COMPOSITION COMPRISING THE ANTI-SENSE OLIGONUCLEOTIDE AND ITS USE FOR THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY.

Abstract

The present invention relates to a composition containing an antisense oligonucleotide and its use for treating Duchenne muscular dystrophy. The present invention relates in particular to the composition described above, which is effective for the treatment of Duchenne muscular dystrophy when administered at a treatment dose and for use thereof.

Description

COMPOSITION COMPRISING THE ANTISENSOR OLIGONUCLEOTIDE AND ITS USE FOR THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY FIELD OF INVENTION The present invention relates to the dosing and administration of an antisense oligomer capable of omitting exon 53 of a human dystrophin gene, and a pharmaceutical composition comprising the oligomer. BACKGROUND OF THE INVENTION Duchenne muscular dystrophy (DMD) is the most common inherited progressive muscular atrophy, affecting approximately one in 3,500 boys at birth. In infancy, individuals with DMD exhibit motor functions nearly identical to those of healthy individuals, but muscle weakness begins around age four or five. This weakness then progresses, and most individuals are unable to walk by age 12. Patients typically die from heart or respiratory failure by age 20. Therefore, DMD is a very serious disease. Currently, there are no effective treatments for DMD, and the development of a new therapeutic agent has been highly desired. DMD is known to be caused by a mutation in the dystrophin gene. The dystrophin gene is a very large gene consisting of 2,200,000 base pairs of DNA located on the X chromosome. This DNA is transcribed into a precursor mRNA, and then the introns are removed by splicing, resulting in mRNA containing 79 exons. From this mRNA, 3,685 amino acids are translated, generating the dystrophin protein. The dystrophin protein is associated with maintaining the stability of muscle cell membranes and is necessary to prevent muscle cell destruction. Because the dystrophin gene in DMD patients is mutated, little functional dystrophin protein is expressed in the muscle cells. Therefore, in the bodies of DMD patients, the structure of muscle cells cannot be maintained, and a large amount of calcium ions flows into the muscle cells.As a result, an inflammation-like reaction occurs, fibrosis progresses, and therefore, muscle cells do not regenerate. Becker muscular dystrophy (BMD) is also caused by a mutation in a dystrophin gene. Symptoms of BMD also include muscle weakness due to muscle atrophy, but the weakness is generally milder than in DMD, and it progresses slowly. In most cases, BMD develops in adulthood. These differences in clinical symptoms between DMD and BMD have been thought to be caused by whether the amino acid reading frame is disrupted due to a mutation or preserved when dystrophin mRNA is translated into a dystrophin protein (non-patent literature 1). That is, in DMD, there is an amino acid reading frame shift mutation, and therefore, almost no functional dystrophin proteins are expressed.On the other hand, in DMO, although some exons are eliminated by mutation, the amino acid reading frame is maintained and thus functional dystrophin proteins are generated, although the function is insufficient. As a therapeutic approach for DMD, an exon skipping method is anticipated. According to this method, the amino acid reading frame of dystrophin mRNA is restored by modifying the splicing process, and the expression of a dystrophin protein with partially recovered function is induced (non-patent literature 2). The portion of the amino acid sequence targeted by the exon skipping is lost. As such, the dystrophin protein expressed as a result of this treatment becomes shorter than a normal dystrophin protein. However, since the amino acid reading frame is maintained, the function of stabilizing muscle cells is partially preserved. Consequently, it is expected that, as a result of exon skipping, DMD will present with symptoms similar to those of a milder form of MODS.The exon skipping method has been tested in animal experiments with mice or dogs, and clinical studies are now being conducted in human patients with DMD. Exon omission can be induced by the binding of antisense nucleic acids to one or both of the 5' and 3' splice sites, or to the interior of an exon. An exon is incorporated into mRNA only when both splice sites are recognized by a spliceosome complex. Therefore, targeting the splice sites with antisense nucleic acids can induce exon omission. Furthermore, for exon recognition by the splicing mechanism, it has been considered necessary for the SR protein to bind to an exon splicing enhancer (ESE), and exon omission can also be induced by targeting the ESE. The dystrophin gene mutation varies among individual DMD patients. Therefore, customized antisense nucleic acids are needed depending on the position or type of genetic mutation. To date, antisense nucleic acids that induce exon skipping to all 79 exons have been produced by Steve Wilton et al. at the University of Western Australia (Non-Proprietary Literature 3), and antisense nucleic acids that induce exon skipping to all 39 exons have also been produced by Annemieke Aartsma-Rus et al. in the Netherlands (Non-Proprietary Literature 4). It has been estimated that approximately 10% of all DMD patients can be treated by omitting exon 53 (hereafter referred to as exon 53). In recent years, several research institutions have reported studies on omitting exon 53 of a dystrophin gene (Patent Literatures 1 to 4; and Non-Patent Literatures 5 and 6). List of Appointments Patent Literature: Patent Literature 1: International Publication No. WO 2006 / 000057 Patent Literature 2: International Publication No. WO 2004 / 048570 Patent Literature 3: US Patent Publication No. US 2010 / 0168212 Patent Literature 4: International Publication No. WO 2010 / 048586 Non-Patent Literature: Non-Patent Literature 1: Monaco AP et al., Genomics 1988; 2: pp. 90-95 Non-Patent Literature 2: Matsuo M., Brain Dev 1996; 18: pp.167-172 Non-Patent Literature 3: Wilton SD et al., Molecular Therapy 2007:15: pp. 1288-96 Non-Patent Literature 4: Annemieke Aartsma-Rus et al., (2002) Neuromuscular Disorders 12: S71-S77 Non-Patent Literature 5: Linda J. Popplewell et al., (2010) Neuromuscular Disorders, vol. 20, no. 2, pp. 102-10 Non-Patent Literature 6: Bladen CL et al., Human Mutation (2015) 36:395-402 BRIEF DESCRIPTION OF THE INVENTION The present invention is as follows, but is not limited to it. <1> A pharmaceutical composition for the treatment of a human patient with Duchenne muscular dystrophy, the pharmaceutical composition comprising an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' terminus of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the treatment comprises intravenous administration to the human patient of the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive. <2> The pharmaceutical composition according to the above <1> in which the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient at a dose of 40 mg / kg / week. <3> The pharmaceutical composition according to the above <1> in which the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient at a dose of 80 mg / kg / week. <4> The pharmaceutical composition according to the above <1>, wherein the human patient has a mutation resulting in a deficiency of any exon selected from the group consisting of exons 43-52, 45-52, 47-52, 48-52, 49-52, 50-52 or 52, in a dystrophin gene. <5> The pharmaceutical composition according to the above <1> , in which the expression of a dystrophin protein in the human patient before treatment is 1% or less compared to that of a healthy subject, as measured by Western Blot or mass spectrometry. <6> The pharmaceutical composition in accordance with the 5 above, in which the expression of a dystrophin protein is not found in the human patient before treatment. The pharmaceutical composition according to the above <1> , wherein the antisense oligomer base sequence consists of the sequence established in SEQ ID NO: 3. <8> The pharmaceutical composition according to the above <1> wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is Viltolarsen or an equivalent thereof. <9> The pharmaceutical composition according to the above <1> comprising the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of between 2.5 mg / ml inclusive and 500 mg / ml inclusive, or between 10 mg / ml inclusive and 100 mg / ml inclusive. <10> The pharmaceutical composition according to the above <1> , comprising the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of 25 mg / ml. <11> The pharmaceutical composition according to the above <1> , comprising the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of 50 mg / ml. <12> The pharmaceutical composition according to the above <1> , further comprising at least one component selected from the group consisting of a tonicity agent, a pH adjuster, and a solvent. <13> The pharmaceutical composition in accordance with the 12 above, wherein the tonicity agent is at least one selected from the group consisting of sodium chloride, potassium chloride, glucose, fructose, maltose, sucrose, lactose, mannitol, sorbitol, xylitol, trehalose, and glycerin. <14> The pharmaceutical composition according to the 12 or 13 above, wherein the pH adjuster is at least one selected from the group consisting of hydrochloric acid, sodium hydroxide, citric acid, lactic acid, phosphate (sodium hydrogen phosphate, potassium dihydrogen phosphate and potassium dihydrogen phosphate) and monoethanolamine. <15> The pharmaceutical composition in accordance with any of the 12 to 14 above, wherein the solvent is water. <16> The pharmaceutical composition according to the above <1> , comprising the antisense oligomer in a concentration of between 2.5 mg / ml inclusive and 500 mg / ml inclusive, or between 10 mg / ml inclusive and 100 mg / ml inclusive, and sodium chloride in a concentration between 8 mg / ml inclusive and 10 mg / ml inclusive, and which is an aqueous solution with a pH of 7.2 to 7.4. <17> The pharmaceutical composition according to the above <1> , wherein the treatment provides at least one effect selected from the group consisting of the following effects (1)a(6): (1) the mean value of the expression level of a dystrophin protein in the patient's skeletal muscle increases 9 times or more compared to the initial value, after administration of the pharmaceutical composition for 24 weeks; (2) a change in velocity obtained from time to standby (TTSTAND) is 0.055 times / sec or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; (3) a change in speed obtained from the 10-meter run / walk time (TTRW) is -0.025 meters / sec or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; (4) a change in speed obtained from time to climb 4 stairs (TTCLIMB) is -0.060 times / sec or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; (5) a change in the North Star Ambulatory Assessment (NSAA) score is 2.2 scores or more compared to the baseline value, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; and (6) a change in the 6-minute walk test (6MWT) is -7.5 meters or more compared to the baseline value, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks. <18> The pharmaceutical composition according to the above <1> , wherein when the treatment is carried out in human patients aged 7 to 9 years with Duchenne muscular dystrophy for 84 weeks, at least one effect selected from the group consisting of the following effects (1) to (6) is provided: (1) the percentage of patients who lose the ability to grow is less than 20% at week 85 after the start of treatment; (2) the percentage of patients who lose the ability to climb 4 steps is less than 10% at week 85 after the start of treatment; (3) the percentage of patients who lose the ability to walk independently is less than 10% at week 85 after the start of treatment; (4) no reduction in 10-meter running / walking speed due to aging is observed at week 85 after the start of treatment; (5) no reduction in the speed of climbing 4 steps due to aging is observed at the time of week 85 after the start of treatment; and (6) no reduction in the speed of ascent due to aging is observed at the time of week 85 after the start of treatment. <19> The pharmaceutical composition according to the above <1> , wherein when the treatment is performed on human patients aged 10 to 12 years with Duchenne muscular dystrophy for 84 weeks, at least one effect selected from the group consisting of the following effects (1) to (6) is provided: (1) the percentage of patients who lose the ability to grow is less than 60% at week 85 after the start of treatment; (2) the percentage of patients who lose the ability to climb 4 steps is less than 50% at the time of the 85th week after the start of treatment; (3) the percentage of patients who lose the ability to walk independently is less than 50% at week 85 after the start of treatment; (4) no reduction in 10-meter running / walking speed due to aging is observed at week 85 after the start of treatment; (5) a period is observed in which the speed of climbing 4 stairs increases at the time of week 85 after the start of treatment; and (6) a period is observed in which the speed of climbing increases at the time of week 85 after the start of treatment. <20> A method for treating Duchenne muscular dystrophy, comprising the intravenous administration of a pharmaceutical composition comprising an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' terminus of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, to a human patient once weekly at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive of the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof. <20-1 > The treatment method according to the preceding 20, wherein at least one of the effects according to claim 17, at least one of the effects according to claim 18, or at least one of the effects according to claim 19, is provided by the treatment method. <21> An antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' terminal of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, for use in a method for treating a human patient with Duchenne muscular dystrophy, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof is administered intravenously to the human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive. <21-1> The antisense oligomer according to the preceding 21, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein at least one of the effects according to claim 17, at least one of the effects according to claim 18, or at least one of the effects according to claim 19 is provided by administration of the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof. <22> Use of an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' terminal of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, for the manufacture of a pharmaceutical composition for the treatment of a human patient with Duchenne muscular dystrophy, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof is administered intravenously to the human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive. Furthermore, as another aspect, the present invention is as follows, but is not limited to it. [1] A method for treating a subject with Duchenne muscular dystrophy amenable to treatment involving the omission of exon 53, wherein the method comprises a step of intravenously administering NS-065 / NCNP-01 to the subject at a dose of approximately 40 mg / kg / week. [2] A method for treating a subject with Duchenne muscular dystrophy amenable to treatment involving the omission of exon 53, wherein the method comprises a step of intravenously administering NS-065 / NCNP-01 to the subject at a dose of approximately 80 mg / kg / week. [3] A method for treating a subject with Duchenne muscular dystrophy amenable to treatment involving the omission of exon 53, wherein the method comprises a step of intravenously administering NS-065 / NCNP-01 to the subject at a dose of 40 mg / kg / week or more and 80 mg / kg / week or less. [4] The method according to [1] above, wherein what is administered is an aqueous solution comprising: NS-065 / NCNP-01 at a concentration of 2.5 mg / mL or more and 500 mg / mL or less; and sodium chloride as a tonic agent at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less, and wherein the aqueous solution has a pH value of approximately 7.3. [5] The method according to the above [2], in which what is administered is an aqueous solution comprising: NS-065 / NCNP-01 at a concentration of 2.5 mg / mL or more and 500 mg / mL or less; and sodium chloride as a tonic agent at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less, and wherein the aqueous solution has a pH value of approximately 7.3. [6] The method according to the above [3], in which what is administered is an aqueous solution comprising: NS-065 / NCNP-01 at a concentration of 2.5 mg / mL or more and 500 mg / mL or less; and sodium chloride as a tonic agent at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less, and wherein the aqueous solution has a pH value of approximately 7.3. [7] A method for inducing the generation of a dystrophin protein in a subject with Duchenne muscular dystrophy amenable to treatment involving the skipping of exon 53, wherein the method comprises a step of intravenously administering NS-065 / NCNP01 to the subject at a dose of approximately 40 mg / kg / week. [8] A method for inducing the generation of a dystrophin protein in a subject with Duchenne muscular dystrophy amenable to treatment involving the skipping of exon 53, wherein the method comprises a step of intravenously administering NS-065 / NCNP01 to the subject at a dose of approximately 80 mg / kg / week. [9] A method for inducing the generation of a dystrophin protein in a subject with Duchenne muscular dystrophy amenable to treatment involving the omission of exon 53, wherein the method comprises a step of intravenously administering NS-065 / NCNP01 to the subject at a dose of 40 mg / kg / week or more and 80 mg / kg / week or less.

[10] The method according to the above [7], in which what is administered is an aqueous solution comprising: NS-065 / NCNP-01 at a concentration of 2.5 mg / mL or more and 500 mg / mL or less; and sodium chloride as a tonic agent at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less, and wherein the aqueous solution has a pH value of approximately 7.3.

[11] The method according to the above [8], where what is administered is an aqueous solution comprising: NS-065 / NCNP-01 at a concentration of 2.5 mg / mL or more and 500 mg / mL or less; and sodium chloride as a tonic agent at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less, and wherein the aqueous solution has a pH value of approximately 7.3.

[12] The method according to the above [9], wherein what is administered is an aqueous solution comprising: NS-065 / NCNP-01 at a concentration of 2.5 mg / mL or more and 500 mg / mL or less; and sodium chloride as a tonic agent at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less, and wherein the aqueous solution has a pH value of approximately 7.3. In the preceding [1] to

[12] , NS-065 / NCNP-01 (also referred to as Viltolarsen in this description) may also be an equivalent of the same. Furthermore, in the preceding [1] to

