WO2005074921A1

WO2005074921A1 - Treatment of atherosclerosis

Info

Publication number: WO2005074921A1
Application number: PCT/CH2005/000053
Authority: WO
Inventors: Romeo Ricci
Original assignee: University Of Zurich
Priority date: 2004-02-09
Filing date: 2005-02-01
Publication date: 2005-08-18

Abstract

The invention relates to a method of treating atherosclerosis comprising administering a c-jun NH2-terminal kinase (JNK) inhibitor, and the use of JNK inhibitors in such a treatment and in the manufacture of medicaments for treating atherosclerosis. Preferred as a JNK inhibitor is a JNK2 inhibitor. The invention is based on the fact that JNK2 (but not JNK1) is required for substantial plaque formation in atherosclerosis. The invention further relates to a method of screening for a compound effective in the treatment of atherosclerosis comprising contacting a candidate compound with a JNK and choosing candidate compounds which selectively reduce enzyme activity of JNK2.

Description

JNK INHIBITORS FOR THE TREATMENT OF ATHEROSCLEROSIS

Field of the invention

This invention relates to the treatment of atherosclerosis.

Background of the invention

C-jun NHz-terminal kinases (JNK)

In mammals, three major groups of the Mitogen Activated Protein Kinase (MAPK) family have been identified (Schaeffer, H.J. & Weber, M.J., Mol Cell Bi 19, 2435-44 (1999)). The ERK and p38 groups of MAPK are related to enzymes found in budding yeast. The c- jun NH₂-terminal kinases (JNK), also known as stress-activated MAP kinases (SAPK), represent a third group of MAPK that has been identified in mammals. JNK contains the dual phosphorylation motif Thr-Pro-Tyr. The JNK protein kinases are encoded by three genes: JNK1 , JNK2 and JNK3. The JNK1 and JNK2 genes are expressed ubiquitously. In contrast, the JNK3 gene has a more limited pattern of expression and is largely restricted to brain, heart, and testis. Transcripts derived from these genes are alternatively spliced to create four JNK1 isoforms, four JNK2 isoforms, and two JNK3 isoforms (Gupta, S. et al., EMBO J 15, 2760-70 (1996)). Two distinct types of alternative splicing have been described. Firstly, all three JNK genes are expressed as 46 kDa and 55 kDa protein kinases. Functional differences between these 46 kDa and 55 kDa isoforms have not been reported. A second form of alternative splicing is restricted to the JNK1 and JNK2 genes and involves the selection of one of two alternative exons that encodes part of the kinase domain. These resulting isoforms of JNK1 and JNK2 differ in their interactions with protein substrates. The analysis of JNK gene disruption in mice confirms that there is extensive complementation between the JNK genes and that there are also tissue-specific defects in signal transduction that may reflect the JNK isoform profile of individual tissues.

JNK and targeting JNK in the treatment of various diseases has been the subject of an extensive recent review (Manning, A.M. & Davis, R.J., Nature Reviews Drug Discovery 2, 554 (2003)). JNK has been implicated in inflammatory diseases, neurodegenerative diseases, metabolic diseases and cancer, and JNK inhibitors are considered for treating such diseases. Although the JNK signalling cascade is also involved in cardiovascular diseases, the specific use of JNK inhibitors in the treatment of atherosclerosis has not been investigated yet.

Atherosclerosis

Arteriosclerosis comprises any of the diseases characterized by thickening and loss of elasticity of arterial walls; there are three distinct forms: atherosclerosis, Mόnckeberg's arteriosclerosis, and arteriolosclerosis. Atherosclerosis is a common form of arteriosclerosis in which deposits of yellowish plaques (atheromas) containing cholesterol, lipoid material, and lipophages are formed within the intima and inner media of large and medium-sized arteries. Complications of atherosclerosis account for the most common causes of death in Western societies. In broad outline, atherosclerosis can be regarded as a form of chronic inflammation resulting from interaction between modified lipoproteins, monocyte-derived macrophages, T cells, and the normal cellular elements of the arterial wall. Atherosclerosis is a multistep disease initiated by formation of fatty streak lesions, followed by lesion progression and plaque rupture and thrombosis. Many events have been described to exert pro-atherogenic effects: Leukocyte adhesion/migration, formation of foam cells, inflammation caused by the interplay of various cellular components, abnormal smooth muscle proliferation and apoptosis.

