Bioconjugate Chem. 2002, 13, 599−604
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A 99mTc(I)-Postlabeled High Affinity Bombesin Analogue as a Potential Tumor Imaging Agent R. La Bella,†,‡ E. Garcia-Garayoa,*,† M. Ba¨hler,‡ P. Bla¨uenstein,† R. Schibli,† P. Conrath,§ D. Tourwe´,§ and P. A. Schubiger†,‡ Center for Radiopharmaceutical Science, Paul Scherrer Institute, CH-5232 Villigen, Switzerland, Department of Applied Biosciences, Swiss Federal Institute of Technology, CH-8057 Zurich, Switzerland, and Department of Organic Chemistry, Vrije Universiteit Brussels, B-1050 Brussels, Belgium. Received October 26, 2001; Revised Manuscript Received March 22, 2002
The overexpression of neuropeptide receptors observed in many cancers provides an attractive target for tumor imaging and therapy. Bombesin is a peptide exhibiting a high affinity for the gastrin releasing peptide (GRP) receptor, which is overexpressed by a variety of tumors such as breast or prostate cancer. In the present study, we have evaluated if the bombesin analogue [NR-histidinyl acetate]bombesin(7-14), radiolabeled with the novel [99mTc(OH2)3(CO)3]+, has the potential to be used as a diagnostic radiopharmaceutical. Receptor saturation studies, carried out on the GRP receptorexpressing PC-3 human prostate cancer cell line, revealed for [99mTc(CO)3-NR-histidinyl acetate]bombesin(7-14) Kd values in the subnanomolar range. Competitive binding assays, using the cold rhenium(I)-labeled analogue as a surrogate for the 99mTc-conjugate, also showed high affinity binding. Incubation of the radioconjugate with PC-3 cells resulted in a rapid temperature- and time-dependent specific internalization. At 37 °C more than 70% was internalized within the first 15 min and remained constant up to 2 h. Despite the weak proteolytic stability of [99mTc(CO)3-NR-histidinyl acetate]bombesin(7-14) in vitro, biodistribution studies, performed in PC-3 tumor-bearing mice, showed low uptake in the tumor (0.89 ( 0.27% ID/g 30 min pi) but high uptake into the pancreas (7.11 ( 3.93% ID/g 30 min pi), a GRP receptor-positive organ. Blockade experiment (coinjection of 300 µg bombesin/mouse with the radioligand) showed specificity of the uptake. Despite the low tumor uptake, tumor-to-blood ratios of 2.0 and 2.7 and tumor-to-muscle ratios of 8.9 and 8.0 were obtained at 30 min and 1.5 h postinjection, respectively. The promising results merit the future in vivo investigation of 99mTc/188Re-tricarbonyllabeled bombesin analogues.
INTRODUCTION
The discovery that certain tumor types overexpress receptors for neuropeptides dates back to the mid 1980s (1). To date, radiolabeled receptor binding peptides emerged as a new class of radiopharmaceuticals (2). Peptides show some favorable characteristics: they are readily synthesized, inexpensive, and can withstand harsh chemical conditions for modifications and radiolabeling. Furthermore, they offer several advantages over antibodies: they are less likely to induce immunogenic responses and they are expected to have a better pharmacokinetic profile inherent in a faster blood clearance, a better tissue penetration, a higher tumor uptake and therefore more favorable tumor-to-background ratios. The amphibian 14-amino acid peptide bombesin (BB) is part of the superfamily of bombesin-like peptides that also includes the mammalian homologue gastrin releasing peptide (GRP) and the related neuromedin B (NMB). These neuropeptides effect a broad range of physiological responses through G protein-coupled receptors with seven transmembrane domains. Besides being naturally localized in the central nervous system and in peripheral * To whom correspondence should be addressed: Telephone: ++41-56-310-2817, Fax: ++41-56-310-2849, E-mail: elisa.
