U.S. patent application number 13/911727 was filed with the patent office on 2016-08-11 for immobilised biological entities.
This patent application is currently assigned to CARMEDA AB. The applicant listed for this patent is Robert Vestberg. Invention is credited to Robert Vestberg.
Application Number | 20160228572 13/911727 |
Document ID | / |
Family ID | 42261450 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160228572 |
Kind Code |
A9 |
Vestberg; Robert |
August 11, 2016 |
Immobilised Biological Entities
Abstract
There is described inter alia a medical device having a surface
which comprises a coating layer, said coating layer being a
biocompatible composition comprising an anti-coagulant entity
capable of interacting with mammalian blood to prevent coagulation
or thrombus formation, which anti-coagulant entity is covalently
attached to said surface through a linker comprising a
thioether.
Inventors: |
Vestberg; Robert; (Upplands
Vasby, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vestberg; Robert |
Upplands Vasby |
|
SE |
|
|
Assignee: |
CARMEDA AB
Upplands Vasby
SE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130273122 A1 |
October 17, 2013 |
|
|
Family ID: |
42261450 |
Appl. No.: |
13/911727 |
Filed: |
June 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13045769 |
Mar 11, 2011 |
8501212 |
|
|
13911727 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6957 20170801;
A61K 47/58 20170801; A61L 33/0029 20130101; A61P 7/02 20180101;
A61K 47/59 20170801; A61K 47/61 20170801 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2010 |
GB |
1004101.0 |
Claims
1-18. (canceled)
19. A process for the production of a solid object having a surface
which comprises an outer coating layer, said outer coating layer
being a biocompatible composition comprising a polymer and an
anti-coagulant entity capable of interacting with mammalian blood
to prevent coagulation or thrombus formation, which anti-coagulant
entity is covalently attached to said polymer through a linker
comprising a thioether; which process comprises the reaction of a
corresponding anti-coagulant entity carrying an alkene or alkyne
group with a corresponding surface carrying a thiol group, or the
reaction of a corresponding anti-coagulant entity carrying a thiol
group with a corresponding surface carrying an alkene or alkyne
group.
20. A process according to claim 19 comprising: (a) treating a
solid object to present a surface comprising a cationic polymer
outer coating layer which has been functionalized to bear thiol
groups; (b) reacting said cationic polymer outer coating layer
which has been functionalized to bear thiol groups with an
anti-coagulant entity which is functionalized to bear an alkene or
alkyne group; thereby to attach the anti-coagulant entity to the
cationic polymer through a linker comprising a thioether.
21. A process according to claim 19 comprising: (a) treating a
solid object to present a cationic polymer outer coating layer
which has been functionalized to bear alkene or alkyne groups; (b)
reacting said cationic polymer outer coating layer which has been
functionalized to bear alkyne groups with an anti-coagulant entity
which is functionalized to bear a thiol group; thereby to attach
the anti-coagulant entity to the cationic polymer through a linker
comprising a thioether.
22. A process according to claim 19 comprising: (a) treating a
solid object to present a cationic polymer surface layer; (b)
associating with said cationic polymer surface layer a
functionalized cationic polymer bearing a multiplicity of
negatively charged anti-coagulant entities such as heparin moieties
which are attached thereto via a linker comprising a thioether,
said cationic polymer bearing a multiplicity of negatively charged
anti-coagulant entities and said functionalized cationic polymer
having a net negative charge.
23. A process according to claim 19 comprising: (a) treating a
solid object to present an anionic polymer surface layer; (b)
associating with said anionic polymer surface layer a
functionalized cationic polymer bearing a multiplicity of
negatively charged anti-coagulant entities such as a heparin
moieties which are attached thereto via a linker comprising a
thioether, said functionalized cationic polymer bearing a
multiplicity of negatively charged anti-coagulant entities and
having a net positive charge.
24. A process according to claim 23, wherein the anionic polymer is
dextran sulfate or a derivative thereof.
25. A process according to claim 20, wherein the cationic polymer
is a polyamine.
26. A process according to claim 19, wherein the solid object is a
medical device.
27. A process according to claim 19, wherein the solid object has a
surface which comprises one or more layers of polysaccharide and
polyamine, which process comprises the reaction of a corresponding
surface having an outer layer of polysaccharide which has a net
negative charge with a polyamine, carrying a corresponding
anti-coagulant entity through a linker comprising a thioether,
having a net positive charge.
28. A process according to claim 19, wherein the solid object has a
surface which comprises one or more layers of polysaccharide and
polyamine, which process comprises the reaction of a corresponding
surface having an outer layer of polysaccharide which has a net
negative charge with a polyamine carrying an thiol or an alkene or
alkyne group which has a net positive charge and reacting the
resulting product with an anti-coagulant entity carrying an alkene
or alkyne or a thiol group respectively.
29. A process according to claim 19, wherein the solid object has a
surface which comprises one or more layers of polysaccharide and
polyamine, which process comprises the reaction of a corresponding
surface having an outer layer of polyamine having a net positive
charge with a polyamine carrying a multiplicity of corresponding
anti-coagulant entities through a linker comprising a thioether
such that said polyamine has a net negative charge.
30. (canceled)
Description
RELATED APPLICATION
[0001] The present application claims the priority of GB
Application Serial No. 1004101.0, filed Mar. 12, 2010. The
disclosure of the aforementioned application is incorporated by
reference herein in its entirety, and applicants claim the benefits
of this application under 35 U.S.C. .sctn.119.
BACKGROUND OF THE INVENTION
[0002] This invention relates to immobilised biological entities,
surfaces, and solid objects, for example medical devices, coated
with such entities, and processes and intermediates for their
production.
[0003] When a medical device is placed in the body, or in contact
with body fluids, a number of different reactions are set into
motion, some of them resulting in the coagulation of the blood in
contact with the device surface. In order to counteract this
serious adverse effect, the well-known anti-coagulant compound
heparin has for a long time been administered systemically to
patients before the medical device is placed in their body, or when
it is in contact with their body fluids, in order to provide an
antithrombotic effect.
[0004] Thrombin is one of several coagulation factors, all of which
work together to result in the formation of thrombi at a surface in
contact with the blood. Antithrombin (also known as antithrombin
III) ("AT") is the most prominent coagulation inhibitor. It
neutralizes the action of thrombin and other coagulation factors
and thus restricts or limits blood coagulation. Heparin
dramatically enhances the rate at which antithrombin inhibits
coagulation factors.
[0005] However, systemic treatment with high doses of heparin is
often associated with serious side-effects of which bleeding is the
predominant. Another rare, but serious complication of heparin
therapy is the development of an allergic response called heparin
induced thrombocytopenia that may lead to thrombosis (both venous
and arterial). High dose systemic heparin treatment e.g. during
surgery also requires frequent monitoring of the activated clotting
time (used to monitor and guide heparin therapy) and the
corresponding dose adjustments as necessary.
[0006] Therefore solutions have been sought where the need for a
systemic heparinisation of the patient would be unnecessary or can
be limited. It was thought that this could be achieved through a
surface modification of the medical devices using the
anti-coagulative properties of heparin. Thus a number of more or
less successful technologies have been developed where a layer of
heparin is attached to the surface of the medical device seeking
thereby to render the surface non-thrombogenic. For devices where
long term bioactivity is required, heparin should desirably be
resistant to leaching and degradation.
[0007] Heparin is a polysaccharide carrying negatively charged
sulfate and carboxylic acid groups on the saccharide units. Ionic
binding of heparin to polycationic surfaces was thus attempted, but
these surface modifications suffered from lack of stability
resulting in lack of function, as the heparin leached from the
surface.
[0008] Thereafter different surface modifications have been
prepared wherein the heparin has been covalently bound to groups on
the surface.
[0009] One of the most successful processes for rendering a medical
device non-thrombogenic has been the covalent binding of a heparin
fragment to a modified surface of the device. The general method
and improvements thereof are described in European patents:
EP-B-0086186, EP-B-0086187, EP-B-0495820 and U.S. Pat. No.
6,461,665.
[0010] These patents describe the preparation of surface modified
substrates by first, a selective cleavage of the heparin
polysaccharide chain, e.g. using nitrous acid degradation, leading
to the formation of terminal aldehyde groups. Secondly, the
introduction of one or more surface modifying layers carrying
primary amino groups on the surface of the medical device, and
thereafter reacting the aldehyde groups on the polysaccharide chain
with the amino groups on the surface modifying layers followed by a
reduction of the intermediate Schiff's bases to form stable
secondary amine bonds.
[0011] DE 19604173 relates to medical devices with a polymer
surface based on a substituted bis-phenyl monomer to which a
pharmaceutically active agent such as heparin may be attached.
[0012] WO 2008/090555 relates to a medical device coated with a
polymer matrix which incorporates a pharmaceutically active agent.
