U.S. patent application number 08/722659 was filed with the patent office on 2001-07-05 for use of heparinase to decrease inflammatory responses.
Invention is credited to BENNETT, D. CLARK, CAUCHON, ELIZABETH, DANAGHER, PAMELA, FINK, DOMINIQUE, GROUIX, BRIGETTE, HSIA, ARIANE, ZIMMERMANN, JOSEPH.
Application Number | 20010006635 08/722659 |
Document ID | / |
Family ID | 26673252 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006635 |
Kind Code |
A1 |
BENNETT, D. CLARK ; et
al. |
July 5, 2001 |
USE OF HEPARINASE TO DECREASE INFLAMMATORY RESPONSES
Abstract
Heparinase enzymes can be used as a medical treatment to reduce
localized inflammatory responses. Treatment of activated
endothelium with heparinase inhibits leukocyte rolling, adhesion
and extravasation. Most of the heparin and heparan sulfate on
endothelial cell surfaces and in basement membranes is degraded by
exposure to heparinase. In addition, immobilized chemokines, which
are attached to heparin/heparan sulfate on activated endothelium
are solubilized by heparinase digestion. Heparinase can be infused
into the vascular system to inhibit accumulation of leukocytes in
inflamed tissue and decrease damage resulting from localized
inflammations. Targeting of heparinase to activated endothelium can
be accomplished through localized administration and/or use of
genetically engineered heparinase containing endothelium
ligand-binding domains.
Inventors: |
BENNETT, D. CLARK;
(MONTREAL, CA) ; CAUCHON, ELIZABETH; (MONTREAL,
CA) ; FINK, DOMINIQUE; (MONTREAL, CA) ;
GROUIX, BRIGETTE; (MONTREAL, CA) ; HSIA, ARIANE;
(MONTREAL, CA) ; DANAGHER, PAMELA; (MONTREAL,
CA) ; ZIMMERMANN, JOSEPH; (MONTREAL, CA) |
Correspondence
Address: |
HOLLIE L BAKER
HALE AND DORR
1455 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
200041008
|
Family ID: |
26673252 |
Appl. No.: |
08/722659 |
Filed: |
September 27, 1996 |
Current U.S.
Class: |
424/94.1 ;
424/130.1; 424/192.1 |
Current CPC
Class: |
C12N 9/88 20130101; Y10S
424/81 20130101; A61P 29/00 20180101; A61K 38/51 20130101; A61P
43/00 20180101; A61K 47/50 20170801; A61P 9/10 20180101; C07K
2319/00 20130101 |
Class at
Publication: |
424/94.1 ;
424/85.1; 424/130.1; 424/192.1 |
International
Class: |
A61K 038/43; A61K
039/00; A61K 039/395; A61K 045/00 |
Claims
What is claimed is:
1. A method to decrease localized inflammatory responses in a
tissue of a patient comprising administering to said patient
heparinase enzyme in an effective amount sufficient to decrease
said localized inflammatory response.
2. The method of claim 1, wherein said administration of said
heparinase enzyme removes and digests heparin and heparan sulfate
from endothelial cell surfaces and extracellular matrices of said
tissue.
3. The method of claim 1, wherein said administration of said
heparinase enzyme decreases the accumulation of leukocytes in
tissue adjacent to endothelial cell surfaces and extracellular
matrices.
4. The method of claim 1, wherein said administration of said
heparinase enzyme inhibits leukocyte extravasation by releasing
immobilized chemokines and destroying chemokines immobilized to
endothelium.
5. The method of claim 1, wherein said administration of said
heparinase enzyme inhibits leukocyte rolling on endothelium.
6. The method of claim 1, wherein said heparinase enzyme is
overexpressed from a recombinant nucleotide sequence, in
Flavobacterium heparinum.
7. The method of claim 1, wherein said heparinase enzyme is
expressed from a recombinant nucleotide sequence in an organism in
which it does not naturally occur.
8. A method to decrease a localized inflammatory response in a
tissue of a patient comprising administering to said patient a
fusion protein comprising a ligand which binds to activated
endothelial cells and a heparinase enzyme in an amount sufficient
to decrease said localized inflammatory response in said
tissue.
9. The method of claim 8, wherein said fusion protein is made by
genetic engineering techniques.
10. A pharmaceutical composition comprising a heparinase enzyme
together with a pharmaceutically or a veterinarilly acceptable
carrier.
11. A pharmaceutical composition comprising fusion molecule
comprising a ligand which binds to activated endothelium and a
heparinase enzyme.
12. The pharmaceutical composition of claim 11, wherein said ligand
binding domain is selected from the group consisting of cytokines,
antibodies, integrins, and selecting.
13. The pharmaceutical composition of claim 11, wherein said ligand
binding domain are fragments of said cytokines, antibodies,
integrins, and selecting.
14. The pharmaceutical composition of claim 11, wherein said
fragments are selected from the group consisting of cytokine
receptor binding domains, Fab fragments, antibody variable regions,
integrin I-domains, and selectin domains.
15. The pharmaceutical composition of either claims 10 or 11,
wherein said carrier is selected from the group consisting of
liposomes, lipospheres, proteosomes, microspheres, microcapsules,
and biodegradable polymeric matrices.
16. The use of a heparinase enzyme in the preparation of a medicant
for treatment of decreasing localized inflammatory responses in a
patient's tissue.
17. The use of a heparinase enzyme in the preparation of a medicant
comprising a fusion protein comprising a ligand which binds to
activated endothelial cells and a heparinase enzyme for the
treatment of decreasing localized inflammatory responses in a
patient's tissue.
Description
[0001] This application is related to U.S. provisional application
Ser. No. 60/004,622, filed Sep. 29, 1995, which is expressly
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of medical treatments and is
directed to the use of heparinase enzyme as a treatment or
prophylactic for reducing localized inflammatory responses.
BACKGROUND OF INVENTION
[0003] An inflammatory response is local response to cellular
injury that is marked by capillary dilation, leukocytic
infiltration, redness, heat, and pain and serves as a mechanism
initiating the elimination of noxious agents and of damaged
tissue.
[0004] A generalized inflammatory response within a tissue occurs
by the recruitment of leukocytes to the tissue. Destruction of
bacteria, foreign materials and/or damaged cells occurs through
phagocytosis and/or extracellular degranulation (secretion of
degradative enzymes, antimicrobial proteins and myeloperoxidase,
which forms superoxides from secreted H.sub.2O.sub.2). While most
localized inflammatory responses are beneficial, harmful
inflammatory responses can occur. Many harmful inflammatory
responses also involve accumulation of leukocytes within a tissue.
This accumulation results in the destruction of viable cells and
tissue. In addition to damaging tissue, these responses are
detrimental to, or debilitating for, the afflicted individual.
Examples of detrimental inflammatory responses can include the
following; ischemia/reperfusion injury following myocardial
infarction, shock, stroke, organ transplantation, cardiopulmonary
bypass surgery, allograft rejection, rheumatoid arthritis,
antigen-induced asthma, allergic rhinitis, and glomerulonephritis
(see the review in; Harlan, et al., Immunol. Rev., 114:5-12, 1990;
Carlos and Harlan, Blood, 84:2068-2101, 1994.)
[0005] Leukocyte recruitment involves a cascade of cellular events,
beginning with activation of vascular endothelium by damaged or
infected tissue adjacent to the endothelium. Activation of the
endothelium results in enhanced adhesion of leukocytes to the
endothelial cells, and transendothelial migration (extravasation)
by bound leukocytes into the damaged tissue. Endothelial activation
is manifested by a short-term, rapid, and/or a long-term
stimulation of the endothelial cells.
[0006] Activators such as thrombin, chemoattractant leukotrienes,
B.sub.4, C.sub.4 and D.sub.4 (LTB.sub.4, LT C.sub.4 & LT
D.sub.4), and histamine cause rapid, transient (<30 minutes)
endothelial cell activation, independent of protein synthesis, and
can increase endothelial cell surface levels of the
chemoattractants such as platelet activating factor (PAF; a
glycerophospholipid) and LTB.sub.4 (shown for histamine), and
adhesion molecules; ICAM-1 (shown for thrombin) and P-selectin
(Zimmerman, et al., J. Cell Biol., 110:529-540, 1990; Sugama, et
al., J. Cell Biol., 119:935-944, 1992; McIntyre, et al. Proc. Natl.
Acad. Sci. USA, 83:2204-2208, 1986; Lorant, et al., J. Cell Biol.,
115:223-234, 1991). The outcome of rapid activation of endothelial
cells is increased leukocyte adhesion to the endothelium (Hoover,
et al., Proc. Natl. Acad. Sci. USA, 81:2191-2194, 1984; Zimmerman,
et al., J. Cell Biol., 110:529-540, 1990; Hanahan, et al. Ann. Rev.
Biochem., 55:483-509, 1986). However, increased LTB.sub.4 surface
levels have not been shown to directly increase transendothelial
migration of neutrophils (Hughes, et al., Prost. Leuk. Essent. F.
A., 45:113-119, 1992), and in certain situations, PAF is not
necessary for adhesion of leukocytes to activated endothelium
(Kuijpers, et al., J. Cell Biol., 117:565-574, 1992).
[0007] Long-term (hours in duration) protein synthesis dependent,
endothelial cells activation is produced by cytokines, such as
IL-1b and TNF-a, and by lipopolysaccharide (LPS) and results in
maintenance of increased surface levels of adhesion molecules:
E-selectin, P-selectin, ICAM-1 and VCAM-1 (reviewed by Carlos, and
Harlan, Blood, 84:2068-2101, 1994). IL-1b and TNF-a also increase
the synthesis of PAF by endothelial cells (Kuijpers, et al., J.
Cell Biol., 117:565-574, 1992). In addition, endothelial cell
activation by IL-1b, TNF-a, LPS and histamine has been shown to
increase the synthesis and secretion of the chemokine, IL-8
(Strieter, et al., Science, 243:1467-1469,1989; Jeannin, et al.,
Blood, 84:2229-2233, 1994).
[0008] Chemokines, IL-8 and MCP-1, have been shown to be produced
by and to be present on the endothelial cells surface (Huber, et
al., Science, 254:99-102, 1991; Springer, Nature, 346:425-434,
1990). The chemokine, MIP-1b, has been shown to be present on lymph
node endothelium, in vivo (Taub, et al., Science, 260:355-359,
1993; Tanaka, et al., Nature, 361:79-82, 1993). The chemokines;
RANTES, MIP-a, MIP-b, MCP-1 and IL-8 are all heparin binding
proteins, which after being secreted, bind to cell surface and
extracellular matrix proteoglycans possessing heparin and heparan
sulfate moieties (reviewed by Miller, et al., Crit. Rev. Immunol.,
12:17-46, 1992).
[0009] Heparin and heparan sulfate are similar glycosaminoglycan
moieties found interspersed on the same unbranched carbohydrate
chains. They are covalently attached to the protein backbones of
proteoglycans. Despite what these two names imply, heparin is more
highly sulfated than is heparan sulfate. Proteoglycans are present
on cell surfaces and in extracellular matrices (e.g. in the
basement membrane of endothelium). Because of difficulty in
distinguishing regions of heparin and heparan sulfate on the same
carbohydrate chain, little data exists on the binding preference of
chemokines for either heparin or heparan sulfate moieties. There is
some indication that chemokines IL-8 and GRO bind with greater
affinity to heparan sulfate than heparin, and that PF4 and NAP-2
bind with greater affinity to heparin moieties (Witt, and Lander,
Curr. Biol., 4:394-400, 1994). Generally, chemokines are referred
to as heparin binding proteins. C-terminal regions of the
chemokines IL-8, PF4, MCP-1 and NAP-2 have been shown to form an
a-helix, and to bind to heparin/heparan sulfate (Webb, et al.,
Proc. Natl. Acad. Sci. USA, 90:7158-7162, 1993; Zucker, et al.,
Proc. Natl. Acad. Sci. USA, 86:7571-7574, 1989; Matsushima, et al.,
in Interleukins: Molec. Biol. Immunol., ed. Kistimoto, Karger,
Basel, 236-265, 1992). This is likely to be a structure, common to
all of the chemokines.
[0010] All of the molecules mentioned above, which are expressed by
activated endothelial cells (PAF, LTB.sub.4, selectins, CAMs and
chemokines), are present on the endothelial cell surface, and are
localized to vascular endothelium adjacent to sites of damaged
tissue. Blood-borne leukocytes which interact with these molecules
will also be localized in their binding in the area of the damaged
tissue. The outcome of long-term activation of endothelium is
increased adhesion and extravasation of leukocytes and a
significant localized accumulation of leukocytes in adjacent
tissue, which cannot occur during short-term activation (Ebisawa,
et al., J. Immunol., 149:4021-4028, 1992; Huber, and Weiss, J.
Clin. Invest., 83:1122-1129, 1989; Oppenheimer-Marks, et al., J.
Immunol., 145:140-148,1990).
[0011] Adhesion of leukocytes to endothelium is thought to be a two
step process (reviewed by Carlos, and Harlan, Blood, 84:2068-2101,
1994). Initially, leukocytes roll along the surface of blood
vessels. Increased rolling is initially mediated on vascular
endothelium (within the first 30 minutes) by interactions between
Sialyl Lewisx structures on the leukocyte surface and P-selectin
and E-selectin, which are increased on activated endothelial cells
(Ley, et al., Blood, 85:3727-3735, 1995). Increased rolling is also
mediated (after 40 minutes) by interactions between L-selectin on
leukocyte cellular membranes and heparin-like molecules on the
vascular endothelium, which are cytokine-inducable (Karin. et al.,
Science, 261:480-483,1993), or between L-selectin on lymphocytes
and vascular addressins; GlyCAM-1, CD34 and MAdCAM-1 on high
endothelial venules (HEVs) in lymphoid tissue. The second step,
firm adhesion of leukocytes to endothelial cells, is based on
interactions between leukocyte integrins (e.g. LFA-1, Mac-1, VLA-4)
and endothelial cellular adhesion molecules (CAMs; e.g. ICAM-1,
ICAM-2, VCAM-1, MAdCAM-1). Leukocytes flatten on the endothelial
surface, and shed L-selectin, concomitant with firm adhesion
(Kishimoto, et al., Science, 245:1238-1242, 1989; Jutila, et al.,
J. Immunol., 143:3318-3324, 1989; Smith, et al., Clin. Invest.,
87:609-618, 1991).
