Note: Descriptions are shown in the official language in which they were submitted.
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Treatment of severe community-acquired pneumonia by administration of tissue
factor
pathway inhibitor (TFPI)
FIELD OF THE INVENTION
The present invention relates to a method for therapeutically treating severe
community-
acquired pneumonia. More specifically, it relates to administering a tissue
factor pathway
inhibitor protein to attenuate exuberant or amplified physiological pathways
associated with
severe pneumonia.
BACKGROUND OF THE INVENTION
Pneumonia results from an acute infection of one or more functional elements
of the lung,
including alveolar spaces and interstitial tissue. In the USA, about 2 million
people develop
pneumonia each year, and 40,000 to 70,000 of these people die. Pneumonia ranks
sixth
among all disease categories as a cause of death and is the most common lethal
nosocomial
(hospital-acquired) infection. Community-acquired pneumonia (CAP) has a
significant
impact on health care costs in the United States, accounting for an estimated
$14 billion per
year in direct costs and $9 billion in lost wages. (Lynch J P, Martinez F J.
Community-
acquired pneumonia. Curr Opin Pulm Med. 1998; 4:162-172). In developing
countries, lower
respiratory tract infections typically are either the major cause of death or
rank second only to
infectious diarrhea. (The Merck Manual, Sec. 6, Ch. 73, Pneumonia, 2000).
The condition known as "severe pneumonia" is characterized according to
guidelines set forth
by various organizations, including the American Thoracic Society (ATS). (Am J
Respir Crit
Care Med 2001; 163:1730-1754). For example, the ATS requires at least one
major criterion,
such as a need for mechanical ventilation or septic shock, in addition to
other criteria for a
diagnosis of severe pneumonia. Generally, severe pneumonia can result from
acute lung
disease, lung inflammatory disease, or any perturbations in lung function due
to factors such
as inflammation or coagulation. A diagnosis of severe CAP is based on a
patient being
admitted to an ICU specifically for pneumonia. Epidemiologically, this patient
population
comprises approximately 10% of all ICU admissions. Patients in the ICU with
pneumonia
have the highest mortality of all CAP patients (30% to 40%) compared with less
than 15% for
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general hospitalized patients with CAP.
Each year in the United States, community-acquired pneumonia (CAP) is
diagnosed in
approximately 4 million adults, with as many as 600,000 requiring
hospitalization. Fine et al.,
N. Engl. J. Med. 336, 243-50, 1997. Overall, the incidence of CAP increases
with age, with
the greatest prevalence found in those aged 65 years and older. Marston et
al., Arch Intern
Med. 1997;157:1709-1718. The incidence is also increased in patients with
comorbidities,
such as chronic obstructive pulmonary disease, asthma, diabetes mellitus,
alcoholism,
immunosuppression, renal insufficiency, chronic liver disease, and cardiac
disease. Marrie,
Curr Opin Pulm Med. 1996;2:192-197 ; Niedermann et al., Am Rev Respir Dis.
1993;148:1418-1426.
Pneumonia is the leading cause of death from infection in the United States
and the sixth
leading cause of death overall. The pneumonia-related mortality rate increased
by 22% from
1979 to 1992, with elderly patients (65 years and older) accounting for 89% of
all
pneumonia-related deaths in 1992. See Pneumonia and influenza death rates--
United States,
1979-1994 [published correction appears in MMWR. 1995;44:782]. MMWR.
1995;44:535-
537. Fine and colleagues (1997) reported that certain coexisting illnesses
(neoplastic disease,
congestive heart failure (CHF) cerebrovascular disease, renal disease, and
liver disease) and
certain physical examination findings (altered mental status, increased heart
rate, increased
respiratory rate, decreased systolic blood pressure, and abnormally low or
elevated
temperatures) are also associated with increased CAP-related mortality. In
addition, CAP has
a significant impact on health care costs in the United States, accounting for
an estimated $14
billion per year in direct costs and $9 billion in lost wages. Lynch &
Martinez, Curr Opin
Pulm Med. 1998;4:162-172.
Tissue factor pathway inhibitor (TFPI) is a protein and a serine protease
inhibitor present in
mammalian blood plasma. Thomas, Bull. Johns Hopkins Hosp. 81, 26 (1947);
Schneider,
Am. J. Physiol. 149, 123 (1947); Broze & Miletich, Proc. Natl. Acad. Sci. USA
84, 1886
(1987). TFPI is also known as tissue factor inhibitor, tissue thromboplastin
inhibitor, Factor
III inhibitor, extrinsic pathway inhibitor (EPI), and lipoprotein-associated
coagulation
inhibitor (LACI). The name "tissue factor pathway inhibitor" (TFPI) was
accepted by the
International Society on Thrombosis and Hemostasis on Jun. 30, 1991.
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Blood coagulation activation is the conversion of fluid blood to a solid gel
or clot. In
addition, consumption of the coagulation proteases leads to excessive
bleeding. 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 catalyzes the
formation of larger
amounts of the next, and so on, resulting in an amplification that results in
an extremely rapid
formation of fibrin.
Coagulation is believed to be initiated by vessel damage which exposes factor
VIIa to tissue
factor (TF), which is expressed on cells beneath the endothelium. The factor
VIIa-TF
complex cleaves factor X to factor Xa and cleaves factor IX to factor IXa.
TFPI binds to both
factor VIIa and factor Xa. The complex formed between TFPI, factor VIIa (with
its bound
TF), and factor Xa inhibits further formation of factors Xa and IXa, required
for sustained
hemostasis. Broze, Jr., Ann. Rev. Med. 46:103 (1995).
Activation of the coagulation cascade by bacterial products, including
endotoxins, introduced
directly into the bloodstream can result in extensive fibrin deposition on
arterial surfaces, as
well as depletion of fibrinogen, prothrombin, factors V and VIII, and
platelets. In addition,
the fibrinolytic system is stimulated, resulting in further formation of
fibrin degradation
products.
At the same time as coagulation activation is apparently initiated by
bacterial products (e.g.,
endotoxin), contravening mechanisms also appear to be activated by clotting,
namely
activation of the fibrinolytic system. Activated Factor XIII converts
plasminogen pro-
activator, to plasminogen activator that subsequently converts plasminogen to
plasmin,
thereby mediating clot lysis. The activation of plasma fibrinolytic systems
may therefore also
contribute to bleeding tendencies.
Endotoxemia is associated with an increase in the circulating levels of tissue
plasminogen
activator inhibitor (PAI). This inhibitor rapidly inactivates tissue
plasminogen activator
(TPA), thereby hindering its ability to promote fibrinolysis through
activation of plasminogen
to plasmin. Impairment of fibrinolysis may cause fibrin deposition in blood
vessels, thus
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contributing to the DIC associated with septic shock.
Efforts are ongoing to identify satisfactory interventions for the prevention
or treatment of
severe pneumonia and associated coagulopathies. An agent that inteiTupts the
coagulation
pathway is not necessarily effective as a therapeutic or a prophylactic
treatment of severe
pneumonia. For example, heparin is a commonly used anticoagulant. However,
management
of the use of heparin has been difficult because heparin can induce excessive
bleeding and
has been found to attenuate coagulation abnormalities but not offer a survival
benefit. See for
example, Aolci et al.," A Comparative Double-BLIND randomized Trial of
Activated Protein
C and Unfractionated Heparin in the Treatment of Disseminated Intravascular
Coagulation,"
Int. J. Hematol. 75, 540-47 (2002). Several clinical trials, mainly in
meningococcal
endotoxemia where fulminating DIC is a prominent feature, have failed to
demonstrate
reduction of mortality in sepsis by heparin treatment. See, for example,
Corrigan et al.,
"Heparin Therapy in Septacemia with Disseminated Intravascular Coagulation.
Effect on
Mortality and on Correction of Hemostatic Defects," N. Engl. 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).
Patients prone to severe community-acquired pneumonia are those patients with
community-
acquired pneumonia requiring admission to an Intensive Care Unit (ICU).
Patients with
community-acquired pneumonia are clinically identified as having an infection
of lung
parenchyma and/or confirmed via radiographic and clinical signs. Severe
pneumonia
includes severe community-acquired pneumonia, typically have well-defined
pathogens,
including S. pneurnonae, legionella, H. influefaae or various gram-negative
bacilli. The
majority of patients with severe community-acquired pneumonia live in the
community
before the CAP episode, with only about 20% admitted as a trasfer from a
hospital, nursing
home or long term care. Patients in the US with Severe CAP are about 50% male
and about
50% female, but tend to be older. About 17% of the patients with severe CAP in
the US are
under 50; about 24% are between the age of 50 and 64; about 21 % are between
the age of 65
and 74 and about 38% are over 75 years of age. Most patients with severe CAP
have one or
more significant comorbidities. Of the US CAP patients receiving ICU care
treatment in
2003, these patients typically have corresponding heart disease, COPD/cystic
fibrosis,
diabetes, kidney disease, cancer, alcoholism and/or drug abuse.
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Administration of recombinant human ala-TFPI (a TFPI analog) has been shown to
improve
survival rates in animal models of sepsis. See, e.g., U.S. Pat. No. 6,063,764.
As an
endogenous protein, TFPI is well tolerated. TFPI administration by intravenous
infusion or
subcutaneous injection has been shown to reduce clotting ability, which is
manifested as
increased prothrombin time (PT). In studies of animals and humans,
prolongations of PT
were linearly related to the increase of plasma TFPI. A. A. Creasey, Sepsis
3:173 (1999).
