Dendritic Iodinated Contrast Agents with PEG-Cores for CT Imaging

Jul 1, 2006 - The purpose of this study was to design, synthesize, and initially characterize a representative set of novel constructs for large-molec...
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Bioconjugate Chem. 2006, 17, 1043−1056

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Dendritic Iodinated Contrast Agents with PEG-Cores for CT Imaging: Synthesis and Preliminary Characterization Yanjun Fu,§ Danute E. Nitecki,§ David Maltby,‡ Gerhard H. Simon,§ Kirill Berejnoi,§ Hans-Juergen Raatschen,§ Benjamin M. Yeh,§ David M. Shames,§ and Robert C. Brasch§,* Center for Pharmaceutical and Molecular Imaging, Department of Radiology, and Mass Spectra Facility, Department of Pharmaceutical Chemistry, University of CaliforniasSan Francisco (UCSF), San Francisco, California 94143-0628. Received January 26, 2006; Revised Manuscript Received April 20, 2006

The purpose of this study was to design, synthesize, and initially characterize a representative set of novel constructs for large-molecular radiographic/computed tomography (CT) contrast agents, intended for a primarily intravascular distribution. A new assembly of well-known and biocompatible components consists of paired, symmetrical dendritic polylysines initiated from both ends of a poly(ethylene glycol) (PEG) core, yielding an array of multiple free amino groups to which were conjugated highly soluble and stable triiodophthalamide (“triiodo”) moieties. An array of six dendritic contrast agents was synthesized originally, using three different PEG cores (3, 6, 12 kDa) with t-Boc lysine-generated dendrimer “amplifiers” (from three to five generations) containing 16 to 64 amino groups for conjugation with reactive triiodo moieties. A clinically used, nonionic, small molecular CT contrast agent, iobitridol, was derivatized via a hydroxyl protection/deprotection strategy, introducing a new carboxyl group available for conjugation to the lysine amino groups of dendrimers. Final products were purified by size exclusion chromatography and characterized by NMR, UV, HPLC, and elemental analysis. Preliminary evaluations were conducted for physicochemical characterization and in vivo CT contrast enhancement in a rat model. All six iodinated PEG-core dendrimer conjugates were synthesized in good yields, with a high degree of size monodispersity, large apparent molecular weight, favored physicochemical properties. A representative compound, PEG12000-carbamate-Gen4-IOB conjugate, 27% (w%) rich in iodine, demonstrated a desirable strong and persistent intravascular enhancement with a monoexponential blood half-life of approximately 35 min assayed by dynamic CT imaging and also showed high water solubility (>550 mg/mL at 25 °C), large apparent molecular size (comparable to a 143-kDa protein), high hydrophilicity (butanol-water partition coefficient 0.015), and stability to autoclaving conditions. This study showed the synthetic feasibility, desired basic characteristics, and potential utility for CT contrast enhancement achieved with a new type of iodinated, large-molecular PEG-core dendritic construct. Further development of this class of macromolecular contrast agents will be required to define the optimal formulation, pharmacology, safety profile, and the full range of diagnostic applications including tumor microvascular quantitative characterization by CT imaging.

INTRODUCTION New water-soluble radio-dense iodinated pharmaceuticals administered intravenously in conjunction with diagnostic X-ray and computed tomography (CT) have the potential to improve the quality, nature, and yield of resulting diagnostic information. So-called iodinated “contrast media” were first considered in the same year as Wilhelm Roentgen’s discovery of the X-ray and have evolved over the past century to the current state of well tolerated and highly efficacious drugs used in the vast majority of patient CT examinations. Without exception, the CT contrast media currently given parenterally, either intravenously or intra-arterially, belong to a class of relatively small molecular compounds, less than 2000 Da, which quickly equilibrate between the intravascular and extracellular fluid compartments of the body. However, another class of compounds, namely macromolecular contrast media (MMCM), has been described, both for CT and magnetic resonance imaging (MRI) tomography, as being potentially better suited for * Corresponding author. Phone: (415) 476-2275. Fax: (415) 4760616. E-mail: [email protected], yanjun.fu@ radiology.ucsf.edu. § Center for Pharmaceutical and Molecular Imaging, Department of Radiology. ‡ Mass Spectra Facility, Department of Pharmaceutical Chemistry.

quantitative characterization of tissues and blood vessels and certain types of pathologic abnormalities as found in cancer, ischemic injury, and altered states of angiogenesis (1-4). These MMCM, sometimes referred to as “blood pool” contrast media, have in common a prolonged intravascular half-life, an initial volume of distribution approximating the plasma volume, and molecular weights generally greater than 30 kDa. Properties considered desirable for candidate macromolecular CT contrast media, in addition to an intravascular distribution, include relatively high content of iodine or other radiodense atoms, high stability, near or complete monodispersity, low osmolality, high water solubility, limited viscosity, high in vivo tolerance, and complete elimination from the body. Toward these goals, a new group of iodinated MMCM, intended for use as blood pool CT contrast enhancing agents, has been designed, synthesized, and preliminarily tested. This class of agents consists of paired, symmetrical dendritic polylysines in a cascade arrangement initiated from both ends of a poly(ethylene glycol) (PEG) core, yielding an array of multiple (16 to 64) free amino groups for conjugation with tri-iodinated ring intermediates. Six of these tri-iodo PEG dendrimer conjugates with varying sizes of the PEG core (3, 6, and 12 kDa) and varying dendritic generations (3, 4, and 5) were synthesized as examples of the class and compared for physicochemical properties. One selected compound was further

10.1021/bc060019c CCC: $33.50 © 2006 American Chemical Society Published on Web 07/01/2006

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evaluated by in vivo CT imaging in an experimental animal subject for angiographic contrast enhancement.

