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.

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.

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.

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.

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.

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 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.

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.

Bardoxolone methyl decreases megalin and activates nrf2 in the kidney.

Inflammation and oxidative stress are hallmarks and mediators of the progression of CKD. Bardoxolone methyl, a potent activator of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant and anti-inflammatory response, increases estimated GFR and decreases BUN, serum phosphorus, and uric acid concentrations in patients with moderate to severe CKD. However, it also increases albuminuria, which is associated with inflammation and disease progression. Therefore, we investigated whether this bardoxolone methyl-induced albuminuria may result from the downregulation of megalin, a protein involved in the tubular reabsorption of albumin and lipid-bound proteins. Administration of bardoxolone methyl to cynomolgus monkeys significantly decreased the protein expression of renal tubular megalin, which inversely correlated with the urine albumin-to-creatinine ratio. Moreover, daily oral administration of bardoxolone methyl to monkeys for 1 year did not lead to any adverse effects on renal histopathologic findings but did reduce serum creatinine and BUN, as observed in patients with CKD. Finally, the bardoxolone methyl-induced decrease in megalin corresponded with pharmacologic induction of renal Nrf2 targets, including NAD(P)H:quinone oxidoreductase 1 enzyme activity and glutathione content. This result indicates that Nrf2 may have a role in megalin regulation. In conclusion, these data suggest that the increase in albuminuria that accompanies bardoxolone methyl administration may result, at least in part, from reduced expression of megalin, which seems to occur without adverse effects and with strong induction of Nrf2 targets.

Comments on the paper "A statistical assessment of differences and equivalences between genetically modified and reference plant varieties" by van der Voet et al. 2011.

van der Voet et al. (2011) describe statistical methodology that the European Food Safety Authority expects an applicant to adopt when making a GM crop regulatory submission. Key to their proposed methodology is the inclusion of reference varieties in the experimental design to provide a measure of natural variation amongst commercially grown crops. While taking proper account of natural variation amongst commercial varieties in the safety assessment of GM plants makes good sense, the methodology described by the authors is shown here to be fundamentally flawed and consequently cannot be considered fit for purpose in its current form.

Anti-inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin gallate in human corneal epithelial cells.

PURPOSE: To determine the anti-inflammatory and anti-oxidant effects of epigallocatechin gallate (EGCG), the major polyphenol component of green tea, in human corneal epithelial cells (HCEpiC). METHODS: HCEpiC were challenged with interleukin-1ß (IL-1ß) for 18 h or hyperosmolarity (440 mOsm) for 24 h. Luminex technology was used to determine the effects of EGCG (0.3-30 µM) on IL-1ß- or hyperosmolar-induced cytokine release into the medium. Cell metabolic activity was measured using the alamarBlue assay. Effects of EGCG on mitogen-activated protein kinase (MAPK) phosphorylation were determined by cell-based enzyme-linked immunosorbent assay (ELISA) and western blotting. Effects of EGCG on nuclear factor kappa B (NFκB) and activator protein-1 (AP-1) transcriptional activity were assessed by reporter gene assay. The effects of EGCG on glucose oxidase (GO)-induced reactive oxygen species (ROS) production was determined using the ROS probe CM-H2DCFDA. RESULTS: Treatment of HCEpiC with 1 ng/ml IL-1ß for 18 h significantly increased release of the cytokines/chemokines granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1), while hyperosmolarity-induced release of IL-6 and MCP-1. When cells were treated with IL-1ß and EGCG or hyperosmolarity and EGCG there was a dose-dependent reduction in release of these cytokines/chemokines, with significant inhibition observed at 3-30 µM. There was no effect of EGCG on cell metabolic activity at any of the doses tested (0.3-30 µM). EGCG significantly inhibited phosphorylation of the MAPKs p38 and c-Jun N-terminal kinase (JNK), and NFκB and AP-1 transcriptional activities. There was a significant dose-dependent decrease in GO-induced ROS levels after treatment of HCEpiC with EGCG. CONCLUSIONS: EGCG acts as an anti-inflammatory and anti-oxidant agent in HCEpiC and therefore may have therapeutic potential for ocular inflammatory conditions such as dry eye.

A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex.

