This work is based on the functional coupling of integrins, boron (B) transporter (NaBC1), and growth factor receptors (GFR) after their activation. In our previous work, we have demonstrated that active-NaBC1, co-localise with fibronectin-binding integrins (α5β1/αvβ3) and GFR producing a functional cluster that synergistically enhances biochemical signals and crosstalk mechanisms, accelerating local muscle repair in vivo after an injury. Based on the data reporting that increased fibronectin (FN) levels are used as a marker of disease in several muscular dystrophies, here we have studied a combined system composed of boron (for NaBC1 stimulation) and FN (for FN-binding integrin stimulation), and evaluated the effects in restoration of dystrophic phenotypes in in vitro and in vivo models of Duchenne Muscular Dystrophy (DMD).
We have used borax (Sodium tetraborate decahydrate) and bortezomib (a dipeptidyl boronic acid) as two different boron compounds. Bortezomib is an FDA approved drug usually employed for the treatment of hematologic malignancies. Human DMD cells have been cultured with soluble borax or bortezomib, alone or combined with FN. Results show that both, borax and bortezomib, combined with FN improved myotube differentiation and diameter. Considering that dystrophic DMD cells present hallmark features such as reduced fusion capability and smaller and thinner myotubes compared to healthy controls, we concluded that both, borax and bortezomib, together with FN, are restoring myotube fusion capability and myotube physiological function (myotube diameter). Interestingly, although the results obtained with both, borax and bortezomib were similar, the borax doses used were in the range of milimolar while the bortezomib doses were in the range of nanomolar (an ultra-low dose compared to its original therapeutic indication), indicating that we obtained similar effects in myotube restoration using ultra-low doses of a boron compound.
After that, we have investigated the effects of ultra-low doses of bortezomib in a DMD mouse model in vivo. We have engineered and characterised injectable PEG-based hydrogels simultaneously presenting cell adhesion peptide domains (RGD) and controlled local bortezomib-release. The selected compositions were injected in both tibialis anterior (TA) of 8 weeks old D2.B10 (DBA/2-congenic) Dmdmdx male mice. Five experimental groups have been evaluated: i) Wild type control mice, ii) DMD control mice, iii) DMD PEG-RGD, iv) DMD PEG-bortezomib and v) DMD PEG-RGD-bortezomib (n = 6). We monitored the evolution of the body weight and motor impairment in behavioural four limb hanging test over 8 weeks. After euthanasia at 12 weeks old, tissues were analysed by histological and immunofluorescence techniques.
Hematoxylin-eosin-stained sections of PEG-RGD-bortezomib treated group, although still displaying typical DMD histopathology, showed a significant decrease in size variability among muscle fibers, mononuclear cell infiltrates, degenerating fibres, and centrally located nuclei. Immunofluorescence analysis revealed that treatment with PEG-RGD-bortezomib significantly reduced FN levels, suggesting a partial prevention of disease progression characterised by massive increase in FN levels.
Altogether, our findings in this local proof of concept suggest that active-NaBC1 is capable, at least partially, of rescuing dystrophic phenotypes in mdx model, opening to new possibilities to develop therapeutic approaches for the treatment of DMD.