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WO1995008560A1 - Nouveaux peptides destines a inhiber la liaison de lipopolysaccharides (lps) par une proteine de liaison de lipopolysaccharides (lbp) - Google Patents

Nouveaux peptides destines a inhiber la liaison de lipopolysaccharides (lps) par une proteine de liaison de lipopolysaccharides (lbp) Download PDF

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Publication number
WO1995008560A1
WO1995008560A1 PCT/US1994/010760 US9410760W WO9508560A1 WO 1995008560 A1 WO1995008560 A1 WO 1995008560A1 US 9410760 W US9410760 W US 9410760W WO 9508560 A1 WO9508560 A1 WO 9508560A1
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ser
phe
val
lys
asp
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George A. Heavner
Alexander Harrison Taylor
David Sherris
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Janssen Biotech Inc
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Centocor Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to peptides which inhibit binding of lipopolysaccharides (LPS) to lipopolysaccharide binding protein (LBP) .
  • LPS lipopolysaccharides
  • LBP lipopolysaccharide binding protein
  • Such peptides are foreseen to hold great promise as therapeutic applications for Gram-negative sepsis.
  • Gram-negative sepsis one of the most critical outcomes for the host is the activation of monocytic cells and the induction of cytokine production. While mild stimulation of the defense system via monocyte activation results in an appropriate beneficial response, excess production of cytokines or disturbance of the coagulation balance may be detrimental to the host .
  • Endotoxins are one of the biological mediators of Gram-negative sepsis syndrome. Endotoxins comprise a series of related LPS molecules that are present in the outer membrane of Gram-negative bacteria.
  • the LPS molecules of smooth Gram-negative bacteria consist of a hydrophobic moiety, termed lipid A, a core oligosaccharide and a chain of repeating units of 1 to 7 sugars, termed the O chain (Rietschel et al . , In Surface Structures of Microorganisms and their Interaction wi th the Mammalian Host, 1988, V.C.H. , Frankfurt, FRG, p. 1) .
  • LPS molecules of rough Gram-negative bacteria consist of lipid A and a core oligosaccharide (Rietschel et al . , In Surface Structures of Microorganisms and their Interaction wi th the Mammalian Host, 1988, V.C.H., Franfurt, FRG, p. 1) .
  • LPS causes B cells to proliferate and produce surface IgG (Dziarski, R., Eur. J. Immunol . , 1989, 19, 125) .
  • LPS also induces transcription of the HIV1 proviral genome in latently infected human monocytes (Pomerantz et al . , J. Exp . Med. , 1990, 172, 253) .
  • An important early event in LPS induced cell activation seems to be the tyrosine phosphorylation of a number of proteins in murine and human macrophages (Weinstein et al . , Proc. Natl . Acad. Sci . USA, 1991, 88, 4148) .
  • LPS stimulates macrophages to release mediators including cytokines, such as interleukin-1 (IL-1) (Cavaillon, J.M. and N. Haeffner-Cavaillon, Cytokine, 1990, 2, 1) , IL-6 (Fong et al . , J. Immunol . , 1989, 142, 2321), IL-8 (Yoshimura et al . , J. Immunol . , 1987, 139, 788) and tumor necrosis factor (TNF) (Beutler, B. and A.
  • IL-1 interleukin-1
  • IL-6 Flong et al . , J. Immunol . , 1989, 142, 2321
  • IL-8 Yoshimura et al . , J. Immunol . , 1987, 139, 788
  • TNF tumor necrosis factor
  • LPS acts at picomolar concentrations making it one of the most potent macrophage agonists known. Most of the circulating LPS in serum is bound to a protein termed LBP (Tobias et al . , J. Exp . Med. , 1986, 164 , 111 ) . LBP cDNA has been cloned and sequenced (Schumann et al . , Science, 1990, 249, 1429) . LBP is produced in hepatocytes as a 50 kDA protein and is constitutively secreted into the bloodstream as a 60 kDA glycoprotein at a concentration of approximately 500 ng/ml (Ulevitch et al .
  • LBP In Endotoxins, from Pathophysiology to Therapeutic Approaches, 1990, Medicine-Sciences Flammarian, Paris, p. 31
  • the protein concentration rises in the acute phase to 50 ⁇ g/ml
  • LBP which does not have activity by itself, binds to LPS with high affinity.
