WO2005004905A1 - Use of calpain for identifying compounds that modulate pain - Google Patents

Abstract

The invention relates to the use of calpain or functional fragments or derivatives thereof for the preparation of pharmaceutical compounds that modulate pain, and the use of calpain or functional fragments of derivatives thereof for identifying such compounds, and a method of screening pharmaceutical useful for modulating and/or preventing pain.

Description

Use of calpain for identifying compounds that modulate pain

Description

The invention relates to the use of calpain or functional fragments or derivatives thereof for the preparation of pharmaceutical compounds that modulate pain, and the use of calpain or functional fragments of derivatives thereof for identifying such compounds, and a method of screening pharmaceuticals useful for modulating and/or preventing pain.

The effects of peripheral inflammatory stimulation on the protein expression pattern in the rat spinal cord was investigated. By the method of two-dimensional (2D) gel electrophoresis a time-dependent breakdown of neurofilament light chain (NF-L) protein after 24, 48 and 96 h of zymosan treatment was determined, which was corroborated by immunohistochemistry and Western Blot experiments.

Since neurofilaments are substrates of the calcium-dependent cysteine protease calpain the role of calpain in inflammatory and painful processes was further assessed.

It could be shown that peripheral inflammatory stimulation with zymosan leads to an upregulation of calpain activity and protein expression in the rat spinal cord. Inhibition of calpain proteolysis using the specific calpain inhibitor III (MDL-28170; 12.5 mg/kg and 25 mg/kg i.p.) attenuated the zymosan induced neurofilament degradation.

Calpain protein expression was not affected by MDL-28170. The chemical structure of calpain inhibitor III is as follows:

The effects of MDL-28170 on inflammation and nociception have been tested in two different animal models. In the zymosan induced paw inflammation model calpain inhibitor III (25mg/kg) significantly reduced the paw edema compared to control animals. In the thermal hyperalgesia model application of calpain inhibitor III led to 5 antinociceptive responses. Taken together, these data suggest an important role of calpain in inflammatory and painful processes and therefore indicate a therapeutic potential of calpain inhibitors as alternative antiinflammatory and analgesic drugs.

Additionally, calpain inhibitor MDL-102935 has been tested as described for calpain 10 inhibitor III. The chemical formula of MDL-102935 is the following: r-,

The effects of MDL-102935 on inflammation and nociception are the same as those of 20 MDL-28170. MDL-102935 is an inhibitor of calpain.

In spinal nociceptive processing various painful stimuli from the periphery are switched over from the primary afferents to the central nervous system in the dorsal horn of the lumbal spinal cord. Persistent stimulation of primary nociceptors and C-fibres, as

25 occurring with inflammatory stimuli triggers an increased excitability of nociceptive neurons in the spinal cord and thereby results in hyperalgesia and allodynia (Yaksh et al., 1999). Long term alterations in synaptic communication (so called long term potentiation (LTP)) are associated with processes of learning and memory (Bliss et al., 1993) and may be responsible for the synaptic plasticity and chronic pain (Rygh et al.,

30 1999; Vikman et al., 2001 ). Repeated or prolonged noxious stimulation activate a number of intracellular second messenger systems, implying phosphorylation of key membrane receptors and channels by protein kinases, particularly protein kinase C (PKC). This kinase activation results in posttranslational changes that can lead to an increase in synaptic efficacy. Intracellular signal cascades result in an induction of immediate early genes (IEG) (c-fos, Zif 268, Cox-2), and later response genes encoding neuropeptides, neuropeptide and neurotrophin receptors. This protein alterations are considered as the beginning of a widespread change in protein synthesis and furthermore as a general basis for nervous system plasticity (Dubner et al., 1992; Ji et al., 1994; McCarson et al., 1994) (Beiche et al., 1996; Ji et al., 1995; Mannion et al., 1999) (Woolf et al., 1999) . In the current study intracellular changes of the protein expression pattern accompanying peripheral inflammatory conditions within the rat lumbal spinal cord have been investigated by two-dimensional gel electrophoresis (2D PAGE) combined with MALDI-TOF MS. Using this method we detected several protein alterations in the rat zymosan induced paw inflammation model. In this study it was focussed on one of them which was identified as neurofilament light chain (NF-L). NF-L is a cytoskeleton protein occuring in neuronal cells where it is responsible for correct assembly of neurofilaments and maintenance of axonal calibre (Sakaguchi et al., 1993). Neurofilament desorganisation is associated with a variety of neurodegenerative diseases like Parkinson and amyotrophic lateral sclerosis (Julien, 1999). Moreover neuronal death and neurological dysfunction after spinal cord injury comes along with the loss of neurofilament protein (Ray et al., 2000a). The degradation of neurofilament proteins is mostly mediated by the action of the protease calpain (Ray et al., 2000b; Schlaepfer et al., 1985; Stys et al., 2002). Calpains are a family of ubiquitously expressed calcium dependent cysteine proteases that have been implicated in basic cellular processes including proliferation, apoptosis and differentiation. Many calpain substrates are localized in the pre- and postsynaptic compartments of neurons indicating growing evidence for the involvement of calpains in neurodegenerative diseases and in the modulation of synaptic plasticity(Chan et al., 1999). The calpain of rat is described by Thompson and Goll (Meth. Mol. Biol., Vol 144, 3-16, 2000). Two human calpain isoforms are known, namely calpain I (embl locus HSCANPR, accession no. X04366.1 ) and calpain II (Morford et al., Biochem. Biophys. Res. Commun. 295 (2), 540-546, 2002). In the current study the role of calpain in the zymosan induced neurofilament degradation as well the potential anti-inflammatory and antinociceptive properties of a specific calpain inhibitor (MDL 28170) in the well established zymosan-induced paw inflammation model was investigated.

Surprisingly it has now be found that calpain plays a role in connection with pain.

An embodiment of the invention is the use of calpain or functional fragments or derivatives thereof for the preparation of pharmaceutical compounds that modulate pain.

Another embodiment of the invention is the use of calpain or functional fragments of derivatives thereof for identifying compounds that modulate pain.

A further embodiment of the invention is the use as described above, wherein the pharmaceutical compounds are compounds for the prevention or treatment of pain, in particular, wherein the compounds lessen or abolish pain.

Another embodiment of the invention is a method of screening pharmaceuticals useful for modulating and/or preventing pain, comprising the steps a. Providing two samples b. contacting one sample containing calpain or a functional fragment or derivative thereof with a compound, c. determining the calpain activity in the presence of compound, d. determining the calpain activity in the absence of compound, and e. comparing the calpain activity according to c) with that according to d).

Another embodiment of the invention is a use the method as described above, wherein calpain is human calpain I and / or calpain II, in particular wherein calpain is an isolated polypeptide or polynucleotide. Another embodiment of the invention is a use or a method as described above, wherein calpain is a polypeptide, in particular wherein calpain is a polypeptide or functional fragment thereof that comprises or consists of the sequence according to SEQ ID No 1 (calpain I) or a fragment thereof, the sequence according to SEQ ID No 3 (calpain II) or a fragment thereof, or is encoded by a polynucleotide comprising or consisting of the sequence according to SEQ ID No 2 (encoding calpain II) or a fragment thereof, or of the sequence according to SEQ ID No 4 (encoding calpain II) or a fragment thereof.

