Christopher R. Heier

VBP15, a novel anti-inflammatory and membrane-stabilizer, improves muscular dystrophy without side effects.

Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti‐inflammatory signaling and membrane‐stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF‐κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live‐imaging and pathology through both preventive and post‐onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.

TNF-α-Induced microRNAs Control Dystrophin Expression in Becker Muscular Dystrophy.

The amount and distribution of dystrophin protein in myofibers and muscle is highly variable in Becker muscular dystrophy and in exon-skipping trials for Duchenne muscular dystrophy. Here, we investigate a molecular basis for this variability. In muscle from Becker patients sharing the same exon 45-47 in-frame deletion, dystrophin levels negatively correlate with microRNAs predicted to target dystrophin. Seven microRNAs inhibit dystrophin expression in vitro, and three are validated in vivo (miR-146b/miR-374a/miR-31). microRNAs are expressed in dystrophic myofibers and increase with age and disease severity. In exon-skipping-treated mdx mice, microRNAs are significantly higher in muscles with low dystrophin rescue. TNF-α increases microRNA levels in vitro whereas NFκB inhibition blocks this in vitro and in vivo. Collectively, these data show that microRNAs contribute to variable dystrophin levels in muscular dystrophy. Our findings suggest a model where chronic inflammation in distinct microenvironments induces pathological microRNAs, initiating a self-sustaining feedback loop that exacerbates disease progression.

Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice

The over‐expression of NF‐κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti‐inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF‐κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non‐steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF‐κB inhibitory activity of compound A in H‐2Kb‐tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post‐natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin‐B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL‐6, CCL2, IFNγ, TNF and IL‐12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild‐type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild‐type mice. In conclusion, selective glucocorticoid receptor modulation by compound A represents a potential therapeutic strategy to improve dystrophic pathology. Copyright

Δ-9,11 Modification of Glucocorticoids Dissociates Nuclear Factor-κB Inhibitory Efficacy from Glucocorticoid Response Element-Associated Side Effects

Glucocorticoids are standard of care for many inflammatory conditions, but chronic use is associated with a broad array of side effects. This has led to a search for dissociative glucocorticoids—drugs able to retain or improve efficacy associated with transrepression [nuclear factor-κB (NF-κB) inhibition] but with the loss of side effects associated with transactivation (receptor-mediated transcriptional activation through glucocorticoid response element gene promoter elements). We investigated a glucocorticoid derivative with a Δ-9,11 modification as a dissociative steroid. The Δ-9,11 analog showed potent inhibition of tumor necrosis factor-α-induced NF-κB signaling in cell reporter assays, and this transrepression activity was blocked by 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU-486), showing the requirement for the glucocorticoid receptor (GR). The Δ-9,11 analog induced the nuclear translocation of GR but showed the loss of transactivation as assayed by GR-luciferase constructs as well as mRNA profiles of treated cells. The Δ-9,11 analog was tested for efficacy and side effects in two mouse models of muscular dystrophy: mdx (dystrophin deficiency), and SJL (dysferlin deficiency). Daily oral delivery of the Δ-9,11 analog showed a reduction of muscle inflammation and improvements in multiple muscle function assays yet no reductions in body weight or spleen size, suggesting the loss of key side effects. Our data demonstrate that a Δ-9,11 analog dissociates the GR-mediated transcriptional activities from anti-inflammatory activities. Accordingly, Δ-9,11 analogs may hold promise as a source of safer therapeutic agents for chronic inflammatory disorders.

Non-Invasive MRI and Spectroscopy of mdx Mice Reveal Temporal Changes in Dystrophic Muscle Imaging and in Energy Deficits

In Duchenne muscular dystrophy (DMD), a genetic disruption of dystrophin protein expression results in repeated muscle injury and chronic inflammation. Magnetic resonance imaging shows promise as a surrogate outcome measure in both DMD and rehabilitation medicine that is capable of predicting clinical benefit years in advance of functional outcome measures. The mdx mouse reproduces the dystrophin deficiency that causes DMD and is routinely used for preclinical drug testing. There is a need to develop sensitive, non-invasive outcome measures in the mdx model that can be readily translatable to human clinical trials. Here we report the use of magnetic resonance imaging and spectroscopy techniques for the non-invasive monitoring of muscle damage in mdx mice. Using these techniques, we studied dystrophic mdx muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the mdx disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in mdx hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old mdx mice. As the mdx mice progressed from the peak disease stage to the recovery stage of disease, each of these phenotypes was either eliminated or reduced, and the cross-sectional area of the mdx muscle was significantly increased when compared to that of wild-type mice. Histology indicates that hyper-intense MRI foci correspond to areas of dystrophic lesions containing inflammation as well as regenerating, degenerating and hypertrophied myofibers. Statistical sample size calculations provide several robust measures with the ability to detect intervention effects using small numbers of animals. These data establish a framework for further imaging or preclinical studies, and they support the development of MRI as a sensitive, non-invasive outcome measure for muscular dystrophy.

