Cell Therapy for Muscular Dystrophy

*Alok Sharma, Hemangi Sane, Nandini Gokulchandran, Amruta Paranjape, Zubiya Shaikh, Arjun KM and Prerna Badhe*

## **Abstract**

Muscular dystrophy is a major unmet medical need associated with an inevitable progressive muscle damage and loss of function. Currently, treatment is only symptomatic and supportive. This chapter focuses on cell therapy as a potential treatment approach for muscular dystrophy. Mechanism of action of cell therapy and its ability to alter disease pathology have been discussed. A review of preclinical and clinical studies has been presented with the advantages and shortcomings of various cell types. Rationale for our treatment protocol and experience of treating muscular dystrophy patients has been discussed. Our published results have shown the efficacy of the intrathecal and intramuscular administration of autologous bone marrow mononuclear cells in different types of muscular dystrophy patients. The scores on outcome measures such as 6-minute walk distance, North star ambulatory assessment, Brooke and Vignose scale, Functional independence measure, and manual muscle testing either improved or were maintained suggestive of slowing down disease progression. Efficacy and safety of the treatment was also studied using comparative MRI-MSK and EMG showing decreased fatty infiltration in various muscles post-cellular therapy. Thus, it was found that autologous BMMNC transplantation is a safe and effective treatment option and improves the quality of life of MD patients.

**Keywords:** muscular dystrophy, stem cells, cell therapy, autologous, bone marrow mononuclear cells

## **1. Introduction**

The term 'muscular dystrophy' first used by Erb (1891), is used for a heterogeneous group of disorders that are hereditary in nature and are characterized by primary involvement of muscles and a tendency for progressive muscle weakness and wasting [1]. They are inherited as X-linked, autosomal dominant, or recessive disease. Over 30 variants of muscular dystrophy (MD) have been identified using genetic or histochemical testing [2, 3]. Most commonly found types of MDs are Duchenne Muscular Dystrophy

(DMD), Becker Muscular Dystrophy (BMD), Limb Girdle Muscular Dystrophy (LGMD), Emery-Dreifuss Muscular Dystrophy (EDMD), Fascioscapulohumeral Muscular Dystrophy (FSHMD), Oculopharyngeal Muscular Dystrophy (OPMD), and Congenital Muscular Dystrophy (CMD). The overall worldwide prevalence of combined MDs is estimated to be 3.6 per 100,000 individuals [4].

Although the onset of symptoms and the rate at which disease progresses is variable and depends mainly on the gene mutation [5], MD is associated with an inevitable progressive muscle damage and loss of function. Loss of ambulation, contractures and deformities are common. Scoliosis is frequently seen in wheelchair-dependent patients. Although the disorder primarily affects skeletal muscles, structural and functional abnormalities are also known to be seen in cardiac muscle, smooth muscle, and the brain [6–8]. Impairment of respiratory function due to weakness of the respiratory muscles is frequent. Cardiac involvement is a feature commonly seen in DMD, BMD, myotonic dystrophy, LGMD, EDMD and CMD. Functional involvement of the brain has also been seen in CMD, myotonic dystrophy, DMD and in some LGMD variants. About one-third boys with DMD have co-morbid mental retardation and other behavioral or psychiatric comorbidities [9, 10].

Currently, treatment is mainly symptomatic and supportive. Though corticosteroids delay loss of function, it is ineffective in stopping progression in MDs. Steroids only reduce inflammation and long-term use is associated with side effects including stunted growth, cataracts, and osteoporosis [11]. MDs represent a major unmet medical need and are associated with progressive disability causing economic and personal burden. Many of the MDs result from mutations in the genes encoding components of the dystrophin-glycoprotein complex (DGC) [12] that links the extracellular matrix of muscle fiber with the F-actin cytoskeleton. The DGC plays an important role in providing mechanical support to the plasma membrane during muscle fiber contraction and is thought to protect muscle fibers from contraction induced damage [13, 14]. Its disruption leads to altered mechanical and signaling functions resulting in increased entry of calcium, immune cell infiltration, progressive muscle wasting, necrosis, and membrane fragility (**Figure 1**) [15, 16]. Gene therapy using viral and non-viral vectors can be a promising treatment option for MD but shows adverse immune responses to vectors raising concerns regarding safety of the treatment [17]. Antisense oligonucleotide (ASO) mediated exon skipping therapy is also a treatment option for MD which targets removal of introns from pre-mRNA to give functional proteins. Currently ASOs for mutations in dystrophin gene amenable to exons 51,53 and 45 are approved by Food and Drug Administration (FDA) which showed the resumption of dystrophin production in MD patients. But the expense of ASOs, its off-target effects and its delivery are current concerns regarding their use [18].

Although the initial cause of muscle damage is genetic in origin, there is now increasing evidence of stem cell dysfunction in MD [19–21], as a contributing factor to the progression of the disease. Blau et al. reported a defect in the proliferative capacity of resident stem cells in DMD [19]. Also, there is increased inflammatory responses in MD patients that disrupt muscle homeostasis and inhibit muscle repair and regeneration [22–24]. It is observed that in DMD and several other forms of MD, the regenerated muscles are prone to degeneration, producing repeated cycles of degeneration and regeneration. As a result, the resident stem cell population is either exhausted or loses the potential to mediate repair leading to progressive replacement of muscle tissue with adipose and fibrotic tissue [25]. Therefore, gene therapy alone

*Cell Therapy for Muscular Dystrophy DOI: http://dx.doi.org/10.5772/intechopen.108600*

**Figure 1.** *Sequelae of DGC disruption in MD.*

is inadequate as it cannot replenish the stem cell pool and even where applicable and available clinically, needs to be combined with treatment approaches that replenish the stem cell pool.

The objective of any effective treatment lies in restoring dystrophin expression in muscle fibers and in promoting regeneration of muscle fibers. While dystrophin restoration can be achieved by gene therapy or cell therapy or combination of the two, regeneration of muscle can be achieved through cell therapy only and it, therefore, represents an essential treatment approach for MDs.

## **2. Pathophysiology of MD**

As described earlier, the core pathophysiology of MD (**Figure 2**) is genetic mutations resulting in altered expression of proteins in DGC and stem cell dysfunction. DGC is the essential component of the cell wall; its alteration leads to increased cell damage even with minimal contractile stress. Muscle damage is repaired by resident muscle stem cells also known as satellite cells. Progressive damage of the muscle is repaired with continuous satellite cell mediated regeneration process which leads to depletion in the satellite cells pool. Due to which there is an increased muscle damage leading to adipose tissue infiltration causing muscle weakness as shown in **Figure 3** [26]. Increased damage also increases the immune cell infiltration resulting in increased inflammation [27]. Inflammation accelerates cell apoptosis and necrosis in the damaged areas [28]. Also, in MD the muscle function is affected because of impaired blood supply to the muscle leading to chronic ischemia. The DGC is also involved in the formation of synaptic connectivity, abnormal DGC leads to impaired neurotransmission and abnormally formed

**Figure 2.** *Pathophysiology of MD.*

*Cell Therapy for Muscular Dystrophy DOI: http://dx.doi.org/10.5772/intechopen.108600*

**Figure 3.**

*Normal muscle repair having adequate number of satellite cells versus dystrophic muscle repair demonstrating exhaustion of stem cells.*

neural junctions increasing muscle wasting [29, 30]. Hence, the pathophysiology of MD is Multifaceted having multiple contributing factors.

## **3. Role of stem cells in MD**

Stem cells have the ability of self-renewal and migration to the site of damage/ injury and carry out repair and restoration processes. They divide and differentiate to replace the damaged and dead cells [31]. In Vitro experiments have shown that stem cells restore dystrophin expression in duchenne-skeletal muscle cells [32]. Stem cells halt further damage by exerting paracrine mechanisms and stimulate endogenous cells to carry out repair processes. Stem cells secrete various cytokines and chemokines exerting anti-inflammatory, anti-apoptotic, angiogenic and immunemodulatory effects [33]. Effectiveness of cell therapy depends on various factors such as number of cells, route of delivery, myogenic potential, migration and homing capabilities of cells, type of MD and extent of muscle damage. Cell therapy can be a promising long-term solution for MD [34].

## **4. Understanding stem cells**

Stem cells are unique undifferentiated cells characterized by their ability for selfrenewal and for differentiation into specialized cell lineages [35].

## **4.1 Classification of stem cells**

## *4.1.1 Based on potency*

Stem cells can be classified according to their ability to differentiate as (**Figure 4**):


**Figure 4.** *Classification of stem cells.*


Depending on the source, they can be differentiated (**Figure 4**) into


Human ESCs are faced with ethical considerations and are also associated with a risk of tumorigenicity whereas umbilical cord stem cells and adult stem cells do not possess ethical concerns.

Cells procured from the patients themselves are autologous cells. Stem cells acquired from donors are allogenic cells. Autologous cells are safer than allogenic cell as they do not show immune rejection while proper HLA typing of donor and recipient is required in case of allogenic cells.

## **5. Cell therapy as a therapeutic strategy for MD**

## **5.1 Preclinical evidence of effectiveness of cell therapy in MD**

### *5.1.1 Induced pluripotent stem cells*

Therapeutic use of ESCs is restricted due to immune rejection and ethical concerns. These concerns have however been overcome partly by Yamanaka et al. by showing that iPSCs can be generated from somatic cells [37]. Pre-clinical studies have shown the ability of myoblasts and mesenchymal stem cells (MSCs) derived from iPSCs to fuse with mature muscle fibers [38, 39] and improve muscle function in dystrophic mice [40].

#### *5.1.2 Stem cells of muscle tissues*

Another type of cell that is Myoblast cells, naturally residing in the muscles, are the primary skeletal muscle stem cells responsible for maintenance of resident stem cell pool by self-renewal and repair of adult skeletal muscle and were the source of stem cells in the earliest cell-based therapies for treating MDs [41]. Pre-clinical studies demonstrated that myoblasts can be expanded in vitro [42, 43] and are able to regenerate muscle [44–46]. Stem cells other than myoblasts that are present within

muscle and possess myogenic potential include muscle derived stem cells [47–49], mesoangioblasts [50–52], muscle derived stem cells, pericytes, CD 133+ stem cells and muscle side population cells that can contribute to muscle regeneration [53]. Transplantation of allogenic muscle derived stem cells contributed to muscle repair, dystrophin expression, satellite cell replenishment and clinical efficacy in MD dogs [54]. Recently, mesoangioblasts, pericytes and CD 133+ have demonstrated promise as stem cell source in treatment of MDs due to their ability to contribute to muscle regeneration and because they can be delivered systemically. Also, mesoangioblasts can reconstitute the satellite cell pool [55]. The ability of mesoangioblasts to contribute to muscle regeneration and to restore muscle structure and function has been tested in the mouse model of LGMD [56, 57] and in the dog model of DMD [58]. Tedesco et al. demonstrated expression of normal dystrophin in muscle fibers and production of functional muscle fibers in mice model of DMD following intramuscular injection of genetically corrected mesoangioblasts [55]. However, the disadvantage of these cell types is their limited ability to colonize in the muscles due to incomplete adhesion and extravasation of these cells [52, 56]. Pericytes are known to be developmentally derived from mesoangioblasts [50, 59], possess myogenic potential and have shown to promote muscle regeneration in dystrophic mice following intra-arterial delivery [59, 60]. Though promising, further research of the role of mesoangioblasts and pericytes in muscle regeneration are required. Torrente et al. demonstrated that CD 133+ cells contributed to muscle regeneration in dystrophic mice [61]. These cells can migrate through blood vessel walls [62]. Intramuscular and intra-arterial application of genetically corrected CD 133+ cells resulted in significant improvement in muscle function and dystrophin expression in dystrophic mice [63]. Laumonier et al. showed that Pax7+/MyoD− muscle reserve stem cells of human origin in immunodeficient mice intramuscularly promoted lacerated muscle regeneration [64].

