WO2004086051A1 - Test system for discovering active ingredients against prion-induced illnesses and active ingredients for preventing and treating said illnesses - Google Patents

Abstract

The invention relates to a test system for discovering active ingredients which stop the inhibition of axon growth by infectious prions, said test system comprising the following constituents: a) a nerve cell kept in a culture, said nerve cell forming axons in the cell culture, b) a preparation containing an abnormal prion protein, and c) a detection system for the formation of axons. The invention also relates to a method for discovering active ingredients which stop the inhibition of axon growth by infectious prions using one such test system, and to active ingredients that can be determined by means of one such test.

Description

 Test system for finding active substances against prion-induced diseases and active substances for the prevention and treatment of these diseases

Field of the Invention.

The invention relates to a test system and a method for identifying active substances which are suitable for the prophylaxis and / or treatment of prion diseases. The invention further relates to such active substances as well as pharmaceutical compositions and formulations containing such active substances.

State of the art and background.

Prion diseases or transmissible spongiform encephalopathies (TSE) such as scrapie (SC), the bovine spongiform encephalopathy (BSE) and the

Creutzfeldt-Jakob Disease (CJD) are diseases of the central nervous system that are associated with vacuolation, gliosis and an accumulation of a disease-specific, protease-resistant, ß-folded isoform (PrPSc; PrPdJD) of the normal prion protein (PrPc).

The PrPc is a glycoprotein that can be found on the membrane of almost all cells, especially the nerve cells, and is connected to the membrane by a glycosylphosphatidylinositol anchor. The PrPc occurs in higher concentrations in the nerve cells and is particularly found in the presynaptic membranes (Ferrer et al Neuroscience 97: 71-726, 2000). The function of the PrPc has not yet been clearly clarified. There is evidence that PrPc plays a role in copper metabolism and that copper ions stimulate endocytosis of PrPc. Other studies indicate that PrPc plays a role in circadian rhythm (Tobler et al.,

1996), and in the long-term potentiation of synapses (Colinge et al., 1994). Furthermore, PrPc appears to be a high affinity ligand for laminin and thereby to support the adhesion and presence of nerve cells and the growth of neurite segments (Martins et al Braz. J Med Biol Res 34: 585-595; 2001. In addition, the Binding of PrPc to caveolin-1 to activate cellular signal-transmitting kinases, such as, for example, by Fyn (Mouillet-Richard et al., 2000); Kellermann et al CR Acad Sei 325: 9-15, 2002). Since PrPc also activates the cAMP / PKA-dependent signaling pathway, for example after cross-linking via antibodies, the PrPc is also assigned a function as a neurotrophic receptor, the activation of which is said to have a neuroprotective effect (Chiarini et al EMBO 21: 3317-3327; 2002) ,

Infection with abnormal, protease-resistant PrPSC is believed to be the cause of TSE. Findings that a chemical or immunological sy pathectomy according to i.p. Injection of PrPsc can delay the onset of TSE suggest that the CNS may be infected by abnormal prions by neuroinvasion (Glatzel et al Neuron 31: 25-34, 2001)

After infection, PrPSC accumulates in nerve cells

Foothills and in the synapses (Ferrer et al Neuroscience 97: 715-726, 2000). The abnormal PrP (PrPSC; PrPCJD) interacts with the PrPc and leads to a structural reshaping of the PrPc. In the course of this accumulation of abnormal PrP, degenerations of the axon ends and of dendrites have been described (Jeffrey et al Neuropathol Appl Neurobiol 26: 41-54, 2000; Jeffrey et al Neuropathol Appl Neurobiol 23: 93-101, 1997).

However, how the abnormal PrP leads to TSE is largely unknown.

