A New High Affinity Technetium-99m-Bombesin Analogue with Low

Biodistribution studies of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in normal ... for the development of tracers for noninvasive detection and therapy of ...
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Bioconjugate Chem. 2005, 16, 43−50

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A New High Affinity Technetium-99m-Bombesin Analogue with Low Abdominal Accumulation Kuo-Shyan Lin,† Andrew Luu,† Kwamena E. Baidoo,*,† Hossein Hashemzadeh-Gargari,† Ming-Kai Chen,† Kenneth Brenneman,† Roberto Pili,‡ Martin Pomper,§ Michael A. Carducci,‡ and Henry N. Wagner, Jr.† Department of Environmental Health Sciences, The Johns Hopkins Medical Institutions, 615 North Wolfe Street, Baltimore, Maryland 21205, and Department of Oncology, The Sidney Kimmel Comprehensive Oncology Center, The Johns Hopkins Medical Institutions, Cancer Research Building, 1650 Orleans Street, Baltimore, Maryland 21231, and Department of Radiology, The Johns Hopkins Medical Institutions, 600 N. Wolfe Street, Baltimore, Maryland 21287. Received July 23, 2004; Revised Manuscript Received October 6, 2004

99m

Tc-labeled bombesin analogues have shown promise for noninvasive detection of many tumors that express bombesin (BN)/gastrin-releasing peptide (GRP) receptors. 99mTc-labeled peptides, however, have a tendency to accumulate in the liver and intestines due to hepatobiliary clearance as a result of the lipophilicity of the 99mTc chelates. This makes the imaging of lesions in the abdominal area difficult. In this study, we have synthesized a new high affinity 99mTc-labeled BN analogue, [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN, having a built-in pharmacokinetic modifier, DTPA, and labeled with 99mTc using a hydrophilic diaminedithiol chelator (Pm-DADT) to effect low hepatobiliary clearance. In vitro binding studies using human prostate cancer PC-3 cell membranes showed that the inhibition constant (Ki) for [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN was 4.1 ( 1.4 nM. Biodistribution studies of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in normal mice showed very low accumulation of radioactivity in the liver and intestines (1.32 ( 0.13 and 4.58 ( 0.50% ID, 4 h postinjection, respectively). There was significant uptake (7.71 ( 1.37% ID/g, 1 h postinjection) in the pancreas which expresses BN/GRP receptors. The uptake in the pancreas could be blocked by BN, partially blocked by neuromedin B, but not affected by somatostatin, indicating that the in vivo binding was BN/GRP receptor specific. Scintigraphic images showed specific, high contrast delineation of prostate cancer PC-3 xenografts in SCID mice. Thus, the new peptide has a great potential for imaging BN/GRP receptor-positive cancers located even in the abdomen.

INTRODUCTION

Bombesin (BN), the first cancer cell autocrine peptide growth factor to be discovered (1), is a 14 amino acid peptide initially isolated from frog skin (2). The main mammalian members of the bombesin family of peptides are gastrin releasing peptide (GRP), a 27 amino acid peptide first isolated from porcine stomach (3), and neuromedin B (NMB), a 10 amino acid peptide first isolated from porcine spinal cord (4). Bombesin-like peptides produce many physiological activities that include stimulation of the release of numerous gastrointestinal hormones and peptides (5, 6), stimulation of pancreatic enzyme secretion (7), effects on the central nervous system (CNS) such as thermoregulation (8), and inhibition of thyroid-stimulating hormone (TSH) (9). Many tumors such as glioblastoma, small cell lung cancer, prostate, breast, gastric, colon, and pancreatic cancer (10-18) are also known to express cell surface receptors to bombesin-like peptides. The receptor system is accessible as a marker for the development of tracers for noninvasive detection and therapy of the cancers. Radionuclides, such as 111In, 125I, 105Rh, and 99mTc (19-28), have been used to label BN/GRP analogues for noninvasive in vivo imaging of BN/GRP receptor-posi* To whom correspondence should be addressed. Telephone: (410)-955-7706. Fax: (410) 955-6222. E-mail: [email protected]. † Department of Environmental Health Sciences. ‡ Department of Oncology. § Department of Radiology.

