Polymeric Gadolinium Chelate Magnetic Resonance Imaging Contrast

Bioconjugate Chem. 1999, 10, 361−370

361

Polymeric Gadolinium Chelate Magnetic Resonance Imaging Contrast Agents: Design, Synthesis, and Properties David L. Ladd,*,† Robert Hollister,† Xin Peng,‡ Donna Wei, Gang Wu,§ Daniel Delecki,† Robert A. Snow,† John L. Toner,| Kenneth Kellar,† Jennifer Eck,⊥ Vinay C. Desai,† Gemma Raymond,† Lewis B. Kinter,# Terry S. Desser M. D.,3 and Daniel L. Rubin M. D.3 Torsten Alme´n Research Center, Nycomed Amersham Imaging, 466 Devon Park Drive, Wayne, Pennsylvania 19087 . Received July 23, 1998; Revised Manuscript Received February 8, 1999

We have synthesized and evaluated five series of polymeric gadolinium chelates which are of interest as potential MRI blood pool contrast agents. The polymers were designed so that important physical properties including molecular weight, relaxivity, metal content, viscosity, and chelate stability could be varied. We have shown that, by selecting polymers of the appropriate MW, extended blood pool retention can be achieved. In addition, relaxivity can be manipulated by changing the polymer rigidity, metal content affected by monomer selection, viscosity by polymer shape, and chelate stability by chelator selection.

INTRODUCTION

The use of paramagnetic gadolinium chelates to enchance magnetic resonance imaging (MRI) examinations has become increasingly important (Hohenschuh and Watson, 1997). The gadolinium-containing contrast agents that are currently approved for human use (Omniscan, Magnevist, Prohance, and Dotarem) are all low molecular weight chelates. When administered parenterally, these agents rapidly extravasate out of the circulation into the interstitial space. For some imaging applications (Brasch, 1992), it would be desirable to have an agent that was largely retained within the intravascular space during the time frame of an MRI examination, a so-called “blood pool” agent. Several approaches have been explored in the quest for MR blood pool agents including: (1) low MW Gd-chelates that bind to serum proteins (Lauffer et al., 1996; Wallace et al., 1998), (2) iron oxide particles (Engelbrecht et al., 1998), (3) Gd-liposomes (Unger et al., 1990), and (4) polymeric Gd-chelates. While others have prepared polymeric Gd-chelates by grafting chelators onto preformed polymers such as albumin (Schmiedl et al., 1987), polylysine (Schuhmann-Giampieri et al., 1991), dextran (Rebizak et al., 1998; Casali et al., 1998), and dendrimers (Schwickert et al., 1995), we have focused our efforts to obtain MR blood pool agents primarily on wholly synthetic polymers. As contrast agents for MRI, the potential advantages of macromolecular complexes in comparison to the currently available monomeric low molecular weight complexes are well recognized (Brasch, 1991). An increased lifetime of the contrast agent in the blood pool has been * To whom correspondence should be addressed. Phone: 610225-4349. Fax: 610-225-4413. E-mail: [email protected]. † Nycomed Amersham Imaging. ‡ SmithKline Beecham. § Bristol Myers Squibb. | Abbott Laboratories. ⊥ Sanofi Research Division. # Astra Pharmaceuticals. 3 Department of Radiology, Stanford University School of Medicine and VA Palo Alto Health Care System.

identified as an important feature in both blood pool and dynamical tumor imaging (Brasch, 1991; Desser et al., 1994a). However, increasing the lifetime in the blood pool is a necessary, but not sufficient, criteria for attaining a useful contrast agent. The agent must also fulfill additional safety related criteria. The ideal Gd-based MR blood pool contrast agent will reside in the blood pool long enough to allow for convenient MR imaging, then clear from the blood pool rapidly after completion of the MR imaging procedure. For dynamical tumor imaging, the situation is even more complicated (Desser et al., 1994a; Roberts et al., 1997). The uptake and clearance from tumors depends on many factors, including the size and structure of the contrast agent (Roberts et al., 1997). Whatever the application, the agent should be a benign, low-volume parenteral that is completely eliminated from the body within a reasonable time. However, except for possibly the dendrimer-based gadolinium chelates, little attention has been given to the design of macromolecular complexes which have a basic structure that can easily be altered to control the resulting physical properties and size. Such a structure that can easily be altered is important, because it allows optimization of the contrast agent with respect to blood pool lifetime or tumor uptake and clearance. Within the basic structure of these complexes, the molecular weight, as well as other important physical properties including relaxivity, metal content, viscosity, and chelate stability, can be optimized. This report describes the design rationale that we employed and the results that were obtained with five series of polymeric gadolinium chelates. Since the polymers described herein are prototypical, not all of the above parameters were optimized, or even measured, for each polymer. Indeed, optimization of all of the parameters for a given agent is probably not feasible due to the interrelationship of the parameters studied, which necesitates the need to make compromises when designing a new agent. Thus, for each series, one or more features were chosen to be modified to illustrate the effect on a given parameter and to demonstrate the feasibility of modifying the key attributes of an MR blood pool polymer.