Another embodiment of the invention is a use or a method as described above, wherein calpain is a polynucleotide, in particular wherein calpain is a polynucleotide comprising or consisting of the sequence or a part of the sequence coding for a functional fragment of calpain according to SEQ ID No 2 or SEQ ID No 4 or a polynucleotide comprising or consisting of a sequence that is able to hybridize with said polynucleotides under stringent conditions, preferably wherein the functional fragments comprise or consist of amino acids 115 to 272 of SEQ ID No 2, and / or 105 to 262 of SEQ ID No 4.

Another embodiment of the invention is a method as described above, wherein a cell expressing, preferably expressing recombinant calpain is used.

Another embodiment of the invention is a method as described above, wherein a modified cell, having a lower calpain activity as compared to its unmodified state, is used, preferably wherein a calpain knock-out cell is used.

Another embodiment of the invention is a method as described above, wherein the calpain activity is determined directly or indirectly.

A further embodiment of the invention is a method of identifying a compound that modulates pain comprising a. Selecting a compound that modulates the activity of calpain as a test compound, and b. Administering said test compound to a subject to determine whether the pain is modulated.

A further embodiment of the invention is the use of MDL-28170 (calpain inhibitor III) in the process of preparing a medicament for the treatment of pain.

A further embodiment of the invention is the use of MDL-28170 as a medicament for the treatment of pain.

A further embodiment of the invention is the use of MDL-102935 in the process of preparing a medicament for the treatment of pain.

A further embodiment of the invention is the use of MDL-102935 as a medicament for the treatment of pain.

In the following the invention is described by the examples which shall in no way limit the invention to the specific embodiments of the examples.

Examples

Materials and Methods used in the examples

Animals

Male Sprague Dawley rats (Charles River, Sulzbach, Germany) weighing 260-300g were housed in groups of five in standard cages and maintained in climate- and light- controlled rooms (22 ± 0.5°C, 12/12 h dark/Light cycle) with free access to food and water. In all experiments the ethic guidelines for investigations in conscious animals were obeyed and the procedures approved by the local Ethics Committee.

Zymosan-treatment of rats Unilateral hind paw inflammation and paw oedema was induced by subcutaneous injection of 1.25 mg zymosan (Sigma, Mϋnchen, Germany), suspended in 100 μl phosphate buffered saline (12.5 mg/ml), into the midplantar region of the right hind paw. After 24, 48 or 96 h animals were anaesthetized and killed by cardiac puncture. Spinal cords were excised, directly frozen in liquid nitrogen and then kept at -80°C until further analysis.

Paw oedema

Paw oedema was induced by zymosan injection as described above. The paw volume was measured before and 0.25, 0.5, 1 , 2, 4, 6, 8 and 24 h after zymosan injection using a plethysmometer (Ugo Basile, Varese, Italy). Six rats per group were used. At each time point five measurements of the paw volume were taken. The average of these five measurements was used for further analysis.

Thermal hyperalgesia

Nociceptive paw withdrawal latency to radial heat was assessed according to Hargreaves (Hargreaves et al., 1988), using a commercially available special device (Ugo Basile, Varese, Italy). Rats were placed in in transparent plastic chambers (18 x 29 x 12.5 cm) on a metal grid (5 x 5 mm). The heat source consisting of a high projector lamp bulb was focused on the mid-plantar surface of the hind paw. The bulb and an electronic timer were simultaneously activated at the test start, and both were automatically inactivated when a photocell detected a paw withdrawal in response to the heat. Paw withdrawal latencies (PWL) of the right and left paw (treated and untreated paw) were recorded before and every 60 min up to 8 h and 24 h after zymosan injection.

Drug Treatment

Calpain inhibitor III (MDL 28170) (Calbiochem, Schwalbach/Ts., Germany) was dissolved in PEG 400/DMSO (1 :1) at a concentration of 10 mg/ml. Animals received calpain inhibitor III either as a 12.5 mg/kg or a 25 mg/kg intraperitoneal (i.p.) bolus injection 20 min prior to the intraplantar injection of zymosan. Six animals were used for each treatment group. (Controls received 1 ml of vehicle.) Treatments were randomly allocated to animals and the observers were unaware of treatment allocations.

Data analysis and statistics

The percent relative difference in paw withdrawal latency (ΔPWL) between the zymosan treated right and the untreated left hind paw, calculated as: ΔPWL = (right - leftyieft x 100, was used to asses antinociceptive effects. To evaluate anti- inflammatory effects, the relative difference between the paw volume before and after zymosan injection (ΔPV) was used. The areas under the ΔPWL versus time curves (AUCΔPWL) as well as the areas under the ΔPV versus time curves (AUCΔPV) were calculated using the linear trapezoidal rule. Statistical evaluation was done by SPSS 9.02 for Windows. Effects of study medications were assessed by submitting the AUCΔPV from 0-8 h to univariate analysis of variance (ANOVA) with subsequent t-tests employing a Bonferroni D-correction for multiple comparisons. Statistical significance of ΔPWL was calculated by unpaired Student^'s t-test (zymosan-treated rats versus control rats). D was set at 0.05.

2D PAGE

Lumbal spinal cords were homogenized in lysis buffer containing 9 M urea, 2% CHAPS, 1% DTT and 2 mM Pefabloc. After removal of cellular debris extracts were ultracentrifuged at 40O00 rpm for 1 h (15°C) and the supernatent was stored at -70°C till further analysis. Protein concentrations were determined by the Bradford protein assay.

2D PAGE was performed as described previously (Gorg et al., 1995) with slight modifications. In brief, for the first dimension (isoelectric focusing (IEF)) 600 μg of protein were precipitated with 50% TCA at -20°C. The pellet was redissolved in rehydration solution (8 M urea, 2% CHAPS, 2.8% DTT and 0,5% IPG buffer (Amersham Biosciences, Freiburg, Germany)) and applied to 13 cm Immobiline DryStrips, pH 3-10 linear (Amersham Biosciences, Freiburg, Germany). IEF was performed using an Amersham IPGphor isoelectric focusing system and the run was carried out as follows: Rehydration 2 h, 30 V for 6 h, 60 V for 3 h, 200 V for 1 h, 500 V for 1 h, 5000 V for 4 h and 8000 V for 1 h. After IEF the IPG strips were equilibrated for 10 min in equilibration buffer (1 ,5 M Tris- Cl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) with addition of 10% DTT and then for further 10 min in equilibration buffer containing 260 mM iodacetamide. The IPG strips were then placed on the top of a 13% sodium dodecyl sulfate- polyacrylamid gel and sealed with 1% agarose solution. Electrophoresis in the second dimension was performed at 2.5 W/gel for 1 h followed by 5 W/gel for 4 h. Gels were stained with Coomassie Brilliant Blue. The stained gels were imaged on a UMAX PowerLooklll scanner and analysed with Image Master 2D software (Amersham Biosciences, Freiburg, Germany). Coomassie stained spots of interest were cut out of the gels, proteins were digested and then identified by peptide mass fingerprinting using the matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS).

