HK1160925B - Use of cathepsin c - Google Patents

Description

Use of cathepsin C

The present invention relates to the use of cathepsin C. Other aspects of the invention relate to methods for screening drugs, for diagnosing pain susceptibility and for treating pain.

Chronic pain is a major unsolved health problem that impairs the health and well-being of millions of people in western countries. Chronic pain seriously afflicts the health of people experiencing pain, often accompanied or followed by vegetative symptoms, often leading to depression. Chronic pain causes pain to the individual, resulting in a tremendous degree of socio-economic cost. Existing pharmacological pain therapies are not universally satisfactory in both efficacy and safety.

In view of the serious drawbacks associated with the field of pain management, it is highly desirable to select new methods for treating persistent pain and for diagnosis and prognosis related to the potential development of chronic pain. In particular, in view of the tremendous gap between the rapidly evolving understanding of pain neurobiology and the unmet clinical need to provide effective treatment without the drawbacks of existing therapeutic areas, there is a need to focus on the discovery of new targets for novel classes of analgesics.

It is therefore an object of the present invention to provide a novel method for developing and providing a novel class of drugs for modulating pain.

This object is achieved by using cathepsin C or functional fragments or derivatives thereof for the identification of compounds that modulate pain.

The present invention is based on the unexpected findings of the inventors, which demonstrate for the first time that cathepsin C expression is closely correlated with pain susceptibility in a mouse model of neuropathic pain.

Pain, by definition of the international association for pain research, is an unpleasant sensory and emotional experience associated with or described in terms of actual or potential tissue damage. Pain is often the result of activation of the nociceptive nervous system, which is specialized to detect and code for injury or potential tissue damage. Thus, pain is part of a body alarm system that initiates a response that minimizes the actual or potential damage to the body. Pain may be a primary symptom of a disease state, or may be a secondary effect of a disease state that generally has no biological significance.

Pain may be acute or chronic. Acute pain is a physiological signal that represents a potential or actual injury. It occurs with tissue damage, infection, inflammation or other acute causes, altering the individual after physical injury or dysfunction. Acute pain can lead to chronic pain if it is not properly treated.

Chronic pain is a disease state of varying origin, duration, intensity and specific symptoms.

Chronic pain may be of nociceptive origin, inflammatory or neuropathic. Nociceptive pain is determined based on a match with sustained activation of somatic or visceral pain-sensitive nerve fibers. Neuropathic pain is pain that results from any kind of injury to peripheral or central nervous tissue; it is thought to be maintained by abnormal somatosensory processes in the peripheral or central nervous system or both (for a review of pain mechanisms see, e.g., Scholz and Woolf, 2002; Julius and Basbaum 2001; Woolf and Mannion, 1999; Wood, j.d., 2000; Woolf and Salter, 2000).

Chronic neuropathic pain varies from patient to patient. Recent data indicate that an individual's susceptibility to pain plays an important role in the amount of pain an individual suffers, i.e., there is an important genetic predisposition to pain, especially to the development of neuropathic pain. The present invention is based on extensive studies aimed at identifying pain susceptibility genes (i.e. genes that determine the amount of pain experienced in the presence of a given fixed degree of tissue damage) by the present inventors in rodent models of chronic pain. The rodent model and experimental setup used by the inventors was such that the following experimental conditions were present between different individuals: a) the nature and consistency of nerve injury can be accurately controlled; and b) genetic and environmental variability can be minimized.

Cathepsin C (CTSC; alternative names: dipeptidyl (amino) peptidase I, DPPI, (EC3.4.14.1)) is a lysosomal protease.

The cathepsin C locus is located on chromosome 11q14.1-q14.3 (see Rao et al, 1997). The genomic sequence of cathepsin C in the NCBI nucleotide database can be obtained according to the following disclosure: NW _925129 (containing the human cathepsin C sequence of the genomic sequence, SEQ ID NO: 3) or NW _001030863 (containing the mouse cathepsin C genomic DNA sequence).

The coding polynucleotide sequence for cathepsin C is publicly available in the NCBI nucleotide database under several accession numbers, for example: NM _001814 (human cathepsin C transcript 1mRNA), BC113897 (full coding sequence of human cathepsin C (cds), transcript 2, mRNA), BC109386 (human cathepsin C, mRNA, full cds), NM _009982 (mouse cathepsin CmRNA). The skilled person knows how to retrieve the coding sequences for other cathepsins C (cathepsins C from other species; mutants or different isoforms of cathepsins C if present) from the NCBI database. If reference is made below to a cathepsin C coding sequence, this may mean any of the above mRNA or coding sequences; preferably, a sequence complying with reference NM-001814 (human) (SEQ ID NO: 1) or NM-009982 (mouse) is meant.

The protein sequence of cathepsin C can be publicly available in the NCBI protein database according to the following accession numbers, e.g. human (hs): all cds: CAA6067, AAL48191, AAL48192, AAL38195, AAH54028, AAQ08887, AAI00893, AAI00894, AAI00892, AAI00895, AAI09387, AAI10072, AAI 13851; hs isoform CRA _ b: EAW 59364; hs isoform CRA _ a: EAW 59363; hs cathepsin C isoform pre-pro-protein: NP-001805; hs isoform b precursor: AAI13898 or NP _ 680475; mouse (mouse) cathepsin C: AAH 67063; preproprotein: NP-034112; isoform CRA-b (mouse): EDL 06796; isoform CRA-a (mouse): EDL 06795. Furthermore, it can be found in UniProtKB database(s) ((s))www.beta.uniprot.org) The protein sequence was publicly obtained according to the following accession numbers: p53634(HUMAN _ CATC, HUMAN cathepsin C, SEQIDNO: 2) or P97821(CATC _ MOUSE, murine cathepsin C). If reference is made below to the cathepsin C protein or amino acid sequence, this may mean any of the above protein sequences; preferably a sequence corresponding to reference number P53634 (for human sequences) or P97821 (for murine sequences).

NCBI is the national center for Biotechnology information (communications Address: national center for Biotechnology information, national library of medicine, Building38A, Bethesda, MD20894, USA; website address:www.ncbi.nhm.nih.gov). In thatwww.beta.uniprot.orgCan retrieve more sequences (e.g., sequences with SwissProt or EMBL accession numbers).

Paris et al, 1995 disclose the cloning of human cathepsin C; pham et al, 1997, published the cloning of murine cathepsin C. Rao et al, 1997 disclose 5' flanking regions (promoters/enhancers) (see especially page 10263, FIG. 4 and the detailed text in the lower part of this page, which is incorporated herein by reference; e.g.gene sequences starting from-1127 up to + 1). Cathepsin C is expressed at high levels in the lung, kidney and placenta, and to a lesser extent in a variety of other organs including cells of the immune system (Rao et al, 1997).

Cathepsin C is a lysosomal protease capable of removing a dipeptide from the amino terminus of a protein substrate. Cathepsin C is implicated in the activation of granule serine proteases, which are expressed in immune system effector cells derived from bone marrow; cathepsin C is involved in the processing and activation of T-lymphocyte granzymes a (gzma) and b (gzmb) and granzyme C; a further function of cathepsin C is the processing of different lysosomal cathepsins, activation of different serine proteases (chymotrypsin-like serine proteases) by removal of the inhibitory N-terminal dipeptide residue (e.g.the above granzymes, cathepsin C itself, neutrophil elastase, protease-3, mast cell chymotrypsin and tryptase (Toomes et al, 1999; Pham and Ley 1999; Turk et al, 2000; Henningsson et al, 2003; McGuire et al, 1993; Adkik et al, 2002; Wolters et al, 2001; Pham et al, 2004; DeHaar et al, 2004; Sheth et al, 2003, method et al, 2007)). Generally, its functions include: protease activity, peptidase activity (e.g., dipeptidyl peptidase, exopeptidase, or endopeptidase activity); serine proteases (e.g., one or more of elastase, cathepsin G, and granzymes A and B, neuraminidase, and factor XIII) are activated.

