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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.

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.

Graphical abstract, 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 200 μM H2O2 for 6 h. 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 100 nM) 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., Highlights • Oxidative stress contributes to the pathogenesis of AMD. • RTA408 in nanomolar concentrations increased survival and restored the redox state in H2O2-damaged RPE cells. • RTA408 stimulated Nrf2 and downstream antioxidant enzymes Grx1, NQO1, and HO-1. • Inhibition of Nrf2 abolished RTA408’s protective effects. • RTA408 may have the potential to treat AMD and other degenerative eye diseases.

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.

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 Muller 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 Muller 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. Muller 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 Muller 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.

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-β–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.

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.

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.

Purpose To explore the effect of a novel multipurpose contact lens solution (MPS) on the junction protein distribution and barrier function of cultured human corneal epithelial cell monolayers. Methods Cultured human corneal epithelial cells (HCEpiC) were exposed to a novel MPS (MPS A; Biotrue™ multi-purpose solution, Bausch & Lomb Incorporated) at 50%, 75% and 100% for 10 or 30 min. Four commercially available MPS products, MPS B (AQuify, Ciba Vision), MPS C (COMPLETE MPS Easy Rub, AMO), MPS D (OPTI-FREE Express, Alcon) and MPS E (OPTI-FREE RepleniSH, Alcon) were tested in parallel. Tight junction structure and integrity were evaluated by confocal microscopy using ZO-1 antibody and scanning microscopy (SEM). Quantitative evaluation of MPSs on epithelial barrier function was determined by measuring transepithelial electrical resistance (TEER) across HCEpiC grown on Transwell Clear permeable supports and on electric cell-substrate impedance sensing (ECIS) electrode arrays. Results Overall after exposure to the three concentrations (50%, 75%, and 100%) of MPS A, ZO-1 distribution and fluorescent intensity on the cell surface appeared similar to the media control with continuous tight junctions and clear intercellular junctions. At all measured time points after exposure to MPS A (50% or 75%) there was also no effect on the TEER using both resistance methodologies, and SEM showed that MPS A appeared similar to the Hank's balanced salt solution (HBSS) control. In cells exposed to MPS D there was a dose-dependent change in the distribution of ZO-1, some cell detachment, and a decrease in monolayer resistance at all time points measured. Ultrastructurally, MPS D caused gross changes, including damage to cell junctions and plasma membranes. To a lesser extent, the remaining three commercial MPS products demonstrated some effects on tight junction ZO-1 distribution and/or TEER. Conclusions Based on the in vitro measurements of tight junction protein expression, monolayer integrity, and transepithelial electrical resistance, the novel multipurpose contact lens solution (MPS A) did not alter corneal barrier function as compared to media, PBS or HBSS control. Clinical significance of the observed differences in epithelial barrier function among the MPSs tested needs further investigation.

