760
Bioconjugate Chem. 2009, 20, 760–767
Development of a Dendritic Manganese-Enhanced Magnetic Resonance Imaging (MEMRI) Contrast Agent: Synthesis, Toxicity (in Vitro) and Relaxivity (in Vitro, in Vivo) Studies Annabelle Bertin,† Je´roˆme Steibel,‡ Anne-Isabelle Michou-Gallani,§ Jean-Louis Gallani,† and Delphine Felder-Flesch*,† Institut de Physique et Chimie des Mate´riaux de Strasbourg, UMR CNRS/ULP 7504, 23 rue du Lœss BP 43, 67034 Strasbourg Cedex 2, France, Laboratoire d’Imagerie et de Neurosciences Cognitives, UMR CNRS/ULP 7191, 12 rue Goethe, 67000 Strasbourg, France, and siRNA Therapeutics, NIBR Biologics Center, Novartis Institutes for Biomedical Research, Inc., 4002 Basel, Switzerland. Received October 28, 2008; Revised Manuscript Received January 26, 2009
A new dendritic manganese(II) chelate 1 has been evaluated by in vivo (relaxivity) and in vitro (toxicity and relaxivity) experiments as a manganese enhanced magnetic resonance imaging (MEMRI) contrast agent. Also, a comparison with its corresponding gadolinium(III) homologue 2 and the commercially available MEMRI agent MnDPDP (Teslascan, Amersham Health) was achieved in order to determine respectively the real influence of the paramagnetic ion in terms of toxicity and relaxivity for this precise treelike structure and the potential of 1 to be a favorable candidate for brain-targeting MRI. Complexes 1 and 2 displayed high hydrosolubility (0.1 M) and revealed no in vitro neuronal toxicity at concentrations as high as 1 mM. Considering manganese(II) complex 1, the in vivo nontoxicity at 20 mM (100% rats survival) is very likely due to a slow diffusion of the compound, meaning a controlled release of the paramagnetic ions. Finally, T1 relaxivity of 4.2 mM-1.s-1 for 2 and T2 relaxivity of 17.4 mM-1.s-1 for 1 at 4.7 T were measured and are higher than that of the commercial MRI contrast agents GdDTPA and MnDPDP, respectively.
INTRODUCTION The development of the current scientific techniques improves day by day the quality of human health and life. The field of medicine, in particular, has brought considerable improvement. Indeed, magnetic resonance imaging (MRI) is one of the most powerful diagnostic imaging tools in medicine, since it provides not only images with excellent anatomical details based on softtissue contrast, but also functional information in a noninvasive and real-time monitoring manner (1). An MR image contrast is the result of a complex interplay of numerous factors, including the longitudinal (T1) and transverse (T2) relaxation times, proton density of the imaged tissues, and instrumental parameters. However, such contrast can be further improved by the administration of suitable paramagnetic species which affect longitudinal and transverse relaxation rates of the surrounding nuclei, mainly the water solvent protons (2). Shortly after the development and market introduction of magnetic resonance imaging contrast media in 1988, their use became a worldwide established tool for improved medical diagnosis, and nowadays, developing effective, nontoxic, and organ-specific contrast agents (CAs) for in vivo image enhancement is a great current area of research (3-10). Indeed, today, contrast media are applied in approximately 30% of all MRI procedures. Although most CAs are based on extracellular, low molecular weight gadolinium(III) chelates due to the seven unpaired electrons and relatively slow electronic relaxation of Gd(III) * Corresponding author. Tel.: + 33 3 88 10 71 63; fax: + 33 3 88 10 72 46; e-mail:
[email protected]. † UMR CNRS/ULP 7504. ‡ UMR CNRS/ULP 7191. § Novartis Institutes for Biomedical Research, Inc.
(11), a number of novel and more specific MRI contrast media (MRI-CM) containing other metals (Mn(II) chelates or iron oxide nanoparticles) (12-14) have been introduced. Notably, Mn(II) based MRI-CM are of great interest and especially in neuroscience research (15), since manganese (five unpaired electrons) has a natural human biochemistry, which may allow the design of target-specific CAs as a result of known biochemical uptake mechanisms. CAs for manganese enhanced MRI (MEMRI) (16) have been investigated over the past decade, but so far, only two agents, the liver-specific manganese(II)-dipyridoxal diphosphate (MnDPDP, Teslascan, Amersham Health) and an oral agent containing manganese(II) chloride (LumenHance), have been available clinically for human use (17). The present work describes the synthesis, toxicity (in vitro) studies, and relaxivity (in vitro and in vivo) measurements of a novel hydrophilic dendritic Mn(II) complex 1 (Figure 1) derived from diethylenetriamine pentaacetic acid (DTPA) with regard to its potential application in MEMRI dedicated to brain imaging. A dendritic approach to the preparation of such specific contrast agents appears promising, as the diversity of functionalization brought by the well-defined (on the opposite to polymers) and hyperbranched structure answers simultaneously all the criteria of biocompatibility, low toxicity, and specificity. Moreover, in addition to the multifunctionalization of a low molecular weight molecule, the dendrimer chemistry allows a versatility of size (according to the generation) and of physicochemical properties (hydrophilic, lipophilic). The resulting effects on stability (dendrimer effect), contrast qualities, and biodistribution of the contrast agents can then also clearly be identified.
