CA2238167A1 - Method of treating sepsis - Google Patents

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

CA 02238167 1998-0~-20 PCT~US96/18616 METHOD OF TREATING SEPSIS

Field of Invention This invention relates to the method of preventing and treating sepsis using certain 5 chemokines alone or in conjunction with an anti-infective agent.
Back~round of In~ention 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 10 minute; (3) respiratory rate >20 breaths per minute or PaCO2 <32 mm Hg; (4) white blood cell count >12,000/cu mm, <4,000/cu mm, or >10% immature (band) forms; (5) organdysfunction, hypoperfusion, or hypotension. Hypoperfusion and perfusion abnormalities may include, but are not limited to lactic acidosis, oliguria, or an acute alteration in mental states. (Chest 1992; 101: 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 Escher~chia coli, followed by other Gram-negative bacteria such as the Klebsiella-Enterobacter-Serratia group and then Pseudomonas. ~Ithough comprising a somewhat smaller percentage of infection, Gram-20 positive microbes such as Stap~ylococcus 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 ischaracterized by inadequate tissue perfusion, leading to insufficient oxygen supply to tissues, hypotension and oliguria.
Septic shock occurs because bacterial products react with cells and components of the coagulation, complement, fibrinolytic and bradykinin systems to release proteases 30 which inJure cells and alter blood flow, especially in the capillaries.
Microorganisms frequently activate the c~c.ciczll complement pathway, and endotoxin activates the alternative pathway. Complement activation, leukotriene generation and the direct effects of bacterial products on neutrophils lead to accumulation CA 02238167 1998-0~-20 PCT~US96/18616 of these infl~ y cells in the lungs, r~elease of their proteolytic enzymcs 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, granulocyte aggregation, hemorrhage and 5 capillary thrombi.
Septic shock is a major cause of death in intensive care units. There are an estim~ d 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 10 decreased very little since the comprehensive description by Waisbren (Arch. Intern. Med.
88:a67-488 (~51~. Althollgh effect.~,c antibio~ics are availabie, and ihere 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 that other agents must be developed to be 15 used alone or in conjunction with antimicrobials in order to rectify the deficiencies of current established therapy.

Summarv of the lnvention This invention relates to a method of preventing or treating sepsis c~ isillg 20 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 groy. 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], modified human gro~ [amino acids 5-73 of the full length protein, SEQ ID NO: 3~ or modified human gro~ ~amino acids 25 5-73 of the full length protein, SEQ ID NO: 4] or mnl~im~srs 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 ;~lmini~¢ration of an anti-infective agent.
Other aspects and advantages of the present invention are described further in the 3~ following detailed description of the preferred embodiments thereof.