There is currently no possibility of effective therapy for TSE (Collins et al (2002) Ann. Neurol 52: 503-506). The previously attempted strategies for the therapy of TSE essentially included the inhibition of the conversion of PrPc caused by PrPSC or PrPCJD into an abnormal PrPSC by specific RNA aptamers (Proske et al; (2002) Chembiochem 3, 717-725); Quinacrine (Caughey et al (2002) Bioche Soc Trans 30: 565-569; Collins et al (2002) Ann. Neurol 52: 503-506); by fragments of the prion protein isoforms and by

Peptidomimetics (Horiuchi et al (2001) J Biol Chem 276: 15489-15497; Chabry et al (1998) J Biol Chem 273: 13203-13207); by Congo red and analogues thereof (Rudyk et al (2000) J Gen Virol 81: 1155-1164), porphyrins and phthalocyanines (Caughey et al (1998) PNAS USA 95: 12117-12122) tetracycline derivatives (Forloni et al., 2002 ) or by triaminopyridines (Perovic et al (1995), Neurodegeneration 4: 369-374.

In a special research approach, the interaction between PrPc and abnormal PrPSC by antibodies against PrP in vitro (Peretz et al (2001) Nature 16/412: 739-743, (Enari et al., 2001) or in vivo Koller et al J Neuroimmunol (2002) 132: 113-116; White et al (2003), Nature 422: 80-83 or by immunization against PrP in a transgenic mouse model (Heppner et al (2001) Science; 5/294 178-182, Sigurdson et al Neurosci Lett (2003), 336: 185-187) and thereby inhibited one Delay in the occurrence of the TSE in the mouse causes.

However, the finding alone that the neurotrophin p75 receptor appears to be crucially involved in the neurotoxicity of abnormal prion protein (Della-Bianca et al (2002-) J Biol Chem 19/276: 38929-389233) and that cyclooxygenase inhibitors can reduce the neurotoxicity of abnormal prion proteins (Bäte et al (2002) Neuroreport 28/13 1933-1938) shows that the pathophysiology of the neurotoxicity of the abnormal prion protein cannot be reduced to its interaction with the PrPc alone.

In view of this incomplete knowledge of the pathophysiological effect of the abnormal prion protein in the development of a TSE, there is a considerable need for new knowledge in the development of the TSE, on the basis of which test systems can then be built up for the search for causally active substances the prophylaxis and early treatment of a TSE, i

Technical problem of the invention.

The invention is therefore based on the technical problem of specifying means with which active substances for the prophylaxis and / or treatment of a TSE can be identified, as well as such active substances. Findings on which the invention is based.

It was surprisingly found 1) that the growth of axons in cultures of mouse motor neuron cells (Prnp - / -) in which the gene for the PrPc had been genetically deleted was significantly lower than in wild-type mice. This decrease in the axon growth of the motor neurons of Prnp (- / -) mice occurs regardless of which neurotrophic factors (BDNF; CNTF, GDNF) are added to the motor neuroculture, ii) that, in contrast to the reduced growth of the axons, the growth of the moto - neurons and their dendrites are not different between wild-type mice (Prnp + / +) and mice with deletion of the gene for PrPc (Prnp - / -), iii) that in embryonic motor neurons of (Prnp + / +) Mice co-localized the endogenous PrPc with tau, a protein localized in axons, and iv) that the addition of infectious brain homogenates (containing PrpSC) to motor neuron cultures of (Prnp + / +) mice to drastically inhibit axon growth leads to a dose-dependent death of the motor neurons as well.

It can thus be concluded from these results that i) that PrPc is a decisive factor for the growth of axons, ii) that the contact of motor neurons with prions drastically inhibits the axon growth of motor neurons and leads to the death of the motor neurons and iii) that one Inhibiting contact between motor neurons, including their offshoots with abnormal prion protein, prevents the inhibition of axon growth and motor neuron death. Basics of the invention and preferred embodiments.

The invention thus relates to a test system for finding active substances which remove the inhibition of axon growth by prions, consisting of

- a nerve cell kept in culture, in particular a motor neuron, this "nerve cell optionally expressing PrPc and forming axons in the cell culture and

- Infectious brain homogenate, which contains abnormal prion protein, for example PrPSC or PrPCJD - and a detection system for the formation of axons, this detection system recording the length of the axon formed per nerve cell directly or, for example, by detecting tau.

or containing such components.

