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

Brooks, Brian

Abstract

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.