Kim Jae Suk, Beadle James R, Freeman William R, Hostetler Karl Y, Hartmann Kathrin, Valiaeva Nadejda, Kozak Igor, Conner Laura, Trahan Julissa, Aldern Kathy A, Cheng Lingyun
Jacobs Retina Center at Shiley Eye Center, University of California, San Diego, La Jolla, CA 92093-0946, USA.
Mol Vis. 2012;18:1907-17. Epub 2012 Jul 14.
The objectives of this study were to synthesize and characterize two types of cytarabine (Ara-C) lipid produgs and evaluate the prodrugs for sustained intraocular delivery after administration by intravitreal injection.
Hexadecyloxypropyl cytarabine 5'-monophosphate (HDP-P-Ara-C) and hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate (HDP-cP-Ara-C) were synthesized starting from cytarabine (1-β-D-arabinofuranosylcytosine). Their vitreal clearance profile was simulated using a custom dissolution chamber, in vitro cytotoxicity was evaluated using cell proliferation assays, and in vivo ocular properties in rat and rabbit eyes were assessed using biomicroscopy, indirect ophthalmoscopy, tonometry, electroretinography, and histology.
HDP-P-Ara-C was cleared from the dissolution chamber (flow rate 2 µL/min) within 7 days. In contrast, HDP-cP-Ara-C, a much more insoluble prodrug, was still detectable 36 days after the dissolution process was started. HDP-P-Ara-C had a 50% cytotoxicity concentration of 52±2.6 μM in human retinal pigment epithelium (ARPE-19) and 32±2.2 µM in a rat Müller cell line, rMC-1. The 50% cytotoxicity concentration values for HDP-cP-Ara-C in ARPE-19 and rMC-1 cells were 50 µM and 25 µM, respectively. HDP-P-Ara-C was not detectable 2 weeks after the highest intravitreal dose (228 µg/rat eye) was injected, and no ocular toxicity was found. With HDP-cP-Ara-C, the drug depot was visible for 26 weeks following a single intravitreal injection (800 µg/rabbit eye). For both compounds, the electroretinogram, intraocular pressure, and other toxicity studies were negative except for the highest dose of HDP-cP-Ara-C (800 µg/eye), which had focal toxicity from the direct touch of the retina and decreased dark adapted a-waves and decreased flicker electroretinogram amplitudes (generalized estimating equations, p=0.039 and 0.01).
The cyclic monophosphate prodrug, HDP-cP-Ara-C, was found to have physiochemical properties better suited for sustained delivery of cytarabine to posterior segments of the eye. These properties included limited aqueous solubility, in vitro antiproliferative activity, and good tolerability after injection into rabbit eyes.
本研究的目的是合成并表征两种类型的阿糖胞苷（Ara-C）脂质前药，并评估这些前药经玻璃体内注射给药后在眼内的持续递送情况。
从阿糖胞苷（1-β-D-阿拉伯呋喃糖基胞嘧啶）开始合成十六烷氧基丙基阿糖胞苷5'-单磷酸酯（HDP-P-Ara-C）和十六烷氧基丙基阿糖胞苷3',5'-环单磷酸酯（HDP-cP-Ara-C）。使用定制的溶解室模拟它们在玻璃体中的清除情况，使用细胞增殖试验评估体外细胞毒性，并使用生物显微镜检查、间接检眼镜检查、眼压测量、视网膜电图和组织学评估大鼠和兔眼中的体内眼部特性。
HDP-P-Ara-C在7天内从溶解室（流速2 μL/min）中清除。相比之下，HDP-cP-Ara-C是一种更难溶的前药，在溶解过程开始36天后仍可检测到。HDP-P-Ara-C在人视网膜色素上皮细胞（ARPE-19）中的50%细胞毒性浓度为52±2.6 μM，在大鼠Müller细胞系rMC-1中的为32±2.2 μM。HDP-cP-Ara-C在ARPE-19和rMC-1细胞中的50%细胞毒性浓度值分别为50 μM和25 μM。在注射最高玻璃体内剂量（228 μg/大鼠眼）2周后未检测到HDP-P-Ara-C，且未发现眼部毒性。对于HDP-cP-Ara-C，单次玻璃体内注射（800 μg/兔眼）后药物储存库在26周内可见。对于这两种化合物而言，除了最高剂量的HDP-cP-Ara-C（800 μg/眼）外，视网膜电图、眼压和其他毒性研究均为阴性，该剂量因直接接触视网膜而具有局部毒性，并导致暗适应a波降低和闪烁视网膜电图振幅降低（广义估计方程，p=0.039和0.01）。
发现环单磷酸酯前药HDP-cP-Ara-C具有更适合将阿糖胞苷持续递送至眼后段的物理化学性质。这些性质包括有限的水溶性、体外抗增殖活性以及注射到兔眼后良好的耐受性。