EXPERIMENTAL PROCEDURES Chemistry. GdDTPA was provided by Aldrich and Mangafodipir (MnDPDP or Teslascan) was purchased from Amer-
10.1021/bc8004683 CCC: $40.75 2009 American Chemical Society Published on Web 03/09/2009
Development of MEMRI Contrast Agent
Bioconjugate Chem., Vol. 20, No. 4, 2009 761
Figure 1. Structures of dendritic Mn(II) and Gd(III) DTPA complexes 1 and 2, respectively.
sham Health. Reagents and solvents were purchased reagentgrade from Sigma Aldrich and Alfa Aesar and used without further purification. Bromide 3 was prepared according to literature procedures (18). All reactions were performed in standard glassware under Ar, and solvents were, if necessary, purified by standard procedures prior to use. Evaporation and concentration were done at water-aspirator pressure and drying in vacuo at 10-2 Torr. Column chromatography: silica gel 60 (230-400 mesh, 0.040-0.063 mm) from E. Merck. NMR spectra: Bruker AM-300 (300 MHz), solvent peaks as reference, δ in ppm. 4: A mixture of methyl-3,5-dihydroxybenzoate (372 mg, 2.21 mmol), 3 (3.20 g, 4.87 mmol), and K2CO3 (1.53 g,11.06 mmol) in acetone (25 mL) was stirred at 65 °C for 68 h. The reaction mixture was filtered (celite), and the solvent was evaporated. The residue was taken up in a mixture of CH2Cl2/H2Osat.NaCl (1:1) (600 mL). The organic layer was isolated, washed with H2Osat.NaCl (2 × 300 mL), dried (MgSO4), filtered, and evaporated to dryness. Column chromatography (SiO2, acetone) afforded 4 (2.07 g, 1.56 mmol) in 71% yield. Yellow oil. 1H NMR (CDCl3): 3.38 (s, 18H), 3.53-3.87 (m, 60H), 3.91 (s, 3H), 4.13-4.19 (m, 12H), 4.96 (s, 4H), 6.67 (s, 4H), 6.79 (t, 4J ) 2 Hz, 1H), 7.28 (d, 4J ) 2 Hz, 2H). 13C NMR (CDCl3): 52.28, 59.00, 68.91, 69.72, 70.36, 70.53, 70.55, 70.68, 70.81, 71.93, 71.95, 72.32, 107.16, 107.29, 108.34, 131.78, 132.07, 138.30, 152.81, 159.71, 166.69. C64H104O28 · 1/2 H2O: calc. C 57.72, H 7.89, O 34.27; found C 57.74, H 7.92, O 34.34. 5: 7.81 mL of LiAlH4 (1 M in THF) was added dropwise to a solution of methyl ester 4 (7.38 g, 5.58 mmol) in THF (100 mL). After 20 h stirring at room temperature, the reaction mixture was neutralized by careful addition of MeOH (50 mL) and H2O (50 mL), filtered (celite), and evaporated to dryness. Column chromatography (SiO2, CH2Cl2/10% MeOH) afforded 5 (6.50 g, 5.03 mmol) in 90% yield. Yellow oil. 1H NMR (CDCl3): 1.72 (s, 1H OH), 3.38 (s, 18H), 3.53-3.87 (m, 60H), 4.13-4.19 (m, 12H), 4.63 (s, 2H), 4.93 (s, 4H), 6.52 (t, 4J ) 2 Hz, 1H), 6.62 (d, 4J ) 2 Hz, 2H), 6.67 (s, 4H). 13C NMR (CDCl3): 58.98, 61.72, 65.1, 68.85, 69.68, 70.46, 70.49, 70.51, 70.64, 70.76, 71.88, 71.91,72.26, 101.25, 105.66, 107.23, 132.17, 138.17, 143.61, 152.73, 160.00. C63H104O27 · H2O: calc. C 57.70, H 8.15, O 34.16; found C 57.81, H 8.16, O 34.03. 6: Triethylamine (TEA) (3.23 mL, 23.10 mmol)) was added dropwise to a stirred solution of diethylenetriaminepentaacetic dianhydride (0.84 g, 2.31 mmol) and 5 (3.00 g, 2.31 mmol) in dry CH2Cl2 (100 mL) heated to 50 °C. After 70 h stirring, the reaction mixture was concentrated to 10 mL and hexane (50
mL) was added. The resulting suspension was cooled to 6 °C for 24 h. The white precipitate obtained was filtered off, washed with hexane (3 × 50 mL), and dried. No further purification was necessary to yield pure ligand 6 (3.50 g, 2.10 mmol) with 91% yield. 1H NMR (CDCl3): 1.29 (t, 4J ) 7.2 Hz, 8H), 3.05 (m, 10H), 3.32 (s, 18H), 3.46-3.85 (m, 60H), 4.07-4.13 (m, 12H), 4.88 (s, 2H), 4.90 (s, 4H), 6.61 (s, 4H), 6.73 (t, 4J ) 2.4 Hz, 1H), 7.22 (d, 4J ) 2.4 Hz, 2H). 13C NMR (CDCl3):45.56, 49.73, 52.63, 55.59, 56.76, 59.03, 68.89, 69.73, 70.50, 70.53, 70.55, 70.69, 71.92, 72.30, 101.29, 105.70, 107.28, 132.22, 138.22, 143.66, 152.78, 160.03, 168.79, 169.96, 170.62, 173.05. Elemental analysis: calc. C 55.40, H 7.55, O 34.53, N 2.52; found C 55.37, H 7.55, O 34.50, N 2.52. Maldi-MS (negative mode): [M]- 1666.85 obtained for C77H125N3O36 (1667.80). 1: An equimolar solution of 6 (0.50 g, 0.30 mmol) and MnCl2 · 4H2O (0.06 g, 0.30 mmol) in H2O (30 mL) was stirred at room temperature for 12 h while maintaining pH at 6.5 by addition of 1 M aqueous solution of NaOH. The reaction mixture is then lyophilized. Impure complex 1 (presence of inorganic salts like NaCl) is then diluted in CH2Cl2 (30 mL). The organic phase obtained is washed with H2O (2 mL) (decreasing reaction yield), dried (MgSO4), filtered, and evaporated to dryness. Complex 1 is obtained as a yellow solid (0.47 g, 0.27 mmol) with 90% yield. Maldi-MS (negative mode): [M Na]2- 1741.77 obtained for MnC77H121N3O36Na (1741.70). 2: An equimolar solution of 6 (0.50 g, 0.30 mmol) and Gd(ClO4)3 (0.25 g of a 50% solution in H2O, 0.30 mmol) in H2O (30 mL) was stirred at room temperature for 12 h while maintaining pH at 6.5 by addition of a 1 M aqueous solution of NaOH. The solution is then treated with Chelex resin (2 g) for 2 h before filtration and lyophilization. The absence of free gadolinium is checked by the Xylenol orange test (19). Complex 2 is obtained as a white solid (0.47 g, 0.25 mmol) with 89% yield (taking into account remaining perchlorates). Maldi-MS (negative mode): [M Na]- 1861.75 obtained for GdC77H123N3O37Na (1862.69). In Vitro Toxicity Studies. Rat pups were sacrificed by decapitation at 7 days of age, and neurons were isolated from the cerebellum and plated in multiwell plates coated with poly(Dlysine). The plates were incubated for 3 days to allow cell maturation to continue to some extend. At DIV 3 (day in vitro 3), the cells were exposed to 0.001 to 10 mM of the test products or to 0.001 to 100 nM of β-bungarotoxin. β-Bungarotoxin is a known neurotoxin that is not expected to decrease cell counts but has a striking negative effect on neuron-specific parameters. The cells were treated with the compounds for 2 days and then
