Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders

Definitions

• Long chain polyunsaturated fatty acids can include the omega-3 (n-3) and omega-6 (n6) polyunsaturated fatty acids containing 18-22 carbons including: arachidonic acid (ARA, C20:4n6, 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).
• ARA arachidonic acid
• EPA eicosapentaenoic acid
• DPA docosapentaenoic acid
• DHA docosahexaenoic acid
• LC-PUFAs are converted via lipoxygenase-type enzymes to biologically active hydroxylated PUFA derivatives that function as biologically active lipid mediators that play important roles in inflammation and related conditions.
• Most important among these are hydroxylated derivatives generated in certain inflammation-related cells via the action of a lipoxygenase (LO or LOX) enzyme (e.g.15-LO, 12-LO), and result in the formation of mono-, di- or tri- hydroxylated PUFA derivatives with potent actions including anti-inflammatory, pro- resolving, neuroprotective or tissue-protective actions, among others.
• LO or LOX lipoxygenase
• neuroprotectin D1 a dihydroxy derivative from DHA formed in cells via the enzymatic action of 15-lipoxygenase (15-LO) was shown to have a defined R/S and Z/E stereochemical structure (10R,17S-dihydroxy-docosa-4Z,7Z,11E, 13E,15Z,19Z-hexaenoic acid) and a unique biological profile that can includes stereoselective potent anti- inflammatory, homeostasis-restoring, pro-resolving, bioactivity. NPD1 has been shown to modulate neuroinflammatory signaling and proteostasis, and to promote nerve regeneration, neuroprotection, and cell survival.
• aspects of the disclosure are drawn to a method of alleviating a symptom of, treating, or preventing an allergic inflammatory disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a VLC-PUFA.
• aspects of the disclosure are drawn to a method of alleviating a symptom of, treating, or preventing a disease by modulating cellular senescence, ferroptosis, or cellular senescence and ferroptosis, the method comprising administering to the subject a therapeutically effective amount of a VLC-PUFA.
• the disease comprises a neurodegenerative disease.
• the disease comprises an Ab-associated disease.
• the disease can be Alzheimer’s disease or age-related macular degeneration.
• aspects of the disclosure are drawn to a method of alleviating a symptom of, treating, or preventing a metabolic disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a VLC-PUFA.
• the metabolic condition comprises obesity or diabetes.
• aspects of the disclosure are drawn to a method of alleviating a symptom of, treating, or preventing an allergic inflammatory disease in a subject.
• the allergic inflammatory disease is indicated by increased production of pro-inflammatory cytokines and chemokines by a cell.
• the allergic inflammatory disease comprises allergic rhinitis, allergic conjunctivitis, allergic dermatitis, or asthma.
• the pro-inflammatory cytokines and chemokines comprise at least one of IL-6, IL-1b, IL-8/CXCL8, CCL2/MCP-1, CXCL1/KC/GRO, VEGF, ICAM1(CD54).
• the cell comprises an epithelial cell.
• epithelial cells comprise a respiratory epithelial cell (such as a human nasal epithelial cell), a corneal epithelial cell, or a skin epithelial cell.
• the method comprises administering to the subject a therapeutically effective amount of a VLC-PUFA.
• the VLC-PUFA abrogates the production of pro-inflammatory cytokines and chemokines
• the VLC-PUFA compound can be selected from the group consisting of the formula A or B: A B
• the VLC-PUFA compound can be selected from the group consisting of: [014]
• the VLC-PUFA is provided as a pharmaceutical composition.
• the pharmaceutical composition comprises a composition for topical administration, a composition for intranasal administration, a composition for oral administration, or a composition for parenteral administration.
• the VLC-PUFA or pharmaceutical composition is administered topically, orally, intranasally, or parenterally.
• the therapeutically effective amount comprises about 500nM concentration, greater than about 500nM concentration, or less than about 500nM concentration.
• the VLC-PUFA is administered prior to exposure to an allergen, at about the same time as exposure to an allergen, or after exposure to an allergen.
• the allergen causes an allergic inflammatory disease in a subject.
• the allergen causes increased production of pro-inflammatory cytokines and chemokines by a cell, decreased production of anti-inflammatory cytokines and chemokines, or both.
• aspects of the disclosure are also drawn towards a method of treating allergic rhinitis comprising administering to the subject a therapeutically effective amount of a VLC-PUFA.
• the VLC-PUFA is administered intranasally.
• aspects of the disclosure are drawn towards a method of treating allergic conjunctivitis comprising administering to the subject a therapeutically effective amount of a VLC-PUFA.
• the VLC-PUFA is administered topically, such as to the eye using an eye drop.
• aspects of the disclosure are drawn towards a method of treating allergic dermatitis comprising administering to the subject a therapeutically effective amount of a VLC-PUFA.
• the VLC-PUFA is administered topically, such as a cream, spray, or gel.
• aspects of the disclosure are drawn towards a method of treating asthma comprising administering to the subject a therapeutically effective amount of a VLC-PUFA.
• the VLC-PUFA is administered intranasally.
• aspects of the disclosure are also drawn towards a method of alleviating, treating, or preventing inflammatory reaction of epithelial tissue comprising contacting the epithelial tissue with a therapeutically effective amount of a VLC-PUFA.
• the inflammatory reaction is indicated by increased production of pro-inflammatory cytokines and chemokines by a cell.
• the pro-inflammatory cytokines and chemokines comprise at least one of IL-6, IL-1b, IL-8/CXCL8, CCL2/MCP-1, CXCL1/KC/GRO, VEGF, ICAM1(CD54).
• the anti-inflammatory molecule comprises IL-10.
• the VLC-PUFA abrogates the production of pro-inflammatory cytokines and chemokines, enhances the production of anti-inflammatory molecules, or both.
• n-3 VLC-PUFA omega- 3 very-long-chain polyunsaturated fatty acids
• elovanoids endogenous hydroxylated derivatives thereof
• This disclosure provides methods for alleviating a symptom of, treating, or preventing an allergic inflammatory disease in a subject.
• n-3 VLC-PUFAs are converted in vivo to several previously unknown types of VLC- PUFA hydroxylated derivatives named elovanoids (ELVs) that are able to protect and prevent the progressive damage to tissues and organs, whose functional integrity has been disrupted.
• ELVs elovanoids
• the ELVs can abrogate the production of pro-inflammatory cytokines and chemokines and thus alleviate a symptom of, treat, or prevent an allergic inflammatory disease in a subject.
• the production of pro-inflammatory cytokines and chemokines can be effectively suppressed by providing certain compounds related to n-3 VLC-PUFA and their corresponding elovanoids (ELVs). Accordingly, the disclosure is related to the prevention and treatment of allergic inflammatory diseases.
• the present disclosure provides compounds, compositions and methods that can promote the protection, prevention, and treatment of disturbances in many organs triggered by pro-inflammatory cytokines.
• the present disclosure provides compounds, compositions and methods that can abrogate the production of pro-inflammatory cytokines and chemokines from epithelial cells, such as nasal epithelial cells, and thus alleviate a symptom of, treat, or prevent an allergic inflammatory disease.
• epithelial cells such as nasal epithelial cells
• one aspect of the disclosure encompasses embodiments 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.
• the composition comprises at least one n-3 VLC-PUFA having at least 23 carbon atoms in its carbon chain, wherein the n-3 VLC-PUFA can be in the form of a carboxylic acid, carboxylic ester, carboxylate salt, or phospholipid derivative.
• the n-3 VLC-PUFA compound can be selected from the group consisting of the formulas A or B: wherein: m can be 0 to 19 and -CO-OR can be a carboxylic acid group, or a salt or an ester thereof, and wherein: if -CO-OR can be a carboxylic acid group and the compound A or B can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and if -CO-OR can be an ester, then R can be an alkyl group.
• the n-3 VLC-PUFA compound can be in the form of a phospholipid selected from the group consisting of the formulas C, D, E or F, wherein m can be 0 to 19: [033]
• the composition can further comprise a pharmaceutically-acceptable carrier and formulated for delivery of an amount of the at least one omega-3 very long chain polyunsaturated fatty acid effective in reducing a pathological condition of a tissue of a recipient subject or the onset of a pathological condition of a tissue of a recipient subject.
• the pathological condition can be an allergic inflammatory disease of the recipient subject.
• the allergic inflammatory disease can be allergic rhinitis, allergic conjunctivitis, allergic dermatitis, or asthma.
• the composition can be formulated for topical delivery of the 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 an eye drop.
• the composition can be formulated for intranasal delivery of the at least one very long chain polyunsaturated fatty acid to the nasal tissue of a recipient subject.
• the composition can be formulated for oral delivery or parenteral delivery of the at least one very long chain polyunsaturated fatty acid to a recipient subject.
• 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.
• the at least one omega-3 very long chain polyunsaturated fatty acid can have 32 or 34 carbon atoms in its carbon chain.
• the omega-3 very long chain polyunsaturated fatty acid can have in its carbon chain five or six alternating double bonds with cis geometry.
• 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.
• 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.
• Another aspect of the disclosure encompasses embodiments of a composition comprising at least one elovanoid having at least 23 carbon atoms in its carbon chain.
• the composition can further comprise a pharmaceutically-acceptable carrier and can be formulated for delivery of an amount of the at least one elovanoid effective in alleviating a symptom of, preventing, or reducing a pathological condition of a tissue of a recipient subject.
• the pathological condition can be an allergic inflammatory disease of the recipient subject, such as allergic rhinitis, allergic conjunctivitis, allergic dermatitis, or asthma.
• the composition can be formulated for topical delivery of the at least one elovanoid to the skin of a recipient subject or the eye of a recipient subject, such as by an eye drop. [047] In some embodiments of this aspect of the disclosure, the composition can be formulated for intranasal delivery of the at least one elovanoid to the nasal tissue of a recipient subject. [048] In some embodiments of this aspect of the disclosure, the composition can be formulated for oral delivery or parenteral delivery of the at least one elovanoid to a recipient subject.
• the at least one elovanoid can be selected from the group consisting of: a mono-hydroxylated elovanoid, a di-hydroxylated elovanoid, an alkynyl mono-hydroxylated elovanoid, and an alkynyl di-hydroxylated elovanoid, or any combination thereof.
• the at least one elovanoid can be a combination of elovanoids, wherein the combination is selected from the group consisting of: a mono-hydroxylated elovanoid and a di-hydroxylated elovanoid; a mono- hydroxylated elovanoid and an alkynyl mono-hydroxylated elovanoid; a mono-hydroxylated elovanoid and an alkynyl di-hydroxylated elovanoid; a di-hydroxylated elovanoid and an alkynyl mono-hydroxylated elovanoid; a di-hydroxylated elovanoid and an alkynyl di- hydroxylated elovanoid; a mono-hydroxylated elovanoid, a di-hydroxylated elovanoid, and an alkynyl di- hydroxylated elovanoid; a mono-hydroxylated elovanoid
• the mono-hydroxylated elovanoid can be selected from the group consisting of the formulas G, H, I or J: wherein: m can be 0 to 19 and -CO-OR can be a carboxylic acid group, or a salt or an ester thereof, and wherein: if -CO-OR can be a carboxylic acid group and the compound G, H, I or J can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and if -CO-OR can be an ester, then R can be an alkyl group.
• the composition can comprise equimolar amounts of the enantiomers G and H wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group. [053] In some embodiments of this aspect of the disclosure, the composition can comprise amounts of the enantiomers I and J wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group. [054] In some embodiments of this aspect of the disclosure, the composition can comprise one of the enantiomers of G or H in an amount exceeding the amount of the other enantiomer of G or H.
• the composition can comprise one of the enantiomers of I or J in an amount exceeding the amount of the other enantiomer of I or J.
• the di-hydroxylated elovanoid can be selected from the group consisting of the formulas K, L, M, and N: wherein: m can be 0 to 19 and -CO-OR can be a carboxylic acid group, or a salt or an ester thereof, and wherein: if -CO-OR can be a carboxylic acid group and the compound K, L, M, or N can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and if -CO-OR can be an ester, then R can be an alkyl group, and wherein: compounds K and L each have a total from 23 to 42 carbon atoms in the carbon chain, with 4 cis carbon-carbon double bonds starting at positions
• the composition can comprise equimolar amounts of diastereomers K and L wherein the diastereomers have either (S) or (R) chirality at position n-6, and (R) chirality at position n-13.
• the composition can comprise equimolar amounts of one or more diastereomers K and L wherein the diastereomers have either (S) or (R) chirality at position n-6, and either (S) or (R) chirality at position n-13.
• the composition can comprise one of the diastereomers of K or L in an amount exceeding the amount of the other diastereomers of K or L.
• the composition can comprise equimolar amounts of diastereomers M and N wherein the diastereomers have either (S) or (R) chirality at position n-6, and (R) chirality at position n-13.
• the composition can comprise equimolar amounts of one or more diastereomers M and N wherein the diastereomers have either (S) or (R) chirality at position n-6, and either (S) or (R) chirality at position n-13.
• the composition can comprise one of the diastereomers of M or N in an amount exceeding the amount of the other diastereomers of M or N.
• the alkynyl mono-hydroxylated elovanoid can be selected from the group consisting of the formulas O, P, Q or R: wherein: m can be 0 to 19 and -CO-OR can be a carboxylic acid group, or a salt or an ester thereof, and wherein: if -CO-OR can be a carboxylic acid group and the compound O, P, Q or R can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and if -CO-OR can be an ester, then R can be an alkyl group, and wherein: compounds O and P each have a total from 23 to 42 carbon atoms in the carbon chain, with 4 cis carbon-carbon double bonds located at positions starting at n-3, n-12, n-15 and n-18; with a trans carbon- carbon double bond at position starting at n-7, and a carbon-carbon triple bond starting at position n
• the composition can comprise equimolar amounts of the enantiomers O and P wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.
• the composition can comprise equimolar amounts of the enantiomers Q and R wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.
• the composition can comprise one of the enantiomers of O or P in an amount exceeding the amount of the other enantiomer of O or P.
• the composition can comprise one of the enantiomers of Q or R in an amount exceeding the amount of the other enantiomer of Q or R.
• the elovanoid can be an alkynyl di-hydroxylated elovanoid selected from the group consisting of the formulas S, T, U or V: wherein: m can be 0 to 19 and -CO-OR can be a carboxylic acid group, or a salt or an ester thereof, and wherein: if -CO-OR can be a carboxylic acid group and the compound S, T, U or V can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and if -CO-OR can be an ester, then R can be an alkyl group, and wherein: compounds S and T each have a total from 23 to 42 carbon atoms in the carbon chain, with 3 c
• the composition can comprise equimolar amounts of diastereomers S and T wherein the diastereomers have either (S) or (R) chirality at position n-6, and (R) chirality at position n-13.
• the composition can comprise equimolar amounts of one or more diastereomers S and T wherein the diastereomers have either (S) or (R) chirality at position n-6, and either (S) or (R) chirality at position n-13.
• the composition can comprise one of the diastereomers of S or T in an amount exceeding the amount of the other diastereomers of S or T.
• the composition can comprise equimolar amounts of diastereomers U and V wherein the diastereomers have either (S) or (R) chirality at position n-6, and (R) chirality at position n-13.
• the composition can comprise equimolar amounts of one or more diastereomers U and V wherein the diastereomers have either (S) or (R) chirality at position n-6, and either (S) or (R) chirality at position n-13.
• the composition can comprise one of the diastereomers of U or V in an amount exceeding the amount of the other diastereomers of U or V.
• the peptide analog modulates cellular senescence, ferroptosis, or cellular senescence and ferroptosis.
• aspects of the invention are drawn towards a method of treating a disease by administering to a subject the peptide analog as described herein.
• embodiments are drawn towards a method of treating a disease by modulating cellular senescence, ferroptosis, or cellular senescence and ferroptosis.
• the method comprises the steps of administering to a subject afflicted with or at risk of a disease an elovanoid or a peptide analog thereof.
• the elovanoid or peptide analog thereof binds to an epitope of a molecular target as described herein.
• Aspects of the invention are further drawn towards a method of treating a disease associated with cellular senescence, ferroptosis, or cellular senescence and ferroptosis.
• the method comprises targeting at least one molecular target with an elovanoid or peptide analog thereof.
• BRIEF DESCRIPTION OF THE DRAWINGS [079]
• the present disclosure is focused on compounds, compositions and methods for applications in alleviate a symptom of, preventing, or treating a disease in a subject.
• the disease is an inflammatory disease, such as an allergic inflammatory disease.
• the disease is a disease associated with cellular senescence, ferroptosis, or both, such as a disease associated with Ab, including age-related macular degeneration or Alzheimer’s disease.
• the disease is a metabolic disorder, such as diabetes, obesity, or both.
• Fig.2 is a scheme illustrating the biosynthesis of n-3 VLC-PUFA.
• Figs.3A-3K illustrate the generation and structural characterization of elovanoids ELV-N-32 and ELV-N-34 from cultured primary human retinal pigment epithelial cells (RPE).
• Fig.3A is a scheme illustrating ELV-N-32 and ELV-N-34 synthesis from the intermediates (1, 2, and 3), each of which was prepared in stereochemically-pure form. The stereochemistry of intermediates 2 and 3 was pre-defined by using enantiomerically-pure epoxide starting materials.
• Fig.3B illustrates the elution profile of C32:6n-3, endogenous mono-hydroxy- C32:6n-3, and ELV-N-32 shown with ELV-N-32 standard.
• MRM of ELV-N-32 shows two large peaks eluted earlier than the peak when standard ELV-N-32 is eluted, displaying the same fragmentation patterns (shown in the insert spectra), indicating that they are isomers.
• Fig.3C illustrates the chromatogram for full daughter scans for ELV- N-32 and ELV- N-34.
• Fig.3D illustrates the fragmentation pattern of ELV-N-32.
• Fig.3E illustrates the elution profile of C34:6n-3 and ELV-N-34.
• Fig.3F illustrates the UV spectrum of endogenous ELV-N-34 showing triene features analogous to NPD1, with ⁇ max at 275 nm and shoulders at 268 and 285 nm.
• Fig.3G illustrates the fragmentation pattern of ELV-N-32.
• Fig.3H illustrates the full fragmentation spectra of endogenous ELV-N-32.
• Fig.3I illustrates the ELV-N-32 standard shows that all major peaks from standard match to the endogenous peaks. However, endogenous ELV-N-32 has more fragments that don’t show up in the standard, indicating that it can includes different isomers.
• Fig.3J illustrates the full fragmentation spectra of endogenous ELV-N-34 peaks match to standard ELV-N-34.
• Fig.3K illustrates the existence of ELV-N-34 isomers.
• Figs.4A-4K illustrate the structural characterization of elovanoids ELV-N-32 and ELV-N-34 from neuronal cell cultures. Cerebral cortical mixed neuronal cells were incubated with 32:6n-3 and 34:6n-310mM each under OGD conditions.
• Fig.4A is a scheme illustrating ELV-N-32 and ELV-N-34 synthesis from the intermediates (a, b, and c), each of which was prepared in stereochemically-pure form. The stereochemistry of intermediates b and c was pre-defined by using enantiomerically-pure epoxide starting materials.
