WO1996028544A1 - Conjugue proteine-lipide, constitue de la proteine fixant l'heparine et d'un analogue d'un ceramide, et son utilisation pharmaceutique pour le traitement de troubles lies a des lesions causees a des cellules par diverses agressions - Google Patents

Conjugue proteine-lipide, constitue de la proteine fixant l'heparine et d'un analogue d'un ceramide, et son utilisation pharmaceutique pour le traitement de troubles lies a des lesions causees a des cellules par diverses agressions Download PDF

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WO1996028544A1
WO1996028544A1 PCT/DK1996/000099 DK9600099W WO9628544A1 WO 1996028544 A1 WO1996028544 A1 WO 1996028544A1 DK 9600099 W DK9600099 W DK 9600099W WO 9628544 A1 WO9628544 A1 WO 9628544A1
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lipid
containing substance
hbp
protein
ceramide
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PCT/DK1996/000099
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English (en)
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Hans Flodgaard
Poul Baad Rasmussen
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Novo Nordisk A/S
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Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to EP96903943A priority Critical patent/EP0871721A1/fr
Priority to AU47848/96A priority patent/AU716791B2/en
Priority to JP8527186A priority patent/JPH11501636A/ja
Publication of WO1996028544A1 publication Critical patent/WO1996028544A1/fr
Priority to NO974123A priority patent/NO974123L/no
Priority to US08/925,708 priority patent/US5939390A/en

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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a protein-lipid conjugate consisting of heparin binding protein (HBP) and a ceramide analogue, and its pharma ⁇ ceutical use in treatment of conditions involving stress injury to cells
  • the reasons for this increase and high incidence of septic shock are believed to be the increased use of invasive devices such as intravascular catheters, increased use of cytotoxic and immunosuppresive drugs, increased longevity of patients liable to develop sepsis and an increase in infections caused by antibiotic-resistant organisms.
  • bacteremia also known as septicemia
  • sepsis characterized by positive blood cultures
  • sepsis characterized by a systemic response to the infection in the form of tachypnea, tachycardia, hyperthermia or hypothermia
  • sepsis syndrome in which there is clinical evidence of sepsis and signs of altered organ perfusion in the form of an abnormally increased lactate level, oliguria or acutely altered mental status
  • early septic shock in which there is clinical evidence of sepsis syndrome as well as hypotension lasting for less than one hour and responsive to conventional therapy
  • refractory septic shock in which there is clinical evidence of sepsis syndrome and hypotension lasting for more than one hour despite conventional therapy.
  • Mild oxidative stress is a normal feature in higher vertebrates as a result of a persistent stage of oxidative siege. Under normal conditions an efficient defense system consisting of an elaborate arsenal of antioxidants ensure that the organism is able to cope with oxygen free radicals by keeping a balance between oxygen free radicals and antioxidants.
  • numerous aggressive oxidative species oxygen free radicals
  • phagocytes activated neutrophil leukocytes, macrophages and monocytes
  • phagocytes activated neutrophil leukocytes, macrophages and monocytes
  • the generation of oxygen free radicals is far beyond the antioxidant capacity of the surrounding cells, and these cell may be injured or die from necrosis or apoptosis (programmed cell death) mediated by oxygen free radicals.
  • cytokine cascade At the site of infection or injury a cytokine cascade is initiated which, in turn, activates neutrophil leukocytes.
  • the initiator of the cytokine cascade is endotoxin (otherwise known as lipopolysaccharide, abbreviated to LPS) released at the infectious or inflammatory site where it induces the release of tumour necrosis factor ⁇ (TNF ), interleukin-1, interleukin-6, interleukin-8 and platelet- activating factor (PAF) from macrophages and other cells.
  • TNF tumour necrosis factor ⁇
  • interleukin-1 interleukin-1
  • interleukin-6 interleukin-6
  • interleukin-8 platelet- activating factor
  • Interleukin-1 and interleukin-6 activate T-cells to produce interferon- ⁇ , interleukin-2, interleukin-4 and granulocyte-monocyte colony- stimulating factor.
