METHOD OF TREATING SEPSIS

Abstract: The invention relates to the method of preventing and treating sepsis using chemokines selected from mature or modified KC [SEQ ID NO: 1], gro alpha [SEQ ID NO:2], gro beta [SEQ ID NO:3] or gro gamma [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 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, 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 (! Q 5!)) 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 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 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

Inventors: DEMARSH PETER LAWRENCE, JOHANSON KYUNG O, DEMARSH, PETER, LAWRENCE, JOHANSON, KYUNG, O.
Applicants: DEMARSH PETER LAWRENCE, JOHANSON KYUNG O, SMITHKLINE BEECHAM CORP, DEMARSH, PETER, LAWRENCE, JOHANSON, KYUNG, O., SMITHKLINE BEECHAM CORPORATION
Application year: 1996
Main classification: C12N15
IPC codes: A61K31, A61K38, A61P7, C07K14, C12N15