KR20070118646A - Polymyxin b analogs for lps detoxification - Google Patents

Abstract

The invention relates to SAEP II peptide dimers that mimic polymyxin B i.a. in its ability to bind non-covalently the lipopolysaccharide (LPS) of Gram-negative bacteria with high affinity, and therefore to detoxify LPS as polymyxin B does. The dimeric structure is maintained by a pair of disulphide bonds involving the two cystein residues present in the peptide sequence, which does not exceed 17 amino acids and essentially comprises cationic and hydrophobic amino acid residues. In the dimers of the invention, peptides may have a parallel or anti-parallel orientation. As a matter of example, a dimer of the invention is constituted by a peptide of formula NH2-Lys-Thr-Lys-Cysl-Lys-Phe-Leu-Leu-Leu-Cys2-COOH, either in a parallel or antiparallel dimeric form. SAEP II dimers are useful for treating or preventing septic shock and related disorders generated by Gram-negative bacteria infection. The invention also relates to LPS-peptide complexes in which LPS and SAEP II dimers are non-covalently bound together. These complexes are useful as vaccinal agents against Gram-negative bacteria infection.

Description

POLYMYXIN B ANALOGS FOR LPS DETOXIFICATION}

The present invention relates to peptide homologs of polymyxin B useful for LPS translation. In the field of pharmacy, it is used for (i) itself to treat a fatal disease caused by, for example, Gram-negative bacterial infection, for example septic shock; Or (ii) noncovalently bound to LPS and used to decipher the same; These complexes are useful as vaccines against Gram-negative bacterial infections.

Lipopolysaccharide (LPS) is a major component of the cell wall envelope of Gram-negative bacteria. LPS is very toxic in mammals, especially humans, and has been referred to as endotoxin with respect to its biological activity. This is responsible for the results from endotoxin poisoning in septic shock, a life-threatening event that occurs during acute infection (septicemia) by Gram-negative bacteria.

The LPS structure is composed of a lipid moiety called lipid A, covalently linked to a polysaccharide residue.

Lipid A, in particular, contributes to the toxic effects of LPS through interaction with B cells and macrophages. This interaction leads to the secretion of cytokines before inflammation. The inflammatory state can lead to a fatal state of endotoxin shock.

Lipid A is very hydrophobic and anchors LPS in the outer layer of bacterial cell walls. Lipid A comprises (i) a conserved bis-phosphorylated disaccharide region (most commonly N, O-acyl beta-1,6-D-glucosamine 1,4'-bisphosphate) and (ii) It consists of fatty acids that replace various hydrogen atoms belonging to the disaccharide hydroxyl. The number of fatty acids and their composition varies between species. For example, the nose, ceria menin GT display (Neisseria meningitidis ) The two symmetric glucoseamines (GlcN1 and GlcN2) of lipid A each have the following fatty acids: 2N-C14,3OH; C12; And 3O-C12,3OH.

The LPS polysaccharide moiety is composed of carbohydrate chains that contribute to antigenicity. The carbohydrate chain structure itself has a conserved inner core called (i) a KDO (2-keto, 3-deoxy octulonic acid) region bound to lipid A and (ii) a variable outer bound to the KDO region. A core (usually defined as comprising various saccharides), and (iii) a first repeating unit of the outer O-specific chain (which may include up to 10 saccharides).

Of non-gram negative-bacteria in the field, for example a brush Ney ceria, Bordetella under (Bordetella), loose (Haemophilus) and morak Cellar (Moraxella), the O- specific chain does not exist (the first iteration What is defined as a unit is not actually repeated). Thus, LPS of these bacteria is often referred to as lipooligosaccharide (LOS).

LPS is not only toxic but also very immunogenic. In mammals, anti-LPS antibodies are induced during infection and delivery and may be protective. In view of the above, it has already been proposed to decipher LPS and use its decoded form for the prevention of Gram-negative bacterial infections and related diseases.

Several decryption methods are already known. In particular, LPS can be deciphered at the same time using polymyxin B or more suitably its peptide analogs.

Polymyxin B is a molecule that binds lipid A with a high affinity and significantly decodes LPS. When provided therapeutically in an animal model, polymyxin B can prevent septic shock. However, polymyxin B is a multicationic antibiotic that may be somewhat toxic to humans due to its non-biodegradability and thus a tendency to accumulate in the kidneys. Thus, use is not recommended for prophylactic or therapeutic products.

To overcome this limitation, peptide homologues for polymyxin B have been developed. It does not retain polymyxin B toxicity but mimics the primary and secondary structures of polymyxin B and binds to lipid A at the same site where polymyxin B binds, thus forming LPS-peptide complexes. . As a result, the LPS is decrypted. Peptide homologs are described in particular in US Pat. Nos. 5,358,933, WO 93/14115, WO 95/03327, WO 96/38163, EP 842 666 and EP 976 402. One of these, the cyclic monomer SAEP2 (synthetic anti-endotoxin peptide 2) of the formula KTKCKFLKKC, has been studied more specifically (Rustici et al, 1993, Science 259: 361 and Velucchi et al, 1997, J. Endotox. Res. 4 (4). ): 261).

In accordance with the present invention, the SAEP2 peptide, as well as similar peptides comprising a plurality of uncharged polar amino acids surrounded by two adjacent cysteine residues in its sequence and balanced by a short outer tail of cationic amino acids (generally since Is particularly important when the SAEP II peptide is present in dimeric form; The dimer was found to be morphologically produced and maintained by a pair of disulfide bonds between the cysteine residues. Indeed, SAEP II peptide dimers exhibit improved detoxification properties for the corresponding monomers.

Accordingly, the present invention relates to SAEP II peptide dimers of formula I or II:

NH2- A -Cys1- B -Cys2- C -COOH

NH2- A ' -Cys1- B' -Cys2- C ' -COOH

Where

The two Cys1 residues are joined together via disulfide bonds

Two Cys2 residues are joined together via disulfide bonds;

NH2- A -Cys1- B -Cys2- C -COOH

COOH- C '-Cys2- B' -Cys1- A '-NH2

Where

Cys1 residues are linked to Cys2 residues through disulfide bonds;

A and A 'are independently a peptide portion of 2 to 5, preferably 3 or 4 amino acid residues, wherein the two or more amino acid residues are independently selected from Lys, Hyl (hydroxy-lysine), Arg and His; ;

B and B 'are independently peptide portions of 3 to 7, preferably 4 or 5 amino acid residues, at least 2, preferably 3 amino acids independently selected from Val, Leu, Ile, Phe, Tyr and Trp Includes residues;

C and C 'are optional (their positions may or may not be empty) and are independently amino acid residues or peptide portions of two to three amino acid residues; only

The cationic amino acid residue / hydrophobic amino acid residue ratio (cationic / hydrophobic ratio) is 0.4 to 2, advantageously 0.5 to 1.2 or 1.5, preferably 0.6 to 1; Most preferably 0.6 to 0.8; For example, 0.75.

Advantageously, A and A'are independently peptide portions of 2 to 5, preferably 3 or 4 amino acid residues, wherein at least one, preferably 2 amino acid residues are independent from Lys, Hyl, Arg and His. Is selected; Those not selected from Lys, Hyl, Arg and His ("rest amino acid residues") (if present) are selected from the group consisting of uncharged polar or nonpolar amino acid residues, preferably Thr, Ser and Gly; Thr is most preferred.

When the A and A ′ peptide moieties comprise three amino acid residues, they are each cationic residues; Or on the other hand two of the three residues are cationic amino acids, while the remaining residues are selected from the group consisting of uncharged polar or nonpolar amino acid residues, preferably Thr, Ser and Gly; Most preferably Thr.

Where the A and A ′ peptide moieties comprise four amino acid residues, two or three of the four residues are selected from the group of cationic amino acid residues as defined above, while the remaining residue (s) are Selected from the group consisting of uncharged polar or nonpolar amino acid residues as defined.

If the A and A ′ peptide moieties comprise five amino acid residues, three or four of the five residues are selected from the group of cationic amino acid residues as defined above, while the remaining residue (s) are Selected from the group consisting of uncharged polar or nonpolar amino acid residues as defined.

Advantageously, B and B 'are independently a peptide moiety of 3 to 7, preferably 4 or 5 amino acid residues, Val, Leu, Ile, Phe, Tyr and Trp; Preferably comprises at least two, preferably three amino acid residues independently selected from Leu, Ile and Phe; Those not selected from Val, Leu, Ile, Phe, Tyr and Trp ("rest amino acid residues") (if present) are independently selected from the group consisting of Lys, Hyl, Arg and His. As can be readily appreciated, the B and B ′ peptide moieties may comprise up to seven amino acid residues independently selected from Val, Leu, Ile, Phe, Tyr and Trp.

Advantageously, said B and B 'peptide moieties comprise the sequences -X1-X2-X3-, wherein X1 and X2; X2 and X3; Or X 1, X 2 and X 3 are Val, Leu, Ile, Phe, Tyr and Trp; Preferably it is independently selected from Leu, Ile and Phe. In a preferred embodiment, the sequence -X1-X2-X3- comprises a Phe-Leu sync.

Particular embodiments of peptide portions B and B 'include:

(i) -X1-X2-X3- sequence

Wherein X 1 is Lys, Hyl, His or Arg, preferably Lys or Arg; most preferably Lys;

X2 is Phe, Leu, Ile, Tyr, Trp or Val; Preferably Phe or Leu; Most preferably Phe;

X3 is Phe, Leu, Ile, Tyr, Trp or Val; Preferably Phe or Leu; Most preferably Leu); And

(ii) amino acid residues (if present, they are, respectively, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His; preferably Val, Leu, Ile, Phe, Tyr and Trp; more preferably Is independently selected from the group consisting of Leu, Ile and Phe).

When B and B 'include more than four nonpolar amino acid residues, A and A' preferably comprise amino acid residues charged in amounts of three or more.

In the C and C ′ peptide moieties, the amino acid residue may be any amino acid residue, provided that the cationic amino acid residue / hydrophobic amino acid residue ratio must be maintained within a specific range. Advantageously, it is independently selected from uncharged polar or nonpolar amino acid residues; The latter is preferred. However, in a preferred sense, C and C 'are empty positions.

Thus, a preferred class of dimers have the general formula III or IV below:

NH2- A -Cys1- B -Cys2-COOH

NH2- A ' -Cys1- B' -Cys2-COOH

NH2- A -Cys1- B -Cys2-COOH

HOOC-Cys2- B ' -Cys1- A' -NH2

Has:

In the above formulas,

A, A ', B and B' are as described above; only

The cationic amino acid residue / hydrophobic amino acid residue ratio is 0.4 to 2, advantageously 0.5 to 1.2 or 1.5, preferably 0.6 to 1; Most preferably 0.6 to 0.8; For example, 0.75.

