WO2002089779A2 - Preparation combinee pour prevenir et/ou soigner des endommagements de cellules nerveuses et/ou gliales au moyen d'une nouvelle methode de traitement - Google Patents

Preparation combinee pour prevenir et/ou soigner des endommagements de cellules nerveuses et/ou gliales au moyen d'une nouvelle methode de traitement Download PDF

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WO2002089779A2
WO2002089779A2 PCT/DE2002/001049 DE0201049W WO02089779A2 WO 2002089779 A2 WO2002089779 A2 WO 2002089779A2 DE 0201049 W DE0201049 W DE 0201049W WO 02089779 A2 WO02089779 A2 WO 02089779A2
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product according
cells
kinase inhibitor
raf
neurotrophic
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WO2002089779A3 (fr
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Michael Sendtner
Hans-Harald Sedlacek
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MedInnova Gesellschaft für medizinische Innovationen aus akademischer Forschung mbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a product containing at least one kinase inhibitor (A) which has little or no inhibitory activity on Raf, at least one neurotrophic factor (B) and / or at least one substance (C), which in is capable of enhancing and / or causing the cellular formation and / or the release of one or more neurotrophic factors (B) and which can be combined with the kinase inhibitor (A) in addition to or instead of the neurotrophic factor (B) Combination preparation for use in the prophylaxis and / or therapy of nerve cell and / or glial cell damage.
  • A kinase inhibitor
  • B neurotrophic factor
  • C substance
  • nerve cells require trophic support for their survival, and for the growth and maintenance of their nerve cell processes. It is also known that nerve cells, regardless of whether they have a sensory or motor function, can only survive in the presence of a number of so-called neurotrophic factors. Withdrawal of these neurotrophic factors leads, at least in cell culture, to increased expression of pro-optically active proteins, such as, for example, p38, JNK and Fas-L (Le-Niculescu et al Mol Cell Biol 19 (1): 751-763, 1999).
  • pro-optically active proteins such as, for example, p38, JNK and Fas-L (Le-Niculescu et al Mol Cell Biol 19 (1): 751-763, 1999).
  • the neurotrophic factors represent members of different families. They include the neurotrophins, such as, for example, the brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, NT-6 (Götz et al Na- ture 372: 266-269, 1994) and the Nerve Growth Factor (NGF) (Kaplan and Miller, Curr. Opin. Neurobiol.
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4/5 NT-6
  • NGF Nerve Growth Factor
  • HGF hepatocyte growth factor
  • GDNF gliacell derived neurotrophic factor
  • Such neurotrophic factors act by binding to and activating tyrosine kinase receptors.
  • tyrosine kinase receptors For example, tie - NGF with high affinity for the receptor trkA,
  • - CNTF binds to a receptor complex consisting of three subunits, the receptor-a, gpl30 and the LIF receptor-b. LIF and CT-1 also bind to this receptor complex.
  • the activation signal is transmitted from these different receptors for neurotrophic factors to the cell nucleus via signal-transducing proteins present in the cytoplasm.
  • signal transmission paths are known in neurons. They include anti-apoptotic activation pathways, such as the PI-3K-AKT signal transmission path and the Ras-Raf transmission path. Both activation pathways are cross-linked by activated RAS, a protein that plays a key role in the Raf transmission path (Yuan and Yankner, Nature 407: 802-809, 2000).
  • Raf-dependent signaling pathways are the Bag-1-C-Raf signaling path (Wang et al., PNAS USA 93: 7063-7068, 1996) and the Rap-1-B-Raf-cAMP signaling pathway (Grewal et al., J Biol. Chem. 275: 3722-3728, 2000).
  • proapoptotic activation routes such as the p38 or JNK transmission routes stimulated by ASK (Sedlacek Drug 59 (3): 435-476, 2000).
  • neurotrophic factors leads, in nerve cells, at least in cell culture, to increased expression of proapoptotic proteins such as p38, JNK and Fas-L (Le-Niculescu et al., Mol Cell Biol 19 (1): 751-763, 1999).
  • proapoptotic proteins such as p38, JNK and Fas-L
  • the withdrawal of a certain neurotrophic factor can be compensated for by adding another neurotrophic factor (Radorel et al., J Neurobiol 36 (4): 455-467, 1998).
  • the combination of different neurotrophic factors can have an additive or synergistic anti-apoptotic effect on nerve cells (Kobayashi and Matsuoka Neuroreport 1 1: 2541-2545, 2000).
  • NGF has a neurotrophic effect on the peripheral sympathetic and on a subpopulation of sensitive nerve cells, but shows no effect on motor nerve cells.
  • BDNF, NT-3 and NT-4 act on motor nerve cells (Griesbeck et al., J Neurosci Res 42: 21-33, 1995; Henderson et al., Nature 363: 266-270, 1993; Sendtner et al Nature 360 : 757-758, 1992) as well as CNTF (Sendtner et al., 1990), LIF, CT-1, IGF, insulin, GDNF and neurturin.
