Methods of treating inflammatory bowel diseases

Abstract

Methods of treating inflammatory bowel diseases are provided herein. Also provided are methods of preparing EOM613 and pharmaceutical compositions including EOM613.

Description

[0001] METHODS OF TREATING INFLAMMATORY BOWEL DISEASES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 536,325, filed September 1, 2023, which is incorporated by reference in its entirety.

[0004] FIELD

[0005] This relates to methods of treating inflammatory bowel diseases, such as Crohn’ s disease or ulcerative colitis.

[0006] BACKGROUND

[0007] 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 indeterminate colitis. Primary symptoms include persistent diarrhea (lasting longer than 4 weeks), abdominal pain, blood or mucus in the stool, fatigue, and weight loss. The severity of IBD can range from a mild illness to a debilitating condition that can lead to life-threatening complications. Currently available treatments are not well -tolerated or durable. A patient’s response to current IBD therapies can decrease over time until the therapy is no longer effective. Thus, there is a great need for alternative IBD treatments.

[0008] SUMMARY

[0009] Disclosed herein are methods of treating inflammatory bowel disease (IBD) in a subject, including administering a therapeutically effective amount of EOM613 to the subject. In some aspects, the IBD is Crohn’s disease. In some aspects, treatment with EOM613 reduces one or more symptoms of IBD, such as rectal bleeding, abdominal bloating, diarrhea, multiple daily bowel movements, fatigue, weight loss, and abdominal pain or cramps. A subject having IBD can be selected for treatment. In some aspects, the subject has active IBD, or is in remission following a diagnosis of IBD. The methods can further include administering a second IBD therapeutic, such as infliximab, adalibumab, certolizumab pegol, risankizumab, vedozilumib, ustekinumab, upadacitimib, aminosalicylates (e.g., ulfasalazine, mesalamine, or olsalazine), mercaptopurine, or methotrexate. Also disclosed are methods of preparing EOM613, and pharmaceutical compositions including EOM613.

[0010] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description.

[0011] DETAILED DESCRIPTION

[0012] I. Introduction

[0013] In 2015, the Centers for Disease Control and Prevention (CDC) estimated 1.3 % of US adults have been diagnosed with either Crohn’s Disease or ulcerative colitis. Between 2000 and 2018, the prevalence markedly increased among older adults in the United States (see, Fang Xu, et al., Morbidity and Mortality Weekly 70(19)1698-701, 2021).

[0014] The etiology of 1BD is largely driven by 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 principal cytokines implicated in precipitating the symptoms of 1BD are TNF-a and IL-23, thus, several therapeutics have been developed based on antibodies directed towards these cytokines. TNF-alpha inhibitor biologicals approved to treat the symptoms of 1BD include Infliximab (Remicade®), Adalibumab (Humira®), and Certolizumab pegol (Cimizia®). IL-23 blocking antibodies include Risankizumab (Skyrizi®). Other agents used clinically to treat 1BD include Vedozilumib (Entyvio®), a gut-homing alpha4-beta integrin inhibitor and Ustekinumab (Stelara®). All of the monoclonal biological agents are injected intravenously, and many have profound adverse side effects including increasing risk of infection (including upper respiratory tract infections and urinary tract infections), nausea, headache, and joint pain. Allergic reactions may also occur, and the drugs may cause harm to fetal health and thus should be avoided by pregnant patients.

[0015] Non-biologicals used to treat IBD include aminosalicylates, such as ulfasalazine, mesalamine, or olsalazine, however, these treatments work best for IBD affecting the lower colon rather than the small intestine. Immunomoulators, such as mercaptopurine or methotrexate, are also sometimes used to induce remission, but can damage the liver and pancreas.

[0016] Treatment failures are frequently seen with existing IBD therapies, such as those discussed above, for patients who do not tolerate the therapy, or patients who develop tolerance over time, eventually rendering the therapy no longer effective. In addition, despite advances made in the treatment of IBD using antibodies directed against IL-23 and TNF-a cytokines, treatment failures are frequent. In general, between 50% and 60% of patients initially respond to these therapies in terms of improvement in symptoms or inflammation markers. Of those patients, only approximately 20% to 30% go into 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).

[0017] EOM613 is a broad spectrum immunomodulator that modulates the release and action of pro-and anti-inflammatory cytokines. In previous clinical use for treating AIDS, cancer cachexia, and COVID-19, the therapy was safe and well-tolerated using daily injections of up to 2 ml administered subcutaneously (sc) twice daily.

[0018] However, EOM613 was not previously investigated for use in treating IBD, such as Crohn’s disease or ulcerative colitis. In addition, EOM613’s immune regulating activities vary depending on the state of activation of the immune cells. For example, EOM613 increased IL-6, IL-ip, and TNF-a secretion by PHA / lL-2-primed peripheral blood monocytes (PBMCs) in cell culture, but did not alter IL- 12 secretion. In contrast, treatment of LPS-activated monocytes decreased TNF-a and IL- 12, increased IL-6, but did not alter IL-ip, secretion (see, e.g., Hirchman, Advances in Bioscience and Biotechnology, 5: 161-168, 2014). Based on its differential effects on cy tokines, it is not possible to predict a priori whether EOM613 would be beneficial in the treatment of IBD. Yet, the data disclosed herein demonstrates that EOM613 is an effective treatment for IBD.

[0019] II. Summary of Terms

[0020] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin 's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a cytokine” includes singular or plural cytokines and can be considered equivalent to the phrase “at least one cytokine.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0021] To facilitate review of the various aspects, the following explanations of terms are provided:

[0022] About: Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

[0023] Administration: The introduction of an agent, such as EOM613, into a subject by a chosen route. Administration can be local or systemic. Exemplary routes of administration include, but are not limited to, oral, parenteral (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes. In some aspects, administration is parenteral.

