Therapeutic uses and methods

Abstract

Provided are uses of a therapeutically effective amount of oligonucleotide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving one or more markers, signs or symptoms of muscular dystrophy in a subject or for delaying the progression of muscular dystrophy in a subject, and a method for treating muscular dystrophy in a subject, a method of improving muscle function or delaying deterioration of muscle function in a subject suffering from muscular dystrophy, comprising administering to the subject a pharmaceutical composition comprising an antisense oligonucleotide of VLA-4 for a sufficient period of time and under sufficient conditions, or in a subject suffering from muscular dystrophy, or in a subject suffering from muscular dystrophy, or in a subject suffering from muscular dystrophy, or in a subject suffering from muscular dystrophy. One or more markers, signs or symptoms of muscular dystrophy or muscular dystrophy muscle fibers of a subject are improved or the progress of muscular dystrophy is delayed. In one embodiment, the method uses low doses of antisense oligonucleotides less than about 50 mg / dose. In one embodiment, antisense oligonucleotide treatment is used as an adjuvant therapy for corticosteroid treatment of a subject suffering from muscular dystrophy, such as DMD.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880095236.4. Technical Field

[0002] The present description enables compositions and methods for treating muscle disorders, such as muscular dystrophy. Background Art

[0003] Bibliographical details of the references in this specification are also listed at the end of the specification.

[0004] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that the prior art forms part of the common general knowledge in any country.

[0005] Muscular dystrophy (MD) is a group of diseases characterized by progressive weakening and wasting of specific muscle tissue (myonecrosis), with replacement of skeletal muscle by fibrous, bone or fatty tissue. There are several different forms of muscular dystrophy that affect males or both males and females, many of which appear during infancy and childhood until middle age or later. Their form and severity vary with age of onset, with younger subjects in particular often experiencing an acutely progressive disease.

[0006] The most common forms of MD are Duchene muscular dystrophy (DMD), limb-girdle muscular dystrophy (LGMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD, including congenital MD of the Fukuyama type and congenital MD with myosin deficiency), facioscapulohumeral, oculopharyngeal, Emery-Dreifuss, and distal. Almost all types of MD are caused by mutations in a single gene.

[0007] DMD and BMD involve defects in the dystrophin gene on the X chromosome. Dystrophin is used to connect the contractile machinery of muscle cells (sarcomeres) and the cytoskeleton (actin filaments) and the extracellular matrix (ECM), where collagen transmits muscle force (Grounds MD, 2008). The ECM is known to play a complex role in muscle function and muscle regeneration. Dystrophic muscle fibers are associated with necrosis, inflammation, and fibrosis. The exact sequence of events that leads to progressive disease due to dystrophin deficiency is not clear at the molecular level. Children with DMD have dystrophin-deficient muscles that are susceptible to contraction-induced muscle damage, which triggers the immune system and exacerbates muscle damage, as summarized in a publication by the Director of FDA CDER (Rosen et al., 2015). The continued deterioration of muscle strength affects the lower limbs, resulting in impaired mobility, and also affects the upper limbs, resulting in further loss of function and self-care ability. While gene therapy and exon skipping approaches would be ideal, researchers are also focused on understanding the nature of the disease in order to develop strategies and drugs that can lessen its severity and slow its progression. The mdx mouse model is widely used in preclinical studies of mechanisms and interventions. Grounds MD, 2008, identified the need for a two-tiered approach targeting both the chronic and acute phases of the disease in the mdx mouse.

[0008] DMD is a devastating disease that affects primarily boys with an incidence of approximately 1:3,500 live births. Boys lose the ability to walk at an early age, become wheelchair-bound after puberty, and often die of cardiopulmonary failure in the third decade of life. BMD is similar to DMD but milder.

[0009] Current treatment with corticosteroids aims to reduce disease severity by reducing inflammation, thereby preserving muscle mass and function over time. Corticosteroids have an acute anti-inflammatory effect, but are likely short-lived, and their mechanism of action is unclear. They are suboptimal because side effects severely limit their use, and they may also cause atrophy. Prednisolone 0.75 mg / kg / day and deflazacort 0.9 mg / kg / day are standard treatments for ambulatory DMD patients, but there is no consensus on the benefit of CS when boys become nonambulatory, and boys may continue treatment, sometimes at the fixed dose they were on when they lost the ability to walk, a reduced mg / kg / day dose, or they may stop CS. Edasalonexent, an anti-inflammatory NF-κB agent, is in development as a monotherapy for young ambulatory boys with DMD. Several drugs are in clinical trials targeting different aspects of malnutrition. For example, tamoxifen for fibrosis, idebenone for respiratory function, and ataluren for stop codon skipping are in clinical trials for MD. Oligonucleotide therapies that induce targeted skipping of exons of the dystrophin gene have been evaluated for the treatment of DMD with mixed results. Eteplirsen, a morpholino oligonucleotide, has been validated in 13% of DMD children with inherited stop codon mutations in exon 51 to skip exon 51, whereas drispersen, a 2'-O-methyl phosphorothioate oligonucleotide for exon 51 skipping, failed to achieve activity and FDA approval.

[0010] These deficiencies in current treatments suggest a need for alternative therapeutic approaches. Summary of the invention

[0011] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0012] As used herein, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a single composition, as well as two or more compositions; reference to "an agent" includes one agent, as well as two or more agents; reference to "the present invention" includes both single and multiple aspects of the invention, etc.

[0013] In one embodiment, the present invention provides a method for treating muscular dystrophy in a subject, comprising administering an inhibitory oligonucleotide to human CD49d (α4 chain of VLA-4).

[0014] In one embodiment, exemplary inhibitory oligonucleotides include isolated or synthetic antisense RNA or DNA, siRNA or siDNA, miRNA, miRNA mimics, shRNA or DNA, and antisense DNA or RNA or DNA:RNA hybrids.

[0015] In one embodiment, the present invention provides a method for treating muscular dystrophy in a subject in need thereof, the method comprising periodically administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an oligonucleotide comprising the following structure:

[0016] 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3'

[0017] in,

[0018] a) each of the 19 nucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester;

[0019] b) ribonucleosides whose nucleotides 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides;

[0020] c) nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides;

[0021] d) nucleotides 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; and

[0022] e) All cytosines are 5-methylcytosine ( Me C),

[0023] or a pharmaceutically acceptable salt thereof.

[0024] In one embodiment, the administration is performed at a time and under conditions sufficient to improve one or more markers, signs or symptoms of muscular dystrophy in the subject or to delay progression of muscular dystrophy in the subject.

[0025] In one embodiment, administration is in combination with standard corticosteroid treatment.

[0026] In one embodiment, the corticosteroid is administered at a low dose. Low dose corticosteroids include 2 / 3, 1 / 2, 1 / 4 and 1 / 3 of the standard dose.

[0027] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the presence of standard or low dose corticosteroids.

[0028] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the absence of corticosteroids.

[0029] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the absence of corticosteroids and wherein the subject is ambulatory.

[0030] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the absence of corticosteroids and wherein the subject is non-ambulatory.

[0031] In another embodiment, the present specification implements a method for improving muscle function or delaying muscle function decline in a subject with muscular dystrophy, the method comprising periodically administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an oligonucleotide comprising the following structure:

[0032] 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3'

[0033] in,

[0034] a) each of the 19 nucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester;

[0035] b) ribonucleosides whose nucleotides 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides;

[0036] c) nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides;

[0037] d) nucleotides 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; and

[0038] e) All cytosines are 5-methylcytosine ( Me C),

[0039] or a pharmaceutically acceptable salt or stereoisomer thereof, for a duration and under conditions sufficient to improve one or more markers, signs or symptoms of dystrophic muscle fibers or delay progression of muscular dystrophy in the subject.

[0040] In one embodiment, the oligonucleotide is an RNA-DNA hybrid.

[0041] In one embodiment, the subject is unable to walk due to MD.

[0042] In one embodiment, the subject is post-pubertal.

[0043] In one embodiment, the method comprises monitoring CD4+ and / or CD8+ T cell levels. In one embodiment, the method comprises monitoring decreased CD4+ and / or CD8+ T cell levels. In one embodiment, the method comprises monitoring M1 macrophages or HLADR + In one embodiment, the method comprises monitoring the reduction of M1 macrophages or HLADR + Monocytes.

[0044] In one embodiment, the method comprises determining the level or presence of one or more markers of MD or dystrophic myofibers, including the level or number of immune cells or immune regulatory factors, the level of inflammatory markers, or the level of fibrosis markers, or the level of muscle status markers.

[0045] In one embodiment, the one or more markers of MD or MD progression or dystrophic muscle fibers include the level or amount of immune cells or immune regulatory factors, the level of inflammatory markers or the level of fibrosis markers or the level of muscle status markers.

[0046] Markers of muscle status include, but are not limited to, markers of motor muscle function, markers indicative of muscle fibrosis or its absence, markers indicative of muscle degeneration or regeneration, markers of cardiac function, and markers of lung function.

[0047] In one embodiment, improving one or more signs of MD or dystrophic muscle fibers comprises improving limb function, body muscle function, heart and / or lung function.

[0048] In one embodiment, the method comprises determining the level or presence of one or more signs of MD or dystrophic muscle fibers. Illustrative signs include limb function, body muscle function, heart and lung function.

[0049] In one embodiment, one or more symptoms of MD or dystrophic muscle fibers include quality of life factors such as energy level, well-being, perceived walking comfort, upper extremity functional mobility, and the like.

[0050] In one embodiment, a subject in need thereof includes a subject with a genetic and / or clinical diagnosis of MD and relatively low levels of dystrophic muscle fibers and inflammatory markers.

[0051] In one embodiment, the subject shows normal or only slightly elevated levels of inflammatory cells. Inflammatory cells include T cells (CD4, CD8), B cells (CD-19), granulocytes (neutrophils, basophils and eosinophils).

[0052] In one embodiment, the subject exhibits normal or only slightly elevated levels of CD49d cells.

