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Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds, 30 and 27, led to significantly better in vivo exposure compared to ezutromid and alleviation of the dystrophic phenotype in mdx mice. While 30 was found to have dose-limiting hepatotoxicity, 27 and its enantiomers exhibited limited off-target effects, resulting in a safe profile and highlighting their potential utility as next-generation utrophin modulators suitable for progression toward a future DMD therapy.

Ezutromid (SMT C1100) is a small-molecule utrophin modulator that was developed to treat Duchenne muscular dystrophy (DMD). Previous clinical trials of this agent revealed lower exposure in DMD patients compared with healthy volunteers, which may reflect differences in diet. This study evaluated the pharmacokinetics of ezutromid in patients with DMD who followed a balanced diet. This was a multicenter, double-blind, placebo-controlled, ascending single and multiple oral dose study. Twelve pediatric patients were randomly allocated to 1 of 3 treatment sequences within which were 3 treatment periods of 2 weeks each. Each patient received, in a dose-escalating fashion, 1250 mg and 2500 mg twice daily (BID) of ezutromid administered orally as a microfluidized suspension (F3) with placebo in the other treatment period. Throughout the study, patients followed a balanced diet including recommended proportions of major food groups and administration of drug accompanied with 100 mL of full-fat milk. This approach improved the absorption of ezutromid, resulting in higher systemic exposure, with considerable variability in exposure between patients at each dose level. Single and multiple oral doses of 1250 mg and 2500 mg BID were considered safe and well tolerated. No severe or serious adverse events and no study discontinuations due to adverse events were reported. This study provides assurance that, with the formulation tested (F3) and instructions regarding food (balanced diet and whole-fat milk), 2500 mg BID of ezutromid achieves plasma concentrations that, based on preclinical studies, should be able to modulate utrophin expression in future clinical trials.

Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds

Duchenne muscular dystrophy (DMD) is a fatal muscle‐wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first‐in‐class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies., Chemical proteomics and phenotypic profiling illustrate the aryl hydrocarbon receptor (AhR) is a target of ezutromid, the first‐in‐class utrophin modulator for the treatment of Duchenne muscular dystrophy. Ezutromid binds to AhR with an apparent K D of 50 nm and functions as an AhR antagonist. Other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications, could also be exploited in future DMD therapies.

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by the lack of a subsarcolemmal protein, dystrophin. We have previously shown that the dystrophin-related protein, utrophin is able to compensate for the lack of dystrophin in the mdx mouse, the mouse model for DMD. Here, we explore whether utrophin delivered to the limb muscle of dystrophin/utrophin-deficient double knockout (dko) neonatal mice can protect the muscle from subsequent dystrophic damage. Utrophin delivery may avoid the potential problems of an immune response associated with the delivery of dystrophin to a previously dystrophin-deficient host. Dko muscle (tibialis anterior) was injected with a first generation recombinant adenovirus containing a utrophin minigene. Up to 95% of the fibres continued expressing the minigene 30 days after injection. Expression of utrophin caused a marked reduction from 80% centrally nucleated fibres (CNFs) in the uninjected dko TA to 12% in the injected dko TA. Within the region of the TA expressing the utrophin minigene, a significant decrease in the prevelance of necrosis was noted. These results demonstrate that the utrophin minigene delivered using an adenoviral vector is able to afford protection to the dystrophin/utrophin-deficient muscle of the dko mouse. Gene Therapy (2000) 7, 201-204.

PurposeSMT C1100 is a utrophin modulator being evaluated as a treatment for Duchenne muscular dystrophy (DMD). This study, the first in pediatric DMD patients, reports the safety, tolerability and PK parameters of single and multiple doses of SMT C1100, as well as analyze potential biomarkers of muscle damage.MethodsThis multicenter, Phase 1 study enrolled 12 patients, divided equally into three groups (A-C). Group A were given 50 mg/kg on Days 1 and 11, and 50 mg/kg bid on Days 2 to 10. Group B and C received 100 mg/kg on Days 1 and 11; Group B and Group C were given 100 mg/kg bid and 100 mg/kg tid, respectively, on Days 2 to 10. A safety review was performed on all patients following the single dose and there was at least 2 weeks between each dose escalation, for safety and PK review. Adverse events (AEs) were monitored throughout the study.ResultsMost patients experienced mild AEs and there were no serious AEs. Two patients required analgesia for pain (headache, ear pain and toothache). One patient experienced moderate psychiatric AEs (abnormal behaviour and mood swings). Plasma concentrations of SMT C1100 at Days 1 and 11 indicated a high degree of patient variability regardless of dose. Unexpectedly the SMT C1100 levels were significantly lower than similar doses administered to healthy volunteers in an earlier clinical study. In general, individual baseline changes of creatine phosphokinase, alanine aminotransferase, aspartate aminotransferase levels fell with SMT C1100 dosing.ConclusionsSMT C1100 was well tolerated in pediatric DMD patients.Trial registrationClinicalTrials.gov NCT02383511.

