DE10331980A1 - Use of calpain to identify pain modulating compounds - Google Patents

Abstract

The invention relates to the use of calpain or functional fragments or derivatives thereof for the preparation of pain modulating pharmaceutical compounds and to the use of calpain or functional fragments or derivatives thereof for the identification of such compounds and a method for the screening of medicaments for pain modulation and / or prevention are suitable.

Description

The The invention relates to the use of calpain or functional Fragments or derivatives thereof for the production of pain modulating pharmaceutical compounds and the use of calpain or functional fragments or derivatives thereof for identification of such compounds and a method for the screening of medicaments, which are suitable for pain modulation and / or prevention.

The Effects of peripheral inflammatory Stimulation on the protein expression pattern in the spinal cord of the rat were examined. With the method of two-dimensional (2D) Gel electrophoresis was a time-dependent degradation of neurofilament light chain (NF-L) protein after 24, 48 or 96 h of treatment with zymosan, which was confirmed by immunohistochemical and Western Blot experiments.

Since the neurofilaments are substrates of the calcium-dependent cysteine protease calpain, the role of calpain in inflammatory and painful processes was further analyzed. It has been shown that peripheral inflammatory stimulation with zymosan leads to an upregulation of calpain activity and protein expression in the rat spinal cord. By inhibiting calpain protolysis with the specific calpain inhibitor III (MDL-28170, 12.5 mg / kg and 25 mg / kg ip), zymosan-induced neurofilament degradation was attenuated. Calpain protein expression was unaffected by MDL-28170. The chemical structure of calpain inhibitor III is as follows:

The Effects of MDL-28170 on inflammation and nociception were tested in two different animal models. In the model of zymosan-induced paw inflammation was compared with Control animals the paw edema significantly reduced by the calpain inhibitor III (25 mg / kg). in the thermal hyperalgesia model led the application of calpain inhibitor III to antinociceptive reactions. Taken together, these data suggest that calpain may be used in inflammatory and pain processes play and therefore play an important role a therapeutic potential for Calpain inhibitors as an alternative anti-inflammatory and analgesic Drugs on.

Die Wirkungen von MDL-102935 auf Entzündung und Nozizeption sind dieselben wie die von MDL-28170. Bei MDL-102935 handelt es sich um einen Calpain-Inhibitor.In addition, the calpain inhibitor MDL-102935 was tested as described for calpain inhibitor III. MDL-102935 has the following chemical formula:

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

In spinal nociceptive processing, various pain stimuli are switched from the periphery of the primary leads to the central nervous system in the dorsal horn of the lumbar cord. Long-term stimulation of primary nociceptors and C-fibers, as occurs in inflammatory stimulation, triggers an increased excitability of nociceptive neurons in the spinal cord, leading to hyperalgesia and allodynia (Yaksh et al., 1999). Long-term changes in synaptic communication (so-called long-term potentiation, LTP) are associated with learning and memory processes (Bliss et al., 1993) and may be responsible for synaptic plasticity as well as chronic pain (Rygh et al. 1999, Vikman et al., 2001). Repeated or extensive noxious stimulation activates a number of intracellular second messenger systems, indicating the phosphorylation of key receptor membrane receptors and channels by protein kinases, particularly protein kinase C (PKC). This kinase activation leads to post-translational changes that may increase synaptic efficacy. Intracellular signaling cascades lead to immediate early gene (IEG) induction (c-fos, Zif 268, Cox-2) and later response genes, which encode neuropeptides as well as neuropeptide and neurotrophin receptors. These protein alterations are considered to be the beginning of a profound change in protein synthesis and moreover as a general basis for the plasticity of the nervous system (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 In the following study, intercellular changes of the protein expression pattern, the peripheral inflammatory conditions in the lumbar region accompany the rat, using two-dimensional gel electrophoresis (2D PAGE) in combination with MALDI-TOF MS. With this Procedures were several protein changes demonstrated in the rat model of zymosan-induced pawing. This work focused on one of these proteins, called as Neurofilament light chain (NF-L) was identified. NF-L is a cytoskeletal protein found in neuronal cells in which it for the correct assembly of neurofilaments as well as the maintenance Axon inside diameter (Sakaguchi et al., 1993). The disorganization of neurofilaments becomes different neurodegenerative diseases such as Parkinson's and amyotrophic Lateral sclerosis, related (Julien, 1999). moreover are neuronal death and neurological dysfunction after spinal cord injuries of Loss of neurofilament protein (Ray et al., 2000a). Of the Degradation of neurofilament proteins is mainly due to the action of Protease calpain (Ray et al., 2000b, Schlaepfer et al. , 1985; Stys et al., 2002). The Calpainen is one Family ubiquitous expressed calcium-dependent cysteine proteases, those with basic cellular processes, including Propagation, apoptosis and differentiation, associated were. Many calpain substrates are in the pre- and postsynaptic compartments localized by neurons, which is increasingly due to the involvement of Calpainene in neurodegenerative diseases and modulation synaptic plasticity indicates (Chan of al., 1999). The calpain of the rat is by Thompson and Goll (Meth. Mol. Biol., Vol. 144, 3-16, 2000). Two human Calpain isoforms are known, namely calpain I (embl locus HSCANPR, access NR. X04366.1) and calpain II (Morford et al., Biochem. Biophys. Res. Commun. 295 (2), 540-546, 2002).