[0012] Selectin and CAM levels increase on the endothelium surface
in response to many cytokines and chemoattractants. These increases
are dependent on synthesis and/or secretion of additional selectin
and CAM molecules onto the cell surface. In contrast, activation of
leukocytes for firm adhesion has been shown to occur within seconds
(Bargatze, et al., J. Exp. Med., 178:367-373, 1993), through
increased secretion of integrins, and more importantly, through
induction of conformational changes in cell surface integrins
(integrin activation), which permits tight binding of the integrins
to CAMs (reviewed in Zimmerman, Immunol. Today, 13:93-99,
1992).
[0013] PAF and E-selectin can activate integrins for endothelial
cell adhesion (Lorant et al., J. Biol. Chem., 115:223-234,1991; Lo,
J. Exp. Med., 173:1493-1500,1991). The presence of MIP-1b,
immobilized by binding to CD44 (possesses heparin/heparan sulfate
moieties), or a heparin-BSA conjugate, has been shown to be
required for CD8+T-cell binding to immobilized VCAM-1 molecules.
This binding was shown to be blocked by an antibody to VLA-4,
indicating that MIP-1b activates VLA-4 on the T-cell surface
(Tanaka, et al., Nature, 361:79-82, 1993). An increase in the level
of integrin, CD18 (part of Mac-1), on the surface of neutrophils
has been shown to occur when neutrophils contact endothelium, which
has been stimulated with IL-1b. An antibody to IL-8 inhibited the
CD18 up-regulation, and also inhibited neutrophil adhesion (Huber,
Science, 254:99-102, 1991). Thus, chemokines can act as direct
activators of leukocyte adhesion. In contrast, Luscinaskas et al.
(J. Immunol., 149:2163-2171, 1992) has demonstrated that
pretreatment of neutrophils with IL-8 inhibits neutrophil
attachment, and addition of exogenous IL-8 detached neutrophils
adhering to activated endothelial cells. Rot (Immunol. Today,
13:291-294, 1992) has reconciled these contradictory results by
proposing that IL-8 bound to the endothelial cells surface promotes
adhesion, while soluble IL-8 can inhibit it.
[0014] Different chemokines activate different leukocytes. IL-8
activates neutrophils, eosinophils and T cells. RANTES activates
monocytes, eosinophils and T cells. MCP-1 activates monocytes.
MIP-la activates CD4+ T cells, monocytes and B cells, while MIP-1b
activates monocytes and CD8+ T cells (reviewed in Lasky, Current
Biol., 3:366-378, 1993). Different combinations of selectins,
integrins, CAMs and chemokines are thought to select for the
adhesion and migration of the leukocyte subtypes observed in
different inflamed tissues (Butcher, Cell, 67:1033-1039, 1991).
[0015] The importance of interactions of integrins, CD11/CD18
(Mac-1), and ICAMs in adhesion and extravasation of leukocytes has
been demonstrated in numerous systems by the use of antibodies to
these molecules. The antibodies interfere with the function of the
adhesion molecules and block or reduce leukocyte recruitment. The
leukocyte adhesion deficiency (LAD) Type I syndrome results in a
partial or total absence of the integrin, CD18, on the leukocytes
of affected patients. Neutrophil recruitment to sites of
inflammation is negligible. However, monocyte and eosinophil
recruitment is normal, indicating that an alternative set of
adhesion molecules may function for recruitment of these cells,
perhaps VLA-4 and VCAM-1 (Harlan, Clin. Immunol. Immunopath.,
67:S16-S24, 1993). VLA-4 is not expressed by neutrophils (Winn and
Harlan, J. Clin. Invest., 92:1168-1174, 1993). As mentioned
previously, chemokines are important for activation and increased
surface levels of integrins VLA-4 and CD18 on leukocytes. IL-8
immobilized on a polycarbonate filter has been shown to be adequate
for directing migration of neutrophils through the filter (Rot,
Immunol. Today, 13:291-294). Huber, et al. (Science, 254:99-102,
1991) has shown that a transendothelial gradient of bound IL-8,
produced by IL-1b stimulated endothelial cell monolayers, is
necessary for extravasation of neutrophils. These neutrophils were
pre-activated with IL-8 and did bind to the endothelial cells, but
did not migrate until the IL-8 gradient was present. This gradient
extended from the endothelial cells luminal surface through the
basement membrane underlying the endothelial cell monolayer.
Washing bound IL-8 from the basement membrane underlying activated
endothelial cells prevented migration across the monolayer. In
addition, an antibody to IL-8 inhibited 70-80% of the neutrophil
migration. Kuijpers et al. (J. Cell Biol., 117:565-572, 1992) used
an anti-IL-8 antibody to produce a 60% reduction in neutrophil
migration across IL-1b and TNF-a activated endothelium, and
addition of a PAF receptor antagonist produced an 85-90% reduction
in migration. These results are in contrast to experiments which
showed that IL-8 pretreated neutrophils were inhibited in their
ability to migrate through an activated endothelial cell monolayer
(Luscinaskas et al., J. Immunol., 149:2163-2171, 1992). Thus, it is
likely that chemokines not only activate leukocytes for adhesion,
but that a bound gradient of chemokine is important in
extravasation of leukocytes. The presence of soluble chemokine can
interfere with adhesion and migration along a bound chemokine
gradient. As discussed below, in vivo localized concentration
increases in soluble chemokines would be minimized by blood
flow.
[0016] Once activated leukocytes have begun to accumulate within a
damaged tissue, they can augment the accumulation of additional
leukocytes, by synthesis and secretion of cytokines, chemokines,
and LTB.sub.4. LPS has been shown to directly increase monocyte
IL-1b expression (Porat, et al., FASEB J., 6:2482-2489, 1992).
IL-8, IL-1b and TNF-a are produced by neutrophils activated with
GM-CSF, another cytokine produced by activated macrophages,
endothelium and T-cells (McCain, et al., Am. J. Respir. Cell Molec.
Biol., 8:28-34, 1993; Lindemann, et al., J. Immunol. 140:837-839,
1988; Lindemann, et al., J. Clin. Invest., 83:1308-1312, 1989).
IL-1b and TNF-a have been shown to stimulate monocytes, thereby
increasing the expression of the chemokines, IL-8 and MIP-1a
(Lukacs, et al., Blood, 82:3668-3674, 1993). Activated neutrophils
and monocytes have been shown to be a major source of LTB.sub.4
production (Samuelsson, et al., Science, 237:1171-1176, 1987;
Brach, et al., Eur. J. Immunol., 22:2705-2711, 1992). As discussed
previously, LTB.sub.4 is not directly involved in further
recruitment of leukocytes, but because neutrophils stimulated with
LTB.sub.4 produce IL-8, the LTB.sub.4-stimulated neutrophils could
promote further neutrophil recruitment, indirectly, through
formation of an IL-8 gradient (McCain, et al., Am. J. Respir. Cell
Molec. Biol., 10:651-657, 1994). The continued recruitment of
leukocytes by these leukocyte-derived activators would require
using the vascular endothelium as an intermediate. Endothelial
cells and basement membranes would bind and display
neutrophil-derived chemokines, forming a gradient, or
leukocyte-derived cytokines would activate the endothelium, which
would also cause the creation of a chemokine gradient.
[0017] Blood flow in the vasculature would prevent a localized
concentration increase in soluble activation factors (cytokines,
chemokines and chemoattractants), produced by a tissue-localized
inflammatory response. If a local inflammation is producing high
blood concentrations of activators, a systemic activation of
leukocytes could occur (Finn, et al., J. Thorac. J. Surg.,
105:234-241, 1993). The activated leukocytes would then bind
transiently to unactivated endothelium and/or degranulate, causing
sepsis (Sawyer, et al., Rev. Infect. Dis., 11:S1532-1544, 1989). In
situations where some blood-borne leukocytes are activated by a
localized inflammation (not all of the activated leukocytes
extravasate), the activated leukocytes would produce and secrete
additional cytokines, chemokines, and LTB.sub.4 into the blood.
This increase in activator concentration could up-regulate
unactivated cells and amplify the systemic response.
[0018] Although the mechanism of inflammatory responses has been
given in some detail, there is still a need for an effective
treatment and pharmaceutical compositions for reducing or
preventing localized inflammatory responses.
SUMMARY OF INVENTION
[0019] This invention is directed to the discovery that heparinase
degrading enzymes, either separately or in combination, can be used
to decrease localized inflammatory response. The heparinases useful
in this invention can be from a variety of sources: heparinases I,
II, and III from the Gram negative bacterium Flavobacterium
heparinum, heparinase from Bacteroides strains, heparinase from
Flavobacterium Hp206, heparinase from Cytophagia species, and
heparanases from mammalian cells. These enzymes, either singly or
in combination, are referred to herein as heparinase or heparinase
enzyme.
[0020] Heparin and heparan sulfate moieties are degraded on the
surface of endothelial cells and from basement membranes by
administration of heparinase. Removal of heparin and heparan
sulfate moieties from up-regulated proteoglycans on activated
endothelial cells prevents L-selectin, found on leukocytes, from
interacting with the proteoglycans. By decreasing
L-selectin-proteoglycan interactions, leukocyte rolling on
activated endothelium can be inhibited.
[0021] In addition, when heparin and heparan sulfate moieties are
removed from the surface of activated endothelial cells and from
their basement membrane, chemokines, which are bound to the heparin
and heparan sulfate, are released from the cell surfaces and
basement membrane. The loss of bound chemokines decreases the
localized concentration of chemokines and disrupts the chemokine
gradient produced by activated endothelium, thereby inhibiting
chemokine activation of rolling leukocytes, which is required for
firm adhesion, and preventing extravasation of leukocytes along the
chemokine gradient. By this invention, decreased leukocyte rolling,
activation and extravasation can inhibit localized tissue
inflammatory responses by interfering with fundamental mechanisms
of leukocyte recruitment.
[0022] Heparinase enzyme can be targeted to specific cell types,
tissues or organs by the selected method of administration, which
deliver localized high concentrations of the enzymes or physically
limit the dispersal of the enzymes. Additionally, according to this
invention heparinase can be targeted by fusion of the enzymes to
binding domains from antibodies, growth factors or adhesion
molecules. The fusion proteins are produced by construction and
expression of gene fusions in recombinant organisms. As examples,
the binding domains can recognize cell surface molecules on
activated endothelium (e.g., ICAM-1, VCAM-1, P-selectin,
E-selectin), or on endothelial cell subtypes (e.g., GlyCAM-1).
Targeted fusion enzymes can increase the number and specificity of
indications, while decreasing the amounts of enzyme required for
efficacy and possible side-effects resulting from treatments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a graph of the counts of 35S-heparin/heparan
sulfate released from the surface of endothelial cells by 1.0 IU/ml
of heparinase III, which were separated according to size on a gel
filtration column. The diamonds indicate counts released by a 5
minute digest, the squares indicate counts released by a 30 minute
digest, and the triangles indicate counts released by a 60 minute
digest. Background counts from fractionation of mock digests have
been subtracted from the fractions derived from the heparinase III
digests.
[0024] FIG. 2A and 2B are graphs of the percent of heparin/heparan
sulfate present on the unactivated (2A) and IL-1b activated (2B)
human endothelial cell line at the indicated times after treatment
with 0.1 IU/ml heparinase I, II or III for 1 hour. 125I-bFGF
binding to cell surface heparin was used to determine the amount of
heparin/heparan sulfate present. Results for heparinase I, II or
III treated cells are indicated by diamonds, squares or triangles,
respectively. The vertical lines indicate the standard error of the
means.
[0025] FIG. 3A and 3B are graphs of the percent of heparin/heparan
sulfate present on the unactivated (3A) and IL-1b activated (3B)
human endothelial cell line at the indicated times after treatment
with 1.0 IU/ml heparinase I. 125I-bFGF binding to cell surface
heparin was used to determine the amount of heparin/heparan sulfate
present. Results for 1, 3 or 5 hour treated cells are indicated by
diamonds, squares or triangles, respectively. The vertical lines
indicate the standard error of the means.
[0026] FIG. 4A and 4B are graphs of the percent of heparin/heparan
sulfate present on the unactivated (4A) and IL-1b activated (4B)
human endothelial cell line at the indicated times after treatment
with 1.0 IU/ml heparinase III. 125I-bFGF binding to cell surface
heparin was used to determine the amount of heparin/heparan sulfate
present. Results for 1, 3 or 5 hour treated cells are indicated by
diamonds, squares or triangles, respectively. The vertical lines
indicate the standard error of the means.
[0027] FIG. 5 contains graphs displaying the levels of IL-8
released from IL-1b activated human endothelial cell layers by
treatment with 1.0 IU/ml of heparinases; I (5A), II (5B), I+III
(bars containing diagonal lines; 5B), and III (5C). The bars
represent the percent difference in the concentration of IL-8 found
in; supernatants from activated endothelial layers treated with
heparinases, versus untreated supernatants from activated
endothelial layers (containing only secreted IL-8). Standard errors
for these percentage differences are indicated by vertical lines.
The lines overlaid on the bars indicate the concentration of IL-8
in the supernatants from the heparinase treated cell layers. The
standard errors of these measurements are also indicated by
vertical lines (not always visible).
[0028] FIG. 6 is a graph of the level of neutrophil adhesion to
endothelial cells, which were unactivated, IL-1b activated, or
treated with 0.1 IU/ml of heparinases I, II or III after IL-1b
activation. The level of adhesion is expressed as the percent of
added neutrophils, which are adhering.
