WO2016050835A2 - Selective inhibitors of neutrophil elastase for treating neuropathic pain and chronic pain states harbouring a neuropathic component - Google Patents

Abstract

The present invention relates to selective inhibitors of neutrophil elastase, also called leukocyte elastase (LE) for use in the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component. The present invention further relates to pharmaceutical compositions and methods of diagnosing, prognosing, preventing and/or treating neuropathic pain and chronic pain states harbouring a neuropathic component.

Description

Selective inhibitors of neutrophil elastase for treating neuropathic pain and chronic pain states harbouring a neu ropathic component

The present invention relates to selective inhibitors of neutrophil elastase, also called leukocyte elastase (LE) for use in the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component. The present invention further relates to pharmaceutical compositions and methods of diagnosing, prognosing, preventing and/or treating neuropathic pain and chronic pain states harbouring a neuropathic component.

BACKGROUND OF THE INVENTION

Recent demographic studies have suggested that approximately every 5^th European suffers from chronic pain (Breivik et al. 2006). In Europe alone, the financial burden of chronic pain management is estimated at 200 billion€ per annum, in spite of which conventional analgesic therapy remains largely unsatisfactory (>50% of patients receive <50% pain relief) and is associated with debilitating side effects upon long-term usage. Neuropathic pain constitutes a major, intractable clinical problem and its pathophysiology is not well understood.

In a large number of clinical cases, neuropathy-associated pain is resistant to conventional therapeutics or their application is severely limited owing to the widespread side effects. Currently available treatment options are unsatisfactory because of their efficacy only in lesser proportion of patients suffering from neuropathic pain. There is a need in the art of improved means and methods for diagnosing and treating neuropathic pain as well as chronic pain states harbouring a neuropathic component.

Recent gene expression profiling studies in animal models have enabled the identification of novel targets with functions relevant to pain (see e.g. Tegeder et al., 2006; Costigan et al., 2010; Nissenbaum et al., 2010; Sorge et al., 2012). However, classical study designs comparing regulated genes across sham and injury groups are disadvantaged by differential expression of hundreds of genes across the two conditions; it is difficult to identify and functionally validate those that are functionally related to pain modulation versus other responses to injury, such as cell survival, cell metabolism, degeneration and regeneration (Persson et al., 2009).

There is a need in the art of improved means and methods for diagnosing and treating neuropathic pain as well as chronic pain states harbouring a neuropathic component.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by a selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) for use in the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component.

According to the present invention this object is solved by a pharmaceutical composition comprising

at least one selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) according to the present invention,

optionally, a pharmaceutical excipient,

optionally, a further pulmonary medicament.

According to the present invention this object is solved by a method for the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component, comprising the step of

administering a therapeutically amount of at least one selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) or a pharmaceutical composition comprising at least one selective inhibitor of LE to a patient or subject in need thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.

Selective LE inhibitors for neuropathic and chronic pain treatment

As discussed above, the present invention provides a selective inhibitor of neutrophil elastase for use in the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component.

Neutrophil elastase, also called leukocyte elastase (LE) is an important inflammatory serine protease with no previous association with the nervous system. Here we show that inhibitors of LE reversed ongoing neuropathic pain in mice. Our data reveal a novel role for LE in the modulation of neuropathic pain. We also show that LE contributing to neuropathic pain can be expressed not only by neutrophils, but also by T-lymphocytes.

The human leukocyte elastase (HLE) (Horvath et al., 2005) is a serine protease secreted by neutrophils during inflammation and is important in the dysregulation of immune functions, particularly in the context of airway inflammation (Lee & Downey, 2001), but which has not been reported to be associated with the nervous system so far. LE is known to activate MMP- 9 (Jackson et al., 2010; Ferry et al., 1997) which is an enzyme implicated in modulating pain (Kawasaki et al., 2008). However, so far there is no report linking LE to pain modulation. Moreover, until our discovery, it was not known that LE can be expressed in T-lymphocytes.

The term "neuropathic pain" as used herein refers to (any form of) pain resulting from damage to the central or the peripheral nervous system.

The term "chronic pain states harbouring a neuropathic component" as used herein refers to any clinical condition that results from direct or indirect damage or lesions to the central or the peripheral nervous system. Examples of direct damage include trauma to peripheral nerves, spinal cord injury, stroke-induced lesions, multiple sclerosis-induced lesions, amongst others. Examples of indirect damage include changes in peripheral nerves caused by tumor growth, metabolic distress (as is the case with diabetic peripheral neuropathy), amongst others. Preferably, the neuropathic pain and chronic pain states harbouring a neuropathic component is selected from

(1) traumatic, metabolic and genetically-induced neuropathies

e.g. peripheral nerve injury- or trauma-associated neuropathic pain, trigeminal neuralgia etc.,

(2) diabetic neuropathic pain (DPN),

(3) cancer- associated pain (e.g. cancer-induced bone pain (CIBP),

(4) pain associated with spinal cord injury (SCI pain),

(5) pain associated with multiple sclerosis (MS-pain),

(6) pain associated with bone- and cartilage-remodelling and ensuing damage of nerves, osteoarthritic pain, metastases-associated bone pain,

and

(7) pain associated with stroke

e.g. post-stroke pain, thalamic syndrome, amongst others.

In one embodiment, the selective inhibitor is used to alleviate pain associated with the respective disorders.

In one embodiment, the selective inhibitor is used for treating neuropathic pain and/or preventing chronic pain harbouring neuropathic components.

In one embodiment, the selective inhibitor is selected from

- Sivelestat, (Sivelastat sodium and Sivelastat sodium tetrahydrate),

- Elastatinal,

- oral neutrophil elastase inhibitor ONO-6818 (2-(5-amino-6-oxo-2-phenyl-l,6-dihydro- pyrimidin- 1 -yl)-N-[(l R,2R)- 1 -(5-tert-butyl- 1 ,3 ,4-oxadiazol-2-yl)- 1 -hydroxy-3-methylbutan- 2-yl]acetamide),

- BAY 85-8501 ,

- SSR 69071 (2-[[6-Methoxy-4-(l-methylethyl)-l,l-dioxido-3-oxo-l,2- benzisothiazol-2(3 H)-yl]methoxy] -9- [2-( 1 -piperidinyl)ethoxy] -4H-pyrido [ 1 ,2-a]pyrimidin-4- one),

- Alvelestat (AZD9668; AZD-9668; AZD 9668),

- M0398 (N-(Methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone) .

Preferably, the selective inhibitor of LE is not a cephalosporin.

In one embodiment, the selective inhibitor is administered systemically or locally.

Systemic administration can be oral, topical, inhalative, intranasal, intraperitoneally, subcutaneously, sublingually, via suppositories and pessaries, via transdermal application systems, intravenously, intraarterially, intramuscularly.

