Macromolecular MRI Contrast Agents with Small Dendrimers

of Radiology, School of Medicine, Johns Hopkins University, Baltimore, ..... Cong Li, Ying-Xia Li, Ga-Lai Law, Kwan Man, Wing-Tak Wong, and Hao Le...
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Bioconjugate Chem. 2003, 14, 388−394

Macromolecular MRI Contrast Agents with Small Dendrimers: Pharmacokinetic Differences between Sizes and Cores Hisataka Kobayashi,*,† Satomi Kawamoto,‡ Sang-Kyung Jo,§ Henry L. Bryant Jr.,| Martin W. Brechbiel,⊥ and Robert A. Star§ Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, Department of Radiology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, Laboratory of Diagnostic Radiology Research, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and Renal Diagnostics and Therapeutics Unit, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland. Received October 28, 2002; Revised Manuscript Received January 29, 2003

Large macromolecular MRI contrast agents with albumin or dendrimer cores are useful for imaging blood vessels. However, their prolonged retention is a major limitation for clinical use. Although smaller dendrimer-based MRI contrast agents are more quickly excreted by the kidneys, they are also able to visualize vascular structures better than Gd-DTPA due to less extravasation. Additionally, unlike Gd-DTPA, they transiently accumulate in renal tubules and thus also can be used to visualize renal structural and functional damage. However, these dendrimer agents are retained in the body for a prolonged time. The purpose of this study was to obtain information from which a macromolecular dendrimer-based MRI contrast agents feasible for use in further clinical studies could be chosen. Six small dendrimer-based MRI contrast agents were synthesized, and their pharmacokinetics, wholebody retention, and dynamic MRI were evaluated in mice to determine an optimal agent in comparison to Gd-[DTPA]-dimeglumine. Diaminobutane (DAB) dendrimer-based agents cleared more rapidly from the body than polyamidoamine (PAMAM) dendrimer-based agents with the same numbers of branches. Smaller dendrimer conjugates were more rapidly excreted from the body than the larger dendrimer conjugates. Since PAMAM-G2, DAB-G3, and DAB-G2 dendrimer-based contrast agents showed relatively rapid excretion, these three conjugates might be acceptable for use in further clinical applications.

INTRODUCTION

Large macromolecular MRI contrast agents with either albumin or dendrimer cores were initially developed for imaging blood vessels because of their prolonged retention in the circulation compared with Gd-[DTPA] and their enhanced relaxivities (1-3). Smaller PAMAM-based macromolecular MRI contrast agents are not optimal vascular agents because they are rapidly cleared from the blood by glomerular filtration and subsequently excreted by the kidneys without significant retention (35). However, one of the relatively small dendrimer-based contrast agents with a generation-4 polyamidoamine (PAMAM) core [PAMAMG4-(1B4M-Gd)64] (termed herein as PAMAM-G4) transiently accumulated in renal tubules and allowed visualization of renal structural and functional damage in the mouse (6). Unfortunately, many of the previously described dendrimer-based MR contrast agents are retained for lengthy * Corresponding author. Address: Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 4N109, 10 Center Drive, Bethesda, MD 20892-1374. Tel.: 1-301-435-8344. Fax: 301-4969956. E-mail: [email protected]. † Center for Cancer Research, National Cancer Institute. ‡ Johns Hopkins University. § National Institutes of Diabetes and Digestive and Kidney Diseases. | Warren Grant Magnuson Clinical Center. ⊥ Radiation Oncology Branch, National Cancer Institute.

times in the body. Theoretical risk of increased toxicity from unstable Gd(III) chelation potentially releasing toxic Gd(III) ions might prevent their clinical use. For example, only 20% of injected dose (%ID) of the PAMAM-G4 based contrast agent was excreted from the body during the first 2 days (4, 5). In contrast, the liver MRI contrast agent based on a four-generation diaminobutane (DAB) dendrimer core [DAB-G4] was excreted from the body faster than PAMAM-G4 (7) despite its considerable accumulation in the liver. Yet, as previously demonstrated, smaller molecular weight dendrimer-based contrast agents consistently are more readily excreted from the body than are larger agents of similar architecture (3, 4). Therefore, small (MW < 60 kD) macromolecular MRI contrast agents with both PAMAM and DAB dendrimer cores were synthesized and their pharmacokinetic characteristics studied with respect to whole-body clearance rates, renal accumulation, and quality of MRI images that could be obtained. EXPERIMENTAL PROCEDURES