Late stage large atheromas contain areas of necrotic tissue due to ischemia caused by insufficient blood supply which is compensated only partially by neovascularization. Necrosis, destruction of the extracellular matrix as well as an increasing inflammatory component destabilize the plaque and most importantly affects the integrity of the endothelial cell layer. Bared subendothelial structures are known to be highly thrombotic. As a consequence a rapidly growing thrombus can be formed at these unstable sites and fatal immediate occlusion may occur. At this stage plaques can also rupture and large emboli may occlude distal arteries. These mechanisms are the most frequent causes of complete obstruction of coronary arteries as well as cerebral vessels leading to myocardial infarction and apoplexy, respectively.

C-jun NH₂-terminal kinases (JNK) in atherosclerosis

Several in vitro studies suggest pro-atherogenic properties for JNK. However, they have been restricted to one specific cell type in vitro and very specific stimuli have been employed, the importance of which in atherosclerosis is still not clear. Therefore, so far, the role of JNK in the complex interplay of all these processes during atherogenesis in vivo is completely unknown. Furthermore, nobody addressed the question which of the JNK members is specifically required in atheroma formation. As a consequence a possible beneficial effect of JNK inhibitors in the treatment of atherosclerotis is at present highly speculative. Nevertheless, several previous studies should be mentioned in the following:

Leukocyte adhesion: In normal circumstances, the endothelial monolayer in contact with flowing blood resists firm adhesion of leukocytes. However, when endothelial cells undergo activation by pro-inflammatory stimuli, for example by TNFα and IL-1 , they increase their expression of various leukocyte adhesion molecules (VCAM-1 , P-, E- selectin and ICAM-1), which is thought to mainly contribute to the initiation of atherosclerosis (Libby, P., Nature 420, 868-74 (2002)). It seems that the JNK signalling pathway is specifically required for the induction of E-selectin expression by TNFα (Min, W. & Pober, J.S., J Immunol 159, 3508-18 (1997) In addition, the activation of the JNK pathway was shown to lead to intercellular adhesion molecule-1 (ICAM-1) expression (De Cesaris, P. et al., J Biol Chem 274, 28978-82 (1999)).

Leukocyte migration: In the classic inflammatory response, adhesion is followed by transmigration of the leukocytes through the endothelial layer and into the intima. This is governed by chemotactic factors produced in the subendothelial layer. The best- characterized of these chemokines is monocyte chemotactic protein-1 (MCP-1), which promotes the recruitment of monocytes and T cells. Li Y.S. et al. demonstrated that the Ras-JNK pathway regulates expression of MCP-1 induced by shear stress (Mol Cell Biol 16, 5947-54 (1996)).

Formation of foam cells: Scavenger cell receptors (SR-A and CD36) are thought to control oxidized low density lipoprotein (ox-LDL) uptake by macrophages to give rise to foam cells. Oxidative stress induces expression of the scavenger cell receptor SR-A in smooth muscle cells and macrophages via JNK and AP-1. Accordingly, regulatory c-jun/AP-1 elements on the SR-A promoter have been detected (Mietus-Snyder, M. et al., Arterioscler Thromb Vase Biol 18, 1440-9 (1998)).

Involvement of T cells: The CD4 positive helper T cells can polarize into those secreting generally pro-inflammatory cytokines (known as Th1 cells) and those secreting predominantly anti-inflammatory cytokines (denoted Th2 cells). In general, Th1 cells predominate in the atheroma. They are secreting cytokines such as IFNγJL-2, and TNF- α, which cause macrophage activation, vascular activation and inflammation. Th2 cytokines such as IL-4, IL-5, and IL-10 are less abundant than cytokines of the Th1 type in end-stage human lesions (Frostegard, J. et al., Atherosclerosis 145, 33-43 (1999)). The JNK signalling pathway has been implicated in the immune response that is mediated by the activation and differentiation of CD4 helper T (Th) cells into Th1 and Th2 effector cells (Constant, S.L. et al., J Immunol 165, 2671-6 (2000); Dong, C. et al., Nature 405, 91-4 (2000); Yang, D.D. et al., Immunity 9, 575-85 (1998); Dong, C. et al., Science 282, 2092-5 (1998)).