tive cancers. Of these radionuclides, 99mTc is more advantageous because of its ready availability, low cost, excellent imaging properties (141 keV photon energy and 89% photon flux), favorable dosimetry, and high specific activity. Many 99mTc-labeled peptides, however, have a tendency to accumulate in the liver and intestines as a result of hepatobiliary clearance. Hepatobiliary clearance is the preferred excretion route because of the lipophilicity conferred on the labeled peptide by many Tc chelates. The high accumulation of radioactivity in the liver and intestines will jeopardize the imaging of BN/GRP receptor-positive cancers and their metastases in the abdominal area. Radiotracers with higher hydrophilicity tend to be excreted trough the urinary system (29-31), Strategies for decreasing abdominal accumulation have included the use of hydrophilic bifunctional chelating agents (BFCA) (28) or coligands (32). Recently, we reported the use of a builtin pharmacokinetic modifier in the form of diethylenetriaminepentaacetic acid (DTPA) to reduce hepatobiliary clearance (33). In this study, we present a new high affinity, hydrophilic 99mTc-labeled BN/GRP analogue, [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN, having the build-in pharmacokinetic modifier, DTPA, at the N-terminus and labeled with 99mTc using a hydrophilic diaminedithiol (DADT) chelate, Pm-DADT (Figure 1), to direct its clearance through the urinary system. The design and synthesis of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN as well as its initial biological evaluation are discussed here.

10.1021/bc049820h CCC: $30.25 © 2005 American Chemical Society Published on Web 12/31/2004

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Figure 1. The structures of the diaminedithiol (DADT) bifunctional chelating agents (BFCA) and their Tc complexes. Reaction of the bifunctional chelating agents Hx-DADT and PmDADT with amine nucleophiles results in the release of a thiol group to complete the chelating core of the diaminedithiol chelating system for subsequent chelation of Tc from labile Tc complexes such as Tc-glucoheptonate. MATERIALS AND METHODS

BN, neuromedin B, and somatostatin were purchased from Advanced ChemTech (Louisville, KY). [99Tc]NH4TcO4 and [125I-Tyr4]BN were purchased from NEN Life Science Products (Boston, MA). [99mTc]NaTcO4 was purchased as a solution in physiologic saline from Syncor (Timonium, MD). The Glucoscan kit was purchased from the Du Pont Radiopharmaceutical Division (Billerica, MA). All other chemicals were obtained from either Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Company (St. Louis, MO). Solvents and chemicals were reagent grade and used as received without further purification. Pm-DADT was synthesized according to our previously published procedure (34). Male CD-1 mice were purchased from the Charles River Laboratories (Charles River, MA). The PC-3 cell line was obtained from the American Type Culture Collection (Rockville, MD) and grown in the Cell Culture Laboratory, School of Medicine, The Johns Hopkins University (Baltimore, MD). Amino acid analyses and MALDI mass spectral analyses were performed by AnaSpec, Inc. (San Jose, CA). HPLC was performed with a Waters Chromatography Division (Milford, MA) HPLC System equipped with two model 510EF pumps, a model 680 automated gradient controller, a model 490 UV absorbance detector, and a Bioscan NaI scintillation detector connected to a Bioscan Flow-count System. The output from two channels of the UV detector and the output from the Flow-count system were fed into a Gateway 2000 P5-133 computer fitted with an IN/US System, Inc. (Tampa, FL) computer card. HPLC acquisition and analysis were performed with the Winflow software from IN/US. The semipreparative column (C18 Novapak cartridge, 25 mm × 10 cm, 6 µm), the analytical column (C18 Novapak cartridge, 8 mm × 10 cm, 4 µm), and the C18 Light Sep-Pak cartridge were purchased from Waters Chromatography Division. The G3000SW size exclusion column (silica, 7.5 mm × 30 cm, particle size 10 µm, pore size 25 nm) was purchased from TosoHaas (Montogomeryville, PA). HPLC solvents consisted of acetontrile containing 0.1% trifluoroacetic acid (solvent A) and water containing 0.1% trifluoroacetic acid (solvent B). Liquid scintillation counting for quantification of 99Tc activity was performed on an LKB Wallac instrument model 1219 Rackbeta using Ecolume (ICN pharmaceuticals, Inc., Costa Mesa, CA) as cocktail. Filtration of in vitro assay mixtures was performed on a BRANDEL, 48M Harvester. 125I activity on filters from

