Publications by authors named "Kanneboyina Nagaraju"

139 Publications

Biomarker-focused multi-drug combination therapy and repurposing trial in mdx mice.

PLoS One 2021 22;16(2):e0246507. Epub 2021 Feb 22.

AGADA Biosciences, Halifax, Nova Scotia, Canada.

Duchenne muscular dystrophy is initiated by dystrophin deficiency, but downstream pathophysiological pathways such as membrane instability, NFĸB activation, mitochondrial dysfunction, and induction of TGFβ fibrosis pathways are thought to drive the disability. Dystrophin replacement strategies are hopeful for addressing upstream dystrophin deficiency; however, all methods to date use semi-functional dystrophin proteins that are likely to trigger downstream pathways. Thus, combination therapies that can target multiple downstream pathways are important in treating DMD, even for dystrophin-replacement strategies. We sought to define blood pharmacodynamic biomarkers of drug response in the mdx mouse model of Duchenne muscular dystrophy using a series of repurposed drugs. Four-week-old mdx mice were treated for four weeks with four different drugs singly and in combination: vehicle, prednisolone, vamorolone, rituximab, β-aminoisobutyric acid (BAIBA) (11 treatment groups; n = 6/group). Blood was collected via cardiac puncture at study termination, and proteomic profiling was carried out using SOMAscan aptamer panels (1,310 proteins assayed). Prednisolone was tested alone and in combination with other drugs. It was found to have a good concordance of prednisolone-responsive biomarkers (56 increased by prednisolone, 39 decreased) focused on NFκB and TGFβ cascades. Vamorolone shared 45 (80%) of increased biomarkers and 13 (33%) of decreased biomarkers with prednisolone. Comparison of published human corticosteroid-responsive biomarkers to our mdx data showed 14% (3/22) concordance between mouse and human. Rituximab showed fewer drug-associated biomarkers, with the most significant being human IgG. On the other hand, BAIBA treatment (high and low dose) showed a drug-associated increase in 40 serum proteins and decreased 5 serum proteins. Our results suggest that a biomarker approach could be employed for assessing drug combinations in both mouse and human studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246507PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899329PMC
February 2021

Mitochondrial dysfunction and consequences in calpain-3-deficient muscle.

Skelet Muscle 2020 12 11;10(1):37. Epub 2020 Dec 11.

Center for Genetic Medicine Research, Children's National Research Institute, Children's National Hospital, Washington, D.C., USA.

Background: Nonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A). While calpain-3 is implicated in muscle cell differentiation, sarcomere formation, and muscle cytoskeletal remodeling, the physiological basis for LGMD2A has remained elusive.

Methods: Cell growth, gene expression profiling, and mitochondrial content and function were analyzed using muscle and muscle cell cultures established from healthy and calpain-3-deficient mice. Calpain-3-deficient mice were also treated with PPAR-delta agonist (GW501516) to assess mitochondrial function and membrane repair. The unpaired t test was used to assess the significance of the differences observed between the two groups or treatments. ANOVAs were used to assess significance over time.

Results: We find that calpain-3 deficiency causes mitochondrial dysfunction in the muscles and myoblasts. Calpain-3-deficient myoblasts showed increased proliferation, and their gene expression profile showed aberrant mitochondrial biogenesis. Myotube gene expression analysis further revealed altered lipid metabolism in calpain-3-deficient muscle. Mitochondrial defects were validated in vitro and in vivo. We used GW501516 to improve mitochondrial biogenesis in vivo in 7-month-old calpain-3-deficient mice. This treatment improved satellite cell activity as indicated by increased MyoD and Pax7 mRNA expression. It also decreased muscle fatigability and reduced serum creatine kinase levels. The decreased mitochondrial function also impaired sarcolemmal repair in the calpain-3-deficient skeletal muscle. Improving mitochondrial activity by acute pyruvate treatment improved sarcolemmal repair.

Conclusion: Our results provide evidence that calpain-3 deficiency in the skeletal muscle is associated with poor mitochondrial biogenesis and function resulting in poor sarcolemmal repair. Addressing this deficit by drugs that improve mitochondrial activity offers new therapeutic avenues for LGMD2A.
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http://dx.doi.org/10.1186/s13395-020-00254-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730798PMC
December 2020

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

Proc Natl Acad Sci U S A 2020 09 11;117(39):24285-24293. Epub 2020 Sep 11.