  • Neutrophils may be activated directly by most of these mediators. Neutrophil-induced damage may thus occur during degranulation by the release of oxygen free radicals and lysosomal enzymes, and during aggregation at infective or inflammatory sites.
  • cytokines vitD 3 and INF- ⁇ have been shown to stimulate production of ceramide in HL-60 cells by stimulating a membrane-bound neutral sphingomyelinase which hydrolyses membrane sphingomyelin to ceramide and phosphorylcholine (cf. T. Okazaki et al., J. Biol. Chem. 265. 1990, pp. 15823-15831).
  • Ceramide has been found to be a second messenger which, in turn, activates a ceramide-activated protein kinase belonging to the family of X Ser Thr Pro protein kinases (cf. S. Mathias et al., Proc. Natl. Acad. Sci. USA 88, 1991, pp.
  • Ceramide has additionally been shown to activate a ceramide-activated Ser/Thr protein phosphatase (cf. R.T. Dobrowski and Y.A. Hannun, J. Biol. Chem. 267, 1992, pp. 5048-5051). These initial reactions were shown to lead to further downstream signaling in a complex and as yet poorly understood manner, involving activation of the MAP kinase cascade, stimulation of transcription factors such as c-Myc and c-Fos, activation NF-KB and stimulation of PLA, leading to the formation of arachidonic acid derivatives.
  • LBP Lipoprotein-binding protein
  • LPS when conjugated to another protein than LBP is able to mimic the second messenger function of ceramide in a different way than by activating a ceramide-activated protein kinase.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of diseases or conditions involving stress injury to cells, the composition comprising
  • the invention in another aspect, relates to a method of preventing or treating disesases or conditions involving stress injury to cells, the method comprising administering, to a patient in need of such treatment, an effective amount of
  • the invention relates to the use of
  • the composition of the invention contains, as the protein to which the lipid-containing substance is conjugated, a heparin-binding protein (HBP) which, in glycosylated form, has an apparent molecular weight of 28 kD (as determined by SDS-PAGE under reducing conditions), the protein being produced in the azurophil granules of polymorphonuclear leukocytes.
  • HBP heparin-binding protein
  • the proteins lack protease activity.
  • the proteins have been named human heparin-binding protein (hHBP) and porcine heparin-binding protein (pHBP), respectively, owing to their high affinity for heparin;
  • hHBP human heparin-binding protein
  • pHBP porcine heparin-binding protein
  • CAP37 protein cationic antimicrobial protein due to its antimicrobial activity.
  • the protein has also been shown to be chemotactic for monocytes over the range 1.3 x 10 ' ' M - 10* M (H.A. Pereira et al., J. Clin.Invest. 85, 1990, p.1468 ff.), consistent with the results apparent from Flodgaard et al., orx tit..
  • HBP has been shown to mediate detachment and contraction of endothelial cells and fibroblasts when added to such cells grown in monolayer culture. HBP also stimulates monocyte survival and thrombospondin secretion (E. ⁇ stergaard and H. Flodgaard, J. Leukocyte Biol. 5 1992, p 316 ff).
  • a protein with the first 20 N-terminal a ino acid residues identical to those of hHBP and CAP37 called azurocidin has also been isolated (J.E. Gabay et al., Proc. Nail. Acad. Sci. USA 86, 1989, p. 5610 ff.; C.G. Wilde et al., J. Biol. Chem. 265, 1990, p. 2038 ff.) and its antimicrobial properties have been reported (D. Campanelli et al., J. Clin. Invest. 85, 1990, p. 904 ff.).
  • HBP-mediated cell detachment and homotypic aggregation accompanied by a downregulation of cellular metabolism may be another protective mechanism against cell injury during inflammation or infection.
  • the matrix cells surviving oxidative stress due to the action of HBP are ready to re-invade the inflammatory site and contribute to the healing processes which are orchestrated by an elaborate array of growth factors and cytokines secreted from monocytes and macrophages attracted to the site by HBP.
