RU2833560C1 - Compositions containing 15-hepe for treating or preventing hematological disorders and/or related diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and can be used to increase the number of erythrocytes in a subject. Disclosed is a method for increasing the number of erythrocytes in a subject, comprising administering to subject 2 g or more of composition containing 15-hydroxyeicosapentaenoic acid (15-HEPE), wherein the subject shows an increase in the number of erythrocytes, a decrease in the width of distribution of erythrocytes and/or a decrease in the number of reticulocytes compared to the individual in the control group not receiving 15-HEPE or the composition.

EFFECT: method provides an increase in the number of erythrocytes, a decrease in the width of distribution of erythrocytes and/or a decrease in the number of reticulocytes in a subject.

18 cl, 39 dwg, 37 tbl, 4 ex

Description

AREA OF TECHNOLOGY

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

SEQUENCE LIST

[0002] This disclosure includes a sequence listing that was 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 titled Sequence Listing 124318-8014.US00_ST25.txt and is 3 kilobytes in size.

LEVEL OF TECHNOLOGY

[0003] Hematologic disorders include red blood cell disorders and thrombophilia. Red blood cell disorders are a class of conditions that affect the production, lifespan, and shape of red blood cells and their oxygen-carrying molecule, hemoglobin. These conditions interfere with the transfer of oxygen from the lungs to the rest of the body. Thrombophilia is a class of disorders that increase the risk of blood clots in the veins and arteries. These clots can break away from a blood vessel and travel through the bloodstream to an organ, thereby blocking blood flow to the organ, leading to ischemia. These conditions lead to an increased risk of stroke and/or pulmonary embolism.

ESSENCE OF THE INVENTION

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

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

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

[0007] In another aspect, the present invention relates to methods for treating and/or preventing a hematological disease in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition, and the subject has one or more of the following: (a) an increase in red blood cell count; (b) a decrease in red blood cell distribution width; and/or (c) a decrease in reticulocyte count.

[0008] In various embodiments, the hematological 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.

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

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

[0011] In another aspect of the present invention, there are provided methods for treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition.

[0012] In another aspect, the present invention relates to methods for treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition, and the subject exhibits one or more of: (a) an increase in red blood cell count; (b) a decrease in red blood cell distribution width; and/or (c) a decrease in reticulocyte count.

[0013] In various embodiments, the hematological 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.

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

[0015] In some embodiments, the hemolytic anemia is hereditary hemolytic anemia or acquired hemolytic anemia. In another embodiment, acquired hemolytic anemia results from a disorder or condition selected from the group consisting of a condition secondary to a primary infection, drug administration, hematological malignancy, autoimmune disease, hypersplenism, mechanical artificial heart valve, and blood transfusion, or a combination thereof. In another embodiment, sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, splenic sequestration, or a combination thereof.

[0016] In some embodiments, the subject exhibits an increase in red blood cell count, a decrease in red blood cell distribution width, and a decrease in reticulocyte count. In some aspects, the present invention provides methods for treating and/or preventing thrombophilic disorders in a subject in need thereof, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject.

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

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

[0019] In another aspect of the present invention, there are provided methods for treating and/or preventing venous thromboembolism in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition.

[0020] In one aspect of the present invention, methods are provided for treating and/or preventing arterial thrombosis in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

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

[0022] In some embodiments, the subject experiences an increase in prothrombin time, an increase in activated partial thromboplastin time, and a decrease in fibrinogen concentration.

[0023] In another embodiment, 15-HEPE is in the free acid form, in the esterified form, or in the salt form. In another embodiment, the esterified form is the alkyl ester form or the triglyceride form. In some embodiments, 15-HEPE comprises 15(S)-HEPE, 15(R)-HEPE, or combinations 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 from about 10 mg to about 10,000 mg of 15-HEPE. In another embodiment, the composition comprises from about 1 g to about 2 g of 15-HEPE. In another embodiment, the composition comprises about 2 g or more of 15-HEPE. In another embodiment, the composition is formulated to provide the subject with about 5 mg of 15-HEPE per kg body weight (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg. In various embodiments, 15-HEPE comprises 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 1-8 capsules per day.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

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

[0028] FIG. 2 shows the changes in body weight of animals according to the study described in Example 1.

[0029] FIG. 3 shows the body weight on the day of sacrifice of the experimental animals in accordance with the study described in Example 1.

[0030] FIGS. 4A-4D show kidney weight and kidney weight to body weight ratio of animals on the day of sacrifice in accordance with the study described in Example 1.

[0031] FIG. 5 shows the hydroxyproline content in the kidneys of animals according to the study described in Example 1.

[0032] FIGS. 6A-6G show Sirius red staining of animals in accordance with the study described in Example 1.

[0033] FIG. 7 is a graph depicting the positive area (%) in animals stained with Sirius red, according to the study described in Example 1.

[0034] FIGS. 8A-8D show gene expression analyses of α-SMA, TIMP-1, TGF-β, and collagen type 1 in animals according to the study described in Example 1, respectively.

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

[0036] FIG. 10 shows changes in body weight of animals according to the study described in Example 2.

[0037] FIG. 11 shows the body weight per day of the sacrificed experimental animals according to the study described in Example 2.

[0038] FIGS. 12A and 12B show the liver weight and the ratio of liver weight to body weight of animals on the day of sacrifice according to the study described in Example 2, respectively.

[0039] FIG. 13 shows changes in ALT levels in animal serum according to the study described in Example 2.

[0040] FIG. 14 shows changes in total bilirubin in animal serum according to the study described in Example 2.

[0041] FIGS. 15A-15E show Sirius red staining and fibrotic area of animals according to the study described in Example 2.

[0042] FIG. 16 is a graph depicting the positive area (%) in animals stained with Sirius red, in accordance with the study described in Example 2.

[0043] FIGS. 17A-17D show gene expression analyses of α-SMA, TIMP-1, TGF-β, and collagen type 1 in animals according to the study described in Example 2, respectively.

[0044] FIGS. 18A and 18B show DS102 (15-HEPE)-induced inhibition of TGF-β in accordance with the study described in Example 3.

[0045] FIGS. 18C-18G show the effect of DS102 on the membrane translocation and degradation of TGF-β receptor type I, TGF-β receptor type II, TGF-β receptor type III, EGFR, and caveolin-1 according to the study described in Example 3, respectively.

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

[0047] FIG. 20 shows the baseline lipid profile of the patients according to the study described in Example 4.

[0048] FIGS. 21A-21C are graphs depicting changes in insulin, glucose, and free fatty acid levels in patients administered epeleuton (15-HEPE) and placebo, respectively.

[0049] FIGS. 22A and 22B are graphs showing changes in HOMA-IR and apido-IR levels in patients administered epeleuton and placebo, respectively.

[0050] FIGS. 23A and 23B are graphs depicting changes in mean HbA1C levels in patients and the proportion of patients having HbA1C levels ≥ 6.5% at week 16 who were administered epeleuton and placebo, respectively.

[0051] FIGS. 24A and 24B are graphs depicting the mean change and median (%) change in lipid profile in patients administered epeleuton and placebo, respectively.

[0052] FIGS. 25A-25C are graphs depicting changes in cholesterol, triglyceride, and VLDL-C levels in patients administered Epeleuton and placebo, respectively.

[0053] FIG. 26 is a diagram depicting changes in the hepatotoxic lipid profile in patients administered DS102.

[0054] FIG. 27 shows graphs demonstrating that DS102 administration eliminates NASH using the OWL liver care test.

[0055] FIG. 28 shows graphs depicting changes in liver fat content by a controlled attenuation parameter in patients administered DS102 and placebo.

[0056] FIGS. 29A-29C are graphs depicting changes in inflammatory and profibrotic protein levels in patients administered DS102 and placebo.

[0057] FIG. 30 shows graphs showing changes in protein expression, including targets for NASH development, in patients administered DS102 and placebo.

[0058] FIG. 31 is a volcano plot depicting the reduction in inflammatory and pro-fibrotic proteins in patients administered DS102 and placebo.

[0059] FIG. 32 shows graphs depicting changes in vascular adhesion molecules in patients administered DS102 and placebo.

[0060] FIG. 33 shows graphs depicting changes in cardiovascular risk proteins in patients administered DS102 and placebo.

[0061] FIG. 34 shows graphs depicting changes in chemokines in patients administered DS102 and placebo.

[0062] FIG. 35 shows graphs depicting changes in tumor necrosis factor receptor superfamily members in patients administered DS102 and placebo.

[0063] FIGS. 36A and 36B are graphs showing the reduction in ALP levels in patients administered DS102 and placebo.

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

[0065] FIGS. 38A-38C are histograms depicting the mean change and standard deviation of red blood cell count, red blood cell distribution width, and reticulocyte count in rats administered 15(S)-HEPE EE and placebo, respectively.

[0066] FIGS. 39A-39C are histograms depicting the mean change and standard deviation of prothrombin time, activated partial thromboplastin time, and fibrinogen concentration in rats administered 15(S)-HEPE EE and placebo, respectively.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[0067] Although the present invention may be embodied in various forms, the following description of several embodiments is made with the understanding that the present description is to be considered as an example of the invention and is not intended to limit the invention to the particular embodiments illustrated. Headings are provided for convenience only and are not to be construed as limiting the invention in any way. Embodiments given under any heading may be combined with embodiments given under any other heading.

[0068] The numbers in the various quantitative values recited herein, unless expressly stated otherwise, are intended to be approximate, as if the minimum and maximum values within the stated ranges were preceded by the word "about." Thus, minor deviations from a stated value can be used to obtain substantially the same results as the stated value. Furthermore, a range is intended to be a continuous range including every value between the stated minimum and maximum values, as well as any ranges that can be formed from such values. Also described herein are any and all ratios (and ranges of any such ratios) that can be obtained by dividing a stated numerical value by any other stated numerical value. Accordingly, one skilled in the art will appreciate that many such ratios, ranges, and ranges of ratios can be uniquely derived from the numerical values recited herein; and in all cases, such ratios, ranges, and ranges of ratios represent various embodiments of the present invention.

[0069] Unless defined otherwise, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference. In case of conflict, this specification, including definitions, will control. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

Definitions

[0070]As used herein, the term "15-HEPE" is 15-hydroxyeicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid. 15-HEPE, also sometimes referred to as 15-OHEPA, can be synthesized from eicosapentaenoic acid ("EPA", eicosa-5,8,11,14,17-pentaenoic acid or 20:5n-3), an omega-3 fatty acid, according to methods known in the art. 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-hydroxyeicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid) and/or a pharmaceutically acceptable ester, conjugate or salt thereof, or mixtures of any of the foregoing. A derivative of 15-HEPE may be used instead, although this does not include any derivative compounds that lack the hydroxyl group of 15-HEPE. In some embodiments, 15-HEPE is used in its free acid form. Alternatively, pharmaceutically acceptable esters or salts of 15-HEPE are used herein. In some embodiments, 15-HEPE is in its C_1-4alkyl ester, such as a methyl ester, an ethyl ester, or a combination of a methyl ester and an ethyl ester. In another embodiment, the 15-HEPE is in the form of a glyceride (e.g., a diglyceride or a triglyceride). In another embodiment, the 15-HEPE is in the form of an ethyl ester (also referred to herein as E-15-HEPE, 15-HEPE EE, or ethyl-15-HEPE).

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

[0072] As used herein, the term "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, the term "epeleuton" refers to 15-HEPE, 15-HEPE EE, a composition comprising 15-HEPE, a composition comprising 15-HEPE EE, or a composition comprising 15-HEPE and 15-HEPE EE.

[0074] As used herein, the term "treating" a disease, disorder, or condition includes, at least in part: (1) suppressing the disease, disorder, or condition, i.e., stopping or reducing the progression of the disease, disorder, or condition or its clinical symptoms; or (2) alleviating the disease, disorder, or condition, i.e., regressing the disease, disorder, or condition or its clinical symptoms. The term "prevention" with respect to a given disease or disorder means: preventing the onset of the disease if it has not yet occurred, preventing the occurrence of the disease or disorder in a subject who may be predisposed to the disorder or disease who has not yet been diagnosed with the disorder or disease, and/or preventing the further progression of the disease/disorder if it is already present.

[0075] An "effective amount" as used herein refers to the amount of an active composition necessary to provide a therapeutic effect in a subject. The term "therapeutically effective amount" as used herein refers to a sufficient amount of an agent or administered compound that to some extent reduces one or more symptoms of a disease, disorder, or condition being treated. In some embodiments, the result is a reduction and/or alleviation of signs, symptoms, or causes of a disease, or any other desired change in a biological system. For example, in some embodiments, an "effective amount" for therapeutic use is the amount of a composition comprising a compound described herein necessary to provide a clinically significant reduction in the symptoms of a disease without undesirable side effects. In some embodiments, a suitable "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 described herein, such as a compound of formula (A) or formula (I), is an amount effective to achieve the desired pharmacological effect or therapeutic improvement without undesirable side effects. In other embodiments, it is understood that an "effective amount" or "therapeutically effective amount" varies from subject to subject due to differences in the metabolism, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the medical judgment of the treating physician. The term "pharmaceutically acceptable" in this context means that the substance in question does not cause unacceptable toxicity to the subject or interaction with other components of the composition.

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

Compositions

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

[0078] In one embodiment, 15-HEPE is in the form of an ester (also referred to herein as E-15-HEPE, ethyl-15-HEPE, or 15-HEPE EE). In another embodiment, 15-HEPE comprises a C ₁ -C ₅ alkyl ester of 15-HEPE. In another embodiment, 15-HEPE comprises a methyl ester of 15-HEPE, a propyl ester of 15-HEPE, or a butyl ester of 15-HEPE. In another embodiment, 15-HEPE comprises optically active 15( S )-hydroxy-(5 Z ,8 Z ,11 Z ,13 E ,17 Z )-eicosapentaenoic acid. This isomer can be used in any of the forms discussed above.

[0079] In another embodiment, 15-HEPE comprises lithium 15-HEPE, mono-, di- or triglyceride of 15-HEPE, or any other ester or salt of 15-HEPE, or the free acid form of 15-HEPE.

[0080] In various embodiments, the invention relates to pharmaceutical compositions, such as oral compositions, comprising 15-. In one embodiment, the compositions comprise a therapeutically effective amount of 15-HEPE. In one embodiment, the pharmaceutical composition comprises from about 0.1% to about 99%, from about 1% to about 95%, from about 5% to about 90% by weight of 15-HEPE. As used herein, the term "composition" and the phrase "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. In one embodiment, the pharmaceutical composition comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% by weight of 15-HEPE.

[0082] In another embodiment, 15-HEPE is present in the composition of the present invention in an amount of from about 1 mg to about 10,000 mg, from about 25 mg to about 7,500 mg, from about 25 mg to about 5,000 mg, from about 50 mg to about 5,000 mg, from about 50 mg to about 3,000 mg, from about 75 mg to about 2,500 mg, or from about 100 mg to about 1,000 mg, such as about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, or up to 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about 1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg, about 2050 mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150 mg, about 2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about 2275 mg, about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg, about 2400 mg, about 2425 mg, about 2450 mg, about 2475 mg, about 2500 mg, 2525 mg, about 2550 mg, about 2575 mg, about 2600 mg, about 2625 mg, about 2650 mg, about 2675 mg, about 2700 mg, about 2725 mg, about 2750 mg, about 2775 mg, about 2800 mg, about 2825 mg, about 2850 mg, about 2875 mg, about 2900 mg, about 2925 mg, about 2950 mg, about 2975 mg, about 3000 mg, about 3025 mg, about 3050 mg, about 3075 mg, about 3100 mg, about 3125 mg, about 3150 mg, about 3175 mg, about 3200 mg, about 3225 mg, about 3250 mg, about 3275 mg, about 3300 mg, about 3325 mg, about 3350 mg, about 3375 mg, about 3400 mg, about 3425 mg, about 3450 mg, about 3475 mg, about 3500 mg, about 3525 mg, about 3550 mg, about 3575 mg, about 3600 mg, about 3625 mg, about 3650 mg, about 3675 mg, about 3700 mg, about 3725 mg, about 3750 mg, about 3775 mg, about 3800 mg, about 3825 mg, about 3850 mg, about 3875 mg, about 3900 mg, about 3925 mg, about 3950 mg, about 3975 mg, about 4000 mg, about 4025 mg, about 4050 mg, about 4075 mg, about 4100 mg, about 4125 mg, about 4150 mg, about 4175 mg, about 4200 mg, about 4225 mg, about 4250 mg, about 4275 mg, about 4300 mg, about 4325 mg, about 4350 mg, about 4375 mg, about 4400 mg, about 4425 mg, about 4450 mg, about 4475 mg, about 4500 mg, about 4525 mg, about 4550 mg, about 4575 mg, about 4600 mg, about 4625 mg, about 4650 mg, about 4675 mg, about 4700 mg, about 4725 mg, about 4750 mg, about 4775 mg, about 4800 mg, about 4825 mg, about 4850 mg, about 4875 mg, about 4900 mg, about 4925 mg, about 4950 mg, about 4975 mg, about 5000 mg, about 5025 mg, about 5050 mg, about 5075 mg, about 5100 mg, about 5125 mg, about 5150 mg, about 5175 mg, about 5200 mg, about 5225 mg, about 5250 mg, about 5275 mg, about 5300 mg mg, about 5325 mg, about 5350 mg, about 5375 mg, about 5400 mg, about 5425 mg, about 5450 mg, about 5475 mg, about 5500 mg, about 5525 mg, about 5550 mg, about 5575 mg, about 5600 mg, about 5625 mg, about 5650 mg, about 5675 mg, about 5700 mg, about 5725 mg, about 5750 mg, about 5775 mg, about 5800 mg, about 5825 mg, about 5850 mg, about 5875 mg, about 5900 mg, about 5925 mg, about 5950 mg, about 5975 mg, about 6000 mg, about 6025 mg, about 6050 mg, about 6075 mg, about 6100 mg, about 6125 mg, about 6150 mg, about 6175 mg, about 6200 mg, about 6225 mg, about 6250 mg, about 6275 mg, about 6300 mg, about 6325 mg, about 6350 mg, about 6375 mg, about 6400 mg, about 6425 mg, about 6450 mg, about 6475 mg, about 6500 mg, about 6525 mg, about 6550 mg, about 6575 mg, about 6600 mg, about 6625 mg, about 6650 mg, about 6675 mg, about 6700 mg, about 6725 mg, about 6750 mg, about 6775 mg, about 6800 mg, about 6825 mg, about 6850 mg, about 6875 mg, about 6900 mg, about 6925 mg, about 6950 mg, about 6975 mg, about 7000 mg, about 7025 mg, about 7050 mg, about 7075 mg, about 7100 mg, about 7125 mg, about 7150 mg, about 7175 mg, about 7200 mg, about 7225 mg, about 7250 mg, about 7275 mg, about 7300 mg, about 7325 mg, about 7350 mg, about 7375 mg, about 7400 mg, about 7425 mg, about 7450 mg, about 7475 mg, about 7500 mg, about 7525 mg, about 7550 mg, about 7575 mg, about 7600 mg, about 7625 mg, about 7650 mg, about 7675 mg, about 7700 mg, about 7725 mg, about 7750 mg, about 7775 mg, about 7800 mg, about 7825 mg, about 7850 mg, about 7875 mg, about 7900 mg, about 7925 mg, 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, about 8725 mg, about 8750 mg, about 8775 mg, about 8800 mg, about 8825 mg, about 8850 mg, about 8875 mg, about 8900 mg, about 8925 mg, about 8950 mg, about 8975 mg, about 9000 mg, about 9025 mg, about 9050 mg, about 9075 mg, about 9100 mg, about 9125 mg, about 9150 mg, about 9175 mg, about 9200 mg, about 9225 mg, about 9250 mg, about 9275 mg, about 9300 mg, about 9325 mg, about 9350 mg, about 9375 mg, about 9400 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 10000 mg.

[0083] In one embodiment, the 15-HEPE present in the composition of the present invention comprises at least about 90% by weight 15-HEPE (as defined and illustrated herein). The 15-HEPE compositions can comprise 15-HEPE of even higher purity, such as at least about 95% by weight 15-HEPE or at least about 97% by weight 15-HEPE, wherein the 15-HEPE is any form of 15-HEPE as described herein. The purity of the 15-HEPE can be further defined (e.g., impurity profile) by any of the descriptions of 15-HEPE provided herein.

[0084] The amounts of 15-HEPE in the pharmaceutical composition and their purity have been discussed above. The nature of the essential fatty acids and their synthesis are such that the 15-HEPE composition may include residues of other essential fatty acids in the essential fatty acid metabolism cascade.

[0085] In one embodiment, the composition of the present invention comprises no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or no more than about 0.5%, by weight of other omega-3 fatty acids, including alpha-linolenic acid, stearidonic acid, docosahexaenoic acid (DHA), or derivatives thereof. In other embodiments, such other omega-3 fatty acids are substantially or completely absent.

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

[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 have an even higher degree of purity, such as at least 91% by weight, at least 92% by weight, at least 93% by weight, at least 94% by weight, at least 95% by weight, at least 96% by weight, or at least 97% by weight of the salt form of 15-HEPE.

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

[0089] In one embodiment, provided herein is 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-HEPE E or a derivative thereof per day. In one embodiment, the composition is administered to a subject in an amount sufficient to provide from about 4 g to about 8 g, from about 1 g to about 2 g, from about 2 g to about 4 g, from about 3 g to about 8 g, from 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., RXL gelatin or a lower molecular weight bone gelatin). In another embodiment, the capsule shell comprises RXL gelatin that has been treated with a proteolytic enzyme to cut the gelatin structure and effectively reduce its molecular weight. In another embodiment, the pharmaceutical composition comprises 15-HEPE esters of D-sorbitol and 1,4-sorbitan. In one embodiment, the capsule shell comprises (a) gelatin and (b) plasticizers selected from one or more of D-sorbitol and 1,4-sorbitan. In one embodiment, the gelatin is as described in U.S. Patent 7,485,323 and is incorporated herein by reference in its entirety.

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

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

[0093] In some embodiments, the capsule shell comprises modified starch, carrageenan (e.g., red seaweed extract), 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 (e.g., colorless, white, or clear).

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

Additional active agents

[0096] In one embodiment, the pharmaceutical composition further comprises one or more additional active agents. In one embodiment, the pharmaceutical composition comprises an amount of the additional active agent that is less than a recognized therapeutically effective amount for that agent. In one embodiment, the pharmaceutical composition comprises an amount of the additional active agent that is equal to or greater than a recognized therapeutically effective amount for that agent. If it is desired to use an additional active agent, 15-HEPE can be formulated as a single dosage unit or can be formulated as two to a plurality of dosage units for coordinated, combined, or simultaneous administration.

[0097] EPA itself has beneficial properties in the treatment of fatty liver disease and/or cardiovascular diseases, 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 the composition of the present invention or administered together in a weight ratio of 15-HEPE:additional agent of from about 1:1000 to about 1000:1, from about 1:500 to about 500:1, from about 1:100 to about 100:1, from about 1:50 to about 50:1, from about 1:25 to about 25:1, from about 1:10 to about 10:1, from about 1:5 to about 5:1, from about 1:4 to about 4:1, from about 1:3 to about 3:1, from about 1:2 to about 2:1, or about 1:1.

Dosage forms

[0099] A composition for use in accordance with the invention may be formulated as one or more dosage units. The terms "dose unit" and "dosage unit" as used herein refer to a portion of a composition that contains an amount of a therapeutic agent suitable for single administration to provide a therapeutic effect. Such dosage units may be administered from one to multiple (e.g., from 1 to about 10, from 1 to 8, from 1 to 6, from 1 to 4, or from 1 to 2) times per day, or as many times as necessary to obtain a therapeutic response.