Families of 2-arylbenzotriazoles and 2-arylindazoles that show positive effects in screens predictive of endogenous utrophin upregulation have been identified. Synthesis and structure-activity relationships are described leading to compounds with attractive in vitro profiles.

A series of novel 2-arylbenzoxazoles that upregulate the production of utrophin in murine H2K cells, as assessed using a luciferase reporter linked assay, have been identified. This compound class appears to hold considerable promise as a potential treatment for Duchenne muscular dystrophy. Following the delineation of structure-activity relationships in the series, a number of potent upregulators were identified, and preliminary ADME evaluation is described. These studies have resulted in the identification of 1, a compound that has been progressed to clinical trials.

It has been shown previously that when utrophin is highly expressed in mice which lack dystrophin, the muscle pathology is prevented. Immunohistochemical evidence strongly suggests that utrophin in these transgenic mice occupies the position normally filled by dystrophin, although it is not possible to establish this firmly at the level of the light microscope. Using the higher resolution provided by the electron microscope, we demonstrate here by immunogold labelling with both monoclonal and polyclonal antibodies that utrophin, in both its truncated and full-length forms, is indeed specifically located in the subcellular position usually occupied by dystrophin in normal muscle. Moreover, when double-labelling of utrophin and β-dystroglycan was carried out, colocalisation of the two labels was often seen, indicating an association of the two proteins. Furthermore, when both utrophin and dystrophin were labelled in a transgenic line in which both were simultaneously expressed, the sites of both proteins were in the same zone in relation to the plasma membrane. When both proteins were present, the density of labelling of each was reduced compared with when they are expressed individually, suggesting that there is a finite number of binding sites. These results constitute further support for the view that utrophin might be therapeutically substituted for dystrophin in dystrophic muscle.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 μl of AdCMV-Utr (7.0 × 10¹¹ virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 μl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse± human cDNA., UI - 99291783

The modulation of utrophin gene expression in muscle by the nerve-derived factor agrin plausibly involves the trophic factor ARIA/heregulin. Here we show that heregulin treatment of mouse and human cultured myotubes caused a ≈2.5-fold increase in utrophin mRNA levels. Transient transfection experiments with utrophin promoter-reporter gene constructs showed that this increase resulted from an enhanced transcription of the utrophin gene. In the case of the nicotinic acetylcholine receptor δ and ɛ subunit genes, heregulin was previously reported to stimulate transcription via a conserved promoter element, the N-box, which binds the multimeric Ets-related transcription factor GA binding protein (GABP). Accordingly, site-directed mutagenesis of a single N-box motif in the utrophin gene promoter abolished the transcriptional response to heregulin. In addition, overexpression of heregulin, or of the two GABP subunits in cultured myotubes, caused an N-box-dependent increase of the utrophin promoter activity. In vivo , direct gene transfer into muscle confirmed that heregulin regulates utrophin gene expression. Finally, electrophoretic mobility shift assays and supershift experiments performed with muscle extracts revealed that the N-box of the utrophin promoter binds GABP. These findings suggest that the subsynaptic activation of transcription by heregulin via the N-box motif and GABP are conserved among genes expressed at the neuromuscular junction. Because utrophin can functionally compensate for the lack of dystrophin, the elucidation of the molecular mechanisms regulating utrophin gene transcription may ultimately lead to therapies based on utrophin expression throughout the muscle fibers of Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349-353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997) J. Biol. Chem. 272, 8117-8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1. 3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24-48 h. Under these conditions, both muscle- and nerve-derived agrin increased the activity of beta-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5' from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic proteins. Together, these observations are therefore relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction and for the potential use of utrophin as a therapeutic strategy to counteract the effects of Duchenne muscular dystrophy.

