Efficacy and Safety of Vamorolone Over 48 Weeks in Boys With Duchenne Muscular Dystrophy: A Randomized Controlled Trial.

BACKGROUND AND OBJECTIVES: Vamorolone is a dissociative agonist of the glucocorticoid receptor that has shown similar efficacy and reduced safety concerns in comparison with prednisone in Duchenne muscular dystrophy (DMD). This study was conducted to determine the efficacy and safety of vamorolone over 48 weeks and to study crossover participants (prednisone to vamorolone; placebo to vamorolone). METHODS: A randomized, double-blind, placebo-controlled and prednisone-controlled clinical trial of 2 doses of vamorolone was conducted in participants with DMD, in the ages from 4 years to younger than 7 years at baseline. The interventions were 2 mg/kg/d of vamorolone and 6 mg/kg/d of vamorolone for 48 weeks (period 1: 24 weeks + period 2: 24 weeks) and 0.75 mg/kg/d of prednisone and placebo for the first 24 weeks (before crossover). Efficacy was evaluated through gross motor outcomes and safety through adverse events, growth velocity, body mass index (BMI), and bone turnover biomarkers. This analysis focused on period 2. RESULTS: A total of 121 participants with DMD were randomized. Vamorolone at a dose of 6 mg/kg/d showed maintenance of improvement for all motor outcomes to week 48 (e.g., for primary outcome, time to stand from supine [TTSTAND] velocity, week 24 least squares mean [LSM] [SE] 0.052 [0.0130] rises/s vs week 48 LSM [SE] 0.0446 [0.0138]). After 48 weeks, vamorolone at a dose of 2 mg/kg/d showed similar improvements as 6 mg/kg/d for North Star Ambulatory Assessment (NSAA) (vamorolone 6 mg/kg/d-vamorolone 2 mg/kg/d LSM [SE] 0.49 [1.14]; 95% CI -1.80 to 2.78, p = 0.67), but less improvement for other motor outcomes. The placebo to vamorolone 6 mg/kg/d group showed rapid improvements after 20 weeks of treatment approaching benefit seen with 48-week 6 mg/kg/d of vamorolone treatment for TTSTAND, time to run/walk 10 m, and NSAA. There was significant improvement in linear growth after crossover in the prednisone to vamorolone 6 mg/kg/d group, and rapid reversal of prednisone-induced decline in bone turnover biomarkers in both crossover groups. There was an increase in BMI after 24 weeks of treatment that then stabilized for both vamorolone groups. DISCUSSION: Improvements of motor outcomes seen with 6 mg/kg/d of vamorolone at 24 weeks of treatment were maintained to 48 weeks of treatment. Vamorolone at a dose of 6 mg/kg/d showed better maintenance of effect compared with vamorolone at a dose of 2 mg/kg/d for most (3/5) motor outcomes. Bone morbidities of prednisone (stunting of growth and declines in serum bone biomarkers) were reversed when treatment transitioned to vamorolone. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT03439670. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for boys with DMD, the efficacy of vamorolone at a dose of 6 mg/kg/d was maintained over 48 weeks.

Efficacy and Safety of Vamorolone vs Placebo and Prednisone Among Boys With Duchenne Muscular Dystrophy: A Randomized Clinical Trial.