Systemic administration can be by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

Local administration can be via local injections or application in the skin, muscle or body organs, including intrathecally, epidurally, subdurally, via intracerebro ventricular injections or direct injections into peripheral nerves, sensory ganglia, spinal cord or the brain.

In one embodiment, the selective inhibitor is administered in combination with a further pain medication.

Said further pain medication can be: non-steroidal antiinflammatory agents (e.g. Aspirin, Ibuprofen, Diclofenac, Celecoxib etc), Paracetamol and Metamizol, Opioid-analgetics, Gabapentin, Pregabalin, Tapentadol, diverse anti depressive, cannabinoids, local anesthetics, Carbamazepin, Lamotrigin and other sodium channel blockers, Ziconotid and other calcium channel blockers, blockers of Transient Receptor Potential (TRP) channels.

In one embodiment, the selective inhibitor is administered via sustained release, controlled release, or delayed release.

Pharmaceutical compositions

As discussed above, the present invention provides a pharmaceutical composition comprising at least one selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) according to the present invention, optionally, a pharmaceutical excipient,

optionally, a further pain medicament.

Said further pain medicament or medication can be: non-steroidal antiinflammatory agents (e.g. Aspirin, Ibuprofen, Diclofenac, Celecoxib etc), Paracetamol and Metamizol, Opioid- analgetics, Gabapentin, Pregabalin, Tapentadol, diverse antidepressive, cannabinoids, local anesthetics, Carbamazepin, Lamotrigin and other sodium channel blockers, Ziconotid and other calcium channel blockers, blockers of Transient Receptor Potential (TRP) channels.

The pharmaceutical compositions of the invention are formulated to be suitable for systemic or local administration, as discussed above. The excipient(s) and/or carrier(s) will be selected according to the intended administration.

Methods for diagnosis and treatment

As discussed above, the present invention provides a method for the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component.

Said method comprises the step of

administering a therapeutically amount of at least one selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) or a pharmaceutical composition comprising at least one selective inhibitor of LE to a patient or subject in need thereof.

A therapeutically effective amount of the at least one selective LE inhibitor or pharmaceutical composition comprising said at least one selective LE inhibitor of this invention refers to the amount that is sufficient to treat the respective disease or achieve the respective outcome.

Analyses in mouse models of chronic pain with three inhibitors of LE indicate that doses in the order of about 20 mg/kg body weight are effective, or doses in the range from about 10 to about 100 mg/kg body weight, preferably about 20 to 50 mg/kg body weight.

In one embodiment, the at least one selective inhibitor is administered to a subject in need thereof systemically or locally. As discussed above, systemic administration can be oral, topical, inhalative, intranasal, intraperitoneally, subcutaneously, sublingually, via suppositories and pessaries, via transdermal application systems, intravenously, intraarterially, intramuscularly.

Systemic administration can be by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

As discussed above, local administration can be via local injections or application in the skin, muscle or body organs, including intrathecally, epidurally, subdurally, via intracerebroventricular injections or direct injections into peripheral nerves, sensory ganglia, spinal cord or the brain.

As discussed above, the neuropathic pain and chronic pain states harbouring a neuropathic component is preferably selected from

(1) traumatic, metabolic and genetically-induced neuropathies

e.g. peripheral nerve injury- or trauma-associated neuropathic pain, trigeminal neuralgia etc.,

(2) diabetic neuropathic pain (DPN),

(3) cancer-associated pain (e.g. cancer-induced bone pain (CIBP),

(4) pain associated with spinal cord injury (SCI pain),

(5) pain associated with multiple sclerosis (MS-pain),

(6) pain associated with bone- and cartilage-remodelling and ensuing damage of nerves, osteoarthritis pain, metastases-associated bone pain,

and

(7) pain associated with stroke

e.g. post-stroke pain, thalamic syndrome, amongst others.

In one embodiment, the selective inliibitor is used to alleviate pain associated with the respective disorders.

In one embodiment, the selective inliibitor is used for treating neuropathic pain and/or preventing chronic pain harbouring neuropathic components. In one embodiment, the selective inhibitor is selected from

- Sivelestat, (Sivelastat sodium and Sivelastat sodium tetrahydrate),

- Elastatinal,

- oral neutrophil elastase inhibitor ONO-6818 (2-(5-amino-6-oxo-2-phenyl-l ,6-dihydro- pyrimidin- 1 -yl)-N-[(lR,2R)-l -(5-tert-butyl-l ,3,4-oxadiazol-2-yl)- 1 -hydroxy-3-methylbutan- 2-yl]acetamide),

- BAY 85-8501,

- SSR 69071 (2-[[6-Methoxy-4-(l-methylethyl)-l ,l-dioxido-3-oxo-l,2- benzisothiazol-2(3H)-yl]methoxy]-9-[2-(l-piperidinyl)ethoxy]-4H-pyrido[l,2-a]pyrimid one),

- Alvelestat (AZD9668; AZD-9668; AZD 9668),

- M0398 (TNf-(Methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine

chloromethylketone) .

In one embodiment, the selective inhibitor is administered in combination with a further pain medication.

Said further pain medication can be: non-steroidal antiinflammatory agents (e.g. Aspirin, Ibuprofen, Diclofenac, Celecoxib etc), Paracetamol and Metamizol, Opioid-analgetics, Gabapentin, Pregabalin, Tapentadol, diverse antidepressive, cannabinoids, local anesthetics, Carbamazepin, Lamotrigin and other sodium channel blockers, Ziconotid and other calcium channel blockers, blockers of Transient Receptor Potential (TRP) channels.

Preferably, the selective inhibitor of LE is not a cephalosporine.

In one embodiment, the selective inhibitor is administered via sustained release, controlled release, or delayed release.

Further description of preferred embodiments

Leukocyte elastase (LE) is an important inflammatory serine protease with no previous association with the nervous system. Here we show that inhibitors of LE reversed ongoing neuropathic pain in mice. Our data reveal a novel role for LE in the modulation of neuropathic pain. - Results: We observed that mice lacking the gene encoding LE (LE-/- mice) showed significantly reduced magnitude of mechanical hypersensitivity induced by peripheral neuropathy via Spared Nerve Injury (SN1), represented as either change in withdrawal threshold (Figure 1) or as the integral of the entire von Frey stimulus-response function (Figure 1), which was evident over the entire time-frame of testing. Because neuropathy- induced drop in withdrawal threshold is the main indication of a clinically very important manifestation of pain, namely allodynia, our data indicate that a lack of LE protects against allodynia in neuropathic pain.

Furthermore, specific blockade of LE with Sivelestat (Kawabata et al., 1991) given as a single, low-dose i.t. injection (200 pmol) rapidly and significantly reduced nociceptive hypersensitivity when administered on day 8 post-SNI, represented as change in withdrawal threshold (Figure 1) or the integral of the entire von Frey stimulus-response function (Figure 1).

