EP0871732A1 - Method of treating sepsis - Google Patents

Method of treating sepsis

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Publication number
EP0871732A1
EP0871732A1 EP96940554A EP96940554A EP0871732A1 EP 0871732 A1 EP0871732 A1 EP 0871732A1 EP 96940554 A EP96940554 A EP 96940554A EP 96940554 A EP96940554 A EP 96940554A EP 0871732 A1 EP0871732 A1 EP 0871732A1
Authority
EP
European Patent Office
Prior art keywords
chemokine
seq
protein
groβ
association
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96940554A
Other languages
German (de)
French (fr)
Other versions
EP0871732A4 (en
Inventor
Peter Lawrence Demarsh
Kyung O. Johanson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
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SmithKline Beecham Corp
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Publication date
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Publication of EP0871732A1 publication Critical patent/EP0871732A1/en
Publication of EP0871732A4 publication Critical patent/EP0871732A4/en
Withdrawn legal-status Critical Current

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Sepsis is broadly defined to mean situations when the invasion of a host by a microbial agent is associated with the clinical manifestations of infection including but not limited to (1) temperature >38°C or ⁇ 36°C, (2) heart rate >90 beats per minute, (3) respiratory rate >20 breaths per minute or PaC0 2 ⁇ 32 mm Hg, (4) white blood cell count >12,000/cu mm, ⁇ 4,000/cu mm, or >10% immature (band) forms, (5) organ dysfunction, hypoperfusion, or hypotension Hypoperfusion and perfusion abnormalities may include, but are not limited to lactic acidosis, oligu ⁇ a, or an acute alteration in mental stales (Chest 1992, i0_L 1644- 1566) Sepsis can occur in hospitalized patients having underlying diseases or conditions that render them susceptible to bloodstream invasion or in burn, trauma or surgical patents In many cases of sepsis, the predominant pathogen is Escherichia coli, followed by othei Gram-negative bacteria such as the
  • Septic shock occurs because bacterial products react with cells and components of the coagulation, complement, fibrinolytic and bradykinin systems to release proteases which ⁇ n
  • ARDS' adult respiratory distress syndrome
  • Septic shock is a major cause of death in intensive care units
  • mortality for established septic shock has decreased very little since the comprehensive description by Waisbren (Arch Intern Med 88 467-488 (! Q 5!) Although effective antibiotic ai .
  • This invention relales to a method of preventing or treating sepsis comprising administering to a human or non-human animal in need thereof an effective amount of a protein derived from a chemokine selected from KC, gro-a, gro ⁇ , and gro ⁇
  • chemokines used in the method of the invention include modified KC [amino acids 5-72 of the full length protein, SEQ ID NO 1 J, modified human gro ⁇ [amino acids 5-73 of the full length protein, SEQ ID NO 3] or modified human gro ⁇ [amino acids 5-73 of the full length protein, SEQ ID NO 4] or multimers thereof
  • the mature chemokines may be utilized in the method of the invention
  • the method of the invention may be performed alone, or in conjunction with administration of an anti-infective agent
  • chemokines useful in the method of the invention include mature KC [SEQ ID NO 1 ].
  • Particularly desirable are the modified KC [amino acids 5-72 of SEQ ID NO 2], modified gro ⁇ [amino acids 5-73 of SEQ ID NO 3], modified gro ⁇ [amino acids 5- 73 of SEQ ID NO 4], and a dimeric modified gro ⁇ [ammo acids 5-73 of SEQ ID NO 3]
  • proteins useful in preparing medicaments and in the methods of the invention include the mature chemokines, modified chemokines, and multimers thereof
  • ' mature chemokines also known as ' lnterc ⁇ nes
  • the amino acid sequences of the murine protein KC which contains 72 residues is provided in SEQ ID NO 1
  • SEQ ID NO 2 amino acid sequences of the human protein groa
  • SEQ ID NO 3 amino acids 1-73
  • SEQ ID NO 4 The sequences of the human protein groy are provided in SEQ ID NO 4
  • the cDNA and amino acid sequences of groy are also provided in International Patent Application, Publication No WO 92/00326 (Jan 9, 1992) These groy sequences have further been published lnjnternational Patent Application, Publication No WO 94/29341 (December 22, 1994), which is incorporated by reference herein
  • the N terminal nictlnoni ⁇ e wluJi is uiseited into tne protein tor expression purposes, may be cleaved, either during the processing of the protein by a host cell or synthetically, using known techniques Alternatively, if so desired, this ammo acid may be cleaved through enzyme digestion or other known means
  • Particularly desirable modified chemokines include modified KC [amino acids 5 - 72 of SEQ ID NO 1], modified human gro ⁇ [amino acids 5-73 of SEQ ID NO 3] and modified human gro ⁇ [amino acids 5-73 of SEQ ID NO 4]
  • modified chemokine are other analogs or derivatives of KC, gro ⁇ , gro ⁇ , or gro ⁇ which share the biological activity of the mature protein
  • analogs and derivatives include modified proteins also characterized by alterations made in the known amino sequence of the proteins, e g , the proteins provided in SEQ ID NOS 1-4
  • Such analogs are characterized by having an amino acid sequence differing from that of the mature protein by 8 or fewer amino acid residues, and preferably by about 5 or fewer residues. It may be preferred that any differences in the amino acid sequences of the proteins involve only conservative amino acid substitutions
  • amino acid substitutions occur when an amino acid has substantially the same charge as the amino acid for which it is substituted and the substitution has no significant effect on the local conformation of the protein or its biological activity Alternatively, changes such as the introduction of a certain ammo acid in the sequence which may alter the stability of the protein, or permit it to be expressed in a desired host cell may be preferred Another characte ⁇ stic of these modified proteins may be enhanced biological activity in comparison to the mature protein By the term "multime ⁇ c protein' .
  • multime ⁇ c forms of the mature and/or modified proteins useful in this invention e g , dimers, t ⁇ mers, tetramers and other aggregated forms
  • Such multime ⁇ c forms can be prepared by synthesis or recombinant expression and can contain chemokines produced by a combination of synthetic and recombinant techniques as detailed below Multimers may form naturally upon expression or may be constructed into such multiple forms
  • Multime ⁇ c chemokines may include multimers of the same modified chemokine
  • Another multimer may be formed by the aggregation of different modified proteins
  • Still another multimer is formed by the aggregation of a modified chemokine of this invention and a known, mature chemokine
  • a dimer or multimer useful in the invention would contain at least uiie dcsdiniii ⁇ chemokine protein and at least one ottier chemokine or other protein characterized by having the same type of biological activity
  • This other protein may be an additional desamino chemok
  • the chemokines useful in the method of the invention are used in the preparation of medicaments and or are useful in the form of a pharmaceutical composition