It appears that the active agent may be incorporated within the
polymer matrix.
[0013] US 2005/0059068 relates to a chemically active surface able
to covalently react with substances containing a hydroxyl group
and/or an amine group, comprising a solid surface having an
activated dendrimer polyamine covalently bonded to said surface
through a silane containing reagent, wherein the dendrimer
polyamine can covalently bind the substance comprising a hydroxyl
group and/or an amine group.
[0014] However there is still a requirement for surface
modifications that can be performed under mild conditions (e.g.
which do not degrade the heparin) which are more easily
manipulated, are simpler and more efficient to produce and/or where
the bioavailability of the heparin moiety is higher.
[0015] Our earlier application WO 2010/029189 relates to a medical
device having a coating with an anticoagulant molecule such as
heparin covalently attached to the coating via a 1,2,3-triazole
linkage. The document describes the azide or alkyne
functionalisation of a polyamine; the preparation of alkyne or
azide functionalised heparin (both native and nitrous acid degraded
heparin); and the reaction to link the derivatised heparin to the
derivatised polymer via a 1,2,3-triazole linker.
[0016] We have now found a further simple method of covalently
attaching entities capable of interacting with mammalian blood to
prevent coagulation or thrombus formation, e.g. heparin, and
especially full length heparin rather than the degraded heparin of
the prior art, to a surface.
SUMMARY OF THE INVENTION
[0017] According to the invention we provide, inter alia, a solid
object having a surface which comprises an outer coating layer,
said outer coating layer being a biocompatible composition
comprising a polymer and an anti-coagulant entity capable of
interacting with mammalian blood to prevent coagulation or thrombus
formation (herein "anti-coagulant entity"), which anti-coagulant
entity is covalently attached to said polymer through a linker
comprising a thioether. Such solid objects, especially medical
devices, are thereby non-thrombogenic.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1: shows photographs of examples of PVC tubing wherein
the luminal side is coated and stained with toluidine blue as
described in Examples 1.1-1.3.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In general, the outer coating layer comprises a multiplicity
of anti-coagulant entities, each of which is covalently attached to
the polymer through a linker comprising a thioether.
[0020] Anti-coagulant entities are well known to those skilled in
the art and many of them are oligosaccharides or polysaccharides.
Some of the entities are glycosaminoglycans including compounds
containing glucosamine, galactosamine, and/or uronic acid. Among
the most suitable glycosaminoglycans are "heparin moieties" and
especially full length heparin (i.e. native heparin).
[0021] The term "heparin moiety" refers to a heparin molecule, a
fragment of the heparin molecule, or a derivative or analogue of
heparin. Heparin derivatives can be any functional or structural
variation of heparin. Representative variations include alkali
metal or alkaline earth metal salts of heparin, such as sodium
heparin (e.g. Hepsal or Pularin), potassium heparin (e.g. Clarin),
lithium heparin, calcium heparin (e.g. Calciparine), magnesium
heparin (e.g. Cutheparine), and low molecular weight heparin
(prepared by e.g. oxidative depolymerization or deaminative
cleavage, e.g. Ardeparin sodium or Dalteparin). Other examples
include heparan sulfate, heparinoids, heparin based compounds and
heparin having a hydrophobic counter-ion. Other desirable
anti-coagulant entities include synthetic heparin compositions
referred to as "fondaparinux" compositions involving antithrombin
III-mediated inhibition of factor Xa. Additional derivatives of
heparin include heparins and heparin moieties modified by means of
e.g. periodate oxidation (U.S. Pat. No. 6,653,457) and other
modification reactions know in the art. Heparin moieties also
include such moieties bound to a linker or spacer as described
below. De-sulphated heparin is less suitable than other forms of
heparin because of its reduced non-thrombogenicity relative to
other forms of heparin.
[0022] Suitably, the anti-coagulant entity is single point attached
to the linker, particularly end point attached. When the
anti-coagulant entity is an end point attached heparin moiety, it
is suitably connected to the linker through its reducing end
(sometimes referred to herein as position C1 of the reducing
terminal). The advantage of end point attachment, especially
reducing end point attachment, is that the biological activity of
the anti-coagulant entity (for example the heparin moiety) is
maximized due to enhanced availability of the antithrombin
interaction sites as compared with attachment elsewhere in the
anti-coagulant entity (e.g. heparin moiety).
[0023] Where there is a multiplicity of anti-coagulant entities
e.g. heparin moieties it is possible for some or all of them to be
of a different type; however generally they will all be of the same
type.
[0024] The term "thioether" refers to a connection between a sulfur
and two carbon atoms. This connection is sometimes referred to as
"sulfide". The sulphur may be attached to two saturated carbon
atoms (i.e. --C--S--C--) or it may be attached to a saturated and
an unsaturated carbon atom (i.e. --C--S--C.dbd.).
[0025] The term "thiol" refers to an --S--H moiety.
[0026] The solid object may be any object to which it is desirable
to attach anti-coagulant entities. In one embodiment the solid
object is a medical device but other solid objects are also
contemplated, for example analytical devices and separation
devices. Thus, in an alternative embodiment, the solid object is an
analytical device or a separation device.
[0027] The term "medical device" refers to implantable or
non-implantable devices but more usually to implantable medical
devices. Examples of implantable medical devices include catheters,
stents including bifurcated stents, balloon expandable stents,
self-expanding stents, stent-grafts including bifurcated
stent-grafts, artificial blood vessels, blood indwelling monitoring
devices, artificial heart valves, pacemaker electrodes, guidewires,
cardiopulmonary bypass circuits, cannulae, balloons, tissue patch
devices and blood pumps. Further examples include grafts including
vascular grafts and bifurcated grafts, cardiac leads and drug
delivery devices. Examples of or non-implantable medical devices
are extracorporeal devices, e.g. extracorporeal blood treatment
devices, and transfusion devices.
[0028] Medical devices may have neurological, peripheral, cardiac,
orthopedal, dermal and gynecological application, inter alia.
[0029] An analytical device may be, for example, a solid support
for carrying out an analytical process such as chromatography or an
immunological assay, reactive chemistry or catalysis. Examples of
such devices include slides, beads, well plates, membranes etc. A
separation device may be, for example, a solid support for carrying
out a separation process such as protein purification, affinity
chromatography or ion exchange. Examples of such devices include
filters and columns etc.
[0030] A medical device may have many coating layers and the term
"outer coating layer" refers to a coating layer which, when the
device is implanted in a patient, is in contact with the tissues of
the patient or is in contact with body fluids. Thus, the outer
coating layer may be the coating layer on the outer and/or the
inner surface of a hollow device or a device of open structure such
as a stent.
[0031] Like a medical device, an analytical device or separation
device may also have many coating layers and the term "outer
coating layer" refers to a coating layer which comes into contact
with a substance to be analysed, separated or handled.
[0032] At its simplest the linker consists of the thioether only.
However more usually the linker comprises at least one spacer in
addition to the thioether so that the thioether will be separated
by a spacer from either the polymer or the heparin moiety or
both.
[0033] The Mw (molecular weight) of the linker is suitably from
10.sup.2 to 10.sup.6 Da and the length of the linker is suitably
from 10 to 10.sup.3 .ANG.. Suitably, the linkers and/or spacers are
straight chain(s), although it is also possible for several, i.e.
more than one, e.g. from 2 to 100, preferably 30 to 100 entities
(e.g. heparin moieties) to be attached to a single linker thus
producing a branched linker in which there are several heparin
moiety side chains.
[0034] In some embodiments the linker includes one or more aromatic
rings. In other embodiments the linker does not include any
aromatic rings. In some embodiments the linker is hydrophilic, for
example, it may comprise a PEG chain.
[0035] In one aspect of the invention, the linker may be formed
from multiple portions, for example two, three, four or five
portions, more usually three, four or five portions, wherein each
portion comprises or consists of a thioether or a spacer.
[0036] One example of a three-portion linker comprises "spacer A"
between the polymer and the thioether, the thioether itself and
"spacer B" between the thioether and the anti-coagulant entity. The
molecular weight of spacers A and B may be, for example, between
about 10.sup.1 and 10.sup.3 Da. In one embodiment, either or both
of spacers A and B may comprise an aromatic ring and in an
alternative embodiment, neither spacer A nor spacer B comprises an
aromatic ring.
[0037] In this type of linker, either spacer A or spacer B or both
may be a hydrophilic spacer, for example a PEG chain.
[0038] As used herein, the term "PEG chain" refers to a polymeric
chain obtainable by polymerisation of ethylene oxide, typically of
weight between 10.sup.2 and 10.sup.6 Da.