Lin et al.

in vitro assays and 99mTc activity of tissues from biodistribution and blocking studies were counted on an automated gamma counter (Pharmacia-Wallac model 1282). Analyses of in vitro binding assay results were performed using the EBDA/LIGAND programs from Biosoft (Milltown, NJ). 99mTc activity for synthetic reactions was measured in a Capintec CRC-7 Dose Calibrator. Synthesis of [DTPA1, Lys3(Pm-DADT), Tyr4]BN. The basic peptide sequence, [DTPA1, Lys3, Tyr4]BN was assembled in an Advanced ChemTech 90 Tabletop Peptide Synthesizer using the tert-butyloxycarbonyl (Boc) protocol starting with Boc-L-methionine-p-methylbenzhydrylamine resin (3 g, 1 mmol/g). Coupling was performed for 30 min with DIC (9 mmol) and HOBt (9 mmol) using the appropriate Boc-protected amino acid (9 mmol) which included Nim-tosyl-His, Nin-formyl-Trp, N-γ-xanthyl-Gln, N-β-xanthyl-Asn, O-2,6-dichlorobenzyl-Tyr, and N-2-chlorobenzyloxycarbonyl-Lys in methylene chloride/dimethylformamide (1:1). The coupling reactions were monitored with the nynhydrin test and was repeated if the test was positive. The Boc group was deprotected during each cycle with trifluoroacetic acid (25%) in methylene chloride. DTPA was conjugated at the last step of peptide assembly using the method of Mokotoff et al. (35). At the end of peptide assembly, cleavage and side chain deprotection were performed using anhydrous hydrogen fluoride (14 g) at 0 °C for 1.5 h in the presence of anisole (19.7 mmol). After removal of hydrogen fluoride using a slow stream of nitrogen, the residue was washed with ether, dried, and then extracted with 5% aqueous acetic acid (50 mL) and lyophilized. The lyophilized powder was dissolved in water (50 mL), and the solution was adjusted to pH 11 with 2.5 M NaOH. After 5 min, the solution was readjusted to pH 7 with 6 N HCl. The crude product was purified by HPLC using a linear gradient from solvent A (15%)/solvent B (85%) to solvent A (40%)/ solvent B (60%) over the course of 50 min at a flow rate of 6 mL/min using the semipreparative column monitored on-line for UV absorption at 220 nm. Eluates containing the product with a retention time of 26.0 min were collected, pooled, and lyophilized. The purity of [DTPA1, Lys3, Tyr4]BN was >95% as assessed by analytical HPLC. The yield of [DTPA1, Lys3, Tyr4]BN was 968 mg (17%). Pm-DADT was coupled to [DTPA1, Lys3, Tyr4]BN according to Scheme 1. Pm-DADT‚2HCl (9.9 mg, 27.2 µmol) and [DTPA1, Lys3, Tyr4]BN (9.0 mg, 4.7 µmol) were dissolved in borate buffer (500 µL, 0.1 M, pH 9) and acetonitrile (500 µL), and the pH was readjusted to 9 with triethylamine. The mixture was incubated at room temperature for 3 h and then extracted with ether (3 × 2 mL). The aqueous fraction was chromatographed by HPLC with a linear gradient from solvent B (100%) to solvent A (80%)/solvent B (20%) over the course of 60 min at a flow rate of 6 mL/min using the semipreparative column monitored on-line for UV absorption at 220 nm. The product with a retention time of 30.8 min was collected, followed by lyophilization. The synthetic yield of [DTPA1, Lys3(Pm-DADT), Tyr4]BN was 5.2 mg (50%). Amino acid analysis gave the expected amino acid ratios; calc/found Asn (1/0.88), His (1/1.14), Val (1/0.98), Leu (1/0.95), Lys (1/0.81), Gln(2/1.79), Gly (2/2.19), Ala (1/0.98), Tyr (1/1.09), Met (1/0.97). MALDI MS: calc MW for [DTPA1, Lys3(Pm-DADT), Tyr4]BN, 2195.0; found [M + H]+ 2195.1. 99m Tc Labeling of [DTPA1, Lys3(Pm-DADT), 4]BN. 99mTc labeling of [DTPA1, Lys3(Pm-DADT), Tyr Tyr4]BN was performed as depicted in Scheme 1. A glucoheptonate kit (Glucoscan) was reconstituted with water (1.0 mL). From this solution, [99mTc]glucoheptonate