762 Bioconjugate Chem., Vol. 20, No. 4, 2009
analyzed as follows: Measure of the cell leakage resulting from membrane damage: lactate deshydrogenase is normally a cytoplasmic enzyme. When the integrity of the plasma membrane is compromised, it leaks into the culture medium, where it can be measured by a colorimetric assay (Roche, LDH Assay). As a positive control for the test, a few test wells in the plate were exposed to a strong detergent, 1% Triton X100, for 30 min (thus causing immediate and measurable release of LDH); then, 120 µL of culture media was collected from all the wells and analyzed. Immunostaining of the cells: the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X100, blocked with 5% BSA (bovine serum albumine), and incubated with an antibody for neuron-specific beta III tubulin (RandD). The secondary antibody used was a TRIC-labeled mouse antibody. The nuclei were stained with DAPI or Hoechst 33342. High cell content analysis (ArrayScan system, Cellomics Thermofisher): The ArrayScan system is an imaging approach that uses fluorescent probes and automated microscopy to quantify multiple cellular markers at the single-cell level. Fluorescence microscopy coupled to electronic imaging allows quantification of many cell-specific parameters, such as nucleus counts, intensity, size or shape of nuclei, quantification of signals in the cytoplasma, or specific organelles (e.g., neurites). The ArrayScan system consists of an optical system with a spatial resolution of 0.68 µm (Carl Zeiss), a triple band fluorescence emission filter set with matched single band excitation filters for selectively imaging Hoechst, FITC, or TRITC (model XF93, Omega Optical), and a CDD camera. The system is used to scan multiple fields in each well (images acquisition). In our studies, the images acquired were analyzed with an algorithm specialized for neurons, “neuronal profiling”. Commercial MnDPDP (Teslascan, Amersham Health) and 6 were suspended in 5% DMSO at a stock concentration of 500 mM (yellow solution for commercial Mangafodipir, white-yellow solution for 6). A 10-fold serial working dilution down to 0.05 mM was done in 5% DMSO. 1, 2, and commercial GdDTPA were suspended in water at a stock concentration of 500 mM (orange tainted solution for 1, yellow solution for 2) and stored at -20 °C. A fresh 10-fold serial working dilution down to 0.05 mM was done in water. Four microliter aliquots of each working dilution were applied to the cells covered by 200 µL culture medium, i.e., a 50× further dilution step. In summary, the cells were exposed to the following final concentrations of product: 10, 1, 0.1, 0.01, and 0.001 mM. As a control, β-bungarotoxin, was included on each plate of cells at concentrations ranging from 100 nM (because of its neurotoxicity, 104 times less concentrated than the test compounds) down to 0.001 nM. Relaxivity Studies. In vitro: All MRI experiments were carried out at 4.7-T using an MR magnet (Magnex Sci. Ltd., Oxford, United Kingdom). T1 and T2 values were determined for each complex at pH 6.5-7 as a function of concentrations and at various temperature. In vivo: all studies were performed in accordance with French animal protection laws and approved by the responsible governmental authority. The animals (rats and mice) were kept under anesthesia using a mixture of isoflurane and oxygen. After being relaxed, the animals were intubated and artificially ventilated with an animal respirator. Rats and mice were placed in a stereostatic device to immobilize the head, with an integrated facility to maintain the body temperature at 37 °C. An anatomically shaped 1H surface coil for small animals (Rapid Biomedical GmbH, Wu¨rzburg, Germany) was positioned on the head to serve as a receiver for the MR signal. The system was then placed into a 1H resonator for rats and mice, with an inner diameter of 69 mm that fits into the -20 cm MR magnet. Brain saggital and coronal slices were acquired with a repetition time (TR) of 3.8 s (T2-weighted) and 0.5 s (T1-weighted), spin-echo time (TE) of 40 ms (T2-weighted)
Bertin et al.
and 21 ms (T1-weighted). Image acquisitions were based on a T1-weighted spin-echo MRI sequence or T2-weighted sequence, using CPMG sequence. All data were analyzed with Matlab software. As physicochemical properties like pH must be taken into account for potential clinical applications, intraperitoneal (i.p.) or intraveinous (i.v.) injections were performed at physiological pH (7.4) using a saline solution of the tested compound at [10 mM].