In In the following work, the role of calpain in zymosan-induced Degradation of neurofilaments as well as the potential anti-inflammatory and antinociceptive properties of a specific calpain inhibitor (MDL 28170) in the generally introduced Model of zymosan-induced pawing investigated.

Surprisingly it has now been established that calpain plays a role related to pain.

A inventive embodiment consists in the use of calpain or functional fragments or derivatives thereof for the manufacture of pain modulating pharmaceutical Links.

A another embodiment of the invention consists in the use of calpain or functional fragments or derivatives thereof for identifying pain modulating compounds.

A another embodiment of the invention consists in the use as described above, wherein it is in the pharmaceutical compounds to compounds for pain prevention or treatment, and in particular the compounds Relieve or eliminate pain.

• a. zwei Proben bereitgestellt werden,
• b. eine Probe, die Calpain oder ein funktionelles Fragment oder Derivat davon enthält, mit einer Verbindung in Kontakt gebracht,
• c. die Calpainaktivität in Gegenwart der Verbindung bestimmt,
• d. die Calpainaktivität in Abwesenheit der Verbindung bestimmt und
• e. die Calpainaktivität gemäß (c) mit der gemäß (d) verglichen wird.

Another embodiment of the invention is a method for screening drugs suitable for pain modulation and / or prevention in which

• a. two samples are provided
• b. a sample containing calpain or a functional fragment or derivative thereof, contacted with a compound,
• c. determines the calpain activity in the presence of the compound,
• d. determines the calpain activity in the absence of the compound and
• e. the calpain activity according to (c) is compared with that according to (d).

A another embodiment of the invention consists in a use of the method described above, wherein Calpain is human calpain I and / or calpain II, in particular, wherein the calpain is an isolated Polypeptide or polynucleotide is.

A another embodiment of the invention consists of a use as described above or a like method described above, wherein the calpain is a Polypeptide, in particular, wherein it is the calpain is a polypeptide or functional fragment thereof, the 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 consists thereof, or wherein the calpain is derived from a polynucleotide comprising the sequence according to SEQ ID No 2 (encodes calpain II) or a fragment thereof or the sequence according to sequence according to SEQ ID No 4 (coded calpain II) or contains a fragment thereof or consists of is encoded.

A another embodiment of the invention consists of a use as described above or a like method described above, wherein the calpain is a Polynucleotide, in particular, wherein it is the calpain a polynucleotide containing the sequence or part of the sequence, the for a functional fragment of calpain according to SEQ ID No 2 or SEQ ID No 4 contains or a polynucleotide containing a sequence, which hybridize with the polynucleotides under stringent conditions can, contains or consists thereof, preferably the functional fragments the amino acids 115 to 272 of SEQ ID No 2 and / or 105 to 262 of SEQ ID No 4 contain or consist of.

A another embodiment of the invention consists of a method as described above, wherein a calpain expressing, preferably recombinant calpain expressing Cell is used.

