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RELATIONSHIP TO OTHER APPLICATION(S)
This application is a continuation-in-part of PCT application PCT/US
93/05601 filed on Jul. 9, 1993 as a continuation-in-part of U.S. Ser. No.
07/910,941 filed on Jul. 9, 1992, now U.S. Pat. No. 5,296,583 the
disclosure(s) of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to materials which are resistant to in
vivo calcification, and more particularly, to calcification-resistant
biomaterials, suitable for implantation in a living being, comprising a
synthetic biocompatible polymer to which an anticalcification agent(s) is
bound by stable, irreversible covalent bonds.
2. Description of the Related Art
This invention relates generally to materials which are resistant to in
vivo calcification, and more particularly, to calcification-resistant
biomaterials, suitable for implantation in a living being, comprising a
synthetic biocompatible polymer to which an anticalcification agent(s) is
bound by stable, irreversible covalent bonds.
More than 100,000 cardiac valve prostheses are placed in patients each
year. Frequently, valve replacement surgery is the only means of treating
cardiac valve disease. Currently used replacement valves include
mechanical valves which may be composed entirely of a synthetic polymeric
material such as polyurethane; bioprosthetic valves derived from bovine
pericardium or porcine aortic valves; and aortic homografts.
Use of mechanical valves is frequently complicated by thrombosis and tissue
overgrowth leading to valvular failure. Calcification is the most frequent
cause of the clinical failure of bioprosthetic heart valves fabricated
from porcine aortic valves or bovine pericardium. Human aortic homograft
implants have also been observed to undergo pathologic calcification
involving both the valvular tissue as well as the adjacent aortic wall
albeit at a slower rate than the bioprosthetic heart valves. Pathologic
calcification leading to valvular failure, in such forms as stenosis
and/or regurgitation, necessitates re-implantation. Therefore, the use of
bioprosthetic heart valves and homografts has been limited because such
tissue is subject to calcification. Pathologic calcification also further
complicates the use of synthetic vascular grafts and other artificial
heart devices, such as ventricular assist systems, because its affects the
flexibility of the synthetic polymers used to produce the devices.
The mechanism for pathological calcification of cardiovascular tissue is
not understood. Generally, the term "pathologic calcification" refers to
the deposition of calcium phosphate mineral salts in association with a
disease process. Calcification may be due to host factors, implant
factors, and extraneous factors, such as mechanical stress. There is some
evidence to suggest that deposits of calcium are related to devitalized
cells, and in particular, cell membranes, where the calcium pump
(Ca.sup.+2 -Mg.sup.+2 -ATPase) responsible for maintaining low
intracellular calcium levels is no longer functioning or is
malfunctioning. Calcification has been observed to begin with an
accumulation of calcium and phosphorous, present as hydroxyapatite, which
develops into nodules which can eventually lead to valvular failure.
Research on the inhibition of calcification of bioprosthetic tissue has
focussed on tissue pretreatment with either detergents or diphosphonates.
Both of the aforementioned compounds tend to wash out of the bioprosthetic
tissue with time due to blood-material interactions. Thus, these
treatments merely delay the onset of the inevitable calcification process.
To date, long-term prevention of calcification has been an unattainable
result. Accordingly, there is a need for a means of providing long-term
calcification resistance for bioprosthetic or synthetic heart valves and
other implantable, or in-dwelling, devices which are subject to in vivo
pathologic calcification.
Systemic use of anticalcification agents, such as diphosphonates, results
in significant side effects on bone, and overall, growth. Site specific
therapy offers treatment with low regional drug levels and minimal side
effects.
There is a further need in the art for improved biomaterials which are
calcification-resistant and thromboresistant. Attempts have been made to
bond the anticoagulant heparin to the surface of biomaterials. However,
the known heparin binding schemes result in products which exhibit only
temporary surface anticoagulation effects. There is, thus, a need for
biomaterials which offer long-term thromboresistance.
It is, therefore, an object of this invention to provide biomaterials for
implantation in a mammal which have increased resistance to in vivo
pathologic calcification.
It is another object of this invention to provide biomaterials for
implantation in a mammal which have a long-term, or prolonged, resistance
to in vivo pathologic calcification.
It is also an object of this invention to provide biomaterials for
implantation in a mammal which have localized calcification inhibition
and, hence, avoid the toxic side effects associated with systemic
administration of anticalcification agents.
It is additionally an object of this invention to provide a method of
fabricating and/or treating biomaterials for implantation in a mammal to
render the biomaterials resistant to in vivo pathologic calcification.
