G Westerberg

A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntingtons disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD.

An exploratory double‐blind, randomized clinical trial with selisistat, a SirT1 inhibitor, in patients with Huntington's disease

AIMS Selisistat, a selective SirT1 inhibitor is being developed as a potentially disease-modifying therapeutic for Huntingtons disease (HD). This was the first study of selisistat in HD patients and was primarily aimed at development of pharmacodynamic biomarkers. METHODS This was a randomized, double-blind, placebo-controlled, multicentre exploratory study. Fifty-five male and female patients in early stage HD were randomized to receive 10 mg or 100 mg of selisistat or placebo once daily for 14 days. Blood sampling, clinical and safety assessments were conducted throughout the study. Candidate pharmacodynamic markers included circulating soluble huntingtin and innate immune markers. RESULTS Selisistat was found to be safe and well tolerated, and systemic exposure parameters showed that the average steady-state plasma concentration achieved at the 10 mg dose level (125 nm) was comparable with the IC50 for SirT1 inhibition. No adverse effects on motor, cognitive or functional readouts were recorded. While circulating levels of soluble huntingtin were not affected by selisistat in this study, the biological samples collected have allowed development of assay technology for use in future studies. No effects on innate immune markers were seen. CONCLUSIONS Selisistat was found to be safe and well tolerated in early stage HD patients at plasma concentrations within the anticipated therapeutic concentration range.

Safety, pharmacokinetics, pharmacogenomics and QT concentration−effect modelling of the SirT1 inhibitor selisistat in healthy volunteers

AIM Selisistat (SEN0014196), a first-in-class SirT1 inhibitor, is being developed as a disease-modifying therapy for Huntingtons disease. This first-in-human study investigated the safety, pharmacokinetics and pharmacogenomics of single and multiple doses of selisistat in healthy male and female subjects. METHOD In this double-blind, randomized, placebo-controlled study, seven cohorts of eight subjects received a single dose of selisistat at dose levels of 5, 25, 75, 150, 300 and 600 mg and four cohorts of eight subjects were administered 100, 200 and 300 mg once daily for 7 days. Blood sampling and safety assessments were conducted throughout the study. RESULTS Selisistat was rapidly absorbed and systemic exposure increased in proportion to dose in the 5-300 mg range. Steady-state plasma concentrations were achieved within 4 days of repeated dosing. The incidence of drug related adverse events showed no correlation with dose level or number of doses received and was comparable with the placebo group. No serious adverse events were reported and no subjects were withdrawn due to adverse events. There were no trends in clinical laboratory parameters or vital signs. No trends in heart rate or ECG parameters, including the QTc interval and T-wave morphology, were observed. There were no findings in physical or neurological examinations or postural control. Transcriptional alteration was observed in peripheral blood. CONCLUSION Selisistat was safe and well tolerated by healthy male and female subjects after single doses up to 600 mg and multiple doses up to 300 mg day(-1).

Q02 A randomised, double-blind, placebo-controlled phase IB pharmacodynamic study with selisistat (SEN0014196) in HD patients

Background Siena Biotech SpA is developing selisistat (SEN0014196) as a potentially disease-modifying therapy for HD. Selsistat is a potent and selective SirT1 inhibitor (IC50 98 nM) that has shown benefit across a range of preclinical models for HD, from cells and neurons transfected with mutant huntingtin to transgenic Drosophila and R6/2 mice. The compound has shown to be safe and well tolerated in healthy volunteers and has a favourable pharmacokinetic profile. Aim The current study was designed to provide biophase samples for analysis of a series of potential target engagement and disease-modification read-outs, helping to establish a proof-of-principle and aid in dose selection for future safety and efficacy studies. Methods A total of 63 HD patients with a wide range of CAG repeats and disease burden scores were screened across six sites in Germany (Bochum, Ulm), Poland (Krakow, Warsaw) and the UK (London, Manchester). Patients were randomised to receive either 10 or 100 mg of selisistat or placebo for 2 weeks. Safety data collected included vital signs, ECGs, clinical laboratory parameters and type and frequency of adverse events. Clinical assessments included UHDRS and a cognitive battery. Serial blood sampling for pharmacokinetics and pharmacodynamics was performed on days 1 and 14 and at follow-up after a washout phase of 14 days. Results and Conclusions A total of 55 patients completed the treatment as per protocol; there were four screening failures and four patients withdrew consent. No Serious Adverse Events were reported and no patient withdrew from the study as a result of an adverse event. We will present full data on safety, tolerability, pharmacokinetics and clinical assessments, while pharmacodynamic data will be reported elsewhere.

