SIRT1

(R)-selisistat is a 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide that has R configuration. It is the inactive enantiomer. It is an enantiomer of a (S)-selisistat.

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.[Cells. 2020 Apr 29;9(5):1101.]

Class I and II HDAC Fluorimetric Assay. [1]
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. [1]
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. [1]
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. [1]
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.

In Vivo Pharmacokinetic Analysis. [1]
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.

Pharmacokinetic Results [Br J Clin Pharmacol. 2015 Mar;79(3):477-91.]
In male subjects, after a single oral dose of 5 to 600 mg of selisistat in a fasting state, the drug was rapidly absorbed, but the absorption rate appeared to be dose-dependent, with the median time to peak concentration (tmax) of selisistat 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). The elimination of selisistat 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 selisistat increased dose-proportionately in the dose range of 5 to 300 mg, and the increase in AUC(0,∞) was significantly greater than that at dose levels between 300 and 600 mg dose levels, suggesting that one or more clearance mechanisms approach saturation at high doses (Figure 2A and Table 2). In all male subjects, the urinary excretion of the unchanged drug was low, with less than 0.02% excreted within 24 hours after administration at all dose levels. Following multiple administrations, the urinary excretion of the unchanged drug 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 administrations showed no dose- or time-dependent effects on tmax and apparent terminal half-life. At each dose level, the morning trough plasma concentration of selisistat typically reached steady state on day 4. Consistent with single-dose results, steady-state AUC(0,τ) increased proportionally over a 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 coefficients of variation (%CV) for AUC(0,∞) and Cmax between subjects were 35%–71% and 23%–46%, respectively. At all dose levels, the pooled inter-subject variability for AUC(0,∞) and Cmax was 56% and 33%, respectively. In the multiple-dose phase, the inter-subject variability (%CV) was 17%–59% for male subjects and 28%–68% for female subjects. After both single and multiple doses, systemic exposure was higher in women than in men. 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 female subjects than in male subjects, respectively. There were no differences in systemic exposure or pharmacokinetic parameter estimates between Caucasian and non-Caucasian subjects.

Safety [Br J Clin Pharmacol. 2015 Mar;79(3):477-91.]
No serious adverse events were reported during the study, and no subjects withdrew from the study due to adverse events. Single oral doses of selisistat 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 selisistat up to 300 mg once daily for 7 days were also 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 the number of subjects experiencing adverse events did not increase with increasing selisistat 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 of celisstat compared to a single dose (Table 6). Most adverse events reported by both male and female subjects were mild and resolved without treatment. Only one serious adverse event occurred during the study period. An 18-year-old male subject experienced orthostatic syncope 1 hour and 18 minutes after taking 150 mg of celisstat. The investigators considered this event to be likely 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 celisstat was headache, occurring in 12% of male subjects and 83% of female subjects. The incidence of adverse events was lower in male subjects after multiple oral doses of celisstat. Among female subjects, 3 out of 6 subjects reported at least one gastrointestinal upset. Overall, the frequency of adverse event reports was higher in women taking the drug and in men taking 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 observed in the parameters recorded by the 12-lead safety ECG, and no clinically significant abnormalities were found in the ECG morphology at any dose level of celisilatal treatment. Based on the 12-lead safety ECG assessment, no subjects had a QTc interval >480 ms or an increase >60 ms from baseline. No clinically significant abnormalities were found in physical examination, postural control, or neurological examination, and the rocking platform test performance remained unchanged.

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Concentration-effect model of ECG parameters [Br J Clin Pharmacol. 2015 Mar; 79(3):477-91.]
QTc data showed low variability as measured by the standard deviation of ΔQTcF between subjects, with 5.3 ms for the single-increment (SAD) portion and 6.8 ms for the multiple-increment (MAD) portion. Table 7 shows the changes in QTcF relative to baseline for each dose group in Part 1 (highest plasma concentration). Patterns at various time points and dose groups indicated that selisistat had no dose-dependent effect on the QTc interval. Within the observed plasma concentration range, no significant concentration-dependent effect of ΔΔQTcF was observed after a single administration of 5 mg to 600 mg selisistat. Linear models with intercepts fitted the data well, with estimated overall intercepts and slopes 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, at an observed geometric Cmax of 26.6 μm, was −0.9 ms (90% CI −3.3, 1.4). After a once-daily 300 mg dose for 7 consecutive days, with an observed Cmax of 22.5 μM, the predicted ΔΔQTcF effect was approximately 2.8 ms (90% CI −0.1, 5.6). For plasma concentrations exceeding the mean Cmax (e.g., 30 μM), using the same model, the predicted QTcF effect was 3.7 ms (90% CI −0.1, 7.5). In both the single-dose (SAD) and multiple-dose (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).

[1]. Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1. J Med Chem. 2005 Dec 15;48(25):8045-54.

(R)-selisistat is a 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide with the R configuration. It is an inactive enantiomer and also an enantiomer of (S)-selisistat.

C13H13CLN2O

248.7081

248.071

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

848193-69-1

Selisistat;49843-98-3;(S)-Selisistat;848193-68-0

707032

Light yellow to yellow solid powder

1.4±0.1 g/cm3

531.7±38.0 °C at 760 mmHg

275.4±26.8 °C

0.0±1.4 mmHg at 25°C

1.688

2.22

2

1

1

17

323

1

C1C[C@H](C2=C(C1)C3=C(N2)C=CC(=C3)Cl)C(=O)N

FUZYTVDVLBBXDL-SECBINFHSA-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)/t9-/m1/s1

(1R)-6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide

848193-69-1; (R)-selisistat; EX-527 (R-enantiomer); (R)-6-Chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; Selisistat R-enantiomer; (1R)-6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; Selisistat, (R)-; EX-527 R-enantiomer;

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)

DMSO : ~100 mg/mL (~402.07 mM)

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