[0100] In some embodiments, the compositions of the present invention are in the form of orally delivered dosage forms or units. Non-limiting examples of suitable dosage forms include tablets (e.g., suspension tablets, suspension tablets, rapid dispersing tablets, chewable tablets, etc.), drops, capsules (e.g., soft or hard gelatin or HPMC capsule), lozenges, sachets, wafers, pastilles, granules, suspension, elixirs, syrups, or any other solid dosage form suitable for oral administration. The terms "oral delivery" and "oral administration" as used herein include any form of delivery in which the agent or composition is placed in the mouth of the subject being treated, with or without ingestion. Thus, this includes buccal and sublingual administration, as well as delivery through the esophagus.

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

[0102] When discussing the amount of 15-HEPE in the composition of the present invention, it can be divided into several dosage forms. There is a limitation in terms of size for oral administration. If a subject needs to administer from about 1 to about 4 g of 15-HEPE per day, this can be up to 4 capsules, each containing about 1 g of 15-HEPE.

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

[0104] In another embodiment, the compositions of the present invention comprise one or more pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" as used herein means any substance that is not itself a therapeutic agent, used as a carrier or vehicle to deliver a therapeutic agent to a subject, or added to a composition to improve processing or storage properties, or to provide or facilitate the formation of a dosage unit of the composition without causing unacceptable toxicity or interaction with other components of the composition. By way of example only, a pharmaceutical composition according to the present invention may comprise one or more antioxidants, surfactants, preservatives, flavoring agents, cosolvents, viscosity enhancing additives, suspending additives, and lipophilic phases.

[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, catechins, epigallocatechin-3-gallate (EGCG), green tea polyphenols (GTP), silymarin, coffee berries, resveratrol, grape seed extracts, pomegranate extracts, genisten, pycnogenol, niacinamide, etc. In one embodiment, the pharmaceutical composition comprises from about 0.01% w/w to about 2% w/w. antioxidant, for example, about 0.01% by weight, about 0.02% by weight, about 0.03% by weight, about 0.04% by weight, about 0.05% by weight, about 0.06% by weight, about 0.07% by weight, about 0.08% by weight, about 0.09% by weight, about 0.1% by weight, about 0.11% by weight, about 0.12% by weight, about 0.13% by weight, about 0.14% by weight, about 0.15% by weight, about 0.16% by weight, about 0.17% by weight, about 0.18% by weight, about 0.19% by weight, about 0.2% by weight, about 0.21% by weight. , about 0.22% by weight, about 0.23% by weight, about 0.24% by weight, about 0.25% by weight, about 0.26% by weight, about 0.27% by weight, about 0.28% by weight, about 0.29% by weight, about 0.3% by weight, about 0.31% by weight, about 0.32% by weight, about 0.33% by weight, about 0.34% by weight, about 0.35% by weight, about 0.36% by weight, about 0.37% by weight, about 0.38% by weight. .%, about 0.39% by weight, about 0.4% by weight, about 0.41% by weight, about 0.42% by weight, about 0.43% by weight, about 0.44% by weight, about 0.45% by weight, about 0.46% by weight, about 0.47% by weight, about 0.48% by weight, about 0.49% by weight, about 0.5% by weight, about 0.51% by weight, about 0.52% by weight. , about 0.53% by weight, about 0.54% by weight, about 0.55% by weight, about 0.56% by weight, about 0.57% by weight, about 0.58% by weight, about 0.59% by weight, about 0.6% by weight, about 0.61% by weight, about 0.62% by weight, about 0.63% by weight, about 0.64% by weight, about 0.65% by weight, about 0.66% by weight, about 0.67% by weight, about 0.68% by weight, about 0.69% by weight, about 0.7% by weight, about 0.71% by weight, about 0.72% by weight, about 0.73% by weight, about 0.74% by weight, about 0.75% by weight, about 0.76% by weight, about 0.77% by weight. , about 0.78% by weight, about 0.79% by weight, about 0.8% by weight, about 0.81% by weight, about 0.82% by weight, about 0.83% by weight, about 0.84% by weight, about 0.85% by weight, about 0.86% by weight, about 0.87% by weight, about 0.88% by weight, about 0.89% by weight, about 0.9% by weight, about 0.91% by weight, about 0.92% by weight, about 0.93% by weight, about 0.94% by weight, about 0.95% by weight, about 0.96% by weight, about 0.97% by weight, about 0.98% by weight, about 0.99% by weight, about 1% by weight, about 1.1% by weight, about 1.2% mass, about 1.3% mass, about 1.4% mass, about 1.5% mass, about 1.6% mass, about 1.7% mass, about 1.8% mass, about 1.9% mass, or about 2% mass of one or more antioxidants.

Therapeutic methods

[0106] The compositions and formulations described herein can be used to treat and/or prevent a hematological disorder in a subject in need thereof. In some embodiments, the hematological disorder is a hemoglobin disorder, a red blood cell disorder, a hemolytic anemia, a thrombophilic disorder, a venous thromboembolism, an arterial thrombosis, an embolism, or a combination thereof. In some embodiments, the methods comprise 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 hematological disorders, hemoglobin disorders, and/or red blood cell disorders. Hematological disorders refer to a class of conditions that affect the blood and blood-forming organs. While hemoglobin disorders and red blood cell disorders are similar to hematological disorders, hemoglobin disorders and red blood cell disorders are a more specific class of disorders. In particular, hemoglobin disorders refer to defects in the oxygen-carrying protein in red blood cells. Red blood cell disorders refer to defects in the red blood cells themselves. Non-limiting examples of risk factors for hematological disorders, hemoglobin disorders, and red blood cell disorders include decreased red blood cell count, increased red blood cell distribution width, and increased reticulocyte count. Non-limiting examples of hematologic disorders, hemoglobin disorders, and red blood cell disorders include anemia (nutritional anemia and anemia not due to iron deficiency, hereditary hemolytic anemia such as sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis, acquired hemolytic anemia such as that secondary to infection, drugs, hematologic malignancy, autoimmune disease, hypersplenism, mechanical heart valve, and transfusion, Fanconi anemia, iron deficiency anemia), hematologic malignancy (lymphoma, leukemia, and myeloma), coagulation defects (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 sequestration.

[0108] In some embodiments, the subject has a reduced red blood cell count of at least 10% below normal, at least 15% below normal, at least 20% below normal, at least 25% below normal, or at least 30% below normal.

[0109] In some embodiments, the subject has an increased red blood cell distribution width, wherein the red blood cells vary in size 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 cells, at least about 10% of the total red blood cells, at least about 15% of the total red blood cells, or at least about 20% of the total red blood cells.

[0111] In some embodiments, the subject has an elevated baseline triglyceride level of from about 135 mg/dL to about 2000 mg/dL, such as from about 135 mg/dL to about 500 mg/dL, from about 150 mg/dL to about 500 mg/dL, from about 200 mg/dL to about 499 mg/dL, from about 200 mg/dL to <500 mg/dL, from about 300 mg/dL to about 1800 mg/dL, from about 500 mg/dL to about 1500 mg/dL, about 500 mg/dL to about <2000 mg/dL, or from about 500 mg/dL to about 2000 mg/dL. In some embodiments, the subject has a baseline fasting triglyceride level of from about 50 mg/dL to about 2000 mg/dL, such as from about 50 mg/dL to about 1500 mg/dL, from about 80 mg/dL to about 1500 mg/dL, from about 50 mg/dL to about 190 mg/dL, from about 80 mg/dL to about 190 mg/dL, from about 190 mg/dL to about 250 mg/dL, from about 250 mg/dL to about 1400 mg/dL, from about 500 mg/dL to about 1200 mg/dL, from about 500 mg/dL to about 1500 mg/dL, from about 500 mg/dL to about <2000 mg/dL, or from about 500 mg/dL to about 2000 mg/dL. In one embodiment, the subject has a fasting triglyceride level of from about 80 mg/dL to about 1400 mg/dL. In some embodiments, the subject or group of subjects has a fasting or postprandial baseline triglyceride level (or an average baseline triglyceride level in the case of a group of subjects) of about 50 mg/dL, about 55 mg/dL, about 60 mg/dL, about 65 mg/dL, about 70 mg/dL, about 75 mg/dL, about 80 mg/dL, about 85 mg/dL, about 90 mg/dL, about 95 mg/dL, about 100 mg/dL, about 105 mg/dL, about 110 mg/dL, about 115 mg/dL, about 120 mg/dL, about 125 mg/dL, about 130 mg/dL, about 135 mg/dL, about 140 mg/dL, about 145 mg/dL, about 150 mg/dL, about 155 mg/dl, about 160 mg/dl, about 165 mg/dl, about 170 mg/dl, about 175 mg/dl, about 180 mg/dl, about 185 mg/dl, about 190 mg/dl, about 195 mg/dl, about 200 mg/dl, about 205 mg/dl, about 210 mg/dl, about 215 mg/dl, about 220 mg/dl, about 225 mg/dl, about 230 mg/dl, about 235 mg/dl, about 240 mg/dl, about 245 mg/dl, about 250 mg/dl, about 255 mg/dl, about 260 mg/dl, about 265 mg/dl, about 270 mg/dl, about 275 mg/dl, about 280 mg/dl, about 285 mg/dl, about 290 mg/dl, about 295 mg/dl, about 300 mg/dl, 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, about 395 mg/dl, about 400 mg/dl, about 405 mg/dl, about 410 mg/dl, about 415 mg/dl, about 420 mg/dl, about 425 mg/dl, about 430 mg/dl, about 435 mg/dl, about 440 mg/dl, about 445 mg/dl, about 450 mg/dl, about 455 mg/dl, about 460 mg/dl, about 465 mg/dl, about 470 mg/dl, about 475 mg/dl, about 480 mg/dl, about 485 mg/dl, about 490 mg/dl, about 495 mg/dl, about 500 mg/dl, about 1000 mg/dl, about 1100 mg/dl, about 1200 mg/dl, about 1300 mg/dL, 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 more than about 5000 mg/dL. In some embodiments, the baseline triglyceride level (or average baseline triglyceride level in the case of a group of subjects) of the subject or group of subjects after a meal or fasting period is greater than or equal to 80 mg/dL, greater than or equal to about 100 mg/dL, greater than or equal to about 120 mg/dL, greater than or equal to about 150 mg/dL, greater than or equal to about 175 mg/dL, greater than or equal to about 250 mg/dL, or greater than about 500 mg/dL, such as from about 190 mg/dL to about 250 mg/dL, from about 80 mg/dL to about 190 mg/dL, from about 250 mg/dL to about 1400 mg/dL, about 200 mg/dL to about 500 mg/dL, from about 300 mg/dL to about 1800 mg/dL, from about 500 mg/dL to about 1500 mg/dL, from about 80 mg/dL to about 1500 mg/dL, from about 80 mg/dL to less than about 2000 mg/dL, from about 80 mg/dL to about 2000 mg/dL, from about 500 mg/dL to less than about 2000 mg/dL, or from about 500 mg/dL to about 2000 mg/dL.

[0112] In another embodiment, the subject's baseline blood pressure is elevated by 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 elevated baseline fasting glucose level in the subject is 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 reduced baseline HDL-C levels of less than about 60 mg/dL, less than about 55 mg/dL, less than about 50 mg/dL, less than about 45 mg/dL, less than about 40 mg/dL, less than about 35 mg/dL, less than about 30 mg/dL, less than about 25 mg/dL, less than about 20 mg/dL, less than about 15 mg/dL, less than about 10 mg/dL, or less than about 5 mg/dL.

[0115] In some embodiments, the present invention provides a method of treating and/or preventing a hematological disorder in a subject, 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 another embodiment, 15-HEPE comprises at least about 90% by weight of the total fatty acids in the composition.

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

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

[0118] In some embodiments, the present invention provides a method of treating and/or preventing a hematological disorder in a subject, comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining whether the subject has at least one risk factor for a hematological disorder prior to administering 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining the baseline red blood cell count, red blood cell distribution width, and/or reticulocyte count of the subject prior to administering 15-HEPE. In one embodiment, the subject has an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in 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 another embodiment, 15-HEPE is at least about 90% by weight of the total fatty acids in the composition.

[0119] In some embodiments, the present invention provides methods of treating and/or preventing a hematological 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 some embodiments, the method further comprises determining whether the subject has at least one risk factor for a hematological disorder prior to administering the 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining the baseline red blood cell count, red blood cell distribution width, and/or reticulocyte count of the subject prior to administering the 15-HEPE. In one embodiment, the subject has an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in reticulocyte count. In another embodiment, the method comprises administering to the subject up to about 8 g of a composition comprising 15-HEPE. In another embodiment, 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

[0120] In some embodiments, the present invention provides methods for treating and/or preventing a hematological disorder in a subject in need thereof, wherein the method comprises 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 up to about 8 g of a composition comprising 15-HEPE. In another embodiment, 15-HEPE is at least about 90% by weight of all fatty acids in the composition.

[0121] In some embodiments, the present invention provides methods of treating and/or preventing a hematological 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 some embodiments, the method further comprises determining whether the subject has at least one risk factor for a hematological disorder prior to administering the 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining the baseline red blood cell count, red blood cell distribution width, and/or reticulocyte count of the subject prior to administering the 15-HEPE. In one embodiment, the subject has an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in reticulocyte count. In another embodiment, the method comprises administering to the subject up to about 8 g of a composition comprising 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 invention provides methods for treating and/or preventing a red blood cell disorder in a subject in need thereof, wherein the method comprises administering to the subject 15-HEPE or a composition comprising 15-HEPE. In another embodiment, the method comprises administering to the subject up to about 8 g of a composition comprising 15-HEPE. In another embodiment, 15-HEPE is at least about 90% by weight of all fatty acids in the composition.

[0123] In some embodiments, the present invention provides methods of treating and/or preventing a 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 whether the subject has at least one risk factor for a red blood cell disorder prior to administering 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining the baseline red blood cell count, red blood cell distribution width, and/or reticulocyte count of the subject prior to administering 15-HEPE. In one embodiment, the subject has an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in reticulocyte count. In another embodiment, the method comprises administering up to about 8 g of a composition comprising 15-HEPE to the subject. In another embodiment, the 15-HEPE is at least about 90% by weight of the total fatty acids in the composition.

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

[0125] In some embodiments, the present invention provides methods for treating and/or preventing a hematological disorder, a hemoglobin disorder, and/or a 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 whether the subject has at least one risk factor for the hematological disorder, hemoglobin disorder, and/or a red blood cell disorder, prior to administering the 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 has one or more of the following: 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 invention provides methods for treating and/or preventing a hematological disorder, a hemoglobin disorder, and/or a red blood cell disorder in a subject in need thereof, wherein the method comprises 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 a composition comprising 15-HEPE to the subject. In another embodiment, 15-HEPE is at least about 90% by weight of all fatty acids in the composition. In another embodiment, the hematological 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 whether 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 determining the baseline red blood cell count, red blood cell distribution width, and/or reticulocyte count of the subject prior to administration of 15-HEPE. In one embodiment, the subject has an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in reticulocyte count.

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

[0128] In some embodiments, the present invention provides methods of treating and/or preventing hemolytic anemia 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 whether the subject has at least one risk factor for hemolytic anemia prior to administering 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 increase in red blood cell distribution width, and/or an increase in reticulocyte count. In some embodiments, the subject has one or more of the following: an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in reticulocyte count.

[0129] In some embodiments, the present invention provides methods for treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE. In some embodiments, 15-HEPE is 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, hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis. In another embodiment, acquired hemolytic anemia is selected from the group consisting of secondary and secondary to infection, drug therapy, hematological malignancy, autoimmune disease, hypersplenism, mechanical prosthetic heart valve, and blood transfusion. In various embodiments, sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration. In some embodiments, the subject exhibits an increase in red blood cell count, a decrease in red blood cell distribution width, and a decrease in reticulocyte count after administration.

[0130] In some embodiments, the present invention provides methods for treating and/or preventing hemolytic anemia in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE. In some embodiments, 15-HEPE is 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 whether the subject has at least one risk factor for hereditary hemolytic anemia or acquired hemolytic anemia prior to administering the 15-HEPE or the composition comprising 15-HEPE. In another embodiment, the acquired hemolytic anemia is selected from the group consisting of secondary and secondary to infection, drug therapy, hematological malignancy, autoimmune disease, hypersplenism, mechanical prosthetic heart valve, and blood transfusion. In various embodiments, sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration. In some embodiments, the method further comprises determining the baseline red blood cell count, red blood cell distribution width, and/or reticulocyte count of the subject prior to administration of 15-HEPE. In one embodiment, the subject has an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in reticulocyte count.

[0131] The compositions and formulations described herein may additionally be used in the treatment and/or prevention of thrombophilia. Thrombophilic disorders refer to a class of conditions characterized by abnormal blood coagulation, which increases the risk of thrombosis. Non-limiting examples of risk factors for thrombophilic disorders include decreased prothrombin time, decreased activated partial thromboplastin time, and increased fibrinogen concentrations.

[0132] In some embodiments, the subject has a reduced prothrombin time of at least 10% below normal, at least 15% below normal, at least 20% below normal, at least 25% below normal, or at least 30% below normal.

[0133] In some embodiments, the subject has a reduced activated partial thromboplastin time by 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.

[0134] In some embodiments, the subject has an elevated fibrinogen concentration of at least 10% below normal, at least 15% below normal, at least 20% below normal, at least 25% below normal, or at least 30% below normal.

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

[0136] In some embodiments, the present invention provides methods of treating and/or preventing a thrombophilic 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 whether the subject has at least one risk factor for a thrombophilic disorder prior to administration of 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decrease in prothrombin time, a decrease in activated partial thromboplastin time, and/or an increase in fibrinogen concentration. In some embodiments, the subject has one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time, and a decrease in fibrinogen concentration following administration of 15-HEPE or a composition comprising 15-HEPE.

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

[0138] In some embodiments, the present invention provides methods for treating and/or preventing venous thromboembolism 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 whether the subject has at least one risk factor for venous thromboembolism prior to administering the 15-HEPE or the composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decrease in prothrombin time, a decrease in activated partial thromboplastin time, and/or an increase in fibrinogen concentration. In some embodiments, the subject has one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time, and/or a decrease in fibrinogen concentration.

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

[0140] In some embodiments, the present invention provides methods for treating and/or preventing arterial thrombosis 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 whether the subject has at least one risk factor for arterial thrombosis prior to administering the 15-HEPE or the composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decrease in prothrombin time, a decrease in activated partial thromboplastin time, and/or an increase in fibrinogen concentration. In some embodiments, the subject has one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time, and/or a decrease in fibrinogen concentration.

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

[0142] In some embodiments, the present invention provides methods for preventing embolism 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 whether the subject has at least one risk factor for developing embolism prior to administering the 15-HEPE or the composition comprising 15-HEPE. In some embodiments, the at least one risk factor is a decrease in prothrombin time, a decrease in activated partial thromboplastin time, and/or an increase in fibrinogen concentration. In some embodiments, the subject has one or more of an increase in prothrombin time, an increase in activated partial thromboplastin time, and/or a decrease in fibrinogen concentration.

[0143] In some embodiments, the present invention provides a method of treating and/or preventing endothelial dysfunction in a subject, 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 another embodiment, 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition.

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

[0145] In some embodiments, the present invention provides a method of treating, preventing, or reducing apoptosis of cells, wherein the method comprises 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 Bcl-2 family proteins, activated caspase fragments, and/or cleaved PARP-1.

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

[0147] The compositions and formulations described herein can be used to treat or prevent cardiovascular diseases or disorders. In one embodiment, the cardiovascular disease or disorder is selected from: dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, primary hypercholesterolemia, primary hyperlipidemia, total primary hyperlipidemia, prevalent hypercholesterolemia, familial hyperlipidemia, familial primary hyperlipidemia, familial hypercholesterolemia, familial hypertriglyceridemia, combined familial hyperlipidemia, familial apolipoprotein b-100 defect, secondary hyperlipidemia, mixed hyperlipidemia, cardiovascular disease, residual cardiovascular risk, prevention of atherosclerotic plaque formation/progression, microvascular disease, macrovascular disease, atherosclerosis, coronary atherosclerosis, diastolic dysfunction, reduction of cardiovascular risk, prevention of serious 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, prevention of diabetes, diabetes mellitus, insulin resistance, hyperinsulinemia, hyperglycemia, dysglycemia, induction of glycemic control, prevention of impaired glucose tolerance and impaired fasting glycemia. Non-limiting examples of microvascular diseases include retinopathy, nephropathy and neuropathy. Non-limiting examples of macrovascular diseases include stroke, peripheral vascular disease, limb ischemia and cardiac disease. In some embodiments, the subject has non-alcoholic liver disease, cholestatic liver disease, kidney disease or metabolic syndrome. Any of the above examples of cardiovascular disease may also refer to non-limiting examples of cardiometabolic disease.

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

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

[0150] In another embodiment, provided herein is a method for preventing hematological disorders in a subject having diabetes (e.g., Type I or Type II), which comprises administering 15-HEPE to the subject. In some embodiments, the method further comprises determining whether the subject has diabetes prior to administering 15-HEPE. In another embodiment, the method comprises administering to the subject about 10 mg per subject weight (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE. In another embodiment, 15-HEPE comprises at least about 90% by weight of the total fatty acids in the composition.

[0151] In another embodiment, provided herein is a method for preventing hematological disorders in a subject having diabetes (e.g., Type I or Type II), comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining whether the subject has diabetes prior to administering 15-HEPE or a composition comprising 15-HEPE. In another embodiment, the method comprises administering to the subject about 10 mg per subject weight (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg of 15-HEPE or a composition comprising 15-HEPE. In another embodiment, 15-HEPE comprises at least about 90% by weight of the total fatty acids in the composition.

[0152] In another embodiment, provided herein is a method of treating and/or preventing a hematological disorder in a subject suffering from a cardiovascular disease, the method comprising administering 15-HEPE to the subject. In some embodiments, the method further comprises determining whether the subject has a cardiovascular disease prior to administering 15-HEPE. In another embodiment, the method comprises administering to the subject about 10 mg per subject weight (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE. In yet another embodiment, 15-HEPE comprises at least about 90% by weight of the total fatty acids in the composition.

[0153] In another embodiment, provided herein is a method of treating and/or preventing a hematological disorder in a subject suffering from a cardiovascular disease, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE. In some embodiments, the method further comprises determining whether the subject has a cardiovascular disease prior to administering 15-HEPE or a composition comprising 15-HEPE. In another embodiment, the method comprises administering to the subject about 10 mg per subject weight (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg of 15-HEPE or a composition comprising 15-HEPE. In yet another embodiment, 15-HEPE comprises at least about 90% by weight of the total fatty acids in the composition.

[0154] In another embodiment, provided herein is a method of treating and/or preventing a hematological disorder in a subject suffering from high blood pressure, the method comprising administering 15-HEPE to the subject. In some embodiments, the method further comprises determining whether the subject has high blood pressure prior to administering the 15-HEPE. In some embodiments, the subject has high blood pressure of at least about 130 mmHg, at least about 135 mmHg, at least about 140 mmHg, at least about 145 mmHg, at least about 150 mmHg, at least about 155 mmHg, at least about 160 mmHg, at least about 165 mmHg, or at least about 170 mmHg. In another embodiment, the method comprises administering to the subject about 10 mg/kg, about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg 15-HEPE. In yet another embodiment, 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition.