The purpose of this investigation was to evaluate the ocular pharmacokinetic/pharmacodynamic (PK/PD) relationship for besifloxacin, moxifloxacin, and gatifloxacin using rabbit ocular PK data, along with in vitro minimum inhibitory concentration (MIC90) values against methicillin- and ciprofloxacin-resistant Staphylococcus aureus (MRSA-CR) and Staphylococcus epidermidis (MRSE-CR).Rabbits received a topical instillation of Besivance™ (besifloxacin ophthalmic suspension, 0.6%), Vigamox (moxifloxacin hydrochloride ophthalmic solution, 0.5% as base), or Zymar (gatifloxacin ophthalmic solution, 0.3%), and ocular tissues and plasma were collected from 4 animals/treatment/collection time at 8 predetermined time intervals during the 24h after dosing. Ocular levels of each agent were measured by LC/MS/MS, and PK parameters (Cmax, Tmax, and AUC₀₋₂₄) were determined. AUC₀₋₂₄/MIC₉₀ ratios were calculated for tears, conjunctiva, cornea, and aqueous humor using previously reported MIC₉₀values for MRSA-CR and MRSE-CR.All of the fluoroquinolones tested demonstrated rapid penetration into ocular tissues after a single instillation. Besifloxacin demonstrated the highest exposure in tear fluid, while exposure in conjunctiva was comparable for all 3 compounds. Peak concentrations of all fluoroquinolones in aqueous humor were at or below ~1g/mL. In comparison with their MIC₉₀values against MRSE-CR and MRSA-CR, besifloxacin achieved an AUC₀₋₂₄/MIC₉₀ ratio of ~800 in tears, compared with values of ≤10 for moxifloxacin and gatifloxacin. In cornea, conjunctiva, and aqueous humor, the AUC₀₋₂₄/MIC₉₀ ratios were10 for all compounds. However, in these tissues AUC₀₋₂₄/MIC₉₀ ratios for besifloxacin were 1.5- to 38-fold higher than moxifloxacin and gatifloxacin.In rabbits, besifloxacin demonstrates a nonclinical ocular PK profile characterized by high and sustained concentrations in tear fluid, resulting in AUC₀₋₂₄/MIC₉₀ ratios of ~800 for ciprofloxacin-resistant MRSE and MRSA after a single administration. Although besifloxacin had the highest AUC₀₋₂₄/MIC₉₀ratios for intraocular tissues, the ratios for all of the drugs were below the target values needed for effective bacterial killing of ciprofloxacin-resistant MRSE and MRSA. Taken together, these nonclinical data indicate that besifloxacin has a favorable ocular PK/PD profile, consistent with the reported clinical efficacy of besifloxacin in the treatment of bacterial conjunctivitis, and consistent with the profile needed for ocular surface sterilization.

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 1β (IL-1β) 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-1β-induced cytokine release in ocular cells; rather, significant increases in IL-1β-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.

RTA 1701 is an orally-bioavailable, selective RORγt inhibitor. RORγt orchestrates the differentiation of Th17 cells and contributes to autoimmune disease pathogenesis. Inhibition of RORγt suppresses Th17 cells and the associated secretion of the effector pro-inflammatory cytokine IL-17A. As such, RTA 1701 is highly efficacious in rodent models of rheumatoid arthritis and multiple sclerosis. This study was conducted to evaluate the pharmacokinetics of RTA 1701 and its effects on ex vivo stimulation of IL-17A production in whole blood after oral administration to naïve healthy cynomolgus monkeys. Monkeys received a single administration of RTA 1701 (0.3, 3, 30, or 300 mg/kg). After a one-week washout, monkeys received the same doses of RTA 1701 once daily for 14 consecutive days. Blood was collected after a single dose and after 14 days of dosing for analysis of plasma RTA 1701 concentrations through 72 hours post-dose and for evaluation of RTA 1701 effects on ex vivo stimulation of IL-17A at 24, 30, and 72 hours post-dose. RTA 1701 exhibited oral bioavailability in monkeys with dose-dependent increases in exposure over the wide range of doses evaluated. Further, oral administration produced systemic exposure to RTA 1701 at sufficient levels to produce significant and dose-dependent suppression of ex vivo stimulation of IL-17A secretion in whole blood at 24 and 30 hours after single and repeat dosing, with IL-17A levels returning to baseline within 72 hours after a single dose. Collectively, these data demonstrate that RTA 1701 is orally bioavailable in non-human primates and significantly suppresses the IL-17A response. These data also support the future clinical development of RTA 1701 for the potential treatment of autoimmune diseases.

Chronic (+)-methamphetamine (METH) use during pregnancy increases the health risk for both mother and fetus. To provide insights into these risks, the relationship between changes in METH disposition and METH-induced pharmacological effects were studied in Sprague-Dawley rat dams and litters. Timed-pregnant rats (n = 5–6) were given saline or METH (5.6–17.8 mg/kg/day) by continuous sc infusion from gestational day (GD) 7 (before organogenesis) until GD21 (0–2 days before delivery). By GD11, all rats in the 17.8-mg/kg/day group died or were sacrificed for humane reasons. There were significant (p < 0.05) dose- and gestational time-dependent decreases in maternal body weight in the 10- to 13.2-mg/kg/day groups, which slowly recovered to near normal by GD21. Continued METH dosing in the surviving groups did not affect the mean pups/litter weight at the end of the experiment on GD21. While maternal and fetal METH and (+)-amphetamine serum concentrations were similar on GD21, brain concentrations were significantly greater in the dams (p < 0.05). Importantly, brain-to-serum ratios in the dams were 9:1 and 3:1 in the pups. METH systemic clearance (ClS) in dams significantly (p < 0.05) decreased from 52 ± 14 ml/min/kg on GD10 to 28 ± 6 ml/min/kg on GD21 in all dose groups, indicating late-gestational stage reductions in METH ClS. Overall, these findings suggest that there were two periods of increased susceptibility for dams and fetuses during chronic METH treatment. First was the period after the start of METH dosing in which neuroadaptation and tolerance to METH occurs in the adult. The second was at the end of pregnancy when METH clearance was significantly reduced.

