Compositions containing 15-HEPE for treating or preventing blood disorders and / or related diseases

Abstract

The present disclosure relates to methods of treating or preventing blood disorders by administering 15-HEPE or compositions thereof.

Description

[Technical Field]

[0001] This application relates generally to compositions containing 15-HEPE and methods of using the same.

[0002] Sequence Listing

[0001] This disclosure contains a Sequence Listing that has been submitted in ASCII format via EFS Web and is incorporated herein by reference in its entirety. The ASCII copy created on April 1, 2020, is Sequence Listing entitled 124318-8014.US00_ST25.txt and is 3 kilobytes in size. [Background technology]

[0003] Blood disorders include red blood cell disorders and thrombophilia. Red blood cell disorders are a class of diseases that affect the production, lifespan, and shape of red blood cells (RBCs) and their oxygen-carrying molecule, hemoglobin. These diseases inhibit the transport of oxygen from the lungs to other parts of the body. Thrombophilia is a class of disorders that increases a subject's risk of developing blood clots in veins and arteries. These clots can break away from blood vessels and travel to organs via the bloodstream, thereby blocking blood flow to those organs and causing ischemia. These diseases contribute to an increased risk of stroke and / or pulmonary embolism. Summary of the Invention

[0004] This application relates to compositions comprising 15-hydroxyeicosapentaenoic acid (15-HEPE) and methods of using such compositions in the treatment of various diseases and disorders.

[0005] In some aspects, the present disclosure provides methods for treating and / or preventing a hematological disorder in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject.

[0006] In some aspects, the present disclosure provides methods for treating and / or preventing a hematological disorder in a subject in need thereof, the methods comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0007] In yet another aspect, the disclosure provides a method of treating and / or preventing a hematological disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% of all lipids in the composition, and wherein the subject exhibits one or more of: (a) an increased red blood cell count; (b) a decreased red blood cell distribution width; and / or (c) a decreased reticulocyte count.

[0008] In various embodiments, the blood disorder is selected from the group consisting of hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.

[0009] In some aspects, the present disclosure provides methods for treating and / or preventing hemoglobin disorders in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject.

[0010] In another aspect, the present disclosure provides a method for treating and / or preventing an red blood cell disorder in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject.

[0011] In another aspect, the present disclosure provides a method for treating and / or preventing hemoglobin disorders and / or red blood cell disorders in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0012] In yet another aspect, the disclosure provides a method of treating and / or preventing hemoglobin disorders and / or red blood cell disorders in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% of all lipids in the composition, and wherein the subject exhibits one or more of: (a) an increased red blood cell count; (b) a decreased red blood cell distribution width; and / or (c) a decreased reticulocyte count.

[0013] In various embodiments, the blood disorder, hemoglobin abnormality and / or red blood cell disorder is selected from the group consisting of hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.

[0014] In yet another aspect, the present disclosure provides a method of treating and / or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0015] In some embodiments, the hemolytic anemia is hereditary hemolytic anemia or acquired hemolytic anemia. In another embodiment, the acquired hemolytic anemia is caused by an infection. accompanying do Occurred , from drug therapy, from hematological malignancies, from autoimmune diseases, from hypersplenism, from mechanical heartThis occurs after placement of a valve, a blood transfusion, or a combination thereof. In yet another embodiment, sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, splenic blood cell retention, or a combination thereof.

[0016] In some embodiments, the subject exhibits an increased red blood cell count, a decreased red blood cell distribution width, and a decreased reticulocyte count. In some aspects, the present disclosure provides methods of treating and / or preventing a thrombophilic disorder in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject.

[0017] In another aspect, the present disclosure provides a method for treating and / or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0018] In another aspect, the disclosure provides a method of treating and / or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% of all lipids in the composition, and wherein the subject exhibits one or more of: (a) a prolonged prothrombin time; (b) a prolonged activated partial thromboplastin time; and / or (c) a reduced fibrinogen concentration.

[0019] In yet another aspect, the present disclosure provides a method of treating and / or preventing venous thromboembolism in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0020] In one aspect, the disclosure provides a method of treating and / or preventing arterial thrombosis in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0021] In another aspect, the present disclosure provides a method for preventing embolism in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0022] In some embodiments, the subject exhibits a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and a reduced fibrinogen concentration.

[0023] In another embodiment, the 15-HEPE is in the free acid form, an esterified form, or a salt form. In yet another embodiment, the esterified form is an alkyl ester form or a triglyceride form. In some embodiments, the 15-HEPE comprises 15(S)-HEPE, 15(R)-HEPE, or a combination thereof.

[0024] In some embodiments, 15-HEPE is in the form of an ethyl ester (15-HEPE EE), or 15-HEPE is in the form of an optically active ester (15(S)-HEPE EE).

[0025] In some embodiments, the composition comprises about 10 mg to about 10,000 mg of 15-HEPE. In other embodiments, the composition comprises about 1 g to about 2 g of 15-HEPE. In other embodiments, the composition comprises about 2 g or more of 15-HEPE. In yet other embodiments, the composition is formulated to provide a subject with about 5 mg of 15-HEPE per kilogram of body weight (mg / kg), about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg of 15-HEPE. In various embodiments, the 15-HEPE represents at least about 90% by weight of all fatty acids present in the composition.

[0026] In some embodiments, 15-HEPE is administered orally. In various embodiments, the composition is administered in 1 to 8 capsules per day. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram of the study and its duration described in Example 1.

[0028] [Figure 2] 1 shows the changes in animal weight from the study described in Example 1.

[0029] [Figure 3] 1 shows the body weights of the animals on the day of sacrifice according to the study described in Example 1.

[0030] [Figure 4A] 1 shows the kidney weights of animals on the day of sacrifice from the study described in Example 1. [Figure 4B] 1 shows the kidney weight to body weight ratio of animals on the day of sacrifice from the study described in Example 1. [Figure 4C] 1 shows the kidney weights of animals on the day of sacrifice from the study described in Example 1. [Figure 4D] 1 shows the kidney weight to body weight ratio of animals on the day of sacrifice from the study described in Example 1.

[0031] [Figure 5]1 shows the kidney hydroxyproline content of animals from the study described in Example 1.

[0032] [Figure 6A] 1 shows Sirius Red staining of animals from the study described in Example 1. [Figure 6B] 1 shows Sirius Red staining of animals from the study described in Example 1. [Figure 6C] 1 shows Sirius Red staining of animals from the study described in Example 1. [Figure 6D] 1 shows Sirius Red staining of animals from the study described in Example 1. [Figure 6E] 1 shows Sirius Red staining of animals from the study described in Example 1. [Figure 6F] 1 shows Sirius Red staining of animals from the study described in Example 1. [Figure 6G] 1 shows Sirius Red staining of animals from the study described in Example 1.

[0033] [Figure 7] 1 shows a plot showing the % Sirius Red positive area of ​​animals from the study described in Example 1.

[0034] [Figure 8A] 1 shows gene expression analysis for α-SMA in animals from the study described in Example 1. [Figure 8B] 1 shows gene expression analysis for TIMP-1 in animals from the study described in Example 1. [Figure 8C] 1 shows gene expression analysis for TGF-β in animals from the study described in Example 1. [Figure 8D] 1 shows gene expression analysis for type 1 collagen in animals from the study described in Example 1.

[0035] [Figure 9] FIG. 1 is a schematic diagram of the study and its duration described in Example 2.

[0036] [Figure 10] 1 shows the changes in animal weight from the study described in Example 2.

[0037] [Figure 11] 1 shows the body weights of the animals on the day of sacrifice according to the study described in Example 2.

[0038] [Figure 12A] 1 shows the liver weights of the animals on the day of sacrifice according to the study described in Example 2. [Figure 12B] 1 shows the liver weight to body weight ratios of the animals on the day of sacrifice from the study described in Example 2, respectively.

[0039] [Figure 13] 1 shows the changes in serum ALT levels in animals from the study described in Example 2.

[0040] [Figure 14] 1 shows the change in serum total bilirubin levels in animals from the study described in Example 2.

[0041] [Figure 15A] 1 shows Sirius red staining and areas of fibrosis in animals from the study described in Example 2. [Figure 15B] 1 shows Sirius red staining and areas of fibrosis in animals from the study described in Example 2. [Figure 15C] 1 shows Sirius red staining and areas of fibrosis in animals from the study described in Example 2. [Figure 15D] 1 shows Sirius red staining and areas of fibrosis in animals from the study described in Example 2. [Figure 15E] 1 shows Sirius red staining and areas of fibrosis in animals from the study described in Example 2.

[0042] [Figure 16] 1 shows a plot showing the % Sirius Red positive area of ​​animals from the study described in Example 2.

[0043] [Figure 17A] 1A-1C show gene expression analysis for α-SMA in animals from the study described in Example 2, respectively. [Figure 17B] 1 shows gene expression analysis for TIMP-1 in animals from the study described in Example 2, respectively. [Figure 17C] 1A-1C show gene expression analysis for TGF-β in animals from the study described in Example 2, respectively. [Figure 17D] 1A and 1B show gene expression analysis for type 1 collagen in animals from the study described in Example 2, respectively.

[0044] [Figure 18A] 1 shows that DS102 (15-HEPE) induced inhibition of TGF-β, according to the study described in Example 3. [Figure 18B] 1 shows that DS102 (15-HEPE) induced inhibition of TGF-β, according to the study described in Example 3.

[0045] [Figure 18C] 1 shows the effect of DS102 on membrane trafficking and degradation of TGF-β type I receptor, TGF-β type II receptor, TGF-β type III receptor, EGFR, and caveolin-1 from the study described in Example 3. [Figure 18D] 1 shows the effect of DS102 on membrane trafficking and degradation of TGF-β type I receptor, TGF-β type II receptor, TGF-β type III receptor, EGFR, and caveolin-1 from the study described in Example 3. [Figure 18E] 1 shows the effect of DS102 on membrane trafficking and degradation of TGF-β type I receptor, TGF-β type II receptor, TGF-β type III receptor, EGFR, and caveolin-1 from the study described in Example 3. [Figure 18F] 1 shows the effect of DS102 on membrane trafficking and degradation of TGF-β type I receptor, TGF-β type II receptor, TGF-β type III receptor, EGFR, and caveolin-1 from the study described in Example 3. [Figure 18G]1 shows the effect of DS102 on membrane trafficking and degradation of TGF-β type I receptor, TGF-β type II receptor, TGF-β type III receptor, EGFR, and caveolin-1 from the study described in Example 3.

[0046] [Figure 19] FIG. 1 is a schematic diagram of the study and its duration described in Example 4.

[0047] [Figure 20] 1 shows the baseline lipidomic profile of patients from the study described in Example 4.

[0048] [Figure 21A] 1 is a plot showing the change in insulin levels in patients receiving epeleuton (15-HEPE) and placebo, respectively. [Figure 21B] 1 is a plot showing the change in glucose levels in patients administered epeleuton (15-HEPE) and placebo, respectively. [Figure 21C] 1 is a plot showing the change in free fatty acid levels in patients administered epeleuton (15-HEPE) and placebo, respectively.

[0049] [Figure 22A] 1 is a plot showing the change in HOMA-IR levels in patients administered epeleuton and placebo, respectively. [Figure 22B] 1 is a plot showing the change in apido-IR levels in patients receiving epeleuton and placebo, respectively.

[0050] [Figure 23A] 1 is a plot showing the change in mean HbA1C levels in patients, respectively. [Figure 23B] 1A and 1B are plots showing the proportion of patients with HbA1C levels ≧6.5% at 16 weeks, respectively.

[0051] [Figure 24A] 1A-1C are plots showing the mean change in lipid profile in patients receiving epeleuton and placebo, respectively. [Figure 24B] 1 is a plot showing the median % change in lipid profile in patients receiving epeleuton and placebo, respectively.

[0052] [Figure 25A] 1A-1C are plots showing the change in cholesterol levels in patients receiving Epeleuton and placebo, respectively. [Figure 25B] 1 is a plot showing the change in triglyceride levels in patients receiving Epeleuton and placebo, respectively. [Figure 25C] 1A-1C are plots showing the change in VLDL-C levels in patients receiving Epeleuton and placebo, respectively.

[0053] [Figure 26] 1 is a chart showing changes in hepatotoxic lipid profiles in patients receiving DS102.

[0054] [Figure 27] 1 is a plot demonstrating that administration of DS102 resolves NASH using the OWL Liver Care trial.

[0055] [Figure 28] 1 is a plot showing the change in liver fat content by CAP in patients receiving DS102 and placebo.

[0056] [Figure 29A] 1 is a chart showing changes in levels of inflammatory and profibrotic proteins in patients receiving DS102 and placebo. [Figure 29B] 1 is a chart showing changes in levels of inflammatory and profibrotic proteins in patients receiving DS102 and placebo. [Figure 29C]1 is a chart showing changes in levels of inflammatory and profibrotic proteins in patients receiving DS102 and placebo.

[0057] [Figure 30] 1 is a plot showing changes in expression of proteins, including NASH development targets, in patients receiving DS102 and placebo.

[0058] [Figure 31] 1 is a volcano plot showing reductions in inflammatory and profibrotic proteins in patients receiving DS102 and placebo.

[0059] [Figure 32] 1 is a plot showing changes in vascular adhesion molecules in patients receiving DS102 and placebo.

[0060] [Figure 33] 1 is a plot showing changes in cardiovascular risk proteins in patients receiving DS102 and placebo.

[0061] [Figure 34] 1 is a plot showing changes in chemokines in patients receiving DS102 and placebo.

[0062] [Figure 35] 1 is a plot showing changes in tumor necrosis factor receptor superfamily members in patients receiving DS102 and placebo.

[0063] [Figure 36A] 1 is a plot showing the reduction in ALP levels in patients receiving DS102 and placebo. [Figure 36B] 1 is a plot showing the reduction in ALP levels in patients receiving DS102 and placebo.

[0064] [Figure 37]FIG. 1 is a box plot of 15-HEPE ethyl ester trough plasma relative concentrations.

[0065] [Figure 38A] 1 is a bar graph showing the mean change and standard deviation of red blood cell counts in rats administered 15(S)-HEPE EE and placebo, respectively. [Figure 38B] 1 is a bar graph showing the mean change and standard deviation of red blood cell distribution width in rats administered 15(S)-HEPE EE and placebo, respectively. [Figure 38C] 1 is a bar graph showing the mean change and standard deviation of reticulocyte counts in rats administered 15(S)-HEPE EE and placebo, respectively.

[0066] [Figure 39A] 1 is a bar graph showing the mean change and standard deviation of prothrombin time in rats administered 15(S)-HEPE EE and placebo, respectively. [Figure 39B] 1 is a bar graph showing the mean change and standard deviation of activated partial thromboplastin time in rats administered 15(S)-HEPE EE and placebo, respectively. [Figure 39C] 1 is a bar graph showing the mean change and standard deviation of fibrinogen concentration in rats administered 15(S)-HEPE EE and placebo, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0067] While the present invention may be embodied in various forms, the following description of some embodiments is made with the understanding that the present disclosure should be considered as an example of the invention and that it is not intended that the invention be limited to the particular embodiments described. Headings are provided for convenience only and should not be construed as limiting the invention in any way. Embodiments shown under any heading may be combined with embodiments shown under any other heading.

[0068] The use of numerical values ​​in the various quantitative values ​​specified in this application, unless expressly stated otherwise, is stated as an approximation, as if the word "about" were added before both the minimum and maximum values ​​in the stated range. In this manner, slight variations from the stated values ​​can be used to achieve substantially the same results as the stated values. The disclosure of ranges is also intended as a continuous range, including every value between the minimum and maximum recited values, and any range that can be formed by such values. Also disclosed herein are any and all ratios (and any such ratio ranges) that can be formed by dividing a recited numerical value into any other recited numerical value. Accordingly, one of ordinary skill in the art will understand that many such ratios, ranges, and ratio ranges can be clearly derived from the numerical values ​​presented herein, and that in all cases, such ratios, ranges, and ratio ranges represent various embodiments of the present invention.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0070] definition As used herein, "15-HEPE" refers to 15-hydroxy-eicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid. 15-HEPE, also referred to as 15-OHEPA, can be synthesized from the omega-3 fatty acid eicosapentaenoic acid ("EPA," eicosa-5,8,11,14,17-pentaenoic acid or 20:5n-3) according to methods known in the art. For example, 15-HEPE can be synthesized by exposing EPA to the enzyme 15-lipoxygenase. As used herein, the term "15-HEPE" refers to 15-HEPE in its free acid form (e.g., 15-hydroxy-eicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid) and / or its pharmaceutically acceptable esters, conjugates, or salts, or mixtures of any of the foregoing. Alternatively, derivatives of 15-HEPE may be used, but this does not include any derivative compounds lacking the hydroxy group of 15-HEPE. In some embodiments, 15-HEPE is used in its free acid form. Alternatively, a pharmaceutically acceptable ester or salt of 15-HEPE is used in the present disclosure. In some embodiments, 15-HEPE is used in the form of a C ester, such as the methyl ester, the ethyl ester, or a combination of the methyl ester and the ethyl ester. 1~4 In yet another embodiment, 15-HEPE is in the form of an alkyl ester. In yet another embodiment, 15-HEPE is in the form of a glyceride (e.g., a diglyceride or triglyceride). In another embodiment, 15-HEPE is in the form of an ethyl ester (also referred to herein as E-15-HEPE, 15-HEPEEE, or ethyl-15-HEPE).

[0071] 15-HEPE is a chiral molecule and can be used in either the (S)- or (R)-enantiomeric form, or as a racemic mixture. As used herein, "15-HEPE" includes all such forms without limitation regarding stereospecificity. In another embodiment, 15-HEPE includes the (S) form: 15(S)-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid or the (R) form ((R)from): 15(R)-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid. In yet another embodiment, 15-HEPE EE includes the (S) form: 15(S)-HEPE EE.

[0072] As used herein, "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.

[0073] As used herein, "Epeleuton" refers to 15-HEPE, 15-HEPE EE, a composition containing 15-HEPE, a composition containing 15-HEPE EE, or a composition containing 15-HEPE and 15-HEPE EE.

[0074] As used herein, "treating" or "treatment" of a disease, disorder, or condition includes, at least in part, (1) inhibiting the disease, disorder, or condition, i.e., halting or reducing the progression of the disease, disorder, or condition or its clinical symptoms, or (2) alleviating the disease, disorder, or condition, i.e., causing regression of the disease, disorder, or condition or its clinical symptoms. The term "prevention" in reference to a given disease or disorder means preventing the onset of disease progression if none has occurred, preventing the disease or disorder from occurring in a subject who may be susceptible to the disorder or disorder but has not yet been diagnosed with the disorder or disorder, and / or preventing further progression of the disease / disorder if the disease or disorder is already present.

[0075] As used herein, an "effective amount" refers to the amount of an active composition required to confer a therapeutic effect on a subject. As used herein, a "therapeutically effective amount" refers to a sufficient amount of an agent or compound being administered to 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. For example, in some embodiments, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein required to provide a clinically significant reduction in disease symptoms without undue adverse side effects. In some embodiments, 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. In other embodiments, 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 pharmacological effect or therapeutic improvement without undue adverse side effects. In other embodiments, it is understood that an "effective amount" or "therapeutically effective amount" will vary from subject to subject, depending on variations in the subject's metabolism, age, weight, general illness, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. The term "pharmaceutically acceptable" in this context means that the substance in question does not produce unacceptable toxicity to the subject or interact with other components of the composition.

[0076] Other features and advantages of the present disclosure will become apparent from the following detailed description.

[0077] composition In one embodiment, the composition of the present disclosure comprises 15-HEPE as an active ingredient. The term "pharmaceutically acceptable" in this context means that the substance in question does not produce unacceptable toxicity to the subject or interact with other components of the composition.

[0078] In one embodiment, 15-HEPE is in the form of an ester (referred to herein as E-15-HEPE, ethyl-15-HEPE, or 15-HEPE EE). In another embodiment, 15-HEPE comprises a C1-C5 alkyl ester of 15-HEPE. In another embodiment, 15-HEPE comprises 15-HEPE methyl ester, 15-HEPE propyl ester, or 15-HEPE butyl ester. In yet another embodiment, 15-HEPE comprises optically active 15(S)-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid. This isomer may be used in any of the forms described above.

[0079] In another embodiment, 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.

[0080] In various embodiments, the present disclosure provides pharmaceutical compositions, e.g., orally deliverable compositions, comprising 15-HEPE. In one embodiment, the composition comprises a therapeutically effective amount of 15-HEPE. In one embodiment, the pharmaceutical composition comprises about 0.1% to about 99%, about 1% to about 95%, or about 5% to about 90% by weight of 15-HEPE. As provided herein, the phrases "composition" and "pharmaceutical composition" are used interchangeably.

[0081] In one embodiment, the pharmaceutical composition comprises at least about 70%, at least about 80%, or at least about 90% by weight of 15-HEPE, hi 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.

[0082] In another embodiment, 15-HEPE is administered 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, or the like. mg, approx. 24 mg, approx. 25 mg, approx. 50 mg, approx. 75 mg, approx. 100 mg, approx. 125 mg, approx. 150 mg, approx. 175 mg, approx. 200 mg, approx. 225 mg, approx. Approx. 450mg, approx. 475mg, approx. 500mg, approx. 525mg, approx. 550mg, approx. 575mg, approx. 600mg, approx. 625mg, approx. 650mg, approx. 675mg, approx. 700mg, approx. , about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 1075mg, about 1100mg, about 1025mg, about 1050mg, about 1075mg, about 1200mg, about 1225mg, about 1250mg, about 1275mg, Approx. 1300mg, approx. 1325mg, approx. 1350mg, approx. 1375mg, approx. 1400mg, approx. 1425mg, approx. 1450mg, approx. 1475mg, approx. 1500mg, approx. mg, approx. 1700mg, approx. 1725mg, approx. 1750mg, approx. 1775mg, approx. 1800mg, approx. 1825mg, approx. 1850mg, approx. 1875mg, approx. 1900mg, approx. 075mg, about 2100mg, about 2125mg, about 2150mg, about 2175mg, about 2200mg, about 2225mg, about 2250mg, about 2275mg, about 2300mg, about 2325mg, about 2350mg, about 2375mg, about 2400mg, about 2425mg, about 2450mg,about 2475mg, about 2500mg, 2525mg, about 2550mg, about 2575mg, about 2600mg, about 2625mg, about 2650mg, about 2675mg, about 2700mg, about 2725mg, about 2750mg, about 2775mg, about 2800mg, about 2825mg, about 2850mg, about 2875mg, about 2900mg, about 2925mg, about 2950mg, about 2975mg, about 3000mg, about 3025mg, about 3050mg, about 3075mg, about 3100mg, about 3125mg, about 3150mg, about 3175mg, about 3200mg, about 3225mg, about 3 250mg, about 3275mg, about 3300mg, about 3325mg, about 3350mg, about 3375mg, about 3400mg, about 3425mg, about 3450mg, about 3475mg, about 3500mg, about 3525mg, about 3550mg, about 3575mg, about 3600mg, about 3625mg, about 3650mg, about 3675mg, about 3700mg, about 3725mg, about 3750mg, about 3775mg, about 3800mg, about 3825mg, about 3850mg, about 3875mg, about 3900mg, about 3925mg, about 3950mg, about 3975mg, about 4000mg, about 402 5mg, about 4050mg, about 4075mg, about 4100mg, about 4125mg, about 4150mg, about 4175mg, about 4200mg, about 4225mg, about 4250mg, about 4275mg, about 4300mg, about 4325mg, about 4350mg, about 4375mg, about 4400mg, about 4425mg, about 4450mg, about 4475mg, about 4500mg, about 4525mg, about 4550mg, about 4575mg, about 4600mg, about 4625mg, about 4650mg, about 4675mg, about 4700mg, about 4725mg, about 4750mg, about 4775mg, about 4800mg g, about 4825mg, about 4850mg, about 4875mg, about 4900mg, about 4925mg, about 4950mg, about 4975mg, about 5000mg, about 5025mg, about 5050mg, about 5075mg, about 5100mg, about 5125mg, about 5150mg, about 5175mg, about 5200mg, about 5225mg, about 5250mg, about 5275mg, about 5300mg, about 5325mg, about 5350mg, about 5375mg, about 5400mg, about 5425mg, about 5450mg, about 5475mg, about 5500mg, about 5525mg, about 5550mg, about 5575mg,about 5600mg, about 5625mg, about 5650mg, about 5675mg, about 5700mg, about 5725mg, about 5750mg, about 5775mg, about 5800mg, about 5825mg, about 5850mg, about 5875mg, about 5900mg, about 5925mg, about 5950mg, about 5975mg, about 6000mg, about 6025mg, about 6050mg, about 6075mg, about 6100mg, about 6125mg, about 6150mg, about 6175mg, about 6200mg, about 6225mg, about 6250mg, about 6275mg, about 6300mg, about 6325mg, about 6350mg, about 6 375mg, about 6400mg, about 6425mg, about 6450mg, about 6475mg, about 6500mg, about 6525mg, about 6550mg, about 6575mg, about 6600mg, about 6625mg, about 6650mg, about 6675mg, about 6700mg, about 6725mg, about 6750mg, about 6775mg, about 6800mg, about 6825mg, about 6850mg, about 6875mg, about 6900mg, about 6925mg, about 6950mg, about 6975mg, about 7000mg, about 7025mg, about 7050mg, about 7075mg, about 7100mg, about 7125mg, about 715 0mg, about 7175mg, about 7200mg, about 7225mg, about 7250mg, about 7275mg, about 7300mg, about 7325mg, about 7350mg, about 7375mg, about 7400mg, about 7425mg, about 7450mg, about 7475mg, about 7500mg, about 7525mg, about 7550mg, about 7575mg, about 7600mg, about 7625mg, about 7650mg, about 7675mg, about 7700mg, about 7725mg, about 7750mg, about 7775mg, about 7800mg, about 7825mg, about 7850mg, about 7875mg, about 7900mg, about 7925mg g, about 7950 mg, about 7975 mg, about 8000 mg, about 8025 mg, about 8050 mg, about 8075 mg, about 8100 mg, about 8125 mg, about 8150 mg, about 8175 mg, about 8200 mg, about 8225 mg, about 8250 mg, about 8275 mg, about 8300 mg, about 8325 mg, about 8350 mg, about 8375 mg, about 8400 mg, about 8425 mg, about 8450 mg, about 8475 mg, about 8500 mg, about 8525 mg, about 8550 mg, about 8575 mg, about 8600 mg, about 8625 mg, about 8650 mg, about 8675 mg, about 8700 mg,Approx. 8725 mg, approx. 8750 mg, approx. 8775 mg, approx. 8800 mg, approx. 8825 mg, approx. 8850 mg, approx. 8875 mg, approx. 8900 mg, approx. 8925 mg, approx. mg, about 9075mg, about 9100mg, about 9125mg, about 9150mg, about 9175mg, about 9200mg, about 9225mg, about 9250mg, about 9275mg, about 9300mg, about 9325mg, about 9350mg, about 9375mg, about 9 The compound is present in the compositions of the present disclosure in an amount of about 400 mg, about 9425 mg, about 9450 mg, about 9475 mg, about 9500 mg, about 9525 mg, about 9550 mg, about 9575 mg, about 9600 mg, about 9625 mg, about 9650 mg, about 9675 mg, about 9700 mg, about 9725 mg, about 9750 mg, about 9775 mg, about 9800 mg, about 9825 mg, about 9850 mg, about 9875 mg, about 9900 mg, about 9925 mg, about 9950 mg, about 9975 mg, or about 10,000 mg.

