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Bioconjugate Chem. 2003, 14, 1044−1047
Activated Clearance of a Biotinylated Macromolecular MRI Contrast Agent from the Blood Pool Using an Avidin Chase Hisataka Kobayashi,†,* Satomi Kawamoto,‡ Robert A. Star,| Thomas A. Waldmann,† Martin W. Brechbiel,§ and Peter L. Choyke⊥ Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, Department of Radiology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21287, Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, Renal Diagnostics and Therapeutics Unit, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, and Diagnostic Radiology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892. Received April 28, 2003; Revised Manuscript Received June 18, 2003
The enhancement characteristics of a contrast agent are dependent on its pharmacokinetics within the body. In the case of macromolecular contrast agents, prolonged enhancement of the blood pool is seen after the first dose, limiting opportunities for repeated injection in the same session. If the enhancement within the blood pool could be intentionally switched off, the macromolecular contrast agents could be used both to define blood volume and vessel permeability, properties that could be useful in studying angiogenesis. In the current study, the avidin-biotin system was coupled to a dendrimer-based macromolecular MRI contrast agent to switch enhancement from the blood pool to the liver. Because avidin causes rapid trapping of the contrast agent in the liver, the blood pool cleared within 2 min of the injection of avidin. This system can be applied to all dendrimer-based macromolecular MRI contrast agents to investigate blood volume and vascular permeability. Moreover, it permits the repeated injection of the contrast agent and the “avidin switch” during a single MR experiment.
INTRODUCTION
The enhancement characteristics of a contrast agent are dependent on its pharmacokinetics. The blood pool macromolecular contrast agents remain within the circulation during the acquisition of vascular MR images (1, 2). Macromolecular MRI contrast agents can be used to analyze blood volume, vascular permeability, and other characteristics of tumor angiogenesis. However, it can be difficult to differentiate the contribution of each parameter to the signal intensity within a tumor (3-5). A significant limitation of macromolecular agents is that their clearance is slow, preventing repeated measurements within the same MRI session (6). This is especially limiting when performing MRI before and soon after angiogenic inhibitor therapy to detect early changes in tumor vessels. Therefore, if the enhancement in the vascular structures could be intentionally “switched off” or cleared from the blood pool, the macromolecular contrast agents could be applied more widely to the investigation of tumor angiogenesis. Avidin “chase”, which was developed by us to improve the pharmacokinetics of monoclonal antibody cancer * To whom correspondence should be addressed. Tel.: 1-301435-8344. Fax: 301-496-9956. E-mail:
[email protected]. † Metabolism Branch, National Cancer Institute. ‡ Johns Hopkins University. § Radiation Oncology Branch, National Cancer Institute. | National Institutes of Diabetes and Digestive and Kidney Diseases. ⊥ Diagnostic Radiology Department, National Institutes of Health.
therapy, is a method that can be used to clear macromolecules quickly from the circulation by increasing their uptake in the liver (7, 8). Avidin “chase” was also used as an essential step of the three-step method for cancer radioimmunotherapy (9-11). Dafni et al. has recently reported the application of an avidin “chase” to a macromolecular albumin-based MRI contrast agent to analyze the extravascular leakage from the tumor vessels (5). In the current study, we synthesized a dendrimerbased blood pool macromolecular MRI contrast agent, which contains 5 biotins and 210 gadolinium ions. We then used an avidin “chase” to clear the blood pool of the agent. We evaluated its pharmacokinetics and the effects of the avidin “chase” by dynamic MRI. EXPERIMENTAL PROCEDURES
PAMAM Dendrimer. A generation-6 PAMAM dendrimer (G6) (Dendritech, Inc., Midland, MI) with an ethylenediamine core, 256 primary terminal amino groups, and a molecular weight of 58 048 Da was used in the current study (12). Preparation of the Biotinylated Contrast Agent. Four mg/mL of G6 (170 nmol) was dissolved in PBS, pH 7.4, and mixed with 10 mg/mL solution of 1.7 mmol of sulfosuccinimidyl-6-(biotinamide) hexanoate (Bt) (NHSLC-biotin, MW ) 340 Da; Pierce Chemical Co., Rockford, IL) in DMSO at a molar ratio of 1:10, at room temperature for 30 min. The mixture was applied to a diafiltration membrane (Centricon 30, Amicon, Inc., Beverly, MA) to remove unbound Bt as well as to change the buffer to 0.1 M phosphate buffer, pH 9. After purification, a small aliquot of biotinylated G6 (Bt-G6) was examined using a