  • BDNF induces neurite growth in cortical, pyramidal neurons (McAllister et al., Neuron 17: 1057-1064, 1996; Neuron 18: 756-778, 1997) and in Purkinje neurons (Baptista et al., Neuron 12: 243 -260, 1994).
  • receptors for neurotrophic factors is not limited to cells of the nervous system.
  • osteoblasts can form trk-A and trk-B and can thereby be stimulated to proliferation by autocrine and / or paracrine-formed NGF and BDNF (Mogi et al., Life Sei 67 (10): 1197-1206, 2000).
  • Pheochromocytoma cells (Zeil line PC 12) react to NGF with the formation of neurite-like processes, a phenomenon that can be increased by adding inhibitors of cell proliferation, such as for example rapamycin, ciclopirox or flavopiridol (Parker et al Neu- ropharmacology 39 (10): 1913-1919, 2000).
  • inhibitors of cell proliferation such as for example rapamycin, ciclopirox or flavopiridol
  • proteins of the IAP / ITA family inhibit the function of the activated caspases-3, -6 and -7 and can also thereby inhibit the apoptosis caused by these enzymes (Devereaux et al., Nature 388: 300-304, 1997; Roy et al., EMBO J. 16: 6914-6925, 1997).
  • Transfection of I AP expression plasmids into isolated sensory or sympathetic nerve cells can prevent cell death caused by NGF withdrawal.
  • the effect of IAPs on neurite growth is significantly less than that of NGF and other neurotrophic factors (Wiese et al., Nature Neurosci 2: 978-893, 1999).
  • Members of the IAP family can thus mediate a survival effect, but are only slightly involved in the outgrowth and maintenance of nerve processes. This indicates that NGF and other neurotrophic factors activate signaling pathways that are different from those of the IAP family.
  • Akt a protein of the PI-3K-AKT signaling pathway
  • Akt-3 is particularly expressed in neurons
  • C-Raf also called Raf-1
  • A-Raf A-Raf
  • B-Raf B-Raf
  • B-Raf B-Raf
  • B-Raf and C-Raf can be detected not only in neurons but also in glial cells (Mikaly et al., Brain Res. 27: 225-238, 1993; Mikaly and Rapp, Acta Histochem. 96: 155-164, 1994) ,
  • proapoptoti see factors within the nerve cell, for example through a lack of phosphorylation and inactivation of proapoptotic proteins (Bad, but especially from Bax) in the context of the AKT and / or Raf signaling pathways,
  • BDNF can drastically amplify nerve cell damage caused by nitrogen monoxide (Ishikawa et al., J Neurochem 75 (2): 494-502, 2000).
  • inhibitors of cell proliferation such as, for example, those which inhibit cyclin-dependent kinases (such as, for example, flavopiridol, roscovitin or olomoucin (Park et al., J Biol Chemistry 271: 21898-21905, 1996; Maas et al., J Neurochem 70 (4): 1401-1410, 1998) or other types of inhibitors of cell proliferation (such as chloro-phenylthio-cAMP, N-acetylcysteine, deferoxamine, ciclopirox or mimosin, Park et al., 17: 1256-1270, 1997) , or selected immunophilin-binding substances (such as, for example, Rapamycin, Parker et al., Neuropharmacology 39 (10): 1913-1919, 2000), are able to prevent pheochromocytoma cells (PC 12) or nerve cells in cell culture from being influenced by cell-damaging factors Protect substances.
  • Inhibitors of apoptotic proteins can prevent the rapid apoptosis of damaged nerve cells, but not the delayed cell death.
  • Substances that have been used in these experiments to damage nerve cells and lead to their apoptosis are, for example, the cytostatics camptothecin, cytosine arabinoside, etoposide, teniposide or mitoxanthrone (Park et al., J Neuroscience 17: 1256-1270, 1997) , or the proapoptotic B-amyloid protein (Giovanni et al., J Biol Chemistry 274/27: 19011-19016, 1999, Park et al., Neurobiol Aging 21, 771-781, 2000).
  • CDKs cyclin-dependent kinases
  • proapoptotic signal proteins such as p38 and JNK in nerve cells
  • the nerve cell protective effect of inhibitors of cell proliferation is based on a direct inhibition of the CDKs (in particular of cdk4 and cdk6).
  • This inhibition consequently inhibits the phosphorylation of pRB and P107 and thus inhibits the transcriptional activity of E2F / Dp (Sedlacek, Critic. Rev. Onc./Heam, 37 (2001).
  • cycloheximide appears to reduce the apoptosis of nerve cells after treatment with Ara-C (and the increased expression of proapoptotic proteins such as p38 and JNK).