A another embodiment of the invention consists of a method as described above, wherein a modified Cell, which compared to its unmodified state a lower calpain activity which is preferably a calpain knockout cell is used.

A another embodiment of the invention consists of a method as described above, wherein the calpain activity is direct or determined indirectly.

• a. eine Verbindung, die die Aktivität von Calpain moduliert, als Testverbindung ausgewählt und
• b. diese Testverbindung an eine Versuchsperson verabreicht wird, um zu bestimmen, ob die Schmerzen moduliert werden.

Another embodiment of the invention consists of a method of identifying a pain modulating compound in which

• a. a compound modulating the activity of calpain is selected as a test compound and
• b. This test compound is administered to a subject to determine if the pain is modulated.

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

A another embodiment of the invention consists of the use of MDL-28170 as a drug for the treatment of pain.

A another embodiment of the invention consists of the use of MDL-102935 in the manufacturing process a drug for pain treatment.

A another embodiment of the invention consists of the use of MDL-102935 as a drug for the treatment of pain.

The Invention is described below by the examples, by the invention in no way to the specific embodiments limited to the examples should be.

Examples

Used in the examples Materials and methods

animals

male Weighing Sprague-Dawley rats (Charles River, Sulzbach) from 260-300 g were in groups of five Animals in standard cages in rooms with climate and light control (22 ± 0.5 ° C, 12/12 h dark / light cycle) with free access to food and water. In all experiments became the ethical guidelines for animal studies conscious and procedures approved by the local Ethics Committee.

Zymosanbehandlung of the rats

A inflammation the hindpaw and a paw edema were each unilaterally by subcutaneous injection of 1.25 mg Zymosan (Sigma, Munich), suspended in 100 μl phosphate buffered saline (12.5 mg / ml), in the middle area of the sole of the right hind paw induced. After 24, 48 or 96 h, the animals were anesthetized and killed by heart puncture. The spinal cord each was excised, snap frozen in liquid nitrogen and then stored at -80 ° C until further analysis.

paw edema

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

thermal hyperalgesia

The nociceptive latency of withdrawal the paw opposite Radical heat was evaluated according to Hargreaves (Hargreaves et al., 1988), wherein a special, commercially available Device (Ugo Basile, Varese, Italy). The rats were in transparent Plastic chambers (18 × 29 × 12.5 cm) on a metal grid (5 × 5 mm). The heat source consisting of a strong projector bulb was on the surface the sole of the foot the hind paw is focused. At the beginning of the test were the bulb and a electronic timepiece activated simultaneously, and both were automatically inactivated when retracting the hind paw as Reaction to the heat was detected by a photocell. The latencies of withdrawal (paw withdrawal latencies, (PWL)) of the right and left paw (treated or untreated paw) were in each case before and then every 60 min recorded up to 8 h and 24 h after zymosan injection.

drug treatment

Calpain inhibitor III (MDL 28170) (Calbiochem, Schwalbach / Ts.) Was in one concentration of 10 mg / ml dissolved in PEG 400 / DMSO (1: 1). The animals received calpain inhibitor III in the form of an intraperitoneal (i.p.) bolus injection of either 12.5 mg / kg or 25 mg / kg 20 min before the intraplantar injection with zymosan. For Each treatment group used six animals (controls received 1 ml of vehicle.). The animals were randomly assigned to the treatments, the observers were unaware of this assignment.

data analysis and statistics

The relative percentage difference in paw retraction latency (ΔPWL) between the zymosan-treated right and untreated left hind paw, calculated as: ΔPWL = (right-left) / left × 100, was used to evaluate antinociceptive effects. To assess the anti-inflammatory effects, the relative difference between the paw volume before and after zymosan injection (ΔPV) was used. The areas below the ΔPWL time curves (AUC _ΔPWL ) as well as the areas below the ΔPV time curves (AUC _ΔPV ) were calculated using the linear trapezoid _rule . Statistical readings were taken with SPSS 9.02 for Windows. The effects of the medications performed in the work were evaluated by taking the AUC _ΔPV value of 0-8 h of a univari Analysis of variance (ANOVA) with subsequent T tests using Bonferroni ⎕ correction for multiple comparisons. The statistical significance of ΔPWL was calculated using unpaired Student's T-test (zymosan-treated rats versus control rats). ⎕ was set to 0.05.

