SMAD7 inhibitory antisense oligonucleotides (ASOs) for treating pouchitis and methods of using them

Abstract

Methods for treating or preventing pouchitis are provided herein, comprising administering a SMAD7 antisense oligonucleotide or a pharmaceutical formulation comprising a SMAD7 antisense oligonucleotide. In certain embodiments, methods for treating pouchitis in an individual requiring treatment of pouchitis are described herein, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3').

Description

Technical Field

[0001] Cross-reference This application claims the benefit and priority of U.S. Patent Application No. 63 / 492,755, filed on March 28, 2023, which is hereby incorporated by reference in its entirety.

Summary of the Invention

Means for Solving the Problems

[0002] Summary A method for treating or preventing ileitis in an individual who needs it, the method comprising administering a therapeutically effective amount of an SMAD7 antisense oligonucleotide or a pharmaceutical formulation comprising an SMAD7 antisense oligonucleotide is provided herein.

[0003] In certain embodiments, a method for treating pouchitis in an individual requiring treatment for pouchitis is described herein, comprising administering to the individual a therapeutically effective amount of SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'). In some embodiments, all nucleoside bonds are O,O-linked phosphorothioate bonds, where X is 5-methyl2'-deoxycytidine. In some embodiments, the SMAD7 antisense oligonucleotide is administered enterally. In some embodiments, enteral administration is orally, sublingually, gastrically, or rectally. In some embodiments, enteral administration is rectally. In some embodiments, the SMAD7 antisense oligonucleotide is administered parenterally. In some embodiments, parenteral administration is intravenously, intratumorally, intrajejunally, intraileally, intracolonally, intrarectally, or intrasacral. In some embodiments, parenteral administration is intrasacral. In some embodiments, the therapeutically effective dose of SMAD7 ASO includes doses of approximately 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg, 300 mg, 320 mg, 340 mg, 360 mg, 380 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, or 500 mg. In some embodiments, the therapeutically effective dose of SMAD7 ASO includes doses ranging from approximately 20 mg to approximately 500 mg. In some embodiments, the therapeutically effective dose of SMAD7 ASO is administered approximately every 6 hours, every 12 hours, every 24 hours, every 48 hours, every 72 hours, daily, twice a week, once every two weeks, or once a month. In some embodiments, the therapeutically effective dose of SMAD7 ASO includes a dose of approximately 160 mg per day. In some embodiments, SMAD7 ASO downregulates intracellular SMAD7 mRNA and / or protein. In some embodiments, the downregulation of SMAD7 protein expression exceeds 40% compared to untreated cells.In some embodiments, at least one sample from an individual has an elevated SMAD7 level relative to a known control level, where the known control level is the SMAD7 level in a sample taken from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual without pouchitis. In some embodiments, pouchitis is acute pouchitis. In some embodiments, pouchitis is chronic pouchitis. In some embodiments, an individual is determined to have pouchitis using at least five modified pouchitis disease activity index (mPDAI) scores.

[0004] In certain embodiments, a method for treating pouchitis in an individual requiring treatment for pouchitis is described herein, comprising administering to the individual a pharmaceutical formulation comprising: a) a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'); and b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises 100 mmol to 5 moles of SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises 100 mmol to 2 moles, 100 mmol to 900 mmol, 300 mmol to 5 moles, 300 mmol to 2 moles, 300 mmol to 900 mmol, 900 mmol to 5 moles, or 900 mmol to 2 moles of SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises about 300 mmol of SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises about 900 mmol of SMAD7 ASO. In some embodiments, the pharmaceutical formulation contains about 2 moles of SMAD7 ASO. In some embodiments, the pharmaceutical formulation contains more than 300 mmol, more than 900 mmol, or more than 2 moles of SMAD7 ASO. In some embodiments, the pharmaceutical formulation is administered orally. In some embodiments, the pharmaceutical formulation contains about 0.5% to about 30% by weight of SMAD7 ASO; about 20% to about 50% by weight of mannitol; about 10% to about 30% by weight of microcrystalline cellulose; and an enteric coating comprising an ethyl acrylate-methacrylic acid copolymer. In some embodiments, the pharmaceutical formulation comprises an intra-granular phase containing a) i) about 5% to about 10% by weight of SMAD7 ASO; ii) about 40% by weight of mannitol; iii) about 8% by weight of microcrystalline cellulose; iv) about 5% by weight of hydroxypropyl methylcellulose; and v) about 2% by weight of sodium starch glycolate; b) an extra-granular phase containing i) about 17% by weight of microcrystalline cellulose; ii) about 2% by weight of sodium starch glycolate; iii) about 0.4% by weight of magnesium stearate; and c) an enteric coating containing an ethyl acrylate-methacrylic acid copolymer, where the weight percentages are the weight of the components relative to the total weight of the pharmaceutical formulation.In some embodiments, the pharmaceutical formulation comprises about 5% to about 30% by weight of SMAD7 ASO; about 20% to about 50% by weight of mannitol; about 10% to about 30% by weight of microcrystalline cellulose; about 0.5% to about 10% by weight of hydroxypropyl methylcellulose; about 0.5% to about 10% by weight of sodium starch glycolate; about 0.05% to about 1% by weight of magnesium stearate; about 0.5% to about 10% by weight of a moisture barrier film coating; and about 5% to about 20% by weight of a polymer coating. In some embodiments, the pharmaceutical formulation comprises about 8.5% by weight of SMAD7 ASO; about 40% by weight of mannitol; about 25% by weight of microcrystalline cellulose; about 5% by weight of hydroxypropyl methylcellulose; about 4% by weight of sodium starch glycolate; about 0.4% of magnesium stearate; about 4% by weight of a moisture barrier film coating; and about 10% to about 15% by weight of a polymer coating. In some embodiments, the pharmaceutical formulation comprises about 23% by weight of SMAD7 ASO; about 28% by weight of mannitol; about 25% by weight of microcrystalline cellulose; about 5% by weight of hydroxypropyl methylcellulose; about 4% by weight of sodium starch glycolate; about 0.4% of magnesium stearate; about 4% by weight of a moisture barrier film coating; and about 7% to about 12% by weight of a polymer coating. In some embodiments, the pharmaceutical formulation downregulates intracellular SMAD7 mRNA and / or protein. In some embodiments, the downregulation of SMAD7 protein expression exceeds 40% compared to untreated cells. In some embodiments, at least one specimen has elevated SMAD7 levels compared to a known control level, where the known control level is the SMAD7 level in a specimen taken before or during treatment for pouchitis, or the SMAD7 level in a specimen taken from a healthy specimen without pouchitis. In some embodiments, pouchitis is acute pouchitis. In some embodiments, pouchitis is chronic pouchitis.In some embodiments, individuals are determined to have pouchitis using at least five modified pouchitis disease activity index (mPDAI) scores. A method for predicting pouchitis in individuals requiring prediction of pouchitis includes determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, and the known control level is the SMAD7 level in a sample taken from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual without pouchitis.

[0005] In certain embodiments, a method for identifying an individual at risk of pouchitis is described herein, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual at risk of pouchitis, and the known control level is the level of SMAD7 in a sample taken from the individual before or during treatment for pouchitis, or the level of SMAD7 in a sample taken from a healthy individual without pouchitis. In some embodiments, the method comprises administering a pharmaceutical formulation containing SMAD7 ASO to the individual if the SMAD7 level is elevated relative to a known control level, or determining the level of SMAD7 in a second sample derived from the individual if the SMAD7 level is not elevated relative to a known control level. In some embodiments, the second sample is collected immediately after the first sample, approximately 1 hour later, approximately 3 hours later, approximately 6 hours later, approximately 12 hours later, approximately 1 day later, approximately 3 days later, approximately 1 week later, approximately 2 weeks later, approximately 1 month later, approximately 2 months later, approximately 3 months later, approximately 4 months later, approximately 5 months later, approximately 6 months later, approximately 7 months later, approximately 8 months later, approximately 9 months later, approximately 10 months later, approximately 11 months later, or approximately 12 months later.

[0006] In certain embodiments, a method for treating pouchitis in an individual requiring treatment for pouchitis is described herein, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, and the known control level is the level of SMAD7 in a sample taken from the individual before or during treatment for pouchitis, or the level of SMAD7 in a sample taken from a healthy individual not having pouchitis. In some embodiments, the SMAD7 ASO comprises a sequence selected from any one of SEQ ID NOs: 1-6 or a pharmaceutically acceptable salt thereof. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'). In some embodiments, all nucleoside bonds are O,O-linked phosphorothioate bonds, where X is 5-methyl2'-deoxycytidine. In some embodiments, the individual is human.

[0007] To help you understand this disclosure and demonstrate how it can be put into practice, embodiments are described here with reference to the attached drawings as merely non-limiting examples. [Brief explanation of the drawing]

[0008] [Figure 1] Figures 1A–1C show representative images of single and double immunofluorescence staining of fresh mucosal biopsy samples analyzed for SMAD7 expression (green) and DAPI (blue). Figure 1A shows staining of a section of healthy ileum. Figure 1B shows staining of a section of a non-inflamed cystic sac. Figure 1C shows staining of a section of an inflamed cystic sac. The scale bar shown in all panels is 50 μm. This figure is representative of three separate experiments that yielded similar results.

[0009] [Figure 2]Figures 2A and 2B show representative Western blot images of SMAD7 and β-actin in total protein extracted from mucosal biopsy samples, as well as a quantitative graphical analysis of the SMAD7 / β-actin ratio from Western blotting measured by densitometry scanning. Figure 2A shows Western blots (left) probed with SMAD7 (upper panel) and β-actin loading control (lower panel) of total protein extracted from mucosal samples of inflamed pouches (pouchitis, n=10), normal pre-pouch ileum (n=5) from the same patients with pouchitis, non-inflamed pouches (n=3), and normal controls (healthy ileum, n=4). The right panel of Figure 2A shows a quantitative analysis of the SMAD7 / β-actin ratio measured by densitometry scanning of the Western blots. Values ​​are expressed in arbitrary units (au) and represent the mean ± SEM of all samples (*p<0.05, **p<0.01, ***p<0.001). Figure 2B shows Western blots (left) probed with SMAD7 (upper panel) and β-actin loading control (lower panel) of total protein extracted from mucosal samples of inflamed pouches (pouchitis, n=10), inflamed ileum (anterior colitis of the pouch, n=3) from the same patients with pouchitis, non-inflamed pouches (n=5), and normal controls (healthy ileum, n=4). The right panel of Figure 2B shows a quantitative analysis of the SMAD7 / β-actin ratio measured by densitometry scanning of the Western blots. Values ​​are expressed in arbitrary units (au) and represent the mean ± SEM of all samples (*p<0.05, **p<0.01).

[0010] [Figure 3]Figures 3A and 3B show representative images of single and double immunofluorescence staining. Figure 3A shows images of fresh mucosal biopsy samples from patients with chronic pouchitis, analyzed for SMAD7 (green), CD3 (red), and DAPI (blue) expression. Figure 3B shows images of fresh mucosal biopsy samples from patients with chronic pouchitis, analyzed for SMAD7 (green), HLA-DRII (red), and DAPI (blue) expression. The scale bar shown in all panels is 75 μm. This figure is representative of three separate experiments that yielded similar results.

[0011] [Figure 4] Figures 4A-4B show the relative expression levels of various inflammatory and anti-inflammatory molecules in ex vivo organ cultures transfected for 24 hours with SMAD7 sense oligonucleotide (S) or SMAD7 antisense oligonucleotide (AS) (10 μg / ml). Figure 4A shows the relative expression of SMAD7 (y-axis of the graph) as assessed by real-time polymerase chain reaction of SMAD7 RNA transcripts. Levels were normalized to β-actin. Values ​​represent the mean ± SD of three experiments. Differences were analyzed using a two-sided Student's t-test (*p<0.005). Figure 4B shows the analysis of cell-free culture supernatants analyzed using a commercially available protein array kit. The right panel is a heatmap showing differential protein expression based on densitometry scanning of array blots. Log2 (fold change) of ex vivo organ cultures transfected with AS and S. [Modes for carrying out the invention]

[0012] Detailed explanation Pouchitis is inflammation of the lining of the pouch surgically created during the treatment of inflammatory bowel diseases such as ulcerative colitis. Pouchitis is the most common long-term complication of ileal pouch-anal anastomosis. Symptoms of pouchitis include increased bowel movement frequency, abdominal cramps or bloating, lower abdominal pain, or occasionally, bloody stools. Current treatments for pouchitis are limited and generally involve antibiotics.

