(12) United States Patent (10) Patent No.: US 9,006,210 B2

US 9,006,210 B24used herein have the same meaning as commonly understoodby one of ordinary skill in the art to which this inventionbelongs.As used herein, the term “Toll like receptor 4 antagonist' or“TLR4 antagonist” refers to an agent, Such as a small molecule, polypeptide, polynucleotide, that is capable of substantially reducing, inhibiting, blocking, and/or mitigating the3FIG. 9 illustrates a graph comparing neutrophil infiltrate incorneas for different dose responses for mice treated eritorantetrasodium (E) or placebo (P) before and after LPS stimulation.FIG. 10 illustrates a graph comparing stromal haze in corneas for different dose responses for mice treated eritorantetrasodium (E) or placebo (P) before and after LPS stimulaactivation of TLR4 of a cell.tion.FIG. 11 illustrates a graph comparing the dose response fortreating neutrofil infiltrate in corneas with eritoran tetraso10dium after LPS stimulation.FIG. 12 illustrates a graph comparing the dose response fortreating Stromal haze in corneas with eritoran tetrasodiumafter LPS stimulation.FIG. 13 illustrates eritoran tetrasodium (E) or placebo (P)treated LPS-stimulated mice cornea sections stained using aterminal deoxynucleotidyl transferase dOTP nick end labeling (TUNEL) assay, and counterstained with 4,6-diamidino2-phenylindole (DAPI) to identify individual cells.FIG. 14 illustrates a graph showing the IL-8 levels in theculture supernatant of human corneal epithelial cells (HCET) stimulated with LPS in the presence of eritoran tetrasodium (E) versus placebo (P) after 24 hours.FIG. 15 illustrates a graph showing the IL-8 levels in theculture supernatant of human corneal epithelial cells (HCET) stimulated with Pam3cys in the presence of eritoran tetrasodium (E) versus placebo (P) after 24 hours.FIG. 16 illustrates a graph showing the IL-8 levels in theculture supernatant of macrophages (U937) stimulated withLPS in the presence of eritoran tetrasodium (E) versus placebo (P) after 3 hours.FIG. 17 illustrates a graph showing the IL-8 levels in theculture supernatant of macrophages (U937) stimulated withPam3cys in the presence of eritoran tetrasodium (E) versusplacebo (P) after 3 hours.FIG. 18 illustrates a graph showing the IL-8 levels in theculture supernatant of neutrophils (HL60) stimulated withLPS in the presence of eritoran tetrasodium (E) versus placebo (P) after 3 hours.FIG. 19 illustrates a graph showing the IL-8 levels in theculture supernatant of neutrophils (HL60) stimulated withPam3cys in the presence of eritoran tetrasodium (E) versusplacebo (P) after 3 hours.FIG. 20 illustrates a graph comparing neutrophil infiltratein P. aeruginosa-stimulated corneas of C57BL/6, TLR415As used herein, the term “effective amount” refers to a25mation and/or a disease or disorder associated with cornealeffected.304045ciated with corneal inflammation and/or results in therapeutically relevant effect. By way of example, a “therapeuticallyeffective amount” may be understood as an amount of TLR4antagonist required to reduce corneal inflammation in a Subject.As used herein, the terms “parenteral administration' and“administered parenterally refers to modes of administrationother than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapSular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal. Subcutaneous, Subcuticular, intraarticular, Subcapsular, Subarachnoid, intraspinal and intrasternal injectionand infusion.MD-2 mice after 24 hours.50aeruginosa-stimulated corneas of C57BL/6, TLR4 andMD-2 mice after 24 hours.As used herein, the terms “pharmaceutically or pharmacologically acceptable' refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as5560DETAILED