Pharmaceutical and diagnostic use of Serum Amyloid P component

Details Pharmaceutical and diagnostic use of Serum Amyloid P component Pharmaceutical and diagnostic use of Serum Amyloid P component

1999-06-18

2002-04-02

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S014800, C514S016700, C514S017400, C530S300000, C530S327000, C530S328000, C530S329000, C530S380000

Reexamination Certificate

active

06365570

ABSTRACT:

The present invention relates 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 monocytes, macrophages and granulocytes (neutrophils). These are hereby activated and secrete various cytokines, such as tumor necrose factor-&agr; (TNF-&agr;), 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 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 the per se known protein Serum Amyloid P component (SAP) is capable of binding to endotoxin. Binding of the circulating SAP to the phagocytes, and therewith activation thereof, is hereby prevented. In this manner SAP is capable of neutralizing the biological action of endotoxin.
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.
SAP is a member of the family of the pentraxins. Pentraxins are proteins with a characteristic pentameric organization of identical subunits which are ordered as single or double annular discs. Another member of this family is the C-reactive protein (CRP). CRP and SAP are both so-called “acute-phase reactants” (APR), i.e. they are involved in the early phase of an inflammation process. Per species however, it is generally found that only one of the two acts as APR. For humans it is the case that the concentration of SAP in normal human plasma is approximately 30 &mgr;g/ml. During inflammation reactions this level remains roughly the same, while the CRP level may well be increased a thousand-fold to 1 mg/ml, depending on the disease and the seriousness thereof.
It may be considered particularly surprising that a compound which plays no part as APR during inflammation reactions is in fact capable of neutralizing sepsis-causing endotoxin. It could be expected that the body itself would make use of this neutralizing capacity by increasing the plasma level of this compound in the case of infection with Gram-negative bacteria and the inflammation reactions resulting therefrom.
SAP is a relatively large protein. For particular ligands is known which of the amino acids of SAP are involved in binding, such as for instance for CRP, C4-binding protein (C4bp), Clq and Calcium ions. It has thus also been established for endotoxin that determined regions of the SAP molecule are involved in the binding. It may therefore be recommended to use only the specific binding parts of the protein for manufacture of a pharmaceutical preparation.
According to the invention it has now further been found that SAP fragments, which consist of at least a part of the amino acids 27-39 of SAP in the sequence occurring in SAP, are very successful in inhibiting the binding of LPS to monocytes.
According to a second aspect of the invention new peptides (SAP fragments) 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 below shows the amino acid composition of the different peptides.
TABLE 1
Amino Acid
Number
27
28
29
30
31
32
33
34
35
36
37
38
39
SEQ ID NO
SAP
PEP 29-39
Glu
Lys
Pro
Leu
Gln
Asn
Phe
Thr
Leu
Cys
Phe
Arg
Ala
1
PEP 33-38
Phe
Thr
Leu
Cys
Phe
Arg
2
PEP 32-39
Asn
Phe
Thr
Leu
Cys
Phe
Arg
Ala
3
PEP 33-38
Gln
Asn
Phe
Thr
Leu
Cys
Phe
Arg
6
PEP 30-37
Leu
Gln
Asn
Phe
Thr
Leu
Cys
Phe
4
PEP 29-36
Pro
Leu
Gln
Asn
Phe
Thr
Leu
Cys
5
PEP 28-35
Lys
Pro
Leu
Gln
Asn
Phe
Thr
Leu
7
PEP 27-34
Glu
Lys
Pro
Leu
Gln
Asn
Phe
Thr
8
Glu-Glutamate;
Lys-Lysine;
Pro-Proline;
Leu-Leucine;
Gln-Glutamine;
Asn-Asparagine;
Phe-Phenylalanine;
Thr-Threonine;
Cys-Cysteine;
Arg-Arginine;
Ala-Alanine
The advantage of SAP fragments is that they can be manufactured more simply owing to their smaller dimensions and can penetrate more easily into bodily tissues.
In the research which led to the present invention it was further inferred that 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). 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 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 environmental factor, contribute to the development of Alzheimer's disease. It is in fact suspected that the basis for Alzheimer's is the alternative processing of the beta-amyloid precursor protein, which possibly takes place outside the brain i

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