  • Treatment with kinase inhibitors also entails the risk that not only proaptotic kinases (such as the cyclin-dependent kinases involved in cell division [CDK]) are inhibited in nerve cells, but also antiapoptotic kinases, for example signal-transducing kinases such as B-Raf (Wiese et al., Nature Neurobiol 4: 137-142, 2001) or those such as PKC, the inhibition of which leads to a reduction in the function and axon growth of nerve cells (Audesirk et al., Brain Res Dev Brain Res 102 (2): 247-260, 1997).
  • proaptotic kinases such as the cyclin-dependent kinases involved in cell division [CDK]
  • antiapoptotic kinases for example signal-transducing kinases such as B-Raf (Wiese et al., Nature Neurobiol 4: 137-142, 2001) or those such as PKC, the
  • staurosporine an inhibitor of a number of kinases including CDKs
  • kinase inhibitors should rather intensify the damage to nerve cells.
  • staurosporine an inhibitor of a number of kinases including CDKs
  • CDKs CDKs
  • Wortmannin a kinase inhibitor with special activity on phosphatidylinositol 3-kinase, inhibits the anti-apoptotic activity of IGF-1 (Yamaguchi et al., J Biol Chem Oct 27, 2000). Flavopiridol not only inhibits CDKs, but also receptor-associated kinases such as PKC very effectively (Sedlacek et al, Int J Oncol 9: 1 143-1168, 1996; Sedlacek, Crit Rev Oncol Hematol 2000). By inhibiting such anti-apoptotic kinases, the administration of flavopiridol could carry the risk of nerve cell damage. In this context it should be borne in mind that an isolated inhibition of PKC can inhibit neurite growth and thus possibly also trophic Disorders of the nerve cell processes occur, which intensify the neurodegenerative changes.
  • kinase inhibitors As well as for any other substance that may protect nerve cells, it is therefore important to what extent it can influence neurite growth-promoting and / or inhibiting mechanisms in clinically relevant target cells, for example whether it is able to demand or inhibit the axon growth of motor neurons, sensory neurons, dopaminergic neurons, Purkinje cells, cholinergic projection neurons and retinal ganglion cells.
  • a combination of flavopiridol with low concentrations of NGF leads to outgrowth of neurite-like processes on pheochromocytoma cells (such as PC12 cells) (Parker et al., Neuropharmacoly 39 (10): 1913-1919, 2000).
  • flavopiridol as a kinase inhibitor, it could also be seen when using other inhibitors of cell proliferation, such as rapamycin or ciclopirox, but not with those immunophilin-binding substances such as FK506 or GPI-1046, which none Represent inhibitors of cell proliferation.
  • this effect was not transferable to nerve cells.
  • FK506 like rapamycin
  • the protective effect of this combination of kinase inhibitor (A) and / or neurotrophic factor (B) and / or substance (C) on nerve cells is significantly stronger than the effectiveness of the individual substances.
  • kinase inhibitor (A) and / or neurotrophic factor (B) and / or substance (C) on nerve cells is significantly stronger than the effectiveness of the individual substances.
  • B-Raf a neurotrophic factor
  • C substance
  • the protective effect of this combination of kinase inhibitor (A) and / or neurotrophic factor (B) and / or substance (C) on nerve cells is significantly stronger than the effectiveness of the individual substances.
  • B-Raf Especially in the presence of an enzymatically functional B-Raf and, above all, with an additionally increased expression of members of the IAP gene family. Expression is increased via PI-3K, AKT and NFkB-mediated signaling pathways.
  • the combination of kinase inhibitor (A) and neurotrophic factor (B) has little or no effect
  • the present invention therefore relates to a product comprising at least one kinase inhibitor (A) which has little or no inhibitory activity on Raf and a. at least one neurotrophic factor (B) and / or b. at least one substance (C) which is capable of enhancing and / or causing the cellular formation and / or the release of one or more neurotrophic factors (B) as a combination preparation in the form of a mixture or as individual components for simultaneous or temporal use different application at different or the same places in the prophylaxis and / or therapy of nerve cell and / or glial cell damage.
  • A kinase inhibitor
  • B neurotrophic factor
  • C at least one substance which is capable of enhancing and / or causing the cellular formation and / or the release of one or more neurotrophic factors (B) as a combination preparation in the form of a mixture or as individual components for simultaneous or temporal use different application at different or the same places in the prophylaxis and / or therapy of nerve cell and
  • the product with the nerve and / or glial cells at the same or different locations, as individual components or as a mixture, simultaneously or at different times, preferably within 6 hours, is particularly preferred within 12 hours, in particular within 24 hours, especially within 48 hours, for example simultaneously with a mixture of kinase inhibitor (A) with neurotrophic factor (B) and / or substance (C), or first with kinase inhibitor (A) and subsequently with neurotrophic factor (B) and / or substance (C) or first with neurotrophic factor (B) and / or substance (C) and subsequently with kinase inhibitor (A).