[0039] In some cases, the linker may comprise two or more
thioethers. For example, a bifunctional linker moiety (having, for
example an SH group at each end) can be connected at each end,
respectively, to an alkyne/alkene functionalized anti-coagulant
entity and an alkyne/alkene functionalized polymer resulting in the
linker containing two thioethers. Alternatively, use of a
bis-alkyne/alkene linker can be connected at each end,
respectively, to thiol functionalized anti-coagulant entity and a
thiol functionalized polymer also resulting in the linker
containing two thioethers.
[0040] Linkers having two or more thioethers suitably comprise
three, four or five portions where, as set out above, each portion
comprises a thioether or a spacer.
[0041] In one embodiment, the linker has five portions--"spacer A"
between the polymer and a first thioether, the first thioether,
"spacer C" between the first thioether and a second thioether, the
second thioether, and "spacer B" between the second thioether and
the anti-coagulant entity.
[0042] In such cases, the molecular weights of spacers A and B may
be, for example between about 10.sup.1 and 10.sup.3 Da and the
molecular weight of spacer C may be between about 10.sup.2 and
10.sup.6 Da.
[0043] Suitably, one or more of spacer A and/or spacer B and/or
spacer C is hydrophilic for example comprising a PEG chain.
[0044] In one embodiment, the linker between the anti-coagulant
entity such as a heparin moiety and the polymer of the outer
coating is an unbranched linker. In another embodiment, the linker
between the anti-coagulant entity such as a heparin moiety and the
polymer of the outer coating is a branched linker wherein the
branch contains another anti-coagulant entity such as a heparin
moiety.
[0045] The linker can be biodegradable or non-biodegradable but is
more suitably non-biodegradable in order that a coated solid
object, such as a medical device is non-thrombogenic for a long
period of time.
[0046] Where there is a multiplicity of linkers it is possible for
some or all of them to be of a different type; however suitably all
the linkers are of the same type.
[0047] In one embodiment, more than one anti-coagulant entity is
attached to a linker (e.g. more than one anti-coagulant entity is
attached to each linker) (see e.g. Example 1.1). In one embodiment
more than one linker is attached to an anti-coagulant entity (e.g.
more than one linker is attached to each anti-coagulant entity)
(see e.g. Example 1.3).
[0048] The surface may comprise a coating layer on a solid object
such as a medical device. The solid object may have one or more
portions containing void spaces, or pores. The pores may be within
the solid object and/or comprising at least one surface of the
solid object. An example of a porous solid object is expanded
polytetrafluoroethylene (ePTFE).
[0049] The solid object, may carry one or more, e.g. 2 or more, or
3 or 4 or 5 e.g. up to 20 coating layers such that desirably a
portion of the surface (desired to be non-thrombogenic) or the
whole of the surface of the object is covered (Multilayer Thin
Films ISBN: 978-3-527-30440-0). The optimum number of layers will
depend on the type of material from which the object is made, and
the contemplated use of the surface. The surface may, if desired,
be made up layer by layer. The number and nature of the layers
needed to provide a full coverage of the surface can be easily
determined by those skilled in the art. The coating layer(s) may be
formed by adsorbing on the surface of the solid object high average
molecular weight cationic polymer, e.g. a polyamine (e.g. that
known as Polymin available from BASF, see also EP 0086187 Larsson
and Golander) and if needed cross-linking the polyamine with, e.g.
an aldehyde crosslinker such as crotonaldehyde and/or
glutaraldehyde, followed by the application of a solution of an
anionic polymer, e.g. an anionic polysaccharide, e.g. dextran
sulfate, to obtain at least one adsorbed layer of the
polysaccharide. Hence the surface may comprise a layer of high
average molecular weight polyamine and a layer of anionic
polysaccharide. More generally, the surface may comprise one or
more coating bilayers of cationic polymer (e.g. polyamine) and
anionic polymer (e.g. anionic polysaccharide), the innermost layer
being a layer of cationic polymer and the outer layer being a layer
of cationic polymer to which the anti-coagulant entity is
covalently attached via a linker comprising a thioether. This
coating procedure is performed essentially as described in
EP-B-0495820. Thus it is only the outer coating layer which
comprises the anti-coagulant entity. Typically the outer coating
layer to which the anti-coagulant entity is attached is not
cross-linked.
[0050] The procedure of EP-B-0495820 may however be modified so
that the outer layer is the anionic polysaccharide which is then
reacted, as described below, with a polyamine to which is attached
the anti-coagulant entity or a polyamine with functional group(s)
capable of forming a linker comprising a thioether.
[0051] Prior to applying the first coating layer the surface of the
solid object, may be cleaned to improve adhesion and surface
coverage. Suitable cleaning agents include solvents as ethanol or
isopropanol (IPA), solutions with high pH like solutions comprising
a mixture of an alcohol and an aqueous solution of a hydroxide
compound (e.g. sodium hydroxide), sodium hydroxide solution as
such, solutions containing tetramethyl ammonium hydroxide (TMAH),
acidic solutions like Piranha (a mixture of sulfuric acid and
hydrogen peroxide), and other oxidizing agents including
combinations of sulfuric acid and potassium permanganate or
different types of peroxysulfuric acid or peroxydisulfuric acid
solutions (also as ammonium, sodium, and potassium salts).
[0052] Thus an aspect of the invention is a solid object, for
example a medical device having a surface wherein the surface
comprises one or more coating bilayers of cationic polymer and
anionic polymer, the innermost layer being a layer of cationic
polymer and the outermost layer being an outer coating layer of
cationic polymer to which an anti-coagulant entity is covalently
attached through a linker comprising a thioether.
[0053] The polymer of the outer coating layer is typically a
polyamine and the outer coating layer may be formed as described
above, either by using the procedure described in EP-B-0495820 or a
modification of this procedure in which an anionic polymer,
typically a polysaccharide, is reacted with a polyamine to which is
attached the anti-coagulant entity or a functional group capable of
forming a linker comprising a thioether.
[0054] Another aspect of the invention is a non-thrombogenic solid
object, especially a non-thrombogenic medical device having a
surface comprising a functionalized cationic polymer outer coating
layer whereby an anti-coagulant entity is attached to the cationic
polymer outer coating layer by means of a linker comprising a
thioether.
[0055] There are a number of ways of forming a thioether but among
the most suitable is the reaction of a first compound containing a
thiol group with a second compound containing an alkene or an
alkyne group. The first and second compounds can each be the
polymer of which the outer coating layer is comprised and the
anti-coagulant entity as appropriate.
[0056] Where the second compound is derivatised with an alkene, in
one embodiment an activated alkene is used. An example of a
suitable activated alkene is a maleimide derivative.
[0057] As noted below, optionally reaction may take place in the
presence of a reducing agent such as tris(2-carboxyethyl)phosphine
hydrochloride, or alternatively dithiothreitol or sodium
borohydride, to avoid or reverse the effective of undesirable
coupling of two thiol groups through oxidation.
[0058] In one embodiment the reaction is initiated with a radical
initiator. An example of a radical initiator is
4,4'-azobis(4-cyanovaleric acid). Further examples are potassium
persulfate,
2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride and
4-(trimethyl ammoniummethyl) benzophenone chloride.
[0059] In another embodiment, the reaction is not initiated with a
radical initiator. Instead, conditions of higher pH (e.g. pH 8-11)
are used. This type of reaction is more suitable when an activated
alkene or alkyne is used for reaction with the thiol.
[0060] In general, however, it is preferable to employ acid
conditions because these conditions appear most compatible with the
heparin and the coating materials.
[0061] The reaction between a first compound containing a thiol
group and a second compound containing an alkyne group may be
represented as follows:
##STR00001##
where one of R.sup.a and R.sup.b is the polymer and the other of
R.sup.a and R.sup.b is the anti-coagulant entity.
[0062] The reaction is described in Example 1.1, where R.sup.a is
heparin and R.sup.b is a polyamine and in Example 1.3, where
R.sup.a is polyamine and R.sup.b is heparin. The reaction may, for
example, be carried out in the presence of
tris(2-carboxyethyl)phosphine hydrochloride as reducing agent, and
4,4'-Azobis(4-cyanovaleric acid) as radical initiator, and under
acidic conditions.
[0063] If an excess of the compound R.sup.a--SH is present, there
may be further addition across the alkene double bond to produce a
compound containing two R.sup.a groups linked to a single R.sup.b
group. Again this is illustrated in Example 1.1, where some of the
linkers have more than one heparin group attached and in Example
1.3, where some of the heparin is attached to several linkers.
[0064] The reaction between a first compound containing a thiol
group and a second compound containing a maleimide group may be
represented as follows:
##STR00002##
where one of R.sup.a and R.sup.b is the polymer and the other of
R.sup.a and R.sup.b is the anti-coagulant entity.
[0065] This is described in detail in Example 1.2, where R.sup.a is
heparin and R.sup.b is a polyamine. The reaction is generally
carried out in the presence of tris(2-carboxyethyl)phosphine
hydrochloride as reducing agent, and 4,4'-azobis(4-cyanovaleric
acid) as radical initiator, and under acidic conditions.
