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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K38/00—Medicinal preparations containing peptides
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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
  • A61K47/38—Cellulose; Derivatives thereof
• • A—HUMAN NECESSITIES
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  • A61K9/00—Medicinal preparations characterised by special physical form
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  • A61K9/0031—Rectum, anus
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates

Definitions

• This invention relates to antisense oligonucleotide therapeutic compound to modulate the expression of intracellular adhesion molecule-1 (ICAM-1) for the amelioration and/or treatment of inflammatory bowel diseases including Crohn's disease, ulcerative colitis and pouchitis.
• IAM-1 intracellular adhesion molecule-1
• ICAM-1 a member of the immunoglobulin (Ig) superfamily, is an inducible transmembrane glycoprotein constitutively expressed at low levels on vascular endothelial cells and on a subset of leucocytes (Dustin et al., J. Immunol., 137:245-54, 1986; Rothlein et al, J. Immunol., 137:1270-4, 1986; Simmons et al, Nature, 331:624-7, 1988).
• ICAM-1 The primary ligands for ICAM-1 binding are the ⁇ 2 integrins, LFA-1 and Mac-1, both of which are expressed on leukocytes (Marlin and Springer, Cell, 51:813-9, 1987; Diamond et al, J. Cell Biol., 111:3129-39, 1990).
• ICAM-1 serves multiple functions in the propagation of inflammatory processes, the best characterized being the facilitation of leukocyte migration from the intravascular compartment to the extravascular space at sites of inflammation (Butcher, Cell, 67:1033-6, 1991; Furie et al, Blood, 78:2089-97, 1991; Oppenheimer-Marks et a.l., J. Immunol., 147:2913-21, 1991).
• ICAM-1 also appears to provide an important second signal to T-lymphocytes during antigen presentation (Altmann et al., Nature, 338:512-4, 1989; Van Seventer et al., J. Immunol. 144:4579-86, 1990; Kuhlman et al., J. Immunol., 146:1773-82, 1991). It also plays and important faciliatory roll in cytotoxic T-cell (Makgoba et al., Eur. J. Immunol., 18:637-40, 1988), natural killer cell (Allavena et al., Blood, 84:2261-8, 1994), and neutrophil-mediated (Ding et al., J. Immunol., 163:5029-38, 1999) damage to target cells.
• TNF- ⁇ tumor necrosis factor-alpha
• IBD inflammatory bowel disease
• rheumatoid arthritis To et al., Arthritis Rheum., 39:467-77, 1996)
• celiac disease Sturgess et al., Clin, Exp. Immunol. 82 :489-92, 1990
• IgA neuropathy Nguyen et al., Am. J. Nephrol.
• Anti-ICAM-1 monoclonal antibodies have demonstrated beneficial effects in a variety of animal models of disease, including pulmonary inflammation and asthma (Barton et al., J. Immunol. 143:1278-82, 1989; Wegener et al., Science, 247:456-9, 1990), allograft rejection (Cosimi et al., J. Immunol., 144:4604-12, 1990; Isobe et al., Science, 255:1125-7, 1993), nephritis (Harning et al., Clin. Immunol Immunopath., 64:129-34, 1992; Kawasaki et al., J.
• ICAM-1 expression has been demonstrated during inflammatory bowel disease (IBD).
• IBD inflammatory bowel disease
• ICAM-1 expression is increased on the endothelium of colonic submucosal and tunica muscularis veins (Bennett, J. Pharm. Exp. Ther., 280:988-1000,1997).
• Human ICAM-1 expression in Crohn's disease is increased in the lamina limba muscle micorovasculature (both colonic and jejunal) (Sousa et al., Gut, 45:856-63, 1999), gut mononuclear cells (Bernstein et al., Clin.
• IBD Inflammatory bowel disease
• endoscopy either sigmoidoscopy or colonoscopy
• CT computed tomography
• Both diseases are chronic, relapsing/remitting inflammatory diseases of the gastrointestinal (GI) tract.
• GI gastrointestinal
• the regions of the GI tract that are most often affected by Crohn's disease are the small intestine and large intestine, also called the colon, including the rectum; however, Crohn's disease can affect the entire GI tract from the mouth to the anus.
• ulcerative colitis may be a single event, or continuous with unrelenting symptoms.
• aminosalicylates For mild ulcerative colitis, orally or topically (i.e. enema) delivered aminosalicylates are typically the first line of treatment.
• the aminosalicylate class consists of agents that contain 5-aminosalicyclic acid (5-ASA), is one of the oldest anti-inflammatory compounds employed in IBD.
