Stabilization of compounds as cyclodextrin complexes

Abstract

To provide a stable formulation containing nitroalkene fatty acid (NO2-FA). [Solution] A composition comprising a complex of cyclodextrin and nitroalkene is provided. Furthermore, a liquid composition comprising a solid powder suspended or dissolved in (a) water and (b) water, comprising a complex of cyclodextrin and nitroalkene is provided. Another embodiment is a liquid composition comprising a solid powder suspended or dissolved in (a) water and (b) water, comprising a complex of cyclodextrin and an active compound, wherein the active compound is, for example, a nitroalkene having the structure of the following formula. TIFF2026143538000031.tif3461

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 992,036, filed on 19 March 2020 (which is incorporated herein by reference).

[0002] Statement of government support This invention was made with government support under grant numbers GM125944 and DK112854, awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Nitroalkene fatty acids (NO2-FAs) have been shown to play a protective role in many experimental settings, including endotoxin-induced vasculitis, endotoxemia and multi-organ injury, inflammatory bowel disease (IBD), allergic respiratory diseases, tumor cell proliferation, invasion and metastasis, renal ischemia and reperfusion (I / R) injury, as well as diabetic and other forms of chronic kidney disease, pulmonary arterial hypertension (PAH), myocardial I / R injury, hypertension, and atherosclerosis.

[0004] 10-Nitro-octadeca-9-enoic acid (NO2-OA) requires storage at -80°C and is unstable at temperatures above -20°C, in the presence of water, upon exposure to moisture in the atmosphere, and / or in the presence of bases, nucleophiles, nucleophilic amino acids, amines, and proteins. This instability is a result of reversible reactions with nucleophiles via Michael addition reactions and base-catalyzed reactions. The reversible nature of these reactions leads to the decomposition of nitroalkene derivatives via isomerization of the nitroalkene CC double bond, double bond transfer, dimerization between two NO2-OA molecules, and oxidation.

[0005] Conventionally, NO2-OA has been stabilized using oil as a method to reduce the influence of conditions known to cause its decomposition (e.g., water content, bases, nucleophiles, temperature). Conventional approaches used to date require refrigerated storage, have limited shelf life, and are all viscous liquid formulations that must react with components of the hard capsule surface and prevent leakage, which overall increases the costs of their manufacture, storage, distribution and overall clinical development. Oils used for nitroalkene fatty acid solvation have included olive oil, sesame oil, and partially purified or synthetic oil preparations (synthetic triacylglycerols). [[Summary of the Invention]] [[Means for Solving the Problem]]

[0006] Abstract One embodiment disclosed herein is a composition comprising a complex of cyclodextrin and a nitroalkene.

[0007] Another embodiment disclosed herein is a composition comprising a complex of cyclodextrin and an active compound, wherein the active compound is as follows: Structure of Formula I: [[Chemical Formula]] which is a nitroalkene, wherein R 1 is hydrogen, C1-C 24 alkyl, C1-C 24 alkenyl, or C1-C 24 alkynyl; R 2 , R 3 , R 7 , and R 8 are each independently hydrogen, oxygen, C1-C 24 alkyl, NO2, OH, or OOH; R 4 is a terminal COOR 6 group, wherein R 6 is hydrogen, or C1-C 24It is alkyl; R 5 is hydrogen, C1-C 24 Alkyl or R 4 and R 5 The combined = C(R 9 )(R 10 ) forms, and here R 9 C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 Contains alkinyl, or R here 9 This is the end COOR 6 It is a base, R 10 is hydrogen, NO2, OH, or OOH; n is 1 to 24; and Here, nitroalkene fatty acids contain at least one NO2 group. Nitroalkenes; Structure of Equation II: [ka] It is a nitroalken, and here R 1 is hydrogen, C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 It is alkinyl; R 2 , R 4 , R 5 and R 6 Each of them is hydrogen; R 7 This is the end COOR 9 It is a base, and here R 9 is hydrogen or C1-C 24 It is alkyl; R 3 and R 8 These are, independently, hydrogen, oxygen, and C1-C 24 The elements are alkyl, NO2, OH, ONO2, NO, ONO, or OOH, however R 3 Or R 8 At least one of them is NO2, and R 3 Or R8 The other of these is hydrogen, ONO, or ONO2. Nitroalkenes; Structure of Equation III: [ka] A nitro group-containing compound, where R 1 is hydrogen, C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 It is alkinyl; R 2 and R 5 Each of them is hydrogen; R 7 This is the end COOR 6 It is a base, and here R 6 is hydrogen or C1-C 24 It is alkyl; R 3 and R 4 These are, independently, hydrogen, oxygen, and C1-C 24 The elements are alkyl, NO2, OH, ONO2, NO, ONO, or OOH, however R 3 Or R 4 At least one of them is NO2, and R 3 Or R 4 The other of these is hydrogen, ONO, or ONO2. Nitro group-containing compounds; Structure of Equation IV: [ka] A compound containing a dicarboxylic acid, where X is an electron-withdrawing group selected from acyl, carboxylic acid, ester, halogen, fluoromethyl, -CN, sulfonyl, sulfone, sulfonic acid, primary ammonium, secondary ammonium, tertiary ammonium, or -NO2. m is between 1 and 10; n is between 1 and 10. compound; Structure of equation V: [ka] A compound containing a dicarboxylic acid, where X is an electron-withdrawing group selected from acyl, carboxylic acid, ester, halogen, fluoromethyl, -CN, sulfonyl, sulfone, sulfonic acid, primary ammonium, secondary ammonium, tertiary ammonium, or -NO2; Y and Z are each independently hydrogen or C1-C 10 It is alkyl; m is between 1 and 10; n is between 1 and 10. compound; or Structure of Equation VI: [ka] A compound containing a dicarboxylic acid, where X is an electron-withdrawing group selected from acyl, carboxylic acid, ester, halogen, fluoromethyl, -CN, sulfonyl, sulfone, sulfonic acid, primary ammonium, secondary ammonium, tertiary ammonium, or -NO2; Y and Z are each independently hydrogen or C1-C 10 It is alkyl; p and t are each independently between 1 and 10; s is either nonexistent or between 1 and 10. r is 1. It is a compound.

[0008] Further disclosed herein are liquid compositions comprising (a) water and (b) a solid powder suspended or dissolved in water, comprising a complex of cyclodextrin and nitroalkene.

[0009] Another embodiment disclosed herein is a liquid composition comprising (a) water and (b) a solid powder suspended or dissolved in water, comprising a complex of cyclodextrin and an active compound, wherein the active compound has the structure of formulas I to VI.

[0010] Another embodiment disclosed herein is a pharmaceutical composition comprising the above-mentioned complex composition and at least one pharmaceutically acceptable excipient.

[0011] Another embodiment is a complex of nitroalkene fatty acid and cyclodextrin.

[0012] Another embodiment disclosed herein is a method comprising the step of contacting a nitroalkene with a cyclodextrin under conditions that result in the formation of a complex between the nitroalkene and the cyclodextrin.

[0013] Another embodiment disclosed herein is a method comprising the step of contacting a cyclodextrin with an active compound under conditions that result in the formation of a complex of the cyclodextrin and the active compound, wherein the active compound has the structure of formulas I to VI.

[0014] Another embodiment disclosed herein is a method comprising the step of mixing together (a) a liquid carrier and (b) a solid powder containing a nitroalkene and cyclodextrin complex.

[0015] Another embodiment disclosed herein is a method for treating a condition in a subject, the method comprising the step of administering any composition disclosed herein to a subject in need thereof, wherein the condition is an inflammatory condition, an immune disorder, psoriasis, obesity, metabolic syndrome, acute kidney disease, chronic kidney disease, focal segmental glomerulosclerosis, atheroma, lipogenesis, neointimal hyperplasia, renal I / R and xenobiotic injury, focal myocardial I / R injury, Ang II-induced systemic hypertension. These include hypertension, pulmonary hypertension, cancer, cardiac and pulmonary fibrosis, hepatic fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), breast cancer, ovarian cancer, inflammatory bowel disease, nociception, stroke, motor neuron degeneration, diabetes mellitus, aneurysm, aortic sclerosis, systemic lupus erythematosus, infant-onset STING-associated vasculitis (SAVI), asthma, chronic obstructive pulmonary disease (COPD), or focal segmental glomerulosclerosis.

