阅读说明：本技术 脂肪酸衍生物及其用途 (Fatty acid derivatives and uses thereof ) 是由 G·凯斯 C·拉姆斯登 于 2018-07-06 设计创作，主要内容包括：本公开涉及脂肪酸衍生物、包含该脂肪酸衍生物的药物组合物和使用该脂肪酸衍生物例如治疗受试者的炎症、慢性瘙痒、慢性疼痛、自体免疫疾病、动脉粥样硬化、皮肤病症、关节炎、神经退行性疾病或精神疾病的方法。在一些实施方式中,脂肪酸衍生物是具有根据下式的结构的化合物或其立体异构体、互变异构体或可药用盐：<Image he="246" wi="580" file="DDA0002356845570000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>其中X的长度为1-16个碳,Z是长度为1-16个碳的脂族基团或不存在,Y选自：<Image he="191" wi="700" file="DDA0002356845570000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>R<Sup>1</Sup>、R<Sup>2</Sup>和R<Sup>3</Sup>独立地是氢或低级烷基、R<Sup>4</Sup>是低级烷基、羟基、羧基或胺,R<Sup>5</Sup>是氢、低级烷基或卤化物,R<Sup>6</Sup>是羟基或取代巯基,并且每个R<Sup>7</Sup>独立地是氢或氟化物或不存在,并且相邻的碳形成炔。(The present disclosure relates to fatty acid derivatives, pharmaceutical compositions comprising the fatty acid derivatives, and methods of using the fatty acid derivatives, for example, to treat inflammation, chronic pruritus, chronic pain, autoimmune disease, atherosclerosis, skin disorders, arthritis, neurodegenerative disease, or psychiatric disease in a subject. In some embodiments, the fatty acid derivative is a compound having a structure according to the following formula: wherein X is 1 to 16 carbons in length, Z is an aliphatic group 1 to 16 carbons in length or is absent, and Y is selected from: R 1 、R 2 and R 3 Independently is hydrogen or lower alkyl, R 4 Is lower alkyl, hydroxy, carboxy or amine, R 5 Is hydrogen, lower alkyl or halide, R 6 Is hydroxy or substituted mercapto, and each R 7 Independently hydrogen or fluoride or absent, and the adjacent carbons form an alkyne.)

1. A compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, the compound having a structure according to the following formula:

wherein:

x is an aliphatic group of 1 to 16 carbons in length;

z is an aliphatic group of 1-16 carbons in length or absent;

y is selected from:

R¹、R²and R³Independently hydrogen or lower alkyl;

R⁴is lower alkyl, hydroxy, carboxy or amine;

R⁵is hydrogen, lower alkyl or halide;

R⁶is hydroxy or substituted mercapto; and

each R⁷Independently hydrogen or fluoride or absent, and adjacent carbon atoms form an alkyne.

2. The compound of claim 1, wherein Y is selected from any one of the following:

and each R⁷Independently hydrogen or fluoride.

3. The compound of any one of the preceding claims, wherein X is 1-10 carbons in length.

4. The compound of any one of the preceding claims, wherein X is 6 carbons in length.

5. The compound of any one of the preceding claims, wherein Z is 1-10 carbons in length.

6. The compound of any one of the preceding claims, wherein Z is 4 carbons in length.

7. The compound of any one of the preceding claims, wherein X and Z are a total of 8-14 carbons in length.

8. The compound of any one of the preceding claims, wherein X and Z are a total of 10 carbons in length.

9. The compound of any one of the preceding claims, wherein X and Z are independently alkyl, substituted alkenyl, or unsubstituted alkenyl.

10. The compound of any one of the preceding claims, wherein X and Z independently comprise one or more fluoroolefin, difluoroolefin, and/or diallyldeuterium substitutions.

11. The compound of claim 1, having a structure according to any one of formulas (II) - (CII), wherein, if present:

R¹is hydrogen or lower alkyl;

R²is hydrogen or lower alkyl;

R³is hydrogen or lower alkyl;

R⁴is lower alkyl, hydroxy, carboxy or amine;

R⁵is hydrogen, lower alkyl or halide;

R⁶is hydroxy or substituted mercapto;

each R⁷Independently hydrogen or fluoride or absent, and the adjacent carbons form an alkyne;

each R⁸Independently hydrogen or fluoride; and

each R⁹Independently hydrogen or deuterium.

12. The compound of claim 11, wherein each R is⁷Independently hydrogen or fluoride.

13. The compound of any one of the preceding claims, wherein R¹、R²、R³And R⁴Independently a methyl group.

14. The compound of any one of the preceding claims, wherein R¹、R²、R³And R⁴Is methyl.

15. The compound of any one of the preceding claims, wherein R⁵Is hydrogen.

16. The compound of any one of the preceding claims, wherein R⁶Is a hydroxyl group.

17. The compound of any one of claims 1-5, wherein R⁶Is cysteine or glutathione.

18. The compound of any one of the preceding claims, wherein the compound has a structure as shown in any one of compounds 2, 4, 9-12, 16-180, 182-183, 185-330.

19. The compound of any one of claims 1-17, wherein the compound does not have the structure shown as any one of compounds 1,3, 5-8, 13, 15, 181, or 184.

20. The compound of any one of claims 1-17, wherein the compound does not have the structure shown in any one of compounds 1-16.

21. A compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, the compound having a structure according to the following formula:

wherein:

x is an aliphatic group 10 to 25 carbons in length and comprising one or more epoxy, hydroxyl, or carboxyl substitutions;

R¹is hydrogen or lower alkyl; and

each R²Independently methyl or hydrogen.

22. The compound of claim 21, wherein X is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

23. The compound of claim 21 or claim 22, wherein each R is²Is methyl.

24. The compound of claim 21 or claim 22, wherein each R is²Is hydrogen.

25. The compound of claims 21-24, wherein X comprises one or more diallyl deuterium substitutions.

26. The compound of claims 21-25, wherein X is 17 carbons in length.

27. A compound, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, the compound having a structure according to one of the following:

wherein:

R⁵is hydrogen, lower alkyl or halide;

each R⁷Independently hydrogen or fluoride or absent, and the adjacent carbons form an alkyne; and

R¹⁰and R¹¹Independently an aliphatic group.

28. The compound of claim 27, wherein each R⁷Independently hydrogen or fluoride.

29. The compound of claim 27 or claim 28, wherein R⁵Is hydrogen.

30. The compound of any one of claims 27-29, wherein R¹⁰Is methyl.

31. The compound of any one of claims 27-30, wherein R¹⁰And R¹¹Independently is substituted or unsubstituted lower alkyl, substituted or unsubstituted lower heteroalkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower heteroalkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower heteroalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

32. The compound of any one of claims 27-31, wherein R¹¹Is methyl.

33. The compound of any one of claims 27-32, wherein the compound has the structure shown in any one of compounds 263-270.

34. A pharmaceutical composition comprising a compound of any one of claims 1-33 and a pharmaceutically acceptable carrier.

35. The pharmaceutical composition of claim 34, formulated for topical, parenteral, or oral administration.

36. A method of treating a disease or disorder in a subject, comprising:

administering to a subject having or suspected of having the disease or disorder a therapeutically effective amount of the pharmaceutical composition of claim 34 or claim 35,

wherein the disease or condition is selected from one of: inflammation, chronic pruritus, chronic pain, autoimmune disease, atherosclerosis, skin disorders, arthritis, neurodegenerative diseases or psychiatric diseases.

37. The method of claim 36, wherein the pharmaceutical composition is administered topically to a site of inflammation, chronic pain, chronic itch, or a skin condition of the skin or mucosa of the subject.

38. The method of claim 37, wherein the skin disorder is a disorder with water barrier dysfunction or increased epidermal water loss.

39. The method of claim 38, wherein the skin disorder is ichthyosis, eczema, atopic dermatitis, psoriasis and/or xeroderma.

40. The method of any one of claims 36-39, wherein the compound in the pharmaceutical composition comprises 2, 2-dimethyl and is an oxidized derivative of linoleic acid.

41. The method of any one of claims 36-40, wherein the compound in the pharmaceutical composition comprises one or more of compounds 31-36, 38-43, and 66-72.

42. A method of diagnosing a disease or disorder in a subject, comprising:

obtaining a biological sample from a subject;

measuring the level of any one of compounds 1-16 in a biological sample;

diagnosing the subject as a subject having a disease or disorder if an elevated level of any of the compounds is detected in the biological sample as compared to a normal control;

wherein the disease or condition is selected from one of: inflammation, chronic pruritus, chronic pain, autoimmune disease, atherosclerosis, skin disorders, arthritis, neurodegenerative diseases or psychiatric diseases.

43. The method of claim 42, further comprising subjecting the subject to a reduced polyunsaturated fatty acid diet if an elevated level of any of the compounds is detected in the biological sample as compared to a normal control.

Technical Field

The present disclosure relates to fatty acid derivatives and methods of using the same, e.g., for treating inflammation, itch, pain, autoimmunity, and/or atherosclerosis in a subject.

Background

Biological processes such as inflammation, itch, pain, autoimmunity, barrier dysfunction, degeneration (degeneration) and atherosclerosis represent a continuing problem in the medical field, in part in patient treatment and evaluation. Despite the treatments for these diseases and conditions, they often fall short of proven needs. Thus, there is a need for new agents that can be used in methods of treating subjects suffering from one or more of these diseases or disorders.

Summary of The Invention

The present disclosure relates to fatty acid derivatives, pharmaceutical compositions comprising the fatty acid derivatives, and methods of using the fatty acid derivatives to treat, for example, inflammation, itch, pain, autoimmunity, atherosclerosis, and/or skin conditions in a subject.

In some embodiments, the disclosed fatty acid derivatives are derivatives of labile endogenous bioactive compounds designed to maintain the effect of the corresponding endogenous bioactive compound while maximizing stability, activity, and ease of delivery to a subject.

In some embodiments, the fatty acid derivative is a compound having a structure according to the following formula:

wherein X is an aliphatic group of 1-16 carbons in length, Z is an aliphatic group of 1-16 carbons in length or absent, and Y is selected from any one of the following:

R¹、R²and R³Independently is hydrogen or lower alkyl, R⁴Is lower alkyl, hydroxy, carboxy or amine, R⁵Is hydrogen, lower alkyl or halide, R⁶Is hydroxy or substituted mercapto, and each R⁷Independently hydrogen or fluoride or absent, and the adjacent carbons form an alkyne. In some embodiments, X and Z are independently an alkynyl group or a substituted or unsubstituted alkenyl group. In some embodiments, X and Z are independently lower alkenyl and comprise one or more fluoroolefin or difluoroolefin moieties.

In some embodiments, a method of treating a disease or disorder in a subject using the disclosed fatty acid derivatives is provided. The method comprises administering to a subject having or suspected of having the disease or disorder a therapeutically effective amount of a pharmaceutical composition comprising the disclosed fatty acid derivatives. Exemplary diseases or conditions to which the method may be applied include inflammation, chronic pruritus, chronic pain, autoimmune diseases, atherosclerosis, skin conditions, arthritis, neurodegenerative diseases or psychiatric diseases.

In some embodiments, a method of diagnosing a disease or disorder in a subject by measuring the levels of the disclosed fatty acid derivatives in a biological sample from the subject is provided. The method comprises obtaining a biological sample from a subject, measuring the level of any of compounds 1-16 provided herein in the biological sample; and diagnosing the subject as a subject having the disease or disorder if an elevated level of the compound is detected in the biological sample as compared to a normal control. Exemplary diseases or conditions to which the method can be applied include inflammation, chronic pruritus, chronic pain, autoimmune diseases and atherosclerosis.

Also provided are embodiments of a pharmaceutical composition comprising a fatty acid derivative as disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be formulated, for example, for topical, parenteral, or oral administration.

The foregoing and other features and advantages of the present disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

Drawings

Figure 1 free hydroxy-epoxy-and keto-epoxy-octadecenoic acids were elevated in psoriatic skin lesions, especially itchy skin. The concentration of free hydroxy-epoxy-and keto-epoxy-octadecenoic acids in psoriatic lesions and control human skin is shown. The compounds tested were:

11-hydroxy (H) -12, 13-trans-epoxy- (E) -octadecenoic acid (11H-12,13E-LA, Compound 1),

11-hydroxy (H) -9, 10-trans-epoxy- (E) -octadecenoic acid (11H-9,10E-LA, Compound 3),

11-keto (K) -9, 10-trans-epoxy- (E) -octadecenoic acid (11K-9,10E-LA, Compound 4),

9-hydroxy (H) -12, 13-trans-epoxy- (E) -octadecenoic acid (9H-12,13E-LA, Compound 5),

9-keto (K) -12, 13-trans-epoxy- (E) -octadecenoic acid (9K-12,13E-LA, Compound 6), and

13-hydroxy (H) -9, 10-trans-epoxy- (E) -octadecenoic acid (13H-9,10E-LA, Compound 7).

Statistical analysis was performed using the Kruskal-Wallis test (Kruskal-Wallis test). For control skin, psoriatic lesions (no itch) and psoriatic skin (itch), N is 7,3 and 5, respectively.

