Use of boron and its derivatives for the treatment of muscular dystrophies

The invention relates to the use of boron or a boron compound for the treatment of muscular dystrophies. Therefore, the present invention can be included in the field of medicinal chemistry or pharmacology.

STATE OF ART

Boron is an essential metalloid, which plays a key role in plants and animals metabolisms. It has been reported that boron is involved in bone mineralisation, has some uses in synthetic chemistry and its potential has been only recently exploited in medicinal chemistry. Little is known about boron homeostasis and function in animal cells. It has been reported that boron is involved in mouse myogenic differentiation {Tissue Eng Part A. 2015 Nov;21 (21 -22):2662-7).

Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease, caused by X- linked mutations of the dystrophin encoding gene. The lack of dystrophin leads to weakness, degeneration, and consequent fibrosis in skeletal and cardiac muscles. Currently, there is no cure for DMD patients. Preclinical and clinical approaches in the pipeline include exon-skipping, gene editing via viral vectors, and stem cell transplants {Stem Cell Reviews and Reports, Vol. 14, 2018). All of these approaches will take time until they are available, and even when approved maybe only be suitable for a select patient population and/or able to improve symptoms but not to relieve them completely. Therefore, therapies that, either independently or in combination with other treatment strategies, improve disease progression are needed.

Myotonic dystrophy (DM, prevalence 1 in 7400 live births) is characterized by dominantly inherited muscle hyperexcitability (myotonia), progressive myopathy, cataracts, defects of cardiac conduction, neuropsychiatric impairment, and other developmental and degenerative manifestations. This complex phenotype results from the expansion of a CTG repeat in the untranslated region (3 ^' -UTR) of the DMPK gene, which encodes a serine-threonine protein kinase. The transcripts from the mutant allele are retained in the nucleus, and levels of DMPK protein are correspondingly reduced. Currently, there is no cure or effective treatment. DM1 is one of the most variable inherited diseases, since patient phenotypes range from asymptomatic adults to severely affected newborns with a congenital manifestation of the disease. Taking into account the age of onset and the severity of the symptoms, the main forms of DM1 are of late development (mild phenotypes), in adults (classical DM1 ), juvenile and congenital. The predominant form of the disease is that in which symptoms appears in the second or third decade of life presenting the typical symptoms of the pathology. Several therapeutic approaches have been tested, although with no clear success {Drug Discovery Today. 2017; 22: 1740- 1748; Drug Discovery Today. 2018; 00: 1-10). Among them, known drugs for other applications such as mexiletine, an antiarrhythmic that acts on sodium channels, has been used off-label to treat myotonia in DM and in non-dystrophic myotonias. A preliminary study has suggested that recombinant human IGF-1 (rhIGFI ) can improve muscle strength and function in adult patients with DM1 , although this therapy presents important limitations. Another important approach to treat DM is designing anti-sense oligonucleotides to neutralize the mRNA defective in these patients {Drug Discovery Today. 2018; 00: 1-10). Nevertheless, these approaches to treatment of DM are supportive and have failed to slow or halt disease progression. Therefore, there is a need to minimize the physiological effect of the defective mRNA to give a life quality to the patients of this rare disease.

DESCRIPTION OF THE INVENTION

The present invention provides a treatment for muscular dystrophies based on the administration of boron or a compound of boron such as sodium borate (borax). The inventors present experimental results in a DM1 fly model, where the ingestion of borax induced an improvement in muscle atrophy and in flight capacity as shown further below. The inventors also provide experimental data in a DM1 mouse in vivo model. After implantation of hydrogel/PLLA material devices loaded with different amounts of borax, treated mice presented a reduction in muscle myotonia and improved the grip strength to a level similar to the one obtained for wild-type mice. Indeed, histological analysis of the implanted quadriceps showed that after borax treatment the number of muscle fibres with central nuclei (a phenotypic characteristic of DM1 affected muscles) was significantly reduced. Transcriptomic analysis performed in patient derived myoblasts showed that borax treatment recovers totally or partially 1 15 genes of 1333 affected in DM1 cells. Most of integrins expression was modified by borax treatment (almost all integrins implied in cell adhesion and intracellular signalling) and similar results were obtained with ion channels (50% of 328 genes identified), being the most important metabolic pathways affected the GO pathway“Development of skeletal system”, Kegg pathway“Calcium signalling” and Reactome pathway“Extracellular matrix organisation”. Furthermore, additional experimental results obtained in a human immortalised DM1 cell line and DMD primary patient cells are shown below.

