阅读说明：本技术 从生物流体中除去细菌毒素的方法 (Method for removing bacterial toxins from biological fluids ) 是由 A·皮尼 C·法尔恰尼 L·布拉奇 J·布鲁内提 L·奎尔希尼 于 2018-04-19 设计创作，主要内容包括：本发明涉及从生物流体中除去细菌毒素如脂多糖和脂磷壁酸的方法。在所述方法中,将选自KKIRVRLSA、RRIRVRLSA、KRIRVRLSA和RKIRVRLSA的肽通过其C末端、任选地通过插入的连接体与固体载体共价连接,并用于捕获毒素。本发明还涉及这种衍生化的固体载体,以及涉及包含这种衍生化的固体载体的筒、柱和医疗装置。(The present invention relates to a method for removing bacterial toxins such as lipopolysaccharides and lipoteichoic acids from biological fluids. In the method, a peptide selected from the group consisting of KKIRVRLSA, RRIRVRLSA, KRIRVRLSA and RKIRVRLSA is covalently linked via its C-terminus, optionally via an intervening linker, to a solid support and used to capture the toxin. The invention also relates to such derivatized solid supports, and to cartridges, columns, and medical devices comprising such derivatized solid supports.)

1. A method of removing a bacterial toxin selected from LPS and LTA from a biological fluid, the method comprising contacting the biological fluid with a peptide selected from KKIRVRLSA, RRIRVRLSA, KRIRVRLSA and RKIRVRLSA, which peptide is covalently linked by its C-terminus (optionally with an inserted linker) to a solid support, thereby forming a linker-peptide moiety, and wherein all amino acids of the peptide are in the L-configuration or the D-configuration.

2. The method of claim 1, wherein the peptide-linker moiety is a group of the formula:

Wherein

R1-, R2-, R3-, R4-, R5-, R6-, R7-and R8-which may be the same or different, are selected from KKIRVRLSA-, RRIRVRLSA-, KRIRVRLSA-and RKIRVRLSA-;

X1, X2, X3, X4, X5, X6 and X7 are the same or different and are at least bifunctional residues;

n, m, p, q, t, v and w may be the same or different and may be 0 or 1, and at least one of m, n, p and q is 1, with the proviso that if t is 0, then n and m are 0, and at least one of p and q is 1, if v is 0, then p and q are 0, and at least one of m and n is 1, if w is 0, then v is 0, and at least one of m and n is 1;

Y is a bond or a spacer.

3. The method of claim 2, wherein X1, X2, X3, X4, X5, X6, and X7 are the same or different and each comprises at least two functional amino groups.

4. The method of claim 3, wherein X1, X2, X3, X4, X5, X6, and X7 are the same or different and are selected from the group consisting of lysine residues, ornithine residues, n-lysine residues, amino alanine residues, and diaminopropionic acid residues.

5. The method of claims 2-4, wherein at least one of R1-, R2-, R3-, R4-, R5-, R6-, R7-, and R8-is KKIRVRLSA-.

6. A method as claimed in claims 2 to 5 wherein Y is an amino acid residue or a peptide.

7. The method of claim 6, wherein Y is selected from the group consisting of a β -alanine residue, an N- (PEG) Y-CH2-CH2-C (O) residue wherein 1. ltoreq. y.ltoreq.11, a cysteine residue, and a peptide comprising such residue.

8. The method as claimed in claims 2-7, wherein w is 0, v is 0, t is 1, m is 1 and n is 1.

9. The method of claim 8, wherein each of X1, X2, and X5 is a lysine residue.

10. The method of claim 8 or 9, wherein at least one of R1-, R2-, R3-and R4-is KKIRVRLSA-.

11. The method as claimed in claims 1-10, wherein the solid support is selected from the group consisting of cross-linked agarose resins and cross-linked composite resins of hydroxyethyl polystyrene and polyethylene glycol.

12. a solid support covalently linked to the C-terminus of a peptide selected from KKIRVRLSA, RRIRVRLSA, KRIRVRLSA and RKIRVRLSA, optionally with an intervening linker, thereby forming a peptide-linker moiety.

13. The solid support of claim 12, wherein the peptide-linker moiety is as defined in claims 2-11.

14. A medical device for removing a bacterial toxin selected from LPS and LTA from a biological fluid, said medical device comprising a solid carrier as claimed in claim 12 or 13.

15. Use of a medical device comprising a solid carrier according to claim 12 or 13 for removing a bacterial toxin selected from LPS and LTA from a biological fluid.

Background

Sepsis is a clinical syndrome caused by the immune and coagulation systems of the human body. Septic shock is a life-threatening disease characterized by low blood pressure and organ dysfunction or failure despite adequate fluid replacement. Sepsis is an important cause of death in all age groups (Perner et al, 2017).

