NL1004258C2 - New peptide(s) from serum amyloid P for neutralising lipo-polysaccharide - Google Patents

Abstract

New peptides (I), i.e. serum amyloid P component (SAP) fragments, for use in neutralising lipopolysaccharide (LPS), contain at least some of amino acids 27-39 of the SAP sequence. Five (I) are claimed, i.e. EKPLQNFTLCFRA (the 27-39 segment of SAP; Ia); FTLCFR; NFTLCFRA; LQNFTLCF and PLQNFTLC. PROCESS - For diagnosis, (I) or SAP is immobilised on a carrier and this incubated with a blood sample. The carrier is washed and the binding between LPS and (I) detected and/or quantified, e.g. using a labelled antibody against LPS.

Description

PHARMACEUTICAL EM DIAGNOSTIC USE OF SERY AMYLOID P COMPONENT

The present invention relates to a new way of treating sepsis.

Sepsis generally includes the syndromes caused by the presence of multiplying bacteria in blood. The direct cause of the symptoms are toxic substances that are released by the bacteria or released during lysis. Gram negative bacteria, for example, produce lipopolysaccharides (LPS) as part of their cell wall. These lipopolysaccharides are toxic under many circumstances. In principle, they are bound to the cell and are not released until the cell lyses. Lipopolysaccharides are also known as endotoxins.

Infection with Gram-negative bacteria can lead to a life-threatening disease, which is initiated by specific binding of LPS to phagocytes, such as monocytes, macrophages and granulocytes (neutrophils). These are activated by this and secrete several cytokines, such as tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), IL-6, IL-8, and other inflammatory mediators. These compounds initiate a cascade of events, either directly or by activation of secondary mediators, which ultimately result in fever and abnormalities in blood clotting, vasodilation, organ failure and ultimately septic shock.

Several treatments for 30 sepsis have already been proposed. For example, monoclonal antibodies to endotoxin are used for the treatment of specific Gram negative sepsis. Trials are also currently being conducted with recombinant BPI, a product of the neutrophils with a strong lipopolysaccharidal de-neutralizing effect.

Antibodies directed against cytokines are used for treatment of general sepsis or antago 1004258 for the soluble TNF (tumor necrosis factor) receptor or for the interleukin-1 receptor.

Until now, no completely satisfactory result has been obtained with the known methods.

The aim of the present invention is to provide a new method of treatment and diagnosis of sepsis.

It

It has surprisingly been found that the protein Serum Amyloid P component (SAP) known per se is capable of binding to endotoxin. This prevents binding of the circulating SAP to the phagocytes and thus their activation. In this way, SAP is able to neutralize the biological action of endotoxin.

The present invention therefore relates to the use of Serum Amyloid P component (SAP) for the manufacture of a pharmaceutical composition for neutralizing lipopolysaccharide in general and the treatment of sepsis in particular.

20 SAP is a member of the pentraxin family. Pentraxins are proteins with a characteristic pentameric organization of identical subunits arranged as single or double annular disks. Another member of this family is the C-reactive protein 25 (CRP). CRP and SAP are both so-called "acute phase reactants" (APR), ie they are involved in the early phase of an inflammatory process. In general, however, only one appears to work as APR per species. In humans, the concentration of 30 SAP in normal human plasma is approximately 30 Mg / ml. During inflammatory reactions, this level remains approximately the same, while the CRP level can be increased a thousandfold to 1 mg / ml, depending on the disease and its severity.

It is particularly surprising to mention that a compound, which does not play a role as APR during inflammatory reactions, is capable of neutralizing sepsis-causing endotoxin. It would be expected that the body itself would utilize this neutralizing ability by increasing the plasma level of this compound in the case of infection with Gram negative bacteria and the resulting inflammatory reactions.

5 SAP is a relatively large protein. It is known for certain ligands which of the amino acids of SAP are involved in binding, such as, for example, for CRP, C4-t binding protein (C4bp), Clq and Calcium ions. Similarly, for endotoxin, it has been determined that certain regions of the SAP molecule are involved in the binding. It may therefore be preferable to use only the specific binding parts of the protein for the manufacture of a pharmaceutical preparation.

The pharmaceutical compositions which according to the invention contain SAP as an active ingredient will in particular be intended for parenteral, and in particular intravenous use. The pharmaceutical compositions can be prepared by combining (ie mixing, dissolving, etc.) SAP with intra-venous administration suitable pharmaceutically acceptable excipients. 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 mode of administration. The dose of the active ingredient to be administered also depends on the route of administration and application, but may vary, for example, between 0.01 Mg and 1 mg per kg body weight, preferably between 0.1 μς and 100 μq per kg body weight.

In addition to use in a pharmaceutical formulation, SAP can also be used for diagnosis of infection with Gram negative bacteria or sepsis. To this end, the invention provides a diagnostic method for detecting the presence of endotoxin in blood or blood fractions, such as serum or plasma, comprising contacting a carrier with bound SAP and / or endotoxin-binding fragments with a blood sample to be tested in order to allow binding of endotoxin to SAP (fragments), removing unbound material and visualizing and / or quantifying the binding between endotoxin and SAP (fragments).