• Fig.4B illustrates the 32:6n-3, endogenous mono-hydroxy-32:6, ELV-N-32, and ELV-N-32 standard in the insert. MRM of ELV-N-32 shows two large peaks eluted earlier than the peak when standard ELV-N-32 is eluted, but they show the same fragmentation patterns, indicating that they are isomers.
• Fig.4C illustrates the same features as in Fig.4A, were shown in 34:6n-3 and ELV- N-34.
• Fig.4D illustrates the UV spectrum of endogenous ELV-N-32 shows triene features, but these are not definite at this concentration.
• Fig.4E illustrates the full fragmentation spectra of endogenous ELV-N-32.
• Fig.4F illustrates the UV spectrum of endogenous ELV-N-34 showing triene features analogous to NPD1, with ⁇ max at 275 nm and shoulders at 268 and 285 nm.
• Fig.4G illustrates the fragmentation pattern of endogenous ELV-N-34.
• Fig.4H illustrates the full fragmentation pattern of endogenous ELV-N-32.
• Fig.4I illustrates the ELV-N-34 standard shows that all major peaks from the standard match to the endogenous peaks, but not perfectly matched; endogenous ELV-N-34 has more fragments that do not show up in the standard. Without wishing to be bound by theory, this indicates that it can contain isomers.
• Fig.4J illustrates the ELV-N-34 full fragmentation spectra; the endogenous ELV-N- 34 peaks match to the standard ELV-N-34
• Fig.4K illustrates the existence of ELV-N-34 isomers.
• Fig.5A and 5B illustrate the detection of ELV-N-32 and ELV-N-34 in neuronal cell cultures.
• FIG.5A illustrates the 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 prepared in stereochemical pure form via stereocontrolled total organic synthesis.
• MRM of endogenous ELV-N-32 matches well with the MRM of the synthetic ELV-N-32 standard.
• Fig.5B illustrates the same features as in Fig.5A were shown in C34:6n-3 and ELV- N-34, with more peaks in ELV- N-34 MRMs, which indicates isomers.
• Fig.6 illustrates Scheme 1 for the total synthesis of mono-hydroxylated elovanoids G, H, I, J, O, P, Q, R.
• Reagents & Conditions (a) Catechol borane, heat; (b) N-iodo- succinimide, MeCN; (c) 4-chlorobut-2-yn-1-ol, Cs 2 CO 3 , NaI, CuI, DMF; (d) CBr4, PPh3, CH 2 Cl 2 , 0 o C; (e) ethynyl-trimethylsilane, CuI, NaI, K 2 CO 3 , DMF; (f) Lindlar cat., H 2 , EtOAc; (g) Na 2 CO 3 , MeOH; (h) Pd(PPh3) 4, CuI, Et3N: (i) t Bu4NF, THF; (j) Lindlar cat., H2, EtOAc or Zn(Cu/Ag), MeOH; (k) NaOH, THF, H 2 O, then acidification with HCl/H 2 O; (l) NaOH, KOH, or the like.
• Fig.7 illustrates Scheme 2 for the total synthesis of di-hydroxylated elovanoids K, L, S, and T.
• Fig.8 illustrates Scheme 3 for the total synthesis of di-hydroxylated elovanoids M, N, U, and V.
• Fig.9 illustrates Scheme 4 for the total synthesis of 32-carbon di-hydroxylated elovanoids.
• Fig.10 illustrates Scheme 5 for the total synthesis of 34-carbon di-hydroxylated elovanoids.
• Fig.11 shows bright field images showing morphology of HNEpC - 10x and 20x.
• Fig.12 shows house dust mite allergenicity. The 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. [117] Fig.13 shows structures of LPS and Poly(I:C). [118] Fig.14 shows experimental design of challenging HNEpC using several stressors (aeroallergens) [119] Fig.15 shows different elovanoids (ELVs) that were used at 500nM concentration for verifying lipid specificity against HNEpCs stressed with different aeroallergens. [120] Fig.16 shows cytotoxicity assay using CyQuant LDH assay. Damage to the plasma membrane releases LDH into the surrounding cell culture media.
• the extracellular LDH in the media can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH.
• Oxidation of NADH by diaphorase leads to the reduction of a tetrazolium salt (INT) to a red formazan product that can be measured spectrophotometrically at 490 nm.
• INT tetrazolium salt
• the level of formazan formation is directly proportional to the amount of LDH released into the medium, which is indicative of cytotoxicity.
• Fig.17A and Fig.17B shows cytotoxicity assay using CyQuant LDH assay.
• Fig.18A and Fig.18B shows cell viability assay using Presto Blue HS reagent.
• Fig.19A and Fig.19B shows ELISA (IL-6).
• Fig.20A and Fig.20B shows ELISA (IL-1b).
• Fig.21A and Fig.21B shows ELISA (IL-8).
• Fig.22A and Fig.22B shows ELISA (CCL2).
• Fig.23A and Fig.23B shows ELISA (CXCL1).
• Fig.24A and Fig.24B shows ELISA (VEGF).
• Fig.25A and Fig.25B shows ELISA (ICAM1).
• FIG.26A and Fig.26B shows ELISA (IL-10) [131] Fig.27 shows representative drawing from Trends in Molecular Medicine; October, 2011; Vol.17, No.10. Jacquet, 2011. [132] FIG.28 shows the converging mechanisms that regulate senescence for the development of new synthetic non-lipidic analogs to mimic the bioactivity of the lipid mediators. [133] FIG.29 shows oxidative-stress and Erastin induced cell deaths counteracted by NPD1 and ELV-32:6 in human RPE cells. [134] FIG.30 shows elovanoids attenuates Erastin-mediated phosphorylation of PEBP-1 (adapted from Wenzel et al., 2017, Cell, 171:628-641).
• FIG.31 shows upstream regulation by elovanoids of ferroptosis and senescence.
• FIG.32 shows deletion of MFRP (Membrane Frizzled-Related Protein) leads to progressive PRC degeneration.
• FIG.33 shows selective loss of PC44:12 and 56:12 in AdipoR1 –/– and MFRP rd6 .
• FIG.34 shows targets in human retina with AMD.
• FIG.35 shows AMD shows PCs selective differences in cone-rich macula and rod- rich periphery; MALDI MS molecular imaging displays distinct layering.
• FIG.36 shows heat maps of 168 GPCR targets and of 73 orphan GPRCs (antagonists or partial agonists) screened by PathHunter b-arrestin enzyme fragment complementation/b- galactosidase against the orphanMAX Panel (DiscoverX, Eurofins, Fremonst, CA).
• GPCR targets blue arrows
• Microplates read with PerkinElmer EnVision multimode for Chemiluminescence.
• FIG.37 shows induction of the expression and activation of a specific AdipoR1 molecule will compensate deficits of neuroprotective mediators (1) deficiencies in precursors and intermediates of lipid mediators pathways and of ELOVL-4 during the early development of retina pathology in the 5xFAD mouse.
• A The biosynthetic pathway of NPD1, 32:6n-3 and 34:6n-3 elovanoids from their PC 54-12 and PC56-12 precursors. For the mass spectrometry detection, the stable monohydroxy products of VLC-PUFAs are used.
• B The bar chart for 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 layers (bottom).
• C-D Western blots and quantification of 15-lypoxygenase-1 expression in RPE and Retina.
• FIG.38 shows interactions between NPD1, ELV32 and ELV34 with positive screened GPCRs by Path Hunter b-arrestin complementation.
• FIG.39 shows UOS up-regulates pro-inflammatory transcriptome and down regulates pro-homeostatic pathways in RPE single-cells. NPD1 and ELV32-6 suppress these changes. Dots in box plots represent expression of the gene for a single RPEC (total of 96 per sample).
• FIG.40 shows structure of glutathione reductase based on the x-ray structure of human glutathione reductase.
• FIG.41 shows analysis of target candidate protein TXNRD1. Structure shown is based on the x-ray structure of human NADP(H) thioredoxin reductase I.
• FIG.42 shows the converging mechanisms that regulate senescence for the development of new synthetic non-lipidic analogs to mimic the bioactivity of the lipid mediators.
• FIG.43 shows population level electrical activity was recorded from different hypothalamic neurons, using the MEA system.
• FIG.44 shows protection by Elovanoids of hypothalamic neuronal cell death (by Fluoro-Jade B staining) in adult obese diabetic mice (db/db).
• FIG.45 shows senescense associated b- galactosidase activity measured in human neuronal-glial (HNG) cells exposed to oligomeric amyloid beta (Oab) (10mM).
• HNG human neuronal-glial
• Oab oligomeric amyloid beta
• A-G SA-b- Gal activity in HNG cells treated with Oab (10mM) and different Elovanoids (ELVs) or neuroprotectin D1 (NPD1) at a concentration of 500 nM. Micrographs were obtained with bright field microscopy.
• FIG.46 shows senescense associated b-galactosidase activity measured in human neuronal-glial (HNG) cells exposed to Erastin (10mM).
• A-G SA-b-Gal activity in HNG cells treated with Erastin (10mM) and different Elovanoids (ELVs) or neuroprotectin D1 (NPD1) at a concentration of 500nM. Micrographs were obtained with bright field microscopy.
• H Quantification of SA-b-Gal+ cells shown in (A-G). SA-b-Gal+ cells were scored in 3 random fields of at least 150 total cells. Results are expressed as percentage of stained SA-b-Gal+ cells (mean ⁇ SEM). Statistical analysis were done using Graphpad Prism software 8.3. Results compared with one-way ANOVA, followed by Holm’s Sidak post hoc tests and p ⁇ 0.05 was considered statistically significant.
• FIG.47 shows experimental design: Human neuronal glial (HNG) cells were challenged using Oab or Erastin.
• FIG.48 shows ELV34 revert the effect of IL1b in human diabetic adipocytes.
• FIG.49 shows ELV34 reduced the levels of IL6 (marker of SASP) induced by IL1b in Diabetic db/db mice hypothalamus indicating that hypothalamic neurons and astrocytes undergo SP. Different effects have been observed in female and male mice.
• IL6 marker of SASP
• FIG.50 shows characteristics of the db/db mice versus WT.
• FIG.51 shows ELV34 treatment increased the levels of Adiponectin an anti-diabetic systemic hormone secreted by adipocytes and other tissues (hypothalamus) that promotes insulin sensitivity.
• SAT subcutaneous adipose tissue
• VAT visceral adipose tissue
• FIG.52 shows deficiency of VLC-PUFAs in phosphatidylcholine molecular species in the retina of the 5xFAD.
• B PCA analysis for PCs illustrates two populations (WT-black and 5xFAD-red) scatter across the principal component 1. Thus, the loading score for the principal component 1 is essential to identify distinct PCs for the difference between WT and 5xFAD.
• (C) The loading score (absolute values) of PCs to the principal component 1. The higher loading score, the more contribution of the PCs into principal component to distinguish WT and 5xFAD (SI Appendix, Fig. S1A). Ten short chain PUFAs ( ⁇ 48C) contained in PCs are found in the top 12 most loading score PCs (C) while the VLCPUFAs contained in PCs contribute two (58:1 and 58:12).
• D The time used in random forest classification for PCs of WT and 5xFAD. The higher time used, the more valuable of the PCs in WT and 5xFAD difference (SI Appendix, Fig. S1B). VLC-PUFAs contained PCs contribute seven of 12 top time used in this classification (D).
• FIG.53 shows deficiency of VLC-PUFAs in phosphatidylcholine molecular species of the RPE of the 5xFAD.
• B PCA for PCs in RPE of 5xFAD and WT.
• FIG.54 shows deficiencies in precursors and intermediates of lipid mediator pathways and of ELOVL-4 during the early development of retina pathology in the 5xFAD.
• A Biosynthetic pathways of NPD1 and of 32:6n-3 and 34:6n-3 ELVs from PC 54-12 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 ELV N-32 and ELV N-34, respectively.
• D Free DHA.
• FIG.55 shows morphology and function of the 5xFAD retina.
• (A) V log I plot showing maximum ERG b-wave amplitudes for light flashes from 0 to 0.075 cd ⁇ s/m2. 5xFAD achieved maximum amplitude of about 100mV, approximately half that recorded for WT (n 6/group).
• FIG.56 shows ELVs restore RPE morphology and reduce gene expression after subretinal injection of OAb in WT mice.
• mRNA were isolated for Real-Time PCR.
• mice were subjected to OCT and then eyes enucleated and processed for whole mount RPE staining and Western blots.
• B Whole flat mount of RPE with tight junction marker, Zonula occludens-1 (ZO-1) antibody. OAb disrupted RPE morphology.
• C OAb effects on retina and RPE by OCT.
• FIG.57 shows OA-b-mediated activation of senescence-associated secretory phenotype (b-galactosidase, SA-b-Gal) and of gene expression in human RPE cells in primary culture are counteracted by ELVs.
• A In vitro experimental design: Primary human RPE cells were treated with 10mM OAb +/- ELVs. After 3 days, RNA was isolated and qPCR analyzed. After 7 days, cells were subjected to b-Galactosidase staining.
• B Live cell images under bright field microscopy after 7 days.
• C b- Gal staining +/- ELVs. Quantitation of % for the b-Gal positive cells.
• FIG.58 shows summary of ELVs effects on OAb-induced RPE and PRC damage.
• A OAb induces a senescence program and disrupts RPE tight junctions. Without wishing to be bound by theory, OAb penetrates the retina, causing PRC cell death in our in vivo WT mice study reflected in less cell body layer (CBL) nuclei. ELVs restore RPE morphology and PRC integrity.
• FIG.59 shows principal component analysis loading score and random forest time used for all PC species in retina and RPE.
• A The full loading score (absolute values) of all PCs to the Principle Component 1 in the retina. The higher loading score, the more contribution of the PCs into Principle Component 1 to distinguish the retinas from wild type and 5xFAD.
• B The time used in random forest classification for all retinal PCs of wild type and 5xFAD mouse.
• FIG.60 shows the fatty acid compositions of PCs are obtained by full fragmentation. The negative ion modes were used for LC/MS/MS data acquisition.
• the VLC-PUFA- containing PCs are depicted;
• 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).
• 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+CH3COO-) 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 on the second row;
• (D) PC38:6 (m/z 864 (M+CH3COO-) corresponds to m/z 806 (M+H+) in positive mode) is composed of FA16:0 (m/z 255) and FA22:6 (m/z 327).
• 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).
• 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 at the third row;
• 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).
• PC38:4 (m/z 868 (M+CH3COO-) corresponds to m/z 810 (M+H+) in positive mode) is composed of FA18:0 (m/z 283) and FA20:4 (m/z 303).
• PC38:5 (m/z 866 (M+CH3COO-) 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 PC38:6 isotopes (two carbons being naturally C13 labeled). That will produce FA16:0 (m/z 255) and FA22:6(m/z 329 when two carbons are C13), or FA16:0 (m/z 256 when one carbon is C13) and FA22:6 (m/z 328 when one carbon is C13).
• FIG.61 shows full fragmentation spectra of ELVs and pre-cursor molecules and NPD1.
• FIG.62 shows fundus and OCT analysis.
• FIG.63 shows inflammation signal is activated by 5xFAD.
• A The relative normalized expression of AMD-related genes of the RPE in WT and 5xFAD. The RNA from eye cup/choroid of WT and 5xFAD (6 months old) were isolated and reverse transcribed into cDNA and subjected to RT-PCR with AMD-related genes. Briefly, there is the activation of different genes, related to the AMD in 5xFAD.
• FIG.64 shows western blot for Oligomers Ab. After 24 hours of oligomerization, the 2 ⁇ l of the Ab stock was loaded in the tricine gel, without the denaturation.
• FIG.65 shows the unfolded protein response (UPR) genes. After 3 days injection, the RNA from RPE and retina were isolated and reverse transcribed into cDNA and subjected to RT-PCR with UPR primers.
• UPR unfolded protein response
• FIG.66 shows antibodies as utilized herein.
• FIG.67 shows gene primer sequences as utilized herein. DETAILED DESCRIPTION OF THE DISCLOSURE [172]
• alkyl groups can include straight-chained, branched and cyclic alkyl radicals containing up to about 20 carbons, or 1 to 16 carbons, and are straight 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.
• lower alkyl can refer to carbon chains having from about 1 or about 2 carbons up to about 6 carbons. Suitable alkyl groups can be saturated or unsaturated.
• an alkyl can also be substituted one or more times on one or more carbons with substituents selected from a group consisting of C1-C15 alkyl, allyl, allenyl, alkenyl, C3-C7 heterocycle, aryl, halo, hydroxy, amino, cyano, oxo, thio, alkoxy, formyl, carboxy, carboxamido, phosphoryl, phosphonate, phosphonamido, sulfonyl, alkylsulfonate, arylsulfonate, and sulfonamide.
• substituents selected from a group consisting of C1-C15 alkyl, allyl, allenyl, alkenyl, C3-C7 heterocycle, aryl, halo, hydroxy, amino, cyano, oxo, thio, alkoxy, formyl, carboxy, carboxamido, phosphoryl, phosphonate, phosphonamid
• an alkyl group can contain up to 10 heteroatoms, in certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8 or 9 heteroatom substituents. Suitable heteroatoms can include nitrogen, oxygen, sulfur and phosphorous.
• cycloalkyl can refer to a mono- or multicyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments of 3 to 6 carbon atoms.
• the ring systems of the cycloalkyl group can be composed of one ring or two or more rings which can be joined together in a fused, bridged or spiro-connected fashion.
• aryl can refer to aromatic monocyclic or multicyclic groups containing from 3 to 16 carbon atoms.
• aryl groups are aryl radicals, which can contain up to 10 heteroatoms, in certain embodiments, 1, 2, 3 or 4 heteroatoms.
• An aryl group can also be substituted one or more times, in certain embodiments, 1 to 3 or 4 times with an aryl group or a lower alkyl group and it can be also fused to other aryl or cycloalkyl rings.
• Suitable aryl groups can include, for example, phenyl, naphthyl, tolyl, imidazolyl, pyridyl, pyrroyl, thienyl, pyrimidyl, thiazolyl and furyl groups.
• a ring can have up to 20 atoms that can include one or more nitrogen, oxygen, sulfur or phosphorous atoms, provided that the ring can have one or more substituents selected from the group consisting of hydrogen, alkyl, allyl, 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 also contain one or more fused rings, including carbocyclic, heterocyclic, aryl or heteroaryl
• alkenyl and alkynyl carbon chains contain from 2 to 20 carbons, or 2 to 16 carbons, and are straight or branched.
• Alkenyl carbon chains of from 2 to 20 carbons in certain embodiments, contain 1 to 8 double bonds, and the alkenyl carbon chains of 2 to 16 carbons, in certain embodiments, contain 1 to 5 double bonds.
• Alkynyl carbon chains of from 2 to 20 carbons in certain embodiments, contain 1 to 8 triple bonds, and the alkynyl carbon chains of 2 to 16 carbons, in certain embodiments, contain 1 to 5 triple bonds.
