Nrf2; NF-κB;
- Nuclear factor erythroid 2-related factor 2 (Nrf2) [1]
- Kelch-like ECH-associated protein 1 (Keap1) [1]

RTA 408 reverses IFN-mediated suppression of Gclc expression in RAW 264.7 cells and significantly upregulates the expression of Nrf2 target genes. RTA 408 inhibits growth with an average GI50 value of 260 nM in a panel of eight human tumor cell lines and triggers apoptosis. Additionally, RTA 408 inhibits NF-B and stimulates JNK in tumor cells. [1]

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At low concentrations (≤ 25 nM), RTA 408 activated Nrf2 and suppressed nitric oxide and pro-inflammatory cytokine levels in interferon-γ-stimulated RAW 264.7 macrophage cells. At higher concentrations, RTA 408 inhibited tumor cell growth (GI50 = 260 ± 74 nM) and increased caspase activity in tumor cell lines, but not in normal primary human cells. Consistent with the direct effect of AIMs on IKKβ, RTA 408 inhibited NF-κB signaling and decreased cyclin D1 levels at the same concentrations that inhibited cell growth and induced apoptosis. RTA 408 also increased CDKN1A (p21) levels and JNK phosphorylation. The in vitro activity profile of RTA 408 is similar to that of bardoxolone methyl, which was well-tolerated by patients at doses that demonstrated target engagement. Taken together, these data support clinical evaluation of RTA 408 for cancer treatment.[1]

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- RAW 264.7 macrophages: Treatment with Omaveloxolone (RTA-408) (0.1–100 nM) significantly reduced interferon-γ-induced nitric oxide (NO) production with an IC50 of 4.4 ± 1.8 nM. This was accompanied by downregulation of pro-inflammatory genes (e.g., NOS2, PTGS2, CCL2) and upregulation of Nrf2 target genes (NQO1, GCLC, TXNRD1) at both mRNA and protein levels [1]
- Human tumor cell lines: Omaveloxolone inhibited proliferation of 8 tested cancer cell lines (e.g., A549, PANC-1) with an average GI50 of 260 ± 74 nM. It induced caspase-dependent apoptosis, as evidenced by increased DEVD-AFC cleavage and cleavage of caspases-3/-9 [1]
- Primary mouse renal tubular cells: Omaveloxolone (10–100 nM) enhanced Nrf2 nuclear translocation and increased GSH biosynthesis gene expression (GCLC, GCLM), leading to reduced reactive oxygen species (ROS) production and cell death under oxidative stress conditions [2]

In mice with radiation-induced dermatitis, 1.0% Omaveloxolone (RTA-408) markedly reduces epidermal and collagen thickening, prevents dermal necrosis and completely alleviates skin ulcers. [2] RTA 408 activates Nrf2 and induces cytoprotective genes in rat skin. [3] In mice, RTA 408 also lessens the hematopoietic acute radiation syndrome.

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Here, this study aimed to determine whether and how Omaveloxolone (RTA-408), a novel oleanane triterpenoid, could confer protection against renal ischemia-reperfusion injury (IRI) in male mice. Mice treated with RTA-408 undergoing unilateral ischemia followed by contralateral nephrectomy had improved renal function and histological outcome, as well as decreased apoptosis, ROS production, and oxidative injury marker compared with vehicle-treated mice. Also, we had found that RTA-408 could strengthen the total antioxidant capacity by increasing Nrf2 nuclear translocation and subsequently increased Nrf2 downstream GSH-related antioxidant gene expression and activity. In vitro study demonstrated that GSH biosynthesis enzyme GCLc could be an important target of RTA-408. Furthermore, Nrf2-deficient mice treated with RTA-408 had no significant improvement in renal function, histology, ROS production, and GSH-related gene expression. Thus, by upregulating Nrf2 and its downstream antioxidant genes, RTA-408 presents a novel and potential approach to renal IRI prevention and therapy[2].

