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WO1996008562A1 - Molecules biologiquement actives derivees de neurotrophines - Google Patents

Molecules biologiquement actives derivees de neurotrophines Download PDF

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Publication number
WO1996008562A1
WO1996008562A1 PCT/EP1995/003538 EP9503538W WO9608562A1 WO 1996008562 A1 WO1996008562 A1 WO 1996008562A1 EP 9503538 W EP9503538 W EP 9503538W WO 9608562 A1 WO9608562 A1 WO 9608562A1
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WO
WIPO (PCT)
Prior art keywords
poly
peptide
bdnf
amino acid
dna
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PCT/EP1995/003538
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German (de)
English (en)
Inventor
Stefan Jungbluth
Roland Kolbeck
Yves-Alain Barde
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Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin
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Application filed by Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin filed Critical Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin
Priority to AU35645/95A priority Critical patent/AU3564595A/en
Publication of WO1996008562A1 publication Critical patent/WO1996008562A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to nucleic acid sequences which encode naturally non-occurring (poly) peptides with the biological activity of a neurotrophic factor, the (poly) peptides having an N-terminus which is elongated in comparison with mature neurotrophic factors.
  • the invention further relates to recombinant DNA molecules which contain the nucleic acid sequences according to the invention, thereby encoded (poly) peptides, medicaments, diagnostics and kits which contain the (poly) peptides according to the invention, and in vitro methods for the detection of diseases (or Precursors thereof) of the nervous system.
  • RNA or protein synthesis allows the conclusion that apoptical neuronal cell death is an active process that takes place via the expression of certain genes.
  • Nerve growth factor (NGF), BDNF ("brain-derived neurotrophic factor"), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and neurotrophin-6 are members of a gene family commonly known as neurotrophins (neurotrophic factors). All these factors prevent the death of a number of embryonic neurons kept in cell culture (cf., for example, Barde et al., Progress in Growth Factor Research 2 (1990), 237-248) and are therefore able to become neuronal, as described above To prevent cell death leading active process.
  • Mature, biologically active neurotrophins are obtained from precursors by cleaving an N-terminal peptide. The cleavage takes place at a 4-amino acid consensus cleavage site of the furin type (Bresnahan et al., J. Cell. Biol. 111 (1990), 2851-2859).
  • the known neurotrophins have a high degree of homology (Barde, Progress in Growth Factor Research 2 (1990), 237-248), which is of the order of 50%. They are secretory, basic, homodimeric proteins with a relative molecular weight of 27 kD. Six conserved cysteine residues form 3 intramolecular disulfide bridges in NGF and BDNF.
  • EP-A 0544293 describes a method for the genetic engineering production of biologically active NGF in prokaryotes.
  • NGF was extended by 3-5 amino acids of an N-terminal sequence of a protein which is expressed in the host cells used with a high expression rate and by 4-10 amino acids from the prosequence of the NGF precursor.
  • Such an elongated NGF like natural NGF, has an effect on neurons and has the same activity as natural human NGF.
  • the object of the present invention was therefore to provide new molecules with the biological activity of the neurotrophins which are characterized in the body by a longer half-life (length of stay in the body) than the naturally occurring mature neurotrophins and, consequently, can be used effectively as therapeutic agents. This object is achieved by the embodiments characterized in the claims.
  • the invention thus relates to a nucleic acid molecule (DNA or RNA) which encodes a naturally non-occurring (poly) peptide with the biological activity of a neurotrophic factor, the (poly) peptide having an N-terminus which is elongated in comparison with a mature neurotrophic factor with the proviso that the nucleic acid molecule does not encode NGF.
  • the invention further relates to a nucleic acid molecule which codes for NGF and which is N-terminally extended by a peptide fragment of up to approximately 19 amino acids, preferably by 4-19 amino acids. This peptide fragment is a C-terminal fragment of the precursor part of NGF.
  • this precursor contains a consensus cleavage site of the furin type, in which an exchange in the amino acid -1 and / or -2 has taken place.
  • Arginine in position -1 is preferably replaced by another amino acid, particularly preferably by lysine.
  • naturally non-occurring (poly) peptide is understood here to mean a (poly) peptide that does not occur either as a natural precursor of a neurotrophin or as a mature neurotrophin (poly) peptide under physiological conditions.
  • the amino acid sequences of many mammalian and fish neurotrophins and their precursors are known in the art; these are naturally occurring (poly) peptides and therefore do not fall under the term defined above.
  • this expressly includes precursors of neurotrophins which have a mutation in the 4-amino acid consensus cleavage site of the furin type, it being assumed that such mutations occur very rarely or not naturally.
  • the nucleic acid according to the invention preferably a DNA or RNA
  • an N-terminus elongated in comparison with a mature neurotrophic factor relates to any elongation of the N-terminus compared to the N-terminus of a mature neurotrophic factor.
  • the extended N-terminus ie the additional
  • the presence of one or more amino acids N-terminal from the N-terminus of a mature neurotrophin may be an altered sequence of the naturally occurring N-terminal, cleavable peptide of the precursor of the neurotrophin or another neurotrophin. This altered sequence can be shortened or lengthened or have the same length as the cleavable peptide, in which case it has at least one amino acid exchange with the cleavable peptide.
  • the shortened and extended sequences can also have such amino acid exchanges, but are additionally characterized by amino acid insertions, additions or deletions or combinations thereof, which can have been carried out internally and / or terminally.
  • the extended N-terminus cannot be derived from the amino acid sequence of the cleavable N-terminal peptide of the neurotrophin precursors.
  • naturally non-occurring polypeptide is to be understood as a (poly) peptide which is extended at the N-terminal by a peptide fragment.
  • This peptide fragment is a fragment of a precursor portion of a neurotrophin that is up to approximately 19 amino acids long, preferably 4-19 amino acids long.
  • Precursor content in the sense of the invention, is to be understood as meaning a peptide which can be cleaved from the N-terminus of the mature protein and which is naturally contained in the corresponding pro-neurotrophin at the N-terminal.