[0029] FIGS. 7A, 7B and 7C are graphs of the percent inhibition of
neutrophil extravasation through IL-1b activated endothelial cell
layers, which were treated with heparinases I, II or III,
respectively. The bars containing diagonal lines represent results
of one hour treatments with 1.0 IU/ml of heparinase. The white bars
represent results of one hour treatments with 0.1 IU/ml of
heparinase. The black bars represent results of 15 minute
treatments with 0.1 IU/ml of heparinase I or III, and the bar
containing vertical lines represents results of 15 minute
treatments with 1.0 IU/ml of heparinase II. The standard deviations
for the percent inhibitions are indicated by vertical lines. The
small asterisks indicate results of one hour treatments that were
significantly different from the results of the 15 minute treatment
with the same heparinase (P<0.05). The large asterisks indicate
the results of one hour treatments with 1.0 IU/ml of heparinase
that were significantly different from the results of one hour
treatments with 0.1 IU/ml of the same heparinase. The numbers in
parentheses under the bars indicate the number of experiments
included in each data set.
[0030] FIG. 8 is a graph showing the activity of human heparinase
(b-thromboglobulin) on ECM at pH 5.8 and 7. The solid bars
represent the percent difference in .sup.35SO.sub.4 released from
ECM treated with 1 ug of human heparinase versus that released from
untreated ECM. The bars containing diagonal lines represent the
percent difference in .sup.35SO.sub.4 released from ECM treated
with 5 ug of human heparinase versus that released from untreated
ECM. The standard deviation of the means are indicated by vertical
lines.
[0031] FIG. 9 is a graph which displays the change in the level of
neutrophil extravasation upon activation of HUVEC layers with
IL-1b, and after treatment of activated HUVEC layers with human
heparinase (hhep). The standard deviation of the means are
indicated by vertical lines.
[0032] FIG. 10 is a graph of rat plasma heparinase III
concentrations over a five hour infusion period. Time points in the
protocol are indicated by the arrows, with descriptions above the
arrows. The vertical lines indicate the standard error of the
means.
[0033] FIG. 11 is a graph of the level of leukocyte rolling in the
rat microvasculature after 3 hours of ischemia, during reperfusion.
The circles indicate the levels in naive rats, the squares indicate
the levels in sham treated rats which underwent ischemia, and the
triangles indicate the levels in heparinase treated rats which
underwent ischemia. The vertical lines indicate the standard error
of the means.
[0034] FIG. 12 is a graph of the level of leukocyte adhesion in the
rat microvasculature after 3 hours of ischemia, during reperfusion.
The circles indicate the levels in naive rats, the squares indicate
the levels in sham treated rats which underwent ischemia, and the
triangles indicate the levels in heparinase treated rats which
underwent ischemia. The vertical lines indicate the standard error
of the means.
[0035] FIG. 13 is a graph of the level of leukocyte extravasation
in the rat microvasculature after 3 hours of ischemia, during
reperfusion. The circles indicate the levels in heparinase treated
rats which underwent ischemia. The vertical lines indicate the
standard error of the means.
[0036] FIG. 14 is a graph of the level of leukocyte extravasation
in the rat microvasculature after 2 hours of ischemia, during
reperfusion. The open bars are the percent difference in the levels
in sham treated rats versus the levels in naive rats. The bars
containing diagonal lines are the percent difference in the levels
in heparinase treated rats versus the levels in naive rats. The
vertical lines indicate the standard error of the means.
[0037] FIG. 15 is a graph of the level of perfusion in rat
postcapillary venules after 3 hours of ischemia, during
reperfusion. The circles indicate the levels in naive rats, the
squares indicate the levels in sham treated rats which underwent
ischemia, and the triangles indicate the levels in heparinase
treated rats which underwent ischemia. The vertical lines indicate
the standard error of the means.
[0038] FIG. 16 is a graph of the heart rate-blood pressure product
for rabbits during ischemia and reperfusion with or without
heparinase treatment. The open circles and squares are data for
saline pretreated and reperfusion treated rats, respectively. The
open pyramids and solid circles are data for heparinase pretreated
and reperfusion treated rabbits, respectively (25 ug/ml target
plasma levels for heparinase III). The solid squares, pyramids and
diamonds are data for heparinase reperfusion treated rabbits with
5, 1.25 and 0.25 ug/ml target plasma levels of heparinase III,
respectively. BASE indicates baseline levels. 30I indicates the
level at 30 minutes of ischemia. 30R, 60R, 120R and 180R indicates
the levels at 30, 60 120 and 180 minutes of reperfusion. The
vertical lines indicate the standard deviation of the means.
[0039] FIG. 17 is a graph of the percent of the infarct size vs.
risk zone after ischemia and reperfusion in rabbit hearts, which
underwent different heparinase treatments. The solid circles
indicate the average levels for each treatment group. The open
shapes indicate the levels for individual rabbits. CPT and CRT
indicate saline pretreated and reperfusion treated rabbits,
respectively. DPT and DRT indicate heparinase pretreated and
reperfusion treated rabbits, respectively. The numbers below DPT
and DRT indicate the target level of heparinase III in the plasma
(in ug/ml). The vertical lines indicate the standard deviation of
the means.
[0040] FIG. 18 is a graph of the concentration of heparinase III
which was infused into the heparinase treated rabbits (in IU/ml).
DPT and DRT indicate heparinase pretreated and reperfusion treated
rabbits, respectively. The numbers below DPT and DRT indicate the
target level of heparinase III in the plasma (in ug/ml). Con
indicates control rabbits infused with saline. The vertical lines
indicate the standard deviation of the means.
[0041] FIG. 19 is a graph of the concentrations of heparinase III
measured in the rabbit plasma during pretreatment and reperfusion.
The circles indicate the actual concentrations measured in
heparinase pretreated rabbits targeted for 25 ug/ml plasma
concentrations of heparinase III. The squares, pyramids, triangles
and diamonds indicate the actual concentrations measured in
heparinase reperfusion treated rabbits targeted for 25, 5, 1.25 and
0.25 ug/ml plasma concentrations of heparinase III, respectively.
BASE indicates baseline concentrations. 30P and 60P indicate
concentrations at 30 and 60 minutes of pretreatment. 15R, 30R, 60R,
120R and 180R indicate concentrations at 15, 30, 60 120 and 180
minutes of reperfusion, respectively. The vertical lines indicate
the standard deviation of the means.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Interactions between leukocytes and endothelium are critical
to the progress of localized inflammatory responses. These critical
interactions include functional contacts between endothelium bound
chemokines and leukocyte chemokine receptors, and between leukocyte
L-selectin and heparin/heparan sulfate proteoglycans on
endothelium. This invention is based on the discovery and is
directed to the use of heparinase enzyme and heparinase fusion
protein to decrease leukocyte-chemokine and leukocyte-endothelial
cells proteoglycan interactions, and thereby inhibiting localized
inflammation.
[0043] Heparin and heparan sulfate are glycosaminoglycan moieties
of proteoglycans located on the surface of many different cell
types and also found in the extracellular matrices produced by many
cells. Endothelial cells produce extracellular matrix, primarily on
their abluminal side, referred to as basement membrane. Endothelial
cells, activated by certain cytokines or by other inflammatory
response stimulators, increase their surface levels of heparin and
heparan sulfate proteoglycans (excluding high endothelial venules),
which act as inflammatory adhesion molecules, and interact with
L-selectin on rolling leukocytes. This interaction increases
contacts of leukocytes with the endothelium (increased rolling of
leukocytes), which are necessary for subsequent steps in leukocyte
recruitment. Activated endothelium also increases it's synthesis
and secretion of chemokines. The secretion of chemokines in turn
increases the localized concentration of the same chemokines,
because the chemokines bind to the heparin and heparan sulfate
moieties of proteoglycans on the endothelial cells surfaces and in
the basement membranes. This localized concentration gradient is
required for activation of leukocytes for firm adhesion and
extravasation, and results in leukocyte recruitment to inflammatory
sites.
[0044] Heparinase enzymes have been found in microorganisms
including Flavobacterium heparinum (Lohse and Linhardt, J. Biol.
Chem. 267:2437-24355, 1992), Bacteroides strains (Saylers, et al.,
Appl. Environ. Microbiol. 33:319-322, 1977; Nakamura, et al., J.
Clin. Microbiol. 26:1070-1071, 1988), Flavobacterium Hp206
(Yoshida, et al., 10th Annual Symposium of Glycoconjugates,
Jerusalem 1989) and Cytophagia species (Bohn, et al., Drug Res.
41(I), Nr. 4:456-460, 1991). Heparanases from mammalian cells have
also been described (Fuks, et al., U.S. Pat. No. 5,362,641, 1994;
Hoogewerf et al., J. Biol. Chem. 270:3268-3277, 1995). The
heparinases from Flavobacterium heparinum, heparinase I (EC
4.2.2.7), and heparinase II degrade heparin, while heparinase II
also degrades heparan sulfate, as does heparinase III (EC 4.2.2.8).
The products of complete digestion by these enzymes are mainly
disaccharides, though small quantities of tetrasaccharides and
oligosaccharides may persist. These enzymes can be used to remove
cell surface and basement membrane glycosaminoglycans, heparin and
heparan sulfate.
[0045] The removal of heparin and heparan sulfate from endothelial
cells interferes with L-selectin interactions with endothelium,
preventing increased leukocyte rolling. The removal of
glycosaminoglycans from endothelial cells and basement membranes
also removes glycosaminoglycan bound chemokines, which are critical
for leukocyte recruitment. Loss of endothelial cells bound
chemokines decreases activation of leukocyte integrins and inhibits
firm adhesion by the leukocytes. It also inhibits extravasation of
leukocytes, because the leukocytes require the presence of a bound
gradient of chemokine for transmigration. It is believed, without
being limited, that unbound chemoattractants are depleted from the
endothelium layer by blood flow, preventing formation of a
significant soluble chemoattractant gradient.
[0046] Generally, after a one hour heparinase treatment, 50% of the
digested cell surface and basement membrane heparin and heparan
sulfate are replaced within 2 to 4 hours, and it is completely
replaced within 12 to 16 hours. Longer treatment times (3 and 5
hours) greatly extended the time needed to replace the same amount
of heparin/heparan sulfate. Inflammatory responses would be
significantly diminished by a slow rate of replacement of cell
surface heparin/heparan sulfate. Appropriate administration of
heparinase could extend the duration of diminished inflammatory
response.
PREPARATION OF HEPARINASE
[0047] Individual heparinases or a combination thereof, that may be
used in this invention can be prepared from a variety of sources.
Heparinase may be prepared by isolation from bacterial or mammalian
cells, either those which naturally produce the enzymes or have
been genetically engineered to produce the enzymes as described in
by known methods. In addition, mammalian heparanases from human
cells may be isolated according to procedures for purification
described by Fuks, et al. (U.S. Pat. No. 5,362,641, 1994).
Isolation of Heparinases from Flavobacterium heparinum
[0048] Heparinase enzymes can be purified from cultures of
Flavobacterium heparinum as follows. F. heparinum is cultured in 15
L computer controlled fermenters, in a variation of the defined
nutrient medium described by Galliher et al., Appl. Environ.
Microbiol. 41(2):360-365, 1981. For fermentations designed to
produce heparin lyases, semi-purified heparin (Celsus Laboratories)
is included in the media at a concentration of 1.0 g/L as the
inducer of heparinase synthesis. The cells are harvested by
centrifugation and the desired enzymes released from the
periplasmic space by a variation of the osmotic shock procedure
described by U.S. Pat. No. 5,169,772 to Zimmermann, et al.
(1992).
[0049] Proteins from the crude osmolate are adsorbed onto cation
exchange resin (CBX, J. T. Baker) at a conductivity of between one
and seven mhos. Unbound proteins from the extract are discarded and
the resin packed into a chromatography column (5.0 cm
i.d..times.100 cm). The bound proteins elute at a linear flow rate
of 3.75 cm.multidot.min-1 with step gradients of 0.01 M phosphate,
0.01 M phosphate/0.1 M sodium chloride, 0.01 M phosphate/0.25 M
sodium chloride and 0.01 M phosphate/ 1.0 M. sodium chloride, all
at pH, 7.0.+-.0.1. Heparinase II elutes in the 0.1 M NaCl fraction
while heparinases, I and III, elute in the 0.25 M fraction.
Alternately, the 0.1 M sodium chloride step is eliminated and the
three heparinases co-eluted with 0.25 M sodium chloride. The
heparinase fractions are loaded directly onto a column containing
cellufine sulfate (5.0 cm i.d..times.30 cm, Amicon) and eluted at a
linear flow rate of 2.50 cm.multidot.min-1 with step gradients of
0.01 M phosphate, 0.01 M phosphate/0.2 M sodium chloride, 0.01 M
phosphate/0.4 M sodium chloride and 0.01 M phosphate/ 1.0 M. sodium
chloride, all at pH, 7.0.+-.0.1. Heparinases II and III elute in
the 0.2 M sodium chloride fraction while heparinase I elutes in the
0.4 M fraction. The 0.2 M sodium chloride fraction from the
cellufine sulfate column is diluted with 0.01 M sodium phosphate to
give a conductance less than 5 mhos. The solution is further
purified by loading the material onto a hydroxyapatite column (2.6
cm i.d..times.20 cm) and eluting the bound protein at a linear flow
rate of 1.0 cm.multidot.min-1 with step gradients of 0.01 M
phosphate, 0.01 M phosphate/0.35 M sodium chloride, 0.01 M
phosphate/0.45 M sodium chloride, 0.01 M phosphate/0.65 M sodium
chloride and 0.01 M phosphate/ 1.0 M. sodium chloride, all at pH,
7.0.+-.0.1. Heparinase II elutes in a single protein peak in the
0.45 M sodium chloride fraction while heparinase III elutes in a
single protein peak in the 0.65 M sodium chloride fraction.