[0083] In one embodiment, the 15-HEPE present in the compositions of the present disclosure comprises at least about 90% by weight of 15-HEPE (the term "15-HEPE" is defined and exemplified herein). The 15-HEPE compositions may contain even higher purity 15-HEPE, for example, at least about 95% by weight of 15-HEPE, or at least about 97% by weight of 15-HEPE, where 15-HEPE is any of the forms of 15-HEPE described herein. The purity of 15-HEPE may be further defined by any of the descriptions of 15-HEPE provided herein (e.g., impurity profile).

[0084] The above discusses the amount of 15-HEPE in pharmaceutical compositions and their purity. The nature of essential fatty acids and their synthesis is such that 15-HEPE compositions may contain moieties from other essential fatty acids in the essential fatty acid metabolic cascade.

[0085] In one embodiment, the compositions of the present invention contain about 10% by weight or less, about 9% by weight or less, about 8% by weight or less, about 7% by weight or less, about 6% by weight or less, about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less, about 1% by weight or less, or about 0.5% by weight or less of other omega-3 fatty acids, such as alpha-linolenic acid, stearidonic acid, docosahexaenoic acid (DHA), or derivatives thereof. In other embodiments, such other omega-3 fatty acids are substantially absent or absent.

[0086] In another embodiment, 15-HEPE represents at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, at least about 98% by weight, at least about 99% by weight, or 100% by weight of all fatty acids present in the compositions of the present disclosure.

[0087] In one embodiment, the salt form of 15-HEPE present in the 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 may contain even higher purity, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, or at least 97% by weight of the salt form of 15-HEPE.

[0088] Some residual eicosapentaenoic acid from the synthesis of 15-HEPE may be present. There may be less than about 10% by weight, less than about 9% by weight, less than about 8% by weight, less than about 7% by weight, less than about 6% by weight, less than about 5% by weight, less than about 4% by weight, less than about 3% by weight, less than about 2% by weight, less than about 1% by weight, or less than about 0.5% by weight of EPA. Alternatively, there may be substantially no or no EPA in a form that has not been modified to the hydroxyl form.

[0089] In one embodiment, the present disclosure provides a pharmaceutical composition comprising 15-HEPE or a derivative thereof encapsulated in a capsule shell. In one embodiment, the composition is administered to a 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-HEPEE or a derivative thereof per day. In one embodiment, the composition is administered to a 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, or 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 a derivative thereof is encapsulated in the capsule shell.

[0090] In one embodiment, the capsule shell comprises gelatin (e.g., gelatin RXL or lime bone gelatin having a lower molecular weight). In another embodiment, the capsule shell comprises gelatin RXL that has been treated with a proteolytic enzyme to cleave the gelatin pattern and effectively reduce its molecular weight. In another embodiment, the pharmaceutical composition comprises a 15-HEPE ester of D-sorbitol and 1,4-sorbitan. In one embodiment, the capsule shell comprises (a) gelatin and (b) a plasticizer selected from one or more of D-sorbitol and 1,4-sorbitan. In one embodiment, the gelatin is as described in U.S. Pat. No. 7,485,323, the entire contents of which are incorporated herein by reference.

[0091] In one embodiment, the plasticizer comprises 1,4-sorbitan in an amount of about 20% to about 30%, e.g., about 24% to about 28%, 24%, or 28% (on a dry basis), and a D-sorbitol content of about 30% to about 50%, e.g., about 35% to about 45% (on a dry basis).

[0092] In some embodiments, the capsule is a hard gelatin capsule. In other embodiments, the capsule is a soft gelatin capsule.

[0093] In some embodiments, the capsule shell comprises modified starch, carrageenan (e.g., an extract of red algae), disodium phosphate, glycerol, and / or sorbitol. In some embodiments, the capsule shell further comprises water. In some embodiments, the capsule shell is stable up to a temperature of about 65°C and / or a pH of about 12.

[0094] In some embodiments, the capsule shell is odorless and has a neutral color (eg, colorless, white, or clear).

[0095] In some embodiments, the capsule shell further comprises glycerol, purified water, titanium dioxide, medium chain triglycerides, and lecithin.

[0096] Additional activators In one embodiment, the pharmaceutical composition further comprises one or more additional active agents. In one embodiment, the pharmaceutical composition comprises the additional active agent in an amount less than the generally recognized therapeutically effective amount of the active agent. In one embodiment, the pharmaceutical composition comprises the additional active agent in an amount equal to or greater than the generally recognized therapeutically effective amount of the active agent. When an additional active agent is used, 15-HEPE may be formulated together as a single dosage unit, or may be formulated as two or more dosage units for coordinated, concomitant, or simultaneous administration.

[0097] EPA itself has beneficial properties for the treatment of fatty liver disease and / or cardiovascular disease, and in an alternative embodiment, 15-HEPE can be combined with EPA.

[0098] In one embodiment, 15-HEPE and one or more active agents are present in a composition of the present disclosure or co-administered in a weight ratio of 15-HEPE:additional active agent(s) 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.

[0099] Dosage form Compositions for use in accordance with the present disclosure may be formulated as one or more dosage units. As used herein, the terms "dose unit" and "dosage unit" refer to a portion of a composition containing an amount of a therapeutic agent suitable for single administration to provide a therapeutic effect. Such dosage units may be administered one to multiple times per day (e.g., 1 to about 10 times, 1 to 8 times, 1 to 6 times, 1 to 4 times, or 1 to 2 times), or as many times as needed to elicit a therapeutic response.

[0100] In some embodiments, the compositions of the present disclosure are in the form of an orally deliverable dosage form or dosage unit. Non-limiting examples of suitable dosage forms include tablets (e.g., suspension tablets, bite suspension tablets, rapid dispersing tablets, chewable tablets, etc.), caplets, capsules (e.g., soft or hard gelatin capsules or HPMC capsules), lozenges, sachets, cachets, troches, pellets, suspensions, elixirs, syrups, or any other solid dosage form reasonably adapted for oral administration. The terms "oral delivery" and "oral administration" herein include any form of delivery in which a drug or composition is placed in the mouth of a subject being treated, regardless of whether it is swallowed. Thus, this includes buccal and sublingual administration, as well as esophageal administration.

[0101] Alternatively, compositions of the present disclosure may also be formulated for rectal, topical, or parenteral (eg, subcutaneous, intramuscular, intravenous and intradermal or infusion) delivery.

[0102] When considering the amount of 15-HEPE in the compositions of the present disclosure, it can be divided into several dosage forms. There are size limitations for oral administration. If a subject receives about 1 to about 4 g of 15-HEPE per day, this can be up to four capsules, each providing about 1 g of 15-HEPE.

[0103] The compositions of the present disclosure may be in the form of liquid dosage forms or dosage units for direct absorption or may be mixed with food or beverages prior to ingestion. Non-limiting examples of suitable liquid dosage forms include solutions, suspensions, elixirs, syrups, liquid aerosol formulations, and the like.

[0104] In another embodiment, the compositions of the present disclosure include one or more pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" as used herein refers to any substance that is not itself a therapeutic agent, but is used as a carrier or vehicle for delivering a therapeutic agent to a subject, or is added to a composition to improve its handling or storage characteristics, or to enable or facilitate the formation of a unit dose of the composition, and does not produce unacceptable toxicity or interactions with other components in the composition. By way of example only, pharmaceutical compositions according to the present disclosure may include one or more of an antioxidant, a surfactant, a preservative, a flavoring agent, a cosolvent, a viscosity aid, a suspension aid, and a lipophilic phase.

[0105] In one embodiment, the pharmaceutical composition comprises one or more antioxidants such as ascorbic acid, palmitic acid, ascorbyl palmitate, α-tocopherol, idebenone, ubiquinone, ferulic acid, coenzyme Q10, lycopene, green tea, catechin, epigallocatechin 3-gallate (EGCG), green tea polyphenols (GTP), silymarin, coffee berry, resveratrol, grape seed, pomegranate extract, genisten, pycnogenol, and niacinamide. In one embodiment, the pharmaceutical composition comprises about 0.01% to about 2% by weight of an antioxidant, for example, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18% by weight , approximately 0.19% by weight, approximately 0.2% by weight, approximately 0.21% by weight, approximately 0.22% by weight, approximately 0.23% by weight, approximately 0.24% by weight, approximately 0.25% by weight, approximately 0.26% by weight, approximately 0.27% by weight, approximately 0.28% by weight, approximately 0.29% by weight, approximately 0.3% by weight, approximately 0.31% by weight, approximately 0.32% by weight, approximately 0.33% by weight, approximately 0.34% by weight, approximately 0.35% by weight, approximately 0.36% by weight, approximately 0.37% by weight, approximately 0.38% by weight, approximately 0.39% by weight, approximately 0.4% by weight, approximately 0.4 1% by weight, approximately 0.42% by weight, approximately 0.43% by weight, approximately 0.44% by weight, approximately 0.45% by weight, approximately 0.46% by weight, approximately 0.47% by weight, approximately 0.48% by weight, approximately 0.49% by weight, approximately 0.5% by weight, approximately 0.51% by weight, approximately 0.52% by weight Amount %, about 0.53 wt%, about 0.54 wt%, about 0.55 wt%, about 0.56 wt%, about 0.57 wt%, about 0.58 wt%, about 0.59 wt%, about 0.6 wt%, about 0.61 wt%, about 0.62 wt%, about 0.63 wt% , approximately 0.64% by weight, approximately 0.65% by weight, approximately 0.66% by weight, approximately 0.67% by weight, approximately 0.68% by weight, approximately 0.69% by weight, approximately 0.7% by weight, approximately 0.71% by weight, approximately 0.72% by weight, approximately 0.73% by weight, approximately 0.74% by weight, approximately 0.75% by weight, approximately 0.76% by weight, approximately 0.77% by weight, approximately 0.78% by weight, approximately 0.79% by weight, approximately 0.8% by weight, approximately 0.81% by weight, approximately 0.82% by weight, approximately 0.83% by weight, approximately 0.84% ​​by weight, approximately 0.85% by weight, approximately 0.86%, about 0.87%, about 0.88%, about 0.89%, about 0.9%, about 0.91%, about 0.92%, about 0.93%, about 0.94%, about 0.95%, about 0.96%, about 0.97%, about 0.98%, about 0.99%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% by weight of one or more antioxidants.

[0106] Treatment method The compositions and formulations disclosed herein can be used to treat and / or prevent a blood disorder in a subject in need thereof. In some embodiments, the blood disorder is a hemoglobin disorder, a red blood cell disorder, a hemolytic anemia, a thrombophilia disorder, a venous thromboembolism, an arterial thrombosis, an embolism, or a combination thereof. In some embodiments, the method comprises administering 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE) to the subject.

[0107] The compositions and formulations disclosed herein can be used to treat and / or prevent blood disorders, hemoglobin disorders, and / or red blood cell disorders. Blood disorders refer to a category of diseases that affect the blood and blood-forming organs. Hemoglobin disorders and red blood cell disorders are similar to blood disorders, but both hemoglobin disorders and red blood cell disorders define narrower categories of diseases. Specifically, hemoglobin disorders refer to defects in hemoglobin, the oxygen-transporting protein in red blood cells. Erythrocyte disorders refer to defects in the red blood cells themselves. Non-limiting examples of risk factors for blood disorders, hemoglobin disorders, and red blood cell disorders include low red blood cell counts, increased red blood cell distribution widths, and increased reticulocyte counts. Non-limiting examples of blood disorders, hemoglobin disorders, and red blood cell disorders include anemia (nutritional and non-nutritional anemia, sickle cell disease, sickle cell anemia, β-thalassemia, and inherited hemolytic anemias such as hereditary spherocytosis), infections, drug treatments, hematologic malignancies, autoimmune diseases, hypersplenism, mechanical heart Valves, blood transfusions, etc. Occurring concomitantly In some embodiments, the conditions and disorders that may be present include acquired hemolytic anemia (including Fanconi anemia, iron deficiency anemia), blood cancers (lymphoma, leukemia, and myeloma), coagulation disorders (thrombophilia, hemophilia, von Willebrand disease, and thrombocytopenia), folate deficiency, B12 deficiency, and myelodysplastic syndrome. In some embodiments, sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and / or splenic blood retention.

[0108] In some embodiments, the subject has a red blood cell count that is 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.

[0109] In some embodiments, the subject has an increased red blood cell distribution width, where the size of red blood cells varies by about 15-20%, about 20-25%, about 25-30%, about 30-35%, or about 35-40%.

[0110] In another embodiment, the subject has an increased reticulocyte count of at least about 5% of the total red blood cell mass, at least about 10% of the total red blood cell mass, at least about 15% of the total red blood cell mass, or at least about 20% of the total red blood cell mass.

[0111] In some embodiments, the subject has an elevated baseline triglyceride level of between about 135 mg / dL and about 2000 mg / dL, e.g., between about 135 mg / dL and about 500 mg / dL, between about 150 mg / dL and about 500 mg / dL, between about 200 mg / dL and about 499 mg / dL, between about 200 mg / dL and <500 mg / dL, between about 300 mg / dL and about 1800 mg / dL, between about 500 mg / dL and about 1500 mg / dL, between about 500 mg / dL and about <2000 mg / dL, or between about 500 mg / dL and about 2000 mg / dL. In some embodiments, the subject has a fasting baseline triglyceride level of about 50 mg / dL to about 2000 mg / dL, e.g., 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. In one embodiment, the subject has a fasting baseline triglyceride level of about 80 mg / dL to about 1400 mg / dL. In some embodiments, the subject or group of subjects has a fasting baseline triglyceride level 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 160 mg / dL, about 170 mg / dL, about 180 mg / dL, about 190 mg / dL, about 200 mg / dL, about 210 mg / dL, about 220 mg / dL, about 230 mg / dL, about 240 mg / dL, about 250 mg / dL, about 260 mg / dL, about 270 mg / dL, about 280 mg / dL, about 290 mg / dL, about 300 mg / dL, about 310 mg / dL, about 320 mg / dL, about 330 mg / dL, about 340 mg / dL, about 350 mg / dL, about 360 mg / dL, about 370 mg / dL, about 380 mg / dL, about 390 mg / dL, about 400 mg / dL, about 4 mg / dL, approximately 155 mg / dL, approximately 160 mg / dL, approximately 165 mg / dL, approximately 170 mg / dL, approximately 175 mg / dL, approximately 180 mg / dL, approximately 185 mg / dL, approximately 190 mg / dL, approximately 195 mg / dL, approximately 200 mg / dL, approximately 2 05mg / dL, about 210mg / dL, about 215mg / dL, about 220mg / dL, about 225mg / dL, about 230mg / dL, about 235mg / dL, about 240mg / dL, about 245mg / dL, about 250mg / dL, about 255mg / dL,Approximately 260mg / dL, approximately 265mg / dL, approximately 270mg / dL, approximately 275mg / dL, approximately 280mg / dL, approximately 285mg / dL, approximately 290mg / dL, approximately 295mg / dL, approximately 300mg / d L, about 305 mg / dL, about 310 mg / dL, about 315 mg / dL, about 320 mg / dL, about 325 mg / dL, about 330 mg / dL, about 335 mg / dL, about 340 mg / dL, about 345 mg / dL, about 350 mg / dL, about 355 mg / dL, about 360 mg / dL, about 365 mg / dL, about 370 mg / dL, about 375 mg / dL, about 380 mg / dL, about 385 mg / dL, about 390 mg / dL, approximately 395mg / dL, approximately 400mg / dL, approximately 405mg / dL, approximately 410mg / dL, approximately 415mg / dL, approximately 420mg / dL, approximately 425mg / dL, approximately 430mg / dL, approximately 435m g / dL, approximately 440 mg / dL, approximately 445 mg / dL, approximately 450 mg / dL, approximately 455 mg / dL, approximately 460 mg / dL, approximately 465 mg / dL, approximately 470 mg / dL, approximately 475 mg / dL, approximately 48 0mg / dL, approx. 485mg / dL, approx. 490mg / dL, approx. 495mg / dL, approx. 500mg / dL, approx. 1000mg / dL, approx. 1100mg / dL, approx. 1200mg / dL, approx. 1300mg / d have a fed or fasting baseline triglyceride level (or median baseline triglyceride level for a control group) of about 1400 mg / dL, about 1500 mg / dL, about 2000 mg / dL, about 2500 mg / dL, about 3000 mg / dL, about 3500 mg / dL, about 4000 mg / dL, about 4500 mg / dL, about 5000 mg / dL, or greater than about 5000 mg / dL. In some embodiments, the subject or group of subjects has a blood cholesterol level of 80 mg / dL or more, about 100 mg / dL or more, about 120 mg / dL or more, about 150 mg / dL or more, about 175 mg / dL or more, about 250 mg / dL or more, about 500 mg / dL or more, e.g., 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 about 2000 mg / dL,Have a fed or fasting baseline triglyceride level (or median baseline triglyceride level for the control group) of about 500 mg / dL to less than about 2000 mg / dL, or about 500 mg / dL to about 2000 mg / dL.

[0112] In another embodiment, 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.

[0113] In some embodiments, the subject has an elevated baseline fasting blood glucose level 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.

[0114] In some embodiments, the subject has a reduced baseline HDL-C level 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.

[0115] In some aspects, the disclosure provides methods of treating and / or preventing a blood disorder in a subject, the methods comprising administering 15-HEPE to the subject. In another embodiment, the methods comprise administering up to about 8 g of 15-HEPE to the subject. In yet another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0116] In some aspects, the disclosure provides methods of treating and / or preventing a hematological disorder in a subject, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In another embodiment, the method comprises administering up to about 8 g of 15-HEPE or a composition comprising 15-HEPE to the subject. In yet another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0117] In some embodiments, the present disclosure provides methods for treating and / or preventing a blood disorder in a subject in need thereof, the methods comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-HEPE. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0118] In some aspects, the disclosure provides methods for treating and / or preventing a hematological disorder in a subject, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the methods further comprise determining that the subject has at least one risk factor for a hematological disorder prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the methods further comprise measuring the subject's baseline red blood cell count, red blood cell distribution width, and / or reticulocyte count prior to administration of 15-HEPE. In one embodiment, the subject exhibits an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count. In another embodiment, the method comprises administering up to about 8 g of 15-HEPE or a composition comprising 15-HEPE to the subject. In yet another embodiment, 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0119] In some embodiments, the present disclosure provides methods for treating and / or preventing a blood disorder in a subject in need thereof, the methods comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-HEPE. In some embodiments, the methods further comprise determining that the subject has at least one risk factor for a blood disorder prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the methods further comprise measuring the subject's baseline red blood cell count, red blood cell distribution width, and / or reticulocyte count prior to administration of 15-HEPE. In one embodiment, the subject exhibits an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0120] In some embodiments, the present disclosure provides methods for treating and / or preventing hemoglobin disorders in a subject in need thereof, the methods comprising administering to the subject a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE) or 15-HEPE. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0121] In some embodiments, the present disclosure provides methods for treating and / or preventing hemoglobin disorders in a subject in need thereof, the methods comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-HEPE. In some embodiments, the methods further comprise determining that the subject has at least one risk factor for a hemoglobin disorder prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the methods further comprise measuring the subject's baseline red blood cell count, red blood cell distribution width, and / or reticulocyte count prior to administration of 15-HEPE. In one embodiment, the subject exhibits an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0122] In some embodiments, the present disclosure provides methods for treating and / or preventing an erythrocyte disorder in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0123] In some embodiments, the present disclosure provides methods for treating and / or preventing an red blood cell disorder in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has at least one risk factor for an red blood cell disorder prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises measuring the subject's baseline red blood cell count, red blood cell distribution width, and / or reticulocyte count prior to administration of 15-HEPE. In one embodiment, the subject exhibits an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0124] In some embodiments, the present disclosure provides methods for treating and / or preventing blood disorders, hemoglobin disorders, and / or blood red blood cell disorders in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0125] In some embodiments, the present disclosure provides methods for treating and / or preventing a blood disorder, hemoglobin disorder, and / or blood red blood cell disorder in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has at least one risk factor for a blood disorder, hemoglobin disorder, and / or blood red blood cell disorder prior to administering 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a low red blood cell count, an increased red blood cell distribution width, and / or an increased reticulocyte count. In some embodiments, the subject exhibits one or more of an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count.

[0126] In some embodiments, the present disclosure provides methods for treating and / or preventing a blood disorder, hemoglobin disorder, and / or blood red blood cell disorder in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition. In another embodiment, the blood disorder, hemoglobin disorder, and / or red blood cell disorder is selected from the group consisting of hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome. In some embodiments, the method further comprises determining that the subject has at least one risk factor for hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and / or myelodysplastic syndrome prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises measuring the subject's baseline red blood cell count, red blood cell distribution width, and / or reticulocyte count prior to administration of 15-HEPE. In one embodiment, the subject exhibits an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count.

[0127] In some embodiments, the present disclosure provides methods for treating and / or preventing hemolytic anemia in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0128] In some embodiments, the present disclosure provides methods for treating and / or preventing hemolytic anemia in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the methods further comprise determining that the subject has at least one risk factor for hemolytic anemia prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decreased red blood cell count, an increased red blood cell distribution width, and / or an increased reticulocyte count. In some embodiments, the subject exhibits one or more of an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count.

[0129] In some embodiments, the present disclosure provides a method of treating and / or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE. In some embodiments, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition. In some embodiments, the hemolytic anemia is hereditary hemolytic anemia or acquired hemolytic anemia. In another embodiment, the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis. In yet another embodiment, the acquired hemolytic anemia is caused by an infection, drug treatment, hematologic malignancy, autoimmune disease, hypersplenism, or mechanical heart Valves and blood transfusions Occurring concomitantly In various embodiments, sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and / or splenic blood cell retention. In some embodiments, the subject exhibits an increased red blood cell count, a decreased red blood cell distribution width, and a decreased reticulocyte count following administration.

[0130] In some embodiments, the present disclosure provides methods for treating and / or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE. In some embodiments, 15-HEPE represents at least about 90% by weight of all fatty acids in the composition. In some embodiments, the hemolytic anemia is hereditary hemolytic anemia or acquired hemolytic anemia. In another embodiment, the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis. In some embodiments, the method further comprises determining that the subject has at least one risk factor for hereditary hemolytic anemia or acquired hemolytic anemia prior to administration of 15-HEPE or a composition comprising 15-HEPE. In yet another embodiment, acquired hemolytic anemia is caused by an infection, drug treatment, hematologic malignancy, autoimmune disease, hypersplenism, or mechanical heart Valves and blood transfusions Occurring concomitantly In various embodiments, sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and / or splenic blood cell retention. In some embodiments, the method further comprises measuring the subject's baseline red blood cell count, red blood cell distribution width, and / or reticulocyte count prior to administration of 15-HEPE. In one embodiment, the subject exhibits an elevated red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count.