Dimers of formula (I) or (III) having peptides in parallel orientation are referred to as parallel dimers. Dimers of formula (II) or (IV) with peptides in reverse parallel orientation are referred to as reverse parallel dimers.

In formulas (I) to (IV), A and A 'are preferably the same. It is B and B '; And C and C '. A and A '; B and B '; And peptide dimers of Formula (I), (II), (III) or (IV), wherein C and C 'are two identical, are referred to as cognate dimers. Indeed, in this case, the peptide subunits contained within the dimer are identical.

By way of example, the following peptides are cited as suitable for use in the dimers of the invention:

Each cationic / hydrophobic ratio of the corresponding cognate dimer is 2.00, 0.50, 0.75 and 0.67.

Particular examples of the dimers described above are constituted by peptides of Formula (V):

NH2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH

The peptide is hereinafter referred to as SAEP2-L2 peptide. As mentioned above, they may also exist in the form of parallel or antiparallel dimers.

Peptides included in the present invention or dimers of the present invention can be synthesized by conventional methods, for example using a computer-driven automated synthesizer. Methods of envisioning a process for obtaining specific peptides are within the skill of those skilled in the art of peptide synthesis. Of course, the cysteine thiol group can be protected during the synthesis step. Once synthesis is complete, deprotection and oxidation of the thiol groups are carried out to produce the cyclic monomers, parallel or antiparallel dimers.

If both cysteine residues present in the peptide are both deprotected at the same time, in theory, each of the three forms may be produced upon oxidation. Each of the three forms can then be separated from each other by conventional biochemical purification methods. Preparative reverse phase high performance liquid chromatography (RP-HPLC) is cited as a suitable example. Indeed, it can be expected that the three forms will each elute with different residence times. Thus, preparations containing the purified cyclic monomers, or purified parallel and inverse parallel dimers, can be obtained simply by bringing together the respective peak fractions.

The respective ratios for each of the three forms produced upon oxidation vary, for example, depending on the specific amino acid sequence and, importantly, the concentration of the peptide. One or two of the three forms may be predominantly produced; Indeed, one or two forms of predominance may be that no other (s) are formed at all.

By way of example, the SAEP2-L2 peptide is spontaneously oxidized proportionally to cyclic monomers and reverse parallel dimers, depending on the concentration of the peptide in solution. The internal steric hindrance of the "side chain" (NH2-Lys-Thr-Lys- part) of the anti-parallel dimer is clearly lower than that of the parallel dimer and the lower minimum energy is an aqueous solvent compared to the parallel dimer. It can be expected to contribute to the preferential formation of the above antiparallel dimer in. As a direct result of this concentration driving method, the formation of the anti-parallel dimer and, to a lesser extent, the cyclic monomer, is advantageous to exclude the parallel dimer from the equilibrium.

If the parallel dimer cannot be spontaneously produced upon oxidation, it is necessary to adopt specific means for associating the peptide within the parallel orientation. Such means are within the skill of one skilled in the art of peptide synthesis. Nevertheless, by way of example only, it was indicated that the differential protection of the Cys1 and Cys2 amino acids followed by selective deprotection is a convenient way to achieve dimerization with the parallel orientation. The dimer can then be purified by conventional methods, for example RP-HPLC.

Chemically synthesized and purified peptides are commonly obtained in salt form due to the fact that acids and salts are used during the chemical synthesis and purification steps. Acetate is a commonly used salt. Thus, the term "peptide" as used herein, of course, also includes salt forms.

Peptides for use in the dimers of the present invention may be subjected to various techniques, such as ion cyclotron resonance (ICR), mass assisted laser desorption ionization-time-of-flight (MALDI-ToF) spectroscopy and nuclear magnetic resonance (NMR) spectrophotometry It can be characterized by a measurement method. In particular, each of these three forms (cyclic monomers, parallel and antiparallel dimers) can be identified by NMR analysis. MALDI-ToF mass spectrometry allows the identification of only monomers and dimers.

The purity of the compounds of the present invention can be assessed by RP-HPLC. Briefly, compound preparations are run RP-HPLC. Relative purity is calculated by integrating the peak surface. This is shown as compound peak surface / total peak surface. It is common to prepare compounds of the invention that each exhibit a purity of at least 95%, often at least 97%.

The invention also

SAEP II peptides in the form of essentially parallel dimers;

SAEP II peptides in essential antiparallel dimer form; or

Mixtures of these

It relates to a composition comprising a.

“Essentially” means that at least 95%, preferably at least 97%, more preferably at least 98% of the specific forms in the composition are pure.

Mixed compositions in which the SAEP II peptides are present in various forms (parallel dimers, antiparallel dimers and / or monomer forms) may have a single presence, e.g., parallel dimer form, maintained at a suitable temperature over a period of time. It may be spontaneously generated from the occurrence of the composition comprising. This can be found for example by RP-HPLC analysis. Each amount of the various peptide forms can be quantified by the same token.

The SAEP II dimer per se is useful as an antidote for Gram negative bacteria LPS in vitro as well as in vivo. Thus, it can be used to prevent or treat pathological conditions resulting from systemic circulation, eg, release of LPS into the blood, as a result of Gram-negative bacterial infection. Such conditions include endotoxin addiction, bacterial sepsis and septic shock.

Therefore, the present invention

Pharmaceutical use of the compounds or compositions of the invention;

A pharmaceutical composition comprising a compound or composition of the invention together with a pharmaceutically acceptable diluent or carrier;

The use of a compound or composition of the invention in the manufacture of a medicament for the treatment or prevention of septic shock; And

-A therapeutic or prophylactically effective amount of a compound or composition of the invention comprising administering to a subject in need of treatment or prevention of septic shock

It includes.

The compounds or compositions of the present invention can be administered to mammals, ie, humans, when Gram-negative bacterial infections have been diagnosed as causing endotoxin addiction, bacterial sepsis and / or septic shock. Gram-negative bacteria that can cause these deadly disorders include, for example, yen menin GT display, E. coli (E. coli), Salmonella typhimurium (Salmonella typhi), Bordetella Pertussis ( Bordetella pertussis) and Pseudomonas Aru four Saginaw (Pseudomonas in aeruginosa ) . The compounds or compositions of the invention can be administered to individuals in need by the systemic route, preferably by the intravenous route. The dose administered varies depending on various factors, such as the age, weight, physiological state of the patient as well as the state of infection. It may be administered one or several times until it is free of the risk of fatal events.

Since the SAEP II dimer and SAEP2-L2 peptide can also decipher LPS in vitro, the present invention also includes (i) the LPS portion of Gram-negative bacteria, and (ii) the SAEP II peptide dimer or SAEP2-L2 peptide. To the LPS-peptide complex, wherein the LPS moiety and the SAEP II peptide dimer or SAEP2-L2 peptide are non-covalently bound to each other.

LPS detoxification can be evaluated by a number of assays mentioned in the European Pharmacopoeia. These include Rimulus Amebosite Lysate (LAL) analysis; Pyrogenicity testing in rabbits and acute toxicity analysis in D-galactosamine sensitized mice. Such analyzes are exemplified later in the Examples. In each of the above assays, the effect of LPS and the effect of LPS-peptide complex are measured in parallel to establish the readout ratio.

In the LAL assay, the readout ratio is shown as the LPS / LPS-peptide complex ratio. In the pyrogen test and acute toxicity assay, the detoxification ratio is expressed as LPS-peptide complex / LPS ratio.

Significant detoxification is achieved for the detoxification ratio measured as follows:

(i) the LAL assay is 100, preferably 500, more preferably at least 1000;

(ii) the pyrogen assay is 50, preferably 100, more preferably 500 or more; or

(iii) D-galactosamine mice are 50, preferably 100, more preferably 200 or more.

Detoxification can also be assessed by comparing the effects of LPS and LPS-peptide complexes on the release of cytokines such as IL6, IL8 and TNFα prior to inflammation in in vitro or in vivo assays. This analysis is illustrated later in the Examples. Said LPS-peptide complex has at least 25-fold, preferably at least 50-fold, more preferably at least 75-fold, most preferably 100-fold IL6 secretion in an in vivo assay as disclosed in the Examples, section 5.4.1 Significant detoxification is achieved when more than doubled.

The LPS-peptide complex of the present invention is advantageously 1: 1.5 to 1: 0.5, preferably 1: 1.2 to 1: 0.8, more preferably 1: 1.1 to 1: 0.9, most preferably 1: 1 LPS: peptide molar ratio.

For use in the complexes of the invention, the LPS is the yen menin GT advantageously di-scan; E. coli; Salmonella Tipi ; Salmonella typhimurium Farah (paratyphi); Shigella flex Tenerife (Shigella flexneri ); Under (Haemophilus influenza in a brush loose influenzae ); Helicobacter pylori (Helicobacter pylori ); Chlamydia trachomatis (Chlamydia trachomatis); Bordetella Pertussis ; Brucellosis (Brucella); New mopiah Legionella (Legionella pneumophia ); Vibrio cholera (Vibrio cholera ); Morak Cellar Kata LAL-less (Moraxella catharralis ); Pseudomonas aeruginosa ; Yersinia (Yersinia); And kiepsiela LPS of Kiebsiella pneumonia .

As mentioned in the introduction, detoxified LPS may be useful as a vaccine against Gram-negative bacterial infections.

Meningitis is a life-threatening disease of viral or bacterial origin. In H. influenza and Yen menin GT discharge is caused in each of about 40 and 50 percent of bacterial meningitis. Vaccines against H. influenza have been on the market for over 10 years, but vaccines against enmeningitidis are still needed.

Meningococcal invasive diseases can manifest as inflammation of the meninges (meningitis) of the brain and spinal cord or systemic infection of blood (meningococcal sepsis or meningococcal).

Meningococcal is classified using serological methods based on the structure of the polysaccharide capsule. Thirteen antigen, chemically unique polysaccharide capsules have been disclosed. The invasive meningococcal disease is caused by almost all five serogroups, namely A, B, C, Y and W-135. The relative importance of each serogroup varies with geographic location. Serum group B is responsible for most temperate meningococcal disease.

Conjugated polysaccharide vaccines already exist for serogroups A, C, Y and W-135, but there are currently no vaccines available for serogroups predominant in the US and Europe. Indeed, the use of capsular polysaccharides as a vaccine to prevent menB disease has been a problem.

Thus, as a vaccine the use of yen menin GT discharge LPS of sufficient antigenicity and in particular the decrypted form is a promising alternative to provide the desired vaccine coverage, especially in serum group B.

As mentioned in the introduction above, the major components of the cell walls of Gram-negative non-intestinal bacteria such as Neisseria , Bordetella , Haemophilus and Moraxella are lipooligosaccharides (LOS) rather than true LPS. to be. Nevertheless, for the purposes of the present application, the term LPS will be understood as including LOS. LOS constitutes a specific subclass of LPS. The terms "meningococcal LPS" and "meningococcal LOS" are used interchangeably later.

