Synthesis and Characterization of Poly (l-glutamic acid) Gadolinium

Oct 8, 2004 - gadolinium chelates as biodegradable blood-pool MRI contrast agents. Two PG ... of blood-pool contrast agents, imaging using computed...
1 downloads 0 Views 313KB Size
Download PDF
Bioconjugate Chem. 2004, 15, 1408−1415

1408

Synthesis and Characterization of Poly(L-glutamic acid) Gadolinium Chelate: A New Biodegradable MRI Contrast Agent Xiaoxia Wen,† Edward F. Jackson,‡ Roger E. Price,‡ E. Edmund Kim,† Qingping Wu,† Sidney Wallace,† Chusilp Charnsangavej,† Juri G. Gelovani,† and Chun Li*,† Department of Experimental Diagnostic Imaging and the Department of Imaging Physics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030. Received April 12, 2004; Revised Manuscript Received August 10, 2004

Most currently evaluated macromolecular contrast agents for magnetic resonance imaging (MRI) are not biodegradable. The goal of this study is to synthesize and characterize poly(L-glutamic acid) (PG) gadolinium chelates as biodegradable blood-pool MRI contrast agents. Two PG chelates of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) were synthesized through the use of difunctional and monofunctional DTPA precursors. The conjugates were characterized with regard to molecular weight and molecular weight distribution, gadolinium content, relaxivity, and degradability. Distributions of the polymeric MRI contrast agents in various organs were determined by intravenous injection of 111In-labeled polymers into mice bearing murine breast tumors. MRI scans were performed at 1.5 T in mice after bolus injection of the polymeric chelates. PG-Hex-DTPA-Gd, obtained from aminohexylsubstituted PG and DTPA-dianhydride, was partially cross-linked and was undegradable in the presence of cathepsin B. On the other hand, PG-Bz-DTPA-Gd synthesized directly from PG and monofunctional p-aminobenzyl-DTPA(acetic acid-tert-butyl ester) was a linear polymer and was degradable. The relaxivities of the polymers at 1.5 T were 3-8 times as great as that of Gd-DTPA. Both polymers had high blood concentrations and were primarily accumulated in the kidney. However, PG-Bz-DTPA-Gd was gradually cleared from the body and had significantly less retention in the blood, the spleen, and the kidney. MRI with PG-Bz-DTPA-Gd in mice showed enhanced vascular contrast at up to 2 h after the contrast agent injection. The ability of PG-Bz-DTPA-Gd to be degraded and cleared from the body makes it a favorable macromolecular MRI contrast agent.

INTRODUCTION

Noninvasive imaging of the intravascular compartment is of critical importance in clinical medicine. Many diseases, e.g., infection, ulcer, cardiovascular diseases, solid tumors, involve gross hemorrhage, abnormal vascular growth, or vascular occlusion (1, 2). With the help of blood-pool contrast agents, imaging using computed tomography (CT), ultrasonography, gamma scintigraphy, and magnetic resonance imaging (MRI)1 can provide minimally invasive angiography, assess angiogenesis, quantify the number and spacing of blood vessels, and measure blood volume and blood flow. MRI blood-pool agents are often paramagnetic gadolinium chelates of high-molecular-weight polymers that are largely retained within the intravascular space during the time frame of an MRI examination (3). For example, blood-pool agents for contrast-enhanced magnetic resonance angiography allow prolonged imaging times for higher contrast and resolution. Imaging may be performed during the steady state when the contrast agent is distributed through the * To whom correspondence should be addressed: Chun Li, Ph.D., Department of Experimental Diagnostic Imaging, Box 59, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 7925182. Fax: (713) 794-5456. E-mail: [email protected]. † Department of Experimental Diagnostic Imaging. ‡ Department of Imaging Physics. 1 Abbreviations: MRI, magnetic resonance imaging; PG, poly(L-glutamic acid); DTPA, diethylenetriaminepentaacetic acid; Gd, gadolinium.

complete vascular system (4). Recent studies have shown that tumor vascular permeability measurements, derived from polymeric contrast agents enhanced dynamic MRI, correlate with tumor microvessel density counts, suggesting that MRI may be used to characterize tumor angiogenic activity (5, 6). Furthermore, polymeric MRI contrast agents may permit more accurate grading of tumor invasiveness than do small-molecular-weight contrast agents, e.g., Gd-DTPA (7, 8). One approach toward clinically acceptable blood-pool MRI agents takes advantage of a small-molecular-weight Gd3+-based agent (i.e., MS-325) that binds strongly but reversibly to human serum albumin (9, 10). MS-325 is designed in such a way that an equilibrium between free and protein-bound compound exists, ensuring efficient renal excretion. An alternative approach is to develop macromolecular MRI contrast agents. Various polymeric MRI contrast agents, including human serum albumin (11), polylysine (12, 13), dendrimers (16-19), polyamide (15, 20), and grafted copolymers (21), have been synthesized and evaluated as blood-pool imaging agents. Although most of these agents fulfill the criteria of long blood circulation time and high relaxivity, the safety of these agents has yet to be established. The clinical applications of many current macromolecular contrast agents are limited by slow excretion from the body and potential toxicity of free Gd3+ ions released by the metabolism of the contrast agents (12-14, 22, 23). For an albumin-based product, the possibility of immunogenic response makes the use of serum proteins less attractive. Dendrimer-based blood-pool MRI agents have the ad-

10.1021/bc049910m CCC: $27.50 © 2004 American Chemical Society Published on Web 10/08/2004