We confirmed that the inhibition of LE activity by Sivelastat in vitro (Figure 2) at a dose comparable to the dose which was applied in vivo for relief of pain. These results indicate that LE is functionally involved in the maintenance of tactile hypersensitivity (allodynia) and that its pharmacological blockade is effective when peak neuropathic pain is established.

We then attempted to identify the cellular source of LE that could account for its above- described role in promoting neuropathic hypersensitivity. As suggested by its name, LE was originally identified as a serine protease produced mainly by neutrophils (Pham et al., 2006). RT-PCR was performed on various cell/tissue types, with paw tissue from neuropathic mice, which harbours infiltrating neutrophils (Meotti et al, 2006), serving as a positive control (Figure 1). We observed that although LE is expressed in mouse-derived L3/L4 DRGs or spinal cord tissue, neuronal cultures derived from the DRG or the spinal cord do not show expression of LE. LE was also not expressed in non-neuronal cells that have been associated with neuropathic pain, including astrocytes, microglia, macrophages or Schwann cells (McMahon & Malcangio, 2009; Scholz & Woolf, 2007), but was found in T-lymphocytes (see Figure 3). Indeed, using CD3 as marker protein for T-cells in immunohistochemistry, we observed that T-cells are present in the DRG in vivo under basal conditions and their levels increase significantly following nerve injury (Figure 3), consistent with previous reports (Kim &Moal em-Taylor, 201 1 ; Hu et al., 2007). Using granulocyte receptor 1 (Gr-1) as marker protein for neutrophils (with inflamed paw serving as a positive control), we observed that although neutrophils are found in the vicinity of the DRG and increase in levels after nerve injury, they do not appear to invade the parenchyma of the DRG (arrows in Fig. 3).

We then established an in vivo assay for LE activity based on fluorogenic substrates and FRET imaging (Gehrig et al., 2012) on DRGs isolated from SNI and sham- operated mice. We observed that LE activity increases significantly in the DRG 1 day and 7 days post-SNI as compared to sham (Figure 1 ; mice lacking LE were used as controls), consistent with the time-course of the increase in T-cell and neutrophil levels in the DRG.

To further strengthen the causal relationship between LE and neuropathic pain, we performed T-cell transfer experiments in mice genetically lacking T-cells (Rag2-/- mice). We observed that Rag2-/- mice lack neuropathic allodynia, but acquire neuropathic allodynia when T-cells from wild-type mice (LE+/+ mice) are transferred into Rag2-/- mice (Figure 1 ). In contrast, transfer of T-cells from LE-/- mice into Rag2-/- mice does not lead to any neuropathic allodynia (Figure 1). These results thus indicate a causal role for T-cells in neuropathic pain and importantly demonstrate that T-cells can only impart neuropathic pain when LE is expressed in them.

Effects of systemic delivery of Sivelastat on neuropathic pain:

We administered Sivelastat systemically via intraperitoneal injections and tested impact on neuropathic pain which just reaches maximal values at 8 days post-SNI in mice. We observed that a single dose of 20 mg/Kg body weight administered intraperitoneally (i.p.) to mice significantly blocked on-going mechanical hypersensitivity at day 8 post-SNI as compared to the corresponding vehicle (represented as change in withdrawal frequency threshold as well as integral of the entire stimulus-response curve) (Figure 4).

Dose-dependent effects of Sivelastat on neuropathic pain:

We also tested a higher dose, 50 mg/Kg body weight i.p., and observed an even stronger reduction of neuropathic pain at some of the time points tested (Figure 4), indicating dose- dependent reduction in neuropathic pain by systemic Sivelastat. We further tested lower concentrations, 0.2 and 2.0 mg/Kg body weight i.p., and also observed a reduction in neuropathic pain at 3 and 6 hrs following Sivelestat application. Effects of systemic delivery of Sivelastat on late stages of neuropathic pain:

We addressed whether Sivelastat can block neuropathic pain when it has been established and is evident in chronic stages, e.g. at 28 days post-SNI in mice. I.p. delivery of Sivelastat significantly blocked on-going mechanical hypersensitivity at day 28 post-SNI as compared to the corresponding vehicle (represented as change in withdrawal threshold as well as integral of the entire stimulus-response curve) (Figure 4) at doses of 20 mg/Kg as well as 50 mg/Kg, but not at lower doses of 0.2 and 2.0 mg/Kg, indicating that LE-dependent mechanisms are still active at chronic stages of neuropathic pain.

We further tested effect of systemic Sivelestat on cold hypersensitivity and observed that a single application of systemic Sivelestat at 20 mg/Kg body weight significantly reduced cold hypersensitivity (Figure 5), which is highly clinically-relevant feature of neuropathic pain. Thus, taken together, these data demonstrate beneficial effects of LE inhibitors on 2 of the most therapy-resistant aspects of neuropathic pain.

We further asked whether Sivelestat causes any motor defects following systemic application at an effective dosage observed in above experiments, i.e. with a dose of 20 mg/Kg. Observation of mice in open-field exploration test following systemic application of Sivelestat at 20 mg/Kg body weight or vehicle showed that there is no difference in total distance and mean distance travelled following systemic Sivelestat application as compared to the vehicle group (Figure 6). Thus, inihibition of LE neither affects motor behaviour nor does it negatively affect typical behaviors related to natural exploration.

In the next step, we sought to compare the magnitude of protection exerted by Sivelastat and the best therapeutic drug currently used in the treatment of neuropathic pain treatment i.e., Pregabalin. Magnitude of inhibition of mechanical hypersensitivity in neuropathic mice was comparable between systemic Pregabalin or Sivelestat at dosage of 20 mg/Kg body weight at both early and late stages following SNI (Figure 7). Given that new drugs can inhibit LE with a higher efficicacy and potency, it is plausible that other inibitors of LE may afford more protection against neuropathic pain.

Effects of other LE inhibitors on neuropathic pain: We then went on to test 2 other, independent inhibitors of LE in the SN1 model of peripheral neuropathy-induced pain, both at early and at late stages following SNI. Elastatinal (Figure 8) or SSR-69701 (Figure 9) or the corresponding vehicles were injected i.p. to mice with SNI at day 8 or day 28. In all cases, we observed a significant and marked reduction of mechanical hypersensitivity at day 8 and at day 28 post-SNI as compared to the corresponding vehicle (represented as change in withdrawal frequency as well as integral of the entire stimulus- response curve) (Figure 8 and Figure 9).

We also tested i.p. Elastatinal at a higher dose of 50 mg/Kg at day 8 and 28 post-SNI, and observed even stronger reduction of mechanical hypersensitivity in the late phase over this chronic phase of SNI-induced pain in mice as compared to vehicle treatment (Figure 8). Thus, both early and late phases of peripheral neuropathy-induced pain responded strongly to LE blockade via several different drugs.