2D-PAGE

The Lumbar cord tissues were incubated in lysis buffer with 9 M urea, 2% CHAPS, 1 DTT and 2 mM Pefabloc homogenized. After removing the cell debris were the extracts for 1 h at 40,000 rpm ultracentrifuged (15 ° C), and the supernatant was stored at -70 ° C until further analysis. The protein concentrations were determined with the Bradford protein assay.

The 2D PAGE was done with slight modifications as previously described carried out (Gorg et al., 1995). Shortly: for the first dimension (isoelectric focusing (IEF)) was 600 μg of protein precipitated with 50% TCA at -20 ° C. The Pellet was in rehydration solution (8M urea, 2% CHAPS, 2.8% DTT and 0.5% IPG buffer (Amersham Biosciences, Freiburg) and 13 cm long Immobiline DryStrips, pH 3-10 linear (Amersham Biosciences, Freiburg). The IEF was determined using the isoelectric focusing system IPGphor performed by Amersham, 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 placed in equilibration buffer for 10 min (1.5 M Tris-Cl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) Add 10% DTT and then 10 more minutes in equilibration buffer equilibrated with 260 mM iodoacetamide. The IPG strips were then placed on top of a 13% sodium dodecyl sulfate polyacrylamide gel and with 1% agarose solution sealed. Electrophoresis in the second dimension became 1 h at 2.5 W / gel and then 4 h at 5 W / gel performed. The Gels were stained with Coomassie Brilliant Blue. The colored gels were imaged with a UMAX PowerLookIII scanner and with the Software Image Master 2D analyzed (Amersham Biosciences, Freiburg). The Coomassie stained desired spots were cut out of the gels, the proteins were digested and then by use of peptide mass fingerprinting the matrix-assisted Laser Desorption / Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) identified.

Immunoblot analysis

The from homogenized spinal cord tissues extracted equivalents Quantities of total cell proteins were assayed with 4x sample buffer (1x sample buffer consists of 2.5% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 6.8), 2.5% 2-mercaptoethanol, 10% glycerol and a trace of bromophenol blue). The samples were 5 min cooked and the proteins (30 ug per lane) of SDS-PAGE with 10% polyacrylamide gels. The proteins were then transferred to nitrocellulose membranes (Pall Corporation). Blots were made in PBS / 5% skimmed milk / 0.3% Tween 20 over Night at 4 ° C blocked. Then they were treated with primary antibody against neurofilament-L (1: 500) (Sigma) for Incubated for 90 min at RT and then three times with PBS / 0.3% Tween 20 washed and then with an alkaline horseradish peroxidase for 60 min (HRP) labeled secondary antibody (1: 20000) (Santa Cruz Biotechnology). After three times Washing with PBS / 0.3% Tween 20 were associated with specific protein bands the Enhanced Chemiluminescence (ECL) system (Santa Cruz Biotechnology). The ECL films were used with a Umax PowerLook III scanner the software Photoshop (Adobe Systems) and the intensity densitometrometrically determined with the software Quantity One (PDI).

immunohistochemical coloring

For the immunohistochemical coloring Lumbar segments were excised, in liquid nitrogen Shock frozen and stored at -80 ° C. Fresh frozen tissue sections (14 μm) were placed in a cryostat cut, applied to gelatin-coated slides and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min. The slides were washed three times for 10 min each in PBS and 15 min with PBS containing 0.1 % Triton-X-100 treated. The sections were then in 3% BSA in PBS blocks for 1 h to reduce nonspecific binding and then with primary 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 incubated at 37 ° C with Cy3-conjugated for one hour secondary antibody (1: 600, Sigma) fluorescently labeled. The slides were after each incubation three times for each washed for 10 min in PBS. The incubations with primary and secondary antibody were performed in PBS with 1% BSA. The slides were with SlowFade Light anti-fade mounting media according to the manufacturer's instructions prepared.

determination calpain activity

The calpain activity was analyzed with a commercially available calpain activity assay kit (Biocat, Heidelberg) according to the manufacturer's instructions. The assay is based on the fluorometric detection of cleavage of the calpain substrate Ac-LLY-AFC which emits blue light (λ _max = 400 nm). After cleavage of the substrate by calpain, the free AFC radiates with a yellow-green fluorescence (λ _max = 505 nm), which was quantified with a fluorometer (Spectra Fluor Plus, Tecan).