There remains a need in the art for treatment approaches that will inhibit the
lethal effects of
severe pneumonia and simultaneously minimize potentially serious side effects.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a method of treating or preventing
severe
pneumonia coinprising administering TFPI or a TFPI analog to a patient who has
or is at risk
of developing severe pneumonia. In some embodiments, the patient has a
demonstrable
infection.
Another embodiment of the present invention is a method for treating severe
pneumonia,
comprising administering to a patient a continuous intravenous infusion of an
agent selected
from the group consisting of TFPI or a TFPI analog. In some embodiments, the
patient has a
demonstrable infection.
Another embodiment of the present invention is a method for treating or
preventing severe
pneumonia comprising administering TFPI or a TFPI analog, to a human patient
who has or
is at risk of developing severe pneumonia, in an amount that does not induce
excessive
bleeding.
Other embodiments include any of the above embodiments wherein said TFPI or
TFPI analog
is administered by continuous intravenous infusion at a dose rate equivalent
to administration
of reference ala-TFPI at a dose rate of less than about 2.0 mg/kg/hr. In a
preferred
embodiment, said dose rate is equivalent to administration of reference ala-
TFPI at a dose
rate from about 0.00025 to about 2.0, or alternatively, from about 0.001 to
about 1.75
mg/kg/hr. In another preferred embodiment, said dose rate is equivalent to
administration of
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reference ala-TFPI at a dose rate from about 0.005 to about 1.50 mg/kg/hr. In
a more
preferred embodiment said dose rate is equivalent to administration of
reference ala-TFPI at a
dose rate from about 0.010 to about 0.75 mg/lcg/hr. In a still more preferred
embodiment, said
TFPI or said TFPI analog is administered at a dose rate equivalent to
administration of
reference ala-TFPI at a dose rate of about 0.2 mg/kg/hr to about 0.8
mg/lcg/hr. In another
preferred embodiment, said dose rate is administered to provide a total dose
equivalent to
administration of reference ala-TFPI at a total dose from about 0.024 to about
4.8 mg/kg. In
another preferred embodiment, said dose rate is administered to provide a
daily dose
equivalent to administration of reference ala-TFPI at a daily dose of at least
about 0.006
mg/kg and less than about 1.2 mg/kg.
Other embodiments include any of the above embodiments, wherein and said TFPI
or TFPI
analog is administered for at least 72 hours. In a preferred embodiment, said
TFPI or TFPI
analog is administered for at least 96 hours.
Other embodiments include any of the above embodiments wherein said TFPI
analog is non-
glycosylated ala-TFPI.
Other embodiments include any of the above embodiments wherein said TFPI
analog
comprises a first Kunitz domain consisting of amino acids 19-89 of SEQ ID
NO:1. In a
preferred embodiment, said TFPI analog further comprises a second Kunitz
domain
consisting of amino acids 90-160 of SEQ ID NO:1.
Other embodiments include any of the above embodiments wherein said TFPI
analog
comprises amino acids 1-160 of SEQ ID NO:1 or wherein said TFPI analog
comprises a
second Kunitz domain consisting of amino acids 90-160 of SEQ ID NO:1.
Other embodiments include any of the above embodiments wherein said TFPI or
TFPI analog
is prepared from a lyophilized composition comprising TFPI or a TFPI analog.
Other embodiments include any of the above embodiments wherein said TFPI or
TFPI analog
is administered as a formulation comprising arginine.
Other embodiments include any of the above embodiments wherein said TFPI or
TFPI analog
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is administered as a formulation comprising citrate.
Other embodiments include any of the above embodiments wherein said TFPI or
TFPI analog
has a concentration of about 0.15 mg/ml in a formulation comprising about 300
mM arginine
hydrochloride and about 20 mM sodium citrate and having a pH of about 5.5.
Other embodiments include any of the above embodiments, further comprising
administering
at the same time as, or within 24 hours of administering said TFPI or TFPI
analog, an
additional agent selected from the group consisting of an antibiotic, an
antibody, an
endotoxin antagonist, a tissue factor analog having anticoagulant activity, an
immunostimulant, a cell adhesion blocker, heparin, BPI protein, an IL-1
antagonist, pafase
(PAF enzyme inhibitor), a TNF inhibitor, an IL-6 inhibitor, and an inhibitor
of complement.
In a preferred embodiment, said additional agent is an antibody that binds
specifically to an
antigen selected from the group consisting of TNF, IL-6, and M-CSF.
Further embodiments of the present invention are apparent in view of the below-
referenced
drawings in conjunction with the detailed description.
DETAILED DESCRIPTION OF THE INVENTION
Administration of TFPI or analogs of TFPI is effective in the prophylaxis and
treatment of
severe pneumonia. Continuous low dosage administration of TFPI or analogs of
TFPI
(hereinafter "low dose TFPI administration") also is effective in the
prophylaxis and
treatment of severe pneumonia. TFPI or TFPI analog administration,
particularly low dose
administration, inhibits or attenuates acute or chronic inflammation,
particularly severe
pneumonia. When low dose TFPI administration is continued for at least three
days, the risk
of death from severe pneumonia is reduced, while the rate of complications
from adverse side
effects, particularly bleeding disorders, may be minimized. A further
advantage of low dose
TFPI administration is the avoidance of tolerance effects that, at
sufficiently high doses, can
reduce the plasma concentration of TFPI. Tolerance effects are stimulated half-
maximally at
a plasma TFPI concentration of about 850 ng/ml, whereas with low dose TFPI
administration
plasma levels generally stay below 500 ng/ml. Low dose TFPI administration
generally is
carried out by continuous intravenous infusion of TFPI or an analog of TFPI.
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TFPI and TFPI Analogs
"TFPI" as used herein refers to the mature serum glycoprotein having the 276
amino acid
residue sequence shown in SEQ ID NO: 1 and a molecular weight of about 38,000
Daltons. It
is a natural inhibitor of tissue factor activity and thus coagulation
activation. U.S. Pat. No.
5,110,730 describes tissue factor (TF), and U.S. Pat. No. 5,106,833 describes
TFPI. The
cloning of the TFPI cDNA is described in Wun et al., U.S. Pat. No. 4,966,852.
TFPI is a
protease inhibitor and has 3 Kunitz domains, two of which are known to
interact with factors
VII and Xa respectively. The function of the third domain remains unknown.
TFPI is
believed to function in viva to limit the initiation of coagulation by forming
an inert,
quaternary factor X<sub>a</sub>:TFPI:factor VII<sub>a</sub>:tissue factor complex. See
reviews by
Rapaport, Blood 73:359-365 (1989) and Broze et al., Biochemistry 29:7539-7546
(1990).
Many of the structural features of TFPI can be deduced from its homology with
other well-
studied protease inhibitors. TFPI is not an enzyme, so it probably inhibits
its protease target
in a stoichiometric manner, i.e., one of the Kunitz domains of TFPI inhibits
one protease
molecule. Preferably, Kunitz domains 1 and/or 2 will be present in TFPI
molecules of the
instant invention. The function of Kunitz domain 3 is unknown.
A "TFPI analog" is a derivative of TFPI modified with one or more amino acid
additions or
substitutions (generally conservative in nature), one or more amino acid
deletions (e.g., TFPI
fragments), or the addition of one or more chemical moieties to one or more
amino acids, so
long as the modifications do not destroy TFPI biological activity. Methods for
making
polypeptide analogs are known in the art and are described further below. A
preferred TFPI
analog is N-L-alanyl-TFPI (ala-TFPI), whose amino acid sequence is shown in
SEQ ID
NO:2. TFPI analogs possess some measure of the activity of TFPI as determined
by a
bioactivity assay as described below. A preferred bioactivity assay for TFPI
and analogs is
the prothrombin time (PT) assay (see below).
TFPI and TFPI analogs can be either glycosylated or non-glycosylated. Analogs
of TFPI are
described in U.S. Pat. No. 5,106,833. Ala-TFPI is a TFPI analog that is also
known under the
international drug name "tifacogin." Ala-TFPI includes the entire amino acid
sequence of
mature, full-length human TFPI plus an additional alanine residue at the amino
terminus. The
amino terminal alanine residue of ala-TFPI was engineered into the TFPI
sequence to
improve E. coli expression and to effect cleavage of what would otherwise be
an amino
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terminal methionine residue. See U.S. Pat. No. 5,212,091.
Particularly preferred TFPI analogs include substitutions that are
conservative in nature, i.e.,
those substitutions that take place within a family of amino acids that are
related in their side
chains. Specifically, amino acids are generally divided into four families:
(1) acidic--aspartate
and glutamate; (2) basic--lysine, arginine, histidine; (3) non-polar--alanine,
valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged
polar--glycine,
asparagine, glutamine, cysteine, serine threonine, and tyrosine.
Phenylalanine, tryptophan,
and tyrosine are sometimes classified as aromatic amino acids. For example, it
is reasonably
predictable that an isolated replacement of leucine with isoleucine or valine,
an aspartate with
a glutamate, a threonine with a serine, or a similar conservative replacement
of an amino acid
with a structurally related amino acid, will not have a major effect on the
biological activity.
For example, the polypeptide of interest may include up to about 1-70
conservative or non-
conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6-50, 15-25, 5-
10, or any integer
from 1 to 70, so long as the desired function of the molecule remains intact.
One of skill in
the art may readily determine regions of the molecule of interest that can be
modified with a
reasonable likelihood of retaining biological activity as defined herein.