Smooth muscle cells: Abnormal proliferation of vascular smooth muscle cells (VSMCs) is thought to play an important role in the pathogenesis of atherosclerosis. In three recent studies, it has been demonstrated that the JNK-c-jun pathway is required for efficient proliferation and migration induced by PDGF in VSMCs (loroi, T. et al., J Pharmacol Sci 91 , 145-8 (2003); Zhan, Y. et al., Artehoscler Thromb Vase Biol 22, 82-8 (2002); Zhan, Y. et al., Arterioscler Thromb Vase Biol 23, 795-801 (2003)).

Summary of the invention

The present invention relates to a method of treating atherosclerosis comprising administering a JNK inhibitor, and the use of JNK inhibitors in such a treatment and in the manufacture of medicaments for treating atherosclerosis. Preferred as a JNK inhibitor is a JNK2 inhibitor, in particular an inhibitor which is selective for JNK2, i.e. which selectively reduces enzyme activity of JNK2 but not to a reasonable extent of JNK1 or JNK3.

The invention further relates to a method of screening for a compound effective in the treatment of atherosclerosis comprising contacting a candidate compound with a JNK and choosing candidate compounds which selectively reduce enzyme activity of JNK2. The invention further relates to compounds selected by this method of screening.

Brief description of the Figures

Fig. 1 : The absence of JNK2 but not JNK1 markedly reduces atheroma formation in ApoE deficient mice. JNK1/ApoE and JNK2/ApoE double deficient mice and corresponding ApoE knock out control mice are fed a high cholesterol diet (1.25%) for 14 weeks, and the percentage of total plaque area from the total vessel wall area is determined in descending aortas en face. (A): Aortas en face stained with Oil red O to visualize plaques. (B): Quantification, % atheroma of total aorta area.

Fig. 2: SP600125, which blocks kinase activity of JNK1 , JNK2 and JNK3, markedly attenuates plaque formation in ApoE deficient mice. ApoE deficient mice are fed a high cholesterol diet (1.25%) during 14 weeks. SP600125 and placebo control pellets (pi) are implanted after 10 weeks. (A): Aorta en face, Oil red O stain. (B): Quantification, % atheroma of total aorta area.

Fig. 3: Intraperitoneal macrophages from mice with different genotypes are subjected to oxLDL for 2.5 hours after two days in culture. Cells are fixed and stained with Oil red O. The percentages of Oil red O positive foam cells from total cells are determined. Markedly reduced foam cell formation in the absence of JNK2 can be observed. Quantification: % foam cells of total cells, n = number of experiments.

Detailed description of the invention

The present invention relates to a method of treating atherosclerosis comprising administering a JNK inhibitor to a mammal in need thereof, and the use of JNK inhibitors in such a treatment and in the manufacture of medicaments for treating atherosclerosis. In particular the invention relates to a method of treating atherosclerosis comprising administering a JNK2 inhibitor, and the use of JNK2 inhibitors in such a treatment and in the manufacture of medicaments for treating atherosclerosis. Preferred is the use of JNK2 inhibitors which are selective for JNK2, i.e. which selectively reduce enzyme activity of JNK2 but not to a reasonable extent of JNK1 or JNK3.