It is clear that there is a need in treating DM and DMD, and that nowadays, a treatment with an acceptable success has not been obtained. The experimental data supporting the therapeutics effects of the present invention would demonstrate that it fulfils a long- felt need in the field.

Currently, there are two different described types of myotonic dystrophy: type 1 and type 2. Both diseases are clinically similar but they are caused by different genetic mutations. DM1 is originated by an expansion of the CTG triplet in the 3 ^' -untranslated region of the Protein Kinase DM (DMPK) gene, while DM2 is originated by a dynamic mutation consisting on the expansion of CCTG repeats of the CCHC-type zinc finger nucleic acid binding protein (CNBP). DM2 symptoms are similar to those of DM1 , although they are milder and with slower progression. Both DM1 and DM2 show a broad clinical spectrum and they are considered degenerative conditions. The fact that these two dynamic mutations in different genes cause diseases with similar symptomatology suggests a common pathogenesis mechanism based on the toxicity of the RNAs containing the CUG and CCUG expansions as necessary and sufficient factors causing myotonic dystrophy. Since both types of DM present similar symptomatology, and the results obtained in the present invention after addition of borax treatment induced an improvement in muscle recovery in DM1 , we can expect the same borax effects in DM2, due to the similarity of symptoms and the mild characteristic of DM2.

Furthermore, as the authors have obtained similar positive results using borax in two different muscular dystrophies (DM1 and DMD), with extremely diverse aetiological origin, they consider the therapeutic effects of boron expandable and embracing several muscular dystrophies.

Thus, a first aspect of the present invention relates to boron or a boron compound for use in the treatment of muscular dystrophies. In a preferred embodiment, the boron compound referred to in the present invention further comprises sodium.

In another preferred embodiment, said boron compound is borax.

In the present invention, the term “borax” refers to hydrated sodium borate Na ₂ B ₄ O ₇ - 10H ₂ O, that occurs naturally as a mineral or is prepared from other minerals, and normally presents in a white crystalline powder.

Other forms of boron which can be included in the scope of the present invention may be the following boron compounds: boron sodium oxide (B ₄ Na ₂ 0 ₇ ), boric acid as for example zinc salt, perboric acid (HBO(0 ₂ )) as sodium salt, boric acid (H ₃ BO ₃ ), boron lithium oxide (B ₄ l_h0 ₇ ), ammonium boron oxide ((NH^BsOe), boron silver oxide (B ₄ Ag ₂ 0 ₇ ), boron zinc oxide (BeZ^On), boric acid (HBO ₂ ) as lithium salt.

In another preferred embodiment, muscular dystrophies are selected from myotonic dystrophy type 1 , myotonic dystrophy type 2 or Duchenne muscular dystrophy.

In the present invention, the term“muscular dystrophy” refers to a group of diseases that cause progressive weakness and loss of muscle mass. In muscular dystrophy, abnormal genes (mutations) interfere with the production of proteins needed to form healthy muscle. The main sign of muscular dystrophy is progressive muscle weakness. Specific signs and symptoms begin at different ages and in different muscle groups, depending on the type of muscular dystrophy. Examples of these diseases are Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, myotonic muscular dystrophy (type 1 and type 2), limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy or distal muscular dystrophy. Considering that these diseases may have a common physiological mechanism, the scope of the present invention may be applicable to the treatment of the whole group.