## *5.1.3 Bone marrow derived stem cells*

Furthermore, Hematopoietic stem cells (HSCs) and MSCs are the two main cell types that can be isolated from the adult bone marrow. MSCs are multipotent stem cells and possess the ability to differentiate into myoblasts [65]. Additionally, MSCs can induce an anti-inflammatory effect and anti-apoptosis through paracrine function. These cells can be easily isolated from the bone marrow, are relatively safe and have minimal tumorigenicity. Ferrari et al. demonstrated that marrow derived stem cells can migrate to areas of degeneration and participate in muscle regeneration [66]. Intravenous delivery of bone marrow stem cells in the mouse model of DMD, donor derived nuclei were incorporated into muscle with partial restoration of dystrophin expression [67]. Maeda et al. showed intraperitoneal transplantation of bone marrow derived MSCs in DKO (dystrophin-utrophin double-knockout) mice increased satellite cells in mice, improved their locomotion, reduced kyphosis, and increased their longevity [68].

## *5.1.4 Wharton jelly derived stem cells*

Park et al. demonstrated that intravenous transplantation of human Wharton jelly derived MSC regenerated muscles, reduced apoptosis, and fibrosis in mdx mice model [69].

#### **5.2 Clinical evidence**

Only myoblasts, bone marrow derived stem cells and to a lesser extent umbilical cord-MSCs, muscle derived CD 133+ and cardiosphere derived stem cells (CDCs) have been investigated in humans.

Though safety and dystrophin production were seen in a case of DMD following myoblast transplantation [70], dystrophin production was seen in some but not all the later studies [71–80]. Although clinical benefit was observed in many studies, clinical use of these cells is hindered by several disadvantages. They cannot be delivered to all the muscles via systemic route, expansion in culture reduces their regenerative capacity [81], allogenic transplantation requires immunosuppression, and these cells have poor dispersion after intramuscular injection [82]. Also, these cells fail to participate in long term regeneration [83] and rapidly die in the first 72 hours after transplantation [84]. These problems have been tried to be resolved [85], but interest in use of these cells in treatment of MD has waned.

There is an ongoing Phase I/II clinical trial using systemic transfer of allogenic muscle derived mesoangioblasts from HLA identical donors in DMD [86]. Autologous transplantation of muscle derived CD 133+ cells in 8 boys with DMD was found to be safe and demonstrated an increased capillary per muscle fiber ratio with a switch from slow to fast myosin-positive myofibers [87]. 82 patients with progressive MD received double transplantation of autologous bone marrow derived MSCs and umbilical cord MSCs [88]. Treatment efficacy was evident in 68 of the 82 patients with no adverse event during the treatment course. Intravenous transplantation of allogenic cord blood cells in a patient with DMD resulted in improved function in standing, walking, and turning over with a mild graft versus host disease (GVHD) [89]. In another study, umbilical cord derived MSCs intravenously and intramuscularly in 11 DMD patients caused stabilization of muscle power as compared with control group and demonstrated safety without inducing GVHD [90]. A total of 15 studies demonstrated the benefits of intrathecal and intramuscular autologous bone marrow mononuclear cell transplantation in MD [91–105]. Dai et al. demonstrated four times administration of allogenic Wharton jelly-derived MSCs via intra-arterial and intramuscular routes in 9 DMD patients resulted in improved pulmonary function and increased dystrophin level with no adverse effects [106]. Another study revealed that intramuscular transplantation of fetal progenitor cells in 22 DMD patients improved their muscle activity, gait quality and reduced pseudohypertrophy [107]. The safety and efficacy of intravenous administration of human allogenic CDCs was studied by Mcdonald et al. in 26 late stage-DMD patients in a multicentre, randomized, double-blind, placebo-controlled, phase 2 clinical trial. The patients showed improved cardiac function and structure with maintained upper limb function with no major adverse effects [108].

## **6. Our experience of autologous bone marrow mononuclear cell therapy in MD treatment**

#### **6.1 Our treatment protocol**

After careful review of available literature and evidence, we have a protocol that we follow for stem cell transplantation at NeuroGen Brain and Spine Institute.

## *6.1.1 Patient selection*

The patient's selection is based on the World Medical Association's Helsinki declaration of Ethical Principles for medical research involving human subjects. The protocol was reviewed by the Institutional Ethics Committee (IEC) which is registered with the Central Drugs Standard Control Organization (CDSCO).

## *6.1.2 Pre-intervention evaluation*

Before cell therapy intervention, the patients undergo a comprehensive evaluation consisting of neurological examination as well as evaluation on various outcome measures such as 6-minute walk distance, North star ambulatory assessment, Brooke and Vignose scale, Functional independence measure and manual muscle testing (MMT). Motor points of the patients are also identified and plotted for the weak muscles by experienced physiotherapists in which BMMNCs are to be injected.

## *6.1.3 Transplantation of BMMNCs*

Transplantation of autologous bone marrow mononuclear cells intrathecally and intramuscularly is done in 3 steps (**Figure 5**). The protocol includes 300mcg granulocyte colony-stimulating factor (GCSF) administration subcutaneously 48 hours and 24 hours before cell therapy to enhance mobilization of cells [109]. 80–120 ml of bone marrow is aspirated from the anterior superior iliac spine. Mononuclear cells are then separated using the density gradient method. The separated cells are checked for viability under microscope using trypan blue and CD34+ cells are identified using fluorescence-activated cell sorting (FACS) analysis. Separated cells are then transplanted intrathecally and intramuscularly. The cells are diluted in the patient's own cerebrospinal fluid and divided into two parts. One part is transplanted intrathecally by lumbar puncture at the level between 4th and 5th lumbar vertebrae and the other part is further divided and injected intramuscularly at the motor point of muscles that are weak and of functional importance. Motor point is the point at which the innervating nerve enters a muscle, and it has the highest density of myoneural junctions. The

#### **Figure 5.**

*3 step cell transplantation 1. Bone marrow aspiration; 2. Stem cell separation; 3. Intrathecal and intramuscular injection of cell.*

identification of motor points is made by using electrical stimulation. A motor point can be identified as a superficial point overlying a muscle that exhibits a contraction at lowest level of stimulation (faradic stimulation with pulse duration of 1 millisecond).

#### *6.1.4 Neurorehabilitation*

The transplantation is followed with an individualized rehabilitation program incorporating physiotherapy, occupational therapy, speech therapy, aquatic therapy, psychological counseling, and dietary and nutritional advice. Patients are closely monitored for post procedure adverse events during their hospital stay. They are advised to continue the rehabilitation program at home preferably under professional supervision after discharge.

#### *6.1.5 GCSF administration*

After cell therapy, patients are also given one GCSF injection per month for the next 6 month after cell therapy that mobilizes the stem cells and improves muscle strength in MD patients [110, 111].

#### *6.1.6 Follow up and adverse event monitoring*

Patients are monitored for short term adverse reactions during their 4 days hospital stay. Patients are also advised to have regular follow-up at 3 months and 6 months, followed by yearly follow-up. During each follow-up, the patients undergo complete neurological assessment and are monitored for any long-term adverse effects.

#### **6.2 Rationale for the protocol**

#### *6.2.1 Autologous bone marrow mononuclear cells*

Although autologous bone marrow mononuclear cells carry the genetic abnormality in patients of MD, they have shown the potential to alter disease pathology and thereby disease progression. Also, autologous cell transplantation is not faced with the risk of immune rejection and therefore does not need immunosuppression. Bone marrow derived cells are easy to isolate, are excluded from ethical concerns, can be easily accessed, and transplanted [112], sufficient number of cells can be obtained by minimally invasive procedure and are marked by no immunogenicity and tumorigenicity [113, 114]. Pre-clinical studies have shown these cells to possess neurogenic [115] and myogenic potential [68, 116]. Also, they can migrate to the site of muscle degeneration; repopulate the satellite cell pool facilitating muscle regeneration [68, 116] and can survive in the injected muscles for long periods of time [83] promoting long term regeneration. These cells constitute a combination of cells including MSCs, hematopoietic cells, monocytes. Macrophages, stromal cells, very-small embryonic like stem cells, progenitor cells, hemangioblasts, endothelial progenitor cells and tissuecommitted stem cells [117]. These cells are known to exert therapeutic benefits mainly through paracrine effects (**Figure 6**). They secrete a broad spectrum of cytokines and growth factors that exert anti-inflammation and immunosuppression, inhibition of apoptosis, homing of endogenous satellite cells, angiogenesis, and regulation of metabolic pathways [118, 119]. In MD, muscle fiber degeneration is followed by an invasion of inflammatory cells such as macrophages and T-lymphocytes [120]. The latter play

#### **Figure 6.**

*Postulated mechanism of action of bone marrow mononuclear cells in MD.*

a role in fibrosis which further hinders the ability of muscle fibers to regenerate. The anti-inflammatory effect of MSCs may provide protection from damage caused by T-lymphocytes [121]. Also, membrane derived vesicles, called as exosomes, arising from these cells may promote transcript transfer from the stem cells to the injured cells, causing injured cells to re-enter cell cycle further facilitating muscle repair [122, 123]. Though the ideal cell types for transplantation in MD continues to remain elusive, autologous bone marrow mononuclear cells are an attractive interim treatment solution with a potential to slow disease progression (**Figures 7** and **8**).

## *6.2.2 Intrathecal and intramuscular delivery of cells*

Occurrence of co-morbid intellectual disability and cognitive impairment in patients with DMD and BMD is suggestive of nervous system involvement in MDs [9, 10]. Dastur and Razzak found an overlap of pathological changes in muscle biopsy specimens of

#### **Figure 7.**

*Kaplan Meier graph showing comparison of the estimated time till loss of ability to lift the hand to mouth or deteriorate to the score of 5 on Brooke scale in intervention and control group.*

#### **Figure 8.**

*Kaplan Meier graph showing comparison of the estimated time taken till loss of ambulation in intervention and control group.*

patients with MD and patients with anterior horn cell disorders [124]. Histological study of the muscle biopsy specimens revealed small, atrophied muscle fibers in one fourth of the MD patients, suggesting a possible denervation and involvement of the neural systems in MD. These findings support intrathecal administration of stem cells and in our experience; it facilitates nerve repair and tightening of neuromuscular junction. Although intra-arterial and intravenous administration of stem cells is feasible because most of the skeletal muscles in the body are affected, only a small fraction of cells reaches the muscle tissue due to significant filtration of cells into the lungs, kidneys, and spleen [125, 126]. Moreover, the DGC is abundant at the neuromuscular junction and plays a role in neuromuscular homeostasis. Defects in DGC as in MD impair neuromuscular transmission and cause motor end plate abnormalities. Injecting the cells directly at motor points ensures repair of the nerve, muscle and myoneural synapse [30].