Bioconjugate Chem., Vol. 16, No. 1, 2005 45

A New Technetium Analogue of Bombesin Scheme 1. Synthesis of [DTPA1, Lys3(Tc-Pm-DADT), Tyr4]BN

was prepared by addition of an aliquot (200 µL) to a [99mTc]pertechnetate solution (500 µL, 10-20 mCi), and the mixture was vortexed for 1 min. Then, the [99mTc]glucoheptonate solution (300 µL) was added to a solution of the [DTPA1, Lys3(Pm-DADT), Tyr4]BN (0.5 µmol) in water (500 µL), and the mixture was vortexed for 1 min and incubated at room temperature for 10 min. The reaction was followed by HPLC using a linear gradient from solvent A (25%)/solvent B (75%) to solvent A (55%)/solvent B (45%) over the course of 60 min at a flow rate of 2 mL/min using the analytical column monitored on-line for UV absorption at 220 nm and scintillation for radioactivity. [DTPA1, Lys3(99mTc-PmDADT), Tyr4]BN was obtained as a single product peak with a retention time of 35.1 min compared with a retention time of 27 min for [DTPA1, Lys3(Pm-DADT), Tyr4]BN, the unlabeled precursor. The yield was > 90%. Preparation of the 99Tc Analogue of [DTPA1, Lys3(Pm-DADT)Tyr4]BN. A solution of [99Tc]glucoheptonate in water (20 µL, 20 mM) was added to a solution of [DTPA1, Lys3(Pm-DADT), Tyr4]BN (0.8 mg, 0.36 µmol) in methanol (400 µL) and water (400 µL), and the mixture was incubated at room temperature for 10 min. A similar product was obtained as described above for the 99mTc analogue. The product was isolated by HPLC using the same conditions above for the purification of the

99mTc

analogue. The yield of the product was 17%. MALDI MS: calc MW for [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN 2308.0, found [M + H]+ 2307.8. In Vitro Binding Assay of BN Analogues. In vitro binding studies were performed using a modified method of Reile et al. (36). Briefly, membrane protein (10 µg) was incubated at 25 °C for 35 min with [125I-Tyr4]BN (100 pM) in the presence or absence of various concentrations of BN, somatostatin, [DTPA1, Lys3, Tyr4]BN, [DTPA1, Lys3(Pm-DADT), Tyr4]BN or [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN ranging from 10 pM to 10 µM in the assay buffer (50 mM Tris-HCl buffer, pH 7.4, containing 1 mg/mL bovine serum albumin (BSA) and 2 µg/mL bacitracin) (125 µL total volume). Nonspecific binding was determined in the presence of 1 µM bombesin. The reaction was terminated by rapid filtration through Whatman GF/B glass fibers presoaked for 30 min in the wash buffer (50 mM Tris-HCl buffer, pH 7.4, containing 1 mg/mL BSA). The filter was washed 3 × 5 mL with ice cold wash buffer. Radioactivity on the filters was counted using the automated gamma counter. The results of the assay were analyzed by the EBDA/LIGAND computer programs (37). The assays were performed in duplicate and the whole experiment was repeated three times. Biodistribution of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in Normal Mice. The HPLC-purified [DTPA1,

