US20040266991A1 - Biologically active peptides and their use for repairing injured nerves - Google Patents

Biologically active peptides and their use for repairing injured nerves Download PDF

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US20040266991A1
US20040266991A1 US10/492,850 US49285004A US2004266991A1 US 20040266991 A1 US20040266991 A1 US 20040266991A1 US 49285004 A US49285004 A US 49285004A US 2004266991 A1 US2004266991 A1 US 2004266991A1
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kdi
peptide
spinal cord
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neurons
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Paivi Liesi
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • 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
    • A61K38/06—Tripeptides
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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

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  • the present invention relates to biologically active peptides derived from the neurite out-growth-promoting domain of laminin-1, i.e. the ⁇ 1-chain of laminin-1.
  • These peptides include the decapeptide RDIAEIIKDI and the truncated peptides derived therefrom comprising the biologically active domain thereof, the tripeptide KDI.
  • the invention is directed to the biologically active tripeptide motif KDI, and to its use in promoting regeneration of neuronal or non-neuronal tissues and, in specific, to its use in the treatment of spinal cord injuries.
  • Laminin-1 promotes neurite outgrowth of both central and peripheral neurons of the rodent (Liesi, 1990).
  • One of the neurite outgrowth domains of laminin-1 has been mapped to the C-terminal decapeptide RDIAEIIKDI (1543; p20; Liesi et al., 1989) of the ⁇ 1-chain of laminin-1.
  • laminin-1 has been shown to prevent neurotoxicity of the amyloid- ⁇ -peptide involved in neuronal death of Alzheimer disease (Bronfman et al., 1996; Drouet et al., 1999). Laminin-1 further affects development of dendritic spines of the cerebellar Purkinje cells (Seil, 1998), and influences memory processing by modulation of LTP (Nakagami et al., 2000).
  • laminins may have specific and diverse effects on development and mature function of the human CNS.
  • ⁇ 1 laminin antibodies on cultures of central neurons, the neurite outgrowth domain of the ⁇ 1-chain of laminin-1 has been shown to play a major role in neuronal migration and axon guidance.
  • WO publication 93/24155 discloses a medical device, useful as a graft in repairing injured nerve tissues, specifically peripheral nerves, the device containing the decapeptide P1543.
  • WO publication 98/43686 discloses fibrin-based, biocompatible materials for, for instance, peripheral nerve regeneration. The materials contain various bioactive peptides, including the above-indicated decapeptide.
  • Hager et al. (1998) showed that the corresponding peptide derived from mouse laminin-1 modulates the electrical activity the neurons of rat neocortex.
  • WO publication 94/04560 discloses protein factors having Schwann cell mitogenic activity.
  • the peptide sequences disclosed in the application include a sequence comprising the KDI motif. Use of the factors for e.g. neural regeneration is suggested.
  • U.S. Pat. No. 5,780,090 discloses flavoring ingredients for food products, comprising tripeptides having hydrophobic amino acid residues. Preparation of the tripeptide KDI is described in the patent, but no medical use is suggested for the peptide.
  • netrin-1 has been shown to act on central neurons via a G-protein coupled receptor mechanism (Corset et al., 2000).
  • the KDI-sequence is present in the chicken netrin-1 (Serafini et al., 1994), and the human netrin-protein (Meyerhardt et al., 1999) also has this domain, although modified. It is unclear, however, if the presence of this short sequence has any functional significance in these proteins.
  • the KDI sequence may be hidden in netrins either by conformation or glycosylation of the proteins.
  • one object of the invention is the use of the decapeptide RDIAEIIKDI or a truncated peptide derived therefrom comprising the tripeptide motif KDI in the manufacture of a medicament for use in a method for treating spinal cord injuries.
  • one object of the present invention is a truncated peptide derived from the decapeptide RDIAEIIKDI including the biologically active peptide motif KDI for use as a medicament.
  • a specific object of the invention is the tripeptide KDI for use as a medicament.