"Homology" refers to the percent similarity between two polynucleotide or two
polypeptide
moieties. Two polypeptide sequences are "substantially homologous" to each
other when the
sequences exhibit at least about 50%, preferably at least about 75%, more
preferably at least
about 80%-85%, preferably at least about 90%, and most preferably at least
about 95%-98%
sequence homology, or any percent homology between the specified ranges, over
a defined
length of the molecules. As used herein, "substantially homologous" also
refers to sequences
showing complete identity to the specified polypeptide sequence.
In general, "identity" refers to an exact amino acid-to-amino acid
correspondence of two
polypeptide sequences, respectively. Percent identity can be determined by a
direct
comparison of the sequence information between two molecules by aligning the
sequences,
counting the exact number of matches between the two aligned sequences,
dividing by the
length of the shorter sequence, and multiplying the result by 100.
Preferably, naturally or non-naturally occurring TFPI analogs have amino acid
sequences
which are at least 70%, 80%, 85%, 90% or 95% or more homologous to TFPI
derived from
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SEQ ID NO:1. More preferably, the molecules are 98% or 99% homologous. Percent
homology is determined using the Smith-Waterman homology search algorithm
using an
affine gap search with a gap open penalty of 12 and a gap extension penalty of
2, and a
BLOSUM matiix of 62. The Smith-Waterman homology search algorithm is taught in
Smith
and Waterman, Adv. Appl. Math. 2:482-489 (1981).
The biological activity of TFPI and TFPI analogs can be determined by the
prothrombin
assay. Suitable prothrombin assays are described in U.S. Pat. No. 5,888,968
and in WO
96/40784. Briefly, prothrombin time can be determined using a coagulometer
(e.g., Coag-A-
Mate MTX II from Organon Teknika). A suitable assay buffer is 100 mM NaCI, 50
mM Tris
adjusted to pH 7.5, containing 1 mg/ml bovine serum albumin. Additional
reagents required
are normal human plasma (e.g., "Verify 1" by Organon Teknika), thromboplastin
reagent
(e.g., "Simplastin Excel" by Organon Teknika), and TFPI standard solution
(e.g., 20 mu.g of
100% pure ala-TFPI (or equivalent thereof) per ml of assay buffer). A standard
curve is
obtained by analyzing the coagulation time of a series of dilutions of the
TFPI standard
solution, e.g., to final concentrations ranging from 1 to 5µg/ml. For the
determination of
clotting time, the sample or TFPI standard is first diluted into the assay
buffer. Then normal
human plasma is added. The clotting reaction is started by the addition of
thromboplastin
reagent. The instrument then records the clotting time. A linear TFPI standard
curve is
obtained from a plot of log clotting time vs. log TFPI concentration. The
standard curve is
adjusted based on the purity of the TFPI standard to correspond to the
equivalent TFPI
concentration of a 100% pure standard. For example, if the standard is a
preparation of ala-
TFPI that is 97% biochemically pure (i.e., it contains 3% by weight of
molecular species
without biological activity of TFPI), then the concentration of each dilution
of the standard is
multiplied by 0.97 to give the actual concentration of TFPI. Thus, a TFPI
standard that is 1.0
µg/ml based on the actual weight per ml of a preparation that is 97% pure
will be
equivalent to, and treated as, a concentration of 1.0×0.97, or 0.97
µg/ml.
Obtaining TFPI and TFPI Analogs
TFPI and analogs of TFPI used in the methods of the invention can be isolated
and purified
from cells or tissues, chemically synthesized, or produced recombinantly in
either prokaryotic
or eukaryotic cells.
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TFPI can be isolated by several methods. For example, cells that secrete TFPI
include aged
endothelial cells, young endothelial cells that have been treated with TNF for
about 3 to 4
days, hepatocytes, and hepatoma cells. TFPI can be purified by conventional
methods,
including the chromatographic methods of Pedersen et al., 1990, J. Biol. Chem.
265, 16786-
93, Novotny et al., 1989, J. Biol. Chem. 264, 18832-37, Novotny et al., 1991,
Blood 78, 394-
400, Wun et al., 1990, J. Biol. Chem. 265, 16096-101, and Broze et al., 1987,
Proc. Natl.
Acad. Sci. USA 84, 1886-90. TFPI appears in the bloodstream and can be
purified from
blood, see Pedersen et al., 1990.
A TFPI or TFPI variant can be produced using chemical methods to synthesize
its amino acid
sequence, such as by direct peptide synthesis using solid-phase techniques
(Merrifield, J. Am.
Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995).
Protein
synthesis can be performed using manual techniques or by automation. Automated
synthesis
can be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin
Elmer). Optionally, fragments of TFPI or TFPI variants can be separately
synthesized and
combined using chemical methods to produce a full-length molecule.
TFPI and TFPI analogs may be produced recombinantly as shown in U.S. Pat. No.
4,966,852.
For example, the cDNA for the desired protein can be incorporated into a
plasmid for
expression in prokaryotes or eukaryotes. U.S. Pat. No. 4,847,201 provides
details for
transforming microorganisms with specific DNA sequences and expressing them.
There are
many other references known to those of ordinary skill in the art that provide
details on
expression of proteins using microorganisms. Many of those are cited in U.S.
Pat. No.
4,847,201, such as Maniatas et al., 1982, Molecular Cloning, Cold Spring
Harbor Press.
A variety of techniques are available for transforming microorganisms and
using them to
express TFPI and TFPI analogs. The following are merely examples of possible
approaches.
TFPI DNA sequences must be isolated and connected to the appropriate control
sequences.
TFPI DNA sequences are shown in U.S. Pat. No. 4,966,852 and can be
incorporated into a
plasmid, such as pUNC13 or pBR3822, which are commercially available from
companies
such as Boehringer-Mannheim. Once the TFPI DNA is inserted into a vector, it
can be cloned
into a suitable host. The DNA can be amplified by techniques such as those
shown in U.S.
Pat. No. 4,683,202 to Mullis and U.S. Pat. No. 4,683,195 to Mullis et al. TFPI
cDNA may be
obtained by inducing cells, such as hepatoma cells (such as HepG2 and SKHep)
to make
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TFPI mRNA, then identifying and isolating the mRNA and reverse transcribing it
to obtain
cDNA for TFPI. After the expression vector is transformed into a host such as
E. coli, the
bacteria may be fermented and the protein expressed. Bacteria are preferred
prokaryotic
microorganisms and E. coli is especially preferred. A preferred microorganism
useful in the
present invention is E. coli K-12, strain MM294 deposited with the ATCC on
Feb. 14, 1984
(Accession No. 39607), under the provisions of the Budapest Treaty.
It is also, of course, possible to express genes encoding polypeptides in
eukaryotic host cell
cultures derived from multicellular organisms. See, for example, Tissue
Culture, 1973, Cruz
and Patterson, eds., Academic Press. Useful mammalian cell lines include
murine myelomas
N51, VERO, HeLa cells, Chinese hamster ovary (CHO) cells, COS, C127, Hep G2,
and SK
Hep. TFPI and TFPI analogs can also be expressed in baculovirus-infected
insect cells (see
also U.S. Pat. No. 4,847,201, referred to above). See also Pedersen et al.,
1990, J. of
Biological Chemistry, 265:16786-16793. Expression vectors for eukaryotic cells
ordinarily
include promoters and control sequences compatible with mammalian cells such
as, for
example, the commonly used early and later promoters from Simian Virus 40
(SV40) (Fiers,
et al., 1978, Nature, 273:113), or other viral promoters such as those derived
from polyoma,
Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, or
immunoglobulin
promoters and heat shock promoters. General aspects of mammalian cell host
system
transformations have been described by Axel, U.S. Pat. No. 4,399,216. It now
appears also
that "enhancer" regions are important in optimizing expression; these are,
generally,
sequences found upstream of the promoter region. Origins of replication may be
obtained, if
needed, from viral sources. However, integration into the chromosome is a
common
mechanism for DNA replication in eukaryotes. Plant cells are also now
available as hosts,
and control sequences compatible with plant cells such as the nopaline
synthase promoter and
polyadenylation signal sequences (Depicker, A., et al., 1982, J. Mol. Appl.
Gen., 1:561) are
available. Methods and vectors for transformation of plant cells have been
disclosed in WO
85/04899.
Methods which can be used for purification of TFPI and TFPI analogs expressed
in
mammalian cells include sequential application of heparin-Sepharose, MonoQ,
MonoS, and
reverse phase HPLC chromatography. See Pedersen et al., supra; Novotny et al.,
1989, J.
Biol. Chem. 264:18832-18837; Novotny et al., 1991, Blood, 78:394-400; Wun et
al., 1990, J.
Biol. Chem. 265:16096-16101; Broze et al., 1987, PNAS (USA), 84:1886-1890;
U.S. Pat.
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WO 2006/113360 PCT/US2006/013890
No. 5,106,833; and U.S. Pat. No. 5,466,783. These references describe various
methods for
purifying mammalian produced TFPI.
TFPI also can be expressed as a recombinant glycosylated protein using
mammalian cell
hosts, such as mouse C127 cells (Day et al., Blood 76, 1538-45, 1990), baby
hamster kidney
cells (Pedersen et al., 1990), Chinese hamster ovary cells, and human SK
hepatoma cells.
C127 TFPI has been used in animal studies and shown to be effective in the
inhibition of
tissue factor-induced intravascular coagulation in rabbits (Day et al.,
supra), in the prevention
of arterial reocclusion after thrombolysis in dogs (Haskel et al., Circulation
84:821-827
(1991)), and in reduction of mortality in an E. coli sepsis model in baboons
(Creasey et al., J.