FIGS. 2A-2D regioselective increase in calcitonin gene-related peptide (CGRP) release from adult rat Dorsal Root Ganglion (DRG) neurons (blind analysis). Ex vivo CGRP release measured from adult DRG neuron cultures. The compounds tested included: 11H-12,13E-LA (Compound 1), 11H-9,10E-LA (Compound 3), 11K-9,10E-LA (Compound 4), 9H-12,13E-LA (Compound 5), 9K-12,13E-LA (Compound 6), 13H-9,10E-LA (Compound 7), 11-keto (K) -12, 13-trans-epoxy- (E) -octadecenoic acid (11K-12,13E-LA, Compound 2), and 13-keto (K) -9, 10-trans-epoxy- (E) -octadecenoic acid (13K-9,10E-LA, Compound 8). At a concentration of 1 μm, prostaglandins E2(PGE2), 11H-12,13E-LA (FIGS. 2A and 2C) and 11H-9,10E-LA (FIGS. 2B and 2C) significantly increased low-pH-induced and capsaicin-induced CGRP release, and 13H-9,10E-LA (FIGS. 2A and 2C) significantly increased low-pH-induced CGRP release, but had no effect on capsaicin-induced release (FIG. 2D). The 3-hydroxy-Z-pentenyl-E-epoxy moiety, which is unique and common to both lipids, is a proposed pharmacodynamic moiety that mediates the effects of 11H-12,13E-LA and 11H-9, 10E-LA. Denotes p <0.05, ANOVA was performed using Tukey's post hoc test. CGRP, calcitonin gene related peptide.

Figures 3A and 3b pain-related behavioral responses after intradermal hindpaw injection of the disclosed fatty acid derivatives (blind experiments). (FIG. 3A) 11-hydroxy-12, 13-epoxy-octadecenoic acid and PGE2 reduced the C-fiber withdrawal latency reaction compared to the vehicle (vehicle) control. (fig. 3B) PGE2 increased the proportion of withdrawal response following stimulation of a δ fibers. For the medium, 11-hydroxy-12, 13-epoxy-octadecenoic acid and PGE2, N is 12, 11, 10, respectively.

Fig. 4A-4c. itch-related scratching responses after intradermal injection of the disclosed fatty acid derivatives (blind analysis). (FIG. 4A) intradermal injection of the disclosed fatty acid derivatives (100. mu.g) showed increased scratch response to 9K-12,13E-LA and to a mixture of 9K-12,13E-LA +13K-9,10E-LA (100 ug each) but no response to 13K-9,10E-LA alone. For media, 9K-12,13E-LA, 13K-9,10E-LA and mixtures, N is 8,7, 6,8, respectively. (FIG. 4B) the scratching response induced by 9K-12,13E-LA was statistically greater than that of the medium and reached a maximum at 10-15 minutes and then gradually decreased. (FIG. 4C) histamine (50. mu.g) induced scratching responses significantly greater than the vehicle within 5 minutes, reaching a maximum within 5-10 minutes, followed by a sharp decrease. For histamine and control, N ═ 6.

Figures 5A-5d diet-induced reductions in plasma 11H-12,13E-LA were associated with clinical pain relief. Fig. 5A shows that a 12-week reduction in dietary LA reduced plasma concentrations of 11H-12,13E-LA, 13H-9,10E-LA and total hydroxy-epoxy-octadecenoic acid (n ═ 44) in patients with chronic daily headache. FIGS. 5B-5D show that diet-induced 11H-12,13E-LA reductions are associated with: the number of headache hours per day decreases (n-40), the number of headache days per month decreases (n-44), and the effect of headache decreases (n-44). The graph includes headache effect (y-axis) based on Poisson regression model (Poisson regression model) controlling the concentration of fatty acid derivative at each outcome and baseline, and fatty acid derivative concentration at 12 weeks (x-axis). The dotted line in fig. 5A represents the limit of quantitation. The 95% confidence intervals in FIGS. 5B-5D are shown in gray shading.

FIG. 6 Ca in murine dorsal root ganglion sensory neurons for the disclosed endogenous fatty acid derivatives and stable analogs thereof²⁺Response (blind analysis). In a blind selection of 15 compounds (72-273 cells/compound), endogenous lipids and their stable analogs (1 μm) cause Ca in the sensory neurons of the dorsal root ganglia of mice²⁺Transient. The compounds tested included: 13H-9,10E-LA (Compound 7), 2DM-13H-9,10E-LA (Compound 53), 13-methyl-13H-9, 10E-LA (Compound 56), 2 DM-13-methyl-13H-9, 10E-LA (Compound 55), 13K-9,10E-LA (Compound 8), 11H-12,13E-LA (Compound 1), 2DM-11H-12,13E-LA (Compound 17), 11-methyl-11H-12, 13E-LA (Compound 26), 11K-12,13E-LA (Compound 2), 9H-12,13E-LA (Compound 5), 9-methyl-9H-12, 13E-LA (Compound 46), 11H-9,10E-LA (Compound 3) and 11-methyl-11H-9, 10E-LA (Compound 36). PGE2, positive control (919 cells).

FIGS. 7A and 7B, Ca of trigeminal sensory neurons on the disclosed endogenous fatty acid derivatives²⁺Response (blind analysis). (FIG. 7A) in 4 combinationsBlindly selected, 11-hydroxy-epoxide and 11-keto-epoxide induced concentration-dependent Ca in mouse trigeminal sensory neurons²⁺Transient. The concentration-response curves show an increase in cell numbers in response to 11H-12,13E-LA and 11H-9,10E-LA (FIG. 7B).

Fig. 8. itch-related scratching responses following intradermal injection of the disclosed fatty acid derivatives (blinded analysis). Intradermal injection of the disclosed fatty acid derivatives (100 μ g) showed increased scratch response to 9K-12,13E-LA in both wild-type and mast cell knockout mice. Each group N is 5-8.

FIG. 9 Blind ApoA1 cholesterol efflux screening (efflux screen) in human THP-1 cells. 9-hydroxy-octadecenoic acid was increased and 11H-12,13E-LA and 11K-12,13E-LA (50 μm) inhibited apolipoprotein A1(ApoA1) mediated cholesterol efflux from monocytes.

Figure 10 blind ApoA1 cholesterol efflux screening in freshly isolated human PBMC. 11H-12,13E-LA and 11K-12,13E-LA (50 μm) inhibited ApoA 1-mediated cholesterol efflux from monocytes.

FIGS. 11A-11D.11H-12,13E-LA and 11K-12,13E-LA (100 μm) effect on cytokine secretion by human PBMC (measured by ELISA) (blind assay). incubation with 11H-12,13E-LA and 11K-12,13E-LA inhibited native Lipopolysaccharide (LPS) -induced (FIG. 11A) and native (FIG. 11B) Tumor Necrosis Factor (TNF) - α secretion, but had no effect on Interleukin (IL) -1 (FIGS. 11C and 11D).

FIGS. 12A and 12B retention time and mass spectra obtained from LC-MS analysis of 13-hydroxy-9, 10-trans-epoxy- (11E) -octadecenoic acid after incubation under esterification conditions.

FIGS. 13A-13C retention time and mass spectra obtained from LC-MS analysis of 9,10, 13-trihydroxy- (11E) -octadecenoic acid before (FIG. 13C) and after (FIGS. 13A and 13B) incubation under esterification conditions.

Figures 14A and 14b retention time and mass spectra obtained from LC-MS analysis of 2, 2-dimethyl-13-hydroxy-9, 10-trans-epoxy- (11E) -octadecenoic acid after incubation under esterification conditions.

FIG. 15 retention time and mass spectra obtained from LS-MS analysis of 4-hydroxy-DHA and 4-hydroxy-DHA lactone after incubation under esterification conditions.

FIG. 16 retention time and mass spectra obtained from LS-MS analysis of 2-methyl-4-hydroxy-DHA and 2-methyl-4-hydroxy-DHA lactone after incubation under esterification conditions.

FIG. 17 retention time and mass spectra obtained from LS-MS analysis of 2, 2-dimethyl-4-hydroxy-DHA after incubation under esterification conditions.

FIG. 18. endogenous lipids and novel compounds activate primary murine sensory neurons. The Y-axis shows the percentage of murine dorsal root ganglion sensory neurons that respond to endogenous mediators (mediators), stable analogs, and small molecules containing their proposed pharmacophores. All compounds were tested at 1 μm and the reaction was normalized to potassium chloride (KCL). Error bars represent standard deviations of the mean. For each compound, 300 KCl-positive cells from 5 or more mice were tested.

Figures 19A-19d. skin free acid and esterified lipid pool (pool) can be selectively manipulated by topical application of a2, 2-dimethyl stable analog of an oxidized derivative of linoleic acid along with labeled free acid. Topical application of 2, 2-dimethyl derivative of oxidized linolenic acid [2, 2-dimethyl-13-hydroxy-9, 10-epoxy-octadecenoic acid ] to mouse skin selectively increased the 2, 2-dimethyl-13, 9, 10-trihydroxy-octadecenoic acid derivative only in the free acid pool (acid pool) without substantial incorporation into the esterified lipids. This was confirmed by the comparable peak areas of 2, 2-dimethyl-13, 9, 10-trihydroxy-linolenic acid ester in the free acid pool (fig. 19A) and the total (free + esterified) lipid pool (fig. 19B). In contrast, topical application of D5-labeled 13-hydroxy-9, 10-epoxy-octadecenoic acid free acid in total pool (fig. 19D) resulted in a significant increase in its D5-13,9, 10-trihydroxy-octadecenoic acid derivative compared to free pool (fig. 19C).

Detailed Description

The present disclosure relates to a family of fatty acid derivatives that have been shown to have in vivo and in vitro activity in models of inflammation, pain/itch perception sensitization, epidermal barrier integrity, lipoprotein function and atherosclerosis. Thus, the disclosed compounds modulate a number of highly leveraged (highlylever) cellular processes. Several of the identified fatty acid derivatives are endogenously present. Other derivatives are also provided that have a common functional portion of the identified endogenous compounds and are modified to retain or antagonize the effect of the corresponding endogenous biologically active compound while maximizing stability, activity and ease of delivery to the subject.

I. Term(s) for

The following explanations of terms and abbreviations are provided to better describe the disclosure and to guide those of skill in the art in the practice of the disclosure. As used herein, unless the context clearly dictates otherwise, "including/comprising" means "including" and "includes. Unless the context clearly indicates otherwise, the term "or" refers to a single element or a combination of 2 or more elements of the optional elements.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the present disclosure will be apparent from the following detailed description and claims.

Although the steps of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required hereinafter in particular language. For example, in some cases, steps described as sequential may be rearranged or performed concurrently. Further, the specification sometimes uses terms such as "produce" and "provide" to describe the disclosed methods. These terms are high abstractions for the steps that are actually performed. The actual steps corresponding to these terms will vary depending on the particular practice and are readily discernible by one of ordinary skill in the art.

Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, percentages, temperatures, times, and so forth used in the specification or claims are to be understood as being modified by the term "about". Accordingly, unless otherwise implicitly or explicitly stated, the numerical parameters set forth are approximations that can depend upon the desired properties sought and/or the limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from the prior art discussed, the numbering of the embodiments is not an approximation unless the term "about" is set forth. Not all alternatives recited herein are equivalent.

Embodiments of the compounds disclosed herein may contain one or more asymmetric elements, such as stereocenters, stereoaxes, and the like, for example, asymmetric carbon atoms, and thus chemical conjugates may exist in different stereoisomeric forms. For compound embodiments having 2 or more asymmetric elements, these compound embodiments may also be mixtures of diastereomers. For compound embodiments having asymmetric centers, all optical isomers in pure form and mixtures thereof are encompassed by the corresponding general formula unless the context clearly indicates otherwise or provides an express statement excluding isomers. In this case, the individual enantiomers may be obtained by methods known to the person skilled in the art, i.e. in optically active form, such as asymmetric synthesis, synthesis from optically pure precursors or resolution by racemates. Resolution of the racemates can also be carried out, for example, by conventional methods such as crystallization in the presence of a resolving agent or chromatography using, for example, a chiral HPLC column. All isomeric forms are contemplated herein, regardless of the method used to obtain them.

Application: the agent, e.g., the disclosed fatty acid derivative, is provided or administered to the subject by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

"administration" of a compound and "administering" a compound is understood to provide a compound, prodrug of a compound, or pharmaceutical composition as described herein. The compound or composition may be administered to the subject by another person (e.g., intravenously) or it may be administered by the subject itself (e.g., tablet).

Alkyl groups: a hydrocarbon group having a saturated carbon chain. The chain may be cyclic, branched or unbranched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. The term lower alkyl denotes a chain comprising 1-10 carbon atoms. The terms alkenyl and alkynyl refer to hydrocarbon groups having carbon chains containing one or more double or triple bonds, respectively.

Aliphatic group (Aliphatic): substantially hydrocarbon-based compounds or groups thereof (e.g. C)₆H₁₃E.g., hexyl), including alkanes, alkenes, alkynes, including cyclic forms thereof, and further including linear and branched arrangements and all stereoisomers and positional isomers. Unless specifically stated otherwise, aliphatic groups contain 1-25 carbon atoms; for example, 1-15, 1-10, 1-6, or 1-4 carbon atoms. The term "lower aliphatic group" refers to an aliphatic group containing 1 to 10 carbon atoms. The aliphatic radical chain may be substituted or unsubstituted. Unless explicitly indicated as "unsubstituted aliphatic group," an aliphatic group can be unsubstituted or substituted. The aliphatic group may be substituted with one or more substituents (up to 2 substituents per methylene carbon for the aliphatic chain, or up to one substituent per carbon in the-C ═ C-double bond in the aliphatic chain, or up to one substituent for the terminal methine carbon). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, haloalkenyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, mercapto, thioalkoxy, or other functional group.