  • a primary object of this invention is a biologically active tripeptide motif KDI for use in a method for treating spinal cord injuries.
  • a further object of this invention is the tripeptide KDI for use in a method for treating injured nerves, e.g. peripheral nerves.
  • a still further object of the invention is a method for repairing injured nerves in an animal in need of such repairing, said method comprising administering to said animal an efficacious amount of a biologically active peptide comprising the tripeptide motif KDI.
  • a biologically active peptide comprising the tripeptide motif KDI.
  • the most preferable is the tripeptide KDI.
  • Another object of the invention is a method for the treatment of spinal cord injuries in an animal in need of such treatment, said method comprising administering to said animal an efficacious amount of a biologically active peptide comprising the tripeptide motif KDI.
  • Preferable peptides are the decapeptide RDIAEIIKDI and the truncated peptides derived therefrom comprising the tripeptide motif KDI.
  • the most preferable for the purposes of the invention is the tripeptide KDI.
  • peptide motif KDI is a biologically active peptide
  • any peptide comprising the tripeptide motif KDI may be useful in regeneration and renewal of damaged or degenerating tissues, either neuronal or non-neuronal.
  • a peptide with the KDI-motif could be used in these functions in both soluble and substrate-bound forms.
  • a still further object of the invention is a pharmaceutical composition, which comprises as an active ingredient the tripeptide motif KDI, in association with at least one pharmaceutically acceptable carrier and/or diluent.
  • FIG. 1 The effects of fusion proteins containing the P1543 peptide sequence on electrical properties of cerebellar granule neurons on a laminin-1 substratum.
  • FIG. 2A to 2 D The effects of different shorter peptides derived from the P1543 decapeptide on electrical properties of cerebellar granule neurons on a laminin-1 substratum.
  • FIG. 3 Mean numbers of viable human embryonic neocortical neurons on glial cells of the human spinal cord 72 hrs after an injury in the presence or absence of soluble KDI peptide at 1 ⁇ g/ml or at 10 ⁇ g/ml or of the P1543-decapeptide at 1 ⁇ g/ml.
  • (SC+n) spinal cord glial cells
  • (SC+LE+n) lesioned spinal cord glial cells
  • (LE+KDI 10 +n) lesioned cells in the presence of 10 ⁇ g/ml of the KDI peptide
  • (KDI 1 +LE+n) lesioned cells in the presence of 10 ⁇ g/ml of the KDI peptide
  • (p20 1 +LE+n) lesioned cells in the presence of 1 ⁇ g/ml of the P 1543 peptide.
  • FIG. 4A Numbers of human spinal cord neurons extending long neurites (>100 ⁇ m) on monolayers of human spinal cord glial cells after 72 h in vitro.
  • (SC+LE+n) lesioned spinal cord glial cells
  • (SC+LE+n, KDI 0.1 ) lesioned spinal cord glial cells in the presence of 0.1 ⁇ g/ml of the KDI peptide
  • (SC+LE+n, KDI 1.0 ) lesioned spinal cord glial cells in the presence of 1.0 ⁇ g/ml of the KDI peptide.
  • FIG. 4B Numbers of human spinal cord neurons on monolayers of human spinal cord glial cells after 72 h in vitro.
  • (SC+n) spinal cord glial cells
  • (SC+LE+n) lesioned spinal cord glial cells
  • (SC+LE+n, KDI 0.0355 ⁇ KDI 1.0 ) lesioned spinal cord glial cells in the presence of 0.0355 ⁇ g/ml to 1.0 ⁇ g/ml of the KDI peptide.
  • FIG. 5A to 5 D Attachment and neurite outgrowth of TUJ1-positive human embryonic brain neurons on peptides KDI-gc ( 5 A- 5 C) and P1543-gc (SD). Note that “gc” indicates Gly-Cys-addition to allow covalent coupling of the short peptides onto glass as described elsewhere (Matsuzawa et al., 1996b).
