US20110319316A1 - Method for Preventing and Treating Hyperpermeability - Google Patents

Method for Preventing and Treating Hyperpermeability Download PDF

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US20110319316A1
US20110319316A1 US13/254,273 US201013254273A US2011319316A1 US 20110319316 A1 US20110319316 A1 US 20110319316A1 US 201013254273 A US201013254273 A US 201013254273A US 2011319316 A1 US2011319316 A1 US 2011319316A1
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peptide
hyperpermeability
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endothelial cells
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Bernhard Fischer
Rudolf Lucas
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Apeptico Forschung und Entwicklung GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to methods for preventing and treating hyperpermeability in endothelial cells and epithelial cells.
  • Endothelial cells and epithelial cells have decisive functions in all tissues and organs of the human and animal body.
  • the endothelium consists of a thin layer of endothelial cells.
  • the layer of endothelial cells forms, among others, the inner surface of the blood vessels, like veins and capillaries, and the barrier between the blood and the outer wall of the blood vessels. Endothelial cells line the entire blood system, from the large blood vessels up to the smallest capillaries.
  • Epithelial cells form single- or multi-layer cell layers, which cover all inner and outer body surfaces of the human and animal organs. Epithelial cells are in close proximity to each other and are rich in cell contacts. For epithelial cells, a distinction can be made into an outer, apical side facing towards the outside or the lumen, and a basal side.
  • epithelial cells have an adhesion complex (junctional complex), consisting of zonula occludens (tight junction), zonula adhaerens (adhaerens junction) and desmosome (macula adhaerens), which on the one hand represents a physicochemical barrier and on the other hand interconnects adjacent epithelial cells.
  • adhesion complex junctional complex
  • the intactness, in particular of the restricting cells and cell layers, is extremely important. If, for example, there is an injury of the endothelial cells or an injury of the endothelium of the blood vessels, respectively, liquid can escape from the blood vessels and result in massive disturbances in the vitality of the entire organism.
  • An injury of the endothelium and the epithelium may cause a so-called hyperpermeability, i.e. an uncontrolled passage of liquid from blood vessels into vital organs and tissues.
  • Known toxins are, among others, listeriolysin from Listeria monocytogenes or also pneumolysin from Streptococcus pneumoniae. These toxins can result in the formation of reactive oxygen molecules in the cells. The reactive oxygen molecules caused by toxins then result in damages to endothelium and epithelium due to the fact, among others, that the barrier function of the cells is damaged.
  • the cells are interconnected via protein fibers.
  • Components of such protein fibers are e.g. the myosin light chain.
  • stresses are caused in the cells and the cell-cell connections, and intercellular gaps are formed, whereby liquid can penetrate and also leak in an uncontrolled manner.
  • protein kinase C A further component in the regulation of the barrier function of the epithelial cells and endothelial cells is protein kinase C.
  • protein kinases C several isoenzymes are known, e.g. protein kinase alpha and zeta. These protein kinase C isoenzymes are activated by reactive oxygen molecules, hydrogen peroxide, microbial toxins, like pneumolysin and listeriolysin, and hydrophilic coronavirus proteins.
  • Activated protein kinase C additionally results in a reduction of the expression of the epithelial sodium channel (ENaC), which is responsible for the sodium and liquid transport in epithelial cells, and thus, activated protein kinase C essentially contributes to the development of hyperpermeability.
  • ENaC epithelial sodium channel
  • viruses like influenza viruses, the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) or the respiratory syncytial virus, which can result in hyperpermeability of the endothelium and epithelium as well as in atypical pneumonia.
  • SARS-CoV proteins due to the activation of the protein kinase C isoform result in a reduction of the size and activity of the epithelial sodium channel, which promotes the development of hyperpermeability.
  • beta-2 adrenergic agonists show no effect.
  • microbial toxins result in an increased level of reactive oxygen molecules in endothelial and epithelial cells. This causes phosphorylation of the myosin light chain, which again results in a disturbance of the cell-cell interaction and in the development of hyperpermeability.
  • Microbial toxins, reactive oxygen molecules as well as viral proteins result in an activation of protein kinase C isoenzymes.
  • the activation of protein kinase C then results in a decrease of the expression of the epithelial sodium channel (ENaC) and the inhibition of its activity.
  • Hyperpermeability of lung tissues is an essential component of various diseases of the lungs, e.g. acute lung injury, acute respiratory distress syndrome (ARDS), pneumonia.