In another preferred embodiment, boron or the boron compound for use as mentioned is in combination with another active principle. As occurs for example with bortezomib, a boron compound for the inhibition of proteasome activity, or in combination with other current therapies for the treatment of muscular diseases such as gene and oligonucleotide delivery, gene editing etc., or any other drug used for symptom alleviation of muscular diseases.

Another aspect of the invention refers to a pharmaceutical composition comprising boron or a boron compound for use in the treatment of muscular dystrophies.

In a preferred embodiment, in the pharmaceutical composition for use as described the boron compound is borax.

The boron or boron compound must be in a pharmaceutically acceptable form to be part of the composition of the invention. As used herein, "pharmaceutically acceptable" means suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/ risk ratio, and effective for their intended use within the scope of sound medical judgment.

For the purpose of the present invention, boron or boron compound may be in the form of solvates or salts.

As used herein, "solvate" means a complex formed by solvation (the combination of solvent molecules with molecules or ions of the active agent of the present invention), or an aggregate that consists of a solute ion or molecule (the active agent of the present invention) with one or more solvent molecules. In the present invention, the preferred solvate is hydrate. Examples of hydrate include, but are not limited to, hemihydrate, monohydrate, dehydrate, trihydrate, hexahydrate, etc. It should be understood by one of ordinary skill in the art that the pharmaceutically acceptable salt of the present compound may also exist in a solvate form.

The term "salts" include derivatives of an active agent, wherein the active agent is modified by making acid or base addition salts thereof. Preferably, the salts are pharmaceutically acceptable salts. Such salts include, but are not limited to, pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like.

The boron or boron compound may be administered with an excipient, carrier, and vehicle, commonly used in the pharmacology field. Vehicles that can be used, but not limited to, are biodegradable polymeric materials in their different compositions (mixture of co-polymers: PL LA, PLGA etc.) and forms (films, particles, nanoparticles, scaffolds etc), lipid particles (embracing different lipid compositions), hydrogels etc.

In another preferred embodiment, the muscular dystrophies are selected from myotonic dystrophy type 1 , myotonic dystrophy type 2 or Duchenne muscular dystrophy.

In another preferred embodiment, the pharmaceutical composition for use as described comprises another active principle, thus being a combination therapy. The term "combination therapy" refers to a first therapy that includes boron or a boron compound, and/or their derivatives, in conjunction with a second therapy (e.g., therapy, surgery, and/or an additional pharmaceutical agent or a biological agent such as an antibody or an antisense oligonucleotide) useful for treating, stabilizing, preventing, and/or delaying the disease or condition.

For these purposes, the compounds of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intracisternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.

These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions or suppositories.

When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1 ,3- butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectal ly administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the drug.

The compounds of this invention can be administered orally to humans in a dosage range of 1 to 100 mg/kg body weight in divided doses. One preferred dosage range is 0.01 to 10 mg/kg body weight orally in divided doses. Another preferred dosage range is 0.01 to 20 mg/kg body weight orally in divided doses. For combination therapy with nucleoside analogues, a preferred dosage range is 0.01 to 20 mg/kg body weight for the compounds of this invention administered orally in divided doses, and 50 mg to 5 g/kg body weight for nucleoside analogues administered orally in divided doses. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions or suppositories.

A further aspect of the present invention refers to a method of treatment of muscular dystrophies comprising administering a therapeutically effective amount of boron or a boron compound to a patient in need thereof.

"Therapeutically effective amount" means the amount of a compound or a therapeutically active agent that, when administered to a patient for treating a disease or other undesirable medical condition, is sufficient to have a beneficial effect with respect to that disease or condition. The therapeutically effective amount will vary depending on the type of the selected compound or a therapeutically active agent, the disease or condition and its severity, and the age, weight, etc. of the patient to be treated. Determining the therapeutically effective amount of a given compound or a therapeutically active agent is within the ordinary skill of the art and requires no more than routine experimentation.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Experiments in fly model. The figure shows measurements of mean muscle area of cross sectional sections from the indirect flies muscles (IFM) in control, DM1 and DM1 flies treated with borax at the indicated concentrations. DM1 flies treated with 0.59 mM borax presented a significant partial rescue of mean muscle area, meaning that muscle atrophy was alleviated. FIG. 2. Experiments in fly model. The figure shows results of flight assay in control, DM1 and DM1 flies treated with borax at the indicated concentrations. DM1 flies treated with 0.59 mM borax presented a rescue of flight ability, meaning that muscle function was significantly improved.