CA 02238167 1998-0~-20 W O 97/19173 PCT/US96/18616 Detailed DescriDtion 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 5 mature KC [SEQ ID NO: 1], groa [SEQ ID NO:2], groB [SEQ ID NO:3], gro y [SEQ ID
NO:4], or the modified and multimeric proteins derived therefrom, which are described in detail in International Patent Application, Publication No. WOg4/29341, incorporated by reference herein. Particularly desirable are the modified KC [amino acids 5-72 of SEQ ID
NO:2], modified grol~ [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~ [amino acids 5-73 of SEQ ID NO:3].
Although thcse chemoki~.es have b~en previously described, their use in prevention and treatment of sepsis has not been reported. It has now been discovered that mature KC
rSEQ ID NO: 11, human groa [SEQ ID NO:2], human gro~ [SEQ ID NO: 3] or human groy [SEQ ID NO: 4], and, particularly the modified and mnltim~.ric chemokines derived 15 therefrom significantly increase the survival of animals challenged with lethal sepsis causing or~nicmc. 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-operatively for the treatment of sepsis caused by a variety of reasons as outlined 20 previously.
As stated above, the proteins useful in preparing medicaments and in the methodsof the invention include the mature chemokines, modified chemokines, and multimers thereof.
The term "mature chemokines" also known as "intercrines", as used herein defines25 the proteins conventionally referred to in the art as KC, groa, grof3, and groy. For convenience, the amino acid sequences of the murine protein KC which contains 72residues is provided in SEQ ID NO: 1. These sequences are available from Genbank, ~cceccion number J04596. The sequences of the human protein groa (aa 1-73) are provided in SEQ ID NO:2. The sequences of the human proeein groJ~ (amino acids 1-73) 30 are provided in SEQ ID NO: 3. The sequences .of the human protein gro~ are provided in ., SEQ ID NO:4. The cDNA and amino acid sequences of gro~y are also provided in Tntt~rn~fional Patent Application, Publication No. WO 92/00326 (Jan. 9, 1992). These groy , CA 02238167 1998-0~-20 W O 97/19173 . PCT~US96/18616 sequences have further been published ir~nternational Patent Application, Publication No.
WO 94/2~341 (December 22, 19g4), which is incorporated by reference herein.
The term "modified chemokines" is defined as in the above-referenced International Application. The modified chemokines are derived from KC, gro~, gro~, and S groy, more preferably from gro./3, groa, and gro y, and most preferably from groJ3. The modified chemokines include cl~oc~mino proteins characterized 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 characterized by removal of the first 4 amino acids at the amino- ~N-~ terminus. Optionally, particularly when expressed recombinantly, the 10 ~ 5~mino chemokines useful in this invention may contain an inserted N-terminal Met.
~he ~-~:crrninal mcthionihle WhiCil iS inser~ed into the protein for 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 amino acid may be cleaved through enzyme digestion or other known means. Particularly desirable modified chemokines 1~ include modified KC Lamino acids ~ - 72 of SEQ ID NO: 1], modi~led human groB Lamino acids 5-73 of SI~Q ID NO: 3] and modified human gror [amino acids 5-73 of SEQ ID NO:
4].
Also included by the term modified chemokine are other analogs or derivatives ofKC, groa, grol3, or gro~ which share the biological activity of the mature protein. As 20 defined herein, such analogs and derivatives include modified proteins also characterized by alterations made in the known a~nino 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 S or fewer residues. It may be pl~rellc~d that any differences in the ~5 amino acid sequences of the proteins involve only conservative amino acid substitutions~
Conservative amino acid substitutions occur when an amino acid has sllt)st~nti~lly 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 amino acid in the sequence which may alter 3[) the stability of the protein, or permit it to be expressed in a desired host cell may be preferred. Another characteristic of these modified proteins rnay be enhanced biological activity in comparison to the mature protein.