  • the chemokines can be formulated into pharmaceutical compositions and administered in the same manner as described in, e g , International Patent Applications, Publication No WO 90/02762 (Mar 22, 1990) and Publication No W094/29341 (Dec 22, 1994)
  • medicaments or pharmaceutical compositions useful in the method of the invention for preventing or treating sepsis contain an effective amount of a mature, modified or multime ⁇ c chemokine protein derived from KC [SEQ ID NO 1 ], human gro-a [SEQ ID NO 2], human gro ⁇ [SEQ ID NO 3], and human gro ⁇ [SEQ ID NO 4] which is administered to an animal in need thereof
  • Particularly desired embodiments utilize the modified chemokines, or multimers thereof
  • These chemokine compositions may be administered alone or in combination with administration of other anti-infective agents
  • a pharmaceutical composition is prepared using one or more of proteins derived from the KC [SEQ ID NO 1], gro ⁇ [SEQ ID NO 2], gro ⁇ [SEQ ID NO 3] or gro ⁇ [SEQ ID NO 4] proteins Suitable pharmaceutical earners are well known to those of skill in the art and may be readily selected Currently, the preferred carrier is saline
  • the pharmaceutical compositions of the invention may contain other active ingredients or be administered in conjunction with other therapeutics
  • the compositions of the invention are particularly well suited for administration in conjunction with anti- infective agents
  • Suitable anti-infective agents include, without limitation, anti-microbial agents routinely used for the treatment of sepsis such as amino-glycosides (such as amikacin, tobramycin, netilmicin, and gentamicin), cephalosponns such as ceftazidime, related beta lactam agents such as maxalactam, carbopenems such as lmipenem, monobactam agents such as aztreonam, ampicillin and broad- spectrum penicillins, (e g , penicillinase-resistant penicillins, ureidopenicillins or antipseudomonal penicillin or Augmentin) that are active against P aeruginosa, Enterobacter species, indole-positive Proteus species, and Serratia Also included within the definition of anti-infective agents are antifungal agents, amphotericin and tne like as well as anti-viral agents such as famvir and acyclovir
  • chemokines desc ⁇ bed herein are useful in the treatment and prevention of sepsis in humans and other animals such as dairy cattle, horses, calves or poultry
  • a mature, modified or multime ⁇ c KC [SEQ ID NO 1 ], gro ⁇ [SEQ ID NO 2], gro ⁇ [SEQ ID NO 3] or human gro ⁇ [SEQ ID NO 4] or their multimers e.g , a dimeric, truncated gro ⁇ , amino acids 5-73 of SEQ ID NO 3
  • the dose may be in the range of about 10 to about 10,000 fg/kg/dose, or orally in the dose range of about 10 to about 10,000 fg/kg body weight per dose, if administered by infusion or similar techniques, the dose may be in the range of about 10 to about 10,000 fg kg/dose, if administered subcutaneously the dose may be in tne range of about 10 to about 10,000 fg/kg/dose
  • the compounds of this invention can be administered for prophylactic and/or therapeutic treatments
  • the compound is administered to a patient already suffenng from a disease in an amount sufficient to cure or at least partially arrest the disease and its complications It may be given at any time after surgery, preferably pnor to 24 hours after surgery
  • a composition containing mature, modified or multime ⁇ c KC [SEQ ID NO 1 ], gro ⁇ [SEQ ID NO 2J, gro ⁇ [SEQ ID NO 3] or gro ⁇ [SEQ ID NO 4] or a multimer thereof is administered to a patient not already in a disease state to enhance the patient's resistance. It may be given one day or one week pnor to surgery, preferably one to two days prior to surgery It may be administered parenteral iy or orally
  • Single or multiple administrations of the compounds can be carried out with dose levels and pattern being selected by the treating physician In any event, a quantity of the compounds of the invention sufficient to effectively treat the patient should be administered
  • chemokines useful in the methods of this invention may also be administered in conjunction with a separately administered conventional anti-infective as disclosed herein above, such a gentamicin, augmentin or ceftazidime
  • a separately administered conventional anti-infective as disclosed herein above, such a gentamicin, augmentin or ceftazidime
  • the particular anti-infective chosen should be one to which the infective organism is susceptible and is selected or modified during therapy as the infecting microorganism is more particularly identified
  • adjunctive agents in the treatment of septic shock also may be useful in combination with the components of this invention They include sympathomimetic amines (vasopressors) such as norepineph ⁇ ne, epineph ⁇ ne, isoproterenol, dopamine, and dobutamine, anti-inflammatory agents such as methylprednisolone anti-inflammatory agents such as lndomethacin and phenylbutazone, and corticosteroids such as betamethasone, hydrocortisone, methylprednisolone, or dexamethasone, anti-coagulants such as hepa ⁇ n, anti-thrombin III or couma ⁇ n type drugs for certain conditions and schedules, diuretics such as furosemide or ethacrynic acid, and antagonist of opiates and beta-endorphins such as naloxone; an antagonist of tumor necrosis factor or of interleukin- 1 , phenothiazines, antivaso
  • E. coli A clinical isolate of £ coh isolated from sputum was utilized The organisms were tested for antibiotic sensitivity by the disc agar diffusion technique and found to be sensitive to gentamicin, ampicillin, cephalothin, chloramphenicol, kanamycin tetracycline, t ⁇ methop ⁇ n/sulfamethoxazole and resistant to penicillin G, erythromycin, and vancomycin The organism was animal passed in mice and subsequently recovered and plated onto MacConkey's agar The reisolated organisms were grown overnight in brain- heart infusion broth, and then stored frozen at -70°C The inoculate the fibrin clot, organisms from thawed stocks were inoculated into brainheart infusion broth and incubated overnight on a rotary shaker (120 rpm ⁇ an 37°C The F coh was harvested by cent ⁇ fugation, washed 3X and finally resuspended in normal saline The number or organisms was quantified by
  • Fibrin Clot The E colt infected fibrin clots were made from a 1 % solution of bovine fibrinogen (Type 1 -S, Sigma) in sterile saline The clot was formed by adding sequentially human thrombin (Hanna Pharma ) bacteria, and fibrinogen solution to 24 well plastic plates Bacte ⁇ al numbers of 2 0 to 3 0 x I0 9 were used in inoculate the fibrin clots The resulting mixture was then incubated at room temperature for 30 minutes before implantation Animal Model.
  • Bovine fibrinogen Type 1 -S, Sigma
  • the clot was formed by adding sequentially human thrombin (Hanna Pharma ) bacteria, and fibrinogen solution to 24 well plastic plates Bacte ⁇ al numbers of 2 0 to 3 0 x I0 9 were used in inoculate the fibrin clots The resulting mixture was then incubated at room temperature for 30 minutes before implantation Animal Model.
  • the rats were anesthetized with ketamine/xylazine (40 mg/kg/5 mg/kg) after which the abdominal surfaced were shaved and a midlme laparotomy performed Bacterial peritonitis was induced by implanting a fib ⁇ n-thrombin clot containing E co into the abdominal cavity After implantation the muscle layers were closed with 4 0 silk suture, and the wound closed with surgical staples The animals were closely observed, any animals obviously moribund were euthanized