[0016] The above will become clearer from the following detailed explanation, which will proceed with reference to the attached drawings. In embodiments of the present invention, for example, the following items are provided. (Item 1) A composition comprising a complex of cyclodextrin and nitroalkene. (Item 2) A composition comprising a complex of cyclodextrin and an active compound, wherein the active compound is as follows: Structure of formula I: [ka] It is a nitroalken, and here R 1 is hydrogen, C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 It is alkinyl; R 2 , R 3 , R7 and R 8 are each independently hydrogen, oxygen, C1-C 24 alkyl, NO2, OH, or OOH; R 4 is a terminal COOR 6 group, wherein R 6 is hydrogen or C1-C 24 alkyl; R 5 is hydrogen or C1-C 24 alkyl, or R 4 and R 5 together form =C(R 9 )(R 10 ), wherein R 9 is C1-C 24 alkyl, C1-C 24 alkenyl, or C1-C 24 alkynyl, or R 9 is a terminal COOR 6 group, and R 10 is hydrogen, NO2, OH, or OOH; n is 1 to 24; and wherein the nitroalkene fatty acid is a nitroalkene containing at least one NO2 group; nitroalkene; Structure of Formula II:

Chemical Formula

[0017] [Figure 1] Figure 1 is a graph comparing the yields of α-cyclodextrin and β-cyclodextrin inclusion complexes of the fatty acid nitroalkene 10-nitro-octadeca-9-enoic acid (NO2-OA). Area refers to the area established by the UV signal tracked at 210 nm during the HPLC trial. The area under the UV-HPLC trace corresponding to the fatty acid is quantified and represents the amount of fatty acid present in the sample. 10 μl aliquots were injected into HPLC-UV, and the signal was tracked using a diode array spectrophotometer between 190 and 700 nm. The ratio (lipid / cyclodextrin) refers to the molar concentration ratio of fatty acid to cyclodextrin used during the preparation of the above inclusion complexes.

[0018] [Figure 2]Figure 2 is a graph showing the recovery percentage obtained from the process of preparing the above inclusion complex. All three proven ratios resulted in efficient incorporation and stabilization of NO2-OA in the inclusion complex. For the recovery calculation, the inclusion complex was extracted using methanol and injected for evaluation by HPLC-UV (quantification, purity, and integrity) and evaluation by HPLC-MS / MS (confirmation of integrity).

[0019] [Figure 3] Figure 3 is a table showing the stability evaluation scheme for the NO2-OA / cyclodextrin complex powder disclosed herein. Exposure to various temperatures in the presence of air and under the same humidity conditions was tested.

[0020] [Figure 4] Figure 4 is a graph showing the recovery of NO2-OA from β-cyclodextrin inclusion complexes after exposure to various conditions defined in Figure 3. No significant changes were observed in the stability of the NO2-OA / β-cyclodextrin inclusion complexes during the 28-day period. Only the stability of samples subjected to 70°C was tested for up to 14 days. Incubation of pure NO2-OA or oil-stabilized NO2-OA under these conditions resulted in significant degradation, along with dimer formation, double bond isomerization, double bond migration, and oxidation products. These degradation products were not observed in the β-cyclodextrin-stabilized samples.

[0021] [Figure 5A] Figure 5A shows the structure of the first (E)10-NO2-OA isomer present in the test material used to evaluate stability (>99%, structure enclosed in a box). It also shows the structures of previously identified and characterized oxidation, isomerization, and dimerization products of NO2-OA decomposition (in triglyceride-based oil formulations), and the categories to which they belong.

[0022] [Figure 5B]Figure 5B is a chromatogram of the NO2-OA / β-cyclodextrin complex disclosed herein, evaluated 14 days after exposure to 55°C in air. The major peak observed in the chromatogram corresponds to pure 10-NO2-OA, with no apparent formation of oxidation or isomerization products. No dimerization products were observed, and the peak observed in the RT area at 9 min was present with similar intensity in the blank injection.

[0023] [Figure 5C] Figure 5C shows superimposed chromatograms of the NO2-OA / β-cyclodextrin complex disclosed herein and a standard material mixture containing the 10-NO2-8,9-alkene, (E)10-NO2-OA and (Z)10-NO2-OA, evaluated after 14 days of exposure to 55°C in air. The superimposed chromatograms show that no degradation products were present in the samples tested after 14 days of exposure to 55°C.

[0024] [Figure 6A] Figure 6A shows a graph demonstrating the reproducibility of the above process and the complete incorporation of 10-NO2-OA into the NO2-OA / β-cyclodextrin inclusion complex. Independent batches were evaluated and quantified in triplicates.

[0025] [Figure 6B] Figure 6B is a graph showing an external calibration curve used to quantify the level of NO2-OA content in β-cyclodextrin inclusion complexes. The quantification was performed by HPLC-UV using an external calibration curve with various concentrations of pure 10-NO2-OA, which were then injected into the HPLC-UV and the area under the curve was quantified.

[0026] [Figure 7]Figures 7A–7C show graphs illustrating the stability of the NO2-OA / β-cyclodextrin complexes disclosed herein when dissolved in water. NO2-OA that is associated but not present in the NO2-OA / β-cyclodextrin inclusion complex rapidly equilibrates after dissolution in water and disintegrates within the first hour (Figure 7A). Subsequently, the concentration and integrity of the inclusion complex remain stable for the remainder of the tested time. The concentration on day 10 was reassessed as an indicator of stability (Figure 7B). In contrast, the addition of the same molar amount of 10-NO2-OA to water resulted in a rapid loss of 10-NO2-OA in solution (within 4 hours) (Figure 7C).

[0027] [Figure 8] Figures 8A-8C show that 10-NO2-OA / β-cyclodextrin inclusion complexes can be used to administer NO2-OA (an oily fatty acid) in drinking water. Two concentrations were tested in mice (0.31 mg / ml and 1.95 mg / ml). Dissolution of the 10-NO2-OA / β-cyclodextrin inclusion complexes did not cause any changes in drinking habits or significant taste aversion (tracked as changes in daily water intake). This is because the β-cyclodextrin masked the flavor of 10-NO2-OA. Ingestion of an aqueous solution of 10-NO2-OA / β-cyclodextrin inclusion complex produced bioavailable 10-NO2-OA, as evidenced by the detection of 10-NO2-OA and its metabolites in urine (not shown) and feces (chromatograms of major metabolites in feces are shown in Figure 8A, and specific β-oxidation products of NO2-OA and NO2-SA are shown in Figures 8B and 8C, respectively). Figure 8B shows the formation of β-oxidation products of 10-NO2-OA (10-NO2-SA) (gray trace), dino-NO2-SA (green trace), tetranol-NO2-SA (red trace), and hexanol-NO2-SA (blue trace). Figure 8C shows the formation of β-oxidation products of reduced 10-NO2-OA (gray trace), dinol-NO2-OA (green trace), tetranol-NO2-OA (red trace), and hexanol-NO2-OA (blue trace).

[0028] [Figure 9] Figure 9 is a graph showing the absorption and metabolism of NO2-OA when drinking water fortified with a β-cyclodextrin-stabilized inclusion complex, as indicated by its detection in plasma. These graphs also show that the absorption process proceeds via the same pathway previously determined for 10-NO2-OA, including incorporation and biodistribution via plasma triglycerides. This figure shows that significant amounts of 10-NO2-OA and its major metabolite, 10-NO2-SA, are incorporated into triglycerides, as evidenced by the increase observed during hydrolysis. Free acid components were quantified using HPLC-MSMS with a deuterated internal standard for species identification and quantification purposes.

[0029] [Figure 10]Figure 10 shows the plasma metabolite profiles of mice administered 10-NO2-OA / β-cyclodextrin inclusion complexes in drinking water for one day. Two concentrations, 0.31 mg / ml and 1.95 mg / ml, were tested, and the 10-NO2-OA metabolic profile under the 0.31 mg / ml condition is shown. Dissolution of the 10-NO2-OA / β-cyclodextrin inclusion complexes did not cause any changes in drinking habits or significant taste aversion (tracked as changes in daily water intake). Drinking the aqueous solution of 10-NO2-OA / β-cyclodextrin inclusion complexes produced bioavailable 10-NO2-OA, as evidenced by the detection of 10-NO2-OA metabolites in plasma. The upper panel shows the formation of β-oxidation products of reduced 10-NO2-OA (10-NO2-SA) (light blue trace, 18:0), dino-NO2-SA (gray trace, 16:0), tetranol-NO2-SA (green trace, 14:0), and hexanol-NO2-SA (red trace, 12:0), as well as the formation of β-oxidation products of 10-NO2-OA (blue trace, 18:1), tetranol-NO2-OA (green trace, 14:1), and hexanol-NO2-OA (green trace, 12:1) (lower panel). The representative chromatogram on the left shows the profile of free NO2-OA and its metabolites in plasma, while the panel on the right shows the chromatogram after hydrolysis of triglycerides using an acid-based hydrolysis method.