CA 02238167 1998-0~-20 W O 97/19173 PCT~US96/18616 By the term "multimeric protein".or "multimer" is meant herein multimeric forms of the mature and/or modified proteins useful in this invention. e.g., dimers, trimers, tetrame}s and other aggregated forms. Such multimeric 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. Multimeric chemokines may include multimers of the same modified chemokine. Another mnltim~r 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, maturechemokine. Preferably, a dimer or multimer useful in the invention would contain at least Oile ~eiamilll) chemokine protein and at least one other chemolcine or other protein characterized by having the same type of biological activity. This other protein may be an additional ~Ps~mino chemokine, or another known protein. In one particularly desirable embodiment, the method of the invention utilizes a dimeric truncated groB 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 p.~pa.dtion of mf~-lics~m~nts and/or are useful in the form of a pharm~eutic~l composition.
Thus, the chl-.m~-kint~s can be formulated into pharmaceutical compositions and minic~ered in the same manner as described in, e.g., International Patent Applications, Publication No. WO 90/02762 (Mar. 22, 1990) and Publication No. WO94/29341 (Dec. 22, 1994).
These medicaments or pharm~fentir~l compositions useful in the method of the invention for preventing or treating sepsis contain an effective amount of a mature, modified or multimeric chemokine protein derived from KC rSEQ ID NO: 1], human gro-a [SEQ ID NO: 2], human groL~ [SEQ ID NO: 3], and human groy [SEQ ID NO: 4] which is administered to an animal in need thereof. Particularly desired embodiments utilize the modified chemokines, or ml-ltim~rs thereof These chemokine compositions may be lmini~t~red alone or in combination with arlmini~tration of other anti-infective agents.
Thus, a phd~ 'c.~ al composition is prepared using one or more of proteins derived from the KC [SEQ ID NO: 1], groa [SEQ ID NO:2], groR [SEQ ID NO:3] or gro y [SEQ ID NO:4~ proteins. Suitable pharm~eutic:~l carriers are well lcnown to those of skill in the art and may be readily selected. Currently, the preferred carrier is saline. Optionally, J. the pharmaceutical compositions of the invention may contain other active ingredients or be ~lmini~t~red in conjunction with other therapeutics. For example, the compositions of s CA 02238167 1998-0~-20 the invention are particularly well suited ~or ~lrninictration 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, 5 tobramycin, netilmicin, and gentamicin), cephalosporins such as ceft~7i~im~, related beta-lactam agents such as max~l~rt~m, carbopenems such as imipenem, monobactam agents such as aztreonam; ampicillin and broad-spectrum penicillins, (e.g., penicillinase-resistant penicillins, ureidopenicillins or antipseudomonal penicillin or ~llgmentin) that are active against P. aeruginosa, Enterobacter species, indole-positive Proteus species, and Serratia.
10 Also included within the definition of anti-infective agents are antifungal agents, amphotericin and the like as well as anti-viral agents such as famvir and acyclovir.
The chemokines described 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 multimeric KC ~SEQ ID
15 NO: 1], groa ISEQ ID NO: 2], gro~ [SEQ ID NO:3~ or human gror [SEQ ID NO: 4~ or their multimers (e.g., a dimeric, truncated gro~, amino acids 5-73 of SEQ ID NO:3) may be ~minictf~.red by injection in the dose range of about 10 to about ~0,000 fg/lcg/dose, or orally in the dose range of about 10 to about 10,000 fg/kg body weight per dose; if ~lminictered by infusion or similar techniques, the dose may be in the range of about 10 to 2~ bout 10,000 fg/lcg/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 ~lminictered for prophylactic and/or therapeutic treatments. In therapeutic application, the compound is ~lmini.ctered to a patient already suffering from a disease in an amount 25 sufficient to cure or at least partially arrest the disease and its complications. It may be given at any time after surgery, preferab~y prior to 24 hours after surgery. In prophylactic applications, a composition containing mature, modified or multimeric KC [SEQ ID NO:
1], groo~ [SEQ ID NO: 2], grol3 [SEQ ID NO:3] or gro~ [SE~Q ID NO: 4~ or a mnltim~r thereof, is ~lminictered to a patient not already in a disease state to enhance the patient's 30 resict~n~ e It may be given one day or one week prior to surgery, preferably one to two days prior to surgery. It may be ~ mini.e~ered pal~llL, ,ally or orally.
Single or rnultiple ~rlmini.ctrations 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 CA 02238l67 l998-05-20 W O 97/19173 PCTrUS96/18616 compounds of the invention sumcient to ~ffectiveiy treat the patient should be lmini.ct~red.
The chemokines useful in the methods of this invention, may also be administeredin conjunction with a separately administered conventional anti-infective as disclosed t S herein above, such a gentamicin, ~llgmr~ntin 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 norepinephrine, epinephrine, isoproterenol, dopamlne, and dobutamine; anti-inflslmm~tory agents such as methylprednisolone anti-infl~"""~t~ y agents such as indomethacin and phenylbutazone;
and corticosteroids such as bet~mPth~one, hydrocortisone, methylprednisolone, ordexamethasone; anti-coagulants such as heparin, anti-thrombin III or coumarin 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-l; phenothi~7ines; anti-hist~minf~c glucagon; a-adrenergic blocking agents, vasodilators; plasma expanders; packed red blood cells; platelets; cryoprecipitates;
fresh frozen plasma; bacterial permeability protein; clindamycin; and antibodies to (lipid A), the J5 mutant of E. coli or to endotoxin core glycolipids. Methods for preparing such antibodies are described 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 variety 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 mllltimf~ric KC [SEQ ID NO: 1], groa rSEQ ID
NO: 2~, gro~ LSEQ ID NO:3], or gro y ~SEQ ID NO: 4] both prophylactically and after infection provides a new and useful therapy of sepsis.
The biological activity of modified KC rSE~Q ID NO: 1~, modified human groL~
[SEQ ID NO: 3], modified human groy [SEQ ID NO: 4~, and a dimeric modified humangro~ are demonstrated by the following assays. These examples illustrate the preferred - methods of the invention. These examples do not limit the scope of the invention.

