Smad7 inhibitory antisense oligonucleotides (ASO) for treating pouchitis and methods of using the same

Abstract

Provided herein are methods for treating or preventing pouch inflammation comprising administering a SMAD7 antisense oligonucleotide or a pharmaceutical formulation comprising the SMAD7 antisense oligonucleotide.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480022224.4 (22) Application Date 2024.03.27 (30) Priority Data 63 / 492,755 2023.03.28 US (85) PCT International Application Entering National Phase Date 2025.09.26 (86) PCT International Application Application Data PCT / EP2024 / 058390 2024.03.27 (87) PCT International Application Publication Data WO2024 / 200594 EN 2024.10.03 (71) Applicant: Norgra Pharmaceuticals Ltd. Address: Ireland (72) Inventors: S. Bellingvia, F. Vitti, M. McNutrie, S. Dematis (74) Patent Agency: China Council for the Promotion of International Trade Patent & Trademark Office Co., Ltd. 11038 Patent Attorney: Zhang Xiaoyong (51) Int.Cl. C12N 15 / 113 (2006.01) A61K 31 / 7088 (2006.01) (54) Invention Title: SMAD7 Inhibitory Antisense Oligonucleotide (ASO) for the Treatment of Pouchitis and Method of Using Thereof (57) Abstract: This document provides a method for the treatment or prevention of pouchitis, including the administration of SMAD7 antisense oligonucleotide or a pharmaceutical preparation containing the SMAD7 antisense oligonucleotide. Claims 4 pages, Description 24 pages, Drawings 4 pages, CN 120958131 A 2025.11.14 CN 1 20 95 81 31 A 1. A method of treating pouchitis in an individual in need, comprising administering to the individual a therapeutically effective amount of SMAD7 antisense oligonucleotide (ASO) having the sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'). 2. The method of claim 1, wherein all nucleoside linkages are O,O-linked phosphate thioester linkages and X is 5-methyl-2'-deoxycytidine. 3. The method of any one of claims 1-2, wherein the SMAD7 antisense oligonucleotide is administered enterically. 4. The method of claim 3, wherein enteric administration is oral, sublingual, gastric, or rectal administration. 5. The method of claim 3, wherein enteric administration is rectal administration. 6. The method of any one of claims 1-2, wherein the SMAD7 antisense oligonucleotide is administered parenterally. 7. The method of claim 6, wherein parenteral administration is administered intravenously, intratumorally, intrajejunally, intraileally, intracolonally, intrarectally, or via a pouch. 8. The method of claim 7, wherein parenteral administration is administered via a pouch. 9. The method of any one of claims 1-8, wherein the therapeutically effective amount of SMAD7ASO comprises about 20 mg, about...Doses of 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. 10. The method of any one of claims 1-8, wherein the therapeutically effective amount of SMAD7 ASO comprises a dose of about 20 mg to about 500 mg. 11. The method of any one of claims 1-8, wherein the therapeutically effective amount of SMAD7 ASO comprises about 160 mg. 12. The method of any one of claims 1-11, wherein the therapeutically effective amount of SMAD7 ASO is administered every about 6 hours, every about 12 hours, every about 24 hours, every about 48 hours, every about 72 hours, daily, twice weekly, once every two weeks, or once monthly. 13. The method of any one of claims 1-12, wherein the therapeutically effective amount of SMAD7 ASO comprises a daily dose of about 160 mg. 14. The method of any one of claims 1-13, wherein SMAD7 ASO downregulates SMAD7 mRNA and / or protein in cells. 15. The method of claim 14, wherein the downregulation of SMAD7 protein expression is greater than 40% compared to untreated cells. 16. The method of any one of claims 1-15, wherein at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in samples collected from the individual before or during treatment for pouchitis, or the SMAD7 level in samples collected from healthy individuals without pouchitis. 17. The method of any one of claims 1-16, wherein the pouchitis is acute pouchitis. 18. The method of any one of claims 1-16, wherein the pouchitis is chronic pouchitis. 19. The method of any one of claims 1-18, wherein the individual is identified as having pouchitis using a Modified Pouchitis Disease Activity Index (mPDAI) score of at least 5. 20. A method of treating pouchitis in an individual in need, comprising administering a pharmaceutical preparation to the individual, said pharmaceutical preparation comprising: a) a SMAD7 antisense oligonucleotide (ASO) having the sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'); and claims 1 / 4 page 2 CN 120958131 Ab) A pharmaceutically acceptable carrier. 21. The method of claim 20, wherein the pharmaceutical formulation comprises 100 mmol to 5 mol of SMAD7 ASO. 22. The method of claim 20, wherein the pharmaceutical formulation comprises 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 of SMAD7 ASO. 23. The method of claim 20, wherein the pharmaceutical formulation comprises about 300 mmol of SMAD7 ASO. 24. The method of claim 20, wherein the pharmaceutical formulation comprises about 900 mmol of SMAD7 ASO. 25. The method of claim 20, wherein the pharmaceutical formulation comprises about 2 mol of SMAD7 ASO. 26. The method of claim 20, wherein the pharmaceutical formulation comprises greater than 300 mmol, greater than 900 mmol, or greater than 2 mol of SMAD7 ASO. 27. The method of any one of claims 20-26, wherein the pharmaceutical formulation is administered orally. 28. The method of claim 27, wherein the pharmaceutical formulation comprises: 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 ethyl acrylate-methacrylic acid copolymer. 29. The method of any one of claims 27, wherein the pharmaceutical formulation comprises: a) an intragranular phase comprising: 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 carboxymethyl starch; b) an extragranular phase comprising: i) about 17% by weight of microcrystalline cellulose; ii) about 2% by weight of sodium carboxymethyl starch; iii) about 0.4% by weight of magnesium stearate; and c) an enteric coating comprising ethyl acrylate-methacrylic acid copolymer, wherein the percentages by weight are the weight of the components relative to the total weight of the pharmaceutical formulation. 30. The method of claim 27, wherein 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 carboxymethyl starch; about 0.05% to about 1% by weight of magnesium stearate; about 0.5% to about 10% by weight of a moisture-proof film coating; and about 5% to about 20% by weight of a polymer coating.31. The method of claim 27, wherein the pharmaceutical formulation comprises: (Claims 2 / 4, page 3, CN 120958131 A) about 8.5% SMAD7 ASO by weight; about 40% mannitol by weight; about 25% microcrystalline cellulose by weight; about 5% hydroxypropyl methylcellulose by weight; about 4% sodium carboxymethyl starch by weight; about 0.4% magnesium stearate by weight; about 4% moisture-proof film coating by weight; and about 10% to about 15% polymer coating by weight. 32. The method of claim 27, wherein the pharmaceutical formulation comprises: about 23% SMAD7 ASO by weight; about 28% mannitol by weight; about 25% microcrystalline cellulose by weight; about 5% hydroxypropyl methylcellulose by weight; about 4% sodium carboxymethyl starch by weight; about 0.4% magnesium stearate by weight; about 4% moisture-proof film coating by weight; and about 7% to about 12% polymer coating by weight. 33. The method of any one of claims 20-32, wherein the pharmaceutical formulation downregulates SMAD7 mRNA and / or protein in cells. 34. The method of claim 33, wherein the downregulation of SMAD7 protein expression is greater than 40% compared to untreated cells. 35. The method of any one of claims 20-34, wherein at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment of pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis. 36. The method of any one of claims 20-35, wherein the pouchitis is acute pouchitis. 37. The method of any one of claims 20-35, wherein the pouchitis is chronic pouchitis. 38. The method of any one of claims 20-37, wherein the individual is identified as having pouchitis using a Modified Pouchitis Disease Activity Index (mPDAI) score of at least 5. 39. A method for predicting pouchitis in an individual in need, comprising determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment of pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis. 40. A method for identifying an individual at risk of developing pouchitis, comprising: determining a level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual as being at risk of developing pouchitis, wherein the known control level is SMAD7 in a sample collected from the individual before or during treatment for pouchitis.41. The method of claim 39 or 40, comprising: (i) administering a pharmaceutical preparation containing SMAD7 ASO to the individual if the SMAD7 level is elevated relative to a known control level; or (ii) determining the SMAD7 level in a second sample from the individual if the SMAD7 level is not elevated relative to a known control level. 42. The method of claim 41, wherein the second sample is collected immediately after the first sample, about 1 hour after the first sample, about 3 hours after the first sample, about 6 hours after the first sample, about 12 hours after the first sample, about 1 day after the first sample, about 3 days after the first sample, about 1 week after the first sample, about 2 weeks after the first sample, about 1 month after the first sample, about 2 months after the first sample, about 3 months after the first sample, about 4 months after the first sample, about 5 months after the first sample, about 6 months after the first sample, about 7 months after the first sample, about 8 months after the first sample, about 9 months after the first sample, about 10 months after the first sample, about 11 months after the first sample, or about 12 months after the first sample. 43. A method for treating pouchitis in an individual in need, comprising determining the SMAD7 level in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment of pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis. 44. The method of any one of claims 39-42, wherein the SMAD7 ASO comprises a sequence selected from any one of SEQ ID NO:1-6 or a pharmaceutically acceptable salt thereof. 45. The method of any one of claims 39-42, wherein the SMAD7 ASO has a sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'). 46. The method of claim 45, wherein all nucleoside linkages are O,O-linked phosphate thioester linkages and X is 5-methyl-2'-deoxycytidine. 47. The method of any one of claims 1-46, wherein the individual is a human. Claims 4 / 4 Page 5 CN 120958131 A SMAD7 inhibitory antisense oligonucleotide (ASO) for the treatment of pouch inflammation and method of use thereof

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 492,755, filed March 28, 2023.Priority, the entire contents of this application are incorporated herein by reference.

[0003] Overview

[0004] This document provides methods for treating or preventing pouchitis in an individual in need, the methods comprising administering a therapeutically effective amount of a SMAD7 antisense oligonucleotide or a pharmaceutical preparation containing said SMAD7 antisense oligonucleotide.

[0005] In some embodiments, this document describes a method for treating pouchitis in an individual in need, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO) having the sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'). In some embodiments, all nucleoside linkages are O,O-linked phosphate thioester linkages and X is 5-methyl-2'-deoxycytidine. In some embodiments, said SMAD7 antisense oligonucleotide is administered enterically. In some embodiments, enteric administration is oral, sublingual, gastric, or rectal administration. In some embodiments, enteric administration is rectal administration. In some embodiments, said SMAD7 antisense oligonucleotide is administered parenterally. In some implementations, parenteral administration is administered intravenously, intratumorally, intrajejunally, intraileally, intracolonally, intrarectally, or via a pouch. In some implementations, parenteral administration is administered via a pouch. In some embodiments, the therapeutically effective amount of SMAD7 ASO includes doses of about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, the therapeutically effective amount of SMAD7 ASO includes doses from about 20 mg to about 500 mg. In some embodiments, the therapeutically effective amount of SMAD7 ASO is administered every approximately 6 hours, every approximately 12 hours, every approximately 24 hours, every approximately 48 hours, every approximately 72 hours, daily, twice weekly, once every two weeks, or once monthly. In some embodiments, the therapeutically effective amount of SMAD7 ASO comprises a dose of approximately 160 mg daily. In some embodiments, the SMAD7 ASO downregulates SMAD7 mRNA and / or protein in cells. In some embodiments, the downregulation of SMAD7 protein expression is greater than 40% compared to untreated cells. In some embodiments, the SMAD7 ASO is derived from...At least one sample from an individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment of pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis. In some embodiments, the pouchitis is acute pouchitis. In some embodiments, the pouchitis is chronic pouchitis. In some embodiments, the individual is identified as having pouchitis using a Modified Pouchitis Disease Activity Index (mPDAI) score of at least 5.