  • ARDS acute respiratory distress syndrome
  • ARDS acute respiratory distress syndrome
  • the object of the present invention therefore is to provide means and methods, by means of which diseases, for which the prevention of hyperpermeability of epithelial cells and endothelial cells plays an essential role in the treatment, in particular lung diseases, like acute lung injuries, ARDS or viral lung diseases, can be prevented or treated.
  • diseases for which the prevention of hyperpermeability of epithelial cells and endothelial cells plays an essential role in the treatment, in particular lung diseases, like acute lung injuries, ARDS or viral lung diseases, can be prevented or treated.
  • the invention is to provide a biologically effective molecule for the prevention and treatment of hyperpermeability of the endothelium and epithelium and for the prevention and treatment of acute lung damage and the consequences of pneumonia.
  • the present invention relates to a peptide, which consists of 7-17 adjacent amino acids and comprises the hexamer TX EX X E, wherein X, X and X can be any natural or non-natural amino acid, wherein the peptide has no TNF receptor binding activity and is cyclized, for the prevention and treatment of hyperpermeability of epithelial cells and endothelial cells.
  • the present invention relates to a peptide consisting of 7-17 adjacent amino acids and comprising the hexamer TPEGAE (SEQ ID No. 4), wherein the peptide has no TNF receptor binding activity and is cyclized, for the prevention and treatment of hyperpermeability of epithelial cells and endothelial cells.
  • QRETPEGAEAKPWY (SEQ ID No. 5) PKDTPEGAELKPWY (SEQ ID No. 6) CGQRETPEGAEAKPWYC (SEQ ID No. 1) and CGPKDTPEGAELKPWYC (SEQ ID No. 7) and fragments of at least 7 amino acids thereof, which fragments include the hexamer TPEGAE, for manufacturing of a drug for preventing and treating hyperpermeability of epithelial cells and endothelial cells.
  • the peptides according to the invention are preferably used for preventing the outbreak of or for treating pneumonia, acute lung injury, acute respiratory distress syndrome (ARDS) or bacterial or viral lung diseases, in particular infections with Listeria monocytogenes, Streptococcus pneumoniae, influenza viruses, SARS or RSV.
  • the cause of pneumonia which can be treated or prevented according to the invention, is independent of the cause of pneumonia and independent of whether it is an acute or chronic inflammation.
  • pneumonias which are caused by an infection with bacteria, viruses, mycoplasmas, protozoa, worms or fungi, can be treated, but also toxically (e.g. by inhalation of toxic substances) or immunologically caused pneumonias or such ones caused by radiation (e.g.
  • the preventive aspect of the present invention is particularly essential, however, also for bedridden persons, in particular older people, or for immunocompromised persons, like HIV patients or transplant patients.
  • the pneumonia can be fought or prevented at a time, when no damages are recognizable on the X-ray yet.
  • Pathogens of primary pneumonias are mostly pneumococci, staphylococci, Haemophilus influenzae, mycoplasmas, chlamydia, legionella ( Legionella pneumophila ) and viruses like the flu virus, adenovirus and parainfluenza viruses.
  • CMV Herpes viruses
  • fungi fungi
  • Pneumocystis jirovecii protozoa (toxoplasmosis) as well as anaerobic bacteria.
  • pneumonias caused by these pathogens are, according to the invention, particularly preferably treatable or (in particular in respect of secondary pneumonias) preventable, respectively.
  • the peptides according to the invention are for example known from the European patent EP 1 264 599 B1 and were suggested in the state of the art for the treatment of liquid accumulations (lung edema) and in particular for the re-absorption of these liquid accumulations, wherein the edema liquid is returned from the alveoli of the lung tissue into the capillaries, i.e. pumped out of the alveoli.
  • these peptides also influence the opposite liquid flow via the endothelium of the capillaries into the epithelium of the lung, however, in a contrary manner: while for the treatment of edemas, the transporting out of the liquid requires open and fully active pumping mechanisms, according to the invention, the passage of the liquid into the alveoli is stopped; the influx is thus prevented in the first place.
  • amino acids aspartic acid and glutamic acid can be preferably intramolecularly cyclized with serine, threonine, tyrosine, asparagine, glutamine, or lysine). Therefore, further preferred peptides according to the invention are, for example, CGQKETPEGAEAKPWYC (SEQ ID No. 8), CGQRETPEGAEARPWYC (SEQ ID No. 9), CGQRETPEGAEAKPC (SEQ ID No. 10), CQRETPEGAEAKPWYC (SEQ ID No. 11), or CGQRETPEGAEAKFWYC (SEQ ID No. 12).
  • cyclization comprises the intramolecular ring closure as well as the integration of a carrier (from which the bound peptide protrudes (with the N and the C terminus of the peptide being bound to the carrier)), wherein the peptide cyclized in such manner shows the cyclic three-dimensional structure and is respectively stabilized.
