眼内动脉化疗治疗视网膜母细胞瘤的首例小动物（兔）模型的药代动力学、组织定位、毒性和疗效。

PURPOSE: Current intra-arterial chemotherapy (IAC) drug regimens for retinoblastoma have ocular and vascular toxicities. No small-animal model of IAC exists to test drug efficacy and toxicity in vivo for IAC drug discovery. The purpose of this study was to develop a small-animal model of IAC and to analyze the ocular tissue penetration, distribution, pharmacokinetics, and treatment efficacy. METHODS: Following selective ophthalmic artery (OA) catheterization, melphalan (0.4 to 1.2 mg/kg) was injected. For pharmacokinetic studies, rabbits were euthanized at 0.5, 1, 2, 4, or 6 hours following intra-OA infusion. Drug levels were determined in vitreous, retina, and blood by liquid chromatography tandem mass spectrometry. To assess toxicity, angiograms, photography, fluorescein angiography, and histopathology were performed. For in situ tissue drug distribution, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was performed. The tumor model was created by combined subretinal/intravitreal injection of human WERI-Rb1 retinoblastoma cells; the tumor was treated in vivo with intra-arterial melphalan or saline; and induction of tumor death was measured by cleaved caspase-3 activity. RESULTS: OA was selectively catheterized for 79 of 79 (100%) eyes in 47 of 47 (100%) rabbits, and melphalan was delivered successfully in 31 of 31 (100%) eyes, without evidence of vascular occlusion or retinal damage. For treated eyes, maximum concentration (Cmax) in the retina was 4.95 μM and area under the curve (AUC0→∞) was 5.26 μM·h. Treated eye vitreous Cmax was 2.24 μM and AUC0→∞ was 4.19 μM·h. Vitreous Cmax for the treated eye was >100-fold higher than for the untreated eye (P = 0.01), and AUC0→∞ was ∼50-fold higher (P = 0.01). Histology-directed MALDI-IMS revealed highest drug localization within the retina. Peripheral blood Cmax was 1.04 μM and AUC0→∞ was 2.07 μM·h. Combined subretinal/intravitreal injection of human retinoblastoma cells led to intra-retinal tumors and subretinal/vitreous seeds, which could be effectively killed in vivo with intra-arterial melphalan. CONCLUSIONS: This first small-animal model of IAC has excellent vitreous and retinal tissue drug penetration, achieving levels sufficient to kill human retinoblastoma cells, facilitating future IAC drug discovery.

目的：目前用于视网膜母细胞瘤的动脉内化疗（IAC）药物方案具有眼和血管毒性。目前还没有小动物模型可以用于体内测试 IAC 药物的疗效和毒性，以进行 IAC 药物发现。本研究的目的是开发一种小动物 IAC 模型，并分析眼组织穿透性、分布、药代动力学和治疗效果。

方法：选择性眼动脉（OA）导管插入后，注射马法兰（0.4 至 1.2mg/kg）。为了进行药代动力学研究，在 OA 内输注后 0.5、1、2、4 或 6 小时处死兔子。通过液相色谱串联质谱法在玻璃体液、视网膜和血液中测定药物水平。为了评估毒性，进行了血管造影、摄影、荧光素血管造影和组织病理学检查。为了进行组织内药物分布分析，采用基质辅助激光解吸/电离成像质谱（MALDI-IMS）。通过视网膜下/玻璃体内联合注射人 WERI-Rb1 视网膜母细胞瘤细胞来创建肿瘤模型；用动脉内马法兰或生理盐水对肿瘤进行体内治疗；并通过 cleaved caspase-3 活性测量诱导的肿瘤死亡。

结果：在 47 只（100%）兔子的 79 只（100%）眼中成功选择性导管插入 OA，在 31 只（100%）眼中成功输送马法兰，没有血管阻塞或视网膜损伤的证据。对于治疗眼，视网膜中的最大浓度（Cmax）为 4.95μM，曲线下面积（AUC0→∞）为 5.26μM·h。治疗眼玻璃体 Cmax 为 2.24μM，AUC0→∞ 为 4.19μM·h。治疗眼玻璃体 Cmax 比未治疗眼高 100 多倍（P=0.01），AUC0→∞ 高约 50 倍（P=0.01）。基于组织学的 MALDI-IMS 显示药物在视网膜中的定位最高。外周血 Cmax 为 1.04μM，AUC0→∞ 为 2.07μM·h。人视网膜母细胞瘤细胞的视网膜下/玻璃体内联合注射导致视网膜内肿瘤和视网膜下/玻璃体内种子，可通过动脉内马法兰有效杀死体内肿瘤。

结论：这是第一个小动物 IAC 模型，具有出色的玻璃体液和视网膜组织药物穿透性，达到足以杀死人视网膜母细胞瘤细胞的水平，为未来的 IAC 药物发现提供了便利。