PURPOSE: The Schirmer's test is commonly used in the clinic for the diagnosis of dry eye disease by measuring tear volume. This report describes a procedure which can be used to recover tears from the Schirmer strip for the measurement of multiple tear cytokines as well as matrix metalloproteinases (MMPs) by Luminex technology. METHODS: Cytokine and MMP recovery was determined by using spiked Schirmer strips presoaked with known cytokines or MMPs prepared in PBS with 1% BSA. In a clinical study, tears were collected from 5 subjects using Schirmer strips. Strips were stored on ice immediately after removal from the subject and stored dry at -20 °C for 16-24 h. Cytokines were extracted from the Schirmer strip in 0.5 M NaCl with 0.5% Tween-20. Concentrations of cytokines and MMPs in collected tear samples were analyzed by Luminex using both a 10-cytokine and a 5-MMP kit. RESULTS: The standard curves for the assay in both the kit assay buffer and extraction buffer were identical for 9 of the 10 cytokines and all 5 MMPs. In the clinical sample all the cytokines (interleukin 1α [IL-1α], IL-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-10, IL-13, monocyte chemotactic protein-1 [MCP-1], and tumor necrosis factor-α [TNF-α]) and 5 MMPs (MMP-1, MMP-2, MMP-7, MMP-9, and MMP-10) tested were detected in at least 50% of the 10 subject samples. Recoveries from extracted Schirmer strips were >60% for 8 of the 10 cytokines and all MMPs. CONCLUSIONS: Numerous cytokines and MMPs were detected in the tear samples collected using the Schirmer strip, including many that have been implicated in ocular surface disease. This procedure may be used to evaluate the cytokine and MMP content in tear samples in clinical studies, especially for the evaluation of dry eye therapeutics. Because the Schirmer test is routine in the assessment of dry eye, this method offers the opportunity to evaluate both the quantity and quality of the tears.

Eosinophil as a cellular target of the ocular anti-allergic action of mapracorat, a novel selective glucocorticoid receptor agonist.

PURPOSE: Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action. METHODS: With in vitro studies apoptosis was evaluated in human eosinophils by flow cytometry and western blot of caspase-3 fragments. Eosinophil migration toward platelet-activating factor was evaluated by transwell assays. Interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), and the chemokine (C-C motif) ligand 5 (CCL5)/regulated upon activation normal T cell expressed, and presumably secreted (RANTES) were measured using a high-throughput multiplex luminex technology. Annexin I and the chemochine receptor C-X-C chemokine receptor 4 (CXCR4) were detected by flow cytometry. With in vivo studies, allergic conjunctivitis was induced in guinea pigs sensitized to ovalbumin by an ocular allergen challenge and evaluated by a clinical score. Conjunctival eosinophils were determined by microscopy or eosinophil peroxidase assay. RESULTS: In cultured human eosinophils, mapracorat showed the same potency as dexamethasone but displayed higher efficacy in increasing spontaneous apoptosis and in counteracting cytokine-sustained eosinophil survival. These effects were prevented by the glucocorticoid receptor antagonist mifepristone. Mapracorat inhibited eosinophil migration and IL-8 release from eosinophils or the release of IL-6, IL-8, CCL5/RANTES, and TNF-α from a human mast cell line with equal potency as dexamethasone, whereas it was clearly less potent than this glucocorticoid in inducing annexin I and CXCR4 expression on the human eosinophil surface; this was taken as a possible sign of glucocorticoid-dependent transactivation. In the guinea pig, mapracorat or dexamethasone eye drops induced an analogous reduction in clinical symptoms of allergic conjunctivitis and conjunctival eosinophil accumulation. CONCLUSIONS: Mapracorat appears to be a promising candidate for the topical treatment of allergic eye disorders. It maintains an anti-allergic profile similar to that of dexamethasone but seems to have fewer transactivation effects in comparison to this classical glucocorticoid. Some of its cellular targets may contribute to eosinophil apoptosis and/or to preventing their recruitment and activation and to inhibiting the release of cytokines and chemokines.

Ocular pharmacokinetics of mapracorat, a novel, selective glucocorticoid receptor agonist, in rabbits and monkeys.

