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Title:  Regulation of cytokine synthesis and release

United States Patent:  6,242,414

Inventors:  Johnson; Kirk (Moraga, CA); Creasey; Abla A. (Piedmont, CA); Aarden; Lucien A. (Amsterdam, NL)

Assignee:  Chiron Corporation (Emeryville, CA); Central Laboratory of The Netherlands Red Cross Blood Tranfusion Service (Amsterdam, NL)

Appl. No.:  483459

Filed:  June 7, 1995


Methods of treatment and prevention of diseases associated with release of neutrophil elastase and IL-8 by administration of TFPI, and analogs of TFPI are disclosed. Methods of determining efficacy of treatment with TFPI, patient's responsiveness to treatment with TFPI and the ultimate determination of patient prognosis are also disclosed.



As used herein, "TFPI" refers to mature Tissue Factor Pathway Inhibitor. As noted above, TFPI is also known in the art as Lipoprotein Associated Coagulation Inhibitor (LACI), Extrinsic Pathway Inhibitor (EPI) and Tissue Factor Inhibitor (or TFI). Muteins of TFPI which retain the biological activity of TFPI are encompassed in this definition. Further, TFPI which has been slightly modified for production in bacterial cells is encompassed in the definition as well. For example, a TFPI analog have an alanine residue at the amino-terminal end of the TFPI polypeptide has been produced in Escherichia coli. See U.S. Pat. No. 5,212,091. Analogs of TFPI having portions of TFPI and TFPI-2, fragments of TFPI comprising the first and second Kunitz-type domains, as well as fragments of TFPI comprising the first and second Kunitz domains and a heparin binding region may all be useful in the method of the invention. Such analogs and fragments are described in U.S. Pat. No. 5,106,833 as well as U.S. Ser. No. 08/286,521. One such fragment is TFPI(1-160) having the first 160 amino acids of mature TFPI.

As used herein, "pharmaceutically acceptable composition" refers to a composition that does not negate or reduce the biological activity of formulated TFPI, and that does not have any adverse biological effects when formulated TFPI is administered to a patient.

As used herein, "patient" encompasses human and veterinary patients.


TFPI may be prepared by recombinant methods as disclosed in U.S. Pat. No. 5,212,091, the disclosure of which is herein incorporated by reference. Briefly, TFPI is expressed in Escherichia coli cells and the inclusion bodies containing TFPI are isolated from the rest of the cellular material. The inclusion bodies are subjected to sulfitolysis, purified using ion exchange chromatography, refolded by disulfide interchange reaction and the refolded, active TEPI purified by cation exchange chromatography. TFPI may also be produced in yeast as disclosed in co-pending U.S. Ser. No. 08/286,530.

Whole Blood Culture

The whole blood culture system can be carried out as follows. Blood is collected from normal donors into anticoagulant. Venous blood from normal health donors was collected directly into clinical heparin or EDTA (K3) vacutainers (Baxter). Alternatively, venous blood was collected into sterile polypropylene syringes and immediately transferred into microtiter wells containing indicated concentrations of various additives including:

a. 20-50 U/ml heparin (blood fully anticoagulated, even with 10.times.dilution)

b. 50-60 U/ml hirudin (recombinant yeast, American Diagnostica)

c. 10 .mu.g/ml TFPI

d. 1 U/ml heparin (ESI)

e. 10 mM EDTA (for isolated neutrophils)

f. 1 ng/ml LPS (E. coli Rc) (Sigma, St. Louis, Mo.)

g. 3.8% citrate (for isolated PBMC).

TFPI was formulated at 11 mg/ml in 2 M urea, 20 mM sodium phosphate pH 7.2 and 0. 14 M NaCl. Blood collected into vacutainers was quickly transferred into polypropylene tubes prior to addition into culture wells.

Whole blood was cultured in 96 well microtiter plates (Corning) at a volume of 200 .mu.l per well at 37 C, 5% CO2 for 2-48 hours in a humidified atmosphere. The blood will typically be at a final dilution of 1:8 to 1: 10 in RPMI 1640 medium +0.1% "low-endotoxin" fetal calf serum (FCS) (Hyclone, Logan, UT). The cultures were then spun down at 400 .times.g for 1 minute at 4oC. Supernatant liquids were then removed at various time points (typically 2-2.5 hours) for analysis of soluble mediators. In the event that clotting occurred during culturing, the contents of clotted wells and comparative groups were transferred to polypropylene microfuge tubes and briefly spun to pellet cells and fibrin clot prior to harvesting supernatants. Soluble mediators were measured in supernatants by ELISA or other bioassay.

Peripheral Blood Mononuclear Cell (PBMC) Cultures

Whole blood was collected into EDTA vacutainers was layered over a ficoll gradient (NIM medium, Cardinal Assoc.) at a mixture of 7-8 ml blood onto 5 ml NIM medium in 15 ml polystyrene tubes. The tubes were spun at 500 .times.g for 30 minutes and the mononuclear cell layer was isolated as the top band in the gradient. In some experiments, PBMC were also isolated using citrated Cell Preparation Tubes (Becton-Dickinson, Mountain View, Calif.) wherein blood is collected and fractionated in the same tube. Identical results were obtain utilizing PBMC isolated in either manner. Following a sterile saline wash, PBMC were cultured at .about.1.times.105 cells per well in RPMI/0. 1% FCS as described above for whole blood cell cultures.

Assay for Soluble Mediators

ELISA assays for elastase, IL-8, IL-6 and TNF were conducted as follows. 96 well microtiter plates were coated overnight with the appropriate antibodies. Plates were washed and samples were added to each well along with biotin-labelled antibody and serum. The plates were then incubated and washed. Streptavidin-horseradish peroxidase was then added to the wells and allowed to incubate. The wells were again washed and developed with TMB, sodium acetate and peroxide. The reaction was stopped by the addition of 2 M sulfuric acid and plates read at O.D. 450 nm. When assaying for elastase, there is an incubation period between the addition of the samples and the biotinylated anti-elastase antibodies. For TNF, poly-streptavidin-horseradish peroxidase and milk are used instead of Strep-HRP and serum.

Spectrozyme Plasmin assay kits were obtained from American Diagnostica. Quantikine IL-1.beta.ELISA kits were purchased from R&D Systems. The manufacturers'instructions were followed in completing the assays.

Coagulation Activation

Measurement of the extent of coagulation activation was performed in a qualitative manner by observation of clotting and, quantitatively, via immunodetection of thrombin:antithrombin (TAT) complex and fibrinopeptide A levels according to manufacturer's protocols (Diagnostica Stago, France). Supernatants were also routinely analyzed for chromogenic activity against various substrates including Spectrozyme Xa and TH (thrombin) (American Diagnostica) for correlation with the above metrics as well as to confirm activity of purified factors added to isolated PBMC cultures including prothrombin, .alpha.-thrombin, and factor Xa.

Claim 1 of 11 Claims

We claim:

1. A method of treating a patient having a disease associated with increased synthesis and release of neutrophil elastase comprising administering to the patient an agent in an amount effective to ameliorate at least one symptom associated with the disease, wherein

the disease is severe acute pancreatitis, emphysema, rheumatoid arthritis, cystic fibrosis, or multiple organ failure;

the agent is:

Tissue Factor Pathway Inhibitor (TFPI),


a fragment of wild-type TFPI comprising the first and second Kunitz domains,

a fragment of wild-type TFPI comprising the first and second Kunitz domains and a heparin binding region, or

a fragment of wild-type TFPI comprising the first 160 amino acids of mature TFPI;

and the agent is capable of inhibiting coagulation and the release of neutrophil elastase.

If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.



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