Vamorolone targets dual nuclear receptors to treat inflammation and dystrophic cardiomyopathy

Vamorolone is a first-in-class dissociative drug that selectively targets the glucocorticoid receptor to safely treat chronic inflammation and the mineralocorticoid receptor to treat cardiomyopathy, providing efficacy with improved safety in mouse models of Duchenne muscular dystrophy.

As you will see, the reviewers appreciate your analysis and provide constructive input on how to further strengthen your dataset. We would thus like to invite you to provide a revised version, addressing the concerns of the reviewers. The concerns raised by reviewers #1 and #3 seem straightforward to address. Reviewer #2 asks for more experimental data to substantiate your claims. While we don't expect global gene expression analysis in heart tissues of the mdx mice (rev#2, last point) for acceptance here, we would appreciate an analysis of primary cardiomyocytes/another relevant cell type as proposed by this reviewer. We are happy to discuss this point further with you should this be helpful.
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Full guidelines are available on our Instructions for Authors page, http://life-sciencealliance.org/authorguide We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Reviewer #1 (Comments to the Authors (Required)): This manuscript by Heier and Spurney reports a very nice series of analyses that dissect the differential effects of vamorolone versus prednisolone, deflazacort, eplerenone, and spironolactone on activating/inhibiting mineralcortocoid and glucocorticoid receptors mediating the inflammation and cardiomyopathy associated with dystrophinopathy in the mdx mouse model. The experiments are presented in a clear and balanced manner. I think the study is important to the field and will be widely read. Below, however, are a few issues that should be addressed.
1) The data in Fig. 4A appear to report that systolic blood pressure in mdx mice that received no aldosterone was not different from WT controls. However, previous studies from this group have reported that mdx mice have significantly lower systolic (CF Spurney et al, K Uaesoontrachoon et al., 2014), diastolic (CF Spurney et al, 2009), or mean blood pressure (CF Spurney e. al, 2009) compared to WT. Can they explain why blood pressure was not lower in the mdx controls compared to WT? Perhaps the current experiment is underpowered to reproduce their previous findings?
2) The bar graph data in Fig. 2D, 2E, 4D, and 5A-F could be more easily evaluated by readers if presented as dot plots.
3) The statement in line 4 is not supported by the reference provided.
Reviewer #2 (Comments to the Authors (Required)): Treatment with glucocorticoids of patients with Duchenne muscular dystrophy (DMD) is considered as standard of care. The chronic use of glucocorticoids has been shown to substantially preserve heart function and improve survival of DMD patients. In the present study, Heier et al. aim to identify the underlying mechanism(s) of Vamorolone, a glucocorticoid receptor (GR) ligand that has been developed from the same group (Heier et al., 2016 EMBO Molecular medicine). Overall, the concept and results of this manuscript are presented in a very structured manner. The conclusion that vamolorone mediates both anti-inflammatory and anti-fibrotic effects in DMD hearts, however, has to be substantiated by additional methodological approaches. Likewise, the quantification of miR-146 alone is not sufficient to determine anti-inflammatory activities of vamorolone and other drugs being studied here. The use of C2C12 myoblasts is furthermore not the best model to study anti-inflammatory effects of vamorolone, which is supposed to act on local cells of the injured heart. Cultures of primary cardiomyocytes, fibroblasts or even immune cells that are known to invade and accumulate during heart failure progression (e.g. macrophages) should be used instead as an in vitro to study direct effects of vamorolone. A more detailed anaylsis of myocardial samples assessing the degree of inflammation (flow cytometry, histology) and fibrosis is also recommended. Minor comments: -The introduction is too long and contains partly repetitions of content. -The steroidal drugs and metabolic derivates enlisted within the abstract (vamorolone, prednisolone, prednisone, deflazacort) should be clearly referred to its corresponding receptor system they activate (if possible). -The figure legends should not contain a description and interpretation of the results itself, e.g. "Vamorolone acted as an MR antagonist, consistent with eplerenone and spironolactone" (Fig. 1) and "Prednisolone caused an increase in D2-mdx heart size" (Fig.5).
-Could the authors estimate the amount of vamorolone per day that the mice received upon osmotic pump implantation? A clear statement is missing within the results section.
