Introduction

Inflammation is closely related to cancer progression and several disease.¹ NSAIDs (non-steroidal anti-inflammatory drugs) are the most common medications for the treatment of anti-inflammatory actions worldwide. These drugs that bind to inducible enzymes such as COX-1 and COX-2 can be used as inflammatory biomarkers for molecular imaging. Herein GdL1, GdL2 and GdL3 were synthesized from DO3A conjugated to carboxylic acid derivatives of NSAIDs which were diflunisal, fenbufen and sulindac respectively. In this study, relaxivities, kinetic stabilities and animal MR imaging experiments were performed. We expect that these gadolinium complexes conjugated with NSAIDs will potentially be able to target inflammation-induced disease in MRI.

Materials and Methods

All reagents were purchased from commercial sources and used as received. The synthesis of all products were confirmed by microanalysis and spectroscopic techniques such as NMR, HR-FAB mass. The kinetic inertness for gadolinium complexes was determined by the evolution of the normalized paramagnetic longitudinal relaxation rates at the transmetallaiton condition.² T₁ measurements were carried out using an inversion recovery method with variable inversion times (TI) at1.5T (64 MHz). For T₂ measurements, the CPMG (Carr-Purcell-Meiboon-Gill) pulse sequence was adapted for multiple spin-echo measurements. T₁, T₂ relaxation times were obtained from the nonlinear least-squares fit of the mean pixel values at variable TI and TE, respectively. The relaxivities (r₁ and r₂) were then calculated as a slope of linear fit of relaxation rate along with concentrations. The in vivo MR experiments was performed in accordance with the rules of the animal research committee of Kyungpook National University (KNU). For the inflammatory model, 30 µL of turpentine oil was injected into left thigh muscle of ICR mice (27-30 g, six-weed of age, male). Inflammation outbreak was maximized after 3 days of injection. The mice were anesthetized by 1.5% isoflurane in oxygen. MR images were acquired before and after intravascular injection of each MR contrast agents (dose: 0.1 mmol Gd/kg). The images were acquired with a 1.5 T (GE Healthcare, Milwaukee, WI, USA) equipped with a homemade small animal volume coil. The imaging parameters for SE (Spin echo) were as follows: repetition time (TR) = 300 ms; echo time (TE) = 13 ms; 10 mm field of view (FOV); 192×160 matrix size; 1.0 mm slice thickness; number of acquisition (NEX) = 8.

Result and Discussion

Scheme 1 shows the synthesis of GdL1, GdL2 and GdL3. Relaxivities of these complexes are slightly higher than those of Gadovist^® as shown table 1. The kinetic stability of these gadolinium complexes were determined with time-dependent longitudinal relaxation rate (R_1,p(t)/R_1,p(0)) and the relaxivity was maintained above 95% against to initial value during the measurement. This kinetic stability was comparable with Dotarem^® employing the same type of macrocyclic chelate (Figure 1). In vivo MR experiment, all of these complexes exhibit strong enhancement at liver, urinary bladder and gallbladder as seen in Figure 2. Also inflamed tissue of left thigh exhibits strong enhancement more than 2 hours demonstrating inflammation targeting ability of the present agents. The degree of signal enhancement with GdL1, GdL2 and GdL3, as expressed by contrast-to-noise ratio (CNR) in the inflamed tissue, is even higher and persists longer than Gadovist^® confirming inflammation targeting nature of these complexes (Figure 3). Especially, GdL3 shows the most intensive enhancement in the inflamed site for long time compared with GdL1 and GdL2.

Conclusion

In conclusion, we have synthesized three kinds of inflammation targeted MR CAs by conjugating NSAIDs to DO3A-based ligand. The kinetic inertness of these complexes compares well with that of structurally related Dotarem^®. The Gadolinium complexes are able to target inflamed tissue, as confirmed by MR images of mice with inflammation. As anti-inflammatory activities vary depending on the type of conjugated NSAIDs as well as on the structure of the complexes, more detailed studies on structure-targeting activity relationships are likely to deepen insight into the future direction of inflammation targeting MR agents based on gadolinium complexes.

Acknowledgements

No acknowledgement found.