Cyclosporin compositions and methods of use

Abstract

The present invention relates to cyclosporin compositions and methods of use. The present invention discloses a compound having a structure of formula I: (I) a stereoisomer thereof, or a pharmaceutically acceptable salt of any one of the compound or the stereoisomer thereof; wherein: L1 is a C1 to C4 alkylene group optionally substituted by one or more F; r is PEG having 40 to 50 ethylene oxide units, wherein the PEG comprises a linker L2; and L2 is a C1-6 unsubstituted heteroalkylene group having one or two nitrogen atoms. The present invention also discloses the use of the compound, the stereoisomer thereof, or the pharmaceutically acceptable salt of any one of the compound or the stereoisomer thereof in the preparation of a medicament for treating a disease associated with neutrophil-mediated inflammation in a target tissue of a mammalian subject in need thereof.

Description

[0001] This application is a divisional application of Chinese patent application filed on August 20, 2019, with application number 201980068708.1 and invention title "Cyclosporin Composition and Method of Use".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 721,195, filed August 22, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] The technology disclosed herein relates to methods, compounds, and compositions for treating or preventing diseases associated with neutrophil-mediated inflammation. Background Technology

[0005] Inflammation, particularly chronic inflammatory diseases (CIDs), is prevalent globally and is considered a major cause of various diseases, including cardiovascular disease, diabetes, obesity, osteoporosis, rheumatoid arthritis, inflammatory bowel disease, asthma, and CNS-related diseases such as depression and Parkinson's disease. Epithelial cells show significantly increased surface expression of the diaphragm ABC transporter multidrug resistance protein 2 (MRP2) in response to infection with serotype typhimurium (Salmonella typhimurium) or a variety of other pathogens. The intracellular biosynthetic pathway of arachidic acid HXA3 is simultaneously upregulated, and the increased surface MRP2 is used to transport HXA3 into the intestinal lumen. This establishes a concentration gradient of HXA3 in epithelial cells, which guides neutrophil chemotaxis from the basolateral to the lumen, leading to a key inflammatory process. Therefore, inhibiting MRP2 is a pathway for treating or preventing inflammatory diseases.

[0006] Gluten-containing grains, such as wheat, rye, and barley, are an important part of the human diet. However, gliadin, the main component of gluten, is associated with a variety of disorders, including celiac disease, irritable bowel syndrome, non-celiac gluten sensitivity, type 1 diabetes, schizophrenia, and autism. Gliadin has been shown to increase intestinal epithelial permeability and by binding to FPR1 as a neutrophil chemoattractant with similar potency to fMet-Leu-Phe. Therefore, inhibiting FPR1 is a pathway to treat or prevent gliadin-related disorders. Summary of the Invention

[0007] On the one hand, this disclosure relates to a compound having the structure of Formula I, (I) Its stereoisomer or a pharmaceutically acceptable salt of any of the aforementioned substances; wherein L1 is a C0 to C4 alkylene group optionally substituted with one or more F groups; R is selected from the group consisting of -OH and PEG, wherein the PEG optionally includes a linker L2.

[0008] In some embodiments, R is selected from -OH or -NH(CH2). 2-6 (CH2CH2O) 42-46 -O(CH2) 0-5 CH3. In some embodiments, R is selected from CH3O-(CH2CH2O). 44 -CH2CH2CH2NH-(BT051), CH3O-(CH2CH2O) 44 The group consists of -CH2CH2NH-(BT090) and HO-(BT070).

[0009] In some embodiments, L1 is ethylidene or propyleneide.

[0010] In some embodiments, R is a PEG having 40 to 50 ethylene oxide units.

[0011] In some embodiments, L2 is a substituted or unsubstituted heteroalkylene group.

[0012] In some embodiments, L2 is a C having one or two nitrogen atoms. 1-6 Unsubstituted heteroalkylene groups.

[0013] In some embodiments, the compound has the structure of formula IA, its stereoisomers, or a pharmaceutically acceptable salt of any of the aforementioned substances: (IA).

[0014] In some embodiments, this disclosure relates to a pharmaceutical composition comprising the said compound and a pharmaceutically acceptable carrier.

[0015] On one hand, this disclosure relates to a method for treating a disease associated with neutrophil-mediated inflammation in a target tissue of a mammalian subject in need, comprising administering to the subject a therapeutically effective amount of a compound of formula I: (I) Or a pharmaceutically acceptable salt thereof; wherein L1 is a C0 to C4 alkylene group optionally substituted with one or more F groups; R is selected from the group consisting of -OH and PEG, wherein the PEG optionally includes a linker L2 as defined herein. In some embodiments, the PEG group has 40 to 50 ethylene oxide units, and L2 is a substituted or unsubstituted heteroalkylene group, such as an unsubstituted aminoalkylene group. In some embodiments, R is selected from -OH or -NH(CH2). 2-6 (CH2CH2O)42-46 -O(CH2) 0-5 CH3. In some embodiments, R is selected from CH3O-(CH2CH2O). 44 -CH2CH2CH2NH-(BT051), CH3O-(CH2CH2O) 44 The group consists of -CH2CH2NH-(BT090) and HO-(BT070).

[0016] In some embodiments, L1 may be ethylene, and the compound of formula I has formula IA: (IA).

[0017] In some embodiments, the disease is selected from the group consisting of intestinal diseases, colitis, inflammatory lung diseases, inflammatory skin diseases, eye diseases, genitourinary diseases, and sexually transmitted diseases.

[0018] In some embodiments, the intestinal disease is selected from the group consisting of proctitis, orchitis, Crohn's disease, and celiac disease.

[0019] In some embodiments, the colitis is selected from the group consisting of ulcerative colitis (also known as ulcerative colitis), infectious / non-infectious enterocolitis, and inflammatory bowel disease (IBD).

[0020] In some embodiments, the inflammatory lung disease is selected from the group consisting of pneumococcal infection, asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis.

[0021] In some embodiments, the inflammatory skin disease is selected from the group consisting of dermatitis (eczema), rosacea, seborrheic dermatitis, and psoriasis.

[0022] In some embodiments, the eye disease is selected from the group consisting of uveitis, retinitis, keratitis, and macular degeneration.

[0023] In some embodiments, the urogenital disease includes urinary tract infection.

[0024] In some embodiments, the sexually transmitted disease is selected from the group consisting of pelvic inflammatory disease, gonorrhea, chlamydia, herpes, and urethritis.

[0025] In some embodiments, the application step is selected from the group consisting of local application and application to the cavitary surface of the target tissue.

[0026] In some embodiments, the inflammation is non-infectious. In some embodiments, the inflammation is infectious.

[0027] In some embodiments, the method further includes administering to the subject a therapeutically effective amount of one or more second compounds that inhibit one or more multidrug resistance protein 2 (MRP2) and HXA3 synthases, wherein the therapeutically effective amount of the second compound reduces the migration of neutrophils to target tissues.

[0028] In some embodiments, the method further includes administering to the subject a therapeutically effective amount of one or more compounds that increase multidrug resistance protein 1 (MRP1), wherein the therapeutically effective amount of a third compound reduces neutrophil migration to target tissues.

[0029] In some embodiments, the method further includes administering one or more compounds that inhibit one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, wherein the therapeutic amount of the second compound reduces the migration of neutrophils to the target tissue.

[0030] In some embodiments, the method further includes administering to the subject a therapeutically effective amount of one or more compounds that increase one or more N-acylethanolamines (NAEs), wherein the therapeutically effective amount of a third compound reduces the migration of neutrophils to target tissues.

[0031] In some embodiments, the inflammation is associated with Crohn's disease, and the treatment or prevention further includes administration of one or more aminosalicylic acid products, corticosteroid preparations, ileal-release budesonide, glucocorticoid / EEN immunomodulators including azathioprine, 6-mercaptopurine and methotrexate, anti-tumor necrosis factor (TNF) drugs including infliximab, adalimumab and cetrus, polyethylene glycol, anti-α-4β-7 integrin antibody vedolizumab, ABT-494 and finasteride.

[0032] In some embodiments, the inflammation is associated with ulcerative colitis, and the treatment or prevention further includes administration of one or more 5-aminosalicylate, aminosalicylic acid, corticosteroids, multi-matrix budesonide, azathioprine, 6-mercaptopurine, anti-TNF drugs including infliximab, adalimumab and golimumab, vedozizumab, tofacitinib, ABT-494 and finasteride.

[0033] In some embodiments, the method further includes administering one or more antibiotics and / or anti-inflammatory agents selected from the group consisting of: dapavancin, orivancin, cubitril, tebuconazole, cefepime, cefepime, cefoloza-tazobactam, mupirocin, neomycin sulfate bacitracin, polymyxin B, 1-ofloxacin, clindamycin phosphate, gentamicin sulfate, metronidazole, hexylresorcinol, mebenzyl chloride, phenol, quaternized... Compounds, tea tree oil, steroids, such as corticosteroids, such as hydrocortisone, hydroxytriamcinolone alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol propionate, hydroxyprednisolone, desoxymethasone acetate, dexamethasone, diclomethasone, difluralasone acetate, difluralasone valerate, fluocinolone acetonide, fluocinolone acetonide, fluocinolone acetonide, fluocinolone acetate, fluocinolone butyl ester, fluocinolone, fluoride Prednisolone acetate, fluprodoxime, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone, cortisone, todoxacin, fluocinolone acetonide, fluprodoxime, difluralasone diacetate, flutronidazole, methylhydroxysone, ansilafal, ansilafate, betamethasone, chlorprednisolone, chlorprednisolone acetate, chlorcotropin, colocillon, dichloroisocyanurate, difluoroprednisolone, fludichloroisocyanurate, fluoride Nissone, flumethrin, flusperidone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone ester, methylprednisolone, peramisone, prednisolone, prednisone, beclomethasone dipropionate, betamethasone dipropionate, triamcinolone, nonsteroidal anti-inflammatory drugs, such as COX inhibitors, LOX inhibitors, p38 kinase inhibitors, immunosuppressants such as cyclosporine and cytokine synthesis inhibitors, tetracycline, dimethylaminotetracycline and doxycycline or any combination thereof.

[0034] In some embodiments, the method further includes administering one or more antibodies selected from the group consisting of: antibodies targeting Clostridium difficile toxin, antibodies targeting tumor necrosis factor (TNF), antibodies targeting interleukins, and antibodies targeting metalloproteinase-9.

[0035] In some embodiments, the compounds of Formula I reduce the migration of neutrophils to target tissues compared to untreated control tissues. Attached Figure Description

[0036] Figure 1 This is a graph showing the dose-response relationship between BT051 and BT070 in inhibiting neutrophil migration.

[0037] Figure 2A , Figure 2B and Figure 2C . Figure 2AThis is a graph showing the stability of BT090 in simulated intestinal fluid (SIF). Figure 2B This is a graph showing the stability of BT090 in simulated gastric juice (SGF). Figure 2C This is a chart showing the stability of BT090 in feces.

[0038] Figure 3A and Figure 3B It displays BT051 ( Figure 3A ) and BT051 and BT070 ( Figure 3B A graph showing the inhibition of formyl peptide receptor 1 (FPR1), which is measured by inhibiting fMLP-mediated neutrophil migration / activation. Detailed Implementation

[0039] I. Definition

[0040] The following terms are used in this article, and their definitions are for reference only.

[0041] Generally, "substituted" refers to an organic group (e.g., an alkyl group) as defined below, wherein one or more bonds with a hydrogen atom contained therein are replaced by bonds with non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds with a carbon or hydrogen atom are replaced by one or more bonds with a heteroatom, including double or triple bonds. Therefore, unless otherwise stated, a substituted group is substituted by one or more substituents. In some embodiments, the substituted group is substituted by 1, 2, 3, 4, 5, or 6 substituents. Those skilled in the art will understand that the substituted groups of this technology are chemically stable groups that allow for the isolation of compounds in which they appear. Examples of substituent groups include: halogens (i.e., F, Cl, Br, and I); hydroxyl groups; alkoxy, alkenoxy, aryloxy, arylalkoxy, heterocyclic, heterocyclic alkyl, heterocyclic and heterocyclic alkoxy groups; carbonyl (oxo); carboxylates; esters; carbamates; oximes; hydroxyamines; alkoxyamines; arylalkoxyamines; thiols; thioethers; sulfoxides; sulfones; sulfonyl groups; sulfonamides; amines; N-oxides; azides; amides; urea; amidine; guanidine; nitro; nitriles (i.e., CN); and so on.

[0042] Alkyl groups include straight-chain and branched alkyl groups having 1 to 12 carbon atoms (unless otherwise stated), and typically 1 to 10 carbon atoms, or in some embodiments 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups can be substituted or unsubstituted. Examples of straight-chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl. Representative substituted alkyl groups may be substituted once or multiple times with substituents, such as those listed above, and include, but are not limited to, haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxylalkyl, etc. In some embodiments, the alkyl group is substituted with 1, 2, or 3 substituents.

[0043] Alkenyl groups comprise straight-chain and branched alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups can be substituted or unsubstituted. Alkenyl groups have 2 to 12 carbon atoms, typically 2 to 10 carbon atoms, or in some embodiments, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkenyl groups have one, two, or three carbon-carbon double bonds. Examples include, but are not limited to, vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, etc. Representative substituted alkenyl groups can be monosubstituted or substituted more than once, such as, but not limited to, substituted groups, such as the substituents listed above, monosubstituted, disubstituted, or trisubstituted.

[0044] The heteroalkyl group and heteroalkenyl group are alkyl (as defined herein) and alkenyl (as defined herein), respectively, comprising 1 to 6 heteroatoms selected from N, O, and S. It should be understood that each heteroatom present is bonded to at least one carbon atom in the heteroalkyl or heteroalkenyl group. In some embodiments, the heteroalkyl or heteroalkenyl group comprises 1, 2, or 3 heteroatoms. The heteroalkyl and heteroalkenyl groups may be substituted or unsubstituted. Examples of heteroalkyl groups include, but are not limited to, CH3CH2OCH2, CH3NHCH2, CH3CH2N(CH3)CH2, CH3CH2SCH2, and CH3CH2OCH2CH2OCH2CH2. Examples of heteroalkenyl groups include, but are not limited to, CH2=CHOCH2, CH2=CHN(CH3)CH2, and CH2=CHSCH2. The representative substituted heteroalkyl or heteroalkenyl groups may be substituted by substituents, such as those listed above, once or multiple times (e.g., 1, 2 or 3 times), and include, but are not limited to, haloalkyl (e.g., trifluoromethyloxyethyl), carboxylaminoalkyl, methyl acrylate, etc.

[0045] Cycloalkyl groups comprise mono-, di-, or tri-cycloalkyl groups having 3 to 12 carbon atoms in the ring, or in some embodiments, having 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups can be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms is 3 to 5, 3 to 6, or 3 to 7. Bicyclic and tricyclic ring systems comprise both bridging cycloalkyl groups and fused rings, such as, but not limited to, bicyclic [2.1.1]hexane, adamantyl, decahydronaphthyl, etc. Substituted cycloalkyl groups can be substituted once or multiple times with non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also comprise rings substituted with straight-chain or branched alkyl groups as defined above. Representative substituted cycloalkyl groups can be monosubstituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4-, 2,5- or 2,6-disubstituted cyclohexyl groups, which can be substituted by substituents such as those listed above.

[0046] A cycloalkyl group is an alkyl group as defined above, wherein the hydrogen or carbon bond of the alkyl group is replaced by the bond of the cycloalkyl group as defined above. The cycloalkyl group may be substituted or unsubstituted. In some embodiments, the cycloalkyl group has 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. A substituted cycloalkyl group may have its alkyl, cycloalkyl, or alkyl and cycloalkyl portions substituted. Representative substituted cycloalkyl groups may be monosubstituted or substituted more than once, such as, but not limited to, monosubstituted, disubstituted, or trisubstituted by substituents such as those listed above.

