Evaluation of the Human Melanoma Targeting ... - ACS Publications

Oct 31, 2003 - Another class of promising agents for melanoma imaging and therapy are α-melanocyte stimulating hormone (α-MSH) peptide analogues...
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Bioconjugate Chem. 2003, 14, 1177−1184

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Evaluation of the Human Melanoma Targeting Properties of Radiolabeled r-Melanocyte Stimulating Hormone Peptide Analogues Yubin Miao,†,⊥ Donna Whitener,† Weiwei Feng,†,| Nellie K. Owen,§ Jianqing Chen,†,| and Thomas 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 targeting properties 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 melanoma cell lines. All eight human melanoma cell lines tested in this study displayed the MC1 receptor at a density of 900 to 5700 receptors per cell. Receptor affinity and biodistribution properties of 188 Re-(Arg11)CCMSH and 188Re-CCMSH were evaluated in a cultured TXM13 human melanomaxenografted Scid mouse model. Biodistribution results demonstrated that 3.06 ( 0.68 %ID/g of 188 Re-(Arg11)CCMSH accumulated in the tumors 1 h postinjection and greater than 65% of the activity at 1 h postinjection remained in the tumors at 4 h after dose administration. Whole body clearance of 188Re-(Arg11)CCMSH was very rapid, with approximately 82% of injected dose cleared through urinary system at 4 h postinjection. There was very little activity in blood and major organs such as liver, lung, and muscle except for the kidney. 188Re-CCMSH exhibited similar tumor uptake and retention in TXM13 human melanoma-xenografted Scid mice as 188Re-(Arg11)CCMSH. However, the kidney uptake value of 188Re-CCMSH was two times higher than that of 188Re-(Arg11)CCMSH. The results of this study indicate that the MC1 receptor is present on the surface of a large number of human 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 as therapeutic agents for human melanoma.

INTRODUCTION

Malignant melanoma has become a serious public health problem due to its increase in incidence (1). In 2001, it was estimated that there were 51 400 cases of malignant melanoma newly reported and 7800 fatalities in the United States (1). It is estimated that more than 1.3% of Americans will develop malignant melanoma during their lifetimes (2). Moreover, metastatic melanoma deposits are difficult to discover and are resistant to conventional chemotherapy and external beam radiation therapy (2). Therefore, there is a great need to develop novel treatment approaches for metastatic melanoma. Radiolabeled antibodies and antibody fragments have been investigated extensively to target melanoma and its metastases (3-5). However, their success has been limited because of the intrinsic limitations of radiolabeled antibodies and antibody fragments, such as slow circulation clearance (6, 7) and reduced rates of tumor penetra* Corresponding author: Thomas P. Quinn, 117 Schweitzer Hall, Department of Biochemistry, University of Missouris Columbia, Columbia, MO 65211. Phone: (573) 882-6099; Fax: (573) 884-4812; E-mail: [email protected]. † Department of Biochemistry, University of Missouris Columbia. ‡ 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.

tion (8, 9). Recently, 123I-labeled N-(2-diethylaminoethyl)4-iodobenzamide ([123I]BZA) and N-(2-diethylaminoethyl)3-iodo-4-methoxybenzamide ([123I]IMBA) have exhibited high tumor uptake value ranging from 5 to 20 %ID/g in B16 tumor model (10, 11). However, their clinical application may be impeded due to the disadvantages of iodine-123, such as routine availability and high cost. More promising results were obtained with 131I-labeled N-(2-diethylaminoethyl)benzamide derivatives (12). In comparison with [123I]BZA and [123I]IMBA, superior tumor uptake and retention of (4-acetamido-N-(2-diethylaminoethyl)-5-[131I]iodo-2-methoxybenzamide) provided considerable potential for melanoma imaging and radionuclide therapy. However, the high liver uptake (9.86 %ID/g at 6 h pi) and slow clearance (3.72 %ID/g at 24 h pi) might limit its therapeutic application. Another class of promising agents for melanoma imaging and therapy are R-melanocyte stimulating hormone (R-MSH) peptide analogues. Wild-type R-MSH is a small tridecapeptide (Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2), which is involved in the control of skin pigmentation. The biological activity of R-MSH is mediated through interactions with the melanocortin 1 (MC1) receptor (13). The melanocortin receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). At the present time, five melanocortin receptors, namely MC1 receptor to MC5 receptor, have been identified and cloned (37-43). The MC1 receptor is mainly expressed in melanocytes and leukocytes and involved in skin pigmentation and animal coat coloration (13, 37, 44). The MC2 receptor has been found in the

