and purified by RP-HPLC.The identities of peptides were confirmed by electrosprayionization mass spectrometry (Mass Consortium Corp.,San Diego, CA).

Radiolabeling of 188Re-R-MSH peptides was accom-plished by a method previously described (23). Briefly,200 µL of 6 mg/mL SnCl2 in an aqueous 0.2 M sodiumglucoheptonate solution and 200 µL of fresh 188ReO4

-

1178 Bioconjugate Chem., Vol. 14, No. 6, 2003 Miao et al.

eluant (1-4 mCi) were added into a reaction vial. Themixture was incubated at 75 °C for 30 min. Ten micro-liters of a 1 mg/mL peptide solution were added into themixture. After being adjusted to pH 8.5 with 1 N NaOH,the resulting solution was incubated at 75 °C for 30 min.The radiolabeled peptide was purified to single speciesby RP-HPLC. Purified peptide samples were purged withN2 gas for 20 min to remove the acetonitrile. The pH offinal solution was adjusted to 5 with 0.1 N NaOH andnormal saline for animal studies. Quality controls wereperformed by RP-HPLC (Figure 3).

Competitive Binding Assay. The IC50 values for theR-MSH peptide analogues were determined by competi-tive binding assays with 125I-Tyr2-NDP (Amersham Phar-macia Biotech, UK) in TXM13 human melanoma cells.Briefly, TXM13 cells were harvested from the cultureflasks with a 0.02% EDTA solution, seeded into a 24-well cell culture plate (5 × 105/well), and incubated at37 °C overnight. After being washed once with bindingmedia (MEM with 25 mM HEPES, pH 7.4, 0.2% BSA,0.3 mM 1,10-phenathroline), the cells were incubated at37 °C for 2 h with approximately 100 000 cpm of 125I-Tyr2-NDP in the presence of increasing concentrationsof R-MSH analogues in 0.3 mL of binding media. Thereaction media was aspirated after incubation. Cells wererinsed with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 MPBS twice and lysed in 0.5 mL of 1 N NaOH for 5 min.The activity in cells was measured in a NaI well counter.The competitive binding curves were obtained by plottingthe percentage of 125I-Tyr2-NDP bound to cells vs con-centrations of displacing peptides. The IC50 values for thepeptides were calculated by using the Grafit software(Erithacus Software Limited, UK).

In Vivo Pharmacokinetics Studies. The pharma-cokinetics of 188Re-CCMSH and 188Re-(Arg11)CCMSHwere performed in TXM13 human melanoma-xenograftedScid female mice (Harlan, Indianapolis, IN). The Scidmice were housed in sterile microisolator cages in atemperature and humidity-controlled room. The animalswere fed with autoclaved food and water for a week priorto the tumor cell inoculation. The Scid mice were inocu-lated subcutaneously with 5 × 106 TXM13 humanmelanoma cells in the both flanks. After four weeks,when the weight of tumors reached approximately 0.3 g,3-8 µCi of 188Re-labeled peptide was injected into eachmouse through the tail vein for in vivo pharmacokineticsstudies. Groups of five mice per each time point wereused for the biodistribution studies. The mice weresacrificed at 1, 4, and 24 h postinjection, and tumors andorgans of interest were harvested, weighed, and counted.Blood values were taken as 6.5% of the whole bodyweight. The results were expressed as percent injecteddose/gram (%ID/g) and as percent injected dose (%ID).All the animal studies were conducted in compliance withInstitutional Animal Care and Committee Approval.Statistical analysis was performed using the Student’st-test for unpaired data. A 95% confidence level waschosen to determine the significance between compounds,with p < 0.05 being significantly different.

RESULTS AND DISCUSSION

A bank of human melanoma cell lines were examinedfor presence and number of MC1 receptors. The humanmelanoma cell lines were derived from metastatic de-posits from various organs as well as from primarytumors that were either producing melanin (melanonic)or not melanin producing (amelanonic). All of the humanmelanoma cell lines displayed MC1 receptors. The recep-

tor numbers ranged from 900 to 5700 receptors per cell(Table 1). There was no correlation between the experi-mentally determined receptor number and melanonicstate of the cells or the location of the original isolate.All of the human melanoma cell lines exhibited 1.4-8times fewer receptor numbers per cell than the murineB16/F1 cell line. These results suggest that in generalhuman melanoma cells have fewer MC1 receptors thanthe B16/F1 murine melanoma cell line. Lower MC1receptor numbers may translate into lower melanomatumor uptake of radiolabeled R-MSH peptide analoguesin vivo and certainly highlight the need to produce andadminister high specific activity preparations for imagingand therapy.

