Enhanced Binding and Inertness to Dehalogenation of α-Melanotropic

Mar 1, 1996 - Two peptides of potential utility for targeting melanoma cells, R-melanocyte-stimulating hormone. (R-MSH) and its more potent analogue [...
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Bioconjugate Chem. 1996, 7, 233−239

233

Enhanced Binding and Inertness to Dehalogenation of r-Melanotropic Peptides Labeled Using N-Succinimidyl 3-Iodobenzoate Pradeep K. Garg,† Kevin L. Alston, Philip C. Welsh, and Michael R. Zalutsky* Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710. Received July 19, 1995X

Two peptides of potential utility for targeting melanoma cells, R-melanocyte-stimulating hormone (R-MSH) and its more potent analogue [Nle4,D-Phe7]-R-MSH, were radioiodinated in 45-65% yield using N-succinimidyl 3-[125I]iodobenzoate (SIB). To determine whether this labeling method resulted in improved in vitro and in vivo characteristics, these peptides also were labeled with 131I by direct iodination with the iodogen method. For R-MSH, the rapid tissue clearance of both radionuclides in mice was consistent with rapid degradation of the peptide; however, significantly lower levels of 125I were observed in thyroid and stomach, reflecting a greater inertness to deiodination. More extensive comparisons were performed with [Nle4,D-Phe7]-R-MSH. The in vitro binding of [Nle4,D-Phe7,Lys11(125I)IBA]-R-MSH (prepared using SIB) to the murine B-16 melanoma cell line, 34.1 ( 4.7%, was more than twice as high as that for [Tyr2(131I),Nle4,D-Phe7]-R-MSH (15.0 ( 0.1%), and its KD was more than 10-fold lower than that for conventionally labeled peptide (10 ( 5 versus 140 ( 14 pM). The normal tissue clearance of [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH in mice was faster than that of [Tyr2(131I),Nle4,D-Phe7]-R-MSH. The 19-40-fold lower activity concentrations of [Nle4,D-Phe7,Lys11-(125I)IBA]R-MSH in tissues accumulating free iodide (thyroid and stomach) suggest a greater inertness of this peptide to deiodination. The primary urinary catabolite of [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH was the lysine conjugate of iodobenzoic acid, whereas radioiodide was the chief catabolite generated from [Tyr2(131I),Nle4,D-Phe7]-R-MSH. We conclude that further evaluation of [Nle4,D-Phe7,Lys11-(125I)IBA]R-MSH for targeting R-MSH receptors is warranted and that SIB may be a useful method for the radioiodination of peptides.

INTRODUCTION

Radiolabeled peptides, including those radioiodinated directly on constituent tyrosine residues, have been valuable tools for in vitro evaluation of receptor status and other biochemical processes. Recent investigations have extended this approach to determine whether peptides could be exploited for the selective delivery of radionuclides to specific cell populations. Examples of this strategy include the evaluation of chemotactic peptides for imaging infection (Babich et al., 1993a,b) and octreotide analogues (Bakker et al., 1991; Krenning et al., 1992) for the delineation of somatostatin receptor positive tumors. In vivo applications such as these provide additional constraints for peptide labeling; the labeling method should yield a stable bond between the radionuclide and the peptide and result in the generation of labeled catabolites which are excreted rapidly from normal tissues. Iodine radionuclides are attractive for in vivo applications (Larson and Carrasquillo, 1988) because their wide range of decay properties is well-suited for single photon emission tomography (125I), positron emission tomographic imaging (124I), β-particle therapy (131I), and Auger/conversion electron therapy (123I and 125I). Unfortunately, direct radioiodination of tyrosine residues on peptides via electrophilic substitution often yields a * Address correspondence to Michael R. Zalutsky, Ph.D., Department of Radiology, Box 3808, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-7708. Fax: (919) 684-7121. † Present address: Yale University/VA PET Center, 950 Campbell Avenue, West Haven, CT 06516. X Abstract published in Advance ACS Abstracts, March 1, 1996.