[0030] [Figure 11]Figure 11 shows the NO2-OA profile of mouse feces and an analysis of its major reported metabolites. NO2-OA was stabilized as an inclusion complex with β-cyclodextrin as disclosed herein and delivered to mice in drinking water to obtain daily doses of 10 mg / kg and 50 mg / kg. In this case, the 50 mg / kg dose is shown. The fecal metabolite profile shows nitrooleic acid uptake and extensive metabolism. Large amounts of NO2-OA have been reported to be excreted through the feces as NO2-OA and as partially metabolized substances. This further indicates that the stabilized inclusion complex, when solvated and administered, can reach the central circulation and exhibit the expected metabolic profile in both urine and feces. The upper panel shows β-oxidation of the reduced metabolite, while the lower panel shows β-oxidation of the parent compound. [Modes for carrying out the invention]

[0031] Detailed explanation Terminology The following explanations of terms and methods are provided to better describe the compounds, compositions and methods of the present invention and to guide those skilled in the art in carrying out the disclosure. It should also be understood that the terminology used in this disclosure is for the purpose of describing specific embodiments and examples and is not intended to be limiting.

[0032] As used herein, "administration" includes administration to a subject by another individual or self-administration by the subject.

[0033] An "alkenyl" is a cyclic, branched, or linear group containing only carbon and hydrogen atoms, and containing one or more double bonds, which may or may not be conjugated. Alkenyl groups may or may not be substituted. Lower alkenyl groups contain 1 to 6 carbon atoms.

[0034] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc.). An alkyl group may be a "substituted alkyl" in which one or more hydrogen atoms are substituted with substituents (e.g., halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl). For example, lower alkyl or (C1-C6) alkyl may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3-C6) cycloalkyl may be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6) cycloalkyl (C1-C6) alkyl may be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (C1-C6) alkoxy may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, 3-Pentoxy or hexyloxy; (C2-C6) alkenyl may be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-C6 Alkinyl can be ethinyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (C1-C6) alkanoyl can be acetyl, propanoyl, or butanoyl;Halo(C₁-C₆)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(C₁-C₆)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C₁-C₆)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.;

[0035] The term "alkynyl" refers to a cyclic, branched or straight chain group containing only carbon and hydrogen, and containing one or more triple bonds. Alkynyl groups may be unsubstituted or substituted.

[0036] The term "amine or amino" refers to a -NRpRq group, wherein the Rp group and Rq group each independently refer to hydrogen, a (C1-C8)alkyl, (C1-C8)haloalkyl, or (C1-C6)hydroxyalkyl group.

[0037] The term "animal" refers to living multicellular vertebrate organisms (e.g., a category including mammals and birds). The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and non-human subjects, and includes birds and non-human mammals, such as non-human primates, companion animals (e.g., dogs and cats), livestock (e.g., pigs, sheep, cattle), and non-domesticated animals (e.g., big cats). The term subject applies regardless of the stage in the life cycle of the organism. Accordingly, the term subject applies to organisms in utero or in ovo, depending on the organism (that is, whether the organism is a mammal or a bird, such as domesticated poultry or wild birds).

[0038] As used herein, "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, for example phenyl or naphthyl. Unless otherwise indicated, an aryl group is unsubstituted or may be substituted with one or more, particularly 1 to 4 groups independently selected from, for example, halo, alkyl, alkenyl, OCF₃, NO₂, CN, OH, alkoxy, amino, CO₂H, CO₂ alkyl, aryl, and heteroaryl. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, and 2,4-methoxychlorophenyl.

[0039] The term "haloalkyl" refers to a C1-C8 alkyl group in which one or more hydrogen atoms in the C1-C8 alkyl group are replaced with halogen atoms, which may be the same or different. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, pentachloroethyl, and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

[0040] The terms "halogen" and "halo" refer to -F, -Cl, -Br, or -I.

[0041] The term "heteroatom" refers to a compound containing oxygen (O), nitrogen (N), and sulfur (S).

[0042] The term "heteroaryl" is used herein to refer to a monocyclic or bicyclic ring system containing one or two aromatic rings, each containing at least one nitrogen, oxygen, or sulfur atom. Unless otherwise indicated, heteroaryl groups are unsubstituted or may be substituted with one or more, preferably 1 to 4, substituents selected from, for example, halo, alkyl, alkenyl, OCF3, NO2, CN, NC, OH, alkoxy, amino, CO2H, CO2 alkyl, aryl, and heteroaryl groups. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0043] The term "heterocyclic" refers to monocyclic, bicyclic, tricyclic, or polycyclic systems that are either unsaturated or aromatic and contain 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms are oxidized as necessary, and the nitrogen heteroatom is quaternized as necessary (including bicyclic and tricyclic systems). The heterocyclic group may be bonded via any heteroatom or carbon atom. Examples of heterocyclic groups include heteroaryl groups as defined above. Representative examples of heterocyclic groups include, but are not limited to, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl, benzotriazolyl, benzoxazolyl, benzoisoxazolyl, prinyl, indolyl, isoquinolinyl, quinolinyl, and quinazolinyl. The heterocyclic group may be unsubstituted or may be substituted as necessary with one or more substituents.

[0044] "Heterocycloalkyl" refers to a monocyclic or bicyclic ring system containing one or two saturated or unsaturated rings and at least one nitrogen, oxygen, or sulfur atom within those rings. The term "cycloalkyl" refers to a monocyclic or bicyclic ring system containing one or two saturated or unsaturated rings.

[0045] The term "hydroxyalkyl" refers to an alkyl group having the indicated number of carbon atoms, wherein one or more of the hydrogen atoms of the alkyl group are substituted with an -OH group. Examples of hydroxyalkyl groups include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2CH2OH, and their branched versions.

[0046] The term "oxo" refers to an =O atom bonded to a saturated or unsaturated (C3-C8) cyclic or (C1-C8) acyclic moiety. The above =O atom may be bonded to carbon, sulfur, and nitrogen atoms that are part of the above cyclic or acyclic moiety.

[0047] The term "subject" includes both human and non-human subjects, such as birds and non-human mammals, e.g., non-human primates, companion animals (e.g., dogs and cats), livestock (e.g., pigs, sheep, and cattle), and undomesticated animals (e.g., large cats). The term "subject" applies regardless of the stage in the organism's life cycle. Therefore, depending on the organism (i.e., whether the organism is a mammal or a bird (e.g., domesticated poultry or wild birds)), the term "subject" applies to organisms in the womb or egg.

[0048] A "therapeutic dose" refers to the amount of a specific drug that is sufficient to achieve the desired effect in a subject being treated with that drug. Ideally, a therapeutic dose of a drug is sufficient to inhibit or treat a disease or condition without causing substantial cytotoxic effects in the subject. The therapeutic dose of a drug depends on the subject being treated, the severity of the affliction, and the mode of administration of the therapeutic composition.

[0049] "Treatment" means a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has started to develop. As used herein, with reference to a disease or pathological condition, the term "ameliorating" means any observable beneficial effect of the treatment. Such beneficial effects may be demonstrated, for example, by delaying the onset of clinical symptoms of the disease in a susceptible subject, by reducing the severity of some or all clinical symptoms of the disease, by slower progression of the disease, by an improvement in the subject's overall health or well-being, or by other parameters well known in the art that are specific to a particular disease. The phrase "treating a disease" means inhibiting the complete development of a disease, for example, in a subject at risk of the disease. "Preventing" a disease or condition means prophylactically administering a composition to a subject who shows no signs of the disease or only early signs, for the purpose of reducing the risk of developing the condition or condition, or reducing the severity of the condition or condition. In certain embodiments, treating a disease means inhibiting the metastasis of the disease.

[0050] A “pharmaceutical composition” is a composition comprising one or more fixed amounts (e.g., a unit dose) of the disclosed compounds together with one or more non-toxic, pharmaceutically acceptable excipients (such as carriers, diluents, and / or adjuvants), and optionally other biologically active components. Such a pharmaceutical composition is Remington's Pharmaceutical Sciences It can be prepared by standard pharmaceutical formulation techniques, such as those disclosed in Mack Publishing Co., Easton, PA (19th edition).

[0051] The compounds of the present invention may exist in various isomeric forms (including stereoisomers, geometric isomers, and conformational isomers), as well as in various tautomeric forms (particularly those with different hydrogen atom bonding sites). The term "isomer" is intended to encompass all isomeric forms of the compounds of the present invention (including the tautomeric forms of the above compounds).

[0052] Certain compounds disclosed herein may have chiral centers and therefore may exist in different enantiomer and diastereomer forms. The compounds of the present invention may exist in the form of optical isomers or diastereomers. Thus, the present invention includes compounds in the form of their optical isomers, diastereoisomers, and mixtures thereof (including racemic mixtures). Optical isomers of the compounds of the present invention may be obtained by known techniques such as asymmetric synthesis, chiral chromatography, or by chemical separation of stereoisomers using an optically active resolving agent. Unless otherwise indicated, “stereoisomer” means one stereoisomer of a compound that substantially does not contain other stereoisomers of that compound. Thus, a stereoisomerically pure compound having one chiral center substantially does not contain the opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers substantially does not contain other diastereomers of that compound. Typical stereoisomerically pure compounds include more than 80% by weight of one stereoisomer of the compound and less than 20% by weight of the other stereoisomer of the compound, for example, more than 90% by weight of one stereoisomer of the compound and less than 10% by weight of the other stereoisomer of the compound, or more than 95% by weight of one stereoisomer of the compound and less than 5% by weight of the other stereoisomer of the compound, or more than 97% by weight of one stereoisomer of the compound and less than 3% by weight of the other stereoisomer of the compound.