Example 1: Chronic inflammation leads to changes with different proteins

To the Evidence of inflammation Reactive proteins became the zymosan-induced model of inflammation in rats with subsequent 2D PAGE of the rat's lumbar spine used. The protein expression patterns of the spinal cord lysate Control was with spinal cord proteins of rats after a zymosan treatment of 24, 48 and 96 h, respectively. More than 500 protein spots were separated on each gel, with the apparent molecular weights in a range of 10 to 100 kDa and the pl values ranged from 3 to 10, as determined by Coomassie Blue staining was detected. The zymosan treatment resulted in the modification of at least 10 protein spots. The effects were most pronounced 96 h after Zymosaninjektion.

Example 2: NF-L becomes after zymosan treatment in the spinal cord the rat is time-dependent downregulated.

Further work in this work focused on Spot 1, identified by MALDI-TOF mass spectrometry as a 61.3 kDa neurofilament light chain (NF-L) and a pS at 4.63 ( 1A ). The 2D-PAGE revealed that the protein expression of NF-L was time-dependent runteneguliert after 24 and 48 h. As the Coomassie staining shows ( 1A ), the protein spot had completely disappeared.

To demonstrate the degradation of NF-L protein expression in the spinal cord following inflammatory stimulation, Western blot analysis was also performed with an anti-NF-L monoclonal antibody that did not cross-react with other intermediate filament proteins. As in 1B demonstrated by Western blot analysis, the NF-L protein was degraded due to the increasing duration of peripheral inflammation.

The densitometric analyzes of the NF-L protein bands in the Western Blot analysis are in 1C shown.

Example 3: Inflammatory Stimulation leads an increase in calpain activity.

It is well known that neurofilament degradation occurs mainly through the activity of the calcium-dependent cysteine protease calpain. Therefore, the effects of peripheral zymosan treatment on calpain activity in the effects of peripheral zymosan treatment on calpain activity in the spinal cord were examined. 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 48 h and significantly after 96 h (p <0.05) ( 2A ). Calpain inhibitor III (carbobenzoxy-valinyl-phenylalaninal, MDL 28170), which is described as a potent, selective and cell-permeable inhibitor of calpain I and II, has been described as an enzyme inhibitor (Chatterjee et al., 1998, Rami et al., 1997). Treatment of rats with calpain inhibitor III (ip) (25 mg / kg) markedly inhibited zymosan-induced protease activation in the spinal cord ( 2 B ).

Example 4: Immunohistochemistry

The Immunohistochemistry was used to assess the protein levels of Neurofilament-L and calpain in spinal cord incisions the rat after zymosan treatment with and without additional Administration of calpain inhibitor III.

The sections treated with a specific NF-L antibody showed a reduced immunofluorescence intensity after 96 h of zymosan treatment, which indicates the degradation of neurofilament. This effect was reversed by the addition of calpain inhibitor III. Treated with the protease inhibitor Sections showed immunofluorescence levels comparable to control sections of untreated rats ( 3A ).

To assess calpain protein expression, an antibody to M-calpain was used. After zymosan treatment (96 h), M-calpain expression was slightly elevated in spinal cord sections. This increase in immunoreactivity was not changed by the use of calpain inhibitor III ( 3B ).

Example 5: Effects a calpain inhibitor in the zymosan-induced inflammatory model in rats

Since calpain activity is up-regulated by zymosan-induced inflammation, potentially causing neurofilament degradation, it has been hypothesized that the specific calpain inhibitor could provide anti-inflammatory properties. This hypothesis was again tested in the model of zymosan-induced paw inflammation in rats. In vehicle-treated rats, intraplantar injection of 1.25 mg zymosan resulted in a maximum increase in paw volume of 129 ± 5.9% (mean ± sem [standard deviation]) at 4 h. As hypothesized, paw inflammation was inhibited dose-dependently by calpain inhibitor III in doses of 12.5 and 25 mg / kg ( 4A ). A statistical comparison of the area under the "paw volume increase" vs. "time" curves (AUC _ΔPV of 0-8 h) showed statistically significant differences between the control rats and the rats treated with 25 mg / kg calpain inhibitor (p <0.001). The results of the post hoc analysis are in 4B shown.