EXPERIMENTAL PROCEDURES Materials and Apparatus. Poly(ethylene glycol) (PEG) 6000 (Mn 6470, Mw 6517, polydispersity index 1.01, measured by MALDI-TOF mass spectroscopy) was purchased from Fluka. PEG12000 (Mn 12163, Mw 12650, polydispersity index 1.04 according to the manufacturer’s data) was purchased from Polymer Labs. These PEGs were dried by azeotropical distillation with anhydrous benzene. Poly(ethylene glycol) bisamine 3400 (Mn 3290, Mw 3321, polydispersity index 1.01) was purchased from Shearwater Polymers Inc., N2,N6-di-tert-butyloxycarbonyl-L-lysine dicyclohexylamine salt (N2,N6-di-t-BocL-Lys‚DCHA) was from Advanced ChemTech, dicyclohexyl carbodiimide (DCC), N-hydroxysuccinimide (HONSu), p-nitrophenyl chloroformate, N,N-dimethylaminopyridine (DMAP), diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), N,Ndimethylformamide (DMF, dried with 4-8 mesh 4 Å molecular sieves), 1,3,5-trinitrobenzenesulfonic acid (TNBS), and other reagents were purchased from Aldrich, Sigma, or Fischer Scientific. All these reagents are of analytical grade or superior. Conventional iodinated contrast agents Iohexol, Iobitridol, and Iodixanol were commercially available, and Iobitridol solution was lyophilized to give a white powder of 99.9% purity (by reversed phase HPLC). H-1 and C-13 NMR spectra were recorded on a Varian 400 NMR spectrometer. Analytical SE-HPLC was performed on a Superdex 200 10/300 GL column (10 mm × 300 mm) using a Rainin HPXL solvent delivery system and a Rainin pressure module (Rainin Instrument Inc., Emeryville, CA) with a Knauer diode array UV detector and a Shimadzu RID-6A as the refractive index detector. A solution of 0.1% TFA and 0.3 M Na2SO4 (pH 2.7) was used as the mobile phase at 0.80 mL/min and 23 °C for purity test of PEG-core dendritic polylysines (UV detection at 210 nm), while a solution of 0.05 M phosphate and 0.15 M NaCl (pH 7.0) was used as the mobile phase at 0.80 mL/min and 23 °C for the purity test of iodinated contrast agents (245 nm). Preparative size exclusion chromatography (SEC) was run on a Sephadex G-100 (or G-75) column (5 cm I. D. × 50 cm) with UV detection. TLC with detection by UV or iodine vapor, or a ninhydrin spray test, was conducted on precoated Merck TLC plates with silica gel G25 UV254 (5 × 20 cm, 250 µm). UV spectra were recorded on a Shimadzu UV260 spectrophotometer. ESI mass spectra were recorded on a Perkin-Elmer SCIEXAPI300 LC/MS mass spectrometer. MALDITOF mass spectra were obtained on a Perkin-Elmer Voyger DESTR mass spectrometer. Melting points were determined on an electrothermal melting point apparatus (uncorrected). Dialysis was carried out using the regenerated cellulose Spectra/Por tubing (cutoff Mw 3500 Da). The CT experiment was performed at 80 kVp and 130 mA on an eight-slice clinical GE LightSpeed QX/I CT Scanner (GE Medical Systems, Milwaukee, WI). One seven-week-old healthy Sprague Dawley rat (180 g, female, Charles River Laboratories, Wilmington, MA) was used for CT imaging. Activation of N2,N6-Di-tert-butyloxycarbonyl-L-lysine. Preparation of Free N2,N6-Di-t-Boc-L-lysine. According to a standard procedure, dicyclohexylamine (DCHA) was removed from N2,N6-di-t-Boc-L-Lys‚DCHA by thoroughly washing its CH2Cl2 solution with 0.5 M citrate buffer (pH 3.5). Yield 92%. TLC: Rf ) 0.70 (CHCl3:CH3OH: HAc ) 90:8:2). 1H NMR (400 MHz, CDCl3): δ (ppm) 1.22-1.28 (t, 4H, CH2CH2), 1.44 (s, 18H, tert-butyl), 1.68 (m, 2H, CH2CH), 3.10 (t, 2H, CH2N), 4.21 (t, 1H, NCHCO), 4.90 (br, 1H, N6-H), 5.42 (d, 1H, N2-H, J ) 7.2 Hz), 10.39 (br, 1H, COOH).

Fu et al.

Carboxy ActiVation of N2,N6-Di-t-Boc-L-lysine. DCC (2.06 g, 10 mmol) was dissolved in 10 mL of CH2Cl2 and cooled to -15 °C. With stirring, to this cold solution was dropwise added a solution of N2,N6-di-t-Boc-L-Lys (3.47 g, 10 mmol) in CH2Cl2 (20 mL). The reaction temperature was kept below -10 °C. Several minutes later, the reaction mixture turned turbid and then was continued to stir for 3-4 min. The resulting solution containing DCC-activated di-t-Boc-lysine should be used immediately in the subsequent coupling reaction with the amines. For comparison, two types of active esters of di-t-Boc-lysine, namely 4-nitrophenyl ester or N-hydoxysuccinimide ester, were also prepared by the reaction of this t-Boc amino acid with 4-nitrophenol (or N-hydroxysuccinimide) and DCC in an equivalent mole ratio in anhydrous chloroform. Synthesis of PEG-Core Dendrimers (with PEG6000 as an example). Preparation of PEG6000 Bis(4-nitrophenyl carbonate (5a). Dried PEG6000 (Mn 6470, 4.0 g, 0.62 mmol) was dissolved in anhydrous CH2Cl2 (15 mL) and dry pyridine (15 mL) and then cooled to 0 °C. To it was added 1.0 g of 4-nitrophenyl chloroformate (4.96 mmol) and 80 mg of DMAP. At 0 °C this mixture was stirred for 8 h. The reaction mixture was evaporated and precipitated by 80 mL of ether. After standing 0.5 h, the syrupy precipitate solidified and was filtered and washed by ether. The crude product was dissolved in 1-2 mL of CH2Cl2 and precipitated in 40 mL of anhydrous ether; this procedure was repeated twice. A white powder (5a, 4.06 g) was finally obtained (97% yield). 1H NMR (CDCl3), δ 3.61 (s, CH2CH2O), 7.3 (d, aromatic H), 8.2 (d, aromatic H). In 13C NMR (CDCl3), δ 155.2 (OCO2). UV spectrophotometry at 400 nm showed, after complete hydrolysis of this carbonate at pH 12, each mole of the carbonate contained 2.05 mol of 4-nitrophenyl group. R,ω-Bis(N-t-Boc-ethylcarbamoyl)-PEG6000. A solution of 5a (2.58 g, 0.38 mmol) in 15 mL of CHCl3 was added dropwise to a solution of 1-N-t-Boc-ethylenediamine (493 mg, 3.08 mmol, prepared according to the literature method) (5) and DIPEA (0.6 g, 4.64 mmol) in 10 mL of CHCl3 at 0 °C. This reaction continued for 24 h at room temperature. The resulting mixture was evaporated and precipitated by 50 mL of ether and purified by three cycles of dissolution-precipitation using CHCl3 and ether. A slightly yellow powder 6 (2.50 g) was obtained in a yield of 97%. 1H NMR (CDCl3), δ 1.41 (s, tert-butyl), 2.92, 3.13 (2 m, CH2N), 3.62 (s, CH2CH2O). PEG6000 Bisamine. Boc-protected biscarbamate 6 (3.02 g, 0.446 mmol) obtained above was dissolved in 8 mL of CH2Cl2 and cooled to 0 °C, to which was added 8 mL of TFA. This mixture was stirred at 0 °C for 30 min and room temperature for another 2 h. Evaporation of the solution under 60 °C gave an oil after adding 50 mL of anhydrous ether. Dissolution/ precipitation of the crude product was performed twice with CH2Cl2/ether. The precipitate solidified while standing at room temperature for several hours, further purified by dialysis (cutoff Mw 3500 Da), and then lyophilized to give 2.93 g of a white powder (yield 97.5%). 1H NMR (CDCl3), δ 1.42 (s, tert-butyl), 2.90, 3.11 (2 m, NCH2), 3.61 (s, CH2CH2O). This PEG bisamine was thus used as an initiator core molecule for subsequent dendrimer synthesis. PEG6000 bisamine can be also regarded as “PEG6000-carbamate-Gen0.0” dendrimer, while “Gen” stands for “generation” and “carbamate” is the chemical linkage between the PEG core and lysine dendrimer component. Dendrimer Propagation (two-step cycle to reach a higher generation). Typical Coupling Step (PEG6000-Gen0.5 preparation). Immediately after the DCC-activated di-t-Boc-lysine was formed as described above, a cooled solution of PEG6000 bisamine (2.07 g, 0.608 mmol NH2) and DIPEA (0.39 g, 3 mmol) in 10 mL of CH2Cl2 was added with stirring at -5 °C. This coupling reaction (COOH/NH2 mole ratio ) 4.0) was