[0053] The term “prodrug” refers to a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions, either in vitro or in vivo, to provide an active compound, particularly a derivative of the compounds of the present invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds of the present invention that contain biohydrolyzable groups such as biohydrolyzable thiol adducts, nitrate esters, amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureids, and biohydrolyzable phosphate analogs (e.g., monophosphate, diphosphate, or triphosphate). For example, a prodrug of a compound having a carboxyl functional group is a lower alkyl of a carboxylic acid. Carboxylic acid esters are conventionally formed by esterifying any of the carboxylic acid moieties present in the molecule. Prodrugs can typically be prepared using well-known methods (e.g., those described in BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY, 6th edition (Wiley, 2001) and DESIGN AND APPLICATION OF PRODRUGS (Harwood Academic Publishers GmbH, 1985)).

[0054] Complexes of active compounds (e.g., compounds containing electron-withdrawing groups, such as nitroalkenes) and cyclodextrins are disclosed herein. Although not bound by any theory, in aqueous solution, cyclodextrins form inclusion complexes with active compounds through a process in which water molecules located in the central cavity are replaced by either the entire active compound molecule or a lipophilic portion of the active compound structure. The three-dimensional structure of the cyclodextrin molecule provides a hydrophobic barrel capable of binding to and protecting the active compound. Once contained within the cyclodextrin cavity (i.e., hydrophobic barrel), the drug molecule can dissociate through complex dilution by replacing the contained drug with another suitable molecule, and the drug can then move to the matrix that has the highest affinity. Importantly, since no covalent bond is formed or is broken during drug-cyclodextrin complex formation, the complex is in a state of dynamic equilibrium with the free drug and the cyclodextrin molecule (RA Rajewski and VJ Stella, "Pharmaceutical applications of cyclodextrins. 2. In vivo drug delivery." J. Pharm. Sci. 85(11), 1142-1169 (1996)).

[0055] The step of contacting the active compound with at least one cyclodextrin may include the step of dissolving or suspending the cyclodextrin in a solvent or mixture of solvents to form a first solution or suspension. Similarly, the active compound may be dissolved or suspended in the same or different solvent or mixture of solvents to form a second solution or suspension. The first solution or suspension may then be combined to form the complex of the present invention between the active compound and the at least one cyclodextrin. The complex may then be separated from the solution, purified if necessary, to yield a stabilized complex.

[0056] The step of contacting the active compound with at least one cyclodextrin may instead include the step of dissolving or suspending at least one cyclodextrin in a solvent or mixture of solvents to form a solution or suspension, and then adding the active compound to the solution or suspension to form the complex of the present invention. The step of contacting the active compound with at least one cyclodextrin may also be carried out by other methods. For example, a solvent that completely dissolves both the active compound and the cyclodextrin may be used. In another embodiment, the cyclodextrin may be dissolved or suspended in a solvent or mixture of solvents and then placed in a rotary evaporator. The active compound may then be sprayed into the solution or suspension, either in neat form or as a solution or suspension of the active compound in a solvent or mixture of solvents. The contacting step may also be achieved by using a two-phase solvent system. For example, the active compound may be combined (either as a suspension or in solution) in a separate immiscible solvent. The immiscible solvent can then be thoroughly mixed until a complex is formed. The complex can then be isolated via one of the isolation techniques discussed herein. It may be desirable to carry out the contact step in the absence of the solvent. For example, in a spray-drying technique, a mist of nitroalkene can be sprayed or atomized onto neat cyclodextrin to produce the complex of the present invention.

[0057] The above cyclodextrin may be dissolved or suspended in a solvent selected from the group comprising non-polar or weakly polar solvents. Exemplary solvents for cyclodextrin include the following: water, methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, tert-butanol, high molecular weight alcohols, dimethylformamide, diethylformamide, ethylene glycol, triethylene glycol, glycerin, polyethylene glycol, formamide, acetone, tetrahydrofuran, dioxane, methyl ethyl ketone, high molecular weight ketones, ethyl acetate, acetonitrile, N,N-dimethylacetimide, dimethyl sulfoxide, carbon disulfide, hexane, hexane isomers, cyclohexane, heptane, heptane isomers, mineral oil, diethyl ether, methyl tert-butyl ether, methylene chloride, chloroform, carbon tetrachloride, benzene, nitrobenzene, toluene, and mixtures thereof. In certain specific embodiments, the above cyclodextrin is dissolved in water.

[0058] The above active compound may be dissolved or suspended in a solvent selected from the group comprising non-polar or weakly polar solvents. Exemplary solvents for the above active compound include the following: methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, tert-butanol, pentanol, high molecular weight alcohols, dimethylformamide, diethylformamide, ethylene glycol, triethylene glycol, formic acid, acetic acid, formamide, acetone, tetrahydrofuran, dioxane, methyl ethyl ketone, high molecular weight ketones, ethyl acetate, acetonitrile, N,N-dimethylacetamide, dimethyl sulfoxide, carbon disulfide, hexane, hexane isomers, cyclohexane, heptane, heptane isomers, mineral oil, diethyl ether, methyl tert-butyl ether, methylene chloride, chloroform, carbon tetrachloride, benzene, nitrobenzene, toluene, and mixtures thereof. In certain specific embodiments, the nitroalkene as the active compound is dissolved in ethanol.

[0059] Optional steps for removing the above complex from the solution or suspension may be carried out by separation techniques. Exemplary separation techniques include one or more of precipitation, filtration, vacuum degassing, freeze-drying, spray drying, and distillation.

[0060] The stabilized active compound / cyclodextrin complex can be stored as a solid at a convenient temperature (e.g., -80 to 30°C, more specifically 4 to 22°C) for a desired period of time. In certain embodiments, the above period may be at least 360 days, more specifically at least 90 days.

[0061] The stabilized active compound / cyclodextrin complex can be stored as a solid for a desired period at a convenient temperature (e.g., -80 to 30°C, more specifically 4 to 22°C) for a desired period of time, so that it can be redissolved using water to obtain a solution or suspension that will be used to administer the active compound. In certain embodiments, the stabilized active compound / cyclodextrin complex is in powder form. In certain embodiments, the period for powder storage may be at least 360 days, more specifically at least 90 days, and the period for powder storage may be at least 14 days, more specifically at least 10 days.

[0062] In certain embodiments, the active compound is a nitroalkene comprising at least one carbon-carbon double bond and at least one nitro group. In certain embodiments, the nitroalkene is a nitroalkene fatty acid. Certain nitroalkene fatty acids are described, for example, in U.S. Patent No. 7,776,916.

[0063] One exemplary embodiment of a nitroalkene is the structure of formula I: [ka] And here R1 is hydrogen, C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 It is alkinyl; R 2 , R 3 , R 7 , and R 8 These are, independently, hydrogen, oxygen, and C1-C 24 It is alkyl, NO2, OH, or OOH; R 4 This is the end COOR 6 It is a base, and here R 6 is hydrogen, or C1-C 24 It is alkyl; R 5 is hydrogen, C1-C 24 Alkyl or R 4 and R 5 The combined = C(R 9 )(R 10 ) forms, and here R 9 C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 Contains alkinyl, or R here 9 This is the end COOR 6 It is a base, R 10 is hydrogen, NO2, OH, or OOH; n is 1 to 24; and Here, the nitroalkene fatty acid contains at least one NO2 group.

[0064] In a particular embodiment of formula I, R 1 C1-C 24 Alkyl, more specifically, C3-C 20 It is alkyl.

[0065] In a particular embodiment of formula I, R 2 It is hydrogen.

[0066] In a particular embodiment of formula I, R 3 or R8 One of them is NO2, and R 3 or R 8 The other of these is hydrogen.

[0067] In a particular embodiment of formula I, n is between 3 and 20.

[0068] In a particular embodiment of formula I, R 4 It is -COOH.

[0069] In a particular embodiment of formula I, R 5 It is hydrogen.

[0070] In a particular embodiment of formula I, R 7 It is hydrogen.

[0071] In a particular embodiment of formula I, R 4 is -COOH; R 5 is methyl; R 7 It is methyl.

[0072] In a particular embodiment of formula I, R 1 C1-C 24 Alkyl, more specifically, C3-C 20 It is alkyl; R 2 R is hydrogen; 3 or R 8 One of them is NO2, and R 3 or R 8 The other of these is hydrogen; R 4 is -COOH; R 5 R is hydrogen; 7 It is hydrogen.