Amine or amino group: a group of formula-NRR ', wherein R and R' can be independently hydrogen or alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, haloalkenyl, or heterocycloalkyl. For example, "alkylamino" or "alkylated amino" refers to-NRR ', wherein at least one of R or R' is alkyl.

Aminoalkyl radical: an alkyl group as defined above in which at least one hydrogen atom is replaced by an amino group (e.g., -CH)₂-NH₂)。

Aryl: monovalent unsaturated aromatic carbocyclic radicals having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthracenyl), which may optionally be unsubstituted or substituted. Heteroaryl is an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thienyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. Aryl or heteroaryl groups may be substituted with one or more groups including, but not limited to: alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl or heteroaryl may be unsubstituted.

Atherosclerosis (Atherosclerosis): progressive narrowing and hardening of the blood vessels over time. Atherosclerosis is a common form of arteriosclerosis in which yellowish plaque deposits containing cholesterol, lipids and phagocytic cells are formed in the intimal and intimal media of large and medium arteries (atheroma). Treatment of atherosclerosis includes reversing or slowing the progression of atherosclerosis, as measured, for example, by the presence of atherosclerotic lesions and/or functional signs of the disease, such as improvement in cardiovascular function as measured by signs (e.g., peripheral capillary refill), symptoms (e.g., chest pain and intermittent claudication), or laboratory evidence (e.g., evidence obtained by EKG, angiography, or other imaging techniques). By "diagnosing atherosclerosis" is meant determining whether a subject has atherosclerosis, determining the prognosis of atherosclerosis in a subject, and/or determining whether a treatment regimen administered to a subject is effective to treat or prevent atherosclerosis in a subject.

In some embodiments, the fatty acid derivatives disclosed herein can be used to treat or prevent atherosclerosis in a subject.

Autoimmune diseases: diseases in which the immune system produces an immune response (e.g., a B cell or T cell response) to endogenous antigens that results in damage to the tissue. For example, rheumatoid arthritis is an autoimmune disease, as are the following diseases: hashimoto's thyroiditis, pernicious anemia, addison's disease, type I diabetes, systemic lupus erythematosus, dermatomyositis, sjogren's syndrome, dermatomyositis, lupus erythematosus, multiple sclerosis, myasthenia gravis, rett's syndrome, graves ' disease, and the like.

In some embodiments, the fatty acid derivatives disclosed herein can be used to treat or prevent an autoimmune disease in a subject.

Carboxyl group: the group-COO^-or-COOH. The carboxyl group may form a carboxylic acid.

Comparison: a sample or standard for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy patient. In other embodiments, the control is a sample obtained from a patient diagnosed with a disease or condition such as: such as inflammation, itching, pain, autoimmunity and/or arteriosclerosis. In some embodiments, a control is a sample obtained from a patient diagnosed with a disease or disorder (such as inflammation, itch, pain, autoimmunity, and/or arteriosclerosis) wherein the patient has not received treatment with a fatty acid derivative disclosed herein. In other embodiments, the control is a historical control or a standard reference value or range of values (such as a previously tested control sample, such as a group of patients with a known prognosis or outcome or a group of samples representing a baseline or normal value).

Reduction (decrease) or reduction (reduce): reducing the number, amount, or intensity of something; such as reducing signs or symptoms of a disease or disorder. In one example, treatment reduces the signs or symptoms of the disease or disorder compared to the response in the absence of treatment. In particular examples, treatment reduces signs or symptoms of a disease or disorder, such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to in the absence of treatment.

Derivative (A): derivatives are molecules that differ in chemical structure from the parent compound, e.g. homologues (differing in chemical structure such as differences in alkyl chain length), molecular fragments, structures differing in one or more functional groups, ionisation changes. In some examples, the derivatives are structurally similar or related to endogenous compounds (e.g., having the same functional groups) that comprise non-natural (or non-biologically derived) modifications intended to impart desirable properties, such as stability, solubility, and/or suitability for delivery in a biological system. The derivatives need not be synthesized from the parent compound. Structural derivatives are commonly discovered using Quantitative Structure Activity Relationships (QSAR), using techniques such as those disclosed in Remington (The science and practice of Pharmacology,19th Edition (1995), chapter 28).

And (3) diagnosis: the course of the disease is identified by signs, symptoms and the results of various tests. The conclusion reached by this process is also referred to as "diagnosis". Commonly performed test formats include blood tests, medical imaging, urinalysis, and biopsy.

Hydroxyl group: a group represented by the formula-OH.

Inflammation: when tissue damage occurs, the body's response to the damage is usually inflammation. For example, the damage may be due to trauma, insufficient blood supply, bleeding, autoimmune attack, transplanted foreign tissue, or infection. This general response of the body involves the release of many components of the immune system (e.g., IL-1 and TNF), the attraction of cells to the site of injury, tissue swelling due to the release of fluid, and other processes. Inflammation can be measured by a number of methods well known in the art, such as the number of leukocytes, the number of polymorphonuclear neutrophils (PMNs), measuring the degree of activation of PMNs (e.g., enhanced chemiluminescence in the lumen), or measuring the amount of cytokines present.

Inflammation can be classified as acute or chronic. Acute inflammation is the initial response of the body to noxious stimuli and is achieved by increased movement of plasma and leukocytes from the blood into the injured tissue. The cascade of biochemical events amplifies and matures the inflammatory response, involving various cells within the local vascular system, immune system, and injured tissue. Persistent inflammation is known as chronic inflammation, which results in a gradual transformation of the cell types present at the site of inflammation, characterized by the simultaneous destruction and healing of tissues during inflammation. An example of chronic inflammation is inflammatory arthritis.

In several embodiments, the fatty acid derivatives disclosed herein can be used to treat or prevent inflammation in a subject.

Pruritus: also known as pruritus, pruritus is a sensation or irritation that induces a subject to scratch the skin in the affected area. Itching may occur systemically or in one location. Itch may or may not be related to histamine.

Pruritus can be divided into the following types: itch affecting inflamed skin with primary disease (e.g., skin affected by inflammation, infection, autoimmune disease, lymphoma, or drug response), itch affecting non-inflamed skin with non-primary disease (e.g., itch associated with neurological disease or psychiatric origin), or itch associated with secondary scratch damage, which is scratch damage caused by the patient responding to the initial itch and includes desquamation, crusting, pimples, nodules, and chronic secondary scratch damage such as nodular prurigo.

Itch is the most common symptom of most inflammatory skin disorders (e.g., atopic dermatitis, contact dermatitis, urticaria, drug reactions, pemphigoid, dermatitis herpetiformis), parasitic or infectious diseases (e.g., scabies, mycoses, chickenpox), insect bites, and cutaneous T-cell lymphoma.

Chronic pruritus is a feeling of pruritus that exists for at least 6 weeks and is particularly prevalent in conditions such as atopic dermatitis, psoriasis, and kidney or liver disease.

In several embodiments, the fatty acid derivatives disclosed herein can be used to treat or prevent itch (such as chronic itch) in a subject.

The method comprises the following steps: the moiety is a molecular fragment or a portion of a conjugate.

Pain: unpleasant sensory and emotional experiences associated with actual or potential tissue damage or described in such damage. The pain experienced by mammals can be divided into 2 main categories: acute pain (or nociceptive) and chronic pain, the latter being classified as chronic inflammatory pain and chronic neuropathic pain. Acute pain is a response to a stimulus that causes tissue damage and is a signal away from the stimulus to minimize tissue damage. On the other hand, chronic pain does not have any biological function and develops due to inflammation caused by tissue damage (inflammatory pain) or damage of the nervous system (such as demyelination) (neuropathic pain). Chronic pain is generally characterized by persistent pain not associated with stimulation or by an abnormal pain sensation triggered by an innocuous stimulation. Non-limiting examples of pain include post-operative pain, pain associated with tissue injury, pain from inflammation, pain from infection (shingles), pain from neurological disorders, and pain from skeletal muscle disorders.

In several embodiments, the fatty acid derivatives disclosed herein can be used to treat or prevent pain (such as chronic pain) in a subject.

Pharmaceutically acceptable: a substance that can be ingested by a subject without significant adverse toxicological effects to the subject. The term "pharmaceutically acceptable form" denotes any pharmaceutically acceptable derivative or variant, such as stereoisomers, mixtures of stereoisomers, enantiomers, solvates, hydrates, isomorphous forms, polymorphs, pseudomorphic forms, neutral forms, salt forms and prodrug agents.

A pharmaceutically acceptable carrier: pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington, The science and Practice of Pharmacy, The University of The Sciences in Philadelphia, Editor, Lippincott, Williams,&Wilkins,Philadelphia,PA,21^stedition (2005) describes compositions and formulations suitable for drug delivery of one or more therapeutic compositions and additional pharmaceutical agents. In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically include as a vehicle a pharmaceutically acceptable salt or solvateInjectable fluids, which include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. In some examples, the pharmaceutically acceptable carrier can be sterile so as to be suitable for administration to a subject (e.g., by parenteral, intramuscular, or subcutaneous injection). In addition to physiologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitol monolaurate. In some examples, the pharmaceutically acceptable carrier is a non-natural or synthetic carrier. The carrier may also be formulated in unit dosage form, e.g., in pills, vials, bottles, or syringes, carrying a preselected therapeutic dose of the active agent.

Pharmaceutically acceptable salts: biocompatible salts of compounds useful as pharmaceuticals derived from a variety of organic and inorganic counterions well known in the art and including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functional group, salts such as organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like are included. Pharmaceutically acceptable acid addition salts are those which retain the biological effectiveness of the free base and are formed from acid partners (acid partners) which are not biologically or otherwise undesirable, e.g., inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric and the like; and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, benzenesulfonic acid (benzenesulfonate), cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Exemplary salts are ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, the following salts: primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. (see, e.g., S.M.Berge, et al, "pharmaceutical salts," J.pharm.Sci., 1977; 66:1-19, which is incorporated herein by reference).

Skin disorders: a disease or condition of the skin, such as an inflammatory, proliferative, sensitizing, skin barrier dysfunction disease or condition. Non-limiting examples of skin conditions include atopic dermatitis, seborrheic dermatitis, acne, rosacea, ichthyosis, erythroderma, alopecia, wrinkles, xeroderma/water barrier function, essential fatty acid deficiency, vitiligo, sebaceous cysts, pilocysts, hypertrophic scars/keloids, seborrheic keratosis, and actinic keratosis. Stereoisomers: isomers that have the same molecular formula and bonding atom order but differ only in the three-dimensional orientation of the atoms in space. Stereoisomers that are not mirror images of each other are referred to as "diastereomers", and those that are not overlapping mirror images of each other are referred to as "enantiomers". When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, there may be one pair of enantiomers. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and can be described by the R-and S-sequence rules of Cahn and Prelog or by the way the molecule rotates the plane of polarized light, either dextrorotatory or levorotatory (i.e., the (+) or (-) isomers, respectively). The chiral compounds may exist as individual enantiomers or as mixtures thereof. Mixtures containing equal proportions of enantiomers are referred to as "racemic mixtures". The E/Z isomers are stereochemically isomeric forms which differ by a double bond. The E isomer (german term from entgegen, denoting "opposite") has a trans configuration at the double bond, with the 2 groups with the highest preference located on different sides of the double bond. The Z isomer (the german term from zusa mmen, denoting "togetherer") has a cis configuration at the double bond, with the 2 groups with the highest preference being located on the same side of the double bond.

Subject: living multicellular vertebrate organisms, including the classes of human and non-human mammals.

Substituted or substituted: the hydrogen atom of the molecule or R-group is replaced by one or more additional R groups. As used herein, unless otherwise defined, the term "optionally-substituted" or "optional substituent" refers to a group that may or may not be further substituted by 1,2, 3, 4 or more groups (preferably 1,2 or 3 groups, more preferably 1 or 2 groups). The substituents may be selected from, for example, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Fluoroalkenyl radical, C_2-6Difluoroalkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, hydroxy, oxo (oxo), C_1-6Alkoxy, aryloxy, C_1-6Alkoxyaryl, halo, C_1-6Haloalkyl (such as CF)₃And CHF₂)、C_1-6Haloalkoxy (such as OCF)₃And OCHF₂) Carboxy, ester, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketone, amide, aminoacyl, substituted amide, disubstituted amide, mercapto, alkylthio, thio (thioxo), sulfate, sulfonate, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfonylamide, substituted sulfonamide, disubstituted sulfonamide, aryl C_1-6Alkyl, heterocyclyl and heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and the groups comprising them may be further optionally substituted. In the case of an N-heterocycle, optional substituents may also include, but are not limited to, C_1-6Alkyl radicals, i.e. N-C_1-3Alkyl, more preferably methyl, especially N-methyl.

Tautomers: the only difference is the position of the protons and electrons and the constitutive isomers of organic compounds that can interconvert by hydrogen atom migration. Tautomers often coexist in equilibrium.

A therapeutically effective amount of: an amount sufficient to provide a beneficial or therapeutic effect to the subject or a specified percentage of subjects. A therapeutically effective amount of a therapeutic agent can be determined in a number of different ways, such as determining a reduction in a disease or disorder, such as atherosclerosis. Therapeutically effective amounts can also be determined by a variety of in vitro, in vivo, or in situ assays. The therapeutic agent may be administered in a single dose or in several doses, for example daily during a course of treatment. However, an effective amount may depend on the source of the application, the subject being treated, the severity and type of the condition being treated, and the mode of administration.

