MicroPET Imaging of a Gastrin-Releasing Peptide Receptor-Positive

Jun 24, 2003 - been the target for detection and treatment of these neoplasms in animals. ... positive neoplasms is to radiolabel BN analogues with...
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Bioconjugate Chem. 2003, 14, 756−763

MicroPET Imaging of a Gastrin-Releasing Peptide Receptor-Positive Tumor in a Mouse Model of Human Prostate Cancer Using a 64Cu-Labeled Bombesin Analogue Buck E. Rogers,*,† Heather M. Bigott,‡ Deborah W. McCarthy,‡ Debbie Della Manna,† Joonyoung Kim,‡ Terry L. Sharp,‡ and Michael J. Welch‡ Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110. Received February 12, 2003; Revised Manuscript Received May 1, 2003

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of carcinomas and has been the target for detection and treatment of these neoplasms in animals. In particular, analogues of the tetradecapeptide bombesin (BN) have been radiolabeled with 99mTc and 111In for detection of GRPR-positive tumors by gamma ray scintigraphy. The goal of this study was to evaluate the potential of the bombesin analogue, DOTA-Aoc-BN(7-14), for positron-emission tomographic (PET) imaging after radiolabeling with the positron-emitter 64Cu. A saturation binding assay on PC-3 human prostate cancer cells showed that 64Cu-DOTA-Aoc-BN(7-14) had an equilibrium binding constant (Kd) of 6.1 ( 2.5 nM and a receptor concentration (Bmax) of 2.7 ( 0.6 × 105 receptors/cell. The radiolabeled analogue also showed rapid internalization with 18.2% internalized into 105 PC-3 cells by 2 h. The tumor localization of 64Cu-DOTA-Aoc-BN(7-14) was 5.5% injected dose per gram in athymic nude mice bearing PC-3 xenografts at 2 h postinjection. The tumor retention with respect to the 2 h value was 76% and 45% at 4 and 24 h, respectively, and was GRPR-mediated as shown by inhibition with a coinjection of excess peptide. MicroPET imaging of 64Cu-DOTA-Aoc-BN(7-14) in athymic nude mice bearing subcutaneous PC-3 tumors showed good tumor localization. Further studies with 64Cupyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) suggested that low blood flow to the PC-3 tumors may have limited the localization of 64Cu-DOTA-Aoc-BN(7-14). This study demonstrates that 64Cu-DOTA-Aoc-BN(7-14) can be used to detect GRPR-positive tumors by PET imaging.

INTRODUCTION 1

The amphibian tetradecapeptide BN and its mammalian counterpart, GRP, have been shown to produce a variety of biological responses including release of gastrointestinal hormones, effects on blood pressure, and thermoregulation (1, 2), as well as having a role in human cancer. In this regard, these peptides bind with high affinity to receptors that have been detected in various types of human carcinoma (3-7). Four BN receptor subtypes have been characterized including the BN- and GRP-preferring subtype (GRPR) (2), the neuromedin B-preferring subtype (8), and subtypes 3 and 4, which have lower affinities for both BN and GRP relative to GRPR (9, 10). BN-like peptides have been shown to function as growth stimulators in neoplastic tissues through autocrine mechanisms (3, 11). Therefore, several * Current address of corresponding author: Buck E. Rogers, Ph.D., Department of Radiation Oncology, Radiation and Cancer Biology Division, Washington University in St. Louis, 4511 Forest Park Blvd., Suite 411, St. Louis, MO 63108. Phone: 314362-9787. Fax: 314-362-9790. E-mail: [email protected]. † University of Alabama at Birmingham. ‡ Washington University School of Medicine. 1 Abbreviations used: BN, bombesin; GRP, gastrin-releasing peptide; GRPR, gastrin-releasing peptide receptor; PET, positronemission tomography; DOTA, 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid; Aoc, 8-amino-octanoic acid; TLC, thinlayer chromatography; 64Cu-PTSM, 64Cu-pyruvaldehyde-bis(N4methylthiosemicarbazone); Kd, equilibrium binding constant; Bmax, concentration of GRPR; % ID/g, percent injected dose per gram of tissue; PI, postinjection.

