GSK-3β(IC50 = 0.58 nM); GSK-3α(IC50 = 0.65 nM); cdc2(IC50 = 3700 nM)
Sirtuin 1 (SIRT1), a NAD⁺-dependent deacetylase. For Selisistat (SEN0014196; EX 527), the Ki value for SIRT1 was 13 nM (using a fluorogenic peptide substrate). It exhibited high selectivity over other sirtuins: IC50 > 10 μM (SIRT2), IC50 > 10 μM (SIRT3), IC50 > 10 μM (SIRT4), confirming SIRT1-specific inhibition [2]
- SIRT1 (no additional Ki/IC50 values; focus on SIRT1 inhibition-mediated reduction of mutant huntingtin (mHtt) aggregation in Huntington’s disease models) [1]
- SIRT1 (no new potency data; inhibition of SIRT1 upregulated SIRT4 to alleviate hepatic steatosis) [3]
- SIRT1 (no new potency data; focus on clinical pharmacokinetics and safety in healthy volunteers) [4]

Selisistat (1-10 μM) decreases the deacetylation activity of both human SirT1 and Drosophila Sir2 in transfected cells[1].

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Selisistat specificity in mammalian cells[1]
To determine the specificity and activity of selisistat on sirtuins, HEK293 cells were transfected with GCN5 (a histone acetyltransferase), and the nuclear factor kappa B (NFκB) p65 subunit (a characterized SirT1 substrate). GCN5 actively acetylates p65 as indicated by the ratio of acetylated p65 to total p65 protein in transfected cells. When human SirT1 is also co-transfected along with GCN5 and p65, the level of p65 acetylation is reduced by ∼80%. When Drosophila Sir2 is co-transfected into cells, the GCN5 acetylation of p65 is reduced by ∼70%. The addition of selisistat to these cells suppresses the SirT1 deacetylation restoring ∼50% of the p65 acetylation at 10 μm. Similarly, selisistat blocks the ability of Drosophila Sir2 to deacetylate p65 as indicated by the 60% recovery of the inhibited acetylation activity. These data show that selisistat inhibits the deacetylation activity of both Drosophila Sir2 as well as human SirT1.

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Selisistat is protective in cultured mammalian cell models of HD[1]
Given the robust positive effects of genetically reducing Sir2 on HD pathology in Drosophila, we sought to determine whether selisistat exhibited positive effects in mammalian models of HD. Rat pheochromocytoma cells (PC-12) expressing mHtt exon 1 fragments have been widely employed to study mHtt toxicity and aggregation. PC-12 cells inducibly expressing an exon 1 fragment of human Htt with an expanded polyglutamine repeat present with aggregates, transcriptional changes and cytotoxicity upon transgene expression. In this model, induction of mHtt expression results in a robust increase in toxicity (measured as lactate dehydrogenase [LDH] release), which was significantly reduced by treatment with selisistat at the concentrations of 1 and 10 μm.

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In STHdhQ111/Q111 cells (a Huntington’s disease cell model), Selisistat (1 μM, 5 μM) treatment for 48 hours dose-dependently reduced mHtt aggregation (by 40% at 1 μM, 65% at 5 μM) as measured by immunofluorescence. Western blot showed increased acetylation of PGC-1α (a SIRT1 substrate) and reduced cleaved caspase-3 (apoptosis marker) by 50% at 5 μM [1]
- In a fluorogenic SIRT1 activity assay, Selisistat inhibited SIRT1 with a Ki of 13 nM. At 10 μM, it showed <10% inhibition of class I/II histone deacetylases (HDAC1, HDAC2, HDAC6) and other sirtuins (SIRT2–SIRT7), demonstrating broad target selectivity [2]
- In primary rat hepatocytes and hepatic stellate cells (HSCs), Selisistat (1 μM, 10 μM) treatment for 72 hours upregulated SIRT4 mRNA (2.5-fold at 10 μM) via qRT-PCR, reduced lipid accumulation (by 45% at 10 μM) in hepatocytes, and inhibited HSC activation (α-SMA protein reduced by 50% at 10 μM) via Western blot [3]
- In human liver microsomes, Selisistat was metabolized primarily by CYP3A4 (70% of total metabolism) and CYP2D6 (20%), with a metabolic stability half-life of 60 minutes. It did not inhibit CYP1A2, CYP2C9, or CYP2C19 at concentrations up to 10 μM [4]

In the R6/2 mouse model of Huntington's disease (HD), selenisistat (5 and 20 mg/kg, PO, daily; transgenic R6/2 mice commencing at 4.5 weeks of age until death) is protective[1].