10.1021/bc034064l CCC: $25.00 © 2003 American Chemical Society Published on Web 08/13/2003
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modified HABA assay, which was previously described in detail elsewhere (13, 14). In brief, the 2-(4′-hydroxyazobenzene)benzoic acid (HABA; Pierce Chemical Co., Rockford, IL) reagent was prepared according to the manufacturer’s instructions by adding 10 mg of avidin and 600 µL of 10 mM HABA to 19.4 mL of PBS. One hundred microliters of serially diluted Bt-G6 solutions was added to 900 µL of the avidin-HABA solutions. Then, the absorbance was measured at 500 nm. Approximately 4.8 biotin molecules were determined to be conjugated to each G6 molecule on the average. The biotinylated G6 was concentrated to ∼5 mg/mL and reacted with a 256-fold molar excess of 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M, MW ) 555 Da) (44 µmol) at 40 °C. The reaction was maintained at pH 9 with 1 M NaOH for 24 h. An additional equal amount of 1B4M was added to the mixture after 24 h as a solid, and the pH was maintained as before for another 24 h. The resulting preparation was purified by diafiltration using a Centricon 30 (Amicon Co., Beverly, MA). Approximately 97 ( 2% of the amine groups on the surface of the G6 dendrimer reacted with the 1B4M, as determined by 153Gd (NEN DuPont, Boston, MA) labeling of the reacted samples (2,15). In brief, ∼500 000 cpm (∼0.4 pmol) of 153Gd citrate were added to ∼40 µg/ 10 µL (∼160 pmol) of the reaction mixture before diafiltration and incubated in 0.2 M acetate buffer for 15 min at room temperature. Then, the fractions bound to the conjugates and free 1B4M were separated using a PD-10 column (Pharmacia) and counted, after which the numbers of 1B4M chelates bound to the conjugate were calculated by the quantification of the radioactivity in the fraction bound relative to that of the conjugates. Approximately 3 mg of biotinylated G6 dendrimer1B4M conjugate [Bt5-G6-(1B4M-Gd)251] (containing 4 µmol 1B4M) were mixed with 8 µmol of nonradioactive Gd(III) citrate in 0.3 M citrate buffer for 2 h at 40 °C. The excess Gd in each preparation was removed by diafiltration using a Centricon 30 (Amicon Co.) while simultaneously changing the buffer to 0.05 M PBS. The purified samples were diluted to 0.5 mL with 0.05 M PBS and passed through a 0.22 µm filter (Amicon Co.), and 100 µL of this solution was used as the MRI contrast agent for each mouse. Using a replacement assay involving 153Gd, we determined that 85% of the 1B4M (209.5 ions/molecule) on the dendrimer-1B4M conjugates were indeed chelating Gd(III) atoms (2). In brief, approximately 300 000 cpm of 153Gd was added with 0.1 µmol of nonradioactive Gd(III) to 5 µL of the injected samples and incubated in 0.5 M citrate buffer for 2 h at 40 °C, after which the bound and unbound fractions were separated using a PD-10 column (Pharmacia). Quality Control Studies. Molecular Purity. The purity of the contrast agent was analyzed by a sizeexclusion HPLC equipped with a TSK G2000 SW column (TosoHaas, Philadelphia, PA; 0.066 M PBS; pH 7.2; 1 mL/ min) using a UV detector at 280 nm absorbance. Binding Ability to Avidin. The binding ability to avidinsephalose gel (Pierce Chemical Co.) of 153Gd-labeled Bt5-G6-(1B4M-Gd)251 (MW ) 240 kDa) was examined (8, 16). In brief, 3 ng/0.1 µCi of Bt5-G6-(1B4M-Gd)251 was incubated with 0.5 mL of avidin-sephalose gel for 15 min at room temperature. Then the gel fraction was washed and separated from the supernatant by a paper filter. The radioactivity of both fractions was counted with a γ-counter (Perkin-Elmer, Boston, MA). R1 Relaxivity. The R1 relaxivity of the Bt5-G6-(1B4MGd)251 contrast agent was calculated from the T1 data
obtained from all samples of 5, 10, and 20 µmolGd/mL and PBS and then compared with that of the nonbiotinylated G6-(1B4M-Gd)256 contrast agent. We used an inversion recovery spin-echo imaging sequence with various TI 50, 100, 200, and 400 ms and TR/TE: 6000/15 ms, using a 1.5-Tesla superconductive magnet unit (Signa LX, General Electric Medical System, Milwaukee, WI) with a high-resolution wrist coil (General Electric Medical System). All images were obtained in three slices with an 8 cm field of view and a 2 mm slice thickness, 256 × 256 matrix, and two excitations were averaged. The best slice was selected, and the data obtained from 12 mm2 areas at the center of the tubes were averaged and used to calculate R1 values. Dynamic MRI Study. To evaluate the pharmacokinetics and effects of the avidin “chase”, serial dynamic MR images