Composition containing antisense oligonucleotide, and use of the same for treatment of duchenne muscular dystrophy

Abstract

To provide a composition and method for treating Duchenne muscular dystrophy.SOLUTION: A pharmaceutical composition for treating Duchenne muscular dystrophy of a human patient contains an antisense oligomer consisting of a base sequence complementary to a sequence consisting of 36-th to 56-th nucleotides from 5' terminal of 53-rd exon of a human dystrophin gene, or pharmaceutically acceptable salt thereof or a hydrate thereof, where the treatment includes intravenously administering 40 mg / kg to 80 mg / kg of the antisense oligomer or pharmaceutically acceptable salt thereof or a hydrate thereof to the human patient once a week.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention enables skipping of the 53rd exon of the human dystrophin gene. The present invention relates to dosage regimens for antisense oligomers and pharmaceutical compositions containing the oligomers. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy, occurring in approximately 1 in every 3,500 male births. It is a hereditary progressive muscular atrophy with a high degree of severity. During infancy, the fate of the child is almost the same as that of normal humans. However, muscle weakness appears from around age 4-5. Muscle weakness continues to progress until around age 12. It is a serious disease that can lead to the inability to walk in the early stages and death from heart or respiratory failure in the 20s. Currently, there is no effective treatment for DMD, and there is a strong demand for the development of new therapeutic drugs.

[0003] DMD is known to be caused by a mutation in the dystrophin gene. The gene is present on the X chromosome and is a huge gene consisting of 2.2 million base pairs of DNA. Transcribed into a precursor, which is then spliced ​​to remove introns and combine 79 exons This mRNA is translated into 3,685 amino acids, and the dystrophin protein is synthesized. The dystrophin protein is involved in maintaining membrane stability in muscle cells. The dystrophin gene in DMD patients is mutated. Therefore, functional dystrophin protein is hardly expressed in muscle cells. As a result, in the bodies of DMD patients, the structure of muscle cells cannot be maintained and a large amount of calcium is produced. Ions flow into muscle cells, causing an inflammation-like reaction and fibrosis, which leads to muscle damage. Cells become less able to regenerate.

[0004] Becker muscular dystrophy (BMD) is also caused by a mutation in the dystrophin gene, Although the symptoms include muscle weakness due to muscle atrophy, they are generally milder than those of DMD. The progression is slow, and in most cases, the disease develops in adulthood. The clinical symptoms of DMD and BMD differ depending on the mutation. Amino acid sequence during translation of dystrophin mRNA into dystrophin protein It is believed that the cause of this is whether the framework is destroyed or maintained (Non-Patent Document 1 In other words, DMD is a disease in which the amino acid reading frame is shifted, resulting in loss of function. In BMD, the dystrophin protein is hardly expressed due to a mutation in one of the exons. Although a portion of the gene is deleted, the amino acid reading frame is maintained, so the gene is functional, albeit incompletely. A dystrophin protein having the gene is produced.

[0005] Exon skipping is a promising treatment for DMD. By modifying the isoform, the amino acid reading frame of dystrophin mRNA was restored, resulting in partial This method is a method for inducing the expression of dystrophin protein with restored function in mice (Non-Patent Document 2 ) The amino acid sequence portion targeted by exon skipping will be lost. The dystrophin protein expressed by the treatment is shorter than normal, but contains amino acids The acid reading frame is maintained, so the function of stabilizing muscle cells is partially retained. Therefore, exon skipping can cause DMD to present symptoms similar to milder forms of BMD. It is expected that exon skipping will be useful in animal experiments using mice and dogs. Following this, clinical trials are currently being conducted on human DMD patients.

[0006] Exon skipping can involve either the 5' or 3' splice site or both, or This can be induced by binding of antisense nucleic acid that targets the inside of the exon. Chthon can be expressed as mR only when both splice sites are recognized by the spliceosome complex. Therefore, targeting splice sites with antisense nucleic acids By using this method, exon skipping can be induced. The splicing enhancer (ESE) must be linked to an exon to be recognized by the splicing machinery. It is believed that R protein binding is necessary, and targeting ESEs may also be involved. Exon skipping can be induced.

[0007] The location and type of dystrophin gene mutations vary among DMD patients. Antisense nucleic acids that correspond to the target gene are needed. lton et al. have developed an antisense oligonucleotide that induces exon skipping for all 79 exons. A synthetic nucleic acid has been prepared by Annemieke Aartsma-Rus et al. in the Netherlands (Non-Patent Document 3). Antisense nucleic acids that induce exon skipping have been created for 39 types of exons. (Non-Patent Document 4).

[0008] Approximately 10% of all DMD patients have a gene that skips the 53rd exon (hereafter referred to as "exon 53"). In recent years, the expression of the dystrophin gene has been Several research institutes have reported on research targeting exon skipping in the 53 gene. It has been reported (Patent Documents 1 to 4; Non-Patent Documents 5 and 6). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication WO 2006 / 000057 [Patent Document 2] International Publication WO 2004 / 048570 [Patent Document 3] U.S. Patent Publication US 2010 / 0168212 [Patent Document 4] International Publication WO 2010 / 048586 [Non-patent literature]

[0010] [Non-Patent Document 1] Monaco AP et al., Genomics 1988; 2: p. 90-95 [Non-Patent Document 2] Matsuo M., Brain Dev 1996; 18: p. 167-172 [Non-Patent Document 3] Wilton SD et al., Molecular Therapy 2007: 15: p. 1288-96 [Non-Patent Document 4] Annemieke Aartsma-Rus et al., (2002) Neuromuscular Disorders 12: S71-S77 [Non-Patent Document 5] Linda J. Popplewell et al., (2010) Neuromuscular Disorders, vol. 20, no. 2, p. 102-10 [Non-Patent Document 6] Bladen CL et al., Human Mutation (2015) 36:395-402 Summary of the Invention

[0011] The present invention is, but not limited to, the following. <1> The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence of nucleotides or A pharmaceutical composition for treating Duchenne muscular dystrophy in a human patient, comprising a pharma- ceutical acceptable salt or hydrate thereof. A pharmaceutical composition for treating trophy, comprising: The treatment comprises administering to a subject a therapeutically effective amount of an antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof. and administering the compound intravenously to the human patient at 40 mg / kg to 80 mg / kg once a week. , the pharmaceutical composition. <2> The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is 0 mg / kg once a week intravenously to said human patient. <1> The pharmaceutical composition according to . <3> The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is 0 mg / kg once a week intravenously to said human patient. <1> The pharmaceutical composition according to . <4> The human patient has exons 43-52, 45-52, 47-5 Any one selected from the group consisting of 2, 48-52, 49-52, 50-52 or 52 The above-mentioned exon-deleted mutation is <1> The pharmaceutical composition described in <5> The expression of dystrophin protein in the human patient before treatment was determined by Western blot analysis. The above is less than 1% of a healthy individual as measured by ELISA or mass spectrometry. <1> The pharmaceutical composition according to composition <6> said human patient exhibiting no expression of dystrophin protein prior to treatment; <5> The pharmaceutical composition described in <7> The sequence of the base portion of the antisense oligomer is the sequence shown in SEQ ID NO: 3. Note <1> The pharmaceutical composition described in <8> The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is iltolarsen or its equivalent. <1> The pharmaceutical composition described in <9> The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is administered in a manner similar to that described above in 2. The above-mentioned contains the compound at a concentration of 5 to 500 mg / ml or 10 to 100 mg / ml. <1> Described in Pharmaceutical compositions. <10> The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is 2. The method according to claim 1, wherein the concentration of <1> The pharmaceutical composition described in <11> The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is added to the mixture for 50 minutes. 2. The method according to claim 1, wherein the concentration of <1> The pharmaceutical composition described in <12> At least one component selected from the group consisting of an isotonicity agent, a pH adjuster, and a solvent is further added. Including the above <1> The pharmaceutical composition described in <13> The isotonicity agent may be sodium chloride, potassium chloride, glucose, fructose, maltose, or the like. Sucrose, lactose, mannitol, sorbitol, xylitol, trehalose and glycerin. <12> Described in A pharmaceutical composition comprising: <14> The pH adjuster is hydrochloric acid, sodium hydroxide, citric acid, lactic acid, phosphate (sodium hydrogen phosphate), thorium, sodium dihydrogen phosphate, potassium dihydrogen phosphate) and monoethanolamine At least one selected from the group consisting of <12> or <13> Described in Pharmaceutical compositions. <15> The solvent is water. <12> ~ <14> 2. The pharmaceutical composition according to claim 1 , <16> Antisense oligos at concentrations of 2.5-500mg / ml or 10-100mg / ml mer, sodium chloride at a concentration of 8-10 mg / ml and a pH of 7.2-7.4 The solution is an aqueous solution. <1> The pharmaceutical composition described in <17> The treatment has at least one effect selected from the group consisting of the following (1) to (6): The above results <1> The pharmaceutical composition according to claim 1, (1) The mean expression level of dystrophin protein in the skeletal muscle of patients was 0.01 at baseline a more than nine-fold increase after 24 weeks of treatment compared with (2) The change in velocity obtained from the rise time (TTSTAND) compared to the baseline Compared to -0.055 / sec or higher at 25 weeks after 24 weeks of treatment; (3) The change in speed obtained from the time to walk (TTRW) per 10 meters is compared to the baseline. -0.025 meters per second or greater at week 25 after 24 weeks of treatment compared to control; (4) The change in speed obtained from the time to climb four steps (TTCLIMB) was compared from baseline In comparison, at 25 weeks after 24 weeks of treatment, the rate was greater than or equal to -0.060 breaths per second; (5) Change in North Star Ambulatory Assessment (NSAA) score compared to baseline and a score of -2.2 or greater at 25 weeks after 24 weeks of treatment; and (6) Change in 6-minute walk test (6MWT) score compared to baseline after 24 weeks of treatment At 25 weeks, the pregnancy should be at least -7.5 metres. <18> A human patient with Duchenne muscular dystrophy aged 7 to 9 years was given the treatment for 84 weeks. In this case, at least one effect selected from the group consisting of the following (1) to (6) is obtained: The above <1> The pharmaceutical composition according to claim 1, (1) Less than 20% of patients lose the ability to stand by 85 weeks after starting treatment thing; (2) Less than 10% of patients will be unable to climb four steps by the 85th week of treatment. To do so; (3) Less than 10% of patients will lose the ability to walk independently by 85 weeks from the start of treatment. and; (4) By the 85th week of treatment, there was no significant age-related decrease in 10-meter running / walking speed. Not seen; (5) From the start of treatment to the 85th week, there was no age-related decrease in 4-step stair climbing speed. thing; and (6) No age-related decrease in standing speed was observed within 85 weeks of treatment. and. <19> Human patients with Duchenne muscular dystrophy aged 10 to 12 years were treated for 84 weeks. When the above-mentioned method is carried out, at least one of the following effects selected from the group consisting of (1) to (6) is obtained: The above <1> The pharmaceutical composition according to claim 1, (1) Less than 60% of patients lose the ability to stand by 85 weeks after starting treatment thing; (2) Less than 50% of patients will be unable to climb four steps within 85 weeks of treatment. To do so; (3) Less than 50% of patients will lose the ability to walk independently by 85 weeks from the start of treatment. and; and (4) By the 85th week of treatment, there was no significant age-related decrease in 10-meter running / walking speed. Not to be seen. (5) A period in which the rate of 4-step stair climbing increased was observed between the start of treatment and 85 weeks. ; and (6) A period of increased rising speed is observed between the start of treatment and 85 weeks. <20> The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence consisting of nucleotides or A pharmaceutical composition containing a pharma- ceutical acceptable salt or hydrate, The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is Duchenne, including intravenously administered to human patients at a dose of 100 mg / kg to 80 mg / kg once weekly. Treatment for type 1 muscular dystrophy. <20-1> The treatment method provides at least one of the effects described in claim 17 and the effect described in claim 18. The method of treatment according to <20>, wherein at least one effect or at least one effect according to claim 19 is produced. <21> A method for treating Duchenne muscular dystrophy in a human patient comprising administering to said patient a human dystrophy From the 36th to 56th nucleotides of the 5' end of the 53rd exon of the ribosome gene or a pharma- ceutical acceptable salt thereof. A salt or hydrate thereof, 40 mg of the antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof / kg to 80 mg / kg is administered intravenously to the human patient once a week; The antisense oligomer or a pharma- ceutically acceptable salt or hydrate thereof. <21-1> The administration results in at least one effect according to claim 17 and at least one effect according to claim 18. The antisense oligomer or a pharma- ceutically acceptable salt or hydrate thereof according to claim <21>, which produces at least one of the effects or at least one of the effects according to claim 19. <22> For the preparation of a pharmaceutical composition for treating Duchenne muscular dystrophy in a human patient. The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence consisting of nucleotides or Use of a pharma- ceutically acceptable salt or hydrate thereof, 40 mg of the antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof / kg to 80 mg / kg is administered intravenously to the human patient once a week; The above uses.

[0012] In addition, in another aspect, the present invention is as follows, but not limited to: [1]: Subjects were administered NS-065 / NCNP-01 intravenously at a dose of approximately 40 mg / kg / week. A method for treating Duchenne myocarditis by exon 53 skipping comprising administering to a subject a subject A method for treating a subject with a strophy. [2]: Subjects were administered NS-065 / NCNP-01 intravenously at a dose of approximately 80 mg / kg / week. A method for treating Duchenne myocarditis by exon 53 skipping comprising administering to said patient a gene encoding said Duchenne myocarditis A method for treating a subject with a strophy. [3]: Subjects were administered NS-065 at a dose of 40 mg / kg / week or more and 80 mg / kg / week or less. / For exon 53 skipping therapy, including intravenous administration of NCNP-01 Suitable methods for treating a subject with Duchenne muscular dystrophy. [4]: What is administered is NS-065 / NCNP-01 at a concentration of ≥2.5 mg / mL and ≤500 mg / mL; and As an isotonicity agent, sodium chloride at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less Um is an aqueous solution containing The method of claim 1, wherein the aqueous solution has a pH of about 7.3. [5]: What is administered is NS-065 / NCNP-01 at a concentration of ≥2.5 mg / mL and ≤500 mg / mL; and As an isotonicity agent, sodium chloride at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less Um is an aqueous solution containing The method of claim 2, wherein the aqueous solution has a pH of about 7.3. [6]: What is administered is NS-065 / NCNP-01 at a concentration of ≥2.5 mg / mL and ≤500 mg / mL; and As an isotonicity agent, sodium chloride at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less Um is an aqueous solution containing The method of claim 3, wherein the aqueous solution has a pH of about 7.3. [7]: Subjects were administered NS-065 / NCNP-01 intravenously at a dose of approximately 40 mg / kg / week. A method for treating Duchenne myocarditis by exon 53 skipping comprising administering to said patient a gene encoding said Duchenne myocarditis For inducing dystrophin protein production in subjects with dystrophin method. [8]: Subjects were administered NS-065 / NCNP-01 intravenously at a dose of approximately 80 mg / kg / week. A method for treating Duchenne myocarditis by exon 53 skipping comprising administering to said patient a gene encoding said Duchenne myocarditis For inducing dystrophin protein production in subjects with dystrophin method. [9]: NS-065 was administered to subjects at a dose of 40 mg / kg / week to 80 mg / kg / week. / For exon 53 skipping therapy, including intravenous administration of NCNP-01 In a suitable subject having Duchenne muscular dystrophy, Methods for inducing protein production.

[10] : What is administered? NS-065 / NCNP-01 at a concentration of ≥2.5 mg / mL and ≤500 mg / mL; and As an isotonicity agent, sodium chloride at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less Um is an aqueous solution containing The method according to [7], wherein the aqueous solution has a pH of about 7.3.

[11] : What is administered? NS-065 / NCNP-01 at a concentration of ≥2.5 mg / mL and ≤500 mg / mL; and As an isotonicity agent, sodium chloride at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less Um is an aqueous solution containing The method of claim 8, wherein the aqueous solution has a pH of about 7.3.