F02 Selisistat: soluble HTT protein levels as a potential pharmacodynamic readout

Background Siena Biotech SpA is developing selisistat (SEN0014196) as a potentially disease-modifying therapy for HD. Selsistat is a potent and selective SirT1 inhibitor (IC50 98 nM) that has shown benefit across a range of preclinical models for HD, from cells and neurons infected with mutant huntingtin to transgenic Drosophila and R6/2 mice. The compound has shown to be safe and well tolerated in healthy volunteers and with a favourable pharmacokinetic profile. Aim To develop a sensitive and quantitative (ELISA-based) readout for the detection of total soluble HTT protein in biological matrices, and to investigate the effects of selisistat on HTT protein levels in Peripheral Blood Mononuclear Cells (PBMC) samples deriving from a Phase 1B study in HD patients. Methods This assay is based on a sandwich ELISA format and uses an electrochemiluminescent read-out to quantify the HTT protein level in PBMC extracts. The PBMCs are lysed by sonication and extracted by centrifugation. The supernatant is applied to a streptavidin-coated MSD plate which has been previously treated by addition of a biotinylated anti-mouse antibody followed by a mouse monoclonal anti HTT (1844–2131 aa) antibody. After incubation, bound HTT is detected using a rabbit anti-HTT (513–590 aa) secondary antibody followed by a Sulfotag conjugated anti-rabbit detection antibody. The signal is quantified by adding MSD read buffer and reading using the Meso Scale Discovery Sector Imager. Results and Conclusions The effects of two dose levels of selisistat and placebo on total soluble HTT levels in PBMC samples from HD patients in a 14-day Phase1B study will be presented.

F04 Selisistat: potential pharmacodynamic readouts based on transcriptomics

Background Siena Biotech SpA is developing selisistat (SEN0014196) as a potentially disease-modifying therapy for HD. Selisistat is a potent and selective SirT1 inhibitor (IC50 98 nM) that has shown benefit across a range of preclinical models for HD, from cells and neurons transfected with mutant huntingtin to transgenic Drosophila and R6/2 mice. The compound has shown to be safe and well tolerated in healthy volunteers and with a favourable pharmacokinetic profile. Transcriptional modulation mediated by SirT1 through deacetylation of transcriptional factors should be modulated by selisistat, thus generating a compound-specific gene expression profile. Aim To investigate the gene expression modulation of selisistat in whole blood derived from HD patients in a Phase 1B study and employ these transcriptional effects as a candidate pharmacodynamic readout for the compound. Methods By crossing results obtained from a microarray study performed on blood samples of healthy volunteers treated with a single dose of selisistat (Phase 1) and from investigation of different literature hypotheses, a candidate transcriptional signature for the compound, comprised of 10 genes, has been identified and validated by real-time PCR (RT-qPCR) in blood samples of healthy subjects treated with multiple doses of selisistat during Phase 1. This candidate signature has been investigated by RT-qPCR in total RNA isolated from whole blood derived from HD patients treated with selisistat or placebo and collected at different time points during the Phase Ib study. Results and Conclusions The effects of selisistat on gene expression from blood samples from a 14-day Phase1b study in HD patients at two dose levels of selisistat and placebo will be presented.

Q01 SIRT 1 mediated modulation of circulating cytokines in huntington's disease- pharmacodynamics results from phase 1B study of selisistat—A SIRT 1 inhibitor

Background Evidence suggests simultaneous dysfunction of CNS and peripheral inflammatory pathways in Huntingtons disease (HD). A pattern of pro-inflammatory cytokine elevation has been observed in plasma in HD, with IL-6 significantly elevated in a group of subjects predicted to be, on average, 16 years from disease, with a parallel post-mortem cytokine expression profile seen in HD striatum. Furthermore, LPS stimulation of HD monocytes reveals an inherent hyper-reactivity that is similar to that seen in microglia, suggesting a cell-autonomous effect of mutant huntingtin in peripheral myeloid cells as well as in the CNS. Sirtuins, which are deacetylating enzymes, have been implicated as potential targets in ageing, metabolism and neurodegeneration. Selisistat, a member of the sirtuin deacetylase family and an inhibitor of SirT1 (Silencing information regulator T1), has a novel mode of action that may be pathologically relevant for HD. A Phase 1B study encompassing six EU sites (Germany, Poland and the UK) was carried out in 55 early-stage HD patients over 14 days at two dose levels (10 and 100 mg OD), with a randomised, double-blind, placebo-controlled, parallel group design. Aim To determine if the innate immune system is a potential target for Selisistat. Methods Cytokine assays were carried out using the MesoScale Discovery (MSD) multiplex platform, as per the manufacturers recommendations, and analysed on a SECTOR 2400 instrument. The cytokines of interest included IL-1b, IL-6, IL-8 and TNF-α. The operator was blinded to the disease state of each sample during processing and statistical analysis was performed independently. Results and Conclusions Here we will present comparative analysis of the plasma cytokine profile of Huntingtons disease patients at four time points, screening, baseline, day 14 and day 28, in each of the three treatment arms of the study.