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  • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
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  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • 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
• • 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
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • 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
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • 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
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• TLR-mediated response is generally seen as a typically antimicrobial host defence response designed to start pro-inflammatory innate immune responses and —eventually- antimicrobial adaptive immune responses.
• the present invention relates to a method for the treatment and/or prophylaxis of a degenerative inflammatory process in a tissue of a subject, said method comprising increasing the activity of TLR3 in cells of said tissue.
• stathmin and stathmin-like proteins can act as activator or agonist for TLR3 or can activate TLR3-mediated signalling.
• the inventors discovered this activity in astrocytes, a major cell type in the mammalian CNS.
• the stathmin-activated TLR3-mediated response of astrocytes includes production of a range of neuroprotective, antiinflammatory, angiogenic and chemotactic mediators that -together- support regenerative responses rather than polarised pro-inflammatory host-defence responses.
• the degenerative inflammatory process is associated with a chronic or acute neurodegenerative disorder.
• the neurodegenerative disorder is preferably selected from the group consisting of stroke, hypovolemic shock, traumatic shock, reperfusion injury, multiple sclerosis, AIDS-associated dementia, neuron toxicity, Alzheimer's disease (AD), head trauma, acute spiral cord injury, Huntington's disease (HD), Parkinson's Disease (PD) and related synucleinopathies, dystonia, Tourette Syndrome, Frontotemporal Dementias (FTDs) and related tauopathies, prion disorders such as Fatal Familial Insomnia (FFI) and other disorders related to Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis (ALS), trinucleotide repeat diseases, Motor Neurone disease, progressive supranuclear palsy, REM Sleep Behavior Disorder (RBD) and cancer.
• Particularly preferred neurogenerative disorders that may be treated or prevented by the method of the invention include stroke, multiple stroke, multiple a aor
• - proteinaceous compounds comprising a polypeptide chain (preferably in alpha helix form) having an amino acid sequence similarity of at least 60% with an amino acid sequence selected from the group consisting of the amino acid sequences of stathmin (SEQ ID NO: 1), SCG-IO (SEQ ID NO:2), SCLIP (SEQ ID NO: 3) and RB3 (SEQ ID NO: 4).
• amino acid sequence similarity relates to the similarity of the polypeptide to the complete reference sequences. In a preferred embodiment, however, the amino acid sequence similarity relates to the stathmin-like alpha helix domain corresponding to residues 44 to 138 of SEQ ID NO: 1.
• stathmin-like protein used in aspects of the present invention is preferably selected from SCGlO, SCLIP and RB3; human stathmin is most preferred, and combinations of stathmins and stathmin-like proteins are also envisaged.
• a method of the present invention may further comprise the use of the TLR3- agonist in combination with compounds that promote stabilisation of the stathmin- like alpha helix domain corresponding to residues 44 to 138 of SEQ ID NO: 1. It should be noted that the method of the invention is not necessarily medical, but may also comprise non-medical embodiments. For instance, the method may be for preventing or retarding the onset of a neurodegenerative disorder in a subject, in the absence of any disorder, disease, or injury.
• the present invention relates to a pharmaceutical composition for the treatment and/or prophylaxis of a degenerative inflammatory process in a tissue of a subject, wherein said composition comprises a therapeutically effective amount of at least one compound capable of increasing the activity of TLR3 in cells of said tissue; and b) a pharmaceutically acceptable carrier.
• the pharmaceutically active compound in a composition of the invention may for instance be:
• composition is for preventing or retarding the onset of degenerative inflammatory disorders and neurodegenerative disorders as described above in a subject, in the absence of any disorder, disease, or injury.
• TLR3 By activating TLR3 or TLR4, also some induction is observed of TLR2, but this is an indirect effects as it is blocked by the protein synthesis-blocking agent cycloheximide, whereas TLR3 is not blocked by such a treatment (Panel B; showing data for a single representative donor).
• Figure 8 summarises the currently documented expression of TLR3 in different human tissues and cell types under normal (non-diseased) conditions.
• Figure 9 lists all gene products represented on a macroarray used in the example study whose expression levels were found to be reproducibly increased at least two-fold following stimulation of astrocytes with either the TLR3 agonist poly LC, the TLR4 agonist LPS or the mixture of both.