[0053] In one embodiment, the subject exhibits normal or only slightly elevated levels of CD49d T cells.

[0054] In one embodiment, the subject exhibits normal or only slightly elevated levels of immune cell markers such as CD3, CD4, CD8, CD49d, CD29, and HLA-DR.

[0055] Suitable methods for markers include cellular or protein / nucleic acid or lipid analysis are known in the art and include, but are not limited to, flow cytometry, microbead techniques and ELISA-based methods, chromatography and / or MS methods, hybridization- or sequencing-based methods.

[0056] In another embodiment, a subject diagnosed with MD exhibits significantly elevated or acute levels of severely dystrophic muscle fibers, accompanied by severe muscle necrosis and inflammation.

[0057] In one embodiment, the subject exhibits significantly elevated levels of CD49d T cells relative to normal healthy controls.

[0058] In one embodiment, the form of MD in the subject is selected from the group consisting of Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy (LGMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), including congenital MD of the Fukuyama type and congenital MD with myosin deficiency, facioscapulohumeral, oculopharyngeal, Emery-Dreifuss, and distal muscular dystrophy.

[0059] In one embodiment, the subject has DMD or BMD and is non-ambulatory.

[0060] In one embodiment, the subject has DMD or BMD and is postpubertal.

[0061] In another form of the invention, embodiments involving; when the pharmaceutical composition is used in the methods or uses currently described, the use of the composition described herein in the preparation of a medicament for treating or preventing muscular dystrophy in a subject, the pharmaceutical composition used in the methods currently described.

[0062] Therefore, in one embodiment, the present invention provides the use of an oligonucleotide comprising the following structure:

[0063] 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3'

[0064] in,

[0065] a) each of the 19 nucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester;

[0066] b) ribonucleosides whose nucleotides 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides;

[0067] c) nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides;

[0068] d) nucleotides 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; and

[0069] e) All cytosines are 5-methylcytosine ( Me C),

[0070] or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing muscular dystrophy in a subject or delaying the progression of muscular dystrophy in a subject.

[0071] In another embodiment, the present description enables an oligonucleotide comprising the following structure:

[0072] 5'- Me C Me UG AGT Me CTG TTTMe U Me C Me CA Me U Me U Me C Me U-3'

[0073] in,

[0074] a) each of the 19 nucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester;

[0075] b) ribonucleosides whose nucleotides 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides;

[0076] c) nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides;

[0077] d) nucleotides 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; and

[0078] e) All cytosines are 5-methylcytosine ( Me C),

[0079] or a pharmaceutically acceptable salt or stereoisomer thereof, for treating or preventing muscular dystrophy in a subject or delaying the progression of muscular dystrophy in a subject.

[0080] In one embodiment, the present invention enables a method of treating muscular dystrophy in a subject in need thereof, the method comprising periodically administering to the subject a therapeutically effective amount of a human CD49d inhibitory oligonucleotide to improve one or more markers, signs or symptoms of muscular dystrophy in the subject or delay its progression.

[0081] In one embodiment, antisense oligonucleotides are administered in combination with or as an adjunct to standard or low-dose corticosteroid therapy.

[0082] In one embodiment, the corticosteroid is administered in a low dose.

[0083] In one embodiment, administration of the antisense oligonucleotide is effective in the absence of corticosteroid treatment.

[0084] In another embodiment, disclosed is the use of an inhibitory oligonucleotide of human CD49d in the preparation of a medicament for improving one or more markers, signs or symptoms of muscular dystrophy or delaying the progression of muscular dystrophy in a subject suffering from muscular dystrophy.

[0085] In one embodiment, a pharmaceutical composition comprising an inhibitory oligonucleotide against human CD49d is provided for treating muscular dystrophy in a subject or delaying the progression of muscular dystrophy in a subject.

[0086] The above summary is not, and should in no way be construed as, an exhaustive listing of all embodiments of the invention.

[0087] Many modifications will be apparent to those skilled in the art without departing from the scope of the invention.

[0088] It will be appreciated by those skilled in the art that various changes and / or modifications may be made to the above embodiments without departing from the broad scope of the present disclosure. Therefore, the present embodiments are considered in all aspects to be illustrative and not restrictive.

[0089] Sequence Listing Keywords

[0090] SEQ ID NO: 1 Human α4 integrin antisense complex (ATL1102)

[0091] SEQ ID NO: 2 Murine α4 integrin antisense complex (ISIS348574) DETAILED DESCRIPTION

[0092] The present invention is not limited to specific screening procedures for medicaments, specific formulations of medicaments and various medical methods, as these may vary. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by one of ordinary skill in the art to which the present invention belongs. Any materials and methods similar to or equivalent to those described herein may be used to practice or test the present invention. For definitions and terminology in the art and other methods known to those skilled in the art, practitioners are particularly referred to Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Remington's Pharmaceutical Sciences, 18th ed., McEaston, Pa., (1990)

[0093] The term "subject" includes human subjects or individuals diagnosed with MD or clinical research model animals.

[0094] For example, DMD is usually diagnosed clinically when infant motor milestones are delayed at 18 months. Early features of muscle weakness include a broad gait, toe walking, excessive lordosis, frequent falls, hypertrophy of muscles such as the gastrocnemius, deltoids, quadriceps, tongue and masseter muscles, difficulty standing up, and arm weakness. Loss of walking ability in DMD patients usually occurs between 7 and 13 years of age, while walking after that time is characteristic of BMD. Cardiopulmonary defects may also be evident. Fatigue and speech development may also be delayed. However, no upper motor neuron signals or muscle fasciculations are observed.

[0095] The diagnosis of DMD can be shown by dystrophin immunofluorescence detection and / or immunoblotting to lack dystrophin, and the clinical manifestations are consistent with typical DMD. Alternatively, the gene deletion test of the dystrophin gene is positive (missing one or more exons), wherein the reading frame can be predicted as "outside the frame", and the clinical manifestations consistent with typical DMD are indicative. In one embodiment, the complete dystrophin gene sequencing can be shown as point mutations, duplications or other mutations, which cause stop codon mutations to be clearly associated with DMD. In brothers and sisters or uncles, a positive family history of DMD confirmed by one of the above criteria is also useful. It is also used to assess the characteristic clinical symptoms or signs of DMD (for example, proximal muscle weakness, Gowers sign (Gowers'manoeuvre), increased serum creatine kinase levels).

[0096] Suitable markers, signs and symptoms of MD or dystrophic muscle fibers / improved muscle function are known to those skilled in the art.

[0097] Appropriate tests include tests of movement, muscle, heart, blood flow, and lung function, which are increased over time during treatment.

[0098] In subjects with preclinical cardiomyopathy, cardiac efficacy can be determined based on serum biomarker responses. This can be achieved by measuring the levels of one or more markers, such as myostatin ratio, cardiac troponin, myocardial BNP, etc. Changes in eGFR can also be monitored. Other cardiac functions can be assessed by telemetry, or rhythm abnormalities can be assessed by continuous mobile telemetry monitoring.

[0099] Further testing included measuring muscle oxygenation parameters and mitochondrial phenotype.

[0100] Reduced fibrosis can be assessed by MRI. Reduced muscle fat, reduced cardiac fibrosis, increased contractility, grip strength, and improved heart and lung function tests. Other assessments attempt to slow the rate of decline in these functions.

[0101] Quality of life questionnaires are very useful in determining how well treatment is working.

[0102] Clinical outcomes may include, for example, determining the percent change in normalized upper extremity accessible surface area, the percent change in cardiac circumferential strain assessed by MRI, and the percent change in cardiac lateral and posterior wall strain. Another useful test is to measure forced vital capacity, delayed loss of respiratory function, such as the change in FVC 5p from baseline as measured by spirometry.

[0103] Motor function tests included the mean change of 4 standard stair climbing tests before and after treatment, the time to rise from the ground, the mean change of magnetic resonance magnetic spectroscopy of the fat part of the vastus lateralis muscle, quadriceps muscle power test, knee extensor peak torque measurement, and forearm ultrasound muscle microvascular blood supply.

[0104] Important clinical assessments include time to walk / run 6 or 10 meters, time to climb 4 floors, time to descend 4 floors, and time to stand from a supine position. Changes in weight, height, and BMI may also be assessed.

[0105] Alternatively or additionally, biomarkers assessed from muscle biopsies, changes in pharmacodynamic marker measurements in a plasma biomarker panel measured by ELISA or proteomics, or changes in circulating immune cell markers are assessed.

[0106] As used herein, the term "antisense compound" refers to an oligomeric compound that hybridizes with a nucleic acid molecule encoding the α4 integrin chain (α4β1) and / or α4β7 integrin of VLA-4. The human α4 integrin chain is CD49d. Antisense compounds can interfere with the expression of CD49d, β1 integrin and / or β7 integrin.

[0107] As used herein, the term "nucleic acid molecule encoding α4 integrin" includes DNA encoding the α4 integrin chain or α4β7 integrin of VLA-4, RNA transcribed from such DNA (including pre-mRNA and mRNA or portions thereof), and further, cDNA derived from such RNA.

[0108] As used herein, the term "VLA-4" refers to a heterodimer of α4 integrin and β1 integrin. VLA-4 is expressed at significant levels on normal peripheral blood B and T cells, thymocytes, monocytes and other cells, as well as hematopoietic stem and progenitor cells. VLA-4 is also expressed on mesenchymal and endothelial progenitor cells, mesenchymal stem cells and potential endothelial stem cells. Ligands for VLA-4 include vascular cell adhesion molecule-1 (VCAM-1) and CS-1, an alternative splicing domain within the Hep II region of fibronectin.

[0109] As used herein, the term "α4β7 integrin" refers to a heterodimer of α4 integrin and β7 integrin. α4β7 integrin identifies a subset of memory T cells that are targeted to the intestine. α4β7 integrin is also expressed on subsets of mast cells, lymphocytes, and NK progenitor cells. α4β7 integrin is expressed on some stem cells and progenitor cells. Ligands for α4β7 integrin include MAdCam-1 and VCAM-1.

