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WO1997000081A1 - Procede empechant la septicemie et la dissemination des bacteries - Google Patents

Procede empechant la septicemie et la dissemination des bacteries Download PDF

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Publication number
WO1997000081A1
WO1997000081A1 PCT/US1996/010803 US9610803W WO9700081A1 WO 1997000081 A1 WO1997000081 A1 WO 1997000081A1 US 9610803 W US9610803 W US 9610803W WO 9700081 A1 WO9700081 A1 WO 9700081A1
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trp
antagonist
peptide
cys
lps
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PCT/US1996/010803
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English (en)
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Sanna M. Goyert
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Goyert Sanna M
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Priority to AU63929/96A priority Critical patent/AU6392996A/en
Publication of WO1997000081A1 publication Critical patent/WO1997000081A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • 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 a method of preventing or treating bacteremia, and subsequent dissemination of microorganisms from the blo dstream into other tissues.
  • Bacterial infection occurs when a pathogenic bacterium enters the host and multiplies inside the body. Most infections begin on the mucous membranes of the respiratory, alimentary, or genitourinary tracts, or in tissues exposed by wounds. After initial entry, the bacteria may remain localized, or they may spread through the blood and lymph systems, resulting in the infection of diverse tissues.
  • Dissemination may be inhibited either by interfering with the transfer of the bacteria from the local seat of infection to the systemic circulation, or by speeding the clearance of the bacteria from the blood and lymph systems.
  • bacteria If bacteria are allowed to disseminate into the circulation, and are not cleared quickly enough, the bacterial levels in the bloodstream will become higher than normal, a condition known as "bacteremia" .
  • LPS lipopolysaccharides
  • bacteremia persists, the patient may develop "septicemia". This is a life-threatening condition attributable to the body's reaction to high levels of bacterial endotoxins.
  • Cytokines such as interleukin-6 (IL-6) and tumor necrosis factor (TNF-o.) are known to promote the invasiveness of pathogenic bacteria, particularly gram-negative bacteria. It is known that IL-6 and TNF- ⁇ , as well as other inflammatory mediators, are released by the body in response to activation of the immune system by LPS endotoxins. Tracey, et al. , Ann. Rev. Med. 45: 491-503 (1994) ; Akira, et al. , Adv. Immunol. 54: 1-78 (1993) ; Bone, R.C., Ann. Intern. Med. 115: 457 (1991) .
  • IL-6 interleukin-6
  • TNF-o. tumor necrosis factor
  • Activation of the immune system by endotoxin/LPS can result in a cascade of reactions including the production of pro-inflammatory cytokines such as IL-6 and TNF-a.
  • Activation of the immune system by endotoxin/LPS can result in a cascade of reactions including the production of proinflammatory cytokines such as IL-6, IL-1 and TNF-o..
  • This initial production of cytokines may lead to a cascade of events which can included some or all of the following: bacteremia and bacterial dissemination, procoagulant activity, acute respiratory distress syndrome and death.
  • This activation is mediated in part by interactions of various molecular receptors with foreign antigen.
  • monocyte/macrophage surface markers that possess receptor and signal transduction functions have been identified. Many of them are cell differentiation markers, i.e., characteristically present in defined stages of development, especially the end stages of cells of defined lineage and function.
  • CD14 is a 55-kD glycoprotein expressed strongly on the surface of monocytes and weakly on the surface of neutrophils. Goyert, et al., J. Immunol. 137: 3909 (1986) ; Haziot, et al. , J. Immunol. 141: 547-552 (1988); Goyert, et al. , Science 239: 497 (1988) . CD14 is linked by a cleavable phosphinositol tail (Haziot, et al . , J. Immunol. 141: 547-552
  • CD14 has been shown to bind to LPS. Haziot, et al.,J. Immuno. 151: 1500 (1993) .
  • the binding causes cells to become highly activated and release interleukins, TNF-o., and other substances that promote the growth and invasiveness of bacteria, enabling the dissemination of bacteria into the bloodstream or into the peritoneal cavity.
  • Another mechanism by which bacteria can disseminate into the blood stream involves interference with the body's normal clearing mechanisms to remove bacteria.
  • the peritoneal cavity can be an important pathway for bacterial dissemination.
  • the parietal peritoneum is a membrane lining the walls of the abdominal and pelvic cavities.
  • the visceral peritoneum is a similar membrane investing the contained viscera. Together the two membranes define an enclosed space known as the peritoneal cavity.
  • Bacteria can enter the peritoneal cavity as a result of perforation of the GI tract, infection of an intraabdominal organ, and direct contamination from an external source, such as by trauma, burns or surgery. Once bacteria enter the peritoneal cavity, dissemination is rapid. Within 6 minutes of intraperitonal inoculation of bacteria in dogs, thoracic lymph is culture-positive; within 12 minutes, there is bacteremia (bacteria at elevated levels in the bloodstream) .
  • Peritonitis is an inflammation of the peritoneum, often attributable to a severe local infection. It may be caused by a number of pathogenic microorganisms, and can result from gastrointestinal trauma, including surgery or peritoneal dialysis. Typical medical treatments for the prevention of peritonitis include antibiotic therapy, especially prior to surgical procedures, radio therapy or chemotherapy. This approach is hindered, however by the multiple drug resistance of many of the bacteria known to cause peritonitis. Moreover, since peritonitis may be caused by both gram-positive and gram-negative microorganisms, the choice of antibiotic may not be sufficient.
  • antibiotic treatment is non-specific, eliminating many non-pathogenic indigenous microorganisms which normally prevent bacterial disease through bacterial antagonism, especially in the gastrointestinal tract.
  • broad spectrum antibiotics such as tetracyclines
  • antibiotic resistant strains of pathogenic microorganisms normally held in check by the antagonistic action of the coliforms and other organisms, multiply freely and can actually foster peritonitis, rather than prevent it.
  • the present invention provides a method for controlling severe local infections, interfering with bacterial dissemination from local infections to other parts of the body, and preventing and treating bacteremia and septicemia.
  • the patient is given a drug which acts as an antagonist to cellular CD14.
  • the CD14 antagonist may be a substance which competes with cellular CD14 for CD14 ligands, such as LPS, or it may be a substance which binds to cellular CD14 and interferes with the latter's binding to relevant ligands, such as LPS.
  • the antagonist is one that competes with cellular CD14, it is preferably a soluble fragment of CD14, or a mutant thereof.
  • the antagonist is a substance which binds CD14, it may be an antibody, a peptide, or other CD14-binding molecule.
  • Figure IA is a bar graph of percent survival of control and CD14-deficient mice following intraperitoneal (i.p.) inoculation of Salmonella minnescia LPS.
  • Figure IB is a bar graph of the amount of TNF- a (ng/ml) in the blood stream of control and CD14-deficient mice following i.p. inoculation of S_j_ minnescia LPS.
  • Figure IC is a bar graph of the amount of serum IL-6 (ng/ml) in control and CD14-deficient mice following i.p. inoculation of S_j_ minnescia LPS.
  • Figure 2A is a bar graph of the per cent survival of control and CD14 deficient mice following i.p. inoculation of viable E. coli.
  • Figure 2B is a bar graph of the amount of serum TNF-o. (ng/ml) present in control and CD14-deficient mice following i.p. inoculation of viable E. coli.