[0024] Control: A reference standard. A control can be a positive or negative control. In some examples, the control is a measure or sample obtained from a healthy subject. In some examples, the control is a measure or sample obtained from a subject diagnosed with IBD. In some examples, the control is a measure or sample obtained from a subject prior to EOM613 administration. In still other examples, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients diagnosed with a disease or condition, for example IBD, that have a known prognosis or outcome, or group of samples that represent baseline or normal values). A practitioner can select a suitable control.

[0025] Detecting: To identify the existence, presence, or fact of something.

[0026] Increase or Decrease: A positive or negative change from a reference value. An increase is a positive change, such as an increase at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500%, as compared to a 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% decrease as compared to a reference value. In some examples, the reference value is a control. In some examples, the increase or decrease is statistically significant.

[0027] Inflammatory Bowel Disease (IBD): A gastrointestinal autoimmune disorder characterized by chronic inflammation of the gastrointestinal tract. IBD includes Crohn’s disease, ulcerative colitis, and indeterminate colitis. Crohn’s disease is characterized by inflammation of the lining of the digestive tract and most commonly affects the small intestine. It can also affect the large intestine and uncommonly, the upper gastrointestinal tract. Ulcerative colitis is characterized by inflammation and ulcers along the lining of the large intestine (colon) and rectum. Indeterminate colitis refers to IBD that has features of both Crohn’s disease and ulcerative colitis.

[0028] IBD symptoms include diarrhea, rectal bleeding, abdominal bloating, abdominal pain, fatigue, and weight loss. Symptoms can range from mild to severe. Affected individuals typically have periods of active IBD (when symptoms are present, also known as a flare-up or flare) followed by periods of remission (when symptoms are not present, or greatly decreased). IBD-related inflammation can cause damage to the gastrointestinal tract, including abscesses, strictures, and fistulas, and can also increase the risk of colon cancer. The exact cause of IBD is unknown, but there may be a genetic component. It has been hypothesized that it may result from a weakened immune system in response to environmental triggers, such as sequelae to a viral or bacterial infection that causes a general state of inflammation in the gastrointestinal tract.

[0029] Nucleotides: Organic molecules consisting of a nucleoside and a phosphate. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. They serve as monomeric units of the nucleic acid polymers deoxyribonucleic acid and ribonucleic acid.

[0030] Nucleic Acid Molecule: A polymeric form of nucleotides, which may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- 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 a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

[0031] Pharmaceutically Acceptable Carriers: The pharmaceutically acceptable carriers of use in the methods disclosed herein are known. In general, the nature of the carrier depends on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions (such as immunogenic compositions) to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, 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.

[0032] Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

[0033] Subject: Living multi-cellular vertebrate organism, a category that includes human and non-human mammals, such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, and the like. 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., dog, cat, or other domestic mammal). In some aspects, a subject in need of 1BD treatment is selected, for example, the subject has 1BD and in need of treatment.

[0034] Therapeutically Effective Amount: The amount of an agent, such as EOM613, that is sufficient to prevent (including prophylaxis), treat, and / or decrease a symptom or underlying cause of a disease or pathological condition, such as IBD. In some aspects, a therapeutically effective amount of EOM613 is an amount that treats IBD, such as decreasing or eliminating one or more symptoms of IBD.

[0035] The exact amount and timing of administration can depend on a number of factors, including the subject being treated, the severity and type of the condition being treated, and the manner of administration. A therapeutically effective amount can be determined by a practitioner through clinical trials. Effective amounts also can be determined through various in vitro, in vivo or in situ assays. In some aspects, a therapeutically effective amount of EOM613 is 2mls twice daily for a sufficient period of time to achieve a therapeutic benefit (e.g., at least 2 weeks, for example, 2 to 4 weeks or 2 to 8 weeks).

[0036] A therapeutically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attain a 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 for two weeks). Treating or Inhibiting (a Disease or Condition): Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a condition (e.g., 1BD) after the condition has begun to develop. The term “ameliorating,” with reference to a disease, syndrome, or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of symptoms in a subject, a reduction in severity of some or all symptoms, a slower progression, reduced recurrence or flare-ups, an improvement in the quality of life of the subject, or by other parameters that are specific to the particular disease, syndrome, or other pathological condition. Treatment may be assessed by objective or subjective parameters; including the results of a physical examination, blood or other clinical tests, self-evaluations by the subject, and the like.

[0037] Inhibiting a disease or condition includes preventing or reducing the risk of the disease or condition, such as reducing the risk of severe symptoms or IBD flare. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease (e.g., a subject in remission) or exhibits only early signs for the purpose of decreasing the risk of developing pathology. Prophylactic treatment can reduce the risk of an IBD flare. In some aspects, the disclosed methods are prophylactic and / or therapeutic. In some aspects, the disclosed methods are therapeutic and not prophylactic.

[0038] III. EOM613

[0039] EOM613, also known as Product R or AVR118, is a composition comprising nucleotides and peptides having molecular weights of no more than 14 KDa, primarily not more than 8 KDa. The nucleotides and peptides are breakdown products of casein, peptone, RNA and serum albumin. EOM613 and its production are described, for example, in US Patents 6,528,098, 6,921,542, 7,074,767, 7,524,661, and 8,084,239, which are incorporated by reference herein in their entireties. In these patents, EOM613 is referred to as Product R. A brief description of EOM613 and its production is provided herein.

