US 2006O160867A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0160867 A1 Freedman (43) Pub. Date: Jul. 20, 2006

(54) METHODS FOR MODULATING PPAR (60) Provisional application No. 60/466,672, filed on Apr. BOLOGICAL ACTIVITY FOR THE 30, 2003. TREATMENT OF DISEASES CAUSED BY MUTATIONS IN THE CFTR GENE Publication Classification (76) Inventor: Steven D. Freedman, Chestnut Hill, MA (US) (51) Int. Cl. A6II 3/426 (2006.01) Correspondence Address: A6II 3L/98 (2006.01) CLARK & ELBNG LLP (52) U.S. Cl...... 514/369; 514/567 101 FEDERAL STREET BOSTON, MA 02110 (US) (21) Appl. No.: 11/262,645 (57) ABSTRACT (22) Filed: Oct. 31, 2005 This invention features methods for treating diseases asso ciated with mutations in the CFTR gene by administering Related U.S. Application Data PPAR agonists, specifically PPARy, PPARC, and PPARö agonists, PPAR inducers, and/or antioxidants. Also dis (63) Continuation-in-part of application No. PCT/US04/ closed are screening methods for identifying therapeutically 13412, filed on Apr. 30, 2004. useful candidate compounds. Patent Application Publication Jul. 20, 2006 Sheet 1 of 19 US 2006/0160867 A1 sg

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METHODS FOR MODULATING PPAR SUMMARY OF THE INVENTION BOLOGICAL ACTIVITY FOR THE TREATMENT OF DISEASES CAUSED BY MUTATIONS IN THE 0007. The invention features a method for treating a CFTR GENE disease in a human patient that has a mutation in the CFTR gene by administering to the patient a therapeutically effec CROSS-REFERENCE TO RELATED tive amount of a peroxisome proliferator-activated receptor APPLICATIONS (PPAR) inducer, a PPAR agonist, an AP-1 inhibitor, a STAT inhibitor, an NFkB inhibitor, or an LXR agonist. PPARs 0001. This application is a Continuation-in-part applica generally include PPARC, PPARö, and PPARy. Diseases tion of and claims priority to International Application No. caused by mutations in a CFTR gene include, for example, PCT/US2004/013412, filed Apr. 30, 2004, which was pub cystic fibrosis, pancreatitis, chronic obstructive pulmonary lished in English under PCT Article 21(2), and which claims disease (COPD), asthma, chronic sinusitis, primary Scleros the benefit of U.S. provisional application No. 60/466,672, ing cholangitis, liver disease, bile duct injury, and congenital filed Apr. 30, 2003, both of which are incorporated herein by bilateral absence of the was deferens. The diseases that are reference in their entirety. treatable by the therapeutic methods of the invention include any disease caused by any of the 1,300 or more mutations in STATEMENT AS TO FEDERALLY SPONSORED the CFTR protein. See for example, J. Zielenski, Canadian RESEARCH CF registry database; Cutting et al., Nature 346:366-369, 0002 This invention was funded by grant R01 DK52765 1990; Dean et al., Cell 61:863-870, 1990; Kerem et al., from the National Institute of Health. The government may Science 245:1073-1080, 1989: Kerem et al., Proc. Natl. Acad. Sci. USA 87:8447-8451, 1990; and Welsh et al., have certain rights in the invention. “Cystic Fibrosis.” Metabolic and Molecular Basis of Inher ited Disease (8" Ed. 2001), pp. 5121-88. Particularly ame FIELD OF THE INVENTION nable to treatment are diseases caused by a deletion of the 0003. The present invention relates to the treatment of phenylalanine normally present at amino acid residue 508 of cystic fibrosis and other diseases associated with mutations the CFTR protein (AF508). The patients being treated in the CFTR gene. according to the methods of this invention may be heterozy gous or homozygous for a CFTR mutation. BACKGROUND OF THE INVENTION 0008 Useful PPAR inducers and agonists affect any 0004 Approximately one in 2000 Caucasians have cystic PPAR, but particularly PPARy, (e.g., PPARy1 and PPARy2), fibrosis (CF), a genetic disorder caused by inactivating PPARC, and PPARö. Examples include eicosapentaenoic mutations in the cystic fibrosis transmembrane conductance acid; any of the thiazolidinediones, but particularly piogli regulator (CFTR) gene. The CFTR protein, a member of the taZone (ACtosTM, Takeda Pharmaceuticals), rosiglitazone ABC transporter family, forms a chloride channel localized (AvandiaTM, GlaxoSmithKline), thioglitazone and analogs to the plasma membrane. The protein consists of five thereof; L-tyrosine derivatives such as fluoromethyloxycar domains: two membrane-spanning domains that form the bonyl; non-steroidal anti-inflammatory drugs such as chloride ion channel, two nucleotide-binding domains that indomethacin, ibuprofen, naprosyn, and fenoprofen; and hydrolyze ATP, and a regulatory domain. Expression of the anti-oxidants such as vitamin E. vitamin C, S-adenosyl CFTR gene is highest in cells that line passageways of the methionine, selenium, idebenone, cysteine, dithioerythritol, lungs, pancreas, colon, ileum, and genitourinary tract. dithionite, dithiothreitol, and pyrosulfate. Additional examples of PPARC agonists and inducers include DHA. 0005. In addition to CF, defects in the CFTR gene are WY14643, and any of the fibrates, particularly, fenofibrate, associated with diseases including, for example, pancreati beZafibrate, gemfibrozil, and analogs thereof. tis, chronic obstructive pulmonary disease (COPD), asthma, chronic sinusitis, primary Sclerosing cholangitis, and con 0009. In one example, the method includes the use of a genital bilateral absence of the vas deferens (CBAVD). PPARC. antagonist for the treatment of bile duct injury or 0006 The most common inactivating mutation of the cystic fibrosis liver disease associated with a mutation in a CFTR gene, detected in about 70% of CF patients, is a CFTR gene. deletion of the three base pairs encoding the phenylalanine 0010. The invention also features a method for treating a at amino acid residue 508 (AF508). The F508 residue is disease in a human patient that has a mutation in the CFTR located in a membrane spanning domain and its deletion gene by administering to the patient a therapeutically effec causes incorrect folding of the newly synthesized protein. As tive amount of a PPARC agonist and a therapeutically a result, misfolded protein is degraded in the endoplasmic effective amount of a PPARY agonist, including but not reticulum shortly after synthesis. Patients having a homozy limited to, the compounds described herein. gous AF508 deletion tend to have the most severe symptoms of cystic fibrosis, resulting from a loss of chloride ion 0011. The invention also features a method for treating a transport. The disturbance in the sodium and chloride ion disease in a human patient that has a mutation in the CFTR balance in the cells lining the respiratory tract results in a gene by administering to the patient a therapeutically effec thick, Sticky mucus layer that is not easily removed by the tive amount of a dual PPARC/PPARY agonist. Examples of cilia. The altered mucus also traps bacteria, resulting in dual PPARC/PPARY agonists include muraglitazar (Bristol chronic infections. Accordingly, most CF therapy is directed Myers Squibb), tesaglitazar (AstraZeneca)), AR-HO39242 to controlling persistent and often fatal lung infections. (Astra/Zeneca), GW-409544 (Glaxo-Wellcome), KRP297 There is a need for improved therapies that treat the under (Kyorin Merck). See U.S. Pat. No. 6,414,002 for additional lying causes of CF and other CFTR-related diseases. exemplary dual PPARC/PPARY agonist compounds. US 2006/01 60867 A1 Jul. 20, 2006

0012 Useful AP-1 inhibitors include, for example, nor cells, bile duct cells, or macrophages. Alternatively, cells dihydroguaiaretic acid, SP600125, SR11302, pyrrolidine engineered to express a recombinant PPAR gene are also dithiocarbamate, curcumin, PD98059, and the spiro com useful. Particularly useful PPARY isoforms include, for pounds. example, PPARy1 and PPARy2. 0013 Useful STAT inhibitors include, for example, the 0020. The invention also features a method for treating a SSI-1, SSI-2, and SSI-3 proteins. These proteins may be disease in a human patient that has a mutation in the CFTR administered by any Suitable route (e.g., inhalation, intra gene by administering to the patient a therapeutically effec venous, intramuscular, or Subcutaneous injection). Alterna tive amount of an antioxidant. Antioxidants useful in the tively, they can be expressed by the target cells in the patient methods of this invention include, for example, Vitamin E, using gene therapy techniques. Useful STAT inhibitors Vitamin C, S-adnenosyl methionine, selenium, beta-caro include, for example, tripeptides having the sequence pro tene, idebenone, cysteine, dithioerythritol, dithionite, dithio line-tyrosine-leucine or alanine-tyrosine-leucine, wherein threitol, and pyrosulfite. said tyrosine is phosphorylated (phospho-tyrosine). 0021 Any of the therapeutic compounds of the invention 0014) Useful NFkB inhibitor include, for example, can be used alone or in combination with one or more 2-chloro-N-3,5-di(trifluoromethyl)phenyl)-4-(trifluorom additional compounds (e.g., another compound of the inven ethyl)pyrimidine-5-carboxamide (SP-100030): 3,4-dihydro tion) for the treatment methods of the invention. 4,4-dimethyl-2H-1,2-benzoselenazine (BXT-51072); declo 0022. By “PPAR’ is meant peroxisome proliferator-acti pramide (Oxi-104), dexlipotam, a salicylanilide (see, U.S. vated receptor. PPARs generally serve as receptors for two Pat. No. 6,492.425, hereby incorporated by reference), 2-hy classes of drugs: the hypolipidemic fibrates and the insulin droxy-4-trifluoromethylbenzoic (HTB) acid and its deriva sensitizing thiazolidinediones. PPARs are ligand-activated tives (e.g., triflsal); see U.S. Pat. No. 6,414,025, hereby transcription factors that increase transcription of target incorporated by reference). genes by binding to a specific nucleotide sequence in the 0015 Useful LXR agonists include GX3965 and gene's promoter. The three preferred PPAR isotypes for the T0901317 (Cayman Chemical Co., Ann Arbor, Mich.). methods of the invention are PPARy, PPARC, and PPARö. 0016. The invention also features methods for identifying 0023. By “biological activity,” when referring to PPARs compounds useful for treating a disease in a patient having (e.g., PPARy, PPARC, and PPAR8), is meant any effect on a mutation in the CFTR gene and wherein said mutation is cell physiology normally associated with the activation of associated with said disease. In one aspect, the method has this receptor. One important PPAR biological activity is its the steps of: (i) providing cells that express a PPAR (e.g., translocation to the nucleus of the cell. Other assays for PPARy, PPARC, and PPAR8), (ii) contacting the cells with PPAR biological activity are based on the ability of PPAR to a candidate compound, and (iii) assessing the level of PPAR bind to the RXR receptor. Alternatively, PPAR biological expression in the cells relative to the level of PPAR expres activity can be measured using a reporter gene operably sion in the absence of the candidate compound, wherein a linked to a PPAR-inducible promoter and assessing expres candidate compound that increases the level of PPAR sion of the reporter gene. Biological activity can be mea expression is identified as a compound useful for treating the Sured using any appropriate methodology known in the art disease. PPAR expression may be assessed using any appro (see, for example, Kliewer et al., Proc. Natl. Acad. Sci. USA priate technique known to those skilled in the art. Tech 94: 4318-4323, 1997). niques include, for example, western blotting and RNA 0024. By “PPAR inducer is meant any compound that analysis (e.g., northern blotting). increases the biological activity or expression of one or more PPARs (e.g., PPARy, PPAR8, and PPARC) in a cell. PPAR 0017. In another aspect, the method has the steps of: (i) inducers may increase biological activity by post-transcrip providing cells that express a PPAR protein (e.g., PPARY, tionally activating PPAR. One example of a PPAR inducer PPARC, and PPAR8), (ii) contacting the cells with a candi is eicosapentaenoic acid. date compound, and (iii) assessing the half life of the PPAR protein in the cells relative to the half life of the PPAR 0025 By “PPAR agonist' is meant any compound that protein in the absence of the candidate compound, wherein increases binding to one or more PPARs (e.g., PPARY, a candidate compound that increases the half life is identi PPARC, and PPARö) and increases its biological activity fied as a compound useful for treating the disease. (i.e., causes translocation of the PPAR to the nucleus). Examples of PPARY agonists include any of the thiazo 0018. In another aspect, the method has the steps of: (i) lidinediones, but particularly rosiglitazone (AvandiaTM, providing cells that express a PPAR (e.g., PPARy, PPARC. GlaxoSmithKline), thioglitaZone, and pioglitaZone and PPAR8), (ii) contacting the cells with a candidate (ActosTM, Takeda Pharmaceuticals), and