EP4125854A1 - Compositions comprising 15-hepe for treating or preventing hematologic disorders, and/or related diseases - Google Patents

Compositions comprising 15-hepe for treating or preventing hematologic disorders, and/or related diseases

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Publication number
EP4125854A1
EP4125854A1 EP20717175.2A EP20717175A EP4125854A1 EP 4125854 A1 EP4125854 A1 EP 4125854A1 EP 20717175 A EP20717175 A EP 20717175A EP 4125854 A1 EP4125854 A1 EP 4125854A1
Authority
EP
European Patent Office
Prior art keywords
hepe
subject
composition
baseline
visit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20717175.2A
Other languages
German (de)
English (en)
French (fr)
Inventor
John Climax
Moayed HAMZA
Markus WEISSBACH
David Coughlan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Afimmune Ltd
Original Assignee
Afimmune Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Afimmune Ltd filed Critical Afimmune Ltd
Publication of EP4125854A1 publication Critical patent/EP4125854A1/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the present application relates generally to compositions comprising 15- HEPE, and to methods of using the same.
  • Red blood cell disorders are a class of conditions that affect the production, life-span, and shape of red blood cells (RBCs) and its oxygen transport molecule, hemoglobin. These conditions inhibit the transport of oxygen from the lungs to the rest of the body.
  • throombophilia is a class of disorders that increase a subject’s risk of developing blood clots in veins and arteries. These clots can break loose from the blood vessel and travel through the blood stream to an organ thereby preventing blood flow to that organ resulting in ischemia. These conditions lead to an increased risk of stroke and/or pulmonary embolism.
  • the application relates to compositions comprising 15- hydroxyeicosapentaenoic acid (15-HEPE) and to methods of using such compositions in the treatment of a variety of diseases and disorders.
  • 15-HEPE 15- hydroxyeicosapentaenoic acid
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15- HEPE.
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition, and the subject exhibits one or more of: (a) an increase in red blood cell count; (b) a decrease in red cell distribution width; and/or (c) a decrease in reticulocyte count.
  • the hematologic disorder is selected from the group consisting of inherited hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.
  • the present disclosure provides methods of treating and/or preventing a hemoglobin disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15- HEPE.
  • the present disclosure provides methods of treating and/or preventing a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15- HEPE.
  • the present disclosure provides methods of treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition, and the subject exhibits one or more of: (a) an increase in red blood cell count; (b) a decrease in red cell distribution width; and/or (c) a decrease in reticulocyte count.
  • the hematologic disorder, the hemoglobin disorder, and/or the red blood cell disorder are selected from the group consisting of inherited hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.
  • the present disclosure provides methods of treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the hemolytic anemia is inherited hemolytic anemia or acquired hemolytic anemia.
  • the acquired hemolytic anemia is acquired as secondary to infection, from a medication, from a hematological malignancy, from autoimmune disease, from hypersplenism, following placement of a mechanical heart valve, a blood transfusion, or a combination thereof.
  • the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, a vaso-occlusive crisis, a splenic sequestration, or a combination thereof.
  • the subject exhibits an increase in red blood cell count, a decrease in red blood cell distribution width, and a decrease in reticulocyte count.
  • the present disclosure provides methods of treating and/or preventing thrombophilia disorders in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the present disclosure provides methods of treating and/or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides method of treating and/or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition, and the subject exhibits one or more of: (a) an increase in prothrombin time; (b) an increase in activated partial thromboplastin time; and/or (c) a decrease in fibrinogen concentration.
  • the present disclosure provides methods of treating and/or preventing a venous thromboembolism in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing an arterial thrombosis in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of preventing an embolism in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the subject exhibits an increase in prothrombin time, an increase in activated partial thromboplastin time, and a decrease in fibrinogen concentration.
  • the 15-HEPE is in a free acid form, esterified form, or salt form.
  • the esterified form is an alkyl ester form or a triglyceride form.
  • the 15-HEPE comprises 15(S)-HEPE, 15(R)- HEPE, or combinations thereof.
  • the 15-HEPE is in the form of an ethyl ester (15- HEPE EE) or the 15-HEPE is in the form of an optically active ester (15(S)-HEPE EE).
  • the composition comprises about 10 mg to about 10,000 mg of 15-HEPE. In another embodiment, the composition comprises about 1 g to about 2 g of 15-HEPE. In another embodiment, the composition comprises about 2 g or more of 15-HEPE. In yet another embodiment, the composition is formulated to provide about 5 mg 15-HEPE per kg of bodyweight (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg to the subject. In various embodiments, the 15-HEPE represents at least about 90%, by weight, of all fatty acids present in the composition.
  • the 15-HEPE is orally administered.
  • the composition is administered in 1 to 8 capsules per day.
  • FIG. 1 is a schematic diagram of the study described in Example 1 and its duration.
  • FIG. 2 shows the body weight changes of the animals according to the study described in Example 1.
  • FIG. 3 shows the body weight on the day of sacrifice of the animals according to the study described in Example 1.
  • FIGs. 4A-4D show the kidney weight and kidney-to-body weight ratio of the animals on the day of sacrifice according to the study described in Example 1.
  • FIG. 5 shows the kidney hydroxyproline content for the animals according to the study described in Example 1.
  • FIGs. 6A-6G show the Sirius red staining of the animals according to the study described in Example 1.
  • FIG. 7 shows a plot depicting the Sirius red-positive area (%) of the animals according to the study described in Example 1.
  • FIGs. 8A-8D show the gene expression analyses for a-SMA, TIMP-1, TGF-b, and Collagen Type 1 of the animals according to the study described in Example 1, respectively.
  • FIG. 9 is a schematic diagram of the study described in Example 2 and its duration.
  • FIG. 10 shows the body weight changes of the animals according to the study described in Example 2.
  • FIG. 11 shows the body weight on the day of sacrifice of the animals according to the study described in Example 2.
  • FIGs. 12A and 12B show the liver weight and liver-to-body weight ratio of the animals on the day of sacrifice according to the study described in Example 2, respectively.
  • FIG. 13 shows the changes of serum ALT levels of the animals according to the study described in Example 2.
  • FIG. 14 shows the changes of serum total bilirubin levels of the animals according to the study described in Example 2.
  • FIGs. 15A-15E show the Sirius red staining and the fibrosis area of the animals according to the study described in Example 2.
  • FIG. 16 shows a plot depicting the Sirius red-positive area (%) of the animals according to the study described in Example 2.
  • FIG. 17A-17D show the gene expression analyses for a-SMA, TIMP-1 , TGF-b, and Collagen Type 1 of the animals according to the study described in Example 2, respectively.
  • FIGs. 18A and 18B show DS102 (15-HEPE) induced inhibition of TGF-b according the study described in Example 3.
  • FIGs. 18C-18G show DS102 effects on membrane translocation and degradation of Type I TGF-b receptor, Type II TGF-b receptor, Type III TGF-b receptor, EGFR and Caveolin-1 according to the study described in Example 3, respectively.
  • FIG. 19 is a schematic diagram of the study described in Example 4 and its duration.
  • FIG. 20 shows the baseline lipidomic profile of patients according to the study described in Example 4.
  • FIGs. 21A-21 C are plots depicting the changes in insulin, glucose, and free fatty acid levels in patients administered Epeleuton (15-HEPE) and placebo, respectively.
  • FIGs. 22A and 22B are plots depicting the changes in HOMA-IR and apido-IR levels in patients administered Epeleuton and placebo, respectively.
  • FIGs. 23A and 23B are plots depicting the changes in mean HbA1 C levels in patients and proportion of patients having HbA1 C levels > 6.5% at week 16 administered Epeleuton and placebo, respectively.
  • FIGs. 24A and 24B are plots depicting the mean change and median (%) change in the lipid profile of patients administered Epeleuton and placebo, respectively.
  • FIGs. 25A-25C are plots depicting the changes in cholesterol, triglyceride, and VLDL-C levels in patients administered Epeleuton and placebo, respectively.
  • FIG. 26 is a chart depicting the changes in hepatoxic lipid profile of patients administered DS102.
  • FIG. 27 are plots that validate that administration DS102 resolves NASFI using the OWL liver care test.
  • FIG. 28 are plots depicting the changes in hepatic fat content by CAP in patients administered DS102 and placebo.
  • FIGs. 29A-29C is a chart depicting that changes in inflammatory and pro- fibrotic protein levels in patients administered DS102 and placebo.
  • FIG. 30 are plots depicting the changes in the protein expression including of NASH development targets in patients administered DS102 and placebo.
  • FIG. 31 is a volcano plot depicting a reduction in inflammatory and pro- fibrotic proteins in patients administered DS102 and placebo.
  • FIG 32 are plots depicting the changes in vascular adhesion molecules in patients administered DS102 and placebo.
  • FIG 33 are plots depicting the changes in cardiovascular risk proteins in patients administered DS102 and placebo.
  • FIG 34 are plots depicting the changes in chemokines in patients administered DS102 and placebo.
  • FIG 35 are plots depicting the changes in tumor necrosis factor receptor superfamily members in patients administered DS102 and placebo.
  • FIGs. 36A and 36B are plots depicting the reduction in ALP levels in patients administered DS102 and placebo.
  • FIG. 37 is a boxplot of 15-HEPE ethyl ester trough plasma relative concentrations.
  • FIG. 38A-38C are bar graphs depicting mean change and standard deviation of red blood cell counts, red blood cell distribution width, and reticulocyte counts in rats administered 15(S)-FIEPE EE and placebo, respectively.
  • FIG. 39A-39C are bar graphs depicting mean change and standard deviation of prothrombin time, activated partial thromboplastin time, and fibrinogen concentration in rats administered 15(S)-FIEPE EE and placebo, respectively.
  • ⁇ 5-HERE is 15-hydroxy-eicosa-5Z,8Z,11Z,13E,17Z- pentaenoic acid.
  • 15-HEPE also occasionally referred to as 15-OHEPA, can be synthesized from eicosapentaenoic acid (“EPA,” eicosa-5, 8,11 ,14,17-pentaenoic acid or 20:5n-3), an omega-3 fatty acid according to methods known in the art.
  • 15-HEPE can be synthesized by exposure of EPA to the enzyme 15- lipoxygenase.
  • the term ⁇ 5-HERE refers to 15-HEPE in its free acid form (e.g., 15-hydroxy-eicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid) and/or a pharmaceutically acceptable ester, conjugate or salt thereof, or mixtures of any of the foregoing.
  • a derivative of 15-HEPE may be used instead, though this does not include any derivative compound missing the hydroxy group of 15-HEPE.
  • the 15-HEPE is used in the free acid form.
  • pharmaceutically acceptable esters or salts of 15-HEPE are used in the disclosure.
  • the 15-HEPE is in the form of a C1-4 alkyl ester such as methyl ester, ethyl ester, or a combination of methyl ester and ethyl ester.
  • the 15-HEPE is in a form of a glyceride (e.g., diglyceride or triglyceride).
  • the 15-HEPE is in the form of an ethyl ester (also referred to herein as E-15-HEPE, 15-HEPE EE, or ethyl-15-HEPE).
  • 15-HEPE is a chiral molecule and may be used in the (S)- or (R)- enantiomeric form, or as a racemic mixture. Used herein, “15-HEPE” includes all such forms, with no limitation as to stereospecificity. In another embodiment, the 15-HEPE comprises the (S) form: 15(S)-hydroxy-(5Z,8Z, 11 Z, 13E, 17Z)-eicosapentaenoic acid or the (R) from: 15(R)-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid.
  • the 15-HEPE EE comprises the (S) form: 15(S)-HEPE EE.
  • DS102 refers to 15-HEPE, 15-HEPE EE, a composition comprising 15-HEPE, a composition comprising 15-HEPE EE, or a composition comprising 15-HEPE and 15-HEPE EE.
  • Epeleuton refers to 15-HEPE, 15-HEPE EE, a composition comprising 15-HEPE, a composition comprising 15-HEPE EE, or a composition comprising 15-HEPE and 15-HEPE EE.
  • treating or “treatment” of a disease, disorder, or condition includes at least partially: (1) inhibiting the disease, disorder, or condition, i.e. , arresting or reducing the development of the disease, disorder, or condition or its clinical symptoms; or (2) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, or condition or its clinical symptoms.
  • prevention in relation to a given disease or disorder means: preventing the onset of disease development if none had occurred, preventing the disease or disorder from occurring in a subject that may be predisposed to the disorder or disease but has not yet been diagnosed as having the disorder or disease, and/or preventing further disease/disorder development if already present.
  • an “effective amount,” as used herein, refers to the amount of an active composition that is required to confer a therapeutic effect on the subject.
  • a “therapeutically effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease, disorder, or condition being treated. In some embodiments, the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • an “effective amount” for therapeutic uses is the amount of the composition including a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms without undue adverse side effects.
  • an appropriate “effective amount” in any individual case is determined using techniques, such as a dose escalation study.
  • the term “therapeutically effective amount” includes, for example, a prophylactically effective amount.
  • an “effective amount” of a compound disclosed herein, such as a compound of Formula (A) or Formula (I) is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects.
  • an effect amount” or “a therapeutically effective amount” varies from subject to subject, due to variation in metabolism, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.
  • pharmaceutically acceptable in the present context means that the substance in question does not produce unacceptable toxicity to the subject or interaction with other components of the composition.
  • compositions of the disclosure comprise 15-HEPE as an active ingredient.
  • pharmaceutically acceptable in the present context means that the substance in question does not produce unacceptable toxicity to the subject or interaction with other components of the composition.
  • the 15-HEPE is in the form of an ester (also referred to herein as E-15-HEPE, ethyl-15-HEPE, or 15-HEPE EE).
  • the 15-HEPE comprises a Ci - Cs alkyl ester of 15-HEPE.
  • the 15-HEPE comprises 15-HEPE methyl ester, 15-HEPE propyl ester, or 15-HEPE butyl ester.
  • the 15-HEPE comprises the optically active 15(S)-Hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid. This isomer may be used in any of the forms discussed above.
  • the 15-HEPE comprises lithium 15-HEPE, mono, di- or triglyceride 15-HEPE or any other ester or salt of 15-HEPE, or the free acid form of 15-HEPE.
  • the disclosure provides pharmaceutical compositions, for example orally deliverable compositions, comprising 15-
  • the compositions comprise a therapeutically effective amount of 15- HEPE.
  • the pharmaceutical composition comprises about 0.1 % to about 99%, about 1 % to about 95%, about 5% to about 90%, by weight, of 15-HEPE.
  • composition and the phrase “pharmaceutical composition” are used interchangeably.
  • the pharmaceutical composition comprises about at least about 70%, at least about 80% or at least about 90%, by weight, of 15-HEPE. In one embodiment, the pharmaceutical composition comprises at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, by weight, of 15-HEPE.
  • 15-HEPE is present in a composition of the disclosure in an amount of about 1 mg to about 10,000 mg, about 25 mg to about 7500 mg, about 25 mg to about 5000 mg, about 50 mg to about 5000 mg, about 50 mg to about 3000 mg, about 75 mg to about 2500 mg, or about 100 mg to about 1000 mg, for example about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg
  • 15-HEPE present in a composition of the disclosure comprises at least about 90%, by weight, 15-HEPE (as the term ⁇ 5-HERE” is defined and exemplified herein).
  • 15-HEPE compositions can comprise even higher purity 15- HEPE, for example at least about 95%, by weight, 15-HEPE or at least about 97%, by weight, 15-HEPE, wherein the 15-HEPE is any form of 15-HEPE as set forth herein.
  • the purity of 15-HEPE can further be defined (e.g., impurity profile) by any of the descriptions of 15-HEPE provided herein.
  • the amounts of the 15-HEPE in the pharmaceutical composition are discussed.
  • the nature of the essential fatty acids and their synthesis is such that the 15-HEPE composition may include moieties from other essential fatty acids in the essential fatty acid metabolic cascade.
  • a composition of the disclosure contains not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1 %, or not more than about 0.5%, by weight of other omega-3 fatty acids including alpha linolenic acid, stearidonic acid, docosahexaenoic acid (DHA) or derivatives thereof. In other embodiments there is substantially no, or no such other omega-3 fatty acids present.
  • DHA docosahexaenoic acid
  • 15-HEPE represents at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%, by weight, of all fatty acids present in a composition of the disclosure.
  • the salt form of 15-HEPE present in a composition of the invention comprises at least 90%, by weight, of the salt form of 15-HEPE.
  • Compositions containing the salt form of 15-HEPE can comprise even higher purity, for example at least 91 % by weight, at least 92% by weight, at least 93% by weight, at least 94% by weight, at least 95% by weight, at least 96% by weight or at least 97% by weight of the salt form of 15-HEPE.