Biodegradable MRI Contrast Agent

vantage of extremely narrow molecular weight distribution. However, these agents are not biodegradable. Ideally, a polymeric MRI contrast agent will reside in the blood long enough to allow for thorough MRI examinations and then be degraded and cleared from the body after completion of the imaging procedures. Poly(Lglutamic acid) (PG) is a synthetic polyamino acid that can be readily degraded by lysosomal enzymes to its basic component, L-glutamic acid, a nontoxic natural compound (24). The polymer contains a large number of carboxyl functional groups for drug attachment. Conjugation of paclitaxel to PG has resulted in a water-soluble paclitaxel conjugate with reduced systemic toxicity and enhanced antitumor efficacy (25). In various studies in rodents, PG used alone as a control agent at doses from 200 to 800 mg/kg did not cause apparent toxic effects. Phase III clinical trials of PG-paclitaxel (Xyotax) as a chemotherapeutic agent are now in progress, underscoring the safety of this polymeric carrier. Recently, Lu et al. (26) reported a PG-based MRI agent using cystamine as the cleavable spacer. Gd3+ chelates were cleaved from the polymer backbone through a disulfide-thiol exchange reaction. However, potential interaction of the polymer with endogenous thiol-containing biomolecules raises concerns over the safety of this type of agents. Also, it appears that the release and clearance of DTPA-Gd from the polymer backbone through in vivo disulfide-thiol exchange reaction is a rapid process, which may limit its application in steady-state MRI applications. In this study, we describe the synthesis, characterization, and preliminary evaluation of Gd3+-chelated PG polymers for blood-pool imaging applications. We demonstrate that the resulting polymeric conjugate is cleared from the body as a result of polymer backbone degradation. EXPERIMENTAL PROCEDURES

Materials. PG sodium salt, N-tert-butoxycarbonyl-1,6diaminohexane hydrochloride (t-Boc-Hex-NH2), 1,3-diisopropylcarbodiimide, pyridine, 4-(dimethylamino)pyridine, trifluoroacetic acid (TFA), diethylenetriaminepentaacetic acid (DTPA) dianhydride, gadolinium(III) chloride hexahydrate, 2,4,6-trinitrobenzenesulfonate (TNBS), 4-(2pyridylazo)resorcinol, PBS (0.01 M phosphate-buffered saline containing 138 mM NaCl and 2.7 mM KCl, pH 7.4), cathepsin B, and all the other reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO). p-Aminobenzyldiethylenetriaminepenta(acetic acid tert-butyl ester) was obtained from Macrocyclics (Dallas, TX). 111 InCl3 was obtained from Perkin-Elmer Life Sciences (Boston, MA). Spectra/Pro 7 dialysis tubing with molecular weight cutoff (MWCO) of 10 000 and Centricon Plus20 centrifugal filters were purchased from Fisher Scientific (Pittsburgh, PA). PD-10 columns came from Amersham-Pharmacia Biotech (Piscataway, NJ). Analytical Methods. Gel permeation chromatography (GPC) was performed on a Waters (Milford, MA) highperformance liquid chromatography (HPLC) system consisting of a 600 controller, a 717 plus auto sampler, and a Viscotek E-Zpro triple detector (Viscotek, Houston, TX) that records refractive index, viscosity, and light-scattering signals. The samples were separated using an TSK-G4000PW 4.6 mm × 30 cm column (TosoHaas, Montgomeryville, PA) eluted with PBS containing 0.1% LiBr at a flow rate of 1.0 mL/min. Number-average molecular weights of the polymer conjugates were calculated using Viscotek TriSEC GPC software. Radio-GPC

Bioconjugate Chem., Vol. 15, No. 6, 2004 1409

was performed with a HPLC unit equipped with a LDC pump (Laboratory Data Control, Rivera Beach, FL) and a LUDLUM radiometric detector (Measurement Inc., Sweetwater, TX). The samples were separated using a Phenomenex Biosep SEC-S3000 7.8 mm × 30 cm column eluted with PBS containing 0.1% LiBr at a flow rate of 1.0 mL/min. 1H NMR was recorded at 300 MHz on a Bruker Avance 300 spectrometer (Billerica, MA) using D2O as a solvent. Elemental analysis was performed by Galbraith Laboratories, Inc. (Knoxville, TN). Free amino groups in the polymer conjugates were quantified using a TNBS assay following a reported protocol (27). Synthesis of 6-Aminohexyl Poly(L-glutamine) [PGNH(CH2)6NH2]. The aqueous solution of sodium salt of poly-L-glutamic acid (number-average molecular weight Mn, 17 500; degree of polymerization, 116) was converted to its acid form by acidifying with 1 N HCl to pH 3-4. The polymer precipitate was collected by centrifugation, washed with deionized water, and lyophilized. Into a solution of PG (0.66 g, 5 mmol [COOH]) in 15 mL of anhydrous dimethylformamide were added t-Boc-HexNH2 hydrochloride (1.28 g, 5 mmol), 1,3-diisopropylcarbodiimide (0.76 g, 6 mmol), 0.5 mL of pyridine, and a trace amount of 4-(dimethylamino)pyridine. The reaction mixture was stirred at room-temperature overnight. The precipitate was filtered off, and the solvent was removed under vacuum. The residue was dissolved in 10 mL of trifluoroacetic acid in an ice bath and stirred at 4 °C overnight. Excess of trifluoroacetic acid was removed under vacuum. After dissolving of the residual solid in ice-water followed by neutralization with 1 N NaOH, the solution was dialyzed against PBS followed by deionized water (MWCO 10 000). The dialysate was lyophilized to yield 0.65 g (66%) of a spongelike solid. About 75% (87 of 116) of the glutamic acid residues in PG were converted to 6-aminohexyl glutamine based on the ratio of the integrals of 6-CH2 protons adjacent to the amine in the 6-aminohexyl group (δ ) 2.88 ppm, t) to the R-CH proton in the PG backbone (δ ) 4.17 ppm, s). Synthesis of PG-NH(CH2)6NH-DTPA-Gd (PG-HexDTPA-Gd). DTPA-dianhydride (1.43 g, 4 mmol) was added in portions over a period of 30 min into a solution of PG-NH(CH2)6NH2 (500 mg, ∼1.1 mmol [NH2]) in 15 mL of 0.1 M NaHCO3. The pH of the reaction solution was adjusted to 8 by adding aliquots of 0.1 N NaOH solution. After being stirred at room temperature for 2 h, the reaction mixture was dialyzed against PBS and deionized water (MWCO 10 000). The resulting solution was concentrated to 5 mL on a centrifugal filter (MWCO 10 000) and stored at 4 °C for future use. The amount of DTPA attached to PG-NH(CH2)6NH2 was determined by quantifying unreacted amino groups with the TNBS assay. Approximately 60% (52 of 87) of the amino groups in PG-NH(CH2)6NH2 were conjugated to DTPA. To chelate with Gd3+, a solution of GdCl3‚6H2O in water (100 mg/mL) was added dropwise to the aqueous solution of PG-NH(CH2)6NH-DTPA (PG-Hex-DTPA). The presence of trace amounts of unchelated Gd3+ ion in the mixture was monitored with a Gd3+ indicator 4-(2pyridylazo)resorcinol. The reaction solution was dialyzed against water (MWCO 10 000) until no free gadolinium was detected in the receiving medium. The product was further purified by gel filtration on a PD-10 column. Fractions that showed only a single polymer peak on GPC were pooled and lyophilized to give 1.0 g of product (91% yield based on PG-Hex-NH2). The compound contained 11.9% (w/w) of gadolinium as determined by elemental analysis.

