GSK-3β(IC50 = 0.58 nM); GSK-3α(IC50 = 0.65 nM); cdc2(IC50 = 3700 nM)

Selisistat (1-10 μM) 可降低转染细胞中人 SirT1 和果蝇 Sir2 的脱乙酰化活性[1]。

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Selisistat在哺乳动物细胞中的特异性研究[1]
为了确定selisistat对sirtuins的特异性和活性，我们用GCN5（一种组蛋白乙酰转移酶）和核因子κB (NFκB) p65亚基（一种特征的SirT1底物）转染HEK293细胞。通过在转染细胞中乙酰化的p65与总p65蛋白的比例可以看出，GCN5积极地使p65乙酰化。当人类SirT1也与GCN5和p65共转染时，p65乙酰化水平降低了约80%。当果蝇Sir2共转染到细胞中时，p65的GCN5乙酰化降低了约70%。在这些细胞中添加selisistat可抑制SirT1去乙酰化，在10 μm处恢复50%的p65乙酰化。同样，selisistat阻断果蝇Sir2对p65去乙酰化的能力，被抑制的乙酰化活性恢复60%。这些数据表明，selisistat抑制果蝇sirr2和人类SirT1的去乙酰化活性。

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Selisistat对培养的HD动物细胞模型具有保护作用[1]
考虑到基因降低Sir2对果蝇HD病理的积极作用，我们试图确定selisistat是否在哺乳动物HD模型中表现出积极作用。表达mHtt外显子1片段的大鼠嗜铬细胞瘤细胞（PC-12）被广泛用于研究mHtt的毒性和聚集性。PC-12细胞诱导表达人类Htt外显子1片段，扩增聚谷氨酰胺重复序列，在转基因表达后出现聚集、转录变化和细胞毒性。在该模型中，诱导mHtt表达导致毒性（以乳酸脱氢酶[LDH]释放来测量）的显著增加，而用浓度为1 μm和10 μm的selisistat处理可显著降低毒性。

在亨廷顿病 (HD) 的 R6/2 小鼠模型中，硒尼司他（每日口服 5 和 20 mg/kg；转基因 R6/2 小鼠从 4.5 周龄开始直至死亡）具有保护作用[1]。

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在本研究中，Selisistat (SEN0014196；ex527)(5µg/kg)，每周给药两次，连续10周，降低血清甘油三酯（TG）、总胆固醇、丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）水平，减轻肝纤维化，马松三色和肝脂肪油红- o染色证实。EX-527上调HFD喂养大鼠肝脏中SIRT2、SIRT3和SIRT4的表达，下调转化生长因子-β1 （TGF-β1）和α-平滑肌肌动蛋白（α-SMA）的表达。它降低了血清中促炎细胞因子的产生和羟脯氨酸水平以及SMAD4的表达，恢复了凋亡蛋白（Bcl-2、Bax和cleaved caspase-3）的表达。这些数据表明SIRT4/SMAD4轴在肝纤维化中起关键作用。SIRT4上调有可能对抗hfd诱导的脂质积累、炎症和纤维形成。我们证明EX-527在抑制hfd诱导的肝纤维化进展方面是一个有希望的候选药物。[3]

I类和II类HDAC荧光测定。[2]
使用含有I类和II类HDAC的HeLa细胞提取物和H4- k16 （Ac）底物（代表赖氨酸16上乙酰化的组蛋白H4残基12−16），在上述荧光法中测量了I类和II类HDAC去乙酰化酶的活性。

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烟酰胺释放试验。[2]
SIRT1的活性是用非荧光法测定的，使用的是代表赖氨酸382乙酰化残基368 - 386的p53肽底物。如前所述，该实验测量了[羰基-14C]-NAD中[14C]烟酰胺的释放。 在未标记烟酰胺浓度为52 μM的情况下，使用上述方法测量烟酰胺交换。添加的烟酰胺通过酶催化交换促进[14C]烟酰胺从标记的NAD释放。[14C]烟酰胺从NAD释放后，未标记的烟酰胺与酶结合并转化为未标记的NAD。
NAD糖水解酶（NADase）酶活性在烟酰胺释放试验中测量，如上所述。采用阴离子交换色谱法纯化猪脑NADase粗酶。每孔含有0.5 μg纯化酶和NAD，浓度为18.55 μM （KM的70%）。

