Very long chain polyunsaturated fatty acid, elongin-like (elowanoid) hydroxylated derivatives and methods of use
阅读说明：本技术 极长链多不饱和脂肪酸、类延长素（elovanoid）羟基化衍生物和使用方法 (Very long chain polyunsaturated fatty acid, elongin-like (elowanoid) hydroxylated derivatives and methods of use ) 是由 N·G·巴赞 N·A·佩塔西斯 于 2018-03-19 设计创作，主要内容包括：提供了化合物、药物组合物、化妆品和皮肤病学组合物或营养补充组合物,其包括ω-3极长链多不饱和脂肪酸(n-3 VLC-PUFA)和/或其内源性羟基化衍生物(被称为类延长素)。本公开提供了用于神经保护、器官和组织保护或恢复、预防或减缓衰老相关疾病和病状以及在衰老过程期间维持功能的方法。(Compounds, pharmaceutical, cosmetic and dermatological or nutraceutical compositions are provided that include omega-3 very long chain polyunsaturated fatty acids (n-3 VLC-PUFAs) and/or their endogenous hydroxylated derivatives (known as elongatoids). The present disclosure provides methods for neuroprotection, organ and tissue protection or restoration, prevention or slowing of aging-related diseases and conditions, and maintaining function during the aging process.)
1. A composition comprising at least one omega-3 very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.
2. The composition of claim 1, further comprising 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 to reduce the onset of or the pathological condition of a tissue of a recipient subject.
3. The composition according to claim 1, wherein said pathological condition is aging or inflammation of the tissue of the recipient subject.
4. The composition of claim 2, wherein the composition is formulated for topical delivery of the at least one omega-3 very long chain polyunsaturated fatty acid tissue to the skin of a recipient subject.
5. The composition of claim 2, wherein the pathological state is a pathological state of a neural tissue of the recipient subject.
6. The composition of claim 1, wherein the composition further comprises at least one nutritional component, and the composition is formulated for oral or parenteral delivery of the at least one omega-3 very long chain polyunsaturated fatty acid to a recipient subject.
7. The composition of claim 1, wherein the at least one omega-3 very long chain polyunsaturated fatty acid has from about 26 to about 42 carbon atoms in its carbon chain.
8. The composition of claim 7, wherein the at least one omega-3 very long chain polyunsaturated fatty acid has 32 or 34 carbon atoms in its carbon chain.
9. The composition of claim 1, wherein the omega-3 very long chain polyunsaturated fatty acid has five or six double bonds in its carbon chain with cis geometry.
10. The composition of claim 1, wherein the omega-3 very long chain polyunsaturated fatty acid is 14Z,17Z,20Z,23Z,26Z,29Z) -docosahexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z) -triacontahc-16, 19,22,25,28, 31-hexaenoic acid.
11. A composition comprising at least one elongatin-like having at least 23 carbon atoms in its carbon chain.
12. The composition of claim 11, wherein the composition further comprises a pharmaceutically acceptable carrier and is formulated for delivery of an amount of the at least one carotenoid effective to reduce the pathological state of the tissue of the recipient subject or delay at least one aging effect in the tissue of the recipient subject.
13. The composition according to claim 11, wherein the pathological condition is aging or inflammation of the tissue of the recipient subject.
14. The composition of claim 11, wherein the composition is formulated for topical delivery of the at least one elongatoid to the skin of a recipient subject.
15. The composition of claim 11, wherein the pathological state is a pathological state of a neural tissue of the recipient subject.
16. The composition of claim 11, wherein the composition further comprises at least one nutritional component, and the composition is formulated for oral or parenteral delivery of the at least one elongatoid to a recipient subject.
17. The composition of claim 11, wherein the at least one elongatoid is selected from the group consisting of: monohydroxylated elongatins, dihydroxylated elongatins, alkynyl monohydroxylated elongatins and alkynyl dihydroxylated elongatins or any combination thereof.
18. The composition of claim 11, wherein the at least one elongatoid is a combination of elongatoids, wherein the combination is selected from the group consisting of: monohydroxylated and dihydroxylated elongases; monohydroxylated elongatoids and alkynyl monohydroxylated elongatoids; monohydroxylated elongatins and alkynyl-dihydroxylated elongatins; dihydroxylated elongatins and alkynyl monohydroxylated elongatins; dihydroxylated elongatins and alkynyl dihydroxylated elongatins; monohydroxylated elongatins, dihydroxylated elongatins and alkynyl monohydroxylated elongatins; monohydroxylated extenders, dihydroxylated extenders and alkynyl-dihydroxylated extenders; and monohydroxylated elongatins, dihydroxylated elongatins, alkynyl monohydroxylated elongatins and alkynyl dihydroxylated elongatins, wherein each elongatoid is independently a racemic or diastereomeric mixture, a separated enantiomer, or a combination of enantiomers wherein the amount of one enantiomer is greater than the amount of the other.
19. The composition of claim 11, wherein the composition further comprises at least one omega-3 very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.
20. The composition of claim 19, wherein the at least one omega-3 very long chain polyunsaturated fatty acid has from about 26 to about 42 carbon atoms in its carbon chain.
21. The composition of claim 19, wherein the at least one omega-3 very long chain polyunsaturated fatty acid has five or six double bonds in its carbon chain with cis geometry.
22. The composition according to claim 19, wherein the at least one very long chain polyunsaturated fatty acid is 14Z,17Z,20Z,23Z,26Z,29Z) -docosahexa-14, 17,20,23,26, 29-hexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z) -triacontahy-16, 19,22,25,28, 31-hexaenoic acid.
23. The composition of claim 17, wherein the monohydroxylated elongatoid is selected from the group consisting of formulas G, H, I or J:
wherein:
n is 0 to 19 and-CO-OR is a carboxylic acid group OR a salt OR ester thereof,
and wherein:
if-CO-OR is a carboxylic acid group and the compound G, H, I OR J is a salt thereof, then the cation of the salt is a pharmaceutically acceptable cation, and
if-CO-OR is an ester, then R is an alkyl group.
24. The composition of claim 23, wherein the pharmaceutically acceptable cation is an ammonium cation, an iminium cation, or a metal cation.
25. The composition of claim 24, wherein the metal cation is a sodium, potassium, magnesium, zinc, or calcium cation.
26. The composition of claim 23, comprising equimolar amounts of enantiomers G and H, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
27. The composition of claim 23, comprising equimolar amounts of enantiomers I and J, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
28. The composition of claim 23, wherein the composition comprises one enantiomer of G or H in an amount greater than the other enantiomer of G or H.
29. The composition of claim 23, wherein the composition comprises one enantiomer of I or J in an amount that exceeds the amount of the other enantiomer of I or J.
30. The composition of claim 23, wherein the monohydroxylated elongatoids are selected from the group consisting of: (S,14Z,17Z,20Z,23Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,23,25, 29-hexaenoic acid methyl ester (G1), (S,14Z,17Z,20Z,23Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,23,25, 29-hexaenoic acid sodium salt (G2), (S,16Z,19Z,22Z,25Z,27E,31Z) -29-hydroxytetradecyl-16, 19,22,25,27, 31-hexaenoic acid methyl ester (G3); and sodium (S,16Z,19Z,22Z,25Z,27E,31Z) -29-hydroxytetradeca-16, 19,22,25,27, 31-hexaenoate (G4), each having the formula:
31. the composition of claim 17, wherein the dihydroxylated elongatinoid is selected from the group consisting of formulas K, L, M and N:
wherein:
m is 0 to 19 and-CO-OR is a carboxylic acid group OR a salt OR ester thereof,
and wherein:
if-CO-OR is a carboxylic acid group and the compound K, L, M OR N is a salt thereof, then the cation of the salt is a pharmaceutically acceptable cation, and
if-CO-OR is an ester, then R is an alkyl group.
32. The composition of claim 31, wherein the pharmaceutically acceptable cation is an ammonium cation, an iminium cation, or a metal cation.
33. The composition of claim 32, wherein the metal cation is a sodium, potassium, magnesium, zinc, or calcium cation.
34. The composition of claim 31, comprising equimolar amounts of diastereomers K and L, wherein the diastereomers have (S) or (R) chirality at the n-6 carbon bearing a hydroxyl group.
35. The composition of claim 31, comprising equimolar amounts of diastereomers M and N, wherein the diastereomers have (S) or (R) chirality at the N-6 carbon bearing a hydroxyl group.
36. The composition of claim 31, wherein the composition comprises one diastereomer of K or L in an amount that exceeds the amount of the other diastereomer of K or L.
37. The composition of claim 31, wherein the composition comprises one diastereomer of M or N in an amount that exceeds the amount of the other diastereomer of M or N.
38. The composition of claim 31, wherein the dihydroxylated elongatinoid is selected from the group consisting of: (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxytridecane-14, 17,21,23,25, 29-hexaenoic acid methyl ester (K1), (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxytridecane-14, 17,21,23,25, 29-hexaenoic acid sodium ester (K2), (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxytrinetradecanone-16, 19,23,25,27, 31-hexaenoic acid methyl ester (K3) and (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxytetradecane-16, 19,23,25,27, 31-hexaenoic acid sodium ester (K4), respectively having the formula:
39. the composition of claim 17, wherein the alkynyl monohydroxylated elongatoid is selected from the group consisting of formula O, P, Q or R:
wherein:
m is 0 to 19 and-CO-OR is a carboxylic acid group OR a salt OR ester thereof,
and wherein:
if-CO-OR is a carboxylic acid group and the compound O, P, Q OR R is a salt thereof, then the cation of the salt is a pharmaceutically acceptable cation, and
if-CO-OR is an ester, R is an alkyl group,
and wherein:
the compounds O and P each have a total of 23 to 42 carbon atoms in the carbon chain, 4 cis-carbon double bonds starting at positions n-3, n-12, n-15 and n-18, one trans-carbon bond starting at position n-7 and one carbon-carbon triple bond starting at position n-9; and is
Compounds Q and R each have a total of 23 to 42 carbon atoms in the carbon chain, 3 cis carbon-carbon double bonds starting at positions n-3, n-12 and n-15, one trans carbon-carbon bond starting at position n-7 and one carbon-carbon triple bond starting at position n-9.
40. The composition of claim 39, wherein the alkynyl monohydroxylated elongatinoid is selected from the group consisting of: (S,14Z,17Z,20Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,25, 29-pentaen-23-ynoic acid methyl ester (O1); (S,17Z,20Z,25E,29Z) -27-hydroxytridecano-17, 20,25, 29-tetraen-23-ynoic acid sodium salt (O2); (S,16Z,19Z,22Z,27E,31Z) -29-hydroxytetradecyl-16, 19,22,27, 31-pentaene-25-ynoic acid methyl ester (O3); and sodium (S,16Z,19Z,22Z,27E,31Z) -29-hydroxytetratetradec-16, 19,22,27, 31-pentaen-25-ynoate (O4), each having the formula:
41. the composition according to claim 39, wherein the pharmaceutically acceptable cation is an ammonium cation, an iminium cation, or a metal cation.
42. The composition of claim 41, wherein the metal cation is a sodium, potassium, magnesium, zinc, or calcium cation.
43. The composition of claim 39, comprising equimolar amounts of enantiomers O and P, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
44. The composition of claim 39, comprising equimolar amounts of enantiomers Q and R, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
45. The composition of claim 39, wherein the composition comprises one enantiomer of O or P in an amount that exceeds the other enantiomer of O or P.
46. The composition of claim 39, wherein the composition comprises one enantiomer of Q or R in an amount that exceeds the other enantiomer of Q or R.
47. The composition of claim 17, wherein the elongatinoid is an alkynyl dihydroxylated elongatinoid selected from the group consisting of formula S, T, U or V:
wherein:
m is 0 to 19 and-CO-OR is a carboxylic acid group OR a salt OR ester thereof,
and wherein:
if-CO-OR is a carboxylic acid group and the compound S, T, U OR V is a salt thereof, the cation of the salt is a pharmaceutically acceptable cation, and
if-CO-OR is an ester, R is an alkyl group,
and wherein:
compounds S and T each have a total of 23 to 42 carbon atoms in the carbon chain, 3 cis-carbon double bonds starting at positions n-3, n-15 and n-18, 2 trans-carbon double bonds starting at positions n-9 and n-11 and one carbon-carbon triple bond starting at position n-7; and is
Each of the compounds U and V has a total of 23 to 42 carbon atoms in the carbon chain, 3 cis-carbon double bonds starting at positions n-3 and n-15, 2 trans-carbon double bonds starting at positions n-9, n-11 and one carbon-carbon triple bond starting at position n-7.
48. The composition according to claim 47, wherein the pharmaceutically acceptable cation is an ammonium cation, an iminium cation, or a metal cation.
49. The composition of claim 48, wherein the metal cation is a sodium, potassium, magnesium, zinc, or calcium cation.
50. The composition of claim 47, wherein the alkynyl monohydroxylated elongatinoid is selected from the group consisting of: (14Z,17Z,20R,21E,23E,27S,29Z) -20, 27-dihydroxy-tridodecen-14, 17,21,23, 29-pentaene-25-ynoic acid methyl ester (S1); (14Z,17Z,20R,21E,23E,27S,29Z) -20, 27-dihydroxy-tridodecen-14, 17,21,23, 29-pentaen-25-ynoic acid sodium salt (S2); (16Z,19Z,22R,23E,25E,29S,31Z) -22, 29-dihydroxy trinetradeca-16, 19,23,25, 31-pentaene-27-ynoic acid methyl ester (S3); and sodium (16Z,19Z,22R,23E,25E,29S,31Z) -22, 29-dihydroxy-trinetradeca-16, 19,23,25, 31-pentaen-27-ynoate (S4), each having the formula:
51. the composition of claim 46, comprising equimolar amounts of diastereomers S and T, wherein the diastereomer has (S) or (R) chirality at position n-6 and (R) chirality at position n-13.
52. The composition of claim 47, comprising equimolar amounts of diastereomers U and V, wherein the diastereomer has (S) or (R) chirality at position n-6 and (R) chirality at position n-13.
53. The composition of claim 47, wherein the composition comprises one diastereomer of S or T in an amount that exceeds the amount of the other diastereomer of S or T.
54. The composition of claim 47, wherein the composition comprises one diastereomer of U or V in an amount that exceeds the amount of the other diastereomer of U or V.
55. A composition comprising a phospholipid compound selected from the group consisting of formula C, D, E or F and an acceptable carrier:
56. the composition of claim 55, further comprising a pharmaceutically acceptable carrier and formulated for delivery in an amount effective to reduce the pathological state of the tissue of the recipient subject or the onset of the pathological state of the tissue of the recipient subject.
57. The composition of claim 55, wherein the composition further comprises at least one nutritional component, and the composition is formulated for oral or parenteral delivery of the at least one elongatoid to a recipient subject.
Technical Field
The present disclosure relates to organ protective, disease preventive, health recovery and anti-aging compounds, compositions and methods of use related to omega-3 very long chain polyunsaturated fatty acids (n-3 VLC-PUFAs) and their hydroxylated derivatives (known as elongatoids).
Background
Although the aging process in humans is inevitable, recent findings have identified factors that disrupt homeostasis and/or accelerate cellular and tissue damage and promote the onset of age-related diseases and conditions. For example, prolonged exposure to uncompensated oxidative stress, accumulation of damaged cells and cell debris, and delay in tissue repair and clearance are all associated with the onset of aging-related diseases and conditions (e.g., chronic inflammation, cancer, neurodegenerative diseases, cardiovascular disease, and cerebrovascular disease). By reducing key factors that disrupt homeostasis, accelerate cell damage and cell death (cellular senescence), age-related diseases and conditions can be prevented or delayed, improve quality of life and extend human life.
Age-related and non-age-related inflammatory, degenerative, neurodegenerative, traumatic, dermatological and cardiovascular diseases comprise a large number of diseases affecting many people worldwide. In most cases, these diseases and related conditions and disorders are difficult to treat, leading to impaired quality of life and/or shortened life span, and remain unmet major medical needs.
Inflammatory, degenerative or neurodegenerative diseases and conditions within the scope of the present disclosure include acute and chronic disorders in which homeostasis is disrupted by an abnormal or deregulated inflammatory response. These conditions are initiated and mediated by a variety of inflammatory factors, including uncompensated oxidative stress, chemokines, cytokines, disruption of the blood/tissue barrier, autoimmune diseases, calcium imbalance (including calcium overload in cells), mitochondrial dysfunction, genetic factors (genetic susceptibility, polymorphism) or genetic conditions, epigenomic modifications, or other conditions involving leukocytes, monocytes/macrophages, microglia, astrocytes, or parenchymal cells that induce excessive protocellular damage, pro-inflammatory/destructive cellular and/or organ homeostasis. These diseases occur in a variety of tissues and organs, including skin, muscle, stomach, intestine, liver, kidney, lung, eye, ear, and brain. These diseases are currently treated by anti-inflammatory agents, such as corticosteroids, non-steroidal anti-inflammatory drugs, TNF modulators, COX-2 inhibitors, and the like.
Systemic inflammatory or degenerative diseases and conditions may affect vital organs (e.g., heart, muscle, stomach, intestine, liver, kidney, and lung) and may lead to age-related chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, and lupus.
Inflammatory or degenerative diseases and conditions of the eye generally affect the cornea, optic nerve, trabecular meshwork, and retina. Without effective prevention or treatment, they can lead to blinding eye diseases such as glaucoma, cataract, diabetic retinopathy and age-related macular degeneration (AMD).
Brain-related inflammatory, degenerative or neurodegenerative diseases and conditions, such as alzheimer's disease, parkinson's disease, multiple sclerosis, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis, often lead to premature aging, cognitive dysfunction and death.
Inflammatory or degenerative diseases and conditions of the skin are often caused by damage to the skin caused by sun exposure or other factors including skin inflammation (dermatitis or eczema), atopic dermatitis (atopic eczema), dehydration of the skin, or by abnormal skin cell proliferation resulting in excessive exfoliation. Skin damage from sun exposure or other factors is associated with a variety of diseases and conditions (e.g., eczema, psoriasis, atopic dermatitis, or neurodermatitis) and may result from exposure to ultraviolet light and other types of contact dermatitis. In addition, pruritus resulting from certain systemic diseases and conditions can lead to skin pruritus from various inflammatory and other types of irritation, and to the need for scratching, which can lead to further skin damage or changes in skin appearance.
Despite great advances, little is known about the pathophysiology of inflammatory, neuroinflammatory, degenerative or neurodegenerative diseases and conditions that often lead to organ damage, chronic disease and accelerated aging. Thus, protection of organs, prevention of aging-related diseases and conditions, and overall restoration of health remain unmet major medical needs. There is also a major gap in effectively protecting skin tissues from inflammatory, neuroinflammatory, hyperproliferative or dehydrated skin conditions. In particular, our understanding of the pathophysiology of skin damage, skin appearance changes, and skin aging remains unclear. As the largest organ of the human body, the skin can provide protection and support and play a key role in the overall appearance and well-being of humans.
In view of the overall importance of skin health, skin function, and skin appearance, efforts have been directed to developing methods to protect skin and overall skin health. Most current treatments involve dermal delivery of corticosteroids, or the use of oils and lotions containing vitamin, mineral or herbal ingredients, which are generally not effective in preventing or treating many types of skin damage and also have side effects such as skin thinning and muscle loss. While such formulations may provide some protection, there remains an unmet need to develop compounds, compositions, and methods that are effective in protecting damaged skin, preventing skin damage, restoring skin health, improving skin appearance, and delaying skin aging.
The present disclosure also relates to previously unknown organ-protecting, disease-preventing, health-restoring, nutrient-enhancing and anti-aging compounds, compositions and methods. In particular, the disclosure relates to the protection of cells and organ function in the face of disease episodes, to the restoration of healthy tissues and organs, and to the delay of aging and the prevention of aging-related diseases and conditions. The present disclosure also relates to the treatment or prevention of a variety of diseases and conditions (including inflammatory, degenerative, neurodegenerative, traumatic, cardiovascular, aging-related diseases and conditions), and for the prevention and treatment of damaged or deformed skin resulting from sun exposure, aging, or other causes.
With the discovery of the anti-inflammatory and pro-regressive effects of omega-3 long chain polyunsaturated fatty acids 20 or 22 carbon omega-3polyunsaturated fatty acids (n-3 or n3 PUFA), i.e. eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or docosapentaenoic acid (DPA), these beneficial lipid molecules and certain types of enzymatically hydroxylated derivatives thereof are increasingly used for therapeutic purposes and as nutritional or dietary supplements to prevent or manage excessive inflammation.
The present disclosure relates to previously unknown beneficial effects of omega-3 very long chain polyunsaturated fatty acid compounds (n3 VLC-PUFAs) containing carbon chains of at least 23 carbons.
The present disclosure also relates to the previously unknown discovery that n3 VLC-PUFAs are endogenously converted to therapeutically beneficial hydroxylated derivatives (known as elongatoids) that exhibit previously unknown biological activities, act as beneficial modulators of inflammatory responses and promote the restoration of disruptive function.
In summary, the present disclosure relates to compounds, compositions and methods involving omega-3 very long chain polyunsaturated fatty acids (n3 VLC-PUFAs or n-3 VLC-PUFAs) and their hydroxylated derivatives (elongatoids) for use in prophylactic, protective, restorative, therapeutic or nutritional applications.
Disclosure of Invention
Compounds, pharmaceutical, cosmetic and dermatological or nutraceutical compositions are provided that include omega-3 very long chain polyunsaturated fatty acids (n-3 VLC-PUFAs) and/or their endogenous hydroxylated derivatives (known as elongatoids). The present disclosure provides methods for neuroprotection, organ and tissue protection or restoration, prevention or slowing of aging-related diseases and conditions, and maintaining function during the aging process.
n3VLC-PUFA are converted in vivo into several previously unknown types of VLC-PUFA hydroxylated derivatives, known as Elongatoids (ELV), which protect and prevent progressive damage to tissues and organs whose functional integrity is compromised.
N3 VLC-PUFAs in neuronal cells and tissues in the brain and retina are released locally in response to neuronal stress states and are enzymatically converted to elongatoids, providing local neuroprotection to ensure neuronal survival.
By providing certain compounds related to n3 VLC-PUFAs and their corresponding Elongatinoids (ELVs), senescence-associated cells can be effectively suppressed.
Accordingly, the present disclosure relates to the prevention and treatment of health afflicting conditions and aging-related diseases and conditions. In particular, the present disclosure provides compounds, compositions and methods that protect and prevent damage that threatens the function and integrity of vital tissues and organs, prevent accelerated aging and retard cellular aging.
The present disclosure provides compounds, compositions and methods that can facilitate the protection, prevention and treatment of disorders in various organs triggered by persistent inflammation, injury or trauma.
Accordingly, one aspect of the present disclosure encompasses embodiments of compositions comprising at least one omega-3 very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.
In some embodiments of this aspect of the present disclosure, the composition comprises at least one n3VLC-PUFA having at least 23 carbon atoms in its carbon chain, wherein the n3VLC-PUFA may be in the form of formic acid, a formate ester, a formate salt, or a phospholipid derivative.
In some embodiments of this aspect of the present disclosure, the n3VLC-PUFA compound may be selected from the group consisting of formula a or B:
wherein: m may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group.
In some other embodiments of the present disclosure, the n3VLC-PUFA compound may be in the form of a phospholipid selected from the group consisting of formula C, D, E or F, wherein m may be 0 to 19:
in some embodiments of the present aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and is formulated for delivery of an amount of the at least one omega-3 very long chain polyunsaturated fatty acid effective to reduce the pathological state of the tissue of the recipient subject or the onset of the pathological state of the tissue of the recipient subject.
In some embodiments of this aspect of the disclosure, the pathological condition may be aging or inflammation of the tissue of the recipient subject.
In some embodiments of the present aspect of the present disclosure, the composition may be formulated for topical delivery of the at least one very long chain polyunsaturated fatty acid tissue to the skin of a recipient subject.
In some embodiments of this aspect of the present disclosure, the pathological state may be a pathological state of neural tissue of the recipient subject.
In some embodiments of the present aspect of the disclosure, the composition may further comprise at least one nutritional component, and the composition may be formulated for oral or parenteral delivery of the at least one very long chain polyunsaturated fatty acid to a recipient subject.
In some embodiments of this aspect of the present disclosure, the at least one omega-3 very long chain polyunsaturated fatty acid can have from about 26 to about 42 carbon atoms in its carbon chain.
In some embodiments of this aspect of the present disclosure, the at least one omega-3 very long chain polyunsaturated fatty acid can have 32 or 34 carbon atoms in its carbon chain.
In some embodiments of this aspect of the present disclosure, the omega-3 very long chain polyunsaturated fatty acid can have five or six alternating cis-geometry double bonds in its carbon chain.
In some embodiments of this aspect of the disclosure, the omega-3 very long chain polyunsaturated fatty acid is 14Z,17Z,20Z,23Z,26Z,29Z) -docosahexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z) -triacontahexaenoic acid-16, 19,22,25,28, 31-hexaenoic acid.
In some embodiments of this aspect of the disclosure, the at least one omega-3 very long chain polyunsaturated fatty acid can be 14Z,17Z,20Z,23Z,26Z,29Z) -docosahexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z) -triacontahexaenoic acid.
Another aspect of the disclosure encompasses embodiments of a composition comprising at least one type of auxin having at least 23 carbon atoms in its carbon chain.
In some embodiments of the present aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and may be formulated for delivery of an amount of the at least one carotenoid effective to reduce the pathological state of or delay at least one aging effect in a tissue of a recipient subject.
In some embodiments of this aspect of the disclosure, the pathological condition may be aging or inflammation of the tissue of the recipient subject.
In some embodiments of this aspect of the present disclosure, the composition may be formulated for topical delivery of the at least one elongatin to the skin of a recipient subject.
In some embodiments of this aspect of the present disclosure, the pathological state may be a pathological state of neural tissue of the recipient subject.
In some embodiments of this aspect of the present disclosure, the composition may further comprise at least one nutritional component, and the composition may be formulated for oral or parenteral delivery of the at least one elongatin to a recipient subject.
In some embodiments of this aspect of the present disclosure, the at least one auxin may be selected from the group consisting of: monohydroxylated elongatins, dihydroxylated elongatins, alkynyl monohydroxylated elongatins and alkynyl dihydroxylated elongatins or any combination thereof.