1410 Bioconjugate Chem., Vol. 15, No. 6, 2004

Synthesis of PG-Benzyl-DTPA (PG-Bz-DTPA). PG (Mn, 42 000; 130 mg, 1.0 mmol of carboxylic unit) and p-aminobenzyldiethylenetriaminepenta(acetic acid tertbutyl ester) (281 mg, 0.36 mmol) were dissolved in 5 mL of anhydrous DMF, followed by the addition of 1,3diisopropylcarbodiimide (52 mg, 0.4 mmol), 0.16 mL of pyridine, and a trace amount of 4-(dimethylamino)pyridine. The reaction mixture was stirred at 4 °C overnight. To remove the protecting groups, the reaction mixture was treated with TFA at 4 °C overnight. After removal of TFA under vacuum, 10 mL of ice-cold 1 M NaHCO3 was added into the residual solid. The pH of the solution was brought up to 7.5 with 1 M NaOH, and the solution was dialyzed against PBS and water sequentially (MWCO 10 000). The resulting solution was filtered through 0.2 µm membrane filters and lyophilized to yield 230 mg of spongelike powder (yield 88%). About 55 of 278 glutamic acid residues were coupled to benzylDTPA, determined by measurement of UV/Vis absorbance of Bz-DTPA at 250 nm. Synthesis of PG-Bz-DTPA-Gd. Into a PG-Bz-DTPA (110 mg) solution in 10 mL of sodium acetate-buffered aqueous solution (0.1 M, pH 5.5) was added 0.37 mL of GdCl3‚6H2O (100 mg/mL, 0.1 mmol) in 0.1 M sodium acetate solution in small fractions. The solution was dialyzed against water (MWCO 10 000) until no free Gd3+ was detectable in the receiving vessel. The solution was lyophilized to yield 100 mg of white powder (yield of polymer 81%). The number-average molecular weight of Gd3+-chelated polymeric conjugate was about 101 200 as measured by GPC. The compound contained 12.3% (w/ w) of gadolinium. Approximately 24% of Glu repeating units contained Gd. Determination of Relaxivity. Solutions of PG-HexDTPA-Gd and PG-Bz-DTPA-Gd were prepared in water at gadolinium concentrations of 0.005, 0.01, 0.02, 0.04, 0.08, and 0.16 mM. Spin lattice (T1) and spin-spin (T2) relaxivities were measured at 1.5 T on a GE Signa LX scanner (Milwaukee, WI) using inversion recovery and mutiecho pulse sequences, respectively. Relaxivities (R1 or R2 in mM-1 s-1) were obtained from linear leastsquares determination of the slopes of 1/T1 vs [Gd] or 1/T2 vs [Gd] plots. Biodegradation of Gd-Chelated PG Polymers. Gadolinium chelated PG polymers were dissolved in PBS buffer (pH 5) at a concentration of 5.3 mg/mL. Cathepsin B was added to the solutions to a final concentration of 10 units/mL. The solutions were incubated at 37 °C. At predetermined time intervals, aliquots of the polymer solution were removed for GPC analysis. 111 In-Labeling of Gd-Chelated PG Polymers. An aqueous solution of either PG-Hex-DTPA-Gd or PG-BzDTPA-Gd (10-15 mg) in 1 M sodium acetate buffer (pH 5.5) was incubated with 300 µCi of 111InCl3 at room temperature for 15 min. Free 111In was removed by gel filtration on a PD-10 column eluted with PBS. The purity of the radiolabeled compounds was analyzed by radioGPC. Biodistribution Studies. Female C3Hf/Kam mice (20-25 g) were bred and maintained in a specific pathogen-free mouse colony in the Department of Experimental Radiation Oncology at the University of Texas M. D. Anderson Cancer Center. All experiments involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. Solid murine MCa-4 tumors were generated in the muscles of the right thigh of mice by inoculating 5 × 105 viable tumor cells in suspension in PBS. When tumors had grown to 8 mm in average diameter, mice were

