Nrf2; NF-κB

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]

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].

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

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.

Absorption, Distribution and Excretion
Between 50 mg (0.33 times the recommended dosage) and 150 mg, the AUC of omaveloxolone increased in a dose-dependent and dose-proportional manner. However, at the same dose range, the Cmax increased in a less than dose-proportional manner in healthy fasted subjects. The median time to achieve peak plasma concentration was 7 to 14 hours. Compared to fasted conditions, the AUC0-inf and Cmax of omaveloxolone were 350% and 15% higher with a high-fat meal (800 to 1000 calories, with 150, 250, and 500 to 600 calories coming from protein, carbohydrate, and fat, respectively).
Omaveloxolone is mainly excreted in feces. In healthy subjects given a single oral dose of radiolabeled omaveloxolone (150 mg), about 92% and 0.1% of the dose were recovered in feces and urine, respectively. Approximately 91% of the omaveloxolone found in feces was recovered within 96 hours after administration.
Omaveloxolone has an apparent volume of distribution of 7361 L.
Omaveloxolone has an apparent plasma clearance of 109 L/hr.

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Metabolism / Metabolites
Omaveloxolone is mainly metabolized by CYP3A, although CYP2C8 and CYP2J2 play also a minor role.

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Biological Half-Life
Omaveloxolone has a terminal half-life of 57 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of omaveloxolone during breastfeeding. Because it is 97% protein bound, exposure of the breastfed infant is likely to be low. Until more data become available, omaveloxolone should be used with caution during breastfeeding, especially while nursing a newborn or preterm infant
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
The protein binding of omaveloxolone is 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.

Omaveloxolone is a semi-synthetic triterpenoid drug. It is an Nrf2 activator that is approved for the treatment of Friedreich's ataxia in adults and adolescents aged 16 years and older. It has a role as an antioxidant, an anti-inflammatory agent, a cardioprotective agent and an antineoplastic agent. It is a pentacyclic triterpenoid, a secondary carboxamide, a nitrile, an organofluorine compound and a cyclic terpene ketone. It derives from a hydride of an oleanane.
Omaveloxolone (RTA-408) is a semisynthetic oleanane triterpenoid with antioxidant and anti-inflammatory properties. Omaveloxolone acts as an activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor that mitigates oxidative stress. In patients with Friedreich's ataxia, a genetic disease involving mitochondrial dysfunction, the Nrf2 pathway is impaired, and Nrf2 activity is lower. Therefore, the use of Nrf2 activators such as omaveloxolone represents a therapeutic advantage in this group of patients. In February 2023, omaveloxolone was approved by the FDA for the treatment of Friedreich's ataxia in adults and adolescents aged 16 years and older. The use of omaveloxolone for the treatment of conditions involving mitochondrial dysfunction and oxidative stress has also been evaluated.
The mechanism of action of omaveloxolone is as a Cytochrome P450 3A4 Inducer, and Cytochrome P450 2C8 Inducer.
Omaveloxolone is a member of the synthetic oleanane triterpenoid class of compounds and an activator of nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2, Nfe2l2), with potential chemopreventive activity. Upon administration, omaveloxolone activates the cytoprotective transcription factor Nrf2. In turn, Nrf2 translocates to the nucleus, dimerizes with a small Maf protein (sMaf), and binds to the antioxidant response element (ARE). This induces the expression of a number of cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), sulfiredoxin 1 (Srxn1), heme oxygenase-1 (HO1, HMOX1), superoxide dismutase 1 (SOD1), gamma-glutamylcysteine synthetase (gamma-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, a leucine zipper transcription factor, plays a key role in the maintenance of redox balance and cytoprotection against oxidative stress.

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Drug Indication
Omaveloxolone is indicated for the treatment of Friedreich's ataxia in adults and adolescents aged 16 years and older.
Treatment of Friedreich's ataxia

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Mechanism of Action
The mechanism of action of omaveloxolone has not been fully elucidated; however, its therapeutic effect in patients with Friedreich's ataxia may be linked to its ability to activate the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. Nrf2 is a transcription factor that mitigates oxidative stress. In normal conditions, Nrf2 levels are regulated by Kelch-like ECH-associated protein 1 (KEAP1), which binds to Nrf2, prevents Nrf2 translocation to the nucleus, and degrades it by ubiquitination. In the presence of oxidative stress, the ubiquitination system is disrupted. Nrf2 accumulates and translocates to the nucleus, where it promotes the expression of genes against oxidative stress. In patients with Friedreich's ataxia, the Nrf2 signaling pathway is dysfunctional. In Friedreich's ataxia models, Nrf2 has a lower activity; therefore, Nrf2 activators such as omaveloxolone represent a therapeutic opportunity. A study has shown that omaveloxolone binds to KEAP1, inhibiting its interaction with Nrf2 and promoting the translocation of Nrf2 to the nucleus. Aside from activating Nrf2, omaveloxolone also inhibits the NF-κB signalling pathway, promoting antioxidative, anti-inflammatory, and antiapoptotic mechanisms.

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Pharmacodynamics
The MOXIe study showed that after 4 weeks of administration, the use of omaveloxolone led to robust dose‐dependent changes at 80 ‐300 mg/day.. The effect of omaveloxolone on QTc interval has yet to be defined. The use of omaveloxolone can lead to an elevation in hepatic transaminases (both aspartate transaminase and alanine transaminase) and an increase in B-type natriuretic peptide (BNP), a marker of cardiac function. It can also cause changes in cholesterol.

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.)