[0047] In the compounds described herein, groups having two or more linkage sites (i.e., divalent, trivalent, or polyvalent) are indicated by the suffix "ene". For example, divalent alkyl is alkylene, divalent cycloalkyl is cycloalkylene, divalent heteroalkyl is heteroalkylene, divalent alkenyl is alkenylene, and so on. Substituted groups having a single linkage site in the compounds of this technology are referred to as not having the "ene" designation. Thus, for example, chloroethyl is not referred to herein as chloroethylene.

[0048] The term "administering" a molecule to a subject refers to delivering the molecule to the subject. "Administration" includes prophylactic administration of the composition (i.e., before the disease and / or one or more symptoms of the disease are detectable) and / or therapeutic administration of the composition (i.e., after the disease and / or one or more symptoms of the disease are detectable). Methods of this technology involve administering one or more compounds. If more than one compound is to be administered, these compounds may be administered substantially simultaneously together, and / or in any order at different times. Furthermore, compounds of this technology may be administered before, simultaneously with, or after another type of medicine or treatment procedure (e.g., surgery).

[0049] When referring to any molecule (e.g., multidrug resistance protein 2 (MRP2), multidrug resistance protein 1 (MRP1), hepoxilin A3 (HXA3) synthase, N-acylethanolamine (NAE), amino acid sequence, nucleic acid sequence, antibody, etc.), cellular and / or phenomenon levels (e.g., activity levels of multidrug resistance protein 2 (MRP2) and / or multidrug resistance protein 1 (MRP1) and / or hepoxilin A3 (HXA3) synthase and / or N-acylethanolamine (NAE), gene expression levels, disease symptoms, binding levels of two molecules (such as the binding of a hormone ligand to its hormone receptor), specificity of binding of two molecules, affinity of binding of two molecules, disease symptoms, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc.) in the first sample (or in the first subject) relative to the second sample (or relative to the second subject), the terms “alteration” and “modification” refer to an increase and / or a decrease.

[0050] "Cannabinoid receptor type 2" ("CB2") is a G protein-coupled receptor of the cannabinoid receptor family, encoded by the CNR2 gene in humans. The primary endogenous ligand of the CB2 receptor is 2-arachidonic acid glycerol (2-AG).

[0051] The use of the terms "comprising," "including," or similar terms to describe or define embodiments of compounds, compositions, or methods having one or more elements should be understood to also disclose embodiments "composed of elements" or "essentially composed of elements," and vice versa. In other words, disclosure of embodiments open to elements other than those listed ("comprising") should also be understood to disclose embodiments closed to additional elements ("composed of elements"), or embodiments that may contain only additional elements that do not substantially affect the characteristics of the embodiment ("essentially composed of elements"). Similarly, embodiments composed of or substantially composed of the listed elements should be understood to disclose embodiments that include those elements.

[0052] The term "conjugation" and its grammatical equivalent, when referring to the conjugation of a target molecule and a polymer, means the covalent attachment of the target molecule to the polymer. The attachment can be direct. Alternatively, the attachment can be performed via a linker or partially indirectly. Methods of conjugating with polymers are known in the art, including methods of conjugating peptides to produce fusion proteins (Pasut, Polymers 6:160-178 (2014); Medscape, Nanomedicine 5(6):915-935 (2010)). In some embodiments, the conjugate comprises cyclosporine A conjugated with a PEG polymer. The precursor of this cyclosporine A conjugate comprises a cyclosporine A modified, for example, having a linker group, but without the PEG polymer. The linker group connects the polymer to cyclosporine A.

[0053] As used herein, the terms "effective amount," "therapeutic effective amount," or "pharmaceutically effective amount" refer to an amount sufficient to achieve the desired therapeutic and / or preventative effect, such as an amount that causes complete or partial relief of inflammation (e.g., inflammation associated with neutrophil migration to target tissues) or inflammation-related disease or disorder or symptoms in a subject in need. In the context of therapeutic or preventative application, the amount of composition administered to a subject will vary depending on the type and severity of the disease and individual characteristics such as general health status, age, sex, weight, and tolerance to the drug. It will also depend on the extent, severity, and type of the disease. A skilled technician will be able to determine the appropriate dosage based on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In some embodiments, multiple doses are administered. Additionally or as another option, in some embodiments, multiple therapeutic compositions or compounds are administered. In the methods described herein, a therapeutic compound may be administered to a subject having one or more signs or symptoms of a disease or disorder associated with inflammation (e.g., inflammation associated with increased neutrophil migration into tissues).

[0054] Endocannabinoids (“ECs”) are compounds that bind to cannabinoid receptors CB1 and CB2, as well as the recently described atypical receptors GPR55 and GPR119. The two main classes of arachidic ECs are… N α-Acylethanolamine ("NAE") and monoacylglycerol (MAG) are metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. N "-Acylethanolamine" is an endocannabinoid and a fatty acid amide formed when one of several acyl groups is attached to the nitrogen atom of an ethanolamine. N -Acylethanolamine is metabolized by fatty acid amide hydrolase (FAAH). Example N -Acylethanolamine endocannabinoids include ethanolamine and arachidonic acid amide (AEA) N -Arachidonic ethanolamine), which is arachidonic acid (20:4 ω-6) amide, oleoylethanolamide (OEA) and α-linolenic acid acylethanolamide (α-LEA).

[0055] "Fatty acid amide hydrolase," "FAAH," and "EC 3.5.1.99" interchangeably refer to a member of the serine hydrolase family. It was first shown to break down arachidonic acid. In humans, it is produced by a gene... FAAH coding.

[0056] “Hepoxilin A3 synthase”, “HXA3 synthase”, “ALOX12”, “12-lipoxygenase”, “arachidonic acid 12-lipoxygenase”, “12S-lipoxygenase”, “12-LOX” and “12S-LOX” interchangeably refer to lipoxygenase-type enzymes (i.e., enzymes that catalyze the dioxygenation reaction of polyunsaturated fatty acids in lipids containing cis- or cis-1,4-pentadiene structures), which are encoded in the human body by the ALOX12 gene, which is located together with other lipoxygenases on chromosome 17p13.3.

[0057] When referring to compounds, for example N When referring to α-acylethanolamine, the term "increase" means increase. N - Levels and / or activities of acylethanolamine. When referring to any molecule (e.g., multidrug resistance protein 2 (MRP2), multidrug resistance protein 1 (MRP1), hepoxilin A3 (HXA3) synthase, in the first sample (or in the first subject) relative to the second sample (or relative to the second subject), N -Acylethanolamine (NAE), amino acid and nucleic acid sequences, antibodies, etc.), levels of cells and / or phenomena (e.g., multidrug resistance protein 2 (MRP2) and / or multidrug resistance protein 1 (MRP1) and / or hepoxilin A3 (HXA3) synthase and / or NWhen referring to the activity level of acylethanolamine (NAE), gene expression level, disease symptoms, binding levels of two molecules (such as the binding of a hormone ligand to its hormone receptor), specificity of binding between two molecules, affinity of binding between two molecules, disease symptoms, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc., the terms "increase," "enhance," "improve," and grammatically equivalent forms (including "higher," "greater," etc.) mean that the number of molecules, cells, and / or phenomena in the first sample (or in the first subject) is higher than the number in the second sample (or in the second subject) by any amount that is statistically significant using any statistical analysis method accepted in the art. In one embodiment, the number of molecules, cells, and / or phenomena in the first sample (or the first subject) is at least 10%, at least 25%, at least 50%, at least 75%, and / or at least 90% greater than the number of the same molecules, cells, and / or phenomena in the second sample (or the second subject). This includes, but is not limited to, a quantity of molecules, cells, and / or phenomena in the first sample (or in the first subject) that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and / or at least 95% greater than the quantity of the same molecules, cells, and / or phenomena in the second sample (or in the second subject). In one embodiment, an example of the first sample (or the first subject) is, but is not limited to, a sample (or subject) manipulated using compositions and / or methods of the present technology. In another embodiment, an example of the second sample (or the second subject) is, but is not limited to, a sample (or subject) manipulated without using compositions and / or methods of the present technology. In alternative embodiments, an example of a second sample (or a second subject) is, but not limited to, a sample (or subject) manipulated using the compositions and / or methods of the present technology at a different dose and / or for a different duration and / or via a different route of administration than the first subject. In one embodiment, the first and second samples (or subjects) may be the same, with the effects of different schemes of the compositions and / or methods of the present technology (e.g., dose, duration, route of administration, etc.) intended to be determined on one sample (or subject). In another embodiment, the first and second samples (or subjects) may be different, such as when comparing the effects of the compositions and / or methods of the present technology on one sample (subject), for example, a patient participating in a clinical trial and another individual in a hospital.

[0058] The term "inhibition" in the context of binding compounds, such as multidrug resistance protein 2 (MRP2), hepoxilin A3 (HXA3) synthase, etc., refers to the inhibition of the activity and / or level of HXA3. When referring to any molecule (e.g., multidrug resistance protein 2 (MRP2), multidrug resistance protein 1 (MRP1), hepoxilin A3 (HXA3) synthase, etc., in the first sample (or in the first subject) relative to the second sample (or relative to the second subject), it is used in this context. N -Acylethanolamine (NAE), amino acid and nucleic acid sequences, antibodies, etc.), levels of cells and / or phenomena (e.g., multidrug resistance protein 2 (MRP2) and / or multidrug resistance protein 1 (MRP1) and / or hepoxilin A3 (HXA3) synthase and / or NWhen considering the activity level of acylethanolamine (NAE), gene expression level, disease symptoms, binding levels of two molecules (such as the binding of a hormone ligand to its hormone receptor), specificity of binding between two molecules, affinity of binding between two molecules, disease symptoms, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc., the terms “inhibition,” “reduction,” “decrease,” and grammatically equivalents (including “lower,” “smaller,” etc.) refer to any amount by which the number of molecules, cells, and / or phenomena in the first sample (or in the first subject) is lower than the number in the second sample (or in the second subject), said amount being statistically significant using any statistical analysis method accepted in the art. In one embodiment, the number of molecules, cells, and / or phenomena in the first sample (or the first subject) is at least 10%, at least 25%, at least 50%, at least 75%, and / or at least 90% lower than the number of the same molecules, cells, and / or phenomena in the second sample (or the second subject). In another embodiment, the number of molecules, cells, and / or phenomena in the first sample (or the first subject) is 5% to 100% lower than the number of the same molecules, cells, and / or phenomena in the second sample (or the second subject), such as, but not limited to, any numerical percentage of 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, and 90% to 100%. In one embodiment, an example of the first sample (or the first subject) is, but not limited to, a sample (or subject) manipulated using compositions and / or methods of the present technology. In another embodiment, an example of the second sample (or the second subject) is, but not limited to, a sample (or subject) manipulated without using compositions and / or methods of the present technology. In an alternative embodiment, an example of the second sample (or the second subject) is, but not limited to, a sample (or subject) manipulated using compositions and / or methods of the present technology at a different dose and / or for a different duration and / or via a different route of administration than the first subject. In one embodiment, the first and second samples (or subjects) may be the same, such as the effects of different regimens of the composition and / or method of the present technology (e.g., dosage, duration, route of administration, etc.) are intended to be determined on one sample (or subject). In another embodiment, the first and second samples (or subjects) may be different, such as when comparing the effects of the composition and / or method of the present technology on one sample (subject), for example, a patient participating in a clinical trial and another individual in a hospital.

[0059] "Multidrug resistance-associated protein 2" (MRP2), "tubule multispecific organic anion transporter 1" (cMOAT), and "ATP-binding cassette subfamily C member 2" (ABCC2) are interchangeable terms referring to proteins in humans that are associated with multidrug resistance-associated protein 2 (MRP2), "multidrug resistance protein 2" (cMOAT), and "tubule multispecific organic anion transporter 1" (ABCC2). ABCC2 Proteins encoded by genes.

[0060] “Multidrug Resistance Protein 1,” “MRP1,” and “ABCC1” are used interchangeably to refer to a single-way efflux transporter with broad substrate specificity, containing important therapeutic drugs. Some of the main functions of this transporter are: (i) efflux of exogenous and endogenous metabolites; (ii) transport of inflammatory mediators (e.g., LTC4); and (iii) defense against oxidative stress. The 190-kDa MRP1 has a core structure consisting of two transmembrane domains (TMDs), each followed by a nucleotide-binding domain (NBD). Like MRP2, 3, 6, and 7, MRP1 contains a third TMD (TMD0) with five predicted trans-membrane segments and an additional cytoplasmic NH2 terminus (Rosenberg) linked to the core structure via a linker region (L0). et al ., J. Biol.Chem. 276(19):13076-16082 (2001)). TMD0 appears to be important for the transport of MRP1 to the plasma membrane (Bakos et al ., J. Cell Sci .113(Pt 24):4451-4461 (2000)), and the precise role, mechanism, and dependence of TMD0 and L0 are important research topics (Westlake) et al . Mol. Biol. Cell 16(5):2483-2492(2005)). MRP1 exhibits broad substrate specificity, transporting hydrophobic and anionic molecules, glucuronides and glutathione conjugates, as well as endogenous glutathione. Although many MRP1 substrates are conjugated with glutathione, cotransport of free glutathione is frequently observed, and it appears to stimulate the transport of, for example, vincristine and daunorubicin (Hooijberga). et al ., FEBS Letters 469:47-51(2000)). Glutathione itself is a low-affinity substrate of MRP1 (Km = 1-5 mM). The assumption of multiple allosteric, cooperating, non-overlapping substrate binding sites could explain why various substrates both cross-inhibit and cross-stimulate (Bakos). et al ., Pflugers Arch - Eur J Physiol453:621-641 (2007). The inflammatory cytokine LTC4 and its major metabolite LTD4 are some of the most affinity substrates for MRP1, suggesting that MRP1 plays a crucial role in the release of cytokines from LTC4-producing cells. Indeed, intracellular LTC4 accumulation has been observed in mrp1 (- / -) mice (Robbiani). et al ., Cell 103:757-768 (2000)). Furthermore, although mrp1 knockout (- / -) mice survive, are healthy, fertile, and phenotypically normal, they are highly sensitive to cytotoxic drugs (Wijnholds). et al ., Nat. Med 3:1275-1279 (1997)). An example of MRP1 is the human protein sequence encoded by the DNA sequence NCBI reference sequence: NG_028268.1 NCBI reference sequence: NP_004987.2. At least 15 naturally occurring mutations have been identified in MRP1, many of which have been found to affect its in vitro transport activity. Polymorphism and mutagenesis studies have been conducted in He... et al ., Curr. Med. Chem. A review was conducted in 18:439-481 (2011). Although many MRP1 SNPs are known, their incidence in the population has been reported to be relatively low. In some populations, the allele frequencies of MRP1 polymorphisms Cys43Ser (128G>C), Thr73Ile (218C>T), Arg723Gln (2168G>A), and Arg1058Gln (3173G>A) were 0.5%, 1.4%, 5.8%, and 0.5%, respectively (Ji-YeYin). et al ., Pharmacogenet.Genomics 19(3):206-216 (2009)).

[0061] "P-glycoprotein" ("P-gp") is an efflux membrane transporter responsible for restricting cellular uptake and distribution of exogenous and toxic substances.