Mercapto group: a group-SH. Substituted mercapto being hydrogen atoms substituted by, for example, C_1-6Alkyl ("-S (C)_1-6Alkyl) "), aryl (" -S (aryl) "), arylalkyl (" -S (alkyl) (aryl) "), and the like.

Treatment (treatment or treatment): with respect to a disease or disorder, either term includes (1) preventing the disease or disorder, e.g., causing clinical symptoms of the disease or disorder not to develop in a subject that may be exposed to the disease or disorder or predisposed to the disease or disorder but does not yet experience or display symptoms of the disease or disorder, (2) inhibiting the disease or disorder, e.g., arresting the development of the disease or disorder or clinical symptoms thereof, or (3) relieving the disease or disease, e.g., causing regression of the disease or disorder or clinical symptoms thereof.

Fatty acid derivatives

Embodiments of fatty acid derivatives are disclosed. As discussed herein, the disclosed fatty acid derivatives have utility in the treatment of a variety of diseases and disorders, including inflammation, itch, pain, autoimmune disorders, and atherosclerosis. In several embodiments, the disclosed fatty acid derivatives have increased activity, lower toxicity, fewer side effects, higher stability, longer half-life, or a combination thereof in human patients as compared to existing agents used to treat inflammation, itch, pain, autoimmune disorders, and/or atherosclerosis. Advantageously, certain embodiments of the disclosed fatty acid derivatives are capable of crossing the blood brain barrier.

In certain embodiments, the fatty acid derivative is a compound having a structure according to formula I or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

in formula I, X is an aliphatic group of 1-16 carbons in length (such as any of 1,2, 3, 4,5, 6, 7,8, 9,10, 11,12, 13, 14, 15, or 16), Z is an aliphatic group of 1-16 carbons in length (such as any of 1,2, 3, 4,5, 6, 7,8, 9,10, 11,12, 13, 14, 15, or 16) or is absent, Y is selected from any of the following:

R¹is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl); r²Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl); r³Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl); r⁴Is lower alkyl (such as methyl, ethyl, propyl or butyl), hydroxy, carboxy or amine; r⁵Is hydrogen, lower alkyl or halide, R⁶Is hydroxy or substituted mercapto, and each R⁷Independently hydrogen or fluorine or absent, and the adjacent carbons form an alkyne. Y may be inserted into formula I in the orientation shown above or in the opposite orientation to produce the compound of formula I.

In some embodiments of formula I, Y is selected from any one of the following:

R¹is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl); r²Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl); r³Is hydrogen or lower alkyl (such as methyl, ethyl, propyl)Alkyl or butyl); r⁴Is lower alkyl (such as methyl, ethyl, propyl or butyl), hydroxy, carboxy or amine; r⁵Is hydrogen, lower alkyl or halide, R⁶Is hydroxy or substituted mercapto, and each R⁷Independently hydrogen or fluorine. Y may be inserted into formula I in the orientation shown above or in the opposite orientation to produce the compound of formula I.

In some embodiments of formula I, X and Z are independently selected from one of the following: the length is 1-10, 4-8, 2-6, 5-10, 4-12, or 10-16 carbons. In some embodiments, Z is 1 to 10 carbons in length and X is 1 to 10 carbons in length. In some embodiments, Z is absent. In some embodiments of formula I, X is selected from 4 to 8 carbons in length and Z is selected from 1 to 6 carbons in length. In some embodiments of formula I, X is 6 carbons in length, and/or Z is 4 carbons in length. In some embodiments of formula I, X and Z are a total of 8 to 14 carbons in length. In some embodiments of formula I, X and Z are a total of 7 to 12 carbons in length. In some embodiments of formula I, X and Z are a total of 10 carbons in length. In several embodiments, X and Z are independently alkyl or alkenyl, or haloalkenyl, particularly fluoroalkenyl or difluoroalkenyl. In some embodiments, X and Z independently comprise one or more fluoroalkene or difluoroalkene moieties.

In some embodiments of formula I, R¹、R²、R³And/or R⁴Is methyl. In some embodiments of formula I, R¹、R²、R³And R⁴Is methyl. In some embodiments of formula I, R⁵Is hydrogen. In some embodiments of formula I, R⁶Is a hydroxyl group. In some embodiments of formula I, R⁶Is cysteine or glutathione. In some embodiments of formula (I), R¹Is hydrogen, R²、R³And R⁴Is methyl, R⁵Is hydrogen and R⁶Is a hydroxyl group. In some embodiments of formula (I), R¹、R²、R³And R⁴Is methyl, R⁵Is hydrogen and R⁶Is a hydroxyl group.

In certain embodiments, the fatty acid derivative is a compound having a structure according to any one of formulas II-XVII, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

in the formula (1)II) - (CII) if present, R¹Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl), R²Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl), R³Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl), R⁴Is lower alkyl (such as methyl, ethyl, propyl or butyl), hydroxy, carboxy or amine, R⁵Is hydrogen, lower alkyl or halide, R⁶Is hydroxy or substituted mercapto, each R⁷Independently hydrogen or fluorine or absent, and adjacent carbons form an alkyne, each R⁸Independently is hydrogen or fluorine, and each R⁹Independently hydrogen or deuterium. Replacement of hydrogen with fluorine or deuterium in a portion of 1, 4-cis, cis-pentadiene allows further enzymatic oxidation of these undesirable substrates, thereby enhancing stability.

In some embodiments of any of formulas (II) - (CII), R, if present¹Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl), R²Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl), R³Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl), R⁴Is lower alkyl (such as methyl, ethyl, propyl or butyl), hydroxy, carboxy or amine, R⁵Is hydrogen, lower alkyl or halide, R⁶Is hydroxy or substituted mercapto, each R⁷Independently hydrogen or fluorine, and adjacent carbons form an alkyne, each R⁸Independently is hydrogen or fluorine, and each R⁹Independently hydrogen or deuterium. Replacement of hydrogen with fluorine or deuterium in a portion of 1, 4-cis, cis-pentadiene allows further enzymatic oxidation of these undesirable substrates, thereby enhancing stability.

In some embodiments of any of formulas (II) - (CII), R¹、R²、R³And/or R⁴Is methyl. In some embodiments of any of formulas (II) - (CII), R¹、R²、R³And R⁴Is methyl. In some embodiments of any of formulas (II) - (CII), R⁵Is hydrogen. In some embodiments of formulas (II) - (XVII), R⁶Is a hydroxyl group. In some embodiments of any of formulas (II) - (XVII), R⁶Is cysteine or glutathione. In some embodiments of any of formulas (II) - (CII), R¹Is hydrogen, R²、R³And R⁴Is methyl, R⁵Is hydrogen and R⁶Is a hydroxyl group. In some embodiments of any of formulas (II) - (CII), R¹、R²、R³And R⁴Is methyl, R⁵Is hydrogen and R⁶Is a hydroxyl group.

Exemplary compound structures of the present disclosure falling within the scope of formula (I) include, but are not limited to, the following:

linoleic acid derivatives

Octadecenoic acid derivatives

Mead [ (5Z,8Z,11Z) -eicosa-5, 8, 11-triene ] acid derivatives

Arachidonic acid derivatives

Didodecene (adrenaline) acid derivatives

Docosahexaenoic acid derivatives

Any of the carboxyl-containing compounds disclosed herein (e.g., compounds 1-330) can be prepared by replacing the carboxyl group with a methyl ester group. In addition, any of the alkenyl-containing compounds disclosed herein can be prepared by replacing a hydrogen on a carbon of any carbon-carbon double bond with a fluorine.

In certain embodiments, the fatty acid derivative is a compound having a structure according to any one of formulas (CIII) - (CX) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

formulas (CIII) - (CX) encompass the putative active sites of compounds 1-16 and are therefore believed to modulate the activity of endogenous targets of compounds 1-16. In the formulae (CIII) - (CX), R⁵Is hydrogen, lower alkyl or halide, each R⁷Independently hydrogen or fluorine or absent, adjacent carbons form an alkyne, and R¹⁰And R¹¹Independently an aliphatic group.

In some embodiments of formulae (CIII) - (CX), R¹⁰And R¹¹Independently is substituted or unsubstituted lower alkyl, substituted or unsubstituted lower heteroalkyl, substituted or unsubstituted lower alkenyl (such as haloalkenyl, e.g., fluoroalkenyl or difluoroalkenyl), substituted or unsubstituted lower heteroalkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower heteroalkynyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

In some embodiments of any of formulas (CIII) - (CX), R⁵Is hydrogen. In some embodiments of any of formulas (CIII) - (CX), R¹⁰Is methyl. In some embodiments of any of formulas (CIII) - (CX), R¹¹Is methyl. In some embodiments of any of formulas (CIII) - (CX), R⁵Is hydrogen, R¹⁰Is methyl and R¹¹Is methyl.

Exemplary compound structures of the present disclosure falling within the scope of formulas (CIII) - (CX) include, but are not limited to, the following:

compounds according to formula (I) - (CX), such as compounds 1-330, can be synthesized by conventional methods, optionally supplemented with the synthetic methods provided herein (see examples). One of ordinary skill in the art will recognize that compounds may exhibit tautomerism, conformational isomerism, geometric isomerism and/or optical isomerism. For example, certain disclosed compounds may contain one or more chiral centers and/or double bonds, and thus may exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), enantiomers, diastereomers, and mixtures thereof, such as racemic mixtures. As another example, certain disclosed compounds may exist in several tautomeric forms, including the enol form, the keto form, and mixtures thereof. Since the nomenclature, structural formulae, and compound diagrams of the various compounds in the specification and claims can represent only one of the possible tautomeric, conformational isomeric, enantiomeric, or geometric isomeric forms, it is to be understood that the disclosed compounds encompass any tautomeric, conformational isomeric, enantiomeric, and/or geometric isomeric form of the compounds described herein, as well as mixtures of such various isomeric forms.

Other Compound embodiments

In certain embodiments, the fatty acid derivative is an oxidized fatty acid having a2, 2-dimethyl group, which reduces esterification of the oxidized fatty acid, as described herein. In several embodiments, the 2, 2-dimethyl modified oxidized fatty acid embodiments have an increased half-life under physiological conditions (such as in blood or in phosphate buffered saline) as compared to the corresponding unmodified oxidized fatty acid compounds. In some embodiments, the 2, 2-dimethyl modified oxidized fatty acid compound has a structure according to formula CXI or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

in formula CXI, X is an aliphatic group of 10 to 25 carbons in length (such as longDegree of any of 10,11, 12,13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 carbons) and includes one or more epoxy, hydroxyl, or carbonyl substitutions, or combinations thereof, R¹Is hydrogen or lower alkyl (such as methyl, ethyl, propyl or butyl), and each R²Independently methyl or hydrogen. In some embodiments of formula CXI, X is a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In several embodiments, X is alkyl or alkenyl or haloalkenyl, particularly fluoroalkenyl or difluoroalkenyl. In some embodiments, X comprises one or more fluoroalkene or difluoroalkene moieties. In some embodiments, X is alkyl or alkenyl. In some embodiments, X comprises one or more diallyldideuterium substitutions, e.g., X is deuterated diallenyl or dideuterodiallylenyl. In some embodiments, each R is²Is methyl. In some embodiments, each R is²Is hydrogen. In some embodiments, each R is²Is methyl and the compound is an oxidised derivative of linoleic acid. In some embodiments, the compound of formula CXI comprises one or more deuterium substitutions of a hydrogen at an oxidation-sensitive site of a fatty acid or at a site that becomes oxygen-sensitive upon further conversion (e.g., at a diallyl position).

Pharmaceutical composition

The present disclosure also includes pharmaceutical compositions comprising at least one fatty acid derivative disclosed herein or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof. In some embodiments, the fatty acid derivative is a compound according to any one of structures 1-330. Some embodiments of the pharmaceutical composition comprise at least one fatty acid derivative and at least one other pharmaceutically acceptable additive other than the selected molecule, such as a pharmaceutically acceptable carrier, thickener, diluent, buffer, preservative, surfactant, and the like. Useful pharmaceutically acceptable carriers and excipients are known in the art.

Pharmaceutical compositions comprising one or more fatty acid derivatives may be formulated in a variety of ways depending, for example, on the mode of administration and/or the location of imaging. Parenteral formulations can include injectable fluids which are pharmaceutically and physiologically acceptable fluid media such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol and the like. Excipients may include, for example, nonionic solubilizers, such as

Polyethoxylated detergents; or a protein, such as human serum albumin or a plasma preparation. If desired, the pharmaceutical composition to be administered may also contain non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitol monolaurate.

The form of the pharmaceutical composition will be determined by the mode of administration selected. Embodiments of the disclosed pharmaceutical compositions can take a form suitable for use in virtually any form of administration, including, for example, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, and the like, or a form suitable for administration by inhalation or insufflation. Typically, embodiments of the disclosed pharmaceutical compositions will be administered parenterally (e.g., by intravenous, intraarterial, subcutaneous, intramuscular, or intraperitoneal injection), intrathecally, or orally.

Useful injectable formulations include sterile suspensions, solutions or emulsions of the active compounds in aqueous or oily media. The composition may also comprise formulating agents such as suspending, stabilizing and/or dispersing agents. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, and may include an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous media, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. For example, parenteral administration may be by bolus injection (bolus injection) or continuous infusion. Alternatively, the fatty acid derivative may be in the form of a powder for reconstitution with a suitable medium (e.g. sterile water) prior to use.