Heparinase I is further purified by loading material from the
cellufine sulfate column, diluted to a conductivity less than 5
mhos, onto a hydroxyapatite column (2.6 cm i.d..times.20 cm) and
eluting the bound protein at a linear flow rate of 1.0
cm.multidot.min-1 with a linear gradient of phosphate (0.01 to 0.25
M) and sodium chloride (0.0 to 0.5 M). Heparinase I elutes in a
single protein peak approximately mid-way through the gradient.
[0050] The heparinase enzymes obtained by this method are greater
than 98.5% pure as estimated by reverse phase HPLC analysis
(BioCad, POROS II). Purification results for the heparinase enzymes
are shown in Table A.
1TABLE A Purification of heparinase enzymes from Flavobacterium
heparinum fermentations specific activity activity yield sample
(IU) (IU/mg) (%) fermentation heparin degrading 94,500 100 heparan
sulfate 75,400 ND 100 degrading osmolate heparin degrading 52,100
55 heparan sulfate 42,000 ND 56 degrading cation exchange heparin
degrading 22,600 24 heparan sulfate 27,540 ND 37 degrading
cellufine sulfate heparin degrading 19,200 20 heparan sulfate 9,328
30.8 12 degrading hydroxylapatite heparinase 1 16,300 115.3 17
heparinase 2 2,049 28.4 3 heparinase 3 5,150 44.5 7
Isolation of Recombinant Enzymes
[0051] Glycosaminoglycan degrading enzymes also can be isolated
from recombinant expression systems such as the heparinase I
expression system described by Sasisekharan, et al., Proc. Natl.
Acad. Sci. USA 90:8660-8664, 1993; the heparinase, II and III,
expression systems described in co-pending U.S. patent application
Ser. No. 08/258,639, "Nucleic Acid Sequences and Expression Systems
for Heparinase II and Heparinase III Derived From Flavobacterium
heparinum" by Su, et al., filed Jun. 10, 1994, the teachings of
which are incorporated herein. In these expression systems, the F.
heparinum genes are isolated and cloned into plasmids downstream
from an inducable promoter. The plasmids are introduced into E.
coli and the expression of the desired enzyme directed by a
suitable induction method such as temperature shift or addition of
IPTG to the medium.
[0052] The enzymes can be recovered in a purified form by a
modification of the methods described herein. Cell disruption is
achieved by homogenization, sonication or enzymatic treatment to
break the cell wall and release cytoplasmic components. If enzyme
synthesis results in aggregation, the aggregate can then be
dissolved by a denaturing agent, 3 to 6 M guanidine HCl or 4 to 8 M
urea and the protein refolded by removal of the denaturing agent
through dialysis or dilution. The refolded enzyme can be further
purified using the liquid chromatographic methods described
above.
Construction of Fusion Proteins
[0053] Fusion proteins incorporating heparinase enzyme fused to
proteins with specific binding properties can be created by
recombinant molecular biology techniques. By choosing an
appropriate binding protein, heparinase activity can be targeted to
specific sites, in vivo. ICAM-1 has been shown to be preferentially
expressed on the surface of activated endothelial cells (Dustin, et
al., J. Immunol., 137:245-254, 1986). As examples of fusion
proteins; an antibody, Fab fragment or variable region, specific
for ICAM-1, VCAM-1 or P-selectin, when fused to heparinase enzyme
or an active portion thereof, localizes heparinase activity near
the luminal and abluminal surfaces of activated endothelium.
Heparin and heparan sulfate moieties are removed in this area,
causing breakdown of the chemokine gradient produced by the
endothelium. As other examples, fusion of heparinase enzyme, or an
active portion thereof, to the I-domain of LFA-1 or Mac-1 (both
bind to ICAM-1) targets activated endothelium for removal of
heparin and heparan sulfate, inhibiting leukocyte rolling and
chemokine gradient formation. Receptors for cytokines such as
IL-1b, are up-regulated on activated endothelium and provide
another target for binding of fusion proteins. By fusing IL-1b, or
the receptor binding domain of IL-1b to heparinase targeting can
also be achieved. The fusion proteins can decrease inflammatory
responses at lower blood concentrations than is required for
comparable decreases using unfused heparinase. In addition, other
cells in the vascular system will be less affected by the enzyme
activity of the fusion protein, reducing possible side effects of
treatments.
[0054] Heparinase fusion proteins created by genetic engineering
retain the binding and catalytic properties of heparinase and of
the protein to which it is fused. Three heparinases have been
purified to homogeneity from Flavobacterium heparinum, and have
been produced in a recombinant form in Escherichia coli. Fusion
proteins consisting of heparinase enzyme combined with binding
domains from antibodies or adhesion molecules can be produced with
a gene fusion in a recombinant host, while retaining the
functionalities of binding and the enzymatic activity of the
separate proteins. These molecules can also be purified to
homogeneity by procedures normally used for purification of the
individual parts of the fusion protein (e.g. affinity
chromatography, heparinase purification protocols). Unlike the
natural heparinase purified from Flavobacterium heparinum, the
recombinant enzyme may not contain amino-terminal pyroglutamate or
carbohydrate moieties. All recombinant heparinase may contain
deletions, additional and/or altered amino acids, which modify the
enzymatic activity of the natural enzyme or the functioning of the
binding domains. Heparinase and fusion heparinase can be stabilized
for in vivo use, by complexing with polyethylene glycol, cross
linking agents, and by microencapsulation.
[0055] For example, the gene for heparinase I was isolated from F.
heparinum as described by Sasisekharan, et al., Proc. Natl. Acad.
Sci. 90:3660-3664, 1993, and an EcoR I restriction site was
inserted 5' to the codon encoding the glutamine-21 residue by
polymerase chain reaction. A fragment containing the heparinase I
gene was prepared by digestion with restriction endonucleases; EcoR
I and BamH I, and ligated to the EcoR I/BamH I cleaved pMALc2
plasmid (New England Biolabs). The resulting plasmid contained a
hybrid gene encoding a 82,000-85,000 Dalton protein incorporating
the maltose binding protein (Ma1B) fused 5' to the heparinase I
gene. This plasmid was inserted into Escherichia coli HB101 cells
using the calcium chloride mediated method described by Cohen et
al., Proc. Natl. Acad. Sci. 69:2110-211. These cells exhibited
heparinase activity under the control of the tac promoter, allowing
synthesis of the fusion protein by addition of 0.1 mM of the
inducing agent IPTG to the growth medium.
[0056] The HB101(pMALc2-HEP1Q21) cells were grown to a cell density
of 1.0 g/L dry cell weight in 500 ml, M9 medium containing 0.1 mM
IPTG at 37.infin.C. and concentrated by centrifugation, 10,000
g.times.10 minutes. The cell pellet was suspended in 10 ml 0.025 M
Tris, pH 7.7, and the cells disrupted by sonication using a Heat
Systems Model XL2020, 4.5 minutes, power level 3, 30 second on 30
second off cycles. Cell debris was removed by centrifugation,
10,000 g.times.10 minutes, and the supernatant applied to an
amylose affinity resin column (1.0 i.d..times. 2 cm, New England
Biolabs). The bound protein was eluted with a step gradient of
0.025 M Tris containing 0.01 M maltose at pH 7.5. The fusion
protein eluted in a protein peak which displayed a heparinase
specific activity of 23.77 IU/mg.
[0057] The heparinase-maltose binding fusion protein also can be
purified by standard protein separation techniques based on
heparinase properties. Cell sonicates were fractionated by ammonium
sulfate precipitation. Non-specific proteins were removed with a
precipitation step at 1.7 M ammonium sulfate and the supernatant
precipitated by raising the ammonium sulfate concentration to 3.2
M. The precipitated material contained the fusion protein and was
resuspended in 0.025 M sodium phosphate, pH 6.5. The material was
applied to a weak cation exchange column (1.6 i.d..times.10 cm,
CBX, J. T. Baker) and eluted with sequential step gradients of 0.0
M sodium chloride, 0.01 M sodium chloride, 0.25 M sodium chloride
and 1.0 M sodium chloride, all in 0.025 M sodium phosphate. The
fusion protein eluted in the 0.25 M sodium chloride elution
fraction and displayed a heparinase specific activity of 29.95
IU/ml. These two purification procedures demonstrate that
functional heparinase fusion proteins can be made by genetically
linking a protein with desired binding properties to the N-terminal
end of heparinase and the resulting fusion protein retains the
functionality of both heparinase and the protein to which it is
fused.
[0058] As another example of a fusion protein, a BamH I/Sal I
restriction fragment from pGBH3, which contains the heparinase III
gene from Flavobacterium heparinum was inserted into pMALc2 to form
a gene for fusion of a maltose binding protein with heparinase II.
Extracts of the E. coli strain DH5a containing the fusion gene
plasmid were produced as described in the last example, and these
extracts contained 18.7 IU/ml/O.D. of heparinase III activity. The
extract was also combined with amylose affinity resin and the resin
was then separated from the extract by centrifugation. The resin
was washed once with 0.025M Tris (pH 7.5) solution and proteins
bound to the resin were resuspended in SDS-PAGE sample buffer and
separated according to size on a 7.5% SDS-polyacrylamide gel.
Western blot analysis of the gel with anti-heparinase III specific
antibody identified a 116,000 Da. protein, which corresponds to the
expected size of the fusion protein. This analysis indicates that
the fusion protein has a functional maltose binding domain. This
example demonstrates that the heparinase III protein can also be
fused to a binding domain to produce a bifunctional fusion
enzyme.
Protection of Proteins In Vivo
[0059] Methods for extending the in vivo half-life are known and
routinely used, especially in the case of enzymes. One example of a
suitable method is the attachment of polyethylene glycol moieties
to the protein, which inhibits uptake by the reticuloendothelial
system. Preparation and characterization of such non-immunogenic
proteins is described by Lu, et al. (Pept. Res. 6(3), 140-146,
1993), Delgado, et al. (Critical Rev. Ther. Drug Carrier Syst.
9(3-4), 249-304, 1992) and Davis et al. (U.S. Pat. No. 4,179,337,
1979), the teachings of which are incorporated herein. Another
example of a suitable method is the use of bifunctional
cross-linking agents to stabilize the enzyme against proteolytic
degradation. Glutaraldehyde is one type of bifunctional
cross-linking agent. PCT WO95/00171, by Novo Nordisk A/S contains a
listing of other useful bifunctional cross-linking agents, and
teaches the use of these, which is incorporated herein.
Preparation of Pharmaceutical Compositions
[0060] Heparinase enzyme can be administered either locally or
systemically. Local administration can provide greater control.
Heparinase is mixed with an appropriate pharmaceutical or
veterinary carrier, then administered in an effective amount to
produce the desired effect on the treated cells using methods known
to those skilled in the art, for example, for local application,
use of perfusion, injection or a catheter.
[0061] Targeting and effective concentration dosages can be
achieved by preparation of targeted enzymes as described above, or
by the use of targeting vehicles, such as a catheter or localized
injection, to achieve controlled site specific delivery of
enzyme.
Administration of Enzymes via Controlled Release Matrices or
Injection
[0062] Heparinase enzyme can be formulated in a carrier for
administration by injection, for example, in saline or an aqueous
buffer, using standard methodology, or encapsulated in a polymeric
matrix. Encapsulation of heparinase in controlled release
formulations is well known; materials include but not limited to
liposomes, lipospheres, biodegradable polymeric matrices, and
vesicles. These encapsulants are typically microparticles having a
diameter from 60 nm to 100 microns, but preferably less than ten
microns, and more preferably one micron or less in diameter.
[0063] Proteosomes are prepared from outer membrane proteins of the
Meningococcal bacteria and been reported to bind proteins
containing hydrophobic anchors by Lowell, et al., Science, 240:800
(1988). Proteosome proteins are highly hydrophobic, reflecting
their role as transmembrane proteins and porins. When isolated,
their hydrophobic protein-protein interactions cause them to form
naturally multimolecular, membranous 60 to 1000 nm vesicles or
membrane vesicle fragments, depending on the strength of the
detergent used in their isolation. Heparinase can also be
encapsulated within a proteoliposome as described by Miller et al.,
J. Exp. Med. 176:1739-1744 (1992) and incorporated by reference
herein, as described above with reference to proteosomes.
Alternatively, heparinase can be encapsulated in lipid vesicles
such as Novasome.TM. lipid vesicles (Micro Vescular Systems, Inc.,
Nashua, N.H.). Another carrier is described in PCT US90/06590 by
Nova Pharmaceuticals, the teachings of which are incorporated
herein, which is referred to as a liposphere, having a solid core
and an outer shell layer formed of phospholipid.
[0064] The carrier may also be a polymeric delayed release system.
Biodegradable synthetic polymers are particularly useful to effect
the controlled release of heparinase. Microencapsulation has been
applied to the injection of microencapsulated pharmaceuticals to
give a controlled release. A number of factors contribute to the
selection of a particular polymer for microencapsulation. The
reproducibility of polymer synthesis and the microencapsulation
process, the cost of the microencapsulation materials and process,
the toxicological profile, the requirements for variable release
kinetics and the physicochemical compatibility of the polymer and
the antigens are all factors that must be considered. Examples of
useful polymers are polycarbonates, polyesters, polyurethanes,
polyorthoesters and polyamides, particularly those that are
biodegradable.
[0065] A frequent choice of a carrier for pharmaceuticals is poly
(d,l-lactide-co-glycolide) (PLGA). This is a biodegradable
polyester that has a long history of medical use in erodible
sutures, bone plates and other temporary prostheses, where it has
not exhibited any toxicity. A wide variety of pharmaceuticals
including peptides and antigens have been formulated into PLGA
microcapsules. The PLGA microencapsulation process uses a phase
separation of a water-in-oil emulsion. Heparinase is prepared as an
aqueous solution and the PLGA is dissolved in a suitable organic
solvents such as methylene chloride and ethyl acetate. These two
immiscible solutions are co-emulsified by high-speed stirring. A
non-solvent for the polymer is then added, causing precipitation of
the polymer around the aqueous droplets to form embryonic
microcapsules. The microcapsules are collected, and stabilized with
one of an assortment of agents (polyvinyl alcohol (PVA), gelatin,
alginates, polyvinylpyrrolidone (PVP), methyl cellulose) and the
solvent removed by either drying in vacuo or solvent extraction.