  • Such neurotrophin precursor sequences are known and are described, for example, for:
  • NGF Suter et al., EMBO J. 10 (1991) 2395-2400 and
  • NT-4 Hallboek et al., Neuron 6, 845-858
  • NT-5 Ip et al., Neuron 10 (1983) 137-149
  • these precursor sequences contain a furin-type consensus cleavage site in which an exchange in the amino acid position -1 and / or -2 has taken place.
  • Arginine in position -1 is preferably replaced by another amino acid, particularly preferably by lysine.
  • the DNA encoding the (poly) peptide according to the invention can be of synthetic, semisynthetic origin or can be produced recombinantly. All of the embodiments discussed above have in common that the (poly) peptide encoded by the DNA according to the invention is non-toxic when applied in pharmaceutically customary concentrations, for example in humans.
  • the (poly) peptide encoded by the DNA according to the invention offers the advantage over all neurotrophins known and available in the prior art to be used effectively for therapeutic purposes.
  • Another significant advantage of the longer half-life in vivo is that, with the same therapeutic efficacy, a smaller amount of the (poly) peptide encoded by the DNA according to the invention can be applied compared to the amount of the corresponding wild-type neurotrophin, and thus undesirable side effects are avoided can.
  • the increased stability of the dimers which are formed by the (poly) peptides encoded by the DNA according to the invention and the advantages associated therewith are of particular importance insofar as it has surprisingly also been found that the neurotrophin monomers have no biological activity at all under physiological conditions .
  • Another advantage of the (poly) peptides according to the invention is that they can be marked much better with 1251 than the mature neurotrophic factors, since the N-terminal peptide, which is usually split off when the neurotrophin precursor is converted to mature neurotrophin, in particular there are also several tyrosine residues in the BDNF precursor, which are radiolabelled when the enzyme lactoperoxidase is used.
  • This labeling option has particularly advantageous effects when using the (poly) peptides according to the invention in diagnostics. It also has advantages for the isolation of the labeled (poly) peptide.
  • a preferred embodiment according to the invention relates to a DNA sequence, the neurotrophic factor originating from vertebrates.
  • a particularly preferred embodiment of the invention relates to a DNA sequence, the neurotrophic factor originating from fish or mammals.
  • the neurotrophic factor is a human neurotrophic factor.
  • Another preferred embodiment of the invention relates to a DNA sequence, the factor being NGF, NT-3, NT-4/5, NT-6 or BDNF.
  • the DNA sequence encodes a (poly) peptide, the N-terminus, which is elongated in comparison with a mature neurotrophic factor, being a mutein of the peptide cleaved off when the neurotrophin precursor changes to the mature neurotrophin.
  • mutant of the peptide split off during the transition from the neurotrophin precursor to the mature neurotrophin means that part of the neurotrophin precursor that is normally split off at the 4 amino acid cleavage site of the furin type and is not converted into the mature neurotrophin and that into its amino acid sequence is preferably genetically modified compared to the naturally occurring precursor in such a way that it is not cleaved off by a corresponding protease.
  • the protease cleavage can either be prevented by exchanging at least one amino acid in the recognition sequence for the protease, or by changing the three-dimensional structure of the N-terminus of the precursor in such a way that binding and / or cleavage by the protease is avoided becomes.
  • muteins are also understood to mean those amino acid sequences which are extended or shortened compared to the cleavable peptide of the precursor. Such an extension or shortening can be achieved genetically by adding, inserting or deleting amino acids or by combinations thereof, within and / or terminally on the cleavable peptide.
  • the processes required for this are standard molecular biology processes and are described, for example, in Sambrook, loc. Cit.
  • the mutein has at least one amino acid exchange in the 4 amino acid consensus cleavage site of the furin type. This cleavage site has the amino acid sequence
  • R-X-R-R (SEQ ID NO: 2) or preferably R-V-R-R (SEQ H> NO: 3) (R: arginine, X: any amino acid, K: lysine, V: valine).
  • This exchange in the amino acid sequence means that the N-terminal peptide can no longer be cleaved off by a protease cleaving at the cleavage site mentioned.
  • further changes already discussed above in the amino acid sequence of the N-terminal peptide can also occur.
  • the change in the amino acid sequence is preferably limited to the exchange / exchanges in the consensus cleavage sequence, the exchanges preferably occurring in the amino acid position -1 and / or -2.
  • the mutein has the 19 C-terminal amino acids of the N-terminal peptide which can be cleaved in the native BDNF precursor and also contains the naturally occurring potential glycosylation site.
  • the 4 amino acid consensus cleavage site of the arginine furin type in position -1 is replaced by lysine.
  • Another particularly preferred embodiment of the invention relates to a DNA sequence which encodes a (poly) peptide which has the 19 C-terminal amino acids of the N-terminal peptide which can be cleaved in the native BDNF precursor, with the exception of the arginine in position -1 which is replaced by lysine.
  • this amino acid exchange in the BDNF precursor prevents cleavage of the N-terminal peptide, thereby producing a molecule which is 19 amino acids longer than the mature BDNF and has a greater dimer stability in solution.
  • the N-terminus which is elongated in comparison with a mature neurotrophic factor, is glycosylated.
  • the glycosylation of the asparagine residue conserved at this or a corresponding position in all neurotrophins in the N-terminal sequence of the (poly) peptide according to the invention leads to the dimer shown here in the examples having a molecular weight which is almost twice that of the wild-type BDNF -Dimer. It seems possible that the glycosylation has an influence on the longer half-life of the (poly) peptide encoded by the DNA according to the invention, it also offers the possibility of using the sugar group for labeling, for example with biotin.
  • the N-terminus which is elongated in comparison with a mature neurotrophic factor, comprises a detectable and / or biologically active amino acid sequence.
  • This amino acid sequence can either start N-terminally from the amino acid sequence encoding the mature neurotrophic factor. In another embodiment, it connects N-terminally to the mutein of the peptide cleaved off when the neurotrophin precursor changes to the mature neurotrophin or to the extended N-terminus, which cannot be derived from the amino acid sequence of the cleavable N-terminal peptide of the neurotrophin precursors is.
  • the amino acid sequence can be any sequence that is detectable and or biologically active.
