DE10260264A1 - Screening for agents that inhibit or promote cell proliferation or have antiinflammatory activity, useful e.g. for treating tumors, based on activation or inhibition of the promoter of the guanylate-binding protein-1 gene - Google Patents

Abstract

Identifying agents (I) with proliferation-inhibiting, antiangiogenic, antiinflammatory and/or invasion-inhibiting activity, is new. Method for identifying agents (I) with proliferation-inhibiting, antiangiogenic, antiinflammatory and/or invasion-inhibiting activity comprises: (a) applying test compounds to either eukaryotic cells (A) that include a nucleic acid (II) comprising the promoter of a GBP-1 (guanylate-binding protein-1)-encoding gene, or of some other gene selectively induced by inflammatory cytokines, or their functionally equivalent fragments or derivatives, and a reporter gene (RG), or an extract or culture supernatant of (A); and (b) measuring the expression level of the RG-encoded transcript or protein. An increase in expression level in presence of a test compound indicates that this is a (I). Independent claims are also included for: (1) similar method for identifying agents (Ia) with proliferation-promoting, proangiogenic, proinflammatory, invasion-promoting and/or angiogenic-sensitizing activities, based on detecting a reduction in the specified expression level; and (2) eukaryotic cells that contain (II).

Description

The invention relates to a method to identify an active ingredient with anti-proliferation, antiangiogenic, invasive and / or anti-inflammatory effect, wherein one (a) eukaryotic cells that have a nucleic acid that contains the promoter sequence a gene encoding human guanylate binding protein-1 (GBP-1), the promoter sequence of another selectively by inflammatory Cytokine inducible gene or a fragment or derivative thereof with corresponding promoter activity functionally linked with contain a reporter gene or extracts of such eukaryotic Cells or cell culture supernatants thereof, in contact with one or more drug candidates; and (b) the level of expression of the transcript or (poly) peptide encoded by the reporter gene measures, with an increase in the level of expression after the addition of one or of the several drug candidates whose proliferation-inhibiting, anti-angiogenic, anti-inflammatory and / or an invasive effect is indicated. In an alternative embodiment The invention relates to a method for identifying an active substance with proliferation enhancing, pro-angiogenic, pro-inflammatory and / or invasive, angiogenic sensitizing (angiogenic sensitizing, invasive) Effect, where (a) in eukaryotic cells which have a nucleic acid, the gene coding for the promoter sequence of a GBP-1, the promoter sequence another selectively inducible by inflammatory cytokines Functionally gene or a fragment or derivative thereof with appropriate promoter activity connected containing a reporter gene or extracts of such eukaryotic Cells or cell culture supernatants thereof, in contact with one or more drug candidates; and (b) the level of expression of the transcript or (poly) peptide encoded by the reporter gene measures, with a decrease in the level of expression after the addition of one or of the several drug candidates whose proliferation-promoting and / or pro-angiogenic (angiogenesis-sensitizing, invasive) effect indexed. The invention further relates to a eukaryotic cell, which is a nucleic acid which has the promoter sequence of a gene coding for GBP-1, the promoter sequence of another selectively by inflammatory Cytokine inducible gene or a fragment or derivative thereof with corresponding promoter activity functionally linked with contains a reporter gene.

There is a number in the description of documents from the prior art mentioned. The revelation content of these documents, including of the operating instructions from manufacturers, is hereby in its entirety incorporated into the description by reference.

The endothelium is a key organ in numerous physiological and pathophysiological processes such as cell-controlled immune response, menstruation, wound healing, inflammation, allergy, Cardiovascular disease and tumor growth. The pathofunction of the Endothelium is inseparable from the activation of endothelial cells connected.

The activation of the endothelium is a complex process that is controlled by a variety of different soluble factors that circulate in the blood or are released by neighboring cells ( 1A ). As a result of this process, the physiology and morphology of the endothelial cells are adapted to the respective requirements in the tissue. The focus here is on the control of cell proliferation, apoptosis, invasion, migration and leukocyte adhesion ability of endothelial cells, which regulates the new and regression of vessels and the extravasation of leukocytes ( 1A ).

The large number of factors involved suggests that several factors control the same phenotype and can be grouped into groups with the same effect ( 1B ). For example, the angiogenic growth factors basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) activate endothelial cell proliferation, whereas the inflammatory cytokines interleukin (IL) -1α, IL-1β, tumor necrosis factor (TNF) -α and interferon (IFN) -γ inhibit proliferation and increase the leukocyte adhesiveness of endothelial cells.

There are currently no suitable ones Methods that can be used to determine where and when the various Factors in inflammatory Tissues act on the endothelial cells. Therefore, is about the spatial and temporal distribution of the different activation states of Endothelial cells in the context of inflammatory Very little known diseases.

Initial work to identify a molecular marker that indicates activation of endothelial cells by the above-mentioned inflammatory cytokines in the tissue led to comparative studies on gene expression in cultured endothelial cells in the presence of different activation conditions. With this approach it was possible to isolate a gene whose expression in endothelial cells is selectively induced by inflammatory cytokines ( 2A ) (Guenzi et al., 2001; Lubeseder-Martellato et al. 2002). This gene codes for the guanylate binding protein-1 (GBP-1), which belongs to the protein family of the large GTPases.

In order to determine whether GBP-1 indicates activation by inflammatory cytokines as in cultured cells in vascular endothelial cells in human tissues, immunohistochemical tests for the detection of GBP-1 using specific monoclonal antibodies were carried out. Histological sections of healthy skin and skin diseases with an inflammatory component, such as Example psoriasis, drug backlash and Kaposi's sarcoma examined ( 2 B ) All of the skin diseases mentioned have in common that there are numerous inflammatory cells concentrated in the lesions, which release the same inflammatory cytokines, which also lead to increased GBP-1 expression. In no case could GBP-1 be detected in the vessels of the healthy skin. In contrast, in all inflammatory diseases examined, individual vessels were clearly positive for GBP-1 ( 2 B , Arrows). These results showed that GBP-1 actually shows an inflammatory activation of endothelial cells in human tissues and can be used as a molecular marker for the detection of this activation in tissues (Lubeseder-Martellato, Guenzi et al.).

The induction of GBP-1 expression by inflammatory cytokines is associated with an inhibition of cell proliferation in endothelial cells. It was therefore investigated whether GBP-1 mediates the inhibition of proliferation induced by inflammatory cytokines. For this purpose, endothelial cells were transduced with retroviral vectors, which cause the constitutive expression of GBP-1 (GBP-1 vector) or an antisense GBP-1 RNA (AS vector) ( 3A ). Western blot analyzes showed that endothelial cells transduced with the GBP-1 vector expressed GBP-1 very strongly ( 3B ). In cells transduced with the AS vector, the induction of GBP-1 expression by IL-1β was efficiently blocked ( 3B ). Subsequent proliferation experiments with the various transduced cell cultures showed that GBP-1 actually inhibits cell proliferation induced by angiogenic growth factors ( 3C , white bars) and it is also necessary that inflammatory cytokines can inhibit the proliferation of endothelial cells ( 3D ). The latter manifests itself in the fact that in cell cultures expressing antisense GBP-1 RNA, the inhibitory effect of inflammatory cytokines on cell proliferation is significantly reduced ( 3D , black bars) (see Guenzi et al. 2001).

Further studies on the structure / function relationship Interestingly, the proliferation-inhibiting effects of GBP-1 showed that not the GTPase function (Prakash et al. 2000), but a section at the C-terminal End of protein (helical domain) inhibits cell proliferation (Guenzi et al. 2001; Töpolt et al. 2002). The adhesiveness of endothelial cells for Leukocytes, also induced by inflammatory cytokines was not affected by GBP-1 (Guenzi et al. 2001). GBP-1 is therefore a new, molecular marker for inflammatory vascular activation that is selective the antiproliferative effect of inflammatory cytokines on endothelial cells controls.

Inhibition of blood vessel supply represents a principle of tumor therapy (Folkman 1995). From ongoing clinical studies on antiangiogenic tumor therapy is known that the effect is present Therapeutics used in tumors of different organs very much varies widely (Carmeliet and Jain 2000). it is supposed that heterogeneity of the endothelial cells in the different areas of the body are responsible for this is (Carmeliet and Jain 2000). GBP-1 inhibits micro-proliferation and macrovascular Endothelial cells (Guenzi et al. 2001, Töpolt et al. 2001) and could therefore regardless of endothelial heterogeneity in different areas of the body develop an antiangiogenic effect.

So far it has been shown that GBP-1 is selectively induced by inflammatory cytokines and that this process with an anti-angiogenic effect correlated to the cells in question (Lubeseder-Martellato et al., 2002). While looking at induction from GBP-1 over its anti-proliferative effect in principle for medical purposes in the context anti-angiogenic therapy could be beneficial, the use of inflammatory Cytokines for such purposes because of the pleiotropic and therefore disadvantageous Effects of these cytokines are beyond dispute.

Furthermore, it could be shown that GBP-1 not only inhibits the proliferation of endothelial cells, but also inhibits the expression of metalloproteinase-1 in these cells efficiently ( 4A ). This effect is also anti-angiogenic and prevents the invasion of endothelial cells into the interstitial matrix ( 4B ) and the formation of capillaries ( 4C ). Both are indispensable processes in the context of angiogenesis. GBP-1 is therefore double-anti-angiogenic: by inhibiting proliferation (regulated by the helical domain of the molecule, regardless of the GTPase function) and by inhibiting MMP-1 expression (regulated by the GTPase function required).

Object of the present invention it was therefore necessary to provide processes with which active substances were identified can be such anti-inflammatory or anti-proliferative and in particular have anti-invasive anti-angiogenic effects, but not the discussed adverse pleiotropic effects of the prior art factors known in the art.

This task is accomplished by providing the in the claims featured embodiments solved.

The invention thus relates to a method for identifying an active substance with proliferation-inhibiting, anti-angiogenic, anti-inflammatory and / or invasive-inhibiting activity, wherein (a) eukaryotic cells which have a nucleic acid which comprise the promoter sequence of a gene coding for GBP-1, the Promoter sequence of another gene selectively inducible by inflammatory cytokines or a fragment or derivative thereof with corresponding promoter activity functionally linked to a reporter gene or extracts of such eukaryotic cells or cell culture supernatants thereof, with a or contacting multiple drug candidates; and (b) measures the level of expression of the transcript or (poly) peptide encoded by the reporter gene, an increase in the level of expression indicating the addition of the one or more drug candidates to the anti-proliferation, anti-angiogenic, anti-inflammatory and / or invasive effect after addition of the one or more drug candidates.

In connection with the present Invention, the term "antiperspirant effect" means that proliferation, i.e. the cellular Propagation by mitotic division, preferably at least 20% at least 40%, stronger preferably at least 60%, such as 75% or 80%, even more preferred at least 90% and particularly preferably at least 95% inhibited becomes. Ideally, proliferation is at least 99% inhibited.

The term "anti-inflammatory effect" means that the Effect on inflammation, that is, the effect on the reaction of a tissue to damage, which is particularly due to redness, Swelling, pain and / or overheating are marked is more preferred, at least 20%, preferably at least 40% at least 60%, such as 75% or 80%, more preferably at least 90% and particularly preferably at least 95% is inhibited. Ideally becomes the inflammation inhibited to at least 99%.