[0013] Improved treatments are needed for the management or prevention of pouchitis. A method for managing or preventing pouchitis by administering an oligonucleotide (e.g., SMAD7 antisense oligonucleotide) is described herein.

[0014] definition The term "oligonucleotide" refers to a short DNA or RNA molecule. Oligonucleotides can be composed of 2'-deoxyribonucleotides (oligodeoxyribonucleotides), which can be modified at the phosphate backbone or the 2' sugar position.

[0015] As used herein, “antisense oligonucleotide” (“ASO”) refers to a short synthetic oligonucleotide sequence complementary to a messenger RNA (mRNA) encoding a target protein (e.g., SMAD7). While not bound by any particular theory, an antisense oligonucleotide sequence can hybridize with a complementary region within the mRNA molecule, thereby producing a double-stranded hybrid that can result in the activation of a universal catalytic enzyme such as RNase H, which degrades the DNA / RNA hybrid strand and thus inhibits protein translation. While not bound by any theory, the antisense oligonucleotides provided herein can hybridize with their target sequence as RNA or DNA. Therefore, even if a DNA sequence is provided as the target, the corresponding RNA sequence (containing uracil instead of thymine) is included as the ASO. “Phosphorothioate antisense oligonucleotide” or “PS ASO” is an antisense oligonucleotide modified to have a phosphorothioate backbone.

[0016] As used herein, “Mothers against decapentaplegic homolog 7” (also known as “SMAD7”, CRCS3, FLJ16482, MADH7, MADH8, MAD (Mothers against decapentaplegic, Drosophila) homolog 7, MAD homolog 8, SMAD, Maternal homolog 7 for DPP, Maternal homolog 8 for DPP) means either a human protein or mRNA transcript encoded by the gene identified by Entrez GeneID number 4092, and their allele variants.

[0017] As used herein, “SMAD7 antisense oligonucleotide” or “SMAD7 ASO” is understood to refer to an oligonucleotide containing a nucleic acid sequence complementary to the nucleic acid sequence in an mRNA molecule transcribed from the SMAD7 gene. More specifically, such an oligonucleotide may be complementary to the nucleic acid sequence in the coding region of such mRNA.

[0018] As used herein, the term “pouchitis” refers to a disease or disorder characterized by inflammation of the pouch. As used herein, the term “pouch” may refer to an artificial rectum surgically created from the ileum (the last part of the small intestine) in an individual who has undergone a colectomy or total colectomy (removal of the colon and rectum) to manage a disease such as ulcerative colitis, unspecified colitis, familial adenomatous polyposis, cancer, or other colitis. An individual may be identified as having pouchitis using the Pouchitis Ileal Disease Activity Index (PDAI) or Modified Pouchitis Ileal Disease Activity Index (mPDAI) scoring system. An individual assessed with a total PDAI score of 7 or higher is classified as having pouchitis. An individual assessed with a total mPDAI score of 5 or higher is classified as having pouchitis.

[0019] The term "acute ileitis" refers to ileitis in which the symptoms persist for a period of less than or equal to 4 weeks, and the disease responds to a 2-week course of antibiotics.

[0020] The term "chronic ileitis" refers to ileitis in which the symptoms persist for 4 weeks or longer and / or despite a standard course of antibiotic treatment.

[0021] The terms "recipient", "individual", "subject", "host", and "patient" are used interchangeably herein and in some embodiments to refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. For the purposes of treatment, a "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as laboratory animals, zoo animals, sport animals, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. None of these terms require the supervision of a medical professional.

[0022] The terms "disease", "disorder", and "condition" are used interchangeably herein.

[0023] As used herein, terms such as “treatment” and “to treat” mean administering a drug or performing a procedure for the purpose of obtaining an effect. This effect may be preventive in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it results in a partial or complete cure of the disease and / or its symptoms. As used herein, “treatment” may include treatment of a disease or disorder in mammals, in particular humans (e.g., pouchitis of the ileum), and may include (a) preventing the occurrence of a disease or its symptoms in a subject that may be susceptible to the disease but has not yet been diagnosed with it (e.g., including diseases that may be associated with or caused by a primary disease); (b) suppressing the disease, i.e., stopping the onset of the disease; and (c) reducing the disease, i.e., causing regression of the disease. Treatment may refer to any indication of success in treating, improving, or preventing cancer, and may include any objective or subjective parameters such as relief of symptoms; remission; reduction; or making the disease state more tolerable to the patient; slowing the rate of degeneration or decline; or making the final stages of degeneration less debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Thus, the term “treatment” includes the administration of any compound or agent of this disclosure to prevent, delay, alleviate, stop, or suppress the onset of symptoms or conditions associated with a disease (e.g., pouchitis). The term “therapeutic effect” refers to the reduction, elimination, or prevention of a disease, symptoms of a disease, or side effects of a disease in a subject.

[0024] As used herein, "preventing" or "to prevent" refers to reducing or eliminating the onset of symptoms or complications of a disease, condition, or disorder. The term "preventing" when used in connection with a condition such as inflammation or recurrence of ileitis is recognized in the art and refers to the ability of a formulation, composition, and / or device to reduce the frequency of signs and / or symptoms of a medical condition in a subject or delay its onset as compared to a subject not receiving the formulation, composition, and / or device. To the extent the methods of the present disclosure relate to preventing a disorder, it is understood that the term "preventing" does not require that the disease state be completely precluded.

[0025] The terms "therapeutically effective amount" or "therapeutic amount" generally refer to the amount of an antibody or drug effective to "treat" a disease or disorder in a subject or mammal. In some embodiments, the compositions described herein are administered to a subject in an amount effective to produce some desired therapeutic effect by suppressing the disease or disorder described herein at a reasonable benefit / risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that at least partially achieves the desired therapeutic or prophylactic effect in an organ or tissue. The amount of antibody or drug required to effect the prevention and / or therapeutic treatment of a disease or disorder is not fixed per se. In some embodiments, the amount of antibody or drug administered varies depending on the type of disease, the spread of the disease, and the size of the mammal afflicted with the disease or disorder. When used in conjunction with a method of treatment that includes administration of a therapeutic agent after a subject exhibits symptoms of a disease or disorder, the term "therapeutically effective" means that one or more signs or symptoms of the disease or disorder are improved or eliminated after treatment.

[0026] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0027] As used herein, all numbers or numerical ranges include all integers within or encompassing such a range, and fractions of values ​​or integers within or encompassing such a range, unless the context clearly indicates otherwise. Thus, for example, a reference to the range 90–100% includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and others. In another example, a reference to the range of 1 to 5,000 times includes 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, etc., and 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, etc., 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, etc., and others.

[0028] As used herein, "approximately" a number means the range that includes that number, and the range from 10% below that number to 10% above that number. "Approximately" range means the range from 10% below the lower limit of that range to 10% above the upper limit of that range.

[0029] Throughout this specification and the claims, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” mean, for example, other additives, components, integers, or steps, but are not intended to exclude them.

[0030] "Identity percentage (%)" refers to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same positions in their alignment. For example, "an amino acid sequence is X% identical to sequence number Y" refers to the identity percentage of the amino acid sequence with respect to sequence number Y, and explains that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure pertains. In case of any conflict, this specification shall prevail. Similar or equivalent methods and materials may be used in carrying out or testing the disclosure, but suitable methods and materials are described below.

[0032] SMAD7 oligonucleotide In certain embodiments, a method for treating or preventing pouchitis by administering a therapeutically effective amount of SMAD7 oligonucleotide (e.g., SMAD7 antisense oligonucleotide (ASO)) or a pharmaceutical formulation containing SMAD7 oligonucleotide is described herein. In some embodiments, the SMAD7 oligonucleotide is SMAD7 antisense oligonucleotide (ASO).

[0033] In some embodiments, SMAD7 ASO contains DNA. In some embodiments, SMAD7 ASO contains RNA.

[0034] In some embodiments, SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), where X is 5-methyl2'-deoxycytidine. In some embodiments, SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), where X is 5-methyl2'-deoxycytidine. In some embodiments, SMAD7 ASO has a sequence that follows SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), where X is 5-methyl2'-deoxycytidine.

[0035] In some embodiments, SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO: 2 (5'-GTXGCCCCTTCTCTCXGCAGC-3'), where X is 5-methyl-2'-deoxycytidine. In some embodiments, SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO: 2. In some embodiments, SMAD7 ASO has a sequence that follows SEQ ID NO: 2.

[0036] In some embodiments, SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO: 3 (5'-GTXYCCCCTTCTCCCXYCAG-3'), where X is a nucleotide containing a nitrogenous base of cytosine, 5-methylcytosine, or 2'-O-methylcytosine, and Y is a nucleotide containing a nitrogenous base of guanine, 5-methylguanine, or 2'-O-methylguanine, wherein at least one of X and Y contains a methylated nitrogenous base. In some embodiments, SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, SMAD7 ASO has a sequence that follows SEQ ID NO: 3.

[0037] In some embodiments, SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO: 4 (5'-GTXGCCCCTTCTCCCXGCAG-3'), where X is 5-methyl-2'-deoxycytidine. In some embodiments, SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, SMAD7 ASO has a sequence that follows SEQ ID NO: 4.

[0038] In some embodiments, SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO: 5 (5'-GTC*GCCCCTTCTCCCC*YCAGC-3'), where C* is 5-methyl-2'-deoxycytidine and Y is a nucleotide containing a nitrogenous base of guanine, 5-methylguanine, or 2'-O-methylguanine. In some embodiments, SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, SMAD7 ASO has a sequence that follows SEQ ID NO: 5.

[0039] In some embodiments, SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO: 6 (5'-GTC*GCCCCTTCTCTCC*YCAGC-3'), where C* is 5-methyl-2'-deoxycytidine and Y is a nucleotide containing a nitrogenous base of guanine, 5-methylguanine, or 2'-O-methylguanine. In some embodiments, SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO: 6. In some embodiments, SMAD7 ASO has a sequence that follows SEQ ID NO: 6.

[0040] In some embodiments, SMAD7 ASOs include naturally occurring nucleic acid bases, sugars, and covalent nucleoside-to-backbone (skeletal) bonds, as well as moieties that do not exist naturally. In some embodiments, the SMAD7 ASOs of the present disclosure, for example, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, include nucleotides comprising deoxycytidine and / or 5-methyl-2'-deoxycytidine, including, but not limited to, 5-methyl-2'-deoxycytidine 5'-monophosphate and 5-methyl-2'-deoxycytidine 5'-monophosphorothioate.