In some embodiments of this aspect of the present disclosure, the at least one elongatoid may be a combination of elongatoids, wherein the combination is selected from the group consisting of: monohydroxylated and dihydroxylated elongases; monohydroxylated elongatoids and alkynyl monohydroxylated elongatoids; monohydroxylated elongatins and alkynyl-dihydroxylated elongatins; dihydroxylated elongatins and alkynyl monohydroxylated elongatins; dihydroxylated elongatins and alkynyl dihydroxylated elongatins; monohydroxylated elongatins, dihydroxylated elongatins and alkynyl monohydroxylated elongatins; monohydroxylated extenders, dihydroxylated extenders and alkynyl-dihydroxylated extenders; and monohydroxylated elongatoids, dihydroxylated elongatins, alkynyl monohydroxylated elongatins and alkynyl dihydroxylated elongatins, wherein each elongatoid is independently a racemic mixture, a separate enantiomer or a combination of enantiomers (wherein the amount of one enantiomer is greater than the amount of the other); and wherein each type of elongator is independently a diastereomeric mixture, separated diastereomers or combination of diastereomers (wherein the amount of one diastereomer is greater than the amount of the other diastereomer).
In some embodiments of this aspect of the present disclosure, the monohydroxylated elongatoids may be selected from the group consisting of formulas G, H, I or J:
wherein: m may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers G and H, wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers I and J, wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of G or H in an amount that exceeds the other enantiomer of G or H.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of I or J in an amount that exceeds the amount of the other enantiomer of I or J.
In some embodiments of this aspect of the present disclosure, the dihydroxylated elongatinoid may be selected from the group consisting of formulas K, L, M and N:
wherein: m may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group, and wherein: the compounds K and L each have a total of 23 to 42 carbon atoms in the carbon chain, 4 cis-carbon double bonds starting at positions n-3, n-7, n-15 and n-18 and 2 trans-carbon double bonds starting at positions n-9 and n-11; and compounds M and N each have a total of 23 to 42 carbon atoms in the carbon chain, with 3 cis carbon-carbon double bonds starting at positions N-3, N-7 and N-15; and 2 trans carbon-carbon double bonds starting at positions n-9 and n-11.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of diastereomers K and L, wherein the diastereomer has (S) or (R) chirality at position n-6 and (R) chirality at position n-13.
In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of one or more diastereomers K and L, wherein the diastereomer has (S) or (R) chirality at position n-6 and (S) or (R) chirality at position n-13.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of K or L in an amount that exceeds the amount of the other diastereomer of K or L.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of diastereomers M and N, wherein the diastereomer has (S) or (R) chirality at position N-6 and (R) chirality at position N-13.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of one or more diastereomers M and N, wherein the diastereomer has (S) or (R) chirality at position N-6 and (S) or (R) chirality at position N-13.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of M or N in an amount that exceeds the amount of the other diastereomer of M or N.
In some embodiments of this aspect of the present disclosure, the alkynyl monohydroxylated elongatinoid may be selected from the group consisting of formula O, P, Q or R:
wherein: m may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group, and wherein: compounds O and P each have a total of 23 to 42 carbon atoms in the carbon chain, with 4 cis carbon-carbon double bonds located at the beginning of n-3, n-12, n-15 and n-18; having a trans carbon-carbon double bond at the position starting at n-7 and a carbon-carbon triple bond starting at position n-9; and compounds Q and R each have a total of 23 to 42 carbon atoms in the carbon chain, 3 cis carbon-carbon double bonds starting at positions n-3, n-12 and n-15, a trans carbon-carbon double bond starting at n-7 and a carbon-carbon triple bond starting at position n-9.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers O and P, wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers Q and R, wherein the enantiomers have (S) or (R) chirality at the n-6 carbon bearing the hydroxyl group.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of O or P in an amount that exceeds the other enantiomer of O or P.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of Q or R in an amount that exceeds the amount of the other enantiomer of Q or R.
In some embodiments of this aspect of the present disclosure, the elongatinoid may be an alkynyl dihydroxylated elongatinoid selected from the group consisting of formula S, T, U or V:
wherein: m may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group, and wherein: compounds S and T each have a total of 23 to 42 carbon atoms in the carbon chain, 3 cis-carbon double bonds at the positions where n-3, n-15 and n-18 begin, 2 trans-carbon double bonds at the positions where n-9 and n-11 begin and a carbon-carbon triple bond at the position n-7 begin; and compounds U and V each have a total of 23 to 42 carbon atoms in the carbon chain, 2 cis-carbon double bonds starting at positions n-3 and n-15, 2 trans-carbon double bonds starting at positions n-9, n-11 and a carbon-carbon triple bond starting at position n-7.
In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of diastereomers S and T, wherein the diastereomers have (S) or (R) chirality at position n-6 and (R) chirality at position n-13.
In some embodiments of this aspect of the disclosure, the composition can include equimolar amounts of one or more diastereomers S and T, wherein the diastereomer has (S) or (R) chirality at position n-6 and (S) or (R) chirality at position n-13.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of S or T in an amount that exceeds the amount of the other diastereomer of S or T.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of diastereomers U and V, wherein the diastereomer has (S) or (R) chirality at position n-6 and (R) chirality at position n-13.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of one or more diastereomers U and V, wherein the diastereomer has an (S) or (R) chirality at position n-6 and an (S) or (R) chirality at position n-13.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of U or V in an amount that exceeds the amount of the other diastereomer of U or V.
In certain advantageous embodiments, the present disclosure provides an effective amount of at least one provided compound and/or provided composition to exert effective neuroprotective, tissue protective, and neurorestorative effects suitable for neuroprotection, organ protection, and tissue protection.
In other embodiments, the present disclosure provides compounds and dermatological or cosmetic compositions for effectively protecting, preventing, or treating skin that has been damaged by sunlight or other causes or that has been affected by skin aging. The compounds, compositions, and methods provided induce the survival and normal function of skin cells, protect the skin, improve skin health and appearance, and delay skin aging.
Drawings
The present disclosure focuses on compounds, compositions and methods for application in skin diseases, retinal diseases, cardiovascular diseases, gastrointestinal/liver diseases and brain diseases. The unique structure, biosynthesis, and function of the provided compounds and compositions were initially studied in the brain and retina, as summarized in the following figures. Further aspects of the present disclosure will be readily appreciated when the detailed description of the various embodiments of the present disclosure described below is reviewed in conjunction with the figures.
FIG. 1 is a scheme showing the putative biosynthesis of elongin-like (ELV) from omega-3 (n-3 or n3) very long chain polyunsaturated fatty acids (n3 VLC-PUFA).
FIG. 2 is a scheme showing the putative biosynthesis of n3 VLC-PUFA.
FIGS. 3A-3K show the production of the elongationlike ELV-N32 and ELV-N34 from primary human retinal pigment epithelial cells (RPE) in culture and structural characterization thereof.
FIG. 3A is a scheme showing the synthesis of ELV-N32 and ELV-N34 from intermediates (1, 2, and 3), each of which is prepared in stereochemically pure form. The stereochemistry of intermediates 2 and 3 was predefined by using enantiomerically pure epoxide starting materials. The final ELV (4) was assembled via iterative coupling of intermediates 1,2, and 3, and was isolated as the methyl ester (Me) or sodium salt (Na).
FIG. 3B shows elution profiles of C32:6N3, endogenous monohydroxy-C32: 6N3, and ELV-N32 shown with ELV-N32 standard. The MRM of ELV-N32 shows two large peaks eluting earlier than the peak eluting with standard ELV-N32, which show the same cleavage pattern (shown in the inset), indicating that they are isomers.
FIG. 3C shows the full subscanning chromatograms of ELV-N32 and ELV-N34.
FIG. 3D shows the cleavage pattern of ELV-N32.
FIG. 3E shows the elution profiles of C34:6N3 and ELV-N34.
FIG. 3F shows a UV spectrum of endogenous ELV-N34, which exhibits triene characteristics similar to NPD1, with λ max at 275nm and shoulders at 268 and 285 nm.
FIG. 3G shows the cleavage pattern of ELV-N32.
FIG. 3H shows the total lytic profile of endogenous ELV-N32.
Figure 3I shows the ELV-N32 standard, which shows that all major peaks from the standard match the endogenous peaks. However, endogenous ELV-N32 has more fragments not shown in the standard, indicating that it contains different isomers.
FIG. 3J shows a full lytic profile of endogenous ELV-N34 matched to the standard ELV-N34 peak.
FIG. 3K shows the presence of the ELV-N34 isomer.
FIGS. 4A-4K show structural characterization of the elongationlike ELV-N32 and ELV-N34 from neuronal cell cultures. Cortical mixed neurons were incubated with 32:6n3 and 34:6n3 (10 μ M each) under OGD conditions.
FIG. 4A is a scheme showing the synthesis of ELV-N32 and ELV-N34 from intermediates (a, b, and c), each of which is prepared in stereochemically pure form. The stereochemistry of intermediates b and c is predefined by using enantiomerically pure epoxide starting materials. The final elv (d) was assembled via iterative coupling of intermediates a, b, and c, and was isolated as the methyl ester (Me) or sodium salt (Na).
FIG. 4B shows in inset 32:6N3, endogenous monohydroxy-32: 6, ELV-N32, and ELV-N32 standards. The MRM of ELV-N32 showed that two large peaks eluted earlier than the peak eluted with standard ELV-N32, but they showed the same cleavage pattern, indicating that they are isomers.
FIG. 4C shows that the same features as in FIG. 4A are exhibited in 34:6N3 and ELV-N34.
FIG. 4D shows a UV spectrum of endogenous ELV-N32, which exhibits triene characteristics, but these are not determined at this concentration.
FIG. 4E shows the total lytic profile of endogenous ELV-N32.
FIG. 4F shows a UV spectrum of endogenous ELV-N34, which exhibits triene characteristics similar to NPD1, with λ max at 275nm and shoulders at 268 and 285 nm.
FIG. 4G shows the cleavage pattern of endogenous ELV-N34.
FIG. 4H shows the global cleavage pattern of endogenous ELV-N32.
FIG. 4I shows an ELV-N34 standard, which shows that all major peaks from the standard match the endogenous peaks, but do not match completely; endogenous ELV-N34 has more fragments not shown in the standard, indicating that it may contain isomers.
FIG. 4J shows an ELV-N34 total lysis map; endogenous ELV-N34 matched with standard ELV-N34 peak
FIG. 4K shows the presence of the implied ELV-N34 isomer.
FIGS. 5A and 5B show the detection of ELV-N32 and ELV-N34 in neuronal cell cultures. Cells were incubated with C32:6n3 and C34:6n3 (5. mu.M each) under OGD conditions.
FIG. 5A shows VLC-PUFA C32:6N3, endogenous 27-hydroxy-32: 6N3, endogenous 27, 33-dihydroxy-32: 6N3(ELV-N32), and synthetic ELV-N32 prepared in a stereochemically pure form via a stereocontrolled, all-organic synthesis. The MRMs of endogenous ELV-N32 closely match the MRMs of synthetic ELV-N32 standards.
FIG. 5B shows that the same features as in FIG. 5A are exhibited in C34:6N3 and ELV-N34, with more peaks in the ELV-N34MRM, suggesting possible isomers.
FIG. 6A shows confocal images of immunostaining of primary human RPE cells with the specific markers ZO-1 (occludin-1), RPE65, MITF (microphthalmia-associated transcription factor), and β -catenin.
Fig. 6B shows light microscopy images depicting primary human RPE cell morphology at different passages in culture. Scale up, 50 μm.
FIGS. 7A-7L show cytoprotection by 32:6n3 and 34:6n3 in human RPE cells under UOS.
FIG. 7A shows the concentration-dependent anti-apoptotic activity of C32:6n3 and C34:6n3 in human RPE cells (ARPE-19 cells). Confluent (80%) ARPE-19 cells in 12-well plates were serum starved for 8 hours, induced to UOS, and then challenged with 50-500nMC32:6n3 or 34:6n3 free acid for 16 hours. Treated cells were harvested and Hoechst positive pycnotic cells were detected. Data are the average of 15-well counts of Hoechst positive pycnotic cells from three independent experiments.
FIG. 7B shows a comparison of DHA (100nM) and 32:6n3 and 34:6n3 (250nM each) for 16 hours of cytoprotection. UOS was introduced into serum-starved ARPE-19 cells and apoptotic cell death was detected as described in fig. 7A. Results are the average of three independent experiments.
FIG. 7C shows SIRT1 upregulation by C32:6n3 and C34:6n3 in RPE under UOS. The effect of PD146176, a 15-LOX-1 inhibitor, on C32:6n3 and C34:6n3 induced upregulation of SIRT1 in RPE under the influence of UOS. Unless otherwise stated, results are the average of three independent experiments (9 wells per experiment).
FIG. 7D shows that 15-LOX-1 inhibitor PD146176 attenuates C32:6n3 or C34:6n 3-induced Iduna upregulation in RPE cells under UOS.
FIGS. 7E-7I show the effect of C32:6n3 or C34:6n3 against apoptotic and pro-apoptotic proteins in ARPE-19 cells under UOS. Western blot assay of the up-and down-regulation effects of C32:6n3 and C34:6n3 on the above proteins in ARPE-19 cells under UOS.
FIG. 7E shows the effect of C32:6n3 or C34:6n3 on the anti-apoptotic protein Bcl-2.
FIG. 7F shows the effect of C32:6n3 or C34:6n3 on anti-apoptotic proteins Bc-xL.
Figure 7G shows the effect of C32:6n3 or C34:6n3 on pro-apoptotic protein Bax.
Figure 7H shows the effect of C32:6n3 or C34:6n3 on pro-apoptotic protein Bim.
Figure 7I shows the effect of C32:6n3 or C34:6n3 on pro-apoptotic protein Bid.
FIG. 7J shows concentration-dependent (100 and 250nM) upregulation of inhibin (type 1) by C32:6n3 and C34:6n3 in RPE cells under UOS.
FIG. 7K shows the effect of NPD1(200nM), C32:6n3, or C34:6n3(3 μ M) on primary human RPE cell survival.
Fig. 7L shows the cytoprotection of primary human RPE cells by C32:6n3 or C34:6n3 in the presence of 10 μ M PD 146176. Error bars, SEM; p < 0.05.
FIG. 8A (panels a-d) shows that VLC-PUFA 32:6n3 and 34:6n3 improve Uncompensated Oxidative Stress (UOS) -induced death of primary human RPE cells. In a figure: untreated (control) RPE cells; and (b) figure: UOS 16h RPE cells; and (c) figure: UOS RPE cells at 16h +32:6n 3; FIG. d: UOS 16 hr +34:6n3 RPE cells. Cell death was prevented when either 32:6n3 or 34:6n3 was added (panels c and d). Typical regions of cell culture are presented in the right panel of each figure. Nuclei were marked by Hoechst staining and dead cells highlighted. These are separated using an intensity threshold algorithm and counted using Image J macros (left column).
Fig. 8B shows quantification of live cells (control cells) and dead cells (UOS cells) based on nuclear size. Error bars, SEM; p < 0.05.
FIG. 8C shows that cell death was prevented when either 32:6n3 or 34:6n3 was added.
FIGS. 9A-9C show that 15-LOX-1 inhibitor was unchanged on primary human RPE cells by 32:6n3 and34:6n3 mediated cytoprotection against UOS. Human RPE cells were incubated with 15-lipoxygenase-1 (15-LOX-1) inhibitor (10. mu. mol, PD146176) for 1 hour in serum deprivation (FIG. 9A, FIGS. a and b) and low serum (FIG. 9A, FIGS. c and d), and then subjected to oxidative stress (H)2O2TNF α) for 16 hours to induce apoptosis (FIG. 9A, FIGS. a-d, FIG. 9C).
The addition of 32:6n3 and 34:6n3 protected human RPE cells (fig. 9A, fig. b and d, fig. 9C) from cell death. Typical regions of cell culture are presented in FIG. 9A, panels a-d, right columns. Nuclei were marked by Hoechst staining and dead cells highlighted. These were separated using an intensity threshold algorithm and counted using Image J macros (fig. 9A, panels a-d, left column).
Fig. 9B shows quantification of live cells (control cells) and dead cells (UOS cells) based on nuclear size. Error bars, SEM; p < 0.05.
FIGS. 10A-10H show that ELV-N32 and ELV-N34 enhance the abundance of homeostatic proteins and decrease the abundance of cell injury proteins in RPE cells under UOS.
FIG. 10A shows concentration-dependent (100 and 250nM) upregulation of SIRT1 in ARPE-19 cells under UOS. Results are the average of three independent experiments.
FIG. 10B shows the effect of PD146176 in RPE cells under UOS on the Iduna upregulation induced by ELV-N32 and ELV-N34.
FIG. 10C shows the cytoprotective ability of ELV-N32 and ELV-N34 in RPE cells under UOS. Effect of lipoxygenase inhibitors on apoptosis inhibition.
FIG. 10D shows the effect of ELV-N32Na or ELV-N32 Me against the apoptotic proteins Bcl-2, Bcl-xL and the pro-apoptotic protein Bax in ARPE-19 cells under UOS.
FIG. 10E shows a comparison of the effect of ELV-N32Na or ELV-N32 Me and ELV-N34 Na or ELV-N34Me in RPE under UOS on the induction of pro-apoptotic protein bim (E).
FIG. 10F shows a comparison of the effect of ELV-N32Na or ELV-N32 Me and ELV-N34 Na or ELV-N34Me in RPE under UOS on the induction of pro-apoptotic protein bid (F).
FIG. 10G shows the concentration-dependent decrease of ELV-N32Na or ELV-N34Me in ARPE-19 cells on UOS-induced apoptosis (50, 100, 250, and 500 nM).
FIG. 10H shows that the up-regulation of inhibin (type 1) by ELV-N32 or ELV-N34 in ARPE-19 cells under UOS depends on concentration (100 and 250 nM). Error bars, SEM; p < 0.05.
FIGS. 11A-11D show that genetic ablation of adiponectin receptor 1 results in the consumption of VLC-PUFA and derivatives thereof in the retina.
Fig. 11A shows that dietary DHA or dietary DHA derived from diet 18:3n3 was provided by the liver and captured by AdipoR1, subsequently extended to VLC-PUFAs in the inner segment of PRC by ELOVL4 and incorporated into phosphatidylcholine molecular species (which also contains DHA). During the daily renewal of the PRC outer segment, these phosphatidylcholine molecular species interact with rhodopsin and become part of the RPE cells after shedding and phagocytosis. UOS or other homeostatic disruptors trigger the release of VLC-PUFA. It is depicted that C32:6N3 and C34:6N3 produce hydroperoxy forms, followed by ELV-N32 or ELV-N34, respectively.
FIG. 11B shows a reduction in pool size of free C32:6n3 in the retina of an AdipoR1 Knockout (KO) mouse compared to that in WT. Inset 1 shows ELV-N32 in KO and Wild Type (WT); inset 2 shows the absence of detectable signal in monohydroxy 32:6N3 (a stable derivative of hydroperoxy precursor of ELV-N32) in WT, as well as in KO.
FIG. 11C shows a similar pool size reduction in free 34:6n3 in the retina of an AdipoR1 KO mouse compared to that in WT. Inset 1 shows ELV-N32 in KO and WT; inset 2 shows the monohydroxy group C34:6N3 (stable derivative of hydroperoxy precursor of ELV-N34) in WT and the lack of detectable signal in KO.
Fig. 11D shows RPE cells maintaining PRC functional integrity (left); right, ablation of adiponectin receptor 1 (AdipoR1) turns off DHA availability with consequent PRC degeneration.
Figures 12A and 12B show that the elongatinoids of the present disclosure act as neuroprotection and maintain photoreceptor cell integrity in retinal degeneration.
Figure 12A shows that the elongin-like pathway is reduced in pluripotent stem cell iPSC-RPE derived from a family affected by late onset retinal degeneration (L-ORD). The disease is due to a mutation in CTRP5 (S163R). Pluripotent stem cells (ipscs) from L-ORD patients and unaffected siblings that differentiated into retinal pigment epithelial cells (RPEs) were used. Cells were incubated in serum-free medium and fed at approximately 5 photoreceptor extracellular segments (POS) per cell for 4 hours to summarize shedding and disc phagocytosis, or1 μ M32: 6 and 34:6 free fatty acids (VLC-PUFA) in medium containing 0.5% serum for 24 hours. Media and cell lysates were collected for LC-MS/MS-based metabolomics analysis. Metabolic lipidomics analysis showed that either POS or VLC-PUFA fed with iPSC-RPE secreted stable elongationlike biosynthetic intermediates (27 s-hydroxy 32:6n-3 and 29 s-hydroxy 34:6n-3) predominantly in a polarized fashion on the apical side of the cell. The control iPSC-RPE secreted significantly more of the elongin-like ELV-N32 and ELV-N34 than the patients.
FIG. 12B shows that 27 s-hydroxy 32:6n-3 was still detected in the control but not in patient iPSC-RPE in the absence of POS or VLC-PUFA. Likewise, the DHA-derived lipid mediator neuroprotective D1(NPD1) was found to be secreted in significantly higher amounts in the control (p < 0.05). Since NPD1 and the elongatinoid are derived from the same phospholipid precursor, a lack of both lipid mediators may occur in L-ORD.
FIGS. 13A-13D show an in vitro model of oxygen-sugar deprivation (OGD) in primary cortical neurons. Omega-3 VLC-PUFAs and elongatoids are released locally in response to neuronal stress and provide protection of cortical neurons exposed to oxygen-sugar deprivation.
Figure 13A shows that OGD media has more free fatty acid (FA32:6 and FA34:6) release than control (no OGD) media.
FIG. 13B shows that 27-S-hydroxy 32:6 was detected in OGD medium (red), but the control medium had a negligible amount of 27-S-hydroxy 32:6 (blue). A sub-scan of ELV-N32 is shown (in the inset), which presents a possible ELV-N32 peak.
FIG. 13C shows that 29-S-hydroxy 34:6 was detected in OGD medium, but the control medium had negligible amounts of 29-S-hydroxy 34: 6.
FIG. 13D shows the full cleavage pattern of 27-S-hydroxy 32:6 that closely matches the theoretical values of possible sub-molecules.
Figures 14A-14C show that the elongatinoids repress key factors that accelerate cell damage and cell death (cellular senescence), resulting in the delay of age-related cell death and related diseases and conditions, thereby extending human life.
Figure 14A shows that the elongatinoid suppresses activation of the senescence signal transduction pathway.
Figure 14B shows that the elongatinoids activate autophagy by inducing expression of autophagy factors (ATG3, ATG5, ATG7, Beclin-1), resulting in autophagy clearance and removal of oligomeric a β peptides, thereby suppressing cellular senescence progression. Autophagy involves more than 100 genes. The following genes are elevated in AMD disease:
FIG. 14C shows that elongatinoids decrease the gene expression of pro-inflammatory factors (CHF, IL-1. beta.) induced by oligomeric A.beta.peptides.
FIGS. 15A-15L show that ELV-N32 and ELV-N34 elicit protection of mixed cortical neuronal cultures exposed to oxygen deprivation (OGD) or NMDA excitotoxicity.
Figure 15A shows bright field images (10X) of cerebral cortex and hippocampal neurons in cultures exhibiting morphology.
Figure 15B shows representative immunofluorescence images of cerebral cortical neurons (DIV12) in β III tubulin, GFAP, and Hoechst stained in vitro 12 day cultures.
FIGS. 15C and 15F show exposure to NMDA (50. mu.M) excitotoxicity (FIG. 15C) or OGD (FIG. 15F) and neuroprotective cerebral cortical neurons elicited by ELV-N32-Na or ELV-N34-Me (500nM concentration), as assessed by fixation and staining of cells with Hoechst 33258. (p <0.0001, p <0.001, and p <0.05, n-9, one-way ANOVA, followed by Holm-Sidak multiple comparison test).
Fig. 15D and 15G show unbiased image analysis methods for calculating frequency distributions of Hoechst positive nuclei and both pycnotic and non-pycnotic nuclei in the presence of NMDA (fig. 15D) or OGD (fig. 15G), respectively.
FIGS. 15E and 15H show representative images showing thresholding and size exclusion of Hoechst positive nuclei challenged with NMDA (FIG. 15E) or OGD stress (FIG. 15H), respectively.
FIGS. 15I-15L show neuroprotection elicited by ELV-N32-Na or ELV-N34-Me following exposure to NMDA (FIGS. 15I and 15J) or OGD (FIGS. 15K-15L), as assessed by calcein positive cell counts.
FIGS. 16A-16I show that ELV-N32 and ELV-N34 induced protection of cortical mixed and hippocampal cultures exposed to uncompensated oxidative stress, oxygen deprivation (OGD) or NMDA excitotoxicity.
FIG. 16A shows cell survival after exposure of mixed cortical neurons in culture to NMDA (100 μ M) excitotoxicity in the presence of the uncompetitive NMDA receptor antagonists MK801 maleate (dezocyclopine) (10 μ M) or neuroprotective D1(NPD1) (100nM) or ELV-N32-Na (200nM) or ELV-N32-Me (200nM), as assessed by the MTT assay. (# p <0.001, n ═ 6, one-way ANOVA, then Holm-Sidak multiple comparison test).
FIGS. 16B and 16C show the exposure of cortical neurons to TNF α (10ng/ml) plus H, respectively2O2Neuroprotective effects of ELV-N32-Na or ELV-N32-Me (200nM concentration) following uncompensated oxidative stress or NMDA excitotoxicity (25. mu.M, 50. mu.M or 100. mu.M) (FIG. 16B) (FIG. 16C). Cell survival was assessed by unbiased image analysis and Hoechst positive nuclear counts. (# p)<0.0001, and p<0.05, n-9, one-way ANOVA, followed by Holm-Sidak multiple comparison test).
FIGS. 16D and 16E show cell survival assessed by Hoechst staining (FIG. 16D) and MTT assay (FIG. 16E) following OGD exposure in the presence of ELV-N32-Na, ELV-N32-Me, ELV-N34-Na, or ELV-N34-Me (1 μ M concentration). (# p <0.0001, # p <0.001, and # p <0.05, n-9, one-way ANOVA, then Holm-Sidak multiple comparison tests).