CA 02238167 1998-0~-20 W O97119173 PCT~US96/18616 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 ~ likitum.
Modified KC rSEQ ID NO: 1], modified human gro~ ~SEQ ID NO: 2] or modified human groy [SEQ ID NO: 3] or multimers thereof, was prepared in E. coli 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 10 on the same schedule. Starting two hours after infection the rats were treated twice daily with subcutaneous gentamicin.

E. coli. A clinical isolate of E. coli isolated from sputum was utilized. The organisms were tested for antibiotic sensitivity by the disc-agar diffusion technique and found to be 15 sensitive to gent~ in, ampicillin, cephalothin, chloramphenicol, kanamycin, tetracycline, trimethoprin/sulfamethoxazole and resistant to penicillin G, erythromycin, and vancomycin. The organism was animal passed in mice and sr-t sequently recovered and plated onto Mac~onkey'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, 20 organisms from thawed stocks wtre inoculated into brainheart infusion broth and incubated overnight on a rotary shaker (120 rpm~ an 37~C. The E. coli was harvested by centrifugation, 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 by scoring colony 25 forming units on MacConkeys agar.

Fibrin Clot. The E. coli infected fibrin clots were made from a l ~o solution of bovine fibrinogen (Type l-S, Sigma) in sterile saline. The clot was formed by adding sequentially human thrombin (Hanna Pharma.) bacteria, and fibrinogen solution to 24 well plastic 30 plates. Bacterial numbers of 2.0 to 3.0 x 109 were used in inoculate the fibrin clots. The resulting mixture was then incubated at room temperature for 30 minutes before implantation.

CA 02238167 1998-0~-20 W O 97/19173 PCT~US96/18616 Animal Model. The rats were anestheti~éd with kt~t~min~lxylazine (40 mg/kg/5 mg/lcg) after which the abdominal surfaced were shaved and a midline laparotomy performed.
Bacterial peritonitis was induced by implanting a fibrin-thrombin clot containing E. coli into the abdominal cavity. After implantation the muscle layers were closed with 4-0 silk 5 suturc, and the wound closed with surgical staples. The animals we}e closely observed, any animals obviously moribund were euth~ni7~-d ?

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 st7lti.~ti('~ y significant at pcO.05.
Example 1- Production oî Tr--nr~t~(l KC and c~o,a A. Expression of recombinant truncated KC and truncated gro~.
When truncated murine KC (amino acids 5-72 of SEQ ID NO:I) and human gro~ (amino acids 5-73 of SEQ ID NO:3) were expressed intracellularly in E. coli, 20 the KC (anlino acids 5-72 of SEQ ID NO: I ) retained the initiator Met. In order to produce the authentic N-terminal 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 grol3 (amino acids 5-73 of SEQ ID
NO:3) were each amplified by polymerase chain reaction (PCR) from plasmids containing 25 complimentary DNA sequences using both a forward primer encoding an NdeI site and a reverse primer containing an XbaI 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. coli LPlL-dependent expression vector pEAKn (pSKF301 derivative) between Ndel and XbaI sites. Each polypeptide was 30 expressed by chemical induction o~ the LPL promoter in a Iysogenic strain of E. coli containing the wild type (ind+) repressor gene (cI~) AR120.