  • FIGS. 6A and 6B TUJ1-immunoreactive neurons from the human embryonic spinal cord on top of the injured monolayers of the human spinal cord glial cells after 48 hrs in vitro.
  • 6 A 0.1 ⁇ g/ml of the KDI peptide added into the culture medium;
  • 6 B No KDI peptide present in the culture medium.
  • FIG. 7 Mean numbers of embryonal bodies (EBS; open columns) and EBS extending long neurites (black columns) on white matter of cryostat sections of the adult human spinal cord.
  • KDI KDI tripeptide added;
  • p20 decapeptide P1543 added;
  • CtR control, no peptide added.
  • FIG. 8 Effect of the KDI peptide on the number of long neurites extending from embryonal bodies onto white matter of the adult human spinal cord.
  • KDI KDI tripeptide added;
  • CtR control, no peptide added.
  • FIG. 9A to 9 D Expression of neurofilament proteins and extension of neurofilament-positive neurites from human spinal cord embryonal bodies on white matter of the adult human spinal cord after 10 days in vitro.
  • 9 A Control cultures with no KDI peptide added
  • 9 B Higher magnification photograph of the same embryonal body as in 9 A. The schematic drawing of the experimental situation indicates where on coronally cut sections of adult human spinal cord the embryonal bodies were placed; 9 C: 5-10 ⁇ g/ml of the KDI peptide added; 9 D: Higher magnification of the embryonal body of 9 C.
  • FIG. 10A to 10 D Stereomicroscopic images of placebo-treated ( 10 A and 10 B) and KDI peptide-treated ( 10 C and 10 D) spinal cords of adult rats three months after injury.
  • 10 A and 10 B the ventral spinal cord is photographed.
  • B and D the dorsal pole is shown.
  • the connective tissue scar at the dorsal pole is present in both placebo and KDI-treated spinal cords, but the scar is considerably smaller in the KDI-treated case (D).
  • the present invention provides biologically active peptides for medical use.
  • the peptides are useful in soluble or substrate-bound forms in repairing injured nerves, for instance peripheral nerves, or injured or degenerating central nervous system.
  • the peptides are particularly useful for the treatment of spinal cord injuries.
  • the KDI-domain may enhance regeneration of injuries in the adult mammalian CNS.
  • this peptide also enhanced viability of human CNS neurons regardless the brain region, we propose that this sequence could be used to prevent neuronal degeneration and death in neurodegenerative diseases.
  • the KDI peptide could be applied either in soluble form or attached to biodegradable polymers, that slowly release the peptide and simultaneously provide a direction for the growing axons. The re-growth of axons would be monitored during a period of 3 months by testing of the motor function of the operated animals.
  • the tissues After 3 months the tissues would be collected and subjected to histological, molecular biological and immunohistochemical analysis to verify the effects of the tripeptide and the extent of regeneration. The extent of axon growth through the injured area could then be monitored using DiI-labelling of the nerve fibres.
  • the KDI peptide could also be used in treating neurodegenerative diseases, such as Parkinson's or Alzheimer's disease.
  • a suitable pharmaceutical composition for that purpose is an injectable liquid to be administered to the intrathecal space or injected to the brain tissue.
  • the peptides of the present invention may be prepared using conventional methods for peptide synthesis, as described, for instance, by Liesi et al. (1989).
  • compositions containing the peptides of the invention for the treatment of spinal cord injuries are preferably liquid preparations suitable for injection.
  • the peptides may be dissolved in sterile saline or water.
  • a pharmaceutical composition may include a modification of the KDI peptide that allows its direct access to the CNS through the blood-brain-barrier, and also include biodegradable polymers, which slowly release the peptide and simultaneously, as an additional advantage, provide a direction for the growing axons.
  • the peptides of the present invention may thus be administered in an efficacious amount within a wide dosage range.
  • the efficacious amount depends on the age and condition of the tissue in question.
