WO1993025230A1 - Prophylaxie et traitement de la septicemie et des troubles de coagulation associes a la septicemie - Google Patents

Prophylaxie et traitement de la septicemie et des troubles de coagulation associes a la septicemie Download PDF

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WO1993025230A1
WO1993025230A1 PCT/US1993/005409 US9305409W WO9325230A1 WO 1993025230 A1 WO1993025230 A1 WO 1993025230A1 US 9305409 W US9305409 W US 9305409W WO 9325230 A1 WO9325230 A1 WO 9325230A1
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sepsis
laci
tfpi
factor
patient
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PCT/US1993/005409
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English (en)
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George J. Broze
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G.D. Searle & Co.
Monsanto Company
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Priority to KR1019940704533A priority Critical patent/KR950701820A/ko
Priority to AU44084/93A priority patent/AU4408493A/en
Publication of WO1993025230A1 publication Critical patent/WO1993025230A1/fr
Priority to NO944714A priority patent/NO944714L/no
Priority to DK140594A priority patent/DK140594A/da

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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
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to prophylaxis and treatment of sepsis and sepsis-associated coagulation disorders such as disseminated intravascular coagulation (DIC) due to sepsis. More particularly, the present invention relates to prophylaxis and treatment of sepsis, particularly gram negative sepsis, or sepsis-associated disorders, such as DIC due to sepsis, by administering a therapeutically-effective amount of lipoprotein-associated coagulation inhibitor (LACI) to a patient afflicted with or susceptible to sepsis or sepsis- associated disorders.
  • LACI lipoprotein-associated coagulation inhibitor
  • LACI Lipoprotein-associated coagulation inhibitor
  • TF tissue factor
  • Factor III tissue thromboplastin
  • EPI extrinsic pathway inhibitor
  • TFPI tissue factor pathway inhibitor
  • Blood coagulation means the conversion of fluid blood to a solid gel or clot.
  • the main event is the conversion of soluble fibrinogen to insoluble strands of fibrin, although fibrin itself forms only 0.15% of the total blood clot.
  • This conversion is the last step in a complex enzyme cascade.
  • the components (factors) are present as zymogens, inactive precursors of proteolytic enzymes, which are converted into active enzymes by proteolytic cleavage at specific sites. Activation of a small amount of one factor catalyses the formation of larger amounts of the next, and so on, giving an amplification which results in an extremely rapid formation of fibrin.
  • the coagulation cascade which occurs in mammalian blood is divided by in vitro methods into an intrinsic system (all factors present in the blood) and an extrinsic system which depends on the addition of thromboplastin.
  • the intrinsic pathway commences when the first zymogen, factor XII or ⁇ ageman Factor', adheres to a negatively charged surface and in the presence of high molecular weight kininogen and prekallikrein, becomes an active enzyme, designated Xlla.
  • the activating surface may be collagen which is exposed by tissue injury.
  • Factor Xlla activates factor XI to give Xla
  • factor Xla activates factor IX to IXa and this, in the presence of calcium ions, a negatively charged phospholipid surface and factor Villa, activates factor X.
  • the negatively charged phospholipid surface is provided by platelets and in vivo this serves to localize the process of coagulation to sites of platelet deposition.
  • Factor Xa in the presence of calcium ions, a platelet-derived negatively charged phospholipid surface and a binding protein, factor V, activates prothrombin to give thrombin (lla) - the main enzyme of the cascade.
  • Thrombin acting on gly-arg bonds, removes small fibrinopeptides from the N-terminal regions of the large dimeric fibrinogen molecules, enabling them to polymerize to form strands of fibrin.
  • Thrombin also activates the fibrin stabilizing factor, factor XIII, to give Xllla, a fibrinoligase, which, in the presence of calcium ions strengthens the fibrin-to-fibri ⁇ links with intermolecular ⁇ -glutamyl- ⁇ -lysine bridges.
  • thrombin acts directly on platelets to cause aggregation, and release of subcellular constituents and arachidonic acid.
  • a further function of thrombin is to activate the coagulation inhibitor, protein C.
  • Factors Xlla, Xla, IXa, Xa, and thrombin are all serine proteases.