Wen et al.

randomly allocated into groups, with each group consisting of 3-4 mice. Mice in each group were injected intravenously with either 111In-labeled PG-Hex-DTPAGd (0.5 mg, 2 µCi per mouse) or 111In-labeled PG-BzDTPA-Gd (0.4 mg, 15 µCi per mouse). Animals were killed at 5 min, 2 h, 24 h and 48 h after injection. Blood, liver, spleen, kidney, muscle, and tumor tissues were removed, weighed, and counted for radioactivity with a gamma counter (Packard, Downers Grove, IL). Uptakes of contrast agent in various tissues were calculated as the percentage of the injected dose per gram of tissue. Student’s t test was used to compare differences in tissue uptakes between the two agents, with p values less than 0.05 considered significant. Magnetic Resonance Imaging. For MRI studies, mice were anesthetized with 1-2% isoflurane in a 1 L/min O2 flow and placed prone within a custom sled. A custom catheter made from 2 Fr silicon tubing and a 27 gauge needle was inserted in the tail vein of the mice. Gd-DTPA (0.1 mmol Gd/kg), PG-Hex-DTPA-Gd (0.008 mmol Gd/ml, 0.04 mmol Gd/kg), or PG-Bz-DTPA-Gd (0.008 mmol Gd/ml, 0.04 mmol Gd/kg) was injected through the catheter and the catheter flushed with normal saline. Two-dimensional coronal section spinecho images were obtained before and at 10 min, 2 h, and 24 h after contrast agent injection using a 2.5 cm quadrature birdcage coil on a 1.5 T Signa LX MR scanner (GE Medical Systems, Milwaukee, WI). Six 1.5 mm contiguous sections were obtained using a 6 cm field-ofview, a 256 × 192 acquisition matrix, and four averages. A dynamic T1-weighted image set was acquired using a custom multiphase fast spin-echo sequence with an echo train length of 4, echo time of 15 ms, and repetition time of 400 ms with a temporal resolution of 13.6 s/imaging set. After baseline images were acquired, polymeric contrast agent (0.04 mmol/kg) was rapidly injected through a tail vein catheter and followed by a saline flush. The total scan time for the dynamic sequence was 600 s. RESULTS

Synthesis and Characterization of Gd-Chelated PG Polymers. Two methods were used to synthesize Gdchelated PG polymers. The synthetic schemes for both synthetic routes were shown in Figure 1. In the first method, DTPA was conjugated to PG through 6-aminohexyl linkers previously introduced to the side chains of PG. Thus, t-Boc-Hex-NH2 was coupled to the side chains of PG using carbodiimide-mediated coupling reaction, followed by removal of t-Boc protecting groups and subsequent reaction of the resulting PG-NH(CH2)6NH2 with DTPA-dianhydride to give PG-Hex-DTPA (Figure 1). About 87 of the 116 carboxyl groups in PG were converted to 6-aminohexyl glutamine based on 1H NMR analysis. Sixty percent of these 87 aminohexyl groups were further coupled to DTPA when reacted with DTPAdianhydride on the basis of consumption of NH2. Thus, the degree of DTPA substitution on the PG polymer was about 0.45 mol DTPA/mol of COOH (Table 1). On the basis of elemental analysis, the Gd content in PG-HexDTPA-Gd was 11.9%, indicating that about 0.28 mol COOH in PG was substituted with Gd-DTPA for each mole of COOH units (Table 1). In the second method, monofunctional, tert-butyl esterprotected p-aminobenzyldiethylenetriaminepenta(acetic acid) was directly reacted with carboxylic groups of PG, followed by a deprotection step to yield PG-Bz-DTPA (Figure 1). Fifty-five out of 278 glutamic acid units, or

Bioconjugate Chem., Vol. 15, No. 6, 2004 1411

Biodegradable MRI Contrast Agent Table 1. Physicochemical Properties of Gd-DTPA-Bound Poly(L-glutamic acid) property Mn calculateda measuredb polydispersityb degree of substitution (mol/mol of COOH) NH2c DPTA Gd content, % (w/w)f relaxivity,g (mM-1 s-1) R1 R2

PG-Hex-DTPA-Gd

PG-Bz-DTPA-Gd

53700 144600 3.34

77000 101200 1.23

0.75 0.45d 11.9 12 18

N/A 0.20e 12.3 21 27

DTPA-Gd 560

28.5 4.1g 5.2g

a Calculated on the basis of degree of substitution and the molecular weight of parent PG. For PG-Hex-DTPA-Gd and PG-Bz-DTPAGd, the number-average molecular weights (Mn) of parent PG were 17 500 Da and 42 000 Da, respectively. b Measured by gel permeation chromatography with a Viscotek E-Zpro triple detector system as described in Materials and Methods section. Polydispersity ) Mw/Mn. c Measured by 1H nuclear magnetic resonance. d Determined by quantifying [NH ]. e Determined by UV/Vis measurement of Bz-DTPA 2 attached to PG. f Measured by elemental analysis. g All data were obtained at 1.5T in phosphate-buffered saline. Data for Gd-DTPA were taken from ref 20.

Figure 1. Reaction scheme for the synthesis of PG-Hex-DTPAGd and PG-Bz-DTPA-Gd.