Furthermore, the invention provides a diagnostic kit for carrying out the method, comprising a carrier with bound SAP and / or endotoxin-binding fragments thereof, and means for visualizing and / or quantifying binding between endotoxin and SAP (fragments).

The support can take various forms, such as a microtiter plate, a column, a membrane or granules. The latter can be, for example, magnetic beads, such as Dynabeads ™.

Binding of endotoxin to SAP can be detected in various ways. For example, a labeled antibody to endotoxin can be used.

Binding of SAP to endotoxin was found by "fishing" in plasma with magnetic beads with endotoxin bound thereto. The neutralizing activity was tested on monocytes, neutrophils and in mice. Details of these tests are described in the following examples, which are for illustrative purposes only and are not intended to limit the invention in any way.

EXAMPLE 1 EXAMPLE 1

Identification and isolation of SAP as an endotoxin-binding molecule 1.1 Materials and method

A 1 mg / ml solution of LPS from Salmonella Re595 (Sigma; L9764) in carbonate buffer (0.1 M, pH 9.5) is incubated with 5xl07 with Tosyl activated 35 DynaBeads ™ (M450, Dynal AS, Oslo, Norway) for 24 hours. . The beads loaded with LPS are washed 3x and stored in HBSS + 0.1% Na-azide (5x107 b / ml).

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A 10% dilution (1 in 10 dilution) serum from healthy volunteers is mixed with 5x106 LPS-Beads for 30 min at 22 ° C. Optionally, the serum is pre-incubated with different concentrations of free LPS. The beads are washed 3x with HBSS

(Gibco BRL, Gaithersburg, MD, USA) and then included in 25 μΐ SDS-PAGE sample buffer (with 2% SDS and 2.5% DTT). After boiling for 3 min at 100 ° C, the sample is analyzed on a 12.5% SDS-PAGE (Mini-10 Proteanll; Bio-Rad). The proteins present are stained with Coomassie Blue. For reference, a sample containing marker proteins of known sizes (Bio-Rad). For identification, the proteins after SDS-PAGE are transferred to nitrocellulose paper using a Mini Trans-Blotter ™. The proteins are stained with Coomassie Blue, and the protein around 30 kDa is excised and dissolved in buffer.

This sample is sequenced.

SAP is isolated from normal human serum using the Ca dependent binding to Sepharose. With delipidated serum, which is prepared by centrifuging serum at 16,000 rpm for 4 minutes, first two precipitation steps are performed (successively with BaCl 2 and NH 4 SO 4) followed by anion exchange chromatography on a

Mono-Q column (Pharmacia). The eluate at 0.2 to 0.3 M NaCl is finally passed over a Sepharose-4B column and 95% pure SAP is obtained by elution with EDTA (analysis on SDS-PAGE and with a specific ELISA). For the purification method, see also Skinner &

Cohen, Methods in Enzymology, vol. 163, pages 523-536 (1988).

1.2 Results 35 1. Figure 1A shows an example of an SDS-PAGE gel of serum incubated LPS beads. For specificity of the reaction, the serum was first incubated with different concentrations (100, 10, 1 and 0 1004258 6

Mg / ml) free ReLPS (L9764, Sigma Chemicals, St. Louis. MO, USA) before incubating the mixture with the LPS Beads. This demonstrates that when SAP is saturated with free LPS, binding to the LPS beads no longer occurs (lanes 3 and 4). It follows that SAP is also able to bind with free LPS.

2. Sequencing of the first 17 amino acids (N-terminal end) of the ± 30 kDa protein resulted in a sequence 100% similar to Serum 10 Amyloid P-Component (SAP). Therefore, the probability that the protein is SAP is 89%. This protein and SAP were used to demonstrate its neutralizing effect.

3. Binding of commercially obtained SAP (Calbiochem; 1565190) to LPS-Beads by analysis with SDS-PAGE is shown in Fig. 1B. This also shows that the component from serum, which binds to the LPS-Beads, is indeed SAP.

20 EXAMPLE 2

Inhibition of endotoxin-induced monocvt activation

by SAP

2.1 Materials and method LPS of Salmonella Re595 (ReLPS, supra) is coated with FITC (Sigma; F7250) under conditions under which LPS is present as a monomer, resulting in a FITC-LPS preparation with a ratio of 1 molecule of FITC per molecule of LPS. After extensive desalination and dialysis against PBS, the stock solution of FITC-LPS 30 is stored at -20eC. Mononuclear leukocytes (monocytes and lymphocytes) are isolated from Heparin blood from healthy volunteers via a Ficoll (Pharmacia) gradient according to standard method. After washing, the cells are taken up in isotonic HEPES buffer 35 (carbon black 1 mM CaCl2 and 2% BSA). SAP preparations are mixed with FITC-LPS and then added to 3x105 mononuclear cells and 30 ng / ml recombinant LBP (obtained from Dr. Lichtenstein) in a total volume 1004258 of 50 μΐ and a FITC-LPS concentration 2.5 ng / ml. After an incubation period of 30 min at 37 ° C, the samples are kept on ice. Monocyte-associated FITC-LPS binding analysis is performed with FACScan ™ 5 Flow cytometer. Forward and Sideward Scatter parameters are used to identify the monocyte population. LysisII ™ software ^ (Beckton & Dickinson) calculates the average fluorescence value of 5000 monocytes. See also Weering A.J.L. et 10 al., J. Immunol. 145, 318-324 (1990)).