JNK inhibitors are compounds which reduce the enzyme activity of a JNK, e.g. JNK1 , JNK2 or JNK3, or reduce the expression of a JNK isotype. For example, compounds which inhibit JNK enzyme activity bind to an ATP binding site in JNK or bind to a catalytic domain of JNK. The compound to be used in the method of the invention preferentially inhibits JNK2, more preferably inhibits JNK2 much better than JNK1 and/or JNK3. JNK inhibitors of the invention may belong to the class of inorganic compounds or organic compounds, and are e.g. polypeptides or small organic compounds, preferably small heterocyclic compounds, for example heterocyclic compounds comprising five- or six- membered heterocycles containing one or two nitrogen atoms, or also sulfonamides or sulfonamide derivatives. JNK inhibitors are widely known. JNK inhibitors are, for example, disclosed in Patent Applications WO 00/35906, WO 00/35909, WO 00/35921 , WO 00/64872, WO 01/12609, WO 01/12621 , WO 01/23378, WO 01/23379, WO 01/23382, WO 01/47920, WO 01/91749, WO 02/010137, WO 02/028856, WO 02/046170, WO 02/062792, WO 02/066450, WO 02/079197, WO 02/081475, WO 02/083648, WO 02/083667, WO 03/010164, WO 03/018020, WO 03/024967, WO 03/051277, WO 03/068750, WO 03/072550, WO 03/101968, and WO 03/103698, which are incorporated herewith by reference. However, the invention is not restricted to the JNK inhibitors disclosed therein, but extends to all JNK inhibitors.

In particular, in International Patent Application WO 01/12609, pyrazoloanthrone and derivatives thereof having a structure of formula (I) are disclosed wherein R¹ and R² are optional substituents placed at any position in the rings, are the same or different, and independently represent alkyl, halogen, nitro, trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-or dialkylaminoalkoxy, optionally substituted amino, optionally substituted aminoalkylamino, optionally substituted acylamino or optionally substituted sulfonylamino.

A preferred JNK inhibitor is the compound of formula (I) wherein R¹ and R² are hydrogen and which is known under the code name SP600125.

One aspect of the invention relates to a method of treating atherosclerosis comprising administering a JNK inhibitor in a quantity effective against atherosclerosis to a mammal in need thereof, for example to a human requiring such treatment. The treatment may be for prophylactic or therapeutic purposes. For the administration, the JNK inhibitor is preferably in the form of a pharmaceutical preparation comprising the JNK inhibitor in chemically pure form and optionally a pharmaceutically acceptable carrier and optionally adjuvants. The JNK inhibitor is used in an amount effective against atherosclerosis. The dosage of the active ingredient depends upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, the mode of administration, and whether the administration is for prophylactic or therapeutic purposes. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 1 mg to approximately 500 mg, preferably from approximately 10 mg to approximately 100 mg, of a JNK inhibitor.

Pharmaceutical compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous administration, are especially preferred. The pharmaceutical compositions comprise from approximately 1% to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient.

For parenteral administration preference is given to the use of solutions of the JNK inhibitor, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example, can be made up shortly before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.

For oral pharmaceutical preparations suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, and also binders, such as starches, cellulose derivatives and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, flow conditioners and lubricants, for example stearic acid or salts thereof and/or polyethylene glycol. Tablet cores can be provided with suitable, optionally enteric, coatings. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient. Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The capsules may contain the active ingredient in the form of granules, or dissolved or suspended in suitable liquid excipients, such as in oils. Transdermal application is also considered, for example using a transdermal patch, which allows administration over an extended period of time, e.g. from one to twenty days.

Another aspect of the invention relates to the use of JNK inhibitors in the treatment of atherosclerosis and in the manufacture of medicaments for treating atherosclerosis. Such medicaments are manufactured by methods known in the art, especially conventional mixing, coating, granulating, dissolving or lyophilizing.

A JNK inhibitor can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of fixed combinations of a JNK inhibitor and one or more other therapeutic agents known in the treatment of atherosclerosis, the administration being staggered or given independently of one another, or being in the form of a fixed combination.

Possible combination partners considered are statins and ACE inhibitors, or also acetylsalicylic acid, anti-hypertensive agents such as diuretics, calcium antagonists, beta- blockers and angiotensin-ll-receptor blockers, and anti-diabetic agents.