Department of Biochemistry, Emory University, Atlanta, GA 30322;

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.
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http://dx.doi.org/10.1073/pnas.2006890117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533876PMC
September 2020

Improving translatability of preclinical studies for neuromuscular disorders: lessons from the TREAT-NMD Advisory Committee for Therapeutics (TACT).

Dis Model Mech 2020 02 7;13(2). Epub 2020 Feb 7.

Unit of Pharmacology, Department of Pharmacy and Drug Sciences, University of Bari Aldo Moro, 70125 Bari, Italy

Clinical trials for rare neuromuscular diseases imply, among other investments, a high emotional burden for the whole disease community. Translation of data from preclinical studies to justify any clinical trial must be carefully pondered in order to minimize the risk of clinical trial withdrawal or failure. A rigorous distinction between proof-of-concept and preclinical efficacy studies using animal models is key to support the rationale of a clinical trial involving patients. This Review evaluates the experience accumulated by the TREAT-NMD Advisory Committee for Therapeutics, which provides detailed constructive feedback on clinical proposals for neuromuscular diseases submitted by researchers in both academia and industry, and emphasizes that a timely critical review of preclinical efficacy data from animal models, including biomarkers for specific diseases, combined with adherence to existing guidelines and standard protocols, can significantly help to de-risk clinical programs and prevent disappointments and costly engagement.
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http://dx.doi.org/10.1242/dmm.042903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044444PMC
February 2020

Muscle Weakness in Myositis: MicroRNA-Mediated Dystrophin Reduction in a Myositis Mouse Model and Human Muscle Biopsies.

Arthritis Rheumatol 2020 07 31;72(7):1170-1183. Epub 2020 May 31.

George Washington University and Children's National Hospital, Washington, DC.

Objective: Muscle inflammation is a feature in myositis and Duchenne muscular dystrophy (DMD). Autoimmune mechanisms are thought to contribute to muscle weakness in patients with myositis. However, a lack of correlation between the extent of inflammatory cell infiltration and muscle weakness indicates that nonimmune pathologic mechanisms may play a role. The present study focused on 2 microRNA (miRNA) sets previously identified as being elevated in the muscle of patients with DMD-an "inflammatory" miRNA set that is dampened with glucocorticoids, and a "dystrophin-targeting" miRNA set that inhibits dystrophin translation-to test the hypothesis that these miRNAs are similarly dysregulated in the muscle of patients with myositis, and could contribute to muscle weakness and disease severity.

Methods: A major histocompatibility complex class I-transgenic mouse model of myositis was utilized to study gene and miRNA expression and histologic features in the muscle tissue, with the findings validated in human muscle biopsy tissue from 6 patients with myositis. Mice were classified as having mild or severe myositis based on transgene expression, body weight, histologic disease severity, and muscle strength/weakness.

Results: In mice with severe myositis, muscle tissue showed mononuclear cell infiltration along with elevated expression of type I interferon and NF-κB-regulated genes, including Tlr7 (3.8-fold increase, P < 0.05). Furthermore, mice with severe myositis showed elevated expression of inflammatory miRNAs (miR-146a, miR-142-3p, miR-142-5p, miR-455-3p, and miR-455-5p; ~3-40-fold increase, P < 0.05) and dystrophin-targeting miRNAs (miR-146a, miR-146b, miR-31, and miR-223; ~3-38-fold increase, P < 0.05). Bioinformatics analyses of chromatin immunoprecipitation sequencing (ChIP-seq) data identified at least one NF-κB consensus element within the promoter/enhancer regions of these miRNAs. Western blotting and immunofluorescence analyses of the muscle tissue from mice with severe myositis demonstrated reduced levels of dystrophin. In addition, elevated levels of NF-κB-regulated genes, TLR7, and miRNAs along with reduced dystrophin levels were observed in muscle biopsy tissue from patients with histologically severe myositis.

