A New High Affinity Technetium Analogue of Bombesin Containing

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Bioconjugate Chem. 2004, 15, 1416−1423

1416

A New High Affinity Technetium Analogue of Bombesin Containing DTPA as a Pharmacokinetic Modifier Kuo-Shyan Lin,† Andrew Luu,† Kwamena E. Baidoo,*,† Hossein Hashemzadeh-Gargari,† Ming-Kai Chen,† Roberto Pili,‡ Martin Pomper,§ Michael Carducci,‡ and Henry N. Wagner, Jr.† Department of Environmental Health Sciences, The Johns Hopkins Medical Institutions, 615 North Wolfe Street, Room E6632, Baltimore, Maryland 21205, 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 North Wolfe Street, Baltimore, Maryland 21287. Received July 20, 2004; Revised Manuscript Received August 27, 2004

The bombesin (BN)/gastrin-releasing peptide (GRP) receptor is expressed in high density on the cell surface of a variety of tumors. This makes the receptors accessible as a molecular target for the detection of lesions in which they are expressed. In this study, we describe a high affinity hydrophilic 99m Tc-labeled BN analogue, [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN, having diethylenetriaminepentaacetic acid (DTPA), as a build-in pharmacokinetic modifier, to direct its excretion through the urinary system in order to lower abdominal background activity. In vitro binding studies using [125I-Tyr4]BN (Kd, 0.1 nM) and human prostate cancer PC-3 cell membranes showed that the inhibition constant (Ki) of [DTPA1, Lys3(99Tc-Hx-DADT), Tyr4]BN was 19.9 ( 8.0 nM. Biodistribution studies in normal mice showed fast blood clearance (0.15 ( 0.01% ID/g, 4 h postinjection), low intestinal accumulation (9.16 ( 2.35% ID/g, 4 h postinjection), and significant uptake in BN/GRP receptor rich tissues such as the pancreas (21.83 ( 2.88% ID/g, 15 min postinjection). The pancreas/blood, pancreas/muscle, and pancreas/liver ratios were highest at 2 h postinjection at 23, 74, and 8.4, respectively. The uptake in the pancreas could be blocked by BN (11.96 ( 1.17 vs 0.65 ( 0.16% ID/g), partially blocked by neuromedin B (11.96 ( 1.17 vs 6.66 ( 0.51% ID/g), but not affected by somatostatin (11.96 ( 1.17 vs 12.91 ( 2.53% ID/g), indicating that the binding of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN to the receptors was specific. Scintigraphic imaging of human PC-3 prostate cancer xenografts in SCID mice gave a high target to nontarget ratio on the image. Thus, [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN has the potential for imaging BN/GRP receptor-positive lesions.

INTRODUCTION

Bombesin (BN), an amphibian tetradecapeptide originally isolated from frog skin (1), and its mammalian analogue, gastrin-releasing peptide (GRP), share the same C-terminal heptapeptide, Trp-Ala-Val-Gly-His-LeuMet-NH2, and bind with high affinity to the BN/GRP receptor. BN-like peptides have a wide range of physiological activities including stimulation of the release of numerous gastrointestinal hormones (2), stimulation of pancreatic enzyme secretion (3, 4), effects on the central nervous system such as thermoregulation (5), and inhibition of thyroid-stimulating hormone (6). The BN/GRP receptor has also been shown to be overexpressed on many cancer cells including small cell lung cancer, glioblastoma, gastric, pancreatic, prostate, breast, and colon cancer (7-14) as well as premalignant cells in the case of prostate, gastric, and lung cancers (13-15). BN/GRP receptor expression could be used as a potential marker for early diagnosis as well as the staging of BN/GRP receptor-positive cancers and their metastasis if suitable tracers could be developed for in vivo scintigraphy of the receptors. Radionuclides, such * To whom correspondence should be addressed. Telephone: (410)-955-7706. Fax: (410) 955-6222. E-mail: [email protected]. † Department of Environmental Health Sciences. ‡ The Sidney Kimmel Comprehensive Oncology Center. § Department of Radiology.

