Targeting the NRF2 pathway: A promising approach for corneal endothelial dysfunction.

Maintaining corneal endothelial function is required for vision, and corneal endothelial dysfunction is a major cause of visual deficits and blindness worldwide. To date there has been a dearth of innovation for therapeutics targeting the corneal endothelium. However, recent advances in understanding the role of oxidative stress and mitochondrial dysfunction have revealed potential avenues for the development of new therapies. This review summarizes recent developments in elucidating the role of the NRF2 pathway in corneal endothelial health and disease, focusing specifically on Fuchs' endothelial corneal dystrophy and the loss of corneal endothelial cells associated with cataract surgery. The pro-mitochondrial and antioxidant phenotype elicited by NRF2 activation offers a promising opportunity for new therapeutics for the diseased corneal endothelium.

Pharmacokinetics and pharmacodynamics of the novel Nrf2 activator omaveloxolone in primates.

BACKGROUND: Omaveloxolone is a synthetic oleanane triterpenoid that pharmacologically activates Nrf2, a master transcription factor that regulates genes with antioxidative, anti-inflammatory, and mitochondrial bioenergetic properties, and is being evaluated in patients with Friedreich's ataxia. METHODS: The present study evaluated the pharmacokinetics (PK) and tissue distribution of omaveloxolone in monkeys after single and multiple oral doses, and then compared these data to initial results in Friedreich's ataxia patients. Pharmacodynamic (PD) evaluations in monkeys consisted of Nrf2 target gene mRNA expression in peripheral blood mononuclear cells (PBMCs), liver, lung, and brain. A PK/PD model was generated with the monkey data, and used to further evaluate the Friedreich's ataxia patient PK profile. RESULTS: Oral administration of omaveloxolone to monkeys was associated with dose-linear plasma PK and readily measureable and dose-proportional concentrations in liver, lung, and brain. Dose-dependent induction of Nrf2 target genes in PBMCs and tissues was also observed. Clinically, oral administration of omaveloxolone to Friedreich's ataxia patients at incremental doses from 2.5 to 300 mg produced dose-proportional systemic exposures. Clinical doses of at least 80 mg were associated with meaningful improvements in neurological function in patients and generated plasma omaveloxolone concentrations consistent with those significantly inducing Nrf2 target genes in monkeys, as shown with the monkey PK/PD model. CONCLUSION: Overall, the monkey data demonstrate a well-characterized and dose-proportional PK and tissue distribution profile after oral administration of omaveloxolone, which was associated with Nrf2 activation. Further, systemic exposures to omaveloxolone that produce Nrf2 activation in monkeys were readily achievable in Friedreich's ataxia patients after oral administration.

Making the best use of experts' estimates to prioritise monitoring and management actions: A freshwater case study.

Under limited time and resources, ecological managers are under increasing pressure to demonstrate tangible impact of monitoring activities. Value of Information (VOI) has been advocated as an ideal tool to evaluate whether more data is required to improve expected management outcomes. Yet, despite several recent works explaining its value, VOI remains seldom used in practice. Here we provide an example of a successful ecological application of VOI. We apply VOI to a novel multi-objective freshwater management problem and show how to make the best use of expert data through a robust sensitivity analysis. Unlike previous VOI approaches, our analysis provides statistical confidence to our recommendations. We apply our approach to the recovery of Moira grass (Pseudoraphis spinescens) plains, a threatened vegetation community at the Ramsar-listed Barmah Forest on the Murray River, Australia. Working closely with managers, we discovered that although many threats may impede Moira grass recovery, reducing grazing pressure and applying ideal depth and duration of flooding were most likely to lead to recovery. We found that learning from monitoring can significantly increase the existing extent of Moira grass, although these gains are modest compared to immediate management action. Our study shows how VOI can be used to demonstrate efficient use of limited environmental water to maximise ecological impact and increase transparency when making monitoring or management decisions. More broadly, the study methods will be of interest to any environmental manager who needs to prioritise monitoring and evaluation activities subject to a limited research budget. At a time where researchers and managers are asked to be more accountable for their decision-making, VOI provides a very accessible tool that can speed up the decision of whether to wait and collect more data or act immediately despite uncertainty.

