WO2004084929A1 - UTILISATION DE proNGF NOTAMMENT POUR LA GUERISON DE PLAIES DE LA CORNEE ET DE LA PEAU - Google Patents

UTILISATION DE proNGF NOTAMMENT POUR LA GUERISON DE PLAIES DE LA CORNEE ET DE LA PEAU Download PDF

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Publication number
WO2004084929A1
WO2004084929A1 PCT/EP2004/002899 EP2004002899W WO2004084929A1 WO 2004084929 A1 WO2004084929 A1 WO 2004084929A1 EP 2004002899 W EP2004002899 W EP 2004002899W WO 2004084929 A1 WO2004084929 A1 WO 2004084929A1
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prongf
cells
use according
skin
ngf
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PCT/EP2004/002899
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German (de)
English (en)
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Susan Lorey
Bernhard Janowski
Gabriele Proetzel
Ulrike Fiedler
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Scil Proteins Gmbh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • proNGF especially for wound healing of the cornea and skin
  • the invention relates to new uses of proNGF, derivatives of proNGF and functional parts of proNGF.
  • the cornea has the function of refraction of light as an essential prerequisite for sharp vision.
  • the cornea consists of 3 layers, an outer layer of epithelium, the stroma and an inner layer of endothelium.
  • the stroma is delimited on the outside by the Bowman membrane and on the inside by the desertion membrane. Corneal lesions can appear in all layers. Injuries to the epithelial layer are compensated for by migration processes of the epithelial cells in early phases and proliferation processes in late phases of wound healing.
  • Stroma injuries are compensated for by the proliferation and migration processes of keratinocytes of the stromal tissue and after their fibroblastic transformation by the production of various collagens, essentially type I and type VI, lesions of the endothelium are compensated for depending on the species by proliferation and / or migration of endothelial cells.
  • Corneal injuries can impair this function and, in severe cases, lead to loss of eyesight.
  • Various causes of corneal injuries are described: viral-induced keratitis, neurotrophic and neuroparalytic keratitis, post-traumatic keratitis, post-infectious keratitis, post-surgical keratitis, dystrophic, degenerative or post-traumatic keratitis but also comea transplants (lamellar, penetrating).
  • comeal impairments are associated with reduced tear production (e.g. from medication, contact lenses, disturbed hormone balance etc.), as a result of laser treatment of the cornea, as a result of chemical or physical burns (Burn) and as a result of non-traumatic pathological conditions (e.g. B. Autoimmune diseases, diabetes, infections).
  • the healing of stringy corneal wounds is often restricted and can make postoperative or posttraumatic recovery difficult. It is not uncommon for corneal ulcerations to result in corneal perforations.
  • Stimulation or induction of dermal wound healing is important when there are wound healing disorders that can often lead to chronic wounds.
  • the risk of chronic wounds, if not treated, is the need for limb amputation and an increased mortality rate.
  • Impaired dermal wound healing is often found in connection with circulatory disorders in peripheral tissues (e.g. the skin) and / or reduced peripheral sensitivity and inflammatory processes. Examples of dermal wound healing disorders are the diabetic foot ulcer, the venous ulcer or the pressure ulcer.
  • the conventional treatment of dermal wounds consists in the use of various wound dressings, which are intended to maintain a moist milieu of the wound, but also compression bandages and the administration of antibiotics to curb or prevent infections.
  • additional agents promoting wound healing are used, e.g. B. protease inhibitors, but also wound dressings based on growth factor level (OASIS TM) or skin equivalents (Apligraf, De ⁇ nagraft).
  • OASIS TM growth factor level
  • a PDGF-BB based product is currently on a gel basis the market that stimulates wound healing in patients with diabetic foot ulcers.
  • the number of amputations necessary due to a diabetic foot ulcer is enormous and amounts to 60,000 amputations per year in the United States alone.
  • Growth factors are proteins that, due to their specific interaction with a receptor, are able to stimulate or influence cell division, migration, differentiation and maturation processes.
  • the proteins can be produced on a large scale both in genetically modified eukaryotes and in prokaryotes (EP0994188, WO0022119). The importance of growth factors in wound healing of the skin has already been demonstrated.
  • NGF from the salivary glands of the mouse (WO9848002, US2002037584) has been described as an active ingredient for the treatment of corneal epithelial damage.
  • US6063757 describes the use of recombinant human NGF for healing corneal and also dermal damage.
  • WO0044396 describes an increase in intraocular pressure both in the animal model (rabbit) and in patients with reduced intraocular pressure. Against the background of a therapeutic application of NGF for corneal defects, such an effect is to be assessed negatively as a possible cause of the development of damage to the fundus (glaucoma).
  • ProNGF is the precursor protein of the NGF.
  • Human NGF is synthesized in vivo as preproNGF.
  • the pre-sequence has a length of 21 amino acids and is cleaved after the translocation of the protein into the endoplasmic reticulum.
  • the resulting proprotein is then proteolytically processed at the N- and C-terminal, making the mature NGF accessible.
  • ProNGF has a molecular weight of 49 kDa, consists of 221 amino acids and appears in its mature form as a homodimer.
