WO2004091651A1 - Prongf as a pharmaceutically active agent for treating demyelinating diseases - Google Patents

Abstract

The invention relates to the use of proNGF, to proNGF derivatives, with the exception of those in which the pro-sequence has been completely deleted, and/or to an effective quantity of a functional part of proNGF, together with carrier materials and/or diluents for the prophylaxis and/or therapy of demyelinating diseases.

Description

 proNGF as a pharmaceutically effective agent for the treatment of demyelinating diseases

The invention describes the use of proNGF as a pharmaceutically active agent for the treatment of demyelinating diseases. These are, in particular, neurodegenerative diseases whose prophylaxis and therapy use proNGF, in particular to maintain vitality and to stimulate proliferative and myelinating processes of glial cells. The invention relates in particular to the use of proNGF, derivatives of proNGF and functional parts of proNGF for the prophylaxis and therapy of demyelinating diseases with the aim of supporting and stimulating regeneration processes of the glial cells. According to the invention, the proNGF can also be used in gene therapy processes.

General state of the art

Definition of glial cells

Glial cells are cells that surround nerve cells. The functions of the glial cells include the supply of nutrients, the breakdown of dead neurons, isolation and physical support. Glial cells include astrocytes, microglia, oligodendrocytes, Schwann cells, satellite cells and Müller glial cells.

Diseases

For normal nerve function in the central nervous system (CNS), the nerves must be surrounded with myelin. The myelin layer is produced by oligodendrocytes (OL). OL are small and have extensions. As so-called "satellite cells" of the nerve cells, they form the medullary sheaths. The myelin layer, which is created by oligodendrocytes, mediates normal nerve impulse transmission in the CNS, with the nerves being electrically isolated from their surroundings. In neurodegenerative diseases, such as. B. multiple sclerosis (MS), the myelin layer of the axons is damaged or destroyed. In many cases, the OL itself is injured. In some cases there is scar tissue in MS that permanently prevents nerve signal transmission in certain cases. It was also observed that practically no remyelination takes place in the central nervous system (CNS), which leads to an increasing deterioration of the condition in MS. In contrast, the phenomenon of remyelination has been observed in the peripheral nervous system. For example, in the Guillain-Barre syndrome, the myelin of the peripheral nervous system is affected, with the myelin layer regenerating itself within 4 to 6 months in 85% of the cases. Demyelination of the peripheral nervous system also occurs in Charcot-Marie-Tooth (CMT) disease. In this case, the defect is genetically inherited and can be attributed to various mutations. The most common form of CMT is type 1, where the peripheral myelin protein gene 22 (PMP-22) is mutated. Other forms of CMT with demyelination are type 1A (CMT 1A), 1B (CMT 1B), type 2 (CMT 2), type 3 (CMT 3) or Dejerine Sottas disease, type 4 (CMT 4) and CMTX. Another progressive demyelizing disease is adrenoleukodystrophy (ALD), which can also lead to blindness and death. The spinal cord is particularly affected in amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease).

Current treatment methods

Current treatments for MS include interferon beta, which inhibits T lymphocytes and inflammation. However, interferon beta has no healing function and can therefore only slow down the course of the disease. Interferon beta also does not work in all MS patients (no permanent remission). 10-20% of MS patients have a relatively benign course, so that not every patient needs such a "disease-modifying therapy".

Another treatment is glatiramer acetate (Copaxone®), a synthetic copolymer composed of alanine, glutamine, lysine and tyrosine. Copaxone® can alleviate the symptoms of MS, but it cannot cause permanent remission. Since cytostatics inhibit leukocyte proliferation and thus also have an immunosuppressive and anti-inflammatory activity, they are also used in MS. However, a clear improvement in the course of the disease in MS has not yet been shown. In the best case, the MS flare-ups (number and intensity) are weakened, but there is no cure for demyelination.

Steroids are also used as an additional treatment, which slow down antibody production and inflammatory reactions, reduce myelin swelling and thus

Symptoms alleviate. Due to the many side effects, the steroids are only suitable for short-term use, but not for continuous treatment.

Furthermore, the use of cytokines, especially interleukin-12 and interleukin-10, is discussed in the literature.

Another approach is to find active ingredients that can cause remyelination.

These substances include growth factors in particular.

Growth factors are proteins that, due to their specific interaction with a receptor, are able to stimulate or influence cell division, migration, differentiation and maturation processes. The proteins can be produced on a large scale both in genetically modified eukaryotes and in prokaryotes (EP0994188, WO00221 19). The importance of growth factors in myelination and demyelination has been examined in several studies (Du and Dreyfus, 2002; Webster, 1997; Woodruff and Franklin, 1997). Positive effects for fibroblast growth factor (FGF), glial growth factor 2 (GGF2), insulin growth factor (IGF) and neurotrophins (e.g. NGF, BDNF, CNTF, GDNF, NT-3, NT-4/5, NT- 6 and NT-7). (US 6268340, US 5885584)

ProNGF is the precursor protein of the NGF. Human NGF is synthesized in vivo as preproNGF. The pre-sequence has a length of 21 amino acids and is cleaved after the translocation of the protein into the endoplasmic reticulum. The resulting proprotein is then proteolytically processed at the N- and C-terminal, making the mature NGF accessible. ProNGF has a molecular weight of 49 kDa, consists of 221 amino acids and appears in its mature form as a homodimer. The pro sequence is located on the surface of the folded NGF, almost unstructured (Rattenholl et al., Eur. J. Biochem. 2001, 268, 3296-3303; J. Mol. Biol. 2001, 305, 523-533) and comprises 103 amino acids at the N-terminus.

The effect of growth factors is mediated through their binding to specific receptors. Two receptors for NGF have been described, TrkA, a high-affinity receptor with tyrosine kinase activity, and P75, a low-affinity receptor to which other neurotrophins (e.g. BDNF, CNTF, GDNF, NT-3, NT-4/5, NT- 6 and NT-7). TrkA expression was detected on oligodendrocytes (Althaus et al., 1997; Althaus and Richter-Landsberg, 2000; Nataf et al., 1998). In addition to the high affinity receptor, the expression of P75 in myelinating cells, such as. B. detect the oligodendrocytes (Althaus et al., 1997; Ladiwala et al., 1998; Nataf et al., 1998; Starkey et al., 2001).