The biodistribution and tumor targeting properties oftwo 188Re-cyclized R-MSH peptide analogues (188Re-(Arg11)CCMSH and 188Re-CCMSH) were determined inthe TXM13 human melanoma-xenografted Scid mice. TheTXM13 human melanoma xenograft model is robust andwell characterized for tumor establishment and meta-static potential, making it attractive for biodistributionanalyses as well as future therapy studies (47). Initially,the binding affinities of the Re-cyclized and apo R-MSHpeptide analogues were examined in cultured TXM-13human melanoma cells. The R-MSH peptide analogues,namely CCMSH, (Arg11)CCMSH, Re-CCMSH and Re-(Arg11)CCMSH were synthesized and purified by RP-HPLC, and the identities of peptides were confirmed byelectrospray ionization mass spectrometry. An illustra-tion of the Re-(Arg11)CCMSH structure is shown inFigure 1. Results from in vitro competitive binding assayof peptides performed in TXM13 human melanoma cellsrevealed that both the peptides with or without nonra-dioactive rhenium cyclization bound to the human mela-noma cells with nanomolar range affinities (Figure 2).The introduction of the ReO core in the peptide sequenceslightly decreased the binding affinity of the peptide(Table 2). A list of the peptide sequences, IC50 values,and molecular weights of CCMSH, (Arg11)CCMSH, andtheir Re-cyclized derivatives is presented in Table 2. The

Figure 1. Structure of Re-(Arg11)CCMSH; Ac-Cys-Cys-Glu-His-D-Phe-Arg-Trp-Cys-Arg-Pro-Val-NH2.

Table 1. MC1 Receptor Numbers on Human MelanomaCells

cell line receptor number (sites/cell)

TXM13 5700UACC62 1000M14 1500SKMEL5 1000SKMEL28 9003M 5000UACC257 2800LOX 1000

Radiolabeled R-MSH Peptide Analogues Bioconjugate Chem., Vol. 14, No. 6, 2003 1179

low nanomolar binding affinities of the R-MSH peptideanalogues for the human MC1 receptors on the TXM-13cells were similar to the affinity values determined forthe murine MC1 receptor on B16/F1 cells. Similarity inligand affinities between the human and mouse MC1receptors was consistent with their shared amino acididentity of 75% (37). However, sequence alignments alsorevealed gaps and stretches of nonconservative aminoacid substitutions that could result in ligand selectivitydifference between the two receptors.

The pharmacokinetics and tumor targeting propertiesof 188Re-(Arg11)CCMSH and188Re-CCMSH were deter-mined in TXM13 human melanoma-xenografted Scidmice. CCMSH and (Arg11)CCMSH were labeled with 188-Re through a glucoheptonate transchelation reaction,using stannous chloride as a reducing agent. 188Re-CCMSH and 188Re-(Arg11)CCMSH were separated com-pletely from their nonlabeled counterparts by RP-HPLC.Quality control of the purified 188Re-CCMSH and 188Re-(Arg11)CCMSH for animal studies was performed by RP-HPLC (Figure 3). The biodistribution of 188Re-CCMSHand 188Re-(Arg11)CCMSH in TXM13 tumor-bearing Scidmice at 1 h, 4 h, and 24 h postinjection is shown in Table3. Substantial tumor uptake of 188Re-(Arg11)CCMSH wasexhibited in TXM13 human melanoma-xenografted Scidmice. For example, there was 3.06 ( 0.68%ID/g of 188Re-(Arg11)CCMSH accumulated in the tumors 1 h after doseadministration. Greater than 65% of the activity at 1 hpostinjection remained in the tumors at 4 h after doseadministration. Even 24 h later, there was 0.93 ( 0.49%ID/g of 188Re-(Arg11)CCMSH remaining in the tumors.The tumor uptake of 188Re-(Arg11)CCMSH was similarto the tumor uptake values of 111In-labeled bombesin (53)