as 111In, 125I, and 105Rh (16-18), have been used to label BN/GRP analogues for in vivo scintigraphy of BN/GRP receptor-bearing tumors. However, due to the advantages of 99mTc, such as ready availability, low cost, excellent imaging properties (141 keV photon energy and 89% photon flux), favorable dosimetry, and high specific activity, the development of 99mTc-labeled BN/GRP analogues for cancer imaging would be even more promising. For his reason several investigators have investigated 99m Tc-labeled analogues of bombesin for imaging cancer (19-25). To label biomolecules with 99mTc, it is necessary to provide a site on the substrate for the chelation of the radiometal. The diaminodithiol (DADT) chelating system forms one of the most stable complexes with Tc. Tc complexes of this system, however, tend to be highly lipophilic. Therefore, upon the use of the ligand system to label substrates with 99mTc, the resultant tracers tend to be excreted primarily through the hepatobiliary system. For example, in a previous study (19), we reported a 99mTc-labeled BN analogue, Lys3(99mTc-Hx-DADT)BN in which the diaminedithiol bifunctional chelating agent, Hx-DADT (Scheme 1) was used to chelate the Tc. This technetium analogue showed high affinity for the BN/ GRP receptor. However, due to its lipophilicity, the radioactivity was excreted mainly through the hepatobiliary pathway as indicated by the high percent injected dose (% ID) in the liver and intestines (96% ID) at 1 h

10.1021/bc0498267 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/22/2004

Bioconjugate Chem., Vol. 15, No. 6, 2004 1417

High Affinity Technetium Analogue of Bombesin Scheme 1. Synthesis of [DTPA1, Lys3(Tc-Hx-DADT), Tyr4]BN

postinjection. This high accumulation of radioactivity in the liver and intestines would jeopardize the imaging of BN/GRP receptor-positive cancers and their metastases in the abdominal area. Since radiotracers with higher hydrophilicity tend to be excreted through the urinary system (26-28), different strategies, such as the use of a water-soluble bifunctional chelating agent (29), more hydrophilic coligands (30), or cleavable linkers (31), have been investigated as ways to increase the hydrophilicity of 99mTc-labeled peptides. In this study, we report the design, synthesis, and initial evaluation of a high affinity, hydrophilic 99mTc-labeled BN/GRP analogue, [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN (Scheme 1), modified with a build-in pharmacokinetic modifier, diethylenetriaminepentaacetic acid (DTPA), at the N-terminus to direct its clearance through the urinary system. EXPERIMENTAL PROCEDURES

Materials and General Methods. BN, neuromedin B, somatostatin, 1,3-diisopropylcarbodiimide (DIC), hydroxybenzotriazole (HOBt), Boc-L-methionine-p-methylbenzhydrylamine resin, and Boc-protected amino acids were purchased from Advanced ChemTech (Louisville, KY). [99Tc]NH4TcO4 and [125I-Tyr4]BN were purchased

from PerkinElmer Life Science (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 the 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. The DADT bifunctional chelating agent, Hx-DADT (Scheme 1), was synthesized according to our previously published procedures (31). Male CD-1 mice were purchased from the Charles River Laboratories (Charles River, MA). All in vivo studies on mice were approved by the Institutional Animal Care and Use Committee. 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 spectroscopy were performed by AnaSpec, Inc. (San Jose, CA). FAB mass spectroscopy was performed by the Mass Spectrometry Service Laboratory of the University of Minnesota (Minneapolis, MN). HPLC was performed with a Waters Chromatography Division (Milford, MA) HPLC System equipped with two

