WO2005034978A2

WO2005034978A2 - MEDICAL USE OF IKKϵ OR OF INHIBITORS THEREOF

Info

Publication number: WO2005034978A2
Application number: PCT/EP2004/010994
Authority: WO
Inventors: Christian Korherr
Original assignee: Xantos Biomedicine Ag
Priority date: 2003-10-02
Filing date: 2004-10-01
Publication date: 2005-04-21
Also published as: EP1670908A2; WO2005035746A2; WO2005035746A3; WO2005034978A3; CA2540775A1

Abstract

The present invention relates to a novel angiogenic factor, IKKϵ, as well as to inhibitors thereof and to their use in pharmaceutical or diagnostic compositions.

Description

Medical use of IKKε or of inhibitors thereof

The present invention relates to the medical use of IKKε or of inhibitors thereof. Especially, the present invention relates to the use of these molecules in promoting or inhibiting angiogenesis.

Angiogenesis, the growth of new capillaries from pre-existing ones, is critical for normal physiological functions in adults [Carmeliet, P. , Mechanisms of angiogenesis and arteriogenesis. Nat Med, 2000 6 (4) 389-95]. Abnormal angiogenesis can lead to impaired wound healing, poor tissue regeneration in ischemic conditions, cyclical growth of the female reproductive system, and tumor development [Carmeliet, P. and R. K. Jain, Angiogenesis in cancer and other diseases].

Promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis. The angiogenic process is highly complex and involves the maintenance of the endothelial cells in the cell cycle, degradation of the extracellular matrix, migration and invasion of the surrounding tissue and finally, tube formation. Because of the crucial role of angiogenesis in so many physiological processes, there is a need to identify and characterize factors which will promote angiogenesis.

The administration of growth factors such as VEGF-A and FGF-2 has been considered as a possible approach for the therapeutic treatment of ischemic disorders.

VEGF is an endothelial cell-specific mitogen and an angiogenesis inducer that is released by a variety of tumor cells and expressed in human tumor cells in situ.

However, both animal studies and early clinical trials with VEGF angiogenesis have encountered severe problems [Carmeliet, Nat Med, 2000 6 1102-3;Yancopoulos et al., Nature, 2000 407 242-8; Veikkola et al., Semin Cancer Biol 1999 9 211-20; Dvorak et al., Semin Perinatal 2000 24 75-8; Lee et al., Circulation, 2000 102 898-901]. VEGF-A stimulated microvessels are disorganized, sinusoidal and dilated, much like those found in tumors [Lee et al., Circulation 2000 102 898-901; and Springer et al., Mol. Cell 1998 2 549-559]. Moreover, these vessels are usually leaky, poorly perfused, torturous and likely to rupture and regress. Thus, these vessels have limited ability to improve the ischemic conditions. In addition, the leakage of blood vessels induced by VEGF-A (also known as Vascular Permeability Factor) could cause cardiac oedema that leads to heart failure.

VEGF not only stimulates vascular endothelial cell proliferation, but also induces vascular permeability and angiogenesis. Angiogenesis, which involves the formation of new blood vessels from preexisting endothelium, is an important component of a variety of diseases and disorders including tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, retinopathy, hemangiomas, immune rejection of transplanted tissues, and chronic inflammation.

In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment to the growing solid tumor. [Folkman, et al., Nature 339:58 (1989)]. Angiogenesis also allows tumors to be in contact with the vascular bed of the host, which may provide a route for metastasis of the tumor cells. Evidence for the role of angiogenesis in tumor metastasis is provided, for example, by studies showing a correlation between the number and density of microvessels in histologic sections of invasive human breast carcinoma and actual presence of distant metastases. [Weidner, et al., New Engl. J. Med. 324:1 (1991)].

In view of the importance of angiogenesis in various diseases, there is a continuous need for means interfering with angiogenesis. Therefore, the problem underlying the present invention resides in providing such means. hi the context of the present invention, it has been surprisingly found that IKKε expression in human and animal cells induces the production of pro-angiogenic factors. Furthermore, it has been found that IKKε exhibits a proliferation inducing activity for endothelial cells. IKKε (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon) is a homologue of IK -1 and IKK-2 (Kishore, N. et al, J. Biol. Chem. 277:13840, WO 00/73469, US 2003/0143540) and is known to be involved in inflammatory and immunologic processes. Furthermore, it is known that IKKε plays a role in NF-κB- activation [Peters, R.T. et al, Mol Cell. 2000 Mar;5(3):513-22], a transcription factor which is involved in many physiological processes like immunologic and inflammatory responses. It is activated e.g. by TNFalpha, IX- 1, LPS and various growth factors.

The protein sequence of human IKKε and the corresponding nucleic acid sequence are given in SEQ ID NO: 1 and 2, respectively. A role of IKKε in angiogenic processes has not been suggested in the art.

