Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-11-02
2001-09-25
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S975000, C435S004000, C435S017000, C424S009100, C424S277100, C424S553000, C424S551000
Reexamination Certificate
active
06294350
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for treatment of disease and particularly to methods for treating diseases characterized by undesirable levels of phosphorylation of c-Jun.
BACKGROUND OF THE INVENTION
Collagenous colitis was first described by Lindstrom as chronic watery diarrhea (
Pathol Eur
11(1):87-89, 1976). Collagenous colitis is characterized by collagen deposition, likely resulting from an imbalance between collagen production by mucosal fibroblasts and collagen degradation. Very little is understood, however, regarding the mechanism by which collagenous colitis results in secretory diarrhea.
The incidence of collagenous colitis is similar to primary biliary cirrhosis. This disease has an annual incidence of 1.8 per 100,000 and a prevalence of 15.7 per 100,000, which is similar to primary biliary cirrhosis (12.8 per 100,000) and lower than ulcerative colitis (234 per 100,000), Crohn's disease (146 per 100,000) or celiac disease (5 per 100,000). In patients with chronic diarrhea, about 0.3 to 5% have ollagenous colitis.
In efforts to characterize the patients under study, sera and monocyte conditioned media (MCM) from patients with collagenous colitis have been assessed for their ability to stimulate fibroproliferation. Cytokine antibodies were used to characterize the fibroproliferative component of patient samples.
Previous studies have suggested that MCM samples obtained from patients with liver disease are capable of stimulating proliferation of fibroblasts (see Peterson and Isbrucker, in
Hepatol.
15(2):191-197, 1992). It has also been established that several genes involved in proliferation possess AP-1 binding sites, and thus would be expected to be susceptible to regulation by the immediate early genes c-fos and c-jun (see, for example, Schafer et al., in
Biochem Biophys Res Commun
221:111-116, 1996) and Bamberger et al., in
Proc Nat Acad Sci. USA
9:6169-6174, 1996).
In order to determine whether these immediate early genes are upregulated by MCM obtained from patients with liver disease, the effect of MCM from patients with liver disease was investigated. MCM exerts many effects due to PDGF (see, for example, Peterson and Isbrucker, supra, 1992) and Peterson and Tanton in
Can. J. Gastroenterol.
10:S76 1996)). Indeed, it has been established that PDGF itself stimulates proliferation of fibroblasts (see T. C. Peterson, in
Hepatol.
173):486-493, 1993) and Peterson et al., in
Immunopharmacol.
28:259-270, 1994). Thus, the question of whether PDGF upregulates the expression of c-fos and c-jun was addressed.
Prior studies have indicated that pentoxifylline inhibits PDGF and MCM stimulated proliferation (see, for example, T. C. Peterson, supra, 1993), Peterson et al., in
Immunopharmacol.
28:259-270, 1994) and Peterson and Neumeister in
Immunopharmacol.
31:183-193, 1996)). The mechanism for this effect of pentoxifylline remains unclear, but does not appear to involve competing for the PDGF receptor or adenosine receptor activation (see T. C. Peterson, in
Biochem Pharmacol
52:597-602, 1996)).
It is known that c-Jun forms heterologous signaling protein combinations with a number of entities. For instance, c-Jun forms heterologous signaling protein complexes with ATF2 (Y. Sano, et al.,
J Biol Chem,
273(44):29098-105, 1998) and the activity of ATF2 is enhanced after phosphorylation occurs with c-Jun N-terminal kinase. Recent evidence also suggests that linkage between ATF2 and c-Jun is important in expression of cyclin kD1, a gene of critical importance in breast tumors where over-expression of cyclin D1 gene has been implicated (R. Lee, et al.,
J Biol Chem,
274(11):7341-50, 1999).
c-Jun also forms a complex with CREB. This protein complex plays a particularly important role in TNF&agr; gene expression. TNF
&agr;
expression has been implicated in endotoxic shock, multiple sclerosis, cerebral malaria and other inflammatory diseases (Delgado-M, Munoz-Elias-Ej, Kan-Y, Gozes-I, Fridkin-M, Brenneman-D E, Gomariz-R P, Ganea-D,
J
-
Biol
-
Chem
273(47):31427-36,1998). The complex CREB/c-Jun has also been implicated in squamous cell differentiation and plays a role in transglutaminase I activity in tracheal epithelial cells (A. Medvedev, et al.,
J Biol Chem,
274(6):3887-96, 1999).
