Introduction

Most clinical contrast agents are based on Gd(III) complexes of polyamino polycarboxylic acids. However, in view of the recent discovery that Gd(III) could possibly be involved in nephrogenic systemic fibrosis (NSF), much emphasis is now being put on alternative approaches based on non-lanthanide metals, in particular manganese, for MR imaging. Although there is no direct relation has been found between Mn and NSF so far, the potential neurotoxicity of free Mn ions is still remaining as major safety concern when used as contrast agent. Indeed, MnDPDP (Mangofodipir; Teslascan^TM), which was FDA approved as T₁ MR contrast agent for liver imaging, showed strong dechelation in plasma after intravenous (IV) injection. Therefore, there have been several efforts to design stable Mn(II) complexes for MR imaging. Herein, we report the design and the synthesis of a new highly stable Mn(II)-based complexes (Mn-DO2A-BTA) for use as a new class of hepatobiliary MRI CA.

Materials and Methods

All reagents were purchased from commercial sources and used as received. DO2A (= 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid) and BTA derivative were synthesized in our lab according to literature method¹. T₁ measurements were carried out using an inversion recovery method with a variable inversion time (TI) at 1.5 T (64MHz, GE Healthcare, Milwaukee, WI, USA). For T₂ measurements, the CPMG (Carr-Purcell-Meiboon-Gill) pulse sequence was adapted for multiple spin-echo measurements. T₁ and T₂ relaxation times were obtained from the non-linear least squares fit of the mean pixel values for the multiple spin-echo measurements at each TI value and echo time. Relaxivities (r₁ and r₂) were then calculated as an inverse of relaxation time per mM. MR images of anaesthetized mice were obtained pre- and post- MnL (0.1 mmol Mn/kg) injection by tail vein with a 1.5 T MR unit (GE Healthcare, Milwaukee, WI, USA) with home-made small animal RF coil. The imaging parameters for SE (Spin echo) were as follows: repetition time (TR) = 300 ms; echo time (TE) = 12 ms; 8 mm field of view (FOV); 192×128 matrix size; 1.2 mm slice thickness; number of acquisition (NEX) = 8. HEK-293 (human embryonic kidney) cells were pated 1 x 10⁴ in 96-well plate. The DMEM serum depletion media for 24 h. Then the live cell count was assayed using CCK-8 (Dojindo, Sunnyvale, CA) according to the manufacturer’s protocol.

Results and Discussion

Scheme 1 shows the synthesis of manganese based DO2A-benzothiazole conjugated complex (Mn-DO2A-BTA). After each step, the synthesized complexes confirmed by ¹H NMR or high resolution mass spectroscopy. The synthesized complex (3.2 mM^-1s^-1) shows good relaxivity in HSA solution compared with MnDPDP® (3.6 mM^-1s^-1)². HSA binding test (m-titration, Figure 1) shows that the corresponding binding constant K_a provide the evidence for the noncovalent lipophilic interaction between the chelate Mn-DO2A-BTA. In vivo MR images of Mn-DO2A-BTA are represented by T₁-weighted MR images of 8-week-old male Institute of Cancer Research (ICR) mice after bolus injection of Mn-DO2A-BTA through tail vein (Figure 2). It shows strong signal enhancement in liver, kidney, gallbladder, heart and aorta. These results show that MnL is able to act as not only liver targeting agent but also blood pool agent. The cytotoxicity of Mn-DO2A-BTA was tested by empoying HEK-293, a normal cell line. Figure 3 shows the Mn-DO2A-BTA has negligible cytotoxicity.

Acknowledgements

No acknowledgement found.