[0131] The compositions and formulations disclosed herein can further be used for the treatment and / or prevention of thrombophilia disorders. Thrombophilia disorders refer to a class of diseases characterized by abnormal blood clotting, which increases the risk of thrombosis. Non-limiting examples of risk factors for thrombophilia disorders include reduced prothrombin time, reduced activated partial thromboplastin time, and increased fibrinogen levels.

[0132] In some embodiments, the subject has a reduced prothrombin time that is 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.

[0133] In some embodiments, the subject has a reduced activated partial thromboplastin time that is at least 10% below the standard, at least 15% below the standard, at least 20% below the standard, at least 25% below the standard, or at least 30% below the standard.

[0134] In some embodiments, the subject has an increased fibrinogen concentration that is 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.

[0135] In some embodiments, the present disclosure provides methods for treating and / or preventing a thrombophilic 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. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0136] In some embodiments, the present disclosure provides methods for treating and / or preventing a thrombophilia disorder in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has at least one risk factor for a thrombophilia disorder prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decreased prothrombin time, a decreased activated partial thromboplastin time, and / or an increased fibrinogen concentration. In some embodiments, the subject exhibits one or more of an increased prothrombin time, an increased activated partial thromboplastin time, and / or a decreased fibrinogen concentration after administration of 15-HEPE or a composition comprising 15-HEPE.

[0137] In some embodiments, the present disclosure provides methods for treating and / or preventing venous thromboembolism in a subject in need thereof, the methods comprising administering to the subject a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE) or 15-HEPE. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0138] In some embodiments, the present disclosure provides methods for treating and / or preventing venous thromboembolism in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the methods further comprise determining that the subject has at least one risk factor for venous thromboembolism prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decreased prothrombin time, a decreased activated partial thromboplastin time, and / or an increased fibrinogen concentration. In some embodiments, the subject exhibits one or more of a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a decreased fibrinogen concentration.

[0139] In some embodiments, the present disclosure provides methods for treating and / or preventing arterial thrombosis in a subject in need thereof, the methods comprising administering to the subject 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-HEPE. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0140] In some embodiments, the present disclosure provides methods for treating and / or preventing arterial thrombosis in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the methods further comprise determining that the subject has at least one risk factor for arterial thrombosis prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decreased prothrombin time, a decreased activated partial thromboplastin time, and / or an increased fibrinogen concentration. In some embodiments, the subject exhibits one or more of a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a decreased fibrinogen concentration.

[0141] In some embodiments, the present disclosure provides a method of preventing embolism in a subject in need thereof, the method comprising administering to the subject a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE) or 15-HEPE. In another embodiment, the method comprises administering to the subject a composition comprising up to about 8 g of 15-HEPE. In another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0142] In some embodiments, the present disclosure provides methods for preventing embolism in a subject in need thereof, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the methods further comprise determining that the subject has at least one risk factor for developing an embolism prior to administering 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decreased prothrombin time, a decreased activated partial thromboplastin time, and / or an increased fibrinogen concentration. In some embodiments, the subject exhibits one or more of a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a decreased fibrinogen concentration.

[0143] In some aspects, the disclosure provides methods of treating and / or preventing endothelial dysfunction in a subject, the methods comprising administering 15-HEPE to the subject. In another embodiment, the method comprises administering up to about 8 g of 15-HEPE to the subject. In yet another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0144] In some aspects, the present disclosure provides a method for treating a subject hand Methods for treating and / or preventing endothelial dysfunction are provided, comprising administering 15-HEPE or a composition comprising 15-HEPE to a subject. In another embodiment, the method comprises administering up to about 8 g of 15-HEPE or a composition comprising 15-HEPE to a subject. In yet another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0145] In some aspects, the present disclosure provides methods of treating, preventing, and / or reducing cell stress apoptosis, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to a subject. In some embodiments, the subject exhibits a reduction in markers associated with apoptosis, such as proteins from the Bcl-2 family, activation fragments of caspases, and / or cleaved PARP-1.

[0146] In some embodiments, the present disclosure provides methods of treating, preventing, and / or reducing inflammation, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to a subject. In some embodiments, the subject exhibits reduced T cell activation, B cell activation, and / or chemotaxis.

[0147] The compositions and formulations disclosed herein can be used to treat or prevent cardiovascular disease or cardiovascular disorders. In one embodiment, the cardiovascular disease or cardiovascular disorder is selected from the group consisting 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, atherosclerotic plaque, and the like. The therapeutic effects of the present invention are selected from the group consisting of prevention of coronary heart disease, microvascular disease, macrovascular disease, atherosclerosis, coronary atherosclerosis, diastolic dysfunction, cardiovascular risk reduction, 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 endothelial function, diabetes, diabetes mellitus, insulin resistance, hyperinsulinemia, hyperglycemia, dysglycemia, induction of glycemic control, impaired glucose tolerance, and impaired fasting glucose. 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. In some embodiments, the subject has non-alcoholic liver disease, cholestatic liver disease, kidney disease, or metabolic syndrome. Any of the aforementioned examples of cardiovascular disease may be referred to as non-limiting examples of cardiometabolic disease.

[0148] In some aspects, the present disclosure provides methods of treating and / or preventing cardiovascular disease in a subject, the methods comprising administering 15-HEPE to the subject.

[0149] In some aspects, the present disclosure provides methods of treating and / or preventing cardiovascular disease in a subject, the methods comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject.

[0150] In another embodiment, the disclosure provides a method for preventing a blood disorder in a subject with diabetes (e.g., type I or type II), the method comprising administering 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has diabetes prior to administering 15-HEPE. In another embodiment, the method comprises administering about 10 mg per kg of body weight (mg / kg), about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg of 15-HEPE to the subject. In yet another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0151] In another embodiment, the disclosure provides a method for preventing a blood disorder in a subject with diabetes (e.g., type I or type II), the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has diabetes prior to administering 15-HEPE or a composition comprising 15-HEPE. In another embodiment, the method comprises administering about 10 mg / kg, about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg of 15-HEPE or a composition comprising 15-HEPE to the subject. In yet another embodiment, 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0152] In another aspect, the present disclosure provides methods for treating and / or preventing a blood disorder in a subject with cardiovascular disease, the method comprising administering 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has cardiovascular disease prior to administering 15-HEPE. In another embodiment, the method comprises administering about 10 mg, about 50 mg, about 250 mg, or about 500 mg / kg of 15-HEPE to the subject per body weight (mg / kg). In yet another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0153] In another aspect, the present disclosure provides methods for treating and / or preventing a blood disorder in a subject with cardiovascular disease, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has cardiovascular disease prior to administering 15-HEPE or a composition comprising 15-HEPE. In another embodiment, the method comprises administering about 10 mg / kg, about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg of 15-HEPE or a composition comprising 15-HEPE to the subject. In yet another embodiment, 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0154] In another aspect, the present disclosure provides methods for treating and / or preventing a blood disorder in a subject with hypertension, the method comprising administering 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has hypertension prior to administering 15-HEPE. In some embodiments, the subject has hypertension 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. In another embodiment, the method comprises administering about 10 mg, about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg of 15-HEPE to the subject. In yet another embodiment, the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0155] In another aspect, the disclosure provides methods for treating and / or preventing a blood disorder in a subject with hypertension, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining that the subject has hypertension prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the subject has hypertension 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. In another embodiment, the method comprises administering about 10 mg, about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg of 15-HEPE or a composition comprising 15-HEPE to the subject. In yet another embodiment, 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0156] The compositions and formulations disclosed herein can also be used to reduce cytokines and / or chemokines in subjects with cardiovascular and / or hematological disorders, including, but not limited to, alpha-smooth muscle action (α-SMA), metallopeptidase inhibitor-1 (TIMP-1), transforming growth factor beta-β (TGF-β), and type 1 collagen.

[0157] In another embodiment, a subject or group of subjects, upon treatment with a composition of the present invention, exhibits one or more of the following outcomes:

[0158] (a) no increase or decrease in serum aminotransferase (ALT) and / or aspartate aminotransferase (AST) levels relative to baseline, placebo control, and / or untreated patients;

[0159] (b) no increase or decrease in bilirubin (BUN) levels relative to baseline, placebo control, and / or untreated patients;

[0160] (c) no increase or reduction in the area of ​​fibrosis relative to baseline, placebo control, and / or untreated patients;

[0161] (d) no increase or reduction in fasting blood glucose levels relative to baseline, placebo control, and / or untreated patients;

[0162] (e) no increase or decrease in insulin levels relative to baseline, placebo control, and / or untreated patients;

[0163] (f) no increase or decrease in alkaline phosphate (ALP) levels relative to baseline, placebo control, and / or untreated patients;

[0164] (g) no increase or decrease in hemoglobin A1C (HbA1C) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0165] (h) no increase or decrease in homeostatic model assessment of insulin resistance (HOMA-IR) versus baseline, placebo control, and / or untreated patients;

[0166] (i) no increase or reduction in adipose tissue insulin resistance (adipo-IR) levels relative to baseline, placebo control, and / or untreated patients;

[0167] (j) no increase in, or a reduction in, total cholesterol levels relative to baseline, placebo control, and / or untreated patients;

[0168] (k) no increase or reduction in triglyceride levels relative to baseline, placebo control, and / or untreated patients;

[0169] (l) no increase or decrease in diglyceride levels relative to baseline, placebo control, and / or untreated patients;

[0170] (m) no increase or reduction in very low density lipoprotein cholesterol (VLDL-C) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0171] (n) no increase or decrease in remnant-like particle cholesterol (RLP-C) levels relative to baseline, placebo control, and / or untreated patients;

[0172] (o) no increase or reduction in non-high-density lipoprotein cholesterol (non-HDL-C) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0173] (p) no reduction or increase in high-density lipoprotein cholesterol (HDL-C) levels relative to baseline, placebo control, and / or untreated patients;

[0174] (q) no increase or reduction in low-density lipoprotein cholesterol (LDL-C) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0175] (r) no increase or decrease in renal hydroxyproline levels relative to baseline, placebo control, and / or untreated patients;

[0176] (s) no increase or decrease in interleukin-33 (IL-33) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0177] (t) no increase or decrease in interleukin-17 (IL-17) levels relative to baseline, placebo control, and / or untreated patients;

[0178] (u) no increase or decrease in aryl hydrocarbon receptor (AhR) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0179] (v) no increase or decrease in TNF-like ligand 1A (TL1A) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0180] (w) no increase or decrease in tumor necrosis factor (TNF-α) levels relative to baseline, placebo control, and / or untreated patients;

[0181] (x) no increase or decrease in interleukin-13 (IL-13) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0182] (y) no increase or decrease in interleukin-6 (IL-6) levels relative to baseline, placebo control, and / or untreated patients;

[0183] (z) no increase or decrease in interleukin-1 beta-β (IL-1β) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0184] (aa) no increase or decrease in type 1 collagen levels relative to baseline, placebo control, and / or untreated patients;

[0185] (bb) no increase or decrease in transforming growth factor beta (TGF-β) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0186] (cc) no increase or decrease in alpha-smooth muscle activity (α-SMA) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0187] (dd) no increase or decrease in tumor necrosis factor ligand superfamily member 11A (TNRSF11A) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0188] (ee) no increase or decrease in serine protease 2 (PRSS2) levels relative to baseline, placebo control, and / or untreated patients;

[0189] (ff) no increase or decrease in amine oxidase, copper-containing 3 (AOC3) levels relative to baseline, placebo control, and / or untreated patients;

[0190] (gg) no increase or decrease in leukocyte immunoglobulin-like receptor B1 (LILBR1) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0191] (hh) no increase or decrease in transferrin receptor protein 1 (TR) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0192] (ii) no increase or decrease in elafin (PI3) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0193] (jj) no increase or decrease in serum amyloid A4 (SAA4) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0194] (kk) no increase or decrease in monocyte chemoattractant protein-1 (MCP-1) levels relative to baseline, placebo control, and / or untreated patients;

[0195] (ll) no increase or decrease in chemokine (CC motif) ligand 16 (CCL16) levels relative to baseline, placebo control, and / or untreated patients;

[0196] (mm) No increase or decrease in trem-like transcript 2 (TLT2) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0197] (nn) no increase or decrease in dipeptidyl peptidase 4 (DPP4) levels relative to baseline, placebo control, and / or treatment-naive patients; and

[0198] (oo) no increase or decrease in TIMP-1 levels relative to baseline, placebo control, and / or treatment-naive patients;

[0199] (pp) no increase or decrease in plasminogen activator inhibitor-1 (PAI-1) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0200] (qq) no increase or decrease in NAFLD score (NFS) relative to baseline, placebo control, and / or untreated patients; and

[0201] (rr) no increase or decrease in fibrosis-4 (FIB-4) levels relative to baseline, placebo control, and / or untreated patients;

[0202] (ss) no increase or reduction in liver stiffness levels relative to baseline, placebo control, and / or treatment-naive patients;

[0203] (tt) no increase or reduction in liver fat content relative to baseline, placebo control, and / or untreated patients;

[0204] (uu) no reduction or increase in glycerophospholipid levels relative to baseline, placebo control, and / or untreated patients;

[0205] (vv) no increase or reduction in blood pressure relative to baseline, placebo-controlled, and / or untreated patients;

[0206] (ww) no increase or reduction in Enhanced Liver Fibrosis (ELF) score relative to baseline, placebo control, and / or untreated patients;

[0207] (xx) no increase or decrease in collagen production in lung and / or skin fibroblasts relative to baseline, placebo control, and / or untreated patients;

[0208] (yy) no reduction or increase in glucose uptake relative to baseline, placebo control, and / or untreated patients;

[0209] (zz) increased lung and / or skin fibroblast viability relative to baseline, placebo control, and / or untreated patients;

[0210] (ab) no change or reduction in interleukin-8 (IL-8) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0211] (ac) no change or reduction in interleukin-23 (IL-23) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0212] (ad) no change or a decrease in interleukin-11 (IL-11) levels relative to baseline, placebo control, and / or treatment-naive patients; and / or

[0213] (ae) no change or reduction in interferon gamma (IFNγ) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0214] (af) no change or reduction in T and / or B cell activation relative to baseline, placebo control, and / or untreated patients;

[0215] (ag) no change or reduction in chemotaxis relative to baseline, placebo control, and / or untreated patients;

[0216] (ah) no change or reduction in phosphorylated B-cell lymphoma 2 (Bcl-2) family members relative to baseline, placebo control, and / or treatment-naive patients;

[0217] (ai) no change or reduction in activated caspase fragment levels relative to baseline, placebo control, and / or untreated patients;

[0218] (aj) no change or reduction in cleaved poly(ADP-ribose) polymerase-1 (PARP-1) levels relative to baseline, placebo control, and / or treatment-naive patients;

[0219] (ak) no change or reduction in waist circumference relative to baseline, placebo-controlled, and / or untreated patients;

[0220] (a) no change or reduction in AST to platelet partition index (APRI) relative to baseline, placebo control, and / or treatment-naive patients;

[0221] (am) no change or reduction in liver inflammation and fibrosis (LIF) score relative to baseline, placebo control, and / or untreated patients;

[0222] (an) no change or reduction in Lok score relative to baseline, placebo control, and / or treatment-naive patients;

[0223] (ao) no change or reduction in fibrosis score relative to baseline, placebo control, and / or untreated patients;

[0224] (ap) no change or reduction in King score relative to baseline, placebo-controlled, and / or untreated patients;

[0225] (aq) No change or reduction in Bonacini score relative to baseline, placebo control, and / or treatment-naive patients;

[0226] (ar) No change or reduction in transient elastography (TE) scores relative to baseline, placebo control, and / or treatment-naive patients.

[0227] (as) no increase or decrease in vascular adhesion molecules relative to baseline, placebo control, and / or untreated patients;

[0228] (at) no increase or reduction in cardiovascular risk proteins relative to baseline, placebo control, and / or untreated patients;

[0229] (au) no increase or decrease in chemokines relative to baseline, placebo control, and / or untreated patients;

[0230] (av) no increase or decrease in tumor necrosis factor receptor superfamily members relative to baseline, placebo control, and / or treatment-naive patients;

[0231] (aw) no change or increase in red blood cell counts relative to baseline, placebo control, and / or untreated patients;

[0232] (ax) no change or increase in white blood cell counts relative to baseline, placebo control, and / or treatment-naive patients;

[0233] (ay) no change or increase in platelet counts relative to baseline, placebo control, and / or treatment-naive patients;

[0234] (az) no change or increase in prothrombin time relative to baseline, placebo control, and / or untreated patients;

[0235] (ba) no change or increase in activated partial thromboplastin time relative to baseline, placebo control, and / or treatment-naive patients;

[0236] (bc) no change or increase in hemoglobin production relative to baseline, placebo control, and / or untreated patients;

[0237] (bd) no change or increase in synthesis of the beta globin chain of the hemoglobin tetramer relative to baseline, placebo control, and / or untreated patients;

[0238] (be) no change or reduction in red blood cell hemolysis relative to baseline, placebo control, and / or treatment-naive patients;

[0239] (bf) no change or reduction in shortness of breath relative to baseline, placebo-controlled, and / or treatment-naive patients;

[0240] (bg) no change or reduction in heart rate relative to baseline, placebo-controlled, and / or untreated patients;

[0241] (bh) no change or reduction in spleen size relative to baseline, placebo control, and / or treatment-naive patients;

[0242] (bi) no change or reduction in liver size relative to baseline, placebo control, and / or untreated patients;

[0243] (bj) no change or reduction in dactylitis relative to baseline, placebo-controlled, and / or treatment-naive patients;

[0244] (bk) no change or reduction in pain crises relative to baseline, placebo-controlled, and / or treatment-naive patients;

[0245] (bl) no change in or a reduction in the occurrence of abnormal blood clots relative to baseline, placebo control, and / or untreated patients;

[0246] (bm) no change or reduction in red blood cell distribution width relative to baseline, placebo control, and / or treatment-naive patients;

[0247] (bn) no change or reduction in reticulocyte count relative to baseline, placebo-controlled, and / or treatment-naive patients; and / or

[0248] (bo) No change or reduction in fibrinogen concentration relative to baseline, placebo control, and / or untreated patients.

[0249] In one embodiment, the method of the invention comprises measuring a baseline level of one or more markers or parameters described in (a)-(bo) above prior to administration to a subject or group of subjects. In another embodiment, the method comprises administering a composition disclosed herein to a subject after measuring a baseline level of one or more markers or parameters described in (a)-(bo), followed by an additional measurement of the one or more markers.

[0250] In another embodiment, upon treatment with a composition of the invention over a period of, for example, about 1 to about 12 weeks, about 1 to about 8 weeks, or about 1 to about 4 weeks, a subject or group of subjects exhibits any 5 or more, any 10 or more, any 15 or more, any 20 or more, any 25 or more, any 30 or more, any 35 or more, any 40 or more, any 45 or more, any 50 or more, any 55 or more, any 60 or more, any 65 or more, any 70 or more, any 75 or more, any 80 or more, any 85 or more, any 90 or more, or all 91 of outcomes (a)-(bo) described immediately above.

[0251] In another embodiment, a subject or group of subjects, upon treatment with a composition of the present invention, exhibits one or more of the following outcomes:

[0252] (a) no increase in, or a reduction 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 patients;

[0253] (b) no increase in BUN levels or a reduction 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 patients;

[0254] (c) no increase in fibrotic area or a reduction 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 patients;

[0255] (d) no increase in fasting blood glucose levels or a reduction 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 patients;

[0256] (e) no increase in insulin or a reduction 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 patients;

[0257] (f) no increase in ALP levels or a reduction 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 patients;

[0258] (g) no increase in, or a reduction 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 patients;

[0259] (h) no increase in, or a reduction 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 patients;

[0260] (i) no increase in adipo-IR levels or a reduction 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 patients;

[0261] (j) no increase in total cholesterol levels or a reduction 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 patients;

[0262] (k) no increase in triglyceride levels or a reduction 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 patients;

[0263] (l) no increase in diglyceride levels or a reduction 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 patients;

[0264] (m) no increase in VLDL-C levels or a reduction 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 patients;

[0265] (n) no increase in RLP-C levels or a reduction 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 patients;

[0266] (o) no increase in non-HDL-C levels or a reduction 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 patients;

[0267] (p) no reduction in HDL-C levels or an increase 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 patients;

[0268] (q) no increase in LDL-C levels or a reduction 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 patients;

[0269] (r) no increase in, or a reduction 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 patients;

[0270] (s) no increase in, or a reduction 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 patients;

[0271] (t) no increase in, or a reduction 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 patients;

[0272] (u) no increase in AhR levels or a reduction 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 patients;

[0273] (v) no increase in TL1A levels or a reduction 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 patients;

[0274] (w) no increase in, or a reduction 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 patients;

[0275] (x) no increase in, or a reduction 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 patients;

[0276] (y) no increase in, or a reduction 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 patients;

[0277] (z) no increase in, or a reduction 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 patients;

[0278] (aa) no increase in, or a reduction 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 patients;

[0279] (bb) no increase in, or a reduction 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 patients;

[0280] (cc) no increase in α-SMA or a reduction 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 patients;

[0281] (dd) no increase in TNRSF11A or a reduction 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 patients;

[0282] (ee) No increase in PRSS2 levels or a reduction 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 patients.

[0283] (ff) no increase in AOC3 levels or a reduction 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 patients;

[0284] (gg) no increase in LILBR1 levels or a reduction 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 patients;

[0285] (hh) no increase in TR levels or a reduction 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 patients;

[0286] (ii) no increase in PI3 or a reduction 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 patients;

[0287] (jj) no increase in SAA4 levels or a reduction 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 patients;

[0288] (kk) no increase in MCP-1 levels or a reduction 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 patients;

[0289] (ll) no increase in CCL16 levels or a reduction 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 patients;

[0290] (mm) No increase in TLT2 levels or a reduction 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 patients;

[0291] (nn) no increase in DPP4 levels or a reduction 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 patients;

[0292] (oo) no increase in TIMP-1 levels or a reduction 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 patients;

[0293] (pp) no increase in PAI-1 levels or a reduction 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 patients;

[0294] (qq) no increase in NFS or a reduction 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 patients;

[0295] (rr) no increase in FIB-4 levels or a reduction 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 patients;

[0296] (ss) no increase in liver stiffness or a reduction 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 patients;

[0297] (tt) no increase in liver fat content or a reduction 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 patients;

[0298] (uu) no reduction in glycerophospholipid levels or an increase 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 patients;

[0299] (vv) no increase in blood pressure or a reduction 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 patients;

[0300] (ww) no increase in ELF score or a reduction 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 patients;

[0301] (zz) no increase in, or a reduction 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% in collagen production score in lung and / or skin fibroblasts relative to baseline, placebo control, and / or untreated patients;

[0302] (yy) no reduction in glucose uptake or an increase 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 patients;

[0303] (zz) an increase 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% in lung and / or skin fibroblast viability relative to baseline, placebo control, and / or untreated patients;

[0304] (ab) no change in IL-8 level score or a reduction 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 patients;

[0305] (ac) no change in, or a reduction 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% in IL-23 levels relative to baseline, placebo control, and / or untreated patients; and / or

[0306] (ad) no change in IL-11 level score or a reduction 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 patients;

[0307] (ae) no change in, or a reduction 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% in IFNγ levels relative to baseline, placebo control, and / or untreated patients;

[0308] (af) no change in, or a reduction 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 patients;

[0309] (ag) no change in chemotaxis or a reduction 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 patients;

[0310] (ah) no change or 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% reduction in Bcl-2 family members relative to baseline, placebo control, and / or untreated patients;

[0311] (ai) no change in, or a reduction 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% in levels of activated fragments of caspases relative to baseline, placebo control, and / or untreated patients;

[0312] (aj) no change in, or a reduction 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% in cleaved PARP-1 levels relative to baseline, placebo control, and / or untreated patients;

[0313] (ak) no change in waist circumference or a reduction 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 patients;

[0314] (a) no change in ARPI or a reduction 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 patients;

[0315] (am) no change in, or a reduction 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% in LIF score relative to baseline, placebo control, and / or untreated patients;

[0316] (an) no change in Lok score or a reduction 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 patients;

[0317] (ao) no change in fibrosis score or a reduction 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 patients;

[0318] (ap) no change in King score or a reduction 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 patients;

[0319] (aq) no change in, or a reduction 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% in Bonacini score relative to baseline, placebo control, and / or untreated patients; and / or

[0320] (ar) No change in, or 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% reduction in TE score relative to baseline, placebo control, and / or untreated patients.

[0321] (as) an increase 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% in vascular adhesion molecules relative to baseline, placebo control, and / or untreated patients;

[0322] (at) an increase 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% in a cardiovascular risk protein relative to baseline, placebo control, and / or untreated patients;

[0323] (au) an increase 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% in chemokines relative to baseline, placebo control, and / or untreated patients;

[0324] (av) an increase 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% in tumor necrosis factor receptor superfamily members relative to baseline, placebo control, and / or untreated patients.

