Caspase; caspase-3

Emricasan, also known as IDN-6556 or PF-03491390, is a sub- to nanomolar active inhibitor of activated caspases. While Emricasan exhibits neuroprotective activity for hNPCs, ZIKV replication is not prevented[2].

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Emricasan, a pan-caspase inhibitor, was identified as the most potent anti-death compound with IC50 values of 0.13 – 0.9 μM in both caspase activity and cell viability assays for SNB-19 cells against three ZIKV strains: MR766 (1947 Ugandan strain), FSS13025 (2010 Cambodian strain), and PRVABC59 (2015 Puerto Rican strain) (Fig. 1a). It was also effective for all three cell types tested (Supplementary Fig. 1f and Supplementary Table 5). In addition, Emricasan reduced the number of active (cleaved) caspase-3-expressing forebrain-specific hNPCs infected by FSS13025 in both monolayer and 3D organoid cultures (Fig. 1b–c). Emricasan treatment of ZIKV-exposed brain organoids did not appear to affect hNPC proliferation compared to the mock treatment, as evaluated by phospho-Histone3 (PH3) expression (106 ± 10%; n = 8; P = 0.7; One-way ANOVA). Notably, ZIKV antigen persisted in both 2D and 3D cultures after Emricasan treatment (Fig. 1b–c). Therefore, Emricasan displays neuroprotective activity for hNPCs, but does not suppress ZIKV replication.[2]

Emricasan reduces NASH-related liver damage but not metabolic disturbance. It lessens inflammation as well. The pan-caspase inhibitor Emricasan reduces hepatic fibrogenesis and stellate cell activation in the murine NASH model[1]. Phase 2 clinical trials are currently being conducted on emricasan to determine whether it can lessen the hepatic damage and liver fibrosis brought on by chronic HCV infection[2].

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Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-β, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. Conclusion: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.[2]

A quantitative high-throughput screening (qHTS), in which each compound was assayed in four concentrations (0.37, 1.84, 9.2, and 46 μM), was performed in singlet for the primary compound screen. While a single compound concentration (in singlet) has been traditionally used for HTS of large compound collections (such as 1 to 3 million compounds), the qHTS format with multiple compound concentrations has recently been used for medium or small compound collections such as approved drug library. Specifically, SNB-19 cells and hNPCs were seeded onto PDL coated 1536-well assay plates at 250 cells per 3 μl/well and incubated at 37°C in 5% CO2 for 16 hours. Test compounds dissolved in DMSO were transferred to assay plates at a volume of 23 nl/well by an automated pintool workstation. Compounds were incubated with cells for 30 minutes at 37°C in 5% CO2, immediately followed by the addition of 2 μl/well of ZIKV (2 FFU/cell). Incubation time of compound-treated cells with ZIKV varied based on assay format. Experiments measuring virus-induced caspase-3/7 activity required a 6-hour incubation of ZIKV in the presence compounds at 37°C in 5% CO2. Following this incubation, 3.5 μl/well of caspase-3/7 reagent mixture was added to assay plates. The plates were incubated for 30 minutes at room temperature, and the resultant luminescence signal was measured using a ViewLux plate reader. Experiments measuring virus-induced cell death required a 72-hour incubation of ZIKV in the presence of compounds at 37°C in 5% CO2. Following this incubation, 3.5 μl/well of ATP content detection reagent was added to assay plates. The plates were incubated for 30 minutes at room temperature, and the resultant luminescence signal was measured in a ViewLux plate reader. Step-by-step assay protocols are listed in Supplementary Tables 1 and 2.[2]

Astrocytes are mock-infected, treated with DMSO, or treated with 2 M niclosamide, 92 M PHA-690509, 9 μM emricasan, or a combination of 92 μM PHA-690509 and 9 M emricasan for 1 h prior to infection with PRVABC59 (MOI = 0.5). Following infection for 24 hours, cells are fixed and stained for ZIKVE and nuclei.

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ATP content assay for cell viability and compound cytotoxicity[2]
The ATPlite luminescence assay system assay kit was used to determine cell viability. The reagent was reconstituted and prepared as described by the manufacturer. In order to measure the cell death caused by ZIKV infection, cells were cultured for 16 hours at 37°C with 5% CO2 in assay plates, followed by addition of ZIKV solution and incubation at 37°C with 5% CO2 for 72 hours. ATPlite, the ATP monitoring reagent, was then added to the assay plates and they were incubated for 15 minutes. The resulting luminescence was measured using the ViewLux plate reader. Data were normalized using wells without cells as a control for 100% cell killing, and cell-containing wells without ZIKV infection as full cell viability (0% cell killing). For analysis of potential toxicity of select compounds, cells were seeded in 96-well plates. One day later, cells were treated with indicated compounds and concentrations for 24–48 hours prior to the addition of Cell Titer-Glo substrate and measured according to manufacturer instructions.