[0155] In another embodiment, provided herein is a method of treating and/or preventing a hematological disorder in a subject suffering from high blood pressure, the method comprising administering 15-HEPE or a composition comprising 15-HEPE to the subject. In some embodiments, the method further comprises determining whether the subject has high blood pressure prior to administering the 15-HEPE or the composition comprising 15-HEPE. In some embodiments, the subject has high blood pressure of at least about 130 mmHg, at least about 135 mmHg, at least about 140 mmHg, at least about 145 mmHg, at least about 150 mmHg, at least about 155 mmHg, at least about 160 mmHg, at least about 165 mmHg. or at least about 170 mmHg. In another embodiment, the method comprises administering to the subject 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. In yet another embodiment, 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition.

[0156] The compositions and formulations described herein can also be used to reduce the level of cytokines and/or chemokines in a subject suffering from a cardiovascular disease and/or a hematological disease. Non-limiting examples of cytokines and/or chemokines include alpha-smooth muscle actin (α-SMA), inhibitor of metallopeptidase-1 (TIMP-1), transforming growth factor beta-β (TGF-β), and collagen type 1.

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

[0158] (a) no increase or decrease in serum alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0159] (b) no increase or decrease in bilirubin (BUN) levels compared to baseline, placebo control, and/or the patient before treatment;

[0160] (c) no increase or decrease in fibrosis area compared to baseline, placebo control, and/or patient before treatment;

[0161] (d) no increase or decrease in fasting blood glucose compared to baseline, placebo control, and/or the patient before treatment;

[0162] (e) no increase or decrease in insulin levels compared to baseline, placebo control, and/or the patient before treatment;

[0163] (f) no increase or decrease in alkaline phosphatase (ALP) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0164] (g) no increase or decrease in hemoglobin A1C (HbA1C) levels compared with baseline, placebo control, and/or the patient before treatment;

[0165] (h) no increase or decrease in homeostatic model insulin resistance (HOMA-IR) compared to baseline, placebo control, and/or patient before treatment;

[0166] (i) no increase or decrease in adipose tissue insulin resistance (adipo-IR) levels compared to baseline, placebo control, and/or the patient before treatment;

[0167] (j) no increase or decrease in total cholesterol levels compared to baseline, placebo control, and/or patient pre-treatment;

[0168] (k) no increase or decrease in triglyceride levels compared to baseline, placebo control, and/or patient pre-treatment;

[0169] (l) no increase or decrease in diglyceride levels compared to baseline, placebo control, and/or patient pre-treatment;

[0170] (m) no increase or decrease in very low density lipoprotein cholesterol (VLDL-C) levels compared with baseline, placebo control, and/or patient pre-treatment;

[0171] (n) no increase or decrease in remnant particle cholesterol (RLP-C) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0172] (o) no increase or decrease in non-HDL cholesterol levels compared to baseline, placebo control, and/or patient pre-treatment;

[0173] (p) no decrease or increase in high-density lipoprotein cholesterol (HDL-C) levels compared with baseline, placebo control, and/or patient pre-treatment;

[0174] (q) no increase or decrease in low-density lipoprotein cholesterol (LDL-C) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0175] (r) no increase or decrease in renal hydroxyproline levels compared to baseline, placebo control, and/or patient pre-treatment;

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

[0177] (t) no increase or decrease in interleukin-17 (IL-17) levels compared to baseline, placebo control, and/or patient pre-treatment;

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

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

[0180] (w) no increase or decrease in tumor necrosis factor (TNF-α) levels compared to baseline, placebo control, and/or patient pre-treatment;

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

[0182] (y) no increase or decrease in interleukin-6 (IL-6) levels compared to baseline, placebo control, and/or patient pre-treatment;

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

[0184] (aa) no increase or decrease in type 1 collagen levels compared to baseline, placebo control, and/or patient before treatment;

[0185] (bb) no increase or decrease in transforming growth factor-β (TGF-β) levels compared to baseline, placebo control, and/or patient pre-treatment;

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

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

[0188] (ee) no increase or decrease in serine protease 2 (PRSS2) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0189] (ff) no increase or decrease in amine oxidase, copper 3 (AOC3) levels compared to baseline, placebo control, and/or patient pre-treatment;

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

[0191] (hh) no increase or decrease in transferrin receptor (TR) protein 1 levels compared with baseline, placebo control, and/or patient pre-treatment;

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

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

[0194] (kk) no increase or decrease in monocyte chemoattractant protein-1 (MCP-1) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0195] (ll) no increase or decrease in chemokine (CC motif) ligand 16 (CCL16) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0196] (mm) no increase or decrease in tremor-like transcript 2 (TLT2) levels compared to baseline, placebo control, and/or patient pre-treatment;

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

[0198] (oo) no increase or decrease in TIMP-1 inhibitor levels compared to baseline, placebo control, and/or patient pre-treatment;

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

[0200] (qq) no increase or decrease in NAFLD score (NFS) compared with baseline, placebo control, and/or patient pre-treatment; and

[0201] (rr) no increase or decrease in fibrosis-4 (FIB-4) levels compared with baseline, placebo control, and/or patient pre-treatment;

[0202] (ss) no increase or decrease in liver elasticity levels compared to baseline, placebo control and/or patient before treatment;

[0203] (tt) no increase or decrease in liver fat content compared to baseline, placebo control, and/or patient before treatment;

[0204] (uu) no decrease or increase in glycerophospholipid levels compared to baseline, placebo control, and/or patient before treatment;

[0205] (vv) no increase or decrease in blood pressure compared with baseline, placebo control, and/or patient before treatment;

[0206] (ww) no increase or decrease in the end-point liver fibrosis (ELF) score compared with baseline, placebo control, and/or patient pre-treatment;

[0207] (xx) no increase or decrease in collagen production in lung and/or dermal fibroblasts compared to baseline, placebo control, and/or patient before treatment;

[0208] (yy) no decrease or increase in glucose absorption compared to baseline, placebo control, and/or patient before treatment;

[0209] (zz) increased viability of lung and/or skin fibroblasts compared to baseline, placebo control and/or patient before treatment;

[0210] (ab) no change or decrease in interleukin-8 (IL-8) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0211] (ac) no change or decrease in interleukin-23 (IL-23) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0212] (ad) no change or decrease in interleukin-11 (IL-11) levels compared to baseline, placebo control, and/or patient pre-treatment; and/or

[0213] (ae) no change or decrease in interferon γ (IFNγ) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0214] (af) no change or decrease in T and/or B cell activation compared to baseline, placebo control and/or patient before treatment;

[0215] (ag) no change or decrease in chemotaxis compared to baseline, placebo control and/or patient before treatment;

[0216] (ah) no change or decrease in phosphorylated B-cell lymphoma family 2 (Bcl-2) members compared to baseline, placebo control, and/or patient before treatment;

[0217] (ai) no change or decrease in levels of activated caspase fragments compared to baseline, placebo control and/or patient before treatment;

[0218] (aj) no change or decrease in cleaved poly(ADP-ribose) polymerase-1 (PARP-1) levels compared to baseline, placebo control, and/or patient pre-treatment;

[0219] (ak) no change or decrease in waist circumference compared to baseline, placebo control and/or untreated patient;

[0220] (al) no change or decrease in AST to platelet ratio index (ARPI) compared to baseline, placebo control, and/or patient before treatment;

[0221] (am) no change or reduction in liver inflammation and fibrosis (LIF) score compared to baseline, placebo control, and/or patient before treatment;

[0222] (an) no change or decrease in Lok score compared to baseline, placebo control and/or patient before treatment;

[0223] (ao) no change or decrease in fibrosis score compared to baseline, placebo control and/or patient before treatment;

[0224] (ap) no change or decrease in King's scale score compared to baseline, placebo control and/or patient before treatment;

[0225] (aq) no change or decrease in Bonacini score compared to baseline, placebo control and/or patient before treatment;

[0226] (ar) no change or decrease in transient elastography (TE) compared to baseline, placebo control and/or patient before treatment.

[0227] (as) no increase or decrease in vascular adhesion molecules compared to baseline, placebo control, and/or patient before treatment;

[0228] (at) no increase or decrease in cardiovascular risk proteins compared to baseline, placebo control, and/or patient pre-treatment;

[0229] (au) no increase or decrease in chemokine levels compared to baseline, placebo control, and/or patient pre-treatment;

[0230] (av) no increase or decrease in the number of tumor necrosis factor receptor superfamily members compared with baseline, placebo control, and/or the patient before treatment;

[0231] (aw) no change or increase in red blood cell count compared to baseline, placebo control, and/or patient pre-treatment;

[0232] (ax) no change or increase in white blood cell count compared to baseline, placebo control, and/or patient before treatment;

[0233] (ay) no change or increase in platelet count compared to baseline, placebo control, and/or patient before treatment;

[0234] (az) no change or increase in prothrombin time compared to baseline, placebo control and/or patient before treatment;

[0235] (ba) no change or increase in activated partial thromboplastin time compared to baseline, placebo control, and/or patient before treatment;

[0236] (bc) no change or increase in hemoglobin production compared to baseline, placebo control, and/or patient before treatment;

[0237] (bd) no change or increase in hemoglobin tetramer beta-globin chain synthesis compared to baseline, placebo control, and/or patient before treatment;

[0238] (be) no change or decrease in red blood cell hemolysis compared to baseline, placebo control, and/or patient before treatment;

[0239] (bf) no change or improvement in dyspnea compared with baseline, placebo control, and/or patient pre-treatment;

[0240] (bg) no change or decrease in heart rate compared to baseline, placebo control and/or patient before treatment;

[0241] (bh) no change or decrease in spleen size compared to baseline, placebo control and/or patient before treatment;

[0242] (bi) no change or decrease in liver size compared to baseline, placebo control and/or patient before treatment;

[0243] (bj) no change or improvement in dactylitis compared to baseline, placebo control, and/or patient before treatment;

[0244] (bk) no change or reduction in pain crises compared to baseline, placebo control and/or patient before treatment;

[0245] (bl) no change or reduction in abnormal blood clot formation compared to baseline, placebo control, and/or patient before treatment;

[0246] (bm) no change or decrease in red blood cell distribution width compared to baseline, placebo control and/or patient before treatment;

[0247] (bn) no change or decrease in reticulocyte count compared to baseline, placebo control, and/or patient before treatment; and/or

[0248] (bo) no change or decrease in fibrinogen concentration compared to baseline, placebo control and/or patient before treatment.

[0249] In one embodiment, the methods of the present invention comprise measuring baseline levels of one or more markers or parameters specified in paragraphs (a)-(bo) above prior to administering a dose to a subject or group of subjects. In another embodiment, the methods comprise administering to a subject a composition described herein after determining baseline levels of one or more markers or parameters specified in paragraphs (a)-(bo) and then further measuring said one or more markers.

[0250] In another embodiment, when treated with a composition of the present invention, such as for a period of about 1 to about 12 weeks, about 1 to about 8 weeks, or about 1 to about 4 weeks, the subject or 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) through (bo) described immediately above.

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

[0252] (a) no increase or decrease in serum ALT and/or AST levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0253] (b) no increase or decrease in serum urea levels by 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 60%, 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 the patient before treatment;

[0254] (c) no increase or decrease in fibrosis area by 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 60%, 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 the patient before treatment;

[0255] (d) no increase or decrease in fasting blood glucose 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 60%, 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 the patient before treatment;

[0256] (e) no increase or decrease in insulin levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0257] (f) no increase or decrease in ALP levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0258] (g) no increase or decrease in HB1Ac levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0259] (h) no increase or decrease in HOMA-IR levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0260] (i) no increase or decrease in adipo-IR levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0261] (j) no increase or decrease in total cholesterol 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 60%, 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 the patient before treatment;

[0262] (k) no increase or decrease in triglyceride levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0263] (l) no increase or decrease in diglyceride levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0264] (m) no increase or decrease in LDL-C levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0265] (n) no increase or decrease in RLP-C levels by 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 60%, 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 the patient before treatment;

[0266] (o) no increase or decrease in non-HDL cholesterol levels by 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 60%, 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 the patient before treatment;

[0267] (p) no reduction in HDL-C levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0268] (q) no increase or decrease in LDL-C levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0269] (r) no increase or decrease in renal hydroxyproline levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0270] (s) no increase or decrease in IL-33 levels by 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 60%, 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 the patient before treatment;

[0271] (t) no increase or decrease in IL-17 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0272] (u) no increase or decrease in AhR levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0273] (v) no increase or decrease in TL1A levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 pre-treatment patient; relative to baseline, placebo control, and/or pre-treatment patient;

[0274] (w) no increase or decrease in TNF-α levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0275] (x) no increase or decrease in IL-13 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0276] (y) no increase or decrease in IL-6 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0277] (z) no increase or decrease in Il-1β levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0278] (aa) no increase or decrease in type 1 collagen levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0279] (bb) no increase or decrease in TGF-β levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0280] (cc) no increase or decrease in α-SMA levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0281] (dd) no increase or decrease in TNRSF11A levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0282] (ee) no increase or decrease in PRSS2 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0283] (ff) no increase or decrease in AOC3 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0284] (gg) no increase or decrease in LILBR1 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0285] (hh) no increase or decrease in TR levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0286] (ii) no increase or decrease in PI3 level 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 60%, 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 the patient before treatment;

[0287] (jj) no increase or decrease in SAA4 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0288] (kk) no increase or decrease in MCP-1 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0289] (ll) no increase or decrease in CCL16 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0290] (mm) no increase or decrease in TLT2 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0291] (nn) no increase or decrease in DPP4 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0292] (oo) no increase or decrease in TIMP-1 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0293] (pp) no increase or decrease in PAI-1 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0294] (qq) no increase or decrease in NFS of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0295] (rr) no increase or decrease in FIB-4 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0296] (ss) no increase or decrease in liver elasticity levels by 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 60%, 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 the patient before treatment;

[0297] (tt) no increase or decrease in liver fat content by 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 60%, 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 the patient before treatment;

[0298] (uu) no decrease or increase in glycerophospholipid levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0299] (vv) no increase or a decrease in blood pressure of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0300] (ww) no increase or decrease in ELF 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 60%, 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 the patient before treatment;

[0301] (zz) no increase or decrease in collagen production in lung and/or skin fibroblasts 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 60%, 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 the patient before treatment;

[0302] (yy) no decrease or an increase in glucose absorption 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 60%, 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 the patient before treatment;

[0303] (zz) increasing the viability of lung and/or skin fibroblasts by 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 60%, 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 the patient before treatment;

[0304] (ab) no change or a decrease in IL-8 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0305] (ac) no change or a decrease in IL-23 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment; and/or

[0306] (ad) no change or a decrease in IL-11 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0307] (ae) no change or decrease in IFNγ levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0308] (af) no change or a decrease in T and/or B cell activation 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 60%, 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 the patient before treatment;

[0309] (ag) no change or a decrease in chemotaxis 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 60%, 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 the patient before treatment;

[0310] (ah) no change or a decrease in the number of Bcl-2 family members 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 60%, 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 the patient before treatment;

[0311] (ai) no change or a decrease in the levels of activated caspase fragments by 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 60%, 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 the patient before treatment;

[0312] (aj) no change or a decrease in cleaved PARP-1 levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0313] (ak) no change or a decrease in waist circumference 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 60%, 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 the patient before treatment;

[0314] (al) no change or a decrease in ARPI 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 60%, 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 the patient before treatment;

[0315] (am) no change or a decrease in LIF 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 60%, 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 the patient before treatment;

[0316] (an) no change or a decrease in Lok 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 60%, 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 the patient before treatment;

[0317] (ao) no change or a decrease in fibrosis score of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0318] (ap) no change or a decrease in King's score of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0319] (aq) no change or a decrease in the Bonacini score of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment; and/or

[0320] (ar) no change or a decrease in TE 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 60%, 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 the patient before treatment;

[0321] (as) an increase in vascular adhesion molecules of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 pre-treatment patient;

[0322] (at) an increase in cardiovascular risk proteins of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0323] (au) an increase in chemokines 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 60%, 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 pre-treatment patient; and

[0324] (av) an increase in the number of tumor necrosis factor receptor superfamily members 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 60%, 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 pre-treatment patient;

[0325] (aw) no change or an increase in red blood cell count 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 60%, 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 the patient before treatment;

[0326] (ax) no change or an increase in white blood cell count 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 60%, 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 the patient before treatment;

[0327] (ay) no change or an increase in platelet count 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 60%, 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 the patient before treatment;

[0328] (az) no change or an increase in prothrombin time 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 60%, 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 the patient before treatment;

[0329] (ba) no change or an increase in activated partial thromboplastin time 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 60%, 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 the patient before treatment;

[0330] (bc) no change or an increase in hemoglobin production 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 60%, 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 the patient before treatment;

[0331] (bd) no change or an increase in hemoglobin tetramer beta-globin chain synthesis 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 60%, 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 the patient before treatment;

[0332] (be) no change or a decrease in red blood cell hemolysis 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 60%, 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 the patient before treatment;

[0333] (bf) no change or a reduction in dyspnea 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 60%, 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 the patient before treatment;

[0334] (bg) no change or a decrease in heart rate 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 60%, 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 the patient before treatment;

[0335] (bh) no change or a decrease in spleen size 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 60%, 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 the patient before treatment;

[0336] (bi) no change or a decrease in liver size 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 60%, 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 the patient before treatment;

[0337] (bj) no change or a reduction in dactylitis 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 60%, 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 the patient before treatment;

[0338] (bk) no change or a reduction in pain crises 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 60%, 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 the patient before treatment;

[0339] (bl) no change or a reduction in abnormal thrombus formation 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 60%, 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 the patient before treatment;

[0340] (bm) no change or a decrease in red blood cell distribution of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 60%, 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 the patient before treatment;

[0341] (bn) no change or a decrease in reticulocyte count 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 60%, 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 the patient before treatment; and/or

[0342] (bo) no change or a decrease in fibrinogen concentration 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 60%, 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 the patient before treatment;

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

EXAMPLES

Example 1. Unilateral renal interstitial fibrosis caused by ureteral obstruction

[0344] The aim of this study was to investigate the effects of DS109 (15-HETrE) and DS102 (15-HEPE) on renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO).

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

1.1 Materials and methods

[0346] Test Substances: The following substances were tested in this study: DS109 (15-HETRE) and DS102 (15-HEPE). To prepare dosing solutions of each substance, DS109 was first weighed and then dissolved in a vehicle containing 0.5% hydroxypropyl methylcellulose (HPMC), and DS102 was diluted in a vehicle containing 0.5% HPMC.

[0347] Surgery for OOM: On day 0 of the study, mice were surgically treated under sodium pentobarbital anesthesia. The hair was shaved and then an abdominal incision was made to expose the left ureter. The ureter was ligated with 4-0 nylon suture at two points. The peritoneum and skin were then sutured and the mouse was transferred to a clean cage until recovery from anesthesia. In sham-operated mice, the left ureter was isolated but not ligated.

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

[0349] Dosage: DS109 was administered at three doses: 5, 50, and 250 milligrams (mg)/kg once daily from Day 0 to Day 13 of the study. DS102 was administered at two doses: 50 and 500 mg/kg once daily from Day 0 to Day 13 of the study.

[0350] Animals: Seven-week-old female C57BL/6 mice (i.e., animals) were provided by Japan SLC, Inc. (Japan) and were housed and fed a regular diet (CE-2; CLEA Japan, Japan) under controlled conditions. Animals were maintained in a specific-pathogen-free (SPF) room under controlled conditions of temperature (e.g., 23±2 °C), humidity (e.g., 45±10%), lighting (e.g., 12-hour artificial light and dark cycle, lights on from 8:00 a.m. to 8:00 p.m.) and air exchange. The experimental room was maintained at high pressure to prevent contamination. Animals were housed in TPX cages (CLEA Japan) with a maximum of 4 mice per cage. Sterilized Paper-Clean (Japan SLC) bedding was used and changed weekly. Sterilized solid regular diet was provided ad libitum, placed in a metal lid on top of the cage. Clean water was also provided ad libitum from a water bottle fitted with a rubber stopper and a drinking tube. Water bottles were replaced weekly, cleaned, autoclaved and reused. Mice were identified by ear piercing and each cage was labelled with a specific identification code.

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

[0352] Histopathological analysis: To visualize collagen deposits, kidney sections were stained with picrosirius red (Waldeck, Germany). To quantify the area of interstitial fibrosis, bright field images of the corticomedullary region were obtained using a digital camera (e.g., DFC295; Leica Microsystems, Germany) at 200x magnification, and positive areas in 5 fields/sections 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. One 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 RNase inhibitor (Toyobo, Japan), 0.5 mM dNTP (Promega, USA), 6.28 μM random hexamer (Promega), 5x first strand buffer (Promega), 10 mM dithiothreitol (Invitrogen, USA), and 200 U MMLV-RT (Invitrogen) in a final volume of 20 μl. The reaction was carried out for 1 hour at 37 °C, then 5 minutes at 99 °C. Real-time PCR was performed using DICE Real-Time PCR and TB Green™ Premix Ex Taq™ II (Takara Bio). To calculate the relative expression level of microRNAs (mRNA), the expression of each gene (e.g., α-SMA, TIMP-1, TGF-β, and collagen type 1) was normalized to the level of the reference gene 36B4 (gene symbol: Rplp0). Information about the PCR primer sets and plate layout are provided in Tables 1 and 2.

[0354] Table 1. PCR primers for quantitative RT-PCR measurement

SEQ ID NO. Gene Installed ID Subsequence 1
2 36B4 MA057856 direct/
back 5'-TTCCAGGCTTTGGGCATCA-3'
5'-ATGTTCAGCATGTTCAGCAGTGTG-3' 3
4 Alpha-SMA MA057911 direct/
back 5'-AAGAGCATCCGACACTGCTGAC-3'
5'-AGCACAGCCTGAATAGCCACATAC-3' 5
6 TIMP-1 MA098519 direct/
back 5'-TGAGCCCTGCTCAGCAAAGA-3'
5'-GAGGACCTGATCCGTCCACAA-3' 7
8 TGF-β MA030397 direct/
back 5'-GTGTGGAGCAACATGTGGAACTCTA-3'
5'-TTGGTTCAGCCACTGCCGTA-3' 9
10 Collagen type 1 MA075477 direct/
back 5'-CCAACAAGCATGTCTGGTTAGGAG-3'
5'-GCAATGCTGTTCTTGCAGTGGTA-3' 36B4: Ribosomal protein, large, P0 (Rplp0)
Alpha-SMA: Actin, alpha-2, smooth muscle, aorta (Acta2)
TIMP-1: Tissue inhibitor of metalloproteinase 1 (Timp1)
TGF-β: Transforming growth factor beta 1 (Tgfb1)
Collagen, type 1: collagen, type I, alpha 2 (Col1a2)

[0355] Table 2. Features of PCR plates for quantitative RT-PCR analysis

Tablet
Mouse ID 1
101-308 2
401-608 3
701-708 Alpha-SMA Tablet 1-2 Tablet 2-2 Tablet 3-2 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1 TIMP-1 Tablet 1-3 Tablet 2-3 Tablet 3-2 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1 TGF-β Tablet 1-4 Tablet 2-4 Tablet 3-3 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1 Collagen type 1 Tablet 1-5 Tablet 2-5 Tablet 3-3 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1

[0356] Specimen collection: For serum samples, non-fasting blood was collected into separate serum tubes without anticoagulant by direct cardiac puncture and centrifuged at 3500 x g for 4 min at 4 °C. The supernatant was collected and stored at -80 °C for transport. For kidney samples, the left kidney was collected and cut into 2 halves horizontally. The superior 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 inferior part of the left kidney was cut into 2 halves in the coronal plane. The anterior part of the left kidney was snap frozen in liquid nitrogen and stored at -80 °C for gene expression analysis. The posterior part of the left kidney was snap frozen in liquid nitrogen and stored at -80 °C for renal biochemical parameters.