and minigastrin (54) evaluated in human tumor xe-nograft models. For example, 111In-labeled bombesin andminigastrin exhibited up to 3.63 %ID/g and 5 %ID/g at 1h postinjection (53, 54) compared to 188Re-(Arg11)CCMSHthat displayed 3.06 %ID/g at the same time point. Thecorresponding 90Y-labeled bombesin and minigastrinanalogues displayed therapeutic efficacy in their respec-tive human tumor xenograft models (55, 56). It isexpected that the uptake of 188Re-(Arg11)CCMSH will besufficient to show therapeutic effects in human melanoma-xenografted Scid mouse model.

Whole body clearance of 188Re-(Arg11)CCMSH was veryrapid, with approximately 82% of injected dose clearedthrough urinary system at 4 h postinjection. Greater than98% of the injected dose was washed out of body by 24 hpostinjection. There was very little activity in blood andmajor organs such as liver, lung, and muscle except forthe kidneys. High tumor/blood and tumor/normal tissueuptake ratios were demonstrated as early as 1 h postin-jection (Table 4). Although the majority of 188Re-(Arg11)-CCMSH in the kidneys cleared rapidly, there was still6.24 ( 1.14 %ID/g of activity present in the kidneys at 4h after injection. Hence, nephrotoxicity would likely bea dose-limiting factor if 188Re-(Arg11)CCMSH was usedfor melanoma therapy trials. In comparison with 188Re-(Arg11)CCMSH, 188Re-CCMSH exhibited similar tumoruptake and retention properties. However, the kidneyuptake value of 188Re-CCMSH was two times higher thanthat of 188Re-(Arg11)CCMSH at 1 h and 4 h postinjection,which would compromise its application for melanomatherapy. The biodistribution data of 188Re-CCMSH dem-onstrated that whole body clearance of 188Re-CCMSH wasrapid as well. Approximately 91% of injected dose waswashed out of body through urinary system at 4 hpostinjection. Greater than 99% of the injected dose wascleared out of body by 24 h postinjection. The accumula-tion of 188Re-CCMSH activity in blood and blood richorgans such liver and lung was similar to that of 188Re-(Arg11)CCMSH. In contrast to 188Re-CCMSH, slightly

Table 2. Molecular Weights (MW), Sequences, and IC50 Values of r-MSH Peptide Analogues

peptide sequence IC50 (nM) calcd MW measured MW

CCMSH Ac-CCEHdFRWCKPV-NH2 2.1 1448.7 1448ReCCMSH Ac-(ReO)CCEHdFRWCKPV-NH2 3.2 1646.9 1648(Arg11)CCMSH Ac-CCEHdFRWCRPV-NH2 1.0 1476.7 1476Re(Arg11)CCMSH Ac-(ReO)CCEHdFRWCRPV-NH2 4.4 1674.9 1676NDP Ac-SYSNleEHdFRWGKPV-NH2 0.21a 1647a 1647a

125I-Tyr2-NDP Ac-S(125I)YSNleEHdFRWGKPV-NH2 NDb 1772b NDb

a Data cited from Chen, J. Q., et al. (2000) Cancer Res. 60, 5649-5658. The IC50 was determined in B16/F1 murine melanoma cells.b ND, data not determined.

Figure 2. Competitive binding curves of CCMSH, Re-CCMSH,(Arg11)CCMSH, and Re-(Arg11)CCMSH in TXM13 human mela-noma cells.

Figure 3. HPLC quality control of 188Re-CCMSH and 188Re-(Arg11)CCMSH.

1180 Bioconjugate Chem., Vol. 14, No. 6, 2003 Miao et al.

more 188Re-(Arg11)CCMSH activity was excreted throughGI system, indicating that the lipophilicity of 188Re-(Arg11)CCMSH was higher than that of 188Re-CCMSH.This hypothesis was consistent with HPLC results, inwhich 188Re-(Arg11)CCMSH exhibited a greater retentiontime than 188Re-CCMSH under the same reverse-phaseelution gradient (Figure 3).