  • LBP Upon binding to the lipid A moiety of LPS (Mathison et al . , J. Immunol . , 1992, 149, 200; Tobias et al . , J. Biol . Chem. , 1989, 264 , 10867) , LBP does not suppress or block the effects of LPS, but enhances endotoxin effects.
  • LPS-induced TNF production and TNF mRNA expression in rabbit peritoneal macrophages is strongly enhanced when LPS is complexed to LBP (Schumann et al . , Science, 1990, 249, 1429) .
  • Macrophages detect and bind LPS much more rapidly when it is complexed with LBP (Schumann et al . , Science, 1990, 249, 1429) .
  • Binding of LBP to LPS on the surface of bacteria or to LPS inserted into erythrocyte membranes dramatically enhances their interaction with macrophages (Wright et al . , J. Exp. Med. , 1989, 170, 1231) .
  • LBP acts as an opsonin for Gram-negative bacteria (Wright et al . , J. Exp . Med. , 1989, 170, 1231) .
  • CD14 is a 53 kDa glycoprotein found on the surface of myeloid cells (Goyert et al . , J. Immunol . , 1986, 137, 3909) . CD14 is linked to the cell surface via a phosphatidylinositol (PI) anchor (Haziot et al . , J. Immunol .
  • CD14 is located on chromosome 5 in a region known to encode for several cytokines and loss of this region (q23-31) is associated with certain forms of leukemia (Goyert et al . , Science, 1988, 239, 497) .
  • Complexes of LPS and LBP are recognized and specifically bound by CD14 on myeloid cells (Wright et al . , Science, 1990, 249, 1431; Couturier et al . , J " .
  • Antibody to CD14 has been shown to inhibit LBP/LPS-mediated effects, including TNF production and opsonin function (Wright et al . , Science, 1990, 249, 1431) .
  • Antibody to CD14 has also been shown to block LPS-induced tyrosine phosphorylation (Weinstein et al . , J. Leuk. Biol . , 1991) .
  • LBPfp immunoglobulin fusion protein
  • This invention therefore relates to novel peptides having the Formula: R ⁇ X-A-B-C-D-E-F-G-H-I-J-Y-R 2 wherein:
  • A is selected from the group consisting of D- or L- lysine, D- or L-arginine, D- or L-alanine, and D- or L- histidine;
  • B is selected from the group consisting of D- or L- serine, D- or L-threonine, D- or L-alanine, and D- or L- arginine;
  • C is selected from the group consisting of D- or L- phenylalanine, D- or L-alanine, D- or L-tyrosine, D- or L- serine, and D- or L-proline;
  • D is selected from the group consisting of D- or L- phenylalanine, D- or L-tyrosine, D- or L-leucine, and D- or L-alanine;
  • E is selected from the group consisting of D- or L- lysine, D- or L-arginine, D- or L-alanine, and D- or L- histidine;
  • F is selected from the group consisting of D- or L- leucine, D- or L-alanine, and D- or L-methionine;
  • G is selected from the group consisting of D- or L- glutamine, D- or L-asparagine, D- or L-lysine, D- or L- serine, and D- or L-alanine;
  • H is selected from the group consisting of D- or L- glycine, and D- or L-alanine;
  • I is selected from the group consisting of D- or L- serine, D- or L-threonine, D- or L-asparagine, and D- or L- alanine;
  • J is selected from the group consisting of D- or L- phenylalanine, D- or L-alanine, and D- or L-tyrosine;
  • X is a linear chain of from zero to four amino acids
  • Y is a linear chain of from zero to five amino acids
  • R 1 is H (signifying a free-terminal amino group) , formyl, lower alkyl, aryl, lower alkanoyl, aroyl, biotinyl, alkyloxycarbonyl, aryloxycarbonyl or desamino (signifying no alpha amino group on the N-terminal amino acid) ;
  • Peptides of the Formula have as their core region portions of the 95-104 amino acid sequences of the LBP, with residue 1 defined as the N-terminus of the mature proteins after the cleavage of the signal peptides.
  • Tests indicate that peptides of the Formula inhibit the binding of LPS in concentrations of peptide ranging from about 1 to about 1500 ⁇ m. Tests also indicate that certain alterations within the core sequence, including the addition or deletion of amino acids, do not result in loss of biological activity.
  • This invention relates not only to the novel peptides of the Formula, but also to pharmaceutical compositions comprising them, to diagnostic and therapeutic methods utilizing them, and to methods of preparing them.
  • Figure 1 is a graph showing the ability of different concentrations of LBPfp to bind different concentrations of LPS-HRP.