[0041] In some embodiments, SMAD7 ASO (e.g., SMAD7 ASO of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6) contains one or more methylated cytosines. In some embodiments, any cytosine in SMAD7 ASO is methylated. In some embodiments, at least one cytosine in SMAD7 ASO is replaced with 5-methylcytidine. In some embodiments, SMAD7 ASO of SEQ ID NO: 1 contains one or more methylated cytosines. In some embodiments, any cytosine in SMAD7 ASO of SEQ ID NO: 1 is methylated. In some embodiments, at least one cytosine in SMAD7 ASO of SEQ ID NO: 1 is replaced with 5-methylcytidine. In some embodiments, SMAD7 ASO of SEQ ID NO: 2 contains one or more methylated cytosines. In some embodiments, any cytosine in SMAD7 ASO of SEQ ID NO: 2 is methylated. In some embodiments, at least one cytosine in SMAD7 ASO of SEQ ID NO: 2 is replaced with 5-methylcytidine. In some embodiments, SMAD7 ASO of SEQ ID NO: 3 contains one or more methylated cytosines. In some embodiments, any cytosine in SMAD7 ASO of SEQ ID NO: 3 is methylated. In some embodiments, at least one cytosine in SMAD7 ASO of SEQ ID NO: 3 is replaced with 5-methylcytidine. In some embodiments, SMAD7 ASO of SEQ ID NO: 4 contains one or more methylated cytosines. In some embodiments, any cytosine in SMAD7 ASO of SEQ ID NO: 4 is methylated. In some embodiments, at least one cytosine in SMAD7 ASO of SEQ ID NO: 4 is replaced with 5-methylcytidine. In some embodiments, SMAD7 ASO of SEQ ID NO: 5 contains one or more methylated cytosines. In some embodiments, any cytosine in SMAD7 ASO of SEQ ID NO: 5 is methylated. In some embodiments, at least one cytosine in SMAD7 ASO of SEQ ID NO: 5 is replaced with 5-methylcytidine. In some embodiments, SMAD7 ASO of SEQ ID NO: 6 contains one or more methylated cytosines.In some embodiments, any cytosine in SMAD7 ASO of SEQ ID NO: 6 is methylated. In some embodiments, at least one cytosine in SMAD7 ASO of SEQ ID NO: 6 is replaced with 5-methylcytidine.

[0042] In some embodiments, SMAD7 ASO is chemically modified. In some embodiments, SMAD7 ASO contains one or more internucleoside linkers modified from natural phosphodiesters. In some embodiments, the internucleoside linkers of SMAD7 ASO, or all of its adjacent nucleotide sequences, are modified. For example, in some embodiments, the internucleoside linkage, such as a phosphorothioate internucleoside linkage, contains sulfur (S).

[0043] In some embodiments, SMAD7 ASO comprises modifications to ribose sugar or nucleic acid bases. In some embodiments, SMAD7 ASO comprises one or more nucleosides containing a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with hexose rings (HNAs), bicyclic rings having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNAs)), or unbound ribose rings typically lacking a bond between the C2 and C3 carbons (e.g., UNAs). In some embodiments, SMAD7 ASO comprises one or more LNAs. In some embodiments, the sugar-modified nucleoside comprises bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, the modified nucleoside includes a nucleoside in which the sugar moiety is replaced by a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0044] In some embodiments, SMAD7 ASO comprises one or more modified sugars. In some embodiments, the sugar modification includes modifications carried out by changing a substituent on the ribose ring to a group other than hydrogen, or to a 2'-OH group naturally found on DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', or 5' position, or a combination thereof. In some embodiments, the nucleoside having a modified sugar moiety includes 2'-modified nucleosides, e.g., 2'-substituted nucleosides. In some embodiments, the 2'-sugar-modified nucleoside is a nucleoside having a substituent other than -H or -OH at the 2' position (2'-substituted nucleoside), or includes a 2'-linked biradical, and includes 2'-substituted nucleosides and LNA (2'-4' biradical-bridged) nucleosides. Examples of 2'-substituted nucleosides include, but are not limited to, 2'-O-alkyl, 2'-O-methyl, 2'-alkoxy, 2'-O-methoxyethyl (MOE), 2'-amino, 2'-fluoro, and 2'-F-ANA nucleosides. In some embodiments, the modification at the ribose group includes the modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl). In some embodiments, SMAD7 ASO comprises one or more nucleosides having 2'-O-methyl (2'-OMe). In some embodiments, SMAD7 ASO comprises one or more nucleosides having 2'-O-methoxyethyl (MOE). In some embodiments, SMAD7 ASO comprises one or more nucleosides having 2'-O-ethyl (cEt).

[0045] In some embodiments, SMAD7 ASO contains a 2'-deoxyribonucleotide (oligodeoxyribonucleotide). In some embodiments, SMAD7 ASO contains a 2'-deoxyribonucleotide (oligodeoxyribonucleotide) that is modified at the phosphate backbone or at the 2' sugar position. In some embodiments, SMAD7 ASO is a phosphorothioate (PS) oligonucleotide in which one of the non-bridged oxygen atoms in the phosphate backbone is replaced with a sulfur atom. In some embodiments, SMAD7 ASO has at least one internucleoside bond that is an O,O-linked phosphorothioate bond (i.e., a phosphorothioate bond). In some embodiments, SMAD7 ASO has all internucleoside bonds that are O,O-linked phosphorothioate bonds (i.e., phosphorothioate bonds).

[0046] The phosphorothioate (PS) bond in oligonucleotides is chiral and has dextrorotatory (Rp) and levorotatory (Sp) isomers called diastereomers. These two PS diastereomers can have different effects on the physicochemical and biological properties of the oligonucleotide, such as nuclease sensitivity, thermodynamic stability of the double-stranded structure, and interactions with various enzymes involved in antisense and RNAi pathways. In some embodiments, SMAD7 ASO has one or more diastereomerically pure nucleoside bonds with respect to the chiral phosphorus. In some embodiments, SMAD7 ASO is prepared by using stereoselective oligonucleotide synthesis to form one or more pre-designed diastereomerically pure nucleoside bonds with respect to the chiral phosphorus.

[0047] In some embodiments, pharmaceutically acceptable salts of SMAD7 ASO are provided herein. In some embodiments, sodium salts of SMAD7 ASO of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 are provided herein, optionally containing 1 to 20 O,O-linked phosphorothioate nucleoside bonds (i.e., phosphorothioate bonds). In some embodiments, SMAD7 ASO is an antisense oligonucleotide comprising the free acid form, salt form, or an anionic form without a counterion of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, where each of the 20 nucleoside bonds is an O,O-linked phosphorothioate bond. In some embodiments, the phosphorothioate skeleton of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 is fully or partially protonated to form the acid form of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. The intended oligonucleotide salts include those that are completely neutralized, for example, each phosphorothioate bond is Na + It associates with ions such as . In some embodiments, the salts of SMAD7 ASO of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 are only partially neutralized, and for example, not all phosphorothioate bonds associate with ions (e.g., less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, or less than 1%).

[0048] In some embodiments, the SMAD7 ASO of this disclosure (e.g., SEQ ID NO: 1) or the pharmaceutical formulations disclosed herein (e.g., compositions comprising SEQ ID NO: 1) downregulate SMAD7 expression. In some embodiments, SMAD7 expression is the expression of SMAD7 protein and / or mRNA. In some embodiments, SMAD7 protein expression is downregulated in cells by more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, or more than about 65% compared to untreated cells. In some embodiments, SMAD7 protein expression is downregulated in cells by more than about 40% compared to untreated cells.

[0049] In some embodiments, SMAD7 ASO has unique functional properties that target the SMAD7 gene, its RNA or protein product, or another molecular entity whose activity or expression exclusively or with high specificity affects the activity or expression of SMAD7 or its product. In some embodiments, SMAD7 ASO reduces SMAD7 expression when introduced into cells (e.g., epithelial cells, mucosal lamina propria mononuclear cells, or PBMCs, dendritic cells, or immune cells such as B cells). In some embodiments, SMAD7 ASO reduces the expression of mRNA transcribed from the gene. In some embodiments, SMAD7 ASO reduces the expression of the protein encoded by the gene. In some embodiments, SMAD7 ASO reduces the secretion of the protein encoded by the gene from cells into which SMAD7 ASO has been introduced.

[0050] In some embodiments, SMAD7 ASO modulates inflammation. In some embodiments, SMAD7 ASO modulates the expression or activity of inflammatory genes. In some embodiments, SMAD7 ASO reduces the expression or activity of inflammatory genes. In some embodiments, SMAD7 ASO modulates the expression or activity of ENA-78, G-CSF, GM-CSF, GRO, GRO-alpha, I-309, IL-1-alpha, IL-1-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IFN-gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIG, MIP-1-delta, RANTES, SCF, SDF-1, TARC, TGF-beta-1, TNF-alpha, TNF-beta, EGF, IGF-1, angiogenin, oncostatin M, TPO-VEGF, PDGF-BB, leptin, or combinations thereof. In some embodiments, SMAD7 ASO reduces the expression or activity of ENA-78, G-CSF, GM-CSF, GRO-alpha, IL-1 beta, IL-10, IL-12, IL-13, IL-15, IFN-gamma, MIG, MIP-1-delta, RANTES, SCF, SDF-1, TARC, TGF-beta-1, TNF-alpha, angiogenin, oncostatin M, TPO, VEGF, PDGF-BB, leptin, or combinations thereof. In some embodiments, SMAD7 ASO increases the expression or activity of GRO, I-309, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, MCP-1, MCP-2, MCP-3, M-CSF, MDC, TNF-beta, EGF, IGF-1, or combinations thereof.

[0051] How to treat pouchitis In some embodiments, a method for treating pouchitis in an individual requiring treatment for pouchitis is described herein, comprising administering a therapeutically effective amount of SMAD7 antisense oligonucleotide (ASO) to the individual. Furthermore, in some embodiments, a method for treating pouchitis in an individual requiring treatment for pouchitis is described herein, comprising administering a therapeutically effective amount of SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3') to the individual. Furthermore, in some embodiments, a method for treating pouchitis in an individual requiring treatment for pouchitis is described herein, comprising administering a pharmaceutical composition to the individual comprising a) SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'); and b) a pharmaceutically acceptable carrier.

[0052] In some embodiments, an individual is determined to have pouchitis using a Pyralitis Disease Activity Index (PDAI) score of at least 7. In some embodiments, an individual is determined to have pouchitis using a Modified Pyralitis Disease Activity Index (mPDAI) score of at least 5.

[0053] In some embodiments, the individual has acute pouchitis. In some embodiments, the individual has chronic pouchitis.

[0054] Furthermore, in some embodiments, a method for predicting pouchitis in an individual requiring prediction of pouchitis is described herein, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, and the known control level is the SMAD7 level in a sample taken from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual without pouchitis. Furthermore, in some embodiments, a method for identifying an individual at risk of pouchitis is described herein, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual at risk of pouchitis, and the known control level is the SMAD7 level in a sample taken from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual without pouchitis.

[0055] In some embodiments, if the SMAD7 level is elevated compared to a known control level, the individual is administered a pharmaceutical composition containing SMAD7 ASO. In some embodiments, if the SMAD7 level is not elevated compared to a known control level, the level of SMAD7 in a second sample derived from the individual is determined. In some embodiments, the second sample is collected immediately after the first sample, about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 1 day, about 3 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months later.

[0056] Furthermore, in some embodiments, a method for treating pouchitis in an individual requiring treatment for pouchitis is described herein, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, and the known control level is the SMAD7 level in a sample taken from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual without pouchitis.

[0057] In some embodiments, individuals with pouchitis have a reduction of at least about 5%, about 10%, about 20%, about 30%, about 40%, or even more than 50%, or higher, in their pouchitis disease activity index (PDAI) score or modified pouchitis disease activity index (mPDAI) score after administration of the SMAD7 antisense oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a pharmaceutically acceptable salt thereof, for example, after 1 day, 2 days, 1 week, 1 month, or 6 months, or longer. In some embodiments, administration of the SMAD7 antisense oligonucleotide is, for example, at least once daily. In some embodiments, as a result of administration of the SMAD7 antisense oligonucleotide, the delay in the onset of clinical symptoms of pouchitis in an individual is at least, for example, 6 months, 1 year, 18 months, or even more than 2 years, or longer, compared to an individual not administered the SMAD7 ASO.

[0058] Pharmaceutical preparations In some embodiments, pharmaceutical formulations containing oligonucleotides such as SMAD7 ASO as described herein, presented in dosage unit form and prepared by any suitable method, are described herein. Pharmaceutical formulations should be formulated to be compatible with their intended route of administration. Useful formulations can be prepared by methods well known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0059] In some embodiments, the pharmaceutical formulation (e.g., an intracapsular formulation) comprises an aqueous injectable composition containing an antisense oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) as described herein. For example, the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation and a liquid solution or suspension are blended together. In some embodiments, a pharmaceutical formulation in solid form suitable for use in preparing a solution or suspension by adding a liquid before injection is prepared; the preparation is emulsified. In some embodiments, the pharmaceutical formulation suitable for injectable use comprises a sterile aqueous solution or dispersion. In some embodiments, the pharmaceutical formulation suitable for injectable use comprises sesame oil, peanut oil, or aqueous propylene glycol. In some embodiments, the pharmaceutical formulation suitable for injectable use comprises a sterile powder.