FIG. 3. Experiments in fly model. The figure shows survival rates of control, DM1 and DM1 flies treated with borax at the indicated concentrations. DM1 flies treated with borax 0.59 and 1.47 mM presented a significant increase in the survival rate of both, male and female flies, meaning that borax increases the longevity of DM1 subjects.

FIG. 4. Experiments in human immortalised DM1 cell line. The figure shows myogenic differentiation of human control and DM1 cells. Myotube formation was significantly enhanced after borax 0.59 mM and 1.47 mM addition, meaning that borax restores the loss of the DM1 cell capacity to differentiate from myoblast to myotube, as can be seen in the images (FIG 4.1 ), and the quantification values obtained (FIG. 4.2).

FIG. 5. Experiments in human immortalised DM1 cell line. The figure shows results from transcriptomic analysis obtained using immortalised wild type and DM1 cells. Borax treatment participates in differential gene expression in both wild type and DM1 cells (FIG. 5.1 ). The percentage of partial or total gene recovery is shown in figure 5.2.

FIG. 6. Experiments in an in vivo murine model of DM1 . The figure shows grip strength test performed in the healthy (FVB) and DM1 mice forelimb after implantation of PLLA/hydrogel material system empty (DM1 ) or loaded with 0.59 or 1.47 mM of borax (DM1 + Borax 0.59 mM or DM1 + Borax 1.47 mM). Borax treatment increased forelimb mouse force at levels higher than wild type. Indeed, figure shows results of electromyography performed in the implanted DM1 mice before (bt) and after the treatment (at). Results indicate that borax treatment decreased myotonia grade after the implantation of the PLLA/hydrogel material system loaded with borax. Overall, these results indicate that borax addition is acting in the recovery of DM1 symptoms such as myotonia and myopathy, meaning that muscle function was significantly improved.

FIG. 7. Experiments in an in vivo murine model of DM1. Figure shows muscle histology of wild type (FVB) and DM1 mice after the implanted PLLA/hydrogel material empty (DM1 ) and loaded with Borax 0.59 mM (DM1 + Borax 0.59 mM) or Borax 1.47 mM (DM1 + 1.47 mM). Representative images are transverse frozen sections of quadriceps muscle. Haematoxylin and eosin-stained muscle is normal in wild type (FVB) but shows increased variability in fibre size and central nuclei in DM1 muscles. After borax treatment, central muscle nuclei and cross sectional area of fibres decreased significantly while the ratio of muscle nuclei per muscle fibre remained similar to DM1. These results indicate that borax treatment is really acting in the muscle recovery, in concordance with the results obtained in DM1 human cells, restoring the loss of DM1 cell capacity to differentiate from myoblast to myotube at physiological and anatomical level and improving the muscle function.

FIG. 8. Figure 8.1 . shows myogenic differentiation of human control and DMD primary cells. Although myotube formation was not completely accomplished, the cells treated with borax presented a relevant increase in cell number and cell fusion as a preliminary step of complete myogenesis, meaning that borax strongly enhances cell migration and fusion. Figure 8.2. shows the same myogenic differentiation experiment but using an immortalised and well characterised human DMD cell line model. Figure 8.3. shows quantification of images represented in FIG. 8.2.