  • Figure 2 is a graph showing the competitive inhibition of LPS-HRP binding to Fc-captured LBPfp. The dashed lines indicate the IC50 values for the competitors. Vertical bars indicate the standard error of the mean.
  • Figure 3 is a graph showing the IC50 values for the peptides of Examples 1-8 in the ELISA competitive assay.
  • Figure 4 provides graphs showing the results of LAL experiments. The dotted lines indicate the IC50 values for each of the inhibitors.
  • Figure 4a compares the neutralization potency of the peptides of Examples 1, 4 and 5 and polymyxin B in a 0111LPS (smooth challenge) .
  • Figure 4b shows the ability of the peptide of Example 1 and polymyxin B to block the LAL reaction to 1 pg/ml rough LPS.
  • Figure 4c shows the ability of the three tested peptides to inhibit the LAL reaction induced by 500 pg/ml lipid A.
  • Figure 5 is a graph showing the blocking of PBMC response to LPS challenge by the peptide of Example 1.
  • the amount of TNF found in cultures (vertical axis) stimulated with increasing concentrations of LPS (horizonal axis) , an index of monocyte activation by LPS, is shown.
  • the different curves represent LPS dose responses in the presence of different amounts of the peptide.
  • the dotted lines show the amount of LPS each peptide concentration tested can inhibit by 50%.
  • Figure 6 is a graph showing the ability of the peptide of Example 6 to inhibit PBMC response to 1% normal human serum-potentiated LPS challenge. Shown is the ability of different concentrations of the peptide (horizontal axis) to inhibit Ing/mL and 10 ng/mL 0111 LPS challenge in the constant presence of 1% normal human serum. The dotted lines indicate the IC50 of the peptide when tested with Ing/mL and 10 ng/mL LPS.
  • Preferred peptides of this invention are those of the Formula as previously defined, wherein X is selected from the following group (or analogs thereof wherein one or more of the amino acids are replaced with the analogous D-amino acid) :
  • peptides having the Formula as previously defined wherein Y is selected from the following group (or analogs thereof wherein one or more of the amino acids are replaced with the analogous D-amino acid) : Asp Asp-Val-Ser-Val
  • Specific preferred peptides include the following "Preferred Peptides” (SEQ ID NOS:l-64) :
  • Trp-Lys-Val-Arg-Lys-Ser-Phe-Phe-Lys-Leu-Gln-Gly- Ser-Phe-Asp-Val-Ser-Val-NH 2 SEQ ID NO: 2
  • Trp-Lys-Val-Arg-Lys-Ser-Phe-Phe-Lys-Leu-Gln-Gly- Ser-Phe-Asp-NH 2 (SEQ ID NO: 3)
  • Val-Ser-Val-NH 2 (SEQ ID NO: 8)
  • Val-Ser-Val-NH 2 (SEQ ID NO: 13) Arg-Lys-Ala-Phe-Phe-Lys-Leu-Gln-Gly-Ser-Phe-Asp- Val-Ser-Val-NH 2 (SEQ ID NO: 14)
  • Val-Ser-Val-NH 2 (SEQ ID NO: 31) Arg-Lys-Ser-Phe-Phe-Lys-Leu-Gln-Ala-Ser-Phe-Asp- Val-Ser-Val-NH 2 (SEQ ID NO: 32)
  • Val-Ser-Tyr-NH 2 (SEQ ID NO: 49) Arg-Lys-Ser-Phe-Phe-Lys-Leu-Gln-Gly-Ser-Phe-Asp- Val-Ser-Ala-NH 2 (SEQ ID NO: 50)
  • Trp-Lys-Val-Arg-Lys-Ser-Phe-Phe-Lys-Leu-Gln-Gly- Ser-Phe-Asp-NH 2 (SEQ ID NO: 58)
  • Trp-Lys-Val-Arg-Lys-Ser-Phe-Phe-Lys-Leu-Gln-Gly- Ser-Phe-NH 2 (SEQ ID NO: 59)
  • alkyl includes branched, straight-chain, and cyclic saturated hydrocarbons.
  • lower alkyl means an alkyl having from one to six carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, cyclopentylmethyl and hexyl .
  • alkanoyl means
  • aroyl means O
  • aryl means an aromatic or heteroaromatic structure having between one and three rings, which may or may not be ring fused structures, and are optionally substituted with halogens, carbons, or other heteroatoms such as nitrogen (N) , sulfur (S) , phosphorus (P) , and boron (B) .