Nrf2 Activation Is a Potential Therapeutic Approach to Attenuate Diabetic Retinopathy.

Purpose: Oxidative stress is a causal factor in the development of diabetic retinopathy; however, clinically relevant strategies to treat the disease by augmenting antioxidant defense mechanisms have not been fully explored. We hypothesized that boosting nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant capacity with the novel Nrf2 activator dh404, would protect the retina in diabetes including vision-threatening breakdown of the blood-retinal barrier (BRB) and associated damage to macroglial Müller cells. Methods: Sprague-Dawley rats were randomized to become diabetic or nondiabetic and administered dh404 by gavage for 10 weeks. Complementary in vitro studies were performed in cultured Müller cells exposed to hyperglycemia. Results: In diabetes, dh404 prevented vascular leakage into the retina and vitreous cavity as well as upregulation of the vascular permeability and angiogenic factors, VEGF, and angiopoietin-2, and inflammatory mediators, including TNF-α and IL-6. Müller cells, which maintain BRB integrity and become gliotic in diabetes with increased immunolabeling for glial fibrillary acidic protein, were protected by dh404. In diabetes, dh404 bolstered the antioxidant capacity of the retina with an increase in hemeoxygenase-1, nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) quinine oxidoreductase-1, and Nrf2. Further, dh404 attenuated the diabetes-induced increase in oxidative stress as measured by dihydroethidium and 8-oxo-2'-deoxyguanosine (8-OHdG) immunolabeling as well as NADPH oxidase isoform expression. Studies in Müller cells supported these findings with dh404 attenuating the hyperglycemia-induced increase in vascular permeability, angiogenic and inflammatory mediators, and oxidative stress. Conclusions: Our data demonstrate the ability of dh404 to protect the retina against diabetes-induced damage and potentially prevent vision loss.

The NRF2 activator DH404 attenuates adverse ventricular remodeling post-myocardial infarction by modifying redox signalling.

BACKGROUND: The novel synthetic triterpenoid, bardoxolone methyl, has the ability to upregulate cytoprotective proteins via induction of the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway. This makes it a promising therapeutic agent in disease states characterized by dysregulated oxidative signalling. We have examined the effect of a Nrf2 activator, dihydro-CDDO-trifluoroethyl amide (DH404), a derivative of bardoxolone methyl, on post-infarct cardiac remodeling in rats. METHODS/RESULTS: DH404, administered from day 2 post myocardial infarction (MI: 30min transient ischemia followed by reperfusion) resulted in almost complete protection against adverse ventricular remodeling as assessed at day 28 (left ventricular end-systolic area: sham 0.14±0.01cm2, MI vehicle 0.29±0.04cm2 vs. MI DH404 0.18±0.02cm2, P<0.05); infarct size (21.3±3.4% MI vehicle vs. 10.9±2.3% MI DH404, P<0.05) with associated benefits on systolic function (fractional shortening: sham 71.9±2.6%, MI vehicle 36.2±1.9% vs. MI DH404 58.6±4.0%, P<0.05). These structural and functional benefits were associated with lower myocardial expression of atrial natriuretic peptide (ANP, P<0.01 vs. MI vehicle), and decreased fibronectin (P<0.01 vs. MI vehicle) in DH404-treated MI rats at 28 days. MI increased glutathionylation of endothelial nitric oxide synthase (eNOS) in vitro - a molecular switch that uncouples the enzyme, increasing superoxide production and decreasing nitric oxide (NO) bioavailability. MI-induced eNOS glutathionylation was substantially ameliorated by DH404. An associated increase in glutaredoxin 1 (Grx1) co-immunoprecipitation with eNOS without a change in expression was mechanistically intriguing. Indeed, in parallel in vitro experiments, silencing of Grx1 abolished the protective effect of DH404 against Angiotensin II-induced eNOS uncoupling. CONCLUSION: The bardoxolone derivative DH404 significantly attenuated cardiac remodeling post MI, at least in part, by re-coupling of eNOS and increasing the functional interaction of Grx1 with eNOS. This agent may have clinical benefits protecting against post MI cardiomyopathy.

