EP1658090A2 - Agent et methode de traitement et de prevention de l'encephalopathie spongiforme transmissible (est) et procede de production dudit agent - Google Patents

Agent et methode de traitement et de prevention de l'encephalopathie spongiforme transmissible (est) et procede de production dudit agent

Info

Publication number
EP1658090A2
EP1658090A2 EP04762583A EP04762583A EP1658090A2 EP 1658090 A2 EP1658090 A2 EP 1658090A2 EP 04762583 A EP04762583 A EP 04762583A EP 04762583 A EP04762583 A EP 04762583A EP 1658090 A2 EP1658090 A2 EP 1658090A2
Authority
EP
European Patent Office
Prior art keywords
thr
ser
pro
trp
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04762583A
Other languages
German (de)
English (en)
Inventor
Dieter Willbold
Jörg SMOLINSKI
Karen Olga HÄNEL
Michael Andreas Wolff
Detlev Riesner
Carsten Korth
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Juelich GmbH
Original Assignee
Forschungszentrum Juelich GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forschungszentrum Juelich GmbH filed Critical Forschungszentrum Juelich GmbH
Publication of EP1658090A2 publication Critical patent/EP1658090A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • 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 invention relates to an agent and a method for the treatment and prevention of TSE and a method for producing the agent.
  • Prions have been identified as the main pathogen. Prions mainly consist of prion protein (PrP) in a pathogenic conformation (PrP Sc ). The healthy organism also produces prion protein, but in a non-pathogenic, harmless conformation (PrP c ). PrP c and PrP Sc have the same amino acid sequence, but show a significant difference in the secondary structure. PrP c contains a relatively high ⁇ -helical component and almost no ⁇ -sheet elements.
  • PrP Sc has a secondary structure that is different from PrP c , with a higher proportion of ß-leaflets and a lower proportion of o! Accordingly, PrP c and PrP Sc are structural isomers. In addition, unlike PrP, PrP is resistant to Protemase K digestion. PrP Sc seems to be formed post-translationally from PrP c . The emergence and development of the disease depend on the continued transformation development of PrP c in PrP Sc . The factors responsible for converting PrP c to PrP Sc are not all known yet.
  • the PrP Sc formation can be slowed down and even inhibited if certain peptides are supplied to prion-infected cells. It is assumed here that the peptides according to the invention attach to the PrP c and thus prevent the conversion of naturally occurring PrP c into the pathogenic PrP Sc . According to the invention, various peptides with partially characteristic amino acid sub-sequence blocks were identified which result in a relief of the symptoms or even a cure of the disease TSE. Exemplary peptides are given in sequences 1 to 27. The following sequences were identified as peptides which can be used according to the invention and can be read in the direction of amino to carboxyl terminus, one of which Component or a mixture of at least two components can be used for treatment:
  • G at least two components from at least one of groups A) to F);
  • Sequences 1-27 of the sequence listing which comprise or contain all 12 amino acids with positions 1-12. These sequences can have any length, preferably a length of 6-40, particularly preferably 8-30, 10-20 or even more preferably 10-12 amino acids. In some specific embodiments, the following sequences may be present, which are preferably 12 amino acids long.
  • amino acid sequences which contain the amino acids Leu, Lys, Ala, and Thr in positions 1, 2, 3 and 4;
  • K amino acid sequences which contain a combination of features I) and J); L) amino acid sequences which have the amino acids Gin, Trp and Thr in positions 4, 5 and 6;
  • N amino acid sequences which have the amino acids Thr and Tyr in positions 11 and 12;
  • Sequence sections I) -R) can be contained in sequences which, for example, have a length of 6-40, preferably 8-30, 30-20 and particularly preferably 10-12 amino acids.
  • FIG. 1 shows the amino acid sequences (in the one-letter code) of the four active peptides which were identified in the phage display screening:
  • VDMINDVQPLTP Seq.Nr.2
  • VYSSTTRPLPSP Seq.Nr.10
  • the peptides according to the invention which bring about a healing of TSE or a relief can be produced by known processes.
