Elovanoid hydroxylated derivatives as very-long-chain polyunsaturated fatty acids, and methods of use

Abstract

To provide elovanoid hydroxylated derivatives as very-long-chain polyunsaturated fatty acids, and methods of use.SOLUTION: The present invention provides methods of use related to omega-3 very-long-chain polyunsaturated fatty acids (n-3 VLC-PUFA) and their hydroxylated derivatives known as elovanoids to alleviate a symptom of, treat, or prevent a disease, and further provides compositions and methods for modulating VLC-PUFA bioactivity and availability.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 895,737, filed September 4, 2019, U.S. Provisional Patent Application No. 62 / 923,770, filed October 21, 2019, U.S. Provisional Patent Application No. 62 / 924,359, filed October 22, 2019, and U.S. Provisional Patent Application No. 62 / 964,995, filed January 23, 2020, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Contracts EY005121, NS104117 and NS109221 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] Field of Disclosure This disclosure relates to methods of use related to omega-3 very long chain polyunsaturated fatty acids (n-3 VLC-PUFAs) known as aerovanoids and their hydroxylated derivatives for alleviating symptoms of, treating, or preventing disease. Additionally, the present invention is directed to compositions and methods for modulating the biological activity and availability of VLC-PUFAs. [Background technology]

[0004] background Long-chain polyunsaturated fatty acids (LC-PUFAs) include omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids containing 18–22 carbons, including arachidonic acid (ARA, C20:4n-6, i.e., 20 carbons, 4 double bonds, omega-6), eicosapentaenoic acid (EPA, C20:5n-3, 20 carbons, 5 double bonds, omega-3), docosapentaenoic acid (DPA, C22:5n-3, 22 carbons, 5 double bonds, omega-3), and docosahexaenoic acid (DHA, C22:6n-3, 22 carbons, 6 double bonds, omega-3). LC-PUFAs are converted via lipoxygenase-type enzymes to biologically active hydroxylated PUFA derivatives that function as biologically active lipid mediators that play an important role in inflammation and related conditions. Among these, the most important are hydroxylated derivatives generated in certain inflammation-related cells through the action of lipoxygenase (LO or LOX) enzymes (e.g., 15-LO, 12-LO), resulting in the formation of mono-, di-, or trihydroxylated PUFA derivatives with potent activities, including anti-inflammatory, inflammation-resolving, neuroprotective, or tissue-protective activities, among others. For example, neuroprotectin D1 (NPD1), a dihydroxy derivative of DHA formed intracellularly through the enzymatic action of 15-lipoxygenase (15-LO), has been shown to possess a unique biological profile that may include a defined R / S and Z / E stereochemical structure (10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) and stereoselective potent anti-inflammatory, homeostatic restoration, inflammation-resolving, and bioactivity. NPD1 has been shown to regulate neuroinflammatory signaling and proteostasis, promoting neuroregeneration, neuroprotection, and cell survival. Summary of the Invention

[0005] Disclosure Overview An embodiment of the present disclosure is directed to a method for alleviating symptoms of, treating, or preventing an allergic inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of a VLC-PUFA to the subject.

[0006] Furthermore, aspects of the present disclosure are directed to a method for treating or preventing disease symptoms by regulating cellular senescence, ferroptosis, or cellular senescence and ferroptosis, comprising administering a therapeutically effective amount of VLC-PUFA to a subject.In embodiments, the disease comprises neurodegenerative disease.In embodiments, the disease comprises Aβ-related disease.For example, the disease can be Alzheimer's disease or age-related macular degeneration.

[0007] Furthermore, embodiments of the present disclosure are directed to a method for alleviating symptoms of, treating, or preventing a metabolic disorder in a subject, the method comprising administering a therapeutically effective amount of a VLC-PUFA to the subject. For example, the metabolic condition includes obesity or diabetes.

[0008] Aspects of the present disclosure are directed to a method for alleviating the symptoms of, treating, or preventing allergic inflammatory disease in a subject.In embodiments, the allergic inflammatory disease is indicated by the increased production of proinflammatory cytokines and chemokines by cells.For example, the allergic inflammatory disease includes allergic rhinitis, allergic conjunctivitis, allergic dermatitis, or asthma.

[0009] For example, the proinflammatory cytokines and chemokines include at least one of IL-6, IL-1β, IL-8 / CXCL8, CCL2 / MCP-1, CXCL1 / KC / GRO, VEGF, ICAM1 (CD54).

[0010] For example, the cell comprises an epithelial cell. Non-limiting examples of epithelial cells include respiratory epithelial cells (such as human nasal epithelial cells), corneal epithelial cells, or skin epithelial cells.

[0011] In embodiments, the methods include administering to the subject a therapeutically effective amount of a VLC-PUFA, hi embodiments, the VLC-PUFA inhibits the production of pro-inflammatory cytokines and chemokines.

[0012] In an embodiment, the VLC-PUFA compound may be selected from the group consisting of Formula A or B: TIFF2025143368000002.tif35128

[0013] In embodiments, the VLC-PUFA compound may be selected from the group consisting of: TIFF2025143368000003.tif72128

[0014] In embodiments, the VLC-PUFA is provided as a pharmaceutical composition, including, for example, a topical, intranasal, oral, or parenteral composition.

[0015] In embodiments, the VLC-PUFA or pharmaceutical composition is administered topically, orally, intranasally, or parenterally.

[0016] In embodiments, a therapeutically effective amount includes a concentration of about 500 nM, a concentration greater than about 500 nM, or a concentration less than about 500 nM.

[0017] In embodiments, the VLC-PUFA is administered before exposure to the allergen, at about the same time as exposure to the allergen, or after exposure to the allergen.

[0018] In embodiments, the allergen causes an allergic inflammatory disease in the subject, for example, the allergen causes cells to increase the production of pro-inflammatory cytokines and chemokines, decrease the production of anti-inflammatory cytokines and chemokines, or both.

[0019] Embodiments of the present disclosure are also directed to methods of treating allergic rhinitis, comprising administering a therapeutically effective amount of a VLC-PUFA to a subject, for example, the VLC-PUFA is administered intranasally.

[0020] Additionally, aspects of the present disclosure are directed to methods of treating allergic conjunctivitis, comprising administering a therapeutically effective amount of a VLC-PUFA to a subject. In embodiments, the VLC-PUFA is administered topically, such as to the eye using eye drops.

[0021] Furthermore, aspects of the present disclosure are directed to methods for treating allergic dermatitis, comprising administering to a subject a therapeutically effective amount of a VLC-PUFA. In embodiments, the VLC-PUFA is administered topically, such as in a cream, spray, or gel.

[0022] Aspects of the present disclosure are also directed to methods of treating asthma comprising administering a therapeutically effective amount of a VLC-PUFA to a subject. In embodiments, the VLC-PUFA is administered intranasally.

[0023] Aspects of the present disclosure are also directed to methods for alleviating, treating, or preventing an inflammatory response in epithelial tissue, comprising contacting the epithelial tissue with a therapeutically effective amount of VLC-PUFA. In embodiments, the inflammatory response is indicated by increased production of pro-inflammatory cytokines and chemokines by cells, decreased production of anti-inflammatory cytokines and chemokines, or both. For example, the pro-inflammatory cytokines and chemokines include at least one of IL-6, IL-1β, IL-8 / CXCL8, CCL2 / MCP-1, CXCL1 / KC / GRO, VEGF, and ICAM1 (CD54). For example, the anti-inflammatory molecule includes IL-10.

[0024] In embodiments, the VLC-PUFAs inhibit the production of pro-inflammatory cytokines and chemokines, enhance the production of anti-inflammatory molecules, or both.

[0025] Provided herein are compounds and pharmaceutical compositions containing omega-3 very long chain polyunsaturated fatty acids (n-3 VLC-PUFAs) and / or their endogenous hydroxylated derivatives known as aerovanoids. The present disclosure also provides methods for alleviating symptoms, treating, or preventing allergic inflammatory diseases in a subject.

[0026] In vivo, n-3 VLC-PUFAs are converted into several unknown types of VLC-PUFA hydroxylated derivatives called elovanoids (ELVs), which can protect and prevent progressive damage to tissues and organs whose functional integrity has been disrupted. Without wishing to be bound by theory, ELVs can suppress the production of pro-inflammatory cytokines and chemokines, and thus alleviate the symptoms of, treat, or prevent allergic inflammatory diseases in subjects.

[0027] By providing specific compounds related to n-3 VLC-PUFAs and their corresponding elovanoids (ELVs), the production of pro-inflammatory cytokines and chemokines can be effectively suppressed. Therefore, the present disclosure is related to the prevention and treatment of allergic inflammatory diseases.

[0028] The present disclosure provides compounds, compositions, and methods that can promote protection, prevention, and treatment of damage in many organs caused by proinflammatory cytokines. For example, the present disclosure provides compounds, compositions, and methods that can suppress the production of proinflammatory cytokines and chemokines from epithelial cells, such as nasal epithelial cells, thereby alleviating the symptoms of, treating, or preventing allergic inflammatory diseases.

[0029] Thus, one aspect of the present disclosure includes an embodiment of a composition comprising at least one omega-3 very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.

[0030] In some embodiments of this aspect of the disclosure, the composition comprises at least one n-3 VLC-PUFA having at least 23 carbon atoms in its carbon chain, and the n-3 VLC-PUFA may be in the form of a carboxylic acid, a carboxylic acid ester, a carboxylic acid salt, or a phospholipid derivative.

[0031] In some embodiments of this aspect of the disclosure, the n-3 VLC-PUFA compound can be selected from the group consisting of Formula A or B: TIFF2025143368000004.tif35128In the formula, m can be 0 to 19, -CO-OR can be a carboxylic acid group, or a salt or ester thereof, and when -CO-OR can be a carboxylic acid group, compound A or B can be a salt thereof, and the cation of the salt can be a pharmaceutically acceptable cation, and when -CO-OR can be an ester, R can be an alkyl group.

[0032] In some other embodiments, the n-3 VLC-PUFA compound in the present disclosure can be in the form of a phospholipid selected from the group consisting of Formula C, D, E, or F, wherein m can be 0-19. TIFF2025143368000005.tif71128

[0033] In some embodiments of this aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and be formulated for delivery of at least one omega-3 very long chain polyunsaturated fatty acid in an amount effective to reduce tissue pathology in a recipient subject or the development of tissue pathology in the recipient subject.

[0034] In some embodiments of this aspect of the disclosure, the pathological condition can be an allergic inflammatory disease of the recipient subject. For example, the allergic inflammatory disease can be allergic rhinitis, allergic conjunctivitis, allergic dermatitis, or asthma.

[0035] In some embodiments of this aspect of the disclosure, the composition may be formulated for local delivery of at least one very long chain polyunsaturated fatty acid to the skin of a recipient subject or to the eye of a recipient subject, such as in eye drops.

[0036] In some embodiments of this aspect of the disclosure, the composition may be formulated for intranasal delivery of at least one very long chain polyunsaturated fatty acid to the nasal tissues of a recipient subject.

[0037] In some embodiments of this aspect of the disclosure, the composition may be formulated for oral or parenteral delivery of at least one very long chain polyunsaturated fatty acid to a recipient subject.

[0038] In some embodiments of this aspect of the disclosure, the at least one omega-3 very long chain polyunsaturated fatty acid can have from about 26 to about 42 carbon atoms in its carbon chain.

[0039] In some embodiments of this aspect of the disclosure, the at least one omega-3 very long chain polyunsaturated fatty acid can have 32 or 34 carbon atoms in its carbon chain.

[0040] In some embodiments of this aspect of the disclosure, the omega-3 very long chain polyunsaturated fatty acid can have five or six alternating double bonds in its carbon chain with a cis configuration.

[0041] In some embodiments of this aspect of the disclosure, the omega-3 very long chain polyunsaturated fatty acid is (14Z,17Z,20Z,23Z,26Z,29Z)-dotriaconta-14,17,20,23,26,29-hexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z)-tetratriaconta-16,19,22,25,28,31-hexaenoic acid.

[0042] In some embodiments of this aspect of the disclosure, the at least one omega-3 very long chain polyunsaturated fatty acid can be (14Z,17Z,20Z,23Z,26Z,29Z)-dotriaconta-14,17,20,23,26,29-hexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z)-tetratriaconta-16,19,22,25,28,31-hexaenoic acid.

[0043] Another aspect of the present disclosure includes an embodiment of a composition comprising at least one erovanoid having at least 23 carbon atoms in its carbon chain.

[0044] In some embodiments of this aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and may be formulated for delivery of an amount of at least one elastinoid effective to alleviate the symptoms of, prevent, or relieve a pathological condition in the tissue of a recipient subject.

[0045] In some embodiments of this aspect of the disclosure, the pathological condition can be an allergic inflammatory disease of the recipient subject, such as allergic rhinitis, allergic conjunctivitis, allergic dermatitis, or asthma.

[0046] In some embodiments of this aspect of the disclosure, the composition may be formulated for local delivery of at least one elastinoid to the skin of a recipient subject or to the eye of a recipient subject, such as by eye drops.

[0047] In some embodiments of this aspect of the disclosure, the composition may be formulated for intranasal delivery of at least one estrogen to the nasal tissues of a recipient subject.

[0048] In some embodiments of this aspect of the disclosure, the composition may be formulated for oral or parenteral delivery of at least one elastinoid to a recipient subject.

[0049] In some embodiments of this aspect of the disclosure, the at least one elovanoid can be selected from the group consisting of a monohydroxylated elovanoid, a dihydroxylated elovanoid, an alkynyl monohydroxylated elovanoid, and an alkynyl dihydroxylated elovanoid, or any combination thereof.

[0050] In some embodiments of this aspect of the disclosure, the at least one elovanoid can be a combination of elovanoids, such as a monohydroxylated elovanoid and a dihydroxylated elovanoid; a monohydroxylated elovanoid and an alkynyl monohydroxylated elovanoid; a monohydroxylated elovanoid and an alkynyl dihydroxylated elovanoid; a dihydroxylated elovanoid and an alkynyl monohydroxylated elovanoid; a dihydroxylated elovanoid and an alkynyl dihydroxylated elovanoid; a monohydroxylated elovanoid, a dihydroxylated elovanoid, and an alkynyl monohydroxylated elovanoid; a monohydroxylated elovanoid, a dihydroxylated elovanoid, and an alkynyl monohydroxylated elovanoid; a monohydroxylated elovanoid, a dihydroxylated elovanoid, and an alkynyl monohydroxylated elovanoid. and alkynyl dihydroxylated erovanoids; and monohydroxylated erovanoids, dihydroxylated erovanoids, and alkynyl monohydroxylated erovanoids, alkynyl dihydroxylated erovanoids, wherein each erovanoid is independently a racemic mixture, an isolated enantiomer, or a combination of enantiomers in which the amount of one enantiomer exceeds the amount of another; and each erovanoid is independently a diastereomeric mixture, an isolated diastereomer, or a combination of diastereomers in which the amount of one diastereomer exceeds the amount of another diastereomer.

[0051] In some embodiments of this aspect of the disclosure, the monohydroxylated erovanoid can be selected from the group consisting of formula G, H, I, or J. TIFF2025143368000006.tif37128In the formula, m can be 0 to 19, -CO-OR can be a carboxylic acid group, or a salt or ester thereof, and when -CO-OR can be a carboxylic acid group, compound G, H, I, or J can be a salt thereof, and the cation of the salt can be a pharmaceutically acceptable cation, and when -CO-OR can be an ester, R can be an alkyl group.

[0052] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of enantiomers G and H, where the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.

[0053] In some embodiments of this aspect of the disclosure, the composition can include amounts of enantiomers I and J, where the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.

[0054] In some embodiments of this aspect of the disclosure, the composition can include one of the enantiomers of G or H in an amount that exceeds the amount of the other enantiomer of G or H.

[0055] In some embodiments of this aspect of the disclosure, the composition can include one of the I or J enantiomers in an amount that exceeds the amount of the other of the I or J enantiomers.

[0056] In some embodiments of this aspect of the disclosure, the dihydroxylated erovanoid can be selected from the group consisting of formulas K, L, M, and N. wherein m is 0 to 19; -CO-OR is a carboxylic acid group, or a salt or ester thereof; when -CO-OR is a carboxylic acid group, compounds K, L, M, or N may be a salt thereof, and the cation of the salt may be a pharmaceutically acceptable cation; when -CO-OR is an ester, R is an alkyl group; compounds K and L each have a total of 23 to 42 carbon atoms in a carbon chain having four cis carbon-carbon double bonds beginning at positions n-3, n-7, n-15, and n-18 and two trans carbon-carbon double bonds beginning at positions n-9 and n-11; and compounds M and N each have a total of 23 to 42 carbon atoms in a carbon chain having three cis carbon-carbon double bonds beginning at positions n-3, n-7, and n-15 and two trans carbon-carbon double bonds beginning at positions n-9 and n-11.

[0057] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers K and L, where the diastereomers have either (S) or (R) chirality at position n-6 and (R) chirality at position n-13.

[0058] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of one or more diastereomers K and L, where the diastereomers have either (S) or (R) chirality at position n-6 and either (S) or (R) chirality at position n-13.

[0059] In some embodiments of this aspect of the disclosure, the composition can include one of the K or L diastereomers in an amount that exceeds the amount of the other of the K or L diastereomers.

[0060] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers M and N, where the diastereomers have either (S) or (R) chirality at position n-6 and (R) chirality at position n-13.

[0061] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of one or more diastereomers M and N, where the diastereomers have either (S) or (R) chirality at position n-6 and either (S) or (R) chirality at position n-13.

[0062] In some embodiments of this aspect of the disclosure, the composition can include one of the diastereomers of M or N in an amount that exceeds the amount of the other diastereomer of M or N.

[0063] In some embodiments of this aspect of the disclosure, the alkynyl monohydroxylated erovanoid can be selected from the group consisting of formula O, P, Q, or R. TIFF2025143368000008.tif37128 wherein m can be 0 to 19; -CO-OR can be a carboxylic acid group, or a salt or ester thereof; when -CO-OR can be a carboxylic acid group, compound O, P, Q, or R can be a salt thereof, and the cation of the salt can be a pharmaceutically acceptable cation; when -CO-OR can be an ester, R can be an alkyl group; compounds O and P can be at positions n-3, n-12, n-15, and and n-18, four cis carbon-carbon double bonds beginning at position n-7, and a carbon-carbon triple bond beginning at position n-9, for a total of 23 to 42 carbon atoms in the carbon chain; and compounds Q and R each have three cis carbon-carbon double bonds beginning at positions n-3, n-12, and n-15, a trans carbon-carbon double bond beginning at position n-7, and a carbon-carbon triple bond beginning at position n-9, for a total of 23 to 42 carbon atoms in the carbon chain.

[0064] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of enantiomers O and P, where the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.

[0065] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of enantiomers Q and R, where the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.

[0066] In some embodiments of this aspect of the disclosure, the composition can include one of the O or P enantiomers in an amount that exceeds the amount of the other O or P enantiomer.

[0067] In some embodiments of this aspect of the disclosure, the composition can include one of the enantiomers of Q or R in an amount that exceeds the amount of the other enantiomer of Q or R.

[0068] In some embodiments of this aspect of the disclosure, the erovanoid can be an alkynyl dihydroxylated erovanoid selected from the group consisting of formula S, T, U, or V. TIFF2025143368000009.tif34128m can be 0 to 19, -CO-OR can be a carboxylic acid group, or a salt or ester thereof, and when -CO-OR can be a carboxylic acid group, compounds S, T, U, or V can be a salt thereof, and the cation of the salt can be a pharmaceutically acceptable cation, and when -CO-OR can be an ester, R can be an alkyl group, and compounds S and T can each have three aryl groups starting at positions n-3, n-15, and n-18. Compounds U and V each have a cis carbon-carbon double bond, two trans carbon-carbon double bonds beginning at positions n-9 and n-11, and a carbon-carbon triple bond beginning at position n-7, totaling 23 to 42 carbon atoms in the carbon chain; compounds U and V each have two cis carbon-carbon double bonds beginning at positions n-3 and n-15, two trans carbon-carbon double bonds beginning at positions n-9, n-11, and a carbon-carbon triple bond beginning at position n-7, totaling 23 to 42 carbon atoms in the carbon chain.

[0069] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers S and T, where the diastereomers have either (S) or (R) chirality at position n-6 and (R) chirality at position n-13.

[0070] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of one or more diastereomers S and T, where the diastereomers have either (S) or (R) chirality at position n-6 and either (S) or (R) chirality at position n-13.

[0071] In some embodiments of this aspect of the disclosure, the composition can include one of the S or T diastereomers in an amount that exceeds the amount of the other of the S or T diastereomers.

[0072] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers U and V, where the diastereomers have either (S) or (R) chirality at position n-6 and (R) chirality at position n-13.

[0073] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of one or more diastereomers U and V, where the diastereomers have either (S) or (R) chirality at position n-6 and either (S) or (R) chirality at position n-13.

[0074] In some embodiments of this aspect of the disclosure, the composition can include one of the diastereomers of U or V in an amount that exceeds the amount of the other diastereomer of U or V.

[0075] Aspects of the present invention are further directed to peptide analogs that bind to epitopes of the molecular targets in the tables contained herein.

[0076] In embodiments, the peptide analog modulates cellular senescence, ferroptosis, or cellular senescence and ferroptosis.

[0077] Further aspects of the present invention are directed to methods for treating a disease by administering a peptide analog described herein to a subject. For example, embodiments are directed to methods for treating a disease by modulating cellular senescence, ferroptosis, or cellular senescence and ferroptosis. In embodiments, the method comprises administering an elovanoid or a peptide analog thereof to a subject suffering from or at risk of a disease. In embodiments, the elovanoid or peptide analog binds to an epitope of a molecular target as described herein.

[0078] Embodiments of the present invention are further directed to methods for treating cellular senescence, ferroptosis, or diseases associated with cellular senescence and ferroptosis, for example, the methods comprising targeting at least one molecular target with an erovanoid or peptide analog thereof. [The present invention 1001] A method for alleviating symptoms of, treating, or preventing an allergic inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of a VLC-PUFA. [The present invention 1002] A method for alleviating symptoms of, treating, or preventing a disease by regulating cellular senescence, ferroptosis, or cellular senescence and ferroptosis, the method comprising administering a therapeutically effective amount of a VLC-PUFA to a subject. [The present invention 1003] A method for alleviating the symptoms of, treating, or preventing a metabolic disorder in a subject, comprising administering to the subject a therapeutically effective amount of a VLC-PUFA. [The present invention 1004] The VLC-PUFA compound has Formula A or B: The method of the present invention 1001, the method of the present invention 1002, or the method of the present invention 1003, which may be selected from the group consisting of TIFF2025143368000010.tif38128. [The present invention 1005] The VLC-PUFA compound is selected from the group consisting of: The method of the present invention 1001, the method of the present invention 1002, or the method of the present invention 1003, which may be selected from the group consisting of TIFF2025143368000011.tif73128. [The present invention 1006] The method of invention 1001, invention 1002, or invention 1003, wherein the VLC-PUFA is provided as a pharmaceutical composition. [The present invention 1007] 1006. The method of claim 1006, wherein said pharmaceutical composition comprises a topical, intranasal, oral, or parenteral composition. [The present invention 1008] 1007. The method of claim 1007, wherein the topical composition comprises a cream. [The present invention 1009] The method of invention 1001, invention 1002, or invention 1003, wherein the VLC-PUFA is administered topically, orally, intranasally, or parenterally. [The present invention 1010] The method of invention 1001, invention 1002, or invention 1003, wherein said therapeutically effective amount comprises a concentration of about 500 nM, a concentration greater than about 500 nM, or a concentration less than about 500 nM. [The present invention 1011] The method of claim 1006, wherein said pharmaceutical composition further comprises one or more additional active agents. [The present invention 1012] 1011. The method of claim 10, wherein said one or more additional active agents comprises at least one antioxidant. [The present invention 1013] 1001. The method of claim 1001, wherein said allergic inflammatory disease is indicated by increased production of pro-inflammatory cytokines and chemokines by cells. [The present invention 1014] 1013. The method of claim 1013, wherein said proinflammatory cytokines and chemokines comprise at least one of IL-6, IL-1β, IL-8 / CXCL8, CCL2 / MCP-1, CXCL1 / KC / GRO, VEGF, ICAM1 (CD54). [The present invention 1015] The method of claim 1013, wherein said VLC-PUFAs inhibit the production of pro-inflammatory cytokines and chemokines. [The present invention 1016] 1014. The method of claim 1013, wherein said cells comprise epithelial cells. [The present invention 1017] 1016. The method of claim 1016, wherein said epithelial cells comprise human nasal epithelial cells. [The present invention 1018] 1017. The method of claim 1017, wherein said epithelial cells comprise nasal epithelial cells, corneal epithelial cells, skin epithelial cells, or respiratory epithelial cells. [The present invention 1019] 1001. The method of claim 1001, wherein said VLC-PUFA is administered before exposure to an allergen, at about the same time as exposure to an allergen, or after exposure to an allergen. [The present invention 1020] The method of claim 1019, wherein said allergen causes an allergic inflammatory disease in the subject. [The present invention 1021] The method of claim 1019, wherein said allergen causes an increase in the production of proinflammatory cytokines and chemokines by cells. [The present invention 1022] 1021. The method of claim 1021, wherein said cells comprise epithelial cells. [The present invention 1023] 1023. The method of claim 1022, wherein said epithelial cells comprise human nasal epithelial cells. [The present invention 1024] 1021. The method of claim 1021, wherein said epithelial cells comprise nasal epithelial cells, corneal epithelial cells, skin epithelial cells, or respiratory epithelial cells. [The present invention 1025] The method of invention 1001 or invention 1020, wherein said allergic inflammatory disease comprises allergic rhinitis, allergic conjunctivitis, allergic dermatitis, asthma. [The present invention 1026] 1002. The method of claim 1002, wherein the disease comprises a neurodegenerative disease. [The present invention 1027] 1002. The method of claim 10, wherein the disease comprises an Aβ-related disease. [The present invention 1028] 1002. The method of claim 1002, wherein the disease comprises Alzheimer's disease or age-related macular degeneration. [The present invention 1029] The method of claim 1003, wherein the metabolic condition comprises obesity or diabetes. [Brief explanation of the drawings]

[0079] The present disclosure focuses on compounds, compositions, and methods for administering to alleviate the symptoms of, prevent, or treat disease in subjects.For example, this disease is an inflammatory disease such as allergic inflammatory disease.In another example, this disease is a disease related to cellular senescence, ferroptosis, or both, including Aβ-related disease, including age-related macular degeneration or Alzheimer's disease.In yet another example, this disease is a metabolic disorder such as diabetes, obesity, or both.

[0080] Further aspects of the present disclosure will be readily appreciated from consideration of the following detailed description of various embodiments thereof when taken in conjunction with the accompanying drawings.