Effects of Sivelastal on other forms of neuropathic pain (caused by cancer or diabetes):

We then went on to test the impact of LE inhibtion on other forms of neuropathic pain. First, we utilized a model based on injection of murine osteolytic breast cancer cells into the intermedullary space of the mouse femur bone, which has been reported to closely mimic pain caused by metastases of sarcomas and carcinomas to large skeletal bones in human cancer patients (Bloom et al., 2011 ; Mantyh, 2006; Mantyh, 2013). Cancer growth in the bone is also associated with osteolysis and damage to intramedullary and periosteal nerves (Bloom et al, 2011 ; Mantyh, 2006; Mantyh, 2013). At day 28 after tumor cell implantation in the femur bone, when the chronic phase of tumor-associated pain is establised, we administered Sivelastat (20 mg/Kg body weight) or vehicle i.p. and observed that Sivelastat significantly attenuated mechanical hypersensitivity as compared to vehicle (Figure 10). Thus, cancer pain responds well to LE inhibition.

Similarly, in a model of Type II diabetes induced by multiple treatments with Streptozotocin (STZ), mice demonstrated significant mechanical hypersensitivity at 8 weeks after STZ treatment. Administration of Sivelastat (20 mg/Kg body weight) i.p. significantly attenuated hypersensitivity as compared to vehicle control (represented as change in withdrawal threshold as well as integral of the entire stimulus-response curve). Thus, diabetic neuropathic pain also responds favourably to LE inhibition.

- Discussion: Besides neurons and resident glia, immune cells invading the nervous system, such as macrophages, neutrophils and T lymphocytes have recently emerged as significant modulators of neuropathic pain transmission (Costigan et al. 2009, Barclay et al 2007, Perkins & Tracey 2000). Our observations suggest that these cells may impact on nociceptive processing in the DRG via release of LE.

We present genetic as well as pharmacological evidence for a role for LE in promoting nociceptive hypersensitivity in 3 independent types of neuropathic pain. Our data indicate that LE is not associated with neuron-glial signaling, but represents a novel effector for nociceptive modulation in the DRG via T-lymphocytes and neutrophils, which accumulate in or surrounding the DRG, following nerve injury.

Importantly, although our experiments have focused on the DRG and spinal cord so far, it is also plausible that LE exerts similar functions at other anatomical sites involved in chronic pain, namely the site of injury itself (e.g. peripheral tissues) as well as the brain. Indeed, we observed very strong reduction of nociceptive hypersensitivity upon systemic delivery, which could be targeting peripheral as well as central loci of action of LE.

Because relief of established neuropathic hypersensitivity was afforded by the intrathecal delivery of a low dose of Sivelestat, a drug which is currently awaiting FDA approval and is already in clinical usage in some countries for the therapy of airway inflammation, use of specific LE inhibitors will present opportunities for therapeutic interventions in several forms of neuropathic pain (of peripheral and central origin) and other types of chronic pain which harbour at least partially a neuropathic component (e.g. cancer pain, osteoarthritic pain). Here, our data clearly show that the therapeutic effects are not limited to Sivelastat, but can be attributed to LE blockade as such since other LE inhibitors demonstrated very strong blockade of neuropathic pain. Moreover, we have demonstrated efficacy in bone metastatic pain, which is clinically very difficult to treat. Moreover, diabetic neuropathic pain, which is widespread and therapy-resistant, was attenuated by systemically delivered LE inhibitors.

The impact of a low, single dose of Sivelastat as well as other inhibitors of LE on change in mechanical hypersensitivity is particularly striking because this is one of the most relevant clinical manifestations of neuropathic pain in patients. Indeed, our data provide evidence that all 3 inhibitors of LE tested significantly attenuated neuropathic pain following a single systemic application, indicating a strong potential for any drug with LE inhibitory capacity in the treatment of neuropathic pain. Moreover, we also demonstrate efficacy against cold hypersensitivity, which is a major debilitating problem associated with neuropathic pain in countries with cooler/cold climates.

In summary, our data lay the basis for the efficacy of LE inhibitors in several forms of chronic pain, i.e. either resulting from direct traumatic damage to nerves, or harbouring a neuropathic component, i.e. a cause indirectly leading to neuropathy, such as pain caused by cancer growth or metabolic disorders, such as diabetes.

The following examples and drawings illustrate the present invention without, however, limiting the same thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : The Leukocyte elastase (LE) plays a key role in neuropathic pain.

(a, b) Analysis of SNI-induced neuropathic mechanical hypersensitivity in LE null mice {LE -

/-) as compared to wild-type controls represented as paw withdrawal thresholds (a) and summary of paw withdrawal responses to von Frey forces from 0.02 g to 1.4 g (b) (n=8-9 each; */^><0.05; repeated measures 2-way ANOVA followed by Tukey's post-hoc test).

(c, d) Paw withdrawal thresholds (c) and summary of paw withdrawal responses to von Frey forces from 0.02 g to 1.4 g (d) in wild-type mice that received a single i.t. injection (200 pmol) of the LE blocker, Sivelestat, or vehicle 8 days post-SNI (n=8-9 each; * <0.001 as compared to 8 days post-SNI; t O.001 as compared to vehicle at the corresponding time point; repeated measures 2-way ANOVA followed by Tukey's post-hoc test).

(e) Screening of LE mRNA expression in different cell types at the neuroimmune interface via reverse-transcriptase PCR using Id post-SNI paw as a positive control, 18sRNA as a housekeeping gene and reverse-transcription reactions performed without (-) cDNA as a negative control.

(£, g) Quantitative summary (f) and typical examples (g) of analysis of LE activity in L4 DRGs derived from LE +/+ and LE -/- mice at 1 and 7 days post-SNI. Upon isolation, L4 DRGs were incubated with an LE substrate, washed and fixed, sectioned and imaged for FRET signals. (n=4-5 different experiments on at least 4 mice per data point;†P<0.05 as compared to LE -/-; * <0.05 as compared to sham; 1-way ANOVA followed by post-hoc Tukey's test). Values in (f) are calculated as % change over values derived from sham treated wild-type mice and negative values in LE -I- mice are indicative of basal LE activity in wild- type sham-treated mice. Lower panels in (g) represent corresponding DRG sections in the transmission microscopy mode. Scale bar: 100 μιη.

(fa) Comparison of paw withdrawal threshold (40% positive responses) to plantar application of graded von Frey hair mechanical forces over time between 4 groups of mice: Rag2+/+ and Rag2-/-mice without T-cell transfer and Rag2-/-mice transferred with LE+/+ or LE-/- T-cells (lxl 0⁷ cells per mouse) 6 d prior to SNI operations. n=10 animals per group;† <0.05 and *P<0.05 as compared to Rag2-/- mice without T-cell transfer at corresponding time points; repeated measures 2-way ANOVA followed by Tukey's post-hoc test).

Figure 2. Analysis of effects of LE inhibitor Sivelestat on LE activity in vitro.