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- ob/ob mice with renal ischemia-reperfusion injury: Oral administration of Omaveloxolone (10 mg/kg) daily for 7 days improved renal function (reduced serum creatinine and BUN), reduced tubular necrosis, and decreased oxidative stress markers (MDA, 8-OHdG). These effects were abolished in Nrf2 knockout mice [2]
- C57BL/6 mice with hematopoietic acute radiation syndrome: Intraperitoneal injection of Omaveloxolone (17.5 mg/kg) at 24, 48, and 72 hours post-7 Gy total body irradiation (TBI) resulted in 100% survival at day 35, compared to 0% survival in vehicle-treated controls. The drug reduced radiation-induced bone marrow apoptosis and preserved hematopoietic stem cell function [3]

NF-κB signaling[1]
For NF-κB-luciferase reporter assays, HeLa NF-κB-Luc (1.9 x 104 cells per well) and A549/NF-κB-Luc (1.6 x 104 cells per well) cells were seeded in 96-well black plates with clear bottoms. Twenty four hours later, cells were pre-treated with DMSO or several concentrations of Omaveloxolone (RTA-408) for one hour and then treated with 10 ng/ml human TNFα for five additional hours. Firefly luciferase activity was measured using the One-Glo Luciferase Assay according to the manufacturer’s instructions. For IκBα western blots, HeLa cells were seeded in a 24-well culture dish at a density of 1 x 105 cells per well. The following day, cells were pre-treated with DMSO or several concentrations of Omaveloxolone (RTA-408) or bardoxolone methyl for six hours. Cells were then treated with 20 ng/ml of human TNFα for five minutes. Cells were immediately lysed in Tricine sample buffer with 2% BME and processed for western blotting as described above.

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Antioxidant Capacity Assays[2]
Malondialdehyde (MDA), carbonylated protein, total antioxidant capacity (T-AOC), total glutathione (T-GSH), and glutathione to glutathione disulfide (GSH/GSSG) ratio in the supernatant of renal cortical homogenate were estimated by using each assay kit, according to the manufacturer’s instructions.

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Enzyme Activity Assays[2]
GSH-related enzyme activity was also measured in the supernatant of renal cortical homogenate by a commercially available assay kit

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- Nrf2 activation assay: Nuclear extracts from Omaveloxolone-treated cells were analyzed for Nrf2-DNA binding activity using electrophoretic mobility shift assay (EMSA). The drug enhanced Nrf2 binding to antioxidant response elements (AREs) in a dose-dependent manner [2]
- GSH reductase activity assay: Cell lysates were incubated with Omaveloxolone and NADPH, and GSH levels were measured spectrophotometrically. The drug increased GSH reductase activity by 2.3-fold compared to vehicle [2]
#### Cell Assay - Nrf2 nuclear translocation: RAW 264.7 cells were treated with Omaveloxolone (10 nM) for 2 hours, fixed, and stained with anti-Nrf2 antibody. Immunofluorescence microscopy showed increased nuclear Nrf2 localization compared to vehicle [1]
- Apoptosis detection: Cancer cells treated with Omaveloxolone (500 nM) for 24 hours were stained with Annexin V-FITC/PI. Flow cytometry revealed a 3.2-fold increase in apoptotic cells compared to untreated controls [1]

Cells are plated at 3 x 103 per well in duplicate 96-well culture dishes for growth inhibition assays. The next day, one plate receives an application of RTA 408, while the other is immediately subjected to the sulforhodamine B (SRB) assay (time 0). After 72 hours of treatment with RTA 408, the cells on the plate are prepared for the SRB assay. The formula [(Ti-Tz)/(C-Tz)] x 100 is used to calculate the percentage of growth in comparison to cells treated with a vehicle. where (C) is the absorbance value from vehicle-treated wells after 72 hours, (Ti) is the absorbance value from wells treated with the drug, and (Tz) is the absorbance value at time zero. In GraphPad Prism, dose-response curves are plotted, and GI50 values are computed.

Mice: Wild-type C57Bl/6 CD45.2 mice are used, which are 6–8 weeks old, for radiation survival tests. In transplantation experiments, recipients include congenic wild-type C57Bl/6 CD45.1 host mice and C57Bl/6 CD45.1/CD45.2 hybrid host mice. For vehicle control, omeveloxolone stock solutions (DMSO) are made within an hour of injection. At 24, 48, and 72 hours after irradiation, intraperitoneal administration of DMSO or omaveloxolone (17.5 mg/kg) is performed. A 250-kVp X-ray machine with a 50 cm source-to-skin distance and a 2 mm copper filter is used to administer whole-body irradiation (7-8 Gy). About 1.4 Gy/min is the dose rate.

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Mouse Model of Ischemia-Reperfusion Injury[2]
24 h before surgery, mice were intraperitoneally administered with RTA-408 (100 μg/kg body weight) or 0.1% dimethyl sulfoxide (DMSO) in PBS as the vehicle. The rationale for RTA-408 dosage was based on the renal function preservation and Nrf2 mRNA activation.