#### *6.2.3 Combination of rehabilitation with cell therapy*

Studies have investigated rehabilitation as a method to optimize cell therapy. Treadmill running following systemic transplantation of bone marrow derived MSCs in mouse models enhanced the contribution of donor cells in muscle fiber regeneration [127]. Endurance exercise results in an increase in blood levels of cytokines and an increase in bone marrow derived progenitor cells in humans. In response to exercise, there is an increase in secretion of vascular endothelial growth factor (VEGF) which is known to stimulate angiogenesis and satellite cell proliferation [128] and migration [129]. Intramuscular injection cannot be given in all the muscles and can only be given in selective muscles based on strength and accessibility. Widespread distribution and recruitment of circulatory stem cells is important which may be achieved through exercise. Long term, low-intensity, and low-load weight-bearing exercise programs may cause a shift in type II fibers to type I muscle fibers which are less susceptible to degeneration in MD [130]. Exercise increases expression of utrophin which is a homolog of dystrophin and can increase dystrophic muscle function [131]. Exercise also helps improve respiratory and cardiac function which is frequently affected in MD.

#### *6.2.4 GCSF injection after cell therapy*

A preclinical study showed higher numbers of normal muscle fibers and reduced inflammation in mdx mice treated with GCSF than the untreated mice [132]. A study in mdx mice model also demonstrated that GCSF has positive effect on the satellite cell proliferation and helps in muscle fiber regeneration [133]. It was also observed that periodic GCSF administration induces mobilization of stem cells including cells having proangiogenic potential such as endothelial progenitor cells and monocytes in MD patients [110]. The mobilized monocytes are recruited at the site of ischemia which in turn stimulate angiogenesis via paracrine mechanisms [134]. A prospective, non-randomized clinical trial demonstrated that repeated GCSF injections were safe and resulted in increased muscle strength and ambulation in 19 MD patients of age ranging from 5 to 15 [111].

#### *6.2.5 Musculoskeletal magnetic resonance imaging*

Skeletal muscle histopathology is a widely used tool in monitoring disease progression in MD cases; however, it is invasive, painful, gives limited information and might not be representative of the entire muscle [135]. In contrast, musculoskeletal MRI is a noninvasive technique and free of ionizing radiation. MRI-MSK provides information about all aspects of muscle structure and function and gives high resolution images of soft tissues and muscle fatty infiltrations [136]. MRI-MSK is sensitive to disease progression in MD and comparative MRI can serve as a biomarker for disease progression and to assess treatment efficacy [91–105, 137].

The efficacy of autologous bone marrow mononuclear cells in muscular dystrophy patients was studied and MRI-MSK was used as an outcome measure. After cell therapy patients showed increased muscle fibers in vastus medialis and lateralis (**Figure 9**), semitendinosus (**Figure 10**), tibialis (**Figure 11**), gastrocnemius (**Figures 12** and **13**), peroneus longus and brevis (**Figures 14** and **15**), triceps (**Figure 16**) and soleus (**Figure 17**) muscles.

#### **6.3 Published results**

A total of 15 studies (3 Cohort studies and 12 case reports) have been published that demonstrated the efficacy of autologous bone marrow mononuclear cell transplantation followed by rehabilitation in MD [91–105].

#### **Figure 9.**

*(a) MRI scan of vastus medialis and lateralis muscles (arrows) pre-cell therapy. (b) MRI scan of vastus medialis and lateralis muscles (arrows) post- cell therapy showing muscle regeneration.*

#### **Figure 10.**

*(a) MRI scan of semitendinosus muscles (arrow) pre-cell therapy. (b) MRI scan of semitendinosus muscles (arrow) post-cell therapy showing muscle regeneration.*

#### **Figure 11.**

*(a) MRI scan of tibialis anterior (arrow) muscles pre-cell therapy. (b) MRI scan of tibialis anterior (arrow) muscles post-cell therapy showing muscle regeneration.*

#### **Figure 12.**

*(a) MRI scan of medial and lateral head of gastrocnemius muscle (arrow) muscles pre-cell therapy. (b) MRI scan of medial and lateral head of gastrocnemius muscle (arrow) muscles post-cell therapy showing muscle regeneration.*

### *6.3.1 Results of published cohort studies*

In 2012, a clinical study demonstrating the effect of bone marrow mononuclear cell transplantation in neurological and neuromuscular disorders in the pediatric population was published [97]. At a mean follow up of 15 ± 1 months post transplantation, 37 of the total38 patients with MD showed improvement in muscle strength (**Figure 18**). 73% showed improved trunk strength, 42% patients showed improvement in upper limb function and 71% patients showed improvement in lower limb function (**Figure 19**). Comparative musculoskeletal magnetic resonance imaging (MRI-MSK) done post intervention in two of the patients revealed decrease in fatty

#### **Figure 13.**

*(A) MRI scan of lateral head of gastrocnemius (arrow) pre-cell therapy. (B) MRI scan of lateral head of gastrocnemius (arrow) post-cell therapy showing muscle regeneration.*

#### **Figure 14.**

*(A) MRI scan showing peroneus longus and brevis (arrow) pre-cell therapy. (B) MRI scan showing peroneus longus and brevis (arrow) post cell therapy demonstrating muscle regeneration.*

#### **Figure 15.**

*Upper row. T1 weighted axial MRI images of peroneus longus and brevis, before cell therapy; lower row: T1 weighted axial MRI images of peroneus longus and brevis, 6 months post cell therapy showing increased isointense areas suggesting muscle regeneration.*

#### **Figure 16.**

*(A) MRI scan of long, medial, and lateral head of triceps (arrow) pre-cell therapy. (B) MRI scan of long, medial, and lateral head of triceps (arrow) post-cell therapy showing muscle regeneration.*

#### **Figure 17.**

*Upper row: T1 weighted axial MRI images of gastrocnemius and soleus, before cell therapy. Lower row: T1 weighted axial MRI images gastrocnemius and soleus, 6 months after cell therapy showing increased isointense areas suggesting muscle regeneration.*

#### **Figure 18.**

*Graph showing improvement in patients with MD post cell therapy.*

#### **Figure 19.**

*Graph representing symptom-wise improvements in MD patients post cell therapy.*

#### **Figure 20.**

*Graph showing symptom wise improvements in muscular dystrophy patients after stem cell therapy. Number of patients showing improvements with respect to trunk strength, upper limb (UL) strength, lower limb (LL) strength, gait, and standing are shown.*

infiltration with minimal muscle regeneration seen mostly in the muscles that had received cells (**Figures 9**–**12**). Improved muscle electrical activity was noted in 3 patients on comparative EMG done post intervention.

In 2013 an open label study that included 150 patients with MD was published [91]. On a mean follow up of 12 ± 1 months, 86.67% cases showed strength improvement with 53% patients showed improvement in trunk strength and 60% patients improving in lower limb strength (**Figure 20**). Improvements were seen on EMG in 9 cases and 6 cases showed improvement on MRI-MSK (**Figures 13**, **14** and **16**).

A longitudinal study of 65 LGMD patients was published in 2015 [98]. Depending on the number of transplants, the patients were divided into 3 groups. Group 1 included patients that underwent single transplantation, group 2 included patients that underwent 2 transplantations and group 3 included patients that

#### *Cell Therapy for Muscular Dystrophy DOI: http://dx.doi.org/10.5772/intechopen.108600*

underwent 3 transplantations. 97% of patients displayed improved function on FIM scale in group 1. Statistically significant strength improvements were noted 6 months post transplantation. In group 2, 96% of the patients displayed improved function on FIM scale. Statistically significant strength improvements were noted 6 months post intervention. In group 3, of the 4 patients, 1 patient deteriorated in FIM score, 2 patients improved and in 1 patient, the FIM score was maintained. Most patients had maintained muscle strength. The patient who showed deterioration in FIM score also showed deterioration in muscle strength. This patient had come for the cell therapy at an advanced stage of the disease, where the muscle strength was minimal, and he was completely dependent for all his activities of daily living.

#### *6.3.2 Results of published case reports*

Though at a variable rate, progressive skeletal muscle weakness is consistent with all the MD variants. The improvements after cellular therapy was measured on various outcome measures which are as follows-

#### a.6-minute walk test

Improvement was seen in the case of BMD in the 6-minute walk test. Maintenance and even improvement of function on the 6-minute walk test, over a follow up period of 1 year, was reported in two individual cases of DMD post intervention [94, 101]. Improvement also observed in a case of LGMD patient (94a). Considering the progressive nature of the disease, the natural decline shown by MD ambulant patients is 22.7 meters in the first year and 64.7 meters in the second year [138] which is slowed down because of cellular therapy.

## b.North star ambulatory Assessment (NSAA)

Improvement was observed in DMD and BMD patients in North star ambulatory Assessment scale [92, 101, 102]. In a published case report, a DMD patient of 10 years of age showed a maintained score on NSAA after 13 months of follow up [93] which shows positive effects of cellular therapy as there is continuous decrease in the scores of NSAA in DMD patients after 7 years of age [138].

## c.Brooke and Vignos Scales

The improvements are also observed in Brooke and Vignos scales that measure the strength of upper and lower extremity respectively. The improvement in ambulation and gait contributed toward a positive shift on Brooke and Vignos Scales in MD patients [91, 97]. Improved scores also observed in a case of DMD [103]. The scores are maintained on two cases of BMD which shows the efficacy of SCT considering the progressive nature of the disease.

## d.Functional Independence Measure

The improved functional independence measure (FIM) score is observed after cellular therapy in most of the BMD, DMD and LGMD patients and is evident by improved quality of life in those patients [91, 93, 97–99, 102, 103]. In two BMD and one LGMD patient the score was maintained demonstrating the halted disease progression [92, 95, 100].

e.Manual muscle Testing (MMT)

The efficacy of cellular therapy was also assessed by MMT score. There are improvements observed in MMT grading which is attributed to improved muscle strength in many DMD, BMD and LGMD patients [91, 95, 96, 98–104]. In a DMD patient, though the grading did not change but the control and quality of movement had improved, grip strength and pinch strength also showed minimal changes on both sides [100]. This indicates alteration in the disease progression, as the natural course of the disease shows reduction of muscular strength by 0.3 MMT units/year and 3.9% reduction in muscle strength every year [103].

#### f. MRI-MSK

Comparative MRI-MSK was done to study the efficacy of cellular therapy. The comparative MRI-MSK findings showed no increase in the fatty infiltration in BMD, DMD and LGMD patients [96, 99, 101–103, 105]. BMD is associated with progressive increase in fatty infiltration of muscle tissue and the comparison of MRI scans of children with DMD also suggests a 5% increase of fatty infiltration every year [139]. Thus, no significant increase in the fatty infiltration shows the effectiveness of cellular therapy in halting the disease progression. Decrease in fatty infiltration was reported in a case of BMD in bilateral peroneus longus and brevis muscle fibers (**Figures 14** and **15**) 6 months post intervention [100].

g.Electromyography (EMG)

Comparative EMG-NCV (EMG-nerve conduction velocity) showed no increase in the dystrophic changes of the muscles, suggesting maintained muscle integrity suggesting altered disease progression [99]. In a DMD patient, EMG studies showed improvement in the recruitment of the vastus medialis muscle, which is a key muscle in patellar stability and knee stability while walking which was functionally reflected as the ability to stand and walk independently, maintained over 3 years [103]. A decrease in extramyocellular lipid (EMCL) resonance peak was seen in a case of LGMD [96]. The EMCL quantifies fat content in a diseased muscle. Improvement in electrical activity of muscle on EMG also observed in 9 patients post cellular therapy [91].

#### *6.3.3 Adverse events*

All these preliminary studies demonstrated safety of cell therapy using intrathecal and intramuscular transplantation of autologous bone marrow mononuclear cells. These studies encountered no procedure related major complications. 3 studies reported minor adverse events which included headache, nausea, vomiting, backache, and pain at injection site [91, 97, 98]. These were self-limiting and resolved within a week.