[0110] Nucleic Acids

[0111] The present invention encompasses the use of various oligonucleotides, also referred to as nucleic acids. Exemplary nucleic acids include DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA)), RNA (e.g., messenger RNA (mRNA), short hairpin RNA (shRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, DNA or RNA analogs (e.g., containing base analogs, sugar analogs and / or non-natural backbones, etc.), RNA / DNA hybrids, and polyamide nucleic acids (PNA), all of which can be in single-stranded or double-stranded form. In one example, isolated nucleic acids. As used herein, the term "isolated nucleic acid" refers to nucleic acids that have been altered or removed from their natural state by human intervention.

[0112] The term "oligonucleotide" refers broadly to short nucleic acid molecules. Oligonucleotides are easily combined with their respective complementary oligonucleotides, DNA or RNA in a sequence-specific manner to form duplexes. In one embodiment, the oligonucleotide length is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides or longer.

[0113] In one embodiment, the oligonucleotide of the present invention is an inhibitory oligonucleotide. In one example, the term "inhibitory oligonucleotide" refers to any oligonucleotide that reduces the production, expression or biological activity of one or more proteins. For example, an inhibitory oligonucleotide can interfere with the translation of mRNA into protein in the ribosome. In another example, an inhibitory oligonucleotide can be fully complementary to a gene or mRNA encoding one or more proteins to bind (hybridize) a target gene or mRNA, thereby reducing the expression or biological activity of a target protein. In another example, an inhibitory oligonucleotide suppresses the biological activity of a nucleic acid in a cell that does not encode a protein. For example, an inhibitory oligonucleotide can suppress the biological activity of a non-coding RNA.

[0114] As used herein, the term "antisense" means a nucleotide sequence that is complementary to and thus capable of binding to a coding sequence, which may be a nucleotide sequence of a DNA double helix strand undergoing transcription, or a nucleotide sequence of a messenger RNA molecule. Antisense DNA is a non-coding strand that is complementary to the coding strand in a double-stranded DNA.

[0115] The term "short hairpin RNA" or "shRNA" refers to an RNA structure having a duplex region and a loop region.

[0116] The term small interfering RNA (siRNA), sometimes referred to as short interfering RNA or silencing RNA, is a class of double-stranded or single-stranded RNA molecules with a length of about 19-25 base pairs. siRNAs that inhibit or prevent translation into specific proteins are represented by the protein name coupled to the term siRNA. Typically, in various embodiments, siRNA is a double-stranded or single-stranded nucleic acid molecule having about 19 to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides).

[0117] The term "microRNA" (abbreviated as miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, which plays a role in RNA silencing and post-transcriptional regulation of gene expression. The prefix "miR" is followed by a dash and a number, the latter usually indicating the order of nomenclature. Different miRNAs with almost identical sequences except one or two nucleotides are marked with additional lowercase letters. Many miRNAs are known in the art (miRBase V.21 naming; see Kozomara et al. 2013; Griffiths-Jones, S.2004). The sequences of these miRNAs are well known in the art and can be found, for example, on the World Wide Web at mirbase.org.

[0118] In one embodiment, an "inhibitory oligonucleotide" simulates the activity of one or more miRNAs. As used herein, the term "miRNA mimics" refers to a small double-stranded RNA molecule designed to simulate an endogenous mature miRNA molecule when introduced into a cell. MiRNA mimics can be obtained from various suppliers, such as Sigma Aldrich and Thermo Fisher Scientific.

[0119] In embodiments, "inhibitory oligonucleotides" inhibit the activity of one or more miRNAs. Various miRNA species are suitable for this purpose. Examples include, but are not limited to, antagonists, interfering RNA, ribozymes, miRNA sponges, and miR-masks. The term "antagonist" is used in the context of the present invention to refer to chemically modified antisense oligonucleotides that bind to target miRNA and inhibit miRNA function by preventing miRNA from binding to its cognate gene target. Antagonists can include any base modification known in the art. In an example, the length of the miRNA species cited above is about 10 to 50 nucleotides. For example, the antagonist can have an antisense portion that is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0120] In one embodiment, miRNA species are chimeric oligonucleotides containing two or more chemically distinct regions, each region consisting of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confer one or more beneficial properties (e.g., increased nuclease resistance, increased cellular uptake, increased binding affinity to a target) and a region that is a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids.

[0121] In one embodiment, the nucleic acids encompassed by the present invention are synthetic. The term "synthetic nucleic acid" means that the nucleic acid does not have the chemical structure or sequence of a naturally occurring nucleic acid. Synthetic nucleotides include engineered nucleic acid molecules. In another example, the nucleic acid structure can also be modified into a locked nucleic acid (LNA), which has a methylene bridge between the 2' oxygen and the 4' carbon to lock the ribose in the 3'-endo (N) conformation in the A-type conformation of the nucleic acid (Lennox et al. 2011; Bader et al. 2011). In the case of miRNA, this modification can significantly increase the target specificity and hybridization properties of the molecule.

[0122] Conventional methods can be used to design nucleic acids for methods disclosed herein as needed. For example, in the context of inhibitory oligonucleotides, a target fragment of 5, 6, 7, 8, 9, 10 or more nucleotides in length is considered suitable for targeting genes, and the target fragment includes a sequence of at least five (5) continuous nucleotides in or immediately adjacent to the seed sequence. An exemplary target fragment may include a sequence including at least 5 continuous nucleotides, 5 continuous nucleotides from the 5' end of one of the seed sequences (the remaining nucleotides are continuous fragments of the same RNA, starting from the upstream of the 5' end of the seed sequence, and continuing until the nucleic acid contains about 5 to about 30 nucleotides). In another example, the target fragment is represented by an RNA sequence, and the RNA sequence includes at least 5 continuous nucleotides starting from the 3' end of one of the seed sequences (the remaining nucleotides are continuous fragments of the same RNA, starting from the downstream of the 3' end of the target fragment, and continuing until the nucleic acid contains about 5 to about 30 nucleotides). The term "seed sequence" is used in the context of the present invention to refer to a 6-8 nucleotide (nt) long substring within the first 8 nt of the 5-end of the miRNA (i.e., the seed sequence), which is an important determinant of target specificity. Once one or more target regions, fragments or sites are identified, an inhibitory nucleic acid compound that is sufficiently complementary to the target, i.e., sufficiently well hybridized and has sufficient specificity (i.e., substantially not binding to other non-target nucleic acid sequences), is selected to produce the desired effect.

[0123] Antisense compounds of α4 integrin

[0124] In one embodiment, the methods of the present invention rely on the use of antisense compounds of α4 integrin. Such antisense compounds target nucleic acids encoding the α4 integrin chain (α4β1) or α4β7 integrin of VLA-4. In one embodiment, the antisense compound is an oligonucleotide. However, other oligomeric antisense compounds are contemplated, including but not limited to oligonucleotide mimetics.

[0125] The hybridization of antisense compounds with their target nucleic acids is generally referred to as "antisense". The hybridization of antisense compounds with their target nucleic acids inhibits the function of the target nucleic acid. Such "antisense inhibition" is generally based on the hybridization of antisense compounds with target nucleic acids based on hydrogen bonding, so that the target nucleic acid is cut, degraded or otherwise becomes inoperable. The function of the disturbed target DNA may include replication and transcription. For example, replication and transcription may come from endogenous cell templates, vectors, plasmid constructs or other. The function of the disturbed RNA may include the following functions, such as translocation of RNA to a site for protein translation, translocation of RNA to a site away from the RNA synthesis site in the cell, translation of protein from RNA, splicing of RNA to produce one or more RNA species, and the formation of catalytic activity or complexes related to RNA that RNA participates in or promotes.

[0126] As used herein, "hybridization" refers to the pairing of complementary bases of an oligonucleotide and a target nucleic acid. Base pairing generally involves hydrogen bonding between complementary nucleosides or nucleotide bases (nucleobases), which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding. Guanine (G) and cytosine (C) are examples of complementary nucleobases that pair by forming three hydrogen bonds. Adenine (A) and thymine (T) are examples of complementary nucleobases that pair by forming two hydrogen bonds. Hybridization can occur in various situations.

[0127] "Nucleoside" is a base-sugar combination. The base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. "Nucleotide" is a nucleoside that further includes a phosphate group covalently attached to the sugar portion of the nucleoside. For those nucleosides that include pentofuranosyl, the phosphate group can be attached to the 2' hydroxyl portion, the 3' hydroxyl portion, or the 5' hydroxyl portion of the sugar.

[0128] "Specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity to allow stable and specific binding to occur between an antisense compound and a target nucleic acid. It should be understood that an antisense compound need not be 100% complementary to its target nucleic acid sequence to be specifically hybridized. An antisense compound is specifically hybridizable when the binding of the antisense compound to the target nucleic acid interferes with the normal function of the target molecule to cause a loss of activity, and under conditions where specific binding is desired, such as physiological conditions for therapeutic treatment, there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target sequences.

[0129] As used herein, "complementarity" refers to the ability of the nucleobase of the antisense compound to accurately match with the target nucleic acid. For example, if the nucleobase at a certain position of the antisense compound can hydrogen bond with the nucleobase at a certain position of the target nucleic acid, the position of the hydrogen bonding between the antisense compound and the target nucleic acid is considered to be a complementary position. The antisense compound can hybridize on one or more fragments so that the insertion or adjacent fragment does not participate in the hybridization event (e.g., loop structure or hairpin structure). In one embodiment, the antisense compound includes at least 70% sequence complementarity with the target region in the target nucleic acid.

[0130] For example, 18 of the 20 core bases are complementary to the target region in the target nucleic acid, and therefore the antisense compound of specific hybridization will represent 90% complementarity. In this example, the remaining non-complementary core bases can be clustered or interspersed with complementary core bases, and do not need to be adjacent to each other or adjacent to complementary core bases. Therefore, a length of 18 core bases, with 4 non-complementary core bases, the antisense compound in the 2 regions of the side joints that are completely complementary to the target nucleic acid will have an overall complementarity of 77.8% with the target nucleic acid, therefore will fall within the scope of the present invention. The complementarity percentage of antisense compounds and target nucleic acid regions can be routinely determined using BLAST programs known in the art (basic local alignment search tool) and PowerBLAST programs (Altschul et al., 1990; Zhang and Madden, 1997).