  • Figure 2C is a bar graph of the amount of serum IL-6 (ng/ml) present in control and CD14-deficient mice following i.p. inoculation of viable E. coli.
  • Figure 3A is a graph of serum concentrations of TNF- ⁇ (ng/ml) in E. coli infected control and CD14-deficient mice over a seven hour period.
  • Figure 3B is a graph of serum concentration of IL-6 (ng/ml) in E. coli infected control and CD14-deficient mice over a seven hour period.
  • Figure 4A is a bar graph of E. coli CFU/ml blood in control and CD14 deficient mice seven hours after i.p. inoculation of E. coli (IO 7 CFU) .
  • Figure 4B is a bar graph of E. coli CFU/ml blood in control and CD14 deficient mice seven hours after i.p. inoculation of E. coli (10 5 CFU) .
  • Figure 4C is a bar graph of E. coli CFU/ml lung in control and CD14 deficient mice seven hours after i.p. inoculation of E. coli (IO 7 CFU) .
  • endotoxin/LPS a component of the outer membrane of gram-negative bacteria activates the immune system.
  • Several receptors for endotoxin/LPS have previously been identified and characterized, including CD14, the cell surface glycoprotein expressed on the surface of monocytes and neutrophils.
  • CD14 the cell surface glycoprotein expressed on the surface of monocytes and neutrophils.
  • CD14 deficient mice are more resistant than normal mice to intraperitoneally administered purified LPS (Example 1) and gram-negative bacteria (Example 2) , and exhibit reduced serum levels of TNF-alpha, IL-6 and IL-1.
  • bacteremia can be prevented or treated by administering to a patient a CD14 "antagonist", that is, a substance that interferes with the ability of the bacteria to interact, directly or indirectly, with CD14 and thereby trigger a metabolic or immunologic reaction that leads (e.g., through cytokine release) to increased bacterial dissemination.
  • a CD14 "antagonist” that is, a substance that interferes with the ability of the bacteria to interact, directly or indirectly, with CD14 and thereby trigger a metabolic or immunologic reaction that leads (e.g., through cytokine release) to increased bacterial dissemination.
  • the CD14 antagonist is a soluble analogue (sCD14) of cellular CD14.
  • the analogue acts as an antagonist by "decoying", that is, the bacterial LPS binds the analogue rather than cellular CD14.
  • This analogue may be a soluble fragment of a naturally occurring cellular CD14, or a substantially homologous mutant or other functional derivative of CD14 (or a fragment thereof) .
  • the analogues retain at least 10% of the LPS (or other relevant ligand) -specific binding activity of cellular CD14.
  • sCD14 may be naturally occurring human soluble CD14 purified by methods known in the art or it may be recombinant sCD14, made by the method described in Applicant's co-pending application Serial No. 08/254,095 or any other art-known method.
  • naturally occurring is meant any form of CD14 found in the human population that is biologically functional.
  • Functional derivatives of sCD14 include any sCD14 molecules that have been modified by such means as, for example, alteration in amino acid sequence or alteration in the glycosylation pattern thereof, but which molecules retain the ability to bind to LPS.
  • Fragments of CD14 useful in the present invention include all soluble fragments of CD14 that retain the relevant biological properties of the parent molecule, e.g., ability to bind to LPS present on the outer membrane of bacteria.
  • fragment refers to an isolated polypeptide sequence corresponding to a portion of the CD14 amino acid sequence. The fragments may be generated by direct proteolytic digestion of CD14 or by expression of genetically engineered nucleic acid sequence that encodes a portion of the full size CD14 polypeptide. Alternatively, fragments of sCD14 may be synthesized from amino acids by art known methods of peptide synthesis. A preferred fragment of sCD14 is the peptide comprising the amino acid sequence:
  • CD14 is a glycophosphoinositol (GPI) anchored membrane protein.
  • GPI glycophosphoinositol
  • a soluble CD14 analogue will omit the GPI anchor. It may contain the full-length amino acid sequence of CD14, or only a portion thereof. It may also differ from the native amino acid sequence by substitution, insertion, or internal deletion mutations, or by fusion of additional amino acid sequences to either terminus.
  • the carboxy terminal of CD14 contains the signal (believed to be at least about 15 amino acids long) which directs mammalian cells to add on the GPI anchor after translation of CD14.
  • the identity of the attachment signal is readily determined by systematically expressing truncation mutants of CD14 and testing for the presence of the GPI anchor.
  • Some soluble CD14 occurs naturally, most likely as a result of proteolytic cleavage of the membrane CD14, leaving the GPI anchor behind and releasing a soluble fragment.
  • Soluble CD14 can be prepared by a number of means, including (1) nonbiological synthesis of CD14 (including full-length CD14) without the GPI anchor, (2) chemically or enzymatically removing the GPI anchor from GPI-linked CD14, e.g., with a glycosidase and/or lipidase; (3) semi-synthesis, wherein the carboxy terminal
  • Functional soluble fragments are readily identified by systematically fragmenting CD14 and testing the fragments for LPS or other relevant CD14-like ligand-binding activity. It is not necessary that every possible fragment be prepared and tested. Rather, the skilled practitioner will designate candidate fragmentation points within the CD14 molecule. These fragmentation points may be spaced uniformly or positioned in the light of the known sequence. For example, the fragmentation points may be chosen to lie between conserved regions, regions of high (or low) hydrophilicity, or regions likely to have significant secondary structure. If none of the fragments tested has activity, the skilled practitioner will test larger fragments. Once a functional fragment has been identified, subfragments may be tested to more finely delineate the smallest fragment which is functional.
  • At least one of the fragments tested retains the sequence corresponding to amino acids 143-168, which applicant has found to inhibit LPS binding, by CD14, and/or 57-64, a putative LPS-binding site of CD14 identified by Juan, et al . , J. Biol. Chem., 270:5219-24 (1995) .
  • a putative LPS-binding site of CD14 identified by Juan, et al . , J. Biol. Chem., 270:5219-24 (1995) .
  • one may start -.'ith the putative LPS-binding site, and extend in either the N- or C- direction, or both, until a functional fragment is obtained.
  • Any desired fragment may be made by expressing a corresponding gene. If the fragments are generated by treating a full length CD14 with proteolytic enzymes, the fragments obtained will be a function of the enzyme selected.
  • the enzymes vary in site specificity, and hence in the number and position of the fragments generated.
  • the analogues are not limited to derivatives of human, or even mammalian, CD14s.
  • the CD14 sequence may be mutated provided the mutant substantially retains the desired solubility and binding activity.
  • the amino acid sequence is at least 50%, more preferably at least 80%, identical with the corresponding CD14 sequence, the compared sequences being at least 20 amino acids long.
  • a protein is more likely to tolerate a mutation which
  • (d) affects a part of the molecule distal to the binding site; (e) is a substitution of one amino acid for another of similar size, charge, and/or hydrophobicity; and (f) is at a site which is subject to substantial variation among a family of homologous proteins to which the protein of interest belongs . These considerations can be used to design functional mutants.
  • Surface residues may be identified experimentally by various labeling techniques, or by 3-D structure mapping techniques like X-ray diffraction and NMR. A 3-D model of a homologous protein can be helpful.