[12] : What is administered is NS-065 / NCNP-01 at a concentration of ≥2.5 mg / mL and ≤500 mg / mL; and As an isotonicity agent, sodium chloride at a concentration of 8.55 mg / mL or more and 9.45 mg / mL or less Um is an aqueous solution containing The method of claim 9, wherein the aqueous solution has a pH of about 7.3. In the above [1] to

[12] , NS-065 / NCNP-01 (referred to herein as "Viltolarsen" may be an equivalent product. In any of the above items

[12] to

[13] , the subject may be a human patient.

[0013] According to the present invention, a stable composition of viltolarsen is provided for the treatment of Duchenne muscular dystrophy. A pharmaceutical composition for treating trophy is provided. Also provided is a pharmaceutical composition comprising Viltolarsen. The composition has an effective therapeutic effect for Duchenne muscular dystrophy and is effective in treating human patients. The dosage regimen of Viltolarsen is provided within a safe range. It can effectively relieve the symptoms of Duchenne muscular dystrophy with low side effects. do. [Brief description of the drawings]

[0014] [Figure 1] The design of the US Phase 2 dose-finding study NS-065 / NCNP-01-201 is shown. [Diagram 2] 1 shows the results of measuring the expression level of de novo dystrophin protein in skeletal muscle using Western blotting. [Diagram 3] The design of the US / Canadian Phase 2 dose-finding study, NS-065 / NCNP-01-201, is shown. [Figure 4] Comparison of changes from baseline in chronofunctional tests over 24 weeks is shown. In the five graphs, "Viltolarsen" is the International Nonproprietary Name (INN) of NS-065 / NCNP-01. [Diagram 5] The results of stability evaluation of viltolarsen in Britton-Robinson buffer (pH 3 to 11) are shown. [Figure 6] The results of a stability evaluation of viltolarsen in potassium phosphate-borax buffer (pH 6-9) are shown. [Figure 7] The results of a study of pH adjusters at 121°C are shown. [Figure 8] Graphs showing changes from baseline in the results of each motor function test. [Figure 9] 1 is a graph showing the results of the 6-minute walking test in each subject in the viltolarsen administration groups (40 mg / kg administration group and 80 mg / kg administration group) up to 85 weeks (84 weeks after the first administration). [Figure 10] This is a graph showing the North Star Walking Test scores for each subject in the Viltolarsen administration groups (40 mg / kg group and 80 mg / kg group) up to Week 85 (84 weeks after the first administration). [Figure 11] This is a graph showing the results of the rise time test speed for each test patient in the viltolarsen administration groups (40 mg / kg administration group and 80 mg / kg administration group) up to 85 weeks (84 weeks after the first administration). [Figure 12] This is a graph showing the results of the speed of the 4-step stair climbing time test for each subject in the Viltolarsen administration groups (40 mg / kg administration group and 80 mg / kg administration group) up to 85 weeks (84 weeks after the first administration). [Figure 13] This is a graph showing the results of the 10 m walking / running test speed for each subject in the viltolarsen administration groups (40 mg / kg administration group and 80 mg / kg administration group) up to 85 weeks (84 weeks after the first administration). [Figure 14] This is a graph showing the correlation between the change in dystrophin expression level from baseline in the dystrophin quantification value by WB and the change in rise time rate from baseline in each patient in the viltolarsen administration group (40 mg / kg administration group and 80 mg / kg administration group) at 49 weeks (48 weeks after the first administration). [Figure 15]This is a graph showing the correlation between the change in dystrophin expression level from baseline in the dystrophin quantification value by WB and the change in speed from baseline in the 4-step stair climbing test in each patient in the viltolarsen treatment groups (40 mg / kg treatment group and 80 mg / kg treatment group) at 49 weeks (48 weeks after the first administration). [Figure 16] This is a graph showing the correlation between the change in dystrophin expression level from baseline in dystrophin quantification values ​​by WB and the change in speed from baseline in a 10-m walk / running test in each patient in the viltolarsen treatment groups (40 mg / kg treatment group and 80 mg / kg treatment group) at 49 weeks (48 weeks after the first administration). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below. The following embodiments are illustrative only for explaining the present invention. However, it is not intended that the present invention be limited to the embodiment. Unless otherwise specified, it can be implemented in various forms. All documents cited in this specification, as well as publications, patent publications and other patents, The documents are incorporated herein by reference. The present application claims priority from a U.S. provisional patent application (US62 / 690,270) filed in October 2018. Details of the U.S. provisional patent application (US62 / 739,386) on which this application claims priority, filed on the 1st of This includes the contents described in the documents and drawings.

[0016] I. First Form In a first aspect, the present invention provides a pharmaceutical composition for treating Duchenne muscular dystrophy. The present invention provides a pharmaceutical composition comprising a nucleic acid sequence encoding the fifth locus of the human dystrophin gene. Complementary to the sequence consisting of 36-56 nucleotides from the 5' end of the third exon Antisense oligomers consisting of the above base sequence (hereinafter also referred to as "oligomers of the present invention"). or a pharma- ceutical acceptable salt or hydrate thereof for use in treating Duchenne encephalopathy in a human patient. A pharmaceutical composition for treating muscular dystrophy, comprising: The treatment comprises administering to the patient an antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof. and administering to the human patient intravenously at 40 mg / kg to 80 mg / kg once a week. The pharmaceutical composition is provided.

[0017] [Exon 53 of the human dystrophin gene] In the present invention, the term "gene" includes not only genomic genes, but also cDNA, mRNA precursors, and mRNA. Preferably, the gene is a precursor mRNA, i.e., pre-mRNA. In the human genome, the human dystrophin gene is located at the locus Xp21.2. The strophin 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. The coding region is distributed within the dystrophin gene as 79 exons (Roberts, RG., et al., Genomics, 16: 536-538 (1993)). A pre-mRNA is spliced ​​to generate a mature mRNA of 14 kb. The nucleotide sequence of the mature mRNA of the trophin gene is known (GenBank Accession No. NM_0 04006). The nucleotide sequence of exon 53 of the human wild-type dystrophin gene is shown in SEQ ID NO:1.

[0018] The pharmaceutical composition of the present invention comprises a 5' end of the 53rd exon of the human dystrophin gene. An antisense oligonucleotide having a base sequence complementary to the sequence consisting of nucleotides 36 to 56 of oligomer (oligomer of the present invention) or a pharma- ceutical acceptable salt or hydrate thereof Contains substances. Here, the oligomer of the present invention is a skipping inhibitor of exon 53 of the human dystrophin gene. By using this technique, the protein encoded by the DMD dystrophin gene was detected in BMD dystrophin. Therefore, the present invention is Exon 53 of the dystrophin gene, which is the target of oligomeric exon skipping, , including not only wild-type but also mutant types.

[0019] Specifically, exon 53 of the mutant human dystrophin gene has the nucleotide sequence of SEQ ID NO:1. For columns, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher , 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, A gene that has an identity of 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more As used herein, the term "polynucleotide" refers to DNA or means RNA.

[0020] The identity of the base sequence was evaluated using the BLAST algorithm by Carlin and Arthur ( ic Local Alignment Search Tool)(Proc. Natl. Acad. Sci. USA 872264-2268, 1990; P roc Natl Acad Sci USA 90: 5873, 1993). Programs called BLASTN and BLASTX based on this method have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing nucleotide sequences using BLASTN, use the following parameters: For example, the score is 100 and the word length is 12. When used, the default parameters of each program are used.

[0021] In the present specification, the term "complementary base sequence" refers to a base sequence that is complementary to the target base sequence and has a Watson-Crick pair structure. The base sequence is not limited to those that form a wobble base pair. Watson-Crick pairs include base sequences that consist of adenine-thymine, adenine The term refers to base pairs in which hydrogen bonds are formed between guanine and uracil and between guanine and cytosine. The base pairs are guanine-uracil, inosine-uracil, inosine-adenine and inosine-cytosine. A "complementary base sequence" refers to a base pair in which a hydrogen bond is formed between the nucleotides. It is not necessary to have 100% complementarity with the target base sequence. For example, The base sequence may contain 1, 2, 3, 4 or 5 non-complementary bases. , and are 1, 2, 3, 4, or 5 bases shorter than the target base sequence. It may be a base sequence.

[0022] A sequence consisting of the 36th to 56th nucleotides from the 5' end of exon 53 (SEQ ID NO: 2) An example of the base sequence and its complementary base sequence (SEQ ID NO: 3) are shown in Table 1 below.

[0023] [Table 1]

[0024] Here, thymine (T) and uracil (U) are interchangeable, and even if "T" is "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 shown in the SEQ ID NO is "U", the same SEQ ID NO Therefore, the sequences disclosed in this application are necessarily represented by the sequences "T" and " It contains both the "U" sequence.

[0025] Therefore, the oligomer of the present invention has a base sequence which is different from the sequence shown in SEQ ID NO:3. The oligomer of the present invention may be an oligonucleotide having an endonucleases of the human dystrophin gene. 100% complementary bases to the target sequence, as long as they allow skipping of chthon 53 For example, the oligomer of the invention may have a target sequence, It may contain 1, 2, 3, 4 or 5 non-complementary bases to SEQ ID NO:2. Often, the target base sequence is 1, 2, 3, 4 or 5 bases long. It may be a short base sequence.

[0026] Whether exon 53 skipping of the human dystrophin gene occurs or not is a question of whether dystrophin The oligomer of the present invention is introduced into a dysplasia-expressing cell (e.g., a human rhabdomyosarcoma cell), and the dysplasia-expressing cell is then subjected to the transfection. The total RNA of dystrophin-expressing cells was used to identify exon 53 of the mRNA of the human dystrophin gene. The surrounding region is amplified by RT-PCR, and the PCR amplified products are subjected to nested PCR or sequence analysis. Alternatively, it can be confirmed by the patient receiving the oligomer of the present invention. Exogenous PCR, Western blot, and mass spectrometry were used to identify the exogenous DNA in previous samples. It is also possible to determine whether skipping has occurred by measuring the amount of son 53. Cut. The skipping efficiency was evaluated by recovering human dystrophin gene mRNA from test cells and The polynucleotide amount of the band with skipped exon 53 is "A" and the polynucleotide amount of the band with skipped exon 53 is "B". The amount of polynucleotide "B" in the non-skipped band was measured, and these "A" and "B" Based on the measured values, it can be calculated according to the following formula: Skipping efficiency (%) = A / ( A + B ) × 100

[0027] The oligomer of the present invention may be an oligonucleotide, a morpholino oligomer, or a peptide. The oligomer of the present invention is a peptide nucleic acid (PNA) oligomer. , morpholino oligomers are preferred.

[0028] The oligonucleotide (hereinafter referred to as "the oligonucleotide of the present invention") is a nucleic acid. The oligomer of the present invention is composed of nucleotides as building blocks. Such nucleotides are ribonucleic acids. The nucleotides may be nucleotides, deoxyribonucleotides or modified nucleotides. Modified nucleotides are nucleotides that are ribonucleotides or deoxyribonucleotides. This refers to a molecule in which the acid-base, sugar moiety, and phosphate binding moiety are all or partially modified.

[0029] Examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, and thymine. Examples of such modified bases include uracil, uracil, and modified bases thereof. For example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g. For example, 5-methylcytosine), 5-alkyluracil (for example, 5-ethyluracil), 5-halo Uracil (5-bromouracil), 6-azapyrimidines, 6-alkylpyrimidines (6-methyluracil), uracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydro 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil 1-Methyladenine, 1-Methylhypoxanthine, 2,2-Dimethylaminomethyluracil, Methylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyl Adenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylamino 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl 2-methylthiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetone Acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, isothiocytosine Examples of suitable amines include, but are not limited to, benzoyl peroxide, imidazole, xanthine, etc. stomach.

[0030] Modifications of the sugar moiety include, for example, modifications of the 2'-position of ribose and modifications of other parts of the sugar. Examples of the modification at the 2' position of ribose include -O Modifications that replace H groups with OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, and I are listed. Here, R represents alkyl or aryl, and R' represents alkylene. Modifications of other parts of the sugar include, for example, O-4′ of ribose or deoxyribose. The 2' and 4' sugar positions are linked together, e.g., LNA (Locked Nucleic Acid Examples of such compounds include 2'-O,4'-C-Ethylene-bridged Nucleic Acid (ENA), and However, the present invention is not limited to these.

[0031] Modification of the phosphate bond moiety can be, for example, a phosphorothioate bond instead of a phosphodiester bond. bond, phosphorodithioate bond, alkylphosphonate bond, phosphoramidate bond , boranophosphate bond (Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (for example, Patent Republished Publication No. 2006 (see Nos. 2006 / 038608 and 2006 / 129594).

[0032] The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. Examples of the aryl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, se c-Butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n- The alkyl may be substituted, and such substitution may be Examples of the substituent include halogen, alkoxy, cyano, and nitro. These may be substituted by 1 to 3 groups.

[0033] The cycloalkyl is preferably a cycloalkyl having 5 to 12 carbon atoms. For example, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl Examples of the alkyl groups include cyclohexyl and cyclododecyl.

[0034] Examples of halogen include fluorine, chlorine, bromine, and iodine.

[0035] The alkoxy is a straight or branched alkoxy having 1 to 6 carbon atoms, for example, meth Oxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy oxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isopropyloxy Particularly preferred is an alkoxy having 1 to 3 carbon atoms. stomach.

[0036] The aryl is preferably an aryl having 6 to 10 carbon atoms. Specifically, for example, phenyl Examples of the aryl include aryl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl may be substituted, and such substituents include, for example, alkyl, halogen, etc. Examples of the aryl group include aryl, alkoxy, cyano, and nitro. These may be substituted with 1 to 3 substituents. good.

[0037] The alkylene is preferably a straight-chain or branched-chain alkylene having 1 to 6 carbon atoms. Specifically, for example, methylene, ethylene, trimethylene, tetramethylene, pentamethylene Examples include ethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene. It is possible.

[0038] Acyl includes linear or branched alkanoyl or aroyl. Examples of the alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-Dimethylacetyl, Propionyl, Butyryl, Isobutyryl, Pentanoyl, 2 , 2-dimethylpropionyl, hexanoyl, etc. Examples of the aroyl include Examples of the aroyl include benzoyl, toluoyl, and naphthoyl. It may be substituted at any position possible, and may be substituted with alkyl.

[0039] In an embodiment in which the oligomer of the invention is an oligonucleotide, Preferably, the -OH group at the 2' position of the ribose is replaced by methoxy, and the phosphate binding moiety is phospho The compound may have a group represented by the following general formula, which is a holothioate bond, as a structural unit. stomach. [ka] (In the formula, Base represents a nucleic acid base.)

[0040] Such oligonucleotides can be synthesized using a variety of automated synthesizers (e.g., AKTA oligopilot plus 10 / 100 (GE Healthcare)) or Alternatively, the preparation can be outsourced to a third party organization (e.g., Promega or Takara).

[0041] When the oligomer of the present invention is a morpholino oligomer, the morpholino oligomer is The compound may have a group represented by the following general formula as a constituent unit. [ka] (In the formula, Base has the same meaning as defined above; W represents a group represented by any of the following formulas: [ka] (Wherein, X is -CHR 1 , -O-CH2R 1 , -S-CH2R 1 , -NR 2 R 3 or F; R 1 represents H, alkyl; R 2 and R 3 are the same or different and represent H, alkyl, cycloalkyl, or aryl. death; Y1 is 0, S, CH2 or NR 1 represents; Y2 is 0, S or NR 1 represents; Z represents 0 or S.

[0042] The morpholino oligomer is preferably an oligomer having a group represented by the following formula as a constituent unit: phosphorodiamidate morpholino oligomer (hereinafter referred to as "PMO") . [ka] (In the formula, Base, R 2 , R 3 has the same meaning as above.)

[0043] Morpholino oligomers can be prepared, for example, from the compounds described in International Publication No. WO 1991 / 009033 or WO 2006 / 013666. In particular, the PMO can be prepared according to WO 2009 / 064471. It can be prepared according to the method described in No. 471 or according to the method described below. Cut.

[0044] One embodiment of the PMO is, for example, a compound represented by the following general formula (I) (hereinafter referred to as PMO(I)): Examples of such cases include: [ka] [In the formula, each Base, R 2 , R 3 is as defined above; n is any integer in the range of 1 to 99, and preferably any integer in the range of 18 to 28. is an integer.]