DESCRIPTIONUnless defined otherwise, all technical and scientific termsAs used herein, the term “therapeutically effectiveamount refers to that amount of a TLR4 antagonist administered alone and/or in combination with additional therapeutic agents that results in amelioration of symptoms associatedwith corneal inflammation and/or a disease or disorder asso35Paeruginosa-stimulated corneas of C57BL/6, TLR4 andFor convenience, certain terms employed in the specification, examples, and appended claims are collected here.dosage of a TLR4 antagonist administered alone or in conjunction with any additional therapeutic agents that are effective and/or sufficient to provide treatment of corneal inflaminflammation. The effective amount can vary depending onthe Subject, the disease being treated, and the treatment beingFIG. 21 illustrates a graph comparing stromal thickness inFIG. 23 illustrates a graph comparing neutrophil infiltratein Paeruginosa-stimulated corneas of C57BL/6 mice treatedwith eritoran tetrasodium or placebo after 24 hours.FIG. 24 illustrates a graph comparing stromal thickness inP. aeruginosa-stimulated corneas of C57BL/6 mice treatedwith eritoran tetrasodium or placebo after 24 hours.FIG. 25 illustrates a graph comparing stromal haze in Paeruginosa-stimulated corneas of C57BL/6 mice treated witheritoran tetrasodium or placebo after 24 hours.of inhibition of, reduction of elimination of, or the amelioration of a disease or pathological condition (e.g. cornealinflammation) including, for example, preventing cornealinflammation from developing, inhibiting corneal inflammation development, arresting development of clinical Symptoms associated with corneal inflammation, and/or relievingthe symptoms associated with corneal inflammation.and MD-2 mice after 24 hours.FIG. 22 illustrates a graph comparing stromal haze in PAs used herein, the term “subject” refers to any warmblooded organism including, but not limited to, humanbeings, pigs, rats, mice, dogs, goats, sheep, horses, monkeys,apes, rabbits, cattle, etc.As used herein, the terms “treatment,” “treating,” or “treat'refers to any specific method or procedure used for the cure65appropriate. Veterinary uses are equally included within theinvention and “pharmaceutically acceptable' formulationsinclude formulations for both clinical and/or veterinary use.As used herein, “pharmaceutically acceptable carrier'includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of Such media andagents for pharmaceutical active Substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeutic compositions is contemplated. For human administration, preparations should meet sterility, pyrogenicity, generalsafety and purity standards as required by FDA Office ofBiologics standards. Supplementary active ingredients canalso be incorporated into the compositions.

US 9,006,210 B25As used herein, “Unit dosage' formulations are those con-6-continuedtaining a dose or Sub-dose of the administered ingredientOadapted for a particular timed delivery. For example, “unitOldosage" formulations are those containing a daily dose or unitJ-G-K, andor daily sub-dose or a weekly dose or unit or weekly sub-dose 5Ol ulJKand the like.As used herein the term “alkyl refers to aliphatic organicgroups which may be branched or straight and which mayoptionally be substituted with one or more halogen atoms atwhere each J, K, and Q, independently, is straight orbranched C1 to C15 alkyl; L is O, NH, or CH; Mis O or NH;and G is NH, O, S, SO, or SO;salt includes salts of compounds derived from the combination of the compound and an organic or inorganic acid or base.The present invention relates generally to methods of treatbranched C5 to C18 alkyl,any position along the alkyl chain.10As