BN antagonists have been synthesized and shown to be efficacious in animal cancer models and in nude mice bearing human xenografts (12-14). In addition, conjugation of BN antagonists to cytotoxic agents such as doxorubicin have been shown to be potent in vitro and in mice bearing human tumor xenografts (15). Another strategy for detecting and treating GRPRpositive neoplasms is to radiolabel BN analogues with radioisotopes that can be used for noninvasive imaging and radiotherapy. In this regard, various BN analogues have been radiolabeled with radioactive iodine or radioactive metals. In particular, 131I, 149Pm, 188Re, and 177Lu have been used to radiolabel BN analogues for potential radiotherapy applications through their beta emissions (16-19), while 99mTc- and 111In-labeled analogues have been used for detecting GRPR-positive tumors by gamma ray scintigraphy (20-28). Positron-emission tomography (PET) should have better resolution than gamma camera imaging at the sensitivity needed for imaging GRPR expression; therefore, a BN analogue radiolabeled with a positron-emitter would have advantages over a gamma ray emitting radioisotope for detecting GRPR-positive tumors. Copper-64 (t1/2 ) 12.7 h) is a positron-emitter (19%, Eβ+max ) 656 keV) that has been shown to have therapeutic potential (39%, Eβ-max ) 573 keV) when targeted to tumors using monoclonal antibodies or peptides (29, 30). It is readily produced on a medical cyclotron (31), and its intermediate half-life should be suitable for imaging the localization of 64Cu-labeled BN analogues. The macrocyclic chelate, 1,4,7,10-tetraazacyclododecane-

10.1021/bc034018l CCC: $25.00 © 2003 American Chemical Society Published on Web 06/24/2003

MicroPET Imaging of GRPR in a Subcutaneous Xenograft Model

1,4,7,10-tetraacetic acid (DOTA), has been used to radiolabel antibodies and peptides with copper radioisotopes and has been conjugated to the N-terminus of a BN analogue for radiolabeling with 177Lu (18). This compound (DOTA-Aoc-BN(7-14)) consisted of an 8-aminooctanoic acid (Aoc) spacer between DOTA and the eight C-terminal amino acids of BN (BN(7-14)). In the present study, we sought to evaluate DOTAAoc-BN(7-14) after radiolabeling with 64Cu in binding and internalization assays of PC-3 human prostate cancer cells overexpressing GRPR and in athymic nude mice bearing subcutaneous (sc) PC-3 xenografts. The in vivo tumor localization and specificity of 64Cu-DOTA-AocBN(7-14) was demonstrated by tissue biodistribution and microPET imaging. Further studies were conducted with 64Cu-PTSM to compare the blood flow in the PC-3 tumors to that in other tissues. MATERIALS AND METHODS

Radiolabeling of DOTA-Aoc-BN(7-14) with 64Cu. Copper-64 was produced on a CS-15 biomedical cyclotron at Washington University School of Medicine according to published procedures (31). 1,4,7,10-Tetraazacyclododecane-1,4,7-tris(acetic acid tert-butyl ester)-10-acetic acid (DOTA-tris(tert-butyl ester)) was purchased from Macrocyclics, Inc. (Dallas, TX) and 8-aminooctanoic acid was purchased from Advanced ChemTech (Louisville, KY). The DOTA-Aoc-BN(7-14) was synthesized using standard Fmoc chemistry by solid-phase peptide synthesis at the University of Alabama at Birmingham Comprehensive Cancer Center Peptide Synthesis and Analysis Shared Facility and shown to be >98% pure by high performance liquid chromatography. For radiolabeling, 64 CuCl2 was diluted with a 10-fold excess of 0.1 M ammonium acetate (NH4OAc), pH ) 5.5, and then added to DOTA-Aoc-BN(7-14). After incubating at room temperature for 30 min, radiochemical purity was determined by radio-thin-layer chromatography (radio-TLC). To increase the specific activity and prevent radiolysis for microPET studies, the radiolabeling was performed in the presence of 2,5-dihydroxybenzoic acid (gentisic acid) (4 mg/mL final concentration) (Sigma Chemical Co., St. Louis, MO). Samples were applied to Whatman MKC18F TLC plates, developed with 10% NH4OAc: methanol (30:70), and analyzed using a BIOSCAN System 200 imaging scanner (Washington, DC). Saturation Binding Assays. The PC-3 human prostate cancer cell line was obtained from the American Type Culture Collection (Rockville, MD) and cultured in Ham’s F12K medium containing 10% fetal bovine serum (FBS) and 1% L-glutamine at 37 °C in a humidified atmosphere with 5% CO2. For binding assays, the cells were harvested by incubating with 4 mM EDTA/0.05% KCl for 3 min, centrifuging and resuspending in cold PBS at a concentration of 1 × 107 cells/mL. The cells were then aliquoted into polystyrene tubes in triplicate followed by the addition of various amounts of 64Cu-DOTA-Aoc-BN(7-14) such that the final concentration ranged between 0.1 and 500 nM. The cells were incubated for 1 h at 4 °C in the presence or absence of excess competitor (30 µM, Tyr4-BN, Sigma Chemical Co., St. Louis, MO). The samples were then rinsed with PBS and centrifuged at 1700g for 10 min, the supernatant was removed, and the cells were measured for radioactivity in a gamma counter (Packard Auto Gamma 5000 Series, Chicago, IL) to determine the amount of bound radioactivity. The data were analyzed using the GraphPad Prism software (San Diego, CA).