• 5-ASA 5-aminosalicyclic acid
• the 5-ASA sulfasalazine was developed in the 1930's for the treatment of rheumatoid arthritis, and its utility in the treatment of IBD was established in the 1970's (Anthonisen et al., Scan. J. Gastroenterol., 9:549-554, 1974).
• 5-ASAs Used in high doses, 5-ASAs can induce remission in acute attacks.
• 5-ASAs have not been demonstrated to be effective in maintaining remission.
• 5-ASA formulations include sulfasalazine, oral and topical mesalamine, olsalazine and balsalazide.
• Various formulations are modified to provide available active drug to the site of interest (e.g. small or large intestine).
• Side effects are not uncommon with 5-ASAs.
• Sulfasalazine has a relatively high toxicity (approximately one third of patients), associated with the sulfa group, including headache, nausea, dyspepsia and anorexia.
• Less common side effects include fever, rash, arthralgia, hemolysis, neutropenia, exacerbation of colitis, hypersensitivity reactions and nephrotoxicity.
• Mesalamine, olsalazine and balsalazide which do not contain sulfa groups result in substantially fewer side effects, but still can cause rash, headaches and fever. Other more severe side effects have also been reported.
• Mesalamine enema has been implicated in the production of an acute intolerance syndrome characterized by cramping, acute abdominal pain and bloody diarrhea, sometimes headache, fever and a rash. While using mesalamine enema, some patients have developed pancolitis; however, the frequency was lower than with a placebo treated group. The extent of absorption of mesalamine from enema is largely dependent on retention time and therefore varies. Systemic exposure is substantially reduced by enema administration as compared to oral administration; however, 10 to 30% of the daily dose can be recovered in 24-hour urine collection suggesting that the systemic exposure is not insubstantial.
• Corticosteroids are among the most effective agents for inducing remission in IBD attacks and are typically the second therapeutic option upon failure of treatment with 5-ASAs.
• the compounds are delivered first either orally or rectally, with or without concomitant administration of 5-ASAs. Upon failure of oral delivery, the compounds are administered intravenously. Ideally, corticosteroids are used for only a short course of treatment and tapered off upon remission of disease.
• Corticosteroids commonly used for the treatment of IBD include prednisone, budesonide and hydrocortisone.
• the use of corticosteroids is limited by the number of severe and significant side effects associated with their use. Common side effects of short term use include insomnia, night sweats, mood changes and altered glucose metabolism. Prolonged maintenance therapy is typically reserved only for severe, refractory cases. Prolonged therapy can lead to adrenal atrophy, whereas abrupt cessation can cause adrenal insufficiency, hypotension, and even death. Other side effects include acne, abnormal fat deposition, excessive hair growth and osteoporosis. In Crohn's disease, corticosteroids can thwart the healing of fistula, exacerbating the disease state.
• Immunosuppressant interventions are not without undesirable side effects.
• 6-Mercaptopurine and azathioprine can cause fever, rash, nausea and headache, with more severe side effects including leucopenia, pancreatitis, severe infections and bone marrow suppression.
• Cyclosporine can have more severe side effects including paresthesias (abnormal sensations like burning or tingling), excessive hair growth, hypertension, tremor, renal insufficiency, headache and opportunistic infections.
• Antibiotics typically ciprofloxacin or metronidazole, are used as add on therapies to 5-ASA or corticosteroids, especially with patients with fistulizing or colonic disease. As with all of the other therapies, there are side effects of long term treatment with antibiotics.
• Infliximab is currently the pharmacotherapy of last resort in IBD. It is a chimeric monoclonal antibody composed of 75% human and 25% mouse protein. Infliximab is an inhibitor of tumor necrosis factor-alpha (TNF- ⁇ ), a potent inflammatory cytokine. The drug acts as a sink by binding both soluble and membrane bound TNF- ⁇ . By inhibiting an activator high in the inflammatory cascade, a number of inflammatory pathways can be inhibited. The drug is administered intravenously first for treatment and subsequently as a maintenance drug every eight (8) weeks as indicated on the product label. However, as it is a biological agent, an immune response can limit utility of the drug.
• TNF- ⁇ tumor necrosis factor-alpha
• TNF- ⁇ plays an important role in the eradication of neoplastic cells; therefore, its suppression can lead to opportunistic infections, malignancies and other complications, especially as a long term strategy (Brown et al., Arthritis Rheum., 46:3151-8, 2002; Lee et al., Arthritis Rheum., 46:2565-70, 2002; Nahar et al., Ann. Pharmacotherapy, 37:1256-65, 2003).
