DE102005020798A1 - Pharmaceutical for treatment of central nervous system diseases, comprises toll-like receptor ligand and/or Nod2 ligand - Google Patents

Abstract

At least one toll-like receptor (TLR) ligand and/or a Nod2 ligand is used for a pharmaceutical for the treatment of diseases affecting the central nervous system. The ligand is selected from the group comprising lipopolysaccharides (LPS), R848, polyinosinic acid-polycytidylic acid, N-acetylmuramyl dipeptide or their derivatives.

Description

The The present invention relates to the use of at least one TLR and / or Nod2 ligands for the manufacture of a medicament for Treatment of a disease of the central nervous system as well as pharmaceutical Compositions containing at least one TLR and / or Nod2 ligand for the treatment of a disease of the CNS.

diseases of the central nervous system (CNS) include a variety of neurological and neuropsychiatric diseases, such as neurodegenerative diseases, behavioral disorders, mood disorders and cognitive disorders. These diseases are often associated with genetic predispositions, Allergies, drug side effects, traumatic injuries or cerebrovascular operations such as aneurysms or strokes associated. There are also some CNS disorders that have no obvious cause triggered become.

Lots neurological disease states However, you go with one due to illness or injury triggered Loss of certain cell populations of the nervous system is accompanied, wherein the destroyed Cells are not functional Cells can be replaced. Many neuronal diseases, such as Parkinson's, Alzheimer's, multiple Sclerosis or amyotrophic lateral sclerosis are due to a destruction of neuronal Cables. The aim of a therapy must therefore be this neuronal Regenerate or replace lines.

One Approach to treating such neurological diseases the transplantation of cells through which the neural Circuits are to be restored at least partially. After that it should be possible be injured by transplantation of neural stem cells or to regenerate or replace dead nerve tissue.

Around However, achieving success with this approach requires many different ones Factors taken into account from disease to disease and from patient to patient are different and must be taken into account, and making this therapy very complex, costly and complicated do.

One Another approach is drug treatment, in particular such medicines are used which increase the availability of neurotransmitters increase.

This Approach, however, is very limited in its spectrum of action and often associated with severe side effects.

newer Findings showed that neuronal progenitor cells persist in the brain and spinal cord, in the case of neuronal injury to the regeneration of the injured Tissue can contribute. Stem / progenitor cells are for the tissue homeostasis and repair responsible. In the spinal cord provides the present there Population of neural progenitor cells (neural progenitor cells, NPC) for these regeneration processes. Furthermore, it could be shown that neural stem cells are dead and dying cells as well as dead neural leads in the degenerative CNS.

outgoing From this prior art, it is therefore an object of the present Invention, new therapeutic approaches and in particular to provide substances with which neural Diseases can be effectively and easily controlled.

These The object is achieved by the Use of TLR and / or Nod2 Ligands for the Preparation of a Drug solved, which is used to treat neuronal CNS diseases becomes.

The Task is further solved by providing a pharmaceutical composition for the treatment of CNS disorders, wherein the pharmaceutical composition contains at least one TLR and / or Nod2 ligand.

Further the task is solved through the use of a TLR and / or Nod2 ligands for the manufacture of a medicament for the treatment CNS produced by stimulating growth, the proliferation or differentiation of neuronal precursor cells is treatable.

The The object underlying the invention is thereby perfect solved.

The Inventors have shown in their own experiments that by administration TLR or Nod2 Ligands Proliferation of Neuronal Stem Cells could be achieved. This creates the opportunity through the proliferation and differentiation of neuronal precursor cells - eg. in the spinal cord, destroyed tissue to regenerate or replace. In this way can diseases treated by the CNS, causing destruction or impairment underlying neuronal conduits or neuronal tissue.

Of the Nod2 receptor and the Toll-like receptors (or tolllike receptors, hereafter TLR) are transmembrane proteins of immune cells that play a key role in the innate immune system play. About that In addition, they show a connection between the innate and the adaptive immune system in vertebrates. The receptors were first However, in the fruit fly Drosophila melanogaster are discovered to corresponding receptors in immune cells of mammals strong homologous. Their function is the detection of pathogens and the release an immune response of cells directed against these pathogens is.