Dendrimers. A brief description of dendrimer terminology has been published by Tomalia (8). Polypropylenimine diaminobutyl (DAB) dendrimers [DAB-Am16, polypropylenimine hexadecaamine dendrimer, generation-2 (DAB-G2), DAB-Am32, polypropylenimine dotriacontaamine dendrimer, generation-3 (DAB-G3), and DABAm64, polypropylenimine tetrahexacontaamine dendrimer generation-4 (DAB-G4) (Aldrich Chemical Co., Milwau-

10.1021/bc025633c CCC: $25.00 © 2003 American Chemical Society Published on Web 02/20/2003

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Table 1. Contrast Agents Used in the Current Studya

a

name

MW (kD)

Gd atoms

core MW (kD)

R1 relaxivity (mM-1 s-1)

commercial name

PAMAM-G4 PAMAM-G3 PAMAM-G2 DAB-G4 DAB-G3 DAB-G2 Gd-[DTPA]-dimeglumine

59 29 14 51 25 12 0.8

64 32 16 64 32 16 1

14.2 6.9 3.5 7.1 3.5 1.7 N/A

28 25 20 29 17 12 5.5

PAMAM Ga4 PAMAM Ga3 PAMAM Ga2 DAB-Am64 (Ga5) DAB-Am32 (Ga4) DAB-Am16 (Ga3) Magnevist

G: generation.

Figure 1. Schematic representation of the two core types of dendrimers. The third- and fourth-generation dendrimer shells are not explicitly illustrated.

kee, WI) were used possessing a diaminobutane core and 16, 32, or 64 terminal primary amino groups, respectively (Table 1; note that Aldrich defines the DAB-Am16, -Am32, and -Am64 dendrimers as generations-3, -4, and -5, respectively. However, in this paper these are defined to be DAB generations-2, -3, and -4 to parallel the naming convention of the PAMAM dendrimers, thereby allowing for direct comparison of the number of surface amines). Generations-2, -3, and -4 (PAMAM-G2, -G3, and -G4) PAMAM dendrimers (Aldrich Chemical Co., Milwaukee, WI) were used possessing an ethylenediamine core and 16, 32, or 64 terminal primary amino groups (8), respectively (Figure 1, Table 1). Conjugation of the Chelating Agent to Dendrimers. The dendrimers were concentrated to 10 mg/mL and diafiltrated against 0.1 M pH 9 sodium phosphate buffer and reacted at 40 °C with a 16-, 32-, or 64-fold molar excess of 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M) for the generation2, -3, and -4 dendrimers, respectively. The reaction solutions were maintained at pH 9 with 1 M NaOH over the reaction time of 48 h. Additional 1B4M equal to the initial amount was added as a solid after 24 h to each reaction. The resulting preparation was purified by diafiltration using a Centricon 30 (Amicon Co., Beverly, MA) for the generation-4 dendrimers and a Centricon 10 (Amicon Co.) for the generation-2 and -3 dendrimers. Over 98% of the amine groups on the dendrimers were reacted with the 1B4M as well as found not to contain free 1B4M chelates as determined by both a 153Gdlabeling assay as previously described (9) and an analysis by size-exclusion HPLC (SE-HPLC) using a TSK G3000 SW column (TosoHaas, Philadelphia, PA; 0.1 M PBS; 0.01 M KCl; pH 7.4; 1 mL/min) using a UV detector at 280 nm absorbance. Radiolabeling of Dendrimer-1B4M.153GdCl3 containing no other stable isotope of Gd(III) was purchased from NEN DuPont (Boston, MA). Approximately 1500 µg of each dendrimer (containing 2 µmol of 1B4M) was reacted with 1.11 MBq (30 µCi) of 153Gd citrate (46 pmol)