1418 Bioconjugate Chem., Vol. 15, No. 6, 2004

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 (Bioscan, Inc., Washington, DC). 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 cartridges were purchased from Waters Chromatography Division. The G3000SW size exclusion column (7.5 mm × 30 cm, particle size 10 µm, pore size 25 nm) was purchased from TosoHaas (Montogomeryville, PA). Reversed phase HPLC solvents consisted of acetonitrile 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 (Biomedical Research and Development Laboratories Inc., Gaithersburg, MD). 125I activity on filters from in vitro assays and 99mTc activity of tissues from biodistribution and blocking studies were counted on an automated gamma counter (PharmaciaWallac 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 and doses for biodistribution were measured in a Capintec (Pittsburgh, PA) CRC-7 Dose Calibrator. Synthesis of [DTPA1, Lys3, Tyr4]BN. [DTPA1, Lys3, Tyr4]BN was synthesized in an Advanced ChemTech 90 Tabletop Peptide Synthesizer using the tert-butyloxycarbonyl (Boc) protocol starting with Boc-L-methionine-pmethylbenzhydrylamine resin (3 g, 1 mmol/g). Each coupling step was performed with DIC (9 mmol), HOBt (9 mmol), and the appropriate Boc-protected amino acid (9 mmol) including Nim-tosyl His, Nin-formyl Trp, N-γxanthyl Gln, N-β-xanthyl Asn, O-2,6-dichlorobenzyl Tyr, and N-2-chlorobenzyloxycarbonyl Lys in methylene chloride/dimethylformamide (1:1). The coupling reaction was usually complete in 30 min and was repeated if the nynhydrin test was positive. Trifluoroacetic acid (25%) in methylene chloride was used for Boc group deprotection during each cycle. DTPA was conjugated as the last step using the method of Mokotoff et al. (32). After assembly, peptide 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 drying, the peptide was extracted into 5% aqueous acetic acid (50 mL) and purified by HPLC with 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%). Amino acid analysis gave the expected amino acid ratio; calcd/found Asn (1/0.98), His (1/1.01), Val (1/ 1.00), Leu (1/1.00), Lys (1/1.02), Gln(2/1.96), Gly (2/2.03),

Lin et al.

Ala (1/1.02), Tyr (1/0.96), Met (1/1.00). MALDI MS: calcd MW for [DTPA1, Lys3, Tyr4]BN 1904.9, found [M + H]+ 1905.8. Coupling of Hx-DADT to [DTPA1, Lys3, Tyr4]BN. Hx-DADT was coupled to [DTPA1, Lys3, Tyr4]BN according to Scheme 1. Hx-DADT‚2HCl (3.6 mg, 9.1 µmol) and [DTPA1, Lys3, Tyr4]BN (3.3 mg, 1.73 µmol) were dissolved in a mixture of borate buffer (0.4 mL, 0.1 M, pH 9) and acetonitrile (0.4 mL), and the pH was adjusted to 9 with triethylamine. The mixture was incubated at room temperature for 2 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 33.0 min was collected and then lyophilized. The yield of [DTPA1, Lys3(Hx-DADT), Tyr4]BN was 1.7 mg (44%). Amino acid analysis gave the expected amino acid ratios; calcd/found Asn (1/0.88), His (1/1.02), Val (1/1.04), Leu (1/1.04), Lys (1/0.99), Gln(2/1.87), Gly (2/2.18), Ala (1/1.01), Tyr (1/1.00), Met (1/0.87). MALDI MS: calc MW for [DTPA1, Lys3(Hx-DADT), Tyr4]BN 2209.1, found [M + H]+ 2211.3. 99mTc Labeling of the [DTPA1, Lys3(Hx-DADT), Tyr4]BN. 99mTc labeling of [DTPA1, Lys3(Hx-DADT), 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 was prepared by addition of an aliquot (200 µL) to a [99mTc]pertechnetate solution (500 µL, 10-20 mCi). The mixture was vortexed for 1 min and allowed to stand at room temperature for 15 min. An aliquot of the [99mTc]glucoheptonate solution (300 µL) was added to a solution of the [DTPA1, Lys3(Hx-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 labeling reaction was followed by reversed phase 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 on the analytical column. HPLC was monitored on-line for UV absorption at 220 nm and scintillation for radioactivity. A single product designated as [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN with a retention time of 37.0 min was obtained. The yield of the product was >90%. Preparation of the 99Tc Analogue of [DTPA1, Lys3(Hx-DADT), Tyr4]BN. To a solution of [DTPA1, Lys3(Hx-DADT), Tyr4]BN (2.6 mg, 1.18 µmol) in a methanol/water mixture (1:1, 800 µL) was added a solution of [99Tc]glucoheptonate in water (60 µL, 20 mM). The mixture was stirred at room temperature for 1 h. A similar product was obtained as described above for the 99m Tc 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 43%. MALDI MS: calcd MW for [DTPA1, Lys3(99Tc-Hx-DADT), Tyr4]BN 2321.0, found [M + H]+ 2321.0. Stability Test of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN in Human Serum. The purified ethanolic solution (300 µL) of [DTPA1, Lys3(99mTc-HX-DADT), Tyr4]BN was diluted with saline (3 mL), and then 100 µL of this diluted solution was mixed with 900 µL of human serum. The mixture was incubated in a 37 °C water bath. After 2, 4, and 6 h, an aliquot (100 µL) was removed and analyzed by isocratic size exclusion HPLC. The HPLC eluent flow rate was 1 mL/min using the size exclusion column monitored on-line for UV absorption at 220 and 280 nm and scintillation for radioactivity.