• Surgical interventions are a method of treatment of IBD, not a cure. Due to the chronic nature of IBD and the relatively early age of onset, multiple surgeries can be required over the lifetime of patients who are not responsive to pharmacological interventions. Removal of short portions of the intestine is possible without substantial side effects. However, removal of larger or multiple segments of the intestine can result in short bowel syndrome in which individuals are unable to absorb nutrients. Removal of portions of the large intestine can result in the need for colostomy or other further surgical procedures. Therefore, surgery is not a preferred method of treatment of IBD.
• an ileal pouch may be constructed from the small intestine by the surgeon to allow removal of feces through the anus rather than requiring a permanent ostomy.
• Pouchitis is a non-specific inflammation of the ileal pouch which typically occurs within the first two years after reconstruction. Symptoms include steadily increasing stool frequency that may be accompanied by incontinence, bleeding, fever and/or a feeling of urgency. Of those who have ulcerative colitis, approximately 20 to 30 percent experience at least one episode. Antibiotics can be sufficient to treat pouchitis; however, other more aggressive therapies similar to those used in IBD are required.
• Antisense oligonucleotides offer an ideal solution to the problems encountered in prior art approaches. They can be designed to selectively inhibit expression of a given nucleic acid or protein, and avoid non-specific mechanisms of action by interacting with a nucleic acid target based on nucleotide sequence, allowing for the inhibition of a specific isoform of a family of similar protein structure or activity. A complete understanding of target mechanisms or macromolecular interactions is not needed to design specific inhibitors.
• ICAM-1 Human ICAM-1 is encoded by a 3.3-kb mRNA (SEQ ID NO 1) resulting in the synthesis of a 55,219 dalton protein. ICAM-1 is heavily glycosylated through N-linked glycosylation sites. The mature protein has an apparent molecular mass of 90 kDa as determined by SDS-polyacrylamide gel electrophoresis (Staunton et al., Cell, 52:925-933, 1988). ICAM-1 is a member of the immunoglobulin (Ig) superfamily. It contains five immunoglobulin-like domains at the amino terminus, followed by a transmembrane domain and a cytoplasmic domain. The primary binding site for LFA-1 and rhinovirus are found in the first immunoglobulin-like domain. However, the binding sites appear to be distinct (Staunton et al., Cell, 61:243-354, 1990).
• ICAM-1 exhibits a broad tissue and cell distribution, and may be found on white blood cells, endothelial cells, fibroblast, keratinocytes and other epithelial cells.
• the expression of ICAM-1 can be regulated on vascular endothelial cells, fibroblasts, keratinocytes, astrocytes and several cell lines by treatment with bacterial lipopolysaccharide and cytokines such as interleukin-1, tumor necrosis factor, gamma-interferon, and lymphotoxin (See, e.g., Frohman et al., J. Neuroimmunol., 23:117-124, 1989).
• oligonucleotides were tested for the ability to inhibit the expression of human ICAM-1 (SEQ ID NO 1) using both in vitro and in vivo experiments (see e.g., U.S. Pat. No. 5,514,788). From these experiments, the oligonucleotide ISIS 2302 (SEQ ID NO 2) which is targeted to nucleotides 2114 to 2133 of human ICAM-1 was selected for further development.
• ISIS 2302 is a 20-base phosphorothioate oligodeoxynucleotide designed to specifically hybridize to a sequence in the 3′-untranslated region of the human ICAM-1 mRNA. Studies strongly suggest that ISIS 2302 functions by specifically binding to the ICAM-1 mRNA resulting in cleavage of the mRNA by the enzyme RNaseH1 (Crooke, Biochim. Biophys. Acta., 1489:31-44, 1999), one of a ubiquitous family of RNaseH nucleases. However, the method of the invention is not limited by the mechanism of action of ISIS 2302.
• Phosphorothioate modification of the oligodeoxynucleotide by substituting a sulfur molecule for a non-bridging oxygen molecule in each phosphodiester linkage, significantly increases exonuclease resistance relative to unmodified DNA and prolongs the drug half life (Geary et al., Anti - Cancer Drug Design, 12:383-94, 1997).