10.1021/bc980086+ CCC: $18.00 © 1999 American Chemical Society Published on Web 03/19/1999

362 Bioconjugate Chem., Vol. 10, No. 3, 1999 EXPERIMENTAL PROCEDURES

General Procedures. The identity of all intermediates was confirmed by 1H NMR spectroscopy; purity was assessed by silica gel TLC. Molecular weight, polydispersity, and purity of polymers were determined by sizeexclusion-HPLC. MolecularWeightDeterminations.Molecularweights and molecular weight distributions were obtained by size exclusion-HPLC comparing the polymers to a set of PEG standards obtained from American Polymer Standards Corp. A Perkin-Elmer series 200 analytical HPLC fitted with a series of three Supelco 30 cm × 7.8 mm size-exclusion columns comprised of a Progel-TSK G2000 SWXL, a Progel-TSK G3000 SWXL, and a Progel-TSK G4000 SWXL were employed. Detection was with a refractive index detector. The method involved 20 µL injections of 1 mg/mL solutions of the polymer samples, a mobile phase comprised of 30% acetonitrile:70% pH 6.8 phosphate buffer, a flow rate of 0.6 mL/min, and a 60 min isocratic program. Turbochrome software was used to control the program and Turbogel software to do the MW analysis. Rabbit Blood Pool Retention Measurements. Formulations of polymers in isotonic saline were injected into the lateral ear vein of New Zealand white rabbits via a 21-gauge Butterfly needle at doses of 0.06 or 0.10 mmol of Gd/Kg. Arterial blood samples for relaxivity measurement were obtained via a catheter placed percutaneously into the lateral ear artery. Blood samples were withdrawn and collected into heparin tubes at 0, 5, 15, 30, 60, 120, 360, 600, and 1440 min following the injection (note: in some experiments every time point was not collected). T1 measurements were made on the blood samples using a Bruker Mini-Spec operating at 20 MHz and 40 °C. 153Gd Labeling of Polymers. Unmetalated polymers were dissolved in 0.1 M, pH 5.0, sodium acetate buffer and treated with 153Gd by addition of 157GdCl3 containing approximately 0.1% (w/w) 153GdCl3 (Amersham, Arlington Heights, IL). The DTPA polymers were reacted for 15 min at room temperature, and the DOTA polymers were reacted for 2 h at 50 °C. The radiolabeling efficiencies as determined by instant thin-layer chromatography (ITLC) and HPLC were found to be >99%. The labeled polymers were purified by preparative SEC-HPLC, and the test article was diluted in 0.9% sterile saline, pH 6.5, to a final concentration of approximately 30 mM Gd. Rat Blood Pool Retention Measurements. Six rats (0.35-0.45 Kg) previously prepared with polyurethane catheters were injected intravenously with 0.08 mmol of Gd/Kg (approximately 0.5 mL containing 1 mCi) of test article. Blood samples (100 mL) were withdrawn at 0.042, 0.08, 0.17, 0.33, 0.5, 0.75, 1, 1.25, 1.5, 2, 4, 6, 24, 48, and 168 h and the radioactivity quantitated using a γ counter. Viscosity Measurements. The viscosities were measured in water with a Brookfield viscometer (model LVCP DVII-plus) equipped with a cone and plate spindle and a jacketed sample cell for temperature control. The temperature was controlled with a LAUDA RM6 circulating water bath. The viscometer was calibrated using Brookfield viscosity standards that ranged from 5 to 100 cP. Relaxation Rate Measurements. The proton longitudinal relaxation rate (1/T1) for aqueous solutions of the polymers was measured by the inversion-recovery method using a Bruker Minispec PC 120/125/10 Vts NMR Process Analyzer operating at 0.47 T and 40 °C. The relaxivity of a given polymer was calculated by subtracting the 1/T1 of pure water from the 1/T1 measured for the polymer-

Ladd et al.