[0006] In some embodiments, a method for treating pouchitis in an individual in need is described herein, comprising administering a pharmaceutical preparation to the individual comprising: a) an SMAD7 antisense oligonucleotide (ASO) having the sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'); and b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical preparation comprises 100 mmol to 5 mol of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises 100 mmol to 2 mmol, 100 mmol to 900 mmol, 300 mmol to 5 mmol, 300 mmol to 2 mmol, 300 mmol to 900 mmol, 900 mmol to 5 mmol, or 900 mmol to 2 mmol of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises about 300 mmol of the SMAD7 ASO described in the specification (page 1 / 24, 6 CN 120958131 A). In some embodiments, the pharmaceutical formulation comprises about 900 mmol of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises about 2 mmol of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises more than 300 mmol, more than 900 mmol, or more than 2 mmol of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation is administered orally. In some embodiments, the pharmaceutical formulation comprises: about 0.5% to about 30% by weight of the 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 ethyl acrylate-methacrylic acid copolymer. In some embodiments, the pharmaceutical formulation comprises: a) an intragranular phase comprising: i) about 5% to about 10% by weight of the 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 carboxymethyl starch; b) an extragranular phase comprising: i) about 17% by weight of microcrystalline cellulose.The pharmaceutical formulation comprises: ii) about 2% sodium carboxymethyl starch by weight; iii) about 0.4% magnesium stearate by weight; and c) an enteric coating comprising ethyl acrylate-methacrylic acid copolymer, wherein the percentages by weight 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% of the SMAD7 ASO by weight; about 20% to about 50% mannitol by weight; about 10% to about 30% microcrystalline cellulose by weight; about 0.5% to about 10% hydroxypropyl methylcellulose by weight; about 0.5% to about 10% sodium carboxymethyl starch by weight; about 0.05% to about 1% magnesium stearate by weight; about 0.5% to about 10% moisture-proof film coating by weight; and about 5% to about 20% polymer coating by weight. In some embodiments, the pharmaceutical formulation comprises: about 8.5% by weight of the 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 carboxymethyl starch; about 0.4% by weight of magnesium stearate; about 4% by weight of a moisture-proof 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 the 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 carboxymethyl starch; about 0.4% by weight of magnesium stearate; about 4% by weight of a moisture-proof film coating; and about 7% to about 12% by weight of a polymer coating. In some embodiments, the pharmaceutical formulation downregulates SMAD7 mRNA and / or protein in cells. In some embodiments, the downregulation of SMAD7 protein expression is greater than 40% compared to untreated cells. In some embodiments, at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment of pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis. In some embodiments, the pouchitis is acute pouchitis. In some embodiments, the pouchitis is chronic pouchitis. In some embodiments, the individual is identified as having pouchitis using a Modified Pouchitis Disease Activity Index (mPDAI) score of at least 5. A method for predicting pouchitis in an individual in need, the method comprising determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, wherein the known control level is...The SMAD7 level in samples collected from the individual before or during treatment of pouchitis, or the SMAD7 level in samples collected from a healthy individual without pouchitis.

[0007] In some embodiments, a method for identifying an individual at risk of pouchitis is described herein, the method comprising: determining the SMAD7 level in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual as being at risk of pouchitis, wherein the known control level is the SMAD7 level in samples collected from the individual before or during treatment of pouchitis, or the SMAD7 level in samples collected from a healthy individual without pouchitis. In some embodiments, the method comprises: administering a pharmaceutical preparation containing SMAD7 ASO to the individual if the SMAD7 level is elevated relative to a known control level; or determining the SMAD7 level in a second sample from the individual if the SMAD7 level is not elevated relative to a known control level. In some implementations, the second sample is collected immediately after the first sample, approximately 1 hour after the first sample, approximately 3 hours after the first sample, approximately 6 hours after the first sample, approximately 12 hours after the first sample, approximately 1 day after the first sample, approximately 3 days after the first sample, approximately 1 week after the first sample, approximately 2 weeks after the first sample, approximately 1 month after the first sample, approximately 2 months after the first sample, approximately 3 months after the first sample, approximately 4 months after the first sample, approximately 5 months after the first sample, approximately 6 months after the first sample, approximately 7 months after the first sample, approximately 8 months after the first sample, approximately 9 months after the first sample, approximately 10 months after the first sample, approximately 11 months after the first sample, or approximately 12 months after the first sample.

[0008] In some embodiments, a method for treating pouch inflammation in an individual in need is described herein, the method comprising determining the SMAD7 level in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouch inflammation, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment of pouch inflammation, or the SMAD7 level in a sample collected from a healthy individual without pouch inflammation. In some embodiments, the SMAD7 ASO comprises a sequence selected from any one of SEQ ID NO: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 linkages are O,O-linked phosphate thioester linkages and X is 5-methyl-2'-deoxyCytidine. In some embodiments, the individual is a human.

[0009] Brief Description of the Drawings

[0010] In order to understand this disclosure and demonstrate how to put it into practice, embodiments are now described by way of non-limiting example only, referring to the accompanying drawings, in which:

[0011] Figures 1A-1C depict representative images showing single and double immunofluorescence staining of fresh mucosal biopsy samples, analyzing the expression of SMAD7 (green) and DAPI (blue). Figure 1A shows the staining of a section of healthy ileum. Figure 1B shows the staining of a section of uninflamed reticulum. Figure 1C shows the staining of a section of inflamed reticulum. The scale bar shown in all figures is 50 μm. The figure represents three separate experiments in which similar results were obtained.

[0012] Figures 2A-2B depict representative Western blot images showing SMAD7 and β-actin in total protein extracted from mucosal biopsy samples, and quantitative graphical analysis of the SMAD7 / β-actin ratio of the Western blot measured by densitometric scanning. Figure 2A shows the Western blot (left) of total protein detected using SMAD7 (top) and a β-actin loading control (bottom), extracted from mucosal samples of inflamed pouches (pouchitis, n=10), normal pouches and pre-ileum from the same patient with pouchitis (n=5), uninflamed pouches (n=3), and normal controls (healthy ileum, n=4). The right panel of Figure 2A shows the quantitative analysis of the SMAD7 / β-actin ratio as measured by density assay scanning of the Western blot. Values ​​are expressed in arbitrary units (a.u.) and indicate the mean ± SEM of all samples (*p<0.05, **p<0.01, ***p<0.001). Figure 2B shows a Western blot (left) of total protein detected using SMAD7 (top) and a β-actin loading control (bottom), extracted from mucosal samples of inflamed pouches (pouchitis, n=10), inflamed ileum from the same patient with pouchitis (pre-pouch ileitis, n=3), uninflamed 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 density assay scanning of the Western blot. Values ​​are expressed in arbitrary units (a.u.) and indicate the mean ± SEM of all samples (*p<0.05, **p<0.01).

[0013] Figures 3A-3B depict representative images showing single and double immunofluorescence staining. Figure 3A shows images of the expression of SMAD7 (green), CD3 (red), and DAPI (blue) in fresh mucosal biopsy samples from patients with chronic pouchitis. Figure 3B shows the analysis instructions for fresh mucosal biopsy samples from patients with chronic pouchitis, page 3 / 24, 8 CN.120958131 A Images of the expression of SMAD7 (green), HLA-DRII (red), and DAPI (blue). Scale bars shown in all figures are 75 μm. This figure represents three separate experiments in which similar results were obtained.

[0014] Figures 4A-4B show the relative expression levels of various inflammatory and anti-inflammatory molecules in ex vivo organ cultures transfected with SMAD7 sense oligonucleotides (S) or SMAD7 antisense oligonucleotides (AS) for 24 hours. Figure 4A shows the relative expression of SMAD7 in SMAD7 RNA transcripts as assessed by real-time polymerase chain reaction (y-axis). Levels are normalized to β-actin. Values ​​indicate the mean ± SD of the three experiments. Differences were analyzed using a two-tailed Student's t-test (*p<0.005). Figure 4B shows the analysis of cell-free culture supernatant analyzed using a commercial protein array kit. The right-hand heatmap shows differential protein expression scans based on array blot density measurements. Log2 (fold change) of ex vivo organ cultures transfected with AS and S.

[0015] Detailed Description

[0016] Pouchitis is an inflammation of the lining of a surgically created pouch for 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 frequency of bowel movements, abdominal cramps or bloating, lower abdominal pain, or sometimes blood in the stool. Current treatments for pouchitis are limited and usually involve antibiotics.

[0017] There is a need for improved treatments or preventative treatments for pouchitis. This article describes methods for treating or preventing pouchitis by administering oligonucleotides (e.g., SMAD7 antisense oligonucleotides).

[0018] Definition

[0019] The term “oligonucleotide” refers to a short DNA or RNA molecule. Oligonucleotides may consist of 2'-deoxyribonucleotides (oligodeoxyribonucleotides) that may be modified at the phosphate backbone or the 2' sugar position.

[0020] 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). Not bound by any particular theory, antisense oligonucleotide sequences can hybridize with complementary regions in mRNA molecules to produce double-stranded hybrids that can lead to activation by ubiquitous catalytic enzymes (e.g., RNase H) that degrade the DNA / RNA hybrid chain, thereby preventing protein translation. Not bound by any theory, the antisense oligonucleotides provided herein can hybridize with their target sequences as either RNA or DNA. Therefore, even when a DNA sequence is provided as a target, the corresponding RNA sequence (including uracil but not thymine) is included as an ASO. “Phosphothioester antisense oligonucleotide” or “PS…”"ASO" is an antisense oligonucleotide modified to have a phosphate thioester backbone.

[0021] As used herein, "Mothers against decapentaplegic homolog7" ("SMAD7", also known as CRCS3, FLJ16482, MADH7, MADH8, MAD (mothers against decapentaplegic, Drosophila) homolog 7, MAD homolog 8, SMAD, mothers against DPP homolog 7, mothers against DPP homolog 8) refers to the human protein encoded by the gene identified by Entrez GeneID No. 4092 or any mRNA transcript thereof and its allele variants.

[0022] As used herein, "SMAD7 antisense oligonucleotide" or "SMAD7 ASO" is understood to mean an oligonucleotide containing a nucleic acid sequence complementary to a nucleic acid sequence in an mRNA molecule transcribed from the SMAD7 gene. More specifically, such an oligonucleotide may be complementary to a nucleic acid sequence in the coding region of such mRNA.

[0023] As used herein, the term “pouch inflammation” refers to a disease or condition characterized by inflammation of the pouch. As used herein, the term “pouch” can refer to an artificial rectum surgically created from the ileum (the last part of the small intestine) in an individual who has undergone a total colectomy (proctocolectomy or total colectomy) (removal of the colon and rectum) to manage conditions such as ulcerative colitis, undifferentiated colitis, familial adenomatous polyposis, cancer, or other colitis. Individuals can be identified as having pouch inflammation using the Pouch Inflammation Disease Activity Index (PDAI) or the Modified Pouch Inflammation Disease Activity Index (mPDAI) scoring system. Individuals assessed with a total PDAI score of 7 or higher are classified as having pouch inflammation. Individuals assessed with a total mPDAI score of 5 or higher are classified as having pouch inflammation.

[0024] The term “acute pouchitis” refers to pouchitis in which symptoms persist for less than or equal to four weeks and the disease responds to a two-week course of antibiotics.

[0025] The term “chronic pouchitis” refers to pouchitis in which symptoms persist for four weeks or longer and / or despite treatment with a standard course of antibiotics.

[0026] The terms “receptor,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and in some embodiments refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. For treatmentPurpose: “mammal” means any animal classified as a mammal, including humans, domestic and farm animals, as well as laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. These terms do not require medical supervision.

[0027] The terms “disease,” “symptom,” and “condition” are used interchangeably herein.

[0028] As used herein, the terms “treatment,” “treating,” etc., refer to the administration of an agent or the performance of a procedure to achieve an effect. This effect may be preventative, in relation to the complete or partial prevention of the disease or its symptoms, and / or may be therapeutic, in relation to the partial or complete cure of the disease and / or its symptoms. As used herein, “treatment” can include treating a disease or condition (e.g., pouchitis) in mammals (particularly humans) and includes: (a) preventing the occurrence of the disease or its symptoms in subjects who may be susceptible to the disease but have not yet been diagnosed with it (e.g., including diseases that may be related to or caused by a primary disease); (b) suppressing the disease, i.e., halting its development; and (c) alleviating the disease, i.e. causing its regression. Treatment can refer to any sign of success in treating, alleviating, or preventing cancer, including any objective or subjective parameter such as reduction; relief; lessening of symptoms; or making the disease condition more tolerable for the patient; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. Treatment or alleviation of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term “treatment” includes administering the compounds or agents of this disclosure to prevent, delay, alleviate, halt, or suppress the development of symptoms or conditions associated with a disease (e.g., pouchitis). The term “treatment effect” refers to a reduction, elimination, or prevention of the disease, its symptoms, or its side effects in a subject.