[0045] PMO(I) can be produced according to known methods, and the compounds and reagents used are The present invention is not particularly limited as long as it is a method generally used for the production of O. This can be carried out by the solid-phase synthesis method (manual or using a commercially available solid-phase automated synthesizer). When producing PMOs using the solid-phase method, an automated synthesizer is used to simplify the operation procedure and ensure the accuracy of the synthesis. The method using the above is preferable.

[0046] Peptide nucleic acid is an oligomer of the present invention having a constituent unit represented by the following general formula: . [ka] [In the formula, Base has the same meaning as defined above.]

[0047] Peptide nucleic acids can be prepared, for example, according to the following documents: 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. Vulpi us, KH Petersen, RH Berg, PE Nielsen, O. Buchardt,J. Org. Chem., 59, 5 767 (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, HF Hansen, P. Andersen, T. Larsen, HG Batz, K. Otteson, H. Or um, J. Pept. Res., 49, 80 (1997)

[0048] The oligomer of the present invention further comprises a 5'-terminus which is a group represented by any one of the following chemical formulas (1) to (3): It may be present. Preferably, it is (3)-OH. [ka] Hereinafter, the groups represented by (1), (2), and (3) above will be referred to as "group (1)," "group (2)," and "group (3)," respectively. 3)".

[0049] Examples of pharma- ceutical acceptable salts of the oligomers of the invention include sodium salts, potassium salts, and the like. , alkali metal salts such as lithium salts, calcium salts, magnesium salts, etc. Earth metal salts; aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts and other metals Salts; Ammonium salts; t-Octylamine salts, Dibenzylamine salts, Morpholine salts, Gluco samine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucan amine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diene ethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt ammonium salts, organic amine salts such as tris(hydroxymethyl)aminomethane salts; hydrogen fluoride hydrohalides such as acid salts, hydrochlorides, hydrobromides, hydroiodides; nitrates, persalts Inorganic acid salts such as perchlorates, sulfates, and phosphates; methanesulfonates, trifluoromethanesulfonates lower alkane sulfonates such as sulfonates and ethanesulfonates; benzenesulfonates arylsulfonates such as p-toluenesulfonates; acetates, malates, fluorides Organic salts such as malates, succinates, citrates, tartrates, oxalates, and maleates salts; glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, asparagus salts and amino acid salts such as phosphates. These salts can be prepared by known methods. Alternatively, the oligomer of the present invention may be in the form of its hydrate.

[0050] In another embodiment, the oligomer of the present invention is viltolarsen or its equivalent. This is also fine. Viltolarsen is the International Nonproprietary Name (INN) of NS-065 / NCNP-01. In the present specification, NS-065 / NCNP-01 is -01, NS-065 / NCNP-01(Viltolarsen) or Viltol It is also called arsen.

[0051] NS-065 / NCNP-01 (Viltolarsen) is an exon 53 skipping for the treatment of Duchenne muscular dystrophy (DMD) patients suitable for treatment with It is an antisense oligonucleotide drug. NS-065 / NCNP-01 (Vilt olarsen) in U.S. Patent No. 9,079,934B2, "PMO No. 8" The compound disclosed in the present invention has a base sequence of SEQ ID NO: 35 (5'-CCTCCGGTTC T GAAGGTGTTC-3', SEQ ID NO: 3 herein), having a 5' end of -OH. No. 9,079,934 B2 is incorporated herein by reference in its entirety. US Patent No. 9,079,934 B2 is also incorporated herein by reference in its entirety. That is, a method for synthesizing NS-065 / NCNP-01 (Viltolarsen) is disclosed. do.

[0052] NS-065 / NCNP-01 (Viltolarsen) is a phosphorothioate oligonucleotide. They have a morpholino backbone that is expected to offer greater safety than oligonucleotides. For example, the phosphorothioate oligonucleotide drisapersen (BioM Development of the morpholino oligonucleotides (M. arin) has been suspended due to safety concerns, but The nucleotide eteplirsen (Sarepta) from Exondys51 (registration The registered trademarks of drisapersen and etepl are approved by the FDA. Both irsen and exon 51 skipping are suitable for DMD patients who are eligible for treatment. Eteplirsen is disclosed in U.S. Patent No. 9,506,058 B2. The entire contents of which are incorporated herein by reference.

[0053] As disclosed in U.S. Pat. No. 9,079,934 B2, NS-065 / NCN P-01 (Viltolarsen) is exon 43-52, 45-52, 47-52, Specific exons, including 48-52, 49-52, 50-52, or 52, are deleted To generate functional dystrophin protein in DMD patients, It was designed to exhibit exon skipping activity by skipping specific exons. An example of a DMD-causing mutation that could theoretically be treated by The contents of the paper by Aartsma-Rus et al. are shown in Table 3 of the paper by Aartsma-Rus et al. The entirety of this document is incorporated by reference. AM Janson, Johan T. den Dunnen, Gert-Jan B. van Ommen, and Judith CT van Deutek. om, "Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy," Neuromuscular Disorders, Vol.12, pp.S71-S77(2002)).

[0054] "Equivalent" of Viltolarsen means a generic version of Viltolarsen or The compound that is the active ingredient in this drug is Viltolarsen. Based on the safety and effectiveness confirmed in the test, the equivalent product itself will undergo clinical trials. It has been approved for manufacture and sale under the Pharmaceutical Affairs Law without any prior notice and is an exon similar to Viltolarsen. It is expected that the Viltolars 53 skipping activity. An "equivalent" of en has the same base sequence as Viltolarsen and its nucleobases, The sugar moiety and the phosphate binding moiety are all or partly modified in the same manner as Viltolarsen. These modified forms include those which have been modified in the manner described above. The "equivalent" to Viltolarsen is a free, medicamentously approved It may be in the form of an acceptable salt or hydrate.

[0055] 2. Pharmaceutical Compositions The pharmaceutical composition of the present invention may be in the form of an aqueous solution. or a pharma- ceutical acceptable salt or hydrate thereof, / ml, 5~450mg / ml, 10~400mg / ml, 15~350mg / ml, 2 0~300mg / ml, 20~250mg / ml, 20~200mg / ml, 20~15 0mg / ml, 20~100mg / ml, 20~50mg / ml, 20~40mg / ml , 20-30mg / ml, 23-27mg / ml, 24-26mg / ml or 25mg / Alternatively, the pharmaceutical composition of the present invention may contain the oligomer of the present invention at a concentration of 1000 ml. or a pharma- ceutical acceptable salt or hydrate thereof, 5mg / ml, 20~80mg / ml, 25~75mg / ml, 30~70mg / ml, 35~65mg / ml, 40~60mg / ml, 45~55mg / ml, 47~53mg / ml, 48-52mg / ml, 49-51mg / ml, or 50mg / ml It's okay to do that.

[0056] In the pharmaceutical composition of the present invention, the concentration of viltolarsen in the aqueous solution is changed. To prepare an aqueous solution of viltolarsen, for example, 250 mg of Vit ltolarsen is mixed in 0.5 mL to 100 mL of water (2.5 mg / mL to 500 mg / mL). (corresponding to a viltolarsen concentration of 100 mg / mL), more preferably 1 mL to 50 mL Mix in 1 L of water (equivalent to a viltolarsen concentration of 5 mg / mL to 250 mg / mL) Most preferably, it is mixed with 5 mL to 10 mL of water (25 mg / mL to 50 mg / mL). (equivalent to the NViltolarsen concentration) may be used.

[0057] The administration route of the pharmaceutical composition of the present invention is intravenous administration. The dosage form is, for example, an injection solution (including a drip infusion solution).

[0058] The pharmaceutical composition of the present invention contains at least one selected from an isotonicity agent, a pH adjuster, and a solvent. It may further contain the component.

[0059] The isotonicity agent contained in the pharmaceutical composition of the present invention is sodium chloride, potassium chloride, glucose , fructose, maltose, sucrose, lactose, mannitol, sorbitol, At least one selected from xylitol, trehalose, and glycerin may be used. .

[0060] A 10 mL solution suitable for injection containing 250 mg of viltolarsen is prepared using isotonicity agent As a result, sodium chloride concentration is 72.0 mg or more and 108.0 mg or less (sodium chloride concentration 7 .2mg / mL to 10.8mg / mL), more preferably 81.0mg or more to 99. 0 mg or less of sodium chloride (sodium chloride concentration 8.1 mg / mL to 9.9 mg / mL ), and most preferably 85.5 mg or more but not exceeding 94.5 mg of sodium chloride (equivalent to sodium chloride It may contain thorium (corresponding to a thorium concentration of 8.55 mg / mL to 9.45 mg / mL).

[0061] As the isotonicity agent, a phosphate buffer may be used. Examples of the buffer include citrate buffer, lactate buffer, and acetate buffer. Alternatively, sugars (other than glucose) may be used as isotonicity agents. Examples of such sugars include sorbitol and mannitol. In preparing the composition, multiple tonicity agents may be used as well.

[0062] The pH adjuster contained in the pharmaceutical composition of the present invention is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, Sodium, potassium hydroxide, triethanolamine, citric acid, lactic acid, phosphates (phosphate water) sodium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate), monoethanolamine It may be at least one selected from the above.

[0063] When a phosphate buffer is used for the oligomer of the present invention, such as Viltolarsen, In this case, the concentration of the phosphate buffer is preferably less than 100 mM. The pharmaceutical composition has a phosphate buffer concentration of 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, M or less, 50mM or less, 40mM or less, 30mM or less, 20mM or less, 10mM or less, or The concentration may be adjusted to 5 mM or less, or it may not contain a phosphate buffer.

[0064] The solvent contained in the pharmaceutical composition of the present invention may be water.

[0065] The pH of the aqueous solution suitable for injection containing viltolarsen is between 6.0 and 8.5. More preferably, it is 6.5 or more and 8.0 or less, and most preferably, it is 7.0 or more and 7.5 or less. good. Although the oligomers of the invention are stable over a wide range of pH, the pharmaceutical compositions of the invention , pH 7.0~7.5, 7.0~7.4, 7.1~7.5, 7.1~7.4, 7.2~ Adjusted to 7.5, 7.2-7.4, 7.3-7.5, 7.3-7.4 or 7.3 It is preferred.

[0066] The pharmaceutical composition of the present invention may be administered at a concentration of 2.5 to 500 mg / ml or 10 to 100 mg / ml. 10 mg / ml of the oligomer of the present invention, 8 to 10 mg / ml of sodium chloride, The aqueous solution may have a pH of 7.2 to 7.4. Alternatively, the pharmaceutical composition of the present invention comprises an oligomer of the present invention at a concentration of 25 mg / ml. and the pH may be adjusted to 7.3 and may be an aqueous solution containing no buffer. In this preparation, pH adjustment is accomplished using hydrochloric acid and / or sodium hydroxide.

[0067] As an example of the pharmaceutical composition of the present invention, The composition for injection containing 250 mg of viltolarsen is shown in Table 2 below. The pharmaceutical composition shown in 2 is hereinafter referred to as "NS-065 / NCNP-01 (Viltolars It is called "Injection 250 mg".

[0068] [Table 2]

[0069] In addition, "NS-065 / NCNP-01 (Viltolarsen) Injection 250mg In the composition of ", the amount of water for injection and the amount of sodium chloride in the isotonicity agent were reduced by half, and the total amount was reduced by 5 Such a composition may be used with viltolarsen at a concentration of 50 mg / mL. Pharmaceutical compositions containing the compound in any of the above dosages are also included in the present invention.

[0070] The volume of the aqueous solution containing viltolarsen was NS-065 / NCNP-01 and and the concentration of the tonicity agent used, as well as Viltolarsen and the tonicity agent used may be increased or decreased as long as the weight ratio between remains the same as described above.

[0071] Compositions containing viltolarsen also reduce the delivery of viltolarsen to muscle tissue. A carrier may be included to facilitate delivery. Such a carrier may be a pharma- ceutically acceptable There is no particular limitation as long as it is possible, and examples thereof include cationic carriers (e.g., cationic liposomes, Examples of carriers include those using soluble cellulose (e.g., cellulose acylates and cationic polymers) and viral envelopes. Examples of cationic liposomes include 2-O-(2-diethylaminoethyl) caprylamide gels. A lipid product consisting of rubamoyl-1,3-O-dioleoylglycerol and phospholipids as essential components. posomal, Oligofectamine® (Thermo Fisher Scientific (manufactured by Thermo Fisher Scientific), Lipofectin (registered trademark) (manufactured by Thermo Fisher Scientific) , Lipofectamine (registered trademark) (Thermo Fisher Scientific), Pofectamine® 2000 (Thermo Fisher Scientific) , DMRIE-C (Thermo Fisher Scientific), GeneSile ncer (registered trademark) (Gene Therapy Systems, Inc.), Trans Messenger (registered trademark) (manufactured by QIAGEN), TransIT-TKO (registered trademark An example of a cationic polymer is JetSI® (GeneX Indi a Bioscience) and Jet-PEI (polyethyleneimine (GeneX India Bioscience) and others. An example of a carrier using an envelope is GenomeOne (registered trademark) (HVJ -E liposome, manufactured by Ishihara Sangyo Kaisha, Ltd.

[0072] Furthermore, the pharmaceutical composition of the present invention may contain an emulsifying aid (e.g., a fatty acid having 6 to 22 carbon atoms or its pharmaceutical agent). Pharmacologically acceptable salts, albumin, dextran) and stabilizers (e.g., cholesterol , phosphatidic acid).

[0073] In the pharmaceutical composition of the present invention comprising viltolarsen and a carrier, The weight ratio of virtolarsen to carrier (i.e., carrier / virtolarsen) varies depending on the carrier used. The ratio may be changed depending on the type of the compound, and is suitably in the range of 0.1 to 100, preferably 1 to 50. More preferably, it is in the range of 10 to 20.

[0074] Aqueous solutions containing viltolarsen are administered to patients by intravenous infusion or infusion. It can be given.

[0075] 3. Efficacy, Effects and Dosage When the pharmaceutical composition of the present invention is used for treatment, the oligomer of the present invention or a pharma- ceutical acceptable salt thereof is Tolerable salts or their hydrates are administered intravenously to human patients at 40 mg / kg to 80 mg / kg once a week. The dosage of the present invention is administered (hereinafter referred to as "the dosage of the present invention"). Alternatively, the dosage of the present invention is , the oligomer of the present invention or a pharma- ceutical acceptable salt or hydrate thereof is 40 mg / kg g of the compound may be administered intravenously to a human patient once a week. The amount is 80 mg of the oligomer of the present invention or a pharma- ceutical acceptable salt or hydrate thereof. / kg once a week to a human patient. In "mg / kg," the "mg" of the molecule refers to the amount of the oligomer of the invention or a pharma- ceutical acceptable salt thereof. The amount of each possible salt or hydrate in milligrams, with the "kg" in the denominator representing the amount of each possible salt or hydrate in milligrams for a human patient. represents 1 kilogram of body weight.

[0076] NS-065 / NCNP-01 (Viltolarsen) Injection 250mg is Weekly intravenous infusion for the treatment of DMD patients suitable for treatment with son53 skipping The US / Canadian Phase 2 clinical program is arsen: designed to evaluate 40 mg / kg / week and 80 mg / kg / week Here, kg is the unit of patient weight. For example, 40m for a patient weighing 40kg. g / kg / week, give the patient 1,600 mg (=40 mg x 40) once a week. This means administering Viltolarsen to patients with rheumatoid arthritis.

[0077] 4. Disease DMD is a muscle disease caused by loss-of-function mutations in the dystrophin gene. This disease results in the loss of the dystrophin protein in the muscles of patients (Hoffman et al., 2013). The dystrophin gene is located on the X chromosome and has a high spontaneous mutation rate. In all world populations studied, the incidence of DMD was 5,000 in men with live births. The prevalence is 1 in 10,000. A recent evaluation showed that 7,000 patients were diagnosed with the disease in a global patient database. 10.1% of 149 DMD patients were suitable for treatment with exon 53 skipping Clinical symptoms typically appear at a young age. Presenting during the school years (age 4–6), affected boys have proximal muscle weakness (e.g., difficulty climbing stairs) Difficulty walking or running (difficulty interacting physically with children of the same age) Difficulty in rising from the floor is particularly evident using the typical Gower movement. Most patients achieve standing by using their legs, knees, and using hand support on the thigh) (Hoffman EP, Brown RH, and Kunkel LM. (1987). Dy strophin: 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, Ca mpbell C, Dai Y, Wang J, Barisic N, Brabec P, Lahdetie J, Walter MC, Schreiber-K atz O, Karcagi V, Garami 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, B loetzer C, Jeannet PY, Joncourt F, Diaz-Manera J, Gallardo E, Karaduman AA, Topa loglu 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, Bero ud C, Lochmuller H. (2015). The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum Mutat. 36(4): 395-402) .