The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activator dh404 protects against diabetes-induced endothelial dysfunction.

BACKGROUND: Vascular dysfunction is a pivotal event in the development of diabetes-associated vascular disease. Increased inflammation and oxidative stress are major contributors to vascular dysfunction. Nrf2, a master regulator of several anti-oxidant genes and a suppressor of inflammatory NF-κB, has potential as a target to combat oxidative stress and inflammation. The aim of this study was to investigate the effects of a novel Nrf2 activator, the bardoxolone methyl derivative dh404, on endothelial function in vitro and in vivo. METHODS: dh404 at 3 mg/kg was administered to male Akita mice, an established diabetic mouse model of insulin insufficiency and hyperglycemia, from 6 weeks of age. At 26 weeks of age, vascular reactivity was assessed by wire myography, pro-inflammatory expression was assessed in the aortas by qRT-PCR and immunohistochemistry, and systemic and vascular oxidative stress measurements were determined. Additionally, studies in human aortic endothelial cells (HAECs) derived from normal and diabetic patients in the presence or absence of dh404 included assessment of pro-inflammatory genes by qRT-PCR and western blotting. Oxidative stress was assessed by three methods; L-012, DCFDA and amplex red. Static adhesion assays were performed to determine the leukocyte-endothelial interaction in the presence or absence of dh404. RESULTS: Dh404 significantly attenuated endothelial dysfunction in diabetic Akita mice characterized by reduced contraction in response to phenylephrine and the downregulation of inflammatory genes (VCAM-1, ICAM-1, p65, IL-1ß) and pro-oxidant genes (Nox1 and Nox2). Furthermore, reduced systemic and vascular oxidative stress levels were observed in diabetic Akita mice. dh404 exhibited cytoprotective effects in diabetic HAECs in vitro, reflected by significant upregulation of Nrf2-responsive genes, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), reduction of oxidative stress markers (O 2·- and H2O2), inhibition of inflammatory genes (VCAM-1 and the p65 subunit of NF-κB) and attenuation of leukocyte-endothelial interactions (P < 0.05 for all in vitro and in vivo parameters; one or two-way ANOVA as appropriate with post hoc testing). CONCLUSION: These studies demonstrate that upregulation of Nrf2 by dh404 represents a novel therapeutic strategy to limit diabetes-associated vascular injury.

Authors response on Schick et al. 2017 "An experiment of the impact of a neonicotinoid pesticide on honey bees; the value of a formal analysis of the data". Environ Sci Eur (2017).

Whilst a formal statistical analysis of any experimental data is always preferable in principle, in the case of Pilling et al. (PLoS ONE 8:e77193, 2013), it is hard to see how the results of any formal analysis-including those provided by Schick et al.-could be considered reliable. Regardless of the issue of statistical analysis, there was a wealth of valuable and novel biological and chemical residue data generated under field conditions of use in Pilling et al., which when taken into consideration alongside other relevant available published data and information (i.e. expert judgement) demonstrated a low risk to honeybees from thiamethoxam when used as a seed treatment on oilseed rape. Indeed, similar conclusions have been reported in subsequent published honeybee field studies using thiamethoxam seed-treated oilseed rape, thus supporting the original conclusions of Pilling et al.

A potent Nrf2 activator, dh404, bolsters antioxidant capacity in glial cells and attenuates ischaemic retinopathy.