Clin. Invest. 91:2850 (1993)). Ala-TFPI can be expressed as a recombinant non-
glycosylated
protein using E. coli host cells. Methods have been described which yield a
highly active ala-
TFPI by in vitro refolding of the recombinant protein produced in E. coli.
See, e.g., WO
96/40784.
TFPI and TFPI analogs also can be produced in bacteria or yeast and
subsequently purified.
Generally, the procedures shown in U.S. Pat. Nos. 5,212,091; 6,063,764; and
6,103,500 or
WO 96/40784 can be employed. Ala-TFPI and other TFPI analogs can be purified,
solubilized, and refolded according WO 96/40784 and Gustafson et al., Prot.
Express. Pur.
5:233 (1994), which are incorporated herein by reference. For example, when
prepared
according Example 9 of WO 96/40784, preparations of ala-TFPI may be obtained
that
contain from about 85% to 90% of the total protein by weight as mature,
properly-folded,
biologically active ala-TFPI, about 10% to 15% of which has one or more
oxidized
methionine residues. These oxidized forms have biological activity that is
equivalent to the
biological activity of underivatized ala-TFPI, as determined by prothrombin
assay, and are
expected to be active in the invention disclosed herein. The remaining
material comprises
various modified forms of ala-TFPI, including dimerized, aggregated, and
acetylated forms.
TFPI and TFPI analogs can have a significant number of cysteine residues, and
the procedure
shown in U.S. Pat. No. 4,929,700 is relevant to TFPI refolding. TFPI and
analogs can be
purified from the buffer solution by various chromatographic methods, such as
those
mentioned above. If desired, the methods shown in U.S. Pat. No. 4,929,700 may
be
employed. Any method may be employed to purify TFPI and TFPI analogs that
results in a
degree of purity and a level of activity suitable for administration to
humans.
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WO 2006/113360 PCT/US2006/013890
Therapeutic Methods and Compositions
Generally, TFPI and TFPI analogs are useful to treat or prevent those diseases
that occur due
to the up-regulation of tissue factor expression and hence TF activity brought
on by TNF, IL-
1 or other cytokines. TFPI administration, and particularly low dose TFPI
administration,
may lower the concentration of cytolcines such as IL-6 in a patient. Low dose
TFPI
administration is useful for treating inflammation and coagulation
abnormalities generally,
including both acute and chronic inflammatory conditions such as severe
pneumonia.
"Severe pneumonia" is defined according to the guidelines set forth by the
American
Thoracic Society. Specifically, severe pneumonia requires a diagnosis of
pneumonia and the
existence of either a) one of two major criteria, b) two of three minor
criteria, or c) two of the
four criteria from the British Thoracic Society (Thorax 2001; 56 [suppl IV]:1-
64). The major
criteria are 1) need for mechanical ventilation and 2) septic shock or need
for pressors for>4
hours. The minor criteria are 1) systolic blood pressure.ltoreq.90 mmHg, 2)
multi-lobar
pneumonia, and 3) hypoxemia criterion (PaO<sub>2</sub>/FiO<sub>2</sub>)<250. The criteria
from the
British Thoracic Society are 1) respiratory rate.gtoreq.30 breaths/minute, 2)
diastolic blood
pressure.Itoreq.60 mmHg, 3) blood urea nitrogen (BUN)>7.0 mM (>19.6 mg/dL) and
4)
confusion. As is understood in the art, the hypoxemia criterion
(PaO<sub>2</sub>/FiO<sub>2</sub>) refers
to the partial pressure of arterial oxygen to the fraction of inspired oxygen
and indicates the
level of impairment of gas exchange.
Preferably, patients with severe pneumonia have an infection demonstrable by
any means
known in the art. These methods include, but are not limited to, detection of
a pathogenic
organism in a culture of blood or other normally sterile body fluid or tissue
by, for example,
GRAM stain, culture, histochemical staining, inununochemical assay, or nucleic
acid assays.
A demonstrable infection also can be evidenced by a chest radiograph
consistent with a
diagnosis of pneumonia that constitutes the reason for systemic anti-infective
therapy, as well
as any clinical symptom of infection, including, but not limited to, an
increase in respiratory
rate>/=30/min or PaCO<sub>2</sub>/FiO<sub>2</sub><250, a decrease in blood pressure, and
an increase in
body temperature.
Formulations of TFPI and TFPI Analogs
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WO 2006/113360 PCT/US2006/013890
Formulations of TFPI and TFPI analogs preferably are administered by
intravenous infusions.
Essentially continuous intravenous infusion is preferred. Methods to
accomplish this
administration are known to those of ordinary skill in the art. Infusion can
be performed via a
central line or a peripheral line. While large fluctuations in the dose rate
are to be avoided,
short-term deviations from the dose rates of the invention are acceptable
provided the
resulting plasma level of administered TFPI is within 20% of that expected
from a continuous
infusion at a constant dose rate according to the preferred embodiments of
invention.
Before administration to patients, formulants may be added to TFPI and TFPI
analogs. A
liquid formulation is preferred. TFPI and TFPI analogs may be formulated at
different
concentrations, using different formulants, and at any physiologically
suitable pH compatible
with the route of administration, solubility, and stability of the TFPI
protein. A preferred
formulation for intravenous infusion includes ala-TFPI at up to about 0.6
mg/ml, arginine
hydrochloride at up to 300 mM, and sodium citrate buffer at pH 5.0-6Ø
Certain solutes such
as arginine, NaCI, sucrose, and mannitol serve to solubilize and/or stabilize
ala-TFPI. See
WO 96/40784. An especially preferred formulation for intravenous infusion
contains about
0.15 mg/ml ala-TFPI, 300 mM arginine hydrochloride, and 20 mM sodium citrate
at pH 5.5.
TFPI and TFPI analogs also can be formulated at concentrations up to about
0.15 mg/ml in
150 mM NaCl and 20 mM sodium phosphate or another buffer at pH 5.5-7.2,
optionally with
0.005% or 0.01% (w/v) polysorbate 80 (Tween 80). Other formulations contain up
to about
0.5 mg/ml TFPI, or TFPI analog in 10 mM sodium acetate at pH 5.5 containing
either 150
mM NaCl, 8% (w/v) sucrose, or 4.5% (w/v) mannitol. TFPI and TFPI analogs can
also be
formulated at higher concentrations up to several mg/ml using high salt. For
example, one
formulation contains up to about 6.7 mg/ml ala-TFPI in 500 mM NaCI and 20 mM
sodium
phosphate at pH 7Ø In addition, the TFPI formulation may contain methionine,
preferably at
a range of about 1 to about 10 mM methione.
A preferred embodiment of a TFPI formulation is ala-TFPI at about 0.1 to about
0.7 mg/ml,
200 to 500 mM L-arginine, 1 to 7 mM methionine, 5 to 50 mM sodium citrate
buffer at pH
5.0-6Ø A preferred embodiment of a TFPI formulation is ala-TFPI at about 0.1
to about 0.5
mg/ml, 250 to 400 mM L-arginine, 3 to 6.5 mM methionine, 15 to 30 mM sodium
citrate
buffer at pH 5.0-6Ø In a preferred embodiment of a TFPI formulation contains
ala-TFPI at
about 0.15mg/ml, L-arginine hydrochloride at about 300 mM, 5 mM methionine, 20
mM
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WO 2006/113360 PCT/US2006/013890
sodium citrate buffer at pH 5.5. Another preferred embodiment of a TFPI
formulation
contains ala-TFPI at about 0.45 mg/ml, L-arginine hydrochloride at about 300
mM, 5 mM
methionine, 20 mM sodium citrate buffer at pH 5.5.
Further examples of formulants for TFPI and TFPI analogs include oils,
polymers, vitainins,
carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking
agents. Preferably
carbohydrates include sugar or sugar alcohols such as mono, di, or
polysaccharides, or water
soluble glucans. The saccharides or glucans can include fructose, dextrose,
lactose, glucose,
mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha
and beta
cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcelloluose,
or mixtures
thereof. Sucrose is most preferred. Sugar alcohol is defmed as a C<sub>4</sub> to
C<sub>8</sub>
hydrocarbon having an --OH group and includes galactitol, inositol, mannitol,
xylitol,
sorbitol, glycerol, and arabitol. Mannitol is most preferred. These sugars or
sugar alcohols
mentioned above may be used individually or in combination. There is no fixed
limit to the
amount used as long as the sugar or sugar alcohol is soluble in the aqueous
preparation.
Preferably, the sugar or sugar alcohol concentration is between 1.0 w/v % and
7.0 w/v %,
more preferable between 2.0 and 6.0 w/v %.
Preferably amino acids include levorotary (L) forms of carnitine, arginine,
and betaine;
however, other amino acids may be added. Preferred polymers include
polyvinylpyrrolidone
(PVP) with an average molecular weight between 2,000 and 3,000, or
polyethylene glycol
(PEG) with an average molecular weight between 3,000 and 5,000. It is also
preferred to use
a buffer in the composition to minimize pH changes in the solution before
lyophilization or
after reconstitution. Most any physiological buffer may be used, but citrate,
phosphate,
succinate, and glutamate buffers or mixtures thereof are preferred.
Preferably, the
concentration of the buffer is from 0.01 to 0.3 molar. Surfactants that can be
added to the
formulation are shown in EP Nos. 270,799 and 268,110.
Additionally, TFPI and TFPI analogs can be chemically modified, for example by
covalent
conjugation to a polymer to increase its circulating half-life. Preferred
polymers and methods
to attach them to peptides are taught in U.S. Pat. Nos. 4,766,106, 4,179,337,
4,495,285, and
4,609,546. Preferred polymers are polyoxyethylated polyols and polyethylene
glycol (PEG).