[12] , the subject may also be a human patient. According to the present invention, a pharmaceutical composition is provided for use in the treatment of Duchenne muscular dystrophy, having a stable composition of Viltolarsen. Furthermore, with respect to the pharmaceutical composition comprising Viltolarsen, dosage and method of administration of Viltolarsen are provided, which exhibit effective therapeutic effects on Duchenne muscular dystrophy and are within a safe range for human patients. Using the pharmaceutical composition, the symptoms of noor Ln / nznz / E / γΐΛΐ Low secondary effects of Duchenne muscular dystrophy can be effectively reduced with BRIEF DESCRIPTION OF FIGURES Figure 1 Shows a design of the US Phase 2 dose-finding study NS065 / NCNP-01-201. Figure 2 Shows the results of measuring the de novo expression level of a dystrophin protein in skeletal muscle using a Western blot method. Figure 3 Shows a design of the US / Canada Phase 2 dose-finding study NS065 / NCNP-01-201. Figure 4 Shows comparisons with respect to changes from baseline in the results of timed function tests performed over 24 weeks. In all five graphs, the term Viltolarsen means the international nonproprietary name (INN) of NS-065 / NCNP-01. Figure 5 shows the results of the stability evaluation of Viltolarsen in a Britton-Robinson buffer (pH 3 to 11).Figure 6 shows the results of the stability evaluation of Viltolarsen in a potassium phosphate-borax buffer (pH 6 to 9). Figure 7 shows the results of the pH adjuster examination at 121°C. Figure 8 includes graphs showing changes from baseline in individual motor function test results. Figure 9 is a graph showing the results of a 6-minute walk test performed on individual patients assessed from the Viltolarsen-administered groups (40 mg / kg dose group and 80 mg / kg dose group) at week 85 (84 weeks after initial administration). Figure 10 is a graph showing the North Star Ambulatory Assessment scores performed on individual patients assessed from the Viltolarsen-administered groups (40 mg / kg dose group and 80 mg / kg dose group) at week 85 (84 weeks after initial administration).Figure 11 is a graph showing the results of a standing time test performed on individual patients from the Viltolarsen-administered groups (40 mg / kg dose group and 80 mg / kg dose group) at week 85 (84 weeks after initial administration). Figure 12 is a graph showing the results of a 4-stair climb test performed on individual patients from the Viltolarsen-administered groups (40 mg / kg dose group and 80 mg / kg dose group) at week 85 (84 weeks after initial administration). noor Ln / nznz / E / γΐΛΐ Figure 13 Figure 14 Figure 15 Figure 16 It is a graph showing the speed results of a 10-meter run / walk time test performed on individual patients evaluated from the administered groups of Viltolarsen (40 mg / kg dose group and 80 mg / kg dose group) at the time of week 85 (after 84 weeks from initial administration). It is a graph showing a correlation between a change in dystrophin expression level from baseline of a quantitative dystrophin value measured by WB and a change in the speed of a time to resist test from baseline, in individual patients assessed from Viltolarsen administered groups (40 mg / kg dose group and 80 mg / kg dose group) at the time of week 49 (after 48 weeks from initial administration). It is a graph showing a correlation between a change in the level of dystrophin expression from baseline of a quantitative dystrophin value measured by WB and a change in the speed of a timed 4-stair climb test from baseline, in individual patients assessed from the groups administered with Viltolarsen (40 mg / kg dose group and 80 mg / kg dose group) at week 49 (after 48 weeks from initial administration). It is a graph showing a correlation between a change in the level of dystrophin expression from baseline of a quantitative dystrophin value measured by WB and a change in the speed of a 10-meter run / walk time test from baseline, in individual patients assessed from the groups administered Viltolarsen (40 mg / kg dose group and 80 mg / kg dose group) at week 49 (after 48 weeks from initial administration). DETAILED DESCRIPTION OF THE INVENTION The present invention will now be described in detail. The following embodiments are provided as examples to illustrate the present invention and are therefore not intended to limit the present invention to only these embodiments. The present invention can be carried out in various embodiments without departing from the spirit of the invention. All patent publications and filings, such as open patent filings or patent applications cited in this description, are incorporated herein by reference in their entirety. In addition, this description includes the content described in the specifications and drawings of U.S. Provisional Patent Application (US62 / 690,270) filed June 26, 2018, and U.S. Provisional Patent Application (US62 / 739,386) filed October 1, 2018, to both of which this application claims priority. L First Realization In a First Embodiment of the present invention, a pharmaceutical composition for treating Duchenne muscular dystrophy is provided. Specifically, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of a human patient with Duchenne muscular dystrophy, the pharmaceutical composition comprising an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5'-terminal exon 53 of a human dystrophin gene (hereinafter also referred to as the oligomer of the present invention), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the treatment comprises the intravenous administration to the human patient of the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive. 53v0 Human Dystrophin Gene Exon In the present invention, the term gene includes cDNA, an mRNA precursor and mRNA, as well as a genomic gene. The gene is preferably an mRNA precursor, i.e., premRNA. In the human genome, a human dystrophin gene is located at the Xp21.2 gene locus. The human dystrophin gene is 3.0 Mbp in size and is the largest known human gene. However, the coding region of the human dystrophin gene is only 14 kb, and this region is dispersed as 79 exons (Roberts, R.G., et al., Genomics, 16: 536-538 (1993)). The pre-mRNA transcriptional product of the human dystrophin gene yields 14 kb mature mRNA as a result of splicing. The base sequence of the mature mRNA of a wild-type human dystrophin gene is known (GenBank Accession No. NM_004006). The base sequence of exon 53 of the wild-type human dystrophin gene is the one shown in SEQ ID NO: 1. The pharmaceutical composition of the present invention comprises an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' terminal of exon 53 of a human dystrophin gene (the oligomer of the present invention), or a pharmaceutically acceptable salt thereof, or a hydrate thereof. In this document, the oligomer of the present invention is produced for the purpose of modifying a protein encoded by a DMD-type dystrophin gene to a BMD-type dystrophin protein by omitting exon 53. Accordingly, exon 53 of a dystrophin gene as the target of exon omission with the oligomer of the present invention includes not only wild-type exon 53 but also mutant-type exon 53. A specific example of such a mutant-type exon 53 of a human dystrophin gene may be a polynucleotide having an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, with respect to the base sequence as set out in SEQ ID NO: 1. In the present description, the term polynucleotide means DNA or RNA. Furthermore, the identity of base sequences can be determined using the BLAST (Basic Local Alignment Search Tool) algorithm of Carlin and Arthur (Proc. Nati. Acad. Sci. USA 872264-2268, 1990; Proc Nati Acad Sci USA 90: 5873, 1993). Based on the BLAST algorithm, programs called BLASTN or BLASTX have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, the parameters are set to, for example, score = 100 and word length = 12. When using the BLAST and Gapped BLAST programs, the default parameters of the individual programs are used. In this description, the complementary base sequence is not limited to a base sequence that forms a Watson-Crick base pair with the target base sequence, but also includes a base sequence that forms a wobble base pair. In this document, the term Watson-Crick base pair means a base pair in which hydrogen bonds are formed between adenine-thymine, adenine-uracil, and guanine-cytosine, while the term wobble base pair means a base pair in which hydrogen bonds are formed between guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine. Furthermore, the complementary base sequence may not have 100% complementarity with the target base sequence and, for example, may comprise 1, 2, 3, 4, or 5 bases that are not complementary to the target base sequence.In addition, the complementary base sequence can also be a base sequence that is shorter than the target base sequence by 1, 2, 3, 4, or 5 bases. The following table shows examples of the sequence consisting of nucleotides at positions 36 to 56 of the 5' terminal of exon 53 (SEQ ID NO: 2) and a sequence of bases complementary to the aforementioned sequence (SEQ ID NO: 3) 1. Table 1 5'-GAACACCTTCAGAACCGGAGG-3' SEQ ID NO: 2 5'-CCTCCGGTTCGAAGGTGTTC-3' SEQ ID NO: 3 In this case, thymine (T) and uracil (U) can be interchanged. Whether the base is T or U does not substantially affect the exon skipping activity of the oligomer of the present invention. Therefore, in this application, even if the T in the base sequence with a certain sequence number is U, it is shown with the same sequence number. Thus, the sequence described in this application inevitably includes both a T sequence and a U sequence. In view of the foregoing, the base sequence of the oligomer of the present invention may consist of the sequence established in SEQ ID NO: 3. Furthermore, the oligomer of the present invention may not have a base sequence that is 100% complementary to the target sequence, provided that it permits the omission of exon 53 of a human dystrophin gene. For example, the oligomer of the present invention may comprise 1, 2, 3, 4, or 5 bases that are not complementary to SEQ ID NO: 2 as the target sequence. Alternatively, the oligomer of the present invention may be a base sequence that is 1, 2, 3, 4, or 5 bases shorter than the target base sequence. Whether or not the omission of exon 53 of a human dystrophin gene has occurred can be confirmed by: introducing the oligomer of the present invention into dystrophin-expressing cells (e.g., human rhabdomyosarcoma cells), amplifying a peripheral region of exon 53 of the mRNA of a human dystrophin gene, from the total RNA of the dystrophin-expressing cells described above, by RT-PCR; and then performing nested PCR or sequence analysis on the PCR-amplified product. Alternatively, whether such an omission has occurred or not, it can also be confirmed by measuring the amount of exon 53 using a method such as RT-PCR, Western Blot, or mass spectrometry in a sample derived from a patient to whom the oligomer of the present invention has been administered. Skipping efficiency can be obtained by retrieving mRNA from a human dystrophin gene from test cells, then measuring the amount of polynucleotide A from a band involving skipping of exon 53 and the amount of polynucleotide B from a band not involving skipping of exon 53 in the mRNA, and then calculating the skipping efficiency according to the following equation based on the A and B measurement values. Efficiency omission (%) = A / (A + B) x 100 The oligomer of the present invention may include an oligonucleotide, a morpholline oligomer, and a peptide nucleic acid (PNA) oligomer. The oligomer of the present invention is preferably a morpholline oligomer. The oligonucleotide described above (hereinafter referred to as the oligonucleotide of the present invention) is an oligomer of the present invention comprising a nucleotide as its constituent unit, and said nucleotide can be any of a ribonucleotide, a deoxyribonucleotide, or a nucleotide. Modified nucleotide means a ribonucleotide or a deoxyribonucleotide, in which all or part of the nucleic acid bases, sugar portions, and phosphate bond portions that constitute the ribonucleotide or deoxyribonucleotide are modified. [Examples of nucleic acid bases may include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and a modified nucleotide of the same.An example of such a modified nucleotide may include, but is not limited to, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracyl (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouratilaminomethylcarboxyl, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytesine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole and xanthine. Examples of modification of a sugar moiety can include modification of the 2' position of ribose and modification of other positions within a sugar moiety. Modification of the 2' position of ribose can be, for example, by substituting the -OH group at the 2' position of ribose with OR, R, R', OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, or I. Here, R indicates alkyl or aryl. R' indicates alkylene. Examples of modification of other positions of a sugar portion may include, among others, the substitution of O at the 4' position of ribose or deoxyribose by S, and crosslinking between the 2' and 4' positions of a sugar portion, such as LNA (Blocked Nucleic Acid) or ENA (Nucleic acids with 2'-O, 4'-C ethylene bridges). Examples of modification of a phosphate bond portion can be the modification by substitution of a phosphodiester bond with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or a boranophosphate bond (Enya et al.: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (see, for example, the Reissue of International Publications PCT Nos. 2006 / 129594 and 2006 / 038608). As an alkyl group, a linear or branched alkyl containing 1 to 6 carbon atoms is preferred. Specific examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, and isohexyl. The alkyl group may be substituted. Examples of such substituents include halogen, alkoxy, cyano, and nitro. The alkyl group may be substituted with 1 to 3 of these substituents. As a cycloalkyl, a cycloalkyl containing 5 to 12 carbon atoms is preferred. Specific examples of such a cycloalkyl include cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl. Examples of halogens can include fluorine, chlorine, bromine, and iodine. Examples of alkoxy include linear or branched alkoxy containing 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, and isohexyloxy. Among others, an alkoxy containing 1 to 3 carbon atoms is preferred. As an aryl group, one containing 6 to 10 carbon atoms is preferred. Specific examples of such an aryl group include phenyl, α-naphthyl, and β-naphthyl. Phenyl is preferred, among others. The aryl group may be substituted. Examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro. The aryl group may be substituted with 1 to 3 of these substituents. As an alkylene, a linear or branched alkylene containing 1 to 6 carbon atoms is preferred. Specific examples of such an alkylene may include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene. Examples of an acyl group include linear or branched alkanoyls and aroyls. Examples of alkanoyls include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, and hexanoyl. Examples of aroyls include benzoyl, toluyl, and naphthoyl. Such an aroyl group may be substituted at a replaceable position and may be substituted with an alkyl group. In one aspect where the oligomer of the present invention is an oligonucleotide, the oligonucleotide may preferably comprise, as a constituent unit, a group represented by the following general formula, wherein the -OH group at the 2' position of the ribose is substituted with a methoxy group and the phosphate bond portion is a phosphorothioate bond: Base <3 OCH3 Formula 1 where Base indicates a nucleic acid base. This oligonucleotide can be easily synthesized using various types of automated synthesizers (e.g., AKTA oligopilot plus 10 / 100 (GE Healthcare)). Alternatively, the oligonucleotide can also be produced by outsourcing its synthesis to a third-party organization (e.g., Promega or Takara), etc. When the oligomer of the present invention is a morpholino oligomer, the morpholino oligomer may comprise, as a constitutional unit, a group represented by the following general formula: £ 'WI Formula 2 where Base is as defined above; and W represents a group represented by any of the following formulas: Formula 3 where X represents -CH2R1, -O-CH2R1, -S-CH2R1, -NR2R3, or F; R1 represents H or alkyl; R2 and R3, which are the same or different, each represents H, alkyl, cycloalkyl, or aryl; Yi represents O, S, CH2, or NR1; Y2 represents O, S, or NR1; and Z represents O or S. The morpholino oligomer is preferably an oligomer comprising, as a constitutional unit, a group represented by the following formula (i.e., a phosphorodiamidate morpholino oligomer (hereafter referred to as PMO)): VWVWUW R2 N' O R31^0 Base i Formula 4 where Base, R2 and R3 are as described above. The morpholino oligomer can be produced, for example, according to International Publication No. WO 1991 / 009033 or International Publication No. WO 2009 / 064471. In particular, PMO can be produced according to the method described in International Publication No. WO 2009 / 064471, or according to the method described below. In one respect, PMO may include, for example, a compound represented by the following general formula (I) (hereafter referred to as PMO (I)): H--O (YO) Formula 5 where each Base, R2 and R3 are as defined above; yn represents any given integer in the range of 1 to 99, and preferably represents any given integer in the range of 18 to 28. PMO(I) can be produced according to a known method, and the compounds and reagents used in its production are not particularly limited, provided they are commonly used in PMO production. Furthermore, production can be carried out using a liquid-phase method or a solid-phase method (in which manual or commercially available automated solid-phase synthesizers are used). When PMO is produced using a solid-phase method, the use of an automated synthesizer is desirable from the standpoint of simplifying operating procedures and improving the accuracy of the synthesis. The peptide nucleic acid is an oligomer of the present invention comprising, as a constitutional unit, a group represented by the following general formula: Base Formula 6 where Base is as described above. The peptide nucleic acid can be produced, for example, according to the following publications: 1) PE Nielsen, M. Egholm, RH Berg, O. Buchardt, Science, 254, 1497 (1991) 2) M. Egholm, O. Buchardt, PE Nielsen, RH Berg, Jacs., 114, 1895 (1992) 3) KL Dueholm, M. Egholm, C. Behrens, L. Christensen, HF Hansen, T. Vulpius, KH Petersen, RH Berg, PE Nielsen, O. Buchardt, J. Org. Chem. 59, 5767 (1994) 4) L. Christensen, R. Fitzpatrick, B. Gildea, KH Petersen, HF Hansen, T. Koch, M. Egholm, O. Buchardt, PE Nielsen, J. Coull, RH Berg, J. Pept. Sci., 1, 175 (1995) 5) T. Koch, H.F. Hansen, P. Andersen, T. Larsen, H.G. Batz, K. Otteson, H. Orum, J. Pept. Res., 49, 80 (1997) Likewise, the 5' terminal of the oligomer of the present invention can be a group represented by any of the following chemical forms (1) to (3). Preferably it is OH shown in (3). Formula 7 From now on, the groups represented by the formulas (1), (2) and (3) above are referred to as group (1), group (2) and group (3), respectively. Examples of pharmaceutically acceptable salts of the oligomer of the present invention may include: alkali metal salts, such as sodium salts, potassium salts, or lithium salts; alkaline earth metal salts, such as calcium salts or magnesium salts; metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, or cobalt salts; ammonium salts; organic amine salts, such as t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzylphenethylamine salts, piperazine salts, tetramethylammonium salts or tris(hydroxymethyl)aminomethamine salts;Salts of hydrohalogenic acids, such as hydrofluoride, hydrochloride, hydrobromide, or hydroiodide; salts of inorganic acids, such as nitrate, perchlorate, sulfate, or phosphate; lower alkanesulfonates, such as methanesulfonate, trifluoromethanesulfonate, or ethanesulfonate; arylsulfonates, such as benzenesulfonate or p-toluenesulfonate; salts of organic acids, such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, or maleate; and salts of amino acids, such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamate, or aspartate. These salts may be produced according to a known method. Otherwise, the oligomer of the present invention may be in the form of a hydrate thereof. In another aspect, the oligomer of the present invention may be Viltolarsen or an equivalent thereof. Viltolarsen es el nombre común internacional (DOI) de NS-065 / NCNP-01. En la presente descripción, NS-065 / NCNP-01 también se denomina NS-065 / NCNP-01 (Viltolarsen) o Viltolarsen, así como NS-065 / NCNP-01. NS-065 / NCNP-01 (Viltolarsen) is an antisense oligonucleotide drug for the treatment of patients with Duchenne muscular dystrophy (DMD) who are eligible for treatment involving the omission of exon 53. NS-065 / NCNP-01 (Viltolarsen) is a compound described as PMO No. 8 in U.S. Patent No. 9,079,934 B2. The base sequence of NS-065 / NCNP-01 (Viltolarsen) is as stated in SEQ ID No. 35 (5'-CCTCCGGTTC TGAAGGTGTTC-3'; SEQ ID No. 3 in this description), and the 5' terminal is therefore -OH. The contents of U.S. Patent No. 9,079,934 B2 are incorporated herein by reference in their entirety. In addition, U.S. Patent No. 9,079,934 B2 describes a method for synthesizing PMO No. 8, namely NS-065 / NCNP-01 (Viltolarsen). NS-065 / NCNP-01 (Viltolarsen) has a morpholino backbone that is expected to provide greater safety than the phosphorothioate oligonucleotide. For example, the development of a phosphorothioate oligonucleotide, drisapersen (from BioMarin), has been suspended due to safety concerns. On the other hand, the FDA approved a morpholino oligonucleotide, eteplirsen (Exondys51® from Sarepta). Both drisapersen and eteplirsen are for DMD patients who are candidates for treatment involving the omission of exon 51. Eteplirsen is described in U.S. Patent No. 9,506,058 B2, and its contents are incorporated herein by reference in their entirety. As described in U.S. Patent No. 9,079,934 B2, NS065 / NCNP-01, (Viltolarsen) has been designed to exhibit specific exon 53 skipping activity in order to produce a functional dystrophin protein in DMD patients with specific exon deficiencies, including exons 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, or 52. Examples of mutations causing theoretically curable DMD by skipping a specific exon are given in Table 3 of Aartsma-Rus et al., 2002. is incorporated by reference in its entirety (Annemieke Aartsma-Rus, Mattie Bremmer-Bout, Anneke AM Janson, Johan T. den Dunnen, Gert-Jan B. van Ommen and Judith CT van Deutekom, “Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy”, Neuromuscular Disorders, Vol. 12, pp. S71-S77 (2002)). A Viltolarsen equivalent is a compound that is either a generic version of Viltolarsen or an active ingredient thereof. Such equivalents have been approved for manufacture and sale under the Pharmaceutical Affairs Act based on the safety and efficacy confirmed by clinical trials of Viltolarsen, without undergoing clinical trials for the equivalents themselves. Equivalents are expected to have exon 53 skipping activity similar to that of Viltolarsen. In some respects, a Viltolarsen equivalent has the same base sequence as Viltolarsen. A Viltolarsen equivalent includes equivalents in which all or part of the nucleic acid bases, sugar portions, and phosphate bond portions of the equivalent are modified in the same way as those in Viltolarsen, or are modified differently from Viltolarsen. The appearance of such a modification is the same as that described above.In addition, the equivalent of Viltolarsen may be in the form of a free body, a pharmaceutically acceptable salt, or a hydrate. 