  • Peptides of the present invention may be administered either as a single dose, or as continuous administration using, for instance, a mini pump system. In the latter case, the daily dosage will not exceed the dose of a single injection, and must be predetermined by animal experimentation.
  • concentrations of a peptide of the invention in a pharmaceutical composition are generally between 0.01 and 100 ⁇ g/ml.
  • concentration of the KDI peptide may be domain dependent or tissue dependent, and therefore pre-testing of the dosage is of utmost importance. Determining of the suitable dosage for individual treatments is within the skills of those familiar with the art.
  • a useful pharmaceutical preparation containing the KDI peptide motif can be prepared and administered, for example, as described for the P1543 decapeptide in WO 93/24155.
  • the pharmaceutical composition of the present invention can be administered by any means that achieve the intended purpose.
  • the composition can be administered to the injury site via a catheter.
  • a most preferable way of administration is using a mini pump system to administer the peptide composition directly to the trauma area of the spinal cord. This can be easily carried out in connection with orthopaedic surgery for disclosing the trauma area.
  • compositions of the present invention can be administered to any animal that can experience the beneficial effects of the peptides of the invention.
  • Human beings are foremost among such animals, although the invention is not intended to be limited to the medical treatment of human beings.
  • KDI is the biologically active domain of the neurite outgrowth decapeptide (P1543) of the ⁇ 1 laminin.
  • An advantageous feature of the KDI peptide is the fact that it is a short peptide, being not immunogenic, and therefore risks for immunological reactions are minimal. Furthermore, as the peptide has previously been disclosed as a flavoring ingredient, it should be safe for human use.
  • Human fetal CNS tissues were obtained from 6-12 week old fetuses after legal abortion and after informed consent from the patients. The tissues were collected by the permission of the Ethics Committee of the Helsinki University Central Hospital. The CNS tissues, identified under a stereomicroscope, were first placed in cold saline and processed for tissue culture experiments. Normal adult spinal cord tissues were from the Neurological Specimen Bank (Baltimore, USA).
  • the CNS tissues were first placed in cold saline.
  • the spinal cord tissues were identified under a stereomicroscope, and carefully freed of meninges.
  • To obtain monolayer cultures of human spinal cord glial cells the cells were dissociated by mechanical trituration using a Pasteur-pipette, and placed in Petri dishes (Corning, N.Y., USA) containing 10% fetal calf serum (Hyclone, Logan, UH) in DMEM-F12 (Gibco, U.K.) supplemented with penicillin and streptomycin as described (Liesi et al. 2001). In this manner, cultures with 100% TUJ1-positive glial cells were obtained, which indicated that the glial cells were precursors of astrocytes.
  • the cells were dissociated by trituration in a sterile culture medium (RPMI 1640) containing penicillin (100 U/ml), streptomycin (100 ⁇ g/ml) and 200 ⁇ M L-glutamine.
  • the dissociated cells were plated on glass coverslips pre-coated (Liesi et al., 1989) with mouse laminin-1 (Boehringer-Mannheim, Germany) or with the peptides KDI-gc and RDIAEIIKDI-gc, as described elsewhere (Matsuzawa et al., 1996b).
  • the peptides were from the Multiple Peptide Systems (La Jolla, Calif.). The cultures were maintained for 24 hrs, fixed in 2% paraformaldehyde, and processed for immunocytochemistry.
  • Immunocytochemistry for neuron specific tubulin isoform TUJ1 was performed as described elsewhere (Liesi et al., 2001). In short, the cells were permeabilized in methanol for 5 min at ⁇ 20° C., washed in PBS and incubated with monoclonal TUJ1-antibodies for 1 hr at room temperature. The antibody was of high specificity and was used at 1:500 dilution. After immunocytochemistry, the coverslips were mounted in PBS:glycerol (1:1) and viewed with Olympus Provis fluorescence microscope with appropriate filter combinations.
  • the cells were incubated in an atmosphere of 95% air/5% CO 2 at +37° C. in RPMI 1640 culture medium supplemented with penicillin and streptomycin and 200 ⁇ M L-glutamine. After 24 hrs, the cells were used for electrophysiology.