  • the peptides according to the invention may preferably be used for protecting epithelial cells and endothelial cells against hyperpermeability caused by reactive oxygen molecules or by bacterial toxins.
  • the peptides according to the invention may also be used for inhibiting the phosphorylation of the myosin light chain, for inhibiting the activation of protein kinase C or for increasing the expression of the epithelial sodium channel.
  • the peptides according to the invention can be used for treating hyperpermeability caused by reactive oxygen molecules, microbial toxins, gram-positive microorganisms or pulmonary virus infections.
  • the present invention relates to a pharmaceutical composition containing a peptide according to the invention (or a mixture of various peptides according to the invention) and a pharmaceutical carrier.
  • this pharmaceutical composition is used for preventing and treating hyperpermeability, as described above, in particular for preventing and treating pneumonia, acute lung injury, acute respiratory distress syndrome (ARDS) or viral lung diseases, in particular infections with Listeria monocytogenes, Streptococcus pneumoniae, SARS, RSV or influenza viruses, in particular influenza A viruses.
  • ARDS acute respiratory distress syndrome
  • influenza viruses in particular influenza A viruses.
  • a pharmaceutical composition refers to any composition comprising a peptide as defined above, which prevents, enhances or heals the conditions described herein.
  • a pharmaceutical composition refers to a composition having a peptide as described above and a pharmaceutically acceptable carrier or excipient (both terms may be used interchangeably).
  • suitable carriers or excipients known to the expert are saline solution, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline solution, substances improving isotonia and chemical stability, buffers and preservative agents.
  • Further suitable carriers include any carrier, which does not induce the production of antibodies itself, which are harmful for the individual receiving the composition, like proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers.
  • the peptide according to the invention may also be cyclized to these carriers via a direct covalent bond.
  • This pharmaceutical composition may (as a drug) be administered using any suitable method known by the expert.
  • the preferred administration path is parenteral, in particular by inhalation (with aerosols) or intravenous administration.
  • the drug of this invention is formulated in an injectable unit dosage form, like a solution, suspension or emulsion, in connection with the pharmaceutically acceptable excipient defined above. Dosage and type of administration, however, depend on the individual.
  • the drug is administered such that the peptide of the present invention is administered at a dose of between 1 ⁇ g/kg and 10 ⁇ g/kg, more preferably between 10 ⁇ g/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg.
  • it is administered as a bolus dose.
  • a continuous infusion may be used as well.
  • the drug may be infused at a dose of between 5 and 20 ⁇ g/kg/minute, more preferably between 7 and 15 ⁇ g/kg/minute.
  • a particularly preferred peptide according to the invention has the following amino acid sequence: SEQ ID No. 1 (NH2)Cys-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Cys(COOH).
  • hyperpermeability can also be induced in human epithelial cells by microbial toxins.
  • the incubation of human epithelial cells with 1 ⁇ g/ml of listeriolysin results in clear hyperpermeability.
  • the hyperpermeability is inhibited with the addition of a peptide according to the invention, in particular 50 ⁇ g/ml of peptide SEQ ID No. 1.
  • a peptide according to the invention in particular 50 ⁇ g/ml of peptide SEQ ID No. 1, inhibits the phosphorylation of the myosin light chain caused by the toxins listeriolysin and pneumolysin.
  • FIG. 1A shows the HPLC chromatogram of the protein with the amino acid sequence SEQ ID No. 1. Units: Y axis “Absorption in mAU”; X axis “Time in minutes”.
  • FIG. 1B shows the HPLC chromatogram of the protein with the amino acid sequence SEQ ID No. 2. Units: Y axis “Absorption in mAU”; X axis “Time in minutes”.
  • FIG. 1C shows the HPLC chromatogram of the protein with the amino acid sequence SEQ ID No. 3. Units: Y axis “Absorption in mAU”; X axis “Time in minutes”.
  • FIG. 2A shows the electron paramagnetic resonance (EPR) spectra of endothelial cells, which were cultivated at either 21% oxygen (normoxic gas mixture) or 0.1% oxygen (hypoxic gas mixture) with and without the addition of peptide SEQ ID No. 1 or peptide SEQ ID No. 3, respectively.
  • EPR electron paramagnetic resonance
  • FIG. 2B shows the relative content of reactive oxygen molecules (superoxide) in endothelial cells, which were cultivated at either 21% oxygen (normoxic gas mixture) or 0.1% oxygen (hypoxic gas mixture) with and without the addition of peptide SEQ ID No. 1, or at 0.1% oxygen (hypoxic gas mixture) with and without the addition of peptide SEQ ID No. 3.