This study was conducted to investigate putative antagonism of integrin receptors αMβ2 and αLβ2 by a novel coumarin derivative (BOL-303225-A), its efficacy in-vivo after retinal ischaemia-reperfusion injury, and its bioavailability in rat plasma. A cellular adhesion assay in Jurkat and U937 cells, and a flow cytometry assay with an antibody against the β2 subunit were conducted. BOL-303225-A bioavailability in rat plasma and the retinal levels of myeloperoxidase (MPO) after ischaemia- reperfusion injury were evaluated after oral administration (10 mg kg−1). In-vitro cell viability assays revealed no cytotoxicity for BOL-303225-A over a wide dose range, and IC50 values of 32.3 ± 1.5 μM and 84.95 ± 2.3 μM were found for Jurkat and U937 cells, respectively. The drug showed specific binding to the αMβ2 and αLβ2 integrin receptors expressed by U937 and Jurkat cells, respectively, producing a fluorescence shift towards lower values in a concentration-dependent manner. The pharmacokinetic profile of BOL-303225-A exhibited rapid absorption following oral administration in the rat. A significant reduction of retinal MPO levels was observed in drug-treated rats. This study demonstrated that BOL-303225-A acts as an antagonist of the integrin αLβ2 and αMβ2 receptors, suggesting that this drug could be used for ocular diseases such as diabetic retinopathy.

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).

Purpose. To provide a review of the literature on rabbit models of contact lens–associated corneal hypoxia. Methods. The literature was searched for articles describing corneal hypoxia in the rabbit coincident with contact lens wear. The articles were identified by key word searches for rabbit models, contact lens, hypoxia, and cornea. Results. Designing a rabbit model of contact lens–associated corneal hypoxia requires identification of the key determinants of the physiologic response. The retention of soft contact lenses, particularly high-water content lenses, is problematic in the rabbit eye, in which blink frequency is on the order of minutes. An intact nictitating membrane may be beneficial in the hydration and, therefore, retention of soft contact lenses. Although tarsorrhaphy improves lens retention, lid closure also enhances the hypoxic response and may obscure the benefits of the greater oxygen transmissibility of soft contact lenses. The duration of lens wear in most reviewed studies was 24 hours or less, with only a few studies evaluating the hypoxic effect of lens wear of 1 week or more. Methods commonly used to characterize corneal hypoxia in rabbits include pachymetry, tear film lactate dehydrogenase measurements, clinical observations of limbal hyperemia, and more invasive methods of assessing cell proliferation and apoptosis. In summary, the ideal rabbit model of contact lens–associated corneal hypoxia depends on the question to be answered. Conclusions. This review tabulates the known studies, highlights key considerations in study design, and describes useful methods in characterization of the hypoxic response.

OBJECTIVES To determine the vitreous volume, and retinal and lens wet weights in male and female Gottingen minipigs of different age groups. PROCEDURES Vitreous, lens and retina were isolated from fresh minipig eyes. Vitreous volume, lens and retina weight were measured and allometric scaling was utilized to predict these parameters. RESULTS There were no gender differences in body weights or retina and lens weights in the three age groups examined in this study. For vitreous volumes, females had a significantly larger volume (approximately 10%-12%) than males in the 4-6 and 6-8 month, but not in the 8-11-month group. The mean body weight increased from 12.2 +/- 2.6 kg (4-6 months) to 19.4 +/- 4.0 kg (8-11 months). In the same period, the mean vitreous volume increased from 2.00 +/- 0.28 to 2.67 +/- 0.31 mL, while the mean retinal and lens weights increased from 114 +/- 22 to 126 +/- 17 mg, and from 298 +/- 26 to 392 +/- 15 mg, respectively. Allometric analysis between six species for vitreous volume and four species for lens weight covering a weight range of 260-fold was not able to estimate the vitreous volume, but it did predict the lens weight in 8-11 months minipigs. CONCLUSIONS Overall these measurements add important context to intraocular pharmacology studies, and will help in the improved design and interpretation of such experiments.