(a, b) Dose-dependent inhibitory effect of the specific LE inhibitor Sivelestat on rLE activity. Recombinant LE (rLE, 1 nM) was preincubated with increasing concentrations of Sivelestat and then its flurometric activity was measured over time. AFU: arbitrary fluorescence units. RFU: relative fluorescence units. Panels at the right (b) show the quantification of percentage of rLE activity compared to 1 nM rLE at indicated time points (a). n=3; * <0.05 as compared to activity of enzyme alone at the corresponding time point; repeated measures 2-way ANOVA followed by Tukey's post-hoc test).

Figure 3. Sources of LE in neuropathic pain.

(a-e) Immunohistochemical characterization of infiltration of T-cells into the DRG after SNI using an anti-CD3 antibody in naive mice (a), at days 1 (b), 3 (c) and 8 post-SNI (c) and in paw skin punches isolated 8 days after SNI operations (e). DAPI (blue) was used as counterstain for cell nuclei.

(f) Quantification of CD3-positive T-cells in L3-L4 DRGs of naive and 1, 3 and 8 days post- SNI mice compared to sham controls (* <0.001 as compared to sham control; n= 3-6 mice; regular 2-way ANOVA followed by Bonferroni's post-hoc test).

(g) Immunohistochemical characterization of neutrophil infiltration into L4 DRG using an anti-Gr-1 antibody, at 3 and 8 days after SNI or sham operations. Arrows indicate positive immunoreactivity for Gr-1. Paw skin punches isolated 8 days after SNI operations were used as positive control for stainings. Scale bars represent 100 pm.

Figure 4. Dose-dependent effects of systemic delivery of Sivelestat on neuropathic pain. Analysis of SNI-induced neuropathic mechanical hypersensitivity following intraperitoneal application of LE inhibitor, Sivelestat, as compared to the vehicle-injected group. A single dose of 0.2 or 2.0 or 20 or 50 mg/kg body weight Sivelestat was injected i.p. on day 8 (POD8) or day 28 (POD28) post-SNI (blue arrow).

(a) Paw withdrawal responses to von Frey force of 0.16g at 1, 3, 6 and 24h following each dosage of i.p. Sivelestat.

(b) An integral of responsivity to mechanical stimuli over all von Frey forces tested (0.02 g to 1.0 g) in Sivelestat-injected group as compared to vehicle-injected group of mice, represented as area under the curve (AUG). In all panels, * denotes P < 0.05 as compared to basal ,† represents P < 0.05 as compared to the vehicle treated group at respective time-point , Two- way ANOVA of repeated measures followed by Tukey's post hoc /est; n = dX least 6 mice per group.

Figure 5. Effects of systemic delivery of Sivelastat on neuropathic pain.

Analysis of SNI-induced neuropathic cold hypersensitivity (4°C) following intraperitoneal application of LE inhibitor, Sivelestat, as compared to the vehicle-injected group. A single dose of 20 mg/kg body weight Sivelestat was injected i.p. on day 10 (POD 10). * denotes P < 0.05 as compared to basal ,† represents P < 0.05 as compared to the vehicle treated group at respective time-point , Two-way ANOVA of repeated measures followed by Tukey's post hoc /est; n = at least 6 mice per group.

Figure 6. Effects of systemic delivery of Sivelastat on motor function in neuropathic pain. Analysis of (a) total distance travelled or (b) mean speed in the open field exploration experiment following intraperitoneal application of LE inhibitor, Sivelestat, as compared to the vehicle-injected group. A single dose of 20 mg/kg body weight Sivelestat was injected i.p. on day 10 (POD 10).

Figure 7. Comparison of effects of systemic delivery of Sivelestat or Pregabalin on neuropathic pain.

Analysis of SNI-induced neuropathic mechanical hypersensitivity following intraperitoneal application of LE inhibitor, Sivelestat or Pregabalin as compared to the vehicle-injected group. A single dose of 20 mg/kg body weight Sivelestat or Pregabalin was injected i.p. on day 8 (POD8) or day 28 (POD28) post-SNI. (a) Paw withdrawal responses to von Frey force of 0.16g at 1, 3, 6 and 24h following each dosage of i.p. Sivelestat or Pregabalin.

(b) An integral of responsivity to mechanical stimuli over all von Frey forces tested (0.02 g to 1.0 g) in Sivelestat or Pregabalin-injected group as compared to vehicle-injected group of mice, represented as area under the curve (AUG). In all panels, * denotes P < 0.05 as compared to basal , † represents P < 0.05 as compared to the vehicle treated group at respective time-point , Two-way ANOVA of repeated measures followed by Tukey's post hoc test; n - dX least 6 mice per group.

Figure 8. Dose-dependent Effects of another LE inhibitor, Elastatmal, on neuropathic pain.

Analysis of SNI-induced neuropathic mechanical hypersensitivity following intraperitoneal application of Leucocyte elastase inhibitor, Elastatinal, as compared to the veliicle-injected group. A single dose of 20 or 50 mg/kg body weight Elastatinal was injected i.p. on day 8 (POD8) or day 28 (POD28) post-SNI.

(a) Paw withdrawal responses to von Frey force of 0.16g at 1 , 3, 6 and 24h following i.p. Elastatinal are presented.

(b) An integral of responsivity to mechanical stimuli over all von Frey forces tested (0.02 g to 1.0 g) in Elastatinal-injected group as compared to vehicle-injected group of mice, represented as area under the curve (AUG). In all panels, * denotes P < 0.05 as compared to basal ,† represents P < 0.05 as compared to the vehicle treated group at respective time-point, Two-way ANOVA of repeated measures followed by Tukey's post hoc test; n = at least 6 mice per group

In both panels, * denotes P < 0.05 as compared to basal ,† represents P < 0.05 as compared to the vehicle treated group at respective time-point or Two-way ANOVA of repeated measures followed by Tukey's post hoc test; n = 6 mice per group.

Figure 9. Effects of another LE inhibitor, SSR-69071, on neuropathic pain.

Analysis of SNI-induced neuropathic mechanical hypersensitivity following intraperitoneal application of LE inhibitor, SSR-69071 , as compared to the vehicle-injected groups. A single dose of 20 mg/kg body weight SSR-69071 or DMSO or PBS were injected i.p. into three different groups of mice on day 8 (POD8) or day 28 (POD28) post-SNI

(a) Paw withdrawal responses to von Frey force of 0.16g at 1, 3, 6 and 24h following i.p.

SSR-69071. ( b) An integral of responsivity to mechanical stimuli over all von Frey forces tested (0.02 g to 1.0 g) in SSR-69071 -injected group as compared to vehicle-injected groups of mice, represented as area under the curve (AUG).

In both panels, * represents P < 0.05 as compared to basal ,† represents P < 0.05 as compared to corresponding data point in the DMSO group and † denotes P < 0.05 as compared to corresponding data point in the PBS group; at respective time-point or Two-way ANOVA of repeated measures followed by Tukey's post hoc test; n - at least 6 mice per group.