[email protected]. † Paul Scherrer Institute. ‡ Swiss Federal Institute of Technology. § Vrije Universiteit Brussel.
tissue (3, 4), the GRP receptor is also expressed in a number of neuroendocrine tumors. Prostate and breast cancer might be of clinical significance as recent findings indicated a massive receptor overexpression in neoplastic transformed prostate and breast tissue (5, 6). Bombesin has been shown to cause through the GRP receptor a tumor growth effect in cancer cell lines in culture or as xenografts in nude mice (7-9). The mechanism by which tumor growth stimulation occurs does not appear to be constant but in general seems to involve transactivation and up-regulation of epidermal growth factor receptors (10, 11). Importantly, the tumor growth-stimulating effect of bombesin can be blocked by GRP receptor antagonists (9, 12, 13). Therefore, the use of GRP receptor antagonists and/or GRP receptor-targeting cytotoxic peptide conjugates could be an efficient chemotherapeutic approach. In nuclear medicine, 99mTc-labeled bombesin analogues could probably allow early noninvasive diagnosis of GRP receptor-positive tumors by γ-scintigraphy. Application of therapeutic radionuclides likes the β--emitting rhenium-186 and rhenium-188 might even allow radiotherapy of these types of tumors. The aim of the present work was the in vitro and in vivo characterization of a new bombesin analogue functionalized with a NR-histidinyl acetate bifunctional chelator (BFC) and radiolabeled with the organometallic precursor [99mTc(OH2)3(CO)3]+. The study involved the synthesis of [99mTc(CO)3-NR-histidinyl acetate]bombesin(7-14), 1, as well as the pharmacological evaluation in
10.1021/bc015571a CCC: $22.00 © 2002 American Chemical Society Published on Web 04/17/2002
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vitro and in vivo including binding properties, internalization, chemical stability, metabolic stability, and biodistribution of the radioconjugate in nude mice bearing PC-3 prostate adenocarcinoma xenografts. MATERIAL AND METHODS
Reagents and materials were obtained from following sources: Fmoc-protected amino acids and 4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)-phenoxy resin were purchased from NovaBiochem (La¨ufelingen, Switzerland). Bombesin and Neuromedin B were obtained from Bachem (Bubendorf, Switzerland). [125I-Tyr4]bombesin was purchased from NEN Life Science Products (Zaventem, Belgium). The 99Mo/99mTc generator was obtained from Mallinckrodt (Petten, The Netherlands). Culture medium Dulbeccos’s MEM with GLUTAMAX I, fetal bovine serum, antibiotic/antimycotic solution, trypsin/EDTA, bovine serum albumin and soybean trypsin inhibitor were obtained from GIBCO BRL Life Technologies AG (Basel, Switzerland). HEPES, chymostatin, and bacitracin were from Sigma (Buchs, Switzerland). The cold binding buffer contained 50 mM Hepes, 125 mM NaCl, 7.5 mM KCl, 5.5 mM MgCl2, 1 mM EGTA, 5 g/l BSA, 2 mg/L chymostatin, 100 mg/L soybean trypsin inhibitor, 50 mg/L bacitracin, pH 7.4. Further chemicals were obtained from Fluka (Buchs, Switzerland). The peptides were synthesized on a semiautomated Labortec peptide synthesizer SP640B. Correct mass was identified by electron spray-mass spectrometry (Micromass VG Trio 2000, Manchester, England). RP-HPLC analyses were performed on a Merck-Hitachi L-7000-system equipped with an L-7400 tunable absorption detector and a Berthold LB 506 B radiometric detector. For analyses and purification a Macherey-Nagel CC Nucleosil 100-5 C18 reverse phase column (10 µm, 250 × 4.6 mm) was used. HPLC solvents consisted of (A) water with 0.1% trifluoroacetic acid and (B) acetonitrile with 0.1% trifluoroacetic acid. The HPLC system started with a linear gradient 95% A/5% B to 40% A/60% B from 0 to 30 min. The gradient remained at 40% A/60% B for 5 min before switching back to 95% A/5% B for another 5 min. The flow rate was 1.5 mL/min. Radioactive samples from in vitro and in vivo experiments were measured with a NaIγ-counter (Packard Canberra Cobra II). Cell Culture. The human PC-3 adenocarcinoma cell line was obtained from European Collection of Cell Cultures (Salisbury, United Kingdom). Cells were maintained in Dulbecco’s MEM with GLUTAMAX I supplemented with 1% fetal bovine serum, 100 units/mL penicilline G sodium, 100 µg/mL streptomycin sulfate and 0.25 µg/mL amphotericin B. Cells were cultured at 37 °C in a humidified incubator under an atmosphere containing 7.5% CO2 and passaged weekly. Peptide Synthesis. Solid-phase synthesis of [NRhistidinyl acetate]bombesin(7-14) was carried out using Fmoc-strategy. The cold rhenium(I)-labeled conjugate was obtained by mixing 100 µL of 1 mM [NR-histidinyl acetate]bombesin(7-14), 20 µL of 5.5 mM (TEA)2Re(CO)3Br3, 20 µL of 15 mM NaOH, and adding up to 200 µL with water. The mixture was heated for 6 h at 75 °C. The peptides were analyzed by ES-MS and RP-HPLC. Radiolabeling. [99mTc(OH2)3(CO)3]+ was prepared according to earlier descriptions (14). Depending on the needs, 0.1 to 0.3 mL of the neutralized technetium-99m tricarbonyl solution were mixed with 10-30 µL of 1 mM [NR-histidinyl acetate]bombesin(7-14) and again heated to about 75 °C for 1 h. The product was analyzed and purified (for determination of Kd) by RP-HPLC.
