Methods for treating inflammatory bowel disease

Abstract

A method for treating inflammatory bowel disease is provided herein. A method for preparing EOM613 and a pharmaceutical composition comprising EOM613 are also provided. The inflammatory bowel disease may be Crohn's disease, ulcerative colitis, or colitis unclassified, and the method is a method for treating inflammatory bowel disease (IBD) in a subject, comprising the step of administering a therapeutically effective amount of EOM613 to the subject. Symptoms of IBD may include one or more of rectal bleeding, abdominal distension, diarrhea, frequent bowel movements, fatigue, weight loss, and abdominal pain or sudden abdominal pain.

Description

[Technical Field]

[0001] Cross-reference to Related Application This application claims priority based on U.S. Provisional Application No. 63 / 536,325, filed on September 1, 2023, which is incorporated by reference in its entirety.

[0002] Field The present invention relates to a method for treating inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis). [Background Art]

[0003] Background Inflammatory bowel disease (IBD) is a condition characterized by chronic inflammation of the gastrointestinal tract. Types of IBD include Crohn's disease, ulcerative colitis, and unclassified colitis. Main symptoms include persistent diarrhea (lasting longer than 4 weeks), abdominal pain, blood or mucus in feces, fatigue, and weight loss. The severity of IBD can range from mild illness to debilitating conditions that can lead to life-threatening complications. Currently available treatments are neither well tolerated nor sustained. Patient response to current IBD treatments can decrease over time until the treatments are no longer effective. Therefore, there is a great need for alternative IBD treatments. [Summary of Invention] [Means for Solving the Problems]

[0004] Abstract A method for treating inflammatory bowel disease (IBD) in a subject is disclosed herein, the method comprising the step of administering a therapeutically effective amount of EOM613 to the subject. In some cases, the IBD is Crohn's disease. In some cases, treatment with EOM613 reduces one or more symptoms of IBD, such as rectal bleeding, abdominal distension, diarrhea, frequent bowel movements, fatigue, weight loss, and abdominal pain or sudden abdominal pain. Subjects having IBD may be selected for treatment. In some cases, the subject has active IBD or is in remission after a diagnosis of IBD. The above method may further include the step of administering a second IBD treatment agent (e.g., infliximab, adalibumab, certolizumab pegol, risankizumab, vedozilumib, ustekinumab, upadacitimib, aminosalicylic acid (e.g., sulfasalazine, mesalamine, or orsalazine), mercaptopurine, or methotrexate). Methods for preparing EOM613 and pharmaceutical compositions containing EOM613 are also disclosed.

[0005] The aforementioned and other objects, features, and advantages of the present invention will become more apparent from the following detailed description. [Modes for carrying out the invention]

[0006] Detailed explanation I. Introduction In 2015, the U.S. Centers for Disease Control and Prevention (CDC) estimated that 1.3% of adults in the United States were diagnosed with either Crohn's disease or ulcerative colitis. Between 2000 and 2018, the prevalence among older adults in the United States increased significantly (see Fang Xu, et al., Morbidity and Mortality Weekly 70(19):698-701, 2021).

[0007] The pathogenesis of IBD is primarily driven by the release of pro-inflammatory cytokines by macrophages in the gastrointestinal tract (see, e.g., Sanchez-Munoz et al., World J Gastroenterol. 14(27):4280-4288, 2008). The main cytokines thought to be involved in promoting IBD symptoms are TNF-α and IL-23, and therefore, several therapeutic agents based on antibodies directed against these cytokines have been developed. Biologic TNF-α inhibitors approved to treat IBD symptoms include infliximab (Remicade®), adalimumab (Humira®), and certolizumab pegol (Cimizia®). An example of an IL-23 blocking antibody is risankizumab (Skyrizi®). Other drugs clinically used to treat IBD include vedolizumab (Entyvio®), gastrointestinal homing α4-β integrin inhibitors, and ustekinumab (Stelara®). All monoclonal biological agents are administered intravenously and many have significant adverse side effects, including an increased risk of infection (including upper respiratory tract and urinary tract infections), nausea, headache, and arthralgia. Allergic reactions can also occur, and these drugs should be avoided in pregnant patients as they can cause harm to the health of the fetus.

[0008] Non-biological agents used to treat IBD include aminosalicylic acids (e.g., sulfasalazine, mesalamine, or orsalazine), which work best for IBD affecting the descending colon rather than the small intestine. Immunomodulatory factors (e.g., mercaptopurine or methotrexate) are also sometimes used to induce remission, but they can damage the liver and pancreas.

[0009] Treatment failure is frequently observed in patients who are resistant to existing IBD treatments (e.g., those discussed above) or who develop resistance over time, ultimately rendering the treatment ineffective. In addition, treatment failure remains frequent despite advances in IBD treatment using antibodies targeted against IL-23 and TNF-α cytokines. Generally, between 50% and 60% of patients initially respond to these treatments in terms of improvement in symptoms or inflammatory markers. Of these patients, only about 20% to 30% achieve remission, and of those in remission, at least half remain in remission over time, based on clinical trial data (AC Moss, Gastroenterol.Hepatol.(NY), 2022:18(6)360-363).

[0010] EOM613 is a broad-spectrum immunomodulator that modulates the release and action of pro-inflammatory and anti-inflammatory cytokines. In previous clinical use to treat AIDS, cancer cachexia, and COVID-19, the above treatment was safe and well-tolerated using daily subcutaneous (sc) injections of up to 2 ml twice daily.

[0011] However, EOM613 has not been previously investigated for use in the treatment of IBD (e.g., Crohn's disease or ulcerative colitis). In addition, the immunomodulatory activity of EOM613 varies depending on the activation state of immune cells. For example, EOM613 increased IL-6, IL-1β, and TNF-α secretion by peripheral blood monocytes (PBMCs) stimulated with PHA / IL-2 in cell cultures, but did not alter IL-12 secretion. In contrast, treatment of LPS-activated monocytes decreased TNF-α and IL-12 secretion and increased IL-6 secretion, but did not alter IL-1β secretion (see, e.g., Hirchman, Advances in Bioscience and Biotechnology, 5:161-168, 2014). Based on its differential effects on cytokines, it is not possible to deductively predict whether EOM613 is beneficial in the treatment of IBD. However, the data disclosed herein demonstrate that EOM613 is an effective treatment for IBD.

[0012] II. Overview of Terminology Unless otherwise noted, technical terms are used according to their conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (ed.), Lewin's Genes XII, published by Jones & Bartlett Learning, 2017. Where used herein, the singular forms "a," "an," and "the" refer to both singular and plural forms unless the context clearly indicates otherwise. For example, the term "cytokine (a cytokine)" encompasses one or more cytokines and may be considered equivalent to the phrase "at least one cytokine." Where used herein, the term "comprises" means "includes." It should be further understood that any and all base sizes or amino acid sizes, as well as all molecular weight or molecular mass values, given with respect to nucleic acids or polypeptides are approximations and provided for descriptive purposes unless otherwise indicated. Many methods and materials similar to or equivalent to those described herein may be used, but certain suitable methods and materials are described herein. In case of any conflict, this specification shall prevail, including the definition of terms. In addition, the materials, methods, and examples described herein are illustrative and not intended to limit the scope of this specification.

[0013] To facilitate the examination of various aspects, the following terms are provided for explanation.

[0014] About: Unless otherwise specified in the context, "about" refers to a range of ±5% of the reference value. For example, "about" 100 refers to a range of 95 to 105.