Cathepsin C is a 200kD tetrameric protein (Paris et al, 1995), as opposed to other cathepsins (e.g., cathepsins B, H, L and S) being small monomeric enzymes. Cathepsin C protein is processed from its prepro form into a proteolytically active enzyme. The mature monomeric form of cathepsin C consists of a heavy chain, a light chain and a propeptide residue linked to the active enzyme (propeptidermaining) (Wolters et al, 1998; Cigic et al, 2000); four such cathepsin C monomers form a proteolytically active 200kD cathepsin C tetramer.

Pham and Ley, 1999 disclose the generation of cathepsin C knockout mice (Dppi-knockutome) (see materials and methods section, from pages 8627 to 8629, especially the second part of the right column at page 8627 to the first part of the left column at page 8628 for the construction of DPPI targeting vectors and the generation of DPPI-/-mice; see also Heusel et al, 1994; Mansour et al, 1988 and Soriano et al, 1991, see embryonic stem cell production for Pham and Ley, 1999).

The use according to the invention makes it possible to identify novel substances for the prophylaxis and/or treatment of pain, in particular neuropathic pain. The use of the invention includes identifying compounds having the desired property (i.e., reducing pain perception) and identifying compounds having the undesired property (i.e., increasing pain perception). Furthermore, the present invention allows for further characterization of compounds that have been identified for the prevention and/or treatment of any disease or disease state. In this case, the invention can be used, for example, to exclude active compounds which have been identified as having unwanted side effects (i.e. increasing pain perception): candidate compounds for a given disease can be characterized, for example, with respect to their effect on cathepsin C (protein and/or nucleic acid, expression and/or function, etc.).

The compound/test compound/active compound used in the various aspects of the invention may be any biological or chemical substance or natural product extract purified, partially purified, synthesized or prepared by biochemical or molecular biological methods.

The compounds believed to be active in modulating pain in the sensations of the various aspects of the invention may be any substance that has an effect on: one of the cathepsin C functions or the amount of cathepsin C in the cell (protein or nucleic acid), cathepsin C expression, post-translational modifications such as N-glycosylation or processing (e.g. cleavage of an exclusion domain at e.g. position 58 or 61), oligomerization of monomers, protein folding or activation.

To this end, the substance may modulate any function of cathepsin C (e.g. the functions listed above or below). Cathepsin C protein activity may be modulated by substances which, for example, act directly on or interfere with cathepsin C polypeptides/proteins or fragments thereof. The substance may also regulate cathepsin C expression, e.g. at the level of transcription (initiation, extension, processing, etc.), transcriptional stability, translation. Furthermore, the substance may modulate cathepsin C in the following respects: post-translational modification, processing from inactive to active form (cleavage of the pro form into three polypeptides forming active monomers); and/or oligomerisation from monomer to tetrameric form; and protein folding, etc. The substance may exert the above effects directly or indirectly (indirect meaning by interfering (positively or negatively) with the natural signal transduction cascade that has an effect on cathepsin C function/protein activity/expression etc.).

The functions of cathepsin C include those enumerated above, such as: protease activity; the ability to remove dipeptides from the amino terminus of one or more protein substrates; the ability to specifically interact with one or more protein substrates (interactions between proteins), such as those listed above; the ability to cleave and/or activate one or more protein substrates, such as those listed above; the ability to process proteinaceous substrates, such as those listed above.

Cathepsin C function also typically includes the ability of the cathepsin C protein or nucleic acid or fragment thereof to interact with other molecules (including but not limited to proteins, nucleic acids, synthetic molecules), preferably with respect to its ability to interact and the ability to cleave protein substrates.

According to the present application, an enzyme substrate is understood to be any molecule that can be modified by an enzyme. Naturally occurring substrates within the scope of the present invention are molecules which correspond to the form in which they occur in the natural physiological or pathological environment (e.g.granule serine proteases, GZMA, GZMB) and which can also be modified by the corresponding enzymes.

Pain modulation may be either decreased or increased.

According to one aspect of this related group of inventions, a fragment or derivative of cathepsin C can be used. The fragments may be protein, polypeptide or polynucleic acid fragments.

Protein or polypeptide fragments are proteins or polypeptides that carry one or more terminal (n-and/or C-terminal) and/or one, two or more internal amino acid deletions when compared to full-length cathepsin C; fragments include, for example:

1. cathepsin C fragments carrying an n-terminal deletion of the dipeptides Xaa-Yaa, Zaa-in their amino acid chain, with particular exception when Xaa is Arg or Lys, or Yaa or Zaa is Pro;

2. a cathepsin C fragment in which a signal peptide (amino acids 1 to 24 of the amino acid chain) is deleted, or a cathepsin C fragment consisting of amino acids 25 to 463;

3. comprises SEQ ID NO: 2 (dipeptidyl-peptidase 1 excluding domain chain) amino acid chain from position 25-134 or a cathepsin C fragment consisting thereof;

4. a cathepsin C fragment in which the propeptide (amino acids 135-230) is deleted;

5. a cathepsin C fragment comprising or consisting of the dipeptidyl peptidase 1 heavy chain (231-394 amino acids);

6. a cathepsin C fragment comprising or consisting of the dipeptidyl peptidase 1 light chain (amino acids 395-463);

7. a cathepsin C fragment comprising the dipeptidyl peptidase 1 light chain (395-463 amino acids) and the heavy chain (231-394 amino acids);

8. a cathepsin C fragment comprising the dipeptidyl peptidase 1 light chain (395-463 amino acids) and the heavy chain (231-394 amino acids);

the positions of the above fragments are referred to SEQ ID NO: 2; preferred examples of the above fragments relate to seq id no: 2.

A functional fragment of a cathepsin C protein is any fragment of the protein having at least one or more of the functional properties of the full-length protein, in particular the properties listed above.

A polynucleotide fragment is a polynucleotide or oligonucleotide that carries one or more termini (5 '-and/or 3' -termini) and/or one, two or more internal nucleotide deletions when compared to the full-length genomic or coding sequence. A functional fragment of a cathepsin C nucleic acid is any fragment having at least one or more functional properties of a full-length polynucleic acid (mRNA, genomic or coding sequence).

The term cathepsin C derivative includes, in comparison to naturally occurring forms, in particular the cathepsin C derivatives of seq id nos: 1 or seq id no2 any modification type of cathepsin C other than deletion. A functional derivative of cathepsin C is any derivative of the protein which has the functional properties of at least one and preferably two or more unmodified proteins. Derivatives include, for example, modifications of amino acids or nucleotide sequences or any other kind of modification, e.g. chemical or biological modifications leading to, for example, stabilization of the polypeptide or polynucleotide (e.g. phosphorothioate modifications of the nucleic acid backbone or exchange of bonds between amino acids etc.), or modifications that specifically target the polypeptide or polynucleotide to or facilitate its entry into or uptake by certain cells (e.g. cell permeant phosphopeptides, ortho-coupled to (orthosteric) cell permeant peptide carriers, e.g. antennapedia/permeant, TAT and signal-peptide based sequences, or coupled to ligand moieties directed against specific transporters or inputs (importers)).

