Br J Pharmacol. 2015 Mar;172(5):1237-49. doi: 10.1111/bph.12999

Novel Nrf2 activators from microbial transformation products inhibit blood-retinal barrier permeability in rabbits.

Nakagami Y, Masuda K, Hatano E, Inoue T, Matsuyama T, Iizuka M, Ono Y, Ohnuki T, Murakami Y, Iwasaki M, Yoshida K, Kasuya Y, Komoriya S.

Daiichi Sankyo Co., Ltd., Tokyo, Japan.

 

Abstract

BACKGROUND AND PURPOSE: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well-known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator.

EXPERIMENTAL APPROACH: The potent novel Nrf2 activator, RS9, was obtained from microbial transformation products. Its Nrf2 activity was evaluated by determining NADPH:quinone oxidoreductase-1 induction activity in Hepa1c1c7 cells. We also investigated the effects of RS9 on oxygen-induced retinopathy in rats and glycated albumin-induced blood-retinal barrier permeability in rabbits because many ocular diseases are associated with oxidative stress and inflammation.

KEY RESULTS: Bardoxolone methyl doubled the specific activity of Nrf2 in Hepa1c1c7 cells at a much higher concentration than RS9. Moreover, the induction of Nrf2-targeted genes was observed at a one-tenth lower concentration of RS9. Interestingly, the cytotoxicity of RS9 was substantially reduced compared with bardoxolone methyl. Oral and intravitreal administration of RS9 ameliorated the pathological scores and leakage in the models of retinopathy in rats and ocular inflammation in rabbits respectively.

CONCLUSION AND IMPLICATIONS: Nrf2 activators are applicable for treating ocular diseases and novel Nrf2 activators have potential as a unique method for prevention and treatment of retinovascular disease.

PMID:25363737

 

Supplement:

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that positively regulates many phase 2 detoxifying enzymes and antioxidant enzymes such as NADPH:quinone oxidoreductase-1 (Nqo1) and heme oxygenase-1 (Hmox1) (1). Among well-known Nrf2 activators are the triterpenoids, which interact with cysteine residues of an Nrf2 adaptor protein, the kelch-like ECH-associated protein 1 (Keap1) (2). This interaction disrupts the Nrf2-Keap1 complex, allowing Nrf2 to penetrate into the nucleus.

One of the triterpenoids, bardoxolone methyl [2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) methyl ester/CDDO-Me/RTA 402] was tested for chronic kidney disease with type 2 diabetes. The Phase 3 was halted due to cardiovascular mortality (3); however Kyowa Hakko Kirin announced the commencement of Phase 2 of RTA 402 in chronic kidney disease patients with type 2 diabetes in Japan on March 5, 2015. Another compound, RTA 408, is currently being tested in clinical trials such as radiation-induced dermatitis with breast cancer (ClinicalTrials.gov Identifier: NCT02142959) (4), post-surgical corneal endothelial cells loss (NCT02128113), Friedreich’s ataxia (NCT02255435), mitochondrial myopathies (NCT02255422) and melanoma (NCT02259231). In this study, we have also succeeded in obtaining a novel Nrf2 activator, RS9, from microbial transformation products (Figure 1).

 

WBF-Fig1-Nakagami

Figure 1. Chemical structures of Nrf2 activators.

 

We have demonstrated that RS9 inhibits blood-retinal barrier permeability in rabbits. Additional experiments showed that intravitreal injection of RS9 inhibited glycated albumin-induced blood retinal barrier permeability (Figure 2A) and the inhibitory effect was significant at a dose of 0.03 mM. A vascular endothelial growth factor (VEGF) antibody (Avastin®, bevacizumab) and triamcinolone acetonide also showed inhibitory effects (Figure 2B).

There is increasing evidence that growth factors such as VEGF play an important role in the pathogenesis of retinovascular diseases; however the influence of oxidative stress is not clear yet. Our novel Nrf2 activators inhibited neovascularization and blood-retinal barrier permeability, and we have most recently reported that RS9 is effective in a model of retinitis pigmentosa, rhodopsin Pro347Leu rabbits (5). These series of data imply that inhibition of oxidative stress is also an attractive approach for treating neovascularization-related ocular diseases, especially retinopathy of prematurity, wet-type aged macular degeneration, diabetic macular edema and diabetic retinopathy.

 

WBF-Fig2-Nakagami

Figure 2. Effects of RS9 on blood-retinal permeability in rabbits. (A) Comparison of the efficacy of RS9 and RTA 402. (B) Effects of a VEGF antibody and triamcinolone acetonide. * p < 0.05, ** p < 0.01 vs. albumin and vehicle-injected rabbits, Dunnett’s test, n = 3-8.

 

References

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  2. Sporn MB, Liby KT, Yore MM, Fu L, Lopchuk JM et al. New synthetic triterpenoids: potent agents for prevention and treatment of tissue injury caused by inflammatory and oxidative stress. J. Nat. Prod. 2011; 74: 537-545.
  3. de Zeeuw D, Akizawa T, Audhya P, Bakris GL, Chin M et al. Bardoxolone methyl in type 2 diabetes and stage 4 chronic kidney disease. N. Engl. J. Med. 2013; 369: 2492-2503.
  4. Reisman SA, Lee CY, Meyer CL, Proksch JW, Sonis ST. Topical application of the synthetic triterpenoid RTA 408 protects mice from radiation-induced dermatitis. Radiat. Res. 2014; 181: 512-520.
  5. Nakagami Y, Hatano E, Inoue T, Yoshida K, Kondo M et al. Cytoprotective effects of a novel Nrf2 activator, RS9, in rhodopsin Pro347Leu rabbits. Curr. Eye Res. (in press).

 

 

 

 

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