Figure 1 shows a graphical representation of the yen menin structure of GT discharge LOS. LOS is composed of branched oligosaccharides consisting of 5 to 10 monosaccharides bound to lipid A by KDO. The inner core consisting of lipid A and two KDOs, two heptoses (Hep I and II), and N-acetylated glucoseamine (GlcNAc) is conserved intraspecies. The rest of the oligosaccharide chain constituting the outer core (α-chain bound to HepI; β-chain bound to position 3 of HepII; and γ-chain bound to position 2 of HepII) are immunogenic (IT Variable).

Enmeningitidis LPS can be classified into 13 immunotypes based on its responsiveness to a series of monoclonal antibodies (Achtman et al, 1992, J Infect. Dis. 165: 53-68). Differences between immunotypes result from changes in the composition and form of the oligosaccharide chains. This is shown in the table below.

As indicated in the table above, phospho ethanol amine (PEA) substitutes the Glc of the β-chain at position 3 of HepII at LOS L1, L3, L7 and L8. PEA binds to positions 6 or 7 in LOS L2, L4 and L6. LOS L2, L3, L4, L5, L5, L7 can also be siallyylated with N-acetyl neuramic acid on the terminal galactose (Gal) of the α-chain.

Immunotype L1-L8 is essentially associated with serogroups B and C, while immunotype L9-L12 is predominantly found in serogroup A.

Although any LOS can be deciphered evenly, it may be advantageous to use LOS L8 in the complex of the present invention, since the latter is further intended for vaccines. Indeed, the complete structure of the LOS L8 α-chain is common for all immunotypes whose structure has been identified so far (Kahler & Stephens, 1988, Crit. Rev. Microbiol. 24: 281).

Meningococcal strains often express several immunotypes, the presence of which may be affected by the culture conditions. If particular interest is in LOS L8, it may be desirable to extract the LOS from strains known to predominantly express the L8 immunotype, or even better, exclusively express it. Strain A1 of serotype A (also referred to as 2E), strain M978 of serotype B (Mandrell & Zollinger, 1977, Infect. Immun. 16: 471; Gu et al, 1992, J. Clin. Microbiol. 30: 2047- 2053; Zhu et al, 2001, FEMS Microbiol. Lett. 203: 173), strain 8680 (Dominique Caugeant collection) of serotype B and strain 8532 (US Pat. No. 6,476,201) are suitable for this purpose. These strains can be obtained from the scientific community (US Pat. No. 6,531,131).

Monoclones specific for LOS L8 include Mab 2-1-18 (Moran et al, 1994 Infect Immun. 62: 5290-5295; Mandrell et al, 1986, Infect Immun. 54: 63-69) Mab 6E7-10 (Braun et. al, 2004, Vaccine 22: 898-908) Mab 4387A5 and 4385G7 (Andersen et al, 1995, Microb. Pathog. 19: 159-168; Gu et al (supra)).

For use in the complexes of the present invention, LPS can be obtained by conventional means; In particular, it can be extracted from Gram-negative bacterial cultures and then purified according to traditional procedures. Numerous explanations for such a process can be found in the literature. This is described, for example, in Gu & Tsai, 1993, Infect. Immun. 61 (5): 1873, Wu et al, 1987, Anal. Biochem. 160: 281 and US Pat. No. 6,531,131, all of which are incorporated by way of example only. LPS formulations can also be quantified according to procedures well known in the art. A convenient method is KDO preparation using high performance anion exchange chromatography (HPAEC) PAD.

LPS can be complexed to the compounds of the invention by themselves or in conjugated form. LPS conjugates typically comprise LPS to a carrier molecule, such as a polypeptide or peptide; It can be prepared via direct covalent bonds or by covalent bonds using chemical spacers / linker molecules. Examples of the carrier molecule has a whooping cough, diphtheria or tetanus toxin, and modified outer membrane protein (OMP), for example, yen menin GT discharge of OMP1 or OMP2 / 3. Numerous descriptions of such bonding processes can be found in the literature. US Patent No. 6,531,131 is cited by way of example only.

When used in conjugate form, the LPS is advantageously conjugated before complexing to the compounds of the present invention. Unconjugated LPS is also suitable.

The invention also

a method for deciphering gram negative bacteria LPS, comprising (i) mixing gram negative bacteria LPS and (ii) a compound of the present invention; And

a process for preparing an LPS-peptide complex comprising (i) mixing gram negative bacteria LPS and (ii) a compound of the present invention together

It is about.

For use in the process of the invention, the two components are advantageously in a liquid medium, suitably in water. LPS and compound solution are advantageously sterilized before mixing. The preparation method is advantageously carried out under sterile conditions. Upon mixing, a precipitate containing the complex is formed. This may be recovered by centrifugation and one or more washing steps may be performed as needed.

As mentioned above, the LPS-peptide complexes of the present invention are useful in that they can be safely administered to mammals. Indeed, LPS is so detoxified that no side effects occur upon administration. As an example, LPS-peptide complexes which exhibit an exotherm limit of greater than 1, preferably 10 ng / ml / kg, in rabbit pyrogen assays are suitable. Alternatively or in addition, mention may be made of LAL analysis. Since vaccines containing LPS ranging from 3,000 to 5,000 LAL endotoxin units have already been approved for human administration (Frederiksen et al, 1991, NIPH Annals 14 (2): 67), the dose of the vaccine of the invention is 5,000 LAL endotoxins. It can be foreseen that less than units, for example, less than 3,000, 2,000, 1,000 or 500 LAL endotoxin units can be safely represented.

Thus, as an example, complexes representing, for example, 100 endotoxin units (EU) / μg in the LAL assay may be allowed to administer 20 μg doses. This can be achieved by the complex as the complex of the present invention may exhibit LAL activity of less than 50 EU / μg, often less than 20 EU / μg.

Moreover, the LPS-peptide complexes of the present invention are stable even under physiological conditions. "Stable" means that the detoxification state of the LPS in the complex remains constant over time, for 3, 6, 12 or 18 months. This can be monitored by evaluating the readability ratio from time to time, ie in one or more of the assays listed above. No significant difference in readability ratio with time was observed.

The LPS-peptide complex of the present invention is also useful in that it can induce an immune response against Gram-negative bacteria. This can be demonstrated by administering the complex to a mammal, such as a rabbit, mouse or human, followed by ELISA analysis of the serum to reveal the presence of antibodies (eg immunoglobulin G or M) specific for LPS. Advantageously, the immune response (antibody induced) can have bactericidal and / or opsonin activity.

The ability of the immune response induced by the complex of the invention to protect against Gram-negative bacterial infection can be assessed in a suitable animal model specific for the current bacterial species or disease. Selecting a known animal model for a particular bacterium or disease is within the skill of those skilled in the vaccine art.

For example, the Yen menin GT the ability of the immune response induced by the complexes of the invention to protect against discharge can be evaluated in a mouse intraperitoneal infection model (Schryvers et al, 1989, Infect Immun 57 (8):.. 2425 And Danve et al, 1993, Vaccine 11 (12): 1214). This can also be assessed in humans by measuring the bactericidal activity of human serum after complex administration. Indeed, this test has been proposed as a surrogate protection test for at least enmeningitidis serogroup B (Holst et al, 2003, Vaccine, 21: 734). Human serum bactericidal activity (SBA) titers of 4 or higher have been shown to correlate with protection.

In view of the above, the present invention also

(i) the use of the LPS-peptide complex of the invention for the treatment or prevention of Gram-negative bacterial infections;

(ii) a pharmaceutical (vaccine) composition comprising the LPS-peptide complex of the invention and a pharmaceutically acceptable diluent or carrier;

(iii) use of the LPS-peptide complex of the present invention in the manufacture of a medicament for the treatment or prevention of Gram-negative bacterial infections;

(iv) inducing an immune response against Gram-negative LPS or Gram-negative bacteria in the mammal, comprising administering to the mammal an effective amount of the LPS-peptide complex of the present invention; And

(v) administering a therapeutically effective amount of the LPS-peptide complex of the present invention to an individual in need thereof for treating or preventing a Gram-negative bacterial infection.

It is about.

The vaccine composition of the present invention may be administered by any conventional route, in particular by systemic or intramuscular route; It may be administered in a single dose or in one or several repeated doses, for example two or three times, sometimes at intervals of, for example, 1, 2, 3, 6, 10, 12 months. Vaccine compositions of the invention may be formulated, typically in liquid form. If necessary, an adjuvant may be added to the vaccine composition of the present invention; It has been pointed out that the complexes of the present invention may be immunogenic enough to not require the presence of adjuvant in the vaccine composition.

Suitable dosages vary depending on various parameters, such as the individual being treated (adult or child), the manner and number of administrations, and the state of LPS detoxification as can be determined by one skilled in the art. In general, the dosage for human adults should not exceed 10,000; Advantageously 8,000; Preferably 5,000; More preferably 1,000; Most preferably 500 LAL endotoxin units are indicated. In the LAL assay, the value measured for the complex of the present invention may typically be as low as 10-20 EU / μg. Thus, the dose may contain 1 to 500, advantageously 2.5 to 100, preferably 10 to 50, more preferably 15 to 30 μg.

It should be understood that by convention, the amount of complex is always expressed as the LPS content. Thus, by way of example only, “50 μg of complex” actually means 50 μg of LPS in the complex formulation.

The examples reported below further illustrate the invention with reference to the following figures.

Figure 1A shows the structure of the LPS L8 yen of menin GT display. Kdo represents 2-keto, 3-deoxy octulonic acid; Hep is heptose; Glc is glucose; Gal is galactose; GlcNAc represents N-acetylated glucoseamine.

Figure 1B shows the reaction occurring when acetic acid and LPS treatment.

2A-2C show HPLC chromatograms obtained at 214 nm using a composition essentially comprising SAEP2-L2 peptide in monomeric form (2A), parallel dimer form (2B) and inverse parallel dimer form (2C). Indicates. The coordinates are time (minutes) and absorbance unit (AU).

FIG. 3 shows the HPLC chromatogram obtained at 214 nm using a composition comprising SAEP2-L2 peptide in monomeric form, parallel dimer form and inverse parallel dimer form.

4A-4C show ¹ H NMR spectra obtained using a composition comprising essentially SAEP2-L2 peptides in monomeric form (4A), parallel dimer form (4B), and inverse parallel dimer form (3C). In all of these, a peak at 1.9 ppm indicates that the peptide is present in acetate salt form.

5A-5C show magnified views of the ¹ H NMR spectral portions of FIGS. 4A-4C contained between 6.5-7.5 ppm.

FIG. 6 shows the 6.5-7.5 ppm region of the ¹ H NMR spectrum obtained using the composition comprising SAEP2-L2 peptide in monomeric form, parallel dimer form and inverse parallel dimer form.