It is yet a further object of this invention to provide a novel method of
covalently bonding anticalcification agents, specifically polyphosphonates
or other anticalcification agents beating functionalities capable of
epoxide derivatization, to biomaterials.
It is also another object of this invention to provide a novel method of
irreversibly binding polyphosphonates to synthetic biomaterials for
permanent calcification inhibition.
It is still a further object of this invention to provide a novel method of
irreversibly binding heparin to the synthetic biomaterials for permanent
thromboresistance.
SUMMARY OF THE INVENTION
The foregoing and other objects are achieved by this invention which
provides, in one aspect thereof, a biomaterial for implantation in the
interior of the body of a living being. The biomaterial has irreversibly
bound thereto an effective amount of an anticalcification agent for
rendering said biomaterial resistant to in vivo pathologic calcification.
The anticalcification agent(s) is bound to the synthetic biomaterial by a
novel epoxide-based derivatization scheme, herein referred to as
"epoxy-bridge incorporation," which results in stable, irreversible
covalent bonding of the anticalcification agent to the synthetic
biomaterial through epoxide linkages. FIG. 1 shows an illustrative
reaction scheme and the resulting product, which, in this embodiment, is a
phosphonated polyurethane wherein the polyphosphonate anticalcification
agent is linked to the soft segment of a polyurethane via the phosphonate
hydroxy groups. FIG. 5 shows an alternative reaction scheme, and resulting
product, in which the polyphosphonate anticalcification agent is linked to
the soft segment of a polyurethane by a less reactive alcoholic hydroxy
functional group of the polyphosphonate anticalcification agent.
The term "biomaterial" as used herein denotes any synthetic biocompatible
polymeric material which is known, or becomes known, as being suitable for
in-dwelling uses in the body of a living being, i.e., which is
biologically inert and physiologically acceptable, non-toxic, and
insoluble in the environment of use.
Illustrative biomaterials suitable for use in the practice of the invention
include naturally-derived polymers, such as cellulose or collagen-based
materials, or synthetic polymers, whether hydrophilic or hydrophobic,
including without limitation, polyurethane, polydimethylsiloxane, ethylene
vinyl acetate, polymethyl methacrylate, polyamide, polycarbonate,
polyester, polyethylene, polypropylene, polystyrene, polyvinyl chloride,
polytetrafluoroethylene, polysulfone, or cellulose acetate. It is to be
understood that the term polymer is to be construed to include copolymers,
such as the copolymer of polyurethane and silicone.
In preferred embodiments, the anticalcification agent is an amino
diphosphonate or other polyphosphonate.
Exemplary diphosphonates include 3-amino-1-hydroxypropane-1,1-diphosphonic
acid (AHDP) and ethanehydroxydiphosphonate (EHDP), also known as
2-hydroxyethane bisphosphonic acid. In certain embodiments, other
polyphosphonates, such as diethylentriaminepenta(methylenephosphonic acid)
and aminotri(methylenephosphonic acid) are preferred. As used herein the
term "polyphosphonate" includes compounds having two or more phosphonates
per molecule. Such polyphosphonates are commercially available or can be
synthesized by those of skill in the art. Additional illustrative examples
include, without limitation, hexamethylenediaminetetra(methylenephosphonic
acid) and diethylenetriaminopenta(methylenephosphonic acid). Of course,
other amino-containing anticalcification agents, such as amino derivatives
of phosphocitrate, would be suitable for incorporation into the practice
of the invention.
The polyphosphonates may, thus, have any alkyl, aralkyl or aryl backbone
structure and reactive hydroxy groups on the phosphonate moieties and less
reactive hydroxy groups on the alkyl, aralkyl or aryl. In certain
embodiments of the invention, the epoxide linkage is through the reactive
hydroxy groups on the phosphonate moieties. In other embodiments, the
epoxide linkage is through the hydroxy group on the alkane.
In an alternative embodiment, the calcification-resistant biomaterials of
the present invention can be rendered resistant to in vivo thrombus
formation by having an effective amount of the anticoagulant heparin
irreversibly bound thereto by the epoxy-bridge incorporation techniques
described herein.
In a method aspect of this embodiment of the invention, a thromboresistant
polymeric material may be fabricated by:
forming a solution of heparin and a reactive polyfunctional epoxide to form
a heparinepoxide monoadduct;
adding a solution of a prepolymerized biocompatible polymer to the
monoadduct to form a mixture; and
polymerizing the mixture.