Immunoblot Analysis

Equivalent amounts of total cellular proteins, extracted from homogenized spinal cords, were diluted with 4x sample buffer (1x sample buffer consists of 2.5% sodium dodecyl sulfate (SDS), 50 mM Tris-HCI (pH 6.8), 2.5% 2-mercaptoethanol, 10% glycerol and a trace of Bromphenol Blue). Samples were boiled for 5 min, and proteins (30 μg per lane) were subjected to SDS-PAGE using 10% polyacrylamide gels. Proteins were then transferred onto nitrocellulose membranes (Pall Corporation). Blots were blocked in PBS/5% skimmed milk/0.3% Tween 20 overnight at 4°C. Afterwards they were incubated with primary antibody against Neurofilament-L (1:500) (Sigma) for 90 min at RT followed by 3 washes with PBS/0.3% Tween 20 and then 60 min with alkaline horse radish peroxidase (HRP)-labeled secondary antibody (1 :20000) (Santa Cruz Biotechnology). After 3 washes with PBS/0.3% Tween 20 specific protein bands were detected by the enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology). ECL films were imaged on an Umax PowerLooklll scanner using Photoshop software (Adobe Systems), and band intensities were determined densitometrically using Quantity One software (PDI).

Immunohistochemical staining

For immunohistochemical staining, the lumbal spinal cord segments were excised, directly frozen in liquid nitrogen and stored at -80°C. Fresh-frozen tissue sections (14 μm) were cut in a cryostat, mounted on gelatin-subbed slides, and fixed for 10 min in 4% paraformaldehyd in PBS (pH 7.4). Slides were washed with three 10-min washes in PBS and treated for 15 min with PBS containing 0.1% Triton-X 100. The sections were then blocked in 3% BSA in PBS for 1 h to reduce nonspecific binding and then incubated with pimary monoclonal antibody, anti-NFL (1 :100; Sigma) and anti-m- calpain (1 :50; Affinity BioReagents) for 1 h at 37°C. NF-L and m-calpain were flourescently labelled with a 1 h incubation at 37°C with Cy3-conjugated secondary antibody (1 :600; Sigma). Slides were washed with three 10-min washes in PBS after each incubation. Primary and secondary antibody incubations were performed in PBS containing 1 % BSA. Slides were mounted with SlowFade Light Antifade mounting media according to manufacturer's protocol.

Determination of Calpain activity

Calpain activity was analysed by a commercially available calpain activity assay kit (Biocat, Heidelberg, Germany), according to the manufactures instructions. The assay is based on fluorometric detection of the cleavage of the calpain substrate Ac-LLY- AFC, which emits blue light (λm_ax = 400 nm). Upon cleavage of the substrate by calpain, free AFC emits a yellow-green fluorescence (λ_maχ = 505 nm), which was quantified using a fluorometer (Spectra Fluor Plus, Tecan). Example 1 : Chronical inflammation leads to alterations in a variety of proteins

For the detection of inflammation responsive proteins, the zymosan induced inflammation model in rats with subsequent 2D-PAGE of the rat lumbal spinal cord has been used. Protein expression patterns of control spinal cord lysate were compared to spinal cord proteins of rats after 24, 48 or 96 h zymosan treatment, respectively. More than 500 protein spots were separated on each gel with an apparent range of molecular masses from 10 to100 kDa and pi values from 3 to 10, as detected by Coomassie Blue staining. Zymosan treatment led to the modification of at least 10 protein spots. The effects were most pronounced 96h after the zymosan injection.

Example 2: NF-L is time dependently down regulated in the rat spinal cord after zymosan treatment

Further investigations in this study were focussed on Spot 1 that was identified by MALDI-TOF mass spectrometry as neurofilament light chain (NF-L) with a molecular mass of 61.3 kDa and a pi at 4.63 (Fig. 1 A). 2D-PAGE revealed that protein expression of NF-L was time dependently downregulated after 24 and 48 h. After 96 h the protein spot was completely diminished as shown by Commassie staining (Fig. 1A).

In order to prove the degradation of NF-L protein expression in the spinal cord after an inflammatory stimulus we also performed Western Blot analysis using a monoclonal anti-NF-L antibody which did not cross-react with other intermediate filament proteins. As shown in FigJ B by Western Blot analysis, the NF-L protein was degraded due to the increasing duration of the peripheral inflammation. Densitometric analysis of NF-L protein bands in the Western Blot analysis are shown in Fig 1 C.

Example 3: Inflammatory stimulation leads to an increase of calpain activity

It is well known that neurofilament degradation occurs mainly by the activity of the calcium dependent cystein-protease calpain. Therefore we examined the effects of peripheral zymosan treatment on calpain activity in the spinal cord. Calpain activity was determined by fluorometric detection of the cleavage of a synthetic calpain substrate. Peripheral inflammation increased calpain activity in the spinal cord slightly after 24 and 48h and significantly after 96h (p< 0.05)(Fig.2A). As enzyme inhibitor we used calpain inhibitor III (Carbobenzoxy-valinyl-phenylalaninal, MDL 28170) which is described as a potent, selective and cell-permeable inhibitor of calpain I and II (Chatterjee et al., 1998; Rami et al., 1997). Treatment of rats with the calpain inhibitor 111 (i.p.) (25mg/kg) significantly inhibited the zymosan-induced protease activation in the spinal cord (Fig. 2B).

Example 4: Immunohistochemistry

Immunohistochemistry was applicated to determine the protein levels of neurofilament- L and calpain in rat spinal cord slices after zymosan treatment with and without additional administration of calpain inhibitor III. Slices treated with a specific NF-L-antibody showed reduced immunoflourescence intensity after 96h of zymosan treatment indicating the neurofilament breakdown. This effect was reversed by addition of calpain inhibitor III. Slices treated with the protease inhibitor showed immunofluorescence levels comparable to control slices of untreated rats (Fig 3A). To assess calpain protein expression an antibody against m-calpain was used, m- calpain expression in spinal cord slices was slightly enhanced after zymosan treatment (96h). This increase in immunoreactivity was not altered by application of the calpain inhibitor III (Fig. 3B)

Example 5: Effects of a calpain inhibitor in the zymosan induced inflammation model in rats

Since calpain activity is upregulated by zymosan induced inflammation and thereby probably causes neurofilament degradation we hypothesized that the specific calpain inhibitor might provide anti-inflammatory properties. We tested this hypothesis again in the zymosan-induced paw inflammation model in rats. In vehicle treated rats intraplantar injection of 1.25 mg zymosan led to a maximum increase of the paw volume of 129 ± 5.9 (mean ± sem) % after 4h. As hypothesized calpain inhibitor III inhibited paw inflammation dose-dependently at doses of 12.5 and 25 mg/kg (Figure 4A). Statistical comparison of the area under the "paw volume increase" versus "time" curves (AUC_Δpvfi"om 0-8h) revealed statistically significant differences between control rats and rats treated with 25mg/kg calpain inhibitor (p<0.001). Results of the post hoc analysis are shown in figure 4B.