[0357] Statistical Analysis: Statistical analysis was performed using the Bonferroni multiple comparison test in GraphPad Prism 6 (GraphPad Software Inc., USA). P values <0.05 were considered statistically significant. Direction or trend was assumed when a one-tailed t-test returned P values <0.1. Results were expressed as mean ± standard deviation.

1.2 Experimental and treatment design

[0358] The study design included the following research groups:

Group 1 (sham-operated control): Eight sham-operated mice were kept without any treatment until sacrificed.

Group 2 (vehicle): Eight mice with OOM were orally administered vehicle [0.5% HPMC] at 10 ml/kg once daily from day 0 to day 13.

Group 3 (DS109, low): Eight mice with OOM were orally administered vehicle supplemented with DS109 at 5 mg/kg once daily from day 0 to day 13.

Group 4 (DS109, medium): Eight mice with OOM 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 mice with OOM were orally administered vehicle supplemented with DS109 at 250 mg/kg once daily from day 0 to day 13.

Group 6 (DS102, low): Eight mice with OOM were orally administered vehicle supplemented with DS102 at 50 mg/kg once daily from day 0 to day 13.

Group 7 (DS102, high): Eight mice with OOM were orally administered vehicle supplemented with DS102 at 500 mg/kg once daily from day 0 to day 13.

[0359] Table 3 summarizes the treatment schedule for each of Groups 1-7 during the study.

[0360] Table 3. Brief description of the treatment schedule

Group Number of mice Model Test substance Dose (mg/kg) Volume (ml/kg) Scheme Coloring (day) 1 8 False operation - - - day 0 - day 13 14 2 8 OOM Carrier - 10 p/o, 1 time/day,
day 0 - day 13 14 3 8 OOM DS109 5 10 p/o, 1 time/day,
day 0 - day 13 14 4 8 OOM DS109 50 10 p/o, 1 time/day,
day 0 - day 13 14 5 8 OOM DS109 250 10 p/o, 1 time/day,
day 0 - day 13 14 6 8 OOM DS102 50 10 p/o, 1 time/day,
day 0 - day 13 14 7 8 OOM DS102 500 10 p/o, 1 time/day,
day 0 - day 13 14

[0361] Animal Monitoring and Euthanasia: Mice were monitored daily for viability, clinical signs, and behavior. Individual body weights were measured daily prior to treatment and throughout the study period. Mice were observed for significant clinical signs of toxicity, moribundity, and mortality for approximately 60 minutes following each administration. Animals were euthanized by direct cardiac puncture under isoflurane anesthesia (Pfizer Inc.) on day 14.

1.3 Results

[0362] Body Weight Changes and Overall Findings: FIG. 2 shows the body weight changes for all animals. All animals experienced a loss of body weight following surgery and gradually recovered during the treatment period. The mean body weight of the vehicle group was significantly lower than that of the sham-operated control group from Day 2 to Day 5 and from Day 10 to Day 11. There were no significant changes in mean body weight on any day during the treatment period between the vehicle and treatment groups. No animals in any group died during the treatment period. No deterioration in general condition was observed in any animal in the present study.

[0363] Body and Kidney Weights on the Day of Sacrifice: FIG. 3 and Table 4 show the body weights 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-operated control group and the vehicle-treated group. There were no significant differences in the mean body weight on the day of sacrifice between the vehicle-treated group and the treatment groups.

[0364] Table 4. Body weight on the day of sacrifice

Group Body weight (g) Right kidney weight (mg) Right kidney to body mass ratio (%) Left kidney mass (mg) Left kidney to body weight ratio (%) Sham-operated control (n=8) 19.2±0.7 122±8 0.64±0.05 115±14 0.60±0.08 Carrier (n=8) 18.9±0.5 139±15 0.73±0.09 488±150 2.56±0.77 DS109, low (n=8) 19.1±0.9 137±7 0.71±0.04 533±138 2.79±0.65 SD109, mean (n=8) 18.6±0.6 135±6 0.73±0.05 523±91 2.83±0.50 DS109, high (n=8) 18.6±0.7 130±13 0.70±0.05 425±105 2.26±0.53 DS102, low (n=8) 18.8±0.6 133±10 0.70±0.05 469±60 2.49±0.30 DS102, high (n=8) 18.7±0.5 136±6 0.71±0.04 487±108 2.61±0.57

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

[0366] Kidney Chemistry: FIG. 5 and Table 5 show the hydroxyproline content of the kidneys of the animals. The vehicle group showed a significant increase in the hydroxyproline content of the kidneys compared to the sham-operated control group. The DS109 low, DS109 high, DS102 low, and DS102 high groups showed a significant decrease in the hydroxyproline content of the kidneys compared to the vehicle group. There was no significant difference in the hydroxyproline content of the kidneys between the vehicle group and the DS109 medium group.

[0367] Table 5. Hydroxyproline content

Group Hydroxyproline in kidney (mcg/mg total protein) Sham-operated control (n=8) 3.02±1.89 Carrier (n=8) 16.67±7.83 DS109, low (n=8) 9.94±2.02 SD109, mean (n=8) 13.94±6.44 DS109, high (n=8) 8.30±1.04 DS102, low (n=8) 8.47±2.08 DS102, high (n=8) 8.59±1.99

[0368] Histological analysis: FIGS. 6A-6G and Table 6 show Sirius red staining and FIG. 7 shows the fibrotic area in the animals. FIG. 6A shows representative photomicrographs of kidney sections stained with Sirius red. The vehicle group showed a significant increase in the percentage of fibrotic area (positive area stained with Sirius red) compared to the sham-operated control group. Bonferroni multiple comparison showed that the fibrotic area in the middle DS109 group tended to decrease compared to the vehicle group. No significant differences were observed in fibrotic area between the vehicle group and the other treatment groups. Due to the presence of significant outliers, the Mann-Whitney U test was used, which showed a trend toward a decrease in fibrosis area (p<0.1) in the DS109 low, DS109 medium, DS109 high, DS102 low, and DS102 high groups compared to the vehicle group.

[0369] Table 6. Fibrosis area in animals

Group Sirius red, positive region (%) Sirius red, positive area (%), median Sham-operated control (n=8) 0.63±0.22 0.51±0.15 Carrier (n=8) 5.00±1.37 4.55±0.94 DS109, low (n=8) 4.23±1.90 3.22±1.47 SD109, mean (n=8) 3.58±1.04 3.28±1.41 DS109, high (n=8) 4.71±0.96 3.76±0.81 DS102, low (n=8) 5.01±3.11 4.19±3.33 DS102, high (n=8) 4.01±1.92 3.08±1.16

[0370] Gene Expression Analyses. Gene expression analysis for alpha-SMA, TIMP-1, TGF-β, collagen type 1 is shown in FIGS. 8A-8D and Table 7.

[0371] Table 7. Gene expression analysis

Group Alpha-SMA TIMP-1 TGF-β Collagen type 1 Sham-operated control (n=8) 1.00±0.18 1.00±0.14 1.00±0.09 1.00±0.13 Carrier (n=8) 3.82±0.58 133.20±26.86 3.88±0.30 11.52±2.15 DS109, low (n=8) 3.87±0.82 153.60±37.12 4.29±0.93 10.76±1.45 SD109, mean (n=8) 4.61±3.15 167.70±122.60 6.01±4.77 15.23±11.52 DS109, high (n=8) 4.85±1.22 180.90±23.12 5.48±0.36 13.88±1.91 DS102, low (n=8) 3.74±0.81 138.30±30.39 4.31±0.69 11.80±1.78 DS102, high (n=8) 5.54±1.54 124.80±28.60 5.37±0.76 14.09±2.88

[0372] Alpha-SMA: A significant increase in α-SMA mRNA expression was observed in the vehicle-treated group compared to the sham-operated control group. No significant differences in α-SMA mRNA expression were observed between the vehicle-treated and treatment groups.

[0373] TIMP-1: A significant increase in TIMP-1 mRNA expression was observed in the vehicle group compared to the sham-operated control group. No significant differences in TIMP-1 mRNA expression were observed between the vehicle and treatment groups.

[0374] TGF-β: A significant increase in TGF-β mRNA expression was observed in the vehicle group compared to the sham-operated control group. No significant differences in TGF-β mRNA expression were observed between the vehicle and treatment groups.

[0375] Collagen Type 1: An increase in collagen type 1 mRNA expression was observed in the vehicle group compared to the sham-operated control group. No significant differences in collagen type 1 mRNA expression were observed between the vehicle group and the treatment groups.

1.4 Brief description

[0376] As shown by Sirius red staining and hydroxyproline content in kidneys, renal fibrosis was found in the vehicle-treated group in the present study.

[0377] DS109: Low dose DS109 treatment showed a significant reduction (p<0.05) in renal hydroxyproline content and a trend toward a decrease (p<0.1) in fibrosis area compared to the vehicle group. Medium dose DS109 treatment showed a trend toward a decrease (p<0.1) in fibrosis area compared to the vehicle group. Finally, high dose DS109 treatment showed a significant reduction (p<0.05) in renal hydroxyproline content and a trend toward a decrease (p<0.1) in fibrosis area compared to the vehicle group.

[0378] DS102: Low dose DS102 treatment showed a significant reduction in renal hydroxyproline content (p<0.05) and a trend toward a decrease (p<0.1) in fibrosis area compared to the vehicle group. High dose DS102 treatment showed a significant reduction in renal hydroxyproline content (p<0.05) and a trend toward a decrease (p<0.1) in fibrosis area compared to the vehicle group.

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

Example 2. Bile Duct Ligation (BDL) Study in Cholestatic Liver Disease and/or Liver Fibrosis

[0380] The aim of this study was to investigate the effect of DS012 on BDL-induced cholestasis.

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

1.1 Materials and methods

[0382] Test substance: The test substance for this study was DS102. To prepare dosing solutions of each substance, DS102 was diluted in a 0.5% hydroxypropyl methylcellulose (HPMC) vehicle.

[0383] Surgery for BDL: On day 0 of the study, surgery was performed under pentobarbital anesthesia (Kyoritsu Seiyaku, Japan). The mice were first shaved, an abdominal incision was made, and two ligatures were placed on the common bile duct with 7-0 surgical silk. The peritoneum and skin were sutured, and the mice were transferred to a clean cage (e.g., resting cage) until recovery from anesthesia. In sham-operated mice, the common bile duct was exposed but not ligated.

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

[0385] Dosage: DS102 was administered at three doses: 50, 250, and 500 milligrams (mg)/kg once daily from Day 0 to Day 13 of the study.

[0386] Animals: Pathogen-free male C57BL/6J mice, 6 weeks old, were provided by Japan SLC, Inc. (Japan). Animals were maintained in a specific pathogen-free (SPF) facility under controlled conditions of temperature (e.g., 23 ± 2 °C), humidity (e.g., 45 ± 10%), lighting (e.g., 12-hour artificial light and dark cycle, lights on from 8:00 a.m. to 8:00 p.m.) and air exchange. The experimental room was maintained at high pressure to prevent contamination. Animals were housed in TPX cages (CLEA Japan) with a maximum of 4 mice per cage. Sterilized Paper-Clean (Japan SLC) was used as bedding and changed weekly. Sterilized solid regular diet was provided ad libitum in a metal lid on top of the cage. Clean water was also provided ad libitum from a water bottle fitted with a rubber stopper and a drinking tube. Water bottles were replaced weekly, cleaned, autoclaved and reused. Mice were identified by ear piercing and each cage was labelled with a specific identification code.

[0387] Measurement of serum biochemical parameters: To visualize collagen deposition, fixed liver sections were stained with picrosirius red (Waldeck, Germany) according to Bouin. For quantitative analysis of fibrosis area, bright-field images of sirius red-stained sections were obtained at 100x magnification using a digital camera (DFC295; Leica, Germany), and positive areas in 5 fields/sections were measured using ImageJ software (National Institutes of Health, USA).

[0388] Histological analysis: To visualize collagen deposits, kidney sections were stained with picrosirius red (Waldeck, Germany). To quantify the area of interstitial fibrosis, bright field images of the corticomedullary region were obtained using a digital camera (e.g., DFC295; Leica Microsystems, Germany) at 200x magnification, and positive areas in 5 fields/sections 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. One μg of RNA was reverse transcribed using a reaction mixture containing 4.4 mM magnesium chloride (MgCl ₂ ) (F. Hoffmann-La Roche, Switzerland), 40 U RNase inhibitor (Toyobo, Japan), 0.5 mM dNTP (Promega, USA), 6.28 μM random hexamer (Promega), 5x first strand buffer (Promega), 10 mM dithiothreitol (Invitrogen, USA), and 200 U MMLV-RT (Invitrogen) in a final volume of 20 μl. The reaction was carried out for 1 hour at 37 °C, then 5 minutes at 99 °C. Real-time PCR was performed using DICE Real-Time PCR and TB Green™ Premix Ex Taq™ II (Takara Bio). To calculate the relative mRNA expression level, the expression of each gene (e.g., α-SMA, TIMP-1, TGF-β, and collagen type 1) was normalized to the level of the reference gene 36B4 (gene symbol: Rplp0). Information on the PCR primer sets and plate layout are provided in Tables 8 and 9.

[0390] Table 8. PCR primers for quantitative RT-PCR measurement

SEQ ID NO. Gene Installed ID Subsequence 1
2 36B4 MA057856 direct/
back 5'-TTCCAGGCTTTGGGCATCA-3'
5'-ATGTTCAGCATGTTCAGCAGTGTG-3' 3
4 Alpha-SMA MA057911 direct/
back 5'-AAGAGCATCCGACACTGCTGAC-3'
5'-AGCACAGCCTGAATAGCCACATAC-3' 5
6 TIMP-1 MA098519 straight/
back 5'-TGAGCCCTGCTCAGCAAAGA-3'
5'-GAGGACCTGATCCGTCCACAA-3' 7
8 TGF-β MA030397 direct/
back 5'-GTGTGGAGCAACATGTGGAACTCTA-3'
5'-TTGGTTCAGCCACTGCCGTA-3' 9
10 Collagen type 1 MA075477 direct/
back 5'-CCAACAAGCATGTCTGGTTAGGAG-3'
5'-GCAATGCTGTTCTTGCAGTGGTA-3' 36B4: Ribosomal protein, large, P0 (Rplp0)
Alpha-SMA: Actin, alpha-2, smooth muscle, aorta (Acta2)
TIMP-1: Tissue inhibitor of metalloproteinase 1 (Timp1)
TGF-β: Transforming growth factor beta 1 (Tgfb1)
Collagen, type 1: collagen, type I, alpha 2 (Col1a2)

[0391] Table 9. Features of PCR plates for quantitative RT-PCR analysis

Tablet
Mouse ID 1
101-308 2
401-608 3
701-708 Alpha-SMA Tablet 1-2 Tablet 2-2 Tablet 3-2 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1 TIMP-1 Tablet 1-3 Tablet 2-3 Tablet 3-2 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1 TGF-β Tablet 1-4 Tablet 2-4 Tablet 3-3 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1 Collagen type 1 Tablet 1-5 Tablet 2-5 Tablet 3-3 36B4 Tablet 1-1 Tablet 2-1 Tablet 3-1

[0392] Specimen collection: For serum samples, non-fasting blood was collected into separate serum tubes without anticoagulant by direct cardiac puncture and centrifuged at 3500 x g for 4 min at 4 °C. The supernatant was collected and stored at -80 °C for biochemical analysis (30 μL) and transport (remainder). For liver samples, the left lateral lobe was collected and cut into 6 pieces. Two left lateral lobe pieces were fixed in Bouin's solution and then embedded in paraffin. The samples were stored at room temperature for histological analysis. The other two left lateral lobe pieces were embedded in OCT reagent and snap frozen in liquid nitrogen. The samples were stored at -80 °C. The remaining left lateral lobe pieces were snap frozen in liquid nitrogen and stored at -80 °C for gene expression analysis. The right middle lobe, left middle lobe, right lobe and caudate lobe were rapidly frozen in liquid nitrogen and stored at -80°C for transport.

[0393] Statistical tests: Statistical analysis was performed using the Bonferroni multiple comparison test in GraphPad Prism 6 (GraphPad Software Inc., USA). P values <0.05 were considered statistically significant. Direction or trend was assumed when a one-tailed t-test returned P values <0.1. Results were expressed as mean ± standard deviation.

1.2 Experimental and treatment design

[0394] The study design included the following research groups:

Group 1 (sham-operated control): Eight sham-operated mice were kept without any treatment until sacrificed.

Group 2 (vehicle): Fifteen BDL-operated mice were orally administered vehicle [0.5% HPMC] at 10 ml/kg once daily from day 0 to day 13.

Group 3 (DS102, low): Fifteen BDL-operated mice were orally administered vehicle supplemented with DS102 at 50 mg/kg once daily from day 0 to day 13.

Group 4 (DS102, medium): Fifteen BDL-operated mice were orally administered vehicle supplemented with DS102 at a dose of 250 mg/kg once daily from day 0 to day 13.

Group 5 (DS102, high): Fifteen BDL-operated mice were orally administered vehicle supplemented with DS102 at a dose of 500 mg/kg once daily from day 0 to day 13.

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

[0396] Table 10. Brief description of the treatment schedule

Group Number of mice Mice Test substance Dose (mg/
kg) Volume (ml/
kg) Scheme Killing 1 8 False operation - - - - Day 14 2 15 BDL Carrier - 10 p/o, 1 time/day, days 0-13 Day 14 3 15 BDL DS102 50 10 p/o, 1 time/day, days 0-13 Day 14 4 15 BDL DS102 250 10 p/o, 1 time/day, days 0-13 Day 14 5 15 BDL DS102 500 10 p/o, 1 time/day, days 0-13 Day 14

[0397] Animal Monitoring and Euthanasia: Mice were monitored daily for viability, clinical signs, and behavior. Individual body weights were measured daily prior to treatment and throughout the study period. Mice were observed for significant clinical signs of toxicity, moribundity, and mortality for approximately 60 minutes following each administration. Animals were sacrificed on postoperative day 14 by exsanguination via direct cardiac puncture under isoflurane (Pfizer Inc.) anesthesia.

1.3 Results

[0398] Body Weight Changes and Overall Conclusions: FIG. 10 shows the body weight changes for all animals. The average body weight in all groups, except the sham-operated control group, gradually decreased over the study period. The average body weight of the vehicle group was significantly lower than that of the sham-operated control group from day 2 to day 14. There were no significant changes in average body weight at any day during the study period between the vehicle and DS102-treated groups.

[0399] During the treatment period, the following mouse deaths were observed before day 14: three of 15 mice in the vehicle group; seven of 15 mice in the DS102 low dose, DS102 medium dose, and DS102 high dose groups. In this model, the percentage of deaths would be expected simply due to disease induction, and the observed mortality rate is consistent with historical data.

[0400] Body and Liver Weights on the Day of Sacrifice: FIG. 11 and Table 11 show the body weights of the animals on the day of sacrifice. The vehicle-treated group showed a significant decrease in the average body weight on the day of sacrifice compared to the sham-operated control group. No significant differences were observed in the average body weight on the day of sacrifice between the vehicle-treated group and the DS102-treated groups.

[0401] Table 11. Body weight on the day of sacrifice

Group Body weight (g) Liver weight (mg) Liver to body weight ratio (%) Sham-operated control (n=8) 23.1±0.7 1132±90 4.9±0.4 Carrier, (n=12) 16.8±1.8 1438±180 8.7±1.4 DS102, low (n=8) 17.4±2.9 1392±328 8.3±2.3 DS102, average (n=8) 17.0±2.4 1513±182 9.0±1.2 DS102, high (n=8) 16.4±1.6 1231±255 7.6±1.5

[0402] FIGS. 12A and 12B and Table 11 show the liver weight and liver to body weight ratio of the animals on the day of sacrifice. The vehicle-treated group showed a significant increase in the average liver weight compared to the sham-operated control group. The average liver weight in the high dose DS102 group tended to decrease compared to the vehicle group. No significant differences in the average liver weight were observed between the vehicle group and the other treatment groups. The vehicle group showed a significant increase in the average liver weight to body weight ratio compared to the sham-operated control group. The average liver weight to body weight ratio in the high dose DS102 group tended to decrease compared to the vehicle group. No significant differences in the average liver weight to body weight ratio were observed between the vehicle group and the other treatment groups.

[0403] Biochemistry: FIG. 13 and Table 12 show the serum aminotransferases (ALT) of the animals. The vehicle group had a significant increase in serum ALT levels compared to the sham-operated control group. No significant differences in serum ALT levels were observed between the vehicle and DS102-treated groups. However, historical data for this model show that ALT levels decrease at day 14 without treatment. Thus, this may affect the ability to detect differences between groups.

[0404] Table 12. Biochemistry

Group Serum ALT (U/L) Total serum bilirubin (mg/dL) Sham-operated control (n=8) 22±2 0.4±0.1 Carrier, (n=12) 335±185 24.9±9.6 DS102, low (n=8) 402±112 26.8±6.9 DS102, average (n=8) 351±155 23.5±9.8 DS102, high (n=8) 383±147 26.2±6.1

[0405] FIG. 14 and Table 12 show the total bilirubin in the serum of the animals. A significant increase in the total bilirubin level in the serum was observed in the vehicle-treated group compared to the sham-operated control group. No significant differences in the total bilirubin level in the serum were observed between the vehicle-treated group and the DS102-treated groups.

[0406] Histological analysis: FIGS. 15A-14E and Table 13 show Sirius red staining and FIG. 16 shows the fibrotic area in the animals. FIG. 14A shows representative photomicrographs of liver sections stained with Sirius red. Liver sections from the vehicle-treated group showed increased collagen deposition in the portal region of the liver lobule and bridging fibrosis of the PV-CV or PV-PV compared to the sham-operated control group. The vehicle-treated group showed a significant increase in the fibrotic area (positive area stained with Sirius red) compared to the placebo control group. The DS102 mid-dose group showed a significant decrease in the fibrotic area compared to the vehicle-treated group. The fibrotic area in the high dose DS102 group was generally reduced compared to the vehicle-treated group. There was no significant difference in fibrosis area between the vehicle group and the low dose DS102 group.

[0407] Table 13. Histological analysis

Group Sirius red, positive region (%) Sham-operated control (n=8) 0.46±0.25 Carrier, (n=12) 1.93±0.67 DS102, low (n=8) 1.70±0.63 DS102, average (n=8) 1.06±0.48 DS102, high (n=8) 1.42±0.60

[0408] Gene Expression Analyses Gene expression analyses for α-SMA, TIMP-1, TGF-β, and collagen type 1 are shown in FIGS. 17A-17D and Table 14.

[0409] Table 14. Gene expression analysis

Group Alpha-SMA TIMP-1 TGF-β Collagen type 1 Sham-operated control (n=8) 1.00±0.24 1.00±0.82 1.00±0.08 1.00±0.24 Carrier, (n=12) 2.98±2.16 33.09±13.75 1.65±0.44 4.77±2.53 DS102, low (n=8) 3.21±1.67 43.82±26.19 1.48±0.48 4.94±2.76 DS102, average (n=8) 2.66±1.33 30.65±16.80 1.50±0.44 4.21±1.90 DS102, high (n=8) 1.42±0.75 16.09±10.16 1.00±0.23 2.51±1.39

[0410] α-SMA: A significant increase in the expression level of α-SMA mRNA was observed in the vehicle-treated group compared to the sham-operated control group. The expression level of α-SMA mRNA in the high-dose DS102 group tended to decrease compared to the vehicle-treated group. No significant differences were observed in the expression level of α-SMA mRNA between the vehicle-treated group and the DS102-treated groups.