[0015] Administration: The process of introducing a drug (e.g., EOM613) into a subject via a selected route. Administration may be local or systemic. Exemplary routes of administration include, but are not limited to, oral, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes. In some aspects, administration is parenteral.

[0016] Control: Reference standard. The control may be a positive control or a negative control. In some cases, the control is a scale or sample obtained from healthy subjects. In some cases, the control is a scale or sample obtained from subjects diagnosed with IBD. In some cases, the control is a scale or sample obtained from subjects before EOM613 administration. In yet other cases, the control is a historical control or a standard reference value or range of values ​​(e.g., a previously tested control sample such as a group of patients diagnosed with a disease or condition with a known prognosis or outcome (e.g., IBD), or a group of samples representing baseline or normal values). A person skilled in the art will be able to select an appropriate control.

[0017] To detect: To identify the existence, presence, or fact of something.

[0018] Increase or Decrease: A positive or negative change in the reference value. An increase is a positive change, such as an increase of at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the reference value. A decrease is a negative change, such as a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% compared to the reference value. In some cases, the above reference value is the control. In some cases, the above increase or decrease is statistically significant.

[0019] Inflammatory bowel disease (IBD) is an autoimmune disorder of the gastrointestinal tract characterized by chronic inflammation of the digestive tract. IBDs include Crohn's disease, ulcerative colitis, and unclassified colitis. Crohn's disease is characterized by inflammation of the inner lining of the digestive tract, most commonly affecting the small intestine. It can also affect the large intestine and, rarely, the upper digestive tract. Ulcerative colitis is characterized by inflammation and ulcers along the inner lining of the large intestine (colon) and rectum. Unclassified colitis refers to IBD that exhibits characteristics of both Crohn's disease and ulcerative colitis.

[0020] Symptoms of IBD include diarrhea, rectal bleeding, abdominal distension, abdominal pain, fatigue, and weight loss. Symptoms can range from mild to severe. Individuals with IBD typically experience periods of active IBD (when symptoms are present, also known as relapses or flares) followed by periods of remission (when symptoms are absent or significantly reduced). IBD-related inflammation can cause damage to the gastrointestinal tract (including abscesses, strictures, and fistulas) and may also increase the risk of colon cancer. The exact cause of IBD is unknown, but a genetic component may exist. It is hypothesized that it may arise from a weakened immune system in response to environmental triggers (e.g., sequelae of viral or bacterial infections that cause a general inflammatory state of the gastrointestinal tract).

[0021] Nucleotides are organic molecules consisting of a nucleoside and a phosphate group. The term "nucleotide" refers to any modified form of ribonucleotide, deoxyribonucleotide, or nucleotide. They function as monomer units, deoxyribonucleic acid, and ribonucleic acid in nucleic acid polymers.

[0022] Nucleic acid molecule: A polymeric form of nucleotides that may include RNA, cDNA, both sense and antisense strands of genomic DNA, as well as synthetic and mixed polymers thereof. The term “nucleic acid molecule” is, as used herein, synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is typically at least 10 nucleotides long unless otherwise specified. The above terms include single-stranded and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. “cDNA” refers to DNA that is complementary to or identical to mRNA in either a single-stranded or double-stranded form. "Encoding" refers to the specific properties of a particular sequence of nucleotides within a polynucleotide (e.g., a gene, cDNA, or mRNA) in order to act as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties derived therefrom.

[0023] Pharmaceutically acceptable carrier: Pharmaceutically acceptable carriers useful in the methods disclosed herein are known in the art. In general, the nature of said carrier will depend on the particular route of administration employed. For example, parenteral preparations usually comprise injectable fluids comprising pharmaceutically and physiologically acceptable fluids as a vehicle, such as water, physiological saline solution, balanced salt solution, aqueous dextrose, glycerol and the like. With regard to solid compositions, for example in the form of powders, pills, tablets or capsules, conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to a biologically neutral carrier, the pharmaceutical composition to be administered, for example an immunogenic composition, can comprise minor amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Remington's Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020), describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein.

[0024] Polypeptide: any chain of amino acids, regardless of length or post-translational modification, for example glycosylation or phosphorylation. The term "polypeptide" covers naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids, for example artificial chemical mimetics of the corresponding naturally occurring amino acid. The term "residue" refers to an amino acid or amino acid mimetic incorporated into a polypeptide through an amide bond or an amide bond mimetic. A polypeptide has an amino terminus (N-terminus) and a carboxy terminus (C-terminus). The term "polypeptide" is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

[0025] Subject: A living multicellular vertebrate organism, a category that includes human and non-human mammals (e.g., non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, etc.). In some aspects, the subject is a mammal. In some aspects, the subject is a human. In some aspects, the subject is a human or veterinary subject (e.g., a dog, a cat, or other domesticated mammal). In some aspects, a subject in need of IBD treatment is selected, for example, the subject has IBD and is in need of treatment.

[0026] Therapeutically effective amount: An amount of an agent (e.g., EOM613) sufficient to prevent (including prophylaxis), treat, and / or reduce the symptoms or underlying causes of a disease or pathological condition (e.g., IBD). In some aspects, a therapeutically effective amount of EOM613 is an amount that treats IBD, for example, reduces or eliminates one or more symptoms of IBD.

[0027] The exact amount and timing of administration can depend on many factors including the subject being treated, the severity and type of the condition being treated, and the mode of administration. A therapeutically effective amount can be determined by those skilled in the art through clinical trials. An effective amount can also be determined through various in vitro, in vivo, or in situ assays. In some aspects, the therapeutically effective amount of EOM613 is 2 ml twice daily over a period sufficient to achieve a therapeutic benefit (e.g., at least 2 weeks, such as 2 to 4 weeks or 2 to 8 weeks).

[0028] A therapeutically effective amount includes divided doses that contribute in combination with previous or subsequent administrations to achieve the desired response. For example, a therapeutically effective amount of an agent can be administered in a single dose, or in several doses during a course of treatment (e.g., twice daily over 2 weeks).

[0029] Treating or inhibiting (a disease or condition): Treatment refers to a therapeutic intervention that improves the signs or symptoms of a condition (e.g., IBD) after the condition has started. With reference to a disease, syndrome, or pathological condition, the term "ameliorating" refers to any observable beneficial effect of the above treatment. Such beneficial effects may be demonstrated, for example, by delaying the onset of symptoms in the subject, reducing the severity of some or all symptoms, slower progression, reduced relapse or recurrence, improvement in the subject's quality of life, or by other parameters specific to a particular disease, syndrome, or other pathological condition. Treatment may be evaluated by objective or subjective parameters, including the results of a physical examination, blood tests or other clinical tests, and self-assessments by the subject.

[0030] Inhibition of disease or condition includes preventing or reducing the risk of the disease or condition described above (e.g., reducing the risk of severe symptoms or IBD flares). Prophylactic treatment is a treatment administered to subjects who show no signs of the disease (e.g., subjects in remission) or subjects showing very early signs for the purpose of reducing the risk of developing the disease. Prophylactic treatment may reduce the risk of IBD flares. In some aspects, the methods of this disclosure are prophylactic and / or therapeutic. In some aspects, the methods of this disclosure are therapeutic and not prophylactic.

[0031] III.EOM613 EOM613 (also known as Product R or AVR118) is a composition comprising nucleotides and peptides having molecular weights of 14 kDa or less, mainly 8 kDa or less. These nucleotides and peptides are degradation products of casein, peptone, RNA, and serum albumin. EOM613 and its production are described, for example, in U.S. Patents 6,528,098, 6,921,542, 7,074,767, 7,524,661, and 8,084,239 (which are incorporated herein by reference in their entirety). In these patents, EOM613 is referred to as Product R. A brief description of EOM613 and its production are provided herein.