The effect that is mediated through the interaction of the NGF with the respective receptors has not yet been clearly elucidated. P75 is described as a receptor through which apoptotic processes are mediated (Lee et al., 2001; Starkey et al., 2001). However, it was also shown that the processes mediated by TrkA such as proliferation and survival and differentiation of cells can be enhanced in the presence of P75. The relationship of the two receptors to each other, the cell type, but also the cellular development status, therefore play a decisive role with regard to the effect mediated by NGF. It is known from the literature that proNGF induces cell death of oligodendrocytes after spinal cord injuries (Neuron 2002; 36 (3): 375-86). Apoptosis is mediated by the p75 receptor.

ProNGF was detected in various tissues in vivo. In addition, the biological activity of NGF precursors and peptide sequences of the precursor protein in various in vitro and in vivo systems (induction of neurite growth or redistribution of F-actin in PC12 cells, increase in the activity of cholinergic enzymes after intracerebroventricular injection in neonatal Rats) (Chen et al., Mol. Cell. Endocrinol. 1997, 127, 129-136; Clos et al., Develop. Brain Res. 1997, 99, 267-270). The biological significance of proNGF, which due to sequence homologies of the pro sequences within the neurotrophins and the localization in different areas weaving is not yet clear. The proproteins are discussed as a reservoir of the mature active protein. In addition, the pro sequence acts as a helper in the folding of the mature protein (Rattenholl et al., Eur. J. Biochem. 2001, 268, 3296-3303; J. Mol. Biol. 2001, 305, 523-533) or a role in the sorting of the neurotrophins by anabolic or catabolic routes (Farhadi et al., J. Neurosci. 2000, 20, 4059-4068).

The pharmaceutical activity of proNGF was first shown in EP 0 994 188. In example 4f, the stimulability and survival of sensory neurons from dissociated dorsal root ganglia are examined on the basis of the formation of neurites. In these experiments, the biological activity for the recombinant proNGF was half that of the recombinant mature ß-NGF. Based on these test results, it was proposed to use recombinant proNGF for the manufacture of a medicament for the treatment of neuropathies, the effect of proNGF relating to the neurons / nerves. An effect on other cell types, in particular on myelinating cells, is not described.

WO 96/08562 describes NGF, which N-terminal is extended by a peptide fragment of up to approx. 19 amino acids, this peptide fragment being the C-terminal fragment of the precursor part of NGF. This precursor contains a furin-type consensus site at the C-terminus, in which an exchange in amino acid 1 and / or -2 has taken place. It is proposed to use such NGF variants in medicaments for the treatment of diseases of the nervous system, for example of degenerative diseases of the nervous system, such as the retinal degeneration, in the treatment of injuries or damage to the nervous system, etc., the action always taking place via the nerve cells themselves , For example, it is described that the survival of dopaminergic and cholinergic neurons is promoted by the NGF derivatives mentioned with a deleted pro portion. An application to myelinating cells, such as. B. oligodendrocytes and Schwann cells are not shown. On the contrary, it is pointed out that the NGF derivatives with the deleted pro portion prevent the multiplication of astroglial cells, that is to say they exert a suppressive effect on myelinating cells. Also in the publication by Beattie et al. (Neuron 2002, 36, 375-386) it is shown that the neurotrophin receptor P75, which is induced in various nervous system injuries, promotes cell death of oligodendrocytes through its connection with proNGF. The apoptotic effect of the proNGF-P75 receptor complex was prevented by proNGF-specific antibodies. These data show that proNGF plays a crucial role in the elimination of damaged nerve cells by activating P75-mediated apoptosis. The activation of P75 by proNGF thus causes the cell death of oligodendrocytes. The interplay between the P75 neurotrophin receptor and proNGF is therefore a central point in apoptosis processes after injuries to the nervous system.

Further indications of P75-mediated apoptosis processes are discussed in connection with an increased proNGF concentration in the brain of Alzheimer's patients (Fahnestock et al. Mol. Cell. Neurosci. 2001, 18, 210-220).

EP 0786520 describes N-terminally elongated β-NGF which, like natural β-NGF, is said to have an effect on neurons. An effect on the indication areas proposed here is not discussed.

It is an object of the invention to provide new, pharmaceutically active agents for the treatment of demyelinating diseases, in particular those diseases in which the proliferative and myelinating processes of glial cells are to be stimulated and the vitality of glial cells is to be maintained in order, for. B. to support or stimulate the regeneration processes of the glial cells.

This object is achieved according to the invention by the use of proNGF, derivatives of proNGF, with the exception of those in which the pro portion has been completely deleted and / or a functional part thereof in an effective amount for the prophylaxis and / or therapy of demyelinating diseases , These substances stimulate glial cell proliferation, maintain glial cell vitality, and support and stimulate glial cell regeneration and migration to achieve myelination and inhibit demyelinating processes. A preferred use is the prophylaxis and therapy of multiple sclerosis, Charcot-Marie-Tooth (CMT) disease, of adrenoleukomyeloneuropathies, for example adrenoleukodystrophy, and of amyotrophic lateral sclerosis (ALS).

According to the invention, it was found that proNGF, its derivatives, with the exception of those in which the pro portion was completely deleted, and / or functional parts thereof, improve and promote the survival and regeneration of glial cells, for example of oligodendrocytes, in particular humans. The invention thus enables the use of proNGF alone or in conjunction with other pharmaceutically active ingredients to prevent or cure diseases in which glial cells are damaged or can be damaged if they are accompanied by demyelination of nerves, in particular multiple sclerosis.

The invention also encompasses derivatives of proNGF in which one or more amino acids have been exchanged, deleted, added and / or chemically modified, always while maintaining the pharmaceutical, medical and biological activity described above.

Amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic (amphiphilic) nature of the residues involved. Examples of nonpolar (hydrophobic) amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, thyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. And negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

"Insertions" or "deletions" typically range from one to five amino acids. The permitted degree of variation can be experimentally determined by systematic insertions, deletions and / or substitutions of amino acids in a polypeptide molecule using DNA recombination techniques and by looking for the resulting recombinant variants with regard to their biological activity.

