EP1073678A1 - Peptide possedant une activite de neutralisation de l'endotoxine - Google Patents

Peptide possedant une activite de neutralisation de l'endotoxine

Info

Publication number
EP1073678A1
EP1073678A1 EP99916076A EP99916076A EP1073678A1 EP 1073678 A1 EP1073678 A1 EP 1073678A1 EP 99916076 A EP99916076 A EP 99916076A EP 99916076 A EP99916076 A EP 99916076A EP 1073678 A1 EP1073678 A1 EP 1073678A1
Authority
EP
European Patent Office
Prior art keywords
peptide
endotoxin
lps
binding
blood
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99916076A
Other languages
German (de)
English (en)
Inventor
Cornelis Petrus Maria Van Kessel
Johannes Antonius Gerardus Van Strijp
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rijksuniversiteit Utrecht
Original Assignee
Rijksuniversiteit Utrecht
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rijksuniversiteit Utrecht filed Critical Rijksuniversiteit Utrecht
Publication of EP1073678A1 publication Critical patent/EP1073678A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a new peptide with endotoxin-neutralizing activity and to a new method of treating sepsis.
  • the invention further provides possibilities for therapeutic and preventive treatment of Alzheimer's disease.
  • Sepsis very generally comprises the clinical pictures which result from the presence of bacteria multiplying in blood.
  • the direct cause of the symptoms are toxic substances which are released by the bacteria or released during lysis thereof.
  • Gram-negative bacteria for instance produce lipopolysaccharides (LPS) as a component of their cell wall .
  • LPS lipopolysaccharides
  • These lipopolysaccharides are toxic in many circumstances. In principle they are bound to the cell and are only released when the cell lyses. Lipopolysaccharides are also referred to as endotoxins .
  • Infection with Gram-negative bacteria can result in a life-threatening disease which is initiated by specific binding of LPS to phagocytes, such as onocytes, macrophages and granulocytes (neutrophils) .
  • phagocytes such as onocytes, macrophages and granulocytes (neutrophils) .
  • TNF- ⁇ tumor necrose factor- ⁇
  • IL-1 interleukin 1
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • cytokines used for treatment of general sepsis are antibodies aimed against cytokines or antagonists for the 2 soluble TNF (tumor necrose factor) receptor or for the interleukin-1 receptor.
  • the first object of the present invention to provide a new method of treatment and diagnosis of sepsis.
  • a peptide with the amino acid sequence QALNYEIRGYV IKP is capable of binding to endotoxin.
  • This peptide which will be further referred to as "the peptide” corresponds with the amino acids 186-200 of the per se known protein Serum Amyloid P component (SAP) .
  • SAP Serum Amyloid P component
  • the peptide prevents binding of the circulating LPS to the phagocytes, and thereby activation thereof. In this way the peptide is able to neutralize the biological activity of endotoxin.
  • the present invention relates not only to the peptide itself but also to the use of the peptide for the preparation of a pharmaceutical composition for neutralizing lipopolysaccharide (s) in general and the treatment of sepsis in particular.
  • the peptide is very successful in inhibiting the binding of LPS to monocytes .
  • Alzheimer's disease coincides with particular forms of cancer, rheumatoid arthritis, diabetes and Down's syndrome under the denominator ' amyloidosis ' .
  • This is a collection of diseases which are characterized by extracellular deposits of normal or mutated proteins.
  • the amyloid deposits in Alzheimer's are ordered in a characteristic three-dimensional pattern of so-called "beta-pleated sheets".
  • the subunit protein component consists of the amyloid beta-protein (A beta- P) . This is a small fragment of approximately 40 amino acids which is released by enzymes from the transmembrane beta-amyloid precursor protein (beta-APP) .
  • amyloid beta-protein is therefore in itself a normal physiological event and the existence of A beta-P can be demonstrated in the cerebral fluid (CFS) of healthy humans.
  • CFS cerebral fluid
  • amyloid beta-protein is the primary event causing Alzheimer's disease.
  • SAP and serum amyloid A proteins are also associated with the amyloid deposits, including SAP and serum amyloid A
  • SAP SAP and SAA can bind to LPS and are capable of neutralizing the biological activity of LPS. As such these two amyloid associated plasma proteins have no structural affinity.
  • LPS enters into a binding with the serum amyloid proteins SAP and SAA, whereby the role of SAP and SAA in the initiation of amyloid deposits is influenced. It is suspected that through binding of LPS to SAP and SAA
  • LPS also plays a part in the development of Alzheimer's disease via an indirect route.
  • Cytokines such as interleukin 1 (IL-1) and interleukin 6 (IL-6), lead to over-expression of beta-amyloid precursor protein in the vessel wall and in microglia and astrocytes in the brain. This points to a role for the acute-phase response in the development of Alzheimer's disease.
  • LPS is a potent initiator of the acute-phase response and also initiates the production of IL-1 and IL-6. Because both cytokines result in more beta-amyloid precursor protein, an indirect role of LPS is assumed.
  • the peptide according to the invention derived from SAP and having a strong LPS-binding and neutralizing activity can therefore be of importance in eliminating the part played by LPS in the development of Alzheimer's disease .
  • the present invention therefore provides the use of the peptide for the preparation of a pharmaceutical composition for the therapeutic and preventive treatment of Alzheimer's disease .