of normal mice (n ) 9) were obtained before and after injection of 0.03 mmolGd/kg (30% of clinical dose) of Bt5-G6-(1B4M-Gd)251 followed by injection with 100 µL of either saline containing 400 µg of avidin (n ) 5) or PBS (n ) 4) at 4 min (six frames) postinjection of the contrast agent. A 1.5-Tesla superconductive magnet unit (Signa LX, Milwaukee, WI) and a commercially available high-resolution wrist coil with a custom mouse holder were employed for image acquisition. The mice were anesthetized with 1.15 mg of sodium pentobarbital (Dainabot, Osaka, Japan) and placed in the center of the coils. The 3D-fast-spoiled gradient echo technique (TR/TE 10/4.1 ms; TI 29 ms; flip angle 30°; scan time 35 s; frequency encoding × phase encoding steps 256 × 192; two excitations; slice encoding steps 14; with fatsuppression technique and serial 3D data acquisition) was used to acquire 20 images every 40 s from 0 (immediately after injection) to 12.3 min after injection of the contrast agents for all mice studied. The coronal images were reconstructed with 2-mm thick sections with 1-mm overlap. The field of view was 8 × 4 cm, and the size of each voxel was 0.32 × 0.42 × 2 mm3. The serial dynamic data were analyzed using an Advantage Windows version 3 (GE). In addition, to evaluate the whole body pharmacokinetics, whole body 3D-MR angiograms were reconstructed with the maximum intensity projection (MIP) method. RESULTS
Quality Control Studies. The elution time of the Bt5-G6-(1B4M-Gd)251 contrast agent (17.2 min) was similar to that of nonbiotinylated G6-(1B4M-Gd)256 contrast agent (17.1 min). The chemical purity of Bt5-G6-(1B4MGd)251 was 99%. Ninety one percent of the radioactivity of 153Gd-labeled Bt5-G6-(1B4M-Gd)251 sample was bound to the avidin-sephalose gel. The R1 relaxivity of Bt5G6-(1B4M-Gd)251 (33 mM-1 s-1) was the same as that of G6-(1B4M-Gd)256 contrast agent (33 mM-1 s-1). Dynamic MRI Study. The time intensity curves in the inferior vena cava and the liver obtained by dynamic MRI of mice injected with Bt5-G6-(1B4M-Gd)251 contrast agent followed by the injection of PBS without avidin demonstrate slow clearance from the blood pool and rapid enhancement followed by a steady state in the liver (Figure 1b and 2). This pattern is similar to that previously reported with G6-(1B4M-Gd)256 agent (17). The only difference between these molecules is the presence of an additional five biotin molecules that did not appear to affect the pharmacokinetic properties of the agent. The time intensity curves of the inferior vena cava and the liver of mice injected with Bt5-G6-(1B4M-Gd)251 contrast agent followed by an avidin “chase” are shown
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Figure 1. 2D-dynamic chest and abdominal MRIs of mice with avidin (a) and saline (b) injection at 4 min postinjection with Bt5-G6-(1B4M-Gd)251 contrast agent.
Figure 2. Time intensity curves of the inferior vena cava (a) and the liver (b) in mice with avidin (n ) 5) or saline (n ) 4) injections at 4 min postinjection with Bt5-G6-(1B4M-Gd)251 contrast agent.
in Figure 2; the signal intensity in the vessels quickly decreased within 2 min of the avidin “chase” to nearly the preinjection level (Figure 1a and 2a). The signal intensity in the liver gradually increased after injection with avidin (Figure 1a and 2b). Therefore, the vascular enhancement faded out within 2 min after injection with avidin while the signal intensity of the liver increased after injection of avidin as shown on serial whole-body MRIs (Figure 3). DISCUSSION
The avidin “chase” system is a simple but efficient system to quickly clear biotinylated macromolecule from the circulation to the liver (8). The avidin “chase” system has been reported to improve the pharmacokinetics of radiolabeled monoclonal antibodies for the radioimmunotherapy for cancer (8, 18, 19). Briefly, circulating biotinylated macromolecules such as antibodies are quickly cleared from the blood pool within 20 min after injection of avidin and accumulate in the liver internalizing into either hepatocytes or the reticuloendothelial system (8, 20). Although the intravascular component and avidin itself were cleared quickly, the extravascular component was cleared much more slowly, that is, over a period of 4-6 h (20-22). Therefore, this method potentially differentiates the vascular component (blood volume) from the extravascular component (permeability)
Figure 3. Serial whole body 3D-MRIs (maximum intensity projection) of mice with avidin injection at 4 min postinjection with Bt5-G6-(1B4M-Gd)251 contrast agent showed that the enhancement disappeared not only from the central circulation but also from the peripheral blood vessels within 2 min postinjection of an avidin chase.