  • the pretreatment is preferably carried out with kinase inhibitor (A) or the simultaneous treatment with kinase inhibitor (A) and / or neurotrophic factor (B) and / or substance (C).
  • the first substance is still effective in the nerve cell to be treated when the second substance is administered.
  • the interval between the administration of the first substance and the second substance is preferably up to 6 hours, particularly preferably up to 12 hours, in particular up to 24 hours, especially up to 48 hours.
  • the contacting can take place by adding the substances, for example, into the nutrient medium of a nerve cell or glial cell culture, or by local or systemic administration of the substances into an organism.
  • Local administration within the meaning of this invention can, for example, in a wound cavity, on the skin, in a body opening, in a body cavity, such as in the chest or abdominal cavity, or in an organ, in particular in the Gehim, into the spinal canal, into a joint, into the connective or muscle tissue.
  • Systemic administration can be oral, rectal or in the circulation, for example intravenously or intraarterially.
  • Function of nerve cells is understood to mean, for example, the stimulus conduction and all the biochemical and / or electrochemical processes involved. Furthermore, the function of nerve cells includes the morphologically detectable growth of neurites or axons and of dendrites. In particular, the term nerve cell function encompasses the survival of nerve cells.
  • the death of nerve cells can be observed, for example, by in vitro test methods with which apoptosis can be detected.
  • test methods are, for example, "tunnel assay” (Gavrielli et al., J. Cell Biol., 1 19: 493-501, 1992; Gold et al., Lab.Invest. 71: 219-225, 1994), chromatin fragmentation ( Götz et al., Hum. Mol. Genet. 9: 2479-2489, 2000), enumeration of surviving and dying nerve cells (Arakawa et al., J. Neurosci.
  • Cells of the nervous system in the sense of the present invention are glial cells or neuronal cells, for example sensory and sympathetic neuronal cells, motor neuronal cells, cholinergic neurons of the basal forebrain, dopaminergic nerve cells of the midbrain (substantia nigra), granule cells and Purkinje cells of the cerebellum and the hippocampus , retinal ganglion cells and photoreceptors as well as neuronal stem cells.
  • glial cells or neuronal cells for example sensory and sympathetic neuronal cells, motor neuronal cells, cholinergic neurons of the basal forebrain, dopaminergic nerve cells of the midbrain (substantia nigra), granule cells and Purkinje cells of the cerebellum and the hippocampus , retinal ganglion cells and photoreceptors as well as neuronal stem cells.
  • the kinase inhibitors (A) with little or no inhibitory activity on B-Raf are kinase-inhibiting derivatives of 4H-1-benzopyran.
  • Examples of such derivatives are described in detail in the patents EP 0137193 and EP 366061 and the structure-activity relationship for these derivatives has been described in detail by Sedlacek et al., Int J Oncol 9: 1 143-1168, 1996).
  • the flavopiridol derivatives and the flavopiridol itself (Sedlacek et al., Int J Oncol 9: 1143-1168, 1996) exemplified in these patents are expressly the subject of this invention.
  • the kinase inhibitors (A) are flavopiridol derivatives, represented by Kim et al., J Med Chem 43 (22): 4126-4134, 2000, in particular thio-flavopiridol and oxy-flavopiridol.
  • examples of kinase inhibitors (A) are the 2- (2-amino-3-methoxyphenyl) -4-oxo-4H- (1) benzopyran shown by Ebendal (WO 00/50030).
  • the kinase inhibitors (A) are kinase inhibitors (A).
  • Kinase inhibitors such as, for example, 7OH-staurosporins and their kinase-inhibiting derivatives, butyrolactones, roscovitines, Purvalanol A, emodin, anilinoquin azolines (PD-168393 and PD 169414) and phenylamino- pyrimidines (STI 571, CGP 78850, CP 358774, CP59326 and CGP 60474), trioylimidazoles (1-779450), as a whole by Meijer et al., Parmacol. Ther. 82 (2-3): 297-284, 1999 and by Sedlacek et al., Int J Oncol 9: 1143-1168, 1996 or by Sedlacek, Drug 59 (3): 435-476, 2000),
  • Tetrapyrrolic macrocycles such as 5,10,15,20-tetraarylporphyrin and 5,10,15-triarylcorrol (Aviezer et al., WOOO / 27379) and / or
  • the kinase inhibitor (A) is preferably administered in a single dose, particularly preferably in a multiple dose, which are less than the maximum tolerable dose (MTD) of the respective substance for humans.
  • MTD maximum tolerable dose
  • one Dose chosen which is half the MTD.
  • the MTD on the tumor patient is 50 mg / m 2 / dx3 (Senderowicz et al., J Clin Oncol 16 (9): 2986-2999, 1998).
  • flavopiridol is thus preferably at a dose of 0.1 50 mg / m 2, more preferably from 5 to 30 mg / m 2, in particular from 25 mg / m 2 administered daily.