  • alkoxycarbonyl means 0
  • R 8 -0-C-0- wherein R 8 is a lower alkyl group.
  • aryloxycarbonyl means 0 9 -0-C-0- wherein R 9 is an aryl and arylmethyl group.
  • Halogen refers to fluorine, chlorine, bromine or iodine .
  • the term "terminal ⁇ -amino group of X" refers to the Q!-amino group of the N-terminal amino acid of X.
  • the peptides of the Formula can be used in the form of the free peptide or a pharmaceutically acceptable salt.
  • Amine salts can be prepared by treating the peptide with an acid according to known methods.
  • Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid.
  • Carboxylic acid groups in the peptide can be converted to a salt by treating the peptide with a base according to known methods.
  • Suitable bases include inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, and tri-alkyl and aryl amines (e.g., triethylamine, diisopropylamine, methylamine, and dimethylamine and optionally substituted mono-, di, and tri-ethanolamines.
  • amino acid components of the peptides and certain materials used in their preparation are identified by abbreviations for convenience. These abbreviations are as follows:
  • amino acids preceded by L- or D- refer, respectively, to the L- or D- enantiomer of the amino acid, whereas amino acids not preceded by L- or D- refer to the L-enantiomer.
  • Common derivatives of any of the amino acids may also be incorporated into the peptides of this invention (e.g., Lys (Biotin) for Lys) .
  • the peptides can generally be prepared following known techniques, as described, for example, in the cited publications, the teachings of which are specifically incorporated herein. In a preferred method, the peptides are prepared following the solid-phase synthetic technique initially described by Merrifield in J.Amer.Chem.Soc. , 85, 2149-2154 (1963) . Other techniques may be found, for example, in M. Bodanszky, et al, Peptide Synthesis, second edition, (John Wiley & Sons, 1976) , as well as in other reference works known to those skilled in the art.
  • the C-terminal amino acid is attached to the appropriately functionalized resin.
  • This can be a resin such as 4-methylbenzhydrylamine- polystyrene-divinylbenzene resin where the C-terminal amide is desired, BOC protection of the alpha amino groups is desired and HF cleavage of the peptide from the resin is desired or HMP-resin (4-hydroxymethylphenoxymethyl-styrene- divinylbenzene resin) where the C-terminal acid is desired, FMOC protection of the alpha amino group is desired and TNF cleavage of the peptide from the resin is desired.
  • the selection of the appropriate resin, protection strategy and cleavage strategy is done according to procedures by those skilled in the art of peptide synthesis.
  • the alpha amino protecting group is removed to expose the free alpha amino group.
  • the desired protected, partially protected or unprotected amino acid is then coupled to the free amino group using reagents or techniques that will form the desired amide bond. This can involve preactivation of the carboxyl and/ or amino groups or their conversion into chemical moieties which, upon reaction, will give the desired amides.
  • Such techniques involve the use of carbodiimides, formation of active esters, active anhydrides or other such procedures employed in the synthesis of peptides.
  • an appropriately protected amino acid is coupled to a second appropriately protected amino acid by forming an amide bond between them.
  • Such techniques involve the use of carbodiimides, formation of active esters, active anhydrides or other such procedures employed in the synthesis of peptides.
  • the amine bond may also be formed through the use of enzymes, functioning in an manner described as reverse proteolysis.
  • additional appropriately protected amino acids or preformed blocks of amino acids are covalently linked through the formation of amide bonds.
  • the selection and removal of appropriate functional group protection is according to procedures known to those skilled in the art of peptide chemistry.
  • the peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art.
  • the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, translating the DNA into RNA and the RNA into the peptide.
  • the peptide is then purified using chromatographic or electrophoretic techniques, or by means of a carrier protein which can be fused to, and subsequently cleaved from, the peptide by inserting into the expression vector in phase with the peptide encoding sequence a nucleic acid sequence encoding the carrier protein.
  • the fusion protein-peptide may be isolated using chromatographic, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the carrier protein) .
  • the peptide can be cleaved using chemical methodology or enzymatically, as by, for example, hydrolases.
  • compositions of this invention comprise a pharmaceutically acceptable carrier or diluent and an effective quantity of one or more of the peptides of the Formula or an acid or base salt thereof.
  • the carrier or diluent may take a wide variety of forms depending on the form of preparation desired for administration, e.g., sublingual, rectal, nasal, oral, transdermal or parenteral.