The invention also includes functional derivatives of cathepsin C fragments.

Another aspect of the invention relates to the use of a non-human transgenic animal heterologously expressing cathepsin C or a functional fragment thereof for identifying or analyzing compounds that modulate pain.

The non-human animal can be any non-human animal. Preferably a rodent, such as a rat or mouse.

A transgenic animal is an animal that carries in its genome exogenous DNA specifically introduced into it. Exogenous DNA can be introduced into the genome of an animal according to standard procedures (see, e.g., the editor Transgenic animal technology biology laboratory handbook.C.A.Pinkert; academic Press Inc., san Diego, Calif., 1994 (ISBN: 0125571658).

The term heterologous expression refers to an expression which is different from the normal gene expression in the host organism (relating to the steady-state level, amount, timing or tissue distribution of the expressed gene, or to the type of gene expressed (i.e. the gene is not normally expressed at all in the host)). Heterologous expression may be constitutive or inducible. Suitable inducible expression systems are well known in the art (e.g., Tetracycline (Tetracycline) inducible systems, etc.). The organism may be a cell or a non-human animal.

In another aspect, the invention relates to the use of a non-human transgenic animal heterologously expressing cathepsin C or a functional fragment thereof as a model system for increased pain sensitivity.

Yet another aspect of the invention relates to the use of a non-human cathepsin C knock-out animal for identifying or analyzing compounds that modulate pain.

A knockout organism (e.g., an animal or cell) refers to an organism in which gene expression or function is partially or fully deleted, including genomic knockouts as well as functional knockouts, inducible knockouts, and constitutive knockouts. The generation of knockout organisms, and cells or animals that can be used to generate knockout organisms, are well known in the art. The generation of cathepsin C-knockout mice is described in Pham and Ley, 1999.

Yet another aspect of the invention relates to the use of a non-human cathepsin C knock-out animal as a model system for reducing pain sensitivity.

The use of cells heterologously expressing cathepsin C or functional fragments thereof for the identification of compounds that modulate pain is a further aspect of the invention.

The cell can be any prokaryotic or eukaryotic cell, for example, a cell that is capable of being transfected with a nucleic acid vector and expressing a reporter gene. These cells include substantially primary cells and cells from cell cultures, such as eukaryotic cell cultures comprising cells derived from multicellular organisms and tissues (e.g., HeLA, CHO, COS, SF9 or 3T3 cells) or unicellular organisms such as cells of yeast (e.g., schizosaccharomyces (s.pombe) or saccharomyces cerevisiae), or prokaryotic cell cultures (preferably Pichia (Pichia) or escherichia coli). Cells and samples derived from tissue can be obtained by well-known techniques, such as blood collection techniques, tissue penetration techniques, or surgical techniques.

According to one embodiment, modified cells are used which have a lower cathepsin C activity compared to their unmodified state. Thus, if the chemical compound to be tested is capable of increasing or reverting to a reduced or completely abolished cathepsin C activity, it may, for example, be tested. Alternatively, in the case of reduced pain sensitivity, the substance may be tested regardless of whether it is capable of performing its function (e.g., pain modulation or even function in the case of another disease state or disease).

The modification may be of any type (stable or transient, preferably stable) which results in a reduction of cathepsin C activity, cathepsin C transcriptional homeostasis (i.e.by activation of cathepsin C transcription or transcript stability) or cathepsin C protein homeostasis (i.e.by activation of cathepsin C translation or its post-translational processing; by modulation of cathepsin C post-translational modification or by activation of its stabilization or by inhibition of its degradation). This can be achieved, for example, by: using dominant negative mutants of cathepsin C, antisense oligonucleotides, RNAi constructs of cathepsin C, generating functional or genomic cathepsin C knockouts (which may, for example, be inducible), or other suitable techniques known in the art. For a review of the above techniques, see, for example: currentprotocolchicobiology (2000) j.g. seidman, chapter 23, appendix 52, john wiley and Sons, inc; GeneTargeting: apracticalapproach (1995), editions: joyner, IRLPress; genetic regulatory of receptorexpression and function, 2000; antisense therapeutics, 1996; scherr et al, 2003.

According to one embodiment, cathepsin C knock-out cells are used. Suitable cell lines for generating knockouts are well known in the art, see, e.g., currentprotocolin molecular biology (2000) j.g. seidman, chapter 23, appendix 52, john wiley and Sons, Inc; or GeneTargetingapracticallappacach (1995) eds A.L. Joyner, IRLPress. Pham and Ley, 1999 (generation of a DPPI murine embryonic stem cell knockout clone, see page 8628, left column, top) also discloses generation of cathepsin C (DPPI) knockout cells.

Thus, another aspect of the invention relates to the use of a cathepsin knock-out cell for identifying or analyzing compounds that modulate pain.

Furthermore, the use of cathepsin knock-out cells as model systems for reducing pain sensitivity is another aspect of this related group of inventions.

According to another embodiment of the invention, the cell may have a higher amount of cathepsin C compared to a reference cell (e.g. the same cell in its unmodified state). This cell system can be used to mimic the state of increased pain sensitivity, since the amount of cathepsin C expression is linked to pain sensitivity.

The invention therefore also relates to the use of cells heterologously expressing cathepsin C or functional fragments thereof as model systems for increasing pain sensitivity.

Use of a cell heterologously expressing a reporter gene linked to the expression of a cathepsin C promoter and/or enhancer or a functional fragment thereof for the identification or analysis of compounds that modulate pain.

The above aspects of the invention are based on typical reporter assays generally known in the art. To this end, the selected promoter is inserted into an expression vector appropriate for the selected host cell type, upstream of the selected reporter gene in a manner that allows expression of the reporter gene when the promoter is active. The construct is then introduced into the selected host cell. Suitable transformation or transfection methods and conditions for cell culture and detection of reporter gene expression are well known in the art (see, e.g., the standard literature listed below). Suitable conditions and vectors, reporter genes and necessary reagents (which are also commercially available) are well known in the art.

The vector is a circular or linear polynucleotide molecule, such as a DNA plasmid, phage or cosmid, by means of which a polynucleotide fragment (e.g., a polynucleotide fragment excised from another vector or amplified by PCR and inserted into a cloning vector) can be specifically amplified in a suitable cell or organism. Expression vectors enable heterologous expression of a gene of interest (e.g., a reporter gene) in a host cell or organism. The cell or organism type is largely dependent on the purpose and the choice thereof is within the knowledge of the skilled person. Suitable organisms for amplifying nucleic acids are, for example, predominantly unicellular organisms with a high reproduction rate, such as bacteria or yeasts. Suitable organisms may also be cells isolated and cultured from multicellular tissue, such as cell lines produced from a variety of organisms (e.g., SF9 cells from Spodoptera frugiperda, etc.). Suitable cloning vectors are known in the art and are available from various biotech suppliers, such as Roche diagnostics, NewEngland Biolabs, Promega, Stratagene and many more. Suitable cell lines are purchased, for example, from the American Type Culture Collection (ATCC).