[0073] Another exemplary embodiment of a nitroalkene is the structure of formula II: [ka] And here R 1 is hydrogen, C1-C 24Alkyl, C1-C 24 Alkenyl, or C1-C 24 It is alkinyl; R 2 , R 4 , R 5 and R 6 Each of them is hydrogen; R 7 This is the end COOR 9 It is a base, and here R 9 is hydrogen or C1-C 24 It is alkyl; R 3 and R 8 These are, independently, hydrogen, oxygen, and C1-C 24 Alkyl, NO2, OH, ONO2, NO, ONO, or OOH, but at least R 3 or R 8 One of them is NO2, and R 3 or R 8 The other of these is hydrogen, ONO, or ONO2.

[0074] In a particular embodiment of formula II, R 1 C1-C 24 Alkyl, more specifically, C3-C 20 It is alkyl; R 9 R is hydrogen; 3 It is NO2 and R 8 is either ONO2 or R 8 It is NO2 and R 3 This is ONO2.

[0075] A further exemplary embodiment of another nitro group-containing compound is the structure of formula III: [ka] And here R 1 is hydrogen, C1-C 24 Alkyl, C1-C 24 Alkenyl, or C1-C 24 It is alkinyl; R 2 and R5 Each of them is hydrogen; R 7 This is the end COOR 6 It is a base, and here R 6 is hydrogen or C1-C 24 It is alkyl; R 3 and R 4 These are, independently, hydrogen, oxygen, and C1-C 24 The elements are alkyl, NO2, OH, ONO2, NO, ONO, or OOH, however R 3 Or R 4 At least one of them is NO2, and R 3 Or R 4 The other of these is hydrogen, ONO, or ONO2.

[0076] In a particular embodiment of Equation III, R 1 C1-C 24 Alkyl, more specifically, C3-C 20 It is alkyl; R 6 R is hydrogen; 3 It is NO2 and R 4 is either ONO2 or R 4 It is NO2 and R 3 This is ONO2.

[0077] Another exemplary compound that can be stabilized as described herein has the structure of formula IV: [ka] It is a compound containing a dicarboxylic acid, and here X is an electron-withdrawing group selected from acyl, carboxylic acid, ester, halogen, fluoromethyl, -CN, sulfonyl, sulfone, sulfonic acid, primary ammonium, secondary ammonium, tertiary ammonium, or -NO2. m is between 1 and 10; n is between 1 and 10.

[0078] In a particular embodiment of formula IV, X is -NO2.

[0079] Further exemplary compounds that can be stabilized as described herein have the structure of formula V: [ka] It is a compound containing a dicarboxylic acid, and here X is an electron-withdrawing group selected from acyl, carboxylic acid, ester, halogen, fluoromethyl, -CN, sulfonyl, sulfone, sulfonic acid, primary ammonium, secondary ammonium, tertiary ammonium, or -NO2; Y and Z are each independently hydrogen or C1-C 10 It is alkyl; m is between 1 and 10; n is between 1 and 10.

[0080] In a particular embodiment of formula V, X is -NO2, and at least one Y and Z are C1-C4 alkyl groups.

[0081] Further exemplary compounds that can be stabilized as described herein have the structure of formula VI: [ka] It is a compound containing a dicarboxylic acid, and here X is an electron-withdrawing group selected from acyl, carboxylic acid, ester, halogen, fluoromethyl, -CN, sulfonyl, sulfone, sulfonic acid, primary ammonium, secondary ammonium, tertiary ammonium, or -NO2; Y and Z are each independently hydrogen or C1-C 10 It is alkyl; p and t are each independently between 1 and 10; s is either nonexistent or between 1 and 10. r is 1.

[0082] In a particular embodiment, the nitroalkene fatty acid is 10-nitro-octadeca-9-enoic acid (10-NO2-OA).

[0083] In a particular embodiment, the nitroalkene fatty acid is 9-nitro-octadeca-9-enoic acid (9-NO2-OA).

[0084] In a particular embodiment, the nitroalkene fatty acid is 8-nitro-nonadeca-9-enoic acid.

[0085] In a particular embodiment, the nitroalkene fatty acid is 7-NO2-nonadeca-7-enoic acid.

[0086] In certain embodiments, the nitroalkene fatty acid is 5-NO2-eicosa-5-enoic acid or 6-NO2-eicosa-5-enoic acid. In certain embodiments, the nitroalkene fatty acid is 9-nitrooctadeca-9,11-dienoic acid, and in certain embodiments, the nitroalkene fatty acid is 12-nitrooctadeca-9,11-dienoic acid.

[0087] In a particular embodiment, the nitroalkene fatty acid is 9-nitro-12-(nitrooxy)octadeca-10-enoic acid.

[0088] In a particular embodiment, the nitroalkene fatty acid is 12-nitro-9-(nitrooxy)octadeca-10-enoic acid.

[0089] In certain embodiments, the nitroalkene is substantially pure. In this context, the stereochemistry of the carbon-carbon double bond is substantially cis (or Z) or substantially trans (or E).

[0090] Examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, and methyl-β-cyclodextrin. β-cyclodextrin is a preferred cyclodextrin.

[0091] The amount of the active compound mixed with the cyclodextrin may vary. In certain embodiments, the molar ratio of nitroalkene fatty acid / cyclodextrin may range from 1:2 to 1:12, more specifically from 1:2 to 1:8, and most specifically from 1:2 to 1:4.

[0092] In certain embodiments, the active compound and the cyclodextrin may be brought into contact at temperatures of 10–90°C, more particularly 20–50°C, and most particularly 30–50°C, in order to form the complex.

[0093] In certain embodiments, the active compound and the cyclodextrin may be in contact with each other for 1 to 48 hours, more specifically 8 to 16 hours, and most specifically 10 to 16 hours, in order to form the complex.

[0094] In certain embodiments, the complex disclosed herein (via the active compound of the complex) can be used to treat a condition in a subject requiring it. Conditions treated may include, for example, inflammatory conditions, immune disorders, psoriasis, obesity, metabolic syndrome, acute kidney disease, chronic kidney disease, focal segmental glomerulosclerosis, atherosclerosis, lipogenesis, neointimal hyperplasia, renal I / R and xenobiotic injury, focal myocardial I / R injury, Ang II-induced systemic hypertension, pulmonary hypertension, cancer, cardiac and pulmonary fibrosis, hepatic fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), breast cancer, ovarian cancer, inflammatory bowel disease, nociception, stroke, motor neuron degeneration, diabetes mellitus, aneurysms, aortic sclerosis, systemic lupus erythematosus, infant-onset STING-associated vasculitis (SAVI), asthma, chronic obstructive pulmonary disease (COPD), or focal segmental glomerulosclerosis.

[0095] In certain embodiments, the inflammatory condition may be: organ preservation for transplantation, osteoarthritis, chronic obstructive pulmonary disease (COPD), atherosclerosis, hypertension, allograft rejection, pelvic peritonitis, ulcerative colitis, Crohn's disease, allergic inflammation in the lungs, cachexia, stroke, congestive heart failure, pulmonary fibrosis, hepatitis, glioblastoma, Guillain-Barré syndrome, systemic lupus erythematosus, viral myocarditis, post-transplant organ protection, acute pancreatitis, irritable bowel disease, general inflammation. Inflammation, autoimmune diseases, autoinflammatory diseases, arterial stenosis, organ graft rejection and burns, chronic lung injury and respiratory distress, insulin-dependent diabetes, non-insulin-dependent diabetes, hypertension, obesity, arthritis, neurodegenerative disorders, lupus, Lyme disease, gout, sepsis, hyperthermia, ulcers, enteritis, osteoporosis, viral or bacterial infections, cytomegalovirus, periodontal disease, glomerulonephritis, sarcoidosis, lung diseases, pneumonia, pulmonary fibrosis, asthma, acquired respiratory distress syndrome Syndrome), smoking-induced lung disease, granuloma formation, hepatic fibrosis, graft-versus-host disease, postoperative inflammation, coronary and peripheral restenosis after angioplasty, stent placement or bypass graft, coronary artery bypass graft (CABG), acute and chronic leukemia, B lymphocyte leukemia, neoplasms, arteriosclerosis, atherosclerosis, myocarditis, psoriasis, immunodeficiency, disseminated intravascular coagulation, systemic sclerosis, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, encephalomyelitis, edema, inflammatory bowel disease, hyper-IgE syndrome, cancer metastasis or proliferation, adoptive immunotherapy, reperfusion syndrome, radiation burns, alopecia areata Areta, ischemia, myocardial infarction, arterial stenosis, rheumatoid arthritis, coronary restenosis, cognitive decline, and insulin resistance.

[0096] In embodiments described herein, methods for treating inflammation, obesity, metabolic syndrome, acute kidney disease, and chronic kidney disease include administering an effective amount of the complex and, if necessary, a pharmaceutically acceptable excipient to a subject in need thereof.