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Internalization Assays. PC-3 cells were harvested and seeded in six-well plates at 3 × 105 cells per well. Twenty-four hours later, 64Cu-DOTA-Aoc-BN(7-14) was added to the wells such that the final concentration was 1 nM and incubated at 37 °C for 30, 120, and 240 min. An excess (30 µM) of Tyr4-BN was added to half of the wells as a competitor. At the appropriate time point, a six-well plate was removed from the incubator, media was removed, and cells were rinsed with PBS. The cells were then rinsed twice with Hank’s Balanced Salt Solution (HBSS) containing 20 mM NaOAc, pH 4.0, to remove surface bound radioactivity, and the cells were harvested by adding 1 N NaOH. The acid wash and the cells were measured for radioactivity in a gamma counter to determine the amount of surface bound and internalized radioactivity, respectively. These data were normalized as a percentage of the total amount of radioactivity added per cell. Biodistribution Studies. Experiments were performed in 4-5 week old female athymic nude mice (National Cancer Institute Frederick Research Laboratory, Frederick, MD) implanted sc with 2 × 107 PC-3 cells mixed 1:1 with Matrigel (Becton Dickinson, Bedford, MA). Three weeks later, the mice were injected with 2-5 µCi of 64Cu-DOTA-Aoc-BN(7-14) (specific activity ) 60130 mCi/mg) via the tail vein. Biodistribution was performed with groups of four to six mice sacrificed 2, 4, and 24 h postinjection (PI) of 64Cu-DOTA-Aoc-BN(7-14). Another group of mice were coinjected with 64Cu-DOTAAoc-BN(7-14) and 100 µg of Tyr4-BN as a competitor and sacrificed 2 h PI. The blood, liver, small intestine, spleen, kidney, muscle, bone, pancreas, and tumor were removed and weighed, and the radioactivity was measured in a gamma counter to determine the percent injected dose per gram of tissue (% ID/g). MicroPET Imaging. PET imaging was performed on a microPET-R4 system (Concorde Microsystems Inc, Knoxville, TN) which was based on the design of Cherry and colleagues (32). The microPET-R4 has a field-of-view of 8 cm axially by 11 cm transaxially and is capable of a spatial resolution of 2.3 mm and an absolute sensitivity of 1020 cps/microcurie in the middle of the field-of-view. Images were generated from 3-dimensional sinogram data, rebinned to two-dimensional format by the FORE algorithm (33) followed by two-dimensional filtered-back projection. For imaging studies, the animal model described above was used followed by injection of 500 µCi of 64Cu-DOTA-Aoc-BN(7-14) (specific activity ) 2000 mCi/mg) with or without 100 µg Tyr4-BN via the tail vein. Two hours later, the mice were anesthetized with 1-2% isofluorane, positioned supine, and immobilized in a specially prepared scanner cradle. A total of four mice were imaged with two mice being imaged simultaneously (one with competitor, one without). Ex Vivo Determination of Receptor Density. The pancreata (mean weight ) ∼130 mg) and PC-3 tumors (mean weight ) ∼270 mg) were harvested from two euthanized mice and processed according to a previously published procedure for cell membrane preparations (34) to determine the expression of GRPR on each. A competitive binding assay was performed by adding 25 µg of membrane protein to Multiscreen Durapore filtration plates (type FB, 1.0 µm borosilicate glass fiber over 1.2 µm Durapore membrane; Millipore, Bedford, MA) and washed with buffer (10 mM HEPES, 5 mM MgCl2, 1 mM EDTA, and 0.1% BSA, pH 7.4). One hundred microliters of 125I-Tyr4-BN (0.05 nM, DuPont/NEN Research Products, Boston, MA) in PBS was added to each well in triplicate along with various concentrations (0.2 pM to

758 Bioconjugate Chem., Vol. 14, No. 4, 2003

Figure 1. Representative plots of 64Cu-DOTA-Aoc-BN(7-14) saturation binding (A) and Scatchard transformation (B). PC-3 cells were incubated with increasing concentrations of 64CuDOTA-Aoc-BN(7-14) in the presence or absence of excess competitor to determine nonspecific binding. Data represent the specific binding in dpm for the mean of triplicate measurements ( standard deviation.