  • Gentamicin Rats were treated subcutaneously with gentamicin sulfate (Elkins Sinn, NJ) 5 mg/kg twice a day for five days
  • E colt cell pellet was lysed in pH 6 0 buffer containing 20 mM dithiothreitol (DTT) to avoid the nonspecific air oxidation
  • the majority of truncated gro ⁇ was m the insoluble lysate pellet which was solubilized in 2 M GdnHCI, pH 8 0 buffer containing 20mM DTT
  • the solubilized truncated gro ⁇ was dialyzed against pH 6 0 buffer containing 2 mM EDTA
  • the majo ⁇ ty of £ colt proteins were precipitated during dialysis while truncated gro ⁇ stayed in solution as a monomeric form at >95 % purity
  • the truncated gro ⁇ solution was adjusted to pH 8 5, stirred overnight for air oxidation
  • the refolded truncated gro ⁇ solution was adjusted to 0 1 % TFA solution, and applied to
  • DET-DDDDK chemokines were purified and refolded as described for truncated gro ⁇
  • the refolded DET-DDDDK chemokines were digested with enterokinase to remove the N-terminal DET-DDDDK and the undigested molecules were removed using anti DET Mab column
  • the digested molecules were further purified using C18 RP-HPLC as described above D Characterization
  • N-termmal sequencing and MALD-MS for molecular weight were performed and confirmed that the molecules are intact from N-terminus to C-termmus, either monomeric or dime ⁇ c form Concentration of each chemokine was determined by amino acid analysis and endotoxin level of each prep was ⁇ 0 05 U/ml
  • E coh LW cells 400 g were lysed in 4 liters of lysis buffer containing 50 mM sodium citrate pH 6.0, 40 mM NaCl, 2 M EDTA, 5% glycerol, 005% Tween 80, 0 2 mM PMSF, 1 mg/ml each of leupeptin and pepstatm A, by two passages through a
  • Typical yield of truncated gro ⁇ dimer was 0 15 mg/g of ⁇ ells when refolding was performed at 0 1 mg/ml or 0 7 mg/g of cells when refolding at 3 mg/ml E Characterization
  • the molecular weight of the truncated gro ⁇ dimer as determined on nonreducing SDS PAGE was approximately twice that of truncated gro ⁇ Upon reduction, both forms migrated to the same spot indicating that truncated gro ⁇ dimer is a disulfide linked dimer
  • the molecular weight of truncated gro ⁇ dimer, as determined b> MALD-MS analysis was 15,069 Da (predicted 15,073 Da), while that of truncated gro ⁇ dimer was 7,536 Da (predicted 7,537 Da) N-terminal sequencing of truncated gro ⁇ dimer showed that 5-10% of the final products retained the initiatory Met Disulfide pairing pattern of truncated gro
  • mice were dosed intraperitoneally with truncated KC [ammo acids 5-72 of SEQ ID NO 1] at doses of 10, 33, 100 or 333 fg/kg 24 hours and 2 hours before infection
  • Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin On day 0 the rats were implanted with an E coh containing fib ⁇ n-thrombin clot Starting two hours after infection the rats were treated with gentamicin twice daily
  • the rats prophylactically treated with truncated KC at 33 or 100 fg kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone
  • the rats were implanted with an E coh containing fib ⁇ n-thrombin clot
  • the animals were dosed intraperitoneally with truncated KC [amino acids 5-72 of SEQ ID NO.1] at doses of 33, 100, 333, or 1,000 fg/kg as a single injection 2 hours after infection
  • Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin
  • the rats therapeutically treated with truncated KCat 100 or 333 fg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone Results
  • Example 5 Therapeutically Administered Truncated KC in S. aureus Sepsis.
  • mice On day 0 the rats were implanted with a S. aureus containing fib ⁇ n-thrombin clot The animals were dosed intraperitoneally with truncated KC [amino acids 5-72 of SEQ ID NO 1 ] at doses of 33, 100, 333, or 1 ,000 fg/kg as a single injection 2 hours after infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with truncated KC at 100 or 333 fg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone. Results
  • the rats were implanted with an E co containing fib ⁇ n-thrombin clot
  • the animals were dosed mtrape ⁇ toneally with truncated gro ⁇ [amino acids 5-73 of SEQ ID NO.3] at doses of 33, 100, 333, or 1,000 fg/kg as a single injection 2 hours after infection
  • Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin
  • the rats therapeutically treated with t ⁇ lesed gro ⁇ at 100 or 333 fg/kg followed by gentamicin treatment demonstrated signi ificantly improved survival rates over the diluent treated control rats receiving gentamicin therapy ⁇ one
  • the rats were implanted with an S aureus containing fib ⁇ n-thrombin clot
  • the animals were dosed intrape ⁇ toneally with truncated gro ⁇ [amino acids 5-73 of SEQ ID NO 3] at doses of 33, 100, 333, or 1 ,000 fg/kg as a single injection 2 hours after infection
  • Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin
  • the rats therapeutically treated with truncated gro ⁇ at 100 or 333 fg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone
  • Results Results
  • Example 8 Therapeutical Subcutaneously Administered Truncated gro ⁇ in E. coli Sepsis On day 0 the rats were implanted with an E. coh containing fib ⁇ n-thrombin clot
  • mice were dosed subcutaneously with truncated gro ⁇ [amino acids 5-73 of SEQ ID N0.3J at doses of 0 1 , 0.3, 1 0, or 3.3 pg/kg as a single injection 2 hours after infection
  • Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin
  • the rats therapeutically treated with truncated gro ⁇ at 0.3 or 1.0 pg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone.
  • Example 9 Therapeutical Subcutaneously Administered Truncated gro ⁇ in S. aureus Sepsis On day 0 the rats were implanted with an S. aureus containing fibrin-thrombin clot
  • mice were dosed subcutaneously with truncated gro ⁇ [amino acids 5-73 of SEQ ID NO.3] at doses of 0.1 , 0 3, 1.0, or 3.3 pg/kg as a single injection 2 hours after infection
  • Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin
  • the rats therapeutically treated with truncated gro ⁇ at 0.3 or 1.0 pg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone.
  • mice were dosed subcutaneously with dimer formed of two truncated gro ⁇ proteins [amino acids 5-73 of SEQ ID NO:3] at doses of 0 1, 0.3, 1.0 or 3.3 pg/kg 24 hours before infection
  • Control animals were doses with dilution buffer on the same schedule
  • the rats were implanted with an E colt containing fib ⁇ n-thrombin clot Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin
  • the rats prophylactically treated with truncated gro ⁇ dimer at 0 3 or 1 0 pg kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone
  • mice On day 0 the rats were implanted with an S aureus containing fib ⁇ n-thrombin clot The animals were dosed subcutaneously with a dimer formed of two truncated groB proteins [ammo acids 5-73 of SEQ ID NO 3] at doses of 0 03, 0 1, 0 3, 1 0, 3 3, or 10 pg/kg as a single injection 2 hours after infection Control animals were doses with dilution buffer on the same schedule
  • Ala Pro lie Ala Asn Glu Leu Arg Cys Gin Cys Leu Gin Thr Met 1 5 10 15
  • Lys lie Val Gin Lys Met Leu Lys Gly Val Pro Lys
  • Lys lie lie Glu Lys Met Leu Asn Ser Asp Lys Ser Asn 65 70
  • Lys lie lie Glu Lys Met Leu Lys Asn Gly Lys Ser Asn
  • Lys lie lie lie Glu Lys lie Leu Asn Lys Gly Ser Thr Asn

