- AIMs influence the expression of hundreds of genes by activating Nrf2, a transcription factor that promotes normal mitochondrial function, increases production of antioxidant and detoxification enzymes, reduces oxidative stress, and reduces pro-inflammatory signaling.
- AIMs also inhibit the major pro-inflammatory transcription factor NF-κB and the production of pro-inflammatory cytokines including TNFα, IL-6, and IL-1.
- Extensive non-clinical research has documented a variety of pharmacological effects exerted by AIMs. A number of these effects have also been documented in clinical trials.
Promoting Energy Metabolism and Mitochondrial Function: Mitochondrial dysfunction, which is manifested through decreased cellular energy production and increased production of ROS, is a feature of many chronic inflammatory diseases1. Nrf2 activation reduces mitochondrial ROS, promotes the availability of fatty acids and glucose for mitochondrial ATP production, and increases mitochondrial biogenesis2,3,4.
Reducing Oxidative Stress: Activation of Nrf2 increases the production of over 200 different proteins in cells, which increase the antioxidant and detoxification capacity of cells in response to increased levels of ROS from inflammation, environmental toxins, or mitochondrial ATP production5.
Reducing Inflammation and Inflammatory Signaling: Inflammation is a protective response of the body to harmful stimuli such as invading pathogens, damaged cells, or irritants. Central features of inflammation are mitochondrial dysfunction and the production of ROS. In many diseases, inflammation does not resolve normally, which leads to chronic excessive ROS, tissue damage, and impaired ATP production6,7,8.
The foundational biology of AIMs underlies our two lead product candidates, bardoxolone methyl and omaveloxolone, and certain of our preclinical programs.
Many diseases have three features in common: inflammation, oxidative stress, and mitochondrial dysfunction. Healthy cells normally sense the presence of pathogenic organisms and other toxic stimuli. When these danger/damage signals are sensed, cells rapidly respond by increasing the production of reactive oxygen species (ROS) and pro-inflammatory cytokines. To facilitate production of these inflammatory mediators, mitochondria adopt an “inflamed” state, wherein normal mitochondrial function is temporarily suppressed. This shift-in-state allows mitochondrial resources to be diverted away from ATP production and toward other pathways that orchestrate normal cellular defense and repair processes. Reorientation of mitochondria from energy production to ROS production allows the affected cells to kill invading pathogens with molecules such as hydrogen peroxide and superoxide. Some of the ROS produced by inflamed mitochondria are released from the mitochondria into the cytoplasm and promote the activity of multiple key pro-inflammatory signaling complexes, including the IKK/NF-κB complex and the ASC/NLRP3 inflammasome. In a normal disease process, after the pathogens have been eliminated the resolution phase of inflammation can begin, and naturally occurring molecules promote the resolution of inflammation, in part, by activating the Keap1/Nrf2 pathway. As a result, pro-inflammatory mediators are reduced, reactive oxygen species are neutralized, and normal mitochondrial function is restored. However, in many chronic inflammatory and genetic diseases, this resolution process does not occur or is inadequate, leading to mitochondrial dysfunction, oxidative stress, and chronic inflammation, all of which can ultimately lead to tissue damage.
AIMs mimic the activity of the endogenous molecules that promote the resolution of inflammation and restore homeostasis by binding to Keap1, a protein that coordinates the cellular response to ROS and other stimuli, each of which can cause cellular damage (generally referred to as oxidative stress). Binding to Keap1 activates Nrf2, a transcription factor that increases the levels of antioxidants, detoxification enzymes, and transporters, thereby reducing the levels of oxidative stress caused by excess ROS. Nrf2 also restores normal mitochondrial function by increasing the availability of substrates and reducing equivalents that are required to support ATP production. Nrf2 activation inhibits inflammation by reducing ROS levels, restoring normal mitochondrial function, and directly inhibiting pro-inflammatory signaling.
Because mitochondrial dysfunction, oxidative stress, and inflammation are features of many diseases, including diseases of genetic mutation, fibrotic cardiovascular diseases, and chronic inflammatory diseases, AIMs have many potential clinical applications and have been the subject of more than 350 peer-reviewed scientific papers.