1043-1802/96/2907-0233$12.00/0

molecule which is susceptible to deiodination in vivo (Bakker et al., 1990, 1991). In addition, the oxidants used in the labeling reaction can alter amino acids such as methionine and, as a result, sometimes have a deleterious effect on the affinity of labeled peptide binding to receptor (Eberle et al., 1991). Previous work from our laboratory (Zalutsky and Narula, 1987, 1988; Zalutsky et al., 1989a; Schuster et al., 1991) and other groups (Wilbur et al., 1989; Badger et al., 1990) has demonstrated that use of N-succinimidyl 3- or 4-iodobenzoate (SIB) for labeling proteins significantly reduced thyroid uptake (an in vivo indicator of dehalogenation) in mice compared with proteins labeled by direct iodination methods. In some cases, monoclonal antibodies (MAbs) labeled using SIB also exhibited better in vitro binding characteristics, higher accumulation in human tumor xenografts, and enhanced therapeutic efficacy (Zalutsky and Narula, 1988; Zalutsky et al., 1989a; Schuster et al., 1991). The present study was undertaken to determine the potential utility of SIB for the radioiodination of peptides. These investigations were performed with the melanotropic peptide R-melanocyte-stimulating hormone (RMSH) (Eberle, 1988) and its more stable analogue [Nle4,D-Phe7]-R-MSH (Sawyer et al., 1980). The amino acid sequences of these peptides are shown in Figure 1. Because of the presence of R-MSH receptors on human melanoma cell lines (Seigrist et al., 1989; Solca et al., 1989) and biopsy tissue (Ghanem et al., 1989; Tatro et al., 1990), R-MSH and its analogues are of interest for the development of specific diagnostic and therapeutic approaches for melanoma. Also relevant to the current study are the difficulties noted in the literature in production of a radioiodinated R-MSH analogue with © 1996 American Chemical Society

234 Bioconjugate Chem., Vol. 7, No. 2, 1996

Figure 1. Amino acid sequence for R-MSH and its [Nle4,DPhe7]-R-MSH analogue. Radioiodination is assumed to occur at Tyr2 with the iodogen method and at Lys11 with SIB reagent.

suitable properties for routine use in receptor assays (Heward et al., 1979; Eberle et al., 1991). In the present study, R-MSH and [Nle4,D-Phe7]-R-MSH were radioiodinated using SIB and the iodogen method. The labeled peptides were evaluated with regard to binding to B-16 murine melanoma cells, tissue distribution in normal mice, and urinary catabolites associated with the two labeling methods. Our results suggest that the SIB reagent offers distinct advantages for the radioiodination of these peptides. EXPERIMENTAL PROCEDURES

Materials. Sodium [131I]iodide (1200 Ci/mmol) and sodium [125I]iodide (no-carrier-added; 2200 Ci/mmol calculated specific activity) in pH 7-11 NaOH were obtained from DuPont-New England Nuclear. Iodogen was purchased from Pierce Chemical Co. The peptides R-MSH and [Nle4,D-Phe7]-R-MSH were obtained from Bachem California (Torrence, CA). The lysine and glycine conjugates of 3-iodobenzoic acid (IBA) were synthesized using previously described procedures (Garg et al., 1995). All other chemicals were purchased from Aldrich Chemical Co. Radioiodination of Peptides Using Iodogen. A slight variation of the original iodogen method was used for the radioiodination of R-MSH (Fraker and Speck, 1978). A solution of R-MSH (100 µg, 5 mg/mL in phosphate buffer, pH 7.4) was added to sodium [131I]iodide in a glass vial coated with 10 µg of iodogen. After a 3 min incubation at room temperature, the radioiodinated peptide was isolated by performing high-performance liquid chromatography (HPLC) using a reversephase column (Alltech adsorbosphere C-18 10 µm) eluted at a flow rate of 1.2 mL/min using a 5 to 60% linear gradient of solvent A [70% acetonitrile in water and 0.1% trifluoroacetic acid (TFA)] in solvent B (0.1% TFA in water) over 45 min. Using this system, R-MSH eluted at 35.0 min and the radioiodinated peptide, [Tyr2(131I)]R-MSH, eluted at 38.2 min. [Nle4,D-Phe7]-R-MSH was radioiodinated in 75-85% radiochemical yield following the method described above and was isolated using the same reverse-phase HPLC system. Under these conditions, [Nle4,D-Phe7]-R-MSH eluted at 36.6 min and the radioiodinated peptide [Tyr2(131I),Nle4,D-Phe7]-R-MSH eluted at 39.4 min. Synthesis of [Nle4,D-Phe7,Lys11-IBA]-r-MSH. This compound was synthesized to serve as a reference standard in the HPLC catabolite analysis studies and to aid in the confirmation of the lysine residue as the site of IBA conjugation. SIB was synthesized by reaction of 3-iodobenzoic acid with N-hydroxysuccinimide and dicyclohexylcarbodiimide as described in a prior publication (Garg et al., 1995). To SIB (0.19 mg) dissolved in 15 µL of dimethylformamide (DMF) were added 2 µL of triethylamine and [Nle4,D-Phe7]-R-MSH (0.8 mg in 160 µL of

Garg et al.