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

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adrenal gland and regulates glucocorticoneogenesis (37). The MC3 receptor and MC4 receptor have been identified in the brain for the control of feeding behavior and energy homeostasis (45). The MC5 receptor is expressed in a variety of peripheral tissues and participated in mediating exocrine gland function (46). Among the members of melanocortin receptors, the MC1 receptor has been the most widely studied (13-16, 44). The MC1 receptor has been identified both on human and mouse melanoma cells (14, 15). More than 80% of human metastatic melanoma tumor samples have been found to bear MC1 receptor (16). Nanomolar receptor affinity of R-MSH and many of its analogues for the MC1 receptor make MC1 receptor an attractive target for the development of new melanoma targeting peptide pharmaceuticals. Recently, several R-MSH analogues have been examined for their abilities to target melanoma (17-20, 5253). In our lab, a novel class of metal-cyclized R-MSH analogues has been developed for melanoma imaging and therapy (21-23). Three cysteine were introduced into the amino acid sequence of CCMSH (Ac-Cys3-Cys4-Glu5-His6D-Phe7-Arg8-Trp9-Cys10-Lys11-Pro12-Val13-NH2) to sitespecifically coordinated radiometals such as 99mTc and 188 Re. Peptide cyclization with 99mTc and 188Re made the molecules resistant to chemical and proteolytic degradation in vivo while retaining high bioactivities (22, 23). Both 99mTc-CCMSH and 188Re-CCMSH exhibited excellent tumor uptake and retention and rapid whole body clearance in B16/F1 murine melanoma bearing C57 mice (22, 23). However, the presence of nonspecific kidney activity associated with 188Re-CCMSH administration indicated that nephrotoxicity might be a problem with high doses used in melanoma therapy trials. Therefore two strategies, chemical modification of the peptide and amino acid coinfusion, were investigated to reduce renal uptake of 188Re-CCMSH in our previous report (23). The substitution of Lys at 11th position of 188Re-CCMSH with Arg yielded 188Re-(Arg11)CCMSH, which possessed superior tumor uptake and lower renal activity accumulation in B16/F1 murine melanoma bearing C57 mice. The renal uptake value of 188Re-CCMSH was significantly decreased by amino acid coinfusion as well (approximately 50%); however, the tumor/kidney ratio of 188Re-(Arg11)CCMSH was higher than that of 188Re-CCMSH with amino acid coinfusion (23). The superior tumor uptake and lower kidney accumulation of 188Re-(Arg11)CCMSH greatly enhanced its potential as a melanoma therapeutic agent. The receptor binding characteristics and in vivo biodistribution properties of radiolabeled R-MSH peptides are most often analyzed in the murine melanoma B16 series of cell lines and in the C57-B16/F1 syngenic murine melanoma mouse model (48, 57). However, if radiolabeled R-MSH peptides are to be effective in melanoma imaging and therapy, they must be able to bind a wide variety of human melanoma cells and show favorable biodistribution properties in human melanoma xenografts. Differences between the human and murine MC1 receptor, tumor morphology, and physiology could have a significant impact on the imaging and therapeutic potential of radiolabeled MSH peptide analogues. In this study, the prevalence of the MC1 receptor on human melanoma was determined in a bank of human melanoma cell lines. The tumor targeting properties of two 188 Re cyclized R-MSH peptide analogues, namely 188Re(Arg11)CCMSH and 188Re-CCMSH, were determined in a TXM13 human melanoma-xenografted, severely compromised immunodeficient (Scid) mouse model. The in vitro receptor binding affinities of (Arg11)CCMSH, CC-

Miao et al.

MSH, and nonradioactive rhenium-conjugated Re-(Arg11)CCMSH and Re-CCMSH in TXM13 human melanoma cells were examined by a competitive displacement cell binding assay using 125I-Tyr2-NDP ([Nle4, D-Phe7]R-MSH). Pharmacokinetic properties of 188Re-(Arg11)CCMSH and 188 Re-CCMSH were determined in TXM13 human melanoma-xenografted Scid mice to evaluate their potential for human melanoma therapy. Results presented in this study demonstrated that both 188Re-(Arg11)CCMSH and 188 Re-CCMSH exhibited substantial tumor uptake and good retention in TXM13 human melanoma-xenografted Scid mice, which highlighted their potential as therapeutic agents for human melanoma. MATERIALS AND METHODS