Promoting Energy Metabolism and Mitochondrial Function
- ATP production: ROS are produced in the mitochondria as a byproduct of ATP production. Nrf2 activation improves mitochondrial efficiency by making antioxidant enzymes available to reduce or neutralize ROS2. The management of these reducing equivalents is a constant and critical balancing act within the mitochondria. Disease processes that increase ROS deplete reducing equivalents available for ATP production1. Accordingly, the induction of antioxidant proteins through Nrf2 activation augments mitochondrial ATP production2,14.
- Efficient consumption of fats and sugars: Nrf2 activation promotes the transport of fatty acids to the mitochondria where they are converted into reducing equivalents used to produce ATP1. Nrf2 also promotes the transport of glucose from the bloodstream into the cells where it is converted into reducing equivalents used to produce ATP. AIMs, through Nrf2 activation, have been shown to promote glucose uptake and oxygen consumption in animal models of diet-induced obesity and diabetes15,16.
- Mitochondrial biogenesis: PGC1α is a protein that increases the number of mitochondria in a cell. Activation of Nrf2 has been shown to increase PGC1alpha expression in skeletal muscle, which may increase ATP production4,17.
Reducing Oxidative Stress
- Reactive oxygen species: ROS are chemically reactive molecules that contain oxygen and have important roles in cell signaling and balancing cellular systems. ROS are formed during mitochondrial ATP production and by a variety of other cellular processes. ROS increase inflammatory signaling, and excessive ROS can cause cellular damage to tissues in critical organs including the muscles, lung, heart, liver, brain, and eyes. Excessive ROS and chronic inflammation have been shown to be the cause of cellular damage in many diseases8. Nrf2 activation increases the cellular content of antioxidant and detoxification enzymes, which makes reducing equivalents available to neutralize ROS9. This suppresses the pro-inflammatory signaling effects of ROS and protects tissues from the damaging effects of excessive ROS.
- Chemical stress: Nrf2 protects cells from externally introduced toxicants by increasing the production of proteins that metabolize and eliminate these chemicals from the body. These proteins include glutathione-S-transferase, sulfotransferases, and multidrug resistance proteins that work together to modify and target toxicants for excretion10,11. Additionally, Nrf2 increases the rate of bile flow which is an important conduit for the elimination of toxicants10.
- Stress from improperly folded proteins: To function, a protein must assume a proper shape. Mutations or environmental stressors can disrupt this process, which is known as protein folding. Cells have multiple proteins, known as chaperones, which facilitate proper protein folding. When stress from improperly formed proteins is sensed, Nrf2 increases the production of a series of these protective chaperones, which assist misfolded proteins to adopt their proper shape, prevent the formation of toxic aggregates, and facilitate disaggregation and elimination of terminally misfolded proteins12,13.
Reducing Inflammation and Inflammatory Signaling
- Inhibition of inflammatory signaling: Mitochondrial ROS, NF-κB, and the NLRP3 inflammasome are important activators and regulators of the inflammatory response. AIMs, through reduction of ROS and inhibition of NF-κB and the inflammasome, suppress production of TNFα, IL-6, IL-1, and other inflammatory cytokines, or cellular messengers. The suppression of these inflammatory cytokines inhibits their downstream proinflammatory signaling pathways9,18,19.
- Reduction of enzymes associated with fibrosis and tissue remodeling: Tissue remodeling and fibrosis can be caused by chronic inflammation due to deposition of collagen and other factors. In a variety of models and settings, suppression of ROS and inhibition of NF-κB has been observed to reduce the expression of enzymes associated with tissue remodeling that are implicated in the progression of PH, certain types of cancer, arthritis, and many other diseases6,9.
- Inhibition of cellular proliferative pathways: Inhibition of NF-kB and the NLRP3 inflammasome by AIMs inhibits a number of signaling pathways that promote hyperproliferation (harmful excessive growth) of cells. The antiproliferative effects of AIMs have been demonstrated in a variety of models of cancer and inflammatory or fibrotic diseases20,21,22.