  • eicosapentaenoic acid from the synthesis of the 15-HEPE.
  • the present disclosure provides a pharmaceutical composition comprising 15-HEPE or derivative thereof encapsulated in a capsule shell.
  • the composition is administered to the subject in an amount sufficient to provide up to about 1 g, about 2 g, about 3 g, about 4 g, about 5 g, about 6 g, about 7 g, about 8 g, about 9 g, or about 10 g of 15-HEPE E or a derivative thereof per day.
  • the composition is administered to the subject in an amount sufficient to provide about 4 g to about 8 g, about 1 g to about 2 g, about 2 g to about 4 g, about 3 g to about 8 g, about 4 g to about 6 g of 15-HEPE or a derivative thereof per day. In one embodiment, about 500 mg to about 1 g of 15-HEPE or derivative thereof is encapsulated in the capsule shell.
  • the capsule shell comprises gelatin (for example, Gelatin RXL or lime bone gelatin with a lower molecular weight).
  • the capsule shell comprises Gelatin RXL that has been treated by proteolytic enzyme to cut the gelatin pattern and effectively decrease its molecular weight.
  • the pharmaceutical composition comprises 15-HEPE esters of D-Sorbitol and 1 ,4-sorbitan.
  • the capsule shell comprises (a) gelatin and (b) plasticizers selected from one or more of D-Sorbitol and 1 ,4- sorbitans.
  • the gelatin is as described in U.S. 7,485,323, and is hereby incorporated by reference herein in its entirety.
  • the plasticizer comprises 1 ,4-sorbitans in an amount from about 20% to about 30%, for example, about 24% to about 28%, 24%, or 28% (on a dry basis), and a D-Sorbitol content of about 30% to about 50%, for example, about 35% to about 45% (on a dry basis).
  • the capsule is a hard gelatin capsule. In another embodiment, the capsule is a soft gelatin capsule.
  • the capsule shell comprises modified starch, carrageenan (e.g., extract of red seaweed), disodium phosphate, glycerol and/or sorbitol.
  • the capsule shell further comprises water.
  • the capsule shell is stable up to a temperature of about 65°C and/or pH of about 12.
  • the capsule shell is odorless and has a neutral color (e.g., colorless, white, or transparent).
  • the capsule shell further comprises glycerol, purified water, titanium dioxide, medium chain triglycerides and lecithin.
  • the pharmaceutical composition further comprises one or more additional active agent(s).
  • the pharmaceutical composition comprises an amount of the additional active agent that is less than the generally recognized therapeutically effective amount for that agent. In one embodiment, the pharmaceutical composition comprises an amount of the additional active agent that is equal to or greater than the generally recognized therapeutically effective amount for that agent. If an additional active agent is to be used, the 15- HEPE can be co-formulated as a single dosage unit or can be formulated as two to a plurality of dosage units for coordinated, combination or concomitant administration.
  • EPA itself has beneficial properties in treating fatty liver disease and/or cardiovascular disease and it is possible to combine the 15-HEPE with EPA in an alternative embodiment.
  • 15-HEPE and one or more active agent(s) are present in a composition of the disclosure, or are co-administered in a weight ratio of 15-HEPE: additional agent of about 1 :1000 to about 1000:1 , about 1 :500 to about 500: 1 , about 1 : 100 to about 100: 1 , about 1 :50 to about 50: 1 , about 1 :25 to about 25: 1 , about 1 : 10 to about 10:1 , about 1 :5 to about 5: 1 , about 1 :4 to about 4: 1 about 1 :3 to about 3: 1 , about 1 :2 to about 2: 1 or about 1 :1.
  • a composition for use in accordance with the disclosure can be formulated as one or more dosage units.
  • dose unit and “dosage unit” herein refer to a portion of a composition that contains an amount of a therapeutic agent suitable for a single administration to provide a therapeutic effect.
  • dosage units may be administered one to a plurality (e.g., 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.
  • compositions of the disclosure are in the form of orally deliverable dosage forms or units.
  • suitable dosage forms include tablets (e.g., suspension tablets, bite suspension tablets, rapid dispersion tablets, chewable tablets, etc), caplets, capsules (e.g., a soft or a hard gelatin capsule or HPMC capsule), lozenges, sachets, cachets, troches, pellets, suspension, elixirs, syrups or any other solid dosage form reasonably adapted for oral administration.
  • oral delivery and “oral administration” herein include any form of delivery wherein the agent or composition is placed in the mouth of the subject under treatment, whether swallowed or not. This therefore includes buccal and sublingual administration, as well as esophageal administration.
  • compositions of the disclosure can also be formulated for rectal, topical, or parenteral (e.g., subcutaneous, intramuscular, intravenous and intradermal or infusion) delivery.
  • parenteral e.g., subcutaneous, intramuscular, intravenous and intradermal or infusion
  • compositions of the disclosure can be in the form of liquid dosage forms or dose units to be imbibed directly or they can be mixed with food or beverage prior to ingestion.
  • suitable liquid dosage forms include solutions, suspensions, elixirs, syrups, liquid aerosol formulations, and the like.
  • compositions of the disclosure comprise one or more pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipient herein means any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.
  • a pharmaceutical composition according to the present disclosure may comprise one or more of: antioxidants, surfactants, preservatives, flavoring agents, co-solvents, viscosity aids, suspension aids, and lipophilic phases.
  • the pharmaceutical composition comprises one or more antioxidants such as ascorbic acid, palmitic acid, ascorbyl palmitate, a- tocopherol, idebenone, ubiquinone, ferulic acid, coenzyme Q10, lycopene, green tea, catechins, epigallocatechin 3-gallate (EGCG), green tea polyphenols (GTP), silymarin, coffeeberry, resveratrol, grape seed, pomegranate extracts, genisten, pycnogenol, niacinamide, and the like.
  • antioxidants such as ascorbic acid, palmitic acid, ascorbyl palmitate, a- tocopherol, idebenone, ubiquinone, ferulic acid, coenzyme Q10, lycopene, green tea, catechins, epigallocatechin 3-gallate (EGCG), green tea polyphenols (GTP), silymarin, coffeeberry, resveratrol, grape seed, pome
  • the pharmaceutical composition comprises about 0.01 wt.% to about 2 wt.% of an antioxidant, for example about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.11 wt.%, about 0.12 wt.%, about 0.13 wt.%, about 0.14 wt.%, about 0.15 wt.%, about 0.16 wt.%, about 0.17 wt.%, about 0.18 wt.%, about 0.19 wt.%, about 0.2 wt.%, about 0.21 wt.%, about 0.22 wt.%, about 0.23 wt.%, about 0.24 wt.%, about 0.25 wt.%,
  • compositions and formulations disclosed herein may be used in the treatment and/or prevention of a hematologic disorder in a subject in need thereof.
  • the hematologic disorder is a hemoglobin disorder, a red blood cell disorder, hemolytic anemia, a thrombophilia disorder, a venous thromboembolism, an arterial thrombosis, an embolism, or a combination thereof.
  • the methods comprise administering to the subject 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE).
  • compositions and formulations disclosed herein may be used in the treatment and/or prevention of hematologic disorders, hemoglobin disorders, and/or red blood cell disorders.
  • Hematologic disorders refer to a class of conditions that affect the blood and blood-forming organs. While hemoglobin disorders and red blood cells disorders are similar to hematologic disorders, both hemoglobin disorders and red blood cell disorders define a narrower class of disorders. Specifically, hemoglobin disorders refer to defects in hemoglobin, the oxygen transport protein in red blood cells. And red blood cell disorders, refer to defects in the red blood cell itself.
  • risk factors of hematologic disorders, hemoglobin disorders, and red blood cell disorders include reduced red blood cell counts, increased red blood cell distribution widths, and increased reticulocyte counts.
  • Non-limiting examples of hematologic disorders, hemoglobin disorders, and red blood cell disorders include anemia (nutritional anemias and non-nutritional anemias, inherited hemolytic anemia such as sickle cell disease, sickle cell anemia, b-thalassemia, and hereditary spherocytosis, acquired hemolytic anemia such as secondary to infection, medication, hematological malignancy, autoimmune disease, hypersplenism, mechanical heart valves, and blood transfusions, Fanconi anemia, iron deficiency anemia), blood cancer (lymphoma, leukemia, and myeloma), coagulation defects (thrombophilia, hemophilia, Von Willebrand disease, and thrombocytopenia), folate deficiency, B12 deficiency, and myelodysplastic syndrome.
  • sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration.
  • the subject has a reduced red blood cell count of at least 10% below normal, at least 15% below normal, at least 20% below normal, at least 25% below normal, or at least 30% below normal.
  • the subject has an increased red blood cell distribution width, where the red blood cells vary in size by about 15-20%, by about 20-25%, by about 25-30%, by about 30-35%, or by about 35-40%.
  • the subject has an increased reticulocyte count of at least about 5% of the total amount of red blood cells, at least about 10% of the total amount of red blood cells, at least about 15% of the total amount of red blood cells, or at least about 20% of the total amount of red blood cells.
  • the subject has elevated baseline triglyceride levels of about 135 mg/dL to about 2000 mg/dL, for example about 135 mg/dL to about 500 mg/dL, about 150 mg/dL to about 500 mg/dL, about 200 mg/dL to about 499 mg/dL, about 200 mg/dL to ⁇ 500 mg/dL, about 300 mg/dL to about 1800 mg/dL, about 500 mg/dl_ to about 1500 mg/dl_, about 500 mg/dl_ to about ⁇ 2000 mg/dl_, or about 500 mg/dl_ to about 2000 mg/dl_.
  • the subject has a fasting baseline triglyceride level of about 50 mg/dL to about 2000 mg/dL, for example about 50 mg/dL to about 1500 mg/dL, about 80 mg/dL to about 1500 mg/dL, about 50 mg/dL to about 190 mg/dl, about 80 mg/dL to about 190 mg/dl, about 190 mg/dL to about 250 mg/dL, about 250 mg/dL to about 1400 mg/dL, about 500 mg/dL to about 1200 mg/dL, about 500 mg/dL to about 1500 mg/dL, about 500 mg/dL to about ⁇ 2000 mg/dL, or about 500 mg/dL to about 2000 mg/dL.
  • a fasting baseline triglyceride level of about 50 mg/dL to about 2000 mg/dL, for example about 50 mg/dL to about 1500 mg/dL, about 80 mg/dL to about 1500 mg/dL, about 50 mg/dL to about 190 mg/dl,
  • the subject has a fasting baseline triglyceride level of about 80 mg/dL to about 1400 mg/dL.
  • the subject or subject group has a baseline triglyceride level (or median baseline triglyceride level in the case of a subject group), fed or fasting, of about 50 mg/dL, about 55 mg/dL, about 60 mg/dL, about 65 mg/dL, about 70 mg/dL, about 75 mg/dL, about 80 mg/dL, about 85 mg/dL, about 90 mg/dL, about 95 mg/dL, about 100 mg/dL, about 105 mg/dL, about 110 mg/dL, about 115 mg/dL, about 120 mg/dL, about 125 mg/dL, about 130 mg/dL, about 135 mg/dL, about 140 mg/dL, about 145 mg/dL, about 150 mg/dL, about 155 mg/dL, about 160 mg/dL, about 165 mg/d
  • the subject or subject group has a baseline triglyceride level (or median baseline triglyceride level in the case of a subject group), fed or fasting, greater than or equal to 80 mg/dL, greater than or equal to about 100 mg/dL, greater than or equal to about 120 mg/dL greater than or equal to about 150 mg/dL, greater than or equal to about 175 mg/dL, greater than or equal to about 250 mg/dL, or greater than equal to about 500 mg/dL, for example about 190 mg/dL to about 250 mg/dL, about 80 mg/dL to about 190 mg/dL, about 250 mg/dL to about 1400 mg/dL, about 200 mg/dL to about 500 mg/dL, about 300 mg/dL to about 1800 mg/dL, about 500 mg/dL to about 1500 mg/dL, about 80 mg/dL to about 1500 mg/dL, about 80 mg/dL to less than about 2000 mg/dL, about 80 mg/dL to
  • the subject has an elevated baseline blood pressure of at least about 100 mmHg, at least about 115 mmHg, at least about 120 mmHg, at least about 125 mmHg, at least about 130 mmHg, at least about 135 mmHg, at least about 140 mmHg, at least about 145 mmHg, at least about 150 mmHg, at least about 155 mmHg, at least about 160 mmHg, at least about 165 mmHg, or at least about 170 mmHg.
  • the subject has elevated baseline fasting glucose levels of at least about 100 mg/dL, at least about 115 mg/dL, at least about 120 mg/dL, at least about 125 mg/dL, at least about 130 mg/dL, at least about 135 mg/dL, at least about 140 mg/dL, at least about 145 mg/dL, at least about 150 mg/dL, at least about 155 mg/dL, at least about 160 mg/dL, at least about 165 mg/dL, or at least about 170 mg/dL.
  • the subject has reduced baseline HDL-C levels of less than about 60 mg/dL, less than about 55 mg/dL, less than about 50 mg/dl, less than about 45 mg/dL, less than about 40 mg/dL, less than about 35 mg/dL, less than about 30 mg/dL, less than about 25 mg/dL, less than about 20 mg/dL, less than about 15 mg/dL, less than about 10 mg/dL, or less than about 5 mg/dL.
  • the present disclosure provides a method of treating and/or preventing hematologic disease in a subject, the method comprising administering to the subject 15-HEPE.
  • the method comprises administering to the subject up to about 8g of 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of treating and/or preventing hematologic disease in a subject, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of 15-HEPE or a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15- HEPE) or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15- HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides a method of treating and/or preventing hematologic disease in a subject, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for hematologic disease before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • the method further comprises determining a baseline red blood cell count, a red blood cell distribution width, and/or a reticulocyte count of the subject before administering the 15-HEPE.
  • the subject exhibits an increase in the red blood cell count, a decrease in the red blood cell distribution width, and/or a decrease in the reticulocyte count.
  • the method comprises administering to the subject up to about 8g of 15-HEPE or a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15- HEPE) or a composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for hematologic disease before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • the method further comprises determining a baseline red blood cell count, a red blood cell distribution width, and/or a reticulocyte count of the subject before administering the 15-HEPE.
  • the subject exhibits an increase in the red blood cell count, a decrease in the red blood cell distribution width, and/or a decrease in the reticulocyte count.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hemoglobin disorder in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15- HEPE) or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15- HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hemoglobin disorder in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15- HEPE) or a composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for the hemoglobin disorder before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • the method further comprises determining a baseline red blood cell count, a red blood cell distribution width, and/or a reticulocyte count of the subject before administering the 15-HEPE.
  • the subject exhibits an increase in the red blood cell count, a decrease in the red blood cell distribution width, and/or a decrease in the reticulocyte count.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE ora composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE ora composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for the red blood cell disorder before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • the method further comprises determining a baseline red blood cell count, a red blood cell distribution width, and/or a reticulocyte count of the subject before administering the 15-HEPE.
  • the subject exhibits an increase in the red blood cell count, a decrease in the red blood cell distribution width, and/or a decrease in the reticulocyte count.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder, a hemoglobin disorder, and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder, a hemoglobin disorder, and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for a hematologic disorder, a hemoglobin disorder, and/or a red blood cell disorder before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • at least one risk factor is reduced red blood cell counts, increased red blood cell distribution widths, and/or increased reticulocyte counts.
  • the subject exhibits one or more of an increase in red blood cell count, a decrease in red blood cell distribution width and/or a reduced reticulocyte count.
  • the present disclosure provides methods of treating and/or preventing a hematologic disorder, a hemoglobin disorder, and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the hematologic disorder, the hemoglobin disorder, and/or the red blood cell disorder are selected from the group consisting of inherited hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.
  • the method further comprises determining that the subject has at least one risk factor for inherited hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and/or myelodysplastic syndrome before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • the method further comprises determining a baseline red blood cell count, a red blood cell distribution width, and/or a reticulocyte count of the subject before administering the 15-HEPE.
  • the subject exhibits an increase in the red blood cell count, a decrease in the red blood cell distribution width, and/or a decrease in the reticulocyte count.
  • the present disclosure provides methods of treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15- HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15- HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15- HEPE. In some embodiments, the method further comprises determining that the subject has at least one risk factor for hemolytic anemia before administering the 15- HEPE, or a composition comprising 15-HEPE. In some embodiments, at least one risk factor is reduced red blood cell counts, increased red blood cell distribution widths, and/or increased reticulocyte counts. In some embodiments, the subject exhibits one or more of an increase in red blood cell count, a decrease in red blood cell distribution width and/or a reduced reticulocyte count.