• Phosphorothioate oligonucleotides are only minimally antigenic, non-cytotoxic and well tolerated, and their pharmacokinetic and pharmacodynamic properties are well characterized (see e.g., Butler et al., Lab. Invest., 77:379-88, 1997; Mirabelli et al., Anti - Cancer Drug Des., 6:647-61, 1991)
• Antisense oligonucleotides targeted to ICAM were shown to be effective in mouse and rat models of inflammatory bowel disease when systemically administered by subcutaneous or intraperitoneal injection (see, e.g., Bennett, U.S. Pat. No. 5,514,788, example 20; and U.S. Pat. No. 6,096,722, example 29, respectively). These data were the foundation for the development of clinical trials for the treatment of Crohn's disease, pouchitis and ulcerative colitis in humans. In the Crohn's trial, ISIS 2302 (Alicaforsen) was administered systemically to individuals in placebo controlled studies.
• ISIS 2302 Phase II studies suggested efficacy of intravenously administered ISIS 2302 in some subsets of patients (Yacshyn et al., Gut, 51:30-36. 2002; Yacyshyn et al., Aliment. Pharmacol. Ther., 16:1761-70. 2002). In two subsequent Phase III trials, intravenously administered ISIS 2302 was found to be ineffective in treating moderate to severe Crohn's disease patients based on clinical remission and disease activity index scores (Chey et al., Gastroenterology, 128:A-112, abst 724. 2002).
• ISIS 2302 An enema formulation of ISIS 2302 was used in a clinical trial for the treatment of pouchitis (US Patent Publication 20040162259, see Example 17, Miner et al., Alimnet. Pharmacol. Ther., 19:281-6.2004). Twelve patients with chronic, unremitting pouchitis and a Pouchitis Disease Activity Index (PDAI) of at least 7 were enrolled in the study.
• PDAI is a clinical score based on a combination of factors including stool frequency, rectal bleeding, fecal urgency, abdominal cramps, fever of greater than 100° F., endoscopic and histologicic scores.
• active pouchitis is defined as having a PDAI score of at least 7.
• ISIS 2302 A small randomized, controlled, double-blind escalating dose study of rectally delivered ISIS 2302 for the treatment of mild to moderate ulcerative colitis (SJH van Deventer et al., Gut, 53:1646-51). Patients were treated with one of four daily doses of ISIS 2302 (6, 30, 120 and 240 mg) for 28 days, and the safety and efficacy of the treatment were monitored after 1, 3 and 6 months for improvement in disease activity index (DAI). DAI is a clinical score based on stool frequency, rectal bleeding, endoscopic appearance and investigator's global assessment. None of the patients in the 240 mg/day group or compared with 50% of the patients in the placebo group required additional surgical or medical intervention over baseline during the six months of the study. The results showed promising acute and long term benefits; however, the results required verification in a larger clinical trial to be conclusive.
• DAI disease activity index
• a method for the sustained amelioration or treatment of ulcerative colitis comprising rectal administration of a therapeutic dose of a composition comprising an antisense oligonucleotide compound 8 to 80 nucleobases in length, preferably formulated in hydroxylpropyl methylcellulose, to an individual wherein the oligonucleotide comprises at least an 8 nucleobase portion that specifically hybridizes with nucleotides 2114 to 2133 of SEQ ID 1, and monitoring the individual for amelioration of disease.
• a method for promoting mucosal healing and a durable response comprising administration of the composition.
• a method for producing disease modifying results comprising administration of the composition.
• Disclosed is a method for the sustained amelioration or treatment of ulcerative colitis comprising rectal administration of a therapeutic dose of a composition comprising an antisense oligonucleotide compound having the sequence (SEQ ID 2):
• oligonucleotide inhibits expression of the ICAM-1 protein, and monitoring the individual for amelioration of disease.
• a method for promoting mucosal healing and a durable response comprising administration of the composition.
• a method for producing disease modifying results comprising administration of the composition.
• composition comprising an antisense oligonucleotide compound 8 to 80 nucleobases in length wherein the oligonucleotide comprises at least an 8 nucleobase portion that specifically hybridizes with nucleotides 2114 to 2133 of SEQ ID NO: 1 for the preparation of a medicament for rectal administration to an individual to promote mucosal healing and a durable response in an individual suffering from ulcerative colitis.
• a further use for the medicament is to produce disease modifying outcomes.
• the oligonucleotide can have the sequence of SEQ ID NO: 2, and that the medicament for rectal administration can be prepared using hydroxypropyl methylcellulose.
• the composition is used for the manufacture of a medicament for the treatment of individuals having moderate to severe ulcerative colitis.