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PEG-Core Dendrimeric CT Contrast Agents Table 1. Synthetic Yields and Structure Data of Six PEG-Core Dendrimeric Polyamines code

yield, % (based on PEG)

8a 8b 9a 9b 11a 11b

50 41 45 45 39 32

a

molecular weight a Mn Mw 8462 13871 10262 16785 7270 11328

polydispersity (Mw/Mn)

NH2 numberb measured (theoretical)

-CH2CH2O-/lysyl ratio NMR-measured (theoretical)

1.012 1.005 1.006 1.028 1.002 1.001

15.3 (16) 15.6 (16) 30.8 (32)c 31.6 (32) 31.8 (32)c 62.9 (64)c

11.03 (10.47) 19.93 (19.72) 5.16 (4.89) 9.05 (9.20) 2.29 (2.48) 1.25 (1.20)

8532 13942 10321 17260 7288 11339

Measured by MALDI-TOF mass spectrometry. b Determined by a classic TNBS method (6). c Determined by a modified TNBS method.

continued at -5 °C for 0.5 h and room temperature for 20 h. A white precipitate was filtered and washed by CH2Cl2, and the combined filtrate was evaporated under reduced pressure to give a thick syrup. After the dissolution of the syrup in 3 mL of CH2Cl2, 120 mL of anhydrous ether was added. Standing 0.5 h, a white precipitate separated and gradually solidified. This procedure was repeated twice so as to remove DIPEA and excess di-t-Boc-lysine from the crude product. The compound was washed profusely by ether and dried in a vacuum. A white, loose solid was afforded in a yield of 87% (1.93 g). Ninhydrin test: negative. Typical Deprotection Step (PEG6000-Gen1.0 preparation). PEG6000-Gen0.5 (1.8 g, 0.247 mmol) was dissolved in 15 mL of CH2Cl2 and cooled to 0-5 °C. With stirring, 15 mL of TFA was added slowly while the reaction temperature was kept below 20 °C. After the TFA addition, the reaction was continued at room temperature for 2-3 h. An anhydrous MgSO4 drying tube was connected to the reaction flask since gaseous byproducts such as CO2 and isobutene were emitted in this deprotection. After the reaction went to completion, the mixture was evaporated under reduced pressure below 40 °C, giving a slightly yellow syrup. Anhydrous ether was added. After standing 1-2 h at room temperature, the syrup solidified. The dissolution/ precipitation procedure was repeated twice. A white precipitate was collected, washed with ether, and dried in a vacuum to give 1.78 g of a white powder (yield 98%, positive for ninhydrin test). Following procedures from Gen1.0 through Gen5.0 are similar. Yields of coupling steps ranged from 85 to 92%, while the yields of deprotection steps varied from 90 to 98%. Every reaction was monitored by ninhydrin test. Of important note, one repeated coupling was conducted in the preparation of Gen4 and Gen5 dendrimers to guarantee a complete capping of those amino groups. Preparative SEC (Sephadex-75 gel, mobile phase 0.1 M trifluoroacetate buffer with pH 3.0, flow rate 14 mL‚min-1‚cm-2) and subsequent dialysis were employed for purification of these full-generation dendrimers (8a through 11b). NMR, MALDI-TOF MS, and UV (for amino quantification using TNBS method) (6) were used for characterization of main intermediates and undecorated dendrimers (data shown in Table 1). For t-Boc-protected half-generation dendrimers including Gen0.5, Gen1.5, Gen2.5, Gen3.5, and Gen4.5, 1H NMR spectra (CDCl3) were given: δ 1.3-1.6 (br, (CH2)3 in lysine), 1.42 (s, tert-butyl), 3.1(br, NCH2), 3.61 (s, CH2CH2O), 4.2 (br, NCHCO). For deprotected full-generation dendrimers including Gen1.0, Gen 2.0, Gen3.0, Gen4.0, and Gen5.0 (TFA salts), 1H NMR spectra (D2O): δ 1.3-1.8 (br, (CH2)3 in lysine), 2.93.2 (br, NCH2), 3.62 (s, CH2CH2O), 4.1-4.3 (d, br, NCHCO). The integration ratio of characteristic peaks (PEG and CH2CH2CH2 in lysine) obtained in 1H NMR confirmed that the t-Boc-protected and deprotected dendrimers had the expected structure. Six synthesized dendrimers for subsequent decoration include PEG6000-carbamate-Gen3.0 (1a), PEG12000-carbamate-Gen3.0 (1b), PEG6000-carbamate-Gen4.0 (2a), PEG12000carbamate-Gen4.0 (2b), PEG3400-amide-Gen4.0 (3a), and PEG3400-amide-Gen5.0 (3b-I).