For heterologous expression of a protein or polypeptide, the cell can be any prokaryotic or eukaryotic cell capable of being transfected with a nucleic acid vector and expressing a gene of interest (e.g., a reporter gene). These cells include substantially primary cells and cells from cell cultures, preferably eukaryotic cell cultures comprising cells derived from multicellular organisms and tissues (e.g., HEK293, RIN-5F, HeLA, CHO, COS, SF9, or 3T3 cells) or unicellular organisms such as cells of yeast (e.g., Schizosaccharomyces or Saccharomyces cerevisiae), or prokaryotic cell cultures (preferably Pichia or Escherichia coli). Cells and samples derived from tissue can be obtained by well-known techniques, such as blood collection techniques, tissue penetration techniques, or surgical techniques.

In the context of this application, the term "transfection" refers to the introduction of a nucleic acid vector into a host cell (prokaryotic or eukaryotic) and thus encompasses the term "transformation".

The transfection may be a stable transfection or a transient transfection.

The cathepsin C promoter is a portion of the cathepsin C gene that is capable of driving expression of a gene product of interest if introduced into a suitable expression vector upstream of the sequence encoding the gene product. Preferably, the cathepsin C promoter comprises or consists of the nucleotide sequence-1127 to +1 as disclosed in FIG. 4 on page 10263 of Rao et al (1997). A functional fragment of a cathepsin C promoter is any fragment of the cathepsin C promoter which, if introduced into a suitable expression vector upstream of the sequence encoding the gene product, is capable of driving expression of the gene product of interest. Preferred fragments comprise a functional fragment of the cathepsin C promoter as disclosed in FIG. 4 on page 10263 of Rao et al (1997).

The reporter gene can be any gene whose gene product can be readily quantified. A large number of reporter genes for eukaryotic or prokaryotic hosts, as well as detection methods and necessary reagents, are known in the art and are commercially available. These reporter genes include, for example, genes for beta lactamase (lacZ), luciferase, green or blue fluorescent protein (GFP or BFP), DsRed, HIS3, URA3, TRP1 or LEU2 or beta-galactosidase, etc. These genes encode proteins (e.g., lacZ, luciferase) that can be easily detected via a visible (color or luminescence) response. These proteins include gene products that can be readily detected by means of a visible (color or luminescence) reaction or gene product that when expressed confers resistance to an antibiotic, such as Ampicillin (ampicilin) or Kanamycin (Kanamycin). Other reporter gene products allow the expressed cells to grow under certain conditions, such as auxotrophic genes.

A functional fragment of a reporter gene is any fragment of a given reporter gene that allows for easy quantitative determination of its gene product.

A functional fragment of a reporter gene is any fragment of a given reporter gene that allows for easy quantitative determination of its gene product.

In the context of this application, the term "transfection" refers to the introduction of a nucleic acid vector into a host cell (prokaryotic or eukaryotic) and thus encompasses the term "transformation".

The transfection may be a stable or transient transfection.

The cell can be any prokaryotic or eukaryotic cell capable of being transfected with a nucleic acid vector and expressing a reporter gene. These cells include substantially primary cells and cells from cell cultures, preferably eukaryotic cell cultures including cells derived from multicellular organisms and tissues (e.g., HeLA, CHO, COS, SF9 or 3T3 cells) or cells of unicellular organisms such as yeast (e.g., schizosaccharomyces or saccharomyces cerevisiae), or prokaryotic cell cultures (preferably pichia or escherichia coli). Cells and samples derived from tissue can be obtained by well-known techniques, such as blood collection techniques, tissue penetration techniques, or surgical techniques.

In the context of the above aspect of the invention, the control vector may be any suitable vector comprising the reporter gene or a functional fragment thereof, but wherein expression of the reporter gene is not driven by the (functional) cathepsin C promoter. This may for example mean that the reporter gene or functional fragment thereof is not operably linked to a functional cathepsin C promoter (i.e. a cathepsin C promoter is completely absent, including a non-functional cathepsin C promoter or promoter fragment or where the linkage of promoter and reporter gene is not functional). Another possibility is that the reporter gene or a functional fragment thereof is operably linked to another promoter (e.g.SV 40 or another standard promoter) than the cathepsin C promoter. Functional and control vectors can also be transfected into the same cell, but in this case a difference in reporter gene is required.

Identification of compounds that meet the above uses can be performed, for example, according to the following or assays known in the art.

The assay is any analytical method or type of system for monitoring a biological process. Suitably, the molecular cascades and mechanisms representing parts of physiological metabolic pathways as well as parts of pathological conditions are reproduced in cellular or biochemical (in vitro) systems. Thus, the pharmacological activity of a potential pharmaceutical compound can be determined by its ability to interfere with or modulate these cascades or mechanisms.

For use in drug screening, especially for high throughput screening of novel drug compounds, the assay must be reproducible and preferably also scalable and stable. In the context of the present invention, high throughput screening means that the method of the invention is carried out on very small scale, for example on 96, 386 or 1536 well plates, with samples containing very small volumes of a few milliliters or even nanoliters or even less. Therefore, a very large amount of samples can be analyzed in a short time. High throughput screening consists primarily of screening approximately 500.000 different compounds for a certain ability with a single assay. The assay is preferably suitable for high throughput screening of chemical substances for the ability to modulate the activity of a target molecule under study. The type of assay depends, for example, on the type of target molecule used (e.g., polypeptide or polynucleotide) and the "read-out", i.e., parameter, on which the activity of the target molecule is to be determined (see below).

Different types of such assays are generally known in the art and are commercially available from commercial suppliers.

Suitable assays for different purposes include radioisotopes or fluorescent assays, such as fluorescence polarization assays (e.g., the assay commercially provided by Panvera), or PackardBiocience (HTRF; ALPHAScreen) for measuring the interaction of a labeled member with a non-labeled member (e.g., the interaction of a labeled protein with its unlabeled protein-ligand)^TM)。

Further examples include cell-based assays in which the cell line stably (inducible or non-inducible; chromosomal or episomal) or transiently expresses the recombinant protein of interest. These assays include, for example, reporter gene assays, in which regulation of certain promoters or signal transduction pathways of members of a signal transduction cascade is measured according to the activity of a reporter enzyme whose expression is under the control of the certain promoters. For this type of assay, the recombinant cell line needs to be constructed to contain a reporter gene under the control of a defined promoter, which is itself being studied or under the control of the signal transduction cascade under study. Suitable reporter enzymes are generally known in the art and include firefly luciferase, renilla (renilla) luciferase (e.g., commercially available from packardreagens), beta-galactosidase. Suitable cell lines depend on the purpose of the assay, but generally include cell lines that are easy to transfect and easy to culture, such as HeLA, COS, CHO, NIH-3T3, and the like.

For determining protease activity, typical protease assay formats are known: for example, with a substrate that carries a reporter tag (e.g., a protein/peptide or entity that emits a luminescent/fluorescent or other signal) at one position of the substrate and a quencher (an entity (e.g., another peptide that inhibits the signaling of the reporter tag, so long as the substrate is intact/uncleaved)) at another position; the substrate is incubated with cathepsin C under suitable conditions such that the substrate is cleaved, resulting in the emission of a detectable signal (e.g., light emission) due to the separation of the quencher and reporter tag.

Other assay types and other "read" types are well known in the art.