The hydropathic index of the amino acids can also be considered when making these changes. Each amino acid can be assigned a specific hydropathic index based on its hydrophobicity and charge properties. Examples of this can be found in WO 00/32039, in US 4,554,101 and in Kyte & Doolittle, 1982, which are referred to here with their content. It is known that certain amino acids can be substituted by other amino acids if they have a similar hydropathic index and, when they are exchanged, bring about a comparable biological activity of the whole molecule. With all derivatizations of the proNGF it is crucial that the biological, medical or pharmaceutical effectiveness is maintained.

The derivatization of proNGF can refer to both the pro portion and the NGF portion. The changes in the pro portion also include amino acid additions at the N-terminus of the pro portion, which are defined by the pre portion. The sequence of the pre-portion, the pro-portion of NGF and of NGF itself are known, and reference is made here, for example, to: http: //www.ncbi. nlm.nih.gov: 80 / entrez / query.fcgi? cmd-Retrieve & db = ^: nucleotide & list uids = 2173196 & dopt = GenBank or http://www.ncbi.nlm.nih.gov: 80 / entrez / query.fcgi? cmd = ^: Retrieve & db-nucleotide & list uid s = 2171804 & dopt = GenBank and EP-A-0 994 188.

"ProNGF" according to the invention, even if not listed separately, always means the derivatives of proNGF, with the exception of those derivatives in which the pro portion has been completely deleted and the functional portions of proNGF as described in the present application to be defined in more detail.

According to the invention, “functional part” of proNGF is understood to mean those parts which have the indications and use claimed in the present application fulfill, with simultaneous deactivation, eg by deletion, those portions of proNGF that do not contribute to the function.

The functional parts can be determined by a person skilled in the art, for example, by deletion analyzes. Systematic deletions of the pro portion, the prepro portion and / or the NGF portion and a subsequent analysis of the remaining molecule for the indications described and claimed here enable the person skilled in the art to determine the desired functional sections. Such functional portions are, for example, the 25 or 71 amino acids of the pro-C terminus of proNGF, in particular complete domains. The functional parts should remain as unchanged as possible in order to ensure the physiological function of the proNGF, for example its binding to the receptor. However, modifications as described below are also possible in the functional parts. The number and type of modifications can be determined experimentally by a person skilled in the art. One or more modifications in the pro, prepro and / or NGF portion can be carried out, for example deletions or substitutions of an amino acid. The protein modified in this way is then examined for its functionality in the context of the present invention, and it is determined whether the change made brings about an improvement or deterioration in the properties.

The active ingredient proNGF includes those of human origin, animal origin, in particular from rodents such as rats, rabbits and mice, from cattle and pigs, and proNGF produced in a recombinant manner. Recombinant forms of proNGF also include proNGF fusion proteins in which proNGF has been linked to another protein or peptide. Furthermore, proNGF can be applied as DNA via expression vectors or by means of cells expressing proNGF. For examples of NGF in which fusions with proteases enable controlled release of the NGF from matrices, we refer to Sakiyama-Elbert et al., FASEB J. 2001, 15, 1300-1302; Park et al, J. Drug Target 1998, 6, 53-64

The derivatives of the pro portion of NGF include in particular those pro forms in which at least 25, more preferably 71 amino acids of the pro-C terminus at the N- Terminus of the NGF are available (Dicou et al., J. Cell. Biol. 1997, 136, 389-398). Preference is furthermore given to those derivatives of proNGF which, at the amino acid level, have a homology to the pro form, to the prepro form and / or to the NGF portion of the proNGF of at least 80%, preferably 85%, 90% or 95% or about that. Homology means that the specified percentage of amino acids, based on the starting form of the proNGF used, for example rh-proNGF, is contained in identical form. This means that preferably less than 15%, particularly preferably less than 10% and even more preferably less than 5% of the number of amino acid residues in the sequence of the proNGF are different from the amino acid residues in the sequence of the corresponding domain of the starting form. Different means that they have been substituted for another amino acid, for example, that another amino acid has been inserted or deleted, or a combination thereof. This also includes changes to the amino acid itself, for example glycosylations, phosphorylations, acetylations, sulphations or amounts of lipid. Connections of the amino acids with a label, for example a dye such as a fluorescent dye, an enzyme, a toxin, etc., are also possible. All these changes are subsumed under the term "derivatives".

The number and type of modifications in the derivatives is always limited by the maintenance of the physiological functions of the proNGF, in particular, of course, by the maintenance of the therapeutic and prophylactic and other functional properties of the proNGF as described in the present invention. In general, no more than 10, preferably no more than 5 and more preferably less than 5, modifications are made in the proNGF. A maximum of 5 modifications are preferably introduced in the pro portion. In particular, the functional sections of the pro portion should remain functional, i.e. positions 143 - 221 and 78 - 221 of the proNGF (numbering of the positions starting at the N-terminus of the proNGF).

A prerequisite for the applicability of the derivatives according to the invention is always that after derivatization, the effectiveness in the areas of use claimed here is retained. Various test systems for determining the activity of the proNGF and its derivatives are given in the examples below. The expert can also identify or develop other exemplary test systems from the literature. (For example the rat Pheochromozytoma PC 12 cell assay Proc. Natl. Acad. Sci. USA 1976; 73 (7): 2424-8); Neurite outgrowth from embryonic dorsal spinal ganglia, Neuroscience Lett. 1999; 262: 29-32; Oligodendrocyte Assay (J. Neurochem Suppl. 2001: 78: 49)

In a preferred embodiment of the invention, the amino acid sequence of the pro portion is changed so that under physiological conditions the pro portion is not split off in the cell or only at a later point in time than under physiological conditions. The natural furin cleavage site can be mutated by means of site-directed mutagenesis or PCR mutagenesis, so that the NGF is not cleaved off (Hempstead et al., Science 2002, 294, 1945-1948). For this purpose, the consensus sequence for the furin convertase, Arg-X- (Arg / Lys) -Arg, is mutated, which occurs at the amino acid position 104 in human proNGF.