Figure 10. Impact of systemic delivery of Sivelastat on neuropathic pain of cancer origin. Analysis of cancer-induced mechanical hypersensitivity following intraperitoneal application of Leucocyte elastase inhibitor, Sivelestat, as compared to the vehicle-injected group. A single dose of 20 mg/kg body weight Sivelestat or equal volume of vehicle was injected i.p. on day 28 (PID28) following tumor cell implantation in the femur.

(a) Paw withdrawal responses to von Frey forces of 0.16g at l h, 3h, 6h and Id following i.p. Sivelestat.

(g) An integral of responsivity to mechanical stimuli over all von Frey forces tested (0.02 g to 1.0 g) in Sivelestat-injected group as compared to vehicle-injected group of mice, represented as area under the curve (AUG).

In all panels, * denotes P < 0.05 as compared to basal and† denotes P < 0.05 as compared to corresponding data point in the vehicle treated group; Two-way ANOVA of repeated measures followed by Tukey's post hoc test, n = 8 mice per group.

Figure 11. Impact of systemic delivery of Sivelestat on neuropathic pain caused by diabetes.

Analysis of diabetic neuropathy-induced mechanical hypersensitivity following systemic application of LE inhibitor, Sivelestat, as compared to the vehicle-injected group. A single dose of 20 mg/kg body weight Sivelestat or equal volume of vehicle was injected i.p. in the 8th week following induction of diabetes via Streptozotocin (8W diabetic).

(a) Paw withdrawal responses to von Frey force of 0.16g at lh, 3h, 6h and Id following i.p. Sivelestat or vehicle.

(b) An integral of responsivity to mechanical stimuli over all von Frey forces tested (0.02 g to 1.0 g) in Sivelestat-injected group as compared to vehicle-injected group of mice, represented as area under the curve (AUC). In both panels, * denotes P < 0.05 as compared to basal and† denotes P < 0.05 as compared to corresponding data point in the vehicle treated group; Two-way ANOVA of repeated measures followed by Tukey's post hoc /est; n = 10 mice per group.

EXAMPLE

1. Methods

Mice lacking the leucocyte elastase (referred to here as LE^' mice) (B6.129X1- Elane^tm^dsi ) (Belaaouaj et al., 1998) were bought commercially (Jackson Laboratories) and genotyped by PCR on mouse genomic DNA using the primers oIMR7064 (wild type forward), oIMR7065 (common) and oIMR8162 (mutant reverse). Homozygote mice were identified by a 310 bp fragment corresponding to the mutant LE gene. Wild-type mice were identified by a 230 bp fragment corresponding to the wild type allele. Heterozygote animals were identified by the presence of both fragments. Primer sequences are available on the corresponding website of the source (Jackson Laboratories).

Mice deficient in the Rag2 gene (referred to here as Rag2^~/~ mice) were provided by Prof. Dr. Bernd Arnold from the German Cancer Research Center in Heidelberg (DKFZ).

Spared Nerve Injury (SNI).

In the 'Spared Nerve Injury' (SNI) model for neuropathic pain (Decosterd & Woolf, 2000), mice were anesthetized under 2% isofluran and the fur of the lateral part of the left tight was removed. The skin on the lateral surface of the thigh was incised and a section was made directly through the biceps femoris muscle exposing the sciatic nerve and its three terminal branches: sural, common peroneal and tibial nerves. The common peroneal and tibial nerve were tightly ligated with 5.0 silk and sectioned distal to the ligation, removing 2-4 mm of the distal nerve stump. These two nerves were subsequently cut and the sural nerve was left intact. Muscle and skin were closed in two layers. The mice were housed under standard conditions in cages for 3 days before the isolation of tissues or perfusion of animals.

Cancer pain model.

Cells of the murine osteolytic breast carcinoma cell line, 4T1-Luc, which express the luciferase reporter gene, were used as described previously (Srivastava et al., 2014). All experiments were performed on Balb/c strain of mice, as described previously (Bloom et al., 201 1 ; Srivastava et al., 2014). Following an arthroton y, the condyles of the left distal femur of the mouse was exposed. A hole was then drilled to create space for the injection needle- Using a 10 μΐ Hamilton syringe. 1.5 x 10⁵ cells were directly injected into the intermedullary space of the mouse femur. The injection hole was sealed with dental cement in order to prevent leakage of the tumor cells. Different cohorts of mice were treated with 1) Sivelastat (20 mg/kg body weight); 2) PBS in a volume of 40 μΐ intraperitoneally at day 28 post tumor- implantation (PID).

Model of STZ-indiiced diabetic neuropathy.

Diabetes is induced by administering 6 consequent dosages of Streptozotocin (STZ, SO 130 Sigma), dissolved in 0.05 M citrate buffer, at the concentration of 60 mg/kg body weight intraperitonially with 24 hrs interval. Mice receiving equal volume of 0.05 M citrate buffer served as sham controls. Blood glucose was monitored in regular intervals and appropriate units of insulin are supplemented subcutaneously to keep the blood glucose levels under control.

Intrathecal delivery of drugs.

To enable intrathecal delivery of pharmacological agents at the level of lumbar spinal segments, mice were deeply anaesthetized with an intraperitoneal injection of 0.65 μΐ/g body weight of sleep mix [0.23 g/μl Sedator (Euro vet International), 3.08 μg/ l Dormicum (Roche), 0.01 μg/μl Fentanyl-Janssen (Janssen-Cilag)] and a polyethylene catheter (Biomedical Instruments) was stereotactically inserted through an opening in the cisterna magna into the lumbar subarachnoid space at the L3-L4 intervertebrae. The tip of the catheter was located near the lumbar enlargement of the spinal cord. The volume of dead space of the i.t. catheter was 10 μΐ. Mice were allowed to recover for 3 days after surgery and only animals showing complete lack of motor abnormalities were used for further experiments. The correct placement of the catheter was verified at the end of the experiment by i.t injection of 5 μΐ 1% Evans blue and performing a laminectomy. Drugs and recombinant proteins were administered intrathecally in the indicated dosage using a microinjection syringe (Hamilton) in a volume of 5 μΐ separated from an 8 μΐ volume of saline through a 1 μΐ air bubble. intraperitoenal delivery of drugs.

Sivelestat (S7198, Sigma) and Elastatinal (sc-201272, Santa Cruz biotechnologies) were dissolved in IX PBS and injected intraperitoneally (i.p.) at required concentration in 40 μΐ volume. Equal volume of IX PBS was injected as vehicle control into separate group of mice. SSR-69071 (sc-203702, Santa Cruz biotechnologies or 2506, RandD systems) was dissolved in 100% DMSO and injected i.p. at required concentration in 40 μΐ volume. Equal volume of 100% DMSO was injected as vehicle control into separate group of mice.

Nociceptive tests.