DMF). The reaction mixture was incubated at room temperature and was monitored for the disappearance of SIB by thin layer chromatography on a silica plate with hexane/ethyl acetate/acetic acid (30/70/0.12) as the solvent. The desired product was isolated from the reaction mixture by HPLC with a reverse-phase C18 column (Alltech adsorbosphere C-18 10 µm) eluted at a flow rate of 1.2 mL/min with a 5 to 60% linear gradient of solvent A [acetonitrile/water (70/30) containing 0.1% TFA] in solvent B (0.1% TFA in water) over 45 min. Retention times for IBA, SIB, and [Nle4,D-Phe7,Lys11-IBA]-R-MSH were 38.1, 43.6, and 48.5 min, respectively. Mass spectra were obtained on a Finigan MAT 700 TSQ machine (Oneida Research Services, Whitsboro, NY) to confirm the molecular weight for the desired peak: MS m/z (electron spray, direct infuse) 1894 (16%, M+), 1878 (47%, M+ - 17). Radioiodination of Peptides Using SIB. The radioiodination agent [125I]SIB was prepared according to a previously reported method (Garg et al., 1989). Briefly, 10 µL of 5% acetic acid, 20 µL of tert-butylhydroperoxide, and 5 µmol of N-succinimidyl 3-(tri-n-butylstannyl)benzoate were added to 100-500 µCi of sodium [125I]iodide. After a 10 min reaction at room temperature, [125I]SIB was isolated by HPLC using a silica column (Alltech adsorbosphere-10 10 µm, 250 × 4.6 mm) eluted with ethyl acetate/hexane/AcOH (30/70/0.12). After solvent evaporation, R-MSH (100 µg, 5 mg/mL in DMF) and triethylamine (2 µL) were added to the [125I]SIB residue and the reaction mixture was incubated for 15-90 min at room temperature or at 37 °C. After incubation, [Lys11-(125I)IBA]-R-MSH was isolated in 45-65% radiochemical yield using the same reverse-phase HPLC system described for the iodogen procedure. Unlabeled R-MSH eluted at 35.0 min, and [Lys11-(125I)IBA]-R-MSH eluted at 46.2 min. Using the same procedure, [Nle4,DPhe7,Lys11-(125I)IBA]-R-MSH was labeled in 45-61% radiochemical yield. The retention time for [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH was 48.5 min, identical to that seen for the cold compound. In Vitro Binding of Radioiodinated Peptides to B16-F1 Melanoma Cells. The binding of the two radioiodinated [Nle4,D-Phe7]-R-MSH analogues to the murine melanoma line B16-F1 (ATCC, Rockville, MD) was determined in vitro. Cells were grown in 150 cm2 tissue culture flasks at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in modified essential media (MEM) with Earle’s salts supplemented with 10% heatinactivated fetal calf serum, 2 mM L-glutamine, 1% MEM nonessential amino acids, 1.5% MEM vitamin solution, 50 units/mL penicillin, and 50 µg/mL streptomycin. Competitive binding experiments were performed using assay conditions described by Siegrist et al. (1989). Briefly, the binding media consisted of MEM supplemented with 25 mM HEPES, 0.2% bovine serum albumin, and 1.0 mM 1,10-phenanthroline. Cells (2 × 106 per tube) were incubated in Eppendorf tubes with approximately 15 000 cpm of [Tyr2(131I),Nle4,D-Phe7]-R-MSH or [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH. Binding in the presence of 10-6 M peptide was considered to be nonspecific. Incubations were performed in triplicate in the presence or absence of varying concentrations (10-6 to 10-13 M) of [Nle4,D-Phe7]-R-MSH. After a 3 h incubation at 15 °C, media was removed and the cells were washed two times with pH 7.4 phosphate-buffered saline. The cell pellets along with input standards were counted for 125 I and 131I activity using an automated γ counter. Binding data were analyzed using the RADLIG computer program developed by G. A. McPherson (Biosoft, Cambridge, U.K.).

