# Patent

## Title
Use of a granulin or a granulin-like compound in the therapy or prophylaxis of chronic pains

## Abstract

The invention relates to the use of a granulin or a granulin-like compound for producing a pharmaceutical composition for the therapy or prophylaxis of chronic pain, in particular for neuropathic pain.

## Background
CROSS-REFERENCE TO A RELATED APPLICATION
This application is a National Stage Application of International Application Number PCT/DE2008/001138, filed Jul. 14, 2008; which claims priority to German Application No. 10 2007 033 359.7, filed Jul. 16, 2007, which are incorporated herein by reference in their entirety, including sequences, tables and figures.
The invention relates to the use of a granulin or a granulin-like compound for the production of a pharmaceutical composition for the therapy or prophylaxis of chronic pain, in particular for the production of pharmaceutical compositions for the therapy or prophylaxis of chronic pain. Furthermore, the invention relates to a respective pharmaceutical composition.
In patients that suffered from neuronal damages, whether traumatic, inflammatory, infectious, tumor-related, ischemic, degenerative, metabolic or toxic, often secondary neuronal damages, such as cellular death and chronic pain, in particular neuropathic pain, take place as a cause of these neuronal damages.
Neuropathic pain can emerge from manifold damages of peripheral and central neuronal cells Pain is common in, amongst others, diabetic or toxic (such as, for example, due to a chemo-therapeutical therapy) polyneuropathy, phantom pains following amputation, postzosteric neuralgia, trigeminus neuralgia, compression syndrome, such as the carpal tunnel syndrome, and ischialgia in spinal prolapse, pain in multiple sclerosis and post-ischemic neuralgia. The non-invasive medicamentous possibilities that are known from the state of the art are limited to the use of different antiepileptics (e.g. gabapentin, pregabalin), anti-depressives (e.g. amitrytilin) and opioids, which, nevertheless, can only achieve a sufficient analgetic effect with simultaneously tolerable side effects in only about 30% of the patients, and do not influence the neuronal damage.
The mechanisms that contribute to the generation of chronic, in particular neuropathic, pain are diverse. Experimental studies in recent years show that the transsynaptic destruction of inhibitory neurons and the activation of glial cells essentially add to this.
Accordingly, it is an object of the present invention to provide a method or a means, respectively, which can be used for the therapy or prophylaxis of a neuronal damage, in order to particularly favour the regeneration in partially peripheral neuronal lesions, and to prevent the secondary cellular death.
The quintessence of the present invention consists in the reduction of the loss of inhibitory neurons and the block of the glial-mediated inflammatory reaction through the therapeutic use of a neuroprotective compound, and thus to avoid a progressing damage through continuous overexcitement. This reduces chronic, in particular neuropathic, pain, and supports regenerative processes.
The object underlying the invention is solved by the use of a granulin or a granulin-like compound for producing a pharmaceutical composition for the therapy and/or prophylaxis of chronic pain. Therein, the term “chronic pain”, amongst others, comprises neuropathic pain, and inflammatory and tumor-related pain.
The use of a granulin or a granulin-like compound for the therapy or prevention of chronic, in particular neuropathic, pain in an acute or chronic neuronal damage represents a new therapeutic principle that, in contrast to the available medicamentous possibilities (antiepileptics, antidepressives, opioids, and cannabioids), positively influences the pathophysiologically underlying mechanisms.
According to the invention, it is possible to avoid the destruction of inhibitory interneurons. In doing so, the insufficient blocking of excitatory impulses, and thus the overactivity of pain-transmitting tracts, is avoided. The chronification of neuropathic pain is therefore reduced through the resulting limitation of the remodelling processes and misconnections that occur due to the apoptosis of inhibitory neurons. In contrast to this, the medicaments that are available until now lead to a general damping of the neuronal activity and do not influence the pathophysiological mechanisms in neuropathic pain resulting from peripheral or central neuronal damage.
In a preferred embodiment of the invention, the granulin or a granulin-like compound is at least one molecule selected from the group containing progranulin, granulin 1, granulin 2, granulin 3, granulin 4, granulin 5, granulin 6, granulin 7, paragranulin, progranulin signaling molecule, and fragments of these molecules that are functionally equivalent to these molecules, and an agonist that is functionally equivalent to these molecules. As will be further discussed below, the agonist particularly can be a peptidomimetic.