  • sequences are antibody chains or parts thereof, or sequences which are recognized by antibodies available in the prior art, e.g. c-myc or HA, furthermore biotin, enzymes or catalytically active parts thereof or enzyme substrates, or labels, such as radioactive, fluorescent, chemiluminescent or bioluminescent labels.
  • the amino acid sequence is detected by binding to an antibody or an enzyme.
  • the detecting antibody is preferably labeled. Labeling methods for antibodies are known in the prior art; see. e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor 1988. If an enzyme is used for the detection, the reaction catalyzed thereby is preferably a color reaction.
  • the biologically active amino acid sequence comprises biotin.
  • Biotin has long been known in the prior art as a component of enzymatic detection systems.
  • Another object of the invention is an RNA sequence which is complementary to the DNA sequence according to the invention or the DNA sequence complementary thereto.
  • RNA sequence according to the invention can be of synthetic, semi-synthetic or recombinant origin. It can be used, for example, for in vitro translation or as anti-sense RNA or as a ribozyme.
  • Another object of the invention is a pair of primers that hybridize to the DNA or RNA sequence according to the invention.
  • the Prime ⁇ aar according to the invention preferably hybridizes under suitable conditions at the two ends of complementary strands of the DNA sequence according to the invention and can thus be used for the specific amplification of the DNA sequence according to the invention by PCR (cf. e.g. Sambrook et al., Loc. Cit.).
  • the invention further relates to a recombinant DNA molecule which contains at least one DNA sequence according to the invention.
  • the recombinant DNA molecule according to the invention is preferably a recombinant vector which is suitable for expressing the DNA sequence according to the invention.
  • One or more DNA sequences according to the invention can be cloned into the recombinant DNA molecule according to the invention. If several DNA sequences according to the invention are cloned into the recombinant DNA molecule, these can originate from genes which code for the same or different neurotrophins.
  • the recombinant DNA molecules according to the invention can furthermore contain genes which encode other (poly) peptides, e.g. Wild type precursors of neurotrophins.
  • the recombinant DNA molecules according to the invention can code fusion proteins of the (poly) peptide according to the invention, for example with ⁇ -galactosidase.
  • the recombinant DNA molecule can also only be used to multiply it.
  • the invention further relates to a host that is transformed with at least one recombinant DNA molecule according to the invention.
  • This recombinant DNA molecule / these recombinant DNA molecules can have the same factor or different Encode factors or, if it codes the same factor, have different amino acid sequences.
  • the transformed host is a bacterium, a fungal, yeast or mammalian cell or a transgenic animal.
  • the host according to the invention is a bacterium, E. coli is preferred. In contrast, if the host is, for example, a transgenic animal, mice are preferred.
  • the production of transgenic animals is now one of the standard methods in modern biology (see e.g. Hanahan, Science 246 (1989), 1265-1275) and is therefore not described in detail here.
  • the expression of the (poly) peptides according to the invention can be examined in a tissue-specific manner in transgenic animals (cf. e.g. Barinaga, Science 265 (1994), 26-28).
  • the invention further relates to a (poly) peptide which is encoded by the DNA sequence according to the invention, the RNA sequence according to the invention or the recombinant DNA molecule according to the invention.
  • the (poly) peptide according to the invention can be produced, for example, by translation of an mRNA which has been transcribed by the DNA molecule according to the invention.
  • the (poly) peptide according to the invention can be produced by chemical processes known in the art or else by a combination of chemical and biological (e.g. translation) processes.
  • (poly) peptides that have been post-translationally modified.
  • the respective post-translational modification of the (poly) peptides depends on the organism used for expression or on the cell used for expression.
  • Another object of the invention is a homodimer which is formed by two (poly) peptides according to the invention which have the biological activity of the same neurotrophic factor.
  • the amino acid sequence of the (poly) peptides according to the invention can differ within the homodimer as long as they are still capable of dimerization and the biological activity does not change in their nature.
  • the two (poly) peptides forming the homodimer may have been transcribed or translated by different DNA-RNA sequences that are in the same vector or in different vectors. ren can be located and in the latter case produced by different hosts / host cells and thus can also be modified differently post-translationally.
  • the neurotrophin dimers have a significantly higher biological activity than the corresponding monomers, and the dimers according to the invention also have a greater stability than the wild-type neurotrophin dimers, which predestines them for therapeutic use.
  • improved stability of the dimers according to the invention is essentially achieved by N-terminal extension of the protein and modification of the consensus cleavage site of the furin type in such a way that the N-terminal peptide can no longer be split off.
  • the dimers according to the invention show improved stability both in the N-terminal peptide glycosylated and in the non-glycosylated form.
  • the neurotrophin is preferably extended by up to about 19 amino acids, preferably by about 4 to 19 amino acids, N-terminally. An improvement in stability is obtained if the N-terminal peptide does not contain a furin-type cleavage site.
  • a non-glycosylated protein according to the invention can be obtained recombinantly in eukaryotes if there is no potential glycosylation site, for example asparagine, in the sequence of the N-terminal extension. If the neurotrophin is produced in prokaryotes, there is no glycosylation, even if the sequence contains asparagine.
  • the N-terminal peptide preferably corresponds to up to approximately 19 amino acids of the C-terminal end of the neurotrophin prosequence, which is naturally cleaved off when the mature neurotrophin is formed. Preference is also given to sequences which differ from this sequence by up to 20%, preferably by up to 10%.
  • the invention further relates to heterodimers which are formed by two (poly) peptides according to the invention which have the biological activity of different neurotrophic factors.
  • the statements made for the homodimers according to the invention apply analogously to the heterodimers according to the invention. It is also important here that the (poly) peptides are still capable of dimerization. Because of the relatively large homologies between the individual neurotrophic factors, it is assumed according to the invention that heterodimers can be formed between the (poly) peptides according to the invention derived from all neurotrophins. The formation of heterodimers of various neurotrophic factors has already been shown by Radziejewski et al., Biochemistry 32 (1993), 13350-13356. Another object of the invention is a method for producing a (poly) peptide, homodimer or heterodimer according to the invention, wherein
  • the culture medium used for culturing the host according to the invention can be any medium which allows the expression of the (poly) peptides according to the invention.