RESULTS AND DISCUSSION Chemistry. The Mn(II) and Gd(III) DTPA-derived complexes 1 and 2 were synthesized starting from the previously described bromo derivative 3 (18) as depicted in Scheme 1. The bromide 3, in the presence of methyl 3,5-dihydroxybenzoate and potassium carbonate in acetone under reflux, led to the formation of methyl ester 4 with 71% yield. Then, reduction of ester 4 by LiAlH4 in THF allowed the preparation of the corresponding benzyl alcohol 5 with 90% yield. Finally, the ligand 6 is obtained by reaction of benzyl alcohol 5 with commercial diethylenetriaminepentaacetic dianhydride in the presence of triethylamine in dichloromethane. After precipitation in hexane, the pure dendritic ligand 6 is obtained with 91% yield. Then, preparing the manganese(II) complex, an equimolar solution of ligand 6 and MnCl2 in water is stirred at room temperature while maintaining the pH of the solution at 6.5. After workup, the manganese complex 1 is then obtained with 90% yield. The same procedure is followed to prepare the gadolinium complex (III) using Gd(ClO4)3. After treatment with Chelex resin, the lack of free gadolinium is checked by the Xylenol orange test (19). After lyophilization, the complex 2 is obtained with 93% yield. Solubility and log P. The distribution of a molecule between aqueous and organic phases (partition coefficient P) determines in part its biological properties such as transport, membrane crossing, and bioavailability (distribution and accumulation). We measured the log P of our contrast agents by the so-called “shake flask” method recommended by the European Union (20). The results are presented in Table 1, as well as their hydrosolubility estimated by the turbidimetric method (21). As it can be noticed, the DTPA complexation induces a huge variation of the partition coefficient: indeed, if the log P is -0.47 for ligand 6, it increases to -2.66 and -3.27 for gadolinium(III) and manganese(II) complexes 1 and 2, respectively. Complexation therefore increases hydrophilicity, which is confirmed by hydrosolubility measurements: concentrations as high as 0.1 M in water can be reached for complexes 1 and 2. Such values are extremely important and encouraging, knowing that the contrast is function of the paramagnetic species concentration. Moreover, as will be explained in the following, our compounds are still nontoxic in vitro and in vivo at such concentrations. In Vitro Toxicity. Toxicity, which is primarily due to the action of manganese as a calcium channel blocker with effects on muscle electrophysiology and contractility, has limited the use of manganese salts as paramagnetic contrast agents (22, 23). Early research in MR imaging employed the use of MnCl2 in dogs (24) and rats (25) as well as in vitro systems involving human blood (26). Nevertheless, MnCl2 is the active component of Lumenhance, a commercial contrast agent used for gastrointestinal imaging (27). Manganese complexes are relatively unstable in vivo and are dissociated in biological media. Indeed, manganese can be displaced by other divalent cations such as calcium, magnesium, or zinc. Due to this release, some concern exists about the potential long-term toxicity associated with the development and use of Mn-based contrast agents (28). Free manganese was shown to be teratogenic and mutagenic in several models (29).
Development of MEMRI Contrast Agent
Bioconjugate Chem., Vol. 20, No. 4, 2009 763
Scheme 1a
a Reagents: (i) Methyl-3,5-dihydroxybenzoate, K2CO3, acetone, 60 °C; (ii) LiAlH4, THF, Ar, 0 °C; (iii) diethylenetriamine pentaacetic dianhydride, CH2Cl2, TEA; (iv) for Mn complex 1: MnCl2 · 4H2O, pH 7; for Gd complex 2: Gd(ClO4)3, H20, pH 7.
Table 1. Hydrosolubility and Log P Values of the Dendritic Ligand 6 and Its Corresponding Mn(II) and Gd(III) Complexes 1 and 2, Respectively compound hydrosolubility (mg.mL-1) hydrosolubility (mmol.L-1) log P
dendritic ligand 6
Mn(II) complex 1
Gd(III complex 2
62 ( 7
172 ( 17
182 ( 18
37 ( 4
98 ( 10
97 ( 10
-0.47
-3.27
-2.66
The teratogenic effect was also demonstrated after commercial MnDPDP injection (30, 31). It was thus of great importance to show that our MEMRI complex 1 is nontoxic both in vitro and in vivo at concentrations as high as 1 to 10 mM, knowing that commonly used concentrations for MRI studies and in vivo experiments are 0.5 mM. Cells used in this test were rat cerebellar neurons isolated from 7-day-old pups. These cells are useful tools for toxicity tests as they are primary cells, unmodified and without any genomic aberration. They represent a great intermediate between cell lines and tests on tissue sections or in vivo tests. Since our final aim is to target the brain and perform chemical engineering to increase such percentage of contrast agents reaching the brain, it appeared important to perform the in vitro toxicity tests on cerebellar neurons. Moreover, as those cells are the most sensitive cells, it becomes possible to conclude that if they are not affected then other less sensitive cells will also not be affected.