  • the extrinsic pathway in vivo is initiated by a substance generated by, or exposed by, tissue damage and termed 'tissue factor', interacting with Factor VII in the presence of calcium ions and phospholipid to activate factors X and IX, after which the sequence proceeds as already described.
  • tissue factor occurs in the plasma membranes of perturbed endothelial cells of blood vessels and also in atheromatous plaques.
  • Sepsis is a toxic condition resulting from the spread of bacteria, or their products (collectively referred to herein as bacterial endotoxins) from a focus of infection.
  • Septicemia is a form of sepsis, and more particularly is a toxic condition resulting from invasion of the blood stream by bacterial endotoxins from a focus of infection.
  • Sepsis can cause shock in many ways, some related to the primary focus of infection and some related to the systemic effects of the bacterial endotoxins.
  • septacemia bacterial endotoxins, along with other cell-derived materials, such as IL-1 , IL-6 and TNF, activate the coagulation system and initiate platelet aggregation. The process leads to blood clotting, a drop in blood pressure and finally kidney, heart and lung failure.
  • Activation of the coagulation cascade by bacterial endotoxins introduced directly into the bloodstream can result in extensive fibrin deposition on arterial surfaces with depletion of fibrinogen, prothrombin, factors V and VIII, and platelets.
  • the fibrinolytic system is stimulated, resulting in further formation of fibrin degradation products.
  • Disseminated intravascular coagulation (DIC) is a complex coagulation disorder resulting from widespread activation of the clotting mechanism or coagulation cascade which, in turn, results from septicemia.
  • DIC Disseminated intravascular coagulation
  • the process represents conversion of plasma to serum within the circulation system. Such process represents one of the most serious acquired coagulation disorders.
  • Intravascular Coagulation Effect on Mortality and on Correction of Hemostatic Defects", N. En ⁇ l. J. Med.. 283, 778- 782 (1970); Lasch et al., Heparin Therapy of Diffuse Intravascular Coagulation (DIC)", Thrombos. Diathes. Haemorrh.. 33, 105 (1974); Straub, "A Case against Heparin Therapy of Intravascular Coagulation", Thrombos. Diathes. Haemorrh.. 33, 107 (1974).
  • DI Diffuse Intravascular Coagulation
  • Wun et al, EP 473,564 discloses that a combination of LACI and heparin can be used to inhibit both the intrinsic and extrinsic pathways of coagulation and therefore may be effective for treatment of DIC.
  • Nordfang [W091/19514, published Dec. 26, 1991] discloses combining heparin with EPI and suggests that such combination would be effective to treat coagulation disorders or cancer.
  • factor X a derivative for treatment of DIC due to sepsis Such derivative was prepared by blocking factor X a in the active center with [5-(dimethylamino) 1 -naphthalene-sulfonyl]- glutamylglycylarginyl chloromethyl ketone.
  • factor X a in the active center with [5-(dimethylamino) 1 -naphthalene-sulfonyl]- glutamylglycylarginyl chloromethyl ketone.
  • the inflammatory, coagulant, and cell injury responses to E. coli of both the treated and control groups were lethal.
  • the present invention is directed to a method of using LACI comprising administering LACI to a patient afflicted with or susceptible to sepsis. More particularly, the present invention is directed to a method for prophylaxis or treatment of sepsis or sepsis-associated coagulation disorders comprising administering to a patient in need thereof a therapeutically-effective amount of LACI.
  • a preferred embodiment is a method for treatment or prophylaxis of disseminated intravascular coagulation comprising administering to a patient susceptible to or afflicted with sepsis a therapeutically-effective amount of LACI.
  • a most preferred embodiment is a method for treatment or prophylaxis of sepsis comprising administering to a patient susceptible to or afflicted with sepsis a therapeutically-effective amount of LACI.
  • the present invention is directed to a method of using LACI which comprises administering to a patient susceptible to or afflicted with sepsis a therapeutically- effective amount of LACI.
  • the invention is directed to a method for treatment or prophylaxis of sepsis- associated coagulation disorders.
  • the term "sepsis" means a toxic condition resulting from the spread of bacterial endotoxins from a focus of infection.
  • sepsis-associated coagulation disorder means a disorder resulting from or associated with coagulation system activation by a bacterial endotoxin, a product of such bacterial endotoxin or both.
