U.S. patent application number 14/154923 was filed with the patent office on 2014-05-08 for novel heparin entities and methods of use.
This patent application is currently assigned to W. L. Gore & Associates, Inc.. The applicant listed for this patent is W. L. Gore & Associates, Inc.. Invention is credited to Roy Biran, Charles D. Claude, Robert L. Cleek, Paul D. Drumheller.
Application Number | 20140128544 14/154923 |
Document ID | / |
Family ID | 43730811 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140128544 |
Kind Code |
A1 |
Biran; Roy ; et al. |
May 8, 2014 |
Novel Heparin Entities and Methods of Use
Abstract
The present invention relates to immobilized biologically active
entities that retain a significant biological activity following
manipulation. The invention also comprises a medical substrate
comprising a heparin entity bound onto a substrate via at least one
heparin molecule, wherein said bound heparin entity is heparinase-1
sensitive.
Inventors: |
Biran; Roy; (Flagstaff,
AZ) ; Claude; Charles D.; (Flagstaff, AZ) ;
Cleek; Robert L.; (Flagstaff, AZ) ; Drumheller; Paul
D.; (Flagstaff, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
W. L. Gore & Associates, Inc. |
Newark |
DE |
US |
|
|
Assignee: |
W. L. Gore & Associates,
Inc.
Newark
DE
|
Family ID: |
43730811 |
Appl. No.: |
14/154923 |
Filed: |
January 14, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12579308 |
Oct 14, 2009 |
|
|
|
14154923 |
|
|
|
|
12561927 |
Sep 17, 2009 |
8591932 |
|
|
12579308 |
|
|
|
|
Current U.S.
Class: |
525/54.1 ;
525/54.2; 530/317; 530/319; 536/13.2; 536/21 |
Current CPC
Class: |
A61L 31/16 20130101;
A61P 25/00 20180101; A61L 33/08 20130101; A61P 9/00 20180101; A61L
29/16 20130101; A61L 2300/236 20130101; A61L 2300/42 20130101; A61L
33/128 20130101; A61L 27/54 20130101; A61L 31/048 20130101; A61L
31/022 20130101; A61L 33/0011 20130101; A61L 31/10 20130101; A61L
31/048 20130101; C08L 27/12 20130101 |
Class at
Publication: |
525/54.1 ;
530/319; 536/13.2; 530/317; 536/21; 525/54.2 |
International
Class: |
A61L 33/00 20060101
A61L033/00; A61L 33/08 20060101 A61L033/08; A61L 33/12 20060101
A61L033/12 |
Claims
1. A heparin entity comprising: at least one heparin molecule and
at least one core molecule such that when said heparin entity is
bound onto a substrate via said at least one heparin molecule, said
heparin entity is heparinase sensitive.
2. The heparin entity of claim 1, wherein said substrate is
selected from the group consisting of polyethylene, polyurethane,
silicone, polyamide-containing polymers, polypropylene,
polytetrafluoroethylene, expanded-polytetrafluoroethylene and
biocompatible metals.
3. The heparin entity of claim 1, wherein said substrate is
expanded-polytetrafluoroethylene.
4. The heparin entity of claim 2, wherein said biocompatible metal
is Nitinol.
5. The heparin entity of claim 1, wherein said substrate is a
component of a medical device.
6. The heparin entity of claim 5, wherein said medical device is
selected from the group consisting of grafts, vascular grafts,
stents, stent-grafts, bifurcated grafts, bifurcated stents,
bifurcated stent-grafts, patches, plugs, drug delivery devices,
catheters, cardiac leads, balloons and indwelling filters.
7. The heparin entity of claim 6, wherein said stents can be used
in cardiac, peripheral or neurological applications.
8. The heparin entity of claim 6, wherein said stent-grafts can be
used in cardiac, peripheral or neurological applications.
9. The heparin entity of claim 1, wherein said core molecule is
either cyclic, linear, branched, dendritic, T, Y or star
shaped.
10. The heparin entity of claim 1, wherein said core molecule is
selected from the group consisting of proteins, polypeptides,
hydrocarbons, polysaccharides, aminoglycosides, and polymers.
11. The heparin entity of claim 10, wherein said protein is
selected from the group consisting of albumin, colistin and
polylysine.
12. The heparin entity of claim 10, wherein said polysaccharide is
selected from the group consisting of cyclodextrin, cellulose, and
chitosan.
13. The heparin entity of claim 10, wherein said polymer is
selected from the group consisting of polyethylene glycol (PEG) and
co-polymers of tetrafluoroethylene.
14. The heparin entity of claim 1, wherein said heparin is derived
from bovine or porcine sources.
15. The heparin entity of claim 1, wherein after heparinase
treatment heparin or fragments thereof will not be detected on said
substrate.
16. The medical substrate of claim 1, wherein after heparinase
treatment heparin or fragments thereof will be detect at a
significantly lower level than before heparinase treatment.
17. The heparin entity of claim 15, wherein heparin or fragments
thereof is detected by a label that binds to heparin or fragments
thereof.
18. The heparin entity of claim 17, wherein said label that binds
to heparin or fragments thereof is selected from the group
consisting of dyes, antibodies, and proteins.
19. The heparin entity of claim 18, wherein said dye is toluidine
blue.
20. The heparin entity of claim 1, wherein after heparinase
treatment an insignificant amount of toluidine blue will bind to
residual heparin or fragments thereof but will not be visually
detected on said substrate.
21. The heparin entity of claim 1, wherein after heparinase
treatment an insignificant amount of toluidine blue will bind to
residual heparin or fragments thereof, and wherein detector
readings will be about background levels or be insignificantly
different from background levels when compared to a substrate
without heparin entities.
22. The heparin entity of claim 1, wherein said heparin entity is
bound onto a substrate via at least one heparin molecule and
wherein said bound heparin molecule is attached to said substrate
via end point attachment.
23. The heparin entity of claim 1, wherein said heparin entity is
bound onto a substrate via at least one heparin molecule and
wherein said bound heparin molecule is attached to said substrate
via loop attachment.
24. The heparin entity of claim 1, wherein said heparin entity is
bound onto a substrate via at least one heparin molecule and
wherein said bound heparin molecule is attached to said substrate
via end point aldehyde.
25. The heparin entity of claim 1, wherein said heparin entity is
bound onto a substrate via at least one heparin molecule and
wherein said bound heparin molecule is attached to said substrate
via aldehydes along the length said heparin.
26. An ATIII binding entity comprising; a core molecule, a
polysaccharide chain attached to the core molecule, and a free
terminal aldehyde moiety on the polysaccharide chain.
27. The ATIII binding entity of claim 26, wherein said
polysaccharide chain is heparin.
28. The ATIII binding entity of claim 26, wherein said core
molecule is selected from the group consisting of a protein, a
hydrocarbon, an aminoglycoside, a polysaccharide and a polymer.
29. The ATIII binding entity of claim 28, wherein said protein is
selected from the group consisting of albumin, colistin, and
polylysine.
30. The ATIII binding entity of claim 28, wherein said
polysaccharide is selected from the group consisting of
cyclodextrin, cellulose, and chitosan.
31. The ATIII binding entity of claim 28, wherein said polymer is
selected from the group consisting of polyethylene glycol (PEG) and
co-polymers of tetrafluoroethylene.
32. The ATIII binding entity of claim 27, wherein said heparin is
derived from bovine or porcine sources.
33. The ATIII binding entity of claim 27, wherein said heparin is
bound onto the core molecule via end point attachment.
34. The ATIII binding entity of claim 27, wherein said heparin is
bound onto a substrate via end point attachment.
35. The ATIII binding entity of claim 34, wherein said substrate is
selected from the group consisting of polyethylene, polyurethane,
silicone, polyamide-containing polymers, and polypropylene,
polytetrafluoroethylene, expanded-polytetrafluoroethylene and
biocompatible metals.
36. The ATIII binding entity of claim 35, wherein said substrate is
expanded-polytetrafluoroethylene.
37. The ATIII binding entity of claim 35, wherein said
biocompatible metal is Nitinol.
38. The ATIII binding entity of claim 34, wherein said substrate is
a component of a medical device.
39. The heparin entity of claim 38, wherein said medical device is
selected from the group consisting of grafts, vascular grafts,
stents, stent-grafts, bifurcated grafts, bifurcated stents,
bifurcated stent-grafts, patches, plugs, drug delivery devices,
catheters and cardiac leads.
40. The heparin entity of claim 39, wherein said stents can be used
in cardiac, peripheral or neurological applications.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to medical substrates having
immobilized biologically active entities that maintain their
biological activity after sterilization. Specifically the present
invention relates to new heparin entities and their method of
use.
BACKGROUND OF THE INVENTION
[0002] Medical devices which serve as substitute blood vessels,
synthetic and intraocular lenses, electrodes, catheters and the
like in and on the body or as extracorporeal devices intended to be
connected to the body to assist in surgery or dialysis are well
known. However, the use of biomaterials in medical devices can
stimulate adverse body responses, including rapid thrombogenic
action. Various plasma proteins play a role in initiating platelet
and fibrin deposition on biomaterial surfaces. These actions lead
to vascular constriction that hinder blood flow, and the
inflammatory reaction that follows can lead to the loss of function
of the medical device. Biologically active entities that reduce or
inhibit thrombus formation on the surface of a biomaterial and/or
covering material are of particular interest for blood contacting
devices. Glycosaminoglycans are generally preferred anti-thrombotic
agents; with heparin, heparin analogs, and derivatives being
particularly preferred.
[0003] Immobilization of glycosaminoglycans, such as heparin, to
biomaterials has been researched extensively to improve bio- and
hemocompatibility. The mechanism responsible for reducing
thrombogenicity of a heparinized material is believed to reside in
the ability of heparin to speed up the inactivation of serine
proteases (blood coagulation enzymes) by anti-thrombin III (ATIII).
In the process, ATIII forms a complex with a well defined
pentasaccharide sequence in heparin, undergoing a conformational
change and thus enhancing the ability of ATIII to form a covalent
bond with the active sites of serine proteases, such as thrombin.
The formed serine protease-ATIII complex is then released from the
heparin, leaving said heparin behind for subsequent rounds of
inactivation via a catalytic process.
[0004] Immobilization of biologically active entities, such as
heparin, on biomaterials in a biologically active form involves an
appreciation of the respective chemistries of the entity and the
biomaterial. In the field of medical devices, ceramic, polymeric,
and/or metallic materials are common biomaterials. These materials
can be used for implantable devices, diagnostic devices or
extracorporeal devices. Modification of the chemical composition of
a biomaterial is often required to immobilize a biologically active
entity thereon. This modification is usually accomplished by
treating surfaces of the biomaterial to generate a population of
chemically reactive moieties or groups, followed by immobilization
of the biologically active entity with an appropriate protocol.
With other biomaterials, surfaces of a biomaterial are covered, or
coated, with a material having reactive chemical groups
incorporated therein. Biologically active entities are then
immobilized on the biomaterial through the reactive chemical groups
of the covering material. A variety of schemes for covering, or
coating, biomaterials have been described. Representative examples
of biologically active entities immobilized to a biomaterial with a
covering, or coating, are described in U.S. Pat. Nos. 4,810,784;
5,213,898; 5,897,955; 5,914,182; 5,916,585; and 6,461,665.
[0005] When biologically active compounds, compositions, or
entities are immobilized, the biological activity of these
"biologics" can be negatively impacted by the process of
immobilization. The biological activity of many biologics is
dependent on the conformation and structure (i.e., primary,
secondary, tertiary, etc.) of the biologic in its immobilized
state. In addition to a carefully selected immobilization process,
chemical alterations to the biologic may be required for the
biologic to be incorporated into the covering material with a
conformation and structure that renders the biologic sufficiently
active to perform its intended function.
[0006] Despite an optimized covering and immobilization scheme,
additional processing, such as sterilization, can degrade the
biological activity of the immobilized biologic. For implantable
medical devices, sterilization is required prior to use.
Sterilization may also be required for in vitro diagnostic devices
having sensitivity to contaminants. Sterilization of such devices
often requires exposure of the devices to elevated temperature,
pressure, and humidity, often for several cycles. In some
instances, antimicrobial sterilants, such as ethylene oxide gas
(EtO) or vapor hydrogen peroxide, are included in the sterilization
process. In addition to sterilization, mechanical compaction and
expansion, or long-term storage of an immobilized biologic can
degrade the activity of the biologic.
[0007] There exists a need for medical devices having biologically
active entities immobilized thereon that can be subjected to
sterilization, mechanical compaction and expansion, and/or storage
without significant loss of biological activity. Such a medical
device would have biologically compatible compositions or compounds
included with the immobilized biological entities that serve to
minimize degradation of the biological activity of the entities
during sterilization, mechanical compaction and expansion, and/or
storage. In some instances, the additional biologically compatible
compositions or compounds would increase the biological activity of
some biologically active entities following a sterilization
procedure.