In addition, the use of combinations of different molecules from the above-mentioned group is included in the present invention.
Basically, the invention relates to both proteins or peptides, respectively, and nucleic acids, such as DNA, cDNA or RNA, as well as their derivatives.
Accordingly, in one embodiment of the invention the granulin or the granulin-like compound can be a nucleic acid encoding for one of said molecules. In particular, the nucleic acid can be a nucleic acid according to one of SEQ. ID. NOs. 1 to 10 or a nucleic acid that is identical to at least 50%, preferably to 60%, 70% or 80%, in particular preferably to 85% or 90%, most preferred to 95% or 98% to one of these sequences. Thereby, the peptide or protein that is encoded by the nucleic acid is advantageously functionally equivalent to a granulin or a granulin-like compound according to SEQ. ID. NO. 11 to 20.
A fragment of a nucleic acid shall mean an arbitrary sequence of at least 50, preferably of at least 150, particularly preferred of at least 180 continuous nucleotides from the sequence of granulin or a granulin-like compound found in a nucleic acid encoding granulin or a granulin-like compound.
The nucleic acid encoding for a functionally equivalent fragment is identical to at least 50%, preferably to 60%, 70% or 80%, in particular to 85% or 90%, most preferably to 95% or 98% with respect to the nucleic acid encoding for said fragment from SEQ. ID. NO. 11 to 20.
“Functionally equivalent” shall mean a fragment that has the same qualitative effect on neurons and glial cells as a granulin or a granulin-like compound according to SEQ. ID. NOs. 11 to 20, wherein the quantitative effect can be different.
Alternatively to this, in another embodiment of the invention the granulin or the granulin-like compound can be a protein or a peptide. In particular, the peptidomimetic as already mentioned above has to be mentioned here.
In a particularly preferred embodiment the granulin or the granulin-like compound is a protein or a peptide according to one SEQ. ID. NOs. 11 to 20 or a peptide or protein that is identical to at least 50%, preferably to 60%, 70% or 80%, in particularly preferred to 85% or 90%, most preferably to 95% or 98% to one of these sequences.
A fragment of proteins or peptides shall mean an arbitrary sequence present in a granulin or a granulin-like compound of at least 5, preferably of at least 10 or at least 20, particularly preferably of at least 30, 40, most preferably of at least 50 continuous amino acids from the sequence of granulin or a granulin-like compound.
The above described compound is suitable for producing pharmaceutical compositions for the therapy or prophylaxis of chronic, in particular neuropathic pain, in mammals, in particular in a human, but also in pets and livestock. The present invention is described with respect to human nucleic acid and protein/peptide sequences. Nevertheless, the person of skill, based on the human sequences and using known methods, can also detect the sequences of other species, as long as they are not contained in the publicly available databases.
The use as described above is also suitable for the production of pharmaceutical compositions for the reduction of neuropathic pain, the avoidance of cellular death of damaged neurons, and/or for the promotion of the neuronal regeneration.
The underlying problem of the invention is also solved by a pharmaceutical composition comprising and/or containing a granulin or a granulin fragment functioning equivalently, or a granulin-agonist.
Thereby, the granulin or the granulin-like compound is preferably at least one molecule selected from a group containing progranulin, granulin 1, granulin 2, granulin 3, granulin 4, granulin 5, granulin 6, granulin 7, paragranulin, progranulin signaling molecule, fragments of the aforementioned molecules that are functioning equivalently, and agonists (e.g. a peptidomimetic that is not one of the aforementioned fragments) of these molecules that are functioning equivalently.
In analogy to the above description, the granulin or the granulin-like compound of the pharmaceutical composition can be a nucleic acid encoding for one of said molecules, in particular a nucleic acid according to one of the SEQ. ID. NOs. 1 to 10 or a nucleic acid that is at least 50%, preferably to 60%, 70% or 80%, in particular preferably to 85% or 90%, most preferably to 95% or 98% identical to one of these sequences. Thereby, the encoded peptide or protein is equivalently effective in the sense of the above definition.
