Identification of Pathway-Specific Serum Biomarkers of Response to Glucocorticoid and Infliximab Treatment in Children with Inflammatory Bowel Disease

Objective: Serum biomarkers may serve to predict early response to therapy, identify relapse, and facilitate drug development in inflammatory bowel disease (IBD). Biomarkers are particularly important in children, in whom achieving early remission and minimizing procedures are especially beneficial. Methods: We profiled protein and micro RNA (miRNA) in serum from patients pre- and post-therapy, to identify molecular markers of pharmacodynamic effect. Serum was obtained from children with IBD before and after treatment with either corticosteroids (prednisone; n=12) or anti-tumor necrosis factor-α biologic (infliximab; n=7). Over 1,100 serum proteins were assayed using aptamer-based SOMAscan proteomics, and 22 miRNAs analyzed by quantitative real time PCR. Concordance of longitudinal changes between the groups was used to identify markers responsive to treatment. Bioinformatic analysis was used to build insight into mechanisms of changes in response to treatment. Results: We identified 18 proteins and three miRNAs responsive to both prednisone and infliximab. Eight markers that decreased are associated with inflammation and have gene promoters regulated by nuclear factor (NF)-κB. Several that increased are associated with resolving inflammation and tissue damage. We also identified six markers that appear to be steroid-specific, three of which have glucocorticoid receptor binding elements in their promoter region. Conclusions: Serum markers regulated by the inflammatory transcription factor NF-κB are potential candidates for pharmacodynamic biomarkers that, if correlated with later outcomes like endoscopic or histologic healing, could be used to monitor treatment, optimize dosing, and enhance drug development. The pharmacodynamic biomarkers identified here hold potential to improve both clinical care and drug development. Further studies are warranted to investigate these markers as early predictors of response, or possibly surrogate outcomes

The corticosteroid compounds prednisolone and vamorolone do not alter the nociception phenotype and exacerbate liver injury in sickle cell mice.

Clinicians often hesitate prescribing corticosteroids to treat corticosteroid-responsive conditions in sickle cell disease (SCD) patients because their use can be associated with complications (increased hospital readmission, rebound pain, strokes, avascular necrosis, acute chest syndrome). Consequently, SCD patients may receive suboptimal treatment for corticosteroid-responsive conditions. We conducted a preclinical trial of dissociative (vamorolone) and conventional (prednisolone) corticosteroid compounds to evaluate their effects on nociception phenotype, inflammation, and organ dysfunction in SCD mice. Prednisolone and vamorolone had no significant effects on nociception phenotype or anemia in homozygous mice. Conversely, prednisolone and vamorolone significantly decreased white blood cell counts and hepatic inflammation. Interestingly, the effects of vamorolone were milder than those of prednisolone, as vamorolone yielded less attenuation of hepatic inflammation compared to prednisolone. Compared to controls and heterozygotes, homozygotes had significant liver necrosis, which was significantly exacerbated by prednisolone and vamorolone despite decreased hepatic inflammation. These hepatic histopathologic changes were associated with increases in transaminases and alkaline phosphatase. Together, these results suggest that, even in the setting of decreasing hepatic inflammation, prednisolone and vamorolone were associated with significant hepatic toxicity in SCD mice. These findings raise the possibility that hepatic function deterioration could occur with the use of corticosteroids (conventional and dissociative) in SCD

Lung Inflammation, Injury, and Proliferative Response after Repetitive Particulate Hexavalent Chromium Exposure