7A-7C show the MALDI-ToF spectra of graduation standards 7A, parallel dimers 7B and inverse parallel dimers 7C.

8 shows an HPEAC-PAD chromatogram of LPS hydrolyzed by acetic acid treatment.

Example  One: SAEP2 - L2  parallel Dimeric  Produce

1.1. synthesis

Synthesis of the corresponding linear monomers is carried out on resin supports such as polyoxyethylene glycol activated polystyrene, or activated polyacrylamides (these are Atheron & Shepard: in Solid phase peptide synthesis, 1989, IRL press, Oxford U). Solid phase using a computer-driven automated synthesizer Milligen 9050 (Milipore Inc.), which is run using a column containing), as reported by, which is activated according to the selection of the first amino acid of the selected peptide sequence. Perform.

The synthesis period proceeds step by step according to the reported linear sequence. This is done in pure solvent dimethylformamide (DMF). Side protected, activated amino acids are used.

The thiol group of the Cys residue at position 10 (Cys-10) is protected with acid labile group trityl (triphenyl-methyl derivative, Trt). The thiol group of position 4 Cys residue (Cys-10) is protected with acid resistant group S-acetamido-methyl (Acm).

All amino acids are activated on the -COOH side by O-penta-fluorophenyl-phosphate esters (O-Pfp-derivatives). This is temporarily protected on the -NH ₂ side by 9-fluorenyl-methyloxy-carbonyl ester (Fmoc-derivative).

Once synthesized, the protected peptide is cleaved from the resin support using 95% TFA in the presence of 2-5% (v / v) of remover ethanedithiol. Under these conditions, the thiol group of Cys-10 is deprotected while the thiol group of Cys-4 remains Acm-protected. The free, Acm-protected peptide is concentrated by vacuum evaporation and then recovered by precipitation with ether at 80% (v / v) final concentration.

The Cys-4 protected, Cys-10 deprotected peptide is dried under vacuum, then dissolved in water at a concentration of 1-10 mg / ml and adjusted to pH 7.50 with 0.1M aqueous ammonia. To achieve dimerization through the Cys 10 residue, oxidation is carried out by vigorously stirring the aqueous solution at 4 ° C. for 18-24 hours under a pressure of 1 Atm. Complete oxidation of the thiol group is determined by Elman colorimetric analysis.

The partially oxidized peptide in the solution at the concentration of 1-10 mg / ml is then treated for deprotection of the remaining Cys-4 S-Acm functional groups. To this end, the peptide solution is added with mercury acetate at a final concentration of 0.1 M, using 2 to 5% (v / v) phenol as the remover. The solution is again vigorously stirred at 20 ° C. for 18 to 24 hours under a pressure of 1 Atm. Complete oxidation of the thiol group is determined by Elman colorimetric analysis.

1.2. refine

In order to remove the low-MW molecules contained in the peptide formulation (removal agent, mercury acetate, etc.), the latter is applied on a reverse phase column Sep-Pack (Millipore) running under a pressure of 1 Atm. In an aqueous solvent, the peptide is held on the column by hydrophobic binding force while flowing all of the water soluble low-MW molecules. The peptide is then eluted with a mixture of methanol-water 50-70% (v / v). Peptides eluted in the alcoholic solvent are recovered by vacuum concentration and again dissolved in water to the desired concentration.

The final purification was performed in HPLC using a 0-100% linear gradient of solvent A (0.1% TFA (trifluoroacetic acid) in water) and solvent B (80% nitrile acetate in water) (dimensions = 250 x 4 Mm). Under these conditions, the parallel dimer elutes as a single sharp peak. Recover the peak fractions.

The formulation is maintained in lyophilized form at +2 to + 6 ° C. under neutral gas, argon or nitrogen.

1.3. Refined Peptide  Specialization

1.3.1. Amino acid composition

The amino acid composition is analyzed by the Pico-Tag method (Millipore). The results are reported in the table below.

amino acid Theoretical value (mol / mol) Actual value (mol / mol) Lee Sin 6.0 5.90 Threonine 2.0 2.00 Phenylalanine 2.0 2.05 Leucine 6.0 6.10 Cysteine 4.0 3.85

1.3.2. Molecular mass

Molecular mass is measured by ion cyclotron resonance (ICR). Found 2,387.33 ± 0.3 AMU, consistent with elemental structure C ₁₁₀ H ₁₉₀ O ₂₄ N ₂₆ S ₄ of the peptide formula.

Example  2: SAEP2 - L2  Monomer and Reverse Parallel Dimeric  Produce

2.1. synthesis

Synthesis of the linear monomer is carried out as in Example 1 except that different methods are used to protect thiol groups of cysteine residues: both Cys-4 and -10 are acid labile group trityl (triphenyl-methyl, Trt) By protecting from his -SH group.

The protected peptide is cleaved from the resin support by 95% TFA in the presence of 2-5% (v / v) of remover ethanedithiol. Under these conditions, the thiol groups of both Cys-4 and 10 residues are deprotected. The cleaved and deprotected peptides are then concentrated under vacuum evaporation and recovered by precipitation with 80% (v / v) ether.

The deprotected peptide is dissolved in water at a concentration of 1-10 mg / ml and the pH is adjusted to 7.50 with 0.1M aqueous ammonia.

The oxidation is then carried out by vigorously stirring the aqueous solution at 4 ° C. for 18-24 hours under a pressure of 1 Atm. Complete oxidation of the thiol group is determined by Elman colorimetric analysis.

2.2. Peptide  refine

The peptide in solution actually constitutes a mixture of cyclic monomers (about 40%) and antiparallel dimers (about 60%). Each form is purified by preparative reverse phase HPLC chromatography. Indeed, the forms elute at different residence times, each with a single sharp peak, so that the cyclic monomer can be separated from the antiparallel dimer. The antiparallel dimer elutes at a lower residence time. This is consistent with the different molecular symmetry of the two dimers. The anti-parallel peptide can take lower minimum energy in an aqueous solvent, thanks to its lower side chain internal steric hindrance, similar to the "trans" to "cis" form of any other isomeric presence.

All formulations are kept in lyophilized form at +2 to + 6 ° C. under neutral gas, argon or nitrogen.

2.3. Inverse parallel Dimeric  Specialization

2.3.1. Amino acid composition

The amino acid composition is analyzed by the pico-tag method (Millipore). The results are reported in the table below.

amino acid Theoretical value (mol / mol) Actual value (mol / mol) Lee Sin 6.0 6.10 Threonine 2.0 1.95 Phenylalanine 2.0 1.90 Leucine 6.0 6.05 Cysteine 4.0 3.90

2.3.2. Molecular mass

Molecular mass is measured by ion cyclotron resonance (ICR). Found 2,387.30 ± 0.3 AMU, consistent with elemental structure C ₁₁₀ H ₁₉₀ O ₂₄ N ₂₆ S ₄ of the peptide formula.

Example  3: HPLC - Reverse , NMR  And MALDI - ToF  Monomer, parallel and antiparallel by mass spectrometry Dimeric  Further characterization

Dimeric parallel peptides as prepared in Example 1 and monomer and dimeric reverse parallel peptides as prepared in Example 2 were HPLC-reversed phases (FIGS. 2A-2C) and NMR (FIGS. 4A-4C and 5A-5C). It is characterized by.

3.1. HPLC - In reverse  Characterization by

Experimental conditions

This technique is performed on HPLC chains (Waters ^™ ) using Millennium software 32 V30501 (Waters ^™ ) for data acquisition. The analytical column Macherey Nagel ^™ ref720014.6 (Nucleosil 5 μm C18 100 Angstroms 250 × 4.6 mm) is run at 25 ° C.

30-40 μg of each lyophilized peptide was first diluted with 30 μl of water; To this was added 30 μl of 0.1% trifluoroacetic acid (TFA) in water.

A mixture of the monomeric, dimeric parallel and antiparallel peptides was also prepared by mixing 40 μl of powdered preparation of each peptide in 60 μl of water; To this was added 60 μl of 0.1% TFA in water.

The column is equilibrated with 20% mobile phase B (0.1% TFA in water, 80% CH ₃ CN). Once the sample was applied to the equilibrated column, the phase B gradient flows from 20 to 60% (1% B / min) in 40 minutes at a flow rate of 1 ml / min.

Detection is performed at 214 nm. The results are shown in Figures 2A-2C.

result

Each peptide is eluted with a different residence time. Under the experimental conditions described above, elution occurs with the following residence time (RT):

Monomer: RT = 28.283 min

Parallel Dimer: RT = 29.708 min

Reverse Parallel Dimer: RT = 22.059 min

The purity of each peptide formulation is confirmed using the HPLC-RP technique. The relative purity of each peptide is calculated by integrating the peak surface. This is shown as the peptide peak surface / surface of the entire peak.

In Figures 2A to 2C, it can be seen that the monomers and parallel and inverse parallel dimer formulations exhibited purity of 98, 96.9 and 97%, respectively.

3 shows the HPLC chromatogram of the mixture.

3.2. NMR Characterization by

Experimental conditions

¹ H NMR analysis (500 MHz, 25 ° C., HOD presaturation) is performed using a sample of peptide diluted in a H ₂ O / D ₂ O mixture (90/10 v / v). Brooker ^TM DRX500 spectrometer and associated software are used for data acquisition.

In more detail, peptide preparations maintained at −70 ° C. are used for analysis. A 0.5 mM dimer peptide solution is prepared with dilution of 1.33 g in 1 ml of H ₂ O. 144 μl of this solution is mixed with 16 μl of 99.9% DD ₂ O in a 3 mm NMR tube. For calibration, 0.075% (w / w) TSP-d4 (3- (trimethylsilyl) propionic acid-2,2,3,3- in H ₂ O / D ₂ O mixture (90/10 v / v) Sodium salt of d4; Aldrich ref 29304-0) external solution is used. The spectrometer is calibrated so that the unique resonance signal of the TSP-d4 is at 0 ppm.

result

In the experimental conditions used, the ¹ H NMR spectrum of the monomers and dimers ranges from 0 to 9.5 ppm and consists of three main regions:

-6.5 to 7.5 ppm;

-5.5 to 2.5 ppm; And

-2 to 0.3 ppm.

This is shown in Figures 4A-4C.

The ¹ H NMR spectrum of this monomer is characterized by the NMR pattern of five aromatic protons expected between 7.25 and 7.45 ppm under the experimental conditions reported above. In the experiments reported in FIG. 5A, the NMR pattern itself has a first multiplet of 7.25 to 7.35 ppm with an integral curve corresponding to 3H and a second multiplet line around 7.39 ppm with an integral curve of 2H. (Pseudo-triple line). The latter signal is characteristic of the monomer only.