The said nucleic acids can be administered to a patient through transfer of a construct comprising said sequences, such as, for example, viruses or plasmids, into at least one cell. The transfection of the constructs can take place by means of known methods, such as lipofection, ballistic transfer (“gene gun”), etc.
In an alternative embodiment, the granulin or the granulin-like compound of the pharmaceutical composition is a protein or a peptide. Thereby, it is advantageous if the granulin or the granulin-like compound is a protein or a peptide according to one of the SEQ. ID. NOs. 11 to 20 or a protein or peptide that is identical to one of the sequences to at least 50%, preferably to 60%, 70% or 80%, in particular preferably to 85% or 90%, most preferably to 95% or 98%.
The pharmaceutical composition as described can preferably be used for producing pharmaceutical compositions for the therapy or prophylaxis of chronic, in particular neuropathic, pain in mammals, in particular in pets and livestock, and in the human. Although the sequences as given here are all derived from the human, based on the sequences as given here the skilled person can identify and isolate the corresponding molecules in other organisms.
According to the invention, the pharmaceutical composition can also serve for producing pharmaceutical compositions for the reduction of chronic, in particular neuropathic pain, avoidance of the cellular death of damaged neurons, and/or the promotion of the neuronal regeneration.
The pharmaceutical composition according to the invention can be present in the form of tablets, dragees, pills, granulates, aerosols, infusion solutions, emulsions, suspensions or solutions.
The use of the agents or the pharmaceutical composition according to the invention, respectively, can take place using suitable known formulations.
The use of the agents according to the invention can be converted in a known manner into the common formulations, such as tablets, dragees, pills, granulates, aerosols, emulsions, suspensions and solutions, using inert, non toxic, pharmaceutically acceptable carriers or solvents. Hereby, the therapeutically effective concentration of agent in relation to the granulin or the granulin-like compounds shall be present in the overall mixture in a concentration of about 0.1 wt.-% to 95 wt.-%, preferably of about 0.5 wt.-% to 90 wt.-%, respectively, i.e. in amounts that are sufficient in order to achieve the concentration of agent as required in the tissue as targeted.
The formulations are, for example, produced by stretching of the agents with solvents and/or carriers, optionally using emulsifiers and/or dispersants, wherein, for example in case of using water as a diluent, optionally organic solvents can be used as auxiliary agents.
Mentioned as auxiliary agents shall be, for example, water, non-toxic solvents, such as paraffin (e.g. crude oil fractions), plant oils (e.g. peanut, sesame oil), alcohols (e.g. ethyl alcohol, glycerol), carriers, such as natural stone dusts (e.g. kaolins, alumina, talcum, chalk), synthetic stone dusts (e.g. highly disperse silica, silicates), sugars (e.g. cane, milk and grape sugar), emulgators (e.g. polyoxyethylene fatty acid esters, polyoxyethylene fatty acid ethers), dispersants (e.g. lignin, spent sulfite liquors, methyl cellulose, starch and polyvinyl pyrrolidone) and lubricants (e.g. magnesium stearate, talcum, stearic acid and sodium sulfate).
The application takes place in a common way, preferably orally or parenterally, in particular perlingually or intravenously. In case of the oral use of the medicaments according to the invention, tablets can naturally also contain additives, such as sodium citrate, calcium carbonate and dicalcium phosphate, together with several additives, such as starch, preferably potato starch, gelatine, and the like, in addition to the carriers as mentioned. Furthermore, lubricants, such as magnesium stearate, sodium lauryl sulphate and talcum can be included for tabletting. In case of aqueous suspensions, several taste improving agents or colouring agents can be added to the active agents in addition to the above mentioned auxiliary agents. In case of a parenteral administration, solutions of the active agents using suitable liquid carrier materials can be employed.
The invention also relates to the use of a granulin or a granulin-like compound as described here for the therapy or prophylaxis of neuropathic pain, or a granulin or a granulin-like compound as described here and its use for the therapy or prophylaxis of neuropathic pain.
The invention also relates to a method for the therapy or prophylaxis of an individual, in particular of a patient, using a granulin or a granulin-like compound or a pharmaceutical composition according to the description as above.
The invention is further explained based on examples, without being limited to these examples. The results of the experiments as described in the examples are shown in the Figures.