[0097] In embodiments described herein, methods for treating inflammation, obesity, metabolic syndrome, focal segmental glomerulosclerosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), acute kidney disease, lithium-induced nephropathy, and chronic kidney disease include the step of administering to a subject in need thereof an effective amount of the complex and, if necessary, a pharmaceutically acceptable excipient, wherein the complex provides the release of activated fatty acids.

[0098] In certain embodiments, the complex disclosed herein is useful for treating endotoxin-induced vasculitis, endotoxemia and multi-organ injury, inflammatory bowel disease (IBD), allergic airway disease, renal ischemia and reperfusion (I / R) injury, diabetic nephropathy, pulmonary arterial hypertension (PAH), myocardial I / R injury, hypertension, and atherosclerosis.

[0099] In some embodiments, the methods disclosed herein include the step of administering to a subject requiring treatment of a pharmaceutical composition, for example, a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of one or more of the complexes disclosed herein. The complexes may be administered orally, parenterally (including techniques of subcutaneous injection (SC or depo-SC), intravenous (IV), intramuscular (IM or depo-IM), intrasternal injection or infusion), sublingually, intranasally (inhalation), intrathecally, topically, ocularly, or rectally. The pharmaceutical compositions may be administered in dose-unit formulations comprising a conventional non-toxic pharmaceutically acceptable carrier, adjuvant, and / or vehicle. The complexes are preferably formulated into appropriate pharmaceutical preparations (e.g., tablets, capsules, or elixirs for oral administration) or in sterile solutions, emulsions, or suspensions for parenteral or topical administration or inhalation.

[0100] One embodiment disclosed herein is a pharmaceutical composition comprising a complex of cyclodextrin and an active compound, which can be resuspended in water and administered orally.

[0101] Also disclosed is a pharmaceutical composition comprising a complex of cyclodextrin and an active compound in powder form, which can be solvated as a liquid pharmaceutical for administration to infants, toddlers and children (e.g., 12 years of age and under).

[0102] In some embodiments, one or more of the disclosed complexes are mixed or combined with a suitable pharmaceutically acceptable carrier to prepare a pharmaceutical composition. Suitable pharmaceutically acceptable carriers or vehicles for the administration of the complexes provided herein include any such carriers known to be suitable for a particular mode of administration. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition (2005) describes exemplary compositions and formulations suitable for the pharmaceutically acceptable delivery of the complexes disclosed herein. Furthermore, the complexes may be formulated as the sole pharmaceutically active ingredient in the compositions, or combined with other active ingredients.

[0103] When the complex is mixed with or added to a pharmaceutically acceptable carrier, the resulting mixture may be a liquid, suspension, emulsion, dry powder, or pill. Liposome suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the complex in the selected carrier or vehicle. If the complex exhibits insufficient solubility, methods for solubilization may be used. Such methods are known and include, but are not limited to, the use of a cosolvent such as dimethyl sulfoxide (DMSO), the use of a surfactant such as Tween®, and dissolution in aqueous sodium bicarbonate. The disclosed complexes may also be prepared with a carrier that protects them from rapid elimination from the body (e.g., time-release formulations or coatings). Such carriers include, but are not limited to, controlled-release formulations (e.g., microencapsulated delivery systems). The disclosed complexes and / or compositions may be contained in multi-dose or single-dose containers. The above-mentioned complexes and / or compositions may also be provided in a kit (including, for example, component parts that can be assembled for use). For example, one or more of the disclosed complexes may be provided in lyophilized form, and a suitable diluent may be provided as a separate component for combination before use. In some examples, the kit may include the disclosed complexes and a second therapeutic agent for co-administration. The above-mentioned complexes and second therapeutic agents may be provided as separate component parts. The kit may include a plurality of containers, each container holding one or more unit doses of the above-mentioned complexes. The above-mentioned containers are preferably adapted to a desired mode of administration (including, but not limited to, tablets, gel capsules, sustained-release capsules, etc. for oral administration; depot products, pre-filled syringes, ampoules, vials, etc. for parenteral administration; and patches, plasters (medipad), creams, etc. for topical administration).

[0104] The above-mentioned pharmaceutical compositions may be in the form of dosage units such as injection solutions, oral delivery solutions (e.g., liquids or suspensions), nasal delivery fluids (e.g., for delivery as aerosols or vapors), semi-solid forms (e.g., topical creams), or solid forms (e.g., powders, pills, tablets, or capsules).

[0105] The above-mentioned complex is contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically beneficial effect on the subject being treated in the absence of undesirable side effects. The therapeutically effective concentration can be empirically determined by testing the complex in known in vitro and in vivo model systems with respect to the disorder being treated. In some examples, the therapeutically effective amount of the complex is the amount in which the complex reduces or improves at least one symptom of the disorder to which it is administered. Typically, the composition is formulated for administration in a single dose. The concentration of the complex in the drug composition depends on the absorption, inactivation, and efflux rates of the active compound, the administration schedule, and the amount administered, as well as other factors known to those skilled in the art.

[0106] In some examples, a disclosed complex, a mixture of such complexes, or a physiologically acceptable salt or ester thereof in amounts ranging from approximately 1 mg to 5000 mg is formulated with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc., in a unit dosage form. The amount of active substance in these compositions or preparations is such that an appropriate dose is obtained within the indicated range. The term “unit dosage form” means a physically discontinuous unit suitable as a unit dosage for human subjects and other mammals, each unit containing a predetermined amount of active substance calculated to associate with appropriate pharmaceutical excipients to produce a desired therapeutic effect. In some examples, the above compositions are formulated in unit dosage forms, each dose containing one or more of the compound ranging from approximately 1 mg to approximately 5000 mg (e.g., approximately 5 mg to approximately 1000 mg, approximately 10 mg to approximately 500 mg, approximately 30 mg to approximately 300 mg, or approximately 50 mg to approximately 100 mg). In other examples, the above unit dosage forms include approximately 0.1 mg, approximately 1 mg, approximately 5 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 150 mg, approximately 200 mg, approximately 250 mg, approximately 300 mg, approximately 500 mg, approximately 700 mg, approximately 800 mg, approximately 1000 mg, approximately 2000 mg, approximately 3000 mg, approximately 5000 mg, or more disclosed complexes.

[0107] The disclosed complex or composition may be administered as a single dose or divided into many smaller doses to be administered at time intervals. The therapeutic composition may be administered in single-dose delivery, by continuous delivery over a long period, or in a repeated-dose protocol (e.g., multiple times a day, once a day, once a week, or once a month). It is understood that the exact dose, timing, and duration of treatment are functions of the disease being treated and may be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It is noted that the concentration and dose values ​​may also vary with the severity of the condition to be alleviated. Furthermore, it is understood that, for a particular subject, the dose regimen may be adjusted over time according to the individual's needs and the professional judgment of the individual administering or supervising the administration of the composition, and that the concentration ranges shown herein are illustrative only.

[0108] When administered orally as a suspension, these compositions may be prepared according to well-known techniques in the field of pharmaceutical formulation and may contain microcrystalline cellulose for bulking, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners / flavoring / odorizing agents. As immediate-release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, and lactose and / or other excipients, binders, bulking agents, disintegrants, diluents, and lubricants. When oral administration is desired, the above complexes are typically provided in a composition that protects them from the acidic environment of the stomach. For example, the above compositions may be formulated in an enteric coating that maintains their integrity in the stomach and releases the active compound in the intestines. The above compositions may also be formulated in combination with antacids or other such components.

[0109] Oral compositions generally contain an inert diluent or food carrier and can be compressed into tablets or encapsulated in gelatin capsules. For the purpose of administering other therapeutic agents, the complex may be incorporated with excipients and used in the form of tablets, capsules, or lozenges. Pharmaceutically compatible binders and adjuvants may be included as part of the composition. The tablets, pills, capsules, lozenges, etc. may contain any of the following components or compounds of similar nature: binders (e.g., tragacanth gum, acacia, corn starch, or gelatin); excipients (e.g., microcrystalline cellulose, starch, or lactose); disintegrants (e.g., alginic acid and corn starch, but not limited to these); lubricants (e.g., magnesium stearate, but not limited to these); gildants (e.g., colloidal silicon dioxide, but not limited to these); sweeteners Flavoring agents (e.g., sucrose or saccharin); and flavoring agents (e.g., peppermint, methyl salicylate, or fruit flavorings).

[0110] The dosage unit form may include various other substances (e.g., sugars and other enteric coatings) that modify the physical form of the dosage unit. The complex may also be administered as a component of elixirs, suspensions, syrups, wafers, chewing gums, etc. In addition to the active ingredient, the syrup may contain sucrose as a sweetener, preservatives, colorants and coatings, and flavoring and odor-modifying agents.