Chemicals and Reagents. Amino acids and resin were purchased from Advanced ChemTech Inc (Louisville, KY). 188ReO4- was obtained from a 188W/188Re generator from Oak Ridge National Laboratory. All chemicals used in this study were purchased from Fischer Scientific and used without further purification. The human melanoma cell lines were obtained from National Cancer Institute except for the TXM13 cell line was supplied by Dr. Isaiah J. Fidler and Dr. Janet Price from the Cell Biology Department, University of Texas M. D. Anderson Cancer Center. MC1 Receptor Quantitation Assay. The Bmax of the human melanoma cell lines were determined by a method previously described (23). Briefly, 1 × 106 human melanoma cells were incubated at 37 °C for 1.5 h in the presence of an increasing concentration of 125I-(Tyr2)-NDP (1.56 nCi to 200 nCi) in 0.5 mL of binding media (MEM with 25 mM HEPES, pH 7.4, 0.2% BSA, 0.3 mM 1,10phenathroline). The reaction media was aspirated after incubation. Cells were rinsed with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M PBS three times. The activity in cells was measured in a NaI well counter. Nonspecific binding was determined by incubating cells and 125I(Tyr2)-NDP with nonradioactive NDP at a final concentration of 10 µM. Scatchard plots were obtained by plotting the ratio of specific binding to free 125I-Tyr2-NDP vs concentration of specific binding (fmole/million cells). The Bmax was the X intercept of linear regression line. Peptide Purification. High performance liquid chromatography (HPLC) analysis was performed on an ISCO system (Lincoln, NE) equipped with absorption detector and Packard radiometric detector (Meriden, CT). Water containing 5 mM hydrogen chloride and acetonitrile were used as HPLC solvents A and B, respectively. A C-18 reverse phase column (218TP54, Vydac, Hesperia, CA) was used to purify 188Re conjugates with a flow rate of 1.0 mL/min. A 20-min gradient of 18-28% acetonitrile in H2O/5 mM HCl was used for radiolabeled peptide purification. Preparation of 188Re-Labeled R-MSH Peptides. The R-MSH peptide analogues, CCMSH and (Arg11)CCMSH, were synthesized by using Fmoc chemistry on amide resin with a Synergy 432A desktop solid-phase peptide synthesizer (Applied Biosystems, Foster City, CA) as previously described (23). The peptides were deprotected, cleaved from the resin, and purified by RP-HPLC. The identities of peptides were confirmed by electrospray ionization mass spectrometry (Mass Consortium Corp., San Diego, CA). Radiolabeling of 188Re-R-MSH peptides was accomplished by a method previously described (23). Briefly, 200 µL of 6 mg/mL SnCl2 in an aqueous 0.2 M sodium glucoheptonate solution and 200 µL of fresh 188ReO4-

Radiolabeled R-MSH Peptide Analogues

eluant (1-4 mCi) were added into a reaction vial. The mixture was incubated at 75 °C for 30 min. Ten microliters of a 1 mg/mL peptide solution were added into the mixture. 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 species by RP-HPLC. Purified peptide samples were purged with N2 gas for 20 min to remove the acetonitrile. The pH of final solution was adjusted to 5 with 0.1 N NaOH and normal saline for animal studies. Quality controls were performed by RP-HPLC (Figure 3). Competitive Binding Assay. The IC50 values for the R-MSH peptide analogues were determined by competitive binding assays with 125I-Tyr2-NDP (Amersham Pharmacia Biotech, UK) in TXM13 human melanoma cells. Briefly, TXM13 cells were harvested from the culture flasks with a 0.02% EDTA solution, seeded into a 24well cell culture plate (5 × 105/well), and incubated at 37 °C overnight. After being washed once with binding media (MEM with 25 mM HEPES, pH 7.4, 0.2% BSA, 0.3 mM 1,10-phenathroline), the cells were incubated at 37 °C for 2 h with approximately 100 000 cpm of 125ITyr2-NDP in the presence of increasing concentrations of R-MSH analogues in 0.3 mL of binding media. The reaction media was aspirated after incubation. Cells were rinsed with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M PBS 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 plotting the percentage of 125I-Tyr2-NDP bound to cells vs concentrations of displacing peptides. The IC50 values for the peptides were calculated by using the Grafit software (Erithacus Software Limited, UK). In Vivo Pharmacokinetics Studies. The pharmacokinetics of 188Re-CCMSH and 188Re-(Arg11)CCMSH were performed in TXM13 human melanoma-xenografted Scid female mice (Harlan, Indianapolis, IN). The Scid mice were housed in sterile microisolator cages in a temperature and humidity-controlled room. The animals were fed with autoclaved food and water for a week prior to the tumor cell inoculation. The Scid mice were inoculated subcutaneously with 5 × 106 TXM13 human melanoma 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 each mouse through the tail vein for in vivo pharmacokinetics studies. Groups of five mice per each time point were used for the biodistribution studies. The mice were sacrificed at 1, 4, and 24 h postinjection, and tumors and organs of interest were harvested, weighed, and counted. Blood values were taken as 6.5% of the whole body weight. The results were expressed as percent injected dose/gram (%ID/g) and as percent injected dose (%ID). All the animal studies were conducted in compliance with Institutional Animal Care and Committee Approval. Statistical analysis was performed using the Student’s t-test for unpaired data. A 95% confidence level was chosen to determine the significance between compounds, with p < 0.05 being significantly different. RESULTS AND DISCUSSION