For two representative dendrimers, 13C NMR data were given. For PEG3400-carbamate-Gen5.0: δ (ppm) 22.7, 26.5, 28.0, 30.9 (CH2CH2CH2CH), 39.4 (CH2N), 53.4, 54.6 (CH), 69.7 (PEG), 112.2, 115.1, 118.0, 120.9 (CF3), 162.7, 163.1 (CF3COO-), 174.7, 179.0 (N6-CO and N2-CO); for PEG6000-carbamateGen4.0, δ (ppm) 25.7, 26.5, 28.1 (CH2CH2CH2), 39.1, 39.5 (CH2N), 53.2, 53.8 (CH), 69.7 (PEG), 112.2, 115.0, 117.9, 120.7 (CF3), 162.5, 163.0 (CF3COO-), 174.4, 178.7 (N6-CO and N2CO in dendritic lysine). Quantification of Periphery Amino Groups in the “PEG Amplifiers”. The NH2 analysis was conducted according to a typical TNBS method (6). To 1 mL of a standard solution (a series of L-lysine mono hydrochloride solution with known concentration, 20-100 µg/mL) or 1 mL of a dendrimer sample solution (0.2-1.5 mg/mL) were added 1.0 mL of 4% sodium bicarbonate solution (pH 8.5) and 1.0 mL of 0.1% freshly prepared TNBS solution. The reaction was continued at 40 °C for 4 h, and 1.0 mL of 10% sodium dodecylsulfonate (SDS) solution and then 0.5 mL of 1 N HCl were added, consecutively. Against a blank control solution the absorbance at 335 nm of standard or sample solutions were recorded. Amino concentration in solutions and hence amino contents in dendrimers were determined (results shown in Table 1). For 9a, 11a, and 11b, their trinitrophenyl conjugates obtained above were redissolved in 1,4-dioxane for UV quantification at 415 nm. Synthesis of an Amino-Reactive “Triiodo” Derivative. Hydroxyl Protection of Iobitridol by Ketalization (compound 13 synthesis). Method A. A powder of Iobitridol 12 (16.70 g, 20 mmol) was added in 50 mL of DMF. With stirring, 10.4 g of 2,2′-dimethoxylpropane (DMP, 100 mmol) and 100 mg of p-toluenesulfonic acid monohydrate were then added. The resulting solution was brought up to 90 °C and stirred at this temperature for 1 h in which the acetalization reaction was shown complete by TLC (CHCl3/CH3OH 15:1, material Rf ) 0, product Rf ) 0.64, 0.50, isomers). DMF and excess DMP were removed by vacuum distillation. A crude product was obtained as a yellow syrup. Further purification was conducted by silica gel chromatography using CHCl3 and then CHCl3/ CH3OH 8:1 as the eluting solvent, 18.7 g of slightly yellow crystalline 13 was obtained (yield 98%). M.p. 88-90 °C. ESIMS: m/z ) 956.0 (M + H)+, 978.0 (M + Na)+, 898.0 (M CH3COCH3 + H)+ for C29H40O9I3N3. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.35, 1.40, 1.46 (3s, 18H, 3 isopropylidene groups), 2.96, 2.97 (2 s, 6H, 2 N-CH3), 3.22 (m, 1H, CHCd O), 3.36, 4.03 (2 br, 4 H, 1:1, CH(CH2O)2), 3.85, 4.17 (2m, 4H, 1:1, 2 NCH2), 4.25, 4.29 (2 m, 4 H, 1:1, 2 OCH2CHO), 4.53 (m, 2 H, 2 OCH2CHO), 8.52, 9.45 (2 br, 1 H, 0.45:0.55, PhNHCdO). 13C NMR (75 MHz, CDCl3): δ (ppm) 21.0, 25.7, 27.2, 31.7, 36.7, 37.9, 38.2, 41.9, 42.2, 50.3, 61.0, 61.3, 67.2, 67.8, 68.0, 74.2, 74.5, 98.3, 98.8, 109.6, 143.6, 149.0, 162.8, 169.9, 170.6. Method B. A powder of Iobitridol 12 (2.64 g, 3.16 mmol) was suspended in 60 mL of dry acetone. With vigorous stirring, 3 g of freshly fused zinc chloride (finely ground) was added, the resulting mixture was allowed to react for 24 h at room temperature. During this period, Iobitridol was converted to a new derivative, which was monitored by TLC (CHCl3/CH3OH

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) 15:1). Sodium carbonate (5 g) in 25 mL of water was added to the reaction mixture. Vigorous stirring was continued for 1-2 h to completely precipitate the zinc catalyst. After filtering the white precipitate, the filtrate was evaporated to remove acetone. The residue was neutralized to pH 7 and extracted with 3 × 25 mL of CHCl3, the organic phase was then dried overnight (anhydrous MgSO4) and evaporated. Following chromatographic purification was similar to Method A above. Slightly yellow product 13 was obtained in a yield of 83% (2.56 g). Subsequent N-Alkylation by Ethyl Bromoacetate (compound 14 synthesis). Sodium methoxide solution was freshly prepared from sodium and methanol, with a concentration of 1.90 M titrated by potassium hydrogen phthalate. This NaOCH3/CH3OH solution (8.27 mL, 15.72 mmol) was added to a 150-mL anhydrous THF solution of 13 (10.00 g, 10.47 mmol). Upon addition, the clear yellow solution turned orange. Ten minutes after NaOCH3 addition, ethyl bromoacetate (2.85 g, 15.70 mmol) was added. White precipitate appeared in minutes, and the reaction mixture was stirred at room temperature for 1 h as shown to be complete by TLC (CHCl3/CH3OH 15:1). In this procedure, compound 13 (Rf ) 0.64 and 0.50) was completely converted into 14 (Rf ) 0.96 and 0.85, isomers), and the color of solution turned back to yellow. After filtration, the tetrahydrofuran solution was washed to neutral by water and dried over anhydrous MgSO4. The solution was evaporated to give a yellow oil as crude product. Purification was conducted by silica gel chromatography, eluting first with CHCl3 to remove excess ethyl bromoacetate, and then with CHCl3/CH3OH (10:1, v/v) to give the pure product 14 as slightly yellow crystals. The yield was 95% (10.30 g). M.p. 112-4 °C. ESI-MS: m/z ) 1042.0 (M + H)+, 1064.0 (M + Na)+, 983.9 (M - CH3COCH3 + H)+ for C33H46O11I3N3. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.28 (t, J ) 5.2 Hz, 3 H, CH2CH3), 1.35, 1.40, 1.46 (3s, 18H, three isopropylidene groups), 2.63 (m, 2 H, CH2CH3), 2.95, 3.03 (2 s, 6H, 2 N-CH3), 3.25 (m, 1 H, CHCdO), 3.36 (m, 2 H, Od CCHCH2O), 3.83 (m, 2 H, CH3NCH2), 3.98-4.42 (m, 10 H, NCH2CdO, CH3NCH2, OdCCHCH2O and 2 OCH2CHO), 4.54 (m, 2 H, 2 OCH2CHO). 13C NMR (75 MHz, CDCl3): δ (ppm) 19.5, 20.3, 25.6, 27.2, 28.7, 31.5, 35.4, 36.9, 37.8, 41.6, 50.3, 62.0, 62.4, 67.8, 74.3, 74.6, 98.3, 98.7, 109.6, 150.5, 151.4, 162.9, 168.5, 170.0, 170.6. Hydrolysis of Ethyl Ester (compound 15 synthesis). A 2 N NaOH solution (18 mL) was added slowly to a 20-mL solution of ester 14 (10.50 g, 10.09 mmol). After the addition, the reaction was continued for 2.5 h at room temperature. The solution was neutralized to pH 7 and evaporated to remove most methanol under 40 °C. The residue was dissolved in 80 mL of water, and the solution pH was adjusted to 3.5 by 2 N HCl at which time an abundant white precipitate appeared. Saturated NaCl solution (80 mL) was added, and then 3 × 100 mL of chloroform was used to extract the carboxylic acid. The extracts were dried over anhydrous MgSO4 overnight and evaporated to give 9.37 g of yellowish crystalline 15 (yield 91.7%). TLC (CHCl3/CH3OH 15:1): Rf ) 0.33 and 0.20 (isomers). M.p. 118120 °C. ESI-MS: m/z ) 1014.0 (M + H)+, 1036.0 (M + Na)+ and 956.0 (M - CH3COCH3 + H)+ for C31H42O11I3N3. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.34, 1.40, 1.46 (3s, 18H, 3 isopropylidene groups), 2.95, 3.03 (2 m, 6H, 2 N-CH3), 3.21 (m, 1H, CHCdO), 3.35 (m, 2 H, OdCCHCH2O), 3.82 (m, 2 H, CH3NCH2), 4.02 (s, 2 H, NCH2CdO), 4.1-4.4 (m, 8 H, CH3NCH2, OdCCHCH2O and 2 OCH2CHO), 4.52 (m, 2 H, 2 OCH2CHO). 13C NMR (75 MHz, CDCl3): δ (ppm) 18.9, 19.4, 25.7, 27.2, 28.4, 28.9, 31.1, 37.1, 37.6, 41.2, 50.4, 62.2, 62.5, 67.8, 74.4, 74.6, 98.3, 98.6, 109.7, 150.8, 151.3, 162.9, 168.2, 170.2, 170.9.