containing solution and dividing this quantity by the concentration of gadolinium as determined by ICP. The relaxivities were found to be independent of gadolinium concentration. Polyoxyethylene Bis(chloride) (3). To a solution of poly(ethylene glycol) [2, average MW, 1.45K] (1000 g, 0.69 mol) in 1.5 L of toluene (60-80 °C) were added SOCl2 (200 mL, 2.76 mol) and DMF (10 mL) dropwise over 10 min. The reaction mixture was heated on a steam bath for 1 h. An analysis by TLC (CH2Cl2:MeOH; 6:1) indicated that a very small amount of starting material still remained. An additional 20 mL of SOCl2 was added to the reaction, and it was heated on the steam bath for another 20 min to drive the reaction to completion. After cooling the reaction to 0 °C with an ice bath, 1 N NaOH (2.5 L) was carefully introduced to neutralize the solution and the layers were separated. The aqueous layer was washed with CH2Cl2 (3 × 1 L), and the combined CH2Cl2 layers were washed with water (2 × 1 L) and dried with MgSO4. It was filtered and concentrated under reduced pressure to give a light yellow oil. The product was precipitated by adding TBME (2 L) to the residue with cooling and stirring. The filtered product was dried in the oven under vacuum overnight to yield 941 g (94%) of the titled compound as a white solid. An analysis by HPLC indicated that the purity of the product was 99.86% with no oligomer present. Anal. calcd for C64H128O31Cl2: C, 52.49; H, 8.81; Cl, 4.84. Found: C, 51.94; H, 8.43; Cl, 5.00. Polyoxyethylene Bis(azide) (4). To a suspension of polyoxyethylene bis(chloride) (3, 500 g, 0.336 mol) and KI (139 g, 0.841 mol) in 1500 mL of DMF was added NaN3 (109 g, 1.68 mol). The suspension was heated on a steam bath at 70 °C for 12 h to give a yellow solution. After cooling the reaction to room temperature, 2.5 L of water was added and the solution was extracted with CH2Cl2 (3 × 1 L). The combined CH2Cl2 layers were washed with water (3 × 1 L), dried over MgSO4, filtered, and concentrated under reduced pressure to give a light yellow oil. The product was precipitated out by adding TBME (1 L) to the residue with cooling and stirring. The filtered product was dried in the oven under vacuum overnight at room temperature to yield 452 g (90%) of the PEG diazide as a white solid. An analysis by HPLC indicated that the purity of the product was 99.2% with no oligomers present. Anal. calcd for C64H128O31N6: C, 52.09; H, 8.73; N, 5.69. Found: C, 52.50; H, 8.58; N, 5.11. Polyoxyethylene Bis(amine) (5). To a solution of polyoxyethylene bis(azide) (4, 176 g, 0.117 mol) in 1 L of 1 N HCl was added Pd/C (17.6 g). The suspension was hydrogenated at 45 psi for 15 h. An aliquot withdrawn from the reaction indicated no more starting material by TLC (CH2Cl2:MeOH; 4:1), and the catalyst was removed by carefully filtering through a short plug of Celite. The filtrate was neutralized with 10% NaOH to pH 5-6 and extracted with CH2Cl2 (3 × 600 mL) to remove the impurities. The aqueous solution was neutralized with 35% NaOH to pH >10 and extracted with CH2Cl2 (2 × 500 mL). The CH2Cl2 layer was washed with water (500 mL), brine (500 mL), and dried over MgSO4. The filtrate was concentrated under reduced pressure to give a light yellow oil. The product was precipitated by adding TBME (1 L) to the residue with cooling and stirring. The filtered product was dried in the oven under vacuum at roomtemperature overnight to give 138 g (81% yield) of the titled compound as a white solid. Analysis by HPLC indicated that the purity of the product was 96.9% with no oligomer detected by gel chromatography. Anal. calcd

Polymeric Gadolinium Chelate Contrast Agents

Bioconjugate Chem., Vol. 10, No. 3, 1999 363

for C64H132O31N2: C, 53.92; H, 9.33; N, 1.96. Found: C, 53.24; H, 9.35; N, 1.66. Preparation of Polymer NC 66368 (1a). [Note: WFI (water for injection) is used for this entire process.] To a solution of polyoxyethylene bis(amine) (5, 550 g, 0.380 mol) in 5500 mL of water was added triethylamine (159 mL, 1.14 mol) and DTPA-dianhydride (6, 149 g, 0.418 mol). The suspension was stirred at room temperature and gave a clear solution after 10 min. The reaction was stirred for an additional 50 min, and a solution of GdCl3‚ 6H2O (156 g, 0.418 mol) in 2000 mL of water was added. The reaction mixture was checked with PAR reagent to be sure only a slightly excess of GdCl3‚6H2O was added. The complexed solution (pH 2) was neutralized to pH ∼5 with 10% NaOH and then diafiltered using a Pellicon diafiltration unit with a 10K cutoff filter for 10 turnovers: A 10% NaCl (USP grade) solution (filtered through a 0.22 µ filter) was used for the first four turnovers and water was used for the remaining six turnovers. At the end of the diafiltration, the solution was concentrated to half of its original volume (pH 7), filtered through a 0.22 µ filter and then lyophilized for 3 days leaving (532 g, 80%) a sponge-like product. MW ) 20 200; p ) 1.63. Total Gd ) 7.09% w/w. KF moisture ) 2.55%. Elemental analysis; anal. calcd for [C80H152GdN5O40]x: C, 48.50; H, 7.73; N, 3.53. Found (corrected for H2O): C, 48.14; H, 7.91; N, 3.29. Preparation of Polymer NC 22181 (10a). (Note: WFI is used for this entire process). To a solution of hexamethylenediamine (8, 200 g, 1.72 mol) in 4 L of DMSO was added triethylamine (810 mL, 5.52 mol) and DTPAdianhydride (6, 664 g, 1.84 mol). The suspension was stirred at room temperature for 45 h, and a light yellow solution was obtained. Four liters of EtOAc was added to the solution, and product (9) was precipitated as an oil. Solvent was decanted, and the oil product was further washed with EtOAc (2 × 2 L). After the oil product was dried under vacuum for 1 h, it was dissolved in 4 L of water and GdCl3‚6H2O (312 g) was added (PAR reagent was used to ensure that no excess Gd3+ was added). The solution was neutralized to pH ∼5 with NaOH and filtered through a 0.45 µ filter. The filtered solution was diafiltered using a Pellicon diafiltration unit with a 10K cutoff filter for 10 turnovers: A 10% NaCl (USP grade) solution (filtered through a 0.22 µ filter) was used for the first five turnovers and water was used for the remaining five turnovers. At the end of the diafiltration, the solution was concentrated to half of its original volume (pH 7), filtered through a 0.22 µ filter and then lyophilized for 3 days leaving (203 g) a sponge-like product (10a). MW ) 18 900; p ) 1.51. Total Gd ) 22.00% w/w. KF moisture ) 10.68%. Elemental analysis; anal. calcd for [C20H32GdN5O8]x (corrected for H2O and NaCl): C, 33.54; H, 5.70; N, 9.78. Found: C, 33.44; H, 5.77; N, 9.63; Na, 0.69; Cl, 0.98. In a similar manner polymers 10a of different MWs were prepared using the following conditions: molecular mmol mmol DMSO reaction mass (kDa)/ expt of 8 of 6 base/mmol (mL) time (h) polydispersity 1 2 3 4