Another example of a c-Jun complex associated with disease is the Nrf1 complex with c-Jun. The Nrf1/c-Jun complex is associated with signaling by the TNF
&agr;
promoter in stimulated mast cells (V. Novotny, et al.,
Nucleic Acids Res,
26(23):5480-5, 1998).
Accordingly, in view of the numerous combinations in which c-Jun participates, there is a need in the art to achieve a better understanding of the mechanism by which c-Jun and complexes of c-Jun with signaling proteins contribute to fibrotic proliferative disorders and for methods of treatment of such diseases.
BRIEF DESCRIPTION OF THE INVENTION
The present invention is based upon the discovery that complexes containing activated (i.e. phosphorylated ) c-Jun function as signaling entities in connection with fibrotic proliferative disorders. It has further been discovered that compounds that block the activity of c-Jun kinase, the enzyme active in phosphorylation of c-Jun, are effective in modulating the phosphorylation of c-Jun and, hence, of the formation of such c-Jun complexes.
Accordingly, in accordance with the present invention, there are provided methods for treatment of a subject afflicted with a disease or condition characterized by one or more of the following symptoms:
increased levels of c-Jun homodimers,
increased heterodimerization of c-Jun with another signaling peptide,
increased levels of phosphorylated c-Jun, and
increased presence of Jun kinase.
The invention treatment method comprises administering to the subject an amount of a compound effective to ameliorate one or more of the symptoms of the disease or condition. Such diseases and conditions are often further characterized by the presence of one or more of the following additional symptoms:
increased levels of PDGF,
increased levels of c-Jun,
activation of NF-kappaB or NF-kappaB p65,
neutrophil infiltration, and
elevated levels of inflammatory cytokine(s).
Exemplary compounds suitable for use in the invention treatment methods are generally antifibrotic and/or antiproliferative agents, with the most preferred compounds being pentoxifylline and functional derivatives or metabolites thereof, such as 1-(5-oxohexyl)-3,7-dimethylxanthine(pentoxifylline), 1-(5-hydroxyhexyl)-3,7-dimethylxanthine(metabolite-1), propentofylline, and the like, and combinations of any two or more thereof. In addition, antisense c-Jun kinase is also contemplated for treatment of fibrotic proliferative disorders according to the present invention because c-Jun kinase facilitates phosphorylation of c-Jun at serine 73, which produces activated c-Jun having the ability to form homodimers and heterodimers with other signaling peptides.
In another embodiment according to the present invention, there are provided assay(s) for identifying whether a test compound is useful for treatment of a subject afflicted with a fibroproliferative disease. The invention assay(s) comprise determining the levels of Jun kinase activity in the presence and absence of the test compound in test cells maintained in the presence of serum obtained from a subject afflicted with a fibroproliferative disease.
In another embodiment according to the present invention, there are provided in vitro assay(s) for identifying whether a test compound that reduces c-Jun phosphorylation is likely to be effective for treatment of a subject afflicted with a fibroproliferative disease. Invention in vitro assay(s) comprise determining the uptake by test cells in suitable media therefor, of a labeled building block indicative of cell proliferation, wherein said uptake is determined in the presence of serum obtained from a subject afflicted with a fibroproliferative disease, and in the presence and absence of the test compound. A reduction of uptake by the test cells of the labeled building block
Dalhousie University
Foley & Lardner
Leary Louise N.
Reiter Stephen E.
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