The value of cynomolgus and rhesus monkeys to predict human pharmacokinetic parameters has been well established in recent years. However, practical limitations on cost and accessibility can often be a deterrent to obtain data in these valuable species, and the characterization of the predictive power of other nonhuman primates would be useful. Therefore, the present investigation was designed to evaluate the pharmacokinetics of a test set of marketed compounds in the African green monkey, to compare the pharmacokinetics of these agents between nonhuman primate species, and to validate the ability of the African green monkey to predict human pharmacokinetics. Intravenous pharmacokinetics were evaluated for 11 test compounds in this study and compared with data from rats, dogs, cynomolgus/rhesus monkeys, and humans. The results from this investigation indicate that African green monkeys deliver reasonable prediction of human clearance and mean residence time and volume of distribution, although somewhat less accurately than cynomolgus and rhesus monkeys, particularly for volume of distribution, potentially because of body size or composition or experimental design differences. Furthermore, use of an optimized clearance prediction algorithm from the literature enhanced predictivity over a simple liver blood flow-based extrapolation methodology. The data from this study show that African green monkeys have the potential to be used as a surrogate for cynomolgus or rhesus monkeys in preclinical pharmacokinetic studies, particularly for the study of clearance processes, and should be considered as an alternate nonhuman primate test species.

Sodium fluorescein permeability assay, alamarBlue assay, and scanning electron microscopy were performed to study the effects of various contact lens disinfecting multipurpose solutions (MPS) on the integrity of the ocular surface epithelium by using corneal epithelial cells.The sodium fluorescein permeability and alamarBlue activity of monolayer cultures of human corneal epithelial cells were compared after exposure to ReNu MultiPlus, OPTI-FREE Express, AQuify 5 Minute, SOLO-care Plus With Aqualube, and Complete Moisture Plus contact lens care solutions for 15 minutes. Additional cell monolayers were prepared for each treatment and were analyzed with a scanning electron microscope after a 10-minute exposure.The sodium fluorescein permeability assay, alamarBlue assay, and scanning electron microscopy showed that OPTI-FREE Express was significantly more damaging to the human corneal epithelial cell monolayer than ReNu MultiPlus, SOLO-care Plus With Aqualube, Complete Moisture Plus, and AQuify 5 Minute contact lens solutions. Cell monolayers treated with OPTI-FREE Express were more permeable to sodium fluorescein and showed lower metabolic activity than cell monolayers treated with the other multipurpose solutions.This experiment shows that ReNu MultiPlus, SOLO-care Plus With Aqualube, Complete Moisture Plus, and AQuify 5 Minute contact lens solutions have a minimal effect on human corneal epithelial cells in culture, whereas OPTI-FREE Express has a higher negative effect on tight junctions, cell membranes, and overall metabolism of these human corneal epithelial cells.