Example 6: Effects of calpain inhibitor III in zymosan-induced thermal Hyperalgesia in rats

To evaluate the effect of calpain inhibition on hyperalgesia, the latency of retraction of the paw in the thermal hyperalgesia model was determined. The results in 5A suggest that rats treated with calpain inhibitor III showed significantly higher latency of paw retraction compared to zymosan-treated rats, suggesting that the rats treated with the inhibitor experienced less pain. The results of the post hoc analysis are in 5B shown.

discussion of the results the examples:

The Results of the present work showed by means of 2D gel electrophoresis, that the Neurofilament light chain degraded after peripheral inflammatory stimulation becomes. The neurofilaments represent an important group of cytoskeletal proteins The control of the axon inner diameter and the Axonaufbaus and the axoplasmic flow (Perrone Capano et al., 2001). Neurofilament anomalies trigger selective degradation and Death of motor neurons and occur in neurodegenerative diseases on (Julien, 1999). The neurofilament degradation was in spinal cord injuries as well as under anoxic and ischemic conditions by activation the calcium dependent Cysteine protease calpain (Banik et al., 1997, Leski et al., 2001; Stys et al., 2002).

As in 6 In particular, the excitatory amino acid glutamate and the neuropeptide substance P lead to the activation of voltage-gated calcium channels as well as ionotropic and metabotropic receptors, thereby causing an influx of calcium from voltage-sensitive ion channels and also cause a release of calcium from intracellular stores. The resulting accumulation of calcium induces activation of calpain as well as upregulation and activation of several other proteins in the spinal cord, eg nNOS, Ca ²⁺ calmodulin kinase (Shields et al., 1998) and adenylyl cyclase. The increase in calpain activity and expression observed here can also be linked to the production of cytokines and the activation of the arachidic acid cascade that occurs during inflammatory processes in astrocytes and inflammatory immune cells. In vitro studies have also shown increased synthesis and activity of calpain in glial cells, neuronal and lymphoid cells in response to stress (Ray et al., 2003).

There are currently two major isoforms of calpain in the central nervous system, μ-calpain (calpain I) and m-calpain (calpain II), which are activated by micromolar and millimolar calcium, respectively. They have been implicated in a variety of physiological and pathological conditions, including neuronal plasticity and neuronal cell death. With regard to this neuronal cell death, activation of calpain results in the protolysis of several cellular proteins that are largely associated with the cell 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, phosphatases and phospholipases) as well as cytokines and transcription factors (Kampfl et al., 1997, Shields et al., 1998). Although many of these calpain effects may be associated with inflammatory mechanisms, the exact role of calpain activation in inflamed tissue remains elucidated.

It indicates that the Activation of calpain leads to the degradation of IκB in the proteasome and therefore a significant step in the translocation of the nuclear factor κB (NF-κB) from the Cytosol leads in the cell nuclei (Chen et al., 2000; Saido et al., 1994). It could be shown, that the Calpain inhibitor I this NF-kB activation and the subsequent Upregulation of iNOS and COX-2 protein prevented, causing the Development of acute and chronic inflammation is reduced (Cuzzocrea et al., 2000). The anti-inflammatory Indomethacin, which has the cyclooxygenase activity and the Inhibiting upregulation of prostaglandin, additionally showed calpain-inhibiting activity (Banik et al., 2000). About that In addition, new results on increased calpain activity in the ischemia, Alzheimer's disease, spinal cord injuries as well as brain trauma, the calpain mediated protolysis with the Destruction and tissue degradation in CNS trauma and disease (Banik et al., 1997; Shields et al., 1998). The uncontrolled Calpain activation leads to degrade cytoskeletal proteins, the consequent loss the structural integrity and to disturbances of axonal transport.