FIGS. 16F and 16G show neuroprotection in hippocampal neurons (DIV12) (FIG. 16F) or cortical neurons (DIV12) (FIG. 16G) in culture assessed by Hoechst positive cell counts after OGD stress in the presence or absence of ELV-N32-Na or ELV-N34-Me (500nM concentration). When added at 500nM concentration, 32:6 or 34:6 also showed neuroprotection.
FIGS. 16H and 16I show cortical mixed neurons (DIV 28) exposed to cultures of NMDA (50. mu.M) (FIG. 16H) or OGD (FIG. 16I) in the presence or absence of ELV-N32-Na or ELV-N34-Na or 32:6 or 34:6(500nM) (assessed by Hoechst staining and cell counting).
FIG. 17 shows cell survival assessed by Hoechst positive nuclear counting and unbiased image analysis after exposure of cortical neurons (DIV12) in culture to NMDA (50 μ M) (panels A-C) or OGD (panels E-G), respectively, in the presence of ELV-N32-Na or ELV-N32-Me (500 nM). Results from three separate experiments. (# p <0.0001, # p <0.001, and # p <0.05, n-9, one-way ANOVA, then Holm-Sidak multiple comparison tests).
32:6(250nM) can attenuate NMDA excitotoxicity (panel D), while 34:6(250nM) elicits neuroprotection (# p <0.0001, and p <0.001) to cortical neurons (DIV 28) in cultures exposed to OGD (panel H).
FIGS. 18A-18I show that ELV-N32 and ELV-N34 elicited protection against cultures of cortical mixed neurons or hippocampal mixed neurons exposed to oxygen deprivation (OGD) or Uncompensated Oxidative Stress (UOS).
Fig. 18A shows a representative image of cortical mixed neuron cultures (DIV12) challenged with 90 min OGD. Fixation and staining of cells with Hoechst 33258 after 12 hours of treatment with ELV-N32Na or ELV-N34Me (500nM concentration) showed both pytosynsis by OGD and neuroprotection triggered by ELV-N32Na or ELV-N34 Me.
Fig. 18B shows a summary of the data from fig. 18A (×) p <0.0001, and × <0.001, n ═ 9, one-way ANOVA, followed by Holm-Sidak multiple comparison tests.
FIGS. 18C and 18D show unbiased image analysis methods for calculating% relative frequency distribution of Hoechst positive nuclei and pycnotic and non-pycnotic nuclei in the presence of OGD + ELV-N32Na (FIG. 18C) or OGD + ELV-N34Me (FIG. 18D), respectively. When subjected to OGD stress, cells undergo pyknosis, as shown by the left shift in the nuclear peak. There was a positive right shift towards the control nuclear population peak after treatment with ELV-N32Na or ELV-N34Me, indicating that these new lipid mediators triggered cell survival. The kernel size limits for defining the pycnotic and non-pycnotic kernels are indicated by the black dashed lines and highlighted by rectangles.
Figure 18E shows neuroprotection elicited by ELV-N32Na or ELV-N34Me following exposure of mixed cortical neuronal cultures (DIV12) challenged with 90 min OGD as assessed by calcein positive cell counts (× p <0.0001, N ═ 3, one-way ANOVA, followed by Holm-Sidak multiple comparative tests).
FIG. 18F shows cell survival after exposure of 90 min OGD challenge cerebral cortical mixed neuron cultures (DIV12) and subsequent treatment with ELV-N32 Me, ELV-N32Na, ELV-N34Me, or ELV-N34 Na (1 μ M concentration), as assessed by MTT assay. (# p <0.0001, # p <0.001, and # p <0.05, n-9, one-way ANOVA, then Holm-Sidak multiple comparison tests).
FIGS. 18G and 18H show neuroprotection elicited by ELV-N32Na, ELV-N34 Na, 32:6 or 34:6 after subjecting hippocampal mixed neuron cultures (DIV12) to OGD stress in the presence or absence of ELV-N32Na or ELV-N34Me (500nM concentration), as assessed by unbiased image analysis followed by Hoechst positive nuclear counts.
When added at 500nM, 32:6 or 34:6 also exhibited neuroprotection (# p <0.0001, n-3, one-way ANOVA, followed by Holm-Sidak multiple comparison testing) (fig. 18G) or cortical mixed neuron culture (DIV 28) (# p <0.0001, p <0.001, and p <0.05, n-3, one-way ANOVA, followed by Holm-Sidak multiple comparison testing) (fig. 18H) were each subjected to 90 min OGD.
FIG. 18I shows that cortical mixed neuron cultures (DIV12) were subjected to TNF α (10ng/mL) and H2O2Addition of (50. mu.M, 100. mu.M or 200. mu.M) induced 12 hNeuroprotection elicited by ELV-N32Na or ELV-N34 Na (200nM concentration) following Uncompensated Oxidative Stress (UOS), as assessed by Hoechst positive nuclear counting followed by unbiased image analysis (# p)<0.0001, and p<0.001, n-9, one-way ANOVA, followed by Holm-Sidak multiple comparison test).
FIGS. 19A-19H show ELV-N32 and ELV-N34 induced protection of mixed cortical neuronal cultures exposed to NMDA excitotoxicity.
Fig. 19A shows representative images of cerebral cortical mixed neuron cultures (DIV12) subjected to 12-hour NMDA excitotoxicity. Fixation and staining of cells with Hoechst 33258 12 hours after treatment with either ELV-N32Na or ELV-N34Me (500nM concentration) and NMDA (100 μ M) indicated both pyknosis due to NMDA excitotoxicity and neuroprotection triggered by ELV-N32Na or ELV-N34 Me.
Fig. 19B shows a summary of data from (fig. 19A) (. x.p <0.0001, and. x.p <0.05, n-9, one-way ANOVA, followed by Holm-Sidak multiple comparison tests).
FIGS. 19C and 19D show unbiased image analysis methods for calculating% relative frequency distribution of Hoechst positive nuclei and solid-condensed nuclei versus non-solid-condensed nuclei in the presence of NMDA + ELV-N32Na (FIG. 19C) or NMDA + ELV-N34Me (FIG. 19D), respectively. When cells are subjected to NMDA excitotoxicity, they undergo a pyknosis, as shown by the left shift in the nuclear peak. After treatment with ELV-N32Na or ELV-N34Me, there was a positive right shift to the control nuclear population peak, indicating cell survival triggered by these classes of elongatins. The kernel size limits for defining the pycnotic and non-pycnotic kernels are indicated by dashed lines and highlighted by rectangles.
Figure 19E shows neuroprotection elicited by ELV-N32Na or ELV-N34Me following exposure to mixed cortical neuronal cultures (DIV12) challenged with 12-hour NMDA (100 μ M concentration), as assessed by calcein positive cell counts (p <0.0001, N-3, one-way ANOVA followed by Holm-Sidak multiple comparative tests).
FIG. 19F shows cell survival following exposure of cerebral cortical mixed neurons (DIV12) in culture to NMDA (100 μ M) excitotoxicity in the presence of the uncompetitive NMDA receptor antagonists MK801 maleate (dezocine) (10 μ M) or ELV-N32-Na (200nM) or ELV-N32-Me (200nM), as assessed by the MTT assay. (# p <0.001, n ═ 6, one-way ANOVA, then Holm-Sidak multiple comparison test).
FIG. 19G shows neuroprotective effects of ELV-N32Na or ELV-N32 Me (200nM concentration) after exposure of mixed cortical neurons (DIV12) to NMDA excitotoxicity (25. mu.M, 50. mu.M, or 100. mu.M). Cell survival was assessed by unbiased image analysis and Hoechst positive nuclear counts. (# p <0.0001, and # p <0.05, n-9, one-way ANOVA followed by Holm-Sidak multiple comparison test).
FIG. 19H shows cortical mixed neurons (DIV 28) exposed to NMDA (50 μ M) in culture in the presence or absence of ELV-N32Na or ELV-N34 Na or 32:6 or 34:6(500nM), assessed by Hoechst staining and cell counts. (# p <0.0001, n-3, one-way ANOVA followed by Holm-Sidak multiple comparison test).
Figures 20A-20D show that ELV-N32 and ELV-N34 improve nerve/behavior scores, preserve penumbra, and reduce MRI lesion volume after ischemic stroke.
Figure 20A shows total neurological scores (normal score 0, max score 12) during MCAo (60 min) and at various times after treatment. All animals scored 11 (possibly 12) at 60 minutes MCAo. Compared to vehicle (cerebrospinal fluid; CSF) treated group, the neuroscore of the elongin-like treated rats was significantly improved at days 1, 3 and 7.
Figure 20B shows that the elongin-like treatment significantly reduced ischemic core, penumbra, and total lesion volume calculated from day 7T 2WI compared to vehicle group.
Fig. 20C and 20D show representative T2WI, pseudo-images, core/penumbra (fig. 20C), and (fig. 20D) calculated 3D infarct volumes from T2WI at day 7. The core and penumbra were extracted from the entire brain. The core and penumbra tissues were automatically identified in vehicle-treated and extensin-like treated animals using a computational MRI method (hierarchical segmentation method for penumbra identification). High intensity of T2 was observed in the ischemic core and penumbra of vehicle treated rats, consistent with edema formation. In contrast, the paraelongin-treated animals had smaller lesion sizes. 3D reconstruction of the same animals in each group from day 7. Values shown are mean ± SD (n-5-6 per group). P <0.05 compared to CSF group (ANOVA was repeated and Bonferroni test was then performed).
FIGS. 21A-20C show that ELV-N32 and ELV-N34 reduced neuronal and astrocytic injury induced by experimental ischemic stroke.
FIGS. 21A and 21B show representative NeuN, SMI-71 and GFAP stained brain sections from all groups. Vehicle (cerebrospinal fluid; CSF) treatment of rats showed extensive neuronal loss, GFAP-reactive astrocytes and SMI-71 positive vascularisation. In contrast, treatment with the elongatinoid increased NeuN, SMI-71 and GFAP positive cells.
FIG. 21C shows a coronary brain map (bregma +1.2mm) showing the location of regions of NeuN, SMI-71 and GFAP positive cell counts in the cortex (columns a, b and C) and striatum (column s). In the ischemic core region (S) and the different penumbra regions (a, b and c), the elongatinoid treatment increased the number of NeuN-positive neurons, SMI-positive vessels and GFAP-positive astrocytes. Values shown are mean ± SD (n-5-6 per group). Significant differences from vehicle (p < 0.05; repeated measures ANOVA followed by Bonferroni test).
FIGS. 22A-22D show that ELV-N32 and ELV-N34 reduce NVU damage and reduce cerebral infarction following ischemic stroke.
Figure 22A shows NVU breakdown assessed by immunoassay of immunoglobulin g (igg) within parenchyma tissue. IgG staining indicated NVU breakdown. Vehicle (cerebrospinal fluid; CSF) treatment of rats showed an increase in IgG immunoreactivity in the cortex and subcortical. Treatment with ELV-N34-Na or ELV-N34-Me showed less IgG staining in the cortex and was mostly located in the infarcted core (subcortical).
Fig. 22B shows a bar graph showing that ELV-N34-Na and ELV-N34-Me significantly reduced (total) IgG immunoreactivity in the cortex, subcortical, and throughout the hemisphere. Values shown are mean ± SD (n-5-6 per group). P <0.05 compared to vehicle group (repeated measures ANOVA followed by Bonferroni test).
Figure 22C shows nissl stained brain sections from rats treated with vehicle and elongatinoid. Vehicle treatment of rats showed greater cortical and subcortical infarctions. In contrast, rats treated with the elongatinoid showed less extensive damage, mainly in the subcortical region.
Figure 22D shows infarct volumes after cortical, subcortical, and total correction. All ELV treatments significantly reduced cortical, subcortical, and total infarct volume compared to vehicle treated groups. Values shown are mean ± SD (n-5-6 per group). Significant differences from vehicle (p < 0.05; repeated measures ANOVA followed by Bonferroni test).
Figures 23A-23C show that the elongatinoid provides neuroprotection and ameliorates neurological deficit following Traumatic Brain Injury (TBI). Total neurological score decreased significantly following treatment with 32-carbon or 34-carbon elongatins methyl or sodium salt. Fig. 23A and 23C: delivered via intravenous injection; FIG. 23B: delivered via epidural injection. SD rats were subjected to a moderate hydraulic impact injury (FPI) model and treated with the Methyl Ester (ME) or sodium salt (Na) forms of the 32 and 34 carbon classes of elongatins (ELV-N32-ME, ELV-N34-ME, ELV-32-Na-d2, ELV-N34-alkyne) or vehicle (cerebrospinal fluid; CSF) (1 μ g per rat). Treatment was delivered into the subdural space 1 hour after TBI. Animals were examined for neurobehavioral tests on days 1,2, 3, 7 or 14 (normal 0, max defect 12). All ELV treatments improved neurological scores from day 1, which persisted throughout the 2-week survival compared to the CSF treated group. Values shown are mean ± SEM (n-6-8 per group), with significant differences from the corresponding saline group (P < 0.05; ANOVA was repeatedly measured, followed by Bonferroni testing). ELV-N34-alkyne (IV, 300. mu.g/animal). Neurobehavioral tests were performed on all animals on days 1,2, 3, 7 or 14 (normal 0, max defect 12).
Fig. 24 shows scheme 1 for the total synthesis of monohydroxylated elongatins G, H, I, J, O, P, Q, R.
Reagents and conditions: (a) catechol borane, addHeating; (b) n-iodo-succinimide, MeCN; (c) 4-chlorobut-2-yn-1-ol, Cs2CO3，NaI，CuI，DMF；(d)CBr4，PPh3，CH2Cl2At 0 ℃ C; (e) ethynyl-trimethylsilane, CuI, NaI, K2CO3DMF; (f) lindla catalyst, H2， EtOAc；(g)Na2CO3，MeOH；(h)Pd(PPh3)4，CuI，Et3N；(i)tBu4NF, THF; (j) lindla catalyst, H2EtOAc or Zn (Cu/Ag), MeOH; (k) NaOH, THF, H2O, then with HCl/H2Acidifying O; (I) NaOH, KOH, etc., or amines, imines, etc.
FIG. 25 shows scheme 2 for the total synthesis of dihydroxylated elongatins K, L, S and T.
Reagents and conditions: (a) CuI, NaI, K2CO3DMF; (b) camphorsulfonic acid (CSA), CH2CI2MeOH, room temperature; (c) lindla catalyst, H2，EtOAc；(d)DMSO，(COCl)2，Et3N， -78℃；(e)Ph3P ═ CHCHO, PhMe, reflux; (f) CHI3，CrCl2THF, 0 ℃; (g) catalyst Pd (Ph)3)4CuI, PhH, room temperature; (h)tBu4NF, THF, room temperature; (i) zn (Cu/Ag), MeOH, 40 ℃; (j) NaOH, THF, H2O, then with HCl/H2Acidifying O; (k) NaOH, KOH, etc., or amines, imines, etc.
FIG. 26 shows scheme 3 for the total synthesis of dihydroxylated elongatins M, N, U and V.
Reagents and conditions: (a) cyanuric chloride, Et3N, acetone, room temperature; (b) (3-Methyloxyoxetan-3-yl) methanol, pyridine, CH2Cl2，0℃；(c)BF3.OEt2，CH2Cl2；(d)nBuLi，BF3.OEt2THF, -78 ℃, then 1; (e)tBuPh2SiCI, imidazole, DMAP, CH2Cl2Room temperature; (f) camphorsulfonic acid, CH2Cl2ROH, room temperature; (g) lindla catalyst, H2，EtOAc；(h)DMSO， (COCl)2，Et3N，-78℃；(i)Ph3P ═ CHCHO, PhMe, reflux; (j) CHl3，CrCl2THF, 0 ℃; (k) catalyst Pd (Ph)3)4CuI, PhH, room temperature; (I)tBu4NF, THF, room temperature; (m) Zn (Cu/Ag), MeOH, 40 ℃; (n) NaOH, THF, H2O, then with HCl/H2Acidifying O; (o) NaOH, KOH, etc., or amines, imines, etc.
FIG. 27 shows scheme 4 for the total synthesis of 32-carbodihydroxylated elongatins.
FIG. 28 shows scheme 5 for the total synthesis of 34 carbon-dihydroxylated-type elongatins.
Detailed Description
Before the present disclosure is described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the advantageous methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and were set forth in its entirety herein to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
It will be apparent to those skilled in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any described method may be performed in the order of events described or in any other order that is logically possible.
Unless otherwise indicated, the embodiments of the present disclosure will employ techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, toxicology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings unless a clearly contradictory intention exists.
As used herein, the following terms have the meanings assigned to them unless otherwise indicated. In the present disclosure, "include (comprising)", "contain (containing)" and "have (having)" and the like may have meanings given to them in the us patent law, and may mean "include (including)" and the like. When applied to the methods and compositions encompassed by the present disclosure, "consisting essentially of … … (consistent essentiality of/consistent sesssentiality)" and the like, refers to compositions similar to those disclosed herein, but may contain additional structural groups, composition components, or method steps (or analogs or derivatives thereof as discussed above). However, such additional structural groups, composition components, method steps, or the like do not materially affect the basic and novel characteristics of the compositions or methods, as compared to those of the corresponding compositions or methods disclosed herein.
Before describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.
Definition of
As used herein, the nomenclature alkyl, alkoxy, carbonyl, and the like are used, as is commonly understood by those skilled in the chemical arts. As used in this specification, an alkyl group (group) may include straight, branched and cyclic alkyl groups (radics) containing up to about 20 carbons or1 to 16 carbons and be straight or branched. Alkyl groups herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, and isohexyl.
As used herein, lower alkyl refers to carbon chains having from about 1 or about 2 carbons to about 6 carbons. Suitable alkyl groups may be saturated or unsaturated. Furthermore, the alkyl group may also be substituted one or more times on one or more carbons with a substituent selected from the group consisting of: C1-C15 alkyl, allyl, allenyl, alkenyl, C3-C7 heterocycle, aryl, halo, hydroxy, amino, cyano, oxo, thio, alkoxy, formyl, carboxy, carboxamide, phosphoryl, phosphonate, phosphonamide, sulfonyl, alkylsulfonate, arylsulfonate, and sulfonamide. Additionally, the alkyl group may contain up to 10 heteroatoms, and in certain embodiments 1,2, 3, 4,5, 6, 7, 8, or 9 heteroatom substituents. Suitable heteroatoms include nitrogen, oxygen, sulfur and phosphorus.
As used herein, "cycloalkyl" refers to a monocyclic or polycyclic ring system, in certain embodiments having 3 to 10 carbon atoms, in other embodiments having 3 to 6 carbon atoms. The ring system of the cycloalkyl group may be composed of one ring or two or more rings, which may be joined together in a fused, bridged or spiro-connected manner.
As used herein, "aryl" refers to an aromatic monocyclic or polycyclic group containing 3 to 16 carbon atoms. As used in this specification, an aryl group (group) is an aryl group (radial) which may contain up to 10 heteroatoms, and in certain embodiments 1,2, 3 or 4 heteroatoms. The aryl group may also be optionally 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 may also be fused to other aryl or cycloalkyl rings. Suitable aryl groups include, for example, phenyl, naphthyl, tolyl, imidazolyl, pyridyl, pyrrolyl, thienyl, pyrimidinyl, thiazolyl, and furanyl groups.
As used in this specification, a ring is defined as having up to 20 atoms which may contain one or more nitrogen, oxygen, sulfur or phosphorus atoms, provided that the ring may 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, carboxamide, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, phosphonate, phosphoramidate, and sulfonyl, and with the further proviso that the ring may also contain one or more fused rings, including carbocyclic, heterocyclic, aryl, or heteroaryl rings.
As used herein, alkenyl and alkynyl carbon chains, if not specifically stated, contain 2 to 20 carbons or 2 to 16 carbons and are straight or branched. In certain embodiments, alkenyl carbon chains having 2 to 20 carbons contain 1 to 8 double bonds, and in certain embodiments, alkenyl carbon chains having 2 to 16 carbons contain 1 to 5 double bonds. In certain embodiments, alkynyl carbon chains having 2 to 20 carbons contain 1 to 8 triple bonds, and in certain embodiments, alkynyl carbon chains having 2 to 16 carbons contain 1 to 5 triple bonds.
As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic ring system, in certain embodiments having from about 5 to about 15 members, wherein one or more (in one embodiment, 1 to 3) of the atoms in the ring system is a heteroatom, i.e., an element other than carbon, including but not limited to nitrogen, oxygen, or sulfur. The heteroaryl group may optionally be fused to a benzene ring. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, and isoquinolinyl.
As used herein, "heterocyclyl" refers to a monocyclic or polycyclic non-aromatic ring system, in one embodiment having 3 to 10 members, in another embodiment having 4 to 7 members, and in another embodiment having 5 to 6 members, wherein one or more (in certain embodiments, 1 to 3) of the atoms in the ring system are heteroatoms, i.e., an element other than carbon, including, but not limited to, nitrogen, oxygen, or sulfur. In embodiments where the heteroatom is nitrogen, the nitrogen is optionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, acyl, guanidino, or the nitrogen may be quaternized to form an ammonium group, where the substituents are selected as above.
As used herein, "aralkyl" refers to an alkyl group in which one hydrogen atom of the alkyl group is replaced with an aryl group.
As used herein, "halo," "halogen," or "halide" refers to F, Cl, Br, or I.
As used herein, "haloalkyl" refers to an alkyl group wherein one or more hydrogen atoms are replaced with a halogen. Such groups include, but are not limited to, chloromethyl and trifluoromethyl.
As used herein, "aryloxy" refers to RO-, wherein R is aryl, including lower aryl, such as phenyl.
As used herein, "acyl" refers to a-COR group, including, for example, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, or heteroarylcarbonyl, all of which may be optionally substituted.
As used herein, "n-3" or "n 3", "n-6" or "n 6" and the like refer to the customary nomenclature of polyunsaturated fatty acids or their derivatives, wherein the position of the double bond (C ═ C) is the carbon atom counted from the end of the carbon chain of the fatty acid or fatty acid derivative (methyl end). For example, "n-3" refers to the third carbon atom from the end of the carbon chain of the fatty acid or fatty acid derivative. Similarly, "n-3" or "n 3", "n-6" or "n 6" and the like refer to the position of a substituent, such as a hydroxyl group (OH) at a carbon atom of a fatty acid or fatty acid derivative, where the number (e.g., 3,6, etc.) is counted from the end of the carbon chain of the fatty acid or fatty acid derivative.
As used herein, unless otherwise indicated, any abbreviations for protecting groups and other compounds shall be used in accordance with their common usage, accepted abbreviations, or the IUPAC-IUB Commission on Biochemical nomenclature (see (1972) biochemistry (Biochem), 11: 942-944).
As used herein, wherein in illustrating the chemical structure of a compound of the present disclosure having a terminal carboxyl group "-COOR", R "is intended to mean a group covalently bonded to a carboxyl group, such as an alkyl group. In the alternative, the carboxyl group is further intended to have a negative charge, such as "-COO-", and R is a cation, including metal cations, ammonium cations, and the like.
As used herein, a "subject" is an animal, typically a mammal, including a human, e.g., a patient.
As used herein, a "pharmaceutically acceptable derivative" of a compound includes a salt, ester, enol ether, enol ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate, or prodrug thereof. Such derivatives can be readily prepared by those skilled in the art using known methods for such derivatization. The resulting compounds can be administered to animals or humans without substantial toxic effects, and are pharmaceutically active or prodrugs.
Pharmaceutically acceptable salts include, but are not limited to, amine salts such as, but not limited to, N' -dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidinyl-T-ylmethyl benzimidazole, diethylamine and other alkylamines, piperazine and tris (hydroxymethyl) aminomethane; alkali metal salts such as, but not limited to, lithium, potassium, and sodium; alkaline earth metal salts such as, but not limited to, barium, calcium, and magnesium; transition metal salts such as, but not limited to, zinc; and other metal salts such as, but not limited to, sodium hydrogen phosphate and disodium phosphate; and also include, but are not limited to, salts of mineral acids such as, but not limited to, hydrochlorides and sulfates; and salts of organic acids such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, and fumarate.
Pharmaceutically acceptable esters 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, formic, phosphoric, phosphinic, sulfonic, sulfinic, and boronic acids.
Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of the formula C ═ C (or), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, or heterocyclyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of the formula C ═ C (oc (o) R), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkylaryl, or heterocyclyl.
Pharmaceutically acceptable solvates and hydrates are complexes of the compound with one or more solvent or water molecules, or from 1 to about 100 or from 1 to about 10 or from one to about 2, 3 or 4 solvent or water molecules.
As used herein, "formulation" shall mean and include any collection of components of a compound, mixture, or solution selected to provide optimal performance for a given end use (including product specifications and/or use conditions). The term formulation shall include liquids, semi-liquids, colloidal solutions, dispersions, emulsions, microemulsions and nanoemulsions, including oil-in-water and water-in-oil emulsions, pastes, powders and suspensions. The formulations of the present invention may also be included or packaged with other non-toxic compounds, such as cosmetic carriers, excipients, binders, fillers, and the like. In particular, acceptable cosmetic carriers, excipients, binders and fillers contemplated for use in practicing the present invention are those that render the compounds suitable for oral delivery and/or provide stability such that the formulations of the present invention exhibit a commercially acceptable shelf life.
As used herein, the term "administering" refers to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject using intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transdermal, intradermal, intracranial, topical, etc. administration. The formulations or pharmaceutical compounds of the present invention may be administered alone, but may also be administered with other compounds, excipients, fillers, binders, carriers, or other vehicles based on the chosen route of administration and standard pharmaceutical practice. Administration may be via a carrier or vehicle, such as an injectable solution, comprising a sterile aqueous or nonaqueous solution or salt solution; ointment; a lotion; a capsule; a tablet; particles; a bolus; powder; suspensions, emulsions or microemulsions; a patch; micelles; a liposome; a vesicle; an implant comprising a microimplant; eye drops; other proteins and polypeptides; synthesizing a polymer; microspheres; nanoparticles, and the like.
The formulations or pharmaceutical compositions of the present invention may also be contained or packaged together with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, binders and fillers, including but not limited to glucose, lactose, acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and/or polysaccharides, starch paste, magnesium trisilicate, talc, corn starch, starch fragments, keratan, colloidal silicon dioxide, potato starch, urea, dextran, dextrin and the like. In particular, pharmaceutically acceptable carriers, excipients, binders and fillers contemplated for use in practicing the invention are those that render the compounds of the invention suitable for intravitreal delivery, intraocular delivery, ocular delivery, subretinal delivery, intrathecal delivery, intravenous delivery, subcutaneous delivery, transdermal delivery, intradermal delivery, intracranial delivery, topical delivery, and the like. Further, the packaging material can be biologically inert or lack biological activity (e.g., plastic polymers, silicones, etc.) and can be treated internally by the subject without affecting the effectiveness of the composition/formulation packaged and/or delivered therewith.
