Foundation-Directed Therapeutic Development in Huntington’s Disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease that devastates patients and their families. It is caused by expansion of the CAG repeat in the huntingtin gene (<i>HTT</i>) and characterized pathologically by the loss of pyramidal neurons in several cortical areas, striatal medium spiny neurons, and hypothalamic neurons. Clinically, a distinguishing feature of the disease is uncontrolled involuntary movements (chorea) accompanied by progressive cognitive and psychiatric impairment. Currently there are no effective disease-modifying treatments for HD, although antidepressant and antipsychotic medications are typically utilized to manage HD symptoms, in addition to the only approved drug for the treatment of chorea in HD, tetrabenazine (TBZ). CHDI is a not-for-profit organization focused solely on HD. Herein we describe our foundation-directed therapeutic development efforts highlighting our collaborations and internal programs that are in various stages of development

Influence of D159687 on OR trial performance.

<p>Effects of D159687 on easy (open bars) versus difficult task (black bars). Dose-dependent improvement in (A) mean percent correct first reach on difficult task performance, with modest improvement on easy trial performance. Individual animal performance plot in easy trials (B) and in difficult trials (C). (D–E) Dose-dependent reduction in the total number of reaches (D) and barrier reaches (E) on difficult taks. (A, D, E) Values are listed as mean ± SEM (n = 8 for vehicle, low and mid-dose groups, n = 7 for high dose group). Asterisks denote significant differences from vehicle treatment (* p<0.05, **p<0.01 and ***p<0.001) following repeat measures one-way ANOVA and Dunnett's post-hoc analysis (n = 7, due to non-completer 7A5D).</p

Influence of D159797 on OR trial performance.

<p>Effects of D159797 on easy (open bars) versus difficult tasks (black bars). Dose-dependent improvement in (A) mean percent correct first reach on difficult task performance. Individual animal performance plot in easy trials (B) and in difficult trials (C). (D–E) Dose-dependent reduction in the total number of reaches (D) and barrier reaches (E) on difficult tasks. (A, D, E) Values are listed as mean ± SEM (n = 8 for vehicle and low dose, and n = 7 for mid and high dose group). Asterisks denote significant differences from vehicle treatment (* p<0.05, **p<0.01 and ***p<0.001) following repeat measures one-way ANOVA and Dunnett's post-hoc analysis (n = 7, due to non-completer 7A5D).</p

Object retrieval task schematic and baseline characterization.

<p>(A) Order of object retrieval task sessions for easy and difficult trials. Drawings illustrate the position of the reward in the boxes. (*) Left position will become right and right position will become left and so on in weekly rotation throughout the study. (#) The <b>bold</b> side of the cube represents the open side of the cube. (**) Trial 17 was for reward purposes and was not included in the data analysis; (B) Box (line indicates median, * indicates mean, box represents upper and lower 25 percentiles) and whisker (maximum to minimum) plots of all animal performance during the 4 training sessions and to vehicle administration during the testing phase on both easy (grey) and difficult (black) trials. Dashed lines indicate targeted performance – performance in easy trials >50% correct first reach, and performance in difficult trials <40% correct first reach. (C) Same data as in (B) but with high performer animal 3939 excluded. Criteria are largely met by exclusion of this animal. The outlier value during the rolipram vehicle trial is due to animal 7A5D.</p

Pharmacokinetic analysis of PDE4D NAMs in female Cynomolgous monkeys.

<p>(A–B) Plasma exposure of D159687 (A), or D159797 (B) in female Cynomolgus monkey plasma following a single intravenous administration at 1.0 mg/kg, and on day 1 and day 7 after repeated daily oral administration at 5.0 mg/kg.</p

Summary of plasma pharmacokinetic parameters of D159687 following single intravenous administration at 1.0/kg, and on day 1 and day 7 after repeated daily oral administration at 5.0 mg/kg.

<p>Data presented as mean ± SD of 3 animals.</p><p>*Calculated from n = 2, as due to patency issues in catheter of one animal following IV dosing, one animal was replaced for po dosing, thus, it was not a cross over design.</p

Summary of plasma pharmacokinetic parameters of D159797 following single intravenous administration at 1.0/kg, and on day 1 and day 7 after repeated daily oral administration at 5.0 mg/kg.

<p>Data presented as mean ± SD of 3 animals.</p

Quantifying autophagy using novel LC3B and p62 TR-FRET assays

<div><p>Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.</p></div

Evaluation of LC3B-II and p62 TR-FRET specificity.

<p>Genetic validation of the LC3B-II and p62 readouts was achieved by gene silencing of LC3B and p62 in HEK293T cells using shRNA. <i>LC3B</i> and <i>p62</i> mRNA were reduced after shRNA of each gene, compared to shRNA scramble, as verified by qRT-PCR (N = 3; avg±SD; Student’s t-test (unpaired; two-tailed); *p<0.001), expression levels were calculated using the 2^-ΔΔCT method and expressed relative to scramble control (A). A corresponding reduction of protein levels was observed by western blot (B) and TR-FRET (N = 4; avg±SD; Student’s t-test (unpaired; two-tailed); *p<0.005; expression relative to scramble control; C). ATG4B overexpression in HEK293T cells was confirmed with western blot (D). ATG4B overexpression did not alter LC3B mRNA levels as seen by qRT-PCR (N = 3, avg±SD; Student’s t-test (unpaired, two-tailed) p>0.05, expression levels were calculated using the 2^-ΔΔCT method and expressed relative to scramble control (E) but clearly reduced LC3B-II detection, as measured by TR-FRET (expressed relative to empty vector; N = 3, unpaired t-test, *p<0.001 (F) and western blot (D). LC3B-II quantification by TR-FRET (fluorescence ratio of 665/615 nm) in HEK293 cells showed detection with as few as 2000 cells/well (N = 2; avg±SD; G). Different concentrations of purified p62 were measured and the p62 TR-FRET assay is sensitive enough to detect 1ng/ml purified recombinant p62 protein (signal expressed as fluorescence ratio of 665/615 nm; N = 2; avg±SEM; H).</p

LC3B-II and p62 quantification in response to tool compounds treatment.

<p>HEK293T cells (A), rat cortico-striatal neurons (B) and rat astrocytes (C) were treated with a serially diluted autophagy inhibitor (bafilomycin A1) or upregulator (KU0063794) and examined at 2, 6 and 24 hours post-treatment. LC3B-II and p62 were measured with TR-FRET (A-C). The response to compound treatment is reported as percentage average of three replicates with respect to vehicle (DMSO) treated samples (100%). Cell viability was evaluated by H33342 stained nuclei count for HEK293T (A) and astrocytes (C-E) and by neurite length/soma (morphometric readout) on MAP2 stained neurons (B). Astrocytes were also treated with SU11652 and NVP-TAE684 and LC3B-II, p62 and viability were measured with TR-FRET (D-E). Each data point is the mean±SEM (N = 3).</p