Example 6: Effects of calpain inhibitor III in the zymosan induced thermal hyperalgesia in rats

To assess the influence of calpain inhibition on hyperalgesia we determined the paw withdrawal latency in the thermal hyperalgesia model. The results in Figure 5A suggest that rats treated with calpain inhibitor HI showed significantly higher paw withdrawal latency as compared to rats treated with zymosan alone indicating that the inhibitor treated rats felt less pain. Results of the post hoc analysis are shown in figure 5B. Discussion of results of examples:

The results of the present study showed by the means of 2D gel electrophoresis that neurofilament light chain is degraded after peripheral inflammatory stimulation. Neurofilaments represent an important group of cytoskeleton proteins that are involved in the control of axonal caliber and architecture and axoplasmic flow (Perrone Capano et al., 2001 ). Neurofilament abnormalities provoke selective degeneration and death of motoneurons and come along with neurodegenerative diseases (Julien, 1999). Neurofilament degradation has been observed in spinal cord injury as well as under anoxic andjschemic conditions by activation of the calcium dependent cystein protease calpain (Banik et al., 1997; Leski et al., 2001; Stys et al., 2002).

As summarized in Figure 6 noxious peripheral stimulation, e.g. inflammatory conditions triggers the release of neurotransmitters in the spinal cord, in particular the excitatory amino acid glutamate and the neuropeptide substance P leading to activation of voltage-gated calcium channels as well as ionotropic and metabotropic receptors, thereby provoking a calcium influx from voltage sensitive ion channels and also a calcium release from intracellular stores. The resulting calcium accumulation induces calpain activation as well as upregulation and activation of several further proteins in the spinal cord e.g. nNOS, Ca²⁺-Calmodulin Kinase (Shields et al., 1998) and adenylyl cyclase. The here found increase in calpain activity and expression may also be related to the production of cytokines and activation of the arachidonic acid cascade which occurs during inflammatory processes in astrocytes and inflammatory immune cells. In vitro studies have also shown increases of synthesis and activity of calpain in glia, neuronal and lymphoid cells in response to stress ( Ray et al., 2003). Currently, there are two major isoforms of calpain in the central nervous system, μ- calpain (calpain I) and m-calpain (calpa n II) which are activated by micromolar and millimolar quantities of calcium, respecti vely. They have been implicated in a number of physiological and pathological condit ons including neuronal plasticity and neuronal cell death. With respect to this neuronal cell death, activation of calpain leads to the proteolysis of several cellular proteins, mostly associated with the cellular membrane, including cytoskeletal proteins (eg, neurofilament, spectrin, fodrin, and microtubule- associated proteins), membrane proteins (eg, growth factor receptors, adhesion molecules, and ion transporters), enzymes (eg, kinases, phosphateses, and phospholipases), as well as cytokines and transcription factors (Kampfl et al., 1997; Shields et al., 1998). Although many of these calpain actions may be implicated in mechanisms contributing to inflammation, the exact role of calpain activation in inflamed tissue remains to be clarified. There is evidence that activation of calpain leads to the degradation of lκB in the proteasome and, hence, is an essential step in the translocation of nuclear factor KB (NF-KB) from the cytosol into the nucleus (Chen et al., 2000; Saido et al., 1994). Calpain inhibitor I has been shown to prevent this NF-kB activation and the subsequent upregulation of iNOS and COX-2 protein thereby reducing the development of acute and chronical inflammation (Cuzzocrea et al., 2000). The anti- inflammatory drug indomethacin which inhibits cyclooxygenase activity and prostaglandin upregulation showed additionally calpain inhibiting activity (Banik et al., 2000). In addition, recent findings of increased calpain activity in ischemia, Alzheimer's disease, spinal cord injury, and brain trauma have implicated calpain mediated proteolysis in tissue destruction and degeneration in CNS trauma and diseases (Banik et al., 1997; Shields et al., 1998). Uncontrolled calpain activation leads to cytoskeletal protein breakdown, subsequent loss of structural integrity and disturbances of axonal transport. The findings of increased calpain activity in diseases suggest that calpain inhibitors may be employed as therapeutic agents since they minimize tissue degeneration by preventing degradation of substrate proteins. In our study we used Calpain inhibitor III (MDL 28170), a potent, specific calpain inhibitor that rapidly penetrates the blood-brain barrier following systemic administration. Intraperitoneal application of this inhibitor blocked the degradation of neurofilament protein in the spinal cord which was due to inhibition of the protease activity but not to calpain protein downregulation. Additional effects of calpain inhibition on other calpain substrates are also possible but have not been investigated in this study. For the first time we could show that pre-treatment of rats with calpain inhibitor III attenuates the development of zymosan induced paw inflammation and thermal hyperalgesia in rats. In conclusion, we propose that calpain is an important mediator in nociceptive responses and that calpain inhibitor III may be useful in the therapy of inflammatory diseases.

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Shields, D. C, Tyor, W. R., Deibler, G. E., Hogan, E. L. & Banik, N. L. Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis. Proc Natl Acad Sci U S A, 95, 5768- 72(1998)

Stys, P. K. & Jiang, Q. Calpain-dependent neurofilament breakdown in anoxic and ischemic rat central axons. Neurosci Lett, 328, 150-4(2002) Vikman, K. S., Kristensson, K. & Hill, R. H. Sensitization of dorsal horn neurons in a two-compartment cell culture model: wind-up and long-term potentiation-like responses. J Neurosci, 21 , RC169(2001)

Woolf, C. J. & Costigan, M. Transcriptional and posttranslational plasticity and the generation of inflammatory pain. Proc Natl Acad Sci U S A, 96, 7723-30(1999)

Yaksh, T. L., Hua, X. Y., Kalcheva, I., Nozaki-Taguchi, N. & Marsala, M. The spinal biology in humans and animals of pain states generated by persistent small afferent input. Proc Natl Acad Sci U S A, 96, 7680-6(1999)

Figure legends

Figure 1 :

Zymosan treatment causes a time-dependent decrease of Neurofilament-L

(A) Enlarged regions of 2D gels of control rat spinal cord tissue and after 24 h, 48 h, and 96 h zymosan treatment. Arrows indicate protein 1 , which was identified as neurofilament light chain. (B) Representative western blot analysis of neurofilament light chain (NF-L) expression following zymosan treatment for 24, 48, and 96h.

(C) Densitometric analysis of 3 independent Western Blot experiments (**statistically significant mean difference, p< 0.01).

Figure 2:

Calpain activity is elevated after zymosan treatment and reduced by calpain inhibitor III

(A): Calpain activity of control rat spinal cord extracts and after 24, 48 and 96h zymosan treatment. (B): Calpain activity of spinal cord extracts of zymosan treated control rats and animals that received either 12.5 mg/kg or 25 mg/kg calpain inhibitor III.

The activity is determined by the fluometric detection of the cleavage of the calpain substrate Ac-LLY-AFC.

(^'"', '"'statistically significant mean difference p<0.01 ; p<0.05, respectively)

Figure 3:

Immunohistochemical Cy-3 labeling of lumbal spinal cord sections. Cross sections were taken of control animals as well as animals 96 h after injection of zymosan with and without pre-treatment with calpain inhibitor III. The images represent the dorsal horn of the spinal cord. (A): Sections stained immunohistochemically with monoclonal anti-NF-L antibody. Calpain inhibitor III reduced the loss of NF-L after zymosan treatement. (B): Sections stained immunohistochemically with monoclonal anti-m-calpain antibody. Zymosan treatment led to an increased m-calpain immunopositivity, which was not altered by calpain inhibitor III.