The ¹ H NMR spectrum of the parallel dimer is characterized by a doublet signal between 7.10 and 7.25 ppm corresponding to four aromatic protons and a multiplet between 7.25 and 7.40 ppm with an integral curve of 6H. In the experiments reported in FIG. 5B, the 4H doublet was found to be centered at 7.185 ppm (peaks at 7.18 and 7.19 ppm).

The ¹ H NMR spectrum of the inverse parallel dimer is characterized by a doublet signal between 6.95 and 7.10 ppm corresponding to four aromatic protons and a multiplet between 7.10 and 7.30 ppm with an integral curve of 6H. In the experiments reported in FIG. 5C, the 4H doublet was found to be centered at 7.025 ppm (peak at 7.02 and 7.03 ppm).

As shown in FIG. 4C, the ¹ H NMR spectrum of the inverse parallel dimer also shows two upfields expected between (i) 0.40 to 0.65 (dual line) and (ii) 0.70 to 0.85 ppm (dual line). It is characterized by methyl resonance. In one experiment, these doublets were found centered around 0.42 and 0.68 ppm. They are neither observed in monomers nor in parallel dimers.

3.3. MALDI - ToF  Confirmation by mass spectrometry

Analysis by MALDI-ToF (mass assisted laser desorption ionization-time of flight) mass spectrometry allows the determination of the monoisotopic mass of the peptide. The technique does not distinguish between inverse parallel dimers and parallel dimers.

Experimental conditions

MALDI-ToF analysis is performed in a positive reflector mode using a Biflex III mass spectrometer (Bruker ^™ ) and associated software. Peptides are mixed with a substrate that absorbs laser energy (alpha cyano-4-hydroxy cinnamic acid).

The spectrometer is externally calibrated with a mixture of synthetic peptides (ACTH 18-39 (adrenal cortical stimulation fragments 18-39)) bombesin and somatostatin 28.

Saturated HCCA substrate solution is prepared by diluting 50 mg HCCA with 300 μl of 70% ACN (acetonitrile) 0.1% TFA (trifluoroacetic acid) in water.

1/2 saturated HCCA solution is further prepared with volume: volume dilution with 30% ACN, 0.1% TFA in water.

For calibrating, the first standard solution is first prepared in 0.1% TFA. The above is as follows:

Adrenal cortical stimulation fragment 18-39 (ACTH 18-39): 100 pmol / μl (0.247 mg / ml);

Bombesin: 100 pmol / μl (0.160 mg / ml); And

Somatostatin 28: 100 pmol / μl (0.31 mg / ml).

A second standard solution is prepared as follows:

-ACTH 100 pmol / μl 2 μl

Bombesin 100 pmol / μl 4 μl

Somatostatin 100 pmol / μl 4 μl

ACN 30%, TFA 0.1% 50 μl

The 1 mg / ml peptide solution in water is diluted to 0.02 mg / ml with 30% ACN, 0.1% TFA in water.

Calibrate and peptide samples are diluted volume: volume with 1/2 saturated HCCA solution. About 1 μl of droplets are deposited on a steel target (Bruker ^™ ) and dried by evaporation.

result

The results are shown in Figures 7A-7C.

The theoretical single isotope mass calculated by the software based on the amino acid sequence is as follows:

ACTH 28 M + H ⁺ = 2465.199 Da

Bombesin M + H ⁺ = 1619.823 Da

Somatostatin 28 M + H ⁺ = 3147.471 Da

SAEP2-L2 M + H ⁺ = 2388.35 Da.

Means were adjusted to be ± 2 Da at theoretical mass.

As shown in FIG. 7A, the experimental values found for the graduated peptide are 2465.225, 1619.814 and 3147.454 Da, respectively. (Thus, the internal measurement deviation is (0.026 + 0.009 + 0.017) /7232.493 = 7.2 ppm (approved <50 ppm)).

As shown in FIGS. 7B and 7C, the experimental values found for the parallel and antiparallel dimer formulations are 2388.449 and 2388.532 Da. The value is within the same range (+2 Da) around the theoretical range. This means that the sample contains what is expected.

Example  4: LPS L8 Of Peptide  Preparation of I "Complexes / Agglomerates

4.1. LPS L8 Manufacture

4.1.1. Meninge Culture

Preculture:

The only L8 form only to a strain known to express the LPS yen using frozen samples 2 ㎖ of fermentation seed from menin GT display underwater glucose solution (500 g / ℓ) 4 ㎖ supplemented mu Ella-Hinton (Mueller Inoculate 2 liters of ethylene containing 200 ml of Hinton Broth. Repeat the above procedure four times. Erlens are incubated at 36 ± 1 ° C. for 10 ± 1 h with stirring (100 rpm).

culture:

The erene contents are pooled together and the preculture is supplemented with 400 ml of glucose solution (500 g / l) and 800 ml of amino acid solution. The formulation is used to inoculate the Mueller Hinton broth in a 30 L incubator (B. Braun ^™ ) at an initial OD _{600 nm} close to 0.05. Fermentation is performed overnight at an initial flow rate of 36 ° C., pH 6.8 ± 0.2, 100 rpm, pO ₂ 30%, and 0.75 L / min / L air. After 7 ± 1 hour (OD _{600 nm up to} about 3), the culture is fed by MH broth at a flow rate of 440 g / h. If the glucose concentration is less than 5 g / l, fermentation is stopped. Usually, the final OD _{600 nm} comprises 20-40. Cells are harvested by centrifugation at 7000 g, 4 ° C. for 1 hour 30 minutes. The pellet is kept frozen at -35 ° C.

4.1.2. LPS Tablets

First phenolic extraction

The pellet is thawed and suspended with 3 vol. Phenol 4.5% (v / v) and stirred vigorously at about 5 ° C. for at least 4 hours.

The bacterial suspension is heated at 65 ° C. and then mixed with 90% v / v of phenol at 65 ° C. The suspension is vigorously stirred at 65 ° C. for 50-70 minutes and then cooled to about 20 ° C.

The suspension is centrifuged for 20 minutes at 11 000 g, about 20 ° C. The aqueous phase is collected and stored. The phenol phase and the intermediate phase are recovered and a second extraction is performed.

Second phenolic extraction

The phenol phase and the intermediate phase are heated at 65 ° C. and mixed with a volume of water equivalent to the volume of the aqueous phase previously collected. The mixture is vigorously stirred at 65 ° C. for 50-70 minutes and then cooled to about 20 ° C. The mixture is centrifuged at 11 000 g, about 20 ° C for 20 minutes. The aqueous phase is collected and stored. The phenol phase and the intermediate phase are recovered to carry out a third extraction.

Third Phenol Extract

Repeat the second extraction process.

dialysis

The three aqueous phases are dialyzed separately for 40 liters of water overnight. The dialysates are collected together. The dialysate pool is adjusted to 20 mM Tris, 20 mM MgCl ₂ (1 volume per 9 volumes of the dialysate pool). Adjust pH to 8.0 ± 0.2 with NaOH 4N.

DNAse  process

DNAse 250 UI is added per gram of treated bacterial pellets (wet weight). The formulation is stirred at 37 ± 2 ° C. for 55-65 minutes. Adjust the pH to 6.8 ± 0.2. The formulation is filtered over a 0.22 μm membrane.

Gel filtration

The formulation is purified on Sephacryl S-300 column (5.0 × 90 cm; Pharmacia ^™ ).

first Alcohol  Sedimentation

A powder of MgCl ₂ , 6H ₂ O, was added to the pooled LPS containing fractions to reach a MgCl ₂ concentration of 0.5M and dissolved with stirring.

While stirring at 5 ± 3 ° C., dehydrated absolute alcohol is added to a final concentration of 55% (v / v). Stirring is performed overnight at 5 ± 3 ° C. and then centrifuged at 5,000 g for 30 minutes at 5 ± 3 ° C. Discard the supernatant and extract the pellet a second time.

second Alcohol  Sedimentation

The pellet is resuspended with at least 100 ml of 0.5 M MgCl ₂ with stirring. Repeat the preceding procedure. The pellet is resuspended with at least 150 ml of water.

Final steps

The gel filtration is repeated and the LPS containing fractions collected together are final sterilized by filtration (0.8-0.22 μm) and maintained at 5 + 3 ° C.

As a preliminary control, the LPS preparations are analyzed by SDS-PAGE electrophoresis. When nitric acid is dyed, a single large band appears. This indicates that at least the formulation contains no presence other than LPS L8.

The purification procedure described above makes it possible to obtain about 150 mg of LPS L8 (yield about 50%) per liter of culture.

4.1.3. LPS L8  Quantification: HPAEC - PAD On by KDO  pharmacy

Bibliographical references to this technique are described in [Kiang et al, (1997) High Performance Anion Exchange Chromatography (HPAEC) Determination of 2-keto-3-deoxyoctuulosonic acid (KDO): Stability and Optimal Hydrolysis of KDO Investigate Conditions Anal. Biochem. 245: 7.

As shown in Figures 1A-1B, the LPS contains 2 KDO units in its structure, one in the lateral position.

LPS quantification is achieved through the preparation of flanking KDO units that are liberated upon weak acid hydrolysis (see FIG. 1B).

Acid hydrolysis

A sample of the LPS formulation obtained after the final diafiltration of section 4.1.2. Was recovered and 400 μl in a Dionex ^™ 1.5 mL flask so that the LPS concentration of the sample was within the etalon range (1.4-72.1 μg / ml). Dilute with water to the final volume of.

The KDO etalon range as well as the sample to be quantified are treated as follows: 100 μl of hydrolysis solution (5% acetic acid; 20 μg / ml of glucuronic acid (GlcA)) is added. Hydrolysis is carried out at 100 ° C. for 1 hour. The flask is then dried at 40 ° C. under nitrogen and filled with 400 μl of water.

pharmacy

The technique is performed on HPAEC chains (Dionex ^™ ) using Chromleon Dionex ^™ software for data acquisition. Analytical column Carbopack PA1 4 × 250 mm (Dionex ^™ ) is run at 30 ° C.

The column is equilibrated with elution solution (NaOH 75 mM, AcONa 90 mM). 100 μl of sample is injected into the column. The elution flow rate of 1 ml / min is then run on the column for 22 minutes.

The chromatogram of the LPS sample is shown in FIG. 8. The amount of KDO present in the sample is measured by the integration of the KDO peak. Since 1 mole of KDO liberated by hydrolysis corresponds to 1 mole of LPS, the LPS concentration of the initial formulation can be measured.

4.2. Peptide  Produce:

Peptides are prepared according to the procedures described in Examples 1 and 2 above.

4.3. LPS L8 Of Peptide  Preparation of I "Complexes / Agglomerates

Purified LPS is used as mock-solution at 1 mg / ml in pyrogen-free sterile water (Milli Q quality, adjusted to pH 7.2, Rimulus negative). The translucent mock-solution is sterilized by filtration using a 0.22 μm membrane.