  • HBP HBP has otherwise been termed CAP37 (cf. WO 91/00907) and azurocidin (cf. C.G. Wilde et al., J. Biol. Chem. 265. 1990, p. 2038).
  • HBP in conjunction with a lipid-containing substance which may be LPS or ceramide, is currently believed to be able to downregulate cellular metabolism.
  • LPS conjugated to HBP would appear not to bind to the LPS receptor CD14 but to the cell surface due to the strong heparan sulfate-binding motifs of HBP.
  • New data from measurements of rapid uptake of neutrophil-derived HBP by monocytes also argue for HBP-specific ligands on monocytes that are distinct from CD14 (Heinzelmann, M. et al., Critical Care, 1996 in press).
  • LPS is subsequently docked into the cell membrane and brought into contact with the signaling apparatus of the cell, ultimately activating a ceramide- activated protein phosphatase.
  • Activation of the phosphatase may, in turn, lead to down-regulation of cellular metabolism.
  • addition of exogenous ceramide made water-soluble and membrane permeable by addition of a hexanoyl group to the molecule to Swiss 3T3 cells leads to morphological changes such as contraction, detachment and homotypic aggregation with preserved cell viability.
  • Ceramide seems to be a key regulator of antiproliferative and apoptotic pathways and as an inhibitor of protein makingking and secretion, and these events have been associated with the TNF ⁇ -induced activation of the ceramide- activated protein phosphatase via the 75 kD TNF ⁇ receptor (cf. Y.A. Hannun, J. Biol. Chem. 269. 1994, pp. 3125-3128).
  • LPS as a ceramide analogue, likewise stimulates the ceramide-activated protein phosphatase when conjugated to HBP.
  • a pharmaceutical composition which may be used to adjust the balance between a necessary cytokine-activated defense of the cells (mediated by stimulation of the ceramide-activated protein kinase in monocytes) and a protection of endothelial cells, smooth muscle cells and fibroblasts (mediated by stimulation of a ceramide-activated protein phosphatase) by inhibiting cellular proliferation and activity at inflammatory sites.
  • a necessary cytokine-activated defense of the cells mediated by stimulation of the ceramide-activated protein kinase in monocytes
  • a protection of endothelial cells, smooth muscle cells and fibroblasts mediated by stimulation of a ceramide-activated protein phosphatase
  • HBP/LPS endothelial cells, fibroblasts and smooth muscle cells at the inflammatory focus resulting in a "dormant" phenotype of these cells may protect them from stress injury and keep them ready to take over the repair processes once the infection has been combated.
  • the HBP may suitably be of mammalian, in particular human or porcine, origin.
  • the HBP is human HBP with the amino acid sequence shown in the Sequence Listing as SEQ ID NO:l, or porcine HBP with the amino acid sequence shown in the Sequence Listing as SEQ ID NO:2, or a functional analogue or peptide fragment thereof capable of binding the lipid A portion of LPS.
  • Such functional analogues include derivatives of the native protein obtained by addition of one or more amino acid residues to either or both the C- or N-terminal end of the native protein, substitution of one or more amino acid residues at either or both ends of the native protein, deletion of one or more amino acid residues at either or both ends of the native protein or at one or more sites within the amino acid sequence, or insertion of one or more amino acid residues at one or more sites in the native amino acid sequence.
  • the HBP may suitably be prepared by a method described in DK patent application No. 1452/94. More specifically, a DNA sequence encoding HBP may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H.
  • oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • the DNA sequence may also be of genomic or cDNA origin, for instance obtained by 0 preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of HBP by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, 1989).
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described 5 in US 4,683,202 or R.K. Saiki et al., Science 239. 1988, pp. 487-491.
  • a recombinant expression vector which may be any vector which may conveniently be subjected to recombinant DNA procedures.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding HBP should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA encoding HBP in mammalian cells are the SV 40 promoter (Subramani et al., Mol. Cell Biol. 1, 1981, pp. 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al.. Science 222. 1983, pp. 809-814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (Vasuvedan et al., FEBS Lett. 311. 1992, pp. 7- 11).