  • the present disclosure provides methods of treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the hemolytic anemia is inherited hemolytic anemia or acquired hemolytic anemia.
  • the inherited hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, b-thalassemia, and hereditary spherocytosis.
  • the acquired hemolytic anemia is selected from the group consisting of secondary to infection, medication, hematological malignancy, autoimmune disease, hypersplenism, mechanical heart valves, and blood transfusions.
  • the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration.
  • the subject exhibits an increase in red blood cell count, a decrease in red blood cell distribution width, and a decrease in reticulocyte count following administration.
  • the present disclosure provides methods of treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the hemolytic anemia is inherited hemolytic anemia or acquired hemolytic anemia.
  • the inherited hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, b-thalassemia, and hereditary spherocytosis.
  • the method further comprises determining that the subject has at least one risk factor for inherited hemolytic anemia or acquired hemolytic anemia before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • the acquired hemolytic anemia is selected from the group consisting of secondary to infection, medication, hematological malignancy, autoimmune disease, hypersplenism, mechanical heart valves, and blood transfusions.
  • the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration.
  • the method further comprises determining a baseline red blood cell count, a red blood cell distribution width, and/or a reticulocyte count of the subject before administering the 15-HEPE.
  • the subject exhibits an increase in the red blood cell count, a decrease in the red blood cell distribution width, and/or a decrease in the reticulocyte count.
  • compositions and formulations disclosed herein may further be used in the treatment and/or prevention of a thrombophilia disorder.
  • thrombophilia disorders refer to a class of conditions characterized by abnormal blood coagulation that increases the risk of thrombosis.
  • risk factors of thrombophilia disorders include decreased prothrombin times, decreased activated partial thromboplastin times, and increased fibrinogen concentration.
  • the subject has a reduced prothrombin time of at least 10% below normal, at least 15% below normal, at least 20% below normal, at least 25% below normal, or at least 30% below normal.
  • the subject has a reduced activated partial thromboplastin time of at least 10% below normal, at least 15% below normal, at least 20% below normal, at least 25% below normal, or at least 30% below normal.
  • the subject has an increased fibrinogen concentration of at least 10% below normal, at least 15% below normal, at least 20% below normal, at least 25% below normal, or at least 30% below normal.
  • the present disclosure provides methods of treating and/or preventing a thrombophilia disorder in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15- HEPE) or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15- HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a thrombophilia disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE ora composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for a thrombophilia disorder before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • at least one risk factor is decreased prothrombin times, decreased activated partial thromboplastin times, and/or increased fibrinogen concentration.
  • the subject exhibits one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time and/or a decrease in fibrinogen concentration after administering the 15-HEPE, or a composition comprising 15-HEPE.
  • the present disclosure provides methods of treating and/or preventing a venous thromboembolism in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15- HEPE) or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15- HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing a venous thromboembolism in a subject in need thereof, the method comprising administering to the subject 15-HEPE ora composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for venous thromboembolism before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • at least one risk factor is decreased prothrombin times, decreased activated partial thromboplastin times, and/or increased fibrinogen concentration.
  • the subject exhibits one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time and/or a decrease in fibrinogen concentration.
  • the present disclosure provides methods of treating and/or preventing an arterial thrombosis in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15- HEPE) or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15- HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of treating and/or preventing an arterial thrombosis in a subject in need thereof, the method comprising administering to the subject 15-HEPE ora composition comprising 15-HEPE.
  • the method further comprises determining that the subject has at least one risk factor for an arterial thrombosis before administering the 15-HEPE, or a composition comprising 15-HEPE.
  • at least one risk factor is decreased prothrombin times, decreased activated partial thromboplastin times, and/or increased fibrinogen concentration.
  • the subject exhibits one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time and/or a decrease in fibrinogen concentration.
  • the present disclosure provides methods of preventing an embolism in a subject in need thereof, the method comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • the present disclosure provides methods of preventing an embolism in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining that the subject has at least one risk factor for developing an embolism before administering the 15-HEPE, or a composition comprising 15-HEPE. In some embodiments, at least one risk factor is decreased prothrombin times, decreased activated partial thromboplastin times, and/or increased fibrinogen concentration. In some embodiments, the subject exhibits one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time and/or a decrease in fibrinogen concentration.
  • the present disclosure provides a method of treating and/or preventing endothelial dysfunction in a subject, the method comprising administering to the subject 15-HEPE.
  • the method comprises administering to the subject up to about 8g of 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of treating and/or preventing endothelial dysfunction in a subject, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method comprises administering to the subject up to about 8g of 15-HEPE or a composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of treating, preventing, or reducing cell stress apoptosis, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the subject exhibits a reduction in markers associated with apoptosis such as proteins from the Bcl-2 family, activated fragments of caspases and/or cleaved PARP-1 .
  • the present disclosure provides a method of treating, preventing, and/or reducing inflammation, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the subject exhibits a reduction T cell activation, B cell activation, and/or chemotaxis.
  • compositions and formulations disclosed herein may be used in the treatment or prevention of cardiovascular disease or disorder.
  • the cardiovascular disease or disorder is selected from: dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, primary hypercholesterolemia, primary hyperlipidemia, common primary hyperlipidemia, common hypercholesterolemia, familial hyperlipidemia, familial primary hyperlipidemia, familial hypercholesterolemia, familial hypertriglyceridemia, familial combined hyperlipidemia, familial defective apolipoprotein b-100, secondary hyperlipidemia, mixed hyperlipidemia, cardiovascular disease, residual cardiovascular risk, prevention of atherosclerotic plaque formation/progression, microvascular disease, macrovascular disease, atherosclerosis, coronary atherosclerosis, diastolic dysfunction, reduction of cardiovascular risk, prevention of major coronary events, prevention of major adverse cardiovascular events, prevention of ischemic events, secondary/primary prevention of cardiovascular events, prevention of cardiovascular death, myocardial infarction, stroke, angina, restoration of normal endot
  • Non limiting examples of microvascular disease include retinopathy, nephropathy, and neuropathy.
  • Non-limiting examples of macrovascular disease include stroke, peripheral vascular disease, limb ischemia, and heart disease.
  • the subject has non-alcoholic liver disease, cholestatic liver disease, kidney disease, or metabolic syndrome. Any of the aforementioned examples of cardiovascular disease may also refer to non-limiting examples of cardiometabolic disease.
  • the present disclosure provides a method of treating and/or preventing cardiovascular disease in a subject, the method comprising administering to the subject 15-HEPE.
  • the present disclosure provides a method of treating and/or preventing cardiovascular disease in a subject, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the present disclosure provides a method of preventing hematologic disorder in a subject having diabetes (e.g., Type I or Type II), the method comprising administering to the subject 15-HEPE.
  • the method further comprises determining the subject has diabetes before administering the 15-HEPE.
  • the method comprises administering to the subject about 10 mg per weight of the subject (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of preventing hematologic disorder in a subject having diabetes (e.g., Type I or Type II), the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method further comprises determining the subject has diabetes before administering the 15-HEPE or composition comprising 15-HEPE.
  • the method comprises administering to the subject about 10 mg per weight of the subject (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE or composition comprising 15- HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of treating and/or preventing hematologic disorder in a subject having cardiovascular disease, the method comprising administering to the subject 15-HEPE.
  • the method further comprises determining the subject has cardiovascular before administering the 15-HEPE.
  • the method comprises administering to the subject about 10 mg per weight of the subject (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of treating and/or preventing hematologic disorder in a subject having cardiovascular disease, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • the method further comprises determining the subject has cardiovascular before administering the 15- HEPE or composition comprising 15-HEPE.
  • the method comprises administering to the subject about 10 mg per weight of the subject (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE or composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of treating and/or preventing hematologic disorder in a subject having high blood pressure, the method comprising administering to the subject 15-HEPE.
  • the method further comprises determining the subject has high blood pressure before administering the 15-HEPE.
  • the subject has a high blood pressure of at least about 130 mmHg, at least about 135 mmHg, at least about 140 mmHg, at least about 145 mmHg, at least about 150 mmHg, at least about 155 mmHg, at least about 160 mmHg, at least about 165 mmHg, or at least about 170 mmHg.
  • the method comprises administering to the subject about 10 mg/kg, about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • the present disclosure provides a method of treating and/or preventing hematologic disorder in a subject having high blood pressure, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining the subject has high blood pressure before administering the 15-HEPE or composition comprising 15-HEPE.
  • the subject has a high blood pressure of at least about 130 mmHg, at least about 135 mmHg, at least about 140 mmHg, at least about 145 mmHg, at least about 150 mmHg, at least about 155 mmHg, at least about 160 mmHg, at least about 165 mmHg, or at least about 170 mmHg.
  • the method comprises administering to the subject about 10 mg/kg, about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15- HEPE or composition comprising 15-HEPE.
  • the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • compositions and formulations disclosed herein may also be used for reducing cytokines and/or chemokines in a subject having cardiovascular disease, and/or hematologic disease.
  • Non-limiting cytokines and/or chemokines include a- smooth muscle action (a-SMA), metallopeptidase inhibitor-1 (TIMP-1 ), transforming growth factor beta-b (TGF-b), and Collagen Type 1 .
  • the subject or subject group upon treatment with a composition of the present invention, exhibits one or more of the following outcomes: [0158] (a) no increase or a reduction in serum aminotransferase (ALT) and/or aspartate aminotransferase (AST) levels relative to baseline, placebo control, and/or untreated patient;
  • HbA1C hemoglobin A1C
  • IR IR
  • VLDL-C VLDL-C
  • HDL-C levels relative to baseline, placebo control, and/or untreated patient
  • TNF-like ligand 1A TNF-like ligand 1A
  • TNF-a tumor necrosis factor
  • TGF-b transforming growth factor-b
  • a-SMA a-smooth muscle action
  • TRSF11A tumor necrosis factor ligand superfamily member 11A
  • methods of the present invention comprise measuring baseline levels of one or more markers or parameters set forth in (a) - (bo) above prior to dosing the subject or subject group.
  • the methods comprise administering a composition as disclosed herein to the subject after baseline levels of one or more markers or parameters set forth in (a) - (bo) are determined, and subsequently taking an additional measurement of said one or more markers.
  • the subject or subject group upon treatment with a composition of the present invention, for example, over a period of about 1 to about 12 weeks, about 1 to about 8 weeks, or about 1 to about 4 weeks, the subject or subject group exhibits any 5 or more of, any 10 or more of, any 15 or more of, any 20 or more of, any 25 or more of, any 30 or more of, any 35 or more of, any 40 or more of, any 45 or more of, any 50 or more of, any 55 or more of, any 60 or more of, any 65 or more of, any 70 or more of, any 75 or more of, any 80 or more of, any 85 or more of, any 90 or more of, or all 91 of outcomes (a) - (bo) described immediately above.
  • the subject or subject group upon treatment with a composition of the present invention, exhibits one or more of the following outcomes: [0252] (a) no increase or a reduction in serum ALT and/or AST levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (m) no increase or a reduction in VLDL-C levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • kidney hydroxyproline levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (x) no increase or a reduction in IL-13 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (y) no increase or a reduction in IL-6 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (z) no increase or a reduction IL-1 b levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (bb) no increase or a reduction in TGF-b levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (cc) no increase or a reduction in a-SMA of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (dd) no increase or a reduction in TNRSF11A of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (ff) no increase or a reduction in AOC3 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (gg) no increase or a reduction in LILBR1 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (hh) no increase or a reduction in TR levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (jj) no increase or a reduction in SAA4 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient; [0288] (kk) no increase or a reduction in MCP-1 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about
  • (nn) no increase or a reduction in DPP4 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (qq) no increase or a reduction in a NFS of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (rr) no increase or a reduction in FIB-4 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (ss) no increase or a reduction in liver stiffness of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (tt) no increase or a reduction in hepatic fat content of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (vv) no increase or a reduction in blood pressure of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (ww) no increase or a reduction in an ELF score of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (zz) no increase or a reduction in collagen production in lung and/or dermal fibroblasts score of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (zz) an increase in lung and/or dermal fibroblast viability of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • no change or a reduction in IL-8 levels score of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • no change or a reduction in IL-11 levels score of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • no change or a reduction in T and/or B cell activation at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • an increase in vascular adhesion molecules of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • an increase in cardiovascular risk proteins of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • (bm) no change or a reduction in red blood cell distribution of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% relative to baseline, placebo control, and/or untreated patient;
  • Figure 1 depicts the study design from surgery and treatment to day 14 of the study.
  • test Substances The test substances for this study were DS109 (15- FIETrE) and DS102 (15-HEPE). To prepare dosing solutions of each substance, DS109 was first weighed and then dissolved in a vehicle of 0.5% hydroxypropyl methyl cellulose (HPMC) and DS102 was diluted in a vehicle of 0.5% HPMC.
  • HPMC hydroxypropyl methyl cellulose
  • UUO Surgery On day 0 of the study, UUO surgery was performed on mice under pentobarbital sodium anesthesia. The mouse’s hair was first shaved and then abdomen cut open to exteriorize the mouse’s left ureter. The ureter was ligated 4-0 nylon sutures at two points. The mouse’s peritoneum and skin were then closed with sutures, and the mouse transferred to a clean cage until recovered from the anesthesia. Sham operated mice had their left ureter exposed but not ligated.
  • DS109 and DS102 were administered to the mice orally in a volume of 10 milliliters (mL)/kilogram (Kg).
  • Treatment Doses DS109 was administered at 3 dose levels of 5, 50, and 250 milligram (mg)/Kg once daily from Day 0 to Day 13 of the study. DS102 was administered at 2 doses levels of 50 and 500 mg/kg once daily from Day 0 to Day 13 of the study.
  • mice Seven-week-old female C57BL/6 mice (i.e. , animals) were obtained from Japan SLC, Inc. (Japan) and were housed and fed with a normal diet (CE-2; CLEA Japan, Japan) under controlled conditions. The animals were maintained in a specific-pathogen free (SPF) facility under controlled conditions of temperature (e.g., 23 ⁇ 2°C), humidity (e.g., 45 ⁇ 10%), lighting (e.g., 12-hour artificial light and dark cycles; light from 8:00 to 20:00) and air exchange. A high pressure was maintained in the experimental room to prevent contamination of the facility. The animals were housed in TPX cages (CLEA Japan) with a maximum of 4 mice per cage.
  • SPPF specific-pathogen free
  • Sterilized Paper-Clean (Japan SLC) was used for bedding and replaced once a week.
  • Sterilized solid normal diet was provided ad libitum, being placed in a metal lid on the top of the cage. Pure water was also provided ad libitum from a water bottle equipped with a rubber stopper and a sipper tube. Water bottles were replaced once a week, cleaned, and sterilized in an autoclave and reused. Mice were identified by ear punch and each cage was labeled with a specific identification code.
  • kidney Biochemistry To quantify kidney hydroxy pro line content, frozen left kidney samples were processed by an alkaline-acid hydrolysis method as follows: kidney samples were dissolved in 2 normal (N) sodium hydroxide (NaOH) at 65°C and autoclaved at 121 °C for 20 minutes. The lysed samples (400 pL) were acid-hydrolyzed with 400 pl_ of 6N hydrochloride acid (HCI) at 121 °C for 20 minutes, and neutralized with 400 mI_ of 4N NaOH containing 10 mg/mL of activated carbon.
  • N normal sodium hydroxide
  • HCI 6N hydrochloride acid
  • An AC buffer e.g., 2.2M acetic acid/0.48M citric acid, 400 mI_
  • AC buffer e.g., 2.2M acetic acid/0.48M citric acid, 400 mI_
  • a standard curve of hydroxyproline was constructed with serial dilutions of trans-4-hydroxy-L-proline (Sigma-Aldrich, USA) starting at 16 pg/mL.
  • the prepared samples and standards e.g., each 400 pL
  • 400 mI_ chloramine T solution (Wako Pure Chemical Industries, Japan) and incubated for 25 minutes at room temperature.
  • the samples were then mixed with Ehrlich's solution (e.g., 400 mI_) and heated at 65°C for 20 minutes to develop the color.
  • kidney hydroxyproline was calculated from the hydroxyproline standard curve. Protein concentrations of kidney samples were determined using a bicinchoninic (BCA) protein assay kit (Thermo Fisher Scientific, USA) and used to normalize the calculated hydroxyproline values. Kidney hydroxyproline contents were expressed as microgram (pg) per mg protein.
