IKK assay

To determine the effect of CDDO-Me on TNF-induced IKK activation, IKK was analyzed. Briefly, the IKK complex from whole-cell extracts was precipitated with antibody against IKKα and IKKβ and then treated with protein A/G-Sepharose beads. After 2 hrs, the beads were washed with lysis buffer and then resuspended in a kinase assay mixture containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 2 mmol/L DTT, 20 μCi [γ-32P]ATP, 10 μmol/L unlabeled ATP, and 2 μg of substrate glutathione S-transferase-IκBα (amino acids 1 ~ 54). After incubation at 30℃ for 30 mins, the reaction was terminated by boiling with SDS sample buffer for 5 mins. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with a Storm820. To determine the total amounts of IKK-α and IKK-β in each sample, 50 μg of whole-cell proteins were resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti-IKK-α or anti-IKK-β antibody.

Cell lines

HL-60, KG-1 and NB4 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

~ 5 μM; 72 hrs

Applications

In leukemia cells, such as HL-60, KG-1, and NB4 cells, Bardoxolone Methyl decreased cell viability with the IC50 values of 0.4, 0.4 and 0.27 μM, respectively.

Animal models

Female A/J mice i.p. injected with vinyl carbamate

Dosage form

60 or 400 mg/kg; p.o.; the mice fed Bardoxolone Methyl for 2 weeks and then switched to a week of control diet, for 15 weeks.

Applications

At the dose of 60 mg, Bardoxolone Methyl reduced the number, size, as well as severity of lung tumors in vivo.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Bardoxolone methyl is an activator of the KEAP1-Nrf2 pathway [1] and also inhibits the pro-inflammatory transcription factor NF-kB [2] which can protect kidneys from aristolochic acid (AA)-induced acute kidney injury (AKI) with IC50 value of 1.5 nM and LC50 value of 2.1 μM [3].
Nrf2, a transcription factor, is a basic leucine zipper (bZIP) protein that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation [4], such as NADPH, Glutathione, SRXN1, TXNRD1, HMOX1, GST, UGT and Mrps. Nrf2 plays an important role in the maintenance of homeostasis which can control the basal and inducible expression of a battery of genes with diverse physiological roles, including the preservation of redox balance, the metabolism and detoxification of xenobiotics, and the regulation of multiple metabolic pathways that ensure the provision of cellular energy[5].
Bardoxolone methyl is a synthetic oleanane triterpenoid compound, which has no effect on the function and histology of normal kidneys but increased renal expression of Nrf2, HO-1 and NQO1 by western blotting analysis of mice kidneys and immunofluorescence staining, and can prevent AA-induced acute kidney injury and reduce AAI-induced TI injury in mRNA and protein levels through real-time PCR.[6] In conclusion, Bardoxolone methyl can prevent AAI-induced renal damage, and it may exert this renoprotective effects by activating the Nrf2 signaling pathway and inducing the downstream target genes expression. A phase 3 clinical trial evaluating bardoxolone methyl for the treatment of chronic kidney disease (CKD) was terminated in October 2012 after patients treated with the drug were found to have experienced a higher rate of heart-related adverse events, including heart failure, hospitalizations and deaths.[7] Now in 2014, Kyowa Hakko Kirin announced plans to evaluate both safety and efficacy of bardoxolone methyl in a Phase 2 clinical study to be performed in Japan for the treatment of CKD associated with type 2 diabetes.[8]
References:
1.Yates MS, Tauchi M, Katsuoka F, et al."Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes." Mol Cancer Ther 2007, 6 (1): 154–62.
2.Ahamd R, Raina D, Meyer C, et al.. "Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179.". J Biol Chem, 2006, 281 (47): 35764–9.
3.Ian M. Copple. et al. Chemical Tuning Enhances Both Potency Toward Nrf2 and In Vitro Therapeutic Index of Triterpenoids. TOXICOLOGICAL SCIENCES,2014,140(2), 462–469.
4.Gold R, Kappos L. Et al. Placebo-controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis. N. Engl. J. Med. 2012, 367 (12): 1098–107.
5.Ma, Q. Role of nrf2 in oxidative stress and toxicity. Annu. Rev. Pharmacol. Toxicol. 2013,53:401–426.
6.Juan Wua. et al. Bardoxolone methyl (BARD) ameliorates aristolochic acid (AA)-induced acute kidney injury through Nrf2 pathway. Toxicology. 2014, 318(6):22–31.
7.de Zeeuw D, Akizawa T, Audhya P, et al. "Bardoxolone methyl in type 2 diabetes and stage 4 chronic kidney disease.". N Engl J Med,2013,369 (26): 2492–503.
8.Kyowa Hakko Kirin Co Ltd announces future development direction for bardoxolone methyl (RTA 402).