Mapracorat is a selective glucocorticoid receptor agonist in development for the treatment of a variety of ocular diseases. The purpose of this investigation was to evaluate the ocular pharmacokinetics of mapracorat after topical dosing over a range of dose levels in rabbits and monkeys. Mapracorat was administered over a range of doses from 0.01 to 3000 µg/eye (rabbit) or 50 to 3000 µg/eye (monkey). All animals received a single instillation, and monkeys also received repeated (three times per day for 4 days) instillations. At predetermined intervals through at least 24 h after dosing, ocular tissues and plasma were collected and analyzed for mapracorat by liquid chromatography-tandem mass spectrometry. Mapracorat was rapidly absorbed and widely distributed into ocular tissues after topical ocular administration, with measurable levels sustained through ≥24 h. In both species, mapracorat concentrations were highest in tears followed by conjunctiva and cornea, with lower levels observed in iris/ciliary body and aqueous humor. Mapracorat concentrations in conjunctiva, cornea, and iris/ciliary body increased linearly with increasing dose levels. Ocular exposure was higher after repeated dosing to monkeys than after a single dose. Systemic exposure to mapracorat was low after a single administration, with an average maximal concentration of ≤2.0 ng/ml at the highest dose tested (3000 µg/eye). In comparison with the traditional glucocorticoids, dexamethasone (0.1%) and prednisolone acetate (1%), mapracorat (3%) demonstrated similar or higher levels in ocular tissues with lower systemic exposure. The favorable pharmacokinetic profile of mapracorat supports further clinical investigation and suggests that a convenient daily dosing regimen may be efficacious for this novel ophthalmic anti-inflammatory therapy.

Mapracorat, a novel selective glucocorticoid receptor agonist, inhibits hyperosmolar-induced cytokine release and MAPK pathways in human corneal epithelial cells.

PURPOSE: Increasing evidence suggests that tear hyperosmolarity is a central mechanism causing ocular surface inflammation and damage in dry eye disease. Mapracorat (BOL-303242-X) is a novel glucocorticoid receptor agonist currently under clinical evaluation for use in the treatment of dry eye disease. This study assessed the anti-inflammatory effects of mapracorat in an in vitro osmotic stress model which mimics some of the pathophysiological changes seen in dry eye. METHODS: Human corneal epithelial cells were cultured in normal osmolar media (317 mOsM) or 440 mOsM hyperosmolar media for 24 h. Luminex technology was used to determine the effect of mapracorat on hyperosmolar-induced cytokine release. Effects of mapracorat on mitogen-activated protein kinase (MAPK) phosphorylation were determined by cell based ELISA. Effects of mapracorat on nuclear factor kappa B (NFkappaB) and activator protein-1 (AP-1) transcriptional activity were assessed by reporter gene assay. Dexamethasone was used as a control. RESULTS: Hyperosmolar conditions induced release of the pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) from cultured human corneal epithelial cells, and altered the phosphorylation state of p38 and c-Jun N-terminal kinase (JNK) and transcriptional activity of NFkappaB and AP-1. Incubation of cells with mapracorat inhibited hyperosmolar-induced cytokine release with comparable activity and potency as dexamethasone. This inhibition was reversed by the glucocorticoid receptor antagonist mifepristone (RU-486). Increased phosphorylation of p38 and JNK caused by hyperosmolarity was inhibited by mapracorat. Mapracorat also significantly decreased the hyperosmolar-induced increase in NFkappaB and AP-1 transcriptional activity. CONCLUSIONS: Mapracorat acts as a potent anti-inflammatory agent in corneal epithelial cells challenged with osmotic stress, with comparable activity to the traditional steroid dexamethasone. These in vitro data suggest that mapracorat may be efficacious in the treatment of dry eye disease.

WY-14 643, a selective PPAR{alpha} agonist, induces proinflammatory and proangiogenic responses in human ocular cells.

Peroxisome proliferator-activated receptor α (PPARα) agonism in ocular inflammation has not been thoroughly investigated. The objective of this investigation was to determine the effect of WY-14 643, a selective PPARα agonist, on inflammatory cytokine release in human ocular cells. Stimulation of primary human corneal epithelial cells, keratocytes, and retinal endothelial cells with 1 to 10 ng/mL interleukin 1ß (IL-1ß) resulted in a significant increase in numerous inflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor α (TNF-α); and dexamethasone was able to significantly inhibit these effects. However, WY-14 643 did not effectively block IL-1ß-induced cytokine release in ocular cells; rather, significant increases in IL-1ß-induced inflammatory cytokines were observed in these cells but not in aortic smooth muscle cells. WY-14 643 also significantly upregulated vascular endothelial growth factor (VEGF) expression in corneal epithelial cells and keratocytes. These studies demonstrate for the first time that PPARα agonism may be proinflammatory and proangiogenic in a variety of ocular cells and suggest that therapeutic applications of such agents in ophthalmology may be limited.

BOL-303242-X, a novel selective glucocorticoid receptor agonist, with full anti-inflammatory properties in human ocular cells.