• Numerical values represent the level of mRNA expression in the stimulated astrocyte population relative to their expression in unstimulated astrocytes from the same donor, cultured for the same time under the same culture conditions but without the specific TLR agonist.
• the numerical values represent the maximum stimulation found after analysing levels of expression after 2, 6, 24 and 48 h.
• Figure 11 shows the nucleotide sequence of the cDNA clone (clone F5) found in the screening experiments described below in the experimental section and its corresponding protein sequence encoding a protein capable of activating TLR3- mediated signalling.
• the protein sequence encoded by F5 (excluding the plasmid sequence) is identical to residues 44 (D, aspartic acid) to the C-terminal residue 149 (D, aspartic acid) of human stathmin (SEQ ID NO.l), which stretch fully comprises the conserved stathmin-like alpha helical domain that is located between residues 44 and 138.
• a “neuroprotective” effect is defined herein as an inhibition, prevention or generally control of processes that cause damage or injury to cells of the nervous tissue, in particular to the neurons.
• the neuroprotective effect of TLR3 stimulation by stathmin was observed in astrocytes as described in more detail in the Examples below.
• stathmin-like protein refers to the art-recognized term for a particular group of proteins that has amino acid sequence motives in common with stathmin (stathmin- 1) and is generally used to address the group of compounds consisting of the stathmin-like proteins SCGlO (Superior Cervical Ganglion 10; stathmin-2), SCLIP (SCGlO-like protein; stathmin-3), and RB3 (stathmin-4).
• SCGlO Superior Cervical Ganglion 10
• stathmin-3 SCGlO-like protein
• RB3 stathmin-4
• percentage of sequence similarity for polypeptides such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence similarity may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide sequence in the comparison window may include amino acid deletions, modification or addition of single amino acids or groups of amino acids as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• TLR3 activation of TLR3 or promotion of the expression of TLR3 or promotion of the TLR3 signalling pathway results in retarding of neurodegenerative processes.
• stathmin and stathmin-like proteins provide very suitably such agonists.
• VEGF vascular endothelial growth factor
• VEGF-C vascular endothelial growth factor
• CXCL8 angiogenesis vascular endothelial growth factor
• CCL2 vascular endothelial growth factor receptor 2
• CCL3 vascular endothelial growth factor receptor 3
• TLR3-activating substances or other substances that specifically promote expression of stathmin and/or stathmin-like proteins and/or TLR3 and/or that promote the functional interaction between stathmin or stathmin- like proteins and TLR3 are effective against autoimmunity, transplant or graft rejection, inflammation, allergic responses, other autoimmune, immune-mediated or trauma related tissue degeneration, tissue diseases and exaggerated inflammation.
• the present invention discloses methods of use for permitting or promoting wound healing; controlling neurodegenerative disorders, including damage or injury to neurons.
• stathmin is expressed at relatively high levels in the central nervous system, testis and thymus and its expression is particularly prominent during neurogenesis and elevated in response to damage (see below).
• stathmin is also expressed, but at levels that are at least 10-fold lower than in the above organs.
• stathmin is down- regulated during oligodendrocyte differentiation in vitro and re-expressed in oligodendrocytes in the brains of MS patients. This parallels its elevated expression in animal models of demyelination.
• Stathmin accumulates not only inside the oligodendrocyte cell body but also closely associated with myelin membranes.
• stathmin expression in vertebrates along with accumulating functional data thus strongly suggest that the various members of the stathmin family play complementary roles in the development, maturation, and functional regulation in response to damage or trauma especially of the central nervous system.
• Inhibition of stathmin expression with antisense oligonucleotides prevents nerve growth factor-induced differentiation of rat PC 12 cells into sympathetic-like neurons.
• Reducing the expression of stathmin with an antisense oligonucleotide inhibits migration of new neurons from the SVZ to the olfactory bulb via the rostral migratory stream.
• stathmin In line with an important role of stathmin especially in the CNS, inactivation of the stathmin gene in mice leads to axonopathy in aged (20-month old) mice.
• D-stathmin is only expressed in germ cells and cells of the central and peripheral nervous systems. This pattern that is very similar to that found for vertebrate stathmin.
• inhibiting stathmin expression by siRNA leads to dramatic defects only in the nervous system.
• most of the axons of the ventral nerve cord are organized in a repetitive pattern of commissures.