## **7. Unpublished results**

#### **7.1 DMD**

A total of 296 patients with DMD underwent autologous transplantation of bone marrow mononuclear cells by intrathecal and intramuscular routes followed

#### **Figure 21.**

*Graph showing percentage analysis of symptoms in DMD patients post cell therapy.*

by rehabilitation. There were no major side effects. 5.4% patients experienced minor procedural adverse events which included spinal headache, fever, nausea and vomiting, pain at aspiration site, backache, neck pain, pain in lower limbs and loose motions. These were self-limiting and resolved within a week with medications. 55 patients that only received standard treatment for DMD formed the control group.

On a follow-up period ranging from 6 months to 78 months (median 18 months), 76% patients showed symptomatic and functional maintenance and/or improvements (**Figure 21**). The natural course of DMD being progressive, symptomatic, or functional maintenance or improvement were considered together.

#### *7.1.1 Effect of cell therapy on muscle strength*

Difference between muscle strength on MMT in individual muscle groups at the first and last assessment at mean follow up duration of 12.2 ± 8.6 months was analyzed using Wilcoxon signed rank test. Except for hip extensors, knee extensors and shoulder flexors, all muscle groups had maintained muscle strength. Also, there was a statistically significant increase in muscle strength post intervention in the wrist flexors. Overall muscle strength (%MRC) was calculated for each patient. Difference between %MRC at the first and last assessment was analyzed using the Wilcoxon signed rank test (**Table 1**). We found a statistically insignificant decline in %MRC of 1.04% in patients aged 5–13 years, over mean follow up of 12.2 ± 8.6 months. This was lower than the annual decline of 3.9% in natural controls [140]. There was a statistically insignificant increase of 0.92% in %MRC in patients aged 14 years or more. A 2% annual decline in %MRC in this age group has been reported in previous literature [141].

#### *7.1.2 Comparison of functional decline between treatment and control group*

On comparing the predicted age at which patients could not lift their hand to the mouth and lost their ability to self-feed (Brooke score declining to 5) using Kaplan-Meier analysis, patients that received cell therapy reached score 5 at age 21 years while patients that did not receive cell therapy reached the score at age 18 years (**Figure 7**


#### **Table 1.**

*Results of analysis of overall manual strength (%MRC) before and after intervention in the age group 5–13 years and > 14 years at a mean follow up of 12 months (p < 0.05 was considered statistically significant).*


#### **Table 2.**

*Results of Kaplan Meier analysis in intervention and control group (\* indicates statistically significant difference between the groups).*

and **Table 2**). This difference of 36 months was clinically meaningful however not statistically significant (p = 0.150).

Comparing the predicted age to loss of ambulation using Kaplan-Meier analysis, the predicted time to loss of ambulation was 130 months in the cell therapy group and 117 months in the control group (**Figure 8** and **Table 2**). There was a statistically significant (p < 0.05) increase in time to loss of ambulation by 13 months in the cell therapy group.

#### **Figure 22.**

*Kaplan Meier graph showing comparison of estimated survival duration of the intervention and control group.*

On comparing the survival duration using Kaplan-Meier analysis, the estimated survival duration of patients that received cell therapy was 297 months, while that of the control group was 260 months (**Figure 22**). Though statistically insignificant (p = 0.173), there was an increase in estimated survival duration by 37 months in the cell therapy group.

## **8. Summary of effects of SCT in MD**

MD is considered as stem cell disease as symptoms are visible only after depletion of the stem cell pool. Therefore, replenishment of the stem cell pool is necessary to ameliorate the symptoms. This can be achieved by stem cell therapy which is found to be a safe and effective treatment strategy for MD. SCT delays the progression of the disease, improves functionality and quality of life of MD patients. SCT in combination with rehabilitation and GCSF administration gives better results. The effects of SCT may be further enhanced by other integrative therapies such as hyperbaric oxygen therapy (HBOT), ozone therapy and deep tissue mobilization (DTM). These therapies may help to fasten the process of regeneration and repair because of their therapeutic effects.

## **9. Integrative therapies in MD**

### **9.1 Hyperbaric oxygen therapy (HBOT)**

The pathophysiology of MD patients includes vascular dysfunction, altered angiogenesis, hypoxia, inflammation, and ischemia [142]. Studies have shown that HBOT enhances healing of wounds, ischemia, and inflammations [143]. It also reduces hypoxia and increases angiogenesis by inducing secretion of VEGF (Vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) [144]. Thereby, HBOT is a promising therapy to ameliorate the condition of MD individuals. A study revealed that HBOT promotes muscle regeneration and satellite cell proliferation in mouse skeletal muscles injury models [145]. Thom et al. demonstrated that exposure to HBOT rapidly mobilizes stem progenitor cells in humans and mice [146]. Thus, HBOT in combination with stem cell therapy can show better therapeutic effects. HBOT stimulates neurogenesis and synaptogenesis, thereby improving motor functions and cognitive functions [147]. Leitman et al. demonstrated that HBOT also improves myocardial functions which are profoundly affected in MD patients [148].

#### **9.2 Ozone therapy**

Ozone therapy enhances tissue oxygenation and improves cellular metabolism. It also reduces oxidative stress and inflammation [149]. Preclinical study has shown that dystrophin deficient mdx mice have increased reactive oxygen species (ROS) levels in their heart which play a critical role in the development of dilated cardiomyopathy and inflammation of heart [150]. Also, Myofiber necrosis in MD patients is closely associated with increased inflammation and ROS levels [151]. The anti-inflammatory and antioxidant properties of ozone therapy may help in limiting disease progression by reducing oxidative stress and inflammation in MD patients. Ozone therapy also helps in mobilization of stem cells and homing of stem cells toward injured/ischemic sites, thus, aid in tissue repair [152]. This evidence suggests that stem cell therapy in combination with ozone therapy may show better therapeutic efficacy in MD patients.

#### **9.3 Deep tissue mobilization (DTM)**

Soft tissue mobilization helps in removing scars and soft tissue regeneration after injury [153]. Massage therapy reduces inflammation and promotes angiogenesis in the injured tissues after muscle damage [154]. In 20 DMD patients, calf massage showed increase in calf and hamstring muscle length and reduction in calf muscle stiffness [155]. Thereby, DTM is a beneficial option in cases of MD as they have increased inflammation [151]. An increased level of profibrotic factor TGFβ1 is observed in muscles of DMD patients [156] which is reduced by brief stretching of tissue beyond the habitual range [157]. A preclinical study demonstrated that massage therapy in 32 rats have increased satellite cell number in their gastrocnemius muscle [158]. This finding suggests that DTM along with stem cell therapy is a beneficial option for the treatment of MD.

## **10. Future directions and conclusion**

Clearly, the initiation of disease pathology is due to genetic defect, but progression is due to satellite cell exhaustion and loss of regenerative capacity of satellite cells. Replenishing satellite cell pool and enhancing regenerative capacity of muscle fibers is essential for any treatment to be effective. Cell therapy represents a potential treatment strategy for MD. Goals that need to be fulfilled include delivery of normal protein to affected muscle fibers, effective fusion of donor cells to affected muscle fibers, delivery of large numbers of stem cells, repopulating of the resident stem cell pool, long term survival of stem cells to facilitate long term muscle repair and homeostasis. All these goals may not be met using one cell type and may require a combination of cell therapies. Future studies could explore systemic delivery of cells along with transplantation of different types of autologous/allogeneic cells in larger numbers intramuscularly and/ or with intrathecal administration. Studies could also combine the genetic therapies with stem cell replacement to identify the most effective cure for DMD. Larger randomized studies to determine the ideal cell combination, route of delivery and cell dosage are recommended. Studies can also explore the effects of cellular therapy in combination with integrative therapies such as HBOT, ozone therapy and DTM.

## **Author details**

Alok Sharma, Hemangi Sane, Nandini Gokulchandran, Amruta Paranjape\*, Zubiya Shaikh, Arjun KM and Prerna Badhe Neurogen Brain and Spine Institute, Navi Mumbai, India

\*Address all correspondence to: amrutap.neurogen@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 3**

Perspective Chapter: Multiple Functions of *Fukutin*, the Gene Responsible for Fukuyama Congenital Muscular Dystrophy, Especially in the Central Nervous System

*Tomoko Yamamoto, Yukinori Okamura, Ryota Tsukui, Yoichiro Kato, Hiromi Onizuka and Kenta Masui*

## **Abstract**

Fukuyama congenital muscular dystrophy (FCMD), accompanying central nervous system (CNS) and ocular anomalies, is the second common muscular dystrophy in Japan, and the responsible gene is *fukutin*. The lesions are mainly caused by fragile basement membrane/cell membrane due to hypoglycosylation of α-dystroglycan (α-DG), and astrocytes play a crucial role for CNS malformation. On the other hand, since fukutin is expressed almost ubiquitously, diverse functions of fukutin, besides the glycosylation of α-DG, can be considered. As for the CNS, fukutin possibly upregulates cyclin D1 expression as a cofactor of activator protein-1 in astrocytoma. Moreover, fukutin may be involved in the phosphorylation of tau, one of the key proteins of dementia represented by Alzheimer's disease, in glutamatergic neurons. A presynaptic function in GABAergic neurons is also suggested. Owing to the recent advances of molecular and biochemical techniques, new therapeutic strategies are under consideration, even for brain malformation, which begins to be formed during the first trimester *in utero*. Recovery of hypoglycosylation of α-DG supposed to be a main therapeutic target, but to know various functions and regulation systems of fukutin might be important for developing suitable therapies.

**Keywords:** fukutin, multifunction, cell proliferation, astrocyte, tau phosphorylation, glutamatergic neuron, presynaptic function, GABAergic neuron

### **1. Introduction**

Fukuyama congenital muscular dystrophy (FCMD), an autosomal recessive disease firstly reported by Fukuyama et al. in 1960 [1], is the second common muscular dystrophy in Japan [2]. It is one of the muscular dystrophies accompanies central nervous system (CNS) and ocular anomalies and is included in α-dystroglycanopathy [3, 4]. The responsible gene is *fukutin* [5]. α-dystroglycan (α-DG), a glycoprotein and a component of dystrophin-glycoprotein complex at the cell/basement membrane, binds to some basement membrane proteins, such as laminin-α2, neurexin-α, and agrin [4, 6]. The sugar chain works as a receptor [4, 6]. The expression is not only in the skeletal muscle but also in the CNS [7] and other organs [8]. Hypoglycosylation of α-DG causes fragile basement membrane, which is considered for the major pathogenesis of α-dystroglycanopathy. Several proteins including fukutin are involved in the glycosylation of α-DG [4, 6]. Fukutin has a function of ribitol 5-phosphate (Rbo5P) transferase that transfers Rbo5P from cytidine diphosphate-Rbo to α-DG [9]. α-DG is hypoglycosylated in the skeletal and cardiac muscles of FCMD patients [10]. Like α-DG, fukutin is expressed in various organs, almost ubiquitously [5, 8]. Diverse functions of fukutin can be suggested. To know these functions seems helpful not only for better understanding of FCMD pathology, but also for developing new therapeutic strategies. In this chapter, several intriguing roles of fukutin in the CNS are presented.

## **2. Fukutin gene**

*Fukutin* is localized on chromosome 9q31 and encodes a 461-amino-acid protein [5]. The mRNA of 7349 bp contains an open reading frame of 1383 bp, composed of 10 exons, and a long 3′-untranslated region [5]. Alternative splicing has been found [11]. In FCMD, a common genetic mutation is a retro-transposal insertion of about 3000 bp into the 3′-untranslated region, called founder haplotype [5], but other mutations have been reported [11]. Mutations heavily affecting the coding protein may provoke a severe phenotype resembling Walker-Warburg syndrome [12–14], while those influencing lightly may cause mild phenotypes like limb girdle muscular dystrophy [12, 15, 16].