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Radioiodination of R-Melanotropic Peptides

Biodistribution Studies. Two paired-label experiments were performed in male Balb/c mice weighing 2025 g. In the first, animals were injected intravenously with 4 µCi each of [Tyr2(131I)]-R-MSH and [Lys11-(125I)IBA]-R-MSH; in the second, animals received 4 µCi each of [Tyr2(131I),Nle4,D-Phe7]-R-MSH and [Nle4,D-Phe7,Lys11(125I)IBA]-R-MSH. The radiochemical purity of all preparations used in the biodistribution studies, determined by HPLC, was greater than 98%. Groups of five animals were killed at 0.5, 1, 2, and 4 h postinjection. Tissues of interest were removed, washed with saline, and counted for radioactivity in an automated γ counter. A correction was applied for the presence of 131I in the 125I counting window. After comparison of the tissue activities to injection standards of appropriate count rate, the results were expressed as percent injected dose per gram of tissue (%ID/g) or percent injected dose per organ (%ID). Urine samples were obtained from mice killed 1 h after injection of [Tyr2(131I),Nle4,D-Phe7]-R-MSH and [Nle4,DPhe7,Lys11-(125I)IBA]-R-MSH, combined, and passed through a 0.45 µm filter. The HPLC system consisted of a reverse-phase column (Alltech adsorbosphere C-18 10 µm, 250 × 4.6 mm) eluted with MeOH/H2O/AcOH (45/ 55/0.2) at a flow rate of 1 mL/min. Iodotyrosine and IBA, as well as the IBA conjugates of lysine and glycine, also were analyzed on this system to aid in the identification of these potential catabolites.

Figure 2. Competitive binding of radioiodinated [Nle4,D-Phe7]R-MSH derivatives to the murine B16-F1 melanoma cell line. Binding of [Tyr2(131I),Nle4,D-Phe7]-R-MSH and [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH measured in the presence of varying concentrations of [Nle4,D-Phe7]-R-MSH. Table 1. Paired-Label Tissue Distribution of Radioiodine in Normal Mice Following Injection of [Tyr2(131I)]-r-MSH and [Lys11-(125I)IBA]-r-MSH

RESULTS

Initially, purification of labeled peptides was attempted by Sephadex LH-20 chromatography as described by Eberle and Hu¨bscher (1979). However, when an aliquot from the purified product was analyzed by HPLC, the presence of unlabeled peptide was observed on the UV trace. Reverse-phase HPLC was then adopted as the purification method and permitted good separation of all radioiodinated peptides from unlabeled peptide; no cold peak was observed on the UV trace along with the radioiodinated product, making these essentially nocarrier-added preparations. Using iodogen, yields for the radioiodination of R-MSH and [Nle4,D-Phe7]-R-MSH generally were greater than 75% with an estimated specific activity of about 1200 Ci/mmol. The efficiency for the coupling of [125I]SIB to R-MSH and [Nle4,D-Phe7]-R-MSH was similar and ranged between 45 and 61% for 15 min to 2 h reaction periods. Although there appeared to be a trend for higher conjugation efficiencies at longer reaction times, these differences were not statistically significant. Running the reaction at 37 °C did not improve labeling yield significantly. A specific activity greater than 1500 Ci/mmol was calculated for peptides labeled with [125I]SIB on the basis of the UV detection limit of our HPLC system. In vitro binding of radioiodinated [Nle4,D-Phe7]-R-MSH analogues was investigated using the B16-F1 murine melanoma line. Specific binding of [Nle4,D-Phe7,Lys11(125I)IBA]-R-MSH, 34.1 ( 4.7%, was more than twice as high as that for [Tyr2(131I),Nle4,D-Phe7]-R-MSH (15.0 ( 1.0%). The specific/nonspecific binding ratio for [Nle4,DPhe7,Lys11-(125I)IBA]-R-MSH and [Tyr2(131I),Nle4,D-Phe7]R-MSH was 26/1 and 5/1, respectively. The apparent equilibrium dissociation constants were calculated for these labeled ligands using [Nle4,D-Phe7]-R-MSH as a competitor (Figure 2). The KD for [Tyr2(131I),Nle4,D-Phe7]R-MSH was 140 ( 14 pM compared with a KD of 10 ( 5 pM for [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH. A tissue distribution study was performed with [Tyr2(131I)]-R-MSH and [Lys11-(125I)IBA]-R-MSH to determine whether SIB improved peptide stability. As summarized in Table 1, the tissue clearance of radioiodine was quite

percent injected dose per gram of tissuea 1h

4h

tissue

Lys11-(IBA)

Tyr2(I)

Lys11-(IBA)

Tyr2(I)

liver spleen lungs heart kidney small intestine large intestine muscle blood brain