used herein, the term “pharmaceutically acceptableR is straight or branched C5 to C15 alkyl:R is selected from the group consisting of straight oring corneal inflammation in a subject as well as to methods of 15Omitigating cornealopacities (e.g., corneal haze, stromal haze,Olstromal thickness) associated with corneal inflammation. Inone example, the corneal inflammation can be caused byand/or related to contact lens wear. In other examples, thelA-CH CH-B,A-CHEC-D,Bcorneal inflammation can be associated with uveitis, Scleritis, 20episcleritis, keratitis, ocular or ophthalmic Surgery (e.g., cor-nea Surgery), endophthalmitis, iritis, atrophic macular degeneration, retinitis pigmentosa, iatrogenic retinopathy, retinalQOllA-CEC-B,Otears and holes, cystoid macular edema, diabetic macularA-E-B-CH CH-D, andledema, diabetic retinopathy, sickle cell retinopathy, retinal 25A-E-B-CEC-Dvein and artery occlusion, optic neuropathy, exudative macular degeneration, neovascular glaucoma, corneal neovascularization, cyclitis, sickle cell retinopathy, and pterygium.where E is NH, O, S, SO, or SO; each A, B, and D,According to an embodiment of a method of the present independently, is straight or branched C1 to C15 alkyl:invention, corneal inflammation in a Subject can be substan- 30R4 is selected from the group consisting of Straight ortially reduced and/or mitigated by administering a TLR4 branched C4 to C20 alkyl, andantagonist to the subject’s cornea at an amount effective tosblock, inhibit, and/or mitigate activation of TLR4. One aspectof the present invention therefore relates to a method of treat-Wing corneal inflammation by administering to a Subject a 35Otherapeutically effective amount of at least TLR4 antagonistlsto reduce and/or mitigate corneal inflammation in the Subject.-UIn one embodiment of the present invention, the TLR4antagonist used to treat corneal inflammation in the Subject isa compound of formula (I):40OOA2ONHR6OOR4R2where each U and V, independently, is straight or branchedC2 to C15 alkyl and W is hydrogen or straight or branched C1to C5 alkyl:R is R or R O CH , R being selected from the(I)RVAlgroup consisting of hydrogen, J', -J'-OH, -J'-O-K', -J'-O-NH45 K' OH, and -J'-O-PO(OH), where each J and K', independently, is straight or branched C1 to C5 alkyl:R is selected from the group consisting of hydroxy, halogen, C1 to C5alkoxy and C1 to C5 acyloxy;A' and A, independently, are selected from the group50 consisting of OH,where R' is selected from the group consisting sQ60z--ol. and O-Z-COHOHOOLus M-Q,l lsJKOO-Q -l lsJKOlOQsJKwhere Z is straight or branched C1 to C10 alkyl:or pharmaceutically acceptable salt or phosphate ester65 thereof.In one embodiment, the TLR4 antagonist of formula (I) isa compound of formula (II):

US 9,006,210 ,O'N-1N1 (CH2)6CH3N OOCHor a pharmaceutically acceptable salt or phosphate esterthereof.2OIn another embodiment, the TLR4 antagonist of formula(II) is:OPO(OH)2CHOs(HO).OPO--Seatries--OOHOn 1 N-(CH2)6CHOOCHor a pharmaceutically acceptable salt or phosphate ester 40thereof.In another embodiment, the TLR4 antagonist is eritorantetrasodium (also known as compound E5664). Eritoran tetrasodium is the tetrasodium salt of the compound shown 45immediately above. Eritoran tetrasodium is described in U.S.Pat. No. 5,935,938.Other TLR4 antagonists, which can be used to treat asubject with corneal inflammation in methods of the presentinvention include the following compounds:O50OOOPO(OH)2OCHO(HO).OPOCH3(CH2)6N OH'NH OHYOO5“N ls(CH2)2CH3OQ-1-N-(CH2)6CH6065