Importance: Corticosteroidal anti-inflammatory drugs are widely prescribed but long-term use shows adverse effects that detract from patient quality of life. Objective: To determine if vamorolone, a structurally unique dissociative steroidal anti-inflammatory drug, is able to retain efficacy while reducing safety concerns with use in Duchenne muscular dystrophy (DMD). Design, Setting, and Participants: Randomized, double-blind, placebo- and prednisone-controlled 24-week clinical trial, conducted from June 29, 2018, to February 24, 2021, with 24 weeks of follow-up. This was a multicenter study (33 referral centers in 11 countries) and included boys 4 to younger than 7 years of age with genetically confirmed DMD not previously treated with corticosteroids. Interventions: The study included 4 groups: placebo; prednisone, 0.75 mg/kg per day; vamorolone, 2 mg/kg per day; and vamorolone, 6 mg/kg per day. Main Outcomes and Measures: Study outcomes monitored (1) efficacy, which included motor outcomes (primary: time to stand from supine velocity in the vamorolone, 6 mg/kg per day, group vs placebo; secondary: time to stand from supine velocity [vamorolone, 2 mg/kg per day], 6-minute walk distance, time to run/walk 10 m [vamorolone, 2 and 6 mg/kg per day]; exploratory: NorthStar Ambulatory Assessment, time to climb 4 stairs) and (2) safety, which included growth, bone biomarkers, and a corticotropin (ACTH)-challenge test. Results: Among the 133 boys with DMD enrolled in the study (mean [SD] age, 5.4 [0.9] years), 121 were randomly assigned to treatment groups, and 114 completed the 24-week treatment period. The trial met the primary end point for change from baseline to week 24 time to stand velocity for vamorolone, 6 mg/kg per day (least-squares mean [SE] velocity, 0.05 [0.01] m/s vs placebo -0.01 [0.01] m/s; 95% CI, 0.02-0.10; P = .002) and the first 4 sequential secondary end points: time to stand velocity, vamorolone, 2 mg/kg per day, vs placebo; 6-minute walk test, vamorolone, 6 mg/kg per day, vs placebo; 6-minute walk test, vamorolone, 2 mg/kg per day, vs placebo; and time to run/walk 10 m velocity, vamorolone, 6 mg/kg per day, vs placebo. Height percentile declined in prednisone-treated (not vamorolone-treated) participants (change from baseline [SD]: prednisone, -1.88 [8.81] percentile vs vamorolone, 6 mg/kg per day, +3.86 [6.16] percentile; P = .02). Bone turnover markers declined with prednisone but not with vamorolone. Boys with DMD at baseline showed low ACTH-stimulated cortisol and high incidence of adrenal insufficiency. All 3 treatment groups led to increased adrenal insufficiency. Conclusions and Relevance: In this pivotal randomized clinical trial, vamorolone was shown to be effective and safe in the treatment of boys with DMD over a 24-week treatment period. Vamorolone may be a safer alternative than prednisone in this disease, in which long-term corticosteroid use is the standard of care. Trial Registration: ClinicalTrials.gov Identifier: NCT03439670.

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.

Efficacy and safety of vamorolone in Duchenne muscular dystrophy: An 18-month interim analysis of a non-randomized open-label extension study.

BACKGROUND: Treatment with corticosteroids is recommended for Duchenne muscular dystrophy (DMD) patients to slow the progression of weakness. However, chronic corticosteroid treatment causes significant morbidities. Vamorolone is a first-in-class anti-inflammatory investigational drug that has shown evidence of efficacy in DMD after 24 weeks of treatment at 2.0 or 6.0 mg/kg/day. Here, open-label efficacy and safety experience of vamorolone was evaluated over a period of 18 months in trial participants with DMD. METHODS AND FINDINGS: A multicenter, open-label, 24-week trial (VBP15-003) with a 24-month long-term extension (VBP15-LTE) was conducted by the Cooperative International Neuromuscular Research Group (CINRG) and evaluated drug-related effects of vamorolone on motor outcomes and corticosteroid-associated safety concerns. The study was carried out in Canada, US, UK, Australia, Sweden, and Israel, from 2016 to 2019. This report covers the initial 24-week trial and the first 12 months of the VBP15-LTE trial (total treatment period 18 months). DMD trial participants (males, 4 to <7 years at entry) treated with 2.0 or 6.0 mg/kg/day vamorolone for the full 18-month period (n = 23) showed clinical improvement of all motor outcomes from baseline to month 18 (time to stand velocity, p = 0.012 [95% CI 0.010, 0.068 event/second]; run/walk 10 meters velocity, p < 0.001 [95% CI 0.220, 0.491 meters/second]; climb 4 stairs velocity, p = 0.001 [95% CI 0.034, 0.105 event/second]; 6-minute walk test, p = 0.001 [95% CI 31.14, 93.38 meters]; North Star Ambulatory Assessment, p < 0.001 [95% CI 2.702, 6.662 points]). Outcomes in vamorolone-treated DMD patients (n = 46) were compared to group-matched participants in the CINRG Duchenne Natural History Study (corticosteroid-naïve, n = 19; corticosteroid-treated, n = 68) over a similar 18-month period. Time to stand was not significantly different between vamorolone-treated and corticosteroid-naïve participants (p = 0.088; least squares [LS] mean 0.042 [95% CI -0.007, 0.091]), but vamorolone-treated participants showed significant improvement compared to group-matched corticosteroid-naïve participants for run/walk 10 meters velocity (p = 0.003; LS mean 0.286 [95% CI 0.104, 0.469]) and climb 4 stairs velocity (p = 0.027; LS mean 0.059 [95% CI 0.007, 0.111]). The vamorolone-related improvements were similar in magnitude to corticosteroid-related improvements. Corticosteroid-treated participants showed stunting of growth, whereas vamorolone-treated trial participants did not (p < 0.001; LS mean 15.86 [95% CI 8.51, 23.22]). Physician-reported incidences of adverse events (AEs) for Cushingoid appearance, hirsutism, weight gain, and behavior change were less for vamorolone than published incidences for prednisone and deflazacort. Key limitations to the study were the open-label design, and use of external comparators. CONCLUSIONS: We observed that vamorolone treatment was associated with improvements in some motor outcomes as compared with corticosteroid-naïve individuals over an 18-month treatment period. We found that fewer physician-reported AEs occurred with vamorolone than have been reported for treatment with prednisone and deflazacort, and that vamorolone treatment did not cause the stunting of growth seen with these corticosteroids. This Phase IIa study provides Class III evidence to support benefit of motor function in young boys with DMD treated with vamorolone 2.0 to 6.0 mg/kg/day, with a favorable safety profile. A Phase III RCT is underway to further investigate safety and efficacy. TRIAL REGISTRATION: Clinical trials were registered at www.clinicaltrials.gov, and the links to each trial are as follows (as provided in manuscript text): VBP15-002 [NCT02760264] VBP15-003 [NCT02760277] VBP15-LTE [NCT03038399].