2. Composition of the Pharmaceutical Product The pharmaceutical composition of the present invention may also be in the form of an aqueous solution. The pharmaceutical composition of the present invention may comprise the oligomer of the present invention, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of 2.5 to 500 mg / ml, 5 to 450 mg / ml, 10 to 400 mg / ml, 15 to 350 mg / ml, 20 to 300 mg / ml, 20 to 250 mg / ml, 20 to 200 mg / ml, 20 to 150 mg / ml, 20 to 100 mg / ml, 20 to 50 mg / ml, 20 to 40 mg / ml, 20 to 30 mg / ml, 23 to 27 mg / ml, 24 to 26 mg / ml, or 25 mg / ml. Otherwise, the pharmaceutical composition of the present invention may comprise the oligomer of the present invention, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of 10 to 100 mg / ml, 15 to 95 mg / ml, 20 to 80 mg / ml, 25 to 75 mg / ml, 30 to 70 mg / ml, 35 to 65 mg / ml, 40 to 60 mg / ml, 45 to 55 mg / ml, 47 to 53 mg / ml, 48 to 52 mg / ml, 49 to 51 mg / ml or 50 mg / ml. In the pharmaceutical composition of the present invention, the concentration of Viltolarsen in the aqueous solution can be changed. To prepare an aqueous solution of Viltolarsen, for example, 250 mg of Viltolarsen can be mixed in 0.5 mL to 100 mL of water (corresponding to a Viltolarsen concentration of 2.5 mg / mL to 500 mg / mL), more preferably 1 mL to 50 mL of water (corresponding to a Viltolarsen concentration of 5 mg / mL to 250 mg / mL), and even more preferably 5 mL to 10 mL of water (corresponding to a Viltolarsen concentration of 25 mg / mL to 50 mg / mL). The method of administration of the pharmaceutical composition of the present invention is intravenous administration. A possible dosage form of the pharmaceutical composition of the present invention is, for example, an injection solution (including a drip fluid). The pharmaceutical composition of the present invention may further comprise at least one component selected from a tonicity agent, a pH adjuster, and a solvent. The tonicity agent comprising the pharmaceutical composition of the present invention may be at least one selected from sodium chloride, potassium chloride, glucose, fructose, maltose, sucrose, lactose, mannitol, sorbitol, xylitol, trehalose, and glycerin. My vials of an aqueous solution comprising 250 mg of Viltolarsen suitable for injection may contain, as a tonic agent, 72.0 mg or more and 108.0 mg or less of sodium chloride (corresponding to sodium chloride at a concentration of 7.2 mg / ml at 10.8 mg / mL), more preferably 81.0 mg or more and 99.0 mg or less of sodium chloride (corresponding to sodium chloride at a concentration of 8.1 mg / mL to 9.9 mg / mL), and more preferably 85.5 mg or more and 94.5 mg or less of sodium chloride (corresponding to sodium chloride at a concentration of 8.55 mg / mL to 9.45 mg / mL). A phosphate buffer may be used as a tonic agent. Examples of such a phosphate buffer include citrate buffer, lactate buffer, and acetate buffer. In addition, sugars (other than glucose) may also be used as tonic agents. Examples of such sugars include sorbitol and mannitol. When preparing a composition containing viltolarsen, a plurality of tonic agents may also be used. The pH adjuster comprising the pharmaceutical composition of the present invention may be at least one selected from hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine, citric acid, lactic acid, phosphate (sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate) and monoethanolamine. In the case of using a phosphate buffer for the oligomer of the present invention, for example, for Viltolarsen, the concentration of the phosphate buffer is preferably less than 100 mM. Accordingly, the concentration of the phosphate buffer in the pharmaceutical composition of the present invention may be adjusted to 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 5 mM or less, or the pharmaceutical composition of the present invention may not comprise a phosphate buffer. The solvent included in the pharmaceutical composition of the present invention may be water. The pH value of the aqueous solution comprising Viltolarsen suitable for injection may be pH 6.0 or more and 8.5 or less, more preferably pH 6.5 or more and 8.0 or less, and most preferably 7.0 or more and 7.5 or less. The oligomer of the present invention exhibits stability over a wide range of pH values. The pH value of the pharmaceutical composition of the present invention is preferably adjusted to pH 7.0 to 7.5, 7.0 to 7.4, 7.1 to 7.5, 7.1 to 7.4, 7.2 to 7.5, 7.2 to 7.4, 7.3 to 7.5, 7.3 to 7.4, or 7.3. Furthermore, the pharmaceutical composition of the present invention may be in the form of an aqueous solution comprising the oligomer of the present invention in a concentration of between 2.5 mg / ml inclusive and 500 mg / ml inclusive, or between 10 mg / ml inclusive and 100 mg / ml inclusive, and sodium chloride in a concentration between 8 mg / ml inclusive and 10 mg / ml inclusive, and having a pH value of 7.2 to 7.4. Alternatively, the pharmaceutical composition of the present invention may be in the form of an aqueous solution comprising the oligomer of the present invention at a concentration of 25 mg / ml and having a pH value adjusted to pH 7.3, without containing buffer. In this pharmaceutical composition, the pH value is adjusted using hydrochloric acid and / or sodium hydroxide. As an example of the pharmaceutical composition of the present invention, Table 2 below shows a composition containing 250 mg of Viltolarsen for injection, which was used in the US / Canadian Phase 2 clinical program. The composition shown in Table 2 is referred to hereafter as NS-065 / NCNP-01 (Viltolarsen) 250 mg Injection. iA / a / ¿u <fu / ui ooou Table 2 NS-065 / NCNP-01 (Viltolarsen) Invention 250 mg Composition of (250 mg in 10 mL, or equivalently, 25 mg / mL) Component (USP = U.S. Pharmacopoeia) Function Quantity (per vial) NS-065 / NCNP-01 (Viltolarsen) Drug Substance 250 mg Sodium Chloride (USP Quality) Tonicity Agent 90 mg Hydrochloric Acid (USP Quality) pH Adjuster qs1 Sodium Hydroxide (USP Quality) pH Adjuster qs1 Water for Injection (USP Quality) Medium qs Total Quantity 10 mL The amount of hydrochloric acid and sodium hydroxide is sufficient to adjust the pH value to pH 7.3 and, at the same time, is an amount that does not substantially affect the isotonicity of the composition. Furthermore, in the composition of NS-065 / NCNP-01 (Viltolarsen) Injectable 250 mg, the amounts of water for injection and sodium chloride used as a tonic agent can each be adjusted to approximately half the amount, so that the total amount is set at 5 ml. Also included in the present invention is a pharmaceutical composition comprising Viltolarsen with the aforementioned composition at a concentration of 50 mg / ml. The volume of the aqueous solution containing Viltolarsen may be increased or decreased, provided that the concentrations of NS-065 / NCNP-01 and the tonic agent used, and the weight ratio between Viltolarsen and the tonic agent used, are maintained at the same level, levels as described above. The composition comprising Viltolarsen may also include a vehicle to promote the delivery of Viltolarsen to muscle tissue. Such a vehicle is not particularly restricted, provided it is a pharmaceutically acceptable vehicle. Examples of such a vehicle may include cationic vehicles (e.g., cationic liposomes and cationic polymers) and vehicles using a viral envelope. Examples of cationic liposomes may include liposomes comprising, as essential components, 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids, such as Oligofectamine® (manufactured by Thermo Fisher Scientific), Lipofectin® (manufactured by Thermo Fisher Scientific), Lipofectamine® (manufactured by Thermo Fisher Scientific), Lipofectamine® 2000 (manufactured by Thermo Fisher Scientific), DMRIE-C (manufactured by Thermo Fisher Scientific), GeneSilencer® (manufactured by Gene Therapy Systems), TransMessenger® (manufactured by QIAGEN), and TransIT-TKO®.Examples of cationic polymers include JetSI® (manufactured by GeneX India Bioscience) and Jet-PEI® (polyethyleneimine, also manufactured by GeneX India Bioscience). An example of carriers that use a viral envelope is GenomeOne® (HVJ-E liposome, manufactured by ISHIHARA SANGYO KAISHA, LTD.). Furthermore, the pharmaceutical composition of the present invention may comprise an emulsifying aid (e.g., fatty acid containing 6 to 22 carbon atoms or a pharmaceutically acceptable salt thereof, albumin and dextran) and a stabilizer (e.g., cholesterol and phosphatidic acid). In the pharmaceutical composition of the present invention comprising Viltolarsen and a carrier, the weight ratio between Viltolarsen and a carrier (i.e., carrier / Viltolarsen) can be changed depending on the type of carrier used. The weight ratio is conveniently in the range of 0.1 to 100, preferably in the range of 1 to 50, and more preferably in the range of 10 to 20. The aqueous solution containing Viltolarsen can be administered to patients by intravenous drip infusion or drip perfusion. 3. Indication / Effects and Administration / Posology When the pharmaceutical composition of the present invention is used in a treatment, the oligomer of the present invention, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to a human patient at a dose of between 40 mg / kg / week and 80 mg / kg / week inclusive (hereinafter referred to as the dosage and administration method of the present invention). Alternatively, the dosage and administration method of the present invention may be as follows: the oligomer of the present invention, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to a human patient at a dose of 40 mg / kg / week.Alternatively, the dosage and administration method of the present invention may be as follows: the oligomer of the present invention, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to a human patient at a dose of 80 mg / kg / week. Herein, the numerator mg in the dosage unit mg / kg indicates the quantity of the oligomer of the present invention, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, which is indicated by the unit milligram, while the denominator kg indicates 1 kilogram of body weight of a human patient. The 250 mg injection of NS-065 / NCNP-01 (Viltolarsen) has been developed for use as a once-weekly intravenous drip infusion for the treatment of DMD patients who are candidates for treatment involving exon 53 skipping. The USULJ / Canada Phase 2 clinical program is designed to evaluate Viltolarsen administered at two dosage levels: 40 mg / kg / week and 80 mg / kg / week. In this document, kg is a unit that indicates a patient's body weight. For example, when Viltolarsen is administered at a dose of 40 mg / kg / week to a patient weighing 40 kg, this means that the patient receives 1,600 mg (40 mg x 40) of Viltolarsen once a week. 4. Disease DMD is a muscular disease caused by a loss-of-function mutation in a dystrophin gene, resulting in the loss of a dystrophin protein in the muscles of affected individuals (Hoffman et al., 1987). The dystrophin gene is located on the X chromosome and exhibits a high rate of spontaneous mutation. In all populations studied worldwide, the incidence rate of DMD is approximately one in 5,000 surviving children. According to a recent evaluation, 10.1% of the 7,149 DMD patients assessed in the global patient database were deemed eligible for treatment with exon 53 skipping (Bladen et al., 2015).Clinical symptoms typically present in early school age (between 4 and 6 years old), when affected children show difficulty keeping up physically with their peers due to proximal muscle weakness (such as difficulty climbing stairs or running). Difficulty rising from the floor is observed in most patients, requiring the typical Gower maneuver to achieve a standing position (i.e., using the hands for support on the legs, knees, and thighs to achieve a standing position). (Hoffman EP, Brown RH, and Kunkel LM. (1987). Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51, 919-928.)and Bladen CL, Salgado D, Monges S, Foncuberta ME, Kekou K, Kosma K, Dawkins H, Lamont L, Roy AJ, Chamova T, Guergueltcheva V, Chan S, Korngut L, Campbell C, Dai Y, Wang J, Barisic N, Brabec P, Lahdetie J, Walter Schrei-Karmi, VK, Garbergi, V. M, Viswanathan V, Bayat F, Buccella F, Kimura E, Koeks Z, van den Bergen JC, Rodrigues M, Roxburgh R, Lusakowska A, Kostera-Pruszczyk A, Zimowski J, Santos R, Neagu E, Artemieva S, Rasic VM, Vojinovic D, Posada M, Blourtzer PY, Jean Blourtzer, PY, FY Díaz-Manera J, Gallardo E, Karaduman AA, Topaloglu H, El Sherif R, Stringer A, Shatillo AV, Martin AS, Peay HL, Bellgard MI, Kirschner J, Flanigan KM, Straub V, Bushby K, Verschuuren J, Aartsma-Rus A, Béroud C, Lochüller H (2015). The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum Mutat. 36(4): 395-402). lA / a / zuzu / ui ooou The muscle tissue of patients with DMD shows chronic inflammation, with episodes of muscle degeneration and regeneration that lead to muscle wasting, disability, and premature death. Patients typically lose the ability to walk in their second decade of life and require assistance with many aspects of daily living by their third decade. This disease usually results in death in adolescence or early adulthood, although the use of respiratory support devices can sometimes prolong life into the fourth decade and, very occasionally, the fifth. The DMD gene mutation in a subject can be detected using multiplex ligation-dependent probe amplification (MLPA) (Murugan et al., 2010). The content of this article by Murugan et al. is incorporated as a reference in its entirety (Sakthivel Murugan SM, Arthi Chandramohan and Bremadesam Raman Lakshmi, “Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis”, Indian Journal of Medical Research, vol. 132, pp. 303-311 (September 2010)). The human patient of interest for treatment using the pharmaceutical composition of the present invention (hereinafter referred to as the patient of interest of the present invention) is not particularly limited, provided that the patient is diagnosed with DMD by a medical doctor. In a certain respect, the patient may have a mutation resulting in a deficiency in any selected exon from the group consisting of exons 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, or 52, in a dystrophin gene. Furthermore, the patient of interest of the present invention may be characterized in that the expression of a dystrophin protein prior to treatment using the pharmaceutical composition of the present invention or the oligomer of the present invention is 1% or less compared to that of a healthy subject (100%), as measured by Western blot or mass spectrometry. In this document, a healthy subject is a human being who does not have a disease associated with a dystrophin protein. The expression level of a dystrophin protein in a healthy subject may be a generally known value or a value obtained from an individual healthy subject. Additionally, the patient of interest of the present invention may also be characterized in that the expression of a dystrophin protein is not observed prior to treatment using the pharmaceutical composition of the present invention or the oligomer of the present invention.The phrase "no dystrophin protein expression is seen" means that the expression of a dystrophin protein is almost at the same level as that of a negative control, as measured by Western blot or mass spectrometry, or that the expression of the dystrophin protein is below the lower limit of detection. Furthermore, Western blot and mass spectrometry are not particularly limited, provided they are methods generally used in this technical field. Examples of Western blot and mass spectrometry can be found in the experimental methods applied in the present Examples. 5. Therapeutic Justification DMD is a severe inherited muscle disorder. This disease most often occurs when out-of-frame amino acid translation is caused by the deletion of one or more exons of the dystrophin gene. A patient's functional dystrophin protein, which is important for muscle function, is not expressed due to this out-of-frame amino acid translation. A less severe form of the disorder, Becker muscular dystrophy (BMD), most often occurs when the absence of one or more exons in the dystrophin gene results in in-frame amino acid translation of the remaining exons. Patients with BMD generally have slower disease progression and a lesser degree of disability. Medical treatment for patients with DMD typically includes glucocorticoid therapy to delay the onset of symptoms in the limb muscles or those involved in breathing.There is a significant unmet medical need in patients with DMD, as expressed in the FDA's February 2018 final guidance, Duchenne Muscular Dystrophy and Related Dystrophinopathies: Drug Development for Treatment Guidance for Industry (available at https: / / www.fda.gov / downloads / Drugs / GuidanceComplianceRegulatorylnformation / Guidances / UCM450229.pdf). One therapeutic approach for treating patients with DMD is to employ an exon skipping strategy to produce functional dystrophin protein, which can cause DMD patients to transition to a BMD phenotype. Exon skipping allows for the restoration of the amino acid reading frame due to the induced skipping of the exon adjacent to the missing exon (Cirak et al., 2011; Voit et al., 2014; Yokota et al., 2012). With the skipping of exon 53, a dystrophin protein is expressed that is slightly shorter than normal but retains partial functional activity.It is hoped that the injection of 250 mg of NS065 / NCNP-01 (Viltolarsen) will change the phenotype of DMD to the greater disease of DMO in which the progression of the disease of the patients is slowed down and the quality of life of the patients improves (Cirak S, Arechavala-Gomeza V, Guglieri M, Feng L, Torelli S, Anthony K, Abbs S, Garralda ME, Bourke J, Wells DJ, Dickson G, Wood MJ, Wilton SD, Straub V, Kole R, Shrewsbury SB, Sewry C, Morgan JE, Bushby K, Muntoni F. (2011). after the Systemic treatment with morpholino phosphorodiamidate oligomers: an open phase 2 dose escalation study. Lancet 378: 595-605., Voit T, Topaloglu H, Straub V, Muntoni F, Deconinck N, Campion G, De Kimpe SJ, Eagle M, Guglieri M, Hood S, Liefaard L, Lourbakos A, Morgan A, Nakielny J, Quarcoo N, Ricotti V, Rolfe K, Serváis L, Wardell C, Wilson R, Wright. P, Kraus JE. (2014). Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomized, placebo-controlled phase 2 study. Lancet Neurol. 