Systemic formulations include those designed for administration by injection, for example, subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

Oral formulations may be liquid (e.g., syrup, solution or suspension) or solid (e.g., powder, tablet or capsule). The oral formulation may be coupled to a targeting ligand to cross the endothelial barrier. Some fatty acid derivative formulations may be spray dried, for example, with a disaccharide to form a fatty acid derivative powder. Solid compositions may be prepared in conventional manner using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, mannitol, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art, for example with sugar, film or enteric coating. Methods of making such dosage forms are known or apparent to those skilled in the art.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous media (e.g., almond oil, oily esters, ethanol,

Detergents or fractionated vegetable oils); and preservatives (e.g., methyl or propyl parabens or sorbic acid). The formulations may also contain buffer salts, preservatives, flavoring agents, coloring agents and sweeteners, as desired. Formulations for oral administration may be suitably formulated to provide controlled release of the fluorophore, as is well known.

Certain embodiments of the pharmaceutical compositions comprising fatty acid derivatives as described herein may be formulated in unit dosage forms suitable for individual administration of precise dosages. If desired, the pharmaceutical composition may be presented in a packaging or dispensing device which may contain one or more unit dosage forms comprising the fatty acid derivative. The package may comprise, for example, a metal or plastic foil, such as a blister pack. The packaging or dispensing device may be accompanied by instructions for administration. The amount of fatty acid derivative administered will depend, at least in part, on the subject being treated, the target (e.g., tumor size, location, and characteristics), and the mode of administration, and is known to those skilled in the art. Within this range, the formulation to be administered will comprise an amount of the fatty acid derivative described herein sufficient to provide a therapeutically effective dose of the drug to the subject being treated.

Method IV

In other embodiments, a method of treating a disease or disorder in a subject using the disclosed fatty acid derivatives is provided. The method comprises administering to a subject having or suspected of having a disease or disorder a therapeutically effective amount of a pharmaceutical composition comprising the disclosed fatty acid derivatives. Exemplary diseases or conditions to which the methods can be applied include inflammation, chronic pruritus, chronic pain, autoimmune disorders, atherosclerosis, skin disorders, neurodegenerative disorders, psychiatric disorders, and arthritis. In other embodiments, the disclosed fatty acid derivatives may be used in any composition applied to the skin, such as compositions for cosmetic or personal care purposes or insect repellents.

The pharmaceutical compositions may be administered by any suitable route, such as topically, parenterally or orally. The subject may be a mammal, such as a human or non-human mammal. In certain examples, the subject is a human.

The fatty acid derivative is administered to the subject as follows: single bolus delivery, via continuous delivery over an extended period of time (e.g., continuous intravenous delivery), or using a repeated administration regimen (e.g., an hourly, daily, weekly, or biwekkly repeated administration regimen). A therapeutically effective amount of a fatty acid derivative can be provided as repeated doses in a long-term treatment regimen that will result in a clinically significant outcome of alleviation of one or more symptoms or detectable conditions associated with the disease or disorder. Determination of an effective dose in this context is generally based on animal model studies followed by human clinical trials and is guided by principles of administration that significantly reduce the onset or severity of the targeted disease symptom or condition in the subject. Suitable models in this regard include, for example, rodents, rats, birds, pigs, cats, non-human primates, and other accepted animal model subjects known in the art. Alternatively, the effective dose may be determined using an in vitro model. Using such models, only routine calculations and adjustments are required to determine the appropriate concentration and dose to administer a therapeutically effective amount of the compound (e.g., an amount effective to elicit a desired immune response or to alleviate one or more symptoms of the targeted disease). In alternative embodiments, an effective amount or effective dose of a fatty acid derivative may simply inhibit or enhance one or more selected biological activities associated with the disease or condition being treated, as set forth herein, for therapeutic or diagnostic purposes.

The actual dosage of the fatty acid derivative will vary depending on the following factors: such as the signs and particulars of the disease in the subject (e.g., the subject's age, size, health, extent of symptoms, susceptibility factors, etc.), the time and route of administration, other drugs or treatments concurrently administered, and the particular pharmacology with which the fatty acid derivative elicits the desired activity or biological response in the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response. A therapeutically effective amount is also an amount that clinically outweighs any toxic or detrimental side effects of the fatty acid derivative. Non-limiting ranges for a therapeutically effective amount of a fatty acid derivative in the methods and formulations of the present disclosure can be within the following ranges: 0.01mg/kg body weight to 5g/kg body weight, such as 10mg/kg to 5g/kg body weight or 1g/kg to 5g/kg body weight. In some embodiments, the fatty acid derivative can be administered in an amount effective to provide a serum fatty acid derivative concentration of 0.1-100 μm or 1-5000 μ g/mL.

The dosage may be varied by the attending physician to maintain the desired concentration at the target site (e.g., systemic circulation). Higher or lower concentrations may be selected depending on the mode of delivery (e.g., oral, intravenous, or topical delivery). The dosage can also be adjusted according to the release rate of the administered formulation, e.g., intra-pulmonary spray versus powder, sustained release oral versus injection granules or transdermal delivery formulation, etc.

In some embodiments, the disclosed fatty acid derivatives have use in treating pruritus (such as chronic pruritus) in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with pruritus (such as chronic pruritus) in a subject. For example, fatty acid derivatives may be used to reduce itch (such as chronic itch) associated with inflamed skin, such as skin affected by: inflammatory skin disorders (e.g., atopic dermatitis, psoriasis, contact dermatitis, urticaria, drug reactions, pemphigoid, dermatitis herpetiformis), parasitic or infectious diseases (e.g., scabies, mycoses, chickenpox), autoimmune disorders, lymphomas (e.g., cutaneous T-cell lymphomas), and itch of uninflammated skin affecting non-primary diseases (such as itch associated with neurological or psychiatric origin) or itch associated with secondary scratch injury resulting from a patient's response to the initial itch and including desquamation, crusting, pimples, nodules, and chronic secondary scratch injury such as prurigo nodularis. In some embodiments, administering a therapeutically effective amount of the disclosed fatty acid derivatives to a subject to treat pruritus reduces pruritus in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% compared to no treatment. The activity of the disclosed fatty acid derivatives for treating itch can be demonstrated in animal models, for example, by assessing the itch response in mice against histamine injections in combination with related fatty acid derivatives or controls (see, e.g., example 1 below).

In some embodiments, the disclosed fatty acid derivatives have use in treating pain (such as chronic pain) in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with pain (such as chronic pain) in the subject. For example, fatty acid derivatives can be used to reduce pain associated with inflammation (including inflammation due to tissue injury, inflammatory pain), pain caused by nervous system injury such as demyelination (neuropathic pain), post-surgical pain, pain associated with tissue injury, pain from infection (shingles), pain from neurological disorders, and pain from skeletal muscle disorders. In some embodiments, administration of a therapeutically effective amount of a disclosed fatty acid derivative to a subject to treat pain can reduce pain in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% compared to not using the treatment. For example, the activity of the disclosed fatty acid derivatives for treating pain can be determined in animal models by: mice were evaluated for pain response to PEG2 injection conjugated to related fatty acid derivatives or controls (see, e.g., example 1 below).

In some embodiments, the disclosed fatty acid derivatives have use in treating atherosclerosis in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with atherosclerosis in the subject. For example, administration of a therapeutically effective amount of a fatty acid derivative to a subject can be used to reverse or slow the progression of atherosclerosis, e.g., as measured by the presence of atherosclerotic lesions and/or functional signs of the disease, such as improvement in cardiovascular function as measured by signs (such as peripheral capillary refilling), symptoms (such as chest pain and intermittent claudication), or laboratory evidence (such as obtained by EKG, angiography, or other imaging techniques). In some embodiments, administration of a therapeutically effective amount of a fatty acid derivative increases cholesterol flux in a subject. In some embodiments, administration of a therapeutically effective amount of a fatty acid derivative reduces the LDL cholesterol level of the subject, e.g., as compared to a baseline level of LDL cholesterol. In some embodiments, administration of a therapeutically effective amount of a disclosed fatty acid derivative to a subject to treat atherosclerosis can reduce atherosclerosis in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% as compared to no treatment. In one example, the activity of the disclosed fatty acid derivatives for treating atherosclerosis can be indicated by: the increase in cholesterol flux induced by ApoA1 in combination with a fatty acid derivative was measured relative to a relevant control (see, e.g., example 14 below).

In some embodiments, the disclosed fatty acid derivatives have use in treating an autoimmune disorder in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with the autoimmune disorder in the subject. For example, administration of a therapeutically effective amount of a fatty acid derivative to a subject can be used to treat, prevent and/or ameliorate the symptoms of rheumatoid arthritis, hashimoto's thyroiditis, pernicious anemia, addison's disease, type I diabetes, systemic lupus erythematosus, dermatomyositis, sjogren's syndrome, dermatomyositis, lupus erythematosus, multiple sclerosis, myasthenia gravis, rett's syndrome, or graves ' disease, among others. In some embodiments, administration of a therapeutically effective amount of a disclosed fatty acid derivative to a subject to treat an autoimmune disorder can reduce the autoimmune disorder in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% as compared to not using the treatment.

In some embodiments, the disclosed fatty acid derivatives have use in treating arthritis (such as degenerative arthritis) in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with arthritis in the subject, such as reducing pain and/or swelling of the buttocks, knee, lower lumbar and cervical spine, proximal and distal interphalangeal joints, first carpometacarpal joint, and/or first tarsometatarsal joint of the foot. For example, administration of a therapeutically effective amount of a fatty acid derivative to a subject can be used to treat, prevent and/or ameliorate the symptoms of arthritis. In some embodiments, administration of a therapeutically effective amount of a disclosed fatty acid derivative to a subject to treat arthritis can reduce arthritis in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% as compared to not using the treatment.

In some embodiments, the disclosed fatty acid derivatives have use in treating a neurodegenerative disorder in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with the neurodegenerative disorder in the subject. For example, administration of a therapeutically effective amount of a fatty acid derivative to a subject can be used to treat, prevent and/or ameliorate the symptoms of alzheimer's disease, vascular dementia, parkinson's disease, huntington's chorea, multiple sclerosis, or Amyotrophic Lateral Sclerosis (ALS). In some embodiments, administration of a therapeutically effective amount of the disclosed fatty acid derivatives to a subject to treat a neurodegenerative disorder can reduce the neurodegenerative disorder in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% compared to not using the treatment.

In some embodiments, the disclosed fatty acid derivatives have use in treating a psychiatric disorder in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with the psychiatric disorder in the subject. For example, administration of a therapeutically effective amount of a fatty acid derivative to a subject can be used to treat, prevent and/or ameliorate the symptoms of depression, anxiety and/or confusion. In some embodiments, administration of a therapeutically effective amount of the disclosed fatty acid derivatives to a subject to treat a psychiatric disorder can reduce neurodegenerative disorder in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% compared to not using treatment.

In some embodiments, the disclosed fatty acid derivatives have use in treating a skin condition in a subject. In such embodiments, administration of a therapeutically effective amount of a fatty acid derivative improves at least one sign or symptom associated with the skin condition in the subject. For example, administration of a therapeutically effective amount of a fatty acid derivative to a subject can be used to treat, prevent and/or ameliorate the symptoms of atopic dermatitis, seborrheic dermatitis, acne, rosacea, ichthyosis, erythroderma, alopecia, wrinkles, xeroderma/water barrier function, essential fatty acid deficiency, vitiligo, sebaceous cysts, pili cysts, hypertrophic scars/keloids, seborrheic keratosis, and actinic keratosis. In some embodiments, administration of a therapeutically effective amount of a fatty acid derivative to a subject may be used to treat, prevent and/or ameliorate symptoms of a disorder with water barrier dysfunction or increased epidermal water loss (epidermal water loss), such as ichthyosis, eczema/atopic dermatitis, psoriasis and/or xeroderma. For example, a pharmaceutical composition comprising a disclosed compound that is an oxidized derivative of linoleic acid, and has 2-methyl or 2, 2-dimethyl, and is an oxidized derivative of linoleic acid (such as any of compounds 31-36, 38-43, or 66-72) can be topically applied to a subject to treat, prevent, and/or ameliorate the symptoms of a condition having water barrier dysfunction or increased epidermal water loss. In some embodiments, administration of a therapeutically effective amount of the disclosed fatty acid derivatives to a subject to treat a skin disorder can reduce a neurodegenerative disorder in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% compared to not using the treatment.

In other embodiments, the methods provide for diagnosing a disease or disorder in a subject, e.g., for determining the likelihood that a subject (such as a healthy subject or a subject suspected of having or at risk of having a disease or disorder) has a disease or disorder or is likely to develop a disease or disorder in the future. The method comprises measuring the level of any one of compounds 1-16 in a biological sample from a subject, and diagnosing the subject as a subject having, or at risk of developing, a disease or disorder if an alteration (e.g., an increase in level, e.g., at least 2-fold) in the level of the compound as compared to a corresponding control (such as the level of the compound in a subject that does not have, or is at risk of developing, the disease or disorder) is detected in the biological sample. In several embodiments, the disease or disorder is selected from one of the following: inflammation, chronic itch, chronic pain, autoimmune disorders, skin disorders, and arteriosclerosis. In some embodiments, the measured level of any one of compounds 1-16 in a biological sample from a subject can be used to guide targeted intervention or advice for preventing or controlling a disease or disorder. For example, a subject identified as having an increased amount in any of compounds 1-16 can be subjected to a dietary intervention that reduces the level of the compound, e.g., a diet with reduced polyunsaturated amino acids.