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Abstract

The invention relates to the method of preventing and treating sepsis using chemokines selected from mature or modified KC [SEQ ID NO: 1], groα [SEQ ID NO:2], groβ [SEQ ID NO:3] or groη [SEQ ID NO:4] or multimers thereof, alone or in conjunction with an anti-infective agent.

Description

METHOD OF TREATING SEPSIS
Field of Invention
This invention relates to the method of preventing and treating sepsis using certain chemokines alone or in conjunction with an anti-infective agent Background of Invention
Sepsis, as used herein, is broadly defined to mean situations when the invasion of a host by a microbial agent is associated with the clinical manifestations of infection including but not limited to (1) temperature >38°C or <36°C, (2) heart rate >90 beats per minute, (3) respiratory rate >20 breaths per minute or PaC02 <32 mm Hg, (4) white blood cell count >12,000/cu mm, <4,000/cu mm, or >10% immature (band) forms, (5) organ dysfunction, hypoperfusion, or hypotension Hypoperfusion and perfusion abnormalities may include, but are not limited to lactic acidosis, oliguπa, or an acute alteration in mental stales (Chest 1992, i0_L 1644- 1566) Sepsis can occur in hospitalized patients having underlying diseases or conditions that render them susceptible to bloodstream invasion or in burn, trauma or surgical patents In many cases of sepsis, the predominant pathogen is Escherichia coli, followed by othei Gram-negative bacteria such as the Klebsiella-Enterobacter-Serratia group and then Pseudomonas Although comprising a somewhat smaller percentage of infection, Gram- positive microbes such as Staphylococcus and systemic viral and fungal infections are included by the term sepsis as used herein The genitourinary tract is the most common site of infection, the gastrointestinal tract and respiratory tract being the next most frequent sources of sepsis Other common foci are wound, burn, and pelvic infections and infected intravenous catheters A serious consequence of bacterial sepsis often is septic shock Septic shock is characterized by inadequate tissue perfusion, leading to insufficient oxygen supply to tissues, hypotension and oliguπa
Septic shock occurs because bacterial products react with cells and components of the coagulation, complement, fibrinolytic and bradykinin systems to release proteases which ιn|ure cells and alter blood flow, especially in the capillaries
Microorganisms frequently activate the classical complement pathway, and endotoxin activates the alternative pathway Complement activation, leukotπene generation and the direct effects of bacteπal products on neutrophils lead to accumulation of these inflammatory cells in the lungs, release of their proteolytic enzymes and tOAic oxygen radicals which damage the pulmonary endothelium and initiate the adult respiratory distress syndrome ("ARDS' ) ARDS is a major cause of death in patients with septic shock and is characterized by pulmonary congestion, graπuiocyte aggregation, hemorrhage and capillary thrombi
Septic shock is a major cause of death in intensive care units There are an estimated 200,000 cases per year of septic shock in the United States, and despite advances in technology (i e , respiratory support) and antibiotic therapy, the mortality rate for septic shock remains in excess of 40% In fact, mortality for established septic shock has decreased very little since the comprehensive description by Waisbren (Arch Intern Med 88 467-488 (!Q5!)) Although effective antibiotic ai . available, and there is an increased awareness of the septic shock syndrome, the incidence of septic shock over the last several decades has actually increased With the appreciation that antimicrobial agents have failed to completely abrogate septic mortality, it is clear thai other agents must be developed to be used alone or in conjunction with antimicrobials in order to rectify the deficiencies of current estabhshed therapy
Summary of the Invention
This invention relales to a method of preventing or treating sepsis comprising administering to a human or non-human animal in need thereof an effective amount of a protein derived from a chemokine selected from KC, gro-a, groβ, and groγ Most preferably, the chemokines used in the method of the invention include modified KC [amino acids 5-72 of the full length protein, SEQ ID NO 1 J, modified human groβ [amino acids 5-73 of the full length protein, SEQ ID NO 3] or modified human groγ [amino acids 5-73 of the full length protein, SEQ ID NO 4] or multimers thereof Alternatively, the mature chemokines may be utilized in the method of the invention
The method of the invention may be performed alone, or in conjunction with administration of an anti-infective agent
Other aspects and advantages of the present invention are descπbed further in the following detailed descπption of the preferred embodiments thereof Detailed Description of the Invention
It is the object of this invention to provide a new method of treatment of sepsis comprising administering to an animal in need thereof, including humans, an effective amount of a chemokine The chemokines useful in the method of the invention include mature KC [SEQ ID NO 1 ]. groα [SEQ ID NO 2], groβ [SEQ ID NO 3], groγ [SEQ ID NO 4], or the modified and multimeπc proteins derived therefrom, which are described in detail in International Patent Application, Publication No W094/29341, incoφorated by reference herein Particularly desirable are the modified KC [amino acids 5-72 of SEQ ID NO 2], modified groβ [amino acids 5-73 of SEQ ID NO 3], modified groγ [amino acids 5- 73 of SEQ ID NO 4], and a dimeric modified groβ [ammo acids 5-73 of SEQ ID NO 3]
Although th se chemo ines have been pieviυusiy described, eir use in prevention and treatment of sepsis has not been reported It has now been discovered that mature KC [SEQ ID NO 1 ], human groα [SEQ ID NO 2], human groβ [SEQ ID NO 3] or human groγ [SEQ ID NO 4J, and, particularly the modified and multimeπc chemokines derived therefrom significantly increase the survival of animals challenged with lethal sepsis causing organisms Treatment with a medicament or the compound of this invention, alone or in combination with an anti-infective agent prior to contemplated thoracic or abdominal surgery would be useful in reducing the likelihood of post-operative sepsis It may also be used post-operativcly for the treatment of sepsis caused by a variety of reasons as outlined previously
As stated above, the proteins useful in preparing medicaments and in the methods of the invention include the mature chemokines, modified chemokines, and multimers thereof
The term ' mature chemokines" also known as ' lntercπnes , as used herein defines the proteins conventionally referred to in the art as KC, gro , groβ, and groy For convenience, the amino acid sequences of the murine protein KC which contains 72 residues is provided in SEQ ID NO 1 These sequences are available from Genbank, accession number J04596 The sequences of the human protein groa (aa 1 73) are provided in SEQ ID NO 2 The sequences of the human protein groβ (amino acids 1-73) are provided in SEQ ID NO 3 The sequences of the human protein groy are provided in SEQ ID NO 4 The cDNA and amino acid sequences of groy are also provided in International Patent Application, Publication No WO 92/00326 (Jan 9, 1992) These groy sequences have further been published lnjnternational Patent Application, Publication No WO 94/29341 (December 22, 1994), which is incorporated by reference herein The term "modified chemokines" is defined as in the above-referenced International Apphcation The modified chemokines are derived from KC, groβ, groa, and groy, more preferably from groβ, groa, and groy, and most preferably from groβ The modified chemokines include desamino proteins characteπzed by the elimination of between about 2 to about 8 amino acids at the amino terminus of the mature protein Most preferably, the modified chemokines are characteπzed by removal of the first 4 amino acids at the amino- (N-) terminus Optionally, particularly when expressed recombinantly, the desamino chemokines useful in this invention may contain an inserted N-terminal Met
The N terminal nictlnoniπe wluJi is uiseited into tne protein tor expression purposes, may be cleaved, either during the processing of the protein by a host cell or synthetically, using known techniques Alternatively, if so desired, this ammo acid may be cleaved through enzyme digestion or other known means Particularly desirable modified chemokines include modified KC [amino acids 5 - 72 of SEQ ID NO 1], modified human groβ [amino acids 5-73 of SEQ ID NO 3] and modified human groγ [amino acids 5-73 of SEQ ID NO 4]