Dystrophin-deficient mice (mdx) expressing a truncated (trc) utrophin transgene show amelioration of the dystrophic phenotype. Here we report a multifunctional study demonstrating that trcutrophin expression leads to major improvements of the mechanical performance of muscle (that is, force development, mechanical resistance to forced lengthenings and maximal spontaneous activity) and of the maintenance of the intracellular calcium homeostasis. These are two essential functions of muscle fibers, known to be impaired in mdx mouse muscles and Duchenne muscular dystrophy (DMD) patients. Our results bring strong support to the hypothesis that muscle wasting in dystrophin-deficient DMD patients could be prevented by upregulation of utrophin.

Recently, the use of a transgenic mouse model system for Duchenne muscular dystrophy has demonstrated the ability of utrophin to functionally replace dystrophin and alleviate the muscle pathology (see Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349-353). However, there is currently a clear lack of information concerning the regulatory mechanisms presiding over utrophin expression during normal myogenesis and synaptogenesis. Using in situ hybridization, we show that utrophin mRNAs selectively accumulate within the postsynaptic sarcoplasm of adult muscle fibers. In addition, we demonstrate that a 1.3-kilobase fragment of the human utrophin promoter is sufficient to confer synapse-specific expression to a reporter gene. Deletion of 800 base pairs from this promoter fragment reduces the overall expression of the reporter gene and abolishes its synapse-specific expression. Finally, we also show that utrophin is present at the postsynaptic membrane of ectopic synapses induced to form at sites distant from the original neuromuscular junctions. Taken together, these results indicate that nerve-derived factors regulate locally the transcriptional activation of the utrophin gene in skeletal muscle fibers and that myonuclei located in extrasynaptic regions are capable of expressing utrophin upon receiving appropriate neuronal cues.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disease that causes cardiac or respiratory failure and results in death at about 20 years of age. Replacement of the missing protein, dystrophin, using myoblast transfer in humans or viral/liposomal delivery in the mouse DMD model is inefficient and short-lived. One alternative approach to treatment would be to upregulate the closely related protein, utrophin, which might be able to compensate for the dystrophin deficiency in all relevant muscles. As a first step to this approach, we have expressed a utrophin transgene at high levels in the dystrophin-deficient mdx mouse. Our results indicate that high expression of the utrophin transgene in skeletal and diaphragm muscle can markedly reduce the dystrophic pathology. These data suggest that systemic upregulation of utrophin in DMD patients may lead to the development of an effective treatment for this devastating disorder.

Utrophin is a ubiquitously expressed cytoskeletal protein which is an important structural component of the mammalian neuromuscular junction. It shows extensive sequence similarity to dystrophin leading to postulation that utrophin may be able to compensate for the absence of dystrophin in Duchenne muscular dystrophy (DMD) patients. In order to study the transcriptional control of utrophin expression including its regulation at the neuromuscular junction, and as a first step in the development of a potential DMD therapy, we have cloned the utrophin promoter region from human and mouse. The utrophin promoter is associated with a CpG island at the 5'-end of the gene, and sequence analysis of the 5'-UTR reveals several Sp1 binding sites and the absence of TATA or CAAT motifs. Transcription is initiated at one major and three minor sites. Using deletion constructs, we have defined an active promoter region of 155 bp. The first exon and 900 bp upstream display limited sequence conservation between human and mouse. The core sequence TTCCGG of the N box which regulates synaptic expression of other genes is also present and may be involved in regulating the specific expression of utrophin at the postsynaptic membrane. This study provides the basis for the understanding of the regulatory mechanism that controls utrophin expression and provides the data needed to develop methods for the upregulation of utrophin in DMD patients.

Utrophin is an autosomally-encoded homologue of dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene. Although, utrophin is very similar in sequence to dystrophin and possesses many of the protein-binding properties ascribed to dystrophin, both proteins are expressed in an apparently reciprocal manner and may be coordinately regulated. In normal skeletal muscle, utrophin is found at the neuromuscular junction (NMJ) whereas dystrophin predominates at the sarcolemma. However, during development, and in some myopathies including DMD, utrophin is also found at the sarcolemma. This re-distribution is often associated with a significant increase in the levels of utrophin. At the NMJ utrophin co-localizes with the acetylcholine receptors (AChR) and may play a role in the stabilization of the synaptic cytoskeleton. Because utrophin and dystrophin are so similar, utrophin may be able to replace dystrophin in dystrophin deficient muscle. This review compares the structure and function of utrophin to dystrophin and discusses the rationale behind the use of utrophin as a potential therapeutic agent.