[0078] Muscle tissue from DMD patients exhibits chronic inflammation, with episodes of muscle degeneration and regeneration, leading to muscle wasting. Patients typically lose their ambulation during the second decade of life and progress to ambulation by the third decade. By the time a child reaches adolescence, they may require assistance with many aspects of daily life. This leads to death in early adulthood or in the early stages of life, whereas the use of ventilator support can sometimes extend life expectancy by up to 40 years. However, it is rare for it to be extended to 50 years.

[0079] DMD gene mutations in subjects were identified using multiplex ligation-dependent probe amplification (MLPA) (Murugan et al., 2010). The contents of this paper are incorporated by reference in its entirety (Sakthivel Murugan SM, Arthi Chandr amohan and Bremadesam Raman Lakshmi, “Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis is,” Indian Journal of Medical Research, Vol. 132, pp. 303-311 (September 2010) .).

[0080] A human patient to be treated with the pharmaceutical composition of the present invention (hereinafter, "the subject of the present invention") The term "patient" refers specifically to patients who have been diagnosed by a physician as having DMD. Although not limited, in one embodiment, the dystrophin gene has exons 43-52, 45-52 , 47-52, 48-52, 49-52, 50-52 or 52 The patient may have a mutation resulting in the deletion of any one of the exons.

[0081] In addition, the subject patient of the present invention can be treated with the pharmaceutical composition of the present invention or the oligomer of the present invention. Dystrophin protein expression was measured by Western blotting or mass spectrometry before treatment. The present invention may be characterized in that the level of the pulmonary circulation is 1% or less of that of a healthy individual (100%) when measured by a method using a blood glucose level measuring device. A "healthy individual" is a person who does not have a disease related to the dystrophin protein. The expression level of dystrophin protein in the mouse may be a commonly known value or an individual value. A value obtained from another healthy individual may be used. Prior to treatment with the pharmaceutical composition or oligomer of the invention, expression of dystrophin protein was "Dystrophin protein expression is not observed. "No" means that the expression level of dystrophin protein was not significantly different from that of dystrophin protein by Western blotting or quantitative analysis. The expression level measured by quantitative analysis was similar to that of the negative control or below the detection limit. In addition, Western blot and mass spectrometry are commonly used in the field. There is no particular limitation on the method as long as it can be used. Examples of the method include Western blot and mass spectrometry. For details, please refer to the experimental method in this embodiment.

[0082] 5.Treatment theory DMD is a severe genetic muscle disorder, most commonly caused by a mutation in the dystrophin gene. One or more exons are deleted, resulting in an out-of-frame amplicon. This is caused by the translation of nicotinamide into fatty acids, which is important for muscle function. The less severe form of the disorder, vein dystrophin, is caused by a loss of expression of the dystrophin protein. BMD is a condition in which one or more exons in the dystrophin gene are deleted. Most commonly absent, but with an in-frame amino acid translation of the remaining exon Patients with BMD generally experience slower disease progression and less disability. Targeted treatment generally involves glucocorticoid treatment to delay the onset of symptoms in the limb muscles. The FDA's final guidance in February 2018, "Duchenne Musculoskeletal Support" lar Dystrophy and Related Dystrophinopathies: Developing Drugs for Treatment Gui dance for Industry.”(https: / / www.fda.gov / downloads / Drugs / GuidanceComplianceReg As indicated in the U.S. Department of Laboratories (available at http: / / www.ucm.gov / guidelines / UCM450229.pdf), There is significant unmet medical need in people with DMD.

[0083] One therapeutic approach to treat DMD patients is to inhibit functional dystrophin protein. The aim of this study is to generate exon-specific proteins to convert DMD patients to a BMD phenotype. The strategy is to use a "skipping" strategy. Exon skipping involves the insertion of a gene next to the deleted exon. Induce exon skipping, thereby restoring the amino acid reading frame (Cirak et al., 2011; Voit et al., 2014; Yokota et al., 2012). Skipping of exon 53 results in a gene that is slightly shorter than normal but partially functional. The result is that functional dystrophin protein is expressed. CNP-01 (Viltolarsen) injection 250mg was administered to patients with DMD phenotype and disease Shift towards milder disease in BMD, slowing disease progression and improving patient quality of life (Cirak S, Arechavala-Gomeza V, Guglieri M, Feng L, Torelli S, A nthony 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. (201 1). Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an o pen-label, phase 2, dose-escalation study. Lancet 378: 595-605.、Voit T, Topalo glu H, Straub V, Muntoni F, Deconinck N, Campion G, De Kimpe SJ, Eagle M, Guglie ri M, Hood S, Liefaard L, Lourbakos A, Morgan A, Nakielny J, Quarcoo N, Ricotti V, Rolfe K, Servais L, Wardell C, Wilson R, Wright P, Kraus JE. (2014). Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DE MAND II): an exploratory, randomised, placebo-controlled phase 2 study. Lancet Neurol. 13(10): 987-96.、およびYokota T, Nakamura A, Nagata T, Saito T, Kobayash i M, Aoki Y, Echigoya Y, Partridge T, Hoffman EP, Takeda S. (2012). Extensive a nd Prolonged Restoration of Dystrophin Expression with Vivo-Morpholino-Mediated Multiple Exon Skipping in Dystrophic Dogs. Nucleic Acid Ther 22(5): 306-15.).

[0084] Therefore, the pharmaceutical composition of the present invention is administered to a human patient according to the above-mentioned dosage regimen of the present invention. It is possible to treat DMD by administering Here, "treating" means alleviating the symptoms of DMD in a patient.

[0085] The treatment using the pharmaceutical composition of the present invention according to the dosage and administration method of the present invention includes the following (1) to (6): (The figures in parentheses are the mean ± standard deviation.) Difference: (1) The mean expression level of dystrophin protein in the skeletal muscle of patients was 0.01 at baseline a more than nine-fold increase after 24 weeks of treatment compared with (2) The change in velocity obtained from the rise time (TTSTAND) compared to the baseline Compared with the 24-week treatment, the mean mean was -0.055 or more times per second or 0.024 ± 0.0 at 25 weeks. Must be at least 75 seconds; (3) The change in speed obtained from the time to walk (TTRW) per 10 meters is compared to the baseline. Compared with placebo, at 25 weeks after 24 weeks of treatment, the mean mean score was -0.025 meters per second or 0.2 27±0.251 meters / second or greater; (4) The change in speed obtained from the time to climb four steps (TTCLIMB) was compared from baseline In comparison, at 25 weeks after 24 weeks of treatment, the rate was greater than -0.060 times / second or 0.032 ± 0. 088 times / second or more; (5) Change in North Star Ambulatory Assessment (NSAA) score compared to baseline After 24 weeks of treatment, the score was -2.2 or higher or 0.8 ± 2.9 or higher at 25 weeks. and (6) Change in 6-minute walk test (6MWT) score compared to baseline after 24 weeks of treatment At 25 weeks postmenopausal, the pregnancy test should be greater than -7.5m or greater than 28.9±36.3m.

[0086] In the above (1) to (6), "baseline" refers to the average value in the untreated patient group. In addition, the "change" in (2) to (6) refers to the change in the average value.

[0087] In one embodiment, the pharmaceutical composition of the present invention is administered according to the above-described dosage regimen of the present invention. In a study of human patients with Duchenne muscular dystrophy aged 7 to 9 years for 84 weeks At least one effect selected from the group consisting of the following (7) to (12) may occur: (7) Less than 20% of patients lose the ability to stand by 85 weeks after starting treatment. thing; (8) Less than 10% of patients will be unable to climb four steps within 85 weeks of treatment. To do so; (9) Less than 10% of patients will lose the ability to walk independently within 85 weeks of treatment. and; (10) Age-related decrease in 10-meter running / walking speed by 85 weeks from the start of treatment Absence of (11) From the start of treatment to 85 weeks later, no age-related decrease was observed in the speed of climbing four steps. and (12) No age-related decrease in standing speed was observed within 85 weeks of treatment thing. The effects of (7) to (12) above are calculated based on the first week of treatment, with the first week being defined as the first week. Up to week 5, 1st to 80th week, 1st to 75th week, 1st to 70th week, 1st It may occur at any time between week 1 and week 65, or between week 1 and week 60.

[0088] In one embodiment, the pharmaceutical composition of the present invention is administered according to the above-described dosage regimen of the present invention. When administered to human patients with Duchenne muscular dystrophy aged 10 to 12 years, the following At least one effect selected from the group consisting of (13) to (18) above is produced: (13) Less than 60% of patients lose the ability to stand by 85 weeks after starting treatment. To do so; (14) Less than 50% of patients will be unable to climb 4 steps within 85 weeks of treatment. To be; (15) Less than 50% of patients lose the ability to walk independently within 85 weeks of treatment. thing; and (16) Age-related decrease in 10-meter running / walking speed by 85 weeks from the start of treatment is not observed. (17) A period in which the rate of stair climbing increased by 4 steps was observed between the start of treatment and 85 weeks. and; (18) A period in which the rate of rising increases is observed within 85 weeks of treatment. . The effects of (13) to (18) above are calculated by dividing the week in which treatment was started by the first week into weeks 1 to 18. Up to 85 weeks, 1st to 80th weeks, 1st to 75th weeks, 1st to 70th weeks, It may occur at any time between 1 week and 65 weeks, or between 1 week and 60 weeks.

[0089] That is, the pharmaceutical composition of the present invention has at least one of the effects (1) to (18) described above. may result.

[0090] II. The second form The present invention, in a second aspect thereof, comprises The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence consisting of nucleotides or A pharmaceutical composition containing a pharma- ceutical acceptable salt or hydrate, The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is and administering to said human patient intravenously at a dose of from 100 mg / kg to 80 mg / kg once a week. The present invention provides a method for treating muscular dystrophy (hereinafter referred to as the "treatment method of the present invention"). . The meaning of each component of the treatment method of the present invention has already been described in the section "I. First embodiment" of the present invention. The composition relating to the pharmaceutical composition is the same as that described above. The treatment method of the present invention is described in the subsection "5. Treatment Theory" of the section "I. First embodiment". At least one of the effects (1) to (18) described above can be obtained.

[0091] III. The third form Furthermore, the present invention, in a third aspect thereof, A method for treating Duchenne muscular dystrophy in a human patient comprising administering to said patient a human dystrophy From the 36th to 56th nucleotides of the 5' end of the 53rd exon of the ribosome gene or a pharma- ceutical acceptable salt thereof. A salt or hydrate thereof, 40 mg of the antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof / kg to 80 mg / kg is administered intravenously to the human patient once a week; The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof (hereinafter, The present invention provides a method for producing a semiconductor device using the above-mentioned method. The meaning of each of the configurations relating to the use specific embodiment of the present invention has already been described in the section "I. First embodiment" of the present invention. The composition of the pharmaceutical composition is the same as that described above. In addition, the specific use embodiment of the present invention is described in the sub-item "5. Treatment Theory" of the item "I. First embodiment". At least one of the effects (1) to (18) described above can be obtained.

[0092] IV. The Fourth Form Furthermore, the present invention, in a fourth aspect thereof, For the preparation of a pharmaceutical composition for treating Duchenne muscular dystrophy in a human patient. The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence consisting of nucleotides or Use of a pharma- ceutically acceptable salt or hydrate thereof, 40 mg of the antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof / kg to 80 mg / kg is administered intravenously to the human patient once a week; The above-mentioned use (hereinafter referred to as "the Swiss-type use of the present invention") is provided. The meaning of each of the configurations relating to the Swiss-type use of the present invention has already been described in section "I. First embodiment" of this invention. The configuration of the pharmaceutical composition of the invention is the same as that explained above. In addition, the Swiss-type use of the present invention is described in the sub-item "5. Treatment Theory" of the item "I. First Form." " may produce at least one of the effects (1) to (18) described in the above.

[0093] The present invention will be described in more detail below with reference to examples. The scope of the present invention is not limited to the above. EXAMPLES

[0094] Manufacturing Example

[0095] (1) Aminomethylpolystyrene Resin-Supported 4-{[(2S,6R)-6-(4-Benzo Amido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy Production of}-4-oxobutanoic acid Step 1: 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidine-1(2H)-isopropylacrylamide Preparation of 4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid Under argon atmosphere, N-{1-[(2R,6S)-6-(hydroxymethyl)-4-trimethyl 2-[2-oxo-1,2-dihydropyrimidin-4-yl]benzamide 2.0 g and 7.04 g of 4-dimethylaminopyridine (4-DMAP) were suspended in 269 mL of dichloromethane. 5.76 g of succinic acid was added and stirred at room temperature for 3 hours. 40 mL of methanol was added to the reaction solution and concentrated under reduced pressure. The residue was extracted with ethyl acetate and 0.5M aqueous potassium dihydrogen phosphate solution. The resulting organic layer was washed with 0.5 M aqueous potassium dihydrogen phosphate solution, water, and saturated saline in that order. The resulting organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain 25.9 g of the desired product.

[0096] Step 2: Aminomethylpolystyrene resin-supported 4-{[(2S,6R)-6-(4-Benzyloxy)phenyl]phenyl Amido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy Production of}-4-oxobutanoic acid 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl) 23.5 g of -4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid was dissolved in pyridine ( Dissolve 4.28 g of 4-DMAP and 1-ethyl-3-(3-dimethylaminopropyl)carbamate in 336 mL of dehydrated water. 40.3 g of ruthenium diimide hydrochloride was added. Then, aminomethyl polystyrene resin 1% DVB crosslinker was added. Add 25.0 g of ethylene glycol (Tokyo Chemical Industry Co., Ltd., A1543) and 24 mL of triethylamine, and shake at room temperature for 4 days. After the reaction, the resin was filtered off. The resulting resin was dissolved in pyridine, methanol, and dichloromethane. The resin was washed with 150 mL of tetrahydrofuran (dehydrated) and dried under reduced pressure. 15 mL of aqueous acetic acid and 15 mL of 2,6-lutidine were added and shaken at room temperature for 2 hours. The precipitate was washed with acetone, methanol, and dichloromethane in that order, and dried under reduced pressure to obtain 33.7 g of the desired product. The loading amount of the target compound was determined by a known method using the moles of trityl per gram of resin. The amount of resin was determined by measuring the UV absorbance at 409 nm. The concentration was 97.4 μmol / g.

[0097] UV measurement conditions Equipment: U-2910 (Hitachi) Solvent: methanesulfonic acid Wavelength: 265 nm Epsilon value: 45000

[0098] (2) Manufacturing of PMO No.8 PMO No. 8 targets the 36th to 56th positions in exon 53 and The group is "group (3)" and the sequence of the base portion is represented by SEQ ID NO:3. 4-{[(2S,6R)-6-(4-benzamide)-supported on aminomethylpolystyrene resin do-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]meth 2 g (800 μmol) of 4-oxobutanoic acid (Reference Example 1) was transferred to a reaction vessel, and dichloromethane was added. After adding 30 mL of ethanol and leaving it for 30 minutes, the mixture was washed twice with 30 mL of dichloromethane. The synthesis cycle was started. The desired base sequence was obtained in each cycle. The morpholino monomer compound was added.

[0099] [Table 3] The deblocking solution was a mixture of trifluoroacetic acid (2 equivalents) and triethylamine (1 equivalent). 1% (v / v) ethanol and 10% (v / v) 2,2 The compound was dissolved in a dichloromethane solution containing 2-trifluoroethanol and used. The neutralization solution was 25% N,N-diisopropylethylamine to 5% (v / v). The samples were dissolved in a dichloromethane solution containing 2-propanol (v / v). Pulling solution A was prepared by adding 10% (v / v) morpholino monomer compound to a concentration of 0.15M. 1,3-Dimethyl-2-imidazolidinone containing N,N-diisopropylethylamine The coupling solution B was dissolved in N,N-diisopropylethylamine. amine dissolved in 1,3-dimethyl-2-imidazolidinone to a concentration of 10% (v / v) The capping solution was 20% (v / v) acetic anhydride in dichloromethane. and 30% (v / v) 2,6-lutidine were dissolved in the solution.