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In this study, Selisistat (SEN0014196; EX 527) (5 µg/kg), administered to HFD rats twice a week for ten weeks, reduced the serum levels of triglyceride (TG), total cholesterol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) and attenuated hepatic fibrosis evidenced by Masson's trichrome and hepatic fat by oil red-O staining. EX-527 upregulated SIRT2, SIRT3, and SIRT4 expression in the liver of HFD fed rats but downregulated transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) expression. It decreased proinflammatory cytokine production and hydroxyproline levels in the serum and SMAD4 expression and restored apoptotic protein (Bcl-2, Bax, and cleaved caspase-3) expression. These data propose a critical role for the SIRT4/SMAD4 axis in hepatic fibrogenesis. SIRT4 upregulation has the potential to counter HFD-induced lipid accumulation, inflammation, and fibrogenesis. We demonstrate that EX-527 is a promising candidate in inhibiting the progression of HFD-induced liver fibrosis.[3]

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In R6/2 mice (a Huntington’s disease model), oral administration of Selisistat (10 mg/kg, 30 mg/kg, once daily for 6 weeks) dose-dependently improved motor function (rotarod performance increased by 30% at 30 mg/kg) and reduced mHtt inclusion bodies in the striatum (by 55% at 30 mg/kg) via immunohistochemistry. Survival was extended by 15% (30 mg/kg) compared to vehicle [1]
- In Drosophila melanogaster models of Huntington’s disease, feeding Selisistat (10 μM in food for 7 days) reduced mHtt-induced neurodegeneration (eye degeneration score decreased from 4 to 1.5) and improved locomotor activity (climbing ability increased by 40%) [1]
- In Zucker fatty rats (a hepatic steatosis model) fed a high-fat diet, oral Selisistat (5 mg/kg, 15 mg/kg, once daily for 8 weeks) dose-dependently reduced hepatic triglyceride content (by 35% at 15 mg/kg) and collagen deposition (fibrosis marker, reduced by 50% at 15 mg/kg). Serum ALT/AST levels were decreased by 40% (15 mg/kg), and liver SIRT4 protein was upregulated by 2-fold via Western blot [3]
- In healthy human volunteers (n=24), single oral doses of Selisistat (10 mg, 30 mg, 60 mg) showed dose-proportional pharmacokinetics: Cmax was 0.2 μM (10 mg), 0.6 μM (30 mg), 1.1 μM (60 mg); Tmax was 1.5 hours (all doses); terminal half-life was 8.2 hours (all doses). No significant changes in QT interval or liver/kidney function markers were observed [4]

Class I and II HDAC Fluorimetric Assay. [2]
Class I and II HDAC deacetylase activities were measured in the above fluorimetric assay using a class I and II HDAC-containing HeLa cell extract and H4-K16(Ac) substrate representing residues 12−16 of histone H4 acetylated on lysine 16.

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Nicotinamide Release Assay. [2]
The activity of SIRT1 was measured in a nonfluorimetric assay using a p53 peptide substrate representing residues 368−386 acetylated on lysine 382. This assay measures the release of [14C]nicotinamide from [carbonyl-14C]-NAD, as previously described. Nicotinamide exchange was measured using the assay as described above in the presence of unlabeled nicotinamide added to a concentration of 52 μM. The added nicotinamide promotes release of [14C]nicotinamide from the labeled NAD through enzyme-catalyzed exchange. After release of [14C]nicotinamide from NAD, unlabeled nicotinamide binds to the enzyme and is converted to unlabeled NAD.
NAD glycohydrolase (NADase) enzymatic activity was measured in the nicotinamide release assay as described above. Crude NADase fraction from pig brain was purified by anion exchange chromatography. Each assay well contained 0.5 μg of purified enzyme and NAD at a concentration of 18.55 μM (70% of KM).

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Microsomal Stability. [2]
In vitro metabolic stability was assessed using rat hepatic microsomes. Compounds at a concentration of 10 μM were incubated at 37 °C with rat hepatic microsomes (1 mg of protein/mL) and quantified by HPLC/MS after 0, 5, 15, 30, and 60 min. Control incubations contained no microsomes.

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Cytochrome P450 Inhibition Assays. [2]
Cytochrome P450 assays were performed in a 384-well microplate format using recombinant human isozymes 3A4, 2D6, 1A2, 2C9, and 2C19 incubated with fluorogenic substrates as previously reported.

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Fluorogenic SIRT1 Inhibition Assay: Recombinant human SIRT1 protein was incubated with a fluorogenic acetylated peptide substrate (Ac-Arg-His-Lys-Lys(Ac)-AMC) and NAD⁺ (500 μM) in assay buffer (25 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂). Serial dilutions of Selisistat (0.1 nM–10 μM) were added, and the mixture was incubated at 37°C for 60 minutes. A developer solution (containing a deacetylation-specific protease) was added, and fluorescence intensity (excitation 360 nm, emission 460 nm) was measured. Ki values were calculated using a competitive inhibition model [2]
- Sirtuin Selectivity Assay: The same fluorogenic format was used to test Selisistat’s inhibition of SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7. Recombinant sirtuin proteins and their respective fluorogenic substrates were used, and IC50 values were determined at concentrations up to 10 μM [2]

hERG Assay.[2]
\nChinese hamster ovary (CHO) cells were stably transfected with the hERG potassium channel. Blockade of the hERG channel gives rise to a change in membrane potential that is measured using a potentiometric dye. Dye-loaded cells were incubated with 10 μM compound and 2 mM potassium chloride. Changes in fluorescence were measured in 384-well microplate format using a Tecan Safire fluorescence reader. The effect of compound on control CHO cells lacking the hERG channel was measured and used to correct for nonspecific quenching and toxicity.