## **3. Clinicopathological characteristics of FCMD**

Generally, FCMD patients are born as floppy infants, and peak motor function is achieved around 5 years old [2]. Patients usually die before 20 years old, but milder cases may live around 30 years old. Besides muscular dystrophy of the skeletal muscle, cardiac involvement is known. As clinical manifestations of the CNS lesion, mental retardation is observed, and more than 50% of patients show seizures. Ophthalmologic symptoms, such as myopia and abnormal eye movements, can be seen.

The CNS lesion of FCMD is represented by cobblestone lissencephaly, in other words polymicrogyria, of the cerebrum and cerebellum in post-natal cases [17, 18] (**Figure 1**). Abnormalities in the spinal cord may be found, especially in severe cases [19]. In the cerebrum and cerebellum of fetal cases, the glia limitans, covered with the basement membrane of the brain surface, is disrupted [20, 21]. In the cerebrum, overmigration of glioneuronal tissues through disruptions is obvious (**Figure 1**). The basement membrane/cell membrane at the glia limitans is abnormal, electron microscopically [21, 22]. Immunoreaction against anti-glycosylated α-DG [19] and laminin-α2 [23] antibodies is decreased at the glia limitans.

*Perspective Chapter: Multiple Functions of* Fukutin*, the Gene Responsible for Fukuyama… DOI: http://dx.doi.org/10.5772/intechopen.108063*

#### **Figure 1.**

*Cerebral lesions of FCMD patients. A) Medial view of the cerebrum of 2-year-old case. Polymicrogyri are observed in the cortex. The corpus callosum looks normal. B) Cortical lesion of a fetus of 16 weeks of the gestation. Glioneuronal tissues overmigrate through a defect of glia limitans (arrow, periodic acid-methenamine-silver staining). CC: Corpus callosum, GL: Glia limitans.*

## **4. Functions of fukutin in astrocytes**

#### **4.1 Functions related to the glycosylation of** α**-DG**

The major pathogenesis of the polymicrogyria of FCMD is considered to be a fragile basement membrane due to hypoglycosylation of α-DG, which causes the disruption of the glia limitans [19]. Disruptions are already detectable in a fetus of 16 weeks of the gestation (**Figure 1**). Since astrocytes, expressing both α-DG [24] and fukutin [25], form the glia limitans, astrocytes are mainly involved in the pathogenesis of CNS lesions of FCMD [19]. The cerebrum and cerebellum show different histological appearances in polymicrogyria, owing to the difference of their structures, components, and ways of neuronal migration. In post-natal FCMD cases, the cerebral surface is continuous, exhibiting marked superficial gliosis with obvious elongation of astrocytic endfeet [19]. After maturation, astrocytes may be able to compensate the fragile basement membrane/cell membrane by reactive gliosis.

#### **4.2 Functions other than the glycosylation of** α**-DG: relation to cell proliferation**

In astrocytes, involvement in the glycosylation of α-DG is the most important role for the pathogenesis of CNS lesions of FCMD. However, other function of fukutin has been found, regarding the regulation of cell proliferation. On an astrocytoma cell line (1321 N1) highly expressing cyclin D1, cell proliferation and expression of cyclin D1 are decreased by suppression of fukutin and increased by overexpression (**Figure 2**) [26]. Cyclin D1, one of the proteins controlling the cell cycle, facilitates cells entering into the S phage of cell cycle for cell proliferation, and its expression is regulated by various transcription factors [27]. In the promoter area of cyclin D1, there are multiple binding sites for each transcription factor, including activator protein-1 (AP-1) [27]. It has been found that a complex

#### **Figure 2.**

*Cell proliferation of astrocytoma cells (1321 N1) after transfection of fukutin. Cells increase in number with transfection of pC3.1-FKTN (a), compared with those with pC3.1-GFP (B). FKTN: Fukutin.*

containing fukutin protein binds to the AP-1 binding site of cyclin D1 and fukutin protein and AP-1 are co-localized [26]. Fukutin can take part in the transcription regulation of cyclin D1 as a cofactor of AP-1, independent from the glycosylation of α-DG [26].

Astrocytes play a variety of roles to maintain the function of CNS properly, among which tissue repair is included. Activated astrocytes proliferate and migrate to repair damaged areas [28]. Given that fukutin is involved in the cell proliferation, a loss of fukutin in astrocytes might influence to wound healing in the CNS of FCMD patients, although studies have not been performed from this point of view to the author's knowledge. Apart from the pathogenesis of FCMD, it might be intriguing to investigate about fukutin on the standpoint of cell proliferation of astrocytoma. Fukutin might act as a cofactor of some other transcription factors besides AP-1.

## **5. Functions of fukutin in neurons**

#### **5.1 Functions in immature neurons**

Although astrocytes are considered to play a crucial role to form the CNS lesions of FCMD, fukutin is also expressed in immature and mature neurons [25, 29]. In the cerebrum, immature neurons migrate from the ventricular zone to the cortical plate along with cytoplasmic processes of radial glia during the early fetal period [30, 31]. The migration is almost completed by 20–24 weeks of gestation [31]. Extracellular matrix proteins such as laminin and agrin are indispensable for the attachment of immature neurons and cytoplasmic processes of radial glia [32]. Since the sugar chain of α-DG is a receptor of such extracellular matrix proteins [4, 6], the glycosylated α-DG seems necessary for the neuronal migration [33]. Neurons begin to differentiate after settling to the proper place of the cerebral cortex [31]. Fukutin expression in immature neurons seems reasonable for the neuronal migration, smoothly interacting with radial glial fibers while keeping immaturity [34]. Irregular distribution of immature neurons is observed in the severely affected area of the cerebrum of FCMD, indicating that migration arrest may be apparent when a function of fukutin is seriously damaged [19]. Compensation of fukutin function by other proteins may be more established in neurons than in astrocytes.

*Perspective Chapter: Multiple Functions of* Fukutin*, the Gene Responsible for Fukuyama… DOI: http://dx.doi.org/10.5772/intechopen.108063*

#### **5.2 Functions of fukutin in mature neurons**

#### *5.2.1 Function in glutamatergic neurons, with relation to the phosphorylation of tau*

Fukutin expression is decreased in mature neurons [34], but some functions must exist in mature neurons. In the brain of FCMD patients more than 20 years old, neurofibrillary tangles (NFTs) immunopositive for phosphorylated tau (p-tau) are predominantly observed in the cerebral cortex [35, 36]. In our adult case of FCMD, NFTs are exclusively observed in areas showing polymicrogyria, and not found in the occipital robe showing almost normal appearance. In the CNS, there are excitatory and inhibitory neurons. The majority of neurons that constitute the cerebral cortex are excitatory/glutamatergic neurons, using glutamate as neurotransmitter, and the rests are inhibitory neurons [30]. Among several types of inhibitory neurons, neurons using γ-aminobutyric acid (GABA) as a neurotransmitter, GABAergic neurons, are a main component in the cerebral cortex [30]. GABA is synthesized from glutamate by glutamate decarboxylase (GAD) [37], which is used as a marker of GABAergic neurons. On immunohistochemical examination of the cerebrum of FCMD cases, neurons containing p-tau-positive NFT do not express GAD. NFTs are likely to be formed in excitatory neurons (**Figure 3**) [38]. Abnormal architectures of neurons derived from the disruption of glia limitans may be one of the factors to give rise to NFTs, considering from the distribution of neurons containing NFT. On the other hand, it is imaginable that fukutin itself is involved in the formation of NFTs.

In the adult CNS, there are six tau isoforms, which are divided into three microtubule-binding repeat (3R) and four microtubule-binding repeat (4R), depending on the absence or existence of exon 10 [39, 40]. There are various neurodegenerative diseases exhibiting p-tau-positive inclusions, so-called tauopathy. Three types of p-tau accumulation are known: 3R + 4R tauopathy, in which inclusions contain 3R and 4R tau, is represented by Alzheimer's disease; 3R tauopathy represented by Pick's disease; 4R tauopathy represented by corticobasal degeneration [39]. On FCMD, Western blotting [36] and immunohistochemical examination reveal both 4R and 3R tau in the diseased brain (**Figure 3**). There is something common on formation of p-taupositive NFTs in FCMD and Alzheimer's disease. However, the distribution of NFTs is somewhat different. Alzheimer's disease shows NFTs predominantly distributed in

#### **Figure 3.**

*Immunohistochemistry on the cerebrum of a 27-year-old FCMD patient. On double-immunohistochemical staining, p-tau (brown)-positive NFT is not formed in a glutamate decarboxylase (purple)-positive neuron (A). NFT is positive for both 3R tau (B) and 4R tau (C). NFT: Neurofibrillary tangle, GAD: Glutamate decarboxylase, 3R: Three microtubule-binding repeats, 4R: Four microtubule-binding repeats.*

the limbic system and in the cerebral cortex, accompanying with senile plaques [41]. In contrast, NFTs are tended to be more in the cerebral cortex, and senile plaques are not found in FCMD. Different pathogenesis can be assumed.

Many molecules are involved in the phosphorylation of tau. One of the representative proteins is glycogen synthase kinase-3 (GSK-3β) [42]. Using neuroblastoma cell lines, we have found that the phosphorylation of both tau and GSK-3β is augmented by suppression of fukutin and is reduced by overexpression of fukutin [38]. Moreover, fukutin, tau, and GSK-3β are suggested to form a complex (**Figure 4**) [38]. Fukutin is possibly involved in the phosphorylation of tau, mediated by GSK-3β, which appears to be independent from the glycosylation of α-DG. It is likely that on glutamatergic neurons of the FCMD cerebrum, loss of fukutin accelerates the phosphorylation of tau, which may be augmented by abnormal network of neurons. Implication of GSK-3β is considered for this phosphorylation. However, GSK-3β is involved in the

#### **Figure 4.**

*Double-immunocytochemical staining on neuroblastoma cells (SH-SY5Y). Fukutin and tau are co-localized (A-C). Fukutin and glycogen synthase kinase-3 show similar localization (D-F). No immunoreaction is observed in negative controls (G-I). FKTN: Fukutin, GSK-3β: Glycogen synthase kinase-3 β.*

*Perspective Chapter: Multiple Functions of* Fukutin*, the Gene Responsible for Fukuyama… DOI: http://dx.doi.org/10.5772/intechopen.108063*

abnormal accumulation of amyloid-β as well, a main component of senile plaque [42, 43]. Further studies are required to elucidate the mechanism between fukutin and tau phosphorylation.

#### *5.2.2 Function in GABAergic neurons, with relation to the synaptic function*

In the cerebrum of our adult FCMD patient, immunoreaction against anti-GAD antibody is increased [38]. Several factors can be postulated for the explanation, e.g., compensation toward hyperactivities of glutamatergic neurons, reaction against abnormal postsynaptic or presynaptic functions, etc. It is curious to know whether fukutin itself directly implicated in this phenomenon or not. A loss of fukutin appears to trigger the increase of GAD, because the increase is observed throughout the cerebral cortex, including the occipital robe showing almost normal histological appearance [38]. The dystrophin-dystroglycan complex (DGC) is existed in the postsynapse, and postsynaptic function of α-DG is well known [6, 44]. In contrast, studies about presynaptic functions of the DGC are not so many, but the DGC exists in the presynapse of GABAergic neurons [45]. On neuroblastoma cell lines, fluorescence immunocytochemistry has revealed that expression GAD is increased by suppression of fukutin and decreased by overexpression of fukutin [38]. Co-localization of fukutin, GAD, and synaptophysin is also suggested (**Figure 5**) [38]. Since synaptophysin is a component of presynaptic vesicle [46], co-localization of fukutin and synaptophysin supports presynaptic function of fukutin. From the existence of the DGC at the presynapse, increase of GAD in GABAergic neurons might result from the decreased glycosylation of α-DG, but co-localization of fukutin and GAD might indicate a direct involvement of fukutin.