0.15 ( 0.06 0.04 ( 0.02 0.18 ( 0.13 0.07 ( 0.04 0.33 ( 0.14 0.34 ( 0.15

1.44 ( 0.66 1.41 ( 0.46 2.29 ( 0.76 1.29 ( 0.56 3.30 ( 0.77 2.14 ( 1.17

0.02 ( 0.01 0.02 ( 0.01 0.04 ( 0.01 0.03 ( 0.02 0.09 ( 0.03 0.02 ( 0.01

0.11 ( 0.05 0.09 ( 0.04 0.21 ( 0.08 0.08 ( 0.04 0.79 ( 0.07 0.16 ( 0.03

a

0.29 ( 0.15 1.19 ( 0.53 0.13 ( 0.04 0.23 ( 0.04 0.10 ( 0.04 0.61 ( 0.31 0.03 ( 0.02 0.04 ( 0.02 0.14 ( 0.09 3.01 ( 1.16 0.05 ( 0.02 0.17 ( 0.08 0.02 ( 0.01 0.20 ( 0.08 0.01 ( 0.00 0.01 ( 0.00

Mean ( standard deviation for five animals.

rapid for both labeling methods. At 1 h after injection, the 131I percent injected dose per gram of tissue activity concentrations were significantly higher (P < 0.01) than those of 125I. Differences were particularly apparent in the thyroid and stomach, two tissues known to accumulate radioiodide (Figure 3). For example, thyroid uptake for [Tyr2(131I)]-R-MSH 1 h after injection was 3.26 ( 0.70 %ID compared with 0.02 ( 0.01 %ID for [Lys11(125I)IBA]-R-MSH; similar differences were seen in stomach (1 h; iodogen, 4.50 ( 0.90 %ID; SIB, 0.06 ( 0.02 %ID). A more extensive paired-label experiment compared the tissue distributions of radioiodine following the injection of [Tyr2(131I),Nle4,D-Phe7]-R-MSH and [Nle4,DPhe7,Lys11-(125I)IBA]-R-MSH. The tissue distribution of [Tyr2(131I)Nle4,D-Phe7]-R-MSH (Table 2) was similar to that observed for [Tyr2(131I)]-R-MSH (Table 1), while retention of activity in most normal tissues was higher for [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH (Table 2) compared with that of [Lys11-(125I)IBA]-R-MSH (Table 1). When the biodistribution data for [Nle4,D-Phe7]-R-MSH labeled using SIB and iodogen were compared (Table 2), no clear trend was seen at 0.5 h with regard to normal tissue retention. As time progressed, increased tissue activity concentrations generally were observed for [Tyr2(131I),Nle4,D-Phe7]-R-MSH. Again, 26-40 times lower

236 Bioconjugate Chem., Vol. 7, No. 2, 1996

Figure 3. Percent of injected dose of radioiodine localized in the thyroid and stomach following the injection of radioiodinated R-MSH and [Nle4,D-Phe7]-R-MSH derivatives labeled using the iodogen (cross-hatched bars) and SIB methods (open bars).

thyroid uptake and 19-25 times lower stomach accumulation were observed for [Nle4,D-Phe7,Lys-(125I)IBA]-R-MSH. HPLC analyses were performed on the 1 h urine samples obtained from mice injected with the radioiodinated [Nle4,D-Phe7]-R-MSH analogues. Using the peptide HPLC purification column, there was no evidence for excretion of intact [Tyr2(131I),Nle4,D-Phe7]-R-MSH, while about 10% of 125I was excreted as intact [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH. The reverse-phase HPLC system was used for the analysis of lower molecular weight species. As shown in Figure 4, with [Tyr2(131I),Nle4,DPhe7]-R-MSH, >95% of the 131I activity eluted with a retention time corresponding to radioiodide, while with [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH, the principal labeled species had an elution time corresponding to the IBA-lysine conjugate. In addition, radioiodide (9%), IBA (3%), and the IBA-glycine conjugate (8%) also were observed. DISCUSSION

A wealth of data is available concerning the in vitro binding specificity and mechanisms of interaction of radioiodinated peptides to tumor-associated receptors. Extension of promising peptides to in vivo applications using iodine radionuclides is attractive because it minimizes the need for performing extensive validation studies with chemically dissimilar radiolabels. An additional feature of peptide radioiodination is that methodology often can be adapted for use with other radiohalogens such as 2 h 18F and 7.2 h 211At, which are of interest for positron emission tomographic imaging and R-particle radiotherapy, respectively. For example, SIB analogues

Garg et al.