0.20 mol/mol of COOH, were attached to benzyl-DTPA as measured by UV absorbance of benzyl-DTPA at 248 nm. The degree of Gd-DTPA substitution based on Gd content in PG-Bz-DTPA-Gd (12.3%) was calculated to be 0.22 mol/mol COOH (Table 1). Other physicochemical properties of the Gd3+-chelated PG polymers are also summarized in Table 1. The reported number average molecular weights were estimated from GPC analyses. For comparison, the theoretical number-average molecular weights calculated on the basis of starting molecular weight of PG and the degree of substitution are also listed. The polydispersity of PG-

Figure 2. GPC profiles of PG-Hex-DTPA-Gd (A) and PG-BzDTPA-Gd (B) revealed by refractive index (- - -) and light scattering (___). The viscosity profile is not shown for reasons of clarity. The chromatography was run on an TSK-G4000PW column eluted with PBS containing 0.1% LiBr at a flow rate of 1.0 mL/min.

Hex-DTPA-Gd (Mw/Mn ) 3.34) was much greater than that of the starting PG polymer (Mw/Mn ) 1.2) and PGBz-DTPA-Gd (Mw/Mn ) 1.23) (Table 1). Both Gd3+chelated PG polymers had greater relaxivities than that of small molecular weight DTPA-Gd. GPC chromatograms of PG-Hex-DTPA-Gd and PG-BzDTPA-Gd are presented in Figure 2. A strong peak from the light-scattering profile was observed in the GPC chromatogram of PG-Hex-DTPA-Gd (Figure 2A). Biodegradation. The degradation of Gd-chelated PG polymers in the presence of cathepsin B was studied by analyzing changes in polymer molecular weights at various time intervals. PG-Bz-DTPA-Gd degraded rap-

1412 Bioconjugate Chem., Vol. 15, No. 6, 2004

Wen et al.

Table 2. Biodistribution of Gd3+-Chelated PG Polymersa time

blood

liver

5 min 2h 24 h 48 h

27.19 ( 4.11 16.36 ( 1.61 4.85 ( 1.26 2.19 ( 0.28**

4.33 ( 1.01 8.09 ( 0.60 7.24 ( 1.54 7.67 ( 1.50

5 min 2h 24 h 48 h

28.50 ( 1.66 7.72 ( 0.28 0.96 ( 0.12 0.53 ( 0.06

5.14 ( 0.29 8.30 ( 1.47 11.10 ( 1.72 9.36 ( 0.43

spleen PG-Hex-DTPA-Gd 6.35 ( 1.67 9.36 ( 1.13 7.68 ( 1.68 8.88 ( 1.59 PG-Bz-DTPA-Gd 6.59 ( 0.13 5.09 ( 0.46 5.19 ( 0.73 4.03 ( 0.50

kidney

muscle

tumor

15.54 ( 4.92 53.39 ( 7.47 44.60 ( 4.28 44.53 ( 3.80

0.39 ( 0.04 0.43 ( 0.08 0.44 ( 0.09 0.48 ( 0.11

1.08 ( 0.44 3.04 ( 0.32 3.44 ( 1.12 3.45 ( 0.59

8.40 ( 0.37 20.77 ( 4.70 17.54 ( 2.70 10.07 ( 0.61

0.64 ( 0.01 0.83 ( 0.07 0.55 ( 0.09 0.39 ( 0.10

0.73 ( 0.14 2.42 ( 0.77 1.74 ( 0.54 2.27 ( 0.22

a All data (other than time) are presented as percentage of injected dose per gram of tissue. Each value represents mean ( standard derivation (n ) 3 or 4).

Figure 3. Degradation of Gd3+-chelated PG polymers in the presence of cathepsin B. The polymers were incubated with a PBS solution (pH 5) of cathepsin B (10 units/ml) at 37 °C. GPC chromatograms of PG-Bz-DTPA-Gd at various time intervals revealed rapid degradation of the polymer (A). A comparison of changes in the number-average molecular weights for PG, PGBz-DTPA-Gd, and PG-Hex-DTPA-Gd is presented in B.

idly, with the main polymeric peak disappeared completely within 24 h (Figure 3A). On the other hand, the molecular weight of PG-Hex-DTPA-Gd only decreased 30% over a 48-h period (Figure 3B). Biodistribution. Both Gd3+-chelated polymers were successfully labeled with 111In as demonstrated by the absence of small-molecular-weight 111In-DTPA (retention time, 9.34 min) in the GPC chromatograms of the radiolabeled polymers (data not shown). Biodistribution data for 111In-labeled Gd3+-chelated polymers are summarized in Table 2. Both contrast agents had high blood activity (∼28% injected dose per gram of blood, IND/g) at 5 min after contrast injection. By 2 h after contrast injection, substantial activities were still found in the blood, being 16.4% IND/g and 7.7% IND/g for mice