[0111] When administered orally, the complex may be administered in a standard dosage form for oral administration. These dosage forms include standard solid-unit dosage forms such as tablets and capsules, as well as liquid dosage forms such as solutions, suspensions, and elixirs. When solid dosage forms are used, it is preferable that they are sustained-release so that the compound needs to be administered only once or twice daily. In some examples, the oral dosage form is administered to the subject once, twice, three, four times, or more times daily. In further examples, the complex may be administered orally to humans in single doses or divided doses in a dosage range of 0.1 to 100 mg / kg body weight. One exemplary dosage range is 1 to 200 mg / kg body weight orally in single doses or divided doses (e.g., 0.5 to 100 mg / kg body weight orally). With regard to oral administration, the above compositions may be provided in the form of tablets containing approximately 1 to 1000 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 milligrams of the active ingredient. However, it is understood that the specific dose level and frequency of the dosage for any particular patient may vary and depend on various factors, including the activity of the particular complex used, its metabolic stability and the duration of its action, age, weight, overall health, sex, diet, mode and timing of administration, elimination rate, drug combination, severity of the particular condition, and the host being treated.

[0112] Injectable solutions or suspensions may also be formulated using a suitable non-toxic, parenterally acceptable diluent or solvent (e.g., mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution), or a suitable dispersant or wetting and suspending agent (e.g., sterile and non-irritating non-volatile oils (including synthetic monoglycerides or diglycerides), and fatty acids (such as oleic acid). Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application may contain any of the following components: sterile diluents (e.g., water for injection, saline solution, non-volatile oil, naturally occurring vegetable oils (e.g., sesame oil, coconut oil, peanut oil, cottonseed oil, etc.)), or synthetic fatty acids. Crude (e.g., ethyl oleate), polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents); antimicrobial agents (e.g., benzyl alcohol and methylparaben); antioxidants (e.g., ascorbic acid and sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetic acid, citric acid, and phosphoric acid); and agents for adjusting tonicity (e.g., sodium chloride and dextrose). Parenteral preparations may be contained in ampoules, disposable syringes, or multi-dose vials made from glass, plastic, or other suitable materials. Buffers, preservatives, antioxidants, etc., may be incorporated as needed.

[0113] For intravenous administration, suitable carriers include physiological saline, phosphate-buffered saline (PBS), and solutions containing concentrating and solubilizing agents (e.g., glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof). Liposome suspensions containing tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers.

[0114] The above complex may be administered parenterally, for example, by IV, IM, depo-IM, SC, or depo-SC. When administered parenterally, a therapeutically effective dose of approximately 1 to approximately 5000 mg / day (e.g., approximately 5 mg / day to approximately 1000 mg / day, or approximately 20 mg / day to approximately 200 mg / day) may be delivered. When the depot formulation is used for injection once a month or once every two weeks, the dose may be approximately 1 mg / day to approximately 5000 mg / day, or approximately 30 mg to approximately 15000 mg per month.

[0115] The above complex may also be administered sublingually. When administered sublingually, the complex should be given 1 to 4 times a day in the amounts described above for IM administration.

[0116] The above complex may also be administered intranasally. When administered by this route, the appropriate dosage form is a nasal spray or a dry powder. The dosage of the above complex for intranasal administration is the amount described above with respect to IM administration. When administered by nasal aerosol or inhalation, these compositions may be prepared according to techniques well known in the field of pharmaceutical formulation, and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizers or dispersants.

[0117] The above complex may be administered intrathecally. When administered via this route, the appropriate form of administration may be parenteral. The dosage of the above complex for intrathecal administration is the same as the amount described above for IM administration.

[0118] The above complex can be administered topically. When administered by this route, appropriate dosage forms are creams, ointments, or patches. When administered topically, exemplary doses range from approximately 2 mg / day to approximately 1000 mg / day. Since the amount that can be delivered by a patch is limited, two or three patches may be used.

[0119] The above complex may be administered rectally by suppository. When administered by suppository, exemplary therapeutically effective doses may range from about 2 mg to about 2000 mg. When administered rectally in the form of suppositories, these compositions may be prepared by mixing the above drug with a suitable non-irritating excipient (e.g., cocoa butter, synthetic glyceride ester of polyethylene glycol) that is solid at normal temperatures but liquefies and / or dissolves in the rectal lumen to release the drug.

[0120] It should be obvious to those skilled in the art that the precise dosage and frequency of administration depend on the specific complex being administered, the specific condition being treated, the severity of the condition being treated, the age, weight, overall physical condition of the specific subject, and other pharmacotherapy, and that such an individual may take the medication as is well known to the administering physician or other clinicians proficient in the treatment of retroviral infections, diseases, and related disorders. [Examples]

[0121] Examples As a first step toward inclusion complex formation, conjugated linoleic acid was used as a substitute for the highly unstable NO2-OA to determine whether a preferred form of cyclodextrin exists for higher yields and to evaluate the effect of its molar ratio on inclusion complex formation. NO2-OA requires storage at -80°C, is unstable to temperature, and its degradation rate gradually increases with temperatures above -20°C, the presence of water and humidity levels (e.g., moisture in the air), the presence of nucleophiles, and the presence of protein amino acids. This includes degradation induced by the shell derived from the hard gelatin capsule. Its instability arises from reactions with nucleophiles that promote degradation reactions including isomerization of the nitroalkene CC double bond, double bond transfer, dimerization between two NO2-OA molecules, and oxidation.

[0122] 1. The CLA-cyclodextrin complex was first formed by weighing 499.39 mg of β-cyclodextrin (0.44 mmol), mixing it in 3 ml of water, and dissolving it by heating to 50°C. 61.69 mg of CLA (0.22 mmol) was weighed into a clean tube, dissolved in ethanol, and then slowly added to the solution containing β-cyclodextrin. The mixture was allowed to stand overnight at 37°C with gentle stirring to form an inclusion complex. The mixture was then dried under a nitrogen stream for 20-25 minutes, and the resulting solution was frozen in a -80°C freezer. The frozen solution was placed in a freeze-dryer and freeze-dried overnight. The resulting powder was transferred to a clean tube and maintained at 4°C. The CLA content was quantified by HPLC-UV, showing that the inclusion complex contained approximately 107 μg CLA / mg.

[0123] 2. Secondly, since NO2-OA is nitrated oleic acid, which is our goal for inclusion complex stabilization, we used oleic acid (OA) to test for inclusion complex formation. Again, the β-cyclodextrin complex was superior, and we used the following protocol.

[0124] 3. The above OA-β-cyclodextrin complex was formed by first weighing 499.39 mg of β-cyclodextrin (0.44 mmol), mixing it in 3 ml of water, and dissolving it by heating to 50°C. 62.14 mg of CLA (0.22 mmol) was weighed into a clean tube, dissolved in ethanol, and then slowly added to the solution containing β-cyclodextrin. The mixture was allowed to stand overnight at 37°C with gentle stirring to form an inclusion complex. The mixture was then dried under a nitrogen stream for 20-25 minutes, and the resulting solution was frozen in a -80°C freezer. The frozen solution was placed in a freeze-dryer and freeze-dried overnight. The resulting powder was transferred to a clean tube and maintained at 4°C. The OA content was quantified by HPLC-UV, showing that the inclusion complex contained approximately 99 μg OA / mg.

[0125] β-cyclodextrin was found to encapsulate conjugated linoleic acid more efficiently than α-cyclodextrin. A lower yield of inclusion into the inclusion complex was observed with α-cyclodextrin. The results are shown in Figure 1.

[0126] After it was determined that β-cyclodextrin was superior to α-cyclodextrin, various molar ratios of NO2-OA to β-cyclodextrin were tested. Three independent analyses were performed using three different molar ratios between NO2-OA and β-cyclodextrin. The molar ratios tested were 1:2, 1:4, and 1:8. The data show that increasing the molar ratio of β-cyclodextrin to NO2-OA had only a small effect on total recovery (see Figure 2).

[0127] Complexes of various ratios were obtained by dissolving various required amounts of β-cyclodextrin in the first 3 ml of water. Thus, for 1:4 and 1:8 molar ratios, the amounts were 998.8 and 1997.6 mg, respectively. In some cases, especially when using higher molar ratios, increased heating was necessary to initially dissolve the β-cyclodextrin in water. For the 1:8 molar ratio, a maximum of 75°C was used, along with incubation / mixing times of up to 30 minutes.

[0128] As before, NO2-OA must be maintained at -80°C. To test whether the β-cyclodextrin-NO2-OA inclusion complex disclosed herein provides stability as a dry powder (after freeze-drying), it was subjected to the stability evaluation shown in Figure 3. The results are shown in Figure 4.