CA 02238167 1998-0~-20 W O 97/19173 PCT~US96/18616 B. Purification and refoldin~ of truncated groJ3 (amino acids 5-73 of 3EQ ID
NO:3) E. coli cell pellet was Iysed in pH 6.0 buffer containing 20 mM
dithiothreitol (DTT) to avoid the nonspecific air oxidation. The majority of truncated grol3 S was in the insoluble Iysate pellet which was solubilized in 2 M GdnHCI, pH 8.0 buffer containing 20rnM DTT. The solubilized truncated groB was dialyzed against p3:16.0 buffer containing 2 n1M EDTA. The majority of E. coli proteins were precipitated during dialysis while truncated grol3 stayed in solution as a monomeric forrn at >95 % purity. The truncated groB solution was adjusted to pH 8.5, sti~Ted overnight for air oxidation. The 10 refolded truncated grol3 solution was adjusted to 0.1 % TFA solution, and applied to Ultrasphere C18 (Beckman) column to separate monomeric form from dimeric form. Each form was pooled separately, evaporaRd to remove acetonitrile, concentrated, dialyzed against PBS and stored at -70~.
C. Purification of truncated KC (amino acids 5-72 of SEQ ID NO~
~ET-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 Cl 8 RP-HPLC
as described above.
D. Characterization N-terminal sequencing and MALD-MS for molecular weight were performed and confirrned that the molecuaes are intact from N-terrninus to C-terrninus, either monomeric or dimeric fonn. Concentration o~ each chemokine was determined by amino acid analysis and endotoxin level of each prep was <0.05 U/rnl.
ExamPle 2 - Production of Truncated GRO~ Dimer A. Cell lysis E. coli LW cells, 400 g, were Iysed in 4 liters of Iysis buffer containing 50 mM sodium citrate pH 6.0, 40 mM NaCI, 2 mM EDTA, 5% glycerol, 0.05% Tween 80, 0.2 30 mM PMSF, I mgfml each of leupeptin and pepstatin A, by two passages through aMicrofluidics (model Ml lOY) homogenizer at 11,000 psi. The cell Iysate was centrifuged at 17,000 g (one hour at 4~C) and the supernatant was discarded.

