U.S. patent application number 13/263715 was filed with the patent office on 2012-05-17 for par-1 activation by metalloproteinase-1 (mmp-1).
This patent application is currently assigned to Tufts Medical Center, Inc.. Invention is credited to Georgios Koukos, Athan Kuliopulos.
Application Number | 20120121706 13/263715 |
Document ID | / |
Family ID | 42936908 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120121706 |
Kind Code |
A1 |
Kuliopulos; Athan ; et
al. |
May 17, 2012 |
PAR-1 Activation by Metalloproteinase-1 (MMP-1)
Abstract
Matrix metalloproteases (MMPs) play many important roles in
normal and pathological remodeling processes including
atherothrombotic disease, inflammation, angiogenesis and cancer.
This invention relates to the activation of protease-activated
receptor-1 (PAR-1) by endogenous platelet MMP-1 collagenase on the
surface of platelets. Exposure of platelets to fibrillar collagen
converts the surface-bound pro-MMP-1 zymogen to active MMP-1, which
promotes aggregation through PAR-1, MMP-1 is shown to cleave the
PAR-1 extracellular domain at a novel site, which then strongly
activates Rho-GTP signaling pathways, cell shape change and
motility, and MAPK signaling. Blockade of MMP-PAR 1 suppresses
thrombogenesis under arterial flow conditions and inhibited
thrombosis in animals. These studies provide a link between
matrix-dependent activation of metalloproteases and platelet-G
protein signaling and identify MMP-1/PAR-1 as a new target for the
treatment and prevention of arterial thrombosis and other
thrombotic diseases.
Inventors: |
Kuliopulos; Athan;
(Winchester, MA) ; Koukos; Georgios; (Boston,
MA) |
Assignee: |
Tufts Medical Center, Inc.
Boston
MA
|
Family ID: |
42936908 |
Appl. No.: |
13/263715 |
Filed: |
April 12, 2010 |
PCT Filed: |
April 12, 2010 |
PCT NO: |
PCT/US10/30783 |
371 Date: |
October 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61168360 |
Apr 10, 2009 |
|
|
|
61168353 |
Apr 10, 2009 |
|
|
|
Current U.S.
Class: |
424/484 ; 435/23;
435/7.21; 436/501; 514/13.8; 514/337; 530/300; 530/328 |
Current CPC
Class: |
A61L 2300/434 20130101;
C07K 2317/76 20130101; A61K 2039/505 20130101; A61L 17/005
20130101; A61L 2300/42 20130101; A61P 7/02 20180101; C07K 16/40
20130101; A61K 31/00 20130101; A61K 45/06 20130101; A61K 39/3955
20130101; A61L 31/16 20130101; A61P 7/00 20180101; A61P 9/00
20180101; A61P 9/10 20180101; A61L 27/54 20130101; A61L 2300/436
20130101; A61K 31/65 20130101; A61L 29/16 20130101; A61P 35/00
20180101; A61P 43/00 20180101; A61K 38/08 20130101; A61K 38/10
20130101 |
Class at
Publication: |
424/484 ;
530/300; 530/328; 436/501; 435/7.21; 435/23; 514/13.8; 514/337 |
International
Class: |
A61K 38/55 20060101
A61K038/55; C07K 7/06 20060101 C07K007/06; A61P 7/02 20060101
A61P007/02; C12Q 1/37 20060101 C12Q001/37; A61K 9/00 20060101
A61K009/00; A61K 31/443 20060101 A61K031/443; C07K 2/00 20060101
C07K002/00; G01N 33/566 20060101 G01N033/566 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under Grant
Nos. R01 HL-57905, R01 HL-64701, and R01 CA-122992, awarded by the
National Institutes of Health. The government has certain rights in
this invention.
Claims
1-71. (canceled)
72. An isolated polypeptide, its sequence comprising no less than 5
contiguous amino acid residues of one of the two fragments that
result from a proteolytic cleavage between aspartic acid at
position 39 (D39) and proline at position 40 (P40) of human
protease-activated receptor-1 (PAR-1), said polypeptide further
terminates at one end with a cleavage site that would have resulted
from said proteolytic cleavage.
73. The isolated polypeptide of claim 72, wherein said polypeptide
has a proline at its N terminus.
74. The isolated polypeptide of claim 72, comprising the
polypeptide sequence of PRSFLLRN (SEQ ID NO:1).
75. A method of diagnosing a thrombotic disease state in a patient,
comprising measuring an amount of the polypeptide of claim 72 in
platelets taken from a patient.
76. A method of identifying a PAR-1 antagonist comprising the steps
of: (a) providing the isolated polypeptide of claim 72; (b)
providing a candidate agent; (c) contacting platelets with said
isolated polypeptide in the presence of said candidate agent; (d)
measuring PAR-1 signaling activity, and (e) comparing said PAR-1
signaling activity in the presence of said candidate agent to said
PAR-1 signaling activity in the absence of said candidate agent,
wherein a decrease of at least 10% in PAR-1 signaling activity in
the presence of said candidate agent as compared to PAR-1 signaling
activity in the absence of said candidate agent identifies said
candidate agent as a PAR-1 antagonist.
77. The method of claim 76, wherein said PAR-1 signaling activity
comprises Rho-GTP or MAPK pathway signaling.
78-79. (canceled)
80. A medical device coated with a matrix layer comprising an agent
that substantially inhibits proteolytic cleavage between aspartic
acid at position 39 (D39) and proline at position 40 (P40) of said
patient's protease-activated receptor-1 (PAR-1).
81. A medical device coated with a matrix layer comprising an agent
that substantially inhibits protease-activated receptor-1 (PAR-1)
signaling activity that results from proteolytic cleavage of PAR-1
between aspartic acid at position 39 (D39) and proline at position
40 (P40).
82. The medical device of claim 81, wherein said agent comprises
SCH 530348.
83. The medical device of claim 81, wherein said matrix layer is a
biocompatible peptide matrix.
84. The medical device of claim 81, wherein said device is
implantable.
85. The medical device of claim 81, wherein said agent comprises a
pepducin lipopeptide of a PAR family member.
86. The medical device of claim 85, wherein said pepducin
lipopeptide of a PAR family member comprises a PAR-1 pepducin
lipopeptide.
87. The medical device of claim 86, wherein said PAR-1 pepducin
lipopeptide is selected from the group consisting of P1i3pal-7,
P1i3pal-12, P1i3pal-12S, P1i3pal-10S, P1i1pal-11, P1i2pal-7,
P1i2pal-11, P1i2pal-16, P1i2pal-21, P1i4pal13 and P1i4pal13R.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of
co-pending U.S. provisional patent application Ser. Nos. 61/168,353
and 61/168,360, both of the above title and both filed on Apr. 10,
2009, which applications are incorporated herein by reference in
entirety to the extent allowed by applicable laws.
FIELD OF INVENTION
[0003] The present invention relates to the diagnosis and treatment
of thrombotic conditions including those related to acute coronary
syndrome and atherosclerosis. The invention also relates to means
of preserving platelets for research or clinical uses.
BACKGROUND
[0004] Platelet activation and aggregation, while needed for normal
physiological functions such as hemostasis, can lead to a myriad of
oft-lethal and highly debilitating conditions and pathologies when
her regulatory mechanisms malfunction. These pathological
conditions can be acute or chronic, and include acute coronary
syndrome, myocardial infarction, unstable angina, stroke, coronary
thrombosis, venous thrombosis, atherothrombosis, restenosis and so
on. In the United States, Europe, and other industrialized nations,
myocardial infarction due to rupture of atherosclerotic plaques is
a leading contributor to morbidity and mortality. Acute plaque
rupture exposes subendothelial collagen which promotes platelet
activation and formation of a potentially occlusive thrombus at the
site of vascular damage (Glass and Witztum, 2001; Ruggeri, 2002).
Following their initial tethering to subendothelial collagen and
matrix proteins, activation of transiently adhered platelets by
autocrine mediators is critical for the propagation of the
formative platelet thrombus. Reinforcement of the transient
adhesive contacts by activating G protein-dependent shape change,
granule release, and integrins permits growth of a stable thrombus
that is resistant to the high shear stress of arterial blood flow
(Jackson et al., 2003; Moers et al., 2003). Drugs that target the
secondary autocrine mediators of platelet thrombus formation such
as aspirin and thienopyridines have proven to be beneficial,
however, many patients taking these thugs still sustain thrombotic
events, and, therefore, might benefit from new therapeutics that
interfere with matrix-dependent platelet activation (Bhatt and
Topol, 2003).
[0005] Two distinct pathways act in parallel to activate platelets
during hemostasis (Furie and Furie, 2008). As the blood vessel wall
gets breached, platelets circulating in blood first encounter
collagen embedded in the subendothelial matrix. As a first line of
defense, exposed collagen initiates the accumulation and activation
of platelets and starts the formation of a thrombus. As blood flows
out further, it encounters a second line of defense, the tissue
factor located in the medial and adventitial layers of the vessel
wall, and a second independent pathway is triggered that also
activates platelets to adhere to each other and form part of the
developing thrombus. The tissue factor-initiated pathway generates
thrombin which in turn cleaves protease-activated receptor 1(PAR1)
on the human platelet surface, causing them to release adenosine
diphosphate (ADP), serotonin, and thromboxane A.sub.2. In turn,
these agonists recruit and activate other platelets, amplifying the
signal in order to block off the breach in the vessel wall. The
present invention, however, is based on discoveries that center
around the other, collagen-initiated platelet activation pathway,
i.e., the first line of defense in a thrombotic event.
[0006] Matrix metalloproteases (MMPs) have recently emerged as
important mediators of platelet function and vascular biology.
Initially described as extracellular matrix remodeling enzymes
involved in tissue repair and cancer invasion (Egeblad and Werb,
2002), a renewed focus has centered on MMPs and the related
metalloprotease disintegrins because of their prominence in
vascular wall inflammation (Dollery and Libby, 2006) and thrombotic
thrombocytopenic purpura (Levy et al., 2001). Endogenous platelet
metalloproteases have been shown to damage platelet function by
cleaving cell surface receptors and broad-spectrum metalloprotease
inhibitors improve post-transfusion recovery of platelet
concentrates (Bergmeier et al., 2003; Bergmeier et al., 2004;
Stephens et al., 2004). Platelets express several metalloproteases
including MMP-1, MMP-2, MMP-3, and MMP-14 on their surface (Chesney
et al., 1974; Galt et al., 2002: Kazes et al., 2000: Sawicki et
al., 1997). Notably, endogenous MMP-1 and MMP-2 can actually
promote platelet aggregation but the cell surface target(s) and
mechanism of activation have not been elucidated (Galt et al.,
2002; Sawicki et al., 1997). A recent study that examined the
effects of MMP-1 promoter polymorphisms in 2000 patients, found a
significantly increased risk of myocardial infarction in patients
with high promoter activity haplotypes and a significantly
decreased risk in patients with low promoter activity haplotypes
(Pearce et al., 2005).
[0007] It was recently shown that the G protein-coupled receptor,
PAR1, is directly cleaved and activated on the surface of cancer
cells by fibroblast-derived MMP-1 (Boire et al., 2005). PAR1 is the
major thrombin receptor of human platelets (Coughlin, 2000; Leger
et al., 2006b) and is an important mediator of platelet aggregation
following tissue factor (TF)-dependent generation of thrombin
(Mackman, 2004; Schwertz et al., 2006). However, under
pathophysiologic conditions of acute plaque rupture, exposed
collagen is the most efficient stimulus of the critical early
events of platelet recruitment and propagation under arterial flow
which could trigger metalloprotease activation on the platelet
surface.
SUMMARY OF INVENTION
[0008] The present invention is based on a novel
metalloprotease-dependent pathway of platelet thrombogenesis
through PAR1. Exposure of platelets to collagen caused activation
of MMP-1 which in turn directly cleaved PAR1 on the surface of
platelets. Unexpectedly. MMP-1 cleaved the N-terminal extracellular
domain of PAR1 at a distinct site from the thrombin cleavage site.
This cleavage event generated a longer tethered peptide ligand
which was an angonist of platelet activation and PAR1 signaling,
Blocking the MMP1-PAR1 pathway inhibited physcilogical events such
as collagen-dependent thrombogenesis, arterial thrombosis and clot
retraction. Accordingly, the present invention provides methods and
therapeutics that target this metalloprotease-receptor system in
treatment of patients diagnosed with or at risk of developing a
thrombotic disease state such as acute coronary syndromes.
[0009] In one aspect, the invention provides for a method of
treating a patient diagnosed with or at substantial risk of
developing a thrombotic disease state by administering a
therapeutically effective amount of an agent that substantially
inhibits proteolytic cleavage between aspartic acid at position 39
(D39) and proline at position 40 (P40) of said patient's
protease-activated receptor-1 (PAR-1). The proteolytic cleavage may
require an enzymatic activity by matrix metalloprotease-1
(MMP-1).
[0010] The patient may be exhibiting or has exhibited one or more
symptoms such as chest pain, shortness of breath, tightness around
chest, tightness in left arm, tightness in left angle of jaw,
excessive sweating, nausea, vomiting, palpitation, anxiety, or
atypical sensation. The patient may have one or more ascertainable
or diagnosable risk factors associated with a thrombotic disease
state.
[0011] A thrombotic disease state may be any pathology that results
from platelet aggregation, including but not limited to acute
coronary syndrome, arterial thrombosis, venous thrombosis,
peripheral arterial disease, unstable angina, atrial fibrillation,
first myocardial infarction, recurrent myocardial infarction,
ischemio sudden death, transient ischemic attack, stroke,
atherosclerosis, deep vein thrombosis, thrombophlebitis, arterial
embolism, coronary arterial thrombosis, cerebral arterial
thrombosis, cerebral embolism, kidney embolism or pulmonary
embolism.
[0012] In one embodiment, the method of the invention is used to
treat a patient diagnosed with cancer.
[0013] In one feature, the administration of the agent
substantially inhibits platelet activation in a patient. The agent
may be a ligand-binding molecule that binds to PAR-1, substantially
inhibits the cleavage of PAR-1 by binding over the cleavage site or
substantially inhibits the cleavage of PAR-1 by inducing a
conformational change in PAR-1. The agent may include a
ligand-binding molecule that binds to MMP-1 or an antibody that is
specific for MMP-1 or PAR-1. The agent may also include a small
molecule that binds to MMP-1 or PAR-1.
[0014] In some embodiments, the agent substantially inhibits
activation of matrix metalloprotease-1 (MMP-1) or MMP-1 enzymatic
activity, cleavage of proMMP-1 by a protease, cleavage of proMMP-1
by matrix metailoprotease-2 (MMP-2) or collagen-initiated MMP-1
activation. The agent may be FN-439, tissue inhibitors of
metalloprotease (TIMPs), MMP-200, Cipemastat (Trocade),
Prinomastat, BAY 12-9566, Batimistat, BMS-275291, Marimastat,
MMI270(B), Metastat, Ro 32-3555, RS-130,830, PD 166793,
Ancorinosides B-D, a tetracycline compound or doxycycline.
[0015] The method of the present invention further provides for
administering to the patient a second agent that substantially
inhibits at least one of thromboxane- and ADP-signaling pathways in
patient's platelets, at least some of PAR-1's enzymatic activity or
thrombin-dependent activation of PAR1. The second agent complements
the first agent, e.g., by inhibiting the tissue-factor-initiated
hemostatic pathway.
[0016] The method further provides for the administration of a
second anti-thrombotic agent including anti-platelet drugs,
anti-coagulant drugs, or thrombolytic drugs. The second
anti-thrombotic agent may be thienopyridines, prostaglandin
analogs, COX inhibitors, vitamin K antagonists, glycoprotein
IIB/IIIA inhibitors or thrombin inhibitors.
[0017] In another embodiment, the second agent may be aspirin,
clopidogrel, ticlopidine, prasugrel, heparin, aboiximab,
eptifibatid, tirofiban and bivalirudin.
[0018] In another embodiment, the second agent may be a pepducin
lipopeptide of a PAR family member or a PAR-1 pepducin lipopeptide
such as P1i3pal-7, P1i3pal-12, P1i3pal-12S, P1i3pal-10S,
P1i1pal-11, P1i2pal-11, P1i2pal-16, P1i2pal-21, P1i4pal13 or
P1i4pal13R.
[0019] The method further provides for administration of the agent
by intravenous (I.V.) injection, subcutaneous injection,
intramuscular injection, oral ingestion, nasal, topical, rectal,
vaginal or parenteral intake. The agent may be formulated with a
pharmaceutically acceptable excipient, carrier or diluent.
[0020] In a second aspect, the invention provides for a method of
treating a thrombotic disease state in a patient by administering
to a patient diagnosed with or at substantial risk of developing a
thrombotic disease state a therapeutically effective amount of an
agent that substantially inhibits the patents protease-activated
receptor-1 (PAR-1) signaling activity that results from proteolytic
cleavage of PAR-1 between aspartic acid at position 39 (D39) and
praline at position 40 (P40). In one embodiment, the agent
comprises SCH 530348.
[0021] In another embodiment, the agent comprises a pepducin
lipopeptide of a PAR family member or a PAR-1 pepducin lipopeptide
such as P1i3pal-7, P1i3pal-12, P1i3pal-12S, P1i3pal-10S,
P1i1pal-11, P1i2pal-7, P1i2pal-11, P1i2pal-16, P1i2pal-21,
P1i4pal13 or P1i4pal13R.
[0022] In a third aspect, the invention provides for a method of
treating a patient diagnosed with or at substantial risk of
developing a thrombotic disease state by administering a
therapeutically effective amount of an agent that substantially
inhibits activation of matrix metalloprotease-1 (MMP-1) or MMP-1
enzymatic activity.
[0023] The agent substantially inhibits cleavage of proMMP-1 by a
proteinase, cleavage of proMMP-1 by matrix metailoprotease-2
(MMP-2) or collagen-initiated MMP-1 activation. The agent may be
FN-439, tissue inhibitors of metalloprotease (TIMPs), MMP-200,
Cipemastat (Trocade), Prinomastat, BAY 12-9566, Batimistat,
BMS-275291, Marimastat, MMI270(B), Metastat, Ro 32-3555,
RS-130,830, PD 166793, Ancorinosides B-D, a tetracycline compound
or doxycycline.
[0024] In a fourth aspect, the invention provides for a method of
treating a patient diagnosed with or at substantial risk of
developing atherosclerosis by administering a therapeutically
effective amount of an agent that substantially inhibits
proteolytic cleavage between aspartic acid at position 39 (D39) and
proline at position 40 (P40) of said patients protease-activated
receptor-1 (PAR-1).
[0025] The agent may be administered after an angioplasty
procedure, a coronary bypass procedure, or an open-heart surgery
has been performed on the patient but preferably for no more than
two weeks.
[0026] In a fifth aspect, the invention provides a method of
treating atherosclerosis by administering to a patient diagnosed
with or at substantial risk of developing atherosclerosis a
therapeutically effective amount of an agent that substantially
inhibits the patients protease-activated receptor-1 (PAR-1)
signaling activity that results from proteolytic cleavage of PAR-1
between aspartic acid at position 39 (D39) and praline at position
40 (P40).
[0027] In one aspect, the agent reduces the size of atherosclerotic
plaque within the aorta of the patient,
[0028] In one embodiment, the agent comprises SCH 530348.
[0029] In another embodiment, the agent comprises a pepducin
lipopeptide of a PAR family member or a PAR-1 pepducin lipopeptide
such as P1i3pal-7, P1i3pal-12, P1i3pal-12S, P1i3pal-10S;
P1i1pal-11, P1i2pal-11, P1i2pal-16; P1i2pal-21, P1i4pal13 or
P1i4pal13R.
[0030] In a sixth aspect, the invention provides for a method of
treating a patient diagnosed with or at substantial risk of
developing atherosclerosis by administering a therapeutically
effective amount of an agent that substantially inhibits activation
of matrix metalloprotease-1 (MMP-1) or MMP-1 enzymatic
activity.
[0031] In another aspect, the invention also provides for a medium
for platelet storage or transportation having an effective
concentration of an agent that substantially inhibits proteolytic
cleavage between aspartic acid at position 39 (D39) and proline at
position 40 (P40) of protease-activated receptor-1 (PAR-1) on
platelets contained therein. The medium may be an aqueous solution
further containing glucose and the average half-life of a normal
platelet contained therein is no less than about 5 days or 1 month
or 6 months. The medium may have an effective concentration of an
agent that inhibits activation of matrix nietalloprotease-1 (MMP-1)
or MMP-1 enzymatic activity. The medium may have include a pepducin
lipopeptide of a PAR family member or a PAR-1 pepducin lipopeptide
such as P1i3pal-7, P1i3pal-12, P1i3pal-12S, P1i3pal-10S;
P1i1pal-11, P1i2pal-11, P1i2pal-16, P1i2pal-21, P1i4pal13 or
P1i4pal13R.
[0032] In another aspect, the invention provides for a medium for
platelet storage or transportation, said medium having an effective
concentration of an agent that substantially inhibits
protease-activated receptor-1 (PAR-1) signaling activity that
results from proteolytic cleavage of PAR-1 between aspartic acid at
position 39 (D39) and proline at position 40 (P40).
[0033] In one embodiment, the agent comprises SCH 530348.
[0034] In yet another aspect, the invention provides a method of
diagnosing a risk for suffering a hemorrhagic event in a patient by
determining whether the patient has a genetic defect that
substantially inhibits activation of matrix metailoprotease-1
(MMP-1) or MMP-1 activity inside the patient.
[0035] In a further aspect, the invention provides a method of
diagnosing a hemophilic or coagulopathic condition or a risk
thereof in a patient by determining whether the patient has a
genetic defect that over-stimulates activation of matrix
metalioprotease-1 (MMR-1) or MMP-1 enzymatic activity inside the
patient.
[0036] The invention further provides an isolated polypeptide
having a sequence comprising no less than 5 contiguous amino acid
residues of one of the two fragments that result from a proteolytic
cleavage between aspartic acid at position 39 (D39) and proline at
position 40 (P40) of human protease-activated receptor-1 (PAR-1)
polypeptide that terminates at one end with a cleavage site that
would have resulted from the proteolytic cleavage. The polypeptide
of the invention can have a proline at its N terminus and, e.g.,
have the polypeptide sequence of PRSFLLRN (SEQ ID NO, 1).
Alternately, the polypeptide of the invention can have an aspartic
acid at its C terminus and have at least another four amino acid
residues as shown to the left of 039 in FIG. 9B, which provides the
full polypeptide sequence of human PAR-1 and in which the D39 and
P40 straddling the cleavage site are bolded and underlined.
[0037] The invention also provides for a method of diagnosing a
thrombotic disease state in a patient by measuring the amount of
the polypeptide of the invention in platelets taken from a
patient.
[0038] In yet another aspect, a method of identifying a PAR-1
antagonist is disclosed having the steps of providing an isolated
polypeptide of the invention having a sequence comprising no less
than 5 contiguous amino acid residues of one of the two fragments
that result from a proteolytic cleavage between aspartic acid at
position 39 (D39) and proline at position 40 (P40) of human
protease-activated receptor-1 (PAR-1) polypeptide that terminates
at one end with a cleavage site that would have resulted from the
proteolytic cleavage, providing a candidate agent, contacting
platelets with the isolated polypeptide in the presence of said
candidate agent, measuring PAR-1 signaling activity, and comparing
the PAR-1 signaling activity in the presence of the candidate agent
to the PAR-1 signaling activity in the absence of the candidate
agent, wherein a decrease of at least 10% in PAR-1 signaling
activity in the presence of the candidate agent as compared to
PAR-1 signaling activity in the absence of the candidate agent
identifies the candidate agent as a PAR-1 antagonist.
[0039] The PAR-1 signaling activity may include Rho-GTP or MARK
pathway signaling,
[0040] In a further aspect, a method of identifying a PAR-1
antagonist is disclosed having the steps of providing activated
MMP-1, providing a candidate agent, contacting platelets with the
activated MMP-1 in the presence of the candidate agent under
conditions where MMP-1 cleaves PAR-1, measuring PAR-1 signaling
activity, and comparing the PAR-1 signaling activity in the
presence of the candidate agent to the PAR-1 signaling activity in
the absence of the candidate agent, wherein a decrease of at least
10% in PAR-1 signaling activity in the presence of the candidate
agent as compared to PAR-1 signaling activity in the absence of the
candidate agent identifies the candidate agent as a PAR-1
antagonist.
[0041] The PAR-1 signaling activity may include Rho-GTP or MARK
pathway signaling.