  • FIG. 3A shows the course of the electric resistance of human epithelial cells of the lungs without addition of the toxin listeriolysin as well as following addition of 125 ng/ml of listeriolysin (125 ng/ml of LLO) and following addition of 500 ng/ml of listeriolysin (500 ng/ml of LLO).
  • FIG. 3B shows the course of the electric resistance of human epithelial cells of the lungs without addition of the toxin pneumolysin as well as following addition of 62.5 ng/ml of pneumolysin (62.5 ng/ml of PLY) and following addition of 250 ng/ml of pneumolysin (250 ng/ml of PLY).
  • FIG. 3C shows the course of the electric resistance of human epithelial cells of the lungs without addition of the toxin pneumolysin/peptide SEQ ID No. 1 (control) as well as following addition of 125 ng/ml of pneumolysin (125 ng/ml of PLY) as well as following addition of 125 ng/ml of pneumolysin/50 ⁇ g/ml of peptide SEQ ID No. 1 (125 ng/ml of PLY/50 ⁇ g/ml of peptide SEQ ID No. 1).
  • FIG. 3D shows the course of the electric resistance of human epithelial cells of the lungs without addition of the toxin listeriolysin/peptide SEQ ID No. 1 (control) as well as following addition of 500 ng/ml of listeriolysin (500 ng/ml of LLO) as well as following addition of 500 ng/ml of listeriolysin/50 ⁇ g/ml of peptide SEQ ID No. 1 (500 ng/ml of LLO/50 ⁇ g/ml of peptide SEQ ID No. 1).
  • FIG. 3E shows the course of the electric resistance of human epithelial cells of the lungs without addition of the toxin listeriolysin/peptide SEQ ID No. 1 (control) as well as following addition of 1 ⁇ g/ml of listeriolysin (1 ⁇ g/ml of LLO) as well as following addition of 1 ⁇ g/ml of listeriolysin/50 ⁇ g/ml of peptide SEQ ID No. 1 (1 ⁇ g/ml of LLO/50 ⁇ g/ml of peptide SEQ ID No. 1).
  • FIG. 4A shows the relative content of phosphorylated myosin light chain in human endothelial cells of the lungs depending on the concentration of the toxin listeriolysin (125 ng/ml of LLO, 250 ng/ml of LLO, 500 ng/ml of LLO).
  • FIG. 4B shows the relative content of phosphorylated myosin light chain in human endothelial cells of the lungs depending on the concentration of the toxin pneumolysin (62.5 ng/ml of PLY, 125 ng/ml of PLY, 250 ng/ml of PLY).
  • FIG. 4C shows the relative content of phosphorylated myosin light chain in human endothelial cells of the lungs depending on the addition of 50 ⁇ g/ml of peptide SEQ ID No. 1, 250 ng/ml of the toxin listeriolysin (LLO), 50 ⁇ g/ml of peptide SEQ ID No. 1/250 ng/ml of the toxin listeriolysin (LLO), 50 ⁇ g/ml of peptide SEQ ID No. 3/250 ng/ml of the toxin listeriolysin (LLO).
  • FIG. 4D shows the relative content of phosphorylated myosin light chain in human endothelial cells of the lungs depending on the addition of 50 ⁇ g/ml of peptide SEQ ID No. 1, 125 ng/ml of the toxin pneumolysin (PLY), 50 ⁇ g/ml of peptide SEQ ID No. 1/125 ng/ml of the toxin pneumolysin (PLY), 50 ⁇ g/ml of peptide SEQ ID No. 3/125 ng/ml of the toxin pneumolysin (PLY).
  • FIG. 5A shows the content of Evans blue dye in the lung tissue of mice 5.5 hours following intratracheal administration of the toxin pneumolysin with the doses 250 ng of pneumolysin per mouse (250 ng of PLY) and 500 ng of pneumolysin per mouse (500 ng of PLY).
  • FIG. 5B shows the content of Evans blue dye in the lung tissue of mice 5.5 hours following intratracheal administration of 250 ng of the toxin pneumolysin per mouse as well as following intratracheal administration of 250 ng of the toxin pneumolysin and 50 ⁇ g of peptide SEQ ID No. 1 per mouse.
  • FIG. 5C shows the content of leukocytes in the bronchoalveolar liquid in the lungs of mice 5.5 hours following intratracheal administration of 250 ng of the toxin pneumolysin per mouse as well as following intratracheal administration of 250 ng of the toxin pneumolysin and 50 ⁇ g of peptide SEQ ID No. 1 per mouse.