However, even without changes in the amino acid sequence, proNGF can be detected over a period of time sufficient to develop therapeutic / prophylactic effectiveness. For example, proNGF can be detected in cell culture over a period of up to 8 hours (Mowla et al., 1999).

The derivatization of proNGF can take place at the amino acid level via direct chemical modifications of amino acids. However, modification by mutagenesis of the DNA coding for the protein is preferred. This includes, for example, site-directed mutagenesis, as is known from the prior art and is constantly being further developed (Brannigan and Wilkinson, Nat. Rev. Mol. Cell. Biol. 2002, 3, 964-970).

The production of proNGF and its derivatives by recombinant techniques has been described, and reference is made to this previously published literature here (Rattendoll et al, Eur. J. Biochem. 2001, 268, 3296-3303; Rattenholl et al., J. Mol. Biol. 2001, 305, 523-533). The production of proNGF and the modifications encompassed by the invention, including the DNA encoding the proNGF proteins and the expression vectors containing the DNA, are within the skill of a biotechnologist, in particular a protein chemist. The production of recombinant DNA, vectors, transformed host cells, proteins and protein fragments by genetic engineering methods is known. For example, the following publications are referred to here: WO 97/10365, WO 97/27317, Laboratory Techniques in Biochemistry and Molecular Biology: Theory and Nucleic Acid Preparation, P. Tijssen, ed. Eiservier, NY (1993) Sambrook et al. , Molecular Cloning. A Laboratory Manual (. 2 ^nd ed), Vols 1-3, Cold Spring Harbor Laboratory (1989), or Current Protocols in Molecular Biology, F. Ausübel et al, ed, Breene Publishing and Wiley-Interscience. NY (1987).

In one embodiment of the invention, the pharmaceutically active proNGF active ingredients are administered to the patient by gene therapy methods. In gene therapy, a distinction is made between two basic methods, with the help of which a gene, here proNGF, can be brought into the patient.

In the ex vivo application, the therapeutically active proNGF gene is introduced into a cell, preferably a body cell, particularly preferably a glial cell, of the patient by means of vetors, and the cells treated in this way are returned to the patient, for example by micro- or nanoparticles. A specific integration of the proNGF gene into the cellular genome is particularly preferred here.

In in vivo gene therapy, the proNGF gene is transported via gene transporters to the target cell in the body, for example with the help of viruses that can infect the cells and introduce the therapeutically effective proNGF gene, but are no longer able to reproduce or spread themselves , With this method, too, nanoparticles or microparticles, for example liposomes, which can fuse with the cell membrane, can be used as gene transporters. Somatic gene therapy is therefore also a preferred means of choice for the treatment of the diseases covered by the invention. Exemplary gene therapy methods, including liposomal transfection of nucleic acids in host cells, are described, for example, in US Patents 5,279,833; 5,286,634; 5,399,346; 5,646,008; 5,651,964; 5,641,484 and 5,643,567, to which full reference is made here.

The target cells to be infected, in particular glial cells of the peripheral and central nervous system, are introduced with an effective amount of a proNGF DNA molecule in conjunction with regulatory sequences in a suitable vector which enables the expression of the proNGF gene and its control. The cell is then grown under appropriate conditions and the polypeptide is expressed.

Techniques for transforming and transfecting a cell are known per se. Reference is made here to the literature cited above.

For example, a virus or an antibody can be used as the gene ferry or carrier vehicle for the proNGF gene, which specifically infects the target cell or which enters into an immune reaction with an antigen of the target cell. For example, retroviruses can be used as viral vehicles that have received or already have a host specificity for the glial cells through genetic manipulations and in which the 3-LTR has been inactivated. These 3 'LTRs, which do not have an enhancer, often also referred to as SIN (self-inactivating viruses), are able to infect and multiply the host cell, but after the productive infection, the 3'LTR is at the 5' end transferred and both viral LTRs are inactive with regard to their transcription activity. The gene to be cloned can be inserted between the two LTRs in conjunction with regulatory elements, for example promoters and enhancer elements. An advantage of a viral infection system is that the target cell is infected with high efficiency.

In addition to retroviral vectors, vectors based on adenoviruses and vaccinia can also be used. When using antibodies, particular preference is given to monoclonal antibodies which are coupled, for example, to poly-L-lysine.

With the aid of nano- and microparticles, the nucleic acids carrying the information for the expression of proNGF, and cells with this information, can be transported into the target cells or the target location. For example, reference is made here to Journal of Pharmacological and Pharmaceutical Science 3 (2): 234-258, 2000, Microencapsulation: Methods and Industrial Applications (Drugs and the Pharmaceutical Sciences, Vol 73) by Simon Benita (Editor), publisher: Marcel Dekker; (April 19, 1996), Fundamentals of Animal Cell Encapsulation and Immobilization by Mattheus F. A. Goosen; Publisher: CRC Press; (November 10, 1992) and Microparticulate Systems for the Delivery of Proteins and Vaccines by Smadar Cohen (Editor), Howard Bernstein (Editor), Publisher: Marcel Dekker; (September 1996).

Examples of nanoparticles are micelles based on biocompatible copolymers of polyethylene oxide, copolymerized with poly L-lactic acid (PLA) and with poly (beta-benzyl-L-aspartate) PBLA. The aldehyde groups on the surface of the PEO-PBLA micelles can interact with the lysine residues of the cell proteins. Further examples of nanoparticles are: poly (lactide-co-glycolide) - [(propylene oxide) poly (ethylene oxide), polyphosphazene derivatives, poly (ethylene glycol) coated nanospheres, azidothymidine (AZT) / dideoxycytidine (ddc) nanoparticles, poly (Isobutylcynoacrylate) nanocapsules, poly (γ-benzyl-L-glutamate) / poly (ethylene oxide), chitosan-poly (ethylene oxide) nanoparticles, methotrexate-o-carboxymethylatchitosan, solid lipid nanoparticles.