[0042] In one aspect, the invention discloses a medical device
coated with a matrix layer comprising an agent that substantially
inhibits proteolytic cleavage between aspartic acid at position 39
(D39) and praline at position 40 (P40) of said patient's
protease-activated receptor-1 (PAR-1).
[0043] In another aspect, the invention discloses a medical device
coated with a matrix layer comprising an agent that substantially
inhibits protease-activated receptor-1 (PAR-1) signaling activity
that results from proteolytic cleavage of PAR-1 between aspartic
acid at position 39 (D39) and proline at position 40 (P40). In one
embodiment, the agent comprises SCH 530348.
[0044] In another embodiment, the agent comprises a pepducin
lipopeptide of a PAR family member or a PAR-1 pepducin lipopeptide
such as P1i3pal-7, P1i3pal-12, P1i3pal-12S, P1i3pal-10S,
P1i1pal-11, P1i2pal-7, P1i2pal-11, P1i2pal-16, P1i2pal-21,
P1i4pal13 or P1i4pal13R.
[0045] The matrix layer may be a biocompatible peptide matrix. The
medical device may be implantable. The matrix may further include a
pepducin lipopeptide of a PAR family member or a PAR-1 pepducin
lipopeptide, such as P1i3pal-7, P1i3pal-12, P1i3pal-12S,
P1i3pal-10S, P1i1pal-11, P1i2pal-7, P1i2pal-11, P1i2pal-16,
P1i2pal-21, P1i4pal13 or P1i4pal13R.
[0046] The previously described embodiments have many advantages,
including methods for the discovery and administration of agents
that inhibit the MMP-1 mediated PAR-1 signaling pathway. The
methods, compositions and kits disclosed herein are therefore
particularly useful for treatment of patients diagnosed with or at
risk of acquiring a thrombotic disease state.
[0047] It should be understood that this application is not limited
to the embodiments disclosed in this Summary, and it is intended to
cover modifications and variations that are within the scope of
those of sufficient skill in the field, and as defined by the
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0048] FIG. 1A shows the MMP activity in human platelets after
treatment with either ADP, U-46619, or type-I collagen.
[0049] FIG. 1B shows ELISA measurements of released and
platelet-associated MMP-1 pro-domains in the pellets and
supernatants collected from the platelets of FIG. 1.
[0050] FIG. 1C shows the expression of MMP-1 on the surface of
platelets as determined by flow cytometry.
[0051] FIG. 1D shows proMMP-1 associates with integrins in resting
human platelets.
[0052] FIG. 1E shows MMP-1 and collagen cause the release of an
N-terminal thrombin-cleavage fragment from the extracellular domain
of PAR1.
[0053] FIG. 1F depicts a dot blot analysis that shows MMP-3 and
MMP-7 are unable to release a N-terminal PAR-1 peptide from resting
platelets.
[0054] FIG. 1G depicts a dot blot analysis that shows a MMP-1
specific antibody can block the collagen-induced release of the
N-terminal PAR-1 peptide from resting platelets.
[0055] FIG. 1H depicts a dot blot analysis that shows ADP and
U-46619 can promote the release of the N-terminal PAR-1 peptide
from resting platelets.
[0056] FIG. 1I depicts a dot blot analysis that shows blocking
either the P2Y12 ADP, receptor with AR-069931MX (ARC) or
thromboxane with aspirin (ASA) had no effect on the
collagen-dependent release of the PAR1 N-terminal peptide.
[0057] FIG. 2A shows the identification of the MMP-1 cleavage site
on the TR26 N-terminal peptide region of PAR1.
[0058] FIG. 2B shows the location of the cleavage of PAR1
N-terminal extracellular mutants by thrombin and MMP-1.
[0059] FIG. 2C shows the cleavage of PAR1 N-terminal extracellular
mutants by thrombin.
[0060] FIG. 2D shows the cleavage of PAR1 N-terminal extracellular
mutants by MMP-1.
[0061] FIG. 2E shows RhoA signaling by the different PAR-1 mutants
in the presence of thrombin or MMP-1
[0062] FIG. 2F depicts the chemotactic migration of MCF-7 cells
expressing thrombin and MMP1-cleavage site mutants.
[0063] FIG. 2G shows the cleavage of PAR1 N-terminal extracellular
domain mutants expressed on COS7 Cells using MMP-1 purified from
another source.
[0064] FIG. 2H shows the cleavage of wild-type PAR-2 expressed on
COS7 Cells by MMP-1 or trypsin.
[0065] FIG. 2I shows the cleavage of wild-type PAR-3 expressed on
COS7 Cells by MMP-1 or thrombin.
[0066] FIG. 2J shows the cleavage of wild-type PAR-4 expressed on
COS7 Cells by MMP-1 or thrombin.
[0067] FIG. 3A shows the PRSFLLRN peptide (PR-TRAP) induces
PAR1-dependent RhoA activation in platelets.
[0068] FIG. 3B shows the PRSFLLRN peptide (PR-TRAP) activates p38
MAPK in platelets.
[0069] FIG. 3C shows changes in platelet shape induced by the
PRSFLLRN peptide (PR-TRAP).
[0070] FIG. 4A shows that MMP-1 activates Rho-GTP in platelets.
[0071] FIG. 4B shows MMP-1 can induce changes in platelet
shape.
[0072] FIG. 4C shows MMP-1 induces PAR I-dependent calcium fluxes
in platelets.
[0073] FIG. 4D shows MMP-1 induces platelet aggregation.
[0074] FIG. 4E shows MMP-1 activates p38MAN in platelets.
[0075] FIG. 4F shows MMP-1 activates the downstream MAPKAP-K2 in
platelets.
[0076] FIG. 5A shows the effect of pharmacologic blockage of
metalloproteases or PAR1 on platelet aggregation in the presence of
5 mg/ml collagen.
[0077] FIG. 5B shows the effect of pharmacologic blockage of
metalloproteases or PAR1 on platelet p38 MAPK activity in the
presence of 5 mg/ml collagen.
[0078] FIG. 5C shows the effect of pharmacologic blockage of
metalloproteases or PAR1 on platelet Rho-GTP activity in the
presence of 5 mg/ml collagen.
[0079] FIG. 5D shows the effect of various blocking Abs
(anti-MMP-1, anti-MMP-8 and anti-MMP-13) and various inhibitors
(ARC(P2Y12 antagonist AR-C69931MX), ASA (aspirin)) on platelet
Rho-GTP activity in the presence of 5 mg/ml collagen alone or
together with MMP-1 (Calbiochem or S2, BioMol).
[0080] FIG. 5E shows effect of a particular pepducin (P1pal-7
denoted as "PZ-128") on platelet aggregation in the presence of 5
mg/ml collagen.
[0081] FIGS. 6A-6B show that inhibition of MMP-1 or PAR1 prevents
early micro-thrombus formation on collagen surfaces in the presence
of heparin.
[0082] FIGS. 6C-6D show that inhibition of MMP-1 or PAR1 prevents
early platelet micro-thrombus formation on collagen surfaces
independently of thrombin.
[0083] FIG. 6E shows that P1pal-7 and FN-439 protects against
collagen-induced systemic platelet activation in guinea pigs.
[0084] FIGS. 6F and 6G show that inhibition of PAR1 and/or MMP-1
prevents occlusion of carotid arteries in guinea pigs.
[0085] FIG. 6H shows the detection of MMP-1 activity in guinea pig
platelet supernatant and arterial clot induced by collagen.
[0086] FIG. 6I are photographic depiction of clot retraction assays
with various agents added to platelet-rich human plasma. The top
block were photographs taken after 90 minutes of incubation and the
bottom block after 240 minutes.
[0087] FIG. 7A shows the surface expression of MMP-1 on guinea pig
platelets as determined by flow cytometry.
[0088] FIG. 7B shows the enzymatic activity of active MMP-1 in
guinea pig platelet lysates and supernatants in the presence or
absence of FN-439, control IgG or a MMP-1 blocking antibody.
[0089] FIG. 7C shows the effect of pharmacologic blockage of
metalloproteases or PAR1 on guinea pig platelet aggregation in the
presence of 10 mg/ml collagen.
[0090] FIG. 7D shows Rho GIP activity in the platelets used in FIG.
7C.
[0091] FIGS. 8A-8F show that pharmacologic inhibition of Matrix
Metalloprotease-2 (MMP-2) attenuates collagen-dependent platelet
aggregation to a similar extent as blockade of MMP-1.
[0092] FIG. 9A depicts a proposed model of MMP-1 mediated PAR-1
activation by PAR-1's tethered ligand.
[0093] FIG. 9B shows the human PAR-1 polypeptide sequence (Genbank
Accession No. NP.sub.--001983).
[0094] FIG. 10A shows the average weight of ApoE-deficient mice
after being fed a western diet for 15 weeks.
[0095] FIG. 10B shows the total plasma cholesterol of
ApoE-deficient mice after being fed a western diet for 15
weeks.
[0096] FIG. 11A depicts the atherosclerotic lesion area in
ApoE-deficient mice treated with Vehicle, MMP Inh-I (FN-439), or
P1pal-7 pepducin lipopeptide.
[0097] FIG. 116 depicts atherosclerotic lesion area in the
abdominal/iliac aorta.
[0098] FIGS. 12A-12C show angiogenesis in the abdominal aorta of
ApoE-/- Mice.
[0099] FIGS. 13A-13G shows that P1pal-7 and MMP1 inhibitors reduce
angiogenesis in the abdominal aorta of ApoE-deficient mice.
DETAILED DESCRIPTION
[0100] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art. The following definitions are provided to help
interpret the disclosure and claims of this application. In the
event a definition in this section is not consistent with
definitions elsewhere, the definition set forth in this section
will control.
[0101] As used herein, the term "about" or "approximately" when
used in conjunction with a number refers to any number within 5, 10
or 15% of the referenced number.
[0102] As used herein the terms "administration" "administering,"
or the like, when used in the context of providing a pharmaceutical
or nutraceutical composition to a subject generally refers to
providing to the subject one or more pharmaceutical compositions
comprising the agent, e.g., an angonist or antagonist of the MMP-1
mediated PAR-1 signaling pathway, in combination with an
appropriate delivery vehicle by any means such that the
administered compound achieves one or more of the intended
biological effects for which the compound was administered. By way
of non-limiting example, a composition may be administered
parenteral, subcutaneous, intravenous, intracoronary, rectal,
intramuscular, intra-peritoneal, transdermal, or buccal routes of
delivery.
[0103] In one embodiment, `administration` of the agent, e.g., an
angonist or antagonist of the MMP-1 mediated PAR-1 signaling
pathway, to the patient may require controlled release, i.e., the
release of the active ingredient from the formulation in a
sustained and regulated manner over a longer period of time than an
immediate release formulation containing the same amount of the
active ingredient would release during the same time period. The
dosage administered will be dependent upon the age, health, weight,
and/or thrombotic disease state of the recipient and/or other
associated risk factors, the kind of concurrent treatment, if any,
the frequency of treatment, and/or the nature of the effect
desired.
[0104] As used herein, an "angonist" refers to any natural or
synthetic molecule or combination of molecules that increases a
biological activity by at least or at least about 2 fold, about 3
fold, about 4 fold, about 5 fold, about 7 fold, about 10 fold,
about 20 fold, about 60 fold or about 100 fold or more in a
standard bioassay or in vivo or when used in a therapeutically
effective dose. In one embodiment, an "angonist" "refers to any
natural or synthetic molecule or combination of molecules that
activates MMP-1 mediated PAR-1 signaling.
[0105] An "antagonist" or "inhibitor" may be used interchangeably
herein and refers to any natural or synthetic molecule or
combination of molecules that interferes with a biological activity
by at least or at least about 10%, about 15%, about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, about 95%, about 96%, about 97%, about 98%, about 99%,
or about 100% in a standard bioassay or in vivo or when used in a
therapeutically effective dose. In one embodiment, an "antagonist"
or "inhibitor" refers to any natural or synthetic molecule or
combination of molecules that interferes with MMP-1 mediated PAR-1
activity. In another embodiment, an "antagonist" or "inhibitor"
refers to any natural or synthetic molecule or combination of
molecules that inhibits MMP-1 mediated PAR-1 activation.
[0106] In another embodiment, an "antagonist" or "inhibitor" refers
to a compound that inhibits cleavage between aspartic acid at
position 39 (D39) and praline at position 40 (P40) of the
protease-activated receptor-1 (PAR-1) by at least or at least about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, about
96%, about 97%, about 98%, about 99%, or about 100%.
[0107] In one embodiment, an "antagonist" of the MMP1-mediated
PAR-1 signaling pathway may be identified by its ability to fully
or partially inhibit PAR-I mediated signaling activity, as
measured, for example, by PAR1-dependent Rho and p38 MAPK
signaling. Inhibition occurs when PAR-I intracellular signaling
from a PAR-I receptor exposed to an "agent" of the invention is by
at least or at least about 10%, about 15%, about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, about 95%, about 96%, about 97%, about 98%, about 99%,
or about 100% in comparison to intracellular signaling from a
control PAR-I not exposed to the "antagonist."
[0108] An "angonist" or "antagonist" compound as used herein, may
comprise one or more protecting groups that prevent undesirable
reactions (such as proteolysis) involving unprotected functional
groups. In one embodiment, the present invention contemplates that
the protecting group is an acyl or an amide. In one embodiment, the
acyl is acetate. In another embodiment, the protecting group is a
benzyl group. In another embodiment, the protecting group is a
benzoyl group. The present invention also contemplates combinations
of such protecting groups.
[0109] As used herein, "anti-coagulant" drugs refer to drugs that
prevent coagulation i.e. that stop blood from clotting.
Non-limiting examples of anti-coagulants that may be used in this
invention include, for example, coumarines (vitamin K antagonists,
Warfarin (Coumadin, Acenocoumarol, Phenprocoumon) and synthetic
pentasaccharide inhibitors of factor Xa (Fondaparinux or
Idraparinux).
[0110] As used herein, "anti-platelet drugs" refer to members of a
class of pharmaceuticals that decreases platelet aggregation.
Non-limiting examples of anti-platelet drugs include, for example,
cyclooxygenase inhibitors (Aspirin), adenosine diphosphate (ADP)
receptor inhibitors (Clopidogrel (Plavix). Ticlopidine (Ticlid)),
phosphodiesterase inhibitors (Cilostazol (Pletal), glycoprotein
IIB/IIIA inhibitors and adenosine reuptake inhibitors (Dipyridamole
(Persantine)). In one embodiment, an antiplatelet drug comprises
SCH 530348.
[0111] As used herein, "glycoprotein IIB/IIIA inhibitors" include,
but are not limited to, (Abciximab (ReoPro), Eptifibatide
(Integrilin) and Tirofiban (Aggrastat), Defibrotide. Abciximab
(previously known as c7E3 Fab), manufactured by Centocor and
distributed by Eli Lilly under the trade name ReoPro, is a platelet
aggregation inhibitor mainly used during and after coronary artery
procedures like angioplasty to prevent platelets from sticking
together and causing thrombus (blood clot) formation within the
coronary artery. Eptifibatide (Integrilin, Millennium
Pharmaceuticals, also co-promoted by Schering-Plough/Essex), is an
antiplatelet drug that selectively blocks the platelet glycoprotein
receptor. Eptifibatide is a cyclic heptapeptide derived from a
protein found in the venom of the southeastern pygmy rattlesnake
(Sistrurus miliarius barbouri). It belongs to the class of the so
called arginin-glycin-aspartat-mimetics and reversibly binds to
platelets. Eptifibatide has a short half-life. The drug is the
third inhibitor of GPIIb/IIIa that has found broad acceptance after
the specific antibody abciximab and the non-peptide
tirofibanentered the global market. Tirofiban is a synthetic,
non-peptide inhibitor acting at glycoprotein (GP) IIb/IIIa
receptors in human platelets. It therefore constitutes an
anticoagulant, specifically an inhibitor of platelet aggregation.
The drug is marketed under the brand name AGGRASTAT in the US by
Medicure Pharma and the rest of the world by Iroko
Pharmaceuticals.
[0112] The term "attached" as used herein, refers to any
interaction between a medium (or carrier) and an agent, e.g. an
angonist or antagonist of the MMP-1 mediated PAR-1 signaling
pathway. Attachment may be reversible or irreversible. Such
attachment includes, but is not limited to, covalent bonding, and
non-covalent bonding including, but not limited to, ionic bonding,
Van der Weals forces or friction, and the like. An agent is
attached to a medium (or carrier) if it is impregnated,
incorporated, coated, in suspension with, in solution with, mixed
with, etc.
[0113] As used herein, a medical device is "coated" when a medium
comprising an agent, e.g., an angonist or antagonist of the MMP-1
mediated PAR-1 signaling pathway, becomes attached to the surface
of a medical device. This attachment may be permanent or temporary.
When temporary, the attachment may result in a controlled release
of the agent. Medical devices may be coated with a thin polymer
film loaded with the agent that inhibits platelet activation. The
coating is applied to the medical device prior to insertion into a
blood vessel using methods well known in the art, such as a solvent
evaporation technique. The solvent evaporation technique entails
mixing a polymer and agent in a solvent. The solution comprising
polymer, agent, and solvent can then be applied to the surface of
the medical device by either dipping or spraying. The medical
device is then subjected to a drying process, during which the
solvent is evaporated, and the polymeric material, with the agent
dispersed therein, forms a thin film layer on the medical device.
U.S. Pat. No. 5,837,313 to Ding et al. describes a method of
preparing a heparin containing coating composition. U.S. Pat. No.
5,525,348 Whitbourne discloses a method of complexing
pharmaceutical agents (including heparin) with quarternary ammonium
components or other ionic surfactants and bound with water
insoluble polymers as an anti-thrombotic coating composition. A
general approach to the coating of medical devices as disclosed in
US 2005/0191333 A1, US 2006/0204533 A1, and WO 2006/099514 A2, all
by Hsu, Li-Chien, et al., uses a low molecular weight complex of
heparin and a counter ion (stearylkonium heparin), or a high
molecular weight polyelectrolyte complex, such as dextran, pectin
to form a complex.
[0114] The term "collagen-induced platelet aggregation", as used
herein, refers to platelet aggregation in response to the presence
of the protein, collagen.
[0115] A "homologue" of a MMP-1 polypeptide refers to a polypeptide
having at least about 80%, preferably at least about 85%, more
preferably at least about 90%, most preferably at least about 95%
amino acid sequence identity with human MMR-1 of amino acid
sequence UniProtKB/Swiss-Prot P03956 (MMP1_HUMAN), which is
incorporated herein by reference. In one embodiment, for example, a
MMP-1 homologue includes those variants that are capable of
cleaving PAR-1 between aspartic acid at position 39 (D39) and
proline at position 40 (P40).
[0116] A "homologue" of a PAR-1 polypeptide refers to a polypeptide
having at least about 80%, preferably at least about 85%, more
preferably at least about 90%, most preferably at least about 95%
amino acid sequence identity with the human PAR-1 polypeptide
sequence with Genbank Accession No. NP.sub.--001983. In one
embodiment, for example, a PAR-1 homologue includes those PAR-1
variants that can be cleaved between aspartic acid at position 39
(D39) and proline at position 40 (P40).
[0117] The term, "inhibiting platelet activation", as used herein,
refers to decreasing or slowing platelet aggregation, as well as
completely eliminating and/or preventing platelet aggreagtion.
[0118] The term "ligand-binding", as used herein, refers to a
member of a binding pair, i.e., two different molecules wherein one
of the molecules specifically binds to the second molecule through
chemical or physical means. In addition to antigen and antibody
binding pair members, other binding pairs include, as examples
without limitation, biotin and avidin, carbohydrates and lectins,
complementary nucleotide sequences, complementary peptide
sequences, effector and receptor molecules, enzyme cofactors and
enzymes, enzyme inhibitors and enzymes, a peptide sequence and an
antibody specific for the sequence or the entire protein, polymeric
acids and bases, dyes and protein binders, peptides and specific
protein binders (e.g., ribonuclease, S-peptide and ribonuclease
S-protein), and the like. Furthermore, binding pairs can include
members that are analogs of the original binding member, for
example, an analyte-analog or a binding member made by recombinant
techniques or molecular engineering. If the binding member is an
immunoreactant it can be, for example, a monoclonal or polyclonal
antibody, a recombinant protein or recombinant antibody, a chimeric
antibody, a mixture(s) or fragment(s) of the foregoing, as well as
a preparation of such antibodies, peptides and nucleotides for
which suitability for use as binding members is well known to those
skilled in the art. A ligand-binding member may be a polypeptide
affinity ligand (see, for example, U.S. Pat. No. 6,326,155, the
contents of which are hereby incorporated by reference herein in
its entirety). In one embodiment, the ligand-binding member is
labeled. The label may be selected from a fluorescent label, a
chemiluminescent label or a bioluminescent label, an
enzyme-antibody construct or other similar suitable labels known in
the art.
[0119] In some embodiments, a ligand-binding molecule refers to an
"antibody" including both polyclonal and monoclonal antibodies; and
may be an intact molecule, a fragment thereof (such as Fv, Ed, Fab,
Fab' and F(ab)'2 fragments, or multimers or aggregates of intact
molecules and/or fragments; and may occur in nature or be produced,
e.g., by immunization, synthesis or genetic engineering. An
antibody may be humanized according to methods that are well known
in the art.
[0120] In another embodiment, a "ligand-binding molecule" may refer
to an "aptamer," i.e. oligonucleotides that are able to bind a
target of interest other than by base pair hybridization.
[0121] As used herein, a "matrix layer" refers to the substance,
such as a polymer, that is suitable for attaching the herein
described "angonist" or "antagonist" and can be applied to the
surface of a medical device. Methods of coating a medical device
are described in U.S. Patent Publication No. 2009/0018646, the
contents of which are hereby incorporated herein in their
entirety.
[0122] The term "medical device", as used herein, refers broadly to
any apparatus used in relation to a medical procedure.
Specifically, any apparatus that comes in contact with a patients
blood during a medical procedure or therapy is contemplated herein
as a medical device. Similarly, any apparatus that administers a
drug or compound to a patient during a medical procedure or therapy
is contemplated herein as a medical device. "Direct medical
implants" include, but are not limited to, urinary and
intravascular catheters, dialysis catheters, wound drain tubes,
skin sutures, vascular grafts and implantable meshes, intraocular
devices, implantable drug delivery systems and heart valves, and
the like, "Wound care devices" include, but are not limited to,
general wound dressings, non-adherent dressings, burn dressings,
biological graft materials, tape closures and dressings, surgical
drapes, sponges and absorbable hemostats. "Surgical devices"
include, but are not limited to, surgical instruments, endoscope
systems (i.e., catheters, vascular catheters, surgical tools such
as scalpels, retractors, and the like) and temporary drug delivery
devices such as drug ports, injection needles etc. to administer
the medium.
[0123] Matrix metalloproteinase-1 (MMP-1, aliases: CLG, CLGN, EC
3.4.24.7) is also known as fibroblast collagenase, interstitial
collagenase, matrix metallopeptidase-1 or matrix metalloprotease-1
(HGNC: 71551; Entrez Gene: 43122; UniProtKB: P039563; Ensembl:
ENSG000001966117; GenBank Accession Number: NM.sub.--002421). The
MMP-1 gene encodes a secreted enzyme that can break down
interstitial collagens, types I, II, and Ill. The gene is part of a
cluster of MMP genes, which localize to human chromosome
11q22.3.
[0124] Matrix metalloprotease-2 (MMP-2; aliases: CLG4, CLG4A, EC
3.4.24.24, MMP-II, MONA, TBE-1) is also known as 72 kDa gelatinase,
gelatinase A, matrix metalloproteinase-2, collagenase type IV-A,
matrix metallopeptidase 2, 72 kDa type IV collagenase or neutrophil
gelatinase (HGNC: 71661; Entrez Gene: 43132; UniProtKB: P082533;
Ensembl: ENSG000000872457).
[0125] As used herein, "metailoprotease" or "MMP" refers to a
family of calcium- and zinc-dependent endopeptidases that share
amino-acid sequences, structural domains, and overlapping
substrates. These enzymes are secreted as zymogens and removal of
an activation peptide is required for their proteolytic activity.
MMPs are involved in the breakdown of components of the
extracellular matrix (ECM) and basement membrane such as aggrecan,
collagen, elastin, fibronectin, gelatin, and laminin. The ability
of MMPs to degrade components of the ECM is essential to cell
growth, cell division, bone growth, wound healing, embryogenesis,
and angiogenesis. The MMPs are divided into several different
classes. They are referred to numerically as MMP-1, MMP-2, etc. as
well as by a common name. The MMPs share several structural and
functional properties but differ in their substrate specificities.