BACKGROUND: Chronic inflammation is implicated in the development of several human cancers, including lung cancer. Certain particulate hexavalent chromium [Cr(VI)] compounds are well-documented human respiratory carcinogens that release genotoxic soluble chromate and are associated with fibrosis, fibrosarcomas, adenocarcinomas, and squamous cell carcinomas of the lung. Despite this, little is known about the pathologic injury and immune responses after repetitive exposure to particulate chromates. OBJECTIVES: In this study we investigated the lung injury, inflammation, proliferation, and survival signaling responses after repetitive exposure to particulate chromate. METHODS: BALB/c mice were repetitively treated with particulate basic zinc chromate or saline using an intranasal exposure regimen. We assessed lungs for Cr(VI)-induced changes by bronchoalveolar lavage, histologic examination, and immunohistochemistry. RESULTS: Single exposure to Cr(VI) resulted in inflammation of lung tissue that persists for up to 21 days. Repetitive Cr(VI) exposure induced a neutrophilic inflammatory airway response 24 hr after each treatment. Neutrophils were subsequently replaced by increasing numbers of macrophages by 5 days after treatment. Repetitive Cr(VI) exposure induced chronic peribronchial inflammation with alveolar and interstitial pneumonitis dominated by lymphocytes and macrophages. Moreover, chronic toxic mucosal injury was observed and accompanied by increased airway pro-matrix metalloprotease-9. Injury and inflammation correlated with airways becoming immuno reactive for phosphorylation of the survival signaling protein Akt and the proliferation marker Ki-67. We observed a reactive proliferative response in epithelial cells lining airways of chromate-exposed animals. CONCLUSIONS: These data illustrate that repetitive exposure to particulate chromate induces chronic injury and an inflammatory microenvironment that may promote Cr(VI) carcinogenesis

VBP15, a glucocorticoid analogue, is effective at reducing allergic lung inflammation in mice

Asthma is a chronic inflammatory condition of the lower respiratory tract associated with airway hyperreactivity and mucus obstruction in which a majority of cases are due to an allergic response to environmental allergens. Glucocorticoids such as prednisone have been standard treatment for many inflammatory diseases for the past 60 years. However, despite their effectiveness, long-term treatment is often limited by adverse side effects believed to be caused by glucocorticoid receptor-mediated gene transcription. This has led to the pursuit of compounds that retain the anti-inflammatory properties yet lack the adverse side effects associated with traditional glucocorticoids. We have developed a novel series of steroidal analogues (VBP compounds) that have been previously shown to maintain anti-inflammatory properties such as NFκB-inhibition without inducing glucocorticoid receptor-mediated gene transcription. This study was undertaken to determine the effectiveness of the lead compound, VBP15, in a mouse model of allergic lung inflammation. We show that VBP15 is as effective as the traditional glucocorticoid, prednisolone, at reducing three major hallmarks of lung inflammation--NFκB activity, leukocyte degranulation, and pro-inflammatory cytokine release from human bronchial epithelial cells obtained from patients with asthma. Moreover, we found that VBP15 is capable of reducing inflammation of the lung in vivo to an extent similar to that of prednisone. We found that prednisolone--but not VBP15 shortens the tibia in mice upon a 5 week treatment regimen suggesting effective dissociation of side effects from efficacy. These findings suggest that VBP15 may represent a potent and safer alternative to traditional glucocorticoids in the treatment of asthma and other inflammatory diseases.Supported in part by grants from the NIH (1R41HL104939-01B; 1K26RR032082; 1P50AR060836-01; 1U54HD071601; 2R24HD050846-06, R01 HL033152- 25), DOD grants (W81XWH-11-1-0330; W81XWH-11-1-0782; W81XWH-10-1-0659; W81XWH-11-1-0809; W81XWH-09-1-0599) a translational research grant from MDA, pilot grant from Parent Project Muscular Dystrophy (PPMD), and a contribution from the Clark Family Foundation

Efficacy and Safety of Vamorolone in Duchenne Muscular Dystrophy: A 30-Month Nonrandomized Controlled Open-Label Extension Trial