The term "invasive effect" means that the Rate of migration of cells through natural or artificial extracellular Matrix or components is inhibited. This effect means that the rate of migration is at least 20%, preferably at least 40%, stronger preferably at least 60%, such as 75% or 80%, even more preferred at least 90% and particularly preferably at least 95% inhibited becomes. Ideally, the rate of migration is at least 99% inhibited.

The invasion of cells by natural or artificial extracellular Matrix can be measured using the Boyden Chamber test. The Boydenkammer consists of two chambers (upper and lower) that pass through a porous Membrane are separated. The membrane is made with extracellular matrix or coated components of the extracellular matrix. In a chemoattractant is placed in the lower chamber and in the upper Chamber the cells to be examined. The number of cells after migrated to the lower chamber during the examination period (e.g. 4 h) are in proportion to the total number of cells added as a percentage of the specified invasive cells. 80% invasive cells means that of 100 cells 80 added have migrated into the lower chamber. If in another approach to this example in the presence of a Inhibitor only 50% of the cells immigrated to the lower chamber then the inhibitory effect of this substance corresponds to 50%.

For the purposes of this invention is also Please note that the above Effects such on currently available Proliferation or inflammation could be, alternatively, they can also be prophylactic, i.e. a possible or expected proliferation or inflammation ab initio or in one early inhibit or prevent.

"Eukaryotic cells" close all eukaryotic cells, including plant cells, yeast cells, Mushroom cells etc. independently whether they come from a multicellular organism or one represent unicellular organism.

Preferred eukaryotic cells are mammalian cells and particularly preferred among mammalian cells are human cells and mouse cells. Eukaryotic cells in the sense of this invention conclude also cells that are transformed or not in nature occur and are, for example, cells from cell lines. "Extracts" of such cells can processes established in the prior art are produced; see. e.g. Sambrook et al. 1989. Cell culture supernatants are usually grown with adherent Cells by simple removal, for example by pipetting off and at non-adherent growing cells e.g. by centrifuging the eukaryotic Get cells. However, a separation is not in every case of the supernatant from the cells necessary or desirable. Therefore, the invention also includes embodiments in which Expression tested in a cell culture vessel will, about the special properties of the respective indicator gene (e.g. in the case of luciferase and β-galactosidase Add appropriate substrates, or in the case of green fluorescent proteins by excitation with light in the specific wavelength).

According to the invention, the term “promoter sequence of a gene coding for GBP-1” denotes a promoter sequence which occurs in a naturally occurring GBP-1 gene. The genes coding for GBP-1 comprise a gene family with at least 5 members in humans, at least 5 members in mice and at least 1 member of the chicken These members of the GBP-1 gene family are characterized by an identity level of at least 80% at the amino acid level or 85% at the nucleic acid level, and on the basis of this information the person skilled in the art is able to obtain corresponding promoters from other mammals Such promoters can also be used in the method according to the invention, including methods in which further promoters or fragments or fragments or derivatives thereof to be isolated from other mammals with corresponding promoter activity are used in the present invention. The sequence of the GBP-1 promoter also includes d ie in 5 (A) dargestell sequences around the 237 bp fragment ( 5 (B) ) around. This sequence can be found in the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide). Another example of a corresponding promoter region is the mouse GBP-1 promoter region (accession number gi5106533 / AF109170 (NCBI)). This is characterized, among other things, by a similar / homologous promoter module as in the human GBP-1 promoter.

According to the invention, it was determined that the GBP-1 promoter activity on the interaction of an interferon-regulated binding site (ISRE / GAS) for the interferon regulator factor (IRF-1) and one that can be activated by NF-KB Binding site (cREL) is based. Both elements together are considered Called module. A purely synthetic promoter of these two Wearing elements will behave in the same way in principle. In any case, the person skilled in the process according to the invention determine usable promoters based on this information.

According to the invention, a fragment of the promoter sequence was also identified with 237 bp, which behaves accordingly and which carries the GAS / ISRE and NF-KB binding sites. Any longer fragment which contains these components can, of course, also be used according to the invention. For example, a fragment with 3757 bp behaves in a corresponding manner, and the function of the above components is achieved and in some cases a somewhat higher activity is achieved (see 5 ).

The term “the promoter sequence of a other gene selectively inducible by inflammatory cytokines "naturally affects occurring promoter sequence of a gene that (at least in humans) by at least one (like two or three) inflammatory cytokines) from the group interferon-α, interferon-β, interferon-γ, interleukin-1α, interleukin-1β and tumor necrosis factor-α and preferably all of these cytokines, but not preferred by other cytokines, Chemokines or growth factors are induced in expression unless this has a proliferation inhibiting effect have on endothelial cells. For example, candidates for such genes on the basis of the in Lubeseder-Martellato et al. (2002, hereby incorporated into the description by reference).

The term "in fragment or derivative thereof with corresponding promoter activity "means according to the invention that not the entire promoter region but part or all of it, also referred to above as modules, in the sense of this invention can be used. The parts can by the specialist on the basis of his general specialist knowledge, in any case but can be put together without undue effort so that the function of the natural Promoters by which the initiation point and the initiation frequency the transcription can be fixed, preserved or essentially (i.e. at least 50%, preferably at least 75%, more preferred at least 85% and most preferably at least 95%) remains or is improved. While the term "fragment" implies that at least one and preferably a combination of functional nucleic acid sequences in the same orientation as in the natural gene as described above in the in the method according to the invention nucleic acid used is incarnated, closes the term "derivative" further implies that the functional nucleic acid sequences new combined, genetically modified (e.g. around the efficiency of the binding to increase) or by additional functional sequences that come from other genes can come from added have been. Fragments as well as derivatives (and of course also the complete Promoter sequences) in synthetic, semi-synthetic, recombinant and other ways getting produced. "Appropriate Promoter activity "refers to the activity of the fragment or derivative compared to the activity of the natural Promoters, of course occurring gene. In other words, that is used in the method according to the invention Promoter fragment or derivative the function of the natural Maintain or essentially maintain or maintain a promoter improved activity and increased sensitivity across from Have cytokines, chemokines, etc., but not another tissue specificity. Basically can the promoter fragments and derivatives are designed in such a way that they are inducible or not inducible (= constitutive).

According to the invention, the term “functionally linked” means that the transcription of the reporter gene under the control of the promoter, Fragmentes or derivatives stands.

The term "reporter gene" denotes a gene whose gene product changes after introduction of the gene into a target cell or organism easily and for example biochemically, immunochemically and in particular visually can be demonstrated. According to the invention is usually the repoter gene merely positioned the coding sequence and usual 3 'of it regulatory elements, but not your own promoter region include. The gene product is preferably one that is normally is not produced by the cell or the organism, i.e. a heterologous gene product. Reporter gene in the sense of the present invention However, it can also be the GBP-1 gene or the coding and 3 'of which is arranged regulatory regions. The GBP-1 coding region can do that naturally occurring link with the GBP-1 promoter or a recombinantly modified as long as the promoter and coding region are functionally linked. The term "reporter gene" is in the state of the Technology well understood and continued by preferred and below listed embodiments shown.

According to the invention, the term “bringing into contact” means that the active ingredient candidate (s) are brought into close proximity to the cells in such a way that there is direct contact between the active ingredient candidates with the cell, for example via cellular receptors and possibly an uptake into the cell. The conditions under which the contact takes place preferably resemble in vivo conditions in order to simulate the conditions to which the cell in the cell network is exposed as far as possible. To control the corresponding conditions, (a) cocultivations of the indicator cell with other cells (primary cells, cell lines), (b) cultivations of the cells in two- and three-dimensional matrices (e.g. from protein mixtures that correspond to the extracellular matrix), (c) treatments with conditioned media. In addition, the generation of flow conditions can be taken into account, particularly when applied to endothelial cells. In vitro, the conditions preferably correspond to physiological conditions, such as, for example, by contacting the cells, including the cells derived from cell lines, in phosphate-buffered saline or a conventional cell culture medium. If the active substances are encoded by nucleic acids and these nucleic acids are introduced into the cell, the term “bring into contact that the nucleic acids are introduced into the cells (suitable techniques are described below) and the active substances, ie peptides or polypeptides, in which Cell synthesized and exert their potential effect on the promoter during or after synthesis.

The term (poly) peptide denotes both peptides, usually amino acid chains with up to 30 amino acids are referred to as peptides as well as polypeptides (proteins), where usually as polypeptides amino acid chains from 30 amino acids be designated.

According to the invention, the term “increase in the level of expression” denotes an increase by at least 20%, preferably at least 40%, more preferred at least 60%, even stronger preferably by at least 80% and most preferably by at least 100%. The strenght the increase can, depending measured by the reporter gene, for example by the intensity of the cell staining become. In this context it is worth noting that the expression of the reporter gene, for example Antibody, which are provided with a detectable marking or with a secondary Antibody, directed against the first antibody directed against the reporter gene is and in turn provided with a detectable marking is can be demonstrated. The increase in expression can Principle at the level of translation products (which is preferred) but also at the level of transcription medium in situ hybridization on cells and RT-PCR (Sturzl et al. 1995) can be measured.

According to the invention, the term “indexed” means that the result of the method according to the invention allows a first conclusion that the drug candidate has the desired effect. GBP-1 inhibits cell proliferation and the expression of GBP-1 can therefore be caused by a decrease in proliferation Proliferation assays thus represent a suitable read-out system for confirming the effect of drug candidates which have been indicated as proliferation-inhibiting in the process according to the invention. The effect is preferably to be confirmed in further tests:
An increase in proliferation can be measured (a) by determining the increase / decrease in the number of cells after a corresponding examination period (3-6 days) [is determined by cell counts using the Neubauer counting chamber or Coulter counter] (b) an increase / decrease DNA replication [is determined in the thymidine incorporation experiment after administration of radioactive or non-radioactive labeled nucleotides (eg H3-thymidine)], c) via the increase in metabolic activity that correlates directly with the cell number [is determined via the detection of mitochondrial dehydrogenases ( MTT assay).

This effect is also possible in animal models to test. Therefor e.g. experimentally induced angiogenesis (mouse Matrigel test (Cornali et al. 1996, Am J Pathol) or after injection of tumor cells in the tumor growth test).

The with the inventive method identified active ingredients, if appropriate and according to preferred embodiments of the present invention further modified to e.g. possible Mitigating side effects becomes one in combating and prophylaxis Range of diseases can be used. For example, active ingredients, which an increase in the expression level of the reporter gene in tumor vascular endothelial cells cause to be used in tumor therapy. Active substances that with the method according to the invention can be identified also (a) to prevent vascularization in chronic inflammation / wounds * and allergic reactions * in the sense of anti-inflammatory therapy (Psoriasis, allergic drug reactions), (b) for inhibition hyperproliferation in endothelial tumor diseases (angiosarcoma, Kaposi's sarcoma *) directly anti-tumorigen, (c) to contain vascular hyperproliferations (e.g. hemangioma) with a known or idiopathic genesis in the sense of a cosmetic or therapeutic application, (d) for the treatment of obesity, which can be restricted by inhibiting angiogenesis (* In inflammatory Inflammatory activation is locally restricted in diseases and thus by inducing GBP-1 expression in the non-inflammatory Area about inhibiting proliferation achieves a therapeutic effect become.).