There are at least 25 members of the MMP family, categorized based
on their domain structures and their preferences for macromolecular
substrates (Nelson, A. et al., (2000) J. Clin. Oncol. 18,
1135-1149., Woessner, J. F., and Nagase, H. (2000) Matrix
Metalloproteinases and TIMPs, Oxford University Press, Oxford).
Most MMPs contain a propeptide domain, a catalytic domain, and a
hemopexin/vitronectin-like domain (Woessner, J. F., and Nagase. H.,
supra). The MMP family includes MMP 1 (interstitial cotlagenase,
collagenase 1), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7
(pump 1, matrilysin), MMP-8 (neutrophil collagenase, collagenase
2), MMP-9 (gelatinase B), MMP-10 (stromelysin 2), MMP-11
(stromelysin 3), MMP-12 (metalloelastase, macrophage elastase),
MMP-13 (collagenase 3), five membrane-type MMPs (MT-MMPs) (MMP-14,
MMP-15, MMP-16, MMP-17, MMP-21), MMP-18 (Xenopus collagenase 4),
MMP-19. MMP-20 (enamelysin), MMP-22 (chicken CMMP), MMP-23, MMP-24,
MMP-25, MMP-26 (endometase), MMP-27, and MMP-28 (epilysin). Some
redundancy of MMP family member numbering exists: telopeptidase,
later designated MMP-4, and 3/4-collagenase (MMP-5) are MMP-3 and
MMP-2, respectively; MMP-6 (acid metalloproteinase) was shown to be
MMP-3.
[0126] In this disclosure, reference to metalloproteases in general
or to any individual member of the MMP family, such as MMP-1 or
MMP-2, will be understood to refer to all splice variants, mutants
(including, but not limited to, deletions, insertions or
polymorphisms or amino acid substitutions), isoforms and homologues
thereof.
[0127] As used herein, to "modulate" means to act as an antagonist,
i.e. partially or fully inhibit, reduce, alleviate, block or
prevent; or to increase or stimulate, i.e. to act as an angonist.
The modulation may be direct or indirect.
[0128] Non-encoded amino acids include, but not limited to,
alpha-amino acids, beta-amino acids, gamma-amino acids, delta-amino
acids, and omega-amino acids, and may have R or S chirality at any
chiral atom. Non-encoded amino acids include isomers of the encoded
amino acids such as, e.g., stereoisomers (including, e.g., D-amino
acids and allo-amino acids such as, e.g., alto threonine and
allo-isoleucine) and structural isomers (including, e.g.,
beta-alanine) of the encoded amino acids. Non-encoded amino acids
also include N-methylated amino acids. In general, where no
specific configuration is indicated for an alpha-amino acid, one
skilled in the art would understand that amino acid to be an
L-amino acid. However, in particular embodiments, non-encoded amino
acids may also be in the form of racemic, non-racemic, and
diastereomeric mixtures. Non-encoded amino acids are well known in
the peptide art and include, but not limited to, N-acetylserine,
aloha//o-isoleucine, alpha//o-threonine, beta-alanine
(3-aminopropionic acid), alpha-aminoadipic acid, 2-aminobutanoic
acid, 4-aminobutanoic acid, 3-amino-1-carboxymethylvalerolactam,
1-aminocyclopentanecarboxylic acid, 6-aminohexanoic acid,
2-aminoheptanedioic acid, 7-aminoheptanoic acid, 2-aminoisobutyric
acid, aminomethylpyrrole carboxylic acid,
8-amino-3,6-dioxa-octanoic acid, aminopiperidinecarboxylic acid,
aminoserine, aminotetrahydropyran-4-carboxylic acid, azetidine
carboxylic acid, benzothiazolylalanine, butylglycine, carnitine,
4-chlorophenylalanine, citrulline, cyclohexylalanine,
cyclohexylstatine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic
acid, dihydroxyphenylalanine, dimethylthiazolidine carboxylic acid,
4-guanyl-phenylalanine, homoarginine, homocitrulline, homocysteine,
homophenylalanine, homoproline, homoserine, 4-hydrazinobenzoic
acid, 4-hydroxyproline, isonipecotic acid, methanoproline,
norleucine, norvaline, ornithine, p-aminobenzoic acid,
penicillamine, phenylglycine, (9-phosphoserine, piperidinyialanine,
piperidinylglycine, pyrrolidinylalanine, sarcosine, statine,
tetrahydropyranglycine, thienylalanine,
[epsiv]-N,N,N-trimethyllysine.
[0129] The Human PAR family includes PAR-1 (Genbank Accession
Number AF019616): PAR2 (Genbank Accession Number XM-003671); PAR3
(Genbank Accession Number NM-0041101); and PAR4 (Genbank Accession
Number NM-003950.1), the sequences of which are hereby incorporated
by reference.
[0130] PAR-1 or protease activated receptor 1 (other aliases: CF2R,
HTR 2, PAR1 or TR) is also known in the art as thrombin receptor or
coagulation factor II (thrombin) receptor (HGNC: 35371; Entrez
Gene: 21492; UniProtKB: P251163; Ensembl; ENSG000001811047). The
human PAR-1 polypeptide sequence has Genbank Accession No.
NP.sub.--001983, which is also incorporated herein by reference and
also reproduced in FIG. 9B.
[0131] In this disclosure, reference to PAR family members in
general or to any individual member of the PAR family member, such
as PAR-1, will be understood to refer to all splice variants,
mutants (including, but not limited to, deletions, insertions or
polymorphisms or amino acid substitutions), isoforms and homologues
thereof.
[0132] The term, "patient," as used herein, refers to any
individual organism. For example, the organism may be a mammal such
as a primate (i.e., for example, a human). Further, the organism
may be a domesticated animal (i.e., for example, cats, dogs, etc.),
livestock (i.e., for example, cattle, horses, pigs, sheep, goats,
etc.), or a laboratory animal (i.e., for example, mouse, rabbit,
rat, guinea pig, etc.).
[0133] As used herein, "platelet activation" refers to the series
of changes in platelet function that ultimately leads to platelet
aggregation and the formation of a stable haemostatic plug or
"thrombus." Platelet activation can be triggered by vascular injury
caused, for example, by the rupture of atherosclerotic plaque. The
subsequent exposure of circulating platelets to the sub-endothelial
tissue and various platelet activation molecules, such as collagen,
thromboxane or ADP, initiates a chain of events that results in
changes to platelet metabolic biochemistry, shape, surface
receptors, and membrane phospholipid orientation and thrombus
formation.
[0134] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0135] As used herein "pepducin lipopeptides" are cell-penetrating
peptides that act as intracellular inhibitors of signal
transference from receptors to G proteins. Pepducin lipopeptides
utilize lipidated fragments of intracellular G protein-coupled
receptor loops to modulate GPCR action in targeted cell-signaling
pathways. A pepducin lipopeptide molecule comprises a short peptide
derived from a GPCR intracellular loop tethered to a hydrophobic
moiety. This structure allows pepducin lipopeptides to anchor in
the cell membrane lipid bilayer and target the GPCR/G protein
interface via a unique intracellular allosteric mechanism. Examples
of pepducin lipopeptides are described in U.S. Patent Publication
US2007/0179090, the contents of which are hereby incorporated
herein by reference in its entirety.
[0136] As used herein, the term "peptide" or "polypeptide" is
intended to encompass a single "polypeptide" as well as plural
"polypeptides," and refers to a molecule composed of monomers
(amino acids) linearly linked by amide bonds (also known as peptide
bonds). The term "polypeptide" refers to any chain or chains of two
or more amino acids, and does not refer to a specific length of the
product. A "peptide" or "polypeptide," as used herein, may be
derived from a natural biological source or produced by recombinant
technology, but is not necessarily translated from a designated
nucleic acid sequence. It may be generated in any manner, including
by chemical synthesis. In accordance with this definition, a
"peptide" or "polypeptide" used in the present invention may be of
a size of about 3 or more, about 5 or more, about 10 or more, about
20 or more, about 25 or more, about 50 or more, about 75 or more,
about 100 or more, about 200 or more, about 500 or more, about
1,000 or more, or about 2,000 or more amino acids. One or more of
the amino acids in an inventive polypeptide may be modified, for
example, by the addition of a chemical entity such as a
carbohydrate group, a phosphate group, a farnesyl group, an
isofarnesyl group, a fatty acid group, an acyl group (e.g., acetyl
group), a linker for conjugation, functionalization, or other known
protecting/blocking groups. In a preferred embodiment, the
modifications of the peptide lead to a more stable peptide (e.g.,
greater half-life in vivo).
[0137] A "peptide" or "polypeptide," as used herein, may be
fragments, derivatives, analogs, or variants of the foregoing
polypeptides, and any combination thereof. Fragments of
polypeptides, as that term or phrase is used herein, include
proteolytic fragments, as well as deletion fragments. Variants of
polypeptides, useful in accordance with the present invention,
include fragments and polypeptides with altered amino acid
sequences due to amino acid substitutions, deletions, or
insertions. Variants may occur naturally or be non-naturally
occurring. Non-naturally occurring variants may be produced using
art-known mutagenesis techniques. Examples include fusion proteins,
polypeptides having one or more residues chemically derivatized by
reaction of a functional side group, and peptides that contain one
or more naturally occurring amino acid derivatives of the twenty
standard amino acids. These modifications may also include
cyclization of the peptide, the incorporation of D-amino acids, or
other non-encoded amino-acids. None of the modifications should
substantially interfere with the desired biological activity of the
peptide.
[0138] As used herein, a "reduction in the size of atherosclerotic
plaque" refers to the reduction in size of atherosclerotic plaque
as a result of treatment with an antagonist of the MMP-1 mediated
PAR-1 signaling pathway as compared to the size of atherosclerotic
plaque before the onset of treatment. The artherosclerotic plaque
is reduced in size if the reduction is at least or at least about
5%, about 10%, about 15%, about 20%, about 25%, about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, about 60%, about
65%, about 70%, about 75%, about 80%, about 85%, about 90%, about
95%, about 96%, about 97%, about 98%, about 99%, about 100%, about
200%, about 500% or more as compared to the size of atherosclerotic
plaque before the onset of treatment.
[0139] As used herein, "risk factors" for venous thromboembolism
include, but are not limited to, cancer, prior VTE (DVTIPE),
hypercoaguiability (genetic predisposition for blood dots),
surgery, advanced age (>70 years of age), obesity (BMI>29),
bed rest, or prolonged immobility and oral contraceptives or
hormone replacement therapy.
[0140] As used herein, "risk factors" for myocardial infarction,
stroke or PAD (Peripheral Arterial Disease) include, but are not
limited to, high blood pressure, diabetes, high cholesterol
(including genetic predisposition to hypercholesteremia), age (risk
doubles for each decade over 55 years of age), family history of
stroke, smoking, oral contraceptives, atrial fibrillation, heart
failure, excess alcohol, prior stroke or heart attack, race (for
example, African Americans have almost twice the risk of first-ever
stroke compared with Caucasians) and gender (each year, in the U.S.
about 46,000 more women than men have a stroke).
[0141] In other embodiments, "risk factors" for thrombosis also
refer to those risks created by the implantation of a prosthesis
inside the body, including, but not limited to, artificial hearts,
lungs as well as stents or other medical devices.
[0142] As used herein, the term "small molecule" and analogous
terms include, but are not limited to, peptides, peptidomimetics,
amino acids, amino acid analogs, polynucleotides, polynucleotide
analogs, nucleotides, nucleotide analogs, other organic and
inorganic compounds (i.e., including heteroorganic and
organometallic compounds) having a molecular weight less than about
10,000 grams per mole. In some embodiments, the term refers to
organic or inorganic compounds having a molecular weight less than
about 5,000 grams per mole, less than about 1,000 grams per mole,
less than about 500 grams per mole, less than about 100 grams per
mole. Salts, esters, and other pharmaceutically acceptable forms of
such compounds are also encompassed.
[0143] As used herein, "thrombolytic drugs" refer to drugs that are
used in medicine to dissolve blood cots in a procedure termed
thrombolysis. Non limiting examples of thrombolytic drugs include #
tissue plasminogen activator--t-PA--alteplase (Activase), reteplase
(Retavase), tenecteplase (TNKase), anistreplase (Eminase),
streptokinase (Kabikinase, Streptase) and urokinase
(Abbokinase).
[0144] As used herein, a "thrombotic disease state" refers to any
medical condition in a patient that can lead to thrombosis i.e. the
formation of a blood clot or "thrombus" inside a blood vessel,
obstructing blood flow through the circulatory system. There are
two distinct forms of thrombosis: venous and arterial thrombosis.
Venous thromboembolism (VIE), which is comprised of deep vein
thrombosis (DVT) and pulmonary embolism (PE), and thoracic outlet
syndrome are examples of venous thrombosis. Stroke, heart attack,
and peripheral arterial disease are examples of arterial
thrombosis. Further examples of a thromboembolic disorder include,
but are not limited to, unstable angina, an acute coronary
syndrome, atrial fibrillation, first myocardial infarction,
recurrent myocardial infarction, ischemic sudden death, transient
ischemic attack, stroke, atherosclerosis, peripheral occlusive
arterial disease (PAD), venous thrombosis, deep vein thrombosis,
thrombophlebitis, arterial embolism, coronary arterial thrombosis,
cerebral arterial thrombosis, cerebral embolism, kidney embolism,
pulmonary embolism, thrombotic re-occlusion subsequent to a
coronary intervention procedure, heart surgery or vascular surgery
and thrombosis resulting from medical implants, devices, or
procedures in which blood is exposed to an artificial surface that
promotes thrombosis. The "thrombotic disease state" also refers to
cardiovascular disease resulting from systemic diseases including,
but not limited to, diabetes mellitus, syndrome X (metabolic
syndrome) or cancer.
[0145] The term "therapeutically effective amount" as used herein
means that amount of active compound or pharmaceutical "agent" that
elicits the biological or medicinal response in a tissue, system,
animal or human that is being sought by a researcher, veterinarian,
medical doctor or other clinician.
[0146] As used herein, "thrombin-dependant activation of PAR-1"
refers to the activation of PAR-1 signaling by a serine protease
(such as thrombin or plasmin or APC) that cleaves the N terminus of
PAR-1 between the arginine residue at position 41, and the serine
residue at position 42.
[0147] As used herein, "treating" or "treatment" cover the
treatment of a thrombotic disease-state in a mammal, particularly
in a human, and include, but not limited to (a) preventing the
disease-state from occurring in a mammal, in particular, when such
mammal is predisposed to the disease-state but has not yet been
diagnosed as having it; (b) inhibiting the disease-state, i.e.,
arresting its development; and/or (c) relieving the disease-state,
i.e., causing regression of the disease state.
[0148] The term, "treating a thrombotic disease state," as used
herein, refers to modulating platelet aggregation including, but
not limited to, decreasing the amount of platelet aggregation
and/or slowing platelet aggregation, as well as completely
eliminating and/or preventing platelet aggregation. Diseases and/or
conditions treatable by modulating platelet aggregation include,
but are not limited to, embolus formation, thrombolytic
complications, thrombosis, coronary heart disease, thromboembolic
complications, myocardial infarction, restenosis, atrial thrombosis
induction of atrial fibrillation, chronic unstable angina,
transient ischemic attacks and strokes, peripheral vascular
disease, arterial thrombosis, preeclampsia, embolism, restenosis
and/or thrombosis following angioplasty, carotid endarterectomy,
anastomosis of vascular grafts, and chronic exposure to
cardiovascular devices. Such conditions may also result from
thromboembolism and re-occlusion during and after thrombolytic
therapy, after angioplasty, and after coronary artery bypass.
I The MMP-1-PAR-1 Signalling Pathway
[0149] I--(a) Collagen Generates Active MMP-1 on Platelets which
Cleaves the PAR-1
[0150] Human platelets contain significant amounts of collagenase
activity, which could be released upon exposure to various
agonists. The major platelet collagenase MMP-1 may be able to prime
the aggregatory response to other agonists and cause redistribution
of the .beta..sub.3 integrins to the cell periphery. To determine
if the pro-aggregatory effects of platelet MMP-1 are mediated by
the PAR1 receptor, the amount of in situ activation of endogenous
proMMP-1 on the platelet surface was measured following stimulation
with the primary angonist collagen versus the secondary mediators,
ADP and thromboxane.
[0151] Platelet pellets and their supernatants were prepared as
follows. The IRB of Tufts Medical Center performed phlebotomy on 20
healthy volunteer donors following established informed consent
procedures. 27 ml of blood was drawn using an 18 gauge needle
attached to a 30 cc syringe containing 3 ml of 3.2% sodium citrate
solution (0.32% v/v final). Platelets from platelet-rich plasma
(PRP) were isolated by gel filtration using a Sepharose 2B
(Pharmacia) in modified PIPES buffer (25 mM PIPES, 137 mM NaCl, 4
mM KCl, 0.1% Glucose, pH 6.6) in the presence of 1 mM EDTA and 0.1
U/ml of apyrase. Alternatively, whole blood was obtained from
Hartley Sprague guinea pigs (drawn from the vena cava) into 3%
citrate plus 10 U/ml heparin. Washed platelets from the guinea pigs
were prepared in PIPES buffer. Platelet aggregation was measured
with a Chronolog 560VS/490-2D aggregometer using modified PIPES
buffer as a blank. Samples were incubated for 5 min in the presence
of inhibitors and 1.8 mM CaCl.sub.2 prior to addition of angonist.
All reactions were in final volumes of 250 .mu.l at 37.degree. C.
while stirring at 900 rpm.
[0152] The enzymatic activity of active MMP-1 in supernatants and
platelet lysates was then determined. Human or guinea pig platelets
from PRP were concentrated four-fold by centrifugation at 700 g for
25 min at room temperature, and then resuspended in 0.25 volume of
PIPES containing 1 mM EDTA (final platelet count was 10.sup.9/mL).
Platelets were treated with PBS (buffer), 20 .mu.M ADP, 20 .mu.M
U-46619, or 20 .mu.g/ml collagen in the presence of 2.5 mM
CaCl.sub.2. The platelets were incubated for 15 min at 37.degree.
C. with occasional gentle mixing. Platelets were collected by
centrifugation at 10,000 g for 5 min at 4.degree. C. and
resuspended in lysis buffer (50 mM Iris HCl, 100 mM NaCl, 1 mM NaF,
5 mM EDTA, 0.1% (v/v) Triton X-100, 100 .mu.M PMSF, pH 74) and then
sheared with a 27 gauge needle.
[0153] The enzymatic activity of active MMP-1 in supernatants and
platelet lysates was measured using DQ collagen I (Molecular
Probes) as fluorogenic substrate and reporter of collagenase
activity substrate (Boire et al., 2005) in the presence or absence
of 3 .mu.M FN-439, or 20 .mu.g/ml each of control IgG, MMP-1
blocking Ab, MMP-8 blocking Ab or MMP-13 blocking Ab (preincubated
for 2 h at 37.degree. C.), as indicated with APMA-activated MMP-1
serving as control (FIG. 1A, striped bars). A standard curve
generated with APMA-activated MMP-1 (Boire et al., 2005) and
collagenase activity was reported in units per milliliter, where
one unit is the amount of MMP-1 degrading 1 .mu.g of collagen per
minute.
[0154] Stimulation of platelets with collagen leads to the release
of the platelet collagenase activity into the supernatant (FIG.
1A). The MMP-1 inhibitor, FN-439 completely blocked cleavage of the
fluorogenic collagen substrate. Blocking antibodies against MMP-1
also completely inhibited the platelet collagenase activity
released by collagen, whereas blocking antibodies against the two
other collagenases, MMP-8 and MMP-13 or an IgG control had no
effect. Stimulation of gel-filtered platelets with ADP or the
thromboxane mimetic, U-46619, however, resulted in a majority of
the MMP-1 collagenase activity remaining bound to the platelet.
[0155] Pellets and supernatants were collected from platelets
(250,000/.mu.L) stimulated with the agonists as described above and
in FIG. 1A or with convulxin (1 .mu.g/ml) by centrifuging the
lysate at 12,000 g for 2 min. The concentration of the released and
platelet-associated MMP-1 pro-domains was then measured by EISA
using antibodies that recognized the pro domain of MMP-1 (FIG. 1B).
Treatment of washed platelets with collagen but not ADP or U-46619
led to efficient release of the proMMP-1 domain (and/or
proMMP-1).
[0156] Surface expression of total platelet MMP-1 was then
determined by flow cytometry (FIG. 1C; dashed grey: secondary
antibody alone; solid lines: FACS profiles of platelets treated
with the indicated concentrations of collagen for 15 min at
37.degree. C. and then stained with primary (AB806) plus secondary
antibodies). FACS analysis confirmed that MMP-1 is expressed on the
surface of resting platelets, which could be released by exposure
to collagen. The lectin, convulxin, which ligands specifically with
the GPVI/Fc.gamma.R collagen receptor, also caused full release of
the proMMP-1 domain from the platelet surface (FIG. 1B). Thus,
collagen fibrils per se are not necessary for the release of
pro-MMP-1 from the platelet surface. Other strong platelet agonists
may also trigger the release mechanism.
[0157] One candidate binding site(s) for the platelet-associated
proMMP-1 is the .alpha..sub.2.beta..sub.1 collagen receptor. To
determine if proMMP-1 associates with integrins in resting human
platelets, lysates from gel-filtered platelets were incubated with
4-5 .mu.g/ml anti-.alpha..sub.2 (Gi9 or AK7), .beta..sub.1
(MAB1987), .beta..sub.3 (MAB1957), GPVI (SC20149), GPIB.alpha.
(MM2/174) or mouse IgG control for 2-4 h at 4.degree. C. Protein G
sepharose was added and incubated for an additional 1 h. Beads were
collected and washed 4.times. in lysis buffer supplemented with 200
mM NaCl. Platelet proteins from the lysates were separated by 12%
SDS-PAGE and Western analyses were conducted using a polyclonal Ab
against the C-terminus of MMP-1 (AB8105) or the hinge region
(AB806) which gave similar results. These co-immunoprecipitation
experiments indicate that proMMP-1 forms a stable complex with the
integrin on platelets (see FIG. 1D), MMP-1 (predominantly in the
pro form) was also found to associate with the
.alpha..sub.IIb.beta..sub.3 integrin, as suggested by previously
described co-focal microscopy studies. Conversely, proMMP-1 did not
associate with GPIb.alpha. or GPVI. Therefore, proMMP-1 is likely
to be pre-associated with both collagen and fibrinogen receptors in
resting platelets.
[0158] To further understand how collagen is able to activate
significant amounts of endogenous MMP-1 collagenase activity on the
surface of platelets, experiments were devised to determine if
PAR-1 is cleaved in either an autocrine or paracrine manner
following exposure to collagen. Using a monoclonal antibody raised
against the amino-terminal thrombin-cleavage peptide region of
PAR1, residues 32-46 (Loew et al., 2000), the relative abilities of
thrombin, MMP-1 and collagen to cause cleavage of platelet PAR1 was
assessed. Gel filtered platelets were treated for 10 min with
thrombin (3 nM), MMP-1 (3 nM), collagen (5 .mu.g/ml), in the
presence or absence (PBS buffer) of 0.00013 U hirudin or 5 .mu.M
FN-439 at 37.degree. C. Supernatants were concentrated 20-fold and
applied to nitrocellulose membranes, then probed with the IIaR-A
monoclonal antibody. The PAR1 N-terminal thrombin cleavage peptide
(A.sub.26-R.sub.41) and PAR1 flexible linker peptide
(N-acetyl-T.sub.67-L.sub.87-C) (Kuliopulos et al., 1999) served as
positive and negative controls (100 ng), respectively. As shown in
FIG. 1E, incubation of platelets with thrombin or MMP-1 was able to
cause release of the N-terminal cleavage peptide of PAR1 into the
supernatant, which was blocked by hirudin or FN-439,
respectively.
[0159] To determine if MMP-3 and MMP-7 could cause the release of
the PAR1 N-terminal peptide, gel filtered human platelets were
treated for 10 min with APMA-dialysate buffer or APMA activated
MMP-3 (Chemicon, 3 nM), MMP-7 (Chemicon, 3 nM) or MMP-1 (Biomol, 3
nM). Platelet pellet and supernatant were separated as described
above. Supernatants were concentrated 20-fold and applied to
nitrocellulose membranes, then probed with the IIaR-A monoclonal
antibody to detect cleaved PAR-1 peptide. As shown in FIG. 1F,
MMP-3 and MMR-7 were not able to cause release of the PAR1
N-terminal peptide from the treated platelets (FIG. 1F).