CA 02238167 1998-0~-20 W O 97/19173 PCT~US96/18616 B. Solubilization and Refoldtng of Truncated GRO~ Dimer The insoluble truncated gro~ ~amino acids 5-73 of SEQ ID NO:3] in Iysate pellet was solubi}ized in 1.3 liters of buffer containing 50 mm Tris HCI pH 8.0, 2 M
gn~ni~linP HCI, 20 mM DTT by stirring overnight at room temperature. Soluble truncated groB [amino acids 5-73 of SEQ ID NO:3] was recovered by centrifugation at 25,000 g, from which guanidine HCI and DTT were removed by exhaustic dialysis against 50 mM
c sodium citrate pH 6.0 containing 2 mM EDTA to obtain soluble and reduced form of truncated groB [amino acids 5-73 of SEQ ID NO:3]. Truncated gro~ solution was concentrated to 3 mg/ml (Anicon YM3 membrane) and raised to pH 8.5 with 0.5 M Trizma base. Air oxidation of truncated grol3 [amino acids 5-73 of SEQ ID NO:3~ was performed by stirring overnight at room t~ e,~lul~. 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 adiusted to pH 8.0 with 10% acetic acid and the dimer captured on Toyopearl SP-650 M
equilibrated in 25 mM Tris HCI pH 8.0 (Buffer A). The column was washed with 4 liters buffer A, 2 liters 0.125 M NaCI in buffer A, and eluted with 4 liters of linear gradient of 0.125 - 0.5 M NaCI in buffer A. Flow rate was 40 ml/min. Truncated gro~ dimer was well separated from truncated groB and other oligomer form of truncated grol3 (SDS-PAGE).
Fractions containing truncaeed grol~ dimer were combined, adjusted to pH 3.0 with 10%
TFA solution and applied to Vydac C18 (2.1 x 25 cm) equilibrated with 0.1 % TFA in 4%
acetonitrile. Truncated gro~ dimer was eluted with linear gradient of 20-40% acetonitrile in 0.1 % TFA for 30 min. Truncated grol3 dimer was eluted at ~30% acetonitrile. Fractions containing truncated groB dimer was pooled, Iyophilized to remove acetonitrile, and dialyzed in Spectrapor 3K MWCO dialysis tubing against PBS.
D. Yield Typical yield of truncated groB dimer was 0.15 mg/g of cells 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 in(lic~ting that truncated gro~ dimer is a tlic--lfide linked dimer. The molecular weight of truncated gro~ dimer, as detellllhled by MALD-MS
analysis was 15,069 Da (predicted 15,073 Da), while that of truncated grol3 dimer was CA 02238167 1998-0~-20 W O 97/19173 PCTrUS96/18616 7,536 Da (predicted 7,537 Da). N-terminal sequencing of truncated gro~ dimer showed that 5-1()% of the final products retained the initiatory Met. Disulfide pairing pattern of truncated grof~ dimer was the same as that of truncated groB ~C5-C3 1, C7-C47) [amino acids 5-73 of SEQ ID NO:3~, however, all pairings were intermolecular rather than 5 intramolecular. Gel fi3tration analysis and ultracentrifugation sedimentation equilibrium studies in PBS (pH 7.0) showed that truncated gro~ dimer exhibited reversible assembly of octamer to hex~dec~m--r at 0.25 mg/m], while truncated grol3 [amino acids 5-73 of SEQ ID
NO:3] was a nonconvalent dimer even at 20 mg/ml. Concentration of truncated groB dimer has been determined by quantitative amino acid analysis.
10 Example 3 - Prophyl:~ s~ lly A~minict~ed l'runcated KC in E. coli Sepsis.
The animals were dosed i~ apeliL~neally with truncated KC [amino 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 15 the rats were implanted with an E. coli containing fibrin-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/lcg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone.
Results Dose (fE/k~) survival (alive/dead) Control 8 / 17 ExamPle 4 - Therapeutically ~lmi~ ed Truncated KC in E. coli sepsis.
On day 0 the rats were implanted with an E. coli containing ~lbrin-thrombin clot.
30 The animals were dosed inllal)eli~oneally with'truncated KC [amino acids 5-72 of SEQ ID
NO:I] 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 CA 02238l67 l998-0~-20 3 PCT~US96/18616 therapeutically treated with truncated KCiat 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 SDose ~f~ ) survival (alive / dead) Control 9 / 16 101,000 10/ 15 Example 5 - Therapen~ ly Administered Truncated KC in S. aureus Seps;s.
On day 0 the rats were implanted with a S. aureus con~:~ining fibrin-thrombin clot.
The animals were dosed h~L,aptliloneally 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.
15 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.
Res~lts Dose (fg/lcg) survival (aiive / dead) Control 8 / 17 l~xample 6 - Therapeutically A-lm;~ leled Tr-lne~te-l gron in E. coli Sepsis.
On day 0 the rats were implanted with an E. coli containing fibrin-thrombin clot.
The animals were dosed in~ldp~ oneally with truncated groJ~ ~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 trlmcated groB at 100 or 333 fg/kg followed by gentamicin W O 97119173 PCTAUS96/lB616 treatment demonstrated signif~cantly imp~oved survival rates over the diluent treated control rats receiving gentamicin therapy alone.
l~esults Dose (f~/kg) surviv~l (alive/~