[0062] Pharmaceutically acceptable salts of the compounds described herein are within the scope of this technology and comprise acid or base addition salts that retain the desired pharmacological activity and are not biologically undesirable (e.g., the salt is not excessively toxic, allergenic, or irritating, and is bioavailable). When the compounds of this technology have a basic group, such as an amino group, pharmaceutically acceptable salts can be formed from inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid), or acidic amino acids (such as aspartic acid and glutamic acid). When the compounds of this technology have an acidic group, such as, for example, a carboxylic acid group, they can form salts with metals, such as alkali metals and alkaline earth metals (e.g., Na₂O₃). + Li + K + Ca 2+ Mg 2+ Zn 2+ Ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, methylpyridine, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine, and ornithine). Such salts can be prepared in situ during the separation and purification of the compound, or by reacting the purified compound, in its free base or free acid form, with a suitable acid or base, and then separating the salt thus formed.

[0063] A "polymer" is a substance with a molecular structure, which is mainly or entirely composed of a large number of similar units bonded together. Polymers can be naturally occurring (e.g., cellulose, polypeptides, nucleotide sequences, etc.) or artificial (e.g., plastics, resins, etc.). Polymers can be used as carriers for drugs conjugated with them, improving the solubility of the conjugated drug, enhancing its pharmacokinetic characteristics, protecting the drug from degradation, and releasing the drug under certain conditions, such as changes in pH or the presence of enzymes, such as esterases, lipases, or proteases. Furthermore, targeting portions or solubilizers can also be introduced into the conjugate to improve its therapeutic index (Medscape). Nanomedicine5(6):915-935(2010)). Polymers can also be used to restrict the distribution of drugs conjugated with them, for example by preventing the conjugated drugs from entering specific body sites (e.g., from the gastrointestinal lumen to underlying tissues). Polymers can be natural polymers and / or synthetic linear polymers and contain polyethylene glycol (PEG), dextran, periodate-oxidized dextran, polysialic acid (PSA), hyaluronic acid (HA), dextrin, hydroxyethyl starch (HES), poly(2-ethyl-2-oxazoline) (PEOZ), polyglutamic acid (PGA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolic acid) (PLA / PLGA), poly(hydroxyalkylmethylacrylamide), polyglycerol, 25% polyaminoamide (PAMAM), polyethyleneimine (PEI), and peptides.

[0064] “SipA” and “Salmonella T3SS effector protein” are used interchangeably to refer to proteins produced by Salmonella. An example is the amino acid sequence of serotype subspecies *Salmonella typhimurium* strain SL1344 (GenBank: AAA86618.1), encoded by the DNA sequence (Locus taq) of serotype subspecies *Salmonella typhimurium* strain SL1344_2861 (NCBI reference sequence: NC_016810.1). The SipA sequence is provided in WO 2015 / 089268.

[0065] Target tissues that may be inflamed include, but are not limited to, epithelial tissues and mucous membranes. Examples of epithelial and / or mucosal tissues include the gastrointestinal tract, lungs (e.g., bronchial tissue), liver, stomach, colon, brain, gallbladder, kidneys, female reproductive tract, eyes, urinary tract, etc., leading to "inflammatory diseases," such as intestinal diseases (examples of which are proctitis, orchitis, Crohn's disease, colitis (such as ulcerative colitis, also known as ulcerative colitis), infectious / non-infectious enterocolitis, inflammatory bowel disease (IBD), etc.), inflammatory lung diseases (such as pneumococcal infection, asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis), inflammatory skin diseases (such as dermatitis (eczema), rosacea, seborrheic dermatitis, and psoriasis), eye diseases (examples of which are uveitis, retinitis, keratitis, macular degeneration, etc.), genitourinary diseases (such as urinary tract infections), sexually transmitted diseases (such as pelvic inflammatory disease, including inflammatory diseases exemplified by gonorrhea and / or chlamydia infection, and ulcerative diseases exemplified by herpes), urethritis, etc. The term "target tissue" as used in this article also includes anatomical spaces, such as the intestinal lumen.

[0066] As used herein, “treating (treat, treated, or treatment)” encompasses treatment of a subject, such as a disease or disorder (e.g., inflammation) described herein in humans, and includes: (i) suppressing the disease or disorder, i.e., hindering its development; (ii) alleviating the disease or disorder, i.e., leading to the remission of the disorder; (iii) slowing the progression of the disorder; and / or (iv) suppressing, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. Symptoms can be assessed using methods known in the art, such as biopsy and histology, as well as blood tests to determine relevant enzyme levels, metabolites or circulating antigens or antibodies (or other biomarkers), quality of life questionnaires, patient-reported symptom scores, and imaging tests.

[0067] As used in this article, "prevention" or "preventing" refers to a compound that, in a statistical sample, reduces the occurrence of a disorder or condition in the treated sample relative to a control sample, or delays the onset of one or more symptoms of a disorder or condition relative to a control sample.

[0068] It should also be understood that the various modes of treatment or prevention of medical diseases and conditions described are intended to mean “substantial”, which include all but less than all treatments or preventions, and in which some biological or medically relevant results are achieved.

[0069] As used herein, the terms “subject,” “individual,” or “patient” can refer to an individual organism, a vertebrate, a mammal, or a human. “Mammalian” includes humans, non-human primates, mice (e.g., mice, rats, guinea pigs, hamsters), sheep, cattle, ruminants, rabbits, pigs, goats, horses, dogs, cats, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0070] Subjects “requiring” treatment according to the methods and / or compositions of this technology include subjects “having” inflammation (i.e., subjects experiencing and / or exhibiting clinical and / or subclinical symptoms of one or more types of inflammation) and subjects “at risk of inflammation.” Subjects “requiring” treatment include animal models of inflammation. Subjects “at risk of inflammation” are those who do not currently exhibit inflammatory symptoms but are predisposed to exhibiting symptoms of one or more diseases. This predisposition may be based on family history, genetic factors, environmental factors such as exposure to harmful compounds present in the environment, etc. The intention is not to limit this technology to any particular sign or symptom. Therefore, the intention is that this technology covers subjects experiencing any range of diseases from subclinical symptoms to full-blown inflammatory disease, wherein the subject exhibits at least one marker (e.g., signs and symptoms) associated with inflammatory disease.

[0071] When referring to any molecule (e.g., multidrug resistance protein 2 (MRP2), multidrug resistance protein 1 (MRP1), hepoxilinA3 (HXA3) synthase), N When describing the levels of N-acylethanolamine (NAE), amino acid sequences, nucleic acid sequences, antibodies, etc., cells, and / or phenomena (e.g., the activity levels of multidrug resistance protein 2 (MRP2) and / or multidrug resistance protein 1 (MRP1) and / or hepoxilin A3 (HXA3) synthase and / or N-acylethanolamine (NAE), gene expression levels, disease symptoms, the binding levels of two molecules (such as the binding of hormone ligands to their hormone receptors), the specificity of the binding of two molecules, the affinity of the binding of two molecules, disease symptoms, specificity to the disease, sensitivity to the disease, affinity of binding, enzyme activity, etc.), the terms "substantially the same," "substantially unchanged," "substantially unchanged," and grammatical equivalences mean that, relative to the second sample (or the second subject), the number of molecules, cells, and / or phenomena in the first sample (or the first subject) has neither increased nor decreased by a statistically significant amount. Therefore, in one embodiment, the number of molecules, cells and / or phenomena in the first sample (or in the first subject) is 90% to 100% of the number in the second sample (or in the second subject) (including, for example, 91% to 100%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100% and / or 99% to 100%).

[0072] As used herein, unless otherwise specifically stated otherwise, “percentage by weight” for a component is based on the total weight of the formulation or composition containing the component.

[0073] II. Overview

[0074] On one hand, this technology provides methods, compounds, and compositions for inhibiting formyl peptide receptor 1 (FPR1) and for treating diseases associated with FPR1 activation. In some embodiments, diseases associated with FPR1 activation include celiac disease.

[0075] On one hand, this technology provides methods, compounds, and compositions for treating neutrophil-mediated inflammation and diseases associated with neutrophil-mediated inflammation. Specifically, this technology provides a method for treating neutrophil-mediated inflammation in target tissues of a mammalian subject in need, comprising administering to the subject a therapeutically effective amount of one or more first compounds that increase the level and / or activity of multidrug resistance protein 1 (MRP1), wherein the therapeutically effective amount of the first compound reduces neutrophil migration to the target tissue, and / or administering a therapeutically effective amount of one or more second compounds that inhibit one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, wherein the therapeutically effective amount of the second compound reduces neutrophil migration to the target tissue, and / or administering a therapeutically effective amount of one or more third compounds that increase one or more N-acylethanolamines (NAE), wherein the therapeutically effective amount of the third compound reduces neutrophil migration to the target tissue.

[0076] In one embodiment, this disclosure provides a method for treating neutrophil-mediated inflammation by targeting the pro-inflammatory MRP2 / HXA3 pathway, comprising administering to a subject a therapeutically effective amount of one or more compounds that inhibit the activity and / or level of one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, wherein the therapeutically effective amount of said compound reduces neutrophil migration to target tissues.

[0077] In another embodiment, this disclosure also provides a method for treating neutrophil-mediated inflammation by targeting the anti-inflammatory P-gp / endocannabinoid pathway, comprising administering to a subject a therapeutically effective amount of one or more compounds that increase the level and / or activity of one or more N-acylethanolamines (NAEs), wherein the therapeutically effective amount of said compound reduces neutrophil migration to target tissues.

[0078] In another embodiment, this disclosure further provides a method for treating neutrophil-mediated inflammation, comprising administering to a subject a therapeutically effective amount of one or more second compounds that increase the level and / or activity of multidrug resistance protein 1 (MRP1), wherein the therapeutically effective amount of said compound reduces neutrophil migration to target tissues.

[0079] In yet another embodiment, this disclosure provides a method for treating neutrophil-mediated inflammation by targeting anti-inflammatory P-gp / endocannabinoid and pro-inflammatory MRP2 / HXA3 pathways, the method comprising administering to a subject a therapeutically effective amount of (A) one or more first compounds that inhibit the activity and / or level of one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, and (B) one or more second compounds that increase the level and / or activity of one or more N-acylethanolamines (NAE), wherein the therapeutic amounts of the first and second compounds reduce neutrophil migration to target tissues.

[0080] III. Compounds of this technology

[0081] This technology provides compositions for treating neutrophil-mediated inflammation and related conditions. In some embodiments, this technology provides compositions comprising one or more first compounds that increase the level and / or activity of multidrug resistance protein 1 (MRP1), a second compound that inhibits one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, and / or a third compound that increases one or more N-acylethanolamines (NAE).

[0082] In some embodiments, this technology discloses cyclosporine A-polymer conjugates and their precursors as defined by Formula I: (I) Its stereoisomers or pharmaceutically acceptable salts of any of the aforementioned substances. In Formula I, L1 can be a C0 to C4 alkylene group optionally substituted with one or more F groups. For example, in some embodiments, L1 can be methylene, ethylene, propylene, or butylene. In some embodiments, L1 can be C0. In some embodiments, L1 can be a C1-C4 fluoroalkylene group. The fluoroalkylene group can have 1, 2, 3, 4, or more F groups and / or can be perfluorinated. In Formula I, R can be -OH or a polymer, optionally containing a linker group L2.

[0083] In some embodiments, the polymer of R is selected from the group consisting of: dextran, polyethylene glycol (PEG), periodate-oxidized dextran, polysialic acid (PSA), hyaluronic acid (HA), dextrin, hydroxyethyl starch (HES), poly(2-ethyl-2-oxazoline) (PEOZ), polyglutamic acid (PGA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolic acid) (PLA / PLGA), poly(hydroxyalkylmethylacrylamide), polyglycerol, 25% polyaminoamine (PAMAM), polyethyleneimine (PEI), and polypeptides. In some embodiments, the polymer is PEG. PEG polymers may be functionalized with amines (NH2) and / or aldehydes (CHO), including linear monoamines and monoaldehydes, linear diamines and dialdehydes, multi-armed amines and multi-armed aldehydes, branched mono, di, and multi-armed amines and aldehydes, and multi-armed forked amines and aldehydes. In some embodiments, the polymer is a PEG having 40-50 ethylene oxide subunits, i.e., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ethylene oxide subunits, or a range between any two of the above values ​​and including said values. The polymer may have any molecular weight as described herein.

[0084] In some embodiments, the polymer has an average molecular weight in the range of about 100 kDa to about 800 kDa. (Unless otherwise stated, "average molecular weight" refers to weight average molecular weight.) In some embodiments, the polymer has an average molecular weight in the range of about 1 kDa to about 800 kDa. In some embodiments, the polymer has an average molecular weight of less than 1 kDa. In some embodiments, the polymer has an average molecular weight of less than 10 kDa. In some embodiments, the average molecular weight of the polymer is about 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 125 kDa, 150 kDa, 175 kDa, 200 kDa, 225 kDa, 250 kDa, 275 kDa, 300 kDa, 325 kDa, 350 kDa, 375 kDa, 400 kDa, 425 kDa, 450 kDa, 475 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, or any range between two of these values ​​and includes said values.

[0085] The polymers described herein can have a variety of different geometries. For example, in some embodiments, the polymer is a linear polymer, a branched polymer, a forked polymer, or any combination of these polymers.

[0086] As described above, the R group in the compound of formula I (or IA) optionally includes a linker L2. In some embodiments, the linker L2 is a biodegradable linker. In some embodiments, the biodegradable linker comprises an oligopeptide having 2 to 10 amino acid residues. The residues may be selected from naturally occurring amino acids.

[0087] In some embodiments, the linker L2 includes substituted or unsubstituted C1-C X Alkylene, cycloalkylene, cycloalkylalkylene, heteroalkylene, alkenyl, or heteroalkenyl, wherein x can be any integer from 1 to 12, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. For example, L2 can include C1-Cx fluoroalkyl groups, wherein one or more hydrogen atoms are fluorine atoms, such as 1, 2, or 3 or more fluorine atoms. In some embodiments, L2 can be a heteroalkylene group containing one or two NH groups, including but not limited to (C1-Cx) fluoroalkyl groups. 10 (e.g., CH2CH2NH, CH2CH2CH2NH, CH2CH2CH2CH2NH, CH2CH(CH3)CH(CH3)CH2NH), (C n alkylene)NH(C p Alkylene), where n and p are independent integers from 1 to 10, but n + p does not exceed 10 (e.g., CH2CH2CH2NHCH2CH2), NH-(C1-C 10 Alkylene)NH (e.g., NH(CH2)5NH, NH(CH2)6NH, NH(CH2)8NH) or NH(C n alkylene)NH(C p Alkylene), where n and p are integers as defined above (e.g., NHCH2CH2CH2NHCH2CH2, NH(CH2)6NHCH2). In some embodiments, L2 may be a heteroalkylene containing one or two oxygen atoms, including but not limited to (C1-C1) 10 Alkylene)-O (e.g., CH2CH2O, CH2CH2CH2O, CH2CH2CH2CH2O, CH2CH(CH3)CH(CH3)CH2O), (C n Alkylene)O(C p Alkylene), where n and p are independent integers from 1 to 10, but n + p does not exceed 10 (e.g., CH2CH2CH2OCH2CH2), O-(C1-C 10 Alkylene)O (e.g., O(CH2)5O, O(CH2)6O, O(CH2)8O) or O(C n Alkylene)O(C pAlkylene), where n and p are integers as defined above (e.g., OCH2CH2CH2OCH2CH2, O(CH2)6OCH2). In some embodiments, L2 may be a heteroalkylene containing O and NH groups, including but not limited to NH-(C1- ... 10 Alkylene O (e.g., NH(CH2)5O, NH(CH2)6O, NH(CH2)8O) or NH(C n Alkylene)O(C p Alkylene), where n and p are integers as defined above (e.g., NHCH2CH2OCH2CH2, O(CH2)6NHCH2).