Conclusion: These data demonstrate that an acquired dystrophin deficiency may occur through NF-κB-regulated miRNAs in myositis, thereby suggesting a unifying theme in which muscle injury, inflammation, and weakness are perpetuated both in myositis and in DMD.
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http://dx.doi.org/10.1002/art.41215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384101PMC
July 2020

A Promising Future for Stem-Cell-Based Therapies in Muscular Dystrophies-In Vitro and In Vivo Treatments to Boost Cellular Engraftment.

Int J Mol Sci 2019 Oct 31;20(21). Epub 2019 Oct 31.

Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro (RJ) 21040-900, Brazil.

Muscular dystrophies (MD) are a group of genetic diseases that lead to skeletal muscle wasting and may affect many organs (multisystem). Unfortunately, no curative therapies are available at present for MD patients, and current treatments mainly address the symptoms. Thus, stem-cell-based therapies may present hope for improvement of life quality and expectancy. Different stem cell types lead to skeletal muscle regeneration and they have potential to be used for cellular therapies, although with several limitations. In this review, we propose a combination of genetic, biochemical, and cell culture treatments to correct pathogenic genetic alterations and to increase proliferation, dispersion, fusion, and differentiation into new or hybrid myotubes. These boosted stem cells can also be injected into pretreate recipient muscles to improve engraftment. We believe that this combination of treatments targeting the limitations of stem-cell-based therapies may result in safer and more efficient therapies for MD patients. Matricryptins have also discussed.
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http://dx.doi.org/10.3390/ijms20215433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861917PMC
October 2019

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

Neurology 2019 09 26;93(13):e1312-e1323. Epub 2019 Aug 26.

From ReveraGen Biopharma (E.P.H., J.M.M., K.N., J.v.d.A., L.S.C., J.M.D.), Rockville, MD; Binghamton University-SUNY (E.P.H., K.N.), NY: Camden Group (B.D.S., L.J.M.-G.), LLC, St. Louis, MO; Duke University (E.C.S.), Durham, NC; University of Texas Southwestern (D.C.), Dallas; Alberta Children's Hospital (J.K.M.), Calgary, Canada; University of California Davis (C.M.M.), Sacramento; Ann & Robert H. Lurie Children's Hospital (N.L.K.), Chicago, IL; Nemours Children's Hospital (R.S.F.), Orlando, FL; John Walton Muscular Dystrophy Research Centre (M.G., K.B.), Newcastle University, Newcastle-Upon-Tyne, UK; Queen Silvia Children's Hospital (M.T.), Gothenburg, Sweden; Schneider Children's Medical Center (Y.N.), Tel Aviv University, Israel; Royal Children's Hospital and Murdoch Children's Research Institute (M.M.R.), Melbourne, Australia; The Children's Hospital at Westmead (R.W.), Sydney, Australia; TRiNDS LLC (A.L.S., L.P.M., A.A., M.S., C.S.), Kensington, MD; Summit Analytical (M.J., P.S.), Denver, CO; Children's National Health System (J.v.d.A., L.S.C., A.C., H.G.-D.), Washington, DC; and University of Pittsburgh and Department of Veterans Affairs Medical Center (P.R.C.), PA.

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.
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http://dx.doi.org/10.1212/WNL.0000000000008168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011869PMC
September 2019

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

Inflamm Res 2019 Nov 24;68(11):969-980. Epub 2019 Aug 24.

ReveraGen BioPharma Inc., Rockville, MD, 20850, USA.

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.
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http://dx.doi.org/10.1007/s00011-019-01279-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814568PMC
November 2019

Orthogonal analysis of dystrophin protein and mRNA as a surrogate outcome for drug development.

Biomark Med 2019 10 5;13(14):1209-1225. Epub 2019 Aug 5.

AGADA BioSciences Inc., Halifax, Nova Scotia B3H0A8, Canada.

Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Both western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.
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http://dx.doi.org/10.2217/bmm-2019-0242DOI Listing
October 2019

Discovery of potential urine-accessible metabolite biomarkers associated with muscle disease and corticosteroid response in the mdx mouse model for Duchenne.

PLoS One 2019 16;14(7):e0219507. Epub 2019 Jul 16.

Department of Oncology, Georgetown University Medical Center, Washington, D.C., United States of America.