FIGURES
FIG. 1: Progranulin is a cysteine-rich protein, which is cleaved by yet unknown proteases into seven different granulins (FIG. 1) that are secreted, and like the starting product exhibit growth factor and cytokine-like effects, promote wound healing, regulate tumour growth, and neuronal development. Until now, extra- and intracellular “receptors” are unknown. In the human, a “loss-of-function” mutation of the progranulin-gene causes a fronto-temporal dementia. Intron-exon-structure of the human gene for progranulin Grn, exons are depicted as boxes. The progranulin-protein is cleaved by proteases into 7 different granulins (black boxes) and an N-terminal “signalling peptide”. All granulins have a structure similar to granulin A (Cx-Cx-CCx-CCx-CCx-CCx-Cx-C). C=cysteine, X=variable number of other amino acids.
FIG. 2: Regulation of the progranulin-expression in the spinal cord and DRGs after peripheral lesion of the N. ischiadicus. 
A Neuropathic models, Spared Nerve Injury (SNI, Decosterd & Woolf), Chronic Constriction Injury (CCI, Bennett), Spinal Nerve Ligation (SNL, Chung).
B Microarray analysis of the progranulin mRNA in the dorsal horn and DRGs in the models shown above.
C Quantitative RT-PCR of the progranulin mRNA in the SNI model.
D Western Blot analysis of the progranulin protein expression in the spinal cord in the SNI model.
E Progranulin-protein in DRGs after SNI.
F Progranulin-protein expression in DRGs in STZ induced diabetes polyneuropathy-model, 3 months after diabetes induction.
FIG. 3: Progranulin in situ hybridisation and post-in situ immunofluorescence for glial fibrillary acid protein, GFAP and the transcription factor ATF-3.
A Grn in situ (black) and GFAP immunofluorescence (red) for demonstrating the astrocytes after SNI in the dorsal horn of the spinal cord.
B Higher magnification of A. Progranulin is not expressed in astrocytes.
C Grn in situ (black) in microglial (small cells) and motor neurons (arrow) in the anterior horn of the spinal cord after SNI.
D Anterior horn as in C with demonstration of damaged motor neurons by ATF-3 immunofluorescence (red). ATF-3 is a marker for neurons with damaged axon.
FIG. 4: Overview of the microglial reaction in the spinal cord after SNI. Demonstration of the microglia by immunofluorescence for the receptor of the complement factor 3 (CD11b).
FIG. 5: Microglial reaction in the anterior horn after SNI
A CD11b immunofluorescence for the demonstration of active microglial cells in the ventral horn of the spinal cord after SNI.
B Progranulin mRNA (in situ hybridisation, grey) in activated microglial cells (arrow) next to motor neurons (red, peripherin-immunofluorescence).
FIG. 6: Neurons of the dorsal ganglia (peripherin IR red) with expression of the progranulin mRNA (in situ hybridisation, grey) after SNI.
FIG. 7: Progranulin mRNA expression and behaviour of mice upon continuous spinal infusion of progranulin siRNA after SNI.
A Grn mRNA after SNI in the spinal cord upon treatment with Grn siRNA and control-oligonucleotides. The treatment took place using a subcutaneous implanted osmotic Alzet pump via a spinal catheter, ending at the level of L3. Infusion begins immediately after SNI and ends 4 weeks after SNI.