5.1 5.5 51.0 55.0 645. 678. 129.1 135.5

NaOH/16.5 Et3N/159. Et3N/2030. Et3N/406.6

18 206 2500 250

50 18 168 24

8.8/1.50 14.2/1.54 18.1/1.34 21.0/1.39

In experiment 3, intermediate 9 was precipitated with ether prior to complexation with GdCl3‚6H2O; in experi-

ments 1, 2, and 4, the reaction mixtures were diluted with water and then complexed with GdCl3‚6H2O. Preparation of Polymer NC 22177 (16a). A solution of methoxy-PEG of average MW 550 (11, 66.67 g, 0.1212 mol; Sigma Chemical Co.) in 1000 mL of toluene was refluxed with azeotropic removal of water for several hours. The cooled toluene solution was treated with triethylamine (55.75 mL, 0.4 mol), 4-(dimethylamino)pyridine (2.96 g, 0.0242 mol) and tosyl chloride (69.33 g, 0.3636 mol) and heated in an oil bath at 60 °C for 48 h. The reaction mixture was then cooled and filtered. The filtrate was extracted three times with water. The combined aqueous extracts were washed three times with ether, then extracted three times with chloroform. The chloroform extracts were dried over anhydrous MgSO4 and concentrated to yield 47.58 g (56%) of product 12. A solution of 14.96 g (21.25 mmol) of 12 in 150 mL of absolute EtOH was treated with 31.8 mL (213 mmol) of tris(2-aminoethyl)amine (13). The reaction mixture was heated in a stainless steel bomb at 160 °C for 16 h. The solvent was removed by evaporation and the residue dissolved in 257 mL of water and made basic with 42.5 mL of 1 N NaOH. The basic solution was washed twice with ether, then extracted twice with chloroform. The chloroform extracts were dried over anhydrous MgSO4; solvent evaporation followed by heating at 90 °C under vacuum (1 mm) to remove residual tris(2-aminoethyl)amine (13) yielded 12.14 g (84%) of 14 as a viscous tan oil. A solution of 4.23 g (6.24 mmol) of 14 in 42 mL of acetonitrile was treated with triethylamine (2.61 mL, 18.7 mmol) and diethylenetriaminepentaacetic acid dianhydride (6) (3.34 g, 9.36 mmol). After 1.0 h at room temperature, 168 mL of water was added and the solution was stirred for 30 min. The solution of crude polymer 15 was treated with gadolinium(III) chloride hexahydrate (3.65 g, 9.83 mmol) then diafiltered against water in a diafiltration cell equipped with a 10 000 MW cutoff membrane. The pH of the solution was adjusted to 7 with NaOH, then the solution was filtered through a 0.2 mm nylon filter. Lyophilization yielded 4.15 g of product (16a) of average molecular mass 13 600 Da (p ) 1.36) as determined by SEC-HPLC using PEG molecular mass standards; 15.6% gadolinium by weight. Preparation of Polymer NC 100203 (24a). A solution of 5.00 g of 19 (hydrobromide salt; WO 93/02045), methyl bromoacetate (0.795 mL), tetramethylguanidine (14.0 mL), and 100 mL of acetonitrile was refluxed for 3 h. The reaction mixture was then concentrated and the residual oil taken up in toluene and washed three times with water. The toluene solution was dried over anhydrous magnesium sulfate and concentrated to 4.46 g of product 20. A solution of 4.10 g of 20 in 82 mL of methanol was treated with 41 mL of tris(2-aminoethyl)amine and stirred for 3 days at room temperature under N2. The solution was concentrated under reduced pressure (1 mm) at 60 °C then taken up in CHCl3 and washed four times with water, dried over anhydrous MgSO4, and concentrated to 4.56 g of 21. A solution of 3.989 g of 17 (Shearwater Polymers, Inc.) in 26.8 mL of CH2Cl2 was treated with 270.5 mg of N-hydroxysuccinimide and 2.579 mL of a 1 M solution of dicyclohexylcarbodiimide in CH2Cl2 for 2 h. The above reaction mixture of 18 was treated with a solution of 0.776 g of 21 in 23 mL of CH2Cl2 for 1 h, filtered, and concentrated to give 3.81 g of 22. The tritert-butyl ester 22 (4.78 g) was treated with 48 mL of CH2Cl2 and 5 mL of TFA for 16 h, a second 5 mL portion of TFA was added, and the reaction continued for 16 h; a third 5 mL portion of TFA was added, and the reaction