Microcapsules and microspheres are spherical particles with a size of between 50 nm and 2 mm with a core substance. These include, for example, multiporous chitosan spheres, coated alginate microspheres, for example calcium alginate microspheres, N-aminoalkylchitosan microspheres, chitosan / calcium alginate spheres, polyadipic anhydride microspheres, gellangoglu-lid, poly (spheres) from gellangogmi-lolid) Microspheres, microspheres made from alginate-poly-L-lysine, from cross-linked chitosan compounds, from chitosan and gelatin, lipospheres from triglycerides, polyelectrolyte complexes from sodium alginate chitosan, microcapsules made of polypeptides and albumin. Other examples are known and are constantly being developed.

Depending on the application form, the patient, the disease to be treated and the active substance to be administered, the amount of the dose, the form of preparation, etc., the person skilled in the art will select a suitable form of application, be it a gene therapy method or a "classic" pharmaceutical preparation , The person skilled in the art will determine the suitable form of preparation, the effective dose, etc. on the basis of his specialist knowledge on the basis of experimental investigations by testing. The dose amounts proposed here are only to be understood as examples and can be easily adapted by the person skilled in the art to the given problem. The active ingredients of the present invention are preferably used in the form of a pharmaceutical composition in which they are mixed with suitable carriers in such doses so that the disease is treated and / or alleviated. Such a composition may contain (in addition to the active ingredients and the carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, penetration enhancers and other materials which are well known in the art. The term "pharmaceutically acceptable" is defined as a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. The choice of carrier and other additives depends on the route of administration. Biologically compatible carriers and additives, which can cross the blood-brain barrier if the administration is not intrathecal or intracerebral, were preferably used.

Techniques for formulating or preparing and administering the compounds of the present application can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. A therapeutically effective dose also refers to an amount of the compound sufficient to achieve symptom improvement, such as treatment, healing, prevention, or amelioration of such conditions. Suitable routes of administration are in particular a topical application. For this purpose, the compositions can be in liquid form, for example in the form of emulsions, suspensions and solutions, in spray form, in the form of transdermal systems such as plasters, in the form of a cream, an ointment or a gel. Retard forms are also preferred. Other dosage forms known per se are familiar to the person skilled in the art.

The amount of drug that is adequate to achieve the effects described above is defined as the therapeutically effective dose. The amount that is effective for this use depends on the severity of the patient's condition and disease. Single or multiple administrations according to a daily, weekly or monthly treatment schedule can be combined with dose strengths and patterns selected by the attending physician. The doses to be used are also determined by the doctor, with amounts of from 0.1 microg / kg to 500 microg / kg body weight preferably being administered by infusion and from 2 microg / kg to 2 mg / kg body weight being administered by injection. Of course, other doses of active substance can also be used, which also depend, for example, on the formulation of the active substance, on the presence of further active substances, on the individual patient, on the application site, on the type of disease, etc.

Furthermore, proNGF protein can be implanted encapsulated, or genetically modified cells that secrete proNGF can be implanted directly or encapsulated. Or proNGF can be used for gene therapy using a suitable vector system.

It is pointed out that the term "proNGF" as used in the present context also includes the configurations specified in the patent claims and defined in more detail in the present description, for example also derivatives of proNGF.

The attached pictures show: Fig. 1:

Comparison of the influence of the effect of NGF and proNGF on the proliferation of

Müller's glia cells.

Fig. 2:

Comparison of the course of EAE after administration of proNGF or a placebo.

Fig. 3:

Comparison of long-term administration of NGF and proNGF for pain sensitivity, measured over that of Frey-Haar stimulation.

Fig. 4.

Comparison of long-term administration of NGF and proNGF for pain sensitivity, measured over that of Frey-Haar stimulation.

The invention is described in further detail below, the person skilled in the art being able, on the basis of his specialist knowledge, to modify the invention within the scope of the patent claims, so that embodiments which are not listed separately in the present description are also included.

Description of the invention

The present invention encompasses the use of proNGF to maintain the vitality of glial cells and to stimulate proliferative and myelinating processes of glial cells. ProNGF is suitable for the treatment of demyelinating diseases with the aim of supporting or stimulating regeneration processes of the glial cells.

The underlying use of proNGF and its derivatives to improve demyelinating diseases includes in particular I.) Providing a biological compatible composition containing an effective concentration of proNGF, II.) intrathecal, intracerebral, intravenous, intramuscular or intraperitoneal application of the composition into the brain, which has a preventive or therapeutic effect on the glial cells with the aim of clinically demonstrable improvement and / or To achieve healing of the myelination by stimulating proliferative and / or migratory processes of the glial cells or to stop demyelination, or peripheral administration in peripheral demyelinating diseases.

I Biologically compatible composition containing proNGF

A: The designation proNGF includes: 1.) the amino acid sequence of the NGF, which is extended at the N-terminal by the complete or partial sequence of the proprotein or preproprotein, the extension at least the amino acids from position 1 to 25 of the C-terminus of the Prosequence comprises and 2.) is glycosylated or not glycosylated or differs in the glycosylation status, 3.) is of human or non-human origin, 4.) is obtained recombinantly or non-recombinantly, 5.) as a fusion protein with another heterologous protein as a fusion partner or is not fused, 6.) is unmodified or modified by amino acid exchanges, deletions or insertions or chemically modified derivatives of proNGF. 7) genetically engineered cells, especially mammalian cells, that secrete proNGF; 8) DNA that is encoded for proNGF and used in gene therapy.

B: ProNGF can be obtained using various methods: 1.) isolation from human or animal tissue, 2.) chemical synthesis z. B. solid phase peptide synthesis, 3.) production by genetically modified pro or eukaryotes.