Duchenne muscular dystrophy is a severe X chromosome-linked, muscle-wasting disease caused by lack of the protein dystrophin. The exact function of dystrophin remains to be determined. However, analysis of its interaction with a large oligomeric protein complex at the sarcolemma and the identification of a structurally related protein, utrophin, is leading to the characterization of candidate genes for other neuromuscular disorders.

Two promoters In the distal half of the Duchenne Muscular Dystrophy gene drive transcription of mRNAs which have novel first exons and encode the shortened forms of dystrophin, apo-dystrophln-1 (Dp71) and apodystrophln-2 (Dpi 16). Apo-dystrophin-1 has a G + C rich promoter and Is expressed in a wide range of cell types, whilst apo-dystrophln-2 is confined to peripheral nerve and brain. We have isolated and sequenced the unique 5' exon of rat apo-dystrophln-2 mRNA. Conceptual translation of this sequence Indicates that apo-dystrophin- 2 contains a unique 23 amino acid terminal peptide. Using specific probes derived from sequences at the 5' ends of apo-dystrophln-1 and apo-dystrophln-2 we have determined the expression of these two mRNAs during mouse embryonic development by RNA In situ hybridization. In contrast to full-length dystrophin, neither of these short dystrophin transcripts appear before organogenesis is well established. Apo-dystrophin-1 mRNA is detected in midllne cells of the ventral neural tube and later, in the ependymal cells lining the ventricles of the brain. These results suggest that apo-dystrophin-1 mRNA is associated with glial cells in the CNS. Apo-dystrophln-1 transcripts are also abundant in the teeth primordla throughout their development. In contrast apo-dystrophin-2 mRNA is largely undetectable during development, although transcripts are seen in the newborn brain. Western blots of late human fetal tissue extracts confirm that apo-dystrophin-2 is most abundant in brain and analysis of RNA and protein in cultured cell lines reveal expression of apo-dystrophln-1 and apo-dystrophin- 2 in glioma cells.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fibers leading to the gradual depletion of skeletal muscle. There is significant evidence demonstrating that increasing levels of the dystrophin-related protein, utrophin, in mouse models results in sarcolemmal bound utrophin and prevents the muscular dystrophy pathology. The aim of this work was to develop a small molecule which increases the levels of utrophin in muscle and thus has therapeutic potential. METHODOLOGY AND PRINCIPAL FINDINGS: We describe the in vivo activity of SMT C1100; the first orally bioavailable small molecule utrophin upregulator. Once-a-day daily-dosing with SMT C1100 reduces a number of the pathological effects of dystrophin deficiency. Treatment results in reduced pathology, better muscle physiology leading to an increase in overall strength, and an ability to resist fatigue after forced exercise; a surrogate for the six minute walk test currently recommended as the pivotal outcome measure in human trials for DMD. CONCLUSIONS AND SIGNIFICANCE: This study demonstrates proof-of-principle for the use of in vitro screening methods in allowing identification of pharmacological agents for utrophin transcriptional upregulation. The best compound identified, SMT C1100, demonstrated significant disease modifying effects in DMD models. Our data warrant the full evaluation of this compound in clinical trials in DMD patients.

During the past year significant progress has been made in understanding how dystrophin deficiency leads to muscle cell necrosis in Duchenne muscular dystrophy and Becker muscular dystrophy. Dystrophin interacts with a glycoprotein complex spanning the muscle sarcolemma, effectively linking the actin cytoskeleton to the extracellular matrix. The carboxyl terminus of dystrophin is required for glycoprotein binding. Interestingly, at least three mRNAs transcribed from the distal end of the DMD gene in tissues other than muscle have been shown to encode this domain. Deficiency of a second component of the dystrophin-associated glycoprotein complex has been shown to occur in another muscle-wasting disorder, severe childhood autosomal recessive muscular dystrophy. Sequence analysis of the entire cDNA for the autosomal dystrophin-related protein utrophin has shown that dystrophin and utrophin are closely related. Furthermore, both of these proteins have been shown to bind to the same or a similar glycoprotein complex in muscle.