-The antagonistic effect of vamorolone on MR activation in vivo is restricted to maintain the kidney size upon treatment with aldosterone. This aspect of vamorolone is not directly linked to its mode of action at sites of cardiac dystrophy and thus should be transferred to a supplemental material section.
-It is stated within the result section "Vamorolone shows potent MR antagonist activity in vivo" that "After six weeks, heart function was assayed by echocardiography, blood pressure was measured, and terminal endpoint measures were performed." These data, however, are not presented here. -The authors should employ a more unbiased and global gene expression profiling approach than selective qRT-PCR to characterize changes of gene expression in heart tissues of mdx and vamorolone treated mdx mice. Such an approach would also reflect potential alterations of the degree of inflammation.
Reviewer #3 (Comments to the Authors (Required)): This manuscript provides a comprehensive set of well designed and conducted studies demonstrating that valmorolone works as an antagonist of the mineralocorticoid receptor (MR) in heart in addition to an agonist of the glucocorticoid receptor (GR). These findings are important due to the ongoing clinical trials with valmorolone for Duchenne muscular dystrophy as well as the current standard of clinical care, which are GR agonists. In silico analyses, reporter assays, GR knockout myoblasts, analysis of GR and MR target genes, are shown to demonstrate the molecular mechanisms of valmorolone's action. In vivo studies of the effect of valmorolone versus eplerenone, and spironolactone on aldosterone treated mdx mice and valmorolone versus prednisolone treatment of D2-mdx mice were compared were used to further demonstrate valmorolone's action as an MR antagonist. Overall, this is a well-conducted study and a well written manuscript.
Two minor points in the discussion should be considered. The authors should be careful about their assumption that the glucocorticoid inactivating enzyme HSD11B2 is not expressed in dystrophic heart, since others have shown this enzyme appears to be increased in mdx skeletal muscles (Hum Mol Genet. 2016 Dec 1;25(23):5167). Although it is reasonable that this experiment may be beyond the scope of the current study, the authors should consider both possibilities and be wary of drawing a model that compares cell types with and without this enzyme. It is possible that the upregulation of HSD11B2 actually explains why dystrophin deficiency is the "second hit" that sensitizes mdx hearts to MR activity. Second, in vivo anti-inflammatory gene expression changes similar to prednisone have also been observed with other MR antagonists and could be referenced (Am J Physiol Cell Physiol. 2017 Feb 1; 312(2): C155). It is possible that although the specific target miRNA assessed in these experiments is GR-dependent, that other inflammatory genes come from MR activation.

Reviewer points and responses: Reviewer #1:
This manuscript by Heier and Spurney reports a very nice series of analyses that dissect the differential effects of vamorolone versus prednisolone, deflazacort, eplerenone, and spironolactone... The experiments are presented in a clear and balanced manner. I think the study is important to the field and will be widely read. Below, however, are a few issues that should be addressed.
A) The statement in line 4 is not supported by the reference provided.  There was another reference for a Line 4 on page 9 of the results that may have been referred to, however this was removed when that section was changed in response to a Reviewer # 3 point.
B) The bar graph data in Fig. 2D, 2E, 4D, and 5A-F could be more easily evaluated by readers if presented as dot plots.
 We reformatted these graphs to present dots plotted for the data points in this revised version of our manuscript.
C) The data in Fig. 4A   We believe the reason for the difference is due to the difference in mdx mouse ages / diseases stages between the studies. In our current study we use younger mice that are pre-symptomatic in terms of cardiomyopathy. In those other studies, we used older mdx mice (≥ 10 months of age) which are at an advanced stage of mdx disease where they display symptomatic cardiomyopathy. For the experiments in our current paper, our goal was to introduce aldosterone challenge onto a background of pre-symptomatic disease stage of mdx cardiomyopathy. This was intentionally done by design; we wanted to study a disease stage where aldosterone could effectively exacerbate mild / presymptomatic disease to a more severe / symptomatic state, while also testing the ability of antagonists to counteract its effects to keep disease in a mild cardiac phenotype.