C: Biologically compatible compositions are materials of aqueous, ointment-like or gel-like consistency that contain a pharmaceutically acceptable carrier. The carriers can contain sterile water, various buffers, alcohols, organic solvents, emulsifying or suspending agents or diluents. The carriers can preferably contain further auxiliaries, for example physiologically compatible surface-active ionic or non-ionic substances. The carriers can contain antibacterial, antifungal components and / or preservatives (e.g. parabens, Chlorobutanol, sorbic acid, phenol, thimerosal). The carriers can contain buffers to maintain a pH between 5.0 and 8.0. The buffers can be conventional buffers, e.g. B. phosphate, borate, citrate, bicarbonate or Tris-HCl buffer. The carriers can be isotonic by adding appropriate osmotically active components (e.g. saline, phosphate buffer, sugar). The carriers can contain other solubilizing components such as proteinaceous carriers or solubilizers (e.g. albumin). The carriers can include antioxidants and stabilizers such as e.g. B. sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea, protein inhibitors. Possible wetting agents can e.g. B. Polysorbate 80, Polysorbate 20, Poloxamer 282 or Tyloxapol. The carriers can include viscosity increasing agents such as e.g. B. dextran 40, gelatin, pectin, glycerin, lanolin, cellulose derivatives (e.g. hydroxyethyl cellulose, hydroymethylpropyicellulose, methyl cellulose, carboxymethyl cellulose), vinyl polymers (e.g. polyvinyl alcohol, polyvinylpyrrolidone), hyaluronic acid, polyacrylic acid, petrolatum, polyoxyethylene, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, polyethylene glycol, , Protamine sulfate, collagens, natural rubber and / or vegetable oils to facilitate application and / or for better tolerance. The carriers can contain antibiotics to control infections. The carriers can contain agents for the slow release of the growth factor. All components of the carrier are characterized in that they are biologically compatible and non-toxic.

Preservatives, stabilizers, humectants, emulsifiers and salts for balancing the osmotic pressure are further possible additives.

D: If infusions (intrathecal or intracerebral pump) are used as a biologically compatible solution for the treatment of demyelination, the concentration of proNGF in the carrier is between 0.1 μg / kg, or 0.5 μg / kg body weight and 50 μg / kg or 20 μg body weight. If compatible carriers are used for injection (i.V., i.m. or i.p.) to treat demyelination in the form of solutions, the concentration of the proNGF in the carrier is between 2 μg, 5 μg and 400 μg / injection.

E: proNGF can be used in the biologically compatible carrier alone or in combination with other healing-stimulating, anti-inflammatory or pain-relieving substances. II application

A: The application of the proNGF-containing drug can be preventive or therapeutic after detection of a demyelinating disease in an amount sufficient to achieve clinically demonstrable stimulation of myelinization by stimulating proliferative and / or migratory processes of glial cells.

B: The carrier containing proNGF is applied in an amount between 100 and 1000 μl, preferably 300 to 700 μl.

C: Continuous or single or multiple applications are possible, preferably multiple (daily) applications over 4 weeks. The application can start at any time after a demyelinating disease is detected.

D: Demyelinating diseases are understood to mean all processes and conditions that represent damage to the glial cells in the peripheral or central nervous system.

EP0994188 and WO0022119 deal with the production of NGF (Nerve Growth Factor) from proNGF and at the same time describe the use of proNGF as a medicament. The in vitro assay (dorsal root ganglion assay) published in these patent applications describes a comparable effect of proNGF and NGF with regard to the survival of chicken embryonic neurons, which suggests a comparable interaction with the receptors TrkA and P75 relevant for NGF. Hempstead et al. (Science 2002, 294, 1945-1948), however, were able to show that mutagenized, non-cleavable rh-proNGF binds to the furin cleavage site with a lower affinity to TrkA but with a higher affinity to P75 compared to NGF. The preferred binding of proNGF to P75 and the associated increased activation of the P75-mediated signal cascade is described as the cause of the apoptosis processes induced by proNGF. In this context, the biological effect mediated by this different receptor binding of both proteins is attributed to different mechanisms of action. The induction of apoptosis by proNGF has been demonstrated both for cells that only express P75 and for neurons that carry both receptors and therefore does not appear to be influenced by the coexpression of the high-affinity receptor TrkA. According to these results, proNGF is regarded as a stimulator of apoptosis processes via a P75-mediated signal cascade, while NGF can cause cell survival even in the presence of P75 via its preferred TrkA binding. Against the background of the Hempstead et al. Published apoptosis induction by proNGF seems to be unsuitable for the use of proNGF to stimulate glial cells and for the other indications described here about the induction of proliferative processes.

In investigations of proNGF in in-vitro cell systems it was surprisingly found that the proNGF active ingredients used according to the invention do not induce apoptosis as previously described, but rather stimulate the proliferation and survival of the glial cells in the cells used according to the invention. Compared to NGF, in vitro tests even demonstrated a stronger effect of proNGF on the proliferation of Müller's glial cells. In addition, animal experiments have shown that proNGF has a stronger effect on the remyelination of artificially placed demyelination (mouse EAE model) compared to NGF.

Accordingly, proNGF does not induce apoptosis in the cell systems we use. All of these results indicate that the mechanism of action mediated by proNGF does not only result in apoptotic processes through an interaction with P75, but surprisingly can also initiate proliferative processes.

1.) Effect of proNGF on glial cells

The in vitro test systems described here are glial cells from the retina, Müller's glial cells, which were examined for their effects mediated by proNGF and NGF. According to the above results, an apoptosis induction by proNGF would have been expected due to the interaction with P75. However, a previously described stimulatory activity of proNGF on the proliferation of the glial cells in vitro was found (Example 1, Fig. 1). Surprisingly, in contrast to the previously published data on apoptosis induction by proNGF, no evidence of apoptosis could be provided in this test system. In addition, it could be shown that proNGF triggers a stronger proliferation induction (Fig. 1) compared to NGF. 25 ng / ml to 100 ng / ml NGF and proNGF were tested. Since proNGF has a higher molecular weight than NGF, 31 kD compared to 16 kD, proNGF is active in a lower concentration.

2.) Effect of proNGF on the course of the disease in the EAE-MS model

In the mouse EAE model it was surprisingly shown that the course of the disease after artificial induction of demyelination in the brain is slowed down by proNGF and no signs of apoptosis induction by rh-proNGF can be observed (example 2). At a dose of 200 μg rh-proNGF / kg body weight / day, almost complete remyelination occurs after 18 days, while the vehicle control shows almost complete remyelination only on day 23 (see Figure 2). Unexpectedly, the side effects were reduced when using proNGF. For example, the pain often observed when administering NGF was significantly reduced. The invention is illustrated by the following examples. All examples are for illustration and are not to be understood as restrictive.