The invention further relates to a method for the detection of active substances which remove the inhibition of axon growth by prions, in which

a test substance is brought into contact with a nerve cell, in particular a motor neuron cell, which optionally expresses PrPc

- and it is checked whether, after the addition of prions, this test substance is able to prevent the inhibition of axon growth by prions and the death of the nerve cell, - The length of the in the

Cell culture-trained axons of a nerve cell is detected directly or via the detection of tau.

The invention furthermore relates to active substances which in the test system according to the invention or in the method according to the invention. Prevent the effect of the abnormal prion protein on axon growth and on nerve cell survival and the use of these active substances to prevent or treat TSE.

Such active substances are, for example, substances which can prevent the binding of abnormal prion protein to a nerve cell, in particular a motor neuron, or stimulate endogenous proteases, or prevent the targeting of PrPc in Caveoli domains or the signal cascade, activated by Prpc Modulate prion binding.

These active substances include, for example: antibodies or a fragment of an antibody specific for Prpc, antibodies or a fragment of an antibody specific for an abnormal prion protein, for example

1 for PrPSC or for PrPCJD, peptides which inhibit the binding of the abnormal prion protein to a nerve cell, for example the PrPc, peptidimetics which can prevent the binding of abnormal prion protein to a nerve cell, for example to PrPc Nucleic acids coding for an antibody, an antibody fragment or a peptide, which codes for an amino acid sequence which prevents the binding of the abnormal prion protein to a nerve cell, for example to PrPc, proteases which Degrading PrPSC, substances that inhibit the translocation of PrPC in Caveoli.

Such active substances are used in a preparation which is well known to the person skilled in the art for parenteral, local, aerogenic or also oral administration to a patient or a person who is suspected of being infected with prions, into the bloodstream, into a body cavity, into the Connective tissue, administered into an organ, particularly intra "ventricular or subarachnoidally into the CNS, nasally or bronchially. However, the administration in the minimal effective dose does not exceed the maximum tolerable dose of the respective active substance at least once as soon as possible after an infection or also several times a day or weekly for a period of preferably two weeks to 6 months.

Examples to illustrate the invention.

Example 1: Production of motor neuron cultures from the spinal cord of the embryonic mouse.

The following materials were used: 2-mercaptoet'hanol (Sigma, Taufkirchen, Germany), B27 (In Vitrogen, Karlsruhe, Germany), Brain derived neurotrophic factor (BDNF; Cell Ceoncepts, Umkirch, Germany), Ciliary neurotrophic factor (CNTF , kind gift, M.Sendtner), Depolarizing saline (0.8% NaCl, 35 mM KCl and 1 μM 2-mercaptoethanol), Falcon 15 ml tubes (Sarstedt, Nurbrecht, Germany), Falcon 50 ml tubes (Sarstedt, Nürm- brecht, Germany), Glas cover slips (Hartenstein, Würzburg, Germany), Glutamax I (Invitrogen, Karlsruhe, Germany) goat seru (Invitrogen, Karlsruhe, Germany), Greiner 4-well dishes (Greiner, Nürtingen, Germany), Hanks balanced salt solution (HBSS w / o Ca2 +, Mg2 +, Invitrogen, Karlsruhe, Germany), HEPES (Invitrogen, Karlsruhe , Germany), Horse serum (Linaris, Wertheim, Germany), KC1

(pA, Merck, Darmstadt, Germany), Laminin-1 (Boehringer / Röche, Mannheim, Germany), Merosin (Laminin-2) (Sigma, Tauf irchen, Germany), Na-Borat (Merck, Darmstadt, Germany), NaCl (pA, Merck, Darmstadt, Germany), Neurobasal (InviTtrogen, Karlsruhe, Germany), Nunclon 10cm petri dishes (cell culture grade; Brandt, Germany), Nunclon 24-well dishes, (Brandt, Heidelberg, Germany), Nunclon 3 cm petri dishes (cell culture grade; Brandt, Germany), Poly-DL-Ornithine (Sigma, Taufkirchen, Germany), Rat anti mouse p75 antibody (Chemicon, Hofheim, Germany), Tris Base (pA, Merck, Darmstadt, Germany), Trypsin (Worthington, New Jersey MA, USA via Cell Systems, St. Katharinen, Germany), Trypsin-Inhibitor from soy bean (Sigma, Taufkirchen, Germany), 1% (w / v) mouse brain homogenate (C57B1 / 6), which has been treated with prions or buffers alone.