  • the pro sequence is localized and almost on the surface of the folded NGF unstructured (Rattenholl et al., Eur. J. Biochem. 2001, 268, 3296-3303; J. Mol. Biol. 2001, 305, 523-533).
  • ProNGF was detected in various tissues in vivo.
  • the biological activity of NGF precursors and peptide sequences of the precursor protein in various in vitro and in vivo systems induction of neurite growth or redistribution of F-actin in PC12 cells, increase in the activity of choline enzymes after intracerebroventricular injection in neonatal rats) (Chen et al., Mol. Cell. Endocrinol. 1997, 127, 129-136; Clos et al., Develop. Brain Res. 1997, 99, 267-270).
  • the biological significance of proNGF which is assumed on the basis of sequence homologies of the pro sequences within the neurotrophins and the localization in different tissues, has not yet been clarified.
  • proproteins are discussed as a reservoir of the mature active protein.
  • the pro sequence acts as a helper in the folding of the mature protein (Rattenholl et al., Eur. J. Biochem. 2001, 268, 3296-3303; J. Mol. Biol. 2001, 305, 523-533) or a role in the sorting of neurotrophins by anabolic or catabolic routes (Farhadi et al, J. Neurosci. 2000, 20, 4059-4068).
  • proNGF The pharmaceutical activity of proNGF was first shown in EP 0 994 188.
  • example 4f the stimulability and survival of sensory neurons from dissociated dorsal root ganglia are examined on the basis of the formation of neurites.
  • the biological activity for the recombinant proNGF was half that of the recombinant mature ⁇ -NGF. Based on these test results, it was proposed to use recombinant proNGF for the manufacture of a medicament for the treatment of neuropathies. Use on comea and conjunctiva cells and skin cells is not described.
  • WO 96/08562 describes NGF, which N-terminal is extended by a peptide fragment of up to approx. 19 amino acids, this peptide fragment being the C-terminal fragment of the precursor part of NGF.
  • This precursor contains a furin-type consensus site at the C-terminus, in which an exchange in amino acid 1 and / or -2 has taken place. It is proposed to use such NGF variants in drugs for the treatment of diseases of the nervous system, for example of degenerative diseases of the nervous system such as degeneration of the retina, in the treatment of injuries or damage to the nervous system etc. An application to cornea and conjunctiva cells and skin cells is not shown.
  • EP 0786520 describes N-terminally extended ⁇ -NGF which, like natural ⁇ -NGF, is said to have an effect on neurons. An effect on the indication areas proposed here is not discussed.
  • TrkA a high affinity receptor with tyrosine activity and P75 a low affinity receptor, to which other neurotrophic proteins and cytokines bind in addition to NGF.
  • TrkA was detected in the basal layer of the corneal epithelium as well as in the stroma and endothelium (Lambiase et al., Invest. Ophthalmol. Vis. Sei. 1998, 39, 1272-1275; Lambiase et al., Invest. Ophthalmol. Vis. Sci. 2000, 41, 1063-1069; You et al., Invest. Ophthalmol. Vis. Sci.
  • P75 is expressed by all cell types of the cornea / Touhami et al., Invest. Ophthalmol. Vis. Be. 2002, 43, 987-994).
  • stimulation of NGF receptor expression can lead to increased NGF binding (Lambiase et al., Invest. Ophthalmol. Vis. Sei. 2000, 41, 1063-1069).
  • P75 is described as a receptor through which apoptotic processes are mediated.
  • TrkA processes mediated by TrkA such as proliferation and survival and differentiation of cells can be enhanced in the presence of P75.
  • proNGF The preferred binding to P75 and the associated increased activation of the P75-mediated signal cascade is seen as the cause of the apoptosis processes induced by proNGF.
  • the induction of apoptosis by proNGF has been demonstrated both for cells that only express P75 and for neurons that carry both receptors. According to these results, proNGF is regarded as a stimulator of apoptosis processes via a P75 mediated signal cascade, while NGF via its preferred TrkA binding can cause cell survival even in the presence of P75.
  • apoptosis induction increased by at least a factor of 10 compared to mature NGF. Since P75 is especially expressed in inflammatory processes, degenerative diseases and injuries together with neurotrophins, proNGF seems to play a role in these diseases for the induction of the apoptotic processes taking place here.
  • proNGF progenitor protein
  • derivatives of proNGF with the exception of those in which the pro portion was completely deleted, and or a functional part thereof in an effective amount together with excipients for the prophylaxis and therapy of diseases of skin, Cornea and conjunctiva cells, in particular tissue, can be used.
  • the results obtained according to the invention also allow the conclusion that the active substances mentioned are suitable for the storage and stabilization of skin, comea and conjunctiva cells and for the in vitro reproduction of these cells.
  • the invention also includes proNGF derivatives in which one or more amino acids have been exchanged, deleted, added and / or chemically modified, always while maintaining the pharmaceutical, medical and biological effectiveness described above.
  • Amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, HydiOphilie, and / or the amphipathic (amphiphilic) nature of the residues involved.
  • nonpolar (hydrophobic) amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, thyrosine, asparagine and glutamine.
  • Positively charged (basic) amino acids include arginine, lysine and histidine.
