PPARγ; CB2

VCE-004.8 activates the HIF pathway by stabilizing HIF-1α and HIF-2α proteins in human microglial (HMC3), human dermal microvascular endothelial (HMEC-1), and oligodendrocyte (MO3.13) cell lines, as shown by Western blot. [1]
VCE-004.8 concentration-dependently activates the EPO gene promoter (luciferase reporter assay) in NIH3T3-EPO-Luc cells. This activation is reversible upon compound washout. [1]
VCE-004.8 inhibits the prolyl hydroxylase activity of PHD1 and PHD2 (but not PHD3), thereby reducing HIF-1α hydroxylation and promoting its stabilization, as demonstrated in an in vitro hydroxylation assay using recombinant GST-HIF-1α protein and immunoprecipitated PHDs. [1]
In human brain microvascular endothelial cells (HBMEC), VCE-004.8 (5 µM) upregulates the expression of numerous HIF-dependent genes, including EPO, VEGFA, ADM, PLAU, ANGPTL4, SLC2A1, and NDRG1, as shown by PCR array and qRT-PCR. [1]
VCE-004.8 upregulates VEGFA and EPO mRNA expression and induces VEGFA protein secretion in HMEC-1 and MO3.13 cells in a concentration-dependent manner. [1]
VCE-004.8 (1 µM) promotes angiogenesis in vitro, evidenced by increased endothelial tube formation in HUVEC/fibroblast co-cultures and enhanced branch point formation in HBMEC cells cultured on Matrigel, comparable to the effect of recombinant VEGFA (10 ng/ml). [1]
VCE-004.8 enhances the migration of differentiated MO3.13 oligodendrocytes in a wound-healing assay. Conditioned medium from VCE-004.8-treated HBMEC cells also promotes oligodendrocyte migration, an effect partially blocked by an anti-VEGFA neutralizing antibody. [1]
VCE-004.8 inhibits IL-17-induced M1 macrophage polarization in RAW264.7 cells, reducing the expression of pro-inflammatory markers (TNF-α, IL-6, CCL2, CCL4). [1]
VCE-004.8 induces the expression of arginase 1 (Arg-1) in RAW264.7 and BV2 microglial cells, both alone and in synergy with IL-4. This induction occurs at the transcriptional level and is independent of PPARγ and CB2 receptors, as it is not blocked by their respective antagonists (GW9662 and SR144528). [1]
VCE-004.8 (1-10 µM) inhibits LPS-induced PGE2 release and COX-2 protein expression in primary rat microglial cells. [1]

Etrinabdione (i.p.; 20 mg/kg/day; for 3 weeks) causes a notable decrease in body weight gain, total fat mass, adipocyte volume, and plasma triglyceride levels over the course of three weeks in HFD mice. Additionally, etrinabdione has been shown to significantly improve glucose tolerance, lower levels of leptin, an adiposity marker, and raise levels of adiponectin, incretins (GLP-1 and GIP), and adiponectin[1].

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In the experimental autoimmune encephalomyelitis (EAE) mouse model (C57BL/6), therapeutic administration of VCE-004.8 (10 mg/kg/day, i.p., from day 8 post-immunization) significantly attenuated clinical disease scores compared to vehicle-treated mice. [1]
In the Theiler's murine encephalomyelitis virus (TMEV) model (SJL/J mice), therapeutic administration of VCE-004.8 (10 mg/kg/day, i.p., from day 60 post-infection for 14 days) restored impaired horizontal and vertical motor activity to levels comparable to sham-infected mice. [1]
Histopathological analysis of spinal cords from EAE and TMEV mice treated with VCE-004.8 showed a significant reduction in microgliosis (Iba-1+ cells), immune cell infiltrates (H&E staining), and CD4+ T cell infiltration (TMEV model) compared to vehicle-treated controls. [1]
VCE-004.8 treatment preserved myelin integrity (assessed by cryomyelin staining) and reduced axonal damage (assessed by neurofilament H staining) in the spinal cords of both EAE and TMEV mice. [1]
VCE-004.8 treatment modulated the expression of multiple genes associated with MS pathophysiology in the spinal cord of EAE and TMEV mice, downregulating pro-inflammatory chemokines, cytokines, chemokine receptors, and adhesion molecules. [1]
In normal C57BL/6 mice, administration of VCE-004.8 (10 mg/kg, i.p., for 3 weeks) significantly increased plasma levels of erythropoietin (EPO). [1]