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255. 1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp.
  • Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) or the tpjA promoter.
  • the DNA sequence encoding HBP may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., O ⁇ J. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, orx tit.) or ADH3 (McKnight et al., OEi cit.) promoters.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An examples of such a sequence is the SV 40 origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hygromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the procedures used to ligate the DNA sequences coding for HBP, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.ci .
  • the host cell into which the expression vector is introduced may be any cell which is capable of producing HBP and is preferably a eukaryotic cell, such as invertebrate (insect) cells or vertebrate cells, e ⁇ . Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10) or CHO (ATCC CCL 61) cell lines.
  • Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159. 1982, pp. 601-621; Southern and Berg. J. Mol. Appl. Genet. 1. 1982, pp. 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79, 1982, pp. 422-426; Wigler et al., Cell 14, 1978, p.
  • fungal cells may be used as host cells.
  • suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharomvces spp., in particular strains of Saccharomyces cerevisiae.
  • Other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus nicer.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 238 023.
  • the medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the HBP produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g. ammonium sulphate
  • purification a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
  • the lipid-containing substance is LPS.
  • LPS suitable for inclusion in the composition of the invention may be obtained from the cell wall of gram-negative bacteria.
  • the lipid-containing substance may be the Lipid A portion of LPS.
  • Lipid A may suitable be prepared by synthesizing Lipid X which is a precursor of Lipid A.
  • the synthesis of Lipid X is described in I. Macher, Carbohydrate Res. 162. 1987, pp. 79-84, and K. Ikeda et al., Chem. Pharm. Bull. 35, 1987, pp. 1383-1387.
  • Lipid A is synthesized by reacting UDP- Lipid X in the presence of a crude preparation of Lipid A synthetase from E. coli, as described in P.L. Stuetz et al. in "Cellular and Molecular Aspects of endotoxin reactions", Eds. A. Nowotny, J.J. Spitzer and E.J.
  • the lipid-containing substance may be a ceramide.
  • Ceramide belongs to the group of sphingolipids, a chemically diverse class of biomolecules including compounds, such as ceramide phosphate and galactosylceramide.
  • Preferred ceramides have the structure
  • R 1 is a linear or branched, saturated or unsaturated C j ⁇ alkyl which may be substituted in the ⁇ -position by a hydroxyl group or esterified in the ⁇ -position by a saturated or unsaturated fatty acid;
  • R ⁇ is a hydrogen atom or a phosphate group
  • R 3 is C j 5_26 alkyl which may be saturated or unsaturated in the ⁇ -position or substituted by a hydroxy group in the ⁇ -position and optionally substituted by one or more ⁇ .
  • a alkyl groups, or R- 3 is an aryl group, preferably a phenyl group, which may be substituted by hydroxyl, halogen, including F, Cl, and Br, or methyl;
  • R 4 is hydrogen or a hydroxyl group. More preferred ceramides have the structure wherein R 1 is C 14 alkyl, R : is hydrogen or a hydroxyl or phosphate group, R 3 is C-j-alkyl or phenyl, and R" is hydrogen or a hydroxyl group.
  • a preferred ceramide is N- hexanoylsphingosine (Cg-ceramide).
  • ceramides may be synthesized by, e.g., substitution on carbon 2 (R 1 ) with various chain length fatty acids as described in. P. Van Veldhoven et al., Anal. Biochem. 183. 1989, pp. 177 - 189, who use acylation of D-erythro-spingosine with the anhydride form of the fatty acid wanted.
  • the substitution on carbon 3 (R 3 , R 4 ) has been described in A. Bielawska et al., J. Biol. Chem. 267. 1992, pp. 18493 - 18497, and A. Bielawska et al., J. Biol Chem. 268. 1993, pp 26226 - 26232.
  • the conjugate may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences. 1985.