A bank of human melanoma cell lines were examined for presence and number of MC1 receptors. The human melanoma cell lines were derived from metastatic deposits from various organs as well as from primary tumors that were either producing melanin (melanonic) or not melanin producing (amelanonic). All of the human melanoma cell lines displayed MC1 receptors. The recep-

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Figure 1. Structure of Re-(Arg11)CCMSH; Ac-Cys-Cys-Glu-HisD-Phe-Arg-Trp-Cys-Arg-Pro-Val-NH2. Table 1. MC1 Receptor Numbers on Human Melanoma Cells cell line

receptor number (sites/cell)

TXM13 UACC62 M14 SKMEL5 SKMEL28 3M UACC257 LOX

5700 1000 1500 1000 900 5000 2800 1000

tor numbers ranged from 900 to 5700 receptors per cell (Table 1). There was no correlation between the experimentally determined receptor number and melanonic state of the cells or the location of the original isolate. All of the human melanoma cell lines exhibited 1.4-8 times fewer receptor numbers per cell than the murine B16/F1 cell line. These results suggest that in general human melanoma cells have fewer MC1 receptors than the B16/F1 murine melanoma cell line. Lower MC1 receptor numbers may translate into lower melanoma tumor uptake of radiolabeled R-MSH peptide analogues in vivo and certainly highlight the need to produce and administer high specific activity preparations for imaging and therapy. The biodistribution and tumor targeting properties of two 188Re-cyclized R-MSH peptide analogues (188Re(Arg11)CCMSH and 188Re-CCMSH) were determined in the TXM13 human melanoma-xenografted Scid mice. The TXM13 human melanoma xenograft model is robust and well characterized for tumor establishment and metastatic potential, making it attractive for biodistribution analyses as well as future therapy studies (47). Initially, the binding affinities of the Re-cyclized and apo R-MSH peptide analogues were examined in cultured TXM-13 human melanoma cells. The R-MSH peptide analogues, namely CCMSH, (Arg11)CCMSH, Re-CCMSH and Re(Arg11)CCMSH were synthesized and purified by RPHPLC, and the identities of peptides were confirmed by electrospray ionization mass spectrometry. An illustration of the Re-(Arg11)CCMSH structure is shown in Figure 1. Results from in vitro competitive binding assay of peptides performed in TXM13 human melanoma cells revealed that both the peptides with or without nonradioactive rhenium cyclization bound to the human melanoma cells with nanomolar range affinities (Figure 2). The introduction of the ReO core in the peptide sequence slightly decreased the binding affinity of the peptide (Table 2). A list of the peptide sequences, IC50 values, and molecular weights of CCMSH, (Arg11)CCMSH, and their Re-cyclized derivatives is presented in Table 2. The

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

b

peptide

sequence

CCMSH ReCCMSH (Arg11)CCMSH Re(Arg11)CCMSH NDP 125I-Tyr2-NDP

Ac-CCEHdFRWCKPV-NH2 Ac-(ReO)CCEHdFRWCKPV-NH2 Ac-CCEHdFRWCRPV-NH2 Ac-(ReO)CCEHdFRWCRPV-NH2 Ac-SYSNleEHdFRWGKPV-NH2 Ac-S(125I)YSNleEHdFRWGKPV-NH2

IC50 (nM) 2.1 3.2 1.0 4.4 0.21a NDb

calcd MW 1448.7 1646.9 1476.7 1674.9 1647a 1772b

measured MW 1448 1648 1476 1676 1647a NDb

a Data cited from Chen, J. Q., et al. (2000) Cancer Res. 60, 5649-5658. The IC 50 was determined in B16/F1 murine melanoma cells. ND, data not determined.

Figure 3. HPLC quality control of (Arg11)CCMSH.

