Evaluation of 64Cu-and 125I-Radiolabeled Bitistatin as Potential

988

Bioconjugate Chem. 2004, 15, 988−996

Evaluation of 64Cu- and 125I-Radiolabeled Bitistatin as Potential Agents for Targeting rvβ3 Integrins in Tumor Angiogenesis Paul McQuade,† Linda C. Knight,*,‡ and Michael J. Welch† Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, and Department of Diagnostic Imaging, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 . Received February 18, 2004; Revised Manuscript Received July 19, 2004

The formation of new blood vessels (angiogenesis) is a feature common to all solid tumors. The integrin receptor RVβ3, which is found on endothelial cells lining newly growing blood vessels at a higher density than on mature blood vessels, is being explored as a marker for tumor angiogenesis. Bitistatin, a member of the disintegrin family of polypeptides, has affinity for RVβ3 integrins. To determine whether radiolabeled bitistatin could target tumors, its biodistribution was tested in tumor-bearing mice. For initial validation studies, 125I-bitistatin was injected into BALB/c mice bearing EMT-6 mouse mammary carcinoma tumors, a model that is highly vascular but which lacks RVβ3 directly on tumor cells. Tumor uptake reached maximal values (11.7 ( 4.6 %ID/g) at 2 h. Co-injection of 200 µg of unlabeled bitistatin reduced tumor uptake 62%, suggesting that the binding of 125I-bitistatin is receptor-mediated. This work was extended to include the β+-emitting radionuclide 64Cu, which was attached to bitistatin via 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA). This modification did not significantly alter receptor binding in vitro. MicroPET images obtained with 64Cu-DOTA-bitistatin showed that the tumor could easily be identified 4 h after administering the radiopharmaceutical. The biodistribution of 64Cu-DOTA-bitistatin differed from the 125I analogue, in that maximum tumor uptake was nearly 8-fold lower and took at least 6 h to reach maximal binding (1.6 ( 0.2 %ID/g). As with 125 I-labeled bitistatin, the 64Cu conjugate showed a 50% reduction in tumor uptake with the co-injection of 200 µg of unlabeled bitistatin (0.8 ( 0.2 %ID/g). Competition studies with integrin-specific peptides indicated that the tumor uptake was related to both Rvβ3 and RIIbβ3 integrin binding. To see if tumor uptake could be improved upon, 64Cu was tethered to bitistatin using bromoacetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (BAD). Tumor uptake for 64Cu-BAD-2ITbitistatin was higher than the DOTA conjugate at all time points, reaching a maximum at least 6 h postinjection (5.2 ( 0.6 %ID/g); however, this was accompanied by higher uptake in nontarget organs at all time points. Radiolabeled ligands of this type may be useful in the targeting of tumor angiogenesis, but the choice of radiolabeling approach has a significant impact on the in vivo properties of the radioligand.

INTRODUCTION

Detecting cancer early enough to improve patient prognosis remains a difficult problem. Typically, solid tumors must be larger than 10 mm to be reliably detected by anatomic imaging techniques such as computed tomography (CT). In a similar fashion, traditional nuclear medicine techniques are limited to detection of larger tumors, so many lesions are missed. Thus, there is a need to develop new techniques for the early detection of tumors, so that treatment can be started earlier and improve patient prognoses. One of the ways this can be achieved is by targeting the evolving vasculature that nourishes a growing tumor. As has been documented, the expansion of a tumor beyond a few cubic millimeters is dependent on the formation of new blood vessels (angiogenesis) to provide nutrients and oxygen needed by the growing tumor (1). * Author for Correspondence: Linda C. Knight, Ph.D., Department of Diagnostic Imaging, Temple University School of Medicine, 3401 N. Broad St., Philadelphia, PA 19140. Phone (215) 707 4940. (215) 707 8110 (FAX). E-mail: lknight@ temple.edu. † Washington University School of Medicine. ‡ Temple University School of Medicine.

The vasculature of the tumor differs from that found in the rest of the body in that it is relatively immature and contains large numbers of receptors involved in vessel growth (2). The unique character of tumor vasculature therefore would be a good target for pharmaceuticals in both the detection and treatment of a wide variety of cancerous tumors, as all solid tumors are dependent on angiogenesis. It is possible that targeting of tumor vasculature could allow monitoring of smaller tumors than can be detected with the present nuclear medicine radiotracers. One marker of tumor angiogenesis that may be a suitable target is RVβ3, also known as the vitronectin receptor (3). It is a member of the integrin family of receptors which are involved in adhesion and signaling of many types of cells (4-6), and binds ligands containing an Arg-Gly-Asp (RGD) motif. Molecules containing this RGD motif are of interest as antagonists of angiogenesis. One such family of molecules is the disintegrins, which were first reported as RIIbβ3 antagonists isolated from the venoms of various snakes (7). These low molecular mass (5-9 kDa), cysteine-rich polypeptides are constrained by internal disulfide linkages into multiloop structures and

10.1021/bc049961j CCC: $27.50 © 2004 American Chemical Society Published on Web 08/13/2004