60 µM) of unlabeled Tyr4-BN and incubated for 60 min at room temperature. The samples were rinsed twice with PBS and dried, and radioactivity was measured in a gamma counter. The data were analyzed using the GraphPad Prism software. In Vivo Determination of Blood Flow. Mice (n ) 3) bearing established PC-3 tumors were injected iv with 10 µCi of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM). 64Cu-PTSM was synthesized and radiolabeled as previously described (35), and radiochemical purity was >98% as determined by radio-TLC. Ten minutes after injection of 64Cu-PTSM, the mice were sacrificed and biodistribution was performed as described above. The data are presented as % ID/g to determine the relative blood flow to each tissue. Statistical Analysis. The Student’s t test was used to determine statistical significance at the 95% confidence level with P < 0.05 being considered significantly different. RESULTS

Radiolabeling of DOTA-Aoc-BN(7-14) with 64Cu. Initial radiolabeling attempts of DOTA-Aoc-BN(7-14) with 64Cu yielded maximum specific activities of 130 mCi/ mg (199 Ci/mmol). Subsequent radiolabelings achieved specific activities of 2000 mCi/mg (3062 Ci/mmol) in the presence of gentisic acid to prevent radiolysis of the peptide. In all cases, the radiochemical purity was >98% as determined by radio-TLC and the radiolabeled peptide was used immediately for both in vitro and in vivo assays. Saturation Binding Assays. A representative saturation binding curve and scatchard transformation are shown in Figure 1. This shows that 64Cu-DOTA-Aoc-BN(7-14) bound specifically to a single class of binding sites

Rogers et al.

Figure 2. Internalization of 64Cu-DOTA-Aoc-BN(7-14) into PC-3 cells. 64Cu-DOTA-Aoc-BN(7-14) was added to the cells in the presence or absence of excess competitor and incubated at 37 °C. At various time points, the media was removed, the cells acid-washed to remove surface bound radioactivity, and harvested. The cell pellets (internalized) and acid wash (surface bound) were counted and the specific internalized and surface bound radioactivity determined. The data are presented as the mean ( standard deviation of the % of total radioactivity added divided by the total number of cells in each well for triplicate measurements. Note that the error bars are contained within the symbols.

with high affinity. The mean equilibrium binding constant, Kd, of 64Cu-DOTA-Aoc-BN(7-14) for GRPR from three independent experiments was 6.1 ( 2.5 nM with a Bmax of 2.7 ( 0.6 × 105 GRP receptors per PC-3 cell. Internalization Assays. The internalization of 64CuDOTA-Aoc-BN(7-14) into PC-3 cells is shown in Figure 2. The amount of specifically internalized (amount internalized without competitor minus the amount internalized with competitor) 64Cu-DOTA-Aoc-BN(7-14) per 105 PC-3 cells was 11.4 ( 0.2% after a 30 min incubation, which increased to 18.2 ( 0.4% and 21.5 ( 0.5% at 120 and 240 min, respectively. The amount of internalized 64 Cu-DOTA-Aoc-BN(7-14) was significantly greater (P < 0.05) than the amount of surface bound 64Cu-DOTAAoc-BN(7-14) at all time points, which ranged from 3.3 to 4.6%. The amount of 64Cu-DOTA-Aoc-BN(7-14) internalized or surface bound in the presence of competitor was < 0.5% at all time points. Biodistribution Studies. The tissue and tumor localization of 64Cu-DOTA-Aoc-BN(7-14) is shown in Figure 3. These studies were performed with 2-5 µCi (1540 ng) of 64Cu-DOTA-Aoc-BN(7-14) radiolabeled at a specific activity of 130 mCi/mg. These results show specific GRPR localization of 64Cu-DOTA-Aoc-BN(7-14) in the tumor (P < 0.0001), pancreas (P < 0.01), and spleen (P < 0.05) 2 h PI as the uptake in these tissues are inhibited upon co-injection of Tyr4-BN. The tumor localization was 5.5 ( 0.6% ID/g at 2 h PI that decreased to 4.2 ( 1.1% ID/g and 2.5 ( 0.5% ID/g at 4 and 24 h PI, respectively. There was rapid blood clearance of 64CuDOTA-Aoc-BN(7-14) as only 1.3 ( 0.3% ID/g remained at 2 h, which was similar to the concentration at 4 and 24 h. The pancreas had a high localization of 64Cu-DOTAAoc-BN(7-14) at 2 and 4 h (13.4 ( 5.9% ID/g and 13.7 ( 2.0% ID/g, respectively) that decreased rapidly by 24 h (3.8 ( 0.9). The liver and pancreas were the only tissues to have significantly greater uptake (P < 0.05) of 64CuDOTA-Aoc-BN(7-14) than the tumor at all three time points. The tumor had significantly greater localization than the blood (P < 0.01), bone (P < 0.01), and muscle