[0325] (aw) no change in red blood cell count or an increase 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 patients;

[0326] (ax) no change in, or an increase 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% in white blood cell count relative to baseline, placebo control, and / or untreated patients;

[0327] (ay) no change in platelet count or an increase 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 patients;

[0328] (az) no change in prothrombin time or an increase 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 patients;

[0329] (ba) no change in activated partial thromboplastin time or an increase 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 patients;

[0330] (bc) no change in hemoglobin production or an increase 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 patients;

[0331] (bd) no change in, or an increase 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 patients, in the synthesis of the beta globin chain of the hemoglobin tetramer;

[0332] (be) no change in red blood cell hemolysis or a reduction 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 patients;

[0333] (bf) no change in shortness of breath or at least about a 5%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, at least about a 30%, at least about a 35%, at least about a 40%, at least about a 45%, at least about a 50%, at least about a 55%, at least about a 60%, at least about a 65%, at least about a 70%, at least about a 75%, at least about a 80%, at least about a 85%, at least about a 90%, or at least about a 95% reduction relative to baseline, placebo control, and / or untreated patients;

[0334] (bg) no change in heart rate or a reduction 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 patients;

[0335] (bh) no change in, or at least about a 5%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, at least about a 30%, at least about a 35%, at least about a 40%, at least about a 45%, at least about a 50%, at least about a 55%, at least about a 60%, at least about a 65%, at least about a 70%, at least about a 75%, at least about a 80%, at least about a 85%, at least about a 90%, or at least about a 95% reduction in spleen size relative to baseline, placebo control, and / or untreated patients;

[0336] (bi) no change in liver size or a reduction 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 patients;

[0337] (bj) no change or 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% reduction in dactylitis relative to baseline, placebo control, and / or untreated patients;

[0338] (bk) no change in pain crises or 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% reduction relative to baseline, placebo control, and / or untreated patients;

[0339] (bl) no change in, or at least about a 5%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, at least about a 30%, at least about a 35%, at least about a 40%, at least about a 45%, at least about a 50%, at least about a 55%, at least about a 60%, at least about a 65%, at least about a 70%, at least about a 75%, at least about a 80%, at least about a 85%, at least about a 90%, or at least about a 95% reduction in the occurrence of abnormal blood clots relative to baseline, placebo control, and / or untreated patients;

[0340] (bm) no change in red blood cell distribution or a reduction 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 patients;

[0341] (bn) no change in reticulocyte count or a reduction 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 patients; and / or

[0342] (bo) no change in fibrinogen concentration or a reduction 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 patients.

[0343] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the agents of the present disclosure and practice the claimed methods. The following examples are provided to facilitate the practice of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure. [Example]

[0344] Example 1: Unilateral ureteral obstruction-induced renal interstitial fibrosis The purpose of this study was to investigate the effects of DS109 (15-HETrE) and DS102 (15-HEPE) on UUO-induced renal interstitial fibrosis.

[0345] Figure 1 shows the study design from surgery and treatment through day 14 of the study.

[0346] 1.1 Materials and Methods Test Substances: The test substances in this study were DS109 (15-HETrE) and DS102 (15-HEPE). To prepare the dosing solutions for each substance, DS109 was first weighed and then dissolved in a vehicle of 0.5% hydroxypropylmethylcellulose (HPMC), and DS102 was diluted in a vehicle of 0.5% HPMC.

[0347] UUO surgery: On day 0 of the study, mice underwent UUO surgery under pentobarbital sodium anesthesia. First, the mice were shaved, and then an abdominal incision was made to expose the left ureter. The ureter was ligated at two points with 4-0 nylon sutures. The peritoneum and skin of the mice were then closed with sutures, and the mice were transferred to clean cages until they recovered from anesthesia. In sham-operated mice, the left ureter was exposed but not ligated.

[0348] Drug administration: DS109 and DS102 were orally administered to mice in a volume of 10 milliliters (mL) / kilogram (Kg).

[0349] Therapeutic Doses: DS109 was administered once daily at three dose levels, 5, 50, and 250 milligrams (mg) / kg, on days 0 through 13 of the study. DS102 was administered once daily at two dose levels, 50 and 500 mg / kg, on days 0 through 13 of the study.

[0350] Animals: Seven-week-old female C57BL / 6 mice (i.e., animals) were obtained from Japan SLC, Inc. (Japan) and housed under controlled conditions. They were fed a standard diet (CE-2, CLEA Japan, Japan). Animals were housed 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., a 12-hour artificial light-dark cycle, with lights on from 8:00 AM to 8:00 PM), and ventilation. High pressure was maintained in the laboratory to prevent contamination of the facility. Animals were housed in TPX cages (CLEA Japan) containing a maximum of four mice per cage. Sterile paper clean (Japan SLC) bedding was used and changed weekly. Sterile solid standard diet was provided ad libitum in a metal lid on 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 changed weekly, washed, autoclaved, and reused. Mice were identified by ear punch, and each cage was marked with a specific identification code.

[0351] Renal biochemistry measurements: To quantify renal hydroxyproline content, frozen left kidney samples were subjected to alkaline acid hydrolysis as follows: kidney samples were dissolved in 2N sodium hydroxide (NaOH) at 65°C and autoclaved at 121°C for 20 min. The dissolved samples (400 μL) were acid hydrolyzed with 400 μL of 6N hydrochloric acid (HCl) at 121°C for 20 min and neutralized with 400 μL of 4N NaOH containing 10 mg / mL activated charcoal. AC buffer (e.g., 2.2 M acetic acid / 0.48 M citric acid, 400 μL) was added to the samples, followed by centrifugation and collection of the supernatant. A hydroxyproline standard curve was generated using serial dilutions of trans-4-hydroxy-L-proline (Sigma-Aldrich, USA) starting at 16 μg / mL. The prepared samples and standard solutions (e.g., 400 μL each) were mixed with 400 μL of chloramine T solution (Wako Pure Chemical Industries, Japan) and incubated at room temperature for 25 minutes. The samples were then mixed with Ehrlich's solution (e.g., 400 μL) and heated at 65°C for 20 minutes to develop color. The samples were cooled on ice and centrifuged to remove precipitates. The optical density of each supernatant was measured at 560 nanometers (nm). Hydroxyproline concentrations were calculated from a hydroxyproline standard curve. Protein concentrations of kidney samples were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA) and used to normalize the calculated hydroxyproline values. Kidney hydroxyproline content was expressed as micrograms (μg) per mg of protein.

[0352] Histopathological analysis: To visualize collagen deposition, kidney sections were stained with picrosirius red solution (Waldeck, Germany). For quantification of interstitial fibrosis, bright-field images of the corticomedullary region were captured at 200x magnification using a digital camera (e.g., DFC295, Leica Microsystems, Germany), and the positive areas in five fields / section were measured using ImageJ software (National Institutes of Health, USA).

[0353] Quantitative RT-PCR: Total ribonucleic acid (RNA) was extracted from kidney samples using RNAiso (Takara Bio, Japan) according to the manufacturer's instructions. 1 μg of RNA was reverse transcribed using a reaction mixture containing 4.4 micromolar (mM) magnesium chloride (MgCl2) (F. Hoffmann-La Roche, Switzerland), 40 U of RNase inhibitor (Toyobo, Japan), 0.5 mM dNTPs (Promega, USA), 6.28 μM random hexamers (Promega), 5x first-strand buffer (Promega), 10 mM dithiothreitol (Invitrogen, USA), and 200 U of MMLV-RT (Invitrogen) in a final volume of 20 μL. The reaction was performed at 37°C for 1 h, followed by 99°C for 5 min. Real-time PCR was performed using DICE and TB Green™ Premix Ex Taq™ II (Takara Bio). To calculate relative microRNA (mRNA) expression levels, the expression of each gene (e.g., α-SMA, TIMP-1, TGF-β, and type 1 collagen) was normalized to the expression of the reference gene 36B4 (gene symbol: Rplp0). Information on the PCR primer sets and plate layout is listed in Tables 1 and 2.

[0354] [Table 1]

[0355] [Table 2]

[0356] Sample Collection: For serum samples, non-fasting blood was collected by direct cardiac puncture into anticoagulant-free serum separator tubes and centrifuged at 3,500 x g for 4 minutes at 4°C. The supernatant was collected and stored at -80°C for transport. For kidney samples, the left kidney was harvested and cut horizontally into two pieces. The upper part of the left kidney was fixed in Bouin's solution and then embedded in paraffin. The paraffin blocks were stored at room temperature for histological analysis. The lower part of the left kidney was cut coronally into two pieces. The anterior part of the left kidney was snap-frozen in liquid nitrogen and stored at -80°C for gene expression assays. The posterior part of the left kidney was snap-frozen in liquid nitrogen and stored at -80°C for renal biochemistry.

[0357] Statistical tests: Statistical analyses were performed using Bonferroni's multiple comparison test in GraphPad Prism 6 (GraphPad Software Inc., USA). A P value of <0.05 was considered statistically significant. A trend or tendency was assumed when a one-tailed t-test returned a P value <0.1. Results were expressed as mean ± SD.

[0358] 1.2 Experimental design and treatments The research design included the following study arms: Group 1 (sham control): Eight sham-operated mice remained without any treatment until sacrifice. Group 2 (vehicle): Eight UUO mice were orally administered with vehicle [0.5% HPMC] at a volume of 10 mL / kg once daily from day 0 to day 13. Group 3 (DS109 low): 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 (in DS109): 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 (DS102 low): 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 (DS102 high): 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.

[0359] Table 3 summarizes the treatment schedule for each of the groups 1–7 under study.

[0360] [Table 3]

[0361] Animal monitoring and sacrifice: Mice were monitored daily for viability, clinical signs, and behavior. Individual body weights were measured daily before treatment and throughout the treatment period. Approximately 60 minutes after each dose, mice were observed for significant clinical signs of toxicity, moribundity, and death. Animals were sacrificed on day 14 by exsanguination via direct cardiac puncture under isoflurane anesthesia (Pfizer Inc.).

[0362] 1.3 Results Weight Changes and General Considerations: Figure 2 shows the weight changes of all animals. In all animals, body weight decreased after surgery and gradually recovered during the treatment period. The mean body weight of the vehicle group was significantly lower than that of the sham control group on days 2 to 5 and 10 to 11. There was no significant change in mean body weight between the vehicle and treatment groups on any day during the treatment period. No animals died in any group during the treatment period. None of the animals in this study showed any deterioration in their general condition.

[0363] Body weight and kidney weight on the day of sacrifice: Figure 3 and Table 4 show the body weight of the animals on the day of sacrifice. There was no significant difference in the mean body weight on the day of sacrifice between the sham control group and the vehicle group. There was no significant difference in the mean body weight on the day of sacrifice between the vehicle group and the treatment group.

[0364] [Table 4]

[0365] Figures 4A-4D and Table 4 show the kidney weights and kidney weight-to-body weight ratios of the animals on the day of sacrifice. The vehicle group showed a significant increase in mean right kidney weight compared to the sham control group. However, there was no significant difference in mean right kidney weight between the vehicle and treatment groups. The vehicle group also showed a significant increase in mean right kidney weight-to-body weight ratio compared to the sham control group. There was no significant difference in mean right kidney weight-to-body weight ratio between the vehicle and treatment groups. The vehicle group also showed a significant increase in mean left kidney weight compared to the sham control group. There was no significant difference in mean left kidney weight between the vehicle and treatment groups. Finally, the vehicle group showed a significant increase in mean left kidney weight-to-body weight ratio compared to the sham control group, but there was no significant difference in mean left kidney weight-to-body weight ratio between the vehicle and treatment groups.

[0366] Renal Chemistry: Figure 5 and Table 5 show the renal hydroxyproline content of the animals. The vehicle group showed a significant increase in renal hydroxyproline content compared to the sham control group. The DS109 Low, DS109 High, DS102 Low, and DS102 High groups showed a significant decrease in renal hydroxyproline content compared to the vehicle group. There was no significant difference in renal hydroxyproline content between the vehicle and DS109 Medium groups.

[0367] [Table 5]

[0368] Histological analysis: Figures 6A-6G and Table 6 show Sirius Red staining, and Figure 7 shows the fibrotic areas of the animals. Figure 6A shows representative photomicrographs of kidney sections stained with Sirius Red. The vehicle group showed a significant increase in the percentage of fibrotic area (Sirius Red-positive area) compared with the sham control group. Bonferroni multiple comparisons revealed that the fibrotic area in the DS109 Medium group tended to be reduced compared with the vehicle group. There were no significant differences in fibrotic area between the vehicle group and the other treatment groups. Due to the presence of significant outliers, a Mann-Whitney U test was performed, revealing that the fibrotic area in the DS109 Low, DS109 Medium, DS109 High, DS102 Low, and DS102 High groups tended to be reduced compared with the vehicle group (p<0.1).

[0369] [Table 6]

[0370] Gene Expression Analysis: Gene expression analysis for α-SMA, TIMP-1, TGF-β, and type 1 collagen is shown in Figures 8A to 8D and Table 7.

[0371] [Table 7]

[0372] α-SMA: The vehicle group showed a significant increase in α-SMA mRNA expression levels compared to the sham control group. There was no significant difference in α-SMA mRNA expression levels between the vehicle and treatment groups.

[0373] TIMP-1: The vehicle group showed a significant increase in TIMP-1 mRNA expression levels compared to the sham control group. There was no significant difference in TIMP-1 mRNA expression levels between the vehicle and treatment groups.

[0374] TGF-β: The vehicle group showed a significant increase in TGF-β mRNA expression levels compared to the sham control group. There was no significant difference in TGF-β mRNA expression levels between the vehicle and treatment groups.

[0375] Collagen type 1: The vehicle group showed a significant increase in the mRNA expression level of collagen type 1 compared to the sham control group. There was no significant difference in the mRNA expression level of collagen type 1 between the vehicle and treatment groups.

[0376] 1.4 Summary Renal fibrosis was established in the vehicle group of this study, as indicated by Sirius red staining and renal hydroxyproline content.

[0377] DS109: Treatment with low dose DS109 showed a significant reduction in renal hydroxyproline content (p<0.05) and a trend toward a reduction in fibrotic area (p<0.1) compared to the vehicle group. Treatment with medium dose DS109 showed a trend toward a reduction in fibrotic area (p<0.1) compared to the vehicle group. Finally, treatment with high dose DS109 showed a significant reduction in renal hydroxyproline content (p<0.05) and a trend toward a reduction in fibrotic area (p<0.1) compared to the vehicle group.

[0378] DS102: Treatment with low dose DS102 showed a significant reduction in renal hydroxyproline content (p<0.05) and a trend toward a reduction in fibrotic area (p<0.1) compared to the vehicle group. Treatment with high dose DS102 showed a significant reduction in renal hydroxyproline content (p<0.05) and a trend toward a reduction in fibrotic area (p<0.1) compared to the vehicle group.

[0379] In conclusion, the results obtained from this study suggest that DS109 and DS102 have an inhibitory effect on renal fibrogenesis and may have potential as therapeutic agents for chronic kidney disease that progresses to fibrosis.

[0380] Example 2: Cholestatic Liver Disease and / or Liver Fibrosis Bile Duct Ligation (BDL) Study The aim of this study was to investigate the effect of DS012 on BDL-induced cholestasis.

[0381] Figure 9 shows the study design from surgery and treatment through day 14 of the study.

[0382] 1.1 Materials and Methods Test Substance: The test substance for this study was DS 102. To prepare the dosing solution for each substance, DS 102 was diluted in a vehicle of 0.5% hydroxypropylmethylcellulose (HPMC).

[0383] BDL surgery: On day 0 of the study, BDL surgery was performed under pentobarbital (Kyoritsu Seiyaku, Japan) anesthesia. First, the mice were shaved, the abdominal cavity was opened, and the common bile duct was ligated twice with 7-0 surgical silk. The mouse's peritoneum and skin were closed with sutures, and the mouse was transferred to a clean cage (e.g., a resting cage) until it recovered from anesthesia. Sham-operated mice had their common bile duct exposed but not ligated.

[0384] Drug administration: DS102 was administered orally in a volume of 10 milliliters (mL) / kilogram (Kg).

[0385] Therapeutic Dose: DS102 was administered once daily on days 0-13 of the study at three dose levels: 50, 250, and 500 milligrams (mg) / Kg.

[0386] Animals: Pathogen-free, 6-week-old male C57BL / 6J mice were obtained from Japan SLC, Inc. (Japan). Animals were housed 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., a 12-hour artificial light-dark cycle, with lights on from 8:00 AM to 8:00 PM), and ventilation. High pressure was maintained in the laboratory to prevent contamination of the facility. Animals were housed in TPX cages (CLEA Japan) containing up to four mice per cage. Sterile paper clean (Japan SLC) bedding was used and changed once a week. Sterile solid regular chow was provided ad libitum in a metal lid on top of the cage. Pure water was also provided ad libitum from a water bottle equipped with a rubber stopper and sipper tube. The water bottle was changed once a week, washed, sterilized in an autoclave, and reused. Mice were identified by ear punch and each cage was given a specific identification code.

[0387] Serum biochemistry measurements: To visualize collagen deposition, Bouin's fixed liver sections were stained with picrosirius red solution (Waldeck, Germany). For quantification of fibrotic areas, bright-field images of Sirius red-stained sections were captured at 100x magnification using a digital camera (DFC295, Leica, Germany), and the positive areas in five fields / section were measured using ImageJ software (National Institutes of Health, USA).

[0388] Histological analysis: To visualize collagen deposition, kidney sections were stained with picrosirius red solution (Waldeck, Germany). For quantification of interstitial fibrosis, bright-field images of the corticomedullary region were captured at 200x magnification using a digital camera (e.g., DFC295, Leica Microsystems, Germany), and the positive areas in five fields / section were measured using ImageJ software (National Institutes of Health, USA).

[0389] Quantitative RT-PCR: Total ribonucleic acid (RNA) was extracted from liver samples using RNAiso (Takara Bio, Japan) according to the manufacturer's instructions. 1 μg of RNA was reverse transcribed using a reaction mixture containing 4.4 mM magnesium chloride (MgCl2) (F. Hoffmann-La Roche, Switzerland), 40 U of RNase inhibitor (Toyobo, Japan), 0.5 mM dNTPs (Promega, USA), 6.28 μM random hexamers (Promega), 5x first-strand buffer (Promega), 10 mM dithiothreitol (Invitrogen, USA), and 200 U of MMLV-RT (Invitrogen) in a final volume of 20 μL. The reaction was carried out at 37°C for 1 hour, followed by 99°C for 5 minutes. Real-time PCR was performed using DICE and TB Green™ Premix Ex Taq™ II (Takara Bio). To calculate relative mRNA expression levels, the expression of each gene (e.g., α-SMA, TIMP-1, TGF-β, and type 1 collagen) was normalized to the expression of the reference gene 36B4 (gene symbol: Rplp0). Information on PCR primer sets and plate layouts is listed in Tables 8 and 9.

[0390] [Table 8]

[0391] [Table 9]

[0392] Sample collection: For serum samples, nonfasting blood was collected by direct cardiac puncture into anticoagulant-free serum separator tubes and centrifuged at 3,500 x g for 4 minutes at 4°C. The supernatant was collected and stored at -80°C for biochemistry (30 μL) and transport (all remaining). For liver samples, the left lateral lobe was collected and cut into six pieces. Two pieces of the left lateral lobe were fixed in Bouin's solution and then embedded in paraffin. Samples were stored at room temperature for histological analysis. The other two pieces of the left lateral lobe were embedded in OCT compound and flash-frozen in liquid nitrogen. Samples were stored at -80°C. The remaining portion of the left lateral lobe was flash-frozen in liquid nitrogen and stored at -80°C for gene expression analysis. The right median lobe, left median lobe, right lobe, and caudate lobe were flash-frozen in liquid nitrogen and stored at -80°C for transport.

[0393] Statistical tests: Statistical analyses were performed using Bonferroni's multiple comparison test in GraphPad Prism 6 (GraphPad Software Inc., USA). A P value of <0.05 was considered statistically significant. A trend or tendency was assumed when a one-tailed t-test returned a P value <0.1. Results were expressed as mean ± SD.

[0394] 1.2 Experimental design and treatments The research design included the following study arms: Group 1 (sham control): Eight sham-operated mice remained without any treatment until sacrifice. Group 2 (vehicle): 15 mice that underwent BDL surgery were orally administered with vehicle [0.5% HPMC] at a volume of 10 mL / kg once daily from day 0 to day 13. Group 3 (DS102 low): 15 mice that underwent BDL surgery were orally administered vehicle supplemented with DS102 at a dose of 50 mg / kg once daily from day 0 to day 13. Group 4 (DS102): 15 mice that underwent BDL surgery were orally administered vehicle supplemented with DS102 at a dose of 250 mg / kg once daily from day 0 to day 13. Group 5 (DS102 high): 15 mice that underwent BDL surgery were orally administered vehicle supplemented with DS102 at a dose of 500 mg / kg once daily from day 0 to day 13.

[0395] Table 10 summarizes the treatment schedule for each of the study groups 1-5.

[0396] [Table 10]

[0397] Animal monitoring and sacrifice: Mice were monitored daily for viability, clinical signs, and behavior. Individual body weights were measured daily before treatment and throughout the treatment period. Approximately 60 minutes after each administration, mice were observed for significant clinical signs of toxicity, moribundity, and death. Animals were sacrificed by exsanguination via direct cardiac puncture under isoflurane anesthesia (Pfizer Inc.) 14 days after BDL surgery.

[0398] 1.3 Results Body weight changes and general considerations: Figure 10 shows the body weight changes of all animals. In all groups except the sham control group, the mean body weight gradually decreased over the course of the study. The mean body weight of the vehicle group was significantly lower than that of the sham control group from days 2 to 14. There was no significant change in mean body weight between the vehicle and DS102-treated groups on any day during the study.

[0399] During the treatment period, mice died before day 14: 3 of 15 mice died in the vehicle group, and 7 of 15 mice died in the DS102-low, DS102-medium, and DS102-high groups. This model predicts a rate of death solely due to disease induction, and the observed rate of death is consistent with previous data.

[0400] Body weight and liver weight on the day of sacrifice: Figure 11 and Table 11 show the body weight of the animals on the day of sacrifice. The vehicle group showed a significant reduction in mean body weight on the day of sacrifice compared to the sham control group. There was no significant difference in mean body weight on the day of sacrifice between the vehicle group and the DS102-treated group.

[0401] [Table 11]

[0402] Figure 12 A and Figure 12B and Table 11 shows the liver weights and liver weight-to-body weight ratios of the animals on the day of sacrifice. The vehicle group showed a significant increase in mean liver weight compared to the sham control group. The mean liver weight of the DS102-high group tended to decrease compared to the vehicle group. There was no significant difference in mean liver weight between the vehicle group and the other treatment groups. The vehicle group showed a significant increase in mean liver weight-to-body weight ratio compared to the sham control group. The mean liver weight-to-body weight ratio of the DS102-high group tended to decrease compared to the vehicle group. There was no significant difference in mean liver weight-to-body weight ratio between the vehicle group and the other treatment groups.

[0403] Biochemistry: Figure 13 and Table 12 show the serum aminotransferase (ALT) of the animals. The vehicle group showed a significant increase in serum ALT levels compared to the sham control group. There was no significant difference in serum ALT levels between the vehicle and DS102-treated groups. However, from previous data in this model, it is known that ALT levels decrease by day 14 without treatment; therefore, this may affect the ability to detect differences between groups.

[0404] [Table 12]

[0405] Figure 14 and Table 12 show the serum total bilirubin of the animals. The vehicle group showed a significant increase in serum total bilirubin levels compared to the sham control group. There was no significant difference in serum total bilirubin levels between the vehicle group and the DS102-treated group.

[0406] Histological analysis: Figures 15A-14E and Table 13 show Sirius Red staining, and Figure 16 shows the fibrotic areas of the animals. Figure 14A shows representative photomicrographs of liver sections stained with Sirius Red. Liver sections from the vehicle group showed increased collagen deposition in the portal venous regions of the hepatic lobule and PV-CV or PV-PV bridging fibrosis compared to the sham control group. The vehicle group showed a significant increase in fibrotic area (Sirius Red-positive area) compared to the sham control group. The DS102 medium group showed a significant reduction in fibrotic area compared to the vehicle group. The fibrotic area in the DS102 high group tended to be reduced compared to the vehicle group. There was no significant difference in fibrotic area between the vehicle and DS102 low groups.

[0407] [Table 13]

[0408] Gene Expression Analysis: Gene expression analysis for α-SMA, TIMP-1, TGF-β, and type 1 collagen is shown in Figures 17A-17D and Table 14.

[0409] [Table 14]

[0410] α-SMA: The vehicle group showed a significant increase in α-SMA mRNA expression levels compared to the sham control group. The α-SMA mRNA expression levels in the DS102 high group tended to decrease compared to the vehicle group. There was no significant difference in α-SMA mRNA expression levels between the vehicle group and the DS102 treatment group.