[0131] Antisense Oligonucleotides

[0132] The present invention provides antisense oligonucleotides for inhibiting the expression of α4 integrin and / or VLA-4 and / or α4β7 integrin. Such antisense oligonucleotides target nucleic acids encoding the α4 integrin chain of VLA-4 or α4β7 integrin.

[0133] As used herein, the term "inhibit" refers to any measurable decrease (eg, 10%, 20%, 50%, 90%, or 100%) in the expression of VLA-4 or α4β7 integrin.

[0134] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of RNA or DNA or its mimetics, chimeras, analogs and homologues. The term includes oligonucleotides consisting of naturally occurring nucleobases, sugars and covalent nucleoside (backbone) bonds, as well as oligonucleotides with non-naturally occurring portions of similar functions. Such modified or substituted oligonucleotides are generally superior to native forms because they have desired properties, such as enhanced cellular uptake, enhanced affinity for target nucleic acids and increased stability in the presence of nucleases.

[0135] Oligonucleotides may contain chiral (asymmetric) centers, or the molecule as a whole may be chiral. Individual stereoisomers (enantiomers and diastereomers) and mixtures of these are within the scope of the present invention. For disclosure of antisense oligonucleotides containing chiral phosphorothioate bonds, reference may be made to Wan et al., Nucleic Acids Research 42 (22: 13456-13468, 2014).

[0136] When forming an oligonucleotide, the phosphate group covalently links adjacent nucleosides to form a linear polymeric compound. In turn, the corresponding ends of the linear polymeric compound can further join to form a cyclic compound; however, linear compounds are generally preferred. In addition, linear compounds can have internal core base complementarity, and therefore can be folded in a manner that produces a completely or partially double-stranded compound. For oligonucleotides, the phosphate group generally refers to the internucleoside backbone that forms the oligonucleotide. The normal bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond.

[0137] Antisense oligonucleotides of the present invention include, for example, ribozymes, siRNA, external guide sequence (EGS) oligonucleotides, alternative splicers, primers, probes, and other oligonucleotides that hybridize with at least a portion of a target nucleic acid. Antisense oligonucleotides of the present invention can be applied in the form of single-stranded, double-stranded, circular or hairpin, and can contain structural elements, such as internal or terminal protuberances or loops. Once applied, antisense oligonucleotides can cause the effect of one or more enzymes or structural proteins to achieve the modification of target nucleic acid.

[0138] A non-limiting example of such an enzyme is RNAse H, a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Single-stranded antisense compounds that are "DNA-like" are known in the art to cause RNAse H. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar effects have been hypothesized for other ribonucleases, such as those of the RNase III and ribonuclease L families.

[0139] The introduction of double-stranded RNA (dsRNA) molecules has been shown to induce effective and specific antisense-mediated reductions in the function of genes or their associated gene products. This phenomenon occurs in plants and animals and is thought to have an evolutionary relationship with viral defense and transposon silencing. The first evidence that dsRNA can cause gene silencing in animals came from work in the nematode Caenorhabditis elegans in 1995 (Guo and Kempheus, 1995). Montgomery et al. (1998) have shown that the main interference effect of dsRNA is post-transcriptional. The post-transcriptional antisense mechanism defined in Caenorhabditis elegans caused by exposure to double-stranded RNA (dsRNA) has since been referred to as RNA interference (RNAi). The term has been summarized as meaning antisense-mediated gene silencing, which involves the introduction of dsRNA, resulting in a sequence-specific reduction in endogenous target mRNA levels (Fire et al., 1998). Recently, it has been shown that single-stranded RNA oligomers, which are in fact the antisense polarity of dsRNA, are potent inducers of RNAi (Tijsterman et al., 2002).

[0140] One of ordinary skill in the art can identify antisense oligonucleotides useful in the methods of the present invention without undue experimentation.

[0141] Modified internucleoside bonds (backbone)

[0142] The antisense compounds of the present invention include oligonucleotides with modified backbones or non-natural internucleoside bonds. Oligonucleotides with modified backbones include oligonucleotides that retain phosphorus atoms in the backbone and oligonucleotides that do not have phosphorus atoms in the backbone.

[0143] The oligonucleotide backbone of the modification containing phosphorus atom comprises, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates, comprise 3'-alkylenephosphonates, 5'-alkylenephosphonates and chiral phosphonates, hypophosphite, phosphoramidate, comprise 3'-phosphoramidate and aminoalkylphosphoramidate, thiophosphoramidate, thioalkylphosphonates, thioalkylphosphotriester, selenophosphonate, have normal 3'-5' key boron phosphonate, 2'-5' key analog boron phosphonate and have those of reverse polarity, wherein one or more nucleotides between key is 3' to 3' key, 5' to 5' or 2' to 2' key. Oligonucleotide with reverse polarity comprises single 3' to 3' key at the key between the nucleotide closest to 3' end, i.e. single reversed nucleoside residue, it may be alkaline (core base lacks or has hydroxyl at its position). Various salts, mixed salts and free acid forms are also comprised.

[0144] Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-containing bonds include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, US 5,541,306, US 5,550,111, US 5,563,253, US 5,571,799, US 5,587,361, US 5,194,599, US 5,565,555, US 5,527,899, US 5,721,218, US 5,672,697 and US 5,625,050.

[0145] Modified oligonucleotide backbones that do not include phosphorus atoms include, for example, backbones formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include backbones with morpholino bonds (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; ribose acetyl backbones; olefins containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; those of amide backbones; and others with mixed N, O, S and CH2 component parts.

[0146] Representative U.S. patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, US 5,608,046, US 5,610,289, US 5,618,704, US 5,623,070, US 5,663,312, US 5,633,360, US 5,677,437, US 5,792,608, US 5,646,269 and US 5,677,439.

[0147] Modified sugars and internucleoside linkages

[0148] The antisense compounds of the present invention include oligonucleotide mimetics in which both the sugar and the internucleoside linkage (ie, backbone) of the nucleotide unit are replaced with novel groups, while the nucleobase unit is retained for hybridization with a target nucleic acid.

[0149] Oligonucleotide mimics that have been shown to have excellent hybridization properties are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by a backbone containing amides, particularly an aminoethylglycine backbone. The core base is retained and directly or indirectly bonded to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents for teaching the preparation of PNA compounds include but are not limited to US 5,539,082, US 5,714,331 and US 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., 1991.

[0150] The antisense compounds of the present invention also include oligonucleotides having a thiophosphate backbone and oligonucleotides having a heteroatom backbone, such as -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- [referred to as a methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -ON(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-] of US 5,489,677 and an amide backbone of US 5,602,240.

[0151] The antisense compounds of the present invention also include oligonucleotides having the morpholino backbone structure of US Pat. No. 5,034,506.

[0152] Modified sugar

[0153] Antisense compounds of the invention include oligonucleotides having one or more substituted sugar moieties.

[0154] Examples include oligonucleotides that include one of the following at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups.

[0155] In one embodiment, the oligonucleotide comprises one of the following at the 2' position: O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2 and O(CH2)nON[(CH2)nCH3]2, wherein n and m are 1 to about 10.

[0156] Further examples of modified oligonucleotides include oligonucleotides that include one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides, or group for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties.

[0157] In one embodiment, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3 (also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995), i.e., alkoxyalkoxy. In another embodiment, the modification includes 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE), or 2'-dimethylaminoethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0158] Other modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O-CH2-CH=CH2) and 2'-fluoro (2'-F). The 2' modification can be at the arabinose (upper) position or the ribose (lower) position. In one embodiment, the 2'-arabinose modification is 2'-F.

[0159] Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or the 3' position of the sugar in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide.

[0160] Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0161] Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, U.S. Pat. No. 5,792,747 and U.S. Pat. No. 5,700,920.

[0162] Further modification of sugar includes locked nucleic acid (LNA), wherein the 2'-hydroxyl is connected to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar part. In one embodiment, the bond is a methylene (-CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2. LNA and its preparation are described in WO 98 / 39352 and WO 99 / 14226.

[0163] Natural and modified nucleobases

[0164] Antisense compounds of the present invention include oligonucleotides with nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0165] Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CC-CH3) uracil and cytosine, other alkynyl derivatives of pyrimidine bases, 6-azouracil, , cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0166] Further modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).

[0167] Modified nucleobases may also include those in which the purine or pyrimidine base is substituted with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Other nucleobases include those disclosed in US 3,687,808, JI Kroschwitz (ed.), The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, John Wiley and Sons (1990), Englisch et al. (1991), and YS Sanghvi, Chapter 15: Antisense Research and Applications, pp. 289-302, ST Crooke, B. Lebleu (ed.), CRC Press, 1993.

[0168] Some of these nucleobases are particularly useful for increasing the binding affinity of oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. In one embodiment, these nucleobase substitutions are combined with 2'-O-methoxyethyl sugar modifications.

[0169] Representative U.S. patents that teach the preparation of some of the above modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, and 5,831,654. 5,763,588, US 6,005,096, US 5,681,941 and US 5,750,692.

[0170] Conjugate

[0171] The antisense compounds of the invention may be conjugated to one or more moieties or groups that enhance the activity, cellular distribution or cellular uptake of the antisense compounds.

[0172] These moieties or groups may be covalently bonded to functional groups such as primary or secondary hydroxyl groups.

[0173] Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0174] Moieties or groups that enhance pharmacodynamic properties include those that improve uptake, increase resistance to degradation, and / or increase sequence-specific hybridization to a target nucleic acid.