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微粒体的稳定性。[2]
利用大鼠肝微粒体评估体外代谢稳定性。将浓度为10 μM的化合物与大鼠肝微粒体（1 mg蛋白质/mL）在37°C下孵育，并在0、5、15、30和60 min后用HPLC/MS进行定量。对照组不含微粒体。

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细胞色素P450抑制试验。[2]
细胞色素P450检测在384孔微孔板上进行，使用重组人同工酶3A4、2D6、1A2、2C9和2C19与荧光底物孵育，如前所述。

hERG试验。[2]
用hERG钾通道稳定转染中国仓鼠卵巢（CHO）细胞。hERG通道的阻断引起膜电位的变化，这是用电位测定染料测量的。染料负载细胞用10 μM化合物和2 mM氯化钾孵育。使用Tecan Safire荧光仪测量384孔微孔板格式的荧光变化。测量了化合物对缺乏hERG通道的对照CHO细胞的影响，并用于纠正非特异性猝灭和毒性。

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表达PC-12外显子1的突变/野生型Htt细胞株[1]
从Rubinsztein教授的实验室获得了稳定表达人类HD基因gfp标记的外显子1片段的PC-12 7210（外显子1 mutQ74）细胞（PC12-Q74）（24）。四环素（Tet-on）诱导的mHTT构建物包括人类Htt序列（NM_00211）的核苷酸1-297，并包括74 CAG重复扩增，一旦表达，对细胞具有毒性。将细胞接种于96孔聚d-赖氨酸（MW 70-150 kDa）预涂板上，密度为45K细胞/100 μl培养基/孔，培养液中含有2% HS、1% FBS、100 mU/ml青霉素/链霉素和1%谷氨酰胺，实验前在37℃、90%相对湿度、10% CO2气氛的培养箱中培养24 h。实验当天，将不含血清的相同培养基添加到孔中，以使先前的血清浓度最终稀释为1:3。在无血清培养基中加入强力霉素（终浓度为1 μg/ml）进行转基因诱导。加入selisistat（从DMSO 10 mm原液中加入）以获得结果中描述的最终浓度，在仅接受DMSO的对照组中省略其添加。所有处理和对照的DMSO终浓度均为0.1%。72h时，用LDH- mix细胞毒性测试试剂盒测定培养基中细胞释放的LDH水平，用分光光度计测定490 nm（读数）和720 nm（空白）处的吸光度

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慢病毒感染培养纹状体神经元[1]
采用慢病毒载体实现HD体外模型（27）。这些模型涉及慢病毒介导的纹状体神经元培养中野生型Htt（18个谷氨酰胺重复序列，18Q）或mHtt(82个谷氨酰胺重复序列，82Q) n端171个氨基酸片段的过表达。对于慢病毒介导的蛋白表达，在播种后24小时感染培养物。第4天，将一半培养基替换为添加2x浓度selisistat的新鲜培养基。此后每周进行1次复方处理，加入1倍浓度的复方新鲜培养基。强启动子结构（高表达，5-10倍内源性）在体外2-4周内导致polyq依赖性细胞死亡，通过降低neun阳性和neun阳性细胞数量来评估。在体外1-2周（高表达）或2-4周（中等表达）时，暴露于Htt- n171 - 82q而非Htt- n171 - 18q的细胞也会出现细胞内Htt包涵体。

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HEK293细胞转染及治疗[1]
HEK 293-T细胞在含有10% FBS， 1% Penstrep， 1% G-Max的DMEM中生长，37℃，10% CO2。将8 × 105个细胞接种在MW6板上，24 h后，按照制造商的说明，用Lipofectamine 2000转染2.5µg总质粒DNA。从OriGene Technologies购买表达GCN5 （NM_021078.1）、p65 （NM_021975.3）、human_SirT1 （NM_012238.4）的质粒，从DGRC订购表达果蝇基因Sir2 cDNA （LD07439）的质粒，克隆到pcDNA载体中。转染4小时后，取出Opti-MEM培养基，将selisistat在培养基中稀释至0.1、1和10µm （DMSO 0.1%, v/v，作为对照），加入细胞中。转染24 h后，收集细胞，用RIPA缓冲液（150 mm NaCl, 1.0% NP-40, 0.5%脱氧胆酸钠，0.1% SDS, 50 mm Tris, pH 8.0）和蛋白酶和磷酸酶抑制剂（Complete EDTA-free蛋白酶抑制剂鸡尾酒，Roche和PhosSTOP抑制剂鸡尾酒，Roche）裂解。总裂解物在3000g下离心5分钟澄清，并根据制造商的说明用BCA定量蛋白质量。