[0160] To demonstrate the role of MMP-1, gel filtered platelets
were pre-incubated with IgG (20 .mu.g/ml) or MMP-1 blocking
antibody (20 .mu.g/ml) for 2 hrs at 37.degree. C. These platelets
were then stimulated with collagen (5 .mu.g/ml) for 10 min. at
37.degree. C. Supernatants were concentrated 20-fold and applied to
nitrocellulose membranes, then probed with the IIaR-A monoclonal
antibody. As shown in FIG. 1G, treatment of the resting platelets
with collagen led to the release of the N-terminal peptide. This
release was specifically blocked by incubation with the MMP-1
inhibitor, FN-439, or an MMP1-blocking antibody (20 .mu.g/ml) but
not by thrombin inhibitor, hirudin (see FIG. 1E, FIG. 1G).
[0161] Treatment of the gel filtered platelets for 10 min at
37.degree. C. with different concentrations of ADP (0.3-30 nM),
U46619 (0.3-30 nM) followed by incubation with collagen (5
.mu.g/ml) for 10 min. at 37.degree. C. showed that ADP and U-46619
were also able to cause the release of the N-terminal peptide of
PAR1 albeit at lower efficiency (see FIG. 1H).
[0162] On the contrary, treatment of the gel filtered platelets for
10 min with collagen (5 .mu.g/ml), in the presence or absence (PBS
buffer) of ARC (0.5 .mu.M) or aspirin (ASA, 1 mM, 30 min
pre-incubation) at 37.degree. C. failed to release the PAR1
N-terminal peptide (see FIG. 1I). Hence, blocking either the P2Y12
ADP receptor with AR-C69931MX (ARC) or thromboxane with aspirin
(ASA) had no effect on the collagen-dependent release of the PAR1
N-terminal peptide (see FIG. 1I).
[0163] Together, these data provide direct evidence that the
endogenously-generated MMP-1 collagenase activity is able to cleave
PAR1 on the surface of human platelets independently of
thrombin.
I--(b) Identification of the MMP-1 Cleavage Site on PAR1
[0164] Several studies have demonstrated that serine proteases such
as thrombin, plasmin and APC directly hydrolyze PAR1 at
LDPR.sub.41.dwnarw.S.sub.42 FL (P4P3P2P1.dwnarw.P1'P2'P3') to
generate the S.sub.42FLLRN-tethered ligand (TRAP), which activates
PAR1 in an intramolecular mode (Kuliopulos et al., 1999; Loew et
al., 2000; Parry et al., 1996: Seeley et al., 2003; Vu et al.,
1991). However, matrix metalloproteases such as MMP-1 generally
prefer a hydrophobic amino acid at the P1 site, a basic or
hydrophobic amino acid at P7, and a small residue (alanine, glycine
or serine) at P3' (Netzel-Arnett et al., 1991; Turk et al., 2001).
Therefore, MMP-1 may not efficiently cleave at the
R.sub.41.dwnarw.S.sub.42FL thrombin site. To determine the MMP-1
cleavage site, a 26 amino acid peptide (TR26, PAR1 residues 36-61)
was synthesized corresponding to the N-terminal domain of PAR1
(FIGS. 2A-B). The synthetic 26mer peptides encompassing the
thrombin cleavage site region and flanking region, TR26
(A.sub.36-S.sub.61) or TR26-P40N (A.sub.36-K.sub.61), were
incubated with 10 nM thrombin, 10 nM MMP-1 (APMA activated,
purified from human fibroblasts) or PBS buffer for 10 min at
37.degree. C. Peptide cleavage mixtures were separated by RP-HPLC
and cleavage products identified by MALDI-mass spectroscopy as
described (Kuliopulos 1999), Incubation of the TR26 peptide with
thrombin yielded the expected cleavage peptide, TR20 (residues
42-61), as determined by mass spectrometry. In contrast, incubation
of the TR26 peptide with MMP-1 yielded TR22, which corresponds to
PAR1 residues 40-61 (FIG. 2A-B), This indicates that MMP-1 cleaves
the PAR1 exodomain at LD.sub.39.dwnarw.P.sub.40RSFL, a site which
is located 2-amino acid residues to the N-terminal side of the
thrombin cleavage site at R.sub.41-S.sub.42.
[0165] To verify the location of this putative MMP-cleavage site in
the full-length receptor, the critical P1' residues of both the
MMP-1 and thrombin cleavage sites were mutated.
[0166] To inhibit cleavage by MMP-1, the putative P1' proline was
replaced with asparagine (P40N PAR1), a substitution which had
previously been shown to reduce cleavage of .alpha.1 collagen
peptides to less than 10% (Berman et al., 1992). To inhibit
proteolysis by thrombin, the P1' serine of the thrombin cleavage
site was mutated to aspartate (S42D PAR1), a mutation which was
anticipated to suppress cleavage by thrombin (Chang, 1986), Human
PAR1 was cloned into pcDEF3 as described previously (Kulicpulos et
al., 1999) and was used for generating all mutants. The PAR1
mutants P40N and S42D were generated using the Quick Change
Site-Directed Mutagenesis kit (Stratagene) and sequenced to verify
the fidelity of the mutagenesis. The effects of these mutations on
cleavage rates of a T7-tagged receptor were then measured.
[0167] In FIG. 2D, COS7 cells transiently transfected with
T7-tagged WT, P40N or S42D PAR1, were incubated for 30 min at
37.degree. C. in PBS with 0.3-30 nM Thrombin (FIG. 2C) or
APMA-activated MMP-1.
[0168] In FIG. 2G, COS7 cells transiently transfected with
T7-tagged WT, P40N or S42D PAR1 were incubated for 30 min at
37.degree. C. in PBS with 0.3-10 nM APMA-activated MMP-1 (Biomol,
Cat No. SE 361).
[0169] In FIGS. 2H-2J, COS7 cells transiently transfected with
T7-tagged PAR-2, PAR-3 and PAR-4 were incubated for 60 min at
37.degree. C. in PBS with 0.3-10 nM thrombin for PAR3 or PAR4) or
0.3-10 nM trypsin (for PAR2), or APMA-activated MMP-1. Loss of 17
epitope was analyzed by flow cytometry as described previously
(Boire et al., 2005; Kuliopulos et al., 1999).
[0170] The results show P40N PAR1 mutant was fully cleaved by
thrombin but was poorly cleaved by MMP-1 using two independent
sources of MMP-1 (FIG. 2C-D, FIG. 2G). Conversely, the S42D PAR1
mutant was substantially cleaved by MMP-1 but was poorly cleaved by
thrombin. Identical results were seen for cleavage of a mutant
TR26-P40N peptide, which was cleaved at the R41-S.sub.42 bond by
thrombin but was not cleaved by MMP-1 (FIG. 2A). Functional studies
validated the relative cleavage specificities of the P40N and S42D
mutants for thrombin and MMP-1.
[0171] The level of RhoA signaling of the different PAR-1 mutants
was then measured in the presence of thrombin or MMP-1 (see FIG.
2E). MCF-7 cells transiently transfected with T7-tagged WT, S42D or
P40N PAR1 for 48 h were stimulated with 10 nM thrombin, 10 nM MMP-1
or PBS buffer for 15 min at 37.degree. C. Rho-GTP present in
platelet lysates (mean+/-SD, n=3) was precipitated with glutathione
S-transferase (GST)-rhotekin-reduced glutathione-agarose beads as
described (Kaneider et al., 2007) and Rho-GTP was determined by
probing the Western blots with anti-RhoA (26C4 Ab) monoclonal
antibody. Platelet lysates were also run on a separate gel and
immunoblotted with anti-RhoA to assess total RhoA.
[0172] As shown in FIG. 2F, chemotactic migration of MCF-7 cells
expressing thrombin and MMP1-cleavage site mutants was also
assessed. MCF-7 cells transfected with the PAR1 cleavage mutants
were allowed to migrate overnight toward DMEM/0.1% BSA (buffer)
plus 3 nM thrombin or 3 nM MMP-1 in a Transwell apparatus (8-.mu.m
pore). Cells which migrated toward the bottom side of the membrane
were counted and expressed as % relative to WT PAR1 and
thrombin.
[0173] Thrombin is able to fully activate Rho signaling and
chemotactic migration in MCF-7 cells expressing the P40N mutant,
but had essentially no activity toward the 842D mutant (FIGS.
2E-F). Conversely, MMP-1 was able to induce Rho signaling and
chemotaxis in MCF-7 cells expressing the S42D mutant, but had
little activity towards the P40N mutant. By comparison, 0.3-10 nM
MMP-1 was not able to detectably cleave T7-tagged PAR2, PAR3, nor
PAR4 expressed on COST cells (FIGS. 2H-2J). Together, these
cleavage and signaling data indicate that MMP-1 specifically
activates PAR1 by cleaving at L.sub.039.dwnarw..sub.P40RSFL rather
than at the LDP.sub.R41.dwnarw..sub.S42FL, thrombin cleavage site
and does not cleave the other PARs.
I--(c) Activation of PAR1 Signaling with the MMP1-Generated
Tethered Ligand
[0174] MMP-1 cleavage of PAR1 at LD.dwnarw.P.sub.40RS will generate
a longer tethered ligand, P.sub.40RSFLLRN-, than that produced by
thrombin. To provide further evidence that MMP1-generated tethered
ligand could activate PAR1, the ability of the synthetic peptide,
PR-SFLLRN(PR-TRAP) to stimulate PAR1 signaling was tested.
[0175] As shown in FIG. 3A, the effect of the PRSFLLRN peptide
(PR-TRAP) on PAR1-dependent RhoA activation in platelets was
measured. Gel-filtered human platelets, supplemented with 0.3 mg/mL
fibrinogen, were treated with 0.2% DMSO vehicle, or 30 .mu.M SFLLRN
(TRAP), PR-TRAP or reversed peptide (RP-TRAP), for 5 min at
37.degree. C. in presence or absence of 1 .mu.M RWJ-56110 as
indicated. Platelets were lysed and Rho-GTP and total Rho was
determined by Western analysis as described in the Experimental
Procedures. Western bands were quantified by densitometry and
results expressed relative to fold-increase from basal.
[0176] In FIG. 3B, the effect of the PRSFLLRN peptide (PR-TRAP) on
p38 MAPK in platelets was measured. Platelets were stimulated with
different concentrations of PR-TRAP, RP-TRAP or TRAP as indicated
for 5 min at 37.degree. C. Platelets were lysed with Laemmli sample
buffer and proteins assessed by Western blot of p38 MAPK activity
with phospho-specific p38 MAPK antibody or total p38MAPK
antibody.
[0177] In FIG. 3C, the ability of the PRSFLLRN peptide (PR-TRAP) to
induce a change in platelet shape was determined. Washed human
platelets were pretreated with 2 mM EGTA and then treated with the
indicated agonists in the presence or absence of 1 .mu.M RWJ-56110
while stirring at 1100 rpm. The decrease in light transmittance is
an indication of the platelet shape change reaction.
[0178] The results show PR-TRAP is a full angonist of
PAR1-dependent Rho and p38 MAPK signaling in platelets (FIG. 3A-B).
Addition of the PAR1 antagonist, RWJ-56110, completely blocked
signaling induced by PR-TRAP. PR-TRAP ligand also activated changes
in platelet shape (see FIG. 3C), a critical early event in platelet
activation which is mediated by G.sub.12/13-Rho signaling (Huang et
al., 2007; Offermanns et al., 1994). Again, PR-TRAP-induced
platelet shape change was completely blocked by the PAR1
antagonist, RWJ-56110 (FIG. 3C).
[0179] Two other peptides were tested for angonist activity which
would be generated by putative cleavage at the flanking peptide
bonds: the R-TRAP peptide corresponding to cleavage at
LDP.sub.40.dwnarw.R.sub.41SFLLRN and the DPR-TRAP peptide
corresponding to cleavage at L.sub.38.dwnarw.D.sub.39PRSFLLRN (FIG.
2B). The R-TRAP peptide retained partial angonist activity for
PAR1-dependent platelet shape change, whereas the DPR-TRAP peptide
had nearly no activity (FIG. 3C). Likewise, the control peptide,
RP-TRAP, in which the first two amino acid residues were reversed,
did not stimulate Rho or p38 MAPK, nor platelet shape change (FIGS.
3A-C).
[0180] The ability of exogenously-added MMP-1 to activate
PAR1-dependent signaling in platelets was also confirmed.
[0181] In FIG. 4A, the effect of MMP-1 on Rho-GTP in platelets was
measured. Gel filtered human platelets were exposed to 3 nM
thrombin or 3 nM APMA-activated MMP-1 as indicated for 5 min at
37.degree. C. and Rho-GTP and total Rho was determined as described
above.
[0182] In FIG. 4B, the ability of MMP-1 to induce platelet shape
change was determined. Washed human platelets were pretreated with
2 mM EGTA and then challenged with MMP-1 in the presence or absence
of 1 .mu.M RWJ-56110 while stirring at 1100 rpm, Shape change was
measured as described above.
[0183] In FIG. 4C, the induction of PAR1-dependent calcium fluxes
by MMP-1 was measured. Calcium flux measurements of gel altered
platelets following challenge with MMP-1 in the presence or absence
of RWJ-56110 were performed at 25.degree. C. with emission recorded
at 510 nm and dual excitation at 340 and 380 nm as described
(Kullopulos, 1999).
[0184] In FIG. 4D, the induction of platelet aggregation by MMP-1
was determined. Gel-filtered platelets were challenged with MMP-1
in the presence or absence (0.2% DMSO vehicle) of the PAR1
inhibitor 1 .mu.M RWJ-56110.
[0185] Finally, in FIGS. 4E-4F, platelet PAR1-dependent MAPK
signaling induced by MMP-1 was also measured. Gel filtered
platelets were challenged with the indicated concentrations of
thrombin (Thr) or MMP-1 for 5 min as in FIG. 4A and p38MAPK (FIG.
4E) or downstream MAPKAP-K2 (FIG. 4F) activation was quantified by
densitometry of Western blots using a phospho-p38MAPK or
phospho-MAPKAP-K2 antibody, respectively. Blots were re-probed by
p38MAPK or MAPKAP-K2 to confirm equal loading in each lane (data
not shown).
[0186] The results show MMP-1 (3 nM) was able to stimulate Rho-GTP
activity to the same extent as equimolar thrombin (FIG. 4A). MMP-1
was also able to elicit platelet shape change, calcium
mobilization, and aggregation which was inhibited by the PAR1
antagonist, RWJ-56110 (FIGS. 4B-D). Exogenously added MMP-1 also
activated phospho-p38 MAPK and its substrate, MAPKAP-K2, in an
activity profile similar to thrombin (FIGS. 4E-F). MAPKAP-K2
phosphorylates the small heat shock protein HSP27 involved in
cytoskeletal reorganization (Sundaresan and Farndale, 2002),
further suggesting that MMP-1 may play a role in the initial events
leading to platelet shape change and help prime platelets for
aggregation.
I--(d) Collagen Triggers p38 MAPK Signaling, Rho Activation and
Platelet Aggregation Through MMP1-PAR1
[0187] The effect of pharmacologic blockage of metalloproteases or
PAR1 on collagen-dependent platelet aggregation was then tested
(see FIG. 5), Gel-filtered platelets from healthy individuals
(supplemented with 0.3 mg/ml fibrinogen) were challenged with 5
.mu.g/ml collagen in the presence or absence (0.2% DMSO vehicle) of
the indicated inhibitors and allowed to stir at 900 rpm in an
aggregometer cuvette (250 .mu.L) at 37.degree. C. Platelets were
pre-incubated for 5 min with the thrombin inhibitors PPACK (200
.mu.M) or hirudin (1 U/ml), the Zn-chelator 1,10-phenanthroline
(1,10-PA; 100 .mu.M), the broad spectrum metalloprotease inhibitor
MMP-200 (200 nM), the MMP-1 inhibitor FN-439 (3 .mu.M), the PAR1
ligand binding site inhibitor RWJ-56110 (1 .mu.M), the PAR1
blocking antibody (75 .mu.g/ml), the PAR1 pepducin lipopeptides
P1pal-12 (3 .mu.M) or P1pal-7 (3 .mu.M), the PAR4 pepducin
lipopeptide P4pal-10 (3 .mu.M), MMP-8 inhibitor (25 nM) or MMP9/13
inhibitor (10 nM).
[0188] In FIG. 5A, platelet aggregation was monitored by light
transmittance.
[0189] In FIG. 5B, platelets were treated as in FIG. 5A and then
lysed with Laemmli sample buffer 5 min after addition of collagen.
p38 MAPK activity was then assessed by western blot with a p38 MAPK
phospho-Ab and total p38 loading was determined using a p38 MAPK
antibody.
[0190] In FIG. 5C, platelets were treated as in FIG. 5B and then
Rho GTP activity was assessed by western blot.
[0191] In FIG. 5D, platelets were pre-treated with various blocking
Abs for 2 h or inhibitors (ARC, 0.5 .mu.M P2Y12 antagonist
AR-C69931MX; ASA, 1 mM aspirin for 30 min) and stimulated with one
of 5 .mu.g/ml collagen, 10 nM MMP-1 (Calbiochem), or 10 nM MMP-1
from a second source (S2, BioMol) as indicated and Rho-GTP activity
assessed as in FIG. 5C, Representative blots are shown at the
bottom of FIGS. 5B-D. Data are the mean.+-.s.d. of three
experiments. P*<0.01, #<0.05.
[0192] In FIG. 5E, platelets were pretreated with various
concentrations of P1pal-7 (denoted as "PZ-128" in the figure, and
also known as P1i3pal-7) in 0.2% DMSO vehicle and activated with
SFLLRN, collagen, ADP and ristocetin as indicated. Percent
aggregation was defined at the maximal point 7-15 min following
addition of angonist.
[0193] The results show soluble type I fibrillar collagen
stimulates platelet aggregation with an EC.sub.50 of 5 .mu.g/ml.
Inhibition of metalloproteases with the zinc-chelating agent
1,10-phenanthroline, resulted in 80% loss of aggregation to 5
.mu.g/ml collagen (FIG. 5A). Likewise, the broad spectrum
metalloprotease hydroxamate inhibitor. MMP-200 (IC.sub.50=7 nM for
MMP-1, 2.3 nM for MMP-2, 135 nM for MMP-3, 10-100 nM for MMP-7,
1-10 nM for MMP-13) caused a significant 50-60% inhibition of
collagen-initiated aggregation. Treatment with the MMP-1 inhibitor,
FN-439, inhibited collagen-induced aggregation to the same extent
as MMP-200. Conversely, inhibitors against MMP-8. MMP-9 and MMP-13
had no effect on collagen-induced aggregation (data not shown). The
specific thrombin inhibitor hirudin or the broad-spectrum serine
protease inhibitor, PPACK, had no effect on collagen aggregation
(FIG. 5A). PAR1 was inhibited by three orthogonal approaches to
evaluate its contribution to collagen-dependent aggregation. The
small-molecule inhibitor RWJ-56110 or a PAR1-blocking antibody,
attenuated 50% of collagen (5 .mu.g/ml)-induced aggregation, the
same extent as the MMP-1 inhibitor. Likewise, the cell-penetrating
PAR1 pepducin lipopeptides, P1pal-12 and P1 pal-7 (also known as
P1i3pal-7), which inhibit PAR1 signaling to intracellular G
proteins (Boire et al., 2005; Covic et al., 2002a; Kaneider et al.,
2007), gave identical levels of inhibition as blocking MMP-1 (FIGS.
5A and 5E). Inhibition of the PAR4 thrombin receptor with the
P4pal-10 pepducin lipopeptide (3 .mu.M) had only a slight
(.about.10%) effect on collagen-induced aggregation (FIG. 5A).
[0194] Collagen is known to induce p38 stress-activated protein
kinase (MAPK) pathways in human platelets though the mechanism
remains unclear (Kuliopulos et al., 2004; Sundaresan and Farndale,
2002). As shown in FIG. 5B, addition of collagen causes robust
phosphorylation of p38 MARK. The collagen (5 .mu.g/ml)-induced
phospho-p38 MARK signal was effectively blocked by the PAR1 and
MMP-1 inhibitors, but not with inhibitors against MMP-8, MMP-9/13,
or thrombin. Collagen-dependent activation of the p38 MARK
substrate, MAPKAP-K2 is also dependent on both PAR1 and MMP-1. The
PAR1 antagonists, RWJ-56110, P1pal-7 and FN-439 but not by MMP8
inhibitor blocked collagen-activation of phospho-MAPKAP-K2 (data
not shown).
[0195] The ability of collagen to stimulate Rho-GTP activity
through the MMP1-PAR1 pathway was also tested. Collagen caused
robust activation of Rho-GTP, which was attenuated by 75% with
antagonists against PAR1 and MMP-1, but not by inhibitors or
blocking antibodies against MMP-8, MMP-9/13, or thrombin (FIGS.
5C-D).
[0196] However, at saturating levels of collagen (20 .mu.g/ml)
sufficient to elicit full aggregation of platelets, none of the
PAR1 nor MMP-1 inhibitors had a major effect (.ltoreq.25%) on
collagen-dependent aggregation, the phospho-p38 MAPK signal, or
Rho-GTP activity (data not shown), indicating that the MMP1-PAR1
pathway can be bypassed at super-EC.sub.50 levels of collagen.
[0197] To address whether the observed MMP-1 effects were due to
secondary secretion of ADP or thromboxane after collagen
stimulation, the P2Y12 ADP and thromboxane pathways were inhibited
with ARC and aspirin (ASA) respectively (FIG. 5D). Treatment of
platelets with either ARC or aspirin had no effect on the ability
of 5 or 20 .mu.g/ml collagen or 10 nM MMP-1 to activate Rho-GTP
(FIGS. 5D, 5E and 5H) or phospho-p38 MARK, but the inhibitors could
still suppress aggregation to collagen (FIGS. 5E, 5G and 5H). In
contrast, blockade of the MMP1-PAR1 pathway nearly completely
inhibited activation of p38 and Rho-GTP to 5 .mu.g/ml collagen
(FIGS. 5B-D). This would indicate that at EC50 collagen exposure,
MMP1-PAR1 is essential for activation of p38 and Rho-GTP, and
important for aggregation, whereas the secondary ADP and
thromboxane pathways do not activate p38 and Rho-GTP at any range
of collagen concentration. At saturating collagen, the ADP and
thromboxane contributions appear to compensate for the MMP1-PAR1
pathway in platelet aggregation via mechanisms that do not involve
p38 or Rho-GTP signaling.
I--(e) Early Platelet Thrombus Formation on Collagen Surfaces is
Promoted by MMP-1 and PAR1
[0198] Activation of platelets in ruptured atherosclerotic plaques
occurs under high shear-stress conditions on subendothelial
surfaces enriched in collagen fibrils. The role of MMP-1 and PAR-1
in the initial formation and propagation of platelet-platelet
thrombi on collagen surfaces was investigated by specifically
inhibiting MMP-1 or PAR1 (see FIGS. 6A-6B).
[0199] Whole human blood was anti-coagulated with heparin (It,
Writ), or with corn trypsin inhibitor (CTI, 30 .mu.g/ml final)
before being pretreated for 10 min with vehicle (0.2% DMSO),
MMP-200 (200 nM), MMP-1 inhibitor FN-439 (3 .mu.M). PAR1 ligand
binding site inhibitor RWJ-56110 (1 .mu.M), PAR1 pepducin
lipopeptides P1pal-12 or P1pal-7 (3 .mu.M), or for 30 min with 1 mM
aspirin prior to the assay as indicated. Following incubation with
inhibitors, the blood was perfused over a glass slide coated with
fibrillar collagen type I.
[0200] A flow chamber (Glycotech) with Type-I fibrillar
collagen-coated glass slides was mounted on the stage of an IMT-2
inverted microscope (Olympus) equipped with Retiga 1300 digital
camera (QImaging) and 40.times. objective. One of the flow chamber
inlets was connected to a syringe pump (Harvard Apparatus)
calibrated to create a shear rate of 1,000.sup.s-1. The whole blood
pretreated with the various pharmacologic inhibitors was then
perfused over the collagen-coated glass slide. After 2-15 min of
perfusion, blood was removed from the flow chamber by gentle
displacement with PIPES buffer and images of 8-10 fields were
acquired using OpenLab software (Improvision). Acquired images were
further analyzed using NIH Image 1.63 software. Images were first
sharpened and the edges of separate platelets and platelet
aggregates were determined. Following the adjustment of threshold,
images were converted into the binary format, and the particle
analysis function was activated to highlight the regions covered by
platelets with the sensitivity set to one single adherent
thrombocyte. For the conversion of pixels into square micrometers,
a calibration curve was built using 2.5, 6, 20, 25, and 45 .mu.m
polystyrene beads (Polyscience) using acquisition conditions
identical to the experimental. The mean area of formed thrombus was
determined at 7 min. Area measurements in FIG. 63 represent the
mean.+-.s.e. of three separate experiments from five different
blood donors.