  • the standard pipette solution contained (in mM): CsMeSO 4 100, CsCl 15, BAPTA 5, HEPES 10 (pH KOH-adjusted to 7.2).
  • Tests for reversal potentials were conducted in low K + bath solution containing (in mM): NaCl 150, KCl 5, CaCl 2 1, HEPES 10 (pH adjusted to 7.2 with NaOH) and a high K + bath solution, containing (in mM): NaCl 115, KCl 40, CaCl 2 1, HEPES 10 (pH adjusted to 7.2 with NaOH). All bath solutions contained 300 nM TTX.
  • the pipette solution for reversal potential trails was (in mM): K-aspartate 110, NaCl 10, MgCl 2 2, BAPTA 5, HEPES 10 (pH NaOH adjusted to 7.2). Nernst reversal potentials were calculated according to Hille (1992). Peptides and fusion proteins were added to aliquots of the standard bath solution immediately before the trials. Between recordings, the dish was perfused with standard bath solution (RPMI 1640, buffered with 10 mM HEPES, pH 7.4). System quality checks were conducted by switching between two reservoirs each containing standard bath solution to detect perfusion artifacts and determine if any active peptides had adhered in the tubing.
  • the peptides RDIAEIIKDI, EIIKDI, and KDI were all from Multiple Peptide Systems (San Diego, Calif.).
  • the fusion proteins (B2-3; B2-4; B2-5) and al laminin control peptides (AG10; AJ5; AI12) were from Drs. Yoshi Yamada and Atsusi Utani (National Institute of Dental Research, NIH) and were purified as described (Utani et al., 1994; Nomizu et al., 1995).
  • the control cultures had neurons added either without the peptide or the lesion.
  • the cells were cultured for 72 hrs in 5% CO 2 /95% air at +37° C., and fixed for quantitation of neurons and measurement of their neuritic lengths.
  • the neurons on glial monolayers were visualized by immunostaining using mouse monoclonal antibodies against a neuronspecific tubulin isoform (TUJ1).
  • the results were evaluated by counting neurons in six random fields on 3 different coverslip per experiment. In this way, more than 300 neurons were counted per experiment.
  • Statistical analysis of the results was performed using one-way analysis of varians (ANOVA) and Student-Newman-Keuls multiple comparisons test on the Instat (v2.03) program (GraphPad, San Diego, Calif.).
  • the glial monolayers were injured using a 18G needle prior to plating of the neurons.
  • the cells were cultured in normal adult human serum for 72 hrs.
  • the statistical analysis was performed using one-way-variance analysis (ANOVA), and the Student-Newman-Keuls multiple comparisons test.
  • Embryonal bodies containing immature stem cells from spinal cord, or neocortex were obtained by placing the mechanically dissociated CNS-cells on 10 cm Petri dishes (Corning) in Neurobasal medium (Gibco, U.K.) with B27-supplement (Gibco, U.K.), antibiotics and 500 ⁇ M L-glutamine.
  • the embryonal bodies failed to attach onto the plastic and grew in aggregates that also increased in size and released new embryonal bodies into the culture medium.
  • Viability and neurite outgrowth of human CNS-neurons on the spinal cord glial cells were evaluated by counting 10 random fields of cells in 6 different cultures. A total of 265 cells were counted. The numbers of long neurites (>10 cell soma) were estimated similarly. One-way-variance analysis (ANOVA) was used to evaluate the results.
  • Cryostat sections (10 ⁇ m) of adult normal human spinal cord were cut in coronal plain on SuperFrost Plus slides (Menzel, Germany). Each slide had three sections. The slides with freshly cut sections were immediately placed in sterile Quadriperm-plates (In Vitro Systems & Services, Germany) and 10 ml of culture medium was added. The culture medium was 10% normal adult human serum in DMEM-F12 supplemented with penicillin and streptomycin. Two embryonal bodies were placed on areas of white matter on top of each section, and the cultures were placed in the incubator with 5% CO 2 /95% air at 98% humidity at +37° C.