64Cu/125I-Bitistatin

for Targeting Angiogenesis

Figure 1. Structures of

64Cu-DOTA-, 125I-,

Bioconjugate Chem., Vol. 15, No. 5, 2004 989

and

64Cu-BAD-2IT-labeled

are the most potent known inhibitors of integrin function. A common feature of the disintegrins is that they contain an RGD or analogous sequence at the apex of one of these loops. The disintegrin bitistatin was selected for initial evaluation as a marker for tumor angiogenesis. When labeled with 123I or 99mTc, bitistatin has been shown to be a promising agent for in vivo imaging of experimental pulmonary emboli (PE) and deep venous thrombi (DVT) (8-11). In a canine model, labeled bitistatin images were characterized by high uptake in thrombi and emboli and low background in muscle and most other tissues except for kidneys and spleen. Bitistatin has been shown to bind to both RVβ3 and RIIbβ3 integrins (12). We hypothesize that binding to both integrins may be advantageous, as bitistatin’s reversible binding to RIIbβ3 on blood platelets may increase the blood residence time and therefore the bioavailability for delivery of radioligand to RVβ3 on endothelial cells. This approach differs significantly from previous studies that have focused on small cyclic RGDcontaining peptides which were designed to have the highest possible affinity for RVβ3, while minimizing platelet binding with the lowest possible affinity for RIIbβ3 (13-16). Molecular modeling studies have shown that the five lysine residues of bitistatin are located on the opposite side of the molecule from the putative binding domain (10). This suggests that bitistatin could be conjugated to bifunctional chelators (BFC) for labeling with radiometals without affecting the binding domain, and this has been successfully accomplished with Hynic modification for 99m Tc labeling. The positron-emitting isotope 64Cu (t1/2 ) 12.7 h, β+ ) 0.655 MeV (19.3%), β- ) 0.573 MeV (39.6%)) is an attractive radionuclide and has found use both as a diagnostic agent by utilizing the high sensitivity of PET imaging, and also as a radiotherapy agent (17). Large quantities of high-specific activity 64Cu can be produced on demand using a biomedical cyclotron (18). In this study, bitistatin was first labeled with 125I by direct radioiodination methods and then with 64Cu to test whether it could be used for PET imaging of tumors.

bitistatin.

EXPERIMENTAL PROCEDURES

General Methods and Materials. All chemicals unless otherwise stated were purchased from SigmaAldrich Chemical Co. (St. Louis, MO). Water was distilled and then deionized (18 MΩ/cm2) by passing through a milli-Q water filtration system (Millipore Corp., Milford, MA). Reversed-phase HPLC separation was achieved using a Dynamax C18, 12 µm, 300 Å, 4.6 × 250 mm column (Rainin Instruments, Woburn, MA), eluted with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid over 30 min at a flow rate of 1.0 mL/min. FPLC analysis was performed using a Sephadex G-25 column equilibrated and eluted with 0.1 M NaCl/HEPES buffer pH 7.4 at a flow rate of 1 mL/min. 64Cu was produced on a CS-15 biomedical cyclotron at Washington University School of Medicine, using published methods (18). Radioactivity was counted with a Beckman Gamma 8000 counter containing a NaI crystal (Beckman Instruments, Inc., Irvine, CA). Bitistatin was purified from the crude venom of Bitis arietans (Miami Serpentarium Laboratories, Punta Gorda, FL) by ion-exchange chromatography followed by RP-HPLC yielding a single peak (8, 19). Mass spectrometry and partial amino acid sequence analysis were used to confirm the identity of bitistatin. 125 I-Bitistatin (Figure 1). Bitistatin (30 µg; 3.3 nmol) in 0.2 M Tris buffer, pH 7.8 was combined with 750 µCi 125 I (Perkin-Elmer Life Sciences, N. Billerica, MA) containing 1 nmol of NaI. The mixture was transferred to a tube containing 20 µg of IodoGen (Pierce Chemical Co, Rockford, IL). After 25 min at room temperature, the reaction was removed from the vial, diluted with saline, and allowed to stand for 5 min without addition of reductant. Unreacted 125I was removed by gel filtration on a Econo-Pak 10DG column (Bio-Rad Laboratories, Hercules, CA) (8). The product had radiochemical purity of greater than 99% as determined by reversed phase HPLC. 64 Cu-DOTA-Bitistatin (Figure 1). The activation of 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) and subsequent conjugation to bitistatin was carried out as described previously (20). Briefly, activa-

990 Bioconjugate Chem., Vol. 15, No. 5, 2004

tion of DOTA with EDC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) and sulfo-NHS (N-hydroxy-sulfosuccinimide) was carried out at 4 °C for 30 min. A 10fold excess of DOTA and sulfo-NHS over EDC was used to ensure only one carboxylic acid moiety per DOTA was activated. The pH was then adjusted to 8.5 and enough bitistatin was added to generate a bitistatin:NHS-DOTA ratio of 1:100. The reaction vessel was then kept at 4 °C for 15 h, with continuous end-over-end mixing. The conjugated peptide was then purified using a Centricon-3 centrifugal filter (MW cutoff 3000; Millipore, Billerica, MA). The average number of DOTA units attached to bitistatin was found to be 1.76, as determined by TLC (21). The conjugated bitistatin was labeled with 64Cu as follows: bitistatin-DOTA (25-40 µg) was combined with 64 CuCl2 (6-8 mCi) in 0.03 M ammonium citrate buffer (pH 6.5), to give a total volume of 150 µL. The reaction mixture was heated at 55 °C for 40 min, after which diethylenetriaminepentaacetic acid (DTPA) was added to challenge nonbound radiometals. The radiolabeled peptide was purified with a Bio-Spin 6 column (Bio-Rad, Hercules, CA) centrifuged at 2500 rpm for 6 min. Using this method, specific activities of 250 µCi/g were typically obtained, with radiopurity of around 96% as determined by FPLC. 64 Cu-BAD-2IT-Bitistatin (Figure 1). The activation of bromoacetamidobenzyl-1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid (BAD) (Macrocyclics, Dallas, TX) and subsequent conjugation to bitistatin was carried out as described previously (22). To a solution of bitistatin (0.1 M ammonium phosphate, pH 8.0) was added in this order: excess BAD in aqueous solution and freshly prepared 2-iminothiolane (2IT) in 50 mM triethanolamine hydrochloride, pH 8.7. The overall bitistatin/BAD/ 2IT ratio was 1:100:20, and the pH of the resulting solution was adjusted to 8.0. The reaction was then incubated at 37 °C for 40 min. The conjugated peptide was purified using a Centricon-3 centrifugal filter (MW cutoff 3000). The average number of BAD units attached to bitistatin was found to be 0.55, as determined by TLC (21). The conjugated bitistatin was labeled with 64Cu in a similar manner as the DOTA analogue. Briefly, BAD2IT-bitistatin (120 µg) was combined with 64CuCl2 (1012 mCi) in 0.1 M ammonium citrate buffer (pH 6.5), to give a total volume of 150 µL. The reaction mixture was heated at 55 °C for 40 min, after which DTPA was added to challenge nonbound radiometals. The radiolabeled peptide was purified via gel filtration with a PD-10 column. Specific activities of 50 µCi/µg were obtained, with a radiopurity of 90% as determined by FPLC. Stability of Radiolabel. To measure the in vivo stability of 64Cu-DOTA-bitistatin in mice, plasma samples obtained at 1 h were purified by size exclusion chromatography and the fractions obtained were counted on a gamma counter. In an analogous in vitro experiment, 64 Cu-DOTA-bitistatin was incubated with human serum albumin (HSA) at 37 °C for 20 h, after which the solution was then separated by size exclusion chromatography to determine the amount of 64Cu that remained bound to DOTA-bitistatin. Inhibition of Platelet Aggregation In Vitro. Blood was obtained from aspirin-free human donors and anticoagulated with 0.1 vol of 3.8% sodium citrate. The blood was centrifuged at 160g for 12 min to obtain plateletrich plasma (PRP). The concentration of platelets in human PRP was determined using a hemacytometer, and then diluted with the donor’s own plasma to obtain a