The different forms of the formulations of the present invention may be calibrated to suit different individuals and the different needs of a single individual. The implementation of this concept is very complex and the necessary research is challenging. The present formulation, then, does not have to combat every cause in every individual. Instead, by countering the necessary causes, the present formulation will restore normal function to the body and brain. The body and brain will then correct the remaining defects themselves. No drug can correct every single cause of AD, but the present formulation will maximize the likelihood.
As used herein, the term "therapeutically effective amount" refers to the amount of one embodiment of the composition or pharmaceutical composition administered that will alleviate, to some extent, one or more symptoms of the disease or condition being treated, and/or that will prevent, to some extent, one or more symptoms of the condition or disease at risk of developing or suffering from the subject being treated. As used interchangeably herein, "subject," "individual," or "patient" refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rats, apes, humans, farm animals, sport animals, and pets. The term "pet" includes dogs, cats, guinea pigs, mice, rats, rabbits, ferrets, and the like. The term farm animal includes horses, sheep, goats, chickens, pigs, cattle, donkeys, llamas, alpacas, turkeys and the like.
"pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" refers to excipients, diluents, carriers, and/or adjuvants that can be used to prepare pharmaceutical compositions that are generally safe, non-toxic, and biologically or otherwise undesirable, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary use and/or human pharmaceutical use. As used in the specification and claims, a "pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant" includes one or more of such excipients, diluents, carriers and adjuvants.
As used herein, "pharmaceutical composition" or "pharmaceutical formulation" is meant to encompass a composition or pharmaceutical composition suitable for administration to a subject (e.g., a mammal, especially a human), and refers to the combination of an active agent or ingredient and a pharmaceutically acceptable carrier or excipient such that the composition is suitable for diagnostic, therapeutic or prophylactic use in vitro, in vivo, or ex vivo. In this context, a "pharmaceutical composition" is sterile and preferably free of contaminants capable of causing an adverse reaction in a subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). The pharmaceutical compositions can be designed for administration to a subject or patient in need thereof via a variety of different routes of administration, including oral, intravenous, oral, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, by stent eluting devices, catheter eluting devices, intravascular balloons, inhalants, and the like.
The term "administering" refers to introducing a composition of the present disclosure into a subject. One advantageous route of administration of the composition is topical administration. Any route of administration may then be used, such as oral administration, intravenous administration, subcutaneous administration, peritoneal administration, intraarterial administration, inhalation administration, vaginal administration, rectal administration, nasal administration, introduction into the cerebrospinal fluid, intravascular administration (intravenous or arterial), or instillation into a body cavity.
As used herein, "treatment" refers to any means aimed at combating the condition, management, and care of a disease or disorder in a manner that ameliorates or otherwise beneficially alters one or more symptoms of the disease or disorder. The term is intended to encompass the full range of treatments for a given condition from which a patient is suffering, such as administration of an active compound for the purpose of: ameliorating or alleviating a symptom or complication; delay of progression of the condition, disease or disorder; cure or eliminate a condition, disease, or disorder; and/or preventing a condition, disease or disorder, wherein "preventing" is understood to mean patient management and care with the aim of impeding the development of the condition, disease or disorder, and comprises administering an active compound to prevent or reduce the risk of onset of symptoms or complications.
The patient to be treated is preferably a mammal, in particular a human being. Treatment also encompasses any pharmaceutical use of the compositions herein, e.g., for the treatment of a disease provided herein.
As used herein, the term "nutritional component" refers to, for example, proteins, carbohydrates, vitamins, minerals, and other beneficial nutrients, comprising the functional ingredients of the present invention, i.e., ingredients intended to produce a particular benefit to a person consuming the food. The carbohydrate may be, but is not limited to, glucose, sucrose, fructose, dextrose, tagatose, lactose, maltose, galactose, xylose, xylitol, dextrose, polydextrose, cyclodextrin, trehalose, raffinose, stachyose, fructo-oligosaccharide, maltodextrin, starch, pectin, gum, carrageenan, inulin, cellulose-based compounds, sugar alcohols, sorbitol, mannitol, maltitol, xylitol, lactitol, isomalt, erythritol, pectin, gums, carrageenan, inulin, hydrogenated indigestible dextrins, hydrogenated starch hydrolysates, highly branched maltodextrins, starch, and cellulose.
Commercial sources of nutritional proteins, carbohydrates, etc., and their specifications are known or can be readily determined by one of ordinary skill in the art of processing food formulations.
The compositions of the present disclosure comprising nutritional components may be food preparations that may be, but are not limited to, compositions that are smaller than the "treat size" or "snack size" that is typically considered a food bar. For example, the food bar may be indented or perforated to allow the consumer to fold into smaller portions to eat, or the food "bar" may be a small piece rather than an elongated product.
The smaller pieces may be individually coated or enrobed. They may be packaged individually or in groups. The food may comprise, for example, but not limited to, a solid material that is not ground into a homogeneous mass. The food may be, for example, but not limited to, coated or enrobed with chocolate (including dark, light, milk or white chocolate, carob, yogurt, other confections, nuts or cereals). The coating can be a composite confectionery coating or a non-confectionery (e.g., sugar-free) coating. The coating may be smooth or may contain solid particles or pieces.
Discussion of the related Art
Age-related and non-age-related inflammatory, degenerative, neurodegenerative, traumatic, dermatological and cardiovascular diseases comprise a large number of diseases affecting many people worldwide. In most cases, these diseases and related conditions and disorders are difficult to treat, leading to impaired quality of life and/or shortened life span, and remain unmet major medical needs.
Inflammatory, degenerative or neurodegenerative diseases and conditions that may be reduced or eliminated by the compositions of the present disclosure include, but are not limited to, acute and chronic disorders in which homeostasis is disrupted by an abnormal or dysregulated inflammatory response. These conditions are initiated and mediated by a variety of inflammatory factors, including uncompensated oxidative stress, chemokines, cytokines, disruption of the blood/tissue barrier, autoimmune diseases, calcium imbalance (including calcium overload in cells), mitochondrial dysfunction, genetic factors (genetic susceptibility, polymorphism) or genetic conditions, or leukocyte, monocyte/macrophage, microglia, astrocyte or parenchymal cell other conditions involving proinflammatory/destruction to induce excessive protocell damage, cellular and/or organ homeostasis. These diseases occur in a variety of tissues and organs, including skin, muscle, stomach, intestine, liver, kidney, lung, eye, ear, and brain. These diseases are currently treated by anti-inflammatory agents, such as corticosteroids, non-steroidal anti-inflammatory drugs, TNF modulators, COX-2 inhibitors, and the like.
Degenerative diseases include conditions involving progressive loss of living cells and tissues, which results in progressive functional impairment, such as cartilage loss in the knee, hip, or other joints (e.g., osteoarthritis). Other degenerative diseases involve disturbances of cell and intercellular homeostasis and include heart disease, atherosclerosis, cancer, diabetes, intestinal diseases, osteoporosis, prostatitis, rheumatoid arthritis, and the like.
Neurodegenerative diseases include some of the major diseases of the brain, retina, spinal cord and peripheral nerves, whereby progressive loss of cellular tissue results in impaired function. These are due to an immunological or inflammatory disorder and/or a genetic pathology or to aging. They include multiple sclerosis, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, retinal degenerative diseases (e.g., age-related macular degeneration), hereditary ocular diseases (e.g., retinitis pigmentosa, glaucoma), and the like.
Inflammatory or degenerative diseases and conditions of the eye generally affect the cornea, optic nerve, trabecular meshwork, and retina. Without effective prevention or treatment, they can lead to blinding eye diseases such as glaucoma, cataract, diabetic retinopathy and age-related macular degeneration (AMD).
Retinal degenerative diseases are a major cause of blindness, affecting many people. Retinal degeneration is the degeneration of the retina caused by the progressive and eventual death of the photoreceptor cells of the retina. Examples of common retinal degenerative diseases include retinitis pigmentosa, age-related macular degeneration, and Stargardt disease. Retinitis pigmentosa affects 50,000 to 100,000 people in the united states alone, while macular degeneration is the leading cause of vision loss in people aged 55 and older in the united states, affecting over 1,000 million people. There is no effective treatment for these and other retinal degenerative diseases.
For retinal degenerative diseases, the detailed molecular mechanisms involved in the progressive loss of photoreceptor cells are still unknown, and the treatments available today are not effective in treating these major diseases and preventing vision loss. There is a need for a method for preventing and treating retinal degenerative diseases that ensures the survival of retinal photoreceptor cells.
Systemic inflammatory or degenerative diseases and conditions may affect vital organs (e.g., heart, muscle, stomach, intestine, liver, kidney, and lung) and may lead to age-related chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, and lupus.
Brain-related inflammatory, degenerative or neurodegenerative diseases and conditions, such as alzheimer's disease, parkinson's disease, multiple sclerosis, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis, often lead to premature aging, cognitive dysfunction and death.
Inflammatory or degenerative diseases and conditions of the skin are often caused by damage to the skin caused by sun exposure or other factors including skin inflammation (dermatitis or eczema), atopic dermatitis (atopic eczema), dehydration of the skin, or by abnormal skin cell proliferation resulting in excessive exfoliation. Skin damage from sun exposure or other factors is associated with a variety of diseases and conditions (e.g., eczema, psoriasis, atopic dermatitis, or neurodermatitis) and may result from exposure to ultraviolet light and other types of contact dermatitis. In addition, pruritus resulting from certain systemic diseases and conditions can lead to skin pruritus from various inflammatory and other types of irritation, and to the need for scratching, which can lead to further skin damage or changes in skin appearance.
Despite great advances, little is known about the pathophysiology of inflammatory, neuroinflammatory, degenerative or neurodegenerative diseases and conditions that often lead to organ damage, chronic disease and accelerated aging.
Thus, protection of organs, prevention of aging-related diseases and conditions, and overall restoration of health remain unmet major medical needs. There is also a major gap in effectively protecting skin tissues from inflammatory, neuroinflammatory, hyperproliferative or dehydrated skin conditions. In particular, the pathophysiology of skin damage, skin appearance changes, and skin aging remains unclear.
The treatments available today are not effective in treating these major diseases or slowing their progressive impairment of vital functions. There is a need for a method of ensuring survival of critical cells undergoing oxidative stress or other homeostatic disruption. Thus, there is a major therapeutic gap in the management of inflammatory, neuroinflammatory, degenerative or neurodegenerative diseases.
The present disclosure encompasses embodiments of compounds, compositions, and methods for the prevention and treatment of inflammatory and degenerative diseases, including neurodegenerative diseases and retinal degenerative diseases. This is based on the novel finding of a critical protective effect with respect to certain omega-3 or omega-6 very long chain polyunsaturated fatty acids (n3 or n6 VLC-PUFA) and their related hydroxylated derivatives.
Studies have shown that certain long chain polyunsaturated fatty acids (LC-PUFAs) play an important role in inflammation and related conditions. These comprise omega-3 (n3) and omega-6 (n6) polyunsaturated fatty acids containing 18-22 carbons comprising: arachidonic acid (ARA, C20:4n6, i.e., 20 carbons, 4 double bonds, omega-6), eicosapentaenoic acid (EPA, C20:5n3, 20 carbons, 5 double bonds, omega-3), docosapentaenoic acid (DPA, C22:5n3, 22 carbons, 5 double bonds, omega-3), and docosahexaenoic acid (DHA, C22:6n3, 22 carbons, 6 double bonds, omega-3).
LC-PUFA are converted via lipoxygenase-type enzymes into bioactive hydroxylated PUFA derivatives that are used as bioactive lipid mediators that play an important role in inflammation and related conditions. The most important of these are hydroxylated derivatives produced in certain inflammation-related cells via the action of lipoxygenase (LO or LOX) enzymes (e.g., 15-LO, 12-LO), which lead to the formation of mono-, di-, or tri-hydroxylated PUFA derivatives with potent effects, including anti-inflammatory, pro-resolution, neuroprotective, or tissue protective effects, among others. For example, neuroprotective peptide D1(NPD1), a dihydroxy derivative of DHA formed in cells via the enzymatic action of 15-lipoxygenase (15-LO), is shown to have defined R/S and Z/E stereochemistry (10R, 17S-dihydroxy-docosahexa-4Z, 7Z,11E,13E,15Z, 19Z-hexaenoicacid) and unique biological characteristics (including stereoselective potent anti-inflammatory, homeostatic recovery, pro-resolution, biological activity). NPD1 has been shown to regulate neuroinflammatory signaling and protein homeostasis, and to promote nerve regeneration, neuroprotection and cell survival.
Other important types of fatty acids are n3 and n6 very long chain polyunsaturated fatty acids (n3 VLC-PUFAs, n6 VLC-PUFAs), which are produced in cells containing elongases that extend n3 and n6 LC-PUFAs to n3 and n6 VLC-PUFAs containing 24 to 42 carbon atoms (C24-C42). The most important of these seems to be VLC-PUFAs with 28-38 carbons (C28-C38). Representative types of VLC-PUFAs comprise C32:6n3(32 carbons, 6 double bonds, omega-3), C34:6n3, C32:5n3 and C34:5n 3. These VLC-PUFAs are biogenically (biogenic) derived by the action of elongases, in particular ELOVL4 (very long chain fatty acid elongation factor 4). VLC-PUFAs are also acylated in complex lipids (including sphingolipids and phospholipids), particularly in certain molecular species of phosphatidylcholine.
The biosynthetic role of ELOVL4 and the biological function of VLC-PUFAs have been the subject of many recent studies that have shown potential roles in retina, brain, testis, and skin. These VLC-PUFAs are believed to function in membrane tissue and their importance for health is also increasingly recognized.
The importance of VLC-PUFAs in the retina (components of the central nervous system) as well as in the brain has been shown. For example, autosomal dominant Stargardt-like macular dystrophy (STGD3) is a juvenile onset retinal degenerative disease that is caused by mutations in exon 6 of the ELOVL4 gene, resulting in truncation of the ELOVL4 protein, a key elongase, without Endoplasmic Reticulum (ER) retention/recovery signals, resulting in severe reduction of VLC-PUFA biosynthesis. Low retinal VLC-PUFA levels and abnormally low n3/n6 ratios also were present in the eyes of age-related macular degeneration (AMD) donors compared to age-matched control eye donors. The recessive ELOVL4 mutation shows clinical features of ichthyosis, seizures, mental retardation and spastic quadriplegia, similar to Sjogren-Larsson syndrome (SLS) with severe neural phenotype, suggesting the importance of VLC-PUFA synthesis for central nervous system and skin development.
VLC-PUFAs were found incorporated into phospholipids of the photoreceptor outer membrane and were shown to play an important role in the longevity of photoreceptors and in their synaptic function and neuronal connectivity. Therefore, VLC-PUFA-based bioactive derivatives that can prevent photoreceptor cell apoptosis can provide therapeutic benefits for various types of retinal degenerative diseases, including Stargardt-like macular dystrophy (STGD3) and X-linked juvenile retinoschisis (XLRS), an inherited early-onset retinal degenerative disease caused by mutations in the RS1 gene, which is a major cause of male juvenile macular degeneration. The disease indicates significant photoreceptor synaptic damage, and no treatment for the photoreceptor synaptic damage is available.
The compounds, compositions, and methods encompassed by embodiments of the present disclosure relate to the use of n3VLC-PUFA to induce survival signaling in the brain and retina, particularly in retinal pigment epithelial cells and photoreceptor cells.
Biosynthetic pathway of n3 VLC-PUFA: the biosynthesis of n3 VLC-PUFAs starts from lower carbon PUFAs, the carbon chain of which contains only an even number of carbons, such as docosahexaenoic acid (DHA) containing 22 carbons and 6 alternating C ═ C bonds (C22:6n3) and docosapentaenoic acid (DPA) containing 22 carbons and 5 alternating C ═ C bonds (C22:5n 3). The biosynthesis of n3 VLC-PUFAs requires the use of DHA or other shorter chain PUFAs as substrates, and the presence and action of certain elongases (e.g., ELOVL 4). As summarized in FIGS. 1 and 2, these 22-carbon omega-3 long chain fatty acids (n3 LC-PUFAs) are substrates for elongases (e.g., ELOVL4) that add one 2-carbon CH to the carboxy terminus at a time2CH2A group forming an n3VLC-PUFA, which contains a carbon chain having at least 24 carbons to at least 42 carbons.
Docosahexaenoic acid (DHA, C22:6n3, 1 is incorporated at position 2 of the phosphatidylcholine molecular species (3) and converted to longer chain n3 VLC-PUFAs by the elongase ELOVL4 (very long chain fatty acid elongation factor 4) elongation by the elongase results in the formation of very long chain omega-3polyunsaturated fatty acids (n3 VLC-PUFAs, 2, comprising C32:6n3 and C34:6n3, which are then incorporated at position 1 of the phosphatidylcholine molecular species, 3-DHA is present at position 2 and n3 VLC-PUFAs is present at position 1 may provide redundant, complementary and synergistic cytoprotective effects, amplifying the potential survival of neurons and other critical cell types upon challenge with pathological conditions.
Lipoxidation of N3-VLC-PUFA, 3 results in the formation of enzymatically hydroxylated derivatives of N3-VLC-PUFA, known as elonoids, comprising monohydroxy compounds (e.g., ELV-27S and ELV-29S, 4) and dihydroxy derivatives (e.g., ELV-N32 and ELV-N34, 5). The elonoid ELV-N32 is a 20R, 27S-dihydroxy 32:6 derivative (a 32 carbon, 6 double bond elonoid with the neuroprotective-like 20(R),27(S) -dihydroxy version). The elonoid ELV-N34 is a 22R, 29S-dihydroxy 34:6 derivative (a 34 carbon, 6 double bond type of elongin, with the 22(R),29(S) -dihydroxy version).
FIG. 2 shows the delivery of docosahexaenoic acid (DHA, C22:6n3) to photoreceptors, the renewal of photoreceptor outer segment membranes and the synthesis of elongatoids. DHA or precursor C18:3n3 was obtained from the diet, as was DHA itself (FIG. 1). The systemic circulation (mainly the portal system) carries them into the liver. Once in the liver, stem cells incorporate DHA into DHA phospholipids (DHA-PL), which are then transported to choroidal capillaries (neurovascular units) and to capillaries of other tissues in the form of lipoproteins.
DHA from the choroidal capillaries passes through Bruch's membrane (fig. 2) and is absorbed by Retinal Pigment Epithelium (RPE) cells lining the back of the retina to be transmitted to the intra-photoreceptor segment. The present targeted delivery route from the liver to the retina is called the DHA long loop.
DHA then passes through the interphotoreceptor matrix (IPM) and reaches the inner photoreceptor segment, where it is incorporated into the outer photoreceptor segment, the cell membrane, and the phospholipids of the organelles. Most are used for disc membrane biogenesis (outer segment). As new DHA-rich discs are synthesized at the base of the photoreceptor outer segment, older discs will be pushed apically towards RPE cells. Every day, RPE cells phagocytose the photoreceptor tips, removing the oldest disc. The resulting phagosomes are degraded in RPE cells and DHA is recycled back to the segment within the photoreceptor for new disc membrane biogenesis. This partial loop is referred to as a 22:6 short loop.
The elongatinoids are formed from omega-3 very long chain polyunsaturated fatty acids (n3VLC-PUFA) biosynthesized by ELOVL4 (very long chain fatty acid elongation factor 4) in segments within the photoreceptor. Thus, the species of phosphatidylcholine molecules in the inner segment containing VLC omega-3 FA at C1 (depicted as C34:6n3) and DHA at C2 (C22:6n3) were used for photoreceptor membrane biogenesis. The present phospholipids have been found to be closely related to rhodopsin. Once phagocytosis of the disc in RPE cells is a routine physiological process, when a potential homeostatic disorder occurs, phospholipase A1(PLA1) cleaves the acyl chain at sn-1, releasing C34:6N3 and leading to the formation of an elongatinoid (e.g., elongatinoid-34, ELV-N34). VLC omega-3 fatty acids not used for the synthesis of the elongatoids circulate through short loops.
Thus, for biosynthetic reasons, naturally occurring and biogenetic derived n3 VLC-PUFAs 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). Thus, n3 VLC-PUFAs containing only odd numbers of carbons (ranging from at least 23 to at least 41 carbon atoms (i.e., 23,25,27, 29, 31, 33, 35, 37, 39, 41 carbons))Not naturally occurringThey can be synthesized and manufactured using synthetic chemistry methods and strategies.
Stereocontrolled total synthesis and structural characterization of the elongatoids ELV-N32 and ELV-N34 in the retina and brain: as summarized in fig. 3 and 4, ELV-N32 (27S-and ELV-N34 was synthesized from three key intermediates (1, 2, and 3), each prepared in stereochemically pure form, the stereochemistry of intermediates 2 and 3 was predefined by using enantiomerically pure epoxide starting materials iterative coupling of intermediates 1,2, and 3 produced ELV-N32 and ELV-N34(4), which were isolated as methyl esters (Me) or sodium salts (Na), the synthetic materials ELV-N32 and ELV-N34 were matched to endogenous elongatoids with the same number of carbons in their carbon chains, which were obtained from cultured human retinal pigment epithelial cells (RPE) (fig. 3) and neuronal cell cultures (fig. 4).
Experimental detection and characterization of elongatoids: experimental evidence records the biosynthetic formation of the elongatoids, mono-and dihydroxy n3VLC-PUFA derivatives with molecular structures similar to DHA-derived 17-hydroxy-DHA and the dihydroxy compound NPD1(10R, 17S-dihydroxy-docosal-4Z, 7Z,11E,13E,15Z, 19Z-hexaenoic acid). The elongatinoids are enzymatically produced hydroxylated derivatives of 32-carbon (ELV-N32) and 34-carbon (ELV-N34) N3VLC-PUFA, first identified in cultures of human primary retinal pigment epithelial cells (RPE) (FIGS. 3A-3K) and neuronal cell cultures (FIGS. 4A-4K).
The present disclosure provides compounds having carbon chains related to n3 VLC-PUFAs, which compounds contain one, two or more hydroxyl groups in addition to having 6 or 5C ═ C bonds. Based on the hypothesis that compounds of this type may be responsible for the protective and neuroprotective effects of n3 VLC-PUFAs, we attempted to identify their presence in human retinal pigment epithelial cells in culture with added 32:6n3 and 34:6n 3VLC-PUFA fatty acids. Our results indicate mono-and dihydroxy-type elongin derivatives from 32:6n3 and 34:6n 3VLC-PUFA fatty acids. The structure of these classes of elongatins (ELV-N32, ELV-N34) was compared to standards prepared in stereochemically pure form via stereocontrolled, all-organic synthesis (FIGS. 5A and 5B).
Beneficial use of n3 VLC-PUFAs and elongatoids in ophthalmic diseases and conditions: inflammatory or degenerative diseases and conditions of the eye generally affect the cornea, optic nerve, trabecular meshwork, and retina. Without effective prevention or treatment, they can lead to blinding eye diseases such as glaucoma, cataract, diabetic retinopathy and age-related macular degeneration (AMD). There is increasing evidence that ELOVL4 mutations and/or n3VLC-PUFA reductions in retinal cells and tissues are associated with degenerative, neurodegenerative and retinal degenerative diseases that are associated with an excessive and persistent inflammatory environment. Thus, given the major role of n3 VLC-PUFAs, the structure, properties and potential role of n3 VLC-PUFAs in retinal cells and tissues was assessed.
The experiments were performed using human Retinal Pigment Epithelium (RPE) cells, which are postmitotic cells of the retina of neuroectodermal origin, which are part of the central nervous system. These cells have a rich mechanism that can protect themselves from damage and protect other cells (particularly the photoreceptor cells) from survival. They are the most active phagocytes in the human body, are critical for the health of photoreceptors and vision, and have the ability to secrete neurotrophins and other beneficial substances.
The beneficial effects of n3 VLC-PUFAs in retinal degenerative diseases (e.g., autosomal dominant Stargardt-like macular dystrophy (STGD3) and age-related macular degeneration (AMD)) are supported by: (a) n3VLC-PUFA is biosynthesized in the retina and is known to play a major role in the retina; (b) the elongationlike ELV-N32 and ELV-N34 were found and structurally characterized in primary human retinal pigment epithelial cells (RPE) in culture (FIGS. 1, 6A and 6B); (c) ELOVL4 is a key enzyme involved in the conversion of DHA (C22:6) to n3 VLC-PUFA; (d) certain mutations in the elongase ELVOL4 result in retinal degenerative diseases such as STGD3 and AMD; (e) genetic ablation of the proteins necessary to capture DHA into retinal cells containing the ELOVL4 product resulted in a dramatic decrease in VLC-PUFA levels, resulting in retinal degeneration; (f) uncompensated Oxidative Stress (UOS) in RPE cells is associated with early stages of retinal degenerative diseases; (g) n3 VLC-PUFAC32:6n3 and C34:6n3 provided cytoprotection in human RPE cells exposed to UOS (as shown in FIGS. 7A-8C), which was not altered with lipoxygenase inhibitors (FIGS. 9A-9C); (h) the elongationlike ELV-N32 and ELV-N34 provide cytoprotection of human RPE cells under UOS by up-regulating anti-apoptotic proteins (fig. 10A-10G) and promoting photoreceptor cell survival (fig. 11A-11D); (i) the elongatinoids ELV-N32 and ELV-N34 promoted photoreceptor cell integrity in late onset retinal degeneration (L-ORD) (FIGS. 12A and 12B).