[0081] [Figure 1] FIG. 1 is a scheme illustrating the biosynthesis of elovanoides (ELVs) from omega-3 (n-3 or n-3) very long-chain polyunsaturated fatty acids (n-3 VLC-PUFAs). [Figure 2] 1 is a scheme illustrating the biosynthesis of n-3 VLC-PUFAs. [Figure 3-1]Figures 3A–3K show the production and structural properties of the erovanoids ELV-N-32 and ELV-N-34 from cultured primary human retinal pigment epithelial cells (RPE). Figure 3A is a scheme illustrating the synthesis of ELV-N-32 and ELV-N-34 from intermediates (1, 2, and 3), each of which was prepared in stereochemically pure form. The stereochemistry of intermediates 2 and 3 was predefined using enantiomerically pure epoxide starting materials. The final ELV (4) was coupled via iterative coupling of intermediates 1, 2, and 3 and isolated as a methyl ester (Me) or sodium salt (Na). Figure 3B shows the elution profile of ELV-N-32 as indicated by C32:6n-3, endogenous monohydroxy-C32:6n-3, and ELV-N-32 standards. The MRM of ELV-N-32 showed two large peaks eluting earlier than the peak eluted by standard ELV-N-32, with the same fragmentation pattern (shown in the inset spectrum), indicating that they are isomers. Figure 3C shows the chromatograms of the full daughter scans of ELV-N-32 and ELV-N-34. [Figure 3-2] Figures 3A-3K show the production and structural properties of the erovanoids ELV-N-32 and ELV-N-34 from cultured primary human RPE. Figure 3D shows the fragmentation pattern of ELV-N-32. Figure 3E shows the elution profiles of C34:6n-3 and ELV-N-34. Figure 3F shows the UV spectrum of endogenous ELV-N-34, which exhibits triene characteristics similar to NPD1. The λmax is 275 nm, with shoulders at 268 and 285 nm. [Figure 3-3]Figures 3A-3K show the production and structural properties of the erovanoids ELV-N-32 and ELV-N-34 from cultured primary human RPE. Figure 3G shows the fragmentation pattern of ELV-N-32. Figure 3H shows the full fragmentation spectrum of endogenous ELV-N-32. Figure 3I shows that the ELV-N-32 standard matches all major peaks from the standard with endogenous peaks. However, endogenous ELV-N-32 contains many fragments not visible in the standard, indicating that it may contain various isomers. [Figure 3-4] Figures 3A-3K show the production and structural properties of the erogenous erogenous rhesus monkeys ELV-N-32 and ELV-N-34 from cultured primary human RPE. Figure 3J shows the full fragmentation spectrum of the endogenous ELV-N-34 peak, consistent with standard ELV-N-34. Figure 3K demonstrates the presence of ELV-N-34 isomers. [Figure 4-1] Figures 4A–4K show the structural properties of the erovanoids ELV-N-32 and ELV-N-34 from neuronal cell cultures. Cortical mixed neuronal cells were incubated with 10 μM of 32:6n-3 and 34:6n-3, respectively, under OGD conditions. Figure 4A shows the synthesis of ELV-N-32 and ELV-N-34 from intermediates (a, b, and c), each of which was prepared in stereochemically pure form. The stereochemistry of intermediates b and c was predefined using enantiomerically pure epoxide starting materials. The final ELV (d) was coupled via iterative coupling of intermediates a, b, and c and isolated as a methyl ester (Me) or sodium salt (Na). Figure 4B shows the 32:6n-3, endogenous monohydroxy-32:6, ELV-N-32, and ELV-N-32 standard in the insert. The MRM of ELV-N-32 shows two large peaks eluting earlier than the peaks eluting with standard ELV-N-32, but with the same fragmentation pattern, indicating that they are isomers. Figure 4C shows the same features as Figure 4A for 34:6n-3 and ELV-N-34. [Figure 4-2] Figures 4A–4K show the structural properties of the erovanoids ELV-N-32 and ELV-N-34 from neuronal cell cultures. Cortical mixed neuronal cells were incubated with 10 μM of 32:6n-3 and 34:6n-3, respectively, under OGD conditions. Figure 4D shows that the UV spectrum of endogenous ELV-N-32 exhibits triene features, although these are not clear at this concentration. Figure 4E shows the complete fragmentation spectrum of endogenous ELV-N-32. Figure 4F shows the UV spectrum of endogenous ELV-N-34, which exhibits triene features similar to NPD1. λmax is 275 nm, with shoulders at 268 and 285 nm. Figure 4G shows the fragmentation pattern of endogenous ELV-N-34. [Figure 4-3] Figures 4A-4K show the structural properties of the erovanoids ELV-N-32 and ELV-N-34 from neuronal cell cultures. Cerebral cortical mixed neuronal cells were incubated with 10 μM of 32:6n-3 and 34:6n-3, respectively, under OGD conditions. Figure 4H shows the complete fragmentation pattern of endogenous ELV-N-32. Figure 4I shows that the ELV-N-34 standard matches all major peaks from the standard, but not all, with the endogenous peaks. Endogenous ELV-N-34 contains many fragments not visible in the standard. While not wishing to be bound by theory, this indicates that it may contain isomers. [Figure 4-4] Figures 4A-4K show the structural properties of the erovanoids ELV-N-32 and ELV-N-34 from neuronal cell cultures. Cortical mixed neuronal cells were incubated with 10 μM of 32:6n-3 and 34:6n-3, respectively, under OGD conditions. Figure 4J shows the full fragmentation spectrum of ELV-N-34. The endogenous ELV-N-34 peaks match those of standard ELV-N-34. Figure 4K demonstrates the presence of ELV-N-34 isomers. [Figure 5]Figures 5A and 5B show the detection of ELV-N-32 and ELV-N-34 in neuronal cell cultures. Cells were incubated with 5 μM C32:6n-3 and C34:6n-3, respectively, under OGD conditions. Figure 5A shows that VLC-PUFA C32:6n-3, endogenous 27-hydroxy-32:6n-3, endogenous 27,33-dihydroxy-32:6n-3 (ELV-N-32), and synthetic ELV-N-32 were prepared in stereochemically pure form by stereocontrolled total organic synthesis. The MRM of endogenous ELV-N-32 closely matches that of the synthetic ELV-N-32 standard. Figure 5B shows the same characteristics as in Figure 5A for C34:6n-3 and ELV-N-34, with multiple peaks in the ELV-N-34 MRM indicating isomers. [Figure 6] Scheme 1 shows the total synthesis of monohydroxylated erovanoids G, H, I, J, O, P, Q, and R. Reagents and conditions: (a) catecholborane, heat; (b) N-iodo-succinimide, MeCN; (c) 4-chlorobut-2-yn-1-ol, Cs2CO3, NaI, CuI, DMF; (d) CBr4, PPh3, CHCl2, 0 °C; (e) ethynyl-trimethylsilane, CuI, NaI, K2CO3, DMF; (f) Lindlar's catalyst, H2, EtOAc; (g) Na2CO3, MeOH; (h) Pd(PPh3)4, CuI, Et3N; (i) tBu4NF, THF; (j) Lindlar's catalyst, H2, EtOAc or Zn(Cu / Ag), MeOH; (k) NaOH, THF, HO, then acidified with HCl / HO; (l) NaOH, KOH, etc., or amines, imines, etc. [Figure 7]Scheme 2 shows the total synthesis of dihydroxylated erovanoids K, L, S, and T. Reagents and conditions: (a) CuI, NaI, K2CO3, DMF; (b) camphorsulfonic acid (CSA), CHCl2, MeOH, room temperature; (c) Lindlar catalyst, H2, EtOAc; (d) DMSO, (COCl)2, Et3N, -78 °C; (e) Ph3P = CHCHO, PhMe, reflux; (f) CHI3, CrCl2, THF, 0 °C; (g) catalyst Pd(Ph3)4, CuI, PhH, room temperature; (h) tBu4NF, THF, room temperature; (i) Zn(Cu / Ag), MeOH, 40 °C; (j) NaOH, THF, HO, followed by acidification with HCl / HO; (k) NaOH, KOH, etc., or amines, imines, etc. [Figure 8] Scheme 3 shows the total synthesis of dihydroxylated erovanoids M, N, U, and V. Reagents and conditions: (a) cyanuric chloride, EtN, acetone, room temperature; (b) (3-methyloxetan-3-yl)methanol, pyridine, CHCl, 0 °C; (c) BF, OEt, CHCl; (d) nBuLi, BF, OEt, THF, -78 °C, then 1; (e) tBuPhSiCl, imidazole, DMAP, CHCl, room temperature; (f) camphorsulfonic acid, CHCl, ROH, room temperature; (g) Lindlar catalyst, H 2, EtOAc, (h) DMSO, (COCl)2, Et3N, -78 °C, (i) Ph3P = CHCHO, PhMe, reflux, (j) CHI3, CrCl2, THF, 0 °C, (k) catalyst Pd(Pd3)4, CuI, PhH, room temperature, (l) tBu4NF, THF, room temperature, (m) Zn(Cu / Ag), MeOH, 40 °C, (n) NaOH, THF, H2O, then acidified with HCl / H2O, (o) NaOH, KOH, etc., or amines, imines, etc. [Figure 9] FIG. 4 shows Scheme 4 for the total synthesis of 32-carbon dihydroxylated erovanoids. [Figure 10] FIG. 5 shows Scheme 5 for the total synthesis of 34-carbon dihydroxylated erovanoids. [Figure 11] Brightfield images showing HNEpC morphology are shown - 10x and 20x. [Figure 12]The allergenicity of house dust mites is demonstrated. Various components of HDM and their associated fecal pellets and dust activate the immune system. See Trends in Immunology, September 2011, Vol. 32, No. 9, Gregory, 2011. [Figure 13-1] FIG. 13 shows the structures of LPS and poly(I:C). [Figure 13-2] See description of Figure 13-1. [Figure 14] The experimental design for the challenge of HNEpCs with several stressors (aeroallergens) is shown. [Figure 15] Shown are different aerovanoids (ELVs) used at 500 nM concentrations to verify lipid specificity on HNEpCs stressed with different aeroallergens. [Figure 16] A cytotoxicity assay using the CyQuant LDH assay is shown. Damage to the cell membrane releases LDH into the surrounding cell culture medium. Extracellular LDH in the medium can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via the reduction of NAD+ to NADH. Oxidation of NADH by diaphorase reduces a tetrazolium salt (INT) to a red formazan product that can be measured spectrophotometrically at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the medium and indicates cytotoxicity. [Figure 17A] Figures 17A and 17B show cytotoxicity assays using the CyQuant LDH assay. [Figure 17B] See legend to Figure 17A. [Figure 18A] Figures 18A and 18B show cell viability assays using Presto Blue HS reagent. [Figure 18B] See legend to Figure 18A. [Figure 19A] Figures 19A and 19B show ELISA (IL-6). [Figure 19B] See legend to Figure 19A. [Figure 20A] Figures 20A and 20B show ELISA (IL-1β). [Figure 20B] See legend to Figure 20A. [Figure 21A] Figures 21A and 21B show ELISA (IL-8). [Figure 21B] See legend to Figure 21A. [Figure 22A] Figures 22A and 22B show ELISA (CCL2). [Figure 22B] See legend to Figure 22A. [Figure 23A] Figures 23A and 23B show ELISA (CXCL1). [Figure 23B] See legend to Figure 23A. [Figure 24A] Figures 24A and 24B show ELISA (VEGF). [Figure 24B] See legend to Figure 24A. [Figure 25A] Figures 25A and 25B show ELISA (ICAM1). [Figure 25B] See legend to Figure 25A. [Figure 26A] Figures 26A and 26B show ELISA (IL-10). [Figure 26B] See legend to Figure 26A. [Figure 27] Representative figures from Trends in Molecular Medicine; October, 2011; Vol. 17, No. 10. Jacquet, 2011 are shown. [Figure 28] Convergent mechanisms controlling aging are indicated for the development of new synthetic non-lipid analogs that mimic the biological activity of lipid mediators. [Figure 29] Figure 1 shows oxidative stress and elastin-induced cell death counteracted by NPD1 and ELV-32:6 in human RPE cells. [Figure 30-1]Figure 30 shows that erovanoids attenuate elastin-mediated PEBP-1 phosphorylation (adapted from Wenzel et al., 2017, Cell, 171:628-641). [Figure 30-2] See description of Figure 30-1. [Figure 31] Showing upstream regulation of ferroptosis and senescence by erovanoids. [Figure 32] We show that loss of MFRP (Membrane Frizzled-Related Protein) leads to progressive PRC degeneration. [Figure 33] Shows selective loss of PC44:12 and 56:12 in AdipoR1- / - and MFRPrd6. [Figure 34] Targets in the human retina with AMD are shown. [Figure 35] We show that AMD exhibits PC-selective differentiation in the cone-rich macula and rod-rich periphery. MALDI MS molecular imaging shows distinct stratification. [Figure 36]Heat maps are shown for 168 GPCR targets and 73 orphan GPCRs (antagonists or partial agonists) screened by PathHunter β-arrestin enzyme fragment complementation / β-galactosidase against the orphanMAX panel (DiscoverX, Eurofins, Fremont, CA). While not wishing to be bound by theory, GPCR targets (blue arrows) display activity above threshold. Assays were performed with cells expressing the GPCR panel, incubated at 37°C for 90 minutes, using NPD1, ELV-N-32, or ELV-N-34 (5 μM), or vehicle. Microplates were read in PerkinElmer EnVision Multimode for chemiluminescence. Activity was analyzed using the CBIS data suite (ChemInnovation, CA). Screens were performed twice in a blinded fashion, with identical results in both cases. Rainbow heat maps were generated in GraphPad Prism 8.2 using % activity (agonists) and % inhibition (antagonists) values. Color mapping done using a uniform legend with a minimum value of 0 and a maximum value of 60. Only GPCRs with color intensity at a high cutoff value (green to red) are considered candidates (blue arrows). [Figure 37]These results suggest that induction of specific AdipoR1 expression and activation may compensate for the deficiency of neuroprotective mediators (1) in the lipid mediator pathway and precursors and intermediates of ELOVL-4 during the early development of retinal pathology in 5xFAD mice. (A) Biosynthetic pathway of 32:6n-3 and 34:6n-3 erovanoids from NPD1, PC54-12, and PC56-12 precursors. Stable monohydroxy products of VLC-PUFAs are used for mass spectrometry detection. (B) Bar graphs of free 32:6n-3 and 34:6n-3 VLC-PUFAs, 27-monohydroxy 32:6n-3 and 29-monohydroxy 34:6n-3 VLC-PUFAs, free DHA, and NPD1 in the retina (top) and RPE layer (bottom). (C-D) Western blot and quantification of 15-lipoxygenase-1 expression in the RPE and retina. In the RPE, 15-lipoxygenase-1 expression was lower in 5xFAD than in WT RPE. There was no difference between the two groups in the retina. This explains why NPD1 levels were lower in 5xFAD but unchanged in the retina. ELOVL4 is expressed only in the retina, and its expression was lower in 5xFAD. This resulted in less free 32:6n-3 and 34:6n-3, as well as less monohydroxyl molecules. (NS: not significant, *P<0.05, using Student's t-test comparison). [Figure 38] Interactions of NPD1, ELV32, and ELV34 with positively screened GPCRs by Path Hunter β-arrestin complementation are shown. Receptors for which the lipids demonstrated antagonistic (red) and agonistic (blue) activity are depicted. Circled GPCRs demonstrated activity above the threshold, while the rest were borderline. Because path-hunter is a heterologous system, an alternative approach is to test GPCRs that do not meet but are close to the threshold activity. [Figure 39]These results demonstrate that UOS upregulates the pro-inflammatory transcriptome and downregulates pro-homeostatic pathways in single RPE cells. NPD1 and ELV32-6 suppress these changes. Boxplot points represent the expression of genes in single RPE cells (96 total per sample). *P<0.001 (***), *P<0.01 (**), one-way ANOVA, post-hoc Tukey HSD test for multiple comparisons. [Figure 40] 1 shows the structure of glutathione reductase, based on the X-ray structure of human glutathione reductase. [Figure 41] Analysis of the target candidate protein TXNRD1 is shown. The structure shown is based on the X-ray structure of human NADP(H) thioredoxin reductase I. [Figure 42] Convergent mechanisms controlling aging are indicated for the development of new synthetic non-lipid analogs that mimic the biological activity of lipid mediators. [Figure 43] We demonstrate that population-level electrical activity was recorded from a variety of hypothalamic neurons using the MEA system. [Figure 44] Figure 1 shows protection of hypothalamic neuronal cell death (as measured by Fluoro-Jade B staining) by erovanoids in adult obese diabetic mice (db / db). [Figure 45]Senescence-associated β-galactosidase activity measured in human neuroglial (HNG) cells exposed to oligomeric amyloid beta (Oaβ) (10 μM) is shown. (A-G) SA-β-Gal activity in HNG cells treated with 500 nM Oaβ (10 μM) and different erovanoids (ELVs) or neuroprotectin D1 (NPD1). Photomicrographs were obtained with a bright-field microscope. (H) Quantification of SA-β-Gal+ cells shown in (A-G). SA-β-Gal+ cells were scored in three random fields for a total of at least 150 cells. Results are expressed as the percentage of stained SA-β-Gal+ cells (mean ± SEM). Statistical analysis was performed using Graphpad Prism software 8.3. Results were compared by one-way ANOVA followed by a Holm-Sidak post-hoc test; p < 0.05 was considered statistically significant. [Figure 46] Senescence-associated β-galactosidase activity measured in human neuroglial (HNG) cells exposed to elastin (10 μM) is shown. (A-G) SA-β-Gal activity in HNG cells treated with 500 nM of elastin (10 μM) and different elastinoids (ELVs) or neuroprotectin D1 (NPD1). Photomicrographs were obtained with a bright-field microscope. (H) Quantification of SA-β-Gal+ cells shown in (A-G). SA-β-Gal+ cells were scored in three random fields for a total of at least 150 cells. Results are expressed as the percentage of stained SA-β-Gal+ cells (mean ± SEM). Statistical analysis was performed using Graphpad Prism software 8.3. Results were compared by one-way ANOVA followed by a Holm-Sidak post-hoc test; p < 0.05 was considered statistically significant. [Figure 47] Experimental design: Human neuroglial (HNG) cells were challenged with Oaβ or erastin. [Figure 48]ELV34 reverses the effects of IL1β in human diabetic adipocytes. A) Experimental design. B) Expression levels of TP53 and IL8 in human diabetic and non-diabetic adipocytes by Taqman real-time PCR. [Figure 49] We show that ELV34 reduced IL1β-induced IL6 (a marker of SASP) levels in the hypothalamus of diabetic db / db mice, indicating that hypothalamic neurons and astrocytes produce SP. Different effects were observed in male and female mice. [Figure 50] The characteristics of db / db mice relative to WT are shown. [Figure 51] These results demonstrate that ELV34 treatment increased levels of adiponectin, an antidiabetic systemic hormone secreted by adipocytes and other tissues (hypothalamus) that promotes insulin sensitivity. A) Diabetic hypothalamus treated with ELV34 showed a trend toward increased adiponectin in both females and males. B) Differential effects of ELV34 on subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). SAT and VAT have different capacities for browning. [Figure 52]The retina of 5xFAD mice shows a lack of VLC-PUFAs in phosphatidylcholine molecular species. (A) PC heatmap analysis of 6-month-old 5xFAD (n = 6) and wild-type mice (n = 6). Two major clusters of PCs evolved with different functions. Group 1 shows PCs enriched in 5xFAD, while Group 2 shows PCs predominant in WT mice, with most PCs containing VLC-PUFAs. (B) PCA analysis of PCs shows two populations (WT—black and 5xFAD—red) distributed across principal component 1. Therefore, the loading scores on principal component 1 are essential for identifying individual PCs for the differences between WT and 5xFAD. (C) PC loading scores (absolute values) on principal component 1. The higher the loading score, the greater the PC's contribution to the principal component for distinguishing WT and 5xFAD mice (SI Appendix, Fig. S1A). Ten short-chain PUFAs (<48C) included in the PCs were included in the top 12 PCs (C) with the highest loading scores, and VLCPUFAs included in the PCs contributed to two (58:1 and 58:12). (D) Time used for random forest classification of PCs from WT and 5xFAD. The longer the time used, the more significant the difference in PC value between WT and 5xFAD (SI Appendix, Fig. S1B). VLC-PUFA-containing PCs contributed to seven of the top 12 times used in this classification (D). (E-G) Boxplots of VLC-PUFA-containing PCs (E), DHA (F), and AA (G). WT had more VLC-PUFA- and DHA-containing PCs, while 5xFAD had more AA-containing PCs. (*P < 0.05, Student's t test) [Figure 53]This shows a lack of VLC-PUFAs in phosphatidylcholine molecular species in 5xFAD RPE. (A) Heatmap analysis of PCs in 6-month-old 5xFAD (n = 6) and WT (n = 6) RPE. VLC-PUFA-containing PCs are less abundant in 5xFAD. (B) PCA for PCs in 5xFAD and WT RPE. There are two populations scattered throughout Principal Component 1, but the distribution is less clear than in the retina (Figure 52). For this reason, the loading score of Principal Component 1 is essential for identifying individual PCs for the differences between WT and 5xFAD. (C) Loading scores (absolute values) of PCs onto Principal Component 1. The higher the loading score, the greater the contribution of the PC to Principal Component 1 for distinguishing WT from 5xFAD (Figure 59, panel C). Six short-chain PUFA (<48C)-containing PCs are included in the top 12 PCs with the highest loading scores (C), while VLC-PUFA-containing PCs contribute six (50:8, 50:12, 52:8, 54:12, 56:12, and 58:12). (D) Time used for random forest classification of PCs from WT and 5xFAD. The longer the time used, the more significant the PCs are in the differences between WT and 5xFAD (Figure 59, panel D). VLC-PUFA-containing PCs contribute nine of the top 12 times used in this classification (D). (E) Percentage plots of PC38-6, PC40-6, and PC44-12 in WT and 5xFAD retina (top) and eye cup (RPE, bottom). Surprisingly, eye cup PCs show that 5xFAD has more PC38:6 and less PC44-12, while PC40-6 is equivalent to WT. (NS: not significant, *P<0.05, Student's t-test). [Figure 54]Defects in lipid mediator pathways and precursors and intermediates of ELOVL-4 during the early development of 5xFAD retinal pathology are shown. (A) Biosynthetic pathways of NPD1 and 32:6n-3 and 34:6n-3 ELVs from PC54-12 and PC56-12. (B) Free 32:6n-3 and 34:6n-3. (C) 27-monohydroxy 32:6n-3 and 29-monohydroxy 34:6n-3, stable derivatives of the hydroperoxyl precursors of ELVN-32 and ELVN-34, respectively. (D) Free DHA. (E) NPD1. In B-E, retina (top) and RPE (bottom) (n = 6 per group). (F and H) Western blots of 15 LOX1 and ELOVL4 and quantification in RPE and retina (n = 6 per group). In the RPE, 15-lipoxygenase-1 in 5xFAD was less than that in WT. In the retina, there was no difference between the two groups. This is consistent with the smaller NPD1 pool size in 5xFAD, but there was no change in the retina (E). ELOVL4, expressed only in the retina, was lower in 5xFAD. This resulted in less free 32:6n-3 and 34:6n-3, as well as less monohydroxy stable precursor derivatives. (NS: not significant, *P<0.05, Student's t-test). [Figure 55]Morphology and function of 5xFAD retinas are shown. (A) VlogI plot showing maximum ERG b-wave amplitude in response to light flashes of 0–0.075 cd·s / m². 5xFAD retinas achieved a maximum amplitude of approximately 100 μV, approximately half that recorded in WT retinas (n=6 / group). (B) Electron microscopy of 6-month-old 5xFAD retinas showing similarity to WT retinas. (Bi) Basal side of 5xFAD RPE cells showing membrane folding along Bruch's membrane (Br). (Bii) Disc synthesis region (arrow) in the basal portion of the rod outer segment showing newly formed discs from the connecting ciliary (CC) membrane (WT). (Biii) Similar region in 5xFAD displaying new disc formation (arrow). (Biv) Outer limiting membrane (OLM, arrow) at the scleral end of the cell body layer (N, photoreceptor nuclei) in 5xFAD retinas. The cytoplasm of Müller cells (M) is brighter than that of PRCs. (Bv) Interface between a 5xFAD RPE cell and a rod PRC tip (PR). Two phagosomes (Ph) in the RPE cytoplasm. The lower Ph is retained within the RPE apical process, while the upper, darker Ph is older and only enters the RPE cell body, indicating normal phagocytic function. (Bvi) Inner segment mitochondria (M) in 5xFAD retain highly elongated PRCs. (C) 5-month-old WT and 5xFAD retinal sections showing normal PRC profiles within 5xFAD retinas. (D) Fluorescent staining of retinas from WT and 5xFAD. Blue (DAPI) indicates nuclei, and red indicates Aβ in the RPE layer of 6-month-old 5xFAD RPE cells. (*P<0.05, Student's t-test). [Figure 56]Following subretinal injection of OAβ into WT mice, ELV restored RPE morphology and reduced gene expression. (A) In vivo experimental design: 6-month-old C57BL / 6J WT mice were divided into seven groups (n = 12 / group): uninjected, PBS, OAβ only, OAβ + ELV-N-32, OAβ + ELV-N-34, ELV-N-32 only, and ELV-N-34 only. On day 3, mRNA was isolated for real-time PCR. On day 7, mice were subjected to optical coherence tomography (OCT), followed by enucleation of the eyes and processing for whole-mount RPE staining and Western blot analysis. (B) Whole-mount RPE images using an antibody against Zonula occludens-1 (ZO-1), a tight junction marker. OAβ disrupted RPE morphology. (C) OCT analysis of the effects of OAβ on the retina and RPE. (D) The PRC layer thickness was thinner in the OAβ-injected group. (E) RPE gene expression after Oaβ(1-42) injection and ELV treatment. (E–G) Gene expression of the same functional group plotted: senescence- and AMD-related genes (E), collagenase, gelatinase, stromelysin, and other matrix metalloproteinases (MMPs) (F), and autophagy (G). (H) Western blot of RPE for p16INK4a, a key marker of senescence. (I) Retinal apoptosis gene expression after OAβ(1-42) injection and ELV treatment. (*P<0.05, Student's t-test). [Figure 57] These results demonstrate that OA-β-mediated activation of senescence-associated secretory phenotype (β-galactosidase, SA-β-Gal) and gene expression in primary cultured human RPE cells is counteracted by ELV. (A) In vitro experimental design: Primary human RPE cells were treated with 10 μM OAβ + / - ELV. After 3 days, RNA was isolated and analyzed by qPCR. After 7 days, cells were subjected to β-galactosidase staining. (B) Live cell images under brightfield microscopy after 7 days. (C) β-Gal staining + / - ELV. Quantification of the % of -Gal-positive cells. ELV reduced the number of positive senescent cells. (D) Gene transcription of senescence-, AMD-related, and autophagy genes after OAβ(1-42) exposure + / - ELV. (*P<0.05, Student's t-test). [Figure 58] This figure shows an overview of the effects of ELV on OAβ-induced RPE and PRC damage. (A) OAβ induces the senescence program and disrupts RPE tight junctions. Without wishing to be bound by theory, OAβ penetrates the retina and causes PRC cell death in our in vivo WT mouse studies, reflected by fewer cell body layer (CBL) nuclei. ELV restores RPE morphology and PRC integrity. (B) OAβ induces the expression of senescence-, autophagy-, matrix metalloproteinase-, and AMD-related genes in the RPE and apoptotic genes in the retina. ELV downregulated OAβ gene induction. The pathway of ELV synthesis is shown. [Figure 59] Principal component analysis loading scores and random forest time used for all retinal and RPE PC types are shown. (A) Absolute loading scores of all PCs onto Principal Component 1 for the retina. The higher the loading score, the greater the PC's contribution to Principal Component 1 for distinguishing retina from wild-type and 5xFAD. (B) Time used for random forest classification of all retinal PCs from wild-type and 5xFAD mice. The longer the time used, the greater the PC's value in distinguishing between wild-type and 5xFAD. (C) Absolute loading scores of all PCs onto Principal Component 1 for the RPE. The higher the loading score, the greater the PC's contribution to Principal Component 1 for distinguishing RPE from wild-type and 5xFAD. (D) Time used for random forest classification of all RPE PCs from wild-type and 5xFAD mice. The longer the time used, the greater the PC's value in distinguishing between wild-type and 5xFAD. [Figure 60]This shows that the fatty acid composition of PC can be obtained by complete fragmentation. Negative ion mode was used for LC / MS / MS data acquisition. VLC-PUFA-containing PCs are depicted. (A) PC54:12 (m / z 1076 (M+CH3COO-) corresponds to m / z 1018 (M+H+) in positive mode) is composed of FA32:6 (m / z 467) and FA22:6 (m / z 327). (B) PC56:12 (m / z 1104 (M+CH3COO-) corresponds to m / z 1046 (M+H+) in positive mode) is composed of FA34:6 (m / z 495) and FA22:6 (m / z 327). (C) PC58:12 (m / z 1132 (M+CHCOO-) corresponds to m / z 1074 (M+H+) in positive mode) is composed of FA36:6 (m / z 523) and FA22:6 (m / z 327). DHA-containing PCs are in the second row. (D) PC38:6 (m / z 864 (M+CHCOO-) corresponds to m / z 806 (M+H+) in positive mode) is composed of FA16:0 (m / z 255) and FA22:6 (m / z 327). (E) PC40:6 (m / z 892 (M+CH3COO-) corresponds to m / z 834 (M+H+) in positive mode) is composed of FA18:0 (m / z 283) and FA22:6 (m / z 327.0). (F) PC44:12 (m / z 936 (M+CH3COO-) corresponds to m / z 878 (M+H+) in positive mode) is composed of two FA22:6s (m / z 327.0). AA-containing PCs are in the third row. (G) PC36:4 (m / z 840 (M+CH3COO-) corresponds to m / z 782 (M+H+) in positive mode) is composed of FA16:0 (m / z 255) and FA20:4 (m / z 303). (H) PC38:4 (m / z 868 (M+CH3COO-) corresponds to m / z 810 (M+H+) in the positive mode) is composed of FA18:0 (m / z 283) and FA20:4 (m / z 303).(I) PC38:5 (m / z 866 (M+CHCOO-) corresponds to m / z 808 (M+H+) in positive mode) is composed of FA18:1 (m / z 281) and FA20:4 (m / z 303). This peak is also from the PC38:6 isotope (two carbons are naturally C13-labeled). This produces FA16:0 (m / z 255) and FA22:6 (m / z 329 if two carbons are C13) or FA16:0 (m / z 256 if one carbon is C13) and FA22:6 (m / z 328 if one carbon is C13). [Figure 61] The complete fragmentation spectra of ELV and precursor molecules and NPD1 are shown. (A) The complete fragmentation spectra of the free fatty acids FA32:6n-3 and FA34:6-n-3, which are precursors of elovanoids, show the molecular structure and fragmentation pattern. (B) The endogenous 27-monohydroxy32:6 and 29-monohydroxy34:6, stable precursors of elovanoids, are in good agreement with the theoretical fragmentation patterns shown in the structures. (C) Complete fragmentation of the elovanoids, ELV32 and ELV34. The standards show all peaks shown in the structures and fragmentation patterns. (D) The NPD1 fragmentation spectrum is expressed as theoretical values. [Figure 62] Fundus and OCT analysis are shown. (A) Fundus and optical coherence tomography (OCT) images of wild-type (WT) and 5xFAD mice. Mice are approximately 6 months old. (B) Analysis of photoreceptor layer thickness. There is no difference between WT and 5xFAD. [Figure 63]This shows that inflammatory signaling is activated by 5xFAD. (A) Relative normalized expression of AMD-related genes in the RPE in WT and 5xFAD. RNA from the eyecup / choroid of WT and 5xFAD (6-month-old) mice was isolated, reverse-transcribed to cDNA, and subjected to RT-PCR using AMD-related genes. Briefly, there is activation of various genes associated with AMD in 5xFAD. (B) Relative normalized expression of inflammatory genes in the retina. RNA from the retina of WT and 5xFAD (6-month-old) mice was isolated, reverse-transcribed to cDNA, and subjected to RT-PCR using various inflammatory genes. Briefly, there is activation of inflammatory signaling in 5xFAD. [Figure 64] Western blot of oligomeric Aβ is shown. After 24 hours of oligomerization, 2 μl of Aβ stock was loaded onto a Tricine gel without denaturation. [Figure 65] Unfolded protein response (UPR) genes are shown. Three days after injection, RNA from the RPE and retina was isolated, reverse transcribed to cDNA, and subjected to RT-PCR using UPR primers. There were no changes in these genes in either the RPE (A) or retina (B). [Figure 66] 1 shows the antibodies utilized herein. [Figure 67-1] Figure 67 shows the gene primer sequences utilized herein. [Figure 67-2] See description of Figure 67-1. DETAILED DESCRIPTION OF THE INVENTION

[0082] Detailed Description of Disclosure Before the present disclosure is described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. The scope of the present disclosure will be limited only by the appended claims, and the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0083] Where a range of values ​​is provided, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value within that stated range, is encompassed in the disclosure, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed in the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range can include one or both of the limits, a range excluding either or both of them can include those limits and be included in the disclosure.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are now described.

[0085] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publication is cited. The citation of a publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0086] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0087] Embodiments of the present disclosure employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, toxicology, and the like, which are within the skill of the art and are fully explained in the literature.

[0088] It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" can include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a support" can include a plurality of supports. In this specification and the following claims, reference will be made to several terms that can have the following meanings, unless a contrary intention is apparent.

[0089] As used herein, the following terms have the meanings ascribed to them unless otherwise specified. In this disclosure, "comprise," "comprising," "containing," "having," and the like can have the meanings ascribed to them in U.S. patent law and can mean "can comprise," "comprises," and the like. When applied to methods and compositions included in this disclosure, "consisting essentially of" or "consisting essentially of" and the like can refer to compositions such as those disclosed herein, but which may include additional structural groups, composition components, or method steps (or analogs or derivatives thereof as discussed herein). However, such additional structural groups, composition components, method steps, and the like do not substantially affect the properties of the composition or method compared to the properties of the corresponding composition or method disclosed herein.

[0090] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is also involved. Similarly, "one example," "exemplary," etc. are understood to be non-limiting.

[0091] The term "substantially" permits deviations from the descriptor that do not adversely affect the intended purpose. It is understood that a descriptor is modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0092] As used herein, the term "about" can refer to approximately, roughly, approximately, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20 percent above or below (high or low).

[0093] Before describing various embodiments, the following exemplary description is provided.

[0094] As used herein, the nomenclature alkyl, alkoxy, carbonyl, and the like are used as would be understood by one of ordinary skill in the chemical arts. As used herein, alkyl groups can include straight-chain, branched, and cyclic alkyl radicals containing up to about 20 carbons, or 1 to 16 carbons, and are linear or branched. Alkyl groups herein can include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, and isohexyl.

[0095] As used herein, lower alkyl can refer to a carbon chain having from about 1 or about 2 carbons up to about 6 carbons. Suitable alkyl groups can be saturated or unsaturated. Furthermore, alkyl can also be substituted one or more times on one or more carbons with a substituent selected from the group consisting of C1-C15 alkyl, allyl, arenyl, alkenyl, C3-C7 heterocycle, aryl, halo, hydroxy, amino, cyano, oxo, thio, alkoxy, formyl, carboxy, carboxamido, phosphoryl, phosphonate, phosphonamido, sulfonyl, alkylsulfonate, arylsulfonate, and sulfonamido. Furthermore, alkyl groups can contain up to 10 heteroatoms, and in certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 heteroatom substituents. Suitable heteroatoms can include nitrogen, oxygen, sulfur, and phosphorus.

