WO2007020075A1

WO2007020075A1 - Use of neuropilin-2 antagonists

Info

Publication number: WO2007020075A1
Application number: PCT/EP2006/008088
Authority: WO
Inventors: Rainer H. Straub
Original assignee: Klinikum Der Universität Regensburg
Priority date: 2005-08-16
Filing date: 2006-08-16
Publication date: 2007-02-22
Also published as: EP1915170A1

Abstract

The present invention is related to the use of a neuropilin-2 antagonist for the manufacture of a medicament for the prevention and/or treatment of a disease associated with or involving repulsion of sympathetic nerve fibres.

Description

Use of neuropilin-2 antagonists

The present invention is related to the use of a neuropilin-2 antagonist for the manufacture of a medicament, and to a method for the screening of an agent for the treatment and/or prevention of a disease.

The treatment of chronic inflammatory diseases such as rheumatoid arthritis is complex. It has been observed that during such disease the function of the axis consisting of the hypothalamus, the pituitary gland and the adrenal cortex (HHN axis) becomes functionally inactive. The improper production of adrenocorticotropic hormone (ACTH) and of glucocorticoids correlates to the degree of inflammation. This reaction is consistent with a permanent simulation of the HHN axis, which is characterised by an increased titer of systemic cytokines. In parallel, the hormone production of the suprarenal gland and of the gonads is inhibited. Consequently, a low Cortisol and androgen concentration can be observed which again correlates to the degree of systemic and local inflammation.

Apart from these biochemical changes observed in chronic inflammatory diseases, also anatomical changes occur. More particularly, there is an increased growth of sensory nerve fibres into the inflamed area going along with arterial neovascularisation and withdrawal of sympathetic nerve fibres. This phenomenon can be understood as an inflammatory/proliferative signal as neurotransmitters of the sensory nervous system exhibit an inflammatory/proliferative effect, whereas the neurotransmitters of the sympathetic nerve system, at high concentrations, are active in an anti-inflammatory/anti-proliferative manner. The prevalence of sensory nerve fibres compared to sympathetic nerve fibres results in an accelerated wound healing as sympathetic nerve fibres usually have a negative impact on wound healing (Perez E. et al., Invest Ophthalmol Vis Sci 1987; 28: 221-4; Wucherpfennig AL et al., Arch Oral Biol 1990; 35: 443-8). The prevalence of sensory nerve fibres compared to sympathetic nerve fibres can not only be observed in areas of wound healing but also in rheumatoid arthritis.

The loss of cooperation between the HHN axis and sympathetic nerve fibres in the inflamed area in arthritis contributes to its chronicity. In view of this, the treatment of arthritis and more particularly of chronic arthritis is still difficult and usually makes use of glucocorticoids, methotrexate, anti-TNF antibodies, or other disease-modifying treatment. The problem underlying the present invention is to provide means, which are suitable in the treatment of inflammatory diseases and more particularly chronic inflammatory diseases.

In a first aspect the problem underlying the present invention is solved by the use of a neuropilin- 2 antagonist for the manufacture of a medicament for the prevention and/or treatment of a disease associated with or involving repulsion of sympathetic nerve fibres.

In an embodiment the repulsion of sympathetic nerve fibres occurs at a site normally innervated by sympathetic nerve fibres.

In a preferred embodiment the site is selected from the group comprising synovial tissue, mucosal tissue, Langerhans islets of the pancreas, tumorous tissue, thyroidal tissue, parotid tissue, preferably salivary gland tissue, renal tissue, synovial tissue, fibrotic skin tissue, muscular tissue, and hepatic tissue.

In an embodiment the disease is selected from the group comprising inflammatory diseases, chronic diseases and chronic inflammatory diseases.

In a further embodiment the disease is selected from the group comprising arthritis, rheumatoid arthritis, colitis, Crohn's disease, insulitis, diabetes mellitus type I, solid tumors, thyroiditis, Sjogren syndrome, skleroderma, spondylarthritis, gout, myositis, liver cirrhosis, preferably chronic arthritis, colitis, Crohn's disease, more preferably chronic arthritis.

In a preferred embodiment the medicament is to be administered during a symptomatic phase of the disease.

In an embodiment the neuropilin-2 antagonist is neuropilin-2.

In an embodiment the antagonist is neuropilin-2 Fc.

In a further embodiment the neuropilin-2 comprises an amino acid sequence according to SEQ. ID. No. 1 or a precursor or isoform(s) thereof. In an embodiment the neuropilin-2 antagonist is a polypeptide inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

In an embodiment the neuropilin-2 antagonist is an antibody inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

In an embodiment the neuropilin-2 antagonist is an anticaline inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

In an embodiment the neuropilin-2 antagonist is an aptamer inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

In a preferred embodiment the aptamer is a spiegelmer.

In an embodiment the ligand is a naturally occurring ligand which is preferably selected from the group comprising Semaphorin 3c, VEGF 165 and placental growth factor.

In an embodiment the neuropilin-2 antagonist is a functional nucleic acid directed against the mRNA of neuropilin-2, whereby preferably surface expression of neuropilin-2 is inhibited.

In an preferred embodiment the functional nucleic acid is selected from the group comprising siRNA, RNAi, siNA, antisense oligonucleotides and ribozymes.

In an embodiment the neuropilin-2 is membrane bound neuropilin-2, preferably neuropilin-2 bound on sympathetic nerve fibres. In a second aspect the problem underlying the present invention is solved by the use of neuropilin-2 as a target in the screening of a compound inhibiting the binding of ligands to neuropilin-2, preferably neuropilin-2 bound on sympathetic nerve fibres.

In an embodiment a library of small molecules, of antibodies, of polypeptides, of anticalines, of aptamers and/or of functional nucleic acids is used in the screening of the library.

In a third aspect the problem underlying the present invention is solved by a method for the screening of an agent for the treatment and/or prevention of a disease and/or for the manufacture of a diagnostic agent for the diagnosis of a disease, whereby the disease is any disease as described in any of the preceding claims, comprising the steps:

a) providing a candidate compound,

b) providing an expression system for neuropilin-2, preferably for neuropilin-2 bound on sympathetic nerve fibres and/or a system detecting the activity of neuropilin-2, preferably for neuropilin-2 bound on sympathetic nerve fibres;

c) contacting of the candidate compound with the expression system for neuropilin- 2, preferably for neuropilin-2 bound on sympathetic nerve fibres and/or the system detecting activity of neuropilin-2, preferably for neuropilin-2 bound on sympathetic nerve fibres;

d) determining if the expression and/or the activity of neuropilin-2, preferably for neuropilin-2 bound on sympathetic nerve fibres is changed under the influence of the candidate compound.

In an embodiment of the third aspect the candidate compound is contained in a library of compounds.

In an embodiment of the third aspect the candidate compound is selected from the group of classes of compounds comprising polypeptides, proteins, antibodies, anticalines, functional nucleic acids, natural compounds and small molecules. In a preferred embodiment of the third aspect the functional nucleic acids are selected from the group which comprises aptameres, ribozymes, antisense oligonucleotides, siRNA, siNA and RNAi.

In a fourth aspect the problem underlying the present invention is solves by the use of an agonist of neuropilin-2 for the manufacture of a means for the diagnosis and/or staging and/or for the assessment of a subject o be at risk to develop any of the diseases defined in any of the preceding claims.

In an embodiment of the fourth aspect the agonist is selected from the group comprising Semaphorin 3 c, VEGF 165 and placental growth factor.

In an embodiment of the fourth aspect the agonist comprises a label, whereby preferably the label is selected from the group comprising radioactive labels, isotope labels, and fluorescent labels.

In a fifth aspect the problem underlying the present invention is solved by the use of an agent binding to an agonist of neuropilin-2 for the manufacture of a means for the diagnosis and/or staging and/or for the assessment of a subject o be at risk to develop any of the diseases defined in any of the first to fourth aspect.

In an embodiment of the fifth aspect the agent binding to an agonist of neuropilin-2 is selected from the group comprising antibodies, polypeptides, anticalines, aptamers and spiegelmers.

In an embodiment of the fifth aspect the antibodies, polypeptides, anticalines, aptamers and spiegelmers are specifically binding to an agonist of neuropilin-2.

In an embodiment of the fifth aspect the agonist of neuropilin-2 is selected from the group comprising Semaphorin 3 c, VEGF 165 and placental growth factor. In an embodiment of the fifth aspect the agent binding to an agonist of neuropilin-2 comprises a label, whereby preferably the label is selected from the group comprising radioactive labels, isotope labels, and fluorescent labels.