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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Dissolved in DMSO; ~5 μg/rat; Intracerebroventricular injection

Male Sprague-Dawley rats

PK results [4]
Single oral doses of 5 to 600 mg selisistat were rapidly absorbed by male subjects in the fasted condition, although the rate of absorption appeared to be dose-dependent with a median tmax of selisistat increasing from 1 h post-dose at 5 mg to 4 h post-dose at 600 mg (Figure 1). Elimination of selisistat occurred in a biphasic manner, with an apparent terminal plasma half-life that appeared to increase with dose (mean values ranging from 1.6 h at 5 mg to 6.1 h at 600 mg). The AUC(0,∞) of selisistat increased in a dose proportional manner over the 5 to 300 mg dose range, with a marked increase in supra-proportionality between the 300 and 600 mg dose levels, suggesting that one or more clearance mechanisms are approaching saturation at higher doses (Figure 2A and Table 2). The fraction of unchanged drug excreted in the urine with respect to dose was low for all dose levels in male subjects, with <0.02% being eliminated up to 24 h post-dose at each dose level. Following multiple dosing, the fraction of the dose excreted in the urine remained low, but increased with time, consistent with the plasma accumulation observed. Food had a minimal effect on the single dose pharmacokinetics of selisistat in male subjects. Following a high fat breakfast, the rate of absorption was delayed, whereas the extent of absorption was largely unchanged.
The multiple oral dose pharmacokinetics of selisistat showed no dose or time dependency in tmax or apparent terminal half-life. At each dose level, the morning trough selisistat plasma concentrations for individual subjects showed that steady-state was generally achieved by day 4. Consistent with the single dose finding, a supra-proportional increase in steady-state AUC(0,τ) was observed across the 100 mg once daily to 300 mg once daily range (Figure 2B), whilst the steady-state Cmax increased in a dose-proportional manner. Furthermore, the steady-state AUC(0,τ) was approximately two-fold higher for the 100 mg twice daily dose level as compared with the 100 mg once daily dose level (Table 3).
In the single dose phase, between-subject variability (%CV) in terms of AUC(0,∞) and Cmax was 35–71% and 23–46%, respectively. Across all dose levels, the pooled between-subject variability for AUC(0,∞) and Cmax was 56% and 33%, respectively. In the multiple dose phase, between-subject variability (%CV) was 17–59% in males and 28–68% in females. Systemic exposure following both single and multiple dosing was higher in females than in males. AUC(0,∞), AUC(0,τ) and Cmax values were 1.1-fold, 2.2–2.3-fold and 1.7–1.9-fold higher in females than inmale subjects. There were no differences in systemic exposure or pharmacokinetic parameter estimates between Caucasian and non-Caucasians subjects.

Safety [4]
There were no serious adverse events reported during the study and no subjects were withdrawn due to adverse events. Single oral doses of selisistat were considered to be safe and well tolerated by healthy male subjects when administered at doses up to 600 mg, and by female subjects when administered at a dose of 300 mg selisistat (Table 5). Multiple oral doses of selisistat were also considered to be safe and well tolerated by healthy male subjects at doses up to 300 mg once daily for 7 days and by healthy female subjects when administered doses of 100 mg twice daily for 7 days. There was a low incidence of drug related adverse events in male subjects, with no increase in the number of subjects experiencing adverse events with increasing dose of selisistat. The incidence of adverse events did not exceed that observed in the placebo group. No increase in the number of adverse events reported was observed following administration of multiple doses of selisistat compared with single doses (Table 6). The majority of adverse events reported by male and female subjects were mild in severity and resolved without treatment. Only one adverse event graded as severe in intensity occurred during the study. One 18-year-old male subject experienced an episode of postural syncope 1 h and 18 min after dosing at 150 mg. This event was considered possibly related to the study drug by the investigator. Dietary state had no effect on adverse events. Following single oral doses of selisistat, the most frequent drug-related adverse event was headache, experienced by 12% of male subjects and 83% of female subjects. Following multiple oral doses of selisistat, the incidence of adverse events was low in male subjects. In female subjects, three out of six subjects reported at least one incident of gastrointestinal complaint. Overall, adverse events were more frequently reported in females than in males on drug and on placebo (Tables 5 and 6). There were no dose- or treatment-related trends in terms of clinical laboratory evaluations, including liver function tests, haematological parameters, vital signs or cardiac function. Specifically, no treatment or dose-related trends in parameters recorded on 12-lead safety ECGs were noted and there were no clinically relevant findings in the ECG morphology at any dose level of selisistat. There were no subjects with a QTc interval >480 ms or an increase from baseline >60 ms as assessed from the 12-lead safety ECGs. There were no clinically significant findings in physical examinations, postural control or neurological examinations and no changes in sway platform performance.