[0066] Another aspect of the invention is a process for preparing a
non-thrombogenic solid object, for example a non-thrombogenic
medical device, the process comprising: [0067] (a) treating a solid
object such as a medical device to present a surface comprising a
cationic polymer outer coating layer which has been functionalized
to bear thiol groups; [0068] (b) reacting said cationic polymer
outer coating layer which has been functionalized to bear thiol
groups with an anti-coagulant entity which is functionalized to
bear an alkene or alkyne group; [0069] thereby to attach the
anti-coagulant entity to the cationic polymer through a linker
comprising a thioether.
[0070] The invention also provides a solid object, particularly a
medical device obtainable by this process.
[0071] Another aspect of the invention is a process for preparing a
non-thrombogenic solid object, for example a non-thrombogenic
medical device, the process comprising: [0072] (a) treating a solid
object such as a medical device to present a cationic polymer outer
coating layer which has been functionalized to bear alkene or
alkyne groups; [0073] (b) reacting said cationic polymer outer
coating layer which has been functionalized to bear alkyne groups
with an anti-coagulant entity which is functionalized to bear a
thiol group; [0074] thereby to attach the anti-coagulant entity to
the cationic polymer through a linker comprising a thioether.
[0075] The invention also provides a solid object, particularly a
medical device obtainable by this process.
[0076] Another aspect of the invention is a process for preparing a
non-thrombogenic solid object, for example a non-thrombogenic
medical device, the process comprising: [0077] (a) treating a solid
object such as a medical device to present a cationic polymer
surface layer; [0078] (b) associating with said cationic polymer
surface layer a functionalized cationic polymer bearing a
multiplicity of negatively charged anti-coagulant entities such as
heparin moieties which are attached thereto via a linker comprising
a thioether said cationic polymer bearing a multiplicity of
negatively charged anti-coagulant entities and said functionalized
cationic polymer having a net negative charge.
[0079] The invention also provides a solid object, particularly a
medical device obtainable by this process.
[0080] As described above, the cationic polymer surface may be
prepared by treating the solid object with a high average molecule
weight cationic polymer such as a polyamine and if necessary
cross-linking it with e.g. an aldehyde cross-linker. Further layers
may optionally be built up by successive steps of (i) application
of a solution of anionic polymer (e.g. anionic polysaccharide) to
obtain an adsorbed layer of the anionic polymer and (ii) then
further treating that with functionalized cationic polymer, such as
a polyamine, to provide an adsorbed outer coating layer of
functionalized cationic polymer, the outer coating layer being
functionalized to bear thiol groups or alkene or alkyne groups.
[0081] Typically the first step of treating the object with a high
average molecule weight cationic polymer is preceded by the step of
cleaning the surface of the object with suitable cleaning agents
(e.g. those mentioned above) or other methods of surface
pretreatment known in the art to improve adherence and coverage of
the first layer e.g. the polyamine layer.
[0082] Another aspect of the invention is a process for preparing a
non-thrombogenic solid object, for example a non-thrombogenic
medical device, the process comprising: [0083] (a) treating a solid
object such as a medical device to present an anionic polymer
surface layer; [0084] (b) associating with said anionic polymer
surface layer a functionalized cationic polymer bearing a
multiplicity of negatively charged anti-coagulant entities such as
heparin moieties which are attached thereto via a linker comprising
a thioether, said functionalized cationic polymer bearing a
multiplicity of negatively charged anti-coagulant entities and
having a net positive charge.
[0085] The invention also provides a solid object, particularly a
medical device obtainable by this process.
[0086] As described above, the solid object which presents an
anionic polymer surface layer is typically prepared by treating the
object (e.g. medical device) with a high average molecule weight
cationic polymer, such as a polyamine, optionally with
cross-linking, followed by treating the polyamine surface with a
solution of anionic polymer (e.g. anionic polysaccharide) to obtain
an adsorbed outer layer of the anionic polymer. Further layers may
be built up by successive steps of (i) application of a cationic
polymer (optionally with cross-linking) to provide an adsorbed
layer of cationic polymer and (ii) then treating that with a
solution of anionic polymer (e.g. anionic polysaccharide) to obtain
an adsorbed outer layer of the anionic polymer.
[0087] Suitably the anionic polymer is a polysaccharide such as
dextran sulfate or a derivative thereof.
[0088] As used herein a "polyamine" is a molecule having multiple
(e.g. 10, 100, 1000 or more) free pendant amino groups preferably
containing at least some primary amino groups. Polyamines are
typically polymeric molecules having multiple amino groups of high
average molecular weight, for example having an average molecular
weight of 10.sup.3-10.sup.6 Da. An exemplary polyamine is a
polyethyleneimine such as that known as Polymin available from
BASF.
[0089] The cationic polymer may be functionalized using techniques
known in the art. As illustrated in the Examples below, primary
amino groups on the polyamine may be used as points of attachment
for the alkene, alkyne or thiol group. However a skilled person
would know how to adapt the chemistry to use secondary amino groups
on the polyamine as points of attachment for the alkene, alkyne or
thiol group. Hence polyamines may be functionalized to bear alkene,
alkyne or thiol groups by conventional means e.g. by reacting
pendant primary amino groups on the polyamine with an activated
carboxylic acid (e.g. an N-hydroxy succinimide derivative of a
carboxylic acid) which acid bears an alkene, alkyne or thiol group.
Another way is to react secondary amines with carboxylic acids with
carbodiimide chemistry or to react with carboxylic acid chlorides
where the carboxylic acid portion bears an alkene, alkyne or thiol
group.
[0090] The anti-coagulant entity, e.g. heparin, carrying an alkene,
alkyne or thiol group may be made by conventional methods known per
se. For example an anti-coagulant entity, e.g. heparin, carrying an
alkyne/alkene group may be made by the reaction of an alkoxyamine
of the formula:
R.sup.1--O--NH.sub.2
wherein R.sup.1 is an alkyne/alkene-containing group; with an
aldehyde or hemi-acetal group on the anti-coagulant entity using
conventional techniques known per se. This type of reaction is
illustrated below in Example 3b; the reaction proceeds via
formation of an oxy-imine function to give a compound of the
formula:
R.sup.1--O--N.dbd.R'
in which R.sup.1 is as defined above and R' is the residue of the
anti-coagulant entity.
[0091] Nitrous acid degraded heparin and native heparin bear
reactive groups, an aldehyde group and a hemi-acetal function
respectively, at their reducing end which may be linked in this
way.
[0092] Similarly, an anti-coagulant entity derivatised with a thiol
group may be formed by the reaction of an aldehyde or hemi-acetal
group on the anti-coagulant entity with a compound of the
formula:
HS--X--NH.sub.2
where X is a hydrocarbon linker, for example (CH.sub.2).sub.n where
n is 1 to 8 e.g. 1 to 4, or X is a hydrocarbon linker as just
described in which one or more (e.g. 1 or 2) methylene groups are
replaced by 0; or X comprises a PEG chain containing 1 to 100 (e.g.
1 to 50 such as 1 to 10) ethylene glycol units; to give a product
of the formula
R'--CH.sub.2--NH--X--SH
where X is as defined above and R'--CH.sub.2-- is the residue of
the anti-coagulant entity.
[0093] An example of such a procedure is given in Example 3a
below.
[0094] A suitable functional group must also be introduced into the
polymer of the outer coating layer so that it can be reacted with
the derivatised anti-coagulant entity.
[0095] For example, a polyamine polymer bearing a number of primary
amine groups represented as follows:
R''--NH.sub.2
where R'' is the polymer residue; may be reacted with a compound of
the formula:
##STR00003##
where n is an integer from 1 to 8 e.g. 1 to 4; to give a maleimide
functionalized polyamine of the formula:
##STR00004##
where R'' and n are as defined above. This reaction is illustrated
in more detail in Example 2a below.
[0096] Alternatively, the polyamine polymer may be reacted with an
activated alkyne-containing group of the formula:
##STR00005##
where n is an integer from 1 to 8 e.g. 1 to 4; to give an alkyne
functionalized polymer of the formula:
##STR00006##
where R'' and n are as defined above. This reaction is illustrated
in more detail in Example 2b below.
[0097] Alternatively, if the polymer is intended to be reacted with
an alkene or alkyne-derivatised anti-coagulant entity, it may be
derivatised with a thiol group. In this case, a polyamine polymer
bearing a number of primary amine groups represented as
follows:
R''--NH.sub.2
where R'' is as defined above; may be reacted with an activated
thiol-containing compound, for example a compound of the
formula:
##STR00007##
where n is an integer from 1 to 8 e.g. 1 to 4; to give a
derivatised polymer of the formula:
##STR00008##
where R'' and n are as defined above. This reaction is illustrated
in more detail in Example 2c below.