High Affinity Technetium Analogue of Bombesin

The eluent consisted of 10% acetonitrile in 0.2 M phosphate buffer (pH 7.15) containing 0.05% sodium azide. The serum proteins eluted at retention time ranging from 5 to 10 min, and [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN eluted at the retention time of 15.8 min. Competition of Hydrolyzed Hx-DADT with DTPA for 99mTc. Hx-DADT‚2HCl (2 mg, 5.3 µmol) was dissolved in water (400 µL) and then hydrolyzed by the addition of 2.5 N NaOH (100 µL). After 10 min incubation, the pH was adjusted to 7 with 1 N HCl. DTPA (104 mg, 264 µmol) was added to the mixture, and the pH was readjusted to 7 with 2.5 N NaOH. 99m Tc-glucoheptonate (300 µL, ∼10 mCi) was added to the mixture, vortexed for 1 min, and then incubated at room temperature for 20 min. For comparison another reaction was set up with all the reagents except DTPA. The reactions were followed by reversed phase HPLC. The HPLC conditions used were the same as described above for the purification of [DTPA1, Lys3(99mTc-HxDADT), Tyr4]BN. Both reaction mixtures showed similar HPLC spectra with two products, at retention time of 12.2 min (minor) and 24.8 min (major). The total labeling yield was >95% and the ratio of major/minor product was ∼5. 99Tc complexes of hydrolyzed Hx-DADT were similarly prepared using [99Tc]glucoheptonate (1.05 µmol). Two products were obtained as described above for the 99mTc analogues. The total labeling yield was 56% and the ratio of major/minor product was ∼1.5. FAB MS: calcd MW for 99Tc-Hx-DADT 416.1, found [M + H]+ 417.2 for both major and minor products. In Vitro Binding Assay of BN Analogues. The binding studies were performed using a modified method of Reile et al. (33). Briefly, 10 µg membrane protein was incubated at 25 °C for 35 min with 100 pM [125I-Tyr4]BN in the presence or absence of various concentrations of BN, somatostatin, [DTPA1, Lys3, Tyr4]BN, [DTPA1, Lys3(Hx-DADT), Tyr4]BN or [DTPA1, Lys3(99Tc-HxDADT), Tyr4]BN ranging from 10 pM to 1 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 in the automated gamma counter. The results of the assay were analyzed by the EBDA/LIGAND computer programs (34). The assays were performed in duplicate and the whole experiment was repeated 3 times. Biodistribution of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN in Normal Mice. The HPLC-purified [DTPA1, Lys3(99mTc-Hx-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-Hx-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) followed by elution of the [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN with ethanol (300 µL). The ethanolic solution was diluted with physiologic saline to make a final injectate (10 µCi/mL) containing 96% ID). As a result, Lys3(99mTc-Hx-DADT)BN would not be suitable for imaging BN/GRP receptor-positive tumors and their metastases in the abdominal area. To increase the hydrophilicity of 99mTc-labeled BN analogues, we have synthesized this new BN/GRP analogue, [DTPA1, Lys3, Tyr4]BN with a built-in pharmacokinetic