In this respect, the invention includes the need in the prior art for methods of being able to screen suitable drug candidates in a simple and reliable manner. It is also advantageous that the process according to the invention can be designed as a high-throughput process and thus large amounts of active substance candidates can be tested for the desired activity within a very short time NEN. Another advantage is that the test takes significantly less time (maximum induction after 5 - 12 hours) than conventional methods (cell count, 3-6 days; thymidine incorporation test, 24 hours)

Preferably and as further demonstrated in the examples, the invention can be implemented as follows:
To identify GBP-1 inducing substances, the promoter region of the GBP-1 gene was isolated. The GBP-1 promoter was fused with an indicator gene [luciferase (luc) gene], the expression of which can be detected by a light reaction. Endothelial cells were transfected with the appropriate construct and the luc expression was examined. It was shown that all inflammatory cytokines could induce luc expression ( 6A ). This illustrates that the isolated promoter has the same expression properties as the GBP-1 cellular promoter.

In the next step, the GBP-1-luc construct was used to generate a stable cell line. This cell line is now used for high-throughput processes in which the effects of various chemically produced and natural compounds on GBP-1 expression are examined. Those substances which induce GBP-1 expression the most are subsequently checked for their anti-tumorigenic effect in mice to which tumors have been implanted by injection of tumor cells ( 6B ). The most promising candidates could later provide a possible basis for the treatment of human tumor diseases.

In a preferred embodiment of the method according to the invention the anti-proliferative effect is an anti-angiogenic effect.

According to the invention, the term “anti-angiogenic” means Effect "that the Formation of capillaries and vessels in the frame angiogenetic (sprout from existing vessels) and vasculogenetic (neovascularization processes are suppressed by immigration of endothelial progenitor cells). As oppression indicates that the formation of new vessels is at least 20%, preferably at least 40%, stronger preferably at least 60%, such as 75% or 80%, even more preferred is suppressed at least 90% and particularly preferably at least 95%. Ideally, the formation of new vessels is suppressed at least 99%.

In addition, the stand of technology need to isolate substances that promote proliferation and in particular have angiogenic effects and are also targeted, preferably selective in inflammatory tissues and without the pleiotropic effects the known from the prior art and mediating these effects Factors such as acidic and basic fibroblast growth factor (aFGF, bFGF), vascular endothelial cell growth factor (VEGF), platelet derived growth factor (PDGF) act.

In an alternative embodiment The invention therefore relates to a method for identifying a Active ingredient proliferation-promoting, pro-angiogenic, pro-inflammatory and / or invasive, angiogenic sensitizing effect, whereby one (a) eukaryotic cells which a nucleic acid which have the promoter sequence of a gene coding for GBP-1, the promoter sequence of another selectively by inflammatory Cytokine inducible gene or a fragment or derivative thereof with corresponding promoter activity functionally linked with contain a reporter gene or extracts of such eukaryotic cells or cell culture supernatants thereof, in contact with one or more drug candidates; and (b) the level of expression of the transcript or (poly) peptide encoded by the reporter gene measures, with a decrease in the level of expression after the addition of one or of the several drug candidates whose proliferation-promoting, pro-angiogenic, pro-inflammatory and / or invasive, angiogenic sensitizing effect indicated.

Angionesis is carried out in vivo via the Number of capillaries sprouting into the test tissue defined and in vitro about determines the number of capillaries formed in the capillary formation test. Sensitizing to attack means that an active ingredient per se is not triggers the formation of capillaries, but capillary formation due to angiogenic growth factors (e.g. bFGF, VEGF) in the presence of angiogenesis inhibitors Promotes substances (e.g. inflammatory cytokines). The term “tackled sensitizing effect "means consequently, that by the administration of an active substance in the presence of a given concentration of bFGF and / or VEGF and at the same time Presence of inflammatory cytokines the number of capillary formation formed by 10%, preferably by 20%, more preferably by 50%, in addition preferably increases by 75%, particularly preferably by 100%. The capillary formation test is among others described in Montesano et al. (1983) J Cell Biol. 97: 1658-52, Davis et al. (1996) Exp Cell Res. 224: 39-51 and Yang et al. (1999) Am J Pathol. 155: 887-95.

The term "invasive effect" means that the Migration speed and ability of cells through natural or artificial extracellular Matrix or components is increased. This effect means that the rate and ability to migrate is at least 20%, preferably at least 40%, stronger preferably at least 60%, such as 75% or 80%, even more preferred increased to at least 90% and particularly preferably to at least 95% becomes. Ideally, the rate of migration and ability increased to at least 99%.

First, either (i) Expression is induced experimentally (e.g. by administration of inflammatory Cytokines), or (ii) cells in which constitutive a high basic activity of the Promoter is present, or (iii) the promoter (or the introduced Expression construct) is changed so that there is a high constitutive expression of the reporter gene.

An example of (ii) is the use of a Cell with high endogenous expression of inflammatory cytokines, an example for (iii) is the use of a construct next to the GBP-1 promoter indicator gene Expression unit carries another expression unit that for the constitutive expression of e.g. Interleukin-1α, Interleukin-1β, Interferon-α, Interteron-β, Interferon-γ, or tumor necrosis factor leads.

The term “proliferation-promoting Effect " according to the invention that proliferation by at least 20%, preferably by at least 40%, stronger preferably by at least 60%, such as 75% or 80%, even more preferred increases by at least 90% and particularly preferably by at least 95%. Ideally, proliferation increases by at least 99%. A Increase in proliferation can be measured (a) via the Determination of the increase in cell number after a corresponding investigation period (3-6 Days) [determined by cell counts using the Neubauer counting chamber or coulter counter] (b) an increase DNA replication [is determined in the thymidine incorporation experiment after administration of radioactive or non-radioactive labeled nucleotides (e.g. H3-thymidine)], c) via the increase in metabolic activity that is directly related to the cell count correlated [is determined via detection of mitochondrial dehydrogenases (MTT test).

Furthermore, the term "pro-angiogenic" also describes angionesis sensitizing effect.

This embodiment of the invention will preferably with cells, e.g. B. cells in the tissue composite performed are exposed to inflammatory conditions or a high internal Have GBP-1 expression. In this embodiment, in addition to one corresponding indicator gene when used in certain cells (for example endothelial cells, see table) also the cell's own GBP-1 can be used. The requirement this is that GBP-1 from these cells in the cell culture supernatant is delivered. The determination of the cell's GBP-1 in the cell culture supernatant thus represents a measure of the activity of the GBP-1 promoter Secreted GBP-1 can be by means of an ELISA procedure provided be detected; see attached example 5.

The cells, preferably endothelial cells, be a cocktail of inflammatory cytokines exposed. Contact with these cytokines becomes one Increases in expression of the GBP-1 gene result and the cells become one Convict an inflammatory condition. So treated cells are then subjected to the method according to the invention. According to the above, the method according to the invention can by the one to be carried out in advance Step of contacting the cells with at least one, preferably two or three and particularly preferably with four inflammatory Cytokines as shown above can be expanded.

The one with this embodiment Active substances identified according to the invention are also medical diverse used. Such active substances are particularly suitable for vascular infusion to promote under inflammatory conditions. These include the advancement the formation of colateral vessels ischaemia (e.g. heart attack, smoker's leg) and the promotion of wound healing processes and graft acceptance. By eliminating the anti-angiogenic Blockage by GBP-1 could opposite the endothelial cells the natural Angiogenesis factors present in the tissue can be sensitized. It however, it can also be thought of using the active ingredient along with To administer angiogenesis factors. This would make the active ingredient increase the efficiency of angiogenesis factors.

This is preferably proliferation-promoting Pro-angiogenic effect of proliferation, invasion, angiogen-sensitizing effect.

According to the invention, the term “pro-angiogenic effect” means that the formation of capillaries and / or vessels is promoted. This requires endothelial cells several skills: preferably they have to proliferate and dissolve the extracellular Be enabled by means of released proteases. The inhibition of GBP-1 expression alone is not pro-angiogenic, but it is sensitive Endothelial cells under inflammatory conditions for activation due to angiogenic growth factors, which are usually also inflammatory Tissue present (equilibrium reaction) or simultaneously with the active ingredient can be administered.

This embodiment of the invention is especially preferred because it provides active ingredients can be which is the blood vessel formation induce and so used for example aftercare for infarcts can be.

In a further preferred embodiment of the method according to the invention the eukaryotic cells are primary cells and preferably mammalian cells like human cells. Also preferred are primary cells Endothelial cells, fibroblasts, smooth muscle cells or cell mixtures. These cells represent in the in vivo situation and show no deregulated growth and invasion properties such as. Tumor cells.

In the context of the invention, the term “primary cells” means that cells that are fresh from human Ge webs, organs or blood were isolated without cloning or immortalization or without being tumor cells.

The term "endothelial cells" closes in the sense the invention is not only primary Endothelial cells, but also derived cells that have the essential differentiation markers of endothelial cells (CD31, factor VIIIRA, e-selectin). You can further differentiated, but also dedifferentiated cells. Also included are cell lines, especially tumor cell lines endothelial origin. The same applies to the other in this preferred embodiment cell types shown.

Accordingly, according to one another preferred embodiment of the method according to the invention the eukaryotic cells are part of a cell line.

The term "cell line" defines cells that are fresh from tissue, Blood or organs are obtained and are unlimited outside the organism can multiply or grow.

Preferred cell lines are mammalian cell lines, e.g. human cell lines and tumor cell lines and preferred there Tumor cell lines of endothelial origin. The following are examples and usable according to the invention Cell lines listed: HMEC-1 (human endothelial cell line), SVEC4-10 (mouse endothelial cell line) HEK293 and HEK293T (renal epithelial cells), HeLa (cervical carcinoma), 3T3 (mouse fibroblasts), HepG2 (liver cells), U937 (monocytic cells), HUT78 (T cells). These cell lines are widely and freely available and can about that can also be purchased from recognized depositors.

According to another preferred embodiment of the method according to the invention becomes the nucleic acid by transfection, transduction, microinjection, electroporation, Genegunning or scratch loading introduced into the eukaryotic cell. Appropriate Methods are known to the person skilled in the art and i.a. described in method books; see e.g. Mühlhardt: The experimenter: molecular biology / genomics; 3rd edition, 2002, Spectrum academic publisher.

In a likewise preferred embodiment of the method according to the invention the reporter gene is encoded into the GBP-1 by homologous recombinants Gene or other selectively inducible by inflammatory cytokines Gene introduced. In this case, the reporter construct is under control of course GBP-1 promoter or other equivalent occurring in the cell activatable promoter. The repoter (poly) peptide can act as a fusion protein synthesized with the endogenous GBP-1 protein or a fragment thereof become. Alternatively, and dependent of the chosen chosen homologous to the target flanking sequences, the reporter (poly) peptide can also be free of endogenous GBP-1 sequences can be synthesized. Techniques for homologous Recombination, which can be used in the sense of the invention described in Recombinant DNA. 2nd edition. Watson JD, Gilman M, Witkowski J, Zoller M. Scientific American Books, 1992. WH Freeman and Company.