[0060] In some embodiments, the oligonucleotide formulation (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) (e.g., an intracapsular formulation) comprises an aqueous injectable composition containing a surfactant (e.g., hydroxypropyl cellulose).

[0061] In some embodiments, oligonucleotide formulations (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) (e.g., intracapsular formulations) include dispersions prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil.

[0062] In some embodiments, the pharmaceutical formulation of an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a rectal pharmaceutical formulation (e.g., suppositories, enemas, gels, and foams). In some embodiments, the rectal pharmaceutical formulation contains an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof).

[0063] In some embodiments, the pharmaceutical formulation (e.g., rectal pharmaceutical formulation) of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a suppository. In some embodiments, the suppository contains a lipophilic excipient (e.g., cocoa butter, coconut oil, hydrogenated vegetable oil, hard fat). In some embodiments, the suppository contains a hydrophilic excipient (e.g., glycerin-gelatin and polyethylene glycol). In some embodiments, the suppository contains a surfactant (e.g., polysorbate 80, Tween® 20, Span® 60).

[0064] In some embodiments, a pharmaceutical formulation (e.g., a rectal pharmaceutical formulation) of an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is an enema. In some embodiments, the enema is a solution. In some embodiments, the enema is a suspension. In some embodiments, the enema is an emulsion.

[0065] In some embodiments, the pharmaceutical formulation (e.g., rectal pharmaceutical formulation) of an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a gel. In some embodiments, the gel comprises a cosolvent (e.g., glycerin and propylene glycol). In some embodiments, the gel comprises an electrolyte.

[0066] In some embodiments, the gel formulation has a gel-like viscosity but is sufficiently fluid to allow local or local administration by a catheter, needle, syringe, or other equivalent means of local or rectal administration.

[0067] In some embodiments, the concentration of the thickener used in the gel formulation is an amount or concentration suitable for achieving the desired thickness or viscosity, for example, about 0.05% by weight to about 10% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% by weight to about 8% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% by weight to about 7% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% by weight to about 6% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% by weight to about 5% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% by weight to about 4% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% by weight to about 3% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% to about 2% by weight. In some embodiments, the concentration of the thickener used in such a gel formulation is in the range of about 0.05% to about 1% by weight. In some embodiments, the gel formulation contains methylcellulose at a concentration of about 0.05% to about 2%, while in other embodiments, the gel formulation contains methylcellulose at a concentration of about 1%.

[0068] In some embodiments, the pharmaceutical formulation (e.g., rectal pharmaceutical formulation) of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a foam. In some embodiments, the foam formulation comprises a surfactant / emulsifier having foaming properties and a spray (e.g., spray gas). In some embodiments, the foam formulation also comprises one or more of the following: suspending / solubilizing agents, thickeners, preservatives, chelating agents, buffers, antioxidants, tonicity modifiers, and spreading agents. In some embodiments, the surfactant / emulsifier includes, in non-limiting examples, nonionic surfactants, anionic surfactants, cationic surfactants, and combinations thereof. In some embodiments, the foam formulation is filled into a pressurized container before administration (e.g., rectal administration). In some embodiments, the pressurized container is a can. In some embodiments, the sprays used herein include, in non-limiting examples, hydrocarbons (such as isobutane, N-butane, or propane), fluorocarbons (e.g., dichlorodifluoromethane and dichlorotetrafluoroethane), chlorofluorocarbons, dimethyl ethers, hydrofluorocarbons, compressed gases, Freon (such as Freon 12 and Freon 114), hydrochlorofluorocarbons, hydrofluorocarbons, or mixtures thereof.

[0069] In some embodiments, the maximum amount of spray used is determined by its miscibility with other components in the composition for forming a mixture, such as a homogeneous mixture. In some embodiments, the minimum level of spray used in the composition is determined by the desired foaming properties and its ability to be substantially or completely discharged from the container.

[0070] In some embodiments, the concentration of the spray used in such foam formulations is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 50%, 55% to approximately 60% (w / w).

[0071] In some embodiments, foam is formed at the time of administration (e.g., rectal administration), when the can's dispensing valve allows for rapid expansion of the spray, causing the surfactant to foam and resulting in foam formation in the rectum and colon. In some embodiments, the foam used for administering the compositions described herein is formed in the dispensing container before administration.

[0072] In some embodiments, the pharmaceutical formulation of an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is an oral pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises a granular internal phase containing the antisense oligonucleotide described herein (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable filler. For example, the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) and the filler are blended together with other additives as needed and formed into granules. In some embodiments, the granular internal phase is formed using wet granulation, for example, by adding a liquid (e.g., water) to a blended antisense compound and filler, and then the combination is dried, ground, and / or sieved to produce granules. Other processes in the art may be used to obtain the granular internal phase.

[0073] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) includes an outer granular phase comprising one or more pharmaceutically acceptable additives, which are blended with an inner granular phase to form the disclosed formulation.

[0074] In some embodiments, oligonucleotide formulations (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) include a granular inner phase containing a filler. Exemplary fillers include, but are not limited to, cellulose, gelatin, calcium phosphate, lactose, sucrose, glucose, mannitol, sorbitol, microcrystalline cellulose, pectin, polyacrylate, dextrose, cellulose acetate, hydroxypropyl methylcellulose, partially pregelatinized starch, calcium carbonate, and other combinations thereof.

[0075] In some embodiments, oligonucleotide formulations (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) (e.g., oral pharmaceutical formulations) include an inner granular phase and / or outer granular phase containing a binder for holding the components of the pharmaceutical formulation together. Exemplary binders include, but are not limited to, starch, sugar, cellulose or modified cellulose, e.g., hydroxypropylcellulose, lactose, pregelatinized maize starch, polyvinylpyrrolidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, methylcellulose, ethylcellulose, sugar alcohols, and other combinations thereof.

[0076] In some embodiments, the pharmaceutical formulations described herein (e.g., oral pharmaceutical formulations) comprising a granular inner phase and / or granular outer phase further include, but are not limited to, disintegrants such as starch, cellulose, cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethylcellulose, alginate, corn starch, croscarmellose sodium, cross-linked carboxymethylcellulose, low-substituted hydroxypropylcellulose, acacia, and others, including combinations thereof. For example, the granular inner phase and / or granular outer phase may include a disintegrant.

[0077] In some embodiments, the oligonucleotide formulations described herein (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) comprise a granular inner phase containing the disclosed antisense oligonucleotide, as well as additives selected from mannitol, microcrystalline cellulose, hydroxypropyl methylcellulose, and sodium starch glycolate, or combinations thereof, and a granular outer phase containing one or more of microcrystalline cellulose, sodium starch glycolate, and magnesium stearate, or mixtures thereof.

[0078] In some embodiments, a pharmaceutical formulation of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) (e.g., a rectal pharmaceutical formulation) contains a lubricant, for example, the granular outer phase contains a lubricant. Lubricants include, but are not limited to, talc, silica, fat, stearin, magnesium stearate, calcium phosphate, silicon dioxide, calcium silicate, calcium phosphate, colloidal silicon dioxide, metallic stearate, hydrogenated vegetable oil, corn starch, sodium benzoate, polyethylene glycol, sodium acetate, calcium stearate, sodium lauryl sulfate, sodium chloride, magnesium lauryl sulfate, talc, and stearic acid.

[0079] In some embodiments, the pharmaceutical formulations of this disclosure (e.g., oral pharmaceutical formulations) include an enteric coating. Generally, enteric coatings create a barrier to oral pharmaceuticals and control the site of absorption of the drug along the gastrointestinal tract. In some embodiments, the enteric coating includes polymers that disintegrate at different rates depending on the pH. In some embodiments, enteric coatings include, for example, cellulose acetate phthalate, methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, methyl methacrylate-methacrylic acid copolymer, ethyl acrylate-methacrylic acid copolymer, methacrylic acid copolymer type C, polyvinyl acetate-phthalate, and cellulose acetate phthalate.

[0080] In some embodiments, enteric coatings include anionic, cationic, or neutral copolymers based on methacrylic acid, methacrylic acid / acrylic acid esters, or derivatives thereof. In certain embodiments, enteric coatings include ethyl acrylate-methacrylic acid copolymers. Commercially available enteric coatings include Opadry® AMB, ethyl acrylate-methacrylic acid copolymers (e.g., Acryl-EZE®), dimethylaminoethyl methacrylate-butyl methacrylate-methyl methacrylate copolymers (2:1:1), or poly(methacrylic acid-co-methyl methacrylate) 1:1 and poly(methacrylic acid-co-methyl methacrylate) 1:2 copolymers (e.g., Eudragit®). In some embodiments, the enteric coating comprises about 5% to about 10%, about 5% to about 20%, about 8% to about 15%, about 8% to about 18%, about 10% to about 12%, or about 12% to about 16% of the weight of the intended pharmaceutical formulation (e.g., a tablet). In some embodiments, the enteric coating comprises an ethyl acrylate-methacrylic acid copolymer comprising about 8% to about 15% of the weight of the pharmaceutical formulation (e.g., a tablet). In some embodiments, the enteric coating comprises an ethyl acrylate-methacrylic acid copolymer comprising about 12% of the weight of the pharmaceutical formulation (e.g., a tablet). In some embodiments, the enteric coating comprises an ethyl acrylate-methacrylic acid copolymer comprising about 10% of the weight of the pharmaceutical formulation (e.g., a tablet).

[0081] In some embodiments, the pharmaceutical formulation (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) contains about 0.5% to about 70% by weight, for example, about 0.5% to about 30% by weight, about 1% to about 20% by weight, or about 5% to about 30% by weight of an antisense oligonucleotide or a pharmaceutically acceptable salt thereof. In some embodiments, such a pharmaceutical formulation contains about 0.5% to about 60% by weight of mannitol, for example, about 20% to about 50% by weight of mannitol, for example, about 40% or about 28% by weight of mannitol; and / or about 20% to about 40% by weight of microcrystalline cellulose, or about 10% to about 30% by weight of microcrystalline cellulose. In some embodiments, the pharmaceutical formulation comprises a granular inner phase containing about 30% to about 60% by weight, about 45% to about 65% by weight, or alternatively about 5% to about 10% by weight of SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof; about 30% to about 50% by weight, or alternatively about 5% to about 15% by weight of mannitol; about 5% to about 15% by weight of microcrystalline cellulose; about 0% to about 4% by weight, or about 1% to about 7% by weight of hydroxypropyl methylcellulose; and about 0% to about 4% by weight, for example, about 2% to about 4% by weight of sodium starch glycolate. In some embodiments, the pharmaceutical formulation comprises about 5% to about 10% by weight or about 10% to about 30% by weight of the oligonucleotide of SEQ ID NO: 1, about 20% to about 50% by weight of mannitol, about 10% to about 30% by weight of microcrystalline cellulose, about 0.5% to about 10% by weight of hydroxypropyl methylcellulose, and about 0.5% to about 10% by weight of sodium starch glycolate. In some embodiments, the pharmaceutical formulation is in the form of a tablet.