An imbalance in oxidative stress and antioxidant defense mechanisms contributes to the development of ischaemic retinopathies such as diabetic retinopathy and retinopathy of prematurity (ROP). Currently, the therapeutic utility of targeting key transcription factors to restore this imbalance remains to be determined. We postulated that dh404, an activator of nuclear factor erythroid-2 related factor 2 (Nrf2), the master regulator of oxidative stress responses, would attenuate retinal vasculopathy by mechanisms involving protection against oxidative stress-mediated damage to glia. Oxygen-induced retinopathy (OIR) was induced in neonatal C57BL/6J mice by exposure to hyperoxia (phase I) followed by room air (phase II). dh404 (1 mg/kg/every second day) reduced the vaso-obliteration of phase I OIR and neovascularization, vascular leakage and inflammation of phase II OIR. In phase I, the astrocytic template and vascular endothelial growth factor (VEGF) expression necessary for physiological angiogenesis are compromised resulting in vaso-obliteration. These events were attenuated by dh404 and related to dh404's ability to reduce the hyperoxia-induced increase in reactive oxygen species (ROS) and markers of cell damage as well as boost the Nrf2-responsive antioxidants in cultured astrocytes. In phase II, neovascularization and vascular leakage occurs following gliosis of Müller cells and their subsequent increased production of angiogenic factors. dh404 reduced Müller cell gliosis and vascular leakage in OIR as well as the hypoxia-induced increase in ROS and angiogenic factors with a concomitant increase in Nrf2-responsive antioxidants in cultured Müller cells. In conclusion, agents such as dh404 that reduce oxidative stress and promote antioxidant capacity offer a novel approach to lessen the vascular and glial cell damage that occurs in ischaemic retinopathies.

The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation.

Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is an important factor in the pathogenesis of age-related macular degeneration (AMD). Previous studies have shown that RTA 408, a synthetic triterpenoid compound, potently activates Nrf2. This study aimed to investigate the protective effects of RTA 408 in cultured RPE cells during oxidative stress and to determine the effects of RTA 408 on Nrf2 and its downstream target genes. Primary human RPE cells were pretreated with RTA 408 and then incubated in 200µM H2O2 for 6h. Cell viability was measured with the WST-8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining and Hoechst 33342 fluorescent staining. Reduced (GSH) and oxidized glutathione (GSSG) were measured using colorimetric assays. Nrf2 activation and its downstream effects on phase II enzymes were examined by Western blot. Treatment of RPE cells with nanomolar ranges (10 and 100nM) of RTA 408 markedly attenuated H2O2-induced viability loss and apoptosis. RTA 408 pretreatment significantly protected cells from oxidative stress-induced GSH loss, GSSG formation and decreased ROS production. RTA 408 activated Nrf2 and increased the expression of its downstream genes, such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1. Consequently, the enzyme activities of NQO1, Grx1, and Trx1 were fully protected by RTA 408 pretreatment under oxidative stress. Moreover, knockdown of Nrf2 by siRNA significantly reduced the cytoprotective effects of RTA 408. In conclusion, our data suggest that RTA 408 protect primary human RPE cells from oxidative stress-induced damage by activating Nrf2 and its downstream genes.

Topical application of RTA 408 lotion activates Nrf2 in human skin and is well-tolerated by healthy human volunteers.

BACKGROUND: Topical application of the synthetic triterpenoid RTA 408 to rodents elicits a potent dermal cytoprotective phenotype through activation of the transcription factor Nrf2. Therefore, studies were conducted to investigate if such cytoprotective properties translate to human dermal cells, and a topical lotion formulation was developed and evaluated clinically. METHODS: In vitro, RTA 408 (3-1000 nM) was incubated with primary human keratinocytes for 16 h. Ex vivo, RTA 408 (0.03, 0.3, or 3 %) was applied to healthy human skin explants twice daily for 3 days. A Phase 1 healthy volunteer clinical study with RTA 408 Lotion (NCT02029716) consisted of 3 sequential parts. In Part A, RTA 408 Lotion (0.5 %, 1 %, and 3 %) and lotion vehicle were applied to individual 4-cm(2) sites twice daily for 14 days. In Parts B and C, separate groups of subjects had 3 % RTA 408 Lotion applied twice daily to a 100-cm(2) site for 14 days or a 500-cm(2) site for 28 days. RESULTS: RTA 408 was well-tolerated in both in vitro and ex vivo settings up to the highest concentrations tested. Further, RTA 408 significantly and dose-dependently induced a variety of Nrf2 target genes. Clinically, RTA 408 Lotion was also well-tolerated up to the highest concentration, largest surface area, and longest duration tested. Moreover, significant increases in expression of the prototypical Nrf2 target gene NQO1 were observed in skin biopsies, suggesting robust activation of the pharmacological target. CONCLUSIONS: Overall, these data suggest RTA 408 Lotion is well-tolerated, activates Nrf2 in human skin, and appears suitable for continued clinical development.