#### **Figure 5.**

*Double- immunocytochemical staining on neuroblastoma cells (SH-SY5Y). Fukutin and glutamate decarboxylase are co-localized (A-C). Co-localization is also observed between fukutin and synaptophysin (D-F). FKTN: Fukutin, GAD: Glutamate decarboxylase, Syn: Synaptophysin.*

## **6. Future perspectives**

In addition to the glycosylation of α-DG, fukutin can contribute at least to cell proliferation, tau phosphorylation, and presynaptic function. The Golgi apparatus is considered to be a major subcellular localization [5, 47], because fukutin is involved in the glycosylation of α-DG. However, PSORT II prediction favors localizations of fukutin in the cytoplasm, mitochondria and nucleus rather than the Golgi apparatus. This prediction matches the findings presented in this chapter and suggests more unknown functions of fukutin.

With regard to the phosphorylation of tau, a relation between fukutin and microtubules can be assumed. Tau is a microtubule-binding protein to stabilize microtubules. Phosphorylated tau proteins that cannot bind to the microtubules are accumulated in the cytoplasm, resulting in NFTs [40]. Fukutin could be involved in the stabilization of microtubules by suppressing the phosphorylation of tau. When fukutin is knocked down on neuroblastoma cell lines, cytoplasmic processes are elongated (**Figure 6**) [34]. Elongation of cytoplasmic processes is also observed in fukutin-suppressed astrocytoma cells [48]. Astrocytes express tau, and astrocytic tau pathology has been reported [49]. It has been shown that overexpression of tau disturbs movements of kinesin, one of the representative molecular motors, and tau-stable cells exhibit rather round appearances [50]. Fukutin might relate to functions of microtubules via tau phosphorylation, not only influencing their stabilization but also affecting movements of molecular motors and cell morphology. A relation between fukutin and microtubules is proposed in cardiomyocytes as well [51]. To study more about the relation between fukutin and microtubules appears interesting.

To mention more about glial cells, there are four major types of glial cells in the CNS; astrocyte, oligodendrocyte, microglia, and ependymal cell. Functions of fukutin in astrocytes are indispensable for the pathogenesis of the CNS lesion of FCMD, while functions in other glial cells have not been elucidated. There are only a few observations suggesting functions of fukutin in oligodendrocytes. The cerebral white matter of FCMD exhibits dysmyelination [52], and fukutin-deficient chimera mice show loss of myelination in the peripheral nerve [53]. On microglia and ependymal cells, investigations relating to fukutin have not been found in English literatures to the authors' knowledge.

#### **Figure 6.**

*Morphological alteration of neuroblastoma cells (IMR32) after suppression of fukutin. Elongation of cytoplasmic processes is conspicuous on cells with suppression of fukutin (A), compared with control cells (B). KD: Knockdown of fukutin.*

*Perspective Chapter: Multiple Functions of* Fukutin*, the Gene Responsible for Fukuyama… DOI: http://dx.doi.org/10.5772/intechopen.108063*

As for therapies of FCMD, in addition to conventional treatments, efficacy of steroids [54] and rapamycin [55] has been suggested for muscular dystrophy. A retro-transposal insertion in the 3′-untranslated region of *fukutin* provokes pathogenic exon-trapping, resulting in a production of abnormal fukutin protein [56]. Treatment of antisense oligonucleotide can prevent this pathogenic exon trapping and restore normal fukutin production on human primary myotube obtained from FCMD patients and from FCMD model mice knocked in the retro-transposal insertion [56]. CDP-ribitol prodrug ameliorates muscular dystrophy in mice that lack *isoprenoid synthase domain-containing protein (ISPD)*, one of the causative genes of α-dystroglycanopathy [57]. Surprisingly, recent studies propose novel strategies toward brain malformation, despite the CNS and ocular anomalies begin to be formed during the first trimester *in utero*. Severe brain malformation of *Emx1-fukutin*-cKO mouse is prevented by delivery of fukutin into the brain at E12.5 [58]. In a brain organoid model of FCMD, abnormal radial glial fiber migration is restored by Mannan-007 [59]. It is not easy to overcome a lot of difficulties to apply new molecular or gene-based therapies, especially to fetuses. Unexpected phenomena could happen. On developing new therapeutic strategies, especially of molecular level, a good knowledge about functions and regulation systems of fukutin seems necessary.

### **7. Conclusion**

Fukutin is considered to be multifunctional. In the CNS, fukutin can be involved at least in the cell proliferation, tau phosphorylation, and presynaptic function, some of which seems independent from the glycosylation of α-DG. To see fukutin form various standpoints may be interesting and indispensable, not only for deep understanding of FCMD pathology but also for developing suitable therapies.

### **Acknowledgements**

The authors are grateful to ex-professor of the Department of Pathology, Noriyuki Shibata for valuable comments, and to Mr. Mizuho Karita, Mr. Hideyuki Takeiri, Ms. Noriko Sakayori, Mr. Fumiaki Muramatsu, and Mr. Shuichi Iwasaki for their excellent technical assistances.

The authors declare no conflict of interest.

## **Author details**

Tomoko Yamamoto1,2\*, Yukinori Okamura1,2, Ryota Tsukui3 , Yoichiro Kato2 , Hiromi Onizuka1,2 and Kenta Masui1,2

1 Department of Surgical Pathology, Tokyo Women's Medical University, Tokyo, Japan

2 Division of Human Pathology and Pathological Neuroscience, Department of Pathology, Tokyo Women's Medical University, Tokyo, Japan

3 Department of Anesthesiology, Tokyo Women's Medical University, Tokyo, Japan

\*Address all correspondence to: yamamoto.tomoko@twmu.ac.jp

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Multiple Functions of* Fukutin*, the Gene Responsible for Fukuyama… DOI: http://dx.doi.org/10.5772/intechopen.108063*

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## **Chapter 4**

## The Potential Benefits of Drug-Repositioning in Muscular Dystrophies

*Ioana Lambrescu, Emilia Manole, Laura Cristina Ceafalan and Gisela Gaina*

### **Abstract**

Muscular dystrophies (MDs) are a complex group of rare neuromuscular disorders caused by genetic mutations that progressively weaken the muscles, resulting in an increasing level of disability. The underlying cause of these conditions consists of mutations in the genes in charge of a person's muscle composition and functionality. MD has no cure, but medications and therapy can help control symptoms and slow the disease's progression. Effective treatments have yet to be developed, despite the identification of the genetic origins and a thorough knowledge of the pathophysiological alterations that these illnesses induce. In this scenario, there is an urgent need for novel therapeutic options for these severe illnesses, and drug repositioning might be one feasible answer. In other words, drug repositioning/repurposing is an accelerated method of developing novel pharmaceuticals since the new indication is based on previously accessible safety, pharmacokinetic, and manufacturing data. This is particularly crucial for individuals with life-threatening illnesses such as MDs, who cannot wait for a conventional medication development cycle. This chapter aims to review the challenges and opportunities of drug-repositioning in a variety of MDs to establish novel treatment approaches for these incurable diseases.

**Keywords:** muscular dystrophies, drug-repositioning, drug-repurposing, novel therapies, utrophin

### **1. Introduction**

Muscular dystrophies (MDs) are a diverse array of hereditary muscle disorders defined by gradual weakening in the affected muscles [1]. Even among people with the same condition and genetic abnormalities, there may be differences in the age at which symptoms first appear, the severity of those symptoms, the rate at which they advance, the prognosis, and the most effective treatment [2]. In terms of epidemiology, each form of MD is rather uncommon, but these conditions account for a significant portion of the individuals who suffer from neuromuscular impairment [2].

Over the past decades, significant advancements have been achieved in the treatment of people who suffer from MD. This progress has been made possible by worldwide cooperation, increasing comprehension of the fundamental genetic mechanisms, and clinical consensus standards [3]. Therapeutic advancements have also expanded, first using mutation and gene-directed techniques, leading to commercially accessible medications targeting particular Duchenne muscular dystrophy (DMD) mutations [3]. Although there is real enthusiasm in the therapeutic area, it is crucial to remember that, since MDs are degenerative conditions, finding a permanent solution will be exceedingly difficult. Therefore, there is an urgent need to find and use innovative pharmacological treatments to enhance the clinical care of MD patients.

Drug repositioning, also known as pharmacological repurposing, is a process that may be used to find innovative therapeutic agents from the current drug molecules that the FDA has authorized for use in clinical settings [4]. Costs for novel treatments may be a significant barrier for researchers and patients in general and with rare illnesses in particular. This difficulty may be significantly addressed by experimenting with the usage of chemicals that were initially intended for other situations.

On average, the success rate of developing a new drug is only 2.01%. According to a report by the Eastern Research Group (ERG), it takes 10 to 15 years to generate a new therapeutic molecule [5]. Furthermore, traditional drug development processes generally include five phases, while drug repurposing only requires four (**Figure 1**) [6]. Researchers currently only require 1–2 years to uncover novel therapeutic targets, while it takes an average of 8 years to produce a repositioned medicine, thanks to the rapid growth of bioinformatics [7].

**Figure 1.** *Approaches for drug repurposing.*

This chapter focuses on the most promising options for repositioning medications for three of the most prevalent forms of MDs: both in preclinical investigations and clinical trials.

## **2. DMD/BMD: showing old molecules how to accomplish new things**

The X-linked muscle disorders known as dystrophinopathies include the muscular dystrophies Duchenne (DMD), Becker (BMD), and DMD-associated dilated cardiomyopathy (D). DMD, an X-linked recessive condition that mostly affects men, is characterized clinically by gradual muscular weakening and deterioration that initially affects proximal muscles [8]. The dystrophin gene (*DMD* gene), which is located on chromosome Xp21.2 and encodes for the dystrophin protein via its 79 exons, is the cause of DMD and BMD depending on the mutation [9]. Dystrophin is an essential component of the protein complex that via the cell membrane binds the cytoskeleton of a muscle fiber to the surrounding extracellular matrix, stabilizing it during muscle contraction [8]. A number of potentially useful therapy techniques have been created and studied using DMD animal models. Nevertheless, the results of clinical trials have been far less spectacular. Currently, there are no treatments that can reverse dystrophinopathies underlying etiology. Conventional therapies employing corticosteroids aim to relieve symptoms, but their long-term administration is linked with substantial side effects [10]. Regarding the concept of targeted therapy several approaches have been developed for restoring dystrophin, each customized to a specific type of mutation. Stop-codon read-through, exon skipping, vector-mediated gene therapy, and the emerging CRISPR/Cas9 gene editing are all promising strategies. However, in the context of these therapies, the initial enthusiasm is overshadowed by all the questions regarding treatment effect, safety, and financial burden [11]. Therefore, drug repositioning could be a cost and time-effective approach when it comes to a rare disease such as muscular dystrophy.