McQuade et al.

platelet concentration of 300 000/µL. For each measurement, 400 µL of adjusted PRP was placed in a cuvette and stirred at 37 °C. Vehicle (saline) or various concentrations of peptide was added in a volume of 10 µL and stirred at 37 °C for 1 min while recording light transmission. ADP was then added (final concentration 10 µM) to induce platelet aggregation. Platelet aggregation was determined by change in light transmission through the PRP, which was recorded for 5 min after ADP addition. The IC50 was determined (by least-squares fitting) as the concentration of peptide required to produce 50% inhibition of the response to ADP in the presence of the vehicle. Results were normalized to the results for the tetrapeptide Arg-Gly-Asp-Ser (RGDS). Biodistribution Studies. All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals established by Washington University’s Animal Studies Committee. Biodistribution studies were carried out on female BALB/c mice (20-25 g) that had been implanted with 1 × 106 EMT-6 cells subcutaneously into the nape of the neck. This cell line was chosen as the resulting tumor is fast growing, and presumably contains vasculature that is rich in RVβ3 integrins, while the cells themselves are devoid of RGD binding receptors (23). EMT-6 murine mammary carcinoma cells were obtained from the Yale Cancer Center. Tumors were allowed to grow for 10 days after implantation of cells, after which time the animals received either 1 µCi of 125I-bitistatin (1.1 pmol), 7 µCi 64Cu-DOTAbitistatin (3.1 pmol), or 5 µCi 64Cu-BAD-2IT-bitistatin (11 pmol), in 125 µL of saline via lateral tail vein injection. In an analogous experiment to monitor the effect excess bitistatin would have on tumor uptake, 200 µg (22 nmol) of unlabeled bitistatin was injected into mice implanted with EMT-6 cells immediately prior to administering 125Ior 64Cu-DOTA-labeled bitistatin. This represents a 20 000and 7100-fold increase over the mass of peptide injected with 125I- and 64Cu-DOTA-bitistatin respectively as compared to the nonblocked biodistribution studies. Animals were then euthanized at desired time points (30 min, 2 and 4 h for 125I-bitistatin, 2, 4, and 6 h for 64Cu-DOTAbitistatin, 30 min, 2 h, 4 h, and 6 h for 64Cu-BAD-2ITbitistatin). Tissues and organs of interest were then removed and weighed and radioactivity was measured in a gamma counter. The percent dose per gram (%ID/g) was then calculated. Because bitistatin can bind to both RVβ3 and RIIbβ3 integrins, a separate study was carried out to determine the effect blocking each integrin individually would have on tumor uptake. BALB/c mice bearing EMT-6 tumors were injected with 7 µCi of 64Cu-DOTA-bitistatin (3.1 pmol) in 125 µL of saline via lateral tail vein injection. Immediately prior to injection of the radiopharmaceutical 40 µg (70 nmol) of the RVβ3-specific peptide cyclo[RGDfV] (24) (Bachem Bioscience Inc, King of Prussia, PA), or 55 µg (66 nmol) of the RIIbβ3-specific peptide eptifibatide (25) (GL Biochem (Shanghai) Ltd, Shanghai, China), was injected. After 2 h the animals were euthanized and their organs were counted. Cell Binding Studies. The extent of platelet binding in vivo was assessed by collecting blood samples by cardiac puncture in mice pretreated with 64Cu-DOTAbitistatin and immediately layering over 100 µL of 30% sucrose in a microfuge tube. After centrifugation at 13000g for 5 min, the plasma was carefully removed by pipet to a clean tube for counting. The tip of the microfuge tube containing the cell pellet was snipped off into a second tube for counting. The percentage of total counts in the cell pellet was calculated.



To determine the extent of cell binding directly to EMT-6 cells, 64Cu-DOTA-, 64Cu-BAD-2IT-, and 125I-bitistatin were incubated with EMT-6 cells in vitro. EMT-6 cells were suspended to a concentration of 5 × 106 cells/ mL in PBS. Cell number was determined with a hemocytometer, and viability was determined by trypan blue exclusion. For the assay, the cell suspension was supplemented with CaCl2 and MnCl2 (final concentration 0.5 mM each), then radiolabeled bitistatin was added to a final concentration of 2 nM. Nonspecific binding was determined in the presence of 2.5 mM Na4EDTA to chelate divalent cations which are needed for integrin function (6, 9). After 1 h at 37 °C, 100 µL aliquots of the cell suspension were layered over 100 µL of a 30% sucrose solution in a microfuge tube. The cells were then pelleted from the media by centrifuging at 13000g for 3 min. The supernatant was transferred into a clean tube for counting and the tip containing the cell pellet was snipped from the microfuge tube and placed in a second tube for counting. Both supernatant and pellet were counted, and the percentage of the radioactivity associated with the cells was calculated. This was done in triplicate for each incubation condition. MicroPET Imaging. Whole body microPET images were obtained on a microPET-R4 system (Concorde Microsystems, Knoxville, TN) (26). Imaging studies were carried out on BALB/c mice that had received an implant of EMT-6 cells (1 × 106 cells) in the nape of the neck (n ) 14), with each mouse receiving 250 µCi of 64Cu-DOTAbitistatin (0.1 nmol). At each time point (4, 6, and 15 h) half of the animals were given 200 µg (22 nmol; 200-fold mass increase over 64Cu-DOTA-bitistatin injected) of unlabeled bitistatin prior to the radiopharmaceutical to monitor the effect it would have on tumor uptake and clearance. The mice were imaged in pairs (blocked vs unblocked) side by side, and standardized uptake values (SUV) for the tumor, kidneys, and liver were obtained at all time points. The SUVs were determined by drawing regions of interest (ROI) around the target tissue to generate the radioactive concentration, which was decay corrected to the time of injection, multiplied by the mouse body weight (grams), and divided by the injected dose (nCi) to give the normalized tissue uptake to injected dose per body weight (27). Statistical Methods. To compare differences between the 64Cu- and 125I-labeled bitistatin, a Student’s t-test was performed. Differences at the 95% confidence level (p < 0.05) were considered significant. RESULTS