Consequently, according to one aspect of the invention, the problem is solved by the use of a nucleic acid encoding IKKε or a functional active derivative thereof for the preparation of a pharmaceutical composition for the treatment of diseases with disturbed angiogenesis, especially ischemic or dental diseases, smoker's leg and diabetic ulcers or for the stimulation of wound healing.

In the context of the present invention, the term "IKKε" relates first to a protein with a sequence as shown in SEQ ID NO: 2. In a further aspect this term further relates to functional active derivatives of the protein as shown in SEQ ID NO: 2.

The term "functional active derivative" of a polypeptide within the meaning of the present invention refers to polypeptides which have a sequence homology, in particular a sequence identity, of about at least 25 %, preferably about 40 %, in particular about 60 %, especially about 70 %, even more preferred about 80 %, in particular about 90 % and most preferred of 98 % with the polypeptide. Such derivatives are e.g. the polypeptide homologous to IKKε, which originate from organisms other than the IKKε according to SEQ ID NO: 2. Other examples of derivatives are polypeptides which are encoded by different alleles of the gene, of different individuals, in different organs of an organism or in different developmental phases. Functional active derivatives preferably also include naturally occurring mutations, particularly mutations that quantitatively alter the activity of the peptides encoded by these sequences. Further, such variants may preferably arise from differential splicing of the encoding genes.

"Sequence identity" refers to the degree of identity (% identity) of two sequences, that in the case of polypeptides can be determined by means of for example BLASTP 2.2.5 and in the case of nucleic acids by means of for example BLASTN 2.2.6, wherein the low complexity filter is set on and BLOSUM is 62 (Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402).

"Sequence homology" refers to the similarity (% positives) of two polypeptide sequences determined by means of for example BLASTP 2.0.1 wherein the Filter is set on and BLOSUM is 62 (Altschul et al, 1997, Nucleic Acids Res., 25:3389-3402).

Nucleic acids encoding functional active derivatives can be isolated by using human IKKε gene sequences in order to identify homologues with methods known to a person skilled in the art, e.g. through PCR amplification or hybridization under stringent conditions (e.g. 60 °C in 2.5 x SSC buffer followed by several washing steps at room temperature concentration) with suitable probes derived from e.g. the human IKKε sequences according to standard laboratory methods (Current Protocols, John Wiley & Sons, Inc., New York (2003)).

"Functional active derivative" refers to a polypeptide that has essentially the biological function(s) as the corresponding protein. In the case of IKKε, this may be the expression of a specific angiogenic activity as demonstrated in Example 1. Therefore, the term "functional active derivative" may also refer to a polypeptide which is responsible for the specific induction of endothelial cell proliferation. A test for the determination of the angiogenic activity induced by a putative IKKε derivative is also demonstrated in Example 1. A preferred embodiment for a nucleic acid encoding IKKε is given in SEQ ID NO: 1. As demonstrated for the first time in the context of the present invention, IKKε is an important angiogenic factor. This enables the use of a nucleic acid encoding IKKε in therapy.

The administration of the nucleic acid encoding LKKε may be effected either as recombinant protein or by gene transfer either as naked DNA or in a vector [Kornowski R, Fuchs S, Leon MB, Epstein SE, Delivery strategies to achieve therapeutic myocardial angiogenesis, Circulation, 2000 101 (4) 454-8; Simons M, Bonow RO, Chronos NA, Cohen DJ, Giordano FJ, Hammond HK, et al., Clinical trials in coronary angiogenesis: issues, problems, consensus: An expert panel summary, Circulation, 2000 102 (11) E73- 86; and Isner JM, Asahara T, Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization, J Clin Invest, 1999 103 (9) 1231-36]. If desired, regulatable vectors may be used as described in Ozawa et al, Annu Rev Pharmacol. & Toxicol, 2000 40 295-317.

Administration may be parenterally, intravenously, dermally, intradermally, intracutaneously, percutaneously, subcutaneously, topically or transdermally.

Alternatively, the nucleic acid can be administered by catheterbased myocardial gene transfer. In this technique, a steerable, deflectable 8F catheter incorporating a 27guage needle is advanced percutaneously to the left ventricular myocardium. A total dose of 200 ug/kg is administered as 6 injections into the ischemic myocardium (total, 6. 0 mL).

Injections are guided by NOGA left ventricular electromechanical mapping. See Vale, P.

R., et al., Randomized, single-blind, placebo-controlled pilot study of catheter-based myocardial gene transfer for therapeutic angiogenesis using left ventricular electromechanical mapping in patients with chronic myocardial ischemia, Circulation, 2001 103 (17) 2138-43.