in 0.3 M citrate buffer at pH 5 for 30 min at room temperature. The preparation was then mixed with a 3-fold molar excess of nonradiolabeled Gd(III) citrate (6 µmol) to conjugate 1B4M to fully saturate the chelating groups with Gd(III). This reaction was incubated for 30 min before the addition of ethylenediaminetetraacetic acid (EDTA) and subsequent column purification. To remove any nonincorporated free metal, 10 µL of 0.5 M EDTA (Sigma, St. Louis, MO) was added to minimize formation of Gd(OH)3 precipitate. The product was purified using a PD-10 column (Pharmacia, Uppsala, Sweden), eluting with pH 7.4 PBS. The radiopurity of the preparations was analyzed by SE-HPLC using a TSK G3000SW column (TosoHaas, Philadelphia, PA; 0.1 M PBS; 0.01 M KCl; pH 7.4; 1 mL/min). DTPA (Sigma) was also labeled with 153Gd in 0.3 M citrate buffer for 30 min at room temperature. Preparation of Contrast Agents for MRI with Nonradioactive Gd(III). The dendrimer-1B4M conjugates (containing 4 µmol of 1B4M) were mixed with 6.5 µmol of Gd(III) citrate (Sigma) in 0.3 M citrate buffer for 2 h at 40 °C. The excess Gd(III) in each preparation was removed by diafiltration using a Centricon 30 (Amicon Co., Beverly, MA) for generation-4 dendrimers and a Centricon 10 (Amicon Co.) for the generation-2 and -3 dendrimers while simultaneously changing the buffer to 0.05 M PBS. In short, conjugated samples were applied onto either a Centricon 30 or 10 and centrifuged at ∼3000g for 45 min. Thereafter, 0.05 M PBS (2 mL) was added to 20 µL of the concentrated samples. The samples were again centrifuged at ∼3000g for 45 min. The purified samples were diluted to 1 mL with 0.05 M PBS, and 100 µL of this final solution was used per mouse. A replacement assay using 153Gd showed that the number of 1B4M chelators of the dendrimer-1B4M conjugates chelating Gd(III) atoms ranged from 77% to 87%. In brief, approximately 500 000 counts per min (9.3 kBq [0.25 µCi]) of 153Gd citrate (0.38 pmol) were 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 this time, the bound and unbound fractions were separated as described above using a PD-10 column (Pharmacia). The R1 relaxivities of the agents were approximately calculated from the T1 data obtained from all samples of 5, 10, and 20 µmolGd/mL and PBS. 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 coils (General Electric Medical System) (Table 1). All images were obtained 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.

390 Bioconjugate Chem., Vol. 14, No. 2, 2003

The Biodistribution and Whole-Body Retention of 153Gd-Labeled Dendrimer-1B4M-Gd Conjugates. Seven groups of nude mice (n ) 4 in each group) were injected with 37 kBq (1 µCi)/ 200 µL of 153Gd-labeled dendrimer-1B4M-Gd conjugates or 153Gd-DTPA. The injected samples were added to nonradioactive preparations, and the total Gd(III) dose was adjusted to 0.02 mmolGd/kg, 20% of the dose of Gd-[DTPA]-dimeglumine used clinically. The mice were sacrificed 15 min postinjection of the 153Gd-labeled preparations, and biodistribution studies were performed. The data were expressed as the percentage of the injected dose per gram (%ID/g) of tissue and as the blood-to-normal tissue ratio. Bone marrow was included in bone accretion assessment. The carcasses of mice were also counted to calculate the whole-body retention (%ID). The urine radioactivity was not included in the whole-body retention amount, principally due to the mice urinating when they were sacrificed with inhalation of carbon dioxide. Contrast-Enhanced Dynamic 3D-Micro-MRI of Mice. To evaluate the whole-body pharmacokinetics of the contrast agents, seven groups of 8-week-old female nude mice (n ) 4 or 5 in each group) (NCI, Frederick, MD) were used to obtain contrast-enhanced dynamic 3Dmicro-MR images. In short, either 0.03 mmolGd/kg (30% of clinical dose) of dendrimer-1B4M-Gd conjugates or 0.1 mmolGd/kg of Gd-[DTPA]-dimeglumine (Magnevist, Schering, Berlin, Germany) were intravenously injected into the left tail vein. All images were obtained using the highresolution wrist coil (General Electric Medical System) with a custom mouse holder using a Signa LX (General Electric Medical System). The mice were anesthetized with 1.15 mg of sodium pentobarbital (Dainabot, Osaka, Japan) and placed in the center of the coils. The fast spoiled gradient echo technique (FSPGR; TR/TE 19.4/4.2; flip angle 60°; scan time 1′40′′; phase encoding steps 256 × 256; 3 number of excitations; slab thickness 12) with chemical fat-suppression technique and serial 3D data acquisition was used to acquire images every 2 min from 0 (immediately after injection) to 14 min after injection of the contrast agents for all mice studies. The coronal images for dynamic MRI were reconstructed with 2 mm section thickness without gap reconstruction. In addition, slice data were processed into 3D images with the maximum intensity protection (MIP) method (Advantage Windows, General Electric Medical System). All studies were approved by the Animal Care Committee of National Institutes of Health. Statistical Analysis. Statistical analysis was performed using Student’s t-test (StatView, SAS Institute Inc., Cary, NC). RESULTS