  • the pharmaceutical compositions which according to the invention contain the peptide as active ingredient, will be particularly intended for parenteral, and then particularly intravenous use.
  • the pharmaceutical compositions can be prepared by combining (i.e. mixing, dissolving etc.) the peptide with pharmaceutically acceptable excipients suitable for intravenous administration.
  • the concentration of the active ingredient in a pharmaceutical composition can vary between 0.001% and 100%, depending on the nature of the treatment and the manner of administration.
  • the dose of the active ingredient to be administered likewise depends on the administering route and application but can for instance vary 5 between 0.01 ⁇ g and 1 mg per kg body weight, preferably between 0.1 ⁇ g and 100 ⁇ g per kg body weight.
  • the peptide can also be used for diagnosis of infection with Gram-negative bacteria or sepsis.
  • the invention provides a diagnostic method for demonstrating the presence of endotoxin in blood or blood fractions, such as serum or plasma, comprising of bringing a carrier with peptide bound thereto into contact with a blood sample for testing in order to enable binding of endotoxin to the peptide, removing non- bound material and visualizing and/or quantifying the binding between endotoxin and the peptide.
  • the invention further provides a diagnostic kit for performing the method, comprising a carrier having peptide bound thereto and means for visualizing and/or quantifying binding between endotoxin and the peptide.
  • the carrier can take different forms, such as a microtitre plate, a column, a membrane or beads. These latter can for instance be magnetic beads, such as Dyna- beadsTM.
  • Binding of endotoxin to the peptide can be detected in different ways. Use can thus be made of a labelled antibody against endotoxin.
  • the invention will be further illustrated in the examples following hereinbelow, which are not intended to limit the invention in any way whatever.
  • LPS-binding to monocytes Plotted on the X-axis is the concentration of the peptide.
  • the Y-axis shows the average fluorescence which represents the ReLPS-binding.
  • Figure 2 shows the inhibition of the LPS priming of neutrophils in full blood by the peptide
  • Figure 3 shows the efficiency with which 30 ⁇ M peptide inhibits different concentrations of LPS.
  • Figure 4 shows the mortality in mice under the influence of LPS in the presence of the peptide (peptide + LPS) compared to a control (buffer + LPS)
  • Preparations with the peptide are mixed with FITC-LPS and subsequently added to 3xl0 5 mononuclear cells and 30 ng/ml recombinant LBP (obtained from Dr. Lichtenstein) in a total volume of 50 ⁇ l and a FITC-LPS concentration of 2.5 ng/ml. After an incubation period of 30 minutes at 37°C, the samples are stored on ice. Analysis of monocyte-associated FITC-LPS binding takes place with FACScanTM Flow cytometer. Forward and Sideward Scatter parameters are used to identify the monocyte population.
  • Figure 1 shows the concentration-dependent inhibition of FITC-LPS binding to monocytes in vitro. This shows that the peptide can inhibit the binding of endotoxin to monocytes extremely efficiently. Even the lowest tested concentration of 0.01 ⁇ M still shows 30% inhibition of the FITC-LPS binding.
  • Figure 2 shows the inhibition of the LPS priming of neutrophils by the peptide in a completely full blood system.
  • the inhibition of the priming is peptide concentration-dependent and is maximal at 10 ⁇ M.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Neurology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un nouveau peptide possédant une activité de neutralisation de l'endotoxine, pouvant être utilisé pour la préparation d'une composition pharmaceutique pour la neutralisation de lipopolysaccharide, et notamment pour le traitement de la septicémie. L'invention se rapporte aussi à une méthode diagnostique permettant de rechercher la présence d'endotoxine dans le sang ou dans des fractions du sang, telles que le sérum ou le plasma.
EP99916076A 1998-04-29 1999-04-21 Peptide possedant une activite de neutralisation de l'endotoxine Withdrawn EP1073678A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
NL1009045 1998-04-29
NL1009045A NL1009045C2 (nl) 1998-04-29 1998-04-29 Peptide met endotoxine-neutraliserende activiteit.
PCT/NL1999/000228 WO1999055731A1 (fr) 1998-04-29 1999-04-21 Peptide possedant une activite de neutralisation de l'endotoxine

Publications (1)

Publication Number Publication Date
EP1073678A1 true EP1073678A1 (fr) 2001-02-07

Family

ID=19767059

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99916076A Withdrawn EP1073678A1 (fr) 1998-04-29 1999-04-21 Peptide possedant une activite de neutralisation de l'endotoxine

Country Status (4)

Country Link
EP (1) EP1073678A1 (fr)
AU (1) AU3445299A (fr)
NL (1) NL1009045C2 (fr)
WO (1) WO1999055731A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3668534A1 (fr) * 2017-08-16 2020-06-24 Stellenbosch University Protéine de liaison de lipopolysaccharides destinée à être utilisée dans une méthode de traitement de la maladie d'alzheimer

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5405832A (en) * 1991-11-27 1995-04-11 Immtech International Inc. Method of treating non-streptococcal bacterial infections
WO1998016556A1 (fr) * 1996-10-11 1998-04-23 Universiteit Utrecht Utilisation pharmaceutique et diagnostique d'un composant amyloide p serique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9955731A1 *

Also Published As

Publication number Publication date
AU3445299A (en) 1999-11-16
NL1009045C2 (nl) 1999-11-01
WO1999055731A1 (fr) 1999-11-04

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