Since the anticalcification agents are irreversibly bound to the
biomaterial substrate by epoxide linkages, any anticalcification agent
which has reactive hydrogen functionalities, such as amine, amide,
alcohol, or carboxylic acid functionalities, for example, can be linked to
a biocompatible elastomer via epoxy-bridge incorporation as described
herein. Examples of other anticalcification agents include, without
limitation, sulfaminotricarballyate, alpha amino oleic acid,
pyrophosphate, statherin, polylysine, and polyarginine.
In a method aspect of the invention, synthetic calcification-resistant
biocompatible polymeric materials are made by incorporation of
polyphosphonate during primary polymerization of a biocompatible polymer
or copolymer. In a specific illustrative embodiment, a
calcification-resistant synthetic polyurethane is fabricated by the steps
of:
forming a monoadduct of a polyphosphonate anticalcification agent and a
reactive polyfunctional epoxide;
adding the monoadduct to a prepolymer base, illustratively a macroglycol,
including active hydrogen functional groups, such as a polyol, polyester,
or polyether;
adding diisocyanate as the second component of the polyurethane; and
polymerizing the resultant mixture.
Illustrative polyfunctional epoxides which are suitable for use in the
practice of the invention include diglycidyl butanediol ether, ethanediol
diglycidyl ether, butanediol diglycidyl ether, and polyglycerol
polyglycidyl ethers.
In still further method embodiments of the invention, synthetic
calcification-resistant biocompatible polymeric materials are made by
incorporation of polyphosphonate into prepolymerized biocompatible
polymeric materials. In one specific illustrative embodiment, a solution
of a polyphosphonate anticalcification agent and a reactive polyfunctional
epoxide is formed in a solvent; a pre-polymerized biocompatible polymer
which is soluble in the same solvent is added to the
polyphosphonate/polyfunctional epoxide solution to form a mixture; and the
mixture is polymerized. Of course, the pre-polymerized polymer could be
dissolved in the same, or a compatible, solvent prior to contact with the
polyphosphonate/polyfunctional epoxide solution.
In yet another method aspect of the invention, a calcification-resistant
polymeric material is made by a technique which does not involve
epoxy-bridge formation. In this embodiment, a diisocyanate-terminated
prepolymer is formed from the soft segment and hard segment components of
a polyurethane, and a chain extender to the diisocyanate-terminated
prepolymer. In embodiments where the chain extender is a short chain diol,
such as 1,4-butanediol, the product is an hydroxy-terminated polyurethane.
The hydroxy-terminated polyurethane can be reacted with a polyphosphonate
anticalcification agent via an epoxy-bridge incorporation technique of the
type described herein to produce a polyphosphonate-terminated
polyurethane.
In other embodiments, the chain extender is a polyphosphonate, which forms
directly a phosphonate-terminated polyurethane.
In a still further method aspect of the invention, a
calcification-resistant biomaterial may be made by:
forming a tetraester derivative of a polyphosphonate anticalcification
agent;
reacting the tetraester derivative of a polyphosphonate anticalcification
agent and a reactive polyfunctional epoxide to form a phosphonated epoxide
monoadduct;
reacting the phosphonated epoxide monoadduct with an hydroxy-terminated
polymer to form a tetraester-terminated biocompatible polymer. The
tetraester-terminated biocompatible polymer can be hydrolyzed to a
phosphonate-terminated biocompatible polymer with a mild hydrolyzing
agent, such as bromotrimethyl silane, or by exposure to water. The result
is a phosphonate-terminated polyurethane which is linked to the polymer
via epoxide linkages with the hydroxy groups on the alkyl, aralkyl, or
aryl backbone of the polyphosphonate anticalcification agent.
BRIEF DESCRIPTION OF THE DRAWING
Comprehension of the invention is facilitated by reading the following
detailed description, in conjunction with the annexed drawing, in which:
FIG. 1 is an illustrative reaction scheme for linking an anticalcification
agent to synthetic biomaterials in accordance with the principles of the
invention herein;
FIG. 2 is a graphical illustration showing dissociation of an
anticalcification agent, EHDP, from a calcification-resistant polyurethane
matrix fabricated in accordance with a method aspect of the invention;
FIG. 3 is a graphical illustration showing the release profile of EHDP from
hydroxy-terminated polyurethane matrices fabricated in accordance with a
second method aspect of the invention as a function of drug loading;
FIG. 4 is a graphical illustration of the calcium content (.mu.g/mg) of
several synthetic biomaterial specimens in accordance with the invention
following subdermal implantation in rats for 60 days;
FIG. 5 is another illustrative reaction scheme for linking an
anticalcification agent to synthetic biomaterials in accordance with the
principles of the invention herein.
