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N-Butyryl arginine and 3-Hydroxybutyrate arginine, for the treatment of DMD through oral administration

Duchenne muscular dystrophy is a X-linked progressive neuromuscular disease affecting 1:3500 boys at birth. It is caused by the absence of dystrophin, a subsarcolemmal protein that confers membrane stability linking cytoskeletal actin to the extracellular matrix. It is part of a multi-protein complex called dystrophin associated protein complex (DAPC), which contains, among the other components, -dystroglycan and nitric oxide synthase (NOS).The consequences of the absence of dystrophin are: deregulation of calcium homeostasis, tissues necrosis, progressive accumulation of fat and fibrosis, inability of the movements and cardiac and respiratory failures that lead to patient’s death, around the age of 20-30 years.The objective of my PhD work is to ameliorate different aspects of dystrophic phenotype. In particular I have tested two different ways of administration of arginine butyrate (AB), the reference drug, through feeding-force and intraperitoneal injection. Meanwhile I have studied two new pharmacological molecules, AB derived, which could be administered orally to DMD patients. These compounds are: 3-Hydroxybutyrate arginate (refer as ABE) and N-butyryl arginine (refer as ABA). All of these molecules partially restore dystrophic phenotype activating two independent pathways (both the nitric oxide pathway and the inhibition of the histone deacetilase), which are known to be beneficent for mdx mice.AB, ABE and ABA have been tested in vitro on human DMD myotubes and in vivo on the mdx mice. The first goal of my project is the observation that the positive effects obtained after intraperitoneal injections of AB can be detected also after oral protocol, promoting the idea that the oral way has to be developed for future clinical trials. I have focused my attention on heart defaults; in particular, starting from the 8th month, a monthly study on heart activity based on echocardiography has been performed on mdx mice treated with AB. We addressed the potential profits of the oral administration of arginine butyrate on vertebral column deformation and electromyogram defaults, with a non-invasive automatized method developed in clinic and then applied to animals. The results collected from these experiments show that AB preserve heart activity, reverse vertebral column deformity and all the axonal excitability parameters that were modified in saline-treated mdx mice.In complement, I have tested different concentrations of ABE and ABA in vivo. The positive effects on many structural and functional dystrophic parameters, previously obtained with high dose of AB administered per os (800 mg/kg/d), has been observed with doses 10 times lower with both new compounds.In parallel, both products were tested in vitro on human muscular cells cultures to investigate their capacity to increase utrophin level. Moreover, the potential ability of histone deacetylase inhibitors (byturate, valproic acid, trichostatin A and isobutyramide) to increase the expression of utrophin and related proteins (-dystroglycan and embryonic myosin) has been studied. Finally, the alteration of calcium homeostasis, largely implicated in the cascades resulting in muscle necrosis/degeneration, was investigated. The spontaneous Ca2+ activity recorded in patient myotubes, i.e. without sarcolemmal integrity was strongly reduced after treatment acting on the NO-pathway activation and/or with HDAC inhibitors. All together, these data constitute a proof of principle of the beneficial effects of arginine butyrate and its derivates on muscular dystrophy, by enhancing NO pathway and inhibiting HDAC.