In some embodiments, provided herein are methods of assessing inflammation, e.g., for determining the likelihood that a subject (such as a healthy subject or a subject suspected of having or at risk of having an inflammatory disorder) has or will likely in the future develop an inflammatory disorder. The method comprises measuring the level of any one of compounds 1-16 in a biological sample from the subject, and diagnosing the subject as a subject having, or at risk of having, an inflammatory disorder if an elevated level (such as an elevated level, e.g., at least a 2-fold increase) of the level of the compound compared to a corresponding control (e.g., a corresponding level of the compound in a healthy subject) is detected in the biological sample.

In some embodiments, provided herein are methods of assessing chronic pruritus, e.g., for determining the likelihood that a subject (such as a healthy subject or a subject suspected of having or at risk of having chronic pruritus) suffers from chronic pruritus or will likely develop chronic pruritus in the future. The method comprises measuring the level of any one of compounds 1-16 in a biological sample from the subject, and diagnosing the subject as having or at risk of having chronic pruritus if an elevated level (such as an elevated level, e.g., at least a 2-fold increase) of the level of the compound compared to a corresponding control (e.g., a corresponding level of the compound in a healthy subject) is detected in the biological sample.

In some embodiments, provided herein are methods of assessing chronic pain, e.g., for determining the likelihood that a subject (such as a healthy subject or a subject suspected of having or at risk of having chronic pain) has chronic pain or will likely develop chronic pain in the future. The method comprises measuring the level of any one of compounds 1-16 in a biological sample from the subject, and diagnosing the subject as having or at risk of having chronic pain if an elevated level (such as an elevated level, e.g., at least a 2-fold increase) of the compound compared to a corresponding control (e.g., a corresponding level of the compound in a healthy subject) is detected in the biological sample.

In some embodiments, provided herein are methods of assessing autoimmunity, e.g., for determining the likelihood that a subject (such as a healthy subject or a subject suspected of having or at risk of having an autoimmune disorder) has an autoimmune disorder or will likely develop an autoimmune disorder in the future. The method comprises measuring the level of any one of compounds 1-16 in a biological sample from the subject, and diagnosing the subject as a subject having or at risk of having an autoimmune disorder if an elevated level (such as an elevated level, e.g., at least a 2-fold increase) of the level of the compound compared to a corresponding control (e.g., a corresponding level of the compound in a healthy subject) is detected in the biological sample.

In some embodiments, provided herein are methods of assessing atherosclerosis, e.g., for determining the likelihood that a subject (such as a healthy subject or a subject suspected of having or at risk of developing atherosclerosis) will develop atherosclerosis or will likely develop atherosclerosis in the future. The method comprises measuring the level of any one of compounds 1-16 in a biological sample from the subject, and diagnosing the subject as a subject having or at risk of having atherosclerosis if an elevated level (such as an elevated level, e.g., at least a 2-fold increase) of the level of the compound compared to a corresponding control (e.g., a corresponding level of the compound in a healthy subject) is detected in the biological sample. In some examples, the subject may have elevated cholesterol or triglyceride levels, elevated C-reactive protein levels, diabetes, or hypertension. Thus, the methods disclosed herein can be used to confirm a previous clinical suspicion of a disease.

In some examples, the biological sample is obtained from a subject for evaluation. The biological sample can be any relevant biological sample, such as, but not limited to, serum, blood, plasma, urine, purified cells (e.g., blood cells, such as leukocytes, B cells, T cells, or monocytes), saliva, a biopsy, or a tissue (such as skin) sample, such as a sample comprising blood vessels, adipocytes, cardiac tissue, neural tissue obtained from a subject, for predicting the risk of a disease or disorder in the subject, such as inflammation, chronic itch, chronic pain, an autoimmune disorder, and atherosclerosis.

V. examples

The following examples provide specific features for illustrating certain embodiments, but the scope of the claims should not be limited to those features exemplified.

Example 1

Systematic method for discovering new mediators (mediators) of pain and itch

Chronic pain and itching are common causes of personal distress, disability and social expense. Current treatments often provide only partial or temporary relief and have substantial side effects. There is a need to discover new endogenous mediators and potential mechanisms of pain and itch to facilitate the development of targeted, effective, safe interventions.

As the largest sensory organ, the skin is abundantly innervated by cutaneous nerve endings that can sense the microenvironment. Linoleic acid (LA, 18:2n-6) -the polyunsaturated fatty acid most abundant in the skin to date-is called the "essential fatty acid" because of the small amount (about 0.5% of energy) required in the diet to form an external waxy epidermal barrier that prevents transepidermal water loss. Since itch and pain are common manifestations of skin inflammatory diseases, and LA is an endogenous substrate for conversion to bioactive lipid mediators, LA-derived mediators may have a unique position in regulating skin itch and pain.

It was previously shown in rats that an increase in dietary LA increases the well-known LA derivatives in a dose-dependent manner in many tissues including skin. In humans, low LA dietary intervention reduces headache and reduces circulating LA associated with pain relief, suggesting that LA-derived lipid mediators may contribute to sensory signaling. However, the specific LA derivatives that mediate or modulate the sensations and molecular pathways involved in their biosynthesis and signal transduction are not fully understood.

It is speculated that the new LA-derived auto-actives (autacoids) abundant in skin may play a role in the development of pain and itch. This hypothesis was studied by applying a system-based translation method (systems-based, translational approach) in rats and humans to:

(1) predicting a new lipid mediator based on tissue-specific precursor abundance and gene expression profile of biosynthetic genes;

(2) synthesizing a predicted compound by total chemical synthesis;

(3) identification and quantification of these mediators in rat and human tissues using authentic standards and liquid chromatography tandem mass spectrometry (LC-MS/MS);

(4) determining whether the levels of these compounds can be altered by diet and chronic inflammatory states; and

(5) these new lipids were tested for analgesic and pruritic activity using blind ex vivo sensory neuron culture and in vivo behavioral testing. A review of this method and system to identify biosynthetic genes and their expression identifies novel LA-derived lipid mediators that modulate inflammatory skin disorders, itch, and nociception.

Results

Prediction of mediators based on precursor abundance and biosynthetic gene expression profiles

Gene expression profiles of precursor fatty acid compositions and tissues are used to guide the prediction of new lipid mediators. LA was observed to be the most abundant polyunsaturated fatty acid in rat skin and sciatic nerve, accounting for 27.4% and 24.6% of the total fatty acids, respectively. LA is much less abundant in sensory ganglia and in the dorsal spinal cord.

ALOX12B and ALOX15B genes (which encode enzymes capable of peroxidation of polyunsaturated fatty acids containing the 1, 4-cis, cis-pentadiene system) are well expressed in human skin; ALOX12B, but not ALOX15B, was also well expressed in rat skin. ALOX15B was expressed fairly well in the human tibial nerve and Dorsal Root Ganglion (DRG), but was expressed less or absent in rat neural tissue including the nociceptive circuit (i.e. sciatic nerve, DRG and spinal cord dorsal horn). The ALOXE3 gene, which encodes an enzyme capable of isomerizing fatty acid hydroperoxidase to form specific hydroxy-and-keto-epoxide derivatives, is also well expressed in rat and human skin, but is less expressed or absent in peripheral nerves, sensory ganglia, and dorsal cords. The CYP2S1 gene (which encodes another enzyme capable of isomerizing fatty acid hydroperoxidase) is well expressed in rat skin, particularly the sciatic nerve, but is less expressed or absent in human pain circuit tissues. Overall, these gene expressions and precursor fatty acid data form a template for predicting new lipid mediators.

Tissue-specific distribution of hydroxy-epoxy-and keto-epoxy-octadecenoic acids

Based on the high levels of LA noted above and the moderate to high expression of genes encoding biosynthetic enzymes, two new 11-hydroxy-trans-epoxy-octadecenoic acids were predicted:

11-hydroxy (H) -12, 13-trans-epoxy- (E) -octadecenoic acid (11H-12,13E-LA)

11-hydroxy (H) -9, 10-trans-epoxy- (E) -octadecenoic acid (11H-9,10E-LA)

And two new 11-keto-trans-epoxy-octadecenoic acids:

11-keto (K) -12, 13-trans-epoxy- (E) -octadecenoic acid (11K-12,13E-LA)

11-keto (K) -9, 10-trans-epoxy- (E) -octadecenoic acid (11K-9,10E-LA)

And 4 previously identified 9-or 13-hydroxy-or keto-trans-epoxy-octadecenoic acids:

9-hydroxy (H) -12, 13-trans-epoxy- (E) -octadecenoic acid (9H-12,13E-LA)

13-hydroxy (H) -9, 10-trans-epoxy- (E) -octadecenoic acid (13H-9,10E-LA)

9-keto (K) -12, 13-trans-epoxy- (E) -octadecenoic acid (9K-12,13E-LA)

13-keto (K) -9, 10-trans-epoxy- (E) -octadecenoic acid (13K-9,10E-LA)

Will be present in large amounts in human and rat skin. These compounds are described below:

after these 8 LA derivatives were subjected to total chemical synthesis for use as authentic standards (see materials and methods and examples below), these mediators in rat and human tissues were quantified using ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). 5 of the 8 mediators were found to be present in rat skin, but none were detected in rat dorsal horn, indicating the tissue specificity of the predictive model. All 8 mediators were detected in human skin; 7 of these 8 mediators were confirmed by matching the ion spectra of the authentic standard and human skin extract at the feature retention time.

Increased levels of free mediators in the skin of inflamed psoriatic patients

Psoriatic lesions show higher expression of genes encoding lipase mediated release (PLA2G2A, PLA2G2F), enzymatic peroxidation (ALOX12B) and hydroperoxide isomerization (CYP2S1) compared to non-diseased psoriatic skin. Thus, an increase in local biosynthesis and release of esterified, preformed lipids would potentially contribute to the higher concentrations of hydroxy-epoxy-and keto-epoxy-octadecenoic acids observed in psoriatic lesions.

These mediators were measured in both the unesterified (free) lipid fraction and the total lipid fraction (sum of free and esterified) in human psoriatic skin lesions and in non-psoriatic control skin. There was no significant difference in the total lipid fraction between psoriatic skin lesions and control skin. However, six mediators (11H-12,13E-LA, 11H-9,10E-LA, 11K-9,10E-LA, 9H-12,13E-LA, 9K-12,13E-LA and 13H-9,10E-LA) were significantly elevated as free acids (bioactive pools) in psoriatic skin lesions compared to control skin. The concentrations of free 11H-12,13E-LA and 9K-12,13E-LA were > 6-fold and > 30-fold higher in psoriatic lesions, respectively, compared to control skin. The highest concentration was observed in lesions of psoriasis patients reported to have itching (figure 1).

To gain further insight into the biochemical status of each mediator, the free acid concentration is divided by the total mediator concentration to determine the percentage of each mediator present as bioavailable free acid. The percentage of free acid varies significantly depending on the mediator. The percentage of free acid in control human skin ranged from 0.05% for 13H-9,10E-LA to 44.4% for 11H-12, 13E-LA. In psoriatic skin lesions, the percentage of free acid in 11H-12,13E-LA, 11K-12,13E-LA, 9K-12,13E-LA and 13H-9,10E-LA is significantly higher than in control skin. These findings support the following hypothesis: in chronic epidermal inflammation, enzymatic synthesis and/or release of free acids from esterified lipids is increased.

Mediator concentrations in serum are not related to skin or psoriatic state

To determine whether measurements obtained from circulating blood provide a surrogate marker of skin inflammation, these mediators were next quantified in serum from psoriasis patients and non-psoriasis controls. Unlike skin, the serum concentrations of these 8 mediators do not differ depending on the condition of the disease.

Novel LA derivatives stimulate rat sensory neurons in a regioselective manner

To determine whether these mediators sensitized DRG neurons, each mediator was tested in an adult rat DRG ex vivo Calcitonin Gene Related Peptide (CGRP) release assay, PGE2 was used as a positive control. Neither PGE2 nor any other test compound directly stimulated CGRP release at a neutral pH 1 μm concentration. However, 11H-12,13E-LA and 11H-9,10E-LA significantly enhanced low-pH-induced and capsaicin-induced release of CGRP. 13H-9,10E-LA significantly enhanced low pH-induced release of CGRP but had no effect on capsaicin-induced release. Neither 9H-12,13E-LA nor any of the keto-epoxy-octadecenoic acids tested enhanced low pH-induced or capsaicin-induced CGRP release (FIGS. 2A-2C). These observations indicate that octadecenoic acid-induced sensitization is regioselective, with the most potent effect observed for compounds containing hydroxyl groups at both carbon 11 and the adjacent epoxy group. Both compounds have a 3-hydroxy-Z-pentenyl-E-epoxide moiety, identifying this substructure as a potential pharmacophore mediating nociceptor sensitization (figure 2D).

Intradermal injection of novel mediators to elicit pain and itch-related behaviors in rodents

Next, behavioral responses to intradermal injection of mediators that produce sensitization as measured by the resulting increase in CGRP release from isolated sensory neurons were determined. 11H-12,13E-LA was chosen as the first mediator to test the pain response because this substance is abundant in the free acid form in inflamed human skin and it increases capsaicin and pH stimulated CGRP release in rat sensory neurons.

For these experiments, the effect of the LA derivative was compared to vehicle and the classical inflammation mediator PGE2 (as a positive control). It was observed that C fiber withdrawal latency was reduced by 28% (p-0.03) and 46% (p-0.001), respectively, for 11H-12,13E-LA and PGE2 after injection, indicating nociceptive hypersensitivity (fig. 3). Intradermal injection of PGE2 instead of 11H-12,13E-LA also significantly increased the proportion of withdrawal response after stimulation with a laser tuned to excite a δ fibers.