Stability of 64Cu-DOTA-Bitistatin. When 64CuDOTA-bitistatin was incubated with HSA at 37 °C for 20 h, 90% of the radiopharmaceutical remained in its original form (no breakdown or plasma protein binding) (Figure 2). Size exclusion chromatography of plasma samples obtained from mice at 1 h did not contain enough radioactivity for accurate assessment by the in-line detector. However, subsequent counting of the fractions on a gamma counter showed that in both blocked and unblocked mice >95% of 64Cu in mouse plasma samples was in the form of 64Cu-DOTA-bitistatin. From these results, we can infer that 64Cu-DOTA-bitistatin has a high in vivo stability in plasma. Platelet Binding Studies. To determine whether conjugation of DOTA to bitistatin affected its ability to bind to RIIbβ3 integrin, DOTA-bitistatin and unmodified bitistatin were compared for their ability to inhibit human platelet aggregation via binding to RIIbβ3. The IC50


Figure 2. Radioactive profile of 64Cu-DOTA-bitistatin before (top) and after (bottom) incubation in HSA for 20 h. The Sephadex G-25 column was eluted with 0.1 M NaCl/HEPES buffer, pH 7.4, at 1 mL/min.

Figure 3. Inhibition of ADP-induced human platelet aggregation by unmodified bitistatin (open squares), DOTA-bitistatin (gray filled triangles), and Arg-Gly-Asp-Ser tetrapeptide (filled circles).

for inhibition of human platelet aggregation was 163 ( 27 nM for DOTA-bitistatin, compared with 140 ( 11 nM for the same lot of bitistatin before modification (Figure 3). There was no significant difference between these groups (p ) 0.46). Binding of 64Cu-DOTA-bitistatin to platelets in vivo was found to be about 60% of the total blood activity in blood samples drawn at 1 h after injection. This is similar to the percentage of platelet binding of 125I-bitistatin (64%) found in mice at 1 h. In mice which also received 200 µg of unlabeled bitistatin, the level of platelet-bound 125 I-bitistatin was reduced to 21%. EMT-6 Binding Studies. Binding of 125I-, 64Cu-DOTA-, and 64Cu-BAD-2IT-bitistatin to EMT-6 cells in vitro is shown in Figure 4. Binding of 125I-bitistatin (6.4 ( 0.7%) was not significantly higher than nonspecific binding (4.3 ( 1.7%; p ) 0.22). Similar results were found for both 64Cu-DOTA-bitistatin (control 9.8 ( 0.8%, nonspe-


McQuade et al.

Figure 4. Binding of 64Cu-DOTA-, 125I-, and 64Cu-BAD-2ITlabeled bitistatin to EMT-6 cells after incubation at 37 °C for 1 h. Nonspecific binding was determined in the presence of EDTA. Each point is based on triplicate samples, with error bars representing standard deviations. Table 1. Biodistribution Data for 125I-Bitistatin in BALB/c Mice Implanted with EMT-6 Tumorsa 30 min blood lung liver spleen kidney muscle fat heart tumor abd aorta thyroid

7.31 ( 1.69 14.09 ( 8.80 4.25 ( 1.26 12.35 ( 2.78 57.60 ( 31.08 2.13 ( 0.39 3.28 ( 0.56 2.95 ( 3.09 6.19 ( 2.02 6.84 ( 2.10 14.15 ( 3.19

2h

2 h blk

4h

5.16 ( 1.78 2.32 ( 1.03 2.21 ( 0.45 9.89 ( 5.25 2.29 ( 0.89 2.69 ( 0.43 2.35 ( 0.91 1.14 ( 0.44 0.97 ( 0.26 8.19 ( 2.18 2.98 ( 0.63 2.91 ( 0.29 14.44 ( 6.46 10.14 ( 4.35 4.45 ( 0.70 1.77 ( 0.46 1.62 ( 0.61 0.83 ( 0.25 1.79 ( 0.68 0.95 ( 0.41 0.97 ( 0.21 3.22 ( 3.88 0.76 ( 0.37 0.81 ( 0.75 11.68 ( 4.56 4.39 ( 2.75 5.35 ( 1.97 5.76 ( 0.62 3.33 ( 2.77 1.77 ( 0.33 128.4 ( 74.16 105.36 ( 36.42 235.22 ( 93.48

a Blocked animals given 200 µg of bitistatin. Data presented as %ID/g ( SD (n ) 5).