  • the composition may typically be in a form suited for local or systemic injection or infusion and may, as such, be formulated with sterile water or an isotonic saline or glucose solution.
  • the compositions may be sterilized by conventional sterilization techniques which are well known in the art.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration.
  • the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • concentration of HBP may vary widely, i.e. from less than about 0.5%, such as from 1%, to as much as 15-20% by weight.
  • a unit dosage of the composition may typically contain from about 10 mg to about 1 g of HBP.
  • composition of the invention is contemplated to be advantageous to use for therapeutic applications such as treatment of inflammation, viral infection, ischemic reperfusion syndrome, bacterial endotoxaemia, sepsis, septic shock, disseminated intravascular coagulation or for stimulating a patient's immune system by activation of monocytes.
  • a daily dosage of the conjugate of 1-100 mg/kg body weight is contemplated to be suitable, dependent on the severity of the condition to be treated and the patient's condition.
  • the LPS binding site in HBP is flanked by the two cysteines, Cys 26 and Cys 42 .
  • the two cysteines were altered to serines by PCR mutagenesis:
  • the transfer construct pVL1392-HBP was used as template in the first round of mutagenesis where Cys 42 was altered.
  • HBP 3 x 10 s SF9 cells growing in supplemented Grace's medium (Gibco) with 10% FCS were centrifuged down and resuspended in a sample from the virus stock giving a MOI (multiplicity of infection) of 1.
  • the cells with virus were transferred to a 0.5 1 Bellco spinner flask (#1965-00500), and fresh supplemented Grace's medium with 2% FCS was added to a final volume of 300 ml.
  • the 400 ml volume was centrifuged in 50 ml tubes in a Sorvall Instruments TECHNOSPIN R centrifuge at 300 rpm for 3 min. The supernatants with cells were sucked away and the pelleted heparin Sepharose beads were separated from the remainder of contaminating cells by resuspension in 30 ml 0.9% NaCl added to each tube followed by centrifugation at 300 rpm. The entire procedure was repeated twice. The beads were finally washed in 20 ml sterile 0.5 M NaCl added to each tube.
  • the beads were then collected in one 50 ml tube in a small volume of 0.5 M sterile NaCl (20 - 30 ml) and transferred to a sterile glass filter funnel. The beads were allowed to drain and the HBP mutant was finally eluted from the beads with 30 ml sterile 3 M NaCl. The HBP mutant was purified from the 3 M eluate according to the method described in WO 89/08666.
  • the HBP mutant material was tested for LPS binding capacity using the assay described below, and no LPS binding was observed. Assay for LPS binding capacity.
  • the binding experiments were performed in 155 ⁇ l of sterile 0.9 % NaCl containing Bovine serum albumine (Sigma St. Louis Mo) 1 mg/ml, 4.5 picomol [ 3 H] lipopolysaccharide from Escherichia coli K12 LCD 25 Lot #5102A, specific activity 1.45 x 10 6 dpm/microgram, List Biological Laboratories, Inc., CA, USA and the following amounts of the HBP mutant: 0 pmol (control) 35 pmol, 18 pmol and 3.6 pmol. The mixture was incubated for 20 min at 37°C using a waterbath.
  • the HBP mutant material was tested for its ability to mediate cell detachment and homotypic aggregation on fibroblasts and endothelial cells as described by ⁇ stergaard and Flodgaard. op. tit.
  • HBP human wildtype HBP was produced using a baculovirus expression system in insect cells (SF.9 BRL). HBP was purified as previously described (1). Aiming at perturbing the putative LPS binding site in HBP without altering the overall folding of the molecule, a chimeric form of HBP was constructed.
  • the loop encompassing aa 26-42 (HBP-numbering) in the family of chymotrypsin like proteases is highly variable (2). It has been suggested that the effector site in HBP is situated within this loop, which also contains the LPS binding site (2).
  • Pereira et al (2) have shown that besides the importance of the conserved cysteine bridge in this loop, the RH motif in the sequence QGRHF just prior to the first C in the loop is important for LPS binding as well.