FIG. 6 is an illustrative reaction scheme for the formation of an
hydroxy-terminated polyurethane as the first step in a synthesis of
diphosphonate-derivatized epoxidized polyurethanes;
FIG. 7 is an illustrative reaction scheme for the epoxidation and covalent
binding of EHDP to the soft segment of the hydroxy-terminated polyurethane
prepared in accordance with FIG. 6 as the second step in the synthesis of
diphosphonate-derivatized epoxidized polyurethanes;
FIG. 8 is a graphical representation of the overlaid ATR-FTIR spectra of
polyurethane (PU-2000), epoxidized polyurethane (FIG. 7, Compound 66), and
the EHDP-epoxidized polyurethane (FIG. 7, Compound 68);
FIG. 9 is an illustrative reaction scheme for coupling a hard segment
modifier or spacer group, such as a bifunctional or polyfunctional
isocyanate, to polyurethane followed by binding the anticalcification
agent to the polymer through the isocyanate functional groups; and
FIG. 10 is a graphical representation of the overlaid ATR-FTIR spectra of
polyurethane (PU-2000), the intermediate (PU-MDI) and EHDP-containing,
surface-modified polyurethane (FIG. 9, Compound 93).
DETAILED DESCRIPTION
Given below are several specific illustrative techniques for producing
calcification-resistant synthetic biomaterials in accordance with the
principles of the invention.
Although the examples given are primarily directed to the preparation of
calcification-resistant heart valve components, the techniques described
herein are applicable to the creation of any other device, prosthesis, or
implant comprising biomaterials of the type used for in-dwelling or
surgically implanted devices. Such additional examples include, other
cardiovascular devices, such as artificial hearts and ventricular assist
systems, urinary catheters, and orthopedic devices which are also subject
to pathologic calcification. In its broadest sense, the
calcification-resistant materials can be configured to encompass, inter
alia, knit or woven fabrics, single or plural filaments, extruded, cast or
molded items, coatings on polymeric substrates or biological tissues, etc.
In accordance with the principles of the invention, polyphosphonate
anticalcification agents have been successfully bound to synthetic
biocompatible polymeric materials, such as medical grade polyurethane, by
epoxy derivatization techniques. These techniques, using reactive
bifunctional or polyfunctional epoxides, result in stable, irreversible
covalent bonding of the diphosphonates to the biomaterial substrate (see,
for example, Table I and FIGS. 2 and 3). The following procedures have
resulted in the incorporation of 100 to 500 nM/mg polyphosphonate
anticalcification agent into the polymeric material (see Table II).
It should be noted that the concentration range for the bound diphosphonate
salt is given for purposes of illustration only, and can be varied by
those of skill in the art because it is greatly in excess of the
therapeutically effective amount. The ability to irreversibly bind a high
concentration of anticalcification agent to the biomaterial (see FIG. 3),
thereby directly placing a high concentration of pharmaceutic at the
potential site of calcification over an extended period of time, is a
significant advantage of this invention over the prior art.
Illustrative reactive bifunctional or polyfunctional epoxides suitable for
use in the practice of the invention include, without limitation,
diglycidyl butanediol ether (GAB), ethanediol diglycidyl ether, erythritol
anhydride (EDE), butanediol diglycidyl ether (BDE), or the polyfunctional
epoxides sold under the trademark Denacol by Nagasi Chemicals, Osaka,
Japan. The Denacol epoxides are polyfunctional polyglycerol polyglycidyl
ethers. For example, Denacol 512 has 4 epoxides per molecule and Denacol
521 (see FIG. 1, compound 12) has 5 epoxides per molecule.
Commercially available medical grade elastomers suitable for the practice
of the invention include, in preferred embodiments, polyurethanes, or
block copolymers which contain high molecular weight macroglycols linked
together by a urethane group. Generally, polyurethane elastomers are
produced by the rearrangement polymerization of diisocyanate and
macroglycols. The main constituents are a hard segment which may be a
diisocyanate, such as methylene diphenyl diisocyanate; a soft segment
which may be a long chain, hydroxyl-terminated macroglycol as either a
polyester or a polyether, illustratively polytetramethylene oxide; and a
chain extender, such as a short chain glycol (e.g., 1,4-butanediol) or
diamine.