Another possibility is the injection of a IKKε plasmid in the muscles of an ischemic limb in accordance with procedures described in Simovic, D., et al., Improvement in chronic ischemic neuropathy after intramuscular phVEGF165 gene transfer in patients with critical limb ischemia, ArchNeurol, 2001 58 (5) 76168. Still another technique for effective administration is by intra-arterial gene transfer of the gene using adenovirus and replication defective retroviruses as described for VEGF in Baumgartner I and Isner JM, Somatic gene therapy in the cardiovascular system, Annu. Rev Physiol, 2001 63 427-50. An additional possibility for administering the nucleic acid is by intracoronary and intravenous administration (see Post, M. J., et al., Therapeutic angiogenesis in cardiology using protein formulations, Cardiovasc Res, 2001 49 522-31).

A still further possibility is to use ex vivo expanded endothelial progenitor cells (EPCs) engineered to express JKKε for myocardial neovascularization as described in Kawamoto, A., et al., Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation, 2001 103 (5) 634-37.

Yet another technique which may be used to administer the nucleic acid is percutaneous adenovirus-mediated gene delivery to the arterial wall in injured atheromatous stented arteries. See, for example, Maillard, L., et al., Effect of percutaneous adenovirus-mediated Gax gene delivery to the arterial wall in double-injured atheromatous stented rabbit iliac arteries, Gene Ther, 2000 7 (16) 1353-61 ; and Laham RJ, Simons M, and Sellke F, Gene transfer for angiogenesis in coronary artery disease,Annu Rev Med, 2001 52485-502.

In one advantageous aspect of the invention, a therapeutically effective dose of the nucleic acid is administered by bolus injection of the active substance into ischemic tissue, e. g. heart or peripheral muscle tissue. The effective dose will vary depending on the weight and condition of the ischemic subject and the nature of the ischemic condition to be treated. It is considered to be within the skill of the art to determine the appropriate dosage for a given subject and condition. Furthermore, the pharmaceutical composition can be administered in further conventional manners, e.g. by means of the mucous membranes, for example the nose or the oral cavity, in the form of dispositories implanted under the skin, by means of injections, infusions or gels which contain the medicaments according to the invention. It is further possible to administer the medicament topically and locally, if appropriate, in the form of liposome complexes. Furthermore, the treatment can be carried out by means of a transdermal therapeutic system (TTS), which makes possible a temporally controlled release of the medicaments. TTS are known for example, from EP 0 944398 Al, EP 0 916 336 Al, EP 0 889 723 Al or EP 0 852 493 Al. In accordance with another aspect of the invention, the nucleic acid is administered by continuous delivery, e. g., using an osmotic minipump, until the patient is able to selfmaintain a functional vascular network.

In another advantageous aspect within the scope of the invention, the nucleic acid is effectively adrninistered to an ischemic subject by contacting ischemic tissue with a viral vector, e. g. an adenovirus vector, containing a polynucleotide sequence encoding the protein operatively linked to a promoter sequence.

The nucleic acid may also be effectively administered by implantation of a micropellet impregnated with active substance in the direct vicinity of ischemic tissue.

For the production of the pharmaceutical compositions of the invention, the molecules of the present invention are usually formulated with suitable additives or auxiliary substances, such as physiological buffer solution, e.g. sodium chloride solution, deminerahzed water, stabilizers, such as protease or nuclease inhibitors, preferably aprotinin, ε-aminocaproic acid or pepstatin A or sequestering agents such as EDTA, gel formulations, such as white vaseline, low-viscosity paraffin and/or yellow wax, etc. depending on the kind of administration.

Suitable further additives are, for example, detergents, such as, for example, Triton X-100 or sodium deoxycholate, but also polyols, such as, for example, polyethylene glycol or glycerol, sugars, such as, for example, sucrose or glucose, zwitterionic compounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or a protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or zwitterionic compounds are preferred.

The physiological buffer solution preferably has a pH of approx. 6.0-8.0, expecially a pH of approx. 6.8-7.8, in particular a pH of approx. 7.4, and/or an osmolarity of approx. 200-400 milliosmol/liter, preferably of approx. 290-310 milliosmol/liter. The pH of the medicament is in general adjusted using a suitable organic or inorganic buffer, such as, for example, preferably using a phosphate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (3-morpholino- 1-propanesulphonic acid). The choice of the respective buffer in general depends on the desired buffer molarity. Phosphate buffer is suitable, for example, for injection and infusion solutions.

Injection solutions are in general used if only relatively small amounts of a solution or suspension, for example about 1 to about 20 ml, are to be administered to the body. Infusion solutions are in general used if a larger amount of a solution or suspension, for example one or more litres, are to be administered. Since, in contrast to the infusion solution, only a few millilitres are administered in the case of injection solutions, small differences from the pH and from the osmotic pressure of the blood or the tissue fluid in the injection do not make themselves noticeable or only make themselves noticeable to an insignificant extent with respect to pain sensation. Dilution of the formulation according to the invention before use is therefore in general not necessary. In the case of the administration of relatively large amounts, however, the formulation according to the invention should be diluted briefly before administration to such an extent that an at least approximately isotonic solution is obtained. An example of an isotonic solution is a 0.9% strength sodium chloride solution. In the case of infusion, the dilution can be carried out, for example, using sterile water while the administration can be carried out, for example, via a so-called bypass.