On Reason of specificity the Nod2 and Toll-like Receptors need the receptors recognize determinants that are predominantly of microorganisms whereas host determinants are rarely recognized become. Therefore, the TLR recognize molecules or parts of molecules of one pathogenic organism that are extremely well preserved. Well preserved Examples are e.g. bacterial cell surfaces, lipopolysaccharides (LPS), Lipoproteins, lipopeptides, proteins such as flagellin, double-stranded RNA of viruses, etc.

Nod2 and TLR are key receptors, by which mammals detect the presence of an infection by identifying specific ones biomolecules derived from microorganisms. Put these biomolecules pathogen-associated molecular patterns (pathogen-associated molecular patterns, hereinafter "PAMPs"). The release of PAMPs during a Infection forms the signal for the Nod2 and TLRs and triggers the innate immune response.

Structurally similar TLRs usually respond to the same types of ligands. TLR2 is eg. essential in the detection of peptidoglycans such as N-acetylmuramyl-dipeptide, TLR3 is involved in the recognition of double-stranded RNRs, which are derived from Virus is derived, and TLR4 is activated in particular by LPS. natural Ligands for TLR9 are bacterial oligonucleotides that have CpG motifs. Of the Nod2 receptor recognizes muramyldi peptide (MDP). Activation of the Nod2 and TLR signaling paths by different ligands leads to the expression of many genes, which serve as defense in the host, such as inflammatory Cytokines, chemokines, MHC (major histocompatibility complex) molecules, co-stimulatory molecules and various effector molecules.

Especially is preferred when the drug is used to treat a disease of the CNS, the disease being selected the group comprising amyotrophic lateral sclerosis, multiple sclerosis, Muscular dystrophy, Alzheimer's disease, Parkinson's disease, and other degenerative processes of the CNS, e.g. due to vascular undersupply triggered become.

The Amyotrophic lateral sclerosis is characterized, for example, by a progressive Degeneration of the first and second neurons in the spinal cord and in the extended one Mark out, multiple sclerosis is a markscheidenzerfall to observe. In Parkinson's disease is the substantia nigra degenerated in the midbrain. The possibility of stimulation or Differentiation of intact neuronal precursor cells in these diseases by Nod2 or TLR ligands provides an excellent therapeutic approach for the Treatment of the respective nerve endings.

Further The drug can be used to treat a disease of the CNS The disease is a consequence of a traumatic injury of the CNS.

By Stimulation of the proliferation or differentiation of intact neuronal Precursor cells through Nod2 or TLR ligands can treated spinal cord lesions become. The spinal cord lesions include, for example. Traumatic injuries of the CNS or ischemic lesions, for example, the consequences of a Stroke can be. These injuries can also to a paralysis of the limbs to lead, because motor neurons destroyed are. Therefore, the drug can also be used to treat by a stroke induced ischemic lesions of the CNS are used.

It is preferred if in the preparation of the medicament a Nod2- or TLR ligand selected from the group comprising Lipopolysaccharides (LPS), R848, poly inosine polycytidylic acid (Poly I: C), N-acetylmuramyl-dipeptide, or derivatives thereof.

With some of the mentioned Nod2 and TLR ligands could induce proliferation of neural progenitor cells. The TLR ligands were administered peripherally.

The Lipopolysaccharide is a characteristic component of the complex outer wall layer (outer membrane) Gram-negative bacteria (bacterial cell wall). The lipopolysaccharide molecules become synthesized in the cell at the cytoplasmic membrane and carrier bound outward transported. The lipopolysaccharides, for example of the Enterobacteriaceae are very similar built up. However, other Gram-negative bacteria families can have a very different lipopolysaccharide structure. lipopolysaccharide is detected by TLR4.

R848, an imidazoquinoline derivative, is recognized in humans by TLR7 and TLR8 also called Resquimod.

poly I: C is a synthetic homopolymer that closely resembles viral RNA. poly I: C is recognized by TLR3.