[0029] As used herein, “preventing” or “prevent” describes reducing or eliminating the occurrence of symptoms or complications of a disease, condition, or symptom. The term “prevention” is recognized in the art when used in relation to a condition (e.g., recurrence of inflammation or pouchitis) 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 to delay the occurrence of signs and / or symptoms of a medical condition in a subject relative to a subject who has not received the formulation, composition, and / or device. Given that the methods of this disclosure are intended to prevent a condition, it should be understood that the term “prevention” does not require the complete prevention of a disease state.

[0030] The term “therapeutic effective amount” or “therapeutic dose” generally refers to the amount of an antibody or drug that effectively “treats” a disease or symptom in a subject or mammal. In some embodiments, it is used to achieve a reasonable benefit / advantage suitable for any medical treatment.The risk ratio is the amount of the composition described herein administered to a subject that effectively produces some desired therapeutic effect by inhibiting a disease or condition as described herein. A therapeutically effective amount is the amount that achieves at least a portion of the desired therapeutic or preventative effect in an organ or tissue. The amount of antibody or drug required to bring about preventative and / or therapeutic treatment of a disease or condition is not fixed in itself. In some embodiments, the amount of antibody or drug administered varies depending on the type of disease, the extent of the disease, and the size of the mammal suffering from the disease or condition. When used in conjunction with a treatment method involving the administration of a therapeutic agent after the subject has developed symptoms of a disease or condition, the term "therapeutically effective" means that one or more signs or symptoms of the disease or condition are improved or eliminated after treatment.

[0031] As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents.

[0032] As used herein, unless the context clearly indicates otherwise, all numerical values ​​or ranges include all integers within or covering those ranges, as well as fractions of values ​​or integers within or covering those ranges. Thus, for example, references to the range of 90-100% include 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 so on. In another instance, references to the range of 1-5,000 times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, etc., and 1.1, 1.2, 1.3, 1.4, 1.5 times, etc., 2.1, 2.2, 2.3, 2.4, 2.5 times, etc., etc.

[0033] As used herein, “about” a number means a range including the number and from 10% below the number to 10% above the number. “about” a range means 10% below the lower limit of the range and spanning 10% above the upper limit of the range.

[0034] Throughout the specification and claims of this application, the word “comprise” and other forms of the word (e.g., “comprising” and “comprises”) mean, but are not intended to exclude, for example, other additives, components, integers, or steps.

[0035] "Percentage (%) identity" refers to the degree to which two sequences (nucleic acids or amino acids) have the same residues at the same position in the alignment. For example, "amino acid sequence is identical to SEQ ID NO: YX%" means that the amino acid sequence is identical to SEQ ID NO: YX%.The identity percentage of NO:Y, and states that X% of the residues in the amino acid sequence are identical to the residues of the sequence disclosed in SEQ ID NO:Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs 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).

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the event of conflict, this specification shall prevail. Although this disclosure may be practiced or tested using methods and materials similar to or equivalent to those described and used herein, suitable methods and materials are described below.

[0037] SMAD7 Oligonucleotide

[0038] In some embodiments herein, methods for treating or preventing pouchitis are described by administering a therapeutically effective amount of a SMAD7 oligonucleotide (e.g., a SMAD7 antisense oligonucleotide (ASO)) or a pharmaceutical preparation containing the SMAD7 oligonucleotide. In some embodiments, the SMAD7 oligonucleotide is a SMAD7 antisense oligonucleotide (ASO).

[0039] In some embodiments, the SMAD7 ASO comprises DNA. In some embodiments, the SMAD7 ASO comprises RNA.

[0040] In some embodiments, the SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), wherein X is 5-methyl-2'-deoxycytidine. In some embodiments, the SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), wherein X is 5-methyl-2'-deoxycytidine. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), wherein X is 5-methyl-2'-deoxycytidine.

[0041] In some embodiments, the SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO:2 (5'-GTXGCCCCTTCTCTCXGCAGC-3'), wherein X is 5-methyl-2'-deoxycytidine. In some...In some embodiments, the SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO:2. In some embodiments, the SMAD7 ASO has the sequence according to SEQ ID NO:2.

[0042] In some embodiments, the SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO:3 (5'-GTXYCCCCTTCTCCCXYCAG-3'), wherein X is a nucleotide containing a nitrogenous base including cytosine, 5-methylcytosine, or 2'-O-methylcytosine, and Y is a nucleotide containing a nitrogenous base including guanine, 5-methylguanine, or 2'-O-methylguanine, provided that at least one of X and Y includes a methylated nitrogenous base. In some embodiments, the SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO:3. In some embodiments, the SMAD7 ASO has the sequence according to SEQ ID NO:3.

[0044] In some embodiments, the SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO:4 (5'-GTXGCCCCTTCTCCCXGCAG-3'), wherein X is 5-methyl-2'-deoxycytidine. In some embodiments, the SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO:4. In some embodiments, the SMAD7 ASO has the sequence according to SEQ ID NO:4.

[0045] In some embodiments, the SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO:5 (5'-GTC*GCCCCTTCTCCCC*YCAGC-3'), wherein C* is 5-methyl-2'-deoxycytidine and Y is a nitrogenous nucleotide including guanine, 5-methylguanine, or 2'-O-methylguanine. In some embodiments, the SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO:5. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO:5.

[0046] In some embodiments, the SMAD7 ASO has a sequence that is at least 90% identical to SEQ ID NO:6 (5'-GTC*GCCCCTTCTCTCC*YCAGC-3'), wherein C* is 5-methyl-2'-deoxycytidine and Y is a guanine, 5-methylThe SMAD7 ASO is a nitrogenous nucleotide containing guanine or 2'-O-methylguanine. In some embodiments, the SMAD7 ASO has a sequence that is at least 95% identical to SEQ ID NO:6. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO:6.

[0047] In some embodiments, the SMAD7 ASO comprises naturally occurring nucleobases, sugars, and covalent nucleosides linked together (in the main chain), as well as non-naturally occurring portions. In some embodiments, the SMAD7 ASO of this disclosure, such as the 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, comprises nucleotides including deoxycytidine and / or 5-methyl-2'-deoxycytidine, including but not limited to 5-methyl-2'-deoxycytidine 5'-monophosphate and 5-methyl-2'-deoxycytidine 5'-monothiophosphate.

[0048] In some embodiments, the SMAD7 ASO (e.g., the 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) comprises one or more methylated cytosines. In some embodiments, any cytosine of the SMAD7 ASO is methylated. In some embodiments, one or more cytosines of the SMAD7 ASO are replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO:1 comprises one or more methylated cytosines. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO:1 is methylated. In some embodiments, one or more cytosines of the SMAD7 ASO of SEQ ID NO:1 are replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO:2 comprises one or more methylated cytosines. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO:2 is methylated. In some embodiments, one or more cytosines of the SMAD7 ASO of SEQ ID NO:2 are replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO:3 comprises one or more methylated cytosines. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO:3 is methylated. In some embodiments, one or more cytosines of the SMAD7 ASO of SEQ ID NO:3 are replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO:4 comprises one or more methylated cytosines.Methylated cytosine. In some embodiments, any cytosine of SEQ ID NO:4 SMAD7 ASO is methylated. In some embodiments, one or more cytosines of SEQ ID NO:4 SMAD7 ASO are replaced with 5-methylcytidine. In some embodiments, SEQ ID NO:5 SMAD7 ASO comprises one or more methylated cytosines. In some embodiments, any cytosine of SEQ ID NO:5 SMAD7 ASO is methylated. In some embodiments, one or more cytosines of SEQ ID NO:5 SMAD7 ASO are replaced with 5-methylcytidine. In some embodiments, SEQ ID NO:6 SMAD7 ASO comprises one or more methylated cytosines. In some embodiments, any cytosine of SEQ ID NO:6 SMAD7 ASO is methylated. In some embodiments, one or more cytosines of SEQ ID NO:6 SMAD7 ASO are replaced with 5-methylcytidine.

[0049] In some embodiments, the SMAD7 ASO is chemically modified. In some embodiments, the SMAD7 ASO comprises one or more nucleoside links modified from a natural phosphodiester. In some embodiments, all nucleoside links of the SMAD7 ASO or its sequential nucleotide sequence are modified. For example, in some embodiments, the nucleoside link comprises sulfur (S), such as a phosphate thioester nucleoside link.

[0050] In some embodiments, the SMAD7 ASO comprises a modification of the ribose sugar or nucleobase. In some embodiments, the SMAD7 ASO comprises one or more nucleosides containing a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribonucleic acid (DNA) and RNA. In some embodiments, the modification is located within a ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acid (LNA)), or an unconnected ribose ring that typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, SMAD7 ASO comprises one or more LNAs. In some embodiments, the sugar-modified nucleoside comprises a bicyclic hexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar portion is replaced by a non-sugar portion, such as a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0051] In some embodiments, SMAD7 ASO comprises one or more modified sugars. In some embodiments, sugar modification comprises changing the substituents on the ribose ring to groups other than hydrogen or naturally occurring in DNA and RNA nucleosides.Modifications are made to the 2'-OH group. In some embodiments, substituents are introduced at the 2', 3', 4', or 5' positions, or combinations thereof. In some embodiments, the nucleoside having the modified sugar moiety comprises a 2'-modified nucleoside, such as a 2'-substituted nucleoside. In some embodiments, a 2'-sugar-modified nucleoside is a nucleoside having a substituent at the 2' position other than -H or -OH (a 2'-substituted nucleoside) or containing a 2'-linked diradical and comprising a 2'-substituted nucleoside and an LNA (2'-4' diradical-bridging) nucleoside. Examples of 2'-substituted modified 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, modification of the ribose group comprises modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribosome is selected from 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).

[0052] In some embodiments, SMAD7 ASO comprises 2'-deoxyribonucleotides (oligodeoxyribonucleotides). In some embodiments, SMAD7 ASO comprises 2'-deoxyribonucleotides (oligodeoxyribonucleotides) that are modified at the phosphate backbone or the 2' sugar position. In some embodiments, SMAD7 ASO is a phosphate thioester (PS) oligonucleotide in which one of the non-bridging oxygen atoms in the phosphate backbone is replaced by a sulfur atom. In some embodiments, SMAD7 ASO has at least one internucleotide link that is an O,O-linked phosphate thioester link (i.e., a phosphate thioester link). In some embodiments, all internucleotide links of SMAD7 ASO are O,O-linked phosphate thioester links (i.e., phosphate thioester links). Specification 8 / 24 pages 13 CN 120958131 A

[0053] The phosphate thioester (PS) link in the oligonucleotide is chiral, having dextrorotatory (Rp) and levorotatory (Sp) isomers (called diastereomers). These two PS diastereomers can differentially affect the physicochemical and biological properties of the oligonucleotide, such as nuclease sensitivity, thermodynamic stability of the double-stranded structure, and interaction with various enzymes involved in antisense and RNAi pathways. In some embodiments, SMAD7 ASO has one or more diastereomers of the chiral linking phosphorus.Nucleoside linkages. In some embodiments, SMAD7 ASO is prepared by using stereoselective oligonucleotide synthesis to form one or more pre-designed diastereoisomeric pure nucleoside linkages with respect to chiral linking phosphorus.

[0054] 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 comprising 1 to 20 O,O-linked thiophosphate nucleoside linkages (i.e., thiophosphate bonds). In some embodiments, SMAD7 ASO is an antisense oligonucleotide comprising the free acid form, salt form, or an anionic form without counterions 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, wherein each of the 20 nucleoside linkages is an O,O-linked thiophosphate linkage. In some embodiments, the thiophosphate backbone 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 acidic 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 salts of the oligonucleotides considered include those that are completely neutralized, for example, where each thiophosphate linker is associated with an ion (e.g., Na+). 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, for example, not all thiophosphate linkages are ion-associated (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%) are neutralized.

[0055] In some embodiments, the SMAD7 ASO of this disclosure (e.g., SEQ ID NO:1) or the pharmaceutical formulation disclosed herein (e.g., containing SEQ ID NO:6)The NO:1 composition downregulates SMAD7 expression. In some embodiments, SMAD7 expression is SMAD7 protein and / or mRNA expression. In some embodiments, SMAD7 protein expression in cells is downregulated 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 in cells is downregulated by more than about 40% compared to untreated cells.