[0201] Treatment with either MMP-1 or PAR1 inhibitors did not
affect the primary adhesion of platelets to the immobilized
collagen fibrils. However, the growth rate and size of platelet
aggregate "strings" was significantly attenuated by .about.75% with
the MMP-1 inhibitor, FN-439, or the PAR1 blocking agents P1pal-12,
P1pal-7 or RWJ-56110 (FIGS. 6A-B). By comparison, aspirin
pre-treatment had little or no effect on the growth of the platelet
thrombi. This result is consistent with thromboxane playing a
relatively minor role in thrombogenesis under arterial shear stress
conditions (Jackson et al., 2003) as compared to the MMP1-PAR1
pathway.
[0202] Collagen-activated platelets also provide a pro-coagulant
surface and produce tissue factor, which aids in the production of
thrombin (Giesen et al., 1999; Mackman, 2004; Schwertz et al.,
2006). To evaluate whether MMP1-PAR1 activation of early platelet
thrombi formation is also relevant under conditions in which
thrombin activity is not inhibited, arterial flow experiments were
performed in the presence of corn trypsin inhibitor (CTI) which
blocks factor XIIa and the contact pathway of coagulation but does
not inhibit thrombin generation in whole blood (Mann et al., 2007;
Rand et al., 1996).
[0203] Whole blood was anti-coagulated with CTI (30 .mu.g/mL) to
block the contact pathway, otherwise the experiments were conducted
identically as in FIG. 6A. Hirudin was used as indicated at 0.0013
U/mL.
[0204] The results using the CTI anti-coagulant were very similar
to those conducted with heparin. As shown in FIGS. 6C and 6D,
inhibition of MMP-1 or PAR-1 significantly attenuated the size of
the platelet micro-thrombi on the collagen surfaces, whereas
addition of the thrombin inhibitor, hirudin, had no effect.
Likewise, aspirin pre-treatment of the whole blood did not affect
the extent of platelet-thrombi formation on the collagen surfaces.
Thus, under conditions of arterial shear stress, MMP1-PAR1
significantly promotes early thrombogenesis on collagen
surfaces.
[0205] Clot retraction assays were then performed to examine the
potential role of MMP-1 on the structure of large platelet-rich
clots over time. Platelet receptors trigger clot retraction by
activating myosin-dependent contraction of the cytoskeleton, which
is in turn connected to the extracellular matrix (fibrinogen) via
focal adhesions (see FIG. 6I).
[0206] Platelet rich plasma (PRP) was isolated from human whole
blood anti-coagulated with 4% sodium citrate. Clot retraction
assays were done in 1 ml volumes containing 800 .mu.l PBS and 200
.mu.l PRP plus 10 .mu.l red blood cells to enhance contrast.
Samples were pretreated with FN-439 (5 .mu.M), MMP9/13 Inh (10 nM)
or RWJ-56110 (10 .mu.M) for 15 min and clot formation was initiated
with 1-20 .mu.g/ml type I fibrillar collagen. Samples were
incubated at 37.degree. C. for 90 or 240 min and then photographed
with a digital camera. Each photograph in FIG. 6I, representative
of three independent experiments.
[0207] As shown in FIG. 6I, the MMP-1 inhibitor, FN-439, completely
inhibited clot formation and retraction induced by 2.5-5 .mu.g/ml
collagen. Blockade of PAR1 with RWJ-56110 gave a nearly identical
pattern of inhibition, whereas the negative control MMP9/13
inhibitor had negligible effects over the whole collagen titration.
Therefore, MMP-1 and PAR1 play a significant role in the formation
and retraction of large platelet-rich thrombi initiated by
collagen.
I--(f) Pharmacologic Inhibition of Matrix Metalloprotease-2 (MMP-2)
Attenuates Collagen-Dependent Platelet Aggregation to a Similar
Extent as Blockade of MMP-1.
[0208] To further demonstrate that inhibition of MMP-1 abrogates
collagen-induced platelet activation, a series of experiments were
performed to test the ability of platelets to aggregate in the
presence of the MMP-2 inhibitors, which are known to inhibit
pro-MMP-1 cleavage and activation.
[0209] Human platelets were isolated by gel filtration
chromatography of platelet-rich plasma with the use of a Sepharose
2B column in modified PIPES buffer. After addition of 2.0 mM
CaCl.sub.2 and 300 .mu.g/ml fibrinogen, the platelets were
pre-incubated for 2 minutes with vehicle (0.2% DMSO), 5 .mu.mol/L
MMP2 inhibitor I (FIG. 8A), 51 .mu.mol/L MMP2/3 inhibitor I (FIG.
8B), 15 .mu.mol/L MMP3 inhibitor III (FIG. 8C), 5 .mu.mol/L. FN-439
(FIG. 8D), 200 nM MMP-200 and then stimulated by various
concentrations of collagen. Aggregation was measured for 15 minutes
with the use of a Chronolog 560VS/490-2D aggregometer. The
aggregation assay was repeated with 3-6 healthy blood donors.
[0210] In FIG. 8E, platelets were pre-incubated for 5 min with the
thrombin inhibitor hirudin (1 U/ml), the metalloprotease inhibitors
1,10-phenanthroline (1,10-P; 100 .mu.M) or MMP-200 (200 nM), the
MMP-1 inhibitor FN-439 (3 .mu.M), the PAR1 ligand binding site
inhibitor RWJ-56110 (1 .mu.M), the PAR1 blocking antibody (75
.mu.g/ml), the PAR1 pepducin lipopeptides P1pal-12 (3 .mu.M) or
P1pal-7 (3 mM), or the PAR4 pepducin lipopeptide P4pal-10 (3
.mu.M). Data are the mean.+-.s.d. of three experiments. P*<0.01,
#<0.05.
[0211] Inhibition of MMP-2, but not MMP-3, inhibits
collagen-induced platelet aggregation.
I--(g) Systemic Platelet Activation and Arterial Thrombosis by
MMP-1 and PAR1 in Guinea Pig
[0212] A series of experiments were then performed to determine if
blocking the MMP1-PAR1 pathway would protect against
collagen-mediated systemic platelet activation in vivo. Guinea pigs
serve as an relevant model to test platelet function because like
humans they also express PAR1 on their platelets (Leger et al.,
2006a) and guinea pig MMP-1 shares 90% identity with human MMP-1
(Huebner et al., 1998).
[0213] Animal experiments were performed in accordance with the
National Institutes of Health guidelines and approved by the Tufts
Medical Center Institutional Animal Care and Use Committee. 2-4
week old Hartley guinea pigs (170-260 g) were anesthetized by an
i.p. injection of xylazine (10 mg/kg) plus ketamine (50 mg/kg) and
then catheterized via the left jugular vein and injected (200
.mu.L) with either vehicle (20% DMSO/80% water), P1pal-7 or FN-439.
For determination of collagen-dependent systemic platelet
activation, 10 min after administration of inhibitors, 200 .mu.g
collagen in 200 .mu.L of PBS was delivered via the jugular vein.
Ten min after collagen injection, blood was collected into sodium
citrate (1% v/v final) from the contralateral jugular vein and
platelet counts were measured with a Hemavet850. The enzymatic
activity of active MMP-1 in supernatants and platelet lysates was
determined using DQ collagen I as fluorogenic substrate in the
presence or absence of 3 .mu.M FN-439, or 20 .mu.g/ml of control
IgG or a MMP-1 blocking Ab (preincubated for 2 h at 37.degree. C.),
as indicated.
[0214] FIG. 7A shows the surface expression of MMP-1 on guinea pig
platelets as determined by flow cytometry. Dashed grey line:
secondary antibody alone; Solid lines: FACS profiles of platelets
treated with the indicated concentrations of collagen for 15 min at
37.degree. C. and then stained with primary MMP-1 (AB806) plus
secondary antibodies. FIG. 7B shows the activation of guinea pig
platelets treated for 15 min with 20 .mu.g/ml type-I collagen in
the presence of various inhibitors As described above.
[0215] As shown in FIGS. 7A and 7B, FACS analysis on guinea pig
platelets showed proMMP-1 is expressed on their surface and
addition of collagen causes release of collagenase activity which
is completely blocked by either FN-439 or a MMP1-neutralizing Ab.
Likewise, inhibition of MMP-1 or PAR1 gave 35-50% suppression of
aggregation and complete inhibition of Rho-GTP activity in response
to 10 .mu.g/ml collagen in guinea pig platelets (FIGS. 7C-D), which
was consistent with the previous results using human platelets.
[0216] Intravascular platelet activation was then induced by an
intravenous injection of collagen into the guinea pigs. Vehicle,
P1pal-7 (3 mg/kg) or FN-439 (10 mg/kg) were delivered i.v. to the
internal jugular vein of guinea pigs (n=6) and allowed to circulate
for 10 min, Blood was drawn before and 10 min after collagen (200
.mu.g) induction of systemic activation of platelets. The infused
collagen caused a severe drop in mean systemic platelet counts from
a baseline level of 309,000.+-.50,000/mL to 194,000.+-.20,000/mL.
Strikingly, pre-administration of the PAR1 pepducin lipopeptide,
P1pal-7, almost completely protected against collagen-induced
thrombocytopenia in the guinea pigs (FIG. 6E). The MMP-1 inhibitor,
FN-439, also afforded significant protection against intravascular
platelet activation.
[0217] To assess the efficacy on arterial thrombosis by blockade of
thrombin, MMP-1, or PAR1, a standard carotid artery FeCl.sub.3
injury model was used in guinea pigs. FeCl.sub.3 causes denudation
of the artery and exposure of type I collagen and other
subendothelial matrix proteins. The effects of blocking thrombin
with bivalirudin, MMP-1 with FN-439, and PAR1 with either the small
molecule antagonist RWJ-56110 or the P1pal-7 pepducin on arterial
thrombosis were then compared using a dopier probe.
[0218] For these arterial thrombosis experiments, 10 min after i.v.
administration of vehicle (-), bivalirudin, RMJ-56110, P1pal-7 or
FN-439 (n=4-5) through the jugular vein at the indicated
concentrations, the right carotid arteries were injured for 20 min
using a 24 mm.sup.2 piece of Bio-Rad Trans-Blot paper soaked in 20%
FeCl.sub.3. Arterial flow 5 mm distal to the site of injury was
measured with a 0.5 V Doppler probe (Transonic Systems). An
arterial occlusion was defined as a flow rate of <0.01 V for
.gtoreq.5 min. Doppler measurements were terminated at the 30 min
time point following injury (* designates p<0.05).
[0219] Bivalirudin alone (0.18 mg/kg) prolonged the mean arterial
occlusion time from 13 min to 20 min (FIG. 6F). The small molecule
PAR1 antagonist, 0.5 mg/kg RWJ-56110, did not appreciably affect
the mean occlusion time, however, equimolar amounts of P1pal-7
(0.13 mg/kg, 75% lipopeptide, 25% salt) gave a similar trend of
protection as bivalirudin (p=0.10) (FIG. 6F). Supplementation of
0.13 mg/kg P1pal-7 with 0.18 mg/kg bivalirudin gave no additional
prolongation of the arterial occlusion time, but a slightly higher
dose of P1pal-7 alone (0.3 mg/kg), gave a significant (p<0.05)
80% prolongation of the mean occlusion time (FIG. 6F). Further, the
ammonium acetate salt of P1pal-7 in 100% water gives similar 1050
values and retains specificity for PAR1. Administration of the
MMP-1 antagonist FN-439 alone (at 2.0 mg/kg), gave a similar
prolongation (90%, FIG. 6F) of the mean arterial occlusion time.
Co-administration of the PAR1 and MMP-1 inhibitors did not lead to
further prolongation of the mean occlusion time, consistent with
MMP-1 acting in the same pathway as PAR1. Similar results were also
reported in FIG. 60. These examples provide the validation for one
of the present inventive principles that inhibition of MMP1-PAR1
can be used to provide substantial protection against
collagen-dependent platelet activation and acute arterial
thrombosis independently of blocking thrombin.
[0220] MMP-1 activity with the clots was determined by collagent
zymography (see FIG. 6H), Platelets were isolated from guinea pigs
and stimulated with 20 .mu.g/ml collagen for 15 min and platelet
supernatants and pellets, or whole resting platelets (control),
prepared as described Above. Samples were resolved on a 8% type I
collagen/acrylamide zymography gel and collagenase zymogram was
developed as described by (Gogly et al., 1998) or immunoblotted
with MMP1-Ab, AB806. Arterial clots were also removed from the
Fe--Cl injured carotid arteries from animals (n=5) treated with
FN-439 (FN439 clot) or vehicle (veh clot) as in 6F and 6G and clots
were dissolved in lysis buffer and passed 5.times. through a
21-gauge needle. The lysates were centrifuged and supernatents
resolved on the zymography gel. The two lanes on the left side of
the western blot have 20 ng of proMMP-1 or 20 ng APMA-activated
MMP-1, and the right hand lane in the zymogram has 0.5 .mu.g of
APMA-activated human MMP-1.
[0221] Collagen zymography revealed that the platelet-rich clot
isolated from injured carotid arteries of vehicle-treated animals
(veh clot) had significant MMP-1 activity, which co-migrated with
APMA-activated MMP-1 and with the MMP-1 activity from the
supernatants of collagen-activated platelets (FIG. 6H). Conversely,
resting platelets (control) from whole blood or arterial thrombi
from animals treated with the MMP1-inhibitor (FN439 clot) did not
contain active MMP-1. These data, together with the previous
results, indicate that inhibition of MMP1-PAR1 may provide
substantial protection against collagen-dependent platelet
activation and acute arterial thrombosis in animals.
Materials
[0222] Sodium citrate, EDTA, apyrase, 1,10-phenanthroline, A-23187,
and U-46619 were obtained from Sigma. ADP and fibrillar Type
collagen were from Chronolog Corp. MMP-200 was obtained from Enzyme
Systems Products.
[0223] ProMMP-1 (.gtoreq.90% purity, from human synovial
fibroblasts), proMMP-3, proMMP-7, FN-439 (MMP inh-1), MMP8 inh,
MMP9/13 inh, hirudin, and PMA were from Calbiochem. RWJ-56110 was a
kind gift from Claudia Derian and Particia Andrade-Gordon of
Johnson & Johnson Pharmaceuticals Research and Development.
AR-C69931 MX was a gift from Astra Zeneca.
[0224] The pepducin lipopeptides, P1pal-12. P4pal-10 and P1pal-7
were synthesized with C-terminal amides and purified by RP-HPLC as
before (Covic et al., 2002a).
[0225] SFLLRN, TFLLRN, PRSFLLRN, RPSFLLRN, PSFLLRN, DPRSFLLRN, PAR1
N-terminal thrombin cleavage peptide (N.sub.26-R.sub.43), PAR1
flexible linker peptide (N-acetyl-T.sub.67-L.sub.84-C), and TR26
(A.sub.36-E.sub.60-S) and TR26-P40N (A.sub.36-K.sub.61) were
synthesized with C-terminal amides by the Tufts Peptide Core
Facility and purified to .gtoreq.95% purity by RP-HPLC.
[0226] The IIaR-A monoclonal antibody, which reacts to the
amino-terminal thrombin-cleavage peptide of PAR1, was from
Biodesign (Kennebunk, Me.).
[0227] The PAR1 blocking antibody raised against residues
S.sub.42FLLRNPNDKYEPF.sub.55C was generated from rabbits as
previously described (Kuliopulos 1999). A solid phase proMMP-1
ELISA system from R&D Systems (Quantikine DMP100) utilizes 2
monoclonal antibodies that recognize the pro domain of MMP-1.
[0228] The ELISA assay detects proMMP-1 and soluble pro domain but
does not detect active MMP-1. The MMP-1 blocking Ab (rabbit
polyclonal antibody AB8105) raised against the conserved C-terminus
recognizes both pro and active forms of MMP-1 but do not cross
react with other MMP family members was from Chemicon, the MMP-8
(IM38L) and MMP-13 (IM44L) blocking Abs were from Oncogene, the
anti-.alpha..sub.2 (Gi9 or AK7), .beta..sub.1 (MAB1987),
.beta..sub.3 (MAB1957) were from Chemicon, GPVI (SC20149) was from
Santa Cruz, GPIB.alpha. (MM2/174) was from AbD Serotec and ELISA
kits for Abs against the MMP-1 pro-domain (DMP100, R&D Systems)
of MMP-1 were used according to the manufacturer's
instructions.
[0229] Anti-phospho p38MAPK, p38MAPK, anti-phospho-MAPKAP-K2, and
anti-MAPKAP-K2 were from Cell Signaling, anti-RhoA (clone 2604) was
from Santa Cruz Biotechnology.
[0230] Corn trypsin inhibitor and thrombin were from Haematologic
Technologies, the Quick Change Mutagenesis kit was from
Stratagene.
II Inhibitors of the MMP-1-PAR-1 Signalling Pathway
II--(a) MMP1-PAR1 As a New Target for the Prevention of a
Thrombotic Disease State
[0231] Matrix metalloproteases are implicated in the chronic
pro-inflammatory and tissue-remodeling events leading to cleavage
of interstitial collagen and development of vulnerable
atherosclerotic plaques (Sukhova et al., 1999). Although
patho-anatomic studies of human atherosclerotic lesions suggest
that large plaques can cause ischemic symptoms, the key
contributing factor to the morbidity and mortality associated with
atherosclerosis is excessive platelet thrombus formation on exposed
collagen surfaces following acute plaque rupture (Glass and
Witztum, 2001; Rugged, 2002).
[0232] I This invention discloses a novel collagen-initiated
pathway of thrombogenesis which is mediated by the autocrine action
of platelet MMP-1 on the PAR1 receptor. Exposure of platelets to
collagen caused robust activation of MMP-1 on the platelet surface,
which in turn directly cleaved and activated PAR1 independently of
thrombin. These studies provide a link between matrix-dependent
activation of metalloproteases and platelet G protein signaling and
identify MMP1-PAR1 as a potential new target for the prevent on of
arterial thrombosis.
[0233] Unexpectedly, MMP-1 cleaved PAR1 at a distinct site in its
extracellular domain, which generated a longer tethered ligand than
that produced by thrombin. The MMP1-cleaved receptor or soluble
peptide analog strongly stimulated G.sub.12/13-Rho-dependent
pathways, chemotaxis and MAPK signaling in platelets and other
cells. The MMP-1 cleavage site on PAR1 aligned with an optimized
MMP-1 cleavage site motif determined from mixture-based oriented
peptide libraries (Turk et al., 2001) and by substrate cleavage
studies (Berman et al., 1992; Netzel-Amett et al., 1991). Mutation
of respective P1' residues uncoupled MMP-1 from thrombin cleavage
and generated PAR1 receptors that exhibited protease-specific
activity.
[0234] Collagen signaling in human platelets through the
.alpha..sub.2.beta..sub.1 and GPVI/Fc.gamma.R collagen receptors is
not well understood, but is dependent on G protein signaling
through autocrine stimulation of ADP and thromboxane receptors
(Jackson et al., 2003). Blockade of the P2Y12 G.sub.i-coupled ADP
receptor inhibits collagen-dependent thrombogenesis under arterial
flow conditions, thus establishing an important role for downstream
ADP-G signaling. Thromboxane activates the G.sub.q and
G.sub.12/13-coupled TXA.sub.2 receptor, however, aspirin fails to
prevent thrombogenesis on collagen surfaces under arterial shear
stress and does not prevent occlusive thrombus formation in patents
with severe arterial stenosis (Veen et al., 1993). The current
studies show that MMP-1 is a potent activator of PAR1-G.sub.12/13
pathways involved in platelet shape change and Rho activation and
thus would synergize with P2Y12-G.sub.i signaling.
[0235] Blockade of MMP-1 or PAR1 with pharmacologic inhibitors
significantly attenuated thrombogenesis on collagen surfaces under
arterial shear stress conditions and thrombosis in animals. As
compared to MMP-1 inhibition, antagonism of thrombin had little
effect on early thrombogenesis on the collagen surfaces under high
arterial flow rates. Thrombin may be more important for later
propagation and stability of platelet thrombi, and is not involved
in initiating early thrombus growth at high arterial shear stress
(Fressinaud et al., 1992; Gast et al., 1994; Inauen et al., 1990)
unless tissue factor levels are extremely high (Okorie et al.,
2008). Likewise, thrombin inhibitors such as heparin have
incomplete effects on platelet thrombus formation at high arterial
flow rates, but have a more prominent inhibitory effect on the
growth and overall stability of platelet thrombi at low and
intermediate shear rates (Inauen et al., 1990).
[0236] Unlike direct blockade of MMP-1 or thrombin, downstream
inhibition of PAR1 may hold the potential to prevent both the
initial MMP1-dependent events of platelet thrombi propagation on
blood vessel collagen, along with later thrombin-dependent
propagation and stabilization and could prove beneficial in
preventing arterial thrombosis in acute settings. To that end, the
present invention provides that various agents, e.g., the
cell-penetrating PAR1 pepducin, can provide efficient blockade of
both thrombin-mediated and collagen-MMP1-mediated activation of
PAR1 which could benefit large patient populations being treated
for atherothrombotic heart disease and ischemic stroke.
II--(e) Screening for Novel MMP1-PAR1 Inhibitors
[0237] Discovery of agents acting on the MMP-1/PAR-1 signaling
pathway may be accomplished using methods that are well known in
the art. In a first step, agents that bind to a target molecule
within the MMP-1-PAR-1 signaling pathway are identified. The
efficacy of a selected agent may then be evaluated using in vitro
PAR-1 signaling or cleavage assays, as described herein, and then
in vivo using animal models of thrombotic disease states.
MMP-1/PAR-1 specific agents may act as an angonist or antagonist of
the MMP-1-PAR-1 signaling pathway.
[0238] In a preferred embodiment, an agent acts as a direct or
indirect antagonist of the MMP-1 PAR-1 signaling pathway. Direct
antagonists are compounds that bind directly to their target
molecule, such as MMP-1 or PAR-1, and inhibit the biological
activity of that target molecule.
[0239] For example, a MMP-1 antagonist may be a compound that binds
to and inhibits MMP-1 enzymatic activity, i.e. proteolytic cleavage
between aspartic acid at position 39 (D39) and praline at position
40 (P40) of the protease-activated receptor-1 (PAR-1). In one
embodiment, the MMP-1 antagonist may bind directly to the MMP-1
active site to inhibit MMP-1 enzymatic activity. In another
embodiment, the MMP-1 antagonist may bind to a site other than the
active site and inhibit MMP-1 activity by inducing a conformational
change in MMP-1 or modifying the post-translational state of the
protein, such as phosphorylation, de-phosphorylation,
glycosylation, acylation, alkylation, or lipoylation.
[0240] In other examples, a PARA antagonist may be a compound that
binds to PAR-1 and prevents proteolytic cleavage between aspartic
acid at position 39 (D39) and praline at position 40 (P40) of the
protease-activated receptor-1 (PAR-1). In one embodiment, a PAR-1
antagonist may be a compound, such as an antibody, that binds
directly over the LD.sub.39.dwnarw.P.sub.40RSFL MMP-1 cleavage site
at the N terminal domain of PAR-1 and prevents proteolytic cleavage
at that site. In other embodiments, a PAT-1 antagonist may inhibit
proteolytic cleavage of PAR-1 by inducing a conformational change
in PAR-1 or altering the post-translational modification of PAR-1,
such as phosphorylation, de-phosphorylation, glycosylation,
acylation, alkylation, or lipoylation in such a way that
proteolytic cleavage at the LD.sub.39.dwnarw.P.sub.10RSFL MMP-1
cleavage site is prevented.
[0241] In still other embodiments, a PAR-1 antagonist may interfere
with the MMP-1-generated tethered ligand's ability to interact with
other domains within PAR-1 and thereby inhibit activation of MMP-1
mediated PAR-1 signaling. In still other embodiments, a PAR-1
antagonist may prevent PAR-1 signaling by interfering with the
assembly of protein complexes comprising PAR-1 or MMP-1 or the
dimerization of PAR-1 with other PAR family members.