[0411] TIMP-1: A significant increase in TIMP-1 mRNA expression was observed in the vehicle group compared to the sham-operated control group. The TIMP-1 mRNA expression level tended to decrease in the high dose DS102 group compared to the vehicle group. No significant differences in TIMP-1 mRNA expression level were observed between the vehicle group and the DS102 groups.

[0412] TGF-β: The vehicle-treated group showed a significant increase in TGF-β mRNA expression level compared to the sham-operated control group. The high dose DS102 group showed a significant decrease in TGF-β mRNA expression level compared to the vehicle-treated group. No significant differences in TGF-β mRNA expression level were observed between the vehicle-treated group and the DS102-treated groups.

[0413] Collagen Type 1: An increase in the expression level of collagen type 1 mRNA was observed in the vehicle group compared to the sham-operated control group. The expression level of collagen type 1 mRNA was generally decreased in the high dose DS102 group compared to the vehicle group. No significant differences in the expression level of collagen type 1 mRNA were observed between the vehicle group and the DS102 groups.

1.4 Brief description

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

[0415] Treatment with DS102 at a medium dose showed a significant reduction (p<0.05) in fibrosis area compared to the vehicle group. Treatment with DS102 at a high dose showed a significant reduction (p<0.05) in TGF-β mRNA expression level and a trend towards significance (p<0.01) in the reduction in fibrosis area, liver weight, liver weight to body weight ratio, α-SMA, TIMP-1, and collagen type 1 mRNA expression levels compared to the vehicle group. These results indicate that DS102 inhibits the production of multiple pro-fibrotic cytokines in the liver, has a suppressive effect on liver fibrosis, and has potential as a therapeutic agent for cholestatic liver diseases.

Example 3: Effect of DS102 on TGF-β receptors, signaling and fibrosis-induced proteins

[0416] The aim of this study was to investigate the effects of 15-HEPE and 15-HEPE EE on TGF-β receptor expression, TGF-β-induced intracellular signaling, and profibrotic epithelial mesenchymal transition proteins.

1.1 Materials and methods

[0417] Cytotoxicity study: The cytotoxicity of 15-HEPE as free acid and ethyl ester was tested on various liver cell lines (hepatomas) to understand the concentration range in the test system.

[0418] Transcriptional activity: A promoter (luciferase) assay was performed to measure TGFβ-induced transcriptional activation following 15-HEPE administration.

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

1.2 Results

[0420] The effect of 15-HEPE on TGF-β-induced signaling and cellular responses was determined. To test the effect of 15-HEPE on TGF-β-induced signaling, the activity of 15-HEPE in regulating TGF-β-stimulated Smad2 phosphorylation and nuclear translocation, both of which are key signaling events leading to TGF-β-induced cellular responses, was examined. One of the important biological activities of TGF-β is the transcriptional activation of genes responsible for epithelial-mesenchymal transition (EMT), which is a crucial event in wound healing, tissue fibrosis, and cancer progression. The effect of 15-HEPE on TGFβ-induced expression of epithelial-mesenchymal transition-related proteins (such as fibronectin, PAI-1, N-cadherin, etc.) in HepG2 and LXR cells was evaluated. HepG2 cells (human hepatoma cell line) were treated with increasing doses of stage II 15-HEPE in DMEM medium containing 0.1% FBS for 1 hour and continuously stimulated with or without 200 picomoles (pM) of TGF-β for 48 hours.

[0421] 15-HEPE directly inhibited TGF-β signaling as shown in FIGS. 18A and 18B. Specifically, FIG. 18A shows that 15-HEPE induces the degradation of the TGF-β type II receptor and blocks TGF-β-induced epithelial-mesenchymal transition (EMT) (i.e., pro-fibrotic) protein production. HepG2 cells (a human hepatoma cell line) were treated with increasing doses of stage II 15-HEPE in DMEM containing 0.1% FBS for 1 hour and continuously stimulated with or without 200 picomoles (pM) of TGF-β for 48 hours. The two panels shown in FIG. 18A represent two separate experiments performed under the same experimental conditions. The results of these individual experiments show that 15-HEPE blocks TGF-β-induced EMT protein production and induces the degradation of the TGF-β type II receptor. FIG. 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. FIG. 18B shows that 15-HEPE inhibits TGF-β-stimulated intracellular signaling (e.g., SMAD2/3 phosphorylation) in hepatic stellate cells. The experiment shown in FIG. 18B involved pretreatment of LX2 cells (human hepatic stellate cells) with increasing concentrations of 0 μM to 100 μM DS102 for 24 hours, followed by 30 minutes of TGF-β stimulation. The results of the study showed that 15-HEPE inhibited TGF-β stimulation.

[0422] A 15-HEPE sucrose density gradient assay of TGF-β receptors was performed in plasma membranes of mink lung epithelial cells (Mv1Lu) treated with 100 μM 15-HEPE and an equal volume of dimethyl sulfoxide (DMSO) at 37 °C for 0, 1, 4, and 24 hours, with cell lysates from these treated cells subjected to sucrose density gradient ultracentrifugation. Sucrose gradient fractions were then analyzed by Western blotting using anti-TβR-I (TGF-β receptor type I - Figure 18C), anti-TβR-II (TGF-β receptor type II - Figure 18D), anti-TβR-III (TGF-β receptor type III, betaglycan - Figure 18E), anti-EGFR (epidermal growth factor receptor - Figure 18F) and anti-caveolin-1 antibodies (Figure 18G). Fractions 4 through 5 contained lipid rafts/caveolae, whereas fractions 7 through 10 represented non-lipid rafts. 15-HEPE treatment did not affect the amount of TβR-I proteins, but induced TβR-I translocation to the lipid raft after 24 h of treatment (FIG. 18C). Asterisks (*) indicate increased TβR-I (24 h) fraction abundance by 15-HEPE compared to control and shorter treatment duration (FIG. 18C). 15-HEPE induced TβR-II translocation from 1 to 4 h and further induced degradation at 24 h treatment (FIG. 18D). Triangles (▼) indicate translocation and decreased TβR-II fraction abundance compared to control cells (FIG. 18D). In Figures 18E, 18F, and 18G, 15-HEPE did not alter the localization and abundance of TβR-III, EGFR, and caveolin-1.

1.3 Brief description

[0423] Accordingly, all these results collectively demonstrate that 15-HEPE in both the free acid and ethyl ester forms induces the translocation and degradation of TGF-β type II receptors, blocks TGF-β-induced profibrotic protein production, and inhibits TGF-β-induced intracellular signaling (SMAD2/3) in hepatic stellate cells. Thus, 15-HEPE and 15-HEPE EE can directly inhibit TGF-β signaling, results that support the therapeutic potential of 15-HEPE and 15-HEPE EE in the treatment of multiple fibrotic diseases including non-alcoholic steatohepatitis (NASH), other fibrotic diseases, cardiometabolic diseases, as well as multiple oncology indications.

Example 4: Efficacy of Oral DS102 in Patients with NAFLD

[0424] The objective of the study was to evaluate the safety and clinical efficacy of DS102 oral capsules compared with placebo in the treatment of adult patients with non-alcoholic fatty liver disease (NAFLD).

1.1 Study endpoints

[0425] Primary endpoints: The primary endpoints for this study included clinical efficacy and safety of DS102 administration. Clinical 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 shear wave elastography from baseline to week 16. Safety was assessed by the number of treatment-emergent adverse events (TEAEs) in each treatment group that led to treatment discontinuation.

[0426] Secondary Endpoints: Secondary endpoints for this study included the change in any 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 index-4 (FIB-4) from baseline to week 16; NAFLD fibrosis score (NFS) from baseline to week 16; change in liver fat measured by controlled attenuation parameter (CAP) from baseline to week 16; end-stage liver fibrosis (ELF) score from baseline to week 16; and homeostatic model assessment of insulin resistance (HOMA-IR) and adipose tissue insulin resistance (adipo-IR) from baseline to weeks 2, 4, 8, 12, and 16.

[0427] Exploratory Analysis: Exploratory analyses included analysis of lipid and metabolic parameters including total cholesterol, triglycerides, very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), remnant particle cholesterol (RLP-C), fasting blood glucose, insulin, free fatty acids, and hemoglobin A1C (HbA1C). Additional exploratory analyses included high-throughput lipidomics and proteomics.

1.2 Study design

[0428] This was a randomized, placebo-controlled, double-blind, multicenter, parallel-group, exploratory phase IIa study to investigate the safety and clinical efficacy of orally administered DS102 capsules and the dose-response relationship between two doses of DS102 and placebo in NAFLD in patients aged 18 to 75 years. This study had three parallel groups of patients with established NAFLD comparing two different doses of DS102 with placebo over a 16-week treatment period. The study planned to enroll 96 evaluable patients, with 32 patients randomized per treatment group.

[0429] The study consisted of a 28-day screening period, a 16-week treatment period, and a 4-week observation period. During the screening visit, patients were assessed with screening examinations. Patients who met the inclusion criteria and did not meet the exclusion criteria were included in the study.

[0430] A schematic diagram of the overall time frame of the study is shown in Figure 19. After enrollment in the study, patients were prohibited from using any other NAFLD treatment. Medications (e.g., prescription and over-the-counter) or therapeutic interventions that were considered necessary for the patient and that, in the opinion of the investigator, would not interfere with the assessment of safety and clinical efficacy were continued unless they were included in the “Concomitant Medications” list below.

[0431] Prior to the start of the comparative treatment period, patients visited the center for baseline disease assessment, with eligible patients randomly assigned to one of three parallel group treatment regimens in a 1:1:1 randomization.

Treatment Group A: 2 capsules of placebo at 500 mg orally twice daily (4 capsules daily) for 16 weeks.

Treatment Group B: 1 capsule DS102 500 mg and 1 capsule placebo 500 mg orally twice daily (4 capsules daily) for 16 weeks.

Treatment group C: 2 capsules of DS102 500 mg orally twice daily (4 capsules daily) for 16 weeks.

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

1.3 Patients and screening

[0433] To participate in this study, patients had to meet all of the following inclusion criteria and not meet any of the following exclusion criteria. Compliance with inclusion and exclusion criteria was 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 as follows:

Patients diagnosed with NAFLD based on the presence of hepatic steatosis on imaging or histology in the absence of any secondary causes.

Patients with ALT ≥1.5 ULN and <5 ULN on two measurements 7 or more days apart at screening.

Patients with a history of liver biopsy showing NASH and/or fibrosis ≥ F1, or NFS ≥ -1.455, or Fib-4 ≥1.3, or Fibroscan ≥8 kPa within 3 months of screening.

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 eligible for the study.

Patients whose pre-study laboratory test results did not prevent them from participating in the study, in the opinion of the investigator.

Patients aged 18 to 75 years.

Female patients and male patients with female partners of childbearing potential were required to use appropriate contraception or have a sterilized partner for the duration of the study. Appropriate contraception was defined as: systemic hormonal contraceptives; an intrauterine device or barrier method of contraception in combination with a spermicide; or sexual abstinence, defined as the patient abstaining from heterosexual intercourse for the entire risk period associated with the study treatment, if this is consistent with the patient's preferred and usual lifestyle. Hormonal contraceptives had to be taken at a stable dose for at least one month prior to baseline.

Patients with whom the investigator has established rapport, who understand and agree to comply with the requirements of the study, and who have read and signed written informed consent.

[0435] The exclusion criteria for this study were as follows:

Patients with unstable metabolic status, such as weight change >5% in the 3 months prior to inclusion.

Patients with a medical/surgical history of gastric bypass surgery, orthotopic liver transplant (OLT), or listed for OLT.

Patients with uncontrolled type 2 diabetes mellitus, i.e. HbA1c ≥9% (75 mmol/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's syndrome were eligible for the study if conjugated bilirubin was ≤1.5 x ULN.

Patients with inflammatory bowel disease that was either active or required drug therapy.

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, with the exception of curatively treated skin cancer (basal cell or squamous cell carcinoma).

Patients with significant systemic or serious illness other than liver disease, including ischemic heart disease, cerebrovascular disease, pulmonary disease, renal failure, serious psychiatric illness, respiratory or hypertensive disease, and diabetes and arthritis, which, in the opinion of the investigator, would prevent the patient from participating in and completing the study.

Patients requiring antidiabetic treatment (including insulin sensitizers) and/or lipid-lowering treatment and who had not been on a stable dose for at least 3 months prior to screening were excluded. In insulin-dependent patients, treatment had to be started at least 3 months prior to screening, with dose changes permitted.

Patients with known hypersensitivity to any ingredients of the study drug.

Patients who test positive for human immunodeficiency virus (HIV) antibodies, hepatitis B surface antigen, or hepatitis C antibodies on 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 deficiency (A1AT), or Wilson's disease.

Patients with a significant history of drug abuse/substance abuse, at the discretion of the investigator.

Patients with a history of alcohol abuse, at the discretion of the investigator, or drinking more than 21 units per week (men) or 14 units per week (women), where a unit consists of 10 ml or 8 mg of pure alcohol.

Patients who took dietary supplements enriched with omega-3 or omega-6 fatty acids for 4 weeks before baseline.

Patients who participated in any other clinical trial of the investigational drug within 3 months prior to the first day of administration of the investigational drug.

Pregnant women, planning to become pregnant, breastfeeding and/or unwilling to use appropriate contraception during the study.

Patients who, in the opinion of the investigator, are not suitable for participation in the study.

1.4 Conducting the study

[0436] Ten clinic visits were scheduled during the study following the screening visit: one at the beginning of the comparator/baseline period (Day 0/Visit 2) and eight during the comparator 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, Week 16/Visit 10). The final follow-up visit to assess safety (Visit 11) was conducted four weeks after Visit 10 or two weeks after the last visit if the patient did not complete the study. Table 15 describes the study visits.

[0437] Table 15. Flow chart of study visits

[0438] Screening Visit (Visit 1): The following is provided during the screening visit:

[0439] Screening Visit (Visit 1): After informed consent was obtained, patients were assigned a screening number. Ideally, the patient was screened in a fasting state. The following screening assessments/specimens were performed: review of inclusion/exclusion criteria; demographic data collected; medical history collected; physical examination; 12-lead electrocardiogram (ECG); vital signs (blood pressure, heart rate, and temperature); safety laboratory samples (hematology, serum chemistry, and coagulation profile); virology testing; pregnancy testing (for patients of childbearing potential); ALT, AST (ALT was measured twice during screening); and assessment of concomitant medications. Unscheduled visits occurred when a patient needed to see the investigator between scheduled visit dates due to an adverse event (AE), difficulty complying with the study protocol, or a significant change in their medical condition. All procedures that were medically necessary were followed. If necessary, at the end of the visit, the patient was instructed not to eat breakfast on the day of the next visit, so that the minimum fasting period was 8 hours.

[0440] Treatment Period: After completing a successful screening visit, patients entered the comparative treatment period (16 weeks). At the start of the comparative treatment period, upon confirmation of enrollment, patients were randomly assigned to one of three treatment regimens. Patients took the study medication (SMT) as DS102 capsules or placebo capsules twice daily during the comparative treatment period. Each SMT self-administration was recorded in the patient diary. Patients were instructed to take DS102 in the morning and evening with or after meals (except for morning clinic visits 3, 4, 6, 8, and 10, when patients were instructed to refrain from taking DS102 prior to the visit and to take DS102 as soon as possible after the clinic visit). At 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), the patient was asked if he or she had fasted for at least 8 hours prior to the visit. If not, the duration of the fasting period was recorded and the patient was re-instructed on the duration of the fasting period. The patient was then offered a light breakfast (eg, tea or orange juice and toast). At baseline (Visit 2), week 8 (Visit 8), and week 16 (Visit 10), blood samples were taken before the patient received the light breakfast. Unscheduled visits occurred when a patient needed to see the investigator between scheduled visit dates because of an adverse event (AE), difficulty complying with the study protocol, or a significant change in their medical condition. All medically necessary procedures were followed. For patients who discontinued the study early, study procedures scheduled for Visit 10 were completed as soon as possible after the patient left the study to allow all study-related information to be recorded. At the discretion of the investigator, urine drug and breath alcohol testing was performed at any time during the study.

[0441] Baseline (Visit 2): At Visit 2, patients visited the study center. The first assessment performed was a blood draw. Following blood draw, the following assessments were performed: review of inclusion/exclusion criteria; medical history; physical examination; 12-lead ECG; pharmacokinetic tests; vital signs (blood pressure, heart rate, and temperature); safety laboratory samples (hematology, serum chemistry, and coagulation profile); lipid profile; urinalysis; pregnancy test (for female patients of childbearing potential); ALT, AST; HOMA-IR/Adipo-IR; ELF; liver stiffness and CAP FIB-4; NFS (including BMI); biomarker blood sample; study blood sample; patient randomization; study drug/placebo administration; AE assessment; and concomitant medication assessment. If all study inclusion criteria were met, the investigator randomized the patient to receive the assigned ILP or placebo from one of the patient packs. The first dose of ILP or placebo was administered on site after completion of all baseline assessments. Patients received the second dose of ILP or placebo on the evening of Day 0. The capsules were then administered twice daily. Patients received ILP or placebo in the morning before the return visit (Visit 3). At the conclusion of the visit, the patient was instructed not to eat breakfast on the day of the following visit to ensure a minimum fasting period of 8 hours.

[0442] Week 2 (Visit 3): At Visit 3, patients visited the study site. Patients did not take IMP or placebo on the morning of Visit 3. The following assessments were completed: physical examination; pharmacokinetic tests; vital signs (eg, blood pressure, heart rate, and body temperature); ALT, AST; HOMA-IR/Adipo-IR; AE assessment; and concomitant medication assessment. The patient returned the IMP or placebo and was given an additional dose of IMP or placebo. The patient took the next dose of IMP or placebo once all visit assessments were completed. Capsules were continued twice daily. Following this visit, patients were instructed to return to the study site in two weeks at Visit 4. Patients did not take IMP or placebo on the morning of the return visit (Visit 4). At the end of the visit, the patient was instructed not to eat breakfast on the day of the next visit, so that the minimum fasting period was 8 hours.

[0443] Week 4 (Visit 4): At Visit 4, patients visited the study center. Patients did not take IMP or placebo on the morning of Visit 4. The following assessments were performed: physical examination; pharmacokinetic tests; vital signs (blood pressure, heart rate, and temperature); safety laboratory samples (hematology, serum chemistry, and coagulation profile); pregnancy testing (for patients of childbearing potential); ALT, AST; HOMA-IR/Adipo-IR; AE assessment; and concomitant medication assessment. The patient returned the IMP or placebo and was given additional IMP or placebo. The patient took the next dose of IMP or placebo once all visit assessments were completed. Capsules continued to be taken twice daily. At the end of this visit, patients were instructed to return to the study center in two weeks at Visit 5. Patients did not take IMP or placebo on the morning of the return visit (Visit 5).

[0444] Week 6 (Visit 5): At Visit 5, patients visited the study site. The following assessments were performed: AE assessment and concomitant medication assessment. ILP or placebo was returned to the patient and an additional dose of ILP was given. The patient took the next dose of ILP or placebo once all visit assessments were completed. Capsules were continued to be taken twice daily. At the conclusion of this visit, patients were instructed to return to the study site in two weeks at Visit 6. Patients did not take ILP or placebo on the morning of the return visit (Visit 6). At the conclusion of the visit, the patient was instructed not to eat breakfast on the day of the following visit to ensure a minimum fasting period of 8 hours.

[0445] Week 8 (Visit 6): At Visit 6, patients visited the study center. Patients did not take IMP or placebo on the morning of Visit 6. The first assessment performed was a blood draw. Following blood collection, the following assessments were performed: physical examination; pharmacokinetic tests; vital signs (blood pressure, heart rate, and temperature); safety laboratory samples (hematology, serum chemistry, and coagulation profile); lipid profile; pregnancy test (for patients of childbearing potential); ALT, AST; HOMA-IR/Adipo-IR; biomarker samples; AE assessment; and concomitant medication assessment. The patient returned the IMP or placebo and was given additional IMP or placebo. The patient took the next dose of IMP or placebo once all visit assessments were completed. The capsules continued to be taken twice daily. At the conclusion of this visit, patients were instructed to return to the study center in two weeks at Visit 7. Patients took either ILP or placebo in the morning before the return visit (Visit 7).

[0446] Week 10 (Visit 7): At Visit 7, patients visited the study site. The following assessments were performed: AE assessment and concomitant medication assessment. ILP or placebo was returned to the patient and an additional dose of ILP or placebo was given. The patient took the next dose of ILP or placebo once all visit assessments were completed. Capsules were continued to be taken twice daily. At the conclusion of this visit, patients were instructed to return to the study site in two weeks at Visit 8. Patients did not take ILP or placebo on the morning of the return visit (Visit 8). At the conclusion of the visit, the patient was instructed not to eat breakfast on the day of the following visit to ensure a minimum fasting period of 8 hours.

[0447] Week 12 (Visit 8): At Visit 8, patients visited the study center. Patients did not take IMP or placebo on the morning of Visit 8. The following assessments were performed: physical examination; pharmacokinetic tests; vital signs (blood pressure, heart rate, and temperature); safety laboratory samples (hematology, serum chemistry, and coagulation profile); pregnancy testing (for patients of childbearing potential); ALT, AST; HOMA-IR/Adipo-IR; AE assessment; and concomitant medication assessment. IMP or placebo was returned to the patient and additional IMP or placebo was given. The patient took the next dose of IMP or placebo once all visit assessments were completed. Capsules continued to be taken twice daily. At the end of this visit, patients were instructed to return to the study center two weeks later at Visit 9. Patients did not take IMP or placebo on the morning of the return visit (Visit 9).

[0448] Week 14 (Visit 9): At Visit 9, patients visited the study site. The following assessments were performed: AE assessment and concomitant medication assessment. ILP or placebo was returned to the patient and an additional dose of ILP or placebo was given. The patient took the next dose of ILP or placebo once all visit assessments were completed. Capsules were continued to be taken twice daily. At the conclusion of this visit, patients were instructed to return to the study site in two weeks at Visit 10. Patients were not to take ILP or placebo on the morning of the return visit (Visit 10). At the conclusion of the visit, the patient was instructed not to eat breakfast on the day of the next visit to ensure a minimum fasting period of 8 hours.

[0449] Week 16 (Visit 10) or Early Study Discontinuation: At Visit 10, patients visited the study center. Patients did not receive IMP or placebo on the morning of Visit 10. The first assessment performed was a blood draw. Following blood collection, the following assessments were performed: physical examination; 12-lead ECG; pharmacokinetic tests; vital signs (blood pressure, heart rate, and temperature); safety laboratory samples (hematology, serum chemistry, and coagulation profile); urinalysis; lipid profile; pregnancy test (for patients of childbearing potential); ALT, AST; HOMA-IR/Adipo-IR; ELF; liver stiffness and CAP FIB-4; NFS [including BMI]; biomarker blood samples; study blood sample; AE assessment; and assessment of concomitant medications. Patients returned the IMP or placebo. No further IMP or placebo blister packs or patient diaries were provided. After completion of the study assessment at this visit, study restrictions remained. At the conclusion of this visit, patients were instructed to return to the study site in four weeks at Visit 11 for assessment of any AEs that occurred after this visit and for safety and clinical efficacy. At the conclusion of the visit, the patient was instructed not to eat breakfast on the day of the next visit to ensure a minimum fasting period of 8 hours.