modifier, DTPA, at the N-terminus so as to direct tracer clearance through the urinary system. To test the utility of this approach, we chose the highly lipophilic 99mTc-Hx-DADT chelate for labeling. In the design of [DTPA1, Lys3, Tyr4]BN we sought minimal modification of the structure of BN in order to preserve high affinity binding to the BN/GRP receptor. All modifications were restricted to the N-terminal region in order not to interfere with the C-terminal octapeptide binding site of the peptide. Pyr at the N-terminus was replaced by DTPA, which serves as the pharmacokinetic modifier. The assumption for using DTPA was that, as constructed, the four carboxylic groups introduced to the peptide after coupling DTPA to the peptide would be negatively charged at physiologic pH. Therefore, the peptide would be highly charged, aiding urinary excretion preferentially over hepatobiliary excretion. Arg at the third position of BN was replaced by Lys to provide an -amino group for coupling to the Tc-coordinating moieties such as Hx-DADT. Leu, the fourth residue, was also replaced by Tyr to allow the potential direct iodination of the resulting analogue. The binding site of BN was retained unaltered in the new analogue. The product, [DTPA1, Lys3, Tyr4]BN, was purified by semipreparative reversed phase HPLC to a single species and characterized by both mass spectrometry and amino acid analysis. It is noteworthy that as constructed, the peptide can potentially be labeled with radionuclides other than 99mTc such as 111In, 123/125/131I, 18F and 186/188Re. Coupling of the bifunctional chelating agent, HxDADT, to [DTPA1, Lys3, Tyr4]BN was performed at pH 9 using a Hx-DADT:[DTPA1, Lys3, Tyr4]BN ratio of 5:1 (Scheme 1). No side chain protection was necessary since the indole and imidazole groups of Trp and His, respectively, are weak nucleophiles under the coupling conditions. The purified adduct was also characterized by both mass spectrometry and amino acid analysis. We have prepared both no-carrier-added (99mTc) and milligram quantities (99Tc) of the Tc-labeled BN analogue, [DTPA1, Lys3(Tc-Hx-DADT), Tyr4]BN (Scheme 1). Only one product was obtained in both cases. The yield of 99mTc product was >90% after a 10 min incubation at room temperature as determined by HPLC. The 99Tc product with 43% isolated yield was characterized by mass spectrometry. DTPA is capable of coordinating Tc. Therefore, a pertinent issue in the design of [DTPA1, Lys3(99mTc-HxDADT), Tyr4]BN was whether the Tc was chelated by

Lin et al. Table 1. Results of in Vitro Binding Studies of Bombesin Analogues to PC-3 Cell Membranes compound

Ki a (nM)

BN [DTPA1, Lys3, Tyr4]BN [DTPA1, Lys3(Hx-DADT), Tyr4]BN [DTPA1, Lys3(99Tc-Hx-DADT, Tyr4]BN somatostatin

1.7 ( 0.6 3.1 ( 1.3 11.2 ( 5.4 19.9 ( 8.0 >10000

a

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

Table 2: Biodistribution (% ID/organ)a of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN in normal Mice bloodb brain heart lung kidneys spleen pancreas intestines stomach liver