  • the KDI peptide (Multiple Peptide Systems, La Jolla, Calif.) was added in the medium at 1-10 ⁇ g/ml, and the 10-amino acid precursor (RDIAEIIKDI) was added at equimolar concentrations.
  • the control cultures received no peptide.
  • the cultures were fixed and immunostained for neurofilament proteins and additional neuronal and glial markers to identify the neurites and cells within the embryonal bodies. Numbers of embryonal bodies attached, and extending neurites directly on the adult white matter tissue were counted on each slide and the results analyzed using one-way-variance-analysis (ANOVA).
  • the mini pumps initially provided the drugs for 30 days, and when feasible, additional experiments were carried out with prolonged (up to 3 months) application.
  • the animals in each group were euthanized and spinal cords were either deep frozen for immunocytochemistry, biochemistry and RNA-work, or fixed in 4% paraformaldehyde for in situ hybridization and histological analysis of recovery. DiIinjections of the fixed spinal cord well above the injury site were used to monitor the growth of axons across the injury site.
  • the brains and sciatic nerves of the animals were also either deep frozen or fixed and stored for later analysis.
  • a fusion protein (B2-5; Utani et al., 1994) covering the P1543 region was able to induce currents (Block C in FIG. 1) similar to those induced by the decapeptide P1543.
  • Application of 40 ⁇ g/ml of a fusion protein (B2-3) with a 15 amino acid deletion that cuts off the entire RDIAEIIKDI-sequence failed to induce a current in cerebellar granule neurons (Block A in FIG. 1).
  • a fusion protein (B2-4) that cuts off the DI-end of the active sequence also failed to induce a current in cerebellar neurons (Block B in FIG. 1).
  • FIG. 2A The 6-amino acid peptide EIIKDI induced currents in cerebellar granule neurons (FIG. 2A) that were comparable to those induced by P1543 (See FIG. 2D).
  • the shortest peptide that induced currents in central neurons was the tripeptide KDI (FIG. 2B), whereas the unrelated peptides derived from the ⁇ 1-chain of laminin-1 (AG10; A112; AJ5) failed to induce currents in the cerebellar granule neurons (FIG. 2C).
  • neocortical neurons attached and extended neurites on spinal cord glial cells (SC+n), on lesioned spinal cord glial cells (SC+LE+n) or on spinal cord glial cells in the presence of 10 ⁇ g/ml of the KDI peptide (LE+KDI 10 +n)(FIG. 3).
  • SC+n spinal cord glial cells
  • SC+LE+n lesioned spinal cord glial cells
  • L+KDI 10 +n spinal cord glial cells in the presence of 10 ⁇ g/ml of the KDI peptide
  • KDI 1 +LE+n neocortical neurons
  • KDI peptide promotes survival of human neocortical neurons on injured spinal cord glial cells.
  • results also indicate that the KDI peptide is, in low concentrations, better than P1543-peptide in this function.
  • FIG. 4A shows numbers of human spinal cord neurons extending long neurites (>100 ⁇ m) on monolayers of human spinal cord glial cells after 72 hrs in vitro.
  • KDI peptide In the absence of KDI peptide, few neurons with long neurites were seen (SC+LE+n).
  • Addition of 0.11 g/ml of the KDI peptide significantly increased the numbers of long neurites (p ⁇ 0.001). Note that this concentration was also the best to support survival of the spinal cord neurons.
  • Addition of 1.0 ⁇ g/ml of the KDI peptide also significantly increased the numbers of long neurites as compared to the lesion-control.
  • FIG. 4B shows numbers of human spinal cord neurons on monolayers of human spinal cord glial cells after 72 hrs in vitro.
  • SC+n control cultures
  • SC+LE+n lesioning of the glial monolayers
  • Addition of 0.5 ⁇ g/ml or 1.0 ⁇ g/ml of the KDI peptide did not promote survival of the spinal cord neurons any better than the lesion by itself (nonsignificant).