The invention is based on the fact that JNK2 (but not JNK1) is required for substantial plaque formation in atherosclerosis. ApoE deficient mice develop a rapid-course atherosclerosis when they are fed a high cholesterol diet. JNK1/ApoE and JNK2/ApoE double knock out mice and corresponding control mice are generated and subjected to high cholesterol diet. Evidence is provided in in vivo experiments that JNK2 but not JNK1 plays an important role in atherosclerosis, and the results are shown in Figure 1. Specifically JNK2 but not JNK1 is the main pro-atherogenetic gene in vivo. Levels of total plasma cholesterol and triglycerides are similarly high in all different genotypes.

A JNK inhibitor, for example JNK inhibitor SP600125, very efficiently reduces plaque formation in ApoE deficient mice. The results of this pharmacological approach in vivo shown in Figure 2 provide evidence that a specific inhibitor of JNK activity markedly reduces atherosclerosis. Blockage of the overall JNK activity by SP600125 efficiently reduces atheroma formation in ApoE deficient mice without causing immunodeficiency and whithout adversely affecting organ function. Levels of total plasma cholesterol and triglycerides remain similar in all different genotypes. Importantly, no obvious infections as well as organ dysfunctions (liver and kidney) can be detected in the mice treated with a JNK inhibitor. This excludes major side effects caused by the JNK inhibitor. Foam cell formation is supposed to be a very important step during development of atherosclerotic plaques. Circulating monocytes adhering on activated endothelial cells give rise to macrophages, which subsequently accumulate in the vessel wall. In response to oxidized low density lipoprotein these macrophages differentiate into foam cells, which are known to be important in the pathogenesis of atherosclerosis (Li, A.C. & Glass, C.K., Nat Med 8, 1235-42 (2002)). In an in vitro foam cell formation assay it can be shown that foam cell formation depends specifically on JNK2 (Figure 3).

The invention further relates to a method of screening for a compound effective in the treatment of atherosclerosis comprising contacting a candidate compound with a JNK and choosing candidate compounds which selectively reduce enzyme activity of JNK2 but not to a reasonable extent of JNK1 or JNK3. The invention further relates to compounds selected by this method of screening, in particular compounds effective in the treatment of atherosclerosis which selectively reduce enzyme activity of JNK2 but not to a reasonable extent of JNK1 or JNK3.

Inhibitors of JNK enzyme activity are identified by contacting a JNK (JNK1 , JNK2 or JNK3) with a candidate compound. A control assay with the corresponding JNK in the absence of the candidate compound is run in parallel. Kinase activity is measured using methods known in the art, for example as described by Hibi et al., Genes Dev 7, 2135 (1993). A decrease in enzyme activity in the presence of the candidate compound compared to the level in the absence of the compound indicates that the compound is a JNK inhibitor. Preferred candidate compounds cause marked decrease of JNK2 enzyme activity while having no or only marginal influence on JNK1 and JNK3 enzyme activity.

Examples

Generation of mice JNK1 -/- and JNK2-/- mice (C57/BL6/129SV background) are crossed/mated with ApoE-/- C57/BL6 mice. The obtained JNK1+/-;ApoE+/- and JNK2+/-;ApoE+/- mice are back- crossed on the ApoE-/- background to produce JNK1+/-;ApoE-/- and JNK2+/-;ApoE-/- mice. These mice are then intercrossed to generate homozygous ApoE-/- mice bearing combination of JNK1 and JNK2 +/+, +/-, and -/-. Mouse models

The ApoE knock out mice are widely employed as an in vivo model to study atherosclerosis. These mice develop spontaneous atheromas. By using high cholesterol diet a rapid course atherosclerosis can be induced. To study the function of the single components JNK1 and JNK2 in atheroma formation, double mutant JNK1/ApoE and JNK2/ApoE mice are fed with high cholesterol diet (1.25%) for 14 weeks starting at the age of 6 weeks. Importantly, the JNK1 as well as the JNK2 deficient mice are viable and lack an obvious phenotype.

Implantation of JNK inhibitor pellets

Specially designed pellets containing SP600125 or the carrier medium only, which allow a constant release (30mg/kg/d) of this JNK inhibitor over one month are obtained from Innovative Research of America. These pellets are implanted subcutaneously in the upper back of the mice under xylaxine/ketamine anesthesia. The implantation of SP600125 or placebo pellets in ApoE deficient mice is carried out after 10 weeks of high cholesterol diet.