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

low nanomolar binding affinities of the R-MSH peptide analogues for the human MC1 receptors on the TXM-13 cells were similar to the affinity values determined for the murine MC1 receptor on B16/F1 cells. Similarity in ligand affinities between the human and mouse MC1 receptors was consistent with their shared amino acid identity of 75% (37). However, sequence alignments also revealed gaps and stretches of nonconservative amino acid substitutions that could result in ligand selectivity difference between the two receptors. The pharmacokinetics and tumor targeting properties of 188Re-(Arg11)CCMSH and188Re-CCMSH were determined in TXM13 human melanoma-xenografted Scid mice. CCMSH and (Arg11)CCMSH were labeled with 188Re through a glucoheptonate transchelation reaction, using stannous chloride as a reducing agent. 188ReCCMSH and 188Re-(Arg11)CCMSH were separated completely from their nonlabeled counterparts by RP-HPLC. Quality control of the purified 188Re-CCMSH and 188Re(Arg11)CCMSH for animal studies was performed by RPHPLC (Figure 3). The biodistribution of 188Re-CCMSH and 188Re-(Arg11)CCMSH in TXM13 tumor-bearing Scid mice at 1 h, 4 h, and 24 h postinjection is shown in Table 3. Substantial tumor uptake of 188Re-(Arg11)CCMSH was exhibited in TXM13 human melanoma-xenografted Scid mice. For example, there was 3.06 ( 0.68%ID/g of 188Re(Arg11)CCMSH accumulated in the tumors 1 h after dose administration. Greater than 65% of the activity at 1 h postinjection remained in the tumors at 4 h after dose administration. 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 similar to the tumor uptake values of 111In-labeled bombesin (53)

188Re-CCMSH

and

188Re-

and minigastrin (54) evaluated in human tumor xenograft models. For example, 111In-labeled bombesin and minigastrin exhibited up to 3.63 %ID/g and 5 %ID/g at 1 h postinjection (53, 54) compared to 188Re-(Arg11)CCMSH that displayed 3.06 %ID/g at the same time point. The corresponding 90Y-labeled bombesin and minigastrin analogues displayed therapeutic efficacy in their respective human tumor xenograft models (55, 56). It is expected that the uptake of 188Re-(Arg11)CCMSH will be sufficient to show therapeutic effects in human melanomaxenografted Scid mouse model. Whole body clearance of 188Re-(Arg11)CCMSH was very rapid, with approximately 82% of injected dose cleared through urinary system at 4 h postinjection. Greater than 98% of the injected dose was washed out of body by 24 h postinjection. There was very little activity in blood and major organs such as liver, lung, and muscle except for the kidneys. High tumor/blood and tumor/normal tissue uptake ratios were demonstrated as early as 1 h postinjection (Table 4). Although the majority of 188Re-(Arg11)CCMSH in the kidneys cleared rapidly, there was still 6.24 ( 1.14 %ID/g of activity present in the kidneys at 4 h after injection. Hence, nephrotoxicity would likely be a dose-limiting factor if 188Re-(Arg11)CCMSH was used for melanoma therapy trials. In comparison with 188Re(Arg11)CCMSH, 188Re-CCMSH exhibited similar tumor uptake and retention properties. However, the kidney uptake value of 188Re-CCMSH was two times higher than that of 188Re-(Arg11)CCMSH at 1 h and 4 h postinjection, which would compromise its application for melanoma therapy. The biodistribution data of 188Re-CCMSH demonstrated that whole body clearance of 188Re-CCMSH was rapid as well. Approximately 91% of injected dose was washed out of body through urinary system at 4 h postinjection. Greater than 99% of the injected dose was cleared out of body by 24 h postinjection. The accumulation of 188Re-CCMSH activity in blood and blood rich organs such liver and lung was similar to that of 188Re(Arg11)CCMSH. In contrast to 188Re-CCMSH, slightly

Radiolabeled R-MSH Peptide Analogues Table 3. Pharmacokinetics of Micea

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188Re-CCMSH

and

188Re-(Arg11)CCMSH

1h tissues

CCMSH

tumor brain blood heart lung liver spleen kidneys muscle pancreas

1.98 ( 0.26 0.02 ( 0.04 0.43 ( 0.05 0.35 ( 0.34 0.90 ( 0.08 0.48 ( 0.02 0.12 ( 0.14 19.92 ( 5.30 0.07 ( 0.08 0.18 ( 0.06