[0098] When a coating layer is used, the surface of all and any
solid objects is transformed to present the same functionalized
outer surface for the subsequent attachment of an anti-coagulant
entity capable of interacting with mammalian blood to prevent
coagulation or thrombus formation. Hence a specific advantage of
the processes described herein is that generally a very uniform
non-thrombogenic surface is created (see FIG. 1). This is
particularly useful when the solid object is a medical device.
[0099] The solid object, e.g. medical device may comprise a metal
or a synthetic or naturally occurring organic or inorganic
polymer.
[0100] Thus, for example, it may be formed from a synthetic or
naturally occurring organic or inorganic polymer or material such
as polyethylene, polypropylene, polyacrylate, polycarbonate,
polyamide, polyurethane (PU), polyvinylchloride (PVC),
polyetherketone (PEEK), cellulose, silicone or rubber
(polyisoprene), plastics materials, metals, glass, ceramics and
other known medical materials or a combination of such materials.
Other suitable substrate materials include fluoropolymers, e.g
expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene
(PTFE), fluorinated ethylene-propylene (FEP), perfluorocarbon
copolymers, e.g. tetrafluoroethylene perfluoroalkylvinyl ether
(TFE/PAVE) copolymers, copolymers of tetrafluoroethylene (TFE) and
perfluoromethyl vinyl ether (PMVE), and combinations of the above
with and without crosslinking between the polymer chains.
[0101] Suitable metals include nickel titanium alloy (Nitinol),
stainless steel, titanium, cobalt chromium, gold and platinum.
Nitinol and stainless steel are preferred. Titanium is also
preferred.
[0102] A particularly suitable embodiment of the present invention
relates to a coated medical device.
[0103] A medical device can be implantable or non-implantable.
Examples of implantable or non-implantable medical devices include
catheters, stents, stent-grafts, artificial blood vessels, blood
indwelling monitoring devices, artificial heart valves, pacemaker
electrodes, guidewires, cardiopulmonary bypass circuits, cannulae,
balloons, tissue patch devices, blood pumps, and extracorporeal
devices, e.g. extracorporeal blood treatment devices, and
transfusion devices.
[0104] We prefer the coated surface to which the anti-coagulant
entity (e.g. heparin or other heparin moiety) is attached to be
such that it retains non-thrombogenic properties after
sterilization, e.g. ethylene oxide (EO) sterilization.
[0105] Sterilization may be carried out by means well known to
those skilled in the art. The preferred method of sterilization is
using ethylene oxide gas. Alternatively, other methods such as
radiation, e.g. e-beam or gamma radiation, may be used where such
radiation will not degrade the object or the coating or both.
[0106] A preferred embodiment of the present invention relates to a
coated medical device for implantation e.g. permanent implantation,
or other placement, at an anatomical site. Other preferred
embodiments include temporary use devices such as catheters and
extracorporeal circuits. Examples are sterile (e.g. sterilized)
medical devices for placement inside an anatomical structure
delimiting a void space, or lumen, to reinforce the anatomical
structure or maintain the void space. Suitably the attached
anti-coagulant entity, e.g. heparin or other heparin moiety, does
not elute to any substantial extent and remains with the device.
For example, after 15 hour rinse with NaCl (0.15 M) prior to
testing the retained AT binding activity remains adequate (e.g.
greater than 1 or 2 or 4 or 5 or 10 pmol/cm.sup.2) and when tested
in the Blood loop evaluation test (see Example 1.4) with fresh
blood from a healthy donor the reduction in platelet count of the
blood after the test is substantially lower for the blood exposed
to the coated surface according to the invention than that of an
uncoated control (e.g. the reduction in platelet count after the
test for the blood exposed to the coated surface is less than 20%,
preferably less than 15% and more preferably less than 10%).
[0107] Suitably the biocompatible composition of the invention is
not biodegradable or bioabsorbable. For biodegradable or
bioabsorbable compositions the non-thrombogenic properties may
generally be expected to be limited in time.
[0108] The non-thrombogenic character of solid objects according to
the present invention may be tested by a number of methods. For
example non-thrombogenic character may be associated with having a
high antithrombin binding activity, especially as compared with
solid objects having untreated surfaces.
[0109] For example, we prefer the surface, e.g. of the medical
device, to have an antithrombin (AT) binding activity of at least 1
e.g. at least 5 picomoles AT per square centimeter (pmol/cm.sup.2)
of surface. In other embodiments, the AT binding activity is at
least 6 pmol/cm.sup.2, at least 7 pmol/cm.sup.2, at least 8
pmol/cm.sup.2, at least 9 pmol/cm.sup.2, or at least 10
pmol/cm.sup.2 of surface. In some embodiments, the AT binding
activity is at least 100 pmol/cm.sup.2 of surface. AT binding
activity can be measured by methods known in the art, e.g. those
described in Pasche., et al., in "Binding of antithrombin to
immobilized heparin under varying flow conditions" Artif.-Organs
15:481-491 (1991) and US 2007/0264308. By way of comparison it may
be concluded from Sanchez et al (1997) J. Biomed. Mater. Res. 37(1)
37-42, see FIG. 1, that AT binding values of around 2.7-4.8
pmol/cm.sup.2 (depending on the experimental set up) or more do not
appear to give rise to significant thrombogenic enzymatic activity
upon contact with plasma.
[0110] Alternatively or additionally we prefer the surface to be
non-thrombogenic due to high capacity to suppress coagulation and
other defence systems in the Blood loop evaluation test described
in Example 1.4. According to that test, the surface to be
investigated is applied to a PVC tubing which is rinsed for 15
hours with 0.15M NaCl prior to testing with fresh blood.
Non-thrombogenicity is indicated by a reduction in platelet count
of the blood measured after the test which is substantially lower
for the blood exposed to the surface prepared according the method
described herein than that of an uncoated control (e.g. the
reduction in platelet count after the test for the blood exposed to
the coated surface is less than 20%, preferably less than 15% and
more preferably less than 10%).
[0111] Other similar blood evaluation methods different from the
Blood loop model can be performed by those skilled in the art in
order to assess thrombogenicity/non-thrombogenicity.
[0112] The amount of the anti-coagulant entity bound to a
particular surface area can be controlled and adjusted, e.g. by
adjusting the amount of the reagents used in the synthesis of the
composition.
[0113] The distribution of the anti-coagulant entity on the surface
can be determined by conventional staining techniques which are
known per se, e.g. the distribution of heparin can be determined
using toluidine blue.
[0114] According to the invention we also provide a process for the
production of a solid object, in particular a medical device,
having a surface which comprises an outer coating layer, said outer
coating layer being a biocompatible composition comprising a
polymer and an anti-coagulant entity capable of interacting with
mammalian blood to prevent coagulation or thrombus formation, which
anti-coagulant entity is covalently attached to said polymer
through a linker comprising a thioether which process comprises the
reaction of a corresponding anti-coagulant entity carrying an
alkene or alkyne group with a corresponding surface carrying a
thiol group, or the reaction of a corresponding anti-coagulant
entity carrying a thiol group with a corresponding surface carrying
an alkene or alkyne group.
[0115] This process may be carried out using procedures known per
se.
[0116] The surface carrying a thiol group or an alkene or alkyne
group may be made by conventional methods known per se, e.g. by
reacting a surface, e.g. a surface as described in EP-B-0086186 or
EP-B-0086187 carrying negatively charged sulfate groups with an
appropriate polyamine carrying either a thiol or an alkene or
alkyne group respectively.
[0117] According to the invention we also provide a polyamine
carrying an anti-coagulant entity through a linker comprising a
thioether.
[0118] In one embodiment in which the reaction is used the surface
carries the thiol group. In another embodiment in which the
reaction is used the anti-coagulant entity carries the thiol
group.
[0119] The reaction may be carried out as described briefly above
and in more detail in the Examples below.
[0120] By this new method the anti-coagulant entity, e.g. heparin,
can advantageously be bound to the surface by surface groups that
are not involved in the build up of the surface covering. By
contrast, the prior art described in EP-B-0086186, EP-B-0086187 and
EP-B-0495820 uses the same type of groups (primary amines) in the
layer by layer surface coating process as those used to bind the
heparin to the coating.
[0121] This new process tends to be less sensitive to pH than are
the prior art processes which is also advantageous.
[0122] The reaction may also, if desired, be carried out under flow
conditions.
[0123] According to the invention we also provide an anti-coagulant
entity, e.g. heparin or other heparin moiety, which anti-coagulant
entity carries an alkene or alkyne or a thiol group. We also
provide an anti-coagulant entity, e.g. a heparin moiety capable of
interacting with mammalian blood to prevent coagulation or thrombus
formation, wherein the anti-coagulant entity carries an alkene or
alkyne or a thiol group, which alkene or alkyne or thiol group is
attached to a linker, wherein the linker is end-point attached to
the anti-coagulant entity (e.g. heparin moiety). When the
anti-coagulant entity is a heparin moiety, it may, for example. be
a full length heparin moiety (i.e. native heparin).
