Purpose

Macrophages represent an essential means of sequestration and immune evasion for Mycobacterium tuberculosis. Pulmonary tuberculosis (TB) is characterized by dense collections of tissue-specific and recruited macrophages, both of which abundantly express CSF1R on their outer surface. 4-Cyano-N-(5-(1-(dimethylglycyl)piperidin-4-yl)-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole-2-carboxamide (JNJ-28312141) is a reported high affinity, CSF1R-selective antagonist. We report the radiosynthesis of 4-cyano-N-(5-(1-(N-methyl-N-([¹¹C]methyl)glycyl)piperidin-4-yl)-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole-2-carboxamide ([¹¹C]JNJ-28312141) and non-invasive detection of granulomatous and diffuse lesions in a mouse model of TB using positron emission tomography (PET).

Methods

Nor-methyl-JNJ-28312141 precursor was radiolabeled with [¹¹C]iodomethane to produce [¹¹C]JNJ-28312141. PET/CT imaging was performed in the C3HeB/FeJ murine model of chronic pulmonary TB to co-localize radiotracer uptake with granulomatous lesions observed on CT. Additionally, CSF1R, Iba1 fluorescence immunohistochemistry was performed to co-localize CSF1R target with reactive macrophages in infected and healthy mice.

Results

Radiosynthesis of [¹¹C]JNJ-28312141 averaged a non-decay-corrected yield of 18.7 ± 2.1%, radiochemical purity of 99%, and specific activity averaging 658 ± 141 GBq/µmol at the end-of-synthesis. PET/CT imaging in healthy mice showed hepatobiliary [13.39–25.34% ID/g, percentage of injected dose per gram of tissue (ID/g)] and kidney uptake (12.35% ID/g) at 40–50 min post-injection. Infected mice showed focal pulmonary lesion uptake (5.58–12.49% ID/g), hepatobiliary uptake (15.30–40.50% ID/g), cervical node uptake, and renal uptake (11.66–29.33% ID/g). The ratio of infected lesioned lung/healthy lung uptake is 5.91:1, while the ratio of lesion uptake to adjacent infected radiolucent lung is 2.8:1. Pre-administration of 1 mg/kg of unlabeled JNJ-28312141 with [¹¹C]JNJ-28312141 in infected animals resulted in substantial blockade. Fluorescence microscopy of infected and uninfected whole lung sections exclusively co-localized CSF1R staining with abundant Iba1 + macrophages. Healthy lung exhibited no CSF1R staining and very few Iba1 + macrophages.

Conclusion

[¹¹C]JNJ-28312141 binds specifically to CSF1R + macrophages and delineates granulomatous foci of disease in a murine model of pulmonary TB.

中文翻译：

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结核病小鼠模型中 CSF1R 的 PET/CT 成像

目的

巨噬细胞是结核分枝杆菌隔离和免疫逃避的重要手段。肺结核 (TB) 的特点是组织特异性巨噬细胞和招募的巨噬细胞密集聚集，这两种巨噬细胞在其外表面都大量表达 CSF1R。4-氰基-N-(5-(1-(二甲基甘氨酰)哌啶-4-基)-2',3',4',5'-四氢-[1,1'-联苯]-2-基)- 1H-咪唑-2-甲酰胺 (JNJ-28312141) 是一种已报道的高亲和力、CSF1R 选择性拮抗剂。我们报道了 4-cyano-N-(5-(1-(N-methyl-N-([ ¹¹ C]methyl)glycyl)piperidin-4-yl)-2',3',4',5 的放射合成'-四氢-[1,1'-联苯]-2-基)-1H-咪唑-2-甲酰胺 ([ ¹¹ C]JNJ-28312141) 以及结核病小鼠模型中肉芽肿性和弥漫性病变的无创检测使用正电子发射断层扫描（PET）。

方法

用[ ¹¹ C]碘甲烷放射性标记正甲基-JNJ-28312141前体以产生[ ¹¹ C]JNJ-28312141。在慢性肺结核的 C3HeB/FeJ 小鼠模型中进行 PET/CT 成像，以将放射性示踪剂的摄取与 CT 上观察到的肉芽肿病变进行共定位。此外，还进行了 CSF1R、Iba1 荧光免疫组织化学分析，以将受感染小鼠和健康小鼠中的 CSF1R 靶标与反应性巨噬细胞共定位。

结果

[ ¹¹ C]JNJ-28312141 的放射合成平均非衰变校正产率为 18.7 ± 2.1%，放射化学纯度为 99%，合成结束时比活度平均为 658 ± 141 GBq/μmol。健康小鼠的 PET/CT 成像显示注射后 40-50 分钟的肝胆 [13.39–25.34% ID/g，每克组织注射剂量百分比 (ID/g)] 和肾脏摄取 (12.35% ID/g) 。感染小鼠表现出局灶性肺部病变摄取（5.58-12.49% ID/g）、肝胆摄取（15.30-40.50% ID/g）、颈淋巴结摄取和肾脏摄取（11.66-29.33% ID/g）。感染的病变肺/健康肺摄取的比率是5.91:1，而病变摄取与邻近感染的射线可透过的肺的比率是2.8:1。在受感染的动物中预施用1mg/kg未标记的JNJ-28312141和[ ¹¹ C]JNJ-28312141导致显着的阻断。感染和未感染的全肺切片的荧光显微镜仅将 CSF1R 染色与丰富的 Iba1 + 巨噬细胞共定位。健康的肺部没有表现出 CSF1R 染色和很少的 Iba1 + 巨噬细胞。

结论

[ ¹¹ C]JNJ-28312141 与 CSF1R + 巨噬细胞特异性结合，并在肺结核小鼠模型中描绘出疾病的肉芽肿病灶。