Neurons used in our in vitro assay are exposed to the complexed entities and to an unknown fraction of free ions released from the complexes, while in vivo neurons may be exposed to free ions released from the complexes. However, despite its limitations, the use of a cellular model based on functional primary cells has the advantage to allow monitoring of alterations on various levels: cell death, loss of cells, alteration of cellular membranes, alteration of the ability to form an healthy neuritic network. All these alterations can be quantified, thus allowing a direct comparison of the contrast agents and a basic characterization. The assay is used for analyzing compounds in case of possible brain penetration and can be proposed more generally if there is a concern about potential neurotoxic effects. Cells were stained in order to visualize nuclei (DAPI), cytoplasm, and neurites (anti β-tubulin III): thus, alteration of the neuron-specific parameters such as nuclei number, number of neurons, mean neurite count, mean total neurite length, and mean neurite width and area reflects neurotoxicity. Influence of the Paramagnetic Ion and Comparison with Commercial Products. From the selected images shown Figure 2, it can be seen that the higher concentration (10 mM) of all four tested compounds disrupts the neuritic network. At 1 mM, disruption remains strong in the corresponding selected image for MnDPDP and gadolinium complex 2, while 1 and GdDTPA are better tolerated. In order to better apprehend, and in a quantitative manner, the potential toxicity of these compounds,
764 Bioconjugate Chem., Vol. 20, No. 4, 2009
Bertin et al.
Figure 2. Pictures of treated neurons after staining for complexes 1 and 2 compared to commercial GdDTPA (Magnevist, Schering) and MnDPDP (Teslascan, Amersham Health) at 1 and 10 mM, together with untreated neurons.
Figure 4. EC50 (mM) of all studied compounds.
Figure 3. LDH efflux. Measurements of the cell leakage resulting from membrane damage of neuronal cells treated with increasing concentrations of dendritic complexes 1, 2, and commercial GdDTPA (Magnevist, Schering) and MnDPDP (Teslascan, Amersham Health).
we conducted a LDH release test (Figure 3). Indeed, the LDH measurement in the culture medium is a classical test for detection of overall toxicity: if the cell membranes lose their integrity, LDH, which is an enzyme present in the cytoplasm, is released in the supernatant culture, where it can be measured by a colorimetric test. Cells that have been exposed to the venom β-bungarotoxin also show dose-dependent membrane damage. 2 elicits a striking LDH release at 10 mM, confirming that this compounds compromises the membrane integrity at high
concentration. MnDPDP is also strongly membrane damaging at 10 mM. Supernatants from wells with neurons treated with increasing concentrations of 1 show a loss in LDH activity. LDH release is a parameter that is proportional to cell number. In parallel, we observe that increasing the concentration of 1 induces loss of nuclei (data not shown). We can suggest that these two observations are linked. Nevertheless, the LDH test has shown that our complex 1 does not damage the cell membrane up to a concentration of 10 mM. Thus, our in vitro neurotoxicity studies evidenced qualitatively (Figure 2) and quantitatively (Figures 3 and 4) that the manganese(II) dendritic complex 1 is no more toxic than the commercial GdDTPA at concentration up to 1 mM and is much less toxic than MnDPDP. In Vitro Relaxivity Studies. Influence of the Paramagnetic Ion and Comparison with Commercial Products. The in vitro relaxivity measurements of dendritic complexes 1 and 2 allowed one to draw the linear relationships r1,2 ) f([M]) (Figure 5), which where compared to the those recorded for one Gd-DTPA, GdCl3, and the two principal MEMRI contrast agents, namely, MnCl2 (only for animal experiments) and MnDPDP. The observed relaxivities measured at 37 °C under 4.7 T are summarized in Table 2. The relaxation theory predicts that higher relaxation rates are obtained upon increase of the
Development of MEMRI Contrast Agent
Bioconjugate Chem., Vol. 20, No. 4, 2009 765
Figure 5. In vitro dose-response relaxivity studies of Mn(II) complex 1 and Gd(III) complex 2 at 4.7 T and 20 °C. Table 2. r1 and r2 Relaxivities of 1, 2, GdDTPA, and MnDPDP in H2O at 20 °C under 4.7 T metal complexes
r1 (mM-1. s-1)
r2 (mM-1. s-1)
1 MnDPDPa MnCl2 2 GdDTPAa GdCl3 · 6H2O
1.3 1.5 (1.3-1.7) 3.59 4.2 3.2 (3.0-3.4) 18.63
17.41 2.7 (2.6-2.8) 48.42 4.82 4.0 (3.8-4.2) 28.2
a According to M. Rohrer, H. Bauer, J. Mintorovitch, M. Requardt, H.-J. Weinmann, InVest. Radiol. 40(11), 2005, 715-724 for the 4.7 T values.
