EP0932624A1 - Pharmaceutical and diagnostic use of serum amyloid p component - Google Patents
Pharmaceutical and diagnostic use of serum amyloid p componentInfo
- Publication number
- EP0932624A1 EP0932624A1 EP97944213A EP97944213A EP0932624A1 EP 0932624 A1 EP0932624 A1 EP 0932624A1 EP 97944213 A EP97944213 A EP 97944213A EP 97944213 A EP97944213 A EP 97944213A EP 0932624 A1 EP0932624 A1 EP 0932624A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sap
- endotoxin
- fragments
- binding
- phe
- 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- Withdrawn
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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
Definitions
- Infection with Gram-negative bacteria can result in a life-threatening disease which is initiated by specific binding of LPS to phagocytes, such as monocytes, macrophages and granulocytes (neutrophils) .
- phagocytes such as monocytes, macrophages and granulocytes (neutrophils) .
- phagocytes such as monocytes, macrophages and granulocytes (neutrophils) .
- cytokines such as tumor necrose factor- ⁇ (TNF- ⁇ ) , interleukin 1 (IL-1), IL-6, IL-8, and other inflammation mediators.
- TNF- ⁇ tumor necrose factor- ⁇
- IL-1 interleukin 1
- IL-6 interleukin 6
- IL-8 interleukin-8
- soluble TNF tumor necrose factor
- interleukin-1 receptor soluble TNF (tumor necrose factor) receptor or for the interleukin-1 receptor.
- SAP Serum Amyloid P component
- the present invention therefore relates in a first aspect to the use of Serum Amyloid P component
- SAP for the preparation of a pharmaceutical composition for neutralizing lipopolysaccharide (s) in general and the treatment of sepsis in particular.
- new peptides are therefore provided for use in neutralizing lipopolysaccharides, which peptides consist of a part of the amino acids 27-39 of SAP in the sequence occurring in SAP.
- Preferred peptides are the PEP 27-39, which consists of the amino acids 27-39, PEP 33-38 (amino acids 33-38) , PEP 32-39 (amino acids 32-39) , PEP 30-37 (amino acids 30-37) and PEP 29-36 (amino acids 29- 36) .
- Table 1 shows the amino acid composition of the different peptides
- SAP fragments are manufactured more simply owing to their smaller dimensions and can penetrate more easily into bodily tissues.
- LPS is possibly involved as environmental factor in the development of Alzheimer's disease.
- 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) .
- SAP and serum amyloid A Other proteins are also associated with the amyloid deposits, including SAP and serum amyloid A (SAA) .
- SAP and SAA can bind to LPS and are capable of neutralizing the biological activity of LPS.
- 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 the occurrence of deposits is stimulated.
- (chronic) bacterial infections, and particularly LPS as environmental factor contribute to the development of Alzheimer's disease.
- soluble amyloid beta-protein in plasma is associated with lipoproteins, in particular the VHDL and HDL3 fractions, in which it is complexed with the apolipo protein J (ApoJ) .
- ApoJ apolipo protein J
- the ApoJ/ amyloid beta-protein complex is capable of passing through the blood-brain barrier. In this manner the amyloid beta-protein enters the brain, where it is deposited.
- SAA is likewise an apolipo protein which is associated with the HDL fraction and particularly with the HDL3 subfraction.
- 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.
- SAP itself and fragments derived from SAP (peptides) with a strong LPS-binding and neutralizing action 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 SAP and/or SAP fragments thereof for the manufacture of a pharmaceutical composition for the therapeutic and preventive treatment of Alzheimer's disease.
- compositions which according to the invention contain SAP and/or one or more SAP fragments (peptides) 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.) SAP and/or one or more SAP fragments with pharmaceutically acceptable excipients suitable for intravenous administration.
- 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 between 0.01 ⁇ g and 1 mg per kg body weight, preferably between 0.1 ⁇ g and 100 ⁇ g per kg body weight .
- SAP and/or the SAP fragments 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 SAP and/or endotoxin-binding SAP fragments bound thereto into contact with a blood sample for testing in order to enable binding of endotoxin to SAP ( -fragments) , removing non-bound material and visualizing and/or quantifying the binding between endotoxin and SAP ( -fragments) .
- 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 SAP or SAP fragments can be detected in different ways. Use can thus be made of a labelled antibody against endotoxin.
- the terms 'SAP fragment ⁇ )', 'peptide(s)' and 'endotoxin-binding peptide(s)' are used interchangeably.