  • SUBSTITUTE SHEET Synthesis in liquid medium can be used.
  • the amino acids are linked together in the sequence of the sequence in accordance with the sequence listing and the listings A) - R).
  • Solid phase peptide synthesis consists of three main steps:
  • the peptides can also be produced by expressing the nucleotide sequences coding for them, for example in chromosomes, plasmids or other information carriers, naked DNA or RNA in organisms, in cells or cell-free systems.
  • the invention therefore also relates to the nucleic acids which code for the peptides according to the amino acid sequences A) -R), including all allelic variations and the nucleotide sequences of the sequence sequences 1-27 including all allelic variations.
  • all mammals including humans can be treated or cured and the peptides can also be used for prevention. Treatment of humans is particularly preferred, but cattle and sheep can also be treated.
  • the peptides according to the invention accumulate the PrP c of all mammals so that the effect according to the invention can take place. The effect according to the invention also takes place when at least two of the peptides mentioned bind to the PrP c .
  • the peptides according to the invention can be used to treat the disease TSE both in humans and in animals.
  • Human diseases include Creutzfeld-Jakob syndrome, Kuru, Gerstmann-St Hurssler-Scheinker syndrome and FFI (Fatal Familial Insomnia).
  • TSE diseases are also known in animals, e.g. B. in sheep scrapie, in cattle bovine spongiform encephatlopathy (BSE) in wild animals chronic wasting disease (CWD).
  • BSE cattle bovine spongiform encephatlopathy
  • CWD chronic wasting disease
  • the peptides according to the invention must be applied in such a way that they reach their site of action. This place of action can be the brain, the spinal cord and / or the entire nervous system, but also any other part of the organism.
  • the peptides according to the invention can be introduced into the body in solid form or dissolved in a solvent, preferably water.
  • the peptides can be introduced into the nose as a solid, for example orally, rectally or as a powder.
  • the active ingredient according to the invention and the pharmaceutical composition can be in the form of liquid, semi-solid or solid pharmaceutical forms and in the form of, for. B. injection solutions, drops, juices, syrups, spray, suspensions, granules, tablets, pellets, transdermal therapeutic systems, capsules, plasters, suppositories, ointments, creams, lotions, gels, emulsions or aerosols are present and administered and contain the inventions 10
  • Peptides according to the invention in a physiologically compatible form and depending on the route of application pharmaceutical auxiliaries, such as. B. carrier materials, fillers, solvents, diluents, surface-active substances, dyes, preservatives, disintegrants, lubricants, lubricants, flavors and / or binders.
  • pharmaceutical auxiliaries such as. B. carrier materials, fillers, solvents, diluents, surface-active substances, dyes, preservatives, disintegrants, lubricants, lubricants, flavors and / or binders.
  • auxiliaries can be, for example: water, ethanol, 2-propanol, glycerin, fructose, lactose, sucrose, dextrose, molasses, starch, modified starch, gelatin, sorbitol, inositol, mannitol, microcrystalline cellulose, methyl cellulose, Carboxymethyl cellulose, cellulose acetate, shellac, cetyl alcohol, polyvinyl pyrrolidone, paraffins, waxes, natural and synthetic rubbers, acacia gum, alginates, dextran, saturated and unsaturated
  • Fatty acids Fatty acids, stearic acid, magnesium stearate, zinc stearate, glyceryl stearate, sodium lauryl sulfate, edible oils, sesame oil, coconut oil, peanut oil, soybean oil, lecithin, sodium lactate, polyoxyethylene and propylene fatty acid esters, sorbitan fatty acid esters, sorbitan fatty acid esters, sorbate
  • excipients and the amounts to be used depends on whether the medication is oral, subcutaneous, parenteral, intravenous, pul onal, intraperitoneal, transdermal, intramuscular, nasal, 11
  • the active ingredient can be in a depot in dissolved form or in a plaster, if necessary with the addition of skin penetrations, to promote skin penetration
  • Formulations which can be used rectally, transmucosally, parenterally, orally or percutaneously can release the peptides according to the invention in a delayed manner.