  • FIGS. 4A and 4B show that the KDI peptide is both a survival and neurite outgrowth factor for human spinal cord neurons. Comparison of these data to the results given in FIG. 3 also show that the spinal cord neurons are more sensitive to the dose of KDI than neocortical neurons in this treatment.
  • Two examples of neurite outgrowth of human central neurons on the KDI peptide (10 ⁇ g/ml) indicate that neurons attach and extend long neurites on the tripeptide (FIGS. 5B and 5C).
  • FIGS. 6A and 6B show TUJ1-immunoreactive neurons from the human embryonic spinal cord on top of the injured monolayers of the human spinal cord glial cells after 48 hrs in vitro.
  • a neuron in the lower right corner of the photograph extends a long neurite in the presence of 0.1 ⁇ g/ml of the KDI peptide in the culture medium.
  • a spinal cord neuron fails to extend long neurites when no KDI peptide is present in the culture medium.
  • FIG. 7 shows mean numbers of embryonal bodies (EBS; open columns) and EBS extending long neurites (black columns) on white matter of cryostat sections of the adult human spinal cord.
  • EBS embryonal bodies
  • EBS long neurites
  • FIG. 7 shows mean numbers of embryonal bodies (EBS; open columns) and EBS extending long neurites (black columns) on white matter of cryostat sections of the adult human spinal cord.
  • KDI peptide 5-10 ⁇ g/ml
  • embryonal bodies attached well on white matter and long neurites (>100 ⁇ m) extended out of the embryonal bodies in immediate contact with the myelin of the white matter.
  • Numbers of embryonal bodies attached and sending out neurites were significantly higher in the presence of KDI as compared to the control (CtR; p ⁇ 0.01) or the 10 amino acid precursor peptide P1543 (p20; p ⁇ 0.01).
  • FIG. 8 shows the effect of the KDI peptide on numbers of long neurites extending from embryonal bodies onto white matter of the adult human spinal cord after 10 days in vitro.
  • KDI peptide CtR
  • few neurites extended on sections of human spinal cord white matter.
  • 5-10 ⁇ g/ml of the KDI peptide a large number of long neurites extended from the embryonal bodies and grew in direct contact with the white matter (p ⁇ 0.0001; Mann-Whitney, non-parametric test).
  • FIGS. 9A to 9 D expression of neurofilament proteins and extension of neurofilament-positive neurites from human spinal cord embryonal bodies on white matter of the adult human spinal cord are shown. 10 days in vitro.
  • FIG. 10 shows stereomicroscopic images of placebo-treated ( 10 A and 10 B) and KDI peptide-treated ( 10 C and 10 D) spinal cords of adult rats three months after injury.
  • the ventral (A, C) side of the spinal cord shows the injury site (white arrow).
  • Preliminary motor scores were obtained from animals treated with KDI, and from control animals. The scores given are based on walking scores, which were evaluated by a person who did not know the treatments of individual animals. The scores for each animal were calculated by adding up the walking scores obtained within a 12 week follow-up-time after the operation. The walking score consisted of evaluation of the walking of an animal on a flat surface (on a table). The scores of six animals in placebo-group and six-animals in the KDI-group were analysed and statistically compared. The statistical evaluation of motor scores of the rats with total spinal cord transections was done using a non-parametric Mann-Whitney test using a two-tailed P-value.
  • the P-value obtained was 0.0022, and was considered very significant.
  • the mean motor score for the placebo group was 1 1 ⁇ 2.4 (SEM) and that of the KDI-group was 78 ⁇ 5.8 (SEM).
  • the mean of the motor score of a normal non-operated animal within the same observation period would be 120.