[0040] Sixteen constituent compounds have been identified and characterized in EOM613: three nucleosides, two nucleoside diphosphates and eight nucleoside monophosphates, together with two peptides (one of them 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 31 -amino-acid peptide derived from bovine beta-casein with a molecular weight of 3536.24 Da. The shorter peptide (referred to as “peptide-B”) is a peptide-oligonucleotide conjugate including a 21-amino acid long peptide attached at a serine residue at position 18 to a diadenine (3'-5') diribonucleotide through a diphosphodiester linkage at the 3'-position. This nucleopeptide conjugate has a MW 2215 peptide moiety attached to a 740 MW nonpeptidic adduct to give a total MW of 2955. Peptide- A and peptide-B are present in EOM613 in approximately equal amount by weight. The amount of peptide- A and peptide-B in EOM613 is about 4.4-7.0 mg / mL, preferably 4.8-5.3 mg / mL, as determined by a Lowry protein assay.

[0041] EOM613 can be prepared, for example, according to the following method: i) suspending casein, beef peptone, RNA, BSA, and sodium hydroxide, in distilled water, thereby producing a suspension; ii) autoclaving the suspension; iii) after autoclaving, cooling the suspension; iv) after cooling, filtering the suspension, thereby producing a filtered solution; v) adjusting the filtered solution: a) to a total nitrogen content of 1.65 to 2.10 mg / ml, and b) to a physiological pH, thereby producing an adjusted solution; vi) optionally filtering the adjusted solution; and vii) sterilizing the adjusted solution, thereby producing EOM613.

[0042] The starting materials (casein, beef peptone, RNA, BSA, and sodium hydroxide) are available commercially or can be readily prepared by a practitioner.

[0043] The suspension includes about 35% to about 50% by dry weight (w / w) casein, 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.

[0044] The suspension includes about 15% to about 40% by dry weight (w / w) beef peptone, 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.

[0045] The suspension includes about 10% to about 25% by dry weight (w / w) RNA, for example, 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 may be used, such as plant RNA or yeast RNA. In some aspects, yeast RNA is used for producing EOM613.

[0046] The suspension includes about 1% to about 10% by dry weight (w / w) bovine serum albumin (BSA), 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.

[0047] The suspension includes about 5% to about 25% by dry weight (w / w) sodium hydroxide, 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 sodium hydroxide is at least 95% pure, for example, at least 96%, 97%, 98%, 99% pure. In a non-limiting example, the sodium hydroxide is at least 97% pure. In some aspects, the sodium hydroxide is in the form of pellets (e.g., Sigma- Aldrich catalog number 567530, or Fisher Scientific catalog number S318- 100).

[0048] The dry ingredients are suspended in an appropriate amount of distilled water. The ratio of total protein (grams) to the volume of distilled water (milliliters) is about 1.5g to about 2.5g total protein to about lOOmls distilled water, preferably about 2.2g to about 100ml. Thus, every 1.5-2.5 grams of total protein is suspended in about 100ml of distilled water. The suspension is then autoclaved under conditions in which RNA will be hydrolyzed into nucleotides. Such conditions are described herein and can be optimized by a practitioner. In some aspects, the autoclave pressure is about 5 to about 15 pounds per square inch, for example, 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 aspects, the autoclave temperature is about 60°C to about 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 temperature is about 90°C to about 110°C. In another example, the temperature is about 93°C to 110°C. In some aspects, the duration of autoclaving about 2 to about 10 hours, for example, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, 2.5 to 4, 2.5 to 3.5, 2.5 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, or 8 to 10 hours. In a non-limiting example, the duration of autoclaving is about 3 to about 5 hours. In another non-limiting example, the duration of autoclaving is at least 3 hours. In a specific, non-limiting example, the autoclaving conditions include a pressure of about 8 to about 10 pounds per square inch, a temperature of about 93°C to about 110°C, for about 3 to about 5 hours.

[0049] In some aspects, the suspension is cooled to about 1°C to about 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 9°C, 4°C to 8°C, 4°C to 7°C, 4°C to 6°C, 4°C to 5°C, 5°C to 10°C, 5°C to 9°C, 5°C to 8°C, 5°C to 7°C, 5°C to 6°C, or 8°C to 10°C. In a nonlimiting example, the suspension is cooled to about 3°C to about 8°C.

[0050] In some aspects, the 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 aspects, the suspension is cooled for about 1 hour to about 24 hours, for example, 2 to 24, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 24, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 24, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 24, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 10, 10 to 24, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 24, 14 to 20, 14 to 18, 14 to 16, 16 to 24, 16 to 20, 16 to 18, 18 to 24, 18 to 20, or 20 to 24 hours. In a non-limiting example, the suspension is cooled for at least 6 hours. In another non-limiting example, the solution is cooled for about 6 hours to about 12 hours. In a non-limiting example, the suspension is cooled to about 3°C to about 8°C for about 6 to about 12 hours.