  • the composition of the medium depends on the host used and, if appropriate, on the regulatory sequences present in the vector. For example, when E.coli is used as the host, Luria Broth (Sambrook et al., Op. Cit.) Can be used, but when using mammalian cells, the modified Eagle medium, which is also known in the art, can be used.
  • the (poly) peptides, homodimers or heterodimers according to the invention are secreted into the medium, depending on the properties of their N-terminus, they are isolated from the culture supernatant and purified by standard methods in protein chemistry. If, on the other hand, they are not secreted, they are isolated from the hosts themselves after disruption, also using standard methods.
  • the invention relates to an antibody which is specifically weighted against the (poly) peptide, homodimer or heterodimer according to the invention.
  • the antibody according to the invention does not cross-react with mature neurotrophic factors.
  • the antibody can e.g. be used in diagnostic methods that require a distinction between the mature neurotrophic factor and the (poly) peptide according to the invention.
  • the antibody according to the invention cross-reacts with mature neurotrophic factors and can therefore be used therapeutically and diagnostically wherever antibodies against mature neurotrophic factors can also be used; see. eg WO 91/03568.
  • the antibody according to the invention is a polyclonal antibody.
  • the antibody according to the invention is a monoclonal antibody.
  • the invention relates to a medicament which contains the (poly) peptide, homodimer and / or heterodimer according to the invention, optionally in combination with a pharmaceutically acceptable carrier.
  • the respective indication determines whether the medicament contains a combination of (poly) peptide, homodimer and heterodimer.
  • the medicament according to the invention can be used in any biocompatible pharmaceutical carrier, e.g. physiological saline, dextrose or water. If necessary, the medicinal product can also be used to prevent the diseases listed below.
  • the amount of drug or active ingredient to be administered depends on the type of disease and the condition of the patient to be treated. If possible, dose-response curves should be drawn up for the active substances before the actual administration in vitro. This can be done, for example, with suitable biological test systems. Such test systems can easily be established by a person skilled in the art with neurons that originate from the central or peripheral nervous system of chickens or rats. The data obtained i_ vitro can then be confirmed in the animal model. The treating doctor can determine the optimal dose to be administered for the treatment of the patient from the data obtained in this way, if necessary after carrying out preclinical tests.
  • Methods of administration include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intrathecal, intraventricular and intranasal administrations.
  • Intrathecal and subcutaneous administration are preferred for the purposes of this invention. It may be necessary to introduce the drug of the invention directly into the CNS using any suitable route of administration, including intraventricular and intrathecal.
  • the intraventricular injection can be facilitated by an intrathecal catheter, which is connected, for example, to a reservoir, such as an Ommaya reservoir.
  • the implant can consist of porous, non-porous or gelatinous material, for example of membranes such as sialastic membranes (“sialastic membranes”) or of fibrous materials.
  • the medicaments according to the invention can also be administered via liposomes, microparticles or microcapsules.
  • liposomes for individual embodiments according to the invention it may be useful or necessary to achieve a delayed release of the (poly) peptides according to the invention.
  • cells are administered which contain the (poly) peptides according to the invention, antagonists thereto or antibodies against them.
  • the medicament serves to increase the survival rate of neurons.
  • the increase in the survival rate of sensory neurons e.g. the petrous, knee, or ventrolateral trigeminal ganglia.
  • the medicament serves to improve neuronal growth.
  • the medicament according to the invention can be used, for example, to increase neurite growth, e.g. of sensory neurons.
  • the medicament according to the invention is used to support differentiated cell functions.
  • the medicament according to the invention is used, for example, to treat disorders which relate to the differentiation of the neural crest.
  • the medicament is used for the treatment of diseases of the nervous system.
  • the medicament according to the invention is used for the treatment of degenerative diseases of the nervous system.
  • the medicament according to the invention can be used to treat hereditary spastic paraplegia with degeneration of the retina (Kjellin and Bamard-Scholz syndromes), retinis pigmentosa, Stargardt's disease, Usher syndrome (Retinis pigmentosa with congenital hearing loss) and Refsum syndrome (retinis pigmentosa, hereditary hearing loss and polyneuropathy) can be used.
  • a defect in the synthesis of one or more neurotrophic factors can be the etiology underlying a syndrome, which is characterized by a combination of retinal degeneration and sensory dysfunction.
  • the medicament according to the invention is used for the treatment of diseases of the nervous system which include injury or damage to the nervous system.
  • the medicament according to the invention is used to treat injuries of this type which are attributable to trauma, surgery, an infarction, an infection or a malignant disease.
  • the medicament according to the invention is also used to treat ischemia, nutritional deficiencies or metabolic diseases.
  • the medicament according to the invention can be used for the treatment of neuropathic side effects which are caused by the use of anti-static active substances in cancer patients.
  • the medicament according to the invention is furthermore preferably used in cases where the above-mentioned disease, injury or damage to the nervous system is due to the action of a toxic active substance.
  • the medicament according to the invention can be administered locally to injured or damaged sensory neurons which occur, for example, in dorsal root ganglia, temporal bone, knee joint or gangrion nodosum or in the other tissues mentioned below: the vestibuloacoustic complex of VIII. Cranial nerve, the ventrolateral pole of the maxillomandibular lobe of the trigeminal ganglion and the mesencephalic trigeminal nucleus).
  • the (poly) peptide contained in the medicament is applied to a membrane, for example a sialastic membrane, which can be implanted in the vicinity of the injured nerve.
  • a membrane for example a sialastic membrane
  • the medicament according to the invention can also serve to accelerate the regeneration of patients suffering from diabetic neuropathies, for example mononeuropathy multiplex.
  • the medicament according to the invention is used for the treatment of diseases of the nervous system which comprise an innate disease of the retina.
  • the medicament according to the invention is used to treat a degenerative disease of the nervous system, which is Alzheimer's disease, Huntington's chorea, a retinal disease, Parkinson's disease or a Parkinson's plus syndrome.
  • a degenerative disease of the nervous system which is Alzheimer's disease, Huntington's chorea, a retinal disease, Parkinson's disease or a Parkinson's plus syndrome.