[0411] TIMP-1: The vehicle group showed a significant increase in TIMP-1 mRNA expression levels compared with the sham control group. The TIMP-1 mRNA expression levels in the DS102-high group tended to decrease compared with the vehicle group. There was no significant difference in TIMP-1 mRNA expression levels between the vehicle group and the DS102-treated group.

[0412] TGF-β: The vehicle group showed a significant increase in TGF-β mRNA expression levels compared to the sham control group. The DS102 high group showed a significant decrease in TGF-β mRNA expression levels compared to the vehicle group. There was no significant difference in TGF-β mRNA expression levels between the vehicle group and the DS102 treatment group.

[0413] Type 1 collagen: The vehicle group showed a significant increase in type 1 collagen mRNA expression levels compared to the sham control group. The type 1 collagen mRNA expression levels in the DS102-high group tended to decrease compared to the vehicle group. There was no significant difference in type 1 collagen mRNA expression levels between the vehicle group and the DS102-treated group.

[0414] 1.4 Summary In this study, biochemical parameters (e.g., ALT and total bilirubin), histological collagen deposition (e.g., fibrotic area), and gene expression levels (e.g., α-SMA, TIMP-1, TGF-β, type 1 collagen) in the vehicle group were significantly increased compared with the sham control group. These results suggested that cholestasis and liver fibrosis were established in the vehicle group.

[0415] Treatment with medium-dose DS102 showed a significant reduction in fibrotic area (p<0.05) compared with the vehicle group. Treatment with high-dose DS102 showed a significant reduction in TGF-β mRNA expression levels (p<0.05) compared with the vehicle group, as well as a trend toward near-significant reductions in fibrotic area, liver weight, liver-to-body weight ratio, α-SMA, TIMP-1, and type 1 collagen mRNA expression levels (p<0.01). These results suggest that DS102 inhibits the production of multiple profibrotic cytokines in the liver, has the effect of suppressing liver fibrosis, and has potential as a therapeutic agent for cholestatic liver disease.

[0416] Example 3: Effects of DS102 on TGF-β receptors, signaling, and inducible fibrotic proteins The purpose of this study was to investigate the effects of 15-HEPE and 15-HEPE EE on TGF-β receptors, TGF-β-induced intracellular signaling, and the expression of profibrotic epithelial-mesenchymal transition proteins.

[0417] 1.1 Materials and Methods Cytotoxicity studies: The cytotoxicity of 15-HEPE free acid and ethyl ester was tested in various liver (hepatocellular carcinoma) cell lines to understand the concentration range in the test system.

[0418] Transcriptional activity: Promoter (luciferase) assays were performed to measure TGFβ-induced transcriptional activation after administration of 15-HEPE.

[0419] Sucrose gradient ultracentrifugation and confocal microscopy were used to identify 15-HEPE-induced microdomain migration of TGF-β receptors by sucrose. Sucrose density gradient analysis of TGF-β receptors was performed on the plasma membranes of Mv1Lu (mink lung epithelial cells) cells treated with 100 μM 15-HEPE and an equal volume of DMSO (dimethyl sulfoxide) for 0, 1, 4, and 24 hours at 37°C. Cell lysates from these treated cells were subjected to sucrose density gradient ultracentrifugation. Sucrose gradient fractions were then analyzed by Western blot using anti-TβR-I (type I TGF-β receptor), anti-TβR-II (type II TGF-β receptor), anti-TβR-III (type III TGF-β receptor, betaglycan), anti-EGFR (epidermal growth factor receptor), and anti-caveolin-1 antibodies. The lipid raft / caveolae and nonlipid raft microdomain localization of TβR-I, TβR-II, TβR-III, EGFR, and caveolin-1 in the plasma membrane of untreated or 15-HEPE-treated cells was assessed to determine the effect of 15-HEPE on membrane microdomain localization of TGF-β receptors.

[0420] 1.2 Results The effects of 15-HEPE on TGF-β-induced signal transduction and cellular responses were determined. To test the effect of 15-HEPE on TGF-β-induced signal transduction, we examined the ability of 15-HEPE to modulate TGF-β-stimulated Smad2 phosphorylation and nuclear translocation, both of which are key signaling events leading to TGF-β-induced cellular responses. One of the key biological activities of TGF-β is the transcriptional activation of genes responsible for epithelial-mesenchymal transition (EMT), a key event in wound healing, tissue fibrosis, and cancer progression. We evaluated the effect of 15-HEPE on the expression of TGF-β-induced epithelial-mesenchymal transition-related proteins (e.g., fibronectin, PAI-1, and N-cadherin) in HepG2 and LXR cells. HepG2 cells (a human hepatocellular carcinoma cell line) were treated with increasing doses of 15-HEPE stage II in DMEM containing 0.1% FBS for 1 hour. continuation 200 picomolar (pM) TGF-β With or without it 48 hours Interstitial I was furious.

[0421] 15-HEPE directly inhibited TGF-β signaling, as shown in Figures 18A and 18B. Specifically, Figure 18A shows that 15-HEPE induced degradation of type II TGF-β receptors and blocked the production of TGF-β-induced epithelial-mesenchymal transition (EMT) (i.e., profibrotic) proteins. HepG2 cells (human hepatoma cell line) were treated with increasing doses of 15-HEPE stage II in DMEM containing 0.1% FBS for 1 hour. continuation 200 picomolar (pM) TGF-β With or without it 48 hours Interstitial The two panels shown in Figure 18A are from two separate experiments performed under the same experimental conditions. Results from these separate experiments demonstrate that 15-HEPE blocked the production of EMT proteins induced by TGF-β and induced the degradation of type II TGF-β receptors. Figure 18A also shows the effect of 15-HEPE on plasminogen activator inhibitor-1 (PA-1), a protein induced by TGF-β and associated with increased cardiovascular risk. Figure 18B shows that 15-HEPE inhibits TGF-β-stimulated intracellular signaling (e.g., SMAD2 / 3 phosphorylation) in hepatic stellate cells. The experiment performed in Figure 18B involved pretreating LX2 cells (human hepatic stellate cells) with increasing concentrations of DS102 (0 μM to 100 μM) for 24 hours, followed by 30 minutes of TGF-β stimulation. The results of the experiment demonstrated that 15-HEPE inhibited TGF-β stimulation.

[0422] 15-HEPE sucrose density gradient analysis of TGF-β receptors was performed on plasma membranes of mink lung epithelial cells (Mv1Lu) treated with 100 μM 15-HEPE and an equal volume of dimethyl sulfoxide (DMSO) for 0, 1, 4, and 24 h at 37°C. Cell lysates from these treated cells were subjected to sucrose density gradient ultracentrifugation. Sucrose gradient fractions were then analyzed by Western blot using anti-TβR-I (type I TGF-β receptor—Figure 18C), anti-TβR-II (type II TGF-β receptor—Figure 18D), anti-TβR-III (type III TGF-β receptor, betaglycan—Figure 18E), anti-EGFR (epidermal growth factor receptor—Figure 18F), and anti-caveolin-1 antibodies (Figure 18G). Fractions 4–5 contained lipid rafts / caveolae, whereas fractions 7–10 were non-lipid raft fractions. Treatment with 15-HEPE did not affect TβR-I protein abundance, but 24 h of treatment induced the translocation of TβR-I to lipid rafts (Figure 18C). Stars (★) indicate that 15-HEPE increased the abundance of TβR-I in the fractions (24 h) compared with the abundance in the control and shorter treatment periods (Figure 18C). 15-HEPE induced the translocation of TβR-II from 1–4 h and further degradation with 24 h of treatment (Figure 18D). Triangles (▼) indicate a reduction in the translocation and abundance of TβR-II in the fractions compared with control cells (Figure 18D). In Figures 18E, 18F, and 18G, 15-HEPE did not alter the localization and abundance of TβR-III, EGFR, and caveolin-1.

[0423] 1.3 Summary Therefore, taken together, these results indicate that 15-HEPE, in both its free acid and ethyl ester forms, induced the translocation and degradation of type II TGF-β receptors, blocked the production of TGF-β-induced profibrotic proteins, and inhibited TGF-β-induced intracellular signaling (SMAD2 / 3) in hepatic stellate cells. Therefore, 15-HEPE and 15-HEPE EE can directly inhibit TGF-β signaling, supporting the therapeutic potential of 15-HEPE and 15-HEPE EE in the treatment of multiple fibrotic diseases, including nonalcoholic steatohepatitis (NASH), other fibrotic diseases, cardiometabolic diseases, and multiple manifestations of cancer.

[0424] Example 4: Efficacy of orally administered DS102 in patients with NAFLD The objective of this study was to evaluate the efficacy of orally administered DS102 capsules versus placebo in the treatment of adult patients with nonalcoholic fatty liver disease (NAFLD).

[0425] 1.1 Study endpoints Primary Endpoints: The primary endpoints of this study included the efficacy and safety of DS102 administration. Efficacy was assessed based on the change in serum alanine aminotransferase (ALT) from baseline to week 16 and the change in liver stiffness measured by transient elastography from baseline to week 16. Safety was assessed by the number of TEAEs (treatable adverse events) leading to treatment discontinuation in each treatment group.

[0426] Secondary Endpoints: Secondary endpoints of this study included changes 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 liver fat measured by controlled decay 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.

[0427] Exploratory Analyses: Exploratory analyses included analyses of lipid and metabolic parameters such as 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 A1C (HbA1C). Additional exploratory analyses included high-throughput lipidomics and proteomics.

[0428] 1.2 Research Plan This was a randomized, placebo-controlled, double-blind, parallel-group, multicenter, exploratory Phase IIa study to investigate the safety and efficacy of orally administered DS102 capsules in patients aged 18 to 75 years with NAFLD, as well as the dose-response relationship between two doses of DS102 and placebo. The study investigated three parallel groups of patients with confirmed NAFLD and compared two different doses of DS102 with placebo over a 16-week treatment period. The study was designed to include 96 evaluable patients, with 32 randomized patients per treatment group.

[0429] The study consisted of a 28-day screening period, a 16-week treatment period, and a 4-week follow-up period. At the screening visit, patients were evaluated using a screening test. Patients who met the inclusion criteria and did not meet the exclusion criteria were enrolled.

[0430] A schematic diagram of the overall time frame of the study is provided in Figure 19. Once patients were enrolled in the study, they were restricted from using any other treatments for NAFLD. Patients were permitted to continue any medications (e.g., prescription and over-the-counter (OTC) medications) or therapeutic interventions that were deemed necessary for the patient and that, in the opinion of the investigator, would not interfere with the safety and efficacy assessments, as long as they were not included in the list of "concomitant medications" described below.

[0431] Before starting the comparative treatment period, patients returned to the site for a baseline assessment of their disease, and eligible patients were randomly assigned in a 1:1:1 randomization to one of three parallel-group treatment regimens. Treatment group A: Two 500 mg capsules of placebo were administered orally twice daily (four capsules per day) for 16 weeks. Treatment Group B: One 500 mg capsule of DS102 and one 500 mg capsule of placebo were orally administered twice daily (four capsules per day) for 16 weeks. Treatment Group C: Two 500 mg capsules of DS102 were orally administered twice daily (four capsules per day) for 16 weeks.

[0432] To maintain double-blind conditions, the DS102 and placebo capsules were identical in appearance.

[0433] 1.3 Patients and Screening To participate in this study, patients had to meet all of the following inclusion criteria and none of the following exclusion criteria: Inclusion and exclusion criteria were verified at the screening visit (Visit 1) and at the start of treatment / baseline visit (Visit 2).

[0434] The inclusion criteria for this study were: Patients diagnosed with NAFLD by the presence of hepatic steatosis on imaging or histology in the absence of secondary causes. Patients with ALT ≥ 1.5ULN and < 5ULN on two occasions at least 7 days apart during screening. Patients with a previous liver biopsy within 3 months of screening showing NASH and / or fibrosis ≥ F1, or NFS ≥ -1.455, or Fib-4 ≥ 1.3, or Fibroscan ≥ 8 kPa. Patients with a body mass index (BMI) of 25.0 to 40.0 kg / m². Patients with a history of controlled obesity or controlled diabetes were allowed in the study. Patients whose pre-study clinical laboratory findings did not preclude participation in the study in the opinion of the investigator. Patients aged 18-75 years. Female patients and male patients with fertile female partners were required to use adequate contraception or have their partners sterilized during the study. Adequate contraception was defined as: systemic hormonal contraception, intrauterine device combined with spermicide, or barrier method of contraception, or agreeing to sexual abstinence, defined as patients abstaining from heterosexual intercourse in line with their preferred usual lifestyle for the entire duration of risk associated with study treatment. Hormonal contraception had to be taken at a stable dose for at least 1 month prior to baseline. Patients who were able to communicate satisfactorily with the investigator, understand and comply with the requirements of the study, and understand and sign written informed consent.

[0435] The exclusion criteria for this study were: Patients with unstable metabolic status, such as weight change >5% in the 3 months prior to enrollment. -Patients with a history of gastric bypass surgery, orthotopic liver transplant (OLT), or enrolled in OLT. Patients with uncontrolled type 2 diabetes, i.e., HbA1c ≥ 9% (75mmol / mol) at screening. Patients with decompensated or severe liver disease as evidenced by one or more of the following: confirmed or suspected cirrhosis, esophageal varices, ascites, suspected portal hypertension, hospitalization for liver disease within 60 days of screening, bilirubin ≥ 2 x ULN, or ALT or AST ≥ 5 x ULN. Patients with Gilbert syndrome were eligible if their conjugated bilirubin was ≤ 1.5 x ULN. Patients with active or medically treated inflammatory bowel disease. Patients with diagnosed or suspected autoimmune diseases such as systemic lupus erythematosus (SLE) and / or rheumatoid arthritis (RA). Patients with a history of or active non-hepatic malignancy other than curatively treated skin cancer (basal cell carcinoma or squamous cell carcinoma). Patients with coronary artery disease, cerebrovascular disease, pulmonary disease, renal failure, serious psychiatric illness, respiratory or hypertensive disease, and any significant systemic or serious illness other than liver disease, including diabetes, arthritis, etc., which in the investigator's opinion precludes the patient from participating in and completing the study. Patients were excluded if they required antidiabetic therapy (including insulin sensitizers) and / or lipid-lowering therapy and had not been on a stable dose for at least 3 months before screening. If patients were insulin-dependent, this therapy should have been initiated at least 3 months before screening, although dose modifications were permitted. Patients with known hypersensitivity to any component of the investigational treatment. Patients who test positive for human immunodeficiency virus (HIV) antibodies, hepatitis B surface antigen, or hepatitis C antibodies at screening. Patients with liver diseases of other etiologies, such as drug-induced, autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), hemochromatosis, alpha-1 antitrypsin (A1AT) deficiency, and Wilson's disease. Patients with a significant medical history of drug / solvent abuse in the opinion of the investigator. Patients who, in the opinion of the investigator, have a history of alcohol abuse or drink more than 21 units per week (men) or 14 units per week (women) (1 unit consists of 10 mg or 8 mg of pure alcohol). Patients who used dietary supplements rich in omega-3 or omega-6 fatty acids in the 4 weeks prior to baseline. Patients who participated in any other clinical study using an investigational drug within 3 months prior to the first day of study treatment. Patients who were pregnant, planning pregnancy, breastfeeding, and / or unwilling to use adequate contraception during the study. Patients who, in the opinion of the investigator, were unsuitable for participation in the study.

[0436] 1.4 Conducting the Research Ten clinic visits were scheduled during the study after the screening visit: one at the start / baseline of the comparative treatment period (Day 0 / Visit 2) and eight during the comparative treatment period (Week 2 / Visit 3, Week 4 / Visit 4, Week 6 / Visit 5, Week 8 / Visit 6, Week 10 / Visit 7, Week 12 / Visit 8, Week 14 / Visit 9, and Week 16 / Visit 10). The final safety follow-up visit (Visit 11) occurred 4 weeks after Visit 10 or 2 weeks after the last visit attended if the patient did not complete the study. Table 17 describes the study clinical visits.

[0437] [Table 15] TIFF0007734686000016.tif74170

[0438] Screening Visit (Visit 1): The following screening visits:

[0439] Screening Visit (Visit 1): Once informed consent was obtained, patients were assigned a patent screening number. Ideally, patients were fasting. 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 pressure, heart rate, and temperature), samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests), virology, pregnancy test (for female patients of childbearing potential), ALT, AST testing (ALT measured twice during screening), and assessment of concomitant medications. Unscheduled visits occurred if patients needed to visit between scheduled visit dates due to adverse events (AEs), difficulty complying with study protocol requirements, or significant changes in disease status. All medically necessary procedures were followed. If eligible, before leaving the clinic, patients were instructed not to eat any breakfast before their next visit and were given a minimum 8-hour fasting period.

[0440] Treatment Period: After successfully completing the 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 three treatment regimens. Patients took their assigned investigational medicinal product (IMP), either DS102 capsules or placebo capsules, twice daily during the comparative treatment period. Each self-administration of IMP was recorded on the patient's diary card. Patients were instructed to take DS102 with or after meals in the morning and evening (except for the mornings of clinic visits 3, 4, 6, 8, and 10, when patients were instructed to refrain from taking DS102 before the visit and to take DS102 as soon as possible after the clinic visit). At the beginning of baseline (Visit 2), Week 2 (Visit 3), Week 4 (Visit 4), Week 8 (Visit 6), Week 12 (Visit 8), Week 16 (Visit 10), and Week 20 (Visit 11), patients were asked if they had fasted for at least 8 hours before the visit. If they had not fasted, the duration of the fasting period was documented, and patients were re-instructed regarding the duration of the fasting period. Patients were then offered a light breakfast (e.g., tea or orange juice and toast). At baseline (Visit 2), Week 8 (Visit 8), and Week 16 (Visit 10), blood collection assessments were performed before patients consumed the light breakfast. Unscheduled visits occurred due to adverse events (AEs), difficulty complying with study protocol requirements, or when patients needed to visit between scheduled visit dates due to significant changes in disease status. All medically necessary procedures were followed. Patients who discontinued the study early had the study procedures scheduled for Visit 10 performed as soon as possible after their withdrawal so that all study-related information could be recorded. Urine DOA and alcohol breath tests were administered at any time during the study at the investigator's discretion.

[0441] Baseline (Visit 2): Patients presented to the study site at Visit 2. Blood draw was the first assessment performed. After blood draw, the following assessments were performed: verification of inclusion / exclusion criteria, medical history, physical examination, 12-lead ECG, pharmacokinetic sampling, vital signs (blood pressure, heart rate, and temperature), samples for laboratory safety studies (hematology, serum biochemistry, and coagulation tests), lipid profile, urinalysis, pregnancy test (for female patients of childbearing potential), ALT, AST tests, HOMA-IR / Adipo-IR, ELF, liver stiffness and CAP, FIB-4, NFS (including BMI), biomarker blood samples, exploratory blood samples, patient randomization, study drug / placebo administration, AE assessment, and assessment of concomitant medications. If all study entry criteria were met, the investigator randomized the patient and provided them with the designated IMP or placebo from one of the patient treatment packs. Once all baseline assessments were completed, the first dose of IMP or placebo was administered at the site. Patients received the second dose of IMP or placebo in the evening of Day 0. Thereafter, capsules were administered twice daily. Patients received IMP or placebo on the morning of their return visit to the site (Visit 3). Before leaving the clinic, patients were instructed not to eat any breakfast before their next visit, allowing for a minimum 8-hour fasting period.

[0442] Week 2 (Visit 3): Patients returned to the study site for 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 pressure, heart rate, and temperature), ALT, AST testing, HOMA-IR / Adipo-IR, AE assessment, and assessment of concomitant medications. IMP or placebo was returned, and additional IMP or placebo was provided to the patient. Patients took the next dose of IMP or placebo as soon as all visit assessments were completed. They continued to receive capsules twice daily. At the completion of this visit, patients were informed that they would need to return to the study site in 2 weeks for Visit 4. Patients did not take IMP or placebo on the morning of their return visit to the site (Visit 4). Before leaving the clinic, patients were instructed not to eat any breakfast before their next visit and were given a minimum 8-hour fasting period.

[0443] Week 4 (Visit 4): Patients returned to the study site for 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 pressure, heart rate, and temperature), samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests), pregnancy test (for female patients of childbearing potential), ALT, AST tests, HOMA-IR / Adipo-IR, AE assessment, and assessment of concomitant medications. IMP or placebo was returned, and additional IMP or placebo was provided to the patient. Patients took their next dose of IMP or placebo as soon as all visit assessments were completed. They continued to receive capsules twice daily. At the completion of this visit, patients were informed that they would need to return to the study site in 2 weeks for Visit 5. Patients did not take IMP or placebo on the morning of their return visit to the site (Visit 5).

[0444] Week 6 (Visit 5): Patients returned to the study site at Visit 5. AE assessments and concomitant medication assessments were performed. IMP or placebo was returned, and additional IMP or placebo was provided to the patient. Patients took the next dose of IMP or placebo as soon as all visit assessments were completed. They continued to receive capsules twice daily. At the completion of this visit, patients were informed that they would need to return to the study site in two weeks at Visit 6. Patients did not take IMP or placebo on the morning of their return visit to the site (Visit 6). Before leaving the clinic, patients were instructed not to eat any breakfast before their next visit, allowing for a minimum fasting period of 8 hours.

[0445] Week 8 (Visit 6): Patients returned to the study site at Visit 6. Patients did not receive IMP or placebo on the morning of Visit 6. Blood draw was the first assessment performed. After the blood draw, the following assessments were performed: physical examination, pharmacokinetic sampling, vital signs (blood pressure, heart rate, and temperature), samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests), lipid profile, pregnancy test (for female patients of childbearing potential), ALT, AST tests, HOMA-IR / Adipo-IR, biomarker blood samples, AE assessment, and assessment of concomitant medications. IMP or placebo was returned, and additional IMP or placebo was provided to the patient. Patients took their next dose of IMP or placebo as soon as all visit assessments were completed. They continued to receive capsules twice daily. At the completion of this visit, patients were informed that they would need to return to the study site within two weeks for Visit 7. Patients took IMP or placebo on the morning of their return visit to the site (Visit 7).

[0446] Week 10 (Visit 7): Patients returned to the study site at Visit 7. AE assessments and concomitant medication assessments were performed. IMP or placebo was returned, and additional IMP or placebo was provided to the patient. Patients took the next dose of IMP or placebo as soon as all visit assessments were completed. They continued to receive capsules twice daily. At the completion of this visit, patients were informed that they would need to return to the study site in two weeks at Visit 8. Patients did not take IMP or placebo on the morning of their return visit to the site (Visit 8). Before leaving the clinic, patients were instructed not to eat any breakfast before their next visit, allowing for a minimum fasting period of eight hours.

[0447] Week 12 (Visit 8): Patients returned to the study 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 pressure, heart rate, and temperature), samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests), pregnancy test (for female patients of childbearing potential), ALT, AST tests, HOMA-IR / Adipo-IR, AE assessment, and assessment of concomitant medications. IMP or placebo was returned, and additional IMP or placebo was provided to the patient. Patients took their next dose of IMP or placebo as soon as all visit assessments were completed. They continued to receive capsules twice daily. At the completion of this visit, patients were informed that they would need to return to the study site in 2 weeks for Visit 9. Patients did not take IMP or placebo on the morning of their return visit to the site (Visit 9).

[0448] Week 14 (Visit 9): Patients returned to the study site at Visit 9. AE assessments and concomitant medication assessments were performed. IMP or placebo was returned, and additional IMP or placebo was provided to the patient. Patients took the next dose of IMP or placebo as soon as all visit assessments were completed. They continued to receive capsules twice daily. At the completion of this visit, patients were informed that they would need to return to the study site in two weeks at Visit 10. Patients should not take IMP or placebo on the morning of their return visit to the site (Visit 10). Before leaving the clinic, patients were instructed not to eat any breakfast before their next visit and were given a minimum eight-hour fasting period.

[0449] Week 16 (Visit 10) or Early Withdrawal: Patients returned to the study site at Visit 10. Patients did not take IMP or placebo on the morning of Visit 10. Blood draw was the first assessment performed. After blood draw, the following assessments were performed: physical examination, 12-lead ECG, pharmacokinetic sampling, vital signs (blood pressure, heart rate, and temperature), samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests), urinalysis, lipid profile, pregnancy test (for female patients of childbearing potential), ALT, AST tests, HOMA-IR / Adipo-IR, ELF, liver stiffness and CAP, FIB-4, NFS [including BMI], biomarker blood samples, exploratory blood samples, AE assessment, and assessment of concomitant medications. IMP or placebo was returned. No further IMP or placebo blister packs or patient diary cards were issued. After completion of the study assessments at this visit, study restrictions continued. Upon completion of this visit, patients were informed that they would need to return to the study site within 4 weeks at Visit 11 to evaluate any AEs since this visit and to perform safety and efficacy assessments. Before leaving the clinic, patients were instructed not to eat any breakfast before their next visit and were given a minimum 8-hour fasting period.