SUMMARY OF THE INVENTION
[0008] Thus, the present invention comprises medical substrates
comprising heparin entities immobilized onto a substrate. The
heparin entities of the invention retain significant biological
activity following immobilization, sterilization, mechanical
compaction and expansion, and/or storage, as compared to other
coated medical substrates.
[0009] One embodiment of the invention comprises a medical
substrate comprising a heparin entity bound onto a substrate via at
least one heparin molecule, wherein said bound heparin entity is
heparinase sensitive. In another embodiment, said substrate is
selected from the group consisting of polyethylene, polyurethane,
silicone, polyamide-containing polymers, polypropylene,
polytetrafluoroethylene, expanded-polytetrafluoroethylene,
fluoropolymers, polyolefins, ceramics, and biocompatible metals. In
another embodiment, said substrate is
expanded-polytetrafluoroethylene. In another embodiment, said
biocompatible metal is a nickel-titanium alloy, such as Nitinol. In
another embodiment, said substrate is a component of a medical
device. In another embodiment, said medical device is selected from
the group consisting of grafts, vascular grafts, stents,
stent-grafts, bifurcated grafts, bifurcated stents, bifurcated
stent-grafts, patches, plugs, drug delivery devices, catheters,
cardiac pacemaker leads, balloons, and indwelling vascular filters.
In another embodiment, after heparinase treatment, heparin, or
fragments thereof, will not be detected on said substrate.
[0010] Another embodiment of the invention comprises a heparin
entity comprising at least one heparin molecule and at least one
core molecule such that when said heparin entity is bound onto a
substrate via a least one heparin molecule, said heparin entity is
heparinase sensitive. In one embodiment, said core molecule is
selected from the group consisting of proteins (including
polypeptides), hydrocarbons, aminoglycosides, polysaccharides and
polymers. In another embodiment, said heparin entity is bound onto
a substrate via at least one heparin molecule and wherein said
bound heparin molecule is attached to said substrate via end point
attachment. In another embodiment, said heparin entity is bound
onto a substrate via at least one heparin molecule and wherein said
bound heparin molecule is attached to said substrate via loop
attachment.
[0011] Another embodiment of the invention comprises an ATIII
binding entity comprising a core molecule, at least one
polysaccharide chain attached to the core molecule, and at least
one free terminal aldehyde moiety on the polysaccharide chain. In
one embodiment, said polysaccharide chain is heparin or a heparin
fragment. In another embodiment, said core molecule is selected
from the group consisting of a protein (including polypeptides), a
hydrocarbon, an aminoglycoside, a polysaccharide and a polymer. In
another embodiment, said substrate is selected from the group
consisting of polyethylene, polyurethane, silicone,
polyamide-containing polymers, polypropylene,
polytetrafluoroethylene, expanded-polytetrafluoroethylene,
fluoropolymers, polyolefins, ceramics, and biocompatible metals. In
another embodiment, said ATIII binding entity is bound onto a
substrate via end-point attachment or loop attachment. In another
embodiment, said substrate is a component of a medical device.
[0012] Another embodiment of the invention comprises a method of
determining the structure of a heparin entity bonded to a
substrate, comprising the steps of providing a substrate comprising
a heparin entity, depolymerizing the heparin entity to generate a
mixture of soluble heparin fragments, detecting each soluble
heparin fragment in said mixture using column chromatography,
determining the identity of each detected heparin fragment from the
above step, and deriving the structure of the heparin entity from
the identities of the detected heparin fragments. In one
embodiment, said depolymerizing is by heparinase depolymerization.
In another embodiment, said column chromatography is strong anion
exchange high performance liquid chromatography (SAX-HPLC).
[0013] Another embodiment of the invention comprises a system for
determining the structure of a heparin entity bonded to a
substrate, comprising a depolymerization solution, a labeling
reagent solution, and a detector. In another embodiment, said
depolymerization solution comprises heparinase. In another
embodiment, said labeling reagent solution comprises toluidine blue
and terbium tris(4-methylthio)benzoate. In another embodiment, said
detector comprises SAX-HPLC, an epifluoroscent microscope, and an
absorption spectroscope.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 depicts several heparin entities of the invention and
types of attachment of said heparin entities to a substrate.
[0015] FIG. 2 depicts ATIII binding capacity of various aldehyde
containing heparin entities conjugated onto expanded
polytetrafluoroethylene (ePTFE) and having undergone multiple EtO
sterilizations. Aldehyde containing heparin entities are classified
according to the core molecule used in the synthesis of the heparin
entity. Hence, colistin sulfate as the core refers to Examples 1,
neomycin to Example 2, poly-L-lysine to Example 4, capreomycin to
Example 3, polyethyleneimine (PEI) to Example 5, and ethylene
diamine (EDA) to Example 6. All bars represent mean values of
samples numbers with error bars for the standard deviation.
[0016] FIGS. 3A and B depict light micrographs of heparin entities
comprising free terminal aldehydes immobilized onto an ePTFE
substrate by a single end-point attachment method before (3A) and
after (3B) treatment with heparinase-1 and stained with toluidine
blue. The absence of coloration in Figure B as compared to A,
demonstrates that heparin entities comprising free terminal
aldehydes immobilized onto an ePTFE substrate by a single end-point
attachment method is essentially depolymerized from the surface
after heparinase-1 treatment.
[0017] FIG. 3C depicts the normalized change in luminosity before
and after treatment with heparinase-1 for heparin immobilized
through end-point aldehyde and multi-point attachment, heparin
entities comprising a neomycin core immobilized through end-point
and multi-point attachment through at least one heparin molecule,
and USP heparin immobilized through multi-point attachment. The low
normalized change in luminosity values for the heparin end-point
aldehyde, heparin entity comprising heparin and neomycin core with
end-point aldehyde, and USP heparin, all multi-point attached to
the substrate, indicated that the surfaces are not heparinase-1
sensitive and still have substantial heparin on the surface.
[0018] FIGS. 4A-C depicts light micrographs of heparin entities
comprising heparin and an EDA core immobilized onto an ePTFE
substrate by a single end-point attachment method before (4A and
4B) and after (4C) treatment with heparinase-1 and stained with
toluidine blue. The stained samples demonstrate the presence of the
heparin entity. Samples 4B and 4C were subjected to a round of
sterilization and rinsed only with DI water post sterilization. The
coloration of FIG. 4C after sterilization and heparinase-1
treatment indicates that heparinase-1 did not recognize heparin
entities on the surface.
[0019] FIGS. 4D and E depict light micrographs of heparin entities
comprising heparin and an EDA core immobilized onto an ePTFE
substrate by a single end-point attachment method before (4D) and
after (4E) treatment with heparinase-1 and stained with toluidine
blue. These samples where subjected to a round of sterilization and
rinsed with DI water and boric acid post sterilization. The lack
coloration of FIG. 4E after sterilization indicates that
heparinase-1 did recognize heparin entities on the surface and
depolymerized them.
[0020] FIGS. 5A-C depicts SAX-HPLC chromatograms from heparinase-1
depolymerization of (A) USP heparin, (B) heparin entities
constructed from heparin and colistin sulfate, and (C) heparin
entities constructed from heparin and neomycin sulfate.
[0021] FIGS. 6A and B depicts SAX-HPLC chromatograms from
heparinase-1 depolymerization of ePTFE surface immobilized (a) USP
heparin bound by free terminal aldehyde and (b) heparin entities
constructed from heparin and colistin sulfate bound by free
terminal aldehyde.
DETAILED DESCRIPTION
[0022] The present invention comprises medical substrates
comprising heparin entities immobilized onto a substrate. The
heparin entities of the invention retain significant biological
activity following immobilization and sterilization as compared to
other coated medical substrates.
[0023] In the context of this disclosure, a number of terms are
used. The following definitions are provided. As used herein and in
the appended claims, the singular forms "a", "an", and "the"
include plural reference unless the context clearly dictates
otherwise.
[0024] As used herein the term "heparin entity" means heparin
molecules covalently attached to a core molecule. Said heparin
molecules can be attached to the core molecule by end point
attachment (as described below and as essentially described in U.S.
Pat. No. 4,613,665, incorporated by reference herein for all
purposes) or other methods known in the art (see e.g. G T
Hermanson, Bioconjugate Techniques, Academic Press, 1996; H G Garg
et al., Chemistry and Biology of Heparin and Heparan Sulfate,
Elsevier, 2005.)
[0025] As used herein the term "core molecule" means a
polyfunctional molecule to which heparin is attached. For the
purposes of this invention, said core molecule and a substrate are
not the same, although a core molecule and a substrate can be made
from the same material.
[0026] As used herein, the term "substantially pure" means, an
object species is the predominant species present (i.e., on a molar
basis it is more abundant than any other individual species in the
composition), and preferably a substantially purified fraction is a
composition wherein the object species comprises at least about 50
percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will comprise more than
about 80 to about 90 percent of all macromolecular species present
in the composition. Most preferably, the object species is purified
to essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0027] As used herein, the term "heparinase" means any enzymatic
reaction that depolymerizes (e.g. digests) heparin. Examples of
heparinase include, but are not limited to, heparinase-1,
heparinase-2, heparinase-3, heparanase, exosulphatases, bacterial
exoenzymes, and glycosidases that can depolymerize heparin.
[0028] As used herein the term "heparinase sensitive" means that
after treatment of a substrate comprising heparin entities with
heparinase and staining said substrate with toluidine blue, the
substrate will not be visibly stained (essentially as depicted in
FIG. 3B and FIG. 4E). The term also means that an insignificant
amount of toluidine blue will bind to residual heparin, or
fragments thereof, and a reading from a detector that can measure
the amount of toluidine blue (or other labels) on a substrate, such
as a spectrophotometer, luminometer, densitometer, liquid
scintillation counter, gamma counter, or the like, will be about
background levels, or be insignificantly different from background
levels when compared to a substrate without heparin entities and
stained with toluidine blue, or be below the sensitivity of said
detectors when compared to a substrate comprising heparin entities
and stained with toluidine blue without heparinase treatment. The
term also means that a label that binds to heparin, or fragments
thereof, will not detect a substantial amount of heparin, or
fragments thereof, after treatment of a substrate comprising
heparin entities with heparinase.
[0029] As used herein the terms "bound," "attached," and
"conjugate," and their derivatives, when referring to heparin
entities and/or heparin means covalently bound, unless specified
otherwise.
[0030] Referring to FIGS. 1A-C, one embodiment of the invention
comprises a medical substrate comprising a heparin entity 100 bound
onto a substrate 106 via at least one heparin molecule 104, wherein
said bound heparin entity is heparinase sensitive. Suitable
substrate materials for immobilizing or binding said heparin
entities comprise polymers such as, but not limited to, polyamides,
polycarbonates, polyesters, polyolefins, polystyrene, polyurethane,
poly(ether urethane), polyvinyl chlorides, silicones,
polyethylenes, polypropylenes, polyisoprenes,
polytetrafluoroethylenes, and expanded-polytetrafluoroethylenes
(ePTFE, as described in U.S. Pat. No. 4,187,390). In one
embodiment, expanded, or porous, polytetrafluoroethylene (ePTFE) is
the substrate.
[0031] Additional substrates include, but are not limited to,
hydrophobic substrates such as polytetrafluoroethylene, expanded
polytetrafluoroethylene, porous polytetrafluoroethylene,
fluorinated ethylene propylene, hexafluoropropylene, polyethylene,
polypropylene, nylon, polyethyleneterephthalate, polyurethane,
rubber, silicone rubber, polystyrene, polysulfone, polyester,
polyhydroxyacids, polycarbonate, polyimide, polyamide, polyamino
acids, regenerated cellulose, and proteins, such as silk, wool, and
leather. Methods of making porous polytetrafluoroethylene materials
are described in U.S. Pat. Nos. 3,953,566 and 4,187,390, each of
which is incorporated herein by reference. In another embodiment,
said ePTFE may be impregnated, filled, imbibed or coated with at
least one chemical compound known to cause a bioactive response.
Compounds that cause a bioactive response comprise anti-microbials
(e.g. anti-bacterials and anti-virals), anti-inflammatories (e.g.
dexamethasone and prednisone), anti-proliferatives (e.g. taxol,
paclitaxel and docetaxel) and anti-coagulating agents (e.g.
abciximab, eptifibatide and tirofibran). In one embodiment, said
anti-inflammatory is a steroid. In another embodiment, said steroid
is dexamethasone. Methods of coating substrates are well known in
the art. In another embodiment, said substrate comprises the
heparin entities of the invention and a coating that comprises a
compound that causes a bioactive response. Said substrate comprises
the materials referred to above and below. In one embodiment, said
substrate is ePTFE.
[0032] Other suitable substrates include, but are not limited to,
cellulosics, agarose, alginate, polyhydroxyethylmethacrylate,
polyvinyl pyrrolidone, polyvinyl alcohol, polyallylamine,
polyallylalcohol, polyacrylamide, and polyacrylic acid.
[0033] Additionally, certain metals and ceramics may be used as
substrates for the present invention. Suitable metals include, but
are not limited to, titanium, stainless steel, gold, silver,
rhodium, zinc, platinum, rubidium, and copper, for example.