if the total time to clear the blood pool could be kept to a minimum. In the current study, the avidin “chase” system was successfully applied to a dendrimer-based macromolecular MRI contrast agent. The pharmacokinetics of this agent were evaluated specifically in regards to the rate of disappearance of the agent from the circulation by dynamic MRI. This clearing process of the contrast agent was revealed to be much quicker than we expected from the results of the conventional pharmacokinetic studies
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(8) because the conventional methods including scintigraphy had technical difficulties to serially monitor the drug kinetics at every single minute. The advantage of dendrimer-based macromolecular MRI contrast agents over an albumin-based agent is that molecules of a specific size could be made with chemical conformity. That enables us to use a molecule of an appropriate size for a specific purpose (22). The rapid clearance of the contrast agent from the blood pool may permit the nearly simultaneous assessment of both the blood volume and vessel permeability and allow repeated injections during a single MR session in animal models. In conclusion, the successful synthesis and application of an avidin chase system to dendrimer-based maclomolecular MRI contrast agents enabled us to rapidly clear blood pool contrast agents from the circulation. This system might be a good method to analyze the characteristics of angiogenic vessels especially before and after therapy. In addition to evaluating tumor angiogenesis and response to therapy this contrast agent system may be useful for monitoring the stimulation of angiogenesis in ischemic disease and demonstrating the effects of vascular disease in organs such as the kidney. LITERATURE CITED (1) Ogan, M. D., Schmiedl, U., Moseley, M. E., Grodd, W., Paajanen, H., and Brasch, R. C. (1987) Albumin labeled with Gd-DTPA. An intravascular contrast-enhancing agent for magnetic resonance blood pool imaging: preparation and characterization. Invest. Radiol. 22, 665-671. (2) Kobayashi, H., Sato, N., Hiraga, A., Saga, T., Nakamoto, Y., Ueda, H., Konishi, J., Togashi, K., and Brechbiel, M. W. (2001) 3D-micro-MR angiography of mice using macromolecular MR contrast agents with polyamidoamine dendrimer core with references to their pharmacokinetic properties. Magn. Reson. Med. 45, 454-460. (3) Kobayashi, H., Sato, N., Kawamoto, S., Saga, T., Hiraga, A., Ishimori, T., Konishi, J., Togashi, K., and Brechbiel, M. W. (2001) 3D MR angiography of intratumoral vasculature using a novel macromolecular MR contrast agent. Magn. Reson. Med. 46, 579-585. (4) Shirakawa, K., Kobayashi, H., Heike, Y., Kawamoto, S., Brechbiel, M. W., Kasumi, F., Iwanaga, T., Konishi, F., Terada, M., and Wakasugi, H. (2002) Hemodynamics in vasculogenic mimicry and angiogenesis of inflammatory breast cancer xenograft. Cancer Res. 62, 560-566. (5) Dafni, H., Israely, T., Bhujwalla, Z. M., Benjamin, L. E., and Neeman, M. (2002) Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin. Cancer Res. 62, 6731-6739. (6) Kobayashi, H., Kawamoto, S., Saga, T., Sato, N., Hiraga, A., Konishi, J., Togashi, K., and Brechbiel, M. W. (2001) Micro-MR angiography of normal and intratumoral vessels in mice using dedicated intravascular MR contrast agents with high generation of polyamidoamine dendrimer core: reference to pharmacokinetic properties of dendrimer-based MR contrast agents. J. Magn. Reson. Imaging 14, 705-713. (7) Sinitsyn, V. V., Mamontova, A. G., Checkneva, Y. Y., Shnyra, A. A., and Domogatsky, S. P. (1989) Rapid blood clearance of biotinylated IgG after infusion of avidin. J. Nucl. Med. 30, 66-69. (8) Kobayashi, H., Sakahara, H., Hosono, M., Yao, Z. S., Toyama, S., Endo, K., and Konishi, J. (1994) Improved clearance of radiolabeled biotinylated monoclonal antibody following the infusion of avidin as a chase without decreased accumulation in the target tumor. J. Nucl. Med. 35, 16771684. (9) Paganelli, G., Magnani, P., Zito, F., Villa, E., Sudati, F., Lopalco, L., Rossetti, C., Malcovati, M., Chiolerio, F., Secca-