EXAMPLES

1 . Study of muscle atrophy in DM1 fly model

For all the experiments performed in flies we worked with four experimental groups: “DM1” that consists in a well characterized DM1 fly model previously described in Disease Models and Mechanisms. 2013; 6: 184-196 (Figures 1 , 2, and 3). Briefly, DM1 flies (w ¹¹¹⁸ ; Mhc-Gal4 UAS-(CTG)480/+), that express 480 CUG repeats under the myosin heavy chain promoter. “Control” flies that are flies with the same genetic background as DM1 but do not express the CTG expansions. And finally, two groups of DM1 flies treated with borax named“Borax 0.59 mM” and“Borax 1.47 mM”. Muscle atrophy was analysed in adult females. Thoraces of seven-day-old flies were embedded in Epon resin and semithin sections (1.5 pm) were obtained using an ultramicrotome. After that, tissue was stained with toluidine blue. Finally, images were taken at a 100X magnification. Muscle area was quantified by generating a binary map of a fix section containing the indirect flight muscles (IFMs). Then the percentage area within this section occupied by the tissue was quantified. A significant rescue was observed when food of DM1 flies was supplemented with borax at 0.59 mM (see“Borax 0.59 mM”, Fig.1 ). However, no significant effect was observed after addition of 1.47 mM of borax (see “Borax 1.47 mM” Fig. 1 ). The graphs show mean ± s.d. Six individuals per condition were analysed and six photographs from each individual were quantified. Values obtained for “Control”, and“ DM1 flies supplemented with Borax 0.59 mM” and“Borax 1.47 mM” were compared to untreated“DM1” flies ^* P<0.05, ^*** P<0.001.

The addition of borax partially restores muscle atrophy in DM1 flies, indicating that DM1 model flies treated can repair their muscle atrophy to physiologically healthy levels.

2. Study of flight assay in DM1 fly model

120 male flies (one-day old) were collected in tubes containing standard food or food supplemented with borax. Flies were aliquoted in groups of 30 flies per tube. T ubes were maintained at 25 °C for seven days, when the flight assay was performed. In the assay, flies were dropped through a funnel to the top of a 90 cm height cylinder. Inside the cylinder there was a plastic sheet covered with glue, so flies got immobilized when got in contact with it. After that, the plastic sheet was removed and a photograph was taken for its subsequent analysis with ImageJ software that includes the measurement of the landing height of each individual. Flyers that are more capable will respond to the drop quicker and will land higher up on the walls of the cylinder. Flies with poor response times or those unable to fly will be glued near or at the bottom of the cylinder. Addition of 0.59 mM of borax to the flies ^' food resulted in a significant increase in the flight capacity of flies analysed. However, addition of 1.47 mM of borax produced no significant results (see Fig. 2). The histogram shows mean ± s.e.m. ^*** P<0.001. P-values were obtained using a two-tailed, non-paired t-test (a = 0.05). All comparisons are referred to“DM1” condition.

The addition of borax produced a statistically significant recovery of the flight capacity of DM1 flies, indicating that borax is causing not only the recovery of the correct anatomical structure of the muscle tissue, but also the functionality of this tissue. This statement is of great importance since it indicates that borax treatment allows muscle regeneration at the anatomical level and also at a functional level with an infinity of possible applications in the area of biomedicine.

3. Survival assay To study the survival time of DM1 flies treated with the compounds, 25 flies were collected per tube containing the supplemented food and were maintained at 25° C until death. Four tubes were established per compound as the experiment was performed with 50 males and 50 females. The number of dead flies was scored daily. Flies were shifted to tubes containing fresh food (plus compound) thrice a week. Survival curves were obtained using the Kaplan-Meier method and were statistically compared according to the Gehan-Breslow-Wilcoxon test (a=0.05) and Mantel-Cox test ^* P<0.05, ^** P<0.01 , ^*** P<0.001 . 0.59 mM and 1.47 mM borax addition to the flies’ food resulted in extended survival of both males and females flies analysed (see Fig. 3).

Another characteristic symptom of DM1 in the fly model is its early death. It has been observed that the borax treatment increases the longevity in both DM1 males and females, indicating that the regeneration of the anatomy and muscular function promoted by borax also extends the life expectancy. This statement is of great importance, considering that patients with muscular dystrophies have compromised their life expectancy.