The tumor uptake of 188Re-CCMSH and 188Re-(Arg11)-CCMSH were lower 1 h and 4 h postinjection in theTXM13 human melanoma-xenografted Scid mice than inthe B16/F1 murine melanoma model (23). For example,the uptake of 188Re-CCMSH and 188Re-(Arg11)CCMSH inthe B16/F1 murine melanoma mouse model was 9.78 (2.00 %ID/g and 16.37 ( 3.27 %ID/g at 4 h postinjectionand 1.94 ( 0.47 %ID/g and 3.50 ( 2.32 %ID/g 24 hpostinjection (23) compared to 2.02 ( 0.24 %ID/g and 2.20( 0.27 %ID/g at 4 h postinjection and 0.87 ( 0.33 %ID/gand 0.93 ( 0.49 %ID/g 24 h postinjection in the TXM13human melanoma xenograft model. The significant tumoruptake value difference between B16/F1 murine mela-noma and TXM13 human melanoma models were at-tributed to MC1 receptor density and the in vivo behaviordifferences between two cell lines. In this report, it wasshown that the MC1 receptor density on human mela-noma cells, including TXM13, was lower than that of B16/F1 cells. Moreover, based on the histopathological resultsshowed in our previous report, TXM13 cells formedamelanonic solid tumors with extensive necrotic centersas opposed to the nonnecrotic gelatinous composition ofB16/F1 murine melanoma tumors (22). Differences intumor morphology could significantly affect the tumoruptake of radioactivity on a per gram basis. Necroticcenters present in the TXM13 human melanoma tumorscontribute to the total mass of the tumors but containfew viable melanoma cells capable of 188Re-CCMSH and188Re-(Arg11)CCMSH uptake. Rhenium-188-CCMSH up-take is likely to be localized to rapidly proliferatingmelanoma cells located around the surface of the TXM13

human melanoma tumors (22). Therefore, the overallpercent activity per gram of TXM13 human melanomatumors is reduced when compared to B16/F1 murinemelanoma tumors that lack significant necrotic regionsand are almost entirely composed of viable melanomacells capable of rhenium-188-CCMSH uptake.

Numerous R-MSH analogues, based on the high affin-ity NDP sequence ([Nle4,D-Phe7]-R-MSH), have beensynthesized and characterized as tumor targeting agents(18-20, 50). NDP radiolabeled with 125I or 18F exhibitedhigh receptor affinities in vitro and rapid pharmacoki-netics in vivo; however, they did not display high tumoruptake and retention (20, 50). Recently, Froidevaux etal. (51, 52) described the characterization of a novel 8amino acid DOTA-conjugated MSH analogue, DOTA-MSH(oct), that was based on the high affinity NDP MSHsequence. Indium-111-labeled DOTA- MSH(oct) displayedrapid pharmacokinetics and good tumor uptake, high-lighting its melanoma imaging potential. Tumor uptakevalues were 4.31 ( 0.30 %ID/g at 4 h and 1.17 ( 0.13%ID/g 24 h postinjection in the B16/F1 murine melanomamouse model (52). Murine melanoma uptake of 111In-DOTA-MSH(oct) was 2 times and 1.2 times greater than188Re-(Arg11)CCMSH in human melanoma at 4 and 24 hpostinjection. This difference was expected since weroutinely see a 5-10-fold reduction in xenografted humanmelanoma compared to B16/F1 murine melanoma tu-mors. No characterization of 111In-DOTA-MSH(oct) wasreported for a human melanoma-xenografted model mak-ing direct biodistribution comparisons difficult. However,a comparison of 111In-DOTA-MSH(oct) with 111In-DOTA-ReCCMSH(Arg11) in the B16/F1 murine melanoma mousemodel showed that 111In-DOTA-ReCCMSH(Arg11) dis-played 4 times the tumor uptake 4 h and 7 times theuptake of 111In-DOTA-MSH(oct) at 24 h postinjection (49).Moreover, the kidney uptake of 111In-DOTA-ReCCMSH-(Arg11) was 1.8 and 1.2 times less than 111In-DOTA-MSH(oct) at 4 h and 24 h postinjection (49). Although 111In-