• formation of the commissures fails to occur or is defective.
• peripheral nervous system is affected.
• D-stathmin is crucially important at various stages of differentiation and maturation of the Drosophila nervous system, mostly in ways that are relevant to neurite and axon formation.
• RNA At the present time versions of double -stranded RNA are known for their ability to activate TLR3-mediated signalling. Ways to identify additional compounds that can activate TLR3-mediated signalling pathways involve screening assays that monitor TLR3 signal transduction pathway in cells that express TLR either naturally, or after transfection with an TLR3-encoding gene construct. Such methods have been recently described in detail in the literature.
• the readout for the screening assay is based on the use of native genes or, alternatively, transfected or otherwise artificially introduced reporter gene constructs which are responsive to the TLR3 signal transduction pathway.
• Readouts can also be performed in this multiwell array, preferably using a multiwell plate reader device or the like. Examples of such devices are well known in the art and are available through commercial sources. Sample and reagent handling can be automated to further enhance the throughput capacity of the screening assay, such that dozens, hundreds, thousands, or even millions of parallel assays can be performed in a day or in a week. Fully robotic systems are known in the art for applications such as generation and analysis of combinatorial libraries of synthetic compounds.
• the binding of a test compound to the TLR3 polypeptide can also be determined directly. For example, a radiolabeled test substance can be incubated with the TLR3 polypeptide so that binding of the test substance to the polypeptide can be monitored.
• stathmin and stathmin-like proteins of all possible sources are suitable for use in aspects and embodiments of the present invention.
• a suitable stathmin and stathmin-like proteins may be obtained from plants, from animals or from micro-organisms, such as yeasts. They may be produced in those sources naturally as a metabolite or may be produced therein as recombinant proteins.
• the stathmin and stathmin-like proteins may be isolated from these organisms or can be used in a less pure form, i.e. as an enriched fraction, or in the case of microorganisms such as (recombinant) yeasts by taking the complete organism(s) or fractions thereof.
• stathmin and stathmin-like proteins may be isolated from other suitable sources, such as from milk, egg, soy, yeast, bacteria, algae, plants, meat, brain, etc. or may be synthetically prepared, for use in a pharmaceutical composition according to the invention or for instance in a nutraceutical composition (i.e. a food item or food supplement).
• Pharmaceutically acceptable bases which form carboxylate salts with free carboxylic groups of peptides and functional equivalents, include ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other mono-, di- and trialkylamines, as well as arylamines.
• Pharmaceutically acceptable solvates are encompassed.
• Stathmin, stathmin-like proteins or any of the substances that promote stathmin expression, stabilisation of its helix, TLR3 expression or TLR3-mediated signalling may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art.
• mice with the TLR3 agonist poly LC 5, 7 and 9 days after experimental induction of the disease almost completely blocks subsequent disease development. This effect is mediated largely by anti-inflammatory mediators that are induced by TLR3 activation, and includes IFN-beta.
• the present invention further relates to a method for the preparation of a pharmaceutical composition for the prevention and/or treatment of a neurodegenerative disorder selected from the group consisting of stroke, hypovolemic shock, traumatic shock, reperfusion injury, multiple sclerosis, AIDS-associated dementia, neuron toxicity, Alzheimer's disease (AD), head trauma, acute spiral cord injury, Huntington's disease (HD), Parkinson's Disease (PD) and related synucleinopathies, dystonia, Tourette Syndrome, Frontotemporal Dementias (FTDs) and related tauopathies, prion disorders such as Fatal Familial Insomnia (FFI) and other disorders related to Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis (ALS), trinucleotide repeat diseases, Motor Neurone disease, progressive supranuclear palsy, REM Sleep Behavior Disorder (RBD) and cancer in a subject, comprising processing or incorporating a stathmin or stathmin-like protein, or a derivative or
• astrocytes When stimulated with cytokines, astrocytes were supplied with TNF- ⁇ , IFN- ⁇ , IL- l ⁇ (each at 500 U/mL), IL-4 (at 250 U/mL), IL-6 (at 200 U/mL), IL- 12 (at 50 ng/mL), IL- 10 (at 20 ng/mL) and TGF- ⁇ (at 12.5 ng/mL) (PeproTech, Rocky Hill, NY).