15 min

1h

2h

4h

7.26 ( 1.27 0.10 ( 0.01 0.19 ( 0.02 0.63 ( 0.13 5.82 ( 0.94 0.15 ( 0.04 2.74 ( 0.45 14.3 ( 0.63 1.26 ( 0.10 9.21 ( 1.67

1.38 ( 0.27 0.02 ( 0.01 0.03 ( 0.01 0.22 ( 0.03 2.90 ( 0.47 0.06 ( 0.03 1.69 ( 0.54 19.0 ( 0.49 1.06 ( 0.21 2.41 ( 0.37

0.53 ( 0.14 0.02 ( 0.02 0.01 ( 0.00 0.08 ( 0.02 1.71 ( 0.06 0.02 ( 0.01 1.16 ( 0.29 22.1 ( 2.13 0.66 ( 0.10 1.45 ( 0.13

0.23 ( 0.01 0.00 ( 0.00 0.01 ( 0.00 0.06 ( 0.04 0.85 ( 0.15 0.01 ( 0.00 0.34 ( 0.07 20.3 ( 4.33 0.42 ( 0.03 1.04 ( 0.29

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

DTPA, DADT or both. The result from mass spectrometry of [DTPA1, Lys3(99Tc-Hx-DADT), Tyr4]BN indicated that only one technetium-oxo (TcO) group was chelated to [DTPA1, Lys3(Hx-DADT), Tyr4]BN. Proteins labeled with Tc using DTPA as the bifunctional chelate usually show poor stability both in vitro and in vivo (43) whereas the use of DADT based chelates result in stable products. Results of the stability test of [DTPA1, Lys3(99mTc-HxDADT), Tyr4]BN in human serum showed that less than 6% of radioactivity was transferred to serum proteins after 6 h incubation at 37 °C. This indicates strong binding of Tc in this product and suggests DADT chelation. In the competition study, identical products (99mTcHx-DADT) were obtained in the presence or absence of excess DTPA confirming that DTPA is unable to compete with DADT for Tc. The products had the same mass expected for Tc-Hx-DADT, indicating isomeric forms of this product as expected for Tc-DADT complexes. The combination of these facts strongly suggests that Tc is chelated to [DTPA1, Lys3(Hx-DADT), Tyr4]BN via the DADT core rather than DTPA. As a first step in the evaluation of the biological activity of [DTPA1, Lys3(Tc-Hx-DADT), Tyr4]BN, we investigated the ability of [DTPA1, Lys3(Tc-Hx-DADT), Tyr4]BN to bind to the BN/GRP receptor on human prostate cancer PC-3 cell membranes. These studies involved competitive binding experiments using the authentic BN receptor ligand [125I-Tyr4]BN. Results of analysis of the binding data with the EBDA/LIGAND computer programs are shown in Table 1. The apparent affinity, Ki, of [DTPA1, Lys3, Tyr4]BN for the BN receptor was comparable to that of BN itself (3.1 ( 1.3 vs 1.7 ( 0.6 nM). Coupling of HxDADT to [DTPA1, Lys3, Tyr4]BN and the subsequent labeling of Tc resulted in products, [DTPA1, Lys3(HxDADT), Tyr4]BN and [DTPA1, Lys3(99Tc-Hx-DADT), Tyr4]BN, with slightly lower affinity (Ki ) 11.2 ( 5.4 and 19.9 ( 8.0 nM, respectively). Results of the biodistribution studies of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN in normal mice are summarized in Tables 2 and 3. [DTPA1, Lys3(99mTc-HxDADT), Tyr4]BN showed fast clearance from the blood (0.23 ( 0.01 ID, 0.15 ( 0.01%ID/g, 4 h postinjection) and nontarget tissues. The radioactivity excreted through the hepatobiliary system (liver plus intestines, ∼21% ID,