Figure 4:

Calpain inhibitor III significantly reduces the paw oedema at 25 mg/kg

(A): Time course of the alteration of the paw volume after intraplantar injection of 1.25 mg zymosan in control animals (♦) and in rats treated with 12.5 (■), and 25 mg/kg calpain inhibitor III (A).

(B): For statistical comparison of drug effects the areas under the "paw volume increase" versus "time" curves (AUC_Δpwfrom 0-8h mean ± s.e.m.) were calculated using the linear trapezoidal rule and subjected to univariate analysis of variance with subsequent Bonferroni post hoc tests. (* statistically significant mean difference with p<0.05)

Figure 5:

Calpain inhibitor III significantly reduces thermal hyperalgesia at 25 mg/kg

Time course of paw withdrawal latency in response to thermal stimulation of the plantar surface after intraplantar injection of 1.25 mg zymosan alone (❖) or zymosan and calpain inhibitor III (Δ). Data are expressed as the relative difference between the zymosan treated right and the untreated left hind paw calculated as: ΔPWL = (right -

Ieft)/left x 100.

(A): Paw withdrawal latency after intraperitoneal administration of 25 mg/kg calpain inhibitor III in comparison with vehicle treated rats. (B): Area under the effect (relative decrease of paw withdrawal latency, ΔPWL) versus time curves after intraplantar injection of zymosan and treatment with 25 mg/kg Calpain inhibitor III. (***, statistically significant mean difference, p<0.001)

Figure 6:

Scheme of the potential signal transduction pathway in the dorsal horn of the spinal cord.

SEQUENCE LISTING

<110> Aventis Pharma Deutschland GmbH

<120> Use of calpain for identifying compounds that modulate pain

<130> NAE-004471EN

<140> <141>

<160> 4

<170> Patentln Ver. 2.1

<210> 1

<211> 714

<212> PRT <213> homo sapiens

<400> 1

Met Ser Glu Glu lie lie Thr Pro Val Tyr Cys Thr Gly Val Ser Ala 1 5 10 15

Gin Val Gin Lys Gin Arg Ala Arg Glu Leu Gly Leu Gly Arg His Glu 20 25 30

Asn Ala lie Lys Tyr Leu Gly Gin Asp Tyr Glu Gin Leu Arg Val Arg 35 40 45

Cys Leu Gin Ser Gly Thr Leu Phe Arg Asp Glu Ala Phe Pro Pro Val 50 55 60

Pro Gin Ser Leu Gly Tyr Lys Asp Leu Gly Pro Asn Ser Ser Lys Thr 65 70 75 80

Tyr Gly lie Lys Trp Lys Arg Pro Thr Glu Leu Leu Ser Asn Pro Gin 85 • 90 95

Phe lie Val Asp Gly Ala Thr Arg Thr Asp lie Cys Gin Gly Ala Leu 100 . 105 110

Gly Asp Cys Trp Leu Leu Ala Ala lie Ala Ser Leu Thr Leu Asn Asp 115 120 125

Thr Leu Leu His Arg Val Val Pro His Gly Gin Ser Phe Gin Asn Gly 130 ^' 135 140

Tyr Ala- Gly lie Phe His Phe Gin Leu Trp Gin Phe Gly Glu Trp Val 145 150 155 160

Asp Val Val Val Asp Asp Leu Leu Pro lie Lys Asp Gly Lys Leu Val 165 170 175

Phe Val His Ser Ala Glu Gly Asn Glu Phe Trp Ser Ala Leu Leu Glu 180 185 190

Lys Ala Tyr Ala Lys Val Asn Gly Ser Tyr Glu Ala Leu Ser Gly Gly 195 200 205

Ser Thr Ser Glu Gly Phe Glu Asp Phe Thr Gly Gly Val Thr Glu Trp 210 215 220 Tyr Glu Leu Arg Lys Ala Pro Ser Asp Leu Tyr GΪn lie lie Leu Lys 225 . 230 ■ ^" 235 240

Ala Leu Glu Arg Gly Ser Leu Leu Gly Cys Ser lie Asp lie Ser Ser 245 250 _, 255

Val Leu Asp Met Glu Ala lie Thr Phe Lys Lys Leu Val Lys Gly His 260 265 270

Ala Tyr Ser Val Thr Gly Ala Lys Gin Val Asn Tyr Arg Gly Gin Val 275 280 ^■ 285

Val Ser Leu lie Arg Met Arg Asn Pro Trp Gly Glu Val Glu Trp Thr 290 295 300

Gly Ala Trp Ser Asp Ser Ser Ser Glu Trp Asn Asn Val Asp Pro Tyr 305 310 315 320

Glu Arg Asp Gin Leu Arg _.Val Lys Met Glu Asp Gly Glu Phe Trp Met 325 330 335

Ser Phe Arg Asp Phe Met Arg Glu Phe Thr Arg Leu Glu lie Cys Asn 340 345 350

Leu Thr Pro Asp Ala Leu Lys Ser Arg Thr lie Arg Lys Trp Asn Thr 355 360. 365

Thr Leu Tyr Glu Gly Thr Trp Arg Arg Gly Ser Thr Ala Gly Gly Cys 370 375 380

Arg Asn Tyr Pro Ala Thr Phe Trp Val Asn Pro Gin Phe Lys lie Arg 385 390 395 400 Leu Asp Glu Thr Asp Asp Pro Asp Asp Tyr Gly Asp Arg Glu Ser Gly 405 410 415

5 Cys Ser Phe Val Leu Ala Leu Met Gin Lys His Arg Arg Arg Glu Arg 420 425 430

Arg Phe Gly Arg Asp Met Glu Thr lie Gly Phe Ala Val Tyr Glu Val 435 440 445

10 Pro Pro Glu Leu Val Gly Gin Pro Ala Val His Leu Lys Arg Asp Phe 450 455 ^'460

Phe Leu Ala Asn Ala Ser Arg Ala Arg Ser Glu Gin Phe lie Asn Leu 15465 470 475 480

Arg Glu Val Ser Thr Arg Phe Arg Leu Pro Pro Gly Glu Tyr Val Val 485 490 495

20 Val Pro Ser Thr Phe Glu Pro Asn Lys Glu Gly Asp Phe Val Leu Arg 500 505 510

Phe Phe Ser Glu Lys Ser Ala Gly Thr Val Glu Leu Asp Asp Gin lie 515 520 ^' 525

25 Gin Ala Asn Leu Pro Asp Glu Gin Val Leu Ser Glu Glu Glu lie Asp 530 535 540

Glu Asn Phe Lys Ala Leu Phe Arg Gin Leu Ala Gly Glu Asp Met Glu 30545 550 555 560 lie Ser Val Lys Glu Leu Arg Thr lie Leu Asn Arg lie lie Ser Lys 565 570 575