Illustrative examples include, without limitation, Thiomer or Tecoflex 80A
or 60A (trademarks of Thermedics Corp., Woburn, Mass.); polyurethane
PU-2000 sold by CarboMedics Corporation, Austin, Tex.; Biomer (an aromatic
co(polyetherurea) available from Ethicon, Somerville, N.J.); Cardiothane
(a silicone-urethane copolymer available from Kontron, Inc., Everett,
Mass.); or Pellathane, a polyurethane sold by Dow Chemical, Midland, Mich.
In specific advantageous embodiments of the invention, the
anticalcification agent is a diphosphonate, such as
ethanehydroxydiphosphonate (EHDP) or aminopropanehydroxydiphosphonate
(APDP), or a polyphosphonate, such as aminotri(methylenephosphonic acid),
and diethylentriaminepenta(methylenephosphonic acid). Other phosphonate
anticalcification agents, however, are suitable for use in the practice of
the invention. Moreover, any other anticalcification agent which is known,
or becomes known, and has amine, amide, alcohol, or carboxylic acid
functionalities, or any reactive hydrogen functionality, for example, can
be linked to a biocompatible elastomer via the epoxide derivatization
techniques described herein.
Other such anticalcification agents include sulf-amino-tricarballylate
(Analyt. Biochem., Vol. 132, p. 115, 1983); alpha-amino-oleic acid, Trans.
Soc, Biomat., Vol. XIV. p. 60, 1991); pyrophosphoric acid, Science, Vol.
165, p. 1264, 1969); and the anticalcification protein, statherin and
profamine sulfate (J. Biomed, Mater. Res., Vol. 25, p. 85, 1991);
polylysine; and polyarginine.
In certain preferred embodiments, it is necessary to use the acid form
since salts of polyphosphonates are not soluble in the organic solvents
used in the reactions. Acid EHDP may be purified from a commercially
available acid form or from the disodium salt. Acid EHDP (crude) is
commercially available from Monsanto Chemical, St. Louis, Mo. under the
trademark Dequest 2010. Disodium acid EHDP is commercially available from
Norwich Pharmaceuticals, Norwich, N.Y.
Illustratively, the crude acid form or the disodium salt of EHDP is
purified on a cation exchange resin, Dowex-50W (50x4-400; Dow Chemical
Company, Midland, Mich.). The Dowex-50 resin is conditioned with
alternating washes of 1M sodium hydroxide and 1M hydrochloric acid through
seven cycles in a Buchner funnel. The final washing is done with
hydrochloric acid. The resin is then washed with double distilled water
until the pH of the effluent corresponds to the pH of the double distilled
water. The resin is stored in water until use.
An appropriate ratio of a sodium or calcium salt of EHDP to ion exchange
resin is 1 g of EHDP salt in 100 ml water to 32 g of resin. The resin
mixture is stirred for four hours at room temperature. The Dowex-50 resin
has a high capacity for sodium, and other cationic contaminants, and
completely exchanges these contaminants with hydrogen to yield a pure
solution of acid EHDP. The supernatant is then decanted from the resin and
freeze dried under high vacuum. The purified acid EHDP may be
recrystallized by any known technique, such as solvent evaporation with
seed crystal addition.
I. EPOXY DERIVATIZATION TECHNIQUES
A. Incorporation of Polyphosphonate During Primary Polymerization
In general, a polyphosphonate or other epoxy-reactive anticalcification
agent will be combined with a polyepoxide in a solution under reactive
conditions, which will result in both adduct formation of the
anticalcification agent with the epoxide, and retention of residual
reactive epoxy groups for subsequent reactions with a polyol. The reactive
anticalcification-epoxy-bridge compound will then be combined with a
polyol or polyether prior to polyurethane polymerization via the usual
diisocyanate addition. The unique feature of this general reaction scheme
is the use of the polyepoxy compound as an epoxy-bridge forming agent, to
incorporate anticalcification compounds within the framework of
conventional polyurethane chemistry, or other biocompatible polymer
chemistry in general.
Typically, a polyphosphonate anticalcification agent and a reactive
polyepoxide are combined in a 1:1 molar ratio in a suitable solvent, such
as THF DMAC, or DMF, for a time sufficient to form a monoadduct,
illustratively 12 to 15 minutes. The monoadduct is combined with a
prepolymer base in molar ratios ranging from 1:1 to 5:1 reactive
adduct-epoxy groups per each potential hydroxy-terminus to form a resin
having both the epoxide and the anticalcification agent. The prepolymer
base may be, in the case of polyurethanes, a macroglycol such as a
polyester or polyether. The second polymer component, which may be a
diisocyanate for a polyurethane, is then added to the resin and
polymerization is initiated.