Within the present invention, subjects which may be treated or diagnosed include animals, preferably mammals and humans, dead or alive. These patients suffer from the diseases as mentioned above.

The diseases mentioned above are all characterised by a disturbed angiogenesis and therefore a nucleic acid encoding LKKε leads to a significant improvement in these diseases.

With respect to the wound healing of fractures, the nucleic acid immobilised to a matrix can be administered directly into the site of fracture to promote the angiogenesis and wound healing. As matrices can be used ceramic matrices or bonemeal on which the protein is immobilised. Slow release formulations to have the factor locally enriched can be used as well. In the context of the present invention, it could be shown that IKKε is a angiogenic factor. Therefore, in a preferred embodiment, the nucleic acid encoding IKKε induces the formation of vascular vessels.

The invention further includes a method for the treatment of a patient in need of such treatment, wherein an effective amount of a nucleic acid encoding IKKε is administered to the patient.

With respect to the preparation of this pharmaceutical composition, its administration and other embodiments the same apply as defined above.

The invention therefore relates to the use of a) IKKε, b) a functional active derivative thereof, c) a nucleic acid encoding IKKε, and /or d) means for the detection of the molecules of sections a), b) , c) or d)

for the preparation of a diagnostic agent for the diagnosis of ischemic or dental diseases, smoker's leg and diabetic ulcers, wound healing disorders, cancer, hyperplasia, tumor progression, rheumatoid arthritis, psoriasis, artherosclerosis, retinopathy, osteoarthritis, endometriosis and or chronic inflammation.

This diagnostic agent may be appropriately combined with additional carriers or diluents or other additives which are suitable in this context. With respect to these agents, the same apply as defined above for the pharmaceutical composition of the invention.

The proteins or nucleic acids may be prepared as defined above.

Within the meaning of the present invention, means of detecting IKKε or a function derivative thereof include antibodies which can e.g. applied in Westen Blotting, hnmunohistochemistry, ELISA or functional assays for the proteins (Current Protocols, John Wiley & Sons, Inc. (2003)).

Means for detecting the nucleic acids as defined above include other nucleic acids being capable of hybridizing with the nucleic acids e.g. in Southern Blots or Northern Blots as well as during hi Situ Hybridization (Current Protocols, John Wiley & Sons, Inc. (2003)).

Angiogenesis is generally a phenomenon which occurs in later tumor stages. Since IKKε is an angiogenic factor, it represents therefore a marker for later tumor stages, i.e. for tumors which have already achieved a malignant state. Furthermore, since JKKε is an important angiogenic factor, its lack is indicative for the diseases disclosed above.

For example, IKKε or functional active derivatives thereof may be detected in the tumor tissue via immunohistochemistry. Nucleic acids encoding these molecules, e.g. mRNA, may be detected using quantitative PCR.

In several diseases as mentioned below, an aberrant angiogenesis contributes the clinical symptoms or is even the reason for these symptoms. The present invention relates to IKKε, which is an important inducer of angiogenesis, e.g. in tumors. Therefore, the inhibition of IKKε results in inhibition of angiogenesis which will result in the treatment of these diseases.

In a further aspect, the present invention therefore relates to the use of a JKKε inhibitor for the preparation of a pharmaceutical composition for the treatment of a disease or diseases with increased angiogenesis^ especially cancer, hyperplasia, rheumatoid arthritis, psoriasis, artherosclerosis, retinopathy, osteoarthritis, endometriosis and / or chronic inflammation.

According to the present invention the term "inhibitor" refers to a biochemical or chemical compound which preferably inhibits or reduces the angiogenic activity of IKKε. This can e.g. occur via suppression of the expression of the corresponding gene. The expression of the gene can be measured by RT-PCR or Western blot analysis. Preferably, the JKKε is not a TBK-1 inhibitor. TBK-1 (tank binding kinase 1) is a homologe of JKK-1 and IKK-2 (Kishore, N. et al, J. Biol. Chem. 277:13840, WO 00/73469, US 2003/0143540) and is known to be involved in inflammatory and immunologic processes. Furthermore, it is known that TBK-1 plays a role in NF-κB- activation, a transcription factor which is involved in many physiological processes like immunologic and inflammatory responses (Matsuda, A. et al., Oncogene 22:3307). It is activated e.g. by TNFalpha, IL-1, LPS and various growth factors.

The protein sequence of human TBK-1 and the corresponding nucleic acid sequence are given in SEQ ID NO: 3 and 4, respectively.