Next, to examine the effect of these 8 mediators on pruritus, a mouse model was used to quantify the onset of scratching associated with pruritus within the first 30 minutes after intradermal injection into the back of the neck. In a small scale test of all 8 mediators per group of n-3, 2 mediators (9K-12,13E-LA and 13K-9,10E-LA) all appeared to increase scratching episodes compared to vehicle. With a larger sample size of n-6-8 per group, it was observed that 9K-12,13E-LA, but not 13K-9,10E-LA, induced scratching behavior (p-0.001) associated with pruritus (fig. 4A). In combination, 9K-12,13E-LA +13K-9,10E-LA also significantly increased scratch behavior (p 0.002) compared to the vehicle, but to the same extent as was observed with 9K-12,13E-LA alone. The scratching response by 9K-12,13E-LA is slower in onset than that observed with histamine and gradually weakens.

In combination with results indicating that 9K-12,13E-LA is elevated exclusively in psoriatic lesions of skin of pruritic patients (fig. 2B), these behavioral findings indicate that 9K-12,13E-LA may represent a new mediator of pruritus.

The novel mediator is regulated by dietary LA and the reduced plasma levels are associated with reduced clinical pain

Next, to determine whether these mediators can be reduced by reducing the amount of their precursor LA in the diet, plasma samples from a fully randomized human clinical trial were used to test a 12-week LA-reduced diet in patients with severe chronic daily headache. 5 of these 8 mediators were observed to be present in plasma (FIG. 5). Dietary intervention via LA reduction significantly reduced both mediators (11H-12,13E-LA and 13H-9, 10E-LA); the sum of 4 hydroxy-epoxide-octadecenoic acids was reduced by 41% (p <0.001) (fig. 5A). In addition, a diet-induced decrease in one of these mediators (11H-12,13E-LA), but not the other mediators, was observed to correlate closely with a decrease in the number of headache hours per day and the number of headache days per month (FIGS. 5B and 5C). Each standard deviation reduction in 11H-12,13E-LA correlates with a 25% and 11% reduction in the number of headache hours per day and headache days per month, respectively (both p < 0.001); the reduction in 11H-12,13E-LA also tended to be associated with overall headache effects (FIG. 5D) and improvement in physical function, but not psychological distress.

Discussion of the related Art

This example provides a interdisciplinary translation approach for rodents and humans, thereby discovering and characterizing a new family of endogenous lipid mediators of pain and itch. As predicted, significant concentrations of 11H-12,13E-LA, 11H-9,10E-LA, 11K-12,13E-LA and 11K-9,10E-LA were measured in human skin. This is believed to be the first demonstration of any of these 4 compounds in any species. Notably, in the ex vivo CGRP release assay, 11H-12,13E-LA was elevated in inflamed psoriatic skin, in sensitized primary afferent dorsal root ganglion neurons, and induced C-fiber mediated pain-related hypersensitivity in rats. In addition, plasma 11H-12,13E-LA is associated with headache frequency and effects in humans and is reduced by reducing the amount of its dietary precursor (LA) in the diet. Taken together, these findings suggest that 11H-12,13E-LA may be a pain mediator regulated by diet and inflammation. 11H-9,10E-LA, which has the same 3-hydroxy-Z-pentenyl-E-epoxide moiety as 11H-12,13E-LA, is also elevated in inflamed human skin and sensitized rat sensory neurons, suggesting that it may also contribute to inflammation-related primary afferent sensitization.

In addition to identifying new endogenous LA derivatives, these findings confirm the presence of previously identified hydroxyl and keto-epoxy-octadecenoic acids in human skin and provide new insights into their potential biological effects. Genes encoding 12-R-lipoxygenase (ALOX12B), 15-lipoxygenase-2 (ALOX15B) and hydroperoxide isomerase e-lipoxygenase 3(ALOXE3) are highly expressed in the skin. It has previously been proposed that the sequential action of two specific enzymes, 12-R-lipoxygenase and E-lipoxygenase-3, oxidizes LA esterified in acyl-ceramide to form the specific stereoisomer of 13H-9,10E-LA (13- (R) hydroxy-9 (R),10(R) -trans-epoxy- (11E) -octadecenoic acid and/or its trihydroxy LA derivatives, which are said to play a key role in the formation of the lipid envelope of keratinocytes. The proposed need for these particular LA derivatives to form a functional water barrier may explain the mechanism that dietary LA in small amounts is required to prevent the clinical manifestations of "essential fatty acid deficiency", including dry skin, thickening and desquamation. Consistent with previous findings, relatively high concentrations of 13H-9-E-LA are observed herein in rodent and human skin. In human skin, 13H-9-E-LA is found almost exclusively in the esterified lipid pool (median > 99.5%), consistent with its proposed role in epidermal keratinocyte lipid envelope formation. In addition, it is reported here for the first time that the concentration of free 13H-9-E-LA in psoriatic skin lesions is 9 times higher than that of control human skin. Coupled with the discovery that free 13H-9-E-LA enhances the release of sensory neuronal CGRP in a low pH environment, higher levels of this free acid in psoriatic skin suggest that it may potentially cause hypersensitivity that accompanies skin inflammation.

Identification of 9-keto-12, 13-epoxy-octadecenoic acid as a novel endogenous itch-causing agent (pruritogen)

Another finding of this example is the identification of 9K-12,13E-LA as an endogenous itch-causing agent that is elevated in inflamed human skin of psoriasis patients reporting chronic itch, but not elevated in lesions without the scratchy character. 9K-12,13E-LA has been previously detected in human plasma and reported to stimulate adrenal steroidogenesis, indicating its biological activity. Similar to 13H-9,10E-LA, in control human skin, the vast majority (> 99%) of 9K-12,13E-LA was observed to be found in the esterified lipid fraction. The significantly higher (> 30-fold) concentration of this mediator in the free fatty acid lipid pool of psoriatic skin lesions compared to control skin suggests that 9K-12,13E-LA may be a signaling molecule in skin inflammation. In agreement with this, it was observed that injection of free 9K-12,13E-LA into the mouse dermis caused scratching behavior associated with itching. It is believed that 9K-12,13E-LA is the only fourth lipid mediator reported to induce scratching behavior in the rodent model of pruritus. Unlike other known lipid itch-causing agents (leukotriene B4, thromboxane a2, hydroperoxy-eicosatetraenoic acid), which are believed to exist only in the free acid form, most of the 9K-12,13E-LA was pre-stored in esterified skin lipids. This accumulation in esterified lipids suggests that lipases can release preformed 9K-12,13E-LA to directly stimulate pruritus, thereby avoiding the need for de novo biosynthesis. In this regard, high expression of PLAG2A and PLAG2F that could exert relevant lipase functions was detected in rat skin and especially in inflamed human skin.

Diet-regulated hydroxy-epoxy-octadecenoic acid and chronic headache

It was previously demonstrated in rats that increased dietary LA as a control variable significantly increased the abundance of LA and its well-known oxidized LA derivatives (e.g., hydroxyoctadecanoic acid (HODEs), epoxy-octadecenoic acid, dihydroxy-octadecenoic acid) in tissues associated with idiopathic pain syndrome, including skin. Furthermore, dietary intervention with reduced LA may reduce headache in patients with severe chronic headache, and reduction of circulating LA is associated with reduced clinical pain, which means that LA or its auto-active derivatives may cause pain in humans. In this study, it was found that diet-induced reductions in circulating 11H-12,13E-LA were closely associated with clinical pain relief, increasing the likelihood that high LA intake may contribute to the biochemical susceptibility to developing chronic pain or itch, in part by increasing tissue levels of hydroxy-and keto-epoxy-octadecenoic acids. This report introduces new mediators into the area of developing lipid mediators of pain and itch. Most of the work in this field has focused on mediators derived from long chain (. gtoreq.20 carbon) polyunsaturated fatty acids, in particular mediators derived from Arachidonic Acid (AA). Since LA is much more abundant in skin and certain epithelial tissues than AA and other polyunsaturated fatty acids, and is also a substrate for enzymatic conversion to oxidation mediators, LA-derived mediators are uniquely located in regulating nociceptive and pruritic responses in these tissues. Hargreaves and colleagues (Patwardhan et al, The Journal of clinical regulation 120,1617,2010; Patwardhan et al, P.N.A.S.,106,18820,2009) previously revealed that 9-HODE, 13-HODE and other generally known LA-derivatives have nociceptive responses in both The peripheral and central nervous systems. The inflammatory response of the skin in vivo is characterized by a low pH and a simultaneous rise in many lipid and non-lipid mediators associated with inflammation-related hypersensitivity (Han and Simon, Science signaling 4, er3,2011; Sun and Chen, Jdental research,95,135,2016). Under these conditions, 9-HODE and 13-HODE can potentially be converted by cytochrome p450 epoxidase or lipoxygenase to 9H-12,13E-LA, 13H-9,10E-LA and other biologically active LA-derived mediators.

Materials and methods

Clinical sample preparation, rodent behavior testing, ex vivo CGRP release assay and all laboratory analyses were performed by researchers blinded to clinical data and treatment groups.

Data analysis

Data for normal distributions are expressed as mean ± standard error and compared using Student's t-test (two groups) or one-way analysis of variance (multiple groups) and corrected for multiple comparisons as described in the legend. The data for the non-normal distributions are expressed as median and interquartile range and compared using Wilcoxon rank-sum test (two groups) and Kruskal-Wallis test (groups) and corrected for the multiple comparisons described in the legend. P <0.05 was considered significant after several comparative adjustments.

Rat tissue Collection

Rat tissues analyzed in this study were obtained according to protocols approved by the institutional Animal Care and Use Committee of the national Institute of Dental and Craniofacial and clinical centers (NIH). Male Sprague-Dawley rats were housed in pairs and fed rodent NIH-31M modified formula (Ziegler) and water ad libitum. To obtain hind paw, sciatic nerve, DRG, TG and dorsal horn tissues, rats were anesthetized with isoflurane, decapitated and the tissues were immediately dissected. A section of the plantar surface of the hind paw was collected using a scalpel. The sciatic nerve was dissected starting from the distal sciatic notch and extending just above the sciatic trigeminal nerve. L4 and L5DRG were removed after laminectomy. The spinal cord was expelled from the spine by hydraulic pressure using a syringe and saline, and the left and right dorsal quadrants were separated. The tissue was immediately frozen on dry ice and stored at-80 ℃ until processed. Rat DRG and sciatic nerve RNA-sequencing data were obtained under entry PRJNA313202 in the SRA database.

Precursor fatty acids of rat pain circuit tissue

Tissue fatty acids were analyzed as previously described (11). Briefly, samples were thawed, weighed, and homogenized in Butylated Hydroxytoluene (BHT)/methanol for fatty acid extraction according to the method of Folch et al (Ramsden et al, molecular μ lar pain 12,2016). During operation, BHT was added to methanol to reduce lipid oxidation. An internal standard, methyl eicosatrioate (23:0), was added to each sample. After that, use 14% BF₃Methanol methylation. The hexane extract was concentrated to a small volume under nitrogen flow and then transferred to a microtube for GC analysis. A capillary chromatography column (DB-FFAP, 15 m.times.0.100 mm.i.d.. times.0.10 μm film thickness, J.) equipped with a flame ionization detector (Hewlett-Packard, Palo Alto, Calif.) and fused silica was used&W Scientific, Folsom, Calif.) was analyzed for fatty acid methyl esters by HP-7890A gas chromatography. The detector and injector temperature were set at 250 ℃. The oven temperature program started at 150 ℃ for 0.25 minutes, followed by a 10 ℃/min ramp to 200 ℃, followed by a 3.5 ℃/min ramp to 225 ℃ for 0.5 minutes, and finally a 40 ℃/min ramp to 245 ℃, and finally a 15 minute ramp. Hydrogen was used as a carrier gas and the linear velocity was 50 cm/s. Custom-mixed, 30-component, quantitative methyl ester standards (containing 10-24 carbons and 0-6 double bonds) were used to determine retention times and ensure accuracyQuantification (Nu Chek Prep462, Elysian, MN). Fatty acid data are expressed as% of the total peak area, which corresponds to wt% ± 5%, as verified by quantifying the standard mixture. Tissue fatty acid concentration was calculated using internal standard method.

Gene expression in human pain circuit tissue

Tissue collection and RNA purification for RNA sequencing analysis

4 human L3 DRG were purchased from Anabios (San Diego, Calif.) from four normal organ donors of different genders. As part of the collection procedure of NIMH Human brain collection Core, 3 samples of the dorsal horn of the medulla were collected at the level of the vertebral intersection and the grey of the dorsal horn was isolated from fresh tissue by dissection as described by Goswami et al (molecular μ lar pain 10,44, 2014). Rat and human samples were homogenized in Qiazol reagent (Qiagen Inc, Valencia CA) using either a Fastprep 24 homogenizer (MP Biomedicals, Santa Ana, CA) or a Polytron homogenizer (IKA, Wilmi ngton, NC) and purified by dnase digestion using the RNeasy Mini kit (Qiagen Inc, Valencia CA). RNA Integrity (RIN) was evaluated after gel electrophoresis using an Agilent bioanalyzer (Agilent Technologies, santa clara, CA). For rat tissues, samples with RIN greater than 8.5 were sequenced. For human DRG, samples with RIN higher than 7 were sequenced. For other human samples, the highest possible RIN was obtained. The lowest sample contained in this study was 5.5.

