Pre-clinical and clinical studies of NTBC and other compounds as potential treatments for albinism

我们之前发表的工作已经证实，尼替松可以增加OCA1B小鼠模型中的黑色素沉着，但不能增加OCA1A的黑色素沉着。自上次报告以来，我们侧重于以下次级项目： 1.经NTBC处理的OCA1B小鼠与对照组比较的视觉功能 基于OCA1B成年小鼠和幼鼠在宫内处理后色素沉着增加的成功，我们询问是否可以检测到断奶后后者的眼睛形态、细胞和功能的变化。通过与NEI视觉功能核心中心和神经生物学、神经退化与修复实验室的合作，我们研究了产前尼替松治疗对神经视网膜发育、神经回路发育和空间视力的影响。一小群小鼠的初步数据表明，用尼替松产前治疗OCA1B小鼠后，受试小鼠的视网膜电信号(ERG)和直接耦合(DC)-ERG反应发生了可测量的变化，恢复到正常值。然而，没有观察到光感受器细胞数量、副视路路线以及通过视动和视动反应测量的视觉行为的变化。虽然OCA1B小鼠的光感受器功能似乎被保留了下来，但行为测量似乎表明这些小鼠是盲人的。我们正在对OCA1B小鼠以及保留了虹膜色素沉着而不是RPE色素沉着的小鼠品系进行进一步的研究，以测试虹膜色素沉着是解释OCA1B小鼠视觉运动和视动力反应的混杂因素的假说。 2.烟替酮对OCA3小鼠模型的影响 我们推测，烟替松可能会改善OCA3小鼠模型(Tyrp1棕色小鼠)的黑化，因为已知酪氨酸酶(Tyr)和Tyrp1在黑素体膜上相互作用并稳定。从杰克逊实验室(C57BL/6J-Tyrp1b-J/J)获得的小鼠是我们表征的Tyrp1新突变的纯合子。他们对尼替松治疗的反应是血浆酪氨酸浓度增加，没有明显的毒性。然而，除了虹膜外，没有发现毛皮或有色眼部结构的颜色变化，这表明尼替松治疗OCA3患者不太可能是有疗效的。目前正在修订一份描述这些数据的手稿，以便重新提交。 3.烟替松对OCA4小鼠模型的影响 我们推测尼替松可能会改善OCA4(SLC45A2的“白底”等位基因)小鼠模型中的黑化。来自杰克逊实验室的小鼠，尽管它们都有典型的白下等位基因，但当培育成纯合子时，它们表现出两种截然不同的毛色(“浅”和“深”)。与OCA3小鼠相似，尼替松治疗的OCA4小鼠血浆酪氨酸浓度升高，没有明显的毒副作用。与OCA3小鼠不同，OCA4小鼠的毛皮色素沉着和虹膜色素沉着都有所增加。电子显微镜还证实，治疗组小鼠脉络膜色素沉着略有增加，而对RPE没有影响。我们目前正在使用高通量测序来识别第二个修饰等位基因。 4.外用尼替松和沙拉坦治疗眼部色素沉着 与全身给药相反，我们发现在所测试的方案下，局部滴眼液尼替松或沙拉坦不能改善OCA1B小鼠的黑色素沉着。 5.高通量药物筛选，寻找调节Tyr活性的化合物 在之前发表的工作中，我们已经纯化了重组野生型和突变型人Tyr，并建立了一种测定Tyr活性的荧光分析方法，作为筛选抑制剂和增强剂的读数。此外，我们最近证明了全长和截短的Tyr具有类似的酶活性(Dolinska等人，PCMR，2017)，从而验证了截短的蛋白质在我们的高通量药物筛选中的使用。与NCATS合作，我们成功地从Genesis药物收藏、天然产品图书馆和NCATS药物收藏中筛选出34,000种化合物。我们发现了新的酪氨酸酶抑制剂(&gt；100)和几种酪氨酸酶激活剂。这些激活剂正在更详细的二级酶体外筛选以及斑马鱼等体内模型中得到验证。 6.尼替西酮治疗OCA1B患者的临床方案 我们已经建立了IRB批准的方案，用于测试标准剂量的尼替松对OCA1B患者眼部和全身黑色素沉着的影响。5例患者接受了尼替西酮治疗。与小鼠相似，服用尼替松会增加所有受试者的酪氨酸血浆浓度。成年人的视觉敏锐度并没有像在成熟的视觉系统中预期的那样，通过尼替西酮治疗而改变。目前正在分析虹膜透光、头发和皮肤色素沉着的变化。

Our previously-published work has established that nitisinone can increase melanin pigmentation in a mouse model of OCA1B, but not OCA1A. Since the time of the last report, we have focused on the following sub-projects: 1. Visual function in NTBC-treated OCA1B mice vs. controls Based on the success with increasing pigmentation in OCA1B adult mice and in pups treated in utero, we asked whether we could detect eye morphological, cellular and functional changes in the latter after weaning. Through collaborations with the NEI Visual Function Core and the Neurobiology, Neurodegeneration & Repair Laboratory, we have studied the effect of prenatal nitisinone treatment on neural retina development, neural circuit development and spatial visual acuity. Preliminary data on a small cohort of mice indicated that prenatal treatment of OCA1B mice with nitisinone results in measurable changes in electroretinogram (ERG) and direct coupled (dc)-ERG responses that returned within normal values in the treated mice. However, no change in photoreceptor cell number, accessory optic tract routing, nor visual behavior measured by optokinetic and optomotor response was observed. While photoreceptor function seems to be preserved in OCA1B mice, behavioral measures seem to indicate that the mice are blind. We are pursuing further studies in the OCA1B mice as well as in mouse strains in which pigmentation of the iris is preserved but not pigmentation of the RPE to test the hypothesis that iris pigmentation is a confounding factor in the interpretation of optomotor and optokinetic responses in OCA1B mice. 2. Effect of nitisinone on a mouse model of OCA3 We hypothesized