[0100] The aminomethylpolystyrene resin carrying the synthesized PMO was recovered from the reaction vessel. The resulting mixture was dried under reduced pressure at room temperature for at least 2 hours. MO was placed in a reaction vessel, 200 mL of 28% aqueous ammonia-ethanol (1 / 4) was added, and the vessel was heated at 55°C for 15 hours. The aminomethyl polystyrene resin was filtered off and washed with 50 mL of water-ethanol (1 / 4). The filtrate was concentrated under reduced pressure. The residue was diluted with 20 mM acetate-triethylamine buffer. Dissolve in 100 mL of a mixture of TEAA buffer and acetonitrile (4 / 1) and apply to the membrane filter. The resulting filtrate was purified by reverse phase HPLC. It is.

[0101] [Table 4] Each fraction was analyzed, and the target substance was recovered with 100 mL of acetonitrile-water (1 / 1), and then 200 mL of alcohol was added, and the mixture was concentrated under reduced pressure. It was then dried under reduced pressure to obtain a white solid. The solid was suspended in 300 mL of 10 mM phosphoric acid aqueous solution. 10 mL of 2 M phosphoric acid aqueous solution was added and the mixture was stirred for 15 The mixture was stirred for 10 minutes. Then, 15 mL of 2M aqueous sodium hydroxide solution was added to neutralize the mixture. The solution was made alkaline by adding 15 mL of sodium hydroxide solution, and the membrane filter (0.45 μ The mixture was filtered through a 100 mL filter. The mixture was washed with 100 mL of 10 mM sodium hydroxide solution, and the target product was extracted as an aqueous solution. Got it. The resulting aqueous solution containing the target substance was purified using an anion exchange resin column. is as follows:

[0102] [Table 5] Each fraction was analyzed (HPLC) to obtain the target substance as an aqueous solution. The mixture was neutralized by adding 225 mL of phosphate buffer (pH 6.0). The mixture was then desalted by ultrafiltration under the following conditions.

[0103] [Table 6] The filtrate was concentrated to obtain about 250 mL of an aqueous solution. The resulting aqueous solution was filtered through a membrane filter (0.4 The resulting aqueous solution was lyophilized to give 1.5 g of the target compound as a white, fluffy solid. I got something. ESI-TOF-MS calculated value: 6924.82 Measurement: 6923.54 EXAMPLES

[0104] US Phase 2 study The Phase II dose-finding study "NS-065 / NCNP-01-201" was completed in January 2016. The trial was initiated in February under the name IND127474. The CDC uses the ls.gov identification number "NCT02740972" in the Safety and Dose Finding St. udy of NS-065 / NCNP-01 in Boys With Duchenne Muscular Dystrophy (DMD) The study protocol is available at https: / / www.clinicaltrials.gov / . Enter the link at trials.gov / ct2 / show / study / NCT02740972 This study was conducted using exon 53 skipping In patients with Duchenne muscular dystrophy (DMD) suitable for treatment with ping High-dose (80 mg / kg) and low-dose (40 mg / kg) N delivered as an intravenous infusion The main objective of this study is to evaluate the safety of S-065 / NCNP-01 (Viltolarsen). In addition, tolerability, muscle function and strength, pharmacokinetics, and pharmacodynamics were further investigated. was included as a target.

[0105] More specifically, this is a Phase II, multicenter, two-period, randomized, placebo-controlled, dose-finding study. NS-065 / NCNP-01 (Viltola) was administered to outpatients aged 4 to 10 years with MD. rsen) was administered intravenously once a week for 24 weeks. Two dose-level cohorts were included. Period 1 The study was conducted in a double-blind fashion. Patients were randomized to receive either NS-065 / NCNP-01 (Vilt olarsen) or placebo was administered intravenously once weekly for the first 4 weeks of participation (period 1). NS-065 / NCNP-01 (Viltolarsen) for 5 to 24 weeks ( 20 weeks of active treatment - Period 2) were administered intravenously. Analysis of these safety data was completed prior to enrollment of patients in the 80 mg / kg dose cohort. Patients who completed the 24-week study were eligible to enter an open-label extension study.

[0106] Clinical efficacy was assessed at regularly scheduled study visits. All patients were She underwent a biceps muscle biopsy at line and a second muscle biopsy at 24 weeks.

[0107] Safety was monitored by adverse events (AEs), blood and urine laboratory tests, electrocardiograms (ECGs), vital signs, and The study was evaluated by collecting baseline physical examinations and blood tests. To evaluate the pharmacokinetics of 65 / NCNP-01 (Viltolarsen), Serial blood samples were collected at study visits.

[0108] <Type of clinical trial: Intervention (clinical trial)> Actual registration: 16 participants. Allocation: Randomized. · Intervention model: Parallel allocation. Masking: Quadruple (participant, caregiver, investigator, outcome assessor) valuer). ·Primary purpose: treatment. Actual exam start date: December 2016. · Initial completion date: March 2018.

[0109] Patient population and interventions Experiment: NS-065 / NCNP-01 (Viltolarsen) 40mg / kg DMD with a gene deletion amenable to treatment with exon 53 skipping was identified Six patients received NS-065 / NCNP-01 (Viltolarsen) 40 mg / The patient was administered intravenously at a dose of 1 kg once weekly for 24 weeks. Experimental: NS-065 / NCNP-01 (Viltolarsen) 80mg / kg DMD with a gene deletion amenable to treatment with exon 53 skipping was identified Six patients received NS-065 / NCNP-01 (Viltolarsen) 80 mg / The patient was administered intravenously at a dose of 1 kg once weekly for 24 weeks. Placebo comparator: Placebo Two or three patients in each dose group received placebo intravenously once a week for four weeks. This was followed by 20 weeks of open-label treatment.

[0110] <Selection criteria> Males aged 4 years or older but under 10 years old. -Dystrophin mRNA reading frame by exon 53 skipping therapy have a DMD mutation(s) in the dystrophin gene that is suitable for restoration of It has been confirmed that Able to walk independently without assistance Time to stand up, time to run / walk, and time to increase rating - At least 3 months of stable administration of glucocorticoids

[0111] <Exclusion criteria> -Acute illness within 4 weeks prior to the first dose of the study drug. Evidence of symptomatic cardiomyopathy (without excluding asymptomatic cardiac abnormalities at the time of investigation) ·Severe allergy or hypersensitivity to medications -Severe behavioral or cognitive problems that, in the opinion of the investigator, would prevent participation in the trial - Any changes that, in the opinion of the investigator, may affect safety, treatment and follow-up There are previous or ongoing issues that make it unlikely that the study will be completed successfully or that could compromise the evaluation of the study results. Current medical condition, medical history, physical findings, or abnormal laboratory test results -Currently taking or within 3 months of starting the study drug. Prior to the first anticipated dose of NS-065 / NCNP-01 (Viltolarsen) Patients who had undergone surgery within the past 3 months or were scheduled to undergo surgery during the study period Patients who have previously participated in this study or NS-065 / NCNP-01 (Viltolars Patients who were previously treated with cyclophosphamide and participated in other studies

[0112] To summarize again, patients who are suitable for treatment with exon 53 skipping and have had a course of treatment for ≥3 months should receive Sixteen male patients were treated with 40 mg / kg / week and 80 mg / day of cocorticoids. This was a 24-week, two-cohort study testing 100 mg / kg / week doses. Patients in the 1st 4-week randomization period were treated as controls for adverse events (safety outcomes). Both placebo-treated and active-treated patients were then randomized to a 20-week treatment period. The study continued for NS-065 / NCNP-01-201. 65 / NCNP-01 (Viltolarsen) injection at a low dose of 40 mg / kg / week and a higher dose of 80 mg / kg / week for 20 to 24 weeks. The effect of trophin-protein de novo expression was evaluated (see Figure 1). The goal of the study NS-065 / NCNP-01-201 is to Dystrophin in skeletal muscle measured using a primary surrogate endpoint The aim was to identify a safe and effective dose based on de novo expression of the protein. Secondary surrogate endpoints included de novo dystrophin protein expression. Immunofluorescence (IF) staining and mass spectrometry detection of protein expression and de novo dystrophy The secondary end point of the phase 2 study included RT-PCR detection of fin mRNA levels. As an additional point, several functional endpoints were also included. The endpoints of NCNP-01-201 are:

[0113] <Primary endpoint> Low dose of NS-065 / NCNP-01 (Viltolarsen) injection (40mg The safety and tolerability of 100 mg / kg / week and high-dose (80 mg / kg / week) intravenous (IV) tolerance Western blot analysis of dystrophin induction in muscle after 20-24 weeks of treatment The antibody was measured by blotting and the results were compared with those of NS-065 / NCNP-01 (Viltolarse n) The effect of low and high IV doses of injection.

[0114] In Figure 1, the time series of "High dose: 80 mg / kg / week" shown in the lower half is The time series of the low dose, 40 mg / kg / week, was offset by the time series of the low dose, 40 mg / kg / week, This indicates that high-dose administration was initiated only after safety had been confirmed in a single dose.

[0115] Measurements: 1. By real-time polymerase chain reaction (RT-PCR) for mRNA analysis Induction of dystrophin mRNA in muscle measured (time frame 20-24 weeks of treatment) location) RT-PCR measures altered splicing of dystrophin RNA. In this study, mRNA was isolated from frozen muscle biopsies and reverse transcribed into cDNA. The splicin gene is designed adjacent to exon 53 on the dystrophin mRNA. RT-PCR bands corresponding to specific versions of the dystrophin mRNA were identified. Visualize and compare the amounts of different mRNA isoforms by gel electrophoresis. In case of successful binding to the RNA target, exon 53 is excluded from the resulting mRNA transcript. do. 2. Protein Analysis of Induced Dipeptides in Muscle as Measured by Western Blot Strophin protein. (Time frame: 20-24 weeks of treatment)

[0116] The primary biochemical outcome measures were dystrophy by immunoblot (Western blot). Dystrophin immunoblots were performed to measure drug-induced increases in dystrophin production. The proteins were analyzed by gel electrophoresis (SDS-PAGE), Electroblotting to cellulose and then detection of dystrophin protein. The nitrocellulose was incubated with antibodies to detect the nuclei, which were then separated by molecular weight. The immunoblot signal for dystrophin from the patient biopsy was then , compared with the signal of a dystrophin standard curve on the same gel (mixed DMD and normal controls). This provides a semi-quantitative assessment of dystrophin content in muscle.

[0117] US Phase 2 Study: Efficacy Outcome Measure 1 Outcome measure 1 (i.e., RT-PCR of de novo dystrophin mRNA levels) The results obtained for PCR detection are as follows:

[0118] [Table 7]

[0119] Outcome Measure 2 Outcome measure 2 (i.e., de novo expression in skeletal muscle using Western blot analysis) The results obtained for the in ovo dystrophin protein expression assay were A 19.0-fold increase from baseline (40 mg / kg / week for 24 weeks) and a 9.8-fold increase (80 mg / kg / week for 24 weeks). and a 27.2-fold increase (40 and 80 m) calculated as the average increase rate for each patient. The data obtained are summarized in Table 8 below and in Figure 2. .

[0120] [Table 8]

[0121] Dystrophin restoration by NS-065 / NCNP-01 (Viltolarsen) The dose (40 or 80 mg / kg / week for 24 weeks) was determined by Sarepta Therapeutics in 2016. Exon 51 skipping treatment available from Aetix dys51® (eteplirsen) (30 mg / kg / week, 48 and 1 This is at least 3-7 times the degree of recovery reported previously for Specifically, for comparison, Exondys51 (registered The corresponding Western blot data for Eteplirsen are shown below. 16 patients who received S-065 / NCNP-01 (Viltolarsen) for 24 weeks In three of the patients, dystrophin levels increased by more than 10% from baseline. Patients who received NS-065 / NCNP-01 (Viltolarsen) for 24 weeks Twelve of the 16 patients had a 3% or greater increase in dystrophin levels. Offman et al. reported "Becker dystrophy and abnormal dystrophin phenotypes" Among patients with Duchenne (<3% of normal levels of dystrophin), the clinical manifestations There was a clear correlation between the severity of the disease and the results of the dystrophin assessment." Eric P. Hoffman, et al., “Characterization of Dystrophin in Muscle-Biopsy Speci mens from Patients with Duchenne's of Becker's Muscular Dystrophy.” N. Engl. J. Med., 318: 1363-1368 (1988)).

[0122] eteplirsen Western blotting: 2.8-fold increase (from 0.16% 0.44% (30 mg / kg / week, 48 weeks)). In 12 patients, dystrophin levels were approximately equal to those of healthy subjects before treatment. 0.16% ± 0.12% (mean ± standard deviation) after 48 weeks of EXONDYS51 administration The results were 0.44% ± 0.43% (p < 0.05)" (https: / / www.fda.gov / downloads / loads / AdvisoryCommittees / CommitteesMeetingMaterials / PediatricAdvisoryCommittee / U (Available from CM557917.pdf) Western blotting: 3.1x (0.3% to 0.93%) % change) (30 mg / kg / week, 180 weeks). (From biopsy of extensor digitorum brevis (EDB) Western blots showed that dystrophin levels were on average about 0.3% of normal. The range was from undetectable to about 1% or more of normal. Western blot analysis, the most accurate quantitative method, revealed approximately 10% IgG from eteplirsen treatment. After 3.5 years, the mean dystrophin level was 0.93% ± 0.84% ​​of that in healthy subjects. (mean ± standard deviation) (https: / / www.fda.gov / downloads / advisorycommittees / committ eesmeetingmaterials / drugs / peripheralandcentralnervoussystemdrugsadvisorycommitte e / ucm497063.pdf. Eteplirsen Western blot: 0.93% at 180 weeks, range 0%-2 47% (30 mg / kg / week, 180 weeks) (Kenji Rowel Q Lim, et al., “E teplirsen in the treatment of Duchenne muscular dystrophy.” Drug Des. Devel. Th er., 11: 533-545 (2017) https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5338848 / ) . In addition, "in a 180-week study of eteplirsen, There was no difference in the dose-response of dystrophin production between weekly doses of 50 mg / kg and 50 mg / kg. This suggests that this approach may not be effective for other exons. "Kenji Rowel Q Lim, et al., "Eteplirsen in the treatment of Dutch enne muscular dystrophy.” Drug Des. Devel. Ther., 11: 533-545 (2017) https: / / w www.ncbi.nlm.nih.gov / pmc / articles / PMC5338848 / )

[0123] Morpholino oligonucleotides for DMD patients suitable for exon 53 skipping treatment Compared to the drug SRP-4053 (golodirsen; Sarepta) In comparison, the increase in dystrophin levels observed was similar in Viltolars. en showed better efficacy. Specifically, for comparison purposes, V SRP-α obtained after 48 weeks of iltolarsen treatment, but not after 24 weeks The corresponding Western blot data for 4053 (golodirsen) is shown below.

[0124] Golodirsen Western Blot: Change from 0.095% to 1.019% (30 mg / kg for 48 weeks). (The mean increase in dystrophin protein was In the tan blot assay (the biological primary endpoint of this study), 1.019% of normal compared with a mean baseline of 0.095% (p<0.001). This represents a 10.7-fold increase from baseline.” rrelations.sarepta.com / news-releases / news-release-details / sarepta-therapeutics-a (Available at: nnounces-positive-results-its-study.) Golodirsen Western Blot: Min 0.09%, Max 4.30% (30 2 out of 25 patients showed an increase in dystrophin levels of 3% or more. (22nd International Congress of the World Muscle Society (3-7 October 2017, St. Malo, France), http: / / www.google.co.jp / url?sa=t&source=web&cd=2&ved=2ahUKE wid7ZTQ9OrbAhUETLwKHX3xDk8QFjABegQIARAB&url=http%3A%2F%2Finvestorrelations.sarep ta.com%2Fstatic-files%2F64d8d897-2e4a-4119-80b4-115cbae17993&usg=AOvVaw1Amh1Mq1V auvLkPvYWfVdR).