Disruption of a key ligand-H-bond network drives dissociative properties in vamorolone for Duchenne muscular dystrophy treatment.

Duchenne muscular dystrophy is a genetic disorder that shows chronic and progressive damage to skeletal and cardiac muscle leading to premature death. Antiinflammatory corticosteroids targeting the glucocorticoid receptor (GR) are the current standard of care but drive adverse side effects such as deleterious bone loss. Through subtle modification to a steroidal backbone, a recently developed drug, vamorolone, appears to preserve beneficial efficacy but with significantly reduced side effects. We use combined structural, biophysical, and biochemical approaches to show that loss of a receptor-ligand hydrogen bond drives these remarkable therapeutic effects. Moreover, vamorolone uniformly weakens coactivator associations but not corepressor associations, implicating partial agonism as the main driver of its dissociative properties. Additionally, we identify a critical and evolutionarily conserved intramolecular network connecting the ligand to the coregulator binding surface. Interruption of this allosteric network by vamorolone selectively reduces GR-driven transactivation while leaving transrepression intact. Our results establish a mechanistic understanding of how vamorolone reduces side effects, guiding the future design of partial agonists as selective GR modulators with an improved therapeutic index.

Vamorolone trial in Duchenne muscular dystrophy shows dose-related improvement of muscle function.

OBJECTIVE: To study vamorolone, a first-in-class steroidal anti-inflammatory drug, in Duchenne muscular dystrophy (DMD). METHODS: An open-label, multiple-ascending-dose study of vamorolone was conducted in 48 boys with DMD (age 4-<7 years, steroid-naive). Dose levels were 0.25, 0.75, 2.0, and 6.0 mg/kg/d in an oral suspension formulation (12 boys per dose level; one-third to 10 times the glucocorticoid dose in DMD). The primary goal was to define optimal doses of vamorolone. The primary outcome for clinical efficacy was time to stand from supine velocity. RESULTS: Oral administration of vamorolone at all doses tested was safe and well tolerated over the 24-week treatment period. The 2.0-mg/kg/d dose group met the primary efficacy outcome of improved muscle function (time to stand; 24 weeks of vamorolone treatment vs natural history controls), without evidence of most adverse effects of glucocorticoids. A biomarker of bone formation, osteocalcin, increased in vamorolone-treated boys, suggesting possible loss of bone morbidities seen with glucocorticoids. Biomarker outcomes for adrenal suppression and insulin resistance were also lower in vamorolone-treated patients with DMD relative to published studies of glucocorticoid therapy. CONCLUSIONS: Daily vamorolone treatment suggested efficacy at doses of 2.0 and 6.0 mg/kg/d in an exploratory 24-week open-label study. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that for boys with DMD, vamorolone demonstrated possible efficacy compared to a natural history cohort of glucocorticoid-naive patients and appeared to be tolerated.