Fu et al.

Preparation of N-Hydroxysuccinimde Ester (compound 16 synthesis). Carboxylic acid 15 (9.12 g, 9.0 mmol) and Nhydroxysuccinimide (1.04 g, 9.0 mmol) were dissolved in 40 mL of chloroform. After cooling to -5-0 °C, dicyclohexylcarbodiimide (1.85 g, 9.0 mmol) in 5 mL of chloroform was added. The reaction continued for 0.5 h at 0 °C and r.t. for 24 h. The white precipitate was filtered, and the filtrate was evaporated to give 9.89 g of active ester 16 (99%). TLC (CHCl3/ CH3OH 15:1): Rf ) 0.93 and 0.80 (isomers). M.p. 116-118 °C. ESI-MS: m/z ) 1111.0 (M + H)+, 1133.0 (M + Na)+ and 1053.0 (M - CH3COCH3 + H)+ for C35H45O13I3N4. 1H NMR (400 MHz, CDCl3), δ (ppm) 1.35, 1.40, 1.47 (3 s, 18H, three isopropylidene groups), 2.84 (m, 4 H, CH2CH2), 2.98, 3.04 (2 m, 6 H, 2 N-CH3), 3.21 (m, 1 H, CHCdO), 3.37 (m, 2H, Od CCHCH2O), 3.83 (m, 2 H, CH3NCH2), 3.90-4.45 (m, 10 H, NCH2CdO, CH3NCH2, OdCCHCH2O and 2 OCH2CHO), 4.53 (m, 2 H, 2 × OCH2CHO). 13C NMR (75 MHz, CDCl3): δ (ppm) 18.1, 23.9, 24.2, 24.9, 25.1, 26.6, 28.4, 29.0, 34.1, 40.9, 41.0, 47.2, 49.8, 53.8, 61.7, 67.2, 73.9, 97.7, 109.2, 110.0, 148.1, 150.4, 162.2, 168.1, 168.3, 168.5, 169.8, 172.2. Synthesis of Dendritic Iodinated Contrast Agents. A general procedure is described here with PEG6000-carbamateGen4-triiodo as an example. PEG6000-carbamate-Gen4.0 dendrimer 9a (0.76 g, 1.72 mmol NH2 groups) and DIPEA (0.66 g, 5.13 mmol) were dissolved in 20 mL of DMF. Active ester 16 (8.59 g, 7.74 mmol) was added in two portions 24 h apart (2/3 was added initially and 1/3 added later). The reaction lasted for 96 h at room temperature at which time the reaction was shown to be complete by a negative ninhydrin test. A small amount of 2-aminoethanol (6 mmol) was added to destroy the excess unreacted ester. The solvent was removed by distillation in a vacuum, and the residue obtained was dissolved in 10 mL of chloroform and precipitated by 180 mL of anhydrous ether. A yellowish solid was obtained and subsequently treated by 40 mL of 60% (v/v) aqueous TFA at room temperature for 6 h. After the deprotection, the solution pH was brought to 7 by 2 N NaOH and then dialyzed against 2 × 2 L of deionized water (cutoff Mw of 3500 Da). The dialysate was concentrated and then purified by SEC on a Sephadex G-100 column (mobile phase 0.05 M phosphate and 0.15 M NaCl, pH 7.0, flow rate 12 mL‚min-1‚cm-2). Chromatographically pure macromolecular fractions were combined, dialyzed (to remove buffer salts), and lyophilized to give a white material, PEG6000-carbamateGen4.0-IOB, namely 2a (1.84 g, 90% yield, “IOB” stands for Iobitridol moiety). UV (λmax, water): 245 nm. Iodine analysis by UV: 31.88%. Elemental analysis measured for I, C, H, and N. 1H NMR (400 MHz, D2O): δ (ppm) 1.20-1.80 (m, CH2CH2CH2CH), 2.39 (br, NCH2), 3.02, 3.16 (2 s, 2 N-CH3), 3.2-3.6 (m, CH2 and CH groups of IOB), 3.62 (s, PEG), 4.04.4 (br, other CH groups of IOB, NCHCO of lysine residue). 13C NMR (75 MHz, D O): δ (ppm) 22.9, 25.7, 28.2, 31.0, 37.9, 2 39.4, 43.6, 47.7, 48.0, 50.6, 52.3, 54.4, 59.9, 60.6, 63.6, 64.2, 69.3, 69.8,71.0, 71.8, 90.7, 98.0, 99.6, 148.2, 150.4, 168.9, 171.6, 172.0, 174.2, 175.1. SE-HPLC: retention time 18.41 min. Similarly, other conjugates including PEG6000-carbamateGen3-IOB (1a), PEG12000-carbamate-Gen3-IOB (1b), PEG12000-carbamate-Gen4-IOB (2b), PEG3400-amide-Gen4-IOB (3a), and PEG3400-amide-Gen5-IOB (3b-I) were synthesized and purified. Of note, the feeding [IOB]/[NH2] ratio was 3, 4.5, and 6, respectively, for Gen3, Gen4, and Gen5 dendrimers. The conjugation reaction time was 72, 96, and 120 h for Gen3, Gen4, and Gen5 dendrimers, respectively. Their structures were similarly characterized by 1H (and 13C) NMR. Synthetic yields, iodine analysis, and SE-HPLC results were also given. (Table 2).

Bioconjugate Chem., Vol. 17, No. 4, 2006 1047

PEG-Core Dendrimeric CT Contrast Agents

Table 2. Synthetic Yields and Structure Characterization of Dendritic Iodinated Contrast Agents code

yield (%)a

iodine % (w/w) measured by UV (or microanalysis)

no. of IOBb measured (theoretical)

molecular weight (Mn)c

polydispersity (Mw/Mn)d

retention time (min) in SE-HPLC

1a 1b 2a 2b 3a 3b-I 3b-II

82 90 78 87 80 84 86

26.67 (26.31) 21.67 (nd) 31.88 (31.24) 27.28 (27.05) 33.48 (nd) 32.14 (31.86) 35.64 (nd)

15.2 (16) 15.5 (16) 30.3 (32) 30.5 (32) 29.1 (32) 39.4 (64) 57.5 (64)

21700 27500 36800 42500 32900 46700 66400

1.012 1.014 1.010 1.033 1.008 1.023 1.017

19.30 17.52 18.41 16.08 19.84 19.07 18.35

a Yield based on the PEG dendrimer. b Measured by 1H NMR (IOB stands for iobitridol). c Calculated by microanalysis data (iodine %). d Determined by size exclusion HPLC.