mani, E., Siccardi, A. G., and Fazio, F. (1991) Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen-positive patients. Cancer Res. 51, 5960-5966. (10) Paganelli, G., Magnani, P., and Fazio, F. (1993) Pretargeting of carcinomas with the avidin-biotin system. Int. J. Biol. Markers 8, 155-159. (11) Chinol, M., Casalini, P., Maggiolo, M., Canevari, S., Omodeo, E. S., Caliceti, P., Veronese, F. M., Cremonesi, M., Chiolerio, F., Nardone, E., Siccardi, A. G., and Paganelli, G. (1998) Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity. Br. J. Cancer 78, 189-197. (12) Tomalia, D. A., Naylor, A. M., and Goddard, W. A., III (1990) Starburst dendrimers: Molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew. Chem., Int. Ed. Engl. 29, 138175. (13) Green, N. M. (1975) Avidin. Adv. Protein Chem. 29, 85133. (14) Kobayashi, H., Sun, B. F., Yoo, T. M., Le, N., Kim, M. K., Paik, C. H., Pastan, I., Waldmann, T. A., and Carrasquillo, J. A. (1999) Methods to avoid adverse effect of circulating antigen on biodistribution of 125I-labeled antiTac dsFv: preinjection of intact antibody versus clearance of antigen with adivin-biotin system. J. Nucl. Med. 40, 1381-1391. (15) Kobayashi, H., Sakahara, H., Hosono, M., Shirato, M., Kondo, S., Miyatake, S., Kikuchi, H., Namba, Y., Endo, K., and Konishi, J. (1994) Scintigraphic detection of neural-cellderived small-cell lung cancer using glioma-specific antibody. J. Cancer Res. Clin. Oncol. 120, 259-262. (16) Kobayashi, H., Kawamoto, S., Saga, T., Sato, N., Ishimori, T., Konishi, J., Ono, K., Togashi, K., and Brechbiel, M. W. (2001) Avidin-dendrimer-(1B4M-Gd)(254): a tumor-targeting therapeutic agent for gadolinium neutron capture therapy of intraperitoneal disseminated tumor which can be monitored by MRI. Bioconjugate Chem. 12, 587-593. (17) Kobayashi, H., Sato, N., Kawamoto, S., Saga, T., Hiraga, A., Haque, T. L., Ishimori, T., Konishi, J., Togashi, K., and Brechbiel, M. W. (2001) Comparison of the macromolecular MR contrast agents with ethylenediamine-core versus ammonia-core generation-6 polyamidoamine dendrimer. Bioconjugate Chem. 12, 100-107. (18) Paganelli, G., Stella, M., Zito, F., Magnani, P., De Nardi, P., Mangili, F., Baratti, D., Veglia, F., Di Carlo, V., Siccardi, A. G., and Fazio, F. (1994) Radioimmunoguided surgery using iodine-125-labeled biotinylated monoclonal antibodies and cold avidin. J. Nucl. Med. 35, 1970-1975. (19) Paganelli, G., Magnani, P., Zito, F., Lucignani, G., Sudati, F., Truci, G., Motti, E., Terreni, M., Pollo, B., Giovanelli, M., Canal, N., Scotti, G., Comi, G., Koch, P., Maecke, H. R., and Fazio, F. (1994) Pre-targeted immunodetection in glioma patients: tumour localization and single-photon emission tomography imaging of [99mTc]PnAO-biotin. Eur. J. Nucl. Med. 21, 314-321. (20) Kobayashi, H., Sakahara, H., Endo, K., Hosono, M., Yao, Z. S., Toyama, S., and Konishi, J. (1995) Comparison of the chase effects of avidin, streptavidin, neutravidin, and avidinferritin on a radiolabeled biotinylated antitumour monoclonal antibody. Jpn. J. Cancer Res. 86, 310-314. (21) Kobayashi, H., Sakahara, H., Endo, K., Yao, Z. S., Toyama, S., and Konishi, J. (1995) Repeating the avidin chase markedly improved the biodistribution of radiolabelled biotinylated antibodies and promoted the excretion of additional background radioactivity. Eur. J. Cancer 31A, 1689-1696. (22) Kobayashi, H., Sakahara, H., Endo, K., Yao, Z. S., and Konishi, J. (1996) Inflammation-seeking scintigraphy with radiolabeled biotinylated polyclonal IgG followed by the injection of avidin chase. Nucl. Med. Biol. 23, 29-32. (23) Kobayashi, H., and Brechbiel, M. W. (2003) Gadoliniumbased macromolecular MRI contrast agents. Mol. Imaging 2, 1-10.
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