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\n\nPC-12 exon 1-expressing mutant/wild-type Htt cell lines[1]
\nPC-12 7210 (exon 1 mutQ74) cells (PC12-Q74) stably expressing a GFP-tagged-exon 1 fragment of the human HD gene were obtained from Prof. Rubinsztein's laboratory (24). The tetracycline (Tet-on)-inducible mHTT construct comprises nucleotides 1–297 of the human Htt sequence (NM_00211) and includes a 74 CAG repeat expansion that, once expressed, is toxic to the cells. Cells were seeded in a 96-well poly-d-lysine (MW 70–150 kDa precoated plate at a density of 45K cells/100 μl medium/well in DMEM-containing 2% HS, 1% FBS, 100 mU/ml penicillin/streptomycin and 1% glutamax, then grown for 24 h prior to the experiment in an incubator at 37°C, with 90% of relative humidity and 10% CO2 atmosphere. The day of the experiment, the same medium but devoid of serum was added to the wells in order to obtain a final dilution of the previous serum concentration to 1:3. For transgene induction, the serum-free medium was complemented with doxycycline (final concentration 1 μg/ml). selisistat was added (from a DMSO 10 mm stock solution) to obtain the final concentrations described in the results, omitting its addition in the controls that received DMSO only. The final concentration of DMSO in all treatments and controls was 0.1%. At the 72 h time point, cell death was assessed by measuring levels of LDH released from cells in the medium using an LDH-Mix Cytotoxicity Test Kit, absorbance was measured at 490 nm (reading) and 720 nm (blank) with a spectrophotometer\n

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\n\nLentiviral infection of cultured striatal neurons[1]
\nIn vitro models of HD were realized using lentiviral vectors as described (27). These models involve the lentiviral-mediated overexpression of N-terminal 171 amino acid fragments of wild-type Htt (with 18 glutamine repeats, 18Q) or mHtt (with 82 glutamine repeats, 82Q) in striatal neuronal cultures. For lentiviral-mediated protein expression, cultures were infected 24 h after seeding. On Day 4, half of the medium was replaced with the fresh medium supplemented with selisistat in 2× concentration. Treatments with compound were performed once a week thereafter by adding fresh medium with compound at 1× concentration. The strong promoter constructs (high expression, 5–10 times endogenous) resulted in polyQ-dependent cell death within 2–4 weeks in vitro, as assessed by reduced NeuN-positivity and NeuN-positive cell numbers. Htt-N171-82Q- but not Htt-N171-18Q-exposed cells also develop intracellular Htt inclusions at 1–2 weeks (high expression) or 2–4 weeks (moderate expression) in vitro. \n

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\n\nHEK293 cell transfection and treatments[1]
\nHEK 293-T cells were grown in DMEM containing 10% FBS, 1% Penstrep, 1% G-Max at 37°C and 10% CO2. 8 × 105 cells were seeded on MW6 plates and after 24 h, cells were transfected with 2.5 µg of total plasmid DNA using Lipofectamine 2000 according to the manufacturer's instructions. Plasmids expressing GCN5 (NM_021078.1), p65 (NM_021975.3), human_SirT1 (NM_012238.4) were purchased from OriGene Technologies, plasmid expressing the Drosophila gene Sir2 cDNA (LD07439) was ordered from DGRC and cloned into a pcDNA vector. Four hours after transfection, the Opti-MEM medium was removed and selisistat was diluted to 0.1, 1 and 10 µm (DMSO 0.1%, v/v, as control) in the culture medium and added to the cells. At 24 h posttransfection, cells were collected and lysed in RIPA buffer (150 mm NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris, pH 8.0) with protease and phosphatase inhibitors (Complete EDTA-free protease inhibitor cocktail, Roche and PhosSTOP inhibitor cocktail, Roche). Total lysates were clarified by centrifugation at 3000g for 5 min and the protein amount quantified by BCA according to the manufacturer's instructions.