Also included by the term modified chemokine are other analogs or derivatives of KC, groα, groβ, or groγ which share the biological activity of the mature protein As defined herein, such analogs and derivatives include modified proteins also characterized by alterations made in the known amino sequence of the proteins, e g , the proteins provided in SEQ ID NOS 1-4 Such analogs are characterized by having an amino acid sequence differing from that of the mature protein by 8 or fewer amino acid residues, and preferably by about 5 or fewer residues. It may be preferred that any differences in the amino acid sequences of the proteins involve only conservative amino acid substitutions
Conservative ammo acid substitutions occur when an amino acid has substantially the same charge as the amino acid for which it is substituted and the substitution has no significant effect on the local conformation of the protein or its biological activity Alternatively, changes such as the introduction of a certain ammo acid in the sequence which may alter the stability of the protein, or permit it to be expressed in a desired host cell may be preferred Another characteπstic of these modified proteins may be enhanced biological activity in comparison to the mature protein By the term "multimeπc protein' . or "multimer ' is meant herein multimeπc forms of the mature and/or modified proteins useful in this invention, e g , dimers, tπmers, tetramers and other aggregated forms Such multimeπc forms can be prepared by synthesis or recombinant expression and can contain chemokines produced by a combination of synthetic and recombinant techniques as detailed below Multimers may form naturally upon expression or may be constructed into such multiple forms Multimeπc chemokines may include multimers of the same modified chemokine Another multimer may be formed by the aggregation of different modified proteins Still another multimer is formed by the aggregation of a modified chemokine of this invention and a known, mature chemokine Preferably, a dimer or multimer useful in the invention would contain at least uiie dcsdiniiiυ chemokine protein and at least one ottier chemokine or other protein characterized by having the same type of biological activity This other protein may be an additional desamino chemokine, or another known protein In one particularly desirable embodiment, the method of the invention utilizes a dimeric truncated groβ protein [amino acids 5 73 of SEQ ID NO 3], which is described in more detail below
Desirably, the chemokines useful in the method of the invention are used in the preparation of medicaments and or are useful in the form of a pharmaceutical composition Thus, the chemokines can be formulated into pharmaceutical compositions and administered in the same manner as described in, e g , International Patent Applications, Publication No WO 90/02762 (Mar 22, 1990) and Publication No W094/29341 (Dec 22, 1994)
These medicaments or pharmaceutical compositions useful in the method of the invention for preventing or treating sepsis contain an effective amount of a mature, modified or multimeπc chemokine protein derived from KC [SEQ ID NO 1 ], human gro-a [SEQ ID NO 2], human groβ [SEQ ID NO 3], and human groγ [SEQ ID NO 4] which is administered to an animal in need thereof Particularly desired embodiments utilize the modified chemokines, or multimers thereof These chemokine compositions may be administered alone or in combination with administration of other anti-infective agents
Thus, a pharmaceutical composition is prepared using one or more of proteins derived from the KC [SEQ ID NO 1], groα [SEQ ID NO 2], groβ [SEQ ID NO 3] or groγ [SEQ ID NO 4] proteins Suitable pharmaceutical earners are well known to those of skill in the art and may be readily selected Currently, the preferred carrier is saline Optionally, the pharmaceutical compositions of the invention may contain other active ingredients or be administered in conjunction with other therapeutics For example, the compositions of the invention are particularly well suited for administration in conjunction with anti- infective agents
Suitable anti-infective agents include, without limitation, anti-microbial agents routinely used for the treatment of sepsis such as amino-glycosides (such as amikacin, tobramycin, netilmicin, and gentamicin), cephalosponns such as ceftazidime, related beta lactam agents such as maxalactam, carbopenems such as lmipenem, monobactam agents such as aztreonam, ampicillin and broad- spectrum penicillins, (e g , penicillinase-resistant penicillins, ureidopenicillins or antipseudomonal penicillin or Augmentin) that are active against P aeruginosa, Enterobacter species, indole-positive Proteus species, and Serratia Also included within the definition of anti-infective agents are antifungal agents, amphotericin and tne like as well as anti-viral agents such as famvir and acyclovir
The chemokines descπbed herein are useful in the treatment and prevention of sepsis in humans and other animals such as dairy cattle, horses, calves or poultry To effectively treat a human or other animal a mature, modified or multimeπc KC [SEQ ID NO 1 ], groα [SEQ ID NO 2], groβ [SEQ ID NO 3] or human groγ [SEQ ID NO 4] or their multimers (e.g , a dimeric, truncated groβ, amino acids 5-73 of SEQ ID NO 3) may be administered by injection in the dose range of about 10 to about 10,000 fg/kg/dose, or orally in the dose range of about 10 to about 10,000 fg/kg body weight per dose, if administered by infusion or similar techniques, the dose may be in the range of about 10 to about 10,000 fg kg/dose, if administered subcutaneously the dose may be in tne range of about 10 to about 10,000 fg/kg/dose
Depending on the patient's condition, the compounds of this invention can be administered for prophylactic and/or therapeutic treatments In therapeutic apphcation, the compound is administered to a patient already suffenng from a disease in an amount sufficient to cure or at least partially arrest the disease and its complications It may be given at any time after surgery, preferably pnor to 24 hours after surgery In prophylactic applications, a composition containing mature, modified or multimeπc KC [SEQ ID NO 1 ], groα [SEQ ID NO 2J, groβ [SEQ ID NO 3] or groγ [SEQ ID NO 4] or a multimer thereof, is administered to a patient not already in a disease state to enhance the patient's resistance. It may be given one day or one week pnor to surgery, preferably one to two days prior to surgery It may be administered parenteral iy or orally
Single or multiple administrations of the compounds can be carried out with dose levels and pattern being selected by the treating physician In any event, a quantity of the compounds of the invention sufficient to effectively treat the patient should be administered
The chemokines useful in the methods of this invention, may also be administered in conjunction with a separately administered conventional anti-infective as disclosed herein above, such a gentamicin, augmentin or ceftazidime The particular anti-infective chosen should be one to which the infective organism is susceptible and is selected or modified during therapy as the infecting microorganism is more particularly identified
Additionally, various adjunctive agents in the treatment of septic shock also may be useful in combination with the components of this invention They include sympathomimetic amines (vasopressors) such as norepinephπne, epinephπne, isoproterenol, dopamine, and dobutamine, anti-inflammatory agents such as methylprednisolone anti-inflammatory agents such as lndomethacin and phenylbutazone, and corticosteroids such as betamethasone, hydrocortisone, methylprednisolone, or dexamethasone, anti-coagulants such as hepaπn, anti-thrombin III or coumaπn type drugs for certain conditions and schedules, diuretics such as furosemide or ethacrynic acid, and antagonist of opiates and beta-endorphins such as naloxone; an antagonist of tumor necrosis factor or of interleukin- 1 , phenothiazines, anti-histamines, glucagon, a-adrenergic blocking agents, vasodilators, plasma expanders, packed red blood cells, platelets, cryoprecipitates, fresh frozen plasma, bacterial permeability protein, chndamycin, and antibodies to (lipid A), the J5 mutant of E coh or to endotoxin core glycolipids Methods for preparing such antibodies are descπbed widely in the literature
One of the most important aspects in the treatment of the clinical septic shock syndrome is its apparently intractable resistance to the effects of a vanety of highly potent antimicrobial agents Despite the development of newer antimicrobial agents, the overall incidence of clinical sepsis has increased, and mortality remains unacceptably high, often approaching 60% of diagnosed patients The discovery of the increased survival with the treatment of the full length, modified and multimeπc KC [SEQ ID NO 1 ], groα [SEQ ID NO 2], groβ [SEQ ID NO 3], or groγ [SEQ ID NO 4] both prophylactically and after infection provides a new and useful therapy of sepsis The biological activity of modified KC [SEQ ID NO 1], modified human groβ