Suitable alloys include cobalt-chromium alloys such as L-605,
MP35N, Elgiloy, nickel-chromium alloys (such as Nitinol), and
niobium alloys, such as Nb-1% Zr, and others.
[0034] Suitable materials for ceramic substrates include, but are
not limited to, silicone oxides, aluminum oxides, alumina, silica,
hydroxyapapitites, glasses, calcium oxides, polysilanols, and
phosphorous oxide. In another embodiment, protein-based substrates,
such as collagen can be used. In another embodiment,
polysaccharide-based substrates, such as cellulose can be used.
[0035] Some substrates may have multiplicities of reactive chemical
groups populating at least a portion of its surface to which
heparin entities of the invention can be bound. Said heparin
entities of the invention are covalently bound to the substrate
material through said reactive chemical groups. Surfaces of said
substrates can be smooth, rough, porous, curved, planar, angular,
irregular, or combinations thereof. In some embodiments, substrates
with surface pores have internal void spaces extending from the
porous surface of the material into the body of the material. These
porous substrates have internal substrate material bounding the
pores that often provides surfaces amenable to immobilizing
biologically active entities. Whether porous or non-porous,
substrates can be in the form of filaments, films, sheets, tubes,
meshworks, wovens, non-wovens, and combinations thereof.
[0036] Substrates lacking reactive chemical groups on their
surfaces (or lacking appropriately reactive chemical groups) can be
covered, at least in part, with a polymeric covering material
having a multiplicity of reactive chemical groups thereon to which
said heparin entities can be bound. Polymeric substrates can also
be modified along their surface, or along their polymer backbone
using a variety of methods, including hydrolysis, aminolysis,
photolysis, etching, plasma modification, plasma polymerization,
carbene insertion, nitrene insertion, etc. Said heparin entities
are covalently attached, or bound, to the polymeric covering
material through the reactive chemical groups of the covering
material or directly to a substrate that has been modified. The
polymeric covering material may form at least one layer on at least
a portion of a substrate.
[0037] There are many other surface modifications, such those
described U.S. Pat. No. 4,600,652 and U.S. Pat. No. 6,642,242,
which are based on substrates having a layer of a polyurethane urea
to which heparin modified to contain aldehyde groups through
oxidation with nitrous acid or periodate, may be bound by covalent
links. A similar technology is described in U.S. Pat. No.
5,032,666, where the substrate surface is coated with an amine rich
fluorinated polyurethane urea before immobilization of an
antithrombogenic agent, such as an aldehyde-activated heparin.
Another antithrombogenic surface modification which may be
mentioned is described in publication WO87/07156. The surface of
the device is modified through the coating with a layer of lysozyme
or a derivative thereof to which heparin is adhered. Yet another
surface modification for producing antithrombogenic articles is
described in U.S. Pat. No. 4,326,532. In this case, the layered
antithrombogenic surface comprises a polymeric substrate, a
chitosan bonded to the polymeric substrate and an antithrombogenic
agent bonded to the chitosan coating. Others have reported an
antithrombogenic hemofilter also using a chitosan layer for binding
heparin. Another process for preparing antithrombogenic surfaces is
described in WO97/07834, wherein the heparin is admixed with
sufficient periodate so as not to react with more than two sugar
units per heparin molecule. This mixture is added to a surface
modified substrate of a medical device, wherein said surface
modification contains amino groups. The above listing of processes
for adding reactive groups to substrates are only a small example
of how this can be accomplished. The above listing is by no means
complete. Furthermore, it is clear that the type of process used to
add reactive chemical groups to a substrate will depend on the
properties of the substrate of which a person of skill in the art
will recognize.
[0038] In another embodiment of the invention, said medical
substrate comprising said bound heparin entity via at least one
heparin molecule is a component of a medical device. Medical
devices comprise, but are not limited to, grafts, vascular grafts,
stents, stent-grafts, bifurcated grafts, bifurcated stents,
bifurcated stent-grafts, hernia patches, hernia plugs, periodontal
grafts, surgical fabrics, drug delivery devices, catheters, cardiac
leads balloons and indwelling filters. In one embodiment, said
stents can be used in cardiac, peripheral or neurological
applications. In another embodiment, said stent-grafts can be used
in cardiac, peripheral or neurological applications.
[0039] Another embodiment of the invention comprises a heparin
entity comprising at least one heparin molecule and at least one
core molecule. As shown in FIG. 1, the core molecule 102 is the
"backbone" of the heparin entity 100 to which heparin molecules 104
are bound. Said core molecule 102 can be either cyclic (102a, FIGS.
1A and 1C), linear (102b, FIG. 1B), branched, dendritic, "Y"
shaped, "T" shaped, or "star" shaped as described by Freudenberg,
U., Biomaterials, 30, 5049-5060, 2009 and Yamaguchi, N.,
Biomacromolecules, 6, 1921-1930, 2005. In one embodiment, said core
molecule is selected from the group consisting of proteins
(including polypeptides), hydrocarbons, lipids, aminoglycosides,
polysaccharides and polymers. Proteins include, but are not limited
to, antibodies, enzymes, receptors, growth factors, hormones,
serpins and any globular protein. Specific proteins and
polypeptides include, but are not limited to, albumin, colistin,
collagen, polylysine, antithrombin III, fibrin, fibrinogen,
thrombin, laminin, keratin, and the like. In another embodiment,
said core molecule can be a polypeptide. Said polypeptide need not
be very long and can comprise one or more repetitions of amino
acids, for example repetitions of serine, glycine (e.g.
Ser-Gly-Gly-Ser-Gly), lysine or ornithine residues. Alternatively,
other amino acid sequences can be used, for example colistin,
polylysine, and polymyxin.
[0040] Examples of polysaccharides include, but are not limited to
neutral polysaccharides such as cellulose, starch, agarose,
carboxymethylcellulose, nitrocellulose, and dextran, anionic
polysaccharides such as alginate, heparin, heparin sulfate, dextran
sulfate, xanthan, hyaluronic acid, carrageenan, gum arabic,
tragacanth, arabinogalactan, and pectin; macrocyclic
polysaccharides such as cyclodextrin and hydroxypropyl
cyclodextrin; and polycationic polysaccharides such as chitin and
chitosan.
[0041] Examples of synthetic polymers include, but are not limited
to, polyethylene glycol (PEG) 200, 300, 400, 600, 1000, 1450, 3350,
4000, 6000, 8000 and 20000, polytetrafluoroethylene, polypropylene
glycol, poly(ethylene glycol-co-propylene glycol), copolymers of
polyethylene glycol, copolymers of polypropylene glycol, copolymers
of tetrafluoroethylene with vinyl acetate and vinyl alcohol,
copolymers of ethylene with vinyl acetate & vinyl alcohol,
polyvinyl alcohol, polyethyleneimine, polyacrylic acid; polyols
such as polyvinyl alcohol and polyallyl alcohol; polyanions such as
acrylic acid and poly(methacrylic acid). Polycation polymers
include poly(allylamine), poly(ethyleneimine), poly(guanidine),
poly(vinyl amine), polyethylene glycol diamine, ethylene diamine,
and poly(quaternary amines); polyacrylonitriles such as hydrolyzed
polyacrylonitrile, poly(acrylamide-co-acrylonitrile), and their
copolymers. Other polymers include fluorinated copolymers including
copolymers of tetrafluoroethylene and vinyl alcohol, vinyl acetate,
vinyl formamide, acrylamide, and vinyl amine. In another
embodiment, said core molecule can be an aminoglycoside, including,
but not limited to, amikacin, arbekacin, gentamicin, kanamycin,
neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin,
tobramycin, and apramycin.
[0042] Heparin is a mucopolysaccharide, isolated from pig intestine
or bovine lung and is heterogeneous with respect to molecular size
and chemical structure. Heparin is built up from alternating
glycuronic acid and glucosamine units. The glycuronic acid units
consist of D-glycuronic acid and L-iduronic acid. These are
respectively D- and L-(1,4)-bound to the D-glucosamine units. A
large proportion of the L-iduronic acid residues are sulfated in
the 2-position. The D-glucosamine units are N-sulfated, sulfated in
the 6-position and are .alpha.-(1,4)-bound to the uronic acid
residues. Certain D-glucosamine units are also sulfated in the
3-position. Heparin contains material with a molecular weight
ranging from about 6,000 Daltons to about 30,000 Daltons. The
hydroxyl and amine groups are derivatized to varying degrees by
sulfation and acetylation. The active sequence in heparin
responsible for its anticoagulation properties is a unique
pentasaccharide sequence that binds to the ligand anti-thrombin III
(ATIII). The sequence consists of three D-glucosamine and two
uronic acid residues. Heparin molecules can also be classified on
the basis of their pentasaccharide content. About one third of
heparin contains chains with one copy of the unique pentasaccharide
sequence (see, Choay, Seminars in Thrombosis and Hemostasis
11:81-85 (1985) which is incorporated herein by reference) with
high affinity for ATIII, whereas a much smaller proportion
(estimated at about 1% of total heparin) consists of chains which
contain more than one copy of the high affinity pentasaccharide
(see, Rosenberg et al., Biochem. Biophys. Res. Comm. 86:1319-1324
(1979) which is incorporated herein by reference). The remainder
(approx. 66%) of the heparin does not contain the pentasaccharide
sequence. Thus, so called "standard heparin" constitutes a mixture
of the three species: "high affinity" heparin is enriched for
species containing at least one copy of the pentasaccharide and
"very high affinity" heparin refers to the approximately 1% of
molecules that contain more than one copy of the pentasaccharide
sequence. These three species can be separated from each other
using routine chromatographic methods, such as chromatography over
an anti-thrombin affinity column (e.g., Sepharose-AT; see, e.g.,
Lam et al., Biochem. Biophys. Res. Comm. 69:570-577 (1976) and
Homer Biochem. J. 262:953-958 (1989) which are incorporated herein
by reference).
[0043] In one embodiment, said heparin is derived from an animal.
In another embodiment, said heparin is bovine or porcine derived.
In another embodiment, said heparin is a synthetic heparin, i.e.
not derived from animal sources (e.g. fondaparinux or enoxaparin).
In another embodiment, heparin entities of the invention comprise
heparin that has been enriched and comprises substantially pure
"high affinity" heparin. In another embodiment, heparin entities of
the invention comprise heparin that has been enriched and comprises
substantially pure "very high affinity" heparin. In another
embodiment, heparin entities of the invention comprises heparin has
been enriched and comprises a combination of substantially pure
"high affinity" and "very high affinity" heparin.
[0044] Another embodiment of the invention comprises the binding of
said heparin entity to a medical substrate via at least one heparin
molecule. As shown in FIG. 1, the heparin entities of the invention
are bound to said substrate via at least one heparin molecule.
Thus, in one embodiment, said bound heparin molecule is attached to
said substrate via end point attachment (as depicted in FIGS. 1A
and 1B). In another embodiment, said bound heparin molecule is
attached to said substrate via an end point aldehyde. This can be
accomplished essentially as described in U.S. Pat. No. 4,613,665,
which is incorporated herein by reference in its entirety, and as
described below.
[0045] In another embodiment, said heparin entity is bound onto a
substrate via at least one heparin molecule, wherein said bound
heparin molecule is attached to said substrate via a "loop
attachment." Loop attachment, as depicted in FIG. 1C, is an
attachment of said heparin entity via at least one heparin, wherein
the heparin is attached loosely to the substrate in a small number
of locations, therefore allowing substantial portions of the bound
heparin to be exposed to heparinase (as opposed to more common
methods that attach heparin tightly in a large number of
locations). The more common methods of coupling heparin to a
substrate comprise reacting a majority of functional groups
randomly localized along a heparin molecule's length (e.g. using
coupling agents such as carbodiimides, epoxides, and
polyaldehydes). These methods result in a high probability that the
active sequence (said unique pentasaccharide sequence describe
above) will be bound to the substrate resulting in reduced and/or
lost activity. In loop attachment of heparin, only a few functional
groups on the heparin react and are bound to the substrate. Thus,
there is a high probability that the active sequence of the
attached heparin will not be bound to the substrate, therefore
allowing said active sequence to bind to its ligand. In another
embodiment, the invention comprises a heparin entity with multiple
attachments to a substrate, wherein the active sequence is not
bound to the substrate. In another embodiment, said bound heparin
entity molecule is attached to said substrate via loop
attachment.
[0046] As discussed above, endpoint and loop attachments allow a
substantial portion of at least one heparin molecule (in a heparin
entity) not to be bound to a substrate. As used herein the term
"substantial portion" means that about 50%, about 60%, about 70%,
about 80%, about 90%, about 95%, about 96% about 97%, about 98% and
about 99% of the heparin molecule is not bound to the substrate. In
another embodiment, the term also refers to the at least one
heparin molecule (in a heparin entity) wherein said the at least
one heparin molecule bound to the substrate is not bound to a
substrate via its active sequence. Thus, since the active sequence
is not bound to the surface of the substrate, the active sequence
has a greater probability of interacting with its ligand. In other
words, if the active sequence is bound to the surface of the
substrate then there is a small chance of heparin binding to its
ligand.