  • the medicinal product is used in combination with surgical implantation of tissue in the treatment of Alzheimer's disease or Parkinson's disease.
  • the medicament is used to treat a Parkinson's plus syndrome, which is the progressive supranuclear pulsy (Steele-Richardson-Olszweski) syndrome, olivopontocerebral atrophy, the Shy-Drager syndrome or the Guam-Parkinson's dementia complex is.
  • Parkinson's plus syndrome which is the progressive supranuclear pulsy (Steele-Richardson-Olszweski) syndrome, olivopontocerebral atrophy, the Shy-Drager syndrome or the Guam-Parkinson's dementia complex is.
  • a further preferred embodiment of the invention relates to a medicament with the (poly) peptide according to the invention, which is used to treat the disease of sensory neurons or a tumor.
  • the medicament according to the invention is used to treat a tumor that is a neuroblastoma.
  • the medicament according to the invention is used to increase the survival rate of dopaminergic neurons.
  • the pharmaceuticals according to the invention can be used, for example, to promote the survival rate of dopaminergic (substantia nigra) neurons in a dose-dependent manner.
  • the medicament according to the invention can be used in the treatment of irregularities in the radio tion of dopaminergic neurons of the CNS, which occur, for example, in Parkinson's disease.
  • the medicament according to the invention is used to increase the survival rate of cholinergic neurons.
  • the medicament according to the invention can be used, for example, to support the survival of cholinergic neurons of the CNS, in particular cholinergic basal neurons of the forebrain, and thus for the treatment of Alzheimer's disease. Indeed, it has been shown in the past that about 35% of patients with Parkinson's disease also suffer from Alzheimer's-like dementia.
  • the medicament containing the (poly) peptide according to the invention may prove to be useful in the single-agent therapy of this complex of diseases.
  • the medicament according to the invention can be used for the treatment of Alzheimer's disease in connection with Down syndrome.
  • the medicament according to the invention can also be used in the treatment of a number of dementias and congenital learning difficulties.
  • the medicament according to the invention should prevent the multiplication of astroglial cells and thus contribute to the prevention of scarring in the CNS (which occurs, for example, following trauma, surgery or an infarction).
  • the medicament according to the invention can also be used for the treatment of tumors of the CNS which have arisen from degenerate astroglial cells.
  • the medicament according to the invention can be used to increase the expression of receptors in neurotrophic factors and can thus be administered, for example, before or together with NGF.
  • the medicament according to the invention is thus preferably also used to increase the survival rate of cholinergic neurons which are cholinergic neurons of the basal forehead or cholinergic neurons of the septum.
  • a further preferred embodiment of the invention relates to the use of the medicament in the suppression of the proliferation of astroglial cells.
  • the medicament has at least one further, mature neurotrophic factor.
  • this further factor is NGF, NT-3, BDNF or NT-4/5. It is intended to use the (poly) peptide or medicament according to the invention in conjunction with other cytokines in order to achieve the desired neurotrophic effects.
  • the medicament according to the invention can be used with NGF or skeletal muscle extract in order to exert a synergistic stimulating effect on the growth of sensory neurons.
  • the invention comprises the use of a (poly) peptide, homodimer or heterodimer according to the invention in the manufacture of a medicament for the treatment of the abovementioned.
  • a (poly) peptide, homodimer or heterodimer according to the invention in the manufacture of a medicament for the treatment of the abovementioned.
  • the (poly) peptide, homodimer or heterodimer according to the invention forming a (poly) peptide, homodimer or heterodimer, whose plasma half-life is longer than that of the corresponding natural neurotrophin.
  • Half-life is the time in which the effective concentration of the therapeutic agent (activity) in the plasma is reduced by half. The measurement is usually carried out after administration of a therapeutically effective dose.
  • the neurotrophins according to the invention have an at least two, preferably a two to five times longer half-life.
  • Another object of the invention is a method for improving the stability of a solution or a lyophilizate of a neurotrophin, which is characterized in that the neurotrophin is extended N-terminally by 1-19 amino acids and this extended neurotrophin is dissolved or lyophilized.
  • the neurotrophin is preferably extended by an N-terminal peptide which corresponds 80%, preferably at least 90%, to the C-terminal peptide of the same length which can be split off from the native neurotrophin precursor form.
  • the invention further relates to an in-ro method for diagnosing one of the diseases listed above, wherein
  • a) brings a tissue sample into contact with a labeled DNA or RNA according to the invention under conditions which allow hybridization; and b) the hybridization is established.
  • the tissue sample for hybridization is prepared according to customary conditions.
  • the hybridization takes place under stringent conditions.
  • These hybridization tests can be used to detect and diagnose, predict or monitor the above-mentioned diseases or precursors, provided that these diseases are accompanied by a change in the expression of neurotrophic factors.
  • diseases generally affect e.g. the violation of neurons and the degeneration of neurons of the retina. They also include trauma to the CNS, an infarction, an infection, degenerative nerve diseases, malignant diseases and postoperative changes, Alzheimer's disease, Parkinson's disease, Huntington's chorea and degenerative diseases of the retina.
  • the total RNA in a patient's tissue sample can be examined for the presence or absence of the RNA of a neurotrophic factor, the decrease in the amount of BDNF mRNA, for example, indicating neuronal degeneration.
  • the invention further relates to an in vitro method for diagnosing one of the diseases listed above, wherein
  • Such abnormalities can e.g. inherited changes or changes in neurons caused by toxic influences.
  • the neurotrophin receptor is the receptor for NGF, NT-3, BDNF, NT-4/5 or NT-6.
  • the invention also relates to a medicament which has an antibody according to the invention in combination with a pharmaceutically acceptable carrier.
  • This medicament can be used, for example, to neutralize the action of the (poly) peptides according to the invention after a certain point in time, if this is desired.
  • the antibodies in the medicament according to the invention cross-react with mature neurotrophic factors, they can also be used to combat diseases in which these factors are overexpressed.
  • the invention further relates to a kit for the diagnosis of one of the diseases listed above, which comprises at least
  • the kit according to the invention is usually used to carry out the methods according to the invention described above.