PURPOSE: BOL-303242-X is a novel selective glucocorticoid receptor agonist under clinical evaluation for the treatment of inflammatory skin and eye diseases. Data from in vitro and in vivo studies suggest an improved side-effect profile of this compound compared to classical glucocorticoids. The aim of this study was to determine the anti-inflammatory effect of BOL-303242-X in ocular cells. METHODS: Four primary human ocular cell cultures, including human conjunctival fibroblasts (HConFs), human corneal epithelial cells (HCEpiCs), human optic nerve astrocytes (HONAs), and human retinal endothelial cells (HRECs), as well as a human monocytic cell line, THP-1, were challenged with either lipopolysacharide (LPS) or interleukin-1ss (IL-1ss). Luminex technology was used to determine the effect of BOL-303242-X on LPS- or IL-1ss-induced cytokine release and intercellular adhesion molecule-1 (ICAM-1) levels. Effects of BOL-303242-X on nuclear factor kappa B (NFkappaB) and mitogen-activated protein kinase (MAPK) in HCEpiCs were also assessed by measuring inhibitory kappa B protein-alpha (IkappaB-alpha), phosphorylated p65 NFkappaB, and MAPK levels by western blotting. Dexamethasone (DEX) or triamcinolone acetonide (TA) was used as the control. RESULTS: LPS or IL-1ss induced multiple cytokine release in all cell types studied. BOL-303242-X significantly reduced LPS- or IL-1ss-induced inflammatory cytokine release in a dose-dependent manner, including granulocyte colony-stimulating factor (G-CSF), IL-1ss, IL-6, IL-8, IL-12p40, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha). BOL-303242-X showed activity and potency comparable to that observed for DEX or TA. A statistically significant inhibitory effect of BOL-303242-X was observed at doses ranging from 1 to 100 nM in HConFs, HCEpiCs, HONAs, and THP-1. The IC(50) values for these effects were in the low nM range. BOL-303242-X also significantly reduced LPS-induced IL-1ss release and ICAM-1 levels in HRECs. Furthermore, BOL-303242-X inhibited IL-1ss-induced decreases in IkappaB-alpha levels, as well as IL-1ss-induced phosphorylation of NFkappaB, p38, and c-Jun-N-terminal kinase (JNK) MAPKs in HCEpiCs. CONCLUSIONS: BOL-303242-X acts as a potent anti-inflammatory agent in various primary human ocular cells with similar activity and potency compared to classical steroids. Results also suggest that MAPK (p38 and JNK) and NFkappaB signaling pathways are involved in the anti-inflammatory properties of BOL-303242-X in HCEpiCs. An improved side effect profile of this novel SEGRA compound has been reported recently. Thus, BOL-303242-X may provide a new option for the treatment of ophthalmic conditions with an inflammatory component.

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.

Besifloxacin, a novel fluoroquinolone antimicrobial agent, exhibits potent inhibition of pro-inflammatory cytokines in human THP-1 monocytes.

OBJECTIVES: This study was conducted to evaluate the anti-inflammatory effects of besifloxacin, a novel fluoroquinolone under clinical evaluation for treatment of ophthalmic infections. METHODS: Cytokine expression in human THP-1 monocytes was stimulated by lipopolysaccharide (LPS), and Luminex technology was used to determine the effect of besifloxacin on LPS-induced cytokine expression. Moxifloxacin, a marketed fluoroquinolone used in ophthalmic infections, was used as the control. RESULTS: LPS induced measurable cytokine expression for 14 of the 16 cytokines assayed. Besifloxacin significantly inhibited LPS-stimulated cytokine production in a dose-dependent manner, with a comparable [granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1beta, IL-8, interferon-inducible protein (IP-10), monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha)] or better [granulocyte CSF (G-CSF), IL-1alpha, IL-1 receptor antagonist (IL-1ra) and IL-6] potency compared with moxifloxacin. A significant inhibitory effect of besifloxacin was observed at 0.1 mg/L for IL-1alpha, at 1 mg/L for G-CSF, IL-1ra and IL-6 and at 30 mg/L for GM-CSF, IL-12p40, IL-1beta, IL-8, IP-10, MCP-1 and MIP-1alpha. CONCLUSIONS: Besifloxacin acts as an anti-inflammatory agent in monocytes in vitro; this attribute may enhance its efficacy in ocular infections with an inflammatory component and warrants further investigation.