Quality Control Study. The radiolabeling yields of all preparations before purification ranged from 95% to 98%. The radiopurity of all preparations analyzed by SEHPLC was greater than 97%. The retention times of the PAMAM-G4, -G3, and -G2 agents were 19.8, 20.7, and 21.5 min with a TSK G3000SW column, respectively. In addition, the retention times for the DAB-G4, -G3, and -G2 agents were 20.4, 21.0, and 21.6 min with the TSK G3000SW column, respectively. Therefore, by this method the physical sizes of the DAB agents at physiological pH were found to be smaller than those of the PAMAM agents of the same dendrimer generation. The Biodistribution and Whole-Body Retention of 153Gd-Labeled Dendrimer-1B4M-Gd Conjugates. The amount of the 153Gd-labeled DAB-based agents

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Figure 2. Retention of 153Gd-labeled dendrimer-1B4M-Gd conjugates or 153Gd-DTPA in the blood (a), liver (b), and kidney (c) in a normal nude mice (n ) 4) at 15 min after injection. Values are expressed as the mean percentages of the injected dose per gram of normal tissues and standard deviation. The asterisks indicate significant differences (p < 0.01) between PAMAM and DAB of the same dendrimer generation.

remaining in the blood increased as molecular weight decreased (Figure 2a). In contrast, the amount of 153Gdlabeled PAMAM-based agents remaining in the blood increased as molecular weight increased (Figure 2a). However, all conjugates cleared from the circulation more rapidly than Gd-[DTPA]. The 153Gd-labeled DAB-based agents as a whole accumulated significantly greater in the liver (Figure 2b) and less in the kidney (Figure 2c) than the 153Gd-labeled PAMAM-based agents (p < 0.01; for all values DAB versus PAMAM). The whole-body retention of the DAB-based agents was less than that of the PAMAM-based agents of the same dendrimer generation (Figure 3). Greater than 60% of the injected doses

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Figure 3. Whole-body retention of 153Gd-labeled dendrimer1B4M-Gd conjugates or 153Gd-DTPA in a normal nude mice (n ) 4) at 15 min after injection. Values are expressed as the mean percentages of the injected dose of mice and standard deviation. The asterisks indicate significant differences (p < 0.01) between PAMAM and DAB of the same dendrimer generation.

of the DAB-G3-, DAB-G2-, or PAMAM-G2-based contrast agents were cleared from the body within 15 min after injection. Contrast-Enhanced Dynamic 3D-Micro-MRI of Mice.The 3D-micro-MR images were obtained using the MIP method following intravenous injection of 0.03 mmolGd/kg of dendrimers. Brighter liver images were obtained with DAB-based agents than PAMAM-based agents or Gd-[DTPA]-dimeglumine (Figure 4). The blood signal-intensity values were significantly higher with generation-4 PAMAM-based agents than with DABbased agents (p < 0.01) at time points later than 2 min after injection (Figure 5a). The blood signal-intensity values were significantly higher with generation-2 DABbased agents than with PAMAM-based agents (p < 0.01) at all time points examined (Figure 5c). The liver signalintensity values were significantly higher with generation-3 and -4 DAB-based agents than with PAMAMbased agents of the same dendrimer generation (p < 0.01) at all time points examined (Figure 5d,e). The kidney signal-intensity values obtained from generation-2 and -4 DAB-based agents were also significantly higher than those obtained with the PAMAM-based agents of the same dendrimer generation (p < 0.01) at most of time points examined (Figure 5g,i). DISCUSSION