Responses to paw pressure were determined using von-Frey filaments. Animals were placed in a multi-compartment enclosure with a grid beneath to enable application of mechanical stimuli. Before testing, mice were habituated in a small plastic (7.5 x 7.5 x 15 cm) cage for 1 h. Then mechanical sensitivity was determined with a graded series of nine von Frey filaments that produced a bending force of 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1 , 1.4 and 2 g, respectively. The stimuli were applied within the sural nerve territory (lateral part of the hindpaw). Each filament was tested 10 times in increasing order starting with the filament producing the lowest force. Von Frey filaments were applied at least 3 s after the mice had returned to their initial resting state. For baseline mechanical sensitivity test: all filaments were applied and the number of withdrawals was recorded. For tactile allodynia: the minimal force filament for which animals presented either a brisk paw withdrawal and/or an escape attempt in response to at least 5 of the 10 stimulations determined the mechanical response threshold, which was defined as the minimum pressure required for eliciting 60% of withdrawal responses out of 5 stimulations and measured in grams (force application).

For testing cold hypersensitivity, mice were kept on a heat block set at 4°C and time required for the onset of nocifensive behavior from the SNI operated paw was noted.

Reverse transcription PCR.

Total RNA was extracted from mouse L3-L4 DRGs (dorsal root ganglia), lumbar L3-L4 spinal cord segments, paw skin isolated from SNI-operated mice using the phenol/chloroform extraction method; similar methods were employed for extracting RNA from cultured DRG neurons and cultured embryonic spinal cord neurons. Total RNA from acutely isolated mouse dorsal spinal cord neurons and T-cells and from primary cell cultures of Schwann cells, astrocytes, microglia and macrophages were purchased from 3H Biomedical, Uppsala, Sweden. The RNA was reverse-transcribed using RevertAid™ M-MuLV Reverse Transcriptase (Fermentas), random hexamer and Oligo(dT) primers (Roche and Invitrogen, respectively) according to standard protocols. PCR reactions were then performed on cDNA from the aforementioned cell types and tissues using the following primers:

IE -(forward): 5 ^'-GGC CCT TGG CAG ACT ATC CAG C-3 ^' [SEQ ID NO. 1], LE (reverse): 5 '-ACC TGC ACG TTG GC'G TTA ATG G-3 '

18sRNA (forward): 5 '-AGT TAT GGT TCC TTT GGT CGC TC- 18sRNA (reverse): 5 '-GTT ATT TTT CGT CAC TAG CTC CC-3

Fluorometric measurements of protease activity.

The proteolytic activity of the desired protease was measured through the fluorescence released after protease-induced cleavage of specific fluorogenic substrates. The dose-response assay of sivelestat effect over rLE activity was performed in an Infinite® 200 PRO multimode reader from Tecan and the measurements expressed as RFU (relative fluorescence units). The activity of mouse recombinant LE (R&D Systems) was measured using the fluorogenic substrate MeOSucAAPV-AMC (Bachem) as described by the manufacter. Pre-mixes containing a fixed amount of rLE and increasing concentrations of rSerpinA3N or sivelestat (Sigma) were prepared in assay buffer (50mM Tris, 1M NaCl, 0.05% (w/v) Brij-35, pH 7.5). The solutions containing the proteins were mixed with equal volumes of substrate in assay buffer reaching a final substrate concentration of 100 μΜ. The samples were placed in the multi-well plate and the reading was measured immediately with the fluorescence reader at excitation and emission wavelengths of 380 nm and 460 nm (respectively) for 30 min.

Measurement of LE activity in ex vivo DRG explants.

The activity of LE in DRG was measured using the LE ratiometric probe NEmo-2, a ratiometric FRET-based monitoring LE reporter that detects the activity of membrane- associated LE, but not soluble LE (Gehrig et al., 2012). Briefly, L4 DRGs from operated mice were isolated and incubated immediately with 50 μΜ NEmo-2 dissolved in F12 medium for 60 min at 37°C protected from light. DRGs were shortly washed with PBS, fixed for 48 h in 4% PFA and postfixed for 16 h in 0.5% PFA followed by overnight incubation in 30% sucrose for cryo sectioning. DRGs were cut in 40 μπι sections and images from every section were taken using a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 20x 1.4 dry objective with the following settings: The donor coumarin 343 of NEmo-2 was excited with a 405 nm diode laser and emission was sampled between 470-510 nm. Sensitized emission of the acceptor TAMRA was sampled between 570-610 nm. The pinhole was set to 300 μηι. For the quantification of LE activity in ex-vivo explants of DRG, images were processed with ImageJ 1.38r software (http://rsb.info.nih.gov/ij/) using background subtraction, exclusion of saturated pixels, smoothing with a median filter, thresholding and calculation of donor/acceptor (D/A) ratio images from which the activity was quantified - these were uniformly applied over all images by an observer unaware of the identity of the groups. Because the probe accumulated unspecifically along the outer rim of all sections, this region (corresponding to 20 pixels thickness) was eliminated from each section and the region corresponding to the somata of DRG neurons was selected as region of interest. About 8 slices were analyzed per DRG and FRET intensity was averaged per DRG. The representative images shown in the manuscript were created by transformation to the LUT range.

T-cell experiments.

For purification of splenic T-cells, Macs cell sorting was performed using Dynabeads (untouched mouse T-cells, Invitrogen) and neutrophils were depleted using biotinylated rat anti-Gr-1 antibody (clone RB6-8C5, BD Biosciences). Mice were injected i.v. with 200 μΐ of purified T-cells in dPBS at a concentration of 1x10 /200 μΐ.

Flow cytometry.

Surface staining was performed according to standard procedures. Data acquisition was carried out with an 8-color flow cytometer (Canto II, BD Biosciences) and analyzed with FlowJo (Treestar).

Statistics.

For all measurements, data was calculated and presented as mean ± standard error of the mean (S.E.M.). Two-tailed unpaired T-test, one-way Analysis of Variance (ANOVA) followed by post-hoc Fisher's test or two-way ANOVA for random or repeated measures followed by Tukey's test were used to determine statistical significances. P < 0.05 was considered significant. For all statistical analyses, the appropriate statistical tests were chosen, the data met the assumptions of the test and the variance between the statistically compared groups was similar.

The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof. REFERENCES

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Claims

Claims

1. A selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) for use in the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component.

2. The selective LE inhibitor for use according to claim 1 , wherein the neuropathic pain and chronic pain states harbouring a neuropathic component is selected from

- traumatic, metabolic and genetically-induced neuropathies (e.g. peripheral nerve injury- or trauma-associated neuropathic pain, trigeminal neuralgia etc),

- diabetic neuropathic pain (DPN),

- cancer-associated pain (e.g. cancer-induced bone pain (CIBP),

- pain associated with spinal cord injury (SCI pain),

- pain associated with multiple sclerosis (MS-pain),

- pain associated with bone- and cartilage-remodelling and ensuing damage of nerves (e.g. osteoarthritic pain, metastases-associated bone pain), and

- pain associated with stroke (e.g. post-stroke pain, thalamic syndrome, amongst others).