injected with 111In-labeled PG-Hex-DTPA-Gd and PG-BzDTPA-Gd, respectively. The highest uptakes at 2-48 h period for both 111In-labeled PG-Hex-DTPA-Gd and PG-Bz-DTPA-Gd were found in the kidney. However, the activity of 111In-labeled PG-Bz-DTPA-Gd in the kidney had decreased to 10.07%IND/g by 48 h postinjecition, while that of 111In-labeled PG-Hex-DTPA-Gd remained at 44.6% IND/g. Interestingly, markedly increased uptake in tumors between 5 min and 2 h was observed with both polymers (PG-Hex-DTPA-Gd, 2.8-fold increase; PGBz-DTPA-Gd, 3.3-fold increase). At 48 postinjection, significantly lower activities were found in the blood (p < 0.001), the spleen (p < 0.05), the kidney (p < 0.001), and the tumor in mice injected with 111In-labeled PG-Bz-DTPA-Gd than in mice injected with 111In-labeled PG-Hex-DTPA-Gd (Table 2). No difference was found between their uptakes in the liver (p ) 0.1). While the uptakes of 111In-labeled PG-Hex-DTPA-Gd in the liver, the spleen, the kidney, and the muscle did not have significant change over the period of 24-48 h, the uptakes of 111In-labeled PG-Bz-DTPA-Gd in these organs had decreased 15.7%, 22.4%, 42.6%, and 29.1%, respectively, over the same time period. MRI Findings. Representative normalized MR signal intensity in the major blood vessel (e.g., IVC) and the muscle over time obtained immediately after the injection of PG-Bz-DTPA-Gd is shown in Figure 4. Figure 5 presents MR images obtained at 10 min, 2h, and 24 h after injection of PG-Bz-DTPA-Gd. Blood vessels such as the vena cava as well as myocardial, hepatic, lung, and renal perfusion were clearly visualized at 2 h after contrast injection at an injected dose of 0.04 mmol Gd/ kg (Figure 5). By 24 h after contrast injection, the bloodpool activity had largely returned to the precontrast level. Interesting, the contrast in certain regions of the tumors was clearly enhanced at 24 h after contrast injection, reflecting enhanced permeability and retention of macromolecules in solid tumors. Similar findings were also observed in mice injected with PG-Hex-DTPA-Gd (data not shown). DISCUSSION

The purpose of this study was aimed at development of biodegradable polymeric MRI contrast agents as bloodpool imaging agents. PG was chosen as the polymeric carrier for Gd3+-chelates owing to its degradability and biocompatibility. Two methods were used to conjugate DTPA to the side chains of PG, resulting in two distinctly different Gd3+-chelated PG polymers: PG-Hex-DTPA-Gd and PG-Bz-DTPA-Gd (Figure 1). The two polymers differ in the way in which the metal chelator DTPA is introduced to PG. In PG-Hex-DTPA, DTPA was conjugated to PG through hexylamine spacers previously introduced to the side chains of PG. During

Biodegradable MRI Contrast Agent

Bioconjugate Chem., Vol. 15, No. 6, 2004 1413

Figure 4. Precontrast (A) and postcontrast (B) of T1-weighted images from the dynamic MRI series acquired after bolus injection of PG-Bz-DTPA-Gd. Time course of normalized MR signal intensity from regions indicated in B represents signals obtained from inferior vena cava (IVC) (red), muscle (green), and a standard (blue) (C).

Figure 5. Whole body images of mice before and at 10 min, 2 h, and 24 h after intravenous injection of PG-Bz-DTPA-Gd at a dose of 0.04 mmol Gd/kg. (A) Selected anterior coronal image after injection of contrast agent demonstrates pulmonary and hepatic perfusion. (B) Selected posterior coronal image after injection of contrast agent demonstrates renal perfusion as well as persistent enhancement of the vena cava. Note enhancement of tumor at 24 h postinjection. Lu: lung; Li: liver; IVC, inferior vena cava; K, kidney; T: tumor.

conjugation of DTPA-dianhydride with PG-Hex-NH2, some cross-linking reactions took place. This is supported by the following observations: (1) The measured molec-

ular weight of PG-Hex-DTPA-Gd was much higher than its corresponding to theoretical molecular weights calculated on the basis of degree of substitution. (2) PG-

1414 Bioconjugate Chem., Vol. 15, No. 6, 2004

Hex-DTPA-Gd had much wider molecular weight distribution than the parent PG polymer. (3) The GPC profile of PG-Hex-DTPA-Gd showed a large peak at the dead volume of the column when light scattering signal was monitored, although this peak only represented a small fraction of polymer mass as shown by the GPC chromatogram of refractive index (Figure 2A). Cross-linking reactions occurred because bifunctional DTPA-dianhydride was used to introduce DTPA to PG-Hex-NH2. To minimize cross-linking reaction, DTPA-dianhydride was added into a dilute solution of PG-Hex-NH2 in multiple fractions over a prolonged period. The resulting PG-Hex-DTPAGd conjugate remained water-soluble and could be given safely to mice at a dose of up to 0.32 mmol Gd/kg. Doses higher than 0.32 mmol Gd/kg could not be given because of the limitation on injection volume. In PG-Bz-DTPA-Gd, a monofunctional DTPA precursor p-aminobenzyldiethylenetriaminepenta(acetic acid tertbutyl ester) was directly conjugated to the carboxyl groups on the side chains of PG (Figure 1). This resulted in a linear polymer, as evidenced by its relatively narrow molecular weight distribution, by the close agreement between calculated molecular weight and GPC-measured molecular weight (Table 1), and by the lack of a fraction of cross-linked molecules (Figure 2B). Compared to PGHex-DTPA-Gd, PG-Bz-DTPA-Gd had much higher relaxivity. The reason for this phenomenon is not clear at this time. It is plausible that linear PG-Bz-DTPA-Gd may be more readily accessible to water molecules than crosslinked PG-Hex-DTPA-Gd polymer. Several studies used isolated tissue lysosomal enzymes to investigate the degradability of PG and its derivatives (28, 29). PG was found to be more susceptible to lysosomal degradation than poly(L-aspartic acid) and poly(Dglutamic acid) (29). Recent results by Singer and his colleagues prove that monomeric L-glutamic acid is formed in the lysosomal degradation of PG (30). Evidence obtained from these studies suggests that cystein proteases, particularly cathepsins B, play key roles in the lysosomal degradation of PG (28, 30). Our results show that the difference in the linker groups (hexyl vs benzyl) and perhaps more importantly the presence/absence of polymer cross-link had profound effect on the biodegradability and distribution of Gd-chelated PG polymers. While PG-Hex-DTPA-Gd was essentially not degradable in the presence of cathepsin B, PG-Bz-DTPA-Gd degraded rapidly within 24 h (Figure 3). In vivo, PG-BzDTPA-Gd was cleared from the blood, spleen, and kidney faster than PG-Hex-DTPA-Gd. Significantly less uptakes in these tissues were found in mice injected with PG-BzDTPA-Gd than in mice injected with PG-Hex-DTPA-Gd at 48 h postinjection. (Table 2). It is noted that although the cross-linked polymer PG-Hex-DTPA-Gd had a high level of uptake in the kidney, the kidney uptake of the linear polymer PG-Bz-DTPA-Gd, on the other hand, was only about one-fourth of that of PG-Hex-DTPA-Gd at 48 h after contrast injection. Unlike PG-Hex-DTPA-Gd, the uptake of PG-Bz-DTPA-Gd in the kidney decreased steadily over a period of 48 h. Therefore, PG-Bz-DTPAGd, but not PG-Hex-DTPA-Gd, warrants further development for potential clinical applications. Dynamic MR images obtained with PG-Bz-DTPA-Gd confirm that the agent markedly enhanced vascular contrast without causing significant loss of signal because of its limited diffusion into intravascular fluid space (Figure 4). MR imaging of mice bearing MCa-4 mammary tumors before and at various time points after the injection of PG-Bz-DTPA-Gd revealed significant contrast enhancement of blood vessels at up to 2 h after their