  • BCA bicinchoninic
  • RNA Total ribonucleic acid
  • RNAiso Troponucleic acid
  • MgCh magnesium chloride
  • mRNA microRNA
  • the study design included the following study groups:
  • Group 1 Sham Control
  • Group 2 Eight UUO mice were orally administered vehicle [0.5% HPMC] in a volume of 10 mL/kg once daily from Day 0 to Day 13.
  • Group 3 Eight UUO mice were orally administered vehicle supplemented with DS109 at a dose of 5 mg/kg once daily from Day 0 to Day 13.
  • Group 4 (DS109 Middle): Eight UUO mice were orally administered vehicle supplemented with DS109 at a dose of 50 mg/kg once daily from Day 0 to Day 13.
  • Group 5 (DS109 High): Eight UUO mice were orally administered vehicle supplemented with DS109 at a dose of 250 mg/kg once daily from Day 0 to Day 13.
  • Group 6 Eight UUO mice were orally administered vehicle supplemented with DS102 at a dose of 50 mg/kg once daily from Day 0 to Day 13.
  • Group 7 Eight UUO mice were orally administered vehicle supplemented with DS102 at a dose of 500 mg/kg once daily from Day 0 to Day 13.
  • Table 3 summarizes the treatment schedule for each of Groups 1-7 during the study.
  • mice were monitored daily. Individual body weight was measured daily before treatment during the treatment period. Mice were observed for significant clinical signs of toxicity, moribundity and mortality approximately 60 minutes after each administration. The animals were sacrificed by exsanguination through direct cardiac puncture under isoflurane anesthesia (Pfizer Inc.) at Day 14.
  • FIG. 2 shows the body weight changes for all animals. In all the animals, body weight decreased after surgery, and recovered gradually during the treatment period. Mean body weight of the Vehicle group was significantly lower than that of the Sham control group from Day 2 to Day 5 and from Day 10 to Day 11 . There were no significant changes in mean body weight at any day during the treatment period between the Vehicle group and the treatment groups. There were no dead animals in all groups during the treatment period. In the present study, none of the animals showed deterioration in general condition.
  • FIG. 3 and Table 4 show the body weight of the animals on the day of sacrifice. There was no significant difference in mean body weight on the day of sacrifice between the Sham control group and the Vehicle group. There were no significant differences in mean body weight on the day of sacrifice between the Vehicle group and the treatment groups.
  • Figures 4A-4D and Table 4 show the kidney weight and kidney-to-body weight ratio of the animals on the day of sacrifice.
  • the Vehicle group showed a significant increase in mean right kidney weight compared with the Sham control group. However, there were no significant differences in mean right kidney weight between the Vehicle group and the treatment groups.
  • the Vehicle group also showed a significant increase in mean right kidney-to-body weight ratio compared with the Sham control group. There were no significant differences in mean right kidney-to-body weight ratio between the Vehicle group and the treatment groups.
  • the Vehicle group also showed a significant increase in mean left kidney weight compared with the Sham control group. There were no significant differences in mean left kidney weight between the Vehicle group and the treatment groups.
  • the Vehicle group showed a significant increase in mean left kidney-to-body weight ratio compared with the Sham control group, but there were no significant differences in mean left kidney- to-body weight ratio between the Vehicle group and the treatment groups.
  • Kidney Chemistry Figure 5 and Table 5 show the kidney hydroxyproline content for the animals.
  • the Vehicle group showed a significant increase in kidney hydroxyproline contents compared with the Sham control group.
  • the DS109 low, DS109 high, DS102 low and DS102 high groups showed significant decreases in kidney hydroxyproline contents compared with the Vehicle group. There was no significant difference in kidney hydroxyproline contents between the Vehicle group and the DS109 middle group.
  • Figures 6A-6G and Table 6 show the Sirius red staining and Figure 7, the fibrosis area of the animals.
  • Figure 6A displays representative photomicrographs of Sirius red-stained kidney sections.
  • the Vehicle group showed a significant increase in the percentage of fibrosis area (Sirius red positive area) compared with the Sham control group.
  • Bonferroni Multiple Comparison revealed that the fibrosis area in the DS109 middle group tended to decrease compared with the Vehicle group. There were no significant differences in fibrosis area between the Vehicle group and the other treatment groups.
  • Mann-Whitney U test was conducted due to the presence of notable outliers and revealed that the DS109 low, DS109 middle, DS109 high, DS102 low and DS102 high groups fibrosis area tended to decrease (p ⁇ 0.1) compared with the Vehicle group.
  • Alpha-SMA The Vehicle group showed a significant increase in a-SMA mRNA expression level compared with the Sham control group. There were no significant differences in a-SMA mRNA expression level between the Vehicle group and the treatment groups.
  • TIMP-1 The Vehicle group showed a significant increase in TIMP-1 mRNA expression level compared with the Sham control group. There were no significant differences in TIMP-1 mRNA expression level between the Vehicle group and the treatment groups.
  • TGF-b The Vehicle group showed a significant increase in TGF-b mRNA expression level compared with the Sham control group. There were no significant differences in TGF-b mRNA expression level between the Vehicle group and the treatment groups.
  • Collagen Type 1 The Vehicle group showed a significant increase in Collagen Type 1 mRNA expression level compared with the Sham control group. There were no significant differences in Collagen Type 1 mRNA expression level between the Vehicle group and the treatment groups.
  • DS109 Treatment with DS109 at low dose showed significant decreases (p ⁇ 0.05) in kidney hydroxyproline contents and a reduction trend (p ⁇ 0.1) in fibrosis area compared with the Vehicle group. Treatment with DS109 at middle dose showed reduction trend (p ⁇ 0.1 ) in the fibrosis area compared with the Vehicle group. Lastly, treatment with DS109 at high dose showed significant decreases (p ⁇ 0.05) in kidney hydroxyproline contents and a reduction trend (p ⁇ 0.1 ) in fibrosis area compared with the Vehicle group.
  • DS102 Treatment with DS102 at low dose showed a significant decrease in kidney hydroxyproline contents (p ⁇ 0.05), and a reduction trend (p ⁇ 0.1 ) in fibrosis area compared with the Vehicle group. Treatment with DS102 at high dose showed significant decreases in kidney hydroxyproline contents (p ⁇ 0.05), and a reduction trend (p ⁇ 0.1 ) in fibrosis area compared with the Vehicle group.
  • Figure 9 depicts the study design from surgery and treatment to day 14 of the study.
  • Test Substance The test substance for this study was DS102. To prepare dosing solutions of each substance, DS102 was diluted in a vehicle of 0.5% hydroxypropyl methyl cellulose (HPMC).
  • HPMC hydroxypropyl methyl cellulose
  • BDL Surgery On Day 0 of the study, BDL surgery was performed under pentobarbital (Kyoritsu Seiyaku, Japan) anesthesia. The mouse’s hair was first shaved, the abdominal cavity cut open, and the common bile duct was ligated twice with 7-0 surgical silk. The mouse’s peritoneum and the skin were closed with sutures, and the mice were transferred to a clean cage (e.g., resting cage) until recovered from anesthesia. Sham operated mice had their common bile duct exposed but not ligated.
  • DS102 was administered to orally in a volume of 10 milliliters (mL)/kilogram (Kg).
  • Treatment Doses DS102 was administered at 3 dose levels of 50, 250, and 500 milligram (mg)/Kg once daily from Day 0 to Day 13 of the study.
  • Sterilized solid normal diet was provided ad libitum, being placed in a metal lid on the top of the cage. Pure water was also provided ad libitum from a water bottle equipped with a rubber stopper and a sipper tube. Water bottles were replaced once a week, cleaned, and sterilized in an autoclave and reused. Mice were identified by ear punch and each cage was labeled with a specific identification code.
  • RNA Total ribonucleic acid
  • left lateral lobe was snap frozen in liquid nitrogen and stored at -80°C for gene expression analyses.
  • Right medial lobe, left medial lobe, right lobe and caudate lobe were snap frozen in liquid nitrogen and stored at -80°C for shipping.
  • Group 2 Fifteen BDL-operated mice were orally administered vehicle [0.5% FIPMC] in a volume of 10 mL/kg once daily from Day 0 to Day 13.
  • Group 3 Fifteen BDL-operated mice were orally administered vehicle supplemented with DS102 at a dose of 50 mg/kg once daily from Day 0 to Day 13.
  • Group 4 Fifteen BDL-operated mice were orally administered vehicle supplemented with DS102 at a dose of 250 mg/kg once daily from Day 0 to Day 13.
  • Group 5 Fifteen BDL-operated mice were orally administered vehicle supplemented with DS102 at a dose of 500 mg/kg once daily from Day 0 to Day 13.
  • Table 10 summarizes the treatment schedule for each of Groups 1-5 during the study.
  • mice were monitored daily. Individual body weight was measured daily before treatment during the treatment period. Mice were observed for significant clinical signs of toxicity, moribundity and mortality approximately 60 minutes after each administration. The animals were sacrificed at Day 14 after BDL surgery by exsanguination through direct cardiac puncture under isoflurane anesthesia (Pfizer Inc.)
  • Figure 10 shows the body weight changes for all animals. Mean body weight in all groups, except of the Sham control group, gradually decreased during the study period. Mean body weight of the Vehicle group was significantly lower than that of the Sham control group from Day 2 to Day 14. There were no significant changes in mean body weight at any day during the study period between the Vehicle group and the DS102 treatment groups.
  • mice found dead before reaching Day 14 were as follows: three out of 15 mice were found dead in the Vehicle group; seven out of 15 mice were found dead in the DS102 low, DS102, middle and DS102 high groups. In this model, a percentage of deaths are expected simply due to disease induction and the observed mortality rate is consistent with historical data.
  • FIG. 11 and Table 11 show the body weight of the animals on the day of sacrifice.
  • the Vehicle group showed a significant decrease in mean body weight on the day of sacrifice compared with the Sham control group. There were no significant differences in mean body weight on the day of sacrifice between the Vehicle group and the DS102 treatment groups.
  • Figures 12A and 12B and Table 11 show the liver weight and liver-to-body weight ratio of the animals on the day of sacrifice.
  • the Vehicle group showed a significant increase in mean liver weight compared with the Sham control group.
  • Mean liver weight in the DS102 high group tended to decrease compared with the Vehicle group. There were no significant differences in mean liver weight between the Vehicle group and the other treatment groups.
  • the Vehicle group showed a significant increase in mean liver-to-body weight ratio compared with the Sham control group.
  • Mean liver-to-body weight ratio in the DS102 high group tended to decrease compared with the Vehicle group. There were no significant differences in mean liver-to-body weight ratio between the Vehicle group and the other treatment groups.
  • FIG. 13 and Table 12 show the serum aminotransferase (ALT) for the animals.
  • the Vehicle group showed a significant increase in serum ALT level compared with the Sham control group. There were no significant differences in serum ALT level between the Vehicle group and the DS102 treatment groups. However, from historical data for this model, ALT levels are known to decrease at Day 14 without treatment. As such, this may impact the ability to detect differences between the groups.
  • Figure 14 and Table 12 show the serum total bilirubin for the animals.
  • the Vehicle group showed a significant increase in serum total bilirubin level compared with the Sham control group. There were no significant differences in serum total bilirubin level between the Vehicle group and the DS102 treatment groups.
  • Figures 15A-14E and Table 13 show the Sirius red staining and Figure 16, the fibrosis area of the animals.
  • Figure 14A displays representative of photomicrographs of Sirius red-stained liver sections. Liver sections from the Vehicle group showed increased collagen deposition in the portal region of liver lobule and PV-CV or PV-PV bridging fibrosis compared with the Sham control group. The Vehicle group showed a significant increase in the fibrosis area (Sirius red-positive area) compared with the Sham control group. The DS102 middle group showed a significant decrease in the fibrosis area compared with the Vehicle group. Fibrosis area in the DS102 high group tended to decrease compared with the Vehicle group. There was no significant difference in the fibrosis area between the Vehicle group and the DS102 low group.
  • a-SMA The Vehicle group showed a significant increase in the a-SMA mRNA expression level compared with the Sham control group. a-SMA mRNA expression level in the DS102 high group tended to decrease compared with the Vehicle group. There were no significant differences in a-SMA mRNA expression level between the Vehicle group and the DS102 treatment groups.
  • TIMP-1 The Vehicle group showed a significant increase in the TIMP-1 mRNA expression level compared with the Sham control group. TIMP-1 mRNA expression level in the DS102 high group tended to decrease compared with the Vehicle group. There were no significant differences in TIMP-1 mRNA expression level between the Vehicle group and the DS102 treatment groups.
  • TGF-b The Vehicle group showed a significant increase in the TGF-b mRNA expression level compared with the Sham control group.
  • the DS102 high group showed a significant decrease in the TGF-b mRNA expression level compared with the Vehicle group. There were no significant differences in TGF-b mRNA expression level between the Vehicle group and the DS102 treatment groups.
  • Collagen Type 1 The Vehicle group showed a significant increase in the Collagen Type 1 mRNA expression level compared with the Sham control group. Collagen Type 1 mRNA expression level in the DS102 high group tended to decrease compared with the Vehicle group. There were no significant differences in Collagen Type 1 mRNA expression level between the Vehicle group and the DS102 treatment groups.
  • biochemical parameters e.g., ALT and total bilirubin
  • histological collagen deposition e.g., fibrosis area
  • gene expression levels e.g., a-SMA, TIMP-1 , TGF-b, Collagen Type 1
  • Treatment with DS102 at the middle dose showed a significant decrease (p ⁇ 0.05) in fibrosis area compared with the Vehicle group.
  • Treatment with DS102 at the high dose showed a significant decrease (p ⁇ 0.05) in TGF-b mRNA expression level, and a trend approaching significance (p ⁇ 0.01) for decrease in fibrosis area, liver weight, liver-to-body weight ratio, a-SMA, TIMP-1 and Collagen Type 1 mRNA expression levels compared with the Vehicle group.
  • Example 3 Effects of DS102 on TGF-b receptors, signaling and induced fibrotic proteins
  • the objective of this study was to examine the effects of 15-HEPE and 15-HEPE EE on the expression of TGF-b receptors, TGF-b induced intracellular signaling and pro-fibrotic epithelial mesenchymal transition proteins.
  • Cytotoxicity testing The cytotoxicity of 15-HEPE free acid and ethyl ester was tested in different liver (hepatoma) cell lines to understand the concentration range in the test system.
  • Transcriptional activity A promoter (Luciferase) assay was conducted to measure TGFp-induced transcriptional activation following administration of 15-HEPE.
  • Sucrose gradient ultracentrifugation and confocal microscopy were used to identify 15-HEPE induced microdomain translocation of TGF-b receptors by sucrose.
  • Sucrose density gradient analysis of TGF-b receptors was conducted in the plasma membranes of Mv1 Lu cells (mink lung epithelial cell) treated with 100 mM of 15-HEPE and an equal volume of DMSO (dimethyl sulfoxide) at 37°C for 0, 1 , 4, and 24 hours, and the cell lysates from these treated cells were subjected to sucrose density gradient ultracentrifugation.
  • sucrose gradient fractions were then analyzed by Western blot analysis using anti ⁇ R-l (type I TGF-b receptor), anti ⁇ R-ll (type II TGF-b receptor), anti ⁇ R-lll (type III TGF-b receptor, betaglycan), anti-EGFR (epidermal growth factor receptor), and anti-caveolin-1 antibodies.
  • anti ⁇ R-l type I TGF-b receptor
  • anti ⁇ R-ll type II TGF-b receptor
  • anti ⁇ R-lll type III TGF-b receptor
  • betaglycan anti-EGFR (epidermal growth factor receptor)
  • anti-EGFR epidermatitisepidermal growth factor receptor
  • anti-caveolin-1 antibodies The lipid raft/caveolae, and non-lipid raft microdomain localization of TbR-l, TbR-ll, TbR-lll, EGFR and caveolin-1 in the plasma membrane of untreated cells or cells treated with 15-HEPE were assessed to determine the effects of 15-HEPE on the membrane micro
  • TGF ⁇ -induced signaling The effects of 15-HEPE on TGF ⁇ -induced signaling and cellular responses were determined.
  • 15-HEPE activities of 15-HEPE to regulate TGF ⁇ -stimulated Smad2 phosphorylation and nuclear translocation, both of which are key signaling events leading to TGF ⁇ -induced cellular responses, were tested.
  • One important biological activity of TGF-b is transcriptional activation of genes responsible for epithelial- mesenchymal transition (EMT), which is a crucial event in wound healing, tissue fibrosis, and cancer progression.
  • EMT epithelial- mesenchymal transition
  • HepG2 cells human hepatoma cell line
  • DMEM fetal calf serum
  • Figure 18A shows that 15-HEPE induced degradation of type II TGF-b receptor and blocked TGF-b induced epithelial mesenchymal transition (EMT) (i.e. , pro-fibrotic) protein production.