[0088] In some embodiments of compounds of formula I (including formula IA), R is -OH or a PEG group having 40 to 50 ethylene oxide units and includes a linker L2, which is a substituted or unsubstituted heteroalkylene group, such as an unsubstituted aminoalkylene group. In some embodiments, R is selected from -OH or -NH(CH2). 2-6 (CH2CH2O) 42-46 -O(CH2) 0-5 CH3. In some embodiments, R is selected from CH3O-(CH2CH2O). 44 -CH2CH2CH2NH-(BT051), CH3O-(CH2CH2O) 44 The group consisting of -CH2CH2NH-(BT090) and HO-(BT070). In any such embodiment, m can be 2 and has the structure of formula IA.

[0089] Cyclosporine A-polymer conjugates and precursors can be prepared using standard techniques known in the art. In some embodiments, a bifunctional linker containing at least two functional groups with heteroatoms selected from N, O, and S, one of which is protected, can be conjugated using standard ester, thioester, and amide bond formation techniques. For example, a diamino-alkylene linker, in which one amino group is protected by a urethane protecting group (e.g., Boc. Cbz, etc.), can be coupled to cyclosporine A in the presence of a coupling agent (e.g., DCC, EDC / HOBt, etc.). Alternatively, an active ester, mixed anhydride, or acyl halide derivative of cyclosporine A can be prepared and reacted with a monoprotected diamine. (See, for example, Bodansky, M. & Bodanszky, A., The Practice of Peptide Synthesis(Springer-Verlag, New York, 1984.) The protecting group can be removed, and the free amine reacts with the aldehyde derivative of the polymer under reducing conditions to provide a conjugated product. Similarly, a linker with a protected aldehyde (e.g., 1,1-dimethoxy) and an amine can be coupled to cyclosporine A, deprotected to form an aldehyde, and reductively amination with an amino-containing polymer to form a conjugated product. Variations of these schemes using α,ω-carboxyamines, α,ω-amino alcohols, α,ω-carboxyl alcohols, α,ω-aminothiols, etc., to link cyclosporine A and the polymer will be readily understood by those skilled in the art.

[0090] In some embodiments, the cyclosporine A compound of this technology is used in combination with one or more compounds that increase (multidrug resistance protein 1) MRP1 for the treatment of inflammatory diseases.

[0091] In some embodiments, the cyclosporine A compound of this technology is used in combination with one or more multidrug resistance protein 2 (MRP2) inhibitors to treat inflammatory diseases. In some embodiments, the MRP2 inhibitor is selected from the group consisting of: MRP2RNAi; 3-([3-(2-[7-chloro-2-quinolinyl]vinyl)phenyl-(3-dimethylamino-3-oxopropyl)-thio-methyl]thio)propionic acid (also known as "MK571" and CysLT1 (LTD4) leukotriene receptor inverse agonist) (Tocris, Minneapolis, USA) (Genuuso et al. (2004) PNAS 101:2470-2475); probenecid (also known as "PROBALAN™"), with probenecid inhibiting MRP2 as an example; FUROSEMIDE®; RITONAVIR®; SAQUINAVIR®; LAMIVUDINE®; ABACAVIR®; EMTRICITABINE®; EFAVIRENZ®; DELAVIRDINE®; NEVIRAPINE®; CIDOFOVIR®; ADEFOVIR®; and TENOFOVIR®. In some embodiments, the compound is conjugated with the polymer.

[0092] In some embodiments, the compound that inhibits MRP2 includes one or more compounds that inhibit Hepoxilin A3 synthase, such as Hepoxilin A3 synthase RNAi. In some embodiments, the compound is conjugated with the polymer.

[0093] In some embodiments, the compound that inhibits MRP2 includes one or more compounds that inhibit fatty acid amide hydrolases (FAAH), such as FAAH RNAi; FAAH inhibitor I (PubChem CID: 295380) 4-phenylmethoxyphenyl N3'-Butyl carbamate); URB597 (PubChem CID: 1383884) 3'-Carbamoyl-[1,1'-Biphenyl]-3-ylcyclohexyl carbamate; FAAH inhibitor 1 (PubChem CID: 1190414) N- (4-(6-Methylbenzo[d]thiazolyl)phenyl)-1-(thiophene-2-ylsulfonyl)piperidine-4-carboxamide; FAAH inhibitor, 2l (PubChem CID: 71699786); FAAH inhibitor, 2i (PubChem CID: 71699785) N 4-(dimethylamino)-3-phenylphenyl cyclohexylcarbamate; FAAH inhibitor, 2h (PubChem CID: 71699784) N4-(hydroxymethyl)-3-phenylphenyl ester of cyclohexylcarbamate; FAAH inhibitor, 2j (PubChem CID: 58801136); FAAH inhibitor, 2e (PubChem CID: 58801135); FAAH inhibitor, 2a (PubChem CID: 58801134); FAAH inhibitor, 2b (PubChem CID: 58801129); FAAH inhibitor, 2f (PubChem CID: 58801126) 4-(hydroxymethyl)-3-phenylcarbamate; FAAH inhibitor, 2k (PubChem CID: 58801125); FAAH inhibitor, 2c (PubChem CID: 57582480); FAAH inhibitor, 2g (PubChem CID: 57582480) 44626363); FAAH inhibitor, 2d (PubChem CID:44626362); AM374, palmitoyl sulfonyl fluoride; ARN2508, flurbiprofen derivative; BIA 10-2474; BMS-469908; CAY-10402; JNJ-245; JNJ-1661010; JNJ-28833155; JNJ-40413269; JNJ-42119779; JNJ-42165279; LY-2183240; cannabidiol; MK-3168; MK-4409; M M-433593; OL-92; OL-135; PF-622; PF-750; PF-3845; PF-04457845; PF-04862853; RN-450; SA-47; SA-73; SSR-411298; ST-4068; TK-25; URB524; URB597 (KDS-4103, Kadmus Pharmaceuticals); URB694; URB937; VER-156084; V-158866; and multiple FAAH inhibitors, derived from ChemCruz® Biochemicals, Dallas, Texas. In some embodiments, the compounds are conjugated with the polymer.

[0094] In some embodiments, the compounds that inhibit MRP2 include one or more compounds that inhibit P-glycoprotein (P-gp), such as P-gp RNAi; SipA; and small molecules (e.g., zoquindal trihydrochloride (LY335979); VALSPODAR® (PSC833) (an inhibitor of P-gp-mediated MDR); CP 100356 hydrochloride (Sigma-Aldrich); and ectidone hydrochloride (R&D Systems). See also WO 2004071498 A1; WO 2014106021 A1; WO 2005033101 A1; WO2004009584 A1; WO 2002030915 A2; US 20100029755 A1; and US 20060073196 A1). In some embodiments, the compounds are conjugated with a polymer.

[0095] In some embodiments, cyclosporine A compounds of the present technology (including, but not limited to, compounds of formulas I and IA) are used in combination with one or more compounds that increase N-acylethanolamine (NAE) for the treatment of inflammatory diseases. In some embodiments, the NAE-increasing compounds are cannabinoid receptor type 2 (CB2) "agonists" (i.e., compounds that specifically bind to and activate CB2). Illustrative CB2 agonists include GW-405,833; AM-1241; HU-308; JWH-015; JWH-133; L-759,633; L-759,656; β-caryophyllene; arachidonicylcyclopropylamide; and arachidonic-2'-chloroethylamide. In some embodiments, the compounds are conjugated with polymers.

[0096] IV. Uses of this technical composition

[0097] This technology provides a method for treating, preventing, or improving neutrophil-mediated inflammation in target tissues of mammalian subjects in need, comprising administering to the subject a therapeutically effective amount of one or more first compounds that inhibit one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, wherein the therapeutically effective amount of the first compound reduces neutrophil migration to the target tissue. In some embodiments, the first compound is a cyclosporine A conjugate or a precursor thereof, such as, for example, a compound of formula I or IA. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more compounds that increase one or more NA second compound of -acylethanolamine (NAE), wherein a therapeutic amount of the second compound reduces neutrophil migration to the target tissue. In another embodiment, the method further includes administering to the subject a therapeutically effective amount of one or more second and / or third compounds that increase the level and / or activity of multidrug resistance protein 1 (MRP1), wherein a therapeutic amount of one or more second and / or third compounds reduces neutrophil migration to the target tissue. In another embodiment, the compounds of this technology are applied alone or in combination to a local surface and / or a luminal surface of the target tissue. In another embodiment, a first compound that reduces neutrophil migration to the target tissue is conjugated with a polymer. In another embodiment, the inflammation is non-infectious and / or infectious inflammation.

[0098] This technology also provides methods for treating, improving, or preventing neutrophil-mediated inflammation in target tissues of mammalian subjects in need, including administering a therapeutically effective amount of one or more of the following methods to the subject: N A first compound of acylethanolamine (NAE), wherein a therapeutic amount of the first compound reduces neutrophil migration to target tissues. In one embodiment, the method further includes administering to the subject a therapeutically effective amount of one or more second compounds that inhibit one or more multidrug resistance protein 2 (MRP2) and HXA3 synthases, wherein the therapeutically effective amount of the second compound reduces neutrophil migration to target tissues. In some embodiments, the second compound is a cyclosporine A conjugate or a precursor thereof, such as, for example, a compound of formula I or IA. In another embodiment, the method further includes administering to the subject a therapeutically effective amount of one or more second and / or third compounds that increase the level and / or activity of multidrug resistance protein 1 (MRP1), wherein the therapeutically effective amount of the one or more second and / or third compounds reduces neutrophil migration to target tissues. In another embodiment, the one or more first compounds that increase the one or more NAEs are cannabinoid receptor type 2 (CB2) agonists. In another embodiment, the first compound that reduces neutrophil migration to target tissues is conjugated with a polymer.

[0099] On the one hand, the methods, compounds, and compositions of this technology relate to cyclosporine A-polymer conjugates and precursors as defined by Formula I and their stereoisomers, as well as pharmaceutically acceptable salts of any of the aforementioned substances:

[0100] Wherein L1 and R can be as defined herein and in one or more uses of these compounds (including, but not limited to, formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) to treat, improve, or prevent neutrophil-mediated inflammation in target tissues in subjects of need. In other embodiments, the compounds of formulas I and IA, in combination with one or more compounds (e.g., compounds that increase MRP1 levels and / or activity, or compounds that increase NAE), will show synergistic effects in this regard.

[0101] In some embodiments, the methods, compounds, and compositions of this technology relate to the use of one or more cyclosporine A compounds of formulas I and IA to treat, improve, or prevent inflammatory bowel disease (IBD), such as ulcerative colitis (UC), Crohn's disease (CD), and infectious / non-infectious enterocolitis. In other embodiments, compounds of formula I (including, but not limited to, formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) in combination with one or more compounds (e.g., compounds that increase MRP1 levels and / or activity, or compounds that increase NAE) will exhibit synergistic effects in this regard.

[0102] In some embodiments, the methods and compositions of this technology involve using one or more compounds of Formula I to treat, improve, or prevent infectious and non-infectious inflammatory lung diseases, including but not limited to pneumococcal infection, asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. In other embodiments, the combination of cyclosporine A compounds with one or more compounds (e.g., compounds that increase MRP1 levels and / or activity, or compounds that increase NAE) will show synergistic effects in this regard.

[0103] In some embodiments, the methods, compounds, and compositions of this technology relate to the use of one or more cyclosporine A compounds of Formula I to treat, improve, or prevent inflammatory skin conditions, including but not limited to dermatitis (eczema), rosacea, seborrheic dermatitis, and psoriasis. In other embodiments, the combination of cyclosporine A compounds with one or more compounds (e.g., compounds that increase MRP1 levels and / or activity, or compounds that increase NAE) will show synergistic effects in this regard.

[0104] The methods of this technique can be used to treat "inflammation," a localized bodily condition in which a part of the body responds to injury and / or infection. Typical symptoms of inflammation are fever, redness, swelling, pain, and / or loss of function. These are manifestations of the physiological changes that occur during the inflammatory process. The three main components of this process are: (1) changes in the diameter of blood vessels and the velocity of blood flow through them (hemodynamic changes); (2) increased capillary permeability; and (3) leukocyte exudation. "Neurotrophic inflammation" refers to the leukocyte exudation and inflammatory phase in which neutrophils migrate into the endothelial lining of small blood vessels (marginalization) and arrange themselves in a close-packed manner (paving). Eventually, these neutrophils escape through the endothelial space into the extravascular space (migration). Once they leave the blood vessels, they move freely and are attracted to the site of injury by chemotaxis. The accumulation of neutrophils (and macrophages) in the inflammatory area neutralizes foreign particles through phagocytosis.

[0105] Inflammation includes acute inflammation, which is usually sudden in onset and characterized by typical signs of fever, redness, swelling, pain, and loss of function, in which vascular and exudative processes dominate; catarrhal inflammation, which is a form that primarily affects the mucosal surface and is characterized by the discharge of large amounts of mucus and epithelial debris; chronic inflammation, which is a long-term, persistent inflammation that is primarily characterized by the formation of new connective tissue; it can be a continuation of an acute form or a long-term, lower-level form; interstitial inflammation, which is inflammation that primarily affects the interstitial tissue of an organ; traumatic inflammation, which is inflammation following a wound or injury; and ulcerative inflammation, in which necrosis on or near the surface leads to tissue loss and the formation of local defects (ulcers).

[0106] Inflammation can be infectious or non-infectious. "Infectious" inflammation refers to inflammation associated with and / or caused by the invasion and proliferation of microorganisms such as bacteria, viruses, and parasites that are not normally present in the body. In contrast, "non-infectious" inflammation refers to inflammation that is not related to and / or not caused by the invasion and proliferation of microorganisms such as bacteria, viruses, and parasites that are not normally present in the body.

[0107] In another embodiment, the present technology provides a method for treating neutrophil-mediated inflammation by targeting the pro-inflammatory MRP2 / HXA3 pathway. In a particular embodiment, such a method for treating neutrophil-mediated inflammation in a target tissue of a mammalian subject in need includes administering to the subject a therapeutically effective amount of one or more first compounds that inhibit the activity and / or levels of one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, wherein the therapeutically effective amount of the first compound reduces neutrophil migration to the target tissue. In some embodiments, the compound is a cyclosporine A conjugate or precursor, such as compounds of formula I (including, but not limited to, formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126).

[0108] In various embodiments, suitable in vitro or in vivo assays are performed to determine the efficacy of a particular composition of the present technology and whether its application is for therapeutic purposes. In various embodiments, in vitro assays may be performed using representative cell-based assays, such as neutrophil migration assays. In other embodiments, in vivo models, represented by animal models, may be used to determine whether a given cyclosporine A conjugate (or precursor) exerts the desired effect in treating a disease or condition, alone or in combination with one or more other compounds (e.g., additional compounds that inhibit one or more MRP2 and HXA3 synthases, compounds that increase MRP1 levels and / or activity, or compounds that increase NAE). Compounds for therapeutic use may be tested in suitable animal model systems, including but not limited to rats, mice, chickens, cattle, monkeys, rabbits, etc., prior to testing in human subjects. Similarly, for in vivo assays, any animal model system known in the art may be used prior to administration to human subjects.