PEG is soluble in water at room temperature and has the general formula: R(O--
CH<sub>2--</sub>
CH<sub>2</sub>)<sub>n--O--R</sub> where R can be hydrogen, or a protective group such as
an alkyl or
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WO 2006/113360 PCT/US2006/013890
alkanol group. Preferably, the protective group has between 1 and 8 carbons,
more preferably
it is methyl. The symbol n is a positive integer, preferably between 1 and
1,000, more
preferably between 2 and 500. The PEG has a preferred average molecular weight
between
1000 and 40,000, more preferably between 2000 and 20,000, most preferably
between 3,000
and 12,000. Preferably, PEG has at least one hydroxy group, more preferably it
is a terminal
hydroxy group. It is this hydroxy group which is preferably activated to react
with a free
amino group on the inhibitor. However, it will be understood that the type and
amount of the
reactive groups may be varied to achieve a covalently conjugated PEG/TFPI of
the present
invention.
Water soluble polyoxyethylated polyols are also useful in the present
invention. They include
polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol
(POG), etc.
POG is preferred. One reason is that the glycerol backbone of polyoxyethylated
glycerol is
the same baclcbone occurring naturally in, for example, animals and humans in
mono-, di-,
triglycerides. Therefore, this branching would not necessarily be seen as a
foreign agent in
the body. The POG has a preferred molecular weight in the same range as PEG.
The structure
for POG is shown in Knauf et al., 1988, J. Bio. Chem. 263:15064-15070, and a
discussion of
POG-protein conjugates is found in U.S. Pat. No. 4,766,106.
After a liquid pharmaceutical composition of TFPI or a TFPI analog is
prepared, it can be
lyophilized to prevent degradation and to preserve sterility. Methods for
lyophilizing liquid
compositions are known to those of ordinary skill in the art. Just prior to
use, the composition
may be reconstituted with a sterile diluent (Ringer's solution, distilled
water, or sterile saline,
for example) that may include additional ingredients. Upon reconstitution, the
composition is
preferably administered to subjects by continuous intravenous infusion.
Dosages of TFPI and TFPI Analogs
TFPI or TFPI analogs are administered at a concentration that is
therapeutically effective to
treat and prevent severe pneumonia. Such doses also are effective for other
acute or chronic
inflammations, and generally diseases in which cytokines upregulate tissue
factor expression.
To accomplish this goal, TFPI or TFPI analogs preferably are administered
intravenously.
Methods to accomplish this administration are known to those of ordinary skill
in the art.
Generally, TFPI or TFPI analogs are given at a dose between 1µg/kg and 30
mg/kg, more
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WO 2006/113360 PCT/US2006/013890
preferably between 20µg/kg and 25 mg/kg, most preferably between 1 and 15
mg/kg.
The above dosages are generally administered over a period of at least about
150 hours, and
preferably over a period of at least about 100 hours. In one embodiment,
administration of
TFPI is continued for about 99 to about 90 hours, preferably for about 97 to
about 94 hours,
and more preferably, for about 96 hours. The total daily dose administered to
a host in single
or divided doses may be in ainounts, for example, from about 2 to about 20
mg/kg body
weight daily and preferably from about 2 to about 15 mg/kg body weight daily
from about 4
to about 10 mg/kg. Dosage unit compositions may contain such amounts or
submultiples
thereof to malce up the daily dose. Lower daily dosage amounts may be useful
for
prophylactic or other purposes, for example, from 1µg/kg to 2 mg/kg. 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. Thus, the
dosage
regimen actually employed may vary widely and therefore may deviate from the
preferred
dosage regimen set forth above. Preferably, doses of TFPI or TFPI analogs
should not exceed
a dose rate equivalent to a dose rate of ala-TFPI of about 0.66 mg/kg/hr.
Low Dose Administration
When TFPI or a TFPI analog is given at a dose rate equivalent to
administration of ala-TFPI
at a dose rate of at least about 0.00025 mg/kg/hr (0.00417 µg/kg/min) and
less than about
2.00 mg/kg/hr (0.833 µg/kg/min), efficacy in treating severe pneumonia is
retained and
adverse side effects, such as bleeding, are minimized. In one preferred
embodiment, ala-
TFPI is administered at a dose rate of at least between about 0.02 mg/kg/hr to
about 1.0
mg/kg/hr, more preferably between about 0.24 mg/kg/hr to about 0.8 mg/kg/hr.
For
improved combined efficacy and safety, the dose rate preferably is equivalent
to a dose rate
of ala-TFPI of at least about 0.010 mg/kg/hr (0.167 µg/kg/min) and less
than about 0.045
mg/kg/hr (0.833 µg/kg/min), or equivalent to a dose rate of ala-TFPI of at
least about
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WO 2006/113360 PCT/US2006/013890
0.020 mg/kg/hr and less than about 0.040 mg/kg/hr, and most preferably
equivalent to a dose
rate of ala-TFPI of about 0.025 mg/kg/hr (0.417 µg/kg/min)). The route of
administration
is generally by intravenous administration, with continuous intravenous
infusion preferred.
Infusion can be administered for at least about 72, 96, 120, or 240 hours.
Preferably,
continuous infusion is administered for 3 to 8 days, more preferably 3 to 6
days, and most
preferably for about 4 days.
To administer "by continuous infusion" means that the infusion is maintained
at
approximately the prescribed rate without substantial interruption for most of
the prescribed
duration. Alternatively, intermittent intravenous infusion can be used. If
intermittent infusion
is used, then a time-averaged dose rate should be used which is equivalent to
the dose rates
described above for continuous infusion. In addition, the program of
intermittent infusion
must result in a maximum serum concentration not more than about 20% above the
maximum
concentration obtained using continuous infusion. To avoid adverse reactions
in the patient,
particularly side effects involving bleeding, the dose rate should be less
than a dose rate that
is equivalent to continuous intravenous infusion of ala-TFPI at about 0.050
mg/kg/hr.
All doses described herein, including dose rates and total doses, are subject
to up to 10%
variation in practice due to errors in determining protein concentration and
biological activity,
with the prothrombin assay. Thus, any actually administered dose up to 10%
higher or 10%
lower than a dose stated herein is considered to be equivalent to the stated
dose. For this
reason, all doses have been stated as "about" a specific dose. For example, a
dose described
as "about 0.025 mg/kg/hr" is considered equivalent to any actual dose ranging
from 0.0225 to
0.0275 mg/kg/hr.
A bolus injection or a briefly higher infusion rate of TFPI or an analog of
TFPI may also be
employed in the practice of the present invention if followed by low dose TFPI
administration. For example, a bolus injection or higher infusion rate can be
used to reduce
the equilibration time of administered TFPI or TFPI analog in the circulation
of a patient. In
doing so, the eventual steady state plasma level of TFPI can be reached more
rapidly and
receptors for TFPI can be saturated faster. Administration of ala-TFPI to
humans at about
0.025 mg/kg/hr for 2 hours increases plasma levels of TFPI (plus ala-TFPI)
from about 80
ng/ml to about 125 ng/ml, or an increase of approximately 50%. The same level
will be
reached faster if the infusion rate is increased, or a bolus injection is
used. Higher infusion
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WO 2006/113360 PCT/US2006/013890
rates will result in higher levels if infusion is continued until steady state
is obtained. Steady
state level for administration of ala-TFPI at about 0.050 mg/kg/hr was found
to be about 300
ng/ml, and for administration of ala-TFPI at about 0.33 or about 0.66 mg/kg/hr
was found to
be about at least 2µg/ml in patients suffering from sepsis.
Total daily dose administered to a host in a single continuous infusion or in
divided infusion
doses may be in amounts, for example, equivalent to administration of at least
about 0.006
mg/kg/day to less than about 1.2 mg/kg/day of ala-TFPI, more usually
equivalent to
administration of from about 0.24 mg/kg/day to less than about 1.2 mg/kg/day
of ala-TFPI,
and preferably equivalent to about 0.6 mg/kg/day of ala-TFPI. Lower amounts
within this
range may be useful for prophylactic or other purposes. Higher doses above
this range may
be useful for the treatment of severe CAP. The dosing protocols of the
invention can also be
expressed as the total dose administered to the patient. The total dose is the
mathematical
product of the rate of infusion and the total time of infusion. For example,
at the preferred
dose rate of about 0.025 mg/kg/hr for ala-TFPI and the preferred infusion time
of 96 hours,
the total dose is about 2.4 ing ala-TFPI per kg body weight. In one
embodiment, the preferred
dose rate of about 0.25 mg/kg/hr for ala-TFPI and the preferred infusion time
of 96 hours, the
total dose is about 24 mg ala-TFPI per kg body weight. In one embodiment, the
preferred
dose rate of about 0.75 mg/kg/hr for ala-TFPI and the preferred infusion time
of 96 hours, the
total dose is about 72 mg ala-TFPI per kg body weight. In another embodiment,
the total
dose of TFPI administered according to the invention is equivalent to at least
about 0.75
µg/kg and less than about 4.8 mg/kg of ala-TFPI. Preferably the total dose
is equivalent to
at least about 1 mg/kg and less than about 4.8 mg/kg of ala-TFPI. More
preferably the total
dose is equivalent to about 2.4 mg/kg of ala-TFPI.