In another preferred embodiment of the method according to the invention is the nucleic acid an expression vector / plasmid or a viral nucleic acid.

Expression vectors / expression and their function are known to the person skilled in the art from the prior art. These are common Vector constructs, e.g. For an effective cloning of a desired nucleic acid, here a sequence encoding a reporter (poly) peptide, a replication motif (ori) and have a (multiple) cloning site. For an effective Expression of a coding nucleic acid is a vector sequence necessary expression of the encoded peptide or protein initiated. Therefor a start codon (ATG) is required. They also contain suitable ones Expression vectors / expression plasmid promoters and according to the invention the above described promoter or the fragment or derivative thereof. This are as mentioned Sequence areas through which the transcription of a gene (coding Nucleic acid) is controlled. The specialist distinguishes among other things between weak and strong, as well as between constitutive, inducible and repressible promoters. Particular examples of expression vectors / expression plasmids for the inventive method are set out in the examples. Plasmids for such promoter studies include e.g. pGL3-Basic (Promega), pEGFP-1 (Clontech), pβgal-Basic (Clontech).

Examples of the viral nucleic acids mentioned are also known to the person skilled in the art from the prior art. This viral nucleic acids contain in addition to the coding nucleic acid sequences described above further sequences, e.g. For encode viral proteins and / or for replication of the viral Nucleic acids / the Virus in a host are necessary. Particular examples of viral nucleic acids for the inventive method are vectors which e.g. on parvo, adeno, lenti, herpes, baculo- and retroviruses are based (pBABE-Puro, Guenzi et al. EMBO J 2001).

In a further preferred embodiment of the method according to the invention is transfection or transduction stable or transient Transfection or transduction.

According to the invention, the term “stable transfection” is understood to mean that the nucleic acid introduced into the cell is reproduced during the cell cycle and passed on during cell division. This means that the introduced nucleic acid remains “stable” in a long-term culture of the cell. The introduced nucleic acid preferably additionally contains an expression unit which carries a resistance gene (in the case of stable transfection and transduction) or it is used together with a plasmid for a re genetically encoded (only with stable transfection). If corresponding cells are cultivated in a selection medium which only allows growth for those cells which have taken up a resistance gene, this process has the effect that the introduced nucleic acids are stably manifested in the cell.

Alternatively, for certain Requirements the corresponding nucleic acid also transiently transfected or transduced. In this process, the cells are left behind the introduction of the nucleic acid used in the process without a subsequent selection step. In a long-term culture of appropriately treated cells, this works introduced nucleic acid lost.

There is a large number in the prior art described by reporter genes. All of these reporter genes are in principle for use in the method according to the invention suitable. It is preferred that the reporter gene is a luciferase, rhodamine, Green fluorescent protein or a GFP variant such as CFP, YFP, eGFP, Venus, or β-galactosidase, Encoded SEAP or lactate dehydrogenase. The use of others in the stand Examples of reporter genes described in the art are also given by the method according to the invention comprises; see for further examples of reporter genes e.g. Mühlhardt: The experimenter: Molecular Biology / Genomics; 3rd edition, 2002, Spektrum Akademischer Publishing company.

It is also particularly preferred that endogenous GBP-1 is used as a reporter gene, inter alia in connection with the embodiments described above. Since it was found according to the invention that the GBP-1 protein is secreted from the cell, the expression strength can be detected in a simple manner in the supernatant, for example by means of a receptor which specifically binds the GBP-1 protein. Suitable receptors are primarily antibodies or fragments (such as Fab, F (ab ') ₂ - or Fv fragments or derivatives (such as chimeric, humanized, bispecific antibodies or scFv constructs) thereof, for example monoclonal, but also polyclonal antibodies but also aptamers for the production of suitable antibodies see Harlow and Lane, 1988. The GBP-1 binding receptor can be labeled, for example a radioactive (for example ³ H or ^{1 25} I) label or a chemiluminescent, bioluminescent or other label However, the detection can also be carried out via a further secondary receptor which is specific for the GBP-1-binding receptor, usually a xenogenic antibody, which in turn bears a detectable label.Generally and in particular in this embodiment, the detection can be carried out in suitable standard methods as RIA, ELISA, EIA and other immunological detection methods are carried out; see also Harlow and Lane, 1988. Sekr The soluble GBP-1 can be detected, for example, in immunoprecipitation methods (see example 4) or in an ELISA method (see example 5).

A method is also preferred according to the invention, wherein the one or more drug candidates are polypeptides, Peptides or low molecular weight substances, antibodies or fragments or derivatives of which or are aptamers. The peptides preferably originate from one Peptide library as known in the art. Peptides or Polypeptides can for example on the surface bacteriophages as fusion proteins with their surface proteins, for example, gpIII or gpVIII are expressed (Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR.Making antibodies by phage display technology. Annu Rev Immunol. 1994; 12: 433-55 Review). The peptides or polypeptides can be as shown above encoded nucleic acid by suitable methods and expressed in the cell. In this way, the Test the impact of these (poly) peptides on the GBP-1 promoter. A particularly preferred one embodiment of the method according to the invention takes advantage of the full endogenous GBP-1 gene as Reporter construct chosen becomes.

The low molecular weight substances can be of organic or inorganic origin. Particularly preferred is according to the invention that the low molecular weight substances organic low molecular weight substances are.

A method is also preferred, further comprising the step of: (c) determining cell proliferation in the presence of the one or more drug candidates and Comparison of the determined value with a control approach, whereby a constant or a decrease in the number of cells compared to the control approach is characteristic of an antiperspirant effect and an increase in the number of cells in comparison to the control approach is characteristic of a poliferation increasing Effect.

An influence of an active substance on cell proliferation can be measured (a) by determining the increase / decrease in the number of cells after a corresponding examination period (3-6 days) [determined by cell counts using the Neubauer counting chamber or counter counter] (b) one Increase / decrease in DNA replication [is determined in the thymidine incorporation experiment after administration of radioactive or non-radioactive labeled nucleotides (eg H ³ thymidine)], c) via the increase in metabolic activity, which correlates directly with the cell number [is determined via the Detection of mitochondrial dehydrogenases determined (MTT test).

In an alternative embodiment, a method is further preferred, which further comprises the following step: (c) the determination of the invasion in the presence of the one or more drug candidates and comparison of the determined value with a control approach, whereby a constant or an Ab taking invasive cells in comparison to the control approach is characteristic for an invasive inhibiting effect and an increase in the number of invasive cells in comparison with the control approach is characteristic for an invasive effect.

Another preferred embodiment of the method according to the invention additionally includes to the process steps described above, the step of isolation of the active ingredient or ingredients.

After the desired active ingredient is identified it can be isolated easily and according to conventional methods be, for example from a substance library. But he can also be isolated directly from the test batch and with suitable Methods, for example NMR, MS, MALDI-TOF, sequencing methods etc. or combinations thereof can be further analyzed.

• (a) Testen von jeweils mehreren Wirkstoffkandidaten pro Testgefäß, wobei diejenigen Wirkstoffkandidaten in Reaktionsgefäßen, in denen keine Zunahme oder Abnahme der Expressionshöhe beobachtet wird, für die weiteren Tests nicht mehr in Betracht gezogen werden;
• (b) Aufteilung der in einem Testgefäß, in welchem ein positives Ergebnis beobachtet wurde, befindlichen Wirkstoffkandidaten in neue Testgefäße und Wiederholung des Tests, wobei wiederum diejenigen Wirkstoffkandidaten in Testgefäßen als Wirkstoffe in Betracht kommen, an denen eine Zunahme bzw. Abnahme der Expressionshöhe beobachtet wird; und gegebenenfalls
• (c) Wiederholung von Schritt (b), bis ein einziger Wirkstoff identifiziert ist, bei dem eine anti-angiogene und/oder anti-inflammatorische Wirkung oder eine pro-angiogene Wirkung indiziert ist.

In a further preferred embodiment of the method according to the invention, the following further steps will be carried out, provided that several drug candidates are tested in one and the same test vessel:

• (a) testing of several drug candidates per test tube, with those drug candidates in reaction vessels in which no increase or decrease in the expression level is observed no longer being considered for the further tests;
• (b) Division of the active substance candidates in a test vessel in which a positive result was observed into new test vessels and repetition of the test, again those active substance candidates in test vessels being considered as active substances on which an increase or decrease in the expression level is observed ; and if necessary
• (c) repeating step (b) until a single active substance is identified in which an anti-angiogenic and / or anti-inflammatory effect or a pro-angiogenic effect is indicated.

The method according to the invention is preferred a high throughput process. High throughput processes are in principle have been described in the prior art, see e.g. Takeshita et al, Anal Biochem. (1997) 247 (2), 242-6. Examples of corresponding High-throughput methods include luciferase tests, or automated ones GFP detection (for Luc and GFP as reporter gene). Another example represents the ELISA, the usual in multiwell plates (24 well, 48 well, 96 well and larger). An example of a such ELISA procedure is described in Example 5.

This high-throughput method is further preferred Computer-assisted. The computer assistant can, for example through the use of picking described in the prior art and / or spotting robots or by the computerized Use of a CCD camera to evaluate the test results.

• (a) die Bindungsstelle des Wirkstoffs an seinen Rezeptor identifiziert;
• (b) die Bindungsstelle Wirkstoffs an den Rezeptor durch molekulares Modellieren modifiziert; und
• (c) Wirkstoff dergestalt modifiziert, daß dessen Bindungsspezifität oder Bindungsaffinität oder Bindungsavidität für den Rezeptor durch molekulares Modellieren erhöht wird.

In addition, the invention relates to a method for improving the pharmacological properties of the identified active ingredient, wherein:

• (a) identifies the binding site of the drug to its receptor;
• (b) the binding site of the active substance to the receptor is modified by molecular modeling; and
• (c) Modifies active ingredient in such a way that its binding specificity or binding affinity or binding avidity for the receptor is increased by molecular modeling.

The term "receptors" are used in context understood with the invention all binding partners of an active ingredient via which this active ingredient can be detected by the method according to the invention Has an effect on GBP-1 expression. This includes e.g. proteins, Carbohydrates and lipids. These receptors can be released from the cell or on the cell surface, in the cytoplasm or in the nucleus be localized.

The various method steps of the method according to the invention described above are conventional method steps and can easily be carried out by a person skilled in the art with the usual skills. Based on the properties of the active substances identified in the method according to the invention, further biological assays can be used by the person skilled in the art, with which the specificity and effectiveness of the active substances can be tested. By modifying the activity of the target structures, the specificity and effectiveness of these active substances can be changed and tested with these assays. Steps (a) and (b) described above can be carried out according to conventional protocols. A protocol for site-directed mutagenesis is described in Ling MM, Robinson BH. (1997) Anal. Biochemical 254: 157-178. The use of methods for modulating homology areas in connection with targeted mutagenesis for the analysis of structure and functional properties is summarized in Szklarz and Halpert (1997) Life Sci. 61: 2507-2520. Chimeric proteins can be generated by ligation of appropriate DNA fragments using suitable, unique restriction sites using conventional cloning techniques. These are among others in Sambrook, Fritsch, Maniatis; Molecular Cloning, a laboratory manual; (1989) Cold Spring Harbor Laboratory Press. A fusion of two DNA fragments, which form a chimeric DNA fragment and encode a chimeric protein, can also be generated with the aid of a gateway system (life technologies). This system ba based on DNA fusion by recombination. A well-known example of molecular modeling is the structure-based design of substances that bind to HIV reverse trascriptase; see for example Mao et al. (2000) Biochem. Pharmacol. 60: 1252 to 1265.