• heteroaryl can refer to a monocyclic or multicyclic aromatic ring system, in certain embodiments, of about 5 to about 15 members where one or more, in one embodiment 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or 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.
• heterocyclyl can refer to a monocyclic or multicyclic non-aromatic ring system, in one embodiment of 3 to 10 members, in another embodiment of 4 to 7 members, in a further embodiment of 5 to 6 members, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
• the nitrogen is substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, acyl, guanidino, or the nitrogen can be quaternized to form an ammonium group where the substituents are selected as described herein.
• aralkyl can refer to an alkyl group in which one of the hydrogen atoms of the alkyl is replaced by an aryl group.
• halo can refer to F, Cl, Br or I.
• haloalkyl can refer to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen. Such groups can include, but are not limited to, chloromethyl and trifluoromethyl.
• aryloxy can refer to RO-, in which R is aryl, including lower aryl, such as phenyl.
• acyl can refer to a –COR group, including for example alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, or heteroarylcarbonyls, all of which can be substituted.
• n-3 means the third carbon atom from the end of the carbon chain of the fatty acid or fatty acid derivative.
• n- 3 or “n-3”, “n-6” or “n6”, etc. also can refer to the position of a substituent such as a hydroxyl group (OH) located at a carbon atom of the fatty acid or fatty acid derivative, wherein the number (e.g. 3, 6, etc.) is counted from the end of the carbon chain of the fatty acid or fatty acid derivative.
• OH hydroxyl group
• subject or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
• subject can include a mammal, for example, a human at any age suffering from pathology. In another embodiment, the term encompasses a subject at risk of developing pathology.
• Subjects to which compounds of the present disclosure can be administered will be animals, for example mammals, such as primates, especially humans.
• a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals for example pets such as dogs and cats.
• livestock such as cattle, sheep, goats, cows, swine, and the like
• poultry such as chickens, ducks, geese, turkeys, and the like
• domesticated animals for example pets such as dogs and cats.
• mammals including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.
• rodents e.g., mice, rats, hamsters
• rabbits primates, and swine
• primates primates
• swine such as inbred pigs and the like.
• living subject can refer to a subject noted herein or another organism that is alive.
• a “subject afflicted with a condition” or “a subject having a condition” can refer to a subject with an existing condition or a known or suspected predisposition toward developing a condition.
• the condition can be an inflammatory disease, such as an allergic inflammatory disease.
• the condition can be a disease associated with cellular senescence, ferroptosis, or both, such as a disease associated with Ab, including age-related macular degeneration or Alzheimer’s disease.
• the disease can be a metabolic disorder, such as diabetes, obesity, or both.
• a “subject having an allergic condition” can refer to a subject with an existing allergic condition or a known or suspected predisposition toward developing an allergic condition.
• the subject can have an active allergic condition or a latent allergic condition. It is not necessary that the allergen be known.
• certain allergic conditions are associated with seasonal or geographical environmental factors, which can but need not be apparent to the subject.
• the allergic condition is intentionally induced in the subject for experimental purposes.
• pharmaceutically acceptable derivatives of a compound can 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 of skill in this art using known methods for such derivatization.
• the compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
• Pharmaceutically acceptable salts can include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'- ylmethylbenzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl) aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali 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 also including, but not limited to, salts of mineral acids, such as but not limited to hydro
• 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.
• compositions are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
• "Formulation" as used herein can refer to any collection of components of a compound, mixture, or solution selected to provide optimal properties for a specified end use, including product specifications and/or service conditions.
• the term formulation shall 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 can included, or packaged, with other non-toxic compounds, such as cosmetic carriers, excipients, binders and fillers, and the like.
• the acceptable cosmetic carriers, excipients, binders, and fillers for use in the practice of the present invention are those which render the compounds amenable to oral delivery and/or provide stability such that the formulations of the present invention exhibit a commercially acceptable storage shelf life.
• the term "administering" can refer to introducing a substance, such as a VLC-PUFA, into a subject.
• administering can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject.
• the formulation or pharmaceutical compound of the present invention can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice.
• Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; 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; and the like.
• injectable solutions including sterile aqueous or non-aqueous solutions, or saline solutions
• creams including 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; and the like.
• the formulations or pharmaceutical composition can also be can included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, binders and fillers including, but not limited to, glucose, lactose, gum acacia, 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, and the like.
• pharmaceutically acceptable carriers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and/or polysaccharides, starch paste, magnesium trisilicate, talc, corn starch, starch fragments, kerat
• the pharmaceutically acceptable carriers, excipients, binders, and fillers for use in the practice of the present invention are those which render the compounds of the invention amenable to intranasal delivery, oral delivery, parenteral delivery, intravitreal delivery, intraocular delivery, ocular delivery, subretinal delivery, intrathecal delivery, intravenous delivery, subcutaneous delivery, transcutaneous delivery, intracutaneous delivery, intracranial delivery, topical delivery and the like.
• the packaging material can be biologically inert or lack bioactivity, such as plastic polymers or silicone, and can be processed internally by the subject without affecting the effectiveness of the composition/formulation packaged and/or delivered therewith.
• compositions or pharmaceutical compositions can be calibrated in order to adapt both to different individuals and to the different needs of a single individual. However, the present formulation need not counter every cause in every individual. Rather, by countering the necessary causes, the present formulation will restore the body and brain to their normal function. Then the body and brain themselves will correct the remaining deficiencies.
• therapeutically effective amount can refer to that amount of an embodiment of the composition or pharmaceutical composition being administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the condition or disease that the subject being treated has or is at risk of developing.
• subject can refer to a vertebrate, for example, a mammal, such as a human.
• Mammals can include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
• the term “pet” can include a dog, cat, guinea pig, mouse, rat, rabbit, ferret, and the like.
• farm animal can include a horse, sheep, goat, chicken, pig, cow, donkey, llama, alpaca, turkey, and the like.
• 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 are useful in preparing a pharmaceutical composition that are safe, non-toxic and neither biologically nor otherwise undesirable, and can include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and/or human pharmaceutical use.
• “A pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant” as used herein can include one and more such excipients, diluents, carriers, and adjuvants.
• composition or a “pharmaceutical formulation” can refer to a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, especially a human and that can refer to the combination of an active agent(s), or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.
• pharmaceutical composition can refer to the composition being sterile, and free of contaminants that can elicit an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
• compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, intranasal, topical, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, by stent-eluting devices, catheters-eluting devices, intravascular balloons, inhalational and the like.
• the pharmaceutical composition can comprise a therapeutically effective amount of an elovanoid and a therapeutically effective amount of one or more additional active agents (such as one or more anti-oxidants, anti-allergenics, anti- inflammatory agents, or pain relievers).
• the one or more anti-oxidants can be synthetic antioxidants, natural antioxidants, or a combination thereof.
• the anti-oxidants can protect the double bonds of the elovanoids.
• the term “administration” can refer to introducing a composition of the present disclosure into a subject.
• Advantageous route of administration of the composition is topical administration, oral administration, or intranasal administration.
• any route of administration such as intravenous, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, introduction into the cerebrospinal fluid, intravascular either veins or arteries, or instillation into body compartments can be used.
• treatment can refer to the management and care of a subject for the purpose of combating a condition, disease or disorder, in any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered.
• the term can include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing the condition, disease or disorder, wherein "preventing” or “prevention” can refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and can includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications.
• Skilled artisans will appreciate a variety of methodologies and assays can be used to assess the development of pathology, and similarly, a variety of methodologies and assays can be used to reduce pathology, driveway, or regression.
• the term "preventing” can refer to preventing a disease, disorder, or condition from occurring in a subject that may be at risk for the disease, but is not yet diagnosed as having the disease. Prevention (and effective dose to prevent) can be demonstrated in population studies. For example, an amount effective to prevent a given disease or condition is an amount effective to reduce the incidence in the treated population, compared to an untreated control population.
• the phrase "alleviating a symptom of” can refer to ameliorating, reducing, or eliminating any condition or symptom associated with an allergic inflammatory disease.
• symptoms of allergic inflammatory conditions can include tingling or itching in the mouth; hives, itching, or eczema; swelling of the lips, face, tongue and throat or other parts of the body; wheezing nasal congestion, or trouble breathing; abdominal pain, diarrhea, nausea or vomiting; and dizziness, lightheadedness, or fainting.
• the patient to be treated can be a mammal, such as a human being. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use for treating a disease as provided herein.
• the composition can further comprise one or more "nutritional components".
• the term "nutritional component” as used herein can refer to such as protein, a carbohydrate, vitamins, minerals and other beneficial nutrients including functional ingredients of the disclosure, that is, ingredients that can produce specific benefits to a person consuming the food.
• the carbohydrate can be, but is not limited to, glucose, sucrose, fructose, dextrose, tagatose, lactose, maltose, galactose, xylose, xylitol, dextrose, polydextrose, cyclodextrins, trehalose, raffinose, stachyose, fructooligosaccharide, maltodextrins, starches, pectins, gums, carrageenan, inulin, cellulose based compounds, sugar alcohols, sorbitol, mannitol, maltitol, xylitol, lactitol, isomalt, erythritol, pectin
• compositions described herein that can include nutritional components can be food preparations that can be, but are not limited to, "snack sized", or "bite sized” compositions that is, smaller than what might normally be considered to be a food bar.
• the food bar can be indented or perforated to allow the consumer to break off smaller portions for eating, or the food "bar” can be small pieces, rather than a long, bar- shaped product.
• the smaller pieces can be individually coated or enrobed. They can be packaged individually or in groups.
• the food can include solid material that is not ground to a homogeneous mass, such as, without limitation.
• the food can be coated or enrobed, such as, and without limitation, with chocolate, including dark, light, milk or white chocolate, carob, yogurt, other confections, nuts or grains.
• the coating can be a compounded confectionary coating or a non-confectionary (e.g., sugar free) coating.
• the coating can be smooth or can contain solid particles or pieces.
• An allergy is when one's immune system reacts to a foreign substance, called an allergen. For example, the allergen can be eaten, inhaled into one's lungs, injected into a subject or touched.
• Allergic reactions are treated using anti-allergy medications, such as Brompheniramine (Dimetane), Cetirizine (Zyrtec), Chlorpheniramine (Chlor-Trimeton), Clemastine (Tavist), Diphenhydramine (Benadryl), and Fexofenadine (Allegra).
• anti-allergy medications such as Brompheniramine (Dimetane), Cetirizine (Zyrtec), Chlorpheniramine (Chlor-Trimeton), Clemastine (Tavist), Diphenhydramine (Benadryl), and Fexofenadine (Allegra).
• compositions provided herein comprising VLC-PUFAs do not cause such unwanted side effects, and therefore are an improvement over the known anti-allergy medications.
• Aspects of this invention are drawn to compositions and methods for alleviating a symptom of, preventing, or treating allergic inflammatory diseases. Referring to the Examples included herein, results from a cytotoxicity assay (LDH) show that upon addition of the stressors to a culture of nasal epithelial cells, there is pronounced increase in the formation of red formazan indicating cytotoxicity, which are reduced by the addition of ELVs.
• LDH cytotoxicity assay
• FIG.17A and FIG.17B cell viability assay using PrestoBlue HS reagent also shows more resorufin production in control cells as compared to cells challenged with the different stressors, and that addition of ELVs increases cell viability and gives protection to the HNEpC (FIG.18A and FIG.18B). Still further, when HNEpC were challenged with the different stressors, there is a pronounced production of pro-inflammatory cytokines and chemokines compared to controls and a pronounced decrease in the release of anti- inflammatory cytokines.
• compositions and methods for alleviating a symptom of, preventing, or treating a disease associated with cellular senescence, ferroptosis, or both are also drawn to compositions and methods for alleviating a symptom of, preventing, or treating a disease associated with cellular senescence, ferroptosis, or both.
• the disease is a disease associated with Ab.
• Non-limiting examples of such diseases include age-related macular degeneration or alzheimer’s disease.
• compositions and methods for alleviating a symptom of, preventing, or treating a metabolic disorder include diabetes and obesity.
• metabolic disorders include diabetes and obesity.
• the disclosure encompasses embodiments of compounds, compositions, and methods for the alleviation of a symptom of, the prevention of, and treatment of diseases.
• This is based on new findings described herein regarding surprising biological activities of certain very long chain-polyunsaturated fatty acids (VLC-PUFA) and their related hydroxylated derivatives. For example, such biological activities include anti- inflammatory role of certain VLC-PUFAs, among others.
• VLC-PUFA very long chain-polyunsaturated fatty acids
• Long chain polyunsaturated fatty acids can include the omega-3 (n-3) and omega-6 (n6) polyunsaturated fatty acids containing 18-22 carbons including: arachidonic acid (ARA, C20:4n6, 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).
• ARA arachidonic acid
• EPA eicosapentaenoic acid
• DPA docosapentaenoic acid
• DHA docosahexaenoic acid
• LC-PUFAs are converted via lipoxygenase-type enzymes to biologically active hydroxylated PUFA derivatives that function as biologically active lipid mediators that play important roles in inflammation and related conditions.
• Most important among these are hydroxylated derivatives generated in certain inflammation-related cells via the action of a lipoxygenase (LO or LOX) enzyme (e.g.15-LO, 12-LO), and result in the formation of mono-, di- or tri- hydroxylated PUFA derivatives with potent actions including anti-inflammatory, pro- resolving, neuroprotective or tissue-protective actions, among others.
• LO or LOX lipoxygenase
• neuroprotectin D1 a dihydroxy derivative from DHA formed in cells via the enzymatic action of 15-lipoxygenase (15-LO) was shown to have a defined R/S and Z/E stereochemical structure (10R,17S-dihydroxy-docosa-4Z,7Z,11E, 13E,15Z,19Z-hexaenoic acid) and a unique biological profile that can includes stereoselective potent anti- inflammatory, homeostasis-restoring, pro-resolving, bioactivity. NPD1 has been shown to modulate neuroinflammatory signaling and proteostasis, and to promote nerve regeneration, neuroprotection, and cell survival.
• n-3 VLC-PUFA very-long-chain polyunsaturated fatty acids
• n6 VLC-PUFA very-long-chain polyunsaturated fatty acids
• C24-C42 elongase enzymes that elongate n-3 and n6 LC-PUFA to n-3 and n6 VLC-PUFA containing from 24 to 42 carbons (C24-C42).
• C24-C42 carbons
• Representative types of VLC-PUFA can include C32:6n-3 (32 carbons, 6 double bonds, omega-3), C34:6n-3, C32:5n-3, and C34:5n-3.
• VLC-PUFA are biogenically-derived through the action of elongase enzymes, such as ELOVL4 (ELOngation of Very Long chain fatty acids 4).
• VLC-PUFA are also acylated in complex lipids including sphingolipids and phospholipids such as in certain molecular species of phosphatidyl choline.
• ELOVL4 ELOngation of Very Long chain fatty acids 4
• VLC-PUFA are also acylated in complex lipids including sphingolipids and phospholipids such as in certain molecular species of phosphatidyl choline.
• the biosynthetic role of ELOVL4 and the biological functions of VLC-PUFA have been the subject of a number of recent investigations. See, for example, PCT/US2016/017112, PCT/US2018/023082, and US 16/576,456, each of which are can included herein by reference in their entireties.
• the compounds, compositions and methods encompassed by the embodiments of the disclosure involve the use of n-3 VLC-PUFA for alleviating a symptom of, preventing, or treating a disease.
• DHA docosahexaenoic acid
• DPA docosapentaenoic acid
• the biosynthesis of n-3 VLC-PUFA requires the availability of DHA or other shorter-chain PUFA as substrates, and the presence and actions of certain elongase enzymes, e.g. ELOVL4.
• these 22- carbon omega-3 long-chain fatty acids are substrates to elongase enzymes, such as ELOVL4, which adds a 2-carbon CH 2 CH 2 group at a time to the carboxylic end, forming n-3 VLC-PUFA that contain carbon chains with at least 24 carbons of up to at least 42 carbons.
• elongase enzymes such as ELOVL4
• ELOVL4 elongase enzymes
• Elongation by the elongase enzyme ELOVL4 leads to the formation of very long chain omega-3 polyunsaturated fatty acids (n-3 VLC-PUFA, 2, including C32:6n-3 and C34:6n-3 that are then incorporated at the 1-position of phosphatidyl choline molecular species, 3.
• n-3 VLC-PUFA 2, including C32:6n-3 and C34:6n-3 that are then incorporated at the 1-position of phosphatidyl choline molecular species, 3.
• the presence of DHA at the 2-position and n-3 VLC-PUFA at the 1-position can offer redundant, complementary, and synergistic cytoprotective and neuroprotective actions that amplify the survival of neurons and other key cell types when challenged with pathological conditions.
• elovanoids can include monohydroxy compounds (e.g. ELV-27S and ELV-29S, 4, and dihydroxy derivatives, e.g. ELV-N-32 and ELV-N-34, 5.
• Elovanoid ELV-N-32 is the 20R,27S-dihydroxy 32:6 derivative (32-carbon, 6 double bond elovanoid with a neuroprotectin-like 20(R),27(S)- dihydroxy pattern).
• Elovanoid ELV-N-34 is the 22R,29S-dihydroxy 34:6 derivative (34- carbon, 6 double bond elovanoid with a 22(R),29(S)-dihydroxy pattern).
• FIG.2 illustrates the delivery of docosahexaenoic acid (DHA, C22:6n-3) to photoreceptors, photoreceptor outer segment membrane renewal, and the synthesis of elovanoids.
• DHA or precursor C18:3n-3 are obtained by diet, as is DHA itself (FIG.1).
• the systemic circulation (mainly the portal system) brings them to the liver.
• DHA-PL DHA-phospholipid
• RPE retinal pigment epithelium
• DHA then passes through the interphotoreceptor matrix (IPM) and to the photoreceptor inner segment, where it is incorporated into phospholipids for the photoreceptor outer segments, cell membrane and organelles.
• IPM interphotoreceptor matrix
• the majority is used in disk membrane biogenesis (outer segments).
• disk membrane biogenesis outer segments.
• Photoreceptor tips are phagocytized by the RPE cells each day, removing the oldest disks. The resulting phagosomes are degraded within the RPE cells, and DHA is recycled back to photoreceptor inner segments for new disk membrane biogenesis. This local recycling is referred to as the 22:6 short loop.
• Elovanoids are formed from omega-3 very long chain polyunsaturated fatty acids (n-3 VLC-PUFA) biosynthesized by ELOVL4 (ELOngation of Very Long chain fatty acids-4) in the photoreceptor inner segments.
• ELOVL4 ELOngation of Very Long chain fatty acids-4
• a phosphatidylcholine molecular species in the inner segment that contains VLC Omega-3 FA at C1 (C34:6n-3 is depicted) and DHA (C22:6n-3) at C2 is used for photoreceptor membrane biogenesis. This phospholipid has been found tightly associated to rhodopsin.
• a phospholipase A1 cleaves the acyl chain at sn-1, releasing C34:6n-3 and leads to the formation of elovanoids (e.g. elovanoid-34, ELV-N-34).