Importance: Vamorolone is a synthetic steroidal drug with potent anti-inflammatory properties. Initial open-label, multiple ascending dose-finding studies of vamorolone among boys with Duchenne muscular dystrophy (DMD) found significant motor function improvement after 6 months treatment in higher-dose (ie, ≥2.0 mg/kg/d) groups. / Objective: To investigate outcomes after 30 months of open-label vamorolone treatment. / Design, Setting, and Participants: This nonrandomized controlled trial was conducted by the Cooperative International Neuromuscular Research Group at 11 US and non-US study sites. Participants were 46 boys ages 4.5 to 7.5 years with DMD who completed the 6-month dose-finding study. Data were analyzed from July 2020 through November 2021. / Interventions: Participants were enrolled in a 24-month, long-term extension (LTE) study with vamorolone dose escalated to 2.0 or 6.0 mg/kg/d. / Main Outcomes and Measures: Change in time-to-stand (TTSTAND) velocity from dose-finding baseline to end of LTE study was the primary outcome. Efficacy assessments included timed function tests, 6-minute walk test, and NorthStar Ambulatory Assessment (NSAA). Participants with DMD treated with glucocorticoids from the Duchenne Natural History Study (DNHS) and NorthStar United Kingdom (NSUK) Network were matched and compared with participants in the LTE study receiving higher doses of vamorolone. / Results: Among 46 boys with DMD who completed the dose-finding study, 41 boys (mean [SD] age, 5.33 [0.96] years) completed the LTE study. Among 21 participants treated with higher-dose (ie, ≥2.0 mg/kg/d) vamorolone consistently throughout the 6-month dose-finding and 24-month LTE studies with data available at 30 months, there was a decrease in mean (SD) TTSTAND velocity from baseline to 30 months (0.206 [0.070] rises/s vs 0.189 (0.124) rises/s), which was not a statistically significant change (-0.011 rises/s; CI, -0.068 to 0.046 rises/s). There were no statistically significant differences between participants receiving higher-dose vamorolone and matched participants in the historical control groups receiving glucocorticoid treatment (75 patients in DNHS and 110 patients in NSUK) over a 2-year period in NSAA total score change (0.22 units vs NSUK; 95% CI, -4.48 to 4.04]; P = .92), body mass index z score change (0.002 vs DNHS SD/mo; 95% CI, -0.006 to 0.010; P = .58), or timed function test change. Vamorolone at doses up to 6.0 mg/kg/d was well tolerated, with 5 of 46 participants discontinuing prematurely and for reasons not associated with study drug. Participants in the DNHS treated with glucocorticoids had significant growth delay in comparison with participants treated with vamorolone who had stable height percentiles (0.37 percentile/mo; 95% CI, 0.23 to 0.52 percentile/mo) over time. / Conclusions and Relevance: This study found that vamorolone treatment was not associated with a change in TTSTAND velocity from baseline to 30 months among boys with DMD aged 4 to 7 years at enrollment. Vamorolone was associated with maintenance of muscle strength and function up to 30 months, similar to standard of care glucocorticoid therapy, and improved height velocity compared with growth deceleration associated with glucocorticoid treatment, suggesting that vamorolone may be an attractive candidate for treatment of DMD. Trial Registration: ClinicalTrials.gov Identifier: NCT03038399

VBP15: Preclinical characterization of a novel anti-inflammatory delta 9,11 steroid

Δ9,11 modifications of glucocorticoids (21-aminosteroids) have been developed as drugs for protection against cell damage (lipid peroxidation; lazaroids) and inhibition of neovascularization (anecortave). Part of the rationale for developing these compounds has been the loss of glucocorticoid receptor binding due to the Δ9,11 modification, thus avoiding many immunosuppressive activities and deleterious side effect profiles associated with binding to glucocorticoid and mineralocorticoid receptors. We recently demonstrated that anecortave acetate and its 21-hydroxy analog (VBP1) do, in fact, show glucocorticoid and mineralocorticoid receptor binding activities, with potent translocation of the glucocorticoid receptor to the cell nucleus. We concluded that Δ9,11 steroids showed novel anti-inflammatory properties, retaining NF-κB inhibition, but losing deleterious glucocorticoid side effect profiles. Evidence for this was developed in pre-clinical trials of chronic muscle inflammation. Here, we describe a drug development program aimed at optimizing the Δ9,11 chemistry. Twenty Δ9,11 derivatives were tested in in vitro screens for NF-κB inhibition and GR translocation to the nucleus, and low cell toxicity. VBP15 was selected as the lead compound due to potent NF-κB inhibition and GR translocation similar to prednisone and dexamethasone, lack of transactivation properties, and good bioavailability. Phamacokinetics were similar to traditional glucocorticoid drugs with terminal half-life of 0.35 h (mice), 0.58 h (rats), 5.42 h (dogs), and bioavailability of 74.5% (mice), and 53.2% (dogs). Metabolic stability showed ≥80% remaining at 1 h of VBP6 and VBP15 in human, dog, and monkey liver microsomes. Solubility, permeability and plasma protein binding were within acceptable limits. VBP15 moderately induced CYP3A4 across the three human hepatocyte donors (24–42%), similar to other steroids. VBP15 is currently under development for treatment of Duchenne muscular dystrophy