Table 2. Biodistribution Data for 64Cu-DOTA-Bitistatin in BALB/c Mice Implanted with EMT-6 Tumors 2h

4h

6h

6 h blk

blood 0.53 ( 0.11 0.50 ( 0.03 0.53 ( 0.04 0.22 ( 0.05 lung 1.61 ( 0.03 1.36 ( 0.13 1.98 ( 0.07 1.06 ( 0.37 liver 2.60 ( 0.09 3.45 ( 0.64 4.66 ( 0.44 2.63 ( 0.70 spleen 2.42 ( 0.13 2.29 ( 0.13 3.07 ( 0.28 1.39 ( 0.14 kidney 41.27 ( 4.74 61.27 ( 6.20 57.93 ( 9.91 62.70 ( 5.65 muscle 0.23 ( 0.04 0.24 ( 0.02 0.33 ( 0.03 0.18 ( 0.05 heart 0.45 ( 0.02 0.64 ( 0.04 0.85 ( 0.03 0.45 ( 0.12 tumor 1.13 ( 0.07 1.03 ( 0.25 1.55 ( 0.19 0.78 ( 0.18 abd. aorta 2.10 ( 2.15 1.04 ( 0.21 1.43 ( 0.22 0.51 ( 0.13 a Blocked animals given 200 µg of bitistatin. Data presented as %ID/g ( SD (n ) 4).

cific 9.8 ( 0.5%; p ) 0.94) and 64Cu-BAD-2IT-bitistatin (control 5.0 ( 0.9%, nonspecific 5.0 ( 0.8%; p ) 0.99). Biodistribution Studies. For 125I-bitistatin, tumor uptake reached a maximum at 2 h postinjection (11.7 ( 4.6 %ID/g) and then decreased (5.4 ( 2.0 %ID/g) after 4 h (Table 1). Co-injection of 200 µg of unlabeled bitistatin reduced tumor uptake at 2 h to 4.4 ( 2.8 %ID/g (p ) 0.01), indicating receptor-mediated uptake. At 30 min, kidney uptake was high (57.6 ( 31.1 %ID/g) but cleared rapidly and after 4 h was 4.4 ( 0.7 %ID/g. As with other biomolecules directly radiolabeled with iodine, accumulation in the thyroid was observed. In the studies undertaken with 64Cu-DOTA-bitistatin, the biodistribution differed from that of the 125I analogue in that the maximum tumor uptake was lower (1.6 ( 0.2 %ID/g, p < 0.001) and was achieved 6 h postinjection (Table 2). As with 125I-bitistatin, the co-injection of 200 µg of unlabeled bitistatin reduced tumor uptake 2-fold

Figure 5. Comparative uptake of 64Cu-DOTA-bitistatin in select organs, obtained from BALB/c mice implanted with EMT-6 tumors at 2 h. Control animals received only 64CuDOTA-bitistatin, while blocked animals received either 40 µg of cyclo[RGDfV] or 55 µg of eptifibatide immediately prior to administering tracer. Data presented as %ID/g ( SD (n ) 4).

(0.78 ( 0.18 %ID/g) at 6 h (p ) 0.007). The clearance properties of 64Cu-DOTA-bitistatin also differed significantly from those observed for the 125I analogue. Initially kidney uptake was high (41.3 ( 4.7 %ID/g), and continued to increase up to 4 h (61.3 ( 6.2 %ID/g) before reaching a plateau. Binding to integrins RVβ3 and RIIbβ3 was selectively blocked with the integrin specific peptides cyclo[RGDfV] and eptifibatide. A comparison of tissue uptake in selected organs is shown in Figure 5. Blocking of RVβ3 integrin with cyclo[RGDfV] had no effect on blood level (0.7 ( 0.2 %ID/g) or uptake in blood rich organs such as the lung (2.2 ( 0.5 %ID/g) or abdominal aorta (0.9 ( 0.1 %ID/g) at 2 h compared to controls. Tumor uptake however was significantly reduced (p ) 0.009) to 0.5 ( 0.1 %ID/g, an approximate 2-fold reduction compared to control values. When the RIIbβ3 selective peptide eptifibatide was administered, significant reductions (p < 0.01) were seen in blood (0.6 ( 0.1 %ID/g), lung (1.3 ( 0.5 %ID/ g), aorta (0.5 ( 0.2 %ID/g), and spleen (2.5 ( 0.7 %ID/g) levels at 2 h compared to the control mice. This was accompanied by a similar 2-fold reduction in tumor uptake (0.5 ( 0.1 %ID/g; p ) 0.003), similar to the effect seen with cyclo[RGDfV]. Unexpectedly, kidney levels in the mice receiving eptifibatide (21.5 ( 6.2 %ID/g) was significantly lower (p ) 0.01) than in mice that had not received competing peptide (36.7 ( 5.6 %ID/g) or in the mice that had received cyclo[RGDfV] (35.9 ( 9.9 %ID/g). Because tumor localization of 64Cu-DOTA-bitistatin was lower than expected, a different 64Cu conjugate was tested to see if tumor targeting could be improved. Tumor uptake for 64Cu-BAD-2IT-bitistatin was higher than the DOTA conjugate at all time points examined (p < 0.001), reaching a maximum 6 h postinjection (5.2 ( 0.6 %ID/g) (Table 3). At 6 h kidney and liver uptake had reached 221 ( 63 and 19.1 ( 4.1 %ID/g, respectively. Uptake in these nontarget organs was higher than those seen with the DOTA conjugate and substantially higher than those obtained for 125I-bitistatin (p < 0.001). MicroPET Imaging. Transaxial slices showing the localization of 64Cu-DOTA-bitistatin in EMT-6 tumors as determined by microPET imaging are shown in Figure 6. Four hours after administering 64Cu-DOTA-bitistatin no statistical difference (p ) 0.98) in tumor SUV data was observed between the control (0.15 ( 0.03) and blocked mice (0.15 ( 0.03). It was not until 6 h postin-




Table 3. Biodistribution Data for 64Cu-BAD-2IT-Bitistatin in BALB/c Mice Implanted with EMT-6 Tumors 30 min