[0056] In some embodiments, SMAD7 ASO has an inherent functional property of another molecular entity that specifically or highly specifically affects the activity or expression of SMAD7 or its product by targeting the SMAD7 gene, its RNA or protein product, or its activity or expression. In some embodiments, SMAD7 ASO reduces SMAD7 expression when introduced into cells (e.g., epithelial cells, lamina propria monocytes, or immune cells such as PBMCs, dendritic cells, or B cells). In some embodiments, SMAD7 ASO reduces the expression of mRNA transcribed from a gene. In some embodiments, SMAD7 ASO reduces the expression of proteins encoded by a gene. In some embodiments, SMAD7 ASO reduces the secretion of proteins encoded by a gene from the cell to which SMAD7 ASO is introduced.

[0057] In some embodiments, SMAD7 ASO regulates inflammation. In some embodiments, SMAD7 ASO regulates the expression or activity of inflammatory genes. In some embodiments, SMAD7 ASO reduces the expression or activity of inflammatory genes. In some implementations, SMAD7 ASO regulates the expression or activity of ENA-78, G-CSF, GM-CSF, GRO, GRO-α, I-309, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IFN-γ, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIG, MIP-1-δ, RANTES, SCF, SDF-1, TARC, TGF-β1, TNF-α, TNF-β, EGF, IGF-1, hematopoietic stem cell production protein, oncosin M, TPO, VEGF, PDGF-BB, leptin, or combinations thereof. In some implementations, SMAD7ASO reduces ENA-78, G-CSF, GM-CSF, GRO-α, IL-1β, IL-10, IL-12, IL-13, IL-15, and IFN-γ.γ, MIG, MIP-1-δ, RANTES, SCF, SDF-1, TARC, TGF-β1, TNF-α, angiogenic protein, tumor suppressor M, TPO, VEGF, PDGF-BB, leptin, or combinations thereof. In some embodiments, SMAD7ASO increases the expression or activity of GRO, I-309, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, MCP-1, MCP-2, MCP-3, M-CSF, MDC, TNF-β, EGF, IGF-1, or combinations thereof.

[0058] Methods for treating pouch inflammation

[0059] In some embodiments, methods for treating pouch inflammation in an individual in need are described herein, comprising administering a therapeutically effective amount of SMAD7 antisense oligonucleotide (ASO) to the individual. In some embodiments, a method of treating pouchitis in an individual in need is also described herein, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO) having the sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'). In some embodiments, a method of treating pouchitis in an individual in need is also described herein, comprising administering to the individual a pharmaceutical composition comprising: a) a SMAD7 antisense oligonucleotide (ASO) having the sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'); and b) a pharmaceutically acceptable carrier.

[0060] In some embodiments, a Pouchitis Disease Activity Index (PDAI) score of at least 7 is used to identify the individual as having pouchitis. In some embodiments, a Modified Pouchitis Disease Activity Index (mPDAI) score of at least 5 is used to identify the individual as having pouchitis.

[0061] In some embodiments, the individual has acute pouchitis. In some embodiments, the individual suffers from chronic pouchitis.

[0062] In some embodiments, a method for predicting pouchitis in an individual in need is also described herein, comprising determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis. In some embodiments, a method for identifying an individual at risk of developing pouchitis is also described herein, comprising: determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual as having pouchitis.In the risk, the known control level is the SMAD7 level in a sample collected from the individual before or during treatment of pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

[0063] In some embodiments, if the SMAD7 level is elevated relative to the known control level, a pharmaceutical composition containing SMAD7 ASO is administered to the individual. In some embodiments, if the SMAD7 level is not elevated relative to the known control level, the SMAD7 level in a second sample from the individual is determined. In some implementations, the second sample is collected immediately after the first sample, approximately 1 hour after the first sample, approximately 3 hours after the first sample, approximately 6 hours after the first sample, approximately 12 hours after the first sample, approximately 1 day after the first sample, approximately 3 days after the first sample, approximately 1 week after the first sample, approximately 2 weeks after the first sample, approximately 1 month after the first sample, approximately 2 months after the first sample, approximately 3 months after the first sample, approximately 4 months after the first sample, approximately 5 months after the first sample, approximately 6 months after the first sample, approximately 7 months after the first sample, approximately 8 months after the first sample, approximately 9 months after the first sample, approximately 10 months after the first sample, approximately 11 months after the first sample, or approximately 12 months after the first sample.

[0064] In some embodiments, a method for treating pouchitis in an individual in need is also described herein, comprising determining the level of SMAD7 in a first sample from the individual, wherein an elevated level of SMAD7 relative to a known control level is indicative of pouchitis, wherein the known control level is the level of SMAD7 in a sample collected from the individual before or during treatment of pouchitis, or the level of SMAD7 in a sample collected from a healthy individual without pouchitis.

[0065] In some embodiments, after administration of the SMAD7 antisense oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, to an individual suffering from pouchitis, for example, after 1 day, 2 days, 1 week, 1 month, or 6 months or longer, a pouchitis disease activity index (PDAI) score or a modified pouchitis disease activity index (mPDAI) score is reduced by at least about 5%, about 10%, about 20%, about 30%, about 40%, or even about 50% or more. In some implementations, SMAD7 antisense oligonucleotides are administered at least once daily. In some implementations, as a result of SMAD7 antisense oligonucleotide administration, the clinical manifestations of pouchitis are delayed by at least, for example, 6 months, 1 year, 18 months, or even 2 years or longer compared to individuals who have not received SMAD7 ASO.

[0066] Pharmaceutical Formulation

[0067] In some embodiments, pharmaceutical formulations containing oligonucleotides (e.g., SMAD7 ASO as described herein) are described herein, presented in dose units and prepared by any suitable method. 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 art. See, for example, Remington's Pharmaceutical Sciences, 18th edition (Mack Publishing Company, 1990).

[0068] In some embodiments, pharmaceutical formulations (e.g., pouch formulations) comprise an aqueous injectable composition containing an antisense oligonucleotide as described herein (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof). For example, the oligonucleotide formulation (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is blended with a liquid solution or suspension. In some embodiments, a solid form of the pharmaceutical formulation suitable for addition to a liquid prior to injection to prepare a solution or suspension is prepared, and the formulation is emulsified. 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.

[0069] In some embodiments, the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation (e.g., in-bag formulation) comprises an aqueous injectable composition comprising a surfactant (e.g., hydroxypropyl cellulose).

[0070] In some embodiments, the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation (e.g., in-bag formulation) comprises glycerol, liquid polyethylene glycol, mixtures thereof, and dispersions prepared in oil.

[0071] In some embodiments, the pharmaceutical formulation of the 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., suppository, enema, gel, and foam). In some embodiments, the rectal pharmaceutical formulation comprises an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof).

[0072] In some embodiments, the pharmaceutical formulation of the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt thereof) (e.g., a rectal pharmaceutical formulation) is a suppository. In some embodiments, the suppository contains...Lipophilic matrix excipients (e.g., cocoa butter, coconut oil, hydrogenated vegetable oil, stearin). In some embodiments, the suppository contains hydrophilic matrix excipients (e.g., glycerin gelatin and polyethylene glycol). In some embodiments, the suppository contains surfactants (e.g., polysorbate 80, Tween 20, Span 60).

[0073] In some embodiments, a pharmaceutical formulation of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6 or a pharmaceutically acceptable salt thereof) (e.g., a rectal pharmaceutical formulation) 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.

[0074] In some embodiments, the pharmaceutical formulation of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) (e.g., a rectal pharmaceutical formulation) is a gel. In some embodiments, the gel comprises a co-solvent (e.g., glycerol and propylene glycol). In some embodiments, the gel comprises an electrolyte.

[0075] In some embodiments, the gel has a gel-like consistency but is sufficiently flowable to allow for local or regional application via catheters, needles, syringes, or other equivalent local or regional application means (e.g., to the rectum).

[0076] In some embodiments, the concentration of the thickener used in the gel formulation is an amount or concentration suitable for achieving the desired consistency or viscosity, for example, from about 0.05% to about 10% by weight. In some embodiments, the concentration range of the thickener used in such a gel formulation is from about 0.05% to about 8% by weight. In some embodiments, the concentration range of the thickener used in such a gel formulation is from about 0.05% to about 7% by weight. In some embodiments, the concentration of the thickener used in this gel formulation ranges from about 0.05% to about 6% by weight. In some embodiments, the concentration of the thickener used in this gel formulation ranges from about 0.05% to about 5% by weight. In some embodiments, the concentration of the thickener used in this gel formulation ranges from about 0.05% to about 4% by weight. In some embodiments, the concentration of the thickener used in this gel formulation ranges from about 0.05% to about 3% by weight. In some embodiments, the concentration of the thickener used in this gel formulation ranges from about 0.05% to about 2% by weight. In some embodiments, the concentration of the thickener used in this gel formulation ranges from about 0.05% to about 1% by weight. In some embodiments, the gel formulation includes methylcellulose at a concentration of about 0.05% to about 2%, while in other embodiments, the gel formulation includes methylcellulose at a concentration of about 1%.

[0077] In some embodiments, the pharmaceutical formulation of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) (e.g., a rectal formulation) is a foam. In some embodiments, the foam formulation comprises a surfactant / emulsifier with foaming properties and a propellant (e.g., a propellant gas). In some embodiments, the foam formulation also comprises one or more of the following: a suspending / solvent, a thickener, a preservative, a chelating agent, a buffer, an antioxidant, a tension modifier, and a spreading agent. In some embodiments, the surfactant / emulsifier includes, but is not limited to, nonionic surfactants, anionic surfactants, cationic surfactants, and combinations thereof. In some embodiments, the foam formulation is filled into a pressurized container prior to application (e.g., rectal application). In some embodiments, the pressurized container is a can. In some embodiments, the propellants used herein include, but are not limited to, hydrocarbons (e.g., isobutane, n-butane, or propane), fluorocarbons (e.g., dichlorodifluoromethane and dichlorotetrafluoroethane), chlorofluorocarbons, dimethyl ethers, hydrofluorocarbons, compressed gases, Freons (e.g., Freon 12, Freon 114), hydrochlorofluorocarbons, hydrofluorocarbons, or mixtures thereof.

[0078] In some embodiments, the maximum amount of propellant used is determined by its miscibility with other components in the composition, such as forming a mixture, e.g., a homogeneous mixture. In some embodiments, the minimum level of propellant used in the composition is determined by the desired foam characteristics and its ability to substantially or completely empty the container.

[0079] In some embodiments, the propellant concentration used in this foam formulation is about 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 about 60% (w / w).

[0080] In some embodiments, foam is formed after application (e.g., rectal application), wherein the dispensing valve of the can allows the propellant to expand rapidly, triggering the foaming action of the surfactant, and the resulting foam is formed in the rectum and colon. In some embodiments, foam for application of the composition described herein is formed in the dispensing container prior to application.

[0081] In some embodiments, the pharmaceutical formulation of oligonucleotides (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 the intraparticle package insert (page 12 / 24, CN 120958131 A), which contains the antisense oligonucleotide described herein (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or its pharmaceutical formulation).The pharmaceutical formulation comprises an extragranular phase comprising one or more pharmaceutically acceptable excipients and a pharmaceutically acceptable filler. For example, an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt thereof) and a filler are blended together with optional other excipients to form granules. In some embodiments, wet granulation is used to form an intragranular phase, such as a liquid (e.g., water) being added to the blended antisense compound and filler, and the combination is then dried, ground and / or sieved to produce granules. Other methods in the art can be used to achieve the intragranular phase.

[0082] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises an extragranular phase comprising one or more pharmaceutically acceptable excipients and is blended with the intragranular phase to form the disclosed formulation.

[0083] In some embodiments, the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt thereof) formulation comprises an intragranular phase comprising a filler. Exemplary fillers include, but are not limited to, cellulose, gelatin, calcium phosphate, lactose, sucrose, glucose, mannitol, sorbitol, microcrystalline cellulose, pectin, polyacrylate, dextran, cellulose acetate, hydroxypropyl methylcellulose, partially pregelatinized starch, calcium carbonate, and combinations thereof.

[0084] In some embodiments, oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulations (e.g., oral pharmaceutical formulations) comprise an intragranular phase and / or an extragranular phase comprising a binder to hold the components of the pharmaceutical formulation together. Exemplary binders include, but are not limited to, starch, sugar, cellulose, or modified cellulose such as hydroxypropyl cellulose, lactose, pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, sugar alcohols, and combinations thereof.