Table 3. Pharmacokinetics of 188Re-CCMSH and 188Re-(Arg11)CCMSH in TXM13 Human Melanoma-Xenografted ScidMicea

1 h 4 h 24 h

tissues CCMSH Arg11CCMSH CCMSH Arg11CCMSH CCMSH Arg11CCMSH

Percent Injected Dose/Gramtumor 1.98 ( 0.26 3.06 ( 0.68b 2.20 ( 0.24 2.02 ( 0.27 0.87 ( 0.33 0.93 ( 0.49brain 0.02 ( 0.04 0.07 ( 0.04 0.08 ( 0.09 0.02 ( 0.05 0.01 ( 0.01 0.07 ( 0.08blood 0.43 ( 0.05 0.84 ( 0.32b 0.01 ( 0.00 0.04 ( 0.05 0.04 ( 0.07 0.09 ( 0.11heart 0.35 ( 0.34 0.74 ( 0.62 0.24 ( 0.17 0.20 ( 0.28 0.06 ( 0.09 0.11 ( 0.18lung 0.90 ( 0.08 1.57 ( 0.65 0.16 ( 0.22 0.21 ( 0.13 0.06 ( 0.04 0.03 ( 0.03liver 0.48 ( 0.02 0.83 ( 0.44 0.11 ( 0.04 0.37 ( 0.12c 0.03 ( 0.01 0.04 ( 0.02spleen 0.12 ( 0.14 0.30 ( 0.34 0.06 ( 0.06 0.25 ( 0.19b 0.07 ( 0.10 0.09 ( 0.11kidneys 19.92 ( 5.30 9.70 ( 3.69b 12.56 ( 0.89 6.24 ( 1.14d 0.39 ( 0.12 0.27 ( 0.06b

muscle 0.07 ( 0.08 0.16 ( 0.19 0.04 ( 0.04 0.27 ( 0.22 0.02 ( 0.04 0.06 ( 0.02pancreas 0.18 ( 0.06 0.33 ( 0.24 0.13 ( 0.17 0.17 ( 0.10 0.01 ( 0.01 0.04 ( 0.03b

Percent Injected Dosestomach 0.28 ( 0.05 0.46 ( 0.23 0.16 ( 0.14 0.52 ( 0.46 0.02 ( 0.01 0.04 ( 0.03intestines 3.71 ( 0.49 7.47 ( 1.36 3.51 ( 1.52 10.98 ( 2.21 0.08 ( 0.03 0.19 ( 0.08urine 83.24 ( 1.28 74.30 ( 5.86 91.03 ( 2.30 82.51 ( 3.02 99.51 ( 0.06 98.83 ( 0.54a The data are presented as percent injected dose/gram or as percent injected dose (mean ( SD, n ) 5). b 0.05 > P > 0.01. c 0.01 > P

> 0.001. d 0.001 > P.

Table 4. The Tumor/normal Tissues Uptake Ratio of 188Re-CCMSH and 188Re-(Arg11)CCMSH in TXM13 HumanMelanoma-Xenografted Scid Mice

1 h 4 h 24 h

ratios CCMSH Arg11CCMSH CCMSH Arg11CCMSH CCMSH Arg11CCMSH

tumor/blood 4.60 3.64 220.00 50.50 21.75 10.33tumor/kidney 0.10 0.32 0.18 0.32 2.23 3.44tumor/lung 2.20 1.95 13.75 9.62 14.50 31.00tumor/liver 4.13 3.69 20.00 5.46 29.00 23.25tumor/muscle 28.29 19.13 55.00 7.48 43.50 15.50

Radiolabeled R-MSH Peptide Analogues Bioconjugate Chem., Vol. 14, No. 6, 2003 1181

DOTA-MSH(oct) exhibited high affinity for the MC1receptor, it may be cleared too rapidly from the bloodstream for optimal tumor uptake.