Examples

Example 1: Glial cells in vitro assay

In the laboratory, isolated glial cells are a good model for identifying factors that can stimulate proliferation, survival, and remyelination. Surprisingly, proNGF supports the proliferation of Müller's glial cells. This shows that proNGF does not have an apoptotic effect on glial cells (Figure 1).

Müller's glial cells are isolated from the retina of guinea pigs (Moll et al. Invest Ophthalmol Vis Sei 2002; 43: 766-773.). For this, the animals are z. B. with urethane (2.0 g / kg, ip) anesthetized. Then the animals are decapitated and the eyes are isolated. The retina is removed and incubated in calcium and magnesium-free phosphate buffer with Nagarse (1 mg / ml; subtilisin, EC 3.4.21.14) for 30 minutes at 37 ° C. The cells are then washed with phosphate buffer plus DNase I (200 units / ml) and the individual cells are sown on uncoated coverslips (e.g. diameter 15 mm; Glaswarenfabrik Hecht, Sontheim / Rhön, Germany). The cells are cultivated in minimal medium (MEM, from Sigma) plus 10% fetal calf serum (e.g. from Biochrom, Berlin, Germany) at 37 ° C. and 5% CO ₂ . The medium is changed twice a week. Before the cells are confluent, about 8 days after the start of the culture, the test substances, proNGF, NGF and vehicle, are added to the culture. After an incubation of 16 hours, the cells are analyzed. The substances are tested in serum-free cell culture medium. The influence on proliferation can e.g. B. measured using the BrdU test. A kit from Röche (Cat. No. 144611) can be used. The detection of the BrdU incorporation in the cellular DNA is demonstrated by anti-BrdU specific antibodies (Kodal et al., Luvest Ophthalmol Vis Sei 2000; 41: 4262- 4267).

Example 2: Animal model: Experimental autoimmune encephalomyelitis (EAE)

Experimental autoimmune encephalomyelitis is a generally accepted animal model for multiple sclerosis. This model has been described in detail by (Arredondo et al., 2001; Ratts et al., 1999). EAE can be induced in mice (e.g. in SLJ, C57BL / 6 or NOD mouse strain) by immunization with peptides (e.g. MOG _35-55 or Ac 1-11 MBP peptide). MOG ₃ 5-55 is an encephaloitogenic peptide with the sequence MEVGWYRSPFSRWHLYRNGK. The peptide is immunized in an emulsion from Freund's adjuvant (eg from Sigma), supplemented with 4 mg / ml of Mycobacterium tuberculosis antigen H37RA (Difco). The antigen challenge occurs by IV injection into the tail vein of 350 ng pertussis toxin (Sigma) immediately and 48 hours after the first immunization. The mice are observed for 25 days. The disease can be observed from day 10 to 25. The treatment of the mice with proNGF and vehicle (PBS) takes place after the immunization and is carried out during the entire study period (until day 25). Here proNGF or Vehicle applied in a 0.5 ml solution by intraperitoneal injection in different doses. Alternatively, the active ingredient and vehicle can also be administered intrathecally. For intrathecal application, the animals are anesthetized and an intraventricular catheter is placed using a stereotaxic frame. The catheter is connected to an osmotic pump (Alzet), which is implanted subcutaneously. Using the EAE model, it can be shown that mice to which proNGF was administered at a dose of 200 micrograms kg / day show a more mild course of the disease compared to vehicles. According to the static evaluation (ANOVA and T-Test) this is with a significance of p <0.0001 (see Figure 2).

Example 3: Comparison of the long-term administration of NGF and proNGF and the cause of pain

For the intracerebral long-term administration of NGF and proNGF, the rats, e.g. B. male Sprague-Dawley, approx. 250-300 g, initially anesthetized (e.g. with Nembutal®, 50 mg / kg, i.p.). The rats are fixed in a David head stereotaxic device. A stainless steel cannula (e.g. Alzet Brain infusion kit) is inserted into the lateral ventricle using the stereotaxical coordinates according to the Atlas of Paxinos and Watson (4th edition 1998, Academic Press) (0.8 mm posterior to bregma, + 1.5 mm lateral to Bregma and 5 mm dorsoventral from the surface of the skull). The cannula is fixed with dental cement (e.g. Dental on Plus®), a catheter connects the cannula to a mini pump (e.g. Alzet 3003, flow rate 0.5 μl / hour for 2 weeks), which is connected to the vehicle or the test substance (NGF or proNGF) is filled. The mini pump is implanted subcutaneously.

The pain test is carried out on different days after the operation, e.g. B. 1 day, 7 days and 14 days after the operation. As a pain test, e.g. B. the "tail flick" test, Von Frey hair stimulation, sensitivity to cold and heat using the "hot plate" test.

Surprisingly, the animals treated with proNGF feel less pain compared to NGF. a) From Frey hair stimulation

The animals are placed on a grid in a clear plastic box. After the acclimatization phase, a filament (e.g. dynamic plantar esthesiometer, Ugo basile) is inserted into the box. Irritation is exerted on the back via the filament. The strength of the pressure is increased until the rat vocalizes or until the maximum pressure is reached. The time to vocalization (in seconds) and the severity of the irritation (in grams) are measured. ProNGF behaves like the vehicle control, i.e. does not increase the sensation of protection with mechanical stimulation, while NGF increases the sensation of pain (Figures 3 and 4).

b) sensitivity to cold

Ethyl chloride is sprayed onto the rat's back. The response is assessed according to different degrees: 0 for no response, 1 for localized response, 2 for transient vocalization and shaking, 3 for permanent vocalization and aversive responses.

c) Heat sensitivity: "hot plate" tests

In the "hot plate" test, the reaction to a heat stimulus is measured. For this purpose, a "hot plate" apparatus (Columbus Instruments, USA) is used. The heating plate has a precisely controlled temperature of 54.0 ± 0.2 ° C during the test. It measures the latency the rat needs to either lick or lift the front or back paw. In the event that the rat shows no response, the latency is limited to 60 seconds.

Finally, it is pointed out that for the complete disclosure, the content of the documents named in the application is included in the application. LITERATURE

Althaus, H, Hempel, R., Kloppner, S., Engel, J., Schmidt-Schultz, T., Kruska, L., and Heumann, R. (1997). Nerve growth factor signal transduction in mature pig ohgodendrocytes. J Neurosci Res 50, 729-742.

