Abstract
1087
Objectives: The p38 MAPK pathway is activated by external stimuli such as oxidative stress and pathogen infection to regulate gene expression via phosphorylation of transcription factors. Among the four isoforms (α, β, γ, δ) of p38, p38α, which is expressed in most cell types (including inflammatory cells), induces the production of inflammatory cytokines such as TNF-α, IL-1β and IL-6 in response to stimuli, and is known to be activated during the early stages of inflammation. Thus, p38α is a potential target molecule for qualitative diagnosis of inflammatory diseases such as rheumatoid arthritis and chronic obstructive pulmonary disease. In this study, we designed and synthesized a radioiodinated pyrimidinopyridone derivative, namely 123/125I-FIC, as a novel p38α imaging probe. We evaluated the effectiveness of the probe by in vitro activity estimation, an in vivo biodistribution study, and in vivo SPECT/CT imaging in a mouse model of inflammation.
Methods: We designed 123/125I-FIC based on the structure-activity relationship of a potent p38α inhibitor, namely pamapimod, with p38α. We synthesized FIC and a tin precursor via 6 and 7 steps, respectively. The inhibitory potency of FIC against p38α was measured with a commercially available kinase assay kit. Radiosynthesis was performed by the reaction of 123/125I-NaI with a tin precursor for 15 min at room temperature, followed by purification and purity evaluation by RP-HPLC. 125I-FIC was incubated in mouse plasma for 24 hours at 37°C for stability assessment. Biodistribution studies with 125I-FIC were carried out in a mouse model of inflammation, in which mice were intramuscularly treated with turpentine oil 2-8 days prior to the study. In vivo SPECT/CT imaging with 123I-FIC was also carried out in the mouse model. For in vivo blocking, R1487, a potent p38α inhibitor with a similar structure to that of FIC, was co-administrated with 123/125I-FIC.
Results: FIC was synthesized with a total yield of 21.9%. FIC exhibited high p38α inhibition activity, which was comparable to that of the potent p38α inhibitor SB203580. 125I-FIC was obtained with a radiochemical yield of 96.4%, and a radiochemical purity of 97.4%. TLC analysis revealed that more than 95% of the radioactivity resulted from intact 125I-FIC after incubation in plasma for 24 hours. Biodistribution studies showed that radioactivity accumulation in the inflammation site peaked at 30 min post injection (6.7%ID/g) and cleared thereafter (2.4%ID/g at 2 hours post injection), while radioactivity was rapidly cleared from the blood and muscle (less than 2.6%ID/g over the time period), which led to high inflammation-to-blood (6.6) and inflammation-to-muscle (4.9) ratios obtained at 30 min post injection. Radioactivity was mainly excreted by the hepatobiliary mode and negligible radioactivity was found in the thyroid, indicating no in vivo deiodination of the probe. In addition, 125I-FIC showed 3 times higher radioactivity accumulation during the early stage of inflammation (treatment with turpentine oil 2 days prior to the study) than during the late stage of inflammation (treatment with turpentine oil 8 days prior to the study). In vivo SPECT imaging clearly delineated the inflammation site at 30 min post injection of 123I-FIC, as expected, while high radioactivity was unexpectedly observed in brown adipose tissue. Accumulation of 125I-FIC in the inflammation site was significantly decreased by co-administration of R1487.
Conclusions: Results of this study demonstrate the high synthetic efficiency, high inhibitory potency, high stability, excellent biodistribution profile, and in vivo imaging potency of 123/125I-FIC as a novel p38α imaging probe. 123I-FIC is thus a promising probe for qualitative diagnosis of inflammatory diseases.