[0450] Follow-up Visit (Week 20 / Visit 11): Four weeks after Visit 10 (or two weeks after the early withdrawal visit), patients returned to the study site. The following assessments were performed: physical examination, pharmacokinetic sampling, vital signs (blood pressure, heart rate, and temperature), samples for clinical laboratory safety tests (hematology, serum biochemistry, and coagulation tests), pregnancy test (for female patients of childbearing potential), ALT, AST tests, HOMA-IR / Adipo-IR, biomarkers, AE assessment, and assessment of concomitant medications.

[0451] 1.5 Rating Efficacy assessments included ALT, AST, ALT:AST ratio, HOMA-IR / Adipo-IR, ELF, liver stiffness and CAP, FIB-4 index, and NFS, a detailed description of each is provided below.

[0452] ALT, AST, and ALT:AST ratio: Elevated liver enzymes (ALT and AST) are markers of liver injury and were assessed at Visit 1 / Screening (twice during screening, at least 7 days apart), Visit 2 / Baseline, Visit 3 / Week 2, Visit 4 / Week 4, Visit 6 / Week 8, Visit 8 / Week 12, Visit 10 / Week 16, and Follow-up Visit 11 / Week 20.

[0453] HOMA-IR / Adipo-IR: HOMA-IR / Adipo-IR levels are a measure of insulin resistance. HOMA-IR is calculated by multiplying fasting plasma insulin (FPI) by fasting plasma glucose (FPG) and dividing by a constant of 405. Adipo-IR is calculated by multiplying fasting nonesterified fatty acids (NEFA) by fasting insulin. Blood samples were collected 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 / Week 16, and Follow-Up Visit 11 / Week 20. All subjects were required to fast for a minimum of 8 hours before blood collection. If subjects did not fast for at least 8 hours, the duration of the fasting period was recorded, and subjects were prompted to fast appropriately for the next clinical visit.

[0454] ELF: The ELF score is a set of extracellular matrix markers consisting of tissue inhibitor of metalloproteinase 1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP), and hyaluronic acid (HA). Blood samples were collected at baseline (Visit 2) and at week 16 (Visit 10) for this assessment.

[0455] Liver stiffness and CAP: Liver stiffness and CAP were assessed using transient elastography (e.g., FibroScan® 502 Touch model or equivalent). If possible, patients were fasted and scanned at the same time of day for baseline (week 0) and visit 10 (week 16). For this evaluation, the following conditions were met: the patient lay supine with the right arm maximally abducted behind the head, in a position similar to that used for liver biopsy (LB); the tip of the transducer was placed on the skin between the ribs over the right lobe of the liver (the physician performed measurements with a probe placed in the intercostal space); during the FibroScan® examination, the choice of the M+ or XL+ probe was determined by the Automatic Probe Selection tool (APS) (if the APS tool notified to use the "XL+ probe" or to "switch" continuously between the "M+ probe" and the "XL+ probe", only the XL+ probe was used); the operator identified the location of the part of the liver free of large vascular structures with the help of ultrasound time-video (the depth of measurement was 35-75 mm for the XL+ probe and 25-65 mm for the M+ probe. The volume explored was 3 cm 3 For each patient, the operator performed a test involving at least 10 valid measurements or a maximum of 20 trials at the same location using either the XL+ or M+ probe. The entire test lasted no longer than 10–15 min. Final hardness and CAP values ​​were recorded as the median of the valid measurements.

[0456] FIB-4 Index: The FIB-4 index is based on age, platelet count, ALT level, and AST level and was assessed at baseline (Visit 2) and at Week 16 (Visit 10). The FIB-4 score is determined as shown in the following formula:

number

[0457] NFS: NFS is based on age, hyperglycemia, BMI, platelet count, albumin level, and AST / ALT ratio. NAFLD fibrosis score = -1.675 + 0.037 × age (years) + 0.094 × BMI (kg / m2) + 1.13 × IFG / diabetes (yes = 1, no = 0) + 0.99 × AST / ALT ratio - 0.013 × platelets (× 109 / L) - 0.66 × albumin (g / dL). NFS was assessed at baseline (Visit 2) and at Week 16 (Visit 10).

[0458] Safety assessments included the following: medical history, physical examination, ECG, vital signs, clinical laboratory safety tests (e.g., hematology, serum chemistry, coagulation, lipid profile, and urinalysis), virology, pregnancy test, blood draw, pharmacokinetic sampling, exploratory blood draw, biomarker blood draw, urine DOA and alcohol breath test, adverse event assessment, concomitant medications, bioanalysis, specimens, storage, handling, and shipping, and restrictions. A detailed description of each is provided below.

[0459] Medical History: A complete review of the patient's medical history was performed by the investigator or designee at the screening visit (Visit 1) and baseline (Visit 2) to ensure that no exclusion criteria were met. All intercurrent illnesses, whether considered study-related or not, were reported by the investigator. When possible, the date of diagnosis or duration of illness was recorded.

[0460] Physical Examination: A physical examination (including height and weight) was performed by the investigator according to the study flowchart 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 / Week 16, and Follow-up Visit 11 / Week 20. This examination was completed in full at baseline and was symptom-based thereafter (i.e., a standard panel of body systems was not assessed unless indicated by the patient). For example, if the patient reported the presence of a "rash" to the investigator, the skin was evaluated. There was no need to evaluate additional body systems unless clinically justified. Any abnormal results were recorded. Changes in physical examination findings compared to the baseline examination were recorded as AEs.

[0461] ECG: A 12-lead ECG 10mm / 1mv, 25mm / sec with a 10-second lead II rhythm strip was recorded at each time point. ECGs were recorded using a GE Mac 1200 or equivalent model. Patients rested quietly in a fully supine position for 5 minutes before the ECG was taken. Recordings were performed at Visit 1 / Screening, Visit 2 / Baseline, and Visit 10 / Week 16 on the days indicated in the study flow chart in Table 19.

[0462] Vital Signs: Vital sign measurements were performed 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 / Week 16, and Follow-up Visit 11 / Week 20, according to the study flowchart in Table 19. Vital sign measurements were performed before any blood samples were drawn. If findings were obtained after the patient signed in, any new findings or changes to previous findings considered clinically significant were recorded as AEs. Vital sign measurements included systolic and diastolic blood pressure (mmHg) performed in the supine position (e.g., after at least 5 minutes of rest), heart rate (beats per minute) measured at rest, and temperature measured according to clinical practice.

[0463] Clinical Laboratory Safety Studies: Safety studies were performed for hematology, serum biochemistry, coagulation, lipid profile, and urinalysis. Blood and urine samples were collected according to the study flow chart in Table 19 for routine hematology, serum biochemistry, coagulation, and urinalysis, along with lipid profile. All samples were analyzed at a central laboratory. All subjects fasted for a minimum of 8 hours prior to blood collection. If a subject did not fast for a minimum of 8 hours, the duration of the fasting period was recorded, and the subject was reminded to fast appropriately for the next clinical visit. Hematology: Complete blood count including red blood cell count, hemoglobin, hematocrit, white blood cell count, differential white blood cell count, platelet count, and reticulocyte count. Serum biochemistry: urea (blood urea nitrogen; BUN), creatinine, uric acid, total bilirubin, indirect and direct bilirubin, sodium, potassium bicarbonate, 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). · Coagulation: Prothrombin time (PT), International Normalized Ratio (INR), and Activated Partial Prothrombin Time (APTT). Lipid profile: LDL, HDL, and VLDL-C. Urinalysis: pH, protein, glucose, blood, ketones, leukocytes, leukocyte esterase, bilirubin, specific gravity, urobilinogen, and nitrates. Reflex micro if blood, protein, leukocyte esterase, or nitrates / nitrites are present.

[0464] Virology: Blood samples were collected for virology testing, including HIV, HepC, and HepB, as detailed in the study flow chart in Table 19.

[0465] Pregnancy Testing: For female patients of childbearing potential, pregnancy tests were performed 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 according to the study flow chart in Table 19.

[0466] Blood Collection: Blood samples were obtained and laboratory results were reviewed for clinically significant values ​​by each investigator following sample analysis and validation. Additional blood may be required to repeat safety laboratory tests.

[0467] Pharmacokinetic (PK) Sampling: Blood samples for PK analysis were collected by direct venipuncture at Visit 2 / Baseline, Visit 3 / Week 2, Visit 4 / Week 4, Visit 6 / Week 8, Visit 8 / Week 12, Visit 10 / Week 16, and Follow-up Visit 11 / Week 20, according to the study flow chart in Table 19. A 1 mL blood sample was collected at each time point. After centrifugation, plasma samples were divided into two aliquots, and a reserve sample was stored at the central laboratory until the bioanalytical assays were completed.

[0468] Exploratory Blood Collection: Blood was collected at baseline (week 0) and visits 10 / 16 weeks according to the study flow chart in Table 19 and saved for potential gene array analysis or additional exploratory testing at a later date.

[0469] Biomarker Blood Collection: Blood was collected at baseline (week 0), visits 6 / 8 weeks, visits 10 / 16 weeks, and follow-up visits 11 / 20 weeks according to the study flow chart in Table 19 and stored for potential biomarker analysis.

[0470] Urine DOA and Alcohol Breath Testing: Alcohol breath testing was administered and urine samples were collected from patients at any time during the conduct of the study and tested for amphetamines, barbiturates, benzodiazepines, cocaine, cannabinoids, and opiates as clinically appropriate at the investigator's discretion.

[0471] Concomitant medications: Patients were required to have been taking any concomitant medications at a stable dose for at least 3 months prior to screening, and the dose had to have remained stable throughout the study. If patients were insulin-dependent, this treatment should have been initiated at least 3 months prior to screening, although dose changes were permitted.

[0472] Restrictions: The study included restrictions on diet, alcohol, caffeine, and physical activity. Regarding diet, patients avoided taking dietary supplements rich in omega-3 or omega-6 fatty acids (e.g., cod liver oil capsules) both during the study and for the four weeks prior to baseline. Regarding alcohol, patients avoided consuming more than 21 units (men) or 14 units (women) per week (1 unit consists of 10 mL or 8 mg of pure alcohol). There were no restrictions on caffeine either before or during the study. Regarding physical activity, patients were required to avoid exercise and strenuous physical activity for at least 3-4 hours before safety laboratory tests (e.g., biochemistry).

[0473] 1.6 Investigational Drugs DS102 capsules were white, opaque, hard-shell capsules (size 0) containing 500 mg of 15-HEPE ethyl ester (EE) with 5% w / w colloidal silicon dioxide as a viscosity modifier.

[0474] The placebo (paraffin oil) for DS102 was a white opaque hard shell capsule (size 0) containing an equivalent fill weight of liquid paraffin with 1% w / w colloidal silicon dioxide as a viscosity modifier.

[0475] DS102 and placebo capsules were stored at 2–8°C in a secure location protected from unauthorized use (e.g., a locked cabinet or drug storage room). Labels were dose-blinded and contained the randomization number.

[0476] Dosage and Administration: The study involved a comparison of DS102 administered orally twice daily with placebo for a total of 16 weeks. The last dose of study medication was administered the day before the 16-week / early termination (ET) visit. Patients were required to take the capsules with or after meals. Medications for other conditions allowed in the study were taken as usual. The wallet-attached blister pack consisted of four capsules for seven days, and finally, patients took their assigned medication for 16 consecutive weeks.

[0477] 1.7 Adverse Events and Serious Adverse Events Adverse events (AEs) and serious adverse events (SAEs) in this study were defined as follows:

[0478] Adverse Event (AE): Any untoward experience occurring in a patient who received the first dose of study drug, whether or not considered related to the investigational IMP. AEs were recorded on a case report form defining their relationship to the IMP and severity.

[0479] Serious Adverse Event (SAE): If a patient experienced a serious adverse event after the first dose of investigational drug, the event was recorded as an SAE. An SAE is characterized as an untoward medical occurrence at any dose that either resulted in death, was life-threatening, required the patient's hospitalization or an extension of an existing hospitalization, or resulted in persistent or significant impairment / incapacity. The term "life-threatening" refers to an event in which the patient was at risk of death at the time of the event. It does not refer to an event that, if more severe, could hypothetically cause death. Examples of serious, but non-life-threatening, events included intensive care in the emergency room, allergic bronchospasm, blood dyscrasia or seizures that did not result in hospitalization, or the development of drug dependence or abuse.

[0480] Unexpected Adverse Event (UAE): An experience not previously reported in the Investigator Brochure or similar product information sheet, such as the Summary of Product Characteristics (SPC).

[0481] The intensity of an AE is an estimate of the relative severity of the event, determined by the investigator based on clinical experience. The following definitions were used to assess the severity of an AE: Mild: Adverse events were transient and easily tolerated. Moderate: The adverse event caused discomfort to the patient and interfered with the patient's usual activities. Severe: The adverse event significantly interfered with the patient's usual activities, was incapacitating, or was life-threatening.

[0482] The causal relationship of AEs to the experimental treatment was also established by considering the patient's medical history, recent physical examination findings, and concomitant medications. The following definitions were used to determine the causal relationship of AEs: Unrelated: The temporal relationship of the onset of the AE to the experimental treatment is not reasonable, or another cause could explain the occurrence of the AE. Related: The temporal relationship of the AE to the experimental treatment was reasonable, followed known patterns of response to treatment, and was unlikely to have an alternative cause.

[0483] All AEs were reported throughout the study period up to and including the follow-up period.

[0484] 1.8 Drug-induced liver injury Severe Drug-Induced Liver Injury (DILI): In the event of severe DILI, regardless of the recognized cause, the study drug was discontinued until the symptomatic episode was deemed to have resolved. If the study drug was determined to be the cause of liver injury, the patient was not re-challenged with that drug. Severe DILI defined evidence of liver damage as indicated by total bilirubin >2 × ULN or INR >1.5.

[0485] Patients with abnormal baseline liver biochemistry: When determining abnormal baseline liver biochemistry, doubling was calculated relative to baseline levels instead of using the ULN. Therefore, following a 3x baseline ALT or AST (or >200 IU / L) value, repeat testing was performed within 72 hours to confirm / determine whether the biochemistry had improved or worsened. AE information was collected along with a thorough physical examination. Liver etiology screening and / or other appropriate testing was performed. If liver dysfunction occurred, patients were managed as having severe DILI. If any of the criteria for severe DILI occurred, suspension of drug treatment was considered.

[0486] 1.9 Serious and Unexpected Adverse Reactions Adverse Reaction: All harmful and unintended responses to a drug relevant to any dose were considered adverse drug reactions. The phrase "response to a drug" means that a causal relationship between the drug and the AE is at least reasonably possible (i.e., the relationship cannot be excluded). For marketed drug products, an adverse reaction is a response to a drug that is harmful, unintended, and occurs at doses normally used by humans for the prevention, diagnosis, or treatment of disease or for the modification of physiological function.

[0487] Unexpected adverse reaction: An adverse reaction whose nature or severity was not consistent with the applicable product information.

[0488] Suspected Unexpected Serious Adverse Reaction (SUSAR): Any unexpected serious adverse reaction that may be related to an IMP as defined above.

[0489] 1.10 Statistical Methodology and Data Management This clinical trial employed a randomized, double-blind, placebo-controlled, parallel-group design. Randomization was used to minimize allocation bias and increase the likelihood that known and unknown patient attributes (e.g., demographic characteristics) were evenly balanced between treatment groups. Blinding was used to reduce potential bias during data collection and safety and efficacy assessments. The use of a placebo as a comparator was justified as a reasonable design for assessing safety and efficacy in patients based on the short study duration and the lack of any potential long-term irreversible damage that could result from placebo treatment.

[0490] Sample size estimation: Assuming a 20% delta response rate between the active drug and placebo groups, a 25% standard deviation, and a 20% dropout rate, 32 patients per group were required for statistical testing at a 5% significance level and 80% power. Sample size was re-estimated at interim analyses based on recommendations from unblinded investigators. To achieve 80% conditional power for the primary endpoint, the sample size could be increased to a maximum of 150 patients total.

[0491] Blinding and Decoding Instructions: All study site personnel, as well as those involved in monitoring or conducting the study, were blinded to individual patient treatment assignment. Randomization details were kept strictly confidential and accessible only in emergency situations by authorized personnel until the time of formal unblinding. The study blinding code was decoded only after all patient data had been recorded and verified and the database had been locked.

[0492] Interim Analysis and Data Monitoring: A safety interim analysis was performed to estimate the conditional power when at least 50% of patients completed the Week 16 visit. The interim analysis was based on data collected for the primary and co-primary efficacy endpoints and secondary endpoints and was used to estimate the conditional power to achieve the primary study objective, potentially re-estimate the sample size, and potentially exclude less effective treatment arms.

[0493] Clinically meaningful response: A higher mean or at least a 20% median reduction in ALT or liver stiffness compared to placebo, and a higher mean or at least a 10% median reduction in both ALT and liver stiffness compared to placebo.

[0494] The analysis sets were the registration set, the complete analysis set (FAS), and the protocol Comply with The analysis sets included a Phase 3 Phase 4 Phase 5 Phase 6 Phase 7 Phase 8 Phase 9 Phase 10 Phase 11 Phase 12 Phase 13 Phase 14 Phase 15 Phase 16 Phase 17 Phase 18 Phase 19 Phase 20 Phase 21 Phase 22 Phase 23 Phase 24 Phase 25 Phase 26 Phase 27 Phase 28 Phase 29 Phase 30 Phase 31 Phase 32 Phase 33 Phase 34 Phase

[0495] Enrollment set: patients who signed the informed consent form. Screening failures were patients from the enrollment population who did not meet the eligibility requirements and who withdrew from the study before randomization.

[0496] FAS: randomized patients who received at least one dose of study treatment and had at least one post-baseline measurement. Patients were analyzed according to the treatment assigned at randomization, regardless of the treatment they actually received.

[0497] PPS: A subset of the FAS consisting of patients from the FAS who did not have major protocol violations. All protocol deviations were individually assessed and documented before database lock, and significant deviations were considered to have a serious impact on efficacy outcomes, leading to the exclusion of the relevant patient from the PPS.

[0498] SAS: patients who received at least one dose of study treatment. Patients were analyzed according to the treatment they actually received.

[0499] PK set: patients with SAS who had at least one PK concentration of DS102. Patients were analyzed according to the treatment they actually received.

[0500] Safety Analysis: Demographic, medical history, and physical examination data were listed and summarized descriptively for each patient. All AEs recorded during the study were coded to system organ class and preferred term using the latest version of the Medical Dictionary for Regulatory Activities (MedDRA). AEs were tabulated and summarized by treatment, relationship to treatment, severity, and severity. Laboratory values ​​(e.g., hematology, biochemistry, and urinalysis) were listed for each patient by treatment and day. Laboratory values ​​outside of normal ranges were listed individually with comments regarding clinical significance and summarized by treatment, highlighting potentially clinically significant abnormalities. Laboratory values ​​obtained before dosing were defined as baseline values. Alcohol breath test and DOA test results were listed for each patient. Individual vital sign values ​​were listed and summarized descriptively by treatment and day. 12-lead ECG assessments were listed for each patient with all relevant comments and summarized by treatment and day. Concomitant medications (if any) classified by drug class and subgroup according to the latest version of the World Health Organization's Drug Dictionary were listed and summarized by treatment. Generally, appropriate descriptive statistics depending on the nature of the variables were applied. Categorical variables were presented using numbers and percentages, and continuous variables were shown using mean, standard deviation, median, minimum, maximum, coefficient of variation, and number of patients.

[0501] Pharmacokinetic Analysis: 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.

[0502] Primary Variable: The primary efficacy variable was the change from baseline in serum ALT at Week 16 (Visit 10). Active treatment groups were compared against placebo using an analysis of covariance (ANCOVA) model, including corresponding baseline values ​​as covariates. Comparisons against placebo were performed according to Dunnett's multiple test procedure. For missing Week 16 values, the last available value was used. Replenish A similar approach was applied to liver stiffness. For ALT, longitudinal modeling was additionally considered.

[0503] Secondary variables: Secondary efficacy variables and their changes from baseline to week 16 (visit 10) were summarized with descriptive statistics by treatment group and visit. This applied to AST, AST:ALT ratio, liver fat measured by CAP, liver stiffness measured by transient elastography, FIB-4, NFS, ELF, and HOMA-IR / Adipo-IR. Changes from baseline in active treatment groups were compared against placebo using an ANOVA model including a center effect term. A 5% significance level was used for all treatment comparisons.

[0504] Exploratory Analyses: Exploratory analyses included analyses of lipid and metabolic parameters such as 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 A1C (HbA1C). Additional exploratory analyses included high-throughput lipidomics and proteomics.

[0505] 1.11 Results This study demonstrated that patients diagnosed with NAFLD and administered DS102 at doses of 1 g or 2 g per day showed statistically significant reductions in markers of metabolic overload, as well as improvements in insulin sensitivity and glycemic control.

[0506] Figure 20 shows that the three treatment groups were well balanced at baseline, with similar lipidomic and metabolomic profiles. Baseline characteristics of patients are shown in Table 18. 10-13% of patients were receiving statin therapy at baseline.

[0507] [Table 16]

[0508] Figures 21A-21C show the change in insulin, glucose, and free fatty acid levels from baseline to week 16 in patients receiving either 1 g or 2 g of DS102 per day compared to placebo. Metabolic substrates including glucose, carbohydrates, and free fatty acids were increased. N Because they contribute to the pathogenesis of ASH, the reduction in insulin, glucose, and free fatty acid levels by administration of DS102 is clinically significant.

[0509] Figures 22A and 22B show the change in HOMA-IR and adipo-IR levels from baseline to week 16 in patients receiving either 1 g or 2 g of DS102 per day compared to placebo. Patients were observed in the 2 g DS102 dose protocol. Comply with Both insulin resistance indices (e.g., reductions in HOMA-IR and adipo-IR levels) showed significant improvements in PPS at 16 weeks, along with improvements in both insulin resistance indices (e.g., reductions in HOMA-IR and adipo-IR levels).

[0510] Figures 23A and 23B show the change in glycated hemoglobin (e.g., HbA1c) levels from baseline to week 16 in patients receiving either 1 g or 2 g of DS012 per day compared to placebo. Specifically, Figure 23A shows the change in HbA1c levels, and Figure 23B shows the change in HbA1c levels in the percentage of patients who had elevated HbA1c levels at baseline but achieved normal levels at week 16. Because Hb1Ac is a measure of the amount of glucose attached to the body's red blood cells and is a surrogate for long-term glycemic control, these results indicate that administration of DS102 results in clinically meaningful improvements and normalizes glycemic control in a dose-dependent manner.

[0511] Figures 24A and 24B show the mean and median (%) changes in lipid profiles of patients in the Safety Analysis Set (SAS) at Week 16. These results, further illustrated in Figures 25A-25C, demonstrate that administration of DS102 significantly improved patients' lipid profiles by either maintaining or reducing their levels of total cholesterol, VLDL-C, non-HDL-C, remnant-like particle (RLP) cholesterol, and triglycerides. Importantly, the reductions did not plateau at Week 16, suggesting that administration of DS102 may induce even greater changes in longer-term studies.

[0512] As shown in Figure 26, administration of DS102 reversed the hepatotoxic lipid signature of NASH and improved multiple lipid classes that were altered in patients diagnosed with NASH. Specifically, administration of 2 g of DS102 significantly reduced the levels of multiple hepatotoxic diglycerides and significantly increased the levels of multiple glycerophospholipid groups. This finding is important because patients with NASH have low levels of glycerophospholipids in the liver and plasma.

[0513] Figure 27 shows that administration of DS102 also resolved NASH based on validated diagnostic tests, such as the OWL Liver Care noninvasive diagnostic test for NASH. OWL Liver Care is a test developed based on plasma lipidomics in patients with biopsy-confirmed NASH, providing high predictive value. OWL Liver Care has an area under the curve (AUC) of 0.88 for distinguishing patients with NAFLD from those with normal liver, and an AUC of 0.79 for distinguishing patients with NASH from those with NAFLD without steatohepatitis. Administration of DS102 significantly improved and normalized NASH diagnosed by OWL Liver Care in a dose-dependent manner compared with placebo at Week 16. Table 19 shows baseline test diagnoses by treatment group, showing that most patients were classified as NASH or NAFLD at baseline, with a lower proportion of patients in the 2 g DS102 group.

[0514] [Table 17]

[0515] Administration of DS102 also reduced liver fat content as assessed by CAP in patients diagnosed with NAFLD, as shown in Figure 28. Furthermore, DS102 would be expected to induce greater changes in liver content in longer-term studies and when assessed by more sensitive methods.

[0516] As shown in Table 20, administration of DS102 also reduced triglyceride levels in patients, suggesting that DS102 may also be effective in reducing cardiovascular risk.

[0517] [Table 18]

[0518] Figures 29A-29C show that administration of DS102 reduces inflammatory and profibrotic proteins. For this evaluation, blood samples before and after treatment with DS102 were analyzed for a panel of over 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. After treatment with DS102, a resolution of metabolic overload and lipotoxicity was observed, preventing subsequent cellular stress, inflammation, and fibrosis. The reduction in inflammatory and profibrotic proteins suggests that DS102 may result in the resolution of NASH and the prevention of fibrosis.