have been used to label MAbs with 18F (Vaidyanathan et al., 1992) and 211At (Zalutsky et al., 1989b). Because receptors for R-MSH have been identified on the surface of human melanoma cells and tissue biopsies (Seigrist et al., 1989; Solca et al., 1989; Ghanem et al., 1989; Tatro et al., 1990), these peptides are of practical interest for tumor targeting. Moreover, the less than optimal in vitro and in vivo properties of its radioiodinated analogues make R-MSH a useful paradigm for the development of better peptide radioiodination strategies. Radioiodination of R-MSH using electrophilic iodination has been investigated using a number of procedures (Heward et al., 1979; Eberle and Hu¨bscher, 1979; Ghanem et al., 1982; Eberle, 1988). Unfortunately, the methionine residue at position 4 is easily oxidized so these labeling methods yield a labeled peptide with substantially reduced receptor affinity. In addition, the tissue distribution in mice of R-MSH labeled with 125I using chloramine-T reflects significant degradation and/ or deiodination of the labeled peptide in vivo (Ghanem et al., 1991). Alternative radioiodination methods and/ or analogues of R-MSH are clearly needed to provide a more suitable labeled melanotropic peptide for in vitro and in vivo applications. With regard to radioiodination methodology, SIB has been demonstrated to decrease MAb deiodination and increase immunoreactivity compared with MAb labeled using iodogen or chloramine-T (Zalutsky and Narula, 1988; Zalutsky et al., 1989a; Wilbur et al., 1989). An advantage of the SIB method is that it avoids exposure of the biomolecule to oxidizing or reducing agents. In addition, the iodination site which is created is structurally dissimilar to iodotyrosine, a compound which is susceptible to the action of multiple endogenous deiodinases. The procedure developed for peptide radioiodination with SIB was modified slightly from that used for MAbs. With MAbs, the coupling reaction was performed in pH 8.5 borate buffer in order to minimize protein denaturation even though higher pH results in greater conjugation yields (Zalutsky and Narula, 1987). In the current study, peptides were dissolved in DMF in the presence of triethylamine, conditions which should increase coupling yield due to the availability of a greater proportion of lysine -amino groups for reaction. In addition, the use of nonaqueous media should minimize competitive hydrolysis of the SIB active ester. The conjugation yields for SIB to these peptides (45-61%) were comparable to those obtained with MAbs at similar protein concentrations and reaction times (Zalutsky and Narula, 1988; Zalutsky et al., 1989). Increasing peptide concentration should permit higher R-MSH coupling efficiencies since we have used this strategy to label a chemotactic peptide with the analogous N-succinimidyl 4-[18F]fluorobenzoate in greater than 80% yield (Vaidyanathan et al., 1995). In vitro studies have documented that R-MSH can be degraded under physiological conditions and in the presence of melanoma cell membranes (Deschodt-Lankman et al., 1990), a characteristic which probably prohibits the use of R-MSH for in vivo applications. Nonetheless, we performed a paired-label tissue distribution study to confirm this behavior and to determine whether deiodination was a contributory factor. In a previous study with an 123I-labeled somatostatin analogue, high thyroid uptake was noted, indicating that extensive dehalogenation had occurred (Bakker et al., 1990). An important parameter for the evaluation of the potential utility of a radiolabeling method is the extent to which it minimizes the retention of radioactivity in normal tissues. Previously, we have demonstrated that,

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Radioiodination of R-Melanotropic Peptides

Table 2. Paired-Label Tissue Distribution of Radioiodine in Normal Mice Following Injection of [Tyr2(131I),Nle4,D-Phe7]-r-MSH and [Nle4,D-Phe7,Lys11-(125I)IBA]-r-MSH percent injected dose per gram of tissuea 0.5 h

1h

2h

4h

tissue

Lys11-(IBA)

Tyr2(I)

Lys11-(IBA)

Tyr2(I)

Lys11-(IBA)

Tyr2(I)

Lys11-(IBA)

Tyr2(I)

liver spleen lung heart kidney small intestine large intestine muscle blood brain

2.56 ( 0.84 0.50 ( 0.14 1.40 ( 0.56 0.36 ( 0.10 5.18 ( 1.42 5.43 ( 1.43 0.24 ( 0.06 0.21 ( 0.08 0.58 ( 0.17 0.05 ( 0.02

1.25 ( 0.36 1.39 ( 0.32 2.48 ( 0.80 1.46 ( 0.37 2.99 ( 0.64 2.34 ( 0.35 1.10 ( 0.26 2.36 ( 0.43 2.68 ( 0.68 0.20 ( 0.06

1.11 ( 0.25 0.32 ( 0.03 0.63 ( 0.28 0.13 ( 0.01 1.77 ( 0.37 4.23 ( 1.29 1.94 ( 0.84 0.09 ( 0.03 0.18 ( 0.05 0.03 ( 0.02

1.04 ( 0.41 1.05 ( 0.30 1.99 ( 0.83 1.03 ( 0.25 2.01 ( 0.52 1.79 ( 0.59 1.02 ( 0.24 1.88 ( 0.28 2.22 ( 0.79 0.16 ( 0.02