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  • Psychiatry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
US10/492,850 2001-10-26 2002-10-25 Biologically active peptides and their use for repairing injured nerves Abandoned US20040266991A1 (en)

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US11/127,206 US7659255B2 (en) 2001-10-26 2005-05-12 Methods of inhibiting glutamate receptors by administering the tripeptide KDI
US12/141,129 US7741436B2 (en) 2001-10-26 2008-06-18 Biologically active peptides and their use for repairing injured nerves

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FI20012082 2001-10-26
FI20012082A FI20012082A0 (sv) 2001-10-26 2001-10-26 Biologiskt aktiva peptider för korrigering av nervskador
PCT/FI2002/000831 WO2003035675A1 (en) 2001-10-26 2002-10-25 Biologically active peptides and their use for repairing injured nerves

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US12/141,129 Division US7741436B2 (en) 2001-10-26 2008-06-18 Biologically active peptides and their use for repairing injured nerves

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JP (1) JP4312054B2 (sv)
AT (1) ATE490972T1 (sv)
AU (1) AU2002336124B2 (sv)
CA (1) CA2465245C (sv)
DE (1) DE60238551D1 (sv)
DK (1) DK1438329T3 (sv)
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Cited By (1)

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US20090105157A1 (en) * 2006-05-30 2009-04-23 Obschestvo Ogranichennoi Otvetstennostyu Sia Peptides Peptide substance stimulating regeneration of central nervous system neurons, pharmaceutical composition on its base, and the method of its application

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DE102005029468A1 (de) * 2005-06-24 2006-12-28 Plt Patent & Licence Trading Ltd. Verwendung von Inhibitoren der N-Methyltransferasen in der Therapie des Parkinson-Syndroms
BRPI0812357A2 (pt) * 2007-05-31 2015-01-27 Hertz Corp Sistema e método para gerar automaticamente um passe de embarque em avião para um viajante retornando um automóvel de aluguel
US8008253B2 (en) 2007-07-03 2011-08-30 Andrew Tasker Treatment for anxiety
WO2009114539A2 (en) * 2008-03-10 2009-09-17 University Of Louisville Research Foundation Neuroprotective integrin-binding peptide and angiopoietin-1 treatments
GB201700529D0 (en) 2017-01-12 2017-03-01 Rogers April Laminin receptor peptide

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US3645756A (en) * 1968-08-16 1972-02-29 Heinz Huth Flavoring material prepared from onions
US3590722A (en) * 1969-06-09 1971-07-06 Samuel Leptrone Flavor injector device
US5780090A (en) * 1995-07-26 1998-07-14 Firmenich Sa Flavored products and a process for their preparation
US20020168718A1 (en) * 1997-04-03 2002-11-14 California Institute Of Technology Enzyme-mediated modification of fibrin for tissue engineering

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090105157A1 (en) * 2006-05-30 2009-04-23 Obschestvo Ogranichennoi Otvetstennostyu Sia Peptides Peptide substance stimulating regeneration of central nervous system neurons, pharmaceutical composition on its base, and the method of its application
US8524674B2 (en) 2006-05-30 2013-09-03 Obschestvo S Ogranichennoi Otvetstvennostyu “SIA Peptides” Method of improving the conditioned reflex habit, the muscle tonus, or the motion coordination of a patient after suffering trauma to the brain cortex

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JP4312054B2 (ja) 2009-08-12
ATE490972T1 (de) 2010-12-15
WO2003035675A1 (en) 2003-05-01
JP2005512979A (ja) 2005-05-12
AU2002336124B2 (en) 2008-07-03
US20050288231A1 (en) 2005-12-29
CA2465245A1 (en) 2003-05-01
DE60238551D1 (de) 2011-01-20
US7659255B2 (en) 2010-02-09
EP1438329A1 (en) 2004-07-21
US7741436B2 (en) 2010-06-22
CA2465245C (en) 2012-09-11
DK1438329T3 (da) 2011-03-28
FI20012082A0 (sv) 2001-10-26
US20080249021A1 (en) 2008-10-09
EP1438329B1 (en) 2010-12-08

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