Binding sites of active substances can be identified are through targeted mutagenesis and studies with the above Receptors. The primary sequences of the receptors are modified and subsequently it is analyzed whether this modification affects the binding affinity of the active substance changed; this method usually allows an exact mapping of the binding pocket of an active ingredient.

As an example for step (b), following Protocol proposed: If the effector site of an active ingredient has been mapped the exact residues involved in the interaction of the binding partners, by combining the information obtained from different Mutagenic studies (step a) and computer simulation of the structure the binding site were obtained. Among other things, this procedure enables an accurate three-dimensional representation of the structure. If the Active ingredient itself is a peptide, this can also be mutated like its attachment partner and the results of different Mutagenesis studies can be combined. Step (c) of inventive method allows modification to increase or improve binding affinity, effectiveness or specificity. If, for example, electrostatic interaction between different Remains of the target structure or sections of the drug molecule exist, can change the total load of corresponding sections and thus also changing the binding of the binding partners.

For the identification of binding sites can different computer programs are used. Hence suitable ones Computer programs are used to identify interaction sites of putative inhibitors / enhancers and the target structure through computer-assisted search after complementary Structural motifs (Fasina, Immunomethods 5 (1995), 114-120). Further suitable computer systems for computer-aided design of proteins and peptides are described in the prior art, e.g. in Berry, Biochem. Soc. Trans. 22: 1033-1036 (1994); Wodac, Ann. N.Y. Acad. Sci. 1987, 501: 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. Modifications of active substances can can be obtained, e.g., by peptidomimetics. Other inhibitors / enhancers can likewise through the synthesis of combinatorial peptidomimetic Libraries for successive chemical modification and testing of the resulting substances can be identified. Procedure for Production and use of combinatorial peptidomimetic Libraries are described in the prior art, e.g. in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.

• (a) eine Verstärkung der induzierenden oder hemmenden Wirkung auf die GBP-1 Promotoraktivität;
• (b) geringere Wirkung auf andere Promotoren; und/oder
• (c) geringe Wirkung auf die anti-angiogenen Funktionen des zellulären GBP-1.

Furthermore, in a further preferred embodiment, the invention relates to a method which further comprises improving the pharmacological properties of the identified active ingredient, testing for

• (a) an enhancement of the inducing or inhibitory effect on GBP-1 promoter activity;
• (b) less effect on other promoters; and or
• (c) little effect on the anti-angiogenic functions of cellular GBP-1.

Procedures for studying promoter activities are known to the person skilled in the art from the prior art (see, inter alia, Mülhardt, loc. cit.). Here, the detection of expression according to the invention a reporter gene used to determine the activity of a promoter.

A reinforcement of the inducing or inhibitory effects on the GBP-1 promoter activity can be achieved by chemical modification of the active ingredient (generation of derivatives) and renewed Testing of derivative libraries. Peptides and proteins can also modified derivatives (acetylation, glycosylation, etc.) or fragments of peptides and proteins are tested.

A lower effect on other promoters can be achieved by examining the effect of the active ingredient in analog tests using other promoters fused with an indicator gene (eg viral SV40 promoter, CMV promoter; cellular Bax, Bcl-2 promoter). The secretion of soluble factors can also be determined here (cytokines, chemokines, etc.), for example using known ELISA methods. The cell phenotype can also be examined directly. In this context, both the morphology of the cells can be examined, as well as the induction of apoptosis in the cells (methods for analyzing apoptosis include JAM test, TUNEL test, Cr ^{5 1} release tests and others; for example, described in Dulat et al. Eur J Immunol. 2001, 31 (7): 2217-26).

A little effect on the anti-aniogenic Functions of cellular GBP-1 can be analyzed by analyzing the proliferation activity of a Indicator cell showing GBP-1 by retroviral transduction (vector pBabePuro, S-GBP-1 construct) expressed constitutively, tested. If an appropriate substance the proliferation of this cell reinforced or increases MMP-1 expression, then this indicates a weakening Effect on the GBP-1 function and is considered an active ingredient that does not for a inventive method suitable is.

According to another preferred embodiment of the method according to the invention is the identified or improved active ingredient by peptidomimetics pharmacologically further improved.

• (i) ein modifiziertes aktives Zentrum, ein modifiziertes Aktivitätsspektrum, eine modifizierte Organ-, Gewebe- oder Zellspezifizität;
• (ii) eine verbesserte Aktivität;
• (iii) gesteigerte Toxizität für Tumorzellen (einen verbesserten therapeutischen Index) oder gesteigerte Wirkeffizienz auf Zielzellen;
• (iv) verminderte Nebenwirkungen;
• (v) ein zeitlicher versetzter Beginn der therapeutischen Wirksamkeit, der Länge der therapeutischen Wirksamkeit;
• (vi) veränderte pharmakokinetische Parameter (Resorption, Distribution, Metabolismus oder Exkretion);
• (vii) modifizierte physikochemische Parameter (Löslichkeit, hygroskopische Eigenschaften, Farbe, Geschmack, Geruch, Stabilität, Zustandsform);
• (viii) verbesserte generelle Spezifität, Organ-/Gewebespezifität; und/oder
• (ix) optimierte Verabreichungsform und -route

durch

• (i) Veresterung von Carboxylgruppen oder
• (ii) Veresterung von Hydroxylgruppen mit Carbonsäuren oder
• (iii) Veresterung von Hydroxylgruppen zu beispielsweise Phosphaten, Pyrophosphaten oder Sulfaten oder "Hemisukzinaten" oder
• (iv) Bildung von pharmazeutisch verträglichen Salzen oder
• (v) Bildung von pharmazeutisch verträglichen Komplexen oder
• (vi) Synthese von pharmakologisch aktiven Polymeren oder
• (vii) Einführung von hydrophilen Gruppen oder
• (viii) Einführung/Austausch von Substituenten in Aromaten oder Seitenketten, Veränderung des Substituentenmusters oder
• (ix) Modifikation durch Einführung von isosterischen oder bioisosterischen Gruppen oder
• (x) Synthese von homologen Verbindungen oder
• (xi) Einführung von verzweigten Seitenketten oder
• (xii) Konversion von Alkylsubstituenten zu zyklischen Analogen oder
• (xiii) Derivatisierung von Hydroxylgruppen zu Ketalen oder Acetalen oder
• (xiv) N-Acetylierung zu Amiden, Phenylcarbamaten oder
• (xv) Synthese von Mannich-Basen, Iminen oder
• (xvi) Umwandlung von Ketonen oder Aldehyden in Schiffs-Basen, Oxime, Acetate, Ketale, Enolester, Oxazolidine, Thiozolidine oder Kombinationen davon zu erreichen.

Also preferred is a method in which the active ingredient is further modified to

• (i) a modified active center, a modified activity spectrum, a modified organ, tissue or cell specificity;
• (ii) improved activity;
• (iii) increased toxicity to tumor cells (an improved therapeutic index) or increased efficacy on target cells;
• (iv) reduced side effects;
• (v) a delayed start of therapeutic efficacy, the length of therapeutic efficacy;
• (vi) changed pharmacokinetic parameters (absorption, distribution, metabolism or excretion);
• (vii) modified physicochemical parameters (solubility, hygroscopic properties, color, taste, smell, stability, condition);
• (viii) improved general specificity, organ / tissue specificity; and or
• (ix) optimized administration form and route

by

• (i) esterification of carboxyl groups or
• (ii) esterification of hydroxyl groups with carboxylic acids or
• (iii) esterification of hydroxyl groups to, for example, phosphates, pyrophosphates or sulfates or "hemisuccinates" or
• (iv) formation of pharmaceutically acceptable salts or
• (v) formation of pharmaceutically acceptable complexes or
• (vi) synthesis of pharmacologically active polymers or
• (vii) introduction of hydrophilic groups or
• (viii) introduction / exchange of substituents in aromatics or side chains, change of the substituent pattern or
• (ix) modification by introduction of isosteric or bioisosteric groups or
• (x) synthesis of homologous compounds or
• (xi) introduction of branched side chains or
• (xii) conversion of alkyl substituents to cyclic analogues or
• (xiii) derivatization of hydroxyl groups to ketals or acetals or
• (xiv) N-acetylation to amides, phenyl carbamates or
• (xv) Synthesis of Mannich bases, imines or
• (xvi) conversion of ketones or aldehydes to ship bases, oximes, acetates, ketals, enol esters, oxazolidines, thiozolidines or combinations thereof.

The steps outlined above are generally known in the art. They include or are based on quantitative structure-action relationship analyzes (QSAR; see Kubinyi, "Hausch Analysis and Related Approaches "VCH Verlag, Weinhaim, 1992), combinatorial biochemistry, classical Chemistry and other processes or disciplines; see. e.g. wooden grave and Bechtold, Deutsche Apotheker Zeitung 140 (8), 813-823, 2000.

In accordance with a further preferred embodiment of the method according to the invention, this further comprises the step of producing a medicament or medicinal product, the active ingredient obtained being formulated with a pharmaceutically acceptable carrier or diluent. Active substances identified with the aid of the method according to the invention can be formulated as medicaments for the treatment or prophylaxis of the diseases described above. The corresponding pharmaceutical formulation is optionally carried out in combination with a “pharmacologically acceptable carrier” and / or diluent and / or excipient. Examples of particularly suitable pharmacologically acceptable carriers are known to the person skilled in the art and include buffered saline solutions, water, emulsions such as oil / water emulsions, various types of detergents, sterile solutions, etc. Medicaments comprising such carriers can be formulated by known conventional methods. These medicaments can be administered to an individual in a suitable dose. Administration can be oral or parenteral, for example intravenous, intraperitoneal, Subcutaneously, intramuscularly, locally, intranasally, intrabronchially or intradermally, or via a catheter at one location in an artery The type of dosage is determined by the attending physician in accordance with the clinical factors t the dosage depends on various factors, such as height or weight, body surface area, age, gender or general health of the patient, but also on the agent to be administered, the duration and type of administration, and of other drugs that may be administered in parallel. A typical dose can be, for example, in a range between 0.001 and 1000 μg, doses below or above this exemplary range being conceivable, especially taking into account the factors mentioned above. In general, with regular administration of the appropriate composition, the dose should be in a range between 10 ng and 10 mg units per day or per application interval. If the composition is administered intravenously, the dose should be in a range between 1 ng and 0.1 mg units per kilogram of body weight per minute.