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Insertions” or “deletions” typically range from one to five amino acids. The degree of variation allowed can be determined experimentally by systematically inserting, deleting and / or substituting amino acids in a polypeptide molecule using recombinant DNA techniques and by examining the resulting recombinant variants for their biological activity.
  • the hydropatic index of the amino acids can also be considered when making these changes.
  • Each amino acid can be assigned a specific hydropatic index based on its hydrophobicity and charge properties. Examples of this can be found in WO 00/32039, in US 4,554,101 and in Kyte & Doolittle, 1982, which are referred to here with their content. It is known that certain amino acids can be substituted by other amino acids if they have a similar hydropatic index and, when they are exchanged, bring about a comparable biological activity of the whole molecule. With all derivatizations of the proNGF it is crucial that the biological, medical or pharmaceutical effectiveness is maintained.
  • the derivatization of proNGF can refer to both the pro portion and the NGF portion.
  • the changes in the pro portion also include amino acid additions at the N-terminus of the pro portion defined by the pre portion are.
  • proNGF functional part of proNGF is understood to mean those parts which fulfill the indications and uses claimed in the present application, with simultaneous deactivation, e.g. by deletion, those portions of proNGF that do not contribute to the function.
  • the functional parts can be determined by a person skilled in the art, for example, by deletion analyzes. Systematic deletions of the pro portion, the prepro portion and / or the NGF portion and a subsequent analysis of the remaining molecule for the indications described and claimed here enable the person skilled in the art to determine the desired functional sections.
  • Such functional parts are, for example, the 25 or 71 amino acids of the pro-C terminus of proNGF.
  • the functional parts should remain as unchanged as possible in order to ensure the physiological function of the proNGF, for example its binding to the receptor.
  • modifications as described below are also possible in the functional parts. The number and type of modifications can be determined experimentally by a person skilled in the art.
  • One or more modifications in the pro, prepro and / or NGF portion can be carried out, for example deletions or substitutions of an amino acid.
  • the protein modified in this way is then examined for its functionality in the context of the present invention, and it is determined whether the change made brings about an improvement or deterioration in the properties.
  • the active ingredient proNGF includes those of human origin, animal origin, in particular from rodents such as rats, rabbits and mice, from cattle and pigs, and recombinantly produced proNGF.
  • the recombinant forms of proNGF also include proNGF fusion proteins in which proNGF is combined with another protein or Peptide was linked.
  • proNGF fusion proteins in which proNGF is combined with another protein or Peptide was linked.
  • the derivatives of the pro portion of NGF include, in particular, those pro forms in which at least 25, more preferably 71 amino acids of the pro-C terminus are present at the N terminus of the NGF (Dicou et al, J. Cell. Biol. 1997 , 136, 389-398). Preference is furthermore given to those derivatives of proNGF which, at the amino acid level, have a homology to the pro form, to the prepro form and / or to the NGF portion of the proNGF of at least 80%, preferably 85%, 90% or 95% or about that. Homology means that the specified percentage of amino acids, based on the starting form of the proNGF used, for example rh-proNGF, is contained in identical form.
  • the number and type of modifications in the derivatives is always limited by the maintenance of the physiological functions of the proNGF, in particular, of course, by the maintenance of the therapeutic and prophylactic and other functional properties of the proNGF as described in the present invention.
  • no more than 10, preferably no more than 5 and more preferably less than 5, modifications are made in the proNGF.
  • a maximum of 5 modifications are preferably introduced in the pro portion.
  • the functional sections of the pro portion remain functional, i.e. positions 143 - 221 and 78 - 221 of the proNGF (numbering of the positions starting at the N-terminus of the proNGF).
  • proNGF can take place at the amino acid level via direct chemical modifications of amino acids.
  • modification by mutagenesis of the DNA coding for the protein is preferred. This includes, for example, site-directed mutagenesis, as is known from the prior art and is constantly being further developed (Brannigan and Wilkinson, Nat. Rev. Mol. Cell. Biol. 2002, 3, 964-970).
  • proNGF and its derivatives by recombinant techniques is described and reference is made to this prepublished literature here (Rattenholl et al., Eur. J. Biochem. 2001, 268, 3296-3303; Rattenholl et al., J. Mol Biol. 2001, 305, 523-533).
  • ProNGF and the derivatives described above stimulate the proliferation and migration of cornea cells, conjunctiva cells and dermal cells, promote the maintenance of the vitality of these cells and the wound healing of the skin and the cornea, for example acute and chronic traumatically-induced and non-traumatic- induced wounds.
  • the regeneration processes are also induced and promoted. Further details on effectiveness are described below.
  • the active compounds of the present invention can be used either as a single substance or in conjunction with the proNGF derivatives or in conjunction with other active compounds.
  • Particularly suitable active ingredients are those which stimulate healing, have an anti-inflammatory effect, have antibiotic properties, have antiviral activity, antifungal substances and active substances which relieve or prevent pain.
  • the further active ingredients contained in the pharmaceutical composition are intended to increase the activity of the proNGF active ingredient or to supplement its activity during treatment in a form which is more favorable for the patient.
  • Preferred is a synergistic effect in the drug and a minimization of side effects and undesirable effects.
  • the active ingredients of the present invention are preferably used in the form of a pharmaceutical composition in which they are mixed with suitable carriers in such doses so that the disease is treated and / or alleviated.