  • An example of such sepsis-associated coagulation disorder is disseminated intravascular coagulation.
  • a therapeutically-effective amount means an amount necessary to permit observation of activity in a patient sufficient to alleviate one or more symptoms generally associated with sepsis. Such symptoms include, but are not limited to, death, increased heart rate, and increased respiration, decreased fibrinogen levels, decreased blood pressure, decreased white cell count and decreased platelet count.
  • a therapeutically- effective amount is an amount necessary to attenuate a decrease in fibrinogen levels in a patient being treated.
  • LACI means one or more of the three Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor which are active in treating sepsis.
  • LACI is aiso known as tissue factor pathway inhibitor (TFPI). This name has been accepted by the International Society on Thrombosis and Hemostasis, June 30, 1991 , Amsterdam.
  • TFPI tissue factor pathway inhibitor
  • This name has been accepted by the International Society on Thrombosis and Hemostasis, June 30, 1991 , Amsterdam.
  • TFPI was first purified from a human hepatoma cell, Hep G2, as described by Broze and Miletich; Proc. Natl. Acad. Sci. USA 84. 1886-1890 (1987), and subsequently from human plasma as reported by Novotny et al., J. Biol. Chem. 264.
  • TFPI cDNA have been isolated from placental and endothelial cDNA libraries as described by Wun et al., J. Biol. Chem. 263. 6001 -6004 (1988); Girard et al., Thromb. Res. 55. 37-50 (1989).
  • the primary amino acid sequence of TFPI deduced from the cDNA sequence, shows that TFPI contains a highly negatively charged amino-terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus.
  • the first Kunitz-domain of TFPI is needed for the inhibition of the. factor Vila/tissue factor complex and the second Kunitz-domain of TFPI is responsible for the inhibition of factor Xa according to Girard et al., Nature 328. 518-520 (1989), while the function of the third Kunitz-domain remains unknown. See also U.S. 5,106,833.
  • TFPI is believed to function in vivo to limit the initiation of coagulation by forming an inert, quaternary factor X a : TFPI: factor Vll a : tissue factor complex. Further background information on TFPI can be had by reference to the recent reviews by Rapaport, Blood 73. 359-365 (1989); Broze et al., Biochemistry 29. 7539-7546 (1990).
  • Recombinant TFPI has been expressed as a glycosylated protein using mammalian cell hosts including mouse C127 cells as disclosed by Day et al., Blood 76. 1538- 1545 (1990), baby hamster kidney cells as reported by Pedersen et al.. J. Biol. Chem. 265. 16786-16793 (1990), Chinese hamster ovary cells and human SK hepatoma cells.
  • the C127 TFPI has been used in animal studies and shown to be effective in the inhibition of tissue factor-induced intravascular coagulation in rabbits according to Day et al., su p ra, and in the prevention of arterial reocclusion after thrombolysis in dogs as described by
  • Recombinant TFPI also has been expressed as a non- glycosylated protein using E. coli host cells and obtaining a highly active TFPI by in vitro folding of the protein as described below in Example 1.
  • LACI utilized in the present invention is preferably obtained from E. coli as disclosed below in Example 1.
  • LACI can be obtained from cells as disclosed.
  • This example illustrates a method for obtaining LACI (TFPI).
  • X8 cation exchanger was purchased from Bio Rad. Mono Q HR 5/5 and HiLoad Q Sepharose anion exchange resins, and Mono S HR 5/5 and Mono S HR 10/16 cation exchange resins were obtained from Pharmacia. Thromboplastin reagent (Simplastin Excel) was from Organon Teknika Corp. Bovine factor X a and
  • Spectrozyme X a were supplied by American Diagnostica, Inc. SDS-PAGE 10-20% gradient gel was obtained from integrated Separation Systems.
  • Maelll site is 15bp downstream from the stop codon in the TFPI cDNA.
  • the expression vector contained the recA promoter, a translational enhancer element and ribosome binding site derived from the gene 10 leader of bacteriophage T7 as described by Oiins and Rangwala, J. Biol. Chem. 264. 16973-16976 (1989), and the T7 transcription terminator.
  • This plasmid also contains an irrelevant sequence, i.e. the bST gene (bovine somatotropin).
  • the Ncol/Nsil fragment of pMON9308 was then replaced by a synthetic DNA fragment designed to (1 ) introduce an alanine encoding codon at the second position, (2) increase the A-T richness of the 5' portion of the gene, and (3) improve £. coli codon usage.
  • ECTFPI 2 and 3 were 5' phosphorylated [Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)].