  • the peptides according to the invention can be administered in liquid form, for example, intravenously, orally, as a nasal spray, subcutaneously, intramuscularly, by inhalation or in or next to the spinal cord.
  • the active compounds according to the invention can be applied by means of ointments or creams.
  • the peptides according to the invention can arise at the site of action or at other locations in the organism after nucleic acids (DNA and RNA or a combination thereof) which code for the peptides according to the invention are introduced into the organism.
  • viral vectors naked nucleic acid (DNA, RNA), plasmids, artificial virus particles, liposomes, which are introduced into the body intravenously, intranasally, orally, rectally, subcutaneously, intramuscularly, in or on the spinal cord. 12
  • the peptides according to the invention can also be used to prevent the above-mentioned diseases.
  • the peptides according to the invention must be applied in such a way that they reach their site of action. This place of action can be the brain, the spinal cord and / or the entire nervous system, but also any other part of the organism.
  • the peptides according to the invention can be introduced into the body in solid form or dissolved in a solvent (preferably water).
  • the peptides can be introduced into the nose as a solid, for example orally, rectally or as a powder.
  • the peptides according to the invention can, for example, intravenously, orally, as a nasal spray, subcutaneously, intramuscularly, by inhalation or in or in addition to that
  • the active compounds according to the invention can be applied by means of ointments or creams.
  • the peptides according to the invention can arise at the site of action or at other locations in the organism after nucleic acids (DNA and RNA or a combination thereof) which code for the peptides according to the invention are introduced into the organism. This can be achieved by viral vectors, naked nucleic acids (DNA, RNA), plasmids, artificial virus particles, liposomes, which are introduced into the body intravenously, intranasally, orally, rectally, subcutaneously, intramuscularly, in or on the spinal cord.
  • the peptides according to the invention can also be modified so that the water solubility, 13
  • peptides according to the invention can be linked to antibodies or fragments thereof in the usual way in order to activate components of the immune system when the protein according to the invention is used therapeutically.
  • diseases to be treated include Creutzfeld-Jakob syndrome, Kuru, Gerstmann-St Hurssler-Scheinker syndrome and Fatal Familia Insomnia (FFI).
  • Phage display is a technique that makes it possible to construct peptide libraries with randomized amino acid sequences and to search them for ligands for a specific target molecule.
  • the peptide library is presented as a fusion of peptide and a phage coat protein on the surface of bacteriophages ("phage display").
  • the diversity of the peptides presented is achieved by inserting a combinatorially mutated DNA as a peptide-coding part of the fusion gene. An extremely large number of phages are generated in this way, each phage presenting a different peptide.
  • biopanning The construction, propagation and selection is referred to as "biopanning”.
  • the library comes with an immobilized target 14
  • the enriched population of phages that present a peptide interacting with the target molecule are amplified by infecting them with bacteria.
  • the screening amplification procedure can be repeated several times to further enrich the library members who have a relatively higher affinity for the target molecule. The result is a peptide population that is dominated by the amino acid sequences that bind best to the target molecule.
  • a commercial phage library with 12 randomized amino acids (New England Biolabs, Frankfurt) was used.
  • the grp3 gene coding for the phage coat protein is inserted at the N-terminal according to the signal sequence. This consists of 1.9 x 10 9 independent clones and is amplified and concentrated in such a way that on average 10 ⁇ l each sequence is present in 55 copies.
  • recombinantly produced hamster prion protein in a concentration between 0.01 ⁇ g / ml and 0.1 ⁇ g / ml in PBS with 0.2% SDS, pH 7.2 using the "Protein Immobilizer Kit” (Exiqon) immobilized in a microtiter plate well.