The aim of this study was to determine the anti-inflammatory effects of besifloxacin, a novel fluoroquinolone under clinical evaluation for the treatment of ophthalmic infections, in human corneal epithelial cells (HCEpiC).Cytokine expression in primary HCEpiC was stimulated by interleukin-1beta (IL-1beta), and Luminex technology was used to determine the effect of besifloxacin on IL-1beta-induced cytokine release. Effect of besifloxacin on nuclear factor kappa B (NFkappaB), and mitogen-activated protein kinase (MAPK) was assessed by measuring inhibitory kappa B protein (IkappaB) degradation, NFkappaB nuclear translocation, and MAPK phosphorylation by Western blotting. Moxifloxacin, a marketed fluoroquinolone, was used as the control. Anti-inflammatory efficacy of besifloxacin was also evaluated in rabbits infected with methicillin-resistant Staphylococcus aureus (MRSA).Stimulation of HCEpiC with IL-1beta increased release of 12 of the 29 cytokines measured. Besifloxacin significantly inhibited IL-1beta-induced cytokine release in a dose-dependent manner, with a comparable (IL-8) or better (G-CSF, GM-CSF, IL-6, MCP-1, MIP-1beta, TGF-alpha, and TNF-alpha) efficacy compared to moxifloxacin. A significant inhibitory effect of besifloxacin was observed at 1 or 10 microg/ml. Besifloxacin inhibited IkappaB degradation, NFkappaB nuclear translocation, and activation of p38 and JNK MAPKs. Based on improvement of clinical score, besifloxacin showed statistically significant anti-inflammatory effect compared to saline treatment.Besifloxacin acts as an anti-inflammatory agent in corneal epithelial cells in vitro, by inhibiting the NFkappaB and MAPK pathways. Besifloxacin also exhibits anti-inflammatory efficacy in vivo. The anti-inflammatory attribute may enhance its efficacy in the treatment of ocular infections with an inflammatory component and warrants further investigation.

The prediction of human pharmacokinetics from preclinical species is an integral component of drug discovery. Recent studies with a 103-compound dataset suggested that scaling from monkey pharmacokinetic data tended to be the most accurate method for predicting human clearance. Additionally, interrogation of the two-dimensional molecular properties of these molecules produced a set of associations which predict the likely extrapolative outcome (success or failure) of preclinical data to project human pharmacokinetics. However, a limitation of the previous analyses was the relative paucity of data for typical "discovery-like" molecules (molecular weight >300 and/or clogP >3). The objective of this investigation was to generate preclinical data required for extension of this dataset for additional discovery-like molecules and determine whether the aforementioned findings continue to apply for these molecules. In vivo nonrodent intravenous pharmacokinetic data were generated for 13 molecules, and data for 8 additional molecules were obtained from the literature. Additionally, the various scaling methodologies and molecular features analysis were applied to this new dataset to predict human pharmacokinetics. Whereas the predictive accuracies demonstrated across all of the various methodologies were lower for this higher clearance compound dataset, scaling from monkey liver blood flow continued to be an accurate methodology, and human volume of distribution was similarly well predicted regardless of scaling methodology. Lastly, application of the molecular feature associations, particularly data-dependent associations, afforded an improved predictivity compared with the liver blood flow scaling approaches, and provides insight into the extrapolation of high clearance compounds in the preclinical species to human.

This study was conducted to compare and contrast published in vitro intrinsic clearance values reported for compounds from different laboratories and the predictivity of these data to project in vivo clearance.A total of 103 compounds were selected for investigation and an exhaustive literature search was conducted to identify in vitro intrinsic clearance (CL,i) values for comparative purposes. The simple well-stirred model was used to predict in vivo clearance using these in vitro intrinsic clearance values.Data were available in the literature for10% of the compounds of interest in rat, dog, monkey, or human S9, as well as10% for dog or monkey microsomes or hepatocytes. Therefore, this comparative exercise was limited to rat and human microsomes and hepatocytes. Examination of the available CL,i values indicated a substantial (up to 100 s-fold) variation in values reported in the literature; this variability translated into substantial variation in predicted in vivo clearance.The literature paucity and variability described here demonstrate the importance of generating experimentally consistent de novo CL,i data for the purpose of method validation or in vitro-in vivo scaling.

A novel and highly convergent synthesis leading to 2‐phenyl‐quinolines has been developed. As demonstrated in the preparation of 6‐fluoro‐3‐(3‐oxo‐piperazin‐1‐ylmethyl)‐2‐phenyl‐quinoline‐4‐carboxylic acid [(S)‐1‐cyclohexyl‐ethyl]‐amide (8), the method provides fascile access to this class of analogues via the common intermediate 7.