  • ECTFPI 1 and 2 and ECTFPI 3 and 4 were annealed in the kinase buffer by incubating for 5 minutes at 70°C and slow-cooling to room temperature. These fragments were cloned into pMON9308 which had been digested with Ncol/Nsil. PCR amplification was used to introduce a Hindlll site as well as a TAA termination codon at the 3' end of the TFPI gene. The PCR primers TPFIterm and TPFIterm 2 are shown below. The TFPI gene was then moved as a NCOI/Hindlll fragment into pMON5766. The resultant plasmid was pMON6870.
  • Hindlll TFPIterm ataaca[aagctt]acatatttt
  • Ncol TFPIterm2 atatat[ccatgg]ctgattct
  • pMON6870 was digested with Bglll/Hindlll. This fragment, containing the expression cassette, was cloned into pMON6710 [Obukowicz et al., Biochemistry 29, 9737-9745 (1990)] which had been digested with Bglll/Hindlll.
  • the resultant plasmid, pMON6875 includes the tac promoter, G10 leader from bacteriophage T7, met-ala TFPI, and the p22 transcriptional terminator.
  • the plasmids were transformed into MON105 (rpoD + rpoH358) containing F from JM101 for the expression of TFPI protein.
  • the culture was harvested four hours post-induction by concentration in an Amicon DC10L concentrator followed by centrifugation in a Beckman J2-21 centrifuge.
  • the cell paste was frozen at -80°C for further processing hereinbeiow.
  • Milli-Q water at a concentration of 75 g/l.
  • the cells were thoroughly dispersed with a homogenizer (Ultra-Turrax model SD-45) for 30 minutes on ice.
  • the cells were mechanically lysed by three passes through the Manton-Gaulin homogenizer (model 15M-8TA) at 12,000 psi.
  • inclusion bodies were centrifuged in the Sorvall RC-2B centrifuge in the GSA rotor at 10,000 rpm (16,270 x g) for 20 minutes. The supernatant was discarded.
  • the inclusion body pellets were collected, resuspended in I liter of cold Milli-Q water and dispersed with the Ultra-Turrax homogenizer for 30 minutes on ice.
  • inclusion bodies were cycled through the Manton-Gaulin homogenizer two more times on ice. Inclusion bodies were pelleted in the Sorvall RC-2B centrifuge as before. Approximately 60 mg of inclusion bodies were collected for every gram of E. coli cells lysed. The inclusion bodies were stored at -80°C.
  • All the buffers used for sulfonation and refolding of E. coli TFPI contained high concentrations of urea.
  • Urea solutions were treated with Bio-Rad mixed bed resin AG501 -X8 at room temperature for at least 20 minutes and filtered through 0.2 ⁇ m filter before mixing with buffers. All the solutions used for chromatography were 0.2 ⁇ m filtered and sonicated under house vacuum for about 10 minutes.
  • inclusion bodies was dispersed in 40 ml of a solution containing 50 mM Tris/HCI, pH 8, and 7.5 M urea by homogenization and vortexing. After the inclusion bodies were largely dissolved, 800 mg of sodium sulfite was added and the mixture was shaken at room temperature for 30 minutes. Then, 400 mg of sodium dithionite or 120 mg of sodium tetrathionate was added and the mixture was shaken at 4°C overnight. The solution dialyzed against 800 ml of a solution containing 20 mM Tris/HCI, pH 8, and 6 M urea for more than 5 hours at 4°C using a Spectrapor #2 membrane. The dialyzed solution was centrifuged at 48,400 x g for 1 hour, filtered through a 0.2 ⁇ m filter, divided into aliquots, and stored at
  • the sulfonated and dialyzed inclusion bodies were fractionated on a Mono Q HR5/5 anion exchange column.
  • the column was pre-equilibrated in Q-buffer (20 mM Tris/HCI, pH 8, 6 M urea, 0.01% Brij 35 non-ionic surfactant) containing 0.15 M NaCl.
  • Q-buffer 20 mM Tris/HCI, pH 8, 6 M urea, 0.01% Brij 35 non-ionic surfactant
  • Two ml of sulfonated inclusion bodies were loaded onto the column.
  • the column was washed with 15 ml of the equilibration buffer and eluted with a 30-ml gradient (0.15-0.4 M NaCl) in Q-buffer. Fractions of 1 ml were collected.
  • the sulfonated, full-length TFPI pool from anion- exchange chromatography was diluted to an absorbance of 0.07 O.D. units at 280 nm with Q-buffer containing 0.3 M NaCl.
  • Solid L-cysteine was added to a final concentration of 2 mM.
  • the solution was incubated at room temperature for 24 hours, diluted 1 :1 with water, 1 mM L-cysteine was added, incubated at room temperature for another 24 hours and then incubated at 4°C for up to 4 to 8 days. pH was maintained at 8.5 by addition of 50 mM Tris.
  • Inhibitory activity against bovine factor X a of TFPI samples were assayed by conventional amidolysis of Spectrozme X a as described previously by Wun et al., J. Biol. Chem. 265. 16096-16101 (1990) except that the assay buffer consisted of 0.1 M Tris/HCI, pH 8.4, and 0.1% Triton X-100 non ⁇ ionic surfactant.