N-acetylmuramyl dipeptide is a derived building block of the polysaccharide chains of murein and thus part of the cell walls (Bacterial cell wall) of many bacteria and is recognized by the Nod2 receptor.

The The invention also relates to a pharmaceutical composition which at least one Nod2 and / or TLR ligand for the treatment of a disease of the CNS.

It is preferred when the pharmaceutical composition for treatment a disease of the CNS is selected that is selected from the group comprising amyotrophic lateral sclerosis, multiple sclerosis, Muscular dystrophy, Alzheimer's disease, Parkinson's disease.

The pharmaceutical composition can only one Nod2 or TLR ligands contain, furthermore however, different Nod2 and TLR ligands can also be used in combination.

The In addition, pharmaceutical composition may contain other ingredients and / or carrier usually included in the preparation of a pharmaceutical composition to be administered be used. Such ingredients are e.g. Diluents, Binders, suspending agents, lubricants, stabilizers, etc. These include, but are not limited thereto, e.g. Water, salt solutions, Alcohols, vegetable oils, Polyethylene glycols, monoglycerides, etc. An overview of such ingredients can be found, for example, in A. Kibbe, "Handbook of Pharmaceutical Excipients ", 3rd Edition 2000, American Pharmaceutical Association and Pharmaceutical Press.

ever after administration of the pharmaceutical composition is the Composition be formulated, so for example. When injected in the form of liquid Preparations. The administration may also be dependent systemically or locally directed by the patient and the disease be made.

The Composition may also contain other active ingredients or drugs such as interferons, EGF (epidermal growth factor), FGF (fibublast growth factor) and BDNF (brain-derived neurotrophic factor), as well as cytokines, chemokines, complement system proteins and / or cell adhesion molecules, the the role of neural progenitor cells in tissue homeostasis can modulate.

The Dose of administration depending on the illness and the patient, factors such as age and weight must be considered.

Further Advantages will be apparent from the figures and the example below.

It it is understood that the above and the following yet to be explained features not only in the specified combination, but also in other combinations or alone, without to leave the scope of the present invention.

embodiments The invention will be explained in more detail in the following figures. It demonstrate:

• (A) Anzahl der BrdU⁺-Zellen/m² in der grauen und weißen Substanz bei Verabreichung von N-acetylmuramyldipeptid, LPS, R848, Poly I:C und CpG Oligonucleotid;
• (B) Quantifizierung von BrdU⁺-Zellen im Ependym des Zentralkanals an den Tagen 2 und 3;

1 The distribution and quantification of BrdU ⁺ cells in the spinal cord on days 2 and 3 after injection of different TLR ligands:

• (A) Number of BrdU ⁺ cells / m ² in gray and white matter upon administration of N-acetylmuramyl dipeptide, LPS, R848, poly I: C and CpG oligonucleotide;
• (B) quantification of BrdU ⁺ cells in the ependyma of the central channel on days 2 and 3;

• (A) Markierungszeitraum nach Poly I:C-Injektion in der weißen und grauen Substanz;
• (B) Markierungszeitraum nach R848-Injektion in der weißen und grauen Substanz;

2 Change in BrdU ⁺ cell density after injection of poly I: C or R848:

• (A) labeling period after poly I: C injection in the white and gray matter;
• (B) marking period after R848 injection in the white and gray matter;

• (A) Poly I:C-Injektion
• (B) R848-Injektion;

3 the transient activation of microglial cells in the spinal cord after poly I: C or R848 injection:

• (A) Poly I: C injection
• (B) R848 injection;

• (A) ED-1⁺-Zellen pro mm² Rückenmark
• (B) BrdU⁺-Zellen pro mm² Rückenmark.