Reagents and formulation [1]
Emricasan (formerly named IDN-6556 or PF-03491390) was suspended in vehicle [2% (v/v) DMSO in 0.5% (w/v) methylcellulose] and administered to mice per os daily. The high fat diet (HFD) used was used, which contains 47% of calories from fat (mostly from Milk fat, 50% saturated fat) with 2% of cholesterol, 35% from carbohydrate (78% of carbohydrate from Sucrose) and 18% of calories from protein, and was designed to approximate the typical human diet from patients with NASH.

Animals[1]
Studies were performed in male C57BL/6J mice. All animals were maintained in a temperature (24°C) and light controlled (12:12 h light:dark) facility, and had free access to food and water. Animals were age-matched and used at approximately 12–16 weeks of age. Four groups were studied (n = 60) with 15 mice per group. Groups 1 and 3 received regular chow. Groups 2 and 4 received HFD and 50 g/L (Sucrose) was added to drinking water for 20 weeks. Groups 3 and 4 received Emricasan 0.3 mg/kg/day per os, and Group 1 and 2 received the vehicle. The dosing was based on previous data 21 that demonstrates that oral administration of Emricasan at doses of 0.3 mg/kg corresponded to the ED90 value to prevent liver injury in the model of α-Fas-induced liver injury. Total body weight was measured at 0, 5, 10, 15 and 20 weeks.

Emricasan Pharmacokinetics [https://pmc.ncbi.nlm.nih.gov/articles/PMC6175779/]
A summary of the key pharmacokinetic data is presented in Table 1. The geometric means of the emricasan AUC0–8, Cmax, and AUC0–last increased in an approximately dose-proportional manner between the 5 mg and 50 mg doses on day 1 and day 4. No plasma accumulation was apparent in any of the treatment arms on day 4 compared to day 1. Generally, lower between-subject variability was observed in all PK parameters in the lower dose treatment arms than in the 50 mg arm, with coefficients of variation (CVs) ranging from 28% to 48% in the IDN-6556 5 mg and 25 mg groups, and from 99% to 258% in the IDN-6556 50 mg group.

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Pharmacokinetic analysis in the rat demonstrated rapid clearance after i.v., i.p., and s.c. administration with terminal t(1/2) ranging from 46 to 51 min. Low absolute bioavailability after p.o. administration was seen (2.7-4%), but portal drug concentrations after oral administration were 3-fold higher than systemic concentrations with a 3.7-fold increase in the terminal t(1/2), indicating a significant first-pass effect. Liver concentrations remained constant after oral administration for at least a 4-h period, reaching a C(max) of 2558 ng/g liver at 120 min. Last, 51 +/- 20 and 4.9 +/- 3.4% of IDN-6556 was excreted intact in bile after i.v. and p.o. administration, respectively. This evaluation indicates that IDN-6556 has marked efficacy in models of liver disease after oral administration and thus, is an excellent candidate for the treatment of liver diseases characterized by excessive apoptosis. [https://pubmed.ncbi.nlm.nih.gov/14742742/]

Safety [https://pmc.ncbi.nlm.nih.gov/articles/PMC6175779/]
As demonstrated in Figure 2, there were a total of 10 deaths across all treatment groups. There were 5 on-study deaths, 2 of which were during the one-month follow-up period following the full course of treatment, and a further 5 were registered via serious adverse event (SAE) reporting following discontinuation or study completion. All of these deaths were attributed to progressive liver disease. Adverse events (AEs) were reported by 17 of the 21 patients, of whom 13 patients reported SAEs. None of the SAEs was determined to be treatment-related. The only AEs deemed to be treatment-related were nausea and vomiting which were reported by one placebo subject. The AEs and SAEs are presented in Table 5.

[1]. Liver Int . 2015 Mar;35(3):953-66.

[2]. Nat Med . 2016 Oct;22(10):1101-1107.

Emricasan is the first caspase inhibitor tested in human which has received orphan drug status by FDA. It is developed by Pfizer and made in such a way that it protects liver cells from excessive apoptosis.