13(10):987-96., Y Yokota T, Nakamura A, Na gata T, Saito T, Kobayashi M, Aoki Y, Echigoya Y, Partridge T, Hoffman EP, Takeda S. (2012). Extensive and prolonged restoration of dystrophin expression with multiple exon skipping mediated by vivo-morpholino in dystrophic dogs. Nucleic Acid Ther 22(5):306-15.). Therefore, the pharmaceutical composition of the present invention is administered to a human patient with DMD according to the dosage and administration method of the present invention mentioned above, so that the DMD can be treated. The term "treat" is used herein to mean reducing the symptoms of DMD in a patient. The treatment using the pharmaceutical composition of the present invention according to the dosage and administration method of the present invention can provide at least one effect selected from the group consisting of the following effects (1) to (6) (where the mean ± standard deviation is shown in parentheses): (1) the mean value of the expression level of a dystrophin protein in the patient's skeletal muscle increases 9 times or more compared to the initial value, after administration of the pharmaceutical composition for 24 weeks; (2) a change in velocity obtained from time to rest (TTSTAND) is 0.055 times / sec or more, or 0.024 ± 0.075 times / sec or more compared to baseline, at time 25 weeks after administration of the pharmaceutical composition for 24 weeks; (3) a change in speed obtained from the time to run / walk 10 meters (TTRW) is -0.025 meters / sec or more, or 0.227 ± 0.251 meters / sec or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; (4) a change in velocity obtained from time to climb 4 stairs (TTCLIMB) is -0.060 times / sec or more, or 0.032 ± 0.088 times / sec or more compared to baseline, at time 25 weeks after administration of the pharmaceutical composition for 24 weeks; (5) a change in the North Star Ambulatory Assessment (NSAA) score is 2.2 scores or more, or 0.8 ± 2.9 scores or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; and (6) a change in the 6-minute walk test (6MWT) is -7.5 meters or more, or 28.9 ± 36.3 meters or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks. With respect to effects (1) to (6) described above, the term baseline means an average value in a group of untreated patients. In addition, the term change used in effects (2) to (6) means a change in the mean values. In a certain embodiment, when the pharmaceutical composition of the present invention is administered to human patients aged 7 to 9 years with Duchenne muscular dystrophy according to the dosage and administration method of the present invention mentioned above for 84 weeks, at least one effect selected from the group consisting of the following effects (7) to (12) can be provided: (7) the percentage of patients who lose the ability to grow is less than 20% at week 85 after the start of treatment; (8) the percentage of patients who lose the ability to climb 4 steps is less than 10% at week 85 after the start of treatment; (9) the percentage of patients who lose the ability to walk independently is less than 10% at week 85 after the start of treatment; (10) no reduction in 10-meter running / walking speed due to aging is observed at week 85 after the start of treatment; (11) no reduction in the speed of climbing 4 steps due to aging is observed at the time of week 85 after the start of treatment; and (12) no reduction in the speed of ascent due to aging is observed at the time of week 85 after the start of treatment. The week in which treatment has been initiated is defined as the first week, and the effects described above (7) to (12) can be provided at any time from week 1 to week 85, from week 1 to week 80, from week 1 to week 75, from week 1 to week 70, from week 1 to week 65 and from week 1 to week 60. In a certain embodiment, at least one effect selected from the group consisting of the following effects (13) to (18) is provided by administering the pharmaceutical composition of the present invention to human patients aged 10 to 12 years with Duchenne muscular dystrophy according to the dosage and method of administration of the present invention mentioned above: (13) the percentage of patients who lose the ability to increase is less than 60% at week 85 after the start of treatment; (14) the percentage of patients who lose the ability to climb 4 steps is less than 50% at week 85 after the start of treatment; (15) the percentage of patients who lose the ability to walk independently is less than 50% at week 85 after the start of treatment; (16) no reduction in 10-meter running / walking speed due to aging is observed at week 85 after the start of treatment; (17) a period is observed in which the speed of climbing 4 steps increases at the time of week 85 after the start of treatment; and (18) a period is observed in which the speed of climbing increases at the time of week 85 after the start of treatment. The week in which treatment has been initiated is defined as the first week, and the effects described above (13) to (18) can be provided at any time from week 1 to week 85, from week 1 to week 80, from week 13 to week 75, from week 1 to week 70, from week 1 to week 65, and from week 13 to week 60. That is, the pharmaceutical composition of the present invention can provide at least one of the effects (1) to (18) described above. IL Second Realization In a second embodiment, the present invention provides a method for treating Duchenne muscular dystrophy, comprising the intravenous administration of a pharmaceutical composition comprising an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 from the 5' terminus of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, to a human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive of the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof (hereinafter referred to as the treatment method of the present invention). The meanings of the individual configurations with respect to the treatment method of the present invention are the same as those of the configurations with respect to the pharmaceutical composition of the present invention already explained in section I. First Embodiment. Furthermore, the treatment method of the present invention can provide at least one of the effects (1) to (18) explained in subsection 5. Therapeutic justification” in section I. First Embodiment. IIL Third Realization Furthermore, in a third embodiment, the present invention provides an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 from the 5' terminus of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable gene, a salt thereof, or a hydrate thereof, for use in a method for treating a human patient with Duchenne muscular dystrophy, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive (hereafter referred to as the limited-use embodiment of the present invention). The meanings of the individual configurations with respect to the limited-use embodiment of the present invention are the same as those of the configurations with respect to the pharmaceutical composition of the present invention already explained in Section I. First Embodiment. Furthermore, the limited-use embodiment of the present invention may provide at least one of the effects (1) to (18) explained in subsection 5. Therapeutic Justification of section I. First Embodiment. IV. Fourth Realization Furthermore, in a fourth embodiment, the present invention provides the use of an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 from the 5' terminus of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, for the manufacture of a pharmaceutical composition for the treatment of a human patient with Duchenne muscular dystrophy, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive (hereafter referred to as the Swiss-type use of the present invention). The meanings of the individual configurations with respect to the Swiss-type use of the present invention are the same as those of the configurations with respect to the pharmaceutical composition of the present invention already explained in Section I. First Embodiment. Furthermore, the Swiss-type use of the present invention may provide at least one of the effects (1) to (18) explained in subsection 5. Therapeutic justification of section I. First Embodiment. The present invention will now be described in more detail in the following examples. However, the present invention is not limited to the scope of the invention shown in these examples. EXAMPLES Production Example (1) Production of 4-{[(2S,6R)-6-(4-benzamide-2-oxoprimidine-1-1)-4-trimethylmorpholin2-11methoxy-4-oxobutanoic Acid Supported on Aminomethyl Polystyrene Resin Paso 1: Producción De Ácido 4-n(2S,6R)-6-(4-benzam¡da-2-oxopirim¡d¡n-1(2H)-¡l)-4tritilmorfol¡n-2-il1metoxi)-4-oxobutanoico In an argon atmosphere, 22.0 g of N-{1-[(2R,6S)-6-(hydroxymethyl)-4-trimethylmorpholin-2-1}benzamide were suspended with 7.04 g of 4-dimethylaminopyridine (4DMAP) in 269 mL of dichloromethane. Then, 5.76 g of succinic anhydride were added to the suspension, and the mixture was stirred at room temperature for 3 hours. Subsequently, 40 mL of methanol were added to the reaction solution, and the resulting solution was then concentrated under reduced pressure. The residue was subjected to an extraction operation using ethyl acetate and a 0.5 M aqueous solution of potassium dihydrogen phosphate. The resulting organic layer was successively washed with a 0.5 M aqueous solution of potassium dihydrogen phosphate, water, and a saturated saline solution. The organic layer was dried over sodium sulfate and then concentrated under reduced pressure to obtain 25.9 g of a product of interest. Step 2: Production of 4-n(2S,6R)-6-(4-benzamide-2-oxopyrimidin-1-yl)-4-trimethylmorpholin-2-methylmethoxy)-4-oxobutanoic Acid Supported on Aminomethyl Polystyrene Resin 4-{[(2S,6R)-6-(4-benzamide-2-oxoprimidin-1(2H)-1)-4-triethylmorphol-2-1]methoxy}-4-oxobutanoic acid (23.5 g) was dissolved in 336 mL of pyridine (dehydrated), and then 4.28 g of 4-DMAP and 40.3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to the solution. Subsequently, 25.0 g of DVB-crosslinked aminomethyl polystyrene resin (manufactured by Tokyo Chemical Industry Co., Ltd., A1543) and 24 mL of triethylamine were added to the mixture, and the resulting mixture was stirred at room temperature for 4 days. Once the reaction was complete, the resin was collected by filtration. The resulting resin was washed with pyridine, methanol, and dichloromethane in that order and then dried under reduced pressure. To the resin, 150 mL of tetrahydrofuran (dehydrated), 15 mL of acetic anhydride, and 15 mL of 2,6-lutidine were added, and the mixture was stirred at room temperature for 2 hours.The resin was collected by filtration and then washed with pyridine, methanol, and dichloromethane in that order, followed by drying under reduced pressure, to obtain 33.7 g of a product of interest. Regarding the amount of the product of interest loaded, the molar amount of trityl per gram of resin was determined by measuring the UV absorbance at 409 nm according to a known method. The amount loaded in the resin was found to be 397.4 pmol / g. iA / a / ooou UV Measurement Conditions Device: U-2910 (Hitachi, Ltd.) Solvent: methanesulfonic acid Wavelength: 265 nm ε value: 45000 (2) PMO Production No. 8 PMO No. 8 points to the sequence in positions 36 to 56 in exon 53, the group at the 5' terminal of the same is group (3), and the sequence of a base portion of the same is as set out in SEQ ID NO: 3. 4-{[(2S,6R)-6-(4-benzamide-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 1) (2 g (800 pmol)) supported on an aminomethyl polystyrene resin was transferred to a reaction tank, and then 30 mL of dichloromethane was added, followed by allowing the resulting mixture to stand for 30 minutes. After that, the reaction mixture was further washed with 30 mL of dichloromethane twice, and the following synthetic cycles were started. A desired morpholino monomer compound was added in each cycle to obtain the base sequence of the compound of interest. Table 3 Step Reagent Amount (mL) Time (min) 1 Unblocking Solution 30 2.0 2 Unblocking Solution 30 2.0 3 Unblocking Solution 30 2.0 4 Unblocking Solution 30 2.0 5 Unblocking Solution 30 2.0 6 Unblocking Solution 30 2.0 7 Neutralizing Solution 30 1.5 8 Neutralizing Solution 30 1.5 9 Neutralizing Solution 30 1.5 10 Neutralizing Solution 30 1.5 11 Neutralizing Solution 30 1.5 12 Neutralizing Solution 30 1.5 13 Dichloromethane 30 0.5 14 Dichloromethane 30 0.5 15 Dichloromethane 30 0.5 16 Coupling Solution B 20 0.5 17 Coupling Solution A 6-11 90.0 18 Dichloromethane 30 0.5 19 Dichloromethane 30 0.5 20 Dichloromethane 30 0.5 21 Plugging Solution 30 3.0 22 Plugging Solution 30 3.0 23 Dichloromethane 30 0.5 24 Dichloromethane 30 0.5 25 Dichloromethane 30 0.5 It should be noted that a mixture of trifluoroacetic acid (2 equivalents) and triethylamine (1 equivalent) dissolved in a dichloromethane solution containing 1% (v / v) ethanol and 10% (v / v) 2,2,2-trifluoroethanol to give a 3% (w / v) solution was used as a deblocking solution. As a neutralizing solution, N,N-diisopropylethylamine dissolved in a dichloromethane solution containing 25% (v / v) 2-propanol to give a 5% (v / v) solution was used. As coupling solution A, a morpholino monomer compound dissolved in 1,3-dimethyl-2-imidazolidine containing 10% (v / v) N,N-diisopropylethylamine was used to give a 0.15 M concentration. As coupling solution B,N,N-diisopropylethylamine dissolved in 1,3-dimethyl-2-imidazolidine was used to give a 10% (v / v) concentration. As buffering solution, a mixture of 20% (v / v) acetic anhydride and 30% (v / v) 2,6-lutidine dissolved in dichloromethane was used. The PMO-supported aminomethyl polystyrene resin thus synthesized was recovered from the reaction vessel and then dried at room temperature under reduced pressure for 2 hours or more. The PMO supported on the dried aminomethyl polystyrene resin was placed in a reaction vessel, and 200 mL of 28% ammonia, water-ethanol (1 / 4) was added, followed by stirring of the resulting mixture at 55°C for 15 hours. After this, the aminomethyl polystyrene resin was collected by filtration and then washed with 50 mL of water-ethanol (1 / 4). The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in 100 mL of a 4:1 solvent mixture of 20 mM acetic acid-triethylamine buffer (TEAA buffer) and acetonitrile, and the resulting solution was then filtered through a membrane filter. The filtrate obtained was purified by HPLC in reserve phase. The conditions applied are as follows. Table 4 XTerra MS18 Column (Water, ψ 50 x 100 mm, 1 CV = 200 mL) Flow Rate 60 mL / min Column Temperature Ambient Temperature Solution A 20 mM TEAA buffer Solution B CH3CN Gradient (B) conc. 20 -> 50% / 9 CV iA / a / ¿uzu / ui ooou Each fraction was analyzed, and a product of interest was recovered using 100 mL of acetonitrile-water (1:1) solution. 200 mL of ethanol were added, and the resulting mixture was concentrated under reduced pressure. The concentrate was further dried under reduced pressure to obtain a white solid. 300 mL of 10 mM aqueous phosphoric acid solution were added to the solid, causing it to become suspended. Then, 10 mL of 2 M aqueous phosphoric acid solution were added to the suspension, and the mixture was stirred for 15 minutes. Finally, 15 mL of 2 M aqueous sodium hydroxide solution were added to the reaction mixture for neutralization. Next, 15 ml of a 2 M aqueous sodium hydroxide solution were added to the reaction mixture to alkalize the mixture, and the mixture was then filtered through a membrane filter (0.45 pm).The resulting mixture was thoroughly washed with 100 mL of a 10 mM aqueous sodium hydroxide solution to obtain a product of interest in the form of an aqueous solution. The resulting aqueous solution containing the product of interest was purified using an anion exchange resin column. The conditions applied are as follows. Table 5 Source Column 30Q (GE Healthcare, φ 40 x 150 mm, 1 CV = 200 mL) Flow Rate 80 mL / min Column Temperature Room Temperature Solution A 10 mM aqueous sodium hydroxide solution Solution B 10 mM aqueous sodium hydroxide solution, 1 mM aqueous sodium chloride solution Gradient (B) conc. 5 35% / 15 CV Each fraction was analyzed (HPLC), and a product of interest was obtained as an aqueous solution. To this aqueous solution, 225 mL of 0.1 M phosphate buffer (pH 6.0) were added for neutralization. The resulting mixture was filtered through a membrane filter (0.45 µm). Subsequently, ultrafiltration was carried out under the following conditions for desalination. Table 6 PELLICON2 MINI FILTER PLBC 3K Regenerated Cellulose, Type C Screen Size 0.1 m2 noor Ln / nznz / E / γΐΛΐ The filtrate was concentrated to obtain approximately 250 mL of an aqueous solution. The resulting aqueous solution was filtered through a membrane filter (0.45 pm). The aqueous solution was then lyophilized to obtain 1.5 g of a compound of interest as a white flocculant solid. ESI-TOF-MS calculated value: 6924.82 Measured value: 6923.54 Example 1: EE· LIU Phase 2 Study. The US Phase 2 dose-finding study, Study NS-065NCNP-01-201, was initiated in December 2016, under IND127474. This study was conducted under ClinicalTrials.gov recognition number NCT02740972 with the title NS-065 / NCNP-01 Safety and Dose Determination Study in Children with Duchenne Muscular Dystrophy (DMD). The study implementation protocols for this study are available at https: / / www.clinicaltrials.gov / ct2 / show / study / NCT02740972, and their content is incorporated herein by reference in full. The present study was primarily aimed at evaluating the safety of NS-065 / NCNP-01 (Viltolarsen) to be administered as an intravenous drip infusion at a high dose (80 mg / kg) and a low dose (40 mg / kg) to patients with Duchenne muscular dystrophy (DMD) eligible for treatment with exon 53 skipping.In addition, other objectives of the present study include the evaluation of tolerability, muscle function and muscle strength, pharmacokinetics and pharmacodynamics. More specifically, the present study was a Phase 2, multicenter, two-period, randomized, placebo-controlled, dose-finding study in which NS-065 / NCNP-01 (Viltolarsen) was administered by once-weekly intravenous drip infusion for 24 weeks to ambulatory children aged 4 years and older and younger than 10 years with DMD. Two dose-level cohorts were enrolled. Period 1 of the study was conducted double-blind. Randomized patients received weekly intravenous drip infusions of NS-065 / NCNP-01 (Viltolarsen) or placebo for the first 4 weeks of their participation (Period 1), and then, intravenous drip infusion of NS-065 / NCNP-01 (Viltolarsen) for 5 to 24 weeks (20 weeks of active treatment - Period 2). Analysis of the safety data from period 1 of the 40 mg / kg dose cohort was completed prior to enrolling patients in the 80 mg / kg dose cohort.Patients who completed the 24-week study were eligible for an open-label extension study. Clinical efficacy was assessed at regularly scheduled study visits. All patients underwent a biceps muscle biopsy at baseline and a second muscle biopsy at week 24. Safety was assessed by collecting adverse events (AEs), performing blood and urine laboratory tests, electrocardiograms (ECGs), recording vital signs, and conducting physical examinations throughout the study. Serial blood samples were collected at four of the study visits to evaluate the pharmacokinetics of NS-065 / NCNP-01 (Viltolarsen). <Tipo De Estudio: Intervencionista (Ensayo Clínico)> - Real Registration: 16 participants. - Assignment: Randomized. - Intervention Model: Parallel Assignment. - Masking: Quadruple (Participant, Care Provider, Researcher, Outcome Evaluator). - Main Purpose: Treatment. - Actual Start Date of the Study: December 2016. - Primary School Completion Date: March 2018. <Grupos De Pacientes E lntervenciones> - Experimental: NS-065 / NCNP-01 (Viltolarsen) 40 mg / kg An intravenous drip infusion of NS-065 / NCNP-01 (Viltolarsen) was administered at a dose of 40 mg / kg once a week for 24 weeks to six patients with confirmed DMD with genetic deletions amenable to treatment with exon 53 skipping. - Experimental: NS-065 / NCNP-01 (Viltolarsen) 80 mg / kg Six patients with confirmed DMD with genetic deletions amenable to treatment involving the skipping of exon 53 were administered an intravenous drip infusion of NS-065 / NCNP-01 (Viltolarsen) at a dose of 80 mg / kg once a week for 24 weeks. - Placebo comparator: Placebo Two or three patients from each dose group were given placebo as an intravenous drip infusion once a week for 4 weeks, followed by 20 weeks of open-label treatment. <Criterios De Inclusiór» - Varones > 4 years and <10 years. - Confirmed DMD mutations in the dystrophin gene that are amenable to treatment involving skipping exon 53 to restore the dystrophin mRNA reading frame. - Patients able to walk independently without assistive devices. - Patients with the ability to complete the standing time, running / walking time, and climbing time assessments. - Stable dose of glucocorticoid for at least 3 months. <Criter¡os De Exclusiór» - Acute illness in the 4 weeks prior to the first dose of the study drug. - Evidence of symptomatic cardiomyopathy. (Asymptomatic cardiac abnormality in the investigation would not exclude it). - Severe allergy or hypersensitivity to medications. - Serious behavioral or cognitive problems that prevent participation in the study, at the discretion of the Researcher. - Previous or ongoing medical condition, medical history, physical findings, or laboratory abnormalities that could affect safety, make it unlikely that treatment and follow-up will be completed correctly, or affect the evaluation of the study results, in the investigator's opinion. - The patient was taking any other investigational drug currently or in the 3 months prior to the start of the study treatment. - The patient had undergone surgery within 3 months prior to the first planned administration of NS-065 / NCNP-01 (Viltolarsen) or surgery was planned at any time during the duration of the study. - The patient had previously participated in this study or any other study during which he / she had been administered NS-065 / NCNP-01 (Viltolarsen). In summary, the study was a 24-week, two-cohort study evaluating doses of 40 mg / kg / week and 80 mg / kg / week in 16 male patients who were eligible for treatment involving exon 53 skipping and who had been on a stable glucocorticoid dose for more than 3 years. Patients in the placebo group in each cohort underwent an initial 4-week randomization period to monitor for adverse events (safety outcomes). Thereafter, both placebo and active-treated patients continued the study for a total of 20 weeks. The NS-065 / NCNP-01-201 trial evaluated the effect of NS-065 / NCNP01 (Viltolarsen) injection on de novo dystrophin protein expression after 20-24 weeks of administration in two dose cohorts: 40 mg / kg / week low dose and 80 mg / kg / week high dose (as shown in Figure 1).The objective of the NS065 / NCNP-01-201 trial was to identify a safe and effective dose based on de novo dystrophin protein expression in skeletal muscle, measured by Western blot (WB) (primary surrogate endpoint). Secondary surrogate endpoints included immunofluorescence (IF) staining and mass spectrometry detection of de novo dystrophin protein expression, as well as RT-PCR detection of de novo dystrophin mRNA levels. Several functional endpoints were also included as secondary endpoints in the phase 2 study. The NS-065 / NCNP-01-201 trial endpoints were: <Criterios De Valoración Principales» - Safety and tolerability of low-dose (40 mg / kg / week) and high-dose (80 mg / kg / week) intravenous (IV) administrations of NS-065 / NCNP-01 (Viltolarsen) Injectable. - The effects of low and high intravenous doses of NS-065 / NCNP-01 (Viltolarsen) Injectable were determined as an induction of dystrophin protein in muscle after 20-24 weeks of treatment measured by Western Blot. In Figure 1, the timeline for “High Dose: 80 mg / kg / week” presented in the lower half is shifted to a later time than the timeline for “Low Dose: 40 mg / kg / week” presented in the upper half to indicate that high dose administration was initiated only after safety was confirmed with low dose administration. Content of the Measure: 1. Induction of dystrophin mRNA in muscle measured by real-time polymerase chain reaction (RT-PCR) for mRNA analysis. (Timeframe: 20-24 weeks of treatment) RT-PCR measures the altered splicing of dystrophin RNA. In this method, RNA is isolated from a frozen muscle biopsy section and reverse-transcribed into cDNA. PCR primers are designed to flank the exon 53 site in the dystrophin mRNA. RT-PCR bands corresponding to specific versions of the spliced ​​dystrophin mRNA are visualized by gel electrophoresis, and the amounts of different mRNA isoforms are compared. If the drug successfully binds to the target RNA, exon 53 is excluded from the resulting mRNA transcripts. 