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.

PK results [Br J Clin Pharmacol. 2015 Mar;79(3):477-91.]
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 [Br J Clin Pharmacol. 2015 Mar;79(3):477-91.]
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 [Br J Clin Pharmacol. 2015 Mar;79(3):477-91.]
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]. 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 that has R configuration. It is the inactive enantiomer. It is an enantiomer of a (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.)