  • FIG. 6 states the content of activated protein kinase C alpha in relation to the overall content of protein kinase C alpha, depending on the incubation of human endothelial lung cells with 250 ng/ml of the toxin pneumolysin (250 ng/ml of PLY) and the mixture of 250 ng/ml of the toxin pneumolysin and 50 ⁇ g/ml of peptide SEQ ID No. 1 (250 ng/ml of PLY/50 ⁇ g/ml of peptide SEQ ID No. 1).
  • FIG. 7 shows the expression of the epithelial sodium channel (ENaC) in human epithelial lung cells compared to cell culture conditions without and following addition of 50 ⁇ g/ml of peptide SEQ ID No. 1 as well as following addition of 50 ⁇ g/ml of peptide SEQ ID No. 3.
  • the content of mRNS for ENaC was determined using “real-time PCR”.
  • FIG. 8 shows the change in the body weight of the test animals with viral pneumonia (group 1: negative control (PBS); group 2: positive control (influenza A via nasal); group 3: influenza A via nasal +10 ⁇ g of peptide SEQ ID No. 1 intratracheal).
  • group 1 negative control (PBS)
  • group 2 positive control (influenza A via nasal)
  • group 3 influenza A via nasal +10 ⁇ g of peptide SEQ ID No. 1 intratracheal).
  • FIG. 9 shows the change in the body temperature of test animals of these groups 1 to 3.
  • FIG. 10 shows the survival rate of test animals of these groups 1 to 3.
  • a peptide with the amino acid sequence SEQ ID No. 1 was fully automatically synthesized using Fmoc solid phase synthesis with the following steps:
  • Step Process Product 1 Coupling of the ami- Peptide bound to the no acids solid phase 2 Split-off from the Peptide in solution solid phase 3 Purification Purified peptide as TFA salt 4 Purification/salt Purified peptide as exchange acetate salt 5 Analytical examina- Purified peptide tion
  • the peptide SEQ ID No. 1 was cyclized by oxidative formation of a disulfide bridge between the side chains of the amino acids cysteine (position 1) and cysteine (position 17)
  • SEQ ID No. 2 (NH2)Lys-Ser-Pro-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly- Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu(COOH), wherein an amide bond is formed between the amino group of the side chain of lysine Lys (1) and the carboxyl group of the side chain of glutamic acid Glu (19).
  • a peptide with the amino acid sequence SEQ ID No. 2 was fully automatically synthesized using Fmoc solid phase synthesis with the following steps:
  • Step Process Product 1 Coupling of the ami- Peptide bound to the no acids solid phase 2 Split-off from the Peptide in solution solid phase 3 Purification Purified peptide as TFA salt 4 Purification/salt Purified peptide as exchange/oxidative acetate salt cyclization 5 Analytical examina- Purified peptide tion
  • the cyclization took place by the connection of the epsilon amino group of lysine (position 1) with the gamma carboxyl group of glutamic acid (position 19) forming an amide bond.
  • This is achieved, for example, by transferring the gamma carboxyl group of the glutamine group into an active ester by means of dicyclohexylcarbodiimide (DHC), which active ester subsequently spontaneously reacts with the epsilon amino group of the lysine, forming a ring closure in the peptide.
  • DHC dicyclohexylcarbodiimide
  • a peptide with the amino acid sequence SEQ ID No. 3 was fully automatically synthesized using Fmoc solid phase synthesis with the following steps:
  • Step Process Product 1 Coupling of the ami- Peptide bound to the no acids solid phase 2 Split-off from the Peptide in solution solid phase 3 Purification Purified peptide as TFA salt 4 Purification/salt Purified peptide as exchange acetate salt 5 Analytical examina- Purified peptide tion
  • the peptide SEQ ID No. 3 was cyclized by oxidative formation of a disulfide bridge between the side chains of the amino acids cysteine (position 1) and cysteine (position 17).
  • peptide SEQ ID No. 3 The difference between peptide SEQ ID No. 3 and peptide SEQ ID No. 1 consists in the fact that the amino acids Thr (6), Glu (8) and Glu (11) from peptide SEQ ID No. 1 are replaced by Ala (6), Ala (8) and Ala (11) in peptide SEQ ID No. 3.
  • the cell culture of endothelial cells took place with addition and without addition of 50 ⁇ g/ml of peptide SEQ ID No. 1 or with addition and without addition of 50 ⁇ g/ml of peptide SEQ ID No. 3, respectively.
  • arterial endothelial cells were cultivated in an oxygen-deficient gas mixture of 0.1% oxygen, 5% carbon monoxide and 94.9% nitrogen (hypoxic gas mixture).