[0125] US Phase 2 Study: Safety NS-065 / NCNP-01 (Viltolarsen) was administered at high and low doses (4 Safety data for 16 DMD patients receiving 0 mg / kg / week and 80 mg / kg / week There were no major concerns about the conduct of the study, the quality of the data, or the safety of participants. No participants discontinued treatment at 36 weeks. There were no adverse events leading to discontinuation or drug-related adverse events. All adverse events were It was mild or moderate. EXAMPLES

[0126] US / Canadian Phase 2 Study The Phase II dose-finding study "NS-065 / NCNP-01-201" was completed in January 2016. The trial was initiated in February under the name IND127474. The CDC uses the ls.gov identification number "NCT02740972" in the Safety and Dose Finding St. udy of NS-065 / NCNP-01 in Boys With Duchenne Muscular Dystrophy (DMD) The study protocol is available at https: / / www.clinicaltrials.gov / . Available at trials.gov / ct2 / show / study / NCT02740972 The present study is directed to the exon 53 skipping In patients with Duchenne muscular dystrophy (DMD) who are suitable for treatment with i.v. High-dose (80 mg / kg) and low-dose (40 mg / kg) NS delivered as an i.v. The main purpose of this study was to evaluate the safety of -065 / NCNP-01 (Viltolarsen). Further objectives included tolerability, muscle function and strength, pharmacokinetics, and pharmacodynamics. and was included. More specifically, this is a Phase II, multicenter, two-period, randomized, placebo-controlled, dose-finding study. NS-065 / NCNP-01 (Viltola) was administered to outpatients aged 4 to 10 years with MD. rsen) was administered intravenously once a week for 24 weeks. Two dose-level cohorts were included. Period 1 The study was conducted in a double-blind fashion. Patients were randomized to receive either NS-065 / NCNP-01 (Vilt olarsen) or placebo was administered intravenously once weekly for the first 4 weeks of participation (period 1). NS-065 / NCNP-01 (Viltolarsen) for 5 to 24 weeks ( 20 weeks of active treatment - Period 2) were administered intravenously. Analysis of these safety data was completed prior to enrollment of patients in the 80 mg / kg dose cohort. Patients who completed the 24-week study were eligible to enter an open-label extension study.

[0127] Clinical efficacy was assessed at regularly scheduled study visits. All patients were She underwent a biceps muscle biopsy at line and a second muscle biopsy at 24 weeks.

[0128] Safety was monitored by adverse events (AEs), blood and urine laboratory tests, electrocardiograms (ECGs), vital signs, and The study was evaluated by collecting baseline physical examinations and blood tests. To evaluate the pharmacokinetics of 65 / NCNP-01 (Viltolarsen), Serial blood samples were collected at study visits.

[0129] <Type of clinical trial: Intervention (clinical trial)> Actual registration: 16 participants. Allocation: Randomized. · Intervention model: Parallel allocation. Masking: Quadruple (participant, caregiver, investigator, outcome assessor) valuer). · Primary purpose: treatment. Actual exam start date: December 2016. · Initial completion date: March 2018.

[0130] Patient population and interventions Experiment: NS-065 / NCNP-01 (Viltolarsen) 40mg / kg DMD with a gene deletion amenable to treatment with exon 53 skipping was identified Six patients received NS-065 / NCNP-01 (Viltolarsen) 40 mg / The patient was administered intravenously at a dose of 1 kg once weekly for 24 weeks. Experimental: NS-065 / NCNP-01 (Viltolarsen) 80mg / kg DMD with a gene deletion amenable to treatment with exon 53 skipping was identified Five patients received NS-065 / NCNP-01 (Viltolarsen) 80 mg / The patient was administered intravenously at a dose of 1 kg once weekly 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 randomly assigned to receive Placebo was administered intravenously once a week for 2 weeks, followed by 20 weeks of open-label treatment. The installation was carried out.

[0131] <Selection criteria> -Males aged 4 years or older at the time of consent and less than 10 years old at the time of the first intravenous infusion. -Dystrophin mRNA reading frame by exon 53 skipping therapy have a DMD mutation(s) in the dystrophin gene that is suitable for restoration of It has been confirmed that Able to walk independently without assistance The motor function assessor measures the time to stand (TSTAND), 10 meter run / walk time (T Patients who were judged to be able to perform evaluation of TRW and 4-step stair climbing time (TTCLIMB) - At least 3 months of stable administration of glucocorticoids

[0132] <Exclusion criteria> -Acute illness within 4 weeks prior to the first dose of the study drug. Evidence of symptomatic cardiomyopathy (without excluding asymptomatic cardiac abnormalities at the time of investigation) ·Severe allergy or hypersensitivity to medications -Severe behavioral or cognitive problems that, in the opinion of the investigator, would prevent participation in the trial - Any changes that, in the opinion of the investigator, may affect safety, treatment and follow-up There are previous or ongoing issues that make it unlikely that the study will be completed successfully or that could compromise the evaluation of the study results. Ongoing illness, medical history, physical findings, or abnormal laboratory test results -Currently taking or taking other investigational drugs within 3 months prior to starting the investigational drug Prior to the first anticipated dose of NS-065 / NCNP-01 (Viltolarsen) Patients who had undergone surgery within the past 3 months or were scheduled to undergo surgery during the study period Patients who have previously participated in this study or NS-065 / NCNP-01 (Viltolars Patients who were participating in other studies in which

[0133] To summarize again, patients who are suitable for exon 53 skipping therapy and have been taking glucocorticoids for more than 3 months should Sixteen male patients stable on Ticoid were given 40 mg / kg / week and 80 mg / kg. This was a 24-week, two-cohort study testing 100 mg / week doses of 100 mg / day of placebo in each cohort. Participants underwent the first 4-week randomization period as a control for adverse events (safety outcomes). Both placebo-treated and active-treated patients were then randomized to receive the 20-week treatment period. In the NS-065 / NCNP-01-201 study, NCNP-01 (Viltolarsen) injection 250 mg at a low dose of 40 mg / kg / week The study was administered in two dose cohorts, one at a dose of 100 mg / kg / week and one at a higher dose of 80 mg / kg / week for 20 to 24 weeks. The effect on de novo expression of the dystrophin protein was evaluated (see Figure 3). The goal of study NS-065 / NCNP-01-201 is to Dystrophy in skeletal muscle measured using a surrogate endpoint The aim of this study is to identify safe and effective doses based on de novo expression of the kinase protein. Secondary surrogate endpoints included de novo dystrophin transcription factor (DTT) and cytotoxic T-cell activation. Immunofluorescence (IF) staining and mass spectrometry detection of protein expression and de novo dysplasia The secondary end of the phase 2 trial included RT-PCR detection of lophin mRNA levels. Several functional endpoints were also included as end points. The endpoints of 65 / NCNP-01-201 are as follows:

[0134] <Primary endpoint> NS-065 / NCNP-01 (Viltolarsen) injection 250mg low dose Safety of intravenous (IV) administration of 40 mg / kg / week and high doses (80 mg / kg / week) Sex and Tolerability Western blot analysis of dystrophin induction in muscle after 20-24 weeks of treatment The antibody was measured by blotting and the results were compared with those of NS-065 / NCNP-01 (Viltolarse n) The effect of low and high IV doses of 250 mg injection. Blood drug concentration of NS-065 / NCNP-01 (Viltolarsen)

[0135] <Secondary endpoint> ·NS-065 / NCNP-01 (Viltolarsen) injection 250mg low dose and high-dose intravenous injections increased dystrophin mRNA and The effect of the compound on the induction of protein was examined by RT-PCR mRNA analysis and immunofluorescence. The protein is assessed by staining and mass spectrometry protein analysis. NS-065 / NCNP-01 (Viltolarsen) injection 250mg low dose and high-dose intravenous administration improved muscle strength, mobility, and functional movement after 20 to 24 weeks of treatment. To examine the effects on physical performance, stand-up time (TSTAND), 10-meter sprint Walking / walking time (TTRW), 4-step stair climbing time (TTCLIMB), North Star walking Capacity Assessment (NSAA), 6-minute walk test (6MWT), and quantitative muscle testing (QMT) was measured in comparison with a matched natural history control group.

[0136] In Figure 3, the time series of "High dose: 80 mg / kg / week" shown in the lower half is The time series of the low dose, 40 mg / kg / week, was offset by the time series of the low dose, 40 mg / kg / week, This indicates that high-dose administration was initiated only after safety had been confirmed in a single dose.

[0137] Secondary endpoint of US / Canada Phase 2 study: External no-treatment natural history group (International Neurological Association Comparison with the Comprehensive Intramuscular Research Group (CINRG) Duchenne Natural History Study (DNHS) Time function test in Patients enrolled in the US / Canadian Phase 2 study NS-065 / NCNP-01-201 were Muscle function was clinically assessed at baseline, 13 weeks, and 25 weeks. No adjustment was made for the 4-week placebo period for these purposes. Several types of time-function tests were included: time to rise from supine position (TTSTAN) D); 10 meter run / walk time (TTRW); 4 step climb time (TTCLIMB) ; the 6-minute walk test (6MWT); and the North Star Ambulatory Assessment (NSAA). Changes were compared with disease trajectories in corresponding patients studied in the CINRG DNHS.

[0138] The CINRG DNHS followed each of the 440 DMD patients over several years. The total trial period was about 10 years. CINRG is the International Neuromuscular Research Group. (Cooperative International Neuromuscular Research Group) Academic and research centers that share a common goal: to positively impact the lives of students and their families. A consortium of medical and scientific researchers from rch.org / . DNHS is the Duchenne Natural History Study. e Natural History Study), and CINRG (http: / / / www.cinrgresearch.org / Duchenne-Natural- "Duchenne muscular The largest prospective multicenter natural history study to date of chronic dystrophy (DMD) (McDonald CM, Henricson EK, Abresch RT, Han JJ, Escolar DM, Florenc e JM, Duong T, Arrieta A, Clemens PR, Hoffman EP, and Cnaan A, “CINRG Investiga tors. The cooperative international neuromuscular research group Duchenne natura l history study - a longitudinal investigation in the era of glucocorticoid ther apy: design of protocol and the methods used,” Muscle Nerve., 48(1), 32-54 (201 3).、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 cooperative international neuromuscular research group Duchenne natural hi story study: glucocorticoid treatment preserves clinically meaningful functional milestones and reduces 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).およびMcDonald CM, Henricson EK, Abresch RT, Duong T, Joy ce NC, Hu F, Clemens PR, Hoffman EP, Cnaan A, and Gordish-Dressman H, “CINRG In vestigators. 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)).

[0139] The NS-065 / NCNP-01-201 study is conducted by Standard operating procedures (clinical manuals) and clinical assessor training programs were used. The protocol is based on the NS-065 / NCNP-01-201 clinical trial and the CINRG DNH NS-065 / NCNP-01-201 vs. CINRG DNH Matching of patients enrolled in S was performed using the following criteria: Boys aged 4 to <10.5 years at baseline. Patients with ≥12 months of time function test data. Region: North America (USA, Canada). Steroids have been administered for at least 3 months and are administered throughout the 12 / 24 month follow-up period. Steroids are administered continuously - Not participating in another clinical trial

[0140] Patients matched to those enrolled in the NS-065 / NCNP-01-201 clinical trial For more information about exon 53 skipping, please contact the CINRG DNHS database. Although we did not identify any dystrophin mutations amenable to treatment with , we did identify exon 3-7 deletions. Patients with and without dystrophin deletions amenable to treatment with exon 44 skipping Patients with genomic abnormalities were excluded, resulting in the 69 subjects shown in Table 9 below. Patients with 3-7 deletions and dystrophic syndromes amenable to treatment with exon 44 skipping The reason for excluding patients with α-deletion was that these patients presented with relatively mild symptoms. Because it had been reported.

[0141] [Table 9]

[0142] In the top row of Table 9, "Exon 53 skip" means exon 53 skipping. "non-exon 53 skipping" refers to DMD patients suitable for treatment with In the bottom row of Table 9, "large deletion / "No overlap" means that there are other mutations, such as point mutations (not amenable to treatment with exon skipping) Encompasses categories.

[0143] The number of CINRG DNHS patients with 6MWT data was compared with the number of patients with other endpoints. The reason for this is that 6MWT was followed up later in the CINRG DNHS protocol. This is because the addition of a CT scan limited the amount of data available compared to other time-function tests.

[0144] Sixteen patients enrolled in NS-065 / NCNP-01-201 and CINRG DN A comparison of the disease trajectories over 24 weeks for 69 patients enrolled in HS showed that During the time frame between these two groups, patients in the natural history comparison group showed a decline in function on time function tests. In contrast, 24 patients enrolled in NS-065 / NCNP-01-201 Over the same period of weeks, the subjects showed average improvements in time function tests. Three of them reached statistical significance: TTRW (weeks 13 and 25); TST AND (week 25); and 6MWT (week 25). 40 mg / kg / week and 80 mg The mean dose of NS-065 / NCNP-01 (Viltolarsen) was 1.2 mg / kg / week. No statistically significant differences were observed.

[0145] Figure 4 (a total of five graphs and one table) is based on the data in NS-065 / NCNP-01-201. Patients who were registered (indicated as "Viltolarsen" in the solid blue line) and CINRG 2 in DNHS-matched patients (indicated as “DNHS” with a dashed red line) A comparison of changes from baseline in chronofunction tests over a 4-week period is shown.

[0146] In the five graphs in Figure 4, "Viltolarsen" is NS-065 / NCN This is the international nonproprietary name (INN) of P-01. In addition, in all graphs, These changes were analyzed using a restricted maximum likelihood (REML)-based mixed model for repeated measures (MMRM) analysis. The results were compared between the Viltolarsen group and the CINRG DNHS group using the did.

[0147] (1) Timed Function Test The time function tests were TTSTAND, TTRW, TTCLIMB, and 6MWT. TS TAND and TTRW were prespecified as separate endpoints, measuring time and a 6-point scale. The evaluation was based on a rating scale. TSTAND and TTRW are components of the NSAA. Therefore, they are measured once and the data are used as independent endpoints. These were used together and in combination as part of the NSAA study. The TRW is further described below in relation to the combined NSAA scale. The TTCLIMB was used to assess the time (seconds) required for patients to climb the four steps. According to the clinical protocol described in the "US / Canada Phase 2 Study," the TTCLIMB study is It should be noted that this was erroneously stated as being carried out as part of the SAA.

[0148] The 6MWT is a widely used and accepted test for many diseases. The version used in this study was adapted for DMD. Clinically assess functional motor status, and 2) the integrated overall response to exercise. ,It is considered to be a simple, standardized, low-tech, and cost-effective assessment tool. In conducting this test, two points (cones) were placed 25 meters apart and the patient was asked to Patients were asked to walk quickly and safely between the stations for 6 minutes. The total distance traveled (in metres) was recorded. The clinical assessor then assessed the number of steps the patient took to complete the first 50 metres and The total distance walked (in meters) in 6 minutes was measured (Craig M. McDonald, MD, Erik K. Hen ricson, MPH, Jay J. Han, MD, R. Ted Abresch MS, Alina Nicorici, BS, Gary L. Elfr ing, MS, Leone Atkinson MD, PhD, Allen Reha BS, Samit Hirawat MD, and Langdon L. Miller MD, “The 6-minute Walk Test as a New Outcome Measure in Duchenne Muscul ar Dystrophy,” Muscle & Nerve, Vol. 41, pp. 500-510, April 2010, Wiley Periodic als, Inc. and Craig M. McDonald, MD, Erik K. Henricson, MPH, R. Ted Abresch, MS , Julaine Florence, PhD, Michelle Eagle, PhD, Eduard Gappmaier, PhD, Allan M. Gl anzman, DPT, Robert Spiegel, MD, Jay Barth, MD, Gary Elfring, MS, Allen Reha, MS , and Stuart W. Peltz, PhD, “The 6-minute Walk Test and Other Clinical Endpoint s in Duchenne Muscular Dystrophy: Reliability, Concurrent Validity, and Minimal Clinically Important Differences from a Multicenter Study,” Muscle & Nerve, Vol. 48, pp. 357-368, September 2013, Wiley Periodicals, Inc.).