• elovanoids e.g. elovanoid-34, ELV-N-34.
• VLC omega-3 fatty acids that are not used for elovanoid synthesis are recycled through the short loop.
• n-3 VLC-PUFA contain only an even number 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).
• n-3 VLC-PUFA that contain only an odd number of carbons ranging from at least 23 of up to at least 41 carbons (i.e.23, 25, 27, 29, 31, 33, 35, 37, 39, 41 carbons) are not naturally occurring, but they can be synthesized and manufactured using synthetic chemical methods and strategies.
• ELV-N- 32 and ELV-N-34 Stereocontrolled total synthesis and structural characterization of elovanoids ELV-N- 32 and ELV-N-34 in the retina and the brain: As summarized in FIG.3 and FIG.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 pre-defined by using enantiomerically pure epoxide starting materials. Iterative couplings of intermediates 1, 2, and 3, led to ELV-N-32 and ELV-N-34 (4) that were isolated as the methyl esters (Me) or sodium salts (Na).
• Me methyl esters
• Na sodium salts
• the synthetic materials ELV-N-32 and ELV- N-34 were matched with endogenous elovanoids with the same number of carbons on their carbon chain, obtained from cultured human retinal pigment epithelial cells (RPE) (FIG.3), and neuronal cell cultures (FIG.4).
• RPE retinal pigment epithelial cells
• the elovanoids are enzymatically generated hydroxylated derivatives of 32-carbon (ELV-N-32) and 34-carbon (ELV-N-34) n-3 VLC-PUFA in that were first identified in cultures of primary human retinal pigment epithelial cells (RPE) (FIG.3A-3K) and in neuronal cell cultures (FIG.4A-4K).
• RPE retinal pigment epithelial cells
• FIG.4A-4K neuronal cell cultures
• n-3 VLC-PUFA beneficial use of the provided n-3 VLC-PUFA and/or elovanoid compounds, as therapeutics for the prevention and treatment of diseases.
• the phrase "allergic disease” or "allergic inflammatory disease” can refer to a disease with an allergic reaction. More specifically, an “allergic disease” can be characterized by a strong correlation between exposure to an allergen and the development of pathological changes, and that the pathological changes have an immune mechanism (i.e., an allergic inflammatory disease). For example, the immune mechanism can refer to leukocytes exhibit an immune response to allergen stimulation.
• the immune response can refer to increased production of pro-inflammatory cytokines and chemokines.
• allergens can include mite antigens and pollen antigens.
• Representative allergic diseases can 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 a parent child of an allergic predisposition. Familial allergic diseases are also called atopic diseases, and the causative genetic factor is atopic constitution.
• Atopic dermatitis is a general term for atopic diseases, such as diseases associated with skin inflammation.
• Non-limiting examples can include allergic conditions selected from the group consisting of eczema, allergic rhinitis, hay fever, urticaria, and food allergies. Allergic conditions can include eczema, allergic rhinitis or nasal cold, hay fever, bronchial asthma, urticaria (urticaria (hives), and food allergies, as well as other atopic conditions.
• Allergic conditions can include eczema, allergic rhinitis or nasal cold, hay fever, bronchial asthma, urticaria (urticaria (hives), and food allergies, as well as other atopic conditions.
• “Asthma” can refer to a disorder of the respiratory system characterized by inflammation, airway narrowing, and increased airway responsiveness to inhaled substances or allergens. Asthma is often but not exclusively associated with atopic or allergic symptoms.
• asthma Symptoms of asthma are widely recognized to can include dyspnea, cough, and wheezing; while all three symptoms coexist, their coexistence is not required to make a diagnosis of asthma.
• allergic asthma can refer to the allergic aspect of asthma among asthma symptoms and can includes, for example, mixed type asthma and atopic asthma. Allergic asthma is discriminated from non-allergic asthma such as aspirin asthma.
• a “therapeutic agent for asthma”, for example, can exert a therapeutic effect via the action on the allergic reaction of asthma.
• the therapeutic agent of asthma for example exerts an effect on chronic bronchitis or airway hypersensitivity.
• the therapeutic agent of asthma has an effect on chronic bronchitis and airway hypersensitivity.
• the therapeutic agent of asthma exerts an effect on the late phase response, the delayed-type response, or the late phase and the delayed-type responses of the allergic reaction.
• the therapeutic agent of asthma exerts an effect on the late phase response, the delayed-type response or the late phase and the delayed-type responses, in addition to the immediate-type response.
• "Allergic rhinitis" can refer to any allergic reaction in the nasal mucosa, which can include hay fever (seasonal allergic rhinitis) and perennial rhinitis (non-seasonal allergic rhinitis). Symptoms of allergic rhinitis can be sneezing, rhinorrhea, nasal congestion, pruritis, eye itching, redness and tearing.
• skin disorder can include the skin reactions of urticaria and angioedema. These skin disorders can be triggered by exposure to certain foods, medications, or virus infections.
• Urticarira referred to as hives or welts
• Urticarira are red, itchy, raised areas of the skin of varying shapes and sizes. Urticarira are the result of release of histamine and other compounds from mast cells that cause serum to leak from local blood vessels and thereby cause swelling in the skin.
• Angioedema is a form of tissue swelling similar to urticaria, but involving deeper skin tissues (i.e., “deep hives”) and lasting longer than urticarial [253]
• the term “allergic dermatitis” can refer to dermatitis related with allergic reaction and can includes, for example, atopic dermatitis. Allergic dermatitis is discriminated from non- allergic dermatitis such as dermatitis due to injuries or wounds.
• a “therapeutic agent for atopic dermatitis” those that show therapeutic effect by acting on the allergic reaction occurring in atopic dermatitis are useful.
• the therapeutic agent of atopic dermatitis has an effect on the late phase response, the delayed-type response, or the late phase and delayed-type responses of the allergic reaction.
• the therapeutic agent of atopic dermatitis has an effect on the late phase response, the delayed-type response or the late phase response and the delayed-type response, in addition to the immediate-type response.
• the phrase “allergic conjunctivitis” can refer to an irritation by an allergen of the clear, thin membrane called the conjunctiva that covers the eyeball and the inside of the eyelids. Symptoms can include swollen eyes, itchy/burning eyes, tearing, and ocular redness.
• allergens can include pollen from trees, grass and ragweed, animal skin and secretions such as saliva, perfumes and cosmetics, skin medicines, air pollution and smoke.
• An “allergen” can refer to a substance that can induce an allergic inflammatory disease in a subject. The list of allergens is enormous and can include pollens, insect venoms, animal dander, house dust mite, dust, fungal spores, latex, and drugs (e.g., penicillin).
• Examples of natural, animal and plant allergens can include proteins specific to the following genera: Canis (Canis familiaris); Dermatophagoides (e.g., Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia); Lolium (e.g., Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinosa); Betula (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.
• Allergens also can include peptides and polypeptides such as are used in experimental animal models of allergy and asthma, including ovalbumin (OVA) and Schistosoma mansoni egg antigen.
• OVA ovalbumin
• Schistosoma mansoni egg antigen can refer to a disorder or disease that results in disruption of the normal physiological state of homeostasis due to changes in metabolism (anabolism and / or catabolism). Metabolic changes fail to degrade (catabolize) the substance to be degraded (eg, phenylalanine), resulting in increased levels of the substance and / or intermediate substance, or some essential substances (eg, insulin) cannot be produced (anabolic).
• Metabolic syndrome can refer to the concept of a grouping of metabolic risk factors that gather in a single individual and lead to a high risk of developing diabetes and / or cardiovascular disease.
• the main features of metabolic syndrome include insulin resistance, hypertension (hypertension), cholesterol abnormalities, dyslipidemia, triglyceride abnormalities, increased risk for coagulation and especially in the abdomen and overweight or obesity.
• Metabolic syndrome is also known as Syndrome X, Insulin Resistance Syndrome, Obesity Syndrome, Abnormal Metabolic Syndrome and Reaven's Syndrome. The interrelationship of the various risk factors of metabolic syndrome is shown in FIG. The presence of three or more risk factors in a single individual is indicative of metabolic syndrome.
• metabolic syndrome is diagnosed by the presence of three or more of the following factors: (1) Increased waist circumference (male, 40 inches (102 cm) or greater; female, 35 inches (88 cm or more), (2) triglyceride elevation (150 mg / dL or more), (3) high density lipid or HDL reduction (male, less than 40 mg / dL; female, less than 50 mg / dL); (4) Increased blood pressure (130/85 mmHg or higher); and (5) increased fasting blood glucose (100 mg / dL or higher).
• “Metabolic syndrome related metabolic disorders” can refer to metabolic syndrome and obesity, insulin resistance, type 2 diabetes, atherosclerosis and cardiomyopathy.
• Diabetes can refer to a group of metabolic disorders characterized by hyperglycemia (glucose) levels resulting from deficiencies in insulin secretion or action or both.
• Type 2 diabetes is one of the two major types of diabetes, at least in the early stages of the disease, because the b cells of the pancreas produce insulin, but the cells of the body are resistant to the action of insulin. Later in the disease, beta cells may stop producing insulin. Type 2 diabetes is also known as insulin resistant diabetes, non-insulin dependent diabetes and adult-onset diabetes.
• Prediabetes can refer to one or more early diabetic conditions including impaired glucose utilization, abnormal or impaired fasting plasma glucose, impaired glucose tolerance, impaired insulin sensitivity and insulin resistance.
• Insulin resistance means that a cell has become resistant to the action of insulin (a hormone that regulates glucose uptake into cells) or the amount of insulin produced maintains normal glucose levels. Cells can have a reduced ability to respond to the effects of insulin (ie, loss of sensitivity to insulin) in facilitating the transport of sugar glucose from blood to muscle and other tissues. Eventually, the pancreas produces much more insulin than normal, and the cells remain resistant. As long as enough insulin is produced to overcome this resistance, blood glucose levels remain normal. When the pancreas can no longer be maintained, blood sugar begins to rise, leading to diabetes. Insulin resistance varies from normal (insulin sensitivity) to insulin resistance (IR).
• A-beta associated diseases can refer to those diseases or conditions characterized by A-beta protein aggregates.
• a primary component of amyloid plaques characteristic of A-beta associated diseases is beta amyloid peptide (A-beta), a highly insoluble peptide 39-43 amino acids (aa) in length that has a strong propensity to adopt beta sheet structures, oligomerize and form protein aggregates.
• Non-limiting examples of A-beta associated diseases include neurodegenerative diseases or disorders, Alzheimer's disease, dementia of the Alzheimer type, cerebral amyloid angiopathy (CAA), trisomy 21 (Down's 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 disorder in an elderly human subject, [264] Origin of the compounds of the disclosure: The provided compounds were not isolated from tissues naturally occurring in nature, but from the result of an artificial experiment combining a human cell and a chemically synthesized n-3-VLC-PUFA.
• the general structures of our synthetic elovanoid compounds were matched using HPLC and mass spectrometry with compounds biosynthesized in human retinal pigment epithelial cells or detected in neuronal cell cultures.
• the natural occurrence of the provided mono- and di-hydroxylated elovanoids with specifically defined stereochemistry is not known at this time.
• the provided compounds are not obtained from natural sources, but they are prepared by adapting stereocontrolled synthetic methods known in the art, starting with commercially available materials.
• the provided preparation methods were designed to be suitable to the unique hydrophobic properties of n-3 VLC-PUFA, which differ significantly from compounds that have a total number of carbons of 22 carbons or less.
• the present disclosure encompasses compounds that have stereochemically pure structures and are chemically synthesized and modified to have additional structural features and properties that allow them to exert pharmacological activity.
• the provided compounds are chemically modified pharmaceutically acceptable derivatives in the form of carboxylic esters or salts that enhance their chemical and biological stability and allow for their use in therapeutic applications involving various forms of drug delivery.
• the disclosure also provides pharmacologically effective compositions of the provided compounds that enhance their ability to be delivered to a subject in a manner that can reach the targeted cells and tissues.
• the data described herein also provides support for the beneficial use of the provided n-3 VLC-PUFA and/or elovanoid compounds, as therapeutics for the prevention and treatment of diseases, such as diseases associated with allergies or allergic reactions, by abrogating the production of pro-inflammatory cytokines and chemokines by a cell, such as an epithelial cell.
• a cell such as an epithelial cell.
• Epithelium lines both the outside (skin) and the inside cavities and lumina of bodies. Epithelial tissue is scutoid shaped, tightly packed and form a continuous sheet. It has almost no intercellular spaces. Epithelia is separated from underlying tissues by an extracellular fibrous basement membrane.
• epithelial tissue The lining of the mouth, lung alveoli and kidney tubules are all made of epithelial tissue.
• the lining of the blood and lymphatic vessels are of a specialized form of epithelium called endothelium.
• epithelium The term “epithelial cell” can refer to cells that line the outside (skin), mucous membranes, and the inside cavities and lumina of the body. Most epithelial cells exhibit an apical-basal polarization of cellular components. Epithelial cells are classified by shape and by their specialization.
• the epidermis i.e., skin
• stratified squamous epithelium The epidermis (i.e., skin) consists of keratinized stratified squamous epithelium.
• keratinocytes produce keratin, a protein that hardens and waterproofs the skin. Mature keratinocytes at the skin surface are dead and filled almost entirely with keratin. Melanocytes produce melanin, a pigment that protects cells from ultraviolet radiation. Melanin from the melanocytes is transferred to the keratinocytes. Langerhans cells are phagocytic macrophages that interact with white blood cells during an immune response. Merkel cells occur deep in the epidermis at the epidermal ⁇ dermal boundary. They form Merkel discs, which, in association with nerve endings, serve a sensory function. [271] There are several layers making up the epidermis.
• the five layers can include the stratum corneum contains many layers of dead, anucleate keratinocytes completely filled with keratin. The outermost layers are constantly shed.
• the stratum lucidum contains two to three layers of anucleate cells. This layer is found only in “thick skin” such as the palm of the hand and the sole of the foot.
• the stratum granulosum contains two to four layers of cells held together by desmosomes. These cells contain keratohyaline 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 active in mitosis.
• the stratum basale (stratum germinativum) contains a single layer of columnar cells actively dividing by mitosis to produce cells that migrate into the upper epidermal layers and ultimately to the surface of the skin.
• Nasal epithelial cells for example, form the outermost protective layer against environmental factors. They clean, humidify, and warm inhaled air. They produce mucus, which bind particles that are subsequently transported to the pharynx by cilia on the epithelial cells.
• the corneal epithelium for example, is made up of epithelial tissue and covers the front of the cornea.
• Respiratory epithelium or airway epithelium, is a type of ciliated columnar epithelium found lining most of the respiratory tract as respiratory mucosa.
• the cells in the respiratory epithelium are of four main types: a) ciliated cells, b) goblet cells, and c) club cells, and d) basal cells.
• the respiratory epithelium functions to moisten and protect the airways.
• the omega-3 very long chain polyunsaturated fatty acids have the structures of A or B, or derivatives thereof: wherein: A contains a total from 23 to 42 carbon atoms in the carbon chain, and with 6 alternating cis carbon-carbon double bonds starting at positions n-3, n-6, n-9, n-12, n-15 and n-18, and wherein B contains a total from 23 to 42 carbon atoms in the carbon chain, and with 5 alternating cis carbon-carbon double bonds starting 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 wherein m is a number from 0 to 19.
• the omega-3 very long chain polyunsaturated fatty acids of the disclosure can have a terminal carboxyl group “-COOR” wherein “R” can represent a group covalently bonded to the carboxyl such as an alkyl group.
• the carboxyl group can further have a negative charge as “-COO-“ and R is a cation including a metal cation, an ammonium cation and the like.
• m is a number selected from a group consisting of 0 to 15. Thus, can be a number selected from 1, 3, 5, 7, 9, 11, 13, or 15 where the fatty acid component contains a total of 24, 26, 28, 30, 32, 34, 36 or 38 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 0, 2, 4, 6, 8, 10, 12 or 14, where the fatty acid component contains a total of 23, 25, 27, 19, 31, 33, 35 or 37 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 5 to 15, where the fatty acid component contains a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 carbon atoms in its carbon chain.
• omega- 3 very long chain polyunsaturated fatty acids In some omega- 3 very long chain polyunsaturated fatty acids, m is a number selected from a group consisting of 9 to 11, where the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
• the omega-3 very long chain polyunsaturated fatty acids is a carboxylic acid, i.e. R is hydrogen.
• the omega-3 very long chain polyunsaturated fatty acids is a carboxylic ester, wherein R is methyl, ethyl or alkyl.
• R can be, but is not limited to, methyl or ethyl.
• the omega-3 very long chain polyunsaturated fatty acid is a carboxylic ester, wherein R is methyl.
• the omega-3 very long chain polyunsaturated fatty acid can be a carboxylate salt, wherein R is an ammonium cation, iminium cation, or a metal cation selected from a group consisting of sodium, potassium, magnesium, zinc, or calcium cation.
• R is ammonium cation or iminium cation.
• R can be a sodium cation or a potassium cation.
• R is a sodium cation.
• the omega-3 very long chain polyunsaturated fatty acid or derivative of the disclosure can have 32- or 34 carbons in its carbon chain and 6 alternating cis double bonds starting at the n-3 position, and have the formula A1 (14Z,17Z,20Z,23Z,26Z,29Z)-dotriaconta- 14,17,20,23,26,29-hexaenoic acid) or formula A2 (16Z,19Z,22Z,25Z,28Z,31Z)-tetratriaconta- 16,19,22,25,28,31-hexaenoic acid): [283]
• the carboxyl derivative is part of a glycerol-derived phospholipid, which can be readily prepared starting with the carboxylic acid form of the n-3 VLC-PUFA of structure A or B, by utilizing methods known in the art, and represented by structures C, D, E, or F: wherein
• m is a number selected from a group consisting of 0 to 15. In other embodiments, m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15 where the fatty acid component contains a total of 24, 26, 28, 30, 32, 34, 36 or 38 carbon atoms in its carbon chain. In additional advantageous embodiments, m is a number selected from a group consisting of 0, 2, 4, 6, 8, 10, 12 or 14, where the fatty acid component contains a total of 23, 25, 27, 19, 31, 33, 35 or 37 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 5 to 15, where the fatty acid component contains 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 a group consisting of 5, 7, 9, 11, 13, or 15, where the fatty acid component 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 a group consisting of 4, 6, 8, 10, 12 or 14, where the fatty acid component contains a total of 27, 29, 31, 33, 35 or 37 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 9 to 11, where the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
• the mono-hydroxylated elovanoids of the disclosure can have the structures of G, H, I or J: wherein compounds G and H have a total from 23 to 42 carbon atoms in the carbon chain, with 5 cis carbon-carbon double bonds starting at positions n-3, n-9, n-12, n-15 and n-18 and a trans carbon-carbon double bond starting at positions n-7; and wherein compounds I and J have a total from 23 to 42 carbon atoms in the carbon chain, and with 4 cis carbon-carbon double bonds starting at positions n-3, n-9, n-12 and n-15, and a trans carbon-carbon double bond starting at positions n-7; wherein R is hydrogen, methyl, ethyl, alkyl, or a cation selected from a group consisting of: ammonium
• the compounds of the disclosure are shown having a terminal carboxyl group “-COOR” the “R” can represent a group covalently bonded to the carboxyl such as an alkyl group.