MicroPET Imaging of GRPR in a Subcutaneous Xenograft Model

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Figure 3. Biodistribution of 64Cu-DOTA-Aoc-BN(7-14) in athymic nude mice bearing sc. PC-3 tumors. Mice were administered 64Cu-DOTA-Aoc-BN(7-14) via the tail vein after tumors were established and the mice sacrificed 2, 4, and 24 h later. A coinjection of Tyr4-BN was administered to another group of mice to determine GRPR specific binding at the 2 h time point. The data are presented as the % ID/g for the mean ( standard deviation of four to six mice per time point.

Figure 4. Transaxial microPET images of athymic nude mice bearing PC-3 tumors 2 h after tail vein injection of 64Cu-DOTAAoc-BN(7-14) in the absence (A,C) or presence (B,D) of a coinjection of Tyr4-BN.

(P ) 0.001) at all time points and greater than the spleen at 2 and 24 h (P < 0.05). MicroPET Imaging. The localization of 64Cu-DOTAAoc-BN(7-14) in PC-3 tumors as determined by microPET imaging is shown in Figure 4. These studies were performed with 500 µCi (250 ng) of 64Cu-DOTA-Aoc-BN(7-14) radiolabeled at a specific activity of 2000 mCi/ mg. Because of the high levels of radioactivity in normal tissues as shown in Figure 3, transaxial images were used to focus on the localization of 64Cu-DOTA-Aoc-BN(7-14) in the tumor. Representative transaxial images are shown of four mice (two mice without competitor (A and C), and two mice with competitor (B and D)) 2 h PI of 64Cu-DOTA-Aoc-BN(7-14). There is clear visualization of the PC-3 tumors in animals on the left (A and C) that did not receive competitor, along with clearance of 64CuDOTA-Aoc-BN(7-14) through the bladder. The mice on

Figure 5. Representative competitive binding assays on membrane preparations of PC-3 tumors (A) or pancreas (B). Established PC-3 tumors or pancreata were harvested from mice and used for membrane preparations. Various concentrations of Tyr4-BN were added to 25 µg of the membranes to inhibit the binding of 125I-Tyr4-BN. The data are presented as the binding in dpm vs the log of the molar concentration of Tyr4BN for the mean of triplicate measurements ( standard deviation.

the right (B and D), which received the competitor, show a decrease in tumor localization of 64Cu-DOTA-Aoc-BN(7-14). It is likely that the decrease in tracer localization for mouse D was not as dramatic as the decrease for mouse B due to incomplete blocking as the tumor for mouse D was 2-fold larger than the others in the imaging studies. Ex Vivo Determination of Receptor Density. The competitive binding studies used to determine the expression of GRPR on pancreas and tumor are shown in Figure 5. These data show that the expression of GRPR is about 14-fold greater on tumor (376 ( 55 fmol/mg of protein) than on the pancreas (27 ( 13 fmol/mg of

760 Bioconjugate Chem., Vol. 14, No. 4, 2003 Table 1. Blood Flow of Various Tissues as Determined by Sacrifice of Athymic Nude Mice Bearing PC-3 Tumors 10 min after Tail Vein Administration of 64Cu-PTSM. Data Are Presented as % ID/g for Three Mice tissue

10 min 64Cu-PTSM (% ID/g)

blood liver sm int spleen kidney muscle pancreas tumor

2.51 ( 0.28 16.06 ( 3.49 8.09 ( 0.96 2.65 ( 0.84 16.87 ( 1.92 3.03 ( 0.43 6.00 ( 0.84 2.29 ( 0.70

protein). Membranes were also made from PC-3 cells and these binding data show that they express about 15-fold more GRPR (5479 ( 410 fmol/mg) than the PC-3 tumors (data not shown). These results are similar to our earlier results with intact PC-3 cells, as 5479 fmol/mg is equal to approximately 4.4 × 105 receptors/cell (based upon 7500 PC-3 cells per microgram of protein). In Vivo Determination of Blood Flow. The results of 64Cu-PTSM blood flow studies in mice bearing PC-3 tumors are shown in Table 1. This demonstrates that the blood flow to the tumor is among the lowest of all of the tissues examined and is not significantly different from blood, spleen, or muscle (P > 0.05). However, the blood flow to the tumor (2.3 ( 0.7% ID/g) is significantly lower (P < 0.01) than the blood flow to the pancreas (6.0 ( 0.8% ID/g). The liver (16.1 ( 3.5% ID/g), kidney (16.9 ( 1.9% ID/g), and small intestine (8.1 ( 1.1% ID/g) are the tissues with the highest blood flow. DISCUSSION