  • the concentration of protein was determined by absorbance at 280 nm and by quantitative amino acid analysis after HCI/vapor phase hydrolysis at 110°C for 24 hours.
  • Daiichi precasted 10-20% gradient gels were used for SDS-PAGE. Samples are either unreduced and not boiled or reduced in 3.3% 2-mercaptoethanol and boiled for 3 minutes before electrophoresis. The gels were stained by Coomassie blue.
  • the first construct which contained the original human TFPI cDNA sequence (except the initiating ATG) and the rec A promoter, achieved a very low level of expression ( ⁇ 0.5% of total cell protein).
  • the second construct, pMON6870 which was similar to the first but was altered by introducing an alanine at the second position, by increasing the A-T richness of the 5'-end and by improving E ⁇ coli codon usage, did not significantly raise the expression level.
  • the third construct pMON6875 which was similar to the second but used a tac promoter, achieved an expression level of approximately 5-10% of total cell protein and was used for further tests herein. The majority of TFPI (>90%) appeared to be sequestered in inclusion bodies.
  • the flow- through and early gradient fractions contained much of the contaminants E. coli protein and truncated TFPI protein (the latter are lower in molecular weight and are immuno-reactive against anti-TFPI-lg).
  • the full-length TFPI-S-sulfonate eluted at about 0.28 M NaCl.
  • the fractionation of sulfonated inclusion bodies was scaled up 20 times using a Hiload Q Sepharose
  • TFPI Sulfonated TFPI underwent spontaneous refolding and oxidation upon mixing with a suitable concentration of L-cysteine.
  • the efficiency of refold as reflected in the increase of TFPI activity varies widely depending on the refold conditions. Numerous refold conditions were compared and optimized in terms of temperature, pH, urea, L-cysteine and protein concentration. A 2-stage refold process appeared to be the best.
  • the full-length TFPI-S-sulfonate pool was adjusted to an absorbance at 280 nm of 0.07 O.D. units, 2 mM of fresh L-cysteine was added, and the mixture was incubated at room temperature for 24 hours. During this period, the TFPI activity increased from 0 to about 12% of full-length SK hepatoma TFPI which served as a standard for comparison.
  • the solution was diluted 1 :1 with water, and fresh L-cysteine was added to a final concentration of 1 mM.
  • the mixture was incubated at room temperature for 24 hours, during which time the specific activity increased about 2 fold to about 30% that of SK Hepatoma TFPI.
  • the solution was then left at 4°C for several days during which time the TFPI activity increased.
  • the specific activity of the refold mixture was lower than the purified mammalian SK TFPI which suggests that the former may contain both correctly folded and misfolded molecules or only partially active misfolded molecules.
  • the refold mixture was fractionated on an analytical Mono S cation exchange column. When the UV-absorbing fractions were analyzed for TFPI activity, the highest specific activity was associated with a sharp peak (fraction 52) eluted at 0.52 M NaCl. All the other fractions had a specific activity less than 30% that of fraction 52. SDS-PAGE analysis showed that fraction 52 contained a sharp band and all other fractions, together with pre-column refold mixture, consisted of diffuse, multiple bands under nonreducing condition.
  • the diffuse bands are apparently mainly full-length TFPI in various folded forms since they become sharp-banded upon reduction (see the last two lanes on the right).
  • the resolution of the peaks become better and all the protein peaks appeared to elute at lower NaCl concentrations Further, it was possible to wash out the majority of the low-activity peaks with 10 column volumes of 0.3 M NaCl before eluting the active peak with a shadow gradient.
  • the ability of the active, refolded E. coli TFPI to inhibit tissue factor-induced coagulation in human plasma was compared with that of the purified SK Hepatoma TFPI.
  • the activity of the E. coli TFPI was approximately two fold more active than SK Hepatoma TFPI on a per mol basis as judged from the concentrations of each TFPI that produce the same prolongation of clotting time.
  • SK unit is defined as the amount of activity equivalent to that produced by 1 mA (1x10 -3 absorbance unit at 280 nm or 1.31 ug) of purified full-length SK hepatoma TFPI.
  • This example illustrates the effectiveness of using LACI to treat patients susceptible to or afflicted with sepsis.
  • this example illustrates the effectiveness of using LACI to treat a sepsis-associated coagulation disorder, namely, DIC.