Vamorolone, a dissociative steroidal compound, reduces collagen antibody-induced joint damage and inflammation when administered after disease onset.

OBJECTIVE AND DESIGN: The objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis. ANIMALS: 84 DBA1/J mice were used in this study (n = 12 per treatment group). TREATMENT: Vamorolone or prednisolone was administered orally after disease onset for a duration of 7 days. METHODS: Disease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice. RESULTS: We found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone-sparing property was not observed with prednisolone treatment at any of the tested doses. CONCLUSIONS: While many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.

Population Pharmacokinetics of Vamorolone (VBP15) in Healthy Men and Boys With Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited neuromuscular disorder occurring in boys and caused by mutations in the dystrophin gene. Vamorolone is a first-generation delta-9,11 compound that has favorable efficacy and side effect profiles relative to classical glucocorticoids. The pharmacokinetics (PK) of oral vamorolone were assessed in parallel-group studies in healthy men (phase 1, n = 86) and boys with DMD (phase 2a, n = 48) during 14 days of once-daily dosing with a range of doses. Vamorolone exhibited moderate variability in PK, with the maximum plasma concentration usually occurring at 2-4 hours and a half-life of approximately 2 hours for all doses and days examined. Population PK modeling of all data together indicated that the PK of vamorolone can be well described by a 1-compartment model with zero-order absorption. Both men and boys showed a dose-linearity of PK parameters for the doses examined, with no accumulation of the drug during daily dosing. Ingestion with food resulted in markedly enhanced absorption of the drug, as tested in healthy men. There were similar PK of vamorolone in healthy men and DMD boys with apparent clearance averaging 2.0 L/h/kg in men and 1.7 L/h/kg in boys. Overall, vamorolone exhibited well-behaved linear PK, with similar profiles in healthy men and boys with DMD, moderate variability in PK parameters, and absorption and disposition profiles similar to those of classical glucocorticoids.

Vamorolone targets dual nuclear receptors to treat inflammation and dystrophic cardiomyopathy.

Cardiomyopathy is a leading cause of death for Duchenne muscular dystrophy. Here, we find that the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) can share common ligands but play distinct roles in dystrophic heart and skeletal muscle pathophysiology. Comparisons of their ligand structures indicate that the Δ9,11 modification of the first-in-class drug vamorolone enables it to avoid interaction with a conserved receptor residue (N770/N564), which would otherwise activate transcription factor properties of both receptors. Reporter assays show that vamorolone and eplerenone are MR antagonists, whereas prednisolone is an MR agonist. Macrophages, cardiomyocytes, and CRISPR knockout myoblasts show vamorolone is also a dissociative GR ligand that inhibits inflammation with improved safety over prednisone and GR-specific deflazacort. In mice, hyperaldosteronism activates MR-driven hypertension and kidney phenotypes. We find that genetic dystrophin loss provides a second hit for MR-mediated cardiomyopathy in Duchenne muscular dystrophy model mice, as aldosterone worsens fibrosis, mass and dysfunction phenotypes. Vamorolone successfully prevents MR-activated phenotypes, whereas prednisolone activates negative MR and GR effects. In conclusion, vamorolone targets dual nuclear receptors to treat inflammation and cardiomyopathy with improved safety.

Serum biomarkers of glucocorticoid response and safety in anti-neutrophil cytoplasmic antibody-associated vasculitis and juvenile dermatomyositis.