Sepsis has been defined as a microbial infection that causes fever (or hypothermia), tachycardia, tachypnea and blood leukocyte changes for more than twenty years. Sepsis is now increasingly viewed as a systemic inflammatory and uncontrolled immune response to microbial invasion that causes organ damage. Septic shock is defined as sepsis and hypercalactia and associated hypotension, requiring treatment for vascular pressure increases with hospitalization mortality approaching 30-50%.

Sepsis and related diseases are one of the leading causes of death worldwide, 1900 ten thousand cases per year, and 1,400 deaths per day. In developed countries such as the united states alone, the incidence of sepsis is estimated to be 1,655,000, resulting in over 250,000 deaths per year. This has become a major economic burden in the united states, with a total of $ 167 billion for healthcare (Lakshmikanth et al, 2016).

Patients with sepsis typically receive intravenous antibiotics, oxygen, fluids, and medications to stimulate the heart and maintain acceptable blood pressure levels. In some cases, dialysis is used.

Despite intensive research in this field, no specific medical treatment for sepsis has been found. With the earlier discovery of sepsis and increased compliance with best medical practice, sepsis is no longer a directly life-threatening disease but is more a long-term, chronic, critical disease, often associated with long-term inflammation, immune suppression, organ damage, and lean tissue consumption. In addition, there is a continuing risk of death after discharge from patients who survive sepsis, as well as long-term cognitive and functional deficits. Early confirmation of diagnosis and better implementation of best medical practice has reduced in-hospital mortality, but the results of using immunomodulators have been disappointing to date. Likewise, no biomarker can definitively diagnose sepsis or predict its clinical outcome. Due to its complexity, the improvement in sepsis outcome may continue to be slow and progressive (Hotchkiss et al, 2016).

Lipopolysaccharide (LPS) or endotoxin is a major component of the outer membrane of gram-negative bacteria and is the major bacterial product of the clinical syndrome causing sepsis. Binding of LPS to the host receptor Toll-like receptor 4(TLR4) triggers an inflammatory response characterized by the release of large amounts of inflammatory mediators that enable the host to respond to invading pathogens. When this production becomes uncontrolled and excessive, it can lead to the onset of septic shock (Ianaro et al, 2009).

Lipoteichoic acid (LTA) is the major cell wall component of gram-positive bacteria and is also associated with a variety of inflammatory diseases, from mild skin diseases to severe sepsis. LTA is known to be recognized by Toll-like receptor 2(TLR2), leading to the initiation of the innate immune response and further development of adaptive immunity. However, excessive immune response may lead to inflammatory sequelae that are associated with serious diseases such as sepsis (Kang et al, 2016).

TORAYMYXIN (Rocco and Klein 2014; Shoji et al 1998) is a therapeutic strategy for the removal of circulating LPS by immobilizing polymyxin B (PMX), a conventional antimicrobial peptide that has been used clinically (Roscia et al, 2013), on polystyrene-derived fibers in a blood perfusion apparatus. PMX cartridges were made by covalently immobilizing PMX on polystyrene derived fibers, which can then be used for external hemofiltration using an extracorporeal circuit to remove LPS from circulation by adsorption to the PMX cartridge.

In another strategy, polymyxin B (PMX) was immobilized on a solid phase (4B) and a plasmapheresis system was developed in conscious rats for specific online plasma adsorption of endotoxin by a PMX-Sepharose column (Cohen et al, 1987).

The LPS adsorber (Ala-Kokko et al, 2011) is a medical device used for extracorporeal clearance of LPS during blood perfusion. The biotechnology of this product is based on synthetic custom-made peptides that selectively bind LPS found in the circulation of septic patients.

However, alternative methods are needed to eliminate LPS in the blood of septic patients and possibly also to remove TLA.

Brief Description of Drawings

FIG. 1 depicts a cross-linked hydroxyethylpolystyrene and polyethylene glycol composite resin.

FIG. 2 depicts how covalent bonds are formed between sulfolinkTM resin and free thiols.

FIG. 3 depicts the HPLC profiles of solutions containing Compound B before (A) and after (B) coupling with sulfolinkTM resin.

Fig. 4 depicts LPS content in biological fluids before and after exposure to resin (a) derivatized with compound B and the same underivatized resin (B).

Fig. 5 depicts LPS content in biological fluids before and after exposure to resin (a) derivatized with compound B and the same underivatized resin (B).

FIG. 6 depicts electropherograms of proteins of serum sample (A), serum sample (B) after contact with a resin derivatized with Compound B, serum sample (C) after contact with a resin derivatized with Compound A, and reference values (D) for such proteins

Fig. 7 depicts the LTA content of biological fluids before and after exposure to a resin (a) derivatized with compound B and the same underivatized resin (B).