[0242] In one embodiment, the agent is a PAR-1 pepducin
lipopeptide. In another embodiment, the agent is SCH 530348, a
PAR-1 antagonist developed by Schering-Plough, or derivative
thereof.
[0243] In another embodiment, a inhibitor may interfere with
complex formation between PAR-1 and various nucleotides including,
but not limited to GTP, GDP or GMP.
[0244] In yet another embodiment, a MMP-1 or PAR-1 agent may
modulate the gene expression of other factors that are required for
MMP-1 mediated PAR-1 activation. In this aspect, an "agent" may
include modulators of the gene transcription or translation of
factors required for MMP-1 mediated PAR-1 activation.
[0245] Indirect antagonists are compounds that bind to ancillary
target molecules that are required for MMP-1 mediated activation of
PAR-1 signaling activity. For example, a MMP-1 antagonist may
inhibit MMP-1 activity by inhibiting the proteolytic cleavage of
pro-MMP-1 to active MMP-1. For example, a MMP-1 antagonist may bind
to the pro-MMP-1's proteolytic cleavage site and prevent
cleavage.
[0246] In other embodiments, a PAR-1 antagonist may inhibit PAR-1
signaling activity by preventing the formation of protein complexes
between MMP-1 and associated factors or between PAR-1 and
associated factors. In still other embodiments, a PAR-1 antagonist
may interfere with the biological activity of downstream PAR-1
effector molecules that are required for MMP-1 mediated PAR-1
signaling activity.
[0247] Direct inhibitors may be identified by screening for
compounds using in vitro screening assays that require a purified
target molecule whose activity is required for MMP-1 mediated PAR-1
signaling. In a preferred embodiment, the target protein is native
PAR-1 protein or a PAR-1 deletion mutant in which the N terminal
residues 1 and 39 are deleted. However, the experimental approach
disclosed herein may be applied to any target protein that is
required for PAR-1 function. For example, the target molecule may
be MMP-1 or factors required for MMP-1 activation. In yet other
examples, the target molecule may be a factor, such as a kinase or
phosphatase, that modulates PAR-1 activity by altering the
post-translational modification of PAR-1 or MMP-1.
II-(c) Prokaryotic Expression of Recombinant PAR-1 Polypeptide
[0248] In one embodiment, the invention requires purification of a
target protein or a fragment thereof, e.g. PAR-1.
[0249] PAR-1 may be purified directly from a biological source,
such as human platelets. Alternatively. DNA encoding PAR-1, or
fragment thereof, may be operably linked to genetic constructs,
e.g., vectors and plasmids for expression in a prokaryotic host. In
some cases a nucleic acid is operably linked to a transcription
and/or translation sequence in an expression vector to enable
expression of a PAR-1 polypeptide. By "operably linked," it is
meant that a selected nucleic acid, e.g., a coding sequence, is
positioned such that it has an effect on, e.g., is located adjacent
to, one or more sequence elements, e.g., a promoter and/or ribosome
binding site (Shine-Dalgarno sequence), which directs transcription
and/or translation of the sequence. Some sequence elements can be
controlled such that transcription and/or translation of the
selected nucleic acid can be selectively induced. Exemplary
sequence elements include inducible promoters such as tac, T7,
P(BAD) (araBAD), and beta-D-glucuronidase (uidA) promoter-based
vectors. Control of inducible promoters in E. coli can be enhanced
by operably linking the promoter to a repressor element such as the
lac operon repressor (lac(R)). In the specific case of a repressor
element, "operably linked" means that a selected promoter sequence
is positioned near enough to the repressor element that the
repressor inhibits transcription from the promoter (under
repressive conditions). Typically, expression plasmids and vectors
include a selectable marker (e.g., antibiotic resistance gene such
as Tet(R) or Amp(R)). Selectable markers are useful for selecting
host cell transformants that contain a vector or plasmid.
Selectable markers can also be used to maintain (e.g., at a high
copy number) a vector or plasmid in a host cell. Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0250] In some embodiments, the polypeptide sequence of interest
may be expressed as part of a fusion protein using recombinant DNA
technology. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa or enterokinase. Typical fusion expression vectors
include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K.
S. (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly,
Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0251] The PAR-1 polypeptide may also be engineered to have an
affinity tag fused to its N or C terminal end. For example, the
target protein may be fused to a short tag peptides, such as the
Hexa-His peptide or the HA Epitope Tag (Influenza Hemaglutinin)
synthetic peptide YPYDVPDYA. These tag proteins or peptides also
facilitate subsequent protein purification by affinity
chromatography.
[0252] Other commonly used bacterial host plasmids include pUC
series of plasmids and commercially available vectors, e.g.,
pAT153, pBR, PBWESCRIPT, pBS, pGEM, pCAT, pEX, pT7, pMSG, pXT,
pEMBL. Another exemplary plasmid is pREV2.1, Plasmids that include
a nucleic acid described herein can be transfected or transformed
into bacterial host cells for expression of PAR-1 polypeptides.
Techniques for transformation are known in the art, including
calcium chloride or electroporation. In other embodiments, the
recombinant DNA sequence is cloned into a bacteriophage vector. In
certain embodiments, transformed host cells include non-pathogenic
prokaryotes capable of highly expressing recombinant proteins.
Exemplary prokaryotic host cells include laboratory and/or
industrial strains of E. coli cells, such as BL21 or K12-derived
strains (e.g., C600, DHIalpha, DH5alpha, HBIOI, INVI, JM109, TBI,
TGI, and X-IBlue). Such strains are available from the ATCC or from
commercial vendors such as BD Biosciences Clontech (Palo Alto,
Calif.) and Stratagene (La Jolla, Calif.). For detailed
descriptions of nucleic acid manipulation techniques, see Ausubel
et al., eds., Current Protocols in Molecular Biology, Wiley
Interscience, 2006, and Sambrook and Russell, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY, 2001.
II--(d) Eukaryotic Expression of Recombinant PAR-1 Polypeptide
[0253] In other embodiments, PAR-1 protein or fragments thereof may
be expressed in eukaryotic cells. Using standard recombinant DNA
techniques, a PAR-1 nucleic acid is cloned into a vector in a form
suitable for expression of the nucleic acid in a host cell.
Preferably the recombinant expression vector includes one or more
regulatory sequences operatively linked to the nucleic acid
sequence that facilitates expression within the eukaryotic cell.
The term "regulatory sequence" includes promoters, enhancers and
other expression control elements (e.g., polyadenylation signals).
Regulatory sequences include those, which direct constitutive
expression of a nucleotide sequence, as well as tissue-specific
regulatory and/or inducible sequences. The design of the expression
vector can depend on such factors as the choice of the host cell to
be transformed, the level of expression of protein desired, and the
like. The expression vectors of the invention can be introduced
into host cells to thereby produce proteins or polypeptides,
including fusion proteins or polypeptides, encoded by nucleic acids
as described herein (e.g., PAR-1 proteins, mutant forms of PAR-1
proteins, fusion proteins, and the like). For example, PAR-1
polypeptides can be expressed in insect cells (e.g., using
baculovirus expression vectors), yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, (1990) Gene
Expression Technology Methods in Enzymology 185, Academic Press,
San Diego, Calif.
[0254] In mammalian cells, the expression vector's control
functions can be provided by viral regulatory elements. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40. In some
embodiments, the promoter may be an inducible promoter, e.g., a
promoter regulated by a steroid hormone, by a polypeptide hormone
(e.g., by means of a signal transduction pathway), or by a
heterologous polypeptide (e.g., the tetracycline-inducible systems,
"Tet-On" and "Tet-Off"; see, e.g., Clontech Inc., CA, Gossen and
Bujard (1992) Proc. Natl. Acad. Sci. USA 89: 5547, and Paillard
(1989) Human Gene Therapy 9: 983). In some embodiments, the PAR-1
polypeptide sequence comprises a signal peptide sequence which
promotes the secretion of the PAR-1 polypeptide.
[0255] Mammalian cell lines suitable for protein expression
include, but is not limited to, Chinese hamster ovary cells (CHO)
or COS cells (African green monkey kidney cells CV-1 origin SV40
cells; Gluzman (1981) Cell 123: 175-182)). Other suitable host
cells are known to those skilled in the art.
[0256] Vector DNA can be introduced into host cells via
conventional transfection techniques. As used herein, the terms
"transformation" and "transfection" are intended to refer to a
variety of art-recognized techniques for introducing foreign
nucleic acid (e.g., DNA) into a host cell, including calcium
phosphate co-precipitation, DEAE-dextran-mediated transfection,
lipofection, or electroporation.
II--(e) Screening Assays--Identification of PAR-1/MMP-1
Ligand-Binding Molecules
[0257] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs), which
bind to PAR-1 or MMP-1 proteins, have an inhibitory effect on, for
example, PAR-1 or MMP-1 expression or PAR-1 or MMP-1 activity.
Compounds thus identified can be used to modulate the activity of
target gene products (e.g., PAR-1 or MMP-1 genes) in a therapeutic
protocol, to elaborate the biological function of the target gene
product, or to identify compounds that disrupt normal target gene
interactions.
[0258] In one embodiment, the invention provides assays for
screening candidate or test compounds that bind to or modulate an
activity of a PAR-1 or MMP-1 protein or polypeptide or a
biologically active portion thereof.
[0259] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive: see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem.
37: 2678-85); spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution: the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection.
The biological library and peptoid library approaches are limited
to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam (1997) Anticancer Drug Des. 12:
145).
[0260] In one embodiment, the test compounds may be peptidominetic
compounds of the PR-TRAP peptide, PRSFLLNRN or variant thereof.
[0261] In other embodiments, a test compound refers to a
"recombinant antibody" that is generated using recombinant DNA
technology, such as, for example, an antibody expressed by a
bacteriophage. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody or parts thereof and which DNA
molecule expresses an antibody protein or parts thereof, or an
amino acid sequence specifying the antibody, wherein the DNA or
amino acid sequence has been obtained using synthetic DNA or amino
acid sequence technology which is available and well known in the
art. Recombinant antibodies may be selected for increased or
improved affinity via the screening of a combinatory antibody
library under stringent binding conditions. For example, nucleic
acids encoding a chimeric or humanized chain can be expressed to
produce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat.
No. 4,816,567; Cabilly et al., European Patent No. 0 125 023 B1;
Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent
No 0 120 694 B1; Neuberger et al., International Publication No.
WO86/01533; Neuberger et al., European Patent No. 0 194 276 B1;
issued to Winter et al., U.S. Pat. No. 5,225,539; issued to Winter
et al., European Patent No. 0 239 400 B1; Queen et al., European
Patent No. 0 451 216 B1; and Padlan et al., EP 0 519 596 A1. See
also, Newman et al., BioTechnology, 10: 1455-1460 (1992), regarding
primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and
Bird et al., Science, 242:423-426 (1988)) regarding single chain
antibodies. The contents of these patent documents and references
are hereby incorporated herein in their entirety.
[0262] In other embodiments, test compounds refer to aptamers.
Aptamers typically comprise DNA, RNA, PNA, nucleotide analogs,
modified nucleotides or mixtures of any of the above. Aptamers may
be naturally occurring or made by synthetic or recombinant means.
Aptamer sequences are typically discovered by SELEX (Systematic
Evolution of Ligands by EXponential Enrichment). This method
provides for the in vitro selection of nucleic acid molecules that
are able to bind with high specificity to target molecules and is
further described in U.S. Pat. No. 5,475,096 entitled "Nucleic Acid
Ligands," U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled
"Nucleic Acid Ligands," and more recently "Method for generating
aptamers with improved off-rates," U.S. Patent Application no.
2009/0004667, each of which is specifically incorporated by
reference herein. Nucleic acid aptamers may also be selected by
screening libraries of structurally defined RNA or DNA motifs, as
described in "Methods for identifying ligands that target nucleic
acid molecules and nucleic acid structural motifs," U.S. Patent
Application No. 2008/0188377, the contents of which are hereby
incorporated herein in their entirety.
[0263] A test compound may be a nucleic acid molecule such as a
short oligonucleotide, that is capable of mediating sequence
specific RNAi (RNA interference), for example short (or small)
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), short hairpin RNA (shRNA), short interfering
oligonucleotide, short interfering nucleic acid, short interfering
modified oligonucleotide, chemically-modified siRNA,
post-transcriptional gene silencing RNA (ptgsRNA), translational
silencing and others.
[0264] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci, U.S.A. 90: 6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91: 11422; Zuckermann et al. (1994). J. Med. Chem.
37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al.
(1994) Angew. Chem. Int. Ed, Engl. 33: 2059; Carell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J.
Med. Chem. 37: 1233.
[0265] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13: 412-421), or on beads (Lam (1991)
Nature 354: 82-84), chips (Fodor (1993) Nature 364: 555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S.
Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Aced
Sci USA 89: 1865-1869) or on phage (Scott and Smith (1990) Science
249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87: 6378-6382; Fetid (1991) J. Mol.
Bid. 222: 301-310; Ladner supra.).
[0266] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a PAR-1 and/or MMP-1 protein or biologically
active portion thereof is contacted with a test compound, and the
ability of the test compound to modulate PAR-1 and/or MMP-1
activity is determined. Determining the ability of the test
compound to modulate PAR-1 activity can be accomplished by
monitoring, for example, Rho or MAPK signaling activity as
described above. In other embodiments, MMP-1 activity may be
monitored by determining the level of cleavage at the
LD.sub.39.dwnarw.P.sub.40RSFL MMP-1 cleavage site. The cell, for
example, can be of mammalian origin, e.g., human. In a preferred
embodiment, the cell is a human platelet. In vivo binding of the
test compound to PAR-1 or MMP-1 can also be evaluated. This can be
accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to PAR-1 and/or MMP-1 can be determined by detecting the labeled
compound in a complex. Alternatively, PAR-1 or MMP-1 could be
coupled with a radioisotope or enzymatic label to monitor the
ability of a test compound to modulate MMP-1 cleavage of PAR-1. For
example, compounds can be labeled with 125I, 35S, 14C, or 3H,
either directly or indirectly, and the radioisotope detected by
direct counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0267] The ability of a test compound to interact with PAR-1 or
MMP-1 with or without the labeling of any of the interactants can
be evaluated. For example, a microphysiometer can be used to detect
the interaction of a compound with PAR-1 or MMP-1 without the
labeling of either the compound or the PAR-1 or MMP-1. McConnell,
H. M. et al. (1992) Science 257: 1906-1912. As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between a compound and PAR-1 or MMP-1.
[0268] In yet another embodiment, a cell-free assay is provided in
which a PAR-1 or MMP-1 protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the PAR-1 or MMP-1 protein or biologically
active portion thereof is evaluated. Preferred biologically active
portions of the PAR-1 or MMP-1 proteins to be used in assays of the
present invention include fragments which participate in
interactions between PAR-1 or MMP-1 molecules.
[0269] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected. The
interaction between two molecules can also be detected, e.g., using
fluorescence energy transfer (FRET) (see, for example, Lakowicz et
al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat.
No. 4,868,103). A fluorophore label on the first, `donor` molecule
is selected such that its emitted fluorescent energy will be
absorbed by a fluorescent label on a second, `acceptor` molecule,
which in turn is able to fluoresce due to the absorbed energy.
Alternately, the `donor` protein molecule may simply utilize the
natural fluorescent energy of tryptophan residues. Labels are
chosen that emit different wavelengths of light, such that the
`acceptor` molecule label may be differentiated from that of the
`donor`. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, the spatial
relationship between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be
maximal. An FRET binding event can be conveniently measured through
standard fluorometric detection means well known in the art (e.g.,
using a fluorimeter).
[0270] In another embodiment, determining the ability of PAR-1 or
MMP-1 protein to bind to a test compound can be accomplished using
real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S, and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345
and Szabo et al. (1995) Corr. Opin. Struct. Biol. 5: 699-705).
"Surface plasmon resonance" or "BIA" detects biospecific
interactions in real time, without labeling any of the interactants
(e.g., Moore). Changes in the mass at the binding surface
(indicative of a binding event) result in alterations of the
refractive index of light near the surface (the optical phenomenon
of surface plasmon resonance (SPR)), resulting in a detectable
signal which can be used as an indication of real-time reactions
between biological molecules.
[0271] In one embodiment, the target gene product or the test
compound is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0272] It may be desirable to immobilize either PAR-1 or MMP-1, an
PAR-1 or MMP-1 antibody or its target compound to facilitate
separation of complexed from uncomplexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to a PAR-1 or MMP-1 protein, or
interaction of a PAR-1 or MMP-1 protein with a candidate compound,
can be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtiter plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/PAR-1 or MMP-1 fusion proteins can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtiter plates, which are
then combined with the test compound or the test compound and
either the non-adsorbed target protein or PAR-1 or MMP-1 protein,
and the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtiter plate wells are
washed to remove any unbound components, the matrix immobilized in
the case of beads, complex determined either directly or
indirectly, for example, as described above. Alternatively, the
complexes can be dissociated from the matrix, and the level of
PAR-1 or MMP-1 binding or activity determined using standard
techniques.
[0273] Other techniques for immobilizing either a PAR-1 or MMP-1
protein or test compounds can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
Pierce Chemical).
[0274] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0275] In one embodiment, this assay is performed utilizing
antibodies reactive with PAR-1 or MMP-1 protein but which do not
interfere with binding of the PAR-1 or MMP-1 protein to its test
compound. Such antibodies can be derivatized to the wells of the
plate, and unbound target or PAR-1 or MMP-1 protein trapped in the
wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the PAR-1 or MMP-1 protein, as well
as enzyme-linked assays which rely on detecting an enzymatic
activity associated with the PAR-1 or MMP-1 protein or target
molecule.
[0276] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including but not limited to: differential centrifugation (see, for
example, Rivas, G., and Minton, A. R, (1993) Trends Biochem Sci 18:
284-7); chromatography (gel filtration chromatography, ion-exchange
chromatography); electrophoresis (see, e.g., Ausubel, F. et al.,
eds. Current Protocols in Molecular Biology 1999, J. Wiley: New
York.); and immunoprecipitation (see, for example, Ausubel, F. et
al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley:
New York). Such resins and chromatographic: techniques are known to
one skilled in the art (see, e.g., Heegaard, N.H., (1998) J Mol
Recognit 11: 141-8; Hage, D. S., and Tweed, S. A. (1997) J
Chromatogr B Biomed Sci Appl. 699: 499-525). Further, fluorescence
energy transfer may also be conveniently utilized, as described
herein, to detect binding without further purification of the
complex from solution.
[0277] In a preferred embodiment, the assay includes contacting the
PAR-1 protein or biologically active portion thereof with MMP-1 to
form an assay mixture, contacting the assay mixture with a test
compound, and determining the ability of the test compound
preferentially bind to PAR-1 or biologically active portion
thereof, or to modulate the activity of a PAR-1.
[0278] The target gene products of the invention can interact in
vivo with one or more cellular or extracellular macromolecules,
such as proteins. For the purposes of this discussion, such
cellular and extracellular macromolecules are referred to herein as
"binding partners." Compounds that disrupt such interactions can be
useful in regulating the activity of the target gene product. Such
compounds can include, but are not limited to molecules such as
antibodies, peptides, and small molecules. The preferred target
genes/products for use in this embodiment are the PAR-1 or MMP-1
genes herein identified. In an alternative embodiment, the
invention provides methods for determining the ability of the test
compound to modulate the activity of a PAR-1 or MMP-1 protein
through modulation of the activity of an upstream effector of a
PAR-1 or MMP-1 target molecule. For example, the activity of the
effector molecule on an appropriate target can be determined, or
the binding of the effector to an appropriate target can be
determined, as previously described.
[0279] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), a reaction mixture containing the target gene
product and the binding partner is prepared, under conditions and
for a time sufficient, to allow the two products to form complex.
In order to test an inhibitory agent, the reaction mixture is
provided in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of the target gene
and its cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test compound or with a placebo.
The formation of any complexes between the target gene product and
the cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target gene product
and the interactive binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal target gene product can also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene product. This comparison can be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
products.
[0280] In yet another aspect, the PAR-1 or MMP-1 proteins can be
used as "bait proteins" in a two-hybrid assay or three-hybrid assay
(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:
223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054;
Bartel et al. (1993) Biotechniques. 14: 920-924; Iwabuchi et al.
(1993) Oncogene 8: 1693-1696; and Brent WO94/10300), to identify
other proteins or peptides, which bind to or interact with PAR-1 or
MMP-1 and interfere with PAR-1/MMP-1 function.
[0281] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a PAR-1 or
MMP-1 protein is fused to a gene encoding the DNA binding domain of
a known transcription factor (e.g., GAL-4). In the other construct,
a DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
(Alternatively the: PAR-1 or MMP-1 protein can be the fused to the
activator domain.) If the "bait" and the "prey" proteins are able
to interact, in vivo, forming a PAR-1-dependent complex, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription
of a reporter gene (e.g., lea), which is operably linked to a
transcriptional regulatory site responsive to the transcription
factor. Expression of the reporter gene can be detected and cell
colonies containing the functional transcription factor can be
isolated and used to obtain the cloned gene, which encodes the
protein/peptide which interacts with the PAR-1 protein.
[0282] In another embodiment, modulators of PAR-1 expression are
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of PAR-1 mRNA or
protein evaluated relative to the level of expression of PAR-1 mRNA
or protein in the absence of the candidate compound. When
expression of PAR-1 or MMP-1 mRNA or protein is less (statistically
significantly less) in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of PAR-1/MMP-1 mRNA or protein expression. The level of
PAR-1/MMP-1 mRNA or protein expression can be determined by methods
known in the art. In one embodiment, a test compound may be RNAi or
microRNAs that inhibits PAR-1 or MMP-1 gene expression.
[0283] In yet another embodiment, the assays described herein may
be used to identify the binding site of the MMP-1 generated
tethered ligand (see FIG. 9A). Cell-based or cell-free assays could
be devised to test if PR-TRAP peptide interacts with one or more
extracellular loops of PAR-1. The identity of the target
polypeptide sequence could then be verified by site directed
mutagenesis of the target sequence using well-established methods
known in the art. Libraries of agents can then be screened
compounds that specifically disrupt the interact of the tethered
ligand with this target sequence by using in vivo binding assays
such two hybrid assays in the presence of test compounds.
Alternatively, candidate compounds may be screened for their
ability to abrogate platelet PAR-1 signaling in the presence of
activated MMP-1 or the PR-TRAP peptide ligand as described above.
For example, candidate test compounds can be screened for their
ability to inhibit Rho-GTP or phospho-p38 MAPK signaling activity
in human platelets as described in detail above. In other
embodiments, the ability of candidate test compounds to inhibit
platelet aggregation may also be assayed as described above. A
reporter molecule such as GFP or the like that is responsive to
PAR-1 signaling activity would facilitate screening for relevant
compounds.
[0284] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a PAR-1 protein can be confirmed in vivo, e.g., in an animal
such as an animal model for a thrombotic disease state. In addition
to the guinea pig thrombosis model described above, the efficacy of
test compounds on thrombosis in vivo can be assessed using a wide
variety of known animal models of thrombosis, for example, as
described in U.S. Patent Application Nos. 2005/0025705 and
2005/0120392, the contents of which are hereby incorporated by
reference herein in their entirety.
[0285] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a PAR-1 or MMP-1 modulating agent, an
antisense PAR-1 or MMP-1 nucleic acid molecule, a PAR-1-specific or
MMP-1-specific antibody, or a PAR-1 or MMP-1-binding partner) in an
appropriate animal model to determine the efficacy, toxicity, side
effects, or mechanism of action, of treatment with such an agent.
Furthermore, novel agents identified by the above-described
screening assays can be used for treatments as described
herein.
II--(f) Effects of Blockade of MMP1-PAR1 in the Development of
Atherosclerotic Lesions and Neovascularization of the Vaso Vasorum
Apolipoprotein (apoE) Deficient Mice
[0286] The role of MMP-1 and PAR1 in the development of
atherosclerotic lesions can be studied in an animal model of
atherosclerosis, such as apolipoprotein E (apoE) deficient
mice.