Alignment and quantification of RNA sequencing count data

Rat data were aligned by (version 2.4.2a) (Dobin et al. (biologics 29,15,2013) and rn6 Genome construction (Ensembl.) the use of QoRTs (version 0.3.18) (Hartley and M μ llikin, BMC biologics 16,224,2015) quantitated the bam files obtained from this analysis and converted to raw read counts and FPKM (fragments Per base of transcript Million mapped reads). selection of 8 high quality samples from the GTEx library (RIN-based) evaluated for human skin (calf) and tibial nerve data, the RPKM values were obtained directly from data files available from Cooperation (Consortium, Natgenetics 45,580,2013.) the evaluation of RPKM values was performed from the SRA database (PRA 236547) (Swindell et al., Genome 7, psoriasis 86, psoriasis-3, Zones and Zymology 2016 using Yeast sample Zymmetry 3 and gradient, Zymology 2016-3, genobiology 7Suppl 1, S121,2006) to align and quantify data from SRA with other human data. Genomic target files for MAGIC alignment may be obtained on request. Quantification and normalization of gene counts was performed by MAGIC and reported as sfpkms.

Full chemical synthesis of hydroxy-epoxy-and keto-epoxy-octadecenoic acids

Each compound was completed by a total chemical synthesis. The synthesized compounds were purified by flash chromatography and/or normal phase HPLC. NMR analysis showed that chemical shifts and coupling constants were consistent with each chemical structure. Hydroxy-epoxy-or keto-epoxy-octadecenoic acid in the free acid or methyl ester form as shown was analyzed by proton NMR in deuterated chloroform.

Identification and quantification of hydroxy-and keto-epoxy-octadecenoic acids by LC-MS/MS

Authentic standards prepared by total synthesis were used to identify and quantify these 8 endogenous compounds in human and rat tissues using UPLC-MS/MS. Briefly, the Solid Phase Extraction (SPE) of the alicyclines from the biomatrix was performed using the Strata X cassette (33u,200mg/6mL, Phenomenex, Pa.). The cartridge was conditioned with 6mL of methanol, followed by the addition of 6mL of water, prior to sample extraction. The sample was washed with 6mL of 10% methanol. Alicyclic was eluted with 6mL of methanol into a glass tube containing 10 μ L of 30% glycerol in methanol. The eluate was evaporated to dryness under a stream of nitrogen and reconstituted with 40. mu.L of methanol, and an aliquot (10. mu.L) was injected into the LC/MS/MS system. Qualitative and quantitative analysis were performed using UPLC (Shimadzu Scientific Instruments, Columbia, MD) coupled to Qtrap 5500(AB SCIEX, USA). Briefly, separations were performed on a Zorbax RRHD Eclipse + C18 column (100mM x4 mM; 1.8 μm) (Agilent Corporation, Palo Alto, Calif.) consisting of (A)12mM ammonium acetate solution and acetic acid (100:0.02v/v) and (B)12mM ammonium acetate and acetonitrile/water/acetic acid (90:10:0.02, v/v/v). The flow rate was 0.5 mL/min. The column box temperature was set to 30 ℃. Elution gradient conditions were as follows: 25-40% B at 0-2.0min, 40-46% B at 2-8min, 46-57% B at 8-9min, 57-66% B at 9-20min, 66-76% B at 20-22min, 76-100% B at 22-27min, 100% B at 27-33min, and 100-25% B at 33.1-35 min. The mass spectrometer was run in electrospray negative ionization using a predetermined multiple reaction monitoring (sMRM), with MRM data for each analyte taken over a retention time window of 90 s. The source parameters are set as follows: ion spray voltage, -4500V; atomizing gas (GS1), 65 psi; turbine gas (GS2), 70 psi; and turbine ion spray source Temperature (TEM), 500 ℃. Analytes were quantified using MRM. For hydroxy-epoxy-octadecenoic acid and keto-epoxy-octadecenoic acid with 2 or 3 isomeric peaks in the synthetic standards, quantification was performed by: the total peak area ratio of its associated peak area integral/peak area IS generated by Analyst 1.6.2 and the best fit of the analyte total peak area ratio/peak area IS versus concentration IS plotted in Microsoft Excel and fitted to the equation y ax + b. MS/MS spectra were obtained using the enhanced product ion scan mode at a scan rate of 1000 Da/s. Collision-induced dissociation (CID) was performed using a collision energy of 35V and a collision energy spread of 10. Data processing was performed using analytical software (version 1.6.2, AB Sciex). Identification of 7 of the 8 expected endogenous compounds was confirmed by matching the MS/MS spectra and retention times of the endogenous LA derivatives from psoriatic skin samples to synthetic materials using a full ion pattern.

Human study Using sample Collection

Skin biopsy and serum collection of psoriasis and control participants

The study included eight consecutive psoriasis participants and 7 non-psoriasis controls (age range 26-82 years) enrolled as an ongoing NIH observational study of psoriasis and cardiac metabolism (NCT 01778569). The study procedures were approved by the National institute for cardiopulmonary blood (National Heart and Lung institute Institutional Review Board). All participants submitted written informed consent prior to enrollment. Briefly, dermatologists used the Psoriasis Area Severity Index (PASI) to confirm and quantify the diagnosis of Psoriasis. The presence of substantial itching was recorded using a self-reported questionnaire. Corresponding controls were continuously recruited to perform the same tests as psoriasis participants. All participants did not receive any systemic anti-psoriasis treatment or topical treatment within 2 weeks prior to biopsy. At baseline, a 4mm punch biopsy was obtained under local anesthesia from psoriatic plaques and unaffected skin. Biopsy sites were selected according to active plaque and varied between subjects. However, biopsies of unaffected and control skin were mainly from the buttocks. Whole blood from the same participant was collected in serum separation tubes, centrifuged and immediately stored at-20 ℃ until analysis.

Chronic Daily Headache (CDH) test

The CDH trial was a 12-week randomized trial designed to test the clinical and biochemical impact of a low linoleic diet (L6 intervention) with or without a concomitant increase in n-3 fatty acids (H3-L6 intervention) in people with CDH. The test was performed at church mountain school of University of North Carolina (University of North Carolina at Chapel Hill (UNC)) from 4 months to 11 months of 2011 in 2009. The experimental procedures have been approved by The UNC Institutional Review Board and The protocol, dietary composition and major clinical findings and some biochemical findings have been described previously (Ramsden et al, Trials 12,97, 2011; macintosh al, The British journel of nutrition 110,559,28, 2013). Briefly, adults meeting the CDH headache criteria with headache >4 hours/day and >15 days/month and lasting at least 3 months and a headache history >2 years were enrolled. During the 4-week pre-intervention period, participants continued with daily care and diet, and headache characteristics were recorded in a daily headache diary. After completion of the break-in phase, participants were randomly assigned to one of the two study diets for 12 weeks. LA in the study diet was reduced by limiting the consumption of vegetable oils and other LA-rich sources and replacing them with vegetable oils and foods rich in monounsaturated and saturated fats. Plasma was collected at baseline and at the end of the 12 week diet phase. It has been previously reported that H3-L6 intervention can significantly reduce headache frequency and severity and improve quality of life and function while reducing the use of acute analgesic drugs (Ramsen et al, Pain 154,2441,2013; Ramsen et al, Pain 156,587,2015). Diet-induced changes in one or more families of n-6 or n-3-derived lipid auto-actives may contribute to these clinical benefits; however, the specific mechanisms responsible for these effects are not clear. In this study, pre-and post-intervention plasma samples were used for: (1) the Wilcoxon paired-signs rank test (Wilcoxon matched-pairs signed-rank) was used to investigate whether diet altered plasma levels of hydroxy-and keto-epoxy derivatives of LA; and (2) using a regression model adjusted for each result and baseline value of mediator, to investigate whether a change in mediator concentration correlates with clinical pain relief.

Preparation of solid tissues for LC-MS/MS analysis

Solid tissues (human skin, rat hind paw, rat dorsal horn) were transferred to FastPrep lysis matrix tubes on ice (MP Biomedicals, USA; lysis matrix a for skin and hind paw, lysis matrix D for dorsal horn) and at least 8-fold more volume of ice-cold methanol containing 0.02% BHT and 0.02% EDTA was immediately added to each tube (v/v). A known amount of internal standard was added to each sample and the samples were homogenized using a FastPrep-24 homogenizer (MP Bio). The homogenate was transferred to-80 ℃ and held for 1 hour to precipitate the protein. The homogenate was centrifuged at 17000g for 10 min at 4 ℃ and the supernatant was subsequently transferred to a new tube. Half of the supernatant was stored at-80 ℃ until SPE purification and LC-MS/MS analysis. To analyze the total lipid pool, the other half of the supernatant was saponified with 2.6 wt% sodium carbonate at 60 ℃ for 30 minutes with gentle shaking. The solution was then neutralized with acetic acid (pH 5-7) and stored at-80 ℃ overnight. Lipid extract (total amount of free and saponified) was added to 9 times more volume of ice cold water, followed immediately by purification by SPE and LC-MS analysis.

Preparation of plasma and serum for LC-MS-MS analysis

200 μ L of plasma or serum was transferred to 500 μ L of ice-cold methanol containing 0.02% BHT and 0.02% EDTA and transferred to-80 ℃ to precipitate proteins (as described above). A known amount of internal standard was then added, the sample centrifuged and the supernatant collected as described above. The supernatant was then added to 9 times more volume of ice cold water as described above and purified with SPE and analyzed by LC-MS/MS.

Ex vivo sensory neuron sensitization assay (CGRP Release assay)

For the release experiments, this work was approved by the Animal Care and Use Committee (Animal Care and Use Committee at Indiana University School of medicine, Indianapolis, IN) of the Indianapolis University. Adult rat sensory neuron cultures were prepared as described previously (Burkey, Hingtgen, and Vasko, Methods in molecular μ lar media 99,189,2004; Kelley et al, PloS one 9, e106485,2014). Cells were plated in F-12 medium (Invitrogen, Carlsbad, Calif.) at 3% CO₂10-12 days at 37 ℃ and the medium is supplemented with 10% horse serum, 2mm glutamine, 100. mu.g/ml norocin^TM50 μ g/ml penicillin, 50 μ g/ml streptomycin, 50 μm 5-fluoro-2' -deoxyuridine (Invitrogen), 150 μm uridine, and 30ng/ml NGF (Harlan Bioproducts for Science, Inc. Indianapolis, IN). On the day of the release experiment, the cultures were treated with HEPES buffer (25mm HEPES, 135mm NaCl, 3.5mm KCl, 2.5mm CaCl)₂、1mm MgCl₂3.3mm D-glucose and 0.1% bovine serum albumin, pH 7.4, and maintained at 37 ℃). The culture was then incubated with 0.4ml of the same buffer in the presence or absence of the drug. Basal release was determined by: cells were exposed to HEPES buffer alone for 10 minutes followed by buffer in the presence of mediator for 10 minutes to determine if the compound stimulated release. The culture is then exposed to a buffer containing 30nM capsaicin or a buffer adjusted to pH 6.0 in the presence or absence of a mediator. The cells were then re-exposed to HEPES buffer without drug for 10 min of incubation to re-establish the basal release. After each incubation, the buffer was removed to measure the amount of CGRP using radioimmunoassay as described previously (Chen et al, Peptides 17,31, 1996). At the end of each release experiment, cells were lysed hypotonically by exposing the culture to 0.1M HCl for 10 minutes, and aliquots were taken to measure total CGRP content in the culture using radioimmunoassay. The total content of CGRP was not significantly altered by exposure to inflammatory mediatorsAnd (6) changing. Release data are expressed as fmol/well of cells/10 min, from three independent experiments from different harvest batches. Statistical analysis was performed using ANOVA and Tukey's post hoc test.

Rodent behavior assay

Feeling of itch (pruritus) behavior

Hydroxy-and keto-epoxide derivatives of LA (100 μ g) or histamine (50 μ g) were injected intradermally into the dorsum of the neck of female mice (C57BL/6J, from Jackson Laboratory). LA derivatives (9-keto-12, 13-epoxy- (10E) -octadecenoic acid or 13-keto-9, 10-epoxy- (11E) -octadecenoic acid) were injected independently and [ 9-keto-12, 13-epoxy- (10E) -octadecenoic acid + 13-keto-9, 10-epoxy- (11E) -octadecenoic acid (100. mu.g each) ] was injected in combination. The perception of itching was quantified as the number of scratching episodes assessed over 30 minutes, as described previously (Mishra and Hoon, Science 340,968,2013).

Pain perception (pain) behavior

11-hydroxy-12, 13-trans-epoxy- (9Z) -octadecenoic acid (30 μ g) was injected intradermally into the hind paw of male Sprague-Dawley rats. Baseline measurements were taken for all tests prior to injection. A-delta and C-fiber mediated hindpaw withdrawal responses were measured as previously described (Mitchellel et al, Pain 155,733,2014). In short, a rapid retraction response is produced by stimulating the plantar surface of the paw with a 100ms laser pulse. Laser pulses were delivered by an infrared diode laser (LASS-10M; lased, Mountain View, Calif., USA) and calibrated to 3500mA at 0.5mm diameter and delivered from a distance of 1cm, and C fiber-mediated responses were measured by delivering a slow temperature rise to the plantar surface of the hind paw, with stimulation terminated when the paw was actively retracted. The laser stimulation was adjusted to result in a retraction latency of about 10 seconds (1000mA, 13cm distance).