A small-molecule therapeutic lead for Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse

Abstract

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine expansion within huntingtin protein. The exact pathological mechanisms determining disease onset and progression remain unclear. However, aggregates of insoluble mutant huntingtin (mhtt), a hallmark of HD, are readily detected within neurons in HD brain. Although aggregated polyglutamines may not be inherently toxic, they constitute a biomarker for mutant huntingtin useful for developing therapeutics. We previously reported that the small molecule, C2-8, inhibits polyglutamine aggregation in cell culture and brain slices and rescues degeneration of photoreceptors in a Drosophila model of HD. In this study, we assessed the therapeutic potential of C2-8 in the R6/2 mouse model of HD, which has been used to provide proof-of-concept data in considering whether to advance therapies to human HD. We show that, at nontoxic doses, C2-8 penetrates the blood-brain barrier and is present in brain at a high concentration. C2-8-treated mice showed improved motor performance and reduced neuronal atrophy and had smaller huntingtin aggregates. There have been no prior drug-like, non-toxic, brain-penetrable aggregation inhibitors to arise from cell-based high-throughput screens for reducing huntingtin aggregation that is efficacious in preclinical in vivo models. C2-8 provides an essential tool to help elucidate mechanisms of neurodegeneration in HD and a therapeutic lead for further optimization and development.

Fig. 1.

C2-8 has brain bioavailability at nontoxic doses. (A) Gradient chromatography coupled with 16-channel colorimetric array detection enabled the detection of C2-8 in mouse brain. (Left) Chromatogram showing C2-8 standard (0.8 μg/ml). The peaks were detected in channels 12–15 at ≈90-min retention time. The analytical values were determined from total integrated values of peaks from channel 12–15. (Right) Chromatogram shows brain levels of C2-8 in wild-type mice receiving 50 mg/kg C2-8 p.o. for 5 days. Five hours after the last dose, levels of C2-8 were determined in cortex based on comparison with standards and found to be 25 μM. (B) Brain and plasma levels of C2-8 in wild-type mice after single p.o. administration of 5 (filled circle), 10 (open circle), 25 (filled triangle), and 50 (open triangle) mg/kg C2-8. Levels were determined 1, 4, and 8 h after dosing. n = 5. Error bars represent SD.

Fig. 2.

C2-8 treatment ameliorates behavioral deficits in R6/2 mice. (A) Rotarod performance of C2-8-treated and placebo-treated R6/2 mice at 5, 8, and 11 weeks of age. C2-8-treated mice performed better than vehicle controls on the rotarod at 8 and 11 weeks of age. (B) Wire-hang endurance of R6/2 mice is significantly improved at 9.5 and 12.5 weeks of age with C2-8 treatment. Black, gray, and white bars represent R6/2 mice treated with 0, 100, or 200 mg/kg/12 h C2-8, respectively. Values represent geometric mean ± 95% confidence interval. n = 15; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

C2-8 increases striatal neuronal cell body volume in R6/2 mice. (A and B) C2-8 treatment has no effect on brain weight and striatal volume in R6/2 mice. (C) Thionin-stained sections of neostriatum from a wild-type littermate mouse (Right), a placebo-treated R6/2 mouse (Center), and a C2-8-treated R6/2 mouse (Left) at 13 weeks of age. There is evidence of neuronal atrophy in the untreated R6/2 mouse with relative preservation of neuronal atrophy in the C2-8-treated mouse. (Scale bars, 25 μm.) (D) Stereological quantification of striatal neuronal cell body volume. Neuronal volume is significantly reduced in R6/2 mice as compared with wild-type littermates. C2-8 treatment partially rescues the loss of striatal neuronal cell body volume in a dose-dependent manner. Error bars represent SEM, n = 10; *, P < 0.05.

Fig. 4.

C2-8 decreases size of neuronal intranuclear huntingtin aggregates. (A) Immunodetection of intraneuronal huntingtin aggregates by using EM48 antibody in the neostriatum of 13-week-old R6/2 mice with and without C2-8 treatment. (Scale bars, 25 μm.) (B) Stereological counts of striatal intraneuronal aggregates in R6/2 mice. C2-8 treatment had no effect on aggregate density. (C) C2-8 treatment significantly reduced the striatal nuclear aggregate volume in R6/2 mice. Error bars indicate SEM, n = 10; ***, P < 0.001.