## **2.1 Targeting Utrophin a via repurposed drugs**

Dystrophin's primary purpose in terms of its functional role is to forge a connection between the internal cytoskeletal actin network and the extracellular matrix. Consequently, this will ensure that the sarcolemma of muscle fibers retains its structural integrity [12, 13]. Utrophin, a paralogue of dystrophin, is a protein highly expressed in developing muscle [14]. To complete the connection from the cytoskeleton through the membrane and into the extracellular matrix, utrophin interacts with the dystrophin-associated protein complex [14]. Therefore, techniques based on targeting dystrophin or utrophin may be applied together in dystrophic muscles. There are two full transcription forms for utrophin. The neuromuscular junction (NMJ), tendon, choroid plexus, pia mater, and glomerulus all express utrophin A, whereas endothelial cells produce utrophin B [15]. Muscle-specific trans-factor known as eukaryotic elongation factor 1A2 (eEF1A2) was discovered to interact with utrophin A's 5'UTR, which is why eEF1A2 targeting might be a possible therapeutic approach for DMD patients [16].

In 2020, Peladeau and colleagues published an interesting study that aimed to identify FDA-approved drugs that acted on the eEF1A2-utrophin A pathway in *mdx* mice [17]. The *in vitro* and *in vivo* experiments focused on five leads: Acarbose, Betaxolol, Labetalol, Pravastatin, and Telbivudine. The authors found that the

beta-androgenic blocking medication *Betaxolol* and the cholesterol-lowering medication *Pravastatin* were the most effective activators of both eEF1A2 and utrophin through its 5′UTR internal ribosome entry site. This observation was based on a 7-day drug treatment of transgenic mice harboring the bicistronic reporter construct containing the utrophin 5'UTR. Furthermore, muscle strength was increased, and both muscle fiber shape and sarcolemma integrity were improved after *mdx* mice were treated with these medicines for 4 weeks [17].

#### **2.2 The monoamine oxidase inhibitors**

Although the pathophysiological basis for DMD is yet unknown, oxidative stress and mitochondrial dysfunction are thought to be major contributors to the development of muscle injury [18–20]. In dystrophic muscles, the mitochondrial enzyme known as monoamine oxidase (MAO) is a key generator of reactive oxygen species (ROS). This mitochondrial enzyme has been researched extensively in the central nervous system [21]. In 2010, a group conducted by Menazza demonstrated that oxidative changes of myofibrillar proteins and cell death, which result in a notable decrease in contractile performance, are significantly influenced by MAO-dependent reactive oxygen species (ROS) buildup [21]. *Pargyline*, an inhibitor of both MAO isoforms, was introduced in the US and the UK in 1963 as an antihypertensive drug. This compound was also administered to dystrophic animals, resulting in a reduction in tropomyosin oxidation and an improvement in disease phenotype [21]. Nevertheless, Pargyline's clinical use has been halted due to its considerable adverse effects [22].

*Safinamide* is a potent and specific inhibitor of MAO-B, which is approved for the treatment of mid to late-stage fluctuating Parkinson's disease [23]. Since intracellular signaling requires a small amount of ROS, the specificity of MAO inhibition is a crucial element [24]. In 2018, Vitiello and colleagues analyzed the impact of safinamide on the skeletal muscle of *mdx* mice and cultured muscle cells obtained from DMD patients. Even after a brief (1 week) course of therapy, reducing MAO-B had a beneficial impact *in vivo*, indicating a mechanism lacking significant tissue remodeling. The reduction of reactive oxygen species (ROS) levels in the fibers of treated animals and the oxidative status of a critical component of the contractile apparatus (tropomyosin) has been confirmed by analysis of muscle sections taken from animals that were given safinamide. Given that *in vitro* cultures, the dystrophin gene is expressed in myotubes but not in myoblasts, the in vitro experiments showed that increased susceptibility to oxidative stress in dystrophic cells appeared to be independent of dystrophin expression [24].

The observation that MAO catalyzes catecholamine removal proves the potential benefit of MAO treatment in DMD [21]. Chronic diseases can be associated with an increased level of catecholamines and DMD patients make no exception to this rule, as an excess of urine catecholamine has been documented in connection to age and disease progression [25]. All of this information, along with the benefit of using MAO-B inhibitors to prevent the hypertensive crisis that MAO-A inhibitors might cause, makes safinamide a therapy with a safe profile capable of increasing muscle performance [24].

#### **2.3 The selective estrogen receptor modulator**

*Tamoxifen*, a selective estrogen receptor modulator, has been commonly used to treat breast cancer for decades [26]. Estrogen receptor alpha (ER), the target via *The Potential Benefits of Drug-Repositioning in Muscular Dystrophies DOI: http://dx.doi.org/10.5772/intechopen.110714*

which tamoxifen operates, is present in both skeletal and cardiac muscle [27, 28]. Numerous studies have shown that tamoxifen protects against contraction-induced membrane damage, controls calcium influx, reduces oxidative stress, and prevents fibrosis [29–32], which is why it was hypothesized that DMD patients could benefit from this drug. In 2013, Dorchies and colleagues published an interesting study that assessed the effect of tamoxifen on dystrophic muscle structure and function [33]. Compared to previous research on normal rodents, the effects observed in the current study were attained with tissue levels of TAM and its primary metabolites that were significantly smaller. A daily dose of 10 mg/kg/day delivered to *mdx*5Cv mice for 15 months enhanced whole-body force, causing a change toward a slower phenotype [33].

The main DMD charity in the UK published a statement in July 2022 on preliminary data from the tamoxifen-DMD clinical case–control trial. The collaborating parties were disappointed to conclude that although patients receiving tamoxifen showed less disease progression, the differences between the tamoxifen and placebo group did not reach statistical significance [34]. For more DMD clinical trials please refer to **Table 1**.

#### **2.4 Dantrolene—Then and now**

*Dantrolene sodium* is a postsynaptic muscle relaxant that inhibits calcium release from the sarcoplasmic reticulum, reducing excitation-contraction coupling in muscle cells [35]. Since 1991, researchers have examined how this compound affects DMD, finding that it significantly lowers CK levels during the first year of treatment compared to age-matched historical controls [35]. Dantrolene is the agent of choice for treating and preventing malignant hyperthermia, a condition triggered by general anesthesia [36].

Exon skipping is a novel therapy that employs an antisense oligonucleotide (ASOs) customized to the patient's DNA mutation to target particular exons for exclusion from mRNA. As a result, the out-of-frame DMD mutation is converted to in-frame deletions, which might result in a partly functioning dystrophin protein [37]. Although 30% of patients may benefit from the existing exon skipping, most of the research on these therapies has focused on low-level dystrophin restoration (less than 6%) [38]. Due to differences in increased muscular function, across clinical studies small molecules were used to augment the effect of exon skipping. Dantolene's safety profile is already known due to its use in patients with DMD and malignant hyperthermia. This information, in conjunction with the fact that this drug reduced CK levels in both *mdx* mice and humans, raised the question of whether dantrolene can modulate exon skipping [35, 39]. Kendall and colleagues tried to answer this question by administering ASOs and dantrolene to *mdx* mice. The authors observed that DMD-directed ASOs and dantrolene cooperate to enhance targeted DMD exon skipping probably by interacting with specific molecular targets that subtly influence splicing activity. A further benefit of dantrolene is that it is effective independent of the specific ASO sequence, as seen by the enhancement of exon skipping activity for human exons 50 and 51 and mouse exon 23 [37]. In 2019, the group conducted by Berthelemy looked into the effects of dantrolene on skipping exons 44 and 45 in cultured myotubes from DMD patients' inducible directly reprogrammable myotubes (iDRMs) and induced pluripotent stem cells (iPSCs) [40]. In both exon 44 and 45 skip-amendable DMD cell models, the administration of dantrolene with the suitable ASOs raises the level of skipped mRNA compared with ASO alone. In patient-derived



*The Potential Benefits of Drug-Repositioning in Muscular Dystrophies DOI: http://dx.doi.org/10.5772/intechopen.110714*


**Table 1.**

*\*\*Pilot study.*

*Examples of repurposed drugs for muscular dystrophies.*

iDRM DMD myotube culture, dantrolene increases exon 44 skippings. Even though the improvement is small, it is still important because research shows that even small amounts of dystrophin can affect muscles [40].

#### **2.5 Repositioning cardiological drugs**

Dilated cardiomyopathy (DCM) is a severe consequence of DMD and one of the most important predictors of life expectancy [24]. In the context of this causality, it might be argued that therapy for the DCM serves as "repositioning." In DMD patients, **angiotensin-converting enzyme inhibitors** (ACEi) and **ß-blockers** (BBs) (and less often diuretics) are recommended for the reduction of peripheral circulatory resistance, blood volume, hyperadrenergic activation and oxygen consumption [24, 41, 42]. In addition, it is well known that BBs lower the incidence of potentially life-threatening arrhythmias initiated by foci of myocardial fibrosis [42]. Tamoxifen is another medication that has been demonstrated to impact the heart muscle significantly. The group conducted by Dorchies revealed that tamoxifen reduced cardiac fibrosis by 50% and positively impacted the diaphragm. As a result, there was a significant quantity of contractile tissue made accessible for respiratory function in the *mdx*5Cv mouse model [33]. The question of whether or not these treatments qualify as repositioning drugs is less significant when viewed in the context of the fact that cardiovascular and respiratory disorders are unavoidable complications of DMD. It is for this reason that we have devoted a brief subchapter to discussing them.

Calcium is essential for muscular function, but its intracellular accumulation is toxic, triggering apoptosis. As the literature provides extensive research on the function of calcium in muscle injury, it is fair to speculate that calcium antagonists may be useful in DMD [43].

A Cochrane database of systematic reviews investigating the impact of calcium antagonists on muscular power and function in DMD was published by Phillips and Quinlivan in 2008 [44]. The authors conclude that although *verapamil* significantly increased muscular strength, it also caused several cardiac adverse effects [45]. Additionally, no significant differences in efficacy between *diltiazem, nifedipine*, and *flunarizine* were found in the remaining studies [44]. In 2009, Matsumura and colleagues published an interesting study that evaluated the effect of verapamil and diltiazem in *mdx* mice [46]. The dystrophic phenotype of *mdx* mice was improved as shown by a lower serum CK level and reduced muscle deterioration in the diaphragm. Moreover, between the two calcium antagonists, diltiazem seems to protect against muscle degeneration more effectively [46].

*Nifedipine*, another calcium channel blocker, was the main focus of a 2013 study by Altamirano [47]. The results add to the growing body of data indicating that the calcium level is high in *mdx* muscles and may be regulated by nifedipine. On *mdx* mice, this treatment resulted in a reduction in the basal ATP release from dystrophic fibers and a decreased prooxidative/apoptotic gene expression. Consequently, a reduced muscle injury was observed in *mdx* mice, as shown by a substantial decrease in serum CK and an improvement in muscular strength [47].

#### **2.6 Metformin: A pleiotropic drug**

As monotherapy or in combination with other medications, *metformin*, a biguanide, is now one of the most often prescribed drugs in the world for the treatment of type 2 diabetes (T2D) [48]. Metformin has a wide range of molecular mechanisms of action, which explain why it may be used to treat autoimmune illnesses, prevent cancer, and protect the cardiovascular system [49–51]. Pharmacological actions of metformin include a decrease of glucose and lipid production via inhibition of mitochondrial complex I (NADH: ubiquinone oxidoreductase) and activation of AMP-activated protein kinase (AMPK) [52]. In this context, it was natural for researchers and clinicians to be drawn to the possible use of this medication in MDs.