[0047] However, because said heparin entities are attached via
heparin by endpoint and/or loop attachment, the heparin is
sensitive to heparinase. Thus, after heparinase treatment, there
will be very little, if any, heparin, or fragments thereof, on the
surface of said substrate. In contrast, some of the more common
methods of attaching heparin to the surface of a substrate (which
comprises multiple bonds along the length of the heparin molecule,
as described above), after heparinase treatment, will have a
significant amount of heparin, or fragments thereof, still attached
to the surface of the substrate. Thus, after heparinase treatment,
heparin, or fragments thereof, can be detected on the surface of
said substrate. Without being bound to any particular theory, the
inventors have that discovered that the more sensitive the bound
heparin or heparin entity is to heparinase, the more biological
activity said bound heparin or heparin entity exhibits. This may be
because the active sequence of the bound heparin or heparin entity
is not attached to the surface of the substrate, thus said bound
heparin or heparin entity has a greater chance of binding to its
ligand.
[0048] Heparin must have intact conformation and structure to be
recognized by ATIII, and if said conformation and structure is
lost, heparin will exhibit poor activity. In addition, loss of said
conformation and structure results in poor recognition by other
proteins, such as heparinase-1, resulting in said heparin being
resistant to depolymerization. For example, modification of soluble
heparin with carbodiimide changes the soluble heparin structure in
such a way that it is no longer recognized by heparinase-1, and the
modified soluble heparin has reduced whole blood anticoagulant
activity (see Olivera, G. B., Biomaterials, 24, 4777-4783, 2003).
The inventors have discovered that heparinase sensitivity of
attached heparin or heparin entity is predictive of ATIII binding
activity of said attached heparin or heparin entity. Without
wanting to be constrained by any particular theory, if the attached
heparin or heparin entity retains specificity for specific enzymes
such as heparinase-1, then the attached heparin or heparin entity
retains substantially enough primary/secondary/tertiary structure
for it also to have specificity for ATIII. Thus, the inventors have
discovered that when an attached heparin or heparin entity is
recognized by heparinase-1, said attached heparin or heparin entity
is also recognized by ATIII, as exemplified by high binding
activities.
[0049] The inventors have also shown that a boric acid rinse will
restore heparinase sensitivity to inactivated attached heparin or
heparin entities (inactivated by sterilization, mechanical
compaction and expansion, or long-term storage, for example). Thus,
another embodiment of the invention comprises a method of restoring
heparinase sensitivity to heparin or heparin entities bound onto a
substrate comprising rinsing said substrate in a solution of boric
acid. In one embodiment, said substrate was exposed to a
sterilization cycle. In another embodiment, said substrate was
exposed to mechanical treatments that reduced heparinase-1
activity.
[0050] In another embodiment of the invention, after treating a
medical substrate with bound heparin entities of the invention with
heparinase, heparin, or fragments thereof, will not be detected on
said substrate. In another embodiment, after treating a medical
substrate with bound heparin entities of the invention with
heparinase, heparin, or fragments thereof, will be detected at a
substantially lower level than before heparinase treatment.
Significantly lower level of detection comprises very little
detection after staining and/or labeling for heparin.
[0051] In another embodiment, said heparin, or fragments thereof,
will not be detected visually (macroscopically) after staining or
labeling. Heparin, or fragments thereof, can be detected by a label
that binds directly or indirectly to heparin, or fragments thereof.
In one embodiment, said label that binds to heparin, or fragments
thereof, is selected from the group consisting of dyes, antibodies,
and proteins. Examples of labels include, but are not limited to
proteins including anti-heparin antibodies (polyclonal or
monoclonal) and ATIII; metachromatic dyes including toluidine blue,
azure A, alcian blue, victoria blue 4R, night blue, methylene blue;
radioiodinated labels including radioiodinated toluidine blue,
radioiodinated methylene blue, radioiodinated heparin antibodies,
radioiodinated ATIII; tritiated labels including tritiated
toluidine blue, tritiated azure A, tritiated alcian blue, tritiated
victoria blue 4R, tritiated night blue, tritiated methylene blue;
carbon-14 labels including 14C-toluidine blue, 140-azure A,
14C-alcian blue, 140-victoria blue 4R, 140-night blue,
14C-methylene blue; fluorescent labels including rhodamine-labelled
heparin antibodies, fluorescein-labelled heparin antibodies,
rhodamine-labelled ATIII, fluorescein-labelled ATIII. In another
embodiment, said dye is toluidine blue. In another embodiment,
after heparinase treatment, an insignificant amount of toluidine
blue will bind to heparin, or fragments thereof, but will not be
visually detected on said substrate (essentially as depicted in
FIGS. 3B and 4E). In another embodiment, after heparinase
treatment, a insignificant amount of toluidine blue will bind to
residual heparin, or fragments thereof, and a reading from a
detector that can measure the amount of toluidine blue (or other
labels described above) on a substrate (e.g. a spectrophotometer,
luminometer, densitometer, liquid scintillation counter, gamma
counter, or the like) will be about background levels, or be
insignificantly different from background levels when compared to a
substrate without heparin entities. In another embodiment, after
heparinase treatment, a reading from a detector that can measure
the amount of toluidine blue (or other labels described above) on a
substrate will be significantly different when compared to a
substrate comprising heparin entities and stained with toluidine
blue (or other labels described above) without heparinase
treatment.
[0052] Another embodiment of the invention comprises a heparin
entity comprising at least one heparin molecule attached to a core
molecule, wherein the entity is bound to a substrate via a heparin
molecule, and wherein after exposure to heparinase and toluidine
blue, the substrate macroscopically evidences substantially no
toluidine blue on its surface (as depicted in FIG. 3B and FIG.
4E).
[0053] Another embodiment of the invention comprises a heparin
entity which comprises at least one heparin molecule and at least
one core molecule such that when said heparin entity is bound onto
a substrate via a least one heparin molecule, said heparin entity
is heparinase sensitive. In one embodiment, said substrate is
selected from the group consisting of polyethylene, polyurethane,
silicone, polyamide-containing polymers, polypropylene,
polytetrafluoroethylene, expanded-polytetrafluoroethylene,
biocompatible metals, ceramics, proteins, polysaccharides, and any
substrate described above. In another embodiment, said substrate is
expanded-polytetrafluoroethylene. In another embodiment, said
substrate is a component of a medical device. In another
embodiment, said medical device is selected from the group
consisting of grafts, vascular grafts, stents, stent-grafts,
bifurcated grafts, bifurcated stents, bifurcated stent-grafts,
patches, plugs, drug delivery devices, catheters and cardiac leads.
In another embodiment, said stents can be used in cardiac,
peripheral or neurological applications. In another embodiment,
said stent-grafts can be used in cardiac, peripheral or
neurological applications. In another embodiment, said medical
device can be used in orthopedic, dermal, or gynecologic
applications. In another embodiment, said core molecule comprises a
cyclic, linear, branched, dendritic, "Y", "T", or star molecular
structure. In another embodiment, said core molecule is selected
from the group consisting of proteins, polypeptides, hydrocarbons,
polysaccharides, aminoglycosides, polymers, and fluoropolymers.
[0054] In another embodiment, heparin, or fragments thereof, is
detected by labels that bind to heparin, or fragments thereof. In
another embodiment, said label that binds to heparin, or fragments
thereof, is selected from the group consisting of dyes, polyclonal
antibodies, and proteins. In another embodiment, said dye is
toluidine blue. In another embodiment, after heparinase treatment,
an insignificant amount of toluidine blue will bind to residual
heparin, or fragments thereof, and will not be visually detected on
said substrate. In another embodiment, after heparinase treatment,
a insignificant amount of toluidine blue will bind to residual
heparin, or fragments thereof, and a reading from a detector that
can measure the amount of toluidine blue (or other labels described
above) on a substrate (e.g. a spectrophotometer, luminometer,
densitometer, liquid scintillation counter, gamma counter, or the
like) will be about background levels, or be insignificantly
different from background levels when compared to a substrate
without heparin entities. In another embodiment, after heparinase
treatment, a reading from a detector that can measure the amount of
toluidine blue (or other labels described above) on a substrate
will be significantly different when compared to a substrate
comprising heparin entities and stained with toluidine blue (or
other labels described above) without heparinase treatment. In
another embodiment, said heparin entity is bound onto a substrate
via at least one heparin molecule and wherein said bound heparin
molecule is attached to said substrate via end-point attachment. In
another embodiment, said heparin entity is bound onto a substrate
via at least one heparin molecule, wherein said bound heparin
molecule is attached to said substrate via end-point aldehyde. In
another embodiment, said heparin entity is bound onto a substrate
via at least one heparin molecule, wherein said bound heparin
molecule is attached to said substrate via loop attachment. In
another embodiment, said heparin entity is bound onto a substrate
via at least one heparin molecule, wherein said bound heparin
molecule is attached to said substrate via aldehydes along the
length said heparin.
[0055] Another embodiment of the invention comprises an ATIII
binding entity comprising: a core molecule, a polysaccharide chain
attached to the core molecule, and a free terminal aldehyde moiety
on the polysaccharide chain. This ATIII binding entity can then be
end-point attached to a substrate via a terminal aldehyde. Another
embodiment of the invention comprises an ATIII binding entity
comprising: a core molecule, a polysaccharide chain attached to the
core molecule, and free terminal aldehyde moieties along the length
of the polysaccharide chain. This ATIII binding entity can then be
looped attached to a substrate via the aldehydes along the length
of the polysaccharide chain. In another embodiment, said
polysaccharide chain is heparin. In another embodiment, said core
molecule is selected from the group consisting of a protein, a
polypeptide, a hydrocarbon, an aminoglycoside, a polysaccharide, a
polymer, a fluoropolymer, or any core molecule described herein. In
another embodiment, heparin is bound onto the core molecule via
end-point attachment. In another embodiment, the substrate is
selected from the group consisting of polyethylene, polyurethane,
silicone, polyamide-containing polymers, and polypropylene,
polytetrafluoroethylene, expanded-polytetrafluoroethylene and
biocompatible metals, or any of the substrates described herein. In
another embodiment said biocompatible metal is Nitinol. In another
embodiment, said substrate is expanded-polytetrafluoroethylene. In
another embodiment, said substrate is a component of a medical
device. In another embodiment, said medical device is selected from
the group consisting of grafts, vascular grafts, stents,
stent-grafts, bifurcated grafts, bifurcated stents, bifurcated
stent-grafts, patches, plugs, drug delivery devices, catheters and
cardiac leads. In another embodiment, said medical device can be
used in cardiac, peripheral, neurologic, orthopedic, gynecologic,
or dermal applications.
[0056] Another embodiment of the invention comprises an implantable
medical device comprising a medical substrate, wherein said medical
substrate comprises a heparin entity bound onto a substrate via at
least one heparin molecule, wherein said bound heparin entities are
heparinase sensitive. In one embodiment, said medical device is
selected from the group consisting of grafts, vascular grafts,
stents, stent-grafts, bifurcated grafts, bifurcated stents,
bifurcated stent-grafts, patches, plugs, drug delivery devices,
catheters and cardiac leads. In another embodiment, said stent can
be used in cardiac, peripheral or neurological applications. In
another embodiment, said stent can be a balloon expandable and/or a
self expanded stent. Said stents can be made from any biocompatible
material including any polymer or metal as described above. In
another embodiment, said stent is made from Nitinol and/or
stainless steel. In another embodiment, said stent comprises a
graft. In another embodiment, said graft and/or stent comprise
heparin entities of the invention.
[0057] The heparin entities of the invention retain significant
biological activity following immobilization and sterilization as
compared to other coated medical substrates. Thus, in one
embodiment said medical substrate comprises, a heparin entity bound
onto a substrate via at least one heparin molecule, wherein said
bound heparin entity is heparinase sensitive has an ATIII activity
of about 300 pmol/cm.sup.2. In another embodiment; the ATIII
activity is about 250 pmol/cm.sup.2, about 200 pmol/cm.sup.2, about
150 pmol/cm.sup.2, about 100 pmol/cm.sup.2, about 50 pmol/cm.sup.2,
about 40 pmol/cm.sup.2, about 30 pmol/cm.sup.2, about 20
pmol/cm.sup.2, about 10 pmol/cm.sup.2 or about 5 pmol/cm.sup.2. In
another embodiment, after a first round of sterilization the ATIII
activity of said medical substrate is about 250 pmol/cm.sup.2,
about 200 pmol/cm.sup.2, about 150 pmol/cm.sup.2, about 100
pmol/cm.sup.2, about 50 pmol/cm.sup.2, about 40 pmol/cm.sup.2,
about 30 pmol/cm.sup.2, about 20 pmol/cm.sup.2, about 10
pmol/cm.sup.2 or about 5 pmol/cm.sup.2. In another embodiment,
after a second round of sterilization, the ATIII activity of said
medical substrate is about 100 pmol/cm.sup.2, about 90
pmol/cm.sup.2, about 80 pmol/cm.sup.2, about 70 pmol/cm.sup.2,
about 60 pmol/cm.sup.2, about 50 pmol/cm.sup.2, about 40
pmol/cm.sup.2, about 30 pmol/cm.sup.2, about 20 pmol/cm.sup.2,
about 10 pmol/cm.sup.2 or about 5 pmol/cm.sup.2. In another
embodiment, after a third round of sterilization, the ATIII
activity of said medical substrate is above about 50 pmol/cm.sup.2,
or about 70 pmol/cm.sup.2, about 60 pmol/cm.sup.2, about 50
pmol/cm.sup.2, about 40 pmol/cm.sup.2, about 30 pmol/cm.sup.2,
about 20 pmol/cm.sup.2, about 10 pmol/cm.sup.2 or about 5
pmol/cm.sup.2. ATIII activity assays are well known in the art and
at least one is described below. In another embodiment, said
heparin entities of the invention retain significant biological
activity following compression and expansion of a medical device.