The present inventor has surprisingly found that the sympathetic nervous system plays a dual role in chronic inflammatory diseases. In the non-symptomatic or pre-symptomatic phase, the sympathetic nervous system supports local plasma extravasation, vasodilation, and migration and re-distribution of leukocytes to the site of inflammation, thereby promoting such inflammatory diseases. However, in the symptomatic phase the sympathetic neurotransmitters at high concentrations would inhibit the production of TNF, IL-2, IL- 12, and IFN-γ, thereby alleviating such diseases. Accordingly, the sympathetic nervous system and more particularly its neurotransmitters is/are active in an anti-inflammatory/anti-proliferative manner during the chronic phase of an inflammatory disease. This dual behaviour of the sympathetic nervous system also exists in case of and is particularly relevant for chronic inflammatory diseases, which involve repulsion of sympathetic nerve fibres. Repulsion of sympathetic nerve fibres lead to low concentrations of sympathetic neurotransmitter, which is a proinflammatory signal. Because of this relationship, neuropilin-2 which is a receptor on sympathetic nerve fibres for nerve repulsion factors such as semaphorin 3 c, VEGF 165 and placental growth factor (PLGF), can be used as the primary target site for drugs antagonizing the effects of such repulsion factors and thus contributing in the healing of this kind of disease. Accordingly, such drugs will avoid the withdrawal of sympathetic nerve fibres from the inflamed joint and will, by doing so, provide for the presence of sympathetic nerve fibres having an anti-inflammatory activity.

Due to this surprising finding neuropilin-2 antagonists may be used in the treatment of a variety of diseases. More particularly, the following tissues have been reported to show repulsion of sympathetic nerve fibres in connection with some indications which may thus be treated using the neuropilin-2 antagonists in accordance with the present invention: Rheumatoid arthritis in connection with synovial tissue (J Rheumatol. 1990;17:1592-1599, FASEB J. 2000;14:2097- 2107, J Rheumatol. 2002;29:427-35, and Ann Rheum Dis. 2005;64: 13-20), colitis in connection with mucosal tissue (Gut 2005;54: 1098— 1106), and insulitis and diabetes mellitus type I in connection with Langerhans islets of the pancreas (Diabetes. 2002;51:2997-3002). Apart from that any other tissue which might be subject to chronic inflammation and which comprises sympathetic nerve fibres and expresses agonists of neuropilin-2 can be treated in accordance with the present invention using the neuropilin-2 antagonists. Such tissues and respective diseases are, among other, thyroiditis in connection with thyroidal tissue, Sjόrgren syndrome in connection with parotid tissue and more particularly salivary gland tissue, glomerulonephritis in connection with renal tissue, other forms of arthritis, including but not limited to, spondylarthritis or gout, in connection with synovial tissue, myositis in connection with muscular tissue, and liver cirrhosis in connection with hepatic tissue.

Also due the mechanism underlying the present invention, diagnostic methods for the onset of the diseases, in particular chronic diseases, as described herein can the diagnosed. Accordingly, the respective method comprises the step of determining the content of an agonist of neuropilin-2 in a sample, such as those described herein including semaphorin 3c, PLGF and VEGF₁₆₅. The determination of the content or concentration of the neuropilin-2 agonist in said sample can be carried out by using any means which is suitable to detect or determine the neuropilin-2 agonist. Such means comprise, among others, the individual agonist itself, preferably a labelled form thereof, such as a radioactively or fluorescently labelled form thereof, antibodies, polypeptides, anticalines, aptamers, spiegelmers, probes directed against the nucleic acid coding therefore, and the like. Preferably the sample is tissue or tissue extract or bodily fluid such as synovial fluid and others. Direct use of this material in the nerve outgrowth assay - mentioned below - can be taken in order to detect the sympathetic nerve fiber repulsive capacity in vitro for diagnostic purposes.

As used herein a neuropilin-2 antagonist is preferably any compound, which is suitable to avoid or to inhibit the activation or activity of neuropolin-2. Preferably, a neuropilin-2 antagonist avoids or inhibits the activation of membrane-bound neuropilin-2, whereby more preferably such membrane-bound neuropilin-2 is present on a sympathetic nerve ending. Activation of neuropilin-2 is preferably the transfer of a signal represented by a ligand of neuropilin-2, particularly membrane bound neuropilin-2 on a sympathetic nerve fibre, through interaction of the ligand with neuropilin-2. The signal can be, among others, a change in the conformation of neuropilin-2, preferably the membrane-bound neuropilin-2, and/or a change in activity of a factor interacting with neuropilin-2, whereby such factor is preferably different from the ligand and is more preferably any one of other factors present on the surface of the sympathetic nerve ending or a change in concentration of a factor such as a second messenger the concentration of which depends on the activation of neuropilin-2. A ligand to neuropilin-2 as preferably used herein, is any compound which is suitable to interact with neuropilin-2, preferable with membrane bound neuropilin-2. Even more preferably, the ligand is suitable to activate neuropilin-2. Ligands to neuropilin-2 are, among others, semaphorin 3c, VEGF 165 and placental growth factor.

Semaphorin 3c is one of several nerve repulsion factors of the group of semaphorins, which are present in human beings. Semaphorins are ligands to receptors such as neuropilin-1 and neuropilin-2. It is known that the distribution of the various types of neuropilins on the axons of the nerve cells are critical for the effect of the semaphorins. Other nerve repulsion factors include, in human beings, semaphorin 3B, semaphorin 3F, and semphorin 6A, B. Semaphorin 3c is responsible for an increased repulsion of sympathetic nerve fibres. Although abundant in patients suffering from chronic rheumatoid arthritis, semaphorin 3c is not an appropriate target for the treatment of this kind of disease as there are other compounds such as placental growth factor (PLGF) and VEGFi ₆₅ which are binding to neuropilin-2 and are suitable to trigger the neuropilin-2 mediated response consisting in the withdrawal of sympathetic nerve fibres. Insofar, the neuropilin-2 is a promiscuous receptor.

In view of the surprising findings disclosed herein, preferably soluble not membrane - bound neuropilin-2 as such may be used as a medicament for the prevention and/or treatment of the various diseases and diseased conditions as described herein, and for the manufacture of a medicament for such purpose and for the manufacture of a diagnostic agent.

In case neuropilin-2 or a fragment or derivative thereof as defined above is used as a medicament itself, it is preferably used as a competitor to the naturally occurring neuropilin-2 and thus preventing the normal biological function thereof. It is particularly preferred that the neuropilin-2 used for that purpose is not suitable to trigger the activation of neuropilin-2, more precisely the activation of neuropilin-2 bound on the membrane of sympathetic nerve fibres. This kind of soluble neuropilin-2 may either be applied to the organism and cell, respectively, or may be introduced into the organism and respective cells by means of gene therapy. Accordingly, the neuropilin-2 used for such purposes is preferably a soluble neuropilin-2 or, if still membrane- bound, not capable of being activated or at least not being capable to transmit a signal as done by the physiological membrane-bound neuropilin-2 on sympathetic nerve endings. Soluble neuropilin-2 acting as an antagonist to neuropilin-2 and more particularly to membrane- bound neuropilin-2 of sympathetic nerve fibres, as preferably used herein, is a neuropilin which is suitable to antagonize the activation of membrane-bound neuropilin-2, preferably of neuropilin-2 bound on sympathetic nerve fibres. In an embodiment, neuropilin-2 exhibits an amino acid according to SEQ. ID. No. 1. However, it is also within the present invention that neuropilin-2 as used herein also comprises derivatives thereof. A derivative of neuropilin-2 is a neuropilin-2, which is different from the full length neuropilin-2 and different from the neuropilin-2 having or comprising an amino acid according to SEQ. ID. No. 1. More preferably, such derivative is a fragment or a truncated form of the full-length neuropilin-2 and/or of the neuropilin-2 having or comprising an amino acid according to SEQ. ID. No. 1. A fragment as used herein is a derivative, which lacks one or several amino acids compared to the full-length neuropilin-2 or the neuropilin-2 according to SEQ. ID. No. 1. Such lack of one or more amino acids can occur at a single or at several sites of the amino acid sequence of the full-length neuropilin-2 or the neuropilin-2 according to SEQ. ID. No. 1. A truncated form of neuropilin-2 as preferably used herein, is a full length neuropilin-2 or a neuropilin-2 according to SEQ. ID. No. 1 at the N-terminal and/or C-terminal end of which one or several amino acids have been deleted. In any case, a derivative of neuropilin-2 according to the present invention is a neuropilin-2 derivative as defined herein, which is active in antagonizing the effect of ligands to membrane-bound neuropilin-2. A respective assay is described in the example part herein. Derivatives of neuropilin-2 also comprise precursors and isoforms. Precursors are known to the one in the art and typically comprise the mature or full-length protein and a pro or even a pre-pro sequence which is typically cleaved off prior to the mature full length protein exerting its physiological activity. The functions of such pre and pre-pro sequences are diverse and comprise, among others, stabilization during translation, transport into cellular or non-cellular compartments, and the like, hi connection therewith it is to be acknowledged that also pre- and pre-pro forms of a protein may exert a physiological activity. The term isoforms as preferably used herein are different forms of a protein that may be produced from different genes or from the same gene by alternative splicing.