  • the DNA or RNA molecules or sequences complementary thereto can themselves be provided with a marker, such as an enzyme, or radioactive marker. If the DNA or RNA molecule according to the invention is used to detect a desired nucleic acid sequence, a second nucleic acid can also be used to detect the nucleic acid complex. This second nucleic acid sequence is marked with a detectable marker and hybridizes to a different region of the DNA or RNA molecule according to the invention than the region which hybridizes to the nucleic acid sequence to be detected. - 21 -
  • the antibodies can be labeled for diagnostic purposes, for example using conventional methods with 1251.
  • kits according to the invention can be used in other processes, the design of which belongs to the skill of the average person skilled in the art. Such methods can be, for example, ELISAs or RIAs.
  • the person skilled in the art is also able to supplement the kit according to the invention with the respective necessary buffers or to carry out any dilutions of the reagents obtained in the kit according to the invention.
  • the invention relates to a diagnostic composition which comprises at least
  • the areas of application for the diagnostic composition according to the invention essentially correspond to those of the kits according to the invention discussed above.
  • FIG. 2A shows a partial amino acid sequence of mouse wild-type NT-3 and mouse wild-type BDNF and of (R-1- * > K) BDNF.
  • the sequences determined by amino acid sequencing are underlined, the remaining sequences were derived from the corresponding DNA sequence.
  • the consensus sequence for cleavage by furin-like enzymes is printed in bold.
  • the open arrow indicates the glycosylated asparagine residue which is part of an N-glycosylation consensus sequence in all neurotrophins.
  • the asterisks serve to optimally stack the sequences together.
  • FIG. 2B shows that (R-1-> K) -BDNF is glycosylated, whereas wild-type BDNF is not.
  • the sugar residues were detected with anti-digoxigenin antibodies after the proteins were electrophoretically separated on SDS gels and transferred to membranes by vest blot. The molecular weight markers are given on the right in kDa. Transferin (T) was used as a positive control for a glycosylated protein. It is worth considering that the broad (R-1-- K) -BDNF band is glycosylated, but wild-type BDNF is not.
  • Molecular weights of neurotrophins determined by gel filtration chromatography. The retention time was plotted against the molecular weight of standard proteins in a semi-logarithmic diagram and the straight line thus obtained was used to determine the molecular weights of the various forms of recombinant neurotrophins.
  • the neurotrophins injected as early-eluting peaks from the urine phase chromatography elute with a molecular weight which corresponds to that of dimers, while the injection of the same peaks after incubation with guanidine hydrochloride showed that these were quantitatively converted into neurotrophin monomers.
  • NT-3 dimers restored by gel filtration.
  • NT-3 monomers were incubated at a concentration of 150 ng / ml PBS and applied to a gel filtration column after the respective incubation time. The absorption was measured at 280 nm. In each field, the peak with the shorter retention time represents NT-3 dimers ( ⁇ 11.4 minutes) and the one with the longer retention time represents NT-3 dimers ( ⁇ 13.17 minutes). An almost quantitative conversion of the monomers into dimers took place after about 24 hours.
  • Biological activity of recombinant neurotrophins measured by a neuron survival test The values obtained are expressed as a percentage of surviving neurons after a 24-hour incubation period.
  • the neurons were isolated from the ganglia nodosa of E8 chicken embryos.
  • BDNF is set at 50 kDa and for wild-type BDNF at 27 kDa.
  • the values are mean values from 3 experiments ⁇ the standard deviation from 2 independently carried out
  • TrkC-expressing fibroblasts were incubated with NT-3 dimers and monomers for 3 minutes at various concentrations. After immunoprecipitation, SDS gel electrophoresis and a Western blot, the tyrosine phosphorylation was determined with an anti-phosphotyrosine antibody and a fluorescent signal.
  • a conservative amino acid exchange (asparagine for lysine, (R-1-> K)) in the basic processing cleavage site of BDNF was introduced using an oligonucleotide-directed mutagenesis kit (Amersham, version 2; see FIG. 2A).
  • the oligonucleotide 5-ATGAGGGTTCGGAAACACTCCGACCCT-3 '(SEQ ID NO: 4) coding for the site to be mutated was attached to single-stranded DNA and the second strand was produced using the Klenow fragment in the presence of phosphorothioate deoxynucleotides.
  • the template strand was cleaved with a restriction enzyme and removed by digestion with exonuclease m.
  • the mutated second strand was synthesized using DNA polymerase I and the plasmid 11KATA18 (Götz et al., Eur. J. Biochem. 204 (1992), 745-749) was amplified in bacteria.
  • the mutation in the sequence was confirmed by DNA sequencing and the sequence encoding the above-described BDNF precursor derivative was cloned in a suitable vector for recombination with the vaccinia virus genome, as described by Götz et al., Supra.
  • Recombinant neurotrophins were produced in a vaccinia virus expression system and purified by chromatography on glass beads with a controlled pore size. This was followed by a reverse phase high performance liquid chromatography (HPLC) step as described by Götz, supra. described.
  • HPLC reverse phase high performance liquid chromatography
  • Pig brain BDNF was purified as described by Hofer and Barde, Nature 331 (1988) 261-262.
  • Recombinant neurotrophins were applied to a reverse phase C8 chromatography column (Aquapore RP-300, 4.6 mm x 220 mm, Applied Biosystems), which had been equilibrated with 0.1% trifluoroacetic acid (TFA; Spectro-grade, Pierce) and with a linear acetonitrile gradient (21% - 56% acetonitrile in 50 minutes, flow rate 1.0 ml / min) eluted.
  • TFA trifluoroacetic acid
  • BDNF from porcine brain was purified from reverse phase HPLC using the same buffer system, with the exception that a C8 column with a smaller diameter (Aquapore RP-300; 250 mm x 1.0 mm, Applied Biosystems) and a flow rate of 0.2 ml / min. were used.
  • the protein peaks were detected at an absorption of 214 nm or 280 nm and collected by hand.
  • the fractions were dried under vacuum in a rotary vacuum dryer and the protein thus obtained was stored at 4 ° C.