In another embodiment, said heparin entities of the invention
retain significant biological activity following storage conditions
for medical devices either in a compacted and/or expanded
state.
[0058] Another embodiment of the invention comprises methods of
determining the structure of a heparin entity bonded to a
substrate. One method of determining the structure of a heparin
entity bonded to a substrate comprises the steps of: providing a
substrate comprising a heparin entity, depolymerizing the heparin
entity to generate a mixture of soluble heparin fragments,
detecting each soluble heparin fragment in said mixture using
column chromatography, determining the identity of each detected
soluble heparin fragment from above, and deriving the structure of
the heparin entity from the identities of the detected soluble
heparin fragments. In one embodiment, said depolymerization is by
heparinase-1. In another embodiment, column chromatography is
strong anion exchange-high performance liquid chromatography or
SAX-HPLC.
[0059] Another embodiment of the invention comprises an implantable
medical device comprising a medical substrate, wherein said medical
substrate comprises a heparin entity bound onto a substrate via at
least one heparin molecule, wherein said bound heparin entities are
heparinase sensitive. In one embodiment, said medical device is
selected from the group consisting of grafts, vascular grafts,
stents, stent-grafts, bifurcated grafts, bifurcated stents,
bifurcated stent-grafts, patches, plugs, drug delivery devices,
catheters, cardiac leads, balloons and indwelling filters. In
another embodiment, said stent can be used in cardiac, peripheral
or neurological applications. In another embodiment, said stent can
be a balloon expandable and/or a self expanded stent. Said stents
can be made from any biocompatible material including any polymer
or metal as described above. In another embodiment, said stent is
made from Nitinol and/or stainless steel. In another embodiment,
said stent comprises a graft. In another embodiment, said graft
and/or stent comprise heparin entities of the invention.
[0060] Another embodiment of the invention comprises methods of
determining the spatial distribution of a heparin entity bonded to
a substrate. One method of determining the spatial distribution of
a heparin entity bonded to a substrate comprises the steps of:
providing a substrate comprising a heparin entity, depolymerizing
the heparin entity to generate a surface comprising surface-bonded
unsaturated heparin fragments, reacting the surface with a labeling
reagent which introduces a detectable component to said
surface-bonded unsaturated heparin fragments, detecting said
surface-bonded unsaturated heparin fragment via said detectable
component, and deriving the spatial distribution of the heparin
entity from the presence of the surface-bonded unsaturated heparin
fragments. In one embodiment, depolymerization is by heparinase-1.
In another embodiment, said labeling reagent is a lanthanoid
Michael-like addition organo-complex. In another embodiment, said
labeling reagent is terbium tris(4-methylthio)benzoate. In another
embodiment, said organo-complex comprises chemisorbed gold
nanoparticles. In another embodiment, said detecting is by
epifluoroscent microscopy or transmission electron microscopy.
[0061] Another embodiment of the invention comprises a system for
determining the structure of a heparin entity bonded to a
substrate, comprising a depolymerization solution, a labeling
reagent solution, and a detector. A system is an assembly of
reagents and instruments used to detect the structure and type of
binding of heparin entities to a substrate. In one embodiment, said
depolymerization solution comprises heparinase-1. In another
embodiment, said labeling reagent solution comprises toluidine
blue, and terbium tris(4-methylthio)benzoate. In another
embodiment, said detector comprises SAX-HPLC, an epifluoroscent
microscope, and an absorption spectroscope. In another embodiment,
said assembly of reagents can be a kit.
[0062] After enzymatic heparinase-1 depolymerization of heparin
and/or heparin entities that are end-point attached, heparin
fragments are left are on the surface that are unsaturated, i.e.
they comprise a carbon-carbon double bond ("nubs"). Enzymatic
heparinase depolymerization involves cleavage of the non-reducing
terminal uronic acid residue to a 4,5-unsaturated derivative. This
produces residual surface-bonded unsaturated heparin fragments
bonded to the substrate that comprises a carbon-carbon double bond.
Thus, the structure of the residual surface-bonded heparin fragment
is unsaturated, and can react with various detection molecules,
including those that comprise Michael-like addition complexes, such
as thiol-containing compounds and thiol-containing fluorescent
compounds, such as terbium tris(4-methylthio)benzoate. Thus, in
another embodiment of the invention, after enzymatic heparinase-1
depolymerization of an end-point attached heparin entity, said
residual surface-bonded unsaturated heparin fragments bonded to the
substrate comprising a carbon-carbon double bond are detected. This
method can determine if heparin and/or heparin entities were
end-point attached to a substrate. In another embodiment, nub
detection is combined with any of the detection and/or
characterization methods described above.
[0063] This invention is further illustrated by the following
Examples which should not be construed as limiting. The contents of
all Figures and references are incorporated herein by
reference.
EXAMPLES
Example 1
[0064] This example describes the construction of heparin entities
comprising heparin and colistin sulfate as the core. This heparin
entity contains free terminal aldehydes that can be used for
attachment to a surface of a substrate.
[0065] Colistin sulfate (0.10 g, Alpharma, Inc.) was dissolved in
300 ml of deionized (DI) water containing MES buffer (pH 4.7,
BupH.TM. Thermo Scientific). To this was added 10 g USP heparin, 4
g N-hydroxysulfosuccinimide (sulfo-NHS, Thermo Scientific), and 4 g
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC
hydrochloride, Sigma-Aldrich, St. Louis, Mo.). The reaction was
allowed to proceed at room temperature for 4 hours, followed by
dialysis overnight with a 50,000 MWCO membrane (Spectra/Por.RTM.).
The retentate (about 350 ml out of 500 ml) was transferred to a
beaker, and cooled to 0.degree. C. Sodium nitrite (10 mg) and
acetic acid (2 ml) were added and the reaction was allowed to
proceed for 1 hour at 0.degree. C. Dialysis was performed overnight
with a 50,000 MWCO membrane with the addition of 1 g NaCl to the
dialysis liquid. Freezing and lyophilization of the retentate
produced a fine powder.
Example 2
[0066] This example describes the construction of heparin entities
comprising heparin and neomycin sulfate as the core. This heparin
entity contains free terminal aldehydes that can be used for
attachment to a surface of a substrate.
[0067] Neomycin sulfate (0.0646 g, Spectrum Chemical) was dissolved
in 300 ml of DI water containing MES buffer (pH 4.7, BupH.TM.
Thermo Scientific). To this was added 10 g USP heparin, 4 g
N-hydroxysulfosuccinimide (sulfo-NHS), and 4 g of EDC
hydrochloride. The reaction was allowed to proceed at room
temperature for 4 hours, followed by dialysis overnight with a
50,000 MWCO membrane (Spectra/Por.RTM.). The retentate (about 400
ml out of 505 ml) was transferred to a beaker and cooled to
0.degree. C. Sodium nitrite (10 mg) and acetic acid (2 ml) were
added and the reaction was allowed to proceed for 1 hour at
0.degree. C. Dialysis was performed overnight with a 50,000 MWCO
membrane with the addition of 1 g NaCl to the dialysis liquid. The
dialyzed retentate was filtered twice using a 20 micrometer,
0.00079 inches U.S.A. standard testing sieve, A.S.T.M.E.-11
specification NO. 635 to remove small particles. Freezing of the
filtrate and lyophilization produced a fine powder.
Example 3
[0068] This example describes the construction of heparin entities
comprising heparin and capreomycin sulfate as the core. This
heparin entity contains free terminal aldehydes that can be used
for attachment to a surface of a substrate.
[0069] Capreomycin sulfate (0.0501 g, Sigma-Aldrich, St. Louis,
Mo.) was dissolved in 300 ml of DI water containing MES buffer (pH
4.7, BupH.TM. Thermo Scientific). To this was added 10 g USP
heparin, 4 g N-hydroxysulfosuccinimide (sulfo-NHS), and 4 g of EDC
hydrochloride. The reaction was allowed to proceed at room
temperature for 4 hours. The reaction mixture was filtered once
using a 20 micrometer, 0.00079 inches U.S.A. standard testing
sieve, A.S.T.M.E.-11 specification NO. 635 to remove small
particles and the filtrate was dialyzed overnight with a 50,000
MWCO membrane (Spectra/Por.RTM.). The retentate (about 400 ml out
of 515 ml) was transferred to a beaker and cooled to 0.degree. C.
Sodium nitrite (10 mg) and acetic acid (2 ml) were added and the
reaction was allowed to proceed for 1 hour at 0.degree. C. Dialysis
was performed overnight with a 50,000 MWCO membrane with the
addition of 1 g NaCl to the dialysis liquid. The retentate was
filtered twice using a 20 micrometer, 0.00079 inches U.S.A.
standard testing sieve, A.S.T.M.E.-11 specification NO. 635 to
remove small particles. Freezing of the filtrate and lyophilization
produced a fine powder.
Example 4
[0070] This example describes the construction of heparin entities
comprising heparin and poly-L-lysine as the core. This heparin
entity contains free terminal aldehydes that can be used for
attachment to a surface of a substrate.
[0071] Poly-L-lysine (0.1776 g, Sigma-Aldrich, molecular weight
1,000 to 5,000 g/mole) was dissolved in 300 ml of DI water
containing MES buffer (pH 4.7, BupH.TM. Thermo Scientific). To this
was added 10 g USP heparin, 4 g N-hydroxysulfosuccinimide
(sulfo-NHS), and 4 g of EDC hydrochloride. The reaction was allowed
to proceed at room temperature for 4 hours followed by dialysis
overnight with a 50,000 MWCO membrane (Spectra/Por.RTM.). The
retentate (about 400 ml out of 505 ml) was transferred to a beaker
and cooled to 0.degree. C. Sodium nitrite (10 mg) and acetic acid
(2 ml) were added and the reaction was allowed to proceed for 1
hour at 0.degree. C. Dialysis was performed overnight with a 50,000
MWCO membrane with the addition of 1 g NaCl to the dialysis liquid.
Freezing of the retentate and lyophilization produced a fine
powder.
Example 5
[0072] This example describes the construction of heparin entities
comprising heparin and polyethyleneimine (PEI) as the core. This
heparin entity contains free terminal aldehydes that can be used
for attachment to a surface of a substrate.
[0073] PEI (Lupasol, BASF, 1.7756 g) was dissolved in 300 ml of DI
water containing MES buffer (pH 4.7, BupH.TM. Thermo Scientific).
To this was added 10 g USP heparin, 4 g N-hydroxysulfosuccinimide
(sulfo-NHS), and 4 g of EDC hydrochloride. The reaction was allowed
to proceed at room temperature for 4 hours followed by dialysis
overnight with a 50,000 MWCO membrane (Spectra/Por.RTM.). The
retentate (about 400 ml out of 505 ml) was transferred to a beaker
and cooled to 0.degree. C. Sodium nitrite (10 mg) and acetic acid
(2 ml) were added and the reaction was allowed to proceed for 1
hour at 0.degree. C. Dialysis was performed overnight with a 50,000
MWCO membrane with the addition of 1 g NaCl to the dialysis liquid.
Freezing of the retentate and lyophilization produced a fine
powder.
Example 6
[0074] This example describes the construction of heparin entities
comprising heparin and ethylene diamine (EDA) as the core. This
heparin entity contains free terminal aldehydes that can be used
for attachment to a surface of a substrate.
[0075] EDA (0.0043 g, Sigma-Aldrich, St. Louis, Mo.) was
neutralized to a pH of 4.7 with equal volume dilution of HCl and DI
water, with the use of an ice bath, then dissolved in 300 ml of DI
water containing MES buffer (pH 4.7, BupH.TM. Thermo Scientific).
To this was added 10 g USP heparin, 4 g N-hydroxysulfosuccinimide
(sulfo-NHS), and 4 g of EDC hydrochloride. The reaction was allowed
to proceed at room temperature for 4 hours followed by dialysis
overnight with a 50,000 MWCO membrane (Spectra/Por.RTM.). The
retentate (about 400 ml out of 505 ml) was transferred to a beaker
and cooled to 0.degree. C. Sodium nitrite (10 mg) and acetic acid
(2 ml) were added and the reaction was allowed to proceed for 1
hour at 0.degree. C. Dialysis was performed overnight with a 50,000
MWCO membrane with the addition of 1 g NaCl to the dialysis liquid.