stomach intestines urine

0.28 ( 0.05 3.71 ( 0.49 83.24 ( 1.28

in TXM13 Human Melanoma-Xenografted Scid

4h Arg11CCMSH

24 h Arg11CCMSH

CCMSH

Percent Injected Dose/Gram 2.20 ( 0.24 2.02 ( 0.27 0.08 ( 0.09 0.02 ( 0.05 0.01 ( 0.00 0.04 ( 0.05 0.24 ( 0.17 0.20 ( 0.28 0.16 ( 0.22 0.21 ( 0.13 0.11 ( 0.04 0.37 ( 0.12c 0.06 ( 0.06 0.25 ( 0.19b 12.56 ( 0.89 6.24 ( 1.14d 0.04 ( 0.04 0.27 ( 0.22 0.13 ( 0.17 0.17 ( 0.10 Percent Injected Dose 0.46 ( 0.23 0.16 ( 0.14 0.52 ( 0.46 7.47 ( 1.36 3.51 ( 1.52 10.98 ( 2.21 74.30 ( 5.86 91.03 ( 2.30 82.51 ( 3.02 3.06 ( 0.68b 0.07 ( 0.04 0.84 ( 0.32b 0.74 ( 0.62 1.57 ( 0.65 0.83 ( 0.44 0.30 ( 0.34 9.70 ( 3.69b 0.16 ( 0.19 0.33 ( 0.24

CCMSH

Arg11CCMSH

0.87 ( 0.33 0.01 ( 0.01 0.04 ( 0.07 0.06 ( 0.09 0.06 ( 0.04 0.03 ( 0.01 0.07 ( 0.10 0.39 ( 0.12 0.02 ( 0.04 0.01 ( 0.01

0.93 ( 0.49 0.07 ( 0.08 0.09 ( 0.11 0.11 ( 0.18 0.03 ( 0.03 0.04 ( 0.02 0.09 ( 0.11 0.27 ( 0.06b 0.06 ( 0.02 0.04 ( 0.03b

0.02 ( 0.01 0.08 ( 0.03 99.51 ( 0.06

0.04 ( 0.03 0.19 ( 0.08 98.83 ( 0.54

a 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 Melanoma-Xenografted Scid Mice

188Re-CCMSH

1h

and

188Re-(Arg11)CCMSH

in TXM13 Human

4h

ratios

CCMSH

Arg11CCMSH

tumor/blood tumor/kidney tumor/lung tumor/liver tumor/muscle

4.60 0.10 2.20 4.13 28.29

3.64 0.32 1.95 3.69 19.13

24 h

CCMSH

Arg11CCMSH

CCMSH

Arg11CCMSH

220.00 0.18 13.75 20.00 55.00

50.50 0.32 9.62 5.46 7.48

21.75 2.23 14.50 29.00 43.50

10.33 3.44 31.00 23.25 15.50

more 188Re-(Arg11)CCMSH activity was excreted through GI system, indicating that the lipophilicity of 188Re(Arg11)CCMSH was higher than that of 188Re-CCMSH. This hypothesis was consistent with HPLC results, in which 188Re-(Arg11)CCMSH exhibited a greater retention time than 188Re-CCMSH under the same reverse-phase elution gradient (Figure 3). The tumor uptake of 188Re-CCMSH and 188Re-(Arg11)CCMSH were lower 1 h and 4 h postinjection in the TXM13 human melanoma-xenografted Scid mice than in the B16/F1 murine melanoma model (23). For example, the uptake of 188Re-CCMSH and 188Re-(Arg11)CCMSH in the B16/F1 murine melanoma mouse model was 9.78 ( 2.00 %ID/g and 16.37 ( 3.27 %ID/g at 4 h postinjection and 1.94 ( 0.47 %ID/g and 3.50 ( 2.32 %ID/g 24 h postinjection (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/g and 0.93 ( 0.49 %ID/g 24 h postinjection in the TXM13 human melanoma xenograft model. The significant tumor uptake value difference between B16/F1 murine melanoma and TXM13 human melanoma models were attributed to MC1 receptor density and the in vivo behavior differences between two cell lines. In this report, it was shown that the MC1 receptor density on human melanoma cells, including TXM13, was lower than that of B16/ F1 cells. Moreover, based on the histopathological results showed in our previous report, TXM13 cells formed amelanonic solid tumors with extensive necrotic centers as opposed to the nonnecrotic gelatinous composition of B16/F1 murine melanoma tumors (22). Differences in tumor morphology could significantly affect the tumor uptake of radioactivity on a per gram basis. Necrotic centers present in the TXM13 human melanoma tumors contribute to the total mass of the tumors but contain few viable melanoma cells capable of 188Re-CCMSH and 188Re-(Arg11)CCMSH uptake. Rhenium-188-CCMSH uptake is likely to be localized to rapidly proliferating melanoma cells located around the surface of the TXM13