The early characterisation of drug metabolism and pharmacokinetic (DMPK) properties of new chemical entities plays a key role in the pharmaceutical industry's effort to reduce attrition. Specifically, a major goal of early DMPK studies is to accurately predict the behaviour of new chemical entities in humans, thus allowing likely failures to be terminated rapidly and resource to be placed on molecules most likely to succeed. The present review summarises progress over the past several years in the key technologies used in the pharmaceutical industry to achieve these goals: namely, in vivo, in vitro and in silico/computational tools. The limitations of the various assays are discussed, with attention also given to likely future directions in this field.

This study was conducted to comprehensively survey the available literature on intravenous pharmacokinetic parameters in the rat, dog, monkey, and human, and to compare common methods for extrapolation of clearance, to identify the most appropriate species to use in pharmacokinetic lead optimization, and to ascertain whether adequate prospective measures of predictive success are currently available. One hundred three nonpeptide xenobiotics were identified with intravenous pharmacokinetic data in rat, dog, monkey, and human; both body weight- and hepatic blood flow-based methods were used for scaling of clearance. Allometric scaling approaches, particularly those using data from only two of the preclinical species, were less successful at predicting human clearance than methods based on clearance as a set fraction of liver blood flow from an individual species. Furthermore, commonly used prospective measures of allometric scaling success, including correlation coefficient and allometric exponent, failed to discriminate between successful and failed allometric predictions. In all instances, the monkey tended to provide the most qualitatively and quantitatively accurate predictions of human clearance and also afforded the least biased predictions compared with other species. Additionally, the availability of data from both common nonrodent species (dog and monkey) did not ensure enhanced predictive quality compared with having only monkey data. The observations in this investigation have major implications for pharmacokinetic lead optimization and for prediction of human clearance from in vivo preclinical data and support the continued use of nonhuman primates in preclinical pharmacokinetics.

Our laboratory is engaged in an ongoing analysis of a 103-compound data set containing reliable intravenous pharmacokinetic parameters in the rat, dog, monkey, and human, and we have previously reported our findings regarding extrapolation of clearance. In this article, we report on our findings regarding volume of distribution and mean residence time. Various allometric and nonallometric methods were used to predict human volume of distribution based on preclinical pharmacokinetic data; clearance and volume of distribution values generated by various means were then used to estimate mean residence time. From both a quantitative and qualitative perspective, estimating human volume and mean residence time based on monkey data alone was the most accurate approach evaluated. For volume, estimation based on monkey data alone was quantitatively the least biased of all approaches evaluated. Additionally, prediction of mean residence time based on clearance and volume from the monkey was the only extrapolation method that exhibited a positive, rather than negative, bias. None of the allometric scaling approaches investigated afforded optimal predictivity for either volume or mean residence time, and neither the correlation coefficient nor the allometric exponent allowed a prospective estimation of predictive success or failure. These observations regarding volume and mean residence time confirm our earlier results with clearance, and further confirm the value of the monkey as a species for pharmacokinetic lead optimization.

1. Transporters have been increasingly identified as a factor in limiting the oral bioavailability of certain drugs. Previously, the present authors investigated a compound (SB-265123) with an apparent absolute oral bioavailability (Fapp) consistently > 100%, and excluded likely artefactual causes for this observation, as well as standard considerations of non-stationary or non-linear pharmacokinetics. The data led the authors to believe that SB-265123 might be a transporter substrate in the rat, and it was hypothesized that transporter interactions might be responsible for the observed Fapp > 100%. 2. In the present study, a model was proposed incorporating rapid and complete absorption and elimination by a saturable intestinal secretory pathway. Intestinal secretion was demonstrated for SB-265123 using a rat single-pass intestinal perfusion technique. In addition, in a study employing both independent and simultaneous intravenous and oral administration of SB-265123, exposure to SB-265123 was greater than additive on joint intravenous and oral administration, lending further support to the hypothesis of a saturable transporter. Furthermore, in a study with co-administration of GF120918A, a transporter inhibitor, the observed Fapp for SB-265123 was only 84 +/- 17%, providing additional evidence for transporter involvement in the >100% Fapp phenomenon. 3. Experience with SB-265123 illustrates a counterintuitive impact of transporters on oral bioavailability and highlights the importance of considering transporter interactions in the systemic disposition of xenobiotics, even those not demonstrating low oral bioavailability.