[0175] Part or group that strengthen pharmacokinetic property include those that improve absorption, distribution, metabolism or excretion of compound of the present invention.Representative part or group are disclosed in PCT / US92 / 09196 and US 6,287,860.Part or group include but not limited to lipid part such as cholesterol part, cholic acid, thioether such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chain such as dodecanediol or undecyl residue, phospholipid such as two-hexadecyl-racemization-glycerol or triethylammonium 1,2-two-O-hexadecyl-racemization-glyceryl-3-H-phosphonate, polyamine or polyethylene glycol chain or adamantaneacetic acid, palmityl part or octadecylamine or hexylamino-carbonyl-oxycholesterol part.

[0176] chimeric compounds

[0177] As will be appreciated by those skilled in the art, it is not necessary that all positions in a given compound be uniformly modified, and in fact, more than one of the above modifications may be incorporated into a single oligonucleotide or even at a single nucleoside within an oligonucleotide.

[0178] Antisense compounds of the present invention include chimeric oligonucleotides. "Chimeric oligonucleotides" contain two or more chemically different regions, each region consisting of at least one monomer unit, i.e., nucleotides in the case of oligonucleotide compounds. These oligonucleotides generally contain at least one region, wherein the oligonucleotide is modified to enhance resistance to nuclease degradation, increase cellular uptake, increase stability and / or increase binding affinity with the target nucleic acid. The additional regions of the oligonucleotide can be used as substrates for enzymes capable of cutting RNA:DNA or RNA:RNA hybrids. For example, RNAse H is a cellular endonuclease that cuts the RNA chain of RNA:DNA duplexes. Therefore, the activation of RNase H leads to the cutting of RNA targets, thereby greatly improving the efficiency of oligonucleotide-mediated gene expression inhibition. The cutting of RNA:RNA hybrids can be completed in a similar manner by the action of endoribonucleases such as RNAseL that cuts cell and viral RNA. The cutting of RNA targets can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

[0179] Chimeric antisense compounds of the present invention can be formed into the composite structure of two or more oligonucleotides, modified oligonucleotides and / or oligonucleotide mimetics.Such compounds are also referred to as hybrids or gapmers in the art.Representative U.S. Patents instructing the preparation of such hybrid structures include but are not limited to US 5,013,830, US 5,149,797, US 5,220,007, US 5,256,775, US 5,366,878, US 5,403,711, US 5,491,133, US 5,565,350, US 5,623,065, US 5,652,355, US 5,652,356 and US 5,700,922.

[0180] Exemplary Oligonucleotides

[0181] Exemplary antisense platforms known in the art include, but are not limited to, morpholinos, first gene oligonucleotides (1 st genoligos), second gene oligonucleotide (2 nd gen oligo's), gapmers, siRNA, LNA, BNA or oligonucleotide mimics like peptide nucleic acids. The oligonucleotides can be naked or formulated in liposomes. The oligonucleotides can be linked to a cellular delivery system or not. The oligonucleotides can be used with or without an endosomal release agent.

[0182] In one embodiment, the antisense compound is a second generation phosphorothioate backbone 2'-MOE modified chimeric oligonucleotide gapmer designed to hybridize to the 3'-untranslated region of VLA-4 mRNA. In one embodiment, the oligonucleotide selectively inhibits VLA-4 expression in primary human cells and several human cell lines by hybridizing to RNA encoding CD49, which is the α4 integrin subunit of VLA-4 and the α4β7 integrin.

[0183] In one embodiment, the oligonucleotide is the 19-sodium salt of a 3'→5' phosphorothioate oligonucleotide 20mer, also known as a 3-9-8MOE gapmer, having a molecular weight of 7230 Daltons, wherein the nucleotides at positions 1-3 at the 5' end are 2'-O-(2-methoxyethyl) (2'MOE) modified ribonucleosides (2'-O-(2-methoxyethyl ribose); the nucleotides at positions 4-12 at the 5' end are 2'-deoxyribonucleosides, and all cytosines are 5-methylcytosine; and the nucleotides at positions 13-20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides.

[0184] In one embodiment, the sequence of the oligonucleotide is (SEQ ID NO: 1):

[0185] 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3'.

[0186] The empirical formula of the oligonucleotide is:

[0187] C 233 H 327 N 60 O 129 P 19 S 19 Na 19 .

[0188] The antisense oligonucleotide ATL1102 has previously been shown to be effective in the central nervous system disorder, MS, and at doses significantly higher than those proposed herein (Limmroth et al.). The ability of antisense oligonucleotides directed against the CD49d α chain of VLA-4 to selectively inhibit VLA-4 in immune cells prevents significant safety events, such as PML, which is characterized by the administration of antibodies and small molecule inhibitors of VLA-4, and is a pan-VLA-4 inhibitor that affects all cells expressing VLA-4.

[0189] In one embodiment, all uracils are 5-methyluracil (MeU). Typically, oligonucleotides are synthesized using 2-methoxyethyl modified thymidine rather than 5-methyluracil.

[0190] In one embodiment, all pyrimidines are C5 methylated (ie, U, T, C are C5 methylated).

[0191] In one embodiment, the sequence of the oligonucleotide may be named by accepted oligonucleotide nomenclature showing each OO-linked phosphorothioate internucleotide linkage:

[0192] 2'-O-methoxyethyl-5-methylcytidylyl-(3'→5'O,O-thiophosphoryl)-2'-O-methoxyethyl-5-methyluridylyl-(3'→5'O,O-thiophosphoryl)-2'-O-methoxyethylguanylyl-(3'→5'O,O-thiophosphoryl)-2'-O-deoxyadenylyl-(3'→5'O,O-thiophosphoryl)-2'-O-deoxyguanylyl-(3'→5 'O,O-thiophosphoryl)-thymidyl-(3'→5'O,O-thiophosphoryl)-2'-deoxy-5-methylcytidylyl-(3'→5'O,O-thiophosphoryl)-thymidyl-(3'→5'O,O-thiophosphoryl)-2'-deoxyguanylyl-(3'→5'O,O-thiophosphoryl)-thymidyl-(3'→5'O,O-thiophosphoryl)-thymidyl-(3'→5'O,O-thiophosphoryl)-thymidyl-(3'→5'O,O-thiophosphoryl)- thiophosphoryl)-thymidylyl-(3'→5'O,O-thiophosphoryl)-2'-O-methoxyethyl-5-methyluridyl-(3'→5'O,O-thiophosphoryl)-2'-methoxyethyl-5-methylcytidyl-(3'→5'O,O-thiophosphoryl)-2'-methoxyethyl-5-methylcytidyl-(3'→5'O,O-thiophosphoryl)-2'-methoxyethyl-5-methylcytidyl-(3'→5'O,O-thiophosphoryl)-2'-O-methoxyethyl-5-adenyl Uridyl-(3'→5'O,O-thiophosphoryl)-2'-O-methoxyethyl-5-methyluridyl-(3'→5'O,O-thiophosphoryl)-2'-O-methoxyethyl-5-methyluridyl-(3'→5'O,O-thiophosphoryl)-2'-O-methoxyethyl-5-methylcytosine (3'→5'O,O-thiophosphoryl)-2'-O-methoxyethyl-5-methyluridyl-19 sodium salt.

[0193] Oligonucleotides can be synthesized by a multistep process that can be divided into two different operations: solid phase synthesis and downstream processing. In the first operation, the nucleotide sequence of the oligonucleotide is assembled by a computer-controlled solid phase synthesizer. Subsequent downstream processing includes a deprotection step, preparative reverse phase chromatography purification, separation and drying to obtain the oligonucleotide drug substance. The chemical synthesis of oligonucleotides utilizes phosphoramidite coupling chemistry, followed by oxidative sulfurization, and involves sequential coupling of activated monomers with extended oligomers, the 3'-end of the extended oligomer being covalently attached to a solid support.

[0194] Detritylation (reaction a)

[0195] Each cycle of solid phase synthesis begins with the removal of the acid-labile 5'-O-4,4'-dimethoxytrityl (DMT) protecting group of the 5' terminal nucleoside of the support-bound oligonucleotide. This is accomplished by treatment with an acid solution (e.g., dichloroacetic acid (DCA) in toluene). After detritylation, excess reagents are removed from the support by washing with acetonitrile to prepare for the next reaction.

[0196] Coupling (reaction b)

[0197] Chain extension is achieved by reacting the 5'-hydroxyl group of the support-bound oligonucleotide with a phosphoramidite solution corresponding to the specific base position (e.g., for base 2: MOE-MeC amide) in the presence of an activator (e.g., 1H-tetrazole). This results in the formation of a phosphite triester bond between the introduced nucleotide synthon and the support-bound oligonucleotide chain. After the coupling reaction, excess reagents are removed from the support by washing with acetonitrile in preparation for the next reaction.

[0198] Sulfurization (reaction c)

[0199] The newly formed phosphite triester bonds are converted to the corresponding [O,O,O)-trialkylthiophosphoric acid triesters by solution treatment with a sulfur transfer reagent such as phenylacetyl disulfide. After sulfurization, excess reagents are removed from the support by washing with acetonitrile in preparation for the next reaction.

[0200] Wearing a hat (reaction d)

[0201] A small proportion of the 5'-hydroxyl groups available in any given cycle fail to be extended. Coupling of these groups in any subsequent cycle will lead to the formation of process-related impurities ("DMT-on(nl)-mers") that are difficult to separate from the desired product. To prevent the formation of these impurities and to facilitate purification, "capping agents" (e.g., acetic anhydride and N-methylimidazole / acetonitrile / pyridine) are introduced into the reactor vessel to obtain capped sequences. The resulting failed sequences ("DMT-off shortmers") are separated from the desired product by reverse phase HPLC purification. After the capping reaction, excess reagents are removed from the support by washing with acetonitrile in preparation for the next reaction.

[0202] Repeating this basic four-step cycle using the appropriate protected nucleoside phosphoramidites can assemble the entire protected oligonucleotide sequence.

[0203] Backbone deprotection (reaction e)

[0204] After the assembly portion of the process is complete, the cyanoethyl group protecting the (O,O)-trialkylphosphorothioate triester internucleotide linkage is removed by treatment with a solution of triethylamine (TEA) in acetonitrile. The reagents and acrylonitrile generated during this step are removed by washing the column with acetonitrile.