  • EMT epithelial mesenchymal transition
  • FlepG2 cells human hepatoma cell line
  • the two panels shown in Figure 18A are two separate experiments run under the same experimental conditions. The results from these separate experiments indicate that 15-FIEPE blocks TGF-b induced EMT protein production and induced degradation of type II TGF-b receptor.
  • Figure 18A also shows the effects of 15-FIEPE on plasminogen activator inhibitor-1 (PA-1 ), a protein induced by TGF-b and associated with increased cardiovascular risk.
  • Figure 18B shows that 15-FIEPE inhibits TGF-p-stimulated intracellular signaling (e.g., SMAD2/3 phosphorylation) in liver stellate cells.
  • the experiment conducted in Figure 18B included pretreating LX2 cells (human liver stellate cells) with increasing concentrations between 0 mM to 100 mM of DS102 for 24 hours followed by 30 min of TGF-b stimulation. The results of the experiment indicated that 15-FIEPE inhibit TGF-b stimulation.
  • a 15-FIEPE sucrose density gradient analysis of TGF-b receptors was conducted in the plasma membranes of mink lung epithelial cell (Mv1 Lu) cells treated with 100 mM of 15-FIEPE and an equal volume of dimethyl sulfoxide (DMSO) at 37°C for 0, 1 , 4, and 24 hours, and the cell lysates from these treated cells were subjected to sucrose density gradient ultracentrifugation.
  • Mv1 Lu mink lung epithelial cell
  • DMSO dimethyl sulfoxide
  • sucrose gradient fractions were then analyzed by Western blot analysis using anti-TpR-l (type I TGF-b receptor - Figure 18C), anti ⁇ R-ll (type II TGF-b receptor - Figure 18D), anti ⁇ R-lll (type III TGF-b receptor, betaglycan - Figure 18E), anti-EGFR (epidermal growth factor receptor - Figure 18F), and anti-caveolin-1 antibodies (Figure 18G).
  • Fractions 4 to 5 contained lipid rafts/caveolae whereas fractions 7-10 were non-lipid raft fractions.
  • Treatment with 15-FIEPE did not affect the abundance of TbR-I proteins but induced translocation of TbR-I to lipid-raft at 24 hours treatment (Figure 18C).
  • Stars(* ⁇ ) indicate 15-FIEPE increased abundance of TbR-I (24 hours) in the fraction in comparison with that of the control and shorter treatment durations (Figure 18C).
  • the triangles (T) indicate translocation and decreased abundance of TbR-II in the fraction in comparison with control cells ( Figure 18D).
  • 15-FIEPE did not change the localization and abundance of TpR-lll, EGFR and caveolin-1 .
  • Example 4 The Efficacy of Orally Administered DS102 in NAFLD Patients
  • NAFLD Non-Alcoholic Fatty Liver Disease
  • the primary endpoints for this study included the efficacy as well as the safety for administering DS102.
  • the efficacy was evaluated based on change in serum alanine aminotransferase (ALT) from baseline to Week 16 and change in liver stiffness measured by transient elastography from baseline to Week 16.
  • the safety was evaluated on the number of treatment emergent adverse events (TEAEs) in each treatment group leading to treatment discontinuation.
  • Secondary Endpoints The secondary endpoints for this study included a change in any one of the following: serum ALT from baseline to Weeks 2, 4, 8 and 12; aspartate aminotransferase (AST) from baseline to Weeks 2, 4, 8, 12 and 16; AST:ALT ratio from baseline to Weeks 2, 4, 8, 12 and 16; fibrosis-4 (FIB-4) index from baseline to Week 16; NAFLD fibrosis score (NFS) from baseline to week 16; change in hepatic fat measured by controlled attenuation parameter (CAP) from baseline to Week 16; enhanced liver fibrosis (ELF) score from baseline to Week 16; and homeostatic model assessment insulin resistance (HOMA-IR) and adipose tissue insulin resistance (adipo-IR) from baseline to Weeks 2, 4, 8, 12 and 16.
  • AST aspartate aminotransferase
  • AST aspartate aminotransferase
  • AST aspartate aminotransferase
  • AST aspartate aminotransferase
  • AST aspart
  • Exploratory analysis included analysis of lipid and metabolic parameters including total cholesterol, triglycerides, very low-density lipoprotein cholesterol (VLDL-C), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), non-high-density lipoprotein cholesterol (non- HDL-C), remnant-like particle cholesterol (RLP-C), fasting glucose, insulin, free fatty acids and hemoglobin A1 C (HbA1 C). Additional exploratory analysis included high throughput lipidomics and proteomics.
  • VLDL-C very low-density lipoprotein cholesterol
  • LDL-C low density lipoprotein cholesterol
  • HDL-C high density lipoprotein cholesterol
  • RLP-C remnant-like particle cholesterol
  • HbA1 C hemoglobin A1 C
  • Additional exploratory analysis included high throughput lipidomics and proteomics.
  • the study consisted of a screening period of 28 days, a 16-week treatment period and a 4 week follow up period. At the screening visit, patients were assessed using the screening examinations. Patients who meet the inclusion criteria and who do not meet the exclusion criteria were enrolled.
  • FIG. 19 A schematic diagram of the overall timeframe of the study is provided in Figure 19. Once patients were enrolled in the study they were restricted from using any other treatment for NAFLD. Any medication (e.g., prescription as well as over the counter (OTC) drugs) or therapeutic intervention deemed necessary for the patient, and which in the opinion of the Investigator do not interfere with the safety and efficacy evaluations, were continued unless they are included in the list of ‘Concomitant Medications’ provided below.
  • OTC over the counter
  • Treatment group B 1 x DS102500 mg capsule & 1 x Placebo 500 mg capsule orally administered twice a day (4 capsules daily) for 16 weeks.
  • Treatment group C 2 x DS102 500 mg capsules orally administered twice a day (4 capsules daily) for 16 weeks.
  • the DS102 capsule and placebo capsule were identical in appearance.
  • BMI body mass index
  • Adequate contraception is defined as: systemic hormonal contraceptives; intrauterine device or barrier method of contraception in conjunction with spermicide; or agree to sexual abstinence, defined as a patient refraining from heterosexual intercourse during the entire period of risk associated with the study treatments and in line with their preferred and usual lifestyle.
  • Hormonal contraceptives were required to be on a stable dose for at least one month before baseline.
  • OLT orthotopic liver transplant
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • liver disease including coronary artery disease, cerebrovascular disease, pulmonary disease, renal insufficiency, serious psychiatric disease, respiratory or hypertensive disease, as well as diabetes and arthritis that, in the opinion of the Investigator, precluded the patient from participating in and completing the study.
  • HIV human immunodeficiency virus
  • liver disease Patients with liver disease of other etiologies such as drug-induced, autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), haemochromatosis, alpha-1 antitrypsin (A1AT) deficiency or Wilson’s disease.
  • PBC primary biliary cholangitis
  • PSC primary sclerosing cholangitis
  • haemochromatosis haemochromatosis
  • alpha-1 antitrypsin (A1AT) deficiency or Wilson’s disease patients with liver disease of other etiologies such as drug-induced, autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), haemochromatosis, alpha-1 antitrypsin (A1AT) deficiency or Wilson’s disease.
  • PBC primary bil
  • Screening Visit 1 At the screening visiting the following
  • Screening Visit (Visit 1 ): Once informed consent was obtained, patients were assigned a Patent Screen Number. Ideally the patient was fasted. The following screening assessments/sample collections were performed: verification of inclusion/exclusion criteria; demographic data; medical history; physical examination; 12-lead electrocardiogram (ECG); Vital signs (blood pressures, heart rate and body temperature); samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests); virology; pregnancy test (for female patients of child-bearing potential); ALT, AST tests (ALT measured on two occasions during screening); and concomitant medication assessment.
  • ECG electrocardiogram
  • hematology, serum biochemistry, and coagulation tests samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests); virology; pregnancy test (for female patients of child-bearing potential); ALT, AST tests (ALT measured on two occasions during screening); and concomitant medication assessment.
  • AE adverse event
  • Treatment Period Following completion of a successful screening visit, patients began the comparative treatment period (16 weeks). At the start of the comparative treatment period, after confirmation of continued eligibility, patients were randomly assigned to one of the three treatment regimens. Patients took the allocated investigation medicinal product (IMP) of a DS102 capsule or placebo capsule twice- daily throughout the comparative treatment period. Each self-administration of IMP was recorded in a patient diary card. Patients were instructed to take DS102 in the morning and in the evening with or after food (except on the mornings of clinic visits 3, 4, 6, 8 and 10 when patients were instructed to abstain from taking DS102 prior to the visit and to take DS102 as soon as possible after the clinic visit).
  • IMP investigation medicinal product
  • AE adverse event
  • Baseline (Visit 2): Patients attended the investigational site at Visit 2. Blood sampling was the first assessment carried out. After the blood sampling, the following assessments were performed: verification of inclusion/exclusion criteria; medical history; physical examination; 12-lead ECG; pharmacokinetic sampling; vital signs (blood pressures, heart rate and body temperature); samples for clinical laboratory safety tests (haematology, serum biochemistry, and coagulation tests); lipid profile; urinalysis; pregnancy test (for female patients of child-bearing potential); ALT, AST tests; HOMA-IR/Adipo-IR; ELF; liver stiffness and CAP; FIB-4; NFS (including BMI); biomarkers blood sample; exploratory blood sample; patient randomization; study drug/placebo administration; AE assessment; and concomitant medication assessment.
  • the Investigator randomized the patient and provided the patient with the designated IMP or placebo from one of the patient treatment packs.
  • the first dose of IMP or placebo was administered at site once all baseline assessments had been completed.
  • the patient took their second dose of IMP or placebo in the evening of Day 0.
  • the capsules were then administered twice-daily. Patients did take IMP or placebo on the morning of their return site visit (Visit 3). Before leaving the clinic, the patient was instructed not to have any breakfast before the next visit to allow a minimum fasting period of 8 hours.
  • Week 2 (Visit 3): Patients returned to the investigational site at Visit 3. Patients did not take IMP or placebo on the morning of Visit 3. The following assessments were performed: physical examination; pharmacokinetic sampling; vital signs (e.g., blood pressures, heart rate and body temperature); ALT, AST tests; HOMA-IR/Adipo-IR; AE assessment; and concomitant medication assessment.
  • the IMP or placebo was returned, and further IMP or placebo was supplied to the patient.
  • the patient took their next dose of IMP or placebo as soon as all visit assessments had been completed.
  • the capsules continued to be administered twice-daily.
  • patients were advised that they were required to return to the investigational site in two weeks at Visit 4. Patients did not take IMP or placebo on the morning of their return site visit (Visit 4).
  • the patient was instructed not to have any breakfast before the next visit to allow a minimum fasting period of 8 hours.
  • Week 4 (Visit 4): Patients returned to the investigational site at Visit 4. Patients did not take IMP or placebo on the morning of Visit 4. The following assessments were performed: physical examination; pharmacokinetic sampling; vital signs (blood pressures, heart rate and body temperature); samples for clinical laboratory safety tests (haematology, serum biochemistry and coagulation tests); pregnancy test (for female patients of child-bearing potential); ALT, AST tests; HOMA- IR/Adipo-IR; AE assessment; and concomitant medication assessment. The IMP or placebo was returned, and further IMP or placebo was supplied to the patient. The patient took their next dose of IMP or placebo as soon as all visit assessments had been completed. The capsule continued to be administered twice-daily. On completion of this visit, patients were advised that they were required to return to the investigational site in two weeks at Visit 5. Patients did not take IMP or placebo on the morning of their return site visit (Visit 5).
  • Week 6 (Visit 5): Patients returned to the investigational site at Visit 5. The following assessments were performed: AE assessment and Concomitant medication assessment. The IMP or placebo was returned and further IMP or was supplied to the patient. The patient took their next dose of IMP or placebo as soon as all visit assessments had been completed. The capsule continued to be administered twice-daily. On completion of this visit, patients were advised that they were required to return to the investigational site in two weeks at Visit 6. Patients did not take IMP or placebo on the morning of their return site visit (Visit 6). Before leaving the clinic, the patient was instructed not to have any breakfast before the next visit to allow a minimum fasting period of 8 hours.
  • Week 8 (Visit 6): Patients returned to the investigational site at Visit 6. Patients did not take IMP or placebo on the morning of Visit 6. Blood sampling was the first assessment carried out. After the blood sampling, the following assessments were performed: physical examination; pharmacokinetic sampling; vital signs (blood pressures, heart rate and body temperature); samples for clinical laboratory safety tests (haematology, serum biochemistry and coagulation tests); lipid profile; pregnancy test (for female patients of child-bearing potential); ALT, AST tests; HOMA- IR/Adipo-IR; biomarker blood samples; ae assessment; and concomitant medication assessment. The IMP or placebo were returned and further IMP or supplied to the patient.
  • Week 10 (Visit 7): Patients returned to the investigational site at Visit 7. The following assessments were performed: AE assessment and concomitant medication assessment. The IMP or placebo were returned, and further IMP or placebo was supplied to the patient. The patient took their next dose of IMP or placebo as soon as all visit assessments had been completed. The capsule continued to be administered twice-daily. On completion of this visit, patients were advised that they were required to return to the investigational site in two weeks at Visit 8. Patients did not take IMP or placebo on the morning of their return site visit (Visit 8). Before leaving the clinic, the patient was instructed not to have any breakfast before the next visit to allow a minimum fasting period of 8 hours.
  • Week 12 (Visit 8): Patients returned to the investigational site at Visit 8. Patients did not take IMP or placebo on the morning of Visit 8. The following assessments were performed: physical examination; pharmacokinetic sampling; vital signs (blood pressures, heart rate and body temperature); samples for clinical laboratory safety tests (haematology, serum biochemistry, and coagulation tests); pregnancy test (for female patients of child-bearing potential); ALT, AST tests; HOMA- IR/Adipo-IR; AE assessment; and concomitant medication assessment. The IMP or placebo was returned, and further IMP or placebo supplied to the patient. The patient took their next dose of IMP or placebo as soon as all visit assessments had been completed. The capsule continued to be administered twice-daily.
  • Week 14 (Visit 9): Patients returned to the investigational site at Visit 9. The following assessments were performed: AE assessment and concomitant medication assessment. The IMP or placebo were returned, and further IMP or placebo supplied to the patient. The patient took their next dose of IMP or placebo as soon as all visit assessments had been completed. The capsule continued to be administered twice-daily. On completion of this visit, patients were advised that they were required to return to the investigational site in two weeks at Visit 10 Patients should not take IMP or placebo on the morning of their return site visit (Visit 10). Before leaving the clinic, the patient was instructed not to have any breakfast before the next visit to allow a minimum fasting period of 8 hours.
  • Week 16 (Visit 10) or Early Withdrawal: Patients returned to the investigational site at Visit 10. Patients did not take IMP or placebo on the morning of Visit 10. Blood sampling was the first assessment carried out. After the blood sampling, the following assessments were performed: physical examination; 12-lead ECG; pharmacokinetic sampling; vital signs (blood pressures, heart rate and body temperature); samples for clinical laboratory safety tests (haematology, serum biochemistry, and coagulation tests); urinalysis; lipid profile; pregnancy test (for female patients of child-bearing potential); ALT, AST tests; HOMA-IR/Adipo-IR; ELF; liver stiffness and CAP; FIB-4; NFS [including BMI]; Biomarker blood samples; exploratory blood sample; AE assessment; and concomitant medication assessment.
  • ALT, AST, ALT:AST ratio Increased liver enzymes (ALT and AST) are a marker of liver injury and were assessed at Visit 1 /Screening (on two occasions during screening 7 or more days apart), Visit 2/Baseline, Visit 3/Week 2, Visit 4/Week 4, Visit 6/Week 8, Visit 8/Week 12, Visit 10/Week16 and follow up Visit 11 /Week 20.
  • HOMA-IR/Adipo-IR levels are a method of measuring insulin resistance.
  • HOMA-IR is calculated by multiplying fasting plasma insulin (FPI) by fasting plasma glucose (FPG), then dividing by the constant 405.
  • Adipo-IR is calculated by multiplying fasting non-esterified fatty acids (NEFA) c fasting insulin.
  • Blood samples were taken to assess HOMA-IR and Adipo-IR at Visit 2/Baseline, Visit 3/Week 2, Visit 4/Week 4, Visit 6/Week 8, Visit 8/Week 12, Visit 10/Week16 and follow up Visit 11/Week 20. All subjects were required to have been fasted for a minimum of 8 hours prior to blood sampling. If subjects had not fasted for a minimum of 8 hours, the duration of fasting time was recorded, and subjects encouraged to fast appropriately for the next clinical visit.