4. In vitro myogenic differentiation of human DM1 cells

Healthy and well characterised human DM1 (2600 CTG repeats) immortalized (hTERT) skin fibroblasts were transdifferentiated into myoblasts (Disease model described in: Disease models and mechanisms 2017; 10: 487-497). 20.000 cells/cm ² were seeded after coating the different substrates used with 20 pg/ml of fibronectin solution using basal medium (DMEM 4.5g Glucose, 10% FBS, 1 % P/S). We used glass as a control substrate and we also included PLLA spin coated substrates as a model material to test one possible delivery vehicle for borax. Cells were allowed to adhere for 24 h. After that, fibroblasts were induced to transdifferentiate into muscle cells by stimulating the expression of MyoD transcription factor from a carrying vector introduced in the cells using differentiation medium (DMEM 4.5g Glucose, 1 % Insulin-T ransferrin-Selenium, 1 % P/S, 0.02% Doxycycline). Myogenic differentiation measured in terms of myotube formation was significantly enhanced after borax 0.59 mM and 1 .47 mM addition. Indeed, the percentage of differentiated cells augmented monotonically as the concentration of borax does, conversely to results obtained with experiments with DM1 fly model. Images show myotube formation of human transdifferentiated DM1 fibroblasts after osarcomeric actinin muscle marker staining (see Fig. 4.1 , panel of images). Images from the fluorescence microscope (DAPI channel - nuclei, and Cy3 channel - sarcomeric myosin) were acquired at 10 x magnification (n = 10), transformed to an 8-bit grayscale bitmap (Fiji-lmageJ software) and segmented using the Trainable Weka Segmentation plugin to create a binary mask, for both DAPI and Cy3 channels. Total nuclei per image were counted using the particle analysis command. Then, the segmented DAPI channel image was subtracted from the Cy3 channel segmented image, and the remaining nuclei were counted and assigned to non-differentiated cells. The fraction of differentiated cells was calculated subtracting the non-differentiated nuclei from the total nuclei counted (see Fig. 4.2, graphs).

In vitro tests performed on human DM1 cells showed that borax exerts a positive effect on myogenic differentiation. These results confirm those obtained in the DM1 fly model. In contrast to the results obtained in fly in which the optimum concentration of borax was 0.59 mM, the results indicate that in human cells the concentration of borax that exerts greater effects is 1 .47 mM. From this, it follows that in the application of a possible therapy, the dose of borax will be dependent on the organism to be treated. After treating human DM1 cells with borax, they were able to reach levels of myogenic differentiation similar to those of healthy cells. This surprising effect suggests that treatment with borax could stimulate the capacity of muscle regeneration that is compromised in the cells of patients with DM1.

5. Transcriptomic study in human immortalised DM1 cell line

RNA sequencing was performed using the lllumina NextSeq 550 sequencer. Very interesting results were obtained in the data analysis using bioinformatics. The results provide information on the mechanism of action of boron and its therapeutic effect on DM1 disease. Model human immortalised DM1 fibroblasts transdifferentiated to myoblasts as described above were analysed in order to identify the therapeutic effects of the compound. Control human immortalised fibroblasts (WT) were also used with and without borax treatment, in order to identify nonspecific effects of the treatment. Through the analysis of the main components (FIG. 5.1 ) we can observe a significant differential gene expression in both comparisons, between DM1 cells and controls and between non-treated and treated cells, meaning that borax treatment generates a significant effect on both wild type and DM1 cells. The differential expression analysis identified 1333 genes that were differentially expressed (using a p value < 0.05 and a fold change > 1 .5) between the control cells and DM1 , concluding that 1333 genes are related to the DM1 disease. From these data, it was possible to calculate the recovery percentage of the genes involved in the disease (FIG. 5.2). Importantly, the treatment led to a partial or total recovery of 1 15 genes representing the 8.7% of the genes. Specifically, analysing the expression of the integrins, 27 were identified, and 19 of them showed their expression modified after borax treatment. A similar analysis of the ion channels was carried out and it was seen that of the 328 identified almost 50% modified their expression after the borax treatment.