  • Recombinant TFPI was expressed as a glycosylated protein using mouse C127 cells as host and was purified by chromatography on a monoclonal antibody convalentiy attached to Sepharose 4B as described by Day et al. [Blood 76, 1 538(1 990)].
  • Baboons 9 month of age weighing 9-13.9 kg, were randomly selected for LACI or excipient pretreatment (1 hr or 15 min) protocol. Each baboon is immobilized with ketamine hydrochloride, 14 mg/kg intramuscularly on the morning of the study and slowly anesthetized with sodium pentobarbital ( ⁇ 9 mg/kg) via a percutaneous catheter positioned in the cephalic vein and brachial vein.
  • the femoral artery and one femoral vein are cannulated aseptically to measure aortic pressure, obtain blood samples, and for infusion of LACI, live organisms, isotonic sodium chloride and sodium pentobarbital.
  • Group 1 Excipient control (4.1 x 10 1 0 cfu/kg)
  • Group 2 LACI (3.8 x 10 1 0 cfu/kg)
  • Group 3 Excipient control (5.4 x 10 1 0 cfu/kg)
  • Group 4 LACI (4.9 x 10 1 0 cfu/kg)
  • Group 1 Excipient control (4.1 x 10 10 cfu/kg)
  • Group 2 LACI (3.8 x 10 10 cfu/kg)
  • Group 3 Excipient control (5.4 x 10 10 cfu/kg)
  • Group 4 LACI (4.9 x 10 0 cfu/kg)
  • LACI to promote survival in patients which are susceptible to or afflicted with sepsis.
  • this example illustrates the effectiveness of using LACI to treat gram-negative sepsis.
  • LACI was prepared by the method described above in Example 1.
  • Each baboon was immobilized with ketamine hydrochloride, 14 mg/kg intramuscularly on the morning of the study and slowly anesthetized with sodium pentobarbital (-9 mg/kg) via a percutaneous catheter positioned in the cephalic vein.
  • the animals were infused with isotonic saline at a rate of 3.3 ml/kg/hr for 12 hours via the barachial vein in the right leg.
  • LACI or PBS buffer control was administered to the animals through the brachial vein 30 minutes after the administration of bacteria.
  • LACI was administered at a loading dose over fifteen minutes and simultaneously started a continuous infusion of LACI for an additional 675 minutes (counting from start of bacterial infusion, which was defined as time zero).
  • E. coli 086 K61 H were used to inoculate tryptic soy broth agar (2); viability counts of the inoculum were determined by standard dilution techniques. At time zero, baboons received an infusion of 4.5 x 10 1 0 live bacteria per kg body weight (4 mls/kg), administered through a percutaneous catheter in the right cephalic vein by continuous infusion for 2 hours.
  • the femoral artery and one femoral vein were cannulated aseptically to measure mean systemic arterial pressure, obtain blood samples and for antibiotic administaration.
  • Gentamicin was given (9 mg/kg i.v.) at end of E. coli infusion, i.e., at T + 120 min. for 30 minutes and then 4.5 mg/kg at T + 300 min . . and T + 540 minutes for 30 min.
  • Gentamicin (4.5 mg/kg IM) was then given at the end of the experiment and once daily for 3 days. Animals were maintained un ⁇ er anaesthesia and monitored continuously for 12 hours. Blood samples were collected hourly for hematology, clinical chemistry, cytokines (TNF, IL-6) and LACI determinations. Similarly, respiration rate, heart rate, mean systemic arterial pressure and temperature were monitored hourly.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration.
  • the dosage regimen is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient, the severity of the condition, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles, whether a drug delivery system is utilized and whether the compound is administered as part of a drug combination.
  • the dosage regimen actually employed may vary widely and therefore may deviate from the preferred dosage regimen set forth above.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in water.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or digiycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • a preferred injectable preparation solution is LACI in an aqueous solution of 150 mM sodium chloride and 20 mM sodium phosphate.
  • LACI can be administered as the sole active anticoagulation pharmaceutical agent, it can also be used in combination with one or more antibodies useful for treating sepsis, such as, for example, anti-endotoxin, monoclonal antibodies (endotoxin-binding Mabs) and anti-TNF products such as an anti-TNF murine Mab.
  • LACI can also be combined with interleukin-1 receptor antagonists, bactericidal/permeability increasing (BPI) protein, immunostimulant, compounds having anti-inflammatory activity, such as PAF antagonists and cell adhesion blockers.
  • the therapeutic agents can be formulated as separate compositions which are given at the same time or different times, or the therapeutic agents can be given as a single composition.