- Figure 1A shows an SDS-PAGE gel of LPS-Beads which are incubated with serum pre-incubated with free LPS.
- lane 1 markers (respectively from top to bottom 92, 66, 45, 31 and 21 kDa)
- lane 2 without pre-incubation with free LPS
- lane 3 pre-incubation with 100 ⁇ g/ml free LPS
- lane 4 pre-incubation with 10 ⁇ g/ml free LPS lane 5 pre-incubation with 1 ⁇ g/ml free LPS.
- Figure IB shows an SDS-PAGE gel of LPS-Beads incubated with serum (lane 1, negative control) , commercially obtained SAP (lane 2) and markers (lane 3, molecular weights respectively from top to bottom 92, 66,
- Figure 2 shows the effect of SAP on LPS-binding to monocytes. Plotted on the X-axis is the concentration of SAP. The Y-axis shows the average fluorescence which represents the ReLPS-binding.
- Figure 3 shows the inhibition of the LPS priming of neutrophils by SAP in the presence and absence of LPS. In the graph the luminescence counts are plotted against the measurement time (in minutes) .
- Figure 4 shows the effect of peptides (SAP fragments) according to the invention on the binding of LPS to monocytes. Plotted on the X-axis is the concentration of the peptides. The Y-axis shows the average fluorescence, which represents the ReLPS-binding.
- a 1 mg/ml solution of LPS of Salmonella Re595 (Sig- ma; L9764) in carbonate buffer (0.1 M, pH 9.5) is incubated for 24 hours with 5xl0 7 DynaBeadsTM (M450, Dynal A.S., Oslo, Norway) activated with Tosyl .
- the beads loaded with LPS are washed 3x and stored in HBSS+0.1% Na-azide (5xl0 7 b/ml) .
- a 10% dilution (1 to 10 dilution) of serum of healthy volunteers is mixed with 5xl0 6 LPS-Beads for 30 minutes at 22 °C.
- the serum has optionally been incubated beforehand with different concentrations of free LPS.
- the beads are washed 3x with HBSS (Gib- co BRL, Gaithersburg, MD, US) and subsequently resuspended in 25 ⁇ l SDS-PAGE sample buffer (with 2% SDS and 2.5% DTT) . After boiling for 3 minutes at 100°C the sample is analyzed on a 12.5% SDS-PAGE (Mini-Proteanll; Bio-Rad) . The proteins present are stained with Coomassie Blue. A sample with Marker proteins of known sizes (Bio-Rad) serves as refer- ence . For identification the proteins are transferred after SDS-PAGE to nitrocellulose paper by means of a Mini Trans-BlotterTM.
- SAP is isolated from normal human serum by making use of the Ca-dependent binding to Sepharose .
- delipidated serum which is manufactured by centri- fuging serum for 4 minutes at 16,000 rpm, two precipitation steps are first performed (successively with BaCl 2 and NH 4 S0 4 ) followed by anion exchange chromatography on a Mono-Q column (Pharmacia) .
- Figure 1A shows an example of a SDS-PAGE gel of LPS- beads incubated with serum.
- serum was first incubated with different concentrations (100, 10, 1 and 0 ⁇ g/ml) of free ReLPS (L9764, Sigma Chemicals, St. Louis. MO, US) before the mixture was incubated with the LPS-Beads.
- L9764 free ReLPS
- LPS of Salmonella Re595 (ReLPS, supra) is labelled with FITC (Sigma; F7250) under conditions in which LPS is present as monomer, resulting in a FITC-LPS preparation with a ratio of 1 molecule FITC per molecule LPS.
- FITC-LPS preparation with a ratio of 1 molecule FITC per molecule LPS.
- the stock solution of FITC-LPS is stored at -20°C.
- Mononuclear leukocytes (monocytes and lymphocytes) are isolated from heparin blood of healthy volunteers via a Ficoll (Pharmacia) gradient in accordance with a standard method.
- Figure 2 shows the concentration-dependent inhibition of FITC-LPS binding to monocytes in vitro. This shows that the more SAP is added in the assay, the less LPS associates itself with the monocytes. Since association of LPS with the monocyte is the first step in the activation of the monocyte, this is a very strong indication that SAP will also inhibit the monocyte activation in vivo at a very early stage.