The molecular defect in Duchenne muscular dystrophy is well established as being due to mutations at Xp21 which disrupt the normal synthesis of the 14kb dystrophin mRNA. More recently, several groups have identified a 4.8kb transcript from this locus which shares exons with the carboxy-terminal region of the dystrophin gene. In this paper we present evidence for an additional 2.2kb mRNA transcript. The 5' untranslated region and first 7 amino acids are identical to that published for the 4.8kb transcript. The position of the translational stop codon and 3' untranslated region is similar to that previously described as the truncated fetal dystrophin isoform. This 2.2kb mRNA has a similar tissue distribution to that described for the 4.8kb mRNA but unlike the other transcripts from the DMD locus, the 2.2kb mRNA is expressed in early development. The relevance of this transcript in the clinical expression of muscular dystrophy and developmental delay is discussed.

The continual expression of utrophin protein by pharmacological maintenance of utrophin transcription in dystrophin deficient muscle fibres is a potential disease modifying treatment for Duchenne muscular dystrophy (DMD). SMT C1100 is a small molecule utrophin modulator demonstrating significant benefit on the muscular dystrophy in the dystrophin deficient mdx mouse. Assessment of the in vitro and in vivo pharmacology plus the clean toxicology and safety data from the pre-clinical work led to the nomination of SMT C1100 as the first candidate for evaluation in DMD clinical trials. We previously reported that SMT C1100 successfully completed a Phase 1 healthy volunteer trial in which an oral paediatric formulation was deemed safe and well tolerated with plasma levels above those determined to be effective to modulate utrophin levels ex-vivo in DMD myoblasts and dystrophin deficient mdx mice. The first DMD paediatric patient trials of SMT C1100 started with an open-label, single and multiple oral dose finding Phase 1b study in DMD boys earlier this year. The purpose of the study was to determine whether increasing doses of SMT C1100 were safe, well tolerated and to understand the pharmacokinetic properties in patients with DMD. The boys were studied in 3 groups (Groups A to C), with each group consisting of 4 patients aged between 5 to 11 years. The doses for Groups A to C were administered in an escalating manner after safety review for each dose group. We will present the summary data from this trial.

The 14kb dystrophin transcript from the Duchenne muscular dystrophy (DMD) locus, which encodes a 427kDa protein, is differentially spliced at the amino terminal end giving rise to alternative transcripts expressed in muscle and brain. Here we present evidence for a 4.8kb transcript from the DMD locus which is ubiquitously expressed but is particularly abundant in Schwannoma cells where dystrophin could not be detected. The hybridisation of Western blots with dystrophin antibodies also identifies a protein of approximately 80kDa of variable abundance in different human and mdx tissues. Immunocytochemistry studies confirm the expression of this protein in nerve cells, a tissue in which full length dystrophin is not detected. Sequencing of the 5' end of a clone isolated from a rat Schwannoma cDNA library, shows that the 4.8kb transcript shares exons with the carboxy terminal end of dystrophin but the 5' untranslated region is not contained within the dystrophin transcript. We propose that the 4.8kb gene product be called apodystrophin-1 as its expression is distinct from the dystrophin 14kb mRNA but it is transcribed from the same locus.

Altered expression of dystrophin led to a reduction in the myocardial PCr to ATP ratio in MD carriers and BMD patients. Decreased myocardial PCr was also observed in the mouse model of MD, themdx mouse, which had decreased myocardial glucose uptake in response to insulin and during ischaemia, with increased susceptibility to ischaemic damage. However, utrophin transgene expression corrected the functional and metabolic abnormalities in the dystrophin-deficient mouse heart. To our knowledge, this is the first report of beneficial effects resulting from the expression of a dystrophin-related transgene in heart, making the findings important for the use of gene therapy in the treatment of cardiomyopathy in dystrophic patients.

The continual expression of utrophin protein by pharmacological maintenance of utrophin transcription in dystrophin deficient muscle fibres is a potential disease modifying treatment for Duchenne muscular dystrophy (DMD). In order for proof of concept of utrophin modulators to be demonstrated in DMD patients, a multicomponent biomarker strategy has been instigated to quantify utrophin levels and demonstrate evidence of reduced muscle fibre regeneration. To demonstrate an increase in utrophin derived from drug treatment above the natural levels resulting from regeneration, we aim to quantify both fibre numbers and levels of utrophin protein localised to mature fibres in pre- and post-dose muscle biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of regenerating fibres, determined by the presence of neonatal and foetal myosin, will be calculated from biopsies. Serum samples will be analysed to quantify the levels of specific microRNAs (miRs) associated with fibre leakage and peptide markers of active fibrosis which characterise fibre damage and degeneration respectively. We will present data from these candidate biomarkers tested in DMD, Becker and normal samples looking to demonstrate quantifiable changes which are indicative of benefit. In future clinical trials of utrophin modulators, and potentially other DMD therapeutic approaches, these biomarkers may be appropriate to confirm benefit to dystrophin deficient muscle.