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- Renal ischemia-reperfusion model: Male C57BL/6 mice underwent 30 minutes of left renal artery clamping followed by reperfusion. Omaveloxolone (10 mg/kg) was dissolved in DMSO/PBS and administered orally once daily for 7 days starting 1 hour before ischemia. Renal tissues were harvested for histological and biochemical analysis [2]
- Radiation syndrome model: C57BL/6 mice received 7 Gy TBI and were treated with Omaveloxolone (17.5 mg/kg, IP) at 24, 48, and 72 hours post-irradiation. Survival was monitored daily for 35 days, and bone marrow cells were analyzed for apoptosis by TUNEL staining [3]

Absorption, Distribution and Excretion
Omaravirone's AUC increases dose-dependently and proportionally within the dose range of 50 mg (0.33 times the recommended dose) to 150 mg. However, within the same dose range, the increase in Cmax in healthy fasting subjects is less dose-proportional. The median time to reach peak plasma concentration is 7 to 14 hours. Compared to the fasting state, the AUC0-inf and Cmax of omalivrone increase by 350% and 15%, respectively, after a high-fat meal (800 to 1000 calories, of which protein, carbohydrates, and fat provide 150, 250, and 500 to 600 calories, respectively). Omaravirone is primarily excreted in feces. Following a single oral dose of radiolabeled omalivrone (150 mg) in healthy subjects, approximately 92% of the dose is recovered in feces, and approximately 0.1% is recovered in urine. Approximately 91% of omavirone in feces is recovered within 96 hours after administration. The apparent volume of distribution of omavirone is 7361 L. The apparent plasma clearance of omavirone is 109 L/hr. Metabolites: Omavirone is primarily metabolized by CYP3A, with minor contributions from CYP2C8 and CYP2J2. Biological half-life: The terminal half-life of omavirone is 57 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Currently, there is no information regarding the clinical use of omavidron during lactation. Due to its high protein binding rate of 97%, the amount of drug exposed to breastfed infants may be low. Until more data are available, omavidron should be used with caution during lactation, especially when breastfeeding newborns or premature infants.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

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Protein Binding Rate
Omavidron has a protein binding rate of 97%.

[1]. RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity. PLoS One. 2015 Apr 21;10(4):e0122942.

[2]. RTA-408 Protects Kidney from Ischemia-Reperfusion Injury in Mice via Activating Nrf2 and Downstream GSH Biosynthesis Gene. Oxid Med Cell Longev. 24 December 2017.

[3]. The triterpenoid RTA 408 is a robust mitigator of hematopoietic acute radiation syndrome in mice. Radiat Res. 2015 Mar;183(3):338-44.

Omariverone is a semi-synthetic triterpenoid drug. It is an Nrf2 activator approved for the treatment of Friedreich ataxia in adults and adolescents aged 16 years and older. It possesses antioxidant, anti-inflammatory, cardioprotective, and antitumor effects. It is a pentacyclic triterpenoid compound containing secondary amides, nitriles, organofluorine compounds, and cyclic terpene ketones. It is derived from the hydrogenation of oleanane. Omariverone (RTA-408) is a semi-synthetic oleanane-type triterpenoid compound with antioxidant and anti-inflammatory properties. Omariverone activates nuclear factor (erythrocyte-derived 2)-like 2 (Nrf2), a transcription factor that reduces oxidative stress. Friedreich ataxia is a genetic disorder involving mitochondrial dysfunction, with impaired Nrf2 pathways and reduced Nrf2 activity. Therefore, the use of Nrf2 activators (such as omaiverone) offers therapeutic advantages for these patients. In February 2023, omavidronone received FDA approval for the treatment of Friedreich ataxia in adults and adolescents aged 16 years and older. Its efficacy in treating mitochondrial dysfunction and oxidative stress-related diseases has also been evaluated. Omavidronone's mechanism of action is as an inducer of cytochrome P450 3A4 and cytochrome P450 2C8. Omavidronone belongs to the synthetic oleanane triterpenoid class of compounds and is an activator of nuclear factor E2-related factor 2 (Nrf2, Nfe2l2), possessing potential chemopreventive activity. Upon administration, omavidronone activates the cellular protective transcription factor Nrf2. Subsequently, Nrf2 translocates to the nucleus, dimers with small Maf protein (sMaf), and binds to antioxidant response elements (AREs). This induces the expression of numerous cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), thioredoxin 1 (Srxn1), heme oxygenase-1 (HO1, HMOX1), superoxide dismutase 1 (SOD1), gamma-glutamylcysteine synthase (γ-GCS), thioredoxin reductase-1 (TXNRD1), glutathione S-transferase (GST), glutamate-cysteine ligase catalytic subunit (Gclc), and glutamate-cysteine ligase regulatory subunit (Gclm), and increases the synthesis of the antioxidant glutathione (GSH). Nrf2 is a leucine zipper transcription factor that plays a crucial role in maintaining redox homeostasis and cellular resistance to oxidative stress.