2.2 Results 1. 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 with the monocytes. Since association of LPS with the monocyte is the first step in monocyte activation, this is a very strong indication that SAP will also inhibit monocyte activation in very early stages in vivo.

EXAMPLE 3

Inhibition of endotoxin-induced neutrophil activation

by SAP

25 3.1 Materials and method

Neutrophils are isolated from Heparin blood from healthy volunteers via a Histopaque-Ficoll gradient according to a standard method (Van Amersfoort, ES & JAG van Strijp, Cytometry 17, 294-301 30 (1994). The remaining erythrocytes in the neutrophil fraction are lysed with sterile water (for 30 sec.) and, after restoration of isotonicity, the fraction is washed The cells are finally taken up (suspended) in HBSS + 2% Human 35 Serum Albumin (HSA) Cells (1x104) are incubated - Bear with lng / ml ReLPS, 30ng / ml recombinant LPS-binding protein (r-LBP) and a sample of SAP in a total volume of 100 μΐ for 30 min at 37 ° C. Then, the 1 004258 8 g samples were placed in a luminometer (Berthold; Autolumat LB953), after which luminol (180 μΜ) and 1 μΜ fMLP (formylmethyonyl-leucyl-phenylalanine) were added to each tube as stimulus 5. The luminescence response was measured continuously for 15 min. and the results expressed as total luminescence response (area under the graph). See also Weersink et al., Immunology 83, 617-623 (1994).

3.2 Results 1. Figure 3 shows the inhibition of LPS "priming" of neutrophils by SAP. In the graph, the lumine-scoring counts are plotted against the measurement time (in minutes). This shows that the activation of neutrophils by LPS is strongly reduced by SAP. This is a strong indication that the toxicity of LPS by SAP can also be reduced in vivo.

20 EXAMPLE 4

Inhibition of endotoxin-induced sepsis in vivo 4.1 Materials and method.

In vivo protection experiments are performed in a mouse peritonitis model (Cross et al., In-25 feet, and Immunity 61, 2741-2747 (1993)) with ReLPS or intact gram-negative bacteria. The SAP preparation is pre-incubated with ReLPS or 018K5 bacteria (Cross et al., Supra) and then administered intraperitoneally to BALB / c mice in a 3xLD50 dose.

Survival is monitored for 7 days.

LEGEND

Figure 1A shows an SDS-PAGE gel of LPS beads incubated with free LPS pre-incubated serum.

lane 1: markers (from top to bottom 92, 66, 45, 31 and 21 kDa, respectively), lane 2: without pre-incubation with free LPS, 1004258 9 lane 3: pre-incubation with 100 Mg / ml free LPS lane 4: pre-incubation with 10 Mg / ml free LPS lane 5: preincubation with 1 Mg / ml free LPS.

Figure 1B shows an SDS-PAGE gel of LPS-Beads 5 incubated with serum (lane 1, negative control), commercially obtained SAP (lane 2) and markers (lane 3, molecular weights from top to bottom 92, 66, 45, 31, respectively) and 21 kDa).

Figure 2 shows the effect of SAP on LPS binding to monocytes. The concentration of SAP is plotted on the X axis. The Y axis shows the mean fluorescence, which represents the ReLPS bond.

Figure 3 shows the inhibition of LPS "priming ·· of neutrophils by SAP in the presence and absence of LPS. The graph plots the luminescence counts against the measurement time (in minutes).

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Claims (4)

1. Use of Serum Amyloid P component (SAP) and / or endotoxin-binding fragments thereof for the manufacture of a pharmaceutical composition for neutralizing lipopolysaccharide. 2. Use of Serum Amyloid P component (SAP) 'fl and / or endotoxin binding fragments thereof for the manufacture of a pharmaceutical composition for the treatment of sepsis. 3. Diagnostic method for detecting the presence of endotoxin in blood or blood fractions, such as serum or plasma, comprising contacting a carrier with bound SAP and / or endotoxin-binding fragments thereof with a blood sample to be tested to allow binding of endotoxin to SAP, removing unbound material and visualizing and / or quantifying the binding between endotoxin and SAP (fragments). A diagnostic kit for carrying out the method of claim 3, comprising a carrier with SAP bound thereto, and means for visualizing and / or quantifying binding between endotoxin and SAP (fragments). 1004258