[SEQ ID NO. 3], modified human groγ [SEQ ID NO 4], and a dimeric modified human groγ are demonstrated by the following assays. These examples illustrate the prefened methods of the invention These examples do not limit the scope of the invention Rats. Male Fischer 344 rats obtained from Taconic farms weighing 200 to 250 g were utilized The rats were housed 2 per cage in standard plastic caging and fed lab chow and water ad libitum Modified KC [SEQ ID NO 1 J, modified human groβ [SEQ ID NO 2] or modified human groγ [SEQ ID NO 3] or multimers thereof, was prepared in E colt by the method given in Example 1 The compound was dissolved in DPBS containing 0 5 % heat inactivated autologous normal rat serum The animals were dosed intraperitoneally with KC 24 hours and 2 hours before infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated tw ice daily with subcutaneous gentamicin
E. coli A clinical isolate of £ coh isolated from sputum was utilized The organisms were tested for antibiotic sensitivity by the disc agar diffusion technique and found to be sensitive to gentamicin, ampicillin, cephalothin, chloramphenicol, kanamycin tetracycline, tπmethopπn/sulfamethoxazole and resistant to penicillin G, erythromycin, and vancomycin The organism was animal passed in mice and subsequently recovered and plated onto MacConkey's agar The reisolated organisms were grown overnight in brain- heart infusion broth, and then stored frozen at -70°C The inoculate the fibrin clot, organisms from thawed stocks were inoculated into brainheart infusion broth and incubated overnight on a rotary shaker (120 rpmϊ an 37°C The F coh was harvested by centπfugation, washed 3X and finally resuspended in normal saline The number or organisms was quantified by turbidimentry, and the concentration adjusted with normal saline All inoculum sizes were based on viable counts determined b> scoring colony forming units on MacConkeys agar
Fibrin Clot The E colt infected fibrin clots were made from a 1 % solution of bovine fibrinogen (Type 1 -S, Sigma) in sterile saline The clot was formed by adding sequentially human thrombin (Hanna Pharma ) bacteria, and fibrinogen solution to 24 well plastic plates Bacteπal numbers of 2 0 to 3 0 x I09 were used in inoculate the fibrin clots The resulting mixture was then incubated at room temperature for 30 minutes before implantation Animal Model. The rats were anesthetized with ketamine/xylazine (40 mg/kg/5 mg/kg) after which the abdominal surfaced were shaved and a midlme laparotomy performed Bacterial peritonitis was induced by implanting a fibπn-thrombin clot containing E co into the abdominal cavity After implantation the muscle layers were closed with 4 0 silk suture, and the wound closed with surgical staples The animals were closely observed, any animals obviously moribund were euthanized
Gentamicin Rats were treated subcutaneously with gentamicin sulfate (Elkins Sinn, NJ) 5 mg/kg twice a day for five days
Statistics All continuously variable data are expressed as the percent survival from several pooled studies The Fisher's Exact test was used to determine the statistical significance of the differences between the survival rates at 14 days The differences between the groups were considered statistically significant at p<0 05
Example 1 - Production of Truncated KC and GRO ?
A Expression of recombinant truncated KC and truncated groβ
When truncated muπne KC (amino acids 5-72 of SEQ ID NO 1 ) and human groβ (amino acids 5-73 of SEQ ID NO 3) were expressed intracellularly in E co , the KC (aiuino acids 5 72 of SEQ ID NO 1 ) retained the initiator Mel In order to produce the authentic N-termmal recombinant proteins, a specific cleavable tag was engineered at the N-terminus of truncated KC (amino acids 5-72 of SEQ ID NO 1 ) The coding sequences of truncated murine KC and truncated human groβ (amino acids 5 73 of SEQ ID NO 3) were each amplified by polymerase chain reaction (PCR) from plasmids containing complimentary DNA sequences ustng both a forward primer encoding an Ndel site and a reverse primer containing an Xbal site For truncated KC (amino acids 5-72 of SEQ ID NO 3), a defined epitope tag (DET) site and an enterokinase cleavage site were also used These PCR fragments were subcloned into the E coh LPIL-dependent expression vector pEAKn (pSKF301 derivative) between Ndel and Xbal sites Each polypeptide was expressed by chemical induction of the LPL promoter in a lysogenic strain of E coh containing the wild type (ιnd+) repressor gene (cl+) AR120 B Purification and refolding of truncated groβ (amino acids 5-73 of SEQ ID
NO 3)
E colt cell pellet was lysed in pH 6 0 buffer containing 20 mM dithiothreitol (DTT) to avoid the nonspecific air oxidation The majority of truncated groβ was m the insoluble lysate pellet which was solubilized in 2 M GdnHCI, pH 8 0 buffer containing 20mM DTT The solubilized truncated groβ was dialyzed against pH 6 0 buffer containing 2 mM EDTA The majoπty of £ colt proteins were precipitated during dialysis while truncated groβ stayed in solution as a monomeric form at >95 % purity The truncated groβ solution was adjusted to pH 8 5, stirred overnight for air oxidation The refolded truncated groβ solution was adjusted to 0 1 % TFA solution, and applied to
Ultrasphere Cl 8 (Beckman) column to separate monomeric form from dimeric form Each form was pooled separately, evaporated to remove acetonitrile, concentrated, dialyzed against PBS and stored at -70°
C Purification of truncated KC (ammo acids 5 72 of SEQ ID NO J ) DET-DDDDK chemokines were purified and refolded as described for truncated groβ The refolded DET-DDDDK chemokines were digested with enterokinase to remove the N-terminal DET-DDDDK and the undigested molecules were removed using anti DET Mab column The digested molecules were further purified using C18 RP-HPLC as described above D Characterization
N-termmal sequencing and MALD-MS for molecular weight were performed and confirmed that the molecules are intact from N-terminus to C-termmus, either monomeric or dimeπc form Concentration of each chemokine was determined by amino acid analysis and endotoxin level of each prep was <0 05 U/ml
Example 2 - Production of Truncated GRO J Dimer A Cell lysis
E coh LW cells, 400 g, were lysed in 4 liters of lysis buffer containing 50 mM sodium citrate pH 6.0, 40 mM NaCl, 2 M EDTA, 5% glycerol, 005% Tween 80, 0 2 mM PMSF, 1 mg/ml each of leupeptin and pepstatm A, by two passages through a
Microfluidics (model M l 10Y) homogenizer at 1 1,000 psi The cell lysate was centrifuged at 17,000 g (one hour at 4°C) and the supernatant was discarded B Solubilization and Refolding of Truncated GROβ Dimer
The insoluble truncated groβ [amino acids 5-73 of SEQ ID NO 3] in lysate pellet was solubilized in 1 3 liters of buffer containing 50 mm Tris HCl pH 8 0, 2 M guanidine HCl, 20 mM DTT by stirring overnight at room temperature Soluble truncated groβ [amino acids 5-73 of SEQ ID NO 3] was recovered by centrifugation at 25,000 g, from which guanidine HCl and DTT were removed by exhaustic dialysis against 50 mM sodium citrate pH 6 0 containing 2 mM EDTA to obtain soluble and reduced form of truncated groβ [amino acids 5-73 of SEQ ID NO 3] Truncated groβ solution was concentrated to 3 mg/ml (Anicon YM3 membrane) and raised to pH 8 5 with 0 5 M Tπzma base Air oxidation of truncated groβ [amino acids 5-73 of SEQ ID NO 3] was performed by stirring overnight at room temperature Formation of dimer was monitored by Vydac C18 (Nest) using 20-40% linear gradient of acetonitrile in 0 1 % TFA for 30 min C Purification of Dimer
When dimer formation reached maximum, the reoxidation solution was adjusted to pH 8 0 with 10% acetic acid and the dimer captured on Toyopearl SP-650 M equilibrated in 25 mM Tris HCl pH 8 0 (Buffer A) The column was washed with 4 liters buffer A, 2 liters 0 125 M NaCl in buffer A, and eluted with 4 liters of linear gradient of 0 125 - 0 5 M NaCl in buffer A Flow rate was 40 ml/min Truncated groβ dimer was well separated from truncated groβ and other oligomer form of truncated groβ (SDS-PAGE) Fractions containing truncated groβ dimer were combined, adjusted to pH 3 0 with 1 % TFA solution and applied to Vydac C18 (2 1 x 25 cm) equilibrated with 0 1 % TFA in 4% acetonitrile Truncated groβ dimer was eluted with linear gradient of 20-40% acetonitrile in 0 1 % TFA for 30 min Truncated groβ dimer was eluted at -30% acetonitrile Fractions containing truncated groβ dimer was pooled, lyophilized to remove acetonitrile, and dialyzed in Spectrapor 3K MWCO dialysis tubing against PBS D Yield