2. Induction of dystrophin protein in muscle measured by Western blot for protein analysis. (Timeframe: 20-24 weeks of treatment) The primary biochemical outcome measure is the measurement of drug-induced increases in dystrophin production using immunoblotting (Western blot). Dystrophin immunoblotting utilizes cryosections of solubilized muscle biopsies, with proteins fractionated by molecular weight by gel electrophoresis (SDS-PAGE), electroblotting onto nitrocellulose, and subsequent nitrocellulose incubation with antibodies to detect dystrophin protein. The immunoblotting signal for dystrophin from a patient's biopsy is then compared to the signal from a dystrophin standard curve on the same gel (mixed DMD and normal controls). This provides a semi-quantitative assessment of dystrophin content in the muscle. US Phase 2 Study: Efficacy Outcome Measure 1 The results obtained from outcome measure 1 (i.e., RT-PCR detection of de novo dystrophin mRNA levels) were as follows. iA / a / ¿u <fu / ui ooou Table 7 Mean baseline dose % (standard deviation) Mean during treatment % (standard deviation) 40 mg / kg / week 0.0 (0.0) 17.4 (7.2) 80 mg / kg / week 0.0 (0.0) 43.9 (16.7) Outcome Measure 2 The results obtained from outcome measure 2 (i.e., the measurement of de novo dystrophin protein expression in skeletal muscle using Western blot methodology) represented a 19.0-fold increase (for 40 mg / kg / week for 24 weeks) and a 9.8-fold increase (for 80 mg / kg / week for 24 weeks) from baseline compared to the mean baseline and treatment values, and a 27.2-fold increase (for both 40 and 80 mg / kg / week for 24 weeks) calculated as the average rate of increase for each patient. The data obtained are summarized in Table 8 and Figure 2 below. Table 8 Mean Baseline Dose % (range, standard deviation) Mean During Treatment Dose % (range, standard deviation) Increase in times 1) Increase in times 2) 40mg / kg / week 0.3 (0.1-0.4, 0.1) 5.7 (3.2-10.3, 2.4) 19.0 27.2 80mg / kg / week 0.6 (0.1-2.6, 0.8) 5.9 (1.1-14.4, 4.5) 9.8 27.2 1) Comparison between the mean baseline value and the treatment value. 2) Average value of the rate of increase for each patient. The degree of dystrophin rescue by NS-065 / NCNP-01 (Viltolarsen) (40 or 80 mg / kg / week, 24 weeks) was approximately 3 to 7 times or 8.8 to 9.7 times greater than previously reported for Exondys 51® (eteplirsen) (30 mg / kg / week, 48 and 180 weeks) in patients amenable to treatment involving exon 51 skipping in a moderate estimate, which was launched by Sarepta Therapeutics in 2016. Specifically, for comparative purposes, the following are the corresponding Western blot data for Exondys 51® (eteplirsen). In 3 of 16 patients who received NS-065 / NCNP-01 (Viltolarsen) for 24 weeks, the dystrophin level increased by more than 10% from baseline. An increase in dystrophin levels of 3% or more was observed in 12 of 16 patients who received NS-065 / NCNP-01 (Viltolarsen) for 24 weeks. Hoffman et al.They have reported that: Among patients with Duchenne muscular dystrophy (<3% dystrophin of the normal level) or Becker muscular dystrophy and an abnormal dystrophin phenotype, there was a clear correlation between the severity of the clinical phenotype and the results of the dystrophin assessment. (Eric P. Hoffman, et al., Characterization of Dystrophin in Muscle-Biopsy Specimens from Patients with Duchenne's or Becker's Muscular Dystrophy. N. Ingl. J. Med., 318: 1363-1368 (1988)). - Western Blot etiprilsen: a 2.8-fold increase, 0.16% —> 0.44% (30 mg / kg / week for 48 weeks). (In the 12 patients with evaluable results, the dystrophin level before treatment was 0.16% ± 0.12% (mean ± standard deviation) of the dystrophin level in a healthy subject and 0.44% ± 0.43% after 48 weeks of treatment with EXONDYS 51 (p<0.05)'j (available at https: / / www.fda.gov / downloads / AdvisoryCommittees / CommitteesMeetingMaterials / PediatricAdvisoryCommittee / UCM557917.pdf.). - Western Blot eteplirsen: a 3.1-fold increase, 0.3% -> 0.93% (30 mg / kg / week for 180 weeks). (In Western blots of extensor digitorum brevis (EDB) biopsies, dystrophin levels averaged approximately 0.3% of normal, but ranged from undetectable to ≥1% of normal or slightly higher. By Western blot, the most accurate quantitative method used according to the applicant, the mean dystrophin level after ~3.5 years of eteplirsen treatment was 0.93% ± 0.84% ​​of normal (mean ± standard deviation) (available at https: / / www.fda.gov / downloads / advisorycommittees / committeesmeetingmaterials / drugs / peripheralandcentralnervoussystemdrugsadvisorycommittee / ucm497063.pdf.) - Western Blot eteplirsen: 0.93% at week 180, range 0%-2.47% (30 mg / kg / week for 180 weeks) (Kenji Rowel Q Lim, et al., Eteplirsen in the treatment of Duchenne muscular dystrophy.. Des. Devel. Ther., 11: 533-545(2017); https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5338848 / ) - Furthermore: For eteplirsen, no differences in dose-response were observed for the amount of dystrophin produced between a weekly dose of 30 mg / kg and 50 mg / kg in studies conducted at week 180, suggesting that this approach may not be successful for other exons. (Kenji Rowel Q Lim, et al., Eteplirsen in the treatment of Duchenne muscular dystrophy. Des. Devel. Ther., 11: 533-545(2017); https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5338848 / ). It is also shown that Viltolarsen achieved superior effects compared to SRP-4053 (golodirsen; from Sarepta), a morpholino oligonucleotide for DMD patients eligible for treatment involving exon 53 skipping, particularly in the higher level of dystrophin observed. Specifically, for comparison purposes, the following are the corresponding Western blot data for SRP-4053 (golodirsen), obtained after 48 weeks of administration instead of 24 weeks of administration for Viltolarsen. - Western Blot with golodirsen: 0.095% -> 1.019% (30 mg / kg for 48 weeks). (Mean dystrophin protein increased to 1.019% of normal compared to a mean baseline value of 0.095% of normal (p<0.001) as measured by Western Blot, the primary biological endpoint of the study, representing a 10.7-fold increase from baseline) (available at http: / / investorrelations.sarepta.com / news-releases / newsrelease-details / sarepta-therapeutics-announces-positive-results-its-study). - Western blot with golodirsen: Minimum 0.09%, Maximum 4.30% (30 mg / kg / week for 48 weeks). An increase in dystrophin level of 3% or more was observed in 2 out of 25 patients (22nd International Congress of the World Muscle Society (October 3-7, 2017, St. Malo, France) tatic-files%2F64d8d897-2e4a-4119-80b4115cbae17993&usg=AOvVaw1Amh1Mq1VauvLkPvY WfVdR). US Phase 2 Study: Safety Regarding the safety data from 16 patients with DMD treated with high and low doses of NS 065 / NCNP-01 (Viltolarsen) (40 mg / kg / week and 80 mg / kg / week), no significant concerns were raised regarding study execution, data quality, or participant safety. No participants discontinued treatment at the 36-week endpoint. Specifically, there were no serious adverse events, no adverse events leading to treatment discontinuation, and no drug-related adverse events. All adverse events (AEs) were mild or moderate. Example 2: US and Canadian Phase 2 Study The US Phase 2 dose-finding study, Study NS-065 / NCNP-01-201, was initiated in December 2016 under INDI 27474. This study was conducted under ClinicalTrials.gov recognition number NCT02740972 with the title "Safety and Dose-Finding Study of NS-065 / NCNP-01 in Children with Duchenne Muscular Dystrophy (DMD)." The clinical protocol for this study is available at https: / / www.clinicaltrials.gov / ct2 / show / study / NCT02740972, and its contents are incorporated herein by reference. The present study was primarily aimed at evaluating the safety of NS-065 / NCNP-01 (Viltolarsen) to be administered as an intravenous drip infusion at a high dose (80 mg / kg) and a low dose (40 mg / kg) to patients with Duchenne muscular dystrophy (DMD) eligible for treatment with exon 53 skipping.In addition, other objectives of the present study include the evaluation of tolerability, muscle function and muscle strength, pharmacokinetics and pharmacodynamics. More specifically, the present study was a Phase 2, multicenter, two-period, randomized, placebo-controlled, dose-finding study in which NS-065 / NCNP-01 (Viltolarsen) was administered by once-weekly intravenous drip infusion for 24 weeks to ambulatory children aged 4 years and older and younger than 10 years with DMD. Two dose-level cohorts were enrolled. Period 1 of the study was conducted double-blind. Randomized patients received weekly intravenous drip infusions of NS-065 / NCNP-01 (Viltolarsen) or placebo for the first 4 weeks of their participation (Period 1), and then, intravenous drip infusion of NS-065 / NCNP-01 (Viltolarsen) for 5 to 24 weeks (20 weeks of active treatment - Period 2). Analysis of the safety data from period 1 of the 40 mg / kg dose cohort was completed prior to enrolling patients in the 80 mg / kg dose cohort.Patients who completed the 24-week study were eligible for an open-label extension study. Clinical efficacy was assessed at regularly scheduled study visits. All patients underwent a biceps muscle biopsy at baseline and a second muscle biopsy at week 24. Safety was assessed by collecting adverse events (AEs), performing blood and urine laboratory tests, electrocardiograms (ECGs), recording vital signs, and conducting physical examinations throughout the study. Serial blood samples were collected at four of the study visits to evaluate the pharmacokinetics of NS-065 / NCNP-01 (Viltolarsen). <Tipo De Estudio: Intervencionista (Ensayo Clínico)» - Actual enrollment: 16 participants. - Assignment: Randomized. - Intervention model: Parallel allocation. - Masking: Quadruple (participant, care provider, researcher, outcome assessor). - Main purpose: Treatment. - Actual start date of the study: December 2016. - Primary completion date: March 2018. <Grupos De Pacientes E Intervenciones» iA / a / ¿u<fu / ui ooou - Experimental: NS-065 / NCNP-01 (Viltolarsen) 40 mg / kg An intravenous drip infusion of NS-065 / NCNP-01 (Viltolarsen) was administered at a dose of 40 mg / kg once a week for 24 weeks to six patients with confirmed DMD with genetic deletions amenable to treatment with exon 53 skipping. - Experimental: NS-065 / NCNP-01 (Viltolarsen) 80 mg / kg Five patients with confirmed DMD with genetic deletions amenable to treatment involving the skipping of exon 53 were given an intravenous drip infusion of NS-065 / NCNP-01 (Viltolarsen) at a dose of 80 mg / kg once a week for 24 weeks. - Placebo comparator: Placebo Two patients in the 40 mg / kg dose group and three patients in the 80 mg / kg dose group were each given placebo as an intravenous drip infusion once a week for 4 weeks followed by 20 weeks of open-label treatment.<Criterios De lnclusión> - Men >4 years of age at the time of consent and <10 years of age at the time of the first intravenous drip infusion. - Confirmed DMD mutations in the dystrophin gene that are amenable to treatment involving skipping exon 53 to restore the dystrophin mRNA reading frame. - Patients able to walk independently without assistive devices. - Patients with the ability to participate in the assessments of time to stand (TSTAND), time to run / walk 10 meters (TTRW) and time to climb 4 steps (TTCLIMB), which were judged by the motor function evaluator. - Stable dose of glucocorticoid for at least 3 months. <Criterios De Exclusión» - Acute illness in the 4 weeks prior to the first dose of the study drug. - Evidence of symptomatic cardiomyopathy. (Asymptomatic cardiac abnormalities in the investigation are not exclusionary). - Severe allergy or hypersensitivity to medications. - Serious behavioral or cognitive problems that prevent participation in the study, at the discretion of the Researcher. - Previous or ongoing medical condition, medical history, physical findings, or laboratory abnormalities that could affect safety, make it unlikely that treatment and follow-up will be completed correctly, or affect the evaluation of the study results, in the investigator's opinion. - The patient was taking any other investigational drug currently or in the 3 months prior to the start of the study treatment. - The patient had undergone surgery within 3 months prior to the first planned administration of NS-065 / NCNP-01 (Viltolarsen) or surgery was planned at any time during the duration of the study. - The patient had previously participated in this study or any other study during which he / she had been administered NS-065 / NCNP-01 (Viltolarsen). In summary, the study was a 24-week, two-cohort study evaluating doses of 40 mg / kg / week and 80 mg / kg / week in 16 male patients who were eligible for treatment involving exon 53 skipping and who had been on a stable glucocorticoid dose for more than 3 years. Patients in the placebo group in each cohort underwent an initial 4-week randomization period to monitor for adverse events (safety outcomes). Thereafter, both placebo and active-treated patients continued the study for a total of 20 weeks. The NS-065 / NCNP-01-201 study evaluated the effect of 250 mg injection of NS065 / NCNP-01 (Viltolarsen) on de novo expression of dystrophin protein after 20-24 weeks of administration in two dose cohorts: 40 mg / kg / week low dose and 80 mg / kg / week high dose (as shown in Figure 3).The objective of study NS065 / NCNP-01-201 was to identify a safe and effective dose based on de novo dystrophin protein expression in skeletal muscle, measured by Western blot (WB) (primary surrogate endpoint). Secondary surrogate endpoints included immunofluorescence (IF) staining and mass spectrometry detection of de novo dystrophin protein expression, as well as RT-PCR detection of de novo dystrophin mRNA levels. Several functional endpoints were also included as secondary endpoints in the phase 2 study. The endpoints for study NS-065 / NCNP-01-201 were: <Criterios De Valoración Princ¡Dales> - Safety and tolerability of low-dose (40 mg / kg / week) and high-dose (80 mg / kg / week) intravenous (IV) administrations of NS-065 / NCNP-01 (Viltolarsen) Injectable 250 mg. - Effects of low and high IV doses of NS-065 / NCNP-01 (Viltolarsen) Injectable 250 mg on dystrophin protein induction in muscle after 20-24 weeks of treatment measured by Western Blot. - Blood drug concentration of NS-065 / NCNP-01 (Viltolarsen)<Criterios De Valoración Secundarios> - To evaluate the effects of intravenous administration of low and high doses of NS065 / NCNP-01 (Viltolarsen) Injectable 250 mg on the induction of dystrophin mRNA and protein in muscle after 20-24 weeks of treatment, measured by RT-PCR for mRNA analysis and immunofluorescence staining methods and mass spectrometry for protein analysis. - To investigate the effects of intravenous administration of low and high doses of NS065 / NCNP-01 (Viltolarsen) Injectable 250 mg after 20-24 weeks of treatment on muscle strength, mobility and functional exercise capacity, as measured by Time To Stand (TTSTAND), Time To Run / Walk 10 meters (TTRW), Time To Climb 4 Steps (TTCLIMB), North Star Ambulatory Assessment (NSAA), 6 Minute Walk Test (6MWT) and Quantitative Muscle Test (QMT) versus a matched natural history control group. In Figure 3, the timeline for “High Dose: 80 mg / kg / week” presented in the lower half is shifted to a later time than the timeline for “Low Dose: 40 mg / kg / week” presented in the upper half to indicate that high dose administration was initiated only after safety was confirmed with low dose administration. US and Canadian Phase 2 Secondary Endpoints: Timed function tests versus untreated external natural history comparator (International Cooperative Group for Neuromuscular Research (CINRG) Duchenne Natural History Study (DNHS)). Patients enrolled in the US / Canadian phase 2 NS-065 / NCNP-01-201 study were clinically assessed for muscle function at baseline, week 13, and week 25. No adjustment was made for the four-week placebo period for this purpose. These assessments included several types of timed function tests: timed standing (TTSTAND); timed 10-meter run / walk (TTRW); timed four-step climb (TTCLIMB); 6-minute walk test (6MWT); and North Star Ambulatory Assessment (NSAA). Changes over time were compared to disease trajectories in matched patients studied at CINRG DNHS. CINRG DNHS is a study that followed each of 440 patients with DMD over a period of years (with a total study duration of approximately 10 years). CINRG stands for Cooperative International Neuromuscular Research Group, a consortium of medical and scientific researchers from academic and research centers who share the common goal of positively impacting the lives of patients with neuromuscular diseases and their families by conducting well-controlled clinical trials (from http: / / www.cinrgresearch.org / ). DNHS stands for Duchenne Natural History Study and is the largest prospective, multicenter natural history study to date on Duchenne muscular dystrophy (DMD), established by CINRG (from http: / / www.cinrgresearch.org / duchenne-natural-history / ).(McDonald CM, Henricson EK, Abresch RT, Han JJ, Escolar DM, Florence JM, Duong T, Arrieta A, Clemens PR, Hoffman EP and Cnaan A, “CINRG Investigators. The International Cooperative Neuromuscular Research Group study of the natural history of Duchenne: glucocorticoid treatment preserves clinically significant functional milestones and reduces the rate of disease progression, as measured by manual muscle testing and other commonly used clinical trial outcome measures.”, Muscle Nerve, 48(1), 32-54(2013), Henricson EK, Abresch RT, Cnaan A, Hu F, Duong T, Arrieta A, Han J, Escolar DM, Florence JM, Clemens PR, Hoffman EP and McDonald CM, “CINRG Investigators.The International Cooperative Neuromuscular Research Group: Natural History Study of Duchenne: Glucocorticoid treatment preserves clinically significant functional milestones and reduces the rate of disease progression as measured by manual muscle testing and other commonly used clinical trial outcome measures. Muscle Nerve, 48(1), 55-67 (2013), and McDonald CM, Henricson EK, Abresch RT, Duong T, Joyce NC, Hu F, Clemens PR, Hoffman EP, Cnaan A, and Gordish-Dressman H, CINRG Investigators. Long-term effects of glucocorticoids on function, quality of life, and survival in patients with Duchenne muscular dystrophy: a prospective cohort study.”, Lancet, 391(10119), 451-461 (2018). The NS-065 / NCNP-01-201 trial was conducted by clinical centers participating in the CINRG network. Standard operating procedures (clinical manuals) and clinical evaluator training protocols were very similar for the NS-065 / NCNP-01-201 clinical trial and the CINRG DNHS trial. Matching of patients enrolled in NS-065 / NCNP-01-201 versus CINRG DNHS was performed using the following set of criteria. - Children between 4 and <10.5 years old at the start of the study. - Patients with at least 12 months of timed functional test data. - Geographic region: North America (USA and Canada). - With spheroids for at least 3 months and continuous use of spheroids during the 12 / 24 month observation period. - You cannot participate in another clinical trial. A search of the CINRG DNHS database for patients matched with patients enrolled in the NS-065 / NCNP-01-201 clinical trial, without specifying a dystrophin mutation amenable to treatment involving exon 53 skipping, but excluding those with an exon 3-7 deletion and those with a dystrophin deletion amenable to treatment involving exon 44 skipping, resulted in 69 subjects as shown in Table 9 below. Those with an exon 3-7 deletion and those with a dystrophin deletion amenable to treatment involving exon 44 skipping were excluded because these patients have been reported to exhibit comparatively mild symptoms. Table 9: Matched Patients from CINRG DNHS Parameter Exon 53 Omission n = 6 No Exon 53 Omission n = 63 Total n = 69 Mean age, years (range) 6.8 (4.5-10.3) 7.4 (4.0-10.5) 7.3 (4.0-10.5) Mean weight, kg (range) 25.0 (16.6-39.1) 25.6 (15.1 - 50.6) 25.6 (15.1 - 50.6) Type of deletion, n (%) Single deletion Multiple deletions Single exon duplication Multiple exon duplications No large deletions / duplications 2 (33%) 4 (67%) 0 0 0 9 (14%) 31 (49%) 2 (3%) 3 (5%) 16 (25%) 11 (16%) 35 (51%) 2 (3%) 3 (4%) 16(23%) * Omission without exon 53: Two patients had no data on the type of Elimination. In the top row of Table 9, Exon 53 Omission refers to DMD patients who are eligible for treatment involving exon 53 omission, while No Exon 53 Omission refers to all other patients (but with the exclusions noted above). In the bottom row of Table 9, No large deletions / duplications encompasses other categories such as point mutations (which are not eligible for treatment involving exon omission). The number of CINRG DNHS patients with data for the 6MWT was lower than the number of patients for the other outcomes. This is because the 6MWT was added late in the CINRG DNHS protocol, resulting in a limited amount of data relative to the other timed function tests. A comparison of disease trajectories over 24 weeks between the 16 patients enrolled in NS-065 / NCNP-01-201 and the 69 patients enrolled in CINRG DNHS showed that patients in the natural history comparator experienced declines in timed function test performance over the 24-week period. In contrast, patients enrolled in NS-065 / NCNP-01-201 showed an average improvement in timed function tests over the same 24-week period. Three of these improvements reached statistical significance: TTRW (at week 13 and week 25); TTSTAND (at week 25); and 6MWT (at week 25). No statistically significant differences were observed between the 40 mg / kg / week and 80 mg / kg / week doses of NS 065 / NCNP-01 (Viltolarsen). Figure 4 (a total of five graphs and one table) shows comparisons of changes from baseline in timed function tests over a 24-week period in patients enrolled in NS-065 / NCNP-01-201 (indicated as Viltolarsen in solid blue line) and matched patients in CINRG DNHS (indicated as DNHS in dashed red line). In the five graphs in Figure 4, Viltolarsen is the international nonproprietary name (INN) of NS-065 / NCNP-01. Additionally, in all graphs, changes from baseline were compared between the Viltolarsen-administered groups and the CINRG DNHS patient groups using a restricted maximum likelihood (REML) mixed model for repeated measures analysis (MMRM). (1) Timed Function Tests The timed function tests were TTSTAND, TTRW, TTCLIMB, and 6MWT. TTSTAND and TTRW were prespecified as distinct and separate outcome measures and were assessed based on time and a 6-point scale. TTSTAND and TTRW are also components of the NSAA. Therefore, they were measured once, and the data were used both as an independent endpoint and as part of the NSAA combined test. TTSTAND and TTRW are described later in the context of the NSAA combined scale. TTCLIMB was used to assess the time (in seconds) it took a patient to climb 4 steps. It should be noted that the clinical protocol mentioned in the earlier US / Canada Phase 2 Study incorrectly described the