  • Residues forming the binding site may be identified by (1) comparing the effects of labeling the surface residues before and after complexing the protein to its target, (2) labeling the binding site directly with affinity ligands, (3) fragmenting the protein and testing the fragments for binding activity, and (4) systematic mutagenesis (e.g., alanine-scanning mutagenesis) to determine which mutants destroy binding. If the binding site of a homologous protein is known, the binding site may be postulated by analogy.
  • Protein libraries may be constructed and screened that a large family (e.g., 10 8 ) of related mutants may be evaluated simultaneously.
  • CD14 In the case of CD14, one may compare homologous sequences, such as human and mouse CD14, to identify regions likely to be involved in LPS binding.
  • Semi-conservative substitutions are defined to be exchanges between two of groups (I) - (V) above which are limited to supergroup (A) , comprising (I) , (II) , and (III) , above, or to supergroup (B) , comprising (IV_ and (V) above. Also, Gly and Ala are considered to be semi-conservative substitutions for any other residue. Non-conservative substitutions are those which are neither conservative nor semi-conservative.
  • substitutions are not limited to the naturally occurring amino acids.
  • the desired amino acid may be used directly.
  • a genetically encoded amino acid may be modified by reacting it with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Aromatic amino acids may be replaced with D- or L- naphthylalanine, D- or L-phenylglycine, D- or L-thieneylalanine, D- or L-1-, 2- 3-, or 4-pyreneylalanine, D- or L-3- thieneylalanine, D- or L- (2-pyridinyl) -alanine, D- or L-(3- pyridinyl) -alanine, D- or L- (2-pyrazinyl) -alanine, D- or L-(4- isopropyl) -phenylglycine, D- (trifluoromethyl) -phenylglycine, D- trifluoromethyl) -phenylalanine, D-p-fluorophenylalanine, D- or L- p - b ipheny1 pheny1 a 1 an i ne , D- or L-p- methoxybiphenylphen
  • Acidic amino acids can be substituted with non- carboxylate amino acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of
  • substitutions include unnatural hydroxylated amino acids made by combining "alkyl” (as defined and exemplified herein) with any natural amino acid.
  • Basic amino acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulline, or (guanidino) -acetic acid, or other (guanidino)alkyl-acetic acids, where "alkyl” is as defined above.
  • Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • any amino acid can be replaced by the same amino acid but of the opposite chirality.
  • any amino acid which occurs naturally in the L-configuration (which may also be referred to as the R or S configuration, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D-amino acid but which can additionally be referred to as the R- or S-, depending upon its composition and chemical configuration.
  • Such derivatives have the property of greatly increased stability to degradation by enzymes, and therefore are advantageous in the formulation of compounds which have longer in vivo half lives when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • Additional amino acid modifications of amino acids may include the following: Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines) , such as 2- chloroacetic acid or chloroacetamine, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo-beta- (5-imidazoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl; disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-r-nitrophenol, or chloro- 7- nitrobenzo-2-oxa-l,3-diazole.
  • compounds such as bromotrifluoroacetone, alpha-bromo-beta- (5-imidazoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl; disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-
  • Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate, e.g., at pH 5.5-7.0, because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used, e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • compounds such as diethylprocarbonate, e.g., at pH 5.5-7.0, because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used, e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Derivatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoester, e.g., methyl picolinimidate pyridoxal phosphate; pyridoxal chloroborohydride trinitrobenzenesulfonic acid; O-methylisourea; 2,3-pentanedione and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1,2-cyclohexandedione and ninhydrin according to known method steps. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon- mino group.
  • tyrosyl residues per se are well known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidazol and tetranitromethane may be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups may be selectively modified by reaction with carbodiimides, (R'-N-C-N- R' ) suchas 1-cyclohexyl-3- (2-morpholinyl) - (4-ethyl)carbodiimide or 1- ethyl-3- (4-azonia-4,4-dimethyIpentyl) carbodiimide.
  • carbodiimides R'-N-C-N- R'
  • aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues may be readily deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the present invention.
  • Derivatization with bifunctional agents is useful for crosslinking the peptide to a water-insoluble support matrix or to other macromolecular carriers, according to known method steps.
  • Commonly used crosslinking agents include, e.g., 1,1- bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including di succ inimidyl esters such as 3 , 3 ' - dithiobis(succinimidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1, 9-octane.
  • Derivatizing agents such as methyl-3- [ (p-azidophenyl)dithio]pro ioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices can be used, such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440, all of which are hereby incorporated in their entirety by reference.
  • Derivatized moieties may improve the solubility, absorption, biological half life, and the like or eliminate or attenuate any possible undesirable side effects of the molecules. Moieties capable of mediating such effects are disclosed, for example in Remington's Pharmaceutical Sciences, 16th ed. , Mack Publishing Co., Easton, PA (1980).
  • Modifications are not limited to the side chains of the amino acids.
  • the peptides may also comprise isoteres of two or more residues in the original peptide.
  • An isotere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence.
  • the term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the ⁇ -carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed. , 1983) .
  • peptide mimetics whose conformation is similar to that of a peptide but do not have a peptide-like molecular formula.
  • all of the residues of the peptide are replaced by one or more isoteres as defined above.
  • Chimeras form a special class of mutants.
  • the chimeras may be of e.g., different mammalian CD14s, or of CD14 and another endotoxin-binding protein, such as bacterial permeability- increasing protein. See Marra, et al. , Critical Care Medicine 22:559 (1994); Fisher, Jr., et al. , Critical Care Medicine, 22:553 (1994) .
  • Anti-idiotype antibodies may also be used as CD14 analogues when the parental antibody occludes the LPS binding site of CD14.
  • the comments made herein regarding mutation and derivatization of CD14 analogues should be taken to apply, mutatis mutandis, to other CD14 antagonists.
  • the CD14 antagonist may also be a molecule which binds CD14.
  • the antagonistic effect is attributable to the drug occupying, or sterically hindering access to, the LPS (or other relevant ligand) binding site of cellular CD14.
  • One class of such CD14 antagonists are anti-CD14 antibodies.
  • the antibodies of interest include both whole antibodies, and antigen-binding derivatives of whole antibodies, such as F(ab) 2 , Fab', Fab, Fv, V H and V L . In some cases it may be advantageous to construct a hybrid antibody that binds two different epitopes.
  • Anti-CD14 antibodies can be made by any art-known method.
  • polyclonal antibodies may be prepared in a conventional manner, by using the subject polypeptide, e.g. CD14, as an immunogen and injecting the polypeptide into a mammalian host, e.g.,mouse, cow, goat, sheep, rabbit, etc., particularly with an adjuvant, such as, for example, complete Freund 's adjuvant, aluminum hydroxide gel, or the like. The host may then be bled and the blood used for isolation of polyclonal antibodies.
  • a mammalian host e.g.,mouse, cow, goat, sheep, rabbit, etc.
  • an adjuvant such as, for example, complete Freund 's adjuvant, aluminum hydroxide gel, or the like.
  • the host may then be bled and the blood used for isolation of polyclonal antibodies.
  • monoclonal antibodies may be made by fusing peripheral blood lymphocytes (B-cells) of the immunized host with an appropriate myeloma cell to produce an immortalized cell line that secretes monoclonal antibodies specific to CD14.
  • B-cells peripheral blood lymphocytes
  • Additional (yet different) monoclonal antibodies which bind to the same antigen, and, if desired, the same epitope of that antigen, as bound by a lead antibody may be obtained by one or more of the following procedures.