[0051] Filtering can be performed with any filter of suitable size. In some aspects, the filter is about 0.05 to about 5 microns, for example, 0.05 to 4, 0.05 to 3, 0.05 to 2, 0.05 to 1, 0.05 to 0.5, 0.05 to 0.2, 0.05 to 0.1, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 0.1 to 0.5, 0.1 to 0.2, 0.2 to 4, 0.2 to 3, 0.2 to 2, 0.2 to 1, 0.2 to 0.5, 0.375 to 4, 0.375 to 3, 0.375 to 2, 0.375 to 1, 0.357 to 0.5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 1 to 4, 1 to 3, 1 to 2, 2 to 4, or 2 to 3 microns. In some aspects, the filter is a pyrogen retention filter. In some aspects, the suspension is passed through multiple 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, the filters can be the same size (e.g., all about 0.2 microns), or the filters can be different sizes (e.g., about 2 micron, about 0.45 micron, and about 0.2 micron filters). Typically, filtration is performed with the largest filter first, followed by additional filters in descending size (largest to smallest). In some aspects, the suspension is filtered, for example, with a 2 micron, 0.45 micron, and 0.2 micron filter before the diluting step. In some aspects, the 0.2 micron filter is a pyrogen retention filter. The filtered solution can optionally be cooled to about 3°C to about 8°C again, and filtration as described above can be repeated. In some aspects, 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 aspects, the suspension is cooled for about 1 hour to about 24 hours, for example, 2 to 24, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 24, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 24, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 24, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 10, 10 to 24, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 24, 14 to 20, 14 to 18, 14 to 16, 16 to 24, 16 to 20, 16 to 18, 18 to 24, 18 to 20, or 20 to 24 hours. In a non-limiting example, the filtered solution is cooled 1 to 6 hours, and filtration as described above is repeated.

[0052] Filtering can be performed under an inert gas, such as nitrogen or argon. In some aspects, filtering is performed under at inert gas at a pressure of about 1-6 pound per square inch (psi), for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, or 5 to 6 psi. In some aspects, the gas pressure is about 1 to about 6 psi.

[0053] The nitrogen content and pH of the filtered solution is then adjusted. The filtered solution is assayed to determine the total nitrogen content and adjusted to a total nitrogen content of about 1 mg / ml to about 3 mg / ml. Adjusting the nitrogen content can include diluting or concentrating the solution. Typically, the filtered solution is diluted, but may be concentrated if needed. Total nitrogen content can be determined by any known method, such as the Kjeldahl method (see, Kjeldahl, Z. Anal. Chem., Vol. 22, p366, 1883), or related methods based on the Kjeldahl method. In some aspects, the total nitrogen content is about 1.50 mg / ml to about 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 2.00 mg / ml, 1.65 to 1.90 mg / ml, 1.65 to 1.80 mg / ml, 1.65 to 1.70 mg / ml, 1.70 to 2.50 mg / ml, 1.70 to 2.25 mg / ml, 1.70 to 2.10 mg / ml, 1.70 to 2.00 mg / ml, 1.70 to 1.90 mg / ml, 1.70 to 1.80 mg / ml, 1.80 to 2.10 mg / ml, 1.80 to 2.00 mg / ml, 1.80 to 1.90 mg / ml, 1.90 to 2.10 mg / ml, 1.90 to 2.00 mg / ml, or 2.00 to 2.10 mg / ml total nitrogen. In some aspects, the total nitrogen content is adjusted to about 1.65 mg / ml to about 2.10 mg / ml. The filtered solution is also adjusted to a physiological pH. In some aspects, the pH is adjusted to about 6.5 to about 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 a non-limiting example, the pH of the filtered solution is adjusted to 7.3 to 7.6. Adjusting the pH includes raising the pH (adding a base) or lowering the pH (adding acid) as needed. In some aspects, the pH is adjusted with NaOH and / or HC1. In some aspects, the pH is adjusted after the total nitrogen content is adjusted.

[0054] After adjusting the nitrogen content and pH, the adjusted solution can optionally be filtered again, for example, with a 0.05 to 5 micron filter, for example, 0.05 to 4, 0.05 to 3, 0.05 to 2, 0.05 to 1, 0.05 to 0.5, 0.05 to 0.2, 0.05 to 0.1, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 0.1 to 0.5, 0.1 to 0.2, 0.2 to 4, 0.2 to 3, 0.2 to 2, 0.2 to 1, 0.2 to 0.5, 0.375 to 4, 0.375 to 3, 0.375 to 2, 0.375 to 1, 0.357 to 0.5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 1 to 4, 1 to 3, 1 to 2, 2 to 4, or 2 to 3 microns, thereby generating a final filtrate. In some aspects, the filter is a pyrogen retention filter. In a non-limiting example, the adjusted solution is filtered through a 0.2 micron filter (e.g., a 0.2 micron pyrogen retention filter). In some aspects, the suspension is passed through multiple 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, the filters can be the same size (e.g., all 0.2 microns), or the filters can be different sizes (e.g., 2 micron, 0.45 micron, and 0.2 micron filters). Typically, filtration is performed with the largest filter first, followed by additional filters in descending size (largest to smallest).

[0055] The adjusted solution or final filtrate has a light absorption 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 aspects, the total protein content of the final filtrate is measured (e.g., by a total protein Lowry assay) to verify that the peptide content is between 3.5 to 8 mg / ml (e.g., 4.7 to 7.0 mg / ml). The adjusted solution or final filtrate can optionally be filled and sealed into vials, such as 2 ml or 10 ml glass vials, under an inert gas.

[0056] The final step is sterilization of the adjusted solution or final filtrate. Once sterilized, the EOM613 composition is ready for administration. Any suitable sterilization method can be used. In some aspects, filled vials are autoclaved for final sterilization, after which they are ready for administration to a subject. In some aspects, the autoclave conditions for the final sterilization step is about 90-150°C (e.g., 100-125°C or 110-120°C) under 12-18 pounds pressure (e.g., 14-16 pounds pressure) for 15 minutes to 1 hour (e.g., 15-30 minutes, 20-60 minutes, or 20-40 minutes).

[0057] IV. Pharmaceutical Compositions

[0058] EOM613 can be directly administered to a subject or be administered as part of a pharmaceutical composition. Such pharmaceutical compositions include EOM613 and a pharmaceutically acceptable carrier.