[0096] As used herein, "cycloalkyl" can refer to a monocyclic or polycyclic ring system of 3 to 10 carbon atoms in particular embodiments, and 3 to 6 carbon atoms in other embodiments. The ring system of a cycloalkyl group can consist of one ring or two or more rings which can be joined together in a fused, bridged, or spiro-connected manner.

[0097] As used herein, "aryl" can refer to an aromatic monocyclic or polycyclic group containing 3 to 16 carbon atoms. As used herein, an aryl group is an aryl radical that can contain up to 10 heteroatoms, and in certain embodiments, 1, 2, 3, or 4 heteroatoms. An aryl group can also be substituted one or more times, and in certain embodiments, 1 to 3 or 4 times, with an aryl group or a lower alkyl group, and it can also be fused to another aryl or cycloalkyl ring. Suitable aryl groups can include, for example, phenyl, naphthyl, tolyl, imidazolyl, pyridyl, pyrroyl, thienyl, pyrimidyl, thiazolyl, and furyl groups.

[0098] As used herein, a ring can have up to 20 atoms, which can include one or more nitrogen, oxygen, sulfur, or phosphorus atoms, provided that the ring can have one or more substituents selected from the group consisting of hydrogen, alkyl, aryl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, phosphonate, phosphonamido, and sulfonyl, and further provided that the ring can contain one or more fused rings, including carbocyclic, heterocyclic, aryl, or heteroaryl rings.

[0099] As used herein, alkenyl and alkynyl carbon chains, unless specified, contain 2 to 20 carbons or 2 to 16 carbons and are straight or branched. In certain embodiments, alkyl carbon chains of 2 to 20 carbons contain 1 to 8 double bonds, and in certain embodiments, alkenyl carbon chains of 2 to 16 carbons contain 1 to 5 double bonds. In certain embodiments, alkynyl carbon chains of 2 to 20 carbons contain 1 to 8 triple bonds, and in certain embodiments, alkynyl carbon chains of 2 to 16 carbons contain 1 to 5 triple bonds.

[0100] As used herein, "heteroaryl" can, in certain embodiments, refer to an about 5 to about 15-membered monocyclic or polycyclic aromatic ring system in which one or more, in one embodiment, one to three, of the atoms in the ring system are heteroatoms, i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur. The heteroaryl group can be fused to a benzene ring. Heteroaryl groups can include, but are not limited to, furyl, imidazolyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, and isoquinolinyl.

[0101] As used herein, "heterocyclyl" can refer to a 3- to 10-membered, in one embodiment, 4- to 7-membered in another embodiment, and 5- to 6-membered in further embodiments, monocyclic or polycyclic non-aromatic ring system in which one or more, and in certain embodiments, 1 to 3 of the atoms in the ring system are heteroatoms, i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, or sulfur. In embodiments in which the heteroatom is nitrogen, the nitrogen can be substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, acyl, guanidino, or quaternized to form an ammonium group, where the substituents are selected as described herein.

[0102] As used herein, "aralkyl" can refer to an alkyl group in which one of the alkyl's hydrogen atoms is replaced with an aryl group.

[0103] As used herein, "halo," "halogen," or "halide" can refer to F, Cl, Br, or I.

[0104] As used herein, "haloalkyl" can refer to an alkyl group in which one or more hydrogen atoms are replaced with halogen. Such groups can include, but are not limited to, chloromethyl and trifluoromethyl.

[0105] As used herein, "aryloxy" can refer to RO- where R is aryl, including lower aryl such as phenyl.

[0106] As used herein, "acyl" can refer to a -COR group that includes, for example, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl, all of which can be substituted.

[0107] As used herein, "n-3" or "n-3," "n-6," or "n6," etc., can refer to the conventional nomenclature of polyunsaturated fatty acids or their derivatives, in which the position of the double bond (C=C) is on the carbon atom counted from the end (methyl end) of the carbon chain of the fatty acid or fatty acid derivative. For example, "n-3" refers to the third carbon atom from the end of the carbon chain of the fatty acid or fatty acid derivative. Similarly, "n-3" or "n-3," "n-6," or "n6," etc., can also refer to the position of a substituent, such as a hydroxyl group (OH), located on the carbon atom of the fatty acid or fatty acid derivative. The number (e.g., 3, 6, etc.) is counted from the end of the carbon chain of the fatty acid or fatty acid derivative.

[0108] As used herein, abbreviations for protecting groups and other compounds conform to their common usage, recognized abbreviations, or IUPAC-IUB Commission on Biochemical Nomenclature (see (1972) Biochem. 11:942-944) unless otherwise indicated.

[0109] As used herein, in the chemical structures of the compounds of the present disclosure, they are shown to have a terminal carboxyl group "-COOR", where "R" can represent a group covalently bonded to the carboxyl, such as an alkyl group. Alternatively, the carboxyl group can be further represented by "-COO - " and R is a cation, including metal cations, ammonium cations, etc.

[0110] The terms "subject" or "patient" can refer to any organism to which aspects of the present invention can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. The term "subject" can include mammals, e.g., humans of any age, suffering from a pathological condition. In another embodiment, the term encompasses subjects at risk of developing a pathological condition. Subjects to which the compounds of the present disclosure can be administered are animals, mammals such as primates, particularly humans. For veterinary applications, a wide variety of subjects would be suitable, including, for example, livestock such as cattle, sheep, goats, cows, pigs, etc., poultry such as chickens, ducks, geese, turkeys, etc., and domestic animals, e.g., pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals would be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs. The term "living subject" can refer to the above subjects or another living organism. The term "living subject" can refer to a whole subject or organism, as well as a portion (eg, a liver or other organ) excised from a living subject.

[0111] A "subject suffering from a condition" or a "subject having a condition" can refer to a subject with a pre-existing condition or a known or suspected predisposition to developing the condition. In embodiments, the condition can be an inflammatory disease, such as an allergic inflammatory disease. In another embodiment, the condition can be a disease associated with cellular senescence, ferroptosis, or both, such as an Aβ-related disease, including age-related macular degeneration or Alzheimer's disease. In yet another embodiment, the disease can be a metabolic disorder, such as diabetes, obesity, or both.

[0112] As an example, a "subject with an allergic condition" can refer to a pre-existing allergic condition or a known or suspected predisposition to developing an allergic condition. Thus, the subject may have an active allergic condition or a latent allergic condition. The allergen need not be known. However, a particular allergic condition can be associated with seasonal or geographic environmental factors and need not be apparent to the subject. In one embodiment, the allergic condition is intentionally induced in the subject for experimental purposes.

[0113] As used herein, "pharmaceutically acceptable derivatives" of a compound may include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates, or prodrugs thereof. Such derivatives can be readily prepared by those skilled in the art using known methods for such derivatization. The resulting compounds can be administered to animals or humans without substantial toxic effects and are either pharmaceutically active or are prodrugs.

[0114] Pharmaceutically acceptable salts include, but are not limited to, amine salts such as N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; lithium, potassium, sodium, and the like. alkaline earth metal salts such as, but not limited to, barium, calcium, and magnesium; transition metal salts such as, but not limited to, zinc; and other metal salts such as, but not limited to, sodium hydrogen phosphate and disodium phosphate; and salts of mineral acids such as, but not limited to, hydrochlorides and sulfates; and salts of organic acids such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, and fumarate.

[0115] Pharmaceutically acceptable esters can include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids, and boronic acids.

[0116] Pharmaceutically acceptable enol ethers may include, but are not limited to, derivatives of the formula C=C(OR), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, or heterocyclyl. Pharmaceutically acceptable enol esters may include, but are not limited to, derivatives of the formula C=C(OC(O)R), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, or heterocyclyl.

[0117] Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or 1 to about 2, 3, or 4 solvent or water molecules.

[0118] As used herein, "formulation" can refer to any collection of compounds, mixtures, or solution components selected to provide optimal properties for a particular end use, including product specifications and / or conditions of use. The term formulation can include liquids, semi-liquids, colloidal solutions, dispersions, emulsions, microemulsions, and nanoemulsions, including oil-in-water emulsions and water-in-oil emulsions, pastes, powders, and suspensions. The formulations of the present invention can also be contained or packaged with other non-toxic compounds, such as cosmetic carriers, excipients, binders, and fillers. Specifically, acceptable cosmetic carriers, excipients, binders, and fillers for use in the practice of the present invention are those that make the compound suitable for oral delivery and / or provide stability so that the formulations of the present invention exhibit a commercially acceptable shelf life.

[0119] As used herein, the term "administering" can refer to introducing a substance, such as a VLC-PUFA, into a subject. Any route of administration can be used, including, for example, intranasal, topical, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transdermal, intradermal, intracranial, etc. In embodiments, "administering" can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject. The formulations or pharmaceutical compounds of the present invention can be administered alone or with other compounds, excipients, fillers, binders, carriers, or other vehicles selected based on the selected route of administration and standard pharmaceutical practice. Administration can be via a carrier or vehicle, such as an injectable solution, including sterile aqueous or non-aqueous solutions, or saline; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; etc.

[0120] The formulation or pharmaceutical composition may also contain or be packaged with other non-toxic compounds such as pharmaceutically acceptable carriers, excipients, binders and fillers, including, but not limited to, glucose, lactose, acacia gum, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and / or polysaccharides, starch paste, magnesium trisilicate, talc, corn starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextrans, dextrins, etc. Specifically, pharmaceutically acceptable carriers, excipients, binders and fillers for use in the practice of the present invention are those that render the compounds of the present invention suitable for intranasal, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transdermal, intradermal, intracranial, topical delivery, etc. Additionally, the packaging material can be biologically inert or lacking biological activity, such as a plastic polymer or silicone, and can be internally processed by the subject without affecting the efficacy of the packaged and / or co-delivered composition / formulation.

[0121] Different forms of the formulations of the present invention can be modified to accommodate both different individuals and the different needs of a single individual. However, the current formulations do not necessarily address all causes in all individuals. Rather, by addressing the necessary causes, the current formulations restore the body and brain to normal function. The body and brain then correct any remaining deficiencies themselves.

[0122] As used herein, the term "therapeutically effective amount" can refer to that amount of an administered composition or pharmaceutical composition embodiment that relieves to some extent one or more symptoms of the disease or condition being treated and / or prevents to some extent one or more symptoms of a condition or disease that the treated subject develops or is at risk of developing. As used interchangeably herein, "subject," "individual," or "patient" can refer to a vertebrate, e.g., a mammal such as a human. Mammals include, but are not limited to, rodents, monkeys, humans, farm animals, sport animals, and pets. The term "pet" can include dogs, cats, guinea pigs, mice, rats, rabbits, ferrets, etc. The term farm animals can include horses, sheep, goats, chickens, pigs, cows, donkeys, llamas, alpacas, turkeys, etc.

[0123] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" can refer to an excipient, diluent, carrier, and / or adjuvant that is useful in preparing a pharmaceutical composition that is safe, non-toxic, and not biologically undesirable, and can include excipients, diluents, carriers, and adjuvants that are acceptable for use in veterinary and / or human medicine. As used herein, a "pharmaceutically acceptable excipient, diluent, carrier, and / or adjuvant" can include one or more such excipients, diluents, carriers, and adjuvants.

[0124] The phrase "pharmaceutical composition" or "formulation" can refer to a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, particularly a human, and can refer to an active agent or a combination of ingredients, including a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or prophylactic use in vitro, in vivo, or ex vivo. A "pharmaceutical composition" can refer to a composition that is sterile and free of contaminants that may elicit an undesirable response in a subject (e.g., compounds in a pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions can be designed to be administered to a subject or patient in need thereof via many different routes of administration, including oral, intranasal, topical, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, by stent-eluting device, catheter-eluting device, intravascular balloon, inhalation, etc.

[0125] In embodiments, the pharmaceutical composition can include a therapeutically effective amount of an elovanoid and a therapeutically effective amount of one or more additional active agents (e.g., one or more antioxidants, antiallergic agents, anti-inflammatory agents, or analgesics). For example, the one or more antioxidants can be synthetic antioxidants, natural antioxidants, or a combination thereof. In embodiments, the antioxidant can protect the double bond of the elovanoid.

[0126] The term "administration" can refer to introducing the composition of the present disclosure into a subject.The preferred administration route of the composition is topical administration, oral administration or intranasal administration.However, any administration route can be used, such as intravenous, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, introduction into cerebrospinal fluid, intravenous or arterial blood vessels or instillation into body compartments.

[0127] As used herein, "treatment" and "treating" refer to the management and care of a subject in any manner in which one or more symptoms of a disease or disorder are improved or otherwise beneficially altered for the purpose of combating the condition, disease, or disorder. This term encompasses the full range of treatments for a given condition from which a patient is suffering, such as the administration of an active compound to alleviate or relieve symptoms or complications; slow the progression of the condition, disease, or disorder; cure or eliminate the condition, disease, or disorder; and / or prevent the condition, disease, or disorder. "Preventing" or "prevention" refers to the management and care of a patient with the goal of preventing the onset of a condition, disease, or disorder, and can include the administration of an active compound to prevent or reduce the risk of developing symptoms or complications. Those skilled in the art will appreciate that various methodologies and assays can be used to assess the development of pathology, and similarly, various methodologies and assays can be used to reduce pathology, progression, or regression.

[0128] As used herein, the term "prevent" can refer to preventing a disease, disorder, or condition from occurring in a subject who may be at risk of the disease but has not yet been diagnosed with the disease. Prevention (and an effective dose for prevention) can be demonstrated in population studies. For example, an effective amount for preventing a given disease or condition is an amount effective for reducing the incidence in a treated population compared to an untreated control population.

[0129] The phrase "alleviating the symptoms of" can refer to ameliorating, reducing, or eliminating the condition or symptoms associated with an allergic inflammatory disease. For example, symptoms of an allergic inflammatory condition include tingling or itching of the mouth, hives, itching, or eczema; swelling of the lips, face, tongue, throat, or other parts of the body; wheezing, stuffy nose, or difficulty breathing; abdominal pain, diarrhea, nausea, or vomiting; dizziness, lightheadedness, or fainting.

[0130] The patient to be treated can be a mammal, such as a human. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use to treat a disease as provided herein.

[0131] In embodiments, the composition may further comprise one or more "nutritional ingredients." As used herein, the term "nutritional ingredients" may refer to proteins, carbohydrates, vitamins, minerals, and other beneficial nutrients, including functional ingredients of the present disclosure, i.e., ingredients that can produce a particular benefit to the person consuming the food product. The carbohydrate can be, but is not limited to, glucose, sucrose, fructose, dextrose, tagatose, lactose, maltose, galactose, xylose, xylitol, dextrose, polydextrose, cyclodextrin, trehalose, raffinose, stachyose, fructooligosaccharides, maltodextrin, starch, pectin, gum, carrageenan, inulin, cellulose-based compounds, sugar alcohols, sorbitol, mannitol, maltitol, xylitol, lactitol, isomalt, erythritol, pectin, gum, carrageenan, inulin, hydrogenated resistant dextrin, hydrogenated starch hydrolysate, highly branched maltodextrin, starch, and cellulose.

[0132] Commercially available sources of nutritional protein, carbohydrates, etc. and their specifications are known or can be readily ascertained by those skilled in the art of processed food formulation.

[0133] The compositions described herein, which may include nutritional ingredients, may be, but are not limited to, "snack-sized" or "bite-sized" compositions, and may be food preparations smaller than what is typically considered a food bar. For example, the food bar may be scored or perforated to allow the consumer to separate smaller portions for consumption. Alternatively, the food "bar" may be in small pieces rather than a long, rod-like product. The smaller portions may be individually coated or enrobed. They may be packaged individually or in groups.

[0134] The food product can include, but is not limited to, solids that are not ground into a uniform mass. The food product can be coated or enrobed with, but is not limited to, dark, light, milk, or white chocolate, carob, yogurt, other confectionery, chocolate containing nuts or grains, and the like. The coating can be a compound confectionery coating or a non-confectionery (e.g., sugar-free) coating. The coating can be smooth or contain solid particles or pieces.

[0135] An allergy is a reaction of the immune system to foreign substances called allergens. For example, allergens can be eaten, inhaled into the lungs, injected into a subject, or touched. This allergic reaction can cause coughing, sneezing, itchy eyes, runny nose, and a scratchy throat. In severe cases, it can cause rashes, hives, low blood pressure, difficulty breathing, asthma attacks, and even death. Allergies are one of the most common illnesses in the country, yet there is no cure.

[0136] Allergic reactions are treated using anti-allergy medications such as brompheniramine (Dimethane), cetirizine (Zyrtec), chlorpheniramine (Chlortrimeton), clemastine (Tavist), diphenhydramine (Benadryl), and fexofenadine (Allegra). Many of these anti-allergy medications are associated with undesirable side effects, such as drowsiness, dizziness, dry mouth / nose / throat, headache, upset stomach, constipation, and sleep disturbances. The pharmaceutical compositions provided herein containing VLC-PUFAs do not cause such undesirable side effects and, therefore, represent an improvement over known anti-allergy medications.

[0137] Embodiments of the present invention are directed to compositions and methods for alleviating symptoms, preventing, or treating allergic inflammatory diseases. Referring to the examples included herein, results of a cytotoxicity assay (LDH) show that the addition of a stressor to cultures of nasal epithelial cells significantly increases the formation of red formazan, which indicates cytotoxicity, and this is reduced by the addition of ELV (Figures 17A and 17B). Furthermore, cell viability assays using PrestoBlue HS reagent show that control cells produce more resorufin than cells challenged with various stressors, and that the addition of ELV improves cell viability and confers protection to HNEpCs (Figures 18A and 18B). Furthermore, when HNEpCs are challenged with various stressors, significant production of pro-inflammatory cytokines and chemokines and significant reduction in the release of anti-inflammatory cytokines are observed compared to controls. This increased production of pro-inflammatory cytokines and chemokines was abrogated by the addition of ELV at a concentration of 500 nM 30 min after challenge with the respective stressor (Figures 19A and 19B), whereas the decrease in the production of anti-inflammatory cytokines and chemokines was reversed (Figures 26A and 26B).

[0138] The present invention also relates to compositions and methods for alleviating the symptoms, preventing or treating diseases associated with cellular senescence, ferroptosis, or both.For example, this disease is Aβ-related disease.Non-limiting examples of such diseases include age-related macular degeneration or Alzheimer's disease.

[0139] Aspects of the present invention are further directed to compositions and methods for alleviating the symptoms of, preventing, or treating metabolic disorders, non-limiting examples of which include diabetes and obesity.

[0140] The present disclosure includes embodiments of compounds, compositions, and methods for the alleviation of symptoms of, prevention of, and treatment of, diseases.

[0141] This is based on the new discoveries described herein regarding the surprising biological activities of certain very long chain polyunsaturated fatty acids (VLC-PUFAs) and their related hydroxylated derivatives, including, for example, the anti-inflammatory effects of certain VLC-PUFAs, among others.

[0142] Long-chain polyunsaturated fatty acids (LC-PUFAs) include omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids containing 18–22 carbons, including arachidonic acid (ARA, C20:4n-6, i.e., 20 carbons, 4 double bonds, omega-6), eicosapentaenoic acid (EPA, C20:5n-3, 20 carbons, 5 double bonds, omega-3), docosapentaenoic acid (DPA, C22:5n-3, 22 carbons, 5 double bonds, omega-3), and docosahexaenoic acid (DHA, C22:6n-3, 22 carbons, 6 double bonds, omega-3). LC-PUFAs are converted via lipoxygenase-type enzymes to biologically active hydroxylated PUFA derivatives that function as biologically active lipid mediators that play an important role in inflammation and related conditions. Among these, the most important are hydroxylated derivatives generated in certain inflammation-related cells through the action of lipoxygenase (LO or LOX) enzymes (e.g., 15-LO, 12-LO), resulting in the formation of mono-, di-, or trihydroxylated PUFA derivatives with potent activities, including anti-inflammatory, inflammation-resolving, neuroprotective, or tissue-protective activities, among others. For example, neuroprotectin D1 (NPD1), a dihydroxy derivative of DHA formed intracellularly through the enzymatic action of 15-lipoxygenase (15-LO), has been shown to possess a unique biological profile with defined R / S and Z / E stereochemical structures (10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) and stereoselective potent anti-inflammatory, homeostasis-restoring, inflammation-resolving, and bioactivities. NPD1 has been shown to regulate neuroinflammatory signaling and proteostasis, promoting neuroregeneration, neuroprotection, and cell survival.

[0143] Other important types of fatty acids are n-3 and n-6 ​​very long-chain polyunsaturated fatty acids (n-3 VLC-PUFAs, n-6 VLC-PUFAs), which are produced in cells containing elongase enzymes that elongate n-3 and n-6 ​​LC-PUFAs to n-3 and n-6 ​​VLC-PUFAs of 24 to 42 carbons (C24-C42). The most important of these appear to be the 28 to 38 carbon VLC-PUFAs (C28-C38). Representative types of VLC-PUFAs include C32:6n-3 (32 carbons, 6 double bonds, omega-3), C34:6n-3, C32:5n-3, and C34:5n-3. These VLC-PUFAs are biosynthetically derived by the action of elongase enzymes such as ELOVL4 (ELOngation of Very Long chain fatty acids 4). VLC-PUFAs are also acylated in complex lipids, including phospholipids such as sphingolipids and certain species of phosphatidylcholine.

[0144] The biosynthetic role of ELOVL4 and the biological functions of VLC-PUFAs have been the subject of much recent research. See, for example, PCT / US2016 / 017112, PCT / US2018 / 023082, and US16 / 576,456, each of which is incorporated herein by reference in its entirety. These VLC-PUFAs display functions in membrane organization and are increasingly recognized as important for health.

[0145] Embodiments of the present disclosure include compounds, compositions and methods that involve the use of n-3 VLC-PUFAs to alleviate symptoms of, prevent, or treat disease.

[0146] Biosynthetic pathway of n-3 VLC-PUFA: The biosynthesis of n-3 VLC-PUFAs begins with low-carbon PUFAs containing only an even number of carbon atoms in the carbon chain, such as docosahexaenoic acid (DHA), which contains 22 carbons and six alternating C=C bonds (C22:6n-3), and docosapentaenoic acid (DPA), which contains 22 carbons and five alternating C=C bonds (C22:5n-3). The biosynthesis of n-3 VLC-PUFAs requires the availability of DHA or other short-chain PUFAs as substrates and the presence and action of specific elongase enzymes, such as ELOVL4. As summarized in Figures 1 and 2, these 22-carbon omega-3 long-chain fatty acids (n-3 LC-PUFAs) are substrates for elongase enzymes, such as ELOVL4, which add two-carbon CH2CH2 groups at a time to the carboxyl terminus to form n-3 VLC-PUFAs containing carbon chains of at least 24 carbons and up to 42 carbons.

[0147] Docosahexaenoic acid (DHA, C22:6n-3, 1) is incorporated into the 2-position of phosphatidylcholine species (3) and converted into long-chain n-3 VLC-PUFAs by elongase enzymes. Elongation by the elongase enzyme ELOVL4 (ELOngation of Very Long chain fatty acids-4) leads to the formation of very long-chain omega-3 polyunsaturated fatty acids (n-3 VLC-PUFAs, 2, including C32:6n-3 and C34:6n-3) that are incorporated into the 1-position of phosphatidylcholine species (3). The presence of DHA at the 2-position and n-3 VLC-PUFAs at the 1-position can provide redundant, complementary, and synergistic cytoprotective and neuroprotective effects that enhance the survival of neurons and other important cell types in the face of pathological conditions.

[0148] Lipooxygenation of n-3-VLC-PUFAs leads to the formation of enzymatically hydroxylated derivatives of n-3-VLC-PUFAs called erovanoids, which can include monohydroxy compounds (e.g., ELV-27S and ELV-29S), and dihydroxy derivatives (e.g., ELV-N-32 and ELV-N-34). The erovanoid ELV-N-32 is a 20R,27S-dihydroxy 32:6 derivative (a 32-carbon, six double bond erovanoid with a neuroprotectin-like 20(R),27(S)-dihydroxy pattern). The erovanoid ELV-N-34 is a 22R,29S-dihydroxy 34:6 derivative (a 34-carbon, six double bond erovanoid with a 22(R),29(S)-dihydroxy pattern).

[0149] Figure 2 illustrates the delivery of docosahexaenoic acid (DHA, C22:6n-3) to photoreceptors, the regeneration of photoreceptor outer segment membranes, and the synthesis of ergobanoids. DHA or the precursor C18:3n-3, like DHA itself, can be obtained through the diet (Figure 1). The systemic circulation (primarily the portal venous system) transports it to the liver. Once in the liver, hepatocytes incorporate DHA into DHA-phospholipids (DHA-PL), which are transported as lipoproteins to capillaries, neurovascular units, and other tissues.

[0150] DHA travels from the choriocapillaris through Bruch's membrane (Figure 2), is taken up by retinal pigment epithelial (RPE) cells lining the retina, and is delivered to the inner photoreceptor segments. This targeted delivery pathway from the liver to the retina is called the DHA long loop.

[0151] DHA then passes through the interphotoreceptor matrix (IPM) to the photoreceptor inner segment, where it is incorporated into phospholipids in the photoreceptor outer segment, plasma membrane, and organelles. Most of it is used in disc membrane biosynthesis (outer segment). As new DHA-rich discs are synthesized at the base of the photoreceptor outer segment, older discs are extruded apically toward RPE cells. The tips of photoreceptors are phagocytosed daily by RPE cells, removing the oldest discs. The resulting phagosomes are degraded within the RPE cells, and DHA is recycled to the photoreceptor inner segment for new disc membrane biosynthesis. This local recycling is called the 22:6 short loop.

[0152] Erovanoids are formed from omega-3 very long-chain polyunsaturated fatty acids (n-3 VLC-PUFAs) biosynthesized by ELOVL4 (ELOngation of Very Long Chain Fatty Acids-4) in the inner segments of photoreceptors. Thus, inner segment phosphatidylcholine species containing a VLC omega-3 FA at C1 (depicted as C34:6n-3) and DHA (C22:6n-3) at C2 are used for photoreceptor membrane biosynthesis. This phospholipid is known to be closely associated with rhodopsin. During daily physiological processes, RPE cells phagocytose discs, and in the event of homeostatic disturbance, phospholipase A1 (PLA1) cleaves the acyl chain at sn-1, releasing C34:6n-3, forming erovanoids (e.g., erovanoid-34, ELV-N-34). VLC omega-3 fatty acids not used for erovanoid synthesis are recycled via a short loop.

[0153] Thus, for biosynthetic reasons, naturally occurring and biogenetically derived n-3 VLC-PUFAs contain only even numbers of carbons ranging from at least 24 carbons to at least 42 carbons (i.e., 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 carbons). Thus, n-3 VLC-PUFAs containing only odd numbers of carbons ranging from at least 23 to a maximum of 41 carbons (i.e., 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 carbons), while not naturally occurring, can be synthesized and produced using synthetic chemistry methods and strategies.

[0154] Stereocontrolled total synthesis and structural characterization of the erovanoids ELV-N-32 and ELV-N-34 in the retina and brain: As summarized in Figure 3 and Figure 4, ELV-N-32 (27S-) and ELV-N-34 were synthesized from three key intermediates (1, 2, and 3), each of which was prepared in stereochemically pure form. The stereochemistry of intermediates 2 and 3 was predefined by using enantiomerically pure epoxide starting materials. Iterative coupling of intermediates 1, 2, and 3 produced ELV-N-32 and ELV-N-34 (4), which were isolated as methyl esters (Me) or sodium salts (Na). The synthetic materials ELV-N-32 and ELV-N-34 were consistent with endogenous erovanoides with the same number of carbons on the carbon chain, obtained from cultured human retinal pigment epithelial cells (RPE) (Figure 3) and neuronal cultures (Figure 4).

[0155] Experimental detection and characterization of erovanoids: Experimental evidence demonstrates the biosynthetic formation of erovanoids, mono- and dihydroxy n-3 VLC-PUFA derivatives with molecular structures similar to DHA-derived 17-hydroxy-DHA and the dihydroxy compound NPD1 (10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid). erovanoids are enzymatically generated hydroxylated derivatives of 32-carbon (ELV-N-32) and 34-carbon (ELV-N-34) n-3 VLC-PUFAs and were first identified in primary human retinal pigment epithelial cell (RPE) cultures (Figure 3A–3K) and neuronal cell cultures (Figure 4A–4K).

[0156] This disclosure provides compounds with carbon chains related to n-3 VLC-PUFAs that contain one, two, or more hydroxyl groups in addition to six or five C=C bonds. Considering that this type of compound may be involved in the protective and neuroprotective effects of n-3 VLC-PUFAs, we attempted to identify their presence by supplementing cultured human retinal pigment epithelial cells with 32:6n-3 and 34:6n-3 VLC-PUFA fatty acids. Our results demonstrated monohydroxy and dihydroxy errovanoid derivatives from both 32:6n-3 and 34:6n-3 VLC-PUFA fatty acids. The structures of these errovanoids (ELV-N-32, ELV-N-34) were compared with standards prepared in stereochemically pure form via stereocontrolled total organic synthesis (Figures 5A and 5B).

[0157] Beneficial role of n-3 VLC-PUFAs as therapeutic agents: The data described herein provided support for the beneficial use of the provided n-3 VLC-PUFA and / or aerovanoid compounds as therapeutic agents for the prevention and treatment of disease.

[0158] The phrases "allergic disease" and "allergic inflammatory disease" can refer to diseases accompanied by allergic reactions. More specifically, "allergic disease" can be characterized by a strong correlation between exposure to allergens and the occurrence of pathological changes, and by the pathological changes being mediated by immune mechanisms (i.e., allergic inflammatory diseases). For example, immune mechanisms can refer to leukocytes exhibiting an immune response to allergen stimulation. For example, immune responses can refer to increased production of proinflammatory cytokines and chemokines. Examples of allergens include dust mite antigens and pollen antigens. Representative allergic diseases include bronchial asthma, allergic rhinitis, atopic dermatitis or allergic dermatitis, allergic conjunctivitis, and pollen and insect allergies. Allergic predisposition is a genetic factor that can be inherited by parents and children with an allergic predisposition. Familial allergic diseases are also called atopic diseases, and the underlying genetic factor is atopic constitution. "Atopic dermatitis" is a general term for atopic diseases, such as diseases associated with skin inflammation. Non-limiting examples may include allergic conditions selected from the group consisting of eczema, allergic rhinitis, hay fever, urticaria, and food allergies, including eczema, allergic rhinitis or rhinitis, hay fever, bronchial asthma, urticaria (hives), food allergies, and other atopic conditions.

[0159] "Asthma" can refer to a disorder of the respiratory system characterized by inflammation, airway narrowing, and increased airway reactivity to inhaled substances or allergens. Asthma is often associated with, but not limited to, atopic or allergic conditions. It is widely recognized that asthma symptoms can include dyspnea, cough, and wheezing. All three symptoms can coexist, but their coexistence is not required to make a diagnosis of asthma.

[0160] The term "allergic asthma" can refer to the allergic aspect of asthma among asthma symptoms, and can include, for example, mixed asthma and atopic asthma. Allergic asthma is distinguished from non-allergic asthma, such as aspirin asthma. For example, a "therapeutic drug for asthma" can exert its therapeutic effect through its action on the allergic response of asthma. Furthermore, a therapeutic drug for asthma can exert its therapeutic effect on, for example, chronic bronchitis or airway hyperresponsiveness. For example, a therapeutic drug for asthma affects chronic bronchitis and airway hyperresponsiveness. A therapeutic drug for asthma affects the late-phase, delayed-phase, or late and delayed-phase reactions of the allergic response. For example, a therapeutic drug for asthma affects not only the immediate-phase reaction but also the late-phase, delayed-phase, or late and delayed-phase reactions.