364 Bioconjugate Chem., Vol. 10, No. 3, 1999

continued for another 16 h, after which NMR indicated incomplete reaction. A fourth 5 mL portion of TFA was added, and after 4 days, the reaction was concentrated to dryness; NMR still indicated incomplete reaction. The crude product was treated with 25 mL of neat TFA for 16 h, after which NMR indicated complete reaction. The reaction mixture was then concentrated and taken up in 100 mL of water, and the pH was raised to 7.0 with 2 N KOH. The solution was then diafiltered vs water in a stirred cell with a YM-3 membrane (Amicon, Inc., Beverly, MA) and lyophilized to give 3.56 g of 23. A 1.55 g sample of 23 in 31 mL of water was treated with 0.185 g of GdCl3‚6H2O; the pH was maintained at 4.5 with 1 N NaOH for 16 h. The pH was lowered to 3.0 with 1 N HCl, and then the solution was diafiltered vs water in a stirred cell with a YM-3 membrane (Amicon, Inc., Beverly, MA). The pH was adjusted to 7.0, and the solution lyophilized to give to give 1.41 g of 24a of average molecular mass 16 200 Da (p ) 1.36) as determined by SEC-HPLC using PEG molecular mass standards; 2.51% Gd by weight determined by ICP. The R1 relaxivity was determined to be 6.6 mM-1 s-1 using a Bruker Mini-Spec operating at 20 MHz and 40 °C. Preparation of Polymer NC 100372 (34). To a solution of 24.6 g (0.062 mol) of 6-aminocaproic acid benzylester p-toluenesulfonic acid salt and 20 g (0.19 mol) of sodium carbonate in 460 mL of 50% water in methylene chloride was added dropwise over 45 min a solution of 10 mL (0.13 mol) of chloroacetyl chloride in 230 mL of methylene chloride. Stirring was continued at room temperature under a nitrogen atmosphere for 2 h. The organic phase was separated and dried over anhydrous magnesium sulfate. Evaporation of the solvent left 18.4 g of crude product 28 which was further purified by flash chromatography on silica gel eluting with CH2Cl2. A solution of 0.676 g (1.14 mmol) of 19, 0.338 g (1.14 mmol) of 28, and 0.43 mL (3.4 mmol) of 1,1,3,3-tetramethylguanidine in 14 mL of acetonitrile was stirred at room temperature under a nitrogen atmosphere for 18 h. Volatiles were removed under reduced pressure, and the residue was partitioned with a mixture of toluene and water.The toluene phase was separated and washed with water, then dried over anhydrous magnesium sulfate, filtered from the drying agent, and concentrated to yield 0.79 g of product 29. A solution of 5.0 g (6.4 mmol) 29 in 200 mL of methanol was treated with 0.5 g of 10% palladium on carbon and hydrogenated on a PARR shaker for 17 h. After the uptake of hydrogen had ceased, the catalyst was removed by careful filtration through a bed of Celite. The volatiles were removed from the filtrate using reduced pressure, and the residue obtained was triturated well with dry ethyl ether. The ether phase was decanted and concentrated under reduced pressure to yield 2.3 g of the product 30 as a white glass. Another 1.4 g of less pure product remained after the ether trituration. To a stirring solution of 2.25 g (3.30 mmol) of 30 (previously dried via azeotropic distillation from methylene chloride) and 0.43 g (3.6 mmol) of dry N-hydroxysuccinimide in 45 mL of methylene chloride at room temperature and under a nitrogen atmosphere was added dropwise, in 5 min, a solution of 7.4 mL (3.7 mmol) of 0.5 M N,N-dicyclohexylcarbodiimide in methylene chloride. After the addition was complete, stirring was continued at room temperature under nitrogen for about 17 h. The reaction slurry was filtered, and the volatiles were removed from the filtrate under reduced pressure to yield 2.7 g of the product 31 as a white glass. A solution of 0.437 g (0.076 mmol) of third generation PAMAM dendrimer (Meltzer et al., 1992) and 2.6 g (2.7 mmol) of 31 in 20 mL of