[0149] The Quantitative Muscle Testing (QMT) assessment is designed to measure the force produced during a static contraction. It is a well-established method for measuring muscle weakness in neuromuscular diseases. The method used was the CINRG Quantitative Muscle System (CQMS). It includes an audiovisual feedback process that increases the compliance of the affected child. The patient is supported on the back. The patient is placed on an examination table equipped with a mechanism to stabilize the patient's back, eliminating the need for manual back stabilization. After a single practice administration, each patient was scored The QMT evaluation was completed by the patient (two tests were performed and the higher of the two values ​​was used for data analysis). The QMT uses a direct computer interface with strain gauges. The test positions and test sequence were standardized. Bilateral testing was performed on the following muscle groups: (Mayhew JE, Florence JM, Mayhew TP, Hen ricson EK, Leshner RT, Mccarter RJ, et al., “Reliable surrogate outcome measure s in multicenter clinical trials of Duchenne muscular dystrophy,” Muscle & Nerv e, 2007; 35(1):36-42. Epub 2006 / 09 / 14.): Hand grips. Elbow flexors (biceps). ·Elbow extensor (triceps). · Knee flexors (hamstrings). ·Knee extensors (quadriceps).

[0150] For QMT, all parameters were significantly improved over the 24-week treatment period, except for the elbow extensor muscle. A slight decrease in the mean strength of the elbow extensors was observed. There was a significant increase (improvement) in all items. There was no statistically significant difference in the

[0151] Strength and function tests include TTSTAND, TTRW, TTCLIMB, NSAA, and 6MWT , followed by QMT.

[0152] (2) North Star Ambulatory Assessment (NSAA) NSAA is for boys with DMD who should be able to walk at least 10 meters. It is a 17-item functional assessment system designed and scored by clinicians (E.S. Mazzone, S.M. essina, G. Vasco, M. Main, M. Eagle, A. D'Amico, L. Doglio, L. Politano, F. Cav. allaro, S. Frosini, L. Bello, F. Magri, A. Corlatti, E. Zucchini, B. Brancalion, F. Rossi, M. Ferretti, MG Motta, MR Cecio, A. Berardinelli, P. Alfieri, T. Mongini, A. Pini, G. Astrea, R. Battini, G. Comi, E. Pegoraro, L. Morandi, M. Pa ne, C. Angelini, C. Bruno, M. Villanova, G. Vita, MA Donati, E. Bertini, and E. Mercuri, “Reliability of the North Star Ambulatory Assessment in a multicentr ic setting,” Neuromuscular Disorders, Vol. 19, Issue 7, pp. 458-461, Jul 2009, Elsevier BV). This assessment tool evaluates the ability to stand up, get up from the floor, and Functional activities including stepping, hopping, and running are assessed. Ability to successfully perform test content; 1 = Ability to perform test content with modified methods or assistance and 0 = unable to perform the test. Thus, total scores range from 0 (completely unable to walk) to 34 ( Individual test item scores and total scores were recorded. STAND and TTRW were implemented as part of the NSAA. The TTSTAND is a routine test performed as a standard clinical examination for patients with DMD. The test uses the time it takes for a patient to go from lying flat on the floor to standing up. The number of seconds required to perform the test and how the patient reached the upright position are recorded. The scores, rated out of a maximum of one point, were recorded. Using TTRW, assess the time (in seconds) it took the patient to walk / run 10 meters. The subjects were assessed for their walking ability (including a 6-point rating scale for quality of running / walking).

[0153] US / Canadian Phase 2 Study: Safety High and low doses of NS-065 / NCNP-01 (Viltolarsen) (4 Safety data for 16 DMD patients receiving 0 mg / kg / week or 80 mg / kg / week There were no major concerns about the conduct of the study, the quality of the data, or the safety of participants. No participants discontinued treatment at the 48-week mark. TEAEs (treatment-related AEs) that required discontinuation or dose reduction of CNP-01 (Viltolarsen) No adverse events (AEs) were reported. All adverse events (AEs) were mild or It was moderate. EXAMPLES

[0154] Effect of pH on the stability of viltolarsen. (a) pH stability evaluation (1) To investigate the relationship between the liquidity of the solution and the stability of Viltolarsen, a Britton-Roche Britton-Robinson buffer (pH 3, 4, 5, 6, 7, 8, 9, 10 or 11) The stability of viltolarsen in solutions of various pH levels was evaluated.

[0155] [Test conditions] Drug concentration: Viltolarsen 2 mg / mL Lysis solution: Britton-Robinson buffer (pH 3, 4, 5, 6, 7, 8, 9, 10 or 11) Storage conditions: (1) 121°C (autoclaved) for 10, 30, or 60 minutes; or (2) 4 or 7 days in an incubator at 80°C Endpoints: Appearance (visual inspection), pH (pH meter), purity test (HPLC method), recovery rate (residual survival rate) (HPLC method) HPLC conditions: Flow rate: 1.0mL per minute Detector: International spectrophotometer (measurement wavelength: 264 nm) Column: 4.6 mm inner diameter, 15 cm long stainless steel tube with 3.5 μm liquid chromatography The column was packed with octadecylsilylated silica gel for column chromatography (Waters, X-bridge C18). Column temperature: 60℃ Mobile phase: Dissolve 1.42 g of disodium hydrogen phosphate in approximately 750 mL of water and add an aqueous solution of sodium hydroxide. After adjusting the pH to 12.0 by adding water, the total volume was 1000 mL (Solution P). Add 300 mL of acetonitrile to 700 mL of P solution and add 16.12 mL of tetrabutylammonium bromide. g was dissolved in mobile phase A. Add 300 mL of acetonitrile to 300 mL of P solution and add 9.67 g of tetrabutylammonium bromide. was dissolved in mobile phase B. Mobile phase delivery: Mobile phase A / B (100 vol% / 0 vol%) to (0 vol% / 100 vol%) 3 It took 0 minutes to change it. In this and the following examples, the purity test values ​​are the detected amounts of impurities. The area of ​​the main peak of Viltolarsen when the total area of ​​all peaks is 100 The percentage (main peak surface percentage (%)) is shown.

[0156] [Test Results] The test results are shown in Table 10 and Figure 5. "ST" means the same concentration of the drug diluted in purified water. The solution was prepared each time a test was performed (it was not stored). As a result, viltolarsen was unstable in acidic solutions, but was stable in neutral solutions. -Relatively stable in weakly alkaline solutions.

[0157] [Table 10]

[0158] (a) pH-stability evaluation (2) To investigate the stable pH range of viltolarsen in more detail, potassium phosphate-phosphate Using sand buffer (pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 or 9.2), The stability of viltolarsen in solutions at various pH levels was evaluated.

[0159] [Test conditions] Drug concentration: Viltolarsen 2 mg / mL Dissolution medium: 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) 121°C (autoclaved) for 10, 30, or 60 minutes; or (2) 1, 4, 7 or 14 days in an incubator at 80°C Endpoints: Appearance (visual inspection), pH (pH meter), purity test (HPLC method), recovery rate (residual survival rate) (HPLC method) HPLC conditions: Same as (a) except that the column temperature was 50°C.

[0160] [Test Results] The test results are shown in Table 11 and Figure 6. As a result, Viltolarsen was found to be effective at pH 7 to 7.5. It was most stable in solution.

[0161] [Table 11] EXAMPLES

[0162] Effect of pH modifiers on formulation stability In this example, the pH of the viltolarsen injection solution was adjusted to a stable pH range (pH 7 to 7.5). The purpose of this study is to select a pH adjuster for the purpose of the following: , 10 mM KH2PO4-Na2HPO4 or 100 mM KH2PO4-Na2HPO4) was added to the solution to adjust the pH to a stable level. The stability of 100 / mL viltolarsen solution was evaluated.

[0163] [Test conditions] Drug concentration: Viltolarsen 10 mg / mL Drug formulation: See Table 12 below Storage conditions: 121℃ (autoclaved) for 30-60 minutes Endpoints: Appearance (visual inspection), pH (pH meter), purity test (HPLC method), recovery rate (residual survival rate) (HPLC method)

[0164] [Table 12]

[0165] [Test Results] The test results are shown in Table 13 and Figure 7. As a result, Viltolarsen acts as a buffer. Even without phosphate buffer, the stability was equal to or greater than that with phosphate buffer. It was more stable than in phosphate buffer (100 mM KH2PO4-Na2HPO4 in this example).

[0166] [Table 13] EXAMPLES

[0167] Relationship between buffer and multimer formation Viltolarsen may form polymers under certain storage conditions, resulting in a decrease in monomer purity. In this example, 50 mL of 0.9% sodium chloride was added as an isotonicity agent. The formation of polymers when various buffers were added to a 50 mg / mL viltolarsen solution The composition was measured.

[0168] [Test conditions] Drug concentration: Viltolarsen 50 mg / mL Storage conditions: Up to 5 days in a 60℃ incubator Endpoint: Multimer (HPLC method, SEC column) Analysis conditions: Detector: UV spectrophotometer (measurement wavelength: 260 nm) Column: 7.8 mm inner diameter, 30 cm long stainless steel tube with 7 μm liquid chromatography The columns were packed with styrene vinyl polymer gel for use in HPLC, and two of the columns were connected in series (TSK gel G 3000 PWXL, 7 μm, 7.8 mm × 30 cm, Tosoh). Column temperature: 25°C Mobile phase: Dissolve 15.6 g of sodium dihydrogen phosphate dihydrate in 750 mL of water and add sodium hydroxide. After adding sodium TS to adjust the pH to 7.3, water was added to make up 1000 mL. 200 mL of acetonitrile was added. Flow rate: Adjusted so that the retention time of the Viltolarsen monomer was about 21 minutes. . Area measurement range: Range up to the Viltolarsen monomer peak

[0169] [Test Results] The test results are shown in Table 14. As a result, 50 mM phosphate buffer (sodium dihydrogen phosphate, Dihydrate-disodium hydrogen phosphate:NaH2PO4·2H2O-Na2HPO4) and citrate buffer solution (Que The addition of sodium phosphate dihydrate increased the formation of polymers.

[0170] [Table 14] EXAMPLES

[0171] Solubility study (100mg / mL) We investigated increasing the concentration of the injection solution.

[0172] [Test conditions] Chemical solution: The following two types

[0173] [Table 15] Filtration filter: PVDF (Millex GV, 0.22 μm, 33 mm, Millipore) Endpoints: Appearance (visual confirmation), pH (pH meter), purity test (HPLC method), Recovery rate after filtration (HPLC method)

[0174] [Test Results] The test results are shown in Table 16. A 100 mg / mL viltolarsen injection was prepared. There were no problems with solubility or filtration through a sterile filter, and the solution was colorless and clear. After the injection, the solution became viscous, but there was no change in appearance. It was found that it was possible to prepare

[0175] [Table 16] EXAMPLES

[0176] Solubility study (50mg / mL) The solubility of a 50 mg / mL drug solution was evaluated. The drug solution was prepared and the solubility and filtration by a sterilizing filter were evaluated. I passed.

[0177] [Test conditions] Chemical solution:

[0178] [Table 17] Filtration filter: PVDF (Millidisc cartridge filter, 0.22 μm, MCGL40 S03 / 1pc + MCGL40S03 / 2pc, Merck) Endpoints: Appearance (visual confirmation), pH (pH meter), purity test (HPLC method), quantification (HPLC method)

[0179] [Test Results] The test results are shown in Table 18. As a result, the 50 mg / mL solution was a colorless, clear solution, and the There was no change in the content. From the above, it was confirmed that a 50 mg / mL injection solution can be prepared and that a scale There was no problem uploading

[0180] [Table 18]

[0181] In the following Examples 8 to 10, samples obtained from patients who participated in the clinical trial of Example 2 The study was conducted on patients who participated in the clinical trial of Example 2. EXAMPLES

[0182] Mass spectrometric quantification of dystrophin protein (Experimental Method) Overview: Biological analysis methods The samples were subjected to SDS-PAGE electrophoresis to separate proteins, and trypsin was added to the gel. The peptide fragments were extracted from the gel pieces and dried. The peptides were redissolved and analyzed for dystrophin protein by HPLC-MS / MS using a reversed-phase column. The standard curve range was determined based on the normal control dystrophin protein. The concentration ranges from 1% to 25% when the mass is taken as 100%, and filamin C is used as the standard protein. The regression equation was calculated from the peak area ratio using the least squares method, y = mx + b (y: peak The formula for the area ratio of the dystrophin-positive cells (x:% dystrophin) was obtained.

[0183] In-gel digestion The samples to be analyzed by LC-MS / MS were analyzed by SDS-PAGE (SDS: sodium dodecyl sulfate, PAGE: Polyacrylamide gel electrophoresis (PAGE) was used to separate proteins according to molecular weight, and the proteins were then subjected to in-gel digestion. Each gel was prepared by adding calibrators (0.0%, 1.0%, 3.0%, 10.0%, 25.0% dilution) that constitute the standard curve. Strophin, and the TA extracted from human myotube cells cultured with stable isotope-labeled amino acids. The study consisted of a total of 11 samples: 12.5 μg of protein (SILAC), a blank, and 4 clinical trial samples. The gel had 12 lanes, with one lane used for molecular weight markers. The calibrators were five non-DMD The muscle biopsy was prepared by mixing protein extracts from two types of DMD muscle biopsies. The materials were obtained from Ghamton University and had passed ethical review. The amount of dystrophin in each muscle biopsy was measured in advance. Seventy serial sections with a thickness of 10 μm were obtained from the muscle. The muscle sections were stored in a microcentrifuge that had been cooled on dry ice. The mixture was transferred to a chlorotube. Protease and phosphatase inhibitors from Thermo Scientific were added. Proteins were extracted from muscle slices using RIPA buffer containing 100% glycerol. The concentration was quantified using a BCA protein assay kit (Pierce). Each sample contained 50 μg of protein and was adjusted by adding 12.5 μg of SILAC. Calibrators and clinical samples were used as internal standards for determining % dystrophin using SILAC extraction. NuPAGE 3-8% Tris-Acetate gel was electrophoresed at 150V for 75 minutes. Gel electrophoresis was performed using two gels (gel A and gel B) for the same sample in duplicate. After electrophoresis, the gel was washed with methanol:water:acetic acid (50:45:5) for 30 minutes. The gel was fixed and rehydrated twice. Coomassie blue staining was performed for 1 hour. The staining was performed overnight at 4°C. The molecular weight markers ranged from 460 kDa to 268 kDa. The gel containing the protein was excised and washed twice with water:acetonitrile (50:50). In-gel digestion was performed using pSin (Gold mass spectrometry grade, Promega Corporation). The peptide fragments were obtained and dried by centrifugation under reduced pressure. The mixture was stored at ℃ and then analyzed by Q Exactive Nano-LC-MS / MS.

[0184] Test sample Sixteen patients participated in the clinical trial, and muscle biopsies were obtained from each patient before and after treatment. In order to replicate, 64 samples were analyzed. In addition, 8 samples that were duplicates of 4 specimens were analyzed again. The analysis was performed and a total of 72 samples were analyzed.

[0185] Analysis by LC-MS / MS High-performance liquid chromatography system Dionex Ultimate 3000 RSLCnano (ThermoFisher Scientific) and mass spectrometer Q Exactive Plus (HRMS: High Resolution Mass Spectromet) Chromatography-Mass Spectrometry (LC-MS) coupled with a 30-μL ELISA kit (ThermoFisher Scientific) was used. The resulting peptide fragments were analyzed by NMR (NMR spectroscopy / MS). Dry peptide fragments were purified using 2% acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA). The sample was introduced into the LC-MS / MS using a 5 μL injection loop and a Dinoex nanoVi A per sample loop (ThermoFisher Scientific) was used.