HIF-1α hydroxylation assay: HEK293T cells were transfected with HA-tagged PHD1, PHD2, or PHD3 plasmids. After 24 hours, cells were treated with VCE-004.8, CBD (negative control), or DMOG (positive control). The PHDs were then immunoprecipitated using an anti-HA antibody. The immunoprecipitated PHDs were incubated with recombinant human GST-HIF-1α protein in a reaction buffer containing Tris-HCl, DTT, FeSO4, ascorbate, and oxoglutarate for 1 hour at 30°C. The reaction was stopped, and the levels of hydroxylated HIF-1α and total HIF-1α were analyzed by immunoblot. This assay demonstrated that VCE-004.8 strongly inhibited the prolyl hydroxylase activity of PHD1 and PHD2, but not PHD3. [1]

EPO-Luciferase Reporter Assay: NIH-3T3-EPO-luc cells (stably transfected with an EPO hypoxia response element-luciferase reporter) were stimulated with VCE-004.8 at indicated concentrations for 6 hours. Luciferase activity was then measured in cell lysates to assess HIF pathway activation. [1]
Western Blot for HIF and related proteins: Cells (e.g., HMC3, HMEC-1, MO3.13) were treated with VCE-004.8, washed with PBS, and lysed. Proteins (30 µg) were separated by SDS-PAGE, transferred to PVDF membranes, blocked, and incubated overnight at 4°C with primary antibodies against HIF-1α, HIF-2α, PHDs, PPARγ, arginase 1, β-actin, etc. After washing, membranes were incubated with HRP-conjugated secondary antibodies and detected by chemiluminescence. [1]
Quantitative PCR (qRT-PCR): Total RNA was extracted from treated cells or tissues, reverse transcribed to cDNA, and analyzed by real-time PCR using SYBR Green. Gene expression was normalized to housekeeping genes (GAPDH or HPRT) and calculated using the 2^(-ΔΔCt) method. This was used to analyze expression of cytokines, chemokines, HIF-target genes (VEGFA, EPO), and Arg-1. [1]
PCR Array: RNA from HBMEC cells or mouse spinal cord was reverse transcribed and analyzed using commercial PCR arrays (Human Hypoxia Signaling Pathway Plus or Mouse Multiple Sclerosis RT² Profiler). The arrays analyzed the expression of 84 pathway-related genes plus controls. Data were analyzed following the manufacturer's instructions to determine fold changes in gene expression. [1]
VEGF ELISA: HMEC-1 or MO3.13 cells were treated with VCE-004.8 for 24 hours. Culture supernatants were collected, and VEGF levels were quantified using a commercial ELISA kit according to the manufacturer's protocol. [1]
In vitro Angiogenesis Assay: Two methods were used. 1) HUVEC cells pre-labeled with Cytolight Green were co-cultured with human dermal fibroblasts in 96-well plates and treated with VCE-004.8 (1 µM) or VEGFA (10 ng/ml) for 7 days. Tube formation was monitored and quantified using live-cell imaging analysis software. 2) HBMEC cells were seeded on a layer of Matrigel in a 96-well plate in the presence of VCE-004.8 (1 µM) or VEGFA (10 ng/ml). After 5 hours, tube formation was imaged and the number of branch points was quantified using image analysis software. [1]
Cell Migration (Wound Healing) Assay: Differentiated MO3.13 cells were seeded in 96-well ImageLock plates and grown to confluence. A uniform wound was created using a 96-pin WoundMaker. Cells were then treated with mitomycin C (to block proliferation) and stimulated either directly with recombinant VEGFA (10 ng/ml) or with conditioned medium from HBMEC cells that had been treated with VCE-004.8 for 24 hours. Wound closure was imaged every 60 minutes for 18 hours, and migration was quantified as Relative Wound Density using live-cell imaging software. [1]
Macrophage/Microglia Polarization and Arg-1 Promoter Assay: RAW264.7 macrophages were serum-starved and pre-incubated with VCE-004.8 for 18 hours, then stimulated with recombinant mouse IL-17 (50 ng/ml) for 24 hours to induce M1 polarization, or treated with VCE-004.8 and/or recombinant mouse IL-4 (40 ng/ml) for 24 hours to assess M2 markers. mRNA was extracted for qRT-PCR analysis. For the promoter assay, RAW264.7 cells were transiently transfected with a plasmid containing the mouse Arg1 promoter fused to a luciferase gene (pGL3-mArg1) along with a Renilla luciferase control plasmid. After transfection, cells were stimulated with VCE-004.8 for 20 hours, and dual-luciferase activity was measured. [1]
PGE2 Release Assay in Primary Microglia: Primary rat microglial cells were seeded and cultured. Cells were then incubated for 18 hours with LPS (0.5 µg/ml) in the presence or absence of different concentrations of VCE-004.8. Supernatants were collected, centrifuged, and PGE2 levels were quantified using a commercial prostaglandin E2 EIA kit according to the manufacturer's instructions. [1]