A particularly preferred neuropilin-2 derivative is a soluble neuropilifi-2. Such soluble neuropilin-2 derivative is preferably lacking a cell membrane-spanning domain and/or the intracellular domain of neuropilin-2. A particularly preferred neuropilin-2 is the soluble neuropilin-2 as described in Japanese patent application JP 11038920 filed on February 17, 1999. In an embodiment the neuropilin-2 is a modified neuropilin-2. A modified neuropilin-2 is a neuropilin-2, which comprises a modification. Preferably such modification is a modification, which is selected from the group comprising acetylation, phosphorylation, glycosylation and provison of an Fc moiety. A more preferred modified neuropilin-2 is neuropilin-2 Fc as described in more detail in the example part of the present specification

In a further embodiment the neuropilin-2 antagonist comprises a label. The use of such labelled neuropilin-2 antagonists is advantageous for several reasons. One advantage is that the site of administration can be controlled providing for a very efficient delivery of the antagonist. A further advantage is that such labelled neuropilin-2 antagonist provides a means for staging the disease and its treatment as its presence and absence, respectively, can be monitored. Such labelling is particularly advantageous if used in connection with ligands to neuropilin-2 which are also labelled, preferably the way that the two labels provide for a quenching of the individual signals. Respective labels can be, among others, fluorescent labels, protein labels or radioactive labels. Fluorescent labels are preferably selected from the group comprising Cy3, Cy 5, and Texas Red. Radioactive labels are preferably selected from the group comprising ¹⁸O, ³²P, ³³P and ³⁵S. Further labels can selected from the group comprising labels which can be detected by imaging methods such as ³H and ¹³C.

It will be acknowledged that the interaction between neuropilin-2 and its ligands as specified herein, can be inhibited by a number of further classes of antagonists which differ in their chemical nature and which particularly comprise the following classes.

Insofar, apart from being a potential drug itself, neuropilin-2 may be used as the compound against which chemical compounds which may be used as drugs or drug candidates or as diagnostic agents, are directed. These chemical compounds belong to different classes of compounds such as antibodies, polypeptides, anticalines, aptamers, spiegelmers, ribozymes, antisense oligonucleotides and siRNA as well as small molecules. The compounds are designed, selected, screened generated and/or manufactured by either using neuropilin-2 itself as a physical or chemical entity, or information related to neuropilin-2 or known ligands at neuropilin-2 such as semaphorin 3C, VEGF 165, placental growth factor and possibly others. In the design, selection, screening, generation and/or manufacturing process of said classes of compounds neuropilin-2 will also be referred to as the target, which is used in the process rather than in the final application of the respective compound to a patient in need thereof. In the processes, which provide the various classes of compounds, either the protein neuropilin-2 as such or a nucleic acid coding for neuropilin-2 may be used. The term neuropilin-2 as used preferably in this context comprises any fragment or derivative of neuropilin-2 which allows the design, selection, screening, generation and/or manufacture of said classes of compounds of the respective class(es) of compounds which in turn are/is upon their/its application as a medicament or as a diagnostic agent active as such. The term nucleic acid coding for neuropilin-2 as used herein shall comprise any nucleic acid, which contains a nucleic acid, which codes for neuropilin-2 as defined above, or a part thereof. A part of a nucleic acid coding for neuropilin-2 is regarded as such as long as it is still suitable for the design, selection, screening, generation and/or manufacture of said classes of compounds which in turn are/is upon their/its application as a medicament or as a diagnostic agent active as such, preferably active in the treatment and diagnosis, respectively, of the diseases described herein. The nucleic acid coding for neuropilin-2 may be genomic nucleic acid, hnRNA, mRNA, cDNA or part of each thereof.

One class of compounds suitable thereto for are polypeptides. Polypeptides having this kind of characteristic, i. e. inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres, can be generated using a screening process making use of a polypeptide library as described herein in more detail. The selection criterion is that the selected polypeptide is actually blocking the functional interaction of the ligand to the neuropilin-2 and the neuropilin-2 bound on sympathetic nerve fibres. Preferably, such blocking occurs through blocking of the ligand binding site on neuropilin-2 and/or by binding to a different part of the neuropilin-2 thus causing a change in confirmation so as to inhibit the binding of the ligands to the membrane bound neuropilin-2. Most importantly, the binding of the polypeptide must not allow the activation of the membrane-bound neuropilin-2 of the sympathetic nerve fibres and its respective signaling cascades. A respective assay in which such a polypeptide can be identified consists of the neuropilin-2, preferably bound to or on a sympathetic nerve fibre or a system which mimics this kind of system such as, e. g., the neurite outgrowth assay, as also described herein in the example part. Once one or several polypeptides having been identified binding to the neuropilin-2, those are selected which do not trigger the activation of the neuropilin-2. It will be understood that in an embodiment, the first step, i. e. the selection of a polypeptide binding to the neuropilin-2 may be performed in the presence of any of the naturally occurring ligands to neuropilin-2. It will also be understood that the two aforementioned steps may actually be realized in one single step with both screening/identification steps being combined. Such screening/identification step preferably makes use of a neurite outgrowth assay.

More specifically, such polypeptides may be generated by using methods according to the state of the art such as phage display. Basically, a library of peptide is generated, such as in the form of phages, and this kind of library is contacted with the target molecule, in the present case, for example, the neuropilin-2. Those peptides binding to the target molecule are subsequently removed from the respective reaction, preferably as a complex with the target molecule. It is known to the one skilled in the art that the binding characteristics, at least to a certain extent, depend on the particularly realized experimental set-up such as salt concentration and the like. After separating those polypeptides binding to the target molecule with a higher affinity or a bigger force, from the non-binding members of the library, and optionally also after removal of the target molecule from the complex of target molecule and polypeptide, the respective polypeptide(s) may subsequently be characterised. Prior to the characterisation optionally an amplification step is realized such as, e. g., by propagating the polypeptide coding phages. The characterisation preferably comprises the sequencing of the target binding polypeptides and ultimately of those polypeptides acting as antagonists of neuropilin-2 as defined herein. Basically, the polypeptides are not limited in their lengths, however, preferably polypeptides having a length from about 8 to 20 amino acids are preferably obtained in the respective methods. The size of the libraries may be about 10² to 10¹⁸, preferably 10⁸ to 10¹⁵ different polypeptides, however, is not limited thereto.

A similar approach can be followed if the neuropilin-2 antagonist is an antibody. It will be understood that the antibody may be a monoclonal antibody or a polyclonal antibody, the manufacture and generation, respectively, which is well known to the one skilled in the art.

The manufacture of an antibody specific for the protein of neuropilin-2 or for the nucleic acid coding for neuropilin-2, is known to the one skilled in the art and, for example, described in Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, NY,(1988). Preferably, monoclonal antibodies may be used in connection with the present invention, which may be manufactured according to the protocol of Kohler and Milstein and further developments based thereon. Antibodies as used herein, include, but are not limited to, complete antibodies, antibody fragments or derivatives such as Fab fragments, Fc fragments and single-stranded antibodies, as long as they are suitable and capable of binding to neuropilin-2. Apart from monoclonal antibodies also polyclonal antibodies may be used and/or generated. The generation of polyclonal antibodies is also known to the one skilled in the art and, for example, described in Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1988). Preferably, the antibodies used for therapeutic and/or diagnostic purposes are humanized or human antibodies.

The antibodies, which may be used according to the present invention, may have one or several markers or labels. Such markers or labels may be useful for detecting the antibody either in its diagnostic application or its therapeutic application. Preferably the markers and labels are selected from the group comprising avidin, streptavidin, biotin, gold and fluorescein and used, e. g., in ELISA methods. These and further markers as well as methods are, e. g. described in Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, NY₅(1988). Additionally or alternatively, the antibodies as well as any other neuropilin-2 antagonist described herein may be a labelled antagonist as more generally described herein.

It is also within the present invention that the label or marker exhibits an additional function apart from detection, such as interaction with other molecules. Such interaction may be, e.g., specific interaction with other compounds. These other compounds may either be those inherent to the system where the antibody is used such as the human or animal body or to the sample which is analysed by using the respective antibody. Appropriate markers may, for example, be biotin or fluoresceine with the specific interaction partners thereof such as avidin and streptavidin and the like being present on the respective compound or structure to interact with the thus marked or labelled antibody. Again this applies also to the other neuropilin-2 antagonists described herein.