• the carboxyl group can further have a negative charge as “-COO-“ and R is a cation including a metal cation, an ammonium cation and the like.
• m is a number selected from a group consisting of 0 to 15.
• m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15 where the fatty acid component contains 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 a group consisting of 0, 2, 4, 6, 8, 10, 12 or 14, where the fatty acid component contains a total of 23, 25, 27, 19, 31, 33, 35 or 37 carbon atoms in its carbon chain. [288] In some embodiments, m is a number selected from a group consisting of 5 to 15, where the fatty acid component contains a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 5, 7, 9, 11, 13, or 15, where the fatty acid component 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 a group consisting of 4, 6, 8, 10, 12 or 14, where the fatty acid component contains 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 a group consisting of 9 to 11, where the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain. [289] In some embodiments the mono-hydroxylated elovanoids of the disclosure are a carboxylic acid, i.e.
• R is hydrogen. In other embodiments the compound is a carboxylic ester, wherein R is methyl, ethyl or alkyl. In advantageous embodiments the compound is a carboxylic ester, wherein R is methyl or ethyl. In advantageous embodiments the compound is a carboxylic ester, wherein R is methyl. In other advantageous embodiments the compound is a carboxylate salt, wherein R is an ammonium cation, iminium cation, or a metal cation selected from a group consisting of sodium, potassium, magnesium, zinc, or calcium cation. In some advantageous embodiments, R is ammonium cation or iminium cation.
• R is a sodium cation or a potassium cation. In advantageous embodiments, R is a sodium cation.
• the di-hydroxylated elovanoids of the disclosure can have the structures K, L, M, or wherein compounds K and L have a total from 23 to 42 carbon atoms in the carbon chain, with 4 cis carbon-carbon double bonds starting at positions n-3, n-7, n-15 and n-18, and 2 trans carbon-carbon bonds starting at positions n-9, n-11; and wherein compounds M and N have a total from 23 to 42 carbon atoms in the carbon chain, with 3 cis carbon-carbon double bonds starting at positions n-3, n-7, n-12 and n-15, and 2 trans carbon-carbon bonds starting at positions n-9, n-11, wherein R is hydrogen, methyl, ethyl, alkyl, or a cation selected from a group consisting of: ammonium cation, iminium cation, or
• the compounds of the disclosure are shown having a terminal carboxyl group “-COOR” the “R” can represent a group covalently bonded to the carboxyl such as an alkyl group.
• the carboxyl group can further have a negative charge as “-COO-“ and R is a cation including a metal cation, an ammonium cation and the like.
• m is a number selected from a group consisting of 5 to 15, where the fatty acid component contains a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 5, 7, 9, 11, 13, or 15, where the fatty acid component 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 a group consisting of 4, 6, 8, 10, 12 or 14, where the fatty acid component 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 a group consisting of 9 to 11, where the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain. [293] Some di-hydroxylated elovanoids of the disclosure are carboxylic acid, i.e. R is hydrogen.
• the di-hydroxylated elovanoid of the disclosure is a carboxylic ester, wherein R is methyl, ethyl or alkyl. In useful embodiments the compound is a carboxylic ester, wherein R is methyl or ethyl. In useful embodiments the compound is a carboxylic ester, wherein R is methyl. [294] In other embodiments the di-hydroxylated elovanoid of the disclosure is a carboxylate salt, wherein R is an ammonium cation, iminium cation, or a metal cation selected from a group consisting of sodium, potassium, magnesium, zinc, or calcium cation.
• R is ammonium cation or iminium cation. In other useful embodiments, R is a sodium cation or a potassium cation. In useful embodiments, R is a sodium cation.
• the alkynyl mono-hydroxylated elovanoids of the disclosure can have the structures f wherein compounds O and P have a total from 23 to 42 carbon atoms in the carbon chain, with 4 cis carbon-carbon double bonds starting at positions n-3, n-12, n-15 and n-18, a trans carbon- carbon bond starting at position n-7, and a carbon-carbon triple bond starting at position n-9; and wherein compounds I and J have a total from 23 to 42 carbon atoms in the carbon chain, with 3 cis carbon-carbon double bonds starting at positions n-3, n-12 and n-15, a trans carbon- carbon bond starting at position n-7, and a carbon-carbon triple bond starting at position n-9; wherein R is hydrogen, methyl,
• the alkynyl mono- hydroxylated elovanoids of the disclosure are shown having a terminal carboxyl group “- COOR” the “R” can represent a group covalently bonded to the carboxyl such as an alkyl group.
• the carboxyl group can further have a negative charge as “-COO-“ and R is a cation including a metal cation, an ammonium cation and the like.
• m is a number selected from a group consisting of 0 to 15.
• m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15 where the fatty acid component contains a total of 24, 26, 28, 30, 32, 34, 36 or 38 carbon atoms in its carbon chain. [298] In additional embodiments, m is a number selected from a group consisting of 0, 2, 4, 6, 8, 10, 12 or 14, where the fatty acid component contains 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 a group consisting of 5 to 15, where the fatty acid component contains a total of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 5, 7, 9, 11, 13, or 15, where the fatty acid component 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 a group consisting of 4, 6, 8, 10, 12 or 14, where the fatty acid component contains 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 a group consisting of 9 to 11, where the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain. [299] In some embodiments the alkynyl mono-hydroxylated elovanoids of the disclosure are carboxylic acids, i.e.
• R is hydrogen.
• the alkynyl mono-hydroxylated elovanoids of the disclosure are carboxylic esters, wherein R is methyl, ethyl or alkyl.
• the alkynyl mono-hydroxylated elovanoids of the disclosure are carboxylic esters, wherein R is methyl or ethyl. [300] In some embodiments R is methyl.
• alkynyl mono-hydroxylated elovanoids of the disclosure can be a carboxylate salt, wherein R is an ammonium cation, iminium cation, or a metal cation selected from a group consisting of sodium, potassium, magnesium, zinc, or calcium cation.
• R is ammonium cation or iminium cation. In other embodiments, R is a sodium cation or a potassium cation. In embodiments, R is a sodium cation.
• the alkynyl di-hydroxylated elovanoids can have the structures of S, T, U or V: wherein compounds S and T have a total from 23 to 42 carbon atoms in the carbon chain, with 3 cis carbon-carbon double bonds starting at positions n-3, n-12, n-15 and n-18, with 2 trans carbon-carbon double bonds starting at positions n-9 and n-11, and a carbon-carbon triple bond starting at position n-7; and wherein compounds U and V have a total from 23 to 42 carbon atoms in the carbon chain, and with 2 cis carbon-carbon double bonds starting at positions n-3 and n-15, with 2 trans carbon-carbon double bonds starting at positions n-9 and n-11, and a carbon-carbon triple bond starting at position n-7; wherein
• the provided compounds S and T are predominately one enantiomer with a defined (S) or (R) chirality at the carbon bearing the hydroxyl group; and wherein, the provided compounds U and V are predominately one enantiomer with a defined (S) or (R) chirality at the carbon bearing the hydroxyl group.
• the compounds of the invention are shown having a terminal carboxyl group “-COOR” the “R” can represent a group covalently bonded to the carboxyl such as an alkyl group.
• the carboxyl group can further have a negative charge as “-COO-“ and R is a cation including a metal cation, an ammonium cation and the like.
• R is a cation including a metal cation, an ammonium cation and the like.
• m is a number selected from a group consisting of 5 to 15, where the fatty acid component contains 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 a group consisting of 5, 7, 9, 11, 13, or 15, where the fatty acid component contains a total of 28, 30, 32, 34, 36 or 38 carbon atoms in its carbon chain.
• m is a number selected from a group consisting of 4, 6, 8, 10, 12 or 14, where the fatty acid component contains a total of 27, 29, 31, 33, 35 or 37 carbon atoms in its carbon chain. In embodiments, m is a number selected from a group consisting of 9 to 11, where the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
• the provided compound is a carboxylic acid, i.e. R is hydrogen.
• the provided compound is a carboxylic ester, wherein R is methyl, ethyl or alkyl. In embodiments the provided compound is a carboxylic ester, wherein R is methyl or ethyl.
• the provided compound is a carboxylic ester, wherein R is methyl.
• the provided compound is a carboxylate salt, wherein R is an ammonium cation, iminium cation, or a metal cation selected from a group consisting of sodium, potassium, magnesium, zinc, or calcium cation.
• R is ammonium cation or iminium cation.
• R is a sodium cation or a potassium cation.
• R is a sodium cation.
• the present disclosure provides a mono-hydroxylated 32-carbon methyl ester of formula G1, having the name: methyl (S,14Z,17Z,20Z,23Z,25E,29Z)-27- hydroxydotriaconta-14,17,20,23,25,29-hexaenoate; a mono-hydroxylated 32-carbon sodium salt of formula G2, having the name: sodium (S,14Z,17Z,20Z,23Z,25E,29Z)-27- hydroxydotriaconta-14,17,20,23,25,29-hexaenoate; a mono-hydroxylated 34-carbon methyl ester of formula G3, having the name: methyl (S,16Z,19Z,22Z,25Z,27E,31Z)-29- hydroxytetratriaconta-16,19,22,25,27,31-hexaenoate; or a mono-hydroxylated 34-carbon sodium salt of formula G4,
• Scheme 1 shows the detailed approach for the stereocontrolled total synthesis of compounds of type O, wherein n is 9, and the fatty acid chain contains a total of 32 carbon atoms, and the R group is methyl or sodium cation.
• Scheme 1 shows the synthesis of compounds ELV-N-32-Me and ELV-N-32-Na, starting with methyl pentadec- 14-ynoate (S1).
• Scheme 2 (FIG.7) describes the total synthesis of the di-hydroxylated elovanoids K and L and their alkyne precursors S and T, by starting with intermediates 2, 5, and 7 that were also used in Scheme 1.
• the conversion of the protected (R) epoxide 4 to intermediate 15, and the coupling of 7 and 15 followed by conversion into intermediate 17 can be done according to literature procedures (Tetrahedron Lett.2012;53(14):1695-8).
• Catalytic cross-coupling between intermediates 2 or 17 or between intermediates 5 or 17, followed by deprotection leads to the formation of alkynyl compounds S and T, which are then selectively reduced to form di-hydroxylated elovanoids K and L.
• this scheme shows the total synthesis of the 32-carbon alkynyl elovanoid compound ELV-N-32-Me-Acetylenic, and its conversion to elovanoid methyl ester ELV-N-32-Me, the elovanoid carboxylic acid ELV-N-32-H, and the elovanoid sodium salt ELV-N-32-Na.
• Scheme 5 shows the stereocontrolled total synthesis of 34-carbon dihydroxylated elovanoids, starting with alkyne methyl ester 30, and by employing the same sequence of reactions as in Scheme 4.
• this scheme shows the total synthesis of the 34-carbon alkynyl elovanoid compound ELV-N-34-Me-Acetylenic, and its conversion to elovanoid methyl ester ELV-N-34-Me, the elovanoid carboxylic acid ELV-N-34-H, and the elovanoid sodium salt ELV-N-34-Na.
• the chemistry presented in Schemes 1-5 (FIGs.6-10) can be also adapted for the total synthesis of additional mono-hydroxylated and di-hydroxylated elovanoids, having at least 23 carbons and up to 42 carbons in their carbon chain.
• compositions for the treatment of diseases in other embodiments, the present disclosure provides formulations of pharmaceutical compositions containing therapeutically effective amounts of one or more of compounds provided herein or their salts thereof in a pharmaceutically acceptable carrier.
• the provided compositions contain one or more compounds provided herein or their salts thereof, and a pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant.
• the compounds can be formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral, buccal, intranasal, vaginal, rectal, ocular administration, sustained release from intravitreal implanted reservoirs or nano-devices or dendrimers, embedded in collagen or other materials on the eye surface, or in sterile solutions or suspensions for parenteral administration, dermal patches as well as transdermal patch preparation and dry powder inhalers.
• the provided formulations can be in the form of a drop, such as an eye drop, and the pharmaceutical formulation can further contain antioxidants and/or known agents for the treatment of eye diseases.
• Embodiments of the disclosure provide pharmaceutical compositions containing various forms of the provided compounds, as the free carboxylic acids or their pharmaceutically acceptable salts, or as their corresponding esters or their phospholipid derivatives.
• the disclosure provides pharmaceutical compositions containing one or more elovanoid that contains one or two hydroxyl groups at positions located between n-3 to n-18 of the very long chain polyunsaturated fatty acids, as the free carboxylic acids or their pharmaceutically acceptable salts, or as their corresponding esters.
• the disclosure provides a pharmaceutical composition for alleviating the symptom of, treating, or preventing a disease.
• the disease is an allergic inflammatory disease, a disease associated with cellular senescence and/or ferroptosis, or a metabolic disorder.
• effective concentrations of one or more compounds or pharmaceutically acceptable derivatives is (are) mixed with a suitable pharmaceutical carrier or vehicle.
• the compounds can be derivatized as the corresponding salts, esters, enol ethers or esters, acids, bases, solvates, hydrates or prodrugs prior to formulation, as described herein.
• compositions can be prepared by adapting methods known in the art.
• the compositions can be a component of a pharmaceutical formulation.
• the pharmaceutical formulation can further contain known agents for the treatment of inflammatory or degenerative diseases, including neurodegenerative diseases.
• the provided compositions can serve as pro-drug precursors of the fatty acids and can be converted to the free fatty acids upon localization to the site of the disease.
• the present disclosure also provides packaged composition(s) or pharmaceutical composition(s) for prevention, restoration, or use in treating the disease or condition.
• compositions or pharmaceutical compositions provided by the present disclosure further can include indicia including at least one of: instructions for using the composition to treat the disease or condition.
• the kit can further include appropriate buffers and reagents known in the art for administering various combinations of the components listed herein to the host.
• Pharmaceutical formulations can include a composition or pharmaceutical composition as identified herein and can be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, naturally occurring or synthetic antioxidants, and/or adjuvants.
• embodiments of the present disclosure can include a composition or pharmaceutical composition formulated with one or more pharmaceutically acceptable auxiliary substances.
• composition or pharmaceutical composition can be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and/or adjuvants to provide an embodiment of a composition of the present disclosure.
• pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients have been amply described in a variety of 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.
• compositions or pharmaceutical composition can be administered to the subject using any means capable of resulting in the desired effect.
• composition or pharmaceutical composition can be incorporated into a variety of formulations for therapeutic administration.
• the composition or pharmaceutical composition can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, creams, and aerosols.
• Suitable excipient vehicles for the composition or pharmaceutical composition are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
• compositions of the present disclosure can include those that comprise a sustained release or controlled release matrix.
• embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations.
• a sustained-release matrix is a matrix made of materials, for example polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
• a sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers 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, polyvinyl propylene, polyvinylpyrrolidone and silicone.
• biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydr
• Illustrative biodegradable matrices can include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix.
• the pharmaceutical composition of the present disclosure (as well as combination compositions) can be delivered in a controlled release system.
• the composition or pharmaceutical composition can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
• a pump can be used (Sefton (1987). CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al. (1980).
• a controlled release system is placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose.
• a controlled release system is placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic.
• Other controlled release systems are discussed in the review by Langer (1990). Science 249:1527-1533.
• compositions of the present disclosure can include those formed by impregnation of the composition or pharmaceutical composition described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions.
• absorptive materials such as sutures, bandages, and gauze
• solid phase materials such as surgical staples, zippers and catheters to deliver the compositions.
• Other delivery systems of this type will be readily apparent to those skilled in the art in view of the disclosure.
• the compositions or pharmaceutical compositions of the present disclosure 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.
• Embodiments of the composition or pharmaceutical composition can be administered to a subject in one or more doses. Those of skill will appreciate that dose levels can vary as a function of the specific the composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Useful dosages for a given compound are readily determinable by those of skill in the art by a variety of means. [337] In an 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 any of a variety of factors, e.g., severity of the symptoms, and the like.
• the composition or pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), three times a day (tid), or four times a day.
• the composition or pharmaceutical composition is administered 1 to 4 times a day over a 1 to 10-day time period.
• the duration of administration of the composition or pharmaceutical composition analogue can vary, depending on any of a variety of factors, e.g., patient response, etc.
• the composition or pharmaceutical composition in combination or separately can be administered over a period of time of about one day to one week, about one day to two weeks.
• the amount of the compositions and pharmaceutical compositions of the present disclosure that can be effective in treating the condition or disease can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be employed to help identify optimal dosage ranges.
• Embodiments of the present disclosure provide methods and compositions for the administration of the active agent(s) to a subject (e.g., a human) using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
• Routes of administration can include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, intravitreal, topical application, intravenous, rectal, nasal, oral, and other enteral and parenteral routes of administration.
• routes of administration can be combined, if desired, or adjusted depending upon the agent and/or the desired effect.
• An active agent can be administered in a single dose or in multiple doses.
• aspects of the invention can be administered by a nebulizer.
• nebulizer can refer to any device known in the art that produces small droplets or an aerosol from a liquid.
• the composition can be administered in the form of a mist, inhaled into the lungs.
• the n-3 VLC-PUFA and their biogenic derivatives are formed in cells and are not a component of human diet.
• Advantageous routes of administration of the compounds provided herein will can include topical, oral, intranasal, and parenteral administration.
• the provided formulations can be delivered in the form of a drop, such as an eye drop, or any other customary method for the treatment of an allergic inflammatory disease of the eye.
• the provided formulations can be delivered in the form of an intranasal spray or any other customary method for the treatment of an allergic inflammatory disease of the nasal passage or lungs.
• the provided formulations can be delivered in the form of a cream or gel or any other customary method for the treatment of an allergic inflammatory disease of the skin.
• Parenteral routes of administration 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 route of administration other than through the alimentary canal.
• Parenteral administration can be conducted to affect systemic or local delivery of the composition. Where systemic delivery is desired, administration involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
• the composition or pharmaceutical composition can also be delivered to the subject by enteral administration.
• Enteral routes of administration can include, but are not limited to, oral and rectal (e.g., using a suppository) delivery.
• Methods of administration of the composition or pharmaceutical composition through the skin or mucosa can include, but are not limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration.
• a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
• Iontophoretic transmission can be accomplished using commercially available "patches" that deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
• the compounds and compositions provided by this disclosure are able to restore homeostasis and induce survival signaling in certain cells undergoing oxidative stress or other homeostatic disruptions.
• the disclosure also provides methods of use of the provided compounds and compositions containing a hydroxylated derivative of very long chain polyunsaturated fatty acids, as the free carboxylic acids or their pharmaceutically acceptable salts, or as their corresponding esters or other prodrug derivatives.