Detailed Description

We have surprisingly determined that M33, when covalently bound by its C-terminus to a solid support, particularly when bound to the structure by forming a covalent bond with compound a or compound B below, allows selective removal of LPS from biological fluids without altering the protein content of the fluid.

The derivatized solid support may also be used to remove LTA from a biological fluid.

Accordingly, in a first aspect of the invention, there is provided a method of removing a bacterial toxin selected from LPS and LTA from a biological fluid, the method comprising contacting the biological fluid with a peptide selected from KKIRVRLSA, RRIRVRLSA, KRIRVRLSA and RKIRVRLSA, which peptide is covalently linked via its C-terminus (optionally with an intervening linker) to a solid phase carrier, and wherein all amino acids of the peptide are in the L-configuration or the D-configuration.

in one embodiment, all amino acids of the peptides selected from KKIRVRLSA, RRIRVRLSA, KRIRVRLSA and RKIRVRLSA are in the L-configuration.

In another embodiment, all of the amino acids of the peptides selected from KKIRVRLSA, RRIRVRLSA, KRIRVRLSA and RKIRVRLSA are in the D-configuration.

In one embodiment, the peptide-linker moiety is a group of the formula

Wherein

R1-, R2-, R3-, R4-, R5-, R6-, R7-and R8-which may be the same or different, are selected from KKIRVRLSA-, RRIRVRLSA-, KRIRVRLSA-and RKIRVRLSA-;

X1, X2, X3, X4, X5, X6 and X7 are the same or different and are at least bifunctional residues;

n, m, p, q, t, v and w may be the same or different and may be 0 or 1, and at least one of m, n, p and q is 1, with the proviso that if t is 0, then n and m are 0, and at least one of p and q is 1, if v is 0, then p and q are 0, and at least one of m and n is 1, and if w is 0, then v is 0, and at least one of m and n is 1;

Y is a bond or a spacer.

in one embodiment, X1, X2, X3, X4, X5, X6, and X7 are the same or different and each comprises at least two functional amino groups.

In another embodiment, X1, X2, X3, X4, X5, X6, and X7 are the same or different and are selected from the group consisting of lysine residues, ornithine residues, n-lysine (norlysine) residues, amino alanine residues, and diaminopropionic acid residues.

in one embodiment, at least one of R1-, R2-, R3-, R4-, R5-, R6-, R7-, and R8-is KKIRVRLSA-.

In another embodiment, each of R1-, R2-, R3-, R4-, R5-, R6-, R7-, and R8-is KKIRVRLSA-.

In one embodiment, Y is selected from the group consisting of an amino acid residue and a peptide.

In a particular embodiment, Y is selected from the group consisting of amino acid residues, dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, octapeptides, nonapeptides and decapeptides.

In a more specific embodiment, Y is selected from the group consisting of a beta-alanine residue, a N- (PEG) Y-CH2-CH2-C (O) residue wherein 1. ltoreq. y.ltoreq.11, a cysteine residue, and peptides comprising such residues.

In an even more specific embodiment, the peptides of the above embodiments are selected from the group consisting of dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, octapeptides, nonapeptides, decapeptides.

In one embodiment, y is selected from 1, 2, 3, 4, 5, 6,7, 8, 9, 10 and 11.

In one embodiment, w is 0, v is 0, t is 1, m is 1 and n is 1.

In a specific embodiment, w is 0, v is 0, t is 1, m is 1, n is 1 and each of X1, X2, and X5 is a lysine residue.

In a specific embodiment, w is 0, v is 0, t is 1, m is 1 and n is 1, and at least one of R1-, R2-, R3-and R4-is KKIRVRLSA-.

In a specific embodiment, w is 0, v is 0, t is 1, m is 1 and n is 1, and R1-, R2-, R3-and R4-are each KKIRVRLSA-.

In certain embodiments, the solid support is porous.

In one embodiment, the solid support is a cross-linked agarose resin.

In another embodiment, the solid support is a cross-linked composite resin of hydroxyethyl polystyrene and polyethylene glycol.

In some embodiments, the biological fluid is selected from the group consisting of serum, plasma, and blood.

All embodiments of the first aspect of the invention may be combined.

In a second aspect of the invention there is provided a solid support bearing a group as hereinbefore described in an embodiment of the first aspect of the invention.

In a third aspect of the invention there is provided an article selected from a column and a cartridge, each comprising a solid support of the second aspect of the invention.

In a fourth aspect of the invention, there is provided a medical device comprising the solid support of the second aspect of the invention or the article of the third aspect of the invention.

In a particular embodiment, the medical device is a medical device for removing a bacterial toxin selected from LPS and LTA from a biological fluid.

In a fifth aspect of the invention there is provided the use of a medical device according to the fourth aspect of the invention for removing a bacterial toxin selected from LPS and LTA from a biological fluid.

Examples

the invention will now be described by way of non-limiting examples.