  • a composition can contain (in addition to the active ingredients and the carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, penetration enhancers and other materials which are well known in the art.
  • pharmaceutically acceptable is defined as a non-toxic material that does not interfere with the effectiveness of the biological activity of the active substance or substance.
  • carrier and other additives depends on the route of administration. Ophthalmologically and dermatologically compatible carriers and additives were preferably used.
  • a therapeutically effective dose also refers to an amount of the compound sufficient to achieve improvement in symptoms, for example treatment, healing, prevention or improvement in such conditions.
  • Suitable routes of administration are in particular a topical application.
  • the compositions can be in liquid form, for example in the form of emulsions, suspensions and solutions, in spray form, in the form of transdermal systems such as plasters, in the form of a cream, an ointment or a gel. Retard forms are also preferred. Further, known forms of presentation are known to the person skilled in the art.
  • the amount of drug that is adequate to achieve the effects described above is defined as the therapeutically effective dose.
  • the amount that is effective for this use depends on the severity of the patient's condition and disease.
  • Single or multiple administrations according to a daily, weekly or monthly treatment schedule can be combined with dose strengths and mustem, which are selected by the treating doctor.
  • the doses to be used are also determined by the doctor, with amounts of preferably from 1 ng / ml to 1 mg / ml or 1 ng / g to 1 mg / g, preferably from 10 ng / ml to 200 ⁇ g / ml or 10 ng / g to 200 ⁇ g / g can be used.
  • other doses of active substance can also be used, which also depend, for example, on the formulation of the active substance, on the presence of further active substances, on the individual patient, on the application site, on the type of disease, etc.
  • proNGF as used in the present context also includes the configurations specified in the patent claims and defined in more detail in the present description, for example also derivatives of proNGF.
  • the present invention involves the use of proNGF to maintain the vitality of corneal cells and to stimulate proliferative and migratory processes of cells.
  • ProNGF is suitable for the treatment of corneal and dermal injuries with the aim of supporting or stimulating corneal and dermal Regeneration processes.
  • the proNGF-containing compositions are suitable for the positioning and stabilization of comeas and their ability to positively influence the vitality of corneal cells.
  • proNGF and its derivatives to accelerate wound healing further includes: I.) providing an ophthalmologically and / or dermatologically compatible composition that contains an effective concentration of proNGF, II.) Applying the composition to the cornea preventively before an injury the cornea or therapeutically after an injury to the cornea with the aim of achieving a clinically demonstrable stimulation of the healing of the corneal injuries by stimulating proliferative and / or migratory processes of corneal cells of the epithelium, stroma or endothelium or therapeutically for the treatment of dermal wounds with the aim of to achieve a clinically demonstrable stimulation of the healing of the injuries by stimulating proliferative and or migratory processes of dermal cells.
  • proNGF includes: 1.) the amino acid sequence of the NGF, which is extended at the N-terminal by the complete or partial sequence of the proprotein or preproprotein, the extension at least the amino acids from positions 1 to 25, further 1-71 of the C-terminus of the prosequence and 2.) is glycosylated or non-glycosylated or differs in the glycation status, 3.) is of human or non-human origin, 4.) is obtained recombinantly or non-recombinantly, 5.) as a fusion protein with a other heterologous protein is present as a fusion partner or unfused, 6.) is unmodified or modified by
  • B ProNGF can be obtained using various methods: 1.) isolation from human or animal tissue, 2.) chemical synthesis z.
  • C Ophthalmologically compatible compositions are materials of aqueous, ointment-like or gel-like consistency that contain a pharmaceutically acceptable carrier.
  • the carriers can contain sterile water, various organic solvents, emulsifying or suspending agents or diluents.
  • the carriers can contain physiologically compatible surface-active ionic or nonionic substances.
  • the carriers can contain antibacterial, antifungal components and / or preservatives (e.g.
  • the carriers can contain buffers to maintain a pH between 5.0 and 8.0.
  • the buffers can be conventional buffers such as e.g. B. phosphate, borate, citrate, bicarbonate or Tris-HCl buffer.
  • the carriers can be isotonic by adding appropriate osmotically active components (e.g. saline, sugar).
  • the carriers can contain other solubilizing components such as proteinaceous carriers or solubilizers (e.g. albumin).
  • the carriers can include antioxidants and stabilizers such as e.g. B. sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea.
  • Possible wetting agents can e.g. B. Polysorbate 80, Polysorbate 20, Poloxamer 282 or Tyloxapol.
  • the carriers can include viscosity-increasing agents such as e.g. B. dextran 40, gelatin, glycerol, lanolin, cellulose derivatives (e.g. hydroxyethyl cellulose, hydroymethyl propyl cellulose,
  • the carriers can contain antibiotics to control infections.
  • the carriers can contain agents for the slow release of the growth factor. All components of the carrier are characterized in that they are ophthalmologically compatible and non-toxic.
  • the concentration of the proNGF in the carrier is between 1 ng / ml and 1 mg ml. If ointments or gels are used as an ophthalmologically compatible carrier for the treatment of the comea, the concentration is of the proNGF in the carrier between 1 ng / g and 1 mg / g.
