Patent 6,949,512

A therapeutic agent for the treatment of septicemia, its preparation and use, with application in the pharmaceutical industry and medicine. This therapeutic agent contains essential element, the lipopolysaccharide binding protein (LBP). Also disclosed are LBP variants, mutants and hybrid proteins. Murine or rabbit LBP may also be used, besides human LBP.

SUMMARY OF THE INVENTION

The invention is based on the task to develop a therapeutic agent for the treatment of septicemia.

This task was solved by an agent containing the protein LBP binding lipopolysaccharide as basic component.

The characteristic features of the invention are contained claims 1-11. It is possible to use also murine or rabbit LBP apart from human LBP.

DETAILED DESCRIPTION OF THE INVENTION

The structure of LBP is known. It was obtained by isolating a clone from an acute phase cDNA gene bank and subsequently sequencing and deriving the amino acid sequence. Recombinant LBP is prepared by cloning cDNA in an expression vector and co-infection of insect cells with the baculovirus.

The cloned protein (LBP) binds LPS with high affinity and is secreted into serum as acute phase protein during septicemia. As is shown in the examples LBP inhibits the LPS effects and may—if given to mice—suppress septicemia caused by LPS and reduce lethality highly significantly. This applies when using LBP simultaneously with the onset of septicemia and before. Thus LBP may seem to be suited for preventing septicemia in high-risk patients also. As LPS plays a central part also in septicemia caused by gram-positive bacteria and for the systemic inflammatory response syndrome, a clinical picture identical to septicemia caused by trauma, via translocation of gram-negative intestinal flora, the inhibition of LPS effects by LBP may also improve these dramatic clinical syndromes.

Apart from the highly active recombinant LBP optimized mutants the function of which was modified which are also used as therapeutic agents for the treatment of septicemia are equally suited for implementing the invention.

A further basic possibility for implementing the invention is to clone the LBP gene in an adenoviral vector with high activity in the liver behind the strong CMV promoter to achieve high levels of expression in addition to the intrinsic hepatic expression of LBP.

The invention is applicable in the case of

septicemia caused by gram-negative bacteria

septicemia caused by gram-positive bacteria

systemic inflammatory response syndrome (SIRS) caused by trauma and injury.

EXAMPLE

Hereinafter the invention shall be explained in greater detail by an example.

The complete LBP cDNA is cloned in the pACHLT-B vector (Pharmingen, San Diego, USA) behind the strong polyhedrin promoter and behind glutathione S-transferase (GST) cDNA. Thus a GST fusion protein is expressed. Then a 500 ml cell culture of Sf-9—insect cells is infected with this vector and the baculovirus DNA (Baculogold, Baculovirus DNA in a linearized form, also from Pharmingen, San Diego, USA). After 2 days the cells are subjected to lysis and the lysate is coupled to glutathione sepharose in the presence of triton X-100 in a "batch" process. Then LBP is split off by the participant in the fusion by digestion with thrombin followed by a treatment with calbiosorb to remove triton and a treatment with benzamidine sepharose to remove thrombine rests. The resulting concentrations of pure LBP totals 0.3-0.5 mg/ml.

Short Interpretation:

Highly dosed LBP in vitro suppresses the synthesis of an important septicemia mediator molecule caused by the bacterial toxin LPS, namely TNF. The production of this protein and other mediators is suppressed in the mouse by simultaneously adding LBP. In addition, the liver damage induced by the addition of LPS is prevented by LBP and the number of surviving mice goes up significantly. That means, the addition of LBP seams to protect against the effects of LPS during septicemia, representing thus a new therapeutic principle for treating septicemia.

Claim 1 of 3 Claims

1. A process for detoxifying bacterial lipopolysaccharide in a patient with septicemia caused by gram-negative bacteria or by gram-positive bacteria, which comprises administering to said patient an effective amount of lipopolysaccharide binding protein to elevate the serum concentration of lipopolysaccharide binding protein in said patient to a sufficiently high level to suppress lipopolysaccharide induced release of cytokine, thereby detoxifying the bacterial lipopolysaccharide.

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