  • 100 ⁇ l protein solution were incubated for 2 hours with gentle shaking. After removing the solution, it was washed 3 times with 200 ⁇ l PBS.
  • 10 ⁇ l phages from the commercial phage library were incubated in 100 ⁇ l PBST with 0.1% BSA for 10 minutes with gentle shaking in an rPrP-coated microtiter plate well. The unbound phages were discarded and then washed 10 times with 300 ⁇ l PBST. Elution was carried out with 100 ⁇ l of 0.2 M glycine-HCl, pH 2.2 for 10 minutes with gentle shaking. The eluate was neutralized in 15 ⁇ l Tris-HCl, pH 9.1.
  • the eluate was placed in 20 ml of E. coli culture and incubated for 4.5 hours at 37 ° C. and 200 rpm. The culture was then centrifuged at 5000 rpm for 10 minutes. The supernatant was transferred and 1/6 volume of PEG / NaCl was added. The mixture was then precipitated at 4 ° C. overnight. The mixture was then centrifuged at 5000 rpm for 20 minutes, the supernatant was discarded and the pellet was resuspended in 1 ml of PBS. The mixture was then centrifuged at 10,000 rpm for 5 minutes. The supernatant was transferred and 1/6 volume of PEG / NaCl was added. The mixture was precipitated on ice for 1 hour.
  • Prion infected cells are cultured for a week and then examined for the formation of PrP Sc .
  • This works by lysing the cells and treating them with 20 ⁇ g / ml Proteinase K (PK).
  • PK Proteinase K
  • the PK-treated cell lysate is subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a membrane and then stained with a PrP-specific antibody ("Western blot").
  • Figure 1 shows the band pattern of the PK-treated cell lysate.
  • the peptides W1, W2, W3 and W4 correspond to the sequences 8,2,10 and 1 according to the sequence listing. This creates a typical band pattern (see track 1 in the figure below).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un agent ou principe actif pharmaceutique, une méthode de traitement, de prévention et de diagnostic de l'encéphalopathie spongiforme transmissible (EST) ainsi qu'un procédé de production de l'agent ou du principe actif pharmaceutique. Cette invention concerne, en particulier, des peptides ou des séquences nucléotidiques codant pour ces peptides, qui apparaissent dans les protocoles de séquence 1 à 27 et dans des séquences partielles associées. Ces peptides permettent de traiter ou de prévenir l'encéphalopathie spongiforme transmissible, par exemple, chez l'homme, le syndrome de Creutzfeldt-Jakob, le kuru, le syndrome de Gerstmann-StrAussler-Scheinker et l'insomnie fatale familiale (IFF). Chez l'animal, les EST connues sont la tremblante du mouton, l'encéphalopathie spongiforme bovine et la maladie du dépérissement chronique des ruminants sauvages (CWD).
EP04762583A 2003-08-29 2004-08-04 Agent et methode de traitement et de prevention de l'encephalopathie spongiforme transmissible (est) et procede de production dudit agent Withdrawn EP1658090A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10340260A DE10340260A1 (de) 2003-08-29 2003-08-29 Mittel und Verfahren zur Behandlung und Prävention von TSE, sowie Verfahren zur Herstellung des Mittels
PCT/DE2004/001738 WO2005023285A2 (fr) 2003-08-29 2004-08-04 Agent et methode de traitement et de prevention de l'encephalopathie spongiforme transmissible (est) et procede de production dudit agent

Publications (1)

Publication Number Publication Date
EP1658090A2 true EP1658090A2 (fr) 2006-05-24

Family

ID=34223260

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04762583A Withdrawn EP1658090A2 (fr) 2003-08-29 2004-08-04 Agent et methode de traitement et de prevention de l'encephalopathie spongiforme transmissible (est) et procede de production dudit agent

Country Status (4)

Country Link
US (1) US20060281676A1 (fr)
EP (1) EP1658090A2 (fr)
DE (2) DE10340260A1 (fr)
WO (1) WO2005023285A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE59208894D1 (de) * 1991-12-02 1997-10-16 Riemschneider Randolph Prof Dr Wässrige synthetische Organextrakte
US20020052323A1 (en) * 2000-08-30 2002-05-02 Jinhai Wang Quinoline-(C=O)-(multiple amino acids)-leaving group compounds for pharmaceutical compositions and reagents

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005023285A2 *

Also Published As

Publication number Publication date
DE112004002133D2 (de) 2006-07-13
US20060281676A1 (en) 2006-12-14
WO2005023285A3 (fr) 2005-06-30
DE10340260A1 (de) 2005-03-31
WO2005023285A2 (fr) 2005-03-17

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