Control 9 / 16 ExamPle 7 - Therapeutical1y ~(~minictered Tr-1nrs~tP~1 gron in S. aureus Sepsis.On day 0 the rats were implanted with an S. ~ureus containing fibrin-thrombin clot.
The animals were dosed intraperitoneally with truncated gro~ lamino 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.
15 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 ge.nt~mif~in treatment demonstrated signifcantly improved survival rates over the diluent treatèd control rats receiving gentamicin therapy alone.
20 Results Dose ~fp/kg) survival Control 9 / 16 1,000 10 / 15 ExamPle 8 - Therapeutical S~hc~ neo1~c1y ~ n;c~red Trunrate~l grol3 in E. coli Sepsis.
On day 0 the rats were implanted with an E. coli containing fibrin-thrombin clot.
The animals were dosed subcutaneously with truncated grol3 Lamino 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 sched~ . Starting two hours CA 02238167 1998-0~-20 W O 97tl9173 PCT~US96/18616 after infection the rats were treated twice daily with subcutaneous gentamicin. The rats therapeutically treated with truncated groB at 0.3 or 1.0 pglkg 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 0.3 181 7 1.0 20/ 5 3.3 11/14 Example 9 - Therapeutical Subcutaneously ~mini.et~red Tr~lnr~t~d gron 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 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 gent~mi~in. The rats 20 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.
Results Dosc (p~/lcg) survival (alive/dead) Control 8 / 17 0.1 13 / 12 0.3 18/ 7 1.0 20 / 5 3.3 12/13 Example 10 - P~ Lyl~ctic~l1y ~lmi~ t~ . ~,d GRO~ Dimer in E. coli Sepsis.
The animals were dosed subcutaneously with dimer formed of two truncated gro~
proteins lamino acids 5-73 of SEQ ID NO:3] at doses of 0.1, 0.3, 1.0 or 3.3 pg/kg 24 hours W O 97/19173 PCT~US96/18616 before infection. Control animals were doses with dilution buffer on the same schedule.
On day 0 the rats were implanted with an E. colc containing fibrin-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 S gentamicin Llc;aLlllellt demonstrated significantly improved survival rates over the diluent treated control rats receiving gentamicin therapy alone.
Results Dose (p~/kg) survival (alive/dead) Control 8 / 17 0.1 l0/ 15 0.3 18/ 7 1.0 20/ 5 3.3 8/17 15 ExamPle 11 - Therapeutically f~rlmi~ . ed GROh' Dinner in S. aureus Sepsis.
On day 0 the rats were implanted with an S. aureus containing fibrin-thrombin clot.
The animals were dosed subcut:~ne~ously with a dimer formed of two truncated groJ~
proteins lamino acids 5-73 of SEQ ID NO:3] at doses of 0.03, 0.1, 0.3, l .0, 3.3, or l0 pg/kg as a single inJection 2 hours after infection. Control animals were doses with dilution 20 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 grol3 dimer at 0.1, 0.3 or l.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/k~ survival (alive/dead) Control 11 ~ 14 0.03 12/ 13 0.1 l~/ 7 0.3 23l 2 1.0 24/ l 3.3 17/ 8 10.0 12/ 13 CA 02238167 1998-0~-20 W O 97119173 PCT~US96/18616 SEQUENCE LISTING
,~, (1) GENERAL INFORMATION:
(i) APPLICANT: SmithKline Beecham Corporation DeMarsh, Peter L.
Johanson, Kyung O.
(ii~ TITLE OF INVENTION: Method o~ 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) COM~U~l~: 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: P50417-l -(ixj TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5016 (B) TELEFAX: 610-270-5090 W O 97/19173 PCT~US96/18616 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUEMCE 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 Ile Ala Asn Glu Leu Arg Cys Gln Cys Leu Gln Thr Met Ala Gly Ile His Leu Lys Asn Ile Gln Ser Leu Lys Val Leu Pro Ser Gly Pro His Cys Thr Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Arg Glu Ala Cys Leu Asp Pro Glu Ala Pro Leu Val Gln Lys Ile Val Gln Lys Met Leu Lys Gly Val Pro Lys (2) INFORMATIOM 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 Gln Cys Leu Gln Thr ~eu Gln Gly Ile His Pro Lys Asn Ile Gln Ser Val Asn Val Lys Ser Pro Gly Pro His Cys Ala Gln Thr Clu Val Ile Ala Thr Leu Lys ~8 CA 02238167 1998-0~-20 Asn Gly Arg Lys Ala Cys Leu Asn Pro Ala Ser Pro Ile Val Lys Lys Ile Ile Glu Lys Met Leu Asn Ser Asp Lys Ser Asn (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 amino acids (B) TYPE: amino acid (C3 STRANDEDNESS:
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Pro Leu Ala Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu Gln Gly Ile His Leu Lys Asn Ile Gln Ser Val Lys Val Lys Ser Pro Gly Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Gln Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Lys Lys Ile Ile Glu Lys Met Leu Lys Asn Gly Lys Ser Asn (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 ~D NO:4:
Ala Ser Val Val Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu Gln Gly Ile His Leu Lys Asn I~e Gln Ser Val Asn Val Arg Ser Pro Gly Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Lys Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Gln Lys Ile Ile Glu Lys Ile Leu Asn Lys Gly Ser Thr Asn