Effects in Immuno-Oncology Settings
New agents that improve anti-tumor immune responses have recently shown significant clinical activity, and this approach shows great promise as a source of improved cancer therapies. AIMs are uniquely positioned to be useful in this emerging field, because they inhibit a fundamental mechanism (induction of antigen-specific T cell tolerance) used by tumors to evade immune surveillance. A pivotal series of studies has shown that certain immature white blood cells, known as myeloid-derived suppressor cells (MDSCs), play a critical role in the induction of T cell tolerance, and production of ROS/RNS by MDSCs is required for this process. High levels of ROS/RNS induce nitrosylation of tumor antigens, effectively “cloaking” these antigens from T cell recognition23. AIMs inhibit this process and restore anti-tumor immune responses in vivo24. AIMs also promote the maturation of dendritic cells, which are required for presentation of antigens to the immune system25. Reduced expression of inducible nitric oxide synthase (iNOS), an enzyme that plays a major role in the generation of ROS/RNS, has been documented in tumor biopsies from cancer patients treated with AIMs26. Elevated iNOS levels are implicated in MDSC-mediated immune evasion and are correlated with poor clinical outcomes in cancer patients27.
The immunomodulatory effects of AIMs have the potential to be complementary to other modes of cancer immunotherapy, positioning AIMs to be applicable in a wide array of cancer therapy settings. In animal studies, AIMs treatment improved immune cell infiltration of tumors and enhanced the anti-tumor effects of a PD-1 inhibitor and a cancer vaccine24,26.
In addition to their inhibitory effects on MDSCs, AIMs have been extensively documented to have direct anti-cancer effects9,28.
- AIMs inhibit cancer cell proliferation in vitro and in vivo, and promote apoptosis of tumor cells by reducing the expression of Bcl-XL and other anti-apoptotic proteins.
- AIMs inhibit inflammatory signaling pathways, reduce the expression of proteins that promote tissue invasion and metastasis, and potently inhibit tumor-related angiogenesis.
- An extensive body of literature has documented the ability of AIMs to prevent carcinogenesis and inhibit early tumor formation and growth. These effects have potential clinical relevance for the prevention of post-therapy recurrence and suppression of micrometastases.
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- Holmstrom et al. Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration. Biol Open. 2013 Jun 20;2(8):761-70.
- Uruno et al. The Keap1-Nrf2 system prevents onset of diabetes mellitus. Mol Cell Biol. 2013 Aug;33(15):2996-3010.
- Wenz et al. Activation of PPAR/PGC-1α pathway prevents a bioenergetics deficit and effectively improves a mitochondrial myopathy phenotype. Cell Metab. 2008 Sep;8(3):249-56.
- Petri et al. Nrf2/ARE signaling pathway: key mediator in oxidative stress and potential therapeutic target in ALS. Neurol Res Int. 2012;2012:8780303.
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- Reisman et al. CDDO-9,11-dihydro-trifluoroethyl amide (CDDO-dhTFEA) induces hepatic cytoprotective genes and increases bile flow in rats. Xenobioteica. 2013 Jul;43(7):571-8.
- Klaassen and Reisman. Nrf2 the rescue: Effects of the antioxidative/electrophilic response on the liver. Toxicol Appl Pharmacol. 2010 Apr 1;244(1):57-65.
- Hensen et al. Activation of the antioxidant response in methionine deprived human cells results in an HSF1-independent increase in HSPA1A mRNA levels. Biochimie. 2013 Jun;95(6):1245-51.
- Niforou et al. Molecular chaperones and proteostasis regulation during redox imbalance. Redox Biol. 2014 Jan 30;2:323-32.
- Kensler et al. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007;47:89-116.
- Saha et al. The Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic-acid Methyl Ester Has Potent Anti-diabetic Effects in Diet-induced Diabetic Mice and Leprdb/db Mice. J Biol Chem. 2010; 285(52): 40581-40592.
- Shin et al. Role of Nrf2 in prevention of high-fat diet-induced obesity by synthetic triterpenoid CDDO-Imidazolide. Eur J Pharmacol. 2009; 620(1-3): 138-144.
- Whitman et al. Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic atrophy. Exp Cell Res. 2013 Oct 15;391(17):2673-83.
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- Yore et al. The synthetic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole blocks nuclear factor-κB activation through direct inhibition of IκB kinase β. Mol Cancer Ther. 2006 Dec;5(12):3232-9.
- Corzo et al. Mechanism regulating reactive oxygen species in tumor-induced myeloid-derived suppressor cells. J Immunol. 2009; 182(9): 5693-701.
- Nagaraj et al. Anti-inflammatory triterpenoid blocks immune suppressive function of MDSCs and improves immune response in cancer. Clin Cancer Res. 2010; 16(6): 1812-23.
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- Ekmekcioglu et al. Tumor iNOS predicts poor survival for stage III melanoma patients. Int J Cancer. 2006; 119(4): 861-6.
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