The corresponding composition can be administered locally or systemically. Preparations for parenteral Administration include sterile aqueous or non-aqueous solutions, Suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as Olive oil, and organic ester compounds such as e.g. Ethyl oleate used for injections are suitable. Include aqueous vehicles Water, alcoholic-aqueous solutions, Emulsions, suspensions, salt solutions and buffered media. Parenteral carriers include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate and bound oils. intravenous carrier include e.g. Liquid-, Nutrient- and electrolyte supplements (such as those based on Ringer's dextrose). The composition according to the invention can also Preservatives and other additives, such as e.g. antimicrobial Compounds, antioxidants, complexing agents and inert gases. Of can further, depending on the intended use, compounds such as e.g. interleukins, Growth factors, differentiation factors, interferons, chemotactic Contain proteins or a non-specific immunomodulatory agent his.

The invention further relates to a eukaryotic cell that has a nucleic acid that contains the promoter sequence of a gene encoding GBP-1, the promoter sequence of another selectively inducible gene by inflammatory cytokines or functional in the fragment or derivative thereof with appropriate promoter activity connected with a heterologous reporter gene.

The eukaryotic cell according to the invention can preferably for screening for desired drug candidates be used as described above. But it can also for testing the inflammatory or anti-inflammatory and the pro- and anti-angiogenic Potential of natural occurring factors are used, the change under the control of the above promoter or Fragment or derivative standing gene expression of the heterologous Gene or the heterologous coding sequence an indication of such Effect is.

In a preferred embodiment The invention relates to a cell that transfects stably or transiently or transduced.

It is also preferred that the cell a primary Cell is.

In a further preferred embodiment the cell is part of a cell line.

Finally, the invention relates a (non-human) transgenic animal, preferably a transgenic Rodent and particularly preferably a transgenic mouse or a transgenic Plant containing the eukaryotic according to the invention Cell in the germline and / or soma. General techniques for Production of transgenic animals and plants are state of the art known and can be used in the sense of the invention. The transgenic animals and Can plant used in drug validation and testing.

The figures show:

• (A) Die Aktivierung des Endothels bei entzündlichen Prozessen wird durch eine Vielzahl verschiedener löslicher Faktoren aus dem Blut und von benachbarten Zellen gesteuert. Dabei stehen im Rahmen der Neu- und Rückbildung von Gefäßen, sowie der Extravasation von Leukozyten die Steuerung von Zellproliferation, Apoptose, Invasion, Migration und Adhäsionsfähigkeit für Leukozyten im Vordergrund.
• (B) Die Vielzahl der beteiligten Faktoren legt nahe, dass mehrere Faktoren ein- und dieselbe Aktivierung beeinflußen und zu wirkungsgleichen Gruppen zusammengefaßt werden können. Gegenwärtig kann nicht bestimmt werden, wann und wo die verschiedenen Faktoren auf die einzelnen Endothelzellen einwirken. Darüber hinaus sind die Beziehungen der meisten Aktivierungsarten zueinander weitgehend unbekannt. Es ist zu bestimmen, ob alle Aktivierungen gleichzeitig in einer Zelle auftreten können (I) oder aufgrund zellbiologischer Restriktionen zeitlich, beziehungsweise räumlich separiert sein müssen (II).

1 : Complexity and redundancy of inflammatory endothelial cell activation.

• (A) Activation of the endothelium in inflammatory processes is controlled by a variety of different soluble factors from the blood and neighboring cells. In the context of the reconstruction and regression of vessels, as well as the extravasation of leukocytes, the focus is on the control of cell proliferation, apoptosis, invasion, migration and adhesiveness for leukocytes.
• (B) The large number of factors involved suggests that several factors influence one and the same activation and can be grouped into groups with the same effect. It is currently not possible to determine when and where the various factors affect the individual endothelial cells. In addition, the relationships between most types of activation are largely unknown. It has to be determined whether all activations can occur simultaneously in one cell (I) or whether they have to be separated in time or space due to cell biological restrictions (II).

• (A) Western Blot-Analyse der GBP-1 Expression in Endothelzellen, die mit den aufgeführten Faktoren für 24 h stimuliert worden waren. Folgende Konzentrationen wurden eingesetzt: IFN-γ (100 U/ml), 1L-1α (5 ng/ml), IL-1β (200 U/ml), TNF-α (300 U/ml), IL-4 (10 U/ml), IL-6 (50 U/ml), IL-10 (50 ng/ml), IL-18 (100 ng/ml), Oncostatin M (10 ng/ml), MCP-1 (50 ng/ml), PF4 (25 ng/ml), SDF-1α (200 ng/ml), bFGF (10 ng/ml), VEGF (10 ng/ml), Ang-2 (800 ng/ml) und PDGF B/B (100 ng/ml). Der gleichzeitige Nachweis des Zytoskelettproteins Aktin zeigt, dass gleiche Proteinmengen aufgetragen wurden.
• (B) Induktion der GBP-1-Expression in vaskulären Endothelzellen bei Hauterkrankungen mit einer inflammatorischen Komponente. Indirekte Immunfluoreszenzfärbung von GBP-1 (grün) und dem Endothelzell-assoziierten Antigen CD31 (rot) in Gewebeschnitten von gesunder Haut, Kaposi-Sarkom, entzündlicher Arzneimittelgegenreaktion der Haut und Psoriasis. Die Überlagerung der Bilder zeigt eine Ko-Expression (gelb) von GBP-1 und CD31 (weiße Pfeile). (Modifiziert nach (Lubeseder-Martellato, Guenzi et al. 2002))

2 : Expression of GBP-1 in cultured endothelial cells and in inflammatory skin diseases.

• (A) Western blot analysis of GBP-1 expression in endothelial cells that had been stimulated with the listed factors for 24 h. The following concentrations were used: IFN-γ (100 U / ml), 1L-1α (5 ng / ml), IL-1β (200 U / ml), TNF-α (300 U / ml), IL-4 (10 U / ml), IL-6 (50 U / ml), IL-10 (50 ng / ml), IL-18 (100 ng / ml), Oncostatin M (10 ng / ml), MCP-1 (50 ng / ml), PF4 (25ng / ml), SDF-1α (200ng / ml), bFGF (10ng / ml), VEGF (10ng / ml), Ang-2 (800ng / ml) and PDGF B / B (100ng / ml). The simultaneous detection of the cytosque lettproteins actin shows that the same amount of protein was applied.
• (B) Induction of GBP-1 expression in vascular endothelial cells in skin diseases with an inflammatory component. Indirect immunofluorescence staining of GBP-1 (green) and the endothelial cell-associated antigen CD31 (red) in tissue sections from healthy skin, Kaposi's sarcoma, inflammatory drug counter-reaction of the skin and psoriasis. The superimposition of the images shows a co-expression (yellow) of GBP-1 and CD31 (white arrows). (Modified from (Lubeseder-Martellato, Guenzi et al. 2002))

• (A) Schematische Darstellung des retroviralen Expressionsvektors pBabePuro (Kontrollvektor, K-Vektor) in den die cDNA von GBP-1 in beiden Orientierungen für die konstitutive Expression von GBP-1 (GBP-1-Vektor) und für die Expression einer GBP-1-antisense-RNA (AS-Vektor) eingesetzt wurde.
• (B) GBP-1-Expression in K-, GBP-1-, und AS-Vektor transduzierten Endothelzellen, die entweder unbehandelt waren oder über einen Zeitraum von 24 h mit 20 U/ml IL-1β stimuliert wurden. Der Nachweis der GBP-1-Expression erfolgte mittels Western-Blot-Analyse mit einem polyklonalen anti-GBP-1-Antikörper. Die gleichzeitige Färbung mit Aktin zeigt, dass gleiche Proteinmengen aufgetragen wurden.
• (C) Proliferationsexperimente mit K-Vektor- und GBP-1-Vektor-transduzierten Endothelzellen in Anwesenheit steigender Konzentrationen angiogener Wachstumsfaktoren (bFGF und VEGF in Kombination).
• (D) Proliferationsexperimente von K-Vektor- und AS-Vektor-transduzierten Endothelzellen in Anwesenheit angiogener Wachstumsfaktoren und aufsteigender Konzentrationen von IL-1β. (Modifiziert nach (Guenzi et al. 2001))

3 : GBP-1 mediates the antiproliferative effect of inflammatory cytokines in endothelial cells.

• (A) Schematic representation of the retroviral expression vector pBabePuro (control vector, K vector) in which the cDNA of GBP-1 in both orientations for the constitutive expression of GBP-1 (GBP-1 vector) and for the expression of a GBP-1 -antisense-RNA (AS vector) was used.
• (B) GBP-1 expression in K, GBP-1, and AS vector transduced endothelial cells that were either untreated or stimulated with 20 U / ml IL-1β over a 24 hour period. The GBP-1 expression was detected by Western blot analysis with a polyclonal anti-GBP-1 antibody. The simultaneous staining with actin shows that the same amounts of protein were applied.
• (C) Proliferation experiments with K vector and GBP-1 vector transduced endothelial cells in the presence of increasing concentrations of angiogenic growth factors (bFGF and VEGF in combination).
• (D) Proliferation experiments of K vector and AS vector transduced endothelial cells in the presence of angiogenic growth factors and increasing concentrations of IL-1β. (Modified from (Guenzi et al. 2001))

• (A) MMP-1 wird nicht in GBP-1-exprimierenden Zellen exprimiert und die MMP-1-Expression kann auch nicht durch angiogene Wachstumsfaktoren, wie bFGF und VEGF induziert werden. Kontrolle bezeichnet Endothelzellen, die mit einem Kontrollvektor transduziert wurden. S-GBP-1 bezeichnet Endothelzellen, die mit einem Vektor transduziert wurden, der die konstitutive Expression von GBP-1 in diesen Zellen bewirkt. Die Expression von GBP-1 und von MMP-1 wurden mit Western Blot-Analyse nachgewiesen. Der Nachweis von GAPDH zeigt, dass gleiche Proteinmengen aufgetragen wurden. AWF (+) bedeutet, dass die Zellen mit angiogenen Wachstumsfaktoren behandelt wurden, AWF (–) bedeutet, dass die Zellen nicht mit angiogenen Wachstumsfaktoren behandelt wurden.
• (B) GBP-1 hemmt die Einwanderung/Invasion von Endothelzellen in Kollagen-I Matrizes (schwarze Balken), wohingegen die Einwanderungsfähigkeit von Endothelzellen, die mit einem Kontrollvektor transduziert wurden, nicht beeinflusst war (weiße Balken). Kontrolle bezeichnet Endothelzellen, die mit einem Kontrollvektor transduziert wurden. S-GBP-1 bezeichnet Endothelzellen, die mit einem Vektor transduziert wurden, der die konstitutive Expression von GBP-1 in diesen Zellen bewirkt. Invasion wurde im Boyden-kammer Test bestimmt unter Verwendung von porösen Membranen, die mit Kollagen-I beschichtet waren.
• (C) GBP-1 hemmt die Bildung von Kapillaren in Kollagen-I. Zellen die mit dem Kontrollvektor transduziert worden waren bildeten nach 48 Stunden Kapillarähnliche Strukturen aus (Kontrolle). Zellen die mit GBP-1 transduziert worden waren konnten im Gegensatz dazu keine Kapillaren ausbilden. Kontrolle bezeichnet Endothelzellen, die mit einem Kontrollvektor transduziert wurden. S-GBP-1 bezeichnet Endothelzellen, die mit einem Vektor transduziert wurden, der die konstitutive Expression von GBP-1 in diesen Zellen bewirkt.