2h

4h

6h

blood 3.86 ( 2.07 1.36 ( 0.30 1.61 ( 0.95 1.51 ( 0.28 lung 3.54 ( 0.93 3.46 ( 1.02 4.32 ( 2.46 5.09 ( 0.89 liver 9.79 ( 3.66 15.33 ( 6.24 15.69 ( 5.89 19.24 ( 4.12 spleen 3.95 ( 1.72 4.12 ( 0.55 4.66 ( 2.40 4.80 ( 0.97 kidney 168.33( 79.67 229.43( 52.04 184.96( 23.00 221.15( 63.36 muscle 1.50 ( 0.69 1.19 ( 0.31 1.21 ( 0.66 1.07 ( 0.15 fat 1.48 ( 1.06 1.12 ( 0.27 1.34 ( 1.40 0.97 ( 0.10 heart 1.56 ( 0.54 1.65 ( 0.28 1.91 ( 0.77 2.39 ( 0.43 tumor 3.57 ( 1.11 4.70 ( 1.61 4.76 ( 2.23 5.18 ( 0.60 abd aorta 4.16 ( 1.52 3.86 ( 0.64 4.45 ( 1.11 6.00 ( 5.12 a

Data presented as %ID/g ( SD (n ) 4).

jection that tumor uptake in the control and blocked animals were statistically different: SUV ) 0.17 ( 0.02 and 0.09 ( 0.01, respectively (p < 0.02). After 15 h, tumor SUV in the control animals was 0.19 ( 0.01, while in the blocked animals tumor SUV was significantly lower (0.12 ( 0.01, p < 0.001). These data agree with qualitative examination of Figure 6, where blocking is observed at 6 and 15 h. Significant uptake of 64Cu-DOTA-bitistatin in the liver and kidneys was observed at all time points. DISCUSSION

Previous studies for the detection of angiogenesis have focused on small cyclic RGD-containing peptides which bind selectively to RVβ3 integrins, with the goal of minimizing platelet binding via RIIbβ3 (13-16). This approach usually results in fast blood clearance and is usually accompanied by low tumor uptake, possibly because of inadequate contact time with the target receptors. In this study, we have taken the opposite approach by utilizing a ligand that has affinity for Rvβ3 integrins, allowing binding to receptors involved in angiogenesis and to RIIbβ3 integrins, prolonging the bioavailability of the radioligand. A source of naturally occurring high-affinity ligands for integrins is found in the family of molecules known as disintegrins. These cysteine-rich peptides were first isolated from viper venoms and to date close to 50 have been isolated (28). Of these peptides, bitistatin was chosen as the targeting molecule for these studies, as previous work has shown that it binds to both RIIbβ3 and Rvβ3, and has been successfully used for in vivo imaging when directly radiolabeled with 123I (8, 9), or when labeled with 99mTc via hydrazinonicotinate (Hynic) linker (10, 11). In studies in a canine model, labeled bitistatin exhibited high uptake in thrombotic lesions and very low uptake in most background tissues. Another characteristic of bitistatin is that it is displaced slowly from the activated RIIbβ3 receptor (9), and it was hoped that this slow offrate would be observed on the Rvβ3 integrin as well. It should be noted that other disintegrins have been reported to have higher affinity and selectivity for Rvβ3 than bitistatin, and thus may be better for tumor angiogenesis targeting. Studies with these peptides are ongoing. Both in vitro and in vivo studies were performed using EMT-6 murine mammary carcinoma cells. This cell line was chosen as the cells themselves are devoid of RGD binding receptors (23, 29), while immunohistochemical staining of untreated EMT-6 tumors have shown a high concentration of vascular endothelial growth factor (VEGF) (30). This is important as it has been shown that VEGF directly activates Rvβ3 leading to an increased concentration of this integrin on endothelial cells (31). It would be desirable to perform immunohistochemical staining of EMT-6 tumors to confirm the presence of Rvβ3; however,

at present the available antibodies for Rvβ3 do not crossreact with murine tissue. When the three labeled conjugates of bitistatin were incubated with EMT-6 cells, there was no significant binding of any of the conjugates above nonspecific levels, confirming that there is no integrin receptor binding of these conjugates to EMT-6 cells. Slight differences in the level of nonspecific binding were observed, however. The higher binding of the 64Cu-DOTA-bitistatin conjugate may be explained by hydrogen bonding of the negatively charged carboxylate arms present on DOTA to positively charged moieties on the cell surface. It is possible that this was not seen for the BAD conjugate because the number of conjugates attached was 3-fold lower, 1.76 vs 0.55, respectively. In other investigations, the ability of bitistatin to bind to cells expressing Rvβ3 has been demonstrated by the finding that 99mTc- or 125I-bitistatin binds in vitro to cultured human endothelial cells and to two Rvβ3 receptor positive tumor cell lines, and that this binding is completely abolished by competition with cyclo[RGDfV] (32). In most previously published studies, the ability of radiolabeled RGD-containing peptides to image tumor angiogenesis was obscured by the fact that the tumors were grown from cell lines that express Rvβ3 directly on the tumor cells. In these cases, it was impossible to determine whether targeting was to the tumor vasculature or directly to the tumor cells themselves. In this study, we have provided evidence for targeting of tumor vasculature without targeting the tumor cells directly. This is likely to be a more challenging task, because the density of the target receptors may be much lower due to the lower number of tumor-associated endothelial cells compared to the number of tumor cells in the tumor mass. The tumor uptakes achieved in this study (11.7 %ID/g at 2 h for 125I-bitistatin and 5.18 %ID/g at 6 h for 64CuBAD-2IT-bitistatin) compare favorably with uptakes of small RGD-containing peptides which have been evaluated in models employing Rvβ3-positive tumor cell lines such as murine osteosarcoma, M21 melanoma, and OVCAR-3 ovarian carcinoma (33). It is notable that studies with an 18F-labeled glycosylated cyclo[RGDfK], one of the most promising synthetic peptide radioligands, showed good uptake in Rvβ3-positive M21 tumors in mice, but imaging showed no focal tracer accumulation in control tumors using Rvβ3-negative M21-L cells (34). Although the tumor/background ratios obtained with bitistatin conjugates are not as high as have been reported with other tracers binding to tumor cell membranes and definitely could be improved upon, it is encouraging that adequate targeting to vasculature was achieved to enable focal imaging at 4-15 h. Biodistribution studies carried out with 125I-bitistatin in mice implanted with EMT-6 cells showed that maximum tumor uptake was observed 2 h postinjection, with elevated uptake in blood-rich organs such as the lung, spleen, and heart. This appeared to be related to receptor binding, as the co-injection of 200 µg of unlabeled bitistatin substantially reduced uptake in these organs. This work was then extended to positron emitting nuclides so that the sensitivity and high resolution of PET could be used to visually identify tumors. For these studies, 64Cu was chosen due to its availability, energy of positron emission, and the stability of the final product. However, unlike iodine it must be attached via a bifunctional chelator (BFC) which is covalently bound to lysine side chains (35-38). 1,4,7,10-TetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid (DOTA) was chosen as the


McQuade et al.