[0519] As shown in Figure 30, administration of DS102 also reduced the expression of multiple NASH development targets. In particular, administration of 2 g of DS102 reduced NASH drug development targets, including CCR2 / 5 signaling (cenicriviroc-Allergan), galectin 3 (GR-MD-02-Galectin), and AOC3 (Boehringer Ingelheim).

[0520] The volcano plot in Figure 31 (data from Bonferroni and Benjamini-Hochberg tests) shows that administration of 2 g of DS102 reduced inflammatory and profibrotic proteins based on changes in protein expression. Table 21 shows the most significant reductions in inflammatory and profibrotic proteins based on the Bonferroni test.

[0521] [Table 19]

[0522] Administration of 1 g of DS102 also showed a significant reduction in TR. Table 22 shows the most significant reduction in inflammatory and profibrotic proteins based on analysis using a linear model.

[0523] [Table 20]

[0524] This analysis also showed that one protein in the 1g DS107 group and zero proteins in the placebo group exceeded the first threshold based on the Benjamini-Hochberg test.

[0525] As shown in Figure 32, administration of DS102 also reduced the expression of several vascular adhesion molecules. Vascular adhesion molecules are involved in atherosclerosis, and their circulating levels are associated with cardiovascular risk.

[0526] As shown in Figure 33, administration of DS102 also reduced the expression of several proteins associated with increased cardiovascular risk.

[0527] As shown in Figure 34, administration of DS102 also reduced the expression of multiple circulating chemokines, which are important drivers of chronic inflammation in atherosclerosis.

[0528] As shown in Figure 35, administration of DS102 also reduced the expression of multiple tumor necrosis factor receptor superfamily members, which are involved in inflammation and atherosclerosis.

[0529] DS102 also proved to be safe and well-tolerated, with no observed differences in safety or tolerability compared to placebo. There were no drug-related serious adverse events (SAEs), and the incidence of adverse events (AEs) was low and consistent across all treatment groups. Most AEs were mild to moderate and transient, and no patients experienced AEs leading to treatment discontinuation. The safety profile across each treatment group is shown in Table 23.

[0530] [Table 21]

[0531] DS102 was also evaluated for its potential efficacy in improving 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) and multiple markers of liver fibrosis, suggesting that DS102 may also be effective in treating PBC and PSC.

[0532] FIG. 37 is a boxplot of 15-HEPE ethyl ester (EE) trough plasma relative concentrations, showing that, as expected, there was higher systematic exposure in the DS102-treated group at weeks 8 and 16.

[0533] 1.12 Summary Overall, these results indicate that DS102 targets multiple stages of NASH pathology by significantly reducing metabolic burden and improving insulin sensitivity. DS102 administration also improved patients' lipid profiles by reversing lipid accumulation levels associated with NASH. Specifically, patients treated with DS102 showed reduced accumulation of hepatotoxic lipids, including total cholesterol, non-HDL cholesterol, RLP cholesterol, triglycerides, diglycerides, and VLDL-C, as well as increased glycerophospholipid levels. These effects are significant because patients diagnosed with NASH are characterized by elevated levels of total cholesterol, triglycerides, diglycerides, and VLDL-C, and reduced levels of glycerophospholipids and omega-3 PUFAs. Furthermore, alterations to multiple lipids are expected to result in reduced cardiovascular risk and improvement of multiple aspects of metabolic syndrome.

[0534] The results also suggested a dose-dependent effect favoring administration of 2 g or more of DS102, with large, statistically significant improvements compared to placebo.

[0535] In conclusion, DS102 is well suited as either monotherapy or part of a combination therapy for treating NASH and may reduce cardiovascular risk in patients diagnosed with NASH or cardiometabolic diseases, including metabolic syndrome.

[0536] Example 5: 26-week toxicity study of 15(S)-HEPEEE by oral gavage in rats with a 4-week recovery period The objectives of this study were (1) to determine the potential toxicity of 15(S)-HEPEEE, an omega-3 fatty acid used to treat liver and lung disease, given to rats by oral gavage for 26 weeks and to evaluate the potential reversibility of any findings, and (2) to determine the toxicokinetic properties of 15(S)-HEPEEE.

[0537] 1.1 Research design Test Article and control Goods was administered to appropriate animals by oral gavage once daily, 7 days a week for a minimum of 26 weeks. The dose per animal was based on the most recent body weight. The dose was administered using a syringe equipped with a gavage cannula. The first day of dosing was designated as day 1. The dosing formulation was removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulation was continuously stirred during dosing.

[0538] Due to the onset of clinical signs, including hunched posture, ruffled fur and abnormal gait, and weight loss, dosing was discontinued for animals 3206M, 3515F, and 3703F beginning on Day 45, and for the remaining Group 3 animals beginning on Day 48. Dosing for animals will begin at the lower dose level (3000 mg / kg / day) beginning on Day 64. The following table shows the treatments (Table 23) and animal groups (Table 24) used in the study.

[0539] [Table 22] a Due to the onset of clinical signs, including hunched posture, ruffled fur and abnormal gait, and weight loss, dosing of the animals was discontinued on day 48. Dosing of the animals will commence on day 64 at a lower dose level (3000 mg / kg / day). b Batch 2540M - 1801 and 2540M - 1802 adjusted concentration. c Adjusted concentrations for batches 2540M / 1901 and 2540M / 1902.

[0540] [Table 23] The spare animals are 4001M、 4002M 、 and will be numbered 4501F, 4502F.

[0541] Recovery Period: Upon completion of the dosing period, designated recovery animals were held for a minimum of 4 weeks recovery period.

[0542] Route Justification and Dose Levels: The oral route of administration was selected for this study because it is the intended route of human administration. Oral toxicity studies over the past 26 weeks in rats administered once daily showed no adverse effects at the highest dose tested (1 mg / kg / day). See Warren, H (2017). A 26-Week Study of 15(S)-HEPE EE by Oral (Gavage) in Rats with a 4-Week Recovery Period. Charles River Study No. 529123. Given the lack of dose-limiting toxicity in this study, an additional 28-day dose-ranging study determined an oral maximum tolerated dose (MTD) of 6 g / kg / day. See Murie, E (ongoing). A Pilot 28-Day Study of DS102 by Oral Gavage in Rats. Charles River Study No. 506611. This current study was conducted to evaluate the chronic toxicity potential of 15(S)-HEPE EE (DS102) at this MTD in compliance with ICH Harmonised Tripartite Guideline M3(R2): Non-Clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals.

[0543] 1.2 Antemortem procedures, observations, and measurements All main study animals and recovery animals were Previous Procedures, Observations, and Measurements were Performed: Toxicokinetics Animals were weighed and clinical observations were recorded at the same time points as the main study animals.

[0544] Mortality / moribund checks were performed daily. Animals were observed for general health / mortality and moribundity. Animals were not removed from their cages during observations unless necessary to identify or confirm potential findings.

[0545] Clinical Observations:

[0546] Cageside observations were performed daily and animals were not removed from their cages during observations unless to identify or confirm potential findings.

[0547] Detailed clinical observations were made weekly. Animals were removed from their cages for examination.

[0548] Post-dose observations were made periodically throughout the day. All animals were examined for response to treatment. The onset, intensity, and duration of these signs were recorded. Animals were given special attention during and for one hour after dosing.

[0549] Body weights were measured twice during the pretreatment period, once daily during the treatment period, and twice weekly during the recovery period. Animals were weighed individually. Weights were recorded on the first day of scheduled necropsy (main and recovery animals only).

[0550] Food intake was quantitatively measured weekly beginning at week 1 and throughout the dosing and recovery period.

[0551] Water consumption was measured periodically throughout the study by visual inspection of water bottles.

[0552] Ophthalmic examinations were performed using an indirect ophthalmoscope after application of a mydriatic (1% tropicamide, Mydriacy). All main study animals and recovery animals were examined during the pretreatment period. Main study animals (control and high dose) were examined during weeks 13 and 26. At week 30, all recovery animals were examined.

[0553] 1.3 Laboratory Evaluation: clinical pathology

[0554] Sample Collection: Immediately before the autopsy Orbital puncture under irreversible isoflurane anesthesia venous plexus When blood is drawn from In combination Unless 、 Blood was collected from the tail vein. Animals were bled in ascending order unless samples were collected prior to necropsy according to the order of euthanasia. Additional blood samples were obtained (e.g., for clotting of non-serum samples) if the allowed sampling frequency and blood volume were not exceeded. After collection, samples were transported to the appropriate laboratory for processing. Samples were collected according to the table below.

[0555] [Table 24] X = sample to be collected, - = not applicable.

[0556] Hematology: 0.5 mL blood samples were collected using the anticoagulant K2EDTA. Blood smears were labeled, stained, and stored. Blood smears were analyzed according to the following parameters:

[0557] [Table 25]

[0558] Coagulation: 0.5 mL blood samples were drawn using the anticoagulant 3.8% (w / v) trisodium citrate for processing to plasma. To assess coagulation, the following coagulation parameters were measured:

[0559] [Table 26]

[0560] Clinical Chemistry: 0.7 mL blood samples were drawn using lithium heparin anticoagulation for processing to plasma. To assess clinical chemistry, the following parameters were measured:

[0561] [Table 27] aIf total bilirubin is >8.55 μmol / L, indirect and direct bilirubin will also be measured.

[0562] Urinalysis: Urine was collected from animals over a period of 6 hours (+ / - 30 minutes) in the absence of food and the presence of moisture. After collection, samples were transported to the appropriate laboratory for processing. To evaluate the urinalysis, the following parameters were measured:

[0563] [Table 28]

[0564] Bone marrow smear analysis: Bone marrow was collected from all animals and preserved in 10% neutral buffered formalin.

[0565] Bioanalysis and toxicokinetic evaluation

[0566] Bioanalytical sample collection: Using a sterile needle and disposable syringe, 0.5 mL blood samples were collected via the jugular vein (or tail vein if the jugular vein was inaccessible) using the anticoagulant K2EDTA. Upon completion of the blood collection schedule, toxicokinetic animals were euthanized. Carcasses were discarded without examination. Samples were collected according to the table below.

[0567] [Table 29] X = Sample to be collected. - = Not applicable. a Samples are taken prior to administration b Day 1 samples were collected from animal 3702F, but on day 44, this animal was euthanized for humane reasons. An additional sample was collected from animal 3707F (time 0) at week 26. a collection) and animals 370 4 F (4-hour collection) and 370 animals 2 The missing point of F Refill to Ta.

[0568] Bioanalytical Sample Processing: Samples were gently mixed and held on crushed wet ice until centrifugation within 60 minutes of collection. Samples were centrifuged at 1500 g for 10 minutes at 4°C. The resulting plasma was separated, transferred to uniquely labeled clear polypropylene tubes, and frozen as quickly as possible on dry ice or in a freezer set to maintain -20°C.

[0569] living organisms Analytical Sample Analysis: Plasma samples were analyzed for concentrations of non-esterified HEPE and total HEPE using a validated analytical procedure. The following were determined: a) Non-esterified (HEPE occurring in plasma as a free fatty acid or bound to plasma albumin). b) Total (quantifying non-esterified HEPE and esterified HEPE (e.g., triglycerides, cholesterol esters, etc.)) concentrations at each sample time.

[0570] living thing An actual sample reanalysis (ISR) for unesterified HEPE was performed for this study according to the appropriate analytical laboratory standard operating procedures. An ISR for the determination of total HEPE in rat plasma samples was successfully performed during laboratory study number 527943 using the analytical procedure referenced above and does not need to be repeated for this study.

[0571] All residual / retained bioanalytical samples were stored for a minimum of 6 months after publication of the draft report and then destroyed. Alternatively, if publication of the final report occurred before the end of the 6-month retention period, residual / retained samples were destroyed prior to the 6-month period. Earlier destruction of these residual / retained samples may also be requested and approved by the Study Principal Investigator.

[0572] Toxicokinetic Evaluation: Toxicokinetic parameters were estimated using Phoenix pharmacokinetic software. Parameter estimation used a non-compartmental approach consistent with the oral route of administration. All parameters were generated from the concentrations of total and unbound free acid of the 15(S)-HEPEEE composition in plasma at Day 1 and Week 26, where practical. The following parameters were estimated:

[0573] [Table 30]

[0574] Partial AUC (for any given sample time) can be derived and reported to aid interpretation. Phoenix was used to generate descriptive statistics (standard error of Cmax and AUC(0-t)) for appropriate grouping and sorting variables. TK tables and graphs were also generated.

[0575] 1.4 Exit Procedures The table below summarizes the end-of-life procedures used in the study.

[0576] [Table 31] X =Steps to be taken:-=Not applicable a For a list of tissues, see the Tissue Collection and Preservation Table. b Target tissues are liver (males and females), thyroid (males and females), kidney (females only), and sternal bone marrow (males only).

[0577] Unscheduled Deaths: If a main study animal or recovery animal died during the study, a necropsy was performed and certain tissues were preserved. If necessary, animals were refrigerated to minimize autolysis. Main study animals or recovery animals were humanely euthanized in accordance with the study facility's SOPs. Body weights were recorded and, if possible, samples were obtained for evaluation of clinical pathology parameters. These animals were necropsied and certain tissues were preserved. If necessary, animals were refrigerated to minimize autolysis. Toxicokinetic animals that died during the study or were humanely euthanized were subjected to a limited necropsy examination. The examination consisted of evaluation of organs and tissues within the thoracic, abdominal, and pelvic cavities; no tissues were retained. During the limited necropsy examination, special attention was paid to evidence of potential gavage trauma. Body weights were recorded and samples were collected for toxicokinetic analysis evaluation.

[0578] Scheduled Euthanasia: Main study animals and recovery animals that survived until scheduled euthanasia were euthanized by exposing the animals to increasing concentrations of carbon dioxide. Terminal body weights were recorded and then exsanguined. When possible, animals were euthanized sequentially across dose groups so that a similar number of animals from each group, including controls, were necropsied throughout the day. Animals were not fasted prior to scheduled necropsy. Toxicokinetic animals that survived until scheduled euthanasia were euthanized using approved methods. No necropsies were performed and no tissues were collected.

[0579] Necropsy: Main study animals and recovery animals were subjected to a complete necropsy, which included the carcass and musculoskeletal system; all external surfaces and orifices; the cranial cavity and external surface of the brain; and the thoracic, abdominal, and pelvic cavities and their associated organs and tissues. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

[0580] Organ Weights: Organs identified for weighting in the Tissue Collection and Preservation Table were weighed at necropsy (unless otherwise specified) for all scheduled euthanized animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or imminently. Paired organs were weighed together. In the case of gross abnormalities, each organ in the pair was weighed in addition to the total weight and entered as a tissue comment. Terminal body and brain weights were used for organ weight analysis.

[0581] Tissue collection and storage: Tissues collected according to the table below were stored in 10% neutral buffered formalin.

[0582] [Table 32] TIFF0007734686000035.tif225143X=Procedure to be performed. -=Not applicable. (1)=Unilateral. (2)=Bilateral. Gross abnormalities in the listed organs and other organs are sampled at necropsy, processed for histological analysis, and examined microscopically. a Two bone marrow smears are taken from femurs at scheduled and unscheduled necropsies (for possible testing). Smears are not taken from animals found dead or moribund and euthanized, and are stored in a refrigerator prior to necropsy. Bone marrow smears are air-dried and not fixed in formalin. b Eyes and optic nerves are preserved in Davidson's fixative. Testes and epididymides are preserved in modified Davidson's fixative. c From the small intestine: Peyer's patches or isolated lymphoid nodules. d Weighed after fixation

[0583] 1.5 Dose Formulation and Analysis Control item :This study Control item was 0.5% HPMC in Milli-Q water. Control item was prepared and stored in a refrigerator set to maintain 4°C when not in use. Control item The solution was removed from the refrigerator and stirred for at least 30 minutes before administration. Control item was stirred continuously.

[0584] test Goods :Tests in this study Goods The drugs were DS102 or 15(S)-HEPEEE. Goods The dosage formulations were prepared in accordance with established procedures at the testing facility at the appropriate concentrations to meet dosage level requirements. Dosage formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and administered daily. Dosage formulations were removed from the refrigerator and stirred for at least 30 minutes prior to administration. Dosage formulations were continuously stirred during administration.

[0585] Sampling and analysis

[0586] Analyzed samples were submitted within the specified stability period. All analyzed samples were transported to the testing facility's analytical laboratory at ambient temperature. All residual / retained analytical samples were stored for a minimum of six months after release of the draft report and then discarded. Alternatively, if release of the final report occurs before the end of the six-month retention period, residual / retained samples were discarded prior to the six-month period. Premature discard of these residual / retained samples may also be approved by the Investigator after consultation with the Sponsor. Drug product samples were collected for analysis as shown in the table below. Additional samples may be collected and analyzed at the Investigator's discretion.

[0587] [Table 33] N / A=Not Applicable. a The homogeneity results from the top, middle, and bottom of the preparations in Groups 2-3 are averaged and used as the concentration results.

[0588] Analysis method

[0589] The analyses described below were performed by UPLC with UV detection using a validated analytical procedure.

[0590] Concentration analysis: Samples for analysis: Duplicate samples of the upper, middle, and lower sections (two middle sections only for the control) were sent for analysis. When only concentration was to be evaluated, two middle sections only were collected. Reserve samples: Triplicate top, middle, and bottom samples (three middle samples for control) were retained at the testing facility. Reserve samples may be analyzed at the discretion of the principal investigator. If only concentration is being evaluated, three middle samples only will be collected. Collection vessel: A suitable sized volumetric flask. Sample volume: Group 1: 0.5 mL in a 50 mL volumetric flask. Group 2: 0.25 mL in a 50 mL volumetric flask. Group 3: 0.25 mL in a 50 mL volumetric flask. The weight of all samples was recorded. Storage conditions: Stored in a refrigerator set to maintain 4°C. Pass / fail criteria: The pass / fail criteria were mean sample concentrations within ±10% of the theoretical concentration. For homogeneity, the pass / fail criteria were a relative standard deviation (RSD) of concentrations of ≤10% per group.

[0591] Stability Analysis: Stability analysis previously performed in conjunction with laboratory study number 439414 Goods However, the data used in this study Multiple concentrations The stability of the formulation in the vehicle was demonstrated when prepared and stored under identical conditions at the same concentration. See Rogers, E (2018). 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 are available at: same Testing Facility Study Number 43941 of 4 Retained in research records.

[0592] 1.6 Test System Animals: The study used male and female Sprague-Dawley rats purchased from Charles River UK Limited (Margate, Kent, UK). The target age at first dosing was 7-8 weeks. The target weight at first dosing was 175-300 g (males) and 120-250 g (females). Each animal was identified using a cylindrical "glass-sealed" Trovan electronic microchip implanted subcutaneously.

[0593] 1.7 Breeding Housing: Animals were acclimated to the rodent toxicology facilities of the test facility for a period of up to 3 weeks before the start of dosing. Animals were randomly assigned to groups. Sexes were randomized separately. Animals in poor health were not assigned to groups. Animals were housed by sex, 2 or 3 per cage, in appropriately sized suspended polycarbonate / polypropylene cages with stainless steel grid tops and solid bottoms. Sterile white wood chips were used as bedding material.

[0594] Environmental conditions: The target conditions for the animal room environment were: temperature 19-23°C; humidity 40-70%; minimum 10 air changes per hour; and a 12-hour light and 12-hour dark photocycle. 。 Light, temperature, and humidity controls were automatically controlled and continuously monitored and recorded.

[0595] Diet: SDS Rat and Mouse (Modified) No. 1 Diet SQC Expanded was provided ad libitum. Water from the public water supply was provided ad libitum via water bottles. Water used in the test facility was analyzed at regular intervals for dissolved substances, heavy metals, pesticide residues, pH, nitrates, and nitrites. Microbiological screening was also performed.

[0596] Animal Enrichment: Animals were socially housed for mental / environmental enrichment and provided with items such as hiding spots and chew objects, except when interrupted by study procedures / activities.

[0597] Veterinary Care: Veterinary care was provided throughout the course of the study, and animals were examined by veterinary staff as required by clinical signs or other changes. All veterinary examinations and recommended treatments, if any, were documented in the study records.

[0598] 1.8 Histology and Histopathology Histology: Tissues identified in the Tissue Collection and Storage Table were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

[0599] Histopathology: Histopathological evaluation was performed by qualified veterinary pathologists or by veterinary pathologists trained and experienced in laboratory animal pathology. Microscopic examination was performed by the primary investigator. Control Microscopic examination of target tissues from the pivotal study low-dose group and recovery animals was performed by the sponsor if a treatment effect was present in the pivotal study high-dose group. After agreement with the principal investigator on the revised clinical trial protocol I went there.

[0600] 1.9 Computerized Systems Computerized Systems: In the table below Computerized Systems but , was available for use in research.

[0601] [Table 34]

[0602] 1.10 Statistical analysis Data collected during the pre-dose period were tabulated, summarized, and statistically analyzed. Unless otherwise stated, all statistical analyses were performed at each study phase. Numerical data collected at scheduled times (excluding data from toxicokinetic animals) were summarized and statistically analyzed according to sex and time as shown below.

[0603] Constituent variables: Body weight change was calculated between each scheduled interval. Organ weights versus body weight were calculated relative to the weight at the end of the scheduled interval. brain Weights were calculated relative to brain weight at scheduled intervals.

[0604] Descriptive statistical analysis: Mean, standard deviation (or % coefficient of variation or standard error where deemed appropriate), percentage, number, and / or incidence were reported as appropriate for each dataset.

[0605] Inferential statistical analyses: All statistical analyses were performed at the 5% level of significance. All pairwise comparisons were performed using two-tailed tests and reported at the 1% and 5% levels unless otherwise stated. Pairwise comparisons of interest are listed below. ·Group 2 vs. Group 1 ·Group 3 vs. Group 1

[0606] Analyses were performed according to the following matrix where possible, excluding all groups with fewer than three observations.

[0607] [Table 35]

[0608] Parametric / Non-parametric: Levene's test was used to assess homogeneity of group variances. If the Levene's test was not significant or the Kruskal-Wallis test was not significant, groups were compared using a general one-way ANOVA F-test. If the general F-test or Kruskal-Wallis test was found to be significant, pairwise comparisons were performed using Dunnett's test or Tan's test, respectively.

[0609] 1.11 Results Changes in red blood cell count, red blood cell distribution width, and reticulocyte count: Figures 38A-38C show the changes in red blood cell count (RBC), red blood cell distribution width (RDW), and reticulocyte count (RETIC) in rats administered 15(s)-HEPEEE or placebo. 15(S)-HEPEEE and placebo were administered once daily by oral gavage, 7 days per week for a minimum of 26 weeks. The volume of each animal was based on the most recent body weight. As shown in Table 38, the highest dose of 6 g / kg / day was not well tolerated and was replaced with a lower dose of 3 g / kg / day.

[0610] [Table 36]

[0611] RBC Changes: Figure 38A and Table 38 show that administration of the 3 g / kg / day dose resulted in a significant increase in RBC compared to the control.

[0612] Changes in RDW: Figure 38B and Table 38 show that administration of both the 2 g / kg / day and 3 g / kg / day doses resulted in a significant reduction in RDW compared to the control.

[0613] Changes in RETIC: Figure 38C and Table 38 show that administration of both the 2 g / kg / day and 3 g / kg / day doses resulted in a significant reduction in RETIC compared to controls.

[0614] These results suggest that 15-HEPE has therapeutic potential for a number of diseases affecting hemoglobin and red blood cells.

[0615] Changes in prothrombin time, activated partial thromboplastin time, and fibrinogen concentration: Figures 39A-39C show the changes in prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) concentrations in rats administered 15(s)-HEPEEE or placebo. 15(S)-HEPEEE and placebo were administered once daily by oral gavage, 7 days a week for a minimum of 26 weeks. The volume of each animal was based on the most recent body weight measurement. As shown in Table 39, the highest dose of 6 g / kg / day was not well tolerated and was replaced with a lower dose of 3 g / kg / day.

[0616] [Table 37]

[0617] Changes in PT: Figure 39A and Table 39 show that administration of both the 2 g / kg / day and 3 g / kg / day doses resulted in a significant reduction in PT compared to the control.

[0618] Changes in APTT: Figure 39B and Table 39 show that administration of both the 2 g / kg / day and 3 g / kg / day doses resulted in a significant increase in APTT compared to the control.

[0619] Changes in FIB concentrations: Figure 39C and Table 39 show that administration of both the 2 g / kg / day and 3 g / kg / day doses resulted in a significant reduction in FIB concentrations compared to the control.

[0620] These results suggest that 15-HEPE has anticoagulant and antithrombotic effects.

[0621] Various embodiments of the present disclosure are described herein below.

[0622] Section A. A method for treating and / or preventing metabolic syndrome in a subject in need thereof, the method comprising administering to the subject 15-HEPE, 15-HETrE, or a composition comprising 15-HEPE and / or HETrE.

[0623] Section B. A method for treating and / or preventing a cardiometabolic disease in a subject in need thereof, the method comprising administering to the subject 15-HEPE, 15-HETrE, or a composition comprising 15-HEPE and / or HETrE.