[0032] Sixteen constituent compounds were identified and characterized in EOM613: three nucleosides, two nucleoside diphosphates, and eight nucleoside monophosphates, along with two peptides (one of which is a peptide-nucleic acid conjugate) and sodium chloride (resulting from the neutralization of sodium hydroxide with hydrochloric acid during the manufacturing process). The longer peptide (referred to as "peptide-A") is a 31-amino acid peptide derived from bovine β-casein with a molecular weight of 3536.24 Da. The shorter peptide (referred to as "peptide-B") is a peptide-oligonucleotide conjugate containing a 21-amino acid peptide linked to a diadenine (3'-5') diribonucleotide via a diphosphodiester bond at the 3'-position at a serine residue at position 18. This nucleopeptide conjugate has an MW2215 peptide moiety, which, when bound to a 740 MW non-peptide adduct, gives a total MW2955. Peptides A and B are present in EOM613 in approximately equal amounts by weight. The amounts of peptides A and B in EOM613, as determined by the Lowry protein assay, are approximately 4.4–7.0 mg / mL, preferably 4.8–5.3 mg / mL.

[0033] EOM613 can be prepared, for example, by the following method: i) A step of suspending casein, beef peptone, RNA, BSA, and sodium hydroxide in distilled water to produce a suspension; ii) Autoclaving the above suspension; iii) A step of cooling the above suspension after autoclaving; iv) After cooling, the above suspension is filtered to produce a filtered solution; v) The filtered solution described above, a) To the total nitrogen content of 1.65 to 2.10 mg / ml, and b) To physiological pH A process of adjusting and thereby producing a adjusted solution; vi) If necessary, the step of filtering the above-prepared solution; and vii) Sterilizing the above-prepared solution to produce EOM613.

[0034] The starting materials (casein, beef peptone, RNA, BSA, and sodium hydroxide) are commercially available or can be easily prepared by those skilled in the art.

[0035] The above suspension contains approximately 35% to 50% (w / w) casein by dry weight, for example, 35% to 48%, 35% to 45%, 35% to 42%, 35% to 40%, 40% to 50%, 40% to 48%, 40% to 45%, or 40% to 42% w / w casein.

[0036] The above suspensions contain approximately 15% to 40% (w / w) beef peptone by dry weight, for example, 15% to 35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 40%, 30% to 35%, or 35% to 40% w / w beef peptone.

[0037] The above suspensions contain approximately 10% to 25% (w / w) RNA on a dry weight basis, e.g., 10% to 20%, 10% to 15%, 15% to 25%, 15% to 30%, 15% to 25%, 15% to 20%, or 20% to 25% w / w RNA. Any suitable RNA source can be used (e.g., plant RNA or yeast RNA). In some aspects, yeast RNA can be used to produce EOM613.

[0038] The above suspensions contain approximately 1% to 10% (w / w) bovine serum albumin (BSA) by dry weight, for example, 1% to 8%, 1% to 6%, 1% to 4%, 1% to 2%, 2% to 10%, 2% to 8%, 2% to 6%, 2% to 4%, 4% to 10%, 4% to 8%, 4% to 6%, 6% to 10%, 6% to 8%, or 8% to 10% w / w BSA.

[0039] The above suspension contains approximately 5% to approximately 25% (w / w) of sodium hydroxide by dry weight, for example, 5% to 20%, 5% to 18%, 5% to 15%, 5% to 12%, 5% to 10%, 5% to 8%, 10% to 25%, 10% to 20%, 10% to 18%, 10% to 15%, 10% to 12%, 15% to 25%, 15% to 20%, 15% to 18%, or 20% to 25% w / w sodium hydroxide. In some aspects, the above sodium hydroxide is at least 95% pure, for example, at least 96%, 97%, 98%, or 99% pure. In non-limiting examples, the above sodium hydroxide is at least 97% pure. In some cases, the sodium hydroxide is available in pellet form (e.g., Sigma-Aldrich catalog number 567530, or Fisher Scientific catalog number S318-100).

[0040] The dry components are suspended in an appropriate amount of distilled water. The ratio of total protein (grams) to volume of distilled water (milliliters) is approximately 1.5g to 2.5g of total protein per 100ml of distilled water, preferably approximately 2.2g to 100ml. Therefore, 1.5 to 2.5 grams of total protein are suspended in approximately 100ml of distilled water.

[0041] The above suspension is then autoclaved under conditions in which the RNA is hydrolyzed to nucleotides. Such conditions are described herein and can be optimized by those skilled in the art. In some aspects, the autoclave pressure is about 5 to about 15 pounds per square inch, e.g., 1 to 15, 1 to 10, 1 to 5, 5 to 15, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 8 to 15, 8 to 12, 8 to 10, 10 to 15, 10 to 12, or 12 to 15 pounds per square inch. In a non-limiting example, the autoclave pressure is about 8 to about 10 pounds per square inch. In some cases, the autoclave temperature is approximately 60°C to 150°C, for example, 60°C to 125°C, 60°C to 110°C, 60°C to 100°C, 60°C to 90°C, 60°C to 80°C, 80°C to 150°C, 80°C to 140°C, 80°C to 130°C, 80°C to 125°C, 80°C to 110°C, 80°C to 100°C, 80°C to 90°C, 90°C to 150°C, 90°C to 140°C, 90°C to 130°C, 90°C to 120°C, 90°C to 110°C, or 90°C to 100°C. In a non-limiting example, the above temperature is approximately 90°C to 110°C. In another example, the above temperature is approximately 93°C to 110°C. In some cases, the duration of autoclave is approximately 2 to 10 hours, for example, 2 to 9 hours, 2 to 8 hours, 2 to 7 hours, 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 2.5 to 8 hours, 2.5 to 7 hours, 2.5 to 6 hours, 2.5 to 5 hours, 2.5 to 4 hours, 2.5 to 3.5 hours, 2.5 to 3 hours, 3 to 10 hours, 3 to 9 hours, 3 to 8 hours, 3 to 7 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 10 hours, 4 to 9 hours, 4 to 8 hours, 4 to 7 hours, 4 to 6 hours, 4 to 5 hours, 5 to 10 hours, 5 to 9 hours, 5 to 8 hours, 5 to 7 hours, 5 to 6 hours, 6 to 10 hours, or 8 to 10 hours. In non-limiting examples, the duration of autoclave is approximately 3 to 5 hours. In another non-limiting example, the duration of autoclaving is at least 3 hours. In a specific non-limiting example, autoclaving conditions include a pressure of approximately 8 to 10 pounds per square inch, a temperature of approximately 93°C to 110°C, and a duration of approximately 3 to 5 hours.

[0042] In some phases, the above suspension is cooled to approximately 1°C to approximately 10°C, for example, 1°C to 9°C, 1°C to 8°C, 1°C to 7°C, 1°C to 6°C, 1°C to 5°C, 1°C to 4°C, 1°C to 3°C, 1°C to 2°C, 2°C to 10°C, 2°C to 9°C, 2°C to 8°C, 2°C to 7°C, 2°C to 6°C, 2°C to 5°C, 2°C to 4°C, 2°C to 3°C, 3°C to 10°C, 3°C to 9°C, 3°C to 8°C, 3°C to 7°C, 3°C to 6°C, 3°C to 5°C, 3°C to 4°C, 4°C to 10°C, 4°C to 8°C, 4°C to 7°C, 5°C to 6°C, or 8°C to 10°C. In non-specific examples, the above suspension is cooled to approximately 3°C to approximately 8°C.