  • Neurotrophin monomers were generated by incubating the dimers in 6M guanidine hydrochloride (pH 5.9, Sigma) in a concentration of 100 ng / ⁇ l at room temperature for 24 hours. Guanidine hydrochloride was removed by reverse phase chromatography. Wild-type BDNF was derived from the (R-1 »K) BDNF mutant by gel filtration using a TSK G3000SW column (7.5 mm ⁇ 600 mm) containing 0.1% TFA and 30% acetonitrile had been equilibrated, separated.
  • gel filtration was performed using a Pharmacia Superdex TM 75 HR 10/30 column equilibrated with 0.1 M sodium phosphate buffer, pH 7.0 containing 0.5 M NaCl and 10% acetonitrile. Proteins used as molecular weight markers and the neurotrophins were applied in aliquots of 20-40 ⁇ l in concentrations between 100 and 200 ng / ⁇ l. Dextran blue and tyrosine were used in standard runs as markers for the empty volume and the enclosed volume. The samples were run at a flow rate of 1.0 ml min. eluted and detected at 280 nm.
  • the molecular weights of the various neurotrophins were determined by comparing the retention times with those of the proteins used as molecular weight markers. The conversion of dimers to monomers and vice versa was followed using a peak integration program (Maxima 820 software, Millipore).
  • the later eluting peak from NT-3 up to over 90% contained a form of NT-3 which the 5 N-terminal amino acids of mature NT-3 lacked. This form was able to form dimers, had the same biological activity as mature NT-3 and was called des-Penta-NT-3.
  • the BDNF isolated from porcine brain also eluted in two individual peaks; see. Fig. 1. Both peaks contained fully processed, mature BDNF, as could be shown by amino acid analysis and sequencing of the N-terminus.
  • a second chromatography of the first peak of purified NT-3 or BDNF over the same column consistently revealed a second, smaller peak that had the same retention time as the later eluting peaks observed during purification; see. Fig. 1.
  • BDNF with the mutated cleavage site elutes only as a single peak, the retention time being comparable to that of the first NT-3 and BDNF peaks; see. Figure 1.
  • the early and late eluting peaks observed with the native neurotrophins cannot be explained by differences in their primary structure, it was examined whether the two peaks were caused by the buffer conditions (0.1% TFA and acetonitrile) used during the cleaning process, represent structurally different conformations.
  • the early-eluting neurotrophins were incubated for one day at 37 ° C in 6M guanidine hydrochloride, which has been shown to influence the conformation of NGF, BDNF and NT-3 (e.g. Greene et al., Neurobiology 1 (1971), 37-48).
  • NT-3 as well as wild-type BDNF and the BDNF mutant were further analyzed by gel filtration before and after the incubation with guanidine hydrochloride. While wild-type BDNF and wild-type NT-3, which eluted from the reverse phase HPLC with shorter retention times, were detectable as a single peak with a calculated molecular weight of 27 kDa, the forms with longer retention times showed a calculated molecular weight of 13.7 kDa, which corresponds to that of ribonuclease A; see. Fig. 3. The relative molecular weight of the BDNF mutant was also determined in this experiment.
  • the neurotrophin dimers were aliquoted, dried, resuspended in 6 M guanidine hydrochloride and incubated therein for different times before application to a gel filtration column; see. Figure 4A. The areas of the two peaks were calculated for each gel filtration run using the Maxima 820 (Millipore) software program and the extent of dissociation of the dimers was shown as a relative amount of monomers .; see. Figure 4B. These results confirm the results obtained with reverse phase chromatography (Fig. 1): they show that the (R-1-K) BDNF mutant forms dimers which are measurably more stable than the wild-type neurotrophins; see. 4B at the time 1 hour. Mature NT-3, which lacks 5 amino acids at the N-terminal (des-penta-NT-3), on the other hand has a much lower dimer stability than mature NT-3 and BDNF (cf. Example 6 and FIG. 4B).
  • the neurotrophins can subsequently form dimers again.
  • NT-3 monomers 150 ng / ml
  • PBS PBS
  • NT-3 monomers 150 ng / ml
  • gel filtration about half of the monomers were already present as dimers.
  • essentially all of the monomers had been aggregated back into dimers (FIG. 5).
  • These newly formed dimers cannot be distinguished by reverse-phase HPLC from the early-eluting peaks described in FIG. 1.
  • the total amounts of NT-3 monomers and dimers did not change during the entire incubation period, as was determined by the integration of the monomer and dimer peaks after each run. This result leads to the conclusion that the monomers and dimers are freely convertible.
  • neurotrophins can exist as monomers and as dimers in solution. It was therefore of interest to find out whether the neurotrophin monomers have measurable biological activity. Since it is known that neurotrophins prevent the death of neurons in cell culture, this property was used to determine the biological activity of the monomers.
  • ganglia nodosa from 8-day-old chicken embryos (E8) were prepared, incubated for 0.5 hours in a 0.1% trypsin solution (Worthington), twice with medium containing serum (F 14/10% Horse serum) and carefully dissociated in this medium. The biological activity of NT-3 and BDNF monomers and dimers was also investigated on neurons of the dorsal root ganglia (E9).
  • the biological activity of (R-1-> K) BDNF was compared with that of mature BDNF on sensory neurons and sympathetic neurons (express).
  • the cell suspension was first plated out to remove non-neuronal cells. 3 hours later, the neurons were carefully resuspended, counted, and at a density of 2000 neurons per well on plates (48 wells) that had previously been coated with polyornithine and either laminin or conditioned medium from the Schwannona cell line RN22, with or without neurotrophic Factors sown.
  • the NT-3 monomers and dimers were freshly dissolved in PBS and added to the culture medium immediately before the neurons were plated.
  • NTH 3T3 cells that express rat trkC more fully (Tsoulfas et al., Neuron 10 (1993), 975-990) .
  • cell culture plates with about 5 x 106 NIH 3T3 cells were incubated for 3 minutes at 37 ° C. with NT-3 monomers and dimers of various concentrations.