  • messenger RNA is extracted from the hybridoma.
  • the mRNA is sequenced directly, see Griffiths and Mulstein, Hybridoma Technology in the Biosciences and Medicine, Chap. 6, pp. 103-115 (1985) to determine both the light and heavy variable domain encoding sequences.
  • Oligonucleotides hybridizing in the constant domains of the kappa and gamma chains are used as primers to synthesize first strand cDNAs, which are then used as templates for PCR amplification of the variable domain. Suitable PCR primers are chosen based on study of the mRNA sequence.
  • These light and heavy chain-encoding sequences may now be mutated to produce new antibodies of similar but different reactivity relative to the parental antibody.
  • the mutated sequences may be introduced into full-length (or deliberately truncated) heavy and light chain genes by cassette mutagenesis. Vectors carrying these sequences may be used to transform suitable host cells.
  • the heavy and light chains may be co- expressed, or expressed separately and combined in vitro.
  • the constant region of the heavy and light chains may be replaced, if desired, by the corresponding portions of human heavy and light chains.
  • Such chimeric antibodies are likely to elicit a smaller HAMA response than the original mouse antibody.
  • Framework residues in the variable regions may likewise be "humanized.” Merck, EP 438,310; Winter, EP 239,400; Queen, WO92/11018; Celltech, WO91/09967 and WO91/09968; Wellcome Foundation, WO92/07075; Gorman, WO92/05274.
  • General references on recombinant production of monoclonal antibodies include Schering, EP 88,494; Genentech, Inc., EP 125,023; Intl. Gen. Eng'g, WO87/02671; Gillies, USP 4,663,281; Kudo, USP 5,101, 024; Moore, USP 4,642,334; Kudo, USP 4,786,719.
  • a set of single substitution mutants are prepared by site-specific mutagenesis, so that each of the hypervariable residues is conservatively mutated in one of the mutants.
  • a V H or V L gene is randomly mutated at predetermined "variable” codons and the mutant domain is displayed on the surface of phage.
  • These "antibody phage" libraries are then screened for binding activity.
  • the allowed amino acids may be any of 2-20 genetically encoded amino acids, and may (and preferably do) include the wild-type amino acid.
  • the frequence of occurrence may be, e.g., such that all allowed amino acids occur with substantially equal frequency, or such that the wild-type amino acid (or some class of amino acid) predominates.
  • each variable residue may be wild-type, or a single possible conservative substitution, and the frequencies of occurrence are chosen so single substitution mutants will predominate.
  • antibody phage include McCafferty, et al., Nature, 348:552-54 (1990) ; Garrard, et al. , Bio/Technology, 9:1373-77 (1991) ; Huse, et al. , J. Immunol., 149:3914-20 (1992) ; Kang, et al. , PNAS, 88:4363-66 (1991) ; Hoogenboom, et al.
  • a parental antibody (Ab j ) or its specific antigen binding fragments may be used to raise anti-idiotype antibodies (Ab 2 ) which bear the internal image of the epitope recognized by the parental antibody.
  • These antibodies (Ab 2 ) or fragments therefore, may then be used to raise anti-anti-idiotype antibodies (Ab 3 ) which complement that internal image and therefore are cross-reactive with the original epitope.
  • the antagonist may be a CD14-binding molecule other than an antibody.
  • Such molecules may be obtained by determining the 3D-structure of at least the binding site of CD14 and modeling a molecule that theoretically would block that interaction, by modifying a known CD14-binding molecule (such as an antibody or portion thereof) or LPS by preparing and screening a library of potential CD14-binding molecules.
  • LPS binding protein complex a molecule which acts as an antagonist by binding to LPS, or to an LPS: LPS binding protein complex, rather than to CD14.
  • candidate binding molecules can be individually prepared and tested, it is convenient to prepare and screen, essentially simultaneously, a combinatorial library of potential binding molecules.
  • combinatorial library implies that the binding molecules are polymers or oligomers constructed by the at least partially random combination of monomeric units provided during synthesis. This random combination imparts diversity to the library. For example, if there are six randomized positions, and each unit is chosen from a set of twenty possible monomers, the library will have a diversity of 20 6 different molecules.
  • oligomers In some libraries, all positions of the oligomer or monomer are randomized. In other libraries, some positions are held constant, while others are randomized. Typically, libraries of oligomers (e.g., hexapeptides) are fully randomized, while libraries of polymers are randomly varied at selected sites. However, the number of variable positions is limited only by one's ability to construct a library in which each theoretical sequence is reasonably likely to be represented in the library in amount sufficient for its binding to the target, if at an acceptable level, to be detectable.
  • the range of possibilities may differ from one variable position to another, and the library may be prepared so that the frequency of occurrence of the allowed monomers differs from one variable position to another, even when the possibilities are the same. It is not necessary that all possible monomers at a given position be equally likely.
  • the choice of which positions to vary, which monomers to allow at each variable positions, and what proportions to allow, will typically be guided by knowledge of one or more reference molecules of known binding activity.
  • the library may be static (its sequences do not change after initial synthesis) or dynamic.
  • a dynamic library the unbound molecules are exposed, intermittently or continuously, to conditions causing degradation and resynthesis (thus scrambling their sequences) , while the bound molecules are protected from further scrambling. See Venton, USP 5,366,862.
  • the building blocks are amino acids
  • the library is composed of peptides or proteins.
  • the peptides or proteins may be composed solely of genetically encoded amino acids, in which case the library may be prepared by recombinant DNA techniques, or it may include other amino acids (including ones which do not occur in nature) , in which case the library is prepared by in vitro chemical or enzymatic synthesis (or possibly by in vitro chemical or enzymatic library) .
  • the peptide or protein may be displayed on the surface of a virus, (e.g., a Mlamentous phage), a cell (especially a bacterial or yeast cell) , or a bacterial spore. In this manner, it can interact with a target, while remaining physically associated with the encoding DNA.
  • Amino acids are the basic building blocks with which peptides and proteins are constructed. Amino acids possess both an amino group (-NH 2 ) and a carboxylic acid group (-COOH) . Many amino acids, but not all, have the structure NH 2 -CHR-COOH, where
  • R is hydrogen, or any of a variety of functional groups.
  • Twenty amino acids are genetically encoded: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine. Of these, all save Glycine are optically isomeric, however, only the L-form is found in humans. Nevertheless, the D-forms of these amino acids do have biological significance; D-Phe, for example, is a known analgesic.
  • amino acids are also known, including: 2- Aminoadipic acid; 3-Aminoadipic acid; beta-Aminopropionic acid; 2-Aminobutyric acid; 4-Aminobutyric acid (Piperidinic acid) ; 6-Ammocaproic acid; 2-Aminoheptanoic acid; 2-Aminoisobutyric acid, 3-Aminoisobutyric acid; 2-Aminopimelic acid; 2,4-Diaminobutyric acid; Desmosine; 2, 2 ' -Diaminopimelic acid;
  • Peptides are constructed by condensation of amino acids and/or smaller peptides.
  • the amino group of one amino acid (or peptide) reacts with the carboxylic acid group of a second amino acid (or peptide) to form a peptide (-NHCO-) bond, releasing one molecule of water. Therefore, when an amino acid is incorporated into a peptide, it should, technically speaking, be referred to as an amino acid residue.