The cultivation of mouse embryonic motor neurons was carried out as follows. Cultures of. Embryonic spinal motor neurons of the mouse (age of the embryos 12.5 days) are carried out using the panning method using a rat anti mouse p75 monoclonal antibody (Chemicon, Hofheim) (Wiese et al., 1999; Wiese et al., 2001 ). The embryos are removed from the uterus and microdissected under the microscope. The ventrolateral parts of the lumbar spinal cord are transferred to HBSS with 10μM 2-mercaptoethanol, then treated for 10 min with 0.1% trypsin (in HBSS) at 37 ° C and finally triturated and with 0.1% trypsin inhibitor (fin. conc.) treated. The isolated cells are placed on one cell culture plate coated with p75 antibody (10ng / l in 10MM Tris / HCl pH 9.5) (24 well plate for single embryo preparations, 10 cm plate for total embryo preparations) and the panning was carried out for 30 min at room temperature. The individual wells / the cell culture plate were then washed 3 times with HBSS and the motor neurons were detached with depolarization solution (0.8% NaCl, 35 mM KC1). The suspension is supplemented with 1: 1 motor neuron medium (Neurobasal, 1 × B27, 1% Glutamax, 10% horse serum, centrifuged at 400 g for 5 min and the cell sediment is resuspended in motor neuron medium. The cell number is determined and the cells are filled with poly-DL-ornithine and Laminin-1 or Poly-DL-Ornithine and Merosin coated plates were plated at a density of 2000 cells / cm 2. In the case of treating the cells with brain extract from Prpsc-infected and MOCK-infected mice, a 1% brain homogenate was produced in HBSS a 1:10 ³ to 1:10 ⁷ (in steps of 10 down) was used as a dilution (in HBSS) for the final coating of the plates, the coating was carried out for 30 min at room temperature and the motor neurons were treated with BDNF, GDNF or treated with CNTF as a neurotrophic factor (each in 10 ng / ml final concentration in the motor neuron medium.) A medium change was carried out on days 1 and 3 and 5. The motor neurons were counted three hours after plating on day 0 and then on days 1, 3 and 5. 10 fields (1.16 mm 2 / field) were counted in each case.

Example 2: Immunohistochemistry for the Detection of Axon and Dendrites The following materials were used: 85% Glycerin solution (Merck, Darmstadt, Germany), Mowiol 40-88 (Sigma, Taufkirchen, Germany), Paraformaldehyde (Merck, Darmstadt, Germany), 100 mM Phosphate buffered saline (w / o Ca2 +, Mg2 +, PBS, pH 7.5; Invitrogen, Karlsruhe, Germany), Goat serum (Invitrogen, Karlsruhe, Germany), Tris buffered saline (TBS), 100 mM Tris, pH 7.5, 0.7% NaCl), Triton- x- 100 (Sigma, Taufkirchen, Germany), Rabbit anti mouse Tau antibody (Sigma, Taufkirchen, Germany), Mouse anti human MAP-2 "antibody (Chemicon, Hofheim, Germany), goat anti rabbit Cy3 (Dianova, Hamburg, Germany), rabbit anti mouse Cy2 (Dianova, Hamburg, Germany).

The cultivated motor neurons were fixed with 4% parafor aldehyde in 100 mM PBS (30 min room temperature). The cells were washed 3 times with TBS (Tris buffered saline, 100 mM Tris, pH 7.5, 0.7% NaCl, 10 min each at 20 ° C. or room temperature) and then subjected to a blocking step with TBS + 10% goat serum + 0, 05% Triton-x-100 for 30 min at room temperature. The blocking solution was then exchanged for a blocking solution which contained the antibodies against tau (polyclonal, Sigma, Deisenhofen, 1: 400) and MAP-2 (monoclonal mouse anti Map-2; 5 μg / ml). The cells were incubated with the antibody mixture at 4 ° C. overnight. It is then washed 3 times with TBS and blocked again, as indicated above. The cells are then incubated with 2 μg / ml of goat anti rabbit Cy3 and rabbit anti mouse Cy2 in TBS + 10% goat serum + 0.05% Triton-x-100 for 30 min at room temperature. The cells are then washed with TBS (3 times 10 min each at room temperature) and the coverslips are capped with 50% glycerol using Mowiol (10 g in 40 ml PBS). The axon and dendrite lengths were determined as follows. Images of the immunostained motor neurons were taken using a confocal microscope (Leica TCS, Heidelberg, Germany). Axonal processes could be distinguished from dentritic processes (Cy2, green) using the Cy3 (red) staining. The extensions were measured using the Scion image program and the data analyzed for further analysis in GraphPad Prizm.