• Control cDNA array profiling experiments included treatment with 50 ⁇ g/mL cycloheximide for 30 min prior to and during poly LC treatment, or with 200 U/mL IFN- ⁇ (Avonex).
• RNA-BeeTM Total cellular RNA was isolated using RNA-BeeTM as previously described (Bsibsi et al., [2002] supra). Subsequently, RNA was reverse transcribed into cDNA and levels of TLRl-10 and ⁇ -actin as a reference were determined by quantitative real-time PCR.
• the following fluorogenic molecular beacons and primers Biolegio, Nijmegen, The Netherlands) were used: ⁇ -actin: sense primers: 5' GGTCATCACCATTGGCAATGA 3'; anti-sense primer 5' ACGTCACACTTCATGATGGAGTTG 3'; beacon cgtgccGCACTCTTCCAGCCTTCCTTCCTGggcacg;
• beacon 5' ACAGTGATAAGATGTCAGAAGTCCAAAG 3'; beacon: cgtgccATCCACGTTCCTAAAGACCTATCCCAGAggcacg; TLR2: sense primer 5' GAAATGTGAAAATCACCGATGAAAG 3'; anti-sense primer: 5' TCCACTTTACCTGGATCTATAACTCTGTC 3'; beacon CgtgccTTTGATGACTGTACCCTTAATGGAGTTggcacg; TLR3: sense primer 5 ' CAGTACATCGAGTTCTTGGTTTCAAA 3'; anti-sense primer: 5'GAGAAATGTTCCCAGACCCAATC 3'; beacon:
• Cos-7 cells (25,000 cells per well in 96-well plates) were transfected for 5 h with 768 pools of cDNA derived from human brain tumours (5 pooled tumours including meningioma, oligodendroglioma, astrocytoma grades II and IV, and medulloblastoma; NCI CGAP Brn35; Invitrogen) using lipofectamine, after which cells were thoroughly washed. Transfection with green-fluorescent protein-encoding cDNA was used as an internal control for transfection efficiencies. After continued culture of the transfected cells for 48 h, 75 ⁇ L of each culture supernatant was harvested and added to cultured human astrocytes seeded at 1,000 cells per well in 75 ⁇ L.
• TLR-6 tumour necrosis factor-stimulated gene 6
• chemokines include CXCL8, CXCL2 and CCL3.
• Poly LC but not LPS also leads to induction of CCL4, CXCL6, CCL5, CXCL9 and CCL2 albeit to a lesser extent.
• the predominant functional association of chemokines is with inflammation and recruitment of leukocytes.
• both astrocytes and neurons express functional receptors. This emphasises a more particular relevance of these chemokines for regulating neural cell migration and tissue remodelling within the CNS.
• anti-fibrotic drugs or agents hold significant potential as therapeutic substances in a wide variety of human disorders.
• the anti-fibrotic effects of the respond extends to other cell types and tissues as well suggesting that stathmin or stathmin-like proteins could serve as anti-fibrotic substances for a wide variety of applications.
• TLR3 TLR3-mediated signalling activates a powerful anti-inflammatory response, and one that promotes neuroprotection, repair and regenerative processes.
• the only known agonist for TLR3, viz. double-stranded RNA is remarkably scarce as an extracellular substance in the CNS. Since also for other reasons a role of double-stranded RNA as the only TLR3 agonist can be rationalised in the present context only with difficulty we decided to examine the possibility that additional TLR3 agonist molecules could be present in the human CNS itself.
• Purified recombinant stathmin was exposed to two different conditions that differentially affect the extent of helix formation within the protein chain.
• One part of the protein preparation was exposed to 5% trifluoroacetie acid (TFA) in water for 1 h at 37°C, strongly acidic conditions that tend to disrupt an alpha helix.
• another part was exposed for 1 h at 37 0 C to 5% of the helix-promoting solvent trifluoroethanol (TFE) in phosphate-buffered saline (PBS).
• TFE helix-promoting solvent trifluoroethanol
• PBS phosphate-buffered saline
• stathmin reduces levels of the p70 subunit of IL- 12 two-fold. This combination will result in accumulation of the anti-inflammatory p40 subunit without its counterpart to form IL- 12. Thus, this effect in pre-dendritic cells represents another stathmin-induced mechanism to inhibit inflammatory processes.

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