[0186] Liquid chromatography was performed under the following conditions. Analytical column: Reversed phase column Acclaim Pepmap RSLC C18 15cm x 75 μm, particle size 3 μm, EASY SPRAY (Thermo Fisher) Analytical column temperature: 50℃ Mobile phase A: 1% formic acid (HCOOH), Mobile phase B: 0.1% formic acid ACN Flow rate: 0.500 μL / min (NC) Sample injection mode: Partial loop (loop size 5 μL) Sample injection volume: 1.00 - 4.00 μL Autosampler temperature: 10℃ Run Time: 40.00 minutes

[0187] [Table 19]

[0188] The mass spectrometer was used under the following conditions. Ion source: Thermo Fisher EASY-Spray Ion mode: Positive ion Scanning: Parallel reaction monitoring Chromium 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 ms Quadrupole mass detector separation width: 1.0 m / z units Spectrum Data: Profile Mass tolerance: 10 ppm Typical Tunable Parameters (Dystrophin Tune File) Spray voltage: 1.8 kV Capillary Temperature: 275℃ S-Lens RF-Level:70

[0189] [Table 20]

[0190] [Table 21]

[0191] [Table 22]

[0192] Data collection from the chromatograph was performed using Thermo Scientific's LC Quan version 3.0. The mass tolerance was 20 ppm, and the integration algorithm was ICIS. If the peak area of ​​dystrophin for other calibration samples is around 10,000 or less, please pay attention to the following: Compared with 0% calibrator, the peptides obtained for both dystrophin and filamin C were The peak area ratio of the labeled peptides was a reliable value and did not affect the % dystrophin. I confirmed that there was no. The peak areas of dystrophin and filamin C correspond to the product ions. The peak area of ​​dystrophin was calculated by summing the areas of the dystrophin peaks. The peptide fragment (DYST2 amino acid sequence: IFLTEQPLEGLEK (SEQ ID NO: 4)) was obtained from Filami. The peak area of ​​filamin C was determined by the analysis of two peptide fragments of filamin C (FILC1 amino acid sequence: VAVGQEQA FSVNTR (SEQ ID NO: 6), FILC2 amino acid sequence: SPFVVNVAPPLDLSK (SEQ ID NO: 8) It was decided.

[0193] [Table 23]

[0194] Dystrophin protein levels in the calibrators and each clinical sample (dystrophin and The peak area ratio of filamin C was calculated from the following formula. Therefore, the dystrophin protein level values ​​for each standard were calculated as follows: The dystrophin protein levels were corrected by subtracting the values ​​from the corresponding values. The percent dystrophin and dystrophin protein levels were calculated from the calibrators for each gel. A regression line was obtained to estimate the dystrophin protein levels of the clinical samples. % of dystrophin was obtained.

number

[0195] [Table 24]

[0196] (result) The % dystrophin of each analyzed sample was determined based on predefined pass / fail criteria. Of the 72 samples measured, 60 met the criteria. The results are shown in Table 25 and The results are shown in Fig. 26. Duplicate samples were used for each specimen, but the 40 mg / kg dose Four pre- and post-treatment specimens were obtained from two patients in the same group (patients E and F in Table 25). The measurements obtained were from one sample (Gel A) because the other sample (Gel B) did not meet the criteria. In the 40 mg / kg group, 14 out of 16 samples (8 samples) were One sample showed 1% and 11 samples were below the limit of quantification. At 25 weeks, 14 out of 16 samples from 8 specimens were measured. One specimen was quantitative in both measurements. In the remaining cases, dystrophin was detected at 1% or more. In the 80 mg / kg group, all 16 samples from 8 specimens before administration were below the quantification limit. In 16 samples from 8 specimens taken at weekly intervals, dystrophin levels were above the limit of quantification in all samples. was detected, with an average of 4.2%. From the above, administration of 40 mg / kg and 80 mg / kg of viltolarsen was effective in preventing dystrophy. It was confirmed that fin protein expression was restored.

[0197] [Table 25]

[0198] [Table 26] EXAMPLES

[0199] Motor function test (at 13 and 25 weeks (12 and 24 weeks after the first administration)) (Experimental Method) The motor function evaluation of the drug group was compared with that of the natural history group. The Cooperative International Neuromuscular Research Network, a clinical trial network of The Duchenne natural history study (CINRG DNHS) was conducted by the Comprehensive Institute of Neurology and Neurology (CINRG) arch group. Studies were selected based on baseline data from available studies. The CINRG DNHS is a longitudinal natural history study of 440 men with DMD. Data were collected for 16 years at baseline, four times in the first year, two times in the second year, and once a year thereafter for a maximum of 1 year. The subjects were expected to visit the hospital for 10 years. At each visit, a time function test, muscle strength test, and questionnaire were performed. The 201 study was conducted at a CINRG affiliated institution. The standard operating procedures (SOPs) and clinical assessor training procedures were consistent between the two studies. He made him do so.

[0200] As controls for this study, we used data from the CINRG DNHS study 201, including age, steroid use, and region. Patients were selected who met the following criteria, including the main inclusion criteria: - At least 12 months of functional test data (baseline rise time (TTSTAN D), 4-step stair climbing time (TTCLIMB) and 10 m walking / running time (TTRW) are required. Age 4 to <10 years at baseline · North America (USA or Canada) Corticosteroids were administered for at least 3 months and continued throughout the 12-24 month observation period. It is being used. Not simultaneously enrolled in clinical trials of other exon skipping drugs Meet the genetic eligibility criteria listed below. (Selection criteria): Patients with genetic test results Patients with duplication mutations Patients with nonsense or frameshift mutations (Exclusion criteria): Patients with mutations between the promoter and exon 8 (reported to have slow disease progression (1) Hu m Mutat. 2018; 39: 1193-1202 and (2) Hum Mutat. 2008; 29(5): 728-37. excluded) Patients suitable for exon 44 skipping treatment (reports of slow disease progression (1) (Excluded for some reason) Patients with in-frame mutations

[0201] As a result, 65 male DMD patients met the above criteria, of which 9 had exon 53 skipping. DMD patients suitable for treatment with exon 53 skipping (exon 53 skipping group), 56 patients The patients were DMD patients (non-exon 53 skipping group) who were not suitable for treatment with genomic DNA repair.

[0202] [Table 27]

[0203] (result) 16 subjects treated with viltolarsen (viltolarsen group) In 65 patients with CINRG DNHS natural history group (DNHS group), baseline or baseline morbidity at 13 and 25 weeks was The baseline time-function tests [6-minute walk test (6MWT), time to rise (TTSTAND ) speed, 4-step stair climbing time (TTCLIMB) speed, 10 meter running / walking time (TTRW) speed The changes in [gait degree] and North Star Ambulatory Assessment (NSAA) were compared. In the analysis method used in this example, the "pre-treatment value" or baseline does not affect the results. The covariates are background factors that give the mean mean, and the data at 13 and 25 weeks are repeated for a specific subject. The MMRM analysis was performed using the repeated measures. The Viltolarsen group in this example was evaluated for distance traveled and speed per 10 meter walk time. In all other motor function tests, the patients in the drug group showed a significant improvement. The condition had improved according to the historical course.

[0204] Figure 8 shows the results at 13 weeks of administration (12 weeks after the first administration) and 25 weeks of administration (24 weeks after the first administration). The motor function of the Viltolarsen-treated group and the CINRG natural history control group (DNHS) was evaluated. 1 is a graph showing the change from baseline in the results of the functional test. The least squares mean of the change from 13 weeks to 25 weeks is shown. Error bars are standard error. P value is calculated using MMRM. Calculated.

[0205] The viltolarsen administration groups and sample sizes at each DNHS time point were as follows: [Table 28]

[0206] [Table 29] EXAMPLES

[0207] Motor function test (up to 85 weeks (84 weeks after first administration)) (Experimental Method) Time function tests [6-minute walk test (6MWT), time to stand (TTSTAND), time to ascend four steps (TTCLIMB, 10-meter run / walk time (TTRW)] and North Star Ambulation Assessment (NS AA) was administered 1 week before the first administration and at each point 12 weeks after the first administration (1 week) (13, 25, The 201 study was conducted by CINRG-affiliated institutions. The motor function test was performed according to the standard operating procedures (SOPs) and clinical assessor training procedures used in the CINRG DNHS. The test was conducted according to the instructions. Since the subjects were children, their interest in the test was sustained. Some items could not be implemented depending on the test due to reasons such as lack of experience.

[0208] (result) The results are shown in Figures 9 to 16. The North Star Ambulatory Ability Assessment showed that Viltolarse Scores were maintained or improved in many patients aged 5 years or older in the n-treated group. Based on the natural history data from Gillis, we set patient entry criteria like those for clinical trials and after 1-2 years According to a study that investigated the change in NSAA score, the 1-year survival rate of patients treated with exon 53 skipping was The mean change between the two was -4.1 points (J Neurol Neurosurg Psychicary 87, 149-55, 2016). The 48-week follow-up of patients receiving viltolarsen who met the entry criteria of the literature The change over time was +1.3 points. 55-67, 2013), 20% of DMD patients aged 7 to 9 years lost the ability to stand up, There were no patients in the viltolarsen group. Of these, 10% were unable to climb four steps, but this was the case in the Viltolarsen group. There were no patients. The rate decreased with age, but the rate decreased with Viltolarsen administration. Overall, the group showed an increase in speed. Ten percent of 7- to 9-year-olds with DMD were able to walk independently. However, there were no patients in the NSP who lost the ability to walk or run 10 meters. The rate decreased with age, but overall in the viltolarsen group, The rate of dystrophin quantification by WB increased from baseline to 1. Based on the change in the amount of alpha-lin expression and the rise time test, 4-step climbing test, and 10m walking / running test Regarding the change in velocity from the line over 48 weeks, the presence or absence of correlation and the linear regression equation were calculated using Excel. The rise time test and the 10 meter run / walk test were There was a significant correlation between the rate of change and the amount of dystrophin (P < 0.05), and the rate of change increased with an increase in dystrophin. It was suggested that the speed of change in motor function would increase. Industrial Applicability

[0209] According to the present invention, a stable combination of NS-065 / NCNP-01 (Viltolarsen) The present invention provides a pharmaceutical composition for treating Duchenne muscular dystrophy, comprising: Regarding the pharmaceutical composition containing NS-065 / NCNP-01 (Viltolarsen), The drug is provided with a dosage regimen that effectively treats DMD and is safe for human patients. The composition effectively relieves symptoms of Duchenne muscular dystrophy with low side effects. It is possible.

Claims

1. The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence of nucleotides or A pharmaceutical composition for treating Duchenne muscular dystrophy in a human patient, comprising a pharma- ceutical acceptable salt or hydrate thereof. A pharmaceutical composition for treating trophy, comprising: The treatment comprises administering to a subject a therapeutically effective amount of an antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof. and administering the compound intravenously to said human patient at a dose of 40 mg / kg to 80 mg / kg once a week. , the pharmaceutical composition.

2. The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is 2. The pharmaceutical composition of claim 1, wherein the composition is administered intravenously to the human patient at 0 mg / kg once a week.

3. The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is 2. The pharmaceutical composition of claim 1, wherein the composition is administered intravenously to the human patient at 0 mg / kg once a week.

4. The human patient has exons 43-52, 45-52, 47-5 Any one selected from the group consisting of 2, 48-52, 49-52, 50-52 or 52 The pharmaceutical composition according to claim 1, which has a mutation in which an exon is deleted.

5. The expression of dystrophin protein in the human patient before treatment was determined by Western blot analysis. The pharmaceutical composition according to claim 1, wherein the amount of the β-amyloid in ... Composition

6. said human patient exhibits no expression of dystrophin protein prior to treatment; Item 6. The pharmaceutical composition according to item 5.

7. The sequence of the base portion of the antisense oligomer is the sequence shown in SEQ ID NO:

3. The pharmaceutical composition according to claim 1.

8. The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is The pharmaceutical composition of claim 1, which is iltolarsen or an equivalent thereof.

9. The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof, The pharmaceutical composition of claim 1, wherein the composition is present in a concentration of 5 to 500 mg / ml or 10 to 100 mg / ml. Pharmaceutical composition.

10. The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is 2. The pharmaceutical composition of claim 1, comprising:

11. The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is 2. The pharmaceutical composition of claim 1, comprising at a concentration of 0 mg / ml.

12. At least one component selected from the group consisting of an isotonicity agent, a pH adjuster, and a solvent is further added. The pharmaceutical composition of claim 1 , comprising:

13. The isotonicity agent may be sodium chloride, potassium chloride, glucose, fructose, maltose, or the like. Sucrose, lactose, mannitol, sorbitol, xylitol, trehalose 13. The composition according to claim 12, wherein the at least one selected from the group consisting of glycerin and glycerin. Pharmaceutical compositions.

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

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

16. Antisense oligos at a concentration of 2.5-500 mg / ml or 10-100 mg / ml mer, sodium chloride at a concentration of 8-10 mg / ml, and a pH of 7.2-7.

4. The pharmaceutical composition of claim 1 which is an aqueous solution.

17. The treatment has at least one effect selected from the group consisting of the following (1) to (6): The pharmaceutical composition according to claim 1, (1) The mean expression level of dystrophin protein in the skeletal muscle of the patient was ≥ 1.0 times higher than baseline. A more than nine-fold increase after 24 weeks of treatment compared with (2) The change in velocity obtained from the rise time (TTSTAND) compared to the baseline In comparison, at 25 weeks after 24 weeks of administration, the rate is greater than or equal to -0.055 / sec; (3) The change in speed obtained from the time to walk (TTRW) of 10 meters is compared with the baseline. -0.025 meters / second or more at 25 weeks after 24 weeks of treatment compared to control; (4) The change in speed obtained from the time to climb four steps (TTCLIMB) was compared with the baseline. In comparison, at week 25 after 24 weeks of administration, the rate is greater than or equal to -0.060 breaths / second; (5) Change in North Star Ambulation Assessment (NSAA) score compared to baseline and a score of -2.2 or higher at 25 weeks after 24 weeks of treatment; and (6) Change in 6-minute walk test (6MWT) score compared to baseline after 24 weeks of treatment At 25 weeks, the pregnancy should be at least -7.5 metres.

18. In a human patient with Duchenne muscular dystrophy aged 7 to 9 years, the treatment was administered for 84 weeks. In this case, at least one effect selected from the group consisting of the following (1) to (6) is produced:

2. The pharmaceutical composition of claim 1 : (1) Less than 20% of patients lose the ability to stand by 85 weeks after starting treatment thing; (2) Less than 10% of patients will be unable to climb four steps by the 85th week of treatment. To do so; (3) Less than 10% of patients will lose the ability to walk independently within 85 weeks of treatment. and; (4) Within 85 weeks of treatment, there was a significant age-related decrease in 10-meter running / walking speed. Not seen; (5) From the start of treatment to 85 weeks later, there was no age-related decrease in the speed of climbing four steps. and (6) No age-related decrease in standing speed was observed within 85 weeks of treatment. and.

19. Human patients with Duchenne muscular dystrophy aged 10-12 years were treated for 84 weeks. When the above-mentioned method is carried out, at least one of the following effects selected from the group consisting of (1) to (6) is obtained: The pharmaceutical composition according to claim 1, (1) Less than 60% of patients lose the ability to stand by 85 weeks after starting treatment thing; (2) Less than 50% of patients will be unable to climb four steps by the 85th week of treatment. To do so; (3) Less than 50% of patients will lose the ability to walk independently within 85 weeks of treatment. and; and (4) Within 85 weeks of treatment, there was a significant age-related decrease in 10-meter running / walking speed. Not to be seen. (5) A period in which the speed of climbing four steps increased was observed between the start of treatment and 85 weeks. and (6) A period of increased speed of rising is observed between the start of treatment and 85 weeks.

20. The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence consisting of nucleotides or A pharmaceutical composition containing a pharma- ceutical acceptable salt or hydrate, The antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof is and administering the compound intravenously to a human patient at a dose of from about 100 mg / kg to about 80 mg / kg once a week. Treatment for type 1 muscular dystrophy.

21. A method for treating Duchenne muscular dystrophy in a human patient comprising administering to said patient a human dystrophy From the 36th to 56th nucleotides from the 5' end of the 53rd exon of the ribosome gene or a pharma- ceutical acceptable salt thereof. A salt or hydrate thereof, 40 mg of the antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof / kg to 80 mg / kg is administered intravenously to the human patient once a week; The antisense oligomer or a pharma- ceutically acceptable salt or hydrate thereof.

22. For the preparation of a pharmaceutical composition for treating Duchenne muscular dystrophy in a human patient. The 36th to 56th residues from the 5' end of the 53rd exon of the human dystrophin gene An antisense oligomer having a base sequence complementary to a sequence consisting of nucleotides or Use of a pharma- ceutically acceptable salt or hydrate thereof, 40 mg of the antisense oligomer or a pharma- ceutical acceptable salt or hydrate thereof / kg to 80 mg / kg is administered intravenously to the human patient once a week; The above uses.

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