TTCLIMB test as being administered as part of the NSAA. The 6MWT is a widely used and accepted test for numerous diseases; in this study, the version adapted for use in DMD was used. This test is considered a simple, standardized, low-tech, and cost-effective means of clinically assessing: 1) functional motor status; and 2) integrated and global responses to exercise. To perform this test, two markers (cones) were placed 25 meters apart, and patients were asked to walk back and forth between the cones quickly and safely for 6 minutes. The total distance in meters that the patient walked in 6 minutes was recorded. The clinical evaluator measured the number of steps taken by the patient during the first 50 meters and the total meters walked in 6 minutes (Craig M. McDonald, MD, Erik K. Henricson, MPH, Jay J. Han, MD, R. Ted Abresch MS, Alina Nicorici, BS, Gary L. Elfring, MS, Leone Atkinson MD, PhD, Alien Reha BS, Samit Hirawat MD, and Langdon L.Miller MD, The 6-minute walk test as a novel outcome measure in Duchenne muscular dystrophy, Muscle & Nerve, Vol. 41, pp. 500-510, April 2010, Wiley Periodicals, Inc. and Craig M. McDonald, MD, Erik K. Henricson, MPH, R. Ted. Abresch, MS, Julaine Florence, PhD, Michelle Eagle, PhD, Eduard Gappmaier, PhD, Alian M. Glanzman, DPT, Robert Spiegel, MD, Jay Barth, MD, Gary Elfring, MS, Alien Reha, MS, and Stuart W. Peltz, PhD, The 6-minute walk test and other clinical endpoints in Duchenne muscular dystrophy: reliability, concurrent validity, and clinically important minimal differences from a multicenter study, Muscle & Nerve, Vol. 48, pp. 357-368, September 2013, Wiley Periodicals, Inc. Quantitative Muscle Testing (QMT) assessments are designed to measure muscle force production during an isometric contraction and are a well-established method for assessing muscle weakness in neuromuscular disease. The methods used here utilize the CINRG Quantitative Muscle System (CQMS). CQMS incorporates an audiovisual feedback process that enhances compliance among children with DMD. Patients were positioned on an examination table with a backrest system to eliminate the need for manual back stabilization. After a single practice administration, each patient completed a scored QMT assessment (perform two tests; the higher of the two values ​​was used for data analysis). QMT was performed by recording force in pounds via a direct computer interface with a strain gauge. Test positions and testing order were standardized.Bilateral testing of the muscle groups listed below was performed (Mayhew JE, Florence JM, Mayhew TP, Henricson EK, Leshner RT, McCarter RJ, et al., Reliable alternative outcome measures in multicenter clinical trials of Duchenne muscular dystrophy, Muscle & Nerve, 2007; 35(1): 36-42. Epub 2006 / 09 / 14.):. - Handle. - Elbow flexors (biceps). - Elbow extensors (triceps). - Knee flexors (hamstrings). - Knee extensors (quadriceps). For QMT, small mean decreases in strength were observed across all parameters during the 24-week treatment period, with the exception of the elbow extensors. The elbow extensors showed small increases (improvements) in strength. None of these changes from baseline were statistically significant at week 25. The strength and function tests were performed in the following order: TTSTAND, TTRW, TTCLIMB, NSAA, 6MWT and QMT. (2) North Star Ambulatory Assessment (NSAA) The NSAA is a 17-item functional scale rated by a physician originally designed for children with DMD who are able to walk at least 10 meters (ES Mazzone, S. Messina, G. Vasco, M. Main, M. Eagle, AD' Amico, L. Doglio, L. Politano, F. Cavallaro, S. Frosini, L. Bello, F. Magri, A. Corlatti, E. Zucchini, B. Brancalion, F. Rossi, M. Ferretti, M.G. Motta, M.R. Cecio, A. Berardinelli, P. Alfieri, T. Mongini, A. Pini, G. Astrea, R. Battini, G. Comí, E. Pegoraro, L. Morandi, M. Pane, C. Angelini, C. Bruno, M. Villanova, G. Vita, M.A. Donati, E. Bertini, and E. Mercuri, Reliability of the North Star ambulatory assessment in a multicenter setting, Neuromuscular Disorders, Vol. 19, No. 7, pp. July 2009, Elsevier BV). This assessment tool evaluates functional activities, such as standing, rising from the floor, negotiating steps, jumping, and running. The assessment is based on a 3-point rating scale: 2 = ability to perform the test normally; 1 = modified method or aid to perform the test; and 0 = unable to perform the test. Therefore, a total score can range from 0 (completely non-ambulatory) to 34 (no impairment) on these assessments.Scores for individual test items and a total score were recorded. TTSTAND and TTRW were administered as part of the NSAA. The TTSTAND test is routinely performed during standard clinical examinations of patients with DMD. It was used to assess the time it takes a patient to transition from lying on the floor to standing. The number of seconds required to complete the test and a 6-point rating scale for how well the patient achieved the standing position were documented. - TTRW was used to assess the time (in seconds) it took a patient to run / walk 10 meters (including a 6-point rating scale for run / walk quality). US / Canada Phase 2 Study: Safety Regarding the safety data from 16 patients with DMD treated with high and low doses of NS 065 / NCNP-01 (Viltolarsen) (40 mg / kg / week or 80 mg / kg / week), no significant concerns were raised regarding study execution, data quality, or participant safety. No participants discontinued treatment at the 48-week follow-up. Specifically, there were no treatment-emergent adverse events (TEAEs) requiring discontinuation or dose reduction of NS 065 / NCNP-01 (Viltolarsen). All adverse events (AEs) were mild or moderate. Example 3: Influence of pH on the Stability of Viltolarsen (A) Evaluation of pH Stability (1) To examine the relationship between the liquid properties of a solution and the stability of Viltolarsen, a Britton-Robinson buffer (pH 3, 4, 5, 6, 7, 8, 9, 10 or 11) was used to assess the stability of Viltolarsen in solutions having various pH values. Test Conditions Drug solution concentration: Viltolarsen 2 mg / mL Solution: Britton-Robinson buffer (pH 3, 4, 5, 6, 7, 8, 9, 10 or 11) Storage conditions: (1) at 121°C (autoclaved) for 10, 30 or 60 minutes; or (2) at 80°C in an incubator for 4 to 7 days Evaluation criteria: appearance (confirmation by visual observation), pH (pH meter), purity test (HPLC method), recovery rate (survival rate) (HPLC method) HPLC conditions: Flow rate: 1.0 ml / min Detector: Ultraviolet absorptiometer (measurement wavelength: 264 nm) Column: A stainless steel tube with an inner diameter of 4.6 mm and a length of 15 cm, filled with 3.5 pm of octadecylsilylated silica gel for liquid chromatography (Waters, X-bridge C18). Column temperature: 60°C Mobile phase: - 1.42 g of disodium hydrogen phosphate were dissolved in approximately 750 ml of water and then an aqueous solution of sodium hydroxide was added to adjust the pH value of the resulting solution to pH 12.0. Subsequently, water was added to the solution to give a result of 1000 ml (solution P). - 300 mL of acetonitrile were added to 700 mL of solution P, and then 16.12 g of tetrabutylammonium bromide were dissolved in the resulting solution (eluents A). - 300 mL of acetonitrile were added to 300 mL of solution P, and then 9.67 g of tetrabutylammonium bromide were dissolved in the resulting solution (eluents B). - Liquid feeding of the mobile phases: the mobile phases A / B were changed from (100% vol / 0% vol) to (0% vol / 100% vol) for 30 minutes. In the present example and the following examples, the percentage of the Viltolarsen main peak area (main peak area (%)) is shown as a purity test value, when a sum of the total detected peak areas comprising impurities was set at 100. Test Results The test results are shown in Table 10 and Figure 5. ST stands for an aqueous solution of Viltolarsen diluted with purified water to the same concentration as the drug solution, and was prepared with each preparation (and not retained). As a result, Viltolarsen was found to be unstable in an acidic solution, but relatively stable in a neutral to weakly alkaline solution. Table 10 Results of the Stability Evaluation of Viltolarsen in Buffer iA / a / ¿uzu / ui ooou Britton-Robinson (pH 3 to 11) Period of Medicine and Medicinal Element ST pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 During preparation - Calcium carbonate Calcium carbonate Calcium carbonate Calcium carbonate Calcium carbonate pH - 3.15 4.10 5.09 6.09 7.08 8.03 8.99 9.87 10.4 9 Pureza: principal area (%) 86. 53 85.9 4 86.7 5 86.8 1 86.7 0 86.5 8 86.7 4 85.9 9 86.5 6 86.3 4 Rate of recovery(time survived) (%) 10 0 96.6 97.5 97.9 97.6 97.8 98.7 95.1 95.8 96.5 121 °C 10 mi n Apariência - Sin cam bio Sin cam bio Sin cam bio Sin cam bio Sin cam cam bio Sin cam bio Sin cam bio Sin cam bio Pureza: area pico principal (%) 86. 49 29.7 1 68.7 2 81.1 9 84.0 4 84.9 1 85.0 8 - - - Rate of recovery(rate of survival) (%) 10 0 25.3 69.9 89.2 92.4 93.0 95.2 - - - 30 mi n Apariência - Sin cam bio Sin cam bio Sin cam bio Sin cam bio Sin cam bio Sin cam bio Sin cam bio Sin cam bio Sin cam bio pH - 3.51 4.16 5.08 - - - - - - Purity: Main peak area (%) 86.60 15.4 0 56.0 9 77.9 0 81.8 5 82.5 3 82.6 1 81.7 1 78.7 7 75.9 4 Recovery rate (survival rate) (%) 10 0 10.6 57.5 85.1 91.0 91.1 92.4 92.2 87.9 85.2 60 min Appearance - - - - No change No change No change No change No change No change pH - - - - 6.13 7.10 8.10 9.04 9.87 10.3 3 Purity: Peak area 86.61 - - - 78.9 5 79.7 5 79.0 3 77.1 3 73.2 6 68.7 0. Main (%) Recovery Rate (Survival Rate) (%) 10 0 - - - 84.9 85.5 85.7 83.4 77.9 72.4 80°C 4 days Appearance - No change No change No change No change No change No change No change No change No change pH - 3.51 4.16 5.08 - - - - - - Purity: Main Peak Area (%) 85.71 0.38 18.0 1 67.1 6 79.6 4 82.9 4 82.6 5 82.5 8 81.3 2 80.5 2 Recovery Rate (Survival Rate) (%) 10 0 0.1 12.1 64.4 87.7 93.5 91.8 91.7 89.4 88.0 7 days Appearance - - - - No change No change No change No change No change No change pH - - - - 6.12 7.08 8.10 9.10 9.97 10.5 3 Purity: Main peak area (%) 85.06 - - - 71.4 2 77.4 3 79.7 5 77.5 4 76.4 8 70.7 1 Recovery rate (survival rate) (%) 10 0 - - - 78.5 86.5 88.4 84.2 82.7 75.2 (B) Evaluation of pH Stability (2) To examine more specifically a pH region in which Viltolarsen is stable, a potassium phosphate borax buffer (pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 or 9.2) was used to assess the stability of Viltolarsen in solutions, having various pH values ​​(pH 6 to 9). Test Conditions Drug solution concentration: Viltolarsen 2 mg / mL Solution: Potassium phosphate-borax buffer (pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 or 9.2) Storage conditions: (1) at 121°C (autoclaved) for 10, 30 or 60 minutes; or (2) at 80°C in an incubator for 1, 4, 7 or 14 days Evaluation criteria: appearance (confirmation by visual observation), pH (pH meter), purity test (HPLC method), recovery rate (survival rate) (HPLC method) HPLC conditions: The same as those applied in section (A) above, with the exception that the column temperature was set at 502C. Test Results The test results are shown in Table 11 and Figure 6. As a result, Viltolarsen was found to be more stable in a solution with a pH of 7 to 7.5. Table 11 Results of the Stability Evaluation of Viltolarsen in Buffer Potassium Phosphate-Borax (pH 6 to 9) Period o de Medici ón Element o de Medición ST pH 6 pH 6.5 pH 7 pH 7.5 pH 8 PH 8.5 pH 9 pH 9.2 Durant e la preparación Aparien cía - Trans parent ee incolo ro Trans parent ee incolo ro Trans parent ee incolo ro Trans parent ee incolo ro Trans parent ee incolo ro Trans parent ee incolo ro Trans parent ee incolo ro Trans parent ee incolo ro pH - 6.02 6.50 7.00 7.51 7.99 8.49 8.99 9.18 Pureza: area pico principal (%) 86.4 9 85.80 85.87 85.70 86.07 85.73 85.73 85.77 85.81 Rate of recovery (survival rate) (%) 10 0 98.0 98.7 97.5 97.4 98.8 98.5 98.4 99.3 12 Γ c 1 0 m in Pureza: area pico principal (%) 86 .0 7 82.44 83.91 84.74 84.27 83.90 83.87 82.99 82.90 Rate of 10 93.9 95.2 96.7 95.4 95.4 93.5 93.4 95.2 Recovery (survival rate) (%) 0 3 0 min Purity: Main peak area (%) 86.1 6 79.99 80.60 81.13 80.86 80.72 79.69 78.26 77.30 Recovery rate (survival rate) (7°) 10 0 93.0 91.1 93.4 93.0 93.5 92.3 90.1 88.1 6 0 min Appearance - No change 0 No change 0 No change 0 No change 0 No change 0 No change 0 No change 0 No change 0 No change 0 pH - 6.04 6.53 7.01 7.53 8.03 8.52 9.02 9.22 Purity: Main peak area (7°) 85.69 76.22 77.33 77.59 77.47 76.79 75.71 73.03 71.42 Recovery rate (survival rate) (7°) 100 85.5 88.5 88.1 87.0 86.5 85.1 81.1 80.4 801 Purity: Area 86.5 81.47 83.20 84.99 84.94 85.08 84.48 83.81 83.02 Main peak area (%) 1 Recovery rate (survival rate) (%) 10 0 92.5 95.0 96.4 96.7 97.6 96.8 96.0 93.6 4 days Purity: main peak area (%) 86.4 0 67.46 72.62 78.71 77.81 75.79 75.37 71.64 69.70 Recovery rate (survival rate) (%) 10 0 75.6 81.6 89.7 87.9 86.2 85.4 80.5 76.5 7 days Purity: main peak area (%) 86.1 2 59.16 62.98 73.62 74.22 70.30 67.95 66.52 60.86 Recovery rate (survival rate) (%) 10 0 64.1 72.4 83.0 83.1 78.8 74.8 74.0 66.7 1 4 Appearance - No change No change No change No change No change No change No change Days 0 0 ooo 0 o 0 pH - 6.06 6.54 7.03 7.54 8.03 8.52 9.02 9.21 Purity: Main peak area (%) 86.0 8 39.64 50.26 60.10 62.46 55.41 53.98 49.95 - Recovery rate (survival rate) (%) 10 0 40.4 54.2 62.5 68.4 58.9 57.7 50.3 - Example 4: Influence of pH Adjuster on Formulation Stability This example aims to select a pH adjuster to adjust the Viltolarsen injection solution to a stable pH range (pH 7 to 7.5). Several types of pH adjusters (10 mM KH₂PO₄, 10 mM Na₂HPO₄, 10 mM KH₂PO₄-Na₂HPO₄, or 100 mM KH₂PO₄-Na₂HPO₄) were added to a 10 mg / mL Viltolarsen solution to adjust its pH to a stable value. The stability of the solutions was then evaluated. Viltolarsen at 10 mg / ml. Test Conditions Drug solution concentration: Viltolarsen 10 mg / mL Drug solution prescription: as shown in Table 12 below. Storage conditions: at 121 °C (autoclaved) for 30 to 60 minutes. Assessment criteria: appearance (confirmation by visual observation), pH (pH meter), purity test (HPLC method), recovery rate (survival rate) (HPLC method). Table 12 Pharmacological Solution Prescription pH adjuster No buffer (HCI / NaOH) 10 mM KH2PO4 10 mM Na2HPO4 10 mM KH2PO4Na2HPO4 100 mM KH2PO4Na2HPO4 Viltolarsen (mg) 10 10 10 10 10 Sodium chloride (mg) Potassium dihydrogen phosphate (mg) - 1.36 - 0.27 2.72 Disodium hydrogen phosphate (mg) - - 1.42 1.14 11.36 0.1 mol / mL HCl qs* - qs* qs* qs* 0.1 mol / mL sodium hydroxide qs* qs* - qs* qs* Water for injection qsqsqsqsqs Total amount (mL) 1 1 1 1 1 *: The pH value is adjusted to pH 7.3 Test Results The test results are shown in Table 13 and Figure 7. The results showed that, even without buffering, Viltolarsen was stable at a level equivalent to or greater than that achieved with a phosphate buffer. Specifically, without buffering, Viltolarsen was more stable than in a high-concentration phosphate buffer (KH₂PO₄-Na₂HPO₄ 100 mM in this example). Table 13 Results of the pH Adjusters Test Measurement Period Measurement Element ST Without buffer 10mM KH2PO4 10mM Na2HPO4 10mM KH2PO4 Na2HPO4 100mM KH2PO4 Na2HPO4 During preparation Appearance - Clear and colorless Clear and colorless Clear and colorless Clear and colorless Clear and colorless pH - 7.32 7.32 7.33 7.30 7.32 Purity: main peak area (%) 86.52 86.17 85.95 85.98 86.15 86.07 Recovery rate (survival rate) (%) 100 100.0 98.7 98.9 98.8 99.0 121° C 10 min Appearance - No change No change No change No change No change Purity: Main peak area (%) 85.79 84.78 84.47 84.41 84.30 84.01 Recovery rate (survival rate) (%) 100 96.7 96.7 97.3 97.1 95.7 30 mi Appearance - No change No change No change No change No change n Purity: Main peak area (%) 86.52 83.41 83.35 83.42 83.57 82.57 Recovery rate (survival rate) (%) 100 94.5 94.0 94.4 94.5 90.3 60 min n Appearance - No change No change No change No change No change pH - 7.26 7.27 7.28 7.26 7.23 Purity: Main peak area (%) 85.79 80.81 79.72 79.67 79.63 78.07 Recovery rate (survival rate) (%) 100 92.3 89.5 91.2 91.1 87.5 Example 5: Relationship Between Buffer and Multimer Generation Viltolarsen can generate multimers depending on storage conditions and, therefore, can decrease the purity of the monomers. In this example, 0.9% sodium chloride was added as a tonic to a 50 mg / mL Viltolarsen drug solution, followed by the addition of various buffers. Multimer generation was then measured. Test Conditions Drug solution concentration: Viltolarsen 50 mg / mL Storage conditions: at 60°C in an incubator for 5 days Evaluation criteria: multimers (HPLC method, SEC column) Analysis Conditions: Detector: Ultraviolet absorptiometer (measurement wavelength: 260 nm) Column: A stainless steel tube with an inside diameter of 7.8 mm and a length of 30 cm was filled with 7 pm of styrene-based vinyl polymer gel for liquid chromatography; and two such columns were connected in series (TSK gel G3000 PWXL, 7 pm, 7.8 mm x 30 cm, Tosoh Corporation). Column temperature: 25°C Mobile phase: 15.6 g of sodium dihydrogen phosphate dihydrate were dissolved in 750 ml of water, and then a sample solution of sodium hydroxide was added to adjust the pH value of the solution to pH 7.3. Then, water was added to the solution to give a result of 1000 ml. Subsequently, 200 ml of acetonitrile were added to 800 ml of the resulting solution. Flow rate: The flow rate was adjusted so that the retention time of the Viltolarsen monomers was approximately 21 minutes. Area measurement range: up to the peak of the Viltolarsen monomers Test Results The test results are shown in Table 14. As a result, it was found that multimer generation increased by the addition of a 50 mM phosphate buffer (sodium dihydrogen phosphate dihydrate-disodium hydrogen phosphate: NaH2PO4-2H2O-Na2HPO4) or a citrate buffer (trisodium citrate dihydrate). iA / a / ¿uzu / ui ooou Table 14 Multimer Evaluation Results (Stored at 60°C) Buffer Concentration (mM) Sodium Chloride (mM) Amount of Multimers (RRT 0.95) Initial Value 1d 2d 5d Without Buffer 150 NDNDND 0.15 Citrate 10 150 NDND 0.12 0.14 50 150 NDND 0.13 0.16 Tris 10 150 NDNDND 0.14 50 150 NDNDND 0.13 Phosphate 10 150 NDNDND 0.14 50 150 NDNDND 0.20 Example 6: Solubility Studies (100 mq / mL) An increase in the concentration of an injectable solution was studied. Test Conditions Pharmacological solution: the following 2 types Table 15 Pharmacological Solution (2 Types) Concentration Main Drug 100 mg / mL pH Adjuster Unbuffered (HCl, NaOH) 10mM KH2PO4-Na2HPO4 Viltolarsen (mg) 100 100 NaCl (mg) 9 9 KH2PO4 (mg) - 0.27 Na2HPO4 (mg) - 1.14 HCl 0.1 N qs* qs* NaOH 0.1 N qs* qs* Water for Injection qsqs Total Amount (mL) 1 1 *: The pH value is adjusted to pH 7.3 Filtration filter: made of PVDF (Millex GV, 0.22 pm, 33 mm, Millipore). Evaluation criteria: appearance (confirmation by visual observation), pH (pH meter), purity test (HPLC method), recovery rate after filtration (HPLC method). Test Results The test results are shown in Table 16. A 100 mg / mL Viltolarsen injectable solution was prepared. The prepared Viltolarsen injectable solution had no problems with solubility or filtration using a sterile filter and was a clear, colorless solution. After storage in a cool place, the solution became viscous, but there was no change in appearance. Based on these results, it was found that it is possible to prepare a 100 mg / mL injectable solution. Table 16 Results of the High Concentration Injectable Solution Examination Drug Solution Measurement Elements Before Filtration After Filtration (PVDF) 100 mg / mL, No buffer Appearance Clear and colorless No change pH 7.28 7.25 Purity: Main peak area (%) 93.31 93.52 Recovery rate (%) 100.0 99.0 100 mg / mL, 10 mM phosphate buffer Appearance Clear and colorless No change pH 7.28 7.28 Purity: Main peak area (%) 93.28 93.33 Recovery rate (%) 100.0 98.9 [Example 71 Solubility Studies (50 mq / mL) The solubility of a 50 mg / ml drug solution was evaluated. A drug solution was prepared and its solubility and filtration were evaluated using a sterilized filter. Test Conditions Pharmacological solution: Table 17 Main Drug Concentration 50 mg / mL pH Adjuster Without buffer (HCl, NaOH) Viltolarsen (g) 1300 NaCl (g) 234 0.1 N HCl qs* 0.1 N NaOH qs* Water for injection qs Total amount (L) 26 *: The pH value is adjusted to pH 7.3 Filtration filter: made of PVDF (Millidisk cartridge filter, 0.22 pm, MCGL40S03 / one + MCGL40S03 / two, Merck) Evaluation criteria: appearance (confirmation by visual observation), pH (pH meter), purity test (HPLC method), quantification (HPLC method) Test Results The test results are shown in Table 18. The 50 mg / ml drug solution became clear and colorless, with no change in concentration before and after filtration. Based on these results, it was determined that preparing a 50 mg / ml injectable solution is feasible and that there are no issues with dilution. Table 18 Results of the iA / a / ¿uzu / ui ooou Evaluation Test Item Before Filtration After Filtration Appearance Clear, colorless solution Clear, colorless solution pH 7.3 7.4 Purity Test (Related Substances) RRT = 0.80 RRT = 0.85 RRT = 0.91 RRT = 1.07 RRT = 1.24 Other Individuals Total Related Substances 0.22% 1.74% 0.72% 2.09% 0.31% 0.13% 5.39% 0.22% 1.74% 0.70% 1.99% 0.30% 0.12% 5.26% Quantification 100.9% 100.8% In the following Examples 8 to 10, tests were carried out on samples obtained from patients who had participated in the clinical trial in Example 2 or from the patients themselves who had participated in the clinical trial in Example 2. Example 8 Quantification of the Dystrophin Protein According to Mass Spectrometry (Experimental Methods) Outline: Biological analysis method The sample was subjected to SDS-PAGE electrophoresis to separate a protein, and the separated protein was then subjected to gel digestion using trypsin to obtain peptide fragments. The peptide fragments were extracted from the gel section and then dried. The peptides were then redissolved, and the dystrophin protein was subsequently identified and quantified by HPLC-MS / MS using a reversed-phase column. When the amount of dystrophin protein in a normal control was set at 100%, the range of a calibration curve was 1% to 25%. The concentration was calculated from a peak area ratio, using filamin C as the standardized protein. A least squares method was applied to the regression equation, resulting in the equation: y = mx + b (y: peak area ratio, x: % dystrophin). Gel Digestion The sample to be measured by LC-MS / MS analysis was obtained by separating a protein by SDS-PAGE (SDS: sodium dodecyl sulfate, PAGE: polyacrylamide gel electrophoresis), according