The assay of the invention involves:

a method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising the steps of:

a. providing at least two samples;

b. contacting a sample containing cathepsin C or a functional fragment or derivative thereof with a compound;

c. assaying cathepsin C activity in the presence of the compound;

d. assaying cathepsin C activity in the absence of the compound; and

e. comparing the cathepsin C activity of C) with the cathepsin C activity of d).

A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. contacting a cathepsin C protein or functional fragment or derivative thereof with a test compound; and

b. determining whether said test compound modulates the activity of cathepsin C protein or functional fragments or derivatives thereof.

A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. contacting a cell having a detectable amount or activity of cathepsin C or a functional fragment or derivative thereof with a test compound;

b. determining whether the test compound is capable of modulating the amount or activity of cathepsin C or functional fragments or derivatives thereof present in the cell.

A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. contacting a nucleic acid encoding a cathepsin C protein or functional fragment or derivative thereof with a test compound in a transcriptionally active system; and

b. determining the amount of mRNA encoding cathepsin C protein or functional fragments or derivatives thereof present in the system in the presence of the compound; and

c. determining whether the compound is capable of modulating the amount of mRNA encoding cathepsin C protein or functional fragment or derivative present in the system.

A transcriptionally active system is any biochemical or cellular system having at least the ability to carry out a transcription reaction of a transcriptional unit. Such systems are well known in the art and include commercially available cells as well as in vitro transcription systems or kits (e.g., based on cell extracts).

A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. providing a cell transfected with a nucleic acid vector comprising a promoter of a cathepsin C gene or functional fragment thereof operably linked to a reporter gene or functional fragment thereof;

b. providing a cell transfected with a control vector comprising a reporter gene or functional fragment thereof not operably linked to a functional cathepsin C promoter;

c. determining the reporter gene activity of the cells of a) and b) in the presence of the test compound;

d. determining the reporter gene activity of the cells of a) and b) in the absence of the test compound.

A method for identifying or analyzing a compound that modulates pain, the method comprising:

a. selecting a compound that modulates cathepsin C activity as a test compound; and

b. administering the test compound to a subject experiencing pain to determine whether the pain is modulated.

A method of identifying or analyzing a compound that modulates and/or prevents pain in a subject, the method comprising:

c. assaying the biological activity of cathepsin C or a functional fragment or derivative thereof in the presence of one or more test compounds to identify one or more regulatory compounds which modulate the biological activity of cathepsin C; and

d. determining the ability of one or more modulatory compounds to reduce pain, pain sensation, or pain sensitivity in a subject.

Further aspects of the invention relate to pharmacogenomic methods for classifying patient populations and assisting physicians in adjusting/improving their treatment of individual patients, for example:

a method for analyzing the pain threshold of an individual, said method comprising analyzing the amount of cathepsin C in a sample taken from said individual in comparison to one or more reference samples to determine whether the amount of cathepsin CmRNA and/or protein present in said sample differs from the amount of cathepsin CmRNA and/or protein of one or more reference samples, wherein the presence of a higher amount indicates an increased pain sensitivity in said individual and the presence of a lower amount indicates a decreased pain sensitivity in said individual.

A method for adjusting the dosage of a medicament for the prevention and/or treatment of pain in an individual, the method comprising examining a taken individual sample to determine whether the amount of cathepsin CmRNA and/or protein present in the sample differs from the amount of cathepsin CmRNA and/or protein in one or more reference samples, the dosage being adjusted according to whether the amount of protein and/or mRNA in the taken individual sample differs from the amount of protein and/or mRNA in one or more reference samples, wherein a higher amount of cathepsin C in the taken individual sample indicates that a higher dosage is required and a lower amount of cathepsin C in the individual sample indicates that a lower dosage is required.

The term "sampled product" as used herein refers to a biological sample collected/isolated from one or more individual organisms (human or non-human animals). Biological materials and biological samples include, for example, preparations or parts of cells, tissues or organs (e.g., brain, blood, liver, spleen, kidney, heart, blood vessels, etc.), preferably from vertebrates, more preferably from mammals, including humans. Also included are cells from cell cultures, preferably eukaryotic cell cultures comprising cells derived from multicellular organisms and tissues (e.g., HeLA, CHO, COS, SF9, or 3T3 cells) or unicellular organisms such as yeast (e.g., schizosaccharomyces or saccharomyces cerevisiae) or prokaryotic cell cultures (preferably pichia or escherichia coli). Cells and samples derived from tissue can be obtained by well-known techniques, such as blood collection techniques, tissue penetration techniques, or surgical techniques. The preparation of recombinant molecules and the purification of naturally occurring molecules from cells or tissues and the preparation of extracts of cells or tissues are well known to those skilled in the art (see also e.g. the standard literature listed below).

The term "reference sample" refers to a biological sample taken from one or more individuals having a known, established pain phenotype, or to an in vitro biological sample (e.g., a sample derived from in vitro cells or tissue cultures (e.g., cultured cells)) that corresponds in certain properties (e.g., cathepsin C activity, amount, or expression level) to an established pain phenotype (e.g., high pain sensitivity or low pain sensitivity).

A further aspect of the invention relates to the use of means for detecting cathepsin C (means) for determining the increased pain sensitivity of an individual by analyzing a biological sample taken from the body of the individual to be examined.

The means for detecting cathepsin C can be any means capable of specifically detecting cathepsin C polypeptides/proteins or nucleic acids present in a biological sample.

The means for specifically detecting a cathepsin C protein or polypeptide may also be any means capable of specifically detecting a wild-type cathepsin C protein/polypeptide and may also be means for specifically detecting cathepsin C proteins/polypeptides having one or more mutations with respect to size or amino acid sequence compared to the wild-type polypeptide/protein. Preferred examples of such means are antibodies capable of specifically detecting cathepsin C protein, e.g. for use in immunohistological or immunohistochemical techniques (e.g. detection of cathepsin C protein or certain mutations thereof in histological tissue sections or cathepsin C protein immobilized on a suitable carrier, e.g. membrane, chip, ELISA plate etc.). Cathepsin C antibodies are commercially available, for example goat anti-human cathepsin C, cat No. AF1071, R & DSystems (Minneapolis, USA), goat anti-mouse cathepsin C, cat No. BAF1034, R & DSystems (Minneapolis, USA).

The means for specifically detecting cathepsin C nucleic acids may for example be means for detecting cathepsin CmRNA/cDNA or genomic DNA (wild type or also having one or more mutations with respect to its length or its nucleic acid sequence compared to wild type cathepsin C nucleic acids). The means may for example be means for the specific detection and/or quantification of cathepsin CmRNA and preferably comprise or be a specific cathepsin C nucleic acid probe or primer set which is capable of amplifying cathepsin CDNA, or for example for PCR sequencing (for detection of mutations in the nucleotide sequence), or capable of amplifying cathepsin CcDNA, for example for RTPCR (for detection and/or quantification of expression of cathepsin CmRNA). Another tool may for example be a nucleic acid probe capable of specifically hybridizing to cathepsin CmRNA or cDNA under standard conditions, e.g. for RNA hybridization or chip hybridization techniques.

The term wild-type refers to a genotype or phenotype found in nature or in a standard laboratory variety of a given organism. According to a preferred embodiment, the wild type sequence of cathepsin C is seq id no: 1. 2 and 3.

The design and synthesis of suitable primers is well known in the art (see also above). According to a preferred embodiment of the invention, the means is a primer set for amplifying cathepsin C nucleic acids (e.g. human cathepsin C nucleic acids), preferably the primer set comprises at least one of the primers of seq id No.4 and/or 5.