[0124] According to the invention we also provide a functionalized
polyamine surface, e.g. a surface prepared essentially as described
in EP-B-0086186, EP-B-0086187 and, EP-B-0495820, but additionally
carrying one or more thiol or one or more alkene or alkyne groups
on the outer coating layer of polyamine.
[0125] According to the invention we also provide a solid object,
especially a medical device, having a polyamine surface carrying a
thiol or an alkene or alkyne group e.g. a thiol or alkene or alkyne
group which is connected to an amino group of the polyamine surface
via a linker.
[0126] According to a further feature of the invention we also
provide a process for the production of a solid object, especially
a medical device, having a surface which comprises an outer coating
layer, said outer coating layer being a biocompatible composition
comprising a polymer and an anti-coagulant entity capable of
interacting with mammalian blood to prevent coagulation or thrombus
formation, which anti-coagulant entity is covalently attached to
said polymer through a linker comprising a thioether, wherein the
object has a surface which comprises one or more layers of
polysaccharide and polyamine, which process comprises the reaction
of a corresponding surface having an outer layer of polysaccharide
which has a net negative charge (i.e. anionic polysaccharide e.g.
carrying negatively charged sulfate groups) with a polyamine,
carrying a corresponding anti-coagulant entity through a linker
comprising a thioether, having a net positive charge, or the
reaction of a corresponding surface having an outer layer of
polysaccharide which has a net negative charge (i.e. anionic
polysaccharide e.g. carrying negatively charged sulfate groups)
with a polyamine carrying a thiol or an alkene or alkyne group
which has a net positive charge and reacting the resulting product
with an anti-coagulant entity carrying an alkene or alkyne or a
thiol group respectively.
[0127] References to a polyamine carrying an anti-coagulant entity
or a thiol, alkene or alkyne groups include references to a
polyamine carrying one or more i.e. a plurality of such groups.
However a polyamine carrying a corresponding anti-coagulant entity
through a linker comprising a thioether having a net positive
charge will only bear so many negatively charged anti-coagulant
entities as allows the net charge to remain net positive.
[0128] According to a further feature of the invention we also
provide a process for the production of a solid object, e.g. a
medical device, having a surface which comprises an outer coating
layer, said outer coating layer being a biocompatible composition
comprising a polymer and an anti-coagulant entity capable of
interacting with mammalian blood to prevent coagulation or thrombus
formation, which anti-coagulant entity is covalently attached to
said polymer through a linker comprising a thioether, wherein the
object has a surface which comprises one or more layers of
polysaccharide (i.e. anionic polysaccharide e.g. carrying
negatively charged sulfate groups) and polyamine, which process
comprises the reaction of a corresponding surface having an outer
layer of polyamine having a net positive charge with a polyamine
carrying a multiplicity of corresponding anti-coagulant entities
through a linker comprising a thioether such that said polyamine
has a net negative charge.
[0129] This process for putting down the layers of polysaccharide
and polyamine may be carried out using procedures known per se, for
example procedures analogous to those described in
EP-B-0495820.
[0130] The presence of a net positive charge on a surface may be
determined by treatment with Ponceau S which would dye a positively
charged surface a red colour.
[0131] The presence of a net negative charge on a surface may be
determined by treatment with toluidine blue which would dye a
negatively charged surface a blue colour.
[0132] According to the invention we also provide a functionalized
polyamine, e.g. Polymin which carries one or more thiols or one or
more alkenes or one or more alkynes e.g. via a linker.
[0133] According to the invention we also provide a functionalized
polyamine carrying an anti-coagulant entity attached thereto
through a linker comprising a thioether. This polyamine may be made
by procedures known per se, e.g. analogous to those described
elsewhere in this specification.
[0134] The products of the invention may have one or more of the
following advantageous properties: [0135] The degree of
substitution of the anti-coagulant entity on the surface can be
controlled; [0136] Both end-point (single point) attachment and
multi-point attachment of the anti-coagulant entity, e.g. heparin,
can be achieved, although end point (especially reducing end point)
attachment is preferred; [0137] The linker length between the
anti-coagulant entity and the surface can be controlled; [0138]
Full length heparin can be used thus avoiding the cleavage of
heparin and the waste of parts of the cleaved product involved in
the prior art nitrous acid degradation of heparin; [0139] When
cleaving heparin, the antithrombin binding sequence can be
destroyed in some of the fragments, therefore using full-length
heparin or heparin linked via a spacer can also improve the
bioavailability of the bound heparin; [0140] A uniform distribution
of the anti-coagulant entity over the surface can be obtained;
[0141] A uniform coating may be obtained which will mask the
intrinsic properties, for example lower the thromogenic properties,
of a device irrespective of the material of its manufacture; [0142]
A coating may be obtained which is comparatively smooth; [0143] The
biocompatibility of the coating may be enhanced; [0144] A coating
according to the present invention may reduce the need for systemic
heparin and reduce the likelihood of contact activation; [0145] The
bioavailability of the anti-coagulant entity can be controlled,
e.g. by the use of different linkers (length, type); [0146] A
non-thrombogenic surface which does not leach heparin and therefore
has long lifetime can be obtained; [0147] An analytical or
separation device with improved binding capacity to biomolecules
may be obtained; and [0148] An analytical or separation device with
extended heparin activity life time may be obtained.
[0149] Other aspects of the invention include a biocompatible
composition comprising an anti-coagulant entity capable of
interacting with mammalian blood to prevent coagulation or thrombus
formation which anti-coagulant entity is covalently attached to a
surface through a linker comprising a thioether.
[0150] The skilled person will appreciate that the biocompatible
composition may be applied to any solid object, of which a medical
device is just one example. Therefore according to another aspect
of the invention there is provided a solid object having a surface
comprising (e.g. coated with) such a biocompatible composition.
[0151] The invention is illustrated, but in no way limited, by the
following Examples:
Example 1.1
Preparation of a Non-Thrombogenic Surface on PVC
[0152] A surface comprising layers of aminated polymer and sulfated
polysaccharide having a functionalized aminated polymer outer layer
is connected to functionalized heparin thereby forming a
thioether.
[0153] A PVC surface was pretreated using the method described by
Larm et al in EP-B-0086186 and EP-495820 (layer-by-layer;
polyelectrolyte charge interactions) ending with a layer of
sulfated polysaccharide.
[0154] The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned
with isopropanol and an oxidizing agent. The priming was built-up
by alternated adsorption of a positively charged polyamine
(Polymin) and negatively charged sulfated polysaccharide (dextran
sulfate). The polyamine is crosslinked with a difunctional aldehyde
(crotonaldehyde). Every pair of polyamine and sulfated
polysaccharide is called one bilayer. The PVC surface was primed
with 4 bilayers ending with the sulfated polysaccharide.
[0155] Polymin SN (Lupasol SN; Lupasol is an alternative trade name
for Polymin) was diluted with water to prepare a stock solution (5
g Polymin SN was added to 20 mL purified water). (Polymin is a
polyethyleneimine cationic tenside available from BASF).
[0156] 1.0 mL of a 5% solution of alkyne functionalized polyamine
(preparation see Example 2b) was added to 500 mL of a 0.04 M/0.04 M
borate/phosphate buffer at pH 8.0. The adsorption of the alkyne
functional polyamine to the sulfate surface was carried out for 20
minutes at room temperature. A two minute water rinse was performed
after the adsorption to rinse off excess polymer.
[0157] 500 mg of nitrite degraded heparin, with thiol
functionalization at C1 of the reducing terminal (prepared as in
Example 3a), was dissolved in 1000 mL of de-ionized water and 50 mg
tris(2-carboxyethyl)phosphine hydrochloride, 500 mg
4,4'-Azobis(4-cyanovaleric acid), and 2.9 g NaCl were added. The pH
was adjusted to 3.7 with 1 M HCl (aq).
[0158] The reaction between the solution of the thiol
functionalized heparin and the alkyne functionalized surface was
carried out at 70.degree. C. for 3 h. Purification was performed by
rinsing off non-covalently linked heparin for 10 minutes using a
0.04 M/0.04 M borate/phosphate buffer at pH 8.0. A final rinse with
de-ionized water for two minutes was performed to wash away buffer
salt residues.
[0159] The flow used during the entire process was 100 mL/min.
##STR00009## ##STR00010##
[0160] The samples were stained with toluidine blue ("TB") (200
mg/L in water) by immersing in the solution for 2 minutes followed
by extensive water rinse. The TB attaches to the heparin via ionic
interaction. The samples showed intense uniform stain with TB, see
FIG. 1.