[0450] Follow-up Visit (Week 20/Visit 11): Four weeks after Visit 10 (or two weeks after the early discontinuation visit), patients visited the study center. The following assessments were performed: physical examination; pharmacokinetic tests; vital signs (blood pressure, heart rate, and temperature); safety laboratory samples (hematology, serum chemistry, and coagulation profile); pregnancy testing (for patients of childbearing potential); ALT, AST; HOMA-IR/Adipo-IR; biomarkers; AE assessment; and concomitant medication assessment.

1.5 Ratings

[0451] Clinical efficacy assessments included: ALT, AST, ALT:AST ratio; HOMA-IR/Adipo-IR; ELF; liver stiffness and CAP FIB-4 index; and NFS. Detailed descriptions are provided below.

[0452] ALT, AST, ALT:AST ratio: Elevation of liver enzymes (ALT and AST) is a marker of liver injury and was assessed at Visit 1/Screening (twice during Screening 7 or more days apart), Visit 2/Baseline, Visit 3/Week 2, Visit 4/Week 4, Visit 6/Week 8, Visit 8/Week 12, Visit 10/Week 16, and Follow-up Visit 11/Week 20.

[0453] HOMA-IR/Adipo-IR: HOMA-IR/Adipo-IR levels are a method of measuring insulin resistance. HOMA-IR is calculated by multiplying the fasting plasma insulin (FPI) level by the fasting plasma glucose (FPG) level and then dividing by a constant of 405. Adipo-IR is calculated by multiplying the fasting non-esterified fatty acid (NEFA) level by the fasting insulin level. Blood samples for HOMA-IR and Adipo-IR assessment were collected 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 at least 8 hours prior to blood collection. If subjects did not fast for at least 8 hours, the duration of fasting was recorded and subjects were further instructed to arrive at the next visit in a fasting state.

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

[0455] Liver Stiffness and CAP: Liver stiffness and controlled attenuation parameter (CAP) were assessed using transient elastography (eg, FibroScan® model 502 Touch or equivalent). Patients were examined in a fasting state at the same time of day, if possible, for baseline (week 0) and Visit 10 (week 16). The following conditions were met for this assessment: patients were supine with their right arm maximally abducted behind their 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 made measurements with the transducer in the intercostal space); During the FibroScan® examination, the choice of M+ or XL+ probe was determined by the Automatic Probe Selection (APS) tool (if the APS advised to use the “XL+ probe” or to continuously “switch” between “M+ and XL+ probes”, only the “XL+” probe was used); and the operator acquiring the ultrasound dynamic image visualized the part of the liver that does not contain large vascular structures (the measurement depth was 35-75 mm for the XL probe and 25-65 mm for the M probe, and the examined volume was 3 ^cm3 ). For each patient, the operator performed an examination including at least 10 valid measurements or no more than 20 attempts using the XL+ or M+ probe at the same site. The entire examination lasted no more than 10-15 minutes. The final stiffness and CAP values were recorded as the median of the acceptable 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 equation below.

FIB-4 = Age (years) x AST (U/L)

Platelet count (109/L) x √ALT (U/L)

[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 week 16 (visit 10).

[0458] Safety assessments included the following: medical history; physical examination; ECG; vital signs; laboratory tests to assess safety (eg, hematology, serum chemistry, coagulation, lipid profile, and urinalysis); virology tests; pregnancy testing; blood collection; pharmacokinetic tests; research blood collection; biomarker blood collection; urine drug testing and breath alcohol testing; adverse event assessment; concomitant medications; bioassay; sample, storage, handling; and delivery; and containment measures. Detailed descriptions are provided below.

[0459] History: A complete review of the patient's medical history was performed by the investigator or designated person at the screening visit (Visit 1) and baseline visit (Visit 2) to check for compliance with inclusion criteria. Any comorbidity was reported, whether or not considered relevant to the study. Dates of diagnosis or duration of illness were noted where possible.

[0460] Physical examination: Physical examination (including height and weight) was performed by the investigator according to the study flow chart in Table 15 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 in accordance with local guidelines. This examination was completed in full at baseline. Thereafter, examination was symptom-driven only (i.e., a battery of standard body systems tests were not assessed unless otherwise indicated by the patient). For example, if the patient reports a rash to the investigator, then skin examination would be performed. Examination of additional body systems was not required unless clinically warranted. Any abnormal findings were recorded. Changes in physical examination results compared to the baseline examination were recorded as AEs.

[0461] ECG: ECG was performed as a 12-lead 10 mm/1 mV, 25 mm/s with a 10-second lead II rhythm strip at each time point. ECG was recorded using a Mac 1200 or equivalent. Patients rested quietly in the supine position for 5 minutes before recording the ECG. Recordings were made on the days indicated in the study flow chart in Table 15, during Visit 1/Screening, Visit 2/Baseline, and Visit 10/Week 16.

[0462] Vital Signs: Vital sign measurements were performed according to the study flow chart in Table 15, 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. Vital sign measurements were performed prior to any blood collection. Any new values or changes in previous values considered clinically significant were recorded as AEs if detected after informed consent was given. Vital sign measurements included: supine blood pressure (e.g., after at least 5 minutes of rest), systolic blood pressure, and diastolic blood pressure (in mmHg); resting heart rate in beats per minute (bpm); and body temperature in accordance with clinical practice.

[0463] Safety Laboratory Tests: Hematology, serum chemistry, coagulation, lipid profiling, and urinalysis were performed Safety Laboratory Tests: Blood and urine samples were collected according to the study flow chart in Table 15 for standard hematology, serum chemistry, coagulation, and urinalysis, and lipid profiling. All samples were analyzed in a central laboratory. All subjects fasted for at least 8 hours prior to blood collection. If subjects did not fast for at least 8 hours, the duration of fasting was recorded and subjects were further instructed to arrive at the next visit in a fasting state.

Hematology : A complete blood count including red blood cell count, hemoglobin, hematocrit, white blood cell count, differential, 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 (APPT).

Lipid profile : LDL-C, HDL-C, and VLDL-C.

Urinalysis : pH, protein, glucose, blood, ketones, white blood cells, leukocyte esterase, bilirubin, specific gravity, urobilinogen, and nitrates. Reflex Micro is performed if blood, protein, leukocyte esterase, or nitrate/nitrite are present.

[0464] Virology: A blood sample was collected for virologic testing, including HIV, hepatitis C, and hepatitis B, as detailed in the study flow chart in Table 15.

[0465] Pregnancy Test: For patients of childbearing potential, pregnancy testing was performed according to the study flow chart in Table 15 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.

[0466] Blood Collection: After blood samples were obtained, each investigator analyzed the results in the laboratory for clinically significant values. Additional blood samples may be required for repeat testing to assess safety.

[0467] Pharmacokinetic (PK) Analysis Sampling: Blood samples for PK analysis were collected by direct venipuncture according to the study flow chart in Table 15 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. A 1 mL blood sample was collected at each time point. After centrifugation, plasma samples were divided into two portions, and a reserve sample was stored in the central laboratory until completion of the bioanalytical studies.

[0468] Blood collection for research purposes: Blood was collected according to the study flow chart in Table 15 at baseline (week 0) and visit 10/week 16 and stored for potential gene array analysis or additional research testing at a later time point.

[0469] Blood collection for biomarkers: Blood was drawn according to the study flow chart in Table 15 at baseline (week 0), visit 6/week 8, visit 10/week 16, and follow-up visit 11/week 20 and stored for analysis of potential biomarkers.

[0470] Urine Drug Testing and Breath Alcohol Testing: Depending on the clinical situation, at the discretion of the investigator, a breath alcohol test and a urine sample test for the presence of amphetamine, barbiturate, benzodiazepine, cocaine, cannabinoid, and opiate were performed at any time during the study.

[0471] Concomitant medications: Patients were on a stable dose of any concomitant medications for at least 3 months prior to screening, and this dose had to remain stable throughout the study period. In insulin-dependent patients, treatment had to be initiated at least 3 months prior to screening, with dose changes permitted.

[0472] Limitations: The study included restrictions on diet, alcohol, caffeine, and exercise. With respect to diet, patients avoided taking omega-3 or omega-6 fatty acid-rich dietary supplements (eg, fish oil capsules) both during the study and for 4 weeks prior to baseline. With respect to alcohol, patients avoided drinking alcohol in excess of 21 units per week (men) or 14 units per week (women), with a unit consisting of 10 ml or 8 mg of pure alcohol. There were no restrictions on caffeine consumption either before or during the study. With respect to physical activity, patients were instructed to avoid exercise and vigorous physical activity for at least 3 to 4 hours prior to a laboratory safety study (eg, biochemical).

1.6 Study drug

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

[0474] Placebo DS102 (paraffin oil) 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 (e.g., locked cabinet or drug storage room) to protect against unintended use. Labels contained the randomization number but no dose information.

[0476] Dose and Administration. This study compared DS102 to placebo administered orally twice daily for 16 weeks. The last administration of study drug was administered the day prior to the Week 16 visit/early discontinuation visit. Patients were to take the capsules with or after meals. Medications for other conditions that were allowed in the study were taken as usual. Wallet-style blisters were supplied for 7 days at 4 capsules/day. Patients took their assigned medication for 16 consecutive weeks.

1.7 Adverse events and serious adverse events

[0477] Adverse events (AEs) and serious adverse events (SAEs) in this study were defined as follows.

[0478] Adverse events (AEs): Any adverse event occurring in a patient who received the first dose of study drug, regardless of whether it was considered related to the IMP. AEs were recorded in the case report form with an indication of the relationship to the IMP and the severity.

[0479] Serious Adverse Events (SAEs): If a patient experienced an SAE after the first dose of study drug, the event was recorded as an SAE. An SAE is defined as an untoward medical event regardless of dose, including any of the following: death, life-threatening event, hospitalization or prolongation of ongoing hospitalization, or persistent or significant disability or incapacity. The term "life-threatening event" refers to an event in which the patient was at risk of death at the time of the event. The criterion does not apply to an event that could hypothetically have resulted in death if it had been more serious. Examples of important but non-life-threatening events included intensive care in the emergency department, allergic bronchospasm, blood dyscrasia, or seizure that did not result in hospitalization or the development of drug dependence or abuse.

[0480] Unexpected Adverse Event (AE): An event that was not previously reported in the Investigator's Brochure or similar product information sheet, such as the Prescribing Information for the Medicinal Product (PIMP).

[0481] The severity of an AE is the investigator's assessment of the relative seriousness of the event based on clinical experience. The following definitions were used to assess the severity of an AE:

Mild: The adverse event was transient and well tolerated.

Moderate: The adverse event caused discomfort to the patient and interfered with his/her usual activities.

Severe: The adverse event significantly interfered with the patient's normal activities and could have been disabling or life-threatening.

[0482] The causal relationship between the AE and the experimental treatment was also established taking into account the patient's medical history, recent physical examination data, and concomitant medications. The following definitions were used to determine the causal relationship with the AE:

Unrelated: The temporal relationship of the onset of the AE to the study treatment was not justified or the cause of the AE may have been different.

Associated: The time course of onset of AEs in relation to study treatment was reasonable, consistent with the known pattern of treatment response, and an alternative cause was unlikely.

[0483] All AEs were recorded throughout the study period up to and including the observation period.

1.8 Drug-induced liver injury

[0484] Severe drug-induced liver injury (DILI): Regardless of the suspected causal relationship, in the event of severe DILI, study drug was discontinued until the episode was considered resolved. If study drug was suspected to be the cause of liver injury, the patient was not rechallenged with the drug. Severe DILI is defined as evidence of liver failure as defined by a total bilirubin level >2 x ULN or an INR >1.5.

[0485] Patients with abnormal baseline liver biochemistry values: For abnormal baseline liver biochemistry values, fold increases were calculated relative to baseline levels rather than using the ULN. Thus, if ALT or AST increased ≥3-fold from baseline (or >200 IU/L), retesting was performed within 72 hours to confirm/determine improvement or worsening of biochemistry changes. AE information was collected during a thorough physical examination. Screening and/or other appropriate testing for hepatic etiology was performed. In case of liver dysfunction, the patient was receiving treatment for severe DILI. Treatment interruption was considered if any of the criteria for severe DILI occurred.

1.9 Serious adverse reactions and unexpected adverse reactions

[0486] Adverse reactions. All adverse and unexpected drug reactions associated with any dose were considered adverse drug reactions. The phrase "drug reaction" means that a causal relationship between the drug and the AE was at least a reasonable possibility (i.e., an association cannot be excluded). For marketed medicinal products, an adverse reaction is an adverse and unexpected reaction to a drug that occurs at doses typically used to prevent, diagnose, or treat disease or to modify physiological function in humans.

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

[0488] Suspected Unexpected Serious Adverse Reaction (SUSAR): Any serious adverse reaction that could be related to the IMP and was unexpected according to the definition above.

1.10 Statistical Methodology and Data Management

[0489] This clinical trial utilized a randomized, double-blind, placebo-controlled, parallel-group design. Randomization was used to minimize assignment bias and to increase the likelihood that known and unknown patient attributes (eg, demographic characteristics) would be equally balanced across treatment groups. Blinding was used to reduce potential bias in data collection and assessment of safety and clinical efficacy. The use of placebo as a comparator was appropriate for assessing safety and clinical efficacy in patients based on the short duration of the study and the absence of any potential long-term irreversible adverse effects that could result from placebo treatment.

[0490] Sample size estimation: Assuming a delta in the percentage response between the active drug and placebo groups of 20%, a standard deviation of 25%, and a dropout rate of 20%, 32 patients per group were required for the statistical test with a significance level of 5% and a power of 80%. At the interim analysis, the sample size was re-evaluated based on the blinding recommendations. The sample size could have been increased to a maximum of 150 patients to achieve a conditional power of 80% for the primary endpoint.

[0491] Blinding and unblinding instructions: All study site personnel and personnel involved in monitoring or conducting the study were blinded to individual patient treatment assignments. Randomisation details were kept strictly confidential, available only in emergency situations to authorised persons, until formal unblinding. Blinding codes for the trial were unblinded only after all patient data had been recorded and verified and the database was locked.

[0492] Interim Analysis and Data Monitoring: An interim safety analysis was conducted to assess the conditional statistical power of the study 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 clinical efficacy endpoints and secondary endpoints and was used to assess the conditional statistical power of the study to achieve its primary objective, potential overestimation of sample size, and possible elimination of a less clinically effective treatment group.

[0493] Clinically significant response: a higher mean or median reduction of at least 20% in ALT or liver stiffness compared with placebo and a higher mean or median reduction of at least 10% in both ALT and liver stiffness compared with placebo

[0494] The analysis set included the enrolled study population, the full analysis set (FAS), the per-protocol population (PPS), the safety analysis set (SAS), and the pharmacokinetic (PK) analysis set. A detailed description of each analysis set is provided below.

[0495] Intended patient population: Patients who signed the informed consent form. Unscreened patients are those patients in the enrolled patient population who did not meet eligibility requirements and were excluded from the study prior to randomization.

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

[0497] PPS: A subset of FAS consisting of patients without serious protocol violations. All protocol deviations were assessed and documented on a case-by-case basis prior to database closure, and serious deviations considered to have a major impact on clinical efficacy outcomes resulted in exclusion of the corresponding patient from the PPS set.

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

[0499] PK analysis set: patients in the SAS set who received at least one dose of DS102 at PK concentrations. Patients were analyzed according to the actual treatment received.

[0500] Safety analysis: Demographics, medical history, and physical examination data were provided for each patient and summarized narratively. All AEs reported during the study were coded by system organ class and preferred terms using the current version of the Medical Dictionary for Regulatory Activities (MedDRA). AEs were tabulated and summarized by treatment, relationship to treatment, severity, and grade. Clinical laboratory values (eg, hematology, chemistry, and urinalysis) were provided for each patient by treatment and day. Values outside the normal laboratory range were listed separately with appropriate comments regarding their clinical significance, with potentially clinically significant abnormalities highlighted and summarized by treatment. Clinical laboratory values obtained before dosing were defined as baseline values. Urine drug screen and breath alcohol screen results were provided for each patient. Individual vital sign values were listed and summarized descriptively by treatment and day. For each patient, 12-lead ECG assessments with all relevant comments were listed and summarized by treatment and day. Concomitant medications (if any), classified by drug groups and subgroups according to the latest version of the World Health Organization Drug Dictionary, were listed and summarized by treatment. In general, appropriate descriptive statistics were used according to the nature of the variable. Categorical variables were presented as counts and percentages, while continuous variables were presented using mean, standard deviation, median, minimum, maximum, coefficient of variation, and number of patients.

[0501] Pharmacokinetic analysis: Plasma 15(S)-HEPE concentrations were tabulated and summarized descriptively. Individual and mean plasma 15(S)-HEPE concentration-time profiles were presented graphically.

[0502] Primary variables: The primary efficacy variable was change from baseline in serum ALT at week 16 (visit 10). Active treatment groups were compared with placebo using analysis of covariance (ANCOVA), including the corresponding baseline value as a covariate. Comparisons with placebo were performed according to Dunnett's multiple testing procedure. For missing values at week 16, the last available value was carried forward (LOCF). Similar methods were used for liver stiffness assessment. Longitudinal modeling was additionally considered for ALT.

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

[0504] Exploratory Analysis: Exploratory analyses included analysis of lipid and metabolic parameters, including total cholesterol, triglycerides, very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), remnant particle cholesterol (RLP-C), fasting blood glucose, insulin, free fatty acids, and hemoglobin A1C (HbA1C). Additional exploratory analyses included high-throughput lipidomics and proteomics.

1.11 Results

[0505] This study showed that patients diagnosed with NAFLD who were administered DS102 at a dose of 1 g or 2 g daily experienced statistically significant reductions in markers of metabolic overload and improvements in insulin sensitivity and glycemic control.

[0506] FIG. 20 shows that the three treatment groups were well balanced at baseline and had similar lipidomic and metabolomic profiles. Baseline patient characteristics are shown in Table 16, where 10-13% of patients were receiving statin therapy at baseline.

[0507] Table 16. Patient characteristics at baseline

2 g DS102
(n=31) 1 g DS102
(n=32) Placebo
(n=30) Age, median, years 47.0 53.5 50.0 Floor, % Female 32.3 37.5 30.0 Male 67.7 62.5 70.0 Race, % Caucasoid 100,00 93.75 90,00 Other 0,00 6.25 10.00 BMI, median, kg/ ^m2 30.9 33.5 33.9 Statin use, % 9.7 12.5 13.3 Fasting glucose, median, mg/dL 119.1 110.9 120.3 Hemoglobin A1C (%), median 6.3 6.2 6.2 ALT, median, IU/L 74.0 70.5 96.5 Liver stiffness, median value, kPa 7.9 8.2 8.4 HOMA-IR, median 4.4 5.1 5.0 Triglycerides, median, mg/dL 242.0 146.0 150,0

[0508] FIGS. 21A-21C show changes in insulin, glucose, and free fatty acid levels from baseline to week 16 in patients administered DS102 at either 1 g or 2 g per day, compared to placebo. The reduction in insulin, glucose, and free fatty acid levels with DS102 administration is clinically significant because metabolic substrates, including glucose, carbohydrates, and free fatty acids, determine the pathogenesis of NASH.

[0509] FIGS. 22A and 22B show changes in HOMA-IR and adipo-IR levels from baseline to week 16 in patients administered DS102 at either 1 g or 2 g per day, compared to placebo. Patients experienced improvements in both indices of insulin resistance (e.g., reductions in HOMA-IR and adipo-IR levels) at week 16, with significant improvements in the per-protocol population (PPS) observed in patients administered 2 g DS102.

[0510] FIGS. 23A and 23B show the changes in glycosylated hemoglobin levels (e.g., HbA1c) from baseline to week 16 in patients administered DS012 at a dose of 1 or 2 g per day, compared to placebo. Specifically, FIG. 23A shows the change in HbA1c levels and FIG. 23B shows the change in HbA1c levels in the proportion of patients with high HbA1c values at baseline but who achieved normal values at week 16. Because Hb1Ac is a measure of the amount of glucose in the body's red blood cells and a surrogate for long-term glycemic control, these results indicate that administration of DS102 provides clinically meaningful improvements and normalizes glycemic control in a dose-dependent manner.

[0511] FIGS. 24A and 24B show the mean change and median (%) change in patient lipid profile at week 16 in the safety analysis set (SAS). These results are further presented in FIGS. 25A-25C and show that administration of DS102 significantly improved patient lipid profiles by either maintaining or reducing total cholesterol, VLDL-C, non-HDL-C, remnant particle cholesterol (RLP), and triglyceride levels. Notably, the reduction did not stabilize at week 16, suggesting that administration of DS102 may produce even greater changes in longer-term studies.

[0512] As shown in FIG. 26, administration of DS102 also reverses the hepatotoxic lipid features of NASH and improves several lipid classes altered in patients with NASH. In particular, administration of 2 g of DS102 significantly reduced levels of multiple hepatotoxic diglycerides and significantly increased levels of multiple glycerophospholipid groups. This finding is important because patients with NASH have low levels of glycerophospholipids in the liver and plasma.

[0513] FIG. 27 shows that administration of DS102 also resulted in resolution of NASH based on validated diagnostic tests, such as the OWL noninvasive diagnostic test for NASH. OWL Liver Care is a test that was developed based on plasma lipidomics in patients with biopsy-proven NASH and has high predictive value. The OWL Liver Care test has an area under the curve (AUC) of 0.88 for distinguishing between patients with NAFLD and normal liver and an AUC of 0.79 for distinguishing between patients with NAFLD without steatohepatitis and NASH. Administration of DS102 significantly improved and normalized OWL Liver Care scores in patients with NASH in a dose-dependent manner compared with placebo at week 16. Table 17 shows the baseline test diagnoses for each treatment group and shows that the majority of patients were classified as NASH or NAFLD at baseline, with a lower percentage of patients in the 2 g DS102 group.

[0514] Table 17. OWL test diagnoses at baseline

Group Diagnosis Initial level Change in diagnosis
(week 16 vs baseline) Improvement No changes Deterioration Placebo NASH 16 1 15 - Steatosis 7 1 4 2 No NAFLD 3 - 1** 2 1 g DS102 NASH 19 5 14 - Steatosis 5 - 2 3 No NAFLD 4 - 3 1 2 g DS102 NASH 12 5 7 - Steatosis 14 1 9 4 No NAFLD 3* - 2*, ** 1

[0515] Administration of DS102 also reduced liver fat content as assessed by CAP in patients diagnosed with NAFLD, as shown in FIG. 28. In addition, DS102 is expected to produce more significant liver changes in longer term studies and when assessed by more sensitive methods.

[0516] Administration of DS102 also reduced triglyceride levels in patients, as shown in Table 18, and thus it is expected that DS102 will also be effective in reducing cardiovascular risk.

[0517] Table 18. Changes in triglyceride levels in patients administered DS102 at week 16

DS102 (2g) Triglycerides (median placebo-adjusted % change from baseline) Full sample of patients for analysis -40.2 Subgroup: preglycerides > upper limit of normal (>150 mg/dL) -39.0 Subgroup: High triglycerides (>200 mg/dL) -20.4

[0518] FIGS. 29A-29C show that DS102 administration reduces inflammatory and pro-fibrotic proteins. For this evaluation, blood samples before and after DS102 treatment were analyzed using a panel of over 350 different protein biomarkers. Treatment with 2 g of DS102 significantly reduced the expression of over 150 markers associated with inflammation, fibrosis, lipid metabolism, apoptosis, and chemotaxis. Following DS102 treatment, resolution of metabolic overload and lipotoxicity was observed, which prevents subsequent cellular stress, inflammation, and fibrosis. The reduction in inflammatory and pro-fibrotic proteins suggests the ability of DS102 to provide resolution of NASH and prevention of fibrosis.