Other means for encapsulation include spray drying,
co-precipitation, and solvent extraction.
Means for Administration
[0066] Heparinase enzyme can be administered by injection, infusion
or perfusion. Typically, injection is performed using either a
syringe or catheter. Either a syringe or a catheter can be used to
apply heparinase locally to areas of blood vessels, tissues or
organs. Patients diagnosed with localized inflammations can be
treated by introduction of heparinase into their vascular system by
these means. Heparinase can also be administered before or
simultaneously with surgery, to reduce resulting inflammatory
responses. In addition, preceding transplant surgery, the donor
organ can be perfused with a heparinase preparation to reduce
inflammation upon reperfusion after transplantation.
[0067] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLES
Example 1: Treatment of Endothelial Cells with Heparinase to
Release Heparin/Heparan Sulfate
[0068] Heparinase alters the cell surface and basement membrane by
cleaving the heparin and heparan sulfate moieties from the cell
surface and extracellular matrix proteoglycans. Removal of these
glycosaminoglycans will decrease leukocyte-endothelium interactions
(leukocyte rolling) by decreasing binding of L-selectin on
leukocytes to endothelium proteoglycans. In addition, the removal
of heparin and heparan sulfate will decrease binding of chemokines
to the endothelium, which will reduce leukocyte activation,
sticking and extravasation. Production of bovine corneal
endothelial cells with 35S- heparin/heparan sulfate proteoglycans
and subsequent digestion of these radiolabeled proteoglycans with
Flavobacterium heparinase III provides a qualitative assessment of
the effect of the enzyme on the cell surface. Digestion of cell
surface proteoglycans with heparinase III will release both heparin
and heparan sulfate moieties, because these moieties are
interspersed on the same unbranched carbohydrate chains.
Significant quantities of .sup.35S- heparin/heparan sulfate were
solubilized by the heparinase III treatment.
[0069] .sup.35S-sulfate containing endothelial cell layers were
produced by seeding 24 well dishes with primary bovine corneal
endothelial cells. These were grown until 1 day prior to confluence
in DMEM with 10% fetal calf serum and 5% calf serum. One day prior
to confluence the cells were diluted 10- fold into Fisher medium
supplemented with 10% fetal calf serum, 5% calf serum, 4% dextran,
and 25 mCi/ml Na.sub.235SO.sub.4 and cultured for 3 days with the
addition of 0.5 ng/ml per day bFGF. Incorporation of label by
near-confluent cells localizes the label in and on the cell and
minimizes the .sup.35S-label incorporated into the basement
membrane.
[0070] Endothelial cell layers containing .sup.35S-sulfate were
treated with 600 ul phosphate buffered saline or heparinases III,
in phosphate buffered saline, at a concentration of 1.0 IU/ml, in
duplicate wells. The digestions were allowed to proceed for 5, 30
or 60 minutes at 37.infin.C. After the indicated time of digestion,
400 ul of digestion solution was removed from each well and
fractionated on a Bio-sil SEC 125-5 gel filtration column
controlled by a Beckman System Gold HPLC, equipped with an
autosampler. The flow rate was 1 ml/minute and 1 ml fractions were
collected. The amount of 35S- sulfate present in each fraction was
determined by measuring an aliquot of each fraction on a Packard
1600 TR liquid scintillation counter. The labeled, untreated
control solutions were fractionated and measured in the same
manner, and the quantity of radioactive material in each fraction
(background) was subtracted from the amount present in fractions
from the heparinase digested samples. The amount of cell surface
.sup.35S-labeled heparin/heparan sulfate in each fraction released
by heparinase III treatment is shown in FIG. 1.
Example 2: Determination of the Extent of Removal and the Rate of
Replacement of Heparin/Heparan Sulfate Moieties on Endothelial
Cells and in Basement Membranes Treated with Heparinase
[0071] The growth factor, basic Fibroblast Growth Factor (bFGF), is
a well characterized heparin binding protein, which is known to
bind to the heparin moieties of proteoglycans on the cell surface
and in the extracellular matrix (Maccarana, et al., J. Biol. Chem.,
268:23898-23905, 1993). Binding of .sup.125I-labeled bFGF to cell
surface and basement membrane proteoglycans was used to access the
amount of heparin/heparan sulfate removed from unactivated and
IL-1b activated endothelial cell layers and their basement
membranes by heparinases I, II or III. Digestion of the cell
surface and basement membrane with heparinase will remove both
heparin and heparan sulfate moieties, because these moieties are
interspersed on the same unbranched carbohydrate chains.
.sup.125I-labeled bFGF binding was also used in this experiment to
determine the rate at which heparin/heparan sulfate moieties were
replaced on the cell surface and basement membrane of endothelium
treated with heparinases I, II or III.
[0072] Most of the heparin/heparan sulfate can be removed using
heparinase (70 to 90%; FIGS. 2, 3 and 4). This demonstrates that
the endothelium can be almost completely depleted of
heparin/heparan sulfate moieties by use of heparinase. With a 1
hour heparinase treatment the rate of replacement of the
heparin/heparan sulfate is generally biphasic in nature.
Replacement of 40 to 50% of the digested heparin/heparan sulfate
occurs within a few hours. Additional replacement of the depleted
heparin/heparan sulfate occurs at a slower rate during the
remainder of the experiment, with complete replacement occurring 12
to 16 hours. 3 and 5 hour treatments with heparinases caused slower
rates of replacement of heparin/heparan sulfate on the cell surface
(FIGS. 3 and 4). This was most evident for unactivated endothelium.
For the experimental results depicted in FIGS. 3 and 4 for the IL-1
activated endothelium, all three treatment times (1, 3, and 5
hours) gave lower replacement rates than was observed in the
experimental results depicted in FIG. 2.
[0073] This data indicates that significant inhibition of
L-selectin binding and immobilized chemokine gradient formation
will result from heparinase treatment, and this would result in
significant inhibition of localized inflammatory responses. Also, a
treatment period of 3 to 5 hours can greatly extend the period of
diminished inflammatory response by decreasing the rate of
heparin/heparan sulfate replacement.
[0074] A human endothelial cell line was grown to confluency in 48
well dishes in 0.25 ml/well of RPMI medium, containing; 1%
penicillin /streptomycin, and 20% fetal serum. Cells in half of the
wells were activated for 4 hours with 10 ul of 50 ng/ml IL-1b.
After activation all wells were washed 3 times with HBSS and
treated with either Incubation Medium (RPMI medium, 25 mM Tris HCL
pH 8, 25 mM HEPES pH 7.4, and 0.1% BSA) or 0.1 IU/ml of heparinase
I, II or III diluted in Incubation Medium, at 37.degree. C., in 5%
CO.sub.2, for 60 min for experimental results depicted in FIG. 2,
or for the results depicted in FIGS. 3 and 4, 1.0 IU/ml heparinase
I or III diluted in Incubation Medium, at 37.degree. C., in 5%
CO.sub.2, for 60 min with none, 3 or 5 replacements of the enzymes
every hour. After enzyme treatments, the wells were washed 3 times
with HBSS and incubated with RPMI medium, containing; 1% penicillin
/streptomycin and 20% fetal serum, .+-.IL-1b, at 37.degree. C., in
5% CO.sub.2, for the times indicated in FIG. 2. The wells were
again washed 3 times with HBSS and 0.1 ml of Incubation Medium was
added to each well, and the plates were cooled on ice for 5 min. To
all of the wells was added 20 ul of 125 ng/ml .sup.125I-bFGF, and
20 ul of 20 ug/ml unlabeled bFGF. To some of the control wells, 15
ul of 10 mg/ml heparin was also added, to determine nonspecific
background binding. The plates were incubated on ice for 40 minutes
and washed 2 times with cold HBSS. 0.25 ml of LAB (25 mM HEPES pH
7.4 and 2 M NaCl) was added to each well to solubilize the bFGF,
and then it was collected in tubes. This step was repeated, and the
contents of the tubes were counted in a gamma counter. The amount
of nonspecific background binding was subtracted from each
untreated control and treated sample. The sample counts were
divided by the control counts to determine the percent of binding
that occurred. The results.+-.standard errors (SE) are shown in
FIGS. 2, 3 and 4.
Example 3: Treatment of Activated Endothelial Cell Layers and
Basement Membranes with Heparinase to Release Heparin/Heparan
Sulfate Bound Chemokine, IL-8
[0075] Removal of the bound chemokine gradient formed by activated
endothelium adjacent to inflamed tissue will inhibit the
accumulation of neutrophils within this tissue, and will decrease
the inflammatory response. The chemokine, IL-8, is produced by
endothelium activated by IL-1b and other cytokines and
chemoattractants, which are secreted by inflamed tissues. If IL-8
bound to endothelium can be solubilized by treatment with
heparinase, then it would be removed from the area of inflammation
by blood flow and the localized inflammatory response would be
inhibited. The in vitro removal and solubilization of 0.5 to 3 fold
more endogenous, immobilized IL-8 (vs. secreted IL-8) from
activated endothelium by heparinases I, II or III, or by
heparinases I and III demonstrates that the bound chemokine
gradient can be destroyed by heparinase treatment.
[0076] One ml of a 3 mg/ml solution of human collagen was used to
coat the wells of a 12 well plate. Any remaining collagen solution
was removed by aspiration. Human umbilical venous endothelial cells
(HUVEC; used at passages 1 to 8) from a confluent 10 ml plate were
trypsin treated, and diluted 1 to 7 in RPMI medium containing; 20%
fetal serum, 1% penicillin /streptomycin, 100 ug/ml heparin, 10
ug/ml of epidermal growth factor and 200 ug/ml of endothelial cell
growth supplement. One ml of diluted cells was added to each well
of the collagen coated 12 well plates. The cells were grown until
confluent, at 37.degree. C., in 5% CO.sub.2. The culture medium was
changed every other day during the growth period. The day before
the chemokine assay the medium was exchanged for 1 ml of RPMI
medium without heparin.
[0077] To activate the endothelium layer, 50 ng/ml of human
recombinant IL-1b, diluted in RPMI medium (minus fetal serum,
epidermal growth factor, endothelial cell growth supplement and
heparin) and 2% BSA was added to non-control wells to a final
concentration of 2 ng/ml. The multiwell plates were incubated at
37.degree. C., in 5% CO.sub.2, for 4 hours. The medium was removed
from all of the wells and the wells were washed two times with
Hanks Balanced Salt Solution (HBSS). 0.5 ml of RPMI medium (minus
fetal serum, epidermal growth factor, endothelial cell growth
supplement and heparin) and 2% BSA was added to the wells for the
times indicated in FIG. 3. After the indicated times, the wells
were emptied and washed once with 1 ml of HBSS. 0.5 ml of HBSS with
or without 1 IU/ml of heparinase I, II or III; or heparinases I and
III were added to the wells. The plates were incubated at
37.degree. C. on a heat block for 15 minutes with occasional
agitation. After 15 minutes, the supernatants were collected and
assayed for IL-8. An enzyme-linked immunosorbent assay (ELISA)
system from Perseptive Diagnostics was used to determine IL-8
concentrations in the supernatants. The manufacturer's recommended
protocol was followed. 90 ul of supernatant and 10 ul of 5M sodium
chloride were used in each well of the ELISA plate. Each washing
step utilized three repeats of 150 ul of washing solution per well,
with 2-3 minutes of agitation between each repeat. The percent
difference in the IL-8 concentration of supernatants from; IL-1b
induced heparinase I, II or III treated endothelium, versus IL-1b
induced non-treated endothelium are shown in FIG. 5. In addition,
the IL-8 concentrations in the supernatants from IL-1b induced
heparinase I, II or III treated endothelium are shown in FIG.
5.
Example 4: Treatment of Endothelial Cell Layers with Heparinase to
Inhibit Neutrophil Adhesion
[0078] In order to concentrate neutrophils at a site of
inflammation, endothelial cell surface molecules activate rolling
neutrophils for tight binding to the walls of postcapillary
venules. Chemokines bound to heparin/heparan sulfate have been
identified as important signal molecules for activation of
neutrophils for tight binding in the microcapillaries. The in vitro
neutrophil adhesion assay system described below is commonly used
to analyze conditions affecting neutrophil adhesion to endothelial
cells. Treatment of the activated human endothelial cell layers
used in this assay with either heparinase I, II or III resulted in
significant reductions in the level of neutrophil adhesion to the
endothelium. These results demonstrate that heparinase treatment of
the vasculature would inhibit localized neutrophil accumulation in
the microcapillaries, and would inhibit inflammatory responses.
Isolation of Human Neutrophils
[0079] 25 ml of venous blood was drawn from a healthy donor into
{fraction (1/10)} volume of 0.1M sodium citrate, pH 7.4, and was
diluted with 25 ml of Dulbecco's phosphate-buffered saline
containing calcium chloride and magnesium chloride (D-PBS). 10 ml
aliquots of diluted blood were layered on 10 ml of Ficoll-Paque in
50 ml tubes. The tubes were centrifuged at 400 x g for 30 minutes
at 20.degree. C., and were allowed to stop, without braking. The
upper layers were removed and the pellets were resuspended in 3
volumes of a solution of 155 mM NH.sub.4Cl, 10 mM KHCO.sub.3 and
0.1 mM EDTA, pH 7.4 at 4.degree. C., to lyse the erythrocytes. The
tubes were inverted after a few minutes and the contents turn black
after 7 to 8 minutes. After 10 minutes, the tubes were centrifuged
again at 400 x g for 5 minutes at 4.degree. C. The supernatants
were aspirated and the pellets were resuspended in the NH.sub.4Cl
solution containing 0.5% human albumin. The suspensions were pooled
and the volume was brought to 50 ml. The cell suspension was
incubated on ice for 15 minutes and centrifuged at 400 x g for 5
minutes at 4.degree. C. The supernatant was removed and if the
pellet was still red, it was washed again with NH.sub.4Cl solution
containing 0.5% human albumin. Finally, the cells were resuspended
in D-PBS with 5 mg/ml human albumin and refrigerated, until needed.