[0043] In some cases, the above-mentioned suspension is cooled for at least 30 minutes, for example, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, or longer. In some phases, the above-mentioned suspension lasts for approximately 1 to 24 hours, for example, 2 to 24 hours, 2 to 20 hours, 2 to 18 hours, 2 to 16 hours, 2 to 14 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 24 hours, 4 to 20 hours, 4 to 18 hours, 4 to 16 hours, 4 to 14 hours, 4 to 12 hours, 4 to 10 hours, 6 to 8 hours, 8 to 24 hours. The mixture is cooled for 8-20 hours, 8-18 hours, 8-16 hours, 8-14 hours, 8-12 hours, 8-10 hours, 10-24 hours, 10-20 hours, 10-18 hours, 10-16 hours, 10-14 hours, 10-12 hours, 12-24 hours, 12-20 hours, 12-18 hours, 12-16 hours, 12-14 hours, 14-24 hours, 14-20 hours, 14-18 hours, 14-16 hours, 16-24 hours, 16-20 hours, 16-18 hours, 18-24 hours, 18-20 hours, or 20-24 hours. In an unspecified example, the suspension is cooled for at least 6 hours. In another unspecified example, the solution is cooled for about 6 hours to about 12 hours. In a non-specific example, the above suspension is cooled to approximately 3°C to approximately 8°C over a period of approximately 6 to 12 hours.

[0044] Filtration can be performed with any filter of appropriate size. In some cases, the above filters are approximately 0.05 to 5 microns, for example, 0.05 to 4 microns, 0.05 to 3 microns, 0.05 to 2 microns, 0.05 to 1 micron, 0.05 to 0.5 microns, 0.05 to 0.2 microns, 0.05 to 0.1 microns, 0.1 to 4 microns, 0.1 to 3 microns, 0.1 to 2 microns, 0.1 to 1 micron, 0.1 to 0.5 microns, 0.1 to 0.2 microns, 0.2 to 4 microns. The micron sizes are 0.2-3 microns, 0.2-2 microns, 0.2-1 micron, 0.2-0.5 microns, 0.375-4 microns, 0.375-3 microns, 0.375-2 microns, 0.375-1 micron, 0.357-0.5 microns, 0.5-4 microns, 0.5-3 microns, 0.5-2 microns, 0.5-1 micron, 1-4 microns, 1-3 microns, 1-2 microns, 2-4 microns, or 2-3 microns. In some aspects, the above filters are exothermic material retaining filters. In some aspects, the above suspensions are passed through a number of filters, for example, at least 2 filters, at least 3 filters, at least 4 filters, at least 5 filters, at least 6 filters, or more. When multiple filters are used, they may be of the same size (e.g., all about 0.2 microns) or they may be of different sizes (e.g., filters of about 2 microns, about 0.45 microns, and about 0.2 microns). Typically, filtration is performed first with the largest filter, followed by additional filters in decreasing size (from largest to smallest). In some cases, the suspension is filtered with filters of, for example, 2 microns, 0.45 microns, and 0.2 microns before the dilution step. In some cases, the 0.2 micron filter is a filter that retains exothermic substances. The filtered solution may be cooled again to about 3°C ​​to about 8°C as needed, and the filtration as described above may be repeated.In some cases, the filtered solution is cooled for at least 30 minutes, for example, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, or longer. In some phases, the above-mentioned suspension lasts for approximately 1 to 24 hours, for example, 2 to 24 hours, 2 to 20 hours, 2 to 18 hours, 2 to 16 hours, 2 to 14 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 24 hours, 4 to 20 hours, 4 to 18 hours, 4 to 16 hours, 4 to 14 hours, 4 to 12 hours, 4 to 10 hours, 6 to 8 hours, 8 to 24 hours. The solution is cooled for 1 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 8 to 12 hours, 8 to 10 hours, 10 to 24 hours, 10 to 20 hours, 10 to 18 hours, 10 to 16 hours, 10 to 14 hours, 10 to 12 hours, 12 to 24 hours, 12 to 20 hours, 12 to 18 hours, 12 to 16 hours, 12 to 14 hours, 14 to 24 hours, 14 to 20 hours, 14 to 18 hours, 14 to 16 hours, 16 to 24 hours, 16 to 20 hours, 16 to 18 hours, 18 to 24 hours, 18 to 20 hours, or 20 to 24 hours. In non-limiting examples, the filtered solution is cooled for 1 to 6 hours, and the filtration as described above is repeated.

[0045] Filtration can be performed under an inert gas (e.g., nitrogen or argon). In some cases, filtration is performed under an inert gas at a pressure of approximately 1–6 pounds / square inch (psi), e.g., 1–5 psi, 1–4 psi, 1–3 psi, 1–2 psi, 2–6 psi, 2–5 psi, 2–4 psi, 2–3 psi, 3–6 psi, 3–5 psi, 3–4 psi, 4–6 psi, 4–5 psi, or 5–6 psi. In some cases, the above gas pressure is approximately 1–6 psi.

[0046] Next, the nitrogen content and pH of the filtered solution are adjusted. The filtered solution is assayed to determine the total nitrogen content and adjusted to approximately 1 mg / ml to approximately 3 mg / ml. The adjustment of the nitrogen content may involve dilution or concentration of the solution. Typically, the filtered solution is diluted, but it may be concentrated if necessary. The total nitrogen content can be determined by any known method (e.g., the Kjeldahl method (see Kjeldahl, Z. Anal. Chem., Vol. 22, p. 366, 1883) or related methods based on the Kjeldahl method). In some cases, the above total nitrogen content is approximately 1.50 mg / ml to approximately 2.50 mg / ml, for example, 1.50 to 2.25 mg / ml, 1.50 to 2.10 mg / ml, 1.50 to 2.00 mg / ml, 1.50 to 1.90 mg / ml, 1.50 to 1.80 mg / ml, 1.50 to 1.70 mg / ml, 1.65 to 2.50 mg / ml, 1.65 to 2.25 mg / ml, 1.65 to 2.10 mg / ml, 1.65 to The total nitrogen content is 2.00 mg / ml, 1.65-1.90 mg / ml, 1.65-1.80 mg / ml, 1.65-1.70 mg / ml, 1.70-2.50 mg / ml, 1.70-2.25 mg / ml, 1.70-2.10 mg / ml, 1.70-2.00 mg / ml, 1.70-1.90 mg / ml, 1.70-1.80 mg / ml, 1.80-2.10 mg / ml, 1.80-2.00 mg / ml, 1.80-1.90 mg / ml, 1.90-2.10 mg / ml, 1.90-2.00 mg / ml, or 2.00-2.10 mg / ml. In some situations, the above total nitrogen content is adjusted to approximately 1.65 mg / ml to approximately 2.10 mg / ml.

[0047] The filtered solution described above is also adjusted to a physiological pH. In some cases, the pH is adjusted to approximately 6.5 to 8, for example, 6.5 to 7.8, 6.5 to 7.6, 6.5 to 7.4, 6.5 to 7.3, 6.5 to 7.2, 6.5 to 7, 6.5 to 6.8, 6.8 to 8, 6.8 to 7.8, 6.8 to 7.6, 6.8 to 7.4, 6.8 to 7.3, 6.8 to 7.2, 6.8 to 7, 7 to 8, 7 to 7.8, 7 to 7.6, 7 to 7.4, 7 to 7.3, 7 to 7.2, 7.3 to 8, 7.3 to 7.8, 7.3 to 7.6, 7.3 to 7.4, 7.5 to 8, 7.5 to 7.8, or 7.5 to 7.6. In non-limiting examples, the pH of the filtered solution is adjusted to 7.3–7.6. Adjusting the pH involves increasing the pH (by adding a base) or decreasing the pH (by adding an acid) as needed. In some cases, the pH is adjusted with NaOH and / or HCl. In some cases, the pH is adjusted after adjusting the total nitrogen content.