  • any of the usual pharmaceutical media may be employed, for example, waters, oils, alcohols, flavoring agents, preservatives, and coloring agents, to make an oral liquid preparation (e.g., suspension, elixir, or solution) or with carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents, to make an oral solid preparation (e.g., powder, capsule, or tablet) .
  • Controlled release forms or enhancers to increase bioavailability may also be used.
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are employed.
  • tablets may be sugar coated or enteric coated by standard techniques.
  • the carrier will usually be sterile water, although other ingredients to aid solubility or as preservatives may be included.
  • injectable suspensions may also be prepared, in which case appropriate liquid carriers and suspending agents can be employed.
  • the peptides can also be administered locally at a wound or inflammatory site by topical application of a solution or cream.
  • the peptide may be administered in liposomes or microspheres (or microparticles) .
  • Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art.
  • U.S. Patent No. 4,789,734 describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
  • a review of known methods is by G. Gregoriadis, Chapter 14, “Liposomes", Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979) .
  • Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream.
  • the peptide can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Patents Nos. 4,906,474, 4,925,673 and 3,625,214.
  • the peptides are generally active when administered parenterally in amounts above about 1 ⁇ g/kg body weight. Effective doses by other routes of administration are generally those which result in similar blood level to i.v. doses above about 10 ⁇ g/Kg.
  • the peptides may be administered parenterally in amounts from about 0.01 to about 10 mg/kg body weight. Generally, the same range of dosage amounts may be used in treatment of other diseases or of conditions where inflammation is to be reduced. This dosage will be dependent, in part, on whether one or more peptides are administered.
  • Methods for Demonstrating Binding Peptides that are biologically active are those which inhibit binding of LPS to LBP.
  • Peptides can be screened for their ability to inhibit such binding, for example, using ELISA-based assay to detect lipid A binding determinants.
  • Such an assay utilizes a fusion protein, immobilized onto EIA plates, comprised of human LBP fused to human IgG constant domains (LPSfp) .
  • LPSfp human IgG constant domains
  • HLPS-HRP Horseradish peroxidase-labelled LPS bound by the LBP moiety of the LBPfp fusion protein is added, and subsequently is detected by the addition of a chromogenic substrate.
  • LBP is specific for the lipid A portion of LPS molecules
  • a Lipid A-binding test substance is added with the labelled-LPS, it would be expected to inhibit binding of the labelled-LPS to the LBPfp.
  • this screening assay could be used to identify test substances that can competitively inhibit binding of LPS to LBPfp.
  • the peptides of this invention inhibit binding of LPS to LBP, they should be useful for the prevention or treatment of Gram negative septic shock.
  • the criteria for assessing response to therapeutic modalities employing these peptides, and, hence, effective dosages of the peptides of this invention for treatment, are dictated by the specific condition and will generally follow standard medical practices.
  • the criteria for the effective dosage to prevent LPS induced toxicity would be determined by one skilled in the art by looking at cytokine release and by monitoring clinical response.
  • the peptides can also be used for the detection of human disorders in which LPS is implicated. Such elevated LPS levels would, for example, be seen in patients with sepsis.
  • Patient samples can be collected and tested using the peptide to bind to LPS and the resulting peptide-LPS or peptide-LPS-CD14 conjugate detected or quantitated using direct or indirect procedures.
  • the peptide is labeled radioactively, with a fluorescent tag, enzymatically, or with electron dense material such as gold for electron microscopy.
  • the LPS can also be measured with ELISA or radioimmunoassay procedures, using labeled peptide as the trapping or the detecting reagent.
  • EXAMPLE 1 Preparation of arginyl-lysyl-seryl- phenylalanyl-phenylalanyl-lysyl-leucyl-glutamyl-glycyl-seryl- phenylalanyl-aspartyl-valyl-seryl-valine-amide (SEQ ID NO: 1)
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using Version 1.12 of the standard BOC software.
  • 4-methyl benzhydrylamine resin (0.46 g, 0.5 mmol) was used in the synthesis.
  • the final weight of the resin was 1.82 g.