• the provided compounds can be readily prepared by adapting methods known in the art, starting with commercially available materials.
• the bioactivity of the provided compounds as exemplified by the elovanoid derivatives ELV-N-32-Me, ELV-N-32-Na, ELV-N-34-Me and ELV-N-34-Na, is attributed to their ability to reach the targeted human cells and exert their biological actions either by entering into the cell or/ and by acting at a membrane bound receptor.
• the provided compounds can act via intracellular receptors (e.g. nuclear membrane), and thus they would work specifically by affecting key signaling events.
• Administration of a pharmaceutical composition, containing a provided compound and a pharmaceutically acceptable carrier restores the homeostatic balance and promotes the survival of certain cells that are 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 of the initiation and early progression of these conditions by mimicking the specific biology of intrinsic cellular/organs responses to attain potency, selectivity, devoid of side effects and sustained bioactivity.
• one aspect of the disclosure encompasses embodiments of a composition comprising at least one very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.
• the composition can further comprise a pharmaceutically-acceptable carrier and formulated for delivery of an amount of the at least one very long chain polyunsaturated fatty acid effective in reducing a pathological condition of a tissue of a recipient subject or the onset of a pathological condition of a tissue of a recipient subject.
• the pathological condition can be an allergic inflammatory disease or allergic inflammatory condition of a tissue of the recipient subject.
• the pathological condition can be associated with cellular senescence, ferroptosis, or both.
• the pathological condition can be associated with a metabolic disorder.
• the composition can be formulated for topical delivery of the at least one very long chain polyunsaturated fatty acid tissue to the skin or eye of a recipient subject.
• the composition can be formulated for intranasal delivery of the at least one very long chain polyunsaturated fatty acid tissue to the nasal passage and/or lungs of a recipient subject.
• the composition can further comprise at least one nutritional component, and, for example, the composition can be formulated for the oral or parenteral delivery of the at least one very long chain polyunsaturated fatty acid to a recipient subject.
• the at least one very long chain polyunsaturated fatty acid can have from about 26 to about 42 carbon atoms in its carbon chain.
• the at least one very long chain polyunsaturated fatty acid can have 32 or 34 carbon atoms in its carbon chain.
• the very long chain polyunsaturated fatty acid can have in its carbon chain five or six double bonds with cis geometry.
• 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.
• compositions comprising at least one elovanoid having at least 23 carbon atoms in its carbon chain.
• the composition can further comprise a pharmaceutically-acceptable carrier and can be formulated for delivery of an amount of the at least one elovanoid effective in reducing a pathological condition of a tissue of a recipient subject.
• the pathological condition can be an allergic inflammatory disease.
• the at least one elovanoid can be selected from the group consisting of: a mono-hydroxylated elovanoid, a di-hydroxylated elovanoid, an alkynyl mono-hydroxylated elovanoid, and an alkynyl di-hydroxylated elovanoid, or any combination thereof.
• the at least one elovanoid can be a combination of elovanoids, wherein the combination is selected from the group consisting of: a mono-hydroxylated elovanoid and a di-hydroxylated elovanoid; a mono- hydroxylated elovanoid and an alkynyl mono-hydroxylated elovanoid; a mono-hydroxylated elovanoid and an alkynyl di-hydroxylated elovanoid; a di-hydroxylated elovanoid and an alkynyl mono-hydroxylated elovanoid; a di-hydroxylated elovanoid and an alkynyl di- hydroxylated elovanoid; a mono-hydroxylated elovanoid, a di-hydroxylated elovanoid, and an alkynyl di- hydroxylated elovanoid; a mono-hydroxylated elovanoid
• the composition can further comprise at least one very long-chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.
• the at least one very long chain polyunsaturated fatty acid can have from about 26 to about 42 carbon atoms in its carbon chain.
• the at least one very long chain polyunsaturated fatty acid can have in its carbon chain five or six double bonds with cis geometry.
• 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.
• the mono-hydroxylated elovanoid can be selected from the group consisting of the formulas G, H, I or J: wherein: n can be 0 to 19 and -CO-OR can be a carboxylic acid group, or a salt or an ester thereof, and wherein: if -CO-OR can be a carboxylic acid group and the compound G, H, I or J can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and if -CO-OR can be an ester, then R can be an alkyl group.
• the pharmaceutically acceptable cation can be an ammonium cation, an iminium cation, or a metal cation.
• the metal cation can be a sodium, potassium, magnesium, zinc, or calcium cation.
• the composition can comprise equimolar amounts of the enantiomers G and H wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
• the composition can comprise amounts of the enantiomers I and J wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
• the composition can comprise one of the enantiomers of G or H in an amount exceeding the amount of the other enantiomer of G or H.
• the composition can comprise one of the enantiomers of I or J in an amount exceeding the amount of the other enantiomer of I or J.
• the mono-hydroxylated elovanoid can be selected from a group consisting of: methyl (S,14Z,17Z,20Z,23Z,25E,29Z)- 27-hydroxydotriaconta-14,17,20,23,25,29-hexaenoate (G1), sodium (S,14Z,17Z,20Z,23Z,25E,29Z)-27-hydroxydotriaconta-14,17,20,23,25,29-hexaenoate (G2), methyl (S,16Z,19Z,22Z,25Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,25,27,31- hexaenoate (G3); and sodium (S,16Z,19Z,22Z,25Z,27E,31Z)-29-hydroxytetratriaconta- 16,19,22,25,27,31-hexaenoate (G3); and sodium (S,
• the pharmaceutically acceptable cation can be an ammonium cation, an iminium cation, or a metal cation.
• the metal cation can be a sodium, potassium, magnesium, zinc, or calcium cation.
• the composition can comprise equimolar amounts of the diastereomers K and L wherein the diastereomers have either (S) or (R) chirality at position n-6, and (R) chirality at position n-13.
• the composition can comprise equimolar amounts of the diastereomers M and N wherein the diastereomers have either (S) or (R) chirality at position n-6, and (R) chirality at position n-13.
• the composition can comprise one of the diastereomers of K or L in an amount exceeding the amount of the other diastereomer of K or L.
• the composition can comprise one of the diastereomers of M or N in an amount exceeding the amount of the other diastereomer of M or N.
• the di-hydroxylated elovanoid can be selected from the group consisting of: methyl (14Z,17Z,20R,21E,23E,25Z,27S,29Z)- 20,27-dihydroxydotriaconta-14,17,21,23,25,29-hexaenoate (K1), sodium (14Z,17Z,20R,21E,23E,25Z,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,25,29- hexaenoate (K2), 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-
• the alkynyl mono-hydroxylated elovanoid can be selected from the group consisting of: methyl (S,14Z,17Z,20Z,25E,29Z)-27- hydroxydotriaconta-14,17,20,25,29-pentaen-23-ynoate (O1); sodium (S,17Z,20Z,25E,29Z)- 27-hydroxydotriaconta-17,20,25,29-tetraen-23-ynoate (O2); methyl (S,16Z,19Z,22Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,27,31-pentaen-25-ynoate (O3); and sodium (S,16Z,19Z,22Z,27E,31Z)-29-hydroxytetratriaconta-16,19,22,27,31-pentaen-25-ynoate (O3); and sodium (S,16Z,
• the pharmaceutically acceptable cation can be an ammonium cation, an iminium cation, or a metal cation.
• the metal cation can be a sodium, potassium, magnesium, zinc, or calcium cation.
• the composition can comprise equimolar amounts of the enantiomers O and P wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
• the composition can comprise equimolar amounts of the enantiomers Q and R wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
• the composition can comprise one of the enantiomers of O or P in an amount exceeding the amount of the other enantiomer of O or P.
• the composition can comprise one of the enantiomers of Q or R in an amount exceeding the amount of the other enantiomer of Q or R.
• the elovanoid can be an alkynyl di-hydroxylated elovanoid selected from the group consisting of the formulas S, T, U or V: wherein: m can be 0 to 19 and -CO-OR can be a carboxylic acid group, or a salt or an ester thereof, and wherein: if -CO-OR can be a carboxylic acid group and the compound S, T, U or V can be a salt thereof, the cation of the salt can be a pharmaceutically acceptable cation, and if -CO-OR can be an ester, then R can be an alkyl group, and wherein: compounds S and T each have a total from 23 to 42 carbon atoms in the carbon chain, with 3 cis carbon-carbon double bonds starting at positions n-3, n-15 and n-18; 2 trans carbon-carbon double bonds starting at positions n-9, n-11; and a carbon-carbon triple bond starting at position
• the pharmaceutically acceptable cation is an ammonium cation, an iminium cation, or a metal cation.
• the metal cation is a sodium, potassium, magnesium, zinc, or calcium cation.
• the alkynyl mono-hydroxylated elovanoid can be selected from the group consisting of: methyl (14Z,17Z,20R,21E,23E,27S,29Z)-20,27-dihydroxydotriaconta-14,17,21,23,29-pentaen-25- ynoate (S1); sodium (14Z,17Z,20R,21E,23E,27S,29Z)-20,27-dihydroxydotriaconta- 14,17,21,23,29-pentaen-25-ynoate (S2); methyl (16Z,19Z,22R,23E,25E,29S,31Z)-22,29- dihydroxytetratriaconta-16,19,23,25,31-pentaen-27-ynoate (S3); and sodium (16Z,19Z,22R,23E,25E,29S,31Z)-22,29-
• the composition can comprise equimolar amounts of the diastereomers U and V wherein the diastereomers have either (S) or (R) chirality at position n-6, and (R) chirality at position n-13.
• the composition can comprises one of the diastereomers of S or T in an amount exceeding the amount of the other diastereomer of S or T.
• the composition can comprise one of the diastereomers of U or V in an amount exceeding the amount of the other diastereomer of U or V.
• compositions and Methods for Modulating Elovanoid Bioactivity and Availability [402] Cellular senescence is a form of cell cycle arrest linked to aging and diseases. Cellular senescence is a proinflammatory cell fate associated with age-related diseases, including AD and AMD.
• the senescence phenotype expresses in cells undergoing terminal, replicative arrest that displays cell enlargement, chromatin alterations, SASP, and cell cycle regulatory proteins (cyclins and cyclin-dependent kinases). Persistent accumulation of senescence is associated with age-related diseases and functional decay. The clearance of senescent cells from tissue alleviates pathologies related to aging because they propagate degenerative and proinflammatory events in their microenvironment. [403] In the brain, the senescence signature program is triggered in astrocytes, microglia, and neurons (despite being post-mitotic). Senescent cell phenotype consequences (e.g., chronic inflammation) are also key in AMD.
• Cellular senescence is a defense event against cancer, and plays a role in aging and age-related diseases. Senescent cells help create a microenvironment that facilitates tumor progression. These events involve depletion of stem and progenitor cells and the cell deranging consequences of the expression of SASP, including proinflammatory homeostatic-perturbing cytokines and chemokines, growth factors, and matrix metalloproteinases. [404] Moreover, senescent cells have beneficial effects in injury repair and tissue remodeling as well. Thus, without being bound by theory, senescence, besides being a driver of age-dependent diseases and of metabolic syndrome, can remove healthy cells in addition to others that are damaging to the organ/organisms through senescent cell clearance.
• a positive effect of senescent cells and SASP is the acceleration of skin wound healing by early secretion of SASP triggered by PDGF-AA, which is secreted by senescence cells.
• Wounding induces senescence in local fibroblasts and endothelial cells.
• myofibroblast differentiation, granulation tissue formation, and completion of wound healing takes place.
• ELVs modify expression (and protein abundance) of p16INK4a (also known as cyclin-dependent kinase inhibitor 2A, Cyclin-Dependent Kinase 4 Inhibitor A), a tumor suppressor protein. This protein is encoded by the Ink4a/Arf locus or Cdkn2a.
• ELVs target upstream ferroptosis, a form of programed cell death that engages senescence.
• the inventors uncovered a new molecular target of ELVs that demonstrated inhibition of cell death by blocking phosphorylation of scaffold protein PEBP-1 (FIG.29-31). As a result, peroxidized lipids are not formed, and ferroptosis is blocked. Iron, ferritin and markers of oxidative stress are enhanced in senescent cells. These cells display aberrant iron homeostasis and influence iron content in aging tissues.
• SASP senescence-associated secretory phenotype
• Ferroptosis is at the interphase with autophagy and engages senescence (FIGs.2-4)
• An AdipoR1 receptor subtype enhances DHA cellular uptake/retention and availability of the VLC-PUFAs’ ELV precursors. After uptake/retention, this receptor subtype facilitates building in phosphatidylcholine of membrane reservoirs of the VLC- PUFAs that enter into that pathway of ELV biosynthesis upon release by a PLA1.
• the membranes containing VLC-PUFAs are released upon uncompensated oxidative stress (UOS) challenges, trauma, ischemia, and the onset of neurodegenerative diseases.5XFAD on pathways failing before PRC death (FIG.32 and FIG.37).
• UOS uncompensated oxidative stress
• Specific GPCRs for ELV and NPD1 are the base to develop synthetic ligands (peptides small molecules or others) interaction of MFRP with AdipoR1 target to peptide that mimic ELV action by targeting specific receptor/s. GPCR data (FIG.36 and FIG.38).
• Intracellular proteins targeted by ELV as regulatory sites to enhance bioactivity. Identification of cell penetrant (or tissue penetrant) peptides or other small molecules that target GPCRs.
• Examples can include vivo MO-(contain bradykinin analog). Example of no toxicity to retina (FIG.34).
• ELVs beneficial role in GBM see Figs.
• TBI see Figs
• embodiments of the invention comprise compositions and methods that result in the elimination of senescent cells.
• embodiments of the invention are drawn to compositions and methods that modulate availability of senescence cells in cancer (chemotherapy, brain (neurodegenerative diseases)), wound healing (diabetes, cornea- keratinocytes, decubital ulcers), and neurodegenerative diseases, such as AMD and AD.
• chemotherapy chemotherapy, brain (neurodegenerative diseases)
• wound healing wound healing
• diabetes cornea- keratinocytes, decubital ulcers
• neurodegenerative diseases such as AMD and AD.
• Embodiments of the invention are also drawn to compositions and methods that are neuroprotective and/or neurorestorative.
• embodiments are neuroprotective in prodromal targeting of MCA and/or sight disturbances preceding blindness in AMD or retinitis pigmentosa (RP) or other retinal degenerative diseases.
• RP retinitis pigmentosa
• embodiments are neuroprotective or neurorestorative in other diseases, such as AD, AM, CV diseases, metabolic syndrome, obesity, type 2 diabetes, myocardial infarction, stroke, TBI, GBM.
• diseases such as AD, AM, CV diseases, metabolic syndrome, obesity, type 2 diabetes, myocardial infarction, stroke, TBI, GBM.
• senescent cells help create a microenvironment that facilitates tumor progression.
• EBVs elovanoids
• aspects of the invention are drawn to compositions and methods for preventing, treating, ameliorating the symptoms of, or slowing the progression of cancer (such as glioblastoma multiform or GBM), age-related macular degeneration (AMD), Alzheimer’s disease (AD), other neurodegenerative diseases, metabolic syndrome, obesity, type 2 diabetes, neurotrauma, skin and corneal wound healing.
• cancer such as glioblastoma multiform or GBM
• AMD age-related macular degeneration
• AD Alzheimer’s disease
• Other neurodegenerative diseases metabolic syndrome
• obesity type 2 diabetes
• neurotrauma skin and corneal wound healing.
• the elovanoids used in embodiments herein are described in PCT/US2016/017112, PCT/US2018/023082, each of which are incorporated by reference herein in their entireties.
• the elovanoid comprises ELV-N-34 or ELV-N-32, or derivatives thereof.
• elovanoids can include monohydroxy compounds (e.g. ELV-27S and ELV-29S, 4, and dihydroxy derivatives, e.g. ELV-N-32 and ELV-N-34, 5.
• Elovanoid ELV-N-32 is the 20R,27S-dihydroxy 32:6 derivative (32-carbon, 6 double bond elovanoid with a neuroprotectin-like 20(R),27(S)-dihydroxy pattern).
• Elovanoid ELV-N-34 is the 22R,29S-dihydroxy 34:6 derivative (34-carbon, 6 double bond elovanoid with a 22(R),29(S)-dihydroxy pattern).
• Peptide Analogs [419] Further, aspects of the invention are drawn to the development of new synthetic non- lipidic analogs to mimic the bioactivity of the lipid mediators, such as elovanoids.
• the term "analog” can refer to a second organic or inorganic molecule which possesses a similar or identical function as a first organic or inorganic molecule. The analog can be structurally similar to the first organic or inorganic molecule.
• the first molecule is a lipid
• the second molecule i.e., analog
• the first molecule is an elovanoid
• the second molecule is a peptide.
• the peptide can be referred to as a "peptide analog”.
• the peptide analog can be considered a therapeutic peptide.
• therapeutic peptide can refer to a peptide or fragment or variant thereof having one or more therapeutic and / or biological activities.
• the term "peptide " can refer to a molecule comprising two or more amino acid residues linked together by a peptide bond.
• peptide may be monomeric or polymeric.
• a "peptide " is a chain of amino acids in which the alpha carbon can be linked through a peptide bond. The terminal amino acid at one end (amino terminus) of the chain thus has a free amino group, while the terminal amino acid at the other terminus (carboxy terminus) of the chain has a free carboxyl group.
• amino terminal (abbreviated N-terminal) can refer to the free amino group on the amino acid at the amino terminus of the peptide or the Amino group of the amino acid at any other position in the peptide.
• carboxy terminus can refer to the free carboxyl group on the carboxy terminus of the peptide or the carboxyl group of the amino acid in any other position within the peptide.
• Peptides also can include essentially any polyamino acids, including, but not limited to, amino acids linked by an ether as opposed to a peptide mimetic, such as an amide bond.
• the peptide analogs are peptides comprising at least four amino acids linked through peptide bonds or other covalent bonds as described herein.
• the peptide or peptide analog is from about 4 to about 50 amino acids in length. All integer subranges of 4 to 50 amino acids are useful for the peptides herein.
• the peptide or peptide analog is an amino acid of about 5 to about 35 amino acids, 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 to be.
• the peptide or peptide analog is an amino acid of about 6 to about 35 amino acids, 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 to be. In an embodiment, the peptide or peptide analog is an amino acid of about 7 to about 35 amino acids, 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 to be. In an embodiment, the peptide or peptide analog is an amino acid of 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 to be.
• the peptide is an amino acid of about 8 to about 17 or 18 or about 9 to about 16 or 17 amino acids in length. In an embodiment, the peptide is from 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 5-mer, 6-mer, 7-mer, 8-mer, 9-mer-10- mer, 16-mer, 17-mer, 18-mer, 19-mer, or 20-mer. [423] In embodiments, the elovanoid and/or the peptide analog can regulate/modulate ferroptosis, wherein modulation of ferroptosis treats or prevents a disease in a subject.
• ferroptosis means regulated cell death that is iron-dependent.
• Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species.
• Ferroptosis is distinct from apoptosis, necrosis, and autophagy.
• Assays for ferroptosis are as disclosed, for instance, in Dixon et al., 2012.