 Our study had a similar number of mice to the provided references (n = 8-10 per group), so we do not believe differences were due to a difference in power. o "Future studies using tissue-specific knockout mice and receptor-specific ligands will be valuable to further dissect roles of the mineralocorticoids, glucocorticoids, their receptors and their regulatory enzymes. For example we find intracellular, inflammatory NF-κB signaling is inhibited by vamorolone, prednisolone and deflazacort in a GR-specific manner. However, the renin-angiotensin-aldosterone system (RAAS) also impacts inflammation via effects on reactive oxygen species, blood pressure, blood volume, and inflammatory cell infiltration/adhesion. Accordingly, work by other groups indicates MR antagonists can also show a level of anti-inflammatory and membrane-stabilizing effects driven independently from the GR and these may provide separate pathways of efficacy for drugs such as eplerenone (Chadwick et al, 2017). Additionally, a recent report suggests myeloid cells can synthesize aldosterone in a manner that could have local impacts on the dystrophic microenvironment of skeletal muscle (Chadwick et al, 2016). In contrast to skeletal muscle in that study, here we do not detect Hsd11b2 expression in heart tissue; this finding is consistent with our findings that prednisolone can worsen heart phenotypes of mdx mice. It will be intriguing to eventually compare a full dissection of steroidal pathways in muscle, heart, immune and fibroblast tissue types. Doing so will provide valuable information on how to best apply current clinical drugs, on future drug development, and on the biology of muscle versus heart involvement in DMD."

Reviewer #3:
Overall, the concept and results of this manuscript are presented in a very structured manner. The conclusion that vamolorone mediates both anti-inflammatory and anti-fibrotic effects in DMD hearts, however, has to be substantiated by additional methodological approaches.
A) The quantification of miR-146 alone is not sufficient to determine anti-inflammatory activities of vamorolone and other drugs being studied here. The use of C2C12 myoblasts is furthermore not the best model to study anti-inflammatory effects of vamorolone, which is supposed to act on local cells of the injured heart. Cultures of primary cardiomyocytes, fibroblasts or even immune cells that are known to invade and accumulate during heart failure progression (e.g. macrophages) should be used instead as an in vitro to study direct effects of vamorolone. We also a felt it wou in Table 1   As suggested, we have clarified this in the Results section and Figure Legends; the mice received vamorolone at 20 mg/kg via daily oral dosing.
o New text in Results:  "Randomized and blinded treatment groups of wild type and mdx mice were implanted with subcutaneous osmotic pumps that secreted either vehicle or aldosterone, the physiological MR agonist, at a calculated dose of 0.25 mg/kg/day (n ≥ 8 per group). The mdx mice receiving aldosterone via osmotic pump were also treated with vehicle, vamorolone (20 mg/kg/day), eplerenone (100 mg/kg/day), or spironolactone (20 mg/kg/day), using daily oral administration via ingestion of sugar syrup formulations." E) The antagonistic effect of vamorolone on MR activation in vivo is restricted to maintain the kidney size upon treatment with aldosterone. This aspect of vamorolone is not directly linked to its mode of action at sites of cardiac dystrophy and thus should be transferred to a supplemental material section.
 We agree that this is not directly linked to cardiac dystrophy, however as a traditional MR/aldosterone target tissue it is linked to the mechanism of action of MR ligands. We feel these may actually be viewed as a good reasons to include it, because this MRmediated phenotype allows us to also assay effects of compounds on an MR activity that is independent of dystrophic pathology. We have decided to keep this in the main text for now because it is directly linked to the drug mechanism of MR antagonism.
 Additionally, the contrast in effects of prednisolone/aldosterone between kidney and heart is important to address Reviewer 2 Point (A). By showing the effects in both kidney and heart, we can show how heart is specifically sensitized to MR-activation by prednisolone due to its lack of expression for the protective, steroid-metabolizing enzyme HSD11B2.
F) The figure legends should not contain a description and interpretation of the results itself, e.g. "Vamorolone acted as an MR antagonist, consistent with eplerenone and spironolactone" (Fig. 1) and "Prednisolone caused an increase in D2-mdx heart size" (Fig.5).
 We used this descriptive style because we thought it was consistent with other papers in this journal and the directions in the Life Science Alliance guidelines. We can change this if the Editor prefers.
G) The authors should employ a more unbiased and global gene expression profiling approach than selective qRT-PCR to characterize changes of gene expression in heart tissues of mdx and vamorolone treated mdx mice.
 In the future, we will look into performing similar profiling experiments towards the development of MR-specific biomarkers. For now, this is beyond the scope of the current manuscript, as the Editor stated in their response that "we don't expect global gene expression analysis in heart tissues of the mdx mice for acceptance here".