This preferably means that the binding affinity of the IKKε inhibitor is at least 50-fold higher, preferably 100-fold and more preferably 1000-fold higher for IKKε than for TBK- 1. However, it is also preferred that the IKKε inhibitor is also a TBK-1 inhibitor, which preferably means that the binding affinities lies of the inhibitor for JKKε and TBK-1 do not differ by more than a factor of 50

Examples of such JKKε inhibitors are binding proteins or binding peptides directed against IKKε, in particular against the active site of IKKε, and nucleic acids directed against the IKKε gene. Preferably, the inhibitor binds to the ATP-binding site of the kinase domain of IKKε

In a preferred embodiment, the inhibitor of the invention is selected from the group consisting of antisense oligonucleotides, antisense RNA, siRNA, and low molecular weight molecules (LMWs).

LMWs are molecules which are not proteins, peptides antibodies or nucleic acids, and which exhibit a molecular weight of less than 5000 Da, preferably less than 2000 Da, more preferably less than 2000 Da, most preferably less than 500 Da. Such LMWs may be identified in High-Through-Put procedures starting from libraries. Such methods are known in the art. They preferably bind to the ATP-binding site of the kinase domain of IKKε. Nucleic acids which may inhibit IKKε activity may be double-stranded or single stranded DNA or RNA which, for example, inhibit the expression of the IKKε gene or the activity of IKKε and include, without limitation, antisense nucleic acids, aptamers, siRNAs (small interfering RNAs) and ribozymes.

The nucleic acids, e.g. the antisense nucleic acids or siRNAs, can be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhhnann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). Aptamers are nucleic acids which bind with high affinity to a polypeptide, here IKKε or derivatives thereof. Aptamers can be isolated by selection methods such as SELEX (see e.g. Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981) from a large pool of different single-stranded RNA molecules. Aptamers can also be synthesized and selected in their mirror-image form, for example as the L- ribonucleotide (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). Forms which have been isolated in this way enjoy the advantage that they are not degraded by naturally occurring ribonucleases and, therefore, possess greater stability.

Nucleic acids may be degraded by endonucleases or exonucleases, in particular by DNases and RNases which can be found in the cell. It is, therefore, advantageous to modify the nucleic acids in order to stabilize them against degradation, thereby ensuring that a high concentration of the nucleic acid is maintained in the cell over a long period of time (Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94; WO 95/11910; WO 98/37240; WO 97/29116). Typically, such a stabilization can be obtained by introducing one or more mtemucleotide phosphorus groups or by introducing one or more non-phosphorus internucleotides.

Suitable modified internucleotides are compiled in Uhlmann and Peyman (1990), supra (see also Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94; WO 95/11910; WO 98/37240; WO 97/29116). Modified internucleotide phosphate radicals and/or non- phosphorus bridges in a nucleic acid which can be employed in one of the uses according to the invention contain, for example, methyl phosphonate, phosphorothioate, phosphoramidate, phosphorodithioate and/or phosphate esters, whereas non-phosphorus internucleotide analogues contain, for example, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamidate bridges and/or thioether bridges. It is also the intention that this modification should improve the durability of a pharmaceutical composition which can be employed in one of the uses according to the invention.

The use of suitable antisense nucleic acids is further described e.g. in Zheng and Kemeny (1995) Clin. Exp. Immunol., 100, 380-2; Nellen and Lichtenstein (1993) Trends Biochem. Sci., 18, 419-23, Stein (1992) Leukemia, 6, 697-74 or Yacyshyn, B. R. et al. (1998) Gastroenterology, 114, 1142).

The production and use of siRNAs as tools for RNA interference in the process to down regulate or to switch off gene expression, here IKKε gene expression, is e.g. described in Elbashir, S. M. et al. (2001) Genes Dev., 15, 188 or Elbashir, S. M. et al. (2001) Nature, 411, 494. Preferably, siRNAs exhibit a length of less than 30 nucleotides, wherein the identity stretch of the sense strang of the siRNA is preferably at least 19 nucleotides.

Ribozymes are also suitable tools to inhibit the translation of nucleic acids, here the IKKε gene, because they are able to specifically bind and cut the mRNAs. They are e.g. described in Amarzguioui et al. (1998) Cell. Mol. Life Sci., 54, 1175-202; Vaish et al. (1998) Nucleic Acids Res., 26, 5237-42; Persidis (1997) Nat. Biotechnol., 15, 921-2 or Couture and Stinchcomb (1996) Trends Genet., 12, 510-5.

Thus, the nucleic acids described can be used to inhibit or reduce the expression of the JKKε genes in the cells both in vivo and in vitro and consequently act as an IKKε inhibitor in the sense of the present invention. A single-stranded DNA or RNA is preferred for the use as an antisense oligonucleotide or ribozyme, respectively.