[0059] Pharmaceutical compositions including EOM613 can be in any suitable form, such as a lyophilized solid (for reconstitution in a normal saline solution), liquid, or gas (aerosol). Pharmaceutical compositions including EOM613 can be formulated such that EOM613 becomes bioavailable upon administration of the pharmaceutical composition to a subject. Pharmaceutical compositions can take the form of one or more dosage units. For example, a vial of liquid EOM613, or a container of EOM613 in aerosol form, may hold a plurality of dosage units. A syringe containing a single unit dose of EOM613 is also provided.

[0060] Pharmaceutical compositions including EOM613 can be in liquid form, for example, solutions, syrups, or suspensions, or can be presented as a drug product for reconstitution with saline or other suitable vehicle before use. Such liquid preparations can be prepared by known methods with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).

[0061] Pharmaceutical compositions including EOM613 can in solid form, for example, tablets or capsules including pharmaceutically acceptable excipients, such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrates (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Tablets can include coatings, such as an enteric coating. For buccal administration, pharmaceutical compositions can take the form of tablets or lozenges formulated by known methods.

[0062] Pharmaceutical compositions including EOM613 can be delivered in the form of an aerosol, for example, from a pressurized pack or nebulizer. Aerosol formulations can include a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In some aspects, the dosage unit of an aerosol can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use, for example, in an inhaler, nebulizer, or insufflator, can be formulated containing a powder mix of EOM613 and a suitable powder base, such as lactose or starch.

[0063] Pharmaceutical compositions including EOM613 can be formulated for parenteral administration, for example by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. A pharmaceutical composition for parenteral administration can take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient (e.g., EOM613) can be in powder form for constitution with a suitable vehicle, e.g., sterile saline, before use. Pharmaceutical compositions can be formulated for controlled release of an active compound.

[0064] EOM613 can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. In such examples, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are examples of delivery vehicles or carriers for hydrophilic drugs. Pharmaceutical compositions including EOM613 can be formulated in compositions, such as creams, lotions, gels, ophthalmic drops, ointments, solutions, suspensions, shampoos, or other forms known to a practitioner and described, for example, in Remington ’s Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020). Actual methods for preparing pharmaceutical compositions have also been described in detail in, for example, in Remington ’s Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020).

[0065] Pharmaceutical compositions can be in unit-dose or multi-dose containers. In some aspects, a pharmaceutical composition including EOM613 is contained in a sealed ampule or vial. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction of the administered ingredient.

[0066] In some aspects, the pharmaceutical composition includes one or more therapeutics in addition to EOM613, such as one or more IBD therapies. Exemplary IBD therapies include, but are not limited to, infliximab (Remicade®), adalibumab (Humira®), certolizumab pegol (Cimizia®), risankizumab (Skyrizi®), vedozilumib (Entyvio®), ustekinumab (Stelara®), upadacitimib (Rinvoq®), aminosalicylates (e.g., ulfasalazine, mesalamine, or olsalazine), mercaptopurine, or methotrexate.

[0067] V. Methods of Treatment

[0068] Disclosed herein are methods of treating inflammatory bowel disease (IBD) in a subject, including administering a therapeutically effective amount of EOM613 to the subject. In some aspects, the methods include 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 indeterminate colitis. In some aspects, the IBD is Crohn’s disease. Individuals with IBD can experience periods of remission or active IBD. Remission occurs when symptoms arc absent or less severe. Active IBD occurs when symptoms arc present (also referred to as a flare or flare-up). In some aspects, the subject has active IBD, or is in remission following a diagnosis of IBD. In some aspects, the subject is a human. In some aspects, EOM613 is included in a pharmaceutical composition.

[0069] In some aspects, EOM613 treatment decreases the frequency or severity of one or more symptoms of IBD in the subject. Exemplary symptoms of IBD include, but are not limited to: rectal bleeding, abdominal bloating, diarrhea, multiple daily bowel movements, fatigue, weight loss, and abdominal pain or cramps. In some aspects, EOM613 treatment eliminates one or more symptoms of IBD in the subject, for example, treatment with IBD eliminates one or more of: rectal bleeding, abdominal bloating, diarrhea, multiple daily bowel movements, fatigue, weight loss, and abdominal pain or cramps in the subject. Complications related to IBD inflammation include abscesses, strictures, and fistulas of the gastrointestinal tract. In some aspects, EOM613 treatment reduces risk of developing one or more IBD complications, such as abscesses, strictures, and fistulas of the gastrointestinal tract. In some aspects, EOM613 treatment decreases the severity of one or more IBD complications, such as abscesses, strictures, and fistulas of the gastrointestinal tract. In some aspects, the methods disclosed herein decrease one or more symptoms of IBD (or a complication of IBD) by at least 10%, for example, by 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 a symptom or complication), as compared to a suitable control. In some aspects, the methods disclosed herein decrease 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 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 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% as compared to a suitable control. In some aspects, the control is a measure of a symptom in the subject prior to treatment with EOM613. A measurement of a symptom can be quantitative (e.g., a clinical measurement) or qualitative (e.g., a patient’s self-reported perception of a symptom or quality of life).

[0070] In a specific, non-limiting example, abdominal bloating is eliminated, or decreased 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%) relative to abdominal bloating in the subject prior to treatment with EOM613. In another specific, non-limiting example, diarrhea is eliminated, or incidents decreased 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%) relative to the subject prior to treatment with EOM613. In another specific, non-limiting example, fatigue or abdominal pain (or cramps) is eliminated or decreased 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%) relative to fatigue or abdominal pain (or cramps) experienced by the subject prior to treatment with EOM613 (e.g., a patient’s self-reported perception of fatigue or pain).