4 : GBP-1 inhibits MMP-1 expression, invasion and capillary formation ability of endothelial cells in collagen-I.

• (A) MMP-1 is not expressed in GBP-1 expressing cells and MMP-1 expression cannot be induced by angiogenic growth factors such as bFGF and VEGF. Control denotes endothelial cells that have been transduced with a control vector. S-GBP-1 denotes endothelial cells that have been transduced with a vector that causes the constitutive expression of GBP-1 in these cells. Expression of GBP-1 and MMP-1 was demonstrated by Western blot analysis. The detection of GAPDH shows that equal amounts of protein were applied. AWF (+) means that the cells were treated with angiogenic growth factors, AWF (-) means that the cells were not treated with angiogenic growth factors.
• (B) GBP-1 inhibits the immigration / invasion of endothelial cells in collagen I matrices (black bars), whereas the immigration ability of endothelial cells transduced with a control vector was not affected (white bars). Control denotes endothelial cells that have been transduced with a control vector. S-GBP-1 denotes endothelial cells that have been transduced with a vector that causes the constitutive expression of GBP-1 in these cells. Invasion was determined in the Boyden chamber test using porous membranes coated with collagen-I.
• (C) GBP-1 inhibits the formation of capillaries in collagen-I. Cells transduced with the control vector developed capillary-like structures after 48 hours (control). In contrast, cells that had been transduced with GBP-1 were unable to form capillaries. Control denotes endothelial cells that have been transduced with a control vector. S-GBP-1 denotes endothelial cells that have been transduced with a vector that causes the constitutive expression of GBP-1 in these cells.

• (A) Sequenz 1 (3757 bp GBP-1 Promotor): Das 237 bp GBP-1 Promotorfragment ist im 3757 bp Promotor enthalten und ist entsprechend gekennzeichnet (Sequenz unterstrichen, fett, kursiv). Sämtliche Sequenzen sind der NCBI-Nukleotiddatenbank zu entnehmen (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide).
• (B) Sequenz 2 (237 bp GBP-1 Promotor): Das 237 bp GBP-1 Promotorfragment trägt die gekennzeichneten Modulkomponenten ISRE/GAS-Bindungsstelle und NF-KB-Bindungsstelle für die Transkriptionsfaktoren IRF-1 und p65/p50; p65/cRel. Mit Pfeilen sind die 4 möglichen Transkriptionsstartstellen eingezeichnet. Beide Promotoren sind durch die inflammatorischen Zytokine IFN-γ, IL-1β und TNF-α selektiv induzierbar. Des weiteren sind mögliche Kandidaten angegeben (Maus-GBP-1 und GBP-2), die sich so verhalten könnten, wie der humane GBP-1 Promotor.

5 : Sequences of GBP-1 promoters.

• (A) Sequence 1 (3757 bp GBP-1 promoter): The 237 bp GBP-1 promoter fragment is contained in the 3757 bp promoter and is labeled accordingly (sequence underlined, bold, italic). All sequences can be found in the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide).
• (B) Sequence 2 (237 bp GBP-1 promoter): The 237 bp GBP-1 promoter fragment carries the labeled module components ISRE / GAS binding site and NF-KB binding site for the transcription factors IRF-1 and p65 / p50; p65 / cRel. The 4 possible transcription start points are shown with arrows. Both promoters can be selectively induced by the inflammatory cytokines IFN-γ, IL-1β and TNF-α. Furthermore, possible candidates are indicated (mouse GBP-1 and GBP-2), which could behave like the human GBP-1 promoter.

6 : Isolation of the promoter from GBP-1 for use in the high-throughput method for the identification of antiangiogenic substances.

• (A) Luciferase detection to determine the activity of the GBP-1 promoter isolated and fused to the luc gene in endothelial cells after stimulation with inflammatory cytokines.
• (B) Scheme of a high-throughput method for the identification of GBP-1-inducing antiangiogenic substances. A cell line was generated in which the luc indicator gene is located control of the isolated GBP-1 promoter. Based this cell line, the effects of various chemically-generated and natural Compounds examined for GBP-1 expression. Those substances which induce GBP-1 expression the most (yellow) subsequently in mice, to which tumors have been implanted by injection of tumor cells, checked for their anti-tumorigenic effect.

7 : Secretion of GBP-1 from IFN-γ treated HUVEC is not due to an increase in apoptosis and / or cell permeability.
HUVEC cells were cultivated overnight (low medium). The cells were then stimulated with IFN-γ (concentration of 100 U / ml for 24 h). Unstimulated cells (C) were only treated with BSA. (A) The lactate dehydrogenase (LDH) activity test shows that stimulation of HUVEC with IFN-γ does not increase the amount of lactate dehydrogenase in the culture supernatant. LDH activity from media to which 1%, 2.5% or 7.5% cell lysate obtained by freeze-thaw cycles was added as standard. (B) HUVEC staining with "membrane impermeant dye dead-red". Cells from three optical fields were counted and the percentage of cells with increased membrane permeability (black columns) compared to the total number of cells.

8th : Detection of secreted G8P-1 by ELISA.
(Low medium). The cells were then stimulated with IFN-γ (concentration of 100 U / ml for 24 h). Unstimulated cells (C) were only treated with BSA. Supernatants were collected and (A) in an ELISA and (B) analyzed by immunoprecipitation and Western blot, see Example 5. Dilutions of purified GBP-1-His were used as the ELISA standard.

9 : Detection of circulating GBP-1 in plasma from IFN-α treated patients
In melanoma patients treated with IFN-α for 9 or 28 days, the concentration of circulating GBP-1 in the plasma was determined by ELISA. The increase in the GBP-1 concentration from day 9 to day 28 in three patients is shown. ELISA microtiter plates were coated with a monoclonal rat anti-GBP-1 antibody, clone 1B1, for 16 hours at 4 ° C. The plates were then washed with PBS-T (0.1% Tween 20 in PBS) and free binding sites were saturated with PBS-T / BSA 2 (PBS-TB) for 30 minutes at room temperature (RT). After aspiration of the PBS-TB, an incubation (2 h) with 100 μl of different plasma samples (1: 2) was carried out at RT. Purified GBP-1-His protein diluted in cell culture medium (EBM-0.5% FCS) was used as standard. BSA served as a negative control. The samples were washed 4 × with PBS-T, 100 μl polyclonal rabbit anti-GBP-1 antibody (1: 500) were added and incubated for 2 hours at RT. After washing four times with PBS-T, the mixture was incubated (1 h, RT) with 100 μl AP-conjugated anti-rabbit antibody (diluted 1: 500 in PBS-TB). After four further washing steps, the GBP-1 protein / antibody complex was visualized by incubation with 100 μl of P-nitrophenyl phosphate. The absorption was measured at 405 nm in the microplate reader. The concentration was determined using a standard curve for which increasing concentrations of purified GBP-1-His were used. The linearity of the measurement is given for a range of 0.1-100 ng / ml.

The examples illustrate the invention.

Example 1: Production of a vector construct

The 237 bp GBP-1 promoter carrier (pro237-GBP-1) was by means of PCR amplification (PCR 2 Advantage Kit, Clontech) of the construct pro3757-GBP-1 with the oligonucleotides 5'-ATTTGAAGCTTCTGGTTGAG-3 '[insert one Hindlll interface (underlined)] or 5'-TGGCTTCTAGCACTTCTG-3 'generated. The construct pro3757-GBP-1 contains 3757 bp of the 5 'regulatory Sequence up the ATG codon of the GBP-1 gene (gi: 4503938, NM_002053) in the vector pT-Adv (Clontech). The 237 bp fragment was in the antisense orientation ligated to the vector pT-Adv, cut with HindIII and into the pGL3-Basic Vector (Promega) subcloned. All constructs were made with the Endofree Maxi Kit (Qiagen) cleaned and sequenced for verification.

Example 2: Establishment a suitable cell line

HEK 293 T cells (human embryonic epithelial kidney cell line with human adenovirus type 5 (Ad 5) DNA transformed (ATCC CRL 1573), which is additionally transformed with SV40 T antigen) were treated with 3 × 10 ⁵ cells / well (6-multi- Well plate, Corning) sown 24 h before transfection. A total of 0.8 μg plasmid was used per well of the 6-well plate, the test plasmid pro237-GBP-1 in a ratio of 1: 5 with the selection plasmid pBABE-Puro (P. Monini, Laboratory of Virology, Instituto Superiore di Santiá, Rome , Italy) was used. The test plasmid pro237-GBP-1 contains the 237 bp promoter fragment coupled to the indicator gene firefly-luciferase, the selection plasmid pBABE-Puro the resistance gene puromycin is finally selected. The transfection of the cells was carried out in accordance with the manufacturer's instructions with Effectene (Qiagen) and after 24 hours the selection was started with 0.7 μg / ml puromycin (Sigma). The growth of single clones was observed on days 8-10, which were then trypsinized with cloning rings and transferred to single wells of a 96-well plate (Falcon). If the confluence was appropriate, the clones were further passaged and examined for their reporter gene activity in the luciferase assay. The clones were stimulated with inflammatory cytokines IFN-γ, IL-1β and TNF-α, as well as buffers for 5 h and harvested in 1x "passive lysis buffer" (Promega). The lysates were examined for firefly luciferase activity and accordingly stable Clones established.

Example 3: Procedure for screening for drug candidates

The stable cell lines that the GBP-1 promoter are stable in the genome of HEK 293T cells Multi-well plates sown (e.g. 96-well plate) and in succession with different substances incubated that may the GBP-1 promoter activity can induce. These are e.g. inflammatory cytokines such as IFN-γ, IL-1β, or TNF-α, but also chemical substances or natural Agents from natural product banks. As a result, the cells are lysed and their reporter gene activity examined in the luciferase assay.

The procedure described here is, in principle, available in all eukaryotic cell lines carried out as well as with different reporter genes (e.g. β-galactosidase, green fluorescent protein) or different sized promoter fragments (Proof provided for Promoter fragments 237-3757 bp 5'-regulatory Region). Furthermore, you can the substances to be tested applied extracellularly or expressed intracellularly become.

Example 4: Immunoprecipitation

Freshly made cell lysates were made pre-cleaned by incubation with 2 μl of non-reacting GBP-1 Pre-immune serum of rabbits and 25 μl Protein A / G agarose beads for at least 3 h at 4 ° C on a shaking platform. After pelleting the beads, the supernatant was treated with 25 ul protein A / G agarose beads and 1 μl of polyclonal rabbit serum against GPB-1 overnight at 4 ° C on one shaking incubated. The beads were washed four to five times in PBS. Then were the beads in 30 μl Laemmli sample buffer (2X) resuspended and boiled for 5 min. Samples were separated in an SDS-PAGE (10%) and in a Western blot or analyzed in an autoradiography.