Bioconjugate Chem., Vol. 15, No. 6, 2004 1421

High Affinity Technetium Analogue of Bombesin Table 3. Results of the Biodistribution (% ID/g)a of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN in normal Mice at Different Time Points organ

15 min

1h

2h

4h

blood 4.68 ( 0.56 0.89 ( 0.19 0.35 ( 0.10 0.15 ( 0.01 brain 0.22 ( 0.02 0.05 ( 0.02 0.04 ( 0.04 0.009 ( 0.001 heart 1.74 ( 0.19 0.31 ( 0.06 0.12 ( 0.02 0.05 ( 0.01 lung 3.54 ( 0.16 1.05 ( 0.22 0.49 ( 0.19 0.28 ( 0.08 kidneys 18.10 ( 3.28 8.54 ( 1.25 5.44 ( 0.83 2.66 ( 0.70 spleen 1.90 ( 0.24 0.62 ( 0.34 0.30 ( 0.06 0.11 ( 0.03 pancreas 21.83 ( 2.88 12.8 ( 2.97 8.10 ( 1.88 2.62 ( 0.37 intestines 5.65 ( 0.41 7.74 ( 0.67 8.70 ( 1.34 9.16 ( 2.35 stomach 1.45 ( 0.56 1.64 ( 0.56 1.05 ( 0.25 1.16 ( 0.05 liver 5.97 ( 0.66 1.73 ( 0.21 0.97 ( 0.11 0.83 ( 0.23 muscle 0.98 ( 0.19 0.20 ( 0.07 0.11 ( 0.03 0.05 ( 0.01 a

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

4 h postinjection) was much lower compared to the results of Lys3(99mTc-Hx-DADT)BN (>96% ID, 3 h postinjection) in our previous studies (19), indicating the success of the pharmacokinetic modifier, DTPA, in reducing the radioactivity in the gut even with the use of the highly lipophilic 99mTc-Hx-DADT chelate for labeling. Only small amounts of activity remained in the kidneys, stomach and liver at 4 h postinjection (2.66 ( 0.70, 1.16 ( 0.05 and 0.83 ( 0.23% ID/g, respectively). The pancreas, which expresses BN receptors (39), has been used as a target by other investigators (25, 45) for the biological evaluation of newly developed radiolabeled BN analogues. In this study, a significant uptake (21.83 ( 2.88% ID/g, 15 min postinjection) of the radioactivity was found in the pancreas. This demonstrates the ability of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN to target BN/GRP receptor expressing cells in vivo. The pancreas/blood and pancreas/muscle ratios were highest at 2 h postinjection at 23 and 74, respectively. These ratios are similar to the ratios (22 and 80, 4 h postinjection) obtained by Karra et al. (25) using 99mTc-P2S2-BN(7-14), a BN analogue labeled with 99mTc by the use of a hydrophilic dithiadiphospine-based bifunctional chelating agent. However, compared to 99mTc-P2S2-BN(7-14), [DTPA1, Lys3(99mTc-HxDADT), Tyr4]BN showed less radioactivity excreted through the hepatobiliary pathway (liver plus intestines, 21 vs 35% ID, 4 h postinjection) and therefore higher pancreas/liver ratio (8.4, 2 h postinjection vs 2.1, 4 h postinjection). Since the hydrophilic dithiadiphospinebased bifunctional chelating agent also reduced the accumulation of radioactivity in the gut compared to [Lys3(99mTc-Hx-DADT)]BN from our previous study (19), it will be interesting to combine both the built-in pharmacokinetic modifier with a hydrophilic bifunctional chelating agent in the same peptide to investigate the effect on hepatobiliary excretion. Results of the receptor blocking studies with BN, neuromedin B, and somatostatin (Table 4) confirmed the specificity of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN toward the BN/GRP receptors. The difference in the uptake of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN with and without subcutaneous injection of 700 µg of BN are significant in the pancreas (0.65 ( 0.16 vs 11.96 ( 1.17% ID/g), intestines (4.23 ( 0.46 vs 6.90 ( 0.68% ID/g) and stomach (0.64 ( 0.24 vs 1.40 ( 0.47% ID/g). Acini cells of the pancreas, submucosal layer of the small intestine, circular muscle, and submucosal layer of the colon as well as the circular muscle of the gastric fundus and antrum all have been shown to express BN/GRP receptors (44, 46). Neuromedin B (47), 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