Specific examples of prepolymer base components include, without
limitation, polytetramethylene glycol, polethylene glycol, polypropylene
glycol, or polyols containing heteroatoms such as sulfur or nitrogen with
reactive bifunctional hydrogen functionalities. The polyols can range in
molecular weight from a few hundred to a thousands. Exemplary
diisocyanates include toluene diisocyanates, cyclohexyl diisocyanates,
hexamethylene diisocyanates, naphthalene diisocyanate, 1-4-butane
diisocyanate, etc.
In a specific illustrative embodiment, butanediol diglycidyl ether (25
.mu.l) was added to a 0.1M solution of acid EHDP in 3 ml of dried
tetrahydrofuran (THF) and stirred for 30 minutes, and preferably between
12 and 15 minutes. The resulting solution was combined with 3.45 g of
polytetramethylene glycol (1000 mw) and stirred for an additional 30
minutes at room temperature. Polytetramethylene glycol is the prepolymer
base for Tecoflex 80A. The second component of the copolymer, diisocyanate
(0.93 g), was added to the solution and stirred until homogeneity was
obtained. The polymerization reaction was catalyzed by the addition of 200
.mu.l acetone-FeCl.sub.3 (5 mg/ml). The mixture was then poured into a
petri dish to polymerize in a vacuum oven at 100.degree. C. (about 48
hours).
Release studies were conducted by incorporating radioactive EHDP (.sup.14 C
EHDP) into Tecoflex 80A in accordance with the procedure described above.
Referring to FIG. 2, the dissociation of epoxy-bridge linked EHDP from the
resulting calcification-resistant polyurethane into a physiological buffer
(pH 7.4) at 37.degree. C. over a 128 day period is negligible. The data is
expressed as the percentage released of the total bound. Thus,
approximately 97% of the originally bound EHDP remains after at the 128th
day.
The resulting calcification-resistant polyurethane can be dissolved in THF,
dimethylacetamide (DMA), or dimethylformamide (DMF), and cast as films or
used as coatings. In the alternative, the calcification-resistant
polyurethane could be cast into molds.
B. Incorporation of Polyphosphonate Into Prepolymerized Materials
Polyphosphonates can also be irreversibly bound to prepolymerized materials
via epoxy-bridge incorporation, illustratively, with hydroxy-terminated
polyurethanes or amino-terminated polyurethane ureas, such as Mitrathane
MPU5 (a polyetherurethane urea, a trademark of Polymedica, Denver, Colo.)
or Biomer (a trademark of Ethicon, Somerville, N.J.).
In general, a polyphosphonate and a polyepoxide are combined in a 1:1 molar
ratio in a suitable solvent, such as THF, for a time sufficient to form a
monoadduct, illustratively 30 minutes. A prepolymerized polymer, which in
some embodiments may be dissolved in a compatible solvent, is combined
with the polyphosphonate/polyepoxide monoadduct in a ratio of one mole
polymer to one mole epoxy group. The resulting mixture is dried and
reacted in a vacuum oven for a period of time, illustratively 24 to 48
hours, at a temperature of about 50.degree. to 75.degree. C.
Referring to FIG. 1, an illustrative reaction scheme shows epoxy-bridge
incorporation of a polyphosphonate into a polyurethane in accordance with
a method aspect of the invention. A 0.1M solution of acidic EHDP (compound
11) in 2.0 ml THF was made. A reactive epoxide, Denacol 521 (a
polyfunctional epoxide with five reactive groups per molecule sold by
Nagasi Chemical, Osaka, Japan and shown as compound 12) was added to the
EHDP solution in a concentration of 0.1M (148 mg) or 0.02M (29.6 mg). The
mixture was stirred for 12 to 15 minutes at 37.degree. C. to form the
monoadduct, or phosphonated-epoxide compound 13. The biocompatible
polymeric material, in this case an hydroxy-terminated polyurethane
compound 14 (72,000 Mn, 1.52-1.60 MWD, 9.0512 g PU-2000 by CarboMedics,
Inc., Austin, Tex.) was then added to the EHDP-Denacol mixture and stirred
until homogeneous. In other embodiments, amino-terminated polymers, such
as Mitrathane MPU5 (11.68 g) or Biomer (10.19 g) are used. Additional
solvent (5-10 ml THF) was added to dilute the solution. The solution was
then poured into a petri dish and placed in a 60.degree. C. oven.