[0287] Twenty five (25) B6. 129P2-Apoe.sup.tm1Unc/J mice, 7 weeks
old, were separated into 3 groups and fed ad libitum with
high-fat/high-cholesterol diet (21% fat, 0.21% cholesterol)
(D12079B, Research Diets) for 15 weeks. Group I included 8 mice
(n=8) was used as a control group and received Vehicle (20% DMSO in
1.times.PBS). Group II included 8 mice (n=8) that were treated with
MMP Inhibitor I FN-439 (Calbiochem) at a dose of 5 mg/kg. Lastly,
group III included 9 mice (n=9) treated with 10 mg/kg of the
P1pal-7 pepducin lipopeptide. MMP Inhibitor I (FN-439) is an
inhibitor of MMP-1 and MMP-8 (IC.sub.50-1 .mu.M) and can also
inhibit MMP-9 and MMP-3, though at much higher concentrations
(IC50=30 .mu.M and 150 .mu.M, respectively). P1pal-7 is a
cell-penetrating pepducin lipopeptide based on the third
intracellular loop (i3) of human PAR1 and acts as an antagonist of
PAR1 signaling. All mice were injected subcutaneously with 100
.mu.l of the corresponding treatment for 6 days a week. At the end
of the treatment, and after a 4 hours fasting, the mice were
anesthetized with ketamine/xylazine and pressure-fixed with 10%
formalin. The heart and the aorta of each mouse were isolated,
removed and fixed for 2 days in 10% formalin and adventitial fat
was removed. The abdominal area of the aorta was cut and used for
whole mount immunohistochemistry. The "aortic arch/thoracic" area
and the rest of the "abdominal/iliac" area of the aorta were cut
open and pinned on dissecting black wax and used for "en face"
staining with Oil-Red-O.
[0288] During the 15-week period, the weight of each mouse was
measured weekly, after a 4-h fast. There was no significant
difference in the mouse weight between the 3 different treatment
groups (FIG. 10A), indicating that the treatments did not appear to
affect the food intake or health of the mice. The development of
atherosclerotic lesions in the apoE-deficient mouse is induced by
the accumulation of cholesterol in the blood stream. Therefore, it
is of great importance to track the lipid profile of the mice under
different treatments. As shown in FIG. 106, the total plasma
cholesterol of the mice increased in the first week of treatment
from 350 mg/di to approximately 600-800 mg/dl and stayed close to
900 mg/di for the rest of the treatment. Plasma samples were
obtained after a 4 h fasting period in the morning of the same day,
weekly, in order to exclude possible fluctuations due to food
intake. No significant differences were observed between the
different treatment groups, indicating that the inhibition of MMPs
or PAR1 did not affect the lipid metabolism: of the mice.
[0289] To study the extent of artherosclerotic plaque formation,
pinned aortas were first fixed in 10% formalin and kept in
1.times.PBS. The aorta of the mice were then subjected to "en face"
staining with Oil-Red-O, which stains lipids red. Briefly, the PBS
was drained from the samples and the stain solution was added for
45 min. The stain was drained and the samples were washed first
with 70% Ethanol and then with water, in order to remove the
background. Digital pictures of the stained aortas were taken and
the MetaXpress software (Molecular Devices) was used to define and
quantify the total aortic area as well as the lesion areas. The
ratio of lesion area to the total aortic area was calculated for
the "aortic arch/thoracic" and the "abdominal/iliac" area. The
aorta of each mouse was isolated and separated into three sections.
The abdominal section (before and after the renal arteries) was
separated and used for whole mount immunohistochemistry. The
remaining sections were the one from the aortic arch to the
mesenteric artery (aortic) and the other from after the renal
arteries to the iliac arteries (abdominal). These sections of the
aorta were used for the "en face" staining and the estimation of
the atherosclerotic lesion area.
[0290] The lesion area in the aortic and the abdominal sections of
the aorta, expressed as a percentage of the total area of the
sections, is shown in FIG. 11. In the aortic section, the mean
lesion area in the control (vehicle) group is 12.9%, in the FN-439
group is 10.7% and in the P1pal-7 group is 8.1% of the total area.
The lesion area in the P1pal-7 group is significantly smaller
(p<0.005), compared to the control group.
[0291] In the abdominal section of the aorta, the mean lesion area
in the control group is 8.9%, in the FN-439 group is 4.1% and in
the P1pal-7 group is 2.3%. The lesion area in the P1pal-7 group is
significantly smaller (p<0.05), compared to the control group.
In both sections, the FN-439 group tends to have a smaller lesion
area as compared to the control group, though no significance was
reached with the present sample size.
[0292] Overall, the inhibition of PAR1 signaling, using the P1pal-7
pepducin lipopeptide, leads to significantly reduced
atherosclerotic burden in apoE-deficient mice, while inhibition of
the activity of MMPs shows a tendency for smaller atherosclerotic
lesions. These findings strongly suggest that PAR1 plays an
important role in the progression of atherosclerosis in the aortic
vessel. The fact that, unlike humans platelets, mouse platelets do
not express PAR1, leads to the conclusion that the above
observations are not due to anti-thrombotic action of the pepducin
lipopeptide but due to the inactivation of certain pathways in the
vascular tissue,
II--(g) Neovascularization
[0293] Angiogenesis may promote the progression of atherosclerosis
and contribute to plaque instability and rupture. Hence, MMP-PAR1
signaling may be contributing to atherosclerosis formation by
stimulating angiogenesis. Angiogenesis was therefore evaluated in
the adventitia of aortas from apoE-/- mice fed a high fat diet for
15 weeks and treated with either the MMP-1 collagenase inhibitor,
MMP Inh-1 (FN-439) or the PAR1 antagonist, P1pal-7. The mice were
then anesthetized with ketamine/Xylazine and pressure fixed with
10% formalin. The abdominal aorta was isolated, removed and fixed
for 1 hour with 10% formalin and adventitial fat was removed. The
abdominal aorta were then whole mount immunostained for CD31, a
marker for endothelial cells and 3D projection images were
constructed from multiple confocal sections using the following
procedure. Briefly, the tissue was blocked for 1 hour with 5% goat
serum in Tris buffered saline containing 0.3% Triton-X 100 (TBST)
and incubated overnight at 4.degree. C. with primary antibody
diluted 1:1000 in TBST. After several washes with TBST, the tissue
was incubated with fluorescently tagged secondary antibody diluted
in TBST for 4 hours. The tissue was washed and post-fixed with 4%
paraformaldehyde for 10 minutes. The aorta was then cut lengthwise
and splayed open onto a microscope slide with the adventitial side
up. The tissue was whole mounted with Vectashield mounting media
and imaged using a Leica TCS SP2 confocal microscope (Zeiss).
Confocal images were constructed into 3D projections of Z-stacks.
Quantification of images was performed using NIH ImageJ. Antibodies
used were: hamster anti-mouse PECAM-1 (Chemicon) and
Cy3-anti-hamster (Jackson Immunolabs).
[0294] FIG. 12 shows the mount immunostaining for CD31 in the
abdominal aorta of ApoE-/- mice treated with vehicle (FIG. 12A),
P1pal-7 (FIG. 12B), MMP Inh-1 (FN-439) (FIG. 12C) as in FIG. 10.
Both P1pal-7 treatment and FN-439 treatment significantly reduced
the amount of CD31-positive vessels (FIGS. 12B and 12C).
Quantitation of CD31 staining (FIG. 13A) was evaluated in vascular
bundles (FIG. 13B), and branch points (FIG. 13C) of the abdominal
aorta of ApoE-/- mice treated with vehicle, P1pal-7 or MMP1 Inh-1
(FN-439) as in FIG. 10. Angiogenesis was not homogeneous over the
entire adventitia of the aorta and vascular bundles appeared in
distinct locations, possibly corresponding to locations of
atherosclerotic plaques. Both P1pal-7 treatment and FN-439
treatment significantly reduced the number of vascular bundles in
the adventitia of the aorta (FIG. 13). To evaluate the structure of
the nascent vessels, branch points were counted, P1pal-7 and FN-439
treatment significantly inhibited the number of branch points per
vessel area. These findings suggest that both MMP-1 and PAR1 are
promoting plaque angiogenesis.
II--(h) Other Inhibitors of MMP-1/PA-1 Signaling
[0295] The MMP-1/PAR-1 signaling pathway may be inhibited in
platelets using other potential inhibitors of the MMP-1 PAR-1
signaling pathway, including, but limited to, inhibitors of MMP-1
or MMP-2. The efficacy of these compounds in inhibiting platelet
aggregation can be evaluated using the cell-based assays and
animals models of a thrombotic disease state described herein.
[0296] Since MMPs contain a zinc atom in the catalytic domain and
need calcium to function, a chelating compound may inhibit MMP
activity. In addition, synthetic derivatives that mimic natural
substrates have been designed as MMP inhibitors. Several classes of
structures such as carboxylic acid derivatives; heterocyclic
structures; hydroxamate moieties with a peptide, peptidomimetic, or
nonpeptide backbone; biphenyl moieties with nonpeptide backbone;
and tetracycline analogs are the most common low-molecular-weight
compounds that have in vitro inhibitory activity against MMPs.
[0297] Non-limiting examples of MMP-1 inhibitors include FN-439,
tissue inhibitors of metalloprotease (TIMPs), MMP-200, Cipemastat
(rINN, also known as Ro 32-3555 and by the tentative trade name
Trocade marketed by Roche). Ancorinosides B-D (Fujita et al.
Tetrahedron, Vol. 57, Issue 7, 1229-1234, 2001).
[0298] Matrix metalloproteinase inhibitors that have entered
clinical trials for an oncologic indication include Prinomastat
(AG3340; Agouron/Pfizer), BAY 12-9566 (Bayer Corp.), Batimistat
(BB-94; British Biotech, Ltd.), BMS-275291 (formerly D2163;
Celltech/Bristol-Myers Squibb), Marimastat (BB 2516; British
Biotech, Ltd./Schering-Plough), MMI270(B) (formerly CGS-27023A;
Novartis), and Metastat (COL-3; CollaGenex) and Ro 32-3565 &
RS-130,830 (Roche Bioscience).
[0299] Additional MMP inhibitors that may be used with the
compounds and therapeutical applications of this application are
disclosed in Design and Therapeutic Application of Matrix
Metalloproteinase Inhibitors Mark Whittaker, Floyd et al. Chem.
Rev., 1999, 99 (9), 2735-2776 and Prevention of progressive joint
destruction in collagen-induced arthritis in rats by a novel matrix
metalloproteinase inhibitor, FR255031, Ishikawa et al. British
Journal of Pharmacology (2005) 144, 133-143, the contents of which
are incorporated herein by reference in their entirety.
[0300] Additional MMP inhibitors include PD 166793 (available Axon
MedChem; H Leon et al., Br. J. Pharmacol. (2008) 153, 676-683.
[0301] The invention further contemplates other peptide antagonists
that interfere with collagen induced platelet activation and may be
combined with the herein described agents that modulate the MMP-1
mediated cleavage of PAR-1 between aspartic acid at position 39
(D39) and proline at position 40 (P40) of said patient's
protease-activated receptor-1 (PAR-1). MMP peptide inhibitors based
on synthetic triple-helical peptides (THPs) are described in U.S.
Patent Application No. 2008/0125354. PAR-1 antibody or peptide
antagonists are described in the PCT application WO 2008/011107.
The contents of these patent documents are hereby incorporated
herein by reference in their entirety.
[0302] The invention also provides for combination of the herein
described modulators of MMP-1 mediated PAR-1 cleavage with one or
more PAR1 pepducin lipopeptides, including those pepducin
lipopeptides described in U.S. Patent Publication US2007/0179090,
the contents of which are hereby incorporated herein by reference
in its entirety.
[0303] Examples of PAR1 pepducin lipopeptides include pepducin
lipopeptides comprising a polpeptide sequence taken from the i1,
i2, i3 or i4 intracellular loops of PAR-1. In one embodiment,
pepducin lipopeptides used herein may have a N-terminal lipid that
can be a palmitate, myristate, lithocholate, fatty acids, steriods,
etc. In another embodiment, pepducin lipopeptides used herein may
have a C-terminal lipid that can be a palmitate, myristate,
lithocholate, fatty acids, steriods, etc.
[0304] Examples of PAR1 pepducin lipopeptides are depicted in TABLE
1.
TABLE-US-00001 TABLE 1 NAME TARGET LOOPS AMINO ACID SEQUENCE
ATTACHED LIPID P1i3pal-7 PAR1 i3 KKSRALF palmitate (a.k.a. P1pal-7)
(SEQ ID NO. 2) P1i3pal-12 PAR1 i3 RCLSSSAVANRS palmitate (SEQ ID
NO. 3) P1i3pal-12S PAR1 i3 RSLSSSAVANRS palmitate (SEQ ID NO. 4)
P1i3pal-10S PAR1 i3 NRSKKSSALF palmitate (SEQ ID NO. 5) P1i1pal-11
PAR1 i1 ILKMKVKKPAV palmitate (SEQ ID NO. 6) P1i2pal-7 PAR1 i2
TLGRASF palmitate (SEQ ID NO. 7) P1i2pal-11 PAR1 i2 LSWRTLGRASF
palmitate (SEQ ID NO. 8) P1i2pal-16 PAR1 i2 YPMQSLSVVRTLGRASF
palmitate (SEQ ID NO. 9) P1i2pal-21 PAR1 i2 FLAVVYPMQSLSWRTLGRASF
palmitate (SEQ ID NO. 10) P1i4pal13 PAR1 i4 ASSESQRYVYSIL palmitate
(SEQ ID NO. 11) P1i4pal13R PAR1 i4 LISYVYRQSESSA palmitate (SEQ ID
NO. 12)
[0305] In other embodiments, the invention provides for small
molecule inhibitors of PAR-1 signaling activity including, but
limited to the compound SCH 530348, SCH 530348 blocks the platelet
PAR-1 receptor to which thrombin binds, thus inhibiting
thrombin-induced activation of platelets, and is therefore
classified as a thrombin-receptor antagonist (TRA). SCH 530348 is
further described in Chintala et al. J Pharmacol Sci 108, 433-438
(2008), Chackalarnannil et al. J. Med. Chem. 2008, 51, 3061-3064
and the published U.S. patent application, US 2008/0234236, the
content of which are hereby incorporated by reference in their
entireties. For purposes of this disclosure, reference to the
compound SCH 530348 includes all isomers, enantiomers and chemical
derivatives of SCH 530348.
[0306] In yet another embodiment, an antagonist of the MMP-1
mediated PAR-1 signaling pathway may be a tetracycline compound or
tetracycline derivative, such as doxycycline. Tetracyclines are a
group of broad-spectrum antibiotics. They are so named for their
four ("tetra-") hydrocarbon rings ("-cycl-") derivation ("-ine").
More specifically, they are defined as "a subclass of polyketides
having an octahydrotetracene-2-carboxamide skeleton". They are
collectively known as derivatives of polycyclic naphthacene
carboxamide having the basic structure:
##STR00001##
Examples of tetracycline derivatives that may be tested for
inhibitory activity on the MMP-1 mediated PAR-1 signaling pathway
include, but are not limited to, Chlortetracycline,
Oxytetracycline, Demeclocycline, Doxycycline, Lymecycline,
Meclocycline, Methacycline, Minocycline. Rolitetracycline and
Tigecycline. Non-limiting examples of tetracycline derivatives have
been described in U.S. Pat. No. 2,980,584; U.S. Pat. No. 2,990,331;
U.S. Pat. No. 3,062,717; U.S. Pat. No. 3,165,531; U.S. Pat. No.
3,454,697; U.S. Pat. No. 3,557,280: U.S. Pat. No. 3,674,859; U.S.
Pat. No. 3,957,980; U.S. Pat. No. 4,018,889; U.S. Pat. No.
4,024,272; and U.S. Pat. No. 4,126,680, the contents of which are
hereby incorporated herein by reference.
III Drug Administration
[0307] Inhibitors of MMP-1 mediated PAR-1 activation may be
administered to mammals, including humans, either alone or, in
combination with pharmaceutically acceptable carriers, excipients
or diluents, in a pharmaceutical composition, according to standard
pharmaceutical practice. The compounds can be administered orally
or parenterally, including the intravenous, intramuscular,
intraperitoneal, subcutaneous, rectal and topical routes of
administration.
[0308] Inhibitors of MMP-1 mediated PAR-1 activation include known
MMP-1 or PAR-1 inhibitors, as defined herein.
[0309] The compounds or "agents" may be used in combination with
one or more other known anti-thrombotic agents or pharmaceutical
agents, including, e.g., a TP antagonist, a thromboxane antagonist,
an ADP receptor antagonist, or a Factor Xa antagonist. When used in
combination, it is understood that lower dosages of one or more of
the combined anti-thrombotic agents may be utilized to achieve a
desired effect, since the two or more anti-thrombotic agents may
act additively or synergistically. Accordingly, a therapeutically
effective dosage of one or more combined anti-thrombotic agents may
correspond to less than 90%, less than 80%, less than 70%, less
than 60%, less than 50%, less than 40%, less than 30% or less than
20% of the therapeutically effective dosage when the
anti-thrombotic "agent" is administered alone. The two or mare
anti-thrombotic agents may be administered at the same time or at
different times, by the same route of administration or by
different routes of administration. For example, in order to
regulate the dosage schedule, the anti-thrombotic agents may be
administered separately in individual dosage units at the same time
or different coordinated times. The respective substances can be
individually formulated in separate unit dosage forms in a manner
similar to that described above. However, fixed combinations of the
anti-thrombotic agents are more convenient and are preferred,
especially in tablet or capsule form for oral administration. Thus,
the present invention also provides unit dose formulations
comprising two or more anti-thrombotic agents, wherein each
thrombotic "agent" is present in a therapeutically effective amount
when administered in the combination.
[0310] The pharmaceutical compositions containing the active
ingredient may be in a form suitable for oral use, for example, as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules,
or syrups or elixirs. Compositions intended for oral use may be
prepared according to any method known to the art for the
manufacture of pharmaceutical compositions and such compositions
may contain one or more agents selected from the group consisting
of sweetening agents, flavoring agents, coloring agents and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets contain the active ingredient in
admixture with non-toxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
may be for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example,
microcrystalline cellulose, sodium crosscarmellose, corn starch, or
alginic acid; binding agents, for example starch, gelatin,
polyvinyl-pyrrolidone or acacia, and lubricating agents, for
example, magnesium stearate, stearic acid or talc. The tablets may
be uncoated or they may be coated by known techniques to mask the
unpleasant taste of the drug or delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a water soluble taste
masking material such as hydroxypropylmethyl-cellulose or
hydroxypropylcellulose, or a time delay material such as ethyl
cellulose, cellulose acetate buryrate may be employed.
[0311] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water soluble carrier such as
polyethyleneglycol or an oil medium, for example peanut oil, liquid
paraffin, or olive oil.
[0312] Aqueous suspensions contain the active material in admixture
with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl-cellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethylene-oxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose, saccharin or aspartame.
[0313] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as butylated
hydroxyanisol or alpha-tocopherol.
[0314] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending "agent" and one or more preservatives. Suitable
dispersing or wetting agents and suspending agents are exemplified
by those already mentioned above. Additional excipients, for
example sweetening, flavoring and coloring agents, may also be
present. These compositions may be preserved by the addition of an
anti-oxidant such as ascorbic acid.
[0315] The pharmaceutical compositions of the invention may also be
in the form of an oil-in-water emulsion. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring phosphatides, for
example soy bean lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening, flavouring
agents, preservatives and antioxidants.
[0316] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative,
flavoring and coloring agents and antioxidant.
[0317] The pharmaceutical compositions may be in the form of
sterile injectable aqueous solutions. Among the acceptable vehicles
and solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution.
[0318] The sterile injectable preparation may also be a sterile
injectable oil-in-water microemulsion where the active ingredient
is dissolved in the oily phase. For example, the active ingredient
may be first dissolved in a mixture of soybean oil and lecithin.
The oil solution then introduced into a water and glycerol mixture
and processed to form a microemulation.
[0319] The injectable solutions or microemulsions may be introduced
into a patient's blood-stream by local bolus injection.
Alternatively, it may be advantageous to administer the solution or
microemulsion in such a way as to maintain a constant circulating
concentration of the instant compound. In order to maintain such a
constant concentration, a continuous intravenous delivery device
may be utilized. An example of such a device is the Deltec
CADD-PLUS.TM. model 5400 intravenous pump.
[0320] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleagenous suspension for
intramuscular and subcutaneous administration. This suspension may
be formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents which have been
mentioned above. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a nontoxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butane diol. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or diglycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables.
[0321] The compounds for the present invention can be administered
in intranasal form via topical use of suitable intranasal vehicles
and delivery devices, or via transdermal routes, using those forms
of transdermal skin patches well known to those of ordinary skill
in the art. To be administered in the form of a transdermal
delivery system, the dosage administration will, of course, be
continuous rather than intermittent throughout the dosage regimen.
Compounds of the present invention may also be delivered as a
suppository employing bases such as cocoa butter, glycerinated
gelatin, hydrogenated vegetable oils, mixtures of polyethylene
glycols of various molecular weights and fatty acid esters of
polyethylene glycol. The compounds of the present invention can
also be administered in the form of liposome delivery systems, such
as small unilamellar vesicles, large unilamellar vesicles and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as cholesterol, stearylamine or
phosphatidylcholines. Compounds of the present invention may also
be delivered by the use of monoclonal antibodies as individual
carriers to which the compound molecules are coupled. The compounds
of the present invention may also be coupled with soluble polymers
as targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropylmethacrylamide-phenol,
polyhydroxy-ethylaspartamide-phenol, or
polyethyleneoxide-polylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be coupled
to a class of biodegradable polymers useful in achieving controlled
release of a drug, for example, polylactic acid, polyglycolic acid,
copolymers of polyactic and polyglycolic acid, polyepsilon
caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked
or amphipathic block copolymers of hydrogels.
[0322] When a composition according to this invention is
administered into a human subject, the prescribing physician will
normally determine the daily dosage with the dosage generally
varying according to the age, weight, and response of the
individual patient, as well as the severity of the patient's
symptoms. In an embodiment, a suitable amount of an "agent" is
administered to a mammal undergoing treatment for thrombosis.
Administration occurs in an amount of "agent" of between about 0.1
mg/kg of body weight to about 60 mg/kg of body weight per day, or
between 0.5 mg/kg of body weight to about 40 mg/kg of body weight
per day. Another therapeutic dosage that comprises the instant
composition includes from about 0.01 mg to about 1000 mg of agent.
In another embodiment, the dosage comprises from about 1 mg to
about 5000 mg of agent.
IV Combination Therapy
[0323] One intended use of the herein described PAR-1 signaling
agents is the prophylactic treatment of patients at risk of a
thrombotic disease state or a recurrence of a thrombotic disease
state, such as atherosclerosis. Patients presenting with risk
factors such as high blood pressure or high cholesterol levels
would be given a therapeutically effective dose of the agent
according to a physician prescribed daily regimen. Patients would
require close monitoring to ensure the treatment does not incur any
undesirable side effects. Appropriate dosage would depend on the
severity of any risk factors as well as age, gender of the patient
and whether or not the patent has a family history of a thrombotic
disease state or other genetic predisposition to a thrombotic
disease state. In one embodiment, the herein described agent may be
administered prophylacticly to a patient who is at an increased
risk of thrombosis, for example, after surgery or after
implantation of a medical device such as a stent or artificial
organs, such as an artificial heart.
[0324] This application further contemplates the combination
therapy of the herein described "agent" with one or more drugs that
are known to treat one or more risk factors of thrombotic disease
state.
[0325] In one embodiment, the drugs may be other known inhibitors
of platelet activation and aggregation, including, but not limited
to, inhibitors of protease activated (PAR) receptors, inhibitors of
MMP-1 or MMP-2 activity and inhibitors of thrombin-mediated
activation of PAR-1 and combinations thereof.
[0326] For example, combination therapy may include known
inhibitors of platelet aggregation such as those described in U.S.
Pat. No. 4,529,596; U.S. Pat. No. 4,847,265; U.S. Pat. No.
6,429,210B1, U.S. Pat. No. 5,288,726; U.S. Pat. No. 6,693,115 and
U.S. Patent Applications No. 2008/0214599 or 2003/0224999, the
contents of which are herein incorporated herein in their
entirety.
[0327] In another example, combination therapy with the herein
described "agent" may include known inhibitors of matrix
metalloproteinase including, but are not limited to, FN-439,
MMP-200 and tissue inhibitors of metalloproteases (TIMPs including
TIMP1, TIMP2, TIMP3 and TIMP4). MMP inhibitors are further
described in U.S. Pat. No. 3,784,701 and WO 96/15096, MMP peptide
inhibitors are described in U.S. Pat. Nos. 5,300,501; 5,530,128;
5,455,258; 5,552,419; WO 95/13289; WO 96/11209 and U.S. Patent
Publication No. 2004/0127420, all of which are incorporated herein
by reference.