• Antisense oligonucleotides hold great promise as therapeutic agents for the treatment of many human diseases. Oligonucleotides specifically bind to the complementary sequence of either pre-mRNA or mature mRNA, as defined by Watson-Crick base pairing, inhibiting the flow of genetic information from DNA to protein. The properties of antisense oligonucleotides that make them specific for their target sequence also make them extraordinarily versatile. Because antisense oligonucleotides are long chains of four monomeric units they may be readily synthesized for any target RNA sequence.
• oligonucleotide is delivered by rectal administration in an enema formulation comprising the oligonucleotide in hydroxypropyl methylcellulose. Details regarding routes of administration are provided in the subsequent examples and in the parent patent applications which are incorporated herein by reference
• Formulations for the delivery of pharmaceutical compositions are well known to those skilled in the art. The selection of a specific formulation is based on considerations well known to those skilled in the art including, but not limited to, route of administration, solubility of the compound to be administered and quantity of the compound to be administered. Detailed formulations are presented in the examples and in U.S. Pat. Nos. 6,096,722 and 6,747,014 both incorporated herein by reference.
• ISIS 2302 produces a durable response that is more sustained than with the standard of care, mesalamine. This durability of response is critical in a disease such as ulcerative colitis that has a profound effect on the quality of life of individuals who suffer from the disease. By reducing the frequency of flares the quality of life of the patient is improved. Moreover, ISIS 2302 has been shown to have disease modifying activity which has not been observed with any previous therapies for ulcerative colitis.
• oligonucleotide tested in the trials disclosed herein had a fully phosphorothioate modified backbone.
• Other possible backbones, modified sugars or other forms of the same nucleotide sequence can be used in the instant invention.
• oligonucleotide refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, increased stability in the presence of nucleases and increased hybridization affinity.
• oligonucleotide backbones and sugars including mixed or chimeric oligomers
• PNA peptide nucleic acids
• LNA locked nucleic acids
• Methods for synthesis of unmodified and modified oligonucleotides are also provided.
• oligonucleotides contain at least one phosphorothioate and/or heteroatom internucleoside linkage wherein a phosphorothioate linkage is most preferred.
• the oligonucleotides in accordance with this invention preferably comprise from about 8 to about 80 nucleic acid base units. It is more preferred that such oligonucleotides comprise from about 12 to 50 nucleic acid base units, still more preferred to have from about 15 to 30 nucleic acid base units, and most preferred to have from about 18 to 22 nucleic acid base units.
• a nucleic acid base unit is a base-sugar combination suitably bound to an adjacent nucleic acid base unit through phosphodiester or other bonds.
• about 8 to about 80 nucleic acid base units includes 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleobase units.
• the oligonucleotide compound of the invention preferably comprise at least an 8 nucleobase portion, more preferably at least a 12 nucleobase portion, even more preferably at least an 15 nucleobase portion, further more preferably an 18 nucleobase portion, most preferably a 20 nucleobase portion that specifically hybridizes with nucleotides 2114 to 2133 of SEQ ID NO: 1.
• IBD Inflammatory bowel disease
• IBD in the context of the invention means diseases that cause irritation and ulcers in the intestinal tract, with the most common forms of IBD being Crohn's disease and ulcerative colitis.
• Other inflammatory bowel diseases include, but are not limited to pouchitis, irritable bowel syndrome, regional enteritis and regional ileitis.
• “Messenger RNA” is understood by those skilled in the art to be the open reading frames (ORFs) of the DNA from which they are transcribed includes not only the information from the ORFs of the DNA, but also associated ribonucleotides which form regions known to such persons as the 5′-untranslated region, the 3′-untranslated region, and intervening sequence ribonucleotides.
• ORFs open reading frames
• oligonucleotides may be formulated in accordance with this invention, which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides.
• the oligonucleotide is specifically hybridizable with a sequence in the 3′-untranslated region, specifically nucleotides 2114 to 2133 of SEQ ID NO 1.
• Hybridization in the context of this invention, means hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand.
• Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them.
• Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.
• oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
• “Pharmaceutical composition” is a composition comprising a pharmacologically active agent optionally further containing a vehicle to deliver the agent wherein the composition is suitable for administration to an animal, preferably a human animal.
• the vehicle may be inert, such as normal saline or agent to make the composition more palatable in the case of an orally administered composition.
• the vehicle may alternatively be an active agent to increase or modify bioavailability of the pharmacologically active agent.
• Such active vehicles include penetration enhancers or compounds to protect the pharmacologically active in the stomach to allow the pharmacologically active agent to be absorbed in the intestine. Such active vehicles are known to those skilled in the art.