UV Quantification of the “Triiodo” Group in Iodinated Dendrimers. A series of dilute Iobitridol (IOB) solutions in distilled water, with known concentrations ranging from 1.0 to 50 µg /mL, was prepared. Their absorption at 245 nm was measured; thus, the standard absorbance versus concentration curve was set up. Data points for this curve followed Beer’s law in the range of 0-30 µg IOB/mL (r ) 0.9995). UV absorption data of dilute sample solutions (1a to 3b) at 245 nm were measured, yielding the IOB contents; hence, iodine percentages were obtained for these iodinated dendrimers (Table 2). Measurement of Apparent Molecular Weight and Size Distribution by SE-HPLC. Analytical SE-HPLC, performed on a Superdex 200 10/300 GL column (10 mm × 300 mm, Amersham Biosciences, Uppsala, Sweden), was utilized for molecular weight/size characterization of synthesized polymers in this work (see conditions in Apparatus). A molecular weight calibration curve was set up based on the SE-HPLC measurements of five protein standards (cytochrome C, carbonic anhydrase, bovine serum albumin, alcohol dehydrogenase, and amylase) with a molecular weight range of 13 to 200 kDa. Size distribution of each iodinated dendrimer, represented by the polydispersity index (PDI), was calculated by the equation: PDI ) Mw/Mn ) (ΣNiMi2/ΣNiMi)/(ΣNiMi/ΣNi), Mw and Mn are weight- and number-average molecular weights, respectively, while Ni and Mi stand for the molecular number and molecular weight respectively of the i-th fraction of a polymer sample. Basic Physicochemical Characterization. A specific amount of a test iodinated dendrimer containing 300 mg iodine was added to 2 mL of distilled water and allowed to stand overnight with gentle stirring, to observe if the test conjugate was completely dissolved at this concentration (150 mg iodine/mL). The unit, mg iodine/mL, was then converted to “mg contrast agent /mL” since the iodine% was known for each compound. Through the flask-shaking method, the butanol-water partition equilibrium of each iodinated contrast agent was measured. Tris buffer (50 mM, pH 7.4) and 1-butanol were chosen as two immiscible solvents. The contrast agent was dissolved in Tris buffer at a concentration of 10 µg iodine/mL. Eight milliliters of a contrast agent solution was mixed with 8 mL of butanol and shook for 0.5 h in a separation funnel (30 mL). After centrifugation, the water phase was separated and its IOB concentration was determined by UV spectrophotometry at 245 nm. The partition coefficient thus was calculated by (C0 - C)/ C, where C0 and C represent the IOB concentration of the contrast agent prior to and after the partition in aqueous phase. In addition, Iobitridol and Iodixilan were tested as standard controls. For each contrast agent, this measurement was conducted in triplicate. Osmolality of the solutions containing synthesized CT MMCM was measured using a conventional freezing point depression method on a wide range osmometer (Model 3W, Advanced Instruments, Needham Heights, MA), expressed with a unit of “mOsm/kg water”. The osmometer was calibrated with standard NaCl solutions (100 and 900 mOsm/Kg water) before

sample measurements. Viscosity of certain contrast agent solutions (Iohexol at 300 mgI/mL, 2b at 75 mgI/mL) were measured by a standard Ubbelohde viscometer (capillary diameter 0.4 mm) in a water bath (37.0 ( 0.3 °C). Each sample was measured in triplicate. Formulation of Contrast Agents. Prior to CT experiments, lyophilized macromolecular contrast agents were dissolved in distilled water in the presence of a trace amount of stabilizer, i.e. 0.40 mg of tris(hydroxymethyl)methylamine (7) per 100 mg of iodine, followed by sterile filtration twice with Acrodisk 25 mm syringe filters (membrane pore size 0.2 µm, Gelman Laboratory). They were finally formulated as 75 mg iodine/ mL solutions with pH of 6.8-7.4. The formulated contrast media were autoclaved for 30 min at 120 °C, followed by a series of tests including SE-HPLC, inorganic iodide detection, and free iodine detection, respectively, to see if any decomposition occurred in this procedure. Preliminary CT Imaging in a Rat Model. The CT scan was performed using an eight-detector-row clinical CT scanner (Lightspeed QX/I, GE Healthcare, Milwaukee, WI). One anesthetized Sprague Dawley rat was placed supine in the CT scanner. Immediately before, and 2, 10, 22, 32 min after, tailvein intravenous contrast injection (dose 450 mg iodine/kg body weight), CT images of the entire animal were acquired using the following imaging parameters: 80 kVp, 130 mA, 0.5 s/gantry rotation, 1.25 mm slice thickness, 2.5 mm/s table speed (pitch 0.625), and 10 cm field of view. Images were transferred to a picture archiving and communications workstation (Impax, AGFA, Mortsel, Belgium). Representative axial images at the level of liver were chosen for comparison of intravascular CT enhancement over the time.

RESULTS AND DISCUSSION Concept and Molecular Design (Scheme 1). Poly(ethylene glycol) (PEG) was selected as the backbone of the iodinated blood pool contrast agents due to its unique properties. PEG’s each repeating units CH2CH2O has the ability to bind several water molecules. Compared to other water-soluble polymers (either naturally occurring or synthetic) with the same molecular weight, PEG has an extremely simple chemical structure but with an extremely high exclusion volume in water. In aqueous solution, PEG physically repulses all other surrounding macromolecules and is well-known for its nonimmunogenicity and good biocompatibility. Attachment of PEG to biomacromolecules can dramatically increase their blood half-life while markedly reduce the immunogenicity. As early as in 1991, the first PEG-protein conjugate (Adagen) for intravenous use was approved by FDA. Extremely narrow size distribution is another advantage of PEG, which makes it possible to synthesize nearly monodisperse PEG conjugates. In addition, PEG has good solubility not only in water but also in organic solvents including methylene chloride, a useful characteristic in practical synthesis (8-10). For CT contrast media development, radiopaque atoms (e.g. iodine, barium) are necessary due to their outstanding X-ray