Table 4. Blocking (% ID/g)a of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN Distribution in normal Mice by Bombesin,b Neuromedin B and Somatostatin control

bombesin

blood 0.85 ( 0.16 1.17 ( 0.16 heart 0.26 ( 0.05 0.44 ( 0.06 lung 0.59 ( 0.09 0.96 ( 0.09 kidneys 7.86 ( 1.72 10.56 ( 1.47 spleen 0.44 ( 0.06 0.35 ( 0.04 pancreas 11.96 ( 1.17 0.65 ( 0.16 intestines 6.90 ( 0.68 4.23 ( 0.46 stomach 1.40 ( 0.47 0.64 ( 0.24 liver 1.24 ( 0.16 1.64 ( 0.46 muscle 0.20 ( 0.03 0.31 ( 0.02

neuromedin b somatostatin 1.08 ( 0.03 0.41 ( 0.02 1.04 ( 0.22 8.55 ( 0.78 0.60 ( 0.27 6.66 ( 0.51 6.72 ( 0.83 1.28 ( 0.21 1.39 ( 0.16 0.24 ( 0.02

1.16 ( 0.17 0.62 ( 0.25 1.17 ( 0.15 10.94 ( 2.86 0.49 ( 0.04 12.94 ( 2.53 6.56 ( 0.50 2.13 ( 0.64 1.72 ( 0.24 0.25 ( 0.04

a Values represent the mean ( SD (n ) 4). The animals were sacrificed 1 h after the injection of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN. b 700 µg (in 100 µL saline) BN, neuromedin B or somatostatin was injected subcutaneously 30 min before the injection of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN.

Figure 1. Image of a mouse carrying a human prostate PC-3 xenograft 6 h postinjection with [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN. Tumor, T; kidney, K; bladder, B.

radioactivity in pancreas (6.66 ( 0.51 vs. 11.96 ( 1.17% ID/g). In contrast, somatostatin with very low affinity for BN/GRP receptor showed no effect on the biodistribution of [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN. These results suggest that the uptake of [DTPA1, Lys3(99mTc-HxDADT), Tyr4]BN in receptor rich areas in vivo is specific. In a preliminary study, we investigated the capability of using [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN for imaging prostate cancer using the PC-3 xenograft model in SCID mice. A representative image is shown in Figure 1. The site of the tumor could be differentiated in the 6 h image. The target to nontarget ratio on the image was 8:1, suggesting that the labeled peptide has potential for imaging prostate cancer and its metastases. In summary, we have described a new hydrophilic peptide, [DTPA1, Lys3, Tyr4]BN, whose 99mTc-chelatelabeled product [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN showed greatly reduced accumulation of radioactivity in the liver and intestines compared with an analogue lacking the pharmacokinetic modifier. It may be possible to use [DTPA1, Lys3(99mTc-Hx-DADT), Tyr4]BN for imaging BN/GRP receptor-positive tumors. Since DADT ligands are also capable of forming highly stable complexes with Re, the congener of Tc, the 186Re and 188Re analogues of [DTPA1, Lys3(Hx-DADT), Tyr4]BN could be directly applicable for the radiotherapy of many cancers on account of the energetic beta emissions from these radionuclides.

1422 Bioconjugate Chem., Vol. 15, No. 6, 2004

The study demonstrates successful modification of in vivo pharmacokinetics using DTPA even when the highly lipophilic 99mTc chelate core, 99mTc-Hx-DADT, was used for labeling. ACKNOWLEDGMENT

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