• a “therapeutic dose” is a quantity of a composition required to provide amelioration or treatment of the disease to be treated by the composition.
• Methods for monitoring disease are well known to those skilled in the art. Guidance is provided in the examples regarding therapeutic doses and methods for monitoring the severity of disease, specifically IBD.
• “Sustained” is a longer state of substantially reduced clinical manifestations of ulcerative colitis and/or a maintained level of mucosal healing after termination of administration of the compositions disclosed herein without the need for increased therapy using other pharmacological or surgical interventions as compared to the standard of care or placebo.
• Sustained is defined as at least about sixty (60) days, preferably at least about ninety (90) days, even more at least about preferably 120 days, most preferably at least about 180 days of reduced indications of the disease or level of mucosal healing beyond administration of the last dose of the composition disclosed herein.
• a sustained reduction in clinical manifestations is indicative of a more durable response to the intervention.
• a drug with “disease modifying activity” as used herein means a composition with the ability to slow down the rate of progression of disease as defined by standard disease monitoring measures (e.g. DA1), and/or to have effects for an extended period after the clearance of the drug.
• standard disease monitoring measures e.g. DA1
• “Indications of ulcerative colitis” are symptoms or conditions that are associated with the presence of the disease ulcerative colitis.
• the indications may be objective (e.g. number of stools, rectal bleeding, mucosal friability), subjective (e.g. physician's assessment) or a composite of multiple observations (e.g. DAI, or clinical activity index)
• Disease Activity Index for ulcerative colitis is defined by Schroeder et al. (1987. N. Engl. J. Med. 317:1625-9) and is an aggregate score based on stool frequency, rectal bleeding, endoscopic appearance and the investigator's global assessment which includes patient's symptoms, findings of sigmoidoscopy and physical examination, laboratory studies and the patient's overall status. Each of the four aspects of the index is rated from 0-3 with 0 being the least severe and 3 being the most severe.
• a “treatment period” as used herein means a time during which the composition is administered on a relatively frequent basis, at least weekly.
• a treatment period is preferably at least twice weekly, more preferably every other day, most preferably daily, for at least about two weeks, preferably at least about three weeks, more preferably at least about four weeks.
• Minimal systemic exposure means the systemically available dose being less than about 5%, preferably less than about 4%, more preferably less than about 3%, even more preferably less than about 2%, and most preferably less than about 1% of the administered dose.
• AUC po is area under the plasma concentration curve for formulated oligonucleotide tablets administered orally
• AUC iv is area under the plasma concentration curve for oligonucleotide administered as an i.v. solution (control)
• D o is the respective dosages for these two regimens.
• the oligonucleotide was formulated in an water-in-oil emulsion prepared as follows. First, the two phases were prepared. The aqueous phase was prepared by mixing 2 ml of ISIS 2302 solution (100 mg/ml) and 2 ml of water. The oil phase was prepared by gently heating 1 g Grill 3 (sorbitan monostearate) (Croda, US), 3 ml Captex 355 (Abitec Corp., Janesville, Wis.), and 3 ml Labrasol (Gattefosse, France) to about 70° C. The aqueous solution was slowly poured into the oil phase with vigorous mixing.
• ISIS 2302 solution 100 mg/ml
• the oil phase was prepared by gently heating 1 g Grill 3 (sorbitan monostearate) (Croda, US), 3 ml Captex 355 (Abitec Corp., Janesville, Wis.), and 3 ml Labrasol (Gattefosse, France) to about 70° C. The aqueous
• Sprague-Dawley rats weighing 250-300 g were used. After overnight fasting, the rats were anesthetized with 5% pentobarbital (50 mg/kg) by intraperitoneal injection. After a midline abdominal incision was made, the small intestine was pulled out and injection site was located (2 cm after the ligament of Treitz). An aliquot of 1.0 mL drug formulation was then injected via a 27 gauge needle. Muscle was then surgically closed and skin was clipped after injection.
• test rats were first administered a cleansing enema following a period of overnight fasting, and then dosed with a sample of the test formulation.
• the enema formulation was applied via a catheter and held for a period of 1 hour.
• samples are processed and the amount of oligonucleotide present assessed by capillary gel electrophoresis (CGE) and HPLC analyses.
• CGE capillary gel electrophoresis
• the absolute bioavailability of ISIS 2302 was determined following intrajejunal instillation in five Sprague-Daley rats and following rectal administration in seven rats.
• Enema Formulations for analysis of tissue uptake of oligonucleotide.
• formulations were prepared (Table 1).