1. SB-265123 is a novel alphavbeta3 (the vitronectin receptor) antagonist. Previous rat studies with it revealed an apparent absolute oral bioavailability (Fapp) of greater than 100%. The present studies were conducted to investigate the potential causes for this observation. 2. Of 49 SB-265123 analogues evaluated in rat using an identical experimental design, Fapp100% was observed for 22 of them, suggesting that the observed Fapp100% with SB-265123 was not anomalous. All 22 compounds had clearances15 ml min(-1) kg(-1). However, Fapp100% were not recorded for all low-clearance analogues. 3. Using SB-265123 as a model to investigate potential artefacts, it was demonstrated that using a chiral assay did not decrease Fapp. Additionally, qualitative sample analysis demonstrated that no metabolites were present in the plasma that could interfere with the liquid chromatography coupled with tandem mass spectrometry detection assay. The high Fapp was also dose-order-, delivery system- and vehicle-independent, and was not affected by the feeding status of the animals. Furthermore, a linearity experiment and an absorption study indicated that oral administration of SB-265123 does not result in hepatic portal vein concentrations that exceed the pharmacokinetic linearity of SB-265123. 4. These observations suggest that the observed Fapp100% for SB-265123 is not due to an experimental artefact or an obvious pharmacokinetic non-linearity. The mechanism(s) for this phenomenon is explored further in the second part of the present paper.

GF120918A, the HCl salt of GF120918 (9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl) ethyl]phenyl]-4-acridine-carboxamide), has been used both in vitro and in vivo as a tool inhibitor of P-glycoprotein (Pgp) to investigate the role of transporters in the disposition of various test molecules. However, to date, a detailed description of the preclinical pharmacokinetic properties of GF120918A has not been published. This investigation was performed to evaluate in vitro and in vivo pharmacokinetic properties of GF120918A in the mouse, rat, dog, and monkey and to evaluate the in vivo efficacy of GF120918A in modulating absorption and systemic exposure in the monkey. GF120918A demonstrated reasonable absorption and systemic exposure in each of the species studied, however, in rodents, administration of 300 mg/kg afforded a substantially less than linear increase in systemic exposure compared with 30 mg/kg. In accordance with its intestinal and hepatic exposure and potency against P-glycoprotein, GF120918A demonstrated marked modulation of erythromycin systemic exposure in the monkey, with no effect on propranolol, a negative control molecule. In vitro, GF120918A demonstrated high plasma protein binding across species, although a definitive protein binding evaluation was precluded by poor recovery, particularly in buffer and in mouse, rat, and dog plasma. GF120918A did not demonstrate potent inhibition of several human cytochrome P450 enzymes evaluated in vitro, with IC(50) values well above concentrations anticipated to be achieved in vivo. Together, these data confirm the utility of GF120918A as a tool P-glycoprotein inhibitor in preclinical species and offer additional guidance on preclinical dose regimens likely to produce P-glycoprotein-mediated effects.

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.

This study was conducted to compare the ability of two potential microdialysis perfusates to enhance the recovery of SB-265123, a lipophilic, highly protein-bound compound, both in vitro and in vivo. Initial in vitro experiments established that the recovery of SB-265123 by microdialysis using normal saline as a perfusate was poor (1.7%). Different concentrations of Intralipid® and Encapsin® also were evaluated in an identical in vitro setting, to determine enhancement of recovery. In vitro recovery was enhanced to approximately 24 and 65% with 5 and 20% Intralipid®, and to approximately 59 and 62% with 5 and 20% Encapsin®, respectively. A rat in vivo study was conducted with 20% Encapsin® to confirm the in vitro observations. In the in vivo study, 75–80% recovery of free SB-265123 was achieved using 20% Encapsin® as a perfusate. The results from this study indicate that for SB-265123, a lipophilic, highly protein-bound molecule, Encapsin® is an efficient recovery enhancer in vitro. The results from this investigation further demonstrate that a recovery enhancer may be useful for in vivo applications, even with a compound that is highly bound to plasma protein. Copyright © 2002 John Wiley & Sons, Ltd.