[0085] In some embodiments, pharmaceutical formulations (e.g., oral pharmaceutical formulations) comprising an intragranular phase and / or an extragranular phase as described herein further include disintegrants, such as, but not limited to, starch, cellulose, crosylvinylpyrrolidone, sodium carboxymethyl starch, sodium carboxymethyl cellulose, alginate, corn starch, crosylcarboxymethyl cellulose, crosylcarboxymethyl cellulose, low-substituted hydroxypropyl cellulose, gum arabic, and others including combinations thereof. For example, the intragranular phase and / or extragranular phase includes a disintegrant.

[0086] In some embodiments, oligonucleotide formulations (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) as described herein comprise an intragranular phase comprising the disclosed antisense oligonucleotide and an excipient selected from mannitol, microcrystalline cellulose, hydroxypropyl methyl cellulose, and sodium carboxymethyl starch or combinations thereof, and an extragranular phase comprising...The extragranular phase includes one or more of the following: microcrystalline cellulose, sodium carboxymethyl starch, and magnesium stearate, or mixtures thereof.

[0087] In some embodiments, the oligonucleotide formulations described herein (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) include a lubricant, for example, the extragranular phase contains a lubricant. Lubricants include, but are not limited to, talc, silica, fats, glyceryl stearate, magnesium stearate, calcium phosphate, silica, calcium silicate, calcium phosphate, colloidal silica, metal stearates, hydrogenated vegetable oils, corn starch, sodium benzoate, polyethylene glycol, sodium acetate, calcium stearate, sodium lauryl sulfate, sodium chloride, magnesium lauryl sulfate, talc, and stearic acid.

[0088] In some embodiments, the pharmaceutical formulations of this disclosure (e.g., oral pharmaceutical formulations) include enteric coatings. Typically, enteric coatings create a barrier for oral pharmaceutical products that controls the location of absorption of the drug along the digestive tract. In some embodiments, the enteric coating comprises a polymer that disintegrates at different rates depending on the pH. In some embodiments, the enteric coating comprises, for example, cellulose acetate phthalate, methacrylate-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.

[0089] In some embodiments, the enteric coating comprises anionic, cationic, or neutral copolymers based on methacrylic acid, methacrylic acid / acrylate, or derivatives thereof. In some embodiments, the enteric coating comprises ethyl acrylate-methacrylic acid copolymer. Commercially available enteric coatings include AMB, ethyl acrylate-methacrylic acid copolymers (e.g., Acryl-), dimethylaminoethyl methacrylate-butyl methacrylate-methyl methacrylate copolymers (2:1:1), or poly(methacrylate-co-methyl methacrylate) 1:1 and poly(methacrylate-co-methyl methacrylate) 1:2 copolymers (e.g.) grades. 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 pharmaceutical formulation (e.g., tablets) under consideration. In some embodiments, the enteric coating comprises ethyl acrylate-methacrylic acid copolymer, which comprises about 8% to about 15% of the weight of the pharmaceutical formulation (e.g., tablets). In some embodiments, the enteric coating comprises ethyl acrylate-methacrylic acid copolymer, which comprises about 12% of the weight of the pharmaceutical formulation (e.g., tablets). In some embodiments, the enteric coating comprises an ethyl acrylate-methacrylic acid copolymer, which constitutes a portion of the pharmaceutical formulation (e.g., tablets) by weight.About 10% of the amount.

[0090] In some embodiments, the pharmaceutical formulation (e.g., SMAD7 ASO or a pharmaceutically acceptable salt thereof of SEQ ID NO: 1, 2, 3, 4, 5 or 6) comprises about 0.5% to about 70%, for example about 0.5% to about 30%, about 1% to about 20%, or about 5% to about 30% by weight of antisense oligonucleotide or a pharmaceutically acceptable salt thereof. In some embodiments, such a pharmaceutical formulation comprises 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 includes an intragranular phase comprising, by weight, about 30% to about 60%, about 45% to about 65%, or alternatively about 5% to about 10% of SMAD7 ASO or a pharmaceutically acceptable salt thereof of SEQ ID NO: 1, 2, 3, 4, 5, or 6; by weight, about 30% to about 50% or alternatively about 5% to about 15% of mannitol; by weight, about 5% to about 15% of microcrystalline cellulose; by weight, about 0% to about 4% or about 1% to about 7% of hydroxypropyl methylcellulose; and by weight, about 0% to about 4%, for example about 2% to about 4% of sodium carboxymethyl starch. In some embodiments, the pharmaceutical formulation comprises, by weight, about 5% to about 10% or about 10% to about 30% of the oligonucleotide of SEQ ID NO:1, about 20% to about 50% of mannitol, about 10% to about 30% of microcrystalline cellulose, about 0.5% to about 10% of hydroxypropyl methylcellulose, and about 0.5% to about 10% of sodium carboxymethyl starch. In some embodiments, the pharmaceutical formulation is in tablet form.

[0091] Exemplary pharmaceutical formulations include dosage forms comprising or substantially consisting of SMAD7 ASO or a pharmaceutically acceptable salt thereof of SEQ ID NO: 1, 2, 3, 4, 5 or 6, of about 10 mg to about 500 mg or about 20 mg to about 500 mg, for example, amounts including about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg,

[0092] Approximately 110mg, approximately 120mg, approximately 130mg, approximately 140mg, approximately 150mg, approximately 160mg, approximately 170mg, approximately 180mg, approximately 190mg, approximately 200mg, approximately 220mg, approximately 240mg, approximately 260mg, approximately 280mg, approximately 300mg, approximately 320mg, approximately 340mg, approximatelyTablets of SMAD7 ASO or a pharmaceutically acceptable salt thereof in SEQ ID NO: 1, 2, 3, 4, 5 or 6, in doses of 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg or about 500 mg. In some embodiments, the pharmaceutical formulations described herein are formulated as tablets comprising about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg of the oligonucleotide of SEQ ID NO:1. In some embodiments, the pharmaceutical formulation described herein is formulated as a tablet comprising about 40 mg of the oligonucleotide of SEQ ID NO:1. In some embodiments, the pharmaceutical formulation described herein is formulated as a tablet comprising about 160 mg of the oligonucleotide of SEQ ID NO:1.

[0093] In some embodiments, the pharmaceutical formulation described herein is formulated as a tablet for oral administration comprising: about 0.5% to about 30% by weight of the oligonucleotide; about 20% to about 50% by weight of mannitol; and about 10% to about 30% by weight of microcrystalline cellulose.

[0094] In some embodiments, a pharmaceutically acceptable tablet for oral administration is provided, comprising about 5% 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 carboxymethyl starch, about 0.5% to about 10% by weight of magnesium stearate, about 0.5% to about 10% by weight of AMB, and about 5% to about 20% by weight of [unclear text - likely a typo, should be "in some embodiments"]. In some embodiments, a pharmaceutically acceptable tablet for oral administration is provided, comprising about 8.5% by weight of SEQ ID NO:1. The oligonucleotide NO:1, 40% mannitol by weight, approximately 25% microcrystalline cellulose by weight, approximately 5% hydroxypropyl methylcellulose by weight, approximately 4% sodium carboxymethyl starch by weight, approximately 0.4% magnesium stearate by weight, and approximately 4% by weight...AMB, and about 10% to about 15% Acryl- by weight. In some embodiments, pharmaceutically acceptable tablets for oral administration are provided, comprising about 23% by weight of the oligonucleotide of SEQ ID NO:1, 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 carboxymethyl starch, about 0.4% by weight of magnesium stearate, about 4% by weight of AMB, and about 7% to about 12% by weight of Acryl-. In some embodiments, pharmaceutically acceptable tablets for oral administration are formulated according to Tables 1 and 2.

[0095]

[0096] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises an intragranular phase comprising about 50% by weight of SMAD7 ASO or a pharmaceutically acceptable salt thereof of SEQ ID NO: 1, 2, 3, 4, 5 or 6, about 11.5% by weight of mannitol, about 10% by weight of microcrystalline cellulose, about 3% by weight of hydroxypropyl methylcellulose, and about 2.5% by weight of sodium carboxymethyl starch; and an extragranular phase comprising about 20% by weight of microcrystalline cellulose, about 2.5% by weight of sodium carboxymethyl starch, and about 0.5% by weight of magnesium stearate. In some embodiments, the pharmaceutical formulation further comprises an enteric coating.

[0097] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises or is substantially composed of: an intragranular phase comprising, by weight, about 5% to about 10%, for example about 8%, of SEQ ID NO: 1, 2, 3, 4, 5 or 6, page 15 / 24, 20 CN 120958131 A SMAD7 ASO or a pharmaceutically acceptable salt (e.g., a sodium salt), about 40% mannitol, about 8% microcrystalline cellulose, about 5% hydroxypropyl methylcellulose, and about 2% sodium carboxymethyl starch; and an extragranular phase comprising, by weight, about 17% microcrystalline cellulose, about 2% sodium carboxymethyl starch, and about 0.4% magnesium stearate.

[0098] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) includes an enteric coating comprising, by weight, about 13%, about 14%, about 15%, about 16%, or about 17% of, for example, an ethyl acrylate-methacrylic acid copolymer (e.g., ).

[0099] For example, the pharmaceutical formulation is present in the form of a pharmaceutically acceptable tablet for oral administration, comprising an intragranular phase and an extragranular phase, wherein, for example, the intragranular phase comprises, by weight, about 5% to about 10% (e.g., about 8% by weight) of SEQ ID NO.The tablet contains, by weight, oligonucleotides of NO:1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof, about 40% mannitol, about 8% microcrystalline cellulose, about 5% hydroxypropyl methylcellulose, and about 2% sodium carboxymethyl starch, and for example, an extragranular phase comprising about 17% microcrystalline cellulose, about 2% sodium carboxymethyl starch, and about 0.4% magnesium stearate, wherein the tablet further comprises an enteric coating.

[0100] In some embodiments, the formulation of consideration results in a minimum plasma concentration of SMAD7 ASO in an individual when administered orally to an individual. In some embodiments, the formulation of consideration is locally delivered to an individual's reservoir, for example, to an individual's affected or diseased reservoir, when administered orally to an individual. In some embodiments, formulations suitable for oral administration are in the form of capsules, pouches, pills, tablets, lozenges (using a flavoring matrix such as sucrose and gum arabic or astragalus gum), powders, granules, or as solutions 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 pasteurees (using an inert matrix such as gelatin and glycerin, or sucrose and gum arabic), each containing a predetermined amount of its subject composition as an active ingredient. In some embodiments, the formulation is administered as a pellet, ointment, or paste.

[0101] In some embodiments, the pharmaceutical formulations described herein comprise an intragranular phase comprising an antisense oligonucleotide, such as SMAD7ASO of SEQ ID NO: 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 formulations described herein comprise an extragranular phase comprising a pharmaceutically acceptable excipient, such as a disintegrant. In some embodiments, the extragranular phase comprises components including microcrystalline cellulose, magnesium stearate, and mixtures thereof. In some embodiments, the pharmaceutical formulation further comprises about 12% to 16% by weight of an enteric coating.

[0102] In some embodiments, the pharmaceutical formulation described herein (e.g., an oral pharmaceutical formulation) comprises about 0.5% to about 10% by weight of an antisense oligonucleotide, such as SMAD7 ASO of SEQ ID NO: 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 enteric coating comprising ethyl acrylate-methacrylic acid copolymer.

[0103] In some embodiments, the pharmaceutical formulation described herein (e.g., an oral pharmaceutical formulation) comprises an intragranular phase comprising about 5% to about 10% by weight of an antisense oligonucleotide, such as SMAD7 of SEQ ID NO: 1, 2, 3, 4, 5, or 6.ASO, or a pharmaceutically acceptable salt thereof, about 40% by weight mannitol, about 8% by weight microcrystalline cellulose, about 5% by weight hydroxypropyl methylcellulose, and about 2% by weight sodium carboxymethyl starch; and an extragranular phase comprising about 17% by weight microcrystalline cellulose, about 2% by weight sodium carboxymethyl starch, and about 0.4% by weight magnesium stearate; and an enteric coating covering the tablet comprising ethyl acrylate-methacrylic acid copolymer.

[0104] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises an enteric coating comprising, for example, about 13%, about 15%, about 16%, about 17%, or about 18% by weight of (see, for example, PCT Publication No. WO2010 / 054826).