  • ELF An ELF score is an extracellular matrix marker set consisting of tissue inhibitor of metalloproteinases 1 (TIMP-1 ), amino-terminal propeptide of type III procollagen (PIIINP) and hyaluronic acid (HA). Blood samples were taken to perform this assessment at baseline (Visit 2) and Week 16 (Visit 10).
  • TMP-1 tissue inhibitor of metalloproteinases 1
  • PIIINP amino-terminal propeptide of type III procollagen
  • HA hyaluronic acid
  • Liver stiffness and CAP were assessed using transient elastography (e.g., FibroScan® 502 Touch model or equivalent). Patients were fasted and scanned at the same time of the day, if possible, for baseline (Week 0) and Visit 10 (Week 16).
  • transient elastography e.g., FibroScan® 502 Touch model or equivalent.
  • the operator performed an examination including at least 10 valid measurements or a maximum of 20 attempts, with the XL+ or M+ probe, at the same spot. The entire examination lasted no more than 10-15 minutes. The final stiffness and CAP values was recorded as median values of valid measurements.
  • FIB-4 Index is based on age, platelet count, ALT level, and AST level and was assessed at Baseline (Visit 2) and Week 16 (Visit 10). FIB-4 score is determined as shown by the equation below.
  • FIB-4 Age (years) x AST (U/D
  • NFS The NFS is based on age, hyperglycemia, BMI, platelet count, albumin level, and AST/ALT ratio.
  • NFS was assessed at Baseline (Visit 2) and Week 16 (Visit 10).
  • Safety Assessments included the following: medical history; physical examination; ECG; vital signs; clinical laboratory safety tests (e.g., hematology, serum biochemistry, coagulation, lipid profile, and urinalysis); virology; pregnancy test; blood sampling; pharmacokinetic sampling; exploratory blood collection; biomarker blood collection; urine DOA and alcohol breath test; adverse event assessment; concomitant medication; bioanalysis; sample, storage, handling; and shipping; and restrictions. A detailed description of each is provided below.
  • ECG A 12-lead ECG 10 mm/1 mv, 25 mm/s with a 10 second lead II rhythm strip was recorded at each time point. ECGs were recorded using the GE Mac 1200 or equivalent model. Patients were rested quietly in a fully supine position for 5 minutes before the ECG was taken. Recordings were made on the days indicated in Study Flow Chart in Table 19 at Visit 1/Screening, Visit 2/Baseline and Visit 10/Week 16.
  • Vital Signs Vital signs measurements were performed as per the Study Flow Chart in Table 19 at Visit 1/Screening, Visit 2/Baseline, Visit 3/Week 2, Visit 4/Week 4, Visit 6/Week 8, Visit 8/Week 12, Visit 10/Week16 and follow up Visit 11 /Week 20. Vital signs measurements were performed before any blood samples were taken. All new findings or changes to previous findings considered clinically significant were recorded as an AE if the finding was made after the patient had signed. Vital sign measurements included: blood pressure performed as supine (e.g., after at least 5 minutes of rest) systolic and diastolic blood pressure (in mm Fig); heart rate taken at rest in beats per minute (bpm); and temperature taken as per clinical practice.
  • supine e.g., after at least 5 minutes of rest
  • systolic and diastolic blood pressure in mm Fig
  • temperature taken as per clinical practice e.g
  • Hematology Full blood count to include red cell count, hemoglobin, hematocrit, white cell count, differential white cell count, platelet count and reticulocyte count.
  • Serum biochemistry Urea (blood urea nitrogen; BUN), creatinine, uric acid, total bilirubin, Indirect and Direct Bilirubin, sodium, bicarbonate potassium, phosphorus, calcium chloride, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), ALT/AST ratio, lactate dehydrogenase (LDH), creatine phosphokinase (CPK), albumin, total protein, cholesterol, triglycerides, glucose, C-reactive protein (CRP).
  • ALP alkaline phosphatase
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALT/AST ratio lactate dehydrogenase
  • LH lactate dehydrogenase
  • CPK creatine phosphokinase
  • albumin total protein
  • cholesterol cholesterol
  • C-reactive protein CRP
  • PT prothrombin time
  • INR international normalized ratio
  • APTT activated partial prothrombin time
  • Lipid Profile LDL, HDL, and VLDL-C.
  • Urinalysis pH, protein, glucose, blood, ketones, leukocytes, leukocyte esterase, bilirubin, specific gravity, urobilinogen and nitrate. Reflex micro if blood, protein, leukocyte esterase or nitrate/nitrite are present.
  • Virology A blood sample was taken to perform virology tests including HIV, Hep C and Hep B as detailed in the Study Flow Chart in Table 19.
  • Pregnancy Test For female patients of childbearing potential, a pregnancy test was carried out as per the Study Flow Chart of Table 19 at Visit 1 /Screening, Visit 2/Baseline, Visit 4/Week 4, Visit 6/Week 8, Visit 8/Week 12, Visit 10/Week 16 and Visit 11/Week 20.
  • Blood Sampling Blood samples were obtained, and laboratory results reviewed for clinically significant values by each Investigator following sample analysis and verification. Additional blood may have been required for repeats of safety laboratory test.
  • PK sampling Blood samples for PK analysis were collected via direct venipuncture as per the Study Flow Chart in Table 19 at Visit 2/Baseline, Visit 3/Week 2, Visit 4/Week 4, Visit 6/Week 8, Visit 8/Week 12, Visit 10/Week16 and follow up Visit 11/Week 20. A 1 mL blood sample was taken at each timepoint. Following centrifugation, plasma samples were split in two and a back-up sample kept at the central laboratory until bioanalytical assays had been completed.
  • Biomarker Blood Collection Blood was collected as per the Study Flow Chart in Table 19 at baseline (Week 0), Visit 6/Week 8, Visit 10/Week 16 and follow up Visit 11 /Week 20 and was stored for potential biomarker analysis.
  • Urine DOA and Alcohol Breath Test As clinically appropriate at the discretion of the Investigator, an alcohol breath test was performed, and a urine sample taken from patients at any time during the conduct of the trial and testing done to detect the following: amphetamine, barbiturate, benzodiazepine, ***e, cannabinoids, and opiates.
  • Concomitant Medication Patients were on a stable dose of any concomitant medications for at least 3 months prior to screening and that dose should have remained stable for the entire study duration. If patients were insulin dependent this treatment should have commenced at least 3 months prior to screening, however changes in dose were permitted.
  • the study include diet, alcohol, caffeine, and physical activity restrictions.
  • diet patients avoided both during the study and for 4 weeks prior to baseline, ingesting food supplements rich in omega-3 or omega-6 fatty acids (e.g., cod liver oil capsules).
  • alcohol patient avoided alcohol consumption in excess of 21 units per week (males) or 14 units per week (females), whereby a unit consists of 10ml or 8mg of pure alcohol.
  • For physical activity patients were to avoid exercise and strenuous physical activity for at least 3 to 4 hours before the safety laboratory test (e.g., biochemistry).
  • DS102 capsules were white, opaque hard-shelled capsules (size 0) containing 500 mg of 15-HEPE ethyl ester (EE) with 5% w/w of colloidal silicon dioxide as viscosity modifier.
  • DS102 and Placebo capsules were stored at 2 - 8°C in a secure area (e.g. a locked cabinet or drug storage room), protected from unintended use. Labels were blinded to the dose and contained the randomization number.
  • a secure area e.g. a locked cabinet or drug storage room
  • Dosage and administration This study involved a comparison of DS102 with placebo, administered orally twice daily for a total duration of 16 weeks. The last study drug administration occurred on the day preceding Week 16 visit/Early Termination (ET) visit. Patients were required to take the capsules with or after food. Medication(s) for other conditions that were permitted in the study were taken as usual. The walleted blister packs consisted of 7 days of 4 capsules and lastly, the patients took the assigned medication for 16 consecutive weeks.
  • AEs Adverse events
  • SAEs serious adverse events
  • Adverse Events Any undesirable experience occurring to a patient who has taken their first dose of the study drug, whether or not considered related to the investigational IMP(s). AEs were recorded in the case report form, defining relationship to IMP and severity.
  • SAE Serious Adverse Events
  • Unexpected Adverse Event An experience not previously reported in the Investigator's Brochure or similar product information sheet such as the Summary of Products Characteristics (SPC).
  • the intensity of an AE is an estimate of the relative severity of the event made by the Investigator based on his or her clinical experience. The following definitions were used to rate the severity of an AE:
  • Severe drug-induced liver injury Irrespective of perceived causation, in the event of severe DILI the investigational drug was discontinued until the episode was deemed resolved. In the event the investigational drug was deemed to be the cause of the liver injury then the patient was not rechallenged with the drug. Severe DILI stipulates evidence of hepatic impairment as demonstrated by a total bilirubin >2x ULN or INR >1.5.
  • Adverse Reaction All noxious and unintended responses to a medicinal product related to any dose were considered adverse drug reactions.
  • the phrase "responses to a medicinal product” means that a causal relationship between a medicinal product and an AE was at least a reasonable possibility (i.e. , the relationship cannot be ruled out).
  • an adverse reaction is a response to a drug which is noxious and unintended, and which occurs at doses normally used in man for prophylaxis, diagnosis, or therapy of disease or for modification of physiological function.
  • This clinical trial employed a randomized, double-blind, placebo- controlled parallel group design. Randomization was used to minimize assignment bias and to increase the likelihood that known and unknown patient attributes (e.g. demographic characteristics) were evenly balanced across the treatment groups. Blinding was used to reduce potential bias during data collection and evaluation of safety and efficacy. The use of placebo as comparator was justified as a reasonable design to assess safety and efficacy in patients based on the brevity of the study duration and the absence of any possible long-term irreversible damage that may have had the result of placebo treatment.
  • Interim Analysis and Data Monitoring Interim analysis safety was carried out to estimate the conditional power when at least 50% of the patients had completed their Week 16 visit. The interim analysis was based on data collected for the primary and co-primary efficacy endpoints as well as the secondary endpoints and was used to estimate the conditional power to achieve the primary study objective, to potentially re-estimate the sample size and to potentially drop the less effective treatment arm.
  • Clinically Meaningful Response A higher mean or median reduction of at least 20% of ALT or liver stiffness compared to placebo and higher mean or median reduction of at least 10% of both ALT and liver stiffness compared to placebo
  • Analysis Sets included the enrolled set, the full analysis set (FAS), per- protocol set (PPS), safety analysis set (SAS), and the pharmacokinetic (PK) set. A detailed description of each analysis set is provided below.
  • Enrolled Set Patients who signed the informed consent form. Screen failures were patients from the Enrolled Population who did not meet the eligibility requirements and were withdrawn from the study prior to randomization.
  • FAS Randomized patients who received at least one administration of study treatment and had at least one post-baseline measurement. Patients were analyzed according to the treatment they were assigned to at randomization, irrespective of what treatment they actually received.
  • PPS A subset of the FAS consisting of those patients of FAS who had no major protocol violations. All protocol deviations were assessed and documented on a case-by-case basis prior to the database lock, and major deviations considered as having a serious impact on the efficacy results lead to the relevant patient being excluded from the PPS.
  • SAS Patients who took at least one administration of study treatment. Patients were analyzed according to the treatment actually taken.
  • PK Set Patients in the SAS who had at least one DS102 PK concentration. Patients were analyzed according to the treatment actually received.
  • Plasma concentrations of 15(S)-HEPE were tabulated and summarized descriptively. Individual and mean plasma concentration time profiles of 15(S)-HEPE were presented graphically.
  • the primary efficacy variable was the change from baseline in serum ALT at Week 16 (Visit 10).
  • the active treatment groups were compared against placebo via an analysis of covariance (ANCOVA) model, including the corresponding baseline value as covariate.
  • the comparisons against placebo were done according to Dunnett’s multiple testing procedure. For missing Week 16 values, the last value available was carried forward (LOCF). Similar methods were applied for liver stiffness. For ALT, longitudinal modelling was considered in addition.
  • Secondary variables The secondary efficacy variables and their changes from baseline to Week 16 (Visit 10) were summarized with descriptive statistics per treatment group and visit. This applied to the AST, AST: ALT ratio, hepatic fat measured by CAP, liver stiffness measurements by transient elastography, FIB-4, NFS, ELF and HOMA-IR/Adipo-IR. The change from baseline for the active treatment groups was compared against placebo via an ANOVA model, including a term for center effects. The 5% level of significance was used for all treatment comparisons.
  • Exploratory analysis included analysis of lipid and metabolic parameters including total cholesterol, triglycerides, very low-density lipoprotein cholesterol (VLDL-C), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), non-high-density lipoprotein cholesterol (non- FIDL-C), remnant-like particle cholesterol (RLP-C), fasting glucose, insulin, free fatty acids and hemoglobin A1C (HbA1C). Additional exploratory analysis included high throughput lipidomics and proteomics.
  • VLDL-C very low-density lipoprotein cholesterol
  • LDL-C low density lipoprotein cholesterol
  • HDL-C high density lipoprotein cholesterol
  • RLP-C non-high-density lipoprotein cholesterol
  • HbA1C hemoglobin A1C
  • Additional exploratory analysis included high throughput lipidomics and proteomics.
  • Figures 21 A-21 C depict the changes in insulin, glucose, and free fatty acid levels from baseline to Week 16 in patients administered DS102 either 1 g or 2 g per day as compared to a placebo.
  • the reduction in insulin, glucose, and free fatty acid levels upon administration of DS102 is clinically significant as metabolic substrates including glucose, carbohydrates and free fatty acids drive the pathogenesis of NASFI.
  • Figures 22A and 22B show the changes in FIOMA-IR and adipo-IR levels from baseline to Week 16 in patients administered DS102 either 1 g or 2 g per day as compared to a placebo.
  • the patients exhibited an improvement in both insulin resistance indices (e.g., a reduction in FIOMA-IR and adipo-IR levels) at Week 16, with significant improvements in the Per Protocol Set (PPS) observed for those patients administered 2 g of DS102.
  • PPS Per Protocol Set
  • Figures 23A and 23B depict the changes in glycosylated hemoglobin (e.g., FlbAlc) levels from baseline to Week 16 in patients administered DS012 either 1 g or 2 g per day as compared to a placebo.
  • Figure 23A shows the change in HbA1c levels
  • Figure 23B shows the change in FlbAlc levels in the proportion of patients who had high FlbAlc levels at baseline but achieved normal levels at Week 16. Since FlblAc is a measure of the amount of glucose attached to the body’s red blood cells and a surrogate for long-term glycemic control, these results indicate the administration of DS102 provides clinically significant improvements and normalizes glycemic control in a dose-dependent manner.
  • FlbAlc glycosylated hemoglobin
  • Figures 24A and 24B show the mean change and median (%) change in the patient’s lipid profile at Week 16 in the safety analysis set (SAS). These results are further depicted in Figures 25A-25C and illustrate that the administration of DS102 significantly improved patient’s lipid profile by either sustaining or reducing total cholesterol, VLDL-C, non-FIDL-C, remnant-like particle (RLP) cholesterol and triglyceride levels in the patients. Significantly, the reductions did not plateau at Week 16, suggesting that the administration DS102 might induce even larger changes in studies of longer duration.
  • SAS safety analysis set
  • the administration of DS102 also reverses the hepatotoxic lipid signature of NASFI and improves multiple lipid classes that are altered in patients diagnosed with NASFI. Specifically, the administration of 2 g of DS102 significantly decreased levels of multiple hepatotoxic diglycerides and significantly increased levels of multiple glycerophospholipid groups. This finding is important as patients with NASFI have low levels of hepatic and plasma glycerophospholipids.
  • FIG. 27 shows that the administration of DS102 also resolved NASFI based on validated diagnostic tests such as the OWL liver care non-invasive diagnostic test for NASFI.
  • OWL Liver Care is a test that was developed based on the plasma lipidomics in biopsy-confirmed NASFI patients and provides high predictive values.
  • OWL Liver Care has an area under the curve (AUC) of 0.88 for distinguishing NAFLD and normal liver patients and an AUC of 0.79 for distinguishing NAFLD without steatohepatitis and NASFI patients.
  • the administration of DS102 significantly improved and normalized OWL liver care diagnosed NASFI in a dose dependent manner as compared to placebo at Week 16.
  • Table 19 shows the test diagnoses at baseline for each treatment group and demonstrates that most patients were classified as NASFI or NAFLD at baseline with a lower percentage of the patients in the 2 g DS102 group.