  • the daily dose can be administered once a day or divided into several portions over the day at approximately the same time intervals.
  • the kinase inhibitor (A) is preferably administered either locally or systemically, for example only on one day or for several days daily or on every second or third day for several weeks until the therapeutic effect becomes visible.
  • a treatment of two to 25 weeks in duration is preferably selected in combination with the neurotrophic factor (B) and / or with substance (C).
  • neurotrophic factors (B) are for example
  • BDNF neurotrophins BDNF and variants of BDNF
  • NT-3, NT-4/5, NT-6 or variants of the NT and NGF such as, for example the NNT-1 (Chang, US 5,741,772),
  • these neurotrophic factors (B) are preferably produced with the aid of recombinant DNA technology.
  • the neurotrophic factors (B) are preferably administered locally or systemically, in a dose which is below the MTD, particularly preferably about half the MTD.
  • the MTD is around 30 mg / kg body weight.
  • CNTF or CT-1 or CLC / N T-1 is preferably used in a dose of 0.1-30 mg / kg, particularly preferably 2-25 mg / kg, further preferably 5-20 mg / kg, in particular 15 mg / kg administered.
  • the daily dose for the neurotrophic factor (B) is administered once or in partial portions at approximately the same time intervals throughout the day.
  • the neurotrophic factor (B) can be administered once or daily or every other, third, fourth, fifth, or sixth day or once a week. Similar to the kinase inhibitor (A), the treatment duration depends on the clinical effect. A treatment period of 2 weeks to 25 weeks in combination with a kinase inhibitor (A) is preferably selected.
  • the neurotrophic factor (B) for example in the form of a recombinantly produced protein, is preferably introduced into an organ or tissue in the subarachnoid space (as described by Dittrich et al., Exp Neurol 141: 225-239, 1996) by means of suitable medicament pump systems. such as in the brain or in the muscles, or systemically, for example in the blood vessel system.
  • the neurotrophic factor (B) is not present as a protein, but rather as a nucleotide sequence which codes for the neurotrophic factor.
  • nucleotide sequences are generated using methods known to those skilled in the art, either in plasmids or in viral vectors, for example adenoviral vectors (for example described by Gravel et al., Nature Medicine 3: 765-770, 1997; Cayouette et al.
  • retroviral vectors or vectors which are derived from other viruses for example AAV or HIV
  • viruses for example lentiviruses, in particular those viruses which have a tropism for nerve cells (for example HPV) or muscle cells
  • Plasmids or viral vectors are mixed with suitable auxiliaries, for example protease inhibitors, detergents, buffers, and / or transfection reagents, such as, for example, cationic lipids or cationic polymers, and administered to the organism in addition to the neurotrophic proteins or instead of the neurotrophic proteins.
  • substances (C) are all substances which stimulate the intracellular formation and / or the release of neurotrophic factors. These substances include, for example
  • Cytokines and interleukins in particular also those which stimulate macrophages, lymphocytes, nerve cells and / or glial cells, such as IL-1, IL-2, IL-3, IL-4, IL-6, TNF-alpha, Interferon (IFN) alpha, beta, and gamma, GM-CSF, M-CSF, (Hughes et al., Brain Res Mol Brain Res 53: 138-151, 1998; Laurenzi et al., Eur J Biochem 223 : 733-744, 1994),
  • IFN Interferon
  • D1 / D2 dopamine agonists for example apomorphins (Ohta et al., BBRC 272: 18-22, 2000),
  • Beta-adrenergic agonists for example isoproteronol (Barouch et al., J Neuroimmunol 103: 1 12-121, 2000),
  • Neurotransmitters for example substance P (Barouch et al., J Neuroimmunol 103: 1 12-121, 2000),
  • Catechol and catechol derivatives for example 4-methylcatechol (Nitta et al., J Pharmacol Exp Ther 291: 1276-1283, 1999),
  • Diterpenoids for example scabronin-A and scabronin-B (Obara et al., Eur J Pharmacol 370: 79-84, 1999), Forskolin and forskolin derivatives, for example NKH 477 (Morinobu et al., J Neurochem 72: 2198-2205, 1999),
  • Agonists for cholinergic receptors for example nicotine, carbachol and pilocarpine (French et al., Brain Res Mol Brain Res 67: 124-136, 1999),
  • Agonists for the glutamate receptor for example quisqualate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N-methyl-D-aspartate (NMDA) (Bessho et al., Brain Res Mol Brain Res 18: 201-208, 1993),
  • AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate
  • NMDA N-methyl-D-aspartate
  • Ribavirins and ribavirin derivatives for example 1- (beta-D-ribofuranosyl) -1H-1,2,4-triazole (Gage et al., WO 00/30656),
  • Retinoids for example retinoic acid (Wion et al., BBRC 149: 510-514, 1987)
  • Sulfonyl-urea derivatives for example glibenclamide, glimepiride, gliclacid, glibomuride, gliquidon, glisoxide pioglitazone, rosiglitazone maelate and / or repaglinide
  • Substance (C) is preferably administered in a dose which is below the maximum tolerable dose (MTD), for example for humans.