[0071] In some aspects, the methods disclosed herein decrease hospitalization or surgeries due to IBD, or decrease IBD flares. In some aspects, IBD hospitalizations, surgeries, and / or flares are decreased following EOM613 treatment, for example, 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 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 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% as compared to a suitable control.

[0072] In some aspects, the methods disclosed herein increase (improve) the subject’s quality of life as compared to a suitable control (such as a measure of the subject’s quality of life prior to administration of EOM613). Quality of life parameters include, but are not limited to, mood, appetite, alertness, activities of daily living (ADLs), malaise, myalgia, number of relapses post-treatment, bowel movement frequency, social activity limitations, and power / energy. Such parameters can be measured using standard methods. In some aspects, quality of life parameters are measured using a survey 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 aspects, the control is a measure or evaluation obtained from the subject prior to treatment with EOM613, or a historical control or standard reference value or range of values (such as a group of samples that represent baseline or “normal” values).

[0073] In some aspects, the methods disclosed herein improve the subject’s quality of life by at least 10%, such as 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 greater than 500%. In some aspects, the methods disclosed herein improve the subject’s quality of life by about 10% to 500%, such as 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 400%, 50% to 300%, 50% to 200%, 50% to 100%, 50% to 75%, 75% to 500%, 75% to 400%, 75% to 300%, 75% to 200%, 75% to 100%, 100% to 500%, 100% to 400%, 100% to 300%, 100% to 200%, 200% to 500%, 200% to 400%, 200% to 300%, 300% to 500%, 300% to 400%, or 400% to 500%.

[0074] In some aspects, the subject has an elevated level of one or more of: C-reactive protein, IL-6, IL-12, TNF-alpha, IL-23, MiR-223, and procalcitonin, and administering EOM613 decreases the elevated level to a non-detectable or a normal level in the subject. In some aspects, an elevated level of C-reactive protein, IL- 6, IL-12, TNFalpha, IL23, MiR-223 and / or procalcitonin, is decreased following EOM613 treatment, for example, 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 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 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% as compared to a suitable control (e.g., a level prior to EOM613 administration). EOM613 can be administered by any suitable route. In some aspects, EOM613 is administered orally, parenterally, topically, intranasally, by inhalation, or systemically. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intraperitoneal, intrapleural, intrasternal injection, or infusion techniques. In a non-limiting example, EOM613 is administered parenterally by subcutaneous injection. The preferred administration route may vary, for example, with the condition and age of the recipient.

[0075] The dose of EOM613 administered to the subject in the methods provided herein may vary depending on multiple factors, including the amount and timing of administration, the subject being treated, the severity of the condition being treated, and the manner of administration. Generally, an appropriate dosage is used that is sufficient to alleviate IBD symptoms without causing unwanted side effects. An appropriate dose can be determined, for example, in clinical trials.

[0076] In some aspects, about 0.5 microliters to about 200 microliters per kilogram of body weight of EOM613 is administered per day to the subject, for example, 1 to 200 microliters, 1 to 180 microliters, 1 to 160 microliters, 1 to 140 microliters, 1 to 120 microliters, 1 to 100 microliters, 1 to 90 microliters, 1 to 80 microliters, 1 to 70 microliters, 1 to 60 microliters, 1 to 50 microliters, 1 to 40 microliters, 1 to 30 microliters, 1 to 20 microliters, 1 to 10 microliters, 10 to 200 microliters, 10 to 180 microliters, 10 to 160 microliters, 10 to 150 microliters, 10 to 140 microliters, 10 to 120 microliters, 10 to 100 microliters, 10 to 90 microliters, 10 to 80 microliters, 10 to 70 microliters, 10 to 60 microliters, 10 to 50 microliters, 10 to 40 microliters, 10 to 30 microliters, 10 to 20 microliters, 20 to 200 microliters, 20 to 180 microliters, 20 to 160 microliters, 20 to 140 microliters, 20 to 120 microliters, 20 to 100 microliters, 20 to 90 microliters, 20 to 80 microliters, 20 to 70 microliters, 20 to 60 microliters, 20 to 50 microliters, 20 to 40 microliters, 20 to 30 microliters, 40 to 200 microliters, 40 to 180 microliters, 40 to 160 microliters, 40 to 140 microliters, 40 to 120 microliters, 40 to 100 microliters, 40 to 90 microliters, 40 to 80 microliters, 40 to 70 microliters, 40 to 60 microliters, 40 to 50 microliters, 60 to 200 microliters, 60 to 180 microliters, 60 to 160 microliters, 60 to 140 microliters, 60 to 120 microliters, 60 to 100 microliters, 60 to 90 microliters, 60 to 80 microliters, 60 to 70 microliters, 80 to 200 microliters, 80 to 180 microliters, 80 to 160 microliters, 80 to 140 microliters, 80 to 120 microliters, 80 to 100 microliters, 80 to 90 microliters, 100 to 200 microliters, 100 to 180 microliters, 100 to 160 microliters, 100 to 140 microliters, or 100 to 120 microliters per kilogram of body weight of EOM613 is administered per day to the subject. In a non-limiting example, about 1 to about 200 microliters per kg body weight is administered to the subject per day. In another non-limiting example, about 10 to about 150 microliters per kg body weight is administered to the subject per day. In a further non-limiting example, about 20 to about 100 microliters per kg body weight is administered to the subject per day.