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Drug Indication
Investigated for use/treatment in hepatitis (viral, C), liver disease, and transplantation (organ or tissue).
Treatment of non-alcoholic steatohepatitis (NASH)

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Mechanism of Action
IDN-6556 significantly improves markers of liver damage in patients infected with the Hepatitis C virus (HCV), an infection that affects up to 170m patients worldwide. IDN-6556 represents a new class of drugs that protect the liver from inflammation and cellular damage induced by viral infections and other causes. Various studies have also shown that it significantly lowers aminotransferase activity in HCV patients and appeared to be well tolerated.

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Background & aims: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. Methods: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan.
Results: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-β, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR.
Conclusion: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.[1]

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In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ∼6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development.[2]

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Background: Cirrhosis and acute-on-chronic liver failure (ACLF) are associated with systemic inflammation, and caspase-mediated hepatocyte cell death. Emricasan is a novel, pan-caspase inhibitor. Aims of this study were to assess the pharmacokinetics, pharmacodynamics, safety and clinical outcomes of emricasan in acute decompensation (AD) of cirrhosis.
Methods: This was a phase 2, multicentre, double-blind, randomized trial. The primary objective was to evaluate the pharmacokinetics, pharmacodynamics and safety of emricasan in patients with cirrhosis presenting with AD and organ failure. AD was defined as an acute decompensating event ≤6 weeks' duration. Patients were randomized proportionately to emricasan 5 mg bid, emricasan 25 mg bid, emricasan 50 mg bid or placebo. Treatment was continued to 28 days, or voluntary discontinuation.
Results: Twenty-three subjects were randomized, of whom 21 were dosed (placebo n = 4; 5 mg n = 5; 25 mg n = 7; 50 mg n = 5). Pharmacokinetic data showed 5 mg dose was associated with low plasma levels (<50 ng/ml), and 25 mg and 50 mg doses showed comparable pharmacokinetic profiles. Therefore, for analysis of secondary endpoints, placebo and 5 mg groups were merged into a 'placebo/low-dose' group, and 25 mg and 50 mg groups were merged into a 'high-dose' group. Five deaths occurred amongst the 21 patients, all due to progression of liver disease (2 in placebo/low-dose, 3 in high-dose). No statistically significant changes from baseline MELD score or CLIF-C ACLF score were noted between placebo/low-dose and high-dose groups at day 7 (MELD -1 vs -1, CLIF-C ACLF 0.7 vs 0.8). An initial reduction in cleaved keratin M30 fragment was noted between placebo/low-dose and high-dose groups (percent relative change: day 2: -11.6 vs -42.6, P = 0.017, day 4: -3.5 vs -38.9 P = 0.017) although this did not persist to day 7 (-3.1 vs -20.8, P = 0.342).
Conclusion: This study demonstrates that emricasan is safe and well tolerated in advanced liver disease. However, this study fails to provide proof-of-concept support for caspase inhibition as a treatment strategy for ACLF.https://pubmed.ncbi.nlm.nih.gov/30302038/

C26H27F4N3O7

569.5021

569.178

C, 54.83; H, 4.78; F, 13.34; N, 7.38; O, 19.66

254750-02-2

254750-02-2;

12000240

white solid powder

1.4±0.1 g/cm3

1.550

4.63

4

11

11

40

934

2

FC1C(=C([H])C(=C(C=1OC([H])([H])C([C@]([H])(C([H])([H])C(=O)O[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C(C(N([H])C1=C([H])C([H])=C([H])C([H])=C1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)=O)=O)=O)F)F)F

SCVHJVCATBPIHN-SJCJKPOMSA-N

InChI=1S/C26H27F4N3O7/c1-12(31-24(38)25(39)32-16-8-6-5-7-13(16)26(2,3)4)23(37)33-17(10-19(35)36)18(34)11-40-22-20(29)14(27)9-15(28)21(22)30/h5-9,12,17H,10-11H2,1-4H3,(H,31,38)(H,32,39)(H,33,37)(H,35,36)/t12-,17-/m0/s1

(3S)-3-[[(2S)-2-[[2-(2-tert-butylanilino)-2-oxoacetyl]amino]propanoyl]amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid

Emricasan; PF 03491390; PF-03491390; IDN-6556; (S)-3-((S)-2-(2-(2-TERT-BUTYLPHENYLAMINO)-2-OXOACETAMIDO)PROPANAMIDO)-4-OXO-5-(2,3,5,6-TETRAFLUOROPHENOXY)PENTANOIC ACID; (S)-3-((S)-2-(2-((2-(tert-Butyl)phenyl)amino)-2-oxoacetamido)propanamido)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid; C26H27F4N3O7; PF03491390; IDN-6556; IDN6556; IDN 6556

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 (~175.6 mM)
Ethanol: ~25 mg/mL (~43.9 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.39 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (4.39 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (4.39 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: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 5mg/ml

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