PEPTIDE WITH ENDOTOXIN-NEUTRALIZING ACTIVITY

Abstract: The present invention relates to a new peptide with endotoxin-neutralizing activity which can be used for the preparation of a pharmaceutical composition for neutralizing lipopolysaccharide and particularly for the treatment of sepsis. The invention further relates to a diagnostic method for demonstrating the presence of endotoxin in blood or blood fractions, such as serum or plasma.

Description: PEPTIDE WITH E DOTOXIN-NEUTRALIZING ACTIVITY 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 . 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) . These are hereby activated and secrete various cytokines, such as tumor necrose factor-α (TNF-α) , interleukin 1 (IL-1), IL-6, IL-8, and other inflammation mediators. These compounds initiate a cascade of events, either directly or by activation of secondary mediators, which ultimately result in fever and disorders in the coagulation of the blood, vasodilation, organ failure and finally septic shock. Different treatments for sepsis have already been proposed. Thus, for the treatment of specific Gram- negative sepsis, monoclonal antibodies are for instance used against endotoxin. Tests are currently also being carried out with recombinant BPI, a product of the neutrophils with a strong lipopolysaccharide-neutralizing effect. 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. Up to the present a totally satisfactory result has not yet been achieved with the known methods . It is therefore the first object of the present invention to provide a new method of treatment and diagnosis of sepsis. Surprisingly, it has been found that 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) . 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 . In the research which led to the present invention it was further inferred that LPS is possibly involved as ambient 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) . This is a small fragment of approximately 40 amino acids which is released by enzymes from the transmembrane beta-amyloid precursor protein (beta-APP) . The processing of this precursor protein can take place in a number of 3 ways and then results in a normally occurring soluble fragment or, under certain conditions via alternative proteases, an intact beta-fragment . The production of 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. However, the deposit of amyloid beta-protein is the primary event causing Alzheimer's disease. Other proteins are also associated with the amyloid deposits, including SAP and serum amyloid A (SAA) . According to the invention it has now been found that both 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. According to the present invention it is now proposed that 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. The hypothesis now is that (chronic) bacterial infections, and particularly LPS as ambient factor, contribute to the development of Alzheimer's disease. It is in fact sus- pected that the basis for Alzheimer's is the alternative splitting of the beta-amyloid precursor protein, which possibly takes place outside the brain in the circulation. There are indications that 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) . 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. 4 It is further suspected that 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 . This influence can relate to the initiation as well as the further progression of the disease. According to a further aspect 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 μg and 1 mg per kg body weight, preferably between 0.1 μg and 100 μg per kg body weight. In addition to use in a pharmaceutical composition, the peptide can also be used for diagnosis of infection with Gram-negative bacteria or sepsis. For this purpose 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- beads™. 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. Reference will be made in the examples to the following accompanying figures : Figure 1 shows the effect of the peptide on 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 ( "pepl86-200) in the presence of LPS. In the graph the peptide concentration is plotted against the percentage 6 of inhibition, shown as the total luminescence response in 10 minutes (10' AUC) . Figure 3 shows the efficiency with which 30 μM 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) EXAMPLES EXAMPLE 1 Inhibition of endotoxin- induced monocyte activation by the peptide according to the invention 1.1 Materials and method 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. After extensive desalting and dialysis against PBS, 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. After washing the cells are inserted in isotonic HEPES buffer (with 1 mM CaCl 2 and 2% BSA) . 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 μl 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 FACScan™ Flow cytometer. Forward and Sideward Scatter parameters are used to identify the monocyte population. With LysisII™ software (Beckton & Dickinson) the average fluorescence value of 5000 monocytes is calculated. See also Weersing A.J.L. et al . , J. Immunol. 145, 318-324 (1990)). 7 2 . 2 Result s 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 μM still shows 30% inhibition of the FITC-LPS binding. EXAMPLE 2 Inhibition of endotoxin- induced neutrophil activation by the peptide 1. Materials and method Immediately after being taken, full blood (de- coagulated with EDTA) is incubated with lng/ml ReLPS and a sample of peptide in a total volume of 250 μl for 30 min. at 37°C. The samples are then diluted lOx with PBS/glucose (5%) . Of this 100 μl is placed in a luminometer (Berthold; Autolumat LB953), whereafter via automatic injection luminol (180 μM) and 1 μM fMLP (formylmethyonyl-leucyl-phenylalanine) is added as stimulus to each tube. The luminescence response is measured continuously for 10 minutes and the results expressed as total luminescence response ("area under the curve" = AUC) . 2. Results 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 μM. What is exceptional about this finding is that the inhibition of LPS takes place in an ex vivo, non- manipulated full blood environment which most closely approximates the in vivo situation. Figure 3 shows the efficiency of 30 μM peptide in inhibiting different concentrations of LPS in the full blood priming system. There is still 50% inhibition of 10 ng/ml LPS . 8 EXAMPLE 3 Protection against endotoxin- induced mortality in mice by peptide (186-200) 1. Materials and method. In vivo protection experiments are performed in an acutely endotoxaemia mouse model with ReLPS . The peptide preparation is first incubated with ReLPS in an HBSS buffer with 2% human albumin for 30 min. at 37°C. This mixture was subsequently administered intravenously together with actinomycin D (25 μg per mouse) to 8 week- old BALB/c mice. Survival was followed for 5 days. 2. Results The survival of 2 groups of 8 mice injected with 3 pg ReLPS with and without peptide is shown in figure 4. The mice injected only with peptide (therefore without LPS) all survived, and thus displayed no acute adverse effects from the peptide (not shown) . The 8 mice injected with LPS plus buffer are all dead within 12 hours (—♦) . However, of the group of 8 mice injected with LPS plus peptide (30 μM or 5 mg/kg) 50% is still alive after 12 hours and displays as a whole group a clearly slowed mortality. (--■) .

Claims: 9 CLAIMS 1. Peptides with endotoxin-neutralizing activity, which peptide consists of the amino acids: QALNYEIRGYVIIKP. 2. Peptide as claimed in claim 1, for use in neutralizing lipopolysaccharides. 3. Use of the peptide as claimed in claim 1 for the preparation of a pharmaceutical composition for neutralizing lipopolysaccharide. 4. Use of the peptide as claimed in claim 1 for the preparation of a pharmaceutical composition for the treatment of sepsis. 5. Diagnostic method for demonstrating the presence of endotoxin in blood or blood fractions, such as serum or plasma, comprising of bringing a carrier with the peptide as claimed in claim 1 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. 6. Diagnostic kit for performing the method as claimed in claim 5, comprising a carrier having bound thereto the peptide as claimed in claim 1 and means for visualizing and/or quantifying of binding between endotoxin and the peptide. 7. Use of the peptide as claimed in claim 1 for the preparation of a pharmaceutical composition for the therapeutic and/or preventive treatment of Alzheimer's disease .

Inventors: VAN KESSEL CORNELIS PETRUS MAR, VAN STRIJP JOHANNES ANTONIUS G, VAN KESSEL, CORNELIS, PETRUS, MARIA, VAN STRIJP, JOHANNES, ANTONIUS, GERARDUS
Applicants: KESSEL CORNELIS PETRUS MARIA V, STRIJP JOHANNES ANTONIUS GERAR, UNIV UTRECHT, UNIVERSITEIT UTRECHT, VAN KESSEL, CORNELIS, PETRUS, MARIA, VAN STRIJP, JOHANNES, ANTONIUS, GERARDUS
Application year: 1999
Main classification: C07K14
IPC codes: A61K38, C07K14