[0048] After adjusting the nitrogen content and pH as described above, the adjusted solution can be filtered as needed, for example, through a 0.05-5 micron filter, a 0.05-4 micron filter, a 0.05-3 micron filter, a 0.05-2 micron filter, a 0.05-1 micron filter, a 0.05-0.5 micron filter, a 0.05-0.2 micron filter, a 0.05-0.1 micron filter, a 0.1-4 micron filter, a 0.1-3 micron filter, a 0.1-2 micron filter, a 0.1-1 micron filter, a 0.1-0.5 micron filter, a 0.1-0.2 micron filter, a 0.2-4 micron filter, or a 0.2-3 micron filter. The solution may be filtered again through a 0.2-micron filter, a 0.2-2 micron filter, a 0.2-1 micron filter, a 0.2-0.5 micron filter, a 0.375-4 micron filter, a 0.375-3 micron filter, a 0.375-2 micron filter, a 0.375-1 micron filter, a 0.357-0.5 micron filter, a 0.5-4 micron filter, a 0.5-3 micron filter, a 0.5-2 micron filter, a 0.5-1 micron filter, a 1-4 micron filter, a 1-3 micron filter, a 1-2 micron filter, a 2-4 micron filter, or a 2-3 micron filter, thereby producing a final filtrate. In some cases, the above filters are exothermic filter. In an unspecified example, the above-prepared solution is filtered through a 0.2 micron filter (e.g., a 0.2 micron exothermic filter). In some cases, the suspension is passed through multiple filters, e.g., at least two filters, at least three filters, at least four filters, at least five filters, at least six filters, or more. When multiple filters are used, the filters may be of the same size (e.g., all 0.2 microns) or they may be of different sizes (e.g., 2 microns, 0.45 microns, and 0.2 microns). Typically, filtration is performed first with the largest filter, followed by additional filters in decreasing size (from largest to smallest).

[0049] The prepared solution or final filtrate has an absorbance spectrum with typical absorption ratios of 2.0 (±10%) at 260 nm / 280 nm and 1.4 (±10%) at 260 nm / 230 nm. In some cases, the total protein content of the final filtrate is measured (e.g., by a total protein Lowry assay) to confirm that the peptide content is between 3.5 and 8 mg / ml (e.g., 4.7 and 7.0 mg / ml). The prepared solution or final filtrate may be filtered and sealed in vials (e.g., 2 ml or 10 ml glass vials) under inert gas, if necessary.

[0050] The final step is sterilization of the prepared solution or final filtrate. Once sterilized, the EOM613 composition is ready for administration. Any suitable sterilization method may be used. In some cases, the filled vials are autoclaved for final sterilization, after which they are ready for administration to subjects. In some cases, the autoclave conditions for the final sterilization step are approximately 90–150°C (e.g., 100–125°C or 110–120°C) for 15 minutes to 1 hour (e.g., 15–30 minutes, 20–60 minutes, or 20–40 minutes) at a pressure of 12–18 pounds (e.g., 14–16 pounds).

[0051] IV. Pharmaceutical Compositions EOM613 may be administered directly to a subject or as part of a pharmaceutical composition. Such a pharmaceutical composition may include EOM613 and a pharmaceutically acceptable carrier.

[0052] Pharmaceutical compositions containing EOM613 may be in any suitable form (e.g., lyophilized solid (for reconstitution in saline), liquid, or gas (aerosol). Pharmaceutical compositions containing EOM613 may be formulated so that EOM613 becomes bioavailable when administered to a subject. Pharmaceutical compositions may take the form of one or more dose units. For example, a vial of liquid EOM613 or a container of EOM613 in aerosol form may hold multiple dose units. Syringes containing a single unit dose of EOM613 may also be provided.

[0053] Pharmaceutical compositions containing EOM613 may be in liquid form (e.g., liquid, syrup, or suspension) or may be presented as a pharmaceutical product to be reconstituted with a saline solution or other suitable vehicle before use. Such liquid preparations may be prepared by known methods together with pharmaceutically acceptable additives (e.g., suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, or sorbic acid)).

[0054] Pharmaceutical compositions containing EOM613 may be in solid form (e.g., tablets or capsules containing pharmaceutically acceptable excipients (e.g., binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate))). Tablets may include a coating (e.g., enteric coating). For oral administration, pharmaceutical compositions may take the form of tablets or lozenges formulated by known methods.

[0055] Pharmaceutical compositions containing EOM613 can be delivered, for example, in the form of an aerosol from a pressurized pack or nebulizer. Aerosol formulations may contain a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In some aspects, the dosage unit of the aerosol may be determined by providing a valve for delivering a measured amount. For example, capsules and cartridges for use in inhalers, nebulizers, or insufflators may be formulated, comprising a powder mixture of EOM613 and a suitable powder base (e.g., lactose or starch).

[0056] Pharmaceutical compositions containing EOM613 may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dose form, for example, in ampoules or multi-dose containers, with preservatives added. Pharmaceutical compositions for parenteral administration may take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspenders, stabilizers, and / or dispersants. Alternatively, the active ingredient (e.g., EOM613) may be in powder form for composition in a suitable vehicle (e.g., sterile salt solution) before use. Pharmaceutical compositions may be formulated for controlled release of the active compound.

[0057] EOM613 can also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. In such cases, the compound may be formulated with a suitable polymer or hydrophobic substance (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a poorly soluble derivative (e.g., as a poorly soluble salt). Liposomes and emulsions are examples of delivery vehicles or carriers for hydrophilic drugs.

[0058] Pharmaceutical compositions containing EOM613 may be formulated in the form of a composition (e.g., a cream, lotion, gel, eye drops, ointment, solution, suspension, shampoo, or other form known to those skilled in the art), for example, as described in Remington's Pharmaceutical Sciences, 23rd edition, Academic Press, Elsevier, (2020). Practical methods for preparing pharmaceutical compositions are also described in detail, for example, Remington's Pharmaceutical Sciences, 23rd edition, Academic Press, Elsevier, (2020).

[0059] Pharmaceutical compositions may be available in unit dose or multi-dose containers. In some cases, pharmaceutical compositions containing EOM613 are contained in sealed ampoules or vials. Preferred unit dose formulations include a daily dose or unit, a daily sub-dose, or an appropriate proportion of the component to be administered.

[0060] In some aspects, the above pharmaceutical composition comprises EOM613 plus one or more therapeutic agents (e.g., one or more IBD treatments). Examples of IBD treatments include, but are not limited to, infliximab (Remicade®), adalimumab (Humira®), certolizumab pegol (Cimzia®), risankizumab (Skyrizi®), vedolizumab (Entyvio®), ustekinumab (Stelara®), upadacitinib (Rinvoq®), aminosalicylic acid (e.g., sulfasalazine, mesalamine, or orsalazine), mercaptopurine, or methotrexate.