  • the peptide was cleaved from the resin (1.74 g) using
  • EXAMPLE 2 Preparation of tryptophyl-lysyl-valyl-arginyl- lysyl-seryl-phenylalanyl-phenylalanyl-lysyl-leucyl-glutamyl- glycyl-seryl-phenylalanyl-aspartyl-valyl-seryl-valine-amide (SEQ ID NO: 2)
  • the peptide was prepared on an ABI Model 431A Peptide
  • EXAMPLE 3 Preparation of Tryptophyl-lysyl-valyl-arginyl- lysyl-seryl-phenylalanyl-phenylalanyl-lysyl-leucyl-glutamyl- glycyl-seryl-phenylalanyl-aspartic acid-amide (SEQ ID NO: 3)
  • the peptide was prepared on an ABI Model 431A Peptide
  • the peptide was cleaved from the resin (1.76 g) using
  • EXAMPLE 4 Preparation of acetyl-arginyl-lysyl-seryl- phenylalanyl-phenylalanyl-lysyl-leucyl-glutamyl-glycyl-seryl- phenylalanyl-aspartyl-valyl-seryl-valine-amide (SEQ ID NO: 4)
  • the peptide was prepared on an ABI Model 431A Peptide
  • the crude peptide (1.00 g) was purified on a Vydac C- 18 column (15 ⁇ , 5 x 25 cm) eluting with a 35-65% gradient of 80% ethanol in 0.1% TFA over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 132 mg of semipure peptide.
  • EXAMPLE 5 Preparation of acetyl-tryptophyl-lysyl-valyl- arginyl-lysyl-seryl-phenylalanyl-phenylalanyl-lysyl-leucyl- glutamyl-glycyl-seryl-phenylalanyl-aspartyl-valyl-seryl- valine-amide (SEQ ID NO: 5)
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using Version 1.12 of the standard BOC software. 4-methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.26 g.
  • the peptide was cleaved from the resin (2.26 g) using 22 mL of HF and 2.2 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with a
  • EXAMPLE 6 Preparation of D-arginyl-lysyl-seryl- phenylalanyl-phenylalanyl-lysyl-leucyl-glutamyl-glycyl-seryl- phenylalanyl-asparaginyl-valyl-seryl-valine-amide (SEQ ID NO: 6)
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using Version 1.12 of the standard BOC software.
  • 4-methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis.
  • the final weight of the resin was 2.12 g.
  • the peptide was cleaved from the resin (2.06 g) using
  • 4-methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis.
  • the final weight of the resin was 2.03 g.
  • the peptide was cleaved from the resin (2.03 g) using 20 mL of HF and 2.0 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with a
  • EXAMPLE 8 Preparation of D-tryptophyl-lysyl-valyl- arginyl-lysyl-seryl-phenylalanyl-phenylalanyl-lysyl-leucyl- glutamyl-glycyl-seryl-phenylalanyl-aspartyl-valyl-seryl- valine-amide (SEQ ID NO: 8)
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using Version 1.12 of the standard BOC software. 4-methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.26 g.
  • the peptide was cleaved from the resin (2.15 g) using 21 mL of HF and 2.1 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with a 1:1 solution of TFA:CH 2 C1 2 to give 1.31 g of crude peptide.
  • the crude peptide (1.00 g) was purified on a Vydac C- 18 column (15 ⁇ , 5 x 25 cm) eluting with a 20-60% gradient of 80%acetonitrile in 0.1% TFA over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 182 mg of semipure peptide.
  • a fusion protein was assembled consisting of the entire mature human LBP coding region fused to the human IgGl constant region.
  • the LBP-IgH fusion construct was co-expressed with a human kappa chain constant region (i.e., a light chain in which the entire variable region had been deleted) in murine myeloma cells.
  • the resulting LBP-immunoglobulin fusion protein termed LBPfp, was designed such that the antigen binding V-region domains of an IgG molecule were replaced with the entire LBP molecule.
  • LBPfp LBP-immunoglobulin fusion protein
  • an ELISA was carried out to measure the capacity of the LBBfp to bind LPS, and to be recognised by anti-human Fc antibodies.
  • Different dilutions of protein A purified LBPfp were Fc-captured onto EIA plates previously coated with affinity purified anti- human Fc antibodies.
  • PBSM low fat dry milk
  • HRP horseradish peroxidase
  • 0111LPS smooth LPS
  • the LBPfp component appears to saturate at around l ⁇ g/ml, but the LPS- HRP does not appear to saturate the Fc-captured LBPfp, even at 1/2500 dilution, the highest concentration tested. Nevertheless, since a strong signal was obtained using l ⁇ g/ml LBPfp and 1/2500 LPS-HRP, these concentrations were selected as standard for subsequent screening assays.