[0161] "Allergic rhinitis" can refer to any allergic reaction of the nasal mucosa, including hay fever (seasonal allergic rhinitis) and perennial rhinitis (non-seasonal allergic rhinitis). Symptoms of allergic rhinitis include sneezing, rhinorrhea, nasal congestion, itching, and itchy, red, and watery eyes.

[0162] The term "skin disorder" can include urticaria and angioedema skin reactions. These skin disorders can be caused by exposure to certain foods, medications, or viral infections. Urticaria (also called hives or wheals) are red, itchy, raised areas of skin of various shapes and sizes. Urticaria is the result of the release of histamine and other compounds from mast cells, causing serum to leak from local blood vessels, thereby causing skin swelling. Angioedema is a type of tissue swelling similar to urticaria, but involves deeper skin tissues (i.e., "deep urticaria") and lasts longer than urticaria.

[0163] The term "allergic dermatitis" can refer to dermatitis associated with an allergic reaction, and can include, for example, atopic dermatitis. Allergic dermatitis is distinguished from non-allergic dermatitis, such as dermatitis caused by injury or wounds. A "therapeutic agent for atopic dermatitis" that exerts a therapeutic effect by acting on the allergic reaction occurring in atopic dermatitis is useful. Furthermore, a therapeutic agent for atopic dermatitis affects the late-phase reaction, delayed-phase reaction, or late-phase and delayed-phase reaction of the allergic reaction. For example, a therapeutic agent for atopic dermatitis affects not only the immediate-phase reaction but also the late-phase reaction, delayed-phase reaction, or late-phase and delayed-phase reaction.

[0164] The term "allergic conjunctivitis" can refer to allergen irritation of the thin, clear membrane called the conjunctiva, which lines the inside of the eyeball and eyelid. Symptoms can include eye swelling, itchy / burning eyes, watery eyes, and redness. Some allergens can include secretions from trees, grasses, ragweed pollen, animal skin, saliva, perfumes, cosmetics, skin medications, air pollution, and smoke.

[0165] "Allergen" can refer to a substance that can induce an allergic inflammatory disease in a subject. The list of allergens is extensive and can include pollen, insect venom, animal dander, house dust mites, dust, fungal spores, latex, and drugs (e.g., penicillin). Examples of natural, animal, and plant allergens include proteins specific to the following genera: Canis (Canis familiaris), Dermatophagoides (e.g., Dermatophagoides farinae), Felis (Felis domesticus), Ambrosia (ragweed Ambrosia artemiisfolia), Lolium (e.g., Lolium perenne or Lolium multiflorum), Cryptomeria (Cryptomeria japonica), Alternaria (Alternaria alternata), Alder, Alnus (European alder), gultinosa), Birch (Betula verrucosa), Quercus (Quercus alba), Olea (Olea europa), Artemisia (Artemisia vulgaris), Plantago (e.g., Plantago lanceolata), Parietaria (e.g., Parietaria officinalis or Parietaria judaica), Blattella (e.g., Blattella germanica), Apis (e.g., Apis multiflorum),multiflorum), Cupressus (e.g., Cupressus sempervirens, Cupressus arizonica, and Cupressus macrocarpa), Juniperus (e.g., Juniperus sabinoides, Juniperus virginiana, Juniperus communis, Juniperus ashei), Thuya (e.g., Thuya orientalis), Chamaecyparis (e.g., Chamaecyparis obtusa), Periplaneta (e.g., Periplaneta americana), americana), Agropyron (e.g., Agropyron repens), Secale (e.g., Secale cereale), Triticum (e.g., Triticum aestivum), Dactylis (e.g., Dactylis glomerata), Festuca (e.g., Festuca elatior), Poa (e.g., Poa pratensis or Poa compressa), Avena (e.g., Avena sativa), Holcus (e.g., Holcus cereale), lanatus), Anthoxanthum (e.g., Anthoxanthum odoratum), Arrhenatherum (e.g., Arrhenatherum elatius), Agrostis (e.g., Agrostis alba), Phleum (e.g., PhleumAllergens may also include peptides and polypeptides used in experimental animal models of allergy and asthma, including ovalbumin (OVA) and Schistosoma mansoni egg antigen.

[0166] "Metabolic disorders" can refer to disorders or diseases that result in disruption of the normal physiological state of homeostasis due to metabolic alterations (anabolism and / or catabolism), such as an inability to break down (catabolism) substances to be broken down (e.g., phenylalanine), elevated levels of substances and / or intermediate substances, or an inability to produce (anabolism) some essential substances (e.g., insulin).

[0167] "Metabolic syndrome" can refer to the concept of a group of metabolic syndromes that congregate in a single individual and place them at increased risk for developing diabetes and / or cardiovascular disease. Key features of metabolic syndrome include insulin resistance, hypertension (high blood pressure), cholesterol abnormalities, dyslipidemia, triglyceride abnormalities, and increased risk of clotting, particularly abdominal and overweight or obesity. Metabolic syndrome is also known as syndrome X, insulin resistance syndrome, obesity syndrome, dysmetabolic syndrome, and Reaven syndrome. The interrelationships of various risk factors for metabolic syndrome are illustrated in Figure 1. The presence of three or more risk factors in a single individual is indicative of metabolic syndrome. The American Heart Association states that metabolic syndrome is diagnosed by the presence of three or more of the following factors: (1) increased waist circumference (men, 40 inches (102 cm) or more; women, 35 inches (88 cm) or more); (2) elevated triglycerides (150 mg / dL or more); (3) decreased high-density lipids or HDL (men, less than 40 mg / dL; women, less than 50 mg / dL); (4) elevated blood pressure (130 / 85 mmHg or more); and (5) elevated fasting blood glucose (100 mg / dL or more).

[0168] "Metabolic syndrome-related metabolic disorders" can refer to metabolic syndrome and obesity, insulin resistance, type 2 diabetes, atherosclerosis, and cardiomyopathy.

[0169] "Diabetes" can refer to a group of metabolic disorders characterized by high blood sugar (glucose) levels due to a deficiency in insulin secretion or action, or both.

[0170] "Type 2 diabetes" is one of the two main types of diabetes because the beta cells in the pancreas produce insulin, but the body's cells are resistant to the effects of insulin, at least in the early stages of the disease. Later in the disease, the beta cells may stop producing insulin. Type 2 diabetes is also known as insulin-resistant diabetes, non-insulin-dependent diabetes, and adult-onset diabetes.

[0171] "Prediabetes" can refer to one or more early diabetic conditions including impaired glucose utilization, impaired or impaired fasting plasma glucose, impaired glucose tolerance, impaired insulin sensitivity, and impaired insulin resistance.

[0172] "Insulin resistance" means that cells become resistant to the action of insulin (the hormone that controls glucose uptake into cells) or that the amount of insulin produced is insufficient to maintain normal glucose levels. Cells may become less able to respond to the effects of insulin (i.e., loss of sensitivity to insulin) in promoting the transport of the sugar glucose from the blood to muscles and other tissues. Eventually, the pancreas produces much more insulin than normal, and cells maintain resistance. As long as enough insulin is produced to overcome this resistance, blood glucose levels remain normal. When the pancreas can no longer keep up, blood glucose levels begin to rise, leading to diabetes. Insulin resistance can range from normal (insulin sensitive) to insulin resistant (IR).

[0173] "A-beta-related disease" may refer to a disease or condition characterized by A-beta protein aggregates. The primary component of the amyloid plaques characteristic of A-beta-related diseases is the beta-amyloid peptide (A-beta), a highly insoluble peptide 39-43 amino acids (aa) in length that adopts a beta-sheet structure and has a strong tendency to oligomerize and form protein aggregates. Non-limiting examples of A-beta-related diseases include neurodegenerative diseases or disorders, Alzheimer's disease, Alzheimer's dementia, cerebral amyloid angiopathy (CAA), trisomy 21 (Down syndrome), adult Down syndrome, hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), dementia with Lewy bodies, frontotemporal lobar degeneration, glaucoma, age-related macular degeneration, amyotrophic lateral sclerosis, sporadic inclusion body myositis, and anxiety disorders in elderly human subjects.

[0174] Origin of the compounds of the present disclosure: The provided compounds were isolated not from naturally occurring tissues but from the results of artificial experiments combining human cells with chemically synthesized n-3 VLC-PUFAs. Using HPLC and mass spectrometry, the general structures of our synthetic elovanoid compounds were consistent with compounds biosynthesized in human retinal pigment epithelial cells or detected in neuronal cell cultures. However, the natural occurrence of the provided mono- and dihydroxylated elovanoids with specifically defined stereochemistry is currently unknown. Furthermore, the provided compounds are not obtained from natural sources; rather, they are prepared by adapting stereocontrolled synthesis methods known in the art starting from commercially available materials. The provided preparation method is designed to accommodate the inherent hydrophobic properties of n-3 VLC-PUFAs, which differ significantly from compounds with a total carbon number of 22 or fewer.

[0175] The present disclosure encompasses compounds that have stereochemically pure structures and are chemically synthesized and modified to have additional structural features and properties that enable them to exert pharmacological activity. The provided compounds are chemically modified pharmaceutically acceptable derivatives in the form of carboxylic acid esters or salts that enhance chemical and biological stability and enable their use in therapeutic applications, including various forms of drug delivery.

[0176] The present disclosure also provides pharmacologically effective compositions of provided compounds that enhance their ability to be delivered to a subject in a manner that allows them to reach targeted cells and tissues.

[0177] The data described herein also provided support for the beneficial use of provided n-3 VLC-PUFA and / or aerovanoid compounds as therapeutic agents for the prevention and treatment of diseases, such as diseases associated with allergies or allergic reactions, by inhibiting the production of pro-inflammatory cytokines and chemokines by cells, such as epithelial cells.

[0178] Epithelium lines both the outside of the body (skin) and the internal cavities and lumens. Epithelial tissue is scoot-like, tightly packed, and forms a continuous sheet. There are few intercellular spaces. Epithelium is separated from underlying tissues by an extracellular fibrous basement membrane. The lining of the mouth, alveoli, and kidney tubules are all made of epithelial tissue. The lining of blood and lymphatic vessels is a specialized form of epithelium called endothelium.

[0179] The term "epithelial cell" can refer to cells that line the exterior (skin), mucous membranes, and interior cavities and lumens of the body. Most epithelial cells exhibit apical-basal polarization of cellular components. Epithelial cells are classified according to their shape and their specializations.

[0180] The epidermis (i.e., skin) is composed of keratinized stratified squamous epithelium. Four cell types are present: Keratinocytes produce keratin, a protein that hardens and waterproofs the skin. Mature keratinocytes at the skin's surface are dead and almost completely filled with keratin. Melanocytes produce melanin, a pigment that protects cells from ultraviolet light. Melanin from melanocytes is transferred to keratinocytes. Langerhans cells are phagocytic macrophages that interact with white blood cells during the immune response. Merkel cells occur deep in the epidermis, at the border between the epidermis and dermis. They form Merkel's discs and perform sensory functions in association with nerve endings.

[0181] The epidermis is made up of several layers. Thick skin, found on the palms of the hands and soles of the feet, is composed of five layers, while thin skin is composed of only four. The five layers can include the stratum corneum, which contains many layers of dead, anucleated keratinocytes completely filled with keratin. The outermost layer is constantly shed. The stratum lucidum contains two to three layers of anucleated cells. This layer is found only in thick skin, such as the palms of the hands and soles of the feet. The stratum granulosum contains two to four layers of cells connected by desmosomes. These cells contain keratohyalin granules, which contribute to the formation of keratin in the upper layers of the epidermis. The stratum spinosum contains eight to ten layers of cells connected by desmosomes. These cells are moderately mitotically active. The stratum germinativum contains a single layer of columnar cells that actively divide by mitosis to generate cells that migrate to the upper epidermal layers and ultimately to the surface of the skin.

[0182] For example, nasal epithelial cells form the outermost protective layer against environmental factors. They clean, humidify, and warm inhaled air. They also produce mucus, which binds to particles and is then transported to the pharynx by the cilia of the epithelial cells.

[0183] For example, the corneal epithelium, which is composed of epithelial tissue and covers the anterior surface of the cornea, acts as a protective barrier, resisting the free flow of fluid from tears and preventing bacteria from invading the epithelium and corneal stroma.

[0184] Respiratory epithelium, or airway epithelium, is a type of ciliated columnar epithelium found lining most of the respiratory tract as the respiratory mucosa. Respiratory epithelium has four main types of cells: a) ciliated cells, b) goblet cells, c) club cells, and d) basal cells. Respiratory epithelium functions to moisten and protect the airways. It acts as a physical barrier against pathogens and foreign bodies, removes pathogens through the mucociliary clearance mechanism, and prevents infection and tissue damage through mucus secretion and mucociliary clearance.

[0185] compound Described herein are compounds based on omega-3 very long chain polyunsaturated fatty acids and their hydroxylated derivatives, termed "erovanoids."

[0186] Omega-3 very long chain polyunsaturated fatty acids have the structure A or B, or their derivatives. TIFF2025143368000012.tif36128A contains a total of 23 to 42 carbon atoms in the carbon chain and has six alternating cis carbon-carbon double bonds beginning at positions n-3, n-6, n-9, n-12, n-15, and n-18, and B contains a total of 23 to 42 carbon atoms in the carbon chain and has five alternating cis carbon-carbon double bonds beginning at positions n-3, n-6, n-9, n-12, and n-15. R can be hydrogen, methyl, ethyl, alkyl, or a cation such as an ammonium cation, an iminium cation, or a metal cation including, but not limited to, sodium, potassium, magnesium, zinc, or calcium cation, and m is a number from 0 to 19.

[0187] The omega-3 very long chain polyunsaturated fatty acids of the present disclosure can have a terminal carboxyl group "-COOR," where "R" can represent a group covalently attached to the carboxyl, such as an alkyl group. Alternatively, the carboxyl group can be further modified with "-COO - " and R is a cation, including metal cations, ammonium cations, etc.

[0188] In some omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 0 to 15. Thus, it can be a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, where the fatty acid moiety contains a total of 24, 26, 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In other omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 0, 2, 4, 6, 8, 10, 12, or 14, where the fatty acid moiety contains a total of 23, 25, 27, 19, 31, 33, 35, or 37 carbon atoms in its carbon chain. In some omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 5 to 15, where the fatty acid moiety contains a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 carbon atoms in its carbon chain. In some omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 9 to 11, and the fatty acid moiety contains a total of 32 or 34 carbon atoms in its carbon chain.

[0189] In some embodiments, the omega-3 very long chain polyunsaturated fatty acid is a carboxylic acid, i.e., R is hydrogen. In other embodiments, the omega-3 very long chain polyunsaturated fatty acid is a carboxylic acid ester, and R is methyl, ethyl, or alkyl. When the omega-3 very long chain polyunsaturated fatty acid is a carboxylic acid ester, R can be, but is not limited to, methyl or ethyl. In some embodiments, the omega-3 very long chain polyunsaturated fatty acid is a carboxylic acid ester, and R is methyl.

[0190] In some embodiments, the omega-3 very long chain polyunsaturated fatty acid can be a carboxylate salt. R is an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cation. In some advantageous embodiments, R is an ammonium cation or an iminium cation. R can be a sodium cation or a potassium cation. In some embodiments, R is a sodium cation.

[0191] The omega-3 very long chain polyunsaturated fatty acid or derivative of the present disclosure may have 32 or 34 carbons in its carbon chain and six alternating cis double bonds starting at the n-3 position, and has the formula A1 (14Z,17Z,20Z,23Z,26Z,29Z)-dotriaconta-14,17,20,23,26,29-hexaenoic acid) or the formula A2 (16Z,19Z,22Z,25Z,28Z,31Z)-tetratriaconta-16,19,22,25,28,31-hexaenoic acid). TIFF2025143368000013.tif38128

[0192] In some embodiments of the omega-3 very long chain polyunsaturated fatty acids, the carboxyl derivatives are part of glycerol-derived phospholipids, which are known in the art and can be readily prepared starting from the carboxylic acid form of an n-3 VLC-PUFA of structure A or B by utilizing methods represented by structures C, D, E, or F. C or E has a total of 23-42 carbon atoms in its carbon chain and six alternating cis carbon-carbon double bonds beginning at positions n-3, n-6, n-9, n-12, n-15, and n-18; D or E has a total of 23-42 carbon atoms in its carbon chain and five alternating cis carbon-carbon double bonds beginning at positions n-3, n-6, n-9, n-12, and n-15. In advantageous embodiments, m is a number selected from the group consisting of 0-15. In other embodiments, m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, and the fatty acid moiety has a total of 24, 26, 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In further advantageous embodiments, m is a number selected from the group consisting of 0, 2, 4, 6, 8, 10, 12 or 14, and the fatty acid moiety comprises a total of 23, 25, 27, 19, 31, 33, 35 or 37 carbon atoms in its carbon chain.

[0193] In some embodiments, m is a number selected from the group consisting of 5 to 15, and the fatty acid moiety comprises a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 carbon atoms in its carbon chain. In some embodiments, m is a number selected from the group consisting of 5, 7, 9, 11, 13, or 15, and the fatty acid moiety comprises a total of 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In other embodiments, m is a number selected from the group consisting of 4, 6, 8, 10, 12, or 14, and the fatty acid moiety comprises a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In advantageous embodiments, m is a number selected from the group consisting of 9 to 11, and the fatty acid moiety comprises a total of 32 or 34 carbon atoms in its carbon chain.

[0194] The monohydroxylated erovanoids of the present disclosure can have a G, H, I, or J structure. TIFF2025143368000015.tif44128 Compounds G and H have a total of 23-42 carbon atoms in the carbon chain, five cis carbon-carbon double bonds beginning at positions n-3, n-9, n-12, n-15, and n-18, and a trans carbon-carbon double bond beginning at position n-7. Compounds I and J have a total of 23-42 carbon atoms in the carbon chain, four cis carbon-carbon double bonds beginning at positions n-3, n-9, n-12, and n-15, and a trans carbon-carbon double bond beginning at position n-7. R is a cation selected from the group consisting of hydrogen, methyl, ethyl, alkyl, or a metal cation selected from the group consisting of ammonium cation, iminium cation, or sodium, potassium, magnesium, zinc, or calcium cation. m is a number selected from the group consisting of 0-19. Compounds G and H can exist as an equimolar mixture. Compounds I and J can exist as an equimolar mixture. The provided compounds G and H are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group. Compounds G and H are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0195] As used herein and in other structures of the present disclosure, compounds of the present disclosure are designated as having a terminal carboxyl group "-COOR", where "R" can represent a group covalently bonded to the carboxyl, such as an alkyl group. Alternatively, the carboxyl group can be further designated as "-COO - " and R is a cation, including metal cations, ammonium cations, etc.

[0196] In some embodiments of the monohydroxylated erovanoids of the present disclosure, m is a number selected from the group consisting of 0 to 15. In other advantageous embodiments, m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, and the fatty acid moiety comprises a total of 24, 26, 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In other embodiments, m is a number selected from the group consisting of 0, 2, 4, 6, 8, 10, 12, or 14, and the fatty acid moiety comprises a total of 23, 25, 27, 19, 31, 33, 35, or 37 carbon atoms in its carbon chain.

[0197] In some embodiments, m is a number selected from the group consisting of 5 to 15, and the fatty acid moiety comprises a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 carbon atoms in its carbon chain. In some embodiments, m is a number selected from the group consisting of 5, 7, 9, 11, 13, or 15, and the fatty acid moiety comprises a total of 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In other embodiments, m is a number selected from the group consisting of 4, 6, 8, 10, 12, or 14, and the fatty acid moiety comprises a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In advantageous embodiments, m is a number selected from the group consisting of 9 to 11, and the fatty acid moiety comprises a total of 32 or 34 carbon atoms in its carbon chain.

[0198] In some embodiments, the monohydroxylated erovanoid of the present disclosure is a carboxylic acid, i.e., R is hydrogen. In other embodiments, the compound is a carboxylic acid ester, and R is methyl, ethyl, or alkyl. In advantageous embodiments, the compound is a carboxylic acid ester, and R is methyl or ethyl. In advantageous embodiments, the compound is a carboxylic acid ester, and R is methyl. In other advantageous embodiments, the compound is a carboxylate salt, and R is an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cation. In some advantageous embodiments, R is an ammonium cation or an iminium cation. In other advantageous embodiments, R is a sodium cation or a potassium cation. In advantageous embodiments, R is a sodium cation.

[0199] The dihydroxylated elastovanoids of the present disclosure can have the structure K, L, M, or N. TIFF2025143368000016.tif61128 Compounds K and L have a total of 23 to 42 carbon atoms in the carbon chain, with four cis carbon-carbon double bonds beginning at positions n-3, n-7, n-15, and n-18, and two trans carbon-carbon bonds beginning at positions n-9 and n-11. Compounds M and N have a total of 23 to 42 carbon atoms in the carbon chain, with three cis carbon-carbon double bonds beginning at positions n-3, n-7, n-12, and n-15, and two trans carbon-carbon bonds beginning at positions n-9 and n-11. R is a cation selected from the group consisting of hydrogen, methyl, ethyl, alkyl, or a metal cation selected from the group consisting of ammonium cation, iminium cation, or sodium, potassium, magnesium, zinc, or calcium cation. m is a number selected from the group consisting of 0 to 19. Compounds K and L can exist as an equimolar mixture. Compounds M and N can exist as equimolar mixtures. Compounds K and L are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group. Provided compounds M and N are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0200] As used herein and in other structures of the present disclosure, compounds of the present disclosure are designated as having a terminal carboxyl group "-COOR", where "R" can represent a group covalently bonded to the carboxyl, such as an alkyl group. Alternatively, the carboxyl group can be further designated as "-COO - " and R is a cation, including metal cations, ammonium cations, etc.

[0201] In some embodiments of the dihydroxylated erovanoids of the present disclosure, m is a number selected from the group consisting of 5 to 15, and the fatty acid moiety contains a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 carbon atoms in its carbon chain. In useful embodiments, m is a number selected from the group consisting of 5, 7, 9, 11, 13, or 15, and the fatty acid moiety contains a total of 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In other embodiments, m is a number selected from the group consisting of 4, 6, 8, 10, 12, or 14, and the fatty acid moiety contains a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In useful embodiments, m is a number selected from the group consisting of 9 to 11, and the fatty acid moiety contains a total of 32 or 34 carbon atoms in its carbon chain.

[0202] Some dihydroxylated elovanoids of the present disclosure are carboxylic acids, that is, R is hydrogen.In other embodiments, the dihydroxylated elovanoids of the present disclosure are carboxylic acid esters, and R is methyl, ethyl, or alkyl.In useful embodiments, the compound is a carboxylic acid ester, and R is methyl or ethyl.In useful embodiments, the compound is a carboxylic acid ester, and R is methyl.

[0203] In other embodiments, the dihydroxylated erovanoid of the present disclosure is a carboxylate, and R is an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cation. In some useful embodiments, R is an ammonium cation or an iminium cation. In other useful embodiments, R is a sodium cation or a potassium cation. In useful embodiments, R is a sodium cation.

[0204] The alkynyl monohydroxylated elastomeric compounds of the present disclosure can have the O, P, Q, or R structure. TIFF2025143368000017.tif36135 Compounds O and P have a total of 23 to 42 carbon atoms in the carbon chain, four cis carbon-carbon double bonds beginning at positions n-3, n-12, n-15, and n-18, a trans carbon-carbon bond beginning at position n-7, and a carbon-carbon triple bond beginning at position n-9. Compounds I and J have a total of 23 to 42 carbon atoms in the carbon chain, three cis carbon-carbon double bonds beginning at positions n-3, n-12, and n-15, a trans carbon-carbon bond beginning at position n-7, and a carbon-carbon triple bond beginning at position n-9. R is a cation selected from the group consisting of hydrogen, methyl, ethyl, alkyl, or a metal cation selected from the group consisting of ammonium cation, iminium cation, or sodium, potassium, magnesium, zinc, or calcium cation. m is a number selected from the group consisting of 0 to 19. Compounds O and P can be present as an equimolar mixture. Compounds Q and R can be present as an equimolar mixture. Provided compounds O and P are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group. Provided compounds O and P are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0205] As used herein and in other structures of the present invention, the alkynyl monohydroxylated elastomeric compounds of the present disclosure are designated as having a terminal carboxyl group "-COOR", where "R" can represent a group covalently bonded to the carboxyl, such as an alkyl group. Alternatively, the carboxyl group can be further designated as "-COO - " and R is a cation, including metal cations, ammonium cations, etc.

[0206] In some embodiments, m is a number selected from the group consisting of 0 to 15. In other embodiments, m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, and the fatty acid moiety contains a total of 24, 26, 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain.

[0207] In additional embodiments, m is a number selected from the group consisting of 0, 2, 4, 6, 8, 10, 12, or 14, and the fatty acid moiety comprises a total of 23, 25, 27, 19, 31, 33, 35, or 37 carbon atoms in its carbon chain. In some embodiments, m is a number selected from the group consisting of 5-15, and the fatty acid moiety comprises a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 carbon atoms in its carbon chain. In embodiments, m is a number selected from the group consisting of 5, 7, 9, 11, 13, or 15, and the fatty acid moiety comprises a total of 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In other embodiments, m is a number selected from the group consisting of 4, 6, 8, 10, 12, or 14, and the fatty acid moiety comprises a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In some embodiments, m is a number selected from the group consisting of 9 to 11, and the fatty acid moiety contains a total of 32 or 34 carbon atoms in its carbon chain.

[0208] In some embodiments, the alkynyl monohydroxylated elastomeric compounds of the present disclosure are carboxylic acids, i.e., R is hydrogen. In other embodiments, the alkynyl monohydroxylated elastomeric compounds of the present disclosure are carboxylic acid esters, and R is methyl, ethyl, or alkyl. In embodiments, the alkynyl monohydroxylated elastomeric compounds of the present disclosure are carboxylic acid esters, and R is methyl or ethyl.

[0209] In some embodiments, R is methyl. In other embodiments, the alkynyl monohydroxylated erovanoid of the present disclosure can be a carboxylate, and R is an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cation. In some embodiments, R is an ammonium cation or an iminium cation. In other embodiments, R is a sodium cation or a potassium cation. In some embodiments, R is a sodium cation.

[0210] The alkynyl dihydroxylated elastomeric compounds can have the S, T, U, or V configuration. TIFF2025143368000018.tif29145 Compounds S and T have a total of 23 to 42 carbon atoms in the carbon chain, three cis carbon-carbon double bonds beginning at positions n-3, n-12, n-15, and n-18, two trans carbon-carbon double bonds beginning at positions n-9 and n-11, and a carbon-carbon triple bond beginning at position n-7. Compounds U and V have a total of 23 to 42 carbon atoms in the carbon chain, two cis carbon-carbon double bonds beginning at positions n-3 and n-15, two trans carbon-carbon double bonds beginning at positions n-9 and n-11, and a carbon-carbon triple bond beginning at position n-7. R is a cation selected from the group consisting of hydrogen, methyl, ethyl, alkyl, or a metal cation selected from the group consisting of ammonium cation, iminium cation, or sodium, potassium, magnesium, zinc, or calcium cation. m is a number selected from the group consisting of 0 to 19. Compounds S and T can be present as an equimolar mixture. Compounds U and V can be present as an equimolar mixture.

[0211] In some embodiments, provided compounds S and T are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group. Provided compounds U and V are predominantly one enantiomer with defined (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0212] As used herein and in other structures of the invention, the compounds of the invention are depicted as having a terminal carboxyl group "-COOR", where "R" can represent a group covalently attached to the carboxyl, such as an alkyl group. Alternatively, the carboxyl group can be further designated "-COO - " and R is a cation, including metal cations, ammonium cations, etc.

[0213] In some embodiments, m is a number selected from the group consisting of 5 to 15, and the fatty acid moiety comprises a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 carbon atoms in its carbon chain. In embodiments, m is a number selected from the group consisting of 5, 7, 9, 11, 13, or 15, and the fatty acid moiety comprises a total of 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain. In other embodiments, m is a number selected from the group consisting of 4, 6, 8, 10, 12, or 14, and the fatty acid moiety comprises a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In embodiments, m is a number selected from the group consisting of 9 to 11, and the fatty acid moiety comprises a total of 32 or 34 carbon atoms in its carbon chain.

[0214] In some embodiments, provided compounds are carboxylic acids, i.e., R is hydrogen.

[0215] In other embodiments, provided compounds are carboxylic acid esters and R is methyl, ethyl, or alkyl. In embodiments, provided compounds are carboxylic acid esters and R is methyl or ethyl. In embodiments, provided compounds are carboxylic acid esters and R is methyl. In other embodiments, provided compounds are carboxylic acid salts and R is an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cations. In some embodiments, R is an ammonium cation or an iminium cation. In other embodiments, R is a sodium cation or a potassium cation. In embodiments, R is a sodium cation.

[0216] In embodiments, the present disclosure provides a monohydroxylated 32-carbon methyl ester of formula G1, named methyl (S,14Z,17Z,20Z,23Z,25E,29Z)-27-hydroxydotriaconta-14,17,20,23,25,29-hexaenoate; a monohydroxylated 32-carbon sodium salt of formula G2, named sodium (S,14Z,17Z,20Z,23Z,25E,29Z)-27-hydroxydotriaconta-14,17,20,23,25,29-hexaenoate; a monohydroxylated 34-carbon methyl ester of formula G3 named methyl (S,16Z,19Z,22Z,25Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,25,27,31-hexaenoate; or a monohydroxylated 34-carbon sodium salt of formula G4 named sodium (S,16Z,19Z,22Z,25Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,25,27,31-hexaenoate. TIFF2025143368000019.tif79135

[0217] In other embodiments, the present disclosure provides a dihydroxylated 32-carbon methyl ester of formula K1 named methyl (14Z,17Z,20R,21E,23E,25Z,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,25,29-hexaenoate; a dihydroxylated 32-carbon sodium salt of formula K2 named sodium (14Z,17Z,20R,21E,23E,25Z,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,25,29-hexaenoate; or or a dihydroxylated 34-carbon methyl ester of formula K3, named methyl (16Z,19Z,22R,23E,25E,27Z,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,27,31-hexaenoate; or a dihydroxylated 34-carbon sodium salt of formula K4, named sodium (16Z,19Z,22R,23E,25E,27Z,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,27,31-hexaenoate. TIFF2025143368000020.tif62145

[0218] In another embodiment, the present invention provides an alkynyl monohydroxylated 32-carbon methyl ester of formula O1, named methyl (S,14Z,17Z,20Z,25E,29Z)-27-hydroxydotriaconta-14,17,20,25,29-hydroxydotriaconta-23-ynoate; an alkynyl monohydroxylated 32-carbon sodium ester of formula O2, named sodium (S,17Z,20Z,25E,29Z)-27-hydroxydotriaconta-17,20,25,29-tetraen-23-ynoate; alkynyl monohydroxylated 34-carbon methyl ester of formula O3 named methyl (S,16Z,19Z,22Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,27,31-pentaen-25-ynoate; and alkynyl monohydroxylated 34-carbon sodium salt of formula O4 named sodium (S,16Z,19Z,22Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,27,31-pentaen-25-ynoate. TIFF2025143368000021.tif81128

[0219] In another advantageous embodiment, the present invention provides an alkynyl dihydroxylated 32-carbon methyl ester of formula S1, named methyl (14Z,17Z,20R,21E,23E,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,29-pentaen-25-ynoate; an alkynyl dihydroxylated 32-carbon sodium salt of formula S2, named sodium (14Z,17Z,20R,21E,23E,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,29-pentaen-25-ynoate; or an alkynyl dihydroxylated 34-carbon methyl ester of formula S3 named methyl (16Z,19Z,22R,23E,25E,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,31-pentaen-27-ynoate; or an alkynyl dihydroxylated 34-carbon sodium salt of formula S4 named sodium (16Z,19Z,22R,23E,25E,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,31-pentaen-27-ynoate. TIFF2025143368000022.tif98136

[0220] Methods for preparing and manufacturing the compounds provided: The compounds provided in this disclosure can be readily prepared by adapting methods known in the art, starting with commercially available materials as summarized in Schemes 1-5 shown in Figures 6-10.

