Theranostics is a future medical strategy that combines therapeutic and diagnostic capabilities [1]. We are presently attempting to develop theranostics probes that will allow us to better understand the redox biology and potential treatment of the brain diseases. Recently, we have succeeded in synthesizing a material by connecting nitroxide probes, which can be used for T1 contrast agents in MRI, to non-steroidal anti-inflammatory drugs, ibuprofen and ketoprofen, in a high yield. In the present study, these newly synthesized theranostics probes were applied to brain disease model mice for diagnostic and therapy. We examined details concerning the distribution and kinetics of these theranostics probes in mouse brains. Additionally, the therapeutic effects of these probes were evaluated in septic mouse brains by a biochemical assay. The obtained results clearly showed that the nitroxide-based theranostics probes worked as anti-inflammatory drugs in septic mouse brains and as T1 contrast agents in MRI.Theranostics probes: Condensation reactions of ibuprofen or ketoprofen and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3-hydroxymethyl-PROXYL) were carried out in the presence of DCC and DMAP in CH₂Cl₂ at room temperature, and ibuprofen-PROXYL and ketoprofen-PROXYL were purified by silica chromatography. Animals: Male C57BL/6 mice aged 5 to 7 weeks with body weights of 20–25 g were used. Septic mice: Lipopolysaccharide from Escherichia coli serotype 055:B5 (Sigma-Aldrich Chemical Co.) was intraperitoneally injected into mice (100mg/kg body weight). The amount of nitric oxide (NO) generated in septic mouse brains was quantitated through a fluorescence method using 2,3-diaminonaphthalene (Dojindo Lab.). Theranostics probes in saline were injected by tail vein cannulation into the mice under isoflurane anesthesia. MRI measurements: MRI of mouse heads was acquired using an MRmini scanner (MR Technology) with a 0.5-T permanent magnet. EPR measurements: EPR spectra in this study were measured with a JEOL X-band ESR spectrometer.Figure1 shows the chemical structure of theranostics probes, ibuprofen-PROXYL and ketoprofen-PROXYL. To evaluate the stability of these two probes in mice, their resistivity toward an endogenous antioxidant, ascorbic acid, was measured in vitro by an EPR spectrometer. The rate constants of the reduction reaction of ibuprofen-PROXYL and ketoprofen-PROXYL were 7.9 ± 0.05 × 10^-3 min^-1 and 7.8 ± 0.04 × 10^-3 min^-1, respectively (n=4). The reduction reaction of both compounds were about 40% slower than that of 3-hydroxymethyl-PROXYL (rate constant: 1.2 ± 0.04 × 10^-2 min^-1 (n=4)) in the presence of ascorbic acid, indicating that both probes were more resistant to ascorbic acid than 3-hydroxymethyl-PROXYL. To examine if nitroxide-based theranostics probes can act as T1 contrast agents for MRI and are capable of visualizing the distribution of both probes in mouse brains, T1-weighted MRI of mouse heads was taken before and after injection of both probes (Figure 2). Difference MRI (C and F in Figure 2) was obtained by subtracting MRI (A and D) before administration of probes from MRI (B and E) after administration of ibuprofen-PROXYL and ketoprofen-PROXYL, respectively. These difference images clearly indicated that both probes entered the brains passing through the blood brain barrier and were distributed in all regions of mouse heads. Time-course MRI of mouse heads was examined after injection of both theranostics probes, and their rate constants in mouse brains were calculated by plotting the changes in the image intensities of mouse brains as functions of time after injection. The rate constants of ibuprofen-PROXYL and ketoprofen-PROXYL in mouse brains were 1.78 ± 0.02 × 10^-2 and 1.61 ± 0.01 × 10^-2 min^-1 (n=3), respectively. The half-lives of ibuprofen-PROXYL and ketoprofen-PROXYL calculated from their rate constants were 38.2 ± 4.3 min and 43.4 ± 3.8 min, respectively, which suggest that both are relatively stable contrast agents for MRI of mouse brains. To examine if ibuprofen-PROXYL and ketoprofen-PROXYL have anti-inflammatory effects on septic mouse brains, the amount of NO produced in septic mouse brains was measured by a fluorescence method with or without injection of both probes. Percent inhibition of NO generation in septic mouse brains with ibuprofen-PROXYL and ketoprofen-PROXYL was 75.5 ± 5.8% (n=3) and 71.8 ± 7.4% (n=3), respectively, strongly indicating both probes could inhibit NO production in septic mouse brains. All of these results suggest that ibuprofen-PROXYL and ketoprofen-PROXYL can act both as anti-inflammatory drugs and as T1 contrast agents in MRI.We successfully developed two nitroxide-based theranostics probes, ibuprofen-PROXYL and ketoprofen-PROXYL. These theranostics probes appear to have the capability to act as both anti-inflammatory drugs that suppress NO production in septic mouse brains and as T1 contrast agents in brains for MRI. The present results suggest that both therapeutic action and diagnostic imaging of these theranostics probes can be followed by MRI.