His Lys Asp Leu Arg Thr Lys Gly Phe Ser Leu Glu Ser Cys Arg Ser 580 585 590

Met Val Asn Leu Met Asp Arg Asp Gly Asn Gly Lys Leu Gly Leu Val 595 600 605

Glu Phe Asn lie Leu Trp Asn Arg lie Arg Asn Tyr Leu Ser lie Phe 610 615 620

Arg Lys Phe Asp Leu Asp Lys Ser Gly Ser Met Ser Ala Tyr Glu Met 625 630 635 640

Arg Met Ala lie Glu Ser Ala Gly Phe Lys Leu Asn Lys Lys Leu Tyr 645 650 655

Glu Leu lie lie Thr Arg Tyr Ser Glu Pro Asp Leu Ala Val Asp Phe 660 665 670

Asp Asn Phe Val Cys Cys Leu Val Arg Leu Glu Thr Met Phe Arg Phe 675 680 685

Phe Lys Thr Leu Asp Thr Asp Leu Asp Gly Val Val Thr Phe Asp Leu 690 695 700

Phe Lys Trp Leu Gin Leu Thr Met Phe Ala 705 710

<210> 2 <211> 2940

<212> DNA

<213> homo sapiens

<400> 2 aaggagagag ggagggcgga gggcggaggg gcggcgggag gagggcgggg aggagcgctc 60 ttcctggttg ggccctgccc tgagctgcca ccgggaagcc agcctcaggg actgcagcga 120 cccccaaaca cccctccccc aggatgtcgg aggagatcat cacgccggtg tactgcactg 18.0 gggtgtcagc ccaagtgcag aagcagcggg ccagggagct gggcctgggc cgccatgaga 240 atgccatcaa gtacctgggc caggattatg agcagctgcg ggtgcgatgc ctgcagagtg 300 ggaccctctt ccgtgatgag gccttccccc cggtacccca gagcctgggt tacaaggacc 360 tgggtcccaa ttcctccaag acctatggca tcaagtggaa gcgtcccacg gaactgctgt 420 caaaccccca gttcattgtg gatggagcta cccgcacaga catctgccag^' ggagcactgg 480 gggactgctg gctcttggcg gccattgcct ccctcactct caacgacacc ctcctgcacc 540 gagtggttcc gcacggccag agcttccaga atggctatgc cggcatcttc catttccagc 600 tgtggcaatt tggggagtgg gtggacgtgg tcgtggatga cctgctgccc atcaaggacg 660 ggaagctagt gttcgtgcac tctgccgaag gcaacgagtt ctggagcgcc ctgcttgaga 720 aggcctatgc caaggtaaat ggcagctacg aggccctgtc agggggcagc acctcagagg 780 gctttgagga cttcacaggc ggggttaccg agtggtacga gttgcgcaag gctcccagtg 840 acctctacca gatcatcctc aaggcgctgg agcggggctc cctgctgggc tgctccatag 900 acatctccag cgttctagac atggaggcca tcactttcaa gaagttggtg aagggccatg 960 cctactctgt gaccggggcc aagcaggtga actaccgagg ccaggtggtg agcctgatcc. 1020 ggatgcggaa cccctggggc gaggtggagt ggacgggagc ctggagcgac agctcctcag 1080 agtggaacaa cgtggaccca tatgaacggg accagctccg ggtcaagatg gaggacgggg 1140 agttctggat gtcattccga gacttcatgc gggagttcac ccgcctggag atctgcaacc 1200 tcacacccga cgccctcaag agccggacca tccgcaaatg gaacaccaca ctctacgaag 1260 gcacctggcg gcgggggagc accgcggggg gctgccgaaa ctacccagcc accttctggg 1320 tgaaccctca gttcaagatc cggctggatg agacggatga cccggacgac tacggggacc 1380 gcgagtcagg ctgcagcttc gtgctcgccc ttatgcagaa gcaccgtcgc cgcgagcgcc 1440 gcttcggccg cgacatggag actattggct tcgcggtcta cgaggtccct ccggagctgg 1500 tgggccagcc ggccgtacac ttgaagcgtg acttcttcct ggccaatgcg tctcgggcgc 1560 gctcagagca gttcatcaac ctgcgagagg tcagcacccg cttccgcctg ccacccgggg 1620 agtatgtggt ggtgccctcc accttcgagc ccaacaagga gggcgacttc gtgctgcgct 1680 tcttctcaga gaagagtgct gggactgtgg agctggatga ccagatccag gccaatctcc 1740 ccgatgagca agtgctctca gaagaggaga ttgacgagaa cttcaaggcc ctcttcaggc 1800 agctggcagg ggaggacatg gagatcagcg tgaaggagtt gcggacaatc ctcaatagga 1860 tcatcagcaa acacaaagac ctgcggacca agggcttcag cctagagtcg tgccgcagca 1920 tggtgaacct catggatcgt gatggcaatg ggaagctggg cctggtggag ttcaacatcc 1980 tgtggaaccg catccggaat tacctgtcca tcttccggaa gtttgacctg gacaagtcgg 2040 gcagcatgag tgcctacgag atgcggatgg ccattgagtc ggcaggcttc aagctcaaca 2100 agaagctgta cgagctcatc atcacccgct actcggagcc cgacctggcg gtcgactttg 2160 acaatttcgt ttgctgcctg gtgcggctag agaccatgtt ccgatttttc aaaactctgg 2220 acacagatct ggatggagtt gtgacctttg acttgtttaa gtggttgcag ctgaccatgt 2280 ttgcatgagg cagggactcg gtcccccttg ccgtgctccc ctccctcctc gtctgccaag 2340 cctcgcctcc taccacacca caccaggcca ccccagctgc aagtgccttc cttggagcag 2400 agaggcagcc tcgtcctcct gtcccctctc ctcccagcca ccatcgttca tctgctccgg 2460 gcagaactgt gtggcccctg cctgtgccag ccatgggctc gggatggact ccctgggccc 2520 cacccattgc caagccagga aggcagcttt cgcttgttcc tgcctcggga cagccccggg 2580 tttccccagc atcctgatgt gtcccctctc cccacttcag aggccaccca ctcagcacca 2-640 ccggcctggc cttgcctgca gactataaac tataaccact agctcgacac agtctgcagt 2700 ccaggcgtgt ggagccgcct cccggctcgg ggaggccccg gggctgggaa cgcctgtgcc 2760 ttcctgcgcc gaagccaacg ccccctctgt ccttccctgg ccctgctgcc gaccaggagc 2820 tgcccagcct gtgggcggtc ggccttccct ccttcgctcc ttttttatat tagtgatttt 2880 aaaggggact cttcagggac ttgtgtactg gttatggggg tgccagaggc actaggcttg 2940 gggtggggag gtcccgtgtt ccatatagag gaaccccaaa taataaaagg ccccacatct 3000 gtctgtg - 3007