[0205] Cleavage from the support and base deprotection (reaction f)

[0206] Deprotection of the exocyclic amino group and cleavage of the crude product from the support are achieved by incubation with aqueous ammonium hydroxide (reaction f). The crude product is purified by reverse phase HPLC to obtain a 5'-O-DMT protected product. The reverse phase HPLC step removes the DMT-off fault sequence. The elution curve is monitored by UV absorption spectroscopy. The fractions containing the DMT-on oligonucleotide product are collected and analyzed.

[0207] Acidic deprotection (reaction g)

[0208] Collect the reversed-phase HPLC fractions of the oligonucleotides containing 5'-O-DMT protection and transfer to a precipitation tank. The products obtained from the purification of several synthesis will be merged at this stage of the process. The DMT-on oligonucleotides of purification are treated with acid (e.g., acetic acid) to remove the DMT group connected to the 5' end. After the acid exposure prescribed time and neutralization, separate and dry the oligonucleotide drug substance.

[0209] After the final acidic deprotection step, the solution is neutralized by adding aqueous sodium hydroxide and the oligonucleotide drug substance is precipitated from the solution by adding ethanol. The precipitated material is allowed to settle at the bottom of the reaction vessel and the ethanol supernatant is decanted. The precipitated material is redissolved in pure water and the solution pH is adjusted to pH 7.2-7.3. The precipitation step is repeated. The precipitated material is dissolved in water and the solution is filtered through a 0.45 micron filter and transferred to a disposable polypropylene dish, which is then loaded into a lyophilizer. The solution is cooled to -50°C. The primary drying is performed at 25°C for 37 hours. The temperature is raised to 30°C and the secondary drying step is performed for 5.5 hours. After the lyophilization process is completed, the drug is transferred to a high-density polyethylene bottle and stored at -200°C.

[0210] Target nucleic acid

[0211] "Targeting" an antisense compound to a specific nucleic acid can be a multi-step process. The process generally begins with identifying a target nucleic acid whose function is to be modulated. In the context of the present invention, the target nucleic acid encodes the α4 integrin chain of VLA-4 or α4β7 integrin.

[0212] The targeting process also generally includes determining at least one target region, fragment or site in the target nucleic acid, so that antisense interactions occur, thereby producing a desired effect, such as inhibiting expression. As used herein, the term "region" is defined as a portion of a target nucleic acid having at least one identifiable structure, function or feature. Within the region of the target nucleic acid is a fragment. A "fragment" is defined as a smaller or sub-portion of a region within the target nucleic acid. As used herein, a "site" refers to a position within the target nucleic acid.

[0213] Since the "translation start codon" is usually 5'-AUG (in the transcribed mRNA molecule; 5'-ATG in the corresponding DNA molecule), the translation start codon is also called "AUG codon", "start codon" or "AUG start codon". A few genes have translation start codons whose RNA sequences are 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to play a role in vivo. Therefore, the terms "translation start codon" and "start codon" can cover many codon sequences, even though the starting amino acid is usually methionine (in eukaryotes) or formylmethionine (in prokaryotes) in each case. It is also known in the art that eukaryotic and prokaryotic genes can have two or more optional start codons, any of which can be preferentially used for translation initiation in a particular cell type or tissue or under a particular set of conditions. As used herein, the terms "start codon" and "translation start codon" refer to one or more codons used in vivo to initiate translation of mRNA transcribed from a gene encoding, for example, the α4 integrin chain of VLA-4 or the α4β7 integrin, regardless of the sequence of these codons.

[0214] A "translation stop codon", also referred to as a "stop codon", may have one of three RNA sequences: 5'-UAA, 5'-UAG, and 5'-UGA (5'-TAA, 5'-TAG, and 5'-TGA, respectively, in the corresponding DNA molecule). As used herein, the terms "translation stop codon" and "stop codon" refer to one or more codons used in vivo to terminate the translation of mRNA transcribed from a gene encoding the α4 integrin chain or α4β7 integrin of VLA-4, regardless of the sequence of these codons.

[0215] The terms "start codon region" and "translation start codon region" refer to a portion of an mRNA or gene that includes about 25 to about 50 consecutive nucleotides in either direction (i.e., 5' or 3') starting from the translation start codon. Similarly, the terms "stop codon region" and "translation stop codon region" refer to a portion of an mRNA or gene that includes about 25 to about 50 consecutive nucleotides in either direction (i.e., 5' or 3') ending from the translation start codon. Therefore, the "start codon region" or "translation start codon region" and the "stop codon region" or "translation stop codon region" are all regions that can be effectively targeted with the antisense compounds of the present invention.

[0216] "Open reading frame" (ORF) or "coding region" is known in the art to refer to the region between the translation start codon and the translation stop codon, which is also a region that can be effectively targeted. In one embodiment, the intragenic region including the translation start or stop codon of the gene ORF is targeted.

[0217] Other target regions include 5' untranslated regions (5'UTR), which are known in the art to refer to the portion of mRNA in the 5' direction from the translation start codon, thus including the nucleotides between the 5' cap site of the mRNA and the translation start codon (or the corresponding nucleotides on the gene), and 3' untranslated regions (3'UTR) known in the art refer to the portion of mRNA in the 3' direction from the translation stop codon, thus including the nucleotides between the translation stop codon and the 3' end of the mRNA (or the corresponding nucleotides on the gene). The 5' cap site of the mRNA includes an N7-methylated guanosine residue, which is joined to the residue closest to the 5' end of the mRNA by a 5'-5' triphosphate bond. It is believed that the 5' cap region of the mRNA includes the 5' cap structure itself, and the first 50 nucleotides adjacent to the cap site. In one embodiment, the 5' cap region is targeted.

[0218] Although some eukaryotic mRNA transcripts are directly translated, many eukaryotic mRNA transcripts contain one or more regions called "spacers", which are excised from the transcript before translation. The remaining (thus translated) regions are called "exons" and spliced ​​together to form a continuous mRNA sequence. The mRNA transcripts produced by the process of splicing two (or more) mRNAs from different gene sources are called "fusion transcripts". In one embodiment, introns or splice sites are targeted, i.e., intron-exon junctions or exon-intron junctions, or abnormal fusion junctions caused by rearrangement or deletion. Alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally referred to as "variants".

[0219] "Pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in the start or end position and contain intron and exon sequences. When one or more exon or intron regions or parts thereof are removed during splicing, the pre-mRNA variant produces a smaller "mRNA variant". Therefore, mRNA variants are processed pre-mRNA variants, and each unique pre-mRNA variant must always produce a unique mRNA variant due to splicing. These mRNA variants are also called "alternative splice variants". If splicing of the pre-mRNA variant does not occur, the pre-mRNA variant is the same as the mRNA variant.

[0220] Transcription can be started or stopped by using an alternate signal, i.e., by using an alternate start codon or stop codon to produce a variant. Variants originating from a pre-mRNA or mRNA using an alternative start codon are referred to as "alternative start variants" of the pre-mRNA or mRNA. Those transcripts using alternative stop codons are referred to as "alternative termination variants" of the pre-mRNA or mRNA. A particular type of alternative termination variant is a "poly A variant", wherein the multiple transcripts produced are produced by the alternative selection of one of the "poly A termination signals" by the transcription machinery, thereby producing transcripts that terminate at unique poly A sites. In one embodiment, the targeted pre-mRNA or mRNA variant.

[0221] The location on the target nucleic acid to which the antisense compound hybridizes is referred to as a "target fragment." As used herein, the term "target fragment" is defined as at least an 8-nucleobase portion of the target region to which the antisense compound is targeted. While not wishing to be bound by theory, it is currently believed that these target fragments represent portions of the target nucleic acid that are available for hybridization.

[0222] Once one or more target regions, segments or sites are identified, antisense compounds are selected that are sufficiently complementary to the target segment, ie, that hybridize sufficiently well and have sufficient specificity to produce the desired effect.

[0223] The target fragment can also be combined with its corresponding complementary antisense compound to form a stable double-stranded (double-stranded) oligonucleotide. Such double-stranded oligonucleotide portions have been shown in the art to adjust target expression and regulate translation and RNA processing by antisense mechanisms. In addition, the double-stranded portion can be chemically modified (Fire et al., 1998; Timmons and Fire, 1998; Timmons et al., 2001; Tabara et al., 1998; Montgomery et al., 1998; Tuschl et al., 1999; Elbashir et al., 2001a; Elbashir et al., 2001b). For example, it has been shown that such a double-stranded portion inhibits the target by classical hybridization of the antisense strand of the duplex with the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., 2002).

[0224] Composition

[0225] The antisense compounds of the invention may be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures or mixtures of compounds to produce, for example, liposomes, receptor targeting molecules, oral, rectal, topical or other formulations to aid in uptake, distribution and / or absorption. Representative U.S. patents that teach the preparation of such absorption, distribution and / or absorption-aiding formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921, 5,354,844, 5,416,016, 5,459,127, 5,521,291, 5,543,158, 5,547,932, 5,583,020, 5,591,721, 4,426,330, 4,534,899, 5,013,556, 5,108,921, 5,213,804, 5,227,170, 5,264,221, 5,356,633, 5,395,619, 5,416,016, 5,5 5,417,978, US 5,462,854, US 5,469,854, US 5,512,295, US 5,527,528, US 5,534,259, US 5,543,152, US 5,556,948, US 5,580,575 and US 5,595,756.

[0226] The antisense compounds of the present invention can be administered in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a molecular entity that does not produce allergic, toxic or other adverse reactions when administered to a subject (particularly a mammal, more particularly a human). A pharmaceutically acceptable carrier can be solid or liquid. Useful examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, excipients, suspending agents, buffers, lubricants, adjuvants, vehicles, emulsifiers, absorbents, dispersion media, coatings, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetrants, chelating agents, isotonic agents and absorption delay agents that do not affect the activity of the activating agent of the present invention.

[0227] In one embodiment, the pharmaceutical carrier is water for injection (WFI) and the pharmaceutical composition is adjusted to pH 7.4, 7.2-7.6.