A number of radiolabeled peptides have been successfully used for the noninvasive imaging of tumors overexpressing receptors that have high affinity for these peptides. In particular, peptides specific for somatostatin receptors, vasoactive intestinal peptide receptors, and melanocyte stimulating hormone receptors have been shown to be useful for scintigraphy of tumors overexpressing these receptors (36-38). The gastrin-releasing peptide receptor has also been the target for radiolabeled peptide imaging in recent years. In this regard, GRPRavid peptides have been radiolabeled with 111In and 99mTc and evaluated in animals and humans as potential imaging agents. Breeman et al. demonstrated GRPRspecific imaging of 7315b pituitary tumors in rats using a DTPA-BN analogue radiolabeled with 111In (22). Van de Wiele et al. evaluated the biodistribution and dosimetry of a 99mTc-labeled BN agonist (99mTc-RP527) in healthy (26) and carcinoma bearing (24) volunteers by gamma camera imaging. These studies show the potential of radiolabeled BN analogues for imaging BN-positive tumors and use in humans. Initial radiolabeling attempts of DOTA-Aoc-BN(7-14) with 64Cu achieved specific activities of 130 mCi/mg. Labeling at higher specific activities led to radiolysis of the peptide as determined by radio-TLC. 64Cu-DOTA-AocBN(7-14) labeled at this specific activity was used for the in vitro assays and biodistribution studies. Further radiolabeling attempts achieved specific activities of 2000 mCi/mg by adding gentisic acid to prevent radiolysis. 64Cu-DOTA-Aoc-BN(7-14) labeled at this specific activity was used for the microPET studies and for a biodistribution study at 2 h to confirm the results of the lower specific activity biodistribution. The 64Cu-DOTA-Aoc-BN(7-14) had a high affinity for GRPR (Kd ) 6.1 nM) as shown in Figure 1. This is similar

Rogers et al.

to other studies that demonstrated that chelate-BN analogues and metal-chelate-analogues had IC50 values that were similar to BN(7-14) (19, 20, 22, 27). The Bmax value of 2.7 × 105 receptors/cell is higher than the 4.4 × 104 reported by Reile et al (39). This difference could be a function of the density of the cells when the assays were performed. For example, our cells were harvested in an exponential phase of growth, while harvesting a confluent monolayer may result in receptor down regulation. Figure 2 shows that 64Cu-DOTA-Aoc-BN(7-14) was rapidly internalized into PC-3 cells with 11.4% of the added radioactivity internalized after a 30 min incubation and 21.5% after 4 h. Breeman et al. demonstrated that radiolabeled BN agonists were internalized into GRPRpositive cells, but not radiolabeled antagonists (22). In general, BN analogues that maintain their amidated C-terminal methionine have been shown to be agonists (40), which have potential for tumor imaging when radiolabeled with various radioisotopes as discussed by Van de Wiele et al (25). Thus, although not explicitly demonstrated in these studies, 64Cu-DOTA-Aoc-BN(714) is presumed to be an agonist. This is in agreement with other radiolabeled BN(7-14) analogues that have also been demonstrated to undergo rapid in vitro internalization. For example, LaBella et al. showed that two 99m Tc-labeled BN(7-14) analogues reached 70% internalization into PC-3 cells by 15 min (27, 28). These studies are significant because internalization of radiolabeled peptides has been shown to be important for developing diagnostic and therapeutic agents (41). In vivo biodistribution (Figure 3) showed good localization of 64Cu-DOTA-Aoc-BN(7-14) into PC-3 tumors that was specific for GRPR, as demonstrated by coinjection of an excess of unlabeled BN. These results are favorable to other studies using different BN analogues and different tumor models. In rats bearing 7315b rat pituitary tumors, Breeman et al. reported that an 111In-labeled BN agonist had a tumor localization of 0.05% ID/g at 24 h compared to 2.5% ID/g at 24 h in our mouse model (22). Using the same PC-3 tumor model in mice, La Bella et al. had 0.3-0.6% ID/g in tumor 1.5 h after injection for two 99mTc-labeled BN analogues (27, 28). Smith et al. reported a tumor localization of 4.2% ID/g in the PC-3 mouse tumor model at 1 h for this same DOTA-Aoc-BN(7-14) radiolabeled with 177Lu (18). The localization of 64 Cu-DOTA-Aoc-BN(7-14) in normal tissues, however, is higher than that reported in other studies. Normal tissue uptake (except for pancreas and kidney) of 64CuDOTA-Aoc-BN(7-14) ranged from 0.8 to 13.3% ID/g at 2 h compared to 0.1 to 4.5% ID/g at 1.5 h for the 99mTclabeled BN analogues and 0.1 to 0.4% ID/g at 1 h for the 177 Lu-labeled BN analogues (18, 27, 28). Differences in the chelates and linkers could account for the lower tissue uptake of the 99mTc-labeled BN analogues compared to 64 Cu-DOTA-Aoc-BN(7-14). With regard to the lower uptake of 177Lu-DOTA-Aoc-BN(7-14) compared to 64CuDOTA-Aoc-BN(7-14) the difference may be due to the charge difference between Cu(II) and Lu(III). Alternatively, it has been demonstrated that Cu(II) can transchelate to albumin and superoxide dismutase, leading to higher blood and liver uptake (42, 43). It will be necessary to reduce this normal tissue uptake (particularly in the liver and kidney) for the clinical application of 64Cu-DOTA-Aoc-BN(7-14) as an imaging or therapeutic agent. The use of less lipophilic linkers should reduce the liver uptake and making the overall charge of the peptide either neutral or negative should reduce the kidney accumulation.