One factor that can be used to adjust the dosage regimen is the individual
patient's
coagulation function, which is typically measured using a prothrombin time
(PT) assay, or
the International Normalized Ratio (INR). INR is the standardization of the PT
assay in
which the assay is calibrated against an international reference
thromboplastin reagent. See,
e.g., R. S. Riley et al., J. Clin. Lab. Anal. 14:101-114 (2000). The INR
response to ala-TFPI
in healthy human volunteers is approximately linear over the range of plasma
concentrations
seen (FIG. 3). The overall change in INR is 1.2 units per 1µg/ml increase
of plasma ala-
TFPI concentration.
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In a pharmacodynamic model, the INR response to ala-TFPI is best described by
a log-linear
model in which log INR was linearly related to ala-TFPI plasma concentration.
The log-linear
nature of the response means that subjects with elevated INR at baseline are
likely to
experience greater anticoagulant responses than subjects with low baseline
values who have
similar levels of circulating ala-TFPI.
The dosing regimens described above, including dosing rate on a mg/kg/hr basis
and total
daily dose, are expressed as a dose "equivalent to administration of reference
ala-TFPI." This
means that they are determined quantitatively by normalization to a dose of
"reference ala-
TFPI" which is defined as mature, 100% pure (on a protein basis), properly
folded,
biologically active, non-glycosylated ala-TFPI. Ala-TFPI is an analog of TFPI
whose amino
acid sequence is depicted in SEQ ID NO:2. Other forms of TFPI can also be used
in the
invention, including mature, full-length TFPI and analogs thereof. To
determine the
appropriate dosing range for practicing the invention with forms of TFPI other
than ala-TFPI
and with preparations of ala-TFPI or another TFPI analog that are less than
100% pure, the
dosing ranges described herein for reference ala-TFPI can be adjusted based on
the intrinsic
biological activity of the particular form of TFPI and further adjusted based
on the
biochemical purity of the preparation.
In a preferred embodiment, the patient has not received an anticoagulant
within 10 days of
receiving the first administration of TFPI. In a preferred embodiment, the
patient has not
received an anticoagulant within 7 days of receiving the first administration
of TFPI.
Preferably, the patient has not received a foim of heparin within 24 hours of
receiving the
first administration of TFPI. In one embodiment, the patient has not received
unfractioned
heparin within 10 hours, preferably within 12 hours of receiving the first
administration of
TFPI. In one embodiment, the patient has not received low molecular weight
heparin within
20 hours, preferably within 24 hours of receiving the first administration of
TFPI. In one
embodiment, the patient has not received drotrecogin-alpha within 10 hours,
preferably
within 12 hours of receiving the first administration of TFPI.
The intrinsic biological activity of TFPI or a TFPI analog refers to the
specific activity, as
defined by the prothrombin assay, of the mature, 100% pure, properly folded
TFPI or TFPI
analog. Thus, the equivalent dose is calculated as (reference ala-TFPI
dose)/((relative
intrinsic activity)×(biochemical purity)), where relative intrinsic
activity refers to
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(intrinsic activity of analog)/(intrinsic activity of reference ala-TFPI). For
example, if a
particular TFPI analog has an intrinsic biological activity which is 80% that
of reference ala-
TFPI, then the equivalent dose for the particular TFPI analog are obtained by
dividing the
dose values for reference ala-TFPI by 0.8. Further, if the formulation
administered to a
patient is, for example, only 90% biochemically pure, i.e., comprising 10% of
molecular
species which lack biological activity of TFPI, then an additional correction
of the reference
dose values for ala-TFPI is performed by dividing the dose values by 0.9.
Thus, for a
hypothetical TFPI analog that has 80% of the intrinsic activity of ala-TFPI
and is 90%
biochemically pure as administered, a dose rate equivalent to administration
of reference ala-
TFPI at 0.025 mg/kg/hr would be 0.0347 mg/kg/hr (i.e., 0.025/(0.8×0.9)).
Equivalent doses can also be determined without knowing either intrinsic
activity or
biochemical purity by determining relative biological activity. Relative
biological activity can
be determined by comparing a particular TFPI analog to a TFPI biological
activity standard
using the prothrombin time assay. For example, ala-TFPI produced according to
the method
of Example 9 of WO 96/40784, which contains about 85% biologically active TFPI
molecular species, can be used as a TFPI biological activity standard. Ala-
TFPI produced
according to the method of Example 9 of WO 96/40784 has about 85% of the
activity of
reference ala-TFPI in the prothrombin assay. In plotting a prothrombin time
standard curve,
the log of clotting time is plotted against the log of TFPI concentration. If
the TFPI biological
activity standard possesses 85% of the activity of reference ala-TFPI, then a
standard curve
can be prepared which is equivalent to that for reference ala-TFPI if the
concentrations of the
TFPI biological activity standard are multiplied by 0.85 prior to plotting, so
that the activity
plotted is equivalent to the activity of 100% pure reference ala-TFPI. When
the clotting time
for a particular TFPI analog is compared to the standard curve, the equivalent
concentration
of reference ala-TFPI can be read off the curve. Alternatively, if the slope
of the linear
portion of the standard curve is obtained by linear regression analysis, then
the slope can be
corrected based on the activity of the TFPI biological activity standard
relative to reference
ala-TFPI. The relative biological activity of a particular TFPI analog is thus
equal to the ratio
of reference ala-TFPI activity to the activity of the analog. For example, if
a particular analog
requires 1.43 mu.g to produce the same prothrombin time activity as 1.00 mu.g
of reference
ala-TFPI, then the relative biological activity of the analog is 1.00/1.43, or
0.7. For that
analog, the equivalent dose to a reference ala-TFPI dose is obtained by
dividing the reference
ala-TFPI dose by the relative biological activity of the analog. For example,
a 0.025 mg/kg/hr
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dose for reference ala-TFPI would be equivalent to 0.0357 mg/kg/hr of the
analog (i.e.,
0.025/0.7).
While TFPI or a TFPI analog can be administered as the sole active
anticoagulation
pharmaceutical agent, these molecules also can be used in combination with one
or more
additional therapeutic agents to provide a combination therapy for the
treatment of sever
pneumonia. Such additional therapeutic agents include antibodies such as, for
example, anti-
endotoxin, monoclonal antibodies (e.g., endotoxin-binding Mabs) and anti-TNF
products
such as an anti-TNF murine Mab. TFPI and TFPI analogs 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 (e.g., antiplatelet agents such as GPIIb/IIIa
inhibitors). When
administered as a combination, the therapeutic agents can be formulated as
separate
compositions that are given at the same time or different times. Preferably,
additional
therapeutic agents are given either at the same time (i.e., during the
administration period of
TFPI or TFPI analogs) or within 24 hours of the administration period of TFPI
or TFPI
analogs (i.e., within 24 hours prior to the start of, or within 24 hours after
the end of, the
administration period of TFPI or TFPI analogs). Additional therapeutic agents
can also be
given as a single composition together with the TFPI or TFPI analogs.
TFPI or a TFPI analog also can be given in combination with other agents that
would be
effective to treat severe pneumonia. For example, the following may be
administered in
combination with TFPI or a TFPI analog: antibiotics that can treat the
underlying bacterial
infection, monoclonal antibodies that are directed against bacterial cell wall
components,
receptors that can complex with cytokines that are involved in the severe
pneumonia
pathway, and generally any agent or protein that can interact with cytokines
or other activated
or amplified physiological pathways including complement proteins to attenuate
severe
pneumonia and/or its symptoms.
Useful antibiotics include those in the general category of: beta-lactam rings
(penicillin),
amino sugars in glycosidic linkage (aminoglycosides), macrocyclic lactone
rings
(macrolides), polycyclic derivatives of napthacenecarboxanide (tetracyclines),
nitrobenzene
derivatives of dichloroacetic acid, peptides (bacitracin, gramicidin, and
polymyxin), large
rings with a conjugated double bond system (polyenes), sulfa drugs derived
from
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sulfanilamide (sulfonamides), 5-nitro-2-furanyl groups (nitrofurans),
quinolone carboxylic
acids (nalidixic acid), and many others. Other antibiotics and more versions
of the above
specific antibiotics may be found in Encyclopedia of Chemical Technology, 3rd
Edition,
Kirk-Othymer (ed.), Vol. 2, pages 782-1036 (1978) and Vol. 3, pages 1-78,
Zinsser,
MicroBiology, 17th Edition W. Joldik et al. (Eds.) pages 235-277 (1980), or
porland's
Illustrated Medical Dictionary, 27th Edition, W. B. Saunders Company (1988).
Other agents that may be combined with TFPI or a TFPI analog include endotoxin
antagonists such as E5531 (a Lipid A analog, see Asai et al., Biol. Pharm.
Bull. 22:432
(1999)), TF analogs with anticoagulant activity (see, e.g., Kelley et al.,
Blood 89:3219 (1997)
and Lee & Kelley, J. Biol. Chem. 273:4149 (1998)), monoclonal antibodies
directed to
cytokines, such as those monoclonal antibodies directed to IL-6 or M-CSF, see
U.S. Ser. No.
07/451,218, filed Dec. 15, 1989, and monoclonal antibodies directed to TNF
(see Cerami et
al., U.S. Pat. No. 4,603,106), inhibitors of protein that cleave the mature
TNF prohormone
from the cell in which it was produced (see U.S. Ser. No. 07/395,253, filed
Aug. 16, 1989),
antagonists of IL-1 (see U.S. Ser. No. 07/517,276, filed May 1, 1990),
inhibitors of IL-6
cytokine expression such as inhibin (see U.S. Pat. No. 5,942,220), and
receptor based
inhibitors of various cytokines such as IL-1. Antibodies to complement or
protein inhibitors
of complement, such as CR<sub>1</sub>, DAF, and MCP also can be used.