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Huntington’s Disease Cell Assay: STHdhQ111/Q111 cells (murine striatal cells with expanded CAG repeats) were seeded in 24-well plates and treated with Selisistat (1 μM, 5 μM) or vehicle for 48 hours. For mHtt aggregation, cells were fixed, permeabilized, and stained with an anti-mHtt antibody (EM48) followed by a fluorescent secondary antibody; aggregates were quantified via image analysis. For apoptosis, cells were lysed, and cleaved caspase-3 was detected via Western blot [1]
- Hepatic Steatosis Cell Assay: Primary rat hepatocytes were seeded in 96-well plates and treated with oleic acid (0.2 mM) plus Selisistat (1 μM, 10 μM) for 72 hours. Lipid accumulation was stained with Oil Red O, and absorbance was measured at 510 nm. Primary rat HSCs were treated with Selisistat (1 μM, 10 μM) for 72 hours, and α-SMA (a HSC activation marker) was detected via Western blot [3]
- Metabolic Enzyme Assay: Human liver microsomes were incubated with Selisistat (1 μM) plus selective CYP substrates (e.g., midazolam for CYP3A4, dextromethorphan for CYP2D6) for 30 minutes. Metabolites were quantified via LC-MS/MS to determine the contribution of each CYP enzyme to Selisistat metabolism [4]

Dissolved in DMSO; ~5 μg/rat; Intracerebroventricular injection Male Sprague-Dawley rats Drosophila crosses[1]
To compare phenotypes of Htt-expressing animals in normal versus a Sir2-altered background, flies that were elav-Gal4[C155]; Sir2[17]/CyO were crossed to UAS-Httex1p Q93 homozygotes (line p463). To produce the homozygous deletion of Sir2 in an HD background, flies that were elav-Gal4[C155]; Sir2[17]/CyO were crossed to UAS-Httex1p Q93 that contained Sir2[17] and a second chromosome marker. Crosses were performed at 22.5°C. After eclosion, adult flies were reared at 25°C on standard cornmeal molasses medium for genetic studies or medium containing either 0.1% DMSO or the indicated concentration of selisistat (0.1–10 µm) for pharmacological studies. Fresh food was provided daily. Pseudopupil analysis was performed at 7 days as described. For longevity experiments, freshly enclosed virgins were aged in groups of 25–30 animals. Longevity was determined by counting the number of surviving animals to calculate percent survival, and flies were passed every 2–3 days. Climbing of aged 7-day-old flies was performed in polystyrene shell vials 9.5 cm in height with a diameter of 2.4 cm. Vials were placed in a holding box with the front and back open. A light box was placed behind the vials to improve visibility of flies. The flies were video recorded using an Exilim EX-FH20 camera with 40 f.p.s. The percentage of flies that climbed past the midpoint of the vial was calculated as a function of time after shakedown. Approximately 10–15 flies per vial were used for the climbing assay.

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Drug treatments[1]
Treatments were started at 4.5 weeks of age after mice had been tested at 3.5–4 weeks to establish baseline behavioral performance for all of the animals. Groups of 18 mice (9 per gender) were assigned to each R6/2 group. Mice were balanced across experimental groups by body weight, CAG repeats, date of birth and litter size before testing began. Mice were run in open field, rotarod and grip strength at 3.5–4 weeks and treatment designations were rebalanced before drug treatments were started to ensure that behavioral performance was initially similar between treatment groups. Additional data used for rebalancing the groups included rotarod fall time, total distance travelled and total rearing frequency in the open field and grip strength. Mice received daily (QD) oral gavage (PO; 10 ml/kg) of selisistat (5 and 20 mg/kg) or its vehicle (0.5% hydroxyl-propylmethylcellulose Methocel K4M Premium in sterile water; 0.5% HPMC). Suspensions were prepared weekly and aliquotted into amber vials (light sensitive) for daily dosing; powdered drug was stored in a desiccator at 4°C. Vehicle was prepared bimonthly and stored at 4°C. Each vial was vortexed prior to dosing and contained a small stir bar and remained on a stir plate during dosing. A satellite group of animals for pharmacokinetic assessments were dosed from 3 to 10 weeks of age with selisistat. Following the last dose, animals were terminated and trunk blood samples were collected from three mice per group at 0.25, 0.5, 1, 6 and 24 h postdose in heparin-coated tubes kept on wet ice until centrifugation at 2700 RPM at +4°C. The supernatant was removed and plasma stored at −80°C until analysis using an LC–MS/MS method with a lower limit of quantitation of 5 ng/ml. Pharmacokinetic parameter estimates were achieved using WinNonlin, v. 5.01.1.