  • a dose in the range of 5% -90% of the MTD is particularly preferred, furthermore preferably 10% -60%, in particular 50% of the MTD.
  • One or more different kinase inhibitors) (A) and neurotrophic factor (s) (B) and / or substance (s) (C) are preferably administered in each case.
  • the product containing the kinase inhibitor (A) and the neurotrophic factor (B) and or the substance (C) is preferably administered in the event of disorders of the function of cells of the central or peripheral nervous system, for example in cerebral ischemia (stroke), amyeotrophic lateral sclerosis (ALS), Alzheimer's disease, nerve lesions, multiple sclerosis, Parkinson's disease, diabetic neuropathy, spinal muscular atrophy and / or prion diseases, such as Creutzfeld-Jakob disease.
  • stroke cerebral ischemia
  • ALS amyeotrophic lateral sclerosis
  • Alzheimer's disease nerve lesions
  • multiple sclerosis multiple sclerosis
  • Parkinson's disease diabetic neuropathy
  • spinal muscular atrophy and / or prion diseases, such as Creutzfeld-Jakob disease.
  • a preferred embodiment is a method for producing a product according to the invention, in which at least one kinase inhibitor (A) is combined in a suitable manner with at least one neurotrophic factor (B) and / or at least one substance (C).
  • a further embodiment is the use of a product according to the invention for the prophylaxis and or therapy of nerve cell and / or glial cell damage.
  • a preferred embodiment is the use of a product according to the invention in which at least one kinase inhibitor (A) has at least one neurotrophic factor (B) and or at least one substance (C) are brought into contact with nerve cells and / or glial cells.
  • at least one kinase inhibitor (A) has at least one neurotrophic factor (B) and or at least one substance (C) are brought into contact with nerve cells and / or glial cells.
  • a further embodiment is a method by which a product according to the invention is brought into contact with nerve cells and / or glial cells in vitro.
  • ventrolateral parts of the lumbar spinal cord were mechanically crushed, transferred into hepespuffer solution (containing 10 ⁇ M 2-mercaptoethanol) and incubated with trypsin (0.05%, 10 min).
  • trypsin 0.05%, 10 min.
  • the single cell suspension in the supernatant was transferred to a culture dish coated with the anti p75 antibody and at room temperature for 30 min. incubated.
  • the individual culture dishes were then washed, and then the adhering cells were removed from the culture plate by 0.8% saline solution containing 35 mM KC1 and 1 ⁇ M 2-mercaptoethanol.
  • the cells obtained in this way were sown with a density of 2000 cells cm 2 in culture plates (Greiner, Nuertingen, Germany), which were precoated with polyomithine and laminin.
  • the cells were in 37 ° C in neurobasal medium (Life Technologies, supplemented with B27 supplement, 10%> horse serum, 500 ⁇ M glutamax and 50 ⁇ g / ml apotransferrin) and kept in a 5% CO 2 atmosphere. 50% o of the cell culture medium was replaced on day 1 and subsequently every other day.
  • the primer sequences for amplifying IAP-1, IAP-2, x-IAP and t-IAP (survivin) were as follows:
  • IAP-lf 5'-TACTACATAGGACCTGGAGA-3 ⁇
  • IAP-lr 5 '-CCCACCATCACAGCAAAA-3', annealing: 55 ° C;
  • IAP-2f 5 '-GGAGAAGAAAATGCTGACCC-3'
  • IAP-2r 5 ' -GCTTGTAAGGGTATCTGTGT-3' annealing 55 ° C;
  • x-IAPf 5 ' -TGCAAGAGCTGGATTTTATG-3',
  • x-IAPr 5'-CCCGATCTGGCAGCTGTACC-3 ' annealing 55 ° C;
  • tlap (survivin) f 5 '-CCA GAT CTG GCA GTA GCT CC-3', tlap (survivin) r: 5'-GCC TGC AGC TCA ATT GAC TG-3 ', annealing 64 ° C.
  • part of the ⁇ -actin mRNA was amplified with the following primers: ⁇ -actinf: 5 ' -GTGGGCCGCCCTAGGCACCAG-3', ß-actinr: 5 ' -CTCTTTAATGTCACGCACGATTTC-3 ⁇ annealing 64 ° C.
  • the RT-PCR was carried out according to the manufacturer's protocol with random hexamer primers. The PCR amplification was carried out as follows: 94 ° C, 30 sec, indicated annealing temperatures, 1 min, 72 ° C, 1 min.