[0161] Antithrombin binding activity of bound heparin: 2.2
pmol/cm.sup.2
[0162] The antithrombin binding activity of bound heparin was
measured essentially as described in Pasche., et al., in "Binding
of antithrombin to immobilized heparin under varying flow
conditions" Artif.-Organs 15:481-491 (1991).
[0163] Non-thrombogenic as tested by the blood loop--see Example
1.4
Example 1.2
Preparation of a Non-Thrombogenic Surface on PVC
[0164] The luminal surface of a PVC tubing (internal diameter 3 mm)
was cleaned with isopropanol and an oxidizing agent. It was then
primed with four bilayers of a positively charged polyamine
(Polymin) and a negatively charged sulfated polysaccharide (dextran
sulfate) ending with the sulfated polysaccharide.
[0165] Then next coating step used a solution of 10 mL of a 1%
solution of maleimide functionalized polyamine (prepared as in
Example 2a) in 1000 mL of a 0.04 M/0.04 M borate/phosphate buffer
at pH 8.0. The adsorption of the maleimide functional polyamine to
the sulfate surface was carried out for 20 minutes at room
temperature. A two minute water rinse was performed after the
adsorption to rinse off excess polymer.
[0166] 500 mg of nitrite degraded heparin, with thiol
functionalization at C1 of the reducing terminal (prepared as in
Example 3a), was dissolved in 1000 mL of de-ionized water and 50 mg
tris(2-carboxyethyl)phosphine hydrochloride, 500 mg
4,4'-Azobis(4-cyanovaleric acid), and 2.9 g NaCl were added. The pH
was adjusted to 3.7 with 1 M HCl (aq).
[0167] The reaction between the solution of the thiol
functionalized heparin and the maleimide functionalized surface was
carried out at 70.degree. C. for 3 h. Purification was performed by
rinsing off non-covalently linked heparin for 10 minutes using a
0.04 M/0.04 M borate/phosphate buffer at pH 8.0. A final rinse with
de-ionized water for two minutes was performed to wash away buffer
salt residues.
[0168] The flow used during the entire process was 100 mL/min.
##STR00011## ##STR00012##
[0169] Staining with TB (as described in Example 1.1) showed an
intense uniform stain after coating, see FIG. 1.
[0170] Antithrombin binding activity of bound heparin: 8.0
pmol/cm.sup.2
[0171] Non-thrombogenic as tested by the blood loop--see Example
1.4
Example 1.3
Preparation of a Non-Thrombogenic Surface on PVC
[0172] The luminal surface of a PVC tubing (internal diameter 3 mm)
was cleaned with isopropanol and an oxidizing agent. It was then
primed with four bilayers of a positively charged polyamine
(Polymin) and a negatively charged sulfated polysaccharide (dextran
sulfate) ending with the sulfated polysaccharide.
[0173] Then next coating step used a solution of 5 mL of a 1%
solution of thiol functionalized polyamine (prepared as in Example
2c) and 125 mg of tris(2-carboxyethyl)phosphine hydrochloride in
500 mL of a 0.04 M/0.04 M borate/phosphate buffer at pH 8.0. The
adsorption of the thiol functional polyamine to the sulfate surface
was carried out for 20 minutes at room temperature. A two minute
water rinse was performed after the adsorption to rinse off excess
polymer.
[0174] 250 mg of nitrite degraded heparin, with alkyne
functionalization at C1 of the reducing terminal (prepared as in
Example 3b), was dissolved in 500 mL of de-ionized water and 25 mg
tris(2-carboxyethyl)phosphine hydrochloride, 250 mg
4,4'-Azobis(4-cyanovaleric acid), and 1.4 g NaCl were added. The pH
was adjusted to 3.7 with 1 M HCl (aq).
[0175] The reaction between the solution of the alkyne
functionalized heparin and the thiol functionalized surface was
carried out at 70.degree. C. for 3 h. Purification was performed by
rinsing off non-covalently linked heparin for 10 minutes using a
0.04 M/0.04 M borate/phosphate buffer at pH 8.0. A final rinse with
de-ionized water for two minutes was performed to wash away buffer
salt residues.
[0176] The flow used during the entire process was 100 mL/min.
##STR00013## ##STR00014##
[0177] Staining with TB (as described in Example 1.1) showed an
intense uniform stain after coating, see FIG. 1.
[0178] Antithrombin binding activity of bound heparin: 1.0
pmol/cm.sup.2
[0179] Non-thrombogenic as tested by the blood loop--see Example
1.4
Example 1.4
Blood Loop Evaluation Test
[0180] Blood loop evaluation was performed on the luminaly coated
PVC tube samples from Examples 1.1-1.3 to show the preserved
heparin bioactivity of the non-thrombogenic surface. First the
luminal side of the coated tubings were washed with 0.15 M NaCl for
15 hours at a flow of 1 mL/min to ensure that all loosely bound
heparin was rinsed off and a stable surface remains. Then the
washed tubings were incubated in a Chandler loop model performed
essentially according to Anderson et al. (Andersson, J.; Sanchez,
J.; Ekdahl, K. N.; Elgue, G.; Nilsson, B.; Larsson, R. J Biomed
Mater Res A 2003, 67(2), 458-466) at 20 rpm. The platelets, from
fresh blood and from the blood collected from the loops, were
counted in a cell counter to measure the loss of platelets which
indicates thrombosis. As references were included a
non-thrombogenic control (i.e Carmeda.RTM. BioActive Surface
applied to PVC, which is prepared essentially as described in
EP-B-0495820), an uncoated PVC tube, and a thrombogenic control
(i.e. a three bilayer coating with an outer layer of sulfated
polysaccharide not binding antithrombin).
[0181] As seen in the table below, there is virtually no platelet
loss (platelet loss indicates thrombosis) seen for the coatings
prepared as described in Examples 1.1-1.3. The uncoated PVC tubing
and the surface with an outer layer of sulfated polysaccharides
(not binding antithrombin) show significant platelet loss in this
experiment.
TABLE-US-00001 Loss in Platelet count .times. platelet Evaluated
surfaces 10.sup.9/L count % Initial value, blood 202 before
Chandler loop Evaluated surfaces From Example 1.1 206 0 according
to the From Example 1.2 190 6 invention From Example 1.3 199 1
Reference surfaces Non-thrombogenic 194 4 control Uncoated PVC tube
57 72 Thrombogenic 9 96 control
[0182] These results demonstrate the non-thrombogenic properties of
the surface prepared according to the invention.
Example 2a
Maleimide Functionalization of Polymin SN
##STR00015##
[0184] Polymin SN (Lupasol SN; Lupasol is an alternative trade name
for Polymin) was diluted with water to prepare a stock solution (5
g Polymin SN was added to 20 mL purified water). (Polymin is a
polyethyleneimine cationic tenside available from BASF).
[0185] 4-maleimidobutyric acid (0.50 g, 2.7 mmol) and
N-hydroxysuccinimide (NHS) (0.32 g, 2.7 mmol) was dissolved in 3 mL
of dichloromethane and stirred at 0.degree. C. A solution of
N,N'-dicyclohexylcarbodiimide (0.56 g, 2.7 mmol) in 3 mL of
dichloromethane was added slowly to the reaction mixture at
0.degree. C. The reaction mixture was stirred over night and the
byproducts were filtered of and the NHS activated
4-maleimidobutyric acid was concentrated and dried under
vacuum.
[0186] The dried NHS activated 4-maleimidobutyric acid was
dissolved in 30 mL of purified water and mixed with 7.6 mL of the
Polymin SN stock solution at 0.degree. C. and left to react
overnight at room temperature to obtain a 1% solution of the
maleimide functionalized polymin.
Example 2b
Alkyne Functionalization of Polymin SN
##STR00016##
[0188] A solution of N-hydroxysuccinimide-(4-pentynoate) (Ref:
Salmain, M.; Vessieres, A.; Butler, I. S.; Jaouen, G. Bioconjugate
Chemistry 1991, 2(1), 13-15) (3.90 g, 19.0 mmol) in 20 mL of
purified water was mixed with 24 mL of the Polymin SN stock
solution (see example 2a) and left to react overnight at 70.degree.
C. The reaction mixture was then diluted with water and isopropanol
(min 99%, PhEur quality, Merck) until the polymer precipitated. The
isopropanol was decanted off and the residual isopropanol of the
resulting slurry was evaporated off.
Example 2c
Thiol Functionalization of Polymin SN
##STR00017##
[0190] 3-mercaptopropionic acid (1.00 g, 9.4 mmol) and
N-hydroxysuccinimide (NHS) (1.09 g, 9.4 mmol) was dissolved in 1 mL
of dichloromethane and stirred at 0.degree. C. under inert
atmosphere (Ar).