A 10 .mu.l aliquot of suspension was diluted with 10 .mu.l of
trypan blue and the cells were counted on a hemacytometer to
determine the number of viable cells per volume of suspension.
Labeling of Neutrophils
[0080] A suspension of 10.times.106 neutrophils was made in 2 ml of
PBS (no Ca, no Mg) with 5 mg/ml human albumin. BCECF-AM (Molecular
Probes) was added to the suspension for a final concentration of 46
uM. The neutrophils were incubated in a water bath at a set
temperature of 37.degree. C. for 30 minutes after which they were
centrifuged and rinsed twice with PBS (no Ca, no Mg) with 5 mg/ml
human albumin. They were finally resuspended in 10 ml of RPMI+20%
bovine fetal serum at 37.degree. C.
Treatment of Endothelial Cells with Heparinases
[0081] HUVEC at passage number 3 were trypsinized and counted. The
cells were plated in RPMI+20 % bovine fetal serum+95 ug/ml
heparin+ECGS+EGF at 5.times.104 cells per well in 96 well plates.
They were grown at 37.degree. C., 5% CO2 for 18 hours. At that
point the growth medium was replaced with RPMI+20% bovine fetal
serum ( 2 ng/ml IL-1b for 4 hours. The cells were then rinsed with
HBSS and treated with heparinase I, II or III at 0.1 IU/ml in HBSS
for 1 hour at 37.degree. C., 5% CO2.
Neutrophil Adhesion Assay
[0082] After the treatment period, HUVEC were rinsed once with
HBSS. 200 ul of the neutrophil suspension (which corresponds to
200,000 neutrophils) was added to each well of treated or control
HUVEC. The plate was put at 37.degree. C., 5% CO2 for 30 minutes.
The adhesion period was stopped by centrifuging the plate upside
down at 250 x g for 5 minutes. 200 ul of PBS (no Ca, no Mg)+5 mg/ml
human albumin was added to each well and the plate was read with a
Fluorolite 1000 fluorescence plate reader at a voltage of 2.5 V.
The emission filter was at 535 nm.+-.35 and the excitation filter
at 485.+-.22.
Analysis of Data
[0083] A standard curve was generated by putting known amounts of
BCECF-AM-stained neutrophils, resuspended in PBS (no Ca, no Mg)+5
mg/ml human albumin in wells of another 96 well plate which
contained confluent HUVEC layers. The fluorescence units were
plotted against the quantity of neutrophils and a slope was
calculated. The standard curve was used to determine the number of
bound neutrophils in the control and heparinase treated wells. The
percent differences between IL-1b activated HUVEC layers and
comparable HUVEC layers treated with heparinases I, II and III are
shown in FIG. 6.
Example 5: Treatment of Endothelial Cell Layers and Basement
Membranes with Heparinase to Inhibit Neutrophil Extravasation
[0084] Leukocytes from the blood accumulate in inflamed tissues by
transmigration across the adjacent endothelium (extravasation). The
endothelial cell layer is activated by the inflamed tissue (via
cytokines and chemoattractants), and the affected endothelium
directs and localizes the accumulation of leukocytes in the
inflamed tissue. In order to activate leukocytes for extravasation,
and to direct the migration of the leukocytes into the inflamed
tissue, the activated endothelium forms an immobilized chemokine
gradient on its cell surface and in its basement membrane. The in
vitro neutrophil transmigration assay system described below is
commonly used to analyze conditions affecting neutrophil
extravasation. Treatment of the activated human endothelial cell
layers used in this system with either heparinase I, II or III
resulted in significant reductions in the level of neutrophil
migration across the endothelium. These results demonstrate that
heparinase treatment of the vasculature would inhibit localized
neutrophil accumulation and inflammatory responses.
Assay of Neutrophil Extravasation
[0085] Neutrophils were isolated as described in example 4. Human
fibronectin was dissolved at 0.4 mg/ml in RPMI medium without
serum. Filter inserts (6.25 mm) of pore size, 3 .mu.m or 8 .mu.m,
were coated with 4 .mu.g/cm2 of human fibronectin for one hour, at
room temperature, and were rinsed with distilled water. Wells of a
24 well plate were filled with 0.3 ml of RPMI medium with 20% fetal
bovine serum, 95 .mu.g/ml heparin, 200 .mu.g/ml ECGS and 10 ng/ml
EGF. The coated filter inserts were seated in the wells, and
8.times.104 human umbilical venous endothelial cells (HUVEC; used
at passages 1 to 7) in 0.3 ml of complete RPMI medium, were plated
on the coated filter inserts. The cells were allowed to grow for 48
to 65 hours, at 37.degree. C., in 5% CO.sub.2. The culture medium
from the filter inserts and the wells was changed once during the
growth period for RPMI medium lacking heparin. After the growth
period, the culture medium underneath all inserts, except negative
control inserts, was removed and replaced with fresh culture medium
lacking heparin and growth factors, but containing 2 ng/ml of human
recombinant IL-1b. The culture medium under negative control
inserts was replaced with fresh culture medium. The multiwell
plates were incubated at 37.degree. C., in 5% CO.sub.2, for 4
hours. The medium was removed from the inserts and wells and the
cells were rinsed once with Hank's Balanced Salt Solution (HBSS).
The filters and wells were filled with 0.3 ml of a solution of
HBSS; treated inserts received HBSS containing heparinase I, II or
III instead of HBSS. This treatment was performed for the times
indicated in FIG. 1, at 37.degree. C., in 5% CO.sub.2. The solution
was removed and the cells were rinsed once with HBSS. 0.3 ml of
fresh culture medium without heparin and growth factors was added
to the wells and 1.5.times.106 of freshly prepared human
neutrophils in 0.3 ml of culture medium without heparin and growth
factors were added to all of the inserts. The plates were incubated
at 37.degree. C., in 5% CO.sub.2, for the times indicated in FIG.
3. After this time, the inserts were removed and the bottoms were
rinsed once with 0.3 ml D-PBS. The contents of the well were
removed and the well was washed with 0.3 ml of D-PBS. The rinses
were added to the well contents and the combined contents were
brought to a 1 ml volume. These samples were frozen for up to 16
hours before assaying for myeloperoxidase activity.
Myeloperoxidase Assay
[0086] Standards containing between 1.times.105 and 1.times.106
neutrophils in a 1 ml volume were produced by diluting an aliquot
of the neutrophil suspension in D-PBS. 4 ml of 50 mM potassium
phosphate, pH 6.7, containing 0.5% hexadecyltrimethylammonium
bromide and 0.5% triton was added to each standard and thawed
sample. 0.1 ml of each sample or standard was added to a plastic
cuvette. 2.9 ml of 50 mM phosphate buffer, pH 6.0 containing 0.167
mg/ml o-dianisidine hydrochloride and 0.0005% hydrogen peroxide
were added to the cuvettes. The change in absorbance at 460 nm was
monitored every 30 seconds for 3 minutes by use of a
spectrophotometer.
[0087] The rate changes obtained from the standards were used to
produce a curve of the rate of increase in absorbance versus
numbers of neutrophils. This curve was used to quantitate the
number of neutrophils in each sample, which had migrated through,
either a treated, or an untreated endothelial layer. The number of
migrating neutrophils was divided by 1.5.times.106 to determine the
percentage of neutrophil migration.
Analysis of Data
[0088] The effectiveness of the heparinase treatment in this assay
depended on the extent to which the HUVEC were covering the filter
surface. The extent of coverage was based on dye exclusion analysis
performed after an extravasation experiment, and it varied somewhat
from filter to filter in a single experiment. Generally, if the
filter was densely covered with a tightly packed HUVEC layer, the
percentage of extravasating neutrophils was low (<10%), and the
differences between treated and untreated wells were not
statistically significant (large well to well variability). If
large areas of the filter were not covered with a HUVEC layer
(estimated at 30-40% of the filter) large numbers of neutrophils
(40-60%) would migrate through the filter, but a 1 hour heparinase
treatment would not be as effective in inhibiting the migration.
This migration is not comparable to extravasation, which can be
functionally defined as neutrophils squeezing between neighboring
endothelial cells. If 75 to 90% of the filter was covered with
HUVEC, generally, 15 to 30% of the neutrophils extravasated, and
the 1 hour heparinase treatment was found to be most effective
under these conditions.
[0089] Using the Student's t-test, experiments were analyzed to
determine if a significant effect for the 1 hr heparinase
treatments had been observed (P<0.05). The data from these
experiments was combined and is displayed in FIG. 7. The data for
the 15 minute heparinase treatments shown in FIG. 7 is derived from
the same experiments as the 1 hour treatments. The
Student-Newman-Keuls test was used to determine the significance of
the differences (P<0.05) between different treatments with the
same enzyme. Significant differences are indicated by asterisks (*)
in FIG. 7.
Example 6: Treatment of Endothelial Cell Layers and Basement
Membranes with Human Heparinase (b-thromboglobulin) to Inhibit
Neutrophil Extravasation
[0090] Commercial preparations of b-thromboglobulin are a mixture
of the chemokines CTAP-III and NAP-2. At non-physiological pH (pH
5.8-6), these chemokines have heparinase activity, while at pH 7,
they bind heparin and act as chemotactic cytokines for leukocytes.
The heparinase activity of commercial b-thromboglobulin
preparations were analyzed at pH 5.8 and pH 7, by digestion of
radioactive 35SO.sub.4-labeled ECM. These preparations showed
significant human heparinase activity only at pH 5.8. They were
then used in the in vitro extravasation assay system described in
example 5, in order to determine if the human heparinase activity
could prevent extravasation of neutrophils across an endothelial
cell layer. The treatment of activated human endothelial cell
layers with human heparinase (b-thromboglobulin) resulted in a
significant reduction in neutrophil extravasation. These results
demonstrate that heparinase treatment of the vasculature with human
heparinase would inhibit localized neutrophil accumulation and
inflammatory responses.
Activity of b-thromboglobulin on Labeled Matrix
[0091] Commercial b-thromboglobulin was tested for heparinase
activity at pH 5.8 and pH 7 by release of 35SO.sub.4-labeled
heparin/heparan sulfate from ECM. Bovine corneal endothelial cells
at passage 1 to 8 were split 1:10 form a confluent plate and seeded
in 4 well plates in DMEM low glucose with 10% fetal bovine serum,
5% calf serum and 4% Dextran added. The dishes received 1 ng/ml
bFGF 3 times a week prior to reaching confluency. Just prior to
reaching confluency, Na.sub.235SO.sub.4 in H.sub.2O diluted to 1
mCi/ml with DMEM low glucose was added to the cells at a final
concentration of 25 uCi/ml, in Fisher medium with 10% fetal bovine
serum, 5% calf serum and 4% Dextran. 3 to 4 days later, the label
was given again. The plates were left undisturbed 12 to 14 days
post confluency. To harvest the matrix, the medium was removed and
replaced with a solution of 0.5% Triton, 0.02M NH.sub.4OH in PBS
(no Ca, no Mg). This solution was removed and the matrix was washed
3 times with PBS (no Ca, no Mg). The plates were stored at
4.degree. C., covered with PBS. These matrices were used within a
year.
[0092] b-thromboglobulin from Wellmark (product #41705) or
Calbiochem (product #605165) were used for digestion of the labeled
ECM. 100 ug of enzyme was dissolved in 1 ml of water (Wellmark) or
PBS (Calbiochem). A further dilution was done in PBS at pH 5.8 or
7. The matrices were covered with 250 ul of PBS alone or PBS with 1
or 5 ug of .beta.-thromboglobulin. The matrices were incubated in a
CO.sub.2 incubator for 3 hours. Aliquots of 100 ul were taken from
every well and counted. The amount of radioactivity released from
each enzyme-treated matrix was compared to an untreated matrix, and
the results are displayed in FIG. 8.
Migration of Neutrophils Across HUVEC Treated with
.beta.-thromboglobulin
[0093] HUVEC were grown on filter inserts and activated as
described in example 5. Five ug of .beta.-thromboglobulin in PBS at
pH 5.8 or PBS alone were applied to the HUVEC layer for one hour.
Neutrophils were added above the filters and the number of
extravasating neutrophils were quantitated as described in example
5. The effect of the human heparinase treatments on neutrophil
extravasation are shown in FIG. 9.
Example 7: Treatment of Rats with Heparinase Inhibits
Leukocyte-Endothelial Cell Interactions Following
Ischemia/Reperfusion
[0094] This example illustrates the effect of heparinase III on
leukocyte behavior in vivo. Three key mechanisms of leukocyte
accumulation in inflamed tissues; leukocyte rolling, sticking, and
extravasation, were analyzed in rat skeletal muscle
microvasculature following ischemia. Pretreating the vasculature
with heparinase III prior to ischemia, and maintaining the plasma
concentration of heparinase III constant during reperfusion was
found to significantly decrease leukocyte rolling, sticking and
extravasation. This example demonstrates that heparinase treatment
of vasculature would inhibit neutrophil accumulation in
microcapillaries and in the surrounding tissues. In vivo heparinase
treatment would result in decreased inflammatory responses. As also
demonstrated by this example, heparinase treatment significantly
increased microvascular perfusion within reperfused muscle
following ischemia. In addition to decreased neutrophil
accumulation, increased microvascular perfusion would positively
affect the recovery of muscle tissue and positively affect the
outcome of ischemia/reperfusion (i.e. inflammatory) events.
General Comments
[0095] In order to establish a plasma level of approximately 1
IU/ml, we undertook pilot studies to test the effect of infusing
Heparinase over the 5 hour period of time required for our in vivo
studies. A previous publication on the leukocyte behavior following
3 hr ischemia (Forbes et al., 1996, Microvascular Research
51:275-287) contains data on naive and sham treated rats. Because
the methods and timing were the same for this previous study and
the present study of 3 hr ischemia, the effect of heparinase
treatment will be compared to the naive and sham results obtained
from the previously published results. For the 2 hr ischemia
protocol, additional naive and sham treated rats were analyzed.