[0221] Scheme 1 (Figure 6) shows a detailed approach for the stereocontrolled total synthesis of type O compounds, where n is 9, the fatty acid chain contains a total of 32 carbon atoms, and the R group is a methyl or sodium cation. For example, Scheme 1 illustrates the synthesis of compounds ELV-N-32-Me and ELV-N-32-Na starting from methyl pentadec-14-ynoate (S1). Starting from heptadec-16-ynoate (T1), this process affords compounds ELV-N-34-Me and ELV-N-34-Na. The alkynyl precursors of ELV-N-32-Me and ELV-N-32-Na, i.e., 13a, 13b, 15a, and 15b, are also included among compounds X and Z provided in the present disclosure. Scheme 1 provides reagents and conditions for the preparation of the provided compounds using reaction conditions typical for this type of reaction.

[0222] Scheme 2 (Figure 7) describes the total synthesis of dihydroxylated erovanoids K and L and their alkyne precursors S and T by starting from intermediates 2, 5, and 7, which were also used in Scheme 1. The conversion of the protected (R) epoxide 4 to intermediate 15, as well as the coupling of 7 and 15 to intermediate 17, can be accomplished according to literature procedures (Tetrahedron Lett. 2012;53(14):1695-8).

[0223] Catalytic cross-coupling between intermediates 2 or 17, or between intermediates 5 or 17, followed by deprotection, forms alkynyl compounds S and T, which are then selectively reduced to form dihydroxylated elovanoids K and L. Hydrolysis and acidification affords the corresponding carboxylic acids, which can be converted to carboxylate salts by adding an equivalent amount of the corresponding base. By varying the number of carbons in the alkyne starting material 7, dihydroxylated elovanoids of types K, L, S, and T with at least 23 carbons and up to 42 carbons in the carbon chain can be similarly prepared.

[0224] Scheme 3 (Figure 8) shows the total synthesis of dihydroxylated erovanoids with five unsaturated double bonds of type M and N, and their alkyne precursors U and V, by utilizing the same alkynyl intermediates 2 and 5 also used in Scheme 1. (Tetrahedron Lett. 2012;53(14):1695-8).

[0225] The synthesis of intermediate 22 begins with carboxylic acid 18, which is converted to orthoester 19 using known methodology (Tetrahedron Lett. 1983, 24(50), 5571-4). Reaction of lithiated alkynes with epoxide 1 affords intermediate 21, which is converted to iodide intermediate 22, similar to the conversion to 16-17. Catalytic cross-coupling between intermediates 2 or 5 and 22, followed by deprotection, leads to the formation of alkynyl dihydroxyerovanoids U and V, which are then selectively reduced to form dihydroxylated erovanoids M and N.

[0226] Hydrolysis and acidification afford the corresponding carboxylic acid, which can be converted to the carboxylate salt by adding an equivalent amount of the corresponding base. By varying the number of carbon atoms in the alkyne carboxylic acid 18, dihydroxylated elastomeric esters of types M, N, U, and V, with at least 23 carbons and up to 42 carbons in the carbon chain, can be similarly prepared.

[0227] Scheme 4 (Figure 9) shows the stereocontrolled total synthesis of 32-carbon dihydroxylated elovanoids starting from alkyne methyl ester 23, intermediate 15, and alkyne intermediate 2. For example, this scheme shows the total synthesis of the 32-carbon alkynyl elovanoid compound ELV-N-32-Me-acetylene and its conversion to elovanoid methyl ester ELV-N-32-Me, elovanoid carboxylic acid ELV-N-32-H, and elovanoid sodium salt ELV-N-32-Na.

[0228] Scheme 5 (Figure 10) shows the stereocontrolled total synthesis of the 34-carbon dihydroxylated erovanoids by starting from alkyne methyl ester 30 and using the same sequence of reactions as in Scheme 4.

[0229] For example, this scheme shows the total synthesis of the 34-carbon alkynyl erovanoid compound ELV-N-34-Me-acetylene and its conversion to erovanoid methyl ester ELV-N-34-Me, erovanoid carboxylic acid ELV-N-34-H, and erovanoid sodium salt ELV-N-34-Na.

[0230] The chemical reactions shown in Schemes 1-5 (Figures 6-10) can also be adapted for the total synthesis of additional monohydroxylated and dihydroxylated elastomeric cyclohexanoides with at least 23 carbons and up to 42 carbons in the carbon chain.

[0231] Pharmaceutical compositions for the treatment of diseases: In other embodiments, the present disclosure provides for the formulation of a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds provided herein or salts thereof in a pharmaceutically acceptable carrier.

[0232] The provided compositions comprise one or more compounds or salts thereof provided herein and pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. The compounds may be formulated into suitable pharmaceutical formulations, such as solutions, suspensions, tablets, powders, pills, capsules, powders, intravitreal implanted reservoirs or nanodevices embedded in collagen or other materials on the ocular surface, sustained-release formulations or elixirs for oral, buccal, intranasal, vaginal, rectal, or ocular administration, or sterile solutions or suspensions for parenteral administration, transdermal patches, transdermal patch formulations, and dry powder inhalers. The provided formulations may be in the form of droplets, such as eye drops, and may further include antioxidants and / or known drugs for the treatment of ocular diseases. The compounds described herein are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel, Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).

[0233] Embodiments of the present disclosure provide pharmaceutical compositions containing various forms of the provided compounds, such as the free carboxylic acid or its pharmaceutically acceptable salt, or its corresponding ester, or its phospholipid derivative. In other useful embodiments, the present disclosure provides pharmaceutical compositions containing one or more elastomeric compounds containing one or two hydroxyl groups at positions n-3 to n-18 of a very long chain polyunsaturated fatty acid, as the free carboxylic acid or its pharmaceutically acceptable salt, or its corresponding ester.

[0234] In further embodiments, the present disclosure provides pharmaceutical compositions for alleviating symptoms, treating, or preventing a disease, such as an allergic inflammatory disease, a disease associated with cellular senescence and / or ferroptosis, or a metabolic disorder.

[0235] In the provided composition, the effective concentration of one or more compounds or pharmaceutically acceptable derivatives is mixed with suitable pharmaceutical carrier or vehicle.Compound can be derivatized as corresponding salt, ester, enol ether or ester, acid, base, solvate, hydrate or prodrug before formulation as described herein.The concentration of compound in the composition is effective to deliver the amount that treats, prevents or alleviates one or more symptoms of disease, disorder or condition when administered.

[0236] As described herein, the composition can be prepared by adapting methods known in the art. The composition can be a component of a pharmaceutical preparation. The pharmaceutical preparation can further include a known agent for the treatment of inflammatory or degenerative diseases, including neurodegenerative diseases. The provided composition can function as a prodrug precursor of fatty acids, and can be converted into free fatty acids when localized at the site of disease.

[0237] The present disclosure also provides packaged compositions or pharmaceutical compositions for the prevention, amelioration, or use in treating a disease or condition. Other packaged compositions or pharmaceutical compositions provided by the present disclosure can further include a label containing at least one of instructions for using the composition to treat a disease or condition. The kit can further include appropriate buffers and reagents known in the art for administering various combinations of the components described herein to a host.

[0238] Pharmaceutical preparations: Embodiments of the present disclosure may include compositions or pharmaceutical compositions identified herein and may be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, naturally occurring or synthetic antioxidants, and / or adjuvants. Additionally, embodiments of the present disclosure may include compositions or pharmaceutical compositions formulated with one or more pharmaceutically acceptable auxiliary substances. For example, compositions or pharmaceutical compositions may be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants to provide embodiments of the compositions of the present disclosure.

[0239] A wide variety of pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients are fully described in various publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are generally available. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusting agents and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, etc., are generally readily available.

[0240] In one embodiment of the present disclosure, composition or pharmaceutical composition can be administered to the subject by any means that can produce desired effect.Therefore, composition or pharmaceutical composition can be incorporated into various preparations for therapeutic administration.For example, composition or pharmaceutical composition can be formulated into pharmaceutical composition by combining with suitable pharmaceutically acceptable carrier or diluent, and can be formulated into solid, semi-solid, liquid or gas form preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, creams and aerosols.

[0241] Suitable excipient vehicles for the composition or pharmaceutical composition are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, if necessary, the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, antioxidants, or pH buffering agents. Methods for preparing such dosage forms are known to those skilled in the art or will become apparent upon consideration of this disclosure. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the composition or pharmaceutical composition sufficient to achieve the desired state in the treated subject.

[0242] Compositions of the present disclosure may include those that contain sustained-release or controlled-release matrices. Furthermore, embodiments of the present disclosure may be used in combination with other therapies that utilize sustained-release formulations. As used herein, a sustained-release matrix is ​​a material, such as a polymeric matrix, that can be degraded by enzymatic or acid-based hydrolysis or dissolution. Once inserted into the body, the matrix is ​​subjected to the action of enzymes and bodily fluids. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. Exemplary biodegradable matrices include polylactide matrices, polyglycolide matrices, and polylactide-co-glycolide (a copolymer of lactic acid and glycolic acid) matrices. In another embodiment, the pharmaceutical compositions (and combination compositions) of the present disclosure can be delivered in a controlled-release system. For example, the composition or pharmaceutical composition can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980). Surgery 88:507; Saudek et al. (1989). N. Engl. J. Med. 321:574). In another embodiment, a polymeric material is used. In yet another embodiment, the controlled-release system is placed near the therapeutic target, thus requiring only a fraction of the systemic dose. In yet another embodiment, the controlled-release system is placed near the therapeutic target, thus requiring only a fraction of the systemic dose. Other controlled release systems are discussed in the review by Langer (1990). Science 249:1527-1533.

[0243] In another embodiment, the compositions of the present disclosure (and compositions separately or combined together) may include those formed by impregnating the compositions or pharmaceutical compositions described herein into absorbent materials such as sutures, bandages, and gauze, or coated onto solid materials such as surgical staples, zippers, catheters, etc. Other delivery systems of this type will be readily apparent to those of skill in the art in view of the present disclosure.

[0244] In another embodiment, the compositions or pharmaceutical compositions of the present disclosure (as well as the compositions combined separately or together) can be part of a delayed-release formulation. Delayed-release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets", eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al. (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules, as well as delayed-release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment, and processes for preparing tablets and capsules, as well as delayed-release dosage forms of tablets, capsules, and granules.

[0245] The embodiment of composition or pharmaceutical composition can be administered to the subject in one or more doses.Those skilled in the art will understand that dosage level can vary according to the function of the specific composition or pharmaceutical composition to be administered, the severity of symptoms and the subject's sensitivity to side effects.The useful dosage of a given compound can be easily determined by those skilled in the art by various means.

[0246] In one embodiment, multiple doses of the composition or pharmaceutical composition are administered. The frequency of administration of the composition or pharmaceutical composition can vary depending on various factors, such as the severity of the symptoms. For example, in one embodiment, the composition or pharmaceutical composition can be administered once a month, twice a month, three times a month, every other week (QOW), once a week (QW), twice a week (BIW), three times a week (TIW), four times a week, five times a week, six times a week, every other day (QOD), daily (QD), twice a day (QID), three times a day (TID), or four times a day. As discussed herein, in one embodiment, the composition or pharmaceutical composition is administered one to four times a day for a period of one to ten days.

[0247] The administration period of the composition or pharmaceutical composition analog, e.g., the period during which the composition or pharmaceutical composition is administered, can vary depending on any of a variety of factors, e.g., patient response, etc. The compositions or pharmaceutical compositions, in combination or separately, can be administered for a period of about 1 day to 1 week, about 1 day to 2 weeks.

[0248] The amount of the compositions and pharmaceutical compositions of the present disclosure that can be effective in treating a condition or disease can be determined by standard clinical techniques.In addition, in vitro or in vivo assays can be used to identify optimal dosage ranges.The exact dosage used can also depend on the route of administration, and can be determined according to the judgment of the practitioner and the circumstances of each patient.

[0249] Route of administration: Embodiments of the present disclosure provide methods and compositions for administering active agents to a subject (e.g., a human) using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local administration routes. Administration routes may include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, intravitreal, topical application, intravenous, rectal, nasal, oral, and other enteral and parenteral routes. Administration routes may be combined as needed or adjusted depending on the agent and / or the desired effect. Active agents may be administered in a single dose or multiple doses.

[0250] In embodiments, aspects of the present invention can be administered by a nebulizer. The term "nebulizer" can refer to any device known in the art that produces small droplets or aerosols from a liquid. For example, the composition can be administered in the form of a mist that is inhaled into the lungs.

[0251] n-3 VLC-PUFA and its biological derivatives are formed intracellularly and are not components of human diet.Advantageous administration routes of compounds provided herein can include topical administration, oral administration, intranasal administration, and parenteral administration.For example, provided formulations can be delivered in the form of drops such as eye drops or any other conventional method for treating allergic inflammatory diseases of the eye.For example, provided formulations can be delivered in the form of intranasal sprays or any other conventional method for treating allergic inflammatory diseases of the nasal cavity or lungs.For example, provided formulations can be delivered in the form of creams or gels or any other conventional method for treating allergic inflammatory diseases of the skin.

[0252] Parenteral administration routes other than inhalation administration can include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any administration route other than the digestive tract. Parenteral administration can be carried out to affect systemic or local delivery of the composition. When systemic delivery is desired, administration includes invasive or systemically absorbed local or mucosal administration of pharmaceutical formulations. In one embodiment, the composition or pharmaceutical composition can also be delivered to a subject by enteral administration. Enteral administration routes can include, but are not limited to, oral and rectal delivery (e.g., using a suppository).

[0253] Methods for administering compositions or pharmaceutical compositions through the skin or mucosa may include, but are not limited to, topical application of appropriate pharmaceutical preparations, transdermal delivery, injection, and epidermal administration. For transdermal delivery, absorption enhancers or iontophoresis are suitable methods. Iontophoretic delivery can be achieved using commercially available "patches" that continuously deliver products through continuous skin via electrical pulses for periods of several days or more.

[0254] The compounds and compositions provided by the present disclosure can restore homeostasis and induce survival signaling in certain cells that are experiencing oxidative stress or other homeostasis disruptions.The present disclosure also provides methods for using the provided compounds and compositions, including hydroxylated derivatives of very long chain polyunsaturated fatty acids, as free carboxylic acids or their pharmaceutically acceptable salts, or as their corresponding esters or other prodrug derivatives.The provided compounds can be easily prepared by starting from commercially available materials and adapting methods known in the art.

[0255] As exemplified by the erovanoid derivatives ELV-N-32-Me, ELV-N-32-Na, ELV-N-34-Me, and ELV-N-34-Na, the biological activity of provided compounds is due to their ability to reach and enter target human cells and / or exert their biological effects by acting on membrane-bound receptors. Alternatively, provided compounds can act via intracellular receptors (e.g., nuclear membranes), thus functioning specifically by influencing key signaling events. Administration of pharmaceutical compositions containing provided compounds and a pharmaceutically acceptable carrier restores homeostatic balance and promotes the survival of specific cells essential for maintaining normal function. The provided compounds, compositions, and methods can be used for the preventive and therapeutic treatment of inflammatory, degenerative, and neurodegenerative diseases. This disclosure targets critical steps in the initiation and early progression of these conditions by mimicking the specific biology of endogenous cellular / organ responses to achieve potent, selective, side-effect-free, and sustained biological activity.

[0256] Thus, one aspect of the present disclosure includes an embodiment of a composition comprising at least one very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.

[0257] In some embodiments of this aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and be formulated for delivery of at least one very long chain polyunsaturated fatty acid in an amount effective to reduce tissue morbidity in a recipient subject or the development of tissue morbidity in the recipient subject.

[0258] In some embodiments of this aspect of the disclosure, the pathological condition can be an allergic inflammatory disease or condition of tissue of the recipient subject.

[0259] In some embodiments of this aspect of the disclosure, the pathological condition may be associated with cellular senescence, ferroptosis, or both.

[0260] In some embodiments of this aspect of the disclosure, the pathological condition may be associated with a metabolic disorder.

[0261] In some embodiments of this aspect of the disclosure, the composition may be formulated for topical delivery of at least one very long chain polyunsaturated fatty acid component to the skin or eye of a recipient subject.

[0262] In some embodiments of this aspect of the disclosure, the composition may be formulated for intranasal delivery of at least one very long chain polyunsaturated fatty acid composition to the nasal cavity and / or lungs of a recipient subject.

[0263] In some embodiments of this aspect of the disclosure, the composition can further comprise at least one nutritional component, e.g., the composition can be formulated for oral or parenteral delivery of at least one very long chain polyunsaturated fatty acid to a recipient subject.

[0264] In some embodiments of this aspect of the disclosure, the at least one very long chain polyunsaturated fatty acid can have from about 26 to about 42 carbon atoms in its carbon chain.

[0265] In some embodiments of this aspect of the disclosure, the at least one very long chain polyunsaturated fatty acid can have 32 or 34 carbon atoms in its carbon chain.

[0266] In some embodiments of this aspect of the disclosure, the very long chain polyunsaturated fatty acid can have five or six double bonds in its carbon chain that have a cis configuration.

[0267] In some embodiments of this aspect of the disclosure, the very long chain polyunsaturated fatty acid is (14Z,17Z,20Z,23Z,26Z,29Z)-dotriaconta-14,17,20,23,26,29-hexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z)-tetratriaconta-16,19,22,25,28,31-hexaenoic acid.

[0268] Another aspect of the present disclosure includes an embodiment of a composition comprising at least one erovanoid having at least 23 carbon atoms in its carbon chain.

[0269] In some embodiments of this aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and may be formulated for delivery of an amount of at least one elastinoid effective to reduce a pathological condition in a tissue of a recipient subject.

[0270] In some embodiments of this aspect of the disclosure, the pathological condition can be an allergic inflammatory disease.

[0271] In some embodiments of this aspect of the disclosure, the at least one elovanoid can be selected from the group consisting of a monohydroxylated elovanoid, a dihydroxylated elovanoid, an alkynyl monohydroxylated elovanoid, and an alkynyl dihydroxylated elovanoid, or any combination thereof.

[0272] In some embodiments of this aspect of the disclosure, the at least one elovanoid can be a combination of elovanoids, such as a monohydroxylated elovanoid and a dihydroxylated elovanoid; a monohydroxylated elovanoid and an alkynyl monohydroxylated elovanoid; a monohydroxylated elovanoid and an alkynyl dihydroxylated elovanoid; a dihydroxylated elovanoid and an alkynyl monohydroxylated elovanoid; a dihydroxylated elovanoid and an alkynyl dihydroxylated elovanoid; a monohydroxylated elovanoid, a dihydroxylated elovanoid, and an alkynyl monohydroxylated elovanoid; a monohydroxylated elovanoid, a dihydroxylated elovanoid, and an alkynyl monohydroxylated elovanoid; a monohydroxylated elovanoid, a dihydroxylated elovanoid, and an Alkynyl dihydroxylated elovanoids; monohydroxylated elovanoids, dihydroxylated elovanoids, and alkynyl monohydroxylated elovanoids, alkynyl dihydroxylated elovanoids; each elovanoid is independently selected from the group consisting of a racemic mixture, an isolated enantiomer, or a combination of enantiomers in which the amount of one enantiomer exceeds the amount of another; and each dihydroxylated elovanoid is independently selected from the group consisting of a diastereomeric mixture, an isolated diastereomer, or a combination of diastereomers in which the amount of one diastereomer exceeds the amount of another.

[0273] In some embodiments of this aspect of the disclosure, the composition can further comprise at least one very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.

[0274] In some embodiments of this aspect of the disclosure, the at least one very long chain polyunsaturated fatty acid can have from about 26 to about 42 carbon atoms in its carbon chain.

[0275] In some embodiments of this aspect of the disclosure, the at least one very long chain polyunsaturated fatty acid can have five or six double bonds in its carbon chain that have a cis configuration.

[0276] In some embodiments of this aspect of the disclosure, the at least one very long chain polyunsaturated fatty acid can be (14Z,17Z,20Z,23Z,26Z,29Z)-dotriaconta-14,17,20,23,26,29-hexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z)-tetratriaconta-16,19,22,25,28,31-hexaenoic acid.

[0277] In some embodiments of this aspect of the disclosure, the monohydroxylated erovanoid can be selected from the group consisting of formula G, H, I, or J. TIFF2025143368000023.tif37128In the formula, n can be 0 to 19, -CO-OR can be a carboxylic acid group, or a salt or ester thereof, and when -CO-OR can be a carboxylic acid group, compound G, H, I, or J can be a salt thereof, and the cation of the salt can be a pharmaceutically acceptable cation, and when -CO-OR can be an ester, R can be an alkyl group.

[0278] In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable cation can be an ammonium cation, an iminium cation, or a metal cation.

[0279] In some embodiments of this aspect of the disclosure, the metal cation can be a sodium, potassium, magnesium, zinc, or calcium cation.

[0280] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of enantiomers G and H, where the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0281] In some embodiments of this aspect of the disclosure, the composition can include amounts of enantiomers I and J, where the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0282] In some embodiments of this aspect of the disclosure, the composition can include one of the enantiomers of G or H in an amount that exceeds the amount of the other enantiomer of G or H.

[0283] In some embodiments of this aspect of the disclosure, the composition can include one of the I or J enantiomers in an amount that exceeds the amount of the other of the I or J enantiomers.

[0284] In some embodiments of this aspect of the disclosure, the monohydroxylated erovanoid has the following formula, respectively: (S,14Z,17Z,20Z,23Z,25E,29Z)-27-hydroxytriaconta-14,17,20,23,25,29-methyl hexaenoate (G1); (S,14Z,17Z,20Z,23Z,25E,29Z)-27-hydroxytriaconta-14,17,20,23,25,29-hexaenoate (G2); It can be selected from the group consisting of sodium enoate (G2); (S,16Z,19Z,22Z,25Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,25,27,31-hexaenoic acid methyl ester (G3); and (S,16Z,19Z,22Z,25Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,25,27,31-hexaenoic acid sodium ester (G4). TIFF2025143368000024.tif76128

[0285] In some embodiments of this aspect of the disclosure, the dihydroxylated erovanoid can be selected from the group consisting of formulas K, L, M, and N. TIFF2025143368000025.tif36132m may be 0 to 19, and -CO-OR may be a carboxylic acid group, or a salt or ester thereof; When -CO-OR can be a carboxylic acid group, compound K, L, M, or N can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and when -CO-OR can be an ester, R can be an alkyl group.

[0286] In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable cation can be an ammonium cation, an iminium cation, or a metal cation.

[0287] In some embodiments of this aspect of the disclosure, the metal cation can be a sodium, potassium, magnesium, zinc, or calcium cation.

[0288] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers K and L, where the diastereomers have either (S) or (R) chirality at position n-6 and (R) chirality at position n-13.

[0289] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers M and N, where the diastereomers have either (S) or (R) chirality at position n-6 and (R) chirality at position n-13.

[0290] In some embodiments of this aspect of the disclosure, the composition can include one of the K or L diastereomers in an amount that exceeds the amount of the other of the K or L diastereomers.

[0291] In some embodiments of this aspect of the disclosure, the composition can include one of the diastereomers of M or N in an amount that exceeds the amount of the other diastereomer of M or N.

[0292] In some embodiments of this aspect of the disclosure, the dihydroxylated elaeovanoid has the following formula: (14Z,17Z,20R,21E,23E,25Z,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,25,29-hexaenoic acid methyl (K1); (14Z,17Z,20R,21E,23E,25Z,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,25,29-hexaenoic acid methyl (K2), respectively. methyl (16Z,19Z,22R,23E,25E,27Z,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,27,31-hexaenoate (K3); and sodium (16Z,19Z,22R,23E,25E,27Z,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,27,31-hexaenoate (K4). TIFF2025143368000026.tif76131

[0293] In some embodiments of this aspect of the disclosure, the alkynyl monohydroxylated erovanoid can be selected from the group consisting of formula O, P, Q, or R. TIFF2025143368000027.tif37128 wherein m may be 0 to 19, and -CO-OR may be a carboxylic acid group, or a salt or ester thereof; when -CO-OR may be a carboxylic acid group, compound O, P, Q, or R may be a salt thereof; the cation of the salt may be a pharmaceutically acceptable cation; and when -CO-OR may be an ester, R may be an alkyl group; compounds O and P may be at positions n-3, n-12, n-15, and four cis carbon-carbon double bonds beginning at position n-18, a trans carbon-carbon double bond beginning at position n-7, and a carbon-carbon triple bond beginning at position n-9, for a total of 23 to 42 carbon atoms in the carbon chain, and compounds Q and R each have three cis carbon-carbon double bonds beginning at positions n-3, n-12, and n-15, a trans carbon-carbon double bond beginning at position n-7, and a carbon-carbon triple bond beginning at position n-9, for a total of 23 to 42 carbon atoms in the carbon chain.

[0294] In some embodiments of this aspect of the disclosure, the alkynyl monohydroxylated erovanoid has the formula: (S,14Z,17Z,20Z,25E,29Z)-27-hydroxydotriaconta-14,17,20,25,29-hydroxydotriaconta-23-ynoic acid methyl (O1); (S,17Z,20Z,25E,29Z)-27-hydroxydotriaconta-17,20,25,29-tetraene-2 3-ynoic acid sodium salt (O2); (S,16Z,19Z,22Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,27,31-pentaen-25-ynoic acid methyl salt (O3); and (S,16Z,19Z,22Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,27,31-pentaen-25-ynoic acid sodium salt (O4). TIFF2025143368000028.tif76128

[0295] In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable cation can be an ammonium cation, an iminium cation, or a metal cation.

[0296] In some embodiments of this aspect of the disclosure, the metal cation can be a sodium, potassium, magnesium, zinc, or calcium cation.

[0297] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of enantiomers O and P, where the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0298] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of enantiomers Q and R, where the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0299] In some embodiments of this aspect of the disclosure, the composition can include one of the O or P enantiomers in an amount that exceeds the amount of the other O or P enantiomer.

[0300] In some embodiments of this aspect of the disclosure, the composition can include one of the enantiomers of Q or R in an amount that exceeds the amount of the other enantiomer of Q or R.

[0301] In some embodiments of this aspect of the disclosure, the elovanoid can be an alkynyl dihydroxylated elovanoid selected from the group consisting of formula S, T, U, or V. TIFF2025143368000029.tif34128m may be 0 to 19, and -CO-OR may be a carboxylic acid group, or a salt or ester thereof. When -CO-OR can be a carboxylic acid group, compounds S, T, U, or V may be a salt thereof, and the cation of the salt may be a pharmaceutically acceptable cation. When -CO-OR can be an ester, R may be an alkyl group. Compounds S and T may be 3 or 4 alkyl groups starting at positions n-3, n-15, and n-18, respectively. Compounds U and V each have two cis carbon-carbon double bonds beginning at positions n-3 and n-15, two trans carbon-carbon double bonds beginning at positions n-9 and n-11, and a carbon-carbon triple bond beginning at position n-7, totaling 23 to 42 carbon atoms in the carbon chain; compounds U and V each have two cis carbon-carbon double bonds beginning at positions n-3 and n-15, two trans carbon-carbon double bonds beginning at positions n-9 and n-11, and a carbon-carbon triple bond beginning at position n-7, totaling 23 to 42 carbon atoms in the carbon chain.

[0302] In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable cation is an ammonium cation, an iminium cation, or a metal cation.

[0303] In some embodiments of this aspect of the disclosure, the metal cation is a sodium, potassium, magnesium, zinc, or calcium cation.

[0304] In some embodiments of this aspect of the disclosure, the alkynyl monohydroxylated erovanoid has the formula: (14Z,17Z,20R,21E,23E,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,29-pentaen-25-ynoic acid methyl (S1); (14Z,17Z,20R,21E,23E,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,29-pentaen-25-ynoic acid methyl (S1); 25-ynoic acid sodium salt (S2); (16Z,19Z,22R,23E,25E,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,31-pentaen-27-ynoic acid methyl salt (S3); and (16Z,19Z,22R,23E,25E,29S,31Z)-22,29-dihydroxytetratriaconta-16,19,23,25,31-pentaen-27-ynoic acid sodium salt (S4). TIFF2025143368000030.tif77128

[0305] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers S and T, where the diastereomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.

[0306] In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers U and V, where the diastereomers have either (S) or (R) chirality at position n-6 and (R) chirality at position n-13.

[0307] In some embodiments of this aspect of the disclosure, the composition can include one of the S or T diastereomers in an amount that exceeds the amount of the other of the S or T diastereomers.

[0308] In some embodiments of this aspect of the disclosure, the composition can include one of the diastereomers of U or V in an amount that exceeds the amount of the other diastereomer of U or V.

[0309] Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one of ordinary skill in the art upon examination of the following figures, detailed description, and examples, and all such additional compositions, compounds, methods, features, and advantages may be included in this description and may be within the scope of the present disclosure.

[0310] Compositions and methods for modulating the bioactivity and availability of erovanoids - Patent Application 20070122999 Cellular senescence is a form of cell cycle arrest associated with aging and disease. It is the fate of inflammatory cells associated with age-related diseases, including AD and AMD. The senescent phenotype manifests in cells undergoing terminal replication arrest, exhibiting cellular enlargement, chromatin alterations, SASP, and cell cycle regulatory proteins (cyclins and cyclin-dependent kinases). The sustained accumulation of senescent cells is associated with age-related diseases and functional decline. Clearance of senescent cells from tissues alleviates aging-related pathologies by propagating degenerative and proinflammatory events in the microenvironment.

[0311] In the brain, the senescence signature program is triggered in astrocytes, microglia, and neurons (albeit postmitotic). The consequences of the senescent cell phenotype (e.g., chronic inflammation) are also key to AMD. Cellular senescence is a protective event against cancer and plays a role in aging and age-related diseases. Senescent cells help create a microenvironment that promotes tumor progression. These events include the depletion of stem and progenitor cells and the cytotoxic consequences of the expression of SASP, including cytokines and chemokines, growth factors, and matrix metalloproteinases, which disrupt inflammatory homeostasis.

[0312] Furthermore, senescent cells also have beneficial effects on injury repair and tissue remodeling. Therefore, without being bound by theory, senescence, in addition to being a driver of age-dependent diseases and metabolic syndrome, can also remove healthy cells, in addition to other cells that damage organs / organisms, through senescent cell clearance. For example, a positive effect of senescent cells and SASP is the acceleration of skin wound healing due to the premature secretion of SASP caused by PDGF-AA secreted by senescent cells. Wounding induces senescence in local fibroblasts and endothelial cells. As a result, myofibroblast differentiation, granulation tissue formation, and completion of wound healing occur. ELV alters the expression (and protein abundance) of the tumor suppressor protein p16INK4a (cyclin-dependent kinase inhibitor 2A, also known as cyclin-dependent kinase 4 inhibitor A). This protein is encoded by the Ink4a / Arf locus or Cdkn2a. p16 plays a role in cell cycle control by slowing cell progression from G1 to S phase.