A similar approach can be pursued using the so-called "anticalines", which are a particular form of target binding polypeptides. Anticalines and their method of manufacture are, among others, described in German patent application DE 197 42 706. Also, the same approach of using libraries of compounds can also be realised when screening for so-called aptamers. In connection therewith, the library consists of nucleotides as will be described in more detail in the following. Aptamers are D-nucleic acids which are either single stranded or double stranded and which specifically interact with a target molecule such as neuropilin-2. The manufacture or selection of aptamers is, e. g., described in European patent EP 0 533 838. Basically the following steps are realized. First, a mixture of nucleic acids, i. e. potential aptamers, is provided whereby each nucleic acid typically comprises a segment of several, preferably at least eight subsequent randomised nucleotides. This mixture is subsequently contacted with the target molecule, whereby the nucleic acid(s) bind to the target molecule, such as based on an increased affinity towards the target or with a bigger force thereto, compared to the candidate mixture. The binding nucleic acid(s) are/is subsequently separated from the remainder of the mixture. Optionally, the thus obtained nucleic acid(s) is amplified using, e. g. polymerase chain reaction. These steps may be repeated several times giving at the end a mixture of nucleic acids or polypeptides having an increased ratio of nucleic acids specifically binding to the target from which the final binding nucleic acid is then optionally selected, whereby preferably the neurite outgrowth assay is used. These specifically binding nucleic acid(s) are referred to aptamers. It is obvious that at any stage of the method for the generation or identification of the aptamers samples of the mixture of individual nucleic acids may be taken to determine the sequence thereof using standard techniques. It is within the present invention that the aptamers may be stabilized such as, e. g., by introducing defined chemical groups which are known to the one skilled in the art of generating aptamers. Such modification may for example reside in the introduction of an amino group at the 2 '-position of the sugar moiety of the nucleotides. Aptamers are currently used as both therapeutic and diagnostic agents. However, it is also within the present invention that the thus selected or generated aptamers may be used for target validation and/or as lead substance for the development of medicaments, preferably of medicaments based on small molecules. This is actually done by a competition assay whereby the specific interaction between the target molecule and the aptamer is inhibited by a candidate drug whereby upon replacement of the aptamer from the complex of target and aptamer it may be assumed that the respective drug candidate allows a specific inhibition of the interaction between target and aptamer, and if the interaction is specific, said candidate drug will, at least in principle, be suitable to block the target and thus decrease its biological availability or activity in a respective system comprising such target. The thus obtained small molecule may then be subject to further derivatisation and modification to optimise its physical, chemical, biological and/or medical characteristics such as toxicity, specificity, biodegradability and bioavailability. A special form of aptamers are Spiegelmers. The generation or manufacture of spiegelmers which may be used or generated according to the present invention using neuropilin-2 or a nucleic acid coding for neuropilin-2, is based on a similar principle. The manufacture of Spiegelmers is described in the international patent application WO 98/08856. Spiegelmers are L-nucleic acids, which means that they are composed of L-nucleotides rather than aptamers which are composed of D-nucleotides as aptamers are. Spiegelmers are characterized by the fact that they have a very high stability in biological system and, comparable to aptamers, specifically interact with the target molecule against which they are directed. In the purpose of generating Spiegelmers, a heterogenous population of D-nucleic acids is created and this population is contacted with the optical antipode of the target molecule, in the present case for example with the D-enantiomer of the naturally occurring L-enantiomer of neuropilin-2. Subsequently, those D-nucleic acids are separated which do not interact with the optical antipode of the target molecule. However, those D-nucleic acids interacting with the optical antipode of the target molecule are separated, optionally determined and/or sequenced and subsequently the corresponding L-nucleic acids are synthesized based on the nucleic acid sequence information obtained from the D-nucleic acids. These L-nucleic acids which are identical in terms of sequence with the aforementioned D-nucleic acids interacting with the optical antipode of the target molecule, will specifically interact with the naturally occurring target molecule rather than with the optical antipode thereof. Similar to the method for the generation of aptamers it is also possible to repeat the various steps several times and thus to enrich those nucleic acids specifically interacting with the optical antipode of the target molecule.

A further group of neuropilin-2 antagonists which can be used for the purpose described herein, are functional nucleic acids. Said functional nucleic acids are preferably directed against the mRNA of neuropilin-2. In principle, using different mode of actions, said functional nucleic acids bind to and degrade the intracellular mRNA of neuropilin-2. It is known that such functional nucleic acids have to be transfected or transferred into the nerve fibre. Due to the transitory nature of this kind of functional nucleic acids the expression of neuropilin-2 is down- regulated for a period of time which can be controlled by, among others, the extent of the transfection of the respective cell and by the stability of the functional nucleic acids. Basically, the knock-down of the neuropilin-2 shall last until the inflammatory process going along with an increased concentration of nerve repulsion factors is returned to a lower level allowing for a balance between the various nerve types usually observed in a non-diseased condition.

It is thus a common feature of all of this functional nucleic acids that they do not interact with the target molecule at the level of the translation product which is in the present case the neuropilin-2, but rather interact with the transcription product, i. e. the nucleic acid coding for neuropilin-2 such as the genomic nucleic acid or any nucleic acid derived therefrom such as the corresponding hnRNA, cDNA and mRNA, respectively. Insofar, the target molecule of the aforementioned classes of compounds is preferably the mRNA of neuropilin-2. Examples of such functional nucleic acids are ribozymes, antisense oligonucleotides, and siRNA.

Ribozymes are catalytically active nucleic acids which preferably consist of RNA which basically comprises two moieties. The first moiety shows a catalytic activity, whereas the second moiety is responsible for the specific interaction with the target nucleic acid, in the present case the nucleic acid coding for neuropilin-2. Upon interaction between the target nucleic acid and the second moiety of the ribozyme, typically by hybridisation and Watson-Crick base pairing of essentially complementary stretches of bases on the two hybridising strands, the catalytically active moiety may become active which means that it catalyses, either intramolecularly or intermolecularly, the target nucleic acid in case the catalytic activity of the ribozyme is a phosphodiesterase activity. Subsequently, there may be a further degradation of the target nucleic acid which in the end results in the degradation of the target nucleic acid and ultimately reduces the amount of the protein derived from the said target nucleic acid which in the present case is neuropilin-2, due to a lack of newly synthesized neuropilin-2 and a turn-over of prior existing neuropilin-2. Ribozymes, their use and design principles are known to the one skilled in the art, and, for example described in Doherty and Doudna (Ribozym structures and mechanism. Annu ref. Biophys. Biomolstruct. 2001 ; 30 :457-75) and Lewin and Hauswirth (Ribozyme Gene Therapy: Applications for molecular medicine. 2001 7: 221-8).

The use of antisense oligonucleotides for the manufacture of a medicament and as a diagnostic agent, respectively, is based on a similar mode of action. Basically, antisense oligonucleotides hybridise based on base complementarity, with a target RNA, preferably with a mRNA, thereby activating RNase H. RNase H is activated by both phosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupled DNA, however, is rapidly degraded by cellular nucleases with the exception of phosphorothioate-coupled DNA. These resistant, non-naturally occurring DNA derivatives do not inhibit RNase H upon hybridisation with RNA. hi other words, antisense polynucleotides are only effective as DNA RNA hybride complexes. Examples for this kind of antisense oligonucleotides are described, among others, in US-patent US 5,849,902 and US 5,989,912. hi other words, based on the nucleic acid sequence of the target molecule which in the present case is the nucleic acid coding for neuropilin-2, either from the target protein from which a respective nucleic acid sequence may in principle be deduced, or by knowing the nucleic acid sequence as such, particularly the mRNA, suitable antisense oligonucleotides may be designed base on the principle of base complementarity.

Particularly preferred are antisense-oligonucleotides which have a short stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNA bases is required for activation of bacterial RNase H and a minimum of 5 bases is required for mammalian RNase H activation. In these chimeric oligonucleotides there is a central region that forms a substrate for RNase H that is flanked by hybridising "arms" comprised of modified nucleotides that do not form substrates for RNase H. The hybridising arms of the chimeric oligonucleotides may be modified such as by 2'-O-methyl or 2'-fluoro. Alternative approaches used methylphosphonate or phosphoramidate linkages in said arms. Further embodiments of the antisense oligonucleotide useful in the practice of the present invention are P-methoxyoligonucleotides, partial P- methoxyoligodeoxyribonucleotides or P-methoxyoligonucleotides.

Of particular relevance and usefulness for the present invention are those antisense oligonucleotides as more particularly described in the above two mentioned US patents. These oligonucleotides contain no naturally occurring 5 '-^3 '-linked nucleotides. Rather the oligonucleotides have two types of nucleotides: 2'-deoxyphosphorothioate, which activate RNase H, and 2 '-modified nucleotides, which do not. The linkages between the 2 '-modified nucleotides can be phosphodiesters, phosphorothioate or P-ethoxyphosphodiester. Activation of RNase H is accomplished by a contiguous RNase H-activating region, which contains between 3 and 5 2'-deoxyphosphorothioate nucleotides to activate bacterial RNase H and between 5 and 10 2'-deoxyphosphorothioate nucleotides to activate eucaryotic and, particularly, mammalian RNase H. Protection from degradation is accomplished by making the 5 ' and 3 ' terminal bases highly nuclease resistant and, optionally, by placing a 3 ' terminal blocking group. Also an antisense oligonucleotide may be used wherein not the 5 ^' terminal nucleoside is attached to an RNase H-activating region but the 3 ' terminal nucleoside as specified above. Also, the 5 ' terminus is selected from the particular group rather than the 3 ^' terminus of said oligonucleotide.

Suitable and useful antisense oligonucleotides are also those comprising a 5 ' terminal RNase H activating region and having between 5 and 10 contiguous deoxyphosphorothioate nucleotides; between 11 to 59 contiguous 5 ^'->3 '-linked 2^'-methoxyribonucleotides; and an exonuclease blocking group present at the 3' end of the oligonucleotide that is drawn from the group consisting of a non-5 '-3 '-phosphodiester-linked nucleotide, from one to three contiguous 5 '-3 '- linked modified nucleotides and a non-nucleotide chemical blocking group.