Ladd et al. Scheme 1

anhydrous dimethyl sulfoxide was stirred at room temperature under a nitrogen atmosphere for 3 days. Most of the solvent was removed under reduced pressure at 60 °C, and the viscous residue was triturated with absolute ethyl ether three times, decanting each time, leaving a residue which was dried under high vacuum yielding 1.6 g of product 32. The average number of unreacted amines on the PAMAM dendrimer moiety of compound 32 was determined to be approximately four by a ninhydrin assay (Curotto and Aros, 1993). Elemental analysis calcd for C905H1,573N191O225, corrected for water of hydration: C, 51.14%; H, 7.46%; N, 12.6%. Found: C, 51.44%; H, 7.66%; N, 12.18%. Treatment of 1 g (0.5 mmol) of 32 with 50 mL of a 25% trifluoroacetic acid solution in methylene chloride at 0 °C under a nitrogen atmosphere was followed by allowing the stirring reaction mixture to warm to ambient temperature during 1 h after which the volatiles were removed under reduced pressure. The residue was triturated with absolute ethyl ether, the ether decanted, and the residue dried under vacuum. The reaction was repeated with 50% trifluoroacetic acid for overnight reaction time. The volatiles were removed under reduced pressure, and the resulting residue stirred with absolute ethyl ether for 2 h, then filtered, washed with ether, and dried in a vacuum oven at 50 °C to give 0.9 g. The 0.9 g was dissolved in 10 mL of deionized water and filtered, and the pH of the filtrate was raised to 8 with 2 N potassium hydroxide solution. This solution was then diafiltered in an Amicon stirred cell against water using a 5000 Da cutoff membrane for 30 turnovers totaling 300 mL. The purified solution was filtered through a 0.2 µ filter and lyophilized yielding a tan foam which was dried under high vacuum and 40 °C to afford 0.4 g of 33



Table 1. Physicochemical and Biological Data for Polymeric Gadolinium Chelates compound

molecular mass (kDa)

magnevist 1a (NC 66368) 10a (NC 22181) 16a (NC 22177) 24a (NC 100203) 34 (NC 100372)

0.9 20.2 18.9 13.6 16.2 18.2e

T1/2 n rabbit 0.77 ( 0.05 6.22 ( 0.13i,j 1.64 ( 0.20k 9.38 ( 0.68i

T1/2 n rat 0.33g

2.96 ( 0.13l 0.53 ( 0.06

%Gd (w/w) 16.8 7.1 ( 0.28 22.0 ( 0.88 15.6 ( 0.62 2.5 ( 0.10 17.3e

R1 (mM-1 s-1)d

viscosity (cP)a (100 mM Gd)

rat 7 day bone retentionm

3.8 ( 0.15 6.0 ( 0.24 9.5 ( 0.39 10.1 ( 0.41 6.6 ( 0.31

2.9f 56.1b

0.21 ( 0.05g,h 3.50 ( 0.68 4.91 ( 0.83

2.2c

0.76 ( 0.30 0.62 ( 0.08

a 1a, 45.3 cP at 20 °C for 210 mg of polymer/mL; 16a, 5.1 cP at 25 °C for 199 mg of polymer/mL. b 20 °C. c 25 °C. d 20 MHz, 40 °C. Calculated value; Gd chelate was prepared in situ from 153GdCl3/157GdCl3. f 500 mM Gd, 37 °C. g Weinmann et al. (1984). h Whole body minus liver, spleen and kidney. i Significantly different from Magnevist, p < 0.01. j Significantly different from 10a, p < 0.01. k Significantly different from 16a, p < 0.01. l Significantly different from 34, p < 0.05. m Percent injected dose. n Mean ( standard deviation.

e

(polydispersity of 1.06). Elemental analysis calcd for C665H1193N191O205 (corrected for water and potassium): C, 48.07; H, 7.96; N, 16.10; K, 2.35. Found: C, 48.17, 48.32; H, 7.70, 7.52; N, 15,48, 15.99; K, 2.88, 3.17.

Scheme 2

RESULTS AND DISCUSSION

Initially, we prepared (Desser et al., 1994a) a series of copolymers of diethylenetriaminepentaacetic acid (DTPA) and poly(ethylene glycol) (PEG) diamines chelated to gadolinium (1, Gd-DTPA-PEGs) ranging in MW from 10.8 to 83.4 kDa as shown in Scheme 1. PEG-diamine (5) was prepared in two steps from poly(ethylene glycol) (2) and polymerized with the dianhydride of DTPA (6) and the resultant polymer (7) chelated with gadolinium to produce the Gd-DTPA-PEG polymer (1a). DTPA was selected as the chelator based upon its successful use in the marketed agents Omniscan and Magnevist; poly(ethylene glycol) diamines were chosen based upon the well-established biocompatability, hydrophilicity, and synthetic accessibility of PEG derivatives (Zalipsky, 1995). The Gd-DTPA-PEG polymers were evaluated (Desser et al., 1994a) in a rabbit model to (1) identify the molecular weight above which Gd-DTPA-PEG polymers behave as blood pool agents and (2) to analyze the tumor enhancement dynamics as a function of polymer molecular weight in a rabbit tumor model. This study showed that linear Gd-DTPA-PEG polymers with a molecular mass of 20 kDa and higher exhibited significant blood pool retention over a 60 min time frame. Therefore, we focused upon a Gd-DTPA-PEG polymer (1a, NC 66368) with a molecular mass of ∼20 kDa prepared from polyethene glycol of MW 1450 (Scheme 1) as our initial lead structure. While NC 66368 proved to be efficacious in the rabbit tumor model (Desser et al., 1994a) and a model demonstrating time-of-flight magnetic resonance angiography (TOF-MRA) (Desser et al., 1994b), it possessed some shortcomings. The low R1 relaxivity (6.0) combined with the low gadolinium content (7.1%) made it necessary to administer large amounts of polymer in order to achieve adequate doses of gadolinium to produce the required t1 shortening. In addition, as the formulation concentration of NC 66368 was increased to minimize the injection volume, the solution viscosity became unacceptably high (56.1 cP for a 100 mM Gd formulation, see Table 1). One approach to addressing the shortcoming of NC 66368 was to design an analogous series of copolymers (10) of diethylenetriaminepentaacetic acid with shortchain alkyldiamines in place of the longer chain poly(ethylene glycol) diamines (Scheme 2). While these polymers had the expected increase in gadolinium content, from 7.1% for 1a to 22.0% for 10a, they also exhibited (Kellar et al., 1997) unexpected increases in R1 relaxivity, from 6.0 for 1a to 9.5 for 10a. A subset of this series, analogues of 10 in which the spacer length (n) was