[0109] In some embodiments, the method of this technology further includes the administration of one or more antibiotics and / or anti-inflammatory agents. Examples of antibiotics / anti-inflammatory agents used alone or in combination in the method of this technology include, but are not limited to, dabavancin (DALVANCE©, XYDALBA©), orbactam (ORBACTIVE©), dapoxetine (Cubicin©), tepidizol (SIVEXTRO©), cefepime (ZEVTERA©, MABELIO©), cefoloza-tazobactam (ZERBAXA©), mupirocin, neomycin sulfate bacitracin, polymyxin B, 1-oxo Flufloxacin, clindamycin phosphate, gentamicin sulfate, metronidazole, hexylresorcinol, mebenzyl chloride, phenol, quaternary ammonium compounds, tea tree oil, steroids such as corticosteroids, such as hydrocortisone, hydroxytriamcinolone acetonide, dexamethasone phosphate, beclomethasone dipropionate, clobetasol propionate, hydroxyprednisolone, desoxymethasone, deoxycorticosterone acetate, dexamethasone, diclomethasone, difluralasone acetate, difluralasone valerate, fluocinolone acetone, fluocinolone, flumethasone new Valerate, Fluocinolone Acetone, Fluocinolone Acetate, Flucobutyl Acetate, Flucodone, Fluprednylidene Acetate, Fluhydrocortisone, Halcinonide, Hydrocortisone Acetate, Hydrocortisone Butyrate, Methylprednisolone, Triamcinolone, Cortisone, Cortisone, Fluocinolone Acetate, Fluhydrocortisone, Difluroxone Diacetate, Fluronnoxone Acetone, Methoxysone, Ansifar, Ansifil, Betamethasone, Chlorprednisolone, Chlorprednisolone Acetate, Chlorcodone, Colocillon, Dichloroisocyanurate Prednisolone, difluprednisolone, fludiclofenac, flunisolone, flumethrin, flusperidone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone, methylprednisolone, peramisone, prednisolone, prednisone, beclomethasone dipropionate, betamethasone dipropionate, triamcinolone, nonsteroidal anti-inflammatory drugs, such as COX inhibitors, LOX inhibitors, p38 kinase inhibitors, immunosuppressants such as cyclosporine and cytokine synthesis inhibitors, tetracycline, dimethylaminotetracycline and doxycycline or any combination thereof.

[0110] In some embodiments, the method of this technology further includes administering one or more antibodies that target one or more of Clostridium difficile toxin, tumor necrosis factor (TNF), interleukin, and metalloproteinase-9 (such as antibody GS-5745 (Gilead)).

[0111] For example, in Crohn's disease, any approach of this technique may also be desirable to include administration of one or more aminosalicylic acid products, corticosteroid preparations, conventional corticosteroids and ileal-release budesonide, glucocorticoid / EEN immunomodulators (such as azathioprine, 6-mercaptopurine and methotrexate), anti-tumor necrosis factor (TNF) drugs (such as infliximab (Remicade, Janssen), adalimumab (Humira, AbbVie and Cimzia, UCB)), anti-α-4β-7 integrin antibody vedozazumab (Entyvio, Takeda), JAK inhibitor ABT-494 (AbbVie) and finasteride (GLP0634, Galapagos and Gilead) (Sandborn, The Present and Future of Inflammatory Bowel Disease Treatment Gastroenterology & Hepatology, Volume 12, Issue 7, July 2016).

[0112] For ulcerative colitis, it may be desirable for any approach of this technique to also include administration of one or more 5-aminosalicylates, aminosalicylates, conventional corticosteroids or multi-matrix budesonide (Uceris, Salix) (which delivers the drug to the colon), azathioprine, 6-mercaptopurine, anti-TNF drugs (such as infliximab, adalimumab and gonimumab (Simponi, Janssen)), vedozizumab, Janus kinase (JAK) inhibitors (such as tofacitinib (Xeljanz, Pfizer), ABT-494 (AbbVie), and finasteride (GLPG0634, Galapagos and Gilead)) (Sandborn 2016).

[0113] V. Combination therapy

[0114] In some embodiments, the cyclosporine A compound of this technology may be combined with one or more adjunctive therapeutic agents for the prevention, improvement or treatment of diseases or conditions.

[0115] In one embodiment, an additional therapeutic agent is administered to a subject in combination with a cyclosporine A conjugate or precursor of the present technology (e.g., compounds of formula I, including but not limited to formula IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) to produce a synergistic therapeutic effect.

[0116] In some embodiments, cyclosporine A compounds of this technology (e.g., compounds of formula I, including but not limited to formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) are combined with one or more methods or compounds for the treatment or prevention of celiac disease or celiac disease-related symptoms. In some embodiments, the one or more compounds include anti-inflammatory agents. In some embodiments, the one or more compounds include infliximab. In some embodiments, the one or more methods include a gluten-free diet.

[0117] In some embodiments, the cyclosporine A compound of the present technology (e.g., compounds of formula I, including but not limited to formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) is combined with one or more compounds that increase the level of the aforementioned multidrug resistance protein 1 (MRP1).

[0118] In some embodiments, the cyclosporine A compound of the present technology (e.g., compounds of formula I, including but not limited to formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) is combined with one or more additional compounds that inhibit one or more of the above-mentioned multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases.

[0119] In some embodiments, the cyclosporine A compound of the present technology (e.g., compounds of formula I, including but not limited to formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) is combined with one or more additional compounds that increase the N-acylethanolamine (NAE) mentioned above.

[0120] In some embodiments, the cyclosporine A compound of the present technology (e.g., compounds of formula I, including but not limited to formulas IA, BT-051, BT-090, BT122, BT123, BT125, and BT126) is combined with one or more other therapeutic agents for the treatment of neutrophil-mediated inflammation and related conditions, including but not limited to ulcerative colitis and Crohn's disease. In some embodiments, the present technology provides a composition comprising one or more first compounds that increase the level and / or activity of multidrug resistance protein 1 (MRP1), a second compound such as a cyclosporine A conjugate that inhibits one or more multidrug resistance protein 2 (MRP2) and hepoxilin A3 (HXA3) synthases, and / or a third compound that increases one or more N-acylethanolamines (NAE).

[0121] Multiple therapeutic agents (e.g., cyclosporine A conjugates, compounds that increase MRP1 levels and / or activity, additional MRP2 and HXA3 synthase inhibitors, and / or compounds that increase NAE) can be administered in any order or even simultaneously. If administered simultaneously, multiple therapeutic agents can be provided in a single, uniform form, or in multiple forms (for example only, as a single formulation or as two separate formulations). One therapeutic agent can be administered in multiple doses, or both can be administered in multiple doses. If not administered simultaneously, the time between multiple doses may vary from more than zero weeks to less than four weeks. Furthermore, the combined methods, compositions, and formulations are not limited to using only two agents.

[0122] In some embodiments, the method of this technology further includes administering to a subject a therapeutically effective amount of at least one compound that increases the level and / or activity of one or more N-acylethanolamines (NAE), wherein the therapeutic amount of said compound reduces the migration of neutrophils to target tissues.

[0123] In some embodiments, the compound that increases NAE is a cannabinoid receptor type 2 (CB2) "agonist" (i.e., a compound that specifically binds to and activates CB2). Examples of CB2 agonists include GW-405,833; AM-1241; HU-308; JWH-015; JWH-133; L-759,633; L-759,656; β-caryophyllene; arachidonicylcyclopropylamide; and arachidonicyl-2'-chloroethylamide.

[0124] In some embodiments, the method of this technology may further include administering one or more antibiotics and / or anti-inflammatory agents. Examples of antibiotics / anti-inflammatory agents used alone or in combination in the method of this technology include, but are not limited to, dabavancin (DALVANCE©, XYDALBA©), oribactam (ORBACTIVE©), dapoxetine (Cubicin©), tebuconazole (SIVEXTRO©), cefepime (ZEVTERA©, MABELIO©), cefepime (ZEVTERA©, MABELIO©), cefoloza-tazobactam (ZERBAXA©), mupirocin, etc. Neomycin sulfate, bacitracin B, 1-ofloxacin, clindamycin phosphate, gentamicin sulfate, metronidazole, hexylresorcinol, mebenzyl chloride, phenol, quaternary ammonium compounds, tea tree oil, steroids such as corticosteroids, such as hydrocortisone, hydroxytriamcinolone acetonide, α-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol propionate, hydroxyprednisolone, desoxymethasone, deoxycorticosterone acetate, dexamethasone, diclomethasone, difluralasone acetate, difluralasone valerate, fluocinolone acetonide, fluocinolone propionate. Ketones, fludrocortisone, flumethasone neopentyl ester, fluocinolone acetonide, fluocinolone acetate, flucodone butyl ester, flucolone, fluprednylidene acetate, fludrocortisone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone, cortisone, todoxacin, fluocinolone acetate, fludrocortisone, difluroxone diacetate, fludroxonolone acetonide, methylhydroxysone, ansil, ansiloferrin, betamethasone, chlorprednisolone, chlorprednisolone acetate, chlorcodone, cod... Losilone, diclofenac, difluprednisolone, fludiclofenac, flunisolone, flumethrin, flusperidone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone, methylprednisolone, peramisone, prednisolone, prednisone, beclomethasone dipropionate, betamethasone dipropionate, triamcinolone, nonsteroidal anti-inflammatory drugs, such as COX inhibitors, LOX inhibitors, p38 kinase inhibitors, immunosuppressants such as cyclosporine and cytokine synthesis inhibitors, tetracycline, dimethylaminotetracycline and doxycycline or any combination thereof.

[0125] In some embodiments, the method of this technology may further include administering one or more antibodies that target one or more of Clostridium difficile toxin, tumor necrosis factor (TNF), interleukin, and metalloproteinase-9 (such as antibody GS-5745 (Gilead)).

[0126] In some embodiments, this disclosure includes methods for treating, improving, or preventing Crohn's disease, comprising administering one or more compounds of the present technology in combination with at least one or more of the following: aminosalicylic acid products, corticosteroid preparations, conventional corticosteroids and ileal-release budesonide, glucocorticoid / EEN immunomodulators (such as azathioprine, 6-mercaptopurine, and methotrexate), anti-tumor necrosis factor (TNF) drugs (such as infliximab (Remicade, Janssen), adalimumab (Humira, AbbVie), and cetuzumab (Cimzia, UCB)), the anti-α-4β-7 integrin antibody vedolizumab (Entyvio, Takeda), the JAK inhibitor ABT-494 (AbbVie), and fenaglinib (GLPG0634, Galapagos and Gilead) (Sandborn, Gastroenterology & Hepatology 12(7) (2016)).

[0127] In some embodiments, this disclosure includes methods for treating, improving, or preventing ulcerative colitis, comprising administering one or more compounds of the present technology in combination with at least one or more of the following: 5-aminosalicylate, aminosalicylate, conventional corticosteroids or multi-matrix budesonide (Uceris, Salix) (which delivers the drug to the colon), azathioprine, 6-mercaptopurine, anti-TNF drugs (such as infliximab, adalimumab, and gonimumab (Simponi, Janssen)), vedozizumab, Janus kinase (JAK) inhibitors (e.g., tofacitinib (Xeljanz, Pfizer), ABT-494 (AbbVie), and finasteride (GLPG0634, Galapagos and Gilead)) (Sandborn 2016).

[0128] VI. Application Method

[0129] Any method known to those skilled in the art for contacting cells, organs, or tissues with the compounds of this technology may be used. Suitable methods include in vitro, ex vivo, or in vivo methods.

[0130] In vitro methods typically involve culturing a sample. For example, cells may be placed in a reservoir (e.g., a tissue culture plate) and incubated with a compound under appropriate conditions suitable for obtaining the desired results. Those skilled in the art can readily determine suitable incubation conditions.

[0131] In vitro methods typically involve removing cells, organs, or tissues from mammals such as humans. For example, the cells, organs, or tissues may be incubated with a compound under appropriate conditions. The contacted cells, organs, or tissues are usually returned to the donor, placed in a recipient, or stored for future use. Therefore, the compound is typically in a pharmaceutically acceptable carrier.

[0132] In vivo methods typically involve administering the compound of this technology to mammals such as humans. When used in vivo for therapy, the amount of the compound of this technology administered to the mammal is sufficient to effectively achieve the desired outcome, such as treating the mammal. The effective amount is determined during preclinical and clinical trials using methods familiar to physicians and clinicians. Dosage and administration regimens will depend on the severity of the subject's disease or condition, the characteristics of the specific compound of this technology used, such as its therapeutic index, the subject, and the subject's medical history.

[0133] An effective amount of the compound of this technique, which is applicable to this method, can be administered to a mammal in need by any of a variety of well-known methods for administering pharmaceutical compositions or medicines, such as in pharmaceutical composition or medicine form. The compound of this technique can be administered systemically or topically.

[0134] The compounds of this technology described herein can be incorporated into pharmaceutical compositions for administration, alone or in combination, to a subject to treat or prevent the disorders described herein. Such compositions typically comprise an active agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Additional active compounds may also be incorporated into the composition.

[0135] In some embodiments, the pharmaceutical compositions disclosed herein contain pharmaceutically acceptable carriers and / or excipients suitable for administering the compound or mixture orally in the form of tablets, capsules or pills, or for parenteral, intravenous, intradermal, intramuscular or subcutaneous or transdermal administration.

[0136] Pharmaceutical compositions are typically formulated to be compatible with the intended route of administration. Administration of the pharmaceutical compositions disclosed herein can be performed in any manner known to those skilled in the art. Routes of administration include, but are not limited to, parenteral, intravenous, intramuscular, intradermal, intraperitoneal, intratracheal, subcutaneous, oral, intranasal / inhalation (e.g., inhalation), transdermal (topical), sublingual, intraocular, vaginal, rectal, and transmucosal administration. Systemic routes include oral and parenteral administration. Several types of devices are commonly used for inhalation administration. These types of devices include metered-dose inhalers (MDIs), respiratory-driven MDIs, dry powder inhalers (DPIs), spacers / compartments combined with MDIs, and nebulizers.

[0137] For oral administration, the compound can be readily formulated by combining the active compound with a pharmaceutically acceptable carrier well known in the art. This carrier enables the compounds of this disclosure to be formulated into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by a subject to treatment. Oral pharmaceutical formulations can be obtained as solid excipients, optionally by grinding the resulting mixture and processing the granular mixture (if desired) with the addition of suitable adjuvants to obtain tablets or sugar-coated pill cores. Suitable excipients are specifically fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose formulations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as croscarmellose, agar, or alginate or its salts, such as sodium alginate, can be added. Alternatively, oral formulations can also be formulated in saline or buffer solutions to neutralize acidic conditions in the body, or can be administered without any carrier.

[0138] Orally administered pharmaceutical formulations comprise compressed capsules made of gelatin, and soft-sealable capsules made of gelatin and plasticizers such as glycerin or sorbitol. Compressed capsules may contain a mixture of the active ingredient and fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. The dosage of all orally administered formulations should be appropriate for this administration.

[0139] For buccal application, the composition can be in the form of tablets or lozenges formulated in a conventional manner.

[0140] For inhalation administration, the compounds used according to this disclosure can be conveniently delivered as an aerosol from a pressurized package or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by a valve that provides the amount of delivery. Capsules and cartridges, such as gelatin, for use with inhalers or blowpipes can be formulated into a powder mixture containing the said compound and a suitable powder matrix, such as lactose or starch.

[0141] When systemic delivery of the compound is required, the compound may be formulated for parenteral administration via injection, such as via pill injection or continuous infusion. Formulations for injection may be present in unit dosage forms, such as in ampoules or multi-dose containers, with added preservatives. The composition may be present in an oily or aqueous medium as a suspension, solution, or emulsion, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0142] Parenteral drug formulations comprise an aqueous solution of the active compound in its water-soluble form. Alternatively, suspensions of the active compound can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or mediators include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions.

[0143] Alternatively, the active compound can be in powder form before use, for use with a suitable medium, such as sterile, pyrogen-free water.