rotational correlation time of complexes (32). Since the molecular rotation correlation time (τR) is proportional to the molecular size, the attachment of low molecular weight paramagnetic gadolinium chelate to macromolecules can considerably enhance the relaxivity of the chelate unit and can extend the residence time in the cardiovascular system, with potential applications in MRI angiography (MRA) (33, 34). However, macromolecular contrast agents tend to have less complete body elimination. In order for the complex to be excreted by kidneys and cleared from the blood pool quickly after being injected, the complex should have a molecular mass lower than 3.5 kD and also be hydrophilic. Thus, our PEGbased dendritic contrast agents may meet the satisfying conditions of both high relaxivity and low toxicity as confirmed by the following results. In the case of the Mn(II)-DTPA complex, no water molecule is directly bound to the metal ion, which makes the outer-sphere
relaxivity become the only relaxivity component. The in vitro relaxivities measured for 1 at 4.7 T are extremely encouraging: r1 ) 1.3 (mM.s)-1 and r2 ) 17.41 (mM.s)-1. One can see in Table 2 that, as expected, the highest relaxivities are obtained for MnCl2 (r1 ) 6.9 (mM.s)-1 and r2 ) 37.5 (mM.s)-1), but the important point is that our dendritic Mn(II)-based complex 1 shows higher r2 relaxivity (4 times higher) than that of the relative small molecular complex Mn-DPDP. One can also point out that r2 values measured at 4.7 T for the dendritic Gd(III)-based complex 2 are much lower than that of the corresponding Mn(II)-based complex 1 and r1 and r2 are of the same range as (as predicted by theory) and very close to the values recorded for Gd-DTPA. Thus, the dendritic architecture seems to have no substantial beneficial effect on the relaxivities. In Vivo Relaxivity Studies. Given the fact that for a great number of MRI examinations the sensitivity is too low, the use of synthetic contrast agents is not only paramount but even mandatory. However, with the aim of rendering MRI an even more powerful technique, these contrast agents are confronted by the improvement of three factors: the spatial accuracy, the contrast resolution, and the examination time. The manganese(II) complex 1 has been synthesized in order to develop a nontoxic Mn2+ ion vehicle, knowing that these ions are highly toxic but for which the highest intracellular contrasts can be obtained. Moreover, as manganese is an essential nutrient for mammals and organisms, they have developed special carrier systems and selective cellular uptake mechanisms. A possible strategy to reduce the in vivo toxicity of manganese(II) is to deliver Mn2+ to a specific site at an appropriate concentration (greatly reduce its concentration) and via a time-efficient manner (maintain a long diffusion). However, even if the MRI studies conducted on Gd(III) complex 2 showed the inability of the product to cross the healthy blood-brain barrier (BBB), they also highlighted a high vascular remanence (the contrast agent is circulating in the blood flow for at least 72 h), which is a behavior of good omen for use in functional imaging or for monitoring treatment. The ability of Mn2+ to accumulate in active regions of the brain was first demonstrated in MRI by activity-induced manganese-enhanced MRI (AIM-MRI) (35). Unfortunately, AIM-MRI is a quite invasive method, as the contrast agent is infused after BBB opening with an hyperosmolar agent. Here, we chose a simple intraperitoneal (i.p) administration of 1 leading to interesting and useful anatomical contrast (Figure 6). An aqueous isotonic saline solution (0.9% NaCl) of dendritic complex 1 was administered to a Long Evans male rat of 300 g via intraperitoneal injection (ip) at a 0.4 mmol/kg scale. As shown Figure 6, this injection led to a contrast increase in the rat brain on both the T1- and T2-weighted images. At 24 h after administration, the contrast is improved throughout the brain but heterogeneously, and details of the cytoarchitecture can be visualized as a result of the presence of Mn2+. Similarly to other studies, on the T1-weighted images, the contrast in the parenchyma is better at 24 h (b) while the contrast in the ventricle is maximal at 15 h (a) and gradually decreases after 48 h (c). The concentration of Mn2+ is minimal in the corpus callosum (a,b,d,e). The maximum concentration of Mn2+ is observed in the hippocampus region, the thalamus, and the cerebellum center (b,e). These results are consistent with previous studies performed on rats using intravenously injected MnCl2 and would mean that Mn2+ ions enter the brain by release. However, on the opposite to MnCl2, manganese chelation and probably the presence of the dendritic structure abolish the cardiovascular adverse side effects and effectively eliminate the problem of the Mn2+ retention in the tissues. In the case of our complex 1, the toxicity disappearance is very likely due to a slow diffusion
766 Bioconjugate Chem., Vol. 20, No. 4, 2009
Bertin et al.
Figure 6. T1 (a,b,c) and T2 (d,e,f) MEMRI images obtained with complex 1. (a,d) 15 h; (b,e) 24 h; (c,f) 72 h. Rat brain, at 0.4 mmol/kg, IP of a 20 mM solution of 1 in NaCl (150 mM); SMIS 4.7 T, 1 mm thickness, resolution 256 × 256, field of view 20 mm.
of the product, meaning a controlled release of the paramagnetic ions. In return, this follows a major loss of the relaxivity compared to MnCl2. But still, the measured relaxivities for 1 are comparable to those obtained for commercial MnDPDP. All these data support the view that, in spite of the lower effective magnetic moment of manganese, complex 1 and welldesigned Mn(II) complexes can therefore be considered as viable alternatives to the currently used gadolinium(III) complexes as contrast agents for (ME)MRI. Considering brain imaging, this is mainly due to the rich biochemistry of manganese allowing absorption and transport of Mn2+ to the brain: the uptake of extracellular Mn2+ by a neuron is directly coupled to its physiological activity and function. This suggests potential applications not only for basic neurosciences but also for managing clinical neurological diseases such as Alzheimer or Parkinson, as well as other diseases showing disturbances in neuronal cell structures without disturbing the BBB.