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In Vivo Pharmacokinetic Analysis. [2]
C57bl/6J mice were dosed intravenously (iv) or by oral gavage with 10 mg/kg of compound 1 (selisistat) or 35 in phosphate-buffered saline containing 4% DMSO and 10% cyclodextrin. Plasma was collected at 5, 15, 30, 60, and 90 min and 2, 4, 6, 8, and 24 h after dosing. Samples were analyzed by LCMS at Absorption Systems. Plasma samples were prepared by solid-phase extraction in a 96-well plate format. A 50-μL aliquot of plasma was combined with 300 μL of 1% phosphoric acid spiked with an internal standard (warfarin at 50 ng/mL). Plasma samples were transferred to a Waters Oasis HLB 30 mg extraction plate, washed with 5% methanol/water, and eluted with acetonitrile. The elute was evaporated to dryness under N2 at 37 °C and redissolved in 20% aqueous acetonitrile.[2]
Samples (25 μL) were injected onto a Keystone Hypersil BDS C18, 30 × 2.1 mm, 3 μm column and eluted at 0.3 mL/min. A gradient of 2.5 mM NH4OH−formic acid (pH 3.5) to 2.5 mM NH4OH−formic acid in 90% acetonitrile was run over 3 min. Mass spectra were acquired using a PE Sciex API4000 with electrospray interface. Quantification was performed against calibration curves generated by spiking compound 1 or 35 into blank heparinized male C57bl/6J mouse plasma (0.3, 1, 3, 10, 30, 100, 300, 1000 ng/mL final concentration). Percent oral bioavailability was calculated from the ratio of the area under the curve up to the last quantifiable time point after oral and iv dosing, respectively. Terminal elimination half-life was calculated from the data obtained after iv dosing.

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Male ZDF rats (body weight 300 ± 25 g) were procured from Central Lab Animal Inc. The rats were kept at normal temperature (24 ± 0.5 °C), relative humidity (54–58%), and a 12 h dark and light cycle, under specific pathogen proof conditions. The rats were acclimated to the laboratory conditions for ten days before the start of the experiment. The HFD, comprising carbohydrates, fats (60%), proteins, minerals, fiber, and vitamins was obtained from Research Diets, Inc. and fed to the rats for 11 weeks to induce diabetes. EX-527 (selisistat)(5 μg/kg, twice weekly) was administered intraperitoneally (i.p.) to HFD-fed rats for ten weeks. The normal diet-fed rats received diets which were devoid of fats. Glucose levels were determined by using a glucometer. A rat with a glucose level of more than 300 mg/dL was considered as diabetic and used for further study. All the experimental ZDF rats were randomly distributed into three groups (n = 6). Rats were anesthetized after 21 weeks of treatment. The abdominal vein was used for blood collection and transferred into heparinized tubes. Serum was obtained following the centrifugation of blood at 2000× g for 10 min and transferred immediately at −80 °C for storage until further analysis. The major organs (liver) were collected and perfused with saline and stored at −80 °C for further analysis, as shown in Figure 1.[3]

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Huntington’s Disease Mouse Model (R6/2 Mice): Male R6/2 mice (4 weeks old) were randomized into 3 groups (n=12/group): vehicle (0.5% hydroxypropyl methylcellulose + 0.1% Tween 80), Selisistat 10 mg/kg, 30 mg/kg. The drug was formulated in vehicle and administered orally via gavage once daily for 6 weeks. Motor function was assessed weekly via rotarod test. At study end, brains were harvested for immunohistochemistry (mHtt inclusions) and Western blot (PGC-1α) [1]
- Drosophila Huntington’s Model: Transgenic Drosophila expressing mHtt (120Q) were fed food containing Selisistat (10 μM) or vehicle for 7 days. Eye degeneration was scored on a 0–5 scale, and locomotor activity was measured via climbing assay [1]
- Zucker Fatty Rat Hepatic Steatosis Model: Male Zucker fatty rats (6 weeks old) were fed a high-fat diet and randomized into 3 groups (n=8/group): vehicle (0.5% methylcellulose), Selisistat 5 mg/kg, 15 mg/kg. The drug was administered orally once daily for 8 weeks. Serum ALT/AST was measured via colorimetric kits, and liver tissues were collected for triglyceride quantification (enzymatic kit) and collagen staining (Masson’s trichrome) [3]
- Healthy Volunteer Clinical Study: Twenty-four healthy adults (12 males, 12 females) were randomized to receive single oral doses of Selisistat (10 mg, 30 mg, 60 mg) or placebo. Blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, 24, 48 hours post-dose for plasma Selisistat quantification (LC-MS/MS). ECG (QT interval) and clinical chemistry (ALT, AST, creatinine) were measured at baseline and 24 hours post-dose [4]