  • IAP-1 and t-IAP were for 33 and 35 cycles, IAP-2 and x-IAP for 28 and 30 cycles and ß-actin for 26 and 28 cycles.
  • the RT-PCR on RNA from El 2.5 brains of b-raf and c-raf +/- pairings showed a clear decrease by an average of 60% or 55%> for IAP-1 for b-raf and c-raf - / - embryos compared to the wild-type control, of 52%> for IAP-2 for b-raf - / - and 46% > for x-IAP in b-raf - / - embryos compared to the wild-type control.
  • Sensory neurons were also isolated from embryos 12.5 days old that were homozygous for b-raf (- / -) or c-raf (- / -).
  • dorsal root ganglia were isolated, in PBS and with trypsin (0.05% in hepespuffer) for 30 min. incubated. The trypsin digestion was stopped by adding L15 medium containing 10% horse serum and then the cells were plated in culture plates for 3-4 hours. Cells in the supernatant were centrifuged (10 min. 400 g) and the cell sediment was kept in the neurobasal medium in the same way as described for spinal motor neurons.
  • Neural stem cells were isolated from the brain from normal, b-raf ' (- / -) or c-raf (- / -) deficient mouse embryos. The area of the forehead was removed under a dissecting microscope, and in more developed embryos the area of the hippocampus and the periventricular zone.
  • HBSS Hanks balanced salt solution
  • trypsin inhibitor from egg yolk sack (Sigma, Deisenhofen)
  • HBSS horse yolk sack
  • HEPES trypsin inhibitor from egg yolk sack
  • 1% in HBSS / 25 mM HEPES trypsin inhibitor from egg yolk sack
  • the cells were triturated 10X with a 200 ⁇ l pipette and placed in medium [ (Neurobasal Medium (Life Technologies), B27 Supplement (Life Technologies Stock 50x, EK lx) Glutamax II (Life Technologies Stock lOOx, EK lx), basicFGF (20ng / ml), EGF (20 ng / ml) l] transferred to a volume of 5 ml.
  • the dissociated cells were cultivated in Sarstedt dishes (50 ml) (incubator, 37 ° C., 5%> CO 2 moisture-saturated atmosphere), the medium was changed every two days. The cells grew as embroid bodies and did not attach, so the cells were transferred to a Falcon tube to change the medium and centrifuged at 400 g for 5 min. The supernatant was aspirated and the cell sediment was triturated and taken up in fresh medium. At the latest after 3 passages, large embroid bodies formed, which could be trypsinized (see above) and in low cell density (max. 10,000 cells / plate) on 10 cm dishes (Sarstedt). Individual cells were then picked and expanded first in 96-well plates, later in 24-well and 12-well plates. These single cell clones of neural stem cells could then be examined for their differentiation capacity and then used in test procedures.
  • Freezing of the neural stem cells was carried out according to the standard protocol, ie after centrifugation, the cells were taken up in medium with 10%> DMSO and initially cooled to -86 C at 1 C / min (in MrFrosti) and then in the liquid N 2 at - 186 C to be stored.
  • Example 3 Effect of neurotrophic factors in combination with a kinase inhibitor on neurons
  • flavopiridol causes a slight increase in the survival rate (depending on the concentration, 20 - 40%> of the cells survive). The greatest increase in the survival rate can be seen with the combination of growth factors, flavopiridol and pilocarpine or with the combination of flavopiridol with pilocarpine (depending on the concentration, survival between 40-90% of the cells).
  • neurotrophic growth factor neurotrophic factor (neurotrophic factor (B)
  • flavopiridol kinase inhibitor (A)
  • pilocarpine substance (C)
  • flavopiridol kinase inhibitor (A)
  • pilocarpine substance (C)
  • flavopiridol kinase inhibitor (A)
  • pilocarpine substance (C)
  • Example 4 Effect of the method on b-raf (- / -) cells into which b-raf was transfected
  • kinase inhibitor (A) and NGF (B) were added under otherwise identical conditions and the survival rate was determined.
  • Such a kinase inhibitor (A) is considered to be slightly inhibitory if the survival rate is at least 10%, better 30%, optimally 50%, of the reference test.
  • concentrations and the concentration ratios of components (A) to (C) can be in the following ranges: i) A: 0.001-100 ⁇ M, in particular 0.05-1 ⁇ M, for example 0.05-0.5 ⁇ M, ii) B: 0.001-100 ng / ml, in particular 0.01-10 ng / ml, for example 0.05-0.5 ng / ml, iii) C: 0.001-100 ⁇ M, in particular 0.005-10 ⁇ M, for example 0.01 - 0.5 ⁇ M.
  • A 0.001-100 ⁇ M, in particular 0.05-1 ⁇ M, for example 0.05-0.5 ⁇ M
  • C 0.001-100 ⁇ M, in particular 0.005-10 ⁇ M, for example 0.01 - 0.5 ⁇ M.
