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Bioconjugate Chem. 2001, 12, 510−514
Paramagnetic Water-Soluble Metallofullerenes Having the Highest Relaxivity for MRI Contrast Agents Masahito Mikawa,† Haruhito Kato,‡ Masafumi Okumura,† Michiko Narazaki,† Yoko Kanazawa,† Naoto Miwa,† and Hisanori Shinohara*,‡ Basic Research Institute, Nihon Schering K.K., 6-64 Nishimiyahara 2-chome Yodogawa-ku, Osaka, 532-0004, Japan, and Department of Chemistry, Nagoya University, Nagoya, 464-8602, Japan. Received December 14, 2000; Revised Manuscript Received March 23, 2001
Water-soluble gadolinium (Gd) endohedral metallofullerenes have been synthesized as polyhydroxyl forms (Gd@C82(OH)n, Gd-fullerenols) and their paramagnetic properties were evaluated by in vivo as well as in vitro for the novel magnetic resonance imaging (MRI) contrast agents for next generation. The in vitro water proton relaxivity, R1 (the effect on 1/T1), of Gd-fullerenols is significantly higher (20-folds) than that of the commercial MRI contrast agent, Magnevist (gadolinium-diethylenetriaminepentaacetic acid, Gd-DTPA) at 1.0 T close to the common field of clinical MRI. This unusually high proton relaxivity of Gd-fullerenols leads to the highest signal enhancement at extremely lower Gd concentration in MRI studies. The strong signal was confirmed in vivo MRI at lung, liver, spleen, and kidney of CDF1 mice after i.v. administration of Gd-fullerenols at a dose of 5 µmol Gd/kg, which was 1/20 of the typical clinical dose (100 µmol Gd/kg) of Gd-DTPA.
INTRODUCTION
With the advent of gadolinium-chelate complex for a magnetic resonance imaging (MRI)1 contrast agent, Magnevist (Gd-DTPA) (1, 2), where the contrast agent can induce extra proton spin relaxation due to the paramagnetic property, many efforts have been given for the design and development of new compounds, such as Eovist (Gd-3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(4ethoxybenzyl)undecandicarboxylic acid, Gd-EOB-DTPA), ProHance (Gd-1,4,7-tris(carbonylmethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazacyclododecane, Gd-HP-DOTA) or Omnicsan (Gd-diethylenetriaminepentaacetate-bis(methylamide), Gd-DTPA-BMA) (3-5). These agents have been used as a routine clinical tool to reveal anatomical details in vivo and to detect lesions for diagnosis. For the future real-time in vivo imaging of molecular/cellular diagnosis, many efforts have been given by using the fluorescent (6, 7) and nuclear imaging techniques (8, 9) as well as MRI technique (10-13). Although fluorescent technique can have excellent temporal resolution, a common shortcoming is limited depth penetration. The nuclear techniques are free from this limitation but suffer from the inherently lower spatial resolution. In contrast, the MRI technique has much less * To whom correspondence should be addressed: Department of Chemistry, Nagoya University, Nagoya, 464-8602, Japan.Tel: (+81) 52-789-2482, Fax: (+81) 52-789-2962. E-mail:
[email protected]. † Nihon Schering K.K. ‡ Nagoya University. 1 1Abbreviations: MRI, magnetic resonance imaging; Gd, gadolinium; DTPA, diethylenetriaminepentaacetic acid; EOBDTPA, 3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(4-ethoxybenzyl)undecandicarboxlic acid; HP-DOTA,1,4,7-tris(carbonylmethyl)10-(2′-hydroxypropyl)-1,4,7,10-tetraazacyclododecane; DTPABMA, diethylenetriaminepentaacetate-bis(methylamide); TBAH, tetrabutylammonium hydroxide; ICP-AES, inductively coupled plasma atomic emission spectroscopy; RES, reticular-endothelial system.
limitation on the observation depth and spatial resolution. As compared to fluorescent and nuclear imaging techniques, however, molecular probe detection by magnetic resonance is several orders of magnitude less sensitive (14, 15). It is necessary, therefore, to accumulate more targeting probes or enforce its potential enhancing ability for molecular/cellular diagnosis by MRI. Thus, novel materials having strong proton relaxivity and a higher capacity for MR signal enhancement at a significantly lower concentration are required for the next generation. Here we have investigated the possibility of application of novel materials, water-soluble Gd metallofullerenes (Gd@C82(OH)n, Gd-fullerenols) (16-20), for MRI contrast agents, and have evaluated them in vivo as well as in vitro. Extremely high water proton relaxivity, R1 (the effect on 1/T1), was obtained with Gd@C82(OH)40, 81 (mM-1 s-1) at 1.0 T and 25 °C which is more than 20 times as high as that of the commercial Gd-DTPA. This unusually high proton relaxivity of Gd@C82(OH)40 led to the highest signal enhancement at significantly lower Gd concentration by in vitro and in vivo MRI studies so far reported. MATERIALS AND METHODS
Gd@C82. Soot containing Gd endohedral metallofullerenes such as Gd@C82 was produced by the direct current arc evaporation method (350 A, 21 V) of Gd/ graphite composite rods (0.8 wt % Gd atom, Toyo Tanso Co.) under a 17 l/min flow of helium at 50 Torr, followed by Soxhlet extraction with carbon disulfide for 12 h (21, 22). The purification and isolation of Gd@C82 was achieved by the two-stage high performance liquid chromatography (HPLC) (23) with a Buckyprep column (20 mm dia × 300 mm, Nacalai Tesque). The purity of Gd@C82 (