Example 3: Preparation of the Prpsc and MOCK brain extract.

RML (Rocky Mountain Laborarory), a mouse-adapted

Scrapie isolate (Chandler, 1961) was passaged in CD-1 mice (Charles River Laboratories). Inocula stocks were 10% (w / v) homogenates of RML-infected, terminally diseased CDI mouse brains in 0.32 M sucrose. Mice were treated with 30 ul of a 10-fold dilution of the stock in HBSS with 5% bovine serum albumin (BSA). The infectivity titers were determined by infection of PrP overexpressing tga20 / tga20 mice (Fischer et al., 1996) with 30 μl of the diluted homogenate injected into the right par'ietal hemisphere. Prion titers were calculated based on the time it took the infected mice to reach terminal disease. (Prusiner et al., 1982; Brandner et al., 1996). MOCK extract was produced in parallel batches with homogenate from non-infected brains. Example 4: Experiments and results on the influence of abnormal prions on axons and survival rates.

In different experiments, lumbar spinal cord motor neurons in single embryo preparations were first isolated from mating pairs of Prnp +/- X Prnp +/- (kept on a C57B1 / 6 / 129SV background) (Bueler et al., 1992). The genotyping of the embryos was carried out by DNA extraction from the residual tissue of the prepared embryos and the genotyping was carried out in accordance with the information from Büeler H, Aguzzi A, Sailer A, et al. 1993 "Mice devoid of PrP are resistant to scrapie" Cell 73: 1339-47. The motor neurons cultured on poly-DL-ornithine and laminin-1 or poly-DL-ornithine and merosin were counted on days 0, 1, 3 and 5 and fixed on day 7 for the subsequent staining against MAP-2 and tau (dendrites or axon-specific (Bommel et al., 2002). The axon lengths and the dendrite lengths were then measured using the computer program Scion image.

Subsequently, lumbar motor neurons were isolated from CD-1 mouse embryos and cultured. As already stated above, the plates were coated first with poly-DL-ornithine, laminin-1 and then brain extract from RML-infected mice or MOCK-infected mice. The brain extracts were applied to the coated plates in different concentrations (see below). The cultured motor neurons were counted on days 0, 1, 3 and 5 and fixed on day 7 for the subsequent staining against MAP-2 and tau (dendrites or axon-specific). Then the axon lengths and measure the dendrite lengths using the computer program Scion Image.

The following results were obtained. The survival rates for cultured lumbar Prnp ko motor neurons compared to Prnp +/- or Prnp + / + (wt) showed no significant differences in the survival of these motor neurons. The measurement of dendrite lengths of the cultured lumbar Prnp ko motor neurons compared to Prnp + / + (wt) ^" showed no significant differences in the dendrite lengths of these cultured lumbar Prnp ko motor neurons compared to Prnp + / + (wt ) showed significant differences in the axon lengths of these motor neurons both on cover slips coated with Poly-DL-Ornithine and Laminin-1 and on cover slips coated with Poly-DL-Ornithine and Merosin.