Typical yield of truncated groβ dimer was 0 15 mg/g of ^ells when refolding was performed at 0 1 mg/ml or 0 7 mg/g of cells when refolding at 3 mg/ml E Characterization The molecular weight of the truncated groβ dimer as determined on nonreducing SDS PAGE was approximately twice that of truncated groβ Upon reduction, both forms migrated to the same spot indicating that truncated groβ dimer is a disulfide linked dimer The molecular weight of truncated groβ dimer, as determined b> MALD-MS analysis was 15,069 Da (predicted 15,073 Da), while that of truncated groβ dimer was 7,536 Da (predicted 7,537 Da) N-terminal sequencing of truncated groβ dimer showed that 5-10% of the final products retained the initiatory Met Disulfide pairing pattern of truncated groβ dimer was the same as that of truncated groβ (C5-C 1 , C7-C47) [ammo acids 5-73 of SEQ ID NO 3], however, all pairings were intermolecular rather than intramolecular Gel filtration analysis and ultracentnfugation sedimentation equilibrium studies in PBS (pH 7 0) showed that truncated groβ dimer exhibited reversible assembly of octamer to hexadecamer at 0 25 mg/ml, while truncated groβ [amino acids 5-73 of SEQ ID NO 3] was a nonconvalent dimer even at 20 mg/ml Concentration of truncated groβ dimer has been determined by quantitative amino acid analysis Example 3 - Prophylactically Administered Truncated KC in E. coli Sepsis.
The animals were dosed intraperitoneally with truncated KC [ammo acids 5-72 of SEQ ID NO 1] at doses of 10, 33, 100 or 333 fg/kg 24 hours and 2 hours before infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin On day 0 the rats were implanted with an E coh containing fibπn-thrombin clot Starting two hours after infection the rats were treated with gentamicin twice daily The rats prophylactically treated with truncated KC at 33 or 100 fg kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone Results
Dose (fg kg) survival (alive/dead)
Control 8 / 17
10 10 / 15
33 17 / 8 100 18 / 7
333 10 / 15
Example 4 - Therapeutically Administered Truncated KC in E. coli sepsis
On day 0 the rats were implanted with an E coh containing fibπn-thrombin clot The animals were dosed intraperitoneally with truncated KC [amino acids 5-72 of SEQ ID NO.1] at doses of 33, 100, 333, or 1,000 fg/kg as a single injection 2 hours after infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with truncated KCat 100 or 333 fg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone Results Dose (fg/kg) survival (alive / dead)
Control 9 / 16
33 1 1 / 14
100 17 / 8
333 18 / 7 1 ,000 10 / 15
Example 5 - Therapeutically Administered Truncated KC in S. aureus Sepsis.
On day 0 the rats were implanted with a S. aureus containing fibπn-thrombin clot The animals were dosed intraperitoneally with truncated KC [amino acids 5-72 of SEQ ID NO 1 ] at doses of 33, 100, 333, or 1 ,000 fg/kg as a single injection 2 hours after infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with truncated KC at 100 or 333 fg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone. Results
Dose (fg/kg) survival (alive / dead)
Control 8 / 17
33 1 1 / 14
100 17 / 8 333 21 / 4
1000 12 / 13
Example 6 - Therapeutically Administered Truncated groβ in E. coli Sepsis
On day 0 the rats were implanted with an E co containing fibπn-thrombin clot The animals were dosed mtrapeπtoneally with truncated groβ [amino acids 5-73 of SEQ ID NO.3] at doses of 33, 100, 333, or 1,000 fg/kg as a single injection 2 hours after infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with tπincated groβ at 100 or 333 fg/kg followed by gentamicin treatment demonstrated signi ificantly improved survival rates over the diluent treated control rats receiving gentamicin therapy Ϊ one
Results
Dose (fg/kg) survival (alive/dead)
Control 9 / 16
33 12 / 13
100 20 / 5
333 18 / 7
1000 10 / 15
Example 7 - Therapeutically Administered Truncated groβ in 5. aureus Sepsis
On day 0 the rats were implanted with an S aureus containing fibπn-thrombin clot The animals were dosed intrapeπtoneally with truncated groβ [amino acids 5-73 of SEQ ID NO 3] at doses of 33, 100, 333, or 1 ,000 fg/kg as a single injection 2 hours after infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with truncated groβ at 100 or 333 fg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone Results
Dose (fg/kg) survival
Control 9 / 16
33 12 / 13
100 20 / 5 333 18 / 7
1 ,000 10 / 15
Example 8 - Therapeutical Subcutaneously Administered Truncated groβ in E. coli Sepsis On day 0 the rats were implanted with an E. coh containing fibπn-thrombin clot
The animals were dosed subcutaneously with truncated groβ [amino acids 5-73 of SEQ ID N0.3J at doses of 0 1 , 0.3, 1 0, or 3.3 pg/kg as a single injection 2 hours after infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with truncated groβ at 0.3 or 1.0 pg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone Results
Dose (pg/kg) survival (alive/dead)
Control 10 / 15
0 1 12 / 13
0.3 18 / 7 1.0 20 / 5
3.3 1 1 / 14
Example 9 - Therapeutical Subcutaneously Administered Truncated groβ in S. aureus Sepsis On day 0 the rats were implanted with an S. aureus containing fibrin-thrombin clot
The animals were dosed subcutaneously with truncated groβ [amino acids 5-73 of SEQ ID NO.3] at doses of 0.1 , 0 3, 1.0, or 3.3 pg/kg as a single injection 2 hours after infection Control animals were dosed with dilution buffer on the same schedule Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with truncated groβ at 0.3 or 1.0 pg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone. Results
Dose (pg/kg) survival (alive/dead) Control 8 / 17
0.1 13 / 12
0.3 18 / 7
1.0 20 / 5
3.3 12 / 13
Example 10 - Prophylactically Administered GRO/J Dimer in E. coli Sepsis
The animals were dosed subcutaneously with dimer formed of two truncated groβ proteins [amino acids 5-73 of SEQ ID NO:3] at doses of 0 1, 0.3, 1.0 or 3.3 pg/kg 24 hours before infection Control animals were doses with dilution buffer on the same schedule On day 0 the rats were implanted with an E colt containing fibπn-thrombin clot Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats prophylactically treated with truncated groβ dimer at 0 3 or 1 0 pg kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone Results
Dose (pg kg) survival (alive/dead)
Control 8 / 17 0 1 10 / 15
0.3 18 / 7
1 0 20 / 5
3 3 8 / 17
Example II - Therapeutically Administered GRO/J Dimer in 5. aureus Sepsis
On day 0 the rats were implanted with an S aureus containing fibπn-thrombin clot The animals were dosed subcutaneously with a dimer formed of two truncated groB proteins [ammo acids 5-73 of SEQ ID NO 3] at doses of 0 03, 0 1, 0 3, 1 0, 3 3, or 10 pg/kg as a single injection 2 hours after infection Control animals were doses with dilution buffer on the same schedule
Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin The rats therapeutically treated with truncated groβ dimer at 0 1 , 0 3 or 1 0 pg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone Results
Dose (pg/kg) survival (alive/dead)
Control 1 1 / 14
0.03 12 / 13
0 1 18 / 7 0 3 23 / 2
1 0 24 / 1
3.3 17 / 8
10.0 12 / 13 SEQUENCE LISTING
) GENERAL INFORMATION:
(i) APPLICANT: SmithKline Beecham Corporation DeMarsh, Peter L. Johanson, Kyung 0.
(ii) TITLE OF INVENTION: Method of Treating Sepsis
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corporation -
Corporate Patents
(B) STREET: 709 Swedeland Road
(C) CITY: King of Prussia
(D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19406-2799
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: WO
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/007,425
(B) FILING DATE: 21-NOV-1995
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hall, Linda E.
(B) REGISTRATION NUMBER: 31,763
(C) REFERENCE/DOCKET NUMBER: P50 17-1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5016
(B) TELEFAX: 610-270-5090 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
Ala Pro lie Ala Asn Glu Leu Arg Cys Gin Cys Leu Gin Thr Met 1 5 10 15
Ala Gly lie His Leu Lys Asn lie Gin Ser Leu Lys Val Leu Pro
20 25 30
Ser Gly Pro His Cys Thr Gin Thr Glu Val lie Ala Thr Leu Lys
35 40 45
Asn Gly Arg Glu Ala Cys Leu Asp Pro Glu Ala Pro Leu Val Gin
50 55 60
Lys lie Val Gin Lys Met Leu Lys Gly Val Pro Lys
65 70
(2) INFORMATION FOR SEQ ID NO: 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :
Ala Ser Val Ala Thr Glu Leu Arg Cys Gin Cys Leu Gin Thr Leu 1 5 10 15
Gin Gly lie His Pro Lys Asn lie Gin Ser Val Asn Val Lys Ser
20 25 30
Pro Gly Pro His Cys Ala Gin Thr Glu Val lie Ala Thr Leu Lys
35 40 45 Asn Gly Arg Lys Ala Cys Leu Asn Pro Ala Ser Pro lie Val Lys
50 55 60
Lys lie lie Glu Lys Met Leu Asn Ser Asp Lys Ser Asn 65 70
(2) INFORMATION FOR SEQ ID NO: 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 amino acids
(B) TYPE: amino acid { C ) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 :
Ala Pro Leu Ala Thr Glu Leu Arg Cys Gin Cys Leu Gin Thr Leu 1 5 10 15
Gin Gly lie His Leu Lys Asn lie Gin Ser Val Lys Val Lys Ser
20 25 30
Pro Gly Pro His Cys Ala Gin Thr Glu Val lie Ala Thr Leu Lys
35 40 45
Asn Gly Gin Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Lys
50 55 60
Lys lie lie Glu Lys Met Leu Lys Asn Gly Lys Ser Asn
65 70
(2) INFORMATION FOR SEQ ID NO:4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ TD NO: 4 :
Ala Ser Val Val Thr Glu Leu Arg Cys Gin Cys Leu Gin Thr Leu 1 5 10 15 Gin Gly lie His Leu Lys Asn lie Gin Ser Val Asn Val Arg Ser
20 25 30
Pro Gly Pro His Cys Ala Gin Thr Glu Val lie Ala Thr Leu Lys
35 40 45
Asn Gly Lys Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Gin
50 55 60
Lys lie lie Glu Lys lie Leu Asn Lys Gly Ser Thr Asn
65 70