  • the gas concentrations were 21% oxygen, 5% carbon monoxide and 74% nitrogen (normoxic gas mixture).
  • the endothelial cells were cultivated with 21% oxygen for a further 30 minutes. Thereafter, 20 ⁇ l of a solution consisting of 20 uM 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine HCl (CHM), 20 ⁇ M DPBS, 25 ⁇ M desferrioxamine and 5 ⁇ M diethyldithiocarbamate as well as 2 ⁇ l of DMSO were added to the cells.
  • CHM 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine HCl
  • the cells were individualized in a manner common in the laboratory by adding a trypsin solution.
  • the endothelial cells were washed and suspended in 35 ⁇ l of a solution consisting of DPBS and 25 ⁇ M desferrioxamine and 5 ⁇ M diethyldithiocarbamate.
  • EPR electron paramagnetic resonance
  • the previously treated cells were placed into 50 ⁇ l capillaries and examined in a MiniScope MS200 ESR of the company Magnettech (Berlin, Germany) at 40 mW microwaves, 3000 mG modulation amplitude, 100 kHz modulation frequency.
  • FIGS. 2A and 2B show, with a normal oxygen concentration of 21% (normoxic gas mixture), there only is a low formation of reactive oxygen molecules. Under oxygen deficiency (0.1% oxygen, hypoxic gas mixture), there is a 3-fold higher formation of reactive oxygen molecules. If, however, the peptide SEQ ID No. 1 is added to endothelial cells cultivated under oxygen deficiency (oxygen content 0.1%, hypoxic gas mixture), then no reactive oxygen molecules are formed by the endothelial cells.
  • peptide SEQ ID No. 3 The difference between peptide SEQ ID No. 3 and peptide SEQ ID No. 1 is that the amino acids Thr (6), Glu (8) and Glu (11) of peptide SEQ ID No. 1 are replaced with Ala (6), Ala (8) and Ala (11) in SEQ ID No. 3.
  • Human epithelial cells of the lungs of type H441 were acquired from the company ATTC.
  • LLO listeriolysin
  • PLY pneumolysin
  • Epithelial cells of the lungs of type H441 were cultivated in a manner common in the laboratory in a commercial RPMI 1640 medium with the additives 2 mM L-glutamine, 1.5 g/l of sodium carbonate, 4.5 g/l of glucose, 10 mM HEPES buffer pH 7.4, 10% bovine serum. The ECIS experiments took place in serum-free medium.
  • the human epithelial cells of the lungs as well as the human endothelial cells of the lungs were cultivated in a manner common in the laboratory up to the formation of a continuous cell layer, and subsequently, the toxins listeriolysin or pneumolysin, respectively, were added.
  • the electric resistance of the cell layer was determined by means of electrical cell-substrate impedance analysis.
  • FIG. 3A shows the electric resistance decreases with an addition of 125 ng/ml of listeriolysin to cultivated human endothelial cells. Hyperpermeability is developed. This effect is even more significant with a higher amount of 500 ng/ml of listeriolysin.
  • FIG. 3B shows that the electric resistance decreases with an addition of 62.5 ng/ml of pneumolysin to cultivated human endothelial cells. Hyperpermeability is developed. This effect is even more significant with a higher amount of 250 ng/ml of pneumolysin.
  • FIG. 3C shows, the electric resistance decreases with an addition of 125 ng/ml of pneumolysin to cultivated human endothelial cells. Hyperpermeability is developed. However, the hyperpermeability caused by the addition of the toxin pneumolysin is inhibited by addition of 50 ⁇ g/ml of peptide SEQ ID No. 1.
  • FIG. 3D shows, the electric resistance decreases with an addition of 500 ng/ml of listeriolysin to cultivated human endothelial cells. Hyperpermeability is developed. However, the hyperpermeability caused by the addition of the toxin listeriolysin is inhibited by addition of 50 ⁇ g/ml of peptide SEQ ID No. 1.
  • FIG. 3E shows, the electric resistance decreases with an addition of 1 ⁇ g/ml of listeriolysin to cultivated human epithelial cells. Hyperpermeability is developed. However, the hyperpermeability caused by the addition of the toxin listeriolysin is inhibited by addition of 50 ⁇ g/ml of peptide SEQ ID No. 1.
  • LLO listeriolysin
  • PLY pneumolysin
  • the cell contents was lysed by incubation of the cells with a solution of 20 mM tris buffer (pH 7.4), 150 mM mol/l of NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodiumpyrophosphate, 1 mM beta-glycerophosphate, 1 mM sodiumvanadate, 1 ⁇ g/ml of leupeptine, 1 mM phenylmethylsulfonylfluoride.