Oral bioavailability measurements in rats for over 1100 drug candidates studied at SmithKline Beecham Pharmaceuticals (now GlaxoSmithKline) have allowed us to analyze the relative importance of molecular properties considered to influence that drug property. Reduced molecular flexibility, as measured by the number of rotatable bonds, and low polar surface area or total hydrogen bond count (sum of donors and acceptors) are found to be important predictors of good oral bioavailability, independent of molecular weight. That on average both the number of rotatable bonds and polar surface area or hydrogen bond count tend to increase with molecular weight may in part explain the success of the molecular weight parameter in predicting oral bioavailability. The commonly applied molecular weight cutoff at 500 does not itself significantly separate compounds with poor oral bioavailability from those with acceptable values in this extensive data set. Our observations suggest that compounds which meet only the two criteria of (1) 10 or fewer rotatable bonds and (2) polar surface area equal to or less than 140 A(2) (or 12 or fewer H-bond donors and acceptors) will have a high probability of good oral bioavailability in the rat. Data sets for the artificial membrane permeation rate and for clearance in the rat were also examined. Reduced polar surface area correlates better with increased permeation rate than does lipophilicity (C log P), and increased rotatable bond count has a negative effect on the permeation rate. A threshold permeation rate is a prerequisite of oral bioavailability. The rotatable bond count does not correlate with the data examined here for the in vivo clearance rate in the rat.

1. Inhibition of p38 MAP kinase has been investigated extensively as a potential therapy for cytokine-mediated diseases such as autoimmune and inflammatory diseases. SB-242235 (1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl) imidazole) is a potent and selective p38 MAP kinase inhibitor; the preclinical pharmacokinetics of SB-242235 have been described previously. The present studies were conducted to describe the in vitro metabolic rates and routes of SB-242235 metabolism, to characterize its in vivo preclinical metabolism, and to use these data to aid in the prediction of the pharmacokinetic behaviour of SB-242235 in man. 2. SB-242235 was metabolically stable in rat, dog, monkey and human hepatic microsomes, isolated hepatocytes and liver slices in vitro. The in vivo preclinical metabolism studies were consistent with the in vitro findings; SB-242235 was minimally metabolized, and was primarily excreted unchanged in the urine (45 and 67% of the administered dose in the rat and monkey, respectively). 3. Allometric scaling using various correction factors predicted that SB-242235 would have low clearance in man with a predicted half-life ranging from 11.5 to 18.7h. This prediction was consistent with the observed mean half-life of 16.4h in the first-in-man study for SB-242235. An allometric scaling method with a correction for interspecies differences in glomerular filtration rate provided the most accurate prediction of the pharmacokinetic behaviour of SB-242235 in humans, although the clinical data also highlight potential difficulties in conducting prospective allometry.

1. SB-242235 (1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl) imidazole) is a potent and selective p38 MAP kinase inhibitor that may be an effective therapy for cytokine-mediated diseases such as autoimmune or inflammatory diseases. The present studies were conducted to evaluate the pharmacokinetics of SB-242235 in several preclinical species, including rat, dog and monkey. 2. SB-242235 demonstrates generally favourable pharmacokinetic properties in all species examined. Systemic plasma clearance was high in rat, but in the non-rodent species SB-242235 demonstrated low to moderate clearance with plasma half-lives > 4h. Oral bioavailability in each preclinical species was high. In rat and monkey, SB-242235 demonstrated non-linear elimination kinetics that manifested as a decrease in clearance with increasing dose and apparent oral bioavailability > 100% at high oral doses. Furthermore, SB-242235 displayed concentration-dependent plasma protein binding over a concentration range of 1000-10,000 ng ml(-1). 3. In conclusion, SB-242235 demonstrates high oral bioavailability across the major preclinical species, and may thus be a useful tool compound for investigation of the role of p38 inhibition in various disease states. However, the observations of non-linear protein binding and disposition also suggest the need for caution in the design of and data interpretation from such studies.

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. Mol Cancer Ther; 13(12); 2968–77. ©2014 AACR.

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.

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-1β), while minimally regulating the expression of interleulin-6 (IL-6), IL-1β, 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.

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.