• the elovanoid and/or the peptide analog can regulate/modulate cellular senescence, wherein modulation of cellular senescence treats a disease in a subject.
• modulate modulating
• grammatical variations thereof mean to change, such as increasing or decreasing a biological activity.
• the elovanoid and/or peptide analog can bind to an epitope on one or more of the molecular targets, such as those as identified in FIG.28 and FIG.42.
• the elovanoid and/or peptide analog can bind to an epitope on one or more of the molecular targets as identified in the table below (for example, to a contiguous or non-contiguous amino acid sequence depicted by the protein NCBI reference number listed in the Table below):
• the elovanoids and the peptide analogs interact with the same or similar epitope on the molecule target.
• the elovanoids and the peptide analogs can interact with different epitopes on the molecular target.
• epitope can refer to a portion of the molecular target, such as those identified in the Table can included herein, to which an elovanoid and/or peptide analog specifically binds.
• an elovanoid or peptide analog thereof can target an epitope on LTB4R, GPR37, GPR52, GPR132, CNR2, BAI2, TXNRD1, PEBP1, and/or GSR to modulate cellular senescence and/or ferroptosis.
• the epitope can include a "target site” or “target sequence”, which can refer to a sequence that is bound by the binding partner, such as the elovanoid or the peptide analog.
• the target site can comprise one or more amino acids.
• the proteins in Table herein can be referred to as "molecular targets”.
• the term "target” or “molecular target” can refer to any molecule, for example a molecule within a cell or associated with a cell membrane, that is being examined for interaction with a candidate compound (e.g., a drug, an elovanoid, or a peptide analog thereof).
• Non-limiting examples of molecular targets can include DNA, RNA and proteins such as receptors (e.g., cell surface, membrane-bound or nuclear), components of signal transduction pathways, transcription factors or functional fragments thereof.
• a molecular-targets can also comprise a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the molecules described herein, or any complex comprising one or more of the molecules described herein.
• a compound, elovanoid, or peptide analog “interacts” with a molecular target when it affects, directly or indirectly, the molecular target.
• the compound can act directly on the molecular target, for example when the molecular target is a protein, the compound may directly interact with the protein by binding to it or may directly regulate expression of the protein via action on transcriptional regulatory elements. Similarly, the compound can also act indirectly on the molecular target, for example by blocking or stimulating a separate molecule that in turn acts on the molecular target. 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.
• binding can refer to the determination by standard assays, including those described herein, that a binding polypeptide recognizes and binds reversibly to a given target. Such standard assays can include, but are not limited to, equilibrium dialysis, gel filtration, and the monitoring of spectroscopic changes that result from binding.
• the elovanoid or peptide analog can have specificity for a molecular target in Table 1.
• specificity can refer to a binding polypeptide having a higher binding affinity for one target over another. Binding specificity may be characterized by a dissociation equilibrium constant (K D ) or an association equilibrium constant (K a ) for the two tested target materials.
• Example 1 Primary human nasal epithelial cells (HNEpC) as used in Examples herein
• HNEpC Primary human nasal epithelial cells
• Cryopreserved human nasal epithelial cells HNEpC were purchased from PromoCell GmbH, Heidelberg, Germany. (Catalog# C-1260, Lot# 436Z028).
• the cells used for our experiments are primary nasal epithelial cells obtained from the nasal mucosa of a 50-year-old Caucasian male.
• Cells were received in passage (P1) and were sub-culture to passage (P3), which were used for all the experiments.
• HNEpC were grown to 80% confluency in Promocell’s Airway epithelial cell growth medium (Catalog# C-21060) which was supplemented with airway epithelial cell growth medium supplement pack (Catalog# C-39160) and Penicillin/Streptomycin.
• Example 2 - HNEpC were challenged using several stressors – aeroallergens ⁇ Lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Catalog# L4391) were obtained from Sigma-Aldrich. LPS is the principal component of Gram-negative bacteria that activates the innate immune system through its recognition by Toll-like receptor 4 (TLR4).
• TLR4 Toll-like receptor 4
• LPS we used for our experiments is a preparation of smooth (S)-form LPS purified from the Gram-negative E. coli 0111:B4 that was used at 30 mg/mL to challenge HNEpC.
• Polyinosinic-polycytidylic acid (abbreviated as poly(I:C) or poly(rI):poly(rC)) is a synthetic analog of double-stranded viral RNA (dsRNA), a molecular pattern associated with viral infection such as loss of epithelial integrity, increased production of mucus and inflammatory cytokines.
• Poly(I:C) a TLR3 agonist activates the antiviral pattern recognition receptors TLR3, RIG-I/MDA5 and PKR, thereby inducing signaling via multiple inflammatory pathways, including NF-kB and IRF.
• High Molecular Weight Poly(I:C) comprises long strands of inosine poly(I) homopolymer annealed to strands of cytidine poly(C) homopolymer.
• the average size of Poly(I:C) HMW is from 1.5 kb to 8 kb.
• Poly(I:C)(Catalog# P1530) were obtained from Sigma-Aldrich and used at 100 mg/mL to challenge HNEpC.
• the highly purified resazurin that is used for PrestoBlue HS results in a reagent with a >50% decrease in background fluorescence and a >100% increase in the signal to background ratio.
• the cellular reducing environment reduces resazurin to resorufin a compound that is red and highly fluorescent.
• Viable cells continuously convert resazurin to resorufin increasing the overall fluorescence and color of the media surrounding the cells. Also, the conversion of resazurin to resorufin results in a pronounced color change, therefore cell viability can be detected using absorbance-based plate readers.
• Example 6 Elovanoids for allergic rhinitis, allergic conjunctivitis, allergic dermatitis and asthma [457] The following are the experimental conditions that trigger inflammation/allergy in the human nasal mucosa (in primary culture) and that elovanoids contracted, protecting the integrity of these cells.
• dsRNA double-stranded viral RNA
• TLR4 Toll-like receptor 4
• the LPS that we used for our experiments is a preparation of smooth (S)-form LPS purified from the Gram-negative E. coli 0111:B4 that was used at 30 mg/mL to challenge HNEpC; c) House Dust Mite extract from Dermatophagoidespteronyssinus (D.P.) (Catalog# 3033) – pure lyophilized extract were obtained from Chondrex, Inc. D.P. was used at 30 mg/mL to challenge HNEpC. Allergen – Der p1, Der p2; d) House Dust Mite extract from Dermatophagoides farina (D.F.) (Catalog# 3040) – pure lyophilized extract were obtained from Chondrex, Inc.
• Example 7 [465] (1) can be a target in prodromal conditions before disease is evident. Structure and function of the 5xFAD retina.
• A V log I plot showing maximum ERG b-wave amplitudes for light flashes from 0 to 0.075 cd ⁇ s/m2. The 5xFAD mice achieved maximum amplitude of about 100 mV, approximately half that recorded for the wild-type mice.
• B Electron microscopy of 5-month-old 5xFAD retinas illustrating morphological similarity to the wild- type retina. i. Basal side of a 5xFAD RPE cell showing membrane infoldings along Bruch’s membrane (Br). ii.
• Disk synthesis region at the basal portion of a wild-type rod outer segment showing newly formed disks from the connecting cilium (CC) membrane.
• CC connecting cilium
• iii Similar region in a 5xFAD retina showing new disk formation (arrow).
• OLM outer limiting membrane
• N photoreceptor nucleus
• the cytoplasm of the Müller cells (M) are lighter than that of the photoreceptors (PR).
• PR The interface between a 5xFAD RPE cell and a rod photoreceptor tip (PR).
• phagosomes Two phagosomes (Ph) are visible just within the RPE cytoplasm; the lower Ph is held within the RPE apical processes, while the upper, darker Ph is older and just entering the RPE cell body, illustrating normal phagocytic function.
• C Five-month-old wild-type and 5xFAD retinal sections illustrating normal photoreceptor profiles within the 5xFAD retina.
• D Fluorescent staining of the retina from WT and 5xFAD. The blue (DAPI) is the nuclei and red Ab in the RPE layer at 6 months old in 5xFAD.
• ELVs restore RPE morphology and reduce gene expression after subretinal injection of OAb in WT mice.
• A Mice were divided into 7 groups: non-injected, PBS, OAb only, OAb + ELV-N-32, OAb + ELV-N-34, ELV-N-32 only and ELV-N-34 only. On day 3, mRNA were isolated for RT-PCR. On day 7, mice were subjected to OCT and then eyes were enucleated and processed to whole mount RPE staining and Western blot.
• B Whole flat mount of RPE. OAb disrupted RPE morphology.
• E senescence- and AMD-related genes
• MMP matrix metalloproteinases
• G autophagy
• E senescence- and AMD-related genes
• MMP matrix metalloproteinases
• G autophagy
• E senescence- and AMD-related genes
• MMP matrix metalloproteinases
• G autophagy
• E senescence- and AMD-related genes
• MMP matrix metalloproteinases
• G autophagy
• H p16INK4a western blots of RPE/Choroid.
• ELV-N-32 and ELV-N-34 down regulated the expression of the key senescence marker, p16INK4a, which was elevated by OAb injection.
• Example 9 [471] OAb toxicity is counteracted by ELVs in primary hRPE.
• A Primary hRPE cells were treated with 10 ⁇ M OAb with or without adding ELVs. After 3 days, the total RNA was isolated and q-PCR analyzed. After 7 days, cells were subjected to b-Galactosidase staining.
• B Live cell images of primary hRPE under bright field microscope imaging after 7 days.
• C b-Galactosidase staining of primary hRPE, +/- ELVs. Quantitation of % for the b-Gal positive cells.
• ELVs decreased positive senescent cells.
• D Transcription of senescence genes, AMD-related genes and autophagy genes in primary hRPE under OAb (1-42) exposure and treatment with ELVs. (*P ⁇ 0.05, using student t-test comparison).
• OAb induces senescence and disrupts the tight junction of RPE. Next, OAb penetrates the retina, causing cell death of photoreceptors reflected in less cell body layer (CBL) nuclei. The Elovanoids restore the morphology of the RPE layer upon OAb exposure and, as a consequence, the retina structure is preserved.
• OAb induces the senescence, autophagy, matrix metalloproteinases, and AMD-related genes in the RPE and apoptosis genes in retina. ELVs downregulated the OAb-gene inductions. Pathways for the synthesis of ELVs are depicted.
• ELVs are dihydroxylated derivatives of the very long-chain polyunsaturated fatty acids (VLC-PUFAs) 32:6n-3 and 34:6n-3.
• VLC-PUFAs are biosynthesized by elongation of a 22:6n-3 fatty acid and catalyzed by ELOVL4 (elongation of very-long-chain fatty acids–4).
• ELOVL4 elongation of very-long-chain fatty acids–4
• ELVs are low-abundance, high-potency, neuroprotective, pro-homeostatic mediators that arrest senescence gene programming and the senescence-activated secretory phenotype (SASP) in neural cells upon homeostasis disruptions 3.
• SASP senescence-activated secretory phenotype
• the HT is also a target because, although it is comprised of terminally differentiated cells and originates from the neuro-epithelium, senescent neurons in aged mice, models of AD 4, and astrocytes 5,6, also expresses senescence and develops secretory SASP that fuels neuroinflammation in nearby cells 7–9.
• Our recent study shows neighboring cells are targeted by neurotoxic actions of SASP, inducing retinal paracrine senescence 3.
• Aim 1) Validate that Elovanoids counteract in human adipocytes from diabetic patients’ senescence programming activation upon induction by TNFa, IL1b or other inducers.
• Active brown/beige AT plasticity increases energy expenditure and is linked to reduced hyperglycemia and hyperlipidemia; on the other hand, its atrophy and inactivation are associated with obesity and aging. Thus, a chronic slow-going local inflammatory condition would disrupt the regulation of internal signals as well as connections with the HC.
• Aim 2) Validate that the HT of genetically diabetic mice develops SP that in turn impairs synaptic connectivity and neuronal dysfunctions.
• ELVs mediate protection in neuronal cultures undergoing either oxygen/glucose deprivation or N methyl-D-aspartate receptor–mediated excitotoxicity, as well as in experimental ischemic stroke 10.
• the methyl ester or sodium salt of ELV-N-32 and ELV-N-34 resulted in reduced infarct volumes, promoted cell survival, and diminished neurovascular unit disruption when administered 1 hour following 2 hours of ischemia by middle cerebral artery occlusion.
• Elovanoids as a therapy is supported by the following: 1) Our data on AT and HT (see herein) 2) Our data on human neurons in culture demonstrate that SASP activation is blocked by ELVs (see herein). ELVs counteract senescence in human neural cells. 3) We have reported that oligomeric A-beta peptide activates the SP, SASP followed by retinal cell death, and that Elovanoids arrest the expression of the SP genes p16INK4a, MMP1, p53, p21, p27, Il-6, and MMP1 as well as of the SASP secretome and of p16 protein by Western blot 3.
• Autophagy is a key event of brown/beige adipocytes plasticity by regulating intracellular remodeling during brown/beige adipogenesis, thermogenic activation, and inactivation. This can include autophagic degradation of mitochondria critical for the inactivation of brown adipocytes and the transition from beige-to-white adipose tissue 3.
• Elovanoids modulate the matrix metalloproteinases transcriptome (MMP1a,MMP2, MMP3, MMP8, MMP9, MMP12, and MMP13), and we indicated that, with SASP, this mechanism contributes to alter the extracellular matrix 3. So, in both AT and HT, SASP is autocrine and paracrine, modifying the homeostasis of the extracellular matrix microenvironment in both the AT and the HT as a consequence, creating an inflammatory milieu that contributes to impaired function in insulin sensitivity. Elovanoids regulate slow- going, chronic, sterile inflammation (i.e., inflammaging). This is even an important tenant of the rationale described herein.
• TNF-a directly prevents insulin action in the adipocyte 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 in addition to IL-6, IFN-g, and CCL2.
• Hypothalamus is a target of metabolic syndrome and critical for obesity and diabetes type 2.
• Various aspects of physiological deterioration including obesity and diabetes type 2 are controlled by the hypothalamus, a critical brain region that connects the neuroendocrine system to physiological functions.
• a set of agouti-related peptide/neuropeptide Y (AgRP/NPY) and proopiomelanocortin (POMC) neurons a set of growth hormone-releasing hormone (GHRH) and somatostatin (SST) neurons, a set of arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) neurons, and a set of gonadotropin-releasing hormone (GnRH) and kisspeptin/ neurokinin B/dynorphin (KNDy) neurons contribute to age-related physiological decline in energy metabolism, hormone regulation, circadian rhythm, and reproduction.
• AgRP/NPY agouti-related peptide/neuropeptide Y
• POMC proopiomelanocortin
• GHRH growth hormone-releasing hormone
• SST somatostatin
• AVP arginine vasopressin
• VIP vasoactive intestinal peptide
• KNDy gonadotropin-releasing hormone
• the underlying cellular mechanism for the hypothalamus-mediated dysfunctions progression comprises dysregulation of nutrient sensing, altered intercellular communication, stem cell exhaustion, senescence programming activation, loss of proteostasis, and epigenetic alterations.
• One of the functions of the arcuate (ARC) hypothalamic neurons is to appropriately respond to hormones and neuropeptides both locally and peripherally and involved in energy homeostasis.
• Arcuate hypothalamic neurons have projections to the PVN and upon stimulation of the ARC, there is a co-release of dopamine and GABA to the neurons in the PVN where dopamine excited orexigenic neurons synthesize AgRP and NPY and inhibit anorexigenic neurons that synthesize POMC.
• VMH Ventromedial nucleus of the hypothalamus
• WT C57BL/6J
• Leprdb db/db diabetic mice
• MEA Maestro microelectrode arrays
• MEA Microelectrode array
• MEA measurements were carried out using a 48-well Microelectrode array (MEA) plate (M768-tMEA-48W) from Axion Biosystems, GA. Each well of the MEA plate, contained a 4 ⁇ 4 grid of 30 nm circular nanoporous PEDOT electrodes embedded in the cell culture substrate, with a pole-to-pole electrode spacing of 200 mM.
• the wells were treated with 0.1% polyethylenimine (PEI) in sodium borate buffer, pH 8.4. Wells were then pre-coated in laminin (6 mg/mL) and the hypothalamic slices (200 mm in thickness) were plated on the electrode grid.
• PEI polyethylenimine
• hypothalamus slices were plated and maintained for 4 days in culture in complete neurobasal medium supplemented with B27TM and N2 supplements along with GlutaMaxTM and Pen Strep (Thermo Fisher, GibcoTM) at 37°C and 5% CO2.
• Extracellular recordings of spontaneous action potentials were performed in culture medium at 37°C using the standard neural setting of the MaestroPro MEA system and AxIS software version 1.5.1.12. (Axion Biosystems). Data were sampled at 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 band-pass filer to remove high frequency noise before spike detection.
• the threshold for spike detection was set to 6 times the rolling standard deviation of the filtered field potential on each electrode. Five-minutes recording spans were used to calculate average spike rate for the well, and the number of active electrodes in a well (“Active Electrodes”), which was defined as the number with spike rates 3 0.5/min. Recorded spike number per electrode were averaged after disregarding noisy electrodes from analysis. Spike time stamps were exported to Neuroexplorer 5.0 (NEX Technologies) for creation of spike raster plots. [501] Using the MEA system, population level electrical activity was recorded from different hypothalamic neurons.
• Spontaneous activity was evident in hypothalamic slices from normal C57Bl6 WT controls, while the middle and right top panels, show the raster plots for the Leprdb (db/db diabetic) male and female mice respectively. These plots show asynchronous field potential and spike/burst activity.
• the low-level spiking could arise from either a cell-autonomous deficiency in excitability or a lack of synaptic drive from neighboring cells. All these spikes were induced with the addition of Dopamine (50nM) followed by GABA-a antagonist, bicuculline (10mM).
• the adult obese diabetic mice showed increased FJb signal in organotypic slices of Hypothalamus while the wild type mice depicted negligible amounts indicating that db/db mice hypothalamus is undergoing neurodegeneration.48h incubation with 500nM ELV34 elicited neuroprotection that was reflected in the decrease of F-Jb signal (a).
• A-G SA-b-Gal activity in HNG cells treated with Erastin (10mM) and different Elovanoids (ELVs) or neuroprotectin D1 (NPD1) at a concentration of 500 nM. Micrographs were obtained with bright field microscopy.
• H Quantification of SA-b-Gal+ cells shown in (A-G). SA-b-Gal+ cells were scored in 3 random fields of at least 150 total cells. Results are expressed as percentage of stained SA-b-Gal+ cells (mean ⁇ SEM). Statistical analysis were done using Graphpad Prism software 8.3. Results compared with one-way ANOVA, followed by Holm’s Sidak post hoc tests and p ⁇ 0.05 was considered statistically significant.
• HNG Human neuronal glial
• Aim 1 Test the prediction that Elovanoids counteract in human adipocytes from diabetic patients’ senescence programming activation upon induction by TNFa, IL1b or other inducers.