Wen et al.

intravenous injection. Indeed, vena cava and pulmonary, myocardial, hepatic, and renal perfusion of mice were clearly visualized (Figure 5). The signal intensities in the blood vessels gradually decreased over the time. By 24 h postinjection, the signal intensities in the blood pool had returned to the preinjection level, suggesting degradation and gradual clearance of the contrast agent from the body. Interestingly, the signal intensity in the tumor had increased during the same time period. These results indicate that PG-Bz-DTPA-Gd was retained in the MCa-4 tumors, possibly a result of the increased permeability and retention effect of macromolecules. In summary, we have demonstrated that the degradability and biodistribution pattern of Gd3+-chelated PG polymers are strongly affected by the way Gd3+ chelators are coupled to PG carrier. PG-Bz-DTPA-Gd and its ability to be cleared from the body make it a favorable macromolecular MRI contrast agent. The dose of PG that would need to be introduced into the body to obtain an equivalent Gd dose of 0.04 mmol/kg is estimated to be less than 52 mg PG/kg. These doses are 10- to 20-fold less than the dose of PG given to rodents without causing apparent toxic effect (25). Detailed studies are necessary to fully assess the toxicity of PG-Bz-DTPA-Gd before it is introduced into clinical applications. Further evaluation of both PG-Hex-DTPA-Gd and PG-Bz-DTPA-Gd is ongoing. ACKNOWLEDGMENT

We would like to thank the National Institutes of Health (grant U54 CA90810), the Department of Defense Breast Cancer Research Program (grant DAMD17-00-10314), and the John S. Dunn Foundation for financial support of this work. LITERATURE CITED (1) Kroft, L. J. and de Roos, A. (1999) Blood pool contrast agents for cardiovascular MR imaging. J. Magn. Reson. Imaging 10, 395-403. (2) Bogdanov Jr., A. A., Lewin, M., and Weissleder, R. (1999) Approaches and agents for imaging the vascular system. Adv. Drug Delivery Rev. 37, 279-293. (3) Brasch, R. C. (1992) New directions in the development of MR imaging contrast media. Radiology 183, 1-11. (4) van Bemmel, C. M., Wink, O., Verdonck, B., Viergever, M. A., and Niessen, W. J. (2003) Blood pool contrast-enhanced MRA: Improved arterial visualization in the steady state. IEEE Trans. Med. Imaging 22, 645-652. (5) Su, M. Y., Muhler, A., Lao, X., and Nalcioglu, O. (1998) Tumor characterization with dynamic contrast-enhanced MRI using MR contrast agents of various molecular weights. Magn. Reson. Med. 39, 259-269. (6) van Dijke, C. F., Brasch, R. C., Roberts, T. P., Weidner, N., Mathur, A., Shames, D. M., Mann, J. S., Demsar, F., Lang, P., and Schwickert, H. C. (1996) Mammary carcinoma model: Correlation of macromolecular contrast-enhanced MR imaging characterizations of tumor microvasculature and histologic capillary density. Radiology 198, 813-818. (7) Gossmann, A., Okuhata, Y., Shames, D. M., Helbich, T. H., Roberts, T. P. L., Wendland, M. F., Huber, S., and Brasch, R. C. (1999) Prostate cancer tumor grade differentiation with dynamic contrast-enhanced MR imaging in the rat: Comparison of macromolecular and small-molecular contrast media-preliminary experience. Radiology 213, 265-272. (8) Daldrup, H., Shames, D. M., Wendland, M., Okuhata, Y., Link, T. M., Rosenau, W., Lu, Y., and Brasch, R. C. (1998) Correlation of dynamic contrast-enhanced MR imaging with histologic tumor grade: Comparison of macromolecular and small-molecular contrast media. Am. J. Roentgenol. 171, 941949.