For the context of these diseases, IKKε inhibition aims at preventing the formation of vascular vessels which support the diseased tissue. This, in turn, will reduce the amount of diseased or malignant cells (e.g. cancer cells).

The pharmaceutical composition may be prepared and administered as discussed above. As it is shown in Example 3, IKKε is frequently expressed at significant levels in tumors. It is known in the art that during the growth of solid tumors, often hypoxic conditions are found, which in turn result in the induction of new vascular vessels. IKKε may be an important factor in this physiological process. In turn, inhibition of U Kε function may result in maintaining the hypoxic conditions in the tumor, resulting in a suppression of tumor growth or even in a regression of tumor size.

Therefore, in a preferred embodiment of the use of the invention, the inhibitor prevents the formation of vascular vessels in the tumor tissue.

According to a preferred embodiment, the disease is cancer, preferably selected from the group consisting of brain cancer, pancreas carcinoma, stomach cancer, colon carcinoma, skin cancer, especially melanoma, bone cancer, kidney carcinoma, liver cancer, lung carcinoma, ovary cancer, mamma carcinoma, uterus carcinoma, prostate cancer and testis carcinoma.

The invention further includes a method for the treatment of a patient in need of such treatment, wherein an effective amount of an inhibitor of IKKε or of a functional active derivative thereof is administered to the patient.

The invention further relates to a method for the identification of an anti-cancer drug, wherein a) a potential IKKε interactor is brought into contact with JKKε or a functional derivative thereof, and b) binding of the potential interactor to IKKε or the functional derivative thereof is determined, and c) the anti-angiogenic capacity of the potential interactor is determined. In this method of the invention, in general, JKKε or the corresponding gene are provided e.g. in an assay system and brought directly or indirectly into contact with a test compound, in particular a biochemical or chemical test compound. Then, the influence of the test compound on JKKε or the corresponding gene is measured or detected by measuring whether the LKKε phenotype is reversed by addition of the potential inhibitor. Thereafter, suitable inhibitors can be analyzed and/or isolated. For the screening of compound libraries, the use of high-throughput assays are preferred which are known to the skilled person or which are commercially available.

Suitable assays may be based on the gene expression of IKKε or on the physiological activity of JKKε, i.e. the angiogenic properties.

For example, the following assay may be used for the identification of an inhibitor of the invention:

• transfection of IKKε into HEK293 cells (see also sequence listing) • transfer of supernatants of HEK 293 cells onto HUVEC cells (as described for the screen in example 1) • addition / incubation of HUVEC cells with LMW (low molecular weight) compound library or other potential inhibitors • screening for inhibition of proliferating activity (reversion of phenotype) • definition of lead structures • analysis of specificity: inhibition of IKKε The experimental steps transfection of 293 cells, transfer of supernatant onto HUVEC cells and screening for proliferation or inhibition of proliferation, repectively, can be carried out according to Example 1.

In a preferred embodiment, in the method of the invention the anti-angiogenic capacity is measured by measuring the inhibition of pro-angiogenic factors.

Preferably, in the method of the invention the potential interactor is provided in the form of a chemical compound library. According to the present invention the term "chemical compound library" refers to a plurality of chemical compounds that have been assembled from any of multiple sources, including chemically synthesized molecules and natural products, or that have been generated by combinatorial chemistry techniques.

In a further preferred embodiment, in the method of the invention the chemical compound library consists of a group of molecules or substances that bind to the ATP binding site of the kinase domain of IKKε.

More preferred, the method of the invention is carried out on an array. Methods for preparing such arrays using solid phase chemistry and photolabile protecting groups are disclosed, for example, in US 5,744,305. These arrays can also be brought into contact with test compound or compound libraries and tested for interaction, for example binding or changing conformation.

In another embodiment of the present invention, the method is carried out in form of a high-through put screening system. In such a system advantageously the screening method is automated and miniaturized, in particular it uses miniaturized wells and microfluidics controlled by a roboter.

The following Figures and Examples are intend to illustrate further the invention without limiting it.

Short Description of the Figures:

Figure 1 indicates proliferation of HUVEC cells following transfer of supernatants from HEK 293 cells transfected with indicated expression plasmids. The relative fluorescence units (RFU) are given as mean value from three independent experiments. Vector represents the negative control resulting from transfection of the cloning vector pCMV-Sport6 into HEK 293 cells and measurement of Alamar Blue to determine background proliferative effect of the supernatant derived from HEK 293 cells. VEGF was derived from the same clone collection to ensure compatibility of expression systems. The bar (20 RLUs) indicates the threshold, above which a proliferative activity was called in our experiments. Please note that JKKα, despite showing comparable NF-κB activation (see Figure 2), showed no proliferative action towards HUVEC cells.