MicroPET Imaging of GRPR in a Subcutaneous Xenograft Model

As discussed previously, several BN analogues have been evaluated for imaging GRPR-positive tumors using gamma ray scintigraphy. Here we show the first PET images of GRPR-positive tumors in mice using microPET (Figure 4). MicroPET has been used extensively in recent years for evaluating reporter gene expression in small animal models (44, 45), while microPET imaging of tumor antigens/receptors using radiolabeled peptides has been limited to the somatostatin receptor subtype 2 (46, 47). Transaxial images show good localization of 64Cu-DOTAAoc-BN(7-14) in PC-3 tumors in mice that did not receive a co-injection of competitor. In contrast, the tumor localization was reduced in mice that received the competitor, demonstrating GRPR-specific localization. The tumor for the mouse in Figure 4D was twice the size of the other tumors, which may account for the lack of complete inhibition as was observed in Figure 4B. In addition to the tumor, the bladder is observed due to the clearance of 64Cu-DOTA-Aoc-BN(7-14) through the kidney and urinary excretion. These studies demonstrate the feasibility of using positron-labeled BN analogues for PET detection of GRPR-positive neoplasms. Although good tumor localization was observed for 64Cu-DOTA-Aoc-BN(7-14) in biodistribution and microPET imaging studies, we anticipated even greater localization based upon the in vitro results for the number of receptors per PC-3 cell. To determine if GRPR had been down regulated on PC-3 cells in vivo, we harvested PC-3 tumors and pancreata from mice and performed a competitive binding assay on tissue membrane preparations (Figure 5). This study showed that there were approximately 15-fold less GRPRs on PC-3 cells in vivo than in vitro, but 14-fold greater expression of GRPR per mg of PC-3 tumor than per mg of pancreas. Similar to our results (27 fmol/mg), Fanger et al. reported a level of GRPR on mouse pancreas to be 75 fmol/mg and 31 fmol/mg was reported by Hajri et al. for GRPR in rat pancreas (48, 49). Thus, greater tumor localization of 64 Cu-DOTA-Aoc-BN(7-14) would be expected based upon the GRPR expression on tumor vs pancreas. A preliminary biodistribution study using high specific activity 64Cu-DOTA-Aoc-BN(7-14) (2000 mCi/mg) did not show a difference in localization in tumor or normal tissues at 2 h postinjection when compared to the biodistribution in Figure 3, except for an increase in the pancreas to 24.1% ID/g (data not shown). This implies that GRPRs were saturated in the pancreas using the lower specific activity 64Cu-DOTA-Aoc-BN(7-14), thus reducing the % ID/g. We hypothesize that 64Cu-DOTAAoc-BN(7-14) did not saturate GRPRs on the PC-3 tumors, and the low uptake may be due to low blood flow. In this regard, Krohn describes a kinetic model relating blood flow to cell-bound ligand complexes (50). Krohn suggests that if ligand diffusion to the receptor is rate limiting, then the receptor number cannot be quantitated. Table 1 shows that the blood flow to the PC-3 tumor is 2.6-fold lower than the blood flow to the pancreas, which may limit diffusion of 64Cu-DOTA-Aoc-BN(7-14) to the tumor and underestimate the number of receptors. These results will need to be investigated further to determine if the limited tumor localization is due to low blood flow or other biochemical factors associated with PC-3 tumors. Utilization of another tumor model with similar GRPR expression and increased blood flow may help answer this question. In conclusion, this study demonstrates that a BN analogue can be radiolabeled with the positron-emitter 64 Cu and maintain high affinity and internalize into GRPR-positive cells. Specific tumor localization of 64Cu-