Regime shifts, thresholds and multiple stable states in freshwater ecosystems; a critical appraisal of the evidence.

The concepts of ecosystem regime shifts, thresholds and alternative or multiple stable states are used extensively in the ecological and environmental management literature. When applied to aquatic ecosystems, these terms are used inconsistently reflecting differing levels of supporting evidence among ecosystem types. Although many aquatic ecosystems around the world have become degraded, the magnitude and causes of changes, relative to the range of historical variability, are poorly known. A working group supported by the Australian Centre for Ecological Analysis and Synthesis (ACEAS) reviewed 135 papers on freshwater ecosystems to assess the evidence for pressure-induced non-linear changes in freshwater ecosystems; these papers used terms indicating sudden and non-linear change in their titles and key words, and so was a positively biased sample. We scrutinized papers for study context and methods, ecosystem characteristics and focus, types of pressures and ecological responses considered, and the type of change reported (i.e., gradual, non-linear, hysteretic or irreversible change). There was little empirical evidence for regime shifts and changes between multiple or alternative stable states in these studies although some shifts between turbid phytoplankton-dominated states and clear-water, macrophyte-dominated states were reported in shallow lakes in temperate climates. We found limited understanding of the subtleties of the relevant theoretical concepts and encountered few mechanistic studies that investigated or identified cause-and-effect relationships between ecological responses and nominal pressures. Our results mirror those of reviews for estuarine, nearshore and marine aquatic ecosystems, demonstrating that although the concepts of regime shifts and alternative stable states have become prominent in the scientific and management literature, their empirical underpinning is weak outside of a specific environmental setting. The application of these concepts in future research and management applications should include evidence on the mechanistic links between pressures and consequent ecological change. Explicit consideration should also be given to whether observed temporal dynamics represent variation along a continuum rather than categorically different states.

Radiation protection of the gastrointestinal tract and growth inhibition of prostate cancer xenografts by a single compound.

Normal tissue toxicity markedly reduces the therapeutic index of genotoxic anticancer agents, including ionizing radiation. Countermeasures against tissue damage caused by radiation are limited by their potential to also protect malignant cells and tissues. Here, we tested a panel of signal transduction modifiers for selective radioprotection of normal but not tumor tissues. These included three inhibitors of GSK3 (LiCl, SB216763, and SB415286) and two inhibitors of NF-κB (ethyl pyruvate and RTA 408). Among these, the thiol-reactive triterpenoid RTA 408 emerged as a robust and effective protector of multiple organ systems (gastrointestinal, skin, and hemopoietic) against lethal doses of radiation. RTA 408 preserved survival and proliferation of intestinal crypt cells in lethally irradiated mice while reducing apoptosis incidence in crypts and villi. In contrast, RTA 408 uniformly inhibited growth of established CWR22Rv1, LNCaP/C4-2B, PC3, and DU145 xenografts either alone or combined with radiation. Antitumor effects in vivo were associated with reduced proliferation and intratumoral apoptosis and with inhibition of NF-κB-dependent transcription in PC3 cells. Selective protection of normal tissue compartments by RTA 408 critically depended on tissue context and could not be replicated in vitro. Collectively, these data highlight the potential of RTA 408 as a cytoprotective agent that may be safely used in chemoradiation approaches.

Mechanisms contributing to adverse cardiovascular events in patients with type 2 diabetes mellitus and stage 4 chronic kidney disease treated with bardoxolone methyl.