B. Behaviour of the mice (C57Black6) after SNI upon treatment with Grn siRNA or control oligonucleotides in different nociceptive tests and in the rotarod test that measures the motoric performance. Mechanic allodynia was measured by measuring the “withdrawal”-latency with a dynamic von Frey aesthesiometer, the shorter the latency, the stronger the allodynia (top left). Cold-allodynia was recorded in the acetone assay (top right) and with a cold plate at 10° C. (bottom right). In the acetone assay the duration of the reaction (licking, shaking, lifting) of the paw after application of a drop of acetone on the sole of the foot was measured, the longer the reaction, the stronger the allodynia. The reaction latency after exposition against cold is measured with the cold plate, the shorter the stronger the allodynia. It is measured in the rotarod test, how long the mouse runs on a turning cylinder without falling down. Measured is the motoric coordination and exhaustion as well as ability for compensation after neuronal lesion. The behavioural experiments show an increase of the allodynia and impaired motoric recovery upon treatment with Grn siRNA. The treatment of mice by spinal infusion of progranulin siRNA blocks the up-regulation of the progranulin occurring after SNI and leads to an increase of the pain-like behaviour in mice. In addition that recovery of the motoric function in the rotarod test is slightly slower than in control animals that were treated with control-oligonucleotides.

FIG. 8: Progranulin/granulin protein expression in cells and mouse tissues. Western blot with N19-antibody (SantaCruz Biotechnology) directed against progranulin, granulin 1, 2, and 7. Molecular weight of the glycosylated progranulin 88 kDa, the individual granulins 6 kDa. Tricine gels were used for separating the small peptides. Abbreviations: MF macrophages, LPS lipopolysaccharide, SC spinal cord, DRG dorsal ganglia, HEK293 human embryonic kidney cells, MCF-7 breast cancer cells, PC 12 phaeochromocytoma cells (neuron-like), PAGE polyacrylamide-gel-electrophoresis.



EXAMPLES
In the following the invention is explained by means of the experiments as described here with reference to the results as depicted in the Figures.
Methods Progranulin
Models of Neuropathy and RNAi Treatment
Adult C57B16 mice were used. In the spared nerve injury (SNI) model two branches of the nervus (N.) ischiadicus, namely the N. peronaeus and N. tibialis were ligated and distally cut through. The N. suralis remains intact. Using an osmotic pump (Alzet osmotic pump 2004; 0.25 μl/h) silencer RNA (stealth RNAi, Invitrogen) for acrogranin was intrathecally infunded continuously via a spinal catheter (PTFE sub-lite wall tubing; OD/ID 0.15 mm/0.05 mm). The tip of the catheter was placed at the level of the lumbal cord, the osmotic pump was fixed in the subcutaneous tissue. The infusion started directly after SNI operation and lasted for 4 weeks. A mixture of three different acrogranin RNAis (1 nmol per each mouse for the duration of 4 weeks) or control RNAis, respectively, was used. In addition, an FITC labelled dummy-RNAi (20 nmol) was infunded for a later immunohistological localisation. The infusion solution consisted of Ringer solution with 1:40 diluted lipofectamin (195 μl Ringer+5 μl lipofecatmin per pump).
Nociceptive Behaviour
The animals were familiarized with the test cages for 3×30 min. The measurement of the behaviour was performed without the knowledge about the treatment. The reaction latency for mechanic stimulation of the hindpaw was measured using a dynamic von Frey device (Dynamic aesthesiometer, UgoBasile, Italien). Using a “cold plate” (4° C.) the pain sensitivity against cold was measured by counting the adverse reactions (licking, jumping, lift leg) over a period of 90 sec. Using a 52° C. “hot plate” the pain sensitivity against heat was determined by measuring the latency until the first reaction. The cut-off latency was 30 sec, in order to avoid issue damages. In the rotarod test the motoric coordination or the motoric recovery after neuronal lesion, respectively, was measured (90 sec cut-off).
Microarrays
The total-RNA was extracted from homogenised spinal cord or DRG tissue by means of Qiagen RNA-extraction kit, and transcribed into DNA. cDNA fragments were amplified by means of PCR and ligated into the pCR4 vector (TA Cloning Kit, Invitrogen). Biotinylated cRNA was produced through in vitro transcription and used for the hybridisation of the Affymetrix RGU34A chips.
Quantitative RT-PCR
The total-RNA was extracted from homogenised spinal cord or DRG tissue by means of Qiagen RNA-extraction kit, and transcribed into DNA. The quantitative real-time rt-PCR was performed using the Sybr-Green detection systems in accordance with the manual of the provider (Applied Biosystems). Primer sets were selected with Primer Express. The amplification of specific PCR-products was verified by agarose gelelectrophoresis.