[0077] In some aspects, about 1 milliliter (ml) to about 10 ml of EOM613 is administered to the subject per day, for example, 1 to 9 ml, 1 to 8 ml, 1 to 7 ml, 1 to 6 ml, 1 to 5 ml, 1 to 4 ml, 1 to 3.5 ml, 1 to 3 ml, 1 to 2.5 ml, 1 to 2 ml, 1.5 to 10 ml, 1.5 to 9 ml, 1.5 to 8 ml, 1.5 to 7 ml, 1.5 to 6 ml, 1.5 to 5 ml, 1.5 to 4 ml, 1.5 to 3.5 ml, 1.5 to 3 ml, 1.5 to 2.5 ml, 1.5 to 2 ml, 2 to 3.5 ml, 2 to 3 ml, 2 to 2.5 ml, 2 to 10 ml, 2 to 9 ml, 2 to 8 ml, 2 to 7 ml, 2 to 6 ml, 2 to 5 ml, 2 to 4 ml, 2 to 3.5 ml, 2 to 3 ml, 2 to 2.5 ml, 3 to 10 ml, 3 to 9 ml, 3 to 8 ml, 3 to 7 ml, 3 to 6 ml, 3 to 5 ml, 3 to 4 ml, 3 to 3.5 ml, 4 to 10 ml, 4 to 9 ml, 4 to 8 ml, 4 to 7 ml, 4 to 6 ml, or 4 to 5 ml EOM613 is administered to the subject per day. The desired dose may be administered once a day, or in two, three or more sub-doses at appropriate intervals, generally equally spread in time, throughout the day. In a nonlimiting example, about 1 ml to about 4 ml EOM613 is administered once or twice per day to the subject. In another non-limiting example, about 1.5 ml to about 2.5 ml EOM613 is administered to the subject twice per day. In a further non-limiting example, about 2 ml of EOM613 is administered to the subject twice a day.

[0078] In some aspects, EOM613 is administered for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, about 12 weeks, or longer. In some aspects, 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 aspects, EOM613 is administered for about 1 to about 12 weeks, for example, 1 to 8 weeks, 1 to 4 weeks, 2 to 8 weeks, or 2 to 4 weeks. In some aspects, EOM613 is administered while a patient has active IBD or has symptoms of IBD.

[0079] The methods disclosed herein can further include administered a second IBD therapeutic, such as infliximab (Remicade®), adalibumab (Humira®), certolizumab pegol (Cimizia®), risankizumab (Skyrizi®), vedozilumib (Entyvio®), ustekinumab (Stelara®), upadacitimib (Rinvoq®), aminosalicylates (e.g., ulfasalazine, mesalamine, or olsalazine), mercaptopurine, or methotrexate.

[0080] In some aspects, EOM613 is prepared by a method described herein, for example: i) suspending 35-50% by dry weight (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, cooling the suspension; iv) after cooling, filtering the suspension, thereby producing a filtered solution; v) adjusting the filtered solution: a) to a total nitrogen content of 1.65 to 2.10 mg / ml, and b) to a physiological pH, thereby producing an adjusted solution; vi) filtering the adjusted solution, thereby producing a final filtrate; and vii) sterilizing the final filtrate, thereby producing EOM613.

[0081] In some aspects, EOM613 is prepared according to the method described in Example 1. VI. Clauses

[0082] [Clause 1] A method of treating inflammatory bowel disease (IBD) in a subject, comprising administering a therapeutically effective amount of EOM613 to the subject.

[0083] [Clause 2] The method of clause 1, wherein the IBD is Crohn’s disease, ulcerative colitis, or indeterminate colitis.

[0084] [Clause 3] The method of any one of the prior clauses, wherein treating the subject decreases the frequency or severity of one or more symptoms of IBD in the subject.

[0085] [Clause 4] The method of any one of the prior clauses, wherein the one or more symptoms of IBD comprise one or more of: rectal bleeding, abdominal bloating, diarrhea, multiple daily bowel movements, fatigue, weight loss, and abdominal pain or cramps.

[0086] [Clause 5] The method of any one of the prior clauses, further comprising selecting the subject having IBD for treatment.

[0087] [Clause 6] The method of any one of the prior clauses, wherein the subject has active IBD or is in remission following a diagnosis of IBD.

[0088] [Clause 7] The method of any one of the prior clauses, wherein the therapeutically effective amount of EOM613 is administered parenterally, topically, by inhalation, or systemically.

[0089] [Clause 8] The method of any one of the prior clauses, wherein the therapeutically effective amount of EOM613 is administered parenterally.

[0090] [Clause 9] The method of any one of the prior clauses, wherein the therapeutically effective amount of EOM613 is administered subcutaneously.

[0091] [Clause 10] The method of any one of the prior clauses, wherein the administering comprises administering about 1 ml to about 4 ml EOM613 once or twice per day to the subject.

[0092] [Clause 11] The method of any one of the prior clauses, wherein the administering comprises administering: i) about 1.5 ml to about 2.5 ml twice per day; or ii) about 2 ml twice per day; of EOM613 to the subject.

[0093] [Clause 12] The method of any one of the prior clauses, wherein the administering comprises administering about 1 microliter to about 200 microliters EOM613 per kilogram of body weight per day to the subject.

[0094] [Clause 13] The method of any one of the prior clauses, wherein the administering comprises administering about 10 microliters to about 150 microliters of EOM613 per kilogram of body weight per day to the subject.

[0095] [Clause 14] The method of any one of the prior clauses, wherein the administering comprises administering about 20 microliters to about 100 microliters of EOM613 per kilogram of body weight per day to the subject.

[0096] [Clause 15] The method of any one of the prior clauses, wherein EOM613 is administered for: i) at least 1 week; ii) at least 2 weeks; iii) 1 to 12 weeks; or iv) 2 to 8 weeks

[0097] [Clause 16] The method of any one of the prior clauses, further comprising administering to the subject a second IBD therapeutic.