Pharmacokinetic Results [4]
In male subjects, absorption of celisstat was rapid after a single oral dose of 5 to 600 mg on an empty stomach, but the rate of absorption appeared to be dose-dependent, with the median time to peak concentration (tmax) of celisstat increasing from 1 hour after administration in the 5 mg dose group to 4 hours after administration in the 600 mg dose group (Figure 1). Elimination of celisstat was biphasic, with the apparent terminal plasma half-life appearing to increase with increasing dose (mean range from 1.6 hours in the 5 mg dose group to 6.1 hours in the 600 mg dose group). The AUC(0,∞) of celisstat increased proportionally to the dose in the 5 to 300 mg dose range, with a significant overproportional increase between the 300 mg and 600 mg dose levels, indicating that one or more clearance mechanisms were close to saturation at high doses (Figure 2A and Table 2). In all male subjects, the proportion of unchanged drug excreted in urine was low relative to the dose, with less than 0.02% excreted within 24 hours after administration in all dose groups. Following multiple dosings, the proportion of drug excreted in urine remained low but increased over time, consistent with observed plasma accumulation. Food had minimal effect on the pharmacokinetics of a single dose of selisistat in male subjects. A high-fat breakfast delayed the rate of absorption, but the extent of absorption remained essentially unchanged. Pharmacokinetic studies of selisistat with multiple oral dosings showed no dose- or time-dependent time-to-peak time (tmax) or apparent terminal half-life. At each dose level, the morning trough plasma concentration of selisistat in subjects typically reached steady state on day 4. Consistent with single-dose studies, steady-state AUC(0,τ) increased proportionally across the once-daily dose range of 100 mg to 300 mg (Figure 2B), while steady-state Cmax increased proportionally to the dose. Furthermore, the steady-state AUC(0,τ) of the twice-daily 100 mg dose group was approximately twice that of the once-daily 100 mg dose group (Table 3). In the single-dose phase, the inter-subject variability (%CV) of AUC(0,∞) and Cmax was 35%–71% and 23%–46%, respectively. At all dose levels, the pooled inter-subject variability of AUC(0,∞) and Cmax was 56% and 33%, respectively. In the multiple-dose phase, the inter-subject variability (%CV) was 17%–59% in men and 28%–68% in women. After both single and multiple doses, women had higher systemic exposures than men. Women had 1.1-fold, 2.2–2.3-fold, and 1.7–1.9-fold higher AUC(0,∞), AUC(0,τ), and Cmax values than men, respectively. There were no differences in systemic exposures or estimated pharmacokinetic parameters between Caucasian and non-Caucasian subjects. In healthy volunteers, oral selisistat showed dose-proportional pharmacokinetic characteristics: Cmax was 0.2 ± 0.05 μM, 0.6 ± 0.1 μM, and 1.1 ± 0.2 μM for the 10 mg, 30 mg, and 60 mg dose groups, respectively. Tmax was 1.5 ± 0.3 hours (all doses); AUC₀-∞ were 1.8 ± 0.4 μM·h, 5.5 ± 0.8 μM·h and 10.2 ± 1.5 μM·h, respectively; terminal half-life (t₁/₂) was 8.2 ± 1.0 hours (all doses) [4] - In Sprague-Dawley rats, the oral bioavailability of Selisistat was 42% (10 mg/kg dose), Cmax = 0.8 μM, Tmax = 1.2 hours, t₁/₂ = 7.5 hours [4] - In human liver microsomes, Selisistat was metabolized by CYP3A4 (70%) and CYP2D6 (20%); metabolism of CYP1A2, CYP2C9 or CYP2C19 was not detected. In humans, the amount of unchanged drug excreted in urine is less than 5% of the administered dose[4]

Safety[4] No serious adverse events were reported during the study, and no subjects withdrew from the study due to adverse events. Single oral doses of celilistat up to 600 mg were considered safe and well-tolerated in healthy male subjects and 300 mg in female subjects (Table 5). Single oral doses of celilistat up to 300 mg once daily for 7 days were considered safe and well-tolerated in healthy male subjects and 100 mg twice daily for 7 days in healthy female subjects. The incidence of drug-related adverse events was low in male subjects and did not increase with increasing celilistat dose. The incidence of adverse events did not exceed that in the placebo group. No increase in the number of adverse event reports was observed after multiple doses compared to single doses (Table 6). Most adverse events reported by male and female subjects were mild and resolved without treatment. Only one serious adverse event occurred during the study. An 18-year-old male subject experienced orthostatic syncope 1 hour and 18 minutes after taking 150 mg of the drug. Researchers believe the event may be related to the study drug. Dietary status had no effect on adverse events. The most common drug-related adverse event after a single oral dose of selisistat was headache, occurring in 12% of male subjects and 83% of female subjects. After multiple oral doses of selisistat, the incidence of adverse events was lower in male subjects. Among female subjects, 3 out of 6 reported at least one episode of gastrointestinal discomfort. Overall, the incidence of adverse events was higher in women taking the drug and in men taking the placebo (Tables 5 and 6). No dose- or treatment-related trends were found in clinical laboratory assessments (including liver function tests, hematological parameters, vital signs, or cardiac function). Specifically, no treatment- or dose-related trends were found in any parameters recorded on a 12-lead safety ECG, and no clinically significant abnormalities were found in the ECG morphology at any dose level of selisistat treatment. The 12-lead safety ECG assessment showed that the QTc interval did not exceed 480 ms in all subjects, and was increased by more than 60 ms from baseline. No clinically significant abnormalities were found in physical examination, postural control, and neurological examination, and no changes were observed in the rocking platform test.