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Abstract

L'invention décrit un procédé d'utilisation d'un inhibiteur de la coagulation associé à une lipoprotéine LACI, dont on administre une quantité efficace thérapeutiquement à un malade souffrant de septicémie.
PCT/US1993/005409 1992-06-11 1993-06-11 Prophylaxie et traitement de la septicemie et des troubles de coagulation associes a la septicemie WO1993025230A1 (fr)

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KR1019940704533A KR950701820A (ko) 1992-06-11 1993-06-11 패혈증 및 패혈증-연관 옹고장애의 예방 및 치료(prophylaxs and treatment of sepsis and sepsis-associated coagulation disorders)
AU44084/93A AU4408493A (en) 1992-06-11 1993-06-11 Prophylaxis and treatment of sepsis and sepsis-associated coagulation disorders
NO944714A NO944714L (no) 1992-06-11 1994-12-07 Profylakse og behandling av sepsis og sepsis-assosierte koagulasjonsforstyrrelser
DK140594A DK140594A (da) 1992-06-11 1994-12-08 Forebyggelse og behandling af sepsis og sepsis-forbundne koagulationsforstyrrelser

Applications Claiming Priority (4)

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US89713592A 1992-06-11 1992-06-11
US07/897,135 1992-06-11
US2047293A 1993-02-22 1993-02-22
US08/020,472 1993-02-22

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WO1993025230A1 true WO1993025230A1 (fr) 1993-12-23

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AU (1) AU4408493A (fr)
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WO (1) WO1993025230A1 (fr)

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EP0559632A2 (fr) * 1992-03-02 1993-09-08 Monsanto Company Procédé pour la production de l'inhibiteur du sentier du facteur de tissu
WO1996001649A1 (fr) * 1994-07-07 1996-01-25 Washington University Procede permettant d'attenuer les stenoses arterielles
WO1996004378A2 (fr) * 1994-08-05 1996-02-15 Chiron Corporation Proteines chimeriques et muteines des inhibiteurs des voies du facteur tissulaire de la coagulation tfpi et tfpi-2
US5772629A (en) * 1995-10-23 1998-06-30 Localmed, Inc. Localized intravascular delivery of TFPI for inhibition of restenosis in recanalized blood vessels
US5824644A (en) * 1994-07-07 1998-10-20 G. D. Searle & Co. Method of attenuating arterial stenosis
US5888968A (en) * 1995-06-07 1999-03-30 Chiron Corporation TFPI formulation
US5914316A (en) * 1994-12-16 1999-06-22 Washington University Method of inhibiting intimal hyperplasia
US6063764A (en) * 1992-06-01 2000-05-16 Washington University & Chiron Corp. Method for using lipoprotein associated coagulation inhibitor to treat sepsis
US6242414B1 (en) 1995-06-07 2001-06-05 Chiron Corporation Regulation of cytokine synthesis and release
AU759412B2 (en) * 1995-06-07 2003-04-17 G.D. Searle & Co. Method of solubilizing, purifying and refolding protein and compositions comprising proteins and solubilizing agents
US6939716B2 (en) 2001-09-19 2005-09-06 Washington University Method for detecting conditions indicative of sepsis
US7022672B2 (en) 1995-06-07 2006-04-04 Chiron Corporation Formulation, solubilization, purification, and refolding of tissue factor pathway inhibitor
AU2003200506B2 (en) * 1995-06-07 2007-01-18 G.D. Searle Llc Method of solubilizing, purifying and refolding protein and compositions comprising proteins and solubilizing agents
US7662774B2 (en) 1992-06-01 2010-02-16 Novartis Vaccines And Diagnostics, Inc. Method for using lipoprotein associated coagulation inhibitor to treat sepsis
US7674769B2 (en) 2001-10-15 2010-03-09 Novartis Vaccines And Diagnostics, Inc. Treatment of severe pneumonia by administration of tissue factor pathway inhibitor (TFPI)
EP2206785A1 (fr) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Expression améliorée de polypeptides HIV et production de particules de type virus
US7851433B2 (en) 2003-08-13 2010-12-14 Novartis Vaccines And Diagnostics, Inc. Method of purifying TFPI and TFPI analogs