[0129] NO2-OA / β-cyclodextrin inclusion complexes in a 1:2 molar ratio exhibited remarkable thermal stability. Without β-cyclodextrin stabilization, NO2-OA would likely have suffered significant loss over 4 weeks at 70°C. Conventionally, NO2-OA is stabilized using oil, the only way to slightly improve its stability. All conventional approaches used to stabilize NO2-OA are based on solvation in oily, viscous liquid formulations. This is unsuitable for use as a human formulation, given its low stability and resulting short shelf life. Oils used included olive oil, sesame oil, and synthetic oils (synthetic triacylglycerols).

[0130] The degradation of NO2-OA at high temperatures is a process that yields several degradation products, including isomerization from the E isomer to the Z isomer (trans), and the formation of OH-NO2-OA, oxo-NO2-OA, and NO2-OA dimers. None of these degradation products, shown in Figure 5A, were identified in the chromatogram evaluated 14 days after exposure of NO2-OA / β-cyclodextrin to 55°C (see Figure 5B). A small peak appearing at approximately 9 minutes was also present in the initial NO2-OA stock solution, but its intensity did not change after incubation and is not consistent with dimer formation during incubation.

[0131] To confirm the fact that the β-cyclodextrin inclusion complex is protected against temperature-dependent isomerization from E to Z, or even double bond transfer, chromatographic analysis was performed. Figure 5A shows the structures of possible degradation products, and Figure 5B shows the chromatograms. A mixture containing (E)10-NO2-OA, (Z)10-NO2-OA, and 10-NO2-octadeca-8-enoic acid was subjected to C 18 When separated using a Polaris column and tracked at 210 nm, a major peak for (E)10-NO2-OA was observed, with a shoulder corresponding to 10-NO2-octadeca-8-enoic acid before the major peak and a more linear shoulder corresponding to (Z)10-NO2-OA after the major peak (see Figure 5C). As shown in Figures 5B-5C, the blue traces corresponding to 10-NO2-OA obtained from the inclusion complex after exposure to 55°C for 14 days did not show evidence of these degradation products.

[0132] The method used to generate the inclusion complex yielded high yields and was highly reproducible. Three independent batches provided 100% of the total yield compared to the initial material used in the preparation (left column) (see Figure 6A). The incorporated yield was quantified by performing an external calibration curve using HPLC-UV at 210 nm with a 10-NO2-OA standard in ethanol, as shown in Figure 6B.

[0133] The decrease in total amount observed after 14–28 days of exposure at higher temperatures was also observed at lower temperatures and at 4°C (Figure 4). This may be a result of changes in the physical properties of the complex, but not related to the degradation of the substance, as indicated by the absence of major oxidation / decomposition products. During the development of the method, it was observed that the quantification of these complexes was greatly influenced by the extraction method used to quantify the residual NO2-OA. In this regard, different extraction methods containing different organic solvents yielded different overall results, even when starting from the same inclusion complex stock material.

[0134] An important consideration when working with nitroalkene fatty acids is their high sensitivity to water and humidity. To demonstrate that NO2-OA / β-cyclodextrin complexes can be used to develop stable solutions of NO2-OA, NO2-OA / β-cyclodextrin inclusion complexes were resuspended in water and their stability was measured. The stability was compared to the total amount added to an aqueous solution.

[0135] Figures 7A and 7B show that NO2-OA remains perfectly stable in water for longer than 10 days after a small-scale disintegration caused by dissolution occurring in the first hour. When these stability assays were performed at room temperature, the data were similar between the two tested concentrations of 0.31 mg / ml and 1.95 mg / ml. Since no degradation products were observed in aqueous solution, it is proposed that the initial decomposition is related to a loosely bound inclusion complex that decomposes under aqueous conditions. A new equilibrium is rapidly achieved, and the sample is stable after this initial disintegration.

[0136] For comparison, the addition of NO2-OA to water resulted in the complete decomposition of NO2-OA in water within 4 hours of addition (see Figure 7C). Therefore, while inclusion complexes stabilize NO2-OA (as a stable powder) for storage / formulation, they can also be used as a vehicle to effectively formulate NO2-OA as a stable liquid drug when added to water.

[0137] The NO2-OA / β-cyclodextrin complex was administered to mice as a suspension in water. To check if this was a viable vehicle, taste aversion was tested by measuring water consumption. Nitrated fatty acids are activators of TRP channels that normally detect the presence of pungent compounds in spicy foods containing capsaicin or related electrophiles. Therefore, nitrated fatty acids are compounds that can induce strong taste aversion due to the spicy and pungent sensations they can induce. The β-cyclodextrin inclusion complex is effective in masking this response and does not alter water consumption by mice.

[0138] To check that β-cyclodextrin inclusion complexes are absorbed when orally consumed from drinking liquids containing suspended NO2-OA / β-cyclodextrin complexes, plasma and fecal analysis after mice ingested suspensions at daily doses of 10 and 50 mg / kg resulted in the detection of the major metabolite. This indicates that β-cyclodextrin complexes are an effective method not only for stabilizing metabolites in capsule or pill formulations but also for producing liquid pediatric formulations suitable for administration to children (e.g., 12 years and under). The β-cyclodextrin inclusion complexes are effectively broken down and released during digestion, and then absorbed, metabolized, and excreted.

[0139] NO2-OA was stabilized as an inclusion complex with β-cyclodextrin, as disclosed herein, and delivered to mice in drinking water. The mice readily drank the water without any changes in their drinking habits or daily liquid intake. This indicates taste masking by the β-cyclodextrin inclusion complex. Nitrooleic acid levels were measured in plasma before and after hydrolysis of the complex lipids (mostly triglycerides) (NO2-OA). See Figure 9. This indicates that the absorbed nitrated fatty acid follows a similar incorporation and biodistribution into the complex lipids as previously observed with oral NO2-OA dissolved in oil. Higher levels of NO2-SA were detected, as previously observed (right figure).

[0140] Figure 10 shows the nitrooleic acid profile and analysis of its major reported metabolites. NO2-OA was stabilized as an inclusion complex with β-cyclodextrin as disclosed herein and delivered to mice in drinking water to obtain daily doses of 10 and 50 mg / kg. The mice readily drank water at both concentrations without any change in their drinking habits or daily liquid intake. The plasma metabolite profile shows the absorption and metabolism of NO2-OA. The upper panel shows the metabolites obtained after reduction of the nitroalkene double bond (18:0) and after β-oxidation of the terminal carboxylic acid (NO2-12:0, NO2-14:0, NO2-16:0). The lower panel shows the metabolites corresponding to two and three β-oxidation cycles of the carboxylic acid terminus of NO2-OA (14:1 and 12:1). The left panel shows NO2-OA and its metabolites as free acid in plasma. The right panel shows all metabolites after complete hydrolysis of plasma lipids.

[0141] Figure 11 shows the NO2-OA profile of mouse feces and an analysis of its major reported metabolites. NO2-OA was stabilized as an inclusion complex with β-cyclodextrin as disclosed herein and delivered to mice in drinking water to obtain daily doses of 10 and 50 mg / kg. In this case, the dose of 50 mg / kg is shown. The fecal metabolite profile shows nitrooleic acid uptake and extensive metabolism. Large amounts of NO2-OA have been reported to be excreted through the feces as NO2-OA and as partially metabolized substances. This further indicates that the stabilized inclusion complex can be solvated and administered to reach the central circulation and exhibit the expected metabolic profile in both urine and feces. The upper panel shows β-oxidation of the reduced metabolite, while the lower panel shows β-oxidation of the parent compound.

[0142] An example of preparing the NO2-OA / β-cyclodextrin complex is described below: 1. Weigh 71.94 mg of nitrated oleic acid (NO2-OA) (0.22 mmol) into a clean tube. 2. Weigh out 499.39 mg of β-cyclodextrin (0.44 mmol). 3. Place the β-cyclodextrin in a tube, add 3 ml of water, and mix thoroughly using a vortex mixer for 1 minute to obtain a suspension. Heat to 60°C for 15 minutes. This will dissolve the β-cyclodextrin. 4. Take the weighed NO2-OA, add 1 ml of ethanol, and mix thoroughly using a vortex mixer for 1 minute. 5. Add the NO2-OA solution (NO2-OA in ethanol) to a tube containing β-cyclodextrin and water. 6. Allow the inclusion complex to form using rotary stirring at 37°C for 16 hours. 7. Take the tube containing the inclusion complex (NO2-OA / β-cyclodextrin). 8. Dry under a nitrogen stream until the ethanol is evaporated. This step is performed at room temperature and takes approximately 20-25 minutes. 9. Take the inclusion complex and freeze it at -80°C, then leave it to stand for 1 hour. 10. Take the tube containing the frozen inclusion complex and freeze-dry it overnight. 11. Take the powder and maintain it at 4°C.

[0143] In light of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the exemplary embodiments are merely preferred examples of the invention and should not be understood as limiting the scope of the invention.

Claims

[Claim 1] The invention described herein.

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