  • the cells were digested with ultrasound.
  • the cell lysate was centrifuged in order to obtain the soluble components.
  • the soluble cell lysate was subsequently applied to denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis in a manner common in the laboratory, and the proteins were separated according to their masses. Thereafter, the proteins were transferred onto nitrocellulose membranes.
  • the protein blots were treated with a solution of 0.1% Tween 20 and 5% dry milk powder for 1 hour in a manner common in the laboratory. Subsequently, the protein blots were incubated with antibodies directed against either the myosin light chain or the phosphorylated myosin light chain.
  • the antibodies were made visible on diagnostic film using chemiluminescence in a manner common in the laboratory. The signal strength was determined with densitometry, and the ratio of myosin light chain to phosphorylated myosin light chain was determined.
  • FIG. 4A shows, an addition of 125 ng/ml of the toxin listeriolysin to human endothelial lung cells results in an increase in the relative content of phosphorylated myosin light chain. This effect is still enhanced by a toxin concentration of 250 ng/ml of listeriolysin.
  • FIG. 4B shows, an addition of 62.5 ng/ml of the toxin pneumolysin to human endothelial lung cells results in an increase in the relative content of phosphorylated myosin light chain. This effect is still enhanced by a toxin concentration of 125 ng/ml of pneumolysin.
  • FIG. 4C shows, an addition of 125 ng/ml of the toxin listeriolysin to human endothelial lung cells results in an increase in the relative content of phosphorylated myosin light chain.
  • An addition of 50 ⁇ g/ml of peptide SEQ ID No. 1 has no influence on the content of phosphorylated myosin light chain.
  • the increase in the content of phosphorylated myosin light chain by 250 ng/ml of the toxin listeriolysin is inhibited by an addition of 50 ⁇ g/ml of peptide SEQ ID No. 1.
  • a peptide SEQ ID No. 3 has no influence on the increase in the content of phosphorylated myosin light chain mediated by the toxin listeriolysin.
  • FIG. 4D shows, an addition of 125 ng/ml of the toxin pneumolysin to human endothelial lung cells results in an increase in the relative content of phosphorylated myosin light chain.
  • An addition of 50 ⁇ g/ml of peptide SEQ ID No. 1 has no influence on the content of phosphorylated myosin light chain.
  • the increase in the content of phosphorylated myosin light chain by 125 ng/ml of the toxin pneumolysin is inhibited by an addition of 50 ⁇ g/ml of peptide SEQ ID No. 1.
  • a peptide SEQ ID No. 3 has no influence on the increase in the content of phosphorylated myosin light chain mediated by the toxin pneumolysin.
  • peptide SEQ ID No. 3 The difference between peptide SEQ ID No. 3 and peptide SEQ ID No. 1 is that the amino acids Thr (6), Glu (8) and Glu (11) of peptide SEQ ID No. 1 are replaced with Ala (6), Ala (8) and Ala (11) in SEQ ID No. 3.
  • mice were intratrachealy treated with a mixture of isoflurane/oxygen prior to preparation of the lungs, as well as with 100 ⁇ l per mouse of a mixture of ketamine/rompun (1.33:1). Following anesthesia, a venous catheter was implanted into the mice. For induction of hyperpermeability of the lungs, 25 ⁇ l of liquid were subsequently nebulized into the lungs with a fine syringe. The liquid either contained 0.9% saline solution or 250 ng of the toxin pneumolysin or 250 ng/ml of pneumolysin/50 ⁇ g/ml of peptide SEQ ID No. 1.
  • the absorptions were then determined photometrically at 620 nm and at 740 nm.
  • the Evans blue dye content in the lung tissue was determined on the basis of a reference curve for Evans blue dye dissolved in formalin solution, deducting the content of hemoglobin pigments.
  • the discharge of Evans blue dye from the capillaries into the lung tissue due to hyperpermeability induced by the toxin pneumolysin was compared to the amount of dye in the blood serum.
  • FIG. 5A shows, an intratracheal application of the toxin pneumolysin with doses of 250 ng and 500 ng per mouse results in hyperpermeability, which is determined by the fact that blood with the Evans blue dye passes from the lung capillaries into the lung tissue and can be verified in the lung tissue.
  • FIG. 5B shows, an intratracheal application of the toxin pneumolysin with a dose of 250 ng per mouse results in hyperpermeability, which is determined by the fact that blood with the Evans blue dye passes from the lung capillaries into the lung tissue and can be verified in the lung tissue.
  • the intratracheal application of 50 ⁇ g of peptide SEQ ID No. 1 there is an inhibition of the toxin-mediated development of hyperpermeability.