Two classes of particularly preferred antisense oligonucleotides can be characterized as follows:

The first class of antisense oligonucleotides comprises a total of 23 nucleotides comprising in 5 ' -^ 3' direction a stretch of seven 2'-0-methylribonucleotides, a stretch of nine T- deoxyribonucleotides, a stretch of six 2 '-O-methy .ribonucleotides and a 3 '-terminal T- deoxyribonucleotide. From the first group of seven 2'-O-methylribonucleotides the first four are phosphorothioate linked, whereas the subsequent four 2'-O-methylribonucleotides are phosphodiester linked. Also, there is a phosphodiester linkage between the last, i. e. the most 3 '- terminal end of the 2'-O-methylribonucleotides and the first nucleotide of the stretch consisting of nine 2'-deoxyribonucleotides. All of the 2'-deoxyribonucleotides are phosphorothioate linked. A phosphorothioate linkage is also present between the last, i. e. the most 3 '-terminal T- deoxynucleotide, and the first 2'-O-methylribonucleotide of the subsequent stretch consisting of six 2'-O-methylribonucleotides. From this group of six 2'-O-methylribonucleotides the first four of them, again in 5' -^ Y direction, are phosphodiester linked, whereas the last three of them, corresponding to positions 20 to 22 are phosphorothioate linked. The last, i. e. terminal 3'- terminal 2'-deoxynucleotide is linked to the last, i. e. most 3 '-terminal 2 -0- methylribonucleotide through a phosphorothioate linkage.

The second class of particularly preferred antisense oligonucleotides also comprises a total of 17 to 23 nucleotides with the following basic structure (in 5 ' -> 3 ' direction). At the 5 '-terminal end there is an inverted abasic nucleotide which is a structure suitable to confer resistance against exonuclease activity and, e. g., described in WO 99/54459. This inverted abasic is linked to a stretch of five to seven 2'-O-methylribonucleotides which are phosphodiester linked. Following this stretch of five to seven 2'-O-methylribonucleotides there is a stretch of seven to nine 2'-deoxyribonucleotides all of which are phosphorothioate linked. The linkage between the last, i. e. the most 3 '-terminal 2'-O-methylribonucleotide and the first 2'-deoxynucleotide of the 2'-deoxynucleotide comprising stretch occurs via a phosphodiester linkage. Adjacent to the stretch of seven to nine 2'-deoxynucleotides a stretch consistent of five to seven 2'-O-methylribonucleotides is connected. The last 2 -deoxynucleotide is linked to the first 2'-O-methylribonucleotide of the latter mentioned stretch consisting of five to seven 2 -0- methylribonucleotides occurs via a phosphorothioate linkage. The stretch of five to seven 2'-O- methy lribonucleotides are phosphodiester linked. At the 3 '-terminal end of the second stretch of five to seven 2'-O-methylribonucleotide another inverted abasic is attached.

A further class of compounds which are also functional nucleic acids and which may be generated based on the technical teaching given herein and which may be used as medicaments and/or diagnostic agents are small interfering RNA (siRNA) directed to the nucleic acid, preferably mRNA, coding for neuropilin-2. siRNA is a double stranded RNA having typically a length of about 19 to about 25 nucleotides. The sequence of one of the two RNA strands corresponds to the sequence of the target nucleic acid such as the nucleic acid coding for neuropilin-2, to be degraded, hi other words, knowing the nucleic acid sequence of the target molecule, in the present case neuropilin-2, preferably the mRNA sequence, a double stranded RNA may be designed with one of the two strands being complementary to said, e. g. mRNA of neuropilin-2 and, upon application of said siRNA to a system containing the gene, genomic DNA, hnRNA or mRNA coding for neuropilin-2, the respective target nucleic acid will be degraded and thus the level of the respective protein be reduced. The basic principles of designing, constructing and using said siRNA as medicament and diagnostic agent, respectively, is, among others, described in international patent applications WO 00/44895 and WO 01/75164.

According to the design principles well known in the art, respective siRNA can be generated. Accordingly, the siRNA claimed herein comprises a stretch of preferably any nucleotide length from 19 to 25 consecutive nucleotides which is either at least partially complementary to the sense or to the antisense strand encoding neuropilin-2, and a second ribonucleotide strand which is at least partially complementary to the first one and thus to the antisense strand and sense strand respectively, encoding neuropilin-2. Any design principle known in the art of generation or manufacture of siRNA may be applied to this kind of duplex structure. The siRNA space disclosed herein comprises siRNA molecules the antisense strand of which starts with a nucleotides which corresponds to nucleotide no. 1 of a neuropilin-2 encoding sequence as specified above. Further such siRNA molecules start with a nucleotide which corresponds to nucleotide no 2 of a neuropilin-2 encoding sequence as specified above, and so on. This kind of scanning over the neuropilin-2 encoding sequence is repeated so as to provide all possible siRNA molecules which can be directed against PKN-beta. The length of any of the siRNA molecules thus generated may be any length suitable for siRNA, more particularly any length as specified above. Preferably, the various siRNA molecule of the siRNA molecule space disclosed herein, overlap except the most 5' terminal nucleotide of the antisense strand or sense strand. It is obvious that the thus obtained antisense sequences have to complemented through base pairing so as to form the at least partially double-stranded structure required for a functionally active siRNA. It will be understood that the term siRNA as used herein also comprises siNA and RNAi molecules as known to the ones skilled in the art, including any stabilization pattern such as modifying distinct bases in the sequence based on their chemical nature, or modifying the bases forming the siRNA molecules at distinct positions of the sequence. Also, the term siRNA comprises blunt end siRNA molecules as well as molecules having an overhang at the 3 'end and/or the 5' end of either the sense-strand or the antisense strand or both.

Based on the aforementioned design principles, it is thus possible to generate such siRNA, antisense oligonucleotide and ribozyme, respectively, once the nucleic acid sequence coding for neuropilin-2 is known. This is also true for precursor molecules of nucleic acid such as hnRNA, cDNA and the like, including genomic nucleic acid. Of course, also knowing the respective antisense strand may allow the design of such nucleic acid based compounds given the basic principle of base pair complementarity, preferably based on Watson-Crick base pairing.

Based on the mode of action of the aforementioned classes of compounds, such as antibodies, peptides, anticalines, aptamers, spiegelmers, ribozymes, antisense oligonucleotides as well as siRNA, it is thus also within the present invention to use any of these compounds targeting neuropilin-2 and the nucleic acid coding therefore, respectively, for the manufacture of a medicament or a diagnostic agent for any of the diseases as described herein and any of the diseased conditions described herein. Furthermore, these agens may be used to monitor the progression of said diseases and diseased conditions and the success of any therapy applied, respectively. This applies also to the small molecules the identification of which is also disclosed herein.

As outlined above it is within the present invention that apart from neuropilin-2 or a part or derivative thereof or a nucleic acid sequence therefore, as described herein, also other means or compounds may be used in order to create or to suppress the effects arising from neuropilin-2 or the nucleic acid coding neuropilin-2. Put in more general terms, such means may be determined or selected in a screening method. More generally, in such screening method a first step is to provide one or several so called candidate compounds. Candidate compounds as used herein are compounds the suitability of which is to be tested in a test system for treating or alleviating the various diseases as described herein and diseased conditions as described herein or to be used as a diagnostic means or agent for this kind of diseases and diseased conditions. If a candidate compound shows a respective effect in a test system said candidate compound is a suitable means or agent for the treatment of said diseases and diseased conditions and, in principle, as well a suitable diagnostic agent for said diseases and diseased conditions. In a second step the candidate compound is contacted with a neuropilin-2 expression system or a neuropilin-2 gene product, preferably a respective gene expression product, such as a hnRNA or mRNA, or a neuropilin-2 activity system or a neuropilin-2. The neuropilin-2 activity system is also referred to herein as and/or is preferably also active in the meaning of a system detecting the activity of neuropilin-2.

A further category of molecules which might be suitable as antagonists to neuropilin-2, are small molecules. An aspect of the present invention is thus related to the screening of such molecules using neuropilin-2 as the target and which preferably is a more preferred embodiment of the above described screening approach. Similar to the situation discussed herein for other neuropilin-2 antagonists, the small molecule identified in the screening process according to the present invention has to make sure that the membrane-bound neuropilin-2 of the sympathetic nerve fibre is not activated by its naturally occurring ligands such as, among others, semaphoring 3c, VEGFi₆₅ and placental growth factor. Insofar, it is within the present invention that the neuropilin-2 protein as well as the nucleic acid coding for neuropilin-2 may be used as the target for the manufacture or development of a medicament for the treatment of the diseases described herein and of the diseased conditions described herein, as well as for the manufacture and/or development of means for the diagnosis of said diseases and said conditions, in a screening process, whereby in the screening process small molecules or libraries of small molecules are used. For such purpose the neurite outgrowth assay can be used in preferred embodiments. This screening comprises the step of contacting the target molecule with a single small molecule or a variety of small molecules at the same time or subsequently, preferably those from the library as specified above, and identifying those small molecules or members of the library which bind to the target molecules which, if screened in connection with other small molecules may be separated from the non-binding or non-interacting small molecules. It will be acknowledged that the binding and non-binding may strongly be influenced by the particular experimental set-up. In modifying the stringency of the reaction parameters it is possible to vary the degree of binding and non-binding which allows a fine tuning of this screening process. If in such screening method the neurite outgrowth assay is used, such assay is intended to represent as closely as possible the conditions existing in vivo.