Polymerization was permitted to take place under vacuum over about a 48
hour period. However, the vacuum was not applied until the air bubbles in
the solution had disappeared. The result is phosphonated polyurethane
compound 15.
Table I below shows the amount of EHDP incorporated (nM/mg) in the
polyurethane biomaterial via epoxy-bridge incorporation and the percent
released in vitro after 35 days in an isotonic HEPES buffer at pH 7.4 at
37.degree. C. under perfect sink conditions. It should be noted that all
EHDP which was not irreversibly covalently bound to the matrix material
was released within 48 hours.
TABLE I
______________________________________
EHDP % Released
Incorporated
After
Polyurethane
Epoxy (nM/mg) 35 Days
______________________________________
Tecoflex GAB 71 3.9%
Tecoflex BDE 72 2.0%
Biomer Denacol 521 81 2.2%
Mitrathane MPU5
Denacol 521 68 35.5%
______________________________________
*unbound drug was released within 48 hours
In still further illustrative embodiments of the invention, the
diphosphonate EHDP and the polyphosphonates, aminomethyltriphosphonic acid
and butylpentaphosphonic acid, were incorporated into pre-polymerized
elastomers, specifically polyurethanes and silicone-polyurethane
copolymers, in accordance with the procedure set forth above using Denacol
512 as the polyepoxide. Table II shows the amount of incorporated
polyphosphonate in nM/mg.
TABLE II
______________________________________
TYPE OF AMOUNT OF
BASE POLY- POLY-
POLYMER PHOSPHONATE PHOSPHONATE (nM/mg)
______________________________________
PU-Si ATMP 100
PU-Si ATMP 500
PU-2000 EHDP 100
PU-2000 EHDP 200
PU-2000 EHDP 300
PU-2000 EHDP 400
PU-2000 EHDP 500
PU-Si EHDP 100
PU-Si EHDP 400
PU-Si EHDP 500
PU-2000 DTMP 100
PU-2000 ATMP 100
______________________________________
Notes:
1) PU2000: solvent cast polyurethane (Carbomedics, Inc., Austin, Tx)
2) PUSi: polyurethanesilicone rubber copolymer (Dow Corning, Midland, MI)
3) EHDP: ethanehydroxydiphosphonate
4) ATMP: aminomethyltriphosphonic acid (Monsanto Chemical, St. Louis, MO)
5) DTMP: butylpentaphosphonic acid (Monsanto Chemical)
In addition to the foregoing, an in vitro radioactive diphosphonate
(.sup.14 C EHDP) release study was conducted with several of the
polyurethane-EHDP derivatives, formed by the epoxy-bridge incorporation
technique, to evaluate release of EHDP from the polyurethane-EHDP matrix
over time, and as a function of drug loading. FIG. 3 illustrates the
release profile of EHDP from hydroxy-terminated polyurethane matrices as a
function of drug loading (45.4 nM/mg to 398.7 nM/mg). As can be seen,
there is virtually no significant dissociation of the covalently linked
diphosphonate incorporated via this reactive scheme.
In the long-term binding stability studies (over 365 days) shown in FIG. 3,
more than 75% of the total covalently bound EHDP was retained in the
polymer matrix. The initial 25% unbound EHDP was released during the first
two weeks. In contrast, a specimen of EHDP (458.17.+-.1.3 nM) physically
dispersed in a hydroxy-terminated polyurethane lost 26% of the EHDP in the
first 24 hours, followed by release of 34% more in the next 60 days. The
remaining 40% leached out slowly and continuously until exhausted.
The higher phosphonate content polyphosphonates are particularly
advantageous for incorporation into biomaterials. Each molecule of a
pentaphosphonate, for example, will have 2.5 times more phosphonate, on a
molar basis, than EHDP. Thus, a greater amount of anticalcification agent
can be irreversibly bound to the substrate material.
The calcification-resistant synthetic biomaterials of the present invention
can be cast into molds; dissolved in solvents, such as DMA and THF, and
cast into thin films or flexing leaflet membranes; combined with other
compatible polymers; dip-coated on surfaces of other materials, including
tissue-derived biomaterials to improve their biophysical stability.
The epoxy-bridge incorporation method can be adapted to irreversibly bind
the anticoagulant heparin to a biocompatible polymeric material, such as
polyurethane. The result is a thromboresistant biomaterial which is
particularly suited for manufacturing devices for intravascular
implantation. The heparin immobilized polymer can be mixed with the
phosphonated polyurethanes described herein so as to produce a biomaterial
which is both thromboresistant and calcification-resistant. Of course, the
heparinized polyurethane can be used alone or homogeneously mixed with
other polymers to render them thromboresistant. Other anticoagulants
having structures similar to heparin, such as low molecular weight
heparins or synthetic heparins may be immobilized by the epoxy-bridge
incorporation technique.