  • a side chain-protected amino acid is coupled by its carboxy terminal to a support material, such as a resin.
  • a side chain and amino terminal protected amino acid reagent is adde r , and its carboxy terminal reacts with the exposed amino terminal of the insolubilized amino acid to form a peptide bond.
  • the amino terminal of the resulting peptide is then deprotected, and a new amino acid reagent is added.
  • the cycle is repeated until the desired peptide has been synthesized.
  • the amino acid reagent is made as pure as possible.
  • the amino acid reagent employed in one or more of the cycles may be a mixture of amino acids, and this mixture may be the same or different, from cycle to cycle.
  • the amino acid reagent employed in one or more of the cycles may be a mixture of amino acids, and this mixture may be the same or different, from cycle to cycle.
  • Ala were coupled to the resin, and a mixture of Glu, Cys, His and Phe were added, the dipeptides Ala-Glu, Ala-Cys, Ala-His and Ala-Phe will be formed.
  • the resulting residue in the peptide molecules being synthesized is called a constant residue.
  • the added residue is called a variable residue.
  • the component amino acids of the mixture which are the only amino acids which can occupy that variable residue position, are called the "set" of that variable residue.
  • the set for one variable residue may be different from that of the next one.
  • a peptide library may consist essentially only of peptides of the same length, or it may include peptides of different length.
  • the peptides of the library may include, at any variable residue position, any desired amino acid.
  • Possible sets include, but are not limited to: (a) all of the genetically encoded amino acids, (b) all of the genetically encoded amino acids except cysteine (because of its ability to form disulfide crosslinks) ,
  • the peptide library may include branched and/or cyclic peptides.
  • the linkages between the amino acid residues may be peptide bonds (-NHC0-) , as in a classical peptide or protein, or another type of linkage, such as a peptide bond analogue (e.g., -CH 2 CH 2 -, -NRC0-, -NHCS-) .
  • a peptide bond analogue e.g., -CH 2 CH 2 -, -NRC0-, -NHCS-
  • the library will usually be described as a peptoid library. See Simon, et al., P.A., "Peptoids: A modular approach to drug discovery.” Proc. Natl . Acad. Sci . USA 89:9367-9371, 1992, and cf. Gilon, et al, "Backbone cyclization: A new method for conferring conformation constraint on peptides.” Biopolymers 31:745-750, 1991.
  • the peptides or peptoids may be linear, branched, or cyclic.
  • One of the advantages of the chemical approach to peptide libraries is the ability to produce and test cyclic and branching peptides.
  • Early examples in the literature for such structures are found in the use of multiple antigenic peptides (MAP) as immunogens, in MAPs, peptide haptens are attached to a branching "tree" of lysines.
  • MAP MAP containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite protein. Bur. J. Immunol . 21:3015-3020, 1991.
  • Cyclization is a common mechanism for stabilization of peptide conformation thereby achieving improved association of the peptide with its ligand and hence improved biological activity. Cyclization is usually achieved by intra-chain Cystine formation, by formation of peptide bond between side chains or between N- and C- terminals. Cyclization was usually achieved by peptides in solution, but several publications have appeared recently that describe cyclization of peptides on beads (see references below) . These published techniques may be directly applicable to our library approach. 1. Spatola, A.F., Anwer, M.K. and Rao, M.N. Phase transfer catalysis in solid phase peptide synthesis. Preparation of cycle [Xxx-Pro-Gly-Yyy-Pro-Gly] model peptides and their conformational analysis. Int. J. Pept . Protein Res . 40:322-332, 1992
  • Trazoak A. Synthesis of 'head-to-tail' cyclized peptides on solid supports by Fmoc chemistry. Tetrahedron Lett . 33:4557- 45560, 1992. 4. Wood, S. J. and Wetzel, R. Novel cyclization chemistry especially suited for biologically derived, unprotected peptides, Int. J. Pept . Protein Res . 39:533-539, 1992.
  • the polymer or oligomer is composed of nucleic acids.
  • the usual bases are the purines adenine and guanine and the pyrimidines thymidine (uracil for RNA) and cytosine. Such bases may be combined by conventional DNA and RNA synthesis methods. Unusual bases, such as those listed below, may be incorporated into the synthesis or produced by post synthetic treatment with mutagenic agents.
  • 5-methylaminomethyluridine 5-methoxyaminomethy1-2-thiouridine. beta,D-mannosylqueosine.
  • DNA may be synthesized by the stepwise addition of nucleotides to a nascent chain.
  • the first step of the synthesis may be the coupling of a nucleoside, via a succinyl linkage, to a suitable support, such as cellulose. This nucleoside represents the 3' end. Chain elongation proceeds from 3' to 5 ' ; each cycle being composed (in one conventional method) of the following steps :
  • a protected nucleotide is coupled to the exposed 5 ' end.
  • the protected nucleotides may be 5 ' -O- dimethoxytrityl-N 6 - (benzoyl) -2 ' -deoxyadenosine, 5 ' - dimethoxytrityl-N 4 - (anisoyl) -2 ' -deoxycytidine, 5 ' -0- dimethoxytrityl-N 6 - (N' ,N' , -di-n-butyl formadine) -2 ' - deoxyadenosine, and 5 ' -O-dimethoxytrityl-N 2 -
  • Unreacted 5 ' -hydroxyl groups are protected, e.g., by acylation.
  • the traditional method for DNA sequencing by chemical cleavage depends on the parallel execution of four base-specific or base-selective modification protocols and the parallel electrophoretic resolution of the hydrolysates in four lanes. It is also possible to analyze DNA based on a single base modification procedure, if it produces some degree of backbone cleavage at all bases in the DNA but the rates of cleavage at the four canonical bases (A, T, G, C) are clearly different. See Ambrose and Pless, Meth. Enzymol., 152:522 (1987) (modification with 0.5M aqueous piperidine, 0.3M NaCl, 90°C , pH > 12, 5 hrs) . The single reagent method is faster but less accurate.
  • the building blocks are monosaccharides.
  • Polysaccharides are larger polymers of monosaccharides in a branched or unbranched chain.
  • Oligosaccharides are shorter polymers of monosaccharides, such as di-, tri-, tetra-, penta-, and hexasaccharides.
  • polymeric carbohydrate will be used to cover both poly- and oligosaccharides.
  • Monosaccharides in a polymeric carbohydrate library may be aldoses, ketoses, or derivatives. They may be tetroses, pentoses, hexoses or more complex sugars. They may be in the D- or the L-form.
  • Suitable D-sugars include D-glyceraldehyde, D- erythrose, D-threose, D-arabinose, D-ribose, D-lyxose, D-xylose, D-glucose, D-mannose, D-altrose, D-allose, D-talose, D-galactose, D-idose, D-gulose, D-rhamnose, and D-fucose.
  • Suitable L-sugars include the L-forms of the aforementioned D-sugars.
  • a sugar hemiacetal may be reacted with a hydroxyl group of another sugar to form a disaccharide, and the reaction may be repeated.
  • carbohydrate synthesis methods see Kanie, 0. and Hindsgaul, 0., "Synthesis of Oligosaccharides, Glycolipids and Glycopeptides," Curr. Opin Struc. Bio. , 2:674-681, (1992) .