  • Fig 2 The specificity of the ELISA is demonstrated in Fig 2. It can be seen that either solution-phase LBPfp or unlabelled 0111 LPS can competitively inhibit binding of LPS-HRP to fc- captured LBPfp, reaching 50% inhibition in the 1 ⁇ g/ml concentration range. Moreover, certain members of a panel of 26 different anti-human LBP monoclonal antibodies can also inhibit the binding of LPS-HRP to Fc-captured LBPfp (data not shown) . Together, the results show that the Fc-captured LBPfp can specifically bind LPS-HRP in a way that can be inhibited by different specific blocking molecules, including monoclonal antibodies, unlabelled homologous LPS, or solution- phase LBPfp.
  • LBPfp was captured onto 96-microwell ELISA plates that had previously been coated with affinity purified anti-human Fc antibodies.
  • Fig. 4a compares the neutralization potency of the peptides of Ex. 1, 5, 4, and polymyxin B in a 0111LPS (smooth) challenge. These results show that the peptides
  • FIG. 4b shows that the peptide of Example 1 can also inhibit the LAL reaction to J5 LPS, a rough form. In this case, polymyxin B appears to be even more potent, demonstrating a 2500-fold greater relative potency than the peptide of Example 1.
  • Panel 4c compares the ability of the peptides to block challenge of LAL with lipid A. In each case, the peptides could substantially inhibit the LAL reaction to lipid A. Hence, the peptides appear to neutralize both smooth and rough LPS as well as lipid A, and with similar potency in the IC50 lO ⁇ M range.
  • PBMC peripheral blood mononuclear cells
  • Figure 5 shows the ability of the peptide of Example 1 to block PBMC response to LPS challenge in serum free, purified LBP- potentiated cultures.
  • the results show that in the absence of the peptide, PBMC cultures respond maximally to stimulation with about lng/ml LPS. As little as 3 ⁇ M of the peptide can completely block the TNF response to this maximal LPS stimulation. It is interesting that the peptide mediated LPS neutralization could be overwhelmed by the addition of increased LPS doses, in a roughly stoichiometric fashion. This observation suggests that the ability of the peptide to neutralize the PBMC LPS response reflects its LPS binding properties, and in particular does not reflect toxicity to the PBMC.
  • the effect of the peptide on viability of several different cell types including PBMC was tested, and was found to have no measurable effect, even at a 5-fold greater concentration than the maximum used here (not shown) .
  • the results show that, in vitro, the peptide can completely inhibit the normal human PBMC TNF response to LPS (up to 10 ng/ml LPS in this experiment) and which, notably, are substantially greater LPS levels than ypically found in the clinical setting.
  • the peptide of Example 6 is otherwise identical to that of Example 1, but has the normal n-terminal residue L- Arg replaced with a D-Arg residue.
  • This analog was tested in the PBMC format for the ability to neutralize LPS in the presence of normal human serum.
  • Figure 6 shows the result of one such experiment in which the peptide of Example 6 was tested for LPS neutralization potency in the presence of 1% normal human serum. The results show that this peptide can completely inhibit stimulation with lng/ml LPS, with IC50 values of less than lO ⁇ M. While this potency is somewhat less than found for the peptide of Example 1 in the serum free, purified LBP potentiated format, it nevertheless demonstrates a large degree of serum stability.
  • the results indicate the peptides of this invention retain LPS neutralization properties when tested in complex biological milieux, including normal human serum. Even more importantly, these peptides can completely inhibit cellular recognition of LPS in vitro, abrogating cytokine secretion, known to play a central role in the pathology of gram negative sepsis syndrome.
  • MOLECULE TYPE Peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:1 :

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Abstract

Nouveaux peptides dérivés de parties de la séquence d'acides aminés 95-104 de la protéine de liaison de lipopolysaccharides (LBP). Cette invention se rapporte également à des compositions pharmaceutiques comprenant ces nouveaux peptides, et à des procédés diagnostiques et thérapeutiques utilisant les peptides et les compositions pharmaceutiques décrites.
