Specific Aim #1: To test the effect of clinically-relevant doses of NTBC in a mouse model of oculocutaneous albinism, type 1a, (C57BL/6J-Tyr c-2J/c-2J ) using predefined ocular, systemic and biochemical outcome variables. C57BL6/J-Tyr c-2J/c-2J mice are phenotypically albino due to a G291T (Arg77Leu) mutation in the Tyr gene that is functional null at the protein level. These mice have a white coat color and pink eyes, and lack any significant fundus pigmentation. As such, they are a reasonable model for OCA1a. Although these mice are completely albino, the mutation in tyrosinase is a missense mutation. This leaves open the possibility that elevated tyrosine may stabilize the enzyme and improve flux through pigment production pathways. To minimize the effect of additional genetic factors on phenotype, both lines of mice used in these experiments are on the same, inbred backgroundC57BL6/J. Baseline plasma tyrosine, coat color (gross and microscopic), anterior segment pigment, and posterior segment pigment will be documented for each mouse prior to initiating experiments. We are proposing to treat ten C57BL6/J-Tyr c-2J/c-2J mice, age 3-4 months, with 100g NTBC in a volume of 0.2-0.3mL every other day via oral gavage for a four week period. These will be compared to an age- and gender-matched cohort of C57BL6/J-Tyr c-2J/c-2J mice over the same time period. The efficacy and safety of NTBC at this dose have been demonstrated in other murine models. A possible side effect of NTBC treatment is corneal irritation, which will be monitored for daily. At the start of treatment, an area of hair will be plucked from each mouses back;this will stimulate new hair growth and possibly new pigment deposition in the hair shaft. At the end of four weeks time, plasma tyrosine concentrations will be assessed in treated and control animals. Our initial experiments revealed that the initial dose of NTBC raised plasma tyrosine levels to approximately two-fold of normal. Because previous work has indicated that closer to millimolar concentrations of plasma tyrosine are necessary for pigmentation changes, we obtained approval to increase the dose three-fold--which is still within safe parameters for use in humans on a per killogram basis. This increase resulted in plasma tyrosine levels at approximately 600 micromolar at 2 weeks treatment. We have completed the thirty days of treatment in the Tyr (c-2J/c-2J) mice and did not observe any overt change in eye or coat color. We have performed photodocumentation and are processing samples for analysis of melanosome number and size in the RPE and choroid. There were no adverse events on the mice during the course of this trial.
Specific Aim #2 : To test the effect of clinically-relevant doses of NTBC in a mouse model of oculocutaneous albinism, type 1b, (C57BL/6J-Tyr c-h/c-h, carrying a temperature-sensitive mutation in tyrsosinase) using predefined ocular, systemic and biochemical outcome variables. The Himalayan, temperature sensitive allele of tyrosinase, Tyrc-h, (MGI Accession ID# 72456) spontaneously arose in 1958 and has since been inbred into the C57BL/6 background. The maximum activity of the tyrosinase produced from this allele occurs at temperatures below normal body temperature (37C) because the mutant protein (c.A1259G, p.H420R) is heat labile. In homozygotes, the first coat is a uniform light tan. At the first molt, the body hair becomes lighter and the ears, nose, tail, and scrotum become dark as in Siamese cats. Eyes are slightly pigmented and appear red. Mice will be housed in standard conditions, at room temperature. Because the Himalayan allele retains some residual enzymatic activity, we feel that these mice are a reasonable model of OCA1b. We plan to carry out an identical set of experiments on mice homozygous for the c-h allele of tyrosinase, as detailed in Specific Aim #1. Ten mice will be treated and ten will be controls. These experiments are currently in progress. Given our experience with the Tyr (c-2J/c-2J) mice, we started this portion of the pre-clinical trial at the higher dose of NTBC. We are currently assaying interim plasma tyrosine levels to assess if this is the appropriate dose. To date, one mouse in the experimental group has died unexpectantly and is currently in autopsy. Note that, in the event that we do not see an effect on pigmentation using the parameters set above, we intend to modify the animal protocol to include longer treatment regiments, a still higher dose of drug, and/or pre-natal treatment of animals.

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