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Concentration−effect modelling of ECG parameters [4]
The variability of the QTc data measured as the standard deviation of the between-subject ΔQTcF was low, 5.3 ms and 6.8 ms in the single ascending dose (SAD) and multiple ascending dose (MAD) parts, respectively 15. The change from baseline QTcF across dose groups in part 1, in which the highest plasma concentrations were achieved, is shown in Table 7. The pattern across time points and dose groups did not suggest a dose-dependent effect of selisistat on the QTc interval. No significant concentration-dependent effect on ΔΔQTcF was seen after single doses from 5 mg to 600 mg of selisistat within the observed plasma concentration range. A linear model with an intercept provided an acceptable fit of the data and the estimated population intercept and slope were 0.9 ms (90% CI −0.2, 2.0) and −0.00026 ms per ng ml−1 (90% CI −0.00063, 0.00010), respectively (Figure 3A). The analysis of data from the MAD part provided similar results with an intercept of 2.8 ms (90% CI −0.16, 5.71) and an estimated slope of −0.00011 ms per ng ml−1 (90% CI −0.00087, 0.00066; Figure 3B). The ΔΔQTcF effect at the observed geometric Cmax of 26.6 μm after a single dose of 600 mg using this model can be predicted to −0.9 ms (90% CI −3.3, 1.4). A ΔΔQTcF effect of approximately 2.8 ms (90% CI −0.1, 5.6) can be predicted for the observed Cmax level of 22.5 μm after 7 days of dosing of 300 mg once daily. For plasma concentrations exceeding the mean Cmax level, e.g. 30 μm, a QTcF effect of 3.7 ms (90% CI −0.1, 7.5) can be predicted using the same model. The upper bound of the 90% CI of the projected ΔΔQTcF effect was below 10 ms for all plasma concentrations observed in both the SAD and the MAD part of the study (Figure 3A, B).

[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 member of the class of carbazoles that is 2,3,4,9-tetrahydro-1H-carbazole which is substituted at position 1 by an aminocarbohyl group and at position 6 by a chlorine. It is a member of carbazoles, a monocarboxylic acid amide and an organochlorine compound.
Selective inhibitor of SIRT1 that does not inhibit histone deacetylase (HDAC) or other sirtuin deacetylase family members (IC50 values are 98, 19600, 48700, > 100000 and > 100000 nM for SIRT1, SIRT2, SIRT3, HDAC and NADase respectively). Enhances p53 acetylation in response to DNA damaging agents.

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High-throughput screening against the human sirtuin SIRT1 led to the discovery of a series of indoles as potent inhibitors that are selective for SIRT1 over other deacetylases and NAD-processing enzymes. The most potent compounds described herein inhibit SIRT1 with IC50 values of 60-100 nM, representing a 500-fold improvement over previously reported SIRT inhibitors. Preparation of enantiomerically pure indole derivatives allowed for their characterization in vitro and in vivo. Kinetic analyses suggest that these inhibitors bind after the release of nicotinamide from the enzyme and prevent the release of deacetylated peptide and O-acetyl-ADP-ribose, the products of enzyme-catalyzed deacetylation. These SIRT1 inhibitors are low molecular weight, cell-permeable, orally bioavailable, and metabolically stable. These compounds provide chemical tools to study the biology of SIRT1 and to explore therapeutic uses for SIRT1 inhibitors.[2]

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Sirtuin (SIRT) is known to prevent nonalcoholic fatty liver disease (NAFLD); however, the role of SIRT4 in the progression of hepatic fibrosis remains unknown. We hypothesize that EX-527, a selective SIRT1 inhibitor, can inhibit the progression of high-fat diet (HFD)-induced hepatic fibrosis. We found that SIRT4 expression in the liver of NAFLD patients is significantly lower than that in normal subjects. In this study, 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] Aim: Selisistat (SEN0014196), a first-in-class SirT1 inhibitor, is being developed as a disease-modifying therapy for Huntington's 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).[4]

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

2.5

58.9

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (10.05 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (10.05 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (10.05 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 1% DMSO+30% polyethylene glycol+1% Tween 80:14mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。