  • sequencing see Y.C Lee, "Review: High-Performance Anion- exchange Chromatography for Carbohydrate Analysis," Anal . Biochem.
  • the libraries of the present invention are not limited to the foregoing types. Any set of organic compounds which can be, essentially interchangeably, ligated to form an oligomer or polymer in reasonable yields can be used to construct a combinatorial library. In some cases, it is advantageous to first screen a peptide library, and then construct a library of peptide mimetics. For further insights, see the preceedings of the Fourth Approval Program on Development of Small molecule Mimetic Drugs (Cambridge Healthtech Institute; May 2-3, 1996) and the Fifth Annual International Conference on High-Throughput Screening (Intermembrane Business Communications; April 25-26, 1996) .
  • the target is insolubilized, and unbound library molecules are removed. This is analogous to a heterogeneous affinity assay.
  • the target is labeled, and either the detectability of the label is altered by binding (as in a homogeneous assay) or the library molecules are immobilized, so that the target molecules "marks" the successful members of the library. After successful binding molecules are recovered, it is necessary to characterize them. This may be done by sequencing the molecule; this is a standard practice with oligopeptide and nucleic acid libraries.
  • the primary sequence of amino acids in a peptide or protein is commonly determined by a stepwise chemical degradation process in which amino acids are removed one-by-one from one end of the peptide, and identified. In the
  • the N-terminal amino acid of the peptide is coupled to phenylisothiocyanate to form the phenylthiocarbamyl
  • the PTC peptide is then treated with strong acid, cyclizing the PTC L -tptide at the first peptide bond and releasing the N-terminal amino acid as the anilino- thiozolinoe (ATZ) derivative.
  • the ATZ amino acid which is highly unstable, is extracted and converted into the more stable phenylthiohydantoin (PTH) derivative and identified by chromatography.
  • the residual peptide is then subjected to further stepwise degradation.
  • the cleavage reagent may be a carboxypeptidase.
  • the present invention is not limited to any particular method of sequencing.
  • protein libraries it is common to express the protein so that it remains physically associated with the corresponding DNA (e.g., the protein is integrated into the coat of a phage encapsulating DNA encoding the protein) .
  • sequence the DNA rather than the protein.
  • attach a specific tag not expected to participate in binding
  • the library may be synthesized so that the spatial position of the binding molecule is fixed, and assists in identifying the binding molecule.
  • a new library may be designed which takes advantage of the knowledge of the structure-activity relations! , .ps gained from the previous library.
  • the compound that is used to block or neutralize CD14 is a random peptide that binds to LPS, LPS-binding protein or LPS-LPS binding protein complex and thereby blocks or neutralizes the interaction of LPS with CD14.
  • Random peptides may be generated by art-known methods of peptide synthesis. Generally, the random peptides contain about four to ten amino acids, preferably five to eight amino acids, and most preferably six amino acids.
  • Random peptides useful in the invention method include six amino acid long peptides having the following combinations of amino acids at the first two positions of the peptide, i.e., at the amino terminal end of the peptide: ALA-HIS, ARG-TRP, ALA-TRP, ALA-GLN, VAL-TRP, THR-TRP, ARG-TRP, ILE-TRP, SER-ARG, TYR-TRP, PHE-TRP, HIS-TRP, CYS-TRP, THR-CYS, ARG-CYS, ILE-CYS, TYR-CYS, HIS-CYS, ' CYS-CYS, and CYS-ARG.
  • the pharmaceutical composition of the present invention comprise one or more drugs as previously defined, and are effective, when administered according to an effective pharmacological schedule to a patient, of providing "protection".
  • "Protection”, as used herein, is intended to include prevention, suppression, and treatment. Prevention involves administration of the protective composition prior to the induction of the disease. Treatment involves administration of the protective composition after the appearance of the disease. It will be understood that in medicine, it is not always possible to distinguish between preventing and suppressing, since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term protection as distinct from treatment to encompass both preventing and suppressing as defined herein. The term protection, as used herein, is meant to include prophylaxis.
  • the protection provided need not be absolute, provided that it is sufficient to carry clinical value.
  • An agent which provides protection to a lesser degree than do competitive agents may still be of value if the other agents are ineffective for a particular individual, if it can be used in combination with other agents to enhance the overall level of protection, or if it is safer than competitive agents.
  • the treatment method may be applied prior to, during or after other medical procedures, such as, for example, surgery, especially gastrointestinal surgery, radiotherapy, chemotherapy or peritoneal dialysis.
  • the present method may also be applied to a patient thought to be at risk of bacteremia to thereby prevent dissemination of bacteria.
  • the composition may be administered parentally or orally, and, if parentally, either systemically or topically.
  • Parenteral routes include subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. One or more such routes may be employed.
  • Parenteral administration can be, e.g., by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route.
  • the suitable dose of a composition according to the present invention will depend upon the age, sex, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This typically involves adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.
  • a drug Prior to use in humans, a drug is first evaluated for safety and efficacy in laboratory animals. In human clinical trials, one begins with a dose expected to be safe in humans, based on the preclinical data for the drug in question, and on customary doses for analogous drugs, if any. If this dose is effective, the dosage may be decreased to determine the minimum effective dose, if desired. If this dose is ineffective, it will be cautiously increased, with the patients monitored for signs of side effects. See, e.g., Berkow et al. , eds., The Merck Manual. 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et al.
  • the total dose required for each treatment may be administered in multiple doses (which may be the same or different) or in a single dose, according to a pharmacological schedule, which may be predetermined or ad hoc.
  • the schedule is selected so as to be pharmaceutically effective, i.e., so as to be sufficient to elicit a response which protective is in itself or which enhances the protection provided by other agents.
  • the doses adequate to accomplish this are defined as "therapeutically effective doses.” (Note that a schedule may be effective even though an individual dose, if administered by itself, would not be effective, and the meaning of "therapeutically effective dose" is best interpreted in the context of the schedule. ) Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • Dosage forms include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments, and parenteral depots. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.
  • the drug is dissolved or suspended in an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • the drugs are preferably supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of drugs are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the pharmaceutical composition may instead comprise a vector comprising an expressible gene encoding such drug.
  • genes encoding naturally occurring proteins, or peptide fragments thereof one may, but need not, use the DNA sequence which encodes the proteins or peptides in nature.
  • composition and method would then be chosen so that the vector was delivered to suitable cells of the subject, so that the gene would be expressed and the drug produced in such a manner as to elicit a protective effect.
  • a preferred vector would be a Vaccinia virus.
  • a nonpathogenic bacterium could be genetically engineered to express the drug.
  • the drug must, of course, either be secreted, or displayed on the outer membrane of the bacterium (or the coat of a virus) in such a manner that it can interact with bacterial LPS or cellular CD14, as appropriate.
  • the amount of CD14 antagonist used in the present method may vary depending on the type of antagonist.
  • the amount administered is generally in the range of from about 1 to about 50-fold excess of the amount of SCD14 found in normal healthy individuals, i.e., about 2 to about 4 ⁇ g/ml sCD14 is found in blood serum of healthy humans.
  • an amount of from about 6 to about 100 ⁇ g/ml, preferably, about 10 to about 90 ⁇ g/ml and most preferably about 40 to about 80 ⁇ g/ml are administered to the patient.