According to another preferred embodiment of the invention, said means are probes for detecting cathepsin C nucleic acids, preferably probes having the sequence of seq id No. 6. The design and synthesis of suitable probes is well known in the art (see also standard literature below).

According to a further preferred embodiment of the invention, the means is an antibody for the specific detection of a cathepsin C protein or polypeptide. The preparation of suitable antibodies or functional fragments thereof is well known in the art, e.g.by immunizing a mammal such as a rabbit with cathepsin C protein or fragments thereof in the presence of e.g.Freund's adjuvant and/or aluminium hydroxide gel, where appropriate (see e.g.Diamond, B.A. et al (1981) the New England journal of medicine: 1344-1349). The polyclonal antibodies formed in the animal as a result of the immune reaction can then be isolated from the blood by well-known methods, e.g., purification by means of column chromatography. May e.g. be in accordance with Winter&Milstein(Winter，G.&Milstein, C. (1991) Nature, 349, 293-299) was performed. Suitable procedures for generating monoclonal antibodiesAre also well known in the art (see, e.g., the literature for the standard methods listed below). In the context of the present invention, the term antibody or antibody fragment also includes antibodies or antigen-binding portions thereof, which have been prepared recombinantly and, where appropriate, may be modified, e.g., chimeric, humanized, multifunctional, bispecific or multispecific antibodies (oligoprocedentification), single-chain antibodies and F (ab) or F (ab)₂Fragments (see, for example, EP-B1-0368684, U.S. Pat. No.4,816,567, U.S. Pat. No.4,816,397, WO88/01649, WO93/06213 or WO 98/24884).

Another aspect of the invention relates to a diagnostic kit for determining pain sensitivity in an individual, the test kit comprising at least one means for detecting cathepsin C in a biological sample.

In the context of the present invention, a test kit is to be understood as any combination of components identified in the present application, which co-exist in a functional unit in spatial association and may contain further components.

In the present case, the test kit comprises at least means for detecting cathepsin C (e.g. amount/or mutation) in a biological sample, suitably together with suitable buffers and/or reagents for performing the detection reaction (e.g. immunological detection of cathepsin C by means of antibodies, enzymatic reactions, etc. for determining cathepsin C activity), and/or sample preparations and optionally an operator's manual for performing the respective detection technique.

Other aspects of the invention relate to methods of treatment, for example:

a method for treating pain in a subject experiencing pain, said method comprising administering to said subject a therapeutically effective amount of a composition that reduces the amount or activity of cathepsin C in said subject. This may be the total amount or activity of cathepsin C, or an amount or activity in certain tissues, such as neural tissue, in lymphoid tissue or cells of the immune system (e.g., mast cells, macrophages, neutrophils, T-cells (e.g., CD8+ T-cells), etc.), wherein a therapeutically effective amount includes an amount sufficient to improve the pain perception or sensitivity of an individual.

Another aspect of the invention relates to a method for reducing pain sensitivity in a subject, comprising administering to the subject a therapeutically effective amount of a composition that reduces the amount (e.g., expression, half-life) or activity of cathepsin C in the subject (e.g., in lymphoid or neurological tissue or immune system cells).

Furthermore, the present invention relates to a method for modulating pain sensitivity in the offspring of a non-human female subject, said method comprising transferring (e.g. electroporation) a nucleic acid conferring modulated cathepsin C expression into a fertilized egg, transferring said fertilized egg into a non-human surrogate mother, and selecting offspring which meet their cathepsin C expression profile (reduced or abolished cathepsin C expression compared to a wild-type subject, e.g. a mouse).

Another aspect of the invention relates to compounds capable of reducing cathepsin C activity and/or expression for the treatment of pain.

Cathepsin C inhibitors are known in the art, e.g. peptide nitrile (peptenitile) inhibitors (see e.g. method et al, 2007, which is hereby incorporated by reference, especially see page 20839 for the different cathepsin C-inhibitor structures of figure 1, e.g. Gly-Phe-DMK, Gly4- (I) Phe-DMK and Compound 1 of said figure^*And compound 2^*)。

For the production of a medicament, the cathepsin C modulators of the invention may be formulated with suitable additives or auxiliary substances, such as physiological buffer solutions, e.g. sodium chloride solution, demineralized water; stabilizers, for example protease or nuclease inhibitors, preferably aprotinin, -aminocaproic acid or pepstatin a; or sequestering agents, such as EDTA; gel formulations, such as white petrolatum, low viscosity paraffin and/or yellow wax, and the like, depend on the type of administration.

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

The physiological buffer preferably has a pH of about 6.0-8.0 (especially a pH of about 6.8-7.8, especially a pH of about 7.4) and/or an osmolality of about 200-400 milliosmoles/l, preferably about 290-310 milliosmoles/l. The pH of the medicament is generally adjusted with suitable organic or inorganic buffers, for example preferably phosphate buffer, tris buffer (tris (hydroxymethyl) aminomethane), HEPES buffer ([4- (2-hydroxyethyl) piperazino ] ethanesulfonic acid) or MOPS buffer (3-morpholino-1-propanesulfonic acid). The choice of each buffer is generally dependent on the desired molar concentration of the buffer. Phosphate buffers are suitable, for example, for injection solutions and infusion solutions.

The medicament may be administered in conventional manner, for example by means of an oral dosage form (e.g. tablets or capsules); by mucosa (e.g., nasal or oral); in the form of subcutaneous implant placement; by means of injections, infusions or gels, which contain the medicament according to the invention. For the treatment of the above-mentioned specific arthropathies, the drug may be administered topically or locally, if appropriate in the form of a liposome complex. Furthermore, the treatment can be carried out by means of a Transdermal Therapeutic System (TTS), which allows the drug release to be controlled over time. TTS are known, for example, from EP0944398a1, EP0916336a1, EP0889723a1 or EP0852493a 1.

Injectable solutions are typically used if only relatively small amounts (e.g., about 1 to about 20ml) of the solution or suspension are administered to the body. Infusion solutions are typically used if large amounts of solution or suspension (e.g., 1 or more liters) are administered. Since only a few milliliters are administered in the case of injection solutions, in contrast to infusion solutions, small differences in the pH and osmotic pressure of the blood or tissue fluid at the time of injection cannot be noticed per se, or can be noticed per se to an insignificant extent only in terms of pain perception. Therefore, it is generally not necessary to dilute the formulations of the invention prior to use. However, in the case of administration of relatively large amounts, the formulations of the invention should be simply diluted to such an extent that an at least approximately isotonic solution is obtained immediately before administration. An example of an isotonic solution is a 0.9% strength sodium chloride solution. In the case of infusion, dilution can be carried out, for example, with sterile water, while administration can be carried out, for example, via the so-called bypass (bypass).

According to a preferred embodiment of the different aspects of the invention, cathepsin C, derivatives or fragments thereof may be used as isolated molecules.

In the context of the present invention, the term "isolated molecule" (especially when referring to cathepsin C) refers to cathepsin C polynucleotides or polypeptides purified from natural sources as well as purified recombinant molecules (wherein the term purified includes partial purification as well as complete purification).

The preparation of recombinant polypeptide or polynucleotide molecules and the purification of naturally occurring molecules from cells or tissues, as well as the preparation of extracts of cells or tissues, is well known to those skilled in the art (see also, for example, the standard literature listed below).