to molecular weight, and subjecting the separated protein to gel digestion. Each gel was composed of a total of 11 samples, namely, analytes that constitute a standard curve (dystrophins at 0.0%, 1.0%, 3.0%, 10.0% and 25.0%), 12.5 pg of a protein (SILAC) extracted from human myotube cells to which amino acids labeled with stable isotopes have been added and which have subsequently been cultured (SILAC), one blank and 4 clinical trial samples. There were 12 lanes for the gel, and the remaining lane was used for a molecular weight marker. The analyte was produced by mixing protein extracts from five types of muscle biopsies without DMD and two types of muscle biopsies with DMD. The DMD muscle biopsies were acquired from Binghamton University, and their ethical review was completed. Prior to this example, the amount of dystrophin in each muscle biopsy had been previously measured by Western blot. Using a co-section preparation device, 70 serial sections, each 10 µm thick, were obtained from the muscle biopsy. The muscle section was transferred to a microtube that had been pre-cooled on dry ice. Using RIPA buffer comprising the Thermo Scientific protease / phosphatase inhibitor, a protein was extracted from the muscle section.The protein concentration in the extract was quantified using the BOA protein assay kit (Pierce). Each sample subjected to electrophoresis contained 50 pg of a protein and was prepared by adding 12.5 pg of SILAC. A SILAC extract was added to the analyte and the clinical sample as an internal standard to determine the dystrophin percentage. Using NuPAGE 38% Tris-acetate gel, electrophoresis was performed at 150 V for 75 minutes. Gel electrophoresis was performed in duplicate, using two gels (gel A and gel B) for each sample. The gel subjected to electrophoresis was immobilized using methanol:water:acetic acid (50:45:5) for 30 minutes, followed by water exchange, and then rehydration was performed twice. The gel was then stained with Coomassie blue for 1 hour. The gel bleaching was carried out at 42C overnight.The gel, containing dystrophin protein in the 460 kDa to 268 kDa range as determined by molecular weight marker, was sectioned and washed twice with water:acetonitrile (50:50). Gel digestion was performed using trypsin (Gold mass spectrometry grade, Promega Corporation) to obtain peptide fragments, which were then dried by vacuum centrifugation. The dystrophin peptide fragments were stored at -80°C and subsequently analyzed according to Q. Exactive Nano-LC-MS / MS. Test Sample Sixteen patients participated in the clinical trial. Muscle biopsy samples were obtained from each of the 16 patients before and after administration. Sixty-four samples were obtained from 32 specimens as a result of duplication and were then analyzed. In addition, since 8 samples obtained from 4 specimens were re-analyzed as a result of duplication, a total of 72 samples were analyzed. Analysis according to LC-MS / MS The peptide fragments obtained were analyzed by a liquid chromatography mass spectrometry method (LC-MS / MS), in which a high-resolution liquid chromatography system, Dionex Ultimate 3000 RSLCnano (Thermo Fisher Scientific), was combined with a mass spectrometry device, Q Exactive Plus (HRMS: high-resolution mass spectrometer) (Thermo Fisher Scientific). The dried peptide fragments were redissolved in 2% acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA). A 5 pL injection loop, Dinoex nanoViper sample loop (Thermo Fisher Scientific), was used for sample introduction into LC-MS / MS. Liquid chromatography was carried out under the following conditions. Analysis column: reversed phase column, Acclaim Pepmap RSLC C18, 15 cm x 75 pm, particle size: 3 pm, EASY SPRAY (Thermo Fisher). Analytical column temperature: 50°C Mobile phases A: 1% formic acid (HCOOH); Mobile phases B: 0.1% formic acid in ACN Flow rate: 0.500 pL / min (NC) Sample injection mode: partial loop (loop size: 5 pL) Sample injection quantity: 1.00 - 4.00 pL Automatic injector temperature: 10°C Execution time: 40.00 minutes Table 19 NC Pump Gradient Configuration Time (min) Flow Rate % Mobile Phase A % Mobile Phase B 0.00 0.500 99.0 1.0 6.50 0.500 99.0 1.0 26.50 0.500 65.0 35.0 26.60 0.500 10.0 90.0 29.00 0.500 10.0 90.0 29.10 0.500 99.0 1.0 40.00 0.500 99.0 1.0 The mass spectrometry device was used under the following conditions: Ion source: Thermo Fisher EASY-Spray Ionic mode: cation Scanning: monitoring of reactions in parallel Chrome peak width (full width at half maximum): 10 seconds Minimum total cycle time: 40 minutes Resolution: 17,500 Automatic gain control target: 1e5 Maximum sample injection time: 50 milliseconds Quadrupole mass spectrometer separation width: unit of 1.0 m / z Spectrum data: profile Mass tolerance: 10 ppm Typical adjustable parameters (dystrophin tuning file) Spraying voltage: 1.8 kV Capillary temperature: 275°C Lens RF level S: 70 Table 20 Dough Selection List Peptide ID Peptide Sequence Mass (m / z) Number of Charges (z) Retention Time: Start (min) Retention Time: Stop (min) DYST 2 IFLTEQPLEGLEK 758.9165 2 18.80 20.40 DYST 2 IS IFLTEQPLEGLE KL 762.9236 2 18.80 20.40 FilC_1 VAVGQEQAFSVNT R 753.3890 2 15.40 17.40 FÍIC1IS VAVGQEQAFSVNT RA 756.3990 2 15.40 17.40 FilC_2 SPFVVNVAPPLDL SK 791.9456 2 20.30 22.20 FÍIC2IS SPFVVNVAPPLDL SKA 795.9527 2 20.30 22.20 ΚΛ= Lys (13C6,15N2); RA= Arg (13C6,15N4) - isotope-labeled amino acids Table 21 Predicted Retention Time Peptide ID Retention Time (min) DYST 2 19.95 DYST 2 IS 19.95 FilC 1 16.24 FilC 1 IS 16.24 FilC 2 20.68 FilC 2 IS 20.68 The retention time changes within the range of ±1.5 minutes. Table 22 Invention Order of Samples Subjected to Electrophoresis with the Same Gel in LC-MS / MS Order Sample 1 Sample excluded from analysis 2 Sample excluded from analysis 3 Blank 4 Blank 5 SILAC 6 Dystrophin analyte 0% 7 Dystrophin analyte 1% 8 Dystrophin analyte 3% 9 Dystrophin analyte 10% 10 Dystrophin analyte 25% 11 Sample excluded from analysis 12 Clinical sample 1 13 Sample excluded from analysis 14 Clinical sample 2 15 Sample excluded from analysis 16 Clinical samples 17 Sample excluded from analysis 18 Clinical sample 4 Sample excluded from analysis: SILAC diluted with blank samples. Using LO Quan version 3.0 manufactured by Thermo Scientific, chromatogram data were collected. The mass tolerance was set to 20 ppm, and the integration algorithm was set to ICIS. When the peak area of ​​dystrophin in the 1% analyte or other analytes was 10,000 or less, the following was noted. Specifically, it was confirmed that, for both dystrophin and filamin C, the peak area ratio between the obtained peptides and the labeled peptides is a reliable numerical value that does not alter the dystrophin percentage compared to the 0% analyte. The peak areas for dystrophin and filamin C were calculated by summing the peak areas corresponding to the product ions. The dystrophin peak area was obtained from one type of dystrophin peptide fragment (i.e., amino acid sequence DYST2: IFLTEQPLEGLEK (SEQ ID NO: 4)). The filamin C peak area was established as an average value of two types of filamin C peptide fragments (i.e., amino acid sequence FILC1: VAVGQEQAFSVNTR (SEO ID NO: 6) and amino acid sequence FILC2: SPFVVNVAPPLDLSK (SEC ID NO: 8)). Table 23 Dystrophin Peptide Sequence MS1 (m / z) Product Ion DYST2 IFLTEQPLEGLEK (SEQ ID NO: 4) 758.9165 785.4387, 1042.5400, 1143.5870, 1256.6697 DYST2IS IFLTEQPLEGLEKA 762.9236 793.4521, 1050.5500, 1151.5932, 1264.6708 Filamin C MS1 Peptide Sequence (m / z) FilC_1 Product Ion VAVGQEQAFSVNTR (SEQ ID NO: 5) 756.3990 729.3985, 800.4356, 928.4942, 1057.5368 F¡IC_2 SPFVVNVAPPLDLSK (SEQ ID NO: 6) 791.9456 769.4454, 840.485, 1935, 1152.6623 FilC_2_IS SPFVVNVAPPLDLSK Λ 795.9527 777.4596, 848.4974, 1061.6081,1160.6765 The dystrophin protein level (the ratio of peak area between dystrophin and filamin C) in the analytes and individual clinical samples was calculated according to the following equation. Since dystrophin was detected even in the 0% analyte, the correction was made by subtracting the numerical value of the dystrophin protein level in the 0% analyte from the numerical value of the dystrophin protein level in each analyte. Using the Excel template, the regression line of the numerical values ​​of the dystrophin percentage and dystrophin protein levels of the analytes was obtained for each gel, and then the dystrophin percentage in clinical samples was obtained from the numerical values ​​of the dystrophin protein levels in the clinical samples. Equation 1 Dystrophin rate Dystrophin protein level = ---——;—lasaFnamwau Dystrophies of each / Dystrophies of iafsnna marked with SILAC item C of each teacher / Filamin C of the form marked with SILAC Table 24 LCcuan Excel 1 DYST 2 peak area Calculate the ratio between the peak area of ​​Dist 2 and the peak area labeled with Dist 2 Ratio between the peak area of ​​Dist 2 and the peak area labeled with Dist 2 % Dystrophin (Calculated from the numerical value of the dystrophin protein level obtained by dividing the ratio between Dist 2 and the peak area labeled with Dist 2 by the ratio between FilC and the peak area labeled with FilC, and the regression line of the % dystrophin of analytes) 2 DYST2IS peak area 3 F¡IC_1 peak area Calculate the ratio between the peak area of ​​FilC 1 and the peak area labeled with FilC 1 Ratio between the peak area of ​​FilC and the peak area labeled with FilC (average of the two remaining values) 4 F¡IC_1_IS peak area 5 F¡IC_2 peak area Calculate the ratio between the peak area of ​​FilC 2 and the peak area marked with FilC 2 6 F¡IC_2_IS peak area Results The percentage of dystrophin in the analyzed individual samples was determined to be acceptable or unacceptable according to predetermined acceptance criteria. Of the 72 samples measured, 60 met the criteria. The measurement results are shown in Tables 25 and 26. Individual samples were analyzed using duplicate samples. However, with respect to four samples obtained from two patients in the 40 mg / kg dose group (patients E and F in Table 25) before and after administration, since one sample (gel B) did not meet the criteria, the measured value was only one from the other sample (gel A). That is, in the 40 mg / kg dose group, among 16 samples from eight specimens before administration, 14 samples could be measured. One sample showed 1% dystrophin, and 11 samples were found to be below the lower limit of quantification. In addition, among 16 samples of 8 specimens in week 25, 14 samples could be measured.One sample was found to be below the lower limit of quantification for both measurements. In addition, 1% or more dystrophin was detected. In the 80 mg / kg dose group, all 16 samples from 8 pre-administration were found to be below the lower limit of quantification. Furthermore, in all 16 samples from 8 at week 25, dystrophin was detected at a level above the limit of quantification, with an average detected amount of 4.2%. Based on the previous results, it was confirmed that the expression of a dystrophin protein is recovered by administering Viltolarsen at doses of 40 mg / kg and 80 mg / kg. Table 25 Dystrophin Protein Concentrations in Extracts from Clinical Trial Samples of Human Muscle Biopsies Calculated with DIST 2 • Cohort 1 (40 mg / kg dose group) Patients to be evaluated Date of Visit % Dystrophin (Gel A) % Dystrophin (Gel B) Patient A Before BLQ administration BLQ Patient A Week 25 2.9 2.2 Patient B Before BLQ administration BLQ Patient B Week 25 2.4 2.1 Patient C Before BLQ administration BLQ Patient C Week 25 2.2 1.5 Patient D Before BLQ administration 1.0 Patient D Week 25 3.2 3.4 Patient E Before BLQ administration NR Patient E Week 25 1.3 NR Patient F Before BLQ administration NR Patient F Week 25 3.3 NR Patient G Before BLQ administration BLQ Patient G Week 25 2.1 1.8 Patient H Before BLQ administration BLQ Patient H Week 25 BLQ BLQ NR: Impossible to describe in reports. (Since the dystrophin peak area was obtained for only two analytes, it did not meet the inspection approval criteria.) BLQ: below the lower limit (1%) of quantification Table 26 Dystrophin Protein Concentrations in Extracts from Clinical Trial Samples of Human Muscle Biopsies Calculated with DIST 2 • Cohort 2 (80 mg / kg dose group) Patients to be evaluated Date of Visit % Dystrophin (Gel A) % Dystrophin (Gel B) Patient I Before BLQ administration BLQ* Patient I Week 25 4.5 3.8 Patient J Before BLQ administration BLQ* Patient J Week 25 1.8 3.5 Patient K Before BLQ administration BLQ Patient K Week 25 2.2 2.6 Patient L Before BLQ administration BLQ Patient L Week 25 1.1 2.2 Patient M Before BLQ administration BLQ Patient M Week 25 1.3 1.3 Patient N Before BLQ administration BLQ Patient N Week 25 12.2 9.3 Patient O Before BLQ administration BLQ Patient O Week 25 10.2 8.6 Patient P Before BLQ administration BLQ Patient P Week 25 1.6 1.3 BLQ: below the lower limit (1%) of quantification*: Since the peak area value of the dystrophin 1% analyte was low, the lower limit of quantification became less than 3%. Example 9 Motor Function Test (At Week 13 and at Week 25 (12 and Weeks Since Initial Administration) (Experimental Method) The motor function of this drug class was evaluated by comparing it to a natural history group used as a control. The natural history group was selected based on baseline data from the Duchenne Natural History Study (CINRG DNHS) conducted by the Cooperative International Neuromuscular Research Group (CINRG), a US-based muscular dystrophy clinical trial network. The CINRG DNHS is a longitudinal, natural history study that followed 440 male patients with DMD over a period of years. Data were collected from 2006 to 2016, and patients were found to have attended the hospital at baseline, four times in the first year, twice in the second year, and then once a year for the maximum 10-year period. At each hospital visit, patients underwent a timed function test, a muscle strength test, a questionnaire-based function test, a pulmonary function test, and a quality of life assessment. Trial 201 was conducted at a CINRG facility, and standard operating procedures (SOPs) and clinical evaluator training protocols were standardized across both trials. As a control for the present study, patients who met the following criteria, including major registration criteria such as age, spheroid use status, and trial area 201, were selected from CINRG DNHS. - Have timed performance test data for at least 12 months [time to stand (TTSTAND), time to climb 4 stairs (TTCLIMB) and time to run / walk 10 meters (TTRW) data were required at baseline]. - Patients between 4 and <10 years old at the start of the study. - Geographic region: North America (USA and Canada). - Administration of corticosteroids for at least 3 months and continuous use of corticosteroids during the observation period of 12 to 24 months. - Patients not simultaneously enrolled in other clinical studies with respect to other exon bypass agents. - Patients meet the following genetic eligibility criteria. Inclusion Criteria: - Patients with genetic test results - Patients with duplication mutation - Patients who have a nonsense mutation or a tiny mutation that causes a frameshift. Exclusion Criteria: - Patients with mutation between the promoter and exon 8 (These patients were excluded because they were reported to have slow disease progression ((1) Hum Mutat.2018; 39: 1193-1202 and (2) Hum Mutat.2008; 29(5):728-37). - Patients likely to receive treatment with exon 44 skipping (these patients were excluded because they were reported to have slow disease progression ((1) Hum Mutat.2018; 39:1193-1202.) - Patients with a mutation in structure. Consequently, 65 male patients with DMD met the criteria described above. Among these patients, 9 DMD patients were eligible for treatment involving the omission of exon 53 (exon 53 omission group), and 56 DMD patients were not eligible for treatment involving the omission of exon 53 (no exon 53 omission group). Table 27 Parameters of the Group Administered by Viltolarsen and of the Natural History Control Group (DNHS) of the CINRG Viltolarsen Parameters n = 16 DNHS Exon 53 Omission n = 9 No DNHS Exon 53 Omission n = 56 Total DNHS n = 65 Age (years) Average value 7.4 6.3 7.2 7.1 (min. - max.) (4.3-9.8) (4.5-7.8) (4.2-9.6) (4.2-9.6) Body weight (kg) Average value 23.0 21.6 24.4 24.0 (min. - max.) (14.9-35.4) (16.6-28.1) (14.8-38.7) (14.8-38.7) Mutation type, n (%) - susceptible to exon 53 treatment omission Single exon deletion 16 (100) 9 (100) 0 9 (13.8) Multiple exon deletions 0 3 (33.3) 0 3 (4.6) - not susceptible to 16 (100) 6 (66.7) 0 6 (9.2) treatment omission 0 0 56(100) 56 (86.2) of exon 53 Single exon deletion 0 0 4(7.1) 4 (6.2) 0 0 30 (53.6) 30 (46.2) Multiple exon deletions 0 0 3 (5.4) 3 (4.6) 0 0 3 (5.4) 3 (4.6) Single exon duplication 0 0 16(28.6) 16 (24.6) Multiple exon duplications Minute mutation Results Changes in timed function tests [i.e., 6-minute walk test (6MWT), speed versus time to stand (TTSTAND), speed versus time to climb 4 stairs (TTCLIMB), and speed versus time to run / walk 10 meters (TTRW)] and North Star Ambulatory Assessment (NSAA) at week 13 and week 25 before administration or from baseline were compared between 16 subjects treated with Viltolarsen (Viltolarsen-administered group) and 65 patients in the CINRG DNHS natural history group (DNHS group). In the analytical method applied in this example, the pre-administration or baseline value was established as a covariate, a background factor influencing the results. Data obtained at Week 13 and Week 25 were used as repeated measures, measured from specific subjects, and MMRM analysis was performed. As a result, the group administered Viltolarsen in this example showed significant improvement in 6-minute walk distance and 10-meter sprint speed. Furthermore, the current drug group showed improvement over the natural history group in all other motor function tests. Figure 8 includes graphs showing changes in individual motor function test scores for the Viltolarsen administered group and the CINRG natural history control (DNHS) group at week 13 (12 weeks after baseline administration) and week 25 (24 weeks after baseline administration) from each baseline. The changed amount indicates the least squares mean of a change in the Week 13 or Week 25 score relative to baseline, the error bar indicates the standard deviation, and the P-value was calculated using MMRM. The number of samples from the group administered by Viltolarsen and the DNHS group at individual time points was as follows. Table 28 Group managed by Viltolarsen Baseline Week 13 Week 25 6-minute walk test 16 15 15 North Star ambulatory assessment 16 15 16 Time to stand 16 16 16 Time to climb 4 steps 16 16 16 Time to run / walk 10 meters 16 16 16 Table 29 DNHS Baseline Week 13 Week 25 6-Walk Test 21 12 13 minutes North Star ambulatory assessment 22 11 15 Time to stand 65 39 42 Time to climb 4 steps 65 40 42 Time to run / walk 10 meters 65 40 43 Example 10 Motor Function Tests (At Week 85 (84 Weeks Passed Since Initial Administration)) (Experimental Method) With respect to the timed functional tests [i.e., 6-minute walk test (6MWT), time to stand (TTSTAND), time to climb 4 stairs (TTCLIMB), and time to run / walk 10 meters (TTRW)] and North Star Ambulatory Assessment (NSAA), measurement was performed 1 week prior to initial administration and at each 12-week time point following initial administration (week 1) (i.e., at time points in weeks 13, 25, 37, 49, 61, 73, and 85). Trial 201 was conducted at a CINRG-owned facility, and the motor function tests were performed in accordance with the standard operating procedures (SOPs) and clinical evaluator training protocols used at CINRG DNHS. Since the subjects were children, some elements could not be carried out in some tests because, for example, the subjects did not remain interested in the implementation of the tests. ResultsThe results are shown in Figures 9 through 16. In the North Star Ambulatory Assessment, many patients aged 5 or more years in the Viltolarsen-administered group maintained or improved their scores. According to the publication on studies that established patient entry criteria similar to those of clinical trials based on natural history data from Italy and England, and studied changes in NSAA scores 1 or 2 years later, the mean change over the 1-year course in patients targeted by exon 53 skipping treatment was -4.1 points (J Neurol Neurosurg Psychiatry 87, 149-55, 2016). The change at week 48 for patients in the Viltolarsen-administered group who met the publication's entry criteria was +1.3 points.According to the publication reporting the results of DHNS (Muscle Nerve 48, 55-67, 2013), 20% of DMD patients of 7 to 9 years lose their ability to increase muscle strength, but there were no such patients in the group administered with Viltolarsen. Furthermore, 10% of DMD patients with 7 to 9 years of history lose the ability to climb 4 steps, but there were no such patients in the group treated with Viltolarsen. Speed ​​decreases with age, but an increase in speed was observed in the group treated with Viltolarsen as a whole. Additionally, 10% of patients with Children with DMD aged 7 to 9 years lose the ability to walk independently, but no such patients were found in the NSP group. Speed ​​relative to the 10-meter run / walk time decreases with age, but an increase in speed was observed in the group administered viltolarsen as a whole. Regarding changes in dystrophin expression levels from baseline (quantitative dystrophin value measured by Western blot) and changes in speeds in the time to stand, time to climb 4 stairs, and time to run / walk 10 meters tests at week 48 from baseline, the presence or absence of correlation and a linear regression equation were calculated using Excel. For the time to stand and time to run 10 meters tests, there was a significant correlation between dystrophin expression levels and changes in speed (p < 0.00).05), and it was suggested that a change in the speed of motor function increases with an increase in dystrophin. Industrial Applicability According to the present invention, a pharmaceutical composition is provided for use in the treatment of Duchenne muscular dystrophy, having a stable composition of NS-065 / NCNP-01 (Viltolarsen). Furthermore, dosage and administration methods are provided with respect to a pharmaceutical composition comprising NS-065 / NCNP-01 (Viltolarsen), which exhibits effective DMD treatment and is within a safe range for human patients. Using the pharmaceutical composition, the symptoms of Duchenne muscular dystrophy can be effectively reduced with minimal side effects. NOVELTY OF THE INVENTION