[0061] V. Treatment Method A method for treating inflammatory bowel disease (IBD) in a subject is disclosed herein, the method comprising the step of administering a therapeutically effective amount of EOM613 to the subject. In some aspects, the method comprises a first step of selecting a subject having inflammatory bowel disease for treatment with EOM613. In some aspects, the IBD is Crohn's disease, ulcerative colitis, or unclassified colitis. In some aspects, the IBD is Crohn's disease. Individuals with IBD may experience remission or active IBD. Remission occurs when symptoms are absent or not very severe. Active IBD occurs when symptoms are present (also called a flare or relapse). In some aspects, the subject has active IBD or is in remission after a diagnosis of IBD. In some aspects, the subject is human. In some aspects, EOM613 is contained in a pharmaceutical composition.

[0062] In some cases, EOM613 treatment reduces the frequency or severity of one or more symptoms of IBD in the subjects described above. Exemplary symptoms of IBD include, but are not limited to, rectal bleeding, abdominal distension, diarrhea, frequent bowel movements, fatigue, weight loss, and abdominal pain or sudden abdominal pain. In some cases, EOM613 treatment eliminates one or more symptoms of IBD in the subjects described above; for example, treatment associated with IBD eliminates one or more of the following in the subjects: rectal bleeding, abdominal distension, diarrhea, frequent bowel movements, fatigue, weight loss, and abdominal pain or sudden abdominal pain. Complications associated with IBD inflammation include gastrointestinal abscesses, strictures, and fistulas. In some cases, EOM613 treatment reduces the risk of developing one or more IBD complications (e.g., gastrointestinal abscesses, strictures, and fistulas). In some cases, EOM613 treatment reduces the severity of one or more IBD complications (e.g., gastrointestinal abscesses, strictures, and fistulas).

[0063] In some aspects, the methods disclosed herein reduce one or more symptoms of IBD (or complications of IBD) by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of symptoms or complications) compared to appropriate controls. In some aspects, the methods disclosed herein reduce one or more symptoms (or complications) of IBD by 5% to 100%, for example, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 30% to 90%. Reduce by 80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 75%~100%, 75%~90%, 75%~80%, 80%~100%, 80%~90%, 90%~100%, 95%~100%, or 90%~95%. In some aspects, the above controls are measures of symptoms in the subjects prior to treatment with EOM613. Symptom measurement may be quantitative (e.g., clinical measurement) or qualitative (e.g., patient-reported perception of symptoms or quality of life).

[0064] In specific, non-limiting examples, abdominal distension is eliminated or reduced by 50% to 100% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 90%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95%) compared to the abdominal distension in the subjects before treatment with EOM613. In another specific, non-limiting example, diarrhea is eliminated or reduced in incidence by 50% to 100% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 90%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95%) compared to the subjects before treatment with EOM613. In another specific, non-limiting example, fatigue or abdominal pain (or sudden abdominal pain) is eliminated or reduced by 50% to 100% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95%) compared to the fatigue or abdominal pain (or sudden abdominal pain) experienced by the subject before treatment with EOM613 (e.g., the patient's self-reported perception of fatigue or pain).

[0065] In some cases, the methods disclosed herein reduce hospitalizations or surgeries due to IBD, or reduce IBD flares. In some cases, IBD hospitalizations, surgeries, and / or flares are reduced after EOM613 treatment by, for example, 5% to 100%, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 3% It is reduced by 80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 75%~100%, 75%~90%, 75%~80%, 80%~100%, 80%~90%, 90%~100%, 95%~100%, or 90%~95%.

[0066] In some cases, the methods disclosed herein increase (improve) the quality of life of the subject compared to an appropriate control (e.g., a measure of the subject's quality of life before administration of EOM613). Parameters of quality of life include, but are not limited to, mood, appetite, alertness, activities of daily living (ADL), fatigue, muscle pain, number of relapses after treatment, frequency of bowel movements, limitations in social activities, and physical strength / energy. Such parameters may be measured using standard methods. In some cases, parameters of quality of life are measured using surveys from the subject before and / or after treatment with EOM613. Useful quality of life measures include, but are not limited to, the Inflammatory Bowel Disease Questionnaire (IBDQ) (see Guyatt et al., Gastroenterology 96(3):804-810, 1989) and the Perceived Stress Scale (PSS; including versions PSS-14, PSS-10, and PSS-4; see Cohen et al., J Health Soc Behav 24(4):385-396, 1983). In some contexts, the above controls are scales or ratings obtained from the above subjects prior to treatment in EOM613, or historical controls or standard reference values ​​or ranges of values ​​(e.g., a sample group representing baseline or "normal" values).

[0067] In some aspects, the methods disclosed herein improve the quality of life of the subject by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, or more than 500%). In some aspects, the methods disclosed herein reduce the quality of life of the subject by approximately 10% to 500% (e.g., 10% to 400%, 10% to 300%, 10% to 200%, 10% to 100%, 10% to 75%, 10% to 50%, 10% to 25%, 25% to 500%, 25% to 400%, 25% to 300%, 25% to 200%, 25% to 100%, 25% to 75%, 25% to 50%, 50% to 500%, 50% to 50%). It improves by 400%, 50%~300%, 50%~200%, 50%~100%, 50%~75%, 75%~500%, 75%~400%, 75%~300%, 75%~200%, 75%~100%, 100%~500%, 100%~400%, 100%~300%, 100%~200%, 200%~500%, 200%~400%, 200%~300%, 300%~500%, 300%~400%, or 400%~500%.

[0068] In several aspects, the subjects exhibit elevated levels of one or more of the following: C-reactive protein, IL-6, IL-12, TNF-α, IL-23, MiR-223, and procalcitonin; administration of EOM613 reduces these elevated levels in the subjects to undetectable or normal levels. In several aspects, elevated levels of C-reactive protein, IL-6, IL-12, TNFα, IL-23, MiR-223, and / or procalcitonin were observed after EOM613 treatment compared to appropriate controls (e.g., levels before EOM613 administration), e.g., 5%-100%, 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, and 20%-40%. , 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, It is reduced by 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 75%~100%, 75%~90%, 75%~80%, 80%~100%, 80%~90%, 90%~100%, 95%~100%, or 90%~95%.

[0069] EOM613 can be administered by any suitable route. In some cases, EOM613 is administered orally, parenterally, topically, intranasally, by inhalation, or systemically. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intraperitoneal, intrathoracic, intrasternal injection, or infusion techniques. In non-limited cases, EOM613 is administered parenterally by subcutaneous injection. The preferred route of administration may vary, for example, depending on the recipient's condition and age.

[0070] The dose of EOM613 administered to the subject in the method provided herein may vary depending on a number of factors, including the amount and timing of administration, the subject being treated, the severity of the condition being treated, and the mode of administration. Generally, an appropriate dose sufficient to alleviate IBD symptoms without causing undesirable side effects is used. The appropriate dose may be determined, for example, in a clinical trial.