Glucocorticoids are standard of care for many chronic inflammatory conditions, including juvenile dermatomyositis (JDM) and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We sought to define pharmacodynamic biomarkers of therapeutic efficacy and safety concerns of glucocorticoid treatment for these two disorders. Previous proteomic profiling of patients with Duchenne muscular dystrophy (DMD) and inflammatory bowel disease (IBD) treated with glucocorticoids identified candidate biomarkers for efficacy and safety concerns of glucocorticoids. Serial serum samples from patients with AAV (n = 30) and JDM (n = 12) were obtained during active disease, and after treatment with glucocorticoids. For AAV, 8 of 11 biomarkers of the anti-inflammatory response to glucocorticoids were validated (P-value ≤0.05; CD23, macrophage-derived cytokine, interleukin-22 binding protein, matrix metalloproteinase-12, T lymphocyte surface antigen Ly9, fibrinogen gamma chain, angiopoietin-2 [all decreased], and protein C [increased]), as were 5 of 7 safety biomarkers (P-value ≤0.05; afamin, matrix metalloproteinase-3, insulin growth factor binding protein-5, angiotensinogen, leptin [all increased]). For JDM, 10 of 11 efficacy biomarkers were validated (P-value ≤0.05; all proteins except fibrinogen gamma chain) and 6 of 7 safety biomarkers (P-value ≤0.05; AAV proteins plus growth hormone binding protein). The identified efficacy biomarkers may be useful as objective outcome measures for early phase proof-of-concept studies when assessing novel anti-inflammatory drugs in JDM and AAV, and likely in other inflammatory disorders. Similarly, safety biomarkers may also be helpful assessing toxicity of alternatives to glucocorticoids.

Phase IIa trial in Duchenne muscular dystrophy shows vamorolone is a first-in-class dissociative steroidal anti-inflammatory drug.

We report a first-in-patient study of vamorolone, a first-in-class dissociative steroidal anti-inflammatory drug, in Duchenne muscular dystrophy. This 2-week, open-label Phase IIa multiple ascending dose study (0.25, 0.75, 2.0, and 6.0 mg/kg/day) enrolled 48 boys with Duchenne muscular dystrophy (4 to <7 years), with outcomes including clinical safety, pharmacokinetics and pharmacodynamic biomarkers. The study design included pharmacodynamic biomarkers in three contexts of use: 1. Secondary outcomes for pharmacodynamic safety (insulin resistance, adrenal suppression, bone turnover); 2. Exploratory outcomes for drug mechanism of action; 3. Exploratory outcomes for expanded pharmacodynamic safety. Vamorolone was safe and well-tolerated through the highest dose tested (6.0 mg/kg/day) and pharmacokinetics of vamorolone were similar to prednisolone. Using pharmacodynamic biomarkers, the study demonstrated improved safety of vamorolone versus glucocorticoids as shown by reduction of insulin resistance, beneficial changes in bone turnover (loss of increased bone resorption and decreased bone formation only at the highest dose level), and a reduction in adrenal suppression. Exploratory biomarkers of pharmacodynamic efficacy showed an anti-inflammatory mechanism of action and a beneficial effect on plasma membrane stability, as demonstrated by a dose-responsive decrease in serum creatine kinase activity. With an array of pre-selected biomarkers in multiple contexts of use, we demonstrate the development of the first dissociative steroid that preserves anti-inflammatory efficacy and decreases steroid-associated safety concerns. Ongoing extension studies offer the potential to bridge exploratory efficacy biomarkers to clinical outcomes.

Muscle miRNAome shows suppression of chronic inflammatory miRNAs with both prednisone and vamorolone.

Corticosteroids are highly prescribed and effective anti-inflammatory drugs but the burden of side effects with chronic use significantly detracts from patient quality of life, particularly in children. Developing safer steroids amenable to long-term use is an important goal for treatment of chronic inflammatory diseases such as Duchenne muscular dystrophy (DMD). We have developed vamorolone (VBP15), a first-in-class dissociative glucocorticoid receptor (GR) ligand that shows the anti-inflammatory efficacy of corticosteroids without key steroid side effects in animal models. miRNAs are increasingly recognized as key regulators of inflammatory responses. To define effects of prednisolone and vamorolone on the muscle miRNAome, we performed a preclinical discovery study in the mdx mouse model of DMD. miRNAs associated with inflammation were highly elevated in mdx muscle. Both vamorolone and prednisolone returned these toward wild-type levels (miR-142-5p, miR-142-3p, miR-146a, miR-301a, miR-324-3p, miR-455-5p, miR-455-3p, miR-497, miR-652). Effects of vamorolone were largely limited to reduction of proinflammatory miRNAs. In contrast, prednisolone activated a separate group of miRNAs associated with steroid side effects and a noncoding RNA cluster homologous to human chromosome 14q32. Effects were validated for inflammatory miRNAs in a second, independent preclinical study. For the anti-inflammatory miRNA signature, bioinformatic analyses showed all of these miRNAs are directly regulated by, or in turn activate, the inflammatory transcription factor NF-κB. Moving forward miR-146a and miR-142 are of particular interest as biomarkers or novel drug targets. These data validate NF-κB signaling as a target of dissociative GR-ligand efficacy in vivo and provide new insight into miRNA signaling in chronic inflammation.