A 1 mg / ml peptide SAEP2-L2 solution in pyrogen-free sterile water (Milli Q quality, adjusted to pH 7.2, Rimulus negative) is also sterilized by filtration using a 0.22 μm membrane.

All next steps are performed under sterile conditions.

One volume of the LPS mock-solution is added to one volume of the peptide SAEP2-L2 solution. A precipitate (modified toxin complex) appears immediately. Stirring is performed at room temperature for 5 minutes. The precipitate is allowed to stand overnight at +4 ° C.

The precipitate (denaturated toxin) is then recovered by centrifugation at 3000 rpm for 10 minutes. Discard the supernatant.

The pellet is washed with 1 volume of pyrogen-free sterile water (Milli Q quality, adjusted to pH 7.2, Rimulus negative). Repeat the centrifuge / wash step five times.

Finally, the pellet is resuspended in a pyrogen-free sterile water (Milli Q quality, pH 7.2) at a concentration of about 1 mg / ml based on the wet weight of the precipitate. The suspension is stored at +4 ° C. KDO preparations are achieved for the determination of the LPS content and the suspension is adjusted to a complex of 0.50 mg / ml, represented as an LPS content, for example.

Below In the embodiment Tested LPS Peptide  The complex is as obtained in section 4.3 unless otherwise indicated. LPS  Inverse parallel Dimer  It is a complex. Thus, the specific complex LPS Peptide  It is called a complex.

In a similar manner, as obtained in section 4.2 LPS Simply LPS It is called.

LPS Wow LPS - Peptide  Comparison of the complexes was also used in the preparation of the complex LPS  Achieve using lots.

Example  5: LPS Peptide  Assessment of the Detoxification of Complexes

Several analyzes are used to assess detoxification.

5.1. Rimulus Amebosite Lysate ( LAL ) analysis

In this assay, the ability of SAEP2-L2 antiparallel and parallel dimers and SAEP2-L2 cyclic monomers to decode LPS is compared. To this end, LPS peptide complexes carrying the parallel dimers or monomers are prepared as reported in Example 4 for the LPS antiparallel peptide complex.

LAL is a very sensitive test used to detect and quantify endotoxins of Gram-negative bacteria. The test is based on the properties of amebosite lysate protein from the crab ( Limulus polyphemus ) which causes coagulation in the presence of endotoxin.

Assessment of the LPS endotoxin activity is performed using endpoint endpoints, as described in the European Pharmacopeia techniques, Edition 5.0, paragraph 2.6.14. Because of this, kit QCL-1000 ref 50-647 U (Cambrex-BioWhittaker ^™ ) (linear band of the kit: 0.1 to 1 UI / ml) as well as a positive control (E. coli endotoxin, 4 10 ³ EU / ml, Sigma ).

Dilution of (i) the sample to be tested, (ii) standard and (iii) positive control was achieved using dilution buffer (Cambrex-BioWhittaker ^™ ), each range: 1/10 to 1/10 ⁵ ; 0.5 to 0.31 EU / ml and 1/10 ⁴ to 1.8 10 ⁴ .

50 μl of sample, standard and positive control dilutions are dispensed per well of 96 flat bottom well ELISA plates. 50 μl of lysate is added per well. Incubation is carried out at 37 ° C. for 10 minutes. 100 μl of p-nitroaniline chromogen substrate is then added. Incubation is carried out at 37 ° C. for 6 minutes. The chromogen reaction is stopped by adding 100 [mu] l of 25% frozen acetic acid in water. The plate is read by spectrophotometry at 405 nm.

Results are expressed in μg of endotoxin unit (EU) / complex. The above is shown in the following table. Detoxification ratios are set by LPS / LPS peptide complex ratio and expressed in logarithm units.

Range EU / μg Average value EU / μg Detoxification Ratio Represented as algebra LPS (6 times analysis) 1-12 10 ⁴ 25,000 LPS-antiparallel peptide complex (13 assays) 5-32 12-20 1,250-2,000 3.5 LPS-Parallel Peptide Complex 30-40 30-40 600-800 3 LPS-monomer peptide complex > 2000 > 2000 <12.5 <1

As can be seen, the dimeric form of the SAEP-L2 peptide is more effective for decoding LPS than the cyclic monomeric form.

5.2. In the rabbit Heating source  exam

Rabbits are known to be animal species that are equivalent to humans, with susceptibility to the pyrogenic effects of LPS. The pyrogen test measures the rise in body temperature induced in three rabbits by intravenous (IV) injection of a sterile solution of the substance to be tested. The tests, readings and calculations are carried out in accordance with the European Pharmacopoeia (Edition 5.0, paragraph 2.6.8). The temperature rise is determined according to the response of the combined body temperatures; If the combined reaction does not exceed 1.15 ° C., it is a match, and if the combined reaction exceeds 2.65 ° C., it is a mismatch. For this test, the exothermic limit is set low between 1.15 and 2.65 ° C.

As found, the limiting exothermic dose (IV) in rabbits corresponds to 0.025 ng / kg (LPS) and 10 to 25 ng / kg (LPS-peptide complex). These results indicate that the LPS-peptide complex is less pyrogenic than LPS when provided by the intravenous route. As measured in this test, the detoxification ratio (LPS-peptide complex / LPS) is between 400 and 1,000.

5.3. Acute Toxicity Assay: D- Galactosamine Sensitized  In the mouse LD50

References to this analysis are described in Galanos et al, 1979, PNAS 76: 5939; Baumgartner et al, 1990, J. Exp. Med. 171 (3): 889 and US Pat. No. 6,531,131.

A group of 8 main female inbred mice was injected by the intraperitoneal (IP) route with increasing doses of LPS or LPS-peptide complex (0.5 mL), followed immediately by D-galactosamine (15) by the IP route. Mg / 0.2 ml) (toxicity of LPS increases approximately 1,000-fold with D-galactosamine treatment, making the model very sensitive). Track mortality for 4 days.

LD50 observed for LPS was 3.6 ng / mouse (1.91 to 6.70 ng / mouse); For LPS-peptide complexes, what was observed was 1 μg / mouse (0.2-5 μg / mouse), indicating that the detoxification ratio (LPS-peptide complex / LPS) is about 250 (100-1000).

5.4. Peptides and  At ignition LPS Weakening of the pre-inflammatory effect of

In order to assess the extent to which the LPS-peptide complex attenuates the LPS-induced toxic effects, the effect of the LPS-peptide complex on the release of pre-inflammatory cytokines is monitored (evaluated) in vitro and in vivo assays.

In vivo: Cytokine (IL6 and TNF α) release in mouse serum immunized with LPS or LPS-peptide complex is compared by ELISA. Blood samples are collected 90 minutes after SC immunization, which is the optimal time for release of the cytokines. C3H / HeOuJ, TLR4-/-, C3H / HeN and CD1 mouse strains are tested. The first two are not sensitive to LPS or LPS-peptide complexes. Both third and fourth were found to be LPS-sensitive. CD1 mice have been found to be more LPS-peptide complex-sensitive than others and are therefore selected for further experiments to maintain the most stringent conditions.

Ex vivo: Cytokines (IL6, IL8 and TNFα) are released from human whole blood cell cultures stimulated at 37 ° C. for 24 hours and compare different concentrations of LPS or LPS-peptide complexes.

5.4.1. In vivo analysis

CD1 mice are administered subcutaneously (SC) with either (i) 10 μg LPS or (ii) 10 μg LPS-peptide complex. The mice are bled 90 minutes after infusion. IL6 and TNFα release are measured in these sera by ELISA.

Cytokines  For secretion ELISA  detection

ELISA is performed using OptEIA mouse IL6 and TNFα sets (Pharmingen), which sets capture antibodies (anti mouse cytokines), detection antibodies (biotinylated anti mouse cytokines), avidin-horseradish peroxidase conjugates, and Includes standard (recombinant cytokines) (all from Pharmingen).

Anti-mouse IL6 and TNFα antibodies are diluted 1/250 with 0.1 M carbonate buffer, pH 9.5 (Sigma). For each assay, 100 μl of antibody dilution is dispensed per well of Maxisorp NUNC 96 flat bottom well ELISA plate. Incubate the plate overnight at +4 ° C.

Plates are washed with 0.05% Tween 20 in PBS. 200 μl of PBS, 0.5% bovine serum albumin (BSA) saturated buffer is then added per well. Incubation is performed at room temperature for 1 hour. Plates are washed with 0.05% Tween 20 in PBS.

Recombinant IL6 or TNFα cytokine dilutions are prepared with (i) 10% FCS in 1% RPMI medium within the 4,000 to 62.5 pg / ml standard range. Dispense 100 μl of each dilution per well to establish a standard curve.

Serum dilutions were collected in 1% P.S. Prepared with glu 10% FCS. Serum from LPS injected mice is diluted 1/25 and 1/125. Serum of mice injected with LPS-peptide complexes is diluted 1/5 and 1/25. Dispense 100 μl of each dilution per well.

Incubation is carried out at room temperature for 2 hours.

Plates are washed with 0.05% Tween 20 in PBS. Biotinylated anti mouse cytokine antibodies and enzymes are diluted 1/250 with 10% calf fetal serum in PBS, respectively. 100 μl of each dilution is added per well. Incubation is performed at room temperature for 1 hour.

Plates are washed with 0.05% Tween 20 in PBS. 100 μl of tetramethylbenzidine (TMB) substrate (mixed TMB solution A and B (KPL) vol / vol) is dispensed into the wells. Incubation is carried out at room temperature for 10-30 minutes.

The reaction is stopped by adding 100 μl of 1M H ₃ PO ₄ per well. The plate is read at 450 nm. The results are shown in the table below.

Product injected into mouse IL6 release TNFα release Mean (pg / mL) n = 6 (log) Detoxification Ratio (Log Units) Mean (pg / mL) n = 6 (log) Detoxification Ratio (Log Units) LPS 4.7 4.1 LPS Peptide Complex 2.2 2.5 <1 > 3.1 Peptide <1 <1

The peptide alone does not induce IL6 or TNFα. The LPS-peptide complex allows about 100-fold translation (100-fold decreases IL6 secretion).

5.4.2. In vitro analysis

Preparation of Test Substance

LPS preparation (1 mg / ml) and LPS-peptide complex (500 μg / ml) are diluted to a concentration of 50 μg / ml with 10 mM Tris, NaCl 150 mM, 0.05% Tween 20, 5% sucrose, respectively. This is further diluted to 5 μg / ml with physiological saline.

A series of 1/5 dilutions are performed in AIM-V medium (Gibco (Invitrogen)) at a concentration of 2.56 10 ^-3 pg / ml for each test substance.

stimulus

Human blood collected on sodium heparin (25,000 U / 5 ml; sanofi-synthelabo) is diluted 1: 4 (vol: vol) with AIM-V medium and dispensed into Micronics ^™ tubes (400 μl / tube) do. 100 μl of test substance dilution is added. Peptide and buffer controls are tested at 1/20 dilution. The tube is incubated at 5% CO ₂ , 37 ° C. for 24 hours in a humid atmosphere.