  • Human umbilical vein endothelial cells were isolated and cultured as previously described (3) with some modifications (4). Briefly, umbilical cords were collected in Ca 2+ and Mg 2+ -free PBS and stored at 4°C until cell isolation. The umbilical cords were used within 24 h. The veins were rinsed with Ca 2 * and Mg 2+ -free PBS prior to incubation with collagenase diluted in PBS at a final concentration of 70 U/ml at 37°C.
  • Released cells were centrifuged, suspended in medium 199 supplemented with fetal bovine serum (8%), calf serum (8%), heparin (16 U/ml), endothelial cell growth supplement (25 ⁇ g/ml) and antibiotics (penicillin 83 U/ml, streptomycin 83 ⁇ g/ml and fungizone 83 ⁇ g/ml) and seeded into 83-cnr flasks precoated with 2% gelatin in PBS. After 3-7 days of culture, the cells were detached using trypsin-EDTA (0.05%:0.5 mM) and seeded into 48-well plated. In some experiments, the cells were passed once before seeded into 48-well plates. The cells were used when expressing cobblestone morphology.
  • HUVECs grown in 48-well plates were washed twice with phosphate-free 5 buffer consisting of glucose (5.56 mM), NaCl (117.2 mM), CaCl 2 (1.8 mM), MgCl 2 (0.81 mM), KC1 (5.36 mM), NaHCO, (17.9 M) and HEPES (10 mM) at pH 7.4 and then incubated with 25 ⁇ Ci ,2 P0 4 in phosphate-free buffer supplemented with 10% human heat-inactivated serum for 30 min at 37°C. The reagents were added to the HUVECs and allowed to incubate for another 30 min. Okadaic acid was diluted in 10% dimethyl
  • Electrophoresis sample buffer (190 ⁇ l) (14) containing 5% SDS was added to the wells and HUVEC were lysed overnight at room temperature on a shaker.
  • the plates were washed extensively by distilled and deionized water, allowed to dry at 37°C and then blocked for 30 min at 37°C with 5 mg/ml very low endotoxin bovine serum albumin prepared in pyrogen-free PBS. The plates were then washed four times in assay buffer which consisted of pyrogen-free 50 mM Tris (pH 7.4), 500 mM NaCl, 1 mg/ml very low endotoxin bovine serum albumin and 0.05% Tween-20.
  • MOLECULE TYPE protein
  • ORIGINAL SOURCE (A) ORGANISM: porcine

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Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies ou de troubles liés à des lésions causées à des cellules par diverses agressions. Cette composition contient (a) une substance contenant un lipide ayant une portion lipidique qui est identique du point de vue structure ou analogue à celle d'un céramide, conjuguée à (b) une protéine capable de fixer cette substance contenant un lipide de manière à ce que, quand le conjugué est en contact avec des cellules vivantes, ladite substance contenant un lipide active une protéine phosphatase activée par un céramide, ce qui provoque une diminution du métabolisme cellulaire et (c) un diluant ou vecteur acceptable sur le plan pharmaceutique.