BACKGROUND: Bardoxolone methyl, an Nrf2-activating and nuclear factor-κB-inhibiting semisynthetic oleanane triterpenoid compound, was evaluated in a phase 3 trial (BEACON) in patients with type 2 diabetes mellitus (T2DM) and stage 4 chronic kidney disease (CKD). The trial was terminated because of an increase in heart failure events in the bardoxolone methyl group, many of which appeared related to fluid retention. Thus, additional analyses were conducted to explain these serious adverse events. METHODS: Patients (n = 2,185) were randomized to receive once-daily bardoxolone methyl (20 mg) or placebo. Twenty-four-hour urine collections were analyzed in a subset of the BEACON population and from a separate, open-label pharmacology study in patients with stage 3b/4 CKD and T2DM administered 20 mg bardoxolone methyl once daily for 56 consecutive days. RESULTS: Bardoxolone-methyl-treated patients in the BEACON substudy had a clinically meaningful reduction in urine volume and sodium excretion at week 4 relative to baseline (p < 0.05), and a separate study revealed that decreased sodium excretion and urine output occurred in some patients with stage 4 CKD but not those with stage 3b CKD. The clinical phenotype of fluid overload and heart failure in BEACON was similar to that observed with endothelin receptor antagonists in advanced CKD patients, and preclinical data demonstrate that bardoxolone methyl modifies endothelin signaling. CONCLUSIONS: The totality of the evidence suggests that through modulation of the endothelin pathway, bardoxolone methyl may pharmacologically promote acute sodium and volume retention and increase blood pressure in patients with more advanced CKD.

Targeting Nrf2 by dihydro-CDDO-trifluoroethyl amide enhances autophagic clearance and viability of ß-cells in a setting of oxidative stress.

Nrf2 appears to be a critical regulator of diabetes in rodents. However, the underlying mechanisms as well as the clinical relevance of the Nrf2 signaling in human diabetes remain to be fully understood. Herein, we report that islet expression of Nrf2 is upregulated at an earlier stage of diabetes in both human and mice. Activation of Nrf2 suppresses oxidative stress and oxidative stress-induced ß-cell apoptosis while enhancing autophagic clearance in isolated rat islets. Additionally, oxidative stress per se activated autophagy in ß-cells. Thus, these results reveal that Nrf2 drives a novel antioxidant independent autophagic clearance for ß-cell protection in the setting of diabetes.

Topical application of the synthetic triterpenoid RTA 408 protects mice from radiation-induced dermatitis.

Free radicals produced during cancer radiotherapy often leads to dermatitis, with the insult ranging from mild erythema to moist desquamation and ulceration. This toxicity can be dose limiting and promote chronic complications, such as fibrosis and wound recurrence. The purpose of this study was to evaluate if RTA 408, a synthetic triterpenoid that potently activates the antioxidative transcription factor Nrf2 and inhibits the proinflammatory transcription factor nuclear factor-kappa b (NF-κB), could protect skin from radiation-induced dermatitis. Mice were irradiated (10 Gy/day) on days 0-2 and 5-7, and RTA 408 (0.01%, 0.1% and 1.0%) was topically applied once daily starting on day 5 or up to day 40. Dermatitis severity was evaluated using a scale ranging from 0 (normal) to 5 (frank ulceration), as well as histologically. The mRNA expression of Nrf2 and NF-κB target genes in skin was also evaluated. RTA 408 (0.01%, 0.1% and 1.0%) reduced the percentage of animal-days with scores ≥2 by 11%, 31% and 55% and scores ≥3 by 16%, 60% and 80%, respectively. Dose-dependent improvements in the appearance of skin were also manifestly visible, with RTA 408 at 1.0% eliciting a normal macroscopic appearance by the end of the treatment period on day 40, including substantial hair regrowth. Moreover, 1.0% RTA 408 markedly reduced epidermal and collagen thickening, prevented dermal necrosis and completely alleviated skin ulcers. These improvements were associated with significant increases in Nrf2 target genes and significant decreases in NF-κB target genes. Together, these data indicate that RTA 408 represents a potentially promising new therapy for the treatment of radiation-induced dermatitis.

Derivative of bardoxolone methyl, dh404, in an inverse dose-dependent manner lessens diabetes-associated atherosclerosis and improves diabetic kidney disease.