LaBella et al.
Receptor Binding Studies. Inhibition assays with PC-3 cells were performed as previously described (15). In brief, cells were placed at confluence in 48-well plates with 25000 cpm of [125I-Tyr4]bombesin in the presence of increasing concentrations of the different bombesin analogues (0-3000 nM). After 1 h at 37 °C, cells were washed twice with cold binding buffer and then solubilized with 1 N NaOH. Radioactivity was determined using a NaIγ-counter. For saturation studies, cells were incubated in triplicate with increasing concentrations (1 to 500 pM, equivalent to 2 to 1000 kBq of 99mTc activity) of the 99mTc(I)-labeled bombesin analogue for 1 h at 37 °C. The concentrations of active 99mTc were calculated as described by Bauer and Pabst (16) and Hill et al. (17). After two washing steps with binding buffer, cells were solubilized with 1 N NaOH at 37 °C. Nonspecific binding was determined with 1 µM bombesin. Internalization Studies. Internalization of the 99m Tc(I)-labeled bombesin analogue was performed as previously described (18, 19). Briefly, PC-3 cells were incubated in six-well plates in triplicate with about 200000 cpm of compound 1 with or without excess of 1 µM cold bombesin for 2 h at 4 °C. Then they were washed three times with ice-cold culture medium to discard free peptide and incubated with previously warmed culture medium for 0, 5, 15, 30, and 120 min at 37 °C for internalization. After these times, cell-surface bound radioligand was removed by two steps of acid wash (50 mM glycine-HCl/100 mM NaCl, pH 2.8) at room temperature for 5 min. Subsequently, the cells were solubilized by incubation with 1 N NaOH at 37 °C to determine internalized radioligand. Release of internalized radioconjugated peptide was determined by incubating PC-3 cells at confluence in tissue culture dishes (60 × 15 mm) with about 2 × 105 cpm of 1 for 1 h at 37 °C. The cells were then washed three times with ice-cold culture medium and cell-surface bound radioligand was removed by two steps of acid wash. After having washed twice with culture medium, the cells were incubated for different time periods at 37 °C. At each time point (30 min, 1, 2.5, 5, and 24 h) the radioactivity present in the medium (released) as well as the acid wash (cell-surface bound) and the acid wash resistant (internalized) were determined with a NaI-γ-counter. Results were expressed as percentage of total activity in the wells (released + cell-surface bound + internalized). Histidine and Cysteine Challenge. Challenging assays were done according to the method of Stalteri et al. (20) with some modifications. A 10 µL amount of the 99mTc(I)-labeled peptide were added to 90 µL of 0.01 M histidine or cysteine (1000-fold molar excess of challenging agents). The resulted solutions were incubated at 37 °C, and progress of the reaction was followed by RPHPLC with radiometric detection. In Vitro Stability. Studies of metabolic stability were performed with compound 1 in human plasma and whole blood. Plasma and blood from healthy donors were incubated at 37 °C with the 99mTc(I)-labeled peptide at a concentration of 0.2 pmol peptide/mL for different time periods. After incubation, plasma samples were precipitated with acetonitrile/ethanol and then centrifuged (4 °C, 2 × 104 g), whereas the blood samples were centrifuged previously to the precipitation to remove first blood cells. After repeated precipitation and centrifugation, the supernatants were then analyzed by RP-HPLC. In Vivo Stability. To evaluate the in vivo degradation of the conjugate, blood of sacrificed PC-3 xenografted CD-1 nu/nu mice (1.5 h pi) was collected. The samples were centrifuged, and the supernatant was precipitated
99mTc(I)-Postlabeled
Bombesin Analogue
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Figure 2. Displacement curves of [125I-Tyr4]bombesin binding to GRP receptors on the human prostate cancer cell line PC-3 by bombesin (O), [NR-histidinyl acetate]bombesin(7-14) (3), [Re(I)-NR-histidinyl acetate]bombesin(7-14) (×), and neuromedin B (9).