For the immunoprecipitation of GPB-1 or MMP-1 from cell culture supernatants, 10 ml of culture medium was placed on ice, centrifuged at 1000 rpm for 5 min, filtered through a filter with a pore size of 45 μm and in some cases a proteinase inhibitor cocktail (0.02 mg / ml pancreatic extract, 5 μg / ml pronase, 0.5 μg / ml thermolysin, 3 μg / ml chymotrypsin and 0.33 μm / ml papin) were added. The pre-cleaning was carried out by incubation with 10 μl of rabbit pre-immune serum which did not react with GBP-1 and 120 μl of protein A / G agarose beads for more than 3 h at 4 ° C. on a shaking platform. After pelleting the beads, the supernatant was incubated with 120 μl protein A / G agarose beads and 6 μl of polyclonal rabbit serum against GPB-1 at 4 ° C. on a shaking platform. The beads were washed four to five times in PBS. The beads were then resuspended in 60 μl PBS + 60 μl Laemmli sample buffer (2X) and boiled for 5 min. Usually 15 μl of each sample were separated in an SDS-PAGE (10%) and analyzed in a Western blot. A corresponding experiment is in 8B shown.

Example 5: GBP-1 ELISA

The following buffers were used for the developed GBP-1 ELISA:
PBS containing 0.1% Tween 20 (PBS-T) and PBS containing 0.1% Tween 20 and 2% BSA (PBS-TB).

96-well ELISA plates (Nunc-Immuno Plates) were coated with 100 μl / well anti-GBP-1 hybridoma supernatant, diluted 1: 5 with PBS or with purified antibody in the concentrations of 1-5 μg / ml ( Incubation for 16 h at 4 ° C). The plates were washed with PBS-T and blocked with PBS-TB for at least 30 min at room temperature. The wells were aspirated and incubated as doublets for 2 h at room temperature with 100 μl of the standard (GBP-1-His, diluted in cell culture medium with 5% FBS), the same concentration of BSA as a control or with 100 μl of a sample in a suitable dilution (diluted in PBS). The wells were washed four times with PBS-T and incubated with 100 μl of a polyclonal antibody against GPB-1, diluted in 1: 500 in PBS-TB for 2 h at room temperature. The wells were then washed four times with PBS-T and diluted 1: 500 in PBS-TB with 100 μl of an alkaline phosphatase conjugated to an anti-rabbit antibody (Zymed, Berlin, Germany) for 1 h Incubated at room temperature. The wells were then washed four times with PBS-T and incubated with 100 μl of p-nitrophenyl phosphate (Zymed). The absorption was determined at 405 nm in a "microplate reader" (BioRad). The concentration of GPB-1 in the sample was calculated using the standard curve The method showed a linearity of 0.1 to 100 ng / ml of GBP-1 / well. The variability of the results in different assays was between 2.3 and 6%. Corresponding experiments are in the 8th and 9 shown.

Example 6: Proliferation Assay

The increase / decrease in proliferation was measured by determining the cell count. Cells were therefor Sown in multiwell plates in a hunger medium (low medium) and overnight treated with the active ingredient. After 3 to 6 days, the cell count by counting determined and the cell number compared with the cell number in control batches.

literature

• Carmeliet. and Jain: Nature 407 (6801): 249-57 (2000).
• Folkman: Nat Med 1 (1): 27-31 (1995).
• Guenzi, Töpolt, Cornali, Lubeseder-Martellato, Jörg, Matzen, Zietz, Kremmer, Nappi, Schwemmte, Hohenadl, Barillari, Tschachler, Monini, Ensoli, and Falls: Embo J 20 (20): 5568-77. (2001).
• Harlow and Lane, "Antibodies, a laboratory manual ", CSH Press 1988, Cold Spring Harbor
• Töpolt, Guenzi, Lubeseder-Martellato, Jörg, Naschberger, falls: Proceedings of the 22nd Meeting of the European Society of Microcirculation (2002).
• Lubeseder-Martellato, Guenzi, Jörg, Töpolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmte, Matzen, Grimm, Ensoli and crashes: Am J Pathol 161 (5): 1749-59 (2002).
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SEQUENCE LISTING

Claims (28)

Method of identifying an active substance with anti-proliferation, anti-angiogenic, anti-inflammatory and / or invasive effect, wherein one (a) eukaryotic Cells that contain a nucleic acid which have the promoter sequence of a gene coding for GBP-1, the promoter sequence of another selectively by inflammatory Cytokine inducible gene or in fragment or derivative thereof with corresponding promoter activity functionally linked with contain a reporter gene or extracts of such eukaryotic Cells or cell culture supernatants thereof, in contact with one or more drug candidates; and (B) the level of expression of the transcript or (poly) peptide encoded by the reporter gene measures, with an increase in the level of expression after the addition of one or of the several drug candidates whose proliferation-inhibiting, antiangiogenic, anti-inflammatory and / or invasive Effect indicated. The method of claim 1, wherein the anti-proliferative Effect is an anti-angiogenic effect. Method for identifying an active ingredient with proliferation-promoting, pro-angiogenic, pro-inflammatory and / or invasive, concerned sensitizing effect, whereby one (a) eukaryotic Cells that contain a nucleic acid which have the promoter sequence of a gene coding for GBP-1, the promoter sequence of another selectively by inflammatory Cytokine inducible gene or in fragment or derivative thereof with corresponding promoter activity functionally linked with contain a reporter gene or extracts of such eukaryotic Cells or cell culture supernatants thereof, in contact with one or more drug candidates; and (B) the level of expression of the transcript or (poly) peptide encoded by the reporter gene measures, with a decrease in the level of expression after the addition of one or of the several drug candidates whose proliferation-promoting, pro-angiogenic, pro-inflammatory and / or invasive, concerned sensitizing effect indicated. The method of claim 3, wherein the proliferative Pro-angiogenic effect of proliferation, invasion, angiogen-sensitizing effect. Method according to one of claims 1 to 4, wherein the eukaryotic Cells primary Are cells. The method of claim 5, wherein the primary cells Endothelial cells, fibroblasts, smooth muscle cells or cell mixtures are. Method according to one of claims 1 to 6, wherein the eukaryotic Cells are part of a cell line. Method according to one of claims 1 to 7, wherein the nucleic acid by Transfection, transduction, microinjection, electroporation, a Genegun or scratch loading is introduced into the eukaryotic cell. A method according to any one of claims 1 to 8, wherein the reporter gene by homologous recombinants in the gene coding for GBP-1 or another gene selectively inducible by inflammatory cytokines is introduced. Method according to one of claims 1 to 9, wherein the nucleic acid Expression vector / expression plasmid or a viral nucleic acid. Method according to one of claims 8 to 10, wherein the transfection or transduction a stable or transient transfection or Transduction is. A method according to any one of claims 1 to 11, wherein the reporter gene a luciferase, rhodamine, green fluorescent protein or a GFP variant, β-galactosidase, Encoded SEAP or lactate dehydrogenase. Method according to one of claims 1 to 12, wherein the one or the multiple drug candidates peptides, polypeptides, low molecular weight Substances, antibodies or fragments or derivatives thereof or aptamers. The method of claim 13, wherein the low molecular weight Substances are organic low-molecular substances. The method of any one of claims 1 to 14, further comprising the step (c) Determination of cell proliferation in the presence of the one or more drug candidates and comparison of the of a certain value with a control approach, whereby one remains the same or characteristic of a decrease in cell number compared to the control approach is for an antiperspirant effect and an increase in the number of cells in comparison to the control approach is characteristic of a poliferation-enhancing effect. The method of any one of claims 1 to 15 further comprising the step of isolating the active ingredient or ingredients. Method according to one of claims 1 to 16, wherein the further following steps provided there are several drug candidates in one and the same Test vessel tested become: (a) Testing of multiple drug candidates per test tube, whereby those drug candidates in reaction vessels in which no increase or decrease in expression level was observed is for the further tests are no longer considered; (B) Distribution of the in a test vessel in which a positive result was observed, drug candidates into new test tubes and repetition of the test, whereby again those drug candidates in test vessels as Active substances come into consideration, in which an increase or decrease the level of expression is observed; and if necessary (c) repeating step (b) until a single drug is identified in which a anti-angiogenic and / or anti-inflammatory effect or a pro-angiogenic effect is indicated. A method according to any one of claims 1 to 17, which is a high throughput method is. The method of claim 18, which is computer assisted is. The method of any one of claims 1 to 19 further comprising the improvement in the pharmacological properties of the identified Active ingredient, where one (a) the binding site of the active ingredient to which its receptor is identified; (b) the binding site Active ingredient modified at the receptor by molecular modeling; and (c) Modifies active ingredient in such a way that its binding specificity or binding affinity or binding avidity for the receptor is increased by molecular modeling. The method of any one of claims 1 to 19 further comprising the improvement in the pharmacological properties of the identified Active ingredient, whereby one tests on (a) reinforcing the inducing or inhibiting effect on GBP-1 promoter activity; (B) less effect on other promoters; and or (c) minor Effect on the anti-angiogenic functions of cellular GBP-1. A method according to any one of claims 1 to 21, wherein the identified one or enhanced drug through peptidomimetics pharmacologically is further improved. A method according to any one of claims 1 to 22, wherein the active ingredient is further modified to (i) a modified active center, a modified activity spectrum, a modified organ, tissue or cell specificity; (ii) improved activity; (iii) increased toxicity to tumor cells (an improved therapeutic index) or increased efficacy on target cells; (iv) reduced side effects; (v) a delayed start of therapeutic efficacy, the length of therapeutic efficacy; (vi) changed pharmacokinetic parameters (absorption, distribution, metabolism or excretion); (vii) modified physicochemical parameters (solubility, hygroscopic properties, color, taste, smell, stability, condition); (viii) improved general specificity, organ / tissue specificity; and / or (ix) optimized administration form and route through (i) esterification of carboxyl groups or (ii) esterification of hydroxyl groups with carboxylic acids or (iii) esterification of hydroxyl groups to, for example, phosphates, pyrophosphates or sulfates or "hemisuccinates" or (iv) formation of pharmaceutically acceptable salts or (v) formation of pharmaceutically acceptable salts Complexes or (vi) synthesis of pharmacologically active polymers or (vii) introduction of hydrophilic groups or (viii) introduction / exchange of substituents in aromatics or side chains, change of the substituent pattern or (ix) modification by introduction of isosteric or bioisosteric groups or (x ) Synthesis of homologous compounds or (xi) introduction of branched side chains or (xii) conversion of alkyl substituents to cyclic analogues or (xiii) derivatization of hydroxyl groups to ketals or acetals or (xiv) N-acetylation to amides, phenyl carbamates or (xv) synthesis of Mannich bases, imines or (xvi) To convert ketones or aldehydes to ship bases, oximes, acetates, ketals, enol esters, oxazolidines, thiozolidines or combinations thereof. The method of any one of claims 1 to 23 further comprising the step of manufacturing a drug or medicinal product, wherein the active ingredient obtained with a pharmaceutically acceptable carrier or diluent or excipients. Eurkaryotic cell that has a nucleic acid the gene coding for the promoter sequence of a GBP-1, the promoter sequence another gene selectively inducible by inflammatory cytokines or a fragment or derivative thereof with appropriate promoter activity functional connected containing a reporter gene. The cell of claim 25 which is stable or transient is transfected or transduced. The cell of claim 25 or 26 which is a primary cell is. A cell according to any one of claims 25 to 27 which is part of a Cell line is.