Figure 2 indicates activation of a NF-kB luciferase reporter construct in HEK 293 cells co- transfected with the reporter plasmid and the individual expression plasmids as indicated. The relative luciferase activity is given as mean value from three independent experiments. Vector represents the negative control resulting from co-transfection of the cloning vector pCMV-Sport6 into HEK 293 cells and measurement of luciferase activity to determine background activity. Activity is indicated as relative luciferase activity as a NF- B dependent firefly luciferase activity normalised to constitutive renilla luciferase activity.

Figure 3: Expression of IKKε in tumor vs normal tissue by quantitative RT-PCR Total RNA from colon, lung, prostate and breast tissue was transcribed into cDNA and relative expression of IKKε versus 18SrRNA was calculated after quantitative real-time

PCR. Absolute expression levels have been analysed by quantitative real-time PCR for a panel of cDNAs from mammary gland and ovary tissue.

Overexpression of IKKε was observed in colon, lung, prostate and breast cancer compared to normal tissue.

Examples

Example 1 : Proliferation inducing-activity

An expression screen was conducted in order to isolate novel cDNAs that encode secreted proteins which stimulate endothelial cell proliferation. Plasmid DNAs were prepared on Xantos' proprietary high-throughput robot assembly according to standard Xantos protocols (see WO 03/014346).

To facilitate the production of the proteins encoded by individual cDNA clones, 2.2x10⁴ 293 HEK cells were seeded in 96-well tissue culture plates (Costar) in lOOμl DMEM medium containing 5% FCS (Invitrogen). Transfection of ca. 10000 cDNAs from a clone collection (Human Full-Length Clone Collection", OriGene Technologies Inc., Rockville, MD, U.S.A.) on 293 cells was performed 24hrs post seeding using calcium phosphate co- precipitation. Precipitates were removed after 4 hours and cells were switched to nutrient deficient DMEM (DMEM, 1.5%FCS, 1% Na-pyruvate, 1% Glutamine, lOOμg/ml gentamycin, 0.5μg/ml amphotericin B). Human umbilical cord vein endothelial cells (HUVEC) were cultured in ECGM with supplements (Promocell Heidelberg, single quots) containing 1 % serum, 50μg/ml gentamycin, 0.4μg/ml amphotericin B and 50U/ml nystatin. HUVECS were plated at 2.5 x 10³ cells /well on day 3. Before transfer of supernatants on day 4, 90μl of medium was removed, HUVECS were washed once with 200μl of PBS, then 75μl of nutrient deficient medium (ECBM, with supplements, Promocell, Heidelberg) containing lμg/ml hydrocortisol, 50μg/ml gentamycin, 0.4μg/ml amphotericin B and 50U/ml nystatin was added following 25 μl of supernatants from the transfected 293 cells. Supernatants were incubated for 4 days on HUVEC cells. Read-out was performed using Alamar Blue (Biosource, California USA). For each well of a 96well plate, 1 lμl of Alamar Blue reagent were mixed with 9μl of ECBM and the resulting 20μl were added directly to the HUVEC cells without removal of medium. Incubation was performed at 37°C for 4 hours. Alamar Blue fluorescence was measured at 530nm excitation and 590nm emission.

As a positive control for proliferation of HUVECs, a supernatant from cells tranfected with VEGF cDNA derived from the same clone collection was used.

Negative controls were supernatants from vector-transfected HEK 293 cells.

This screen led to the isolation of a cDNA which will be referred to as inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKε).

For the verification or specification of the proliferation-inducing activity of IKKε, JKKε and controls were transfected into HEK293 cells and supernatants were transferred onto HUVEC as described for the screen above except that all manipulations were carried out manually. Figure 1 shows the proliferation-inducing activity of JKKε in comparison to LKKα and VEGF. The results of these analysis showed that JKKε but not LKKα have proliferation-inducing activity on endothelial cells (HUVEC).

Example 2: Induction of proliferative activity by IKKε is independent of activation of NF- KB

For members of the IKK famitiy (IKKα, IKKβ, IKKε) is described that they activate the transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF- B) . Therefore we tested the activation of the transcription factor in a NF-κB dependent reporter gene assay and whether the induction of proliferative activity is dependent on this transcription factor activation. For that 2.4 x 10⁴ HEK293 cells were co- transfected with 28ng of a constitutive active expression plasmid for renilla luciferase, 42ng of a NF-κB dependent firefly luciferase reporter construct and 210ng of the individual expression plasmids for the indicated kinases. 24 hours after transfection cells were lysed and NF-kB dependent firefly luciferase activity was measured. Activity of renilla luciferase was determined to normalise for transfection efficiencies. As shown in figure 2 IKKα and IKKε are activating the NF-κB dependent reporter gene which is consistent with the activation of NF-κB described in the literature. As shown in figure 1, IKKε but not IKKα is inducing proliferative activity in supernatants of HEK293 cells overexpressing the kinase. Therefore we conclude that induction of proliferative activity on endothelial cells (e.g. HUVEC) is independent of the activation of NF-κB.