  • proNGF can be used in the ophthalmologically compatible carrier alone or in combination with other healing-stimulating, anti-inflammatory or pain-relieving substances.
  • Dermatologically compatible compositions are materials with an aqueous, ointment-like, emulsion-like, cream-like or gel-like consistency that contain a pharmaceutically acceptable carrier.
  • the carriers can contain sterile water, various organic solvents, emulsifying or suspending agents or diluents.
  • the carriers can be physiologically compatible surface-active ionic or nonionic substances.
  • the carriers can contain antibacterial, antifungal components and / or preservatives (e.g. parabens, chlorobutanol, sorbic acid, phenol, thimerosal).
  • the carriers can contain buffers to maintain a defined pH.
  • the buffers can be conventional buffers such as e.g. B. phosphate, borate, citrate, bicarbonate or Tris-HCl buffer.
  • the carriers can contain other solubilizing components.
  • the carriers can contain antioxidants and stabilizers.
  • the carriers can include viscosity-increasing agents / polymers such as e.g. B. dextran 40, gelatin, glycerol, cellulose derivatives (e.g. hydroxyethyl cellulose, hy droymethyl propyl cellulose, methyl cellulose, carboxymethyl cellulose),
  • Polyethylene glycol Polyethylene glycol, vinyl polymers (e.g. polyvinyl alcohol, polyvinylpolyvinylpyrrolidone), polyacrylic acid and its derivatives, polyoxyethylenes, polyoxypropylene and their copolymers (e.g. Pluronic), polysaccharides (e.g. hyaluronic acid, heparin) collagen and / or vegetable oils or fatty acids or contain fatty acid esters or derivatives.
  • the carriers can contain antibiotics to control infections. All components of the carrier are characterized in that they are dermatologically compatible and non-toxic.
  • the concentration of the proNGF in the carrier is between 1 ng / g and lmg / g.
  • the proNGF concentration in the carrier is between 1 ng / ml and 1 mg / ml.
  • Wound dressings can with an effective dose of lyophilized proNGF's are prepared in a range between 1 ng / cm 2 and 1 mg / cm 2 .
  • proNGF can be used alone in the dermatologically compatible carrier or in combination with other healing-stimulating, anti-inflammatory or pain-relieving substances.
  • the application of the proNGF-containing carrier to the cornea can take place preventively before an injury to the cornea or therapeutically after an injury to the cornea in an amount that is clinically demonstrable stimulation of the healing of the corneal injuries by stimulating proliferative and / or migratory Processes of corneal cells.
  • the proNGF-containing carrier is applied to the cornea in an amount between 10 and 500 ⁇ l, preferably 50 to 100 ⁇ l.
  • Corneal injuries are understood to mean all processes and conditions that cause or characterize damage to all areas of the comea, the epithelium, the stroma and the endothelium (including the Bowman and membrane membranes). This includes both superficial injuries and deep wounds on the entire area of the comea. Examples of superficial wounds of the epithelium and stroma are viral-induced, neurotrophic and neuroparalytic, post-traumatic, post-infectious, post-surgical, dy strophic, degenerative keratitis, but also corneal transplants and corneal impairments in connection with reduced tear production (e.g.
  • Deep injuries up to the endothelium can e.g. B. by diseases that are associated with the development of abnormalities of the endothelium (e.g. Fuchs dystrophy, aphakic bullous keratopathy, pseudoaphakic bullous keratopathy, viral endothelitis, ulceris) or by surgery in the anterior eye area, e.g. B. after cataract surgery, penetrating keratoplasty, intraocular lens insertion, intraocular lens replacement, iridoplasty, pupiloplasty, trabeculectomy arise.
  • diseases that are associated with the development of abnormalities of the endothelium e.g. Fuchs dystrophy, aphakic bullous keratopathy, pseudoaphakic bullous keratopathy, viral endothelitis, ulceris
  • cataract surgery e.g. B. after cataract surgery, penetrating keratoplasty, intraocular lens insertion, intraocular lens replacement, iridoplasty, pupiloplasty, trabeculectomy arise.
  • proNGF-containing carrier to dermal wounds takes place therapeutically after an injury in an amount which is sufficient to achieve clinically demonstrable stimulation of the healing of the dermal injury by stimulating proliferative or migratory processes of dermal cells.
  • the proNGF-containing carrier is applied to the skin wound in an amount sufficient to cover the wound area.
  • Skin means the epidermis and the corium with their individual layers. For details, see the keywords “Cutis”, “Epidermis” and “Korium” in Pschyrembel, Kim. Dictionary, de Gruyter Verlag, Berlin-New York, last edition, noted.
  • Skin injuries are understood to mean all processes and conditions that cause or characterize damage to all areas of the skin. This includes both superficial injuries and deep skin wounds. Examples of dermal wounds are the diabetic foot ulcer, the venous ulcer and the pressure ulcer, Skin wounds associated with systemic diseases (e.g. systemic sclerosis, rheumatoid arthritis), skin injuries due to chemical or thermal effects, accidents, microorganisms such as viruses, fungi and bacteria etc. Dermal wounds can be acute or chronic wounds.
  • systemic diseases e.g. systemic sclerosis, rheumatoid arthritis
  • Dermal wounds can be acute or chronic wounds.