• Active brown/beige AT plasticity increases energy expenditure and is linked to reduced hyperglycemia and hyperlipidemia; on the other hand, its atrophy and inactivation are associated with obesity and aging.
• IL1b is a cytokine that induce insulin resistance in adipocytes 18 •
• IL8 was elevated by IL1b and brought down around 40 folds by 500nM ELV34 in both diabetic and non-diabetic adipocytes. Even though the mechanisms are unknown ELV34 can be a therapeutic agent to stop or halt damaging signaling observed in diabetic patients.
• ELV34 revert the effect of IL1b in human diabetic adipocytes.
• ELV34 reduced the levels of IL6 (marker of SASP) induced by IL1b in Diabetic db/db mice hypothalamus indicating that hypothalamic neurons and astrocytes undergo SP. Different effects have been observed in female and male mice.
• ELV34 treatment increased the levels of Adiponectin an anti-diabetic systemic hormone secreted by adipocytes and other tissues (hypothalamus) that promotes insulin sensitivity.
• A) Diabetic hypothalamus treated with ELV34 showed a trend of increase in adiponectin in female and males.
• SAT subcutaneous adipose tissue
• VAT visceral adipose tissue
• SAT and VAT possess differential ability to browning 19.
• APPROACH [517] Specific Aim 1. Test the prediction that elovanoids counteract in human adipocytes from diabetic patients’ senescence programming activation upon induction by TNFa, IL1b or other inducers. [518] Rationale. TNFa is circulating in obese patients and has an important role in insulin resistance pathogenesis20,21. In addition, Interleukin 1b signaling mediates the effects of macrophages on the adipose tissue 18.
• Circulating IL1b induce disruption in the function of adipose tissue 22.
• systemic IL1b induce senescence in human adipocytes.
• the loss of functionality of the adipocytes exposed to cytokines can include the incapability of the cells for browning.
• Active brown/beige AT plasticity increases energy expenditure and is linked to reduced hyperglycemia and hyperlipidemia; on the other hand, its atrophy and inactivation are associated with obesity and aging.
• a chronic slow going local inflammatory condition would disrupt the regulation of internal signals as well as connections with the HC.
• other hormones produced and secreted by fat tissue like adiponectin, may be reduced when the adipocytes undergo SP.
• adiponectin has anti-diabetic and anti-inflammatory effects, and it also functions as an insulin sensitizer 23.
• EMV34 Elovanoid 34
• senescence markers To test for the expression of senescence markers, differentiated adipocytes from diabetic and non-diabetic patients will be exposed to IL1b and/or TNFa for 6 days plus and minus ELV34, harvested and their RNA will be extracted and used as template for cDNA synthesis. The expression levels of p16, p21, p27 and p53 will be assessed by Real time PCR using Taqman probes. The results will be normalized by the expression of housekeeping genes: PPIA, GAPDH, b-actin, B2M, TBP and TFRC. The activity of these markers will be measured by western blot assay to detect phosphorylation and increase in the total protein content.
• the b-galactosidase activity assay is based on the overexpression and accumulation of the endogenous lysosomal beta-galactosidase specifically in senescent cells 24. Similarly to the detection of the expression of the senescence markers, human diabetic and non-diabetic adipocytes will be exposed to IL1b and/or TNFa for 6, 9 and 12 days plus and minus ELV34 and then stained using a b-galactosidase substrate that produces fluorescent signal when hydrolyzed by the endogenous enzyme. The samples will be imaged by two methods: confocal microscopy and flow cytometry.
• the third part consists on determining the Senescence-Associated Secretory Phenotype.
• the senescence of the adipocytes will be induced as mentioned described herein in the presence or absence of EL34 and the resulting culture medium will be collected, concentrated and tested for candidates via western blot or ELISA assays.
• Some of the candidates to be tested are: IL-6, IL-7, IL-1a, -1b, IL-13, IL-15, IL-8, GRO-a,-b,-g, MCP-2, MIP-1a, Eotaxin, Eotaxin-3, TECK, ENA-78, I-309 and MMP-1, -3, -10, -12, -13, -14.
• adipocytes undergoing SP and or SASP will be then treated with ELV34 and the parameters described herein will be tested.
• Experiment 3 to assess the ability of diabetic adipocytes exposed to IL1b and/or TNFa for 6, 9 and 12 days to synthesize and release Adiponectin.
• Adipocytes treated with IL1b and/or TNFa will be tested for expression of adiponectin mRNA via real time PCR ( Figure X), western blot assay and the culture medium will be collected and subjected to ELISA and/or western blot assay.
• the effects of the ELV34 on the production of Adiponectin will be tested using the procedures described herein.
• the diabetic adipocytes that become senescent will show lower expression of BAT markers; CD137, TMEM26 (transmembrane protein 26) and TBX1 (T-box 1) concomitantly with low activity of the main transcription factors responsible for the genetic signature observed in brown adipocytes.
• BAT markers CD137, TMEM26 (transmembrane protein 26) and TBX1 (T-box 1) concomitantly with low activity of the main transcription factors responsible for the genetic signature observed in brown adipocytes.
• BAT markers CD137
• TMEM26 transmembrane protein 26
• T-box 1 T-box 1
• mice This strain of mice is used to model phases I to III of diabetes type II and obesity. We will dissect out the brain of these animals and slice it to isolate the hypothalamus, the hippocampus and the cortex. Each organotypic slice will be set up in an individual well with Neurobasal medium supplemented with B27.48h post-plating medium will be supplemented with 500 nM ELV and 24h later, the organotypic slices will be recorded. Neuronal activity will be determined using the Axion BioSystems’ Maestro multielectrode array (MEA) technology.
• MEA Maestro multielectrode array
• hypothalamic neurons identity that we are testing by adding 50 nM dopamine and 10 ⁇ M bicuculine, which will indicates the presence of the ventro-medium (VTM) and arcuate nuclei that are important for satiety and feeding.
• VTM ventro-medium
• Comparison of the neuronal activity of control and diabetic mice hypothalamus with and without ELV will provide us the baseline activity of this brain structure and help us evaluate the functionality of the neurons. After the recording, the hypothalamus slice will be retrieved and total RNA will be extracted.
• First- strand cDNA will be reversed transcribed, and the expression of genes involved in senescence programming, as well as insulin signaling and sensitivity and glucose metabolism will be examined.
• the up- and downregulation of candidates (p53, p21, p16ink4a, and Bmi- 1) will be further confirmed by Western Blot or capillary Western Blot for a larger panel of targets.
• Outcome Neuronal activity of the HT of diabetic mice has a decreased sensitivity for dopamine and bicculine, indicating malfunctioning neurons. By investigating the causality, an upregulation of markers for SP and SASP in diabetic mice as compared to control will be observed.
• mice The intended effects can be systemic.
• Experimental Design We will evaluate our findings with BKS.Cg-Dock7m+/+ Leprdb/J mice and controls C57BLKS/J. This strain of mice is used to model phases I to III of diabetes type II and obesity. Samples from both genders will be included. All animal experiments will be carried out in accordance with the approved IACUC protocol issued by Louisiana State University Health Sciences Center. Intranasal administration will be performed on lightly anesthetized mice. Each mouse will be placed on a sterile surgical pad and lightly stretched out to better hold the scruff. With a firm grip on the scruff, the mouse will be turned on its back while still allowing the mouse to breathe and be comfortable.
• the tip of the pipettor containing the Elovanoids dispersed in 0.9% saline will be placed near the left nostril of the mouse at a 45- degree angle, and about 5 mL of the drug will be administered to the nostril with a 2–3 sec interval in between for a total of 10 mL/nostril.
• the mouse will be held in this position for 5 sec or until it regained consciousness, then the administration step will be repeated for the other nostril for a total of 20 mL/mouse. After the mouse receives all drops, the animal will be kept restrained on its back until the material disappears into the nares and then will be returned to its cage.
• VAT and SAT visceral and subcutaneous adipose tissues
• adipocytes will be plated in 6 well-plates and the brain dissected out to collect the hypothalamus, hippocampus and cerebral cortex.
• SP and SASP will halt in VAT, SAT and HT of Leprdb/J mice.
• amyloid b-peptide accumulates in subretinal drusen.
• ERG early functional deficiencies
• PRC photoreceptor cell
• NPD1 neuroprotectin D1
• ELVs elovanoids
• ELVs prevented OAb-induced changes in gene expression engaged in senescence, inflammation, autophagy, extracellular matrix remodeling and AMD.
• OAb target the RPE we used primary human RPE cell cultures and demonstrated that OAb caused cell damage, while ELVs protected and restored gene expression as in mouse.
• Our data show OAb activates senescence as reflected by enhanced expression of p16INK4a, MMP1, p53, p21, p27 and Il-6 and of senescence-associated secretory phenotype (SASP) secretome, followed by RPE and PRC demise and that elovanoids 32 and 34 blunt these events and elicits protection.
• SASP senescence-associated secretory phenotype
• ELVs counteracted OAb-induced expression of genes engaged in AMD, autophagy and extracellular matrix (ECM) remodeling.
• ECM extracellular matrix
• Neuroprotectin D1 a docosanoid derived from an omega-3 essential fatty acid, is neuroprotective by arresting inflammation initiation and thus sustaining photoreceptor cell (PRC) integrity (1) and is deficient in the hippocampus CA1 area of early Alzheimer’s disease (AD) (2). Ab accumulates in AD. In the 5xFAD retina, Ab also accumulates and, although Ab in AMD sets in motion homeostasis disturbances that can include inflammation and contribute to PRC death (3, 4), it is not known how to limit Ab-mediated cell damage.
• PRC photoreceptor cell
• VLC-PUFAs very long-chain PUFAs (VLC-PUFAs, C>28) are synthesized by ELOVL4 (elongation of very long chain fatty acid–4) (5, 6) and are necessary for rhodopsin function (7). Mutations on the ELOVL4 gene (5) cause Stargardt macular dystrophy type 3 with central vision loss. Recessive ELOVL4 mutations cause seizures, mental retardation, and spastic quadriplegia, indicating the importance of VLC-PUFAs in brain development and physiology as well (8).
• ELOVL4 elongation of very long chain fatty acid–4
• VLC- PUFAs are incorporated into specific phosphatidylcholine molecular species (PCs) of the photoreceptor cells outer segments, they arrive to the retinal pigment epithelium (RPE) after daily PRC disk shedding and phagocytosis.
• ELVs with 32 and 34C are enzymatically synthesized in RPE cells from PC-released VLCPUFAs by a phospholipase A1 (9, 10). These new lipid mediators have the ability to protect RPE cells from uncompensated oxidative stress by upregulating pro-homeostatic and prosurvival protein abundance with attenuation of apoptosis in photoreceptor cells (9, 10) as well as in neurons (11).
• Ab42 is a component of drusen in AMD and of senile plaques of AD (12, 13). In AMD, Abeta contributes to inflammation, perturbed RPE morphology and function, and PRC integrity (14, 15).
• the 5xFAD transgenic mouse carries mutations associated with early-onset familial AD, and although it displays several unspecific changes, it shows PRC degeneration (16, 17).
• OAb oligomeric Ab
• WT wild- type mice
• ECM extracellular matrix
• elovanoids modify OAb-induced gene expression, including the senescence program and senescence-associated secretory phenotype (SASP) to, in turn, protect the RPE and sustain photoreceptor cell integrity.
• PCs were presented in three groups: (i) DHA and VLC-PUFA containing PCs, (ii) DHA containing PCs, and (iii) AA containing PCs. Structures and m/z of PCs (FIG.60). The 5xFAD depicted decreases of both DHA and VLC-PUFA containing PCs, including PC54:12, PC56:12, and PC58:12 (FIG.52, panel E), and DHA containing PCs, including PC36:8, PC38:8, and PC44:12 (FIG.52, panel F).
• PCs containing AA were increased in the 5xFAD retina (FIG.52, panel G), indicating that the balance of n-6/n-3 (AA, DHA, and VLC-PUFA) was altered.
• DHA and VLC-PUFA contained in PCs were deficient in 5xFAD retina (Fig. 1A-G) unlike in RPE (FIG.53).
• the PC38:6 content was higher in the RPE of 5xFAD (contrast to the retina – Fig.2E), and the PC40:6 was similar in 5xFAD and WT (Fig.2E).
• PC44:12 was lower in the 5xFAD RPE as in the retina.
• Non-injected, PBS-injected and ELV- 32, ELV-34-injected mice did not yield PRC degeneration (FIG.56, panels C, D).
• the ZO-1 staining of flat mounted RPE revealed that oligomeric b-Amyloid disrupted tight junctions and triggered cell damage.
• the mice injected with PBS or ELVs alone showed a small reduction of ONL, due to mechanical stress following subretinal injection (FIG.56, panels C and D).
• ELVs counteract OAb-induced senescence, autophagy, AMD and ECM remodeling gene expression disruptions in RPE and of apoptotic gene expression in retina.
• qPCR quantitative PCR
• SASP matrix metalloproteinases
• chemokines e.g., TNF-a, IL-6, and IL-8
• cytokines e.g., TNF-a, IL-6, and IL-8
• the senescence genes studied are P16 INK4a (Cdkn2a), p21CIP1(Cdkn1A), p27 KIP (Cdkn1B), p53 (Tp53 or TRP53), IL6 and MMP1.
• ELV-N-32 and ELV-N-34 reverted these effects (FIG.57, panels B-D.
• Ab-based antibody as well as anti-inflammatory therapies for AD have been largely unsuccessful, therefore there is a need to understand mechanisms and identify specific agents that limit Ab neurotoxicity (25–28).
• RPE sustains PRC integrity and its dysfunction sets in motion PRC death in retinal degenerative diseases, including AMD.
• Oab drives RPE and PRC pathology, both in vivo in a rodent and in primary human RPE cell culture.
• 5xFAD PRC degeneration we report deficits in precursors and pathways for NPD1 and ELV biosynthesis. These deficits precede ECM and histology signs of PRC damage while ERG already displays impairments.
• Elovanoids counteracted the cytotoxicity of OAb subretinally administered in WT mice leading to RPE tight junction disruptions followed by PRC cell death.
• Our data show that OAb activates a senescence program reflected by enhanced gene expression of p16INK4a, MMP1, p53, p21, p27, Il-6, MMP1 and SASP secretome, followed by RPE and PRC demise, and that ELV-N-32 and ELV-N-34 blunt these events and elicit protection to both cells.
• the RPE cell is terminally differentiated and originated from the neuroepithelium.
• senescent neurons in aged mice and models of AD (50) and astrocytes (51,52) also express senescence and develop secretory SASP that fuel neuroinflammation in nearby cells (53–55).
• neighbor cells can be targeted by SASP neurotoxic actions, inducing photoreceptor paracrine senescence. Therefore, SASP from RPE cells may be autocrine and paracrine, altering the homeostasis of the interphotoreceptor matrix microenvironment as a consequence and creating an inflammatory milieu the contributes to loss of function associated with ageing (56), age- related pathologies (56) and AMD.
• ELVs restore expression of ECM remodeling matrix metalloproteinases altered by OAb treatment, pointing to an additional disturbance in the interphotoreceptor matrix.
• the inflammation set in motion can be a low- grade, sterile, chronic pro-inflammatory condition similar to inflammaging that is also linked to senescence of the immune system (56, 57).
• elovanoids counteracted OAb- induced expression of genes engaged in AMD and autophagy. Without wishing to be bound by theory, the elovanoids targeted event(s) on gene transcription (FIG.58) to inform new unifying regulatory mechanisms to sustain health span during aging and neurodegenerative diseases (56, 58). Although further research is needed, our results, overall, show ELVs as a therapeutic avenue of exploration for AMD.
• Genotyping was performed by PCR of tail DNA. All analyses were carried out blind with respect to the mice genotype.
• 6-month-old C57BL/6J mice were anesthetized by an intraperitoneal injection of ketamine/xylazine, pupils dilated with 1.0% tropicamide (Akorn, IL, USA); and 0.5% Proparacaine Hydrochloride (Akorn) was applied for topical anesthesia. Eyes were punctured with a 30-gauge needle between the corneoscleral junction and the ora serrata into the vitreous cavity without disturbing the lens.
• samples were dried under N2 and then resuspended in 20ml of the sample solvent (acetonitrile/chloroform/methanol, 90:5:5 by volume).
• sample solvent acetonitrile/chloroform/methanol, 90:5:5 by volume.
• the Acquity UPLC BEH HILIC 1.7- mm, 2.1 ⁇ 100-mm column was used with a mixture of solvent A (acetonitrile/water, 1:1; 10mM ammonium acetate, pH 8.3) and solvent B (acetonitrile/water, 95:5; 10mM ammonium acetate, pH 8.3) as the mobile phase (0.5ml/min).
• Solvent B (100%) was isocratically run for the first 5 min and then run in a gradient to 20% of solvent A for 8 min, increased to 65% of solvent A for 0.5 min, run isocratically at 65% of solvent A for 3 min, and then returned to 100% of solvent B for 3.5 min for equilibration.
• the column temperature was set to 30°C.
• the amount for each PC and PE species was calculated as % of the total PCs and PEs/sample.
• six retinas or six RPE/Choroid were pooled and homogenized as described herein.
• Cells were collected from anonymous donors provided by eye banks. Briefly, globes of a 19- year-old Caucasian male, without eye pathology were obtained from NDRI within 24 hours after death from head trauma. Globes were opened, and RPE cells harvested and cultured (1, 2) and grown in MEM medium supplemented with 10% FBS, 5% NCS, non-essential amino acids, Penicillin-Streptomycin (100U/mL), human fibroblast growth factor 10ng/ml and incubated at 37°C with a constant supply of 5% CO2. Cells integrity was validated as in previous study (3). For oligomeric Ab treatment, cells were seeded in the 6-well plates, 30.000 cells/cm2.
• SA-b-Gal staining [664] Senescence-Associated b-Galactosidase (SA-b-Gal) staining [665] Cells were visualized using SA-b-Gal staining kit (Cat 9860, Cell Signaling Technology, MA, USA). Briefly, RPE cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, then washed again with PBS and incubated in staining solution mix overnight at 37oC (no CO2), the presence of CO2 can cause changes to the pH which may affect staining results. Pictures were taken under brightfield microscope (Nikon Eclipse TS100) 200X magnification after the development of blue color, and cells counted in 10 different random fields per well.
• PFA paraformaldehyde
• RNA isolation and qPCR analysis [669] Cell culture media was removed, cells were wash with PBS 1X and samples were collected using cell scraper. Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany).
• mice were anesthetized with ip ketamine/xylazine, pupil dilated by topical 1.0% tropicamide and placed in a custom-built holder for OCT imaging (body temperature maintained at 38°C with a heat pad).
• Retinas were imaged along the horizontal meridian through the optic nerve head using a Heidelberg Spectralis HRA OCT system (Heidelberg Engineering, Heidelberg, Germany). Axial resolution is 7mm optical and 3.5mm digital.
• the raw OCT B-scans cross-sectional images were exported with the scale in mm and opened in ImageJ (http//imagej.nih.gov/ij).

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