[0105] The rate at which the coating dissolves and the active ingredient is released is its dissolution rate. In some embodiments, the pharmaceutical formulation (e.g., in tablet form) has a melting profile, for example, when tested in a USP / EP Type 2 device (paddle) at 100 rpm and 37°C in phosphate buffer at pH 7.2, approximately 50% to 100% oligonucleotide release occurs after approximately 120 to approximately 240 minutes, or for example, after 180 minutes. In some embodiments, the pharmaceutical formulation (e.g., in tablet form) has a melting profile, for example, when tested in a USP / EP Type 2 device (paddle) at 100 rpm and 37°C in diluted HCl at pH 1.0, there is substantially no oligonucleotide release after 120 minutes. In some embodiments, the pharmaceutical formulation (e.g., in tablet form) has a melting profile, for example, when tested in a USP / EP Type 2 device (paddle) at 100 rpm and 37°C in phosphate buffer at pH 6.6, approximately 10% to approximately 30%, or no more than approximately 50%, of oligonucleotide release occurs after 30 minutes.

[0106] In some embodiments, the pharmaceutical formulation described herein results in a minimum plasma concentration of oligonucleotides in an individual when administered to an individual. In some embodiments, the disclosed formulation, when administered orally to an individual, is locally delivered to the colon or rectum of the individual, for example, to the affected or diseased site of the individual.

[0107] In some embodiments, the pharmaceutical formulation described herein is sterile. In some embodiments, sterilization is achieved, for example, by filtration through a sterile filter membrane. In some embodiments, the composition is lyophilized, and filtration sterilization is performed before or after lyophilization and reconstitution.

[0108] In some embodiments, the pharmaceutical formulation comprising SEQ ID NO:1 comprises greater than 50 mmol, greater than 100 mmol, greater than 200 mmol, greater than 300 mmol, greater than 400 mmol, greater than 500 mmol, greater than 600 mmol, and greater than...The pharmaceutical preparation comprising SEQ ID NO:1 in quantities of 700 mmol, greater than 800 mmol, greater than 900 mmol, greater than 1 mol, greater than 1.5 mol, greater than 2 mol, greater than 2.5 mol, greater than 3 mol, greater than 4 mol, or greater than 5 mol. In some embodiments, the pharmaceutical preparation comprising SEQ ID NO:1 comprises greater than 2 mol of SEQ ID NO:1. In some embodiments, the pharmaceutical formulation comprises 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 of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises more than 100 mmol to about 5 mol of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises more than 100 mmol to about 2 mol of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises more than 300 mmol to about 5 mol of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 300 mmol to about 2 mol of SEQ ID NO:1. In some embodiments, the pharmaceutical formulation comprises greater than 900 mmol to about 5 mol of SEQ ID NO:1. In some embodiments, the pharmaceutical formulation comprises greater than 900 mmol to about 2 mol of SEQ ID NO:1. In some embodiments, the pharmaceutical formulation comprises greater than 300 mmol to about 900 mmol of SEQ ID NO:1. In some embodiments, the pharmaceutical formulation comprises greater than 300 mmol to about 5 mmol, greater than 300 mmol to about 4 mmol, greater than 300 mmol to about 3 mmol, greater than 300 mmol to about 2.5 mmol, greater than 300 mmol to about 2 mmol, greater than 300 mmol to about 1.5 mmol, greater than 300 mmol to about 1 mmol, greater than 300 mmol to about 900 mmol, greater than 300 mmol to about 800 mmol, greater than 300 mmol to about 700 mmol, greater than 300 mmol to about 600 mmol, greater than 300 mmol to about 500 mmol, greater than 300 mmol to about 400 mmol, greater than 400 mmol to about 5 mmol, greater than 400 mmol to about 4 mmol, greater than 400 mmol to about 3 mmol, greater than 400 mmol to about 2.5 mmol, greater than 400 mmol to about 2 mmol, greater than 400 mmol to about 1.5 mmol, and greater than 400 mmol to about 1 mmol.Greater than 400 mmol to about 900 mmol, greater than 400 mmol to about 800 mmol, greater than 400 mmol to about 700 mmol, greater than 400 mmol to about 600 mmol, greater than 400 mmol to about 500 mmol, greater than 500 mmol to about 5 mmol, greater than 500 mmol to about 4 mmol, greater than 500 mmol to about 3 mmol, greater than 500 mmol to about 2.5 mmol, greater than 500 mmol to about 2 mmol, greater than 500 mmol to about 1.5 mmol, greater than 500 mmol to about 1 mmol, greater than 500 mmol to about 900 mmol, greater than 500 mmol to about 800 mmol, greater than 500 mmol to about 700 mmol, greater than 400 mmol to about 600 mmol, greater than 400 mmol to about 500 mmol, greater than 5 ... mmol, greater than 500 mmol to about 4 mmol, greater than 500 mmol to about 3 mmol, greater than 500 mmol to about 2.5 mmol, greater than 500 mmol to about 2 mmol, greater than 500 mmol to about 1 mmol, greater than 500 mmol to about 900 mmol, greater than 500 mmol to about 800 mmol, greater than 500 mmol to about 700 mmol, greater than 400 mmol to about 600 mmol, greater than 4 Greater than 500 mmol to about 600 mmol, greater than 600 mmol to about 5 mmol, greater than 600 mmol to about 4 mmol, greater than 600 mmol to about 3 mmol, greater than 600 mmol to about 2.5 mmol, greater than 600 mmol to about 2 mmol, greater than 600 mmol to about 1.5 mmol, greater than 600 mmol to about 1 mmol, greater than 600 mmol to about 900 mmol, greater than 600 mmol to about 800 mmol, greater than 600 mmol to about 700 mmol, greater than 700 mmol to about 5 mmol, greater than 700 mmol to about 4 mmol, greater than 700 mmol to about 3 mmol, greater than 700 mmol to about 2.5 mmol, greater than 700 mmol to about 2 mmol, greater than 700 mmol to about 1.5 mmol, greater than 700 mmol to about 1 mmol, greater than 700 mmol to about 900 mmol, greater than 700 mmol From approximately 800 mmol, greater than 800 mmol to approximately 5 mmol, greater than 800 mmol to approximately 4 mmol, greater than 800 mmol to approximately 3 mmol, greater than 800 mmol to approximately 2.5 mmol, greater than 800 mmol to approximately 2 mmol, greater than 800 mmol to approximately 1.5 mmol, greater than 800 mmol to approximately 1 mmol, greater than 800 mmol to approximately 900 mmol, greater than 900 mmol to approximately 5 mmol, greater than 900 mmol to approximately 4 mmol, greater than 900 mmol to approximately 3 mmol, greater than 900 mmol to approximately 2.5 mmol, greater than 900 mmol to approximately 2 mmol, greater than 900 mmol to approximately 1.5 mmol, greater than 900 mmol to approximately 1 mmol, greater than 1 mmol to approximately 5 mmol, greater than 1 mmol to approximately 4 mmol, greater than 1 mmol to approximately 3 mmol, greater than 1 mmol to approximately 2.5 mmol, greater than 1 mmol to approximately 2 mmol, greater than 1 mmol to approximately 1 mmol 0.5 mol, greater than 1.5 mol to about 5 mol, greater than 1.5 mol to about 4 mol, greater than 1.5 mol to about 3 mol, greater than 1.5 mol to about 2.5 mol, greater than 1.5 mol to about 2 mol, greater than 2 mol to about 5 mol, greater than 2 mol to about 4 mol, greater than 2 mol to about 3 mol, greater than 2 mol to aboutSEQ ID NO:1 in amounts of 2.5 mol, greater than 2.5 mol to about 5 mol, greater than 2.5 mol to about 4 mol, greater than 2.5 mol to about 3 mol, greater than 3 mol to about 5 mol, greater than 3 mol to about 4 mol, or greater than 4 mol to about 5 mol. In some embodiments, the pharmaceutical formulation comprises about 1 mol, about 900 mmol, about 850 mmol, about 800 mmol, about 750 mmol, about 700 mmol, about 650 mmol, about 600 mmol, about 550 mmol, about 500 mmol, about 450 mmol, about 400 mmol, about 350 mmol, about 300 mmol, about 250 mmol, about 200 mmol, about 150 mmol, or about 100 mmol.

[0109] Route of Administration

[0110] Intestinal Administration

[0111] In some embodiments, the SMAD7 ASO described herein is administered intestinally. In some embodiments, enteric administration is oral, sublingual, gastric, or rectal administration. In some embodiments, enteric administration is rectal administration.

[0112] In some embodiments, the SMAD7 ASO described herein is suitable for rectal delivery (e.g., suppositories, enemas, gels, and foams). In some embodiments, rectal administration of SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof.

[0113] In some embodiments, the formulation of oligonucleotides (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. In some embodiments, the rectal pharmaceutical formulation comprises oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof). In some embodiments, the rectal pharmaceutical formulation comprises SMAD7 ASO (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof).

[0114] In some embodiments, the SMAD7 ASO described herein is suitable for oral delivery (e.g., capsules, tablets, sacs, pills, lozenges, powders, and granules). In some embodiments, oral administration of SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, is permitted.

[0115] In some embodiments, the formulation of the 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 oral pharmaceutical formulation comprises an oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1).In some embodiments, the oral pharmaceutical formulation comprises SMAD7 ASO (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt thereof).

[0116] In some embodiments, the pharmaceutical formulation of the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt thereof) is a tablet or capsule. In some embodiments, the tablet or capsule is encapsulated with an enteric coating.

[0117] In some embodiments, the tablet of the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt thereof) is formulated as a small tablet, granules, microcapsules or microflakes. In some embodiments, the tablet of the oligonucleotide (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt thereof) is formulated as a small tablet. In some embodiments, the tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) are formulated as granules. In some embodiments, the tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) are formulated as microspheres. In some embodiments, the tablets of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) are formulated as microplates. In some embodiments, the tablets, granules, microspheres, or microplates are encapsulated with an enteric coating.

[0118] In some embodiments, the oral pharmaceutical formulations of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) are not encapsulated with an enteric coating. In some embodiments, the oral pharmaceutical formulation of the oligonucleotide (e.g., the oligonucleotide of SEQ ID NO:1 or a pharmaceutically acceptable salt thereof) is not encapsulated with an enteric coating.

[0119] In some embodiments, the oral pharmaceutical formulation comprises a gastric resistance coating, such that the formulation delivers the compound to, for example, the gastrointestinal tract of an individual. In some embodiments, such administration results in a topical effect, for example, by substantially locally and directly applying the antisense compound to the affected portion of the subject's gastrointestinal tract. In some embodiments, such administration substantially avoids unwanted systemic absorption of the antisense compound.

[0120] In some embodiments, the pharmaceutical formulations described herein (e.g., oral pharmaceutical formulations and rectal pharmaceutical formulations) comprise pharmaceutically acceptable excipients. Exemplary pharmaceutically acceptable excipients include, but are not limited to, fillers, binders,Disintegrants and / or lubricants, as well as colorants, release agents, coating agents, sweeteners, flavoring agents such as wintergreen, orange, xylitol, sorbitol, fructose, and maltodextrin, and flavoring agents, preservatives, and / or antioxidants.

[0121] Parenteral Administration

[0122] 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, intraenteric (e.g., intrajejunal, intraileal), intracolonic, or intrarectal, pouch, or intraperitoneal routes. In some embodiments, the SMAD7 ASO or pharmaceutical formulation described herein is administered via pouch. In some embodiments, the SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, is administered via pouch.

[0123] In some embodiments, those skilled in the art will know, based on this disclosure, the preparation of aqueous compositions (e.g., aqueous pharmaceutical formulations containing the disclosed SMAD7 ASO). In some embodiments, these compositions are formulated as injections, as liquid solutions or suspensions. In some embodiments, a solid form suitable for addition to a liquid prior to injection to prepare a solution or suspension is prepared; and the formulation is emulsified. Specification 19 / 24 pages 24 CN 120958131 A

[0124] In some embodiments, pharmaceutical formulations suitable for injection comprise sterile aqueous solutions or dispersions. In some embodiments, pharmaceutical formulations suitable for injection comprise sesame oil, peanut oil, or aqueous propylene glycol. In some embodiments, pharmaceutical formulations suitable for injection are sterile powders. In some embodiments, pharmaceutical formulations are sterile and have sufficient flowability to be readily injectable. In some embodiments, pharmaceutical formulations are stable under manufacturing and storage conditions and must be protected against contamination by microorganisms (e.g., bacteria and fungi).