Determination of total plasma cholesterol and triglycerides

The effect of the absence of JNK1 and JNK2 as well as inhibition of the kinase activity on the plasma lipid profile is measured with a commercially available cholesterol kit (SIGMA).

Assessment of kidney and liver function

Kidney function is checked by measuring plasma levels of creatinine. Liver function is analyzed by measuring plasma levels of the liver specific enzymes AST, ALP and ALT.

Assessment of the extent of atherosclerosis in aortas

Aortas from mice in all different in vivo experiments are harvested after 14 weeks. Mice are given an anesthesia (ketamin/xylazine), the chest is opened and the left ventricle of the heart is punctured to rinse the aortic route with cold PBS. The whole aorta is dissected and put into ice cold PBS. The descending part of the aorta up to the bifurcation is prefixed with paraformaldehyde (1 %) for one hour, cut longitudinally and spread on filter papers. The spread vessels are postfixed with paraformaldehyde (1% over night) and stained with Oil-red O (lipid stain) to distinguish the plaques from the normal vessel wall. A quantitative computer-assisted image analysis (Metamorph) of the plaque staining areas is performed and correlated with the whole vessel wall areas. For the genetic experiments a total amount of 6 aortas from mice from each genotype are used. A total amount of 5 aortas from mice treated with inhibitor or placebo is quantified.

Foam cell formation assay The foam cell formation assay is performed with peritoneal macrophages isolated from ApoE-/-, ApoE-/-;JNK1-/- and ApoE-/-;JNK2-/- mice. Six weeks old mice are injected intraperitoneally with 2 ml of 4% thioglycolate (Becton Dickinson 0256-15). After 4 days macrophages are harvested by peritoneal lavage and plated on 6 cm dishes in RPMI 1640 medium (Invitrogen Corporation 31870-025) supplied with 10% FCS. After 2 h non adhered cells are washed out and macrophages are starved for 48h. After that copper oxidized low densitiy lipoprotein (oxLDL) is added to the medium in a concentration of 50 μg/ml. After 2.5 and 5 h medium is removed and cells are fixed in 4% paraformaldehyde for 15 min at room temperature, then washed twice with PBS and stained over night with Oil-red O at 4°C, washed twice with PBS, co-stained with hematoxilin for 20 sec and mounted with an aqueous mounting medium.

Statistics

Data are presented as mean ± SD. Statistical significance of differences is calculated using an ANOVA with post hoc multiple comparison test and Student unpaired r test respectively.

Bone marrow transplantation experiments

Circulating monocytes adhering on activated endothelial cells give rise to macrophages, which subsequently accumulate in the vessel wall. In response to oxLDL these macrophages differentiate into foam cells, which are known to be important in the pathogenesis of atherosclerosis.

The present data provide evidence that specifically JNK2 is required for foam cell formation. This finding could, at least in part, explain the reduction of atherosclerosis in the absence of JNK2. To proof that the inability of JNK2 deficient macrophages to differentiate into foam cells is a cell-autonomous defect, ApoE/JNK2 double mutant mice and corresponding ApoE control knock out mice are lethally irradiated. Hematopoietic reconstitution is established by transplantation of ApoE deficient bone marrow cells. These mice are fed a high cholesterol diet, sacrificed after 14 weeks, and quantification of atherosclerosis is performed. In case impaired foam cell formation in macrophages lacking JNK2 is a cell-autonomous process, a rescue of the reduction of atherosclerosis in the transplanted ApoE/JNK2 double mutant mice, in which hematopoietic cells should express JNK2 normally, is expected.