Plasma recovery

The tube is then centrifuged at 500 g for 10 minutes. At least 200 μl of supernatant is withdrawn from each tube and kept frozen at −80 ° C. until titration.

Cytokines  Secretory ELISA  detection

ELISA is performed using OptEIA human IL6, IL8 and TNFα sets (from Pharmingen), which capture cells (mouse anti human cytokines), detection antibodies (biotinylated mouse anti human cytokines), avidin-horseradish peroxy, respectively Multidrug conjugates and standards (recombinant cytokines).

Anti-human IL6, IL8 and TNFα antibodies are diluted 1/250 in 0.1 M carbonate buffer, pH 9.5 (Sigma). For each assay, 100 μl of antibody dilution is dispensed per well of the Maxifthor NUNC 96 flat bottom well ELISA plate. Incubate the plate overnight at +4 ° C.

Plates are washed with 0.05% Tween 20 in PBS. 200 μl of PBS, 0.5% bovine serum albumin (BSA) saturated buffer is then added per well. Incubation is performed at room temperature for 1 hour. Plates are washed with 0.05% Tween 20 in PBS.

Recombinant IL6, IL8 or TNFα cytokine diluents were (i) 1,200-18.75 pg / ml; (ii) 800 to 12.5 pg / ml; And (iii) AIM-V media within the respective range of 1,000 to 15.87 pg / ml standard. Dispense 100 μl of each dilution per well to establish a standard curve.

Serum dilutions are prepared in AIM-V. Plasma recovered from LPS-stimulated blood is diluted 1/25 and 1/125. Dilutions from the blood in contact with the LPS-peptide complex are diluted 1/5 and 1/25. Dispense 100 μl of each dilution per well.

Incubation is carried out at room temperature for 2 hours.

Plates are washed with 0.05% Tween 20 in PBS. Biotinylated anti human cytokine antibodies and enzymes are diluted 1/250 with 10% calf fetal serum in PBS, respectively. 100 μl of each dilution is added per well. Incubation is performed at room temperature for 1 hour.

Plates are washed with 0.05% Tween 20 in PBS. 100 μl of tetramethylbenzidine (TMB) substrate (mixed TMB solution A and B (KPL) vol / vol) is dispensed into the wells. Incubation is carried out at room temperature for 10-30 minutes.

The reaction is stopped by adding 100 μl of 1M H ₃ PO ₄ per well. The plate is read at 450 nm.

result

The original result and cytokine release curve = f (LPS or complex concentration) do not allow comparison of different samples. The calculation of the detoxification ratio may exclude variability between blood donors and between trials. Only the linear part of the curve is considered in the calculation of the decoding ratio. The maximum IL6 release, in which linear progression is no longer observed, is measured and then the amount of material needed to elicit 50% of the maximum is calculated by linear regression.

The readout ratio is expressed as the ratio of the concentration of LPS-peptide complexes that induces 50% of the maximum IL6 release (ED ₅₀ expressed in pg / ml) versus that observed for LPS. The larger the ratio, the stronger the detoxification. The detoxification ratio is measured systemically using whole blood from several independent blood donors, so the results are averaged.

The readout ratio observed for the LPS-peptide complex is measured several times. Average data of 6 values obtained in IL6 release assay: 64 ± 20.

The IL6 release is associated with TNFα and IL8 secretion. Thus, the IL6 release assay is chosen to routinely assess the reduction in inflammation observed for LPS-peptide complexes.

5.5 Conclusion

The readout ratio is measured between 10 ² and 10 ³ according to the test. The readout values are summarized in the table below.

analysis LPS L8 LPS-peptide complex Detoxification Ratio LAL 25,000 EU / μg 12-20 EU / μg 1,250-2,000 Limited fever capacity (IV) in rabbits 0.025 ng / kg 10-25 ng / kg 400-1,000 Cytokine Release Test in Mice IL6 = 25,000 pg / ml IL6 = 10,000 pg / ml IL6 = 270 pg / ml IL6 = 100 pg / ml 100 In vitro analysis of IL6 release by human PBMC (ED ₅₀ : concentration of product inducing 50% of maximal IL6 release) ED ₅₀ = 2 pg / ml ED ₅₀ = 880 pg / ml 64 LD50 in Galactosamine-sensitized Mice 4 ng / souris 1 μg / souris (0.2-5) 250

Example  6: LPS Peptide  Complex Stability Study

The stability of the LPS-peptide complex is evaluated by studying for 6 months and measuring the readability ratio in two assays (in vitro IL-6 release by LAL and huPBMC). A pyrogen test can also be accomplished in rabbits.

6.1. Formulated LPS Peptide  In vitro stability of the complex

The stability of the formulated LPS-peptide complex is followed for 6 months at 5 ° C. Measurements are taken on days 0, 90 and 180 (6 months). The results are as follows.

IL6 release from human blood cells (detoxification ratio) LAL assay endotoxin (EU / μg) Pyrogen Test Exotherm limit as selected: 10 ng / ml / kg IV 0 3 months 6 months 0 3 months 6 months 0 3 months 6 months 125 40 163 14 58 10 C ^* C C C: Compliant

The detoxification ratio in the IL6 release test does not differ significantly after 3 and 6 months, indicating the stability of the LPS-peptide complex: LPS complexed with the peptide remains detoxified after 6 months at 5 ° C.

6.2. In physiological liquids LPS Peptide  In vitro stability of the complex

The purpose of the experiment is to demonstrate that LPS is not released when the complex is administered and that the detoxification rate does not decrease after contact with physiological liquids.

1 ml of LPS-peptide complex, mixed with 1 ml of human serum, is incubated at 37 ° C. Detoxification rates are assessed after 1 and 24 hours. Human serum and LPS peptide complexes as prepared in section 4.3. Are also tested in parallel.

No significant difference was observed in the detoxification evaluated by the two assays after 1 and 24 hours contact of the human serum with the LPS-peptide complex at 37 ° C., with results similar to the LPS-peptide complex control.

Example  7: LPS - Peptide  Immunogenicity of the Complex

7.1. LPS - Peptide  By complex In the rabbit  Induced term LPS  Bactericidal Activity of Antibodies

Immunization of three mature New Zealand rabbits was performed by intramuscular (IM) and subcutaneous (SC) routes (2 x 0.5 mL and 5 x 0.2 mL, respectively) using 100 μg of LPS-peptide complex in the presence of adjuvant. Perform. The rabbit is infused three times at three week intervals; The first is injected with complete Freund's adjuvant and the second and third with incomplete Freund's adjuvant. The rabbits are bled 2 weeks after the last injection. Controls are immunized with these peptides with adjuvant (71 μg, equivalent to the amount of peptide in 100 μg of LPS-peptide complex) using the same protocol.

Evaluated for yen menin GT display used for LPS production as described for the serum (SBA) the fungicidal activity of the sample as the external source of complement in the presence of a young rabbit serum described in Example 5.

SBA  analysis

Serum is heat inactivated at 56 ° C. for 30 minutes. The heat inactivated serum is then serially diluted twice (10 times) with Dulbecco's phosphate buffered saline containing Ca ⁺⁺ and Mg ⁺⁺ (volume per well: 50 μl) in wells of 96-well microplates.

Of 25 ㎕, mu Element exerts a log phase culture young rabbit serum of water and 25 of ㎕ Hinton broth (4.10 ³ CFU / ㎖) was yen menin GT discharge grown in each well. The plates are incubated for 1 hour at 37 ° C under shaking.

50 μl of the mixture from each well is plated on Mueller Hinton agar. Petri dishes are incubated overnight at + 37 ° C. under 10% CO ₂ atmosphere.

In each experiment, the controls included (i) a complement source without bacteria and antibodies (complement control), (ii) bacteria and heat inactivated complement, and (iii) bacterial and heat inactivated complement in the presence of antibody. do.

Bactericidal titers are reported as the highest relative serum dilution at which ≧ 50% lethality of bacteria is observed compared to the complement control.

SBA  result

The results are shown in the table below. High SBA titers are obtained by this complex. The specificity of the SBA reaction is confirmed by the disappearance of the reaction upon adsorption on the LPS of the serum (after three administrations).

rabbit# Preliminary immunized serum Immunized serum after 3 doses Immunized serum after 3 doses adsorbed on LPS LPS-peptide complex A 16 512 4 B 16 1,024 8 C 4 128 <4 Peptide D 4 16 4 E 16 16 8

7.2. LPS Peptide  Immune Responses Induced in Mice by Complexes

Ten 6 week old female outbred CD1 mice are immunized by the subcutaneous route (0.2 ml) with a 10 μg dose of LPS-peptide complex. The mice are injected twice at three week intervals. The mice are bled before each infusion and bleed 14 days after the last infusion. Inject control group to the control group.

In the first experiment, evaluated by an ELISA for antibody response and twice the yen menin GT discharge strain used for LPS production as the bactericidal activity of serum samples after the administration of embodiment described in Example 4 (homologous strain) and heterogeneous yen Menin evaluated for discharge GT strain [yen menin GT discharge group B strain RH873 (L4, 7, 8-type immune).

In a second experiment, the antibody response is assessed by ELISA and the opsonin activity of serum samples after two doses is assessed by FACS.

7.2.1. In the mouse LPS - Peptide  Immunogenicity of the Complex

term- LPS  Antibody ELISA Titer

Wells of 96-well microplates are coated with 100 μl of a 10 μg / ml LPS solution in Buffer 1 (PBS + 10 mM MgCl ₂ ). The plate was kept at + 37 ° C. for 2 hours; Then incubate overnight at +5 ° C.

The LPS solution is removed from the plate and the wells are saturated with buffer 2 (PBS + 1% milk + 0.05% Tween 20). Incubate the plate at 37 ° C. for 1 hour; Then wash with buffer 3 (PBS + 0.05% Tween 20).

Serum is diluted 12-fold serially directly in the wells using Buffer 2 (volume: 100 μl / well). Incubate the plate for 90 min at +37 ° C; Then wash with buffer 3.

100 μl of diluted goat anti mouse IgG (γ chain specific) or IgM (μ chain specific) peroxidase conjugate is added to each well. The plate is incubated for 90 minutes at 37 ° C. and then washed with buffer 3.

The reaction proceeds by adding 100 μl of tetramethylbenzidine substrate solution to each well. The plate is incubated at 37 ° C. for 20 minutes. 1 M HCl is added to stop the reaction and the absorbance is measured at 450 nm.

ELISA  result

Results are expressed in arbitrary ELISA units / ml (EU / ml) by comparison to reference serum.