Althaus, H. H, and Richter-Landsberg, C. (2000). Glial cells as targets and producers of neurotrophins. Int Rev Cytol 197, 203-277.

Arredondo, L., Deng, C, Ratts, R., Lovett-Racke, A., Holtzman, D., and Racke, M. (2001). Role of nerve growth factor in experimental autoimmune encephalomyelitis. Eur J Immunol 31, 625-633.

Du, Y, and Dreyfus, C. (2002). Ohgodendrocytes as providers of growth factors. J Neurosci Res 68, 647-654.

Ladiwala, U., Lachance, C, Simoneau, S., Bhakar, A., Barker, P., and Antel, J. (1998). p75 neurotrophin receptor expression on adult human ohgodendrocytes: signaling without cell death in response to NGF. J Neurosci 18, 1297-1304.

Lee, R., Kermani, P., Teng, K.K., and Hempstead, B.L. (2001). Regulation of cell survival by secreted proneuro rophins. Science 294, 1945-1948.

Mowla, S.J., Pareek, S., Farhadi, H.F., Petrecca, K., Fawcett, J.P., Seidah, N.G, Morris, S.J., Sossin, W. S., andMurphy, R.A. (1999). Differential Sorting of Nerve Growth Factor and Brain-Derived Neurotrophic Factor in Hippocampal Neurons. J Neurosci 19, 2069-2080.

Nataf, S., Naveilhan, P., Sindji, L., Darcy, F., Brächet, P., and Montero-Menei, C.N. (1998). Low affinity NGF receptor expression in the central nervous system during experimental allergic encephalomyelitis. J Neurosci Res 52, 83-92. Ratts, R., Arredondo, L., Bittner, P., Perrin, P., Lovett-Racke, A., and Racke, M. (1999). The role of CTLA-4 in tolerance induction and T cell differentiation in experimental autoimmune encephalomyelitis: i.p. antigen administration. Int hnmunol 11, 1881-1888. Starkey, G, Petratos, S., Shipham, K., Butzkueven, H, Bucci, T., Lowry, K., Cheema, S., and Kilpatrick, T. (2001). Neurotrophin receptor expression and responsiveness by post-natal cerebral oligodendroglia. Neuroreport 12, 4081-4086.

Webster, H. (1997). Growth factors and myelin regeneration in multiple sclerosis. Mult Scler 3, 113-120.

Woodruff, R.H. and Franklin, R.J. (1997). Growth factors and remyelination in the CNS. Histol Histopathol 12, 459-466.

Claims

1. Use of proNGF, derivatives of proNGF, with the exception of those in which the pro portion has been completely deleted and / or a functional part thereof in an effective amount for the prophylaxis and / or therapy of demyelinating diseases. 2. Use according to claim 1, wherein the derivatives of proNGF are selected from proNGF in which one or more amino acids are exchanged, deleted, added and / or chemically modified, the pharmaceutical activity being retained. 3. Use according to claim 2, wherein the proNGF derivatives in which one or more amino acids are added include the prepro form of NGF. 4. Use according to one or more of the preceding claims, wherein the derivatives are selected from proNGF with at least 25 or 71 amino acids of the pro-C terminus and / or from derivatives with at least 80%, 85%, 90% or 95% homology to Pro, prepro and / or NGF portion of the proNGF. 5. Use according to one or more of the preceding claims, wherein proNGF is selected from animal or human proNGF, tissue-isolated or recombinantly produced proNGF, and / or proNGF fusion proteins. 6. Use according to one or more of the preceding claims, wherein the pro portion of proNGF is in non-cleavable form, and / or proNGF is provided in the form of a nucleic acid encoding proNGF. 7. Use according to claim 6, wherein the nucleic acid is present in a cell such that the proNGF is secreted by the cell. 8. Use according to claim 6 or 7, wherein the nucleic acid coding for proNGF is introduced ex vivo into a cell for the production of proNGF and the cell thus treated is supplied to the patient, or the nucleic acid is administered directly to the patient in the form of a vector, or the nucleic acid encoding proNGF or the cell containing proNGF encoding nucleic acid is administered to the patient in the form of microparticles or nanoparticles. 9. Use according to one or more of the preceding claims, wherein the nucleic acid is capable of expressing proNGF in glial cells. 10. Use according to one or more of the preceding claims, the use being the stimulation of proliferation, the maintenance of vitality and the support and stimulation of regeneration and / or migration processes for the remyelination of glial cells and an inhibition of demyelinating processes, both in the central and also in the peripheral nervous system. 11. Use according to claim 1 or 10, wherein the diseases are selected from neurodegenerative diseases, in particular multiple sclerosis, Charcot-Marie-Tooth and amyotrophic lateral sclerosis. 12. Use according to claim 10, wherein the glial cells comprise oligodendrocytes, astrocytes, microglia, Schwann cells, Müller cells and satellite cells. 13. Use according to one or more of the preceding claims, wherein the active ingredients are in the form of a pharmaceutically active composition, preferably in combination with carriers, diluents, further active ingredients and / or auxiliaries. 14. Use according to claim 13, wherein the further active ingredients are selected from substances that stimulate healing, anti-inflammatory, immunosuppressive, anti-inflammatory and / or analgesic. 15. Use according to one or more of the preceding claims, wherein the proNGF, the derivatives of the proNGF, with the exception of those in which the pro fraction has been completely deleted, and or the functional parts thereof in an amount of 0.1 μg / kg to 500 μg / kg body weight when administered by infusion and from 2 μg to 2 mg per injection when administered by injection. 16. Use according to claim 15, wherein the amount is 0.5 ug / kg to 20 ug / kg body weight when administered by infusion and from 5 ug to 500 ug per injection when administered by injection. 17. Use according to one or more of the preceding claims, the active ingredients being intrathecal, intracerebral, i.m., i.V., i.p. and / or transdermally applicable composition. 18. Use according to one or more of the preceding claims, wherein the pharmaceutical composition is in the form of a liquid, a cream, an ointment or a gel. 19. Use according to one or more of the preceding claims, wherein the glial cells are in the form of glial tissue.