varied, was found to have relaxivities up to 20.7 (Kellar et al., 1997). This combination of increased gadolinium content and increased relaxivity resulted in significant decreases in the amount of polymer required for imaging studies. From this series of polymers, NC 22181 (10a) was identified as a significantly improved lead. Like NC 66368, NC 22181 has been shown to be an effective contrast agent for MRA imaging in rabbits (T. Desser and D. L. Rubin, unpublished data) and an effective agent for MR urography in pigs (Nolte-Ernsting et al., 1997). While blood pool retention is known to be affected by polymer MW, size, shape, and charge (Petrak and Goddard, 1989), within a given series in which MW and consequently size is the only variable, it would be expected that blood pool retention would be determined by MW. However, this relationship would not necessarily hold up comparing polymers across series as more than one variable could be changing. Therefore, the results from the Gd-DTPA-PEG polymer series could not be used to confidently predict the minimal MW required for NC 22181 to act as a blood pool agent, and thus polymers ranging in molecular mass from 9 to 21 kDa were prepared and evaluated in the rabbit model. The results of this study are summarized in Figures 2 and 3. The data shows that NC 22181 of molecular mass 14 kDa and above has a prolonged blood pool phase reflected in significantly increased blood half-lives and decreased volumes of distribution and thus would qualify as a candidate MR blood pool imaging agent. A third approach to designing blood pool agents was aimed primarily at addressing the relaxivity and viscosity


Ladd et al.

Figure 3. Volume of distribution and overall blood clearance half-life vs molecular weight for NC 22181 of four different molecular weights.

Figure 1. Polymeric gadolinium chelates.

Figure 2. Blood retention vs time for NC 22181 of four different molecular weights and Magnevist.

issues. A series was designed (Ladd, 1996) to produce polymers with a more globular shape than the above two linear polymers. The nonlinear shape was achieved by appending poly(ethylene glycol) chains to the polymer backbone [vs incorporation of poly(ethylene glycol) chains into the polymer backbones of 1 and 10] and incorporating interchain cross-links through the DTPA moieties (Scheme 3). Methoxy-PEG-triamine (14) was prepared from the methoxy-PEG-tosylate (12), polymerized with

1.5 equiv of DTPA dianhydride and chelated with gadolinium to give 16a. It was expected that the more globular shape of 16a would result in lower viscosity solutions and that the increased rigidity resulting from cross-linking would result in higher relaxivities. This was indeed found to be the case. Solutions of 16a had considerably lower viscosities than solutions of 1a when compared on the basis of similar gadolinium concentration or polymer concentration (see Table 1). The relaxivity of 16a (10.1) was found to be 68% higher than for the linear, non-crosslinked polymer 1a. Since blood pool gadolinium polymers, by design, reside in the body for extended periods of time, there is an increased opportunity for in vivo metal dissociation (Cacheris et al., 1990; Wedeking et al., 1992; Duncan et al., 1994; Deal et al., 1996; Corot et al., 1998). This is illustrated by the percentage of the injected dose recovered in the bone at 7 days (Table 1). For example, Magnevist with a short half-life has bone gadolinium retention of 0.21% of the injected dose (% ID), while the two DTPA based polymers (1a and 10a) showed higher levels of retention at 7 days, 3.50 and 4.91%ID, respectively. Only bone data at 7 days is reported in Table 1 as no other major organs retained significant amounts of metal and bone retention is generally considered to be a good measure of long term gadolinium retention. Traditionally, reduction of metal retention has been addressed by the choice of chelator. Therefore, in response to the concern of long term metal retention, a series of polymers was designed which employ the macrocyclic chelator DOTA in place of the linear chelator DTPA. Macrocyclic Gd-DOTA chelates which are found in the marketed agents Prohance and Dotarem are known (Wedeking et al., 1992) to be more thermodynamically and kinetically stable than linear Gd-DTPA chelates. Scheme 4 shows the synthesis of a DOTA polymer (24a). The PEG-bisNHS ester (18) was polymerized with the tri-tert-butyl ester-DOTA-diamine (21), and the resultant polymer (22) deprotected with TFA and chelated with gadolinium. The requirement that the DOTA nucleus maintains at least three carboxylates to maintain the desired tight metal binding necessitated trade offs that resulted in a polymer with less than optimal properties. Appending the chelator to the polymer backbone through a single amide bond resulted in a nonrigid structure and consequently a relatively low R1 relaxivity of 6.6. Because the polymerization of 21 with PEG-bis-NHS esters was not a very efficient process, a high molecular mass PEG monomer