that nitisinone might improve melanization in a mouse model of OCA3 (the Tyrp1-brown mouse), as tyrosinase (Tyr) and Tyrp1 are known to interact and stabilize one another in the melanosome membrane. The mice received from Jackson Labs (C57BL/6J-Tyrp1b-J/J) are homozygous for a novel mutation in Tyrp1 that we have characterized. They respond to nitisinone treatment with increased plasma tyrosine concentrations and no overt toxicity. However, no change in the color of fur or pigmented ocular structures could be discerned with the exception of the iris, suggesting that treatment of OCA3 patients with nitisinone is unlikely to be therapeutic. A manuscript describing these data is currently being revised for resubmission. 3. Effect of nitisinone on a mouse model of OCA4 We hypothesized that nitisinone might improve melanization in a mouse model of OCA4 (the so-called "underwhite" allele of SLC45A2). The mice received from Jackson Lab, although they all harbor the classic underwhite allele, demonstrated two distinct coat colors ("light" and "dark") when bred to homozygosity. Similar to the OCA3 mice, plasma tyrosine concentration was increased in nitisinone-treated OCA4 mice with no overt toxic side-effects. Unlike the OCA3 mice, fur pigmentation was augmented in OCA4 mice as well as iris pigmentation. Electron microscopy also confirmed a small increase in pigmentation of the choroid in treated mice whereas, no effect was observed on the RPE. We are currently using high-throughput sequencing to identify the second, modifying allele. 4. Use of topical nitisinone and Xalatan in ocular pigmentation Contrary to systemic administration, we found that topical eye drops of nitisinone or Xalatan could not improve melanin pigmentation in the OCA1B mouse under the regimen tested. 5. High-throughput drug screening to find compounds that regulate Tyr activity In previous published work, we had purified recombinant wild-type and mutant human Tyr and established a fluorometric assay for measuring Tyr activity as a read-out for screening inhibitors and enhancers. In addition, we have recently demonstrated that full-length and truncated Tyr have similar enzymatic activities (Dolinska et al, PCMR, 2017), thus validating the use of the truncated protein in our high-throughput drug screening. In collaboration with NCATS, we successfully screened 34,000 compounds from the Genesis Drug Collection, the Natural Products Library, and the NCATS Pharmaceutical Collection. We identified new inhibitors (>100) and several activators of tyrosinase. These activators are being validated in a more detailed, secondary enzymatic screen in vitro, as well as in in vivo models such as zebrafish. 6. Clinical Protocol for Studying the Effect of Nitisinone Treatment in Human Subjects with OCA1B We have established an IRB-approved protocol for the testing of a standard oral dose of nitisinone on ocular and systemic melanin pigmentation in patients with OCA1B. Five patients were treated with nitisinone. Similar to the mouse, administration of nitisinone increased tyrosine plasma concentration in all subjects. Visual acuity was not altered by nitisinone treatment in adults, as expected in a mature visual system. Changes in iris transillumination, hair and skin pigmentation are now being analyzed.