[0519] Administration of DS102 also reduced the expression of several NASH developmental targets, as shown in FIG. 30. In particular, administration of 2 g of DS102 reduced targets of drug development for the treatment of NASH, including CCR2/5 (Cenicriviroc - Allergan), Galectin3 (GR-MD-02 - Galectin), and AOC3 (Boehringer Ingelheim) signaling.

[0520] The volcano plot in FIG. 31 (Bonferroni and Benjamini-Hochberg testing data) shows that administration of 2 g DS102 reduced the amount of inflammatory and pro-fibrotic proteins based on changes in protein expression. Table 19 shows the most significant reduction in inflammatory and pro-fibrotic protein based on the Bonferroni test.

[0521] Table 19. Significant proteins based on the Bonferroni test

Protein Description PAI-1 TGF-β-induced protein is elevated in metabolic syndrome, atherosclerosis, fibrosis TIMP-1 Profibrotic DPP4 T cell activation, DPP4 inhibition increases GLP-1 levels TLT2 Enhances T-cell activation, clearance of apoptotic cells CCL16 a chemokine that binds to CCR 1,2,5,8. Cenicriviroc (Allergan) is a CCR2/5 antagonist. MCP-1 (CCL2) a chemokine that binds to CCR 2, 4. Cenicriviroc (Allergan) is a CCR2/5 antagonist. Increased in NASH, atherosclerosis, etc. SAA4 Apolipoprotein PI3/Elafin Increase in graft versus host disease TR Iron homeostasis LILRB1 Receptor for MHC class I antigens TNRSF11A T cell/dendritic cell interactions

[0522] Administration of 1 g DS102 also showed a significant reduction in TR. Table 20 shows the most significant reduction in inflammatory and profibrotic protein based on linear model analysis.

[0523] Table 20. Significant proteins based on the linear model

Protein Description TLT2 Enhances T-cell activation, clearance of apoptotic cells AOC3 Disrupts glucose homeostasis. Increases in NASH, atherosclerosis. PRSS2 Expressed in the pancreas, involved in cell adhesion

[0524] This analysis also showed that 1 protein in the 1 g DS107 group and 0 proteins in the placebo group crossed the 1st threshold based on the Benjamini-Hochberg test.

[0525] Administration of DS102 also reduced the expression of multiple vascular adhesion molecules, as shown in FIG. 32. Vascular adhesion molecules are associated with atherosclerosis, and their circulating levels are associated with cardiovascular risk.

[0526] Administration of DS102 also reduced the expression of several proteins associated with increased cardiovascular risk, as shown in FIG. 33.

[0527] Administration of DS102 also reduced the expression of multiple circulating chemokines, as shown in FIG. 34. Chemokines are important factors in chronic inflammation in atherosclerosis.

[0528] Administration of DS102 also reduced the expression of members of the multiple tumor necrosis factor receptor superfamily, as shown in FIG. 35. Members of the tumor necrosis factor receptor superfamily are associated with inflammation and atherosclerosis.

[0529] DS102 was also shown to be safe and well tolerated, with no observed differences in safety and tolerability compared with placebo. There were no drug-related serious adverse events (SAEs), and the incidence of adverse events (AEs) was similarly low across treatment groups. Most AEs were mild to moderate and transient, and no patient experienced an AE that led to early discontinuation of treatment. The safety profile for each study group is shown in Table 21.

[0530] Table 21. Safety profile for study groups

Patients, n (%) DS102, 2 g (n=30) DS102, 1 g (n=32) Placebo (n=31) ≥1 AE 15 (50.0) 19 (59.4) 17 (54.8) ≥1 AE related to study drug 3 (10.0) 2 (6.3) 4 (12.9) ≥1 SNA 1 (3.3) 0 1 (3.2) AEs leading to treatment discontinuation 0 0 1 (3.2) SAEs related to study drug 0 0 0 Fatal outcome 0 0 0

[0531] DS102 was also evaluated for potential efficacy in improving the related indications of primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), as shown in FIGS. 36A and 36B. Importantly, administration of 2 g DS102 reduced alkaline phosphatase (ALP) levels as well as multiple markers of liver fibrosis, suggesting that DS102 will also be effective in the treatment of PBC and PSC.

[0532] FIG. 37 shows a plot of 15-HEPE ethyl ester (EE) versus relative plasma concentrations, which indicates higher systemic exposure in the DS102 treatment group at weeks 8 and 16, as expected.

1.12 Brief description

[0533] Overall, these results demonstrate that DS102 targets multiple stages of NASH pathology by significantly reducing metabolic burden and improving insulin sensitivity. DS102 administration also improved the lipid profile of patients by reversing the levels of lipid accumulation associated with NASH. Specifically, patients treated with DS102 experienced decreased accumulation of hepatotoxic lipids, including total cholesterol, non-HDL-cholesterol, RLP-cholesterol, triglycerides, diglycerides, and VLDL-C, and increased glycerophospholipid levels. These effects are significant because patients diagnosed with NASH are characterized by high levels of total cholesterol, triglycerides, diglycerides, and VLDL-C and low levels of glycerophospholipids and omega-3 PUFAs. In addition, changes in many lipids are expected to result in reduced cardiovascular risk and improvement in many aspects of metabolic syndrome.

[0534] The results also showed that there was a dose-dependent effect in favor of administration of 2 g or more of DS102, with large and statistically significant improvements compared to placebo.

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

Example 5: 26-week oral toxicity study of 15(S)-HEPE EE in rats with a 4-week recovery period

[0536] The objectives of this study were to determine (1) the potential toxicity of 15(S)-HEPE EE, an omega-3 fatty acid used to treat liver and lung diseases, when administered orally for 26 weeks to rats and to assess the potential reversibility of any changes; and (2) the toxicokinetic characteristics of 15(S)-HEPE EE.

1.1 Study design

[0537] The working and control substances were administered to the respective animals once daily via gavage 7 days per week for a minimum of 26 weeks. The volume for each animal was based on the most recent body weight measurements. Doses were administered via a syringe with an attached cannula via gavage. The first day of dosing was designated as day 1. The drug was removed from the refrigerator and mixed for at least 30 minutes prior to administration. The drug was also continuously mixed during administration.

[0538] Due to the occurrence of clinical signs including hunched posture, protruding fur, abnormal gait and weight loss, animals 3206M, 3515F and 3703F were given a dosing break beginning on Day 45 and the remaining animals in Group 3 were given a dosing break beginning on Day 48. Animals were given lower doses (3000 mg/kg/day) beginning on Day 64. The following tables illustrate the treatments (Table 22) and animal groups (Table 23) used in the study.

[0539] Table 22. Treatment Table

Group No. Treatment Dose level (mg/kg/day) Dose volume (ml/kg) Dose concentration (mg/ml) Adjusted Dose Concentration (mg/mL) Number of animals Main research Recovery Toxicokinetics Males Females Males Females Males Females 1 Costal 0 10 0 0 15 15 5 5 3 3 2 15(S)-HEPE EE 2000 10 200 206 ^b /204 ^c 15 15 5 5 9 9 3- 15(S)-HEPE EE 6000 ^a 10 600 618 ^b 15 15 5 5 9 9 3 15(S)-HEPE EE 3000 ^a 10 300 309b/306c 1.5 15 5 5 9 9 ^a Due to the occurrence of clinical signs including hunched posture, protruding hair, abnormal gait and weight loss, animals were given a dosing break beginning on day 48. Animals were administered lower doses (3000 mg/kg/day) beginning on day 64.
^b Adjusted concentration for batches 2540M-1801 and 2540M-1802.
^c Corrected concentration for lots 2540M/1901 and 2540M/1902.

[0540] Table 23. Animal groups

Group No. Animal numbers Main research Recovery Toxicokinetics Males Females Males Females Males Females 1 1001-1015 1501-1515 1101-1105 1601-1605 1201-1203 1701-1703 2 2001-2015 2504-2515 2101-2105 2601-2605 2201-2209 2701-2709 3 3001-3015 3501-3515 3101-3105 3001-3605 3201-3209 3701-3709 The duplicate animals will have numbers 4002M and 4501F, 4502F.

[0541] Recovery Period: Following completion of the treatment period, assigned convalescent animals were left for a minimum of 4 weeks recovery period.

[0542] Rationale for Route of Administration and Dosing Levels: The oral route of administration was chosen for this study because this route is the expected route of administration in humans. A previous 26-week oral toxicity study in rats dosed once daily revealed no adverse effects at the highest dose tested (1 mg/kg/day). See Warren, H (2017). A 26-Week Oral Study of 15(S)-HEPE EE in Rats with a 4-Week Recovery Period. Charles River Study No. 529123. Given that no dose-limiting toxicity was observed in this study, an additional 28-day dose-ranging study determined that the maximum tolerated dose (MTD) for oral administration was 6 g/kg/day. See Murie, E (pending). A Preliminary 28-Day Oral Study of DS102 in Rats. Charles River Study No. 506611. This ongoing study was conducted to evaluate the chronic toxicity potential of 15(S)-HEPE EE (DS102) at the given MTD according to ICH M3 (R2). See ICH Harmonised Tripartite Guideline M3 (R2). Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals.

1.2 In vivo procedures, observations and measurements:

[0543] In vivo procedures, observations, and measurements were performed for all main study and recovery animals. Animals in the toxicokinetic evaluation group were weighed and clinical observations were recorded at the same time points as animals in the main study groups.

[0544] Mortality/moribundity checks were performed daily. Animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observations unless necessary for identification or confirmation of possible findings.

[0545] Clinical observations:

[0546] Cages were observed daily. Animals were not removed from the cage during observations except for identification or confirmation of possible results.

[0547] Detailed clinical observations were made weekly. Animals were removed from the cage for examination.

[0548] Post-dose observations were made at regular intervals throughout the day. All animals were observed for response to treatment. The onset, intensity, and duration of these signs were recorded, with particular attention paid to animals during and for the first hour after dosing.

[0549] Body weight measurements were made twice during the pre-treatment period; once daily during the dosing period; and twice weekly during the recovery period. Animals were weighed individually. Weights were recorded on the first day of the scheduled necropsy (for primary and recovery animals only).

[0550] Food intake was quantified weekly beginning at week 1 during the introduction and recovery periods.

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

[0552] Ophthalmologic examinations were performed using an indirect ophthalmoscope after administration of a mydriatic (1% tropicamide, mydriasis). All animals in the study groups and the recovery group were examined during the pretreatment period. Animals in the study groups (control and high-dose groups) were examined at weeks 13 and 26. All animals in the recovery group were examined at week 30.

1.3 Laboratory assessments

[0553] Clinical pathology

[0554] Specimen collection: Blood was collected from the tail vein unless immediately prior to necropsy, in which case blood was collected from the orbital sinus under isoflurane anesthesia without recovery. Animals were bled in ascending order unless samples were collected prior to necropsy, in which case the order of sacrifice was followed. Additional blood samples were collected (e.g., due to clotting of non-serum samples) without exceeding the acceptable sampling frequency and blood volume. After collection, samples were transferred to the appropriate laboratory for processing. Samples were collected in accordance with the following table.

[0555] Table 24. Specimens for clinical and pathological evaluation

Group No. Point in time Hematology Coagulation Clinical biochemistry Urine analysis Animals from the main study groups and the recovery group Week 26 - - - X Recovery Day 183 X X X - Main research Just before opening X X X - Animals from the recovery group Week 30 - - - X Just before opening X X X - Unplanned killing (if possible) Before the killing X X X - "X" - sample to be collected; "-" - not applicable.

[0556] Hematology: A 0.5 ml blood sample was collected using the anticoagulant K2EDTA. Blood smears were labeled, stained and stored. Blood smears were analyzed for the following parameters.

[0557] Table 25. Hematological parameters

Red blood cell count
Hemoglobin concentration
Hematocrit
Mean corpuscular volume
Red blood cell distribution width
Mean corpuscular hemoglobin concentration
Mean corpuscular hemoglobin
Reticulocyte count (abs.)
Platelet count White blood cell count
Neutrophil count (abs.)
Lymphocyte count (abs.)
Monocyte count (abs.)
Eosinophil count (abs.)
Basophil count (abs.)
Large unstained cells (abs.)

[0558] Coagulation: A 0.5 mL blood sample was collected using 3.8% (w/v) trisodium citrate anticoagulant for processing into plasma. The following coagulation parameters were measured to assess coagulation.

[0559] Table 26. Coagulation parameters

Activated partial thromboplastin time
Fibrinogen Prothrombin time
Sample quality

[0560] Clinical biochemistry: A 0.7 ml blood sample was collected using the lithium heparin anticoagulant to obtain plasma. The following parameters were determined to evaluate biochemical parameters:

[0561] Table 27. Clinical biochemistry parameters

Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Gamma-glutamyl transferase
Creatine kinase
Total bilirubin ^a
Urea
Creatinine
Calcium
Phosphate Total protein
Albumen
Globulin
Albumin/globulin ratio
Glucose
Cholesterol
Triglycerides
Sodium
Potassium
Chloride
Sample quality ^and if total bilirubin is >8.55 µmol/l, indirect and direct bilirubin will also be measured.

[0562] Urinalysis: Urine was collected from animals over a 6-hour period (+/- 30 min) under conditions of fasting and water availability. After collection, samples were transferred to the appropriate laboratory for processing. The following parameters were measured to evaluate the urine analysis results:

[0563] Table 28. Urine analysis parameters

Color
Appearance/Transparency
Specific gravity
Volume
pH Protein
Glucose
Bilirubin
Ketones
Blood

[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: A 0.5 mL blood sample was collected using K ₂ EDTA anticoagulant from the jugular vein (or tail vein if the jugular was not available) using sterile needles and disposable syringes. Upon completion of blood collection, animals from the toxicokinetic evaluation groups were sacrificed and the carcasses were disposed of without testing. Samples were collected according to the following table.

[0567] Table 29. Sampling schedule for toxicokinetic evaluation

Group No. Animal numbers Number of animals Sample collection time points (time after dosing) on day 1 and week 26 0 h ^a 1 hour 2 h 4 h 8 h 24 h 1 1201-1203
1701-1703 3M/3F - - - X - - 2 2201-2203
2701-2703 X - - X - - 2204-2206
2704-2706 - X - - X - 2207-2209
2707-270927W - - X - - X 3 3201-3203
3701-3706 ^b X - - X - - 3204-3206
3704-3706 ^b - X - - X - 3207-3209
3707-3709 ^b - - X - - X "X" - sample to be collected. "-" - not applicable.
^a The sample will be collected before administration.
^b Samples from animal 3702F were collected on day 1, but this animal was humanely euthanized on day 44. Additional samples will be collected at week 26 from animals 3707F (0 ^h collection) and 3704F (4 h collection) to cover the missing time points for animal 3702F.

[0568] Bioanalytical sample processing: Samples were mixed gently and stored on crushed wet ice until centrifugation within 60 minutes of blood 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 soon as possible over dry ice or in a freezer set to -20 °C.

[0569] Sample bioassay: Plasma samples were analyzed for unesterified and total HEPE concentrations using validated analytical procedures. The following were determined:

a) Unesterified (HEPE is present in plasma as a free fatty acid or bound to plasma albumin).

b) Total (quantitation of unesterified HEPE and esterified HEPE (e.g. as triglycerides, cholesterol esters)) concentration at each sampling time.

[0570] Incurred sample reanalysis (ISR) for unesterified HEPE was performed for this study in accordance with the appropriate bioanalytical laboratory standard operating procedure(s). ISR was successfully performed for total HEPE in rat plasma samples using the assay procedure referenced above during laboratory study #527943 and does not need to be repeated in this study.

[0571] Any residual/retained bioanalytical samples were retained for at least 6 months after publication of the draft report, after which the samples were discarded. Alternatively, residual/retained samples were discarded before the end of the 6-month period if the final report was issued before the end of the 6-month retention period. The Principal Investigator may also request and authorize the disposal of these residual/retained samples earlier than the specified timeframes.

[0572] Toxicokinetic evaluation: Toxicokinetic parameters were evaluated using Phoenix pharmacokinetic software. A single-arm approach consistent with the oral route of administration was used to estimate the parameters. All parameters were derived from the total free acid and unbound free acid concentrations of 15(S)-HEPE EE in plasma from day 1 and week 26, when appropriate. The following parameters were evaluated.

[0573] Table 30. Estimated parameters

Parameter Parameter Description Tmax Time after dosing at which maximum concentration was observed. Cmax Maximum observed concentration measured after dosing. Cmax/D Cmax divided by the administered dose. AUC(0-t) The area under the concentration-time curve from the start of dosing to the last observed concentration that can be quantified using the linear or linear log-trapezoidal method. AUC(0-t)/D AUC(0-t) divided by the administered dose.

[0574] Partial AUCs (between any given sampling time) can be obtained and reported to facilitate interpretation. Descriptive statistics (standard error for Cmax and AUC(0-t)) for relevant grouping and sorting variables were generated using Phoenix. A table and plots of the TDs were also created.

1.4 Killing procedures

[0575] The following table summarizes the euthanasia procedures used in the study.

[0576] Table 31. Euthanasia procedures for animals from the main study groups

Group number Scheduled euthanasia day Autopsy procedures Histology ^a Histopathology ^a Males Females Opening Fabric collection Weight of organs ^a 1 15 15 183/184 X X X Full fabric Full fabric 2 15 15 Macroscopic changes in target tissue ^b Macroscopic changes in target tissue ^b 3 15 15 Full fabric Full fabric 1 5- 5 211 Macroscopic changes in target tissue ^b Macroscopic changes in target tissue ^b 2 5 5 Macroscopic changes in target tissue ^b Macroscopic changes in target tissue ^b 3 5 3 Macroscopic changes in target tissue ^b％ Macroscopic changes in target tissue ^b Unplanned deaths X X - Full fabric Full fabric "X" - procedure to be performed: "-" = not applicable
^a For a list of fabrics, see the table "Collection and Preservation of Fabrics".
^b Target tissue is liver (males and females), thyroid gland (males and females), kidney (females only), sternum bone marrow (males only).

[0577] Unplanned Deaths: If an animal in the main study groups or the recovery group died during the study, a necropsy was performed and certain tissues were saved. When necessary, the body temperature of the animals was lowered to minimize autolysis. Animals in the main study groups or the recovery group were humanely euthanized in accordance with the test facility SOP. Body weights were recorded and, when possible, samples were obtained for evaluation of clinicopathological parameters. Such animals were necropsied and certain tissues were saved. When necessary, the body temperature of the animals was lowered to minimize autolysis. Animals in the toxicokinetic evaluation group that died or were humanely euthanized during the study were subjected to limited postmortem examination. The examination included evaluation of organs and tissues in the thoracic, abdominal, and pelvic cavities without saving tissue. During the limited postmortem examination, particular attention was paid to signs of possible gavage injury. Body weights were recorded and samples were collected for toxicokinetic analysis.

[0578] Scheduled euthanasia: Animals from the study and recovery groups that survived to scheduled euthanasia were euthanized with carbon dioxide, final body weights were recorded, and the animal was exsanguinated. Whenever possible, animals were sacrificed in an alternating order across dose groups such that equal numbers of animals from each group, including controls, were necropsied during the day. Animals were fed prior to scheduled necropsy. Animals from the toxicokinetic evaluation group that survived to scheduled euthanasia were euthanized by appropriate methods. Necropsies were not performed and no tissue was collected.

[0579] Necropsy: Animals from the main study and recovery groups underwent a complete necropsy that included assessment of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities and associated organs and tissues. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and general pathology.

[0580] Organ Weights: Organs specified for weighing in the Tissue Collection and Preservation table were weighed at necropsy (unless otherwise noted) for all animals scheduled for euthanasia. Organ weights were not recorded for animals found dead or euthanized in poor physical condition or moribund. Paired organs were weighed together. In the case of significant anomalies, the weight of each organ in the pair was recorded in addition to the total weight and as a comment on the corresponding tissue sample. The final body and brain weights were used for organ weight analysis.

[0581] Tissue Collection and Preservation: Tissue samples collected according to the table below were preserved in 10% neutral buffered formalin.

[0582] Table 32. Table "Collection and preservation of tissues"

Organ Weight Macroscopic assessment and collection Histological processing Microscopic evaluation Animal ID - X - - Artery, aorta - X X X Body cavity, nasal - X - - Bone marrow, breast - X X X Bone marrow smear - X ^a - - Bone, femur - X (1) X (1) X (1) Bone, sternum - X X X Brain X X X X Cervix - X X X Epididymis X (2) X (2) ^b X (2) X (2) Esophagus - X X X Eye - X (2) ^b X (2) X (2) Gland, adrenal X (2) X (2) X (2) X (2) Gland, clitoral - X (2) - - Gland, lacrimal - X (2, extraorbital) - -

Organ Weight Macroscopic assessment and collection Histological processing Microscopic evaluation Iron, Harderian - X(2) X(1) X(1) Iron, milk - X X X Gland, parathyroid - X(2) X(2) X(2) Pituitary gland X X X Preputial gland - X(2) - - Gland, prostate X X X Gland, submandibular, salivary - X(2) X(1) X(1) Gland, salivary, sublingual - X(2) - - Gland, salivary, parotid - X(2) - - Gland, seminal - X(2) X(2) X(2) Gland, thyroid/gland, parathyroid X(2) ^d - - - Gland, thyroid - X(2) X(2) X(2) Zimbal's gland - X(2) - - Intestinal lymphoid tissue ^c - X X X Heart X X X X Joint, femorotibial - X(1) X(1) X(1) Kidney X(2) X(2) X(2) X(2) Large intestine, cecum - X X X Large intestine, colon - X X X Large intestine, rectum - X X X Larynx - X - - Liver X X X X Easy - X X X Lymph node, submandibular - X(2) X(1) X(1) Lymph node, mesenteric - X X X Muscles, skeletal - X X X Nasopharynx - X - - Second cranial nerve - X(2) ^c X(2) X(2) Sciatic nerve - X(2) X(1) X(1) Ovary X(2) X(2) X(2) X(2) Fallopian tube - X(2) - - Pancreas - X X X Skin - X X X Small intestine, duodenum - X X X Small intestine, ileum - X X X Small intestine, small bowel - X X X Spinal cord - X X X Spleen X X X X Stomach - X X X Testicle X(2) X(2) X(2) X(2) Thymus X X X X Language X X X Trachea X X X Ureter X(2) ^c - - Bladder X X X Uterus/cervix X - - Uterus - X X X Vagina - X X X "X" - procedure being performed "-" - not applicable. (1) - one side. (2) - both sides.
Samples of macroscopic abnormalities in the listed organs and in other organs will be collected at autopsy, processed for histology and examined microscopically.
^a Two bone marrow smears will be collected from the femur at the scheduled and unscheduled necropsy (for possible testing). Smears will not be collected from animals that were found dead or from animals that were killed in moribundity and then refrigerated prior to necropsy. Bone marrow smears are air dried and not fixed in formalin.
^b The eyes and optic nerves are preserved in a Davidson fixator. The testes and epididymis are preserved in a modified Davidson fixator.
^c From the small intestine: Peyer's patches or solitary lymphoid follicles.
^d Weighing after fixation

1.5 Dosage form and its analysis

[0583] Control: The control for this study was 0.5% HPMC in Milli-Q water. The control was prepared as required and stored in a refrigerator at 4°C when not in use. The prepared control was removed from the refrigerator and stirred for at least 30 minutes prior to dosing. The control was also continuously stirred during dosing.

[0584] Working substance: The working substance for this study was DS102 or 15(S)-HEPE EE. The working substance formulation was prepared based on the method established at the trial site at appropriate concentrations to meet the dosage level requirements. The formulations were prepared weekly and stored in a refrigerator at 4°C. The drug was removed from the refrigerator and mixed for at least 30 minutes prior to administration. The formulation was also continuously mixed during dosing.