analogs thereof. compound, and (iii) assessing the level of PPAR transloca Additional examples of PPARY agonists include non-steroi tion to the nucleus of the cells relative to the level of PPAR dal anti-inflammatory drugs, such as indomethacin, ibupro expression in the absence of the candidate compound, fen, naprosyn, and fenoprofen, and antioxidants such as wherein a candidate compound that increases the level of Vitamin E. Vitamin C, S-adnenosyl methionine, selenium, PPAR translocation to the nucleus is identified as a com beta-carotene, idebenone, cysteine, dithioerythritol, dithion pound useful for treating the disease. Immunohistochemistry ite, dithiothreitol, and pyrosulfite. Examples of PPARC. is a particularly useful method for determining PPAR agonists include Docosahexaenoic Acid (DHA), Wy 14643, nuclear translocation. and any of the fibrates (e.g., fenafibrate, beZafibrate, gem 0019. Any cells that express any PPAR (e.g., PPARY, fibrozil, and analogs thereof). PPARC, and PPARö) are useful in these screening methods, 0026. By “LXR" is meant liver X receptor (LXR) which for example, pancreatic exocrine cells, lung cells, intestinal is a member of the family of nuclear receptors that include US 2006/01 60867 A1 Jul. 20, 2006 the farnesoid X receptor, retinoic acid X receptors, and anti-PPARY antibody. Samples from two wild-type and two peroxisome proliferator-activated receptors. LXR is cfir' mice are shown from the colon. involved in the regulation of the transcription of genes involved in lipid and sterol metabolism and balance as well 0033 FIG. 4C is a bar graph showing the densitometric as the regulation of bacterial inflammatory signaling in quantification for colonic samples from 3 wild-type and 3 macrophages. LXR ligands, such as GX3965 and T0901317 cfir mice. Background was subtracted from bands. Values are currently under investigation as potential therapeutic are expressed as the mean-SEM relative to WT, where agents for the treatment of low HDL, a disorder common in 100% is the mean value. both nondiabetic and diabetic humans. 0034 FIGS. 5A-5D are a series of photomicrographs showing the immunohistochemical distribution of PPARY in 0027. By “therapeutically effective amount is meant an intestinal epithelium of cftir mice after rosiglitazone treat amount sufficient to provide medical benefit. ment. Wild-type and cfir' mice were given rosiglitazone 0028 By “substantially pure,” when referring to a natu by gavage for 9 days. Colon (FIGS. 5A and 5B) and ileum rally occurring compound (i.e., EPA) is meant a compound (FIGS. 5C and 5D) were analyzed from wild-type (FIGS. that has been partially or totally separated from the compo 5A and 5C) and cfir (FIGS. 5B and 5D) mice. Magni nents that naturally accompany it. Typically, the compound fication is 200x. is substantially pure when it is at least 50%, 60%, 70%, 80%, 0035 FIGS. 6A-6B are bar graphs showing the PPARY 90% 95%, or even 99%, by weight, free from the organic mRNA expression in the colon (FIG. 6A) and ileum (FIG. molecules with which it is naturally associated. For 6B) following rosiglitazone treatment of wild-type (WT) example, substantially pure EPA may be obtained by extrac and cfir (CF) mice. RNA extracts were subjected to tion from a natural Source Such as fish oil. Alternatively, quantitative RT-PCR. Values are expressed relative to WT. chemical synthesis of EPA may result in a totally pure where 100% is the mean value in each of the respective product. tissues. Data are expressed as means-SEM (n=5). BRIEF DESCRIPTION OF DRAWINGS 0.036 FIGS. 7A-7B are a series of electrophoretic gels showing the differential PPARY binding to PPRE in mouse 0029 FIG. 1 is a bar graph comparing PPARY mRNA colonic mucosa. PPARY DNA binding was analyzed by expression levels in various tissues of cfir' and wild-type EMSA in colonic mucosa of wild type (WT) and cfir (CF) mice. mRNA expression of total PPARY was analyzed in mice. Colon nuclear extracts were used as a source of colon, ileum, fat, liver and lung from wild-type (WT) and protein. Oligonucleotide probes carrying a perfect DR1 cfir (CF) mice. RNA extracts were subjected to quanti motif of the PPRE from the acyl-CoA oxidase promoter tative RT-PCR. Values for cfir tissues are expressed were used as probes. Each lane contains protein sample from relative to WT, where 100% is the mean value in each of the a different mouse and 3 different samples from each geno respective tissues. Data are expressed as means-SEM (n=5). type were used. FIG. 7A shows an electrophoretic mobility *p-0.05. shift assay. FIG. 7B is a competition binding experiment 0030 FIG. 2 is a bar graph illustrating CFTR expression using unlabled oligonucleotide. A 100-fold excess of the in various tissues of the wild-type mice used in FIG. 1. synthetic PPRE was used. CFTR is significantly expressed in mouse tissues showing 0037 FIG. 7C is a supershift assay of samples from WT decreased PPARY expression. mRNA expression of CFTR and CF mice treated with rosiglitazone. A rabbit polyclonal was analyzed in colon, ileum, lung, fat, and liver from anti-PPARY antibody against the C-terminus was used. wild-type mice. RNA extracts were subjected to quantitative 0038 FIG. 8 is a bar graph showing expression of RT-PCR. Values are represented as the ratio between the PPARC. mRNA in response to dextran sodium sulfate (DSS) respective mRNA and 18S ribosomal RNA levels. Data are and DHA. RNA extracts were subjected to quantitative expressed as means-SEM (n=4). RT-PCR. The Y-axis represents mRNA levels normalized to 0031 FIGS. 3A-3F are a series of photomicrographs 18s rRNA. Results are expressed as means-ESE. p-0.05. showing the immunohistochemical distribution of PPARY in Three to five mice were used per group. ileum, colon and lung. PPARY immunohistochemistry was performed on colon (FIGS. 3A and 3B), ileum (FIGS. 3C 0.039 FIG.9 is a bar graph showing expression of PPARY and 3D) and lung (FIGS. 3E and 3F) from wild-type mRNA in response to DSS and DHA. RNA extracts were (FIGS. 3A, 3C, and 3E) and cftri (FIGS. 3B, 3D, and 3F) subjected to quantitative RT-PCR. The Y-axis represents mice. Tissue sections were stained with a rabbit polyclonal mRNA levels normalized to 18s rRNA. Results are anti-PPARY antibody and a biotinylated secondary antibody. expressed as means-ESE. Three to five mice were used per Sections from cfir' and wild-type mice correspond to group. equivalent tissue regions. Magnification is 200x for FIGS. 0040 FIGS. 10A and 10B are western blots showing 3A-D and 100x for FIGS. 3E and 3F. Incubation in the PPARC. protein expression in nuclear and cytosolic com absence of primary antibody showed no staining. partments in response to DSS and DHA in wild type (FIG. 0032 FIGS. 4A and 4B are western blots of PPARY 10A) and cftr (FIG. 10B) mice. Western blot analysis of protein expression in the nuclear and cytosolic compart PPARC. was performed on nuclear (N) and cytosolic (C) ments of colon and fat cells. Western blot analysis of PPARY extracts from liver homogenate. The arrow indicates was performed on nuclear (Nuc) and cytosolic (Cyt) extracts PPARC. Results are representative of three mice per group. from colon (FIG. 4A) and perigonadal fat (FIG. 4B) from 0041 FIGS. 11A-11D are a series of photomicrographs wild type (WT) and cfir (CF) mice. Protein extracts were showing the distribution of PPARC. in the liver by immu Subjected to western blotting using a rabbit polyclonal nohistochemistry. Liver tissue sections from WT mice (FIG. US 2006/01 60867 A1 Jul. 20, 2006

11A) and from cfir (FIG. 11B) were stained with a rabbit DETAILED DESCRIPTION polyclonal anti-PPARC. antibody and a biotinylated second 0050 Ligands (agonists) and inducers of PPARs, particu ary antibody. The black arrows point to normal appearing larly inducers of PPARC. or PPARY, are useful for treating bile ducts. Incubation in the absence of primary antibody diseases caused by mutations in the CFTR gene. Antioxi showed no staining (bottom panels) in WT (FIG. 11C) and dants, alone or in combination with PPAR ligands and cftr-1 mice (FIG. 11D). Magnification is 400x. inducers, are also useful for treating these diseases. Diseases 0.042 FIGS. 12 A-D are a series of photomicrographs amenable to treatment include, for example, cystic fibrosis showing the effect of DSS on liver histology in PPARC. (CF), pancreatitis, chronic obstructive pulmonary disease mice and WT control mice. Representative hematoxylin and (COPD), asthma, chronic sinusitis, primary Sclerosing cho eosin-stained sections of liver are shown from PPARC. langitis, bile duct injury, liver disease, and congenital bilat mice (FIG. 12A) and WT control mice (FIG. 12B), as well eral absence of the was deferens. as from cfir control mice (FIG. 12C). The portal tracts 0051 A fatty acid imbalance in plasma from cystic show normal appearing bile ducts (arrows). In cfir mice fibrosis patients and in tissues from cfir' mice has been treated with DSS (FIG. 12D), there is bile duct proliferation extensively reported, first as an essential fatty acid defi as indicated by the arrows with associated polymorpho ciency (Farrel et al., Pediatr. Res. 19: 104-109, 1985) and nuclear and mononuclear cell infiltrates highlighted by more recently as an increase in arachidonic and a concomi arrowheads. Magnification is 200x. tant decrease in docosahexaenoic acids (Freedman et al., Proc. Natl. Acad. Sci. USA, 96: 13995-14000, 1999). This 0043 FIG. 13 is a bar graph showing TNFC. levels in defect has been suggested to play a role in the increased response to DSS. Liver TNFC. levels were assayed in WT inflammatory response in cystic fibrosis, since arachidonic and cftir mice. The Y-axis represents TNFC. pg/mg of total acid is the precursor of a number of eicosanoids and inflam protein. Results are expressed as +SD. A minimum of 3 mice matory mediators. Both fatty acids have independent bio was used per group. synthetic precursors, and although they use similar pro 0044 FIG. 14 is an electrophoretic mobility shift assay cesses and share some of the enzymes involved, (EMSA) showing that PPAR/RXR binding to the PPAR docosahexaenoic acid synthesis requires an additional response element is reduced in cfir cells but can be 3-oxidation step that takes place in peroxisomes. Impair normalized by DHA treatment. ment in the peroxisomal 3-oxidation of docosahexaenoic 004.5 FIG. 15 is a bar graph showing the increase in acid precursors would lead to low docosahexaenoic acid and NFKB activity in macrophages from cfir mice. Oral DHA high arachidonic acid levels. treatment decreased the activity in both wild-type and Peroxisome Proliferator-Activated Receptors cftr-/- macrophages (p<0.05, * p-0.01). 0052 PPARs are a subfamily of ligand-activated tran 0046 FIGS. 16A and 16B are graphs showing DHA scription factors. They act by binding as heterodimers with treatment in vivo decreased LPS-induced TNFCS ecretion a retinoid-X receptor (RXR) to specific DNA sequences (FIG. 16A) but not IL-6 secretion in CF mouse peritoneal known as peroxisome proliferator responsive elements macrophages. (*, ** p-0.05, 0.01 compared with wild type (PPRE) (reviewed in Berger et al., Annu. Rev. Med., 53: and &, && p<0.05, 0.01 compared with CF). 409-435, 2002). The PPAR genes were discovered in 1990, when found to be activated by peroxisome proliferators. The 0047 FIG. 17 is a bar graph showing the effect of PPAR three PPAR genes most relevant to this invention are PPARY, agonists on TNFC. Secretion in peritoneal macrophages from PPARC, and PPARö. PPARY has two isoforms, 1 and 2. LPS-induced wild-type and CFTR mice. Values are nor PPARy2 is mostly expressed in adipose tissue while PPARY1 malized to secretion in controls stimulated with 100 ng/ml is more widely distributed including Small and large intes LPS (defined as m100%). Incubating the cells with DHA in tine. PPARC. is present in hepatocytes, macrophages, and vitro produced results similar to DHA pretreatment of the monocytes and is activated by polyunsaturated fatty acids CF mice. A combination of DHA and either WY 14643 or and fibrates. rosiglitazone did not further decrease secretion of TNFC. 0053 PPARs are involved in the regulation of lipid Wy 14643 decreased TNFC. secretion in CF macrophages by metabolism, by regulating the expression of a number of 16% (p<0.001) and the PPARY agonist rosiglitazone genes, such as the fatty acid-binding protein aP phospho decreased TNFC. secretion by 33% (p<0.001). A combina enolpyruvate carboxykinase, acyl-CoA synthase, lipoprotein tion of the PPARC. and PPARY agonists had no additive lipase, the fatty acid transport protein-1, CD36, and leptin. effect on TNFC. secretion. In general, PPARY activation augments lipid catabolism and 0048 FIG. 18 is a bar graph showing the effect of PPAR induces differentiation of fibroblasts into adipocytes. agonists on IL-6 secretion in peritoneal macrophages from PPAR-Y also regulates peroxisomal proliferation and lipid LPS-induced wild type and cftr mice. Values are normal metabolism by increasing beta oxidation. Because DHA is ized to secretion in controls stimulated with 100 ng/ml LPS synthesized in peroxisomes by beta oxidation, PPAR induc (defined as 100%). 11-6 secretion was decreased by 16% ers increase DHA levels in cells, attenuating or reversing the (p<0.05) by Wy 14643 in CF macrophages, with rosiglita effects of CFTR deletions. Zone having no effect on IL-6 secretion. Wy 14643 and 0054 PPARC. is activated by free fatty acids including rosiglitaZone together decreased IL-6 secretion by an addi linoleic, arachidonic, and oleic acids. Induction of peroxi tional 13% (p<0.05) compared with Wy 14643 alone. Somes by this mechanism leads to a reduction in blood 0049 FIG. 19 is a western blot showing the decrease in triglyceride levels. LXR (40 kDa) in cfir' macrophages compared with wild 0.055 PPARY and PPARC. are also involved in the regu type macrophages. lation of inflammatory responses and the enhancement of US 2006/01 60867 A1 Jul. 20, 2006

insulin sensitivity. PPARY and PPARC. have also been shown distributed including Small and large intestine, kidney, to regulate cell proliferation and cell differentiation and muscle and liver (Fajas et al., J. Biol. Chem. 272: 18779 PPARC has been shown to suppress apoptosis in hepato 18789, 1997). Lower but detectable expression levels of cytes. PPARY I have also been reported in both mouse and human 0056 PPAR agonists, specifically PPARY and PPARC. lung tissue (Lambe et al., Eur. J. Biochem. 239: 1-7, 1996; agonists, are used as insulin sensitizers and regulators of Zhu et al., J. Biol. Chem. 268: 26817-26820, 1993). Expres lipid homeostasis in the treatment of diabetes. PPARY and sion in lung has been localized in alveolar type-II pneu PPARC agonists also inhibit the expression of the proin mocytes whereas receptor activity has been found in human flammatory and insulin resistance-inducing cytokine TNFC. airway epithelial cells, as well as in several human lung increase other insulin signaling mediators, and block the epithelial cell lines. These results are in agreement with the NF-kB proinflammatory signaling pathway PPARY has been immunohistochemical results seen in the experiments shown to exert anti-inflammatory effects in the colon. For described below. example, fibrates, which are PPARC. agonists, may be of 0060. The mechanism by which PPARY expression is benefit in the treatment of atherosclerotic disease not by decreased in these select CFTR expressing tissues in cfir decreasing serum lipids, but rather by increasing cholesterol mice may be due to either (i) a reduction in transcription and metabolism in foamy macrophages and decreasing inflam translation of PPARY, (ii) shorter half life of the protein, or mation and ulcerations within atherosclerotic plaques (iii) a lack of stimulation by endogenous PPARY ligands. through PPARC. pathways. In another example, the PPARY Different ligands show diverse effects on PPARY mRNA synthetic agonists thiazolidinediones (TZDs) have been expression. Only troglitaZone, unlike rosiglitaZone and other used as anti-diabetic drugs and exert anti-inflammatory high affinity PPARY ligands, has been shown to upregulate effects in the colon. PPARY expression in nonadipose tissues and cell lines (Davies et al., Mol. Cell. Biol. Res. Commun. 2: 202-208, Modulating PPAR Biological Activity for the Treatment of 1999; J. Pharmacol. Exp. Ther. 300: 72-77, 2002). The Diseases Caused by Mutations in the CFTR Gene experiments below demonstrate that rosiglitaZone induces 0057 The results of the experiments described in the nuclear translocation of PPARY but did not increase RNA examples below demonstrate that PPARY mRNA expression expression is in agreement with these findings. The mecha is decreased in those tissues specifically regulated by CFTR nism for troglitazone-induced RNA expression of PPARY (colon, ileum, liver, and lung). This was confirmed at the may occur through its antioxidant potential, since C-toco protein level by western blot analysis of colon. Based on pherol shows a similar effect. immunohistochemistry, the proportion of PPARy-expressing 0061. In contrast to PPARy, where there is low basal cells was not decreased in these particular cystic fibrosis levels in epithelial cells in affected organs from the cfir tissues from cftr mice and hence would not explain the mouse, the experiments described in Examples 7-9, below, lower levels of PPARY. The fact that no significant differ demonstrate the lack of induction of PPARC. in inflamma ences were found in liver or fat where CFTR RNA levels tory diseases in the clfir' mouse model leading to a were found to be extremely low, suggests that CFTR may pro-inflammatory state. Comparing peritoneal macrophages play a role in modulating PPARY expression. It should be from cftr mice to those from wild-type littermates, there pointed out that although there is expression of CFTR in bile is a lack of induction of PPARC. (based on electrophorectic ducts, these cells represent less than 3% of total cells in the mobility gel shift assays) in the CF macrophages, resulting liver. in an increase in NFKB, TNF, and IL-6. In another set of 0.058. The results of western blotting and immunohis experiments, also described below, it was demonstrated that tochemistry also show that the subcellular localization of in the liver of wild-type mice, colitis leads to an induction PPARY is altered in cftr mice. This alteration consists of of PPARC, which prevents bile duct inflammation. In cfir a shift from predominantly nuclear staining in wild-type mice, there is no increase in PPARC. at the RNA or protein animals to a diffuse cytoplasmic staining in cfir mice. level resulting in bile duct inflammation as evidenced by Western blot analysis of colonic mucosal Scrapings demon increased mononuclear cell infiltrates around the bile ducts strated that this is mostly due to a decrease in the nuclear and bile duct proliferation. presence of PPARY, and was supported by the decreased 0062 PPAR activation has been shown to result in binding of the PPARy/RXR complex to PPRE in cfir decreased inflammation through inhibition of AP-1, STAT colon, as revealed by EMSA. This confirms that not only and NFkB pathways in monocytes and macrophages that expression of PPARY, but also its function as a transcription results in a modulatory effect on cytokine Secretion (Jiang et factor is compromised in cfir tissues. The fact that admin al., Nature 391: 82-86, 1998: Nagy et al., Cell 93: 229-240, istration of rosiglitazone, a PPAR ligand, restored both the 1998; Ricote et al., Nature 391: 79-82, 1998), inhibition of nuclear localization of PPARY in ileum and colon based on IL-2 secretion from T cells (Clark et al., J. Immunol. 164: immunohistochemistry, and binding to PPRE in colon cells 1364-1371, 2000), and inhibition of NFkB activity in epi as shown by EMSA, indicates that activation followed by thelial cells (Su et al., J. Clin. Invest. 104: 383-389, 1999). translocation to the nucleus can occur in cftr mice. Thus, a decrease in PPAR expression and function could 0059 PPARY has been shown to be expressed in multiple explain several sequelae that are associated with the cystic tissues. Adipose tissue and colon are the major organs fibrosis phenotype such as an excessive host inflammatory expressing PPARY, while lower levels are present in kidney, response, increased peripheral insulin resistance, and alter liver, skin, ileum and mononuclear blood cells (Dubuquoy et ations in lipid metabolism within the peroxisomal compart al., Lancet 360: 1410-1418, 2002). PPARy2 mRNA is pre ment. dominantly expressed in adipocytes with less significant 0063 Cystic fibrosis is also associated with a high inci amounts in liver, while PPARY 1 mRNA is more universally dence of impaired glucose tolerance and development of US 2006/01 60867 A1 Jul. 20, 2006

diabetes mellitus. A combination of decreased insulin secre are shown in FIG. 1. PPARY expression in colonic mucosa, tion and increased insulin resistance has been proposed. The ileal mucosa, and lung homogenate from cfir mice were former is attributed to pancreatic atrophy and fibrosis char 2-fold lower as compared to wild type mice (p<0.05, n=5). acteristic of cystic fibrosis patients, affecting both exocrine No significant differences in mRNA expression were found and endocrine function. The latter effect on increased either in perigonadal adipose tissue or in liver homogenate. peripheral insulin resistance could be explained by an Accordingly, administration of a PPAR ligand (agonist) or impairment in PPARY function due to decreased production. inducer that acts in a CFTR-independent manner mitigates 0064. Thiazolidinediones (including rosiglitazone and the symptoms associated with CFTR dysfunction. troglitazone), synthetic ligands for PPARY, are extensively 0069. To evaluate a potential association between PPARY used as a treatment for type 2 (non-insulin dependent) levels and tissue-specific regulation by CFTR, CFTR RNA diabetes (Mudaliar et al., Annu. Rev. Med. 52: 239-257, was quantified in parallel in these tissues from wild-type 2001). Other compounds that selectively bind to the PPARY mice. The results shown in FIG. 2 demonstrate that CFTR binding domain, such as GW 1929, have also been proven to is mostly expressed in intestinal mucosa, preferentially in be potent insulin sensitizers in vivo (Brown et al., Diabetes, colon, and at a lower extent in total lung. mRNA expression 48:1415-1424, 1999). Thus, any of these compounds can be of CFTR in adipose tissue and liver was very low and in the used to treat cystic fibrosis or any other disorder caused by latter, near background levels. a mutation in the CFTR gene. EXAMPLE 2 EXAMPLE 1. Immunohistochemical Localization of PPARY in PPARY Expression is Decreased in CFTR Mice CFTR Mice 0065. An established breeding colony of exon 10 CFTR (cftr) knockout mice and wild-type littermates was used 0070 PPARY immunostaining was performed using a for this study. Tail-clip samples of 14-day-old male mice rabbit polyclonal antibody (Cell Signaling, Beverly, Mass.). were processed for genotype analysis. All mice were weaned After pretreatment with 0.3% hydrogen peroxide in absolute at 23 days of age and then placed on Peptamen (Nestle methanol, sections were blocked with 1% BSA for 2 hours Clinical Nutrition, Deerfield, Ill.) and water until 30 days of at room temperature and then incubated with the primary age, and then continued for 10 days with 15 mL/day of antibody (1: 100 dilution) overnight at 4° C. This was Peptamen. Mice were euthanized by CO and the organs followed with washing and incubating with biotinylated harvested. Ileum and colon mucosal samples were prepared secondary antibody (1:200 dilution). Peroxidase activity was by opening up the intestine, removing the lumenal contents visualized with 3,3'-diaminobenzidine (DAB kit; Vector by flushing with PBS, and then scraping the mucosa from Laboratories, Burlingame, Calif.) as a Substrate. Omission of the muscle layers with a razor blade. Tissues were Snap the primary antibody served as a negative control. frozen in RNAlater (Ambion, Austin, Tex.) for RNA extrac 0071 PPARY was predominantly localized to nuclei in tion (Barerett et al., Nat. Genetics 23: 32-33, 1999). For the mucosal layer of colon and ileum in wild-type mice, western blotting nuclear and cytoplasmic extracts were (FIGS. 3A and 3C). In contrast, the colon and ileum mucosa prepared as described below, and for immunohistochemistry from cftr mice showed reduced nuclear labeling and a tissues were fixed in 10% formalin. predominant diffuse cytoplasmic staining (FIGS. 3B and 0066) Total RNA from cftr and wild-type tissues was 3D). Analysis of lung tissue showed a mixed labeling of both prepared using the RNA STAT-60 isolation reagent (Tel nuclei and cytoplasm localized to larger bronchi and a Test, Friendswood, Tex.) and quantified spectrophotometri diffuse lighter staining of the remaining tissue (FIGS. 3E cally. Quantitative PCR was performed in a ABI Prism 7700 and 3F) in both wild-type and cfir mice. Sequence Detector (Applied Biosystems, Foster City, Calif.) using the RT-PCR master mix kit (Applied Biosystems) EXAMPLE 3 according to the manufacturers instructions. PCR primers, PPARY and CFTR FAM-labeled TaqMan probes were pro Reduced PPARY Levels in Colonic Epithelium vided by Integrated DNA Technologies (Coralville, Iowa). Nuclei of CFTR Mice The oligonucleotide sequences used were the following: 0072 For western blotting of total, nuclear, and cytosolic PPARY exon 2 FW: 5'-tca caa gag ctg acc caatgg t-3' (SEQ extracts, tissue samples were harvested, minced, and ID NO: 1), PPARY exon 2 RV: 5'-ata ata agg tgg agatgc agg homogenized in 0.5 ml of hypotonic buffer (20 mM Hepes titc tac-3' (SEQID NO: 2), PPARY probe: 5'-FAM-ctgaagctic pH 7.5, 5 mM NaF, 0.1 mM EDTA, 1 mM NaVO) caa gaa tac caa agt gcg atc-TAMRA-3 (SEQ ID NO: 3), containing 0.01% NP-40 with a pre-chilled Dounce homog CFTR exon 2 FW: 5'-aagaat ccc cag ctt atc cac g-3' (SEQ enizer. The Suspension was incubated 15 minutes on ice ID NO: 4), CFTR exon 3 RV: 5'-tgg acagcc ttggtg act tcc-3' followed by centrifugation for 10 min at 850xg at 4°C. The (SEQ ID NO. 5), and CFTR probe: 5'-FAM-cct tcgg.cg atg Supernatants (cytoplasmic fraction) were transferred and the ctt timt ctggag att-TAMRA-3', (SEQ ID NO: 6). pellets were resuspended in 0.5 ml hypotonic buffer con 0067. The mRNA levels were normalized by 18s riboso taining 0.5% of NP-40, incubated 15 minutes at 4° C. mal RNA expression (ribosomal RNA control reagents, centrifuged 30 sec at 14000xg and the supernatants dis Applied BioSystems) and quantified simultaneously to carded. Pellets, representing the nuclear fraction, were PPARY or CFTR in a multiplex RT-PCR reaction. All resuspended in 50 ul of lysis buffer (20 mM Hepes pH 7.5, samples were analyzed in duplicates. 400 mM NaCl, 20% Glycerol, 0.1% EDTA, 10 mM NaF. 10 uM NaMO 1 mM NavOs, 10 mM PNPP, 10 mM B-glyc 0068 RNA extracts from wild type and cftr mice were erophosphate) containing 1 mM DTT and Complete Mini subjected to quantitative analysis of total PPARY. The results EDTA-free protease inhibitor cocktail (Roche Diagnostics, US 2006/01 60867 A1 Jul. 20, 2006

Indianapolis, Ind.). Protein concentration in all samples was EXAMPLE 5 measured by Bradford protein assay (Bio-Rad, Hercules, Calif.). Equal amounts of proteins were subjected to SDS PPARY DNA Binding is Altered in the Colonic 10% PAGE, electrotransferred onto immobilon-P (Milli Musoca of CFTR Mice pore, Billerica, Mass.), then immunoblotted for PPARY 0.077 Electrophoretic mobility shift assays (EMSA) were (1:2000 dilution). Densitometric analysis was performed performed as described Tzameli et al. (Mol. Cell. Biol. 20: using by National Institutes of Health Image 1.62 program. 2951-2958, 2000). Briefly, double-stranded oligonucleotides containing either a perfect DR1 motif (synthetic 0.073 Western blotting was performed in order to confirm PPARyrecognition element (PPRE): 5' agc tacgtg acc tttgac the findings revealed by immunohistochemistry of a redis ctggt-3' (SEQ ID NO:7)) or the PPRE from the mouse tribution of PPARY from predominantly nuclear to a less acyl-CoA oxidase promoter (5'-acaggggac caggac aaaggt intense but more equal partitioning between nuclear and cac gttcgg gag t-3' (SEQ ID NO:8)) were end-labeled with cytoplasmic compartments in ileum and colon. As shown in Y-32P ATP (PerkinElmer, Boston, Mass.) and incubated FIG. 4A, levels of PPARY in colon from wild-type mice with 10 mg of nuclear extracts, for 20 minutes at room were significantly higher in the nuclear compared to the temperature. To test specificity, a rabbit polyclonal PPARY cytosolic fractions with the band in the cytoplasmic fraction specific antibody against the C-terminal part of the protein exhibiting a slower mobility on SDS-PAGE. In contrast, (Santa Cruz, Biotechnology, Santa Cruz, Calif.) was incu PPARY levels in cfir mice were decreased with similar bated with the nuclear extracts for 30 minutes, prior to the amounts observed between the nuclear and cytosolic frac addition of the probe. Competition for specific binding was tions. This is shown quantitatively in FIG. 4C where the performed by adding excess of unlabeled oligonucleotide to principal difference in cftir mice compared to wild-type the reaction, also 30 minutes prior to the addition of the controls is a decrease in the nuclear fraction with little probe. The complexes were resolved on a 4% nondenaturing change in cytosolic quantities. polyacrylamide gel and visualized by autoradiography. 0078. The decrease in nuclear PPARY protein expression 0074 Perigonadal fat was also examined. As shown in in colon from cfir' mice demonstrated by western blotting, FIG. 4B, both isoforms of PPAR are seen in adipocytes with suggests that an equal decrease in PPARY DNA binding PPARY2 having a higher apparent molecular weight com activity to its consensus site should be observed. EMSA pared to PPARy1. No differences were seen in PPARY levels analysis of nuclear proteins from wild-type and cftir mice in fat comparing cfir mice with wild-type littermates. was performed using both synthetic PPRE and the natural PPRE from the acyl-CoA oxidase promoter, both oligo 0075 PPARY is known to migrate as two different bands nucleotides used, in the cfir mice (FIG. 7A). Reduced by SDS-PAGE and has been attributed to post-translational binding of the PPARy/RXR heterodimer from nuclear modification. The higher apparent molecular weight form is extracts of cftr mice was seen compared to wild-type due to phosphorylation following insulin stimulation in NIH controls. In addition, a faster migrating complex was appar 3T3 cells or in human colorectal HCT-116 cells. In addition, ent in 2 of the 3 cfir' mouse protein samples. In order to nitration of tyrosine residues on PPARY has been demon test for specificity of this shift, competition analysis was strated in macrophage-like RAW 264 cells in response to performed. As shown in FIG. 7B, a 100-fold excess of TNF or lipopolysaccharide resulting in inhibition of ligand unlabeled oligonuleotide efficiently competed for binding of dependent translocation to the nucleus. These postransla the wild-type and the cftr mouse samples to the synthetic tional modifications likely explain the two forms of PPARY PPRE. This suggests that binding is specific and that the seen on our western blots (FIGS. 4A and 4B). The higher faster migrating complex seen in cftr samples may rep apparent molecular weight form (around 3 kDa) seen in the resent a proteolytic fragment of the proteins containing an cytosolic fractions is not PPARY2 based on comparison with intact DNA binding domain. Protein extracts from perigo results obtained with adipose tissue. nadal fat of both wild-type and cftr mice, which contain minimal amounts of CFTR, demonstrated equally strong EXAMPLE 4 binding of the PPARy/RXR heterodimer to the synthetic PPRE. RosiglitaZone Increases Nuclear Localization of PPARy Without Affecting RNA Expression EXAMPLE 6 0076. In order to determine whether rosiglitazone, a Rosiglitazone Treatment Corrects the PPARY synthetic PPARY ligand, increases nuclear localization and/ Binding Defect in CFTR Mice or increases RNA expression, 3 mg/kg of rosiglitaZone 0079. In agreement with the immunohistochemical (GlaxoSmithKline, Philadelphia, Pa.) was administered by analysis of wild-type and cftr mice, rosiglitazone treat gavage once a day for 9 days. Immunohistochemical analy ment also led to a significant increase in the binding of the sis of PPARY showed very strong nuclear labeling in both PPARy/RXRheterodimer to the synthetic PPRE (FIG. 7C). colon and ileum from wild-type mice (FIGS. 5A and C, Colon protein samples from both genotypes produced a respectively). In contrast to the decreased nuclear staining of strong band on the gel. Specificity of this shift was tested by both tissues in cfir' mice (FIGS. 3B and 3D), treatment incubation with a rabbit polyclonal antibody against the with rosiglitaZone led to a dramatic increase in nuclear C-terminal part of PPARY. Addition of antibody reduced the labeling (FIGS. 5B and 5D). As shown in FIGS. 6A and binding in both samples tested. Again, no differences in the 6B, rosiglitazone did not increase RNA expression in either binding of the PPARy/RXR heterodimer to the synthetic the colonic (FIG. 6A) or the ileal (FIG. 6B) mucosa, PPRE between perigonadal fat protein extracts of rosiglita compared to controls. zone treated wild-type and cftr mice were observed. US 2006/01 60867 A1 Jul. 20, 2006

EXAMPLE 7 prevent the development of bile duct injury in cftr mice in response to DSS induced colitis based on histology and Decreased PPARC is Associated with Bile Duct serum alkaline phosphatase levels (Blanco, P. G. et al., Injury in cfir' mice Supra). 0080 Primary sclerosing cholangitis (PSC) is a chronic 0084 Abnormalities in PPAR function would link the liver disease characterized by fibro-obliterative inflamma enhanced innate immune response and the alterations in tion of the biliary tract. Although the etiology and patho fatty acid metabolism seen in CF. In the study described genesis of PSC is not known, it is strongly associated with below, we hypothesized that 1) CFTR dysfunction by reduc ulcerative colitis and Crohn's disease in approximately 80% ing PPARC. and/or 7 expression in the liver leads to bile duct of cases (Broome et al., Gut, 4:610-615, 1996; Farrant et al., injury in cfir mice, and 2) DHA prevents bile duct injury Gastroenterology, 100:1710-1717, 1991). Conversely, 2.5- through an increase in PPARC. or Y expression in the liver. 7.5% of patients with inflammatory bowel disease develop To evaluate this, we examined whether PPARC. or Y expres PSC (Lee et al., N. Engl. J. Med., 332:924-933, 1995) sion is decreased in the liver of cfir' mice compared to WT although the mechanism by which these patients are predis littermates following DSS and whether the protective posed to bile duct inflammation is unknown. mechanism of action of DHA is through modulation of PPARC. ory. The role of PPARC. alone in the genesis of bile 0081 Cholestasis, chronic inflammation, and portal tract duct injury was tested by histologic examination of the liver damage characterize both PSC and cystic fibrosis liver from PPARC mice treated with DSS. disease, with cholangiographic findings of stricturing and beading observed in both diseases (Nagel et al., Lancet, Materials and Methods 2:1422-1425, 1989; O'Brien et al., Gut, 33:387-391, 1992). CFTR expression in the hepatobiliary system is localized to 0085 Breeding of Mice the apical membrane of the intrahepatic and extrahepatic 0086) The Beth Israel Deaconess Medical Center Insti bile duct epithelial cells where it plays a major role in biliary tutional Animal Care and Use Committee approved all fluid secretion (Cohn et al. Gastroenterology, 105:1857 protocols. University of North Carolina heterozygous CFTR 1864, 1993). Hence, mutations in the CFTR gene result in exon 10 C57BL6 transgenic knockout mice (Jackson Labo the formation of inspissated biliary secretions. In addition, ratory, Bar Harbor, Me.) were bred to produce WT and null CFTR dysfunction results in an excessive host inflammatory mice and utilized for all experiments. The tails of 14-day old response (Khan et al., Am. J. Respir. Crit. Care Med., mice were clipped and processed for analysis of genotype as 151:1075-1082, 1995; Muhlebach et al., Am. J. Respir. Crit. previously described (Zeng, W. et al., Am. J. Physiol. Cell Care Med., 160:186-191, 1999). Physiol., 273:C442-C445 (1997)). Cfir' and WT mice were weaned at 23 days and placed on water and Peptamen 0082 Due to the similarities between PSC and the liver (Nestle Clinical Nutrition, Deerfield, Ill.). PPARC mice disease in CF, studies have examined the prevalence of with an Sv/129 genetic background and Sv/129 WT mice as CFTR mutations in adults with PSC. In one study, only 1 of strain controls were also obtained from Jackson Laborato 19 subjects with PSC had neither a CFTR mutation/variant nor the M470V genotype (Sheth et al., Human Genetics, 1S. 113:286-292, 2003). CFTR function in these patients was 0087 Dextran Sodium Sulfate Induced Bile Duct Injury decreased as measured by nasal transmembrane potential difference testing. Another study failed to demonstrate an 0088. On day 40, WT and cfir' mice were fed Peptamen association of common CF disease-causing mutations with alone or Peptamen plus 125 mg DSS/day (MP Biomedicals, PSC (Gallegos-Orozco et al., Am. J. Gastroenterol. 100: Aurora, Ohio) for 5 days followed by 9 days of 85 mg 874-878, 2005). However, exhaustive genotyping as well as DSS/day as previously described for the genesis of bile duct functional analyses were not performed in that study. Further injury (Blanco, P. G. et al., supra). The amount of DSS in support for the concept that CFTR dysfunction in the setting Peptamen was equally measured for WT and cfir' mice. of colitis predisposes to bile duct injury comes from experi The volume of Peptamen administered (20 ml) was mea ments with exon 10 cfir mice where induction of colitis sured on a daily basis. There was no difference in Peptamen with dextran sodium sulfate (DSS) results in a mononuclear intake between WT and cftr mice. The mean weight of cell infiltrate in the portal tracts in conjunction with bile duct WT mice at the start of DSS treatment was 20.5 g (range proliferation (Blanco et al., Am. J. Physiol. Liver Physiol. 14.5-26 g), and the mean weight of cftr mice was 18.9 g (range 15.5-23.5 g). These values were not statistically 287:G491-G496, 2004). This was not observed in wild-type different between WT and cftr mice. The degree of colitis (WT) controls. was similar between WT and cftr mice as determined by 0083. In order to understand the mechanism by which visualization of bloody diarrhea in all animals and histo CFTR dysfunction leads to the phenotypic expression of CF, logical evidence demonstrating features of colitis with multiple studies in both humans and mice have demon mononuclear cell infiltrates, loss of crypts, and mucosal strated a link to abnormalities in fatty acid metabolism. ulcerations in the colonic resection specimens. There was no Arachidonic Acid (AA) is increased and Docosahexaenoic difference in weight lost during DSS treatment comparing Acid (DHA) decreased in lung, pancreas, and ileum from WT and cfir mice. Additional mice from these groups cfir mice (Freedman et al., Proc. Natl. Acad. Sci. U.S.A., were given DHA (Pure Encapsulations, Sudbury, Mass.) 96:13995-14000, 1999) as well as in tissue and plasma from prepared as a stable emulsion in Peptamen, at a dose of 40 humans with CF (Freedman et al., N. Engl. J. Med., mg per day for 5 days before and continued for 14 days 350:560-569, 2004). In cfir mice, oral DHA corrected this during administration of the DSS. Bile duct injury was fatty acid defect and reversed the pathology in CF affected quantified, as previously described, by examining histologi organs. Similarly, oral administration of DHA was found to cal features of epithelial injury, bile duct proliferation, and US 2006/01 60867 A1 Jul. 20, 2006 bile duct angulation (Blanco et al., Supra). To determine natants (cytoplasmic fraction) were transferred and the pel whether a complete absence of PPARC. was responsible for lets were resuspended in 0.5 ml hypotonic buffer containing the bile duct injury, 5 PPARC mice were treated with 5% 0.5% of NP-40, incubated 15 minutes at 4°C., centrifuiged DSS in drinking water for 7 days to induce colitis (Okayasu 30 seconds at 14000xg and the supernatants discarded. et al., Gastroenterology,98:694-702, 1990). PPARY mice Pellets, representing the nuclear fraction, were resuspended are not viable and thus were not tested. in 50 ul of lysis buffer (20 mM Hepes pH 7.5, 400 mM NaCl, 20% Glycerol, 0.1% EDTA, 10 mM NaF. 10 uM NaMoO, 0089 Analysis of PPAR Expression 1 mM NavOs, 10 mM PNPP, 10 mM B-glycerophosphate) 0090. After each specific treatment, the mice were eutha containing 1 mM DTT and Complete Mini EDTA-free nized with carbon dioxide. Tissues were snap frozen in protease inhibitor cocktail (Roche Diagnostics, Indianapolis, RNAlater (Ambion, Austin, Tex.) for RNA extraction (Bar Ind.). Protein concentration was measured in all samples by rett et al., Nat Genet, 23:32-33, 1999). PPAR mRNA analy the Bradford protein assay (Bio-Rad, Hercules, Calif.). ses were performed by quantitative RT-PCR. Total RNA Equal amounts of proteins were subjected to SDS-10% from cfir' and WT tissues was prepared using the RNA PAGE, electrotransferred onto immobilon-P (Millipore, Bil STAT-60 isolation reagent (Tel-Test, Friendswood, Tex.) and lerica, Mass.), then immunoblotted for PPARC. (1:2,000 quantified spectrophotometrically. Quantitative PCR was dilution of rabbit polyclonal primary antibody (Affinity performed in an ABI Prism 7700 Sequence Detector Bioreagents, Golden, Colo.) followed by 1:4,000 of goat (Applied Biosystems, Foster City, Calif.) using a RT-PCR anti-rabbit HRP antibody). Chemiluminescence detection master mix kit (Applied Biosystems) according to the manu was performed using lumiGLO reagent (Cell Signaling). facturers instructions. PCR primers and PPAR FAM-la Densitometric analysis was performed using the National beled TaqMan probes were from Integrated DNA Technolo Institutes of Health Image 1.62 program. gies (Coralville, Iowa). The oligonucleotide sequences used 0093. Analysis of TNFC. Levels for PPARY were the following: PPARY exon 2 FW 5'-TCA CAA GAG CTG ACC CAA TGG T-3' (SEQ ID NO: 1), 0094. The liver tissue was homogenized with 500 ul of PPARY exon 2 RV 5'-ATAATAAGGTGG AGATGC AGG RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.1% SDS, TTC TAC-3' (SEQ ID NO: 2), PPARY probe 5'-FAM-CTG 10 mM Tris-HCl and 1 mM EDTA, pH 7.4 with protease AAG CTCCAAGAATAC CAAAGT GCG ATC-TAMRA inhibitor cocktail). The homogenate was centrifuged at 3' (SEQID NO:3). The sequences used for PPARC. were the 15,000 rpm for 15 minutes to collect supernatants. The following: PPARC. FW 5'-TAT TCG GCT GAA GCT GGT amounts of TNFC. in the samples were quantified using the GTA C-3' (SEQ ID NO: 9), PPARC. RV 5'-CTG GCA TTT sandwich ELISA. GTT CCG GTT CT-3' (SEQ ID NO: 10), PPARC. probe 0.095 Statistical Analysis 5'-CTG AAT CTT GCA GCT CCG ATC ACA CTT G-3 (SEQ ID NO: 11). Levels of mRNA were normalized to 18s 0096 T-tests were performed to determine significance of ribosomal RNA (ribosomal RNA control reagents, Applied TNFC. and densitometry results. The RT-PCR and densito Biosystems) quantified simultaneously to PPAR in a multi metry results are expressed as mean values-ESE. TNFC. plex RT-PCR reaction. Thermocycler conditions used results are expressed as mean values-SD. We used both were Stage 1: 48° C./30 minutes; Stage 2: 95° C./10 ANOVA and Kruskal-Wallis tests to determine overall sig minutes; Stage 3: 40 cycles of 95° C./15 seconds and 60° nificance of individual comparison groups for RT-PCR. Due to small sample size, we used Wilcoxon Rank sum test C./60 seconds. All samples were analyzed in duplicate. post-hoc. 0.091 For immunohistochemistry, tissue samples were fixed in 10% formalin. Samples were embedded in paraffin, Results 5 um sections prepared, and stained with haematoxylin and 0097. In order to determine if PPARC. or 7 mRNA levels eosin (H&E) for light microscopic examinations. PPARC. are decreased in the setting of CFTR dysfunction, RNA immunostaining was performed using a rabbit polyclonal extracts from the liver of WT and cfir mice were sub antibody (Affinity Bioreagents, Golden Colo.). After pre jected to quantitative RT-PCR. As shown in FIG. 8, liver treatment with 0.3% hydrogen peroxide in absolute metha PPARC levels by RT-PCR exhibited no difference between nol, sections were blocked with 1% BSA for 2 hours at room WT and cftr control littermates. WT mice given DSS temperature and then incubated with the primary antibody showed a 9.3 fold increase in PPARC. mRNA (p=0.02). In (1:100 dilution) overnight at 4°C. This was followed with contrast, cftr mice exhibited no significant change in washing and incubating with biotinylated secondary anti mRNA levels. The role of DHA as a potential inducer of body (1:200 dilution). Peroxidase activity was visualized PPAR expression was examined. In the absence of DSS, with 3.3-diaminobenzidine (DAB kit; Vector Laboratories, DHA treatment led to a 12.1 fold increase in liver PPARC. Burlingame, Calif.) as a Substrate. Omission of the primary mRNA levels in WT mice (p=0.02) and 7.0 fold increase in antibody served as a negative control. cfir mice (p=0.007), compared to no DHA. The combi nation of both DSS and DHA led to a 10.4 fold increase in 0092 For western blotting, nuclear and cytoplasmic PPARC. mRNA in WT mice (p=0.02) and a 4.3 fold increase extracts were prepared as described previously (Ollero et al., in cfir mice (p=0.04), compared to no DSS and no DHA. Supra). Briefly, tissue samples were harvested, minced, and The combination of DSS and DHA led to a 2.7 fold increase homogenized with a pre-chilled Dounce homogenizer in 0.5 in PPARC. mRNA in cfir mice compared to cfir mice ml of hypotonic buffer (20 mM Hepes pH 7.5, 5 mM NaF. treated only with DSS, but this increase was not statistically 0.1 mM EDTA, 1 mM NaVO 10 uM Na MoO) contain significant. ing 0.01% NP-40, 1 mM DTT and protease inhibitor. The suspension was incubated 15 minutes on ice followed by 0.098 As shown in FIG. 9, PPARY mRNA levels were centrifugation for 10 minutes at 850xg at 4°C. The super low compared to PPARC. expression and showed little US 2006/01 60867 A1 Jul. 20, 2006

difference between WT and cfir control littermates. There expression. In WT mice, PPARC. mRNA levels increased in was no significant change with the addition of DHA to either the liver and were associated with nuclear translocation group of animals. With DSS treatment in the absence or following induction of colitis. In contrast, neither an presence of DHA, PPARY mRNA levels were markedly increase in PPARC. mRNA nor nuclear translocation was suppressed in both WT and cfir' mice to undetectable observed in the liver of cftr mice following induction of levels. Based on the lack of differences between WT and colitis. DHA, a known PPARC. ligand (Lin et al., Biochem cfir' mice and the low levels of PPARY mRNA expression, istry, 38:185-190, 1999), selectively increased mRNA all subsequent analyses focused on PPARC. expression of PPARC. in cfir mice. In contrast to PPARC, liver PPARY levels were low in both WT and cftr mice in 0099] To determine if the changes in PPARC. mRNA were the presence or absence of DHA and Suppressed equally in reflected at the protein level and whether nuclear transloca both of these groups of animals by the induction of colitis. tion was altered in cftir mice, western blot analyses were Colitis in mice does lead to decreased PPARY expression in performed. Although cfir mice had a greater amount of the colon (Bassaganya-Riera et al., Gastroenterology, PPARC. protein per mg of total liver homogenate, both 127:777-791, 2004; Katayama et al., Gastroenterology, cfir' and WT control littermates exhibited little difference 124:1315-1324, 2003) with our data showing a similar effect in PPARC. nuclear/cytoplasmic ratio, as shown in the rep in the liver. However, this is unrelated to bile duct injury resentative western blots in FIGS. 10A and 10B. By den since WT mice demonstrate no biliary tract inflammation in sitometric quantitation with 3 mice per group, the mean contrast to cfir mice. nuclear/cytoplasmic ratio in WT mice was 0.39+0.08, with similar results seen in cftr mice as evidenced by a 0.104) These results are consistent with previous studies nuclear/cytoplasmic ratio 0.52+0.04 (p=0.22). The admin examining PPAR mRNA and protein expression in other istration of DSS resulted in nuclear translocation of PPARC. inflammatory disorders. In addition to colitis, Dharancy et in WT mice (nuclear/cytoplasmic ratio 1.06+0.05, p=0.002), al. showed that PPARC. mRNA levels are decreased in the but not in cfir mice treated with DSS (nuclear/cytoplas livers of untreated patients with hepatitis C infection com mic ratio 0.61+0.02, p=0.15). DHA did not cause translo pared with controls, suggesting that low PPARC. levels may cation in either WT (0.41+0.09, p=0.90) or cfir' mice play a role in the pathogenesis of chronic inflammation (0.54+0.06, p=0.81). (Dharancy et al., Supra). Functional studies as assessed by electrophoretic mobility shift assay of PPAR response ele 0100. To determine if differences in PPARC. localization ment (PPRE) binding have been shown to parallel protein may explain the previous observations, immunohistochemi levels. For example, a recent study demonstrated that cal studies were performed. Immunohistochemical localiza lipopolysaccharide decreased PPRE binding in the liver is tion of PPARC. in hepatocytes and cholangiocytes from both associated with decreased PPARC. and Y nuclear localization WT and cftr mice showed similar diffuse staining, as by western blot (Romics et al., Hepatology, 40:376-385, shown in FIGS. 11A and 11B. Staining was mostly cyto 2004). Another agonist of PPARC, WY14643, has recently plasmic although nuclear rim localization was observed. been shown to increase PPARC. mRNA levels in rat liver This principally cytoplasmic localization paralleled the dis (Toyama et al., Biochem. Biophys. Res. Commun., 324:697 tribution seen by western blot. Specificity demonstrated was 704, 2004). Based on these studies, impaired PPARC. expres demonstrated by omission of the primary antibody (FIGS. sion plays an important role in pathologic inflammation. 11C and 11D). This distribution is similar to that observed 0105 Much work has been done to understand how in human liver (Dharancy et al., Gastroenterology, 128:334 PPARs control inflammation. PPARC. is expressed primarily 342, 2005). in tissues with a high level of fatty acid catabolism such as 0101 To test the hypothesis that an inability to increase liver, brown fat, kidney, heart, and skeletal muscle consistent PPARC. in the liver upon DSS induced colitis is responsible with its role in lipid metabolism (Cabrero et al., Curr. Drug for bile duct injury, liver histology from PPARC mice Targets Inflamm. Allergy, 1:243-248, 2002). It is also following DSS induced colitis was examined. Administra present in inflammatory cells such as macrophages and T tion of DSS led to a similar degree of bloody diarrhea in both lymphocytes (Marx et al., supra: Wahli et al., Chem Biol PPARC mice and WT controls. However, DSS did not 2:261-266, 1995). Upon binding with a ligand, the cytosolic induce bile duct injury in PPARC mice (FIG. 12A) or receptor translocates to the nucleus whereupon PPARs het controls (FIG. 12B). Cftr mice in the absence of DSS erodimerize with Retinoid-X Receptor (RXR)Cl. The PPAR/ (FIG. 12C) did not show bile duct injury, however when RXR heterodimer binds to a DNA specific sequence called treated with DSS (FIG. 12D) showed periductular mono PPRE and stimulates transcription of target genes. PPARC. nuclear cell infiltrates, some neutrophils, and bile duct activation results in the repression of NFKB signaling and proliferation. inflammatory cytokine production in different cell-types. Consequently, PPARC. agonists modulate mediators of 01.02 Since TNFC can suppress PPARC expression in inflammation such as NFKB, IL-6, and TNFC. (Staels et al., the liver, TNFC. levels were examined as a function of DSS supra; Cunard et al., J. Immunol. 169:6806-6812, 2002). The administration. As shown in FIG. 13, there was no statisti data shown herein indicate that in response to colitis, there cally significant difference in liver TNFO. levels comparing is either a lack of ligand binding and/or a defect in the cfir mice in the absence or presence of DSS (p=0.13). translocation process in cfir mice. The differences in Similarly, there was no difference in TNFC. levels comparing PPARC. expression in these experiments likely play a role in WT mice with or without administration of DSS (p=0.30). regulating bile duct injury. However, it is possible that PPARC. levels are decreased as a result of bile duct injury. Discussion This is supported by studies in rats whereby TNFC. Sup 0103) These results indicate that DSS induced bile duct presses hepatic PPARC. expression (Beier et al., FEBS Lett. injury in cfir mice is associated with impaired PPARC. 412:385-387, 1997). To test whether PPARC. is down regu US 2006/01 60867 A1 Jul. 20, 2006

lated in our model due to increased TNFC, we examined EXAMPLE 8 TNFC. levels in WT and cfir' mice. Although similar trends were observed comparing WT and cftr mice, there was no PPARC. Expression is Decreased in Macrophages significant difference in TNFC. levels in WT and cftr mice from cftr Mice with or without DSS treatment. Hence, this would not 0.108 We have shown that peritoneal macrophages from explain the decreased levels of PPARC. seen in cfir' mice CF mice have decreased PPAR as well as an increased NFKB compared to WT controls. It would seem therefore, that the activity. These are normalized by pretreatment with the n-3 decrease in PPARC. in cftr associated bile duct injury is fatty acid DHA. DHA’s actions could be through its PPAR not secondary to the inflammatory response itself, but may agonist effect or alternatively through a downregulation of play an important role in mediating the injury. proinflammatory arachidonic acid metabolites. The aim of this study was to determine if TNFC. and IL-6 secretion is 0106. In addition to their role in suppressing inflamma increased in CF peritoneal macrophages and whether it is tion, PPARs are known to regulate lipid metabolism. CF in normalized by selective PPAR agonists. Furthermore we both humans (Freedman et al., (2004), Supra) and in mouse wanted to relate this to the fatty acid profile of CF mac models (Freedman et al., (1999), supra) is associated with a rophages. defect in fatty acid metabolism, specifically an increase in Methods AA and a reciprocal decrease in DHA. Since administration of high doses of DHA to cfir' mice ameliorates the 0109) Cftrexon 10 knockout mice and wild-type (WT) pathology in CF affected tissues (Freedman et al., (1999), littermates were maintained on peptamen (as described supra) including the bile duct injury in response to DSS above). Peritoneal macrophages were induced by intraperi toneal injection of 2 ml thioglycollate broth followed by induced colitis in our CF mouse model (Blanco et al., Supra), harvesting of the macrophages 4 days after injection. For we examined whether DHA may mediate its action, in part studies determining the effect of in vivo pretreatment of through PPAR. DHA is a known PPARC. agonist and the DHA, cfir' mice were given 40 mg DHA/day for 10 days. requirement for doses of DHA beyond what is needed to For in vitro experiments, harvested macrophages were correct the fatty acid defect Suggests that it may decrease plated in RPMI medium with 10% serum and incubated for inflammation through modulation of inflammatory media 12 hours with media alone or with DHA (5uM), EPA (5uM) tors. The results presented herein demonstrate that DHA can the PPAR agonist Wy 14643 (50 uM) or the PPAR agonist increase PPARC, mRNA levels, and this may at least in part rosiglitazone (10M). The cells were washed and then stimu explain its mechanism to ameliorate bile duct injury. How lated with 0-10000 ng/ml LPS for 4.5 hours. TNFC. and IL-6 ever, the fact that DHA did not induce translocation of secretion were measured by ELISA. Fatty acids were ana PPARC. in the liver of cftr mice suggests that DHA may lyzed by GC/MS. have other actions including direct activation of Retinoid-X Receptor (de Urquiza et al., Science, 290:2140-2144, 2000). Results In addition, this does not preclude the possibility that the full 0110 Electrophoretic mobility shift assays showed that effect of DHA involves other pathways including modula PPARC/RXR binding to the PPAR response element is tion of prostanoids (Freedman et al., J. Appl. Physiol. reduced in cfir mice but can be normalized by DHA 92:2169-2176, 2002). treatment (FIG. 14). 0107 The mechanism by which CFTR dysfunction and 0111 Furthermore, in cfir' mice, NF-kB activity was low PPARC. expression leads to bile duct injury may involve increased in peritoneal macrophages from cfir mice as a combination of an impaired PPARC. response together with compared to wild type. Oral treatment with DHA decreased other aspects of CFTR dysfunction, such as altered intestinal the activity in both wild type and cfir' macrophages (FIG. flora. In the CF lung, there is an aberrant immune response 15). in the setting of altered bacterial flora, leading to chronic 0112 LPS stimulation of CF peritoneal macrophages led lung disease. Similarly, bacterial overgrowth in the CF to an increase in both TNFC. (3.1 fold) and IL-6 (2.3 fold) intestine, as recently shown in the cftr mouse (Norkina et secretion compared to that seen from WT macrophages al., Infect. Immun., 72:6040-6049, 2004), may play a role in (FIGS. 16A and B). Pretreatment of CF mice with oral DHA predisposing to excessive inflammation. In the setting of decreased TNFC. secretion from peritoneal macrophages by colitis and altered bacterial flora, the combination of CF 43% (p<0.05), with no effect on IL-6 secretion. Similar associated bacterial pathogens in portal blood and abnormal results were seen after incubation of CF macrophages in qualitative and/or quantitative innate immune responses in vitro with DHA (FIG. 17). The n-3 fatty acid eicosapen taenoic acid (EPA) had no effect on TNFC. secretion indi the liver, accompanied by a difference in cytokines/chemok cating a specific effect of DHA unrelated to decreases in the ines may predispose to bile duct injury in cfir mice. This n-6 arachidonate pathway. The PPARC. agonist Wy 14643 mechanism potentially explains why cfir' mice develop decreased LPS induced TNFC. secretion in CF macrophages bile duct injury in the setting of colitis whereas WT mice do by 27% (p<0.01) (FIG. 17) and the PPARYagonist rosigli not. It is clear from the current study that a decrease in tazone decreased TNFC. secretion by 28% (p<0.001) (FIG. PPARC. is not sufficient to cause bile duct injury, as 17), with neither affecting IL-6 secretion (FIG. 18). The PPARC mice did not develop bile duct injury in the effect of DHA and Wy14643 were specific for CF in that setting of colitis. Rather, it appears that the combination of these agonists did not alter secretion from WT macrophages. an impaired PPARC. response together with other aspects of In contrast, rosiglitaZone also affected WT macrophages CFTR dysfunction, such as altered intestinal flora, may be with a decrease in TNFC. secretion by 18%. (p<0.05). Fatty critical. acid analysis demonstrated a selective increase in the arachi US 2006/01 60867 A1 Jul. 20, 2006 12 donate downstream product 22:5n-6 in the CF macrophages thereof. (See Forman et al., Proc. Natl. Acad. Sci. 94:4312 (0.26 mol % compared to 0.42, p<0.05) (Table 1). 4317, 1997 for a list of some of these compounds). For example, agonists of PPARC. include DHA, Wy 14643, and TABLE 1. fibrates (e.g., beZafibrate and beZafibrate analogs such as binifibrate, ciprofibrate, clinofibrate, clofibrate, etofibrate; Fatty acid profile in peritoneal macrophages. fenofibrate, and gemfibrozil. Agonists of PPARY include any of the thiazolidinediones, such as balaglitaZone, ciglitaZone, WT CF englitaZone, rosiglitaZone, darglitaZone, englitaZone, 16:0 313 O.9 29.4 O.9 18:0 18.1 - O.S 18.2 - 1.0 netoglitazone, KRP-297, JTT-501, NC-2100, NIP-223, 18:1 m-9 11.8 O.4 13.6 1.3 MCC-555, L-764486, CS-011, G1262570, GW347845, or n-6 FK614. Preferred thiazolidinediones useful as PPARY ago nists include pioglitaZone and any analogs thereof (Actos, 18:2 11.8 O.8 10.9 O.S 20:3 1.8 O.O6 17 O.11 Takeda Pharmaceuticals, described, for example, in U.S. 20:4 (AA) 13.5 - 0.6 14.4 + 0.6 Pat. Nos. 4,687,777; 5,965,584; 6,150,383: 6,150,384; 22:4 3.8 O.3 4.1 O.3 6,166,043; 6,172,090; 6,211,205; 6,271,243; 6,303,640; and 22:5 O-26 - O.O2 O.42 0.07* 6,329.404), rosiglitaZone and analogs thereof (Avandia, n-3 GlaxoSmithKline, described for example, in U.S. Pat. Nos. 18:3 O.23 - 0.09 O.21 0.08 5,002,953, 5,741,803, and 6.288,095), and troglitazone, and 20:5 O-26 - 0.10 O.23 - 0.03 any analogs thereof. Agonists can also include tyrosine 22:5 1.6 O.14 1.8 O.15 based PPARY modulators (e.g., fluoromethyloxycarbonyl, 22:6 (DHA) 2.4 - 0.3 2.90.3 GI262570; (S)-2-(2-benzoylphenylamino)-3-4-2-(5-me thyl-2-phenyl-2-oxazol-4-yl)ethoxyphenylpropionic acid, and GW347845 (Cobb et al., J Med. Chem. 41:5055-5069, Conclusion 1998)), 3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfo nyl)-3H-benzimidazole-5-carboxamide (FK614)). Addi 0113 LPS induced TNFC. and IL-6 secretion was tional PPARY agonists include prostaglandin J2 and nonste increased in CF peritoneal macrophages with TNFC. secre roidal anti-inflammatory drugs such as indomethacin, tion being selectively decreased by treatment with DHA as ibuprofen, and fenoprofen. Other PPAR agonists, including well as the PPAR agonist Wy14643. The effect of rosigli dual PPARC/PPARY agonists, that may be used in the taZone on the inhibition of TNFC. secretion appears to be methods, of the invention are AA-10090, AD-5075, AMG unrelated to CFTR in that it was observed in both WT and 131, ARH-049020, AR-HO39242 (AstraZeneca), AVE-0847, CF macrophages. The fact that Wy 14643 only had an effect AVE-8134, AY-31637, BAY-549801, bexarotene, in the CF macrophages is consistent with the hypothesis that BM-131246, BM-501050, CLX-0921, CLX-0940, DRF a defect PPARC. is at least partly responsible for the increase 10945, DRF-4832, E-3030, fargilitazar, fenofibrate/met in TNFC. secretion. This is further supported by the data formin, GW-0072, GW-1929, GW-2570, GW-409544 demonstrating that DHA is decreasing TNFC. secretion (Glaxo-Wellcome), GW-409890, GW-501516, GW-5393, through its actions as a PPAR agonist and not by affecting GW-590735, GW-7282, GW-9578, KRP-101, KRP297 the n-6 arachidonate pathway based on normal DHA and (Kyorin Merck), KT-6207, L-764406, LF-200337, arachidonate levels as well as the lack of effect of EPA. LG-101506, LR-90, LY-465608, LY-510929, LY-518674, MBX-102, MK-0767, muraglitazar, naveglitazar, NC-2 100, EXAMPLE 9 NS-220, ONO-5129, oxeglitazar, PD-72953, R-119702, ragaglitazar, reglitazar, SB-219994, tesaglitazar, 641597, Expression of LXR is Decreased in Macrophages and TY-51501. For additional descriptions of dual PPARC/ from CFTR Mice PPARY agonists see U.S. Pat. No. 6,414,002 and Murakami 0114 PPARs exert their effects largely through binding to et al., Diabetes 47: 1841-1847, 1998. RXR in the nucleus. LXR is another member of the family 0116 Combinations of any of these therapeutic com of nuclear receptors and also binds to RXR and downregu pounds are also contemplated by the invention. For example, lates inflammation as well as activates lipid metabolism a combination of a thiazolidinediones, such as rosiglitaZone including cholesterol efflux. To determine expression of or pioglitaZone, with a non-steroidal anti-inflammatory drug LXR in macrophages in the absence of CFTR, peritoneal or an anti-oxidant can be used in the methods of the macrophages were prepared from both wild-type and cfir invention. mice. Equal amounts of protein were run on this SDS 0.117) The therapeutic compounds of the invention may polyacrylamide gel. Western blotting was performed using act as an agonist or an inducer of either PPARY or PPARC. an antibody again LXRC. As can be seen in FIG. 19, LXR or and PPARy- or PPARC-like receptors, or any combination (40 kDa) is markedly diminished in cfir' macrophages (2 thereof. It will be recognized by the skilled artisan that samples) compared with wild-type controls. although a compound may be classified as a regulator of Therapeutic Compounds PPARY or PPARC, this classification is not intended to be limiting. The compound may affect both pathways and may 0115 We have discovered that changes in PPARs, spe also affect additional pathways. For example, DHA is a cifically PPARY and PPARC, expression and activity occur PPARY agonist but can also act as a stimulator of PPARY in tissues specifically regulated by CFTR. Non-limiting activity. examples of the therapeutic compounds useful in the meth ods of the invention include agonists or inducers of PPARs, 0118 Inducers of PPAR include any compound that including PPARy, PPARC, PPARö, or any combination increases the biological activity or expression level of any US 2006/01 60867 A1 Jul. 20, 2006

one or more PPAR genes. For example, there are a variety a mutation in the CFTR gene can be identified by the of natural and synthetic ligands exist that cause PPARY methods of the present invention. A candidate compound activation. For example, arachidonic acid metabolites can be identified for its ability to affect the biological activity including prostaglandin J2 and hydroxyoctadecanoic acid as of a PPAR or the expression of a PPAR gene. While the well as C-linolenic acid, eicosapentaenoic acid (EPA; exemplary methods described below refer to PPARy, it will C20:5n-3), and DHA stimulate PPARY activity. DHA is be understood that the methods can be used as screening synthesized in peroxisomes through beta oxidation and, assays to identify compounds that promote the expression or because PPARY influences beta oxidation, it is likely that biological activity of any PPAR gene or protein. PPARY induction increases DHA synthesis within the cells (see below). Therefore, the low DHA and PPAR levels could 0.125. Any number of methods are available for carrying be due to either low PPAR expression leading to low DHA out Screening assays to identify new candidate compounds synthesis in peroxisomes. Alternatively, low DHA levels that promote the expression of a PPARY gene. In one results in decreased PPAR activation, thereby decreasing example, candidate compounds are added at varying con centrations to the culture medium of cultured cells express PPAR expression. It should be noted, however, that DHA is ing a PPARY gene. Gene expression is then measured, for a more portent ligand for PPARC. than for PPARy. example, by microarray analysis, northern blot analysis (see, 0119) Additional therapeutic compounds useful in the for example, Short Protocols in Molecular Biology, ed. methods of the invention include any LXR agonists. Ausubel, et al., (1989)), or RT-PCR, using any appropriate Examples of LXRagonists include GW3965 and T0901317 fragment prepared from the PPARY nucleic acid molecule as (Cayman Chemical Co., Ann Arbor, Mich.). T0901317 is a a hybridization probe. The level of PPARY gene expression highly selective LXR agonist that has been shown both in in the presence of the candidate compound is compared to vitro and in vivo to regulate LXR target genes such as the level measured in a control culture medium lacking the ABCA1. See, for example, Chisholm, J., Lipid Res. candidate compound. A compound which promotes an 44:2039–2048, 1996; Joseph et al., Proc. Natl. Acad. Sci. increase in the expression of a PPARY gene is considered USA. 99: 7604-7609, 2002; Wu et al., J. Biol. Chem. useful in the invention and may be used as a therapeutic to 278:15565-15570, 2003; and Zaghini et al., J. Biol. Chem. treat a human patient. 277:1324-1331, 2002. 0.126 In another example, the effect of candidate com DHA Reduces Pathology in CF Mice pounds may be measured at the level of PPARY protein 0120) Docosahexaenoic acid (DHA) levels are decreased production using standard immunological techniques, Such in plasma of cystic fibrosis patients (Roulet et al., Eur. J. as western blotting or immunoprecipitation with an antibody Pediatr. 156: 952-956, 1997) as well as in CFTR regulated specific for the PPARY protein. Polyclonal or monoclonal tissues from cfir mice (Freedman et al., Proc. Natl. Acad. antibodies that are capable of binding to a PPARY protein Sci. USA 96: 13995-14000, 1999). Docosahexaenoic acid may be used in any standard immunoassay format (e.g., biosynthesis requires a beta-oxidation step which occurs in ELISA, western blot, or RIA assay) to measure the level of peroxisomes. Since PPARs regulate the expression of acyl the protein. In some embodiments, a compound that pro coenzyme-A oxidase gene, a key element in fatty acid motes an increase in PPARY expression or biological activity B-oxidation, a deficit in PPARY expression would produce an is considered particularly useful. alteration in peroxisomal function possibly resulting in low docosahexaenoic acid levels. 0.127 Expression of a reporter gene that is operably linked to a PPARY promoter can also be used to identify a 0121) Dietary DHA supplementation of cfir mice candidate compound for treating a disease associated with a increases phospholipids-bound DNA levels in the blood and CFTR mutation. Assays employing the detection of reporter reduces lung inflammation following a Pseudomonas LPS gene products are extremely sensitive and readily amenable challenge as measured by the neutrophil concentration in a to automation, hence making them ideal for the design of broncho-alveolar lavage (BAL). No significant effects on high-throughput screens. Assays for reporter genes may TNFO, MIP-2, or KC were measured. Instead, a selective employ, for example, calorimetric, chemiluminescent, or decrease in the eicosanoids PGE 6-keto-PGF, PGF fluorometric detection of reporter gene products. Many and thromboxane B. PGE2 is a potent neutrophil chemoat varieties of plasmid and viral vectors containing reporter tractant and its reduction underlies the reduced neutrophil gene cassettes are easily obtained. Such vectors contain recruitment into the lung following the LPS challenge. cassettes encoding reporter genes such as lac7/B-galactosi 0122 DHA also inhibits apoptosis in tissues normally dase, green fluorescent protein, and luciferase, among oth expressing high CFTR levels. For example, DHA treatment ers. A genomic DNA fragment carrying a PPARy-specific decreases villi height in the ileum of cftr mice. transcriptional control region (e.g., a promoter and/or enhancer) is first cloned using standard approaches (such as 0123 The loss of ion channel function is also mitigated those described by Ausubel et al., supra). The DNA carrying by DHA treatment. DHA, at nanomolar concentrations, the PPARY transcriptional control region is then inserted, by activate fast Sodium channels (plasma membrane) and cal DNA Subcloning, into a reporter vector, thereby placing a cium channels (sarcoplasmic reticulum) in cardiac myo vector-encoded reporter gene under the control of the PPARY cytes. Additionally, in T84 colon cancer cells, DHA transcriptional control region. The activity of the PPARY enhances carbachol-stimulated chloride conductance with transcriptional control region operably linked to the reporter out affecting cAMP-stimulated chloride conductance. gene can then be directly observed and quantified as a Identification of Candidate Compounds function of reporter gene activity in a reporter gene assay. 0.124. A candidate compound that is beneficial in the 0128. In one embodiment, for example, the PPARY tran treatment (reduction or prevention of symptoms) caused by Scriptional control region could be cloned upstream from a US 2006/01 60867 A1 Jul. 20, 2006 luciferase reporter gene within a reporter vector. This could orally, parenterally (e.g. intramuscular, intraperitoneal, be introduced into the test cells, along with an internal intravenous, or Subcutaneous injection), topically, locally, or control reporter vector (e.g., a lacZ gene under the transcrip by intrathecal or intracerebroventricular injection in an tional regulation of the B-actin promoter). After the cells are admixture with a pharmaceutically acceptable carrier exposed to the test compounds, reporter gene activity is adapted for the route of administration. measured and PPAR-Y reporter gene activity is normalized 0.132 Methods well known in the art for making formu to internal control reporter gene activity. lations are found, for example, in Remington: The Science 0129. A candidate compound identified by the methods and Practice of Pharmacy, 20th edition, 2000, ed. A. R. of the present invention can be from natural as well as Gennaro, Lippincott Williams & Wilkins, Philadelphia. synthetic sources. Those skilled in the field of drug discov Compositions intended for oral use may be prepared in Solid ery and development will understand that the precise Source or liquid forms according to any method known to the art for of test extracts or compounds is not critical to the methods the manufacture of pharmaceutical compositions. The com of the invention. Examples of Such extracts or compounds positions may optionally contain Sweetening, flavoring, col include, but are not limited to, plant-, fungal-, prokaryotic-, oring, perfuming, and/or preserving agents in order to pro or animal-based extracts, fermentation broths, and synthetic vide a more palatable preparation. Solid dosage forms for compounds, as well as modification of existing compounds. oral administration include capsules, tablets, pills, powders, Numerous methods are also available for generating random and granules. In Such solid forms, the active compound is or directed synthesis (e.g., semi-synthesis or total synthesis) admixed with at least one inert pharmaceutically acceptable of any number of chemical compounds, including, but not carrier or excipient. These may include, for example, inert limited to, saccharide-, lipid-, peptide-, and nucleic acid diluents, such as calcium carbonate, sodium carbonate, based compounds. Synthetic compound libraries are com lactose, Sucrose, starch, calcium phosphate, Sodium phos mercially available from Brandon Associates (Merrimack, phate, or kaolin. Binding agents, buffering agents, and/or N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alterna lubricating agents (e.g., magnesium Stearate) may also be tively, libraries of natural compounds in the form of bacte used. Tablets and pills can additionally be prepared with rial, fungal, plant, and animal extracts are commercially enteric coatings. available from a number of sources, including Biotics (Sus 0.133 Liquid dosage forms for oral administration sex, UK), Xenova (Slough, UK), Harbor Branch Ocean include pharmaceutically acceptable emulsions, Solutions, graphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. Suspensions, syrups, and soft gelatin capsules. These forms (Cambridge, Mass.). In addition, natural and synthetically contain inert diluents commonly used in the art, Such as produced libraries are produced, if desired, according to water or an oil medium. Besides Such inert diluents, com methods known in the art, e.g., by standard extraction and positions can also include adjuvants, such as wetting agents, fractionation methods. Furthermore, if desired, any library emulsifying agents, and Suspending agents. or compound is readily modified using standard chemical, physical, or biochemical methods. 0.134) Formulations for parenteral administration include sterile aqueous or non-aqueous Solutions, Suspensions, or Administration of Therapeutics emulsions. Examples of Suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydro 0130. The present invention also includes the adminis genated naphalenes, and injectable organic esters, such as tration of a PPAR inducer, PPAR agonist, an antioxidant, or ethyl oleate. Such formulations may also contain adjuvants, any combination thereof, for the treatment of a disease Such as preserving, wetting, emulsifying, and dispersing associated with a CFTR mutation. Therapeutics of this agents. Biocompatible, biodegradable lactide polymer, lac invention may be formulated as pharmaceutically acceptable tide/glycolide copolymer, or polyoxyethylene-polyoxypro salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical pylene copolymers may be used to control the release of the industry. Examples of acid addition salts include organic compounds. Other potentially useful parenteral delivery acids such as acetic, lactic, pamoic, maleic, citric, malic, systems for the proteins of the invention include ethylene ascorbic, succinic, benzoic, palmitic, Suberic, salicylic, tar vinyl acetate copolymer particles, osmotic pumps, implant taric, methanesulfonic, toluenesulfonic, or trifluoroacetic able infusion systems, and liposomes. acids or the like; polymeric acids such as tannic acid, 0.135 Liquid formulations can be sterilized by, for carboxymethyl cellulose, or the like; and inorganic acids example, filtration through a bacteria-retaining filter, by Such as hydrochloric acid, hydrobromic acid, Sulfuric acid incorporating sterilizing agents into the compositions, or by phosphoric acid, or the like. Metal complexes include Zinc, irradiating or heating the compositions. Alternatively, they iron, and the like. One exemplary pharmaceutically accept can also be manufactured in the form of sterile, solid able carrier is physiological Saline. Other physiologically compositions which can be dissolved in sterile water or acceptable carriers and their formulations are known to one some other sterile injectable medium immediately before skilled in the art and described, (see, e.g., Remington: The SC. Science and Practice of Pharmacy, 20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, 0.136 The amount of active ingredient in the composi and Encyclopedia of Pharmaceutical Technology, eds. J. tions of the invention can be varied. One skilled in the art will appreciate that the exact individual dosages may be Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, adjusted somewhat depending upon a variety of factors, New York). including the protein being administered, the time of admin 0131 Pharmaceutical formulations of a therapeutically istration, the route of administration, the nature of the effective amount of a compound of the invention, or phar formulation, the rate of excretion, the nature of the subjects maceutically acceptable salt-thereof, can be administered conditions, and the age, weight, health, and gender of the US 2006/01 60867 A1 Jul. 20, 2006 patient. Generally, dosage levels of between 0.1 g/kg to 100 oped and have been used in clinical settings (Rosenberg et mg/kg of body weight are administered daily as a single dose al., N. Engl. J. Med. 323:370, 1990; Anderson et al., U.S. or divided into multiple doses. Desirably, the general dosage Pat. No. 5,399.346). Most preferably, a viral vector is used range is between 250 ug/kg to 5.0 mg/kg of body weight per to administer the gene of interest to a target endothelial cell. day. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of 0140. Non-viral approaches can also be employed for the administration. For instance, oral administration generally introduction of therapeutic nucleic acids to a cell of a would be expected to require higher dosage levels than patient. For example, a nucleic acid molecule can be intro administration by intravenous injection. Variations in these duced into a cell by administering the nucleic acid in the dosage levels can be adjusted using standard empirical presence of lipofection (Felgner et al., Proc. Natl. Acad. Sci. routines for optimization, which are well known in the art. U.S.A. 84:7413, 1987: Ono et al., Neuroscience Letters In general, the precise therapeutically effective dosage will 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, be determined by the attending physician in consideration of 1989; Staubinger et al., Methods in Enzymology 101.512, the above-identified factors. 1983), asialoorosomucoid-polylysine conjugation (Wu et al., J. Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. 0137 The therapeutics of the invention can be adminis Chem. 264:16985, 1989), or by micro-injection under sur tered in a Sustained release composition, such as those gical conditions (Wolff et al., Science 247: 1465, 1990). described in, for example, U.S. Pat. No. 5,672,659 and U.S. Preferably the nucleic acids are administered in combination Pat. No. 5,595,760. The use of immediate or sustained with a liposome and protamine. release compositions depends on the type of condition being treated. If the condition consists of an acute or Subacute 0.141 Gene transfer can also be achieved using non-viral disorder, a treatment with an immediate release form will be means involving transfection in vitro. Such methods include preferred over a prolonged release composition. Alterna the use of calcium phosphate, DEAE dextran, electropora tively, for preventative or long-term treatments, a Sustained tion, and protoplast fusion. Liposomes can also be poten released composition will generally be preferred. tially beneficial for delivery of DNA into a cell. Transplan tation of normal genes into the affected tissues of a patient Gene Therapy can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous 0138 Gene therapy is another therapeutic approach for or heterologous primary cell or progeny thereof), after which increasing PPAR biological activity. Heterologous nucleic the cell (or its descendants) are injected into a targeted acid molecules encoding a PPAR protein can be delivered to tissue. the affected cells (e.g., lung epithelium). Expression of PPARY proteins in the target cells can ameliorate the symp 0.142 cl DNA expression for use in gene therapy methods toms associated with CFTR dysfuntion. The nucleic acid can be directed from any Suitable promoter (e.g., an endocan molecules must be delivered to those cells in a form in which promoter, Flt-1 promoter, or other tumor endothelial pro they can be taken up by the cells and so that sufficient levels moter identified using methods known in the art), and of protein can be produced to increase the PPAR biological regulated by any appropriate mammalian regulatory element activity. (see for example Davis et al. (1986) Basic Methods In Molecular Biology, Maniatis et al. Molecular Cloning. A 0139 Transducing viral (e.g., retroviral, adenoviral, and Laboratory Manual, 2nd Edition (1989), and Short Proto adeno-associated viral) vectors can be used for Somatic cell cols in Molecular Biology, ed. Ausubel, et al., (1989)). For gene therapy, especially because of their high efficiency of example, if desired, an enhancers known to preferentially infection and stable integration and expression (see, e.g., direct gene expression in a tumor endothelial cell, (e.g., the Cayouette et al., Human Gene Therapy 8:423-430, 1997: 300 base pair Tie-2 intronic enhancer element described Kido et al., Current Eye Research 15:833-844, 1996: herein) can be used to direct the expression of a nucleic acid. Bloomer et al., Journal of Virology 71:6641-6649, 1997: The enhancers used can include, without limitation, those Naldini et al., Science 272:263-267, 1996; and Miyoshi et that are characterized as tissue- or cell-specific enhancers. al., Proc. Natl. Acad. Sci. U.S.A. 94:103.19, 1997). For Alternatively, if a genomic clone is used as a therapeutic example, a full length PPAR gene, or a portion thereof, can construct, regulation can be mediated by the cognate regu be cloned into a retroviral vector and expression can be latory sequences or, if desired, by regulatory sequences driven from its endogenous promoter, from the retroviral derived from a heterologous source, including any of the long terminal repeat, or from a promoter specifically promoters or regulatory elements described above. expressed in a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a 0.143 Another therapeutic approach included in the bovine papilloma virus, or a herpes virus, Such as Epstein invention involves administration of a recombinant nuclear Barr Virus (also see, for example, the vectors of Miller, encoded mitochondrial metabolism or proteasomal polypep Human Gene Therapy 15-14, 1990; Friedman, Science tide, either directly to the site of a potential or actual 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608 disease-affected tissue (for example, by injection into the 614, 1988: Tolstoshev et al., Current Opinion in Biotech ventricles of the brain or into the cerebrospinal fluid) or nology 1:55-61, 1990; Sharp, Lancet 337:1277-1278, 1991; systemically (for example, by any conventional recombinant Cornetta et al., Nucleic Acid Research and Molecular Biol protein administration technique). The dosage of the admin ogy 36:311-322, 1987; Anderson, Science 226:401-409, istered protein depends on a number of factors, including the 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., size and health of the individual patient. For any particular Biotechnology 7: 980-990, 1989; Le Gal La Salle et al., Subject, the specific dosage regimes should be adjusted over Science 259:988-990, 1993; and Johnson, Chest 107: 77S time according to the individual need and the professional 83S, 1995). Retroviral vectors are particularly well devel judgment of the person administering or Supervising the US 2006/01 60867 A1 Jul. 20, 2006

administration of the compositions. Generally, between 0.1 S-adnenosyl methionine, selenium, beta-carotene, ide mg and 100 mg, is administered per day to an adult in any benone, cysteine, dithioerythritol, dithionite, dithiothreitol, pharmaceutically acceptable formulation. and pyrosulfite. 10. The method of claim 1, wherein said PPAR agonist is OTHER EMBODIMENTS a PPARC. agonist. 0144 All publications and patent applications cited in 11. The method of claim 10, wherein said PPARC. agonist this specification are herein incorporated by reference as if is selected from the group consisting of DHA, fibrates, and each individual publication or patent application were spe cifically and individually indicated to be incorporated by Wy 14643. reference. Although the foregoing invention has been 12. The method of claim 11, wherein said fibrate is described in some detail by way of illustration and example selected from the group consisting of fenofibrate, beZafi for purposes of clarity of understanding, it will be readily brate, gemfibrozil, and analogs thereof. apparent to those of ordinary skill in the art in light of the 13. The method of claim 1, wherein said PPAR agonist is teachings of this invention that certain changes and modi a PPARö, agonist. fications may be made thereto without departing from the 14. The method of claim 1, wherein said PPAR agonist is spirit or scope of the appended claims. a PPARC agonist and said disease is bile duct injury or cystic What is claimed is: fibrosis liver disease. 1. A method for treating a disease in a human patient, 15. The method of claim 1, wherein said mutation is a wherein said patient has a mutation in the CFTR gene and deletion of F508. wherein said mutation is associated with said disease, said 16. A method for treating a disease in a human patient, method comprising administering to said patient a therapeu wherein said patient has a mutation in the CFTR gene and tically effective amount of a peroxisome proliferator-acti wherein said mutation is associated with said disease, said vated receptor (PPAR) agonist. method comprising administering to said patient a therapeu 2. The method of claim 1, wherein said disease is cystic tically effective amount of a PPARC. agonist and a thera fibrosis. peutically effective amount of a PPARY agonist. 3. The method of claim 1, wherein said disease is selected 17. A method for treating a disease in a human patient, from the group consisting of pancreatitis, chronic obstruc wherein said patient has a mutation in the CFTR gene and tive pulmonary disease (COPD), asthma, chronic sinusitis, wherein said mutation is associated with said disease, said primary Sclerosing cholangitis, bile duct injury, liver dis method comprising administering to said patient a therapeu ease, and congenital bilateral absence of the vas deferens. tically effective amount of a dual PPARO/PPARY agonist. 4. The method of claim 1, wherein said PPAR agonist is 18. A method for treating a disease in a human patient, a PPARY agonist. wherein said patient has a mutation in the CFTR gene and 5. The method of claim 4, wherein said PPARY is a wherein said disease is associated with said mutation, said PPARy1. method comprising administering to said patient a therapeu 6. The method of claim 4, wherein said PPARY agonist is selected from the group consisting of thiazolidinediones, tically effective amount of an antioxidant. L-tyrosine derivatives, fluoromethyloxycarbonyl, non-ste 19. The method of claim 18, wherein said antioxidant is roidal anti-inflammatory drugs, and anti-oxidants. a PPARy inducer. 7. The method of claim 6, wherein said thiazolidinedione 20. The method of claim 18, wherein said antioxidant is is selected from the group consisting of pioglitaZone, rosigli selected from the group consisting of vitamin E. Vitamin C, taZone, troglitaZone, and analogs thereof. S-adnenosyl methionine, selenium, beta-carotene, ide 8. The method of claim 6, wherein said non-steroidal benone, cysteine, dithioerythritol, dithionite, dithiothreitol, anti-inflammatory drug is ibuprofen or naprosyn. and pyrosulfite. 9. The method of claim 6, wherein said antioxidant is selected from the group consisting of Vitamin E. Vitamin C,