[0125] In some embodiments, a solution of the free base or pharmacologically acceptable salt of the active compound is prepared by suitably mixing it with a surfactant such as hydroxypropyl cellulose in water. In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and in oil. In some embodiments, a sterile fixative oil is used as a solvent or suspension medium. For this purpose, in some embodiments, mild fixative oils are used, including synthetic monoglycerides or diglycerides. In some embodiments, fatty acids such as oleic acid are used to prepare the injection. In some embodiments, the sterile injection formulation is a sterile injection solution, suspension, or emulsion in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution in 1,3-butanediol. In some embodiments, the acceptable vehicle and solvent used are water, Ringer's solution,USP and isotonic sodium chloride solution. In some embodiments, the disclosed SMAD7ASO is suspended in a carrier fluid containing 1% (w / v) sodium carboxymethyl cellulose and 0.1% (v / v) TWEENTM80. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0126] In some embodiments, injectable formulations, such as sterile injectable aqueous or oily suspensions, are formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. In some embodiments, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and other desired ingredients from those described above. In some embodiments, the sterile injectable solutions of this disclosure are prepared by mixing the disclosed SMAD7 ASO in the desired amount with a suitable solvent and, as desired, various amounts of other ingredients from those described above, followed by filtration and sterilization. In the case of sterile powders used to prepare sterile injectable solutions, in some embodiments, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient plus any additional desired components from its previously sterile filtered solution. In some embodiments, the injectable formulation is sterilized by, for example, filtration through a bacterial retention filter.

[0127] In some embodiments, the preparation of more or higher concentration solutions for intramuscular injection is also contemplated. In some embodiments, DMSO is preferably used as a solvent because this will result in extremely rapid permeation, delivering high concentrations of the exposed SMAD7 ASO to a small area.

[0128] In some embodiments, suitable preservatives for such solutions include benzalkonium chloride, benzalkonium bromide, chlorobutanol, thimerosal, etc. 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, sodium dihydrogen phosphate, etc., in amounts sufficient to maintain the pH between about pH 6 and pH 8 and, for example, between about pH 7 and pH 7.5. In some embodiments, suitable tonics include dextran 40, dextran 70, dextran, glycerin, potassium chloride, propylene glycol, sodium chloride, etc., such that the sodium chloride equivalent of the solution is in the range of 0.9 ± 0.2%. In some embodiments, suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea, etc. In some embodiments, suitable wetting agents and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282, and tyloxapine. Suitable thickeners include dextran 40, dextran 70, gelatin, glycerin, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose, etc.

[0129] Dosage regimen

[0130] In some embodiments, pharmaceutical formulations of oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5 or 6) or pharmaceutically acceptable salts thereof provided in this disclosure are administered before and / or after the onset of pouchitis symptoms, or are suitable for administration as per the instructions for use (page 25, CN 120958131 A, page 20 / 24).

[0131] In some embodiments, pharmaceutical formulations of this disclosure (including, for example, SEQ ID NO: 1, 2, 3, 4, 5 or 6 or pharmaceutically acceptable salts thereof) are administered or suitable for administration about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, about every 72 hours, daily, twice a week, once every two weeks or monthly.

[0132] The frequency of administration may vary depending on factors such as the route of administration, dosage, and the disease being treated. Exemplary administration frequencies are once daily, once a week, and once every two weeks. In some embodiments, administration is once daily for 7 days.

[0133] In some embodiments, the pharmaceutical formulation comprises a dosage form consisting of or substantially composed of about 10 mg to about 500 mg or about 20 mg to about 500 mg of an oligonucleotide (e.g., SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5 or 6 or a pharmaceutically acceptable salt thereof). For example, this article considers formulations comprising about 20 mg, about 30 mg, about 35 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg of oligonucleotides (e.g., SMAD7 oligonucleotides of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof). In some embodiments, the formulation comprises about 40 mg, about 80 mg, or about 160 mg of an oligonucleotide, such as SEQ ID NO: 1, 2, 3, 4, 5, or 6, of SMAD7 oligonucleotide. In some embodiments, the formulation comprises at least about 100 μg of an oligonucleotide, such as SEQ ID NO: 1, 2, 3, 4, 5, or 6, of SMAD7 oligonucleotide. For example, the formulation comprises about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg of an oligonucleotide, such as SEQ ID NO: 1, 2, 3, 4, 5, or 6.SMAD7 oligonucleotides NO:1, 2, 3, 4, 5, or 6.

[0134] The amount administered will depend on variables such as the type and severity of the disease or indication to be treated, the individual's overall health and size, the in vivo potency of the oligonucleotide, the pharmaceutical formulation, and the route of administration. In some embodiments, the initial dose is increased above an upper limit level to rapidly reach the desired blood or tissue levels. In some embodiments, the initial dose is less than the optimal value, and the dose is gradually increased during treatment. In some embodiments, such as in a routine Phase I dose escalation study designed to run from 40 mg to 160 mg, the human dose is optimized.

[0135] In some embodiments, an individual with pouchitis will be administered an initial dose of SMAD7 ASO, such as SMAD7 ASO of SEQ ID NO:1. As used herein, "initial dose" refers to the dose of SMAD7 ASO administered to an individual with pouchitis in a series of doses. 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 more than a single dose of SMAD7 ASO. In some embodiments, the initial dose is the dose of SMAD7 ASO administered to the individual prior to any subsequent dose. For example, the initial dose is, but not limited to, the first dose of SMAD7 ASO administered to a treatment-naïve individual. 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 of a first treatment cycle, the first dose of a second treatment cycle, or the first dose of any subsequent treatment cycle.

[0136] In some embodiments, an individual with pouchitis is administered a subsequent dose of SMAD7 ASO, such as the SMAD7 ASO of SEQ ID NO:1. As used herein, "subsequent dose" means the dose of SMAD7 ASO administered to an individual with pouchitis after the administration of a previous dose (e.g., the initial dose). In some embodiments, the subsequent dose is administered to the individual with pouchitis in a series of doses comprising two or more doses. In some embodiments, the amount of the subsequent dose is calibrated relative to the initial dose or the previous dose such that the subsequent dose is greater than, equal to, or less than the previous dose. In some embodiments, the subsequent dose is a dose administered to an individual with pouchitis after the first dose (e.g., the initial dose) of SMAD7 ASO. In some embodiments, the subsequent dose is a dose administered after the prior dose of SMAD7 ASO administered to an individual with pouchitis, for example, in the same treatment cycle or in different treatment cycles (e.g., previous treatment cycles) following the administration of the prior dose.A dose administered after a previous dose. In some embodiments, a subsequent dose is a dose following any previous dose, such as a previous dose immediately preceding a subsequent dose or a previous dose following one or more doses administered before the administration of the subsequent dose.

[0137] The compositions, formulations, or methods of this disclosure provide administration of oligonucleotides (e.g., SMAD7 oligonucleotides of SEQ ID NO: 1, 2, 3, 4, 5, or 6) or pharmaceutically acceptable salts thereof to subjects refractory to a first therapy.

[0138] In some embodiments, subjects refractory to a first therapy are treated concurrently with or subsequently with oligonucleotides (e.g., SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6) or pharmaceutically acceptable salts thereof.

[0139] In some embodiments, the methods provided herein further include administration of at least one other agent relating to the treatment of the diseases and conditions disclosed herein (e.g., pouchitis). In some embodiments, the other agents considered are administered co-administered (e.g., sequentially or simultaneously). Examples

[0140] The following examples further illustrate this disclosure. These embodiments are provided for illustrative purposes only and should not be construed in any way as limiting the scope or content of this disclosure.

[0141] Example 1: Preparation of Patient Mucosal Samples

[0142] The mucosal samples prepared and used in the following examples (i.e., Examples 2-5) were taken from inflamed pouches of 16 patients with chronic pouchitis (CP). The modified pouchitis disease activity index (mPDAI), assessed using clinical and endoscopic findings, was used to diagnose pouchitis, where pouchitis was defined as mPDAI ≥ 5. Samples used as experimental controls included mucosal biopsy samples from uninflamed pouches of patients without clinical / endoscopic evidence of pouchitis, and ileal samples from normal / pre-inflamed pouches of patients with chronic pouchitis and normal controls who underwent colonoscopy as part of a colorectal cancer screening procedure at Tor Vergata University Hospital (Rome, Italy).

[0143] Example 2: Increased SMAD7 in Chronic Pouchitis

[0144] This example assesses SMAD7 expression in various mucosal samples by immunofluorescence staining.

[0145] Briefly, patient mucosal samples were prepared 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 at room temperature for 20 minutes. The sections were then blocked at room temperature for 1 hour (BSA 1%, Tween 0.1%, glycine 2%) and then incubated overnight at 4°C with a mouse primary antibody against SMAD7 (1:150). After overnight incubation, the sections were washed with PBS 1X and treated with the secondary antibody goat anti-mouse Alexa488 (1:1000) at room temperature.1 hour. The slides were then washed with PBS1X, mounted with a long-acting gold anti-fading reagent containing DAPI, and analyzed under a microscope.

[0146] The data are shown in Figures 1A-1C, which show sections of fresh patient mucosal biopsy samples, analyzing the expression of SMAD7 (green) and DAPI (blue). This figure (Figures 1A-1C) represents three separate experiments in which similar results were obtained. Single and double immunofluorescence staining of sections of healthy ileum (Figure 1A), uninflamed pouch (Figure 1B), and inflamed pouch (Figure 1C) show exemplary results. Scale bar is 50 μm.

[0147] The data indicate that the expression of SMAD7, visualized as a green immunofluorescence signal, was increased in the sample sections of inflamed pouches compared to the expression of SMAD7 in sections of healthy ileum or uninflamed pouches.

[0148] Example 3: Increased SMAD7 protein expression in chronic pouchitis

[0149] In this example, SMAD7 protein expression in various mucosal samples was assessed by Western blotting, and the results of Western blotting were quantitatively analyzed by density measurement scanning. Instructions for Use, Pages 22 / 24, 27 CN 120958131 A

[0150] In short, mucosal biopsy samples for protein blotting are lysed on ice in a buffer containing 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, supplemented with 1 mM dithiothreitol (DTT), 10 mg / ml protease inhibitor peptide, 10 mg / ml leucine inhibitor peptide, 1 mM benzyl sulfonyl fluoride (PMSF), 1 mM Na3VO4 and 1 mM sodium fluoride (NaF). The lysate is clarified by centrifugation and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The blots were incubated with antibodies against SMAD7 (1:1000) or β-actin (1:5000), followed by incubation with a secondary antibody conjugated with horseradish peroxidase (1:20000). The intensity of the immunoreaction bands was analyzed using computer-aided scanning density assays.

[0151] The Western blot results depicted in Figures 2A-2B show the protein expression of SMAD7 and β-actin in total proteins extracted from fresh mucosal biopsy samples, and include the corresponding analysis of the SMAD7 / β-actin ratio measured by density assay scanning of the Western blots.

[0152] Data are shown in Figures 2A-2B, which display the results of Western blot detection of SMAD7 and β-actin in total proteins extracted from mucosal biopsy samples, and include the results from the Western blots measured by density assay scanning.The corresponding quantitative graphical analysis of the SMAD7 / β-actin ratio. In Figure 2A, the total protein extract analyzed was isolated from mucosal samples of inflamed pouch (pouchitis), normal pre-pouch ileum of the same patient with pouchitis, uninflamed pouch, and healthy ileum (normal control). In Figure 2B, the total protein extract analyzed was isolated from mucosal samples of inflamed pouch (pouchitis), inflamed ileum of the same patient with pouchitis (pre-pouch ileitis), and healthy ileum (normal control).

[0153] The data showed that SMAD7 protein expression was increased in inflamed pre-pouch and inflamed pouch mucosal samples compared with the SMAD7 protein expression levels observed in uninflamed and normal healthy ileal mucosal samples.

[0154] Example 4: Colocalization of SMAD7 with CD3 and HLA-DRII in pouchitis tissue

[0155] This example uses immunofluorescence staining to assess the colocalization of SMAD7 with CD3 and HLA-DRII in pouchitis tissue samples.

[0156] Briefly, sections of fresh mucosal biopsy samples were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 at room temperature for 20 minutes. The sections were then blocked at room temperature for 1 hour (BSA 1%, Tween 0.1%, glycine 2%) and then incubated overnight at 4°C with 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 PBS1X, the secondary antibody goat anti-rabbit Alexa488 (1:1500) or goat anti-mouse Alexa488 (1:1000) was applied and left at room temperature for 1 hour. The slides were then washed with PBS1X, mounted with a long-acting gold anti-fading reagent containing DAPI, and analyzed under a microscope.