In preliminary immunization experiments, ELISA assays are achieved using a pool of 10 sera. As shown in the table below, LPS-peptide complexes can induce high anti-LPS IgG titers and anti-LPS IgM after a single injection in mice (ELISA). Significant IgG potentiation was observed after the second infusion, while no significant IgM increase was observed.

Anti-LPS IgG (EU / mL) Anti-LPS IgM (EU / mL) After 1 dose After 2 doses After 1 dose After 2 doses LPS-peptide complex 1,800 22,000 280 550 Buffer <40 <40 <40 <40

In further immunization experiments, ELISA assays are performed individually. After the second infusion, 7 of 10 mice show high IgG and IgM titers. The overall mean titers in logarithms are about 3.7 and 2.8, respectively.

7.2.2. Bactericidal Activity of Mouse Serum

Bactericidal activity is measured as described in section 7.1.

2 40% of the time after administration the serum the homologous yen Show bactericidal activity (SBA titers ≧ 16) against Meningitidis strains. Four are bactericidal against heterologous strains.

7.2.3. Mouse serum Opsonin  activation

Opsonization  analysis

Opsonine activity is measured by flow cytometry (FACS) using human promyelocytic cell differentiated HL60 cells as effector and LPS coated latex fluorescent beads as targets.

The effector cells are differentiated into granulocytes after treatment with 100 mM dimethylformamide. The resulting cells are washed, resuspended in Hank's balanced salt solution and the concentration is adjusted to 2.5 × 10 ⁷ cells / ml.

Serum is heat inactivated at 56 ° C. for 30 minutes. In deep 96 well microplates, heat inactivated serum is serially diluted (three times) with Hank's balanced salt buffer containing Ca ⁺⁺ and Mg ⁺⁺ (volume per well: 300 μl).

20 μl of LPS coated latex fluorescent beads and 10 μl of young rabbit serum are added to each well as an external complement source. The plates are incubated for 30 minutes at 37 ° C under shaking.

50 μl of effector cell suspension is added to each well. The plates are incubated for 30 minutes at 37 ° C under shaking.

150 μl from each well is transferred to the second deep well and 400 μl of PBS + 0.02% EDTA is added to stop the reaction. The plate is centrifuged and washed twice with PBS + BSA buffer.

Phagocytosis of LPS coated beads by effector cells in the presence of antisera and external complement sources is measured by FACS.

Opsonine activity is expressed as the inverse of serum dilution giving a phagocytic product (PP) = 200. PP is measured as the ratio of the number of beads / macrophage x the number of fluorescent cells.

Control wells lacking antiserum and positive monoclonal antiserum are included in each experiment.

Opsonization  result

Eight out of ten mice show high opsonin activity (≧ 350).

<110> Sanofi Pasteur          Biosynth <120> Polymixin B analogs for LPS detoxification <130> PM0502 <150> EP05300270.5 <151> 2005-04-11 <150> EP05300319.0 <151> 2005-04-26 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> unidentified <400> 1 Lys Thr Lys Cys Lys Phe Leu Leu Leu Cys   1 5 10  

Claims (32)

SAEP II peptide dimers of formula (I) or (II) Formula I NH ₂ - A -Cys1- B -Cys2- C -COOH NH ₂ - A ' -Cys1- B' -Cys2- C ' -COOH Where The two Cys1 residues are joined together via disulfide bonds Two Cys2 residues are joined together via disulfide bonds; Formula II NH ₂ - A -Cys1- B -Cys2- C -COOH HOOC- C ' -Cys2- B' -Cys1- A ' -NH ₂ Where Cys1 residues are linked to Cys2 residues through disulfide bonds; A and A 'are independently a peptide portion of 2 to 5, preferably 3 or 4 amino acid residues, wherein the two or more amino acid residues are independently selected from Lys, Hyl (hydroxy-lysine), Arg and His; ; B and B 'are independently peptide portions of 3 to 7, preferably 4 or 5 amino acid residues, at least 2, preferably 3 amino acids independently selected from Val, Leu, Ile, Phe, Tyr and Trp Includes residues; C and C 'are optionally, independently amino acid residues or peptide portions of two to three amino acid residues; only The cationic amino acid residue / hydrophobic amino acid residue ratio (cationic / hydrophobic ratio) is 0.4 to 2. The method of claim 1, SAEP II peptide dimer having Formula I or II wherein the cationic / hydrophobic ratio is from 0.5 to 1.2 or 1.5. The method of claim 2, SAEP II peptide dimer having Formula I or II wherein the cationic / hydrophobic ratio is 0.6-1. The method of claim 3, wherein SAEP II peptide dimer with formula I or II wherein the cationic / hydrophobic ratio is from 0.6 to 0.8. The method according to any one of claims 1 to 4, Said B and B 'peptide portions comprise the sequences -X1-X2-X3-, wherein X1 and X2; X2 and X3; Or X 1, X 2 and X 3 are Val, Leu, Ile, Phe, Tyr and Trp; SAEP II peptide dimers having Formula I or II preferably selected independently from Leu, Ile and Phe. The method of claim 5, Said B and B 'peptide moieties (i) -X1-X2-X3- sequence Wherein X 1 is Lys, Hyl, His or Arg, preferably Lys or Arg; most preferably Lys; X2 is Phe, Leu, Ile, Tyr, Trp or Val; Preferably Phe or Leu; Most preferably Phe; X3 is Phe, Leu, Ile, Tyr, Trp or Val; Preferably Phe or Leu; Most preferably Leu); And (ii) amino acid residues (if present, they are, respectively, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His; preferably Val, Leu, Ile, Phe, Tyr and Trp; more preferably Is independently selected from the group consisting of Leu, Ile and Phe) SAEP II peptide dimer having Formula I or II comprising a. The method according to any one of claims 1 to 6, SAEP II peptide dimers having the formula III or IV: Formula III NH ₂ - A -Cys1- B -Cys2-COOH NH ₂ - A ' -Cys1- B' -Cys2-COOH Where The two Cys1 residues are joined together via disulfide bonds Two Cys2 residues are joined together via disulfide bonds; Formula IV NH ₂ - A -Cys1- B -Cys2-COOH COOH-Cys2- B ' -Cys1- A' -NH ₂ Where Cys1 residues are linked to Cys2 residues through disulfide bonds; A, A ', B and B' are as defined in any one of claims 1 to 6. The method according to any one of claims 1 to 7, SAEP II peptide dimer which is a homologous peptide dimer. The method according to any one of claims 1 to 8, SAEP II peptide dimer in the form of an inverse parallel dimer of Formula VI: NH ₂ -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH COOH-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH ₂ Where Cys1 residues are linked to Cys2 residues through disulfide bonds. The method according to any one of claims 1 to 8, SAEP II peptide dimer which is a parallel dimer of formula (VII) NH ₂ -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH NH ₂ -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH Where The two Cys1 residues are joined together via disulfide bonds Two Cys2 residues are joined together via disulfide bonds. A composition comprising a peptide dimer according to any one of claims 1 to 10, wherein said peptide is in essentially dimeric parallel form. A composition comprising a peptide dimer according to any one of claims 1 to 10, wherein said peptide is essentially a dimeric antiparallel form. Use of a peptide dimer according to any one of claims 1 to 10 as an antidote for gram negative bacteria lipopolysaccharide (LPS). A pharmaceutical composition comprising (i) a peptide dimer according to any one of claims 1 to 10 or a composition according to claims 11 or 12 and (ii) a pharmaceutically acceptable diluent or carrier. Use of a peptide dimer according to any one of claims 1 to 10 or a composition according to claim 11 or 12 in the manufacture of a medicament for the treatment or prevention of septic shock. The treatment comprising administering a therapeutically effective amount of a peptide dimer according to any one of claims 1 to 10 or a composition according to claims 11 or 12 to an individual in need of treatment or prevention of septic shock. Or preventive measures. An LPS-peptide complex comprising (i) the presence (part) of LPS negative bacteria and (ii) the SAEP II peptide dimer according to any one of claims 1 to 10, wherein said LPS moiety and SAEP II peptide dimer Complexes in which sieves are non-covalently bound to each other. The method of claim 17, It is the LPS yen menin GT display (N. meningitidis); E. coli (E. coli); Salmonella typhimurium (Salmonella typhi ); Salmonella typhimurium Farah (paratyphi); Shigella flex Tenerife (Shigella flexneri ); Under (Haemophilus influenza in a brush loose influenzae ); Lori H. pie (Helicobacter pylori ); Chlamydia trachomatis (Chlamydia trachomatis); Bordetella Pertussis ( Bordetella pertussis ); Brucellosis (Brucella); New mopiah Legionella (Legionella pneumophia ); Vibrio cholera (Vibrio cholera ); Morak Cellar Kata LAL-less (Moraxella catharralis ); Pseudomonas Pseudomonas aeruginosa ); When Ella and kiep pneumoniae LPS in the LPS- peptide complex (Kiebsiella pneumonia). The method of claim 18, The LPS is Neseria The display of menin GT LPS LPS- peptide complex. The method of claim 19, LPS-peptide complex, wherein the LPS is LPS L8. The method according to any one of claims 17 to 20, LPS-peptide complexes characterized by an LPS: peptide molar ratio of 1: 1.5 to 1: 0.5, preferably 1: 1.2 to 1: 0.8. The method of claim 21, LPS-peptide complexes characterized by an LPS: peptide molar ratio of 1: 1. Use of an LPS-peptide complex according to any one of claims 17 to 22 for the treatment or prevention of Gram-negative bacterial infections. A pharmaceutical composition comprising the LPS-peptide complex according to any one of claims 17 to 22 and a pharmaceutically acceptable diluent or carrier. Use of an LPS-peptide complex according to any one of claims 17 to 22 in the manufacture of a medicament for the treatment or prevention of Gram-negative bacterial infection. A method of treating or preventing a gram-negative bacterial infection comprising administering to the individual in need thereof a therapeutically effective amount of the LPS-peptide complex according to any one of claims 17 to 22. A process for preparing an LPS / peptide complex, comprising (i) mixing LPS of a Gram-negative bacterium and (ii) the peptide dimer according to any one of claims 1 to 10. The method of claim 27, Mixing the LPS and the peptide or salts thereof in an LPS: peptide molar ratio of 1: 1.2 to 1: 0.8. The method of claim 29, Mixing said LPS and peptide or salts thereof in a molar ratio of LPS: peptide of 1: 1. A method for deciphering LPS, comprising mixing together (i) the gram negative bacteria LPS and (ii) the peptide dimer according to any one of claims 1 to 10. The method of claim 30, Mixing the LPS and the peptide or salts thereof in an LPS: peptide molar ratio of 1: 1.2 to 1: 0.8. The method of claim 31, wherein Mixing said LPS and peptide or salts thereof in a molar ratio of LPS: peptide of 1: 1.

Images