4 Investigation on inhibition of poly I: C-mediated cell activation after administration of dexamethasone and indomethacin:

• (A) ED-1 ⁺ cells per mm ² spinal cord
• (B) BrdU ⁺ cells per mm ² spinal cord.

example

1. material and methods

1.1 Animals used

Six to eight week old male Le Wis rats (Charles River, Sulzfeld, Germany) were kept under standardized laboratory conditions, with food and liquid being available in any amount. All procedures performed on the animals were carried out according to the International Health Guide Lines following a protocol approved by various animal welfare committees.

1.2 Reagents

phosphorothioate CpG ODN (5'-tccatgacgttcctgacgtt-3 '; SEQ ID NO: 1) from MWG-Biotech AG (Ebersberg, Germany). Pam3Cys, R848, poly I: C and N-muramyl-dipeptide (MDP) from InVivogen (San Diego, USA), LPS (of the E. coli serotype 026: B6) and indomethacin from Sigma (Deisenhofen, Germany) and BrdU and dexamethasone purchased from Serva (Heidelberg, Germany).

1.3 Animal experiments

Pam3Cys (5 mg / kg), N-acetylmuramyl-dipeptide (1 mg / kg), poly I: C (5 mg / kg), LPS (1 mg / kg), R848 (1 mg / kg) or PS CpG-ODN (5 mg / kg) were intraperitoneally injected. The control group was 1 ml of phosphate-buffered saline (PBS) also injected intraperitoneally. For the studies of inhibition of poly I: C-mediated cell activation, was 2 hours after Poly I: C administration of dexamethasone (5 mg / kg) or indomethacin (2 mg / kg).

For the histological The rats were examined at different times the injection was killed as shown below. Received two days before killing the rats twice daily BrdU injections (50 mg / kg, intraperitoneally) at intervals of 8 hours. Following this, the rats were anesthetized with diethyl ether; 4% paraformaldehyde (PFA) in PBS was then administered intracardially. The spinal cord was respectively removed and post-fixed in 4% PFA overnight at 4 ° C. The spinal cord was divided into 8 mm sections and embedded in paraffin, serial cut (3 μm) and on silanized slides given.

1.4 Immunohistochemistry

After deparaffinization, slides with spinal cord sections were boiled in citrate buffer (2.1 g sodium citrate / L, pH 6) for 15 minutes (in a 600 W microwave oven). Endogenous peroxidase was inhibited with 1% H ₂ O ₂ in methanol for 15 minutes. The slides were incubated with 10% normal porcine serum (Biochem, Berlin, Germany) to block non-specific binding of immunoglobulins and tested with the following mouse monoclonal antibodies: ED-1 (Anti-Rat, Serotec, Oxford, UK, 1 : 100) for the detection of activated microglia, GFAP for the detection of astrocytes, W3 / 13 for the detection of T lymphocytes (all Serotec), Nestin (Chemicon, Planegg-Munich, Germany, 1: 500) for the detection of stem / progenitor cells and BrdU (Anti-BrdU, BD Pharmingen, San Diego, 1: 100) for the detection of proliferating cells.

The binding of the antibodies to animal tissue was visualized by means of biotinylated rabbit anti-mouse IgG F (ab) ₂ antibody fragments (DAKO, Hamburg, Germany, 1: 400). Subsequently, the sections were incubated with a streptavidin-avidin-biotin complex (DAKO, Hamburg, Germany) and subsequently developed with diaminobenzidine (DRB) substrate (Fluka, Neu-Ulm, Germany). Finally, the sections were counterstained with hematoxylin.

1.5 double staining experiments

For double staining experiments were the slides with the spinal cord cuts pretreated as described above and then with the respective monoclonal antibody incubated. Subsequently a reaction with the secondary antibody (biotinylated rabbit Anti-mouse IgG) with a dilution of 1: 400 in TBS-BSA for 30 minutes and with an alkaline phosphatase conjugated ABC complex, diluted with 1: 400 in Tris-BSA for 30 minutes. Subsequently, immunostaining with Fast Blue BB salt-chromogen substrate solution was developed. Subsequently were the slides cooked in citrate buffer for 15 minutes in a microwave and immuno-labeled as described above.

Of the BrdU incorporation by different cell types was by double staining with the antibodies described in 1.4 above.

