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

Michael Ziemba, Molly Barkhouse, Kitipong Uaesoontrachoon, Mamta Giri, Yetrib Hathout, Utkarsh J Dang, Heather Gordish-Dressman, Kanneboyina Nagaraju, Eric P Hoffman, Michael Ziemba, Molly Barkhouse, Kitipong Uaesoontrachoon, Mamta Giri, Yetrib Hathout, Utkarsh J Dang, Heather Gordish-Dressman, Kanneboyina Nagaraju, Eric P Hoffman

Abstract

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.

Conflict of interest statement

Declaration of Interest: KN and EPH hold founder shares and management positions in AGADA Biosciences. MB and KU are employees of AGADA Biosciences. AGADA Biosciences is a contract research organization that received a competitive grant from Foundation to Eradicate Duchenne to perform studies presented in this paper. UD receives consultancy funds from ReveraGen BioPharma. KN and EPH hold founder shares and management positions in ReveraGen BioPharma, the sponsor of vamorolone, a drug in clinical development for Duchenne muscular dystrophy. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Ingenuity Pathway Analysis of prednisolone-responsive…
Fig 1. Ingenuity Pathway Analysis of prednisolone-responsive biomarkers identifies inflammatory cytokine and NFκB pathways shared with vamorolone.
The two most significant enrichment of protein-protein interactions for prednisolone-responsive biomarkers are shown (left panels), with vamorolone-responsive proteins overlaid onto the prednisolone networks. Red indicates upregulation, and green indicates downregulation, and color intensity correlates with the size of expression change. The multicolored elements are protein complexes where the underlying data and documentation are inconsistent with IPAs search algorithms, and therefore, the details are not shown.
Fig 2. Ingenuity Pathway Analysis of BAIBA-responsive…
Fig 2. Ingenuity Pathway Analysis of BAIBA-responsive biomarkers identifies modulation of inflammatory pathways.
The top IPA network identified with high dose BAIBA treatment data is shown.

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