Urine is increasingly being considered as a source of biomarker development in Duchenne Muscular Dystrophy (DMD), a severe, life-limiting disorder that affects approximately 1 in 4500 boys. In this study, we considered the mdx mice-a murine model of DMD-to discover biomarkers of disease, as well as pharmacodynamic biomarkers responsive to prednisolone, a corticosteroid commonly used to treat DMD. Longitudinal urine samples were analyzed from male age-matched mdx and wild-type mice randomized to prednisolone or vehicle control via liquid chromatography tandem mass spectrometry. A large number of metabolites (869 out of 6,334) were found to be significantly different between mdx and wild-type mice at baseline (Bonferroni-adjusted p-value < 0.05), thus being associated with disease status. These included a metabolite with m/z = 357 and creatine, which were also reported in a previous human study looking at serum. Novel observations in this study included peaks identified as biliverdin and hypusine. These four metabolites were significantly higher at baseline in the urine of mdx mice compared to wild-type, and significantly changed their levels over time after baseline. Creatine and biliverdin levels were also different between treated and control groups, but for creatine this may have been driven by an imbalance at baseline. In conclusion, our study reports a number of biomarkers, both known and novel, which may be related to either the mechanisms of muscle injury in DMD or prednisolone treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219507PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634414PMC
March 2020

AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

J Inherit Metab Dis 2019 11 1;42(6):1088-1096. Epub 2019 Aug 1.

Center for Genetic Medicine Research, Children's National Medical Center, Washington, District of Columbia.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.
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http://dx.doi.org/10.1002/jimd.12133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385982PMC
November 2019

Fibroadipogenic progenitors are responsible for muscle loss in limb girdle muscular dystrophy 2B.

Nat Commun 2019 06 3;10(1):2430. Epub 2019 Jun 3.

Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, Washington, DC, 20010, USA.

Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.
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http://dx.doi.org/10.1038/s41467-019-10438-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547715PMC
June 2019

Mitochondrial dysfunction and role of harakiri in the pathogenesis of myositis.

J Pathol 2019 10 18;249(2):215-226. Epub 2019 Jul 18.

Center for Genetic Medicine Research, Children's National Health System, Washington, DC, USA.

The etiology of myositis is unknown. Although attempts to identify viruses in myositis skeletal muscle have failed, several studies have identified the presence of a viral signature in myositis patients. Here we postulate that in individuals with susceptible genetic backgrounds, viral infection alters the epigenome to activate the pathological pathways leading to disease onset. To identify epigenetic changes, methylation profiling of Coxsackie B infected human myotubes and muscle biopsies from polymyositis (PM) and dermatomyositis (DM) patients were compared to changes in global transcript expression induced by in vitro Coxsackie B infection. Gene and protein expression analysis and live cell imaging were performed to examine the mechanisms. Analysis of methylation and gene expression changes identified that a mitochondria-localized activator of apoptosis - harakiri (HRK) - is upregulated in myositis skeletal muscle cells. Muscle cells with higher HRK expression have reduced mitochondrial potential and poor ability to repair from injury as compared to controls. In cells from myositis patient toll-like receptor 7 (TLR7) activates and sustains high HRK expression. Forced over expression of HRK in healthy muscle cells is sufficient to compromise their membrane repair ability. Endurance exercise that is associated with improved muscle and mitochondrial function in PM and DM patients decreased TLR7 and HRK expression identifying these as therapeutic targets. Increased HRK and TLR7 expression causes mitochondrial damage leading to poor myofiber repair, myofiber death and muscle weakness in myositis patients and exercise induced reduction of HRK and TLR7 expression in patients is associated with disease amelioration. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7219509PMC
October 2019

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice.

J Pathol 2019 07 16;248(3):339-351. Epub 2019 Apr 16.

Center for Genetic Medicine, Children's National Health System, Washington, DC, USA.

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579705PMC
July 2019

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

Life Sci Alliance 2019 02 11;2(1). Epub 2019 Feb 11.

Department of Genomics and Precision Medicine, George Washington University School of Medicine and Health Sciences, Washington, DC, USA.

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.
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http://dx.doi.org/10.26508/lsa.201800186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371196PMC
February 2019

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

J Clin Pharmacol 2019 07 11;59(7):979-988. Epub 2019 Feb 11.

Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA.

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.
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http://dx.doi.org/10.1002/jcph.1388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548694PMC
July 2019

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

Steroids 2018 12 21;140:159-166. Epub 2018 Oct 21.

ReveraGen BioPharma, 155 Gibbs St., Suite 433, Rockville, MD 20850, USA; School of Pharmacy and Pharmaceutical Sciences, Binghamton University - SUNY, 4400 Vestal Pkwy E, Binghamton, NY 13902, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.steroids.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640634PMC
December 2018

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

Pharmacol Res 2018 10 13;136:140-150. Epub 2018 Sep 13.

Children's Hospital of Eastern Ontario, Ottawa, ON, Canada.

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.
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http://dx.doi.org/10.1016/j.phrs.2018.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218284PMC
October 2018

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

J Neuromuscul Dis 2018 ;5(4):407-417

Department of Human Genetics, Leiden University Medical Center, the Netherlands.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.
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http://dx.doi.org/10.3233/JND-180324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218134PMC
January 2019

Membrane Stabilization by Modified Steroid Offers a Potential Therapy for Muscular Dystrophy Due to Dysferlin Deficit.

Mol Ther 2018 09 27;26(9):2231-2242. Epub 2018 Aug 27.

Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC 20010, USA; Department of Genomics and Precision Medicine, George Washington University School of Medicine and Health Sciences, Washington, DC 20010, USA. Electronic address:

Mutations of the DYSF gene leading to reduced dysferlin protein level causes limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin facilitates sarcolemmal membrane repair in healthy myofibers, thus its deficit compromises myofiber repair and leads to chronic muscle inflammation. An experimental therapeutic approach for LGMD2B is to protect damage or improve repair of myofiber sarcolemma. Here, we compared the effects of prednisolone and vamorolone (a dissociative steroid; VBP15) on dysferlin-deficient myofiber repair. Vamorolone, but not prednisolone, stabilized dysferlin-deficient muscle cell membrane and improved repair of dysferlin-deficient mouse (B6A/J) myofibers injured by focal sarcolemmal damage, eccentric contraction-induced injury or injury due to spontaneous in vivo activity. Vamorolone decreased sarcolemmal lipid mobility, increased muscle strength, and decreased late-stage myofiber loss due to adipogenic infiltration. In contrast, the conventional glucocorticoid prednisolone failed to stabilize dysferlin deficient muscle cell membrane or improve repair of dysferlinopathic patient myoblasts and mouse myofibers. Instead, prednisolone treatment increased muscle weakness and myofiber atrophy in B6A/J mice-findings that correlate with reports of prednisolone worsening symptoms of LGMD2B patients. Our findings showing improved cellular and pre-clinical efficacy of vamorolone compared to prednisolone and better safety profile of vamorolone indicates the suitability of vamorolone for clinical trials in LGMD2B.
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http://dx.doi.org/10.1016/j.ymthe.2018.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127637PMC
September 2018

The role of nitrite in muscle function, susceptibility to contraction injury, and fatigability in sickle cell mice.

Nitric Oxide 2018 11 14;80:70-81. Epub 2018 Aug 14.

Department of Perioperative Medicine, National Institutes of Health Clinical Center, National Institutes of Health, Bethesda, MD, 20892, USA. Electronic address:

Sickle cell disease (SCD) patients can have limited exercise capacity and muscle dysfunction characterized by decreased force, atrophy, microvascular abnormalities, fiber distribution changes, and skeletal muscle energetics abnormalities. Growing evidence suggests that in SCD there is alteration in nitric oxide (NO) availability/signaling and that nitrate/nitrite can serve as a NO reservoir and enhance muscle performance. Here, we examined effects of nitrite on muscle strength, exercise capacity, and on contractile properties of fast-(extensor digitorum longus, EDL) and slow-twitch (soleus) muscles in SCD mice. Compared to controls, homozygotes (sickling) had decreased grip strength, impaired wheel running performance, and decreased muscle mass of fast-twitch, but not slow-twitch muscle. Nitrite treatment yielded increases in nitrite plasma levels in controls, heterozygotes, and homozygotes but decreases in muscle nitrite levels in heterozygotes and homozygotes. Regardless of genotype, nitrite yielded increases in grip strength, which were coupled with increases in specific force in EDL, but not in soleus muscle. Further, nitrite increased EDL, but not soleus, fatigability in all genotypes. Conversely, in controls, nitrite decreased, whereas in homozygotes, it increased EDL susceptibility to contraction-induced injury. Interestingly, nitrite yielded no changes in distances ran on the running wheel. These differential effects of nitrite in fast- and slow-twitch muscles suggest that its ergogenic effects would be observed in high-intensity/short exercises as found with grip force increases but no changes on wheel running distances. Further, the differential effects of nitrite in homozygotes and control animals suggests that sickling mice, which have altered NO availability/signaling, handle nitrite differently than do control animals.
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http://dx.doi.org/10.1016/j.niox.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186197PMC
November 2018