[0624] Section C. A method for treating and / or preventing metabolic syndrome and / or cardiometabolic disease in a subject in need thereof, the method comprising administering to the subject Best Large (approx. 8g) , 15-HEPE and / or A method comprising administering a composition comprising 15-HETrE, wherein 15-HEPE and / or 15-HETrE represents at least about 90% by weight of all fatty acids in the composition.

[0625] Section D. A method for treating and / or preventing metabolic syndrome and / or cardiometabolic disease in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 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 reduced levels of diglycerides, glycerophospholipids, liver fat, blood pressure, abdominal circumference, and / or free fatty acids, and / or increased glycerophospholipid levels.

[0626] Item E. A method for preventing the progression of a first stage of nonalcoholic steatohepatitis (NASH) to a second stage of NASH in a subject, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE.

[0627] Paragraph F. The method of paragraph E, wherein the first step is metabolic overload, increased liver fat content and lipotoxicity, cellular stress apoptosis, inflammation, and / or fibrogenic remodeling.

[0628] Paragraph G. The method of paragraph E or F, wherein the second step is an increase in liver fat content and lipotoxicity, cell stress apoptosis, inflammation, and / or fibrogenic remodeling.

[0629] Section H. A method for treating and / or preventing cardiovascular disease in a subject with nonalcoholic fatty liver disease (NAFLD), metabolic syndrome, and / or a cardiometabolic disease in need of such treatment and / or prevention, the method comprising administering to the subject 15-HEPE, 15-HETrE, or a composition comprising 15-HEPE and / or HETrE.

[0630] Item I. A method for treating and / or preventing cardiovascular disease in a subject having nonalcoholic fatty liver disease (NAFLD), metabolic syndrome, and / or a cardiometabolic disease in need of such treatment and / or prevention, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0631] Section J. Subject is α-smooth muscle action The method of any one of paragraphs AI, wherein the patient exhibits a reduction in one or more of the following levels: α-SMA, metallopeptidase inhibitor-1 (TIMP-1), transforming growth factor beta-β (TGF-β), and / or type 1 collagen.

[0632] Item K. The method of any one of items A-C or E-J, wherein the subject exhibits reduced levels of diglycerides, liver fat, blood pressure, abdominal circumference, and / or free fatty acids, and / or increased levels of glycerophospholipids.

[0633] Item L. The method of any one of paragraphs AK, wherein the subject exhibits reduced alkaline phosphate (ALP) levels.

[0634] Item M. The method of any one of paragraphs A-L, wherein the subject exhibits reduced levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and / or bilirubin (BUN).

[0635] Item N. The method of any one of items A-M, wherein the subject exhibits a reduced area of ​​fibrosis.

[0636] Item O. The method of any one of items A-N, wherein the subject exhibits reduced levels of hemoglobin A1C (HbA1C), homeostasis model assessment of insulin resistance (HOMA-IR), and / or adipose tissue insulin resistance (adipo-IR).

[0637] Item P. The method according to any one of items H to O, wherein NAFLD is non-alcoholic steatohepatitis (NASH).

[0638] Section Q. Cardiovascular metabolic or cardiovascular diseases include 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 The method of any one of paragraphs B-D or F-P, wherein the treatment is one or more of the following: cardiovascular disease, macrovascular disease, atherosclerosis, coronary atherosclerosis, diastolic dysfunction, cardiovascular risk reduction, 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 endothelial function, diabetes, diabetes mellitus, insulin resistance, hyperinsulinemia, hyperglycemia, dysglycemia, induction of glycemic control, impaired glucose tolerance, and impaired fasting glucose.

[0639] Item R. The method according to item Q, wherein the microvascular disease is retinopathy, nephropathy, neuropathy, or a combination thereof.

[0640] Section S. The method according to section Q, wherein the macrovascular disease is stroke, peripheral vascular disease, limb ischemia, heart disease, or a combination thereof.

[0641] Item T. The method of any one of paragraphs A-S, wherein the subject exhibits reduced levels of 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 high density lipoprotein cholesterol (HDL-C).

[0642] Item U. The method of any one of items A-T, wherein the subject exhibits a reduction in liver stiffness, Fibrosis-4 (FIB-4), Enhanced Liver Fibrosis (ELF) score, and / or NAFLD score (NFS).

[0643] Item V. The method of any one of paragraphs A-U, wherein the subject exhibits a reduction in a pro-inflammatory and pro-fibrotic protein 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).

[0644] Item 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.

[0645] Item X. The method of item W, wherein the cholestatic liver disease is primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis, or a combination thereof.

[0646] Section Y. Cholestatic liver disease is a condition that can be caused by drug-induced liver injury, total parenteral nutrition (TPN), viral and alcoholic hepatitis, and systemic diseases. Occurring concomitantly The method according to paragraphs W or X, wherein the cholestasis is caused by cholestasis, graft dysfunction, post-liver transplant cholestasis, pancreatitis, common bile duct stones, Mirizzi syndrome, a genetic disorder, a malignancy, or a combination thereof.

[0647] Item Z. The method according to Item Y, wherein the malignant tumor is hepatocellular carcinoma, bile duct tumor, pancreatic cancer, or a combination thereof.

[0648] Item AA. The method of any one of items W-Z, wherein the subject exhibits a reduction in cytokines and / or chemokines selected from the group consisting of alpha-smooth muscle activity (α-SMA), metallopeptidase inhibitor-1 (TIMP-1), transforming growth factor beta-β (TGF-β), and type 1 collagen levels.

[0649] Section BB. A method for treating or preventing kidney disease in a subject, the method comprising administering 15-HEPE and / or 15-HETrE, or a composition comprising 15-HEPE and / or 15-HETrE, to the subject, wherein the subject has at least one risk factor for kidney disease.

[0650] Paragraph CC. The method of paragraph BB, wherein the kidney disease is selected from the group consisting of renal fibrosis, tubulointerstitial fibrosis, chronic kidney disease, severe interstitial fibrosis, renal interstitial fibrosis, and end-stage renal disease.

[0651] Item DD. Kidney disease leads to fibrosis, the method according to item CC.

[0652] Item EE. The method of any one of items BB to DD, wherein the at least one risk factor for kidney disease is selected from the group consisting of diabetes, hypertension, cardiovascular disease, glomerulonephritis, and polycystic kidney disease.

[0653] Paragraph FF. The method of any one of paragraphs BB-EE, wherein the subject exhibits reduced kidney hydroxyproline levels.

[0654] Paragraph GG. The method of any one of paragraphs BB-FF, wherein the subject does not exhibit reduced levels of α-SMA, TIMP-1, TGF-β, and / or type 1 collagen.

[0655] Item HH. The method of any one of items BB-GG, wherein the subject exhibits reduced levels of α-SMA, TIMP-1, TGF-β, and / or type 1 collagen.

[0656] Item II. The method of any one of items AA-HH, wherein the subject exhibits a reduction in pro-fibrotic cytokines in the liver.

[0657] The method of paragraph JJ., wherein the profibrotic cytokine is one or more of α-SMA, TIMP-1, TGF-β, type 1 collagen, interleukin 1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-13 (IL-13), tumor necrosis factor (TNF-α), 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).

[0658] Paragraph KK. The method of any one of paragraphs A-JJ, wherein the subject exhibits a reduction in vascular adhesion molecules and / or chemokines and / or tumor necrosis factor receptor superfamily members.

[0659] Item LL. The method of any one of items A to KK, wherein 15-HEPE, 15-HETrE, or a composition comprising 15-HEPE and / or 15-HETrE is administered orally.

[0660] Item MM. The method of any one of paragraphs A-LL, wherein the 15-HEPE and / or 15-HETrE is in a free acid form, an esterified form, or a salt form.

[0661] Item NN. The method according to paragraph MM, wherein the esterified form is an alkyl ester form or a triglyceride form.

[0662] Item OO. The method of any one of items A to NN, wherein 15-HEPE includes 15(S)-HEPE, 15(R)-HEPE, or a combination thereof, and / or 15-HETrE includes 15(S)-HETrE, 15(R)-HETrE, or a combination thereof.

[0663] Item PP. The method according to any one of items A to OO, wherein the composition contains about 1 g to about 2 g of 15-HEPE and / or 15-HETrE.

[0664] Item QQ. The method of any one of items A to PP, wherein the composition contains about 2 g or more of 15-HEPE and / or 15-HETrE.

[0665] Item RR. The method of any one of paragraphs A-QQ, wherein the composition comprises about 1 g or about 2 g of 15-HEPE and / or 15-HETrE.

[0666] Item SS. The method of any one of items A to RR, wherein the composition contains about 10 mg to about 10,000 mg of 15-HEPE and / or 15-HETrE.

[0667] Item TT. The method of any one of items A-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.

[0668] Item UU. The method of any one of paragraphs A through TT, wherein 15-HEPE and / or 15-HETrE represents at least about 90% by weight of all fatty acids present in the composition.

[0669] Item VV. The method of any one of items A-UU, wherein the composition is administered in 1 to 8 capsules per day.

[0670] Item WW. The method of any one of items AC or HVV, wherein the method comprises administering 15-HEPE or a composition comprising 15-HEPE to the subject.

[0671] Item XX. The method of any one of Items AC or HVV, wherein the method comprises administering to the subject 15-HETrE or a composition comprising 15-HETrE.

[0672] Item YY. A method for treating and / or preventing a blood disorder in a subject in need thereof, the method comprising administering 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE) to the subject.

[0673] Item ZZ. The method according to Item YY, wherein the blood disorder is selected from the group consisting of anemia, blood cancer, and coagulation disorder.

[0674] Item AB. The method of paragraph ZZ, wherein the anemia is selected from the group consisting of nutritional anemia and non-nutritional anemia.

[0675] Paragraph AC. The method of paragraph ZZ, wherein the blood cancer is selected from the group consisting of lymphoma, leukemia, and myeloma.

[0676] The method of paragraph ZZ, wherein the coagulation disorder is selected from the group consisting of thrombophilia, hemophilia, von Willebrand's disease, and thrombocytopenia.

[0677] Section AE. A method for treating and / or preventing hemoglobin disorders in a subject in need thereof, the method comprising administering to the subject a composition comprising 15-HEPE or 15-hydroxyeicosapentaenoic acid (15-HEPE).

[0678] Section AF. A method for treating and / or preventing an red blood cell disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising 15-HEPE or 15-hydroxyeicosapentaenoic acid (15-HEPE).

[0679] Section AG. A method for treating and / or preventing hemoglobin disorders and / or red blood cell disorders in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0680] Section AH. A method for treating and / or preventing hemoglobin disorders and / or red blood cell disorders in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition, and the subject: (a) Increased red blood cell count, (b) a decrease in red blood cell distribution width, and / or (c) a decrease in reticulocyte count.

[0681] Section AI. Subjects are controls not administered the composition. subject The method according to paragraph AH, wherein the method 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

[0682] Section AJ. Comparison subject The method described in paragraph AI, wherein the patient has a red blood cell count of approximately 4 x 10 12 red blood cells / L.

[0683] Section AK. Subjects are controls not administered the composition. subject The method according to paragraph AH, wherein the red blood cell distribution width is reduced by at least about 15-20%, at least about 20-25%, at least about 25-30%, at least about 30-35%, or at least about 35-40% compared to the red blood cell distribution width.

[0684] Section AL. Comparison subject The method according to paragraph AK, wherein the red blood cell distribution width is greater than 15%.

[0685] Section AM. Subjects are controls who have not received the composition. subject The method according to paragraph AH, wherein the method has a reduction in reticulocyte count of at least about 5%, at least about 10%, at least about 15%, or at least about 20% compared to

[0686] Section AN.Contrast subject The method according to paragraph AM, wherein the reticulocyte count is greater than 5% of the total red blood cell count.

[0687] Item AO. A method for treating and / or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0688] Item AP. The method according to item AO, wherein the hemolytic anemia is hereditary hemolytic anemia or acquired hemolytic anemia.

[0689] Section AQ. The method according to Section AP, wherein the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, beta-thalassemia, and hereditary spherocytosis.

[0690] Section AR. Acquired hemolytic anemia is caused by infections, drug treatment, hematologic malignancies, autoimmune diseases, hypersplenism, and mechanical heart Valves and blood transfusions Occurring concomitantly The method according to paragraph AP, selected from the group consisting of:

[0691] The method according to paragraph AS. Sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and / or splenic blood cell retention.

[0692] Item AT. The method of any one of paragraphs YY to AS, wherein 15-HEPE is in the form of an ethyl ester (15-HEPE EE) or 15-HEPE is in the form of an optically active ester (15(S)-HEPE EE).

[0693] Item AU. The method of any one of items AE to AN, wherein the hemoglobin abnormality and / or red blood cell disorder is selected from the group consisting of hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.

[0694] Item AV. The method of any one of items YY-AG, wherein the subject exhibits an increased red blood cell count, a decreased reticulocyte count, and a decreased red blood cell distribution width.

[0695] Item AW. A method for treating and / or preventing a blood disorder and / or blood disease in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0696] Item AX. The method according to item AW, wherein 15-HEPE is in the form of an ethyl ester (15-HEPE EE), or 15-HEPE is in the form of an optically active ester (15(S)-HEPE EE).

[0697] Item AY. The method according to item AW, wherein the blood disorder and / or blood disease is selected from the group consisting of hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.

[0698] Item AZ. The method according to Item AY, wherein the hemolytic anemia is hereditary hemolytic anemia or acquired hemolytic anemia.

[0699] Paragraph BA. The method of paragraph AZ, wherein the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, beta-thalassemia, and hereditary spherocytosis.

[0700] Section BC. Acquired hemolytic anemia is caused by infections, drug treatment, hematologic malignancies, autoimmune diseases, hypersplenism, and mechanical heart Valves and blood transfusions Occurring concomitantlyThe method of any of the preceding claims, wherein the method is selected from the group consisting of:

[0701] Paragraph BD. The method according to paragraph BA, wherein sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and / or splenic blood cell retention.

[0702] Item BE. The method of any one of items AW-BD, wherein the subject exhibits an elevated red blood cell count, a reduced reticulocyte count, and a reduced red blood cell distribution width.

[0703] Section BF. A method for treating and / or preventing a thrombophilia disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising 15-HEPE or 15-hydroxyeicosapentaenoic acid (15-HEPE).

[0704] Section BG. A method for treating and / or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0705] Section BH. A method for treating and / or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition, and the subject: (a) Prolongation of prothrombin time (b) prolongation of activated partial thromboplastin time, and / or (c) a decrease in fibrinogen concentration.

[0706] Section BI. A method for treating and / or preventing venous thromboembolism in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0707] Section BJ. A method for treating and / or preventing arterial thrombosis in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0708] Section BK. A method for preventing embolism in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-hydroxyeicosapentaenoic acid (15-HEPE), wherein the 15-HEPE represents at least about 90% by weight of all lipids in the composition.

[0709] Item BL. The method of any one of items BI to BK, wherein the first step is the formation of a thrombus.

[0710] Item BM. The method of any one of Items BF-BG or Items BI-BL, wherein the subject exhibits an increased prothrombin time, an increased activated partial thromboplastin time, and a decreased fibrinogen concentration.

[0711] Item BN. The method of any one of Items YY to BM, wherein 15-HEPE is administered orally.

[0712] Paragraph BO. The method of any one of paragraphs YY to BN, wherein the composition is administered in 1 to 8 capsules per day.

[0713] The method of any one of paragraphs YY to BO, wherein the BP.15-HEPE is in the free acid form, an esterified form, or a salt form.

[0714] Item BQ. The method of any one of Items YY to BP, wherein the composition contains about 1 g to about 2 g of 15-HEPE.

[0715] Paragraph BR. The method of any one of paragraphs YY to BQ, wherein the composition comprises about 2 g or more of 15-HEPE.

[0716] Paragraph BS. The method of any one of paragraphs YY to BR, wherein the composition comprises about 5 mg / kg, about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg of 15-HEPE. The present application provides the following aspects of the invention. (Aspect 1) A method for treating and / or preventing a blood disorder in a subject in need thereof, the method comprising administering 15-hydroxyeicosapentaenoic acid (15-HEPE) or a composition comprising 15-HEPE to the subject. (Aspect 2) A method for treating and / or preventing hemoglobin disorders in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. (Aspect 3) A method for treating and / or preventing an erythrocyte disorder in a subject in need thereof, said method comprising administering 15-HEPE or a composition comprising 15-HEPE to said subject. (Aspect 4) A method for treating and / or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject a composition comprising up to about 8 g of 15-HEPE. (Aspect 5) 2. The method of embodiment 1, wherein the blood disorder is selected from the group consisting of anemia, blood cancer, and coagulation disorder. (Aspect 6) The method of embodiment 5, wherein the anemia is nutritional anemia, non-nutritional anemia, or a combination thereof. (Aspect 7) 6. The method of embodiment 5, wherein the hematological cancer is selected from the group consisting of lymphoma, leukemia, and myeloma. (Aspect 8) 6. The method of claim 5, wherein the coagulation disorder is selected from the group consisting of thrombophilia, hemophilia, von Willebrand's disease, and thrombocytopenia. (Aspect 9) The method of any one of aspects 1 to 8, wherein the subject has an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count compared to a control subject not administered the 15-HEPE or the composition. (Aspect 10) The method of any one of aspects 1 to 9, wherein 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 administered the 15-HEPE or composition. (Aspect 11) The method of any one of Aspects 1 to 10, wherein the subject has a decrease in red blood cell distribution width of about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, or about 35% to about 40% compared to a control subject not administered 15-HEPE or the composition. (Aspect 12) 12. The method of any one of aspects 9 to 11, wherein the control subject has a red blood cell distribution width of greater than 15%. (Aspect 13) 13. The method of any one of aspects 1-12, wherein the subject has a reduction 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 administered the 15-HEPE or composition. (Aspect 14) Aspect 14. The method of any one of aspects 9 to 13, wherein the control subject has a reticulocyte count greater than 5% of the total red blood cell count. (Aspect 15) 15. The method of any one of aspects 2 to 14, wherein the hemoglobin abnormality and / or the red blood cell disorder is selected from the group consisting of hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome. (Aspect 16) 16. The method of aspect 15, wherein the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis. (Aspect 17) 16. The method of aspect 15, wherein the acquired hemolytic anemia results from a disorder or disease selected from the group consisting of a primary infection, drug treatment, hematological malignancy, autoimmune disease, hypersplenism, placement of a mechanical heart valve, and a disease occurring concomitantly with blood transfusion. (Aspect 18) 17. The method of aspect 16, wherein the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and / or splenic blood cell retention. (Aspect 19) A method for treating and / or preventing a thrombophilic disorder in a subject in need thereof, said method comprising administering 15-HEPE or a composition comprising 15-HEPE to said subject. (Aspect 20) A method for treating and / or preventing thrombophilia in a subject in need thereof, said method comprising administering to said subject 15-HEPE or a composition comprising up to about 8 g of 15-HEPE. (Aspect 21) 1. A method for treating and / or preventing venous thromboembolism in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising up to about 8 g of 15-HEPE. (Aspect 22) 1. A method for treating and / or preventing arterial thrombosis in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising up to about 8 g of 15-HEPE. (Aspect 23) 1. A method for preventing embolism in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising up to about 8 g of 15-HEPE. (Aspect 24) 24. The method of any one of aspects 19 to 23, wherein the subject has an increased prothrombin time, an increased activated partial thromboplastin time, and / or a decreased fibrinogen concentration compared to a control subject not administered the composition. (Aspect 25) Aspect 25. The method of any one of aspects 1 to 24, wherein the 15-HEPE is in a free acid form, an esterified form, a conjugate form, or a salt form. (Aspect 26) Aspect 26. The method of any one of aspects 1-25, wherein the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition. (Aspect 27) 27. The method of any one of aspects 1-26, wherein the composition comprises up to about 2 g of 15-HEPE. (Aspect 28) 28. The method of any one of aspects 1-27, wherein the composition is administered in 1 to 8 capsules per day. (Aspect 29) The method of any one of aspects 1-28, wherein the composition comprises at least 2 g of 15-HEPE. (Aspect 30) 30. The method of any one of aspects 1-29, wherein the composition is formulated to provide the subject with about 5 mg 15-HEPE per kilogram of body weight (mg / kg), about 50 mg / kg, about 250 mg / kg, or about 500 mg / kg. (Aspect 31) The method of any one of aspects 1 to 30, wherein the 15-HEPE or the composition comprising 15-HEPE is administered orally. (Aspect 32) Aspect 32. The method of any one of aspects 1 to 31, wherein the 15-HEPE is in a free acid form, an esterified form, a conjugate form, or a salt form. (Aspect 33) Aspect 33. The method of any one of aspects 1 to 32, wherein the esterified form of 15-HEPE is a methyl ester, an ethyl ester, or a combination thereof.

Claims

1. 1. A pharmaceutical composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE) for use in a method for increasing red blood cell count in a subject having a blood disorder, the method comprising administering up to 8 g of the pharmaceutical composition to the subject, wherein the subject administered the pharmaceutical composition has an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count compared to a control subject not administered the pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the blood disorder is selected from the group consisting of anemia, and a subject administered the pharmaceutical composition has an increased red blood cell count, a decreased red blood cell distribution width, and / or a decreased reticulocyte count compared to a control subject not administered the pharmaceutical composition.

3. The pharmaceutical composition of claim 2 , wherein the anemia is nutritional anemia, non-nutritional anemia, or a combination thereof.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein a subject administered the pharmaceutical composition 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 administered the pharmaceutical composition.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein a subject administered the pharmaceutical composition has a reduction in red blood cell distribution width of 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, or 35% to 40% compared to a control subject not administered the pharmaceutical composition.

6. The pharmaceutical composition of claim 4 or 5, wherein the control subject has a red blood cell distribution width of greater than 15%.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein a subject administered the pharmaceutical composition has a reduction in reticulocyte count of at least 5%, at least 10%, at least 15%, or at least 20% compared to a control subject not administered the pharmaceutical composition.

8. The pharmaceutical composition of any one of claims 4 to 7, wherein the control subject has a reticulocyte count of greater than 5% of the total red blood cell count.

9. A pharmaceutical composition described in any one of claims 4 to 8, wherein the blood disorder is selected from the group consisting of hereditary hemolytic anemia, acquired hemolytic anemia, Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome.

10. 10. The pharmaceutical composition of claim 9, wherein the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis.

11. 10. The pharmaceutical composition of claim 9, wherein the acquired hemolytic anemia results from a disorder or disease selected from the group consisting of primary infection, drug treatment, hematological malignancy, autoimmune disease, hypersplenism, placement of a mechanical heart valve, and diseases occurring concomitantly with blood transfusion.

12. 11. The pharmaceutical composition of claim 10, wherein the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and / or splenic blood cell retention.

13. A pharmaceutical composition comprising 15-HEPE for use in a method for treating or preventing a thrombophilic disorder, the method comprising administering up to 8 g of the pharmaceutical composition to a subject, wherein the subject administered the pharmaceutical composition has a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a reduced fibrinogen concentration compared to a control subject not administered the pharmaceutical composition.

14. A pharmaceutical composition comprising 15-HEPE for use in a method for treating or preventing thrombophilia, the method comprising administering up to 8 g of the pharmaceutical composition to a subject, wherein the subject administered the pharmaceutical composition has a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a reduced fibrinogen concentration compared to a control subject not administered the pharmaceutical composition.

15. A pharmaceutical composition comprising 15-HEPE for use in a method for treating or preventing venous thromboembolism, the method comprising administering up to 8 g of the pharmaceutical composition to a subject, wherein the subject administered the pharmaceutical composition has a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a reduced fibrinogen concentration compared to a control subject not administered the pharmaceutical composition.

16. A pharmaceutical composition comprising 15-HEPE for use in a method for treating or preventing arterial thrombosis, the method comprising administering up to 8 g of the pharmaceutical composition to a subject, wherein the subject administered the pharmaceutical composition has a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a reduced fibrinogen concentration compared to a control subject not administered the pharmaceutical composition.

17. A pharmaceutical composition comprising 15-HEPE for use in a method for treating or preventing embolism, the method comprising administering up to 8 g of the pharmaceutical composition to a subject, wherein the subject administered the pharmaceutical composition has a prolonged prothrombin time, a prolonged activated partial thromboplastin time, and / or a reduced fibrinogen concentration compared to a control subject not administered the pharmaceutical composition.

18. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the 15-HEPE is in a free acid form, an esterified form, or a salt form.

19. 19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the 15-HEPE represents at least 90% by weight of all fatty acids in the pharmaceutical composition.

20. 20. The pharmaceutical composition of any one of claims 1 to 19, comprising up to 2 g of 15-HEPE.

21. The pharmaceutical composition according to any one of claims 1 to 20, administered in 1 to 8 capsules per day.

22. 22. The pharmaceutical composition of any one of claims 1 to 21, formulated to provide a subject with 5 mg 15-HEPE per kg body weight (mg / kg), 50 mg / kg, 250 mg / kg, or 500 mg / kg.

23. The pharmaceutical composition according to any one of claims 1 to 22, which is administered orally.

24. 19. The pharmaceutical composition of claim 18, wherein the esterified form of 15-HEPE is a methyl ester, an ethyl ester, or a combination thereof.

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