Freezing of the retentate and lyophilization produced a fine
powder.
Example 7
[0076] The heparin entities containing free terminal aldehydes of
Examples 1 through 6 were immobilized onto the surface of an ePTFE
substrate and tested for ATIII activity.
[0077] An ePTFE substrate material in sheet form was obtained from
W.L. Gore & Associates, Inc., Flagstaff, Ariz. under the trade
name GORE.TM. Microfiltration Media (GMM-406). A covering material
in the form of a base coating was applied to the ePTFE material by
mounting the material on a ten centimeter (10 cm) diameter plastic
embroidery hoop and immersing the supported ePTFE material first in
100% isopropyl alcohol (IPA) for about five minutes (5 min) and
then in a solution of polyethylene imine (PEI, Lupasol, BASF) and
IPA in a one to one ratio (1:1). LUPASOL.RTM. water-free PEI was
obtained from BASF and diluted to a concentration of about four
percent (4%) and adjusted to pH 9.6. Following immersion of the
ePTFE material in the solution for about fifteen minutes (15 min),
the material was removed from the solution and rinsed in DI water
at pH 9.6 for 15 min. PEI remaining on the ePTFE material was
cross-linked with a 0.05% aqueous solution of glutaraldehyde
(Amresco) at pH 9.6 for 15 min. Additional PEI was added to the
construction by placing the construction in a 0.5% aqueous solution
of PEI at pH 9.6 for 15 min and rinsing again in DI water at pH 9.6
for 15 min. The imine formed as a result of the reaction between
glutaraldehyde and the PEI layer is reduced with a sodium
cyanborohydride (NaCNBH.sub.3) solution (5 g dissolved in 1 L DI
water, pH 9.6) for 15 min and rinsed in DI water for thirty minutes
(30 min).
[0078] An additional layer of PEI was added to the construction by
immersing the construction in 0.05% aqueous glutaraldehyde solution
at pH 9.6 for 15 min, followed by immersion in a 0.5% aqueous
solution of PEI at pH 9.6 for 15 min. The construction was then
rinsed in DI water at pH 9.6 for 15 min. The resultant imines were
reduced by immersing the construction in a solution of NaCNBH.sub.3
(5 g dissolved in 1 L DI water, pH 9.6) for 15 min followed by a
rinse in DI water for 30 min. A third layer was applied to the
construction by repeating these steps. The result was a porous
hydrophobic fluoropolymeric base material, or disk having a
hydrophilic cross-linked polymer base coat on substantially all of
the exposed and interstitial surfaces of the base material.
[0079] An intermediate chemical layer was attached to the polymer
base coat in preparation for placement of another layer of PEI on
the construction. The intermediate ionic charge layer was made by
incubating the construction in a solution of dextran sulfate
(Amersham Pharmacia Biotech) and sodium chloride (0.15 g dextran
sulfate and 100 g NaCl dissolved in 1 L DI water, pH 3) at
60.degree. C. for ninety minutes (90 min) followed by rinsing in DI
water for 15 min.
[0080] A layer of PEI, referred to herein as a "capping layer" was
attached to the intermediate layer by placing the construction in a
0.3% aqueous solution of PEI (pH 9) for about forty-five minutes
(45 min) followed by a rinse in a sodium chloride solution (50 g
NaCl dissolved in 1 L DI water) for twenty minutes (20 min). A
final DI water rinse was conducted for 20 min.
[0081] The heparin entities containing free terminal aldehydes of
Examples 1 through 6 were attached, or conjugated, to the PEI
layer(s) by placing the construction in a heparin entity-containing
sodium chloride salt solution (approximately 0.9 g of heparin
entity containing free terminal aldehydes, 5.88 g NaCl dissolved in
200 ml DI water, pH 3.9) and kept for ten minutes (10 min) at
60.degree. C. A 572 .mu.L volume of a 2.5% (w/v) aqueous
NaCNBH.sub.3 solution was added to the (200 ml) heparin entity
solution. Samples were kept for additional one hundred ten minutes
(110 min) at the above temperature.
[0082] The samples were then rinsed in DI water for 15 min, borate
buffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl
dissolved in 1 L DI water, pH 9.0) for 20 min, and finally in DI
water for 15 min followed by lyophilization of the entire
construction to produce a dry construct comprising a heparin entity
bound to the surface of the ePTFE substrate material. The presence
and uniformity of the macromolecular construct of heparin was
determined by staining samples of the construction on both sides
with toluidine blue. The staining produced an evenly stained
surface indicating heparin was present and uniformly bound to the
ePTFE material.
[0083] Samples approximately one square centimeter (1 cm.sup.2) in
nominal size were cut from the construction and assayed for heparin
activity by measuring the ATIII binding capacity of the heparin
entities containing free terminal aldehydes that were end-point
attached onto the surface of the ePTFE substrate. The assay is
described by Larsen M. L., et al., in "Assay of plasma heparin
using thrombin and the chromogenic substrate H-D-Phe-Pip-Arg-pNA
(S-2238)." Thromb Res 13:285-288 (1978) and Pasche B., et al., in
"A binding of antithrombin to immobilized heparin under varying
flow conditions." Artif. Organs 15:281-491 (1991), both of which
are incorporated by reference herein for all purposes. The results
were expressed as amount of ATIII bound per unit surface area
substrate material in picomoles per square centimeter
(pmol/cm.sup.2). All samples were maintained in a wet condition
throughout the assay. It is important to note that while the
approximately one square centimeter (1 cm.sup.2) samples each have
a total surface area of two square centimeters (2 cm.sup.2) if both
sides of the material are considered, only one surface on the
sample (i.e., 1 cm.sup.2) was used for calculating ATIII heparin
entity-binding activity in pmol/cm.sup.2.
[0084] Lyophilized samples representing each conjugated constructs
produced in Examples 1 through 6 were placed in an individual Tower
DUALPEEL.RTM. Self Sealing Pouch (Allegiance Healthcare Corp.,
McGraw Park, Ill.) and sealed for EtO sterilization. Ethylene oxide
sterilization was carried out under conditions of conditioning for
one hour (1 hr), an EtO gas dwell time of 1 hr, a set point
temperature of 55.degree. C., and an aeration time of twelve hours
(12 hrs). Sterilization with EtO was repeated up to 3 times with
samples taken after each EtO sterilization.
[0085] FIG. 2 is a bar graph illustrating the ATIII binding
capacity of heparin entities containing free terminal aldehydes
from Examples 1 through 6 immobilized onto an ePTFE surface and
having undergone up to three EtO sterilization cycles.
Anti-thrombin III binding activity is expressed as picomoles of
bound anti-thrombin III per square centimeter of substrate
material. As seen from the results, all conjugated heparin entities
containing free terminal aldehydes resulted in high anti-thrombin
III binding activity before sterilization and following up to three
EtO sterilizations. All bars represent mean values of sample
numbers with error bars for the standard deviation.
Example 8
[0086] The heparin entities containing free terminal aldehydes
produced in Examples 2, 3, 4, and 6 were analyzed in order to
determine their absolute molecular weights.
[0087] A Waters 2414 RI detector in conjunction with Wyatt ASTRA
5.3.4.10 software was used to determine the dn/dc for USP heparin
in 100 mM NaNO.sub.3 with 0.02% NaN.sub.3 at a laser wavelength of
660 nm. The dn/dc (change in refractive index divided by change in
concentration) for USP heparin was determined by plotting known
concentrations of heparin versus the RI detector response and
calculating the slope.
[0088] The heparin entities were analyzed with a Wyatt-Dawn
Helleos-II 18-angle light scattering detector (Wyatt Technology
Corp.) for measurement of absolute molecular weight, with detectors
1, 2, 3, 4, 17, and 18 not utilized. A stock solution of the
heparin entity was prepared in 100 mM NaNO.sub.3 with 0.02%
NaN.sub.3 mobile phase. From this stock solution the following
concentrations were made: 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0
mg/mL, and 2.5 mg/mL for the heparin entities of Examples 3 and 4.
For the heparin entities of Examples 2 and 6, concentrations of
0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, and 2.0 mg/mL were
made. Each sample was filtered with a 0.02 micron syringe filter
using a 5 ml syringe prior to injection into the light scattering
detector. Batch data analysis was performed on all samples using a
Zimm plot and the do/dc for USP heparin (0.126 L/g). Table 1
depicts the absolute molecular weights.
TABLE-US-00001 TABLE 1 Absolute Molecular Weight Values for Heparin
Entities Core Example # Molecule Mw (g/mol) 2 Neomycin 18,570 3
Capreomycin 17,710 4 Poly-L-Lysine 20,300 6 EDA 21,850 USP Heparin
14,810
[0089] All heparin entities analyzed for absolute molecular weight
showed values larger than USP heparin (14,810 g/mol), with the
values ranging from 17,710 g/mol for the heparin entity comprising
heparin and capreomycin as the core, to 21,850 g/mol for the
heparin entity comprising heparin and EDA as the core.
Example 9
[0090] This example demonstrates a detection method for discerning
the method of attachment of heparin or a heparin entity onto a
surface of a substrate. Specifically, this example looks at
attachment of heparin and heparin entities via immobilization onto
an ePTFE substrate, using a single point attachment comprising
free-terminal aldehydes, and using a multi-point attachment
comprising carbodiimide conjugation.
[0091] Heparin end-point aldehyde was made according to U.S. Pat.
No. 4,613,665 and immobilized onto PEI-ePTFE substrates as
described in Example 7. This produced a surface in which the
heparin was immobilized by end-point attachment. Heparin attachment
was demonstrated by staining a sample with toluidine blue and
noting the coloration, as shown in FIG. 3A.
[0092] A surface was also produced in which the heparin end-point
aldehyde was attached not by the free terminal aldehyde, but by
multiple carboxylic acid residues along the heparin chain length
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). EDC
conjugation of heparin onto a surface is known to bind the heparin
through a multiplicity of sites. A PEI containing disk of Example 7
was immersed into 300 ml of 0.1 MES buffer (pH 4.7). To this
solution, 1 gram of heparin with end-point aldehydes and 4 grams
EDC hydrochloride was added. The reaction was allowed to proceed at
room temperature for 4 hours. The immobilized heparin disk was
rinsed with DI water, borate buffer, and a final DI water
rinse.
[0093] A surface was also produced in which USP heparin containing
no free terminal aldehydes was attached by EDC through multiple
bond sites on the surface. A PEI containing disks of Example 7 was
immersed into 300 ml of 0.1 MES buffer (pH 4.7). To the solution, 1
gram USP heparin and 4 grams EDC hydrochloride was added. The
reaction was allowed to proceed at room temperature for 4 hours.
The immobilized heparin disk was rinsed with DI-water, borate
buffer, and a final DI water rinse.
[0094] The heparin entity of Example 2 immobilized onto ePTFE/PEI
as described in Example 7 was also produced. Alternatively, the
heparin entity of Example 2 was immobilized onto ePTFE/PEI using
carbodiimide conjugation. A PEI containing disk of Example 7 was
immersed into 300 ml of 0.1 MES buffer (pH 4.7). To the solution, 1
gram of the heparin entity of Example 2 and 4 grams EDC
hydrochloride was added. The reaction was allowed to proceed at
room temperature for 4 hours. The immobilized heparin entity disk
was rinsed with DI water, borate buffer, and a final DI water
rinse.
[0095] To demonstrate that heparin and heparin entities were
immobilized by each technique described above, samples
approximately 1.times.1 cm were stained with toluidine blue. It was
noted that all samples stained with toluidine blue similar to FIG.
3A (which depicts USP heparin end-point aldehyde immobilized by
end-point attachment).
[0096] For each of the various ePTFE-PEI disks conjugated with
heparin and heparin entity, a 2.times.2 cm square was cut and
placed in a 1.5 ml vial. To this was added 1 ml of heparinase-1
(from Flavobacterium heparinum, E.C. 4.2.2.7, Sigma-Aldrich, St.
Louis, Mo.) diluted to 1 mg/mL in the following buffer: 20 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 4 mM CaCl2, 0.01% BSA. The sample was
incubated for 30 minute at room temperature, rinsed with DI-water,
and stained with toluidine blue. Samples that where end-point
attached, and not multi-point attached, appeared substantially less
stained, as shown in FIG. 3B for USP heparin end-point aldehyde
immobilized by end-point attachment. Multi-point immobilized
heparin entities retained stain.
[0097] Quantitation of the staining was performed utilizing
luminosity measurements for each of the samples. Samples were
mounted onto glass slides and secured with a single strip of
adhesive tape. Digital images were taken with an Olympus SZX12
microscope (Olympus America Inc.) equipped with an Olympus DP71
digital camera controlled with DP Controller 3.1.1.267 software.