[0098] [Clause 17] The method of any one of the prior clauses, wherein the subject is a human. [Clause 18] The method of any one of the prior clauses, wherein the subject has an elevated level of one or more of: C-reactive protein, IL-6, IL- 12, TNF-alpha, IL-23, MiR-223, and procalcitonin; optionally wherein administering the therapeutically effective amount of EOM613 decreases the elevated level to a non- detectable or a normal level in the subject.

[0099] [Clause 19] A method of treating inflammatory bowel disease (IBD), comprising: selecting a subject having IBD for treatment, and administering parenterally 2 ml of EOM613 twice daily to the subject, thereby treating the IBD.

[0100] [Clause 20] The method of any one of the prior clauses, wherein the EOM613 is prepared by a method comprising: i) suspending 35-50% by dry weight (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, cooling the suspension to 3°C to 8°C for at least 12 hours; iv) after cooling, filtering the suspension, thereby producing a filtered solution; v) adjusting the filtered solution: a) to a total nitrogen content of 1.65 to 2.10 mg / ml, and b) to a physiological pH, thereby producing an adjusted solution; vii) filtering the adjusted solution, thereby producing a final filtrate; and viii) sterilizing the final filtrate, thereby producing EOM613.

[0101] EXAMPLES

[0102] The following examples are provided to illustrate particular features of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0103] Example 1 Exemplary Method of Making EOM613

[0104] About 35g casein, 17.1g beef peptone, 22g yeast RNA, 3.25g BSA, and 16.5g sodium hydroxide were suspended in about 2.5 L distilled water. The suspension was autoclaved at a pressure of about 9 lbs. at 93-110°C until the RNA was completely digested (for example, about 4 hours). After autoclaving, the solution was cooled to room temperature, and then further cooled to 3-8°C for about 6 hours to precipitate insoluble elements. The solution was then filtered through a 2 micron filter and subsequently a 0.45 micron filter under an inert gas at low pressure (e.g., 1-6 psi). The solution was then filtered through a 0.2 micron pyrogen retention filter.

[0105] The filtered solution was assayed for total nitrogen content and diluted with chilled distilled water to an appropriate volume having a total nitrogen content of 1.65-2.10 mg / ml. The pH of the diluted solution was then adjusted with HC1 to a pH of about 7.0 to 7.4. The diluted solution was filtered again through a 0.2 micron filter under an inert gas as described above. The final filtrate was then filled and sealed into vials. The filled vials were autoclaved for final sterilization (about 115°C, 14-16 pounds pressure for about 30 minutes), after which they were ready for administration to a subject. Example 2

[0106] Treatment of Crohn’s Disease Case Study

[0107] Two male subjects, aged 36 and 30, respectively, diagnosed with chronic Crohn’s disease of moderate to severe intensity were treated with EOM613 solution 2 ml injected subcutaneously twice daily. Therapy commenced during an active flare-up. In both subjects, symptoms resolved within 2 weeks of treatment with lessening of stool movements from 4-5 daily to 1-2 movement, with both patients resuming the ability to travel and work uninterrupted. In the case of the 36-year-old with Crohn’ s disease of longer standing, the considerable abdominal bloat was diminished and mitigated with the stomach flattening out as well. Once the daily bowel movement of the 36-year-old patient was reduced and stabilized to 1-2 per day, the dose of EOM613 was reduced to 2mLs once daily. However, upon reduction of the dose, symptoms were not mitigated as well (bowel movement frequency increased to >2 daily) suggesting that the higher dose was more effective in controlling symptoms.

[0108] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:

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

2. The method of claim 1, wherein the IBD is Crohn’s disease, ulcerative colitis, or indeterminate colitis.

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

4. The method of claim 3, wherein the one or more symptoms of IBD comprise one or more of: rectal bleeding, abdominal bloating, diarrhea, multiple daily bowel movements, fatigue, weight loss, and abdominal pain or cramps.

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

6. The method of claim 5, wherein the subject has active IBD or is in remission following a diagnosis of IBD.

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

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

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

10. The method of claim 5, wherein the administering comprises administering about 1 ml to about 4 ml EOM613 once or twice per day to the subject.

11. The method of claim 10, wherein the administering comprises administering: i) about 1.5 ml to about 2.5 ml twice per day; or ii) about 2 ml twice per day; of EOM613 to the subject.

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

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

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

15. The method of claim 5, wherein EOM613 is administered for: i) at least 1 week; ii) at least 2 weeks; iii) 1 to 12 weeks; or iv) 2 to 8 weeks16. The method of claim 1, further comprising administering to the subject a second IBD therapeutic.

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

18. The method of claim 1, wherein the subject has an elevated level of one or more of: C- reactive protein, IL-6, IL- 12, TNF-alpha, IL-23, MiR-223, and procalcitonin; optionally wherein administering the therapeutically effective amount of EOM613 decreases the elevated level to a non-detectable or a normal level in the subject.

19. A method of treating inflammatory bowel disease (IBD), comprising: selecting a subject having IBD for treatment, and administering parenterally 2 ml of EOM613 twice daily to the subject, thereby treating the IBD.

20. The method of any one of the prior claims, wherein the EOM613 is prepared by a method comprising:i) suspending 35-50% by dry weight (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, cooling the suspension to 3°C to 8°C for at least 12 hours; iv) after cooling, filtering the suspension, thereby producing a filtered solution; v) adjusting the filtered solution: a) to a total nitrogen content of 1.65 to 2.10 mg / ml, and b) to a physiological pH, thereby producing an adjusted solution; vii) filtering the adjusted solution, thereby producing a final filtrate; and viii) sterilizing the final filtrate, thereby producing EOM613.