Dendrimers are a class of highly branched spherical polymers. Two types of dendrimers, the polyamidoamine (PAMAM) (8) and diaminobutane core polyaminoamine (DAB), are commercially available (10). Both types are highly soluble in aqueous solutions and possess a unique surface covered by primary amino groups (8, 11) (Figure 1). Almost all the amino groups are uncharged at pH above 9.0, and the defined structure and large number of available surface amino groups of these dendrimers serve as convenient attachment sites for chelating agents or single antibody molecules (11-15). These dendrimers have also been employed for the preparation of MRI contrast agents (1-3, 9, 16). Several studies that focus on the relationship between dendrimer generation and relaxivity of dendrimer-based contrast agents have been previously reported (1, 2). This general class of organ-specific MRI agents showing different pharmacokinetics from Gd-[DTPA] has been actively investigated for years (17, 18), and the first

Figure 4. Whole-body 3D-micro-MR-imaging of mice injected with 0.03 mmol Gd/kg of dendrimer-1B4M-Gd conjugates or 0.1 mmol Gd/kg Gd-[DTPA]-dimeglumine [ PAMAM-G4 (a), PAMAM-G3 (b), PAMAM-G2 (c), DAB-G4 (d), DAB-G3 (e), DABG2 (f), Gd-[DTPA]-dimeglumine (g)]. Images were obtained immediately after injection. Maximum intensity projections are shown.

gadolinium-based liver agent, [Gd(BOPTA)(H2O)]2- (MultiHance, Bracco-BYK Gulden, Germany), was approved in Europe in October 1998 (19-22). The composition and size of dendrimer-based MR imaging agents influences their behavior and, hence, the proposed clinical indications. For example, the presence of hydrophobic groups on metal chelates causes hepatocellular uptake and excretion into the bile ducts, gall bladder, and intestines. Actual physical size is also an important factor. Large macromolecular MRI contrast agents with albumin or dendrimer cores are useful for vascular imaging (1, 3, 23, 24). Smaller macromolecular MRI contrast agents are excreted by the kidneys; however, unlike Gd-[DTPA], they are concentrated in renal tubules and can visualize renal structural and functional damage (6). In the current studies, optimization of the performance of dendrimer-based MRI agents was an objective. Size and internal structure were found to have important effects on whole-body retention and MRI imaging characteristics. A significantly larger amount of DAB-based agents accumulated in the liver as compared to the same generation PAMAM-based agents by pharmacokinetic studies with 153Gd as shown on Figure 2b. That reflected well to the intensity-time curves obtained by the MRI studies as shown in Figure 5d-f. In contrast, a significantly smaller amount of DAB-based agents accumulated in the kidney as compared to the same generation of PAMAM-based agents in the pharmacokinetic studies as shown on Figure 2c. However, DAB-based agents exhibited higher signal intensity than the same generation of PAMAM-based agents in the kidney as shown on Figure 5g-i. DAB-based agents, generation-2 and -3 of which

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Figure 5. Plots of the signal intensity of the blood in the left ventricle of the heart (G4, a; G3, b; G2, c), the liver (G4, d; G3, e; G2, f), and the kidney (G4, g; G3, h; G2, i) obtained from the contrast-enhanced dynamic MRI of mice with 0.03 mmol/kg of dendrimer1B4M-Gd conjugates (PAMAM, open circles; DAB, closed circles) or 0.1 mmol Gd/kg Gd-[DTPA]-dimeglumine (DTPA, closed squear) are shown. A single most appropriate region of interest was set in each organ in each mouse (n ) 4 or 5). Data are expressed as the mean signal intensity and standard deviation. The asterisks indicate significant differences (p < 0.01) between PAMAM and DAB of the same dendrimer generation.