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Lys3(99mTc-Pm-DADT), Tyr4]BN was isolated from the HPLC eluate by solid-phase extraction using the C18 Light Sep-Pak cartridge. The Sep-Pak cartridge was first washed with ethanol (5 mL) followed by a solution of ammonium acetate (0.05 M, 5 mL). The HPLC eluate containing [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN was diluted five times with ammonium acetate (0.05 M) and then passed through the Sep-Pak cartridge. The Sep-Pak cartridge was washed with an ammonium acetate solution (0.05 M, 5 mL) and then [DTPA1, Lys3(99mTc-PmDADT), Tyr4]BN was eluated with ethanol (300 µL). The ethanolic solution was diluted with physiologic saline to make the final injectate (10 µCi/mL) containing 10000

Values represent the mean ( SD (n ) 3).

bifunctional chelating agent, Pm-DADT, to [DTPA1, Lys3, Tyr4]BN was performed at pH 9 using a Pm-DADT: [DTPA1, Lys3, Tyr4]BN ratio of 6:1 (Scheme 1). After 3 h incubation at room temperature, the excess Pm-DADT was removed by extraction with ether and the [DTPA1, Lys3(Pm-DADT), Tyr4]BN adduct purified by semipreparative reversed phase HPLC. Side chain protection was unnecessary since the indole and imidazole groups of Trp and His, respectively, are weak neucleophiles under the coupling conditions. The purified adduct was characterized by both mass spectrometry and amino acid analysis. We prepared both no-carrier-added (99mTc) and milligram quantities (99Tc) of the Tc-labeled BN analogue, [DTPA1, Lys3(Tc-Pm-DADT), Tyr4]BN. These Tc derivatives were prepared in ligand exchange reactions by incubation of a mixture of [DTPA1, Lys3(Pm-DADT), Tyr4]BN with [99mTc]- or [99Tc]glucoheptonate at room temperature (Scheme 1). Only one product was obtained in both cases. The yield of 99mTc product was >90% after incubation for 10 min at room temperature as determined by HPLC. The 99Tc product with 17% isolated yield was characterized by chromatographic comparison to the 99mTc analogue and mass spectrometry. Both DTPA and the DADT chelating ligands can coordinate Tc. Therefore, a major issue in the design of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN was the site of Tc chelation. The mass spectrometry results of [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN indicated that only one [Tc-oxo] center was chelated to [DTPA1, Lys3(Pm-DADT), Tyr4]BN. This could either be at the DADT or DTPA site. In the stability studies, only 6.8% of radioactivity was transferred to serum proteins even after 6 h incubation at 37 °C. This is an indication of the strong binding of Tc to the peptide, suggesting binding at the DADT site. The labeling of proteins with 99mTc via DTPA results in poor stability both in vitro and in vivo (52). Furthermore, we have shown in previous studies that in the presence of the benzylamine derivative of Pm-DADT, more than 100 fold excess DTPA cannot compete for the chelation of 99mTc (34). Taken together, the likelihood is strong that the 99mTc is chelated via the DADT ligand site rather than DTPA. As a first step in the evaluation of the biological activity of [DTPA1, Lys3(Tc-Pm-DADT), Tyr4]BN, we have investigated the ability of [DTPA1, Lys3(Tc-Pm-DADT), Tyr4]BN to bind to the BN/GRP receptor in human prostate cancer PC-3 cell membrane preparations according to a modified method of Reile et al. (36). These studies involved competitive binding experiments using the authentic BN receptor ligand [125I-Tyr4]BN. Results of analysis with the EBDA/LIGAND computer programs are shown in Table 1. The apparent affinities (Ki) of [DTPA1, Lys3, Tyr4]BN (3.1 ( 1.3 nM) and [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN (4.1 ( 1.4 nM) for the BN receptor were comparable to that of BN (1.7 ( 0.6 nM) itself. The unlabeled Pm-DADT adduct, [DTPA1, Lys3(Pm-DADT), Tyr4]BN, had slightly lower affinity (15.5 ( 1.2 nM). Somatostatin which does not bind to the receptor system had a Ki > 10000 nM.