Synthesis, Characterization, and Function of an RNA-Based Transfection Reagent.

Curr Protoc Nucleic Acid Chem 2018 03;72(1):4.81.1-4.81.29

Laboratory of Biological Chemistry, Food and Drug Administration, Silver Spring, Maryland.

A synthetic 8-mer, amphipathic, trans-acting poly-2'-O-methyluridylic thiophosphate triester RNA element (2'-OMeUtaPS) can be prepared using solid-phase synthesis protocols. 2'-OMeUtaPS efficiently mediates the delivery of uncharged polyA-tailed phosphorodiamidate morpholino (PMO) sequences in HeLa pLuc 705 cells, as evidenced by flow cytometry measurements. In this cell line, 2'-OMeUtaPS-mediated transfection of an antisense polyA-tailed PMO sequence induces alternative splicing of an aberrant luciferase pre-mRNA splice site, leading to restoration of functional luciferase, as quantitatively measured using a typical luciferase assay. 2'-OMeUtaPS is also potent at delivering an uncharged antisense polyA-tailed PMO sequence in muscle cells of the mdx mouse model of muscular dystrophy; targeting the polyA-tailed PMO sequence against a splice site of the pre-mRNA encoding mutated dystrophin triggers an alternate splicing event that results in excision of the mutated exon (exon 23) from the pre-mRNA and production of functional dystrophin, as demonstrated by agarose gel electrophoresis. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpnc.51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020023PMC
March 2018

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

Physiol Genomics 2018 09 8;50(9):735-745. Epub 2018 Jun 8.

Center for Genetic Medicine Research, Children's National Medical Center , Washington, District of Columbia.

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.
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http://dx.doi.org/10.1152/physiolgenomics.00134.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172612PMC
September 2018

Risk factors and disease mechanisms in myositis.

Nat Rev Rheumatol 2018 04;14(5):255-268

Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Binghamton, NY, USA.

Autoimmune diseases develop as a result of chronic inflammation owing to interactions between genes and the environment. However, the mechanisms by which autoimmune diseases evolve remain poorly understood. Newly discovered risk factors and pathogenic processes in the various idiopathic inflammatory myopathy (IIM) phenotypes (known collectively as myositis) have illuminated innovative approaches for understanding these diseases. The HLA 8.1 ancestral haplotype is a key risk factor for major IIM phenotypes in some populations, and several genetic variants associated with other autoimmune diseases have been identified as IIM risk factors. Environmental risk factors are less well studied than genetic factors but might include viruses, bacteria, ultraviolet radiation, smoking, occupational and perinatal exposures and a growing list of drugs (including biologic agents) and dietary supplements. Disease mechanisms vary by phenotype, with evidence of shared innate and adaptive immune and metabolic pathways in some phenotypes but unique pathways in others. The heterogeneity and rarity of the IIMs make advancements in diagnosis and treatment cumbersome. Novel approaches, better-defined phenotypes, and international, multidisciplinary consensus have contributed to progress, and it is hoped that these methods will eventually enable therapeutic intervention before the onset or major progression of disease. In the future, preemptive strategies for IIM management might be possible.
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http://dx.doi.org/10.1038/nrrheum.2018.48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745704PMC
April 2018

Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy.

Skelet Muscle 2018 04 6;8(1):13. Epub 2018 Apr 6.

Children's National Heart Institute, Center for Genetic Medicine Research, Children's National Health System, Washington, DC, USA.