Images were captured using a 1.times. lens at 7.times.
magnification with exposure set to 1/350 sec and lighted with an
overhead ring-light. Before capture of final images, images were
examined to ensure saturation was not exceeded. It is important to
note that stained samples, i.e., those that stained substantially
with toluidine blue, produced low luminosity values, while those
that did not stain substantially produced high luminosity values
(the luminosity scale for this example ranged from 0 to 255).
[0098] The luminosity of each captured digital image was assessed
using Adobe Photoshop Elements 2.0 (Adobe Systems Inc., San Jose,
Calif.). Within Adobe Photoshop Elements 2.0 the image was loaded
(resolution of 144 pixels/inch) and a representative rectangular
region of the sample was outlined using the rectangular marquee
tool. From the top tool bar, image was selected followed by
selection of histogram. The histogram window opened, with the
channel set to luminosity. The mean is recorded as the mean
luminosity.
[0099] All samples conjugated with heparin and heparin entity
(tabulated with luminosity values in Table 2) stained substantially
with toluidine blue, indicating dense coverage of attached heparin
and heparin entity on the substrate. Luminosity values after
immobilization and staining ranged from 27.3 for heparin end-point
aldehyde immobilized by end-point attachment to 139.1 for USP
heparin immobilized by multi-point attachment with EDC. After
heparinase-1 treatment and staining, luminosity values increased
for all samples, indicating a loss of heparin and a consequential
decrease in staining and in coloration. For samples more sensitive
to heparinase-1, the change is more significant. This change is
demonstrated in a graph of normalized change in luminosity. The
term "normalized change in luminosity" is defined as the luminosity
value after immobilization subtracted from the luminosity value
after heparinase-1 treatment divided by the luminosity after
immobilization value, with the resultant multiplied by 100, i.e.,
{[(luminosity(post heparinase)-luminosity(pre
heparinase)]=luminosity(pre heparinase)}*100. Normalized change in
luminosity for each of the samples in Table 2 is shown in FIG. 3C,
displayed as a function of heparin entity type and immobilization
attachment method. The normalized change in luminosity of heparin
end-point aldehyde was dependent upon the immobilization attachment
method, with end-point attachment giving a value of 603 and
multi-point attachment giving a value of 66. This dependency was
observed for the heparin entity of heparin and neomycin with an
end-point attachment value of 231 and multi-point attachment of 16.
USP heparin with multi-point attachment also exhibited a low
normalized change in luminosity with a value of 14. Low values in
normalized change in luminosity indicated a surface resistant to
heparinase-1 and hence small quantities of heparin removed.
[0100] The heparinase-1 was effective at removing the heparin or
heparin entity from the surface, as indicated by a lack of
substantial staining by toluidine blue and a consequential lack of
coloration, and a consequential high value for normalized change in
luminosity. Heparinase-1 was utilized to discern whether heparin or
a heparin entity was attached via free terminal aldehydes or via
multi-point attachment using carbodiimide conjugation.
TABLE-US-00002 TABLE 2 Luminosity Values Luminosity after
Luminosity after Immobilization Immobilization & Heparinase-1
Heparin Entity Method Staining & Staining Heparin end-
End-point 27.3 192.3 point aldehyde Heparin end- EDC multi- 73.6
122.8 point aldehyde point USP heparin EDC multi- 139.1 158.9 point
Heparin and EDC multi- 82.8 96.2 neomycin core point Example 2
Heparin and End-point 57.0 189.3 neomycin core Example 2
Example 10
[0101] This example demonstrates a detection method for discerning
the method of attachment of a heparin entity onto a surface of a
substrate after sterilization. Specifically, this example looks at
attachment of heparin entities via immobilization onto an ePTFE
substrate, using a single point attachment comprising free-terminal
aldehydes followed by sterilization and a boric acid rinse.
[0102] The heparin entity of Example 6 was immobilized onto ePTFE
as described in Example 7. Samples where shown to have good heparin
coverage as indicated by toluidine blue staining (as shown in FIG.
4A). Other end-point attached samples were sterilized, as described
in Example 7, via 3 cycles of EtO, as described in Example 7. A
portion of these samples was rinsed in DI water for 15 min, borate
buffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl
dissolved in 1 L DI water, pH 9.0) for 20 min, and finally in DI
water for 15 min after sterilization and before toluidine blue
staining and measuring luminosity as described in Example 9.
[0103] Samples having undergone sterilization but no boric acid
rinse stained with toluidine blue before heparinase-1 treatment
(FIG. 4B) and after (FIG. 4C). Both samples indicate the presence
of heparin entity by coloration and low luminosity values of 31.7
and 85.6, respectively. Sterilization has appeared to diminish the
ability of heparinase-1 to depolymerize the heparin entity bound by
the free terminal aldehyde, as compared to heparin entity that was
not sterilized.
[0104] Samples having undergone sterilization and boric acid rinse
were stained with toluidine blue before heparinase-1 treatment
(FIG. 4D) and after (FIG. 4E). Dense heparin entity coverage was
indicated before heparinase-1 treatment by toluidine blue stain and
a luminosity value of 54.4, while the sample receiving the boric
acid rinse and heparinase-1 had essentially no toluidine blue stain
and a luminosity value of 186.3, indicating substantial heparinase
sensitivity of attached heparin entity.
[0105] This example shows that boric acid restored heparin
conformation of the attached heparin entity, exemplified by high
ATIII specificity and heparinase sensitivity. Without wishing to be
bound by theory, it is hypothesized sterilization altered the
conformation of the immobilized heparin entity layer, substantially
reducing specificity for ATIII (as evidenced by low activity) and
reducing heparinase sensitivity (as evidenced by substantial
staining with toluidine blue). It is further hypothesized the boric
acid rinse restored conformation to the attached heparin entity
layer that was altered by sterilization. Restoration of
conformation resulted in sensitivity of the attached heparin entity
to heparinase-1 depolymerization, as shown by lack of staining in
FIG. 4E. It is further hypothesized that if an attached heparin
entity has a conformation that heparinase-1 recognizes, then ATIII
will recognize the attached heparin entity, and visa versa.
Example 11
[0106] This example demonstrates a detection method for determining
the composition of the heparin entities using oligosaccharide
mapping of heparinase-1 depolymerized heparin entities with strong
anion exchange-high performance liquid chromatography
(SAX-HPLC).
[0107] USP Heparin and the heparin entities of Examples 1 and 2
were dissolved at 0.1 mg in 100 .mu.l of 50 mM acetate buffer, pH
7.3, containing 2.5 mmol of calcium acetate. The USP heparin and
the heparin entities of Examples 1 and 2 were depolymerized to
their constituent oligosaccharides by the addition of 6 milliunits
of heparinase-1 for 15 hrs at 30.degree. C., and flash frozen at
-85.degree. C.
[0108] Analysis of the oligosaccharides from each sample were
performed by SAX-HPLC and quantified at 232 nm using a 5 micron SAX
column (150.times.4.6 mm; Spherisorb, Waters). Isocratic separation
was performed from 0 to 5 min with 50 mM NaCl, pH 4.0, and linear
gradient separation was performed from 5 to 90 min with 100% 50 mM
NaCl, pH 4.0, to 100% 1.2 M NaCl, pH 4.0, at a flow of 1.2
mL/min.
[0109] FIG. 5 shows qualitative maps of (A) depolymerized USP
heparin, (B) the depolymerized heparin entity of Example 1
comprising heparin and a core comprising colistin sulfate, and (C)
the depolymerized heparin entity of Example 2 comprising heparin
and a core comprising neomycin sulfate. The chromatogram for USP
heparin was the base line case and served as a standard of
reference for the heparin entities of Examples 1 and 2. Each peak
in FIG. 5A represents a unique depolymerized oligosaccharide
fragment characteristic of USP heparin. New peaks, as indicated by
the vertical arrows in FIGS. 5B and C, represent novel
oligosaccharides units distinct from USP heparin, and hence,
allowed the identification of heparin entities through these
distinct signature peaks.
[0110] For the heparin entity of Example 1 comprising heparin and a
core comprising colistin sulfate, the chromatogram of FIG. 5B
exhibits at least 3 distinct peaks relative to the USP heparin
chromatogram at 15.581, 24.699, and 35.023 minutes (shown by
vertical arrows). Structurally, these new peaks are related to the
core molecule colistin sulfate utilized in the construction of the
heparin entity. When the heparin entity was depolymerized with
heparinase-1, new structurally distinct polysaccharide units that
contained the core molecule colistin sulfate were produced.
[0111] For the heparin entity of Example 2 comprising heparin and a
core comprising neomycin sulfate, the chromatogram of FIG. 5C
exhibits at least 3 distinct peaks relative to the USP heparin
chromatogram at 8.276, 25.386, and 34.867 minutes (shown by
vertical arrows). Structurally, these new peaks are related to the
core molecule neomycin sulfate utilized in the construction of the
heparin entity. When the heparin entity was depolymerized with
heparinase-1, new structurally distinct polysaccharide units that
contained the core molecule neomycin sulfate were produced.
Example 12
[0112] This example demonstrates a detection method for determining
the composition of heparin entities immobilized on a surface using
oligosaccharide mapping of heparinase-1 depolymerized heparin
entities with strong anion exchange-high performance liquid
chromatography (SAX-HPLC).
[0113] Heparin comprising free-terminal aldehydes was immobilized
onto disks of ePTFE/PEI according to Example 9. The heparin entity
of Example 1 was immobilized onto disks of ePTFE/PEI according to
Example 7. Samples of approximately 4 cm.sup.2 of each disk were
placed in individual tubes. These samples were depolymerized to
their constituent oligosaccharides by the addition of 1 ml of
acetate buffer (consisting of 50 mM sodium acetate, 2.5 mM calcium
acetate, pH 7.3) to each tube along with 60 .mu.l of heparinase-1
solution. The heparinase-1 solution comprised acetate buffer (50 mM
sodium acetate, 2.5 mM calcium acetate, pH 7.3) with heparinase-1
(EC 4.2.2.7, Sigma-Aldrich) at a concentration of 1.67 IU/ml. Tubes
were incubated at 30.degree. C. for 18 hours, and liquid samples of
approximately 0.5 ml were taken and flash frozen at -85.degree. C.
for SAX-HPLC analysis as described in Example 11.
[0114] FIG. 6 shows the qualitative SAX-HPLC maps of surface
depolymerized (A) heparin comprising free-terminal aldehydes
immobilized on ePTFE and (B) a heparin entity constructed of
heparin and a core of colistin sulfate immobilized on ePTFE. The
chromatogram for heparin comprising free-terminal aldehydes
immobilized on ePTFE was the base line case and served as a
standard of reference for the heparin entity of Example 1. Each
peak in FIG. 6A represents a unique oligosaccharide that is
characteristic for heparin comprising free-terminal aldehydes
immobilized on ePTFE. New peaks, as indicated by arrows in FIG. 6B,
represent additional oligosaccharides units distinct from heparin
comprising free-terminal aldehydes immobilized on ePTFE, and hence,
identify the heparin entity of heparin and a core of colistin
sulfate immobilized on ePTFE.
Example 13
[0115] This example describes the construction of a heparin entity
comprising heparin and a core comprising poly-L-lysine. This
heparin entity does not contain free terminal aldehydes that can be
used for attachment to a surface of a substrate. This heparin
entity can be used for attachment to a surface of a substrate
through ionic bonding.
[0116] Poly-L-lysine hydrobromide with molecular weight of 1,000 to
5,000 (0.1776 g, Sigma-Aldrich, St. Louis, Mo.) was dissolved in
300 ml of DI water containing MES buffer (pH 4.7, BupH.TM. Thermo
Scientific) and pH adjusted to 4.7. To this was added, 10 g USP
heparin, 4 g N-hydroxysulfosuccinimide (sulfo-NHS), and 4 g of EDC
hydrochloride. The reaction was allowed to proceed at room
temperature for 4 hours followed by dialysis overnight with 50,000
MWCO membrane (Spectra/Por.RTM.). The retentate was transferred to
50 ml tubes, flash frozen, and lyophilized to produce a fine
powder. This powdered product was further used to immobilize the
construct of heparin and a core of poly-L-lysine on an ePTFE sheet
material through ionic bonding.
Example 14
[0117] The heparin entity of Example 13 was immobilized on the
surface of the substrate ePTFE through ionic bonding.
[0118] Disks of ePTFE/PEI were prepared according to Example 7. The
heparin entity of Example 13 containing a core of poly-L-lysine and
no free terminal aldehydes, was attached, via ionic bonding, to the
PEI layer(s) by placing 5 1.times.1 cm square ePTFE samples of the
construction in a heparin entity-containing sodium chloride salt
solution (approximately 0.247 g of heparin containing a core of
poly-L-lysine containing no aldehydes, 0.16 g sodium citrate tri
basic dehydrate, and 1.607 g NaCl dissolved in 55 ml DI water, pH
3.9) and kept for one hundred and twenty minutes (120 min) at
60.degree. C.
[0119] The samples were then rinsed in DI water for 15 min, borate
buffer solution (10.6 g boric acid, 2.7 g NaOH and 0.7 g NaCl
dissolved in 1 L DI water, pH 9.0) for 20 min, and finally in DI
water for 15 min followed by lyophilization of the entire
construction to produce dry heparin bound to the ePTFE material.