Experimental Autoimmune Encephalomyelitis (EAE) Model: EAE was induced in female C57BL/6 mice (6-8 weeks old) by subcutaneous immunization with MOG35-55 peptide emulsified in Complete Freund's Adjuvant, followed by intraperitoneal injections of pertussis toxin. Treatment began at day 8 post-immunization (curative protocol) when the first symptoms appeared. Mice received daily intraperitoneal (i.p.) injections of VCE-004.8 (10 mg/kg) or vehicle (4% DMSO + 6.4% Tween 80 in phosphate-buffered saline) for 21 days. Mice were monitored daily for clinical signs and scored. All animals were sacrificed at day 28 for tissue collection. [1]
Theiler's Virus-Induced Demyelinating Disease (TMEV-IDD) Model: SJL/J mice were intracranially inoculated with Theiler's virus (DA strain). Sixty days post-infection, mice were treated daily for 14 consecutive days with VCE-004.8 (10 mg/kg, i.p.) or vehicle (4% DMSO + 6.4% Tween 80 in phosphate-buffered saline) (curative protocol). Motor function was evaluated using an activity monitor system from day 60 to day 75 post-infection. [1]
In vivo EPO Induction Study: Eight-week-old C57BL/6 male mice were dosed intraperitoneally with VCE-004.8 (10 mg/kg) for 3 weeks. Blood samples were collected under anesthesia, plasma was separated, and circulating EPO levels were quantified using a commercial mouse EPO ELISA kit. [1]
Tissue Processing for Histology: At the endpoint of EAE or TMEV studies, mice were perfused with saline. Spinal cords were extracted. A portion was frozen for RNA analysis. The remainder was fixed, cryoprotected, and frozen. Serial free-floating sections of the thoracic spinal cord (15 or 30 µm thick) were cut for immunohistochemical staining. [1]

The pharmacokinetic profile of Etrinabdione is being evaluated in clinical studies, with key parameters including maximum observed plasma concentration (Cmax), time to peak drug concentration (Tmax), and area under the plasma concentration-time curve (AUC) as measures of total systemic exposure . In a Phase 2a study in patients with peripheral arterial disease, plasma samples are collected pre-dose and 3 hours post-dose at Day 1 and at months 1, 3, 6, 12, and 13 to assess these parameters. Plasma trough levels are also measured to determine potential drug accumulation . In a Phase 1 healthy volunteer study (NCT03745001), the safety, tolerability, pharmacokinetics, and food effect of Etrinabdione were evaluated . Additionally, in a Phase 2a study for systemic sclerosis, pharmacokinetic analysis over 3 months of treatment did not show apparent drug accumulation in patients' blood .