[0082] Exemplary pharmaceutical formulations include dosage forms containing or essentially derived from SMAD7 ASO of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof, in doses of approximately 10 mg to approximately 500 mg or approximately 20 mg to approximately 500 mg, for example, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg, approximately 160 mg Tablets containing SMAD7 ASO of Sequence ID No. 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, in amounts of mg, approximately 170 mg, approximately 180 mg, approximately 190 mg, approximately 200 mg, approximately 220 mg, approximately 240 mg, approximately 260 mg, approximately 280 mg, approximately 300 mg, approximately 320 mg, approximately 340 mg, approximately 360 mg, approximately 380 mg, approximately 400 mg, approximately 420 mg, approximately 440 mg, approximately 460 mg, approximately 480 mg, or approximately 500 mg are intended herein. In some embodiments, the pharmaceutical formulations described herein are formulated as tablets containing approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg, approximately 160 mg, approximately 170 mg, approximately 180 mg, approximately 190 mg, approximately 200 mg, approximately 220 mg, approximately 240 mg, approximately 260 mg, approximately 280 mg, approximately 300 mg, approximately 320 mg, approximately 340 mg, approximately 360 mg, approximately 380 mg, approximately 400 mg, approximately 420 mg, approximately 440 mg, approximately 460 mg, approximately 480 mg, or approximately 500 mg of the oligonucleotide of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation described herein is formulated as a tablet containing approximately 40 mg of the oligonucleotide of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation described herein is formulated as a tablet containing approximately 160 mg of the oligonucleotide of SEQ ID NO: 1.

[0083] In certain embodiments, the pharmaceutical formulations described herein are formulated as tablets for oral use and contain about 0.5% to about 30% by weight of oligonucleotides; about 20% to about 50% by weight of mannitol; and about 10% to about 30% by weight of microcrystalline cellulose.

[0084] In some embodiments, pharmaceutically acceptable tablets for oral administration are provided, comprising about 5% to about 30% by weight of the oligonucleotide of SEQ ID NO: 1, 20% to about 50% by weight of mannitol, about 10% to about 30% by weight of microcrystalline cellulose, about 0.5% to about 10% by weight of hydroxypropyl methylcellulose, and about 0.5% to about 10% by weight of sodium starch glycolate, about 0.5% to about 10% by weight of magnesium stearate, about 0.5% to about 10% by weight of Opadry® AMB, and about 5% to about 20% by weight of Acryl-EZE®. In some embodiments, a pharmaceutically acceptable tablet for oral administration is provided, comprising about 8.5 wt% of the oligonucleotide of SEQ ID NO: 1, 40 wt% of mannitol, about 25 wt% of microcrystalline cellulose, about 5 wt% of hydroxypropyl methylcellulose, about 4 wt% of sodium starch glycolate, about 0.4 wt% of magnesium stearate, about 4 wt% of Opadry® AMB, and about 10 wt% to about 15 wt% of Acryl-EZE®. In some embodiments, a pharmaceutically acceptable tablet for oral administration is provided, comprising about 23 wt% of the oligonucleotide of SEQ ID NO: 1, 28 wt% of mannitol, about 25 wt% of microcrystalline cellulose, about 5 wt% of hydroxypropyl methylcellulose, about 4 wt% of sodium starch glycolate, about 0.4 wt% of magnesium stearate, about 4 wt% of Opadry® AMB, and about 7 wt% to about 12 wt% of Acryl-EZE®. In some embodiments, pharmaceutically acceptable tablets for oral administration are formulated according to Tables 1 and 2. Table 1 [Table 1] Table 2 [Table 2]

[0085] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises a granular inner phase containing about 50 wt% of SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 11.5 wt% of mannitol, about 10 wt% of microcrystalline cellulose, about 3 wt% of hydroxypropyl methylcellulose, and about 2.5 wt% of sodium starch glycolate; and a granular outer phase containing about 20 wt% of microcrystalline cellulose, about 2.5 wt% of sodium starch glycolate, and about 0.5 wt% of magnesium stearate. In some embodiments, the pharmaceutical formulation further comprises an enteric coating.

[0086] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises, or essentially consists of, an internal granular phase comprising about 5% to about 10% by weight, for example, about 8% by weight of SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof (e.g., sodium salt), about 40% by weight of mannitol, about 8% by weight of microcrystalline cellulose, about 5% by weight of hydroxypropyl methylcellulose, and about 2% by weight of sodium starch glycolate; and an external granular phase comprising about 17% by weight of microcrystalline cellulose, about 2% by weight of sodium starch glycolate, and about 0.4% by weight of magnesium stearate.

[0087] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) includes an enteric coating in about 13% by weight, about 14% by weight, about 15% by weight, about 16% by weight, or about 17% by weight, such as an enteric coating comprising an ethyl acrylate-methacrylic acid copolymer (e.g., AcrylEZE®).

[0088] For example, the pharmaceutical formulation is in the form of a pharmaceutically acceptable tablet for oral use, comprising a granular inner phase and a granular outer phase, for example, the granular inner phase comprising about 5% to about 10% (e.g., about 8% by weight) of an oligonucleotide represented by SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 40% by weight of mannitol, about 8% by weight of microcrystalline cellulose, about 5% by weight of hydroxypropyl methylcellulose, and about 2% by weight of sodium starch glycolate, for example, the granular outer phase comprising about 17% by weight of microcrystalline cellulose, about 2% by weight of sodium starch glycolate, and about 0.4% by weight of magnesium stearate, and the tablet further comprising an enteric coating.

[0089] In some embodiments, the intended formulation, when administered orally to an individual, yields a minimum plasma concentration of SMAD7 ASO in the individual. In some embodiments, the intended formulation, when administered orally to an individual, is delivered topically to the individual's cysts, e.g., to the affected or pathological cysts of the individual. In some embodiments, formulations suitable for oral administration are in the form of capsules, cachets, pills, tablets, lozenges (e.g., using a flavored base such as sucrose and acacia or tragacanth), powders, granules, or as liquids or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as flavored tablets (using gelatin and glycerin, or an inert base such as sucrose and acacia), each containing a predetermined amount of the target composition as the active ingredient. In some embodiments, the formulation is administered as a bolus, lick, or paste.

[0090] In some embodiments, the pharmaceutical formulation described herein comprises a granular inner phase, which comprises an antisense oligonucleotide such as SMAD7 ASO of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof (e.g., a sodium salt), and a pharmaceutically acceptable filler. In some embodiments, the pharmaceutical formulation described herein comprises a granular outer phase, which comprises a pharmaceutically acceptable additive such as a disintegrant. In some embodiments, the granular outer phase comprises components including microcrystalline cellulose, magnesium stearate, and mixtures thereof. In some embodiments, the pharmaceutical formulation further comprises an enteric coating in an amount of about 12% to 16% by weight relative to the pharmaceutical formulation.

[0091] In some embodiments, the pharmaceutical formulations described herein (e.g., oral pharmaceutical formulations) include an enteric coating comprising about 0.5% to about 10% by weight of an antisense oligonucleotide, such as SMAD7 ASO of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 30% to 50% by weight of mannitol, about 10% to 30% by weight of microcrystalline cellulose, and an ethyl acrylate-methacrylic acid copolymer.

[0092] In some embodiments, the pharmaceutical formulations described herein (e.g., oral pharmaceutical formulations) include a granular inner phase comprising about 5% to about 10% by weight of antisense oligonucleotides, e.g., SMAD7 ASO of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 40% by weight of mannitol, about 8% by weight of microcrystalline cellulose, about 5% by weight of hydroxypropyl methylcellulose, and about 2% by weight of sodium starch glycolate; a granular outer phase comprising about 17% by weight of microcrystalline cellulose, about 2% by weight of sodium starch glycolate, and about 0.4% by weight of magnesium stearate; and an enteric coating on a tablet comprising an ethyl acrylate-methacrylic acid copolymer.

[0093] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) includes, for example, an enteric coating containing about 13% by weight, about 15% by weight, about 16% by weight, about 17% by weight, or about 18% by weight of AcyrlEZE® (see, for example, PCT Publication WO2010 / 054826).

[0094] The rate at which the coating dissolves and the active ingredient is released is the dissolution rate. In some embodiments, a pharmaceutical formulation (e.g., in tablet form) has a dissolution profile of approximately 50% to 100% oligonucleotide release after approximately 120 to 240 minutes, for example, after 180 minutes, when tested in a USP / EP Type 2 instrument (paddle) at 100 rpm and 37°C in a phosphate buffer having a pH of 7.2. In some embodiments, a pharmaceutical formulation (e.g., in tablet form) has a dissolution profile of substantially no oligonucleotide release after 120 minutes, when tested in a USP / EP Type 2 instrument (paddle) at 100 rpm and 37°C in dilute HCl having a pH of 1.0. In some embodiments, the pharmaceutical formulation (e.g., in tablet form) has a dissolution profile of oligonucleotide release of approximately 10% to approximately 30%, or less than approximately 50%, after 30 minutes when tested in a USP / EP Type 2 apparatus (paddle) at 100 rpm and 37°C in a phosphate buffer having a pH of 6.6.

[0095] In some embodiments, the pharmaceutical formulations described herein, when administered to an individual, result in a minimum plasma concentration of oligonucleotides in the individual. In some embodiments, when the disclosed formulations are administered orally to an individual, they are delivered locally to the colon or rectum of the individual, for example, to the affected or pathological site of the individual.

[0096] In some embodiments, the pharmaceutical formulations described herein are sterile. In some embodiments, sterilization is achieved, for example, by filtration with a sterile filtration membrane. In some embodiments, if the composition is freeze-dried, sterilization by filtration is performed before or after freeze-drying and reconstitution.