As shown in fig. 14A-14C, Elongatinoids (ELVs) oppose a β peptide-induced progression of retinal pigment epithelial cell senescence. A β 42 is the end product of the amyloidogenic pathway and is a component of drusen in age-related macular degeneration (AMD) and senile plaques in Alzheimer's Disease (AD). To mimic the effect of OA β in vivo, 6-month old mice were used, injected subretinally with OA β only, OA β + ELV, and ELV only, respectively. Non-injected mice served as negative controls, while PBS-injected mice served as false controls. The injection amount was 2. mu.l PBS, 10. mu.M OA. beta. +200ng ELV-32, only 200ng ELV-32, 10. mu.M OA. beta. +200ng ELV-34, or only 200ng ELV-34. On day 3 post-injection, mRNA was isolated from the optic cup and gene expression was analyzed using q-PCR. Then, on day 7, mice were subjected to Optical Coherence Tomography (OCT) analysis, and then eyes were removed and processed for histology, bulk RPE staining, and Western Blotting (WB). OCT and histology (data not shown) showed that the thickness of the retina was thinner in the OA β -induced retinal degeneration compared to the control and ELV treated groups. Bulk staining with ZO-1 showed that tight junctions were also disrupted by OA β. Interestingly, co-injection of ELV and OA β indicated that ELV was able to restore morphology and homeostasis of the RPE layer. Furthermore, the protein level of the aging marker p16INK4a was up-regulated in the OA β group but suppressed in the ELV combination treatment group and the control group in the WB assay. Finally, gene expression analysis showed that ELV reduced the levels of markers of aging and AMD, which were triggered by OA β injections, while inducing expression of RPE functional genes, which were down-regulated in the OA β injection group. This data indicates that ELV-32 and ELV-34 restores retinal architecture and maintains homeostasis by down-regulating aging, AMD, and inflammation-related gene expression and by preserving the expression of RPE functional genes to protect RPE and retina from OA β -induced aging.
An elongin-like (ELV) compound: ELV C32: 6-ethynylmethyl ester; ELV C32:6-NPD 1-like sodium salt; ELV C34: 6-ethynylmethyl ester; ELV C32:6-NPD 1-like methyl ester; ELV C34:6-NPD 1-like methyl esters.
Autophagy involves more than 100 genes. The following genes are elevated in AMD disease:
the ATG3 (autophagy-related 3) gene encodes a ubiquitin-like conjugating enzyme that is a component of the ubiquitination-like system involved in autophagy (the process of degradation, turnover and circulation of cytoplasmic components in eukaryotic cells). The present protein is known to play a role in the regulation of autophagy during cell death.
ATG5 (autophagy-related 5) protein encoded by this gene binds to autophagy protein 12 and functions as an E1-like activating enzyme in a ubiquitin-like conjugation system. The encoded proteins are involved in a number of cellular processes including autophagic vesicle formation, mitochondrial quality control following oxidative damage, down regulation of innate anti-viral immune responses, lymphocyte development and proliferation, MHC II antigen presentation, adipocyte differentiation, and apoptosis.
The ATG7 (autophagy-related 7) gene encodes an E1-like activating enzyme, and is crucial for autophagy and transport of the cytoplasm to the vacuole. The encoded protein is also thought to regulate the p 53-dependent cell cycle pathway during prolonged metabolic stress. It is associated with a variety of functions, including axonal membrane trafficking, axonal homeostasis, mitochondrial autophagy, adipose differentiation, and hematopoietic stem cell maintenance.
BECN1(Beclin 1) the basic gene encodes a protein that regulates autophagy (a metabolic process of degradation induced by starvation). The encoded protein is a component of the phosphatidylinositol 3-kinase (PI3K) complex that mediates vesicle trafficking. The present proteins are thought to play a role in a variety of cellular processes, including tumorigenesis, neurodegeneration, and apoptosis.
Senescent cells have several distinct properties, such as increased cell size, induced enzymatic activity of the lysosomal hydrolase senescence-associated β -galactosidase (SA- β -GAL). (2). In addition to these features, there is activation of the senescence signaling pathway (including p 16. sup. INK4a, p21CIP1, p27KIP and p 53). (3)
p 16. sup. INK4a (also known as cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase 4 inhibitor A or several other synonyms) is a tumor suppressor protein. The protein is encoded by the CDKN2A gene. p16 plays an important role in cell cycle regulation by slowing cellular progression from G1 to S phase.
p21Cip1 (also known as p21Waf1, cyclin dependent kinase inhibitor 1 or CDK interacting protein 1) is a cyclin dependent kinase inhibitor (CKI) which is capable of inhibiting all cyclin/CDK complexes. The protein is encoded by the CDKN1A gene. p21 represents the primary target for p53 activity and is therefore associated with linking DNA damage to cell cycle arrest.
p27Kip1 (also known as cyclin-dependent kinase inhibitor 1B) is an enzyme inhibitor. The protein is encoded by the CDKN1B gene. It encodes a protein belonging to the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitory proteins. The encoded protein binds to and prevents activation of the cyclin E-CDK2 or cyclin D-CDK4 complex, thus controlling the cell cycle progression of G1. It is commonly referred to as a cell cycle inhibitory protein because its primary function is to stop or slow down the cell division cycle.
p53 (also known as tumor protein p53, tumor suppressor p53) is also a cell cycle inhibitor. The protein is encoded by TP53 (human) and Trp53 (mouse). Various stressors may activate p53 directly or indirectly through kinases. The result is inhibition of all cyclins and cell arrest.
The data provide support for therapeutic or prophylactic use of N3 VLC-PUFAs or elongatoids thereof (e.g., ELV-N32, ELV-N34) in the eye, including treatment of retinal degenerative diseases and other ophthalmic diseases and conditions, including glaucoma, cataracts, diabetic retinopathy, Stargardt-like macular dystrophy (STGD3), and age-related macular degeneration (AMD).
Beneficial use of n3 VLC-PUFAs and elongatoids in brain diseases and conditions: VLC-PUFA and elongatinoid pathways are active in the Central Nervous System (CNS), including brain and neuronal cells.
ELV-N32 (in Na or Me form) or ELV-N34 (in Na or Me form) is able to overcome uncompensated oxidative stress, NMDA-induced neuronal excitotoxicity, or the damaging effects of OGD when applied to cortical mixed neuronal cells or hippocampal cells in culture. Most strokes are ischemic in nature and oxygen deprivation leads to a series of events involving mitochondrial damage, ultimately leading to neuronal death. Thus, the in vitro OGD model provides an opportunity to comb cellular events involved in ELV and putative potential neuroprotective signaling pathways. Both ELV-N32 and ELV-N34 can elicit neuroprotection and overcome neuronal cytotoxicity. The 32-carbon omega-3 VLC-PUFA (C32:6n3) precursors of ELV were able to provide neuroprotection to cerebral cortical neurons when applied at a dose of 250nM 2 hours after a re-oxygenation phase following 90 minutes of OGD damage. In summary, endogenously produced elongatoids (ELV-N32 or ELV-N34) ameliorate neuronal damage induced by a variety of stressors (e.g., NMDA, uncompensated oxidative stress or OGD) in cerebral cortex and hippocampal neurons in culture. These novel bioactive lipids belong to a new class of lipid mediators, called Elonoids (ELVs), which are derived from a phospholipid molecular species with two PUFAs at positions C1 and C2.
All ELV treatments performed 2 hours after experimental ischemic stroke 1 hour improved neurological recovery throughout the 7-day survival period. The rapid induction of cerebral edema following focal ischemia is a major cause of morbidity and mortality following stroke. The greatest degree of protection was detected in the cortex (penumbra) as well as in the subcortical region. Histopathology revealed less infarction, less whole cell damage, higher density of eosinophil regions, and neuronal contraction along the infarct margin in the cortical and subcortical regions, all of which were detected in the elongationlike treated rats.
Cerebral ischemia initiates a complex series of cellular, molecular, and metabolic events that result in irreversible brain damage. Dead neurons and damaged tissue are cleared by activated resident microglia and/or macrophages invading the damaged tissue from the bloodstream. The surviving astrocytes and activated microglia in the penumbra may promote the restoration of neuronal integrity by producing growth factors, cytokines and extracellular matrix molecules involved in repair mechanisms. The results indicate that ELV treatment increases the number of NeuN-positive neurons, GFAP-positive reactive astrocytes, and SMI-71-positive vascular density in the cortex. Vascular integrity promotes neurogenesis and synaptogenesis, which in turn contributes to improved functional recovery.
Following cerebral ischemia, the integrity of the BBB is compromised, allowing molecules to enter the brain parenchyma uncontrollably, thus exacerbating the ischemia-induced injury. In patients, loss of BBB integrity is associated with worsening prognosis of stroke. The BBB ischemic destruction of endogenous IgG penetration into brain parenchyma was measured. Treatment with ELV-N34-Na and ELV-N34-Me reduced BBB destruction induced by focal cerebral ischemia.
More recently flagged ELV protected neurons undergoing oxygen deprivation or NMDA receptor mediated excitotoxicity. In addition, ELV reduces infarct volume, rescues ischemic core and penumbra, reduces BBB injury, and promotes cell survival with neuro/behavioral recovery. It is reasonable to suggest that novel ELV therapy has the potential to treat focal ischemic stroke and other conditions that cause inflammatory/homeostatic destruction.
Provided herein are beneficial effects of ELOVL4 and n3 VLC-PUFAs in the CNS: (a) ELOVL4 is expressed in the CNS (including neuronal cells) and is involved in the conversion of DHA (C22:6) to n3 VLC-PUFA; (b) in an oxygen sugar deprivation (OGD) in vitro model of primary cortical neurons, n3VLC-PUFA are released by primary cortical neurons in response to OGD and are enzymatically converted to monohydroxyelongases 27(S) -hydroxy-32: 6n3 (fig. 13B) and 29(S) -hydroxy-34: 6n3 (fig. 13C). However, in control media in which neurons were not exposed to OGD, the levels of n3VLC-PUFA (e.g., C32:6n3 and C34:6n3) and monohydroxy-type elongases were negligible (fig. 13B and 13C).
As shown in fig. 13A-13D, n3 VLC-PUFAs (e.g., C32:6n3 and C34:6n3) were endogenously released by primary cortical neurons from SD rat embryos in response to oxygen sugar deprivation (OGD); and enzymatically converted to an elongatinoid, comprising 29(S) -hydroxy-34: 6n3 and 27(S) -hydroxy-32: 6n 3. In control media in which neurons were not exposed to OGD, levels of VLC-PUFAs (e.g., C32:6n3 and C34:6n3) and elongatoids were negligible. An in vitro OGD model is established, and primary mixed cortical neurons are cultured from SD rat embryos. Cells were washed with Phosphate Buffered Saline (PBS) on DIV12 and incubated with glucose-free neural basal medium (Gibco) for 30 min. Thereafter, the cells were placed in a modular incubator (Billups-Rothenberg Inc.) and in an anaerobic chamber (95% N) at 37 deg.C2And 5% CO2) OGD incubation was performed for 90 minutes. After 90 minutes of OGD exposure, the cells were returned to the original medium [ neural basal Medium (Gibco) containing 2% B27(Gibco) and 2% N-2(Gibco) supplement and 0.5mM glutamine and penicillin-streptomycin (50U/ml) (Gibco)]And in a normal oxygen chamber (37 ℃, 5% CO)2) And keeping for 12 hours. For normoxic (control) conditions, neurons were washed with PBS, but were maintained in conventional media [ nerve basal media (Gibco) containing 2% B27(Gibco) and 2% N-2(Gibco) supplements and 0.5mM glutamine and cyan during a 120 minute period during which other cells were subjected to OGD stressMycin-streptomycin (50U/ml) (Gibco)]In (1). Thereafter, control cells are subjected to subsequent regular media changes to match the timing of cells stressed by OGD.
After 12 hours, both control and OGD plates were washed with ice cold Phosphate Buffered Saline (PBS), cells were scraped off and collected in methanol for LC-MS/MS analysis. Fatty acids were extracted from the collected cell culture medium using liquid-liquid lipid extraction. The extract was loaded on a liquid chromatography tandem mass spectrometer for analysis. We analyzed fatty acids, monohydroxy fatty acid derivatives (27-S-hydroxy 32:6 and 29-S-hydroxy 34:6), ELV-N32(20, 27-dihydroxy fatty acid 32:6N3), and ELV-N34(22, 29-dihydroxy fatty acid 34:6N 3). The samples were normalized to an internal standard (AA-d8) for comparison.
It is speculated that OGD triggers the release of monohydroxyelongases and to a lesser extent ELV-N32 and ELV-N34, which will protect primary cortical neurons. These data indicate the neuroprotective effects of n3 VLC-PUFAs and elongatoids.
The elongatinoids ELV-N32 and ELV-N34 elicit protection against: (a) cerebral cortical neurons exposed to OGD (FIGS. 18A-18I) or NMDA toxicity (FIGS. 15A-15L and FIGS. 19A-19H); (b) cerebral cortical mixed and hippocampal neuronal cultures exposed to Uncompensated Oxidative Stress (UOS), oxygen deprivation (OGD), or NMDA excitotoxicity (fig. 16A-16I), in which cell survival was assessed (fig. 17).
The elongatinoids ELV-N32 and ELV-N34 improved the neurological/behavioral score, protected penumbra and reduced MRI lesion volume after ischemic stroke (fig. 20A-2D).
The elongatinoids ELV-N32 and ELV-N34 reduced neuronal and astrocyte damage induced by experimental ischemic stroke (FIGS. 21A-21C).
The elongatinoids ELV-N32 and ELV-N34 reduced neurovascular unit (NVU) destruction and reduced cerebral infarction following ischemic stroke (FIGS. 22A-22D).
The elongatoids ELV-N32 and ELV-N34 provided neuroprotection and improved neurological deficits following Traumatic Brain Injury (TBI) (fig. 23A-23C).
The data consistently support the potential therapeutic or prophylactic use of N3 VLC-PUFAs and elongatoids (e.g., ELV-N32, ELV-N34) in the brain, including the treatment of brain-related inflammatory, degenerative, or neurodegenerative diseases and conditions (e.g., alzheimer's disease, parkinson's disease, multiple sclerosis, ischemic stroke, traumatic brain injury, epilepsy, amyotrophic lateral sclerosis).
Use of n3 VLC-PUFAs and elongatoids in systemic and/or age-related diseases and conditions: systemic diseases caused by inflammatory, autoimmune, degenerative, neurodegenerative, stress-related, age-related or traumatic conditions may affect vital organs (e.g., heart, muscle, stomach, intestine, liver, kidney, and lung) and may lead to age-related chronic inflammatory diseases, such as rheumatoid arthritis, cardiovascular diseases, cerebrovascular diseases, atherosclerosis, lupus, and other aging-related diseases and conditions. Given its unique beneficial role in protecting the function of key cells and organs that prevent chronic diseases and conditions, the n3VLC-PUFA and/or elongatinoid compounds provided are expected to be effective in treating a variety of these chronic diseases and conditions.
Beneficial effects of n3 VLC-PUFAs and elongatoids in skin diseases and conditions: inflammatory or degenerative diseases and conditions of the skin are often caused by damage to the skin caused by sun exposure or other factors including skin inflammation (dermatitis or eczema), atopic dermatitis (atopic eczema), dehydration of the skin, or by abnormal skin cell proliferation resulting in excessive exfoliation. Skin damage from sun exposure or other factors is associated with a variety of diseases and conditions (e.g., eczema, psoriasis, atopic dermatitis, or neurodermatitis) and may result from exposure to ultraviolet light and other types of contact dermatitis. In addition, pruritus resulting from certain systemic diseases and conditions can lead to skin pruritus from various inflammatory and other types of irritation, and to the need for scratching, which can lead to further skin damage or changes in skin appearance.
In view of the overall importance of skin health, skin function, and skin appearance, efforts have been directed to developing methods to protect skin and overall skin health. Most current treatments involve dermal delivery of corticosteroids, or the use of oils and lotions containing vitamin, mineral or herbal ingredients, which are generally not effective in preventing or treating many types of skin damage and also have side effects such as skin thinning and muscle loss. While such formulations may provide some protection, there remains an unmet need to develop compounds, compositions, and methods that are effective in protecting damaged skin, preventing skin damage, restoring skin health, improving skin appearance, and delaying skin aging.
Although the compounds provided have tremendous protective, neuroprotective, reparative and other beneficial effects on skin and other tissues, they are locally biosynthetic in limited amounts. Over time and due to skin damage and skin aging, the local supply of the compounds provided is insufficient to provide the desired protection to the damaged tissue.
Thus, by providing a composition of the provided compounds in a manner that can be absorbed directly and locally onto the skin, the provided compounds and compositions will provide significant benefits to the damaged or aging affected skin, thereby achieving restoration of skin health, cosmetic improvement in skin appearance, and retardation of skin aging.
By suppressing aging-related skin tissue damage, and by preventing neuronal damage and restoring neuronal function, the compounds, compositions, and methods provided are capable of protecting skin from damage, improving skin health and appearance, and delaying skin aging. Given their unique beneficial role in protecting key cell and organ (including skin) function, the provided n3VLC-PUFA and/or elongin-like compounds are expected to be effective in treating a variety of skin diseases and conditions, including skin-related inflammatory, autoimmune, degenerative or neurodegenerative skin diseases and conditions.
In view of the foregoing, the above data and analysis provide the basis for the present disclosure (i.e., the provided compounds, dermatological or cosmetic compositions, and methods of providing compounds for application to skin tissue can provide protection, prevention, and treatment of damaged skin due to inflammation, dehydration, aging, or other causes).
Beneficial effects of n3VLC-PUFA as potential therapeutic agents: the concepts and data described herein provide support for the beneficial use of provided n3VLC-PUFA and/or elongin-like compounds as potential therapeutic agents for the prevention and treatment of retinal degenerative diseases and diseases associated with the brain, CNS, and other unmet therapeutic needs associated with inflammatory or degenerative diseases and conditions.
Sources of compounds of the present disclosure: the compounds provided are not isolated from tissues naturally occurring in nature, but from the results of artificial experiments combining human cells and chemically synthesizing n 3-VLC-PUFA. Using HPLC and mass spectrometry, the general structure of our synthetic elongin-like compounds was matched to compounds that were either biosynthesized in human retinal pigment epithelial cells or detected in neuronal cell cultures. However, the natural existence of mono-and dihydroxylated elongatins provided with a specifically defined stereochemistry is not known at present. Furthermore, the provided compounds are not obtained from natural sources, but are prepared by starting from commercially available materials using stereocontrolled synthetic methods known in the art. The provided preparation method is designed to suit the unique hydrophobic properties of n3VLC-PUFA, which is significantly different from compounds with 22 carbons or less in total carbon.
The present disclosure encompasses compounds that have stereochemically pure structures and are chemically synthesized and modified to have additional structural features and properties that enable them to exert pharmacological activity. The provided compounds are chemically modified pharmaceutically acceptable derivatives in the form of formate esters or salts that enhance their chemical and biological stability and enable their use in therapeutic applications involving various drug delivery forms.
The present 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 targeted cells and tissues.
Overall beneficial use of n3 VLC-PUFAs and elongatoids: the present disclosure provides compounds and compositions for the prevention and treatment of a variety of systemic inflammatory, degenerative and neurodegenerative diseases, including skin diseases, ophthalmic diseases, brain diseases, including neurotrauma.
The compounds and compositions provided by the present disclosure are capable of restoring homeostasis and inducing cell survival signaling in certain cells undergoing uncompensated oxidative stress or other homeostatic disruption.
The present disclosure also provides methods of using the provided compounds and compositions containing hydroxylated derivatives of omega-3 very long chain polyunsaturated fatty acids in the form of free formic acid or a pharmaceutically acceptable salt thereof, or a corresponding ester or other prodrug derivative thereof. The provided compounds can be readily prepared by employing methods known in the art starting from commercially available materials.
Administration of a pharmaceutical composition containing a provided compound and a pharmaceutically acceptable carrier restores homeostatic balance and promotes survival of certain cells necessary to maintain normal function. The provided compounds, compositions and methods can be used for the prevention and treatment of inflammatory, degenerative and neurodegenerative diseases.
The present disclosure targets key steps in the initiation and early progression of these conditions by mimicking the specific biology of the innate cell/organ response to achieve efficacy, selectivity, no side effects, and sustained biological activity.
Compound (I)
Described herein are compounds based on omega-3 very long chain polyunsaturated fatty acids and their hydroxylated derivatives, which are referred to as "elongatoids".
Omega-3 very long chain polyunsaturated fatty acids have the structure of a or B or derivatives thereof:
wherein: a contains a total of 23 to 42 carbon atoms in the carbon chain and has 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 of 23 to 42 carbon atoms in the carbon chain and has 5 alternating cis carbon-carbon double bonds starting at positions n-3, n-6, n-9, n-12 and n-15. R may be hydrogen, methyl, ethyl, alkyl, or a cation, such as an ammonium cation, an imine cation, or a metal cation, including but not limited to sodium, potassium, magnesium, zinc, or calcium cations, and wherein m is a number from 0 to 19.
The omega-3 very long chain polyunsaturated fatty acids of the present disclosure can have a terminal carboxyl group "-COOR", where "R" is intended to mean a group covalently bonded to a carboxyl group, such as an alkyl group. In the alternative, the carboxyl group is further intended to have a negative charge, such as "-COO-", and R is a cation, including metal cations, ammonium cations, and the like.
In some omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 0 to 15. Thus, m may be a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, wherein 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 omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 0,2, 4, 6, 8, 10, 12 or 14, wherein 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 omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 5 to 15, wherein 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 omega-3 very long chain polyunsaturated fatty acids, m is a number selected from the group consisting of 9 to 11, wherein the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
In some embodiments, the omega-3 very long chain polyunsaturated fatty acid is formic acid, i.e., R is hydrogen. In other embodiments, the omega-3 very long chain polyunsaturated fatty acid is a formate ester, wherein R is methyl, ethyl, or alkyl. When the omega-3 very long chain polyunsaturated fatty acid is a formate, R can be, but is not limited to, methyl or ethyl. In some embodiments, the omega-3 very long chain polyunsaturated fatty acid is a formate ester, wherein R is methyl.
In some embodiments, the omega-3 very long chain polyunsaturated fatty acid may be a formate salt, wherein R is an ammonium cation, an imine cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cations. In some advantageous embodiments, R is an ammonium cation or an iminium cation. R may be a sodium cation or a potassium cation. In some embodiments, R is a sodium cation.
The omega-3 very long chain polyunsaturated fatty acid or derivative of the present disclosure can have 32 or 34 carbons in its carbon chain and 6 alternating cis double bonds starting from position n-3 and have the formula a1(14Z,17Z,20Z,23Z,26Z,29Z) -docosahexaenoic acid, 17,20,23,26, 29Z) or formula a2(16Z,19Z,22Z,25Z,28Z,31Z) -thirty-four carbon-16, 19,22,25,28, 31-hexaenoic acid):
in some embodiments of omega-3 very long chain polyunsaturated fatty acids, the carboxyl derivative is part of a glycerol-derived phospholipid that can be readily prepared by using methods known in the art starting from the formic acid form of the n3VLC-PUFA of structures a or B and represented by structures C, D, E or F:
wherein C or E contains a total of 23 to 42 carbon atoms in the carbon chain and has 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 D or E contains a total of 23 to 42 carbon atoms in the carbon chain and has 5 alternating cis carbon-carbon double bonds starting at positions n-3, n-6, n-9, n-12 and n-15. In an advantageous embodiment, m is a number selected from the group consisting of 0 to 15. In other embodiments, m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, wherein the fatty acid component contains a total of 24, 26, 28, 30, 32, 34, 36, or 38 carbon atoms in the carbon chain. In further advantageous embodiments, m is a number selected from the group consisting of 0,2, 4, 6, 8, 10, 12, or 14, wherein 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 the group consisting of 5 to 15, wherein 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 the group consisting of 5, 7, 9, 11, 13, or 15, wherein 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 the group consisting of 4, 6, 8, 10, 12, or 14, wherein the fatty acid component contains a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In an advantageous embodiment, m is a number selected from the group consisting of 9 to 11, wherein the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
The monohydroxylated elongatoids of the present disclosure may have the structure G, H, I or J:
wherein compounds G and H have a total of 23 to 42 carbon atoms in the carbon chain, 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 position n-7; and wherein compounds I and J have a total of 23 to 42 carbon atoms in the carbon chain and have 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 position n-7; wherein R is hydrogen, methyl, ethyl, alkyl, or a cation selected from the group consisting of: an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of: sodium, potassium, magnesium, zinc or calcium cations, and wherein m is a number selected from the group consisting of 0 to 19; wherein compounds G and H may be present as an equimolar mixture; wherein compounds I and J may be present as an equimolar mixture; wherein the compounds G and H provided are predominantly one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group; and wherein compounds G and H are predominantly one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group.
As used herein and in other structures of the present disclosure, the compounds of the present disclosure are shown having a terminal carboxyl group "-COOR" which is intended to mean a group covalently bonded to a carboxyl group, such as an alkyl group. In the alternative, the carboxyl group is further intended to have a negative charge, such as "-COO-", and R is a cation, including metal cations, ammonium cations, and the like.
In some embodiments of the monohydroxylated elongatoids of the present disclosure, m is a number selected from the group consisting of 0 to 15. In other advantageous embodiments, m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, wherein 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 the group consisting of 0,2, 4, 6, 8, 10, 12, or 14, wherein 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 the group consisting of 5 to 15, wherein 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 the group consisting of 5, 7, 9, 11, 13, or 15, wherein 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 the group consisting of 4, 6, 8, 10, 12, or 14, wherein the fatty acid component contains a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In an advantageous embodiment, m is a number selected from the group consisting of 9 to 11, wherein the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
In some embodiments, the monohydroxylated elongatinoid of the present disclosure is formic acid, i.e., R is hydrogen. In other embodiments, the compound is a formate ester, wherein R is methyl, ethyl, or alkyl. In an advantageous embodiment, the compound is a formate ester, wherein R is methyl or ethyl. In an advantageous embodiment, the compound is a formate, wherein R is methyl. In other advantageous embodiments, the compound is a formate salt, wherein R is an ammonium cation, an imine cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cations. In some advantageous embodiments, R is an ammonium cation or an iminium cation. In other advantageous embodiments, R is a sodium cation or a potassium cation. In an advantageous embodiment, R is a sodium cation.
The dihydroxylated elongatins of the present disclosure may have structure K, L, M or N
Wherein the compounds K and L have a total of 23 to 42 carbon atoms in the carbon chain, 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 of 23 to 42 carbon atoms in the carbon chain, having 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 the group consisting of: an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of: sodium, potassium, magnesium, zinc or calcium cations, and wherein m is a number selected from the group consisting of 0 to 19; wherein compounds K and L may be present as an equimolar mixture; wherein compounds M and N may be present as an equimolar mixture, wherein compounds K and L are predominantly one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group; wherein said provided compounds M and N are predominantly one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group.