0.47 ( 0.26 0.20 ( 0.06 0.46 ( 0.21 0.06 ( 0.02 0.80 ( 0.16 0.55 ( 0.14 1.96 ( 0.57 0.03 ( 0.04 0.07 ( 0.03 0.03 ( 0.02

0.37 ( 0.05 0.49 ( 0.14 0.75 ( 0.12 0.47 ( 0.20 0.91 ( 0.08 0.63 ( 0.15 0.65 ( 0.18 1.24 ( 0.19 0.66 ( 0.13 0.46 ( 0.38

0.22 ( 0.03 0.17 ( 0.04 0.39 ( 0.09 0.03 ( 0.01 0.27 ( 0.07 0.16 ( 0.05 0.63 ( 0.32 0.03 ( 0.01 0.02 ( 0.01 0.01 ( 0.01

0.28 ( 0.08 0.35 ( 0.10 0.57 ( 0.10 0.36 ( 0.10 0.65 ( 0.11 0.54 ( 0.19 0.52 ( 0.20 0.76 ( 0.08 0.60 ( 0.15 0.10 ( 0.08

a

Mean ( standard deviation for five animals.

Figure 4. Reverse-phase HPLC chromatogram of a urine sample obtained from normal mice 1 h after coinjection of [Tyr2(131I),Nle4,D-Phe7]-R-MSH and [Nle4,D-Phe7,Lys11-(125I)IBA]-RMSH.

compared with the Iodogen method, utilization of SIB for the radioiodination of an intact MAb (Zalutsky et al., 1989a) and a F(ab′)2 fragment (Zalutsky and Narula, 1988) resulted in significant reduction in activity levels in most normal tissues. Paired-label tissue distribution experiments were performed to determine whether SIB labeling would confer a similar advantage for the labeling of R-MSH and its more metabolically inert [Nle4,D-Phe7]R-MSH analogue. The clearance of radioiodine activity from normal tissues following the injection of both [Tyr2(131I)]-R-MSH and [Lys11-(125I)IBA]-R-MSH was quite rapid and qualitatively similar to the results reported previously for R-MSH labeled with chloramine-T (Ghanem et al., 1991). The lower levels of radioiodine in the thyroid and stomach for peptide labeled using SIB suggest that this labeling method reduced deiodination significantly. Activity levels in other normal tissues from [Tyr2(131I)]-RMSH and [Lys11-(125I)IBA]-R-MSH were quite similar to those measured previously for iodide and IBA, respectively (Zalutsky and Narula, 1988). Thus, even though SIB appeared to reduce peptide deiodination, rapid degradation of labeled R-MSH was the predominant determinant, rendering this peptide of minimal interest for in vivo applications.

For this reason, subsequent studies focused on the evaluation of radioiodinated [Nle4,D-Phe7]-R-MSH. In this R-MSH analogue, norleucine is substituted for methionine at position 4 to avoid susceptibility to oxidative degradation and a D-phenylalanine is substituted for its L-isomer to increase resistance to proteolytic degradation (Sawyer et al., 1980). The affinity of [Nle4,D-Phe7]-RMSH for the R-MSH receptor has been reported to be 3-5 times higher than that of R-MSH itself (Siegrist et al., 1989; Chhajlani and Wilkberg, 1992). Using the murine B16 melanoma cell line and [Nle4,D-Phe7]-R-MSH as a competitor, a KD of 0.32 ( 0.13 nM for [Tyr2(125I),Nle4,DPhe7]-R-MSH has been measured (Seigrist et al., 1988). The binding characteristics determined in the current study for [Tyr2(131I),Nle4,D-Phe7]-R-MSH compare favorably with those reported in the literature for this tracer. Using [Nle4,D-Phe7]-R-MSH as a competitor, a KD ) 0.140 ( 0.014 nM was determined for [Tyr2(131I),Nle4,D-Phe7]R-MSH binding to B16-F1 murine melanoma cells. The maximum specific binding in the absence of carrier and the nonspecific binding percentage measured in the current study also were slightly more favorable than those determined previously for [Tyr2(131I),Nle4,D-Phe7]R-MSH prepared using chloramine-T (Siegrist and Eberle, 1993; Eberle et al., 1991; Tatro and Reichlin, 1987; Eberle et al., 1993). These results are consistent with those obtained with other proteins and peptides which show that radioiodination using iodogen can provide greater retention of biological activity than that using chloramine-T (Fraker and Speck, 1978). The apparent KD measured for [Nle4,D-Phe7,Lys11(125I)IBA]-R-MSH was 10 ( 5 pM, a value lower than that reported previously for radioiodinated R-MSH analogues. In addition, the maximal specific binding of this tracer to B16 melanoma cells was high, 34.1 ( 4.7%, with a low degree of nonspecific binding. The latter property is important because addition of IBA increased peptide lipophilicity as reflected by its longer retention time on reverse-phase HPLC. The excellent in vitro binding properties of [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH to melanoma cells may in part reflect a greater inertness of this compound to dehalogenation. This is consistent with the observation that after only a 10 min incubation in vitro the fraction of intact [Tyr2(125I),Nle4,D-Phe7]-R-MSH remaining had declined to 92.0 ( 1.0% (Tatro and Reichlin, 1987). Another possibility is the greater proximity of Lys11 compared with Tyr2 to the amino acid residues considered to be involved in receptor binding (Eberle, 1988); as proteolytic breakdown proceeds, a label on Lys11 might be more likely to remain associated with a peptide fragment retaining receptor binding capabilities. Because of their potential for the diagnosis and treatment of melanomas, the two radioiodinated [Nle4,D-Phe7]-