[0096] In order to investigate directly the effect of heparinase,
in vivo, we applied intravital video microscopy to the extensor
digitorum longus muscle in the rat hind limb. This analysis
occurred during a period of 105 or 90 minutes of reperfusion, which
followed 2 or 3 hours, respectively, of no-flow ischemia. Such
periods of ischemia/reperfusion (I-R) result in an inflammatory
response in skeletal muscle sufficient to cause increased
leukocyte-endothelial cell interactions.
Methods
[0097] Male Wistar rats weighing 225 to 250 gm were anesthetized by
inhalation of halothane (1%-1.5%) and the carotid artery and
jugular vein cannulated to monitor blood pressure and permit
infusion of fluids, respectively. The extensor digitorum longus
(EDL) muscle in the rat hind limb was prepared for intravital
microscopy. Briefly, with the anaesthetized rat lying on the stage
of the microscope, the EDL muscle was exposed by reflection of the
overlying skin and separation of the tibialis anterior and
gastrocnemius muscles. A suture was tied around the distal tendon
of the muscle allowing reflection of the muscle into a saline bath
positioned on the microscope stage. Normal muscle and body
temperatures were maintained (i.e., muscle at 32.degree. C.; body
at 37.degree. C.) by heat lamps. The muscle was covered by a glass
cover slip and all exposed tissue covered with Saran wrap to
prevent dehydration. Following the preparation of the EDL muscle
for intravital video microscopy, and a 30 minute period of
recovery, to allow microvascular blood flow to return to normal
following the hyperaemia induced by the preparatory methods, 1-2
fields of view each containing two or more postcapillary venules
were chosen . These fields of view were used throughout the
experiment so that temporal changes in leukocyte flow behavior
could be measured. One minute recordings of these fields of view
were made using low magnification to provide information regarding
RBC flow within individual capillaries. Following the low
magnification recordings, views of the postcapillary venules were
recorded for 1 min at high magnification. The video recording of
such views allows for the "off-line" analysis of microcirculatory
parameters. Thus, control values of the density of perfused
capillaries and the flow behavior of leukocytes (number of stuck,
rolling and extravasating per unit area) were measured.
[0098] Plasma levels of heparinase III are measured using a
heparinase III ELISA. The heparinase III ELISA is a quantitative
two-antibody sandwich assay. Affinity purified anti-heparinase III
rabbit antibodies are coated onto a microtiter plate. Wells are
washed and incubated for 2 hours at 37(C with blocking buffer (TBS,
1% BSA+1% Tween 20). After 3 washes, standards and samples are
added to the wells and any heparinase III present is bound by the
immobilized antibody. Any unbound substance is then washed away and
biotin labeled anti- heparinase III rabbit antibodies are added to
the wells. Excess antibodies are removed by washing.
Peroxidase-conjugated streptavidin is added and binds to any biotin
complex present in the well. After washing away any unreacted
streptavidin, a substrate solution containing hydrogen peroxide and
3, 3', 5, 5' tetramethylbenzidine in aqueous DMF is added to the
wells, according to the procedure described for the TMB Peroxidase
EIA Substrate Kit (Biorad, CA), and color develops in proportion of
the amount of heparinase III in the sample. A 1 N H.sub.2SO.sub.4
solution stops the reaction and the absorbance is measure at 450
nm.
Specific Protocol
[0099] A series of rats were infused with heparinase III via the
venous catheter at a rate of 3 ml/hr for 5 hr. to maintain a
heparinase level of 1.0 IU/ml in the blood. Through such pilot
studies it was determined that 0.33 IU/gm body weight/hr was
adequate for this purpose.
[0100] Microvascular blood flow and leukocyte behavior was recorded
every 15 min for a total of 90 to 105 min of reperfusion. Blood
samples were taken before the administration of heparinase and
during reperfusion to ensure correct plasma concentrations of
heparinase.
Statistical Analysis
[0101] In all cases means are expressed with their standard error
of estimate. Comparisons were made using analysis of variance
(ANOVA) followed where appropriate by Scheff tests as ad hoc
analysis. Significance was assumed at p>0.05.
Results
[0102] A plasma concentration of approximately 1 IU/ml ( adjusted
for activity) was achieved during infusion, and in spite of a trend
toward increased plasma levels during the last 2 hours of infusion,
the plasma concentration remained constant (FIG. 10). Prolonged
infusion of heparinase III appeared to have no adverse side-effects
at least in terms of blood pressure, or rate of respiration.
[0103] Immediately following 3 hr of ischemia, the number of
rolling leukocytes (Lr) significantly increased in sham I-R rats
(14.77.+-.1.33), compared to naive (no I-R) rats (5.66.+-.0.11).
The average number of rolling leukocytes remained constant at these
levels during reperfusion in both the sham and naive rats for the
duration of the 90 minute reperfusion period (FIG. 3). In spite of
3 hr ischemia, no change in leukocyte rolling within postcapillary
venules of heparinase III treated rats was measured. In fact, the
average number of rolling leukocytes following heparinase treatment
(2.81.+-.0.64) tended to be less than in naive rats over the 90
minute observation period (FIG. 11).
[0104] The number of leukocytes stuck to the wall of postcapillary
venules (Ls) progressively increased in sham I-R rats, reaching a
constant level (11.96.+-.0.01) within 45 minutes of the release of
the tourniquet (3 hr ischemia). However, the number of stuck
leukocytes in heparinase III treated rats showed no change
following ischemia, and was not significantly different from naive
rats (FIG. 12).
[0105] As would be expected based on the low numbers of sticking
leukocytes following heparinase III treatment, the number of
extravasated leukocytes (Le) did not change during reperfusion
after 3 hr of ischemia (FIG. 13). Le for sham and naive rats are
not available for the 3 hr ischemia protocol. For a 2 hr ischemia
protocol, the Le for the heparinase III treated rats was higher
than for the naive rats, but was significantly lower than the Le in
the sham treated animals. This data is displayed in FIG. 14, as the
percent difference in the Le for heparinase and sham treated rats
vs. naive rats.
[0106] Following release of the tourniquet after 3 hr of ischemia,
a significant decline in microvascular perfusion (CDper) occurred
in both the sham and heparinase III treated rats, compared to
perfusion measured in naive rats. However, unlike the perfusion in
sham rats microvascular perfusion in heparinase III treated muscles
returned to normal within 30 minutes of the release of the
tourniquet (FIG. 15).
Example 8: Cardioprotective Effects of Heparinase III in a Rabbit
Preparation of Ischemia/Reperfusion Injury
Introduction
[0107] Ischemia produces a significant degree of damage at the
level of myocytes and endothelial cells within the coronary
vascular bed; this can lead to extravasation of plasma and other
blood and cellular components into the interstitial space.
Polymorphonuclear leukocytes can migrate through the endothelial
cell layer; this migration of neutrophils across the connective
tissue barrier is dependent on the actions of neutrophil-derived
proteolytic enzymes even in the presence of plasma antiproteases.
Restoration of blood flow allows rapid access of inflammatory cells
to jeopardized myocardium. Neutrophil adhesion to endothelial cells
is stimulated by endotoxin IL-1b (activates vascular endothelium to
produce adherence molecules for leukocytes) or tumor necrosis
factor. A number of studies have explored the possibility of using
various pharmacologic interventions including monoclonal antibodies
to prevent neutrophil adhesion to vascular or myocardial cells
particularly during the reperfusion phase. Interfering with
neutrophil-cellular interactions (neutrophil rolling, adhesion and
extravasation) has been shown to significantly reduce the extent of
cellular injury following ischemia-reperfusion. This suggests that
inflammatory cells play an important role in the pathophysiology of
ischemia-induced cellular injury; inflammatory cells may also play
a role in extending myocyte injury beyond that which occurs during
the ischemic insult (i.e., reperfusion injury). Because heparinase
treatment is effective for inhibition of neutrophil-endothelium
interactions (see examples above), prevention of reperfusion injury
to rabbit myocardium by treatment with heparinase was investigated
in the experiments described in this example. Heparinase III was
found to attenuate the extent of tissue necrosis when administered
at a target dose of 25 ug/ml either before the onset of coronary
occlusion or at the onset of reperfusion. Lower dosages of
heparinase III did not provide cardioprotection in this animal
preparation of ischemia-reperfusion injury; however, at a target
dose of 5 ug/ml there was a trend toward reduced infarct size.
Protection was obtained without significant changes in cardiac
hemodynamics or transmural blood flow distribution.
[0108] When an inflammatory response is the result of an ischemic
episode as in the non-limiting examples of heart attack and stroke,
this example demonstrates that heparinase treatment before or at
the time of the inflammatory event can reduce tissue injury. In
addition, this example indicates that tissue damage resulting from
leukocyte accumulation during any inflammatory event can be reduced
by heparinase treatment or pretreatment.
Methods
[0109] Male New Zealand White rabbits (2.2-3.0 Kg body weight) were
used for these studies. Rabbits were cared for in accordance with
the Guide to the Care and Use of Experimental Animals (vol. 1 and
2) of the Canadian Council on Animal Care. They were premedicated
with intramuscular acepromazine maleate (5 mg/Kg; Austin
Laboratories) and anesthetized with pentobarbital sodium (25 mg/Kg;
i.v.; MTC Pharmaceuticals). Additional anesthetic was administered
hourly. The trachea was cannulated and rabbits were mechanically
ventilated with room air. The right jugular vein was cannulated for
administration of drugs (Heparinase III or vehicle); the left
jugular vein was cannulated to permit withdrawal of blood for
determinations of plasma heparinase levels. A cannula (PE-90) was
placed in the left carotid artery for withdrawal of reference
arterial blood during injection of radiolabeled microspheres.
[0110] The heart was exposed via a left thoracotomy and a snare
(4-0 silk) placed around the first anterolateral branch of the left
circumflex coronary artery midway between the atrioventricular
groove and the apex. The silk suture was passed through a length of
Tygon tubing to provide a snare for coronary occlusion. Left
ventricular chamber pressure was obtained with a fluid-filled
catheter positioned via the apex. Cardiac hemodynamics were allowed
to stabilize for 20 minutes.
[0111] Regional myocardial ischemia was induced by pulling the
suture through the plastic tubing and clamping with a mosquito
hemostat. Ischemia was verified visually by the appearance of
regional epicardial cyanosis and ST segment elevation on the
electrocardiogram (Lead II). In hearts that developed ventricular
fibrillation normal sinus rhythm was restored by gentle flicking of
the LV (electrical cardioversion was not used in these
experiments); hearts that could not be cardioverted after two
attempts were excluded from the data analysis. Lead II
electrocardiogram and LV pressure were recorded throughout the
experiments with a Gould (TA240) EasyGraph 4-channel physiograph
recorder (Interfax Inc., Montreal, Quebec).
Experimental Protocol
[0112] Rabbits were assigned to seven different treatment groups;
Group 1 rabbits were given saline (i.v.) for 60 minutes prior to
onset of ischemia; Group 2 rabbits were given saline (i.v.) at the
onset of coronary reperfusion; Group 3 rabbits were given
heparinase III (25 ug/ml target level, i.v.) for 60 minutes prior
to onset of ischemia; Group 4 rabbits were given heparinase III (25
ug/ml target level, i.v.) at the onset of coronary reperfusion;
Groups 5, 6 and 7 rabbits were given target levels of either 0.25,
1.25, or 5.0 (g/ml heparinase III, respectively at the onset of
coronary reperfusion. Drug or saline was infused intravenously (4.0
ml/hr) for 60 minutes prior to onset of myocardial ischemia in two
treatment groups (Groups 1 and 3) and then continuously for 2 hours
using a Harvard infusion pump (Ealing Scientific, Montreal,
Canada). In the remaining experimental groups vehicle or drug
infusion was initiated at the time of reperfusion and continued for
3 hours during reperfusion. Rabbits were assigned to a particular
group by rotating drug treatment on succeeding experiments through
the seven treatment protocols. All rabbits were subjected to 45
minutes of regional coronary occlusion followed by 180 minutes of
reperfusion.
Plasma Heparinase III Determinations
[0113] Blood samples were obtained from the right jugular vein at
baseline, 30 and 60 minutes after vehicle or drug infusion in Group
1 and 3 rabbits; blood was also obtained at 15, 30, 60, 120 and 180
minutes of coronary reperfusion. In the remaining experimental
groups blood was obtained at baseline and 15, 30, 60, 120 and 180
minutes of coronary reperfusion. Blood samples were centrifuged for
15 minutes at 1500 rpm at 4.degree. C.; plasma was frozen and
stored at -20.degree. C. for later analysis. Heparinase III plasma
levels were determined as described in example
7. Transmural Blood Flow
[0114] Blood flow to ischemic and non-ischemic vascular beds was
measured using radiolabeled microspheres (.+-.15 um; NEN, Boston,
MA) using the reference withdrawal technique. For each blood flow
measurement, 0.4-0.6.times.106 microspheres labeled with either
113Sn, 46Sc, or 85Sr (agitated mechanically with a vortex mixer
immediately before use) were injected into the left atrium under
hemodynamic steady-state conditions followed by two flushes with 3
ml warmed saline (injection of microspheres into the left atrium
ensures adequate mixing in the LV chamber and prevents streaming
artifacts which occur with direct injections of microspheres into
the coronary circulation). Reference arterial blood samples from
the carotid artery were collected beginning 10 seconds before the
injection of microspheres and continuing for 2 minutes thereafter
at a rate of 2.6 ml/min. Myocardial blood flow distribution was
assessed at; 1-baseline, 2-30 minutes after onset of coronary
reperfusion and 3-180 minutes coronary reperfusion. Tissue and
reference blood radioactivity is measured using a multichannel
pulse-height analyzer (Cobra II, Canberra Packard) with correction
for overlap of isotope spectra.
