Characterisation of skeletal development and the use of anabolic agents in murine models of Duchenne muscular dystrophy

Abstract

Short stature and osteoporosis are common in Duchenne muscular dystrophy (DMD) and its pathophsyiology may include an abnormality of the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis, which is further exacerbated by glucocorticoid (GC) treatment. The mechanisms that underlie the undesirable effects of GCs on skeletal development are unclear and there is no proven intervention. Investigation of compounds to treat the defect in growth and skeletal development in GC treated DMD boys necessitates an appropriate pre-clinical model, but at present there is no established animal model to investigate GC effects on skeletal development. The mdx mouse is commonly used but its phenotype is mild and few medications that have shown benefit in the mdx mouse have also shown efficacy in clinical trials. The mdx:cmah mouse carries a human-like mutation in the Cmah gene and has a more severe muscle phenotype, but its growth and bone characteristics have never been investigated. This thesis tested the overall hypothesis that "GC- treated mouse models of Duchenne muscular dystrophy (DMD) have an abnormality of linear growth and skeletal development that can be rescued by modulation of the growth hormone (GH)/ insulin-like growth factor-1 (IGF-1) axis. Firstly the growth and bone phenotype of the mdx, mdx:utr and mdx:cmah muscular dystrophy mouse models were characterised. No clear intrinsic skeletal defect were observed in the mdx or mdx:utr mice. Furthermore, mdx:cmah mice showed clear evidence of catch-up growth that was also associated with an increase in bone development. This pattern does not mimic the typical DMD growth trajectory. Whilst the utility of the mdx:cmah mouse for studying growth and skeletal development in DMD may be limited, further studies of this model may shed light on the phenomenon of catch-up growth. In the second part of the project, the aim was to identify a suitable GC regimen to induce both growth retardation and osteoporosis in juvenile mdx and mdx:cmah mice and their wildtype C57BL10 controls. C57BL10 mice appeared fairly resistant to GC challenge; despite high doses no biomechanical or trabecular architecture changes were noted. Prednisolone 20mg/kg body weight given by oral gavage appeared to be the most effective regimen to induce growth retardation and osteoporosis over a 28- day period. A GC-sparing agent, VBP-6 was also examined as an alternative to GC, with the aim of further characterising its effects on skeletal development. This thesis has provided extra data to support the clinical trials that are currently investigating the use of VBP-15 (a VBP-6 analogue) as a GC-sparing agent in DMD and provides further evidence that VBP-6 is able to improve muscle function without the deleterious skeletal side-effects. VBP-6 could potentially be used as an alternative in other childhood conditions that necessitate the use of long-term GCs. The final part of the project explored the potential of GH and IGF-1 given in combination to rescue the growth retardation and cortical bone defect seen during 4 weeks of prednisolone treatment in mdx mice. This intervention study determined that growth retardation could be rescued by combined GH/IGF-1 treatment but the osteopenia phenotype could not. As short stature remains one of the most significant concerns for boys with DMD as their life expectancy increases, it may now be appropriate to design a pilot clinical study to evaluate the safety and tolerability of combination GH and IGF-1 therapy in a small group of patients with DMD.