Figure 1. Proposed structure of [99mTc(CO)3-NR-histidinyl acetate]bombesin(7-14).
with acetonitrile/ethanol (1:1) and centrifuged again at 4 °C. The final supernatants were analyzed by RP-HPLC. Animal Studies. Biodistribution of the radioconjugate was estimated in female CD-1 nu/nu mice (6- to 8-weeks old). Human PC-3 prostate adenocarcinoma xenografts were induced by subcutaneous injection of 8 × 106 cells/ mouse. Ten days after the tumor induction the mice received the 99mTc(I)-labeled bombesin analogue (3.5-4 MBq/mouse) intravenously into the tail vein. Organs and tissue (blood, heart, lung, spleen, kidneys, stomach, pancreas, ileum, colon, liver, muscle, bone, brain, and tumor) were excised from the sacrificed animals at 30 min, 1.5, and 24 h postinjection. All tissues were weighed, and the radioactivity was determined by γ-counting. Results were expressed as percentage of injected dose per gram (% ID/g) of tissue. An in vivo receptor blocking study was also carried out. In this study, each animal received 300 µg of cold bombesin by coinjection with the radiolabeled bombesin analogue (in 95% saline/5% ethanol). The animals were sacrificed at 30 min postinjection, all tissues were removed, weighed, and γ-counted, and the % ID/g tissue was calculated. RESULTS
Radiolabeling. HPLC analyses revealed a single main peak (>95% of total activity) after the labeling, which corresponded to the 99mTc-peptide conjugate (Figure 1). Generally, pertechnetate and free 99mTc(I)-tricarbonyl represented only 0-5% and 0-1%, respectively, of the total radioactivity after 1 h of labeling. Receptor Binding Studies. All bombesin analogues tested showed the typical sigmoid curves in the displacement assays (Figure 2). The synthesized conjugates inhibited the binding of [125I-Tyr4]bombesin in the same concentration range as the natural peptide itself (Table 1). In saturation studies, receptor binding of [99mTc-NRhistidinyl acetate]bombesin(7-14) to intact PC-3 cells was found to be saturable (Figure 3, A). Specific binding rapidly increased in the concentration range of 1 to 100 pM radioconjugate and reached a plateau at 250 pM.
Figure 3. A: Binding of [99mTc(CO)3-NR-histidinyl acetate]bombesin(7-14) to PC-3 cells as a function of ligand concentration. The cells were incubated with 1 to 500 pM [99mTc(CO)3NR-histidinyl acetate]bombesin(7-14) in the absence (0, total binding) or presence (×, nonspecific binding) of 1 µM bombesin for 1 h at 37 °C. Specific binding ([) was calculated as the difference between total and nonspecific binding. B: Scatchard analysis of these data. Each point means the value of triplicate determinations. Table 1. Inhibition of [125I-Tyr4]Bombesin Binding to PC-3 Cells by Different Bombesin Analogues compound
IC50 [nM]
bombesin [NR-histidinyl acetate]bombesin(7-14) [Re(I)-NR-histidinyl acetate]bombesin(7-14) neuromedin B
1.5 4.3 0.6 73.9
Scatchard analysis of the binding data indicated the presence of a homogeneous population of binding sites with high affinity for the used cell line (Figure 3, B). At 37 °C, the calculated dissociation constant (Kd) was 2.1 × 10-10 M. Internalization Studies. The rate of internalization of [99mTc(CO)3-NR-histidinyl acetate]bombesin(7-14) was time and temperature dependent (Figure 4). At 4 °C, cellsurface binding toward GRP receptors occurred but no internalization (