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.

Phase 1 trial of vamorolone, a first-in-class steroid, shows improvements in side effects via biomarkers bridged to clinical outcomes.

BACKGROUND: Glucocorticoid drugs are highly effective anti-inflammatory agents, but chronic use is associated with extensive pharmacodynamic safety concerns that have a considerable negative impact on patient quality of life. PURPOSE: Vamorolone (VBP15) is a first-in-class steroidal multi-functional drug that shows potent inhibition of pro-inflammatory NFkB pathways via high-affinity binding to the glucocorticoid receptor, high affinity antagonism for the mineralocorticoid receptor, and membrane stabilization properties. Pre-clinical data in multiple mouse models of inflammation showed retention of anti-inflammatory efficacy, but loss of most or all side effects. EXPERIMENTAL APPROACH: We report first-in-human Phase 1 clinical trials (86 healthy adult males), with single ascending doses (0.1-20.0 mg/kg), and multiple ascending doses (1.0-20 mg/kg/day; 14 days treatment). KEY RESULTS: Vamorolone was well-tolerated at all dose levels. Vamorolone showed pharmacokinetic and metabolism profiles similar to prednisone. Biomarker studies showed loss of side effects of traditional glucocorticoid drugs (bone fragility, metabolic disturbance, immune suppression). Suppression of the adrenal axis was 10-fold less than prednisone. The crystallographic structure of vamorolone was solved, and compared to prednisone and dexamethasone. There was overlap in structure, but differences in conformation at the C-ring where glucocorticoids interact with Asn564 of the glucocorticoid receptor. The predicted loss of Asn564 binding to vamorolone may underlie the loss of gene transcriptional activity. CONCLUSIONS AND INTERPRETATIONS: Vamorolone is a dissociative steroid that retains high affinity binding and nuclear translocation of both glucocorticoid (agonist) and mineralocorticoid (antagonist) receptors, but does not show pharmacodynamic safety concerns of existing glucocorticoid drugs at up to 20 mg/kg/day.

Vamorolone, a dissociative steroidal compound, reduces pro-inflammatory cytokine expression in glioma cells and increases activity and survival in a murine model of cortical tumor.

Corticosteroids, such as dexamethasone, are routinely used as palliative care in neuro-oncology for their anti-inflammatory benefits, however many patients experience dose limiting side effects caused by glucocorticoid response element (GRE)-mediated transcription. The purpose of this study was to use a murine model to investigate a new steroid alternative, vamorolone, which promises to reduce side effects through dissociating GRE-mediated transcription and NF-κB -mediated anti-inflammatory actions. To compare vamorolone to dexamethasone in reducing pro-inflammatory signals in vitro, murine glioma cells were treated with dexamethasone, vamorolone or vehicle control. Changes in mRNA expression were assessed using the nanostring inflammatory platform. Furthermore, drug efficacy, post-treatment behavioral activity and side effects were assessed by treating two cohorts of brain tumor bearing mice with dexamethasone, vamorolone, or vehicle control. Our investigation showed that treatment with vamorolone resulted in a reduction of pro-inflammatory signals in tumor cells in vitro similar to treatment with dexamethasone. Treatment with vamorolone resulted in a better safety profile in comparison to dexamethasone treatment. Vamorolone- treated mice showed similar or better activity and survival when compared to dexamethasone-treated mice. Our data indicate vamorolone is a potential steroid-sparing alternative for treating patients with brain tumors.