Preferably, after the identification of one or several small molecules which specifically interact with the target molecule, this small molecule may be further characterised. This further characterisation may, for example, reside in the identification of the small molecule and determination of its molecule structure and further physical, chemical, biological and/or medical characteristics. Preferably, the natural compounds have a molecular weight of about 100 to 1000

Da. Also preferably, small molecules are those which comply with the Lepinsky rules of five known to the ones skilled in the art. Alternatively, small molecules may also be defined such that they are synthetic-small-molecules, preferably arising from combinatorial chemistry, in contrast to natural products which preferably are non-synthetic. However, it is to be noted that these definitions are only subsidiary to the general understanding of the respective terms in the art.

The screening preferably makes use of an expression system and/or an activity system, respectively. A neuropilin-2 expression system is basically an expression system which shows or displays the expression of neuropilin-2, whereby the extent or level of expression basically may be changed. Preferably, a neuropilin-2 activity system is essentially an expression system whereby the activity or condition of activity or activation is measured rather than the expression of neuropilin-2. In a preferred embodiment such neuropilin-2 activity system is a neurite outgrowth assay. In any of these systems it is tested whether under the influence of a candidate compound the activity or activation of neuropilin-2 or of the nucleic acid coding neuropilin-2 is different from the situation without the candidate compound. Regardless whether the particular system is either an expression system or an activity system, it is within the scope of the present invention that either an increase or a decrease of the activity/activation and expression, respectively, may occur and be measured. Typically, the expression system and/or activity/activation system is an in vitro reaction, such as a cell extract or a fraction of the cell extract such as a nucleus extract or a membrane preparation comprising a membrane-bound neuropilin-2. A neuropilin-2 expression system as used herein may also be a cell, preferably a cell of a tissue or organ involved in the diseases as described herein and diseased conditions as described herein. It is within the present invention to use sympathetic cell lines for such purpose.

Whether there is an increase or decrease in the activity system or expression system may be determined at each level of the expression, for example by measuring the increase or decrease of the amount of nucleic acid coding for neuropilin-2, more particularly mRNA or the increase or decrease of neuropilin-2 more particularly of its activity and activation, respectively, expressed or shown under the influence of the candidate compound. The techniques required for the measurement, more particularly the quantitative measurement of this kind of changes, such as for the mRNA or the protein or its activity/activation are known to the one skilled in the art. Also known to the one skilled in the art are methods to determine the amount of or content of neuropilin-2, e. g. by the use of appropriate antibodies. Antibodies may be generated as known to the one skilled in the art and described, e. g. by Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, NY₅(1988). Also, methods are known to the one skilled in the art to determine the activation of neuropilin-2 the method of which is, e. g. described in Xiao-Mei Xu et al., J. Neuroscience 2000,20:2638-2648; Beate Rohm et al., Mech. Develop. 2000;93:95-104; Shepherd IT et al., Development 1997,124:1377-1385.

In case of a neuropilin-2 expression system an increase or decrease of the activity or activation of neuropilin-2 may be determined, preferably in a functional assay such as a neurite outgrowth factor. Contacting the candidate compound and the expression system and activity or activation system, respectively, usually is performed by adding an aqueous solution of the candidate compound to a respective reaction system which is generally referred to herein as test system. Besides aqueous solutions also suspensions or solutions of the candidate compound in organic solvents may be used. The aqueous solution is preferably a buffer solution.

Preferably, in each run using the expression system and activity or activation system, respectively, only a single candidate compound is used. However, it is also within the present invention that several of this kind of tests are performed in parallel in a high throughput system.

A further step in the method according to the present invention resides in determining whether under the influence of the candidate compound the expression or activity of the expression system and activity or activation system, respectively, in relation to neuropilin-2 or a nucleic acid coding therefore is changed. Typically this is done by comparing the system's reaction upon addition of the candidate compound relative to the one without addition of the candidate compound. Preferably, the candidate compound is a member of a library of compounds.

Basically any library of compounds is suitable for the purpose of this invention regardless of the class of compounds. Suitable libraries of compounds are, among others, libraries composed of small molecules, of peptides, proteins, antibodies, anticalines and functional nucleic acids. The latter compounds may be generated as known to the one skilled in the art and outlined herein. It is also within the present invention that the system described in more detail above in connection with the screening of neuropilin-2 antagonists based on small molecules, is also applicable to any of the other neuropilin-2 antagonists described herein.

It will be acknowledged by the ones skilled in the art that the antagonists according to the present invention can, in principle, be locally administered or systemically administered using the various routes known to the ones skilled in the art. However, local administration is preferred, whereby local administration is particularly preferred at sites normally innervated by sympathetic nerve fibres which, typically, are withdrawn in the chronically diseased state. The term "at a site normally innervated by sympathetic nerve fibres" as used herein, preferably means that without disease related repulsion of the sympathetic nerve fibres the sympathetic nerve fibres would be present at such site.

The various classes of compounds designed according to the present invention such as antibodies, peptides, anticalines, small molecules, aptamers, spiegelmers, ribozymes, antisense oligonucleotides and siRNA may also be contained in a pharmaceutical composition. Preferably such pharmaceutical composition is used for the treatment of the diseases as described herein or the diseased conditions described herein. The pharmaceutical composition may comprise in an embodiment one or several of the aforementioned classes of compounds and/or one or more members of a single class, and optionally a further pharmaceutical active compound, and a pharmaceutically acceptable carrier. Such carrier may be either liquid or solid, for example a solution, a buffer, an alcoholic solution or the like. Suitable solid carriers are, among others, starch, liposomes and the like. It is known to the one skilled in the art to provide respective formulations for the various compounds according to the aforementioned classes of compounds in order to realize the particular route of administrations such as oral, parenteral, subcutaneous, intravenous, intramuscular, intraarticular and the like.

It is within the present invention that also antagonists against neuropilin-1 can be used, whereby such use is the same in all of its aspects as disclosed herein for neuropilin-2 and neuropilin-2 antagonists, respectively. In accordance therewith any class of antagonist described herein which is used as a neuropilin-2 antagonist may in principle also be used as a neuropilin-1 antagonist, whereby the target of such neuropilin-1 antagonist is neuropilin-1. It is also with in the present invention that neuropilin-1 may be used in the various methods described herein, as a target in order to generate, identify or isolate a respective neuropilin-1 antagonist. Neuropilin-1, its nucleic acid sequence and its amino acid sequence is described in Rossignol M, Gagnon ML, Klagsbrun M. Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms. Genomics 2000; 70: 211- 222.

The various compounds of the different classes of compounds as mentioned above, may also be, either alone or in combination, subject to or contained in a kit. Such kit comprises apart from the respective compound(s) additionally one or several further elements or compounds whereby the elements are selected from the group comprising buffers, negative controls, positive controls and instructions on the use of the various compounds. Preferably, the various compounds are present in either dry or liquid form, preferably as a unit dosage for a single administration each. The kit may particularly be used for the therapy, diagnosis or monitoring of the progress of the disease or applied therapies in relation to the diseases and diseased conditions as described herein.

It is within the present invention that the correlation found by the present inventors between the diseases described herein, and more particularly between the onset thereof, and the absence of symphathetic nerve fibres can be used for the diagnosis and staging, respectively, of said diseases. Without wishing to be bound by any theory, the present inventors currently assume that prior to, i.e. preferably prior to the onset, and during said diseases, respectively, in the afflicted tissues which are preferably those recited in connection with the treatment of said diseases, an area can be observed which is free, or essentially free, of sympathetic nerve fibres.

One way to perform such diagnosis and staging, respectively, is to detect such sympathetic nerve fibre free areas in a subject or patient suffering from any of said diseases, suspected of suffering from any of said diseases or having undergone a treatment for any of such diseases. This can be realized by various methods known to the ones skilled in the art. Such methods comprise, among others, imaging techniques. For such imaging techniques, either the agonist to neuropilin-2 or an agent specifically binding to such agonist to neuropilin-2 can be used. Preferably, the agonist to neuropilin-2 or the agent specifically binding to neuropilin-2 is labelled. The label is one of the kind which allows the detecting of the thus labelled compound in imaging procedures. Such label is preferably any label as described herein in connection with the labelling of the neuropilin-2 and neuropilin-2 antagonists as described herein.

Another way to perform such diagnosis and staging, respectively, of any of said diseases as described herein, is to take a sample from or to use a sample taken from a subject or patient suffering from any of said diseases, suspected of suffering from any of said diseases or having undergone a treatment for any of such diseases, and to subject such sample to an analysis. The sample may be taken either from a tissue involved in any of said diseases, or from a more general body fluid. The tissues from which such sample may be taken is preferably a tissue which is one of those recited in connection with the treatment of any of said diseases described herein. The analysis typically comprises the detection or quantification of any of the neuropilin-2 agonists, whereby quantification preferably comprises a mere comparison of the neuropilin-2 agonist with neuropilin-2 agonist found in non-diseases tissues and non-diseased subjects, respectively.

The neuropilin-2 agonist is preferably a nerve repulsion factor. More preferably the neuropilin-2 agonist is selected from the group comprising semaphorin 3 c, VEGF 165 and placental growth factor. Even more preferably, the neuropilin-2 agonist is a ligand, preferably a naturally occurring ligand to neuropilin-2.