This paper reviews the evidence that utrophin, the autosomally encoded protein related to dystrophin, may be capable of performing the same cellular functions as dystrophin. If this is the case, it may be possible to modify the regulation of utrophin expression as an alternative route to dystrophin gene therapy for sufferers of DMD and/or BMD.

DMD is a devastating X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches (e.g. exon skipping, read through of stop codons, gene therapy) are being developed. By pharmacologically modulating the dystrophin-related protein utrophin, we aim to develop a therapy applicable to all DMD patients by targeting the primary defect and restoring sarcolemmal stability. In partnership with Summit plc we previously developed SMT C1100; a small molecule utrophin modulator that reduced dystrophic symptoms in the mdx mouse. As a potential first-in-class molecule, SMT C1100 recently successfully completed a Phase 1a trial. DMD patients are currently being dosed in an ongoing Phase 1b trial for which a multicomponent biomarker strategy has been implemented with a quantification of utrophin level, regeneration, fibrosis, inflammation and analysis of specific miRNAs. The successful clinical progression to date provides crucial proof-of-concept for the strategy we developed. We are now optimising next generation utrophin modulator molecule which were discovered using an improved drug screening assay based on immortalised myoblasts from the utrophin luciferase knock-in mouse. This enables us to screen utrophin in its genomic context, better mimicking the in vivo situation and enabling identification of compounds which modulate utrophin through regulatory pathways outside of the 8.4kb promoter fragment used in our previous screen. Thus, we identified multiple structural classes which significantly modulate utrophin expression in both mouse and human DMD myoblasts. New compounds exhibit favourable solubility, stability, oral absorption and are well tolerated in the mouse and structure–activity studies are underway, with the objective to improve compound effectiveness. The two of the best new utrophin modulators are currently being studied in pre-clinical in vivo studies in mdx mice.

The continual expression of utrophin protein by pharmacological maintenance of utrophin transcription in dystrophin deficient muscle fibres is a potential disease modifying treatment for Duchenne muscular dystrophy (DMD). In order for proof of concept of utrophin modulators to be demonstrated in DMD patients, a multicomponent biomarker strategy has been instigated to quantify utrophin levels and demonstrate evidence of reduced muscle fibre regeneration. To demonstrate an increase in utrophin derived from drug treatment above the natural levels resulting from regeneration, we aim to quantify both fibre numbers and levels of utrophin protein localised to mature fibres in pre- and post-dose muscle biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of regenerating fibres, determined by the presence of neonatal and foetal myosin, will be calculated from biopsies. Serum samples will be analysed to quantify the levels of specific microRNAs (miRs) associated with fibre leakage and peptide markers of active fibrosis which characterise fibre damage and degeneration respectively. We will present data from these candidate biomarkers tested in DMD, Becker and normal samples looking to demonstrate quantifiable changes which are indicative of benefit. In future clinical trials of utrophin modulators, and potentially other DMD therapeutic approaches, these biomarkers may be appropriate to confirm benefit to dystrophin deficient muscle.

DMD is a devastating X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches (e.g. exon skipping, read through of stop codons, gene therapy) are being developed. By pharmacologically modulating the dystrophin-related protein utrophin, we aim to develop a therapy applicable to all DMD patients by targeting the primary defect and restoring sarcolemmal stability. In partnership with Summit plc we previously developed SMT C1100; a small molecule utrophin modulator that reduced dystrophic symptoms in the mdx mouse. As a potential first-in-class molecule, SMT C1100 recently successfully completed a Phase 1a trial. DMD patients are currently being dosed in an ongoing Phase 1b trial for which a multicomponent biomarker strategy has been implemented with a quantification of utrophin level, regeneration, fibrosis, inflammation and analysis of specific miRNAs. The successful clinical progression to date provides crucial proof-of-concept for the strategy we developed. We are now optimising next generation utrophin modulator molecule which were discovered using an improved drug screening assay based on immortalised myoblasts from the utrophin luciferase knock-in mouse. This enables us to screen utrophin in its genomic context, better mimicking the in vivo situation and enabling identification of compounds which modulate utrophin through regulatory pathways outside of the 8.4 kb promoter fragment used in our previous screen. Thus, we identified multiple structural classes which significantly modulate utrophin expression in both mouse and human DMD myoblasts. New compounds exhibit favourable solubility, stability, oral absorption and are well tolerated in the mouse and structure–activity studies are underway, with the objective to improve compound effectiveness. The two of the best new utrophin modulators are currently being studied in pre-clinical in vivo studies in mdx mice.