human melanoma tumors (22). Therefore, the overall percent activity per gram of TXM13 human melanoma tumors is reduced when compared to B16/F1 murine melanoma tumors that lack significant necrotic regions and are almost entirely composed of viable melanoma cells capable of rhenium-188-CCMSH uptake. Numerous R-MSH analogues, based on the high affinity NDP sequence ([Nle4,D-Phe7]-R-MSH), have been synthesized and characterized as tumor targeting agents (18-20, 50). NDP radiolabeled with 125I or 18F exhibited high receptor affinities in vitro and rapid pharmacokinetics in vivo; however, they did not display high tumor uptake and retention (20, 50). Recently, Froidevaux et al. (51, 52) described the characterization of a novel 8 amino acid DOTA-conjugated MSH analogue, DOTAMSH(oct), that was based on the high affinity NDP MSH sequence. Indium-111-labeled DOTA- MSH(oct) displayed rapid pharmacokinetics and good tumor uptake, highlighting its melanoma imaging potential. Tumor uptake values 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 melanoma mouse model (52). Murine melanoma uptake of 111InDOTA-MSH(oct) was 2 times and 1.2 times greater than 188 Re-(Arg11)CCMSH in human melanoma at 4 and 24 h postinjection. This difference was expected since we routinely see a 5-10-fold reduction in xenografted human melanoma compared to B16/F1 murine melanoma tumors. No characterization of 111In-DOTA-MSH(oct) was reported for a human melanoma-xenografted model making direct biodistribution comparisons difficult. However, a comparison of 111In-DOTA-MSH(oct) with 111In-DOTAReCCMSH(Arg11) in the B16/F1 murine melanoma mouse model showed that 111In-DOTA-ReCCMSH(Arg11) displayed 4 times the tumor uptake 4 h and 7 times the uptake 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-DOTAMSH(oct) at 4 h and 24 h postinjection (49). Although 111In-

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DOTA-MSH(oct) exhibited high affinity for the MC1 receptor, it may be cleared too rapidly from the blood stream for optimal tumor uptake. Nonspecific kidney accumulation is a common problem associated with radiolabeled peptide and small protein administration (24-26). Cationic peptides and proteins bind to the negatively charged surface of tubule cells via electrostatic interaction when they are generally filtered in the glomerulus and reabsorbed in the cells of the proximal tubule (24). Therefore, two strategies of chemical modification of the molecule and basic amino acid coinfusion have been employed to decrease renal accumulation by masking the electrostatic interaction between molecule and the surface of tubular cells (2431). For instance, Kim and Kobayashi et al. reported the use of 2,3,5,6-tetrafluorophenyl (TFP)-glycolate to neutralize the positive charges and lower the isoelectric point of anti-Tac disulfide-bonded variable region single-chain Fv fragments (dsFv) and humanized anti-Tac Fab fragments. Their findings indicated that neutralizing the positive charges decreased the kidney accumulation of the glycolated conjugates without impairing their tumor uptake (32-35). Recently, Akizawa et al. investigated the effect of molecular charges on renal uptake of 111In-DTPA-conjugated peptide by substitution of the N-terminal D-phenylalanine of 111In-DTPA-D-Phe1-Octreotide with L-aspartic acid, L-lysine, L-methionine, and L-phenylalanine. They found that the net charges of 111 In-DTPA-D-Phe1-Octreotide derivatives significantly affected their kidney uptake. The strategy of increasing negative charges in peptide molecules might be used to decrease the renal uptake of 111In-DTPA-conjugated low molecular weight peptides (36). An analysis of biodistribution comparison of 188Re-CCMSH and 188Re-(Arg11)CCMSH in this work revealed that the charge distribution of peptide molecule might be another important factor, which affected the renal uptake of peptide. Theoretically, the positive charge of Arg is same as Lys; however, the positive charge distribution is different between the Arg guanidinium group and Lys primary amine. The biodistribution data demonstrated that the kidney uptake value of 188Re-(Arg11)CCMSH was approximately 50% of that of 188Re-CCMSH 1 h and 4 h postinjection. Moreover, coinfusion of positively charged amino acids reduced the kidney retention of 188ReCCMSH by more than 50% while only reducing that of 188Re-(Arg11)CCMSH by 40% in our previous report (23). This supports the assertion that difference in charge distribution on the side chain greatly influences nonspecific charge-charge interaction and could be responsible for reduced kidney retention of 188Re-(Arg11)CCMSH. Arg at 11th position of CCMSH peptide sequence played a critical role in decreasing the renal uptake value of 188Re-labeled CCMSH peptide analogues. The coinfusion of basic amino acid or amino acids combination, such as L-lysine, L-arginine, D-lysine, and combination of L-lysine and L-arginine, have been proved to be effective in decreasing the renal uptake of radiolabeled peptides and antibodies (24-31). It was also clearly demonstrated by our previous report that L-lysine coinjection could significantly decrease the renal uptake of 188Re-CCMSH and 188 Re-(Arg11)CCMSH in B16/F1 murine melanoma bearing C57 mice (23). Hence, the strategy of L-lysine coinfusion could potentially be employed to enhance the therapeutic efficacy of 188Re-(Arg11)CCMSH by further decreasing the nephrotoxicity in human melanoma therapy trials. In conclusion, all eight human melanoma cell lines tested in this study displayed the MC1 receptor. Bio-