Assessment of morphology, cellular composition, proliferation and apoptosis in plaques To show in vivo that foam cells are less abundant in plaques of ApoE/JNK2 double mutant mice, foam cells are quantified by using the Oil red O stain. As described above, JNK is involved in other cellular processes during development of atherosclerosis. Therefore, it is determined whether, apart from a defective foam cell formation, pro-atherogenic processes such as inflammation, apoptotis or increased proliferation are involved in ApoE/JNK2 double mutant mice phenotype. For this purpose, plaque morphology is analyzed on cross-sections of the aortic sinus from mice with all different genotypes by conventional histology (H&E and Collagene stain). The cellular composition is analyzed by immunohistochemistry. Antibodies against the following proteins distinguish the different cell types: • CD3: T cells; CD4: T helper subtype; CD8: T suppressor subtype • F4/80: Macrophages • αSM-actin: Smooth muscle cells • CD31 : Endothelial cells

Proliferation and apoptosis of different cell types in the plaques of the different mice is analyzed by using immunohistochemistry for KI67 as a proliferative marker and TUNEL stain as an indicator for apoptosis.

Cytokine and foam-cell specific gene expression in atherosclerotic plaques

To further analyze the role of JNK in the inflammatory process during atheroma formation, cytokines in plaques are measured at the RNA level by RT-PCR. JNK1 and JNK2 deficient mice fail to efficiently differentiate T helper cells into Th1 effector cells. Therefore, pro-inflammatory Th1 cytokines (IL-2, INFγ) could be reduced. Instead, the Th2 cells secreting anti-inflammatory cytokines (IL-4, IL-5) type could be increased. Genes such as SR-A, CD36 and PPARγ have been shown to be essential in foam cell formation. The expression of these genes in atherosclerotic aortic arches is measured by RT-PCR.

Assessment of overall JNK activity in plaques and macrophages To get an idea how the overall JNK activity correlates with the amount of atherosclerosis in vessels of mice with different genotypes (wild type, ApoE knock out, ApoE/JNK1 and ApoE/JNK2 double mutant mice), Western blots with a phospho-JNK antibody as well as an in vitro kinase assay is performed. To localize activated JNK in plaques immunohistochemistry with a phospho-JNK antibody is done. Efficiency of the inhibition of JNK by SP600125 in vivo is checked by using the in vitro kinase assay with lysates from ApoE knock out mice treated with SP600125 or placebo. To test the specificity of SP600125 the activity of immunoprecipitated ERK1/2 is determined by measuring phosphorylation of the substrate GST-Elk1. Activation of JNK in macrophages with different genotypes upon stimulation with oxLDL is assessed by Western blotting with a phospho-JNK antibody.

Molecular targets regulated by JNK2 during foam cell formation

Expression of possible JNK2 targets such as SR-A, CD36 and PPARγ, which are involved during foam cell formation, is analyzed by Northern blotting.

Claims

1. Use of a c-jun NH₂-terminal kinase (JNK) inhibitor for the manufacture of a medicament for the treatment of atherosclerosis.

2. Use according to claim 1 wherein the JNK inhibitor selectively reduces enzyme activity of JNK2.

3. Use according to claim 1 wherein the JNK inhibitor is a compound of formula (I)

wherein R¹ and R² are optional substituents placed at any position in the rings, are the same or different, and independently represent alkyl, halogen, nitro, trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-or dialkylaminoalkoxy, optionally substituted amino, optionally substituted aminoalkylamino, optionally substituted acylamino or optionally substituted sulfonylamino.

4. Use according to claim 3 wherein the JNK inhibitor is the compound of formula (I) wherein R¹ and R² are hydrogen.

5. A method of treating atherosclerosis in a mammal comprising administering a therapeutically effective amount of a JNK inhibitor to a mammal in need thereof.

6. The method of claim 5 wherein the JNK inhibitor selectively reduces enzyme activity of JNK2.

7. Use of a JNK inhibitor in a method of treating atherosclerosis.

8. A method of screening for a compound effective in the treatment of atherosclerosis comprising contacting a candidate compound with a JNK and choosing candidate compounds which selectively reduce enzyme activity of JNK2.

9. A compound obtained in the method of screening of claim 8.

10. A compound according to claim 9 effective in the treatment of atherosclerosis which selectively reduces the enzyme activity of JNK2 but not to a reasonable extent of JNK1 or JNK3.