Oxidative stress and inflammation are inextricably linked and play essential roles in the initiation and progression of diabetes complications such as diabetes-associated atherosclerosis and nephropathy. Bolstering antioxidant defenses is an important mechanism to lessen oxidative stress and inflammation. In this study, we have used a novel analog of the NFE2-related factor 2 (Nrf2) agonist bardoxolone methyl, dh404, to investigate its effects on diabetic macrovascular and renal injury in streptozotocin-induced diabetic apolipoprotein E(-/-) mice. We show that dh404, at lower but not higher doses, significantly lessens diabetes-associated atherosclerosis with reductions in oxidative stress (in plasma, urine, and vascular tissue) and proinflammatory mediators tumor necrosis factor-α, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemotactic protein-1 (MCP-1). We demonstrate that dh404 attenuates functional (urinary albumin-to-creatinine ratio) and structural (mesangial expansion) glomerular injury and improves renal tubular injury. Liver functional and structural studies showed that dh404 is well tolerated. Complementary in vitro studies in normal rat kidney cells showed that dh404 significantly upregulates Nrf2-responsive genes, heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and glutathione-S transferase, with inhibition of transforming growth factor-ß-mediated profibrotic fibronectin, collagen I, and proinflammatory interleukin-6. Higher doses of dh404 were associated with increased expression of proinflammatory mediators MCP-1 and nuclear factor-κB. These findings suggest that this class of compound is worthy of further study to lessen diabetes complications but that dosage needs consideration.

Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2.

Nuclear factor erythroid 2-related factor (Nrf2) is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems, thus Nrf2 has evolved to be an attractive drug target for the treatment and/or prevention of human disease. Several synthetic oleanolic triterpenoids including dihydro-CDDO-trifluoroethyl amide (dh404) appear to be potent activators of Nrf2 and exhibit chemopreventive promises in multiple disease models. While the pharmacological efficacy of Nrf2 activators may be dependent on the nature of Nrf2 activation in specific cell types of target organs, the precise role of Nrf2 in mediating biological effects of Nrf2 activating compounds in various cell types remains to be further explored. Herein we report a unique and Nrf2-dependent anti-inflammatory profile of dh404 in inflamed macrophages. In lipopolysaccharide (LPS)-inflamed RAW264.7 macrophages, dh404 dramatically suppressed the expression of pro-inflammatory cytokines including inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1ß), while minimally regulating the expression of interleulin-6 (IL-6), IL-1ß, and tumor necrosis factor alpha (TNFα). Dh404 potently activated Nrf2 signaling; however, it did not affect LPS-induced NF-κB activity. Dh404 did not interrupt the interaction of Nrf2 with its endogenous inhibitor Kelch-like ECH associating protein 1 (Keap1) in macrophages. Moreover, knockout of Nrf2 blocked the dh404-induced anti-inflammatory responses in LPS-inflamed macrophages. These results demonstrated that dh404 suppresses pro-inflammatory responses in macrophages via an activation of Nrf2 independently of Keap1 and NF-κB, suggesting a unique therapeutic potential of dh404 for specific targeting a Nrf2-mediated resolution of inflammation.

Topical application of the synthetic triterpenoid RTA 408 activates Nrf2 and induces cytoprotective genes in rat skin.

RTA 408 is a member of the synthetic oleanane triterpenoid class of compounds known to potently activate the cytoprotective transcription factor Nrf2. Because skin is constantly exposed to external oxidative stress, such as that from ultraviolet radiation, from chemical exposure, during improper wound healing, and throughout the course of cancer radiation therapy, it may benefit from activation of Nrf2. This study was conducted to evaluate the transdermal penetration properties and Nrf2 activation potential of RTA 408 in normal rat skin. RTA 408 (0.1, 1.0, or 3.0%) was applied topically to the shaved skin of male Sprague-Dawley rats twice daily for 4 days and once on Day 5. Topical application of RTA 408 resulted in transdermal penetration, with low but dose-dependent plasma exposure with AUC(0-24 h) values of 3.6, 26.0, and 41.1 h ng/mL for the 0.1, 1.0, and 3.0% doses, respectively. Further, topical application of RTA 408 resulted in increased translocation of Nrf2 to the nucleus, dose-dependent mRNA induction of Nrf2 target genes (e.g. Nqo1, Srxn1, Gclc, and Gclm), and induction of the protein expression of the prototypical Nrf2 target gene Nqo1 and increased total glutathione (GSH) in normal rat skin. Immunohistochemistry demonstrated that increased staining for Nqo1 and total GSH of structures in both the epidermis and dermis was consistent with the full transdermal penetration of RTA 408. Finally, topically administered RTA 408 was well tolerated with no adverse in-life observations and normal skin histology. Thus, the data support the further development of RTA 408 for the potential treatment of skin diseases.