Western Blot
Spinal cord or DRG tissue was homogenized in PhosphoSafe extraction buffer (Novagen), the homogenate was centrifuged (40.000 g, 20 min) and the proteins in the supernatant were separated by polyacrylamide or tricine gel electrophoresis, respectively. The proteins were transferred onto nitrocellulose-membranes by means of wet-blot, blocked in PBST-5° A) milk (0.05 Tween 20 in 1 mol/l PBS with 5% low-fat milk powder), incubated over night with antibodies against acrogranin (1:1000 in PBST), washed, incubated with secondary antibodies 2 hours (h) at room temperature and evaluated using the Odyssee Western Blot Scanner.
In Situ Hybridisation
Mice were sacrificed through CO2 and exsanguination, L4/5 DRGs and spinal cord were rapidly removed, embedded in OCT, frozen on dry ice, and cut with a cryotome into 14 μm sections. Sections were postfixed in 4% paraformaldehyde, and acetylated. DIG-labelled ribosamples were generated through in vitro transcription of cDNA fragments and incorporation of digoxigenin-labelled dUTP (Dig-labelling kit, Roche). The sections were prehybridized for 2 h at RT in prehybridization solution (5×SSC, 50% formamide, 2×Denhardt's, 500 μg/ml herring sperm DNA, 250 μg/ml yeast tRNA) and subsequently hybridized with 250 ng/ml riboprobe (antisense and sense) in prehybridization solution for 16 h at 72° C. in the oven. The sections were washed in 0.2×SSC at 68° C. for 2 h, incubated with anti-sig-AP antibody (1:1000 in 1% blocking reagent (Roche) in 0.1 mol/l maleic acid buffer) at 4° C. overnight and developed with NBT/BCIP/Levamisol (Boehringer Mannheim). The sections were embedded in glycerol/gelatine or further developed through post in situ immunofluorescence with respective antibodies.
Immunofluorescence
For a fixation the animals were intracardially perfused with 1×PBS and subsequently 4% paraformaldehyde. The tissue was prepared, post-fixed in 4% PFA for 2 h, protected against cryoartifacts over night in 20% sucrose in 1×PBS, embedded in OCT and 12 or 14 μm sections, respectively, were cut with a cryotome. The sections were blocked in 1×PBS with 0.03% Triton X-100 and 1% blocking reagent (Roche) for 2 h at RT, incubated over night with primary antibodies in blocking-solution (see above) at 4° C., washed, then incubated with species-specific Cy3, Alexa-488 or FITC labelled secondary antibodies in blocking-solution for 2 h at RT, washed again, treated with 0.02% Sudan black for a reduction of the autofluorescence, and embedded with Vectashield. The analysis took place at the fluorescence (Nikon) or laser scanning microscope (Zeiss), respectively.
The following primary antibodies were used: Mouse NF200 1:4000 (Sigma), mouse CGRP 1:1000 (Sigma), rabbit ATF-3 1:300 (SantaCruz). FITC-labeled griffonia simplicifolia isolectin B4 (Sigma) 1:500, rabbit GFAP (1:1000, Chemicon), rabbit anti-peripherin (1:1000, Chemicon), rabbit Iba-1 (1:500, DAKO).
After a peripheral neuronal damage a microglial activation occurs in the ipsilateral spinal cord, which is important for the generation of neuropathic pain. These activated microglial cells exhibit a strong expression of progranulin, as shown by microarray (FIG. 2), QRT-PCR (FIG. 2), Western Blot (FIG. 2), and in situ hybridization (FIG. 3). DRG- and motor neurons that are directly damaged by the neuronal lesions exhibit a weaker expression (FIG. 3-4).

## Claims

1. A method for treating neuropathic pain, wherein said method comprises administering, to a subject in need of such treatment, a therapeutically-effective amount of a neuroprotective granulin-like compound comprising amino acid sequence having at least 90% sequence identity to SEQ ID NO:11, or a fragment of the granulin-like compound, wherein the granulin-like compound and the functional fragment thereof have neuroprotective activity, and wherein neuropathic pain is treated.

2. The method according to claim 1, wherein the neuroprotective granulin-like compound comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:11.

3. The method according to claim 1, wherein the neuroprotective granulin-like compound comprises the amino acid sequence of SEQ ID NO:11.

4. The method according to claim 1, wherein the functional fragment of the granulin-like compound comprises at least 30 amino acids.

5. The method according to claim 1, wherein the functional fragment of the granulin-like compound comprises at least 50 amino acids.

6. The method according to claim 1, wherein the method comprises administering a therapeutically effective amount of a neuroprotective granulin-like compound comprising the amino acid sequence of SEQ ID NO:11.