The agent specifically binding to such agonist to neuropilin-2 is preferably selected from the group comprising antibodies, polypeptides, anticalines, aptamers and spiegelmers. It will be acknowledged by the ones skilled in the art that any of the aforementioned agents specifically binding to such agonist to neuropilin-2 can be generated using standard protocols as known in the art. Such generation is described herein for the generation of antagonists of neuropilin-2 and can also be preformed for the generation of agents specifically binding to agonists to neuropilin- 2. It will also be understood that any of said agents specifically binding to such agonist to neuropilin-2 can be labelled. In an embodiment, such labelling is a labelling as described above for the labelling of the neuropilin-2 agonists.

As preferably used herein, the term specifically binding refers to a condition or characteristic of an agent binding to another agent, whereby the agent is binding such that the binding event is suitable to discriminate the binding to said another agent from the binding to a further agent. More preferably such binding is expressed as the Kd value, whereby the Kd value of the binding of the agent binding to another agent to said another agent differs from the Kd value of the binding of the agent binding to another agent to said further agent by a factor of at least 2, preferably by a factor of at least 5 and more preferably by a factor of at least 10.

The present invention will be further illustrated now by reference to the figures and examples from which further features, embodiments and advantages may be taken. More particularly,

Fig. IA shows a micrograph of a sympathetic nerve fibre in a synovial plica (above) and at a magnification of 400 (below); Fig. IB shows a diagram illustrating the nerve fibre density of control and arthritic animals in a collagen induced arthritis mouse model;

Fig. 2 shows a diagram depicting the degree of arthritis as a function of time in a collagen type II system, whereby the sympathetic fibres have been dissected at different points in time;

Fig. 3 shows a diagram depicting the severity of arthritis expressed as score points as a function of time upon administration and non-administration of the fusion protein neuropilin-2 Fc; and

Fig. 4 shows the structure of a neuropilin-2 Fc fusion protein.

Example 1: Loss of sympathetic nerve fibres in collage-induced mouse arthritis

In a rheumatoid arthritis mouse model the loss of sympathetic nerve fibres was studied. More particularly, in the chronic phase of the disease sympathetic innervation was investigated using immunohistochemistry. For immunohistochemistry a specific antibody against tyrosine hydroxylase, a key marker of sympathetic nerve fibres, was used. These experiments clearly demonstrated the loss of sympathetic nerve fibres in a mouse model of arthritis. In addition, the same phenomenon was observed in human synovial tissue of patients with rheumatoid arthritis as demonstrated by the group of the inventor in Weidler C et al., Ann Rheum Dis. 2005;64:13- 20. Loss of sympathetic nerve fibres was also demonstrated by the inventor in experimental colitis as given in Straub et al., Gut 2005;54: 1098-1106.

The results of the rheumatoid arthritis mouse model are depicted in Figs. IA and IB.

As depicted in Figs. IA and IB, there was a significant loss of sympathetic nerve fibres in this experimental model for collagen type II - induced arthritis. Fig. IA, upper part, represents an overview of a section of a synovial plica in blue fluorescence, whereas the lower part shows the same section at a magnification of 400. The sympathetic nerve fibres are indicated in red fluorescence. For immunohistochemistry a specific antibody against tyrosine hydroxylase, a key marker of sympathetic nerve fibres, was used. Ten visual fields of the lower part of Fig. 1 B were analysed and the number of thyroxine hydroxylase-positive nerve fibres was determined. The result is depicted in Fig. IB. From Fig. IB it can be taken that the density of thyroxine hydroxylase-positive sympathetic nerve fibres is significantly reduced in the collagen type II arthritis model. This completely supports the findings in patients with rheumatoid arthritis as demonstrated by the group of the inventor in Weidler C et al., Ann Rheum Dis. 2005;64: 13-20, and Miller LE et al., FASEB J. 2000;14:2097- 2107.

Example 2: Effect of sympathetic nerve fibres at various stages of arthritis

The effect of sympathetic nerve fibres at various stages of arthritis was studied using the collagen type II arthritis mouse model as investigated by the group of the inventor in Harle P et al., Arthritis Rheum. 2005;52: 1305-1313, whereby the effect of the elimination of the sympathetic nervous system was studied. The collagen type II arthritis was induced using 100 μg collagen type II in complete Freund's adjuvant in DBA/1 mice. The same dose of collagen type II in Freund's adjuvant was administered at day 21 (booster immunization). The results are depicted in Fig. 2. The arrows indicate the point in time where the late stage elimination of the sympathetic nerve system occurred. The immune responses indicated at the bottom of Fig. 2 are immune responses predominant in the respective periods of time, which are quite different with respect to the influence of the sympathetic nerve fibres. In the early period sympathetic nerve fibres promote inflammation because neurotransmitters support the directed migration of immune cells to the inflamed compartment. However, more realistic to the situation in a patient, in the chronic phase of the disease, sympathetic neurotransmitters would inhibit the ongoing inflammation which is maintained by neutrophils and macrophages, which are inhibited by sympathetic neurotransmitters at high concentrations. Due to the lack of sympathetic nerve fibres in the chronic phase of the disease, the concentration of the neurotransmitters is too low to elicit an antiinflammatory effect on present neutrophils and macrophages. Exactly here, the molecules and those in accordance with the present invention will lead to a regrowth/re-entering of sympathetic nerve fibres into the tissue promoting high enough concentrations in order to inhibit this late phase of the chronic inflammation. As may be taken from Fig. 2, if the sympathetic nervous system was first eliminated before the induction of arthritis a significant decrease in inflammation was observed (black circles in Fig. 2). Sympathectomy was achieved using the false neurotransmitter 6-dyroxydopamine which destroys peripheral sympathetic nerves. In contrast thereto, elimination of the sympathetic nervous system in the chronic symptomatic phase of the disease resulted in a significant increase of the severity of arthritis (open circles in Fig. 2). From this, it can be concluded that the sympathetic nervous system exhibits different functions depending on the stage of the disease.

Example 3: Generation of a neuropilin-2 Fc

Neuropilin-2 Fc as used herein, is a fusion protein consisting of a neuropilin-2 moiety and an Fc moiety which can be generated as follows: Neuropilin-2 is a membrane protein and may be retrieved as NM 010939 from the EMBL databank. Using polymerase chain reaction the cDNA encoding for the extracellular parts of the mature protein ranging from the end of the signal sequence to the beginning of the transmembrane region, is generated and subsequently sequenced. The respective primers are designed such as to allow for the subsequent steps. The 5' primer contained an N-terminal marker sequence (FLAG; (DYKDDDDK)), which ensures a subsequent recovery of the fusion protein. The PCR products are digested and fused in frame to a human IgGl-Fc fragment in the vector pSignal (R & D). The open reading frame of the neuropilin-2 Fc fragments are removed and cloned into the vector pMT/BiP/V5/His (Invitrogen) in frame with the BiP signal sequence. A stop codon after the Fc part allows for the production of neuropilin-2 Fc (without the V5 and the His epitopes).

Using this expression vectors, Schneider S2 cells are first transiently transfected in order to assess a successful protein production after stimulation using copper sulfate in the supernatant of the cells. The Fc part allows for immune precipitation using protein A agarose. The detection in the Western Blot occurred by means of antisera against neuropilin-2 (R&D Systems, Wiesbaden, Germany).

Upon confirmation of the correct expression and secretion, S2 cells are co-transfected with the vectors and pHygro (resistance against hygromycine) and selected by hygromycine after 48 hours. Surviving cells are expanded in the presence of hygromycine. The cells are transferred into a serum-free S2 expression medium upon having reached a sufficient cell density, and are stimulated with copper sulphate. The optimum production conditions such as time after stimulation, cell density and the like, are established and the fusion protein containing supernatants collected. The supernatants are cleared by filtration through 0,45μm filters and Protein G Agarose is added. Tubes are agitated for 2 h and the loaded Protein G is collected by centrifugation. The Gel is washed with buffered saline on a column and the fusion protein is eluted at pH 3 using a glycine buffer, and neutralised immediately. The material is stored lyophilised after buffer exchange to PBS. The purity of the protein is analysed using quantitative gel electrophoresis. All experiments are performed under sterile conditions so as to exclude any contamination by lipopolysaccharides.

Fig. 4 shows the structure of the thus generated neuropilin-2 Fc fusion protein which was used in the present examples, if not indicated to the contrary. The fusion protein consists of two identical polypeptides each consisting of two CUB domains and two FA58C domains. Both polypeptides are covalently linked to each other through two S-S-bonds provided by two Cys residues in the FCl portion of each of such polypeptide.

Example 5: Testing of neuropilin-2

The fusion protein neuropilin-2 is tested with regard to its blocking activity. More particularly, such blocking activity is determined in a neurite outgrowth assay of sympathetic neurons.