The survival rates for cultured wild-type lumbar embryonic motor neurons cultured on coverslips coated with poly-DL-ornithine and laminin-1, and either with prions or MOCK, differed significantly in a range from 1: 1000 to 1: 1000,000, ie brain extract used in a range of 0.1% to 0.001% (final concentration in the coating). The prion brain extract resulted in a significantly lower survival of cultured lumbar embryonic motor neurons in culture at a concentration of the prion-containing brain extract of 1: 1,000 and 1: 10,000 compared to motor neurons that were cultivated in the presence of MOCK extract. The dendrite lengths were determined at a prion or MOCK extract final concentration for the coating of 0.1% and did not differ significantly from one another. The axon lengths were determined for a prion brain extract or MOCK extract Final concentration in the coating of 0.1% determined and differed significantly from each other. The prion brain extract resulted in significantly shorter axon lengths of cultured lumbar embryonic motor neurons in culture compared to motor neurons cultured in the presence of MOCK extract.

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639-642.

Claims

claims: 1) Test system for the detection of active substances which remove the inhibition of axon growth by infectious prions, with the following components: a) a nerve cell kept in culture, this nerve cell forming axons in the cell culture, b) a preparation containing abnormal prion protein and c) a detection system for the formation of axons. 2) Method for finding active substances which cancel the inhibition of axon growth by infectious prions, with the following method steps: a) a test substance is brought into contact with a nerve cell, b) a preparation with abnormal prion protein is brought into contact with the nerve cell, c) by means of a detection system for the formation of axons from ^¬ Values determined, indicating whether the test substance is capable of reducing the inhibition of axonal growth by the abnormal prion protein or prevent c ') and / or by means of a viability test, measured values which indicate whether the test substance is able to prevent the nerve cell from dying d) where the measured values are optionally compared with a corresponding measurement under the same conditions, but either without adding a test substance or with adding a placebo substance, e) a test substance is selected as an active ingredient ^" if the measured values indicate at least a reduction in the inhibition of axon growth or a prevention of the death of the nerve cell, process steps a) and b) can be carried out in any order, including simultaneously. 3) Test system or method according to claim 1 or 2, wherein the nerve cell is a motor neuron. Test system or method according to one of claims 1 to 3, wherein the nerve cell expresses PrPc. 5) test system or method according to any one of claims 1 to 4, wherein the preparation is a Ho ogenat a brain tissue of a mammal, which is suffering from a prion disease. 6) Test system or method according to one of claims 1 to 5, wherein the detection system is the length of a formed axons per nerve cell recorded directly or via the detection of tau. 7) Active substance, which in a test system or Method according to one of claims 1 to 6, the effect of the abnormal prion protein on the axon growth and on the survival of the nerve cell is prevented or can be identified therewith. 8) Active substance according to claim 7, which prevents the binding of abnormal prion protein to a nerve cell, in particular to a motor neuron. 9) Active substances according to claim 7, which modulates the cellular signaling cascade, activated by PrPc after binding of prions. 10) active substance according to one of claims 7 to 9, selected from the group consisting of "antibody or a fragment of an antibody specific for PrPc, antibody 'or a fragment of an antibody specific for an abnormal prion protein, for example for PrPsc or for PrPcjd, Peptides which inhibit the binding of the abnormal prion protein to a nerve cell, for example the PrPc, peptidimimetics which inhibit the binding of abnormal Preventing prion protein from affecting a nerve cell, for example from PrPc, nucleic acids coding for an antibody, an antibody fragment or a peptide, which is for an amino acid sequence encodes which prevents the binding of the abnormal prion protein to a nerve cell, for example to PrPc, proteases which degrade abnormal prion protein, substances which prevent the transition of the PrPc into Caveoli or other lipid-enriched regions of cell membranes). 11) Active substances according to one of claims 7 to 10 in a preparation for parenteral, local, aerogenic or oral administration. 12) Preparation containing an active substance according to one of claims 7 to 10 for administration into the bloodstream, into a body cavity, into the connective tissue, into an organ, particularly intraventricularly or subarachnoidally into the CNS, administered nasally or bronchially. 13) Use of an active substance according to one of claims 7 to 10 for the prevention or treatment of a TSE. 14) Use of an active substance for the production of a pharmaceutical composition for the prophylaxis or treatment of a TSE, optionally in a formulation according to claim 11 or 12. 15) A method for treatment of a TSE, wherein a Suckle ^¬ animal, including a human, which is suffering from TSE or the risk of developing TSE is exposed, a pharmaceutical composition according to claim 14) is administered in a physiologically effective dose.