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WO1991019514A1 (fr) * 1990-06-19 1991-12-26 Novo Nordisk A/S Preparation anticoagulante

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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0559632A2 (fr) * 1992-03-02 1993-09-08 Monsanto Company Procédé pour la production de l'inhibiteur du sentier du facteur de tissu
EP0559632A3 (fr) * 1992-03-02 1995-03-29 Monsanto Co
US7662774B2 (en) 1992-06-01 2010-02-16 Novartis Vaccines And Diagnostics, Inc. Method for using lipoprotein associated coagulation inhibitor to treat sepsis
US6063764A (en) * 1992-06-01 2000-05-16 Washington University & Chiron Corp. Method for using lipoprotein associated coagulation inhibitor to treat sepsis
WO1996001649A1 (fr) * 1994-07-07 1996-01-25 Washington University Procede permettant d'attenuer les stenoses arterielles
US5824644A (en) * 1994-07-07 1998-10-20 G. D. Searle & Co. Method of attenuating arterial stenosis
US5589359A (en) * 1994-08-05 1996-12-31 Chiron Corporation Chimeric proteins
WO1996004378A3 (fr) * 1994-08-05 1996-03-14 Chiron Corp Proteines chimeriques et muteines des inhibiteurs des voies du facteur tissulaire de la coagulation tfpi et tfpi-2
WO1996004378A2 (fr) * 1994-08-05 1996-02-15 Chiron Corporation Proteines chimeriques et muteines des inhibiteurs des voies du facteur tissulaire de la coagulation tfpi et tfpi-2
US5563123A (en) * 1994-08-05 1996-10-08 Chiron Corporation Chimeric proteins
US6783960B2 (en) 1994-08-05 2004-08-31 Chiron Corporation Chimeric proteins
US5696088A (en) * 1994-08-05 1997-12-09 Chiron Corporation Chimeric proteins
US5914316A (en) * 1994-12-16 1999-06-22 Washington University Method of inhibiting intimal hyperplasia
US6242414B1 (en) 1995-06-07 2001-06-05 Chiron Corporation Regulation of cytokine synthesis and release
AU759412B2 (en) * 1995-06-07 2003-04-17 G.D. Searle & Co. Method of solubilizing, purifying and refolding protein and compositions comprising proteins and solubilizing agents
US5888968A (en) * 1995-06-07 1999-03-30 Chiron Corporation TFPI formulation
US7022672B2 (en) 1995-06-07 2006-04-04 Chiron Corporation Formulation, solubilization, purification, and refolding of tissue factor pathway inhibitor
AU2003200506B2 (en) * 1995-06-07 2007-01-18 G.D. Searle Llc Method of solubilizing, purifying and refolding protein and compositions comprising proteins and solubilizing agents
US7226757B2 (en) 1995-06-07 2007-06-05 G.D. Searle Llc Formulation, solubilization, purification, and refolding of tissue factor pathway inhibitor
US5772629A (en) * 1995-10-23 1998-06-30 Localmed, Inc. Localized intravascular delivery of TFPI for inhibition of restenosis in recanalized blood vessels
EP2206785A1 (fr) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Expression améliorée de polypeptides HIV et production de particules de type virus
US6939716B2 (en) 2001-09-19 2005-09-06 Washington University Method for detecting conditions indicative of sepsis
US7674769B2 (en) 2001-10-15 2010-03-09 Novartis Vaccines And Diagnostics, Inc. Treatment of severe pneumonia by administration of tissue factor pathway inhibitor (TFPI)
US7851433B2 (en) 2003-08-13 2010-12-14 Novartis Vaccines And Diagnostics, Inc. Method of purifying TFPI and TFPI analogs

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DK140594A (da) 1994-12-08
AU4408493A (en) 1994-01-04
KR950701820A (ko) 1995-05-17

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