  • an anti-CD14 monoclonal or polyclonal antibody is administered to the patient the dosage is generally in the range of from about 10 to about 70 ⁇ g/ml, preferably from about 15 to about 40 ⁇ g/ml and most preferably, from about 20 to about 30 ⁇ g/ml. This range of amount of anti-CD14 antibody has been demonstrated to block binding of endotoxin to CD14 in vitro.
  • the amount of a peptide administered to a patient to effect binding to LPS, LPS-binding protein or LPS-LPS-binding protein complex is in the range of from about 1 to about 50-fold excess over the amount of sCD14 normally found in the blood of healthy individuals and preferably, in the range of from about 1 to about 20-fold excess over the amount of sCD14 normally found in the blood of healthy individuals.
  • CD14 neutralizing or blocking compounds are sufficient to decrease the risk of bacteremia.
  • specific amount of CD14 neutralizing or blocking compound administered for the prevention of bacteremia or peritonitis can be determined readily for any particular patient according to recognized procedures and based on the expertise and experience of the skilled practitioner. Precise dosing for a patient can be determined according to routine medical practice.
  • patient is used herein to mean an animal, including humans and other mammals.
  • present method is useful in veterinary medicine as well as in the treatment of humans.
  • Treatment of a patient with a pharmaceutically effective amount of a CD14 antagonist invention is carried out for a period of time required to prevent bacteremia, subsequent or the systemic dissemination of bacteria from the bloodstream to tissues such as the lung.
  • the treatment regimen will vary depending on such factors as the particular condition to be treated, e.g. ,peritonitis or gastroenteritis, the medical condition underlying the risk of bacteremia , e.g.,the presence of a localized bacterial infection or trauma, such as an invasive medical treatment or surgery, particularly to the area of the gastrointestinal or respiratory tract, or other predisposing medical condition, the overall health of the patient, the route of administration, etc.
  • the present method may be applied to a patient thought to be at risk of bacterial dissemination into or from the blood stream from such underlying medical conditions as peritonitis, physical injury resulting in intestinal perforation, diverticulitis, appendicitis, acute pancreatitis, other trauma (incl. surgery, i.v. lines, or invasive diagnostic procedures) immunosuppression as a result of chemotherapy, preparation for transplant, or HIV infection, pneumonia, gastroenteritis, colitis, dysentery, severe cellulitis, urinary tract infection, inflammatory bowel disease, hemorrhagic shock, burn infection, endocarditis, meningitis, tuberculosis, etc.
  • use of the present method to prevent bacteremia includes application of the method at least once per day until the risk of bacteremia is assessed to be over. The skilled medical practitioner can assess the relative risk of bacteremia according to routine medical practice and procedure.
  • CD14-deficient mice Resistance of CD14-deficient mice to LPS-induced shock.
  • CD14 deficient mice were produced by homologous recombination in embryonic stem cells as described by Thomas, et al . , Cell 51: 503-12 (1987) .
  • the human CD14 gene whose isolation and characterization are described in Applicant 's co-pending application Serial No. 08/254,095 filed June 6, 1995, incorporated in its entirety herein by reference thereto, was disrupted in its first and second exons with a neo gene construct.
  • the defective gene was transfected into W9.5 embryonic stem cells by electroporation.
  • Cells carrying the disrupted CD14 gene were injected into C57BL/6 mouse blastocytes.
  • Male chimeras were bred with C57BL6 mice and the offspring were interbred to produce homozygous CD14-negative mice.
  • the absence of expression of CD14 was confirmed by staining of peritoneal macrophages with polyclonal and monoclonal anti-murine CD14 antibodies.
  • CD14-deficient mice have no obvious abnormalities and are fertile and healthy ( > 1 year) when housed in a clean, non-pathogen-free environment.
  • CD14 The role of CD14 in the in vivo response to dissemination of bacteria into the bloodstream was analyzed by injecting CD14-deficient and control mice intraperitoneally (i.p.) with a dose of LPS purified from wild type Salmonella minnesota corresponding to an LD100 for control mice or with a dose corresponding to ten times the LD100. Mice were monitored for seven days. The results are shown in Figure IA.
  • CD14-deficient mice had a serum level of TNF- ⁇ that was ten times lower than that of control mice. Furthermore, there was no additional release of TNF when CD14-deficient mice were exposed to a ten-fold higher dose of LPS (200 mg/kg) . Similarly, the serum level of IL-6 in CD14-deficient mice was at least ten times lower than in control mice. However, at a ten-fold higher dose of LPS there was a slightly stronger IL-6 response. The IL-6 measurements are shown in Figure IC
  • CD14-deficient mice Resistance of CD14-deficient mice to bacterial-induced shock.
  • E. coli 5 x 10° CFU
  • E. coli 0111 :B4 were grown on tryptic soy broth (Difco) agar plates and single colonies were inoculated in tryptic soy broth medium.
  • Bacteria in mid-logarithmic phase were collected, chilled on ice, the concentration of bacteria determined on the basis of absorbance at 620 nm using a pre-determined calibration curve. For injections bacteria were washed once in non-pyrogenic saline, and serial delusions were prepared in saline.
  • mice were injected i.p. The condition of surviving mice was monitored for 21 days. The results are shown in Figure 2A. Numbers above the bars indicate number of survivors/number of mice per group. Cytokine levels were determined as in Example 1. Results are represented as mean +/- SE in each group, (p ⁇ 0.05) (Mann-Whitney test) .
  • the serum levels of TNF- ⁇ and IL-6 in control and CD14-deficient mice were measured two hours after the injection of gram-negative bacteria. The results are shown in Figure 2B.
  • the mean serum concentrations of TNF- ⁇ in CD14-deficient mice infected i.p. with gram-negative bacteria was ten-fold lower than in control mice.
  • the IL-6 levels after infection were 48-fold lower in CD14-deficient mice than in control mice ( Figure 2C) .
  • Serum concentrations of IL-1 were also depressed in infected CD14-deficient mice.
  • mice Blood and organ counts of bacteria in infected mice. Control and CD14 mice were also examined to determine the level of viable bacteria in the blood, which is a direct measurement of bacteremia.
  • Mice were injected i.p. with (a) 3 x IO 7 CFU (4 mice per group) , (b) 5 x 10° CFU (4 mice per group) and (c) 3 x 10 7 CFU (3 mice per group) of E. coli 0111 :B4. Seven hours after injection mice were sacrificed by C02 inhalation, bled by heart puncture and lungs were aseptically recovered and homogenized. Bacterial counts were determined by plating 10-fold serial dilutions of blood or homogenized lungs on tryptic soy broth agar plates. The results are shown in Figure 4A, 4B and 4C. Results are represented as a mean +/- SE in each group. (p ⁇ 0.05) (Mann- Whitney test) .
  • CD14-deficient mice had 35-fold fewer bacteria in the blood than control mice after a lethal i.p. injection of viable E. coli (3 x IO 7 CFU per mouse) ( Figure 4A) .
  • a dose that was lethal only to control mice was used as inoculum (5 x IO 6 CFU per mouse) a 27-fold lower level of bacteremia was observed in the CD14-deficient mice ( Figure 4B) .
  • the number of live bacteria in the lungs of mice injected with 3 x IO 7 bacteria was also similarly reduced in CD14-deficient mice ( Figure 4C) .