US 9,006,210 B211or pharmaceutically acceptable salts thereof or phosphateester thereof (see U.S. Pat. App. No. 2007/0072824A1).Additional TLR4 antagonists that can be used in the invention include, for example, compound B531 (U.S. Pat. No.5.530,113), as well as other compounds described in thefollowing patents: U.S. Pat. No. 5,935,389 (e.g., substitutedliposaccharides identified by formula I); U.S. Pat. No. 5,612,476 (e.g., lipid A analogs disclosed at columns 2-41); U.S.Pat. No. 5,756,718 (lipid A analogs disclosed at columns2-40); U.S. Pat. No. 5,843.918 (e.g., lipid A analogs disclosedat columns 2-48): U.S. Pat. No. 5,750,664 (e.g., substitutedliposaccharides identified by formula I); U.S. Pat. No. 6,235,724 (e.g., lipid A analogs identified by formulas I and II); U.S.Pat. No. 6,184,366 (e.g., lipid A analogs identified by formulaI), U.S. Pat. No. 5,681,824, U.S. Pat. App. Pub. No.2003.014.4503A1, and U.S. Pat. App. Pub. No.20020028927A1. Methods for making these compounds are12OH(HO)P-OOOCO2HOONHHNOOO10OOOOO15also described within these documents. Additional methodsfor making Such compounds are described, for example, inWO O2/94O19.Still other examples of the TLR4 antagonists, which can beused to treat a Subject with corneal inflammation according tomethods of the present invention include compounds of formula (III):2530(III)OOH (HO)2P-OO-S orOONH35HNOOOOIn other embodiments, the TLR4 antagonist of formula(III) is a compound selected from one of the following:O(CH2)n ) odu, f ?igns (?ign) 0ICH,CHor a pharmaceutically acceptable salt or phosphate esterthereof. The above-identified TLR4 antagonist is commercially available from GlaxoSmithKline (UK) under the tradename CRX 526. (See Fort, Madeline M. et al. Journal ofImmunology, 174: 6416-6423 (2005)).(CH2)naCH40(CH2)ns) oIdH, (CH2)ngda,or a pharmaceutically acceptable salt or phosphate esterthereof; wherein n, n, and ns are the same or different andare positive integers from, for example, 1 to about 10 (e.g.,10); n, n, and n are the same or different and are positiveintegers less than 8. Compounds of formula (III) are syntheticlipid A mimetics that do not stimulate cytokine production orother gene expression in human peripheral blood monocytesin vitro or induce an inflammatory response in vivo. (Stoveretal. (Journal of Biological Chemistry Vol. 27, No. 6)).In one example, at least one of n, n, and n is less than 7so that at least one secondary acyl group of formula (III) isless than 10 carbons. Compounds of formula (III) with at leastone secondary acyl group less than 10 carbons have beenshown to be potent TLR4 antagonists. (Stover et al. (Journalof Biological Chemistry Vol. 27, No. 6)).45OHO50OorSlotHNONHOOOO55OOOO6065In one embodiment, the TLR4 antagonist of formula (III)used in the present method has the following structure:(HO,-oO

US 9,006,210 B2-continued-continuedOHrSlot(HO) -oO(HO)P-OOHNNHCOHor N- 2OHNNHOOOOOOOOOO10O1525(HO-Oor pharmaceutically acceptable salts and phosphate estersthereof. The above identified examples of formula (III) areidentified by Stover et al. (Journal of Biological ChemistryVol. 27, No. 6) as being synthetic lipid A mimetics and weresynthesized as described in Johnson et al. Biorog. Med. Chem.Lett. 9, 2273-2278.30In some embodiments, the TLR4 antagonist is a compoundof formula (I):(I)3540RAOOOAlDCI C.iR'iR'where R' is selected from the group consisting of:45OH(HO-O50-ls, N-1s, Sls,tOsus,where each J, K, and Q, independently, is straight orbranched C1 to C15 alkyl:55R’ is straight or branched C5 to C15 alkyl:R is selected from the group consisting of straight orbranched C5 to C18 acyl andO6065lA-CH CH-B,where A and B are each independently straight or branchedC1 to C15 alkyl:R" is selected from the group consisting of straight orbranched C4 to C20 alkyl, and