Claims

We Claim
1 A method of treating sepsis comprising administering to an animal in need thereof an effective amount of a protein derived from a chemokine selected from the group consisting of (a) KC SEQ ID NO 1 , (b) groα SEQ ID NO 2, (c) groβ SEQ ID NO 3, and (d) groγ SEQ ID NO 4
2 The method according to claim 1 wherein the chemokine is selected from the group consisting of
(a) mature groβ,
(b) modified groβ consisting of amino acids 5 to 73 of SEQ ID NO 3,
(c) a multimeπc chemokine protein which compπses an association of two or more of (a) or (b), and
(d) a multimeπc chemokine protein which comprises an association of (a) or (b) with a second chemokine
3 The method according to claim 2 wherein the chemokine is a dimeric protein consisting of two modified groβ proteins
4 The method according to claim 1 wherein said chemokine is selected from the group consisting of
(a) mature groα,
(b) modified groα consisting of amino acids 5 to 73 of SEQ ID NO 2,
(c) a multimeπc chemokine protein which comprises an association of two or more of (a) or (b), and
(d) a multimeπc chemokine protein which comprises an association of (a) or (b) with a second chemokine
5 The method according to claim 1 wherein said chemokine is selected from the group consisting of
(a) mature groγ, (b) modified groγ consisting of amino acids 5 to 73 of SEQ ID NO 4,
(c) a multimeπc chemokine protein which comprises an association of two or more of (a) or (b), and
(d) a multimeπc chemokine protein which comprises an association of (a) or (b) with a second chemokine.
6 The method according to claim 1 wherein said chemokine is selected from the group consisting of
(a) mature KC,
(b) modified KC consisting of amino acids 5 to 72 of SEQ ID NO I .
(c) a multimeπc chemokine protein which comprises an association of two or more of (a) or (b), and
(d) a muitimeπc chemokine protein which comprises an association of (a) or (b) with a second chemokine
7 A method according to claim 1 wherein said effective amount is from about 10 to about 1 ,000 fg/kg/dose
8 The method according to claim 1 wherein said chemokine is administered 2 hours to 24 hours after surgery
9 The method according to claim 1 wherein said chemokine is administered orally
10 The method according to claim 1 wherein said chemokine is administered subcutaneously
1 1 The method according to claim 1 further comprising the step of administering the chemokine in conjunction with an effective amount of an anti-mfective agent 12 A method according to claim 1 1 wherein the anti-infective agent is selected from the group consisting of gentamicin, augmentin or ceftazidime
13 A method for the prevention of sepsis comprising administering to an animal in need thereof an effective amount of an effective amount of a protein derived from a chemokine selected from the group consisting of (a) KC SEQ ID NO 1 , (b) groα SEQ ID NO 2, (c) groβ SEQ ID NO 3, and (d) groγ SEQ ID NO 4
14 The method according to claim 13 wherein the chemokine is selected from the group consisting of
(a) mature groβ,
(b) modified groβ consisting of amino acids 5 to 73 of SEQ ID NO 3,
(c) a multimeπc chemokine protein which comprises an association of two or more of (a) or (b), and
(d) a multimeπc chemokine protein which comprises an association of (a) or (b) with a second chemokine
15 The method according to claim 14 wherein the chemokine is a dimeric protein consisting of two modified groβ proteins
16 The method according to claim 13 wherein said chemokine is selected from the group consisting of
(a) mature groα,
(b) modified groα consisting of amino acids 5 to 73 of SEQ ID NO 2,
(c) a multimeπc chemokine protein which comprises an association of two or more of (a) or (b), and
(d) a multimeπc chemokine protein which comprises an association of (a) or (b) with a second chemokine
17 The method according to claim 13 wherein said chemokine is selected from the group consisting of
(a) mature groγ, (b) modified groγ consisting of amino acids 5 to 73 of SEQ ID NO 4,
(c) a multimeπc chemokine protein which comprises an association of two or more of (a) or (b), and
(d) a rnultimeric chemokine protein which comprises an association of (a) or (b) with a second chemokine
18 The method according to claim 13 wherein said chemokine is selected from the group consisting of
(a) mature KC,
(b) modified KC consisting of amino acids 5 to 72 of SEQ ID NO: 1 ;
(c) a multimeπc chemokine protein which comprises an association of two or more of (a) or (b), and
(d) a multimeπc chemokine protein which comprises an association of (a) or (b) with a second chemokine.
19 A method according to claim 13 wherein the effective amount is from about 10 to about 1 ,000 fg/kg/dose
20 The method according to claim 13 wherein said chemokine is administered 1 to 2 days prior to surgery
21 The method according to claim 13 further comprising the step of administering the chemokine in conjunction with an effective amount of an anti-infective agent
22 A method according to claim 21 wherein the anti-mfective agent is selected from the group consisting of gentamicin, augmentin or ceftazidime
EP96940554A 1995-11-21 1996-11-20 Method of treating sepsis Withdrawn EP0871732A4 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US742595P 1995-11-21 1995-11-21
US7425P 1995-11-21
PCT/US1996/018616 WO1997019173A1 (en) 1995-11-21 1996-11-20 Method of treating sepsis

Publications (2)

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EP0871732A1 true EP0871732A1 (en) 1998-10-21
EP0871732A4 EP0871732A4 (en) 2002-01-16

Family

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AU (1) AU1020997A (en)
WO (1) WO1997019173A1 (en)
ZA (1) ZA969721B (en)

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US6042821A (en) * 1995-11-21 2000-03-28 Smithkline Beecham Corporation Method of treating sepsis with chemokines
US6290948B1 (en) * 1996-05-14 2001-09-18 Smithkline Beecham Corporation Method of treating sepsis and ARDS using chamohine beta-10
US6100387A (en) 1997-02-28 2000-08-08 Genetics Institute, Inc. Chimeric polypeptides containing chemokine domains
US6852508B1 (en) 1997-02-28 2005-02-08 Genetics Institute, Llc Chemokine with amino-terminal modifications
US6989435B2 (en) 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7067117B1 (en) 1997-09-11 2006-06-27 Cambridge University Technical Services, Ltd. Compounds and methods to inhibit or augment an inflammatory response
US6838435B1 (en) 1997-09-25 2005-01-04 Academisch Ziekenhuis Bij De Universiteit Van Amsterdam Isolated and recombinant antimicrobial peptides thrombocidin-1 (TC-1) and thrombocidin-2(TC-2) or variants thereof
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
AU2002221099A1 (en) * 2000-12-08 2002-06-18 Takeda Chemical Industries Ltd. Combination drugs
CN106632616B (en) * 2016-07-18 2020-12-25 复旦大学附属妇产科医院 Gene delivery system with ovarian tumor targeting D-configuration polypeptide

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ZA969721B (en) 1997-11-28
EP0871732A4 (en) 2002-01-16
WO1997019173A1 (en) 1997-05-29
JP2000504310A (en) 2000-04-11
AU1020997A (en) 1997-06-11

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