The functional study of the genes altered and modified by the treatment with the Gene Onthology, Kegg, Reactome and Disease Onthology databases showed three routes that were of particular interest for the aetiology of the disease and also for our hypothesis about the mechanism of action of borax. Specifically, the term GO“Development of the skeletal system”, the Kegg route“Calcium Signalling” and genes identified in Reactome of “Organization of the extracellular matrix”. These routes are among the most significantly modified by the treatment in the model cells.

6. Effects of borax in an in vivo murine model of DM1. Force and myotonia measurements

For the experiments performed in the in vivo DM1 mice model we have used a transgenic mice well characterized and described in Science. 2000; 289: 1769-1772. Briefly, the mouse was genetically modified using a genomic fragment containing the human skeletal actin (HSA) gene to express an untranslated CUG repeat in the muscle of transgenic mice. An expanded (250) CTG repeats were inserted in the final exon of the HSA gene, midway between the termination codon and the polyadenylation site. This placement is similar to the relative position of the CTG repeat within the human DMPK gene, but the repeat tract is shorter than the highly expanded alleles (1 to 4000 CTG repeats) in DM skeletal muscle. Except for the repeat, the HSA constructs are devoid of sequences from the DM locus. Myotonia is present in HSALR mice as early as 4 weeks of age, when the muscles had a normal histologic appearance. These observations indicate that HSALR mice have a true myotonic disorder, rather than nonspecific hyperexcitability associated with muscle necrosis. These mice that express the long-repeat transgene develop histologically defined myopathy, lines expressing long repeats showed a consistent pattern of muscle histopathology, including increases in central nuclei and ring fibers and variability in fiber size. As a control mouse we have used an FVB/NJ background mouse, a widely used multipurpose inbred strain.

DM1 and FVB mice were implanted in the right quadriceps with empty PLLA/hydrogel material as a control or loaded with Borax 0.59 or 1 .47 mM. Four groups of animals were assayed: Group 1 -FVB healthy control, Group 2-DM1 implanted with empty material, Group 3-DM1 implanted with Borax 0.59 mM-loaded material and Group 4-DM1 implanted with Borax 1.47 mM-loaded material. Electromyography (EMG) was performed in all the mice before and 15 days after the surgery, under halothane anesthesia using 30-gauge concentric needle electrodes, with sampling of at least three proximal and three distal muscle groups in each forelimb.

Figure 6 shows results of electromyography, indicating that borax treatment decreased myotonia grade after the implantation of the P LA/hydrogel material system loaded with borax. Myotonia values represent a specific indicator of the muscle function. That is to say, the higher myotonia values represent the lower muscle relaxation periods after application of electric pulse. The fact that borax treatment is diminishing myotonia values indicate that borax is acting in the recovery of DM1 symptoms such as myotonia and myopathy, meaning that muscle function is significantly recovered, in concordance with the results obtained in the fly model.

15 days after the surgery and before euthanasia of the animals, we also performed a grip strength test. The peak grip force was measured using a grip strength meter (Bioseb - In Vivo Research Instruments), according to the manufacturer’s protocol. Mice were allowed to grasp a wire mesh with their hindlimbs, and then pulled steadily by their tails horizontally until they lost their grip. Measurements were performed 3 times using the same experimental groups described previously.

Figure 6 shows force measurements after grip strength test. Borax treatment increased forelimb mice force at levels higher than wild type. Again, these results suggest that muscle function is significantly recovered after borax treatment. It is interesting to note here that force measurements were obtained from mice hindlimbs and borax-loaded implants were located in the quadriceps (forelimbs) of animals, indicating that borax effects are acting systemically rather than locally in the point of the implant, concordant again with the results obtained in the fly model. 7. Effects of borax in an in vivo murine model of DM1. Muscle histology and analysis

After 15 days of surgery and euthanasia of animals, quadriceps muscles of the four experimental groups were snap-frozen in isopentane chilled with liquid nitrogen. After that, quadriceps muscles were sliced at 10 pm with a cryostat. Haematoxylin and eosin (H&E) staining was done according to the standard procedures.