The result of this analysis showed that induction of the proliferative activity by IKKε is independent of the activation of the transcription factor NF-κB.

Example 3: Increased expression of JKKε in tumor tissue

For the analysis of IKKε expression in tumor tissue, tissue samples of patients (normal and tumor tissue) were analysed for JKKε mRNA expression levels by quantitative real-time PCR. cDNA was synthesized from 1 μg of total RNA in a volume of 20 μl using random hexamers as primer and AMV ReverseTranscriptase (Roche Diagnostics).

Real-time PCR was carried out using a LightCycler (Roche Diagnostics). Reactions were set up in microcapillary tubes using the following final concentrations: 0.5 μM each of JKKε sense (CTG CCC TTC ATC CCC TTT G) and IKKε antisense (TCC GTG GTG ATC CGG TAG A) primers, 3 M MgCl₂, lx SYBR Greenmaster mix and 2 μl of cDNA.

Cycling conditions for IKKε were as follows: denaturation (95° C for 10 min), amplification and quantitation(95°C for 10 s, 58°C for 10 s and 72°C for 13 s, with a single fluorescence measurement at the end of the 72°C for 13 s segment) repeated 45 times. A melting curve program (55-95°C with a heating rate of 0.1 ° C/s and continuous fluorescence measurement) and a cooling step to 40°C followed.

For relative quantification the procedure was repeated for 18S rRNA as reference gene. Data were analyzed using LightCycler analysis software.

Figure 3 shows the results of IKKε expression level in normal tissue and cancer samples via QPCR. For that expression levels of IKKε in RNAs and cDNAs from human colon (normal and cancer), lung (normal and cancer), prostate (normal and cancer) and breast (normal and cancer) were analysed by quantitative real-time PCR.

The results indicate higher expression of human IKKε in most cancer versus normal tissues. One sample of normal lung tissue also shows very high expression of JKKε mRNA (Figure 3, as indicated by an asterisk). This may be due to the activation of inflammatory responses in this sample.

Claims

1. Use of a nucleic acid encoding IKKε or a functional active derivative thereof for the preparation of a pharmaceutical composition for the treatment of ischemic or dental diseases, smoker's leg and diabetic ulcers or for the stimulation of wound healing.

2. The use of claim 1, wherein the nucleic acid induces the production of pro- angiogenic factors.

3. The use according to any of claims 1 or 2, wherein the nucleic acid induces the formation of vascular vessels.

4. Use of a) IKKε, b) a functional active derivative thereof, c) a nucleic acid encoding IKKε, and /or d) means for the detection of the molecules of sections a), b) , c) or d) for the preparation of a diagnostic agent for the diagnosis of ischemic or dental diseases, smoker's leg and diabetic ulcers, wound healing disorders, cancer, hyperplasia, tumor progression, rheumatoid arthritis, psoriasis, artherosclerosis, retinopathy, osteoarthritis, endometriosis or chronic inflammation.

5. Use of a JKKε inhibitor for the preparation of a pharmaceutical composition for the treatment of cancer, hyperplasia, rheumatoid arthritis, psoriasis, artherosclerosis, retinopathy, osteoarthritis, endometriosis or chronic inflammation.

6. The use of claim 5, wherein the inhibitor inhibits the production of VEGF.

7. The use of any of claims 5 or 6, wherein the inhibitor inhibits the formation of vascular vessels.

8. The use of any of claims 5 or 7, wherein the inhibitor is selected from the group consisting of antisense oligonucleotides, antisense RNA, siRNA, aptamers and Low molecular weight molecules (LMWs).

9. The use of claim 8, wherein the LMWs bind to the ATP-binding site of the kinase domain of IKKε.

10. The use of any of claims 4 to 9, wherein the disease is cancer, preferably selected from the group consisting of brain cancer, pancreas carcinoma, stomach cancer, colon carcinoma, skin cancer, especially melanoma, bone cancer, kidney carcinoma, liver cancer, lung carcinoma, ovary cancer, mamma carcinoma, uterus carcinoma, prostate cancer and testis carcinoma.

11. A method for the identification of an anti-cancer drug, wherein a) a potential IKKε interactor is brought into contact with IKKε or a functional derivative thereof, and b) binding of the potential interactor to IKKε or the functional derivative thereof is determined, and c) the anti-angiogenic capacity of the potential interactor is determined.

12. The method of claim 11, wherein the anti-angiogenic capacity is determined by measuring the inhibition of VEGF production.

13. The method of any of claims 11 or 12, wherein the potential interactor is provided in the form of a chemical compound library.

14. The method of claim 13, wherein the chemical compound library consists of a group of molecules or substances that bind to the ATP binding site of the kinase domain of IKKε.

15. The method of any of claims 11 or 14, wherein the method is carried out on an array.