PCT/DK1996/000099 1995-03-09 1996-03-11 Conjugue proteine-lipide, constitue de la proteine fixant l'heparine et d'un analogue d'un ceramide, et son utilisation pharmaceutique pour le traitement de troubles lies a des lesions causees a des cellules par diverses agressions WO1996028544A1 (fr)

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EP96903943A EP0871721A1 (fr) 1995-03-09 1996-03-11 Conjugue proteine-lipide, constitue de la proteine fixant l'heparine et d'un analogue d'un ceramide, et son utilisation pharmaceutique pour le traitement de troubles lies a des lesions causees a des cellules par diverses agressions
AU47848/96A AU716791B2 (en) 1995-03-09 1996-03-11 A protein-lipid conjugate, consisting of heparin binding protein (HBP) and a ceramide analogue, and its pharmaceutical use in treatment of conditions involving stress injury to cells
JP8527186A JPH11501636A (ja) 1995-03-09 1996-03-11 医薬組成物
NO974123A NO974123L (no) 1995-03-09 1997-09-08 Konjugat av protein og lipid bestående av heparinbindende protein (HBP) og en ceramidanalog, og dets farmasöytiske anvendelse ved behandling av tilstander som medförer stresskade på celler
US08/925,708 US5939390A (en) 1995-03-09 1997-09-09 Pharmaceutical composition

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DK0240/95 1995-03-09
DK24095 1995-03-09

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US08/925,708 Continuation US5939390A (en) 1995-03-09 1997-09-09 Pharmaceutical composition

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999026647A1 (fr) * 1997-11-20 1999-06-03 Novo Nordisk A/S Utilisation de proteine de liaison a l'heparine pour la modulation ou la prophylaxie de l'apoptose des cellules de mammiferes
EP1471870A2 (fr) * 2000-01-10 2004-11-03 Yissum Research Development Company Of The Hebrew University Of Jerusalem Utilisation de conjugues de lipides dans le traitement de maladies
US6887470B1 (en) * 1999-09-10 2005-05-03 Conjuchem, Inc. Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components
US7101859B2 (en) 2000-01-10 2006-09-05 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US7141552B2 (en) 2000-01-10 2006-11-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
WO2007045873A2 (fr) * 2005-10-19 2007-04-26 Rutherford, Jodie Utilisations medicales et therapies fondees sur l'action de l'azurocidine sur igfbp-1
US7393938B2 (en) 2000-01-10 2008-07-01 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US7504384B2 (en) 2000-01-10 2009-03-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of infection
US7608598B2 (en) 2000-01-10 2009-10-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of conjunctivitis
US7772196B2 (en) 2000-01-10 2010-08-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US7811999B2 (en) 2000-01-10 2010-10-12 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Use of lipid conjugates in the treatment of diseases
US7893226B2 (en) 2004-09-29 2011-02-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Use of lipid conjugates in the treatment of diseases
US7906482B2 (en) 1999-05-17 2011-03-15 Advanced Diagnostics And Discovery Anti-obesity agents
US8076312B2 (en) 2000-01-10 2011-12-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Use of lipid conjugates in the treatment of disease
US8304395B2 (en) 2000-01-10 2012-11-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Lipid conjugates in the treatment of disease
US8501701B2 (en) 2000-01-10 2013-08-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Use of lipid conjugates in the treatment of disease
US8859524B2 (en) 2005-11-17 2014-10-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Lipid conjugates in the treatment of chronic rhinosinusitis
US8865681B2 (en) 2004-03-02 2014-10-21 Yissum Research Development Company of the Hebrew Unitersity of Jerusalem Use of lipid conjugates in the treatment of diseases or disorders of the eye
US8883761B2 (en) 2001-01-10 2014-11-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases associated with vasculature
US8906882B2 (en) 2005-11-17 2014-12-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Lipid conjugates in the treatment of allergic rhinitis
US8916539B2 (en) 2000-01-10 2014-12-23 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of disease
US9040078B2 (en) 2000-01-10 2015-05-26 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases of the nervous system
US11013811B2 (en) 2009-05-11 2021-05-25 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Lipid-polymer conjugates, their preparation and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1349410A (zh) * 1999-04-29 2002-05-15 诺沃挪第克公司 结合肝素的拮抗剂在缓激肽释放的抑制中的应用
CN110726846B (zh) * 2019-12-04 2022-08-26 南京市儿童医院 Hbp蛋白作为川崎病的诊断标志物的应用