Background: Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease.

Methods: We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation.

Results: P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p < 0.05).

Conclusions: FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies.
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http://dx.doi.org/10.1186/s13395-018-0158-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889611PMC
April 2018

Author Correction: Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

Nat Commun 2018 03 23;9(1):1256. Epub 2018 Mar 23.

Center for Genetic Medicine Research, Children's Research Institute, Children's National Health System, Washington, DC, 20010, USA.

The originally published version of this Article contained an error in Figure 6. In panel b, the top graph (BrdU 21-24d) and the bottom graph (BrdU 28-31d) were inadvertently swapped. This error has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-03709-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865121PMC
March 2018

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

Steroids 2018 06 8;134:43-52. Epub 2018 Mar 8.

ReveraGen BioPharma, Rockville, MD, USA.

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.
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http://dx.doi.org/10.1016/j.steroids.2018.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136660PMC
June 2018

Update on Standard Operating Procedures in Preclinical Research for DMD and SMA Report of TREAT-NMD Alliance Workshop, Schiphol Airport, 26 April 2015, The Netherlands.

J Neuromuscul Dis 2018;5(1):29-34

Biozentrum, University of Basel, Basel, Switzerland.

A workshop took place in 2015 to follow up TREAT-NMD activities dedicated to improving quality in the preclinical phase of drug development for neuromuscular diseases. In particular, this workshop adressed necessary future steps regarding common standard experimental protocols and the issue of improving the translatability of preclinical efficacy studies.
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http://dx.doi.org/10.3233/JND-170288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836406PMC
June 2018

Author Correction: Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

Nat Commun 2018 01 15;9(1):208. Epub 2018 Jan 15.

Center for Genetic Medicine Research, Children's Research Institute, Children's National Health System, Washington, DC, 20010, USA.

In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from the CRI Light Microscopy and Image Analysis Core.
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http://dx.doi.org/10.1038/s41467-017-02206-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768807PMC
January 2018

Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.

FASEB J 2018 02 3;32(2):1025-1043. Epub 2018 Jan 3.

Pharmacology Unit, Department of Pharmacy-Drug Sciences, University of Bari, Bari, Italy.

Muscle fibers lacking dystrophin undergo a long-term alteration of Ca homeostasis, partially caused by a leaky Ca release ryanodine (RyR) channel. S48168/ARM210, an RyR calcium release channel stabilizer (a Rycal compound), is expected to enhance the rebinding of calstabin to the RyR channel complex and possibly alleviate the pathologic Ca leakage in dystrophin-deficient skeletal and cardiac muscle. This study systematically investigated the effect of S48168/ARM210 on the phenotype of mdx mice by means of a first proof-of-concept, short (4 wk), phase 1 treatment, followed by a 12-wk treatment (phase 2) performed in parallel by 2 independent laboratories. The mdx mice were treated with S48168/ARM210 at two different concentrations (50 or 10 mg/kg/d) in their drinking water for 4 and 12 wk, respectively. The mice were subjected to treadmill sessions twice per week (12 m/min for 30 min) to unmask the mild disease. This testing was followed by in vivo forelimb and hindlimb grip strength and fatigability measurement, ex vivo extensor digitorum longus (EDL) and diaphragm (DIA) force contraction measurement and histologic and biochemical analysis. The treatments resulted in functional (grip strength, ex vivo force production in DIA and EDL muscles) as well as histologic improvement after 4 and 12 wk, with no adverse effects. Furthermore, levels of cellular biomarkers of calcium homeostasis increased. Therefore, these data suggest that S48168/ARM210 may be a safe therapeutic option, at the dose levels tested, for the treatment of Duchenne muscular dystrophy (DMD).-Capogrosso, R. F., Mantuano, P., Uaesoontrachoon, K., Cozzoli, A., Giustino, A., Dow, T., Srinivassane, S., Filipovic, M., Bell, C., Vandermeulen, J., Massari, A. M., De Bellis, M., Conte, E., Pierno, S., Camerino, G. M., Liantonio, A., Nagaraju, K., De Luca, A. Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.
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http://dx.doi.org/10.1096/fj.201700182RRRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888399PMC
February 2018