Nonspecific kidney accumulation is a common problemassociated with radiolabeled peptide and small proteinadministration (24-26). Cationic peptides and proteinsbind to the negatively charged surface of tubule cells viaelectrostatic interaction when they are generally filteredin the glomerulus and reabsorbed in the cells of theproximal tubule (24). Therefore, two strategies of chemi-cal modification of the molecule and basic amino acidcoinfusion have been employed to decrease renal ac-cumulation by masking the electrostatic interactionbetween molecule and the surface of tubular cells (24-31). For instance, Kim and Kobayashi et al. reported theuse of 2,3,5,6-tetrafluorophenyl (TFP)-glycolate to neu-tralize the positive charges and lower the isoelectric pointof anti-Tac disulfide-bonded variable region single-chainFv fragments (dsFv) and humanized anti-Tac Fab frag-ments. Their findings indicated that neutralizing thepositive charges decreased the kidney accumulationof the glycolated conjugates without impairing theirtumor uptake (32-35). Recently, Akizawa et al. investi-gated the effect of molecular charges on renal uptake of111In-DTPA-conjugated peptide by substitution of theN-terminal D-phenylalanine of 111In-DTPA-D-Phe1-Oct-reotide with L-aspartic acid, L-lysine, L-methionine, andL-phenylalanine. They found that the net charges of111In-DTPA-D-Phe1-Octreotide derivatives significantlyaffected their kidney uptake. The strategy of increasingnegative charges in peptide molecules might be used todecrease the renal uptake of 111In-DTPA-conjugated lowmolecular weight peptides (36). An analysis of biodistri-bution comparison of 188Re-CCMSH and 188Re-(Arg11)-CCMSH in this work revealed that the charge distribu-tion of peptide molecule might be another importantfactor, which affected the renal uptake of peptide.Theoretically, the positive charge of Arg is same as Lys;however, the positive charge distribution is differentbetween the Arg guanidinium group and Lys primaryamine. The biodistribution data demonstrated that thekidney uptake value of 188Re-(Arg11)CCMSH was ap-proximately 50% of that of 188Re-CCMSH 1 h and 4 hpostinjection. Moreover, coinfusion of positively chargedamino acids reduced the kidney retention of 188Re-CCMSH by more than 50% while only reducing that of188Re-(Arg11)CCMSH by 40% in our previous report (23).This supports the assertion that difference in chargedistribution on the side chain greatly influences nonspe-cific charge-charge interaction and could be responsiblefor reduced kidney retention of 188Re-(Arg11)CCMSH. Argat 11th position of CCMSH peptide sequence played acritical role in decreasing the renal uptake value of 188-Re-labeled CCMSH peptide analogues. The coinfusion ofbasic amino acid or amino acids combination, such asL-lysine, L-arginine, D-lysine, and combination of L-lysineand L-arginine, have been proved to be effective indecreasing the renal uptake of radiolabeled peptides andantibodies (24-31). It was also clearly demonstrated byour previous report that L-lysine coinjection could sig-nificantly decrease the renal uptake of 188Re-CCMSH and188Re-(Arg11)CCMSH in B16/F1 murine melanoma bear-ing C57 mice (23). Hence, the strategy of L-lysine coin-fusion could potentially be employed to enhance thetherapeutic efficacy of 188Re-(Arg11)CCMSH by furtherdecreasing the nephrotoxicity in human melanoma therapytrials.

In conclusion, all eight human melanoma cell linestested in this study displayed the MC1 receptor. Bio-

distribution results demonstrated that both 188Re-(Arg11)CCMSH and188Re-CCMSH exhibited substantialtumor uptake and good retention, coupled with rapidwhole body clearance in TXM13 human melanoma-xenografted Scid mice model. Human melanoma-target-ing properties of 188Re-(Arg11)CCMSH and188Re-CCMSHaccentuated their potential as therapeutic agents forhuman melanoma.

ACKNOWLEDGMENT

The authors would like to thank Drs. Wynn A. Volkert,Susan L. Deutscher, Silvia S. Jurisson, and Timothy J.Hoffman for their helpful discussion, and Gary L. Sieck-man and Dana G. Mazuru for their technical assistance.This work was supported by a grant (ER61661) from theDepartment of Energy (to T.P.Q.) and a grant from theUniversity of Missouri Life Science Mission Enhance-ment Postdoctoral Fellowship (to Y.M.).

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