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
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.
DETAILED DESCRIPTION OF THE INVENTION
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
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
B. GENERAL METHODS
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
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
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.
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.
1 of 11 Claims
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
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
and the agent is capable of inhibiting coagulation and the release of
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