  • the cells were washed for 15 to 20 minutes in 600 ⁇ l lysis buffer (TBS, pH 7.5, containing 1% Triton X100, 10% glycerol, 0.5 mM phenylmethanesulfonyl fluoride (PMSF ), 0.5 mM ortho vanadate, each 5 mg / ml pepstatin and trypsin inhibitor) incubated.
  • TBS 600 ⁇ l lysis buffer
  • PMSF 0.5 mM phenylmethanesulfonyl fluoride
  • ortho vanadate each 5 mg / ml pepstatin and trypsin inhibitor
  • the proteins were transferred to Immobiion-P membranes (Millipore) using a discontinuous buffer system.
  • Buffer 1 was 300 mM Tris-HCl, pH 10.4;
  • Buffer 2 was 25 mM Tris-HCl, pH 10.4;
  • buffer 3 was 40mM norleucine, 25mM Tris-HCl, pH 9.4.
  • Sodium borate was added to each buffer at a final concentration of 10 mM before use.
  • the membrane used for the blot was washed in methanol and water and then incubated in buffer 2. The gels were incubated in buffer 3 for 5 min.
  • the proteins were transferred to the membranes using a semi-dry blotting chamber using a current of 0.9 mA / cm 2 for one hour.
  • the free binding sites on the membranes were blocked by incubating the membranes for ten minutes in a PBS solution containing 2.2% polyvinylpyrrolidone (a 1: 5: 5 mixture (% by weight) of PVP 360, PVP 40 and PVP 10) . After washing with 0.17% PVP and 0.1% Tween, 20 containing PBS, the membranes were incubated with a monoclonal anti-phosphotyrosine mouse antibody (UBI), which was used in a final dilution of 1: 1000 in washing buffer has been. After a further washing step as described above, the membranes were incubated with anti-mouse IgG (1: 10,000 dilution in washing buffer) which was coupled to horseradish peroxidase.
  • UBI monoclonal anti-phosphotyrosine mouse antibody
  • the signals were detected via the ECL Westem Detection System (Amersham) according to the manufacturer's information on chemiluminescence. After three minutes of incubation with various concentrations of the NT-3 dimers, a tyrosine phosphorylation signal could already be observed at a concentration of 10 ng ml. Maximum signal strength was reached at 100 ng / ml; see. 8. In contrast, only an extremely weak phosphorylation signal was achieved even at the highest concentrations of NT-3 monomers (1 ⁇ g / ml); see. Figure 8A. However, when the NT-3 monomer solution was incubated long enough to allow dimer formation, complete tyrosine phosphorylation activity was achieved; see. Figure 8B. These results indicate that the NT-3 monomers are 100 to 1000 times less active than the NT-3 dimers in inducing trkC phosphorylation.
  • NT-3 and BDNF wild-type proteins were routinely determined by SDS-PAGE, subsequent Coomassie blue staining and densitometric quantification (Ultroscan XL Enhanced Laser Densitometer, LKB).
  • known amounts of egg protein lysozyme (Sigma) were used as set standards.
  • the (R-1-> K) -BDNF mutant was quantified by analysis of the amino acid composition using the ninhydrin method known in the art.

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Abstract

L'invention concerne des séquences nucléotidiques codant de (poly)peptides n'apparaissant pas naturellement et dotés de l'activité biologique d'un facteur neurotrophique. Ces (poly)peptides ont une extrémité terminale N plus longue que celle de facteurs neurotrophiques mûrs. L'invention concerne en outre des molécules d'ADN de recombinaison qui contiennent les séquences nucléotidiques décrites, les (poly)peptides codés par celles-ci, des médicaments, des agents de diagnostic et des nécessaires contenant les (poly)peptides décrits, ainsi que des procédés in vitro de dépistage de maladies (ou de leurs prodromes) du système nerveux.
PCT/EP1995/003538 1994-09-12 1995-09-08 Molecules biologiquement actives derivees de neurotrophines WO1996008562A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0994188B1 (fr) * 1998-10-09 2004-01-07 Scil proteins GmbH Procédé d'obtention du NGF-bêta biologiquement actif
WO2008002572A2 (fr) * 2006-06-27 2008-01-03 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Procédé de mesure de facteur neurotrophique dérivé de cerveau mature

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005254A1 (fr) * 1990-09-25 1992-04-02 Genentech, Inc. Nouveau facteur neurotrophique
WO1993010150A1 (fr) * 1991-11-14 1993-05-27 Regeneron Pharmaceuticals, Inc. Expression de facteurs neurotrophiques au moyen de regions 'prepro' heterologues
EP0544293A2 (fr) * 1991-11-27 1993-06-02 Roche Diagnostics GmbH Procédé pour la préparation de bêta-NGF biologiquement actif par génie génétique
WO1994016073A2 (fr) * 1993-01-08 1994-07-21 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University Procedes et reactifs permettant l'inhibition de la furine endoprotease

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005254A1 (fr) * 1990-09-25 1992-04-02 Genentech, Inc. Nouveau facteur neurotrophique
WO1993010150A1 (fr) * 1991-11-14 1993-05-27 Regeneron Pharmaceuticals, Inc. Expression de facteurs neurotrophiques au moyen de regions 'prepro' heterologues
EP0544293A2 (fr) * 1991-11-27 1993-06-02 Roche Diagnostics GmbH Procédé pour la préparation de bêta-NGF biologiquement actif par génie génétique
WO1994016073A2 (fr) * 1993-01-08 1994-07-21 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University Procedes et reactifs permettant l'inhibition de la furine endoprotease

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Title
KOLBECK, ROLAND ET AL: "Characterization of neurotrophin dimers and monomers", EUR. J. BIOCHEM. (1994), 225(3), 995-1003 CODEN: EJBCAI;ISSN: 0014-2956 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0994188B1 (fr) * 1998-10-09 2004-01-07 Scil proteins GmbH Procédé d'obtention du NGF-bêta biologiquement actif
WO2008002572A2 (fr) * 2006-06-27 2008-01-03 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Procédé de mesure de facteur neurotrophique dérivé de cerveau mature
WO2008002572A3 (fr) * 2006-06-27 2008-03-20 Us Gov Health & Human Serv Procédé de mesure de facteur neurotrophique dérivé de cerveau mature

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