  • a patient suffering from an underlying medical condition requiring surgery within the gastrointestinal tract e.g. ,colonostomy
  • the level of viable bacteria in the blood is measured on the day following surgery and at three days following surgery to determine whether there is a bacteremic condition.
  • Antagonists will be defined by screening combinatorial libraries of small organic compounds obtained from a variety of commercial sources by a combination of three methods.
  • b. Positive compounds in assay (a) are then rescreened for their ability to inhibit activation (as measured by release of cytokines, TNF- ⁇ , IL-1, IL-6) of human PBMNC (peripheral blood mononuclear cells) , by LPS or organism (bacterial or fungal) (whole blood assay, ref. 2) .
  • Identification will be performed by binding soluble human CD14 (1) to 96-well plate, incubate separately with each of 400 pools of combinatorial hexamer peptide library (prepared as described by Houghton) , wash, elute peptides with weak acid, filter out soluble CD14 by membrane filter which separates molecules on basis of size or, by affinity chromatography using anti-human CD14 antibody coupled to resin, and determine amino acid sequence of peptide(s) preferentially bound and compare to sequence of peptides in pool to identify optimal amino acids at positions 3- 6.
  • soluble human CD14 covalently bound to resin and select for peptides preferentially bound to soluble human CD14 by conventional affinity chromatography.
  • Peptides with optimal sequence based on 2(a) will be synthesized and rescreened in assays 1(a), (b) , and (c) as described above.
  • Additional antagonists will be identified on the basis of this information by making D-amino acid peptides with the same sequence or by making D-amino acid peptides with the same sequence but in reverse order.
  • Additional antagonists will also be identified by solving NMR structure of peptides identified in (a) and (b) above and designing small organics with similar 3D structure and structure-activity analyses.
  • Example 7 Haziot, A., et al. , (1993), Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide, J. Immunol.. 151:1500-1507. 2. Haziot, A., et al . , (1994) , Recombinant soluble CD14 inhibits LPS-induced Tumor Necrosis Factor- ⁇ production by cells in whole blood, J. Immunol.. 152:5868-5876.

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Abstract

L'invention porte sur procédé empêchant la dissémination des bactéries dans le sang ou à partir du sang qui s'avère utile pour la prévention des péritonites et de la septicémie.
PCT/US1996/010803 1995-06-19 1996-06-19 Procede empechant la septicemie et la dissemination des bacteries WO1997000081A1 (fr)

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AU63929/96A AU6392996A (en) 1995-06-19 1996-06-19 A method for inhibiting bacteremia and bacterial dissemination

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000042994A3 (fr) * 1999-01-21 2003-08-28 Long Island Jewish Res Inst Inhibition de la dissemination de bacteries
US6649746B1 (en) 1999-05-07 2003-11-18 University Of Virginia Patent Foundation Biological production of stable glutamine, poly-glutamine derivatives in transgenic organisms and their use for therapeutic purposes
EP4080216A1 (fr) * 2021-04-23 2022-10-26 Institute of Science and Technology Austria Procédés de détermination du potentiel des médicaments pour le traitement des infections bactériennes et composition pour le traitement des infections bactériennes

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Publication number Priority date Publication date Assignee Title
WO1991001639A1 (fr) * 1989-08-01 1991-02-21 Scripps Clinic And Research Foundation Procedes et compositions ameliorant les symptomes de la septicemie
WO1992004908A1 (fr) * 1990-09-14 1992-04-02 Imtox Privatinstitut Für Immunbiologische Forschung Gmbh Medicament contenant du cd14
WO1993004168A1 (fr) * 1991-08-21 1993-03-04 North Shore University Hospital Research Corporation Modele animal transgenique non humain destine a developper et tester des therapies de traitement de la septicemie
WO1993019772A1 (fr) * 1992-04-06 1993-10-14 North Shore University Hospital Research Corporation Nouvelle therapie permettant de traiter la septicemie a l'aide d'une forme soluble de l'antigene myelomonocytique cd14 recombine
WO1996008272A1 (fr) * 1994-09-16 1996-03-21 The Scripps Research Institute Utilisation d'anticorps pour bloquer les effets des bacteries gram-positives et des mycobacteries

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001639A1 (fr) * 1989-08-01 1991-02-21 Scripps Clinic And Research Foundation Procedes et compositions ameliorant les symptomes de la septicemie
WO1992004908A1 (fr) * 1990-09-14 1992-04-02 Imtox Privatinstitut Für Immunbiologische Forschung Gmbh Medicament contenant du cd14
WO1993004168A1 (fr) * 1991-08-21 1993-03-04 North Shore University Hospital Research Corporation Modele animal transgenique non humain destine a developper et tester des therapies de traitement de la septicemie
WO1993019772A1 (fr) * 1992-04-06 1993-10-14 North Shore University Hospital Research Corporation Nouvelle therapie permettant de traiter la septicemie a l'aide d'une forme soluble de l'antigene myelomonocytique cd14 recombine
WO1996008272A1 (fr) * 1994-09-16 1996-03-21 The Scripps Research Institute Utilisation d'anticorps pour bloquer les effets des bacteries gram-positives et des mycobacteries

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Title
DATABASE MEDLINE FILE SERVER STN KARLSRUHE; BIRKENMAIER ET AL: "MODULATION OF THE ENDOTOXIN RECEPTOR (CD14) IN SEPTIC PATIENTS", XP002016894 *
FERRERO ET AL: "TRANSGENIC MICE EXPRESSING HUMAN CD14 ARE HYPERSENSITIVE TO LIPOPOLYSACCHARIDE", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES,USA, vol. 90, 1993, pages 2380 - 2384, XP002016890 *
GOYERT ET AL: "EXPRESSION AND FUNCTION OF HUMAN CD14 IN TRANSGENIC MICE", JOURNAL OF CELLULAR BIOCHEMISTRY,SUPPLEMENT 16C, 1992, pages 153, XP002016892 *
GOYERT ET AL: "TRANSGENIC MICE EXPRESSING HUMAN CD14: STUDIES OF EXPRESSION AND FUNCTION", THE FASEB JOURNAL, vol. 6, no. 5, 1992, pages A1886, XP002016893 *
HAZIOT ET AL: "RESISTANCE TO ENDOTOXIN SHOCK AND REDUCED DISSEMINATION OF GRAM-NEGATIVE BACTERIA IN CD14-DEFICIENT MICE", IMMUNITY, vol. 4, April 1996 (1996-04-01), pages 407 - 414, XP000608215 *
MALISZEWSKI: "CD14 AND IMMUNE REPONSE TO LIPOPOLYSACCHARIDE", SCIENCE, vol. 252, 1991, pages 1321 - 1322, XP002016891 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000042994A3 (fr) * 1999-01-21 2003-08-28 Long Island Jewish Res Inst Inhibition de la dissemination de bacteries
US6649746B1 (en) 1999-05-07 2003-11-18 University Of Virginia Patent Foundation Biological production of stable glutamine, poly-glutamine derivatives in transgenic organisms and their use for therapeutic purposes
EP4080216A1 (fr) * 2021-04-23 2022-10-26 Institute of Science and Technology Austria Procédés de détermination du potentiel des médicaments pour le traitement des infections bactériennes et composition pour le traitement des infections bactériennes

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