Claims (22)

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) gro.alpha. SEQ ID NO: 2, (c) gro.beta. SEQ ID NO:3, and (d) gro.gamma. SEQ ID NO:4.

2. The method according to claim 1 wherein the chemokine is selected from the group consisting of:
(a) mature gro.beta.;
(b) modified gro.beta. consisting of amino acids 5 to 73 of SEQ ID NO: 3;
(c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b); and (d) a multimeric 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 gro.beta. proteins.

4. The method according to claim 1 wherein said chemokine is selected from the group consisting of:
(a) mature gro.alpha.;
(b) modified gro.alpha. consisting of amino acids 5 to 73 of SEQ ID
NO: 2;
(c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b); and (d) a multimeric 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 gro.gamma.;

(b) modified gro.gamma. consisting of amino acids 5 to 73 of SEQ ID
NO: 4;
(c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b); and (d) a multimeric 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: 1;
(c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b); and (d) a multimeric 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.

11. The method according to claim 1 further comprising the step of administering the chemokine in conjunction with an effective amount of an anti-infective agent.

12. A method according to claim 11 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) gro.alpha. SEQ ID
NO: 2, (c) gro.beta. SEQ ID NO:3, and (d) gro.gamma. SEQ ID NO:4.

14. The method according to claim 13 wherein the chemokine is selected from the group consisting of:
(a) mature gro.beta.;
(b) modified gro.beta. consisting of amino acids 5 to 73 of SEQ ID NO: 3;
(c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b); and (d) a multimeric 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 gro.beta. proteins.

16. The method according to claim 13 wherein said chemokine is selected from the group consisting of:
(a) mature gro.alpha.;
(b) modified gro.alpha. consisting of amino acids 5 to 73 of SEQ ID
NO: 2;
(c) a multimeric chemokine protein which comprises an association of two o} more of (a) or (b); and (d) a multimeric 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 gro.gamma.;

(b) modified gro.gamma. consisting of amino acids 5 to 73 of SEQ ID
NO: 4;
(c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b); and (d) a multimeric 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 multimeric chemokine protein which comprises an association of two or more of (a) or (b); and (d) a multimeric 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 fglkg/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-infective agent is selected from the group consisting of gentamicin, augmentin or ceftazidime.