As used herein and in other structures of the present disclosure, the compounds of the present disclosure are shown having a terminal carboxyl group "-COOR" which is intended to mean a group covalently bonded to a carboxyl group, such as an alkyl group. In the alternative, the carboxyl group is further intended to have a negative charge, such as "-COO-", and R is a cation, including metal cations, ammonium cations, and the like.
In some embodiments of the dihydroxylated elongatoids of the present disclosure, m is a number selected from the group consisting of 5 to 15, wherein 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 advantageous embodiments, m is a number selected from the group consisting of 5, 7, 9, 11, 13, or 15, wherein 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 the group consisting of 4, 6, 8, 10, 12, or 14, wherein the fatty acid component contains a total of 27, 29, 31, 33, 35, or 37 carbon atoms in its carbon chain. In an advantageous embodiment, m is a number selected from the group consisting of 9 to 11, wherein the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
Some dihydroxylated elongatins of the present disclosure are formic acid, i.e., R is hydrogen. In other embodiments, the dihydroxylated elongations of the present disclosure are formate esters, wherein R is methyl, ethyl, or alkyl. In an advantageous embodiment, the compound is a formate ester, wherein R is methyl or ethyl. In an advantageous embodiment, the compound is a formate, wherein R is methyl.
In other embodiments, the dihydroxylated elongatinoid of the present disclosure is a formate salt, wherein R is an ammonium cation, an imine cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cations. In some advantageous embodiments, R is an ammonium cation or an iminium cation. In other advantageous embodiments, R is a sodium cation or a potassium cation. In an advantageous embodiment, R is a sodium cation.
The alkynyl monohydroxylated elongatoids of the present disclosure may have the structure O, P, Q or R:
wherein compounds O and P have a total of 23 to 42 carbon atoms in the carbon chain, 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; wherein compounds I and J have a total of 23 to 42 carbon atoms in the carbon chain, 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, ethyl, alkyl, or a cation selected from the group consisting of: an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of: sodium, potassium, magnesium, zinc or calcium cations, and wherein m is a number selected from the group consisting of 0 to 19; wherein compounds O and P may be present as an equimolar mixture; wherein compounds Q and R may be present as an equimolar mixture; wherein the compounds O and P provided are predominantly one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group; and wherein the compounds O and P provided are predominantly one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group.
As used herein and in other structures of the invention, the alkynyl monohydroxylated elongations of the present disclosure are shown to have a terminal carboxyl group "-COOR" which is intended to mean a group covalently bonded to a carboxyl group, such as an alkyl group. In the alternative, the carboxyl group is further intended to have a negative charge, such as "-COO-", and R is a cation, including metal cations, ammonium cations, and the like.
In some embodiments, m is a number selected from the group consisting of 0 to 15. In other embodiments, m is a number selected from 1, 3, 5, 7, 9, 11, 13, or 15, wherein the fatty acid component contains a total of 24, 26, 28, 30, 32, 34, 36, or 38 carbon atoms in its carbon chain.
In further embodiments, m is a number selected from the group consisting of 0,2, 4, 6, 8, 10, 12, or 14, wherein 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 the group consisting of 5 to 15, wherein 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 the group consisting of 5, 7, 9, 11, 13, or 15, wherein 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 the group consisting of 4, 6, 8, 10, 12, or 14, wherein 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 the group consisting of 9 to 11, wherein the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
In some embodiments, the alkynyl monohydroxylated elongatoids of the present disclosure are formic acid, i.e., R is hydrogen. In other embodiments, the alkynyl monohydroxylated elongations of the present disclosure are formates wherein R is methyl, ethyl or alkyl. In embodiments, the alkynyl monohydroxylated elongatoids of the present disclosure are formates wherein R is methyl or ethyl.
In some embodiments, R is methyl. In other embodiments, the alkynyl monohydroxyelongations of the present disclosure may be formate salts, wherein R is an ammonium cation, an imine cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cations. In some embodiments, R is an ammonium cation or an iminium cation. In other embodiments, R is a sodium cation or a potassium cation. In an embodiment, R is a sodium cation.
The alkynyl dihydroxylated elongatinoid may have the structure S, T, U or V:
wherein the compounds S and T have a total of 23 to 42 carbon atoms in the carbon chain, 3 cis-carbon double bonds starting at positions n-3, n-12, n-15 and n-18, 2 trans-carbon double bonds starting at positions n-9 and n-11 and a carbon-carbon triple bond starting at position n-7; and wherein the compounds U and V have a total of 23 to 42 carbon atoms in the carbon chain and have 2 cis-carbon double bonds starting at positions n-3 and n-15, 2 trans-carbon double bonds starting at positions n-9 and n-11 and a carbon-carbon triple bond starting at position n-7; wherein R is hydrogen, methyl, ethyl, alkyl, or a cation selected from the group consisting of: an ammonium cation, an iminium cation, or a metal cation selected from the group consisting of: sodium, potassium, magnesium, zinc or calcium cations, and wherein m is a number selected from the group consisting of 0 to 19; wherein compounds S and T may be present as an equimolar mixture; wherein compounds U and V may be present as an equimolar mixture.
In some embodiments, provided compounds S and T are predominantly one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group; wherein the compounds U and V are provided predominantly as one enantiomer having a defined (S) or (R) chirality at the carbon bearing the hydroxyl group.
As used herein and in other structures of the invention, the compounds of the invention are shown to have a terminal carboxyl group "-COOR" which is intended to mean a group covalently bonded to a carboxyl group, such as an alkyl group. In the alternative, the carboxyl group is further intended to have a negative charge, such as "-COO-", and R is a cation, including metal cations, ammonium cations, and the like.
In some embodiments, m is a number selected from the group consisting of 5 to 15, wherein 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 the group consisting of 5, 7, 9, 11, 13, or 15, wherein 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 the group consisting of 4, 6, 8, 10, 12, or 14, wherein 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 the group consisting of 9 to 11, wherein the fatty acid component contains a total of 32 or 34 carbon atoms in its carbon chain.
In some embodiments, the provided compound is formic acid, i.e., R is hydrogen.
In other embodiments, provided compounds are formates, wherein R is methyl, ethyl, or alkyl. In embodiments, provided compounds are formates, wherein R is methyl or ethyl. In an embodiment, provided compounds are formates, wherein R is methyl. In other embodiments, provided compounds are formate salts, where R is an ammonium cation, an imine cation, or a metal cation selected from the group consisting of sodium, potassium, magnesium, zinc, or calcium cations. In some embodiments, R is an ammonium cation or an iminium cation. In other embodiments, R is a sodium cation or a potassium cation. In an embodiment, R is a sodium cation.
In an advantageous embodiment, the present disclosure provides a monohydroxylated 32 carbon methyl ester of formula G1, which is designated by the following: (S,14Z,17Z,20Z,23Z,25E,29Z) -27-hydroxytodeca-14, 17,20,23,25, 29-hexaenoic acid methyl ester; a monohydroxylated 32 carbon sodium salt of formula G2, the name: (S,14Z,17Z,20Z,23Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,23,25, 29-hexaenoate sodium; monohydroxylated 34 carbon methyl esters of the formula G3, the name: (S,16Z,19Z,22Z,25Z,27E,31Z) -29-hydroxytetradecanoyl-16, 19,22,25,27, 31-hexaenoic acid methyl ester; or a monohydroxylated 34 carbon sodium salt of formula G4, which is designated by the following: sodium-hexaenoate of (S,16Z,19Z,22Z,25Z,27E,31Z) -29-hydroxytetradecyl-16, 19,22,25,27, 31:
in other advantageous embodiments, the present disclosure provides a dihydroxylated 32-carbon methyl ester of formula K1, entitled: (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxy docosahexenoic acid methyl ester, 17,21,23,25, 29-hexaenoic acid methyl ester; a dihydroxylated 32-carbon sodium salt of formula K2, having the name: sodium (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxy-tridodecen-14, 17,21,23,25, 29-hexaenoate; a dihydroxylated 34-carbon methyl ester of formula K3, designated by the name: (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxy-trinetradecanoic-16, 19,23,25,27, 31-hexaenoic acid methyl ester; or a dihydroxylated 34 carbon sodium salt of formula K4, designated by the name: sodium (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxy-trinetradeca-16, 19,23,25,27, 31-hexaenoate:
in other embodiments, the present invention provides alkynyl monohydroxylated 32 carbon methyl esters of the formula O1, which is designated by the following: (S,14Z,17Z,20Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,25, 29-pentaen-23-ynoic acid methyl ester; an alkynyl monohydroxylated 32-carbon sodium salt of formula O2, the name: sodium (S,17Z,20Z,25E,29Z) -27-hydroxydodecacarbonate-17, 20,25, 29-tetraen-23-ynoate; an alkynyl monohydroxylated 34 carbon methyl ester of the formula O3, the name: (S,16Z,19Z,22Z,27E,31Z) -29-hydroxytetradecyl-16, 19,22,27, 31-pentaen-25-ynoic acid methyl ester; an alkynyl monohydroxylated 34 carbon sodium salt of formula O4, the name: sodium (S,16Z,19Z,22Z,27E,31Z) -29-hydroxytetradecyl-16, 19,22,27, 31-pentaen-25-ynoate:
in other advantageous embodiments, the present invention provides alkynyl dihydroxylated 32-carbon methyl esters of formula S1, entitled: (14Z,17Z,20R,21E,23E,27S,29Z) -20, 27-dihydroxy-tridodecen-14, 17,21,23, 29-pentaene-25-ynoic acid methyl ester; an alkynyl dihydroxylated 32-carbon sodium salt of formula S2, having the name: sodium (14Z,17Z,20R,21E,23E,27S,29Z) -20, 27-dihydroxy-tridodecen-14, 17,21,23, 29-pentaen-25-ynoate; or an alkynyl dihydroxylated 34 carbon methyl ester of formula S3, designated by the following name: (16Z,19Z,22R,23E,25E,29S,31Z) -22, 29-dihydroxy trinetradeca-16, 19,23,25, 31-pentaene-27-ynoic acid methyl ester; or an alkynyl dihydroxylated 34 carbon sodium salt of formula S4, designated by the following name: (16Z,19Z,22R,23E,25E,29S,31Z) -22, 29-dihydroxy-trinetradeca-16, 19,23,25, 31-pentaen-27-ynoic acid sodium salt.
Methods of preparation and manufacture of the provided compounds: the provided compounds of the present disclosure can be readily prepared by employing methods known in the art starting from commercially available materials, as summarized in schemes 1-5 shown in fig. 24-28.
Scheme 1 (fig. 24) shows a detailed method for the stereocontrolled total synthesis of compound form O, where n is 9, and the fatty acid chain contains a total of 32 carbon atoms, and the R group is a methyl or sodium cation. In particular, scheme 1 shows the synthesis of the compounds ELV-N32-Me and ELV-N32-Na starting with pentadecane-14-ynoic acid methyl ester (S1). This procedure gave the compounds ELV-N34-Me and ELV-N34-Na, starting from heptadeca-16-alkynoate (T1). Alkynyl precursors of ELV-N32-Me and ELV-N32-Na (i.e., 13a, 13b, 15a, and 15b) are also in the provided compounds X and Z in the present disclosure. Scheme 1 provides reagents and conditions for preparing the provided compounds by employing reaction conditions typical for this type of reaction.
Scheme 2 (figure 25) describes the total synthesis of dihydroxylated elongatoids K and L and their alkyne precursors S and T by starting with intermediates 2,5 and 7 also used in scheme 1. Conversion of protected (R) epoxide 4 to intermediate 15, as well as coupling of 7 and 15 and subsequent conversion to intermediate 17, can be accomplished according to literature procedures (tetrahedron letters, 2012; 53(14): 1695-8).
Catalytic cross-coupling between intermediates 2 or 17 or between intermediates 5 or 17 followed by deprotection forms the alkynyl compounds S and T which are then selectively reduced to form the dihydroxylated elongatoids K and L. The corresponding formic acid is obtained by hydrolysis and acidification and can be converted into the formate by adding an equivalent amount of the corresponding base. K, L, S and T-type dihydroxylated elongatoids having at least 23 carbons and up to 42 carbons in their carbon chain can be prepared similarly by varying the carbon number in the alkyne starting material 7.
Scheme 3 (fig. 26) describes the total synthesis of dihydroxylated elongatins with five unsaturated double bonds and their alkyne precursors U and V, of the M and N type, by using the same alkynyl intermediates 2 and 5 also used in scheme 1. ("Tetrahedron letters.", (2012); 53(14): 1695-8).
The synthesis of the common intermediate 22 begins with formic acid 18, which is converted to the orthoester 19 using known methods (Tetrahedron letters, 1983,24(50), 5571-4). Reaction of the lithiated alkyne with epoxide 1 gives intermediate 21 which is converted to iodide intermediate 22, similar to the conversion from 16 to 17. Catalytic cross-coupling between intermediates 2 or 5 and 22 followed by deprotection forms alkynyl dihydroxy elongatoids U and V which are then selectively reduced to form dihydroxylated elongatins M and N.
The corresponding formic acid is obtained by hydrolysis and acidification and can be converted into the formate by adding an equivalent amount of the corresponding base. The dihydroxylated elongatoids of form M, N, U and V, having at least 23 carbons and up to 42 carbons in their carbon chain, can be prepared similarly by varying the carbon number in the alkyne formate 18.
Scheme 4 (fig. 27) shows a stereocontrolled total synthesis of 32-carbodihydroxylated elongatenoids starting with alkyne methyl ester 23, intermediate 15 and alkyne intermediate 2. In particular, this scheme illustrates the total synthesis of the 32-carbon alkynyl-like elongin compound ELV-N32-Me-acetylene and its conversion to the methyl ester of the elongin, ELV-N32-Me, the acid of the elongin, ELV-N32-H, and the sodium salt of the elongin, ELV-N32-Na.
Scheme 5 (fig. 28) shows a stereocontrolled total synthesis of 34-carbodihydroxylated elongatins starting with an alkyne methyl ester 30 and employing the same sequence of reactions as in scheme 4.
In particular, this scheme illustrates the total synthesis of the 34-carbon alkynyl-like elongin compound ELV-N34-Me-acetylene and its conversion to the methyl ester of the elongin, ELV-N34-Me, the acid of the elongin, ELV-N34-H, and the sodium salt of the elongin, ELV-N34-Na.
The chemistry presented in schemes 1-5 (fig. 24-28) can also be readily adapted to the total synthesis of additional mono-and dihydroxylated elongations having at least 23 carbons and up to 42 carbons in their carbon chains.
Pharmaceutical compositions for the treatment of diseases: in other embodiments, the present disclosure provides formulations of pharmaceutical compositions containing a therapeutically effective amount of one or more compounds provided herein, or salts thereof, in a pharmaceutically acceptable carrier.
Provided compositions contain one or more compounds provided herein, or salts thereof, and a pharmaceutically acceptable excipient, diluent, carrier, and/or adjuvant. The compounds are preferably formulated in suitable pharmaceutical formulations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs (for oral, buccal, intranasal, vaginal, rectal, ocular administration, intravitreal implanted reservoirs or sustained release nanodevices or dendrimers embedded in collagen or other materials on the ocular surface), or sterile solutions or suspensions (for parenteral administration), skin patches and transdermal patch formulations and dry powder inhalers. The provided formulations may be in the form of drops (e.g., eye drops), and the pharmaceutical formulations may further contain antioxidants and/or known agents for treating ocular diseases. Typically, the above compounds are formulated into Pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel introduction to Pharmaceutical Dosage Forms, fourth edition, 1985, 126).
Advantageous embodiments of the present disclosure provide pharmaceutical compositions containing various forms of the provided compounds (either in the form of free formic acid or a pharmaceutically acceptable salt thereof, or in the form of its corresponding ester or phospholipid derivative thereof). In other advantageous embodiments, the present disclosure provides pharmaceutical compositions containing one or more elongatoids (in the form of free formic acid or a pharmaceutically acceptable salt thereof, or the corresponding ester thereof) containing one or two hydroxyl groups at positions between n-3 and n-18 of very long chain polyunsaturated fatty acids.
In another advantageous embodiment, the present disclosure provides a pharmaceutical composition for maintaining and protecting skin at all ages and for treating a skin disease or condition. In some embodiments, the skin disease or disorder involves inflammation of the skin, hyperproliferation of the skin, or dehydration of the skin.
In embodiments, the present disclosure provides a composition for treating a skin disease or condition selected from the group consisting of: dermatitis, eczema, atopic dermatitis, neurodermatitis, light contact dermatitis, xerotic eczema, seborrheic eczema, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis, neurodermatitis and self-eczematization, radiation-induced skin inflammation or psoriasis.
In the provided compositions, an effective concentration of one or more compounds or pharmaceutically acceptable derivatives is admixed with a suitable pharmaceutical carrier or vehicle. As noted above, the compounds may be derivatized to the corresponding salts, esters, enol ethers or esters, acids, bases, solvates, hydrates, or prodrugs prior to formulation. The concentration of the compound in the composition, when administered, is effective to deliver an amount that treats, prevents, or ameliorates one or more symptoms of the disease, disorder, or condition.
As described herein, the compositions can be readily prepared by employing methods known in the art. The composition may be a component of a pharmaceutical formulation.
The pharmaceutical formulation may further contain known agents for the treatment of inflammatory or degenerative diseases, including neurodegenerative diseases. The provided compositions can be used as prodrugs of fatty acids and can be converted to free fatty acids upon localization to the diseased site.
The present disclosure also provides a packaged composition or pharmaceutical composition for preventing, restoring, or treating a disease or condition. Other packaged or pharmaceutical compositions provided by the present disclosure further comprise an indication comprising at least one of: instructions for using the composition to treat a disease or condition. The kit may further comprise suitable buffers and reagents known in the art for administering various combinations of the above listed components to a host.
Pharmaceutical formulation: embodiments of the present disclosure include the compositions or pharmaceutical compositions identified herein, and may be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, naturally occurring or synthetic antioxidants and/or adjuvants. Additionally, embodiments of the present disclosure include compositions or pharmaceutical compositions formulated with one or more pharmaceutically acceptable auxiliary substances. In particular, the composition or pharmaceutical composition may be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and/or adjuvants to provide one embodiment of the composition of the present disclosure.
A variety of pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients are well described in various publications, including, for example, a.gennaro (2000), "ramendon: science and Practice of Pharmacy (Remington: the science and Practice of Pharmacy, 20 th edition, Lippincott, Williams, & Wilkins; pharmaceutical Dosage Forms and Drug Delivery Systems (Pharmaceutical Dosage Forms and Drug Delivery Systems) (1999), edited by h.c. ansel et al, 7 th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000), edited by A.H.Kibbe et al, 3 rd edition, Amer.pharmaceutical Assoc. Pharmaceutically acceptable excipients (e.g., vehicles, adjuvants, carriers, or diluents) are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances (e.g., pH adjusting and buffering agents, tonicity adjusting agents, stabilizing agents, wetting agents, etc.) are readily available to the public.
In one embodiment of the present disclosure, the composition or pharmaceutical composition may be administered to a subject using any means capable of producing the desired effect. Thus, the composition or pharmaceutical composition may be incorporated into a variety of formulations for therapeutic administration. For example, the composition or pharmaceutical composition may be formulated into a pharmaceutical composition by combining with an appropriate pharmaceutically acceptable carrier or diluent, and may be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
Suitable excipient vehicles for the composition or pharmaceutical composition are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the vehicle may, if desired, contain minor amounts of auxiliary substances, for example wetting or emulsifying agents, antioxidants or pH buffering agents. Methods of making such dosage forms are known to those skilled in the art or will be apparent upon consideration of this disclosure. See, e.g., Remington's pharmaceutical sciences, Mack Publishing Company, Iston, Pa., 17 th edition, 1985. In any event, the composition or formulation to be administered will contain an amount of the composition or pharmaceutical composition sufficient to achieve the desired state in the subject being treated.
The compositions of the present disclosure may comprise those that include a sustained or controlled release matrix. Additionally, embodiments of the present disclosure may be used in conjunction with other therapies using sustained release formulations. As used herein, a sustained release matrix is a matrix made of a material (typically a polymer) that is 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. The sustained release matrix is desirably selected from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolides (polymers of glycolic acid), polyglycolides (copolymers of lactic and glycolic acid), polyanhydrides, poly (ortho) esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, formic acid, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids (e.g., phenylalanine, tyrosine, isoleucine), polynucleotides, polyvinyl propylene, polyvinyl pyrrolidone, and silicone. Illustrative biodegradable matrices include polylactide matrices, polyglycolide matrices, and polyglycolide (a copolymer of lactic and glycolic acid) matrices. In another embodiment, the pharmaceutical compositions (and combination compositions) of the present disclosure may be delivered in a controlled release system. For example, the composition or pharmaceutical composition may be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, a pump (Sefton (1987), CRI (CRC Crit. Ref. biomed. Eng.), 14:201, Buchwald et al (1980), Surgery (Surgery), 88:507, Saudek et al (1989), New England journal of medicine (N.Engl. J. Med.), 321:574) may be used. In another embodiment, a polymeric material is used. In yet another embodiment, the controlled release system is placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose. In yet another embodiment, the controlled release system is placed in proximity to the therapeutic target, thus requiring only a portion of the whole body. Other controlled release systems are discussed in the reviews of Langer (1990), Science (Science), 249: 1527-.
In another embodiment, the compositions of the present disclosure (and combined compositions, separately or together) include those formed by impregnating a composition or pharmaceutical composition described herein into an absorbent material (e.g., sutures, bandages, and gauze) or coated onto the surface of a solid phase material (e.g., surgical staples, zippers, and catheters for delivery of the composition). Other delivery systems of the present type will be apparent to those skilled in the art in view of this disclosure.
In another embodiment, the compositions or pharmaceutical compositions of the present disclosure (as well as separate or together combined compositions) may be part of a delayed release formulation. Delayed release dosage formulations may be prepared as described in standard references such as Pharmaceutical dosage forms-tablets (Pharmaceutical dosage forms), editors by Liberman et al (new york, Marcel Dekker, inc., 1989), remington: science and practice of pharmacy (Remington-the science and practice of pharmacy), 20 th edition, Lippincott Williams & Wilkins, Baltimore, Maryland, 2000, and Pharmaceutical dosage form and drug delivery systems (Pharmaceutical systems for and delivery systems), 6 th edition, Ansel et al, (Meidia, Pa.: Williams and Wilkins, 1995). These references provide information on the excipients, materials, equipment and methods used to prepare tablets and capsules, as well as delayed release dosage forms of tablets, capsules and granules. These references provide information on carriers, materials, equipment and methods for making tablets and capsules, and delayed release dosage forms of tablets, capsules and granules.
Embodiments of the compositions or pharmaceutical compositions may have been administered to a subject in one or more doses. One skilled in the art will readily appreciate that dosage levels may vary depending on the specifics of the composition or pharmaceutical composition being administered, the severity of the symptoms, and the subject's sensitivity to side effects. Advantageous dosages for a given compound can be readily determined by one skilled in the art in a variety of ways.
In one embodiment, multiple doses of the composition or pharmaceutical composition are administered. The frequency of administration of the composition or pharmaceutical composition can vary depending on any of a variety of factors (e.g., severity of symptoms, etc.). For example, in one embodiment, the composition or pharmaceutical composition may be administered monthly, twice monthly, three times monthly, every other week (qow), weekly (qw), twice weekly (biw), three times weekly (tiw), four times weekly, five times weekly, six times weekly, every other day (qod), once daily (qd), twice daily (qid), three times daily (tid), or four times daily. As discussed above, in one embodiment, the composition or pharmaceutical composition is administered 1 to 4 times daily over a period of 1 to 10 days.
The duration of administration of the composition or pharmaceutical composition analog (e.g., the period of time over which the composition or pharmaceutical composition is administered) can vary depending on any of a variety of factors (e.g., patient response, etc.). For example, the compositions or pharmaceutical compositions may be administered in combination or separately over a period 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 to treat a condition or disease can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed may also depend on the route of administration and may be determined at the discretion of the practitioner and the condition of each patient.
The administration route is as follows: embodiments of the present disclosure provide methods and compositions for administering active agents to a subject (e.g., a human) using any available methods and routes suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local routes of administration. Routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, intravitreal, topical, intravenous, rectal, nasal, oral, and other enteral and parenteral routes of administration. The routes of administration can be combined, if desired, or adjusted depending on the agent and/or desired effect. The active agent may be administered in a single dose or in multiple doses.
The n-3VLC-PUFA and its biogenic derivatives are formed in cells and are not part of the human diet. Advantageous routes of administration of the novel compounds provided herein would include oral and parenteral administration (including on the ocular surface), intravitreal and subretinal injection into the eye to bypass intestinal absorption, the intestinal-hepatic and blood-ocular barriers. The provided formulations may be delivered in the form of drops (e.g., eye drops) or by any other conventional method for treating an eye disease.
Parenteral routes of administration other than administration by inhalation 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 performed to affect systemic or local delivery of the composition. Where systemic delivery is desired, administration typically involves local or mucosal administration of the pharmaceutical formulation, either invasive or systemically absorbed. In one embodiment, the composition or pharmaceutical composition may also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not limited to, oral and rectal delivery (e.g., using suppositories).
Methods of administering the composition or pharmaceutical composition through the skin or mucosa include, but are not limited to, topical application, transdermal delivery, injection, and epidermal administration of suitable pharmaceutical formulations. For transdermal delivery, absorption enhancers or iontophoresis are suitable methods, and iontophoretic delivery can be accomplished using commercially available "patches" that continuously deliver their products via an electrical pulse for a continuous period of several days or longer.
The compounds and compositions provided by the present disclosure are capable of restoring homeostasis and inducing survival signaling in certain cells undergoing oxidative stress or other homeostatic disruption. The present disclosure also provides methods of using the provided compounds and compositions containing hydroxylated derivatives of very long chain polyunsaturated fatty acids in the form of free formic acid or a pharmaceutically acceptable salt thereof, or a corresponding ester or other prodrug derivative thereof. The provided compounds can be readily prepared by employing methods known in the art starting from commercially available materials.