<210> 3 <211> 700 <212> PRT <213> homo sapiens

<400> 3

Met Ala Gly lie Ala Ala Lys Leu Ala Lys Asp Arg Glu Ala Ala Glu 1 5 10 15

Gly Leu Gly Ser His Glu Arg Ala lie Lys Tyr Leu Asn Gin Asp Tyr 20 25 30

Glu Ala Leu Arg Asn Glu Cys Leu Glu Ala Gly Thr Leu Phe Gin Asp 35 40 45

Pro Ser Phe Pro Ala lie Pro Ser Ala Leu Gly Phe Lys Glu Leu Gly 50 55 60 Pro Tyr Ser Ser Lys Thr Arg Gly Met Arg Trp Lys Arg Pro Thr Glu 65 70 75 80

lie Cys Ala Asp Pro Gin Phe lie lie Gly Gly Ala Thr Arg Thr Asp 85 90 95

lie Cys Gin Gly Ala Leu Gly Asp Cys Trp Leu Leu Ala Ala lie Ala 100 105 110

Ser Leu Thr Leu Asn Glu Glu lie Leu Ala Arg Val Val Pro Leu Asn 115 120 125

Gin Ser Phe Gin Glu Asn Tyr Ala Gly lie Phe His Phe Gin Phe Trp 130 135 140

Gin Tyr Gly Glu Trp Val Glu Val Val Val Asp Asp Arg Leu Pro Thr 145 150 155 160

Lys Asp Gly Glu Leu Leu Phe Val His Ser Ala Glu Gly Ser Glu Phe 165 170 175

Trp Ser Ala Leu Leu Glu Lys Ala Tyr Ala Lys lie Asn Gly Cys Tyr 180 185 190

Glu Ala Leu Ser Gly Gly Ala Thr Thr Glu Gly Phe Glu Asp Phe Thr 195 200 205

Gly Gly lie Ala Glu Trp Tyr Glu Leu Lys Lys Pro Pro Pro Asn Leu 210 215 220

Phe Lys lie lie Gin Lys Ala Leu Gin Lys Gly Ser Leu Leu Gly Cys 225 230 235 240 Ser lie Asp lie Thr Ser Ala Ala Asp Ser Glu Ala lie Thr Phe Gin 245 250 255

Lys Leu Val Lys Gly His Ala Tyr Ser Val Thr Gly Ala Glu Glu Val 260 265 270

Glu Ser Asn Gly Ser Leu Gin Lys Leu lie Arg lie Arg Asn Pro Trp 275 280 285

Gly Glu Val Glu Trp Thr Gly Arg Trp Asn Asp Asn Cys Pro Ser Trp 290 295 300

Asn Thr lie Asp Pro Glu Glu Arg Glu Arg Leu Thr Arg Arg His Glu 305 310 315 320

Asp Gly Glu Phe Trp Met Ser Phe Ser Asp Phe Leu Arg His Tyr Ser 325 330 335

Arg Leu Glu lie Cys Asn Leu Thr Pro Asp Thr Leu Thr Ser Asp Thr 340 345 350

Tyr Lys Lys Trp Lys Leu Thr Lys Met Asp Gly Asn Trp Arg Arg Gly 355 360 365

Ser Thr Ala Gly Gly Cys Arg Asn Tyr Pro Asn Thr Phe Trp Met Asn ^" 370 375 380

Pro Gin Tyr Leu lie Lys Leu Glu Glu Glu Asp Glu Asp Glu Glu Asp

385 390 395 ^' 400

Gly Glu Ser Gly Cys Thr Phe Leu Val Gly Leu lie Gin Lys His Arg 405 410 415 Arg Arg Gin Arg Lys Met Gly Glu Asp Met His Thr lie Gly Phe Gly 420 425 430

lie Tyr Glu Val Pro Glu Glu Leu Ser Gly Gin Thr Asn lie His Leu 435 440 445

Ser Lys Asn Phe Phe Leu Thr Asn Arg Ala Arg Glu Arg Ser Asp Thr 450 455 460

Phe lie Asn Leu Arg Glu Val Leu Asn Arg Phe Lys Leu Pro Pro Gly 465 . 470 475 480

Glu Tyr lie Leu Val Pro Ser Thr Phe Glu Pro Asn Lys Asp Gly Asp 485 490 495

Phe Cys lie Arg Nal Phe Ser Glu Lys Lys Ala Asp Tyr Gin Ala Val 500 505 510

Asp Asp Glu lie Glu Ala Asn Leu Glu Glu Phe Asp lie Ser Glu Asp 515 520 525

Asp lie Asp Asp Gly Val Arg Arg Leu Phe Ala Gin Leu Ala Gly Glu 530 ^' 535 540

Asp Ala Glu lie Ser Ala Phe Glu Leu Gin Thr lie Leu Arg Arg Val 545 550 555 560

Leu Ala Lys Arg Gin Asp lie Lys Ser Asp Gly Phe Ser lie Glu Thr 565 570 575

Cys Lys lie Met Val Asp Met Leu Asp Ser Asp Gly Ser Gly Lys Leu 580 585 590 Gly Leu Lys Glu Phe Tyr lie Leu Trp Thr- Lys lie Gin Lys Tyr Gin 595 600 605

Lys lie Tyr Arg Glu lie Asp Val Asp Arg Ser Gly Thr Met Asn Ser 610 615- 620

Tyr Glu Met Arg Lys Ala_j Leu Glu Glu Ala Gly Phe Lys Met Pro Cys 625 630 635 640