Bardoxolone methyl analogs RTA 405 and dh404 are well tolerated and exhibit efficacy in rodent models of Type 2 diabetes and obesity.

Bardoxolone methyl and related triterpenoids are well tolerated and efficacious in numerous animal models potentially relevant to patients with Type 2 diabetes and chronic kidney disease. These agents enhance glucose control and regulate lipid accumulation in rodent models of diabetes and obesity, and improve renal function, reduce inflammation, and prevent structural injury in models of renal disease. However, a recent study in Zucker diabetic fatty (ZDF) rats noted poor tolerability with the bardoxolone methyl analog RTA 405 within 1 mo after treatment initiation, although this study was confounded in part by the use of an impure RTA 405 batch. To investigate these discordant observations, the present studies were conducted to further characterize triterpenoids in rodent models of diabetes and obesity. A follow-up study was conducted in ZDF rats with two related triterpenoids (RTA 405 and dh404) for 1.5 mo. Consistent with previous rodent experience, and in contrast to the more recent ZDF report, ZDF rats administered RTA 405 or dh404 exhibited no adverse clinical signs, had laboratory values similar to controls, and exhibited no evidence of adverse liver or kidney histopathology. Additionally, RTA 405 was well tolerated in streptozotocin-induced Type 1 diabetic rats and high-fat-diet-induced obese mice. The present results are consistent with the overall published body of data obtained with triterpenoids and provide further evidence that these molecules are well tolerated without adverse effects on hepatobiliary or renal function in rodent models of diabetes and obesity.

CDDO-9,11-dihydro-trifluoroethyl amide (CDDO-dhTFEA) induces hepatic cytoprotective genes and increases bile flow in rats.

1. The transcription factor Nrf2 is important for hepatoprotection against oxidative stress, as it regulates many cytoprotective genes, including several important for glutathione (GSH) homeostasis. In addition to being an important endogenous antioxidant, GSH is also critical for the maintenance of bile acid-independent bile flow. While it has been well-established that synthetic oleanane triterpenoids pharmacologically activate Nrf2, their effects on bile flow and hepatic cytoprotective capacity have not been fully explored. 2. The present studies were conducted to evaluate the effects of a compound in this class, CDDO-9,11-dihydro-trifluoroethyl amide (CDDO-dhTFEA), on these parameters. CDDO-dhTFEA at 3, 10 or 30 mg/kg was orally administered to bile duct-cannulated rats once daily for 7 days, with bile collected 5 h after each dose for 1 h. Livers were harvested after the final bile collection for the evaluation of histology and Nrf2 targets. 3. CDDO-dhTFEA did not affect liver histology. CDDO-dhTFEA markedly and dose-dependently increased bile flow, as well as the biliary excretion of GSH, cholesterol and phospholipids without affecting biliary excretion of bile acids. This was accompanied by dose-dependent increases in mRNA expression and/or enzyme activity of a broad panel of cytoprotective Nrf2 target genes, including NAD(P)H quinone oxidoreductase 1 (Nqo1), thioredoxin reductase (Txnrd), sulfiredoxin 1(Srxn1), glutamate cysteine ligase catalytic and modifier subunits (Gclc and Gclm), glutathione reductase (Gsr), gamma-glutamyl transpeptidase 1 (Ggt1), heme oxygenase-1 (Ho-1) and epoxide hydrolase-1 (Eh-1). 4. These data further demonstrate the important hepatobiliary attributes of oleanane synthetic triterpenoids and support their continued investigation for liver diseases.