PCT/US1994/010760 1993-09-24 1994-09-22 Nouveaux peptides destines a inhiber la liaison de lipopolysaccharides (lps) par une proteine de liaison de lipopolysaccharides (lbp) Ceased WO1995008560A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038473A3 (fr) * 1995-06-02 1997-04-03 Univ British Columbia Peptides cationiques antimicrobiens
WO1999002178A1 (fr) * 1997-07-11 1999-01-21 Max-Delbrück-Centrum für Molekulare Medizin Agent therapeutique pour le traitement de la septicemie, sa preparation et son utilisation
EP1074561A3 (fr) * 1999-06-06 2002-07-10 Centro De Ingenieria Genetica Y Biotecnologia Analogues de protéine fixatrice de liposacharides (LBP)
GB2380998A (en) * 2001-08-03 2003-04-23 Marc Kvansakul D-amino acid peptide for security tagging
US8003313B2 (en) 2005-01-21 2011-08-23 Hyglos Invest Gmbh Method for detecting and removing endotoxin

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US5334564A (en) * 1990-07-16 1994-08-02 Board Of Trustees Operating Michigan State University Method for the preparation of highly reactive clay composites for the removal of SOx from flue gas streams

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US5334564A (en) * 1990-07-16 1994-08-02 Board Of Trustees Operating Michigan State University Method for the preparation of highly reactive clay composites for the removal of SOx from flue gas streams

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Title
ANALYTICAL BIOCHEMISTRY, Volume 197, issued 1991, VALERIO et al., "Synthesis of Peptide Analogues Using the Multipin Peptide Synthesis Method", pages 168-177. *
ARCHIVES OF SURGERY, Volume 128, issued January 1993, GELLER et al., "Induction of Hepatocyte Lipopolysaccharide Binding Protein in Models of Sepsis and the Acute-phase Response", pages 22-28. *
BIO/TECHNOLOGY, Volume 12, issued August 1994, HIGHFIELD, "Sepsis: the More the Murkier", page 828. *
INFECTION AND IMMUNITY, Volume 61, Number 7, issued July 1993, CROSS et al., "Choice of Bacteria in Animal Models of Sepsis", pages 2741-2747. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 90, issued November 1993, GALLAY et al., "Lipopolysaccharide-binding Protein as a Major Plasma Protein Responsible for Endotoxemic Shock", pages 9935-9938. *
SCIENCE, Volume 249, issued 21 September 1990, SCHUMANN et al., "Structure and Function of Lipopolysaccharide Binding Protein", pages 1429-1433. *
THE EMBO JOURNAL, Volume 12, Number 9, issued 01 September 1993, HOESS et al., "Crystal Structures of an Endotoxin-neutralizing Protein from the Horseshoe Crab, Limulus Anti-LPS Factor, a 1.5 a Resolution", pages 3351-3356. *
THE JOURNAL OF IMMUNOLOGY, Volume 151, Number 7, issued 01 October 1993, GESSANI et al., "Enhanced Production of LPS-induced Cytokines During Differentiation of Human Monocytes to Macrophages", pages 3758-3766. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038473A3 (fr) * 1995-06-02 1997-04-03 Univ British Columbia Peptides cationiques antimicrobiens
US5877274A (en) * 1995-06-02 1999-03-02 University Of British Columbia Antimicrobial cationic peptides
WO1999002178A1 (fr) * 1997-07-11 1999-01-21 Max-Delbrück-Centrum für Molekulare Medizin Agent therapeutique pour le traitement de la septicemie, sa preparation et son utilisation
US6949512B1 (en) * 1997-07-11 2005-09-27 Max-Delbruck-Zentrum Fur Molekulare Medizin Therapeutic agent for the treatment of septicaemia its preparation and use
EP1074561A3 (fr) * 1999-06-06 2002-07-10 Centro De Ingenieria Genetica Y Biotecnologia Analogues de protéine fixatrice de liposacharides (LBP)
US7012127B1 (en) 1999-06-06 2006-03-14 Centro De Ingenicria Genetica Y Biotecnologia Analogues of lipopolysaccharide-binding protein (LBP)-derived peptides that efficiently neutralize lipopolysaccharides (LPS)
GB2380998A (en) * 2001-08-03 2003-04-23 Marc Kvansakul D-amino acid peptide for security tagging
GB2380998B (en) * 2001-08-03 2004-10-20 Marc Kvansakul D-amino acid peptide for security tagging
US8003313B2 (en) 2005-01-21 2011-08-23 Hyglos Invest Gmbh Method for detecting and removing endotoxin
US8329393B2 (en) 2005-01-21 2012-12-11 Hyglos Invest Gmbh Method for detecting and removing endotoxin
US8822641B2 (en) 2005-01-21 2014-09-02 Hyglos Invest Gmbh Method for detecting and removing endotoxin
US9229002B2 (en) 2005-01-21 2016-01-05 Hyglos Invest Gmbh Nucleic acids encoding bacteriophage tail proteins

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