The Results over an increased calpain in diseases suggest that calpain inhibitors can be used as therapeutics, as they through the tissue degradation Minimize prevention of degradation of substrate proteins. In the present work was the Calpain Inhibitor III (MDL 28170), a potent, specific calpain inhibitor used after Systemic administration rapidly penetrates the blood-brain barrier. By intraperitoneal application of this inhibitor was the degradation of neurofilament protein in the spinal cord blocked, indicating the inhibition of protease activity, however was not due to a down-regulation of calpain protein. additional Effects of calpain inhibition on other calpain substrates also possible, however, were not investigated in the following work.

It could be shown for the first time that pretreatment of rats with calpain inhibitor III the development of zymosan-induced paw inflammation and attenuates thermal hyperalgesia in rats. As conclusion is from We suggested that it Calpain is an important mediator in nociceptive Reactions and that the Calpain inhibitor III may be for the treatment of inflammatory diseases suitable is.

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Description of the pictures

1 : Treatment with zymosan causes a time-dependent decrease in neurofilament-L.
(A) Enlarged areas of the 2D gels of rat spinal cord tissue from the control and after 24, 48 and 96 hours of zymosan treatment. The arrows mark protein 1, which has been identified as neurofilament light chain.
(B) Representative Western blot analysis of neurofilament light chain (NF-L) expression after 24, 48 and 96 h zymosan treatment.
(C) Densitometric analysis of 3 independent Western blot experiments (** statistically significant mean difference, p <0.01).

2 : Calpain activity is increased after zymosan treatment and is reduced by calpain inhibitor III.
(A): Calpain activity of rat spinal cord extracts of the control and after 24, 48 and 96 hours of zymosan treatment.
(B): Calpain activity of the spinal cord extracts of zymosan-treated control rats and of animals receiving 12.5 mg / kg or 25 mg / kg calpain inhibitor III.
Activity is determined by fluorometric detection of cleavage of the calpain substrate Ac-LLY-AFC.
(*, ** statistically significant mean difference p <0.01 or p <0.05)

3 : Immunohistochemical labeling of lumbar sections with Cy-3. The cross-sections were taken from control animals as well as from animals 96 h after injection with zymosan with and without pretreatment with calpain inhibitor III. The pictures represent the spine of the spinal cord.
(A): Immunohistochemically stained sections with monoclonal anti-NF-L antibody. Calpain inhibitor III reduces the loss of NF-L after zymosan treatment.
(B): immunohistochemically stained sections with monoclonal anti-m-calpain antibody. Zymosan treatment resulted in an increase in m-calpain immunopositivity, which was not altered by calpain inhibitor III.

Calpain-Inhibitor III behandelten Ratten.
(B): Für den statistischen Vergleich der Wirkstoffwirkungen wurden die Flächen unterhalb der Kurven "Pfotenvolumenanstieg" gegen "Zeit" (AUC_ΔPW von 0–8 h, Mittelwert ± s.e.m.) unter Verwendung der linearen Trapezregel berechnet und einer univariaten Varianzanalyse mit anschließendem Bonferroni-post-hoc-Tests unterzogen (* statistisch signifikanter mittlerer Unterschied mit p < 0.05) 4 : Calpain inhibitor III at a concentration of 25 mg / kg significantly reduces paw edema
(A): Change in paw volume as a function of time after intraplantar injection of 1.25 mg zymosan in control animals (♦) and at 12.5 (∎) and 25 mg / kg, respectively

Calpain inhibitor III treated rats.
(B): For the statistical comparison of drug effects, the areas under the paw volume increase versus time curves (AUC _ΔPW of 0-8 h, mean ± sem) were calculated using the linear trapezoidal rule and a univariate analysis of variance followed by Bonferroni. subjected to post-hoc tests (* statistically significant mean difference with p <0.05)