• Solution and emulsion formulations of ISIS 2302 were prepared.
• Additives used in the formulations included saline, hydroxypropyl methyl cellulose (HPMC), carrageenan, Vitamin E a-tocopheryl polyethyelene glycol 1000 succinate (TPGS), Tween 80 and sorbitol.
• Formulation 1a A solution of ISIS 2302 was prepared in sterile saline at the desired concentration and used for in vivo evaluation.
• Formulation 1b A solution of ISIS 2302 and hydroxypropyl methyl cellulose (HPMC) was prepared such that the final concentration of ISIS 2302 was identical to that in Formulation 2a and the concentration of HPMC was 1.5%.
• Formulation 1c A solution of ISIS 2302 was prepared, as for Formulation 2a, containing 1.0% carrageenan and 2.5% Vitamin E TPGS.
• Formulation 1d A water-in-oil emulsion of ISIS 2302 was prepared following the general methods in the above example.
• Formulation 1e This formulation was prepared by mixing ISIS 2302, Tween 80 and HPMC in the appropriate quantities so as to afford a mixture that had the desired concentration of ISIS 2302, 0.5% Tween 80 and 0.75% HPMC.
• Formulation 1f This formulation was prepared by mixing ISIS 2302, Sorbitol and HPMC in the appropriate quantities so as to afford a mixture that had the desired concentration of ISIS 2302, 5% Tween 80 and 0.75% HPMC.
• ISIS 2302 formulations for topical/enema administration Formulation Composition 1a ISIS 2302 in Saline 1b ISIS 2302 + 1.5% Hydroxypropyl Methyl Cellulose (HPMC) 1c ISIS 2302 + 1.0% Carrageenan + 2.5% Vitamin E a-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS) 1d ISIS 2302 in a water-in-oil emulsion 1e ISIS 2302 + 0.5% Tween 80 + 0.75% HPMC 1f ISIS 2302 + 5% Sorbitol + 0.75% HPMC
• Formulations of oligonucleotide were evaluated via rectal administration as enemas to laboratory beagle dogs. Following a period of overnight fasting, test dogs were first administered a cleansing enema and then dosed with a sample of the test formulation. The enema formulation was applied via a Foley catheter and held for a period of 1 h. In order to assess colonic tissue delivery and uptake of oligonucleotide, colon tissue biopsies were performed on the test animal, 3 h and 24 h after dosing. Tissue samples were processed and the amount of oligonucleotide present in the tissue assessed by capillary gel electrophoresis (CGE) and immunohistochemical (IHC) analyses.
• CGE capillary gel electrophoresis
• IHC immunohistochemical
• ISIS 2302 Analysis of toxicity and pharmacokinetics of intravenously administered ISIS 2302 in humans in a Phase I clinical trial.
• the first clinical trial with ISIS 2302 was to assess the safety and pharmacokinetics of intravenous administration of an anti-ICAM-1 antisense oligodeoxynucleotide in healthy subjects before commencing pilot therapeutic trials in target disease states. This was a double-blind, placebo-controlled, randomized (3:1, drug: placebo) study.
• Four healthy male volunteers were enrolled in each of seven single dose (0.06, 0.12, 0.24, 0.5, 1.0, 1.5 and 2.0 mg/kg) and multiple dose groups (0.2, 0.5, 1.0 and 2.0 mg/kg every other day for four doses).
• ISIS 2302 (or sterile normal saline as placebo) was administered by intravenous infusion in a volume of 80 ml over two hours. Subjects remained recumbent, with continuous ECG monitoring for four hours after the beginning of each infusion. Before and at intervals after each infusion, supine blood pressure and pulse, clotting screen including a PTT, thrombin time, prothrombin time, serum complement split products C3a and C5a, neutrophil count, urine microproteins, and standard laboratory safety screen (hematology, blood biochemistry and urinalysis) were measured. Serum samples were collected from multiple dose groups at 14 and 21 days after the last infusion to be analyzed for the presence of antibodies to ISIS 2302. Blood samples were taken for measurements of plasma concentration of ISIS 2302 before and up to 24 hours after the beginning of infusion.
• Complement split products were measured by commercially available C3a and C5a assay kits (Amersham). Plasma was examined for the presence of anti-ISIS 2302 antibodies using a modification of a previously described ELISA methodology (Lacy and Voss, J. Immunol. Methods, 116:87-98, 1989). Medium from a hybridoma cell line producing monoclonal antibodies which recognize ISIS 2302 served as a positive control. The cell line was produced by immunizing mice with ISIS 2302 conjugated to keyhole limpet hemocyanin as ISIS 2302 does not appear to be immunogenic in mice.