[0071] In several scenarios, EOM613 was administered to the subjects daily at doses ranging from approximately 0.5 microliters to approximately 200 microliters per kilogram of body weight, for example, 1-200 microliters, 1-180 microliters, 1-160 microliters, 1-140 microliters, 1-120 microliters, 1-100 microliters, 1-90 microliters, 1-80 microliters, 1-70 microliters, 1-60 microliters, 1-50 microliters, 1-40 microliters, and 1-30 microliters. Toru, 1-20 microliters, 1-10 microliters, 10-200 microliters, 10-180 microliters, 10-160 microliters, 10-150 microliters, 10-140 microliters, 10-120 microliters, 10-100 microliters, 10-90 microliters, 10-80 microliters, 10-70 microliters, 10-60 microliters, 10-50 microliters, 10-40 microliters, 10-30 microliters, 10-20 microliters, 2 0-200 microliters, 20-180 microliters, 20-160 microliters, 20-140 microliters, 20-120 microliters, 20-100 microliters, 20-90 microliters, 20-80 microliters, 20-70 microliters, 20-60 microliters, 20-50 microliters, 20-40 microliters, 20-30 microliters, 40-200 microliters, 40-180 microliters, 40-160 microliters, 40-140 microliters, 40-120 microliters, 40-100 microliters, 40-90 microliters, 40-80 microliters, 40-70 microliters, 40-60 microliters, 40-50 microliters, 60-200 microliters, 60-180 microliters, 60-160 microliters, 60-140 microliters, 60-120 microliters, 60-100 microliters, 60-90 microliters, 60-80 microliters, 60-70 microliters, 80-200 microliters,EOM613 is administered to the subject at a dose of 80-180 microliters, 80-160 microliters, 80-140 microliters, 80-120 microliters, 80-100 microliters, 80-90 microliters, 100-200 microliters, 100-180 microliters, 100-160 microliters, 100-140 microliters, or 100-120 microliters / kg body weight per day. In non-limiting cases, approximately 1-200 microliters / kg body weight is administered to the subject per day. In another non-limiting case, approximately 10-150 microliters / kg body weight is administered to the subject per day. In yet another non-limiting case, approximately 20-100 microliters / kg body weight is administered to the subject per day.

[0072] In several scenarios, approximately 1 ml to 10 ml of EOM613 was administered to the subject per day, for example, 1-9 ml, 1-8 ml, 1-7 ml, 1-6 ml, 1-5 ml, 1-4 ml, 1-3.5 ml, 1-3 ml, 1-2.5 ml, 1-2 ml, 1.5-10 ml, 1.5-9 ml, 1.5-8 ml, 1.5-7 ml, 1.5-6 ml, 1.5-5 ml, 1.5-4 ml, 1.5-3.5 ml, 1.5-3 ml, 1.5-2.5 ml, 1 EOM613 in amounts of 0.5-2 ml, 2-3.5 ml, 2-3 ml, 2-2.5 ml, 2-10 ml, 2-9 ml, 2-8 ml, 2-7 ml, 2-6 ml, 2-5 ml, 2-4 ml, 2-3.5 ml, 2-3 ml, 2-2.5 ml, 3-10 ml, 3-9 ml, 3-8 ml, 3-7 ml, 3-6 ml, 3-5 ml, 3-4 ml, 3-3.5 ml, 4-10 ml, 4-9 ml, 4-8 ml, 4-7 ml, 4-6 ml, or 4-5 ml is administered to the subject daily. The desired dose may be administered once daily or in two, three, or four divided doses throughout the day, generally evenly distributed over time, at appropriate intervals. In non-limiting cases, approximately 1 ml to 4 ml of EOM613 is administered to the subject once or twice daily. In another non-limiting case, approximately 1.5 ml to 2.5 ml of EOM613 is administered to the subject twice daily. In yet another non-limiting case, approximately 2 ml of EOM613 is administered to the subject twice daily.

[0073] In some cases, EOM613 is administered for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or longer. In some cases, EOM613 is administered for at least 1 week, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 8 weeks, or longer. In some cases, EOM613 is administered for approximately 1 to 12 weeks, for example, 1 to 8 weeks, 1 to 4 weeks, 2 to 8 weeks, or 2 to 4 weeks. In some cases, EOM613 is administered while the patient has active IBD or is experiencing symptoms of IBD.

[0074] The methods disclosed herein may further include administering a second IBD treatment agent (e.g., infliximab (Remicade®), adalimumab (Humira®), certolizumab pegol (Cimzia®), risankizumab (Skyrizi®), vedolizumab (Entyvio®), ustekinumab (Stelara®), upadacitinib (Rinvoq®), aminosalicylic acid (e.g., sulfasalazine, mesalamine, or orsalazine), mercaptopurine, or methotrexate).

[0075] In some aspects, EOM613 is used in the manner described herein, for example, i) A process of suspending 35-50% (w / w) casein, 15-40% w / w beef peptone, 10-25% w / w RNA, 1-10% w / w BSA, and 5-25% w / w sodium hydroxide in distilled water, thereby producing a suspension; ii) Autoclaving the above suspension; iii) A step of cooling the above suspension after autoclaving; iv) After cooling, the above suspension is filtered to produce a filtered solution; v) The filtered solution described above, a) To the total nitrogen content of 1.65 to 2.10 mg / ml, and b) To physiological pH A process of adjusting and thereby producing a adjusted solution; vi) filtering the above-prepared solution to produce a final filtrate; and vii) Sterilizing the final filtrate as described above, thereby producing EOM613. It is prepared by [method].

[0076] In some cases, EOM613 is prepared according to the method described in Example 1.

[0077] VI. Clause [Clause 1] A method for treating inflammatory bowel disease (IBD) in a subject, the method comprising the step of administering a therapeutically effective amount of EOM613 to the subject. [Clause 2] The method according to Clause 1, wherein the IBD is Crohn's disease, ulcerative colitis, or unclassified colitis. [Clause 3] The procedure for treating the subject is the method described in any one of the preceding clauses, which reduces the frequency or severity of one or more symptoms of IBD in the subject. [Clause 4] The method described in any one of the preceding clauses, wherein the one or more symptoms of IBD include rectal bleeding, abdominal distension, diarrhea, frequent bowel movements, fatigue, weight loss, and abdominal pain or sudden abdominal pain. [Clause 5] The method of any one of the preceding clauses, further comprising the step of selecting the subject having IBD for treatment. [Clause 6] The method described in any one of the preceding clauses, wherein the subject has active IBD or is in remission after being diagnosed with IBD. [Clause 7] The therapeutically effective amount of EOM613 is administered parenterally, topically, by inhalation, or systemically, as described in any one of the preceding clauses. [Clause 8] The therapeutically effective amount of EOM613 is administered parenterally as described in any one of the preceding clauses. [Clause 9] The therapeutically effective amount of EOM613 is administered subcutaneously as described in any one of the preceding clauses. [Clause 10] The method according to any one of the preceding clauses, wherein the administration step comprises administering approximately 1 ml to approximately 4 ml of EOM613 to the subject once or twice a day. [Clause 11] The administration step involves administering EOM613 to the subject. i) Approximately 1.5 ml to 2.5 ml twice a day; or ii) Approximately 2 ml twice a day The method described in any one of the preceding clauses, which includes the step of administering the substance. [Clause 12] The method according to any one of the preceding clauses, wherein the administration step comprises administering to the subject approximately 1 microliter to approximately 200 microliters of EOM613 / kilogram body weight per day. [Clause 13] The method according to any one of the preceding clauses, wherein the administration step comprises administering to the subject approximately 10 microliters to approximately 150 microliters of EOM613 / kilogram body weight per day. [Clause 14] The method according to any one of the preceding clauses, wherein the administration step comprises administering to the subject approximately 20 microliters to approximately 100 microliters of EOM613 / kilogram body weight per day. [Clause 15] EOM613 is, i) at least one week; ii) at least two weeks; iii) 1 to 12 weeks; or iv) 2-8 weeks The method described in any one of the preceding clauses, administered over a certain period. [Clause 16] The method according to any one of the preceding clauses, further comprising the step of administering a second IBD therapeutic agent to the subject. [Clause 17] The subject is a human, as described in any one of the preceding clauses. [Clause 18] The subject has elevated levels of one or more of the following: C-reactive protein, IL-6, IL-12, TNF-α, IL-23, MiR-223, and procalcitonin; If necessary, the step of administering the therapeutically effective amount of EOM613 reduces the elevated level in the subject to undetectable or normal levels. The method described in any one of the preceding clauses. [Clause 19] A method for treating inflammatory bowel disease (IBD), the method being: A step of selecting subjects with IBD for treatment, and A step of treating IBD by administering 2 ml of EOM613 parenterally to the subject twice a day. A method of including. [Clause 20] The EOM613 mentioned above is, i) A process of suspending 35-50% (w / w) casein, 15-40% w / w beef peptone, 10-25% w / w RNA, 1-10% w / w BSA, and 5-25% w / w sodium hydroxide in distilled water, thereby producing a suspension; ii) Autoclaving the suspension; iii) After autoclaving, the suspension is cooled to 3°C to 8°C for at least 12 hours; iv) After cooling, the suspension is filtered to produce a filtered solution; v) The filtered solution, a) To the total nitrogen content of 1.65 to 2.10 mg / ml, and b) To physiological pH A process of adjusting and thereby producing a adjusted solution; vii) A step of filtering the prepared solution to produce a final filtrate; and viii) A step of sterilizing the final filtrate and thereby producing EOM613, The method described in any one of the preceding clauses, prepared by a method that includes the following: [Examples]