Dorsal root ganglions and sympathetic ganglions are prepared from 30 DBA-I mice embryos between day E12 and El 7. The explants are cultivated on coverglasses (Chamber Slide, Nunc, Wiesbaden) as follows. The explants are transferred onto a gel of collagen type I of the rat tail (Cultrex Rat Collagen I, Trevigen through Biozol, Eching), and overlaid with Neurobasal medium (supplemented with L-glutamin, D-glucose, NaHCθ₃, B27 supplement; from Gibco Life Technologies Invitrogen, Karlsruhe). Mouse β-NGF (typically 1 - 50 ng/ml, R & D Systems, Wiesbaden) is added to the medium and/or to the collagen gel used for the overlay. The neurite growth is observed over a maximum of three days. The neurites grow from any side of the explant into the surroundings under the above specified culture conditions. As VEGFi ₆₅ (recombinant protein from Alexis Deutschland, Grϋnberg) and semaphorin 3C, respectively, bind to neuropilin-2 a nerve fibre repulsion of these sympathetic fibres is to be observed upon administration by means of VEGFi ₆₅ or semaphorin 3 C loaded onto eroding microlipid particles, which allow for a controlled release of VEGFi ₆₅ and semaphorin 3 C, respectively, over six days. More precisely, nerve fibre repulsion can be observed the way that the neurites only extend from the side opposite to where the microlipid particles are releasing the VEGFi ₆₄ [such micro lipid particles are 2 x 2 mm in size]. At the proximal side, i. e. the side facing the microlipid particle, there is no or only little outgrowth. The distally (d) and proximally (p) growing neurites are counted and the quotient determined as Q = 100 x p/d as a measure for repulsion.

Further details on the neurite outgrowth assay can, among others be taken from Xu XM, Fisher DA, Zhou L, White FA, Ng S, Snider WD, Luo Y. The transmembrane protein semaphorin 6A repels embryonic sympathetic axons. J Neurosci. 2000;20:2638-2648; Rohm B, Ottemeyer A, Lohrum M, Puschel AW. Plexin/neuropilin complexes mediate repulsion by the axonal guidance signal semaphorin 3 A. Mech Dev. 2000;93:95-104; and Shepherd IT, Luo Y, Lefcort F, Reichardt LF, Raper JA. A sensory axon repellent secreted from ventral spinal cord explants is neutralized by antibodies raised against collapsin-1. Development. 1997;124:1377-1385.

Example 6: Impact of neuropilin-2 Fc on the progression of chronic arthritis

Again using the collagen type II induced arthritis mouse model in DBA/1 mice, arthritis was induced at day 0 using 100 μg collagen type II in complete Freund's adjuvant. On day 22, 50 μg LPS were administered i. p. for synchronising arthritis. One group (n = 12) received 12 μg of the neuropilin-2 Fc fusion protein on day 28 and 37. Rat IgGl was used as control (n = 12).

The results are depicted in Fig. 3.

Fig. 3 shows the degree of arthritis as a function of time, whereby the severity of arthritis is clearly reduced compared to the control (closed circles) when 20 μg of neuropilin-2 Fc as produced in example 4 are used (open circles). The injection of 20 μg neuropilin-2 Fc fusion protein per mouse was performed on days 28 and 37 of arthritis, i. e. during the chronic symptomatic phase. The delivery route was intraperitoneal. The result of these experiments proves for the very first time that the repulsion of the sympathetic nerve fibres has an impact on the progress of chronic arthritis. It is obvious that the effect of the neuropilin-2 Fc fusion construct decreases after 14 to 20 days (starting from day 46 on). The reason for this is that the neuropilin-2 Fc fusion protein is either eliminated or degraded at least in a form that it is no longer active in an anti-inflammatory manner and thus acting as an antagonist to the membrane-bound neuropilin-2 on sympathetic nerve fibres. This is not a problem for a potential therapy because other biological drugs such as anti-TNF antibodies used in the treatment of rheumatoid arthritis undergo the same degradation process. Necessarily, anti- TNF antibody infusion in patients with rheumatoid arthritis needs to be repeated every 14 to 21 days depending on the antiinflammatory effects. It will be understood that the generation of a humanised counterpart of such neuropilin-2 Fc fusion protein is within the skills of those of the art.

The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

Claims

1. Use of a neuropilin-2 antagonist for the manufacture of a medicament for the prevention and/or treatment of a disease associated with or involving repulsion of sympathetic nerve fibres.

2. Use according to claim 1, whereby the repulsion of sympathetic nerve fibres occurs at a site normally innervated by sympathetic nerve fibres.

3. Use according to claim 2, whereby the site is selected from the group comprising synovial tissue, mucosal tissue, Langerhans islets of the pancreas, tumorous tissue, thyroidal tissue, parotid tissue, preferably salivary gland tissue, renal tissue, synovial tissue, fibrotic skin tissue, muscular tissue, and hepatic tissue.

4. Use according to any of claims 1 to 3, whereby the disease is selected from the group comprising inflammatory diseases, chronic diseases and chronic inflammatory diseases.

5. Use according to any of claims 1 to 4, whereby the disease is selected from the group comprising arthritis, rheumatoid arthritis, colitis, Crohn's disease, insulitis, diabetes mellitus type I, solid tumors, thyroiditis, Sjogren syndrome, skleroderma, spondylarthritis, gout, myositis, liver cirrhosis, preferably chronic arthritis, colitis, Crohn's disease, more preferably chronic arthritis.

6. Use according to claim 5, whereby the medicament is to be administered during a symptomatic phase of the disease.

7. Use according to any of claims 1 to 6, whereby the neuropilin-2 antagonist is neuropilin- 2.

8. Use according to any of claims 1 to 7, whereby the antagonist is neuropilin-2 Fc.

9. Use according to any of claims 7 to 8, whereby neuropilin-2 comprises an amino acid sequence according to SEQ. ID. No. 1 or a precursor or isoform(s) thereof.

10. Use according to any of claims 1 to 6, whereby the neuropilin-2 antagonist is a polypeptide inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

11. Use according to any of claims 1 to 6, whereby the neuropilin-2 antagonist is an antibody inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

12. Use according to any of claims 1 to 6, whereby the neuropilin-2 antagonist is an anticaline inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

13. Use according to any of claims 1 to 6, whereby the neuropilin-2 antagonist is an aptamer inhibiting the interaction between neuropilin-2 bound on sympathetic nerve fibres and a ligand to neuropilin-2 bound on sympathetic nerve fibres.

14. Use according to claim 14, whereby the aptamer is a spiegelmer.

15. Use according to any of claims 10 to 14, whereby the ligand is a naturally occurring ligand which is preferably selected from the group comprising Semaphorin 3 c, VEGF 165 and placental growth factor.

16. Use according to any of claims 1 to 6, whereby the neuropilin-2 antagonist is a functional nucleic acid directed against the mRNA of neuropilin-2, whereby preferably surface expression of neuropilin-2 is inhibited.

17. Use according to claim 16, whereby the functional nucleic acid is selected from the group comprising siRNA, RNAi, siNA, antisense oligonucleotides and ribozymes.

18. Use according to any of claims 16 to 17, whereby the neuropilin-2 is membrane bound neuropilin-2, preferably neuropilin-2 bound on sympathetic nerve fibres.

19. Use of neuropilin-2 as a target in the screening of a compound inhibiting the binding of ligands to neuropilin-2, preferably neuropilin-2 bound on sympathetic nerve fibres.

20. Use according to claim 19, whereby in the screening a library of small molecules, of antibodies, of polypeptides, of anticalines, of aptamers and/or of functional nucleic acids is used.

21. A method for the screening of an agent for the treatment and/or prevention of a disease and/or for the manufacture of a diagnostic agent for the diagnosis of a disease, whereby the disease is any disease as described in any of the preceding claims, comprising the steps:

a) providing a candidate compound,

22. Method according to claim 21, characterised in that the candidate compound is contained in a library of compounds.

23. The method according to claim 21 or 22, characterised in that the candidate compound is selected from the group of classes of compounds comprising polypeptides, proteins, antibodies, anticalines, functional nucleic acids, natural compounds and small molecules.

24. The method according to claim 23, characterised in that the functional nucleic acids are selected from the group which comprises aptameres, ribozymes, antisense oligonucleotides, siRNA, siNA and RNAi.

25. Use of an agonist of neuropilin-2 for the manufacture of a means for the diagnosis and/or staging and/or for the assessment of a subject o be at risk to develop any of the diseases defined in any of the preceding claims.

26. Use according to claim 25, whereby the agonist is selected from the group comprising Semaphorin 3c, VEGF 165 and placental growth factor.

27. Use according to claim 25 and 26, whereby the agonist comprises a label, whereby preferably the label is selected from the group comprising radioactive labels, isotope labels, and fluorescent labels.

28. Use of an agent binding to an agonist of neuropilin-2 for the manufacture of a means for the diagnosis and/or staging and/or for the assessment of a subject o be at risk to develop any of the diseases defined in any of the preceding claims.

29. Use according to claims 28, whereby the agent binding to an agonist of neuropilin-2 is selected from the group comprising antibodies, polypeptides, anticalines, aptamers and spiegelmers.

30. Use according to claim 29, whereby the antibodies, polypeptides, anticalines, aptamers and spiegelmers are specifically binding to an agonist of neuropilin-2.

31. Use according to claim 30, whereby the agonist of neuropilin-2 is selected from the group comprising Semaphorin 3c, VEGF 165 and placental growth factor.

32. Use according to claim 30 and 31 , whereby the agent binding to an agonist of neuropilin- 2 comprises a label, whereby preferably the label is selected from the group comprising radioactive labels, isotope labels, and fluorescent labels.