1048 Bioconjugate Chem., Vol. 17, No. 4, 2006

attenuation ability under clinical CT conditions. Comparing various elements with relatively high atomic number (39-82) comprehensively in their radio-opacity effectiveness, biocompatibility, and synthetic availability, the iodine atom (atomic number 53) now still remains our first choice as the radiopaque atom. (11-13) In the subsequent screening of iodinated building blocks, tolerability is considered as the criterion with the highest priority. Thus, abandoning the ionic or incompletely substituted triiodobenzene derivatives, we selected a type of nonionic, highly soluble and stable structure containing N,N′-bis(polyhydroxyalkyl)-5-(N-acylamino)-2,4,6-triiodo-1,3-phthalamide moiety for signal enhancement. Specifically, Iobitridol, a new nonionic clinically used contrast agent, was chosen as the initial ‘‘triiodo′′ building block in our syntheses. PEG has only two termini available for derivatization; thus, an appropriate amplifying method needs to be adopted to produce sufficient reactive termini (such as NH2 groups) for following chemical decoration. To carry out this method, dendritic polylysines as “amplifiers” with the varying generations are introduced to two ends of PEG diol (or PEG bisamine) via carbamate (or amide) linkages (see Scheme 2). Different from commonly used pegylation strategies in syntheses of PEG-based macromolecular pharmaceuticals (8, 14, 15), in this design we use the PEG as a macro core to initiate the total synthesis of dendrimeric contrast agents, instead of conjugating PEG to either a synthetic or a naturally occurring macromolecule. Thus, both the number of conjugated PEG (only one here) and conjugation site could be exactly controlled. Rational derivatization of Iobitridol molecules and subsequent conjugation to PEG-core dendrimers were originally designed (see Schemes 3 and 4). The overall molecular size and terminal group number may be precisely controlled by adjusting PEG size and number of generation during the synthesis. On the basis of PEG-core dendrimer structure, we synthesized and preliminarily characterized six macromolecular iodinated contrast agents for CT imaging in this study. Synthesis and Chemical Characterization of PEG-Core Dendritic Polylysines. To synthesize the dendritic polylysine part, t-Boc chemistry was chosen due to the substantially lower cost of the monomer di-t-Boc-L-lysine compared with di-FmocL-lysine. PEG3400 bisamine was used directly as the core molecule due to the high reactivity of terminal NH2 groups. PEG6000 and PEG12000 bisamines were not commercially available and were thus prepared from their corresponding PEG diols. PEG diol was first converted to its bis(4-nitrophenyl) biscarbonate, subsequently reacted with 1-N-t-Boc-ethylenediamine, and then deprotected by TFA, yielding PEG bisamines containing stable linkages (NHCOO) in the backbone. The synthesis of each dendrimeric generation includes two steps: the coupling step and the deprotection step. Coupling reaction between an amino-terminated lower generation dendrimer Gen n (n ) 0, 1, 2, 3 ...) and excess COOH-activated di-t-Boc-lysine gives a t-Boc-protected dendrimer, defined as Gen (n + 0.5), namely the half generation. To prevent the formation of a defected structure, such as missing lysine sequences or coexistence of different generations, we must ensure that every coupling step is complete, or else the defective structure would arise and exponentially accumulate with the increase of generation. To address this extremely important issue, several factors including reactant feeding ratio, reaction time, and reaction solvent need to be considered carefully. The equivalent ratio of activated di-t-Boc-lysine to the dendrimer amino group, namely the [COOH]/[NH2] ratio, needs to be at least 3, and up to 6 in our synthesis. The ratio of 6 did not give further benefits in the reaction completeness. For

Fu et al. Scheme 1. Synthesized PEG-Core Dendrimer Conjugates with Multiple Triiodophthalamide Moieties as Macromolecular Contrast Agents for Computed Tomography (CT)a

a Note: As a typical example, the abbreviation “PEG6000-carbamateGen4-IOB” stands for a macromolecular conjugate of generation-4 (Gen4) dendritic polylysine initiated with a PEG3400 core via carbamate linkages, having iobitridol (IOB) moieties as terminal decorating groups.

lower generations (50 °C) caused formation of minor byproducts with higher molecular weights, likely due to intermolecular cross-linking of unreacted lysine chains. In contrast agent synthesis, the final step, namely the hydroxyl deprotection, was completed in 60% (v%) aqueous TFA solution at room temperature. Other acids such as 2 N HCl in 1:1 methanol/water also worked well.

1054 Bioconjugate Chem., Vol. 17, No. 4, 2006

Fu et al.

Figure 5. 1H NMR spectra of PEG12000-carbamate-Gen4 9b (a, TFA salt) and PEG12000-carbamate-Gen4-IOB 2b (b) in D2O.

Figure 6. SE-HPLC traces of PEG12000-carbamate-Gen4 polyamine 9b (a, TFA salt) and PEG12000-carbamate-Gen4-IOB conjugate 2b (b) on a Superdex 200 column, with flow rate of 0.8 mL/min and each run time of 30 min. Other HPLC conditions: (a) mobile phase 0.1% trifluoroacetic acid and 0.3 M Na2SO4 (pH 2.7), UV detection at 210 nm; (b) 0.05 M phosphate and 0.15 M NaCl (pH 7.0), 245 nm.

All synthesized iodinated dendrimers were first dialyzed and then purified by a Sephadex G-100 column eluted with phosphate buffer at pH 7. An alternative mobile phase includes 0.1 M ammonium acetate (pH 7). Excess small molecular iodinated derivatives and seldom found large-molecular crosslinked byproducts were removed efficiently and completely. Analytical SE-HPLC (Figure 6) showed that PEG12000carbamate-Gen4-IOB gave considerably narrow and symmetric peaks in size exclusion chromatography, indicating a high degree of monodispersity in molecular size. Iodine quantitative analysis (both by UV and elemental analysis), 1H/13C NMR, and UV confirmed that synthesized contrast agents have the expected structure. Both PEG (3.62 ppm in 1H NMR, ∼69.7 ppm in 13C NMR) and the iobitridol derivative (NCH3 at 3.0-3.2 ppm, in 1H NMR, aromatic C in regions of 90-100 ppm and 145-150 ppm in 13C NMR, λmax 245 nm in UV) were present in the final products (Figures 4b and 5b). Due to overlapping of the PEG peak (3.62 ppm) with most IOB side chain peaks (3.24.5 ppm), the degree of dendrimer amino substitution could not be determined using the PEG peak. However, the ratio of IOB/ lysine can be obtained by comparing the integrals of lysine trimethylene group (1.2-1.8 ppm) and characteristic NCH3 groups (3.0-3.2 ppm) in IOB. Thus, the degree of dendrimer amino substitution can be calculated; for example, the conjugation degree was 96% in the case of PEG12000-carbamate-Gen4 dendrimer (Table 2). Iodine percentages (w%) measured by UV or elemental analysis agree with these NMR-measured amino substitution results. These data were used to calculate the actual molecular weights of iodinated dendrimer products (Table 2). To directly measure the absolute molecular weights, MALDITOF mass spectrometry of these iodinated dendrimers were tried but failed under varying conditions (different ratios of matrix/ sample, different spectrometers in different MS labs). Addition-

ally, two derivatives of PEG12000-carbamate-Gen4-IOB, including its ketal precursor (i.e. the intermediate compound prior to hydroxyl deprotection) and its fully acetylated derivative, were isolated or prepared and then underwent MALDI mass spectral analysis; unfortunately there still were no peaks observed because their protonated ions could not fly under test conditions. Compared with our successful MALDI analyses of the undecorated PEG-core dendrimers, this unexpected difficulty must be due to the existence of a large number of heavy iodine atoms in their structure. Preliminary Data of Physicochemical Characteristics of Iodinated PEG-Core Dendrimers. The ideal macromolecular CT contrast agent could be defined as being (a) highly efficient in X-ray attenuation, (b) primarily intravascular in distribution, (c) well-tolerated and nonimmunogenic, (d) monodisperse or near-monodisperse in size, (e) eliminated completely in a timely manner, and (f) with certain desired physicochemical properties including high water solubility, low osmolality, limited viscosity, and good stability to autoclave conditions. The new structures described in this work virtually demonstrated most of these desired features, at least all of those tested in this preliminary study (Table 3). Apparent molecular weight based on protein standards ranged from 35 kDa (PEG3400amide-Gen4-IOB) to 143 kDa (PEG12000-carbamate-Gen4IOB) with a high degree of molecular monodispersity (Table 2, polydispersity index Mw/Mn < 1.04 for all six compounds), and the ratios between apparent and actual molecular weights were as large as 3.4, indicating that the choice of PEG as the macromolecular backbone substantially increased the molecular hydrodynamic size. The largest one of this series, PEG12000carbamate-Gen4-IOB, had a CT-assayed blood half-life (t1/2) of approximately 35 min (monoexponential kinetics) and an estimated volume of distribution (Vd) of 13%, intermediate between the accepted normal intravascular volume (∼5%) and the normal extracellular fluid volume (∼25% of the body weight), as reported somewhere else (11). Of note, both blood t1/2 and Vd might be underestimated due to relatively short time course (∼30 min) at dynamic CT. The same compound was tested by SE-HPLC prior to and after heat sterilization, showing identical HPLC traces, and neither inorganic iodide nor iodine was detected in autoclaved solutions. This contrast agent was proven to be highly stable to autoclaving conditions for 30 min at 120 °C. The six synthesized iodinated dendrimers were effective for producing strong X-ray attenuation due to generally high iodine contents (21-36%, w/w). (Table 2). Water solubility for each of the four compounds tested for this characteristic was >440 mg/mL. These new compounds also showed high levels of hydrophilicity. Butanol-water partition coefficients of the four test compounds were between 0.015 and 0.064, similar to or more hydrophilic than clinically used CT contrast media (Iobitridol, 0.070; Iodixanol, 0.046;