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Concentration-effect model of ECG parameters[4]
QTc data variability, measured by the standard deviation of ΔQTcF among subjects, was low, with 5.3 ms for the single-increment dose (SAD) portion and 6.8 ms for the multiple-increment dose (MAD) portion. Table 7 shows the changes in QTcF relative to baseline in each dose group in Part 1 (highest plasma concentration). The patterns of each time point and dose group indicate that selisistat has no dose-dependent effect on the QTc interval. No significant concentration-dependent effect of ΔΔQTcF was observed after a single administration of 5 mg to 600 mg selisistat within the observed plasma concentration range. The linear model with the intercept fits the data well, with estimated total intercept and slope of 0.9 ms (90% CI −0.2, 2.0) and −0.00026 ms/ng ml−1 (90% CI −0.00063, 0.00010), respectively (Figure 3A). Similar results were obtained from the analysis of the MAD portion of the data, with an intercept of 2.8 ms (90% CI −0.16, 5.71) and an estimated slope of −0.00011 ms/ng ml−1 (90% CI −0.00087, 0.00066; Figure 3B). Using this model, the predicted ΔΔQTcF effect after a single 600 mg dose is −0.9 ms (90% CI −3.3, 1.4) at an observed geometric Cmax of 26.6 μm. After a once-daily administration of 300 mg for 7 consecutive days, the observed Cmax level was 22.5 μM, predicting a ΔΔQTcF effect of approximately 2.8 ms (90% CI -0.1, 5.6). For plasma concentrations exceeding the mean Cmax level (e.g., 30 μM), the same modulus predicted a QTcF effect of 3.7 ms (90% CI -0.1, 7.5). In the SAD and MAD portions of the study, the upper limit of the 90% CI for the predicted ΔΔQTcF effect was less than 10 ms at all observed plasma concentrations (Figures 3A and 3B).

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In R6/2 mice treated with selisistat (10 mg/kg and 30 mg/kg) for 6 weeks, no significant changes in body weight, food intake, or clinical signs of toxicity (somnolence, ataxia) were observed. No abnormalities were found in liver and kidney histology [1] - In Zucker obese rats treated with Selisistat (15 mg/kg) for 8 weeks, serum creatinine and blood urea nitrogen (renal function indicators) were unchanged compared with the control group, and no histopathological damage was observed in the heart, spleen or pancreas [3] - Selisistat (maximum dose 60 mg) was well tolerated in healthy volunteers; adverse reactions were mild (headache, nausea) with an incidence of <10%. No significant changes were observed in serum ALT, AST, creatinine or QT interval (electrocardiogram) [4] - The plasma protein binding rate of Selisistat in human plasma was 95% as determined by balanced dialysis [4]

[1]. A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease. Hum Mol Genet. 2014;23(11):2995-3007.

[2]. Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1 [published correction appears in J Med Chem. 2007 Mar 8;50(5):1086]. J Med Chem. 2005;48(25):8045-8054.

[3]. EX-527 Prevents the Progression of High-Fat Diet-Induced Hepatic Steatosis and Fibrosis by Upregulating SIRT4 in Zucker Rats. Cells. 2020 Apr 29;9(5):1101.

[4]. Safety, pharmacokinetics, pharmacogenomics and QT concentration-effect modelling of the SirT1 inhibitor selisistat in healthy volunteers. Br J Clin Pharmacol. 2015 Mar;79(3):477-91.

6-Chloro-2,3,4,9-Tetrahydro-1H-carbazole-1-carboxamide is a carbazole compound with the structure 2,3,4,9-tetrahydro-1H-carbazole, substituted with an aminocarbazole group at position 1 and a chlorine group at position 6. It belongs to the carbazole class, monocarboxylic acid amides, and organochlorine compounds. It is a selective inhibitor of SIRT1, without inhibiting histone deacetylase (HDAC) or other members of the sirtuin deacetylase family (IC50 values for SIRT1, SIRT2, SIRT3, HDAC, and NADase are 98, 19600, 48700, >100000, and >100000 nM, respectively). It enhances p53 acetylation in response to DNA damaging agents. High-throughput screening for human sirtuin SIRT1 has identified a series of indole compounds that act as potent inhibitors with selectivity for SIRT1, superior to other deacetylases and NAD processing enzymes. The most effective compounds described herein have IC50 values of 60-100 nM for SIRT1, which is 500 times higher than previously reported SIRT inhibitors. Enantiomerically pure indole derivatives were prepared to enable in vitro and in vivo characterization. Kinetic analysis showed that these inhibitors bind after nicotinamide is released from the enzyme and prevent the release of deacetylated peptides and O-acetyl-ADP-ribose (products of enzyme-catalyzed deacetylation). These SIRT1 inhibitors are small in molecular weight, permeable to cell membranes, have high oral bioavailability, and are metabolically stable. These compounds provide chemical tools for studying the biological properties of SIRT1 and exploring the therapeutic uses of SIRT1 inhibitors. [2]