Claims

Having described the present invention as above, the following claims are considered novel and, therefore, are claimed as property: CLAIMS 1. A pharmaceutical composition for the treatment of a human patient with Duchenne muscular dystrophy, the pharmaceutical composition comprising an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' terminus of exon 53 of a human dystrophin gene or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the treatment comprises the intravenous administration to the human patient of the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive. 2.The pharmaceutical composition according to claim 1, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient at a dose of 40 mg / kg / week.

3. The pharmaceutical composition according to claim 1, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient at a dose of 80 mg / kg / week.

4. The pharmaceutical composition according to claim 1, wherein the human patient has a mutation resulting in a deficiency of any exon selected from the group consisting of exons 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, or 52, in a dystrophin gene. 5.The pharmaceutical composition according to claim 1, wherein the expression of a dystrophin protein in the human patient prior to treatment is 1% or less compared to that of a healthy subject, as measured by Western blot or mass spectrometry.

6. The pharmaceutical composition according to claim 5, wherein the expression of a dystrophin protein is not found in the human patient prior to treatment.

7. The pharmaceutical composition according to claim 1, wherein the base sequence of the antisense oligomer consists of the sequence set out in SEQ ID NO:

3.

8. The pharmaceutical composition according to claim 1, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is viltolarsen or an equivalent thereof. 9.The pharmaceutical composition according to claim 1, comprising the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of between 2.5 mg / ml inclusive and 500 mg / ml inclusive, or between 10 mg / ml inclusive and 100 mg / ml inclusive.

10. The pharmaceutical composition according to claim 1, comprising the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of 25 mg / ml.

11. The pharmaceutical composition according to claim 1, comprising the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in a concentration of 50 mg / ml.

12. The pharmaceutical composition according to claim 1, further comprising at least one component selected from the group consisting of a tonicity agent, a pH adjuster, and a solvent. 13.A pharmaceutical composition according to claim 12, wherein the tonicity agent is at least one selected from the group consisting of sodium chloride, potassium chloride, glucose, fructose, maltose, sucrose, lactose, mannitol, sorbitol, xylitol, trehalose, and glycerin.

14. The pharmaceutical composition according to claim 12 or 13, wherein the pH adjuster is at least one selected from the group consisting of hydrochloric acid, sodium hydroxide, citric acid, lactic acid, phosphate (sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate), and monoethanolamine.

15. The pharmaceutical composition according to any one of claims 12 to 14, wherein the solvent is water.

16. The pharmaceutical composition according to claim 1, comprising the antisense oligomer at a concentration of between 2.5 mg / ml inclusive and 500 mg / ml inclusive, or between 10 mg / ml inclusive and 100 mg / ml inclusive, and sodium chloride at a concentration between 8 mg / ml inclusive and 10 mg / ml inclusive, and which is an aqueous solution with a pH of 7.2 to 7.

4.

17. The pharmaceutical composition according to claim 1, wherein the treatment provides at least one effect selected from the group consisting of the following effects (1) to (6): (1) the mean value of the expression level of a dystrophin protein in the skeletal muscle of the patient increases 9 times or more compared to the initial value, after administration of the pharmaceutical composition for 24 weeks; (2) a change in velocity obtained from time to standby (TTSTAND) is 0.055 times / sec or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; (3) a change in speed obtained from the 10-meter run / walk time (TTRW) is -0.025 meters / sec or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; (4) a change in speed obtained from time to climb 4 stairs (TTCLIMB) is -0.060 times / sec or more compared to baseline, at the time of week 25 after administration of the pharmaceutical composition for 24 weeks; (5) a change in the North Star Ambulatory Assessment (NSAA) score of 2.2 scores or more compared to baseline, at week 25 after administration of the pharmaceutical composition for 24 weeks; and (6) a change in the 6-minute walk test (6MWT) score of -7.5 meters or more compared to baseline, at week 25 after administration of the pharmaceutical composition for 24 weeks.

18. The pharmaceutical composition according to claim 1, wherein when treatment is administered to human patients aged 7 to 9 years with Duchenne muscular dystrophy for 84 weeks, at least one effect selected from the group consisting of the following effects (1) to (6) is provided: (1) the percentage of patients who lose the ability to climb is less than 20% at week 85 after the start of treatment; (2) the percentage of patients who lose the ability to climb 4(3) the percentage of patients who lose the ability to walk independently is less than 10% at week 85 after the start of treatment; (4) no reduction in 10-meter running / walking speed due to aging is observed at week 85 after the start of treatment; (5) no reduction in 4-step climbing speed due to aging is observed at week 85 after the start of treatment; and (6) no reduction in stair climbing speed due to aging is observed at week 85 after the start of treatment.

19. The pharmaceutical composition according to claim 1, wherein when the treatment is administered to human patients aged 10 to 12 years with Duchenne muscular dystrophy for 84 weeks, at least one effect selected from the group consisting ofThe following effects (1) to (6) are provided: (1) the percentage of patients who lose the ability to climb is less than 60% at week 85 after the start of treatment; (2) the percentage of patients who lose the ability to climb 4 steps is less than 50% at week 85 after the start of treatment; (3) the percentage of patients who lose the ability to walk independently is less than 50% at week 85 after the start of treatment; (4) no reduction in 10-meter running / walking speed due to aging is observed at week 85 after the start of treatment; (5) a period of increased speed in climbing 4 steps is observed at week 85 after the start of treatment; and (6) a period of increased climbing speed is observed at week 85 after the start of treatment.

20. A method for21. An antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' end of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, for use in a method for treating a human patient with Duchenne muscular dystrophy, comprising the intravenous administration of a pharmaceutical composition comprising an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' end of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, to a human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive of the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.Duchenne, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive.

22. The use of an antisense oligomer consisting of a base sequence complementary to a sequence consisting of nucleotides at positions 36 to 56 of the 5' end of exon 53 of a human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, for the manufacture of a pharmaceutical composition for the treatment of a human patient with Duchenne muscular dystrophy, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, is administered intravenously to the human patient once a week at a dose of between 40 mg / kg / week inclusive and 80 mg / kg / week inclusive.