Scheme 3

(18, molecular mass ) 3.4 kDa) was needed to form a polymer with the requisite MW for blood pool retention resulting in a low metal content for the polymer (2.5%). As anticipated, the 7 day bone retention for 24a (0.76% ID) was significantly lower than for the two DTPA based polymers (1a and 10a). While it is recognized that 24a is structurally dissimilar to 1a and 10a, its significant blood half-life in rats qualifies it as a blood pool agent and therefore the reduced bone retention is meaningful. An additional example of a polymeric Gd-DOTA (34) was prepared (Scheme 5) by grafting DOTAs onto the surface of a dendrimer. While various linkers and chemistries have been employed (Lewis et al., 1994; Li et al., 1994; McCall et al., 1990; Kline et al., 1991; Brasch et al., 1994; Kubomura et al., 1993; Schmitt-Willich et al., 1992) to graft DOTA macrocycles onto a variety of macromolecules, we chose to use the novel intermediate 31. DOTA-tris(tert-butyl ester) activated ester (31) was

designed to couple DOTA to the dendrimer via conversion of one of the four DOTA carboxylates to an amide of aminocaproic acid, thus placing the chelators at a reasonable distance from the surface of the dendrimer. Intermediate 28 was prepared from amino-ester 27 (Goodacre et al., 1977), which was prepared (Callahan et al., 1988) from 25 (Sigma). When activated ester 31 was reacted with a third generation PAMAM dendrimer, approximately 20 of the 24 surface amino groups on the dendrimer were derivatized to form the DOTA grafted dendrimer 32. Deprotection of 32 with TFA followed by metalation with gadolinium chloride produced the chelated polymer 34. Like 24a, 34 was found to have less 7 day bone retention (0.62% ID) than the DTPA-based polymers 1a and 10a. While the blood half-life of globular polymer 34 was determined to be longer than Magnevist, it was considerably shorter than the linear DOTA polymer 24a which likely is a reflection of the influence of shape on polymer retention.


Ladd et al.

Scheme 4

CONCLUSION

We have shown that polymeric gadolinium chelates with extended blood pool retention can be obtained by selecting polymers of the appropriate MW. For example, with linear copolymers of DTPA and PEG-diamines (1), it was found that polymers of 20 kDa and higher are blood pool agents as are linear copolymers of DTPA and alkyldiamines (10) of 14 kDa and higher. For polymer 10a (NC 22181), linear relationships were found for both

overall blood clearance half-lives and volumes of distribution vs MW within the range of MWs studied (Figure 3). In addition, with copolymers 10 a significant decrease in dose was realized as a consequence of these polymer’s increased metal contents and higher relaxivities. It has also been shown that additional features which are important to achieve a pharmaceutically acceptable contrast agent can be controlled by the design of the polymer. Changing the shape of the polymer from linear as are



Scheme 5

series 1 and 10 to the more globular series 16 resulted in decreased viscosity as well as increased relaxivity. Potential in vivo metal dissociation was addressed by designing a polymer series (24) which employs the DOTA chelator which is known to form gadolinium chelates that are both kinetically and thermodynamically more stable than DTPA gadolinium chelates. Finally, we designed a polymer (34) that conceptually incorporates most of the above desirable features. The DOTA-dendrimer construct is globular with tight binding gadolinium chelators capable of incorporating a high percentage of metal. In summary, we have demonstrated that the principal characteristics of polymeric gadolinium chelates including blood retention, metal content, viscosity, chelate stability, and relaxivity can be manipulated by the choice of features incorporated into the polymer. To the best of our knowledge, this report is the first study that addresses the rational design of polymeric MR

blood pool contrast agents. The purpose of this study was not to evaluate every parameter for each class of polymer, but to emphasize the relative importance of each parameter. We hope that this paper serves as the basis for further work to develop agents to satisfy an important unmet medical need. LITERATURE CITED Brasch, R. C. (1991) Rational and applications for macromolecular Gd-based contrast agents. Magn. Reson. Med. 22, 282-287. Brasch, R. C. (1992) New Directions in the Development of MR Imaging Contrast Media. Radiology 183, 1-11. Brasch, R. C., Mann, J. S., and Nitecki, D. E. (1994) Macromolecular Contrast Media for MR Imaging. WO 9427498, Dec. 8, 1994. Cacheris, W., Quay, S. C., and Rocklage, S. M. (1990) The Relationship Between Thermodynamics and the Toxicity of Gadolinium Complexes. Magn. Reson. Imaging 8, 467-481.

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Polymeric Gadolinium Chelate Magnetic Resonance Imaging Contrast

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