Bioconjugate Chem., Vol. 17, No. 4, 2006 1055

PEG-Core Dendrimeric CT Contrast Agents Table 3. Physicochemical Properties of Iodinated Dendritic Contrast Agents with PEG Cores contrast agent

PEG size (kDa)

gen. no.

theor. no. of dendrimer termini

no. of “triiodo” attached

1a 1b 2a 2b 3a 3b-II iohexol

6 12 6 12 3.4 3.4 nae

3 3 4 4 4 5 na

16 16 32 32 32 64 na

15.2 15.5 30.3 30.5 29.1 57.5 1

a

MW (kDa) actual apparent 21.7 27.5 36.8 42.5 32.9 66.4 0.82

43.7 84.0 60.6 142.7 35.8 61.9 na

solubility (mg/mL, 25 °C)

hydrophilicitya

viscosity (cP, 37 °C)

osmolality (mOsm/kg water)

ndd nd >470 >550 >440 >460 >750

nd nd 0.034 0.015 0.042 0.064 0.055

nd nd nd 11.4b nd nd 6.1c

nd nd 306b 253b 268b 237b 684c

Partition coefficient between 1-butanol and 50 mM Tris buffer (pH 7). b 75 mgI/mL. c 300 mgI/mL. d Not determined. e Not applicable.

Figure 7. Serial axial CT images of a normal Sprague-Dawley rat at the level of liver prior to, 2, 10, 22, and 32 min after intravenous injection of PEG12000-carbamate-Gen4-IOB as a CT contrast agent at a dose of 450 mg iodine/Kg body weight. Note the strong and persistent enhancement of the inferior vena cava (see the arrows) and other vessels (hepatic veins) within the liver.

Iohexol, 0.055), likely due to the presence of the PEG core. Of note, taking advantage of the so-called “hydrophilic sphere stabilization” effect (26), Iobitridol molecules were carefully derivatized, chemically activated, and conjugated to the dendrimer with such a strategy that did not sacrifice any of its six hydroxyl groups upon conjugation. This strategy enabled all attached IOB moieties to remain highly soluble and hydrophilic after conjugation. Our initial attempts to conjugate Ioxilan to the same dendrimers via exploitation of one of its five hydroxyl groups yielded poorly water-soluble iodinated dendrimers, indicating that it is mandatory to keep a sufficient number of hydroxyl groups surrounding the hydrophobic triiodobenzene core so as to retain the required hydrophilicity and solubility of attached “triiodo” moieties (11). The optimal concentration chosen for intravenous use will depend not only on water solubility but also on the viscosity and osmolality of the contrast agent. The viscosity of PEG12000carbamate-Gen4-IOB was measured to be 11.4 cP (75 mg I/mL, 37 °C), a value considered acceptable for a macromolecular formulation, but higher than viscosity values obtained for conventional small molecular (30 kDa) for CT enhancement have not advanced to the point of warranting clinical trials. Previously reported macromolecular formulations include iodinated hydroxyethyl starch (28), carboxymethyl dextran derivatives with triiodobenzoic acid (29), and vinyl copolymers from acrylamide and hydrophilic “triiodo” monomer (30). Different designs revealed different shortcomings: some were highly heterogeneous in molecular size. For others, radioopacity was low, and some were too viscose, not sufficiently stable, or poorly tolerated. Of note, there were other efforts reported to achieve pegylated longcirculating vesicles including Iopromide-encapsulated liposome (31), iodinated triglyceride emulsion (32), and iodinated micelles (33). Experimental CT Imaging. As shown in Figure 7, the intravascular CT enhancement in the normal rat through 32 min postinjection of PEG12000-Gen4-IOB (450 mg I/kg dose) was considerably intensive and persistent. Although not shown, it is well-known that for CT imaging obtained after administration of conventional small-molecular contrast media there is exhaustion of the vascular enhancement in less than 5 min. The iodinated PEG-core dendrimers by virtue of their large size remain much longer in the blood pool. The details of the considerable advantages of prolonged intravascular enhancement in CT go beyond the scope of this chemistry-focused report. For the in vivo CT image enhancement available with iodinated PEG-core dendrimers, only one compound was evaluated in this study as an example. Further characterization studies of this new class of CT contrast agents including pharmacology, metabolism, and the safety profile are ongoing.

1056 Bioconjugate Chem., Vol. 17, No. 4, 2006

CONCLUSION To summarize, a new design for macromolecular contrast enhancing agents with potential applications in angiographic and dynamic CT imaging has been developed by the synthesis and preliminary testing of six representative compounds. The basic construct consists of a poly(ethylene glycol) core with two dendritic polylysine amplifiers (dendrons) at both PEG termini, to which amplifiers are conjugated multiple “triiodo” moieties. Basically, the desirable characteristics for such a macromolecular contrast agent could be approximated by one or more of these PEG-core dendrimer conjugates. Demonstrated favorable characteristics include prolonged intravascular enhancement in an animal model, adjustable and large apparent molecular weights (35 to 143 kDa), near monodispersity in size, high X-ray attenuation, high water-solubility and hydrophilicity, low osmolality, and good chemical stability. This new class of iodinated dendrimers has good potential in angiography and, more importantly, in quantitative microvascular characterization of a wide variety of diseases and abnormalities including cancer, ischemic injury, inflammation, and altered states of angiogenesis.

ACKNOWLEDGMENT This work was supported in part by NIH grants CA103850 and CA82923 and by Lucile-Packard Foundation. MALDI-TOF MS and ESI-MS measurements were conducted by the UCSF Mass Spectrometry Facility (Director Dr. A. L. Burlingame) supported by NIH NCRR RR01614. 1H NMR and 13C NMR spectra were recorded on a Varian 400 NMR spectrometer in the NMR Lab, School of Pharmacy at UCSF. Drs. Karl Turetschek and Vincenzo Lucidi are thanked for their generous gifts of Iobitridol. Mr. Jason Kipke, Ms. Christina Hampton, and Ms. Jessica Pfannenstiel are acknowledged for their great assistance in the CT scans.

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