  • the proNGF active ingredients used according to the invention can also be used for the storage, cultivation and production in vitro of comea, conjunctiva and skin cells, cell groups, in particular cell tissues.
  • the active compounds are then added to the culture medium in an amount which promotes storage and cultivation and can be determined by a person skilled in the art; Amounts of 1 ng / ml to 100 ⁇ g / ml are preferably used.
  • the addition of the proNGF active ingredients stimulates proliferation and maintains the vitality of the skin, cornea and conjunctiva cells, promotes the regeneration of injured cells and thus facilitates the positioning and stabilization of these cells and supports the cultivation of the cells in vitro.
  • the cells obtained in this way can be used, for example, for transplantation in patients with damaged skin, comea and conjunctiva cell dressings.
  • the cells can be stored over a longer period of time by using the proNGF active ingredients and thus promote the possibility of transplantation of skin, comea and conjunctiva tissue. Longer transport times from the donor to the recipient of the graft can thus be bridged.
  • the cells can be co-cultivated in connection with carrier tissues and various cell types. It is also possible to produce comea cell mixed tissue in order to use an artificial comea for transplantation (Griffith et al, Comea 2002, 21 (7 Suppl), S54-61; Germain et al., Prog. Retin. Eye. Res. 2000 , 19, 497-527).
  • comea cell mixed tissue in order to use an artificial comea for transplantation (Griffith et al, Comea 2002, 21 (7 Suppl), S54-61; Germain et al., Prog. Retin. Eye. Res. 2000 , 19, 497-527).
  • the differentiation processes of the skin, comea and conjunctiva cells can also be stimulated by the proNGF molecules used according to the invention.
  • proNGF The induction of apoptosis by proNGF has been demonstrated both for cells that only express P75 and for neurons that carry both receptors and therefore does not appear to be influenced by the coexpression of the high-affinity receptor TrkA. According to these results, proNGF is regarded as a stimulator of apoptosis processes via a P75-mediated signal cascade, while NGF can cause cell survival even in the presence of P75 via its preferred TrkA binding. Against the background of the Hempstead et al. Published apoptosis induction by proNGF seems to be unsuitable for proNGF to stimulate comeal and dermal wound healing and for the other indications described here regarding the induction of proliferative and migratory processes.
  • proNGF does not induce apoptosis as previously described, but rather stimulate the proliferation and survival and migration of cells in the cells used according to the invention.
  • various in vitro tests have even demonstrated a stronger effect of proNGF on the proliferation and migration of corneal cells.
  • animal experiments have shown that proNGF has a stronger effect on the healing of artificially placed corneal wounds compared to NGF. Accordingly, proNGF does not induce apoptosis in the cell systems we use. All these results indicate that the mechanism of action mediated by proNGF does not only result in apoptotic processes through an interaction with P75, but surprisingly can also initiate proliferative or migratory processes.
  • the in vitro test system for migration induction described here is mouse fibroblasts (3T3), which were examined for the effects mediated by rh-proNGF and rh-NGF on migration in the Boyden chamber test. If an apoptosis induction by proNGF should be expected according to the above results due to the interaction with P75, no migratory activity of the cells in the presence of rh-proNGF should be demonstrated in this test. Surprisingly, a stimulatory activity of rh-proNGF on the migration of the fibroblasts in vitro, which has not been described so far, could be found in this test system.
  • the effect mediated by rh-proNGF is comparable to the effect mediated by rh-NGF (Example 1, Fig. 1).
  • the in vitro test systems described here are cells of the comea tissue (BCE C / D 1-b), which were examined for the effects mediated by proNGF and NGF. According to the above results, an apoptosis induction by proNGF would have been expected due to the interaction with P75. However, a previously not described stimulatory activity of proNGF on the proliferation and migration of corneal cells in vitro was found (Examples 2 and 3, Figs. 2 and 3). Surprisingly, in contrast to the previously published data on the induction of apoptosis by proNGF, no evidence of apoptosis could be provided in these test systems. In addition, it was shown that proNGF triggers a stronger proliferation induction (Fig. 2) and a significantly stronger migration induction (Fig. 3) than bovine corneal cells (BCE C / D 1-b). 3.) Effect of proNGF on the healing of corneal wounds in the animal model
  • the invention thus also encompasses methods of treating the human and animal body, namely the prophylaxis and / or therapy of diseases of the skin, comea and conjunctiva, a therapeutically effective amount of proNGF and / or derivatives of proNGF, with the exception of those in which the pro portion has been completely deleted, and / or a functional portion thereof is administered together with carriers and / or diluents.
  • Further refinements of the method result from the attached patent claims and from the present description.
  • the dose to be used depends on the type of illness to be treated, on the patient, on the form of preparation, on the type of application, on the severity of the illness and on other parameters known per se. The same applies to prophylaxis.
  • the appropriate preparation forms, application forms and dose-response relationships can be determined by conventional tests in vitro, on animal models and in the context of clinical studies on patients.
  • the cells are grown in full medium (DMEM (Sigma D 5796) with 10% FBS (Sigma, F-2442) and 50 U / ml penicillin and 50 ⁇ g / ml streptomycin (Sigma, P-4333) at 37 ° C, 5% CO .. 2 and 95% humidity cultured Upon reaching a confluence of approximately 60%, the cells are trypsinized the 210 ul of a rh-NGF or rh-proNGF-containing solution of various concentrations (for example rh-NGF.