[0313] Referring to the figures, ELV targets upstream ferroptosis, a form of programmed cell death involved in aging. We discovered a novel molecular target of ELV that inhibited cell death by blocking phosphorylation of the scaffolding protein PEBP-1 (Figures 29-31). As a result, lipid peroxides are not formed and ferroptosis is blocked. Markers of iron, ferritin, and oxidative stress are enhanced in senescent cells. These cells exhibit abnormal iron homeostasis, which affects the iron content of aged tissues. Iron itself induces microglial senescence, but reduction of iron chelators can reduce and prevent the accumulation of iron and ferritin in senescent cells. Without wishing to be bound by theory, the senescence-associated secretory phenotype (SASP) may promote ferritin expression in neurons and glia as an acute phase response, increasing their susceptibility to ferroptosis, an iron-mediated cell death process.

[0314] For example, a specific convergence mechanism is as follows:

[0315] Ferroptosis is an intermediate stage of autophagy that leads to aging (Figures 2–4).

[0316] The AdipoR1 receptor subtype enhances DHA cellular uptake / retention and the availability of ELV precursors of VLC-PUFAs. After uptake / retention, this receptor subtype promotes the construction of membrane reservoirs of VLC-PUFA phosphatidylcholine, which upon release by PLA1 enter the pathway for ELV biosynthesis. Membranes containing VLC-PUFAs are released during uncompensated oxidative stress (UOS) challenges, trauma, ischemia, and the onset of neurodegenerative diseases. 5XFAD, a pathway that fails prior to PRC death (Figure 32 and Figure 37).

[0317] The specific GPCRs of ELV and NPD1 are the basis for developing interactions between MFRP and AdipoR1-targeted synthetic ligands (such as small peptide molecules) for peptides that mimic the action of ELV by targeting specific receptor GPCR data (Figure 36 and Figure 38).

[0318] Targeting intracellular proteins with ELVs as regulatory sites for enhancing biological activity. Identifying cell-permeable (or tissue-permeable) peptides or other small molecules that target GPCRs. Examples include in vivo MOs (including bradykinin analogs). Examples lack retinal toxicity (Figure 34).

[0319] enzymes for signal termination by degrading ELV as a target for novel small molecules that increase the availability of ELV by blocking / attenuating its degradation.

[0320] Beneficial role of ELV in GBM (see figure).

[0321] TBI (see diagram).

[0322] Thus, embodiments of the present invention include compositions and methods that result in the elimination of senescent cells. For example, embodiments of the present invention are directed to compositions and methods that modulate the availability of senescent cells in cancer (chemotherapy, brain (neurodegenerative diseases)), wound healing (diabetes, corneal keratinocytes, decubitus ulcers), and neurodegenerative diseases such as AMD and AD.

[0323] Embodiments of the present invention are also directed to compositions and methods that are neuroprotective and / or neurorestorative. For example, embodiments are neuroprotective in the prodromal targeting of MCA and / or visual impairment that precedes blindness in AMD or retinitis pigmentosa (RP) or other retinal degenerative diseases. Furthermore, embodiments are neuroprotective or neurorestorative in other diseases, such as AD, AM, CV disease, metabolic syndrome, obesity, type 2 diabetes, myocardial infarction, stroke, TBI, and GBM. For example, in cancer, senescent cells help create a microenvironment that promotes tumor progression.

[0324] Embodiments of the present invention are directed to a series of converging mechanisms of erovanoides (ELVs) that regulate ferroptosis and aging, leading to beneficial outcomes in disease. For example, embodiments of the present invention are directed to compositions and methods for preventing, treating, ameliorating, or slowing the progression of cancer (such as glioblastoma multiforme or GBM), age-related macular degeneration (AMD), Alzheimer's disease (AD), other neurodegenerative diseases, metabolic syndrome, obesity, type 2 diabetes, neurotrauma, and skin and corneal wound healing.

[0325] The elovanoids used in the embodiments herein are described in PCT / US2016 / 017112 and PCT / US2018 / 023082, each of which is incorporated herein by reference in its entirety. For example, the elovanoids include ELV-N-34 or ELV-N-32, or derivatives thereof. Lipooxygenation of n-3-VLC-PUFAs leads to the formation of enzymatically hydroxylated derivatives of n-3-VLC-PUFAs called erovanoids, which can include monohydroxy compounds (e.g., ELV-27S and ELV-29S), and dihydroxy derivatives (e.g., ELV-N-32 and ELV-N-34). The erovanoid ELV-N-32 is a 20R,27S-dihydroxy 32:6 derivative (a 32-carbon, six double bond erovanoid with a neuroprotectin-like 20(R),27(S)-dihydroxy pattern). The erovanoid ELV-N-34 is a 22R,29S-dihydroxy 34:6 derivative (a 34-carbon, six double bond erovanoid with a 22(R),29(S)-dihydroxy pattern).

[0326] Peptide analogues Furthermore, aspects of the present invention are directed to the development of new synthetic non-lipid analogs to mimic the biological activity of lipid mediators, such as estrogen-like compounds. The term "analog" can refer to a second organic or inorganic molecule that has a similar or identical function to a first organic or inorganic molecule. The analog may be structurally similar to the first organic or inorganic molecule. In embodiments, the first molecule is a lipid and the second molecule (i.e., the analog) is a non-lipid molecule. For example, the first molecule is an estrogen-like compound and the second molecule is a peptide. In such embodiments, the peptide may be referred to as a "peptide analog."

[0327] In embodiments, peptide analogs can be considered therapeutic peptides. The term "therapeutic peptide" can refer to a peptide or fragment or variant thereof that has one or more therapeutic and / or biological activities.

[0328] The term "peptide" can refer to a molecule containing two or more amino acid residues linked together by peptide bonds. These terms can include, for example, natural and artificial proteins, protein fragments of protein sequences, and polypeptide mimetics (such as muteins, variants, and fusion proteins), as well as peptides that are post-translationally or otherwise covalently or non-covalently modified. Peptides can be monomeric or polymeric. In certain embodiments, a "peptide" is a chain of amino acids whose alpha carbons may be joined via peptide bonds. Thus, the terminal amino acid at one end of the chain (the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) can refer to the free amino group on the amino terminal amino acid of a peptide or the amino group of an amino acid at any other position within the peptide. Similarly, the term "carboxy terminus" can refer to the free carboxyl group at the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides can also include essentially any polyamino acid, including but not limited to amino acids linked by ether bonds, as opposed to peptidomimetics such as amide bonds.

[0329] In embodiments, peptide analogs are peptides comprising at least four amino acids joined via peptide bonds or other covalent bonds, as described herein. In one embodiment, the peptide or peptide analog is about 4 to about 50 amino acids in length. All integer subranges from 4 to 50 amino acids are useful for peptides herein. In one embodiment, the peptide or peptide analog is about 5 to about 35 amino acids in length, about 5 to about 30 amino acids in length, about 5 to about 25 amino acids in length, or about 5 to about 20 amino acids in length. In one embodiment, the peptide or peptide analog is about 6 to about 35 amino acids in length, about 7 to about 30 amino acids in length, about 6 to about 25 amino acids in length, or about 6 to about 20 amino acids in length. In one embodiment, the peptide or peptide analog is about 7 to about 35 amino acids in length, about 7 to about 30 amino acids in length, about 7 to about 25 amino acids in length, or about 7 to about 20 amino acids in length. In one embodiment, the peptide or peptide analog is about 8 to about 35 amino acids in length, about 8 to about 30 amino acids in length, about 8 to about 25 amino acids in length, or about 8 to about 20 amino acids in length. In one embodiment, the peptide is about 8 to about 17 or 18 amino acids in length, or about 9 to about 16 or 17 amino acids in length. In one embodiment, the peptide is about 10 to about 17, or about 12 to about 16 or 17, or about 14 to about 16 amino acids in length. In some embodiments, the peptide is selected from the group consisting of a 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 16-mer, 17-mer, 18-mer, 19-mer, or 20-mer.

[0330] In embodiments, the erobanoids and / or peptide analogs can regulate / modulate ferroptosis, and the modulation of ferroptosis treats or prevents a disease in a subject. As used herein, "ferroptosis" refers to iron-dependent regulated cell death. Ferroptosis is characterized by the overwhelming iron-dependent accumulation of lethal lipid reactive oxygen species. Ferroptosis is distinct from apoptosis, necrosis, and autophagy. Ferroptosis assays are, for example, as disclosed in Dixon et al., 2012.

[0331] In other embodiments, the erovanoids and / or peptide analogs can control / modulate cellular senescence, and the modulation of cellular senescence treats a disease of interest.

[0332] The terms "modulate" and "modulating" and grammatical variations thereof mean to alter, such as increase or decrease, biological activity.

[0333] molecular target In embodiments, the aerovanoids and / or peptide analogs can bind to epitopes on one or more molecular targets, such as those identified in FIG. 28 and FIG.

[0334] In embodiments, the erovanoids and / or peptide analogs can bind to epitopes on one or more molecular targets (e.g., to contiguous or non-contiguous amino acid sequences indicated by the protein NCBI reference numbers listed in the table below), as shown in the table below. TIFF2025143368000031.tif82148

[0335] In embodiments, the elovanoid and peptide analog interact with the same or similar epitopes on the molecular target. In embodiments, the elovanoid and peptide analog can interact with different epitopes on the molecular target.

[0336] As used herein, the term "epitope" can refer to a portion of a molecular target to which an elovanoid and / or peptide analog specifically binds, such as those identified in the Tables.

[0337] For example, in embodiments, the elobanoid or peptide analog can target an epitope on LTB4R, GPR37, GPR52, GPR132, CNR2, BAI2, TXNRD1, PEBP1, and / or GSR to regulate cellular senescence and / or ferroptosis. In embodiments, the epitope can include a "target site" or "target sequence," which can refer to a sequence bound by a binding partner, such as an elobanoid or peptide analog. For example, the target site can include one or more amino acids.

[0338] The proteins in the tables herein may be referred to as "molecular targets." The term "target" or "molecular target" can refer to any molecule being investigated for interaction with a candidate compound (e.g., a drug, an elobanoid, or a peptide analog thereof), for example, a molecule within a cell or a molecule associated with a cell membrane. Non-limiting examples of molecular targets may include proteins such as DNA, RNA, and receptors (e.g., cell surface, membrane-bound, or nuclear), components of signal transduction pathways, transcription factors, or functional fragments thereof. Molecular targets may also include macromolecules such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivative of a molecule described herein, or any complex containing one or more molecules described herein. A compound, an elobanoid, or a peptide analog "interacts" with a molecular target, either directly or indirectly, if it affects the molecular target. A compound can act directly on a molecular target; for example, if the molecular target is a protein, the compound may directly interact with the protein by binding to it or directly regulate the expression of the protein through its action on a transcriptional control element. Similarly, a compound can also act indirectly on a molecular target, for example, by blocking or stimulating a separate molecule that in turn acts on the molecular target. An indirect action of a compound on a molecular target can occur, for example, when the target is a non-protein molecule and the compound interacts with a protein involved in the production, stability, activity, maintenance, and / or modification of the non-protein molecular target.

[0339] The term "binding" can refer to the determination by standard assays, including those described herein, that a binding polypeptide recognizes and reversibly binds to a given target. Such standard assays include, but are not limited to, equilibrium dialysis, gel filtration, and monitoring spectroscopic changes resulting from binding.

[0340] In embodiments, the elastomeric or peptide analogs can have specificity for the molecular targets in Table 1. The term "specificity" can refer to a binding polypeptide that has a higher binding affinity for one target than another. Binding specificity is determined by the dissociation equilibrium constant (K D ) or the binding equilibrium constant (K a ) [Example]

[0341] Example 1 - Primary Human Nasal Epithelial Cells (HNEpCs) Used in the Examples herein Cryopreserved human nasal epithelial cells (HNEpC) were purchased from PromoCell GmbH, Heidelberg, Germany (catalog number C-1260, lot number 436Z028).

[0342] The cells used in our experiments were primary nasal epithelial cells obtained from the nasal mucosa of a 50-year-old Caucasian male.

[0343] Cells were received at passage (P1), subcultured up to passage (P3), and used for all experiments.

[0344] HNEpCs were grown to 80% confluency in Promocell's Airway Epithelial Cell Growth Medium (Cat. No. C-21060) supplemented with Airway Epithelial Cell Growth Medium Supplement Pack (Cat. No. C-39160) and penicillin / streptomycin.

[0345] Example 2 - HNEpCs were challenged with several stressors (aeroallergens) Lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (catalog number L4391) was obtained from Sigma-Aldrich. LPS is a major component of Gram-negative bacteria and activates the innate immune system through recognition by Toll-like receptor 4 (TLR4). This leads to a signaling cascade that ultimately results in the activation of NF-κB and the production of proinflammatory cytokines. The LPS used in the experiments was a preparation of smooth (S) LPS purified from Gram-negative E. coli 0111:B4, and was used at 30 μg / mL to challenge HNEpCs. Polyinosinic-polycytidylic acid (poly(I:C) or abbreviated poly(rI):poly(rC)) is a synthetic analog of double-stranded viral RNA (dsRNA), a molecular pattern associated with viral infection, including loss of epithelial integrity, increased mucus production, and increased production of inflammatory cytokines. Poly(I:C), a TLR3 agonist, activates the antiviral pattern recognition receptors TLR3, RIG-I / MDA5, and PKR, inducing signaling through multiple inflammatory pathways, including NF-κB and IRFs. High-molecular-weight poly(I:C) contains long strands of inosine poly(I) homopolymer annealed to strands of cytidine poly(C) homopolymer. The average size of poly(I:C) HMW is 1.5 kb–8 kb. Poly(I:C) (catalog number P1530) was obtained from Sigma-Aldrich and used at 100 μg / mL to challenge HNEpCs. • House Dust Mite Extract (DP) from Dermatophagoides pteronyssinus (Cat. No. 3033) - Pure lyophilized extract was obtained from Chondrex, Inc. DP was used at 30 μg / mL to challenge HNEpC. Allergens - Der p1, Der p2. • House Dust Mite Extract (DF) from Dermatophagoides farinae (Cat. No. 3040) - Pure lyophilized extract was obtained from Chondrex, Inc. DF was used at 30 μg / mL to challenge HNEpC. Allergens - Der f1, Der f2. • HDM, a mixture of both house dust mite extracts (DP) and (DF), was used at (15 μg / mL + 15 μg / mL) to challenge HNEpC.

[0346] Example 3 - (HNEpC) were challenged with several stressors (aeroallergens) and the following assays were performed: LDH Cytotoxicity Assay - Using Invitrogen's CyQuant LDH Cytotoxicity Assay Kit (Cat. No. C20301). • Cell viability assay - Invitrogen's PrestoBlueHS Cell Viability Assay Kit (Cat. No. C50201) was used. Chondrex, Abcam, and R&D Systems sandwich ELISA assays: 1) Chondrex Human IL-6 Detection Kit (Cat. No. 6802) 2) Chondrex Human IL-1β Detection Kit (Cat. No. 6805) 3) R&D Systems Human IL-8 / CXCL8 Quantikine ELISA Kit (Cat. No. D8000C) 4) Chondrex Human CCL2 / MCP-1 Detection Kit (Cat. No. 6821) 5) Chondrex Human CXCL1 / KC / GRO Detection Kit (Cat. No. 6825) 6) Chondrex Human VEGF Detection Kit (Cat. No. 6810) 7) Abcam Human ICAM1 (CD54) ELISA Kit (Cat. No. ab100640) 8) Chondrex Human IL-10 Detection Kit (Cat. No. 6806)

[0347] Example 4 - Cell viability assay using Presto Blue HS reagent PrestoBlue HS Cell Viability Reagent is a non-toxic, add-and-read reagent that does not require cell lysis. The highly purified resazurin used in PrestoBlue HS results in a reagent with a >50% reduction in background fluorescence and a >100% increase in signal-to-background ratio.

[0348] Once inside living cells, the cellular reducing environment reduces resazurin to resorufin, a red, highly fluorescent compound.

[0349] Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence and color of the medium surrounding the cells. The conversion of resazurin to resorufin produces a noticeable color change, allowing cell viability to be detected using an absorbance-based plate reader.

[0350] Fluorescence is read using a fluorescence excitation wavelength of 560 nm (excitation range is 540-570 nm) and emission of 590 nm (emission range is 580-610 nm).

[0351] Example 5 - Conclusion The results of the cytotoxicity assay (LDH) showed that the addition of stressors (LPS, poly(I:C), HDM extract) significantly increased the formation of red formazan, which indicates cytotoxicity, and this was reduced by the addition of ELV (Figures 17A and 17B).

[0352] Cell viability assays using PrestoBlue HS reagent also show that resorufin production is greater in control cells compared to cells challenged with various stressors (LPS, poly(I:C), HDM extract), and that addition of ELVs improves cell viability and confers protection to HNEpCs (Figures 18A and 18B).

[0353] When HNEpCs were challenged with various stressors (LPS, poly(I:C), HDM extract), there was a significant increase in the production of proinflammatory cytokines and chemokines—IL-6, IL-1β, IL-8 / CXCL8, CCL2 / MCP-1, CXCL1 / KC / GRO, VEGF, and ICAM1 (CD54)—compared to controls. This increased production of proinflammatory cytokines and chemokines was abrogated by the addition of ELV at a concentration of 500 nM 30 min after challenge with the respective stressors (Figures 19A and 19B).

[0354] Conversely, when HNEpCs were challenged with various stressors (LPS, poly(I:C), HDM extract), the release of the anti-inflammatory cytokine IL-10 was significantly reduced compared to controls. This reduced production of anti-inflammatory cytokines was reversed by the addition of ELV at a concentration of 500 nM 30 min after challenge with the respective stressors (Figures 26A and 26B).

[0355] Example 6 - Erovanoids for allergic rhinitis, allergic conjunctivitis, allergic dermatitis, and asthma The following are experimental conditions that induce inflammation / allergy in human nasal mucosa (in primary culture), leading to the contraction of erovanoids and preserving the integrity of these cells. a) Polyinosinic-polycytidylic acid (poly(I:C) or poly(rI):poly(rC)), a synthetic analog of double-stranded viral RNA (dsRNA), a molecular pattern associated with viral infections such as loss of epithelial integrity and increased production of mucus and inflammatory cytokines. b) LPS, a major component of Gram-negative bacteria, activates the innate immune system through recognition by Toll-like receptor 4 (TLR4). This leads to a signaling cascade that ultimately results in the activation of NF-κB and the production of proinflammatory cytokines. The LPS used in the experiments was a preparation of smooth (S) LPS purified from Gram-negative Escherichia coli 0111:B4, used at 30 μg / mL to challenge HNEpCs. c) House Dust Mite Extract (DP) from Dermatophagoides pteronyssinus (Cat. No. 3033) - Pure lyophilized extract was obtained from Chondrex, Inc. DP was used at 30 μg / mL to challenge HNEpC. Allergens - Der p1, Der p2. d) House Dust Mite Extract (DF) from Dermatophagoides farinae (Cat. No. 3040) - Pure lyophilized extract was obtained from Chondrex, Inc. DF was used at 30 μg / mL to challenge HNEpC. Allergens - Der f1, Der f2. e) House dust mite extract - HDM, a mixture of both (DP) and (DF), was used at (15 μg / mL + 15 μg / mL) to challenge HNEpC.

[0356] Allergy treatments are consistently ineffective, causing itching, difficulty breathing, and many people experience drowsiness, dry mouth, and other side effects that make daily functioning difficult. Erovanoids can be delivered intranasally to treat allergic rhinitis, allergic conjunctivitis, allergic dermatitis, and asthma, halting these conditions in their tracks and providing an effective alternative to most over-the-counter medications while minimizing their side effects.

[0357] The results of the cytotoxicity assay (LDH) showed that the addition of stressors (LPS, poly(I:C), HDM extract) significantly increased the formation of red formazan, which indicates cytotoxicity, and this was reduced by the addition of ELV (Figures 17A and 17B).

[0358] Cell viability assays using PrestoBlue HS reagent also show that resorufin production is greater in control cells compared to cells challenged with various stressors (LPS, poly(I:C), HDM extract), and that addition of ELVs improves cell viability and confers protection to HNEpCs (Figures 18A and 18B).

[0359] When HNEpCs were challenged with various stressors (LPS, poly(I:C), HDM extract), there was a significant increase in the production of proinflammatory cytokines and chemokines—IL-6, IL-1β, IL-8 / CXCL8, CCL2 / MCP-1, CXCL1 / KC / GRO, VEGF, and ICAM1 (CD54)—compared to controls. This increased production of proinflammatory cytokines and chemokines was abrogated by the addition of ELV at a concentration of 500 nM 30 min after challenge with the respective stressors (Figures 19A and 19B).

[0360] Conversely, when HNEpCs were challenged with various stressors (LPS, poly(I:C), HDM extract), the release of the anti-inflammatory cytokine IL-10 was reduced compared to controls. This reduced production of anti-inflammatory cytokines was reversed by the addition of ELV at a concentration of 500 nM 30 min after challenge with the respective stressors (Figures 26A and 26B).

[0361] Example 7 (1) Potential prodromal targets before disease manifestations become apparent. Structure and function of the 5xFAD retina. (A) VlogI plot showing maximum ERG b-wave amplitude in response to light flashes of 0–0.075 cd·s / m². 5xFAD mice achieved a maximum amplitude of approximately 100 μV, approximately half that recorded in wild-type mice. (B) Electron microscopy of a 5-month-old 5xFAD retina demonstrating morphological similarities to wild-type retinas. i. Basal side of 5xFAD RPE cells showing membrane folding along Bruch's membrane (Br). ii. Disc synthesis area (arrow) in the basal portion of a wild-type rod outer segment showing newly formed discs from the connecting ciliary (CC) membrane. iii. Similar area (arrow) in the 5xFAD retina showing new disc formation. iv. Outer limiting membrane (OLM, arrow) at the scleral edge of the cell body layer (N, photoreceptor nuclei) in the 5xFAD retina. The cytoplasm of Müller cells (M) is brighter than that of photoreceptors (PR). (v) Interface between a 5xFAD RPE cell and a rod photoreceptor tip (PR). Two phagosomes (Ph) are visible within the RPE cytoplasm. The lower Ph is retained within the RPE apical process, while the upper, darker Ph is older and only enters the RPE cell body, indicating normal phagocytic function. (vi) Inner segment mitochondria (M) in a 5xFAD retina retain healthy photoreceptors with a highly elongated shape. (C) Five-month-old wild-type and 5xFAD retinal sections showing normal photoreceptor profiles within 5xFAD retinas. (D) Fluorescent staining of retinas from WT and 5xFAD. Blue (DAPI) indicates nuclei, and red indicates Aβ in the RPE layer of 6-month-old 5xFAD RPE cells. (*P<0.05, Student's t-test comparison).

[0362] Example 8 ELV restores RPE morphology and reduces gene expression after subretinal injection of OAβ in WT mice. (A) Mice were divided into seven groups: uninjected, PBS, OAβ only, OAβ + ELV-N-32, OAβ + ELV-N-34, ELV-N-32 only, and ELV-N-34 only. On day 3, mRNA was isolated for RT-PCR. On day 7, mice were subjected to optical coherence tomography (OCT), followed by enucleation of the eyes and processing for whole-mount RPE staining and Western blot analysis. (B) Whole flat-mount images of RPE. OAβ disrupted RPE morphology. However, the ELV-treated group showed less RPE damage than the PBS-treated group, and ELV alone did not induce any changes. (C) OCT evaluation of the effects of OAβ on the retina and RPE. (D) PRC thickness was reduced in the OAβ-injected group. OAβ caused PRC cell death, resulting in reduced thickness as measured by OCT. (E) RPE gene expression after OAβ(1-42) injection and ELV treatment. Three days after injection, RNA was isolated from RPE, reverse transcribed into cDNA, and subjected to RT-PCR using different primers. Genes from the same functional group, including senescence- and AMD-related genes (E), collagenases, gelatinases, stromelysins, and other matrix metalloproteinases (MMPs) (F), and autophagy (G), were plotted on the same chart. (H) Western blot of p16INK4a in RPE / choroid. ELV-N-32 and ELV-N-34 downregulated the expression of p16INK4a, an important senescence marker elevated by OAβ injection. (I) Retinal gene expression after OAβ(1-42) injection and ELV treatment. OAβ activates apoptotic genes in the retina. Co-injection of ELV downregulated these genes. (*P<0.05, using Student's t test comparison).

[0363] Example 9 OAβ toxicity is counteracted by ELV in primary hRPE. (A) Primary hRPE cells were treated with 10 μM OAβ with or without ELV. After 3 days, total RNA was isolated and analyzed by q-PCR. After 7 days, cells were subjected to β-galactosidase staining. (B) Live cell images of primary hRPE under bright-field microscopy after 7 days. (C) β-galactosidase staining of primary hRPE + / - ELV. Quantification of the % of β-Gal-positive cells. ELV reduced positive senescent cells. (D) Transcription of senescence, AMD-related, and autophagy genes in primary hRPE exposed to OAβ(1-42) and treated with ELV (*P<0.05, using Student's t-test for comparison).

[0364] Example 10 A working model of ELVs in OAβ-induced RPE and PRC damage. (A) OAβ induces senescence and disrupts tight junctions in the RPE. OAβ then penetrates the retina and causes photoreceptor cell death, reflected in the nuclei of the lesser cell body layer (CBL). ELVs restore the morphology of the RPE layer upon OAβ exposure, resulting in the maintenance of retinal structure. (B) OAβ induces senescence-, autophagy-, matrix metalloproteinase-, and AMD-related genes in the RPE, as well as apoptosis genes in the retina. ELVs downregulated the induction of OAβ genes. The pathway for ELV synthesis is depicted.

[0365] Example 11 - Downregulation of aging programming in the hypothalamus and adipose tissue by erovanoids counteracts the onset and progression of diabetes 1) Research plan a) Specific Purposes The incidence of type 2 diabetes (a consequence of obesity), for example, increases rapidly during aging and is a risk factor for renal dysfunction, cardiovascular disease, stroke, impaired wound healing, infection, depression, anxiety, and cognitive decline. Despite advances in our understanding of the pathogenesis of diabetes, metabolic syndrome, and comorbidities, no effective treatments are available. Cellular senescence has been implicated in the pathogenesis of type 2 diabetes by targeting age-related chronic inflammatory diseases, including metabolic syndrome (hypertension, obesity, and atherosclerosis), pancreatic beta cell function, and causing adipose tissue dysfunction. Senescence programming forms a diabetes loop—a cause and effect of cellular dysfunction. This new class of lipid mediator, erovanoides (ELVs), will be investigated as downregulators of senescence programming to combat diabetes onset and progression. Herein, we propose to test a novel therapeutic approach utilizing specific novel compounds supported by compelling evidence in experimental models of diabetes and our recent mechanistic discoveries.

[0366] ELVs are dihydroxylated derivatives of the very-long-chain polyunsaturated fatty acids (VLC-PUFAs) 32:6n-3 and 34:6n-3. As precursors of ELVs, VLC-PUFAs are biosynthesized by the elongation of 22:6n-3 fatty acids, catalyzed by the elongation of very-long-chain fatty acids-4 (ELOVL4). Our laboratory reported the discovery of ELVs, including detailed structure and stereochemistry established by stereocontrolled total organic synthesis. Recently, our laboratory uncovered ELVs as a low-abundance, highly potent, neuroprotective, and homeostatic mediator that blocks neuronal senescence genetic programming and the senescence-activated secretory phenotype (SASP) upon disruption of homeostasis.

[0367] Without wishing to be bound by theory, erobanoids downregulate slowly progressive inflammation (inflammage-senescence) in adipose tissue (AT) and hypothalamus (HT) primarily through the senescence transcriptome and senescence program (SP), including the SASP. This is validated by data using human adipocytes from diabetic patients, genetic diabetic mouse models, human brain cells in culture, and cutting-edge approaches to address specific functional issues in HT.

[0368] Although HT is composed of terminally differentiated cells and originates from the neuroepithelium, senescent neurons in aged mice, models of AD, and astrocytes are also targets, as they express senescence and generate secreted SASP that promotes neuroinflammation in nearby cells. Our recent studies have shown that neighboring cells are targets of the neurotoxic effects of SASP, inducing paracrine senescence in the retina.

[0369] Objective 1) To verify that erovanoids neutralize the activation of senescence programming in human adipocytes from diabetic patients upon induction with TNFα, IL1β, or other inducers.

[0370] Active brown / beige AT plasticity increases energy expenditure and leads to reduced hyperglycemia and hyperlipidemia, whereas its atrophy and inactivation are associated with obesity and aging. Thus, a chronic, slowly progressive local inflammatory state may disrupt the regulation of internal signals as well as connections with HCs.

[0371] Aim 2) To verify that HT in genetically diabetic mice develops SP, which impairs synaptic connectivity and neuronal dysfunction. This is supported by data showing that HCs of genetically diabetic mice exhibit disturbed electrophysiological activity (in our newly developed Maestro system). Thus, we have a new experimental model and mechanism to utilize, in addition to a model for evaluating therapeutic erobanoids.

[0372] Aim 3) To test the experimental therapeutic effects of elovanoids when administered systemically and / or intranasally in diabetic mice.

[0373] b) Importance and innovation This example is based on the therapeutic use of novel homeostatic and neuroprotective mediators, elovanoids (ELVs), for diabetes. These compounds maintain neuronal integrity, counteract aging programming, and, as supported by current data, block signaling impairments in adipose tissue (human diabetic patients) and the hypothalamus (genetic diabetic mouse models). ELVs mediate protection in neuronal cultures subjected to either oxygen / glucose deprivation or N-methyl-D-aspartate receptor-mediated excitotoxicity, as well as in experimental ischemic stroke. The methyl esters or sodium salts of ELV-N-32 and ELV-N-34, when administered 1 hour after 2 hours of ischemia due to middle cerebral artery occlusion, reduced infarct volume, promoted cell survival, and reduced destruction of the neurovascular unit.

[0374] Erovanoids as treatments for diabetes and obesity Recently, it was discovered that when lean mice become obese due to a high-fat diet, they exhibit increased abundance of senescent cells in the brain and increased anxiety behavior.11 This study also provides the first evidence that obesity-induced anxiety can be blocked by a novel senolytic drug that dissipates senescent cells. Senescent cells release a senescence-associated secretory phenotype (SASP), inducing nearby healthy cells to participate in dysfunction.