Metformin's potential to improve muscular fibrosis and strength was shown in mdx mice via non-AMPK-related pathways [53]. Another preclinical study led by Lai and colleagues hypothesized that metformin could downregulate different chemokines in MD mouse models [54]. Thus, in mdx mice, levels of CXCL12 (C–X–C motif chemokine ligand 12) both a glucocorticoid target and a differentially expressed gene and its receptor CXCR4 (C–X–C motif chemokine receptor 4) were increased. As a result of prednisone therapy, their concentration decreased considerably. Furthermore, CXCL12 and CXCR4 expression was similarly shown to be reduced in mdx mice after treatment with metformin, suggesting that this pathway may be an attractive therapeutic target for DMD [54].

As previously discussed, ASOs-mediated exon-skipping is a promising line of treatment for DMD patients. However, a significant obstacle to its therapeutic use is the poor systemic effectiveness, necessitating drugs that enhance ASOs' activity. Based on previous studies that demonstrated an improvement of phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscle in mdx mice by intravenous administration of glycine, the group conducted by Lin analyzed the effect of oral glycine and metformin alongside PMO in dystrophin/utrophin double knock-out (DKO) mice [55]. Thus, without any toxicity that could be detected and with a life span extension, the scientists demonstrated improvements in the cardio-respiratory and skeletal systems and a phenotypic rescue in DKO mice [55].

Though widely administered in the T2D adult population it is important to mention that metformin has been tested in children and adolescents with neurogenic defects and muscle disorders [56]. In 2010, Casteels and colleagues reported that metformin is an insulin sensitizer capable of limiting weight gain and visceral adiposity in children with a neurogenic or myogenic motor deficit [56]. Thus, exploring the use of metformin as an additional therapy in a variety of illnesses has reached the clinical context of DMD. In 2019, Hafner and colleagues published the results of a randomized double-blind placebo-controlled parallel-group clinical trial that included 47 ambulant male children aged 6.5 to 10 years with DMD that received treatment with a combination of l-citrulline and metformin. Among ambulant patients with DMD, the co-treatment was not associated with an overall halting of the decline in motor function, although the stable subgroup of patients presented a decrease in motor function impairment [57]. Also, Metformin or L-citrulline supplementation in BMD patients results in notable antidromic changes in the arginine glycine amidinotransferase and guanidinoacetate methyltransferase pathways.

Metformin treatment has also been shown to be useful in congenital muscular dystrophy type 1A [58]. Metformin therapy promotes weight growth and energy efficiency, improves muscular function, and improves skeletal muscle histology in female dy2J/dy2J mice (but not in male dy2J/dy2J mice) [58]. Metformin also improved the mobility and walking abilities of people with myotonic dystrophy [59]. Metformin has also been shown to increase autophagy and provide cardioprotection in a mouse model deficient in δ-sarcoglycan, a protein encoded by the SGCD gene and associated with LGMD R6 (LGMD2F) [60].

#### **2.7 Tranilast: From allergies to Duchenne cardiomyopathy**

The life expectancy of DMD patients has improved recently due to advancements in the prevention of respiratory complications, however, there has been a notable increase in advanced cardiomyopathy symptoms [61]. To eventually cure DMD, gene replacement or other correction therapy must also approach the existence of fibrosis [62]. Furthermore, this concept favors DMD patients who develop DCM, marked by inflammation, fibrosis, and necrosis [63]. A compound with anti-fibrotic properties is *tranilast*, an anti-allergic agent and a calcium channel blocker prescribed for over 30 years to adults and children [64]. In mdx mice, improving muscle pathology and motor performance with Ca-handling drugs prevented aberrant intracellular Ca influx through the transient receptor potential cation channel, subfamily V 2 (TRPV2) [65]. The results of a single-arm, open-label, multicenter study on the safety and efficacy of tranilast for heart failure in patients with MDs—of whom the vast majority were DMD patients—were published in 2022 by Matsumura and colleagues [66]. Although there was no significant improvement in cardiac function, the authors concluded that tranilast was safe and effective in inhibiting TRPV2 expression and may represent a viable medication for patients with early heart failure [66].

Even in MD patients with advanced heart failure, tranilast is safe and effective at inhibiting TRPV2 expression.

#### **2.8 Phosphodiesterase type 5 inhibitors**

Increased muscle damage and irregular blood flow after muscle contraction—the condition known as functional ischemia—are hallmarks of DMD [67]. Thus, multiple pathways have been studied in the past decades to alleviate the ischemic picture in MDs. A potential novel therapeutic target for DMD is the nitric oxide (NO) - cyclic guanosine monophosphate (cGMP) pathway. *Phosphodiesterase type 5 (PDE5) inhibitors*, which extend the half-life of cGMP, have been shown to improve the function of the limb, respiratory, and cardiac muscles in mdx mice and to increase the lifespan of dystrophin-deficient zebrafish [68–70]. Since their major indication is erectile dysfunction, PDE5 can be considered as repurposed therapy in MDs. There are four major types of PDE5 inhibitors approved by the FDA [71] of which *sildenafil* and *tadalafil* were the most studied outside of their intended use. According to a 2012 study by Percival and colleagues, sildenafil administration for 14 weeks decreased diaphragm muscle weakness and supported normal extracellular matrix organization in mdx animals [72]. However, a placebo-controlled phase II trial (REVERSE DBMD; [73] including patients with Duchenne and Becker MD was terminated earlier due to worsening cardiomyopathy in some of the patients who received sildenafil [72]. As a PDE inhibitor tadalafil is more selective for PDE5 than sildenafil [74], which is why its evaluation advanced to a randomized, placebo-controlled phase III trial [73] that enrolled 331 patients. Nevertheless, tadalafil did not demonstrate efficacy in reducing the decline of walking ability in the treated group [75].

#### **2.9 A new perspective on antibiotics**

Since the early 1990s, it has been recognized that some antibiotics may reduce premature termination codons in eukaryotic cells [76]. In animal studies, the antibiotic family known as aminoglycosides has been shown to prevent nonsense mutations [24]. Thus, it was only a matter of time before researchers used this compound in

MDs. Barton-Davis and colleagues reported in 1999 that subcutaneous injections of gentamicin restored dystrophin levels in the skeletal muscles of mdx mice, demonstrating the potential of this family of antibiotics for the first time in *in vivo* [77].

Stretch-activated channels (SACs) are another pathway that has been proposed as being relevant in the pathophysiology of DMD [78]. These channels are permeable to Na+ and Ca2 and respond to mechanical stress [79, 80]. Lack of dystrophin increases skeletal muscle SAC activity in mdx mice [81–83]. Therefore, in muscles exhibiting various degrees of the dystrophic phenotype, researchers looked at the transient receptor potential canonical channel 1 (TRPC1) level and the effects of streptomycin, a SAC blocker [78]. In diaphragm and sternomastoid muscles, streptomycin decreased creatine kinase and reduced exercise-induced increases in total calcium and Evans blue dye absorption (a sensitive and early marker of myofiber injury) [78]. However, it must be taken into account when using aminoglycosides, that these drugs present various levels of oto- and/or nephrotoxicity [84, 85].

In 2003, the group led by Politano published the results of a small study on four patients with DMD. Immunohistochemistry and immunoblotting showed that dystrophin re-expression occurred in muscle biopsies performed after gentamicin treatment in three out of four patients that presented a more permissive UGA stop codon [86]. Seven years later, Malik and colleagues published the results of a multicenter trial that evaluated the safety of gentamicin infusions twice a week for 6 months [87]. Except for one patient who received an incorrectly calculated dosage, no patient showed a decreased renal or hearing function. The study's second goal was to see whether gentamicin improved muscular strength and increased dystrophin binding at the muscle membrane. Dystrophin levels significantly improved (p = 0.027) after 6 months of gentamicin treatment, and this was associated with a decrease in serum CK [88].

#### **2.10 Tyrosine kinase inhibitors**

Muscle degeneration and poor muscle regeneration brought on by a lack of dystrophin are major characteristics of DMD pathophysiology [89]. Several variables, including a reduction in satellite cell (SC) capacity (the critical precursors to myogenesis), have been linked to this defective regeneration [90]. SC proliferation and self-renewal in response to resistance training and muscle damage depend on the interleukin-6 (IL-6) cytokine's activation of the signal transducer and activator of transcription 3 (STAT3) [91–93]. Sunitinib is a multi-targeted tyrosine kinase inhibitor and a therapeutic option for treating renal cell carcinoma, gastrointestinal stromal tumors, and progressive, well-differentiated, advanced panNETs [24, 94]. Sunitinib was evaluated for its efficacy in the mdx mice model of DMD, in which it demonstrated the ability to induce muscle regeneration via transient STAT3 activation [90]. Furthermore, in 2022, Oliveira-Santos and colleagues published the results of an interesting study that evaluated how long-term sunitinib use affected heart pathology and function in mdx mouse model. The authors concluded that sunitinib increased cardiac electrical performance and reduced ventricular hypertrophy, cardiomyocyte membrane damage, and fibrotic tissue deposition in the heart muscle of mdx animals via lowered STAT3 phosphorylation [95].

Nintedanib is also a tyrosine kinase inhibitor approved for treating idiopathic pulmonary fibrosis (IPF) [96].

Previous studies on primary lung fibroblasts and dermal fibroblasts from patients with IPF and systemic sclerosis have demonstrated the anti-fibrotic activity of nintedanib, thus rendering this compound a potential aid in DMD [97, 98]. A one-month

course of nintedanib therapy in mdx mice reduced skeletal muscle fibrosis and improved muscle function, thus reinforcing the idea that tyrosine kinase inhibitors could have a potential role for clinical exploration in DMD [99].

## **3. Conclusions**

Recent scientific breakthroughs have enhanced diagnostic skills and permitted experimental research into gene therapy and standard pharmacological therapies, eventually leading to successful treatments for many inherited disorders.

Among them, muscular dystrophies and genetic diseases caused by mutations in over 40 genes result in dystrophic alterations on muscle biopsy and cause progressive weakness and degeneration of skeletal muscles. Due to advances in molecular biology techniques and an understanding of the mechanisms underlying these diseases, the genetic defect of most muscular dystrophies can now be precisely determined. Also, specialized therapy to help patients can be provided for some types of dystrophies, while many others are in the advanced clinical development stage. Although advances in the management and care of people with these disorders have slowed disease progression, more research is needed because the patients still face a lack of effective treatments. As a result, new forms of therapies are required. Repurposing existing intensively studied drugs with well-known pharmacokinetic, pharmacodynamic, tolerability, and safety profiles holds promise for timely effective therapies for patients suffering from life-threatening conditions who cannot wait for a traditional drug development cycle. Quickly redirecting these drugs to other directions can improve life expectancy and quality of life in affected patients. The use of this path is accelerated by incentives, guidance, and protection provided by holding an orphan drug designation status. Drug repurposing is a valuable strategy with enormous potential for delivering long-awaited therapies that may be safeguarded by orphan drug designation status, enabling successful, fair-priced commercialization.

## **Acknowledgements**

This work was funded by the Ministry of Research, Innovation, and Digitalization in Romania, under Program 1—The Improvement of the National System of Research and Development, Subprogram 1.2—Institutional Excellence-Projects of Excellence Funding in RDI, Contract No. 31PFE/30.12.2021. National Program 31 N/2016/PN 16.22.02.05 and National Program 10 N/2023/PN 23.16.01.02.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Ioana Lambrescu1,2, Emilia Manole1,3, Laura Cristina Ceafalan1,2 and Gisela Gaina1 \*

1 Cell Biology, Neurosciences and Experimental Myology Laboratory, Victor Babes Institute of Pathology, Bucharest, Romania

2 Department of Cellular and Molecular Biology and Histology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

3 Pathology Department, Colentina Clinical Hospital, Bucharest, Romania

\*Address all correspondence to: gisela.gaina@ivb.ro

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 5**