ACKNOWLEDGMENT We thank the French Ministry of Research for a fellowship to A. Bertin and CNRS for financial support. Pr. D. Grucker is warmly acknowledged for the in vitro relaxivity measurements. We also thanck E. Couzigne´ and B. Guignard for technical assistance. Supporting Information Available: 1H and 13C NMR spectra of compound 6, mass-spectrometry spectra (Maldi-MS, negative mode) of complexes 1 and 2, in vitro relaxivity theory. This material is available free of charge via the Internet at http:// pubs.acs.org.
LITERATURE CITED (1) Mansson, S., Bjornerud, A. in: Merbach, A. E., Toth, E., Eds. (2001) The Chemistry of contrast agents in medical magnetic resonance imaging, pp 1-44, Wiley, New York. (2) Aime, S., Frullano, L., and Crich, S. G. (2002) Compartmentalization of a gadolinium complex in the apoferritin cavity: a route to obtain high relaxivity contrast agents for MRI. Angew. Chem., Int. Ed. Engl. 41, 1018–1019. (3) Reynolds, C. H., Annan, N., Beshah, K., Huber, J. H., Shaber, S. H., and Lenkinski, R. E. (2000) Gadolinium-loaded nanoparticles: new contrast agents for MRI. J. Am. Chem. Soc. 122, 8940–8945.
(4) Rodriguez, E., Roig, A., Molins, E., Arus, C., Quintero, M. R., Cabanas, M. E., Cerdan, S., Lopez-Larrubia, P., and Sanfeliu, C. (2005) In vitro characterization of an Fe8 cluster as potential MRI contrast agent. NMR Biomed. 18, 300–307. (5) Parmelee, D. J., Walovitch, R. C., Ouellet, H. S., Lauffer, R. B. (2001) InVest. Radiol. 32, 741-747. (6) Lokling, K. E., Fossheim, S. L., Skurtveit, R., Bjornerud, A., and Klaveness, J. (2001) pH-sensitive paramagnetic liposomes as MRI contrast agents: in Vitro feasibility studies. Magn. Reson. Imaging 19, 731–738. (7) Wolfgang, S., Sanjay, S., Jan, P., Ralph, W., Mukesh, H., William, M.-S., and Peter, F. H. (1999) MR imaging of the liver with Gd-BOPTA: quantitative analysis of T1-weighted images at two different doses. J. Magn. Reson. Imaging 10, 80–83. (8) Langereis, S., de Lussanet, Q. G., van Genderen, M. H. P., Meijer, E. W., Beets-Tan, R. G. H., Griffioen, A. W., Engelshoven, J. M. A., and Backes, W. H. (2006) Evaluation of Gd(III)DTPA-terminated poly(propylene imine) dendrimers as contrast agents for MR imaging. NMR Biomed. 19, 133–141. (9) Aime, S., Gianolio, E., Uggeri, F., Tagliapietra, S., Barge, A., and Cravotto, G. (2006) New paramagnetic supramolecular adducts for MRI applications based on non-covalent interactions between Gd(III) complexes and β- or γ-cyclodextrin units anchored to chitosan. J. Inorg. Biochem. 100, 931–938. (10) Shu, C. Y., Gan, L. H., Wang, C. R., Pei, X. L., and Han, H. B. (2006) Synthesis and characterization of a new watersoluble endohedral metallofullerene. Carbon 44, 496–500. (11) Comet, M., and Vidal, M. (1998) Radiopharmaceutiques. Chimie des radiotraceurs et applications biologiques. In Presses UniVersitaires de Grenoble 206–246. (12) Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Vander Elst, L., and Muller, R. N. (2008) Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations and biological applications. Chem. ReV. 108, 2064–2110. (13) Li, Z., Li, W., Li, X., Pei, F., Wang, X., and Lei, H. (2007) Mn(II)-monosubstituted polyoxometalates as candidates for contrast agents in magnetic resonance imaging. J. Inorg. Biochem. 101, 1036–1042. (14) Na, H. B., Lee, J. H., An, K., Park, Y. I., Park, M., Lee, I. S., Nam, D.-H., Kim, S. T., Kim, S.-H., Kim, S.-W., Lim, K.-H., Kim, K.-S., Kim, S.-O., and Hyeon, T. (2007) Development of a T1 contrast agent for magnetic resonance imaging using MnO nanoparticles. Angew. Chem., Int. Ed. Engl. 46, 1–6. (15) Yu, X., Wadghiri, Y. Z., Sanes, D. H., and Turnbull, D. H. (2005) In vivo auditory brain mapping in mice with Mn-enhanced MRI. Nat. Neurosci. 8 (7), 961–966.