[0328] In other examples, combination therapy with the herein
described "agent" may include anticoagulants including, but not
limited to, a thrombin inhibitor (e.g., melagatran, E-5555,
MCC-977, and bivalirudin (Angiomax.TM.)), Factor Xa inhibitor,
tissue factor inhibitor, Factor VIIa inhibitor, Factor IXa
inhibitor, Factor Vs inhibitor, Factor XIa inhibitor, Factor XIIa
inhibitor, TAFI.alpha. inhibitor, .alpha.2-antiplasmin inhibitor,
PAI-1 inhibitor, PAI-2 inhibitor, PAI-3 inhibitor, prothrombinase
inhibitor, tick anticoagulation peptide, protein C, warfarin,
heparin, lepirudin, aspirin, ticlopidine, clopidogrel, tirofiban,
and eptifibatide.
[0329] In other embodiments, combination therapy with the herein
described `agent` may include inhibitors of platelet function,
including, but not limited to, GPIIb/IIIa receptor inhibitors, ADP
receptors (e.g., P2Y.sub.1, and P2Y.sub.12) inhibitors, thrombin
receptor (e.g., PAR-1 and PAR-4) inhibitor, CD40 inhibitors, CD40L
(CD40 ligand) inhibitors, Gas6 inhibitors, Gas6 receptor axl
inhibitors, Gas6 receptor inhibitors Sky, Gas6 receptors Mer
inhibitor, P-selectin inhibitor, P-selectin receptor PSGL-1
inhibitors, thromboxane inhibitors, synthetase inhibitors,
fibrinogen receptor antagonists, prostacyclin mimetics,
phosphodiesterase inhibitors. RANTES inhibitor,
phosphoinositide-3-kinase (PI(3)K) isoform .beta. inhibitors,
phosphoinositide-3-kinase (PI(3)K) isoform .gamma. inhibitors,
eptifibatide, tirofiban, ticlopidine, and clopidogrel.
[0330] In other embodiments, the "agent" described herein may be
combined with known drugs that a physician may use to treat medical
conditions known to increase the risk of cardio-vascular disease.
For example, the herein described "agent" may be combined with a
HMG-CoA reductase inhibitor, otherwise known as a statin,
including, but are not limited to, simvastatin, pravastatin,
rivastatin, mevastatin, fluindostatin, cerivastatin, velostatin,
fluvastatin, dalvastatin, dihydrocompactin, compactin, or
lovastatin; or a pharmaceutically acceptable salt of simvastatin,
pravastatin, rivastatin, cerivastatin, mevastatin, fluindostatin,
velostatin, fluvastatin, dalvastatin, dihydrocompactin, compactin,
lovastatin, or pharmaceutically acceptable salts thereof. Similar
combination therapy regimens using statins are disclosed in U.S.
Patent Publication No. 2005/0020607, the contents of which are
hereby incorporated herein by reference in its entirety.
[0331] In certain embodiments, the combination therapy with the
herein described "agent" may include a known drug that is used to
prevent or treat a thrombotic disease state. Preferably, though not
necessarily, the drug may be one that has already been deemed safe
and effective for use in humans or animals by the appropriate
governmental agency or regulatory body. For example, drugs approved
for human use are listed by the FDA under 21 C.F.R. sections 330.5,
331 through 361, and 440 through 460, incorporated herein by
reference; drugs for veterinary use are listed by the FDA under 21
C.F.R. sections 500 through 589, incorporated harem by
reference.
V Diagnostic Kits
[0332] A variety of methods can be used to determine the level of
activated PAR-1 in platelets taken from a patient. In general,
these methods include contacting an agent that selectively binds to
the MMP-1 mediated PAR-1 peptide (residues 1-39), such as an
antibody with a sample, to evaluate the level of the peptide in the
sample. In another embodiment, the methods detect MMP-1 cleaved
PAR-1 or parameters associated with PAR-1 activation. In a
preferred embodiment, the antibody bears a detectable label,
Antibodies can be polyclonal, or more preferably, monoclonal. An
intact antibody, or a fragment thereof (e.g., Fab or F(ab).sub.2)
can be used. The term "labeled", with regard to the probe or
antibody, is intended to encompass direct labeling of the probe or
antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with a detectable
substance.
[0333] In vitro techniques for detection of PAR-1(1-39) peptide or
MMP-1 cleaved PAR-1 include enzyme linked immunosorbent assays
(ELISAs), immunoprecipitations, immunofluorescence, enzyme
immunoassay (EIA), radioimmunoassay (RIA), and western blot
analysis.
[0334] The invention also includes kits for detecting the presence
of activated PAR-1 in a biological sample. For example, the kit can
include a compound or agent capable of detecting PAR-1 peptide
(1-39) or MMP-1 cleaved PAR-1 in a biological sample and a
standard. The compound or agent can be packaged in a suitable
container. The kit can further comprise instructions for using the
kit to detect PAR-1 peptide (1-39) or MMP-1 cleaved PAR-1.
[0335] For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a PAR-1
(1-39) peptide; and, optionally, (2) a second, different antibody
which binds to either the peptide or the first antibody and is
conjugated to a detectable agent. The kit can also include a
buffering agent, a preservative, or a protein stabilizing agent.
The kit can also include components necessary for detecting the
detectable agent (e.g., an enzyme or a substrate). The kit can also
contain a control sample or a series of control samples, which can
be assayed and compared to the test sample contained. Each
component of the kit can be enclosed within an individual container
and all of the various containers can be within a single package,
along with instructions for interpreting the results of the assays
performed using the kit.
[0336] The diagnostic methods described herein can identify
subjects having, or at risk of developing a thrombotic disease
state. The prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., a
antagonist, peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate).
[0337] In another aspect, the invention features a computer medium
having a plurality of digitally encoded data records. Each data
record includes a value representing the level of activated PAR-1
in a sample, and a descriptor of the sample. The descriptor of the
sample can be an identifier of the sample, a subject from which the
sample was derived (e.g., a patient), a diagnosis, or a treatment
(e.g., a preferred treatment).
[0338] In a preferred embodiment, the data record further includes
values representing the level of other risk factors associated with
a thrombotic: disease state. The data record can be structured as a
table, e.g., a table that is part of a database such as a
relational database (e.g., a sql database of the oracle or sybase
database environments).
[0339] Also featured is a method of evaluating a sample. The method
includes providing a sample, e.g., from the subject, and
determining PAR-1 activation. The method can further include
comparing the value or the profile (i.e., multiple values) to a
reference value or reference profile.
[0340] The method can be used to diagnose and monitor a thrombotic
disease state in a subject wherein an increase or decrease in PAR-1
activation is an indication that the subject has or is disposed to
having a thrombotic disease state. The method can also be used to
monitor treatment of a thrombotic disease state in a subject. The
PAR-1 activation profile can be compared to a reference profile or
to a profile obtained from the subject prior to treatment or prior
to onset of the disorder.
[0341] In another aspect, the invention features, a method of
evaluating a subject. The method includes: a) obtaining a sample
from a subject, e.g., from a caregiver, e.g., a caregiver who
obtains the sample from the subject; b) determining a PAR-1
activation profile for the sample. Optionally, the method further
includes either or both of steps: c) comparing the subject profile
to one or more reference profiles; and d) selecting the reference
profile most similar to the subject reference profile. A variety of
routine statistical measures can be used to compare two reference
profiles.
[0342] The invention also contemplates kits for the detection of
polymorphism(s) in MMP-1 genes and associated factors (such natural
MMP-1 inhibitors such TIMPs) that may predispose a patient to a
thrombotic disease state. Several polymorphisms in the promoters of
a number of MMP genes, including MMP-1, have been well
characterized. These polymorphisms are thought to affect the
respective MMP production in an allele-specific manner. For
example, the promoter region of the MMP-1 gene contains consensus
sequences for DNA-binding proteins such as AP-1, AP-2, Ets/PEA-3,
and responsive elements to glucocorticoids, retinoic acid, and
cyclic AMP (Rutter et al, 1997). A single nucleotide polymorphism
(SNP) has been identified at position -1607 bp within the MMP-1
promoter region, whereby the insertion of an additional guanine (G)
residue creates an extra Ets-binding site (Rutter et al, (1998)
Cancer Res 58: 5321-5325). A promoter containing this SNP (giving
rise to the 2G genotype) displays significantly `higher`
transcriptional activity in normal and malignant cells compared to
cells possessing a 1G allele (Rutter et al, 1998; Wyatt et al,
(2002) Cancer Res 62: 7200-7202) with `lower` transcriptional
activity. Hence, this MMP-1 polymorphism may be a predictor of an
innate predisposition to a thrombotic disease state.
[0343] Kit components for the detection of polymorphism are well
known in the art and may include polymorphism specific primers and
reagent for POT amplification.
VI Platelet Storage Medium
[0344] Platelets can be obtained as a by-product from whole blood
donations and from plateletpheresis. Donated blood is typically
processed to separate various blood components including platelets
that can be separately used. For example, a unit of donated whole
blood can be processed to separate red cells, usually concentrated
as packed red cells (pro), platelets, usually concentrated as
platelet concentrate (PC), and plasma. In accordance with typical
processing protocols, blood can be processed to form, among other
fractions, a platelet-containing fluid, e.g., platelet-rich-plasma
(PRP) or huffy coat, that are further processed (including
centrifugation) to form the PC. Moreover, multiple units of
platelets or buffy coat can be pooled before producing the final
transfusion product.
[0345] In accordance with current conventional blood banking
practice, pc produced in a closed (sterile) system can be stored
for up to only 5 days before being used as a transfusion product.
In some processing protocols, a platelet additive solution is added
to the platelet-containing fluid (e.g., the buffy coat) and the
platelets are resuspended in the additive solution before the
platelets are stored, wherein most of the plasma is removed before
the additive solution is added. Alternatively, platelets can be
stored in their own plasma.
[0346] In order to provide optimal platelet function and viability
during storage, it is recommended that the platelet-containing
fluid (with or without an additive solution) be maintained at a ph
in the range of from 6.8 to 7.4 (European practice), or maintained
at a ph of 6.2 or greater (us practice) during the storage period.
It is also recommended that the platelets be stored in the presence
of glucose or dextrose to maintain platelet quality. In addition,
platelets may become activated during the processing of blood to
concentrate the platelets (including during the subsequent
resuspension of the platelets in the additive solution), leading to
platelet aggregation and loss of viability. Hence, common
components added to the platelet storage medium include an
anticoagulant, typically a citrate.
[0347] Further examples of the medium and methods commonly used in
blood donation, preparation, storage and transportation can be
found in a variety of literatures, e.g., "Textbook Of Blood Banking
And Transfusion Medicine" written by Sally v. Rudmann, and
published by Elsevier Health Sciences, 2005.
[0348] Based on the present discovery of the role of MMP-1
activated PAR-1 in platelet aggregation, an aspect of the present
invention is to provide in a platelet-containing medium, at any
time during the preparation, storage or transportation of such a
medium, the "agent" of the present invention, which substantially
inhibits proteolytic cleavage between the aspartic acid at position
39 (d39) and the proline at position 40 (p40) of the PAR-1 on the
platelets surface. In an embodiment, the "agent" of the invention
inhibits activation of MMP-1 or MMP-1 enzymatic activity. In an
another embodiment, the "agent" may inhibit PAR-1 signaling
activity after proteolytic cleavage between the aspartic acid at
position 39 (d39) and the proline at position 40 (p40) of the
PAR-1. The "agent" of the invention can be added to a
platelet-storage medium in addition to or in place of a more
conventional anti-coagulant. In an embodiment, the storage medium
with the "agent" of the invention prolongs the shelf-life of
platelets contained therein beyond the current 5 days at room
temperature (about 22.degree. C.), e.g., by 0.5, 1, 2, 3, 4, 5, 6,
or even 7 days.
VII Medical Devices
[0349] Surfaces of implantable medical devices such as stents
[0350] The compound described herein made used alone or in
combination with other know anti-thrombotic agents to coat medical
devices.
[0351] Methods of coating are well known in the art. For example.
PCT application WO20051097223 A1-Stucke et al, discloses a method
wherein a mixture of heparin conjugated with photoactive
crosslinkers with dissolved or dispersed with other durabal
polymers such as Poly(butyl methacrylate) and poly(vinyl
pyrrolidone) in a same coating solution and crosslinked with UV
light in the solution or after the coating is applied.
[0352] Another general approach as disclosed in US 2005/0191333 A1,
US 2006/0204533 A1, and WO 2006/099514 A2, all by Hsu, Li-Chien, et
al., uses a low molecular weight complex of heparin and a counter
on (stearylkonium heparin), or a high molecular weight
polyelectrolyte complex, such as dextran, pectin to form a complex
form of an anti-thrombotic entity. These anti-thrombotic complexes
are further dispersed in a polymer matrix that may further comprise
a drug.
[0353] U.S. Published Patent application No. 2008/0269875 also
discloses methods of applying multiple layers of polymeric
compositions to a medical device. One layer may comprise a base
coat that allows additional layers to adhere thereto. An additional
layer(s) can carry bioactive agents within their polymer
matrices.
[0354] The contents of these patent documents are hereby
incorporated herein by reference in their entirety.
VIII Other Therapeutic Applications
[0355] MMP-1/PAR-1 test compounds described herein may also find
uses for the diagnosis and treatments of other medical conditions
associated with PAR-1 activation. For example, medical conditions
that may benefit from the compounds described herein, include, but
not limited to, chronic intestinal inflammatory disorders,
including inflammatory bowel disease (IBD), irritable bowel
syndrome (IBS) and ulcerative colitis and fibrotic disorders,
including liver fibrosis and lung fibrosis (see, for example,
Vergnolle, et al., J Clin Invest (2004) 114(10): 1444; Yoshida, et
al, Aliment Pharmacol Them (2006) 24(Suppl 4):249; Mercer, et al,
Ann NY Aced Sci (2007) 1096:86-88; Sokolova and Reiser, Pharmacol
Ther (2007) PMID: 17532472), ischemia-reperfusion injury, including
myocardial, renal, cerebral and intestinal ischemia-reperfusion
injury (see, for example, Strand % et al., Basic Res. Cardiol
(2007) 102(4):350-8; Sevastos, et al., Blood (2007) 109(2):577-583;
Singe, et al., Proc Natl Aced Sci USA. (2003) 100(22): 13019-24 and
Tsuboi, et al., Am J Physiol Gastrointest Liver Physiol (2007)
292(2):G678-83. Inhibiting PAR1 intracellular signaling can also be
used to inhibit herpes simple virus (HSV1 and HSV2) infection of
cells. See, Sutherland, et al., J Thromb Haemost (2007)
5(5):1055-61), in the pathogenesis of neurodegenerative diseases
including Alzheimer's disease (AD) and Parkinson's disease (see
Nishimura et al. Cell, Vol. 116, Issue 5, 671-682, (2004), Ishida
et al. J Neuropathol Exp Neurol. 2006 January; 65(1):66-77;
Rosenberg (2009) The Lancet Neurology, Vol. 8, 205-216, sepsis
(Kaneider et al., Nature Immunology 8, 1303-1312 (2007)) or
endometriosis (Hirota et al. J Clin Endocrinol Metab 2005;
90(6):3673-3679), cancer and angiogenesis (reviewed by Tsopanoglou
N E and Maragoudakis Me. Semin Thromb Hemost. 2007 October; 33
(7):680-7).
[0356] The biology and pathophysiology of PAR activation in
different tissues, cells, and species was recently reviewed by
Steinhoff et al. Endocrine Reviews, February 2005, 26(1): 1-43.
[0357] Any patent, patent application, publication, or other
disclosure material identified in the specification is hereby
incorporated by reference herein in its entirety. Any material, or
portion thereof, that is said to be incorporated by reference
herein, but which conflicts with existing definitions, statements,
or other disclosure material set forth herein is only incorporated
to the extent that no conflict arises between that incorporated
material and the present disclosure material.
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Sequence CWU 1
1
2518PRTHomo sapiens 1Pro Arg Ser Phe Leu Leu Arg Asn1
527PRTArtificial SequencePAR1 pepducin lipopeptide 2Lys Lys Ser Arg
Ala Leu Phe1 5312PRTArtificial SequencePAR1 pepducin lipopeptide
3Arg Cys Leu Ser Ser Ser Ala Val Ala Asn Arg Ser1 5
10412PRTArtificial SequencePAR1 pepducin lipopeptide 4Arg Ser Leu
Ser Ser Ser Ala Val Ala Asn Arg Ser1 5 10510PRTArtificial
SequencePAR1 pepducin lipopeptide 5Asn Arg Ser Lys Lys Ser Ser Ala
Leu Phe1 5 10611PRTArtificial SequencePAR1 pepducin lipopeptide
6Ile Leu Lys Met Lys Val Lys Lys Pro Ala Val1 5 1077PRTArtificial
SequencePAR1 pepducin lipopeptide 7Thr Leu Gly Arg Ala Ser Phe1
5811PRTArtificial SequencePAR1 pepducin lipopeptide 8Leu Ser Trp
Arg Thr Leu Gly Arg Ala Ser Phe1 5 10916PRTArtificial SequencePAR1
pepducin lipopeptide 9Tyr Pro Met Gln Ser Leu Ser Trp Arg Thr Leu
Gly Arg Ala Ser Phe1 5 10 151021PRTArtificial SequencePAR1 pepducin
lipopeptide 10Phe Leu Ala Val Val Tyr Pro Met Gln Ser Leu Ser Trp
Arg Thr Leu1 5 10 15Gly Arg Ala Ser Phe 201113PRTArtificial
SequencePAR1 pepducin lipopeptide 11Ala Ser Ser Glu Ser Gln Arg Tyr
Val Tyr Ser Ile Leu1 5 101213PRTArtificial SequencePAR1 pepducin
lipopeptide 12Leu Ile Ser Tyr Val Tyr Arg Gln Ser Glu Ser Ser Ala1
5 101336PRTHomo sapiens 13Ala Arg Arg Pro Glu Ser Lys Ala Thr Asn
Ala Thr Leu Asp Pro Arg1 5 10 15Ser Phe Leu Leu Arg Asn Pro Asn Asp
Lys Tyr Glu Pro Phe Trp Glu 20 25 30Asp Glu Glu Lys
351436PRTArtificial SequencePAR1 N-terminal extracellular mutant
14Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Thr Leu Asp Pro Arg1
5 10 15Ser Phe Leu Leu Arg Asn Pro Asn Asp Lys Tyr Glu Pro Phe Trp
Glu 20 25 30Asp Glu Glu Lys 351526PRTArtificial SequencePAR1
N-terminal extracellular mutant 15Ala Thr Leu Asp Pro Arg Ser Phe
Leu Leu Arg Asn Pro Asn Asp Lys1 5 10 15Tyr Glu Pro Phe Trp Glu Asp
Glu Glu Ser 20 251620PRTArtificial SequencePAR1 N-terminal
extracellular mutant 16Ser Phe Leu Leu Arg Asn Pro Asn Asp Lys Tyr
Glu Pro Phe Trp Glu1 5 10 15Asp Glu Glu Ser 201722PRTArtificial
SequencePAR1 N-terminal extracellular mutant 17Pro Arg Ser Phe Leu
Leu Arg Asn Pro Asn Asp Lys Tyr Glu Pro Phe1 5 10 15Trp Glu Asp Glu
Glu Ser 201826PRTArtificial SequencePAR1 N-terminal extracellular
mutant 18Ala Thr Leu Asp Asn Arg Ser Phe Leu Leu Arg Asn Pro Asn
Asp Lys1 5 10 15Tyr Glu Pro Phe Trp Glu Asp Glu Glu Lys 20
251936PRTArtificial SequencePAR1 N-terminal extracellular mutant
19Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Thr Leu Asp Asn Arg1
5 10 15Ser Phe Leu Leu Arg Asn Pro Asn Asp Lys Tyr Glu Pro Phe Trp
Glu 20 25 30Asp Glu Glu Lys 352036PRTArtificial SequencePAR1
N-terminal extracellular mutant 20Met Ala Ser Met Thr Gly Gly Gln
Gln Met Gly Thr Leu Asp Pro Arg1 5 10 15Asp Phe Leu Leu Arg Asn Pro
Asn Asp Lys Tyr Glu Pro Phe Trp Glu 20 25 30Asp Glu Glu Lys
35218PRTArtificial SequencePAR1 N-terminal extracellular mutant
21Arg Pro Ser Phe Leu Leu Arg Asn1 5227PRTArtificial SequencePAR1
N-terminal extracellular mutant 22Arg Ser Phe Leu Leu Arg Asn1
5236PRTArtificial SequencePAR1 N-terminal extracellular mutant
23Ser Phe Leu Leu Arg Asn1 5249PRTArtificial SequencePAR1
N-terminal extracellular mutant 24Asp Pro Arg Ser Phe Leu Leu Arg
Asn1 525425PRTHomo sapiens 25Met Gly Pro Arg Arg Leu Leu Leu Val
Ala Ala Cys Phe Ser Leu Cys1 5 10 15Gly Pro Leu Leu Ser Ala Arg Thr
Arg Ala Arg Arg Pro Glu Ser Lys 20 25 30Ala Thr Asn Ala Thr Leu Asp
Pro Arg Ser Phe Leu Leu Arg Asn Pro 35 40 45Asn Asp Lys Tyr Glu Pro
Phe Trp Glu Asp Glu Glu Lys Asn Glu Ser 50 55 60Gly Leu Thr Glu Tyr
Arg Leu Val Ser Ile Asn Lys Ser Ser Pro Leu65 70 75 80Gln Lys Gln
Leu Pro Ala Phe Ile Ser Glu Asp Ala Ser Gly Tyr Leu 85 90 95Thr Ser
Ser Trp Leu Thr Leu Phe Val Pro Ser Val Tyr Thr Gly Val 100 105
110Phe Val Val Ser Leu Pro Leu Asn Ile Met Ala Ile Val Val Phe Ile
115 120 125Leu Lys Met Lys Val Lys Lys Pro Ala Val Val Tyr Met Leu
His Leu 130 135 140Ala Thr Ala Asp Val Leu Phe Val Ser Val Leu Pro
Phe Lys Ile Ser145 150 155 160Tyr Tyr Phe Ser Gly Ser Asp Trp Gln
Phe Gly Ser Glu Leu Cys Arg 165 170 175Phe Val Thr Ala Ala Phe Tyr
Cys Asn Met Tyr Ala Ser Ile Leu Leu 180 185 190Met Thr Val Ile Ser
Ile Asp Arg Phe Leu Ala Val Val Tyr Pro Met 195 200 205Gln Ser Leu
Ser Trp Arg Thr Leu Gly Arg Ala Ser Phe Thr Cys Leu 210 215 220Ala
Ile Trp Ala Leu Ala Ile Ala Gly Val Val Pro Leu Leu Leu Lys225 230
235 240Glu Gln Thr Ile Gln Val Pro Gly Leu Asn Ile Thr Thr Cys His
Asp 245 250 255Val Leu Asn Glu Thr Leu Leu Glu Gly Tyr Tyr Ala Tyr
Tyr Phe Ser 260 265 270Ala Phe Ser Ala Val Phe Phe Phe Val Pro Leu
Ile Ile Ser Thr Val 275 280 285Cys Tyr Val Ser Ile Ile Arg Cys Leu
Ser Ser Ser Ala Val Ala Asn 290 295 300Arg Ser Lys Lys Ser Arg Ala
Leu Phe Leu Ser Ala Ala Val Phe Cys305 310 315 320Ile Phe Ile Ile
Cys Phe Gly Pro Thr Asn Val Leu Leu Ile Ala His 325 330 335Tyr Ser
Phe Leu Ser His Thr Ser Thr Thr Glu Ala Ala Tyr Phe Ala 340 345
350Tyr Leu Leu Cys Val Cys Val Ser Ser Ile Ser Cys Cys Ile Asp Pro
355 360 365Leu Ile Tyr Tyr Tyr Ala Ser Ser Glu Cys Gln Arg Tyr Val
Tyr Ser 370 375 380Ile Leu Cys Cys Lys Glu Ser Ser Asp Pro Ser Ser
Tyr Asn Ser Ser385 390 395 400Gly Gln Leu Met Ala Ser Lys Met Asp
Thr Cys Ser Ser Asn Leu Asn 405 410 415Asn Ser Ile Tyr Lys Lys Leu
Leu Thr 420 425
* * * * *