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R-MSH derivatives were compared in a paired-label tissue distribution experiment. [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH exhibited significantly lower activity levels compared with [Tyr2(131I),Nle4,D-Phe7]-R-MSH in thyroid and stomach, two tissues known to accumulate free radioiodide. The biodistribution pattern for [Tyr2(131I),Nle4,D-Phe7]-R-MSH was similar to that seen for [Tyr2(131I)]-R-MSH, suggesting that the enhanced stability of the [Nle4,D-Phe7]-R-MSH structure could not be exploited due to the predominating role of deiodination in determining the catabolism of the label. Indeed, more than 95% of the radioactivity excreted in the urine was identified as radioiodide with no evidence for monoiodotyrosine. Although absolute percent injected dose per gram of tissue values were not included, Tatro and Reichlin (1987) reported extensive deiodination of [Tyr2(125I),Nle4,D-Phe7]-R-MSH 10 min after injection. It thus appears that, to be able to exploit the potential advantages of [Nle4,D-Phe7]-R-MSH for in vivo applications, it would be preferable to utilize a labeling method which reduces deiodination. Minimization of retention of radioactivity in normal tissues is another consideration in the selection of a method for labeling peptides since the low molecular weight of these carrier molecules should result in low normal tissue uptake levels. This has not always been the case with 111In-labeled R-MSH analogues where significant activity levels have been observed in liver and kidneys (Bard et al., 1990; Bagutti et al., 1994). In the current study, rapid normal tissue clearance was seen for [Nle4,D-Phe7,Lys11-(125I)IBA]-RMSH, with liver and kidney retaining less than 0.3% ID/g at 4 h. An investigation of the labeled species present in the urine after administration of [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH revealed that Lys-IBA was the primary labeled catabolite. This observation suggests that labeling had occurred at Lys11 on the peptide. Lys-IBA also was determined to be the major catabolite for a MAb F(ab′)2 fragment labeled using SIB (Garg et al., 1995), and Lys-drug and Lys-chelate conjugates have been reported as major catabolites of lysine-modified proteins (Franssen et al., 1992; Duncan and Welch, 1993). Gly-IBA is an additional catabolite which has been observed with MAb fragments labeled using SIB (Garg et al., 1995), and a small amount of Gly-IBA also was seen in the urine when the peptide was labeled by this method. We have previously reported that IBA reacts in vivo with endogenous glycine to form Gly-IBA conjugates (Garg et al., 1989b), an observation consistent with the well-known ability of glycine to form conjugates with benzoic acids (Gatley and Scheratt, 1976). Thus, the observation of low levels of Gly-IBA in the urine of mice injected with [Nle4,D-Phe7,Lys11-(125I)IBA]-R-MSH most likely reflects the cleavage of IBA from the labeled peptide itself or from lower-molecular weight IBAconjugate intermediate catabolites. In summary, [Nle4,D-Phe7,Lys11-IBA]-R-MSH appears to be a promising reagent for R-MSH receptors because of its high affinity and low nonspecific binding and for in vivo applications because of its inertness to deiodination. When labeled with 123I, this tracer might be valuable for the localization of melanoma metastases. Experiments to evaluate the pharmacokinetics of [Nle4,DPhe7,Lys11-(125I)IBA]-R-MSH in a murine melanoma model are planned. In addition, the investigation of the applicability of the SIB reagent for the radioiodination of other peptides appears to be warranted.

Garg et al. ACKNOWLEDGMENT

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