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

1.烟替松(NTBC)治疗人OCA1B的临床方案 我们之前发表的工作已经证实，烟替松(NTBC)可以增加OCA1B小鼠模型中的黑色素沉着。我们对五名患有OCA-1B的成人患者进行了一项开放标签的先导性研究(Adams D.R.等人，JCI Insight。2019年)。在建立了虹膜、皮肤和头发色素沉着的基线测量后，患者接受了超过12个月的口服NTBC治疗，每天2 mg。每隔3个月评估一次色素沉着和视功能的变化。虹膜黑色素在NTBC治疗后保持不变，然而，一些患有OCA-1B的患者的头发和皮肤色素沉着似乎有所增加。这项研究中使用的虹膜透光分级标准被证明是可靠的，有可能在未来的临床试验中使用。 2.NTBC对OCA2、OCA3、OCA4小鼠模型的影响 口服NTBC治疗OCA3小鼠模型不会对黑色素的产生产生有利影响，并且对虹膜基质中有色黑素小体的数量影响最小(Onojafe I.F.等人，Invest Ophthalol Vis Sci)。2019年)。我们的结论是，对患有NTBC的OCA3患者的治疗不太可能是有疗效的。 我们进一步假设，NTBC可能会改善OCA2(黑素细胞特异性转运蛋白)和OCA4(SLC45A2的“白下”等位基因)小鼠模型的黑化。与OCA3小鼠相似，NTBC处理的OCA2和OCA4小鼠在尼替松治疗一个月后血浆酪氨酸浓度升高，没有明显的毒副作用。与OCA3小鼠不同，两个品系的小鼠皮毛色素沉着都增强了。虹膜色素沉着主要在OCA4小鼠身上增强。电子显微镜还证实，只有OCA4治疗的小鼠脉络膜色素沉着略有增加，而在两个品系的RPE中都没有观察到影响。在一篇准备中的手稿中，我们建议NTBC治疗可能对部分恢复OCA4的色素沉积有利，但对OCA2无效。 3.高通量药物筛选，以确定调节Tyr活性的化合物 我们在高通量药物筛选中使用了纯化的、截短的Tyr蛋白(之前经过测试和验证具有与全长蛋白相同的酶活性)。与NCATS合作，我们从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。我们确定了&gt；100种新的酪氨酸酶抑制剂和一些激活剂。在体外和体内的二次酶学筛选中验证之后，我们正在OCA1B小鼠模型上测试两种剂量的首选候选化合物。在测试的最低剂量下，该药物在ip时耐受性良好。30天。 4.OCA体外病盘模型的建立 我们利用诱导多能干细胞(IPSC)技术建立了1A型眼皮肤白化病(OCA1A)和2型眼皮肤白化病(OCA2)的盘中病模型。OCA患者的成纤维细胞被重新编程为IPSCs，并使用发育指导的分化方案向视网膜色素上皮(OCA-RPE)分化。组织形态和生理学以及视觉周期装置的表达和功能与对照着色IPSC来源的RPE相似。此外，体外培养的OCA-RPE细胞中含有未成熟黑素小体的色素沉着缺陷忠实地复制了体内的组织特征。

1. Clinical protocol for studying the effect of nitisinone (NTBC) treatment in human subjects with OCA1B Our previously-published work had established that nitisinone (NTBC) can increase melanin pigmentation in a mouse model of OCA1B. We performed an open-label pilot study with five adult patients with OCA-1B (Adams D.R., et al., JCI Insight. 2019). After establishing baseline measurements of iris, skin, and hair pigmentation, the patients were treated over 12 months with 2 mg/d oral NTBC. Changes in pigmentation and visual function were evaluated at 3-month intervals. Iris melanin remained unchanged after NTBC treatment, however, hair and skin pigmentation appeared to have increased in some patients with OCA-1B. The iris transillumination grading scale used in this study proved robust, with potential for use in future clinical trials. 2. Effect of NTBC in mouse models of OCA 2, OCA3 and OCA4 Treatment of a mouse model of OCA3 with oral NTBC does not have a favorable effect on melanin production and minimally affects the number of pigmented melanosomes in the iris stroma (Onojafe I.F., et al., Invest Ophthalmol Vis Sci. 2019). We conclude that treatment of OCA3 patients with NTBC is unlikely to be therapeutic. We further hypothesized that NTBC might improve melanization in mouse models of OCA2 (melanocyte-specific transporter protein) and of OCA4 (the so-called "underwhite" allele of SLC45A2). Similar to OCA3 mice, plasma tyrosine concentration was increased in NTBC-treated OCA2 and OCA4 mice after a month of nitisinone treatment with no overt toxic side-effects. Unlike the OCA3 mice, fur pigmentation was augmented in both mouse lines. Iris pigmentation was augmented predominantly in OCA4 mice. Electron microscopy also confirmed a small increase in choroidal pigmentation only in treated OCA4 mice, whereas, no effect was observed in the RPE in both lines. In a manuscript in preparation, we suggest that NTBC treatment might be beneficial in partially restoring pigmentation in OCA4 but not OCA2. 3. High-throughput drug screening to identify compounds that regulate Tyr activity We used purified, truncated Tyr protein (previously tested and validated to have equivalent enzymatic activity to full length protein) in a high-throughput drug screening. In collaboration with NCATS, we screened 34,000 compounds from the Genesis Drug Collection, the Natural Products Library, and the NCATS Pharmaceutical Collection. We identified >100 new inhibitors and a few activators of tyrosinase. After validation in a secondary enzymatic screen in vitro and in zebrafish in vivo, we are testing two doses of the top candidate compound on the OCA1B mouse model. At the lowest dose tested, the drug was well tolerated when administered i.p. for 30 days. 4. In vitro disease-in-a-dish modeling of OCA We developed a disease-in-a-dish model of oculocutaneous albinism type 1A (OCA1A) and type 2 (OCA2) using induced pluripotent stem cell (iPSC) technology. OCA patient fibroblasts were reprogrammed to iPSCs and differentiated to retinal pigment epithelium (OCA-RPE) using a developmentally-guided differentiation protocol. Tissue morphology and physiology as well as expression and functionality of the visual cycle apparatus were comparable to control pigmented iPSC-derived RPE. Furthermore, pigmentation defects comprising immature melanosomes in the OCA-RPE cells in vitro faithfully replicated tissue characteristics in vivo.