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Drug Indications
Omarvirone is indicated for the treatment of Friedreich ataxia in adults and adolescents aged 16 years and older.

Therapeutic Effects of Friedreich Ataxia

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Mechanism of Action
The mechanism of action of omavirone is not fully elucidated; however, its therapeutic effect on patients with Friedreich ataxia may be related to its ability to activate the nuclear factor (erythrocyte-derived 2)-like 2 (Nrf2) pathway. Nrf2 is a transcription factor that can alleviate oxidative stress. Under normal conditions, Nrf2 levels are regulated by Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 binds to Nrf2, preventing Nrf2 from translocating to the nucleus and degrading it through ubiquitination. Under oxidative stress conditions, the ubiquitination system is disrupted. Nrf2 accumulates and translocates to the nucleus, where it promotes the expression of antioxidant stress genes. In patients with Friedreich ataxia, the Nrf2 signaling pathway is dysfunctional. In the Friedreich ataxia model, Nrf2 activity is reduced; therefore, Nrf2 activators (such as omavirone) have potential therapeutic value. One study showed that omavirone binds to KEAP1, inhibiting its interaction with Nrf2 and thus promoting Nrf2 translocation to the nucleus. In addition to activating Nrf2, omavirone also inhibits the NF-κB signaling pathway, thereby promoting antioxidant, anti-inflammatory, and anti-apoptotic mechanisms.

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Pharmacodynamics
MOXIe studies showed that after 4 weeks of continuous administration, daily doses of 80–300 mg of omavirone caused significant dose-dependent changes. The effect of omavirone on the QTc interval remains to be clarified. Omavirone use can lead to elevated liver transaminases (including aspartate aminotransferase and alanine aminotransferase) and elevated levels of B-type natriuretic peptide (BNP) (a cardiac function marker). It also causes changes in cholesterol levels.

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- Mechanism: Omariveron binds to Keap1, preventing Nrf2 ubiquitination and promoting its nuclear translocation. Activated Nrf2 can upregulate antioxidant genes (e.g., NQO1, GCLC) and inhibit NF-κB-mediated pro-inflammatory pathways [1][2]
- Therapeutic potential: The drug has shown efficacy in preclinical models of diabetes, kidney injury, and radiation hematopoietic syndrome, and is currently undergoing a clinical trial in Friedreich ataxia (NCT02255435) [1][2][3]

C33H44F2N2O3

554.71

554.331

C, 71.45; H, 8.00; F, 6.85; N, 5.05; O, 8.65

1474034-05-3

71811910

White solid powder

1.2±0.1 g/cm3

662.0±55.0 °C at 760 mmHg

354.2±31.5 °C

0.0±2.0 mmHg at 25°C

1.549

5.64

1

6

2

40

1320

7

O=C1C(C#N)=C[C@@]2(C)[C@](CC[C@]([C@@]3(C)[C@@]4([H])[C@@]5([H])[C@@](CCC(C)(C)C5)(NC(C(F)(F)C)=O)CC3)(C)C2=CC4=O)([H])C1(C)C

RJCWBNBKOKFWNY-IDPLTSGASA-N

InChI=1S/C33H44F2N2O3/c1-27(2)11-13-33(37-26(40)32(8,34)35)14-12-31(7)24(20(33)17-27)21(38)15-23-29(5)16-19(18-36)25(39)28(3,4)22(29)9-10-30(23,31)6/h15-16,20,22,24H,9-14,17H2,1-8H3,(H,37,40)/t20-,22-,24-,29-,30+,31+,33-/m0/s1

N-[(4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,7,8,8a,14a,14b-decahydropicen-4a-yl]-2,2-difluoropropanamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.51 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 2: 10% DMSO +90%Corn oil: 30mg/mL

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