  • either the concentration of component A in the lower half of the above exemplary ranges is at concentrations of components B and / or C in the upper half of the above exemplary ranges, or the concentration of component A is in the upper half of the above exemplary ranges Concentrations of Components B and / or C in the lower half of the above exemplary ranges.
  • the invention also relates to a method for the treatment and / or prophylaxis of diseases with nerve cell and / or glial cell damage, wherein a patient is given a pharmacologically effective dose of the product according to the invention in galenic form for the selected dosage form.
  • the invention further relates to a method for the in vitro efficacy test of a product containing a component A or several different components A and a component B or several different components B and / or a component C or several different components C, raf expressing nerve cells and / or glial cells are cultivated, wherein said cells are mixed with said product, after which the survival rate is determined after a defined incubation period and compared to the survival rate under the same experimental conditions, but with a reference product according to the invention.
  • it is also easy to optimize the concentration ratios of components A, B and / or C for example by comparing the survival rate with reference concentrations or by maximizing the absolute value of the survival rate.

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Abstract

L'invention concerne un produit, qui contient au moins un inhibiteur de kinase (A), lequel n'a qu'une faible activité sur Raf ou pas du tout, et au moins un facteur neurotrophique (B) et/ou au moins une substance (C) apte à renforcer et/ou à provoquer la formation cellulaire et/ou la libération cellulaire d'un ou de plusieurs facteurs neurotrophiques (B), en tant que préparation combinée, destinée à prévenir et/ou soigner des endommagements de cellules nerveuses et/ou gliales.
PCT/DE2002/001049 2001-03-20 2002-03-19 Preparation combinee pour prevenir et/ou soigner des endommagements de cellules nerveuses et/ou gliales au moyen d'une nouvelle methode de traitement WO2002089779A2 (fr)

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DE10113513A DE10113513A1 (de) 2001-03-20 2001-03-20 Kombinationspräparat zur Prophylaxe und/oder Therapie von Nervenzell- und/oder Gliazellschäden durch ein neues Behandlungsverfahren
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DE10313098A1 (de) * 2003-03-24 2004-10-14 MedInnova Gesellschaft für medizinische Innovationen aus akademischer Forschung Testsystem für die Findung von Wirkstoffen gegen prioneninduzierte Erkrankungen und Wirkstoffe zur Verhütung und zur Behandlung dieser Erkrankung
WO2008097861A2 (fr) * 2007-02-02 2008-08-14 Braincells, Inc. Modulation d'une neurogénèse avec des biguanides et des agents gsk3-ss

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0137193A2 (fr) * 1983-08-12 1985-04-17 Hoechst Aktiengesellschaft Alcaloide de chromane, procédé pour son isolation à partir de dysoxylum binectariferum, et son utilisation comme médicament
EP0366061A1 (fr) * 1988-10-28 1990-05-02 Hoechst Aktiengesellschaft Application de dérivés de la 4H-1-benzopyrannone-4, dérivés de la 4H-1-benzopyrannone-4 et médicaments les contenant
EP0651754A1 (fr) * 1992-07-24 1995-05-10 Cephalon Inc DERIVES DE K-252a ET DE BIS-STAUROSPORINE.
GB2337702A (en) * 1998-05-29 1999-12-01 Merck Sharp & Dohme The use of staurosporine analogues for enhancing neurotrophin activity
WO2000033858A1 (fr) * 1998-12-07 2000-06-15 Ecosmart Technologies, Inc. Composition anticancereuse et procede utilisant des huiles essentielles vegetales naturelles avec des modulateurs de transduction de signal
WO2000050030A1 (fr) * 1999-02-25 2000-08-31 Ted Ebendal Nouvelle utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0137193A2 (fr) * 1983-08-12 1985-04-17 Hoechst Aktiengesellschaft Alcaloide de chromane, procédé pour son isolation à partir de dysoxylum binectariferum, et son utilisation comme médicament
EP0366061A1 (fr) * 1988-10-28 1990-05-02 Hoechst Aktiengesellschaft Application de dérivés de la 4H-1-benzopyrannone-4, dérivés de la 4H-1-benzopyrannone-4 et médicaments les contenant
EP0651754A1 (fr) * 1992-07-24 1995-05-10 Cephalon Inc DERIVES DE K-252a ET DE BIS-STAUROSPORINE.
GB2337702A (en) * 1998-05-29 1999-12-01 Merck Sharp & Dohme The use of staurosporine analogues for enhancing neurotrophin activity
WO2000033858A1 (fr) * 1998-12-07 2000-06-15 Ecosmart Technologies, Inc. Composition anticancereuse et procede utilisant des huiles essentielles vegetales naturelles avec des modulateurs de transduction de signal
WO2000050030A1 (fr) * 1999-02-25 2000-08-31 Ted Ebendal Nouvelle utilisation

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