US 9,006,210 B216OOHsusJwhere each Uand V, independently, is straight or branchedC2 to C15 alkyl and W is hydrogen or straight orbranched C1 to C5 alkyl:K,where J is —CH2—and K is straight or branched C10 to C13alkyl. In other embodiments, R' is10R is R O CH , R being selected from the groupconsisting of hydrogen, J', -J'-OH, -J'-O-K', -J'-OK OH, and -J'-O-PO(OH), where each J and K",independently, is straight or branched C1 to C5 alkyl;R is selected from the group consisting of hydroxy, haloOJ15lsK,gen, C1 to C5alkoxy and C1 to C5 acyloxy;A' and A are each independentlywhere J is —CH2—, K is straight or branched C10 to C13alkyl and Q is straight or branched —CH. In further embodiments, R isOl,OH25or a pharmaceutically acceptable salt or phosphate esterthereof.where J is —CH2—and K is straight or branched C10 to C13alkyl.In some embodiments, R is30In some embodiments, R is straight or branched C8 to C12ments, R is straight or branched C10 to C18 acyl, e.g., C18acyl. In other embodiments, R isalkyl, e.g., straight or branched C10 alkyl. In some embodis35where J is straight or branched C10 to C15 alkyl.OIn other embodiments, R' islOHls,where J is straight or branched C1 to C3 alkyl and K is straightwhere A is straight or branched C7 to C12 alkyl and B isstraight or branched C4 to C9 alkyl. For example, in someembodiments, R is45or branched C8 to C15 alkyl. In still other embodiments, R' isOlsOlJA-CH CH-B,40A-CH CH-B,50where A is straight or branched C9 alkyl and B is straight orbranched C6 alkyl.K,where J is straight or branched C1 to C3 alkyl, K is straight orbranched C8 to C15 alkyl and Q is straight or branched C1 to55C3 alkyl. In further embodiments, R' isl,where J is straight or branched C1 to C3 alkyl and K is straightor branched C8 to C15 alkyl.For example, in some embodiments, R' isIn some embodiments, R is straight or branched C8 to C12ments, R isalkyl, e.g., straight or branched C10 alkyl. In other embodi6065where U is straight or branched C2 to C4alkyl, V is straightor branched C5 to C9 alkyl and W is hydrogen or —CH. Forexample, in some embodiments, R is

US 9,006,210 B218OOHor pharmaceutically acceptable salt or phosphate esterwhere U is straight or branched C2 alkyl, V is straight orbranched C7 alkyl and W is hydrogen or —CH.In some embodiments, R is R O CH , where R isembodiments, R is R O CH , where R is —CHs.In some embodiments, R is hydroxyl.thereof.10J'and where J is straight or branched C1 to C5 alkyl. In someIn further embodiments, the TLR4 antagonist is a compound of formula (I):RAOOOIn still further embodiments, the TLR4 antagonist is acompound of formula (I):(I)RAA2II. R' OCI hOA2t R6*R4 R'HR2R'25l,30--Alwhere R iswhere R' is selected fromOO15(I)AlOSls,OH-l,where J is straight or branched C1 to C3 alkyl, K is straightor branched C8 to C15 alkyl and Q is straight orbranched C1 to C3 alkyl;R’ is straight or branched C8 to C12 alkyl35R iswhere J is straight or branched C1 to C3 alkyl, K is straightor branched C8 to C15 alkyl and Q is straight orbranched C1 to C3 alkyl;R’ is straight or branched C8 to C12 alkylOl40A-CH CH-B,R iswhere A is straight or branched C7 to C12 alkyland B isstraight or branched C4 to C9 alkylR isOl45A-CH CH-B,where A is straight or branched C7 to C12 alkyl and B isstraight or branched C4 to C9 alkyl50R is selected from straight or branched C8 to C12 alkylandwhere U is straight or branched C2 to C4alkyl, V is straightor branched C5 to C9 alkyl and W is hydrogen or—CH:55R is R O CH , where R is J and where J isstraight o

contact lens wear. In addition to infectious keratitis, contact lens wear is also associated with sterile, culture-negative clinical manifesta tions, including contact lens associated red eye (CLARE). contact lens peripheral ulcers (CLPU), and contact lens asso ciated corneal infiltrates CLACI (Stapleton et al., Optom Vis Sci. 2007; 84:257-272).