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Table 2. Biodistribution Results (% ID/organ)a of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in normal Mice at Different Time Points bloodb brain heart lung kidneys spleen pancreas intestines stomach liver

15 min

1h

2h

4h

5.07 ( 0.50 0.05 ( 0.02 0.15 ( 0.02 0.37 ( 0.02 9.14 ( 1.47 0.14 ( 0.08 1.85 ( 0.24 5.51 ( 0.56 0.88 ( 0.07 4.26 ( 0.81

2.78 ( 0.50 0.03 ( 0.01 0.07 ( 0.02 0.19 ( 0.04 7.67 ( 1.47 0.07 ( 0.03 1.49 ( 0.40 7.25 ( 0.90 0.74 ( 0.13 1.70 ( 0.31

1.52 ( 0.13 0.02 ( 0.01 0.06 ( 0.03 0.21 ( 0.08 6.11 ( 0.90 0.06 ( 0.02 1.29 ( 0.38 6.15 ( 0.50 0.61 ( 0.08 1.66 ( 0.41

1.22 ( 0.07 0.017 ( 0.004 0.03 ( 0.01 0.16 ( 0.05 3.40 ( 0.76 0.05 ( 0.01 1.33 ( 0.24 4.58 ( 0.50 0.43 ( 0.23 1.32 ( 0.13

Values represent the mean ( SD (n ) 4). The blood volume is estimated to be 6.5% of the body weight. a

b

Table 3. Radioactivity Concentration (% ID/g)a of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in Normal Mice Organs at Different Time Points blood brain heart lung kidneys spleen pancreas intestines stomach liver muscle a

15 min

1h

2h

4h

2.44 ( 0.24 0.12 ( 0.04 0.96 ( 0.15 1.72 ( 0.15 16.4 ( 2.80 1.09 ( 0.37 8.95 ( 0.98 1.62 ( 0.11 1.69 ( 0.74 1.95 ( 0.24 0.67 ( 0.08

1.39 ( 0.24 0.07 ( 0.03 0.46 ( 0.13 0.78 ( 0.13 15.3 ( 3.11 0.68 ( 0.22 7.71 ( 1.37 2.61 ( 0.29 1.61 ( 0.48 0.93 ( 0.16 0.39 ( 0.12

0.74 ( 0.07 0.05 ( 0.03 0.38 ( 0.15 0.73 ( 0.21 11.9 ( 2.58 0.57 ( 0.22 5.99 ( 1.56 2.00 ( 0.17 1.08 ( 0.49 0.84 ( 0.27 0.30 ( 0.09

0.58 ( 0.05 0.04 ( 0.01 0.23 ( 0.02 0.69 ( 0.08 6.66 ( 0.94 0.55 ( 0.15 5.45 ( 0.36 1.47 ( 0.25 0.87 ( 0.42 0.77 ( 0.11 0.11 ( 0.02

Values represent the mean ( SD (n ) 4).