[0144] The compound can also be formulated into rectal or vaginal compositions, such as suppositories or retention enemas, for example containing a conventional suppository base, such as cocoa butter or other glycerides.

[0145] In some embodiments, application is local and / or onto the cavitary surface of the tissue to be treated. “Local” application of the composition means that the composition comes into contact with the skin. “Cavity surface” refers to the internal open space or cavity of a tubular organ, such as the central space inside an artery or vein through which blood flows; the interior of the gastrointestinal tract; the passageway of the bronchi in the lungs; the interior of the renal tubules and urine collecting ducts; and the passageway of the female reproductive tract, which begins with a passageway in the vagina, splits into two cavities within the uterus, both of which continue through the fallopian tubes.

[0146] In some embodiments, the compounds of this technology are applied topically and / or onto the cavitary surface of the target tissue. This helps to reduce the potential systemic toxicity of the compounds.

[0147] Other delivery systems may include timed-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated administration of the compound, increasing convenience for both the subject and the physician. Many types of release delivery systems are available and are known to those skilled in the art. They include polymer-based systems such as poly(lactide-glycolic acid), copolyoxalate, polycaprolactone, polyesteramide, polyorthoester, polyhydroxybutyrate, and polyanhydride. Microcapsules containing the aforementioned polymers of the drug are described, for example, in U.S. Patent No. 5,075,109. Delivery systems also include non-polymer systems, namely: lipids containing sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral fats such as mono, di, and triglycerides; hydrogel release systems; silicone rubber systems; peptide-based systems; wax encapsulations; compressed tablets using conventional adhesives and excipients; partially fused implants; and so on. Specific examples include, but are not limited to: (a) erosion systems in which the agents of this disclosure are contained in a matrix, such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152; and (b) diffusion systems in which the active component permeates from a polymer at a controlled rate, such as those described in U.S. Patent Nos. 3,854,480, 5,133,974, and 5,407,686. Furthermore, pump-based hardware delivery systems may be used, some of which are suitable for implantation.

[0148] Experimental Example

[0149] The following examples further illustrate this technology, and these examples should not be construed as limiting it in any way.

[0150] Example 1: Synthesis of PEG-cyclosporine A conjugate.

[0151] Illustrative examples of the general synthesis of PEG-cyclosporine A conjugates are shown in schemes 1 and 2. As will be understood by those skilled in the art, the length of the side chain to which PEG is attached can be varied by using a suitable homologue of the olefinic ester 1. For example, benzyl pentanoate can be substituted with benzyl propionate (benzyl acrylate), benzyl butyrate, benzyl hexanoate, or benzyl hepta-6-enoate to provide shorter or longer side chains. The use of such homologues is within the scope of the art.

[0152] Option 1

[0153] Synthesis of Intermediate 1A 2 L round-bottom flask equipped with a mechanical stirrer and thermocouple was purged with nitrogen. Sodium hydride (18.9 g, 1.05 eq, 60% dispersion in mineral oil (Sigma)) was added, followed by anhydrous THF (540 mL, 10 vol (Sigma)). The mixture was cooled to < 10 °C in an ice-water bath. Then, benzyl alcohol (50.9 mL, 0.473 mol, 1.05 eq (Sigma)) was added dropwise over 45 min. The internal temperature was maintained below 10 °C during the addition. The ice bath was removed, and the reaction mixture was warmed to ambient temperature and stirred for 15 min. The mixture was cooled again to < 10 °C. 4-Pentenoyl chloride (50 mL, 1 eq, 0.450 mol (Sigma)) was added dropwise over 60 min (the internal temperature was raised from 5 °C to 15 °C during the addition). The reaction product was warmed to ambient temperature and stirred for 20 h. The reaction was quenched with 30 mL of saturated NH4Cl, and EtOAc (250 mL) was added. The layers were separated, and the organic layer was washed with saturated NaHCO3 (200 mL) followed by brine (200 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated by rotary evaporation to give crude product 1 in oil form. The crude product was purified by chromatography (SiO2, 1 kg, 25 cm x 10 cm, 10% EtOAc / heptane) to give intermediate 1 (81.9 g, 95% yield) in clear liquid form. 1 The ¹H NMR spectrum was consistent with the specified structure, and the HPLC purity was 98.9%.

[0154] Synthesis of intermediate 3 A 25 mL round-bottom flask equipped with a magnetic stir bar was placed under a nitrogen blanket. Compound 2 (1.40 g, 1.16 mmol, 1.0 eq) was added, followed by DCM (35 mL, purged with nitrogen to obtain a solution. Intermediate 1 (1.87 g, 9.84 mmol, 8.5 eq) was added to this solution. The mixture was then purged with nitrogen for 5 min. Grubb second-generation catalyst (0.11 g, 0.13 mmol, 0.13 eq (Sigma-Aldrich)) was then added to the flask, and the mixture was again purged with nitrogen for 5 min. The mixture was refluxed under argon and stirred vigorously for 17 h. The reaction mixture was cooled to ambient temperature and evaporated to dryness. The residue was purified by chromatography (ISCO, SiO2, 0-10% MeOH / DCM) to obtain intermediate 3 (1.1 g) in the form of a light yellow solid, which contained 7.3% of compound 2, as determined by HPLC analysis.

[0155] The reaction was repeated under the same conditions on a 5 g scale (compound 2). The crude product was purified by column chromatography.

[0156] Synthesis of Intermediate 4 (BT-070) Intermediate 3 (1.0 g, 0.74 mmol, 1.0 eq), 10% Pd / C (0.40 g, 50% water (Johnson Matthey)), and EtOH (20 mL) were added to a 150 mL Fisher-Porter flask. The flask was evacuated under vacuum and refilled three times with H2, and stirred for 23 h at a hydrogen pressure of 50 PSI. The reaction mixture was filtered through a diatomaceous earth mat, and the mat was washed with methanol (2 x 10 mL). The combined filtrates were concentrated by rotary evaporation to give intermediate 4 (0.95 g) in solid form. Intermediate 4, as used in Scheme 1, also refers to BT-070.

[0157] Synthesis of BT-051 Crude intermediate 4 (0.57 g, AMRI, JWU-B-5-1), MEPA-20H (1.00 g, NOF), and HOBt·H2O (0.096 g (Aldrich)) were added to a 50 mL RB flask purged with nitrogen and equipped with a magnetic stir bar. The mixture was placed under a nitrogen blanket and dissolved in acetonitrile (10 mL (Sigma-Aldrich)). TEA (0.18 mL) was added, and the mixture was cooled in an ice-water bath. EDC·HCl (0.104 g (Sigma-Aldrich)) was added in one go, and the reaction product was removed from the ice bath and gradually heated to ambient temperature. The reaction was considered complete by HPLC analysis after 21 h. The reaction product was diluted with DCM (50 mL) and washed with DI H2O (2 x 20 mL) and brine (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude material was purified by chromatography (SiO2, 5-15% MeOH / DCM) to obtain BT-051 in the form of a white solid (0.40 g, 27% yield). 1 The ¹H NMR spectrum is consistent with the specified structure, and the HPLC purity is 98.2%.

[0158] The general synthesis of the PEG-cyclosporine A conjugate as shown in Scheme 1 also resulted in the production of + / -CH2 derivatives of BT-070 and BT-051, as follows. Not wishing to be bound by theory, it is believed that the + / -CH2 derivatives were generated via Scheme 1 because a Grubb catalyst was used in the first step of the synthesis, which facilitated double bond migration. The derivative of BT-070 with one extra carbon in the linker (L1 = -CH2CH2CH2-) is BT125. The derivative of BT-070 with one less carbon in the linker (L1 = -CH2-) is BT122. The derivative of BT-051 with one extra carbon in the linker (L1 = -CH2CH2CH2-) is BT126. The derivative with one less carbon in the linker (L1 = -CH2-) is BT123.

[0159]

[0160]

[0161]

[0162]

[0163] Option 2

[0164]

[0165] Synthesis of BT-090Reaction conditions: 1.0 eq CsA acid (BT070, also known as intermediate 4), 1.25 eq PLS-269, 1.4 eq EDC·HCl, 1.25 eq HOBt, 1 eq TEA, MeCN (4 mL, 31.7 vol), 0℃–room temperature, 28.5 h (Scheme 2). MEPA-20H (0.230 g, NOF), HOBt·H2O (0.019 g (Aldrich)) and a stir bar were added to 100 mL RB containing HOU-E-63-3 (0.126 g, AMRI). The reagents were dissolved in acetonitrile (3.2 mL (Sigma Aldrich)) under an N2 blanket, followed by the addition of TEA (0.013 mL (Aldrich)) and stirring in an ice bath for 10 min. EDC·HCl (0.027 g (Sigma-Aldrich)) was added in a single batch, and the reaction product was removed from the ice bath and gradually heated to room temperature. After 21 h, the reaction product was diluted with DCM (50 mL (Pride)), extracted with DI H2O (2 x 20 mL) and brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude material was purified by chromatography (SiO2, 5-15% MeOH / DCM) to obtain the product (0.181 g, 61%). The material was dissolved in a small amount of water, uniformly frozen in a dry ice / acetone bath, and lyophilized until dry to obtain a white solid.

[0166] Example 2: Compounds from this technology inhibit neutrophil migration.

[0167] This example demonstrates the efficacy of the compounds of this technology in inhibiting neutrophil migration in vitro, and the compounds of this technology exhibit dose-responsive inhibition of neutrophil migration.

[0168] PMN migration assay This allows T84 colorectal cancer cells to grow to confluence on the underside of a 12-well polycarbonate membrane insert in a 24-well Transwell® plate. The confluence of these epithelial cells forms a physiologically relevant top surface (cavity) and basal lateral surface within the transfer wells.

[0169] Within the dosage range, the top surface of the monolayer was treated with BT051, BT070, BT090, BT122, BT123, BT125, or BT126 for 1 hour. Then, the monolayer was infected with Salmonella typhimurium on the top surface for 2 hours, inducing hepoxilin A3 (HXA3) to efflux through the MRP2 membrane protein and enter the top chamber.

[0170] Bacteria were washed off the monolayer, and the top surface was re-exposed to BT051, BT070, BT090, BT122, BT123, BT125, or BT126. Freshly isolated and prepared human neutrophils were then added to the lateral basal layer and allowed to migrate through the monolayer for 2 hours. The number of neutrophils migrating to the top chamber was then determined by assessing myeloperoxidase activity (a neutrophil biomarker) (due to the HXA3 gradient). Lower MPO activity in the top chamber indicated inhibition of migration.

[0171] result .like Figure 1 As shown, BT051 and BT070 exhibited dose-responsive inhibition of neutrophil migration. A 50% inhibition was observed at a compound concentration of 31.25 nM.

[0172] As shown in Table A, BT051 and BT070 exhibited inhibition of neutrophil migration at a dose of 100 nM. However, BT090 did not show inhibition of neutrophil migration at a dose of 100 nM. For the purposes of Table A, PEG-cyclosporine A conjugates / precursors are represented by compounds having the following formula: (IA).

[0173] Table A. Inhibitory activity of PEG-cyclosporine A conjugates and precursors in neutrophil migration assays (compounds tested at 100 nM).

[0174]

[0175] As shown in Table B, the BT051 derivative, having an extra carbon in the linker (BT126) or one less carbon in the linker (BT123), and the BT070 derivative, having an extra carbon in the linker (BT125) or one less carbon in the linker (BT122), exhibited inhibition of neutrophil migration at a dose of 100 nM. For the purposes of Table B, PEG-cyclosporine A conjugates / precursors are represented by compounds having the following formula: (IB).

[0176]

[0177] These results demonstrate the surprising and unexpected ability of certain PEG-cyclosporine A conjugates / precursors to inhibit neutrophil migration. Therefore, these results demonstrate that compounds of this technology can be used in methods for inhibiting neutrophil migration, such as methods for the prevention or treatment of diseases or conditions caused by, resulting from, or otherwise associated with neutrophil migration.

[0178] Example 3: Stability of PEG-cyclosporine A compound in simulated intestinal fluid, simulated gastric fluid, and excrement.

[0179] This example demonstrates that the PEG-cyclosporine A compound (containing the conjugate) of this technology is stable in simulated intestinal fluid, simulated gastric fluid, and excrement.

[0180] A. BT090

[0181] fecal stability Prior to this study, excrement from male Sprague-Dawley rats was collected overnight on a mixture of dry and wet ice. The excrement was then homogenized in phosphate buffer at pH 6.5 and filtered to remove particles. BT090 (10 μM) was incubated in duplicate at 37°C for a total of 24 hours in freshly prepared rat excrement homogenate. At selected time points (0.5, 2, 6, and 24 hours), 25 μL aliquots were taken from the incubation mixture and quenched with 225 μL of acetonitrile containing an internal standard (IS). The samples were then centrifuged to allow precipitated proteins to settle, and the supernatant was diluted 5-fold. The 0-hour sample was prepared in pre-quenched excrement homogenate near the end of the incubation period. All quenched samples were then subjected to bioanalysis for LC-MS / MS quantification.

[0182] SGF / FeSSIF stability procedure BT090 (10 μM) was incubated in duplicate for a total of 6 hours at 37°C in simulated gastric fluid (SGF) (Ricca, Arlington, TX) and simulated intestinal fluid under feeding conditions (FeSSIF) (Biorelevant, London, UK). Reactions were initiated by adding the test sample (1 mM water stock solution) to their respective buffers. At selected time points (0, 0.5, 1, 2, 4, and 6 hours), 50 μL aliquots of the incubation mixture were removed and frozen at -80°C. At the end of the incubation period, 200 μL of 50:50 acetonitrile:water containing the internal standard (IS) was added to each tube and vortexed thoroughly. Samples were prepared at 0 hours in pre-quenched SGF or FeSSIF. All quenched samples were then subjected to bioanalysis for LC-MS / MS quantification.

[0183] BioanalysisThe BT090 sample was analyzed by LC-MS / MS using a Thermo Accela UPLC and a Thermo Q-Exactive mass spectrometer. [M+H]+ adducts of the compounds and internal standards were monitored using positive mode electrospray ionization in precise mass mode. Analytes were injected onto a C18 column and chromatographic analysis was performed using a reversed-phase gradient of 0.1% formic acid in water and 0.1% formic acid in a 20 / 80 isopropanol / acetonitrile mobile phase. Due to the presence of several substances in the polymer mixture, five most prominent peaks were integrated (tracked masses: 768.8, 779.8, 792.0, 801.8, 813.0; all in a 4+ charge state).

[0184] result .like Figure 2A As shown, BT090 is stable in simulated intestinal fluid. The compound was incubated in FeSSIF for more than 6 hours, and the residual amount of the compound was determined at specified time points. Figure 2B As shown, BT090 is stable in simulated gastric fluid. The compound was incubated in SGF for more than 6 hours, and the residual compound was determined at specified time points. Figure 2C As shown, BT090 is stable in fresh rat excrement homogenate. The compound was incubated in excrement for more than 24 hours, and the residual amount of the compound was determined at specified time points.

[0185] B. BT051 and BT070

[0186] fecal stability Prior to this study, excrement from male Sprague-Dawley rats was collected overnight on a mixture of dry and wet ice. The excrement was then homogenized in phosphate buffer at pH 6.5 and filtered to remove particles. BT051 and BT070 (10 μM) were incubated in duplicate at 37°C for a total of 24 hours in freshly prepared rat excrement homogenates. At selected time points (0.5, 2, 6, and 24 hours), 25 μL aliquots were taken from the incubation mixture and quenched with 225 μL of acetonitrile containing an internal standard (IS). The samples were then centrifuged to allow precipitated proteins to settle, and the supernatant was diluted 5-fold. The 0-hour sample was prepared in a pre-quenched excrement homogenate near the end of the incubation period. All quenched samples were then subjected to bioanalysis for LC-MS / MS quantification.

[0187] SGF / FeSSIF stability procedureBT051 and BT070 (10 μM) were incubated in duplicate for a total of 6 hours at 37°C in simulated gastric fluid (SGF) (Ricca, Arlington, TX) and simulated intestinal fluid under feeding conditions (FeSSIF) (Biorelevant, London, UK). Reactions were initiated by adding the test item (1 mM water stock solution) to their respective buffers. At selected time points (0, 0.5, 1, 2, 4, and 6 hours), 50 μL aliquots of the incubation mixture were removed and frozen at -80°C. At the end of the incubation period, 200 μL of 50:50 acetonitrile:water containing the internal standard (IS) was added to each tube, and the mixture was thoroughly vortexed. Samples were prepared at 0 hours in pre-quenched SGF or FeSSIF. All quenched samples were then subjected to bioanalysis for LC-MS / MS quantification.

[0188] Bioanalysis Samples BT051 and BT070 were analyzed by LC-MS / MS using a Thermo Accela UPLC and a Thermo Q-Exactive mass spectrometer. [M+H]+ adducts of the compounds and internal standards were monitored using positive mode electrospray ionization in precise mass mode. Analytes were injected onto a C18 column and chromatographic analysis was performed using a reversed-phase gradient of 0.1% formic acid in water and 0.1% formic acid in a 20 / 80 isopropanol / acetonitrile mobile phase. Due to the presence of several substances in the polymer mixture, five most prominent peaks were integrated (tracked masses: 768.8, 779.8, 792.0, 801.8, 813.0; all in a 4+ charge state).

[0189] result BT051 and BT070 are expected to be stable in simulated intestinal fluid, simulated gastric fluid, and fresh rat excrement homogenate.

[0190] These results indicate that the compounds of this technology can be used in methods including exposing the compounds to intestinal fluid, gastric fluid, and excretory materials, such as methods for treating gastrointestinal diseases.

[0191] Example 4: Pharmacokinetics of the PEG-cyclosporine A compound of this technology.

[0192] This example demonstrates the pharmacokinetics of the compounds in this technique.

[0193] Three male, cannulated, fasted Sprague-Dawley rats were orally administered 10 mg / kg of BT090, BT051, or BT070 at a dose rate of 10 mL / kg at t=0 h. Blood samples were collected at specified time points and processed into plasma. Plasma concentrations of BT090 were determined by LC-MS / MS. Excrement was collected at t=0 to 4 h, 4–8 h, and 8–24 h, homogenized in buffer, and BT090 concentrations were determined by LC-MS / MS. The total weight of excrement collected at each time interval was recorded. BT090 was prepared in 0.5% methylcellulose in 1% NMP and 0.3% Tween-80.

[0194] Tables 1-4 show the oral bioavailability of compounds BT090, BT051, BT070 (a breakdown product of orally administered BT051), and BT070 (administered alone). As shown in Table 1, BT090 is not orally bioavailable. When administered orally at 10 mg / kg in rats, BT090 was not detected in plasma from 15 minutes to 24 hours. Only rat “C” showed any detectable level at 15 min, and this level was close to the detection limit of the assay (25 ng / mL). NQ - below the detection limit of the bioanalytical assay. NC - not calculated due to lack of data. As shown in Table 2, BT051 is orally bioavailable. When administered orally at 10 mg / kg in rats, BT051 was detectable in plasma at 15 minutes in each rat subject (AE). As shown in Table 3, the breakdown product of BT051 (i.e., BT070) is not orally bioavailable in rats after oral administration of BT051 at 10 mg / kg. As shown in Table 4, when administered orally alone at 10 mg / kg in rats, BT070 was present in the plasma of all tested rats within 15 minutes to 24 hours.

[0195] Table 1. PK of BT090 rats.

[0196]

[0197] NQ: Not quantifiable. No peak or below the limit of quantitation (LOQ = 25.0 ng / mL)

[0198] NC: Cannot be calculated.

[0199] Table 2. PK of BT051 rats.

[0200]

[0201] NQ: Not quantifiable. No peak or below the limit of quantitation (LOQ = 5.0 ng / mL)

[0202] NC: Cannot be calculated.

[0203] Table 3. Rat PK of BT070 (decomposition product of BT051).

[0204]

[0205] NQ: Not quantifiable. No peak or below the limit of quantitation (LOQ = 2.5 ng / mL)

[0206] NC: Cannot be calculated.

[0207] Table 4. BT070 rat PK.

[0208]

[0209] NQ: Not quantifiable. No peak or below the limit of quantitation (LOQ = 10 ng / mL)

[0210] NC: Cannot be calculated.

[0211] Table 5-8 shows the amounts of BT090, BT051, BT070 (a breakdown product of BT051), and BT070 (administered alone) present in the excrement of rats orally administered Spraguedoli. The amounts are assumed to be 1 gram of excrement equal to 1 ml.

[0212] Table 5. The amount of BT090 secreted in excrement remained unchanged after oral administration of a dose of 10 mg / kg.

[0213]

[0214] For concentrations corrected for dilution factor (10-fold for 0-4 h; 50-fold for 4-24 h) Table 6. The amount of BT051 secreted in excrement remained unchanged after oral administration of a dose of 10 mg / kg.

[0215]

[0216] Concentration corrected for dilution factor.

[0217] NS: No samples

[0218] NC: Cannot be calculated.

[0219] NQ: Not quantifiable. No peak or below the limit of quantitation (LOQ = 0.5 µg / mL)

[0220] Table 7. The amount of BT070 (a breakdown product of BT051) secreted in excrement remained unchanged after oral administration of 10 mg / kg dose of BT051.

[0221]

[0222] Concentration corrected for dilution factor.

[0223] NS: No samples

[0224] NC: Cannot be calculated.

[0225] NQ: Not quantifiable. No peak or below the limit of quantitation (LOQ = 0.5 µg / mL)

[0226] Table 8. The amount of BT070 secreted in excrement remained unchanged after oral administration of a dose of 10 mg / kg.

[0227]

[0228] Concentrations corrected for a 1:9 dilution factor.

[0229] NQ: Not quantifiable. No peak or below the limit of quantitation (LOQ = 1000 ng / mL)

[0230] Example 5: The compound of this technology inhibits FPR1.

[0231] This example demonstrates the use of compounds of this technology to inhibit FPR1, as measured by inhibiting fMLP-induced neutrophil migration.

[0232] A. BT051

[0233] T84 colorectal cancer cells were grown to confluence on the underside of a 12-well polycarbonate membrane insert in a 24-well Transwell® plate. The confluence of these epithelial cells formed physiologically relevant top (cavity) and basal lateral surfaces within the transfer wells. Freshly isolated and prepared human neutrophils were pretreated on ice for 1 hour in a compound (BT051 or cyclosporine A).

[0234] BT051 or cyclosporine A was added to the top surface of the monolayer. A potent neutrophil chemical inducer, N-formyl-methionyl-leucyl-phenylalanine (fMLP) (100 nM), which activates neutrophil migration by interacting with the FPR1 protein on neutrophils, was also added to the top side of the monolayer. The compound-treated neutrophils were then added to the basal side and allowed to migrate through the monolayer for 2 hours. The number of neutrophils migrating to the top chamber was then determined by assessing myeloperoxidase activity (a neutrophil biomarker) due to the fMLP gradient. Lower MPO activity in the top chamber indicated inhibition of migration.

[0235] result .like Figure 3A As shown, BT051 inhibits fMLP-mediated migration / activation of polymorphonuclear cells (PMNs). Human PMNs were pretreated with BT051 and added to the “basolateral” epithelial monolayer of the transfer well system. fMLP appeared on the “apical side” of the transfer well as a chemical inducer / neutrophil activator. BT051 significantly blocked PMN migration and activation to the apical side at concentrations of 10 μM and 1 μM, similar to the known FPR1 inhibitor cyclosporine A (CsA).

[0236] These results indicate that the compositions of this technology inhibit FPR1. Therefore, the compounds can be used in methods that include inhibiting FPR1, such as for treating FPR1-mediated diseases, such as celiac disease.

[0237] B. BT070 and BT090

[0238] T84 colorectal cancer cells were grown to confluence on the underside of a 12-well polycarbonate membrane insert in a 24-well Transwell® plate. The confluence of these epithelial cells formed physiologically relevant top (cavity) and basal lateral surfaces within the transfer wells. Freshly isolated and prepared human neutrophils were pretreated on ice for 1 hour in BT070, BT090, or cyclosporine A.

[0239] BT070, BT090, or cyclosporine A were added to the top surface of the monolayer. N-formyl-methionyl-leucyl-phenylalanine (fMLP), a potent neutrophil chemical inducer that activates neutrophil migration, was also added to the top side of the monolayer. Compound-treated neutrophils were then added to the basal lateral side and allowed to migrate through the monolayer for 2 hours. The number of neutrophils migrating to the top chamber was then determined by assessing myeloperoxidase activity (a neutrophil biomarker) due to the fMLP gradient. Lower MPO activity in the top chamber indicated inhibition of migration.

[0240] result .like Figure 3B As shown, BT070 and BT051 inhibit fMLP-mediated migration / activation of polymorphonuclear cells (PMNs). It is anticipated that BT090 will also inhibit fMLP-mediated PMN migration / activation. BT070 inhibited PMN migration / activation by approximately 50%. In this experiment, BT051 inhibited PMN migration / activation by 50% at a concentration of 10 μM.

[0241] These results indicate (or will indicate) that the compositions of this technology inhibit FPR1. Therefore, the compounds can be used in methods that include inhibiting FPR1, such as for treating FPR1-mediated diseases, such as celiac disease.

[0242] Example 6: Compounds of this technology used for the prevention and treatment of colitis

[0243] This example demonstrates the use of compounds of this technology for the prevention and treatment of colitis in animal models and human subjects.

[0244] animal models

[0245] Animal models applicable to this example include, but are not limited to, animals suffering from colitis, such as those described herein. Those skilled in the art will understand that the following description is illustrative and can be appropriately applied to other animal models.

[0246] Overview C57BL / 6 and cnr2- / - The mice will be purchased from Jackson Laboratory; and FVB wt and mdr 1a- / - Mice will be purchased from Taconic. Female mice were used at 6–12 weeks of age, and genotypes were mixed 2–4 weeks prior to the experiment to balance the microbiome. Mice were treated with 3% DSS (molecular weight 36,000–50,000 (MP Biomedicals)) in drinking water for 7 days, then returned to normal water and sacrificed on day 9, which represents the peak of disease. Samples from the intermediate and distal colon were fixed in 10% formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin for histopathological analysis. Each sample was semi-quantitatively graded from 0 to 3 according to four criteria: (1) the degree of epithelial hyperplasia and goblet cell reduction; (2) leukocyte infiltration in the lamina propria; (3) the area of ​​affected tissue; and (4) the presence of severe inflammatory markers such as crypt abscesses, submucosal inflammation, and ulcers. Samples were scored by trained researchers without knowledge of the sample identity, and the median and distal values ​​were averaged to obtain the colonic histopathological score.

[0247] According to the methods described herein, the compound of this technology is administered to a subject, such as by rectal administration. In some embodiments, the compound is administered once daily, once weekly, or once monthly. In some embodiments, the compound is administered multiple times daily, multiple times weekly, or multiple times monthly. A control subject is administered the mediator alone.

[0248] Isolation and flow cytometry of lamina propria leukocytes As previously described, a cell suspension derived from the stratum propria (Buonocore) was prepared. et alIntestinal tissue was cut into small pieces and treated with RPMI of 10% FBS and 5 mM EDTA to remove epithelial cells, and then incubated with 100 U / mL type VIII collagenase (Sigma-Aldrich) for two 1-hour periods. Cells were then applied to a discontinuous 30 / 40 / 75% Percoll gradient (GE Healthsciences) and harvested from the 40 / 70% interface. Cells were washed in PBS / 0.1% BSA, incubated with anti-Fc receptors (αCD16 / 32, eBioscience), stained with Zombie Live / Dead infrared staining (eBioscience), and then surface stained with antibodies against CD45, CD11b, Ly6G, and Ly6C or Gr1. Samples were run on MACSquant Analyzer 10 (Miltenyi Bioscience) and analyzed using Flowjo software version 10 (Treestar).

[0249] Analysis of myeloperoxidase content in mouse samples Myeloperoxidase activity was measured in the samples as described. Colon tissue sections were frozen in liquid N2 and stored at -80°C until use. Sections were placed in cetyltrimethylammonium bromide (HTAB (Sigma)) buffer with lysis matrix D (MP Biomedicals) and homogenized at grade 6 for 40 s using a FastPrep-24 homogenizer. Samples were bound to ABTS, and fluorescence readings were obtained after 8 min. The slope was calculated using linear regression with GraphpadPrism and normalized to the protein content of individual samples as measured by dioctanine assay (BioRad). For analysis of excretory samples, excretory contents were weighed and HTAB buffer was added at a rate of 10 μL / mg, and the calculated slope was used directly.

[0250] HXA in colonic mucosa 3 Mass spectrometry analysis Mice were administered 5% DSS in their drinking water and sacrificed on day 7. Proximal colons were harvested from untreated or DSS-treated mice (n=9 / group), and three intestinal segments were combined. Mucosal scrapings were collected by scraping the intestinal surface in PBS with a rubber broom, and HXA3 content was analyzed as previously described (Mumy, KL). et al. , Infect. Immun. 76:3614-3627 (2008)).

[0251] resultCompared to control animals, intrarectal administration of the compound of this technology is expected to significantly reduce DSS-induced intestinal pathology and colonic shortening. Histopathological analysis of the colon will show that mice treated with the compound have reduced neutrophil infiltration into the colonic lumen, which will be confirmed by a significant reduction in myeloperoxidase in fecal samples.

[0252] Therefore, these results are expected to demonstrate that the compounds of this technology can be used in methods to reduce neutrophil infiltration in vivo, such as in the prevention and treatment of inflammation associated with neutrophil migration, such as colitis.

[0253] human subjects

[0254] Human subjects diagnosed with or suspected of having colitis or a related disorder and currently exhibiting one or more symptoms and / or pathologies of colitis or a related disorder were recruited using selection criteria known and accepted in the art.

[0255] Prevention and treatment methods: The compound of this technology is administered to the subject at a dose and frequency appropriate to the stage and severity of the disease. In some embodiments, the compound is administered once daily, once weekly, or once monthly. In some embodiments, the compound is administered multiple times daily, weekly, or monthly.

[0256] To demonstrate the method of prevention and treatment in human subjects, the compound of this technology is administered to subjects before or after the development of symptoms and / or pathology of colitis or related disorders, and the reversal of symptoms / pathology or the expected reduction of symptoms / pathology is assessed using methods known in the art.

[0257] result It is anticipated that the compounds of this technology will induce a reversal of symptoms and / or pathology of colitis and related disorders in human subjects. These results will demonstrate that the compounds of this technology are useful and effective in the prevention and treatment of these disorders.

[0258] Example 7: Compounds of this technology for the prevention and treatment of neutrophil-mediated skin diseases

[0259] This example demonstrates the use of the compounds of this technology in animal models and human subjects for the prevention and treatment of neutrophil-mediated skin diseases, such as dermatitis (eczema), rosacea, seborrheic dermatitis, and psoriasis. Those skilled in the art will understand that the examples of psoriasis presented below illustrate neutrophil-mediated skin diseases, and the methods are generally applicable to any neutrophil-mediated skin disease.