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.

Serum pharmacodynamic biomarkers for chronic corticosteroid treatment of children.

Corticosteroids are extensively used in pediatrics, yet the burden of side effects is significant. Availability of a simple, fast, and reliable biochemical read out of steroidal drug pharmacodynamics could enable a rapid and objective assessment of safety and efficacy of corticosteroids and aid development of corticosteroid replacement drugs. To identify potential corticosteroid responsive biomarkers we performed proteome profiling of serum samples from DMD and IBD patients with and without corticosteroid treatment using SOMAscan aptamer panel testing 1,129 proteins in <0.1 cc of sera. Ten pro-inflammatory proteins were elevated in untreated patients and suppressed by corticosteroids (MMP12, IL22RA2, CCL22, IGFBP2, FCER2, LY9, ITGa1/b1, LTa1/b2, ANGPT2 and FGG). These are candidate biomarkers for anti-inflammatory efficacy of corticosteroids. Known safety concerns were validated, including elevated non-fasting insulin (insulin resistance), and elevated angiotensinogen (salt retention). These were extended by new candidates for metabolism disturbances (leptin, afamin), stunting of growth (growth hormone binding protein), and connective tissue remodeling (MMP3). Significant suppression of multiple adrenal steroid hormones was also seen in treated children (reductions of 17-hydroxyprogesterone, corticosterone, 11-deoxycortisol and testosterone). A panel of new pharmacodynamic biomarkers for corticosteroids in children was defined. Future studies will need to bridge specific biomarkers to mechanism of drug action, and specific clinical outcomes.

OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages.

NEW FINDINGS: What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full-length human OPN-a isoform is the most pro-inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD-integrin-dependent manner. OPN-a upregulates expression of tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro-inflammatory effects of OPN-a, suggesting that a potential mechanism of OPN action is by promoting TNC-TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro-inflammatory activities of human OPN isoforms (OPN-a, OPN-b and OPN-c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN-a > OPN-b > OPN-c) and dog muscle (OPN-a = OPN-c). In healthy human muscle, mechanical loading also upregulated OPN-a expression (eightfold; P < 0.01), but did not significantly upregulate OPN-c expression (twofold; P > 0.05). In vitro, OPN-a displayed the most pronounced pro-inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN-a upregulated tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN-mediated macrophage cytokine production. In summary, OPN-a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN-a promotes pro-inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC-TLR4 signalling. Together, our findings suggest that specific targeting of OPN-a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance.

VBP15, a novel dissociative steroid compound, reduces NFκB-induced expression of inflammatory cytokines in vitro and symptoms of murine trinitrobenzene sulfonic acid-induced colitis.

OBJECTIVE AND DESIGN: The goal of this study was to assess the capacity of VBP15, a dissociative steroidal compound, to reduce pro-inflammatory cytokine expression in vitro, to reduce symptoms of colitis in the trinitrobenzene sulfonic acid-induced murine model, and to assess the effect of VBP15 on growth stunting in juvenile mice. MATERIALS: In vitro studies were performed in primary human intestinal epithelial cells. Colitis was induced in mice by administering trinitrobenzene sulfonic acid. Growth stunting studies were performed in wild type outbred mice. TREATMENT: Cells were treated with VBP15 or prednisolone (10 µM) for 24 h. Mice were subjected to 3 days of VBP15 (30 mg/kg) or prednisolone (30 mg/kg) in the colitis study. In the growth stunting study, mice were subjected to VBP15 (10, 30, 45 mg/kg) or prednisolone (10 mg/kg) for 5 weeks. METHODS: Cytokines were measured by PCR and via Luminex. Colitis symptoms were evaluated by assessing weight loss, intestinal blood, and stool consistency. Growth stunting was assessed using an electronic caliper. RESULTS: VBP15 significantly reduced the in vitro production of CCL5 (p < 0.001) IL-6 (p < 0.001), IL-8 (p < 0.05) and reduced colitis symptoms (p < 0.05). VBP15 caused less growth stunting than prednisolone (p < 0.001) in juvenile mice. CONCLUSION: VBP15 may reduce symptoms of IBD, while decreasing or avoiding detrimental side effects.

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1ß upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1ß in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex.