WO2004098510A2

WO2004098510A2 - Cystic fibrosis therapy

Info

Publication number: WO2004098510A2
Application number: PCT/US2004/013412
Authority: WO
Inventors: Steven D. Freedman
Original assignee: Beth Israel Deaconess Medical Center
Priority date: 2003-04-30
Filing date: 2004-04-30
Publication date: 2004-11-18
Also published as: WO2004098510A3

Abstract

This invention features methods for treating diseases associated with mutations in the CFTR gene by administering PPAR-Ϝ inducers and/or antioxidants. Also disclosed are screening methods for identifying therapeutically useful candidate compounds.

Description

CYSTIC FIBROSIS THERAPY

This invention was funded by grant D52765 from the National Institute of Health. The government may have certain rights in the invention

Field of the Invention

The present invention relates to the treatment of cystic fibrosis and other diseases associated with mutations in the CFTR gene.

Background of the Invention

Approximately one in 2000 Caucasians have cystic fibrosis (CF), a genetic disorder caused by inactivating mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR protein is a member of the ABC transporter family which forms a chloride channel localized to the plasma membrane. The protein consists of five domains: two membrane-spanning domains that form the chloride ion channel, two nucleotide-binding domains that hydrolyze ATP, and a regulatory domain. Expression of the CFTR gene is highest in cells that line passageways of the lungs, pancreas, colon, ileum, and genitourinary tract.

In addition to CF, defects in the CFTR gene are associated with diseases including, for example, pancreatitis, chronic obstructive pulmonary disease (COPD), asthma, chronic sinusitis, primary sclerosing cholangitis ,and congenital bilateral absence of the vas deferens (CBAVD).

The most common inactivating mutation of the CFTR gene, detected in about 70% of CF patients, is a deletion of the three base pairs encoding the phenylalanine at amino acid residue 508 (ΔF508). The F508 residue is located in a membrane spamiing domain and its deletion causes incorrect folding of the newly synthesized protein. As a result, misfolded protein is degraded in the endoplasmic reticulum shortly after synthesis. Patients having a homozygous ΔF508 deletion tend to have the most severe symptoms of cystic fibrosis resulting from a loss of chloride ion transport. The disturbance in the sodium and chloride ion balance in the cells lining the respiratory tract results in a thick, sticky mucus layer that is not easily removed by the cilia. The altered mucus also traps bacteria, resulting in chronic infections. Accordingly, most CF therapy is directed to controlling persistent and often fatal lung infections. There is a need for improved therapies that treat the underlying causes of CF and other CFTR-related diseases.

Summary of the Invention The invention features a method for treating a disease in a human patient that has a mutation in the CFTR gene by administering to the patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPARγ) inducer, a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, an AP-1 inhibitor, a STAT inhibitor, or an NFkB inhibitor. Diseases caused by mutations in a CFTR gene include, for example, cystic fibrosis, pancreatitis, chronic obstructive pulmonary disease (COPD), asthma, chronic sinusitis, primary sclerosing cholangitis, and congenital bilateral absence of the vas deferens. Particularly amenable to treatment are diseases caused by a deletion of the phenylalanine normally present at amino acid residue 508 of the CFTR protein (ΔF508). The patients being treated according to the methods of this invention may be heterozygous or homozygous for a CFTR mutation. Useful PPARγ inducers and agonists affect any PPARγ, but particularly

PPARγ 1 and PPARγ2. Particularly useful PPARγ inducers include, for example, eicosapentaenoic acid, any of the thiazolidinediones, but particularly troglitazone, L-tyrosine derivatives such as fluoromethyloxycarbonyl, non- steroidal anti-inflammatory drugs such as indomethacin, ibuprofen, and fenoprofen.

Particularly useful PPARγ ligands include the thiazolidinediones, but particularly rosiglitazone.

Useful AP-1 inhibitors include, for example, nordihydroguaiaretic acid, SP600125, SRI 1302, pyrrolidine dithiocarbamate, curcumin, PD98059, and the spiro compounds (U.S. Patent 6,384,065, hereby incorporated by reference).

Useful STAT inhibitors include, for example, the SSI-1, SSI-2, and SSI- 3 proteins. These proteins may be administered by any suitable route (e.g., inhalation, intravenous, intramuscular, or subcutaneous injection). Alternatively, they can be expressed by the target cells in the patient using gene therapy techniques. Useful STAT inhibitors include, for example, tripeptides having the sequence proline-tyrosine-leucine or alanine-tyrosine-leucine, wherein said tyrosine is phosphorylated (phospho-tyrosine).

Useful NFkB inhibitor include, for example, 2-chloro-N-[3,5- di(trifluoromethyl)phenyl]-4-(trifluoromethyl)pyrimidine-5 -carboxamide (SP- 100030); 3 ,4-dihydro-4,4-dimethyl-2H- 1 ,2-benzoselenazine (BXT-51072); declopramide (Oxi-104), dexlipotam, a salicylanilide (see, U.S. Patent 6,492,425, hereby incorporated by reference, 2-hydroxy-4- trifluoromethylbenzoic (HTB) acid and its derivatives (e.g., triflsal); see U.S. Patent 6,414,025, hereby incorporated by reference).

The invention also features methods for identifying compounds useful for treating a disease in a patient having a mutation in the CFTR gene and wherein said mutation is associated with said disease. In one aspect, the method has the steps of: (i) providing cells that express a PPARγ, (ii) contacting the cells with a candidate compound, and (iii) assessing the level of PPARγ expression in the cells relative to the level of PPARγ expression in the absence of the candidate compound, wherein a candidate compound that increases the level of PPARγ expression is identified as a compound useful for treating said disease. PPARγ expression may be assessed using any appropriate technique known to those skilled in the art. Techniques include, for example, Western blotting and RNA analysis (e.g, Northern blotting). In another aspect, the method has the steps of: (i) providing cells that express a PPARγ protein, (ii) contacting the cells with a candidate compound, and (iii) assessing the half life of said PPARγ protein in the cells relative to the half life of said PPARγ protein in the absence of the candidate compound, wherein a candidate compound that increases the half life is identified as a compound useful for treating the disease.

In another aspect, the method has the steps of: (i) providing cells that express a PPARγ, (ii) contacting the cells with a candidate compound, and (iii) assessing the level of PPARγ translocation to the nucleus of the cells relative to the level of PPARγ expression in the absence of the candidate compound, wherein a candidate compound that increases the level of PPARγ translocation to the nucleus is identified as a compound useful for treating said disease. Immunohistorchemistry is a particularly useful method for determining PPARγ nuclear translocation.

Any cells that express any PPARγ are useful in these screening methods. Particularly useful cells include, for example, pancreatic exocrine cells, lung cells, intestinal cells, bile duct cells, or macrophages. Alternatively, cells engineered to express a recombinant PPARγ gene are also useful. Particularly useful PPARγ isoforms include, for example, PPARγ 1 and PPARγ2.

The invention also features a method for treating a disease in a human patient that has a mutation in the CFTR gene by administering to the patient a therapeutically effective amount of an antioxidant. Antioxidants useful in the methods of this invention include, for example, vitamin E, vitamin C, S- adnenosyl methionine, selenium, vitamin C, beta-carotene, idebenone, cysteine, dithioerythritol, dithionite, dithiothreitol, and pyrosulfite. By "biological activity," when referring to PPARγ, is meant any effect on cell physiology normally associated with the activation of this receptor. One important PPARγ biological activity is its translocation to the nucleus of the cell. Other assays for PPARγ biological activity are based on the ability of PPARγ to bind to the RXR receptor. Alternatively, PPARγ biological activity can be measured using a reporter gene operably linked to a PPARγ-inducible promoter and assessing expression of the reporter gene. Biological activity can be measured using any appropriate methodology known in the art (see, for example, Kliewer et al., Proc. Natl. Acad. Sci. USA 94: 4318-4323, 1997). By "PPARγ inducer" is meant any compound that increases the biological activity of PPAR-γ in a cell. PPARγ inducers may increase biological activity by post-transcriptionally activating PPARγ. Alternatively, PPAR-γ inducers may increase the expression of one or more of the PPARγ genes. By "PPARγ agonist" is meant any compound that increases binding to a

PPARγ and increases its biological activity (i.e., causes translocation of the PPARγ to the nucleus).

By "therapeutically effective amount" is meant an amount sufficient to provide medical benefit. By "substantially pure," when referring to a naturally occurring compound (i.e., EPA) is meant a compound that has been partially or totally separated from the components that naturally accompany it. Typically, the compound is substantially pure when it is at least 50%, 60%, 70%, 80%, 90% 95%, or even 99%, by weight, free from the organic molecules with which it is naturally associated. For example, substantially pure EPA may be obtained by extraction from a natural source such as fish oil. Alternatively, chemical synthesis of EPA may result in a totally pure product. Brief Description of Drawings

FIGURE 1 is a bar graph comparing PPARγ mRNA expression levels in various tissues of CFTR^{" "} and wild-type mice. mRNA expression of total PPARγ was analyzed in colon, ileum, fat, liver and lung from wild type (WT) and cftr^"7" (CF) mice. RNA extracts were subjected to quantitative RT-PCR. Values for cftr^{" "} tissues are expressed relative to WT, where 100% is the mean value in each of the respective tissues. Data are expressed as means ± SEM (n=5). *p<0.05.

FIGURE 2 is a bar graph illustrating CFTR expression in various tissues of the wild-type mice used in Figure 1. CFTR is significantly expressed in mouse tissues showing decreased PPARg expression. mRNA expression of CFTR was analyzed in colon, ileum, lung, fat, and liver from wild-type mice. RNA extracts were subjected to quantitative RT-PCR. Values are represented as the ratio between the respective mRNA and 18s ribosomal RNA levels. Data are expressed as means ± SEM (n=4).

FIGURE 3 is a series of photomicrographs showing the immunohistochemical distribution of PPARγ in ileum, colon and lung. PPARγ immunohistochemistry was performed on colon (FIGURES 3 A and 3B), ileum (FIGURES 3C and 3D) and lung (FIGURES 3E and 3F) from wild-type (FIGURES 3A, 3B, and 3E) and cftr^"7" (FIGURES 3B, 3D, and 3F) mice.

Tissue sections were stained with a rabbit polyclonal anti- PPARγ antibody and a biotinylated secondary antibody. Sections from cftr^{" "} and wild type mice correspond to equivalent tissue regions. Magnification is 200x for FIGURES 3A-D and lOOx for FIGURES 3E and 3F. Incubation in the absence of primary antibody showed no staining.

FIGURE 4A and 4B are a Western blots of PPARγ protein expression in the nuclear and cytosolic compartments of colon and fat cells. Western blot analysis of PPARγ was performed on nuclear (Nuc) and cytosolic (Cyt) extracts from colon (FIGURE 4A) and perigonadal fat (FIGURE 4B) from wild type (WT) and cftr^{" "} (CF) mice. Protein extracts were subjected to Western blotting using a rabbit polyclonal anti- PPARγ antibody. Samples from two wild-type and two cftr^"7" mice are shown from the colon. FIGURE 4C is a bar graph showing the densitometric quantification for colonic samples from 3 wild-type and 3 cftr^{" "} mice. Background was subtracted from bands. Values are expressed as the mean ± SEM relative to WT, where 100% is the mean value.

FIGURE 5 is a series of photomicrographs showing the immunohistochemical distribution of PPARγ in intestinal epithelium of cftr^"7" mice after rosiglitazone treatment. Wild-type and cftr^"7" mice were given rosiglitazone by gavage for 9 days. Colon (FIGURE 5 A and 5B) and ileum

(FIGURE 5C and 5D) were analyzed from wild-type (FIGURE 5A and 5C) and cftr^"7" (FIGURE 5B and 5D) mice. Magnification is 200x.

FIGURE 6 is a series of bar graphs showing the PPARγ mRNA expression in the colon (FIGURE 6A) and ilium (FIGURE 6B) following rosiglitazone treatment of wild-type (WT) and cftr^"7" (CF) mice. RNA extracts were subjected to quantitative RT-PCR. Values are expressed relative to WT, where 100% is the mean value in each of the respective tissues. Data are expressed as means ± SEM (n=5).

FIGURE 7 is a series of electrophoretic gels showing the differential PPARγ binding to PPRE in mouse colonic mucosa. PPARγ DNA binding was analyzed by EMS A in colonic mucosa of wild type (WT) and cftr^"7"(CF) mice. Colon nuclear extracts were used as a source of protein. Oligonucleotide probes carrying a perfect DR1 motif of the PPRE from the acyl-CoA oxidase promoter were used as probes. Each lane contains protein sample from a different mouse and 3 different samples from each genotype were used.

FIGURE 7A shows an electrophoretic mobility shift assay. FIGURE 7B is a competition binding experiment using unlabled oligonucleotide. A 100-fold excess of the synthetic PPRE was used. FIGURE 7C is a supershift assay of samples from WT and CF mice treated with rosiglitazone. A rabbit polyclonal anti- PPARγ antibody against the C-terminus was used.

Detailed Description

Ligands (agonists) and inducers of peroxisome proliferator-activated receptors (PPARs), particularly inducers of PPAR-γ, are useful for treating diseases caused by mutations in the CFTR gene. Antioxidants, alone or in combination with PPAR ligands and inducers, are also useful for treating these diseases. Diseases amenable to treatment include, for example, cystic fibrosis (CF), pancreatitis, chronic obstructive pulmonary disease (COPD), asthma, chronic sinusitis, primary sclerosing cholangitis, and congenital bilateral absence of the vas deferens.

A fatty acid imbalance in plasma from cystic fibrosis patients and in tissues from cftr^{" "} mice has been extensively reported, first as an essential fatty acid deficiency (Farrel et al., Pediatr. Res. 19: 104-109, 1985) and more recently as an increase in arachidonic and a concomitant decrease in docosahexaenoic acids (Freedman et al., Proc. Natl. Acad. Sci. USA, 96: 13995-14000, 1999). This defect has been suggested to play a role in the increased inflammatory response in cystic fibrosis, since arachidonic acid is the precursor of a number of eicosanoids and inflammatory mediators. Both fatty acids have independent biosynthetic precursors, and although they use similar processes and share some of the enzymes involved, docosahexaenoic acid synthesis requires an additional β-oxidation step that takes place in peroxisomes. Impairment in the peroxisomal β-oxidation of docosahexaenoic acid precursors would lead to low docosahexaenoic acid and high arachidonic acid levels. Peroxisome Proliferator-activated Receptors (PPARs)

Peroxisome proliferator-activated receptors (PPARs) are a subfamily of ligand-activated transcription factors. They act by binding as heterodimers with a retinoid-X receptor (RXR) to specific DNA sequences known as peroxisome proliferator responsive elements (PPRE) (reviewed in Berger et al., Annu. Rev. Med., 53: 409-435, 2002). The PPAR genes were discovered in 1990, when found to be activated by peroxisome proliferators. PPARγ has two isoforms, 1 and 2. PPARγ2 is mostly expressed in adipose tissue while PPARγ 1 is more widely distributed including small and large intestine. PPARγ is a key element in lipid metabolism, by regulating the expression of a number of genes, such as the fatty acid-binding protein aP, phosphoenolpyruvate carboxykinase, acyl- CoA synthase, lipoprotein lipase, the fatty acid transport protein- 1, CD36, and leptin. In general, PPARγ activation augments lipid catabolism and induces differentiation of fϊbroblasts into adipocytes. PPAR-γ also regulates peroxisomal proliferation and lipid metabolism by increasing beta oxidation. Because DHA is synthesized in peroxisomes by beta oxidation, PPAR inducers increase DHA levels in cells, attenuating or reversing the effects of CFTR deletions.

PPARγ activation has also been suggested to inhibit cell proliferation in some tumors. Other regulatory functions of PPARγ include the modulation of inflammatory response and the enhancement of insulin sensitivity. Thus, PPARγ agonists inhibit the expression of the proinflammatory and insulin resistance-inducing cytokine TNFα, increase other insulin signaling mediators, and block the NFkB proinflammatory signaling pathway. The PPARγ synthetic agonists thiazolidinediones (TZDs) have been used as anti-diabetic drugs and exert anti-inflammatory effects in the colon. Modulating PPAR Biological Activity for the Treatment of Diseases Caused by Mutations in the CFTR Gene

The results of this experiments described below demonstrate that PPARγ mRNA expression is decreased in those tissues specifically regulated by CFTR (colon, ileum and lung). This was confirmed at the protein level by western blot analysis of colon. Based on immunohistochemistry, the proportion of PPARγ-expressing cells was not decreased in these particular cystic fibrosis tissues from cftr^"7" mice and hence would not explain the lower levels of PPARγ. The fact that no significant differences were found in liver or fat where CFTR RNA levels were found to be extremely low, suggests that CFTR may play a role in modulating PPARγ expression. It should be pointed out that although there is expression of CFTR in bile ducts, these cells represent less than 3% of total cells in the liver.

The results of western blotting and immunohistochemistry also show that the subcellular localization of PPARγ is altered in cftr^"7" mice. This alteration consists of a shift from predominantly nuclear staining in wild-type animals to a diffuse cytoplasmic staining in cftr^"7" mice. Western blot analysis of colonic mucosal scrapings demonstrated that this is mostly due to a decrease in the nuclear presence of PPARγ, and was supported by the decreased binding of the PPARγ RXR complex to PPRE in cftr^"7" colon, as revealed by EMS A. This confirms that not only expression of PPARγ, but also its function as a transcription factor is compromised in cftr^"7" tissues. The fact that administration of rosiglitazone, a PPAR ligand, restored both the nuclear localization of PPARγ in ileum and colon based on immunohistochemistry, and binding to PPRE in colon cells as shown by EMS A, indicates that activation followed by translocation to the nucleus can occur in cftr^"7" mice.

PPARγ has been shown to be expressed in multiple tissues. Adipose tissue and colon are the major organs expressing PPARγ, while lower levels are present in kidney, liver, skin, ileum and mononuclear blood cells (Dubuquoy et al, Lancet 360: 1410-1418, 2002). PPARγ2 mRNA is predominantly expressed in adipocytes with less significant amounts in liver, while PPARγ 1 mRNA is more universally distributed including small and large intestine, kidney, muscle and liver (Fajas et al., J. Biol. Chem. 272: 18779-18789, 1997). Lower but detectable expression levels of PPARγ 1 have also been reported in both mouse and human lung tissue (Lambe et al., Eur. J. Biochem 239: 1-7, 1996; Zhu et al, J. Biol. Chem. 268: 26817-26820, 1993). Expression in lung has been localized in alveolar type-II pneumocytes whereas receptor activity has been found in human airway epithelial cells, as well as in several human lung epithelial cell lines. These results are in agreement with the immunohistochemical results seen in the experiments described below.

The mechanism by which PPARγ expression is decreased in these select CFTR expressing tissues in cftr^{" "} mice may be due to either (i) a reduction in transcription and translation of PPARγ, (ii) shorter half life of the protein, or (iii) a lack of stimulation by endogenous PPARγ ligands. Different ligands show diverse effects on PPARγ mRNA expression. Only troglitazone, unlike rosiglitazone and other high affinity PPARγ ligands, has been shown to upregulate PPARγ expression in nonadipose tissues and cell lines (Davies et al, Mol Cell Biol. Res. Commun. 2: 202-208, 1999; J. Pharmacol. Exp. Ther. 300: 72-77, 2002). The experiments below demonstrate that rosiglitazone induces nuclear translocation of PPARγ but did not increase RNA expression is in agreement with these findings. The mechanism for troglitazone-induced RNA expression of PPARγ may occur through its antioxidant potential, since α- tocopherol shows a similar effect. PPARγ activation has been shown to result in decreased inflammation through inhibition of AP-1, STAT and NFkB pathways in monocytes and macrophages that results in a modulatory effect on cytokine secretion (Jiang et al, Nature 391: 82-86, 1998; Nagy et al, Cell 93: 229-240, 1998; Ricote et al, Nature 391 : 79-82, 1998), inhibition of IL-2 secretion from T cells (Clark et al, J. Immunol. 164: 1364-1371, 2000), and inhibition of NFkB activity in epithelial cells (Su et al, J. Clin. Invest. 104: 383-389, 1999). Thus, a decrease in PPAR expression and function could explain several sequelae that are associated with the cystic fibrosis phenotype such as an excessive host inflammatory response, increased peripheral insulin resistance, and alterations in lipid metabolism within the peroxisomal compartment.

Cystic fibrosis is also associated with a high incidence of impaired glucose tolerance and development of diabetes mellitus. A combination of decreased insulin secretion and increased insulin resistance has been proposed. The former is attributed to pancreatic atrophy and fibrosis characteristic of cystic fibrosis patients, affecting both exocrine and endocrine function. The latter effect on increased peripheral insulin resistance could be explained by an impairment in PPARγ function due to decreased production. Thiazolidinediones (including rosiglitazone and troglitazone), synthetic ligands for PPARγ, are extensively used as a treatment for type 2 (non-insulin dependent) diabetes (Mudaliar et al, Annu Rev. Med. 52: 239-257, 2001). Other compounds that selectively bind to the PPARγ binding domain, such as GW1929, have also been proven to be potent insulin sensitizers in vivo (Brown, et al, Diabetes, 48:1415-1424, 1999). Thus, any of these compounds can be used to treat cystic fibrosis or any other disorder caused by a mutation in the CFTR gene.

DHA Reduces Pathology in CF Mice

Docosahexaenoic acid (DHA) levels are decreased in plasma of cystic fibrosis patients (Roulet et al, Eur. J. Pediatr. 156: 952-956, 1997) as well as in CFTR regulated tissues from cftr^"7" mice (Freedman et al, Proc. Natl. Acad. Sci. USA 96: 13995-14000, 1999). Docosahexaenoic acid biosynthesis requires a beta-oxidation step which occurs in peroxisomes. Since PPARs regulate the expression of acyl coenzyme-A oxidase gene, a key element in fatty acid β- oxidation, a deficit in PPARγ expression would produce an alteration in peroxisomal function possibly resulting in low docosahexaenoic acid levels.

Dietary DHA supplementation of CFTR^{" "} mice increases phospholipids- bound DNA levels in the blood and reduces lung inflammation following a Pseudomonas LPS challenge as measured by the neutrophil concentration in a broncho-alveolar lavage (BAL). No significant effects on TNF-α, MIP-2, or KC were measured. Instead, a selective decrease in the eicosanoids PGE₂, 6- keto-PGF_lα, PGF_2α, and thromboxane B₂. PGE2 is a potent neutrophil chemoattractant and its reduction underlies the reduced neutrophil recruitment into the lung following the LPS challenge.

DHA also inhibits apoptosis in tissues normally expressing high CFTR levels. For example, DHA treatment decreases villi height in the ileum of CFTR^"7" mice. The loss of ion channel function is also mitigated by DHA treatment.

DHA, at nanomolar concentrations, activate fast sodium channels (plasma membrane) and calcium channels (sarcoplasmic reticulum) in cardiac myocytes. Additionally, in T84 colon cancer cells, DHA enhances carbachol-stimulated chloride conductance without affecting cAMP-stimulated chloride conductance.

Example 1: PPAR-γ Expression is Decreased in CFTR^"7" Mice

An established breeding colony of exon 10 CFTR (cftr^"7") knockout mice and wild type littermates was used for this study. Tail-clip samples of 14-day- old male mice were processed for genotype analysis. All mice were weaned at 23 days of age and then placed on Peptamen (Nestle Clinical Nutrition,

Deerfield, IL) and water until 30 days of age, and then continued for 10 days with 15 mL/day of Peptamen. Mice were euthanized by C0₂ and the organs harvested. Ileum and colon mucosal samples were prepared by opening up the intestine, removing the lumenal contents by flushing with PBS, and then scraping the mucosa from the muscle layers with a razor blade. Tissues were snap frozen in RNAlater (Ambion, Austin, TX) for RNA extraction (Barerett et al, Nat. Genetics 23: 32-33, 1999). For western blotting nuclear and cytoplasmic extracts were prepared as described below, and for immunohistochemistry tissues were fixed in 10% formalin.

Total RNA from cftr^"7" and wild type tissues was prepared using the RNA STAT-60 isolation reagent (Tel-Test, Friendswood, TX) and quantified spectrophotometrically. Quantitative PCR was performed in a ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA) using the RT-PCR master mix kit (Applied Biosystems) according to the manufacturer's instructions. PCR primers, PPARγ and CFTR FAM-labeled TaqMan probes were provided by Integrated DNA Technologies (Coralville, Iowa). The oligonucleotide sequences used were the following: PPARγ exon 2 FW: 5 '-tea caa gag ctg ace caa tgg t-3' (SEQ ID NO:l), PPARγ exon 2 RV: 5 '-ata ata agg tgg aga tgc agg ttc tac-3' (SEQ ID NO:2), PPARγ probe: 5'- FAM-ctg aag etc caa gaa tac caa agt gcg atc-TAMRA-3' (SEQ ID NO:3),

CFTR exon 2 FW: 5 '-aag aat ccc cag ctt ate cac g-3' (SEQ ID NO:4), CFTR exon 3 RV: 5 '-tgg aca gcc ttg gtg act tcc-3' (SEQ ID NO:5), and CFTR probe: 5'-FAM-cct teg gcg atg ctt ttt ctg gag att-TAMRA-3', (SEQ ID NO:6).

The mRNA levels were normalized by 18s ribosomal RNA expression (ribosomal RNA control reagents, Applied Biosystems) and quantified simultaneously to PPARγ or CFTR in a multiplex RT-PCR reaction. All samples were analyzed in duplicates.

RNA extracts from wild type and cftr^{" "} mice were subjected to quantitative analysis of total PPARγ. The results are shown in Figure 1. PPARγ expression in colonic mucosa, ileal mucosa and lung homogenate from cftr^"7" mice were 2-fold lower as compared to wild type mice (p<0.05, n=5). No significant differences in mRNA expression were found either in perigonadal adipose tissue or in liver homogenate. Accordingly, administration of a PPAR ligand (agonist) or inducer that acts in a CFTR-independent manner mitigates the symptoms associated with CFTR dysfunction. To evaluate a potential association between PPARγ levels and tissue- specific regulation by CFTR, CFTR RNA was quantified in parallel in these tissues from wild-type mice. The results shown in Figure 2 demonstrate that CFTR is mostly expressed in intestinal mucosa, preferentially in colon, and at a lower extent in total lung. mRNA expression of CFTR in adipose tissue and liver was very low and in the latter, near background levels.

Example 2: Immunohistochemical Localization of PPAR-γ in CFTR^"7" Mice

PPARγ immunostaining was performed using a rabbit polyclonal antibody (Cell Signaling, Beverly, MA). After pretreatment with 0.3% hydrogen peroxide in absolute methanol, sections were blocked with 1% BSA for 2 hours at room temperature and then incubated with the primary antibody (1 : 100 dilution) overnight at 4°C. This was followed with washing and incubating with biotinylated secondary antibody (1:200 dilution). Peroxidase activity was visualized with 3,3'-diaminobenzidine (DAB kit; Vector Laboratories, Burlingame, CA) as a substrate. Omission of the primary antibody served as a negative control.

PPARγ was predominantly localized to nuclei in the mucosal layer of colon and ileum in wild-type mice, (Figures 3 A and 3C). In contrast, the colon and ileum mucosa from cftr^{" "} mice showed reduced nuclear labeling and a predominant diffuse cytoplasmic staining (Figures 3B and 3D). Analysis of lung tissue showed a mixed labeling of both nuclei and cytoplasm localized to larger bronchi and a diffuse lighter staining of the remaining tissue (Figures 3E and 3F) in both wild-type and cftr^{" "} mice. Example 3: Reduced PPAR-γ Levels in Colonic Epithelium Nuclei of CFTR^"7 Mice

For Western blotting of total, nuclear, and cytosolic extracts, tissue samples were harvested, minced, and homogenized in 0.5 ml of hypotonic buffer (20 mM Hepes pH 7.5, 5 mM NaF, 0.1 mM EDTA, lmM Na₃V0₄) containing 0.01% NP-40 with a pre-chilled Dounce homogenizer. The suspension was incubated 15 min on ice followed by centrifugation for 10 min at 850xg at 4°C. The supematants (cytoplasmic fraction) were transferred and the pellets were resuspended in 0.5 ml hypotonic buffer containing 0.5% of NP- 40, incubated 15 min at 4°C, centrifuged 30 sec at 14000xg and the supematants discarded. Pellets, representing the nuclear fraction, were resuspended in 50μl of lysis buffer (20 mM Hepes pH 7.5, 400 mM NaCI, 20% Glycerol, 0.1% EDTA, 10 mM NaF, 10 μM Na₂M0₄, lmM NaV0₃, 10 mM PNPP, 10 mM β-glycerophosphate) containing 1 mM DTT and Complete Mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Protein concentration in all samples was measured by Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were subjected to SDS- 10% PAGE, electrotransferred onto immobilon-P (Millipore, Billerica, MA), then immunoblotted for PPARγ (1 :2000 dilution). Densitometric analysis was performed using by National Institutes of Health Image 1.62 program. Western blotting was performed in order to confirm the findings revealed by immunohistochemistry of a redistribution of PPARγ from predominantly nuclear to a less intense but more equal partitioning between nuclear and cytoplasmic compartments in ileum and colon. As shown in Figure 4 A, levels of PPARγ in colon from wild-type mice were significantly higher in the nuclear compared to the cytosolic fractions with the band in the cytoplasmic fraction exhibiting a slower mobility on SDS-PAGE. In contrast, PPARγ levels in cftr^"7" mice were decreased with similar amounts observed between the nuclear and cytosolic fractions. This is shown quantitatively in Figure 4C where the principal difference in cftr^"7" mice compared to wild-type controls is a decrease in the nuclear fraction with little change in cytosolic quantities. Perigonadal fat was also examined. As shown in Figure 4B, both isoforms of PPAR are seen in adipocytes with PPARγ2 having a higher apparent molecular weight compared to PPARγ 1. No differences were seen in PPARγ levels in fat comparing cftr^"7" mice with wild-type littermates.

PPARγ is known to migrate as two different bands by SDS-PAGE and has been attributed to post-translational modification. The higher apparent molecular weight form is due to phosphorylation following insulin stimulation in NIH 3T3 cells or in human colorectal HCT-116 cells. In addition, nitration of tyrosine residues on PPARγ has been demonstrated in macrophage-like RAW 264 cells in response to TNF or lipopolysaccharide resulting in inhibition of ligand-dependent translocation to the nucleus. These postranslational modifications likely explain the two forms of PPARγ seen on our western blots (Figure 4). The higher apparent molecular weight form (around 3 kDa) seen in the cytosolic fractions is not PPARγ2 based on comparison with results obtained with adipose tissue.

Example 4: Rosiglitazone Increases Nuclear Localization of PPAR-γ Without Affecting RNA Expression

In order to determine whether rosiglitazone, a synthetic PPARγ ligand, increases nuclear localization and/or increases RNA expression, 3mg/kg of rosiglitazone (Glaxo SmithKline, Philadelphia, PA) were administered by gavage once a day for 9 days. Immunohistochemical analysis of PPARγ showed very strong nuclear labeling in both colon and ileum from wild-type mice (Figures 5A and C, respectively). In contrast to the decreased nuclear staining of both tissues in cftr^"7" mice (Figures 3B and 3D), treatment with rosiglitazone led to a dramatic increase in nuclear labeling (Figures 5B and 5D). As shown in Figure 6, rosiglitazone did not increase RNA expression in either the colonic (Figure 6 A) or the ileal (Figure 6B) mucosa, compared to controls.

Example 5: PPAR-γ DNA Binding is Altered in the Colonic Musoca of CFTR^"7" Mice

Electrophoretic mobility shift assays (EMSA) were performed as described Tzameli et al. (Mol Cell. Biol. 20: 2951-2958, 2000). Briefly, double-stranded oligonucleotides containing either a perfect DR1 motif (synthetic PPARγ recognition element (PPRE): 5' age tac gtg ace ttt gac ctg gt- 3' (SEQ ID NO: 7)) or the PPRE from the mouse acyl-CoA oxidase promoter (5'-aca ggg gac cag gac aaa ggt cac gtt egg gag t-3' (SEQ ID NO:8)) were end- labeled with [γ-32P] ATP (PerkinElmer, Boston, MA) and incubated with 10 mg of nuclear extracts, for 20 minutes at room temperature. To test specificity, a rabbit polyclonal PPARγ specific antibody against the C-terminal part of the protein (Santa Cruz Biotechnology, Santa Cruz, CA) was incubated with the nuclear extracts for 30 minutes, prior to the addition of the probe. Competition for specific binding was performed by adding excess of unlabeled oligonucleotide to the reaction, also 30 minutes prior to the addition of the probe. The complexes were resolved on a 4% nondenaturing polyacrylamide gel and visualized by autoradiography.

The decrease in nuclear PPARγ protein expression in colon from cftr^{" "} mice demonstrated by western blotting, suggests that an equal decrease in PPARγ DNA binding activity to its consensus site should be observed. EMSA analysis of nuclear proteins from wild-type and cftr^"7" mice was performed using both synthetic PPRE and the natural PPRE from the acyl-CoA oxidase promoter, both oligonucleotides used, in the cftr^'7" mice (Figure 7A). Reduced binding of the PPARγ/RXR heterodimer from nuclear extracts of cftr^"7" mice was seen compared to wild-type controls. In addition, a faster migrating complex was apparent in 2 of the 3 cftr^{" "} mouse protein samples. In order to test for specificity of this shift, competition analysis was performed. As shown in Figure 7B, a 100-fold excess of unlabeled oligonuleotide efficiently competed for binding of the wild-type and the cftr^{" "} mouse samples to the synthetic PPRE. This suggests that binding is specific and that the faster migrating complex seen in cftr^"7" samples may represent a proteolytic fragment of the proteins containing an intact DNA binding domain. Protein extracts from perigonadal fat of both wild-type and cftr^{" "} mice, which contain minimal amounts of CFTR, demonstrated equally strong binding of the PPARγ/RXR heterodimer to the synthetic PPRE.

Example 6: Rosiglitazone Treatment Corrects the PPAR-γ Binding Defect in CFTR^"7" Mice

In agreement with the immunohistochemical analysis of wild-type and cftr^"7" mice, rosiglitazone treatment also led to a significant increase in the binding of the PPARγ RXR heterodimer to the synthetic PPRE (Figure 7C).

Colon protein samples from both genotypes produced a strong band on the gel.

Specificity of this shift was tested by incubation with a rabbit polyclonal antibody against the C-terminal part of PPARγ. Addition of antibody reduced the binding in both samples tested. Again, no differences in the binding of the

PPARγ/RXR heterodimer to the synthetic PPRE between perigonadal fat protein extracts of rosiglitazone treated wild-type and cftr^{" "} mice were observed.

PPAR-γ Inducers Increase Endogenous DHA

A variety of natural and synthetic ligands exist that cause PPAR-γ activation. For example, arachidonic acid metabolites including prostaglandin J2 and hydroxyoctadecanoic acid as well as α-linolenic acid, eicosapentaenoic acid (EPA; C20:5n-3), and DHA stimulate PPAR-γ activity. Synthetic ligands include, for example, thiazolidinediones, L-tyrosine derivatives such as fluoromethyloxycarbonyl, and nonsteroidal anti-inflammatory drugs such as indomethacin, ibuprofen, and fenoprofen, troglitazone, and rosiglitazone.

DHA is synthesized in peroxisomes through beta oxidation and, because PPAR-γ influences beta oxidation, it is likely that PPAR-γ induction increases DHA synthesis within the cells.

Identification of Candidate Compounds

A candidate compound that is beneficial in the treatment (reduction or prevention of symptoms) caused by a mutation in the CFTR gene can be identified by the methods of the present invention. A candidate compound can be identified for its ability to affect the biological activity of a PPAR-γ or the expression of a PPAR-γ gene.

Any number of methods are available for carrying out screening assays to identify new candidate compounds that promote the expression of a PPAR-γ gene. In one example, candidate compounds are added at varying concentrations to the culture medium of cultured cells expressing a PPAR-γ gene. Gene expression is then measured, for example, by microarray analysis, Northern blot analysis (Ausubel et al, supra), or RT-PCR, using any appropriate fragment prepared from the PPAR-γ nucleic acid molecule as a hybridization probe. The level of PPAR-γ gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate compound. A compound which promotes an increase in the expression of a PPAR-γ gene is considered useful in the invention and may be used as a therapeutic to treat a human patient.

In another example, the effect of candidate compounds may be measured at the level of PPAR-γ protein production using standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for the PPAR-γ protein. Polyclonal or monoclonal antibodies that are capable of binding to a PPAR-γ protein may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure the level of the protein. In some embodiments, a compound that promotes an increase in PPAR-γ expression or biological activity is considered particularly useful.

Expression of a reporter gene that is operably linked to a PPAR-γ promoter can also be used to identify a candidate compound for treating a disease associated with a CFTR mutation. Assays employing the detection of reporter gene products are extremely sensitive and readily amenable to automation, hence making them ideal for the design of high-throughput screens. Assays for reporter genes may employ, for example, colorimetric, chemiluminescent, or fluorometric detection of reporter gene products. Many varieties of plasmid and viral vectors containing reporter gene cassettes are easily obtained. Such vectors contain cassettes encoding reporter genes such as lacZ/β-galactosidase, green fluorescent protein, and luciferase, among others. A genomic DNA fragment carrying a PPAR-γ-specific transcriptional control region (e.g., a promoter and/or enhancer) is first cloned using standard approaches (such as those described by Ausubel et al. (supra). The DNA carrying the PPAR-γ transcriptional control region is then inserted, by DNA subcloning, into a reporter vector, thereby placing a vector-encoded reporter gene under the control of the PPAR-γ transcriptional control region. The activity of the PPAR-γ transcriptional control region operably linked to the reporter gene can then be directly observed and quantified as a function of reporter gene activity in a reporter gene assay. In one embodiment, for example, the PPAR-γ transcriptional control region could be cloned upstream from a luciferase reporter gene within a reporter vector. This could be introduced into the test cells, along with an internal control reporter vector (e.g., a lacZ gene under the transcriptional regulation of the β-actin promoter). After the cells are exposed to the test compounds, reporter gene activity is measured and PPAR-γ reporter gene activity is normalized to internal control reporter gene activity.

A candidate compound identified by the methods of the present invention can be from natural as well as synthetic sources. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the methods of the invention. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic-, or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

Administration of Therapeutics

The present invention also includes the administration of a PPAR-γ inducer or an antioxidant for the treatment of a disease associated with a CFTR mutation. Therapeutics of this invention may be formulated as pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, PA.

Pharmaceutical formulations of a therapeutically effective amount of a compound of the invention, or pharmaceutically acceptable salt- thereof, can be administered orally, parenterally (e.g. intramuscular, intraperitoneal, intravenous, or subcutaneous injection), or by intrathecal or intracerebro ventricular injection in an admixture with a pharmaceutically acceptable carrier adapted for the route of administration. Methods well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, PA. Compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions. The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient. These may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents (e.g., magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules. These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate. Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the proteins of the invention include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use. The amount of active ingredient in the compositions of the invention can be varied. One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the protein being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient. Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses. Desirably, the general dosage range is between 250 μg/kg to 5.0 mg/kg of body weight per day. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are well known in the art. In general, the precise therapeutically effective dosage will be determined by the attending physician in consideration of the above identified factors.

The therapeutics of the invention can be administered in a sustained release composition, such as those described in, for example, U.S. Patent No. 5,672,659 and U.S. Patent No. 5,595,760. The use of immediate or sustained release compositions depends on the type of condition being treated. If the condition consists of an acute or subacute disorder, a treatment with an immediate release form will be preferred over a prolonged release composition. Alternatively, for preventative or long-term treatments, a sustained released composition will generally be preferred. Gene Therapy

Gene therapy is another therapeutic approach for increasing PPARγ biological activity. Heterologous nucleic acid molecules encoding a PPARγ protein can be delivered to a the affected cells (e.g., lung epithelium). Expression of PPARγ proteins in the target cells can ameliorate the symptoms associated with CFTR dysfuntion. The nucleic acid molecules must be delivered to those cells in a form in which they can be taken up by the cells and so that sufficient levels of protein can be produced to increase the PPARγ biological activity. Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al, Human Gene Therapy 8:423-430, 1997; Kido et al, Current Eye Research 15:833-844, 1996; Bloomer et al, Journal of Virology 71:6641-6649, 1997; Naldini et al, Science 272:263-267, 1996; and Miyoshi et al, Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a full length PPARγ gene, or a portion thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specifically expressed in a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15- 14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al, BioTechniques 6:608-614, 1988; Tolstoshev et al, Current Opinion in Biotechnology 1:55-61, 1990; Sharp, Lancet 337:1277-1278, 1991; Cometta et al, Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al, Biotechnology 7: 980-990, 1989; Le Gal La Salle et al, Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N Engl J. Med. 323:370, 1990; Anderson et al, U.S. Patent No. 5,399,346). Most preferably, a viral vector is used to administer the gene of interest to a target endothelial cell. Non- viral approaches can also be employed for the introduction of therapeutic nucleic acids to a cell of a patient. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al, Proc. Natl Acad. Sci. U.S.A. 84:7413, 1987; Ono et al, Neuroscience Letters 17:259, 1990; Brigham et al, Am. J. Med. Sci. 298:278, 1989; Staubinger et al, Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al, J. Biol. Chem. 263:14621, 1988; Wu et al, J. Biol Chem. 264:16985, 1989), or by micro- injection under surgical conditions (Wolff et al, Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.

Gene transfer can also be achieved using non- viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. cDNA expression for use in gene therapy methods can be directed from any suitable promoter (e.g., an endocan promoter, Flt-1 promoter, or other tumor endothelial promoter identified using the methods described herein), and regulated by any appropriate mammalian regulatory element. For example, if desired, an enhancers known to preferentially direct gene expression in a tumor endothelial cell, (e.g., the 300 base pair Tie-2 intronic enhancer element described herein) can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

Another therapeutic approach included in the invention involves administration of a recombinant nuclear encoded mitochondrial metabolism or proteasomal polypeptide, either directly to the site of a potential or actual disease-affected tissue (for example, by injection into the ventricles of the brain or into the cerebrospinal fluid) or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Generally, between 0.1 mg and 100 mg, is administered per day to an adult in any pharmaceutically acceptable formulation.

Other Embodiments

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

What is claimed is:

Claims

1. A method for treating a disease in a human patient, wherein said patient has a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising administering to said patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR-γ) inducer.

2. A method for treating a disease in a human patient, wherein said patient has a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising administering to said patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist.

3. A method for treating a disease in a human patient, wherein said patient has a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising administering to said patient a therapeutically effective amount of an AP-1 inhibitor.

4. A method for treating a disease in a human patient, wherein said patient has a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising administering to said patient a therapeutically effective amount of a STAT inhibitor.

5. A method for treating a disease in a human patient, wherein said patient has a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising administering to said patient a therapeutically effective amount of an NFkB inhibitor.

6. The method of any of claims 1-5, wherein said disease is cystic fibrosis

7. The method of any of claims 1-5, wherein said disease is selected from the group consisting of pancreatitis, chronic obstructive pulmonary disease (COPD), asthma, chronic sinusitis, primary sclerosing cholangitis, and congenital bilateral absence of the vas deferens.

8. The method of any of claims 1-7, wherein said PPAR-γ is a PPAR- γl .

9. The method of claim 1 , wherein said PPAR inducer is substantially pure eicosapentaenoic acid.

10. The method of claim 1, wherein said PPAR inducer is selected from the group consisting of the thiazolidinediones, fluoromethyloxycarbonyl, indomethacin, ibuprofen, fenoprofen, and troglitazone.

11. The method of claim 2, wherein said PPAR ligand is rosiglitazone.

12. The method of claim 3, wherein said AP-1 inhibitor is selected from the group consisting of nordihydroguaiaretic acid, SP600125, SRI 1302, pyrrolidine dithiocarbamate, curcumin, PD98059, and spiro compounds.

13. The method of claim 4, wherein said STAT inhibitor is selected from the group consisting of SSI- 1, SSI-2, and SSI-3.

14. The method of claim 13, wherein said STAT inhibitor is expressed in the target cell by gene therapy. "

15. The method of claim 13, wherein said STAT inhibitor is administered to said patient by intravenous injection or inhalation.

16. The method of claim 4, wherein said STAT inhibitor is a tripeptide having the sequence proline-tyrosine-leucine or alanine-tyrosine-leucine, wherein said tyrosine is phosphorylated.

17. The method of claim 5, wherein said NFkB inhibitor is selected from the group consisting of 2-chloro-N-[3,5-di(trifluoromethyl)phenyl]-4- (trifluoromethyl)pyrimidine-5 -carboxamide (SP-100030); 3,4-dihydro-4,4- dimethyl-2H-l,2-benzoselenazine (BXT-51072); declopramide (Oxi-104), dexlipotam, a salicylanilide, 2-hydroxy-4-trifluoromethylbenzoic acid and 2- hydroxy-4-trifluoromethylbenzoic acid derivatives.

18. The method of claim 17, wherein said 2-hydroxy-4- trifluoromethylbenzoic acid derivative is triflsal.

19. The method of any of claims 1-18, wherein said mutation is a deletion of F508.

20. A method for identifying a compound useful for treating a disease in a patient having a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising the steps of:

(i) providing cells that express a PPARγ, (ii) contacting said cells with a candidate compound, and (iii) assessing the level of PPARγ expression in said cells relative to the level of PPARγ expression in the absence of said candidate compound, wherein a candidate compound that increases the level of PPARγ expression is identified as a compound useful for treating said disease.

21. A method for identifying a compound useful for treating a disease in a patient having a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising the steps of:

(i) providing cells that express a PPARγ protein, (ii) contacting said cells with a candidate compound, and (iii) assessing the half life of said PPARγ protein in said cells relative to the half life of said PPARγ protein in the absence of said candidate compound, wherein a candidate compound that increases said half life is identified as a compound useful for treating said disease.

22. A method for identifying a compound useful for treating a disease in a patient having a mutation in the CFTR gene and wherein said mutation is associated with said disease, said method comprising the steps of:

(i) providing cells that express a PPARγ, (ii) contacting said cells with a candidate compound, and (iii) assessing the level of PPARγ translocation to the nucleus of said cells relative to the level of PPARγ expression in the absence of said candidate compound, wherein a candidate compound that increases the level of PPARγ translocation to the nucleus is identified as a compound useful for treating said disease.

23. The method of any of claims 20-22, wherein said cells are pancreatic exocrine cells, lung cells, intestinal cells, bile duct cells, macrophages, or derivates therefrom.

24. The method of any of claims 20-22, wherein said PPARγ is PPARγ 1 or PPARγ2.

25. The method of claim 20, wherein said assessing comprises western blotting.

26. The method of claim 20, wherein said assessing comprises measuring the amount of PPARγ RNA in said cells.

26. The method of claim 22, wherein said assessing comprises immunohistochemistry.

27. A method for treating a disease in a human patient, wherein said patient has a mutation in the CFTR gene and wherein said disease is associated with said mutation, said method comprising administering to said patient a therapeutically effective amount of an antioxidant.

28. The method of claim 27, wherein said antioxidant is a peroxisome proliferator-activated receptor gamma (PPAR-γ) inducer.

29. The method of claim 27, wherein said antioxidant is selected from the group consisting of Vitamin E, Vitamin C, S-adnenosyl methionine, selenium, vitamin C, beta-carotene, idebenone, cysteine, dithioerythritol, dithionite, dithiothreitol, and pyrosulfite.