The biological activity of the provided compounds (exemplified by the elongatinoid derivatives ELV-N32-Me, ELV-N32-Na, ELV-N34-Me and ELV-N34-Na) is attributed to their ability to reach targeted human cells and exert their biological effects by entering the cells or/and by acting at membrane-bound receptors. Alternatively, the provided compounds may act via intracellular receptors (e.g., nuclear membrane), and thus they will specifically act by affecting key signal transduction events. Administration of a pharmaceutical composition containing a provided compound and a pharmaceutically acceptable carrier restores homeostatic balance and promotes survival of certain cells necessary to maintain normal function. The provided compounds, compositions and methods can be used for the prevention and treatment of inflammatory, degenerative and neurodegenerative diseases. The present disclosure targets key steps in the initiation and early progression of these conditions by mimicking the specific biology of the innate cell/organ response to achieve efficacy, selectivity, no side effects, and sustained biological activity.
Accordingly, one aspect of the present 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.
In some embodiments of the present aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and is formulated for delivery of an amount of the at least one very long chain polyunsaturated fatty acid effective to reduce the pathological state of the tissue of the recipient subject or the onset of the pathological state of the tissue of the recipient subject.
In some embodiments of this aspect of the disclosure, the pathological condition may be aging or inflammation of the tissue of the recipient subject.
In some embodiments of the present aspect of the present disclosure, the composition may be formulated for topical delivery of the at least one very long chain polyunsaturated fatty acid tissue to the skin of a recipient subject.
In some embodiments of this aspect of the present disclosure, the pathological state may be a pathological state of neural tissue of the recipient subject.
In some embodiments of the present aspect of the disclosure, the composition may further comprise at least one nutritional component, and the composition may be formulated for oral or parenteral delivery of the at least one very long chain polyunsaturated fatty acid to a recipient subject.
In some embodiments of this aspect of the present disclosure, the at least one very long chain polyunsaturated fatty acid may have from about 26 to about 42 carbon atoms in its carbon chain.
In some embodiments of this aspect of the present disclosure, the at least one very long chain polyunsaturated fatty acid may have 32 or 34 carbon atoms in its carbon chain.
In some embodiments of this aspect of the present disclosure, the very long chain polyunsaturated fatty acid may have five or six double bonds with cis geometry in its carbon chain.
In some embodiments of this aspect of the disclosure, the very long chain polyunsaturated fatty acid is 14Z,17Z,20Z,23Z,26Z,29Z) -docosahexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z) -triacontahexaenoic acid 16,19,22,25,28, 31Z.
Another aspect of the disclosure encompasses embodiments of a composition comprising at least one type of auxin having at least 23 carbon atoms in its carbon chain.
In some embodiments of the present aspect of the disclosure, the composition may further comprise a pharmaceutically acceptable carrier and may be formulated for delivery of an amount of the at least one carotenoid effective to reduce the pathological state of or delay at least one aging effect in a tissue of a recipient subject.
In some embodiments of this aspect of the disclosure, the pathological condition may be aging or inflammation of the tissue of the recipient subject.
In some embodiments of this aspect of the present disclosure, the composition may be formulated for topical delivery of the at least one elongatin to the skin of a recipient subject.
In some embodiments of this aspect of the present disclosure, the pathological state may be a pathological state of neural tissue of the recipient subject.
In some embodiments of this aspect of the present disclosure, the composition may further comprise at least one nutritional component, and the composition may be formulated for oral or parenteral delivery of the at least one elongatin to a recipient subject.
In some embodiments of this aspect of the present disclosure, the at least one auxin may be selected from the group consisting of: monohydroxylated elongatins, dihydroxylated elongatins, alkynyl monohydroxylated elongatins and alkynyl dihydroxylated elongatins or any combination thereof.
In some embodiments of this aspect of the present disclosure, the at least one elongatoid may be a combination of elongatoids, wherein the combination is selected from the group consisting of: monohydroxylated and dihydroxylated elongases; monohydroxylated elongatoids and alkynyl monohydroxylated elongatoids; monohydroxylated elongatins and alkynyl-dihydroxylated elongatins; dihydroxylated elongatins and alkynyl monohydroxylated elongatins; dihydroxylated elongatins and alkynyl dihydroxylated elongatins; monohydroxylated elongatins, dihydroxylated elongatins and alkynyl monohydroxylated elongatins; monohydroxylated extenders, dihydroxylated extenders and alkynyl-dihydroxylated extenders; and monohydroxylated elongatoids, dihydroxylated elongatins, alkynyl monohydroxylated elongatins and alkynyl dihydroxylated elongatins, wherein each elongatoid is independently a racemic mixture, a separate enantiomer or a combination of enantiomers (wherein the amount of one enantiomer is greater than the amount of the other); and wherein each dihydroxylated elongatinoid is independently a diastereomeric mixture, separated diastereomers, or a combination of diastereomers (wherein the amount of one diastereomer is greater than the amount of the other diastereomer).
In some embodiments of this aspect of the present disclosure, the composition may further comprise at least one very long chain polyunsaturated fatty acid having at least 23 carbon atoms in its carbon chain.
In some embodiments of this aspect of the present disclosure, the at least one very long chain polyunsaturated fatty acid may have from about 26 to about 42 carbon atoms in its carbon chain.
In some embodiments of this aspect of the present disclosure, the at least one very long chain polyunsaturated fatty acid may have five or six cis-geometry double bonds in its carbon chain.
In some embodiments of this aspect of the present disclosure, the at least one very long chain polyunsaturated fatty acid can be 14Z,17Z,20Z,23Z,26Z,29Z) -docosahexa-14, 17,20,23,26, 29-hexaenoic acid or (16Z,19Z,22Z,25Z,28Z,31Z) -triacontahete-16, 19,22,25,28, 31-hexaenoic acid.
In some embodiments of this aspect of the present disclosure, the monohydroxylated elongatoids may be selected from the group consisting of formulas G, H, I or J:
wherein: n may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group.
In some embodiments of this aspect of the present disclosure, the pharmaceutically acceptable cation may be an ammonium cation, an iminium cation, or a metal cation.
In some embodiments of this aspect of the present disclosure, the metal cation may be a sodium, potassium, magnesium, zinc, or calcium cation.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers G and H, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers I and J, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of G or H in an amount that exceeds the other enantiomer of G or H.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of I or J in an amount that exceeds the amount of the other enantiomer of I or J.
In some embodiments of this aspect of the present disclosure, the monohydroxylated elongatoids may be selected from the group consisting of: (S,14Z,17Z,20Z,23Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,23,25, 29-hexaenoic acid methyl ester (G1), (S,14Z,17Z,20Z,23Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,23,25, 29-hexaenoic acid sodium salt (G2), (S,16Z,19Z,22Z,25Z,27E,31Z) -29-hydroxytetradecyl-16, 19,22,25,27, 31-hexaenoic acid methyl ester (G3); and sodium (S,16Z,19Z,22Z,25Z,27E,31Z) -29-hydroxytetradeca-16, 19,22,25,27, 31-hexaenoate (G4), each having the formula:
in some embodiments of this aspect of the present disclosure, the dihydroxylated elongatinoid may be selected from the group consisting of formulas K, L, M and N:
wherein: m may be from 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group.
In some embodiments of this aspect of the present disclosure, the pharmaceutically acceptable cation may be an ammonium cation, an iminium cation, or a metal cation.
In some embodiments of this aspect of the present disclosure, the metal cation may be a sodium, potassium, magnesium, zinc, or calcium cation.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of diastereomers K and L, wherein the diastereomer has (S) or (R) chirality at position n-6 and (R) chirality at position n-13.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of diastereomers M and N, wherein the diastereomer has (S) or (R) chirality at position N-6 and (R) chirality at position N-13.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of K or L in an amount that exceeds the amount of the other diastereomer of K or L.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of M or N in an amount that exceeds the amount of the other diastereomer of M or N.
In some embodiments of this aspect of the present disclosure, the dihydroxylated elongatinoid may be selected from the group consisting of: (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxytridecane-14, 17,21,23,25, 29-hexaenoic acid methyl ester (K1), (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxytridecane-14, 17,21,23,25, 29-hexaenoic acid sodium ester (K2), (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxytrinetradecanone-16, 19,23,25,27, 31-hexaenoic acid methyl ester (K3) and (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxytetradecane-16, 19,23,25,27, 31-hexaenoic acid sodium ester (K4), respectively having the formula:
in some embodiments of this aspect of the present disclosure, the alkynyl monohydroxylated elongatinoid may be selected from the group consisting of formula O, P, Q or R:
wherein: m may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group, and wherein: compounds O and P each have a total of 23 to 42 carbon atoms in the carbon chain, with 4 cis carbon-carbon double bonds located at the beginning of n-3, n-12, n-15 and n-18; having a trans carbon-carbon double bond at the position starting at n-7 and a carbon-carbon triple bond starting at position n-9; and compounds Q and R each have a total of 23 to 42 carbon atoms in the carbon chain, 3 cis carbon-carbon double bonds starting at positions n-3, n-12 and n-15, a trans carbon-carbon double bond starting at n-7 and a carbon-carbon triple bond starting at position n-9.
In some embodiments of this aspect of the present disclosure, the alkynyl monohydroxylated elongatinoid may be selected from the group consisting of: (S,14Z,17Z,20Z,25E,29Z) -27-hydroxytridecyl-14, 17,20,25, 29-pentaen-23-ynoic acid methyl ester (O1); (S,17Z,20Z,25E,29Z) -27-hydroxytridecano-17, 20,25, 29-tetraen-23-ynoic acid sodium salt (O2); (S,16Z,19Z,22Z,27E,31Z) -29-hydroxytetradecyl-16, 19,22,27, 31-pentaene-25-ynoic acid methyl ester (O3); and sodium (S,16Z,19Z,22Z,27E,31Z) -29-hydroxytetratetradec-16, 19,22,27, 31-pentaen-25-ynoate (O4), each having the formula:
in some embodiments of this aspect of the present disclosure, the pharmaceutically acceptable cation may be an ammonium cation, an iminium cation, or a metal cation.
In some embodiments of this aspect of the present disclosure, the metal cation may be a sodium, potassium, magnesium, zinc, or calcium cation.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers O and P, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of enantiomers Q and R, wherein the enantiomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of O or P in an amount that exceeds the other enantiomer of O or P.
In some embodiments of this aspect of the disclosure, the composition may include one enantiomer of Q or R in an amount that exceeds the amount of the other enantiomer of Q or R.
In some embodiments of this aspect of the present disclosure, the elongatinoid may be an alkynyl dihydroxylated elongatinoid selected from the group consisting of formula S, T, U or V:
wherein: m may be 0 to 19, and-CO-OR may be a carboxylic acid group OR a salt OR 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, R can be an alkyl group, and wherein: compounds S and T each have a total of 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 n-7; and compounds U and V each have a total of 23 to 42 carbon atoms in the carbon chain, with 2 cis carbon-carbon double bonds starting at positions n-3 and n-15; 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.
In some embodiments of this aspect of the present disclosure, the pharmaceutically acceptable cation is an ammonium cation, an iminium cation, or a metal cation.
In some embodiments of this aspect of the present disclosure, the metal cation is a sodium, potassium, magnesium, zinc, or calcium cation.
In some embodiments of this aspect of the present disclosure, the alkynyl monohydroxylated elongatinoid may be selected from the group consisting of: (14Z,17Z,20R,21E,23E,27S,29Z) -20, 27-dihydroxy-tridodecen-14, 17,21,23, 29-pentaene-25-ynoic acid methyl ester (S1); (14Z,17Z,20R,21E,23E,27S,29Z) -20, 27-dihydroxy-tridodecen-14, 17,21,23, 29-pentaen-25-ynoic acid sodium salt (S2); (16Z,19Z,22R,23E,25E,29S,31Z) -22, 29-dihydroxy trinetradeca-16, 19,23,25, 31-pentaene-27-ynoic acid methyl ester (S3); and (16Z,19Z,22R,23E,25E,29S,31Z) -22, 29-dihydroxy-trinetradeca-16, 19,23,25, 31-pentaene-27-ynoic acid sodium salt (S4), each having the formula:
in some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of diastereomers S and T, wherein the diastereomers have (S) or (R) chirality at the carbon bearing the hydroxyl group.
In some embodiments of this aspect of the present disclosure, the composition can include equimolar amounts of diastereomers U and V, wherein the diastereomer has (S) or (R) chirality at position n-6 and (R) chirality at position n-13.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of S or T in an amount that exceeds the amount of the other diastereomer of S or T.
In some embodiments of this aspect of the disclosure, the composition may include one diastereomer of U or V in an amount that exceeds the amount of the other diastereomer of U or V.
Other compositions, compounds, methods, features and advantages of the disclosure will be, or will become, apparent to one of ordinary skill in the art upon examination of the following figures, detailed description and examples. It is intended that all such additional compositions, compounds, methods, features and advantages be included within this description and be within the scope of the present disclosure.
Examples of the invention
Example 1
Primary cultures of cortical neurons: primary cultures of cortical and hippocampal neurons were harvested from 18-day-old embryos (E18) taken from February Sprague-Dawley (SD) rats (Charles River Lab., Wilmington, Mass.) determined during pregnancy. Briefly, SD rats determined for pregnancy were euthanized and embryos were collected under sterile conditions. Embryonic brains were dissected out on ice with forceps and placed into petri dishes containing ice-cold Hank's Balanced Salt Solution (HBSS) (GIBCO). The meninges were removed under a dissecting microscope and the cortical tissue was cut into small pieces with micro-spring scissors. These tissues were transferred to 15ml tubes containing trypsin-EDTA (0.025% HBSS solution) and DNase I. The tubes were incubated in a 37 ℃ cabinet for 15 minutes with stirring every 5 minutes. After stopping the tryptic digestion with 5ml of 10% FBS, the tissues were ground 15 times with fire-polished pasteur pipettes. The cell pellet was allowed to stand for 2 minutes and the supernatant was transferred to a 15ml microcentrifuge tube. The supernatant was filtered through a 70 μm pore size filter (Corning cell strainer) and centrifuged at 1000rpm for 5 minutes. The cells were then resuspended in a suspension containing 2% B27(GIBCO) and 2% N2(GIBCO) supplement and 0.5mM glutamine, 50U/ml penicillin/streptomycin in neurobasal medium (GIBCO).
Cells were counted using a Neubauer hemocytometer. Will be 1x106The individual cells were seeded on poly-D-lysine coated 12-well cell culture dishes (CORNING) and cultured in an incubator (37 ℃, 5% CO)2). The medium was replaced for the first time after 24 hours and then half of the medium was replaced with fresh medium every three days. As a result, neurons were obtained with 90% purity as determined by class III β tubulin, GFAP and Hoechst 33258 staining. Cells were maintained in culture for about 2 weeks until they were exposed to uncompensated oxidative stress, OGD, or NMDA excitotoxicity.
Example 2
Antibody: the following antibodies were used: beta-catenin (cat # sc-7963, lot # K0812) Santa cruz biotechnology: (use concentration 1: 50); ZO-1 (Cat No. 187430, batch No. 1633993A) Life technologies: (use concentration 1: 100); MITF (Cat No. ab59232, batch No. GR52475-3) ABCAM: (use concentration 1: 250); RPE65 (catalog No. ab78036, lot No. 3GR254004-1), ABCAM: (use concentration 1: 250).
Example 3
Human RPE cell cultures: primary human retinal pigment epithelial cells (RPE) prepared from donor eyes without ocular pathology were plated and transformed after eight passages. Cells were plated in T75 flasks in MEM medium containing 10% FBS, 5% NCS, MEM-NEAA (ThermoFisher Scientific, Waltham, Mass.), 1 XPicillin/streptomycin, and 10ng/ml FGF at 37 deg.C, 5% CO2Incubation at 99% relative humidity for 24-48 hours followed by incubation with 10 μ M free 32:6 and 34:6 fatty acid mixtures for 24 hours.
FIGS. 6A and 6B depict immunostaining of primary human RPE cells using the specific markers ZO-1 (occludin-1), RPE65, MITF (microphthalmia-associated transcription factor), and β -catenin, and light microscopy depicting morphology of primary human RPE cells at different passages in culture. ARPE-19 cells were grown and maintained in T-75mM flasks in DMEM F-12 medium containing 10% FBS, and at 5% CO2The constant supply of (2) was incubated at 37 ℃. Prior to exposure, 75-80% confluent cells (72 hours in DMEM/F12+ 10% FBS) in 6-well plates were serum starved for 8 hours.
Example 4
RPE cells were exposed to UOS and VLC-PUFA: for cell viability assay experiments, hRPE cells were concomitantly treated with NPD1(200nM), 32-6, 34-6 (3. mu.M each) fatty acid, or both 32-6 and 34-6. hRPE medium was supplemented with 3. mu.M 32-6 and 34-6 throughout the duration of the experiment. 15lox-1 inhibitor (10. mu.M) was added to the cells 1 hour prior to OS induction and remained unchanged throughout the experiment. Cells were fixed with 4% PFA and stained with Hoescht.
Prior to exposure, 75-80% confluent ARPE19 cells (grown in DMEM/F12+ 10% FBS for 72 hours) in 6-well plates were serum-starved for 8 hours. Serum-starved cells were treated with TNF-alpha (Sigma-Aldrich, St. Louis, Mo.) (10ng/ml) and H2O2(600 μ M) treatment to induce oxidative stress and simultaneous challenge with increasing concentrations (50-500nM) of VLC-PUFA (C32:6n3 and C34:6n3), where oxidative stress lasted 16 hours (apoptosis) and 6 hours before apoptosis was detected and harvested for protein analysis (Western blot). In some experiments, DHA was added at a concentration of 100nM and PD146176, a 15-LOX-1 inhibitor, at a concentration of 1 μ M, to treat RPE cells under stress. Cell extracts were made and protein concentrations were adjusted by Bio-Rad (herraglesi, ca) protein reagents and used for western blot analysis.
Example 5
Protein analysis: bcl-2 family proteins, SIRT1 and inhibin (type 1) and Iduna proteins were analyzed by Western blot analysis. Briefly, 20-25. mu.g equivalents of each cell extract were electrophoresed on a 4-12% gel (Promega) at 125V for 2 hours. The proteins were transferred to nitrocellulose membranes using an I-blot transfer apparatus. The membranes were treated overnight at 4 ℃ with Bcl-2, Bcl-xL, Bax, Bid, Bim, SIRT1 and statin (type 1) (Santa Cruz Biotechnology) specific primary and Iduna antibodies (Neuro-Mab Lab, UCLA, los angeles, ca) and with secondary antibody, goat anti-mouse lg: horseradish peroxidase and conjugated horseradish peroxidase-conjugated anti-biotin antibody were probed for 45 min and then proteins were assessed using the ECL kit (Amersham).
Example 6
Immunocytochemistry and apoptosis evaluation: immunocytochemistry assays were performed in 8-well slide chambers. Briefly, cells were fixed in 4% paraformaldehyde (FA) for 20 minutes and permeabilized with 0.1% Triton X-100 in PBS. Nonspecific epitopes were blocked in 10% Bovine Serum Albumin (BSA) in 1x PBS for 1 hour at room temperature. Immunostaining was performed by incubating the primary antibody at 4 ℃ overnight. Samples were stained with Alexa Fluor 555 conjugated secondary antibody (meridian life Science inc., menphis, tennessee, usa) diluted with 1/250 for 2 hours at room temperature, and nuclei were stained with Hoechst (2 μ M Hoechst 33258). Photographs were taken with a Zeiss LSM510 confocal microscope and a Zeiss Axioplan-2 deconvolution microscope.
To assess cell death, hRPE and ARPE-19 cells were fixed with methanol for 15 minutes, washed with 1x PBS, then loaded with 2 μ M Hoechst (dissolved in rockwell's solution (Promega)) and incubated for an additional 15 minutes prior to imaging. Cells were then observed under UV fluorescence by using a Zeiss LSM510 confocal microscope. Images were recorded and apoptosis was assessed by using an automated unbiased method.
Example 7
LC-MS/MS of the elongatinoids ELV-N32 and ELV-N34 in RPE cells: human RPE cells (ABC cells p #19) were cultured in T75 flasks for 24-48 hours, followed by incubation with 10. mu.M free 32:6 and 34:6 fatty acid mixtures for 24 hours. Immediately after 24 hours of serum deprivation, cells were treated with 1mM H2O2Incubation was carried out for 24 hours. Fatty acids were extracted from the collected cell culture media using liquid-liquid lipid extraction followed by mass spectrometry. The extract was loaded on a liquid chromatography tandem mass spectrometer (LC-MS/MS) for analysis. Fatty acids, monohydroxy fatty acid derivatives (27-hydroxy fatty acids C32:6N3 and 29-hydroxy fatty acids 34:6N3), as well as ELV-N32(20, 27-dihydroxy fatty acids C32:6N3) and ELV-N34(22, 29-dihydroxy fatty acids C34:6N3) were analyzed. ELV-N32 and ELV-N34 and their deuterium labeled derivatives ELV-N32-d2 and ELV-N34-d2 were prepared by stereocontrolled chemical synthesis and used to match the cell-produced derivatives.
Example 8
Photo-oxidative stress: C57/BI6 wild type and AdipoR1 knockout mice were housed in a temperature controlled room at 21-23 ℃ for 12 hours with a 12 hour light-dark cycle. For light-induced oxidative stress, mice were exposed to 1 hour of intense light (8-lamp array of 10-inch round 22W fluorescent lamps; cold white, FTC8T 9/CW; Electric, Verfield, Connecticut; 18 klux; 270. mu. E m-2 s). Following light exposure, animals were sacrificed by cervical dislocation and eyes were removed. The cornea, iris and lens were discarded and the retina was separated from the rest of the cup. These tissues were then snap frozen. Retinas from animals of the same genotype were pooled. Samples were processed for lipid extraction and LC-MS/MS based lipidomic analysis.
Example 9
Oxygen deprivation (OGD), NMDA excitotoxicity, or Uncompensated Oxidative Stress (UOS) exposure: an in vitro oxygen sugar deprivation (OGD) model was established. Primary cortical neurons were cultured from SD rat embryos. On day 12 in vitro (DIV), cells were washed with phosphate buffered saline and incubated in glucose free neural basal medium (GIBCO) for 30 min. The cells were then placed in a modular incubator and in an anaerobic chamber (95% N) at 37 deg.C2，5％CO2) And (4) incubating for 90 minutes to perform OGD.
After 90 min of OGD exposure, cells were returned to the original medium (containing 2% B27(GIBCO) and 2% N)2(GIBCO) supplement and 0.5mM glutamine, 50U/ml penicillin/streptomycin in nerve basal medium (GIBCO)) were placed in an oxygen chamber (37 ℃, 5% CO)2) For 2 hours. Then, the mixture was mixed with a solution containing ELV-N32 or ELV-N34[500nM ]]The medium was changed and placed in an oxygen chamber (37 ℃, 5% CO)2) For 12 hours, then the cells were sampled and cell viability was determined using different methods as previously described (25-28). By addition of NMDA (25. mu.M, 50. mu.M or 100. mu.M concentration) or by addition of TNF alpha (10ng/ml) and H2O2(50. mu.M, 100. mu.M or 200. mu.M) cerebral cortical mixed neuronal cells or hippocampal cells in culture were exposed to NMDA or Uncompensated Oxidative Stress (UOS) for 12 hours. Cell viability and neuroprotection in the presence of ELV-N32, ELV-N34, 32:6 or 34:6 was determined after 12 hours.
Example 10
Hoechst staining and unbiased image analysis: cells were washed with 1 × Dolbecco Phosphate Buffered Saline (DPBS) (GIBCO) without calcium or magnesium and fixed with ice-cold 4% Paraformaldehyde (PFA) for 10 min followed by incubation in 100% methanol for 15 min. Cells were washed with 1 × Phosphate Buffered Saline (PBS) (GIBCO) pH 7.4 and incubated in PBS containing 20 μ M hoechst 33258(Molecular Probes) for 20 minutes. Cells were then washed 3 times with 1x PBS and stored in 1x PBS at 4 ℃ until imaged for microscopy.
A4X 4 small horse was obtained from the center of each well using a Zeiss 510Meta laser confocal microscope and LSM510 Meta softwareA mosaic pattern. The images were imported into the image analysis software ImageJ (national institutes of health, besistar, maryland) and batch processed using custom macros. The Otsu autothreshold was applied to each image of Hoechst stained nuclei and the area of each detected object was recorded. Area of<10μm2Is excluded from the analysis. To estimate the percentage of non-fixed shrink kernels, a size threshold is chosen, and objects above this value are all assumed to be non-fixed shrink kernels. The pycnotic size limits are selected based on the shape of the nuclear size distribution from the various cell populations. The results were exported to Microsoft Excel and analyzed.
Example 11
Calcein AM-propidium iodide live/dead assay and MTT 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide assay: a 10mL solution was prepared using 20 μ L A component (calcein-AM) and 20 μ L B component (propidium iodide) in combination with the two components of the live/dead cytotoxicity kit (Invitrogen). 50 μ L of this solution was added to each well of a 12-well cell culture plate, and the cells were placed in an normoxic chamber (37 ℃, 5% CO)2) And (4) culturing for 1-2 hours. Cells were then imaged using an olympus fluoview laser confocal microscope. The images were imported into NIH image analysis software ImageJ and the green and red channels were separated. The images were counted using a cell counter to determine the number of live cells (green) and dead nuclei (red). The results were exported to Microsoft Excel and analyzed.
The MTT assay is based on the cleavage of yellow tetrazolium salt MTT into purple formazan crystals by metabolically active cells. The assay was performed to measure the viability of primary cortical neurons in each treatment group. Briefly, thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) (5mg/ml, 100. mu.L per well) was added to cells in 12-well plates in an normoxic chamber (37 ℃, 5% CO)2) And (5) incubating for 2 hours. The blue formazan reduction product generated is then dissolved in 1mL of isopropanol due to the action of succinate dehydrogenase on the dye in living cells, transferred to triplicate wells in a 96-well plate, and its absorbance read at 490nm using a Molecular probes spectra max microplate reader. Results are expressed as percent cell survival.