- Neutrophils are isolated from heparin blood of healthy volunteers via a Histopaque-Ficoll gradient in accordance with a standard method (Van Amers- foort, E.S. & J.A.G. van Strijp, Cytometry 17, 294- 301 (1994) .
- the remaining erythrocytes in the neu- trophil fraction are lysed with sterile water (for 30 seconds) and, after reinstatement of the isotonicity, the fraction is washed.
- the cells are finally inserted (suspended) in HBSS+2% Human Serum Albumin (HSA) .
- HSA Human Serum Albumin
- Cells (lxlO 4 ) are incubated with 1 ng/ml ReLPS, 30 ng/ml recombinant LPS-binding protein (r-LBP) and a sample SAP in a total volume of 100 ⁇ l for 30 minutes at 37°C.
- the samples are subsequently placed in a luminometer (Berthold; Autolumat LB953) , whereafter via automatic injection luminol (180 ⁇ M) and 1 ⁇ M fMLP (formylmethyonyl- leucyl-phenylalanine) are added as stimulus to each tube.
- the luminescence response is measured continuously for 15 minutes and the results expressed as total luminescence response (area under the graph) . See also Weersink et al . , Immunology 83, 617-623 (1994) .
- Figure 3 shows the inhibition of the LPS "priming" of neutrophils by SAP.
- the luminescence counts are plotted against the measurement time (in minutes) . This shows that the activation of neutrophils by LPS is greatly reduced by SAP. This is a strong indication that the toxicity of LPS can also be reduced in vivo by SAP . i i ' 15
Abstract
Description
Claims
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NL1004258A NL1004258C2 (en) | 1996-10-11 | 1996-10-11 | New peptide(s) from serum amyloid P for neutralising lipo-polysaccharide |
NL1004258 | 1996-10-11 | ||
NL1006583 | 1997-07-14 | ||
NL1006583 | 1997-07-14 | ||
PCT/NL1997/000567 WO1998016556A1 (en) | 1996-10-11 | 1997-10-10 | Pharmaceutical and diagnostic use of serum amyloid p component |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0932624A1 true EP0932624A1 (en) | 1999-08-04 |
Family
ID=26642453
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97944213A Withdrawn EP0932624A1 (en) | 1996-10-11 | 1997-10-10 | Pharmaceutical and diagnostic use of serum amyloid p component |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0932624A1 (en) |
AU (1) | AU4575897A (en) |
WO (1) | WO1998016556A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6620414B2 (en) | 1992-03-27 | 2003-09-16 | Smithkline Beecham Biologicals (S.A.) | Hepatitis vaccines containing 3-0-deacylated monophoshoryl lipid A |
EP0915707B1 (en) * | 1996-01-25 | 2002-10-30 | Profylakse ApS | Pharmaceutical composition comprising serum amyloid p component for prophylactic or therapeutic treatment of virus infections and a kit for detecting binding of compositions to virus components |
US6365570B1 (en) * | 1997-10-10 | 2002-04-02 | Universiteit Utrecht | Pharmaceutical and diagnostic use of Serum Amyloid P component |
NL1009045C2 (en) * | 1998-04-29 | 1999-11-01 | Univ Utrecht | Peptide with endotoxin neutralizing activity. |
CN112742067B (en) * | 2020-12-14 | 2022-07-05 | 广州博仁安康医疗科技有限公司 | Method for removing endotoxin from fetal calf serum |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5092876A (en) * | 1989-08-30 | 1992-03-03 | The United States Of America As Represented By The Department Of Health And Human Services | Cell attachment peptides derived from amyloid P component |
US5405832A (en) * | 1991-11-27 | 1995-04-11 | Immtech International Inc. | Method of treating non-streptococcal bacterial infections |
GB9317120D0 (en) * | 1993-08-17 | 1993-09-29 | Royal Postgrad Med School | Human serum amyloid p component |
EP0915707B1 (en) * | 1996-01-25 | 2002-10-30 | Profylakse ApS | Pharmaceutical composition comprising serum amyloid p component for prophylactic or therapeutic treatment of virus infections and a kit for detecting binding of compositions to virus components |
-
1997
- 1997-10-10 WO PCT/NL1997/000567 patent/WO1998016556A1/en not_active Application Discontinuation
- 1997-10-10 AU AU45758/97A patent/AU4575897A/en not_active Abandoned
- 1997-10-10 EP EP97944213A patent/EP0932624A1/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO9816556A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1998016556A1 (en) | 1998-04-23 |
AU4575897A (en) | 1998-05-11 |
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