Figure 7 is a representative of haematoxylin and eosin-stained muscle, normal in wild type (FVB) but showing increased variability in fibre size and central nuclei in DM1 muscles. After borax treatment, central muscle nuclei and cross sectional area of fibres decreased significantly while the ratio of muscle nuclei per muscle fibre remained similar to DM1 . These results showing the analysis of the muscle at anatomical level, indicate that borax treatment is really acting in the muscle recovery, in concordance with the results obtained in fly model and DM1 human cells. Borax treatment restored the lost DM1 capacity to normal myofibre formation at anatomical level, and thus recovers the normal muscle function.

8. In vitro myogenic differentiation of human DMD cells

Healthy and DMD primary cells from patient biopsies were used for this experiment. 20.000 cells/cm ² were seeded after coating the different substrates with 20 pg/ml of fibronectin solution using myogenic differentiation medium (DMEM 4.5g Glucose, 1 % Insulin-Transferrin-Selenium, 1 % P/S). Myogenic differentiation was analysed after 6 days of culture. For this kind of cell culture, using primary cells directly obtained from patients, we have to take into account that they are extremely delicate, normally their growth is slowed and their attachment is limited even in the control healthy cells. For its proper maintenance, growth and differentiation, the addition of a variety of growth factors and other essential supplements in the culture medium is required. We conducted this preliminary experiment in the most restrictive possible conditions, in order to eliminate the effects provided by the various growth factors and other supplements in the media, which could distort the expected effect exerted by borax. Results showed that myoblast alignment was significantly enhanced after borax 0.59 mM and 1 .47 mM addition in both control and DMD cells, being the optimal concentration 1.47 mM, results in line with those obtained with DM1 cells. Cells without borax treatment appeared isolated and without any other cell contact. Borax treated cells, presented clear cell alignment and contact, as a previous step of myotube formation. In this case we couldn ^' t analyse myotube formation due to the restrictive experimental conditions, as we have used a minimal culture medium (DMEM 4.5 g Glucose, 1 % Insulin-Transferrin-Selenium, 1 % P/S). Images show a-sarcomeric actinin muscle marker staining (see Fig. 5). Images from the fluorescence microscope (DAPI channel - nuclei, and Cy3 channel - sarcomeric myosin) were acquired at 20 x magnification (n = 10).

Interestingly the experiments performed with DMD cells showed that borax treatment accelerated cell migration and alignment, indicative of a previous step of myotube formation. This phenomenon occurs under restrictive cell culture conditions and is not present in healthy cells.

Cell cultures derived from muscle or skin biopsies from DMD patients are very difficult to find, because samples are obtained from paediatric patients with a rare disease, and becomes more complicated when a specific mutation of DMD is needed (even more rare). Furthermore, patient ^' s cell cultures are often difficult to expand and do not differentiate well as commented above. For this reason, we employed an immortalised DMD cell line 638A, well characterised, containing the deletion 52 of Dystrophin gene, from a Biobanc Center for Research in Myology (Paris-France).

Following a similar procedure as that described above, we analysed myoblast differentiation in this DMD cell line. The results showed that myotube formation was significantly enhanced after borax 0.59 mM and 1.47 mM addition in both control wild type and DMD cells, being the optimal concentration 1.47 mM, results in line with those obtained with DM1 cells and in the mouse in vivo model (Figure 8.2 and quantification of images Figure 8.3).

Even under the most restrictive conditions, the obtained results with primary cultures from patients corroborated with DMD immortalised cells, indicate that borax exerts a clear effect in myogenic differentiation, not only in myotonic dystrophy type 1 cells, but also in Duchenne ^' s muscular dystrophy. This surprising finding has incredible implications, since both types of dystrophy have a very different aetiological origin. For this reason, the scope of the present invention may be extended to other muscular dystrophies apart from DM and DMD.