Gin Leu His Gin Val lie Val Ala Arg Phe Ala Asp Asp Gin Leu lie 645 650 655

lie Asp Phe Asp Asn Phe Val Arg Cys Leu Val Arg Leu Glu Thr Leu 660^' 665 670

Phe Lys lie Phe Lys Gin Leu Asp Pro Glu Asn ,Thr Gly Thr lie Glu 675 680 685

Leu Asp Leu lie Ser Trp Leu Cys Phe Ser Val Leu 690 695 700

<210> 4 <211> 3419 <212> DNA <213> homo sapiens

<400> 4 ttccctccgg tgaatcatcg ctcgcagcgg cggcgcccgc agtggccgca gcagcgcgcc 60 gggccctggc cgcgccccag ccgagcgcag cgcggagtcg ccccgacctt tctctgcgca 120 gtacggccgc cgggaccgca gcatggcggg catcgcggcc aagctggcga aggaccggga 180 ggcggccgag gggctgggct cccacgagag ggccatcaag tacctcaacc aggactacga 240 ggcgctgcgg aacgagtgcc tggaggccgg gacgctcttc caggacccgt ccttcccggc 300 catcccctcg gccctgggct tcaaggagtt ggggccctac tccagcaaaa cccggggcat 360 gagatggaag cgccccacgg agatctgcgc tgacccccag tttatcattg gaggagccac 420 ccgcacagac atctgccaag gagccctagg tgactgctgg ctgctggcag ccattgcctc 480 cctcaccttg aatgaagaaa tcctggctcg agtcgtcccc ctaaaccaga gcttccagga 540 aaactatgca gggatctttc acttccagtt ctggcaatac ggcgagtggg tggaggtggt 600 ggtggatgac aggctgccca ccaaggacgg ggagctgctc tttgtgcatt cagccgaagg 660 gagcgagttc tggagcgccc tgctggagaa ggcatacgcc aagatcaacg gatgctatga 720 agctctatca gggggtgcca ccactgaggg cttcgaagac ttcaccggag gcattgctga 780 gtggtatgag ttgaagaagc cccctcccaa cctgttcaag atcatccaga aagctctgca 840 aaaaggctct ctccttggct gctccatcga catcaccagc gccgcggact cggaggccat 900 cacgtttcag aagctggtga aggggcacgc gtactcggtc accggagccg aggaggttga 960 aagtaacgga agcctacaga aactgatccg catccgaaat ccctggggag aagtggagtg 1020 gacagggcgg tggaatgaca actgcccaag ctggaacact atagacccag aggagaggga 1080 aaggctgacc agacggcatg aagatggaga attctggatg tctttcagtg acttcctgag 1140 gcactattcc cgcctggaga tctgtaacct gaccccagac actctcacca gcgataccta 1200 caagaagtgg aaactcacca aaatggatgg gaactggagg cggggctcca ccgcgggagg 1260 ttgcaggaac tacccgaaca cattctggat gaaccctcag tacctgatca agctggagga 1320 ggaggatgag gacgaggagg atggggagag cggctgcacc ttcctggtgg ggctcattca 1380 gaagcaccga cggcggcaga ggaagatggg cgaggacatg cacaccatcg gctttggcat 1440 ctatgaggtt ccagaggagt taagtgggca gaccaacatc cacctcagca aaaacttctt 1500 cctgacgaat cgcgccaggg agcgctcaga caccttcatc aacctccggg aggtgctcaa 1560 ccgcttcaag ctgccgccag gagagtacat tctcgtgcct tccaccttcg aacccaacaa 1620 ggatggggat ttctgcatcc gggtcttttc tgaaaagaaa gctgactacc aagctgtcga 1680 tgatgaaatc gaggccaatc ttga.agagtt cga.catcagc gaggatgaca ttgatgatgg 1740 agtcaggaga ctgtttgccc agttggcagg agaggatgcg^' gagatctctg cctttgagct 1800 gcagaccatc ctgagaaggg ttctagcaaa gcgccaagat atcaagtcag atggcttcag 1860 catcgagaca tgcaaaatta tggttgacat gctagattcg gacgggagtg gcaagctggg 1920 gctgaaggag ttctacattc tctggacgaa gattcaaaaa taccaaaaaa tttaccgaga 1980 aatcgacgtt gacaggtctg gtaccatgaa ttcctatgaa atgcggaagg cattagaaga 2040 agcaggtttc aagatgccct gtcaactcca ccaagtcatc gttgctcggt ttgcagatga 2100 ccagctcatc atcgattttg ataattttgt tcggtgtttg gttcggctgg aaacgctatt 2160 caagatattt aagcagctgg atcccgagaa tactggaaca atagagctcg accttatctc 2220 ttggctctgt ttctcagtac tttgaagtta taactaatct gcctgaagac ttctcatgat 2280 ggaaaatcag ccaaggacta agcttccata gaaatacact ttgtatctgg acctcaaaat 2340 tatgggaaca tttacttaaa cggatgatca tagctgaaaa taatgatact gtcaatttga 2400 gatagcagaa gtttcacaca tcaaagtaaa agatttgcat atcattatac taaatgcaaa 2460 tgagtcgctt aacccttgac aaggtcaaag aaagctttaa atctgtaaat agtatacact 2520 ttttactttt acacactttc ctgttcatag caatattaaa tcaggaaaaa aaaatgcagg 2580 gaggtattta acagctgagc aaaaacattg agtcgctctc aaaggacacg aggcccttgg 2640 cagggaatat ttaaagcaac ttcaagttta aaatgcagct gttgattcta ccaaacaaca 2700 gtccaagatt accatttccc atgagccaac tgggaaacat ggtatatcat gaagtaatct 2760 tgtcaaggca tctggagagt ccaggagagg agactcacct ctgtcgcttg ggttaaacaa 2820 gagacaggtt ttgtagaata ttgattggta atagtaaatc gttctcctta caatcaagtt 2880 cttgacccta ttcggcctta tacatctggt cttacaaaga ccaaagggat cctgcgcttg 2940 atcaactgaa ccagtatgcc aaaaccaggc atccaatttg taaaccaatt atgataaagg 3000 acaaaataag ctgtttgcca cctcaaaact ttatgaactt caccaccact agtgtctgtc 3060 catggagtta gaggggacat cacttagaag ttcttataga aaggacacaa gtttgtttcc 3120 tggctttacc ttgggaaaat gctagcaaca ttatagaaat tttgccttgt tgccttatct 3180 tcttccaaat gtactgttaa ataaaaataa agggttaccc catgcaatca caccatgcca 3240 tgttttcctt cctggagggc agccccacag gacggtttat- gagcacacaa ttatagcttg 3300 tttctacttt aacagggtat gctgcctctg taaattcatg tattcaaagg aaaagacacc 3360 ttgcctataa ttaaaatgtg gaactataaa attttttaaa atccaaaaaa aaaaaaaaa

3419

Claims

Claims

1. The use of calpain or functional fragments or derivatives thereof for the preparation of pharmaceutical compounds that modulate pain.

2. The use of calpain or functional fragments of derivatives thereof for identifying pharmaceutical compounds that modulate pain.

3. The use according to any of claims 1 or 2, wherein the pharmaceutical compounds are compounds for the prevention or treatment of pain.

4. The use according to claim 3, wherein the compounds lessen or abolish pain.

5. A method of screening pharmaceuticals useful for modulating and/or preventing pain, comprising the steps a. Providing two samples b. contacting one sample containing calpain or a functional fragment or derivative thereof with a compound, c. determining the calpain activity in the presence of compound, d. determining the calpain activity in the absence of compound, and e. comparing the calpain activity according to c) with that according to

6. The use according to one of the claims 1 to 4 or the method according to claim 5, wherein calpain is human calpain I and / or calpain II.

7. The use or the method according to claim 6, wherein calpain is an isolated polypeptide or polynucleotide.

8. The use or the method according to claim 7, wherein calpain is a polypeptide.

9. The use or the method according to claim 8, wherein calpain is a polypeptide or functional fragment thereof that comprises or consists of the sequence according to SEQ ID No 1 (calpain I) or a fragment thereof, the sequence according to SEQ ID No 3 (calpain II) or a fragment thereof, or is encoded by a polynucleotide comprising or consisting of the sequence according to SEQ ID No 2 (encoding calpain II) or a fragment thereof, or of the sequence according to SEQ ID No 4 (encoding calpain II) or a fragment thereof

10. The use or the method according to claim 7, wherein calpain is a polynucleotide.

11. The use or the method according to claim 10, wherein calpain is a polynucleotide comprising or consisting of the sequence or a part of the sequence coding for a functional fragment of calpain according to SEQ ID No 2 or SEQ ID No 4 or a polynucleotide comprising or consisting of a sequence that is able to hybridize with said polynucleotides under stringent conditions.

12. The use or the method according to one of the above claims, wherein the functional fragments comprise or consist of amino acids 115 to 272 of SEQ ID No 2, and / or 105 to 262 of SEQ ID No 4.

13. The method according to claim 5, wherein a cell expressing, preferably expressing recombinant calpain is used.

14. The method according to claim 5, wherein a modified cell, having a lower calpain activity as compared to its unmodified state, is used.

15. The method according to claim 14, wherein a calpain knock-out cell is used.

16. The method according to one of the claims 5, 13 or 14, wherein the calpain activity is determined directly.

17. The method according to one of the claims 5, 13 or 14, wherein the calpain activity is determined indirectly.

18. A method of identifying a compound that modulates pain comprising 5 a. Selecting a compound that modulates the activity of calpain as a test compound, and b. Administering said test compound to a subject to determine whether the pain is modulated.

10 19. The use of MDL-28170 (calpain inhibitor III) in the process of preparing a medicament for the treatment of pain.

20. The use of MDL-28170 as a medicament for the treatment of pain.

15 21. The use of MDL-102935 in the process of preparing a medicament for the treatment of pain.

22. The use of MDL-102935 as a medicament for the treatment of pain.