[0585] Sample Collection and Analysis

[0586] The assay samples were submitted within the established stability period. All assay samples were transferred at ambient temperature to the test facility analytical laboratory. Any residual/retained assay samples were stored for at least 6 months after preparation of the draft report, after which the samples were discarded. Alternatively, residual/retained samples were discarded before the end of the 6-month period if the final report was released before the end of the 6-month retention period. Earlier disposal of residual/retained samples may also be permitted by the Principal Investigator in consultation with the sponsor. Samples of the dosage form were collected for analysis as outlined in the following table. Additional samples may be collected and analyzed at the discretion of the Principal Investigator.

[0587] Table 33: Dosage Form Sampling Schedule

Interval Concentration Homogeneity Stability Day 1 All groups Groups 2-3 ^a N/A Week 2 All groups Groups 2-3 ^a N/A Week 10 Group 3 Group 3 N/A Week 13 All groups N/A N/A Week 14 All groups All groups ^a N/A Week 26 All groups N/A N/A N/A - not applicable.
^a The homogeneity assessment results obtained in the upper, middle and lower parts of the preparation of groups 2 and 3 will be averaged and used as the concentration assessment results.

[0588] Analytical method

[0589] The analysis described below was performed by UHPLC with UV detection using a validated analytical procedure.

[0590] Concentration Analysis:

Sample for analysis: Two samples each of the top, middle and bottom samples (with a duplicate middle sample as a control only) were sent for analysis. Where only concentration was assessed, only the middle samples were duplicated.

Spare samples: The top three, middle (for control only) and bottom samples are kept in the testing laboratory. Spare samples may be analysed at the discretion of the responsible performer. Where only concentration is being assessed, three samples from the middle will be taken.

Sampling vessel: Volumetric flasks of suitable size.

Sample volume:

Group 1: 0.5 ml in a 50 ml measuring flask.

Group 2: 0.25 ml in a 50 ml measuring flask.

Group 3: 0.25 ml in a 50 ml measuring flask.

The mass of all samples was recorded.

Storage conditions: store in the refrigerator at 4°C.

Acceptance criteria: The acceptance criteria were the mean concentration of the sample within ±10% of the theoretical concentration. For homogeneity, the acceptance criterion for concentrations was ≤10% relative standard deviation (RSD) for each group.

[0591] Stability Analysis: Stability analyses previously conducted in Laboratory Study No. 439414 demonstrated that the working substance was stable in the vehicle when prepared and stored under the same conditions at concentrations outside the limits used in the present study. See Rogers, E (2018). Validation of an Ultra Performance Liquid Chromatography Method for the Determination of 15(S) HEPE EE in Oral Gavage Dosage Products. Charles River Study No. 439414. Stability data were retained in the protocols of Laboratory Study No. 439414. Id .

1.6 - Test system

[0592] Animals: Male and female Sprague Dawley rats purchased from Charles River UK Limited (Margate, Kent, UK) were used in the study. The target age at initial dosing was 7-8 weeks. Target weights at initial dosing were 175-300 g (males) and 120-250 g (females). Each animal was identified by a subcutaneously implanted electronic TROVAN cylindrical glass microchip.

1.7 Animal keeping conditions

[0593] Housing: Animals were allowed to acclimate to the rodent toxicology room for up to 3 weeks prior to dosing. Animals were randomly assigned to groups. Males and females were randomized separately. Animals in poor health were not assigned to groups. Animals were separated by sex and housed 2 or 3 in appropriately sized polycarbonate/polypropylene hanging cages with a stainless steel slatted top and solid bottom. Sterilized white wood shavings were used as bedding material.

[0594] Environmental conditions: The target conditions for the animal housing were: temperature 19-23°C; humidity 40-70%; ventilation of at least 10 air changes per hour; light cycle of 12 hours light and 12 hours dark. Light cycles, temperature and humidity were automatically monitored and recorded on a continuous basis.

[0595] Diet: Rat and mouse diet (modified) #1 was provided ad libitum, and extensive quality control of the special diet was performed. Tap water was provided ad libitum via a drinking bowl. Water used in the testing facility was regularly analyzed for dissolved solids, heavy metals, pesticide residues, pH, nitrates, and nitrites. Microbiological screening was also performed.

[0596] Animal housing: Animals were housed in socialization conditions to meet psychological and environmental needs. Cages were provided with shelter and chewable items. Animals were housed continuously, with breaks for study procedures.

[0597] Veterinary care: Veterinary care was available throughout the study, and a veterinarian examined the animals based on clinical signs and other changes. All veterinary examinations and recommended therapeutic procedures, if performed, were documented in the study log.

1.8 Histology and histopathology

[0598] Histology: Tissues listed in the Tissue Collection and Preservation table were embedded in paraffin, sectioned, mounted on slides, and stained with hematoxylin and eosin.

[0599] Histopathology: Histopathology was performed by a board-certified veterinary pathologist or a veterinary pathologist with training and experience in laboratory animal pathology. Microscopic examination of tissues was performed on animals in the primary control and high-dose groups and in cases of unplanned deaths and major lesions (including animals in the primary, low-dose, and recovery groups). If treatment effects were present in the high-dose primary study group, microscopic examination of target tissues from animals in the low-dose primary and recovery groups was performed after approval by the sponsor and principal investigator through a protocol modification.

1.9 Computerized systems

[0600] Computerized Systems: The following table of computerized systems was available for use in the study:

[0601] Table 34. Critical Computerized Systems

System name Description of data collected and/or analyzed Dispense Receipt of the study subject, reporting and/or actions related to the composition and dosage form. Provantis Suitable for intravital procedures, laboratory studies and autopsies Empower 3 Collection and processing of chromatographic data (composition analysis) Analyst Chromatographic data collection (bioassay) Watson Chromatographic processing (bioanalysis) Phoenix Non-compartmental analysis and descriptive statistics (toxicokinetic assessment) Internal reporting software Nevis 2012 (using SAS) Suitable for intravital procedures, laboratory studies and autopsies Deviation Information Library Deviations Trend 963 Secure Continuous recording of temperature and humidity. Alarm for rooms with animals, refrigerators, freezers and incubators. Share Document Management System Reporting Docusign Collection of signatures in accordance with Part 11

1.10 Statistical analysis

[0602] Data collected during the pre-dose period were tabulated, summarized, and statistically analyzed. All statistical analyses were performed within the appropriate phase of the study unless otherwise noted. Numerical data collected from planned cases (excluding data from animals in the toxicokinetic evaluation group) were summarized and statistically analyzed as follows, by sex and case.

[0603] Design variables: Changes in body weight were calculated between each planned interval. Organ weight to body weight was calculated relative to final body weight for the planned intervals. Organ weight to brain weight was calculated relative to brain weight for the planned intervals.

[0604] Descriptive statistical analysis: Means, standard deviations (or percent coefficient of variation or standard error when considered appropriate), percentages, counts, and/or incidences will be reported as appropriate for the data set.

[0605] Inferential Statistical Analysis: All statistical analyses were performed at the 5% significance level. All pairwise comparisons were performed using two-tailed tests and are 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] The analysis was performed according to the matrix below when possible, but all groups with fewer than 3 observations were excluded.

[0607] Table 35. Statistical matrix

Variables for logical analysis Statistical method parametric/non-parametric Body weight X Weight gain X Hematological variables X Coagulation variables X Biochemical variables X Urine Analysis Variables X Weight of organs X Organ weight relative to body weight X Organ weight relative to brain weight X

[0608] Parametric/nonparametric: Levene's test was used to assess homogeneity of group variance. Groups were compared using an overall one-way ANOVA F-test if Levene's or Kruskal-Wallis test was not significant. If the overall F-test or Kruskal-Wallis test was significant, pairwise comparisons were made using Dunnett's test or Dunn's test, respectively.

1.11 Results

[0609] Change in Red Blood Cell Count, Red Blood Cell Distribution Width, and Reticulocyte Count: FIGS. 38A-38C show the change in red blood cell (RBC) count, red blood cell distribution width (RDW), and reticulocyte (RETIC) count in rats administered 15(s)-HEPE EE or placebo. 15(S)-HEPE EE and placebo were administered once daily by gavage 7 days per week for a minimum of 26 weeks. The volume for each animal was based on the most recent body weight measurements. As noted in Table 36, the highest dose of 6 g/kg/day was poorly tolerated and was changed to a lower dose of 3 g/kg/day.

[0610] Table 36. Change in RBC, RDW and RETIC in rats treated with 15(S)-HEPE EE

STUDY 26-week study in rats (CRL509554) Dose 15 (S)-HEPE EE (mg/kg/day) 0 (carrier) 2000 6000/3000 Red blood cell count (RBC) (10 ^A 12/L)
Average (SD) 8.38
0.42 8.67
0.41 8.81**
0.42 Red blood cell distribution width (RDW) (%)
Average
(SO) 14.98
1.23 13.91**
0.96 13.60**
0.57 Reticulocyte count (RETIC) (10^9/l)
Average (SD) 219.51
40,41 154.60*
32,31 141.46**
30.57 Anova and Dunnett: * - p≤0.05; ** - p≤0.01
All values presented were observed in males.
The highest dose of 6 g/kg/day was poorly tolerated and was replaced by a lower dose of 3 g/kg/day.

[0611] Changes in RBC: FIG. 38A and Table 36 show that administration of the 3 g/kg/day dose resulted in a significant increase in RBC count compared to control.

[0612] Changes in RDW: FIG. 38B and Table 36 show that administration of doses of 2 g/kg/day and 3 g/kg/day resulted in a significant decrease in RDW compared to the control.

[0613] Changes in RETIC: FIG. 38C and Table 36 show that administration of 2 g/kg/day and 3 g/kg/day doses resulted in a significant decrease in reactivity compared to control.

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

[0615] Change in Prothrombin Time, Activated Partial Thromboplastin Time, and Fibrinogen Concentration: FIGS. 39A-39C show the change in prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen concentration (FIB) in rats administered 15(s)-HEPE EE or placebo. 15(S)-HEPE EE and placebo were administered once daily by gavage 7 days per week for a minimum of 26 weeks. The volume for each animal was based on the most recent body weight measurements. As indicated in Table 37, the highest dose of 6 g/kg/day was poorly tolerated and was replaced by a lower dose of 3 g/kg/day.

[0616] Table 37. Change in PT, APTT and FIB concentrations in 15(S)-HEPE EE administered rats

STUDY 26-week study in rats (CRL509554) Dose 15 (S) HEPE EE
(mg/kg/day) 0 (carrier) 2000 6000/3000 Prothrombin time (PT) (sec)
Average (SD) 10.25
0.34 10.69**
0.41 10.81**
0.47 Activated Partial Thromboplastin Time (APTT) (sec) Average
(SO) 12.92
2.12 15.03*
2.64 15.73**
2.00 Fibrinogen (FIB) (g/l)
Average (SD) 2.61
0.26 2.35**
0.29 2.26**
0.24 Anova and Dunnett: * - p≤0.05; ** - p≤0.01
All values presented were observed in males.
The highest dose of 6 g/kg/day was poorly tolerated and was replaced by a lower dose of 3 g/kg/day.

[0617] Changes in PT: FIG. 39A and Table 37 show that administration of 2 g/kg/day and 3 g/kg/day resulted in significant increases in PT compared to control.

[0618] Changes in APTT: FIG. 39B and Table 37 show that administration of doses of 2 g/kg/day and 3 g/kg/day resulted in significant increases in APTT compared to control.

[0619] Change in FIB concentration: FIG. 39C and Table 37 show that administration of doses of 2 g/kg/day and 3 g/kg/day resulted in a significant decrease in FIB concentration compared to the control.

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

[0621] Various embodiments of the present disclosure are set forth below:

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

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

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

[0625] Item D. A method for treating and/or preventing metabolic syndrome and/or cardiometabolic disease in a subject in need thereof, comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein the 15-HEPE is at least about 90% by weight of all fatty acids in the composition, wherein the subject exhibits one or more of: a decrease in diglyceride levels, glycerophospholipids, liver fat, blood pressure, waist circumference, and/or free fatty acid levels; and/or an increase in glycerophospholipid levels.

[0626] Item E. A method for preventing the progression of stage 1 nonalcoholic steatohepatitis (NASH) to stage 2 NASH in a subject, comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE.

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

[0628] Item G. The method described in item E or item F, wherein the second step is increased liver fat and lipotoxicity, cellular stress apoptosis, inflammation and/or fibrogenic remodeling.

[0629] Item H. A method for treating and/or preventing cardiovascular disease in a subject suffering from non-alcoholic fatty liver disease (NAFLD), metabolic syndrome and/or cardiometabolic disease in a subject in need thereof, 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 in need thereof and suffering from non-alcoholic fatty liver disease (NAFLD), metabolic syndrome, or cardiometabolic disease, comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition.

[0631] Item J. The method of any one of items A-I, wherein the subject has a decrease in one or more of: alpha-smooth muscle actin (α-SMA) activity, inhibitor of metallopeptidase-1 (TIMP-1), transforming growth factor beta-β (TGF-β), and/or collagen type I levels.

[0632] Item K. The method of any one of items AC or EJ, wherein the subject has a decrease in diglyceride levels, liver fat, blood pressure, waist circumference, and/or free fatty acid levels and/or an increase in glycerophospholipid levels.

[0633] Item L. The method of any one of Item AK, wherein the subject has a decreased alkaline phosphatase (ALP) level.

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

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

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

[0637] Item P. The method of any one of Item HO, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).

[0638] Item Q. The method of any one of Item B-D or FP, wherein the cardiometabolic disease or cardiovascular disease is one or more of: dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, primary hypercholesterolemia, primary hyperlipidemia, total primary hyperlipidemia, prevalent hypercholesterolemia, familial hyperlipidemia, familial primary hyperlipidemia, familial hypercholesterolemia, familial hypertriglyceridemia, combined familial hyperlipidemia, familial apolipoprotein b-100 defect, secondary hyperlipidemia, mixed hyperlipidemia, cardiovascular disease, residual cardiovascular risk, prevention of atherosclerotic plaque formation/progression, 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 death from cardiovascular diseases, 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 of item Q, wherein the microvascular disease is retinopathy, nephropathy, neuropathy, or a combination thereof.

[0640] Item S. The method of item Q, wherein the macrovascular disease is stroke, peripheral vascular disease, limb ischemia, cardiac disease, or a combination thereof.

[0641] Item T. The method of any one of items A-S, wherein the subject has a decrease in very low density lipoprotein cholesterol (VLDL-C), non-high density lipoprotein cholesterol (non-HDL-C), and/or remnant particle cholesterol (RLP-C), and/or high density lipoprotein cholesterol (HDL-C).

[0642] Item U. The method of any one of AT, wherein the subject has a decrease in liver stiffness, fibrosis-4 (FIB-4), end-stage liver fibrosis (ELF) score, and/or NAFLD score (NFS).

[0643] Item V. The method of any one of Item AU, wherein the subject has a reduction in inflammatory and profibrotic proteins selected from the group consisting of plasminogen activator inhibitor-1 (PAI-1), inhibitor of metallopeptidase-1 (TIMP-1), dipeptidyl peptidase 4 (DPP4), treme-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), human leukocyte immunoglobulin receptor-like B1 (LILBR1), amine oxidase, copper-containing agents 3 (AOC3), serine proteases 2 (PRSS2), and member tumor necrosis factor ligand superfamily 11A (TNRSF11A).

[0644] Item W. A method of treating or preventing cholestatic liver disease in a subject, 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] Item Y. The method of item W or item X, wherein the cholestatic liver disease is caused by drug-induced liver injury, total parenteral nutrition (TPN), viral and alcoholic hepatitis, cholestasis secondary to systemic diseases, graft dysfunction, cholestasis after liver transplantation, pancreatitis, choledocholithiasis, Mirizzi syndrome, genetic diseases, malignancy, or a combination thereof.

[0647] Item Z. The method of item Y, wherein the malignancy is hepatocellular carcinoma, bile duct tumor, pancreatic carcinoma, or a combination thereof.

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

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

[0650] Item CC. The method of item 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. The method of item CC, wherein the kidney disease causes fibrosis.

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

[0653] Item FF. The method of any one of items BB-EE, wherein the subject has a decrease in kidney hydroxyproline levels.

[0654] Item GG. The method of any of items BB-FF, wherein the subject does not exhibit an increase in levels of α-SMA, TIMP-1, TGF-β, and/or collagen type 1.

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

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

[0657] Item JJ. The method of item II, wherein the profibrotic cytokines are one or more of α-SMA, TIMP-1, TGF-β, collagen type 1, interleukin 1β (IL-1β), interleukin-6 (IL-6), interleukin-6 (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] Item KK. The method of any one of items A-JJ, wherein the subject has a reduction in vascular adhesion molecules and/or chemokines and/or tumor necrosis factor receptor superfamily members.

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

[0660] Item MM. The method according to any of items A-LL, wherein 15-HEPE and/or 15-HEtrE are in free acid form, in esterified form or in salt form.

[0661] Item NN. The method of item 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-NN, wherein 15-HEPE comprises 15(S)-HEPE, 15(R)-HEPE, or combinations thereof and/or 15-HETRE comprises 15(S)-HETRE, 15(R)-HETRE, or combinations thereof.

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

[0665] Item RR. The method of any one of items 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-RR, wherein the composition comprises from 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 items A-TT, wherein 15-HEPE and/or 15-HETrE comprises at least about 90% by weight of all fatty acids present in the composition.

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

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

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

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

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

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

[0675] Item AC. The method of item ZZ, wherein the malignant blood disease is selected from the group consisting of lymphoma, leukemia and myeloma.

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

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

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

[0679] Item AG. A method of treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition.

[0680] Item AH. A method of treating and/or preventing a hemoglobin disorder and/or a red blood cell disorder in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition, and wherein the subject exhibits one or more of the following:

(a) increase in the number of red blood cells;

(b) a decrease in the red blood cell distribution width; and/or

(c) decrease in the number of reticulocytes.

[0681] Item AI. The method of Item AH, 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 patient in a control group not receiving the composition.

[0682] Item AJ. The method of item AI, wherein the patient in the control group has a red blood cell count of approximately 4×10 ¹² red blood cells/L.

[0683] Item AK. The method of AH, wherein the subject has a decrease in red blood cell distribution width of about 15-20%, about 20-25%, about 25-30%, about 30-35%, or about 35-40% compared to a control patient who does not receive the composition.

[0684] Item AL. The method of item AK wherein the patient in the control group has a red blood cell distribution width greater than 15%.

[0685] Item AM. The method of Item AH, wherein the subject has a decrease in reticulocyte count of at least about 5%, at least about 10%, at least about 15%, or at least about 20% compared to a patient in a control group not receiving the composition.

[0686] Item AN. The method of item AM wherein the patient in the control group has a reticulocyte count 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 up to about 8 g of a composition comprising 15-HEPE, wherein the 15-HEPE represents at least about 90% by weight of all fatty acids in the composition.

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

[0689] Item AQ. The method of item AP, wherein the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis.

[0690] Item AR. The method of item AP, wherein the acquired hemolytic anemia is selected from the group consisting of secondary and developed against the background of infection, drug therapy, hematological malignancy, autoimmune disease, hypersplenism, artificial mechanical heart valve and blood transfusion.

[0691] Item AS. The method of item AQ, wherein the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration.

[0692] Item AT. The method according to any one of the preceding items YY-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-AN, wherein the hematological 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.

[0694] Item AV. The method of any one of items YY-AG or AO-AT, wherein the subject has an increase in the red blood cell count, a decrease in the reticulocyte count, and a decrease in the red blood cell distribution width.

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

[0696] Item AX. The method of 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 of item AW, wherein the hematological disease 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 of item AY, wherein the hemolytic anemia is hereditary hemolytic anemia or acquired hemolytic anemia.

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

[0700] Item BC. The method of item AZ, wherein the acquired hemolytic anemia is selected from the group consisting of secondary to infection, drug therapy, hematological malignancy, autoimmune disease, hypersplenism, mechanical heart valve, and blood transfusion.

[0701] Item BD. The method of item BA, wherein the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration.

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

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

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

[0705] Item BH. A method of treating and/or preventing thrombophilia in a subject in need thereof, the method comprising administering to the subject up to about 8 g of a composition comprising 15-HEPE, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition, and wherein the subject exhibits one or more of:

(a) increase in prothrombin time;

(b) an increase in activated partial thromboplastin time; and/or

(c) decrease in fibrinogen concentration.

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

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

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

[0709] Item BL. The method according to items BI-BK, in which the first step involves the formation of a thrombus.

[0710] Item BM. The method of any one of items BF-BG or BI-BL, wherein the subject has an increase in prothrombin time, an increase in activated partial thromboplastin time, and a decrease in fibrinogen concentration.

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

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

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

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

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

[0716] Item BS. The method of any one of items YY-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.

Claims (18)

1. A method for increasing the red blood cell count in a subject, the method comprising administering to the subject 2 g or more of a composition comprising 15-hydroxyeicosapentaenoic acid (15-HEPE), wherein the subject exhibits an increase in red blood cell count, a decrease in red blood cell distribution width, and/or a decrease in reticulocyte count compared to a subject in a control group not receiving 15-HEPE or the composition. 2. The method according to claim 1, wherein the subject has a red blood cell disorder. 3. The method of claim 2, wherein the red blood cell disorder is selected from the group consisting of anemia, blood cancer, and a coagulation defect. 4. The method according to claim 3, wherein the anemia is hemolytic anemia. 5. The method according to claim 4, wherein the hemolytic anemia is selected from the group consisting of hereditary hemolytic anemia and acquired hemolytic anemia. 6. The method of claim 3, wherein the anemia is nutritional anemia, non-iron deficiency anemia, or a combination thereof. 7. The method according to claim 3, wherein the blood cancer is selected from the group consisting of lymphoma, leukemia and myeloma. 8. The method of claim 3, wherein the coagulation defect is selected from the group consisting of thrombophilia, hemophilia, von Willebrand disease, and thrombocytopenia. 9. The method of claim 2, wherein the red blood cell disorder is selected from the group consisting of Fanconi anemia, iron deficiency anemia, folate deficiency, B12 deficiency, and myelodysplastic syndrome. 10. The method of claim 5, wherein the hereditary hemolytic anemia is selected from the group consisting of sickle cell disease, sickle cell anemia, β-thalassemia, and hereditary spherocytosis. 11. The method of claim 5, wherein the acquired hemolytic anemia develops as a result of a disorder or condition selected from the group consisting of a condition secondary to a primary infection, drug administration, hematological malignancy, autoimmune disease, hypersplenism, mechanical artificial heart valve, and blood transfusion. 12. The method of claim 10, wherein the sickle cell disease and sickle cell anemia are associated with sickle cell crisis, vaso-occlusive crisis, and/or splenic sequestration. 13. The method of any one of the preceding claims, wherein 15-HEPE comprises at least about 90% by weight of all fatty acids in the composition. 14. The method according to any of the preceding claims, wherein the composition is administered in an amount of from 1 to 8 capsules per day. 15. The method of any one of the preceding claims, wherein the composition is formulated to provide the subject with about 5 mg 15-HEPE per kg body weight (mg/kg), about 50 mg/kg, about 250 mg/kg, or about 500 mg/kg. 16. The method according to any of the preceding claims, wherein the composition containing 15-HEPE is administered orally. 17. The method according to any one of the preceding claims, wherein 15-HEPE is in free acid form, esterified form, conjugate form or salt form. 18. The method according to any one of the preceding claims, wherein the esterified form of 15-HEPE is a methyl ester, an ethyl ester, or a combination thereof.

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