1.6 Evaluation

The spinal tissue sections were divided into groups of eight to twelve sections of different regions of the spinal cord. For immunohistochemistry, the stained sections were examined by light microscopy. The number of BrdU-labeled cells in the gray matter, white matter and around the central channel was determined by manual counting under twenty times magnification. The number of ED-1 ⁺ was counted on average throughout the spinal cord. The region of gray and white matter of the spinal cord sections was evaluated using the Axio Vision LE Rel.4.1 (Carl Zeiss Vision GmbH) software. The results for the white and gray matter are the arithmetic mean of the posi tive cells per mm ² and were standardized. The number of ependymal cells are given and standardized as arithmetic mean BrdU-positive cells per spinal cord incision. Statistical analysis was performed by ANOVA and Dunnett's Multiple Comparison Test (Graph Pad Prism 4.0 software). Data with p <0.05 were considered statistically significant.

2 results

Around the effect of different Nod2 and TLR ligands on proliferation neural progenitor cells within the intact spinal cord To determine, rats became TLR ligands Intraperitoneally injected and proliferating cells by intraperitoneal Injection of BrdU labeled (BrdU is a thymidine analogue that while the cell division is incorporated into DNA). Proliferating cells were carried through Immunohistochemistry detected with anti-BrdU antibodies.

a Day after injection of the Nod2 and TLR ligands, the rats were brdU injected for two days and then the proliferation of the neuronal progenitor cells determined by immunohistology.

In 1A It has been shown that in both white and gray matter, the groups treated with either N-acetylmuramyl-dipeptide, LPS, R848 or poly I: C were able to measure a significantly higher density of BrdU ⁺ cells than in the control group (average density in the gray and white matter of the control group: 9.45 ± 0.83 cells / mm ² and 7.72 ± 0.84 cells / mm ² (p <0.05)). Only the group treated with PS CpG-ODN (recognized by TLR9) showed no significant difference compared to the control group.

BrdU ⁺ cells were distributed in the spinal cord over the white and gray matter. Nevertheless, a higher density of BrdU ⁺ cells in the white matter of all groups could be detected compared to the gray matter (see 1A ).

Around To identify proliferating cells, double staining experiments were performed used. ED-1 and BrdU double stains showed that ED-1 was positive Microglial cells were rarely BrdU positive.

The ependymal cells around the central canal of the spinal cord are considered to be neuronal stem cells. In the experiments of the inventor, proliferation of ependymal cells in the normal spinal cord tissue could be observed, however, the TLR ligands did not induce a significant increase in the number of ependymal BrdU ⁺ cells (see 1B , mean number of ependymal BrdU ⁺ cells in the control group is equal to 1.1 ± 0.40).

To study the time course of NPC proliferation, rats previously treated with poly I: C or R848 were sacrificed at different time points and then cell proliferation was analyzed. As in 2A showed a significant increase in the number of BrdU ⁺ cells per mm ² in rats receiving BrdU on days 2-3 after poly I: C injection. Similar results were observed in both white and gray matter (80.67 ± 3.34 cells / mm ² and 64.19 ± 4.04 cells / mm ² , p <0.05). At later times, the density of BrdU ⁺ cells rapidly decreased to basal levels, both in the white and gray matter.

With respect to the R848-treated groups, the highest density of BrdU ⁺ cells was found to be 111.2 ± 7.67 cells / mm ² in the white matter and 72.22 ± 2.8 cells / mm ² in the gray matter in the spinal cord of Rats were administered to BrdU on days 2 and 3 (compared to the control group the differences are significant (p <0.05)). At later time points, the density of BrdU ⁺ cells rapidly decreased to basal levels (see 2 B ).

In further experiments, the spinal cord sections were stained with the monoclonal antibody ED-1 and examined by light microscopy. A significant increase in ED-1 ⁺ cells was observed four days after poly I: C injection (see 3A , average density of treated and control groups are 9.1 cells / mm ² ± 0.99 and 0.91 cells / mm ² ± 0.24; p <0.05). Even on day 6 post-injection, a significant increase in ED-1 ⁺ cells was observed (5.2 cells / mm ² ± 0.35, p <0.05).