[0191] A solution of N,N'-dicyclohexylcarbodiimide (1.94 g, 9.4
mmol) in 10 mL of dichloromethane was added slowly to the reaction
mixture at 0.degree. C. The reaction mixture was stirred over night
under inert atmosphere (Ar) at room temperature and the byproducts
were filtered of and the NHS activated 3-mercaptopropionic acid was
concentrated and dried under vacuum.
[0192] The dried NHS activated 3-mercaptopropionic acid was
dissolved in 115 mL of purified water and mixed with 28.6 mL of the
Polymin SN stock solution (see example 2a) at 0.degree. C. and left
to react overnight under inert atmosphere (Ar) at room temperature
to obtain a 1% solution of the thiol functionalized polymin.
Example 3a
Preparation of Thiol Functionalized Nitrous Acid Degraded
Heparin
##STR00018##
[0194] Nitrous acid degraded heparin with aldehyde groups (prepared
essentially as in Example 2 of U.S. Pat. No. 4,613,665) (5.00 g,
1.0 mmol), cysteamine hydrochloride (0.57 g, 5.0 mmol) and sodium
chloride (0.6 g) were dissolved in purified water. The pH was
adjusted to 6.0 with 1 M NaOH (aq) and 1 M HCl (aq). To the
solution was added 3.1 ml of 5% (aq) NaCNBH.sub.3 (0.16 g, 2.5
mmol) and the reaction was stirred over night at room temperature.
The pH was adjusted to 11.0 with 1 M NaOH (aq) and the resulting
product was dialyzed against purified water with a SpectraPor
dialysis membrane mwco 1 kD (flat width 45 mm) for three days. The
reaction mixture was then concentrated and freeze dried to obtain
2.6 g of a white fluffy powder.
Example 3b
Preparation of Alkyne Functionalized Nitrous Acid Degraded
Heparin
##STR00019##
[0195] Reagents:
[0196] (i) Nitrous acid degraded heparin with aldehyde groups
(prepared essentially as in Example 2 of U.S. Pat. No. 4,613,665)
3.25 g dry weight (0.65 mmol) [0197] (ii)
O-(prop-2-ynyl)-hydroxylamine hydrochloride (Ref: Xu, R.; Sim, M.
K.; Go, M. L., Synthesis and pharmacological characterization of
O-alkynyloximes of tropinone and N-methylpiperidinone as muscarinic
agonists. J Med Chem 1998, 41, (17), 3220-3231) 0.70 g dry weight
(6.5 mmol) [0198] (iii) Acetic acid (100% Merck) 3 mL [0199] (iv)
Purified water 50 mL
[0200] The compounds were dissolved in the mixed solvents and the
pH adjusted to 4.5 with 4M NaOH.
[0201] The reaction was continued for 3 days at room temperature.
The resulting product was dialyzed against purified water with a
SpectraPor dialysis membrane mwco 1 kD (flat width 45 mm).
[0202] The functionalized product was analyzed by FTIR which showed
a typical signal from the alkyne at 3100 cm.sup.-1.
[0203] The activity of the functionalized heparin was 96 IU/mg
which indicates that the activity of the functionalized heparin is
substantially unaffected by functionalization.
Example 3c
Preparation of Alkyne Functionalized Native Heparin
##STR00020##
[0205] The native heparin (SPL, Scientific Protein Laboratories,
lot no. 1037) was functionalized according to the procedures
described in Example 3b.
[0206] The activity of the functionalized heparin was 211 IU/mg
which indicates that the activity of the functionalized heparin is
substantially unaffected by functionalization.
Example 3d
Preparation of Alkyne Functionalized Native Heparin with Aromatic
Spacer
##STR00021##
[0208] The native heparin (SPL, Scientific Protein Laboratories,
lot no. 1037) (20 mg) was dissolved in 250 .mu.L acetic acid (100%
Merck) and 250 .mu.L purified water and 6 .mu.L
N-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamide from stock solution
(see Example 5 below) was added. The reaction was carried out at
room temperature for 16 hrs. The reaction products were
concentrated and co-evaporated with toluene (3.times.2 mL) to give
a yellowish solid (.about.20 mg).
Preparation of Intermediates
Example 5
Bifunctional Linker
5 a) N-(4-(2-(hydroxy)ethyl)phenyl)pent-4-ynamide
[0209] N-hydroxysuccinimide-(4-pentynoate) (Ref: Malkoch, M.;
Schleicher, K.; Drockenmuller, E.; Hawker, C. J.; Russell, T. P.;
Wu, P.; Fokin, V. V., Structurally Diverse Dendritic Libraries: A
Highly Efficient Functionalization Approach Using Click Chemistry.
Macromolecules 2005, 38, (9), 3663-3678.) (200 mg, 1.0 mmol) and
p-aminophenylethanol (125 mg, 0.9 mmol) were dissolved in 2 mL of
dichloromethane together with triethyl amine (140 .mu.L, 1.0 mmol),
and 5 drops of dimethyl formamide. The reaction mixture was stirred
at room temperature for 2 hours. The crude reaction product was
concentrated, dissolved in 10 mL of ethyl acetate and washed with 5
mL of water followed by, 5 mL of 0.5 M HCl (aq.), 5 mL of 10
NaHCO.sub.3 (aq.) and finally 5 mL of water. The organic phase was
dried with MgSO.sub.4, filtered, and the solvent was evaporated.
The product was further purified by column chromatography on silica
gel eluting with a gradient of toluene (T) and ethyl acetate (E)
from 4:1 to 1:2 (T:E). The product
N-(4-(2-(hydroxy)ethyl)phenyl)pent-4-ynamide was characterized by
NMR and MALDI-TOF.
5 b) N-(4-(2-(methanesulfonate)ethyl)phenyl)pent-4-ynamide
[0210] N-(4-(2-(hydroxy)ethyl)phenyl)pent-4-ynamide (210 mg, 1.0
mmol) was dissolved in 4 mL of pyridine. Methanesulfonyl chloride
(MsCl) (100 .mu.L, 1.3 mmol) was added at 0.degree. C. The stirred
reaction was brought back to room temperature and reacted at room
temperature for 5 min. The solvent was evaporated and the residue
re-dissolved in 10 mL of ethyl acetate and washed with 5 mL of
water followed by 5 mL of 0.1 M HCl (aq.), and finally 5 mL of
water. The organic phase was dried with MgSO.sub.4, filtered, and
the solvent was evaporated to yield the product
N-(4-(2-(methanesulfonate)ethyl)phenyl)pent-4-ynamide.
5 c) N-(4-(2-(N-oxyphthalimide)ethyl)phenyl)pent-4-ynamide
[0211] The N-(4-(2-(methanesulfonate)ethyl)phenyl)pent-4-ynamide
was dissolved in 6 mL of acetonitrile and added to a solution of
N-hydroxyphthalimide (200 mg, 0.9 mmol) and triethyl amine (250
.mu.l, 1.8 mmol) in 2 mL acetonitrile. The reaction mixture was
stirred at 50.degree. C. for 2 days. The reaction mixture was then
diluted with 40 mL of ethyl acetate and washed with 20 mL of 0.5 M
HCl (aq.), 5.times.30 mL of 10 NaHCO.sub.3 (aq.) to remove the red
color, and finally 5 mL of water. The organic phase was dried with
MgSO.sub.4, filtered, and the solvent was evaporated. The crude
product was re-crystallized from 10 mL of toluene to obtain
N-(4-(2-(N-oxyphthalimide)ethyl)phenyl)pent-4-ynamide which was
characterized by NMR and MALDI-TOF.
5 d) N-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamide
[0212] N-(4-(2-(N-oxyphthalimide)ethyl)phenyl)pent-4-ynamide (20
mg, 5.5 .mu.mol) and ethylenediamine (200 .mu.L, 3.0 mmol) was
dissolved in 2 mL of ethanol. The reaction was stirred at
75.degree. C. for 2 hours. The solvent was evaporated and the crude
product purified by column chromatography on silica gel eluting
with a gradient of toluene (T) and ethyl acetate (E) from 2:1 to
1:3 (T:E). The product N-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamide
was characterized by NMR and MALDI-TOF.
##STR00022##
Preparation of Stock Solution:
[0213] N-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamide (2.5 mg) was
placed in a metric flask and acetonitrile (1000 .mu.L) was added to
dissolve the linker.
[0214] Throughout the specification and the claims which follow,
unless the context requires otherwise, the word `comprise`, and
variations such as `comprises` and `comprising`, will be understood
to imply the inclusion of a stated integer, step, group of integers
or group of steps but not to the exclusion of any other integer,
step, group of integers or group of steps.
[0215] All patents and patent applications mentioned throughout the
specification of the present invention are herein incorporated in
their entirety by reference.
[0216] The invention embraces all combinations of preferred and
more preferred groups and suitable and more suitable groups and
embodiments of groups recited above.
* * * * *