  • Table 19 shows the test diagnoses at baseline for each treatment group and demonstrates that most patients were classified as NASFI or NAFLD
  • DS102 also reduced hepatic fat content as assessed by CAP in patients diagnosed with NAFLD as shown in Figure 28. It is further contemplated that DS102 is expected to induce larger changes in hepatic content in studies of longer duration and when assessed by more sensitive methods.
  • Figures 29A-29C shows that the administration of DS102 decreases inflammatory and pro-fibrotic proteins.
  • blood samples before and after treatment with DS102 were analyzed for a panel of greater than 350 different protein biomarkers.
  • Treatment with 2 g of DS102 significantly downregulated the expression of over 150 markers associated with inflammation, fibrosis, lipid metabolism, apoptosis, and chemotaxis.
  • the resolution of metabolic overload and lipotoxicity was observed following treatment with DS102, which prevents subsequent cell stress, inflammation, and fibrosis.
  • the reduction in the inflammatory and pro- fibrotic proteins suggests the potential of DS102 to provide resolution for NASH and the prevention of fibrosis.
  • the administration of DS102 also decreased the expression of multiple NASH development targets as shown in Figure 30.
  • the administration of 2 g of DS102 decreased the NASH drug development targets to include CCR2/5 signaling (Cenicriviroc - Allergan), Galectin3 (GR-MD-02 - Galectin), and AOC3 (Boehringer Ingelheim).
  • DS102 decreased the expression of multiple vascular adhesion molecules as shown in Figure 32.
  • Vascular adhesion molecules are implicated in atherosclerosis and their circulating levels are associated with cardiovascular risk
  • DS102 The administration of DS102 also decreased the expression of multiple tumor necrosis factor receptor superfamily members as shown in Figure 35. Tumor necrosis factor receptor superfamily members are implicated in inflammation and atherosclerosis.
  • DS102 was also proven to be safe and well tolerated, with no observed safety and tolerance differences as compared to placebo. There was no drug related serious adverse events (SAEs) and a low incidence in adverse events (AEs), which was consistent across all treatment groups. Most AEs were mild to moderate as well as transient and no patients experienced an AEs that lead to treatment discontinuation. The safety profile across each treatment group is shown in Table 23.
  • DS102 was also evaluated for potential efficacy in improving the related indications, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) as shown in Figures 36A and 36B. Importantly, administration of 2 g of DS102 reduced alkaline phosphate (ALP) as well as multiple markers for liver fibrosis suggesting that DS102 will also be effective in treating PBC and PSC.
  • PBC primary biliary cirrhosis
  • PSC primary sclerosing cholangitis
  • Figure 37 is a boxplot of 15-HEPE ethyl ester (EE) trough plasma relative concentrations, which indicates that there was a higher systematic exposure in the DS102 treatment group at Weeks 8 and 16 as expected.
  • EE ethyl ester
  • DS102 targets multiple stages of NASH pathology by significantly reducing metabolic load and improving insulin sensitivity.
  • the administration DS102 also improved patient’s lipid profiles by reversing the lipid accumulation levels associated with NASH. Specifically, those patients administered DS102 exhibited a reduction the accumulation of the hepatotoxic lipid levels to include total cholesterol, non-HDL cholesterol, RLP cholesterol, triglycerides, diglycerides, and VLDL-C as well as an increase of glycerophospholipid levels.
  • These effects are significant as patients diagnosed with NASH are characterized by high total cholesterol, triglyceride, diglyceride, and VLDL-C levels and low glycerophospholipid and omega- 3 PUFA levels. Additionally, changes to multiple lipids are expected to confer a reduction of cardiovascular risk and improvement of multiple aspects of metabolic syndrome.
  • DS102 is well suited as either monotherapy or part of combination therapy for treating NASH and is contemplated to reduce cardiovascular risk, including in patients diagnosed with NASH or cardiometabolic diseases including metabolic syndrome.
  • Example 5 A 26 Week Toxicity Study of 15(S)-HEPE EE by Oral Gavage in Rats with a 4 Week Recovery Period
  • the objectives of this study were to determine (1 ) the potential toxicity of 15(S)-HEPE EE, an omega-3 fatty acid used in the treatment of hepatic and pulmonary diseases, when given by oral gavage for 26 weeks to rats and to evaluate the potential reversibility of any findings; and (2) the toxicokinetic characteristics of 15(S)-HEPE EE.
  • test and control items were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 26 weeks.
  • the volume for each animal was based on the most recent body weight measurements.
  • the doses were given using a syringe with attached gavage cannula.
  • the first day of dosing was designated as Day 1 .
  • the dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations were also stirred continuously during dosing.
  • Spare animals will be numbered 4001M, 4002M and 4501F, 4502F.
  • Clinical observations [0546] Cage side observations were made daily. Animals were not removed from the cage during observation, unless for identification or confirmation of possible findings.
  • Body weight measurements were made twice during the pretreatment period; once daily during the dosing period; and twice weekly during the recovery period. Animals were individually weighed. A weight was recorded on the first day of scheduled necropsy (for main and recovery animals only).
  • Ophthalmic examinations were performed using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacy). During pretreatment, all main study and recovery animals were examined. During weeks 13 and 26, main study animals (control and high dose) were examined. During week 30, all recovery animals were examined.
  • a mydriatic agent 1% Tropicamide, Mydriacy
  • Sample Collection Blood was collected via the tail vein unless immediately prior to necropsy, when blood was collected from the orbital sinus under non-recoverable isoflurane anesthesia. Blood was collected in ascending animal order unless samples were collected prior to necropsy where the order of euthanasia was followed. Additional blood samples were obtained (e.g. due to clotting of non serum samples) if permissible sampling frequency and blood volume were not exceeded. After collection, samples were transferred to the appropriate laboratory for processing. Samples were collected according to the following table.
  • Hematology A 0.5 ml_ blood sample was collected using the anticoagulant K2EDTA. Blood smears were labelled, stained, and stored. The blood smears were analyzed according to the following parameters.
  • Coagulation A 0.5 ml_ blood sample was collected using the anticoagulant 3.8% (w/v) trisodium citrate for processing to plasma. To assess coagulation the following coagulation parameters were measured.
  • Urinalysis Urine was collected from animals over a period of 6 hours (+/- 30 min) with the absence of food and the presence of water. After collection, samples were transferred to the appropriate laboratory for processing. To assess the urinalysis, the following parameters were measured:
  • Bone Marrow Smear Analysis Bone marrow was collected from all animals and preserved in 10% neutral buffered formalin.
  • Bioanalytic Sample Collection A 0.5 ml_ blood sample was collected using the anticoagulant K2EDTA via the jugular vein (or the tail vein if the jugular was not assessable) using sterile needles and disposable syringes. At completion of the blood collection schedule, the toxicokinetic animals were euthanized, and the carcasses were discarded without examination. Samples were collected according to the following table.
  • Bioanalytical Sample Processing Samples were mixed gently and kept on crushed wet ice until centrifugation within 60 minutes of blood withdrawal. The samples were centrifuged at 1500g for 10 minutes at 4°C. The resultant plasma was separated, transferred to uniquely labelled clear polypropylene tubes, and frozen as soon as possible over dry ice or in a freezer set to maintain -20°C.
  • Bioanalytical Sample Analysis Plasma samples were analyzed for concentration of unesterified and total HEPE using validated analytical procedures. The following was determined: a) Unesterified (HEPE occurring in plasma as the free fatty acid or bound to plasma albumin). b) Total (quantifies unesterified HEPE and esterified HEPE (e.g. as triglycerides, cholesterol esters)) concentration at each sample time.
  • ISR Incurred sample reanalysis
  • Toxicokinetic parameters were estimated using Phoenix pharmacokinetic software. A non-compartmental approach consistent with the oral route of administration was used for parameter estimation. All parameters were generated from total free acid and unbound free acid of 15(S)-HEPE EE composition concentrations in plasma from Day 1 and Week 26 whenever practical. The following parameters were estimated.
  • Partial AUCs may be derived and reported to aid interpretation.
  • Descriptive statistics standard error for Cmax and AUC(O-t)) for appropriate grouping and sorting variables were generated using Phoenix. TK table and graphs were also generated.
  • Target tissues are liver (male and female), thyroid (male and female), kidney (female only), bone marrow sternum (male only).
  • Unscheduled Deaths If a main study or recovery animal died during the study, a necropsy was performed and specified tissues were saved. If necessary, the animals were refrigerated to minimize autolysis. Main study or recovery animals were euthanized for humane reasons as per Test Facility SOPs. The body weight was recorded and samples for evaluation of clinical pathology parameters, were obtained if possible. These animals were necropsied, and specified tissues were retained. If necessary, the animals were refrigerated to minimize autolysis.
  • Toxicokinetic animals that died during the study or were euthanized for humane reasons were subjected to a limited necropsy examination.
  • the examination consisted of an evaluation of the organs and tissues in the thoracic, abdominal, and pelvic cavities, with no tissues retained.
  • special attention was directed to evidence of possible gavage trauma.
  • the body weight was recorded and samples for evaluation of toxicokinetic analysis were collected.
  • Necropsy Main study and recovery animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.
  • Organ Weights The organs identified for weighing in the Tissues Collection and Preservation table were weighed at necropsy (unless otherwise specified) for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair was taken and entered as a tissue comment. Terminal body and brain weights were used for organ weight analysis.
  • Tissue Collection and Preservation Tissues collected according to the table below were preserved in 10% neutral buffered formalin.
  • Macroscopic afcaonnaJities in file organs fisted and m other organs will be sampl at necropsy, processed for Msiotogy and sanamecl m rostopicaUy.
  • Bone marrow smears are allowed to air dry and are not fixed in fannsln.
  • Control Item The control item for this study was 0.5% HPMC in Milli-Q water.
  • the control item was prepared as required and stored in a refrigerator set to maintain 4°C when not in use.
  • the prepared control item was removed from the refrigerator and stirred for at least 30 minutes before dosing.
  • the control item was also stirred continuously during dosing.
  • Test Item The test item for this study was DS102 or 15(S)-HEPE EE.
  • Test item dosing formulations were prepared based on a method established at the test facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared weekly and stored in a refrigerator set to maintain 4°C and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulation were also be stirred continuously during dosing.
  • the analyzed samples were submitted within the established stability period. All analyzed samples were transferred at ambient temperature to the analytical laboratory at the test facility. Any residual/retained analytical samples were maintained for a minimum of 6 months following issue of the draft report after which samples will be discarded. Alternatively, residual/retained samples were discarded prior to the 6 month period in the situation that the issue of the final report occurred prior to the end of the 6 month retention period. An earlier discard of these residual/retained samples may also be authorized by the study director after consultation with the sponsor. Does formulation samples were collected for analysis as indicated in the following table. Additional samples may be collected and analyzed at the discretion of the study director.
  • Acceptance Criteria The criteria for acceptability was mean sample concentration results within or equal to ⁇ 10% of theoretical concentration. For homogeneity, the criteria for acceptability was a relative standard deviation (RSD) of concentrations of ⁇ 10% for each group.
  • RSS relative standard deviation
  • Stability Analysis Stability analyses performed previously in conjunction with Test Facility Study No. 439414 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. See Rogers, E (2016). Validation of an Ultra High-Performance Liquid Chromatographic Method for the Determination of 15(S) HEPE EE in oral (Gavage) Dosing Formulations. Charles River Study No. 439414. Stability data was retained in the study records for Test Facility Study No. 439414. Id.
  • Animals The study used male and female Sprague Dawley rats purchased from Charles River UK Limited (Margate, Kent, UK). The target age at the initial dosing was 7-8 weeks. The target weight at the initial dosing was 175-300 g (males) and 120-250 g (females). Each animal was identified using a subcutaneously implanted electronic cylindrical, “glass-sealed” TROVAN microchip.
  • Housing The animals were allowed to acclimate to the test facility rodent toxicology accommodation for a period of up to 3 weeks before the commencement of dosing. Animals were randomly assigned to groups. Males and females were randomized separately. Animals in poor health were not assigned to groups. Animals were housed 2 or 3 per cage by sex in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Sterilized white wood shavings were used as bedding material.
  • Environmental Conditions The targeted conditions for animal room environment were as follows: temperature 19-23°C; humidity 40-70%; ventilation a minimum of 10 air changes per hour; light cycle 12 hour light and 12 hour dark. Control of light, temperature, and humidity was automatically controlled and was continuously monitored and recorded.
  • Animal Enrichment Animals were socially housed for psychological/environmental enrichment and were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study procedures/activities.
  • Veterinary Care was available throughout the course of the study and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the study records.
  • Histology Tissues identified in the Tissue Collection and Preservation table were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
  • Histopathology Histopathological evaluations were performed by a board-certified veterinary pathologist or a veterinary pathologist with training and experience in laboratory animal pathology. Microscopic examination was carried out for tissues from main study control and high dose animals, unscheduled deaths and gross lesions (including from the main study low dose group and recovery animals). If treatment effects were present in the main study high dose group, microscopic examination of target tissues from the main study low dose group and recovery animals were carried out after agreement with the Sponsor and Study Director by protocol amendment.
  • Body weight changes were calculated between each scheduled interval. Organ weight relative to body weight was calculated against the terminal body weight for scheduled intervals. Organ weight relative to brain weight was calculated against the brain weight for scheduled intervals.
  • Levene’s Test was used to assess the homogeneity of group variances. The groups were compared using an overall one way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test was not significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
  • Figure 38A-38C shows change in red blood cell count (RBC), red blood cell distribution width (RDW), and reticulocyte count (RETIC) for rats administered 15(s)-HEPE EE or placebo.
  • 15(S)-HEPE EE and placebo were administered once daily by oral gavage 7 days a week for a minimum of 26 weeks. The volume for each animal was based on the most recent body weight measurements. As noted in Table 38, the highest dose of 6 g/kg/day was not well tolerated and was replaced with a lower 3 g/kg/day.
  • Figure 39A-39C shows change in prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) concentration for rats administered 15(s)-FIEPE EE or placebo.
  • 15(S)-FIEPE EE and placebo were administered once daily by oral gavage 7 days a week for a minimum of 26 weeks. The volume for each animal was based on the most recent body weight measurements. As noted in Table 39, the highest dose of 6 g/kg/day was not well tolerated and was replaced with a lower 3 g/kg/day.
  • Table 39 Change in PT, APTT, and FIB concentration in Rats administered 15(S)-HEPE EE
  • Change in PT Figure 39A and Table 39 show that administration of both the 2 g/kg/day and 3 g/kg/day dose resulted in a significant increase in PT compared to control.
  • a method of treating and/or preventing metabolic syndrome in a subject in need thereof comprising administering to the subject 15-FIEPE, 15-FIETrE, or a composition comprising 15-FIEPE and/or HETrE.
  • Para. B A method of treating and/or preventing cardiometabolic disease in a subject in need thereof, the method comprising administering to the subject 15- FIEPE, 15-FIETrE, or a composition comprising 15-FIEPE and/or HETrE.
  • a method of treating and/or preventing metabolic syndrome and/or cardiometabolic disease in a subject in need thereof comprising administering to the subject up to about 8g of a composition comprising 15-HEPE and/or 15-HETrE, wherein the 15-HEPE and/or 15-HETrE represents at least about 90%, by weight, all fatty acids in the composition.
  • Para. D A method of treating and/or preventing metabolic syndrome and/or cardiometabolic disease in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition and, wherein the subject exhibits one or more of: a reduction in diglyceride, glycerophospholipid, hepatic fat, blood pressure, waist circumference, and/or free fatty acid levels; and/or an increase in glycerophospholipid levels.
  • a reduction in diglyceride, glycerophospholipid, hepatic fat, blood pressure, waist circumference, and/or free fatty acid levels and/or an increase in glycerophospholipid levels.
  • a method of preventing a first stage of non-alcoholic steatohepatitis (NASH) from progressing to a second stage of NASH in a subject comprising administering to the subject up to about 8g of a composition comprising 15-HEPE.
  • NASH non-alcoholic steatohepatitis
  • Para. F The method of Para. E, wherein the first stage is metabolic overload, increased hepatic fat content and lipotoxicity, cell stress apoptosis, inflammation, and/or fibrogenic remodeling.
  • Para. G The method as in Para. E or Para. F, wherein the second stage is increased hepatic fat content and lipotoxicity, cell stress apoptosis, inflammation, and/or fibrogenic remodeling.
  • NAFLD non-alcoholic fatty liver disorder
  • HETrE cardiometabolic disease
  • a method of treating and/or preventing cardiovascular disease in a subject having non-alcoholic fatty liver disorder (NAFLD), metabolic syndrome, or cardiometabolic disease in a subject in need thereof comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • NAFLD non-alcoholic fatty liver disorder
  • a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90%, by weight, all fatty acids in the composition.