[0260] animal models

[0261] Animal models applicable to this example include any accepted psoriasis model, including but not limited to models with spontaneous mutations, genetically engineered animals, immunological models, and pharmacological models. Spontaneous mutation models include, but are not limited to, asebia (…). Scd1 ab / Scd1 ab Chronic proliferative dermatitis Sharpin cpdm / Sharpin cpdm ), dry and cracked skin TTC7 fsn / TTC7 fsn Homozygous mice with the mutation. Genetically engineered models include animals that ectopically express or lack key regulatory molecules known in the art. Immunological models include animal subjects who have undergone adoptive transfer or related methods known in the art. Pharmacological models include subjects administered agents that induce psoriasis or psoriasis-related symptoms. For example, subjects may receive topical imiquimod (IMQ), toll-like receptor (TLR)-7, and TLR-8 agonists.

[0262] Those skilled in the art will understand that the following description is illustrative and can be appropriately applied to other animal models.

[0263] Material Imiquimod (IMQ, 5% cream, Beselna®) was purchased from Mochida Pharmaceutical (Tokyo, Japan). Betamethasone butyrate propionate (0.05% ointment, Antebate®) was purchased from Torii Pharmaceutical (Tokyo, Japan). Real-time PCR probes and related reagents were purchased from Applied Biosystems (Massachusetts, USA).

[0264] animal Female BALB / c mice and male CB-17 scid mice aged 7-12 weeks were placed under specific pathogen-free conditions, at a room temperature of 23±3℃ and an air humidity of 55±15%, in a 12-hour light / dark cycle environment, with food and water provided at will.

[0265] Inducing skin inflammationIMQ 5% cream was applied once daily to the inner and / or outer side of the skin of the left ear. The dosage of IMQ was 250 μg on the outer side, 500 μg on the outer side, or 250 μg on both the inner and outer sides of the ear. Betamethasone ointment or a related ointment base was applied twice daily to the left ear, 5 μL on the inner and / or outer side. Before applying IMQ, the thickness of the left ear was measured once daily using a thrombectomy (IDA-112M, Mitutoyo, Kawasaki, Japan) as a quantitative indicator of skin inflammation. Control subjects received the mediator alone.

[0266] For the prevention method, subjects were pretreated with the compound of this technology by topical application within a predetermined time before exposure to IMQ.

[0267] For the treatment method, after confirming IMQ-induced inflammation using methods known in the art, the subject applies the compound of this technology topically within a predetermined time.

[0268] The subject was euthanized with carbon dioxide gas, and the left ear was harvested after examining the gross morphology of erythema and scaling. A portion of the harvested tissue sections were fixed in buffered 10% formalin solution and processed to prepare histological paraffin sections. The sections were stained with hematoxylin and eosin and examined under a light microscope. The remaining tissue was stored at -80°C for mRNA analysis via real-time PCR.

[0269] Real-time PCR assay Total RNA samples from ear tissue were obtained using the RNeasy® Lipid Tissue Microkit (QIAGEN, Venlo, the Netherlands) according to the manufacturer's instructions. Transcriptional levels encoding the target cytokines in this study were measured using the RNA-to-Ct™ 1-Step Kit via TaqMan gene expression assay.

[0270] Illustrative targets include, but are not limited to, IFN-γ, IL-13, L-17, IL-22, IL-23, TNF-α, and IL-1β. Target transcriptional levels were normalized to GAPDH transcriptional levels.

[0271] Statistical analysis Ear thickness values ​​were shown as an increase from the pre-treated values ​​measured on day 1 and are expressed as mean ± standard deviation (SD). Statistical significance was determined by an F-test for ear thickness followed by an Aspin-Welch test. t - The analysis was performed using the Bartlett test and then the Dunnett test or the Steel test, and by performing the Bartlett test at the mRNA transcription level, followed by the Tukey test or the Steel-Dwass test.p A value less than 0.05 is considered statistically significant.

[0272] result It is anticipated that application of compounds of this technology will prevent or reduce IMQ-induced inflammation as measured by tissue thickness, expression of inflammatory genes, and neutrophil infiltration in the skin. These results will suggest that compounds of this technology may be used for the prevention and treatment of conditions associated with inflammation and neutrophil infiltration in the skin, including but not limited to dermatitis (eczema), rosacea, seborrheic dermatitis, and psoriasis.

[0273] human subjects

[0274] Human subjects diagnosed with or suspected of having neutrophil-mediated skin diseases (such as dermatitis (eczema), rosacea, seborrheic dermatitis, or psoriasis) and currently exhibiting one or more symptoms and / or pathologies of the aforementioned disorder were recruited using selection criteria known and accepted in the art.

[0275] Prevention and treatment methods The compound of this technology is administered to the subject at a dose and frequency appropriate to the stage and severity of the disease. In some embodiments, the compound is administered once daily, once weekly, or once monthly. In some embodiments, the compound is administered multiple times daily, weekly, or monthly.

[0276] To demonstrate methods of prevention and treatment in human subjects, a compound of the present technology is administered to a subject before or after the development of symptoms and / or pathology of a neutrophil-mediated dermatitis, and the reversal or expected reduction of symptoms / pathology is assessed using methods known in the art. For example, a compound of the present technology is administered to a subject before or after the development of a neutrophil-mediated dermatitis or its symptoms. The prevention, reversal, or reduction of the subject's impairment or symptoms is then assessed using methods known in the art.

[0277] result It is anticipated that the compounds of this technology will induce the reversal of symptoms and / or pathology in neutrophil-mediated skin diseases such as dermatitis (eczema), rosacea, seborrheic dermatitis, and psoriasis. These results will demonstrate that the compounds of this technology are useful and effective for the prevention and treatment of neutrophil-mediated skin diseases in human subjects.

[0278] Example 8: Compounds of this technology used for the prevention and treatment of celiac disease

[0279] This example demonstrates the use of the compounds of this technology for the prevention and treatment of celiac disease. Those skilled in the art will understand that the examples given below are a general illustration of gluten intolerance disorders, and the methods are generally applicable to celiac disease and related disorders.

[0280] animal models

[0281] Animal models suitable for use in this example include any accepted peritoneal models, including but not limited to spontaneous models such as dog and monkey models known in the art, induced models such as germ-free Wistar AVN rats administered gliadin immediately after birth, and transgenic models such as animals overexpressing IL-15. Those skilled in the art will understand that the following description is illustrative and can be appropriately applied to other animal models.

[0282] Overview Animal models are selected and maintained according to relevant standards known in the art. The compounds of this technology are administered to subjects according to the methods described herein, such as by oral administration. In some embodiments, the compounds are administered once daily, once weekly, or once monthly. In some embodiments, the compounds are administered multiple times daily, multiple times weekly, or multiple times monthly. Control subjects are administered the mediator alone.

[0283] For preventative methods, the compound of this technology is administered to the subject before or after the development of symptoms and / or pathology of celiac disease or related disorders, and the reversal of symptoms / pathology or the expected reduction of symptoms / pathology is assessed using methods known in the art.

[0284] result It is anticipated that the compounds of this technology will induce the reversal of symptoms and / or pathology of celiac disease and related disorders in animal models. These results will demonstrate that the compounds of this technology are useful and effective for the prevention and treatment of celiac disease and related disorders.

[0285] human subjects

[0286] Human subjects diagnosed with or suspected of having celiac disease or a related disorder and currently exhibiting one or more symptoms and / or pathologies of celiac disease or a related disorder were recruited using selection criteria known and accepted in the art.

[0287] Prevention and treatment methods: The compound of this technology is administered to the subject at a dose and frequency appropriate to the stage and severity of the disease. In some embodiments, the compound is administered once daily, once weekly, or once monthly. In some embodiments, the compound is administered multiple times daily, weekly, or monthly.

[0288] For preventative methods, the compound of this technology is administered to the subject before or after the development of symptoms and / or pathology of celiac disease or related disorders, and the reversal of symptoms / pathology or the expected reduction of symptoms / pathology is assessed using methods known in the art.

[0289] resultIt is anticipated that the compounds of this technology will induce the reversal of symptoms and / or pathology of celiac disease and related disorders in human subjects. These results will demonstrate that the compounds of this technology are useful and effective for the prevention and treatment of celiac disease and related disorders.

[0290] Equivalent form

[0291] This technology is not limited to the specific embodiments described in this application, which are intended as a single illustration of various aspects of this technology. Many modifications and variations can be made to this technology without departing from the spirit and scope of the invention, as will be apparent to those skilled in the art. In addition to the methods and apparatuses listed herein, functionally equivalent methods and apparatuses within the scope of this technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This technology is limited only by the terms of the appended claims and the full scope of their equivalents. It should be understood that this technology is not limited to specific methods, reagents, compound compositions, or biological systems, which can, of course, be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0292] Every publication and patent mentioned in the foregoing specification is incorporated herein by reference in its entirety for all purposes. Various modifications and variations to the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the art. Although the art has been described in conjunction with specific embodiments, the claimed art should not be unduly limited to these specific embodiments. In fact, various modifications to the described modes for implementing the art, which will be apparent to those skilled in the art, are within the scope of the appended claims.

Claims

1. A compound having the structure of Formula I, （I） Its stereoisomer, or a pharmaceutically acceptable salt of the compound or any of its stereoisomers; wherein: L1 is a C1 to C4 alkylene group optionally substituted with one or more F; R is a PEG having 40 to 50 ethylene oxide units, wherein the PEG includes a linker group L2; and L2 is a C atom with one or two nitrogen atoms. 1-6 Unsubstituted heteroalkylene groups.

2. The compound according to claim 1, wherein R is -NH(CH2). 2-6 (CH2CH2O) 42-46 -O(CH2) 0-5 CH3.

3. The compound according to claim 1, wherein R is selected from CH3O-(CH2CH2O). 44 -CH2CH2CH2NH- and CH3O-(CH2CH2O) 44 The group consisting of -CH2CH2NH-.

4. The compound according to claim 1, wherein L2 is CH2CH2CH2NH.

5. The compound according to any one of claims 1 to 4, wherein L1 is ethylene or propylene.

6. The compound according to claim 1, having the structure of formula IA, its stereoisomer, or a pharmaceutically acceptable salt of any of the compound or its stereoisomers: (IA).

7. The compound according to claim 6, wherein R is CH3O-(CH2CH2O). 44 -CH2CH2CH2NH-.

8. Use of the compound of claim 1, 6, or 7, its stereoisomer, or a pharmaceutically acceptable salt of any of the compound or its stereoisomers, in the preparation of an agent for treating a disease associated with neutrophil-mediated inflammation in target tissues of mammalian subjects in need, wherein: - The diseases mentioned are selected from the group consisting of intestinal diseases, colitis, inflammatory lung diseases, inflammatory skin diseases, eye diseases, genitourinary diseases, and sexually transmitted diseases; or - The disease is an intestinal disease, selected from the group consisting of proctitis, orchitis, Crohn's disease, and celiac disease; or - The disease is colitis, selected from the group consisting of ulcerative colitis (also known as ulcerative colitis), infectious / non-infectious enterocolitis, and inflammatory bowel disease (IBD); or - The disease is an inflammatory lung disease, selected from the group consisting of pneumococcal infection, asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis; or - The disease is an inflammatory skin disease, selected from the group consisting of dermatitis (eczema), rosacea, seborrheic dermatitis, and psoriasis; or - The disease is an eye disease, selected from the group consisting of uveitis, retinitis, keratitis, and macular degeneration; or - The disease is a urogenital disease, including urinary tract infections; or - The disease is a sexually transmitted disease, selected from the group consisting of pelvic inflammatory disease, gonorrhea, chlamydia, herpes, and urethritis; or - The inflammation is non-infectious; or - The inflammation mentioned is infectious inflammation.

9. The use according to claim 8, wherein the compound is formulated into a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier.

10. The use according to claim 8, wherein the compound is formulated for topical application or for application to the cavitary surface of the target tissue.

11. The use according to claim 8, wherein: - The treatment further includes administering to the subject a therapeutically effective amount of one or more second compounds that inhibit one or more multidrug resistance protein 2 (MRP2) and HXA3 synthases, wherein the therapeutically effective amount of the second compound reduces the migration of neutrophils to the target tissue; or - The treatment further includes administering to the subject a therapeutically effective amount of one or more third compounds that increase multidrug resistance protein 1 (MRP1), wherein the therapeutically effective amount of the third compound reduces neutrophil migration to the target tissue; or - The treatment further includes administering to the subject a therapeutically effective amount of one or more fourth compounds that increase one or more N-acylethanolamines (NAE), wherein the therapeutic amount of the fourth compound reduces the migration of neutrophils to the target tissue.

12. The use according to claim 8, wherein: - The inflammation is associated with Crohn's disease, and the treatment also includes administration of one or more aminosalicylic acid products, corticosteroid preparations, ileal-release budesonide, glucocorticoid / EEN immunomodulators including azathioprine, 6-mercaptopurine, and methotrexate, anti-tumor necrosis factor (TNF) drugs including infliximab, adalimumab, and cetrus, polyethylene glycol, anti-α-4β-7 integrin antibody vedozumab, ABT-494, and finatinib; or - The inflammation is associated with ulcerative colitis, and the treatment also includes administration of 5-aminosalicylate, aminosalicylic acid, corticosteroids, multi-matrix budesonide, azathioprine, 6-mercaptopurine, anti-TNF drugs, including one or more of infliximab, adalimumab and golimumab, vedozizumab, tofacitinib, ABT-494 and finasteride.

13. The use according to claim 8, wherein: - The treatment also includes the administration of one or more antibiotics and / or anti-inflammatory agents selected from the group consisting of: dapavancin, orivancin, cubisacodyl, tebuconazole, cefepime, cefoloza-tazobactam, mupirocin, neomycin sulfate bacitracin, polymyxin B, 1-ofloxacin, clindamycin phosphate, gentamicin sulfate, metronidazole, hexylresorcinol, mebenzyl chloride, phenol, quaternary ammonium compounds, tea tree oil, and steroids. Medications such as corticosteroids, including hydrocortisone, hydroxytriamcinolone acetonide, dexamethasone phosphate, beclomethasone dipropionate, clobetasol propionate, hydroxyprednisolone, desoxymethasone, deoxycorticosterone acetate, dexamethasone, diclomethasone acetate, difluralasone acetate, diflucolone valerate, fluocinolone acetonide, fluocinolone acetonide, fluocinolone acetate, fluocinolone butyl ester, fluocinolone, and fluprednisolone (flucodone). Rednylidene acetate, fluocinonide, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone, cortisone, todoxacin, fluocinolone acetonide, fluocinonide, diflubenzuron diacetate, flutronidazole, methylhydroxyzine, ansil, ansiloferrin, betamethasone, chlorprednisolone, chlorprednisolone acetate, chlorcotropin, colocillon, dichloroisocyanurate, difluoroprednisolone, flunisolone, fluocinonide, fluoxetine, fluoxetine acetate Fluoropyron, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone hydrocortisone, methylprednisolone, peramisone, prednisolone, beclomethasone dipropionate, betamethasone dipropionate, triamcinolone, nonsteroidal anti-inflammatory drugs, such as COX inhibitors, LOX inhibitors, p38 kinase inhibitors, immunosuppressants, such as cyclosporine and cytokine synthesis inhibitors, tetracycline, dimethylaminotetracycline and doxycycline, or any combination thereof; and / or - The treatment also includes administration of one or more antibodies selected from the group consisting of: antibodies targeting Clostridium difficile toxin, antibodies targeting tumor necrosis factor (TNF), antibodies targeting interleukins, and antibodies targeting metalloproteinase-9; and / or - Compared to untreated control tissue, the compound reduces the migration of neutrophils to the target tissue.

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