They include, for example, amplification of a polynucleotide of a desired length via Polymerase Chain Reaction (PCR) based on published genomic or coding polynucleotide sequences, followed by cloning of the resulting polynucleotide into a host cell (see, e.g., the standard literature listed below).

In the context of the present invention, the term "polypeptide" refers to a molecule comprising amino acids linked to each other by peptide bonds, which contains at least 50 amino acids linked to each other in a linear manner to form a polypeptide chain. Shorter such molecules are called peptides. The term "protein" refers to a molecule comprising at least one polypeptide chain, but may also refer to a molecule comprising more than one polypeptide chain associated or bound to each other. Thus, the term "protein" includes the term "polypeptide".

The present invention is explained in more detail below with the aid of examples, without the intention of limiting the invention thereto.

Examples

Materials and methods

Mouse strains used

5 different inbred mouse strains were used: AKR/J (AKR), CBA/J (CBA), C3H/HeJ (C3H), C57BL/6J (B6) and C58/J (C58). Mice were obtained from Jackson laboratories (BarHarbor, ME, USA). These mouse strains have been shown to differ significantly in several in vivo pain measurements (Mogil et al 1999).

Total RNA isolation

With PicoPure^TMRNA isolation kit (Arcturus) total RNA was isolated from DRG (dorsal root ganglion) according to the manufacturer's instructions. Using 2100 bioanalyzer and RNA6000NanoLabChip^TMThe kit (Agilent) assesses RNA quality.

AffymetrixGeneChip ^TM Microarray

500ng of 100pMT7- (dT)24 oligomer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-dT) was used according to Baugh, L.R, Hill, A.A., Brown, E.L., and Hunter, C.P (2001) nucleic acids Res.29, e29₂₄SEQ ID NO: 4) and first strand cDNA was synthesized using SuperScriptII reverse transcriptase following the manufacturer's instructions. Double-stranded cDNA was synthesized and then extracted with a phenol-chloroform followed by an ethanol precipitation step. In vitro transcription reactions were performed with double stranded cDNA samples using the BioArray high yield RNA transcription labeling kit (BioArrayHighyieldRNATransscription Labelingkit) (Enzo) following the manufacturer's instructions. The transcription reactions were incubated at 37 ℃ for 16 hours. Using RNeasy^TMThe cRNA was purified by RNA cleanup using the Mini kit (Qiagen) protocol and quantitated using a spectrophotometer. Biotin-labeled cRNA was fragmented with RNA fragmentation buffer (200mM Tris-acetate, 500mM KOAc, 150mM MgOAc, pH 8.1). Mouse genomes 4302.0GeneChips following manufacturer's instructions^TM(Affymetrix) hybridization and staining. Using GeneChip^TMThe microarray was scanned using a 3000 scanner, and the scanned data was entered and analyzed using Resolverv5.1 expression data analysis software (Rosetta Biosoftware)And (6) analyzing.

L5 spinal nerve transection and sham surgical procedure

The left L5 spinal nerve was exposed in anesthetized mice, and then the transverse process (transverseprocess) was partially removed. After separation from the L4 spinal nerve, the L5 spinal nerve was transected. The Sham procedure is identical to the L5 spinal nerve transection procedure, but does not transect the L5 spinal nerve (see DeLeo et al 2000).

Determination of foot contraction threshold

The Paw Withdrawal Threshold (PWT) was evaluated with a dynamic plantar tactile meter (dynamic plantaaerasthesiometer) (see Szabo et al 2005). After the mice were acclimated to the wire mesh floor isolation room, a stimulator was placed under the animal's hind paw, and a straight wire driven by an electric actuator touched the plantar surface of the foot, applying increasing force until the animal removed the paw (withdrawal threshold, PWT). The PWT of the operated ipsilateral and contralateral hind paw of the body was evaluated. Each animal was used only once. In all animal experiments, the ethical guidelines for the study of conscious animals were followed and approved by the local ethical committee for the procedure.

Correlation analysis

For correlation analysis, the "pain phenotype" of each nerve transection animal (Chung animal) was defined as C1-S1, where C1 ═ ln (ipsilateral PWT/contralateral PWT) and S1 ═ average_{All sham animals of the same strain}ln (ipsilateral/contralateral PWT).

Two differential transcriptional regulation measurements were defined for each Chung animal, each measured gene based on its intensity expression data. For each gene and animal, the "raw intensity measurements" were taken as the intensity measurements calculated by Resolver expression data analysis software (v 5.1). For specific genes and Chung animals, a "log ratio measurement" ln (C2/S2) was calculated, where C2 ═ Chung expression intensity and S2 ═ average_{All sham animals of the same strain}Sham expresses intensity.

Prior to calculating the correlation, the gene set was filtered to exclude genes that expressed below noise levels and had no significant Chung versus sham modulation. A eligible gene should be regulated in at least 60% of Chung animals with a fold change greater than or equal to 1.5 or at least 20% of Chung animals with a fold change greater than or equal to 2.0. Furthermore, the corresponding gene expression should be detectable ("present") in at least 5 animals which are delimited by respective intensity p-values < 0.001.

Using the R software package (http://www.r-project.org/) To calculate a Pearson correlation coefficient for each gene between the pain phenotype score and one of the defined transcriptional regulation measures. Based on this, a statistically significant p-value and corresponding false-discovery rate (FDR) were generated according to the method of Storey et al (2002). Genes with FDR < 0.05 under "log ratio measurements" or "intensity measurements" were considered significantly related.

Drawings

FIG. 1: cathepsin C-related curves

FIG. 1 shows the gene regulation (log ratio (Chung vs Sham control), Y-axis) of the neuropathic pain phenotype (mechanical hypersensitivity, X-axis) and corresponding cathepsin C in L5DRG for each individual mouse. The mouse data were color coded according to the strain used. Pearson correlation analysis was performed showing a significant positive correlation between the pain phenotype and the regulation of the cathepsin C gene. This means that for individual mice, the higher the cathepsin C expression in neurally operated Chung mice L5DRG, the more pronounced the mechanical hyperalgesia, as shown by behavioral testing. This significant correlation indicates a causal relationship of cathepsin C gene expression for the induction of neuropathic pain phenotypes.

FIG. 2: cathepsin C-intensity data

Absolute values of cathepsin C expression in the L5 ganglia of individual mice of AKR, CBA and C57 species after chung or sham surgery.

FIG. 3: mouse cathepsin CmRNA fragment detected by Affymetrix probe set 1416382_ at (mouse genome 4302.0 microarray) (seq id No. 7).

FIG. 4: the cDNA sequence of cathepsin C of NM-001814 (SEQ ID NO. 1).

FIG. 5: the cathepsin C protein sequence of Swiss-ProtHUMAN _ CATCP53634 (SEQ ID NO. 2).

FIG. 6: primer sets (SEQ ID NO4 and 5) for detecting human cathepsin CcDNA of SEQ ID NO. 1.

FIG. 7: a probe (SEQ ID NO.6) for detecting mouse cathepsin CcDNA.

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Claims (48)

1. Use of cathepsin C for the identification of compounds that modulate pain.

2. Use of a non-human transgenic animal heterologously expressing cathepsin C for the preparation of a model system for identifying or analyzing compounds that modulate pain.

3. Use of a non-human transgenic animal heterologously expressing cathepsin C as a model system for increased pain sensitivity.