[0097] In some embodiments, a pharmaceutical formulation containing SEQ ID NO: 1 contains more than 50 millimoles, more than 100 millimoles, more than 200 millimoles, more than 300 millimoles, more than 400 millimoles, more than 500 millimoles, more than 600 millimoles, more than 700 millimoles, more than 800 millimoles, more than 900 millimoles, more than 1 mole, more than 1.5 moles, more than 2 moles, more than 2.5 moles, more than 3 moles, more than 4 moles, or more than 5 moles of SEQ ID NO: 1. In some embodiments, a pharmaceutical formulation containing SEQ ID NO: 1 contains more than 2 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation includes SEQ ID NO: 1 in amounts less than 5 mol, less than 4 mol, less than 3 mol, less than 2.5 mol, less than 2 mol, less than 1.5 mol, less than 1 mol, less than 900 mmol, less than 850 mmol, less than 800 mmol, less than 750 mmol, less than 700 mmol, less than 650 mmol, less than 600 mmol, less than 550 mmol, less than 500 mmol, less than 450 mmol, less than 400 mmol, less than 350 mmol, less than 300 mmol, less than 250 mmol, less than 200 mmol, less than 150 mmol, or less than 100 mmol. In some embodiments, the pharmaceutical formulation includes SEQ ID NO: 1 in amounts greater than 100 mmol up to about 5 mol. In some embodiments, the pharmaceutical formulation includes SEQ ID NO: 1 in amounts greater than 100 mmol up to about 2 mol. In some embodiments, the pharmaceutical formulation includes SEQ ID NO: 1 in amounts greater than 300 mmol up to about 5 mol. In some embodiments, the pharmaceutical formulation contains more than 300 millimoles up to about 2 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation contains more than 900 millimoles up to about 5 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation contains more than 900 millimoles up to about 2 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation contains more than 300 millimoles up to about 900 millimoles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation contains more than 300 millimoles up to about 5 moles, more than 300 millimoles up to about 4 moles, more than 300 millimoles up to about 3 moles, more than 300 millimoles up to about 2.5 moles, more than 300 millimoles up to about 2 moles, and more than 300 millimoles up to about 1.Up to 5 moles, more than 300 millimoles up to approximately 1 mole, more than 300 millimoles up to approximately 900 millimoles, more than 300 millimoles up to approximately 800 millimoles, more than 300 millimoles up to approximately 700 millimoles, more than 300 millimoles up to approximately 600 millimoles, more than 300 millimoles up to approximately 500 millimoles, more than 300 millimoles up to approximately 400 millimoles, more than 400 millimoles up to approximately 5 moles, more than 400 millimoles up to approximately 4 moles, more than 400 millimoles up to approximately 3 moles, more than 400 millimoles up to approximately 2.5 moles , more than 400 mmol up to about 2 mmol, more than 400 mmol up to about 1.5 mmol, more than 400 mmol up to about 1 mmol, more than 400 mmol up to about 900 mmol, more than 400 mmol up to about 800 mmol, more than 400 mmol up to about 700 mmol, more than 400 mmol up to about 600 mmol, more than 400 mmol up to about 500 mmol, more than 500 mmol up to about 5 mmol, more than 500 mmol up to about 4 mmol, more than 500 mmol up to about 3 mmol, more than 500 mmol Up to approximately 2.5 moles, more than 500 millimoles up to approximately 2 moles, more than 500 millimoles up to approximately 1.5 moles, more than 500 millimoles up to approximately 1 mole, more than 500 millimoles up to approximately 900 millimoles, more than 500 millimoles up to approximately 800 millimoles, more than 500 millimoles up to approximately 700 millimoles, more than 500 millimoles up to approximately 600 millimoles, more than 600 millimoles up to approximately 5 moles, more than 600 millimoles up to approximately 4 moles, more than 600 millimoles up to approximately 3 moles, more than 600 millimoles up to approximately 2.5 moles , up to approximately 2 moles (more than 600 mmol), up to approximately 1.5 moles (more than 600 mmol), up to approximately 1 mole (more than 600 mmol), up to approximately 900 mmol (more than 600 mmol), up to approximately 800 mmol (more than 600 mmol), up to approximately 700 mmol (more than 600 mmol), up to approximately 5 moles (more than 700 mmol), up to approximately 4 moles (more than 700 mmol), up to approximately 3 moles (more than 700 mmol), up to approximately 2.5 moles (more than 700 mmol), up to approximately 2 moles (more than 700 mmol), up to approximately 1.Up to 5 moles, more than 700 mmol up to approximately 1 mole, more than 700 mmol up to approximately 900 mmol, more than 700 mmol up to approximately 800 mmol, more than 800 mmol up to approximately 5 moles, more than 800 mmol up to approximately 4 moles, more than 800 mmol up to approximately 3 moles, more than 800 mmol up to approximately 2.5 moles, more than 800 mmol up to approximately 2 moles, more than 800 mmol up to approximately 1.5 moles, more than 800 mmol up to approximately 1 mole, more than 800 mmol up to approximately 900 mmol, more than 900 mmol up to approximately 5 moles, more than 900 mmol up to approximately 4 moles, more than 900 mmol up to approximately 3 moles, more than 900 mmol up to approximately 2.5 moles, more than 900 mmol up to approximately 2 moles, more than 900 mmol up to approximately 1.5 moles, more than 900 mmol Includes Sequence ID 1 for up to 1 mole, more than 1 mole up to approximately 5 moles, more than 1 mole up to approximately 4 moles, more than 1 mole up to approximately 3 moles, more than 1 mole up to approximately 2.5 moles, more than 1 mole up to approximately 2 moles, more than 1 mole up to approximately 1.5 moles, more than 1.5 moles up to approximately 5 moles, more than 1.5 moles up to approximately 4 moles, more than 1.5 moles up to approximately 3 moles, more than 1.5 moles up to approximately 2.5 moles, more than 2 moles up to approximately 5 moles, more than 2 moles up to approximately 4 moles, more than 2 moles up to approximately 3 moles, more than 2 moles up to approximately 2.5 moles, more than 2.5 moles up to approximately 5 moles, more than 2.5 moles up to approximately 4 moles, more than 2.5 moles up to approximately 3 moles, more than 3 moles up to approximately 5 moles, or more than 4 moles up to approximately 5 moles. In some embodiments, the pharmaceutical formulation contains SEQ ID NO: 1 in amounts of approximately 1 mol, approximately 900 mmol, approximately 850 mmol, approximately 800 mmol, approximately 750 mmol, approximately 700 mmol, approximately 650 mmol, approximately 600 mmol, approximately 550 mmol, approximately 500 mmol, approximately 450 mmol, approximately 400 mmol, approximately 350 mmol, approximately 300 mmol, approximately 250 mmol, approximately 200 mmol, approximately 150 mmol, or approximately 100 mmol.

[0098] Route of administration Enteral administration In some embodiments, the SMAD7 ASO described herein is administered enterally. In some embodiments, enteral administration is oral, sublingual, gastric, or rectal. In some embodiments, enteral administration is rectal.

[0099] In some embodiments, the SMAD7 ASOs described herein are suitable for rectal delivery (e.g., suppositories, enemas, gels, and foams). In some embodiments, the SMAD7 ASO of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, is administered rectally.

[0100] In some embodiments, the preparation of an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a rectal pharmaceutical preparation. In some embodiments, the rectal pharmaceutical preparation contains an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof). In some embodiments, the rectal pharmaceutical preparation contains SMAD7 ASO (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof).

[0101] In some embodiments, the SMAD7 ASOs described herein are suitable for oral delivery (e.g., capsules, tablets, caplets, pills, lozenges, powders, and granules). In some embodiments, the SMAD7 ASO of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, is administered orally.

[0102] In some embodiments, the preparation of an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is an oral pharmaceutical preparation. In some embodiments, the oral pharmaceutical preparation contains an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof). In some embodiments, the oral pharmaceutical preparation contains SMAD7 ASO (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof).

[0103] In some embodiments, the pharmaceutical formulation of an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is in the form of a tablet or capsule. In some embodiments, the tablet or capsule is enterically coated.

[0104] In some embodiments, tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are formulated as minitablets, granulates, micropellets, or microtablets. In some embodiments, tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are formulated as minitablets. In some embodiments, tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are formulated as granulates. In some embodiments, tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are formulated as micropellets. In some embodiments, tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are formulated as microtablets. In some embodiments, minitablets, granules, micropellets, or microtablets are enterically coated.

[0105] In some embodiments, oral pharmaceutical formulations of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are not enterically coated. In certain embodiments, oral pharmaceutical formulations of oligonucleotides (e.g., oligonucleotide of SEQ ID NO: 1, or pharmaceutically acceptable salts thereof) are not enterically coated.

[0106] In some embodiments, the oral pharmaceutical formulation includes a gastric-resistant coating, which allows the formulation to deliver the compound to, for example, the gastrointestinal tract of an individual. In some embodiments, such administration produces a local effect by substantially topically and directly applying the antisense compound to, for example, the affected portion of the gastrointestinal tract of the subject. In some embodiments, such administration substantially avoids the undesirable systemic absorption of the antisense compound.

[0107] In some embodiments, the pharmaceutical formulations described herein (e.g., oral and rectal pharmaceutical formulations) include pharmaceutically acceptable additives. Exemplary pharmaceutically acceptable additives include, but are not limited to, fillers, binders, disintegrants, and / or lubricants, as well as colorants, release agents, coatings, sweeteners, flavoring agents, such as wintergreen, orange, xylitol, sorbitol, fructose, and maltodextrin, and perfumering agents, preservatives, and / or antioxidants.

[0108] Parenteral administration In some embodiments, the SMAD7 ASO and pharmaceutical formulations of this disclosure are formulated for parenteral administration, for example, for injection via intravenous, intratumoral, intramuscular, subcutaneous, intralesional, intraintestinal (e.g., intrajejunal, intraileal), intracolonic, intrarectal, intrasacral, or intraperitoneal routes. In some embodiments, the SMAD7 ASO or pharmaceutical formulations described herein are administered intrasacrally. In some embodiments, the SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, is administered intrasacrally.

[0109] In some embodiments, the preparation of aqueous compositions, such as aqueous pharmaceutical formulations containing the disclosed SMAD7 ASO, is known to those skilled in the art in light of this disclosure. In some embodiments, such compositions are prepared as either liquid solutions or suspensions for injection. In some embodiments, a solid form is prepared that is suitable for use in preparing a solution or suspension by adding liquid before injection; the preparation is emulsified.

[0110] In some embodiments, the pharmaceutical formulation suitable for injection comprises a sterile aqueous solution or dispersion. In some embodiments, the pharmaceutical formulation suitable for injection comprises sesame oil, peanut oil, or aqueous propylene glycol. In some embodiments, the pharmaceutical formulation suitable for injection comprises a sterile powder. In some embodiments, the pharmaceutical formulation is sterile and has fluidity to the extent that it is easily injectable (syringability). In some embodiments, the pharmaceutical formulation must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0111] In some embodiments, solutions of the active compound, as a free base or a pharmacokinetically acceptable salt, are prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. In some embodiments, sterile non-volatile oils are used as solvents or suspension media. For this purpose, in some embodiments, non-irritating non-volatile oils containing synthetic monoglycerides or diglycerides are used. In some embodiments, fatty acids such as oleic acid are used in the preparation of injectable preparations. In some embodiments, sterile injectable preparations are sterile injectable solutions, suspensions, or emulsions as solutions in a non-toxic, parenterally acceptable diluent or solvent, for example, in 1,3-butanediol. In some embodiments, acceptable vehicles and solvents used are water, Ringer's solution, USP, and isotonic sodium chloride solution. In some embodiments, the disclosed SMAD7 ASO is suspended in a carrier fluid containing 1% (w / v) sodium carboxymethylcellulose and 0.1% (v / v) TWEEN® 80. Under normal storage and use conditions, these preparations contain preservatives to inhibit microbial growth.

[0112] In some embodiments, injectable preparations, such as sterile aqueous or oily suspensions for injection, are formulated according to known techniques using appropriate dispersants or wetting and suspending agents. In some embodiments, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a base dispersion medium and other required components from those listed above. In some embodiments, sterile injectable solutions of the present disclosure are prepared by incorporating SMAD7 ASO disclosed in the required amount in a suitable solvent, along with various amounts of other components listed above as needed, and subsequently by filtration sterilization. In some embodiments, for sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient plus any additional desired components from the solution that has been pre-sterilized and filtered. In some embodiments, injectable formulations are sterilized, for example, by filtration with a bacterial-retaining filter.

[0113] In some embodiments, the preparation of more or more concentrated solutions for intramuscular injection is also intended. In some embodiments, the use of DMSO as the solvent is preferred because it allows for extremely rapid penetration and delivery of a high concentration of the disclosed SMAD7 ASO to a small area.

[0114] In some embodiments, suitable preservatives for use in such solutions include benzalkonium chloride, benzethonium chloride, chlorobutanol, and thimerosal. In some embodiments, suitable buffers include boric acid, sodium bicarbonate and potassium bicarbonate, sodium borate and potassium borate, sodium carbonate and potassium carbonate, sodium acetate, and sodium dihydrogen phosphate in amounts sufficient to maintain a pH between approximately pH 6 and pH 8, for example, between approximately pH 7 and pH 7.5. In some embodiments, suitable tonicity agents include dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, and sodium chloride, which result in a sodium chloride equivalent in the solution being in the range of 0.9 ± 0.2%. In some embodiments, suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfite, and thiourea. In some embodiments, suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282, and tyroxapole. Suitable viscosity enhancers include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and carboxymethylcellulose.

[0115] Medication regimen In some embodiments, pharmaceutical formulations of oligonucleotides provided in this disclosure (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6) or pharmaceutically acceptable salts thereof are administered before and / or after the onset of symptoms of pouchitis, or are suitable for such administration.

[0116] In some embodiments, the pharmaceutical formulations of the present disclosure (e.g., including SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are administered or are suitable for administration approximately every 6 hours, approximately every 12 hours, approximately every 24 hours, approximately every 48 hours, approximately every 72 hours, daily, twice a week, once every two weeks, or once a month.

[0117] The frequency of medication may vary depending on factors such as the route of administration, the dosage, and the disease being treated. Examples of medication frequencies include once daily, once weekly, and once every two weeks. In certain embodiments, medication is administered once daily for seven days.