  • FIG. 5C shows, an intratracheal application of the toxin pneumolysin with a dose of 250 ng per mouse results in an increased number of leukocytes in the bronchoalveolar liquid of the lungs in mice due to the development of hyperpermeability.
  • the intratracheal application of 50 ⁇ g of peptide SEQ ID No. 1 there is an inhibition of the toxin-mediated development of hyperpermeability and a clear reduction in the number of leukocytes in the bronchoalveolar liquid in the lungs of mice.
  • the microbial toxin pneumolysin (PLY) was acquired from the University of Giessen.
  • Human endothelial cells of the lungs isolated from capillaries of the lungs, were cultivated in a manner common in the laboratory. During cell culture, the toxin pneumolysin was added with a concentration of 250 ng/ml, or the toxin pneumolysin with a concentration of 250 ng/ml and the peptide SEQ ID No. 1 with a concentration of 50 ⁇ g/ml.
  • the content of activated protein kinase C alpha was determined by ELISA measurement using an antibody directed against the activated protein kinase C alpha (phospho-threonine 638 protein kinase C alpha). Simultaneously, the overall content of protein kinase C alpha was determined using a commercially available ELISA assay.
  • FIG. 6 shows, due to the effect of the toxin pneumolysin, there is a strong increase in the content of activated protein kinase C alpha compared to the overall concentration of protein kinase C alpha.
  • peptide SEQ ID No. 1 there is an inhibition of the activation of protein kinase C alpha
  • Human epithelial cells of the lungs of type H441 were acquired from the company ATTC.
  • Epithelial cells of the lungs of type H441 were cultivated in a manner common in the laboratory in a commercial RPMI 1640 medium with the additives 2 mM L-glutamine, 1.5 g/l of sodium carbonate, 4.5 g/l of glucose, 10 mM HEPES buffer pH 7.4, 10% bovine serum.
  • the expression of the sodium channel was determined by means of “real-time PCR”. These examination took place in cells without and with the addition of 50 ug/ml of peptide SEQ ID No. 1, as well as following the addition of 50 ⁇ g/ml of peptide SEQ ID No. 3.
  • peptide SEQ ID No. 3 The difference between peptide SEQ ID No. 3 and peptide SEQ ID No. 1 consists in the fact that the amino acids Thr (6), Glu (8) and Glu (11) from peptide SEQ ID No. 1 are replaced by Ala (6), Ala (8) and Ala (11) in peptide SEQ ID No. 3.
  • mice In each group, 6 BALB/c mice were used.
  • test animals with viral lung infection (group 2) lost approx. 20% of their body weight within 10 days.

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US20150225460A1 (en) * 2012-06-28 2015-08-13 Apeptico Forschung Und Entwicklung Gmbh Pharmaceutical composition for treatment of the pulmonary form of altitude sickness caused by lack of oxygen and reduced air pressure
US11639368B2 (en) 2009-03-05 2023-05-02 Apeptico Forschung Und Entwicklung Gmbh Method for preventing and treating hyperpermeability

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US11161881B2 (en) * 2010-11-18 2021-11-02 Apeptico Forschung Und Entwicklung Gmbh Composition comprising a peptide and an inhibitor of viral neuraminidase
AT510585B1 (de) * 2010-11-18 2012-05-15 Apeptico Forschung & Entwicklung Gmbh Zusammensetzung umfassend ein peptid und ein hemmstoff der viralen neuraminidase
JP6335279B2 (ja) * 2013-04-23 2018-05-30 アペプティコ フオルシユング ウント アントウィクラング ゲーエムベーハー 式ｘ１−ｇｑｒｅｔｐｅｇａｅａｋｐｗｙ−ｘ２の環状ペプチドを含んでなる医薬組成物および体外肺処置のための使用
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US12004506B2 (en) 2013-04-23 2024-06-11 Apeptico Forschung Und Entwicklung Gmbh Pharmaceutical composition comprising a cyclic peptide of formula X1-GQRETPEGAEAKPWY-X2 and use for extracorporeal lung treatment
JP6644695B2 (ja) * 2014-03-04 2020-02-12 アペプティコ フオルシユング ウント アントウィクラング ゲーエムベーハー 肺内炎症の減弱化
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DK4051307T5 (da) 2020-05-08 2024-07-22 Apeptico Forschung & Entwicklung Gmbh Peptid til forebyggelse eller behandling af covid-19

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US20130072444A1 (en) * 2010-01-14 2013-03-21 APEPTICO Forschung und Entwicklung GmbH c/o mingo bueros Organic compound for the regulation of vectorial ion channels
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