The presence and uniformity of the heparin containing a core of
poly-L-lysine was determined by staining samples of the
construction on both sides with toluidine blue. The staining
produced an evenly stained surface indicating heparin was present
and uniformly bound to the ePTFE material.
Example 15
[0120] This example demonstrates a detection method for determining
the conjugation method for immobilizing heparin and heparin
entities on a surface. Specifically, this example looks at the
detection of surface-bonded unsaturated heparin fragments, or
"nubs," on the surface of immobilized heparin or heparin entities
after heparinase-1 depolymerization. Heparinase-1 depolymerization
of heparin involves an enzymatic cleavage of heparin's non-reducing
terminal uronic acid to a 4,5-unsaturated derivative that can react
with various detection molecules, such as a thiol-terbium
fluorescent molecule. A negative control of ionic bound heparin
(Example 14) is included.
[0121] A thiol-terbium based florescent molecule was utilized. 5
grams hydroxypropyl .beta.-cyclodextrin was dissolved into 50 ml
DI-water, and 0.03894 grams terbium tris(4-methylthio) benzoate
[Tb(4MTB.sub.3)] was dissolved into 10 ml N,N-dimethylacetamide
(DMAc). The Tb(4MTB.sub.3) solution was then added drop wise into
the hydroxypropyl .beta.-cyclodextrin solution, yielding a clear
colorless solution. The solution was then filtered through a 0.22
.mu.m Sterix filter cartridge before use.
[0122] Samples of 1 cm.times.1 cm ePTFE coated substrates of
Example 14, and 1cm.times.1 cm samples of the heparin entity of
Example 1, comprising heparin and a core comprising colistin
sulfate, immobilized according to Example 7, were depolymerized
with heparinase-1 before reaction with thiol-terbium. For
comparison, heparin end-point aldehyde (made according to U.S. Pat.
No. 4,613,665) immobilized onto PEI-ePTFE substrates in accordance
with Example 7, was also utilized; this produced a surface in which
the heparin was immobilized by end-point attachment.
[0123] Samples were depolymerized with 100 units heparinase-1 (EC
4.2.2.7, Sigma-Aldrich) diluted in 1 ml of buffer (20 mM tris, 50
mM NaCl, 10 mM CaCl.sub.2, 0.01% BSA, and pH 7.5) for 35 min on a
shaker at room temperature. This resulted in small fragment "nubs"
of surface-bonded unsaturated heparin fragments bound to the
surface of the ePTFE substrate. The samples were then rinsed in DI
water for 15 min, borate buffer solution (10.6 g boric acid, 2.7 g
NaOH and 0.7 g NaCl dissolved in 1 L DI water, pH 9.0) for 20 min,
and stored in DI-water until used for final analysis. Fluorescence
labeling of samples, through a Michael-like addition of the
thiol-terbium compound to the unsaturated heparin fragment bound to
the surface of the ePTFE substrate, was performed by placing each
sample into vials containing Tb(4MTB.sub.3)/hydroxypropyl
.beta.-cyclodextrin solution, purged with nitrogen for 1 minute,
capped, and incubated overnight at 70.degree. C. Samples were
removed from vials, rinsed with 10 wt % hydroxypropyl
.beta.-cyclodextrin in DI-water, and placed on a glass microscope
slide for imaging.
[0124] Imaging of samples was performed with a Nikon E-6000
microscope using an Ocean Optics Deuterium short-wavelength
excitation source at an oblique angle. Both white light and UV
excitation fluorescence images were taken using a FITC filter cube.
All samples were maintained in a wet state during imaging to
minimize background scattering, and imaged with a black and white
camera. Samples excited with UV light were imaged, and green
tinting was artificially added to the image for visualization
purposes.
[0125] Distinct UV fluorescence, and hence the detection of
surface-bonded unsaturated heparin fragments bound to the surface
of the ePTFE substrate ("nubs"), was noted for the end-point
aldehyde heparin and heparin entity comprising heparin and a core
comprising colistin sulfate samples. An absence of UV fluorescence
was noted for the macromolecular construct of ionically bound
heparin and poly-L-lysine containing no aldehydes.
Example 16
[0126] This example describes the construction and utilization of
an embodiment of the present invention in which high heparin
anti-thrombin III (ATIII) binding is present for a heparin entity
comprising heparin and a core comprising an amine-containing
fluoropolymer. This heparin entity contains free terminal aldehydes
that can be used for attachment to a surface of a substrate.
[0127] The amine-containing fluoropolymer was prepared using the
following conditions. A copolymer comprising a mole ratio of 20:80
tetrafluoroethylene and vinyl acetate was prepared. To a nitrogen
purged 1 L pressure reactor under vacuum were added 500 g DI water,
2 g of 20% aqueous ammonium perfluorooctanoate, 30 ml of distilled
vinyl acetate, 10 g of n-butanol, and 0.2 g of ammonium persulfate.
Tetrafluoroethylene monomer was then fed into the reactor until the
reactor pressure reached 1500 KPa. The mixture was stirred and
heated to 50.degree. C. When a pressure drop was observed, 25 ml of
vinyl acetate was slowly fed into the reactor. The reaction was
stopped when the pressure dropped another 150 KPa after vinyl
acetate addition. The copolymer was obtained from freeze-thaw
coagulation of the latex emulsion and cleaned with methanol/water
extraction. The copolymer then was hydrolyzed. To a 50 ml round
bottle flask were add 0.5 g of the copolymer, 10 ml methanol and
0.46 g NaOH in 2 ml DI water. The mixture was stirred and heated to
60.degree. C. for 5 hrs. The reaction mixture was then acidified to
pH 4 and precipitated in DI water. The hydrolyzed copolymer was
then acetalized. The hydrolyzed copolymer was dissolved in methanol
at 2.5% w/v. To 50 g of this solution was added 33 ml of DI water
with vortexing to produce a homogeneous solution. To this solution
was added 0.153 g of aminobutyraldehyde dimethyl acetal, and 0.120
ml of a 37% HCl solution. The solution was reacted with stirring
under nitrogen, 80.degree. C., for 48 hrs. Sodium hydroxide from a
1M solution was added drop wise to a pH of about 9.0. The resulting
copolymer of poly(tetrafluoroethylene-co-vinyl
alcohol-co-vinyl[aminobutyraldehyde acetal]) (TFE-VOH-AcAm) was
recovered by precipitation into copious DI water. The precipitate
was filtered, redissolved into methanol, and reprecipitated into
copious DI water for two more cycles. The final product was dried
under vacuum at 60.degree. C. for 3 hrs. FTIR and carbon NMR
confirmed a polymer structure of poly(tetrafluoroethylene-co-vinyl
alcohol-co-vinyl[aminobutyraldehyde acetal]).
[0128] 48 mg of aldehyde-modified-heparin (made according to U.S.
Pat. No. 4,613,665) was dissolved in 30 ml of DI water. To this
solution was added 86 .mu.l of 2.5% sodium cyanoborohydride
solution (Aldrich) and the pH was adjusted to 3.8 with HCl.
Separately, the TFE-VOH-AcAm copolymer was dissolved in superheated
methanol at 2.5% w/v and then cooled to room temperature. To 20 ml
of the TFE-VOH-AcAm solution was added 13 ml of the heparin
solution drop wise, to produce a slightly milky emulsion. The
emulsion was maintained at 60.degree. C. for 2.5 hrs and then at
room temperature for an additional 2 hrs. The emulsion was dialyzed
against DI water using a 50KDa membrane (SpectraPor) for 18 hrs,
flash frozen at -80.degree. C. and then lyophilized to a powder. 10
mg of the powder was suspended in 2.5 ml of ice cold DI water
supplemented with 0.1 mg sodium nitrite (Sigma) and 20 .mu.l of
acetic acid (Baker). After reacting for 2 hrs at 0.degree. C., the
suspension was dialyzed against DI water using a 10 KDa membrane
(SpectaPor) for 18 hrs, flash frozen at -80.degree. C. and then
lyophilized.
Example 17
[0129] This example describes the construction and utilization of
an embodiment of the present invention in which high heparin ATIII
binding is present for heparin entity comprising heparin and a core
comprising an amine-containing fluoropolymer. This heparin entity
contains aldehydes along the length of the heparin component that
can be used for attachment to a surface of a substrate.
[0130] 48 mg of aldehyde-modified-heparin (made according to U.S.
Pat. No. 4,613,665) was dissolved in 30 ml of DI water. To this
solution was added 86 .mu.l of 2.5% sodium cyanoborohydride
solution (Aldrich), and the pH adjusted to 3.8 with HCl.
Separately, the TFE-VOH-AcAm copolymer of Example 16 was dissolved
in superheated methanol at 2.5% w/v and then cooled to room
temperature. To 20 ml of the TFE-VOH-AcAm solution was added 13 ml
of the heparin solution drop wise to produce a slightly milky
emulsion. The emulsion was maintained at 60.degree. C. for 2.5 hrs
and then at room temperature for an additional 2 hrs. The emulsion
was dialyzed against DI water using a 50 KDa membrane (SpectraPor)
for 18 hrs, flash frozen at -80.degree. C. and then lyophilized to
a powder.
[0131] A solution was prepared containing 100 ml DI water, 0.82 g
sodium acetate, and 0.128 g sodium periodate (ICN). To 12 ml of
this solution was suspended 12 mg of the powder. After reacting for
30 min in the dark, 1.2 ml of glycerol was added to quench the
reaction, the suspension was dialyzed against DI water using a 10
KDa membrane (SpectaPor) for 18 hrs, flash frozen at -80.degree. C.
and then lyophilized.
Example 18
[0132] The heparin entities of Examples 16 and 17, comprising
heparin and a core comprising amine-containing fluoropolymer, were
immobilized onto the ePTFE/PEI substrates, following the method
described in Example 7, except that the samples were not exposed to
EtO. ATIII binding activity was measured following the method
described in Example 7.
TABLE-US-00003 TABLE 3 ATIII binding activity Example # Attachment
type pmol/cm2 16 Free terminal aldehyde 106 17 Aldehyde along chain
length 66 (loop attachment)
Example 19
[0133] This example describes the construction and utilization of
an embodiment of the present invention in which high heparin ATIII
binding is present for heparin entity comprising heparin and a core
comprising an amine containing fluoropolymer. This heparin entity
contains free terminal aldehydes that can be used for attachment to
a surface of a substrate.
[0134] The amine containing fluoropolymer was prepared using the
following conditions. A 4 L reactor was charged with 2 L of
t-butanol. 50 g of tetrafluoroethylene (TFE), 200 g of
perfluoromethylvinylether (PMVE) and 100 g of N-vinyl formamide
(NFA) were added, along with 0.4 g of diisopropyl peroxydicarbonate
as initiator. The solution was stirred at a speed of 800 rpm at
70.degree. C. for 3 hrs. The precipitate was removed from the
reactor, air-dried for 2 hrs, and dried at 40.degree. C. under
vacuum for 24 hrs. Proton and fluorine NMR analysis confirmed a
TFE-PMVE-NFA polymer composition of 46 weight % NFA, 27 weight %
PTFE and 27 weight % PMVE. This polymer was soluble in methanol and
swelled in water.
[0135] 25 g of the TFE-PMVE-NFA polymer was dispersed in 100 mL of
DI water. The mixture was heated to 70.degree. C., and 30 mL of 37%
HCl was slowly added. The solution was kept at 90.degree. C. for 4
hrs. Hydrolyzed polymer was recovered from acetone precipitation,
air-dried for 2 hrs, and dried at 40.degree. C. under vacuum for 24
hrs. FTIR analysis confirmed hydrolysis of the vinyl formamide
groups to vinyl amine (VA) groups. The TFE-PMVE-VA polymer was
water soluble.
[0136] In a vial, 2 g of USP Heparin was dissolved in 50 mL of 0.1M
MES buffer, containing 0.8 g of EDC and 0.8 g sulfo-NHS. In a
second vial, a second solution was prepared consisting of 1 g of
TFE-PMVE-VA polymer and 30 mL of 0.1M MES buffer. The heparin
solution was added drop wise into the polymer solution over 4 hrs
at room temperature, and pH maintained at 4.7 with 1.0N NaOH. The
reaction was kept overnight at room temperature. The solution was
dialyzed in DI water for two days with 10,000 MWCO membrane
(Spectra/Por.RTM.). The retentate was concentrated with rotary
evaporation.
[0137] 0.01 g NaNO2, 100 mL of DI water, and 2 mL of acetic acid
were added to the retentate. The reaction proceeded at 0.degree. C.
for 2 hrs, followed by dialysis against DI water for two days,
flash frozen at -80.degree. C. and then lyophilized.
[0138] It will be apparent to those skilled in the art that various
modifications and variation can be made in the present invention
without departing from the spirit or scope of the invention. Thus,
it is intended that the present invention cover the modifications
and variations of this invention provided they come within the
scope of the appended claims and their equivalents.
* * * * *