[0118] In some embodiments, the pharmaceutical formulation comprises about 10 mg to about 500 mg, or about 20 mg to about 500 mg, of an oligonucleotide (e.g., the SMAD7 oligonucleotide of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof), or a dosage form essentially thereof. For example, formulations containing approximately 20 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg, 300 mg, 320 mg, 340 mg, 360 mg, 380 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, or 500 mg of oligonucleotides (e.g., SMAD7 oligonucleotides of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) are intended herein. In certain embodiments, the formulation contains an oligonucleotide, such as the SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, in amounts of approximately 40 mg, approximately 80 mg, or approximately 160 mg. In certain embodiments, the formulation contains an oligonucleotide, such as the SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, in amounts of at least approximately 100 μg. For example, the formulation contains an oligonucleotide, such as the SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, in amounts of approximately 0.1 mg, approximately 0.2 mg, approximately 0.3 mg, approximately 0.4 mg, approximately 0.5 mg, approximately 1 mg, approximately 5 mg, approximately 10 mg, approximately 15 mg, approximately 20 mg, or approximately 25 mg.

[0119] The dose administered depends on variables such as the type and severity of the disease or indication being treated, the overall health and size of the individual, the in vivo efficacy of the oligonucleotide, the pharmaceutical formulation, and the route of administration. In some embodiments, the initial dose is increased beyond an upper level to rapidly achieve the desired blood or tissue level. In some embodiments, the initial dose is lower than the optimal dose, and the dose is gradually increased during the treatment process. In some embodiments, the human dose is optimized in a conventional Phase I dose escalation study designed to be performed, for example, from 40 mg to 160 mg.

[0120] In some embodiments, an individual with pouchitis is administered an initial dose of SMAD7 ASO, for example, SMAD7 ASO of SEQ ID NO: 1. As used herein, “initial dose” refers to a series of doses of SMAD7 ASO administered to an individual with pouchitis. In some embodiments, a series of doses includes one or more doses. For example, a series of doses includes a single dose of SMAD7 ASO or a dose greater than a single dose of SMAD7 ASO. In some embodiments, the initial dose is a dose of SMAD7 ASO administered to an individual before any subsequent doses administered to the individual. For example, the initial dose is, but is not limited to, the first dose of SMAD7 ASO administered to an individual that has not received treatment. In some embodiments, the initial dose is the first dose of SMAD7 ASO in any treatment cycle. For example, the initial dose is the first dose in the first treatment cycle, the second treatment cycle, or any subsequent treatment cycle.

[0121] In some embodiments, individuals with pouchitis are administered a subsequent dose of SMAD7 ASO, for example, SMAD7 ASO of SEQ ID NO: 1. As used herein, “subsequent dose” refers to the dose of SMAD7 ASO administered to an individual with pouchitis after the administration of a preceding dose, for example, an initial dose. In some embodiments, the subsequent dose is administered to an individual with pouchitis in a series of doses, including two or more doses. In some embodiments, the amount of the subsequent dose is calibrated relative to the initial or preceding dose so that the subsequent dose is greater than, equal to, or less than the preceding dose. In some embodiments, the subsequent dose is the first dose of SMAD7 ASO administered to the individual, for example, the dose administered to an individual with pouchitis after the initial dose. In some embodiments, the subsequent dose is the dose administered after the preceding dose of SMAD7 ASO administered to an individual with pouchitis, for example, the dose administered after the preceding dose in the same round of treatment or a different round of treatment, for example, the dose administered in a previous round of treatment. In some embodiments, the subsequent dose of a subsequent dose is a subsequent dose to any preceding dose, for example, a preceding dose immediately preceding the subsequent dose, or a preceding dose administered before the administration of the subsequent dose, and one or more subsequent doses.

[0122] The compositions, formulations, or methods of the present disclosure provide administration of oligonucleotides (e.g., SMAD7 oligonucleotides of SEQ ID NOs: 1, 2, 3, 4, 5, or 6) or pharmaceutically acceptable salts thereof to subjects refractory to a first treatment.

[0123] In some embodiments, subjects refractory to the first treatment are treated concurrently with or after the first treatment with an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NOs: 1, 2, 3, 4, 5, or 6) or a pharmaceutically acceptable salt thereof.

[0124] In some embodiments, the methods provided herein further include administering at least one other agent relating to the treatment of diseases and disorders disclosed herein (e.g., pouchitis). In some embodiments, the other agents intended are administered concurrently (e.g., sequentially or simultaneously). [Examples]

[0125] This disclosure is further illustrated by the following embodiments. The embodiments are provided for illustrative purposes only and should not be construed as limiting the scope or content of this disclosure.

[0126] (Example 1) Preparation of patient mucosal samples The mucosal samples prepared and used in the following examples (i.e., Examples 2-5) were collected from the inflamed pouch of 16 patients with chronic pouchitis (CP). The modified Pylungitis Disease Activity Index (mPDAI), assessed using clinical and endoscopic findings, was used to diagnose pouchitis, with pouchitis defined as mPDAI ≥ 5. Samples used as experimental controls included mucosal biopsy samples collected from the non-inflamed pouch of patients without clinical / endoscopic evidence of pouchitis, and ileal samples collected from the front of the normal / inflamed pouch of patients with chronic pouchitis and normal controls who underwent colonoscopy for a colorectal cancer screening program at Tor Vergata University Hospital (Rome, Italy).

[0127] (Example 2) SMAD7 increases in chronic pouchitis. This example evaluates SMAD7 expression in various mucosal samples by immunofluorescence staining.

[0128] In short, patient mucosal samples were prepared in the same manner as in Example 1. Sections of fresh mucosal biopsy samples were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton® X-100 for 20 minutes at room temperature. The sections were then blocked at room temperature for 1 hour (BSA 1%, Tween® 0.1%, glycine 2%) and incubated overnight at 4°C with mouse primary antibody against SMAD7 (1:150). After overnight incubation, the sections were washed with PBS 1× and treated with the secondary antibody, goat anti-mouse Alexa488 (1:1000), at room temperature for 1 hour. The slides were then washed with PBS 1×, mounted using a prolonged gold antifade reagent containing DAPI, and analyzed by microscopy.

[0129] The data are shown in Figures 1A–1C, which display sections of fresh patient mucosal biopsy specimens analyzed for SMAD7 expression (green) and DAPI (blue). These figures (Figures 1A–1C) are representative of three separate experiments that yielded similar results. Exemplary results of single and double immunofluorescence staining of sections from a healthy ileum (Figure 1A), a non-inflamed cyst (Figure 1B), and an inflamed cyst (Figure 1C) are shown. The scale bar is 50 μm.

[0130] The data demonstrate that SMAD7 expression, visualized as a green immunofluorescence signal, is increased in inflamed cyst samples compared to SMAD7 expression in healthy ileum or non-inflamed cyst sections.

[0131] (Example 3) SMAD7 protein expression increases in chronic pouchitis. This example evaluates SMAD7 protein expression in various mucosal samples by Western blotting, accompanied by corresponding quantitative analysis of Western blotting results obtained by densitometry scanning.

[0132] In short, mucosal biopsy samples used for Western blotting were dissolved on ice in a buffer containing 1 mM dithiothreitol (DTT), 10 mg / ml aprotinin, 10 mg / ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, and 1 mM sodium fluoride (NaF) supplemented with 10 mM HEPES (pH 7.9), 10 mM potassium chloride (KCl), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 0.5% Nonidet P40. The lysates were clarified by centrifugation and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The blots were incubated with an antibody against SMAD7 (1:1000) or an antibody against β-actin (1:5000), and then incubated with a secondary antibody conjugated to horseradish peroxidase (1:20000). The intensity of the immunoreactive bands was analyzed using computer-aided scanning densitometry.

[0133] The Western blotting results shown in Figures 2A-2B illustrate the expression of SMAD7 and β-actin proteins in total protein extracted from fresh mucosal biopsy samples, and include a corresponding analysis of the SMAD7 / β-actin ratio measured by Western blot densitometry scanning.

[0134] The data shown in Figures 2A and 2B demonstrate the Western blot detection of SMAD7 and β-actin in total protein extracted from mucosal biopsy samples, including corresponding quantitative graph analysis of the SMAD7 / β-actin ratio from the Western blotting results, measured by densitometry scanning. In Figure 2A, the analyzed total protein extracts were isolated from mucosal samples of the inflamed pouch (pilulitis), the normal pre-pilus enterocolitis of the same patient with pouchitis, the non-inflamed pouch, and the healthy ileum (normal control). In Figure 2B, the analyzed total protein extracts were isolated from mucosal samples of the inflamed pouch (pilulitis), the inflamed ileum (pre-pilus enterocolitis) of the same patient with pouchitis, and the healthy ileum (normal control).

[0135] The data demonstrate that SMAD7 protein expression was increased in mucosal samples from the inflamed pre-sacral and inflamed sacral regions compared to the levels of SMAD7 protein expression present in non-inflammatory and normal, healthy ileal mucosal samples.

[0136] (Example 4) SMAD7 co-localizes with CD3 and HLA-DRII in ileal pouchitis tissue. This example evaluates the co-localization of SMAD7, CD3, and HLA-DRII in ileal pouchitis tissue samples using immunofluorescence staining.

[0137] In short, sections of fresh mucosal biopsy specimens were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton® X-100 for 20 minutes at room temperature. The sections were then blocked at room temperature for 1 hour (BSA 1%, Tween® 0.1%, glycine 2%) and incubated overnight at 4°C with either rabbit primary antibody against CD3 (1:100) or rabbit primary antibody against HLA class II (1:100), and mouse primary antibody against SMAD7 (1:150). After washing with PBS 1×, the secondary antibody, goat anti-rabbit Alexa488 (1:1500) or goat anti-mouse Alexa488 (1:1000), was applied at room temperature for 1 hour. The slides were then washed with PBS 1×, mounted using a persistent gold anti-fade reagent containing DAPI, and analyzed by microscopy.

[0138] The data are shown in Figures 3A–3B, which display single and double immunofluorescence staining of sections of fresh mucosal biopsy samples taken from patients with chronic pouchitis, analyzed for SMAD7 (green), CD3 (red; Figure 3A), HLA-DRII (red; Figure 3B), and DAPI (blue). This figure (Figures 3A–3B) is representative of three separate experiments that yielded similar results. The scale bar is 75 μm.

[0139] The data in Figure 3A demonstrates that SMAD7 expression co-localizes with CD3 in pouchitis tissue, and Figure 3B demonstrates that SMAD7 expression co-localizes with HLA DRII in pouchitis tissue.

[0140] (Example 5) SMAD7 knockdown in ex vivo organ cultures alters the production of several inflammatory and anti-inflammatory molecules. This example evaluates the expression levels of SMAD7 and various inflammatory and anti-inflammatory molecules in ex vivo organ cultures transfected with SMAD7 sense oligonucleotides (S) or SMAD7 antisense oligonucleotides (AS). Exemplary results are shown in Figures 4A-4B.

[0141] In short, ex vivo organ cultures were prepared from mucosal samples collected from inflamed cysts and cultured in RPMI 1640 medium on a steel grid in an organ culture chamber at 37°C in a 5% CO2 / 95% O2 atmosphere. Ex vivo organ cultures were transfected with SMAD7 sense oligonucleotides or SMAD7 AS (10 μg / ml) for 24 hours using Opti-MEM medium and Lipofectamine 3000 reagent. Phosphothioate single-chain oligonucleotides matching the human SMAD7 complementary DNA sequence region 107-128 (5'-GCTGCGGGGAGAAGGGGCGAC-3') were synthesized in sense and antisense orientations.

[0142] For the real-time polymerase chain reaction results shown in Figure 4A, total RNA was extracted, and a fixed amount of RNA (1 μg / sample) was retrotranscribed to complementary DNA (cDNA) using oligo(dT) primers and M-MLV reverse transcriptase. This was amplified under the following conditions: denaturation at 95°C for 1 minute; annealing at 59°C for human SMAD7 and at 60°C for human β-actin for 30 seconds; extension at 72°C for 30 seconds. RNA expression was calculated for the β-actin gene using the ΔΔCt algorithm. The primer sequences were as follows: SMAD7 Fw 5'-GCCCGACTTCTTCATGGTGT-3', Rev 5'-TGCCGCTCCTTCAGTTTCTT-3'; β-actin FW 5'-AAGATGACCCAGATCATGTTTGAGACC-3', Rev 5'-AGCCAGTCCAGACGCAGGAT-3'.