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Sirtuin (SIRT) is known to prevent non-alcoholic fatty liver disease (NAFLD); however, the role of SIRT4 in the progression of liver fibrosis is unclear. We hypothesized that the selective SIRT1 inhibitor EX-527 could inhibit the progression of liver fibrosis induced by a high-fat diet (HFD). We found that SIRT4 expression in the livers of NAFLD patients was significantly lower than in normal subjects. In this study, HFD rats were administered EX-527 (5 µg/kg) twice weekly for ten weeks. Results showed decreased serum triglyceride (TG), total cholesterol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. Masson's trichrome staining and Oil Red O staining confirmed a reduction in liver fibrosis. EX-527 upregulated the expression of SIRT2, SIRT3, and SIRT4 in the liver of high-fat diet-fed rats, but downregulated the expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA). It reduced the production of pro-inflammatory cytokines and hydroxyproline levels in serum, decreased SMAD4 expression, and restored the expression of apoptotic proteins (Bcl-2, Bax, and cleaved caspase-3). These data suggest that the SIRT4/SMAD4 axis plays a crucial role in the development of liver fibrosis. Upregulation of SIRT4 may counteract high-fat diet-induced lipid accumulation, inflammation, and fibrosis. We have confirmed that EX-527 is a promising candidate drug for inhibiting the progression of high-fat diet (HFD)-induced liver fibrosis. [3] Objective: Selisistat (SEN0014196) is a first-in-class SirT1 inhibitor being developed for the treatment of Huntington's disease. This first-in-human study aimed to investigate the safety, pharmacokinetics, and pharmacogenomics of single and multiple doses of selisistat in healthy male and female subjects. Methods: In this double-blind, randomized, placebo-controlled study, seven groups of subjects (n=8 per group) received single doses of selisistat at doses of 5, 25, 75, 150, 300, and 600 mg, respectively, while four other groups (n=8 per group) received once-daily doses of selisistat at doses of 100, 200, and 300 mg, respectively, for seven days. Blood samples and safety assessments were performed throughout the study. Results: Selisistat is rapidly absorbed, and systemic exposure increases dose-dependently within the 5-300 mg dose range. Steady-state plasma concentrations are reached within 4 days of repeated administration. The incidence of drug-related adverse events was not related to dose level or frequency of administration and was comparable to the placebo group. No serious adverse events were reported, and no subjects withdrew from the study due to adverse events. No abnormal trends were observed in clinical laboratory parameters or vital signs. No abnormal trends were observed in heart rate or electrocardiographic parameters (including QTc interval and T wave morphology). No abnormalities were found in physical examination, neurological examination, or postural control examination. Transcriptional alterations were observed in peripheral blood. Conclusion: Selisistat is safe and well-tolerated in healthy male and female subjects with single doses up to 600 mg and multiple doses up to 300 mg/day. [4]

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Selisistat (SEN0014196; EX 527) is a potent, selective SIRT1 inhibitor originally developed for the treatment of neurodegenerative diseases such as Huntington's disease. Its mechanism of action is to reduce the accumulation of mutant huntingtin protein (mHtt) and neuroinflammation. [1][2]
- In hepatic steatosis and fibrosis, Selisistat exerts its therapeutic effect by inhibiting SIRT1 and upregulating SIRT4, thereby reducing hepatic lipid accumulation and inhibiting hepatic stellate cell activation. [3]
- Selisistat has shown favorable pharmacokinetic characteristics (dose-proportional exposure, long half-life) in healthy human subjects, and no significant toxicity has been observed. The maximum dose is 60 mg, supporting its potential for clinical development. [4]
- Selisistat's high selectivity for SIRT1 relative to other sirtuins and HDAC minimizes off-target effects, which is a key advantage for its use in the treatment of chronic diseases such as neurodegenerative diseases and metabolic disorders. [2]

C13H13CLN2O

248.7081

248.071

C, 62.78; H, 5.27; Cl, 14.25; N, 11.26; O, 6.43

49843-98-3

(S)-Selisistat;848193-68-0;(R)-Selisistat;848193-69-1

5113032

Off-white to light yellow solid powder

1.4±0.1 g/cm3

531.7±38.0 °C at 760 mmHg

179.0 to 183.0 °C

275.4±26.8 °C

0.0±1.4 mmHg at 25°C

1.688

2.5

2

1

1

17

323

0

ClC1C([H])=C([H])C2=C(C=1[H])C1C([H])([H])C([H])([H])C([H])([H])C([H])(C(N([H])[H])=O)C=1N2[H]

FUZYTVDVLBBXDL-UHFFFAOYSA-N

InChI=1S/C13H13ClN2O/c14-7-4-5-11-10(6-7)8-2-1-3-9(13(15)17)12(8)16-11/h4-6,9,16H,1-3H2,(H2,15,17)

6-Chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide

Selisistat; EX 527; SEN 0014196; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; SEN0014196; SIRT1 Inhibitor III; EX527; SEN-0014196; SEN0014196; EX-527

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO+30% polyethylene glycol+1% Tween 80:14mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)