The safety profile of Etrinabdione has been evaluated in several clinical studies. In a Phase 2a study for systemic sclerosis, an independent Safety Review Committee evaluated blinded safety data from patients receiving 25 mg daily and 25 mg twice daily (50 mg total). The drug was determined to be well-tolerated, with no drug-related serious adverse events reported. Only mild related adverse events were observed . In a Phase 2a study for peripheral arterial disease (CLAUDIA study), the primary safety outcome is the incidence and severity of Treatment Emergent Adverse Events (TEAEs), monitored over 13 months. The study uses a sequential dose-escalation design: 12 patients first receive the low dose (25 mg twice daily) for 3 months; after evaluation of PK and safety data by the regulatory authority, the sponsor, and investigators, the high dose (50 mg twice daily) may be released . Key exclusion criteria for this study include moderate to severe hepatocellular dysfunction, severe hypertension, blood clotting disorders, and positive tests for HIV, hepatitis B, hepatitis C, or syphilis, helping to define the drug's safety context in specific populations .

[1]. Hypoxia mimetic activity of VCE-004.8, a cannabidiol quinone derivative: implications for multiple sclerosis therapy. J Neuroinflammation. 2018 Mar 1;15(1):64.

[2]. VCE-004.8, A Multitarget Cannabinoquinone, Attenuates Adipogenesis and Prevents Diet-Induced Obesity. Sci Rep. 2018 Oct 31;8(1):16092.

VCE-004.8 is an aminoquinone derivative derived from cannabidiol (CBD), a semi-synthetic cannabinoid designed to enhance its bioactivity and druggability. [1]
It is a dual PPARγ and CB2 receptor agonist and exerts additional hypoxia-mimicking activity by inhibiting PHD1/2, making it a multi-target compound for the treatment of multiple sclerosis (MS). [1]
This study suggests that its combined mechanisms—anti-inflammatory (via PPARγ/CB2), immunomodulatory (inhibition of M1 polarization, induction of Arg-1), pro-angiogenic, and neuro/oligodendrocyte protective (through HIF stabilization induction of EPO, VEGFA)—contribute to its efficacy in improving neuroinflammation and demyelination in MS models. [1]
Given that VCE-004.8 is active in both inflammatory EAE models and progressive virus-mediated TMEV models, it suggests potential therapeutic potential for different stages or subtypes of multiple sclerosis. [1]

C28H35NO3

433.582408189774

433.26

C, 77.56; H, 8.14; N, 3.23; O, 11.07

1818428-24-8

1818428-24-8

118465221

Dark purple to black solid powder

6.3

2

4

9

32

839

2

CCCCCC1=C(C(=C(C(=O)C1=O)[C@@H]2C=C(CC[C@H]2C(=C)C)C)O)NCC3=CC=CC=C3

CGGGAXJIRQSRPH-JTHBVZDNSA-N

InChI=1S/C28H35NO3/c1-5-6-8-13-22-25(29-17-20-11-9-7-10-12-20)27(31)24(28(32)26(22)30)23-16-19(4)14-15-21(23)18(2)3/h7,9-12,16,21,23,29,31H,2,5-6,8,13-15,17H2,1,3-4H3/t21-,23+/m0/s1

5-(benzylamino)-4-hydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-pentylcyclohexa-3,5-diene-1,2-dione

etrinabdione; EHP-101; EHP101; VCE-004; EHP 101; VCE004; VCE 004; VCE004.8; VCE 004.8; VCE-004.8

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: ~50 mg/mL (~115.3 mM）

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.77 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 (5.77 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 (5.77 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.)