All patents, patent applications, and references cited in this disclosure are
incorporated herein
by reference in their entireties.
The present invention will now be illustrated by reference to the following
examples that set
forth particularly advantageous embodiments. However, it should be noted that
these
embodiments are illustrative and are not to be construed as restricting the
invention in any
way.
EXAMPLES
Example 1
ala-TFPI Treatment of Severe Pneumonia Patients
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Patients with severe pneumonia were evaluated to explore the potential affect
of treatment
with ala-TFPI in a relatively homogeneous group. Pneumonia patients were
identified if one
source of sepsis documented by the investigator was coded as pneumonia. Other
sites of
infection could also be present. Due to the difficulty in differentiating
infectious from
chemical sequelae, patients with aspiration pneumonia were not included.
Patients identified
as having pneumonia were then classified as being culture positive (any
evidence of infection
such as culture or Gram stain), or culture negative (negative culture or
culture not done).
Patients were treated by continuous intravenous confusion with a preparation
of non-
glycosylated ala-TFPI expressed in E. coli at a dose of 0.025 mg/kg/h
formulated in a buffer
containing 300 mM L-arginine, 20 mM sodium citrate, pH 5.5, osmolarity 560+/-
110 mOsm.
Placebo consisted of the same buffer without ala-TFPI and was infused at the
same rate as the
study drug. Results of these analyses demonstrate a positive effect from ala-
TFPI treatment in
those patients with culture positive pneumonia (Table 1). Those patients
without evidence of
an infectious source demonstrated a negative effect.
Table 1. Mortality By Pneumonia Status
INR _ 1.2 Overall
Placebo TFPI p=
Pneumonia Culture Positive
(N=) 236 268
% Mortality 39.8% 31.3% 0.05
Pneumonia Culture Negative
(N=) 118 122
% Mortality 30.5% 45.1% 0.02
Table 2. Mortality By Pneumonia Status Low INR
INR < 1.2 Overall
Placebo TFPI p=
Pneumonia Culture Pos.
(N=) 33 22
% Mortality 30.3% 13.6% 0.15
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Pneumonia Culture Neg.
(N=) 25 23
% Mortality 32.0% 8.7% 0.08
The increased mortality in the high INR culture negative group appeared to be
present in
patient populations with or without added administration of heparin, although
it should be
noted that the number of subjects in the pneumonia culture negative, non-
heparin group is
relatively small (Table 3). A strong positive treatment effect was observed in
the culture
positive/no heparin cohort.
Table 3. Mortality by Pneumonia Status and Heparin
INR>= 1.2 Pneumonia Culture Positive Pneumonia Culture Negative
Placebo TFPI = Placebo TFPI =
Heparin at Baseline or During Dosing
(N=) 160 187 87 85
% Mortality 32.5% 31.6% 0.84 36.8% 56.5% 0.01
No Heparin at Baseline or During Dosing
(N=) 76 81 31 37
% Mortality 55.3% 30.9% 0.002 32.3% 48.6% 0.17
Example 2
Investigation of Baseline Severity of Illness Variables
A number of baseline severity of illness variables were evaluated to determine
whether there
were group imbalances that could explain the observed outcome. These data
indicate that the
difference in outcomes associated with culture status are not due to baseline
imbalances.
Accordingly, the results appear to represent a differential effect of TFPI
treatment due to
biological differences between patients with and without infection. Despite
the fact that the
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severity indicators(e.g., APACHE II score or organ dysfunction score) were
either equal to
placebo or lower in the TFPI treated pneumonia culture negative group the
culture negative
group demonstrated the highest overall mortality (Table 4).
Table 4. Baseline Severity of Illness by Pneumonia Status
INR >= 1.2 Pneumonia Culture Positive Pneumonia Culture Negative
Placebo TFPI Placebo TFPI
N= 236 268 118 122
% Mortality 39.8% 31.3% 30.5% 45.1%
APACHE II 25.8 25.9 24.3 25.2
INR 1.53 1.50 1.52 1.45
Mean Organ 3.0 3.0 3.0 2.9
Dysfunctions
CV- Hypotension 79% 74% 73% 72%
Acidosis 66% 66% 64% 58%
Oliguria 42% 48% 47% 49%
Pulmonary 93% 91% 91% 90%
Dysfunction
Thrombocytopenia 20% 23% 22% 16%
IL-6 is an inflammatory cytokine that is elevated early in sepsis, reflects
the intensity of the
inflammatory response and is associated with outcome. At baseline, IL-6 levels
are lower in
patients clinically identified as having pneumonia but without evidence of
infection (Table
5). This suggests that there is a biological difference between patients with
a documented
infectious source of pneumonia versus those without an apparent infectious
source.
Paradoxically, the culture negative TFPI group has the lowest baseline IL-6
levels but the
highest mortality rate. In a sepsis population IL-6 levels fall over time. The
rate of fall in IL-6
is reduced in TFPI treated pneumonia culture negative subjects (Table 5). This
suggests that
the biological effect of TFPI may differ in those patients with and without
infection.
Table 5. IL-6 By Pneumonia Status
INR> 1.2 Baseline 6 hrs (%A*) 24 hrs (%A*) 96 hrs (%A*)
Pneumonia
PL TFPI - PL TFPI - PL TFPI - PL TFPI p-
Culture
Positive 494 489 0.96 (25%) (27%) 0.75 (57%) (63%) 0.26 (83%) (84%) 0.69
(n=493)
Culture
Negative 300 195 0.11 (21%) (8%) 0.08 (54%) (32%) 0.03 (98%) (97%) 0.06
(n=236)
Example 3
Analysis of Severe Pneumonia Patients by Type of Documentation of Infection
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As discussed above, an overall benefit from ala-TFPI treatment was observed in
those
patients with the highest certainty of infection, i.e., those with a positive
blood culture. In an
analysis of severe pneumonia patients by type of documentation of infection, a
benefit from
ala-TFPI treatment was seen in both subjects with a positive blood culture and
those with
other evidence (Table 6). The effect was strongest in the bacteremia group,
i.e., the group
with the highest probability of infection or most demonstrable source of
infection.
Table 6. Mortality By Culture Status and Pneumonia Status
INR _ 1.2 Blood Culture Positive Other Culture Positive Culture Negative / ND
Placebo TFPI p= Placebo TFPI p= Placebo TFPI p=
Pneumonia
(N=) 80 107 156 161 110 110
%Mortality 38.8% 26.2% 0.07 40.4% 34.8% 0.30 30.9% 46.4% 0.02
As previously shown, patients with documentation of infection (blood+"other")
benefited
from TFPI treatment in the absence of heparin. This result is mostly due to
the benefit
derived from the pneumonia group (Table 7). This finding seems to indicate
that the benefit
from endogenous anticoagulants is greatest in those patients with severe
pulmonary
infections.
Table 7. Mortality By Infection Status, Pneumonia Status and Heparin Use
INR _ 1.2 Heparin No Heparin
Placebo TFPI I p= Placebo TFPI =
Documented Infection (Blood + "Other")
N= 433 442 211 207
% Morality 31.4% 31.9% 0.89 43.1 % 32.4% 0.02
Pneumonia (Culture Positive)
N= 160 187 76 81
% Mortalit 32.5% 31.6% 0.84 55.3% 30.9% 0.002
Non-Pneumonia Documented Infections (Documented minus Pneumonia)
N= 273 255 135 126% Mortalit 30.8% 32.2% 0.73 36.3% 33.3% 0.62
To further limit heterogeneity, future trials can be focused on community
acquired
pneumonia (CAP). Patients who develop pneumonia while in hospital (nosocomial
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pneumonia) are more likely to be colonized with pathogenic organisms and have
other
pulmonary disorders making the diagnosis of infectious pneumonia more
difficult. In
addition, patients with CAP are less likely to have been exposed to heparin
than patients with
nosocomial pneumonia. When data were analyzed by length of stay in hospital
prior
treatment, a similar benefit was noted for culture positive patients
hospitalized.ltoreq.2 days
(community acquired) versus those hospitalized longer than 2 days
(nosocomial). The
negative effect in the culture negative patients was seen primarily in the
nosocomial group
(Table 8).
Table 8. Mortality by Pneumonia Status and Time from Hospitalization
INR ? 1.2 Pneumonia Culture Positive Pneumonia Culture Negative
Placebo TFPI I p= Placebo TFPI
Community Acquired (> 2 Days)
(N=) 121 143 61 52
% Mortality 38.8% 29.4% 0.10 27.9% 30.8% 0.74
Nosocomial (> 2 days)
(N=) 115 125 57 70
% Mortality 40.9% 33.6% 0.24 33.3% 55.7% 0.01
Example 4
Alveolar fibrin deposition and alteration in fibrinolytic activity is a
hallmark of pneumonia
and acute lung injury. Tissue factor has been considered as an important
initiator of
coagulation in this setting and tissue factor pathway inhibitor (TFPI) has
been demonstrated
to prevent or reduce lung damage in animal models.
Recombinant tissue factor pathway inhibitor (tifacogin) has been investigated
in severe sepsis
and a phase III double-blind, placebo-controlled, randomized trial has been
completed
(TFP007). The study failed to demonstrate a significant reduction in 28-day
all-cause
mortality. A post-hoc analysis was performed to evaluate whether patients
developing severe
sepsis due to CAP might benefit from tifacogin. Also, the possible heparin
interaction and
importance of microbiological documentation was investigated.