Figure 6. Transaxial images of BALB/c containing EMT-6 murine mammary carcinoma tumors 4, 6, and 15 h after tail vein injection of 250 µCi of 64Cu-DOTA-bitistatin. Blocked mice were co-injected with 200 µg of bitistatin (T ) tumor).

metal chelating agent due to its commercial availability and its flexibility to bind a variety of metal ions including Ga3+, In3+, and Y3+. MicroPET images obtained with 64 Cu-DOTA-bitistatin showed that EMT-6 tumors could be identified after 4 h. As with 125I-bitistatin, the coinjection of 200 µg of bitistatin reduced uptake in the tumor and blood-rich organs, indicating receptor mediated uptake. However, tumor uptake of 64Cu-DOTAbitistatin was 87% lower than the 125I analogue and did not reach a maximum until at least 6 h postinjection. In addition, the percentage of 64Cu-DOTA-bitistatin found in blood-rich organs was lower than observed for the 125I analogue. This was surprising as the 64Cu-DOTA conjugate appeared to be stable both in vitro and in vivo, with no significant change in potency for inhibition of human platelet aggregation by DOTA-bitistatin as compared unmodified bitistatin. Furthermore, although total blood levels for 125I-bitistatin and 64Cu-DOTA-bitistatin were different at 1 h postinjection, for both tracers a similar percentage of blood activity was bound to platelets in vivo at this time. A possible explanation of this is that during conjugation of DOTA to bitistatin, some of the conjugation locations resulted in unfavorable alterations in bitistatin’s receptor binding. These alterations may be more pronounced after labeling, which was not tested in the platelet assay in vitro. Conjugates that have impaired binding to platelets may leave the blood faster by renal clearance. At 1 h, if these forms have been substantially cleared, total blood activity would be lower, but the percent of total blood activity which is platelet-bound could be the same as for the 125I-labeled bitistatin. Studies with site-directed mutagenesis to alter the locations of attachment of bifunctional chelating agents to bitistatin have shown that conjugation of Lys35 with Hynic followed by 99mTc labeling results in a slight loss of binding to RIIbβ3 (39). Unfortunately, we were unable to test for differences among the conjugates for binding to Rvβ3 in

vitro in this study because the EMT-6 cells do not express the receptor. Future studies with Rvβ3 positive cells or purified integrin will need to be done. These observations suggest that modification of lysine residues thought to be far from the binding domain could have an effect on bitistatin’s pharmacokinetics. To investigate this further, the BFC bromoacetamidobenzyl1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (BAD) was examined. This chelator was chosen because the copper binding domain of BAD is identical to DOTA, but the linker unit between the macrocycle and the lysine residue is longer, and any steric interactions that may occur are minimized. As with the DOTA conjugate, maximum tumor uptake did not occur until 6 h postinjection; however, tumor levels were 3-4-fold higher at all time points. This demonstrates that modifying the same lysine residues along the polypeptide backbone with differing conjugates can have a profound effect on the pharmokinetics, which alter not only receptor-mediated uptake but also uptake in clearance organs, as can be seen with elevated uptake in nontarget tissue compared to the DOTA analogue. Despite the increased tumor uptake compared to 64Cu-DOTA-bitistatin, the overall tumor-to-tissue ratios obtained with 64Cu-BAD-2IT-bitistatin were similar to those obtained with the 64CuDOTA analogue (Table 4). Kidney uptake and retention of both 64Cu-DOTA- and 64 Cu-BAD-2IT-bitistatin was substantial. Initially, like 125 I-bitistatin, kidney uptake was high, but unlike the 125I analogue they were retained and levels increased over time. The 125I direct label was not as stable in vivo as evidenced by the thyroid uptake. It is possible that reaching maximal uptake in tumor at 2 h reflects the instability of the iodine label in blood preventing higher uptake beyond that time. More work is needed to select the optimal labeling method for best tumor targeting and background clearance.




Table 4. Comparison of Selected Tumor-to-Tissue Ratios (%ID/g) of BALB/c Mice Bearing EMT-6 Tumors