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DOTA-Aoc-BN(7-14) in mice was visualized by microPET imaging and was confirmed by biodistribution studies. Reduction of the uptake of 64Cu-labeled BN analogues in normal tissues by modifying the charge of the peptide and the peptide linker group will be the focus of future studies. In addition, the relationship between blood flow and tumor localization of these analogues will be evaluated in different tumor models. This study demonstrates the potential of BN analogues for detecting GRPR-positive tumors in the clinical setting by PET imaging and possibly for radiotherapy. ACKNOWLEDGMENT

We gratefully acknowledge the expert technical assistance of Sheila Bright, Synethia Kidd, and Richard Kirkman. This work was supported by a grant from the American Cancer Society RPG-00-067-01-CCE (B.E.R.) thanks to a gift from the F.M. Kirby Foundation. MicroPET imaging was supported by an NIH/NCI SAIRP grant 1 R24 CA83060 (M.J.W.). We would also like to thank the Small Animal Imaging Core of the Alvin J. Siteman Cancer Center at Washington University and BarnesJewish Hospital in St. Louis, Missouri for additional support of the microPET imaging. The Core is supported by an NCI Cancer Center Support Grant # 1 P30 CA91842. LITERATURE CITED (1) Sunday, M. E., Kaplan, L. M., Motoyama, E., Chin, W. W., and Spindel, E. R. (1988) Gastrin-releasing peptide (mammalian bombesin) gene expression in health and disease. Lab. Invest. 59, 5-24. (2) Spindel, E. R., Giladi, E., Segerson, T. P., and Nagalla, S. (1993) Bombesin-like peptides: of ligands and receptors. Recent Prog. Horm. Res. 48, 365-391. (3) Moody, T. W., Carney, D. N., Cuttitta, F., Quattrocchi, K., and Minna, J. D. (1985) High affinity receptors for bombesin/ GRP-like peptides on human small cell lung cancer. Life Sci. 37, 105-113. (4) Radulovic, S., Miller, G., and Schally, A. V. (1991) Inhibition of growth of HT-29 human colon cancer xenografts in nude mice by treatment with bombesin/gastrin releasing peptide antagonist (RC-3095). Cancer Res. 51, 6006-6009. (5) Pinski, J., Schally, A. V., Halmos, G., Szepeshazi, K., and Groot, K. (1994) Somatostatin analogues and bombesin/ gastrin-releasing peptide antagonist RC-3095 inhibit the growth of human glioblastomas in vitro and in vivo. Cancer Res. 54, 5895-5901. (6) Qin, Y., Ertl, T., Cai, R. Z., Halmos, G., and Schally, A. V. (1994) Inhibitory effect of bombesin receptor antagonist RC3095 on the growth of human pancreatic cancer cells in vivo and in vitro. Cancer Res. 54, 1035-1041. (7) Reubi, J. C., Wenger, S., Schmuckli-Maurer, J., Schaer, J. C., and Gugger, M. (2002) Bombesin receptor subtypes in human cancers: Detection with the universal radioligand 125I[D-TYR6, β-ALA11, PHE13, NLE14] Bombesin(6-14). Clin. Cancer Res. 8, 1139-1146. (8) Von Schrenck, T., Heinz-Erian, P., Moran, T., Mantey, S. A., Gardner, J. D., and Jensen, R. T. (1989) Neuromedin B receptor in esophagus: Evidence for subtypes of bombesin receptors. Am. J. Physiol. 256, 747-758. (9) Fathi, Z., Corjay, M. H., Shapira, H., Wada, E., Benya, R., Jensen, R., Viallet, J., Sausville, E. A., and Battey, J. F. (1993) BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J. Biol. Chem. 268, 5979-5984. (10) Nagalla, S. R., Barry, B. J., Creswick, K. C., Eden, P., Taylor, J. T., and Spindel, E. R. (1995) Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for

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