Development of MEMRI Contrast Agent (16) Koretsky, A. P., Silva, A. C (2004) Manganese-enhanced magnetic resonance imaging (MEMRI). NMR Biomed, 17, 527531, special issue of NMR in biomedicine on manganeseenhanced magnetic resonance imaging. (17) Elizondo, G., Fretz, C. J., Stark, D. D., Rocklage, S. M., Quay, S. C., Worah, D., Tsang, Y., Chen, M. C., and Ferrucci, J. T. (1991) Preclinical evaluation of MnDPDP: new paramagnetic hepatobiliary contrast agent for MR imaging. Radiology 178, 73–78. (18) Hayek, A., Bolze, F., Nicoud, J.-F., Baldeck, P. L., and Me´ly, Y. (2006) Synthesis and characterization of water-soluble twophoton excited blue fluorescent chromophores for bioimaging. Photochem. Photobiol. Sci. 5, 102–106. (19) Laurent, S., Parac-Vogt, T. N., Kimpe, K., Thirifays, C., Binnemans, K., Muller, R. N., and Vander Elst, L. (2007) Bis(phenylethylamide) derivatives of Gd-DTPA as potential receptor-specific MRI contrast agents. Eur. J. Inorg. Chem. 2061– 2067. (20) Simeonov, A., Matsushita, M., Juban, E. A., Thompson, E. H. Z., Hoffman, T. Z., Beusher, A. E., Taylor, M. J., Wirshing, P., Rettig, W., McCusker, J. K., Stevens, R. C., Millar, D. P., Schultz, P. G., Lerner, R. A., and Janda, K. D. (2000) Bluefluorescent antibodies. Science 290, 307–313. (21) Avdeef, A., and Testa, B. (2002) Physicochemical profiling in drug research: a brief survey of the state-of-the-art of experimental techniques. Cell. Mol. Life Sci. 59, 1681–1689. (22) Langer, G. A., Serena, S. D., and Nudd, L. M. (1975) Localization of contractile-dependent calcium. Comparison of manganese and verapamil in cardiac and skeletal muscle. Am. J. Physiol. 229, 1003–1007. (23) Haworth, R. A., Goknur, A. B., and Berkoff, H. A. (1989) Measurement of calcium channel activity of isolated adult rat heart cells using 54Mn. Arch. Biochem. Biophys. 268, 594–604. (24) Lauterbur, P. C., Mendoca-Dias, M. H., and Rudin, A. M. (1978) Augmentation of tissue water proton spin-lattice relaxation rates by in vivo addition of paramagnetic ions, In Frontiers of Biological Energetics (Dutton, P. L., Leigh, L. S., and Scarpa, A., Eds.) Vol 1, pp 752-759, Academic Press, New York. (25) Mamourian, A. C., Burnett, K. R., Goldstein, E. J., Wolf, G. L., Kressel, H. Y., and Baum, S. (1984) Proton relaxation enhancement in tissue due to ingested manganese chloride: time course and dose response in the rat. Physiol. Chem. Phys. Med. NMR 16, 123–128. (26) Kang, Y. S., Gore, J. C., and Armitage, I. M. (1984) Studies of factors affecting the design of NMR contrast agents: manganese in blood as a model system. Magn. Res. Med. 1, 396–409.
Bioconjugate Chem., Vol. 20, No. 4, 2009 767 (27) Small, W. C., DeSimone-Macchi, D., Parker, J. R., Sukerkar, A., Hahn, P. F., Rubin, D. L., Zelch, J. V., Kuhlman, J. E., Outwater, E. K., Weinreb, J. C., Brown, J. J., De Lange, E. E., Woodward, P. J., Arildsen, R., Foster, G. S., Runge, V. M., Aisen, A. M., Muroff, L. R., Thoeni, R. F., Parisky, Y. R., Tanenbaum, L. N., Totterman, S., Herfkens, R. J., Knudsen, J., Laster, R. E., Duerinckx, A., Stillman, A. E., Spritzer, C. E., Saini, S., Rofsky, N. M., and Bernardino, M. E. (1999) A multisite phase III study of the safety and efficacy of a new manganese chloride-based gastrointestinal contrast agent for MRI of the abdomen and pelvis. J. Magn. Res. Imaging 10, 15–24. (28) Crossgrove, J., and Zheng, W. (2004) Manganese toxicity upon overexposure. NMR Biomed. 17, 544–553. (29) Wolf, G. L., and Baum, L. (1983) Cardiovascular toxicity and tissue proton T1 response to manganese injection in the dog and raddit. AJR Am. J. Roentgenol. 141, 193–197. (30) Gallez, B., Bacic, G. G., and Schwartz, H. M. (1996) Evidence for the dissociation of the hepatobiliary MRI contrast agent MnDPDP. Magn. Reson. Med. 35, 14–19. (31) Federle, M. P., Chezmar, J. L., Rubin, D. L., Weinreb, J. C., Freeny, P. C., Semelka, R. C., Brown, J. J., Borello, J. A., Lee, J. K., Mattrey, R., Dachman, A. H., Saini, S., Harmon, B., Fenstermacher, M., Pelsang, R. E., Harms, S. E., Mitchell, D. G., Halford, H. H., Anderson, M. W., Johnson, C. D., Francis, I. R., Bova, J. G., Kenney, P. J., Klippenstein, D. L., Foster, G. S., and Turner, D. A. (2000) Safety and efficacy of mangafodipir trisodium (MnDPDP) injection for hepatic MRI in adults: results of the U.S. multicenter phase III clinical trials. J. Magn. Reson. Imaging 12, 186–197. (32) Aime, S., Botta, M., Fasano, M., and Terreno, E. (1998) Lanthanide III chelates for NMR biomedical applications. Chem. Soc. ReV. 27, 19–29. (33) Vander Elst, L., Maton, F., Laurent, S., Seghi, F., Chapelle, F., and Muller, R. N. (1997) A multinuclear MR study of GdEOB-DTPA: comprehensive preclinical characterization of an organ specific MRI contrast agent. Magn. Res. Med. 38, 604– 614. (34) Muller, R. N., Raduchel, B., Laurent, S., Platzek, J., Pierart, C., Mareski, P., and Vander Elst, L. (1999) Physicochemical characterization of MS-325, a new gadolinium complex, by multinuclear relaxometry. Eur. J. Inorg. Chem. 1949–1955. (35) Lin, Y. J., and Koretsky, A. P. (1997) Manganese ion enhances T1-weighted MRI during brain activation: an approach to direct imaging of brain function. Magn. Reson. Med. 38, 378–388. BC8004683