  • Para. J The method as in any one of Paras. A to I, wherein the subject exhibits a reduction in one or more of: a-smooth muscle action (a-SMA), metallopeptidase inhibitor-1 (TIMP-1), transforming growth factor beta-b (TGF-b), and/or Collagen Type 1 levels.
  • a-SMA smooth muscle action
  • TGF-b transforming growth factor beta-b
  • Para. K The method as in any one of Paras. A to C or E to J, wherein the subject exhibits a reduction in diglyceride, hepatic fat, blood pressure, waist circumference, and/or free fatty acid levels and/or an increase in glycerophospholipid levels.
  • Para. L The method as in any one of Paras. A to K, wherein the subject exhibits a reduction in alkaline phosphate (ALP) levels.
  • Para. M The method as in any one of Paras. A to L, wherein the subject exhibits a reduction in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and/or bilirubin (BUN) levels.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • BUN bilirubin
  • Para. N The method as in any one of Paras. A to M, wherein the subject exhibits a reduction in fibrosis area.
  • Para. 0. The method as in any one of Paras. A to N, wherein the subject exhibits a reduction in hemoglobin A1 C (HbA1 C), homeostatic model assessment of insulin resistance (HOMA-IR), and/or adipose tissue insulin resistance (adipo-IR) levels.
  • HbA1 C hemoglobin A1 C
  • HOMA-IR homeostatic model assessment of insulin resistance
  • adipo-IR adipose tissue insulin resistance
  • Para. P The method as in any one of Paras. H to O, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).
  • NAFLD non-alcoholic steatohepatitis
  • the cardiometabolic disease or the cardiovascular disease is one or more of: dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, primary hypercholesterolemia, primary hyperlipidemia, common primary hyperlipidemia, common hypercholesterolemia, familial hyperlipidemia, familial primary hyperlipidemia, familial hypercholesterolemia, familial hypertriglyceridemia, familial combined hyperlipidemia, familial defective apolipoprotein b-100, secondary hyperlipidemia, mixed hyperlipidemia, cardiovascular disease, residual cardiovascular risk, prevention of atherosclerotic plaque formation/progression, microvascular disease, macrovascular disease, atherosclerosis, coronary atherosclerosis, diastolic dysfunction, reduction of cardiovascular risk, prevention of major coronary events, prevention of major adverse cardiovascular events, prevention of ischemic events, secondary/primary prevention of cardiovascular events, prevention of cardiovascular death, myocardial infarction, stroke, angina, restoration of
  • Para. R The method of Para. Q, wherein the microvascular disease is retinopathy, nephropathy, neuropathy, or combination thereof.
  • Para. S The method of Para. Q, wherein the macrovascular disease is stroke, peripheral vascular disease, limb ischemia, heart disease, or combination thereof.
  • Para. T The method as in any one of Paras. A to S, wherein the subject exhibits a reduction in very low-density lipoprotein cholesterol (VLDL-C), non-high- density lipoprotein cholesterol (non-HDL-C), and/or remnant-like particle cholesterol (RLP-C) and/or a high-density lipoprotein cholesterol (HDL-C) levels.
  • VLDL-C very low-density lipoprotein cholesterol
  • non-HDL-C non-high- density lipoprotein cholesterol
  • RLP-C remnant-like particle cholesterol
  • HDL-C high-density lipoprotein cholesterol
  • Para. U The method as in any one of Paras. A to T, wherein the subject exhibits a reduction in liver stiffness, fibrosis-4 (FIB-4), enhanced liver fibrosis (ELF) score and/or NAFLD score (NFS).
  • FIB-4 fibrosis-4
  • EEF enhanced liver fibrosis
  • NFS NAFLD score
  • Para. V The method as in any one of Paras. A to U, wherein the subject exhibits a reduction in inflammatory and pro-fibrotic proteins selected from the group consisting of plasminogen activator inhibitor-1 (PAI-1), metallopeptidase inhibitor-1 (TIMP-1), dipeptidyl peptidase 4 (DPP4), trem-like transcript 2 (TLT2), chemokine (C- C motif) ligand 16 (CCL16), monocyte chemoattractant protein-1 (MCP-1), serum amyloid A4 (SAA4), phosphoinositide 3 (PI3), thioredoxin reductase (TR), leukocyte immunoglobulin like receptor B1 (LILBR1), amine oxidase, copper containing 3 (AOC3), serine protease 2 (PRSS2), and tumor necrosis factor ligand superfamily member 11A (TNRSF11A).
  • PAI-1 plasminogen activator inhibitor-1
  • TPP4 dipeptidy
  • Para. W A method of treating or preventing cholestatic liver disease in a subject, the method comprising administering to the subject 15-HEPE, 15-HETrE, or a composition comprising 15-HEPE and/or 15-HETrE.
  • Para. X The method of Para. W, wherein the cholestatic liver disease is primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis, or combination thereof.
  • PBC primary biliary cholangitis
  • PSC primary sclerosing cholangitis
  • progressive familial intrahepatic cholestasis or combination thereof.
  • Para. Y The method of Para. W. or Para. X, wherein the cholestatic liver disease is caused by a drug induced liver injury, total parenteral nutrition (TPN), viral and alcoholic hepatitis, cholestasis secondary to systemic diseases, graft dysfunction, post liver transplant cholestasis, pancreatitis, choledocholithiasis, Mirizzi syndrome, genetic diseases, malignancy, or combination thereof.
  • TPN total parenteral nutrition
  • Para. Z The method of Para. Y, wherein the malignancy is a hepatocellular carcinoma, a bile duct tumor, pancreatic carcinoma, or combination thereof.
  • Para. AA The method as in any one of Paras. W to Z, wherein the subject exhibits a reduction in cytokines and/or chemokines selected from the group consisting a-smooth muscle action (a-SMA), metallopeptidase inhibitor-1 (TIMP-1 ), transforming growth factor beta-b (TGF-b), and Collagen Type 1 levels.
  • a-SMA a-smooth muscle action
  • TGF-b transforming growth factor beta-b
  • Collagen Type 1 levels selected from the group consisting a-smooth muscle action (a-SMA), metallopeptidase inhibitor-1 (TIMP-1 ), transforming growth factor beta-b (TGF-b), and Collagen Type 1 levels.
  • Para. BB A method of treating or preventing kidney disease in a subject, the method comprising administering to the subject 15-HEPE and/or 15-HETrE or a composition comprising 15-HEPE and/or 15-HETrE, wherein the subject has at least one risk factor for kidney disease.
  • Para. CC The method of Para. BB, wherein the kidney disease is selected from the group consisting of kidney fibrosis, tubulointerstitial fibrosis, chronic kidney disease, severe interstitial fibrosis, renal interstitial fibrosis, and end stage renal disease.
  • Para. DD The method of Para. CC, wherein the kidney disease leads to fibrosis.
  • Para. EE The method as in any one of Paras. BB to DD, wherein the at least one risk factor for a kidney disease is selected from the group consisting of diabetes, high blood pressure, cardiovascular disease, glomerulonephritis, and polycystic kidney disease.
  • Para. FF The method as in any one of Paras. BB to EE, wherein the subject exhibits a reduction in kidney hydroxyproline levels.
  • Para. GG The method as in any one of Paras. BB to FF, wherein the subject exhibits no increase in a-SMA, TIMP-1 , TGF-b, and/or Collagen Type 1 levels.
  • Para. HH The method as in any one of Paras. BB to GG, wherein the subject exhibits a reduction in a-SMA, TIMP-1 , TGF-b, and/or Collagen Type 1 levels.
  • Para. II The method as in any one of Paras. AA to HH, wherein the subject exhibits a reduction in pro-fibrotic cytokines in the liver.
  • pro-fibrotic cytokines are one or more of a-SMA, TIMP-1 , TGF-b, Collagen Type 1 , interleukin 1 b (IL-1 b), interleukin-6 (IL-6), interleukin-6 (IL-8), interleukin-13 (IL-13), tumor necrosis factor (TNF-a), TNF-like ligand 1A (TL1A), aryl hydrocarbon receptor (AhR), interleukin-17 (IL-17), interleukin-23 (IL-23), interleukin-11 (IL-11 ), and/or interleukin-33 (IL-33).
  • Para. KK The method as in any one of Paras. A to JJ, wherein the subject exhibits a reduction in vascular adhesion molecules and/or chemokines and/or tumor necrosis factor receptor superfamily members.
  • Para. LL The method as in any one of Paras, A to KK, wherein the 15- FIEPE, 15-FIETrE, or the composition comprising 15-FIEPE and/or 15-FIETrE is orally administered.
  • Para. MM The method as in any one of Paras. A to LL, wherein the 15- FIEPE and/or 15-HETrE is in free acid form, esterified form, or salt form.
  • Para. NN The method of Para. MM, wherein the esterified form is an alkyl ester form or a triglyceride form.
  • Para. OO The method as in any one of Paras. A to NN, wherein the 15- FIEPE comprises 15(S)-HEPE, 15(R)-HEPE, or combinations thereof and/or the 15- FIETrE comprises 15(S)-FIETrE, 15(R)-FIETrE, or combinations thereof.
  • Para. PP The method as in any one of Paras. A to OO, wherein the composition comprises about 1 g to about 2 g of 15-FIEPE and/or 15-FIETrE.
  • Para. QQ The method as in any one of Paras. A to PP, wherein the composition comprises about 2 g or more of 15-FIEPE and/or 15-FIETrE.
  • Para. RR The method as in any one of Paras. A to QQ, wherein the composition comprises about 1 g or about 2 g of 15-FIEPE and/or 15-FIETrE.
  • Para. SS The method as in any one of Paras. A to RR, wherein the composition comprises about 10 mg to about 10,000 mg of 15-FIEPE and/or 15-FIETrE.
  • Para. TT The method as in any one of Paras. A to SS, wherein the composition comprises about 5 mg/kg, about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg of 15-HEPE and/or 15-HETrE.
  • Para. UU The method as in any one of Paras. A to TT, wherein the 15- HEPE and/or 15-HETrE represents at least about 90%, by weight, of all fatty acids present in the composition.
  • Para. W The method as in any one of Paras. A to UU, wherein the composition is administered in 1 to 8 capsules per day.
  • Para. WW The method as in any one of Paras. A to C or H to W, wherein the method comprises administering to the subject 15-HEPE or a composition comprising 15-HEPE.
  • Para. XX The method as in any one of Paras. A to C or H to W, wherein the method comprises administering to the subject 15-HETrE or a composition comprising 15-HETrE.
  • a method of treating and/or preventing a hematologic disorder in a subject in need thereof comprising administering to the subject 15- hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15- hydroxyeicosapentaenoic acid (15-HEPE).
  • Para. ZZ The method of Para. YY, wherein the hematologic disorder is selected from the group consisting of anemia, blood cancer, and coagulation defects.
  • Para. AB The method of Para. ZZ, wherein the anemia is selected from the group consisting of nutritional anemias and non-nutritional anemias.
  • Para. AC The method of Para. ZZ, wherein the blood cancer is selected from the group consisting of lymphoma, leukemia, and myeloma.
  • Para. AD The method of Para. ZZ, wherein the coagulation defect is selected from the group consisting of thrombophilia, hemophilia, Von Willebrand disease, and thrombocytopenia.
  • a method of treating and/or preventing a hemoglobin disorder in a subject in need thereof comprising administering to the subject 15- HEPE or a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE).
  • Para. AF A method of treating and/or preventing a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject 15- HEPE or a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE).
  • Para. AG A method of treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • Para. AH A method of treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition, and wherein the subject exhibits one or more of:
  • Para. Al The method Para. AH, wherein where the subject has an increase in red blood cell count of at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% compared to a control subject not receiving the composition.
  • Para. AJ The method of Para. Al, wherein the control subject has a red blood cell count of about 4x10 12 red blood cells/L.
  • Para. AK The method of Para. AH, wherein the subject has a decrease in red blood cell distribution width of about 15-20%, of about 20-25%, of about 25-30%, of about 30-35%, or of about 35-40% compared to a control subject not receiving the composition.
  • Para. AL The method of Para. AK, wherein the control subject has a red blood cell distribution width greater than 15%.
  • Para. AM The method of Para. AH, wherein the subject has a decrease in reticulocyte count of at least about 5%, at least about 10%, at least about 15%, or at least about 20% compared to a control subject not receiving the composition.
  • Para. AN The method of Para. AM, where the control subject has a reticulocyte count greater than 5% of the total number of red blood cells.
  • Para. AO A method of treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • Para. AP The method of Para. AO, wherein the hemolytic anemia is inherited hemolytic anemia or acquired hemolytic anemia.
  • Para. AQ The method of Para. AP, wherein the inherited hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, b-thalassemia, and hereditary spherocytosis.
  • Para. AR The method of Para. AP, wherein the acquired hemolytic anemia is selected from the group consisting of secondary to infection, medication, hematological malignancy, autoimmune disease, hypersplenism, mechanical heart valves, and blood transfusions.
  • Para. AU A method of any one of Paras. AE to AN, wherein the hemoglobin disorder and/or the red blood cell disorder are selected from the group consisting of inherited hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.
  • Para. AV The method of any one of Paras. YY to AG, or AO to AT, wherein the subject exhibits an increase in red blood cell count, a decrease in reticulocyte count, and a decrease in red cell distribution width.
  • a method of treating and/or preventing a hematologic disorder and/or a blood disorder in a subject in need thereof comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • Para. AX The method of Para. AW, wherein the 15-HEPE is in the form of an ethyl ester (15-HEPE EE) or the 15-HEPE is in the form of an optically active ester (15(S)-HEPE EE).
  • Para. AY The method of Para. AW, wherein the hematologic disorder and/or the blood disorder are selected from the group consisting of inherited hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.
  • Para. AZ The method of Para. AY, wherein the hemolytic anemia is inherited hemolytic anemia or acquired hemolytic anemia.
  • Para. BA The method of Para. AZ, wherein the inherited hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, b-thalassemia, and hereditary spherocytosis.
  • Para. BC The method of Para. AZ, wherein the acquired hemolytic anemia is selected from the group consisting of secondary to infection, medication, hematological malignancy, autoimmune disease, hypersplenism, mechanical heart valves, and blood transfusions.
  • Para. BD The method of Para. BA, wherein the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration.
  • Para. BE The method of any one of Paras AW to BD, wherein the subject exhibits an increase in red blood cell count, a decrease in reticulocyte count, and a decrease in red cell distribution width.
  • Para. BF A method of treating and/or preventing thrombophilia disorder in a subject in need thereof, the method comprising administering to the subject 15- FIEPE or a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE).
  • BG A method of treating and/or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-FIEPE, wherein the 15-FIEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • Para. BH A method of treating and/or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject up to about 8g of a composition comprising 15-FIEPE, wherein the 15-FIEPE represents at least about 90% by weight, of all fatty acids in the composition, and wherein the subject exhibits one or more of: (a) an increase in prothrombin time;
  • a method of treating and/or preventing an arterial thrombosis in a subject in need thereof comprising administering to the subject up to about 8g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • a method of preventing an embolism in a subject in need thereof comprising administering to the subject up to about 8g of a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE), wherein the 15-HEPE represents at least about 90% by weight, of all fatty acids in the composition.
  • 15-HEPE 15-hydroxyeicosapentaenoic acid
  • Para. BL The method of Paras. Bl to BK, wherein the first stage is formation of a thrombus.
  • Para. BM The method of any one of Paras. BF to BG or Bl to BL, wherein the subject exhibits an increase in prothrombin time, an increase in activated partial thromboplastin time, and a decrease in fibrinogen concentration.
  • Para. BN The method of any one of Paras. YY to BM, wherein the 15- HEPE is orally administered.
  • Para. BO The method of any one of Paras. YY to BN, wherein the composition is administered in 1 to 8 capsules per day.
  • Para. BP The method of any one of Pars. YY to BO, wherein the 15- HEPE is in free acid form, esterified form, or salt form.
  • Para. BQ The method of any one Paras. YY to BP, wherein the composition comprises about 1 g to about 2 g of 15-HEPE.
  • Para. BR The method of any one of Paras. YY to BQ, wherein the composition comprises about 2 g to more of 15-HEPE.
  • Para. BS The method of any one Paras. YY to BR, wherein the composition comprises about 5mg/kg, about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg or 15-HEPE.

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EP20717175.2A 2020-04-03 2020-04-03 Compositions comprising 15-hepe for treating or preventing hematologic disorders, and/or related diseases Pending EP4125854A1 (en)

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