Bioconjugate Chem., Vol. 15, No. 6, 2004 1415

Biodegradable MRI Contrast Agent (9) Lauffer, R. B., Parmelee, D. J., Dunham, S. U., Ouellet, H. S., Dolan, R. P., Witte, S., McMurry, T. J., and Walovitch, R. C. (1998) Ms-325: Albumin-targeted contrast agent for MR angiography. Radiology 207, 529-538. (10) Adzamli, K., Yablonskiy, D. A., Chicoine, M. R., Won, E. K., Galen, K. P., Zahner, M. C., Woolsey, T. A., and Ackerman, J. J. (2003) Albumin-binding MR blood pool agents as MRI contrast agents in an intracranial mouse glioma model. Magn. Reson. Med. 49, 586-590. (11) Schmiedl, U., Ogan, M., Paajanen, H., Marotti, M., Crooks, L., Brito, A., and Brasch, R. (1987) Albumin labeled with GdDTPA as an intravascular, blood pool-enhancing agent for MR imaging: Biodistribution and imaging studies. Radiology 162, 205-210. (12) Schuhmann-Giampieri, G., Schmitt-Willich, H., Frenzel, T., Press, W. R., and Weinmann, H. J. (1991) In vivo and in vitro evaluation of Gd-DTPA-polylysine as a macromolecular contrast agent for magnetic resonance imaging. Invest. Radiol. 26, 969-974. (13) Bogdanov, J., Alexei A., Weissleder, R., and Brady, T. J. (1995) Long-circulating blood pool imaging agents. Adv. Drug Delivery Rev. 16, 335-348. (14) Wang, S. C., Wikstrom, M. G., White, D. L., Klaveness, J., Holtz, E., Rongved, P., Moseley, M. E., and Brasch, R. C. (1990) Evaluation of Gd-DTPA-labeled dextran as an intravascular MR contrast agent: Imaging characteristics in normal rat tissues. Radiology 175, 483-488. (15) Rebizak, R., Schaefer, M., and Dellacherie, E. (1997) Polymeric conjugates of Gd(3+)-diethylenetriaminepentaacetic acid and dextran. 1. Synthesis, characterization, and paramagnetic properties. Bioconjugate Chem. 8, 605-610. (16) 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. (17) Adam, G., Neuerburg, J., Spuntrup, E., Muhler, A., Scherer, K., and Gunther, R. W. (1994) Gd-DTPA-cascade-polymer: Potential blood pool contrast agent for MR imaging. J. Magn. Reson. Imaging 4, 462-466. (18) Kobayashi, H. and Brechbiel, M. W. (2003) Dendrimerbased macromolecular mri contrast agents: Characteristics and application. Mol. Imaging 2, 1-10. (19) Kim, Y. H., Choi, B. I., Cho, W. H., Lim, S., Moon, W. K., Han, J. K., Weinmann, H. J., and Chang, K. H. (2003) Dynamic contrast-enhanced MR imaging of vx2 carcinomas after X irradiation in rabbits: Comparison of gadopentetate dimeglumine and a macromolecular contrast agent. Invest. Radiol. 38, 539-549.

(20) Unger, E. C., Shen, D., Wu, G., Stewart, L., Matsunaga, T. O., and Trouard, T. P. (1999) Gadolinium-containing copolymeric chelatessa new potential MR contrast agent. Magma 8, 154-162. (21) Gupta, H., Wilkinson, R. A., Bogdanov, A. A., Jr., Callahan, R. J., and Weissleder, R. (1995) Inflammation: Imaging with methoxy poly(ethylene glycol)-poly-l-lysine-DTPA, a longcirculating graft copolymer. Radiology 197, 665-669. (22) Rebizak, R., Schaefer, M., and Dellacherie, E. (1998) Polymeric conjugates of Gd(3+)-diethylenetriaminepentaacetic acid and dextran. 2. Influence of spacer arm length and conjugate molecular mass on the paramagnetic properties and some biological parameters. Bioconjugate Chem. 9, 94-99. (23) Franano, F. N., Edwards, W. B., Welch, M. J., Brechbiel, M. W., Gansow, O. A., and Duncan, J. R. (1995) Biodistribution and metabolism of targeted and nontargeted proteinchelate-gadolinium complexes: Evidence for gadolinium dissociation in vitro and in vivo. Magn. Reson. Imaging 13, 201214. (24) Li, C. (2002) Poly(l-glutamic acid)- -anticancer drug conjugates. Adv. Drug Delivery Rev. 54, 695-713. (25) Li, C., Yu, D. F., Newman, R. A., Cabral, F., Stephens, L. C., Hunter, N., Milas, L., and Wallace, S. (1998) Complete regression of well-established tumors using a novel watersoluble poly(l-glutamic acid)-paclitaxel conjugate. Cancer Res. 58, 2404-2409. (26) Lu, Z. R., Wang, X., Parker, D. L., Goodrich, K. C., and Buswell, H. R. (2003) Poly(L-glutamic acid) Gd(iii)-DOTA conjugate with a degradable spacer for magnetic resonance imaging. Bioconjugate Chem. 14, 715-719. (27) Hermanson, G. T. (1996) Bioconjugate Techniques, pp 112113, Academic press, San Diego. (28) Chiu, H. C., Kopeckova, P., Deshmane, S. S., and Kopecek, J. (1997) Lysosomal degradability of poly(alpha-amino acids). J. Biomed. Mater. Res. 34, 381-392. (29) Kishore, B. K., Lambricht, P., Laurent, G., Maldague, P., Wagner, R., and Tulkens, P. M. (1990) Mechanism of protection afforded by polyaspartic acid against gentamicin-induced phospholipidosis. Ii. Comparative in vitro and in vivo studies with poly-l-aspartic, poly-l-glutamic and poly-d-glutamic acids. J. Pharmacol. Exp. Ther. 255, 875-885. (30) Singer, J. W., Baker, B., De Vries, P., Kumar, A., Shaffer, S., Vawter, E., Bolton, M., and Garzone, P. (2003) Poly-(l)glutamic acid-paclitaxel (ct-2103) [Xyotax], a biodegradable polymeric drug conjugate: Characterization, preclinical pharmacology, and preliminary clinical data. Adv. Exp. Med. Biol. 519, 81-99.

BC049910M