[0228] In one embodiment, the salt is a sodium salt or a potassium salt.

[0229] An oligonucleotide may contain chiral (asymmetric) centers, or the molecule as a whole may be chiral. Individual stereoisomers (enantiomers and diastereomers) and mixtures of these are within the scope of the present invention.

[0230] The antisense compounds of the present invention may be pharmaceutically acceptable salts, esters, or salts of esters, or any other compound that is capable of providing (directly or indirectly) a biologically active metabolite upon administration.

[0231] As used herein, the term "pharmaceutically acceptable salt" refers to physiologically and pharmaceutically acceptable salts of antisense compounds that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects when administered. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860.

[0232] The antisense compounds of the present invention may be prodrugs or pharmaceutically acceptable salts of prodrugs, or other bioequivalents. The term "prodrug" as used herein refers to a therapeutic agent prepared in an inactive form, which is converted into an active form (i.e., a drug) by the action of endogenous enzymes or other chemicals and / or conditions when administered. In particular, the prodrug form of the antisense compounds of the present invention is prepared as a SATE [(S-acetyl-2-thioethyl) phosphate] derivative according to the methods disclosed in WO 93 / 24510, WO 94 / 26764 and US 5,770,713.

[0233] Prodrugs can be converted into their active forms that have medical effects, for example, in vivo (e.g., hydrolyzed in the blood). Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Volume 14 of the ACSSymposium Series (1976); "Prodrug Design", ed., H. Bundgaard, Elsevier, 1985; and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein by reference. Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are called "solvates". For example, complexes with water are called "hydrates".

[0234] Conventional treatment

[0235] Corticosteroid therapy is the mainstay of outpatient DMD therapy. "Corticosteroid" refers to any of several synthetic or naturally occurring substances that have the general chemical structure of a steroid that simulates or enhances the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including prednisolone's precursors prednisone, methylprednisolone), dexamethasone triamcinolone, budesonide, and betamethasone.

[0236] In one embodiment, the present invention is directed to the treatment of MD in a human subject suffering from MD, comprising administering to the subject an effective amount of a therapeutic agent, such as an antisense oligonucleotide to CD49d (the α chain of VLA-4), and further comprising administering to the subject an effective amount of a second drug, i.e., a corticosteroid. In one embodiment, the corticosteroid is prednisone (or prednisone equivalent), deflazacort (prednisolone derivative). As above, other corticosteroids are known in the art.

[0237] Combined administration herein includes both co-administration using separate formulations (or separate pharmaceutical formulations) and consecutive administration in either order, wherein there is generally a time period while both (or all) active agents simultaneously exert their biological activities.

[0238] DMD patients are treated with standard doses of corticosteroids when they are able to walk, as this has been shown to have a certain effect on the walking ability of some patients. However, long-term treatment with standard doses (0.75mg / kg / day prednisone or 0.9mg / kg / day focort) can lead to muscle atrophy and / or have other side effects. For DMD patients who cannot walk and continue to use CS, there is no standard of care, sometimes a fixed dose of CS used when they lose the ability to walk, that is, a reduced mg / kg / day dose of CS, or they may stop CS treatment because of side effects and / or lack of benefit. As proposed and shown herein, CD49d antisense therapy is combined with corticosteroid therapy, including reducing corticosteroid treatment levels, reducing or delaying the progression of muscle function in subjects who cannot walk. It is unclear whether CS treatment provides any benefit in such subjects, so this supports the treatment of ATL1102 monotherapy or combination.

[0239] As used herein, the term "combination" in the context of administering a therapy refers to the use of more than one therapy or therapeutic agent. The use of the term "combination" does not limit the order in which the therapy or therapeutic agent is administered to the subject. The therapy or therapeutic agent can be administered before, simultaneously with, or after the second therapy or therapeutic agent is administered to the subject.

[0240] Drug administration

[0241] In one embodiment, the antisense compounds of the invention are administered systemically. As used herein, "systemic administration" is an enteral or parenteral route of administration.

[0242] As used herein, "enteral" refers to forms of administration involving any part of the gastrointestinal tract, and includes oral administration of antisense oligonucleotides, for example, in the form of tablets, capsules, or drops; gastric feeding tubes, duodenal feeding tubes, or gastrostomy; and rectal administration of antisense compounds, for example, in the form of suppositories or enemas.

[0243] As used herein, "parenteral" includes administration by injection or infusion. Examples include, intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), subcutaneous (under the skin), intraosseous infusion (into the bone marrow), intradermal (into the skin itself), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical (infusion into the bladder), transdermal (diffusion through intact skin), transmucosal (diffusion through mucous membranes), inhalation.

[0244] In one embodiment, administration of the pharmaceutical composition is subcutaneous.

[0245] The antisense compounds can be administered as a single dose or in repeated cycles, for example, daily, once every two days, every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen days, once a week, twice a week, three times a week, or every two weeks or every three weeks.

[0246] In one embodiment, administration is performed 1 to 3 times per week, or once a week, once every two weeks, once every three weeks, once every four weeks, or once every two months.

[0247] In one embodiment, administration is once a week.

[0248] In one embodiment, low doses are administered for 3 to 6 months, such as about 25-50 mg / week, for at least 3 to 6 months, then up to 12 months and long term.

[0249] Exemplary dosages are between about 10-300 mg. Exemplary dosages include 25, 50, 100, 150, 200 mg. Exemplary dosages include 1.5 mg / kg (about 50-100 mg) and 3 mg / kg (100-200 mg) and 4.5 mg / kg (150-300 mg). In one embodiment, the dosage is administered once a week. Therefore, in one embodiment, a low dose of about 10-30, or 20-40, or 20-28 mg can be given to a subject whose body weight is usually between about 25-65 kg. In one embodiment, the antisense oligonucleotide is administered at a dosage of less than 50 mg, or less than 30 mg, or about 25 mg per dose to produce a therapeutic effect. In one embodiment, the antisense oligonucleotide ATL1102 is administered at a dosage of less than 50 mg, or less than 30 mg, or about 25 mg per dose to produce a therapeutic effect.

[0250] In one embodiment, a therapeutic effect such as a delay in progression is seen within about three months after the first dose of the drug is administered. As used herein, the term "therapeutically effective amount" refers to a dose of an antisense compound that is sufficient, for example, to improve one or more markers, signs or symptoms of muscular dystrophy in a subject or delay the progression of muscular dystrophy in a subject, or to improve one or more markers, signs or symptoms of muscular dystrophy muscle fibers in a subject or delay the progression of muscular dystrophy in a subject under the conditions of administration.

[0251] In another embodiment, the administration is effective to provide a Cmax of the oligonucleotide in the plasma of a human subject of greater than 2890 ng / mL, and in one embodiment, about 10,000-11,000 ng / mL.

[0252] In another embodiment, the administration is effective to provide a Cmin or Ctrough of at least 2.5 ng / mL, in one embodiment at least 20 ng / mL, or at least 45 ng / mL of the oligonucleotide in the plasma of the human subject.

[0253] Studies were conducted to demonstrate that treatment of muscular dystrophy with inhibitory oligonucleotides of the VLA-4 integrin reduced the level of VLA-4 in the blood or muscle of human subjects. The reduction in VLA-4 levels can be detected in cell subsets of one or more organs, including blood, muscle, or lungs. In one embodiment, the subject takes a corticosteroid about 24 hours prior to administration.

[0254] This allowed the evaluation of the effects of inhibitory antisense oligonucleotides to the CD49d alpha chain of the VLA-4 integrin in immune cells in the absence of corticosteroids, which do not reach significant levels in the bloodstream 24 hours after administration, to exert an effect on circulating immune cells.

[0255] Example 1

[0256] In one embodiment, ATL1102 is administered weekly at about 1.5 mg / kg (about 50-100 mg) and 3 mg / kg (100-200 mg) and 4.5 mg / kg (150-300 mg) to 10 years old or older non-ambulatory adolescent (or puberty) boys with DMD for up to 12 weeks. The oligonucleotides administered are measured, for example, for the effects on motor / muscle function and inflammatory markers. Markers of muscle degeneration-regeneration and fibrosis are also assessed. The markers can be detected in situ or in samples, such as plasma, urine or muscle biopsies. Inspiratory and expiratory pressures, maximum cough flow, and forced vital capacity (FVC) are assessed to assess changes in respiratory function. Muscle function is assessed using standardized upper limb accessible surface area percentage change, upper limb function assessment score percentage change, and patient-reported outcome measurement upper limb functional capacity score change percentage (PROM-UL). Quality of life questionnaires can be used to determine the efficacy of treatment.

[0257] Corticosteroids can be given daily, or less frequently. Prednisolone can be given at 0.75 mg / kg / day and deflazacort can be given at 0.9 mg / kg / day as standard therapy for ambulatory DMD patients, at two-thirds, half, or one-third of the standard dose.

[0258] Example 2

[0259] In one embodiment, ATL1102 is administered weekly at about 1.5 mg / kg (about 10-100 mg) and 3 mg / kg (about 20-200 mg) and 4.5 mg / kg (30-300 mg) to ambulatory pediatric boys aged 4-11 with DMD for up to 12 weeks. The effects of the administered oligonucleotides, for example, on motor / muscle function and inflammatory markers are determined. Ambulatory pediatric boys may be in good walking condition or poor walking condition. Maintaining or reducing the loss of walking ability can be assessed by methods known to those skilled in the art.