Duchenne muscular dystrophy (DMD) is an inherited, severe muscle wasting disease caused by the loss of the cytoskeletal protein, dystrophin. Patients usually die in their late teens or early twenties of cardiac or respiratory failure. We have previously demonstrated that the dystrophin related protein, utrophin is able to compensate for the loss of dystrophin in the mdx mouse, the mouse model of the disease. Expression of a utrophin transgene under the control of an HSA promoter results in localization of utrophin to the sarcolemma and prevents the muscle pathology. Here we show that the over-expression of full-length utrophin in a broad range of tissues is not detrimental in the mdx mouse. These findings have important implications for the feasibility of the up-regulation of utrophin in therapy for DMD since they suggest that tissue specific up-regulation may not be necessary.

[b]BACKGROUND[/b]: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. [b]MATERIALS AND METHODS[/b]: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (

DYSTROPHIN-RELATED protein (DRP or 'utrophin'1) is localized in normal adult muscle primarily at the neuromuscular junction2–4. In the absence of dystrophin in Duchenne muscular dystrophy (DMD) patients, DRP is also present in the sarcolemma3–7. DRP is expressed in fetal and regenerating muscle and may play a similar role to dystrophin in early development3,7–9, although it remains to be determined whether DRP can functionally replace dystrophin in adult tissue. Previously we described a 3.5-kilobase complementary DNA clone that exhibits 80 per cent homology to the C-terminal domain of dystrophin10. This sequence identifies a 13-kilobase transcript that maps to human chromosome 6 (refs 2, 11). Antibodies raised against the gene product identify a polypeptide with a relative molecular mass of about 400K in all tissues examined7,8,12. To investigate the relationship between DRP and dystrophin in more detail, we have cloned and sequenced the whole DRP cDNA. Homology between DRP and dystrophin extends over their entire length, suggesting that they derive from a common ancestral gene. Comparative analysis of primary sequences highlights regions of functional importance, including those that may mediate the localization of DRP and dystrophin in the muscle cell.

Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5′ end of the gene and have shown it to be synaptically regulated in vivo . In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3′ to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fiber leading to the gradual depletion of skeletal muscle. The molecular structure of dystrophin is very similar to that of the related protein utrophin. Utrophin is found in all tissues and is confined to the neuromuscular and myotendinous junctions in mature muscle. Sarcolemmal localization of a truncated utrophin transgene in the dystrophin-deficient mdx mouse significantly improves the dystrophic muscle phenotype. Therefore, up-regulation of utrophin by drug therapy is a plausible therapeutic approach in the treatment of DMD. Here we demonstrate that expression of full-length utrophin in mdx mice prevents the development of muscular dystrophy. We assessed muscle morphology, fiber regeneration and mechanical properties (force development and resistance to stretch) of mdx and transgenic mdx skeletal and diaphragm muscle. The utrophin levels required in muscle are significantly less than the normal endogenous utrophin levels seen in lung and kidney, and we provide evidence that the pathology depends on the amount of utrophin expression. These results also have important implications for DMD therapies in which utrophin replacement is achieved by delivery using exogenous vectors.