  • test medium DMEM with 50 U / ml penicillin and 50 ⁇ g / ml streptomycin
  • test medium DMEM with 50 U / ml penicillin and 50 ⁇ g / ml streptomycin
  • each 800 ⁇ l of a cell suspension with a density of 2-10 5 cells / ml are placed in the upper chamber the Boyden crest he given.
  • the chambers are incubated for 4 hours at 37 ° C, 5% CO2 and 95% humidity.
  • the chambers are then screwed apart and the membranes are stained using hemalum / eosin (HEMACOLOR ® fixing solution HEMACOLOR ® 2 staining reagent red HEMACOLOR ® 3 staining reagent blue, VWR International, 1.1 1957.2500).
  • Rh-proNGF shows a significant one at doses 5-10 "7 M and 5T0 " 8 M.
  • Rh-proNGF also demonstrates a migration induction comparable to that of rh-NGF.
  • the cells are grown in complete medium (DMEM (Sigma, D-6046) with 5% FBS (Sigma, F-2442) and 10% calf suspension (Sigma, C-8056), 50 U / ml penicillin and 50 ⁇ g / ml streptomycin ( Sigma, P-4333) and 150 ⁇ M monothioglycerol (Sigma, M-6145) are cultured at 37 ° C., 5% CO 2 and 95% atmospheric humidity, and the cells are trypsinized when a confluence of approximately 80% is reached Cell suspension with a density of 5-10 4 cells / ml is sown in the wells of a 24-hole plate, the cells are cultured in full medium for 24 hours, after which the medium is removed, the cells are washed 3 times with sterile PBS and then in 1 ml each Test medium (DMEM with 1% FBS as well as 50 U / ml penicillin and 50 ⁇ g / ml streptomycin) which contains the rh-NG
  • Rh-proNGF shows a significant at all doses (10 "7 M, 10 ⁇ 9 M and 10 " u M)
  • Rh-proNGF also makes it stronger
  • the cells are grown in full medium (DMEM (Sigma, D-6046) with 5% FBS (Sigma, F-2442) and 10% calf serum (Sigma, C-8056), 50 U / ml penicillin and 50 ⁇ g / ml streptomycin (Sigma , P-4333) and 150 ⁇ M monothioglycerol (Sigma, M-6145) cultivated at 37 ° C., 5% CO 2 and 95% atmospheric humidity, when the confluence of approx. 80% is reached, the cells are trypsinized -NGF or rh-proNGF-containing solution of different concentrations (e.g.
  • NGF 10 "6 , 10 ⁇ 7 , 10 " 8 M
  • proNGF 10 "6 , 10 " 7 , 10 “8 M
  • test medium DMEM with 50 U / ml penicillin and 50 ⁇ g / ml streptomycin
  • Test medium with antibiotic serves as a negative control
  • a porous membrane VWR, 150446, Whatman, Nuclepore Track-Etch Membrane, PC MB, 13 mm, 8.0 ⁇ m pore size, PVPF
  • each 800 ⁇ l of a cell suspension of a density is used of 2T0 5 cells / ml in the upper chamber of the Boyden chamber.
  • the chambers are incubated for 4 hours at 37 ° C, 5% CO2 and 95% humidity.
  • HEMACOLOR ® - fixing solution HEMACOLOR ® 2 - coloring reagent red HEMACOLOR ® 3 - coloring reagent blue, VWR International, 1.11957.2500.
  • the value of the positive control is set to 100% and all other values are related to it.
  • Rh-proNGF shows a significant migration induction of the corneal cells compared to migration in the test medium alone at a dose of 10 "6 M. Furthermore, at this dose rh-proNGF shows a significantly stronger migration induction compared to the migration caused by rh-NGF.
  • New Zealand albino rabbits (2.5 to 3.1 kg body weight, female) are surgically placed bilateral coma wounds.
  • the animals are z. B. with 25 mg / kg of Tiletamine-Zolazepam (Zoletil ® , VIRBAC) and 5 mg / ml of xylazine (Rompun ® 2%, BAYER AG).
  • a topical complementary anesthesia of the eye is done by dropping z.
  • the wound on the comea occurs on day 0 using a bilateral, centrally located 6 mm lamellar keratectomy.
  • the entire treatment is carried out under standardized aseptic conditions.
  • the comea of the animals is treated four times a day by dropping in 50 ⁇ l of a 0.9% saline solution or one containing rh-proNGF or rh-NGF (2 ⁇ g / ml, 20 ⁇ g / ml or 200 ⁇ g / ml protein) 0.9% saline solution starting on day 1 after wounding up to and including day 7 after wounding.
  • the wound healing process is followed daily from day 1 to day 7 by dropping a fluorescein solution into the eyes and digital photo documentation of morphological changes based on the corneal staining.
  • the wound size is determined using appropriate morphometry software (e.g. Focus ® , ESCIL).
  • morphometry software e.g. Focus ® , ESCIL.