[0078] Examples The following embodiments are provided to illustrate certain features of the present disclosure, but the claims should not be limited to those exemplified features.

[0079] Example 1 Exemplary method for producing EOM613 Approximately 35 g of casein, 17.1 g of beef peptone, 22 g of yeast RNA, 3.25 g of BSA, and 16.5 g of sodium hydroxide were suspended in approximately 2.5 L of distilled water. The suspension was autoclaved at a pressure of approximately 9 lbs and 93-110°C until the RNA was completely digested (e.g., approximately 4 hours). After autoclaving, the solution was cooled to room temperature, and then cooled to 3-8°C for approximately 6 hours to precipitate the insoluble elements. The solution was then filtered under inert gas at low pressure (e.g., 1-6 psi) through a 2 micron filter and then a 0.45 micron filter. The solution was then filtered through a 0.2 micron heat-retaining filter.

[0080] The filtered solution was assayed for total nitrogen content and diluted with cooled distilled water to an appropriate volume with a total nitrogen content of 1.65–2.10 mg / ml. The pH of the diluted solution was then adjusted with HCl to approximately 7.0–7.4. The diluted solution was filtered again through a 0.2 micron filter under inert gas as described above. The final filtrate was then filtered and sealed into vials. The filled vials were autoclaved for final sterilization (approximately 115°C, 14–16 lbs pressure, approximately 30 minutes). They were then ready for administration to subjects.

[0081] Example 2 Crohn's disease treatment case study Two male subjects (36 and 30 years old, respectively) diagnosed with moderate to severe chronic Crohn's disease were treated with 2 ml of EOM613 solution twice daily via subcutaneous injection. Treatment was initiated during an active relapse. In both subjects, symptoms resolved within two weeks of treatment, and bowel movements decreased from 4-5 times per day to 1-2 times per day. Both patients were able to resume travel and work without interruption. In the 36-year-old patient, whose Crohn's disease had persisted longer, significant abdominal distension decreased and lessened, and the abdomen became flatter. After the 36-year-old patient's daily bowel movements initially decreased and stabilized at 1-2 times per day, the dose of EOM613 was reduced to 2 ml once daily. However, symptoms did not subside as much with this dose reduction (the frequency of bowel movements increased to >2 times per day). This suggests that higher doses were more effective in controlling symptoms.

[0082] It is apparent that the exact details of the methods or compositions described may be changed or modified without departing from the spirit of this disclosure. The inventors claim all such modifications and variations that fall within the scope and spirit of the following claims.

Claims

1. A method for treating inflammatory bowel disease (IBD) in a subject, the method comprising the step of administering a therapeutically effective amount of EOM613 to the subject.

2. The method according to claim 1, wherein the IBD is Crohn's disease, ulcerative colitis, or unclassified colitis.

3. The method according to claim 1, wherein the step of treating the subject reduces the frequency or severity of one or more symptoms of IBD in the subject.

4. The method according to claim 3, wherein the one or more symptoms of IBD include one or more of rectal bleeding, abdominal distension, diarrhea, frequent bowel movements, fatigue, weight loss, and abdominal pain or sudden abdominal pain.

5. The method according to claim 1, further comprising the step of selecting a subject having IBD for treatment.

6. The method according to claim 5, wherein the subject has active IBD or is in remission after being diagnosed with IBD.

7. The method according to claim 1, wherein the therapeutically effective amount of EOM613 is administered parenterally, topically, by inhalation, or systemically.

8. The method according to claim 7, wherein the therapeutically effective amount of EOM613 is administered parenterally.

9. The method according to claim 1, wherein the therapeutically effective amount of EOM613 is administered subcutaneously.

10. The method according to claim 5, wherein the administration step comprises administering approximately 1 ml to approximately 4 ml of EOM613 to the subject once or twice a day.

11. The above-mentioned administration step involves administering EOM613 to the subject. i) Approximately 1.5 ml to 2.5 ml twice a day; or ii) Approximately 2 ml twice a day The method according to claim 10, comprising the step of administering.

12. The method according to claim 5, wherein the administration step comprises administering to the subject about 1 microliter to about 200 microliters of EOM613 / kilogram body weight per day.

13. The method according to claim 5, wherein the administration step comprises administering to the subject about 10 microliters to about 150 microliters of EOM613 per kilogram of body weight per day.

14. The method according to claim 5, wherein the administration step comprises administering to the subject about 20 microliters to about 100 microliters of EOM613 per kilogram of body weight per day.

15. EOM613 is, i) at least one week; ii) At least two weeks; iii) 1 to 12 weeks; or IV) 2-8 weeks The method according to claim 5, administered over a period of time.

16. The method according to claim 1, further comprising the step of administering a second IBD therapeutic agent to the subject.

17. The method according to claim 1, wherein the subject is a human.

18. The subject has elevated levels of one or more of the following: C-reactive protein, IL-6, IL-12, TNF-α, IL-23, MiR-223, and procalcitonin; If necessary, the step of administering the therapeutically effective amount of EOM613 reduces the elevated level in the subject to undetectable or normal levels. The method according to claim 1.

19. A method for treating inflammatory bowel disease (IBD), wherein the method is: A step of selecting subjects with IBD for treatment, and A step of administering 2 ml of EOM613 parenterally to the subject twice a day to treat the IBD. A method of including.

20. The aforementioned EOM613 is i) A step of suspending 35-50% (w / w) casein, 15-40% w / w beef peptone, 10-25% w / w RNA, 1-10% w / w BSA, and 5-25% w / w sodium hydroxide in distilled water, thereby producing a suspension; ii) Autoclaving the suspension; iii) After autoclaving, a step of cooling the suspension to 3°C to 8°C for at least 12 hours; iv) After cooling, the suspension is filtered to produce a filtered solution; v) The filtered solution, a) To the total nitrogen content of 1.65 to 2.10 mg / ml, and b) To physiological pH The process of adjusting and thereby producing the adjusted solution; vii) filtering the prepared solution to produce a final filtrate; and viiii) Sterilizing the final filtrate and thereby producing EOM613, The method according to any one of the above claims, prepared by a method encompassing the above.

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