[0260] Example 3

[0261] In one embodiment, 10 non-ambulatory DMD patients aged 12 to 18 years received ATL1102 at a starting dose of 3 mg / kg once a week for 4 weeks. The first 5 patients continued to take the drug at 3 mg / kg / week for 4 weeks, and the remaining 5 patients increased their dose to 4.5 mg / kg / week (twice a week, 2.25 mg / kg per week) for a total of 4 weeks. After 8 weeks of treatment, 4 weeks of monitoring were performed. During the treatment and monitoring period, assessments were performed at baseline, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks and 12 weeks, respectively. The main active outcomes were the number and safety of circulating lymphocytes, CD4+ and CD8+ T cells, and hi CD49d T cells evaluated at 4 and 8 weeks of treatment, including injection site reactions, platelet changes, liver enzymes GGT-bilirubin, CRP and albumin, and changes in A / G ratio. Secondary endpoint clinical assessments are measures of strength, upper limb function, functional capacity, quality of life, and respiratory markers, MRI assessments of muscle fibrosis, adipose inflammation edema, and atrophy, and pharmacokinetics. Exploratory outcome measures will include serum / plasma biomarker responses, such as those associated with muscle inflammation, muscle fibrosis, muscle apoptosis / degeneration, and muscle regeneration (including cytokines), as well as proteomics and monocyte RNA arrays and exosomal RNA.

[0262] Example 4

[0263] Low-dose administration of inhibitory oligonucleotides

[0264] In one embodiment, 9 DMD patients aged 10-18 years and weighing 25-60 kg who could not walk received ATL1102 at a starting dose of 25 mg once a week for 24 weeks. After 24 weeks of treatment, 8 weeks of monitoring were performed. During the treatment and monitoring period, assessments were performed at baseline, every 2 weeks during treatment, and every 4 weeks during post-treatment monitoring. The main active outcomes were the number and safety of circulating lymphocytes, CD4+ and CD8+ T cells, and hi CD49d T cells evaluated at 4 and 8 weeks of treatment, including injection site reactions, platelet changes, liver enzymes GGT-bilirubin, CRP and albumin, and changes in A / G ratio. The secondary endpoints of the clinical assessment were to measure strength, upper limb function measured by myoset, functional capacity measured by PUL-2 (performance of the DMD 2.0 upper limb module), quality of life, respiratory markers, and MRI assessments of muscle fibrosis, fat inflammation-edema, atrophy, and pharmacokinetics. Exploratory outcome measures will include serum / plasma biomarker responses, such as those associated with muscle inflammation, muscle fibrosis, muscle apoptosis / degeneration, and muscle regeneration (including cytokines, one or more of which may be markers of muscle damage), as well as proteomics and monocyte RNA arrays and potentially exosomal RNA.

[0265] Example 5

[0266] result

[0267] A 13-year-old, 62kg, non-ambulatory subject was treated with a low dose of 25mg / week of ATL1102 and a 30mg daily dose of corticosteroid (CS) Deflazacort (0.48mg / kg / day) for 12 weeks. ATL1102 effectively reduced the number of circulating CD8+ cells and CD8+ cells expressing CD49d at high levels per microliter at baseline (week 1) before daily CS administration in patients, and reduced muscle damage markers measured by biochemical exploratory markers, muscle strength measured by myoset, and important functions measured by PUL-2. The subject lost the ability to walk about 2.5 years ago, and when able to walk, Deflazacort was used at about 50% of the standard of care, 0.90mg / kg / day for subjects with DMD. When able to walk, the equivalent prednisolone dose used as standard care in DMD is 0.75mg / kg / day.

[0268] Immune cells

[0269] The effects of ATL1102 on immune cells were measured by flow cytometry and hematology at baseline (week 1), week 5 (3 days after dosing with ATL1103), week 8, and week 12 (7 days after dosing at weeks 8 and 12). The effects of ATL1102 on CD8+ cells were relatively selective compared to previous experience with higher doses of ATL1102 in multiple sclerosis (MS) (Limroth et al., 2014). These effects were also longer than the 3 days after dosing measured in previous MS studies, where effects were first shown 7 days after dosing with ATL1102. Certain immune cells were not affected at this dose and the timing of the assessments indicated that the ATL1102 effects were relatively more specific at this low dose. For example, in the MS study, there was no significant reduction in neutrophils or platelets observed at higher doses with ATL1102 (Limmroth et al.).

[0270] Myoset-Muscle Strength Data

[0271] Preliminary data from Myoset in this study for subject 1 showed a decrease in strength in his dominant hand after 12 weeks of treatment compared to baseline, but no decrease in thumb strength (measured in myopinch), nor was there a decrease in finger strength determined by moviplate after 12 weeks compared to baseline when the number of taps was the same. Data on the other hand showed no loss of hand or finger strength at week 12 compared to baseline, and thumb strength was numerically higher. Ricotti et al. (2016) looked at 15 patients (14 treated with CS) treated with CS and Myoset for 12 weeks or more and observed a mean trend of a 0.22kg decrease in myogrip, a 0.1kg decrease in myopinch, and an increase in moviplate compared to baseline.

[0272] PUL-2 - Functional Data

[0273] The PUL-2 for DMD is an updated version of the PUL-1 that measures upper extremity function. The PUL-2 measures higher level shoulder function with a maximum score of 12, moderate elbow function with a maximum score of 17, and distal wrist and hand function with a maximum score of 13. An entry score of 3 to 6 means (6 being the highest score and 1 being the lowest) that the subject's shoulder function can be assessed. Subject 1 had an entry level function of 5, and the recorded measurements indicated that he had been on medication for more than 12 weeks, with scores of 8 at baseline and 12 weeks, with no impairment in shoulder function. Subject 1 gained median elbow function at week 12 with a score of 14 compared to 13 at baseline, and gained distal wrist and hand function at week 12 with a score of 12 compared to 10 at baseline. Compared to baseline, the PUL-2 score was 34 at week 12 compared to 31 at baseline. The patient enrollment score also increased from a baseline level of 5 to a level of 6 consistent with the documented PUL-2 results.

[0274] Add-on therapy with ATL1102 helped preserve muscle strength as measured by myoset and appeared to maintain, if not improve, function as measured by PUL-2 in the subjects. ATL1102 therapy may therefore slow disease progression.

[0275] The results validated the use of CD49d (VLA-4 α chain) antisense nucleic acid and ATL1102 to specifically treat DMD patients, as well as the use of CS alone or in combination to delay disease progression in muscular dystrophy.

[0276] Exploratory pharmacodynamic outcome measure of muscle damage

[0277] Creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are indicators of muscle damage in boys with DMD, primarily related to low or absent dystrophin levels and damage to the muscle during contraction, and secondarily to inflammation and other downstream damage to the muscle. Creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are indicators of muscle damage in young ambulatory DMD patients, who have more muscle mass and inflammation compared with nonambulatory patients.

[0278] Nevertheless, blood and serum samples were collected to investigate changes in markers of muscle damage in Subject 1. In Subject 1, CK, ALT, AST, and LDH decreased at Weeks 8 and 12 compared to Baseline and Week 5. Baseline / Week 5 levels (units / liter) of CK, ALT, AST, and LDH were 5860 / 6881, 304 / 404, not tested / 184, and 632 / 681, respectively, compared to Week 8 / 12 levels of 4606 / 5358, 265 / 250, 116 / 134, and 564 / 498. The LDH level decreased from levels considered high to within the normal range. This suggests that signs of muscle damage associated with loss of dystrophin or inflammation or other damage have been reduced in this patient who was unable to walk.

[0279] Many modifications are known to those skilled in the art.

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[0304] S. Sanghvi, Chapter 15: Antisense Research and Applications, pp. 289-302, ST Crooke, B. Lebleu (eds.), CRC Press, 1993.

Claims

1. Use of a therapeutically effective amount of an oligonucleotide comprising the following structure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving one or more markers, signs or symptoms of muscular dystrophy in a subject or delaying the progression of muscular dystrophy in a subject: 5'- Me C Me UG AGT Me CTG TTT Me HE Me C Me CA Me HE Me HE Me C Me The 3' in, a) each of the 19 nucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides at positions 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides at positions 13-20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; and e) All cytosines are 5-methylcytosine ( Me C).

2. Use of a therapeutically effective amount of an oligonucleotide comprising the following structure or a pharmaceutically acceptable salt or stereoisomer thereof in the preparation of a medicament for improving one or more markers, signs or symptoms of muscular dystrophy muscle fibers in a subject or delaying the progression of muscular dystrophy in a subject: 5'- Me C Me UG AGT Me CTG TTT Me HE Me C Me CA Me HE Me HE Me C Me The 3' in, a) each of the 19 nucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides at positions 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides at positions 13-20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; and e) All cytosines are 5-methylcytosine ( Me C) and wherein the therapeutically effective amount is 25 mg to 100 mg; and wherein the medicament is used in combination with corticosteroid therapy.

3. The use according to claim 1 or 2, wherein The drug is formulated for periodic administration, once, twice or three times a week, every two weeks, or a month.

4. The use according to any one of claims 1 to 3, wherein The therapeutically effective amount is 10 mg to 300 mg.

5. The use according to any one of claims 1 to 3, wherein The therapeutically effective amount is selected from 10 mg to 25 mg, 25 mg to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, and 150 mg to 300 mg.

6. The use according to any one of claims 1 to 5, wherein The oligonucleotide is a sodium salt or a potassium salt.

7. The use according to any one of claims 1 to 6, wherein The pharmaceutical carrier was water for injection WFI and the composition was adjusted to pH 7.2-7.

6.

8. The use according to any one of claims 1 to 7, wherein The muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy (LGMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD including Fukuyama congenital MD and congenital MD with myosin deficiency), facioscapulohumeral, oculopharyngeal, Emery-Dreifuss and distal muscular dystrophy.

9. The use according to claim 8, wherein The muscular dystrophy involves mutations in the dystrophin gene.

10. The use according to any one of claims 1 to 9, wherein The subjects are diagnosed with muscular dystrophy and are either ambulatory or non-ambulatory.

11. The use according to claim 10, wherein The subjects are teenage or adolescent boys 10 years of age or older.

12. The use according to any one of claims 1 to 11, wherein The drug is administered subcutaneously.

13. The use according to any one of claims 1 to 12, wherein The drug is used in combination with corticosteroid therapy.

14. The use according to claim 13, wherein Corticosteroids are given in low doses.

15. The use according to claim 14, wherein Corticosteroid treatment is 10 to 40 mg of prednisolone or deflazacort.