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.

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.

VBP15, a Novel Anti-Inflammatory, is Effective at Reducing the Severity of Murine Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is a chronic disease of the central nervous system characterized by an autoimmune inflammatory reaction that leads to axonal demyelination and tissue damage. Glucocorticoids, such as prednisolone, are effective in the treatment of multiple sclerosis in large part due to their ability to inhibit pro-inflammatory pathways (e.g., NFκB). However, despite their effectiveness, long-term treatment is limited by adverse side effects. VBP15 is a recently described compound synthesized based on the lazeroid steroidal backbone that shows activity in acute and chronic inflammatory conditions, yet displays a much-reduced side effect profile compared to traditional glucocorticoids. The purpose of this study was to determine the effectiveness of VBP15 in inhibiting inflammation and disease progression in experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of multiple sclerosis. Our data show that VBP15 is effective at reducing both disease onset and severity. In parallel studies, we observed that VBP15 was able to inhibit the production of NFκB-regulated pro-inflammatory transcripts in human macrophages. Furthermore, treatment with prednisolone—but not VBP15—increased expression of genes associated with bone loss and muscle atrophy, suggesting lack of side effects of VBP15. These findings suggest that VBP15 may represent a potentially safer alternative to traditional glucocorticoids in the treatment of multiple sclerosis and other inflammatory diseases.

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.

P-226 Pharmacodynamic Serum Biomarker Discovery in Pediatric Inflammatory Bowel Diseases

Background:Non-invasive pharmacodynamic (PD) biomarkers used in conjunction with patient-reported outcome measures are potential endpoints for Inflammatory Bowel Disease (IBD) trials. PD biomarkers may also be used to predict efficacy and ablate placebo effects, thereby supporting extrapolation in pediatric trials. To identify potential serum-based molecules that change with anti-inflammatory therapies, we used 2 candidate-based approaches: a microRNA and a proteomic discovery platform. Methods:Serum samples were obtained pre- and post-corticosteroid therapy for a median duration of 9 weeks (range: 3–18 weeks) in 10 children with Crohns Disease (CD) and 2 with ulcerative colitis (UC). Serum samples were obtained pre-infliximab therapy, and at 6 weeks (post 2 doses) in another set of 12 children with CD and 2 with UC. All patients demonstrated clinical response by PCDAI and PUCAI. MicroRNAs were analyzed by candidate-based qPCR in both cohorts. Proteins were analyzed by SomaScan aptamer-based analysis of the corticosteroid treated cohort. A core set of protein candidates, and encoding genes, was assembled. To determine if changes in expression could reflect treatment effects at either inflammation-regulated or steroid-regulated gene elements in DNA, we surveyed the binding of NF-&kgr;B and glucocorticoid receptor (GRE) proteins to regulatory promoter regions in each gene using ChIP-seq data. Results:Several microRNAs were consistently reduced by short-term treatment with corticosteroids and infliximab, including miR-146a, miR-146b, miR-486, miR-320a, and miR-762. Some of these miRNAs are regulated by the activity of NF-&kgr;B. Several proteins were highly responsive to corticosteroid therapy, including a set associated with inflammation, and another set associated with metabolic activity. Some of these genes were found to have NF-&kgr;B binding regulatory promotor regions. Conclusions:Changes in molecules related to inflammatory pathways were identified in both steroidal and biologic-based treatment groups. Changes in metabolic-related molecules were also noted, which may be indicative of steroid-specific effects. Circulating serum NF-&kgr;B-dependent microRNAs and proteins are potential non-invasive PD biomarkers of therapeutic response in IBD. These biomarkers warrant further study, including correlation with post-treatment endoscopic evaluation.