[0375] Transplantation of senescent cells into young mice causes weakness, frailty, and persistent functional impairment. However, administration of a senolytic cocktail, which may contain dasatinib (an anti-leukemia drug) and quercetin (a plant flavonol), which induces programmed cell death in senescent cells, alleviates these effects and extends both the lifespan and healthspan of aged mice. Several publications have reported that senescent cells accumulate in obesity. Obese mice show increased abundance of senescent cells in the white matter adjacent to the lateral ventricles. 12-17

[0376] Erovanoids as a treatment are supported by the following: 1) Our data on AT and HT (see herein). 2) Our data on human neurons in culture show that activation of the SASP is blocked by ELV (see herein), which counteracts senescence in human neurons. 3) We reported that oligomeric A-beta peptides activate SP and SASP, subsequently causing retinal cell death, and by Western blot analysis, erovanoids inhibited the expression of SP genes p16INK4a, MMP1, p53, p21, p27, Il-6, and MMP1, as well as the expression of SASP secretome and p16 protein. 4) Furthermore, we found that erovanoides inhibited the expression of autophagy genes (ATG3, ATG5, ATG7, and Beclin-1) upon oligomeric A-beta peptide challenge in retinal cells. Autophagy is a key event in brown / beige adipocyte plasticity by regulating intracellular remodeling during brown / beige adipogenesis, thermogenesis activation, and inactivation. This may include autophagic degradation of mitochondria, which is important for brown adipocyte inactivation and the transition from beige to white adipose tissue. 3 5) We also discovered that erovanoids regulate the matrix metalloproteinase transcriptome (MMP1a, MMP2, MMP3, MMP8, MMP9, MMP12, and MMP13) and demonstrated that, together with SASP, this mechanism contributes to changes in the extracellular matrix. Thus, in both AT and HT, SASP acts both autocrine and paracrine, altering the homeostasis of the extracellular matrix microenvironment and creating an inflammatory environment that contributes to impaired insulin sensitivity. erovanoids control slow-progressing, chronic, sterile inflammation (i.e., inflammaging). This is a key tenet of the rationale described herein. 6) Furthermore, another target of erobanoids is unresolved oxidative stress and inflammation in neuronal and neuronal injury models. 1, 2 These changes, such as unresolved inflammation, develop in dysfunctional adipocytes and are one of the most well-studied outcomes of pro-inflammatory signaling in AT and insulin resistance. In addition to IL-6, IFN-γ, and CCL2, TNF-α, produced by immune cells, directly prevents insulin action in adipocytes by downregulating the major insulin-responsive glucose transporter GLUT4 and inhibits insulin-dependent tyrosine phosphorylation of the insulin receptor and IRS-1 through ceramide production. 7) Translational Innovation. Biomimetic therapeutic approaches using synthetically produced molecules of endogenously produced erovanoids: -Restores homeostasis and combats diabetes -Prevents aging programming and neuronal damage in metabolic syndrome / obesity. -Use innovative medicinal chemistry.

[0377] c) Research approach The hypothalamus is a target of metabolic syndrome and is important in obesity and type 2 diabetes.

[0378] Various aspects of physiological deterioration, including obesity and type 2 diabetes, are controlled by the hypothalamus, a key brain region connecting the neuroendocrine system to physiological function. Furthermore, functional changes in the agouti-related peptide / neuropeptide Y (AgRP / NPY) and proopiomelanocortin (POMC) neuron sets, growth hormone-releasing hormone (GHRH) and somatostatin (SST) neuron sets, arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) neuron sets, and gonadotropin-releasing hormone (GnRH) and kisspeptin / neurokinin B / dynorphin (KNDy) neuron sets contribute to age-related physiological declines in energy metabolism, hormone control, circadian rhythms, and reproduction. Cellular mechanisms underlying the progression of hypothalamus-mediated dysfunction include dysregulation of nutrient sensing, altered intercellular communication, stem cell depletion, activation of senescence programming, loss of proteostasis, and epigenetic changes.

[0379] One of the functions of arcuate hypothalamic (ARC) neurons is to respond appropriately to hormones and neuropeptides locally and peripherally and to participate in energy homeostasis. Arcuate hypothalamic neurons project to the PVN, and stimulation of the ARC induces the simultaneous release of dopamine and GABA into neurons in the PVN, where dopamine-induced orexigenic neurons synthesize AgRP and NPY and inhibit anorexigenic neurons that synthesize POMC. The ventromedial nucleus of the hypothalamus (VMH) is involved in detecting hypoglycemic events and initiating physiological counterregulatory responses to overcome them. Therefore, to evaluate the hypothalamus as a target of erovanoids, we used ex vivo organotypic cultures of hypothalamic slices from the brains of C57BL / 6J (WT) and age-matched Leprdb (db / db diabetic) mice and found that they elicit changes in synaptic circuit activity and firing trains of action potentials using the innovative Maestro microelectrode array (MEA) performed on a MaestroPro MEA, Axion Biosystems, GA (see below).

[0380] Microelectrode array (MEA) measurements to evaluate the hypothalamus as a target of erovanoids in diabetes We determined whether ex vivo organotypic cultures of hypothalamic slices (200 μm thick) obtained from the brains of C57BL / 6J (WT) and age-matched Leprdb (db / db diabetic) mice were electrically active and capable of firing trains of action potentials using a Maestro microelectrode array (MEA) run on a MaestroPro MEA system from Axion Biosystems, GA.

[0381] MEA measurements were performed using a 48-well microelectrode array (MEA) plate (M768-tMEA-48W) from Axion Biosystems, GA. Each well of the MEA plate contained a 4 x 4 grid of 30 nm circular nanoporous PEDOT electrodes embedded in a cell culture substrate, with an interelectrode spacing of 200 μM. In preparation for seeding hypothalamic slices, wells were treated with 0.1% polyethyleneimine (PEI) in sodium borate buffer, pH 8.4. Next, the wells were precoated with laminin (6 μg / mL), and hypothalamic slices (200 μm thick) were plated onto the electrode grid. Hypothalamic slices were plated and cultured for 4 days at 37°C and 5% CO2 in complete neurobasal medium supplemented with B27™ and N2 supplements, along with GlutaMax™ and Pen Strep (Thermo Fisher Scientific, Gibco™).

[0382] Extracellular recordings of spontaneous action potentials were performed in culture medium at 37 °C using standard neuronal settings on the MaestroPro MEA system and AxIS software version 1.5.1.12 (Axion Biosystems). Data were sampled at a rate of 12.5 kHz with a hardware frequency bandwidth of 200–5000 Hz and filtered again in software using a 200–2500 Hz single-order Butterworth bandpass filter to remove high-frequency noise prior to spike detection. The threshold for spike detection was set at six times the rolling standard deviation of the filtered field potential for each electrode. Using a 5-minute recording span, we calculated the mean spike rate for the well and the number of active electrodes in the well ("active electrodes"), defined as the number with a spike rate of 0.5 / min or greater. The number of recorded spikes per electrode was averaged after ignoring noisy electrodes from analysis. Spike timestamps were exported to Neuroexplorer 5.0 (NEX Technologies) to create spike raster plots.

[0383] Using the MEA system, population-level electrical activity was recorded from various hypothalamic neurons. Referring to Figure 43, the top left panel shows representative raster plots showing burst activity and spike histograms from a C57Bl6 (WT) male mouse, while the bottom left panel shows raster plots from a C57Bl6 (WT) male mouse treated with ELV (500 nM). Raster traces in each well demonstrated measurable firing by hypothalamic neurons with electrodes in contact with active neurons. Each horizontal row represents an electrode within a well. While spontaneous activity (isolated single spikes and multiple spike bursts) was evident in hypothalamic slices from normal C57Bl6 WT controls, the middle and top right panels show raster plots from Leprdb (db / db diabetic) male and female mice, respectively. These plots demonstrate asynchronous field potentials and spike / burst activity. The low level of spiking could arise from either a cell-autonomous deficiency in excitability or a lack of synaptic drive from neighboring cells. All spikes were induced by dopamine (50 nM) followed by the GABA antagonist bicuculline (10 μM). Addition of NMDA (10 μM) did not induce significant spikes and maintained baseline activity. The middle and bottom right panels show hypothalamic slices from Lepr / db (db / db diabetic) male and female age-matched control mice. Addition of ELV-34-6 Na (500 nM) overcomes asynchronous activity and restores synchronous spikes. ELV34 may protect the hypothalamus from neurodegeneration. Here, we demonstrate that CNS regulatory disorders can be modeled and differences between control and disease states can be delineated using a multielectrode array (MEA) system-based HTS screening method. We demonstrate that ergobanoids may have therapeutic utility in reversing the adverse effects of diabetes and may play an important role in CNS control of glucose metabolism.

[0384] Protection of hypothalamic neuronal cell death (as determined by Fluoro-Jade B staining) by erovanoids in adult obese diabetic mice (db / db) Fluoro-Jade b (F-Jb) stains degenerating neurons. Adult obese diabetic mice (db / db) showed increased FJb signaling in organotypic slices of the hypothalamus, whereas wild-type mice showed negligible amounts, indicating that the hypothalamus of db / db mice is undergoing neurodegeneration. Incubation with 500 nM ELV34 for 48 hours induced neuroprotection reflected by a decrease in F-Jb signaling (a).

[0385] Validation experiments in human brain neurons / astrocytes demonstrate that erovanoids block the senescence-associated secretory phenotype (SASP): β-galactosidase staining of senescent neurons Senescence-associated β-galactosidase activity was measured in human neuroglial (HNG) cells exposed to oligomeric amyloid beta (Oaβ) (10 μM). (A-G) SA-β-Gal activity in HNG cells treated with 500 nM Oaβ (10 μM) and different erovanoides (ELVs) or neuroprotectin D1 (NPD1). Photomicrographs were obtained with a bright-field microscope. (H) Quantification of SA-β-Gal+ cells shown in (A-G). SA-β-Gal+ cells were scored in three random fields for a total of at least 150 cells. Results are expressed as the percentage of stained SA-β-Gal+ cells (mean ± SEM). Statistical analysis was performed using Graphpad Prism software 8.3. Results were compared by one-way ANOVA followed by a Holm-Sidak post-hoc test; p < 0.05 was considered statistically significant.

[0386] Similarly, senescence-associated β-galactosidase activity was measured in human neuroglial (HNG) cells exposed to elastin (10 μM). (A–G) SA-β-Gal activity in HNG cells treated with 500 nM of elastin (10 μM) and different elastinoids (ELVs) or neuroprotectin D1 (NPD1). Photomicrographs were obtained with a bright-field microscope. (H) Quantification of SA-β-Gal+ cells shown in (A–G). SA-β-Gal+ cells were scored in three random fields for a total of at least 150 cells. Results are expressed as the percentage of stained SA-β-Gal+ cells (mean ± SEM). Statistical analysis was performed using Graphpad Prism software 8.3. Results were compared by one-way ANOVA followed by a Holm-Sidak post-hoc test; p < 0.05 was considered statistically significant. Experimental Design: Human neuroglial (HNG) cells were challenged with Oaβ or erastin.

[0387] The next objective is to test the ingredients described herein.

[0388] Objective 1) Test the prediction that erovanoids neutralize the activation of senescence programming in human adipocytes from diabetic patients upon induction with TNFα, IL1β, or other inducers.

[0389] Active brown / beige AT plasticity increases energy expenditure and reduces hyperglycemia and hyperlipidemia, whereas its atrophy and inactivation are associated with obesity and aging.

[0390] Aim 2) To test whether HT in genetically diabetic mice develops SP, which impairs synaptic connectivity and neuronal dysfunction. This is supported by data showing that HCs of genetically diabetic mice exhibit disturbed electrophysiological activity (in our newly developed Maestro system). Thus, we have a new experimental model to test mechanisms in addition to a model for evaluating therapeutic erobanoids.

[0391] Aim 3) To test the experimental therapeutic effects of elovanoids when administered systemically and / or intranasally in diabetic mice.

[0392] Data demonstrating erovanoids in programming aging and combating inflammation ELV34 reduced TP53 mRNA levels to non-diabetic control levels in human adipocytes from diabetic patients treated with IL-1β, a cytokine that induces insulin resistance in adipocytes. Similarly, IL-8 was elevated by IL-1β and reduced approximately 40-fold by 500 nM ELV34 in both diabetic and non-diabetic adipocytes. Although the mechanism is unclear, ELV34 may be a therapeutic agent that interrupts or stops the deleterious signaling observed in diabetic patients.

[0393] ELV34 reverses the effects of IL1β in human diabetic adipocytes. A) Experimental design. B) TP53 and IL8 expression levels in human diabetic and non-diabetic adipocytes by Taqman real-time PCR.

[0394] ELV34 reduced IL1β-induced IL6 (a marker of SASP) levels in the hypothalamus of diabetic db / db mice, indicating that hypothalamic neurons and astrocytes produce SP. Different effects were observed in male and female mice.

[0395] ELV34 treatment increased levels of adiponectin, an antidiabetic systemic hormone secreted by adipocytes and other tissues (hypothalamus) that promotes insulin sensitivity. A) Diabetic hypothalamus treated with ELV34 showed a trend toward increased adiponectin in both females and males. B) Differential effects of ELV34 on subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). SAT and VAT have different capacities for browning.

[0396] approach Specific Objective 1 We test the prediction that erovanoids counteract the activation of senescence programming in human adipocytes from diabetic patients upon induction by TNFα, IL1β, or other inducers.

[0397] Rationale TNFα circulates in obese patients and plays an important role in the pathogenesis of insulin resistance. 20, 21 Furthermore, interleukin-1β signaling mediates the effects of macrophages on adipose tissue. 18 Circulating IL1β induces the disruption of adipose tissue function. 22 Without wishing to be bound by theory, systemic IL1β induces senescence in human adipocytes. The loss of function in cytokine-exposed adipocytes may include the inability of the cells to brown. Active brown / beige AT plasticity increases energy expenditure and leads to reduced hyperglycemia and hyperlipidemia. On the other hand, its atrophy and inactivation are associated with obesity and aging. Therefore, a chronic, slowly progressing local inflammatory state would disrupt the control of internal signals as well as connections with HCs. Furthermore, when adipocytes undergo SP, other hormones produced and secreted by adipose tissue, such as adiponectin, may be reduced. Without wishing to be bound by theory, adiponectin has antidiabetic and anti-inflammatory effects and also functions as an insulin sensitizer. 23 Results showed that elovanoid 34 (ELV34) can reverse some pathological features of diabetic mouse (db / db) and human diabetic adipocytes. Based on these results, it is possible to stop SP and SASP in human diabetic adipocytes.

[0398] Experimental design Experiment 1: To determine whether IL1β induces senescence in adipocytes, 1) test the expression and activity of markers of senescence: p16, p21, p27, and p53, 2) measure β-galactosidase activity, and 3) measure the senescence-associated secretory phenotype (SASP) in human adipocytes from diabetic and non-diabetic patients exposed to IL1β and / or TNFα.

[0399] To test for the expression of senescence markers, differentiated adipocytes from diabetic and non-diabetic patients were exposed to IL1β and / or TNFα for 6 days with or without ELV34, harvested, and their RNA was extracted and used as a template for cDNA synthesis. Expression levels of p16, p21, p27, and p53 were assessed by real-time PCR using TaqMan probes. Results were normalized by the expression of housekeeping genes: PPIA, GAPDH, β-actin, B2M, TBP, and TFRC. The activity of these markers was measured by Western blot assay to detect increases in phosphorylation and total protein content.

[0400] The β-galactosidase activity assay is based on the overexpression and accumulation of endogenous lysosomal β-galactosidase, specifically in senescent cells. 24 Similar to the detection of senescence marker expression, human diabetic and nondiabetic adipocytes are exposed to IL1β and / or TNFα with and without ELV34 for 6, 9, and 12 days and then stained using a β-galactosidase substrate that generates a fluorescent signal when hydrolyzed by endogenous enzymes. Samples are imaged by two methods: confocal microscopy and flow cytometry.

[0401] The third part is the determination of senescence-associated secretory phenotypes. Adipocyte senescence is induced as described herein in the presence or absence of EL34, and the resulting media is collected, concentrated, and tested for candidates via Western blot or ELISA assays. Some of the candidates tested are: IL-6, IL-7, IL-1α, IL-1β, IL-13, IL-15, IL-8, GROVE-α, GROVE-β, GROVE-γ, MCP-2, MIP-1α, eotaxin, eotaxin-3, TECK, ENA-78, I-309, and MMP-1, -3, -10, -12, -13, -14.

[0402] Experiment 2: To evaluate the effects of SP and / or SASP on brown and white adipocyte content, cells from Experiment 1 will be tested for markers of brown tissue, such as CD137, TMEM26 (transmembrane protein 26), and TBX1 (T-box 1), which are known to be abundant in human brown adipose tissue (BAT). PGC-1α, PPARγ, C / EBPα, and PRDM16 will also be assessed using Western blot in diabetic and nondiabetic adipocytes exposed to IL1β and / or TNFα to confirm their content and measure their activity by luciferase reporter assay. Next, adipocytes undergoing SP and / or SASP will be treated with ELV34 and the parameters described herein will be tested.

[0403] Experiment 3: To evaluate the ability of diabetic adipocytes exposed to IL1β and / or TNFα for 6, 9, and 12 days to synthesize and release adiponectin. IL1β and / or TNFα-treated adipocytes were tested for adiponectin mRNA expression via real-time PCR (Figure X), Western blot assay, and culture medium was collected and subjected to ELISA and / or Western blot assay. The effect of ELV34 on adiponectin production was tested using the procedures described herein.

[0404] Without wishing to be bound by theory, we detect senescence and SASP in human adipocytes from diabetic patients exposed to IL1β and / or TNFα, but not in non-diabetic adipocytes. However, when a senescence-like phenotype is induced in cells by overexpressing cell cycle inhibitors such as p16 or p21, the cells undergo growth arrest with many characteristics of senescent cells but do not develop SASP. 25 Therefore, without wishing to be bound by theory, SASP is not observed when p16 or p21 is increased.

[0405] Without wishing to be bound by theory, diabetic adipocytes, upon aging, show lower expression of BAT markers: CD137, TMEM26 (transmembrane protein 26) and TBX1 (T-box 1), as well as lower activity of key transcription factors responsible for the genetic features observed in brown adipocytes.

[0406] In parallel with SP, and without wishing to be bound by theory, a decrease in adiponectin expression and secretion in diabetic adipocytes is observed.

[0407] ELV34 abrogates the SP and SASP of diabetic adipocytes in culture and promotes cell browning and AdipoQ synthesis.

[0408] Specific Objective 2 We demonstrate that HT in genetically diabetic mice generates SP, which impairs synaptic connectivity and neuronal dysfunction. This is supported by data showing that HCs from genetically diabetic mice exhibit disturbed electrophysiological activity (using our newly developed Maestro system). Thus, we have an experimental model for validating the embodiments and mechanisms described herein, in addition to a model for evaluating therapeutic erobanoids.

[0409] Rationale Neuroinflammation in the hypothalamus induces neuronal dysregulation, which contributes to aging and metabolic syndrome.

[0410] Experimental design Findings are evaluated using BKS.Cg-Dock7m+ / +Leprdb / J diabetic mice and control C57BLKS / J mice. Both sexes are included. This strain of mice is used to model phases I to III of type II diabetes and obesity. The brains of these animals are dissected and sliced ​​to isolate the hypothalamus, hippocampus, and cortex. Each organotypic slice is placed in an individual well using Neurobasal medium supplemented with B27. 48 hours after plating, the medium is supplemented with 500 nM ELV, and organotypic slices are recorded 24 hours later. Neuronal activity is determined using Axion BioSystems' Maestro multielectrode array (MEA) technology. Due to the heterogeneity of neurons that make up the hypothalamus, the identity of the hypothalamic neurons being tested is verified by adding 50 nM dopamine and 10 μM bicuculline. This indicates the presence of the peritoneum membranosum (VTM) and arcuate nucleus, which are important for satiety and feeding. Comparison of neural activity in the hypothalamus of control and diabetic mice with and without ELV provides a baseline of activity in this brain structure and helps assess neuronal function. After recording, hypothalamic slices are harvested and total RNA is extracted. First-strand cDNA is reverse-transcribed to examine the expression of genes involved in aging programming, as well as insulin signaling and sensitivity and glucose metabolism. Up- and down-regulation of candidates (p53, p21, p16ink4a, and Bmi-1) is further confirmed by Western blot or capillary Western blot of a larger panel of targets.

[0411] result Neuronal activity in diabetic mice with HT has decreased sensitivity to dopamine and bicuculline, indicating neuronal dysfunction. Causal relationships are investigated by observing an upregulation of SP and SASP markers in diabetic mice compared to controls.

[0412] Specific Objective 3 To examine the therapeutic effects of elovanoids when administered systemically and / or intranasally to diabetic mice.

[0413] Rationale The route of administration affects bioavailability by altering the number of biological barriers a drug must pass through or by altering the drug's exposure to pumps and metabolic mechanisms. We validate that various routes of administration, such as intravenous and intranasal pulmonary, function as effective drug delivery routes. The alveoli represent a large surface area and minimal barrier to diffusion. The lungs also receive total cardiac output via the bloodstream. Therefore, absorption from the lungs is very rapid and complete. Erovanoids dissolved in 0.9% saline (vehicle) are non-irritating and, from previous experiments, have been shown to be delivered very effectively intranasally. The intended effect may be systemic.

[0414] Experimental design The findings will be evaluated using BKS.Cg-Dock7m+ / +Leprdb / J mice and control C57BLKS / J mice. This strain of mice is used to model phases I to III of type II diabetes and obesity. Both genders will be included. All animal experiments will be performed in accordance with the approved IACUC protocol issued by the Louisiana State University Health Sciences Center. Intranasal administration will be performed on lightly anesthetized mice. Place each mouse on a sterile surgical pad and gently stretch it. Hold the scruff of the neck firmly while holding the mouse supine, allowing it to breathe comfortably. With the neck and chin flat and parallel to the pad, place the tip of a pipettor containing erovanoids dispersed in 0.9% saline (vehicle) near the left nostril at a 45-degree angle. Administer approximately 5 μL of the drug into each nostril at 2-3 second intervals, for a total of 10 μL per nostril. The mouse is held in this position for 5 seconds or until consciousness is restored, after which the administration step is repeated for the other nostril, for a total of 20 μL per mouse. After the mouse receives all droplets, the animal remains restrained supine until the material disappears into the nostril, after which it is returned to its cage. After 4, 24, 48, 72, and 120 hours, the mouse is sacrificed. Visceral and subcutaneous adipose tissues (VAT and SAT), as well as the brain, are collected and the hypothalamus dissected to prepare organotypic slices. VAT and SAT are isolated, adipocytes are plated in six-well plates, and the brain is dissected to collect the hypothalamus, hippocampus, and cortex. The tissues are used to test the parameters described in the experiments designed for specific Aims 1 and 2.

[0415] result Without wishing to be bound by theory, SP and SASP are abolished in the VAT, SAT, and HT of Leprdb / J mice. Furthermore, intranasal treatment of db / db mice with ELV-34 restores spontaneous electrical activity in the HT of these mice. Furthermore, VAT and SAT synthesize and release adiponectin, which restores SP reprogramming.

[0416] result 1-Establish a solid foundation for elovanoids as a treatment for obesity and type 2 diabetes, although there is also a case for their use as a treatment for type 1 diabetes.

[0417] References cited in the examples herein TIFF2025143368000032.tif224148TIFF2025143368000033.tif224148TIFF2025143368000034.tif73148

[0418] Example 12 - Erovanoids counteract oligomeric β-amyloid-induced gene expression and protect photoreceptors summary The development of neurodegenerative diseases activates inflammation, leading to progressive neuronal cell death and cognitive impairment (Alzheimer's disease, AD) and visual impairment (age-related macular degeneration, AMD). How neuroinflammation can be counteracted is unclear. In AMD, amyloid-β peptide (Aβ) accumulates in subretinal drusen. In 5xFAD retinas, we found early functional impairment (ERG) without photoreceptor cell (PRC) death and identified early dysfunction of the biosynthetic pathways of the homeostatic / neuroprotective mediators neuroprotectin D1 (NPD1) and elovanoid (ELV). To mimic the inflammatory environment in wild-type (WT) mice, we subretinally injected oligomeric β-amyloid (OAβ) to induce retinal pigment epithelium (RPE) damage and PRC death, and observed that ELV administration counteracted these effects and protected these cells. Furthermore, ELV prevented OAβ-induced changes in gene expression involved in senescence, inflammation, autophagy, extracellular matrix remodeling, and AMD. Furthermore, because OAβ targets the RPE, we used primary human RPE cell cultures to demonstrate that OAβ caused cell damage and ELV protected and restored gene expression, similar to that observed in mice. Our data showed that OAβ activated senescence, as reflected by enhanced expression of p16INK4a, MMP1, p53, p21, p27, and IL-6, and the senescence-associated secretory phenotype (SASP) secretome, followed by RPE and PRC death, and that erovanoids 32 and 34 blunted these events and exerted protective effects. Furthermore, ELV counteracted OAβ-induced expression of genes involved in AMD, autophagy, and extracellular matrix (ECM) remodeling. Overall, our data reveal that ELV suppresses the induction of the OAβ senescence program and inflammatory transcriptional events, protecting RPE cells and PRCs and thus may be useful as a therapeutic tool for AMD.

[0419] This example reveals dysfunction of the biosynthetic pathways of the homeostatic / neuroprotective mediators neuroprotectin D1 and elovanoid in the retina during early pathogenesis in transgenic Alzheimer's disease 5xFAD mice. These changes correlate with their dysfunction, which precedes photoreceptor cell loss. In AMD, amyloid beta (Aβ) peptides accumulate in drusen. Thus, injection of oligomeric beta-amyloid into the retina of wild-type mice induces photoreceptor cell degeneration and disruption of gene expression, which may include upregulation of the senescence program and SASP. Similar changes also occur in human retinal pigment epithelial cells in culture. Elovanoid, a novel lipid mediator, reverses Aβ peptide-induced changes in gene expression and the SASP secretome, protecting these cells. This study paves the way for the therapeutic evaluation of elovanoids for AMD.

[0420] Introduction The onset of neuroinflammatory responses involves the synthesis of endogenous mediators aimed at countering brain and / or retinal damage. Neuroprotectin D1 (NPD1), a docosanoid derived from omega-3 essential fatty acids, exhibits neuroprotective properties by blocking the initiation of inflammation and preserving the integrity of photoreceptor cells (PRCs) (1) and is deficient in the hippocampal CA1 region in early Alzheimer's disease (AD) (2). Aβ accumulates in AD. Aβ also accumulates in 5xFAD retinas. In AMD, Aβ causes homeostatic disturbances, including inflammation, that may contribute to PRC death (3, 4). However, how Aβ-mediated cellular damage is limited is unclear. In PRCs, very long-chain PUFAs (VLC-PUFAs, C>28) are synthesized by ELOVL4 (elongation of very long chain fatty acid-4) (5, 6) and are required for rhodopsin function (7). Mutations in the ELOVL4 gene (5) cause Stargardt retino-macular dystrophy type 3, which is associated with central vision loss. Recessive ELOVL4 mutations cause seizures, mental retardation, and spastic quadriplegia, demonstrating the importance of VLC-PUFAs in brain development and physiology (8). VLC-PUFAs are incorporated into specific phosphatidylcholine molecular species (PCs) in the outer segments of photoreceptor cells and reach the retinal pigment epithelium (RPE) after daily PRC disc shedding and phagocytosis. ELVs containing 32 and 34C are enzymatically synthesized by phospholipase A1 in RPE cells from PC-released VLCPUFAs (9, 10). These novel lipid mediators have the ability to protect RPE cells from uncompensated oxidative stress by attenuating apoptosis in photoreceptor cells (9, 10) and neurons (11) and upregulating the abundance of homeostatic and pro-survival proteins.

[0421] Aβ42 is a component of drusen in AMD and senile plaques in AD (12, 13). In AMD, β-amyloid contributes to inflammation, disruption of RPE morphology and function, and PRC integrity (14, 15). 5xFAD transgenic mice, carrying mutations associated with early-onset familial AD, exhibit several nonspecific changes but also PRC degeneration (16, 17). We first investigated the 5xFAD retinal phospholipid profile to understand the availability of lipid mediator precursors preceding the onset of PRC degeneration in 5xFAD mice. Next, we subretinal-injected oligomeric Aβ (OAβ), one of the most cytotoxic forms of β-amyloid, into wild-type (WT) mice and studied the consequences on the RPE and PRC, as well as the expression of genes involved in senescence, autophagy, AMD, extracellular matrix (ECM) remodeling, and apoptosis. We also exposed human RPE cells in culture to OAβ and assessed similar endpoints. Finally, we assessed whether erovanoids modify OAβ-induced gene expression, including the senescence program and senescence-associated secretory phenotype (SASP), to protect the RPE and maintain photoreceptor cell integrity.

[0422] result The retina and RPE of 5xFAD mice reveal defects in the pathways leading to the biosynthesis of NPD1 and ELV.

[0423] Upon cleavage by PLA2 and PLA1, the acyl chains of phosphatidylcholine containing DHA (sn2) and VLC-PUFA, n-3 (sn1), lead to the synthesis of NPD1 and ergobanoids, respectively (10). To confirm the availability of these PCs in 5xFAD retinas and RPE, heat map analyses were performed. These analyses revealed two clusters of PCs: short-chain (<48C) and saturated (<6 double bonds) (Group 1) and a cluster of low abundance (Group 2) when comparing 5xFAD and WT (Figure 52, Panel A). This indicates that 5xFAD retinas contain relatively few VLC-PUFA-containing PCs. However, principal component analysis (PCA) revealed no significant differences, as all identifiable PCs in 5xFAD and WT mice were short-chain PCs (Figure 52, Panels B and C). Therefore, we performed random forest classification, with the criterion that longer exposure to phosphatidylcholine emphasized the contribution of PCs to the variation in 5xFAD versus WT. The results showed that frequently used PCs were densely distributed in the VLC-PUFA-containing PC region, which confirmed the observations from the heatmap analysis (Figure 52, Panel D). Therefore, PCs were divided into three groups: (i) DHA- and VLC-PUFA-containing PCs, (ii) DHA-containing PCs, and (iii) AA-containing PCs. PC structures and m / z (Figure 60) showed a decrease in both DHA- and VLC-PUFA-containing PCs, including PC54:12, PC56:12, and PC58:12 (Figure 52, Panel E), and DHA-containing PCs, including PC36:8, PC38:8, and PC44:12 (Figure 52, Panel F). In contrast, AA-containing PCs, including PC36:4, PC38:4, and PC36:5, were increased in 5xFAD retinas (Figure 5C, panel G), while n-6 / n-3 (AA, DHA, and VLC-PUFA) content was altered. Next, we observed that DHA and VLC-PUFAs contained in PCs were depleted in 5xFAD retinas (Figure 1A-G), unlike the RPE (Figure 5D). The content of PC38:6 was high in 5xFAD RPE (in contrast to the retina—Figure 2E), while PC40:6 was similar in 5xFAD and WT (Figure 2E).However, PC44:12 was lower in 5xFAD RPE as well as in the retina. Furthermore, the relative abundance of PCs differed between the retina and RPE. In the retina, VLC-PUFA-containing PCs accounted for 3% of total PCs, whereas these PCs accounted for less than 0.3% in the RPE. Similarly, PC44:12 accounted for 5% in the retina and less than 0.5% in the RPE. Thus, DHA- and VLC-PUFA-containing PCs are more abundant in photoreceptor cells than in the RPE. Despite the small contribution of these PCs in the RPE, our results revealed a deficiency of VLC-PUFA-containing PCs in 5xFAD RPE.