31P NMR spectroscopy was used to study the energy metabolism of dystrophin-deficient skeletal muscle of mdx mice, an animal model of Duchenne muscular dystrophy, in which expression of a truncated form of utrophin has been obtained through transgenesis technology. Measurements of ATP, phosphocreatine (PCr), inorganic phosphates (Pi) and intracellular pH (pHi) were made at rest, during a fatigue protocol and during the subsequent recovery. Mechanical fatigue of transgenic muscles was similar to normal muscle, while mdx muscle showed larger force loss. At rest, muscles of all groups had similar values for [ATP], [PCr], [Pi] and pHi. During fatigue, [PCr] decreases mirrored [Pi] increases and were similar in all groups. The major difference between mdx muscles and the group of normal and trc-utrophin muscles concerned the values and evolution of pHi. The mdx muscles showed a more severe intracellular acidosis during exercise and a slower and incomplete post-exercise recovery of normal pHi. In contrast, in trc-utrophin muscles, the kinetics and amplitude of pHi changes were remarkably close to normal behaviour. We conclude that the impaired proton washout which is present in mdx muscles, is corrected to a great extent by the expression of trc-utrophin.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease usually resulting in death of patients by their early twenties. In contrast, mice lacking dystrophin (Dmd(mdx)), appear physically normal despite their underlying muscle pathology. Mice deficient for both dystrophin and the dystrophin-related protein, utrophin, (Dmd(mdx);Utrn-/- mice) die between 6 and 20 weeks of age suffering from severe muscle weakness with joint contractures, pronounced growth retardation and kyphosis, suggesting that dystrophin and utrophin play complementary roles. The exact cause of death in these mice was not determined. Here we show that expression of a truncated utrophin transgene solely within the skeletal muscle of these mutants prevents premature death and the development of any clinical phenotype. In the absence of full-length dystrophin and utrophin, the presence of truncated utrophin also decreases muscle fibre regeneration, relocalizes the dystrophin protein complex to the sarcolemma and re-establishes a normal expression pattern of developmental muscle proteins. These data suggest that Dmd(mdx);Utrn-/- mice succumb to a skeletal muscle defect and that their reduced lifespan is not due to cardiac or neurogenic components. The phenotypic rescue observed demonstrates that the Dmd(mdx);Utrn-/- mice are an ideal model for testing gene delivery protocols for the expression of utrophin or dystrophin in skeletal muscle. To determine the cause of death of the Dmd(mdx):Utrn-/- mice.

Utrophin is a homologue of dystrophin, the protein whose absence is responsible for Duchenne muscular dystrophy (DMD). As a first step toward clarifying if adenovirus (AV)-mediated utrophin transfer is a possible option to treat DMD, we have constructed an AV expressing utrophin (AdCMV-Utr) and studied utrophin expression after intramuscular injection of mdx mice, the mouse DMD model. Overexpression of utrophin by AdCMV-Utr was marked and nontoxic. The recombinant utrophin was distributed homogeneously at the surface of the muscle fibers. Its expression was sufficient to restore the normal histochemical pattern of α-sarcoglycan and β-dystroglycan at this site. these two proteins are members of the dystrophin associated protein complex whose distribution is greatly reduced at the surface of the DMD muscle. These data indicate that AV-mediated utrophin transfer is an efficient way of utrophin upregulation in muscle and has the potential of becoming a treatment for DMD., UI - 98102816

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.

Utrophin modulation i.e. the re-programming of utrophin transcription such that utrophin RNA and protein is continually expressed in mature fibres is expected to be disease modifying in all genetic forms of DMD. SMTC1100 is a small molecule utrophin modulator demonstrating significant benefit on the muscular dystrophy in the dystrophin deficient mdx mouse. These data led to the nomination of SMTC1100 as the candidate for evaluation in DMD clinical trials. In 2012 we reported that SMTC1100 successfully completed a Phase 1 healthy volunteer trial in which an oral paediatric formulation was deemed safe and well tolerated with plasma levels well above those determined to be efficacious in cells and animals. Plans for the first patient trials of SMTC1100 have been developed consisting of two components; a safety and dose finding study in DMD boys in late 2013 followed by a proof of concept study in 2014. In order for proof of concept to be demonstrated in patients, a multicomponent biomarker strategy has been implemented that comprises of two modules; the increase of utrophin levels and evidence of reduction in muscle regeneration. To demonstrate increased utrophin derived from drug treatment above that normally found in regenerating DMD muscle, we aim to quantify utrophin RNA, total utrophin protein and utrophin fibre localisation derived from pre- and post-dose biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of newly regenerating fibres as determined by the presence of neonatal and foetal myosin will be calculated from the biopsies. Using serum and urine samples we will quantify the levels of specific miRNAs associated with fibre leakage and peptide markers of active fibrosis which characterises fibre damage and degeneration respectively. We will present the patient clinical trial plans and data from candidate biomarkers tested both in DMD samples and dystrophin deficient animals.