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. 2013;2(4):443-51.
doi: 10.3233/JHD-130074.

An independent study of the preclinical efficacy of C2-8 in the R6/2 transgenic mouse model of Huntington's disease

Nan Wang  1 , Xiao-Hong Lu  1 , Susana V Sandoval  1 , X William Yang  1
Affiliations

An independent study of the preclinical efficacy of C2-8 in the R6/2 transgenic mouse model of Huntington's disease

Nan Wang et al. J Huntingtons Dis. 2013.

Abstract

Background: C2-8 is a small molecule inhibitor of polyglutamine aggregation and can reduce photoreceptor neurodegeneration in a Drosophila model of Huntington's disease (HD). Further preclinical studies have shown that oral administration of C2-8 in R6/2 HD transgenic mice can penetrate into the brain, reduce mHTT-exon1 aggregation, improve motor performance and diminish striatal neuron atrophy.

Objective: In this independent preclinical study, we aimed to evaluate the pharmacokinetic properties and therapeutic efficacy of C2-8 intraperitoneal (IP) delivery in the R6/2 HD mouse.

Methods: R6/2 mice were IP injected with low dose C2-8 (10 mg/kg), high dose C2-8 (20 mg/kg), or vehicle twice daily from 3 weeks to 3 months old. Longitudinal behavioral tests (accelerating Rotarod and wire-hang) were performed to evaluate the motor deficits, and neuropathology was measured by unbiased stereology.

Results: We confirmed that the compound has good blood-brain-barrier penetration after acute or sub-chronic intraperitoneal delivery. Chronic treatment with C2-8 in R6/2 mice results in a significant reduction of nuclear mHTT aggregate volume in the brains, replicating a key finding of C2-8 as a polyglutamine aggregation inhibitor in vivo. However, by comparing HD mice with C2-8 treatment to those with vehicle treatment, we were unable to demonstrate significant amelioration of motor deficits using Rotarod and wire-hang tests. Moreover, we did not observe improvement in the striatal neurodegenerative pathology, as measured by brain weight, striatal volume, and striatal neuron volume in the C2-8 treated R6/2 mice.

Conclusions: Our study supports the practice of independent preclinical studies for novel molecules in HD therapeutic development and suggests that the use of alternative delivery strategies and full-length HD mouse models are likely needed to further assess whether the aggregate-inhibiting properties of C2-8 can be consistently translated into a preclinical benefit in HD mice.

Keywords: C2-8; Huntington's disease; R6/2; aggregate; huntingtin; preclinical.

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Conflict of interest statement

Conflict of Interest: The authors have no conflict of interest to report.

Figures

Fig. 1
Fig. 1
C2-8 has good brain bioavailability. (A) Plasma and brain levels of C2-8 in wild-type mice after single bolus IP injection administration of 500 mg/kg C2-8 were plotted. Levels were determined 3, 6, 12 and 24 h after dosing. N = 3 per time point. (B) Plasma and brain levels of C2-8 in wild-type mice with 5-day I.P. injection of 20 mg/kg C2-8 b.i.d. at 1, 4 and 8 hours after the last dose. N = 3 per time point. The lines connect the means values for each time point.
Fig. 2
Fig. 2
C2-8 treatment does not show a significant rescue effect on behavioral deficits in R6/2 mice. (A) Rotarod performance of C2-8-treated and vehicle-treated R6/2 mice at 5, 8, and 11 weeks of age. C2-8-treated mice performed similar to vehicle controls on the Rotarod at all three time points (drug effect: F(2,70) = 0.29, P value = 0.7483). (B) Wire-hang endurance of R6/2 mice showed no improvement at 9.5 and 12.5 weeks of age with C2-8 treatment (F(2,35) = 0.14, P value = 0.8738). Values represent mean ± S.E.M. Vehicle: n = 11; C2-8 10 m/kg: n = 14; C2-8 20 mg/kg: n = 13. Data were analyzed with repeated measure two-way ANOVA.
Fig. 3
Fig. 3
C2-8 treatment has no effect on forebrain or cerebellum weight in R6/2 mice. Forebrain: F(2,35) = 0.86, P value = 0.4308; cerebellum: F(2,35) = 1.37, P value = 0.2660. Values represent mean ± S.E.M. Vehicle: n = 11; C2-8 10m/kg: n = 14; C2-8 20 mg/kg: n = 13. Data were analyzed with one-way ANOVA.
Fig. 4
Fig. 4
C2-8 treatment does not affect R6/2 mouse striatal volume. All groups of R6/2 mice have similar striatal volumes (F(2,27) = 1.559, P value = 0.2287), which were all significantly lower than wild-type mice (***P < 0.001). Values represent mean ± S.E.M, n =10 per group. Data were analyzed with one-way ANOVA followed by Tukey post-test.
Fig. 5
Fig. 5
C2-8 treatment does not affect R6/2 mouse striatal neuron volume. All groups of R6/2 mice have similar striatal neuronal volume (F(2,27) = 0.4569, P value = 0.6381). Values represent mean ± S.E.M, n = 10 per group. Data were analyzed with one-way ANOVA followed by Tukey post-test.
Fig. 6
Fig. 6
C2-8 treatment leads to significant reduction of mutant huntingtin aggregate volume in R6/2 mouse striatum. (A) Representative immunodetection of intraneuronal mutant huntingtin aggregates using EM48 antibody in the neostriatum of 13-week-old R6/2 mice with vehicle or 20 mg/kg C2-8 treatment (scale bar = 25 μm). (B) C2-8 treatment significantly reduced the striatal intranuclear aggregate volume in R6/2 mice (F(2,27) = 9.339, = 0.0008). Values represent mean ± S.E.M., n = 10; **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey post-test.
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