For the rats treated with R848, the density of ED-1 ⁺ cells reached a maximum on day 4 post-injection (3.1 cells / mm ² ± 0.35, p <0.05), but decreased on day 5 injection (2.4 cells / mm ² ± 0.25, p <0.05) and decreased to baseline on day 6 post-injection (see 3B ).

For the others Investigation of the regulatory cascade caused by the injection triggered the TLR agonist Poly I: C was used as a stimulus and examined below, whether a treatment with a blood-brain barrier-passing cyclooxygenase blocker, Indomethacin, or a strong synthetic corticosteroid, dexamethasone, cell activation in the spinal cord could be inhibited.

The results of these studies are in 4 shown. Out 4A and 4B shows that poly I: C-mediated cell activation could be inhibited by injection of dexamethasone. Surprisingly, dexamethasone had a strong suppressive effect, whereas indomethacin had no effect.

3. Summary

By The experiments presented above could be shown to be neuronal progenitor cells by the peripheral administration of certain Nod2 and / or TLR ligands temporarily stimulated for proliferation in the spinal cord can be. The Number of proliferating cells was 2-3 days after injection the strongest and then sank quickly at basic level.

In normal spinal cord dividing cells represent only a very small population under physiological circumstances. In the experiments presented above, a low density of BrdU ⁺ cells in the spinal cord of the control groups was observed (9.45 ± 0.83 cells / mm ² and 7.72 ± 0.84 cells / mm ² ). While it is known that under certain conditions, such as, for example, injury, inflammation or autoimmune reactions, the number of proliferating cells may increase dramatically, there is no understanding of the mechanism of proliferation regulation of neuronal progenitor cells.

Limke et al., "Neural stem cell therapy in the aging brain: pitfalls and possibilities ", J. Hematother Stem Cell Res. 2003; 12: 615-23, showed that various factors in the microenvironment of the CNS, such as the epidermal growth factor (EGF) and fibroblast growth factor (FGF) and brain-derived neurotrophic factor (BDNF) to modulate the role of neural progenitor cells in tissue homeostasis can, as well as certain inflammatory cytokines, chemokines, proteins of the complement system or cell adhesion molecules (see Tzeng and Wu, "Responses of microglia and neuronal progenitors to mechanical brain injury ", Neuroreport 1999, 10: 2287-2292).

The The results presented above show for the first time that peripheral Nod2 and TLR ligands transiently delivered neuronal proliferation progenitor cells in the spinal cord induce.

Claims (8)

Use of at least one Nod2 and / or TLR ligands for the preparation of a medicament for treatment a disease of the central nervous system (CNS). Use according to claim 1, characterized that the drug is made to treat a disease of the CNS is caused by the stimulation of growth, proliferation or differentiation of neural progenitor cells is treatable. Use according to claim 1 or 2, characterized that the drug is made to treat a disease of the CNS which is selected is from the group comprising Amyotrophic Lateral Sclerosis, Multiple Sclerosis, muscular dystrophy, Alzheimer's disease, Parkinson's disease. Use according to claim 1 or 2, characterized that the drug is made to treat a disease of the CNS The disease is a consequence of a traumatic injury or ischemia of the CNS. Use according to one of Claims 1 to 4, characterized that the TLR ligand is selected is selected from the group comprising lipopolysaccharide, R848, polyinosin-polycytidic acid, N-acetylmuramyl-dipeptide, or derivatives thereof. Pharmaceutical composition containing at least a Nod2 and / or TLR ligand for the treatment of a disease of the CNS. A pharmaceutical composition according to claim 6 for Treatment of a disease of the CNS selected from the group comprising amyotrophs Lateral sclerosis, multiple sclerosis, muscular dystrophy, Alzheimer's Disease, Parkinson's disease. A pharmaceutical composition according to claim 6 or 7 for parenteral, intracerebral or intrathecal administration.