3. The selective LE inhibitor for use according to claim 1 or 2, wherein the selective inhibitor is used to alleviate pain associated with the respective disorders.

4. The selective LE inhibitor for use according to any one of claims 1 to 3, wherein the selective inhibitor is used for treating neuropathic pain and/or preventing chronic pain harbouring neuropathic components.

5. The selective LE inhibitor for use according to any one of claims 1 to 4, wherein the selective inhibitor is selected from

- Sivelestat, (Sivelastat sodium and Sivelastat sodium tetrahydrate),

- Elastatinal, - oral neutrophil elastase inhibitor ONO-681 8 (2-(5-a.mino-6-oxo-2-phenyl- 1 ,6-dihydro- pyrimidin- 1 -yl)-N- [( 1 R,2R)- 1 -(5 -tert-butyl - 1 ,3 ,4-oxadi azol-2-yl)- 1 -hydroxy- 3 -methylbutan- 2-yl]acetamide),

- BAY 85-8501,

- SSR 69071 (2-[[6-Methoxy-4-(l-methylethyl)-l ,l-dioxido-3-oxo-l,2-benzisothiazol-2(3H)- yl]methoxy]-9-[2-(l-piperidinyl)ethoxy]-4H-pyrido[l ,2-a]pyrimidin-4-one),

- Alvelestat (AZD9668; AZD-9668; AZD 9668),

- M0398 (N-(Methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone).

6. The selective LE inhibitor for use according to any one of claims 1 to 5, wherein the selective inhibitor is administered

- systemically

such as oral, topical, inhalative, intranasal, intraperitoneally, subcutaneously, sublingually, via suppositories and pessaries, via transdermal application systems,

intravenously, intraarterially, intramuscularly

by inhalation spray or rectally in dosage unit formulations containing conventional non toxic pharmaceutically acceptable earners, adjuvants and vehicles,

or

- locally,

such as via local injections or application in the skin, muscle or body organs, including intrathecally, epidurally, subdurally, via intracerebro ventricular injections or direct injections into peripheral nerves, sensory ganglia, spinal cord or the brain.

7. The selective LE inhibitor for use according to any one of claims 1 to 5, wherein the selective inhibitor is administered in combination with a further pain medication,

e.g. non-steroidal antiinflammatory agents (e.g. Aspirin, ibuprofen, Diclofenac, Celecoxib etc), Paracetamol and Metamizol, Opioid-analgetics, Gabapentin, Pregabalin, Tapentadol, diverse antidepressive, cannabinoids, local anesthetics, Carbamazepin, Lamotrigin and other sodium channel blockers, Ziconotid and other calcium channel blockers, blockers of Transient Receptor Potential (TRP) channels.

8. A pharmaceutical composition comprising

at least one selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) according to any of claims 1 to 6, optionally, a pharmaceutical excipient,

optionally, a further pain medicament.

9. A method for the diagnosis, prognosis, prevention and/or treatment of neuropathic pain and chronic pain states harbouring a neuropathic component,

comprising the step of

administering a therapeutically amount of at least one selective inhibitor of neutrophil elastase (also called leukocyte elastase, LE) or a pharmaceutical composition comprising at least one selective inhibitor of LE to a patient or subject in need thereof.

10. The method of claim 9, wherein the at least one selective inhibitor is administered to a subject in need thereof

- systemically

such as oral, topical, inlialative, intranasal, intraperitoneally, subcutaneously, sublingually, via suppositories and pessaries, via transdermal application systems,

intravenously, intraarterially, intramuscularly

by inhalation spray or rectally in dosage unit formulations containing conventional non toxic pharmaceutically acceptable earners, adjuvants and vehicles,

or

- locally,

such as via local injections or application in the skin, muscle or body organs, including intrathecally, epidurally, subdurally, via intracerebroventricular injections or direct injections into peripheral nerves, sensory ganglia, spinal cord or the brain.

1 1. The method of claim 9 or 10, wherein the wherein the neuropathic pain and chronic pain states harbouring a neuropathic component is selected from

- traumatic, metabolic and genetically-induced neuropathies (e.g. peripheral nerve injury- or trauma-associated neuropathic pain, trigeminal neuralgia etc),

- diabetic neuropathic pain (DPN),

- cancer-associated pain (e.g. cancer-induced bone pain (CIBP),

- pain associated with spinal cord injury (SCI pain),

- pain associated with multiple sclerosis (MS-pain),

- pain associated with bone- and cartilage-remodelling and ensuing damage of nerves (e.g. osteoarthritic pain, metastases-associated bone pain), and - pain associated with stroke (e.g. post-stroke pain, thalamic syndrome, amongst others).

12. The method of any of claims 9 to 11, wherein the selective inhibitor is used to alleviate pain associated with the respective disorders.

13. The method of any of claims 9 to 12, wherein the selective inhibitor is used for treating neuropathic pain and/or preventing chronic pain harbouring neuropathic components.

14. The method of any of claims 9 to 13, wherein the selective inhibitor is selected from

- Sivelestat, (Sivelastat sodium and Sivelastat sodium tetrahydrate),

- Elastatinal,

- oral neutrophil elastase inhibitor ONO-6818 (2-(5-amino-6-oxo-2-phenyl-l,6-dihydro- pyrimidin-1 -yl)-N-[(l R,2R)- 1 -(5-tert-butyl- 1 ,3,4-oxadiazol-2-yl)- 1 -hydroxy-3-methylbutan- 2-yl]acetamide),

- BAY 85-8501,

- SSR 69071 (2-[[6-Methoxy-4-(l-methylethyl)-l ,l-dioxido-3-oxo-l ,2-benzisotluazol-2(3H)- yl]methoxy]-9-[2-(l-piperidinyl)ethoxy]-4H-pyrido[l,2-a]pyrimidin-4-one),

- Alvelestat (AZD9668; AZD-9668; AZD 9668),

- M0398 (N-(Methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone).

15. The method of any of claims 9 to 14, wherein the selective inhibitor is administered in combination with a further pain medication,

e.g. non-steroidal antiinflammatory agents (e.g. Aspirin, Ibuprofen, Diclofenac, Celecoxib etc), Paracetamol and Metamizol, Opioid-analgetics, Gabapentin, Pregabalin, Tapentadol, diverse antidepressive, cannabinoids, local anesthetics, Carbamazepin, Tamotrigin and other sodium channel blockers, Ziconotid and other calcium channel blockers, blockers of Transient Receptor Potential (TRP) channels.

16. The selective LE inhibitor for use according to any one of claims 1 to 5 or the method of any of claims 9 to 14, wherein the selective inliibitor of LE is not a cephalosporine.

17. The selective LE inhibitor for use according to any one of claims 1 to 5 or the method of any of claims 9 to 14, wherein the selective inhibitor is administered via sustained release, controlled release, or delayed release.