Material and Methods
Patients (pts) enrolled in TFP007 with CAP as the primary cause of severe
sepsis were kept
for post hoc analysis. 28-day all cause mortality and safety were determined
for the overall
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CAP population. The influence of concomitant heparin administration for DVT
prophylaxis
and importance of microbiological documentation was further analysed.
Results
TFP007 included 1955 pts of whom 435 had CAP (TFPI 217, placebo 218). Baseline
characteristics were similar in the two groups (mean age 62.3 vs 62; mean
APACHE 1124.9
vs 24; mean OD 3 vs 3) in TFPI and placebo respectively (NS). 28-day all cause
mortality
data are provided below in table 9. Mortality tended to be reduced in pts
treated with
tifacogin compared to placebo in particular when no concomitant heparin was
administered
and in the presence of microbiological documentation.
Serious bleeding events occurred in 12 treated pts (5.5%) vs 5 pts (2.2%) in
placebo (NS).
CNS bleeding were observed in 6 tifacogin treated pts (2.7%) vs 2 in placebo
((1%) (NS) for
the first 28-day period.
Table 9 : 28-day all-cause mortality in CAP with regard to concomitant heparin
and
microbiological evidence
TFPI Placebo
N Mortality (%) N Mortality (%) P-value
CAPall 217 27% 218 33% 0.15
CAP + Hep 149 26.2 % 142 28.2 % 0.7
CAP - Hep 68 29.4 % 76 43.4 % 0.08
CAP + ME 153 28% 142 37% 0.09
CAP -ME 64 25% 76 26% 0.85
Hep : Heparin , ME: microbiological evidence
Conclusion
From this post hoc analysis, tifacogin seems to reduce 28-day all cause
mortality in patients
with severe CAP. A bigger benefit seems to be obtained in patients having a
documented
CAP and/or in the absence of concomitant heparin administration. The safety
profile is
acceptable. The potential benefit of tifacogin is currently evalutated in a
phase III clinical
study including pts with severe CAP.
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Occurrences of bleeding and serious bleeding for all patients enrolled in
TFP007, as well as
for various subpopulations, are provided below in tables 9A-9F.
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Tables 9A-9F
Table 9A: TFP007 Bleeding for TFP007 opulation (All Subjects)
Heparin TFPI Placebo P- ~
n (%) N n (%) Value*
Yes 665 154 (23%) 681 1130 (19%)
No 298 81(27%) 311 63 (20%)
* Chisq Test; + Hospital-acquired pneumonia
N=Number of subjects; n (%)=Number of bleeds (rate)
Table 9B: TFP007 Serious Bleeding for TFP007 o ulation (All Subjects)
Heparin TFPI Placebo P- ~
n (%) N n (%) Value*
Yes 665 43 (6.5%) 681 31 (4.6%)
No 298 18 (6.0%) 311 15(4.8%)
* Chisq Test; + Hospital-acquired pneumonia
N=Number of sub'ects; n (%)=Number of serious bleeds (rate)
Table 9C: TFP007 Bleedin for Sub o ulations CAP/HAP(All Subjects)
Heparin Pneumonia TFPI Placebo P-
n (%) N n (%) Value*
Yes CAP 149 38 (26%) 142 28 (20%)
HAP+ 163 40 (25%) 146 27(18%)
No CAP 68 14 (21%) 76 13 (17%)
HAP+ 55 13 (24%) 48 10 (21%)
* Chisq Test; + Hospital-acquired pneumonia
N=Number of subjects; n (%)=Number of bleeds (rate)
Table 9D: TFP007 Serious Bleeding for Sub o ulations CAP/HAP(Al1 Sub'ects)
Heparin Pneumonia TFPI Placebo P-
n (%) N n (%) Value*
.
Yes CAP 149 9(6.0%) 142 3(2.1%)
HAP+ 163 14 (8.6%) 146 8(5.5%)
No CAP 68 3(4.4%) 76 2(2.6%)
HAP+ 55 3(5.5%) 48 3(6.3%)
* Chisq Test; + Hospital-acquired pneumonia
N=Number of subjects; n (%)=Number of serious bleeds (rate)
Table 9E: TFP007 Bleedin for Sub o ulations CAP/HAP b ME doc (All Sub'ects)
Heparin Pneumonia F Pneumonia TFPI Placebo P- *
Status N n(%) N n (%) Value*
Documented 100 31(31%) 88 19 (22%) .1857
Yes Undocumented 49 7(14%) 54 9(17%) .7910
HAP+ Documented 107 25 (23%) 96 18 (19%) .4925
Undocumented 56 15 (27%) 50 9(18%) .3546
No CAP Documented 53 9(17%) 54 11(20%) .8049
Undocumented 15 5(33%) 22 2(9%) .0953
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HAP+ Documented 30 4(13%) 31 6(19%) .7315
Undocumented 25 9 (36%) 17 4 (24%) .5046
Fisher Exact Test; + Hospital-acquired pneumonia
N=Number of subjects; n (%)=Number of bleeds (rate)
Table 9F: TFP007 Serious Bleeding for Sub o ulations (All Subjects)
Heparin Pneumonia Pneumonia TFPI Placebo P-Valuel
Status n (%) N n (%)
CAP Documented 100 9(9%) 88 3(3%) .1430
Yes Undocumented 49 0(0%) 54 0(0%) -
HAP+ Documented 107 9(8%) 96 6(6%) .6018
Undocumented 56 5(9%) 50 2(4%) .4426
CAP Documented 53 2(4%) 54 2(4%) 1.000
No Undocumented 15 1(7%) 22 0(0%) .4054
HAP+ Documented 30 1(3%) 31 2(7%) 1.000
Undocumented 25 2(8%) 17 1(6%) 1.000
* Fisher Exact Test; + Hospital-acquired pneumonia
# N=Nuinber of sub'ects; n (%)=Number of serious bleeds (rate)
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CA 02605057 2007-10-15
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CA 02605057 2007-10-15
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Table 12
tfpi (all pts) placebo (all pts)
N mortality N mortality p-value
CAP 217 27% 218 33% 0.1532
CAP+hep 149 26.20% 142 28.20% 0.7022
CAP-hep 68 29.40% 76 43.40% 0.0818
CAP+ME 153 28% 142 37% 0.0913
CAP-ME 64 25% 76 26% 0.8592
ITFP0007 all 963 32.29% 992 32.56% NS
TFP-007 BL characteristics all INR combined
tfpi placebo
mean age 62.3 62
mean 24.9 24.8
APACHE
mean o/fs 3 3
44
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The present invention has been described with reference to specific
embodiments. However, this
application is intended to cover those changes and substitutions which may be
made by those
skilled in the art without departing fiom the spirit and the scope of the
appended claims.
Sequence CWU 1
2 1 276 PRT Homo sapiens 1 Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp
Thr Glu Leu
15 10 15 Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp 20 25
30 Gly Pro
Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr 35 40 45 Arg Gln Cys
Glu Glu Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn 50 55 60 Arg Phe Glu Ser Leu Glu Glu
Cys Lys Lys
Met Cys Thr Arg Asp Asn 65 70 75 80 Ala Asn Arg Ile Ile Lys Thr Thr Leu Gln
Gln Glu Lys
Pro Asp Phe 85 90 95 Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile
Thr Arg 100
105 110 Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly 115
120 125 Gly
Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys 130 135 140 Asn
Ile Cys Glu
Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly 145 150 155 160 Thr Gln Leu
Asn Ala Val
Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys 165 170 175 Val Pro Ser Leu Phe Glu
Phe His Gly
Pro Ser Trp Cys Leu Thr Pro 180 185 190 Ala Asp Arg Gly Leu Cys Arg Ala Asn
Glu Asn Arg
Phe Tyr Tyr Asn 195 200 205 Ser Val Ile Gly Lys Cys Arg Pro Phe Lys Tyr Ser
Gly Cys Gly
Gly 210 215 220 Asn Glu Asn Asn Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys
Lys 225 230
235 240 Lys Gly Phe Ile Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys 245
250 255 Arg Lys
Arg Lys Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe 260 265 270 Val Lys
Asn Met 275 2
277 PRT Homo sapiens 2 Ala Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp
Thr Glu 15
15 Leu Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp 20 25 30
Asp Gly
Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe 35 40 45 Thr Arg Gln
Cys Glu Glu
Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln 50 55 60 Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys
Lys Met Cys Thr Arg Asp 65 70 75 80 Asn Ala Asn Arg Ile Ile Lys Thr Thr Leu
Gln Gln Glu
Lys Pro Asp 85 90 95 Phe Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr
Ile Thr 100
105 110 Arg Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr 115
120 125 Gly
Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys 130 135 140 Lys
Asn Ile Cys
Glu Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr 145 150 155 160 Gly Thr Gln
Leu Asn Ala
Val Asn Asn Ser Leu Thr Pro Gln Ser Thr 165 170 175 Lys Val Pro Ser Leu Phe
Glu Phe His
Gly Pro Ser Trp Cys Leu Thr 180 185 190 Pro Ala Asp Arg Gly Leu Cys Arg Ala
Asn Glu Asn
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WO 2006/113360 PCT/US2006/013890
Arg Phe Tyr Tyr 195 200 205 Asn Ser Val Ile Gly Lys Cys Arg Pro Phe Lys Tyr
Ser Gly Cys
Gly 210 215 220 Gly Asn Glu Asn Asn Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala
Cys 225 230
235 240 Lys Lys Gly Phe Ile Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr 245
250 255 Lys Arg
Lys Arg Lys Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile 260 265 270 Phe Val
Lys Asn Met
275
46