4. Use of a non-human cathepsin C knock-out animal for the preparation of a model system for identifying or analyzing compounds that modulate pain.

5. Use of a non-human cathepsin C knockout animal as a model system for reducing pain sensitivity.

6. Use of a cell heterologously expressing cathepsin C for the identification of compounds that modulate pain.

7. Use of a cell heterologously expressing cathepsin C as a model system for increasing pain sensitivity.

8. Use of a cathepsin C knockout cell for identifying or analyzing compounds that modulate pain.

9. Use of a cathepsin C knockout cell as a model system for reducing pain sensitivity.

10. Use of a cell heterologously expressing a reporter gene or a functional fragment thereof expressibly linked to a cathepsin C promoter or to a cathepsin C promoter and enhancer for the identification or analysis of compounds that modulate pain.

11. A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising the steps of:

a. providing at least two samples;

b. contacting a sample containing cathepsin C with a compound;

c. measuring cathepsin C activity in the presence of the compound;

d. assaying cathepsin C activity in the absence of the compound; and

e. comparing the cathepsin C activity of C) with the cathepsin C activity of d).

12. A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. contacting cathepsin C protein with a test compound; and

b. determining whether the test compound modulates the activity of cathepsin C protein.

13. A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. contacting a cell having a detectable amount or activity of cathepsin C with a test compound;

b. determining whether the test compound is capable of modulating the amount or activity of cathepsin C present in the cell.

14. A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. contacting a nucleic acid encoding a cathepsin C protein with a test compound in a transcriptionally active system; and

b. determining the amount of mRNA encoding cathepsin C protein present in the system in the presence of the compound; and

c. determining whether said compound is capable of modulating the amount of mRNA encoding cathepsin C protein present in said system.

15. A method for identifying or analyzing a compound that modulates and/or prevents pain, the method comprising:

a. providing a cell transfected with a nucleic acid vector comprising a promoter of a cathepsin C gene operably linked to a reporter gene or functional fragment thereof;

b. providing a cell transfected with a control vector comprising a reporter gene or functional fragment thereof not operably linked to a functional cathepsin C promoter;

c. determining the reporter gene activity of the cells of a) and b) in the presence of the test compound;

d. determining the reporter gene activity of the cells of a) and b) in the absence of the test compound.

16. Use of a means for detecting cathepsin C in the manufacture of a test kit for the analysis of pain threshold in an individual, wherein said means for detecting cathepsin C is selected from the group consisting of: an antibody capable of specifically detecting cathepsin C protein, a specific cathepsin C nucleic acid probe, a primer set capable of amplifying cathepsin CDNA or cathepsin CcDNA, and a cathepsin C-specific anti-DNA antibody.

17. Use of means for detecting cathepsin C for the manufacture of a test kit for adjusting the dosage of a medicament for the prevention and/or treatment of pain in an individual, wherein said means for detecting cathepsin C is selected from the group consisting of: an antibody capable of specifically detecting cathepsin C protein, a specific cathepsin C nucleic acid probe, a primer set capable of amplifying cathepsin CDNA or cathepsin CcDNA, and a cathepsin C-specific anti-DNA antibody.

18. Use of a means for detecting cathepsin C in the manufacture of a test kit for diagnosing an increased pain sensitivity in an individual, wherein the means for detecting cathepsin C is selected from the group consisting of: an antibody capable of specifically detecting cathepsin C protein, a specific cathepsin C nucleic acid probe, a primer set capable of amplifying cathepsin CDNA or cathepsin CcDNA, and a cathepsin C-specific anti-DNA antibody.

19. Use of a composition that reduces the amount or activity of cathepsin C in the manufacture of a medicament for treating existing pain in a subject experiencing pain.

20. Use of a composition that reduces the amount or activity of cathepsin C in the manufacture of a medicament for reducing pain sensitivity in a subject.

21. Use of a compound capable of reducing cathepsin C activity and/or expression in the manufacture of a medicament for the treatment of pain.

22. The use of any one of claims 1, 2, 6, 8 and 10 or the method of any one of claims 11-15, wherein said modulation is reduction, abolishment or prevention of pain.

23. The use of any one of claims 1, 2, 6, 8 and 10 or the method of any one of claims 11-15, wherein said modulation is an increase.

24. The use or method of claim 22, wherein the compound is a compound for the prevention or treatment of pain.

25. The use of any one of claims 1-10 and 16-21 or the method of any one of claims 11-15, wherein cathepsin C is mammalian cathepsin C.

26. The method or use of claim 25, wherein cathepsin C is human cathepsin C.

27. The use according to any one of claims 1-10 and 16-21 or the method according to any one of claims 11-15, wherein the pain is acute or chronic pain.

28. The use of any one of claims 1-10 and 16-21 or the method of any one of claims 11-15, wherein the pain is inflammatory pain or neuropathic pain.

29. The use or method of claim 28, wherein the pain is osteoarthritic pain.

30. The use of any one of claims 1-3, 6, 8 and 10 or the method of any one of claims 11-15, wherein cathepsin C is used as an isolated protein or polynucleotide.

31. The use of any one of claims 1-3, 6, 8 and 10 or the method of any one of claims 11-15, wherein cathepsin C is:

a. a polypeptide consisting of the sequence of seq id No. 2; or

b. A polypeptide encoded by a polynucleotide consisting of the sequence of seq id no 1.

32. The use of any one of claims 1-3, 6, 8 and 10 or the method of any one of claims 11-15, wherein cathepsin C is:

a. a polynucleotide consisting of the sequence of seq id No. 1; or

b. A polynucleotide encoding cathepsin C of seq id No. 2.

33. The method of claim 13 or 14, wherein cells expressing recombinant cathepsin C are used.

34. The method of any one of claims 11 to 15, wherein the determination of cathepsin C activity relates to its protease activity.

35. The use of any one of claims 6 to 10 or the method of any one of claims 13 or 14, wherein the cell is a mammalian cell.

36. The method or use of claim 35, wherein the cell is a rodent cell.

37. The use of any one of claims 2-5, wherein the non-human animal is a mammal.

38. The use of claim 37, wherein the non-human animal is a rodent.

39. The use of claim 10 or the method of claim 15, wherein the reporter gene is a gene for beta lactamase (lacZ), luciferase, green or blue fluorescent protein (GFP or BFP), DsRed, HIS3, URA3, TRP1 or LEU2 or beta-galactosidase.

40. The method of claim 14, wherein changes in the amount of mRNA are analyzed.

41. The method of claim 40, wherein said change in the amount of mRNA is analyzed by using one or more suitable primers for amplifying cathepsin CcDNA or one or more probes suitable for hybridization with cathepsin CcDNA or mRNA under standard conditions.

42. The method of claim 40, wherein the amount of mRNA is analyzed by means of PCR, RNA hybridization, array hybridization or chip hybridization.

43. The method of claim 14, wherein a change in the amount of cathepsin C protein is determined.

44. The method of claim 43, wherein the protein is detected with the aid of at least one antibody.

45. The method of claim 43 or 44, wherein the detection is performed via ELISA, Western blot or protein chip.

46. The method of claim 43 or 44, wherein the detection is performed by means of immunohistochemical or immunoradiochemical detection methods.

47. The use of claim 21, wherein the compound is a reversible peptide nitrile inhibitor of cathepsin C.

48. The use of claim 47, wherein the compound is a dipeptide α -aminoacetonitrile inhibitor of cathepsin C.