Example 12
Middle cerebral artery occlusion and cannula implantation into the right ventricle: male Sprague-Dawley rats weighing 280-340g (Charles River Lab., Wilmington, Mass.) were fasted overnight, but had free access to water. Atropine sulfate (0.5mg/kg, i.p.) was injected 10 minutes before anesthesia. Anesthesia was induced with 3% isoflurane in a mixture of 70% nitrous oxide and 30% oxygen. All rats were intubated orally and mechanically ventilated. During the period of aeration, animals were paralyzed with pancuronium bromide (0.6mg/kg, i.p.). Catheters were implanted in the right femoral artery and vein for blood sampling and drug infusion. Serial analyses of arterial blood gas, blood glucose, arterial blood pressure and heart beat rate were performed before and during surgery. Rectal (CMA/150 temperature controller, CMA/Microdialysis AB, stockholm, sweden) and intracranial (temporal muscle; Omega Engineering, stanford, connecticut) temperatures were monitored closely before, during and after MCAo. Rectal temperature and body weight were monitored daily until sacrifice.
Rats were left with right middle cerebral artery occlusion by endoluminal embolization for 2 hours (MCAo). Briefly, the right Common Carotid Artery (CCA) bifurcation was exposed through a midline cervical incision, and the occipital artery branch of the external carotid artery was isolated, ligated, and dissected. After careful isolation of the Internal Carotid Artery (ICA), the poly-L-lysine coated 3-0 single strand was advanced through the ICA to the MCA until mild resistance was felt. The neck incision was closed with suture and the animal was allowed to recover. After 2 hours MCAo, rats were anesthetized with the same anesthetic combination. The temperature probe was reinserted and the intraluminal sutures were carefully removed. The animals were allowed to drink water for 7 days on their free diets.
Thirty minutes after removal of the sutures, a brain infusion cannula was implanted into the right ventricle for treatment of each rat. Rats were anesthetized with 3% isoflurane and then fixed to a stereotaxic apparatus with the skull placed between bregma and herringbone points. Sterile stainless steel cannulae (5mm long) were implanted into the lateral ventricles (0.2 mm caudal to bregma, 2mm lateral to midline, 5mm subdural) using stereotactic coordinates. After treatment is complete, the cannula is removed.
Example 13
Treatment: the Elongationlike (ELV) in the form of sodium salt (Na) or methyl ester (Me) was dissolved in artificial cerebrospinal fluid (CSF) and administered 1 hour after 2 hours of MCAo into the right ventricle. The following ELV was used: ELV-N32-Na, ELV-N32-Me, ELV-N34-Na and ELV-N34-Me (5. mu.g/50. mu.l) or CSF (50. mu.l). All treatments were performed by researchers blinded to the treatment groups.
Example 14
Nerve/behavior testing: behavioral testing was performed by an observer blinded to the treatment group at 60 minutes (during MCAo) and then on days 1,2, 3 and 7 after MCAo. The test combination consists of two tests previously used to assess various aspects of neural function: (1) a postural reflex test for examining the upper body posture of an animal when suspended with a tail; and (2) a forelimb placement test for examining sensorimotor integration in forelimb placement response to visual, tactile, and proprioceptive stimuli. Neural function was graded on a scale of 0-12 (normal score 0, maximum score 12). Rats that did not show a high contralateral defect (score 10-11) 60 minutes during MCAo were excluded from further studies.
Example 15
Magnetic Resonance Imaging (MRI) acquisition and analysis of total lesion, core and penumbra volumes: on day 7, high resolution ex vivo MRI was performed on 4% paraformaldehyde fixed brains using an 11.7T Bruker Advance 8.9cm horizontal bore hole machine (Bruker Biospin, Billerica, MA) equipped with 89mm (id) receiver coils. T2 weighted images (T2WI), Diffusion Weighted Images (DWI), 3D volumes, and Apparent Diffusion Coefficient (ADC) maps were collected. The T2 and ADC plots were calculated from T2WI and DWI, respectively. Stratified zonal segmentation (HRS) was used to automatically identify core and penumbra volumes from T2 relaxation and water migration (ADC) (total lesion ═ core + penumbra). Penumbra tissue determination by HRS was confirmed by using PWI/DWI subtraction at each brain level. Penumbra was defined as the difference between PWI and aberrant ADC (diffusion-perfusion mismatch) (2 STD increase or decrease compared to normal tissue).
Example 16
Histopathology and immunohistochemistry: 7 days after MCao, rats were re-anesthetized with 3% isoflurane, 70% nitrous oxide, and the balance oxygen, and perfused with 0.9% saline, followed by 4% paraformaldehyde, via heart. The brains were removed and embedded in a gelatin matrix using MultiBrain RTM technology (Neuroscience Associates, Nox Kelvier, Tenn.). To quantify infarct volume, histological sections were digitized with nine standard coronal planes (bregma plane: +5.2, +2.7, +1.2, -0.3, -1.3, -1.8, -3.8, -5.0 and-7.3 mm) using a CCD camera (qiam Fast 1394, qiamaging, british) (30). Brain sections were imaged on an electric microscope BX61VS (olympus, japan) with a 10-fold objective lens. The areas of cortical and subcortical infarctions and left and right hemispheres of each section were noted by a researcher blinded to the experimental group. Infarct volume was calculated as the integral product of cross-sectional area and distance of intersection and corrected for brain swelling.
Brain edema was measured by the difference between ipsilateral and contralateral hemispheres. Immunohistochemistry procedures were performed on adjacent sections to identify specific vascular and neuronal elements in the ischemic core and penumbra. The following antibodies were used: the blood brain barrier of rats (SMI-71, BioLegent, san Diego, Calif.) for use as a vascular marker; glial fibrillary acidic protein (GFAP, Agilent tech., santa clara, ca) for labeling reactive astrocytes; and neuron-specific nucleoproteins (NeuN, Chemicon/Millipore, belerica, ma) and biotinylated anti-rat immunoglobulin (IgG) antibodies (BioLegend, san diego, ca) for detection of BBB breakdown. The number of positive cells and immune positive vessels was counted in the cortex and striatum at the level of the mid-foci (bregma-0.3 mm). Data are presented as the number of positive cells and vessels per high power field (magnification x 40).
Following a specific experimental protocol, images of the sections were obtained using a confocal laser microscope (LSM510, Carl Zeiss microscopy, ltwn, ca). Images were acquired using Zen software (Carl Zeiss microscopy) at a size of 212.3 μmx 212.3 μm. Image analysis was performed using ImageJ software. Analysis was performed by a researcher blinded to the experimental conditions. IgG staining intensity was calculated and averaged at the same level as assessed for ischemic injury as described previously (23, 31). To calculate the intensity of IgG staining, the images were converted to grayscale, and the mean grayscale values were recorded and compared. ImageJ software assigned black pixels with the value "0" and white pixels with the value "1". The gray scale levels are assigned to values in-between that increase as the pixel becomes darker and decrease as the pixel becomes darker. Therefore, for clarity of the figure, the IgG intensity values are expressed as the inverse of the mean gray scale. All slices are imaged simultaneously using the same settings without adjusting brightness or contrast. IgG staining intensity was measured in the entire contralateral and ipsilateral hemisphere as well as cortex and striatum.
Example 16
Statistical analysis: for cell cultures: all results are expressed as mean ± SEM. Data from all experiments were averaged using one-way ANOVA (analysis of variance) and then subjected to Sidak multiple comparison post hoc testing. Statistical analysis was performed using Graphpad Prism software version 7.02. A value of p <0.05 was considered statistically significant.
For ischemic stroke: values are expressed as mean ± SD. Comparison between groups of neurobehavioral scores and infarct size across the coronal plane was performed using repeated measures ANOVA, then corrected for multiple comparisons by the Bonferroni program. Two groups of comparisons were made using the two-tailed student t-test. Differences with p <0.05 were considered statistically significant.
Example 17
Structure and stereochemistry of ELV-N32 and ELV-N34 in mixed neuronal cultures: the complete structure and stereochemistry of the novel elongases ELV-N32 and ELV-N34 were determined by direct comparison with compounds prepared via stereocontrolled total organic synthesis by employing our previously reported method for full synthesis of NPD 1. These structural designations were further determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the synthesized deuterium labeled derivatives.
ELV-N32 and ELV-N34 were prepared by stereocontrolled total chemical synthesis (FIG. 4A). The availability of these synthetic ELVs with well-defined structure and stereochemistry enables us to determine the complete R/S configuration and Z/E geometry of the double bonds in these mixed neuronal cell culture derived ELVs. Synthetic stereochemically pure deuterium labeled ELVs were also generated and their structure and stereochemistry confirmed by matching them with endogenously produced molecules by LC-MS/MS.
ELV and its precursors were detected in cells under OGD stress (fig. 4B-4K). ELV-N32 detection was performed using m/z 499- >93 and 499- >401MRM transitions (FIG. 4B), and ELV-N34 detection was performed using m/z 527- >93 and 527- >429 transitions (FIG. 4C). For their corresponding monohydroxy precursors, m/z 483- >385 for 27-hydroxy-C32: 6n3 (FIG. 4B) and m/z 511- >413 for 29-hydroxy-C34: 6n3 (FIG. 4C) were used. For further identification, ELV was fully split and found to have a good match to the synthetically produced standard. The UV maxima of the two ELVs at 275nm are consistent with the conjugated triene structure (fig. 4D and 4F).
The complete structure and stereochemistry of ELV-N32 and ELV-N34 was established after the synthetic ELVs were matched to the biogenic ELVs derived from mixed neuronal cells in culture. Defined ELV-N32 (prolactin-like neuroprotective agent derived from 32-carbon omega-3polyunsaturated fatty acids: (A) (B))elovanoidneuroprotectin-like,derived from a32-carbon omega-3polyunsaturated fatty acid)) and ELV-N34 (an auxin-like neuroprotective agent derived from a34 carbon omega-3polyunsaturated fatty acid: (I) ((II))elovanoidneuroprotectin-like,derived from a34-carbon omega-3 polymerized failure acid)) is: ELV-N32: (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxy-trideca-14, 17,21,23,25, 29-hexaenoic acid (fig. 1E); ELV-N34: (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxytrinetradeca-16, 19,23,25,27, 31-hexaenoic acid (FIG. 4G).
Example 18
Neuroprotection of ELV in uncompensated oxidative stress, oxygen/sugar deprivation, or NMDA-induced excitotoxicity: at a concentration of 200nM, sodium The salts ELV-N32-Na or methyl ester ELV-N32-Me caused neuroprotection of mixed cortical neuronal cells in cultures exposed to uncompensated oxidative stress for 12 hours by the addition of 10ng/mL tumor necrosis factor alpha (TNF α) and H2O2(50. mu.M, 100. mu.M or 200. mu.M). Dose-dependent increase of ELV-N32-Na or ELV-N32-Me against apoptotic nuclei (FIG. 16B).
To determine the neuroprotective biological activity of ELV-N32 or ELV-N34 on OGD-induced neuronal cell death, cortical mixed neuronal cells in culture or hippocampal neurons in culture were exposed to OGD for 90 minutes. After 2 hours of re-oxygenation, ELV-N32 or ELV-N34 was added at a concentration of 200nM, 500nM or1 μ M, and cell viability was assessed by Hoechst positive nuclear counts, or calcein positive cell counts or MTT assays. Under all different conditions and concentrations, ELV-N32-Na, ELV-N32-Me, ELV-N34-Na, or ELV-N34-Me was found to elicit neuroprotection compared to cells exposed to OGD alone (FIGS. 15F-15H, 15K, and 15L; FIGS. 16D-16G and 16I). In addition, the results also show that when added after OGD exposure, precursor 34:6 can elicit neuroprotection at concentrations as low as 250nM (supplement fig. 1H).
In addition, NMDA exposure at 25. mu.M, 50. mu.M or 100. mu.M concentrations for 12 hours induced neuronal death in mixed cortical and hippocampal cultures (FIGS. 15C-15E and 15-15J; FIGS. 16A, 16C and 16H), which could be compensated by the addition of ELV-N32(Na or Me) or ELV-N34(Na or Me) at 200nM or 500nM concentrations (when added simultaneously with NMDA). There was a dose-dependent increase in apoptotic nuclei when cells were exposed to NMDA at concentrations of 25. mu.M, 50. mu.M or 100. mu.M, which was compensated for in the presence of ELV-N32-Na or ELV-N32-Me. For one experiment, it was tested whether there was a synergistic effect by adding 200nM ELV-N32(Na or Me) and NPD1 at 100nM concentration. Both ELV-N32-Na and ELV-N32-Me showed synergistic neuroprotection against neuroNMDA excitotoxicity at 100. mu.M for 12 hours (FIG. 16A). However, in addition to NPD1, ELV-N32-Me was also more effective than ELV-N32-Na and NPD1 used together. We have also found that NMDA excitotoxicity can be overcome by the addition of the uncompetitive NMDA receptor antagonist MK801 maleate (dezocyclopine, 10 μ M). The addition of MK801 and NPD1 together with ELV-N32-Na or ELV-N32-Me improves neuroprotection elicited by ELV-N32-Na or ELV-N32-Me alone. In addition, precursor 34:6 at 500nM concentration attenuated NMDA receptor-mediated excitotoxicity (fig. 16H).
Example 19
Continued neurological improvement and protection following ELV-induced ischemic stroke: focal ischemic stroke results in impaired sensorimotor and cognitive functions, with 70-80% of patients exhibiting hemiplegia immediately after stroke. ELV was administered into the right ventricle via a stereotactically implanted infusion cannula 1 hour after the 2 hour middle cerebral artery occlusion (MCAo). post-MCAo rodent functional impairment resembles sensorimotor impairment and since the ultimate goal of any stroke therapy is recovery of neuro/behavioral function, a combination of sensorimotor tests is used to detect neurological impairment following experimental ischemic stroke.
All ELV treated animals significantly improved the neurological score in a sustained manner compared to the cerebrospinal fluid (CSF) group until 7 days survival (fig. 20A). CSF-treated rats continued to exhibit severe injury through this time period. T2-weighted imaging (T2WI) showed larger lesions, with high intensity of T2 observed in the ischemic core and penumbra of CSF-treated rats, consistent with edema formation (fig. 20B and 20C). In contrast, all ELV treatments significantly reduced the ischemic core and penumbra volume (calculated from T2WI) (fig. 4B). ELV-N32-Na, ELV-N32-Me, ELV-N34-Na, and ELV-N34-Me significantly reduced the total lesion volume (60%, 56%, 99%, and 91%, respectively) compared to the CSF treated group (fig. 20B). Three-dimensional (3D) lesion volumes were calculated from T2WI on day 7 post MCAo (fig. 20D). With elongatinoid treatment, lesion volume was greatly reduced and mostly localized only in the subcortical region of the brain (fig. 20D).
Example 20
ELV-reduced cell damage, vascular integrity and BBB disruption: on day 7, neurons, astrocytes and blood vessels involved in cerebral infarction were examined using immunohistochemistry. CSF-treated rats showed larger lesions involving the cortical and subcortical regions, characterized by loss of neuronal, glial, and vascular elements (fig. 21A and 21B).
In contrast, ELV-treated rats exhibited less infarction and an increased number of NeuN-positive, GFAP-positive cells and SMI-71-positive vessels in the cortex compared to the CSF-treated group. Cell counts of NeuN, SMI-71 and GFAP (regions depicted in the graph of fig. 21C) indicate that all ELV treatments increased NeuN-positive neurons and GFAP-positive reactive astrocytes and protected vascular integrity (fig. 21C). The result of almost all ELV treatments (except ELV-N32-Na) was that the vascular density (SMI-71) in the penumbra tissue increased and in parallel a denser GFAP-rich scar tissue was formed. Thus, an increase in vascular density may promote neurogenesis and synaptogenesis, thereby contributing to improved repair and ultimately improved functional recovery.
Ischemic destruction of the Blood Brain Barrier (BBB) was initially measured by penetration of endogenous IgG into the brain parenchyma (fig. 22A and 22B). IgG staining intensity was observed in the ipsilateral hemisphere after MCAo (fig. 22A). Staining intensity was similar on day 7 in CSF, ELV-N32-Na and ELV-N32-Me treated groups. In contrast, treatment with ELV-N34-Na and ELV-N34-Me showed significantly less IgG staining in the cortex; staining was mostly located in the core of the infarct (subcortical). In addition, IgG immunoreactivity (total) was reduced throughout the hemisphere (fig. 22B) (all animals survived smoothly). Brains from CSF-treated rats exhibited gross necrotic (pannecrotic) lesions involving the cortical and subcortical regions of the right hemisphere (fig. 22C). In contrast, infarct size in rats treated with ELV compounds showed less extensive damage, mainly in the subcortical region. Compared to the CSF treated group, ELV-mediated protection was extensive in the frontal lobe cortex (tissue salvage 57-96%) and subcortical (73-75%) (fig. 22D). In all ELV treatment groups, the total infarct volume, corrected for brain swelling, was reduced by 55-91% in magnitude (fig. 22D).
Example 21
RPE cells, ELV structure and stereochemistry: the complete structure and stereochemistry of the novel 32-and 34-carbon class of elongatins ELV-N32 and ELV-N34 was determined by direct comparison with compounds prepared via stereocontrolled total organic synthesis by employing previously reported methods for the total synthesis of DHA-derived lipid mediator neuroprotective D1(NPD 1; 10R,17S) -dihydroxydocosac- (4Z,7Z,11E,13E,15Z,19Z) -hexaenoic acid). These structural designations were further determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of synthetic deuterium labeled derivatives (ELV-N32-d2 and ELV-N34-d 2). ELV-N32 and ELV-N34 were prepared by stereocontrolled total chemical synthesis (FIG. 3A). The availability of synthetic materials with well-defined structures and stereochemistry enabled us to determine the complete R/S configuration and Z/E geometry of the double bonds in these human primary RPE cell-derived ELVs. Confocal images of immunostaining of primary human RPE cells with specific markers ZO-1 (occludin-1), RPE65, MITF (microphthalmia-associated transcription factor) and β -catenin and light microscopy morphology at different passages in culture are depicted in fig. 6A and 6B.
Briefly, these cells were cultured for 24-48 hours, followed by incubation with 10. mu.M free 32:6, n6 plus 34:6, n6 for 24 hours. Cells were then deprived of serum for 24 hours and then treated with 1mM H2O2Incubation was carried out for 24 hours. The incubation medium was collected, lipids were extracted and loaded onto a liquid chromatography tandem mass spectrometer (LC-MS/MS) for analysis. Synthetic stereochemically pure deuterium labeled ELVs were also generated and their structure and stereochemistry confirmed by matching them with endogenously produced molecules by LC-MS/MS. The complete structures of ELV-N32 (from 32C omega-3 polyunsaturated fatty acids) and ELV-N34 (from 34C omega-3 polyunsaturated fatty acids) were confirmed after re-matching with human primary RPE cell culture medium-derived elongatoids as follows: ELV-N32: (14Z,17Z,20R,21E,23E,25Z,27S,29Z) -20, 27-dihydroxy-trideca-14, 17,21,23,25, 29-hexaenoic acid; ELV-N34: (16Z,19Z,22R,23E,25E,27Z,29S,31Z) -22, 29-dihydroxytrinetradeca-16, 19,23,25,27, 31-hexaenoic acid.
ELV and its precursor VLC-PUFA were detected in RPE cells under UOS (FIGS. 3B-3K). The m/z 499 → 93 and 499 → 401MRM transition is used for ELV-N32 and the m/z 527 → 93 and 527 → 429 transition is used for ELV-N34 to detect. For the corresponding precursors, m/z 483 → 385 for 27-hydroxy-32: 6n3 and m/z 511 → 413 for 29-hydroxy-34: 6n3 were used. For further identification, complete cleavage of the ELV was performed and found to have a good match with the standard.
Example 22
ELV N32 and N34 elicited potent cytoprotection: it was shown that free 32:6n3 or 34:6n3 elicited protection against UOS in ARPE-19 cells (fig. 7A and 7B) and that lipoxygenase inhibitors blocked this effect (fig. 10C). To test the efficacy of 32:6n3 and 34:6n3 VLC-PUFAs in modulating human RPE cell homeostasis and survival, human RPE cells undergoing UOS were incubated with two VLC-PUFAs (3 μ M each) and NPD1(200nM) for 16 hours. Addition of H2O2Apoptosis (50% cell death) was induced (800 μ M). Both 32:6n3 and 34:6n3 successfully prevented cell death (4% and 18%, respectively); NPD1 reduced apoptosis to 11% (fig. 7K and 7L).
The oxidative stress stimulates the enzymatic oxidation of DHA by activating 15-lipoxygenase-1 (15-LOX-1), resulting in NPD111The biosynthesis of (3). NPD1 is a stress-responsive lipid mediator derived from DHA, and it enhances survival signal transduction in RPE cells exposed to oxidative stress by facilitating the regulation of the activity and content of proteins directly involved in cell fate decisions.
hRPE cells previously deprived of serum for 12 hours were incubated with 15 lipoxygenase 1(15-LOX-1) inhibitor (PD146176) (10 μ M, 1 hour) and then with 600 μ M H2O2A combination of/TNF-alpha with a mixture of 32:6n3 and 34:6n3 (3. mu.M each) was bathed for 16 hours. The 15-LOX-1 inhibitor can sensitize cells. Therefore, lower H was used than in the cytoprotective experiments2O2And (4) concentration. Addition of H2O2TNF-. alpha.induced apoptosis in RPE cells and treatment with the mixture of 32:6n3 and 34:6n3 successfully prevented cell death (FIG. 71), indicating that 15-LOX-1 is not involved in the present free fatty acid cytoprotective mechanism using human primary RPE cells.
Example 23
32:6n3 and 34:6n3 VLC-PUFAs enhance anti-apoptotic and pro-survivin expression: in FIGS. 7A-7K, 32:6n3 and 34:6n3 upregulated survival BcL2 and BcL-xLAnd down-regulates the pro-apoptotic proteins Bax, Bim and Bid (FIGS. 7E and 7F)(FIGS. 7G-7I). Furthermore, the homeostatic effects of 32:6n3 and 34:6n3 were concentration dependent (fig. 7J). In the presence of 15-LOX-1 inhibitors, the expression of SIRT1 (SIRT-1) was increased (fig. 7C), while the effect of the inhibitor on Iduna expression was unaffected (fig. 7D).
Example 24
ELV N32 and N34 attenuated apoptosis in RPE: whether ELV is able to inhibit UOS-induced apoptosis in RPE cells was tested. As shown in FIG. 10C, ELV-N32-Na and ELV-N34-Na mimic the UOS-mediated attenuation of apoptosis in RPE cells at a concentration of 200 nM. Interestingly, two different 1 μ M concentrations of 15-LOX-1 inhibitor (15-LOX-1 inhibitor or PD146176) were able to compensate for ELV-mediated inhibition of apoptosis in RPE cells undergoing UOS (fig. 10C). UOS-induced apoptotic cell death was attenuated in RPE cells by ELV-N32-Na or ELV-N34-Me in a concentration-dependent manner (50-500 nM). The highest inhibition was 500nM (sodium and methyl ester forms) and the lowest was 50nM (FIG. 10G).
Example 25
ELV up-regulates pro-homeostatic and anti-apoptotic proteins: whether ELV enhances survival and homeostatic protein expression in RPE cells undergoing UOS was investigated. FIG. 10Aa shows that ELV-N32-Na and ELV-N34-Na upregulate deacetylase 1(SIRT1) in UOS RPE cells in a dose-dependent manner (100 nM and 200nM) and that ELV-N32-Na is more effective in upregulating SIRT1 than ELV-N34-Na. ELV-N32-Na and ELV-N34-Na enhanced the expression of Iduna in RPE cells under UOS at a concentration of 200nM (FIG. 10B). PD-146176 is a 15-LOX-1 inhibitor that blocks these effects at a concentration of 1 μ M in ARPE-19 cells undergoing UOS. Inhibin (type 1) is a survivin cell that is upregulated in a concentration-dependent manner (100-200nM) by ELV-N32 and ELV-N34 (sodium and methyl ester forms) in RPE cells undergoing UOS (FIG. 10H). In FIG. 10D, it is shown that ELV-N32-Na or ELV-N34-Na enhances the abundance of the anti-apoptotic proteins Bcl-2 and Bcl-xL. On the other hand, ELV-N32 or ELV-N34 (sodium or methyl ester) reduced pro-apoptotic Bax, Bim and Bid (FIGS. 10D-10F). Interestingly, while Bcl-2 and Bcl-xL were up-regulated (FIG. 10D), Bax, Bim and Bid were down-regulated by sodium or methyl esters (FIG. 10D-10F).
Example 26
AdipoR1 regulates DHA uptake and ELV formation: RPE cells maintain PRC functional integrity, and its depletion involves the development of various forms of retinal degeneration (fig. 11D). One function of RPE cells is to recover DHA during PRC renewal and return it to the PRC inner segment through the interphotoreceptor matrix for new outer segment disc membrane biogenesis37. Recently, adiponectin receptor 1(AdipoR1) was found to be essential for the availability of DHA from photoreceptor cells28And single amino acid mutations are responsible for autosomal dominant retinitis pigmentosa38. Genetic ablation of this receptor leads to PRC degeneration and to the turning off of VLC-PUFA synthesis in the retina. Pool sizes of free C32:6n3 and 34:6n3 in retinas of AdipoR1 Knockout (KO) mice (red) were significantly reduced compared to WT (blue). In addition, no ELV-N32 and ELV-N34 were detected in KO (Red). In contrast to wild type (blue), monohydroxy groups 32:6N3 and C34:6N3 (stable derivatives of hydroperoxy precursors of ELV-N32 and ELV-N34, respectively) lacked a detectable signal in KO (red) (FIGS. 11B and 11C).
Example 27
ELV protects RPE cells that maintain PRC integrity: elongation of DHA by ELOVL4 in intra-photoreceptor segments resulted in the biosynthesis of VLC-PUFA and its insertion at C1 position of phosphatidylcholine within PRC discoid membranes. However, under stress conditions, these VLC-PUFAs were cleaved by PLA1 to synthesize monohydroxy and dihydroxy VLC-PUFAs (elv) (fig. 11A). Light-induced oxidative stress in the mouse retina triggered the production of free VLC-PUFAs (32:6n3 and 34:6n3) and their mono-and dihydroxy derivatives (fig. 11A). No detectable amounts of these molecules were found in AdipoR1 KO mice (fig. 5b, c, red curve). Thus, the lack of the VLC-PUFA precursor DHA resulted in retinal degeneration (fig. 11D), followed by significant down-regulation of free VLC-PUFA omega-3 molecular species and ELV biosynthesis.
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