Die Daten werden als relativer Unterschied zwischen der mit Zymosan behandelten rechten und der unbehandelten linken Hinterpfote, berechnet als: ΔPWL = (rechts – links)/links × 100, ausgedrückt.
(A): Latenz des Zurückziehens der Pfote nach intraperitonealer Verabreichung von 25 mg/kg Calpain-Inhibitor 111 im Vergleich mit Vehikel-behandelten Ratten. (B): Fläche unterhalb der Wirkung (relative Abnahme der Latenz des Zurückziehens der Pfote, ΔPWL) gegen Zeit-Kurven nach intraplantarer Injektion mit Zymosan und Behandlung mit 25 mg/kg Calpain-Inhibitor III. (***, statistisch signifikanter mittlerer Unterschied, p < 0,001) 5 : Calpain inhibitor III at a concentration of 25 mg / kg significantly reduces thermal hyperalgesia
Time course of latency of paw retraction in response to thermal stimulation of the plantar surface after intraplantar injection of 1.25 mg zymosan alone (♦) or zymosan and calpain inhibitor III

Data are expressed as the relative difference between the zymosan-treated right and untreated left hind paw, calculated as: ΔPWL = (right-left) / left × 100.
(A): Latency of paw retraction after intraperitoneal administration of 25 mg / kg calpain inhibitor 111 in comparison with vehicle-treated rats. (B): area below effect (relative decrease in paw retraction latency, ΔPWL) versus time curves after intraplantar injection with zymosan and treatment with 25 mg / kg calpain inhibitor III. (***, statistically significant mean difference, p <0.001)

6 : Scheme of the possible signal conduction path in the dorsal horn of the spinal cord

SEQUENCE LISTING

Claims (22)

Use of calpain or functional fragments or derivatives thereof for the manufacture of pain modulating pharmaceutical Links. Use of calpain or functional fragments or derivatives thereof for identifying pain modulating pharmaceutical Links. Use according to claim 1 or 2, wherein it is in the pharmaceutical compounds to compounds for pain prevention or treatment. Use according to claim 3, wherein the compounds Relieve or eliminate pain. Method for screening for pain modulation and / or prevention of suitable drugs in which a. two Be provided samples b. a sample, the calpain or a functional fragment or derivative thereof, with a connection in contact, c. the calpain activity in the presence the connection determines d. the calpain activity in the absence the connection is determined and e. the calpain activity according to (c) with which was compared according to (d) becomes. Use according to one the claims 1 to 4 or method according to claim 5, wherein the calpain is human calpain 1 and / or Calpain II acts. Use or method according to claim 6, wherein Calpain is an isolated polypeptide or polynucleotide is. Use or method according to claim 7, wherein Calpain is a polypeptide. Use or method according to claim 8, wherein Calpain is a polypeptide or functional fragment of which is 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 where the calpain is derived from a polynucleotide, that is the sequence according to SEQ ID No 2 (encodes calpain II) or a fragment thereof or the sequence according to sequence according to SEQ ID No 4 (coded calpain II) or contains a fragment thereof or consists of is encoded. Use or method according to claim 7, wherein it is the calpain is a polynucleotide. Use or method according to claim 10, wherein the calpain is a polynucleotide containing the sequence or a part of the sequence for a functional fragment of calpain according to SEQ ID No 2 or SEQ ID No 4 contains or a polynucleotide containing a sequence, which hybridize with the polynucleotides under stringent conditions can, contains or consists of, acts. Use or method according to any of the above claims, wherein the functional fragments are the Amino acids 115 to 272 of SEQ ID No 2 and / or 105 to 262 of SEQ ID No 4 contain or consist thereof. The method of claim 5, wherein a calpain expressing, preferably recombinant calpain expressing cell is used becomes. The method of claim 5, wherein a modified Cell with a lower calpain activity compared to its unmodified Condition is used. The method of claim 14, wherein a calpain knockout cell is used. Method according to one of claims 5, 13 and 14, wherein the calpain is determined directly. Method according to one of claims 5, 13 and 14, wherein the calpain is determined indirectly. Method for identifying a pain modulating Connection at the a. a compound that inhibits the activity of calpain modulated, selected as test compound and b. this test compound is administered to a subject to determine if the Be modulated in pain. Use of MDL-28170 (calpain inhibitor III) in the process of preparing a medicament for pain treatment. Use of MDL-28170 as a drug for pain treatment. Use of MDL-102935 in the process of manufacture a drug for pain treatment. Use of MDL-102935 as a drug for pain treatment.