showed smaller R1 relaxivity values than PAMAM-based agents of the same generation as shown in Table 1, were excreted into the urine more efficiently than PAMAMbased agents, which showed prolonged retention (14) and possible internalization into renal tubular cells. The high T1-weighted MRI signal obtained with DAB agents may be results of both high accessibility of water to Gd(III) ions and large proportion of DAB agents located in the free water (urine) rather than in the lipid vesicles (tubular cells) (25). Although the MRI imaging characteristics of PAMAM (3, 4, 24) and DAB (7, 26) based contrast agents have been well studied, their toxicity profile remains uncertain. Malik et al. reported that the difference between terminal groups on the surface of the various dendrimer molecules could alter their biological characteristics, especially with respect to their cellular toxicity (10). However, although the internal repeat unit structures

of the dendrimers altered the in vivo pharmacokinetics in these studies, the biological toxicity characteristics were not measurably different (10, 26, 27). For use in clinical practice, macromolecular MR contrast agents must be efficiently and rapidly excreted to minimize the potential toxicity of these agents, mostly caused by free Gd(III). Renal filtration and excretion is usually considered to be the optimal route to remove circulating molecules from the body because they can be eliminated intact without cellular metabolism or processing. However, the filtered macromolecules could then be reabsorbed by the proximal tubules and retained within the cell. Even if the molecules could be catabolized and excreted, most of the chelated metal ions are retained in the cell (28, 29). If free Gd(III) is liberated in the cell, this could contribute to toxicity. Smaller MR contrast agents are generally considered to be safer, because they are more rapidly cleared, and have less cellular uptake,

MRI Contrast Agents for Functional Kidney Imaging

although even in case of DAB-G2, 8% of the injected dose retained in the body at 48 h postinjection, which is much more than that of Gd-DTPA (>2% at 48 h postinjection). In the present study, the smaller dendrimer-Gd conjugates demonstrated lower retention in the whole body and thus could be used to obtain detailed renal structural information. An elegant study has recently been reported with a series of synthetic peglated linear copolymer with molecular weight ranging 30-120 kD (30). The molecular weights of the agents were close to the larger size of the dendrimer-based agents in the current study. However, the excretion rate of the copolymer-based agents appeared to be much slower than that of both classes of dendrimer-based agents. The rapid excretion of the dendrimer-based agents was related to their small molecular sizes induced by their inherent spherical shape. Linear polymers might behave as larger molecules in the body than the spherical polymers of similar molecular weight. Protein molecules with small molecular weights such as β2 microglobulin of the single-chain Fv fragment of antibody (Fv) were quickly filtered through glomerulus mostly at the first pass (31). In case disulfide-stabilized Fv, which has larger molecular weight (25kD) than PAMAM-G2, DAB-G3, and DAB-G2, was examined with the same method as we used in this study: around 80% of its injected dose into mice was stuck or passed through kidney within 15 min after injection (31). Therefore, the quick excretion rates of these contrast agents were reasonable for molecules with sizes in this range, although the excretion might be much slower in human than in mice because of their four times as much heart rates and quick catabolism of drugs. In conclusion, although the dendrimer-1B4M-Gd conjugates with smaller molecular weight exhibited a more rapid excretion from the whole body, all of the dendrimer-1B4M-Gd conjugates visualized the proximal straight tubule in the kidney. Since the PAMAM-G2-, DAB-G3-, and DAB-G2-based contrast agents showed relatively rapid excretion from the body, these three conjugates may be acceptable for use in clinical practice as either blood-pool or functional kidney MRI contrast agents. Further investigations will be required to ascertain any resolution between these three agents in order to chose a final candidate for clinical evaluations. LITERATURE CITED (1) Wiener, E. C., Brechbiel, M. W., Brothers, H., Magin, R. L., Gansow, O. A., Tomalia, D. A., and Lauterbur, P. C. (1994) Dendrimer-based metal chelates: a new class of magnetic resonance imaging contrast agents. Magn. Reson. Med. 31, 1-8. (2) Bryant, L. H., Jr., Brechbiel, M. W., Wu, C., Bulte, J. W., Herynek, V., and Frank, J. A. (1999) Synthesis and relaxometry of high-generation (G ) 5, 7, 9, and 10) PAMAM dendrimer-DOTA-gadolinium chelates. J. Magn. Reson. Imaging 9, 348-352. (3) 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. (4) Sato, N., Kobayashi, H., Hiraga, A., Saga, T., Togashi, K., Konishi, J., and Brechbiel, M. W. (2001) Pharmacokinetics and enhancement patterns of macromolecular MR contrast agents with various sizes of polyamidoamine dendrimer cores. Magn. Reson. Med. 46, 1169-1173.

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