Results of the biodistribution studies of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in normal mice are summarized in Tables 2 and 3. [DTPA1, Lys3(99mTc-PmDADT), Tyr4]BN showed fast clearance from the blood pool (0.58 ( 0.05% ID/g, 4 h postinjection) in good agreement with our previous findings (22). However, the radioactivity excreted through the hepatobiliary system (liver and intestines, ∼6% ID, 4 h postinjection) was much lower compared to the results of Lys3(99mTc-HxDADT)BN (96% ID, 3 h postinjection), Lys3(99mTc-PmDADT)BN (29% ID, 3 h postinjection) and [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN (21% ID, 4 h postinjection) from our previous studies (22, 33). These results indicated the success of the pharmacokinetic modifier, DTPA, and the combination with the hydrophilic Tc-PmDADT chelate, in reducing the radioactivity in the hepatobiliary system. Only small amounts of activity remained in the stomach and liver 4 h postinjection (0.43 ( 0.23 and 1.32 ( 0.13% ID, respectively). The pancreas, which expresses the BN/GRP receptor, showed significant uptake (7.71 ( 1.37% ID/g, 1 h postinjection) of radioactivity. This demonstrated the ability of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4] to target BN/GRP receptor expressing cells in vivo. The pancreas/blood, pancreas/ muscle, and pancreas/liver ratios were highest at 4 h postinjection at 9.1, 50, and 7.1, respectively. To determine if the distribution of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in vivo was specific, we conducted experiments in which bombesin, neuromedin-B, somatostatin, or saline vehicle was preinjected in normal mice prior to the administration of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN. Results of these receptor blocking studies in vivo are shown in Table 4. The difference in the uptake of [DTPA1, Lys3(99mTc-PmDADT), Tyr4]BN with and without subcutaneous injection of 700 µg of BN is significant in the pancreas (0.48 ( 0.07 vs 8.71 ( 0.99% ID/g) and intestines (0.68 ( 0.11

Table 4. Results (% ID/ga) of Blocking Studies in Normal Mice unblocked

bombesinb

blood 1.34 ( 0.17 1.54 ( 0.19 heart 0.54 ( 0.11 0.55 ( 0.06 lung 1.40 ( 0.21 1.10 ( 0.14 kidneys 13.65 ( 0.61 14.32 ( 2.40 spleen 0.56 ( 0.16 0.57 ( 0.16 pancreas 8.71 ( 0.99 0.48 ( 0.07 intestines 2.20 ( 0.01 0.68 ( 0.11 stomach 3.09 ( 0.71 2.42 ( 0.47 liver 1.31 ( 0.16 1.39 ( 0.18 muscle 0.29 ( 0.03 0.34 ( 0.35

neuromedin bb somatostatinb 1.59 ( 0.14 0.59 ( 0.05 1.20 ( 0.14 15.20 ( 1.18 0.74 ( 0.12 4.87 ( 0.51 1.88 ( 0.05 3.28 ( 0.38 1.74 ( 0.08 0.31 ( 0.01

1.42 ( 0.16 0.51 ( 0.03 1.36 ( 0.11 12.26 ( 2.05 0.80 ( 0.06 8.90 ( 1.51 1.96 ( 0.30 4.83 ( 0.59 1.55 ( 0.12 0.30 ( 0.03

a Values represent the mean ( SD (n ) 4). b 700 µg (in 100 µL saline) BN, neuromedin B, or somatostatin was injected subcutaneously 30 min before the injection of [DTPA1, Lys3(99mTc-PmDADT), Tyr4]BN.