64Cu-DOTA

and

64Cu-BAD-2IT-Labeled

64Cu-DOTA

tumor/blood tumor/liver tumor/kidney tumor/muscle

Bitistatin in

64Cu-2IT-BAD

2h

4h

6h

2h

4h

6h

2.2 ( 0.5 0.4 ( 0.1 0.03 ( 0.006 3.2 ( 1.5

2.1 ( 0.7 0.3 ( 0.02 0.02 (0.003 4.3 ( 0.8

2.9 ( 0.2 0.3 ( 0.1 0.03 ( 0.008 4.7 ( 0.8

3.4 ( 0.4 0.3 ( 0.03 0.02 ( 0.002 4.0 ( 0.7

3.2 ( 0.7 0.3 ( 0.03 0.03 ( 0.01 4.0 ( 0.3

3.5 ( 0.4 0.3 ( 0.02 0.03 ( 0.007 4.9 ( 0. 9

These studies demonstrated that tumor uptake of 125Iand 64Cu-DOTA-bitistatin was dependent on binding both to RVβ3 integrins on endothelial cells and RIIbβ3 integrins found in circulating blood platelets. Selectively blocking Rvβ3 with cyclo[RGDfV] or RIIbβ3 with eptifibatide, an antithrombotic drug, each reduced tumor uptake of 64CuDOTA-bitistatin. Blocking with eptifibatide also led to reductions in radiotracer levels in blood and blood-rich organs such as spleen and lung. Blocking with cyclo[RGDfV] led to reductions only in tumor and spleen, an organ that has previously been reported to take up RVβ3targeted peptides (13). These data indicate that while tumor uptake is indeed mediated by RVβ3 receptor binding, the increased blood retention caused by binding to RIIbβ3 has an equivalently large effect, suggesting that the observed tumor uptake of labeled bitistatin is dependent on both Rvβ3 and RIIbβ3 integrins. Unexpectedly kidney uptake in the mice receiving eptifibatide was significantly lower (p ) 0.01) than controls or in the mice that had received cyclo[RGDfV]. One possible explanation is that eptifibatide or some metabolite inhibits the tubular reabsorption of 64CuDOTA-bitistatin, similar to the effect seen when an infusion of amino acids is given (40). In summary, bitistatin, when directly labeled with 125I or with 64Cu via the BFCs DOTA and BAD was shown to accumulate in tumors in which the tumor cells themselves do not express RVβ3 integrin. Uptake was determined to be receptor-mediated by a combination of RIIbβ3 and RVβ3 integrins. 125I-bitistatin achieved a higher tumor uptake than bitistatin labeled with 64Cu using a BFC. The biodistribution data suggest a different timeactivity profile for uptake and washout of 125I-bitistatin from the tumor as compared to its 64Cu analogue. This along with the differing tumor and renal uptake for DOTA and BAD conjugated bitistatin suggest that modification of the side chains can have a dramatic affect on the distribution, stability, and clearance properties of radiolabeled bitistatin. Even though tumor uptake of 64 Cu-DOTA-bitistatin was significantly lower than that seen with 125I-bitistatin, microPET imaging showed the tumor could be identified as quickly as 4 h. Therefore, radiolabeled ligands of this type may be useful in the targeting of tumor angiogenesis, but as demonstrated here the choice of radiolabeling approach has a significant impact on the in vivo properties of the radioligand. ACKNOWLEDGMENT

The authors thank Jan E. Romano for purification of bitistatin and for performing platelet aggregation assays. Preparation of cells for implantation and uptake studies was done by Susan Adams. MicroPET imaging and evaluation was performed by John A Engelbach and Jerrel R. Rutlin, with biodistribution studies done with the assistance of Terry Sharp, Lynne Jones, and Nicole Fettig. This work was supported by NIH R24 CA86307 (M.J.W.), NIH R01 CA96792 (L.C.K.), and NIH R01 HL54578 (L.C.K.). We would also like to thank the Small Animal Imaging Core of the Alvin J. Siteman Cancer

Center at Washington University and Barnes-Jewish Hospital in St. Louis, Missouri for additional support of the microPET imaging and the Statistics Core for statistical analysis. The Core is supported by an NCI Cancer Center Support Grant #1 P30 CA91842. The production of copper radionuclides is supported by a grant from the National Cancer Institute (CA86307). LITERATURE CITED (1) Folkman, J. (1971) Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 285, 1182-1186. (2) Keshet, E., and Ben-Sasson, S. A. (1999) Anticancer drug targets: approaching angiogenesis. J. Clin. Invest. 104, 14971501. (3) Harris, T. D., Kalogeropoulos, S., Nguyen, T., Liu, S., Bartis, J., Ellars, C., Edwards, S., Onthank, D., Silva, P., Yalamanchili, P., Robinson, S., Lazewatsky, J., Barrett, J., and Bozarth, J. (2003) Design, synthesis, and evaluation of radiolabeled integrin alpha v beta 3 receptor antagonists for tumor imaging and radiotherapy. Cancer Biother. Radiopharm. 18, 627-641. (4) Hynes, R. O. (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25. (5) Ruoslahti, E. (1991) Integrins. J. Clin. Invest. 87, 1-5. (6) Cox, D., Aoki, T., Seki, J., Motoyama, Y., and Yoshida, K. (1994) The pharmacology of the integrins. Med. Res. Rev. 14, 195-228. (7) Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S., and Lazarus, R. A. (1990) Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of plateletaggregation inhibitors. Proc. Natl. Acad. Sci. U.S.A. 87, 2471-2475. (8) Knight, L. C., Maurer, A. H., and Romano, J. E. (1996) Comparison of iodine-123-disintegrins for imaging thrombi and emboli in a canine model. J. Nucl. Med. 37, 476-482. (9) Knight, L. C., Romano, J. E., and Maurer, A. H. (1998) In vitro platelet binding compared with in vivo thrombus imaging using alpha(IIb)beta3-targeted radioligands. Thromb. Haemostasis 80, 845-851. (10) Knight, L. C., Baidoo, K. E., Romano, J. E., Gabriel, J. L., and Maurer, A. H. (2000) Imaging pulmonary emboli and deep venous thrombi with 99mTc-bitistatin, a platelet-binding polypeptide from viper venom. J. Nucl. Med. 41, 1056-1064. (11) Knight, L. C. (2001) Radiolabeled peptide ligands for imaging thrombi and emboli. Nucl. Med. Biol. 28, 515-526. (12) Pfaff, M., McLane, M. A., Beviglia, L., Niewiarowski, S., and Timpl, R. (1994) Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition. Cell Adhes. Commun. 2, 491-501. (13) Ogawa, M., Hatano, K., Oishi, S., Kawasumi, Y., Fujii, N., Kawaguchi, M., Doi, R., Imamura, M., Yamamoto, M., Ajito, K., Mukai, T., Saji, H., and Ito, K. (2003) Direct electrophilic radiofluorination of a cyclic RGD peptide for in vivo alpha(v)beta3 integrin related tumor imaging. Nucl. Med. Biol. 30, 1-9. (14) Pfaff, M., Tangemann, K., Muller, B., Gurrath, M., Muller, G., Kessler, H., Timpl, R., and Engel, J. (1994) Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins. J. Biol. Chem. 269, 20233-20238.

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BC049961J

Evaluation of 64Cu-and 125I-Radiolabeled Bitistatin as Potential

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