• CGE capillary gel electrophoresis
• a phosphorothioate standard oligonucleotide composed of 27 thymidine nucleotides (T27) was added to both plasma and urine as an internal standard.
• the linear range of concentrations of oligonucleotides detectable in plasma using this method was 10 nM to 20 ⁇ M (approximately 0.07 to 140 ⁇ g/ml).
• Urine samples from the 1.0 and 2.0 mg/kg multiple dose groups were analyzed for concentrations of intact drug and metabolites. Although very low concentrations of drug or metabolites were excreted in urine (less than 0.5% of the total drug administered was excreted in the first six hours), intact drug and n-1, n-2 and n-3 forms could be measured, and the quantity of shorter forms could be estimated from electropherograms. The amount of intact drug excreted over six hours after the beginning of infusion averaged approximately 0.05% of the administered dose, and the estimated total excretion of parent drug and metabolites in this time period was less than 0.5% of the total dose.
• PK and clinical data from the first 12 patients were analyzed.
• Maximum plasma concentrations (C max ) ranged from 2.2 ng/mL to 38 ng/mL with the average time to maximum plasma concentration of 1.8 hr following the first enema.
• Mean AUC was 0.124 ⁇ g*hr/mL, indicating that less than 1% of the administered dose was systemically available.
• colonic mucosal tissue concentrations of the parent oligonucleotide averaged 4300 ng/g wet tissue. Local tissue exposure was 100-fold higher than maximum drug concentrations observed in plasma. In a population with intestinal inflammation, friability and potential spontaneous bleeding, this low systemic exposure was surprising. Confinement of the drug to the specific region to be treated potentially increases safety and efficacy of the compound.
• ISIS 2302 enemas Steady state and single dose systemic availability of ISIS 2302 enemas is minimal in patients with active colonic inflammation. Effective tissue concentration of alicaforsen was achieved. A relationship between mucosal tissue concentration of alicaforsen and clinical response could not be established in this small cohort of patients.
• ISIS 2302 enema Analysis of the safety and efficacy of ISIS 2302 enema in the treatment of mild to moderate ulcerative colitis.
• the safety and efficacy of four different dosing regimens of ISIS 2302 enema was assessed for the treatment of ulcerative colitis were analyzed in a placebo-controlled, double-blinded Phase 2 study.
• Patient baseline characteristics were as follows: mean age 47.1 year, gender M/F:67/45, mean disease duration 7.5 years, mean baseline DAI 6.9. Baseline characteristics were similar between the study groups. Patients were equally randomized into one of five study arms.
• Primary response end point was a reduction in DAI at 6 weeks with secondary response being acute response, improvement/mucosal healing, remission and relapse.
• ISIS 2302 enema Analysis of the safety and efficacy of ISIS 2302 enema in the treatment of mild to moderate ulcerative colitis.
• the safety and efficacy of ISIS 2302 in a hydroxypropylmethylcellulose formulation was assessed for the treatment of patients with active mild to moderate active ulcerative colitis, left-sided colitis or pancolitis with left sided disease flare in a multicenter, randomized, double-blind, placebo controlled, dose ranging Phase 2 study.
• Patient baseline characteristics were as follows: mean age 45.3 year, gender M/F:86/73, mean duration of disease 9.3 years, mean baseline DAI 7.3. Baseline characteristics were similar between study groups.
• the ISIS 2302 doses were selected for because they showed statistically significant efficacy in initial Phase 2 dose ranging studies.
• the doses were predicted on the basis of animal studies to yield local tissue concentrations shown to be effective in animal pharmacology models for inflammatory bowel disease.
• the primary response endpoint was a reduction in DAI at 6 weeks with secondary responses being acute response, improvement/mucosal healing, remission and relapse.
• the clinical response rates between the ISIS 2302 240 mg and mesalamine enemas were comparable throughout the study and both were superior to the ISIS 2302 120 mg enema.
• the acute response to ISIS 2302 and mesalamine is similar; however, the response to ISIS 2302 is substantially more durable and sustained as determined by percent reduction in DAI, physician's assessment, maintenance of clinical remission and mucosal healing.
• ISIS 2302 240 mg enema demonstrated activity similar to mesalamine enema in the acute left sided UC. However, subjects treated with ISIS 2302 240 mg trended toward a higher remission rate and a longer duration of remission than mesalamine. ISIS 2302 was well generally well tolerated.

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