  • rh-proNGF at a concentration of 2 ⁇ g / ml surprisingly has the strongest stimulatory effect on the healing of corneal wounds (Fig. 4) and also causes a greater acceleration in wound healing compared to rh-NGF. Almost complete healing of the wounds (> 98%) is achieved with this dose as early as the 4th day after wounding. On day 5 after wounding, almost complete healing of the wounds (> 98%) was achieved with rh-proNGF at a dose of 20 ⁇ g / ml and 200 ⁇ g / ml.
  • the EC 5 o-value was determined for rh-proNGF and rh-NGF a dose of 2 micrograms / ml in comparison to the vehicle control. Accordingly, wound size was reduced by 50% after 1.24 days with rh-proNGF treatment, while a 50% reduction was found after 1.28 days in the case of rh-NGF. When treating the eyes with the vehicle control, a 50% reduction in the wound area is only achieved after 1.42 days.
  • the comea of New Zealand albino rabbits (2.5 to 3.1 kg body weight, female) is made 4 times a day by dropping 50 ⁇ l of 0.9% saline or rh-proNGF-containing (200 ⁇ g / ml protein) 0 , 9% saline over a period of 6 days.
  • Diabetic sugar rats (360 to 425 g body weight, male) are surgically B. 2 skin wounds each.
  • the animals are z. B. anesthetized with 50 mg / kg of Tiletamine-Zolazepam (Zoletil ® 100, VIRBAC).
  • Two paravertebral areas are shaved and the skin is disinfected with povidone iodine (e.g. VETEDJNE ® solution, VETOQUINOL).
  • povidone iodine e.g. VETEDJNE ® solution, VETOQUINOL
  • 2 paravertebral symmetrical dermo-epidermal wounds with a size of e.g. B. 2.0 x 2.0 cm placed on the flanks of each animal.
  • the wounds are covered with an approx. 4.4 x 4.4 cm dressing (e.g. TEGADERM ® ) which is fixed by an elastic bandage (e.g. VETRAP ®
  • the skin wounds of the animals are treated four times a day by dropping 50 ⁇ l of a PBS solution or a rh-proNGF or rh-NGF-containing (3.8540 "6 M, 3.85-10 " 7 M) PBS solution. Treatment begins on day 1 after wounding and continues until day 10 after wounding. From day 11 to day 18, wounds are treated once a day. The wounds are all over . Treatment covered.
  • the wound healing process is photo-documented on days 0, 2, 4, 8, 10, 14 and 21 under standardized conditions.
  • the wound size is determined semi-automatically using appropriate morphometry software (e.g. SAMBA TITN image analyzer).
  • SAMBA TITN image analyzer e.g. SAMBA TITN image analyzer.
  • Fig. 5 The results of the morphometric evaluation are shown in Fig. 5 and Tab. 2.
  • the course of the wound healing (Fig. 5) shows that, in comparison to the vehicle control rli-proNGF, surprisingly, in both dosages, it shows a stimulatory effect on the early wound healing phase (day 2) that exceeds the effect mediated by rh-NGF.
  • day 2 On day 4 a stimulatory effect on wound healing was also observed for both rh-proNGF doses compared to the vehicle control. This effect is only surpassed by rh-NGF in the low dosage.
  • rh-proNGF shows the best wound healing stimulation in high doses.
  • the EC 25 and ECc> 5 values of wound healing were determined for both dosages rh-proNGF and rh-NGF in comparison to the vehicle control (Table 2).
  • this stimulation of the early wound healing phase is not significant compared to the vehicle control.
  • An almost complete healing of the wounds (95%) is surprisingly achieved with the high dosage of rh-proNGF after 13.56 days.

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Abstract

L'invention concerne l'utilisation de proNGF, de dérivés de proNGF, à l'exception de ceux dont la fraction pro a été complètement délétée, et/ou d'une partie fonctionnelle de proNGF dans une quantité efficace avec des excipients et/ou diluants pour la prévention et/ou le traitement des maladies des cellules de la peau, de la cornée et de la conjonctive aux fins de stockage et de stabilisation des cellules de la peau, de la cornée et de la conjonctive, et de multiplication in vitro des cellules de la peau, de la cornée et de la conjonctive.
PCT/EP2004/002899 2003-03-25 2004-03-19 UTILISATION DE proNGF NOTAMMENT POUR LA GUERISON DE PLAIES DE LA CORNEE ET DE LA PEAU WO2004084929A1 (fr)

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DE10313341A DE10313341A1 (de) 2003-03-25 2003-03-25 Verwendung von proNGF, insbesondere zur Wundheilung der Cornea und der Haut
DE10313341.0 2003-03-25

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998048002A1 (fr) * 1997-04-24 1998-10-29 Alessandro Lambiase Utilisation du facteur de croissance du tissu nerveux pour conservation, culture ou traitement de la cornee

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998048002A1 (fr) * 1997-04-24 1998-10-29 Alessandro Lambiase Utilisation du facteur de croissance du tissu nerveux pour conservation, culture ou traitement de la cornee

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
IBANEZ, CARLOS F.: "Jekyll-Hyde neurotrophins: the story of proNGF", TRENDS IN NEUROSCIENCES , 25(6), 284-286 CODEN: TNSCDR; ISSN: 0166-2236, 2002, XP002285011 *

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