[0143] The results of the human cytokine expression array assay shown in Figure 4B were obtained using the Human Cytokine Array C3. Briefly, supernatants from inflamed cyst samples transfected with either SMAD7 sense oligonucleotide or SMAD7 AS (10 μg / ml) were added to the membrane and incubated overnight at 4°C. The membrane was then incubated with biotin-conjugated antibody for 2 hours, followed by incubation with HRP-conjugated streptavidin for 30 minutes. Unbound reagents were removed by washing, and bound antibodies on the membrane were visualized using an ECL system. Quantitative analysis was performed by densitometry scanning of array blots.

[0144] In the statistical analysis, differences between groups were compared using Student's t-test. The correlation between SMAD7 and the modified pouchillitis disease activity index (mPDAI) was examined using Pearson's correlation coefficient.

[0145] The data in Figure 4A demonstrate that ex vivo organ cultures transfected with SMAD7 antisense oligonucleotides show reduced SMAD7 RNA expression compared to cultures transfected with SMAD7 sense oligonucleotides. The data in Figure 4B demonstrate that ex vivo organ cultures transfected with SMAD7 antisense oligonucleotides show altered expression profiles for several inflammatory and anti-inflammatory molecules compared to cultures transfected with SMAD7 sense oligonucleotides. In summary, these data indicate that SMAD7 ASO downregulates SMAD7 RNA production and alters the production of several inflammatory and anti-inflammatory molecules in organ cultures derived from inflamed cystic tissue.

[0146] Equal portions This disclosure may be embodied in other specific forms without departing from its essential features. Accordingly, the embodiments described herein should be considered illustrative and not limiting to the disclosures set forth herein. The scope of this disclosure is indicated by the appended claims rather than by the foregoing description, and all modifications within the meaning and equivalence of the claims are intended to be incorporated herein.

Claims

1. A method for treating pouchitis in an individual requiring treatment, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCCCCCXGCAGC-3').

2. The method according to claim 1, wherein all internucleoside bonds are O,O-linked phosphorothioate bonds, and X is 5-methyl2'-deoxycytidine.

3. The method according to any one of claims 1 to 2, wherein the SMAD7 antisense oligonucleotide is administered enterally.

4. The method according to claim 3, wherein the enteral administration is oral, sublingual, gastric, or rectal.

5. The method according to claim 3, wherein the enteral administration is rectal.

6. The method according to any one of claims 1 to 2, wherein the SMAD7 antisense oligonucleotide is administered parenterally.

7. The method according to claim 6, wherein the parenteral administration is intravenous, intratumoral, intrajejunal, intraileal, intracolonic, intrarectal, or intravesical.

8. The method according to claim 7, wherein the parenteral administration is performed intracapsulated.

9. The method according to any one of claims 1 to 8, wherein the therapeutically effective dose of SMAD7 ASO comprises a dose of approximately 20 mg, approximately 30 mg, 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg, approximately 160 mg, approximately 170 mg, approximately 180 mg, approximately 190 mg, approximately 200 mg, approximately 220 mg, approximately 240 mg, approximately 260 mg, approximately 280 mg, approximately 300 mg, approximately 320 mg, approximately 340 mg, approximately 360 mg, approximately 380 mg, approximately 400 mg, approximately 420 mg, approximately 440 mg, approximately 460 mg, approximately 480 mg, or approximately 500 mg.

10. The method according to any one of claims 1 to 8, wherein the therapeutically effective dose of SMAD7 ASO comprises a dose of about 20 mg to about 500 mg.

11. The method according to any one of claims 1 to 8, wherein the therapeutically effective dose of SMAD7 ASO comprises about 160 mg.

12. The method according to any one of claims 1 to 11, wherein the therapeutically effective dose of SMAD7 ASO is administered approximately every 6 hours, approximately every 12 hours, approximately every 24 hours, approximately every 48 hours, approximately every 72 hours, daily, twice a week, once every two weeks, or once a month.

13. The method according to any one of claims 1 to 12, wherein the therapeutically effective dose of SMAD7 ASO comprises a dose of approximately 160 mg per day.

14. The method according to any one of claims 1 to 13, wherein the SMAD7 ASO downregulates intracellular SMAD7 mRNA and / or protein.

15. The method according to claim 14, wherein the downregulation of SMAD7 protein expression is greater than 40% compared to untreated cells.

16. The method according to any one of claims 1 to 15, wherein at least one sample derived from the individual has an elevated SMAD7 level relative to a known control level, the known control level being the SMAD7 level in a sample taken from the individual before or during the treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual not having pouchitis.

17. The method according to any one of claims 1 to 16, wherein the pouchitis is acute pouchitis.

18. The method according to any one of claims 1 to 16, wherein the pouchitis is chronic pouchitis.

19. The method according to any one of claims 1 to 18, wherein the individual is determined to have pouchitis using at least five modified pouchitis disease activity index (mPDAI) scores.

20. A method for treating pouchitis in individuals requiring treatment for pouchitis, a) SMAD7 antisense oligonucleotides (ASOs) having a sequence according to Sequence ID No. 1 (5'-GTXGCCCCTTCCCXGCAGC-3'); and b) Pharmaceutically acceptable carriers A method comprising administering a pharmaceutical preparation containing to the individual.

21. The method according to claim 20, wherein the pharmaceutical preparation contains 100 mmol to 5 mol of the SMAD7 ASO.

22. The method according to claim 20, wherein the pharmaceutical preparation contains SMAD7 ASO in amounts of 100 mmol to 2 mol, 100 mmol to 900 mmol, 300 mmol to 5 mol, 300 mmol to 2 mol, 300 mmol to 900 mmol, 900 mmol to 5 mol, or 900 mmol to 2 mol.

23. The method according to claim 20, wherein the pharmaceutical preparation comprises about 300 mmol of the SMAD7 ASO.

24. The method according to claim 20, wherein the pharmaceutical preparation comprises about 900 millimoles of the SMAD7 ASO.

25. The method according to claim 20, wherein the pharmaceutical preparation comprises about 2 moles of the SMAD7 ASO.

26. The method according to claim 20, wherein the pharmaceutical preparation contains more than 300 mmol, more than 900 mmol, or more than 2 moles of the SMAD7 ASO.

27. The method according to any one of claims 20 to 26, wherein the pharmaceutical preparation is administered orally.

28. The aforementioned pharmaceutical preparation Approximately 0.5% to approximately 30% by weight of the SMAD7 ASO; Approximately 20% to 50% by weight of mannitol; Approximately 10% to 30% by weight of microcrystalline cellulose; and Enteric coating containing ethyl acrylate-methacrylic acid copolymer The method according to claim 27, including the method described in claim 27.

29. The aforementioned pharmaceutical preparation a) i) Approximately 5% to 10% by weight of the SMAD7 ASO; ii) Approximately 40% by weight of mannitol; iii) Approximately 8% by weight of microcrystalline cellulose; iv) Approximately 5% by weight of hydroxypropyl methylcellulose; and v) Approximately 2% by weight of sodium starch glycolate Granular interior phase containing; b) i) Approximately 17% by weight of microcrystalline cellulose; ii) Approximately 2% by weight of sodium starch glycolate, iii) Approximately 0.4% by weight of magnesium stearate Granular external phase including; and c) Enteric coating containing ethyl acrylate-methacrylic acid copolymer Includes, The weight percentage is the weight of the component compared to the total weight of the pharmaceutical preparation. The method according to any one of claims 27.

30. The aforementioned pharmaceutical preparation Approximately 5% to 30% by weight of the SMAD7 ASO; Approximately 20% to 50% by weight of mannitol; Approximately 10% to 30% by weight of microcrystalline cellulose, Approximately 0.5% to 10% by weight of hydroxypropyl methylcellulose; Approximately 0.5% to 10% by weight of sodium starch glycolate; Approximately 0.05% to 1% by weight of magnesium stearate; A moisture-proof film coating of approximately 0.5% to 10% by weight; and Polymer coating of approximately 5% to 20% by weight The method according to claim 27, including the method described in claim 27.

31. The aforementioned pharmaceutical preparation Approximately 8.5% by weight of the SMAD7 ASO; Approximately 40% by weight of mannitol; Approximately 25% by weight of microcrystalline cellulose, Approximately 5% by weight of hydroxypropyl methylcellulose; Approximately 4% by weight of sodium starch glycolate; Approximately 0.4% by weight of magnesium stearate; Approximately 4% by weight moisture-proof film coating; and Approximately 10% to 15% by weight polymer coating The method according to claim 27, including the method described in claim 27.

32. The aforementioned pharmaceutical preparation Approximately 23% by weight of the SMAD7 ASO; Approximately 28% by weight of mannitol; Approximately 25% by weight of microcrystalline cellulose, Approximately 5% by weight of hydroxypropyl methylcellulose; Approximately 4% by weight of sodium starch glycolate; Approximately 0.4% by weight of magnesium stearate; Approximately 4% by weight moisture-proof film coating; and Polymer coating of approximately 7% to 12% by weight The method according to claim 27, including the method described in claim 27.

33. The method according to any one of claims 20 to 32, wherein the pharmaceutical formulation downregulates intracellular SMAD7 mRNA and / or protein.

34. The method according to claim 33, wherein the downregulation of SMAD7 protein expression is greater than 40% compared to untreated cells.

35. The method according to any one of claims 20 to 34, wherein at least one sample derived from the individual has an elevated SMAD7 level relative to a known control level, the known control level being the SMAD7 level in a sample taken from the individual before or during the treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual not having pouchitis.

36. The method according to any one of claims 20 to 35, wherein the pouchitis is acute pouchitis.

37. The method according to any one of claims 20 to 35, wherein the pouchitis is chronic pouchitis.

38. The method according to any one of claims 20 to 37, wherein the individual is determined to have pouchitis using at least five modified pouchitis disease activity index (mPDAI) scores.

39. A method for predicting pouchitis in an individual requiring prediction of pouchitis, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, and the known control level is the SMAD7 level in a sample taken from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual without pouchitis.

40. A method for identifying an individual at risk of pouchitis, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual at risk of pouchitis, and the known control level is the SMAD7 level in a sample taken from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual without pouchitis.

41. (i) If the SMAD7 level is elevated compared to the known control level, administer a pharmaceutical formulation containing SMAD7 ASO to the individual; or (ii) If the SMAD7 level has not risen above the known control level, determine the level of SMAD7 in the second sample derived from the individual. The method according to claim 39 or 40, including the method described in claim 39 or 40.

42. The method of claim 41, wherein the second sample is collected immediately after the first sample, approximately 1 hour later, approximately 3 hours later, approximately 6 hours later, approximately 12 hours later, approximately 1 day later, approximately 3 days later, approximately 1 week later, approximately 2 weeks later, approximately 1 month later, approximately 2 months later, approximately 3 months later, approximately 4 months later, approximately 5 months later, approximately 6 months later, approximately 7 months later, approximately 8 months later, approximately 9 months later, approximately 10 months later, approximately 11 months later, or approximately 12 months later.

43. A method for treating pouchitis in an individual requiring treatment for pouchitis, comprising determining the level of SMAD7 in a first sample derived from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, and the known control level is the SMAD7 level in a sample taken from the individual before or during the treatment for pouchitis, or the SMAD7 level in a sample taken from a healthy individual not having pouchitis.

44. The method according to any one of claims 39 to 42, wherein the SMAD7 ASO comprises a sequence selected from any one of sequence numbers 1 to 6 or a pharmaceutically acceptable salt thereof.

45. The method according to any one of claims 39 to 42, wherein the SMAD7 ASO has a sequence according to sequence number 1 (5'-GTXGCCCCTTCCCXGCAGC-3').

46. The method according to claim 45, wherein all internucleoside bonds are O,O-linked phosphorothioate bonds, and X is 5-methyl2'-deoxycytidine.

47. The method according to any one of claims 1 to 46, wherein the individual is a human.

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