[0424] Erovanoids are generated from VLC-PUFAs stored in PC54-12 and PC56-12 and are present in limited amounts in the retina and RPE (Figures 1 and 2). We found increased free pool sizes of 32:6n-3 and 34:6n-3, reflecting their release from the sn-1 position in PC54-12 and PC56-12, respectively. We next examined the subsequent lipoxygenase-catalyzed enzymatic epoxidation to form an epoxide intermediate, followed by hydrolase-catalyzed enzymatic hydrolysis, resulting in the synthesis of dihydroxylated ELV-N-32 or ELV-N-34 bearing a Z,E,E triene moiety (Figure 5, Panel A). Thus, expression of 15-lipoxygenase-1 in 5xFAD RPE is lower than that in WT, consistent with the abundance of NPD1, which is low in 5xFAD RPE and unchanged in the retina (Figure 54, Panel B). In contrast, ELOVL4, an enzyme that elongates EPA or DHA, is expressed only in PRCs and is low in 5xFAD, correlating with the smaller pool sizes of 32:6n-3 and 34:6n-3, as well as monohydroxystable derivatives of elovanoid hydroperoxide precursors (Figure 54, Panels B-D). Thus, the expression of lipids, as well as two enzymes involved in the ELV and NPD1 pathways, is significantly suppressed in the retina and RPE early during 5xFAD retinal pathogenesis.

[0425] Early abnormal retinal function precedes PRC loss in 5xFAD B-wave ERG analysis of 6-month-old 5xFAD mice revealed a loss of visual sensitivity (Figure 55, Panel A). However, retinal ultrastructure, including the RPE cell / Bruch's membrane interface, the outer segment basal region of disc synthesis, the integrity of the outer limiting membrane (OLM), elongated inner segment mitochondria (without fission profiles), and PRC tip release and phagocytosis by the RPE (Figure 55, Panel B), demonstrated a lack of abnormalities. Furthermore, histology did not demonstrate PRC loss in 5xFAD (Figure 55, Panel C). On the other hand, immunofluorescence microscopy demonstrated that in 5xFAD, Aβ accumulated primarily in the retina beneath the RPE, similar to the AMD phenotype of drusen (Figure 55, Panel D).

[0426] ELVs protect RPE and PRCs from OAβ-induced toxicity Because of the early defects in the pro-homeostatic pathway leading to amyloidosis in 5xFAD retinas and subsequent retinal degeneration, we next asked whether ELV provided protection from the effects of OAβ, the most cytotoxic Aβ peptide (18). Six-month-old WT mice subretinally injected with OAβ exhibited PRC degeneration (Figure 56, panels A and C). Fundus (left) and corresponding optical coherence tomography (OCT) (right) images are depicted. The PRC layer underwent cell loss, from a thickness of 105 μm in uninjected retinas to 35 μm in OAβ-injected retinas. Uninjected, PBS-injected, and ELV-32- and ELV-34-injected mice did not develop PRC degeneration (Figure 56, panels C and D). ZO-1 staining of flat-mounted RPE revealed that oligomeric β-amyloid disrupted tight junctions and caused cell damage. Co-injection of ELV32 or ELV34 with OAβ, followed by topical application of elovanoids for 7 days (Figure 56, Panel A), resulted in restoration of RPE morphology (Figure 56, Panel B) and protection of PRCs (Figure 56, Panels C and D). Mice injected with PBS or ELV alone showed a slight reduction in ONL due to mechanical stress after subretinal injection (Figure 56, Panels C and D). These results indicate that elovanoids maintain the integrity of PRCs, demonstrating the ability of these lipid mediators to counteract cellular injury sustained by OAβ toxicity.

[0427] ELVs counteract OAβ-induced disruption of senescence, autophagy, AMD, and ECM remodeling gene expression in the RPE, and apoptotic gene expression in the retina To investigate the mechanisms involved in ELV protection against OAβ-mediated damage, isolated RPE and retina were subjected to quantitative PCR (qPCR). We chose to investigate genes involved in senescence (19, 20), autophagy (21), AMD (22, 23), and ECM remodeling (24) 3 days after injection of RPE (Figure 56, panels E–G). Furthermore, we examined the cell death-related genes Bax, Bad, Casp3, Dapk1, and Fas in the retina (Figure 56, panel I). OAβ-mediated upregulation of senescence, autophagy, AMD, and several ECM remodeling gene expression was counteracted by OAβ (Figure 56). Specific matrix metalloproteinases (1b, 10, 14, and 7) were not affected by OAβ. Furthermore, in the RPE, the protein abundance of the major senescent p16INK4a (Figure 56, Panel H) correlates with its gene expression (Figure 56, Panel E).

[0428] ELVs protect human RPE cells from OAβ-induced senescence and other gene transcriptional disruptions Because 5xFAD mice exhibit RPE tight junction disruption upon Aβ accumulation (16), we assessed damage and ELV-N-32 or ELV-N-34 protection using primary human RPE cells in culture challenged with Aβ (Figure 57, Panel A). After 7 days of incubation, oligomeric β-amyloid altered RPE cell morphology, activated the SASP, and enhanced the expression of a set of senescence genes (Figure 57, Panel E), AMD, matrix metalloproteinases, and autophagy-related genes (Figure 57, Panel D), as revealed by SA-β-Gal staining (Figure 57, Panels B and C). Interestingly, although some matrix metalloproteinases were affected, not all expressed in RPE cells were affected. In other cells, the SASP has been shown to be primarily pro-inflammatory and involves chemokines, metalloproteinases, proteases, cytokines (such as TNF-α, IL-6, and IL-8), and insulin-like growth factor binding proteins. Senescence genes studied include p16 INK4a (Cdkn2a), p21CIP1 (Cdkn1A), p27 KIP (Cdkn1B), p53 (Tp53 or TRP53), IL6, and MMP1. ELV-N-32 and ELV-N-34 reversed these effects (Figure 57, panels B-D).

[0429] Consideration AMD and Alzheimer's disease manifest as the accumulation of β-amyloid in the retina and brain, respectively. Because Aβ-based antibodies and anti-inflammatory therapies for AD have met with little success, there is a need to understand the mechanisms and identify specific agents that limit Aβ neurotoxicity (25-28). The RPE maintains the integrity of PRCs, and its dysfunction leads to PRC death in retinal degenerative diseases, including AMD. Herein, we show that Oaβ promotes RPE and PRC pathology in both rodent and primary human RPE cell cultures in vivo. Early in the pathogenesis of 5xFAD PRC degeneration, we report defects in precursors and pathways for NPD1 and ELV biosynthesis. These defects precede histological signs of ECM and PRC damage, while ERG already indicates impairment. These findings reveal prodromal alterations in key homeostatic lipid signaling pathways during disease onset and early disease progression. In addition to being used as biomarkers, they can also be explored as therapeutic targets for AMD.

[0430] There is no clear evidence in genetic animal models that blocking Aβ formation reduces AMD pathology. However, some studies have aimed to experimentally inhibit ocular Aβ to protect PRCs. For example, Liu et al. showed that 10-month Aβ vaccination inhibited retinal deposition but caused retinal amyloid angiopathy characterized by microglial infiltration and astrogliosis in AD transgenic mice (29). The drawback of this is the fact that active immunization can cause severe side effects.

[0431] It is unclear how many AD patients will develop AMD, and vice versa. However, there is a correlation between AD and eye diseases other than AMD, which may include glaucoma and diabetic retinopathy (30). Key signaling mechanisms underlying AD may involve CFH, APOE (31-33), and the matrix metalloproteinase pathway (34). Our data show that subretinal Aβ injection in mice causes RPE cell damage and PRC loss after 7 days. To test the robustness of Aβ's deleterious effects on the RPE, we used human RPE cells in primary culture and demonstrated that it induces damage similar to that observed in rodents in vivo. Furthermore, changes in gene expression profiles were similar in both the in vivo rodent model and in vitro human cells. Aβ synthesis occurs in the RPE (35-38) and accumulates in drusen. It is becoming clear that dysfunctional processing of amyloid precursor protein leads to accumulation of the peptide in the inner nuclear layer of the retina, also adjacent to ganglion cells (39-41), and its synthesis, abundance, secretion, and aggregation increase in an age-dependent manner (39). Our subretinal injection of Aβ recapitulates some of the conditions associated with AMD pathology, targeting the RPE.

[0432] The finding that OAβ-induced RPE and photoreceptor cell death in wild-type mice in vivo was counteracted by erovanoids highlights the additional biological activity of these specific downstream mediators from omega-3 fatty acids. Mechanistically, neuroinflammatory destruction is involved in the early stages of AMD pathology, and several studies have used dietary supplements of omega-3 fatty acids (42-46), but have not provided clear benefits because the delivery of these important fatty acids to PRCs and synapses involves complex steps, including the intestine, liver, bloodstream transport, and cellular uptake (47, 48). A rational therapeutic approach for AMD may be to use mediators from omega-3 fatty acids with neuroprotective biological activity.

[0433] This study identifies ELVs 32 and 34 as down-regulating mediators of OAβ-induced senescence, as indicated by the expression of senescence-related genes in the SASP and RPE. Under these conditions, the up-regulated expression of autophagy- and AMD-related genes, including human complement factors (49) and extracellular matrix genes, was also beneficially targeted by erovanoids. Thus, the similarity of oligomeric β-amyloid-induced effects in RPE cells in culture and in RPE and PRCs in vivo, including ELV-targeted protection, demonstrates relevance to the human retina. Surprisingly, we observed that OAβ injection triggered apoptosis-related cell death signaling in photoreceptor cells, rather than senescence. However, it is important to note that erovanoids prevented both OAβ-induced senescence in the RPE and OAβ-induced PRC apoptosis.

[0434] In conclusion, we uncovered early defects in pro-homeostatic pathways prior to PRC death in 5xFAD mice, highlighted by reduced abundance of PC molecular species in the RPE (e.g., those containing VLC-PUFA) and retina (those containing DHA and VLCPUFA). We also found depletion of the pool sizes of free VLC-PUFA and their precursors, 27- and 29-monohydroxybenzoates, as well as the stable derivatives ELV-32 and ELV-34. Furthermore, the retina exhibited deficiencies in key enzymes in the pathways for the synthesis of the pro-homeostatic / neuroprotective NPD1 and ELVs, indicating functional impairment without overt PRC damage or loss. Erovanoids counteracted the cytotoxicity of OAβ administered subretinally to WT mice, which caused disruption of RPE tight junctions and subsequent PRC cell death. Our data show that OAβ activates a senescence program reflected by enhanced gene expression of p16INK4a, MMP1, p53, p21, p27, Il-6, and MMP1, as well as the SASP secretome, leading to the subsequent death of RPE and PRCs. ELV-N-32 and ELV-N-34 blunt these events, eliciting protection for both RPE cells. RPE cells are terminally differentiated and derived from the neuroepithelium. In this context, senescent neurons and astrocytes in aged mice and models of AD (50) and AD (51, 52) also express senescence and generate secretory SASP that promotes neuroinflammation in nearby cells (53-55). Our studies suggest that neighboring cells may be targets of SASP neurotoxicity, inducing paracrine senescence in photoreceptors. Thus, SASP from RPE cells may be autocrine and paracrine, resulting in altered homeostasis of the interphotoreceptor matrix microenvironment, creating an inflammatory milieu and contributing to the loss of function associated with aging (56), age-related pathologies (56), and AMD. Furthermore, ELVs restore the expression of ECM remodeling matrix metalloproteinases altered by OAβ treatment, indicating further impairment of the interphotoreceptor matrix. The ongoing inflammation may represent a low-grade, sterile, chronic proinflammatory state similar to inflammaging, which is also associated with immune aging (56, 57). Furthermore, erovanoids counteracted OAβ-induced expression of genes involved in AMD and autophagy.Without wishing to be bound by theory, elastinoids target gene transcriptional events (Figure 58), providing a novel unifying regulatory mechanism for maintaining a healthy span of life during aging and neurodegenerative diseases (56, 58). Although further research is needed, our results collectively point to elastinoids as a tool for therapeutic exploration in AMD.

[0435] material and method Materials and Methods: This information may include animals, lipid extraction and LC-MS / MS-based lipidomics analysis, primary human RPE cultures, Aβ(1-42) oligomerization, SA-β-Gal staining, protein extraction and Western blot analysis, RNA isolation and quantitative PCR analysis, immunofluorescence and confocal microscopy, and statistics.

[0436] References cited in this example TIFF2025143368000035.tif190149TIFF2025143368000036.tif224150TIFF2025143368 000037.tif217145TIFF2025143368000038.tif224148TIFF2025143368000039.tif94144

[0437] material and method animal All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of LSU Health New Orleans. Six-month-old 5xFAD mice (stock number: 34848-JAX, The Jackson Laboratory, Bar Harbor, ME, USA) co-overexpressed FAD mutant forms of human amyloid precursor protein (Swedish mutation: K670N, M671L; Florida mutation: I716V; and London mutation: V717I) and presenilin 1 (PS1, encoded by Psen1: M146L, L286V) transgenes under the transcriptional control of the neuron-specific mouse Thy1 promoter. 5xFAD mice were hemizygous for the transgene and non-transgenic WT littermates. Genotyping was performed by PCR of tail DNA. All analyses were performed blind to mouse genotype. For subretinal injections, 6-month-old C57BL / 6J mice were anesthetized with an intraperitoneal injection of ketamine / xylazine, and the pupils were dilated with 1.0% tropicamide (Akorn, IL, USA). For topical anesthesia, 0.5% proparacaine hydrochloride (Akorn) was applied. The eye was punctured with a 30-gauge needle into the vitreous cavity between the corneoscleral junction and the ora serrata without disturbing the lens. Under a dissecting microscope, compounds were delivered to the subretinal space using a 33-gauge blunt needle attached to a 5 μl Hamilton syringe (Hamilton Company, Reno, NV, USA). Non-injected mice served as negative controls, and PBS-injected mice were used for sham experiments. Injection volumes were 2 μl containing PBS, 10 μM OAβ, 10 μM OAβ + 200 ng ELV-N-32, 200 ng ELV-N-32 alone, 10 μM OAβ + 200 ng ELV-N-34, or 200 ng ELV-N-34 alone (n = 12 / group). All groups received topical drops: PBS alone, ELV-N-32 (200 nM), or ELV-N-34 (200 nM), twice daily for 3 or 7 days.

[0438] Lipid extraction and LC-MS / MS-based lipidomics analysis The retina or RPE / choroid was homogenized in 3 ml of MeOH, followed by the addition of 6 ml of CHCl3 and 5 μl of a deuterium-labeled lipid internal standard mixture (AA-d8 (5 ng / μl), PGD2-d4 (1 ng / μl), EPA-d5 (1 ng / μl), 15-HETE-d8 (1 ng / μl), and LTB4-d4 (1 ng / μl)). The samples were sonicated for 30 min and stored at -80 °C overnight. The supernatant was then collected, the pellet washed with 1 ml of CHCl3 / MeOH (2:1), centrifuged, and the supernatants combined. 2 ml of distilled water (pH 3.5) was added to the supernatant, vortexed, and centrifuged. The pH of the upper phase was then adjusted to 3.5–4.0 with 0.1 N HCl. The lower phase was dried under N2 and then resuspended in 1 ml of MeOH. LC-MS / MS analysis was performed on a Xevo TQ (Waters, Milford, MA, USA) equipped with an Acquity I-class UPLC with a flow-through needle. For PC and PE molecular species analysis, samples were dried with N2 and then resuspended in 20 μl of sample solvent (acetonitrile / chloroform / methanol, 90:5:5 by volume). An Acquity UPLC BEH HILIC 1.7-μm, 2.1 × 100-mm column was used with a mixture of solvent A (acetonitrile / water, 1:1; 10 mM ammonium acetate, pH 8.3) and solvent B (acetonitrile / water, 95:5; 10 mM ammonium acetate, pH 8.3) as the mobile phase (0.5 mL / min). Solvent B (100%) was run isocratically for the first 5 min, followed by an 8 min gradient to 20% solvent A, increasing to 65% solvent A for 0.5 min, and running at 65% solvent A isocratically for 3 min. The column was then equilibrated back to 100% solvent B for 3.5 min. The column temperature was set at 30°C. The amount of each PC and PE species was calculated as a % of the total PC and PE / sample. For analysis of fatty acids and their derivatives, six retinas or six RPE / choroids were pooled and homogenized as described herein.The sample (1 ml in MeOH) was mixed with 9 ml of H2O at pH 3.5, loaded onto a C18 column (Agilent, Santa Clara, CA, USA), eluted with 10 ml of methyl formate, dried with N2, resuspended in 50 μl of MeOH / H2O (1:1), and injected onto an Acquity UPLC HSS T3 1.8-μm 2.1 × 50-mm column. The mobile phases were 45% solvent A (H2O + 0.01% acetic acid) and 55% solvent B (MeOH + 0.01% acetic acid), initially at a flow rate of 0.4 ml / min, followed by a gradient to 15% solvent A for the first 10 min, a gradient to 2% solvent A for 18 min, an isocratic run at 2% solvent A for 25 min, and then a gradient back to 45% solvent A for 30 min. Lipid standards (Cayman, Ann Arbor, MI, USA) were used for tuning and optimization, and calibration curves for each compound were generated.

[0439] Primary human RPE cultures All experiments using primary human RPE cells were approved by the LSUHNO Institutional Review Board and conducted in accordance with NIH guidelines. Cells were collected from an anonymous donor donated by the eye bank. Briefly, the globes of a 19-year-old Caucasian male with no ocular pathology were obtained from the NDRI within 24 hours of death from head trauma. The globes were opened, and RPE cells were harvested and cultured (1, 2). They were grown in MEM medium supplemented with 10% FBS, 5% NCS, non-essential amino acids, penicillin-streptomycin (100 U / mL), and 10 ng / mL human fibroblast growth factor at 37°C with a constant supply of 5% CO2. Cell integrity was verified as in a previous study (3). For oligomeric Aβ treatment, cells were seeded at 30,000 cells / cm2 in 6-well plates. After 2 days, subconfluent cells were treated with 10 μM Aβ or PBS (vehicle control).

[0440] Aβ(1-42) oligomerization Aβ(1-42) (HFIP-treated, ANASPEC Company, Fremont, CA, USA, Cat. AS-64129) was resuspended in 1% NH4OH / water and DMSO to a concentration of 500 μM and sonicated for 10 min. Oligomerization was then performed by diluting tAβ(1-42) in sterile phosphate buffer in a low-binding polypropylene microcentrifuge tube for 24 h at 4°C. Oligomerization was confirmed by Western blot using mouse monoclonal 6E10 antibody (Figure 64).

[0441] Senescence-associated β-galactosidase (SA-β-Gal) staining Cells were visualized using an SA-β-Gal staining kit (Cat 9860, Cell Signaling Technology, MA, USA). Briefly, RPE cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 minutes, washed again with PBS, and incubated overnight at 37°C (without CO2) in the staining mix. The presence of CO2 can alter the pH, potentially affecting staining results. Photographs were taken at 200X magnification under a bright-field microscope (Nikon Eclipse TS100) after blue staining, and cells were counted in 10 different random fields per well.

[0442] Protein extraction and Western blot analysis Samples were lysed in RIPA buffer, and protein was measured by Bradford assay (Bio-Rad, Hercules, CA, USA). After denaturation, 20 μl of each medium sample or 30 μg of total protein from cell / tissue samples was separated on an SDS-PAGE (4-12% gradient) gel (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in PBST, probed with primary antibody (Figure 66) for 1 h, washed three times with PBST, and probed with secondary antibody (GE Healthcare, Chicago, IL, USA) for 1 h and washed three times with PBST. Protein bands were visualized using an LAS 4000 imaging system (GE Healthcare). Densitometry data were statistically analyzed at a 95% confidence level.

[0443] RNA isolation and qPCR analysis The cell culture medium was removed, the cells were washed with PBS 1X, and samples were collected using a cell scraper. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany).

[0444] For in vivo experiments, eyes were enucleated, the anterior segment including the cornea, lens, and iris was removed, and the retina was separated from the remaining optic cup (RPE / choroid). Total retinal and optic cup (RPE / choroid) RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). 1 μg of total RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad), and reactions were performed using BrightGreen 2X qPCR MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada) and validated primers (SI Appendix, Table S2). Quantitative PCR was performed on a CFX-384 Real-Time PCR System (Bio-Rad). Target gene expression was normalized to the geometric mean of housekeeping genes, and relative expression was calculated by the comparative threshold cycle method (ΔΔCT).

[0445] Immunofluorescence and confocal microscopy For whole-mount RPE staining, eyes were enucleated and prefixed in 4% PFA for 15 minutes. The eyecups containing the RPE sheets were then fixed in 4% PFA for 30 minutes, washed three times with PBS, and then blocked at room temperature for 1 hour. Immunostaining was performed by incubating the primary antibody (ZO-1) at 4°C for 48 hours. The eyecups were then washed three times with PBS and incubated with the secondary antibody for 12 hours at 4°C. Primary human RPE cells and mouse eyecups were embedded in ProLong™ Gold Antifade Mounting Medium (Thermo Fisher Scientific) between two glass coverslips. Photographs were taken with an Olympus FV1200 microscope (Olympus, Japan). Images were analyzed using ImageJ software (rsb.info.nih.gov / ij / ).

[0446] Spectral Domain-Optical Coherence Tomography Imaging and Analysis Seven days after injection, mice were anesthetized with i.p. ketamine / xylazine, their pupils were dilated with topical 1.0% tropicamide, and they were placed in a custom-made holder for OCT imaging (body temperature was maintained at 38°C with a heat pad). The retina was imaged along the horizontal meridian through the optic nerve head using a Heidelberg Spectralis HRA OCT system (Heidelberg Engineering, Heidelberg, Germany). Axial resolution was 7 mm optical and 3.5 mm digital. Raw OCT B-scan cross-sectional images were exported with a scale in μm and opened in ImageJ (http: / / imagej.nih.gov / ij). PRC layer thickness was defined as the width from the tip of the outer nuclear layer, just after the outer plexiform layer, to the outer segment of the PRC. Three measurements were taken on the same scan and averaged. The mean and standard error of the mean (SEM) were calculated (n = 4 / group). Statistical significance was calculated using Student's T-test, and P values ​​less than 0.05 were considered significant.

[0447] statistics Data are expressed as the mean ± SEM of at least three independent experiments. Data were analyzed by one-way analysis of variance followed by Tukey's HSD post-hoc test at a 95% confidence level to compare different groups, with P < 0.05 considered significant. Pearson correlation analysis was used to analyze the relationship between factors. Statistical analysis was performed using BioVinci software (Bioturing INC., San Diego, CA, USA).

[0448] References TIFF2025143368000040.tif48149

[0449] Sequence information SEQUENCE LISTING <110> THE BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AND AGRICULTURAL AND MECHANICAL COLLEGE PELAEZ, Ricardo Palacios <120> VERY-LONG-CHAIN POLYUNSATURATED FATTY ACIDS, ELOVANOID HYDROXYLATED DERIVATIVES, AND METHODS OF USE <150> US 62 / 964,995 <151> 2020-01-23 <150> US 62 / 924,359 <151> 2019-10-22 <150> US 62 / 923,770 <151> 2019-10-21 <150> US 62 / 895,737 <151> 2019-09-04 <160> 115 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> PRT <213> Homo sapiens <400> 1 Ser Tyr Asp Tyr Leu Val Ile Gly Gly Gly Ser Gly Gly Leu Ala Ser 1 5 10 15 Ala Arg <210> 2 <211> 21 <212> PRT <213> Homo sapiens <400> 2 Ala Asp Phe Asp Asn Thr Val Ala Ile His Pro Thr Ser Ser Glu Glu 1 5 10 15 Leu Val Thr Leu Arg 20 <210> 3 <211> 15 <212> PRT <213> Homo sapiens <400> 3 Thr Tyr Ser Thr Ser Phe Thr Pro Met Tyr His Ala Val Thr Lys 1 5 10 15 <210> 4 <211> 19 <212> PRT <213> Homo sapiens <400> 4 His Val Leu Gly Pro Asn Ala Gly Glu Val Thr Gln Gly Phe Ala Ala 1 5 10 15 Ala Leu Lys <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Val Glu Leu Thr Pro Val Ala Ile Gln 1 5 <210> 6 <211> 23 <212> PRT <213> Homo sapiens <400> 6 Val Val Gly Phe His Val Leu Gly Pro Asn Ala Gly Glu Val Thr Gln 1 5 10 15 Gly Phe Ala Ala Ala Leu Lys 20 <210> 7 <211> 13 <212> PRT <213> Homo sapiens <400> 7 Val Leu Asp Phe Val Thr Pro Thr Pro Leu Gly Thr Arg 1 5 10 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 gctcaactac ggtgcagatt c 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 gcacgatgtc ttgatgtccc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 cgagaacggt ggaactttga c 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 ccagggctca ggtagacctt 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 tcaaacgtga gagtgtctaa cg 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 13 ccgggccgaa gagatttctg 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 14 gcgtaaacgc ttcgagatgt t 21 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 15 tttttatggc gggaagtaga ctg 23 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 16 ctgcaagaga cttccatcca g 21 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 17 agtggtatag acaggtctgt tgg 23 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 18 aggctcgtgg tcagaacaac 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 19 gttagacgcc acccattttc c 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 20 cctggtaatg gcccctcctc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 21 ccccattgct ctgtgccttg 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 22 ctgacaggat gcctagccg 19 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 23 cgcaggtaat cccagaagc 19 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 24 gtgatgctca ggtatccatc ca 22 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 cacagttctc aaagcacagc g 21 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 gcaactgttc ctgaactcaa ct 22 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 27 atcttttggg gtccgtcaac t 21 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 gaggaagtcc agtgtccagc 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 ttgctgatgg caacttcaac 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 30 ctcctcctcc atcccttcat 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 31 gagcaacatt catcagcagg 20 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 32 tgtacgcgca caagctagaa tt 22 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 33 tggaaagtgg agtccaggga ga 22 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 34 gatgaacttg gccgcatact 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 35 atgactgtgt tcaggcagga 20 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 36 gatgcacact ctgcgatgaa g 21 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 37 cagtgttcac agccaggaga at 22 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 38 ggtaaataga ttcatgccag aac 23 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 39 ccatgtacta gaatcacggg 20 <210> 40 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 40 agtctttgag gaggaaggcg ata 23 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 41 caaacctagg cctggcagaa 20 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 42 aatccttgcc catgcctttc aacc 24 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 43 ccaaattcat gagcagccac gaga 24 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 44 ttctggtctt ctggcacacg cttt 24 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 45 ccaagctcat gggcagcaac aata 24 <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 46 ccttaaaagt atggagcgac gtca 24 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 47 agcgttccca tactttacgc g 21 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 48 acgacataga cggcatccag tatc 24 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 49 aggtatagtg ggacacatag tggg 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 50 accaacctat tcctggttgc tgct 24 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 51 atggaaacgg gacaagtctg tgga 24 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 52 cactccctgg gtctctttca 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 53 tttgtctggg gtctcaggtc 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 54 ccgccatgca aaagttctat 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 55 gcccacctta ggggtgtaat 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 56 tgcagatcga acagttctgg 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 57 tcgttcgcct ttgaagaagt 20 <210> 58 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 58 tgaatttggc cactctctgg gtct 24 <210> 59 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 59 tctgaatgcc tgcaatgtcg tcct 24 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 60 tagaagcaac tgggcaactg gaca 24 <210> 61 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 61 accgcttcat ccatcttgac ctct 24 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 62 catgttggac agtggtggac 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 63 gaagtggccg agtacctgac 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 64 acagcttctg gatgaaaggc 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 65 tgggactgca gaatgacaga 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 66 tgtggttgct tgcttcagac 20 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 67 gaaggattca ccttcctagc g 21 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 68 ggcgagtttc aataaatggc 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 69 ccaggaactc acagctccat 20 <210> 70 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 70 gtgacgttga catccgtaaa ga 22 <210> 71 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 71 gccggactca tcgtactcc 1...

Claims

1. A method for alleviating the symptoms of, treating, or preventing an allergic inflammatory disease in a subject, comprising administering to said subject a therapeutically effective amount of a VLC-PUFA.

2. A method for alleviating symptoms of, treating, or preventing a disease by modulating cellular senescence, ferroptosis, or cellular senescence and ferroptosis, the method comprising administering a therapeutically effective amount of VLC-PUFA to a subject.

3. A method of alleviating the symptoms of, treating, or preventing a metabolic disorder in a subject, comprising administering to said subject a therapeutically effective amount of a VLC-PUFA.

4. The VLC-PUFA compound has the formula A or B:

4. The method of claim 1, claim 2, or claim 3, wherein the compound may be selected from the group consisting of:

5. The VLC-PUFA compound is selected from the group consisting of:

4. The method of claim 1, claim 2, or claim 3, wherein the compound may be selected from the group consisting of:

6. 4. The method of claim 1, claim 2, or claim 3, wherein the VLC-PUFA is provided as a pharmaceutical composition.

7. 7. The method of claim 6, wherein the pharmaceutical composition comprises a topical, intranasal, oral, or parenteral composition.

8. The method of claim 7 , wherein the topical composition comprises a cream.

9. 4. The method of claim 1, claim 2, or claim 3, wherein the VLC-PUFA is administered topically, orally, intranasally, or parenterally.

10. 4. The method of claim 1, claim 2, or claim 3, wherein the therapeutically effective amount comprises a concentration of about 500 nM, a concentration greater than about 500 nM, or a concentration less than about 500 nM.

11. 7. The method of claim 6, wherein the pharmaceutical composition further comprises one or more additional active agents.

12. The method of claim 11 , wherein the one or more additional active agents comprises at least one antioxidant.

13. 10. The method of claim 1, wherein the allergic inflammatory disorder is indicated by increased production of pro-inflammatory cytokines and chemokines by cells.

14. 14. The method of claim 13, wherein the proinflammatory cytokines and chemokines comprise at least one of IL-6, IL-1β, IL-8 / CXCL8, CCL2 / MCP-1, CXCL1 / KC / GRO, VEGF, ICAM1 (CD54).

15. 14. The method of claim 13, wherein the VLC-PUFA inhibits the production of pro-inflammatory cytokines and chemokines.

16. The method of claim 13 , wherein the cells comprise epithelial cells.

17. The method of claim 16 , wherein the epithelial cells comprise human nasal epithelial cells.

18. 18. The method of claim 17, wherein the epithelial cells comprise nasal epithelial cells, corneal epithelial cells, skin epithelial cells, or respiratory epithelial cells.

19. 10. The method of claim 1, wherein the VLC-PUFA is administered before, at about the same time as, or after exposure to an allergen.

20. 20. The method of claim 19, wherein the allergen causes an allergic inflammatory disease in the subject.

21. 20. The method of claim 19, wherein the allergen causes an increase in the production of pro-inflammatory cytokines and chemokines by cells.

22. 22. The method of claim 21, wherein the cells comprise epithelial cells.

23. 23. The method of claim 22, wherein the epithelial cells comprise human nasal epithelial cells.

24. 22. The method of claim 21, wherein the epithelial cells comprise nasal epithelial cells, corneal epithelial cells, skin epithelial cells, or respiratory epithelial cells.

25. 21. The method of claim 1 or claim 20, wherein the allergic inflammatory disease comprises allergic rhinitis, allergic conjunctivitis, allergic dermatitis, and asthma.

26. The method of claim 2 , wherein the disease comprises a neurodegenerative disease.

27. 3. The method of claim 2, wherein the disease comprises an Aβ-related disease.

28. The method of claim 2 , wherein the disease comprises Alzheimer's disease or age-related macular degeneration.

29. 4. The method of claim 3, wherein the metabolic condition comprises obesity or diabetes.

Images