Omaveloxolone (RTA-408)

Nrf2; NF-κB

体外活性：Omaveloxolone（RTA-408）有效增加 Nrf2 靶基因的表达，并逆转 RAW 264.7 细胞中 IFNγ 介导的 Gclc 表达抑制。在一组八种人类肿瘤细胞系中，RTA 408 抑制生长，平均 GI50 值为 260 nM，并诱导细胞凋亡。 RTA 408 还抑制肿瘤细胞中的 NF-κB 并激活 JNK。细胞测定：对于生长抑制测定，将细胞以每孔 3 x 103 个细胞的密度铺在重复的 96 孔培养皿中。第二天，一个板用 RTA 408 处理，另一个板立即进行磺酰罗丹明 B (SRB) 测定（时间 0）。处理开始 72 小时后，对 RTA 408 处理的板中的细胞进行 SRB 测定。使用以下公式计算相对于载体处理细胞的生长百分比：[(Ti-Tz)/(C-Tz)] x 100，其中 (Tz) 是时间零时的吸光度值，(C) 是载体处理孔的吸光度值72小时后，(Ti)是用药物处理的孔的吸光度值。在 GraphPad Prism 中绘制剂量反应曲线并计算 GI50 值。

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在低浓度（≤25 nM）下，RTA 408激活Nrf2，抑制干扰素-γ刺激的RAW 264.7巨噬细胞中的一氧化氮和促炎细胞因子水平。在较高浓度下，RTA 408抑制了肿瘤细胞生长（GI50=260±74 nM），并增加了肿瘤细胞系中的胱天蛋白酶活性，但在正常原代人类细胞中没有。与AIMs对IKKβ的直接作用一致，RTA 408在抑制细胞生长和诱导凋亡的相同浓度下抑制NF-κB信号传导并降低细胞周期蛋白D1水平。RTA 408还增加了CDKN1A（p21）水平和JNK磷酸化。RTA 408的体外活性曲线类似于甲基巴多索龙，在证明靶向参与的剂量下，患者对其耐受良好。总之，这些数据支持用于癌症治疗的RTA 408的临床评价。[1]

在患有放射性皮炎的小鼠中，1.0%Omaveloxolone（RTA-408） 显着减少表皮和胶原增厚，防止真皮坏死并完全缓解皮肤溃疡。在大鼠皮肤中，RTA 408 激活 Nrf2 并诱导细胞保护基因。 RTA 408 还可以减轻小鼠的造血急性辐射综合征。
本研究旨在确定新型齐墩烷三萜类化合物Omaveloxolone（RTA-408）是否以及如何对雄性小鼠的肾缺血再灌注损伤（IRI）提供保护。与赋形剂治疗的小鼠相比，用Omaveloxolone（RTA-408）治疗的小鼠在单侧缺血后进行对侧肾切除术，肾功能和组织学结果得到改善，凋亡、ROS产生和氧化损伤标志物减少。此外，我们发现RTA-408可以通过增加Nrf2核转位来增强总抗氧化能力，从而增加Nrf2下游GSH相关抗氧化基因的表达和活性。体外研究表明，GSH合成酶GCLc可能是RTA-408的重要靶点。此外，用RTA-408治疗的Nrf2缺陷小鼠在肾功能、组织学、ROS产生和GSH相关基因表达方面没有显著改善。因此，通过上调Nrf2及其下游抗氧化基因，RTA-408为肾脏IRI的预防和治疗提供了一种新的潜在方法[2]。

NF-κB信号传导[1]
对于NF-κB-萤光素酶报告基因检测，将HeLa NF-κB-Luc（每孔1.9 x 104个细胞）和A549/NF-κB-Luc（每孔1.6 x 104个电池）细胞接种在底部透明的96孔黑色平板中。24小时后，细胞用DMSO或几种浓度的Omaveloxolone（RTA-408）预处理1小时，然后用10ng/ml的人TNFα再处理5小时。根据制造商的说明，使用One Glo萤光素酶测定法测量萤火虫萤光素酶活性。对于IκBα蛋白质印迹，将HeLa细胞以每孔1 x 105个细胞的密度接种在24孔培养皿中。第二天，细胞用DMSO或几种浓度的Omaveloxolone（RTA-408）或甲基巴多索龙预处理6小时。然后用20ng/ml的人TNFα处理细胞5分钟。细胞立即在含有2%BME的Tricine样品缓冲液中裂解，并如上所述进行蛋白质印迹处理。

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抗氧化能力测定[2]
根据制造商的说明，使用每个检测试剂盒估算肾皮质匀浆上清液中丙二醛（MDA）、羰基化蛋白、总抗氧化能力（T-AOC）、总谷胱甘肽（T-GSH）和谷胱甘肽与谷胱甘肽二硫化物（GSH/GSSG）的比率。

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酶活性测定[2]
还使用市售的检测试剂盒测量了肾皮质匀浆上清液中GSH相关酶的活性。

将细胞以每孔 3 x 103 个铺板于重复的 96 孔培养皿中，用于生长抑制测定。第二天，一个板接受 Omaveloxolone（RTA-408）的应用，而另一个板立即进行磺基罗丹明 B (SRB) 测定（时间 0）。用 RTA 408 处理 72 小时后，板上的细胞准备用于 SRB 测定。公式 [(Ti-Tz)/(C-Tz)] x 100 用于计算与用载体处理的细胞相比的生长百分比。其中 (C) 是 72 小时后媒介物处理孔的吸光度值，(Ti) 是用药物处理的孔的吸光度值，(Tz) 是零时间时的吸光度值。在 GraphPad Prism 中，绘制剂量反应曲线，并计算 GI50 值。

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

87

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.51 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: 10% DMSO +90%Corn oil: 30mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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