Analysis of Infarct Size
[0115] At the end of the experimental protocol, hearts were
arrested in diastole by intravenous injection of 10 ml of saturated
potassium chloride, quickly extirpated, rinsed in saline and
cannulated via the aorta on a Langendorff perfusion apparatus.
Hearts were perfused ex vivo via the aorta at 75 mm Hg with
2,3,5-triphenyltetrazolium chloride at 37.degree. C. for 20
minutes. Subsequently, the arterial suture was re-tied and
Monastral Blue was injected retrogradely via the aortic cannula to
allow delineation of the anatomic risk zone. Hearts were then
removed from the perfusion apparatus, the atria and right ventricle
were trimmed away, and the left ventricle was weighed and fixed by
immersion in buffered 10% formalin.
Post-mortem Studies
[0116] The principal end-point of this study was the effect of drug
treatment on infarct size (normalized to anatomic risk zone size),
assessed using tetrazolium staining. Hearts were sectioned into 2
mm slices and the outline of the LV slices and the
tetrazolium-negative (i.e., infarct) areas were traced onto clear
acetate sheets. Anatomic risk zone was delineated by the absence of
Monastral blue dye and traced onto clear acetate sheets. Infarcts
were normalized to anatomic risk zone size for each heart. Total LV
cross-sectional area, risk area, infarct area were determined from
enlarged tracings (1.5.times.) by computerized planimetry (Sigma
Scan; Jandel Scientific Inc., Calif.) using a Summagraphics
Summasketch Plus Bitpad connected to an IBM PS/2 computer. Risk
volume, infarct volume, and LV volume for each slice was calculated
as the sum of the area obtained by computerized planimetry and the
thickness of each ventricular slice. The values from the sequential
slices were summed to provide the total volume of the LV, risk zone
and infarct zone.
Data Analysis
[0117] Differences in hemodynamic data before and after coronary
occlusion were examined using one-way ANOVA. Heart rate-blood
pressure product was used as an index for cardiac metabolic demand.
Infarct volume, risk volume, infarct size, and LV volume were
compared by a one-way ANOVA. Where overall group differences were
detected, Dunnett's multiple comparison test was used for
comparison to controls. All statistical comparisons were made with
Statistical Analysis Systems Programs (SAS Institute, Cary, N.C.)
for the personal computer. A probability (p) level of less than
0.05 was considered statistically significant. To establish sample
size for this study, "n/group" values were calculated to provide a
0.90 power to detect a minimum 15 percent reduction (expected
standard deviation of 8%) of infarct size.
Results
[0118] One hundred thirty rabbits were entered into the present
studies; five rabbits died due to respiratory failure (n=1),
surgical error (n=1), or non-convertible ventricular fibrillation
(n=3). Thirty-six rabbits were included in the dose-response
studies and another 28 were assigned to the biochemical evaluations
(cardiac hemodynamic and plasma heparinase III levels were included
in the overall statistical analysis). Consequently, sixty-one
rabbits were included in the infarct size data analyses.
[0119] Cardiac hemodynamic variables are summarized in Table 1.
Heart rate, left ventricular systolic and diastolic pressures and
heart rate-blood pressure product (indicator of myocardial oxygen
demand) before the onset of coronary occlusion were comparable for
all of the treatment groups. Heart rate-blood pressure product
(FIG. 16) appeared to be higher at 60 minutes reperfusion in
rabbits treated with heparinase III (25 ug/ml target level) at the
time of reperfusion (Group 4); however, cardiac hemodynamics were
similar for all animals at the end of the study.
[0120] Data on left ventricular weight, infarct and risk volume,
infarct size and anatomic risk area as percent of LV volume are
summarized in Table 3. Infarct size (FIG. 17), normalized to risk
zone size, was 42.3.+-.4.8 (mean .+-.1 SD) and 40.0.+-.5.3 percent
(p=NS) in controls with/without vehicle pre-treatment,
respectively. Heparinase III pretreatment and heparinase III given
at the time of reperfusion at the 25 ug/ml target level resulted in
a significant reduction (p=0.01 versus vehicle treated controls) in
myocyte necrosis of 26.1.+-.5.2 and 24.7.+-.5.1 percent,
respectively; no differences were detectable between groups treated
either pre-ischemia or at the time of coronary reperfusion.
Treatment with heparinase III at 0.25, 1.25 or 5.0 ug/ml target
levels did not limit infarct size; they were 42.8.+-.6.5,
39.1.+-.5.4, and 37.9.+-.4.6 per cent respectively. There was a
slight trend to smaller infarcts in the 5.0 (g/ml treatment group
with a p value of 0.066.
[0121] Heparinase III injectate levels which were administered to
the respective treatment groups are shown in FIG. 18. The initial
heparinase III target dose (i.e., therapeutic dose) of 25 ug/ml was
investigated followed by the respective drug dilutions of 1:5, 1:20
and 1:100; dilutions were made with saline. A time-course of actual
plasma heparinase III concentrations is shown in FIG. 19;
pre-treatment with heparinase III and treatment at the time of
reperfusion provided similar plasma drug concentrations in Groups 3
and 4. Plasma heparinase III concentrations were considerably lower
after 3 hours coronary reperfusion in rabbits which were initially
pre-treated (drug only administered during first 3 hours of
experimental protocol); most importantly, the drug was on board at
the time of coronary reperfusion. This may account for the similar
results obtained in Groups 3 and 4 with respect to infarct
size.
2TABLE 1 Summary of Cardiac Hemodynamics LVPsys LVPdias RPP (mm Hg
.times. Group HR(bpm) (mm HG) (mm HG) bpm) Baseline 1 229 .+-. 34
63 .+-. 8 2 .+-. 1 14,192 .+-. 3,643 2 245 .+-. 31 58 .+-. 10 2
.+-. 2 13,705 .+-. 2,739 3 246 .+-. 29 64 .+-. 10 3 .+-. 3 15,169
.+-. 3,629 4 242 .+-. 39 70 .+-. 12 3 .+-. 2 16,276 .+-. 4,180 5
254 .+-. 27 67 .+-. 11 2 .+-. 2 16,432 .+-. 2,718 6 231 .+-. 34 63
.+-. 12 1 .+-. 1 14,389 .+-. 4,116 7 233 .+-. 28 67 .+-. 12 1 .+-.
1 15,293 .+-. 3,757 Ischemia(30 minutes) 1 218 .+-. 26 57 .+-. 8 4
.+-. 3 11,518 .+-. 2,904 2 237 .+-. 33 49 .+-. 11 4 .+-. 3 10,833
.+-. 3,372 3 241 .+-. 29 60 .+-. 8 6 .+-. 4 11,586 .+-. 2,634 4 236
.+-. 40 64 .+-. 11 5 .+-. 4 14,216 .+-. 3,832 5 228 .+-. 31 61 .+-.
12 2 .+-. 2 13,421 .+-. 3,545 6 219 .+-. 34 60 .+-. 13 4 .+-. 3
12,639 .+-. 4,551 7 211 .+-. 26 58 .+-. 8 4 .+-. 2 11,434 .+-.
2,704 Reperfusion(30 minutes) 1 206 .+-. 41 57 .+-. 8 3 .+-. 2
11,257 .+-. 3,731 2 229 .+-. 43 50 .+-. 9 3 .+-. 2 11,006 .+-.
3,755 3 201 .+-. 32 60 .+-. 10 5 .+-. 3 11,129 .+-. 3,069 4 238
.+-. 33 64 .+-. 11 5 .+-. 3 14,218 .+-. 4,031 5 206 .+-. 24 61 .+-.
11 3 .+-. 3 12,166 .+-. 2,833 6 197 .+-. 32 59 .+-. 11 2 .+-. 1
11,523 .+-. 3,825 7 191 .+-. 22 57 .+-. 11 3 .+-. 2 10,447 .+-.
2,888 Reperfusion(60 minutes) 1 199 .+-. 26 57 .+-. 7 3 .+-. 2
10,921 .+-. 2,695 2 220 .+-. 42 52 .+-. 10 3 .+-. 2 11,022 .+-.
3,932 3 197 .+-. 28 61 .+-. 13 5 .+-. 3 11,038 .+-. 2,981 4 233
.+-. 34 67 .+-. 12 5 .+-. 3 14,313 .+-. 4,082 5 199 .+-. 27 60 .+-.
11 2 .+-. 2 11,467 .+-. 3,172 6 182 .+-. 31 59 .+-. 11 2 .+-. 2
10,416 .+-. 3,151 7 183 .+-. 22 59 .+-. 10 4 .+-. 3 10,091 .+-.
2,138 Reperfusion(120 minutes) 1 177 .+-. 22 57 .+-. 8 2 .+-. 1
9,701 .+-. 2,499 2 197 .+-. 40 52 .+-. 10 3 .+-. 2 9,745 .+-. 3,870
3 188 .+-. 29 62 .+-. 12 5 .+-. 3 10,736 .+-. 3,180 4 210 .+-. 37
65 .+-. 13 6 .+-. 4 14,313 .+-. 4,082 5 184 .+-. 31 61 .+-. 13 2
.+-. 2 10,967 .+-. 3,624 6 165 .+-. 26 59 .+-. 11 2 .+-. 2 9,366
.+-. 2,587 7 165 .+-. 21 56 .+-. 10 3 .+-. 3 8,926 .+-. 2,164
Reperfusion(180 minutes) 1 156 .+-. 32 53 .+-. 13 2 .+-. 1 8,188
.+-. 3,450 2 187 .+-. 46 49 .+-. 5 4 .+-. 3 8,482 .+-. 2,163 3 178
.+-. 36 58 .+-. 10 4 .+-. 2 9,573 .+-. 2,591 4 206 .+-. 40 65 .+-.
15 7 .+-. 4 11,924 .+-. 4,116 5 172 .+-. 35 58 .+-. 12 2 .+-. 2
9,899 .+-. 3,542 6 154 .+-. 28 59 .+-. 12 2 .+-. 2 9,123 .+-. 3,313
7 154 .+-. 22 55 .+-. 10 2 .+-. 2 8,162 .+-. 2,346
[0122] Values are means .+-.1 SD. HR=heart rate; LVPsys=systolic
left ventricular chamber pressure; LVPdias=diastolic left
ventricular chamber pressure; RPP= heart rate-blood pressure
product.
3TABLE 2 Myocardial Blood Flow in Ischemic and Non-ischemic
Perfusion Beds Ischemic perfusion bed Non-ischemic perfusion bed
Group BASE 30" RP 180" RP BASE 30" RP 180" RP 1 2.17 .+-. 1.90 .+-.
1.02 .+-. 1.91 .+-. 2.20 .+-. 1.22 .+-. 0.99 0.57 0.63 1.11 0.78
0.64 2 2.73 .+-. 1.64 .+-. 0.88 .+-. 2.52 .+-. 1.92 .+-. 1.38 .+-.
1.31 0.75 0.32 1.07 0.71 0.23 3 2.13 .+-. 1.24 .+-. 1.17 .+-. 2.38
.+-. 1.78 .+-. 1.71 .+-. 1.11 0.711 0.96 0.78 0.51 0.69 4 2.33 .+-.
1.68 .+-. 0.84 .+-. 2.52 .+-. 2.24 .+-. 1.42 .+-. 0.83 0.87 0.47
0.71 0.64 0.46 5 2.76 .+-. 1.98 .+-. 1.27 .+-. 2.74 .+-. 1.83 .+-.
1.36 .+-. 1.15 0.65 0.55 1.55 0.55 0.28 6 2.43 .+-. 2.41 .+-. 1.57
.+-. 2.19 .+-. 2.24 .+-. 1.46 .+-. 0.70 0.38 0.58 0.68 0.36 0.29 7
2.38 .+-. 2.14 .+-. 1.13 .+-. 2.41 .+-. 2.20 .+-. 1.39 .+-. 0.45
0.76 0.49 0.58 0.61 0.35
[0123] Values are means .+-.1 SD. 1p(0.05 versus Group 6.
BASE=baseline flow measurement (i.e., before ischemia); RP=coronary
reperfusion. Data are expressed in ml/min/g wet weight.
4TABLE 3 Infarct Size Measurements Htwt AN AR ANAR ARLV Group n (g)
(cm3) (cm3) (%) (%) 1 9 4.46 .+-. 0.51 .+-. 1.22 .+-. 42.3 .+-.
36.5 .+-. 0.28 0.11 0.27 4.8 6.7 2 9 4.53 .+-. 0.41 .+-. 1.04 .+-.
40.0 .+-. 33.9 .+-. 0.63 0.07 0.18 5.3 5.8 3 9 4.71 .+-. 0.26 .+-.
1.03 .+-. 26.2 .+-. 33.3 .+-. 0.69 0.06.sup.1 0.31 4.9.sup.1 8.1 4
9 4.51 .+-. 0.24 .+-. 0.98 .+-. 24.7 .+-. 31.7 .+-. 0.77 0.07.sup.1
0.24 5.1.sup.1 7.2 5 8 4.30 .+-. 0.36 .+-. 0.91 .+-. 42.8 .+-. 29.4
.+-. 0.52 0.12 0.12 6.5 4.6 6 9 4.61 .+-. 0.39 .+-. 1.02 .+-. 39.1
.+-. 32.0 .+-. 0.52 0.08 0.27 5.4 7.9 7 9 4.02 .+-. 0.30 .+-. 0.80
.+-. 37.9 .+-. 27.5 .+-. 0.63 0.05 0.18.sup.1 4.6 6.7
[0124] Values are means .+-.1 SD. .sup.1p(0.05 versus controls.
Htwt=ventricular weight; AN=area of necrosis, AR=area at risk;
ANAR=necrosis normalized to anatomic risk area; ARLV=risk zone
normalized to total LV area.
[0125] These data indicate the utility of compositions containing
heparinase for diminishing localized inflammatory responses.
[0126] Modifications and variations of the compositions and methods
of use of the present invention will be obvious from this detailed
description to those skilled in the art. Such modifications are
intended to come within the scope of the following claims.
* * * * *