vs 2.20 ( 0.01% ID/g). Acini cells of the pancreas, submucosal layer of the small intestine, longitudal and circular muscle, and submucosal layer of the colon all have been shown to express BN/GRP receptors (53, 54). Neuromedin B (55), another mammalian analogue of BN, which shows moderate affinity (Ki, 150-350 nM against [125I-Tyr4]BN) toward BN/GRP receptors in acini cells of rat pancreas partially blocked the uptake of radioactivity in pancreas (4.87 ( 0.06 vs 8.71 ( 0.99% ID/g). In contrast, biodistribution of the group with the preinjection of somatostatin, which does not bind to the BN/GRP receptor, was similar to that of the unblocked group. Thus, blocking in vivo followed the expected rank order with respect to the affinity of the blocking agents to the BN/GRP receptor in the pancreas. These results show the specificity of the uptake of [DTPA1, Lys3(99mTc-PmDADT), Tyr4]BN in receptor rich tissue in vivo. Furthermore, [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN appears to have subtype selectivity on account of the limited ability of neuromedin-B to block its binding in vivo. The incidence of the specific subtype of receptors present in human tumors has begun to be investigated. Preston et al. found the predominant subtype in stomach cancer was the BB2. Recently, Reubi et al. (46) also studied the incidence of bombesin receptor subtypes in human tumors. They reported in their series of 161 human tumors that 12/12 prostate cancers, 41/51 breast cancers, and 5/5 gastrinomas expressed the BB2 receptor subtype predominantly. The study confirmed previous studies that indicate that for prostate and breast cancers, the predominant subtype expressed in high density is the BB2 subtype (17, 40, 41). Other tumors in this series such as small cell lung cancer (3/9) and renal cell carcinomas (6/16) also showed BB2 receptor subtype expression. Lower incidence of expression of the BB1 and BB3 receptor subtypes were reported in bronchial carcinoids, renal cancer, Ewing sarcomas except intestinal carcinoids that showed 11/24 BB1 receptors, and small cell lung cancer that showed 4/9 BB3 receptors in this series. The results of our blocking studies suggest that [DTPA1, Lys3(99mTc-Pm-DADT) may be more specific for the BB2 subtype, the predominant subtype expressed in many tumors. This is because neuromedin-B has higher affinity for the BB1 receptor than the BB2 receptor. Further work is ongoing in our laboratory to investigate the subtype specificity of [DTPA1, Lys3(99mTc-Pm-DADT) Tyr4]. In preliminary studies, we investigated the feasibility of imaging human prostate cancer PC-3 xenografts in SCID mice. In these studies, one group of mice were injected with unlabeled bombesin prior to the injection of the tracer to block the receptors while the other group

A New Technetium Analogue of Bombesin

Figure 2. Representative images of human prostate cancer PC-3 tumors in SCID mice injected with [DTPA1, Lys3(99mTcPm-DADT), Tyr4]BN. Each mouse received a 200 µCi injection of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN. One group of mice received 700 µg of BN subcutaneously (blocked) and another group received saline vehicle (unblocked) 30 min before the intravenous injection of tracer. The mice were imaged at 12 h postinjection. The target/nontarget ratios were 2 and 10 for the blocked versus unblocked mice, respectively.

only received the tracer. Representative images obtained after 12 h postinjection are shown in Figure 2. The tumor could more clearly be seen in the mice that received no blocking dose of bombesin, whereas the tumors could barely be seen in the mice whose receptors were blocked. Analysis of regions of interest drawn over tumor areas compared to contralateral regions in the same mice showed that the target (tumor) to nontarget (contralateral region) ratios were 2 and 10 for the blocked versus unblocked mice, respectively. The results show that the binding of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in vivo in the tumor is specific and indicates the potential of the tracer for imaging prostate cancer. In conclusion, the use of the built-in pharmacokinetic modifier, DTPA, combined with the hydrophilic TcPm-DADT chelate, showed minimum abdominal accumulation and the ability to target BN/GRP receptorpositive tissues. This will enable [DTPA1, Lys3(99mTc-PmDADT), Tyr4]BN to be used for imaging BN/GRP receptorpositive cancers and their metastases located even in the abdominal area. DADT ligands are also capable of forming highly stable complexes with Re, the congener of Tc. Thus, the 186Re and 188Re analogues of [DTPA1, Lys3(Pm-DADT), Tyr4]BN would be directly applicable for the radiotherapy of many cancers. When used together, the Tc and Re analogues would represent a diagnostic and therapeutic pair of radiopharmaceuticals. The Tc analogues could then be used to select the patients that could benefit from therapy using the Re analogues. ACKNOWLEDGMENT

This work was supported by PHS Research Grant numbers CA32845, CA096817, SAIRP grant CA92871, and Training Grant CA09199 support for A.L., H.H.G., and K.B. from the National Cancer Institute. LITERATURE CITED (1) Cuttita, F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko, J., Fischler, A., and Minna, J. D. (1986) Bombesinlike peptides can function as autocrine growth factors in human small cell lung cancer. Nature 316, 823-825. (2) Anastasi, A., Erspamer, V., and Bucci, M. (1971) Isolation and structure of bombesin and alytesin, two analogous active peptides from the skin of the European amphibian Bombina Alytes. Experientia 27, 166-167.

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