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WO1991000907A1 (fr) * 1989-07-05 1991-01-24 Emory University Proteines chimiotactiques monocytes et peptides associes
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J. BIOL. CHEM., Vol. 269, No. 26, 1994, C.K. JOSEPH et al., "Bacterial Lipopolysaccharide Has Structural Similarity to Ceramide and Stimulates Ceramide-Activated Protein Kinase in Myeloid Cells", pages 17606-17610. *
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Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999026647A1 (fr) * 1997-11-20 1999-06-03 Novo Nordisk A/S Utilisation de proteine de liaison a l'heparine pour la modulation ou la prophylaxie de l'apoptose des cellules de mammiferes
US7906482B2 (en) 1999-05-17 2011-03-15 Advanced Diagnostics And Discovery Anti-obesity agents
US6887470B1 (en) * 1999-09-10 2005-05-03 Conjuchem, Inc. Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components
US7256253B2 (en) 1999-09-10 2007-08-14 Conjuchem Biotechnologies Inc. Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components
US7772196B2 (en) 2000-01-10 2010-08-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
EP1471870A2 (fr) * 2000-01-10 2004-11-03 Yissum Research Development Company Of The Hebrew University Of Jerusalem Utilisation de conjugues de lipides dans le traitement de maladies
US9040078B2 (en) 2000-01-10 2015-05-26 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases of the nervous system
US7101859B2 (en) 2000-01-10 2006-09-05 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
EP1471870A4 (fr) * 2000-01-10 2006-02-22 Yissum Res Dev Co Utilisation de conjugues de lipides dans le traitement de maladies
US7393938B2 (en) 2000-01-10 2008-07-01 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US7504384B2 (en) 2000-01-10 2009-03-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of infection
US7608598B2 (en) 2000-01-10 2009-10-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of conjunctivitis
US8916539B2 (en) 2000-01-10 2014-12-23 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of disease
US7811999B2 (en) 2000-01-10 2010-10-12 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Use of lipid conjugates in the treatment of diseases
US8901103B2 (en) 2000-01-10 2014-12-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US7141552B2 (en) 2000-01-10 2006-11-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US8076312B2 (en) 2000-01-10 2011-12-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Use of lipid conjugates in the treatment of disease
US8304395B2 (en) 2000-01-10 2012-11-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Lipid conjugates in the treatment of disease
US8372815B2 (en) 2000-01-10 2013-02-12 Yissum Research Development Company Use of lipid conjugates in the treatment of conjunctivitis
US8383787B2 (en) 2000-01-10 2013-02-26 Yissum Research Development Company Use of lipid conjugates in the treatment of diseases
US8501701B2 (en) 2000-01-10 2013-08-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Use of lipid conjugates in the treatment of disease
US9012396B2 (en) 2000-01-10 2015-04-21 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of conjunctivitis
US8865878B2 (en) 2000-01-10 2014-10-21 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US8883761B2 (en) 2001-01-10 2014-11-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases associated with vasculature
US8865681B2 (en) 2004-03-02 2014-10-21 Yissum Research Development Company of the Hebrew Unitersity of Jerusalem Use of lipid conjugates in the treatment of diseases or disorders of the eye
US7893226B2 (en) 2004-09-29 2011-02-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Use of lipid conjugates in the treatment of diseases
WO2007045873A3 (fr) * 2005-10-19 2007-08-09 Rutherford Jodie Utilisations medicales et therapies fondees sur l'action de l'azurocidine sur igfbp-1
WO2007045873A2 (fr) * 2005-10-19 2007-04-26 Rutherford, Jodie Utilisations medicales et therapies fondees sur l'action de l'azurocidine sur igfbp-1
US8906882B2 (en) 2005-11-17 2014-12-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Lipid conjugates in the treatment of allergic rhinitis
US8859524B2 (en) 2005-11-17 2014-10-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Lipid conjugates in the treatment of chronic rhinosinusitis
US11013811B2 (en) 2009-05-11 2021-05-25 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Lipid-polymer conjugates, their preparation and uses thereof

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CA2214799A1 (fr) 1996-09-19
JPH11501636A (ja) 1999-02-09
NO974123D0 (no) 1997-09-08
AU716791B2 (en) 2000-03-09
NO974123L (no) 1997-11-07
AU4784896A (en) 1996-10-02
CN1181783A (zh) 1998-05-13
EP0871721A1 (fr) 1998-10-21
CN1099462C (zh) 2003-01-22

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