Miao et al.

distribution results demonstrated that both 188Re(Arg11)CCMSH and188Re-CCMSH exhibited substantial tumor uptake and good retention, coupled with rapid whole body clearance in TXM13 human melanomaxenografted Scid mice model. Human melanoma-targeting properties of 188Re-(Arg11)CCMSH and188Re-CCMSH accentuated their potential as therapeutic agents for human 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. Sieckman and Dana G. Mazuru for their technical assistance. This work was supported by a grant (ER61661) from the Department of Energy (to T.P.Q.) and a grant from the University of Missouri Life Science Mission Enhancement Postdoctoral Fellowship (to Y.M.). LITERATURE CITED (1) Greenlee, R. T., Hill-Harmon, M. B., Murray, T., and Thun, M. (2001) Cancer statistics, 2001. CA Cancer J. Clin. 51, 1536. (2) Marghood, A. A., Slade, J., Salopek, T. G., Kopf, A. W., Bart, R. S., and Rigel, D. S. (1995) Basal cell and squamous cell carcinomas are important risk factors for cutaneous malignant melanoma. Cancer 75, 707-714. (3) Larson, S. M., Brown, J. P., Wright, P. W., Carrasquillo, J. A., Hellstorm, I., and Hellstorm, K. E. (1983) Imaging of melanoma with I-131-labeled monoclonal antibodies. J. Nucl. Med. 24, 123-129. (4) Wahl, R. L., Swanson, N. A., Johnson, J. W., Natale, R., Petry, N. A., Mallette, S., Kasina, S., Reno, J., Sullivan, K., and Abrams, P. (1992) Clincal experience with Tc-99m labeled (N2S2) anti-melanoma antibody fragments and single photon emission computed tomography. Am. J. Physiol. Imaging 7, 48-58. (5) Loeffler, K. U., Brautigam, P., Simon, J. C., Althauser, S. R., Wuttig, C., and Witschel, H. (1996) Immunoscintigraphy for ocular melanoma: a reliable diagnostic technique? Graefe’s Arch. Clin. Exp. Ophthalmol. 234, 100-104. (6) Halpern, S. E., and Bartholomew, R. (1995) Pharmacokinetics of an indium-111-labeled IgG monoclonal antibody over a prolonged period. Eur. J. Nucl. Med. 22, 1323-1325. (7) Carrasquillo, J. A., Abrams, P. G., Schroff, R. W., Reynolds, J. C., Woodhouse, C. S., Morgan, A. C., Keenan, A. M., Foon, K. A., Perentesis, P., and Marshall, S. (1988) Effect of antibody dose on the imaging and biodistribution of In-111 9.2.27 anti-melanoma monoclonal antibody. J. Nucl. Med. 29, 39-47. (8) Kwok, C. S., Cole, S. E., and Liao, S. K. (1988) Uptake kinetics of monoclonal antibodies by human melanoma multicell spheroids. Cancer Res. 48, 1856-1863. (9) Ong, G. L., and Mattes, M. J. (1989) Penetration and binding of antibodies in experimental human solid tumors grown in mice. Cancer Res. 49, 4264-4273. (10) Nicholl, C., Mohammed, A., Hull, W. E., Bubeck, B., and Eisenhut, M. (1997) Pharmacokinetics of iodine-123-IMBA for melanoma imaging. J. Nucl. Med. 38, 127-133. (11) Mohammed, A., Nicholl, C., Titsch, U., and Eisenhut, M. (1997) Radioiodinated N-(alkylaminoalkyl)-substituted 4-methoxy-, 4-hydroxy-, and 4-aminobenzamide: Biological investigations for the improvement of melanoma-imaging agents. Nucl. Med. Biol. 24, 373-380. (12) Eisenhut, M., Hull, W. E., Mohammed, A., Mier, W., Lay, D., Just, W., Gorgas, K., Lehmann, W. D., and Haberkorn, U. (2000) Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: Structure-affinity relationships, metabolic fate, and intracellular localization. J. Med. Chem. 43, 3913-3922. (13) Hruby, V. J., Sharma, S. D., Toth, K., Jaw, J. Y., Al-Obeidi, F., Sawyer, T. K., and Hadley, M. E. (1993) Design, synthesis,

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