[0157] The data are shown in Figures 3A-3B, which show single and double immunofluorescence staining of sections of mucosal biopsy samples taken from patients with chronic pouchitis, analyzing the expression of SMAD7 (green), CD3 (red; Figure 3A), HLA-DRII (red; Figure 3B), and DAPI (blue). This figure (Figures 3A-3B) represents three separate experiments in which similar results were obtained. Scale bar is 75 μm.

[0158] The data in Figure 3A show that SMAD7 expression colocalizes with CD3 in pouch inflammation tissue, and Figure 3B shows that SMAD7 expression colocalizes with HLA DRII in pouch inflammation tissue.

[0159] Example 5: SMAD7 knockdown alters the production of several inflammatory and anti-inflammatory molecules in ex vivo organ cultures

[0160] This example evaluated ex vivo organ cultures transfected with SMAD7 sense oligonucleotides (S) or SMAD7 antisense oligonucleotides (AS).Expression levels of SMAD7 and various inflammatory and anti-inflammatory molecules in organ cultures. Exemplary results are shown in Figures 4A-4B.

[0161] In short, ex vivo organ cultures were prepared from mucosal samples taken from inflamed storage bags and placed on a steel mesh in an organ culture chamber at 37°C in a 5% CO2 / 95% O2 atmosphere and cultured in RPMI 1640 medium. The ex vivo organ cultures were transfected with SMAD7 semantic oligonucleotides or SMAD7 AS (10 μg / ml) for 24 hours using Opti-MEM medium and Lipofectamine 3000 reagent, as per the instructions on page 23 / 24 of the manual (CN 120958131 A). Phosphothioester single-stranded oligonucleotides matching region 107-128 (5'-GCTGCGGGGAGAAGGGGCGAC-3') of the complementary DNA sequence to human SMAD7 were synthesized in both sense and antisense directions

[0162] . For the real-time polymerase chain reaction results shown in Figure 4A, total RNA was extracted and a constant amount of RNA (1 μg / sample) was reverse transcribed into complementary DNA (cDNA) using Oligo(dT) primers and M-MLV reverse transcriptase. Amplification was performed under the following conditions: denaturation at 95°C for 1 min; annealing at 59°C for 30 sec for human SMAD7 and annealing at 60°C for 30 sec for human β-actin; extension at 72°C for 30 sec. RNA expression was calculated relative to the β-actin gene using the ΔΔCt algorithm. The primer sequences are as follows: SMAD7 Fw 5'-GCCCGACTTCTTCATGGTGT-3', Rev 5'-TGCCGCTCCTTCAGTTTCTT-3'; β-actin FW 5'-AAGATGACCCAGATCATGTTTGAGACC-3', Rev 5'-AGCCAGTCCAGACGCAGGAT-3'.

[0163] For the results of the human cytokine expression array assay shown in Figure 4B, the human cytokine array C3 was used. In short, the supernatant of the inflamed reservoir sample transfected with SMAD7 semantic oligonucleotides or SMAD7AS (10 μg / ml) was 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 the bound antibodies on the membrane were visualized using the ECL system. Quantitative analysis was performed using density measurement scans of the array imprint.

[0164] For statistical analysis, Student's t-test was used to compare differences between groups. Pearson correlation coefficient was used to assess the correlation between SMAD7 and the modified pouch inflammation disease activity index (mPDAI).

[0165] The data in Figure 4A show that ex vivo organ cultures transduced with SMAD7 antisense oligonucleotides exhibit reduced SMAD7 RNA expression compared to cultures transduced with SMAD7 sense oligonucleotides. The data in Figure 4B show that ex vivo organ cultures transduced with SMAD7 antisense oligonucleotides exhibit altered expression profiles of several inflammatory and anti-inflammatory molecules compared to cultures transduced 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 pouch tissue.

[0166] Equivalents

[0167] This disclosure may be embodied in other specific forms without departing from its essential characteristics. Therefore, the foregoing embodiments are to be considered illustrative rather than limiting of the disclosure described herein. The scope of this disclosure is indicated by the appended claims rather than the foregoing description and is intended to include all variations falling within the meaning and equivalent scope of the claims. Instruction manual, 24 / 24 pages; 29 CN 120958131 A; Figure 1A; Figure 1B; Figure 1C; Instruction manual drawing, 1 / 4 page; 30 CN 120958131 A; Figure 2A; Figure 2B; Instruction manual drawing, 2 / 4 page; 31 CN 120958131 A; Figure 3A; Figure 3B; Instruction manual drawing, 3 / 4 page; 32 CN 120958131 A; Figure 4A; Figure 4B; Instruction manual drawing, 4 / 4 page; 33 CN 120958131 A

Claims

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

2. The method of claim 1, wherein all nucleoside linkages are O,O-linked thiophosphate linkages and X is 5-methyl-2'-deoxycytidine.

3. The method of any one of claims 1-2, wherein the SMAD7 antisense oligonucleotide is administered intestinally.

4. The method of claim 3, wherein intestinal administration is oral, sublingual, gastric, or rectal administration.

5. The method of claim 3, wherein intestinal administration is rectal administration.

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

7. The method of claim 6, wherein the parenteral administration is administered intravenously, intratumorally, intrajejunally, intraileally, intracolonally, intrarectally, or via a pouch.

8. The method of claim 7, wherein parenteral administration is administered via a storage bag.

9. The method of any one of claims 1-8, wherein the therapeutically effective amount of SMAD7ASO comprises a dose of about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg.

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

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

12. The method of any one of claims 1-11, wherein a therapeutically effective amount of SMAD7ASO is applied every about 6 hours, every about 12 hours, every about 24 hours, every about 48 hours, every about 72 hours, daily, twice a week, once every two weeks, or once a month.

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

14. The method of any one of claims 1-13, wherein SMAD7 ASO downregulates SMAD7 mRNA and / or protein in cells.

15. The method of claim 14, wherein the expression of SMAD7 protein is downregulated by more than 40% compared with untreated cells.

16. The method of any one of claims 1-15, wherein at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

17. The method of any one of claims 1-16, wherein pouchitis is acute pouchitis.

18. The method of any one of claims 1-16, wherein pouchitis is chronic pouchitis.

19. The method of any one of claims 1-18, wherein the individual is identified as having pouchitis using a modified pouchitis disease activity index (mPDAI) score of at least 5.

20. A method of treating pouchitis in an individual in need, comprising administering a pharmaceutical preparation to the individual, said pharmaceutical preparation comprising: a) An SMAD7 antisense oligonucleotide (ASO) having the sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'); and b) Pharmaceutically acceptable carriers.

21. The method of claim 20, wherein the pharmaceutical formulation comprises 100 millimoles to 5 moles of SMAD7 ASO.

22. The method of claim 20, wherein the pharmaceutical formulation comprises 100 mmol to 2 mmol, 100 mmol to 900 mmol, 300 mmol to 5 mmol, 300 mmol to 2 mmol, 300 mmol to 900 mmol, 900 mmol to 5 mmol, or 900 mmol to 2 mmol of SMAD7 ASO.

23. The method of claim 20, wherein the pharmaceutical formulation comprises about 300 millimoles of SMAD7 ASO.

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

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

26. The method of claim 20, wherein the pharmaceutical formulation comprises greater than 300 mmol, greater than 900 mmol, or greater than 2 mmol of SMAD7 ASO.

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

28. The method of claim 27, wherein the pharmaceutical formulation comprises: SMAD7 ASO, by weight, is approximately 0.5% to approximately 30%. Mannitol, approximately 20% to approximately 50% by weight; Microcrystalline cellulose, by weight, approximately 10% to approximately 30%; and Enteric coating, which contains ethyl acrylate-methacrylic acid copolymer.

29. The method of any one of claims 27, wherein the pharmaceutical formulation comprises: a) Intragranular phase, which includes: i) Approximately 5% to approximately 10% by weight of SMAD7 ASO; ii) Approximately 40% mannitol by weight; iii) Approximately 8% microcrystalline cellulose by weight; iv) Approximately 5% by weight of hydroxypropyl methylcellulose; and v) Approximately 2% sodium carboxymethyl starch by weight; b) Extragranular phase, which includes: i) Approximately 17% microcrystalline cellulose by weight; ii) Sodium carboxymethyl starch, approximately 2% by weight iii) about 0.4% magnesium stearate by weight; and c) Enteric coating, which contains ethyl acrylate-methacrylic acid copolymer. The percentage by weight is the weight of the component relative to the total weight of the pharmaceutical preparation.

30. The method of claim 27, wherein the pharmaceutical formulation comprises: SMAD7 ASO, by weight, is approximately 5% to approximately 30%; Mannitol, approximately 20% to approximately 50% by weight; Microcrystalline cellulose, by weight, comprises approximately 10% to approximately 30%. Hydroxypropyl methylcellulose, approximately 0.5% to approximately 10% by weight; Sodium carboxymethyl starch, approximately 0.5% to approximately 10% by weight; Magnesium stearate, approximately 0.05% to approximately 1% by weight; A moisture-proof film coating of about 0.5% to about 10% by weight; and The polymer coating comprises approximately 5% to approximately 20% by weight.

31. The method of claim 27, wherein the pharmaceutical formulation comprises: Approximately 8.5% SMAD7 ASO by weight; Approximately 40% mannitol by weight; Approximately 25% microcrystalline cellulose by weight, Approximately 5% hydroxypropyl methylcellulose by weight; Sodium carboxymethyl starch, approximately 4% by weight; Magnesium stearate, approximately 0.4% by weight; Approximately 4% moisture-proof film coating by weight; and The polymer coating comprises approximately 10% to approximately 15% by weight.

32. The method of claim 27, wherein the pharmaceutical formulation comprises: Approximately 23% SMAD7 ASO by weight; Approximately 28% mannitol by weight; Approximately 25% microcrystalline cellulose by weight, Approximately 5% hydroxypropyl methylcellulose by weight; Sodium carboxymethyl starch, approximately 4% by weight; Magnesium stearate, approximately 0.4% by weight; Approximately 4% moisture-proof film coating by weight; and The polymer coating comprises approximately 7% to approximately 12% by weight.

33. The method of any one of claims 20-32, wherein the pharmaceutical formulation downregulates SMAD7 mRNA and / or protein in cells.

34. The method of claim 33, wherein the expression of SMAD7 protein is downregulated by more than 40% compared with untreated cells.

35. The method of any one of claims 20-34, wherein at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

36. The method of any one of claims 20-35, wherein pouchitis is acute pouchitis.

37. The method of any one of claims 20-35, wherein pouchitis is chronic pouchitis.

38. The method of any one of claims 20-37, wherein the individual is identified as having pouchitis using a modified pouchitis disease activity index (mPDAI) score of at least 5.

39. A method for predicting pouch inflammation in an individual in need, comprising determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouch inflammation, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment for pouch inflammation, or the SMAD7 level in a sample collected from a healthy individual without pouch inflammation.

40. A method for identifying individuals at risk of developing pouchitis, comprising: Determine the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual as being at risk of developing pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment for pouchitis, or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

41. The method of claim 39 or 40, comprising: (i) If the SMAD7 level is elevated relative to the known control level, administer a drug formulation containing SMAD7 ASO to the individual; or (ii) If the SMAD7 level is not elevated relative to the known control level, determine the SMAD7 level in the second sample from that individual.

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

43. A method for treating pouch inflammation in an individual in need, comprising determining a SMAD7 level in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level predicts pouch inflammation, wherein the known control level is the SMAD7 level in a sample collected from the individual before or during treatment for pouch inflammation, or the SMAD7 level in a sample collected from a healthy individual without pouch inflammation.

44. The method of any one of claims 39-42, wherein SMAD7 ASO comprises a sequence selected from any one of SEQ ID NO:1-6 or a pharmaceutically acceptable salt thereof.

45. The method of any one of claims 39-42, wherein SMAD7 ASO has a sequence according to SEQ ID NO:1 (5'-GTXGCCCCTTCTCCCXGCAGC-3').

46. ​​The method of claim 45, wherein all nucleoside linkages are O,O-linked thiophosphate linkages and X is 5-methyl-2'-deoxycytidine.

47. The method of any one of claims 1-46, wherein the individual is a person.

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