Heparin (500 nM), such as that derived from porcine intestinal mucosa, is
dissolved in a minimal volume of distilled water. About 0.2 ml dry THF is
added. A polyfunctional epoxide, Denacol 512 (1:1 w/w ratio) is added to
the heparin solution and the resulting mixture is permitted to react for a
period of time, illustratively about 12 minutes, before it is added to a
solution of a hydroxy-terminated polyurethane of known composition (4 mg,
or 1:1 w/w ratio). The resulting mixture is reacted for about 24 hours,
with constant stirring, and then cast into a film.
Release studies were conducted on heparinized polymer samples incorporating
radiolabelled heparin. There was an initial burst phase release of heparin
from the polymer matrix, possibly due to release of unreacted heparin from
the matrix. However, after 4 days, the release stabilized, i.e., there was
no further release, indicating that the remaining heparin was covalently
bound to the polymer.
Heparinized polymer samples (1 mm.times.1 mm) containing 500 nM heparin
were placed in dog plasma and incubated for 1 hour or 24 hours at
37.degree. C. The plasma was then observed for clot formation time by the
standard APPT Test. For comparative purposes, specimens of PU-2000,
alkylated PU-2000, and an alkylated polyurethane-silicone copolymer were
also incubated in plasma. The results are set forth in Table III below.
TABLE III
______________________________________
Ave. Clot
Sample Clotting Time (sec.)
Time (sec.)
______________________________________
Incubated Plasma
14.7 14.4 14.3 14.8 -- 14.22
PU-2000/1 hr
15.8 14.8 15.8 14.3 15.9 15.32
PU-2000/24 hrs
33.3 33.6 33.5 -- -- 33.46
Hep. PU-2000
NO CLOT EVEN AFTER >200
200 sec.
alk. PU-2000/1 hr
14.7 13.8 14.4 13.9 13.9 14.14
alk. PU-2000/24 hrs
15.3 14.3 13.8 23.4 18.3 18.3
alk. PU-Si/1 hr
14.9 15.3 14.8 14.3 15.4 14.94
alk. PU-Si/24 hr
14.9 16.8 15.9 14.4 13.8 13.8
______________________________________
In all of the foregoing method aspects, the irreversible covalent binding
of the polyphosphonate anticalcification agent was achieved through a
reactive phosphonate hydroxy functional group as shown in FIG. 1. In still
a further alternative embodiment of a method aspect of the invention, the
polyphosphonate anticalcification agent is bound to the polymer at the
less reactive alcoholic hydroxy functional group of the polyphosphonate
anticalcification agent using a variation of the epoxy-bridge
incorporation technique as shown in the illustrative preparatory scheme of
FIG. 5. The result is a novel biocompatible polymeric material which can
be hydrolyzed to a phosphonate-terminated polyurethane. In some
embodiments, a deblocking agent, such as the mild hydrolyzing agent
bromotrimethyl silane, is used to hydrolyze the ester groups. In other
embodiments, exposure to an aqueous environment causes hydrolysis of the
ester linkage.
In a specific illustrative embodiment of the invention, a tetraester
derivative of EHDP, specifically tetraethyl
ethane-1-hydroxy-1,1-diphosphonate, is synthesized and then covalently
bound to hydroxy-terminated polyurethane by an epoxy-bridge incorporation
technique as shown in FIG. 5.
a. Synthesis of a tetraester derivative of a polyphosphonate
anticalcification agent
i.) A direct method generally requires the synthesis of dialkyl acetyl
phosphonate from a trialkyl phosphite and acetyl chloride. The dialkyl
acetyl phosphonate is then reacted with dialkyl hydrogen phosphite and
dialkyl amine to yield tetraalkyl 1-hydroxyethylidene bisphosphonic acid
(TEED). A specific illustrative embodiment is given below:
Triethyl phosphite (1.0 mole) was added dropwise with stirring to acetyl
chloride (1.0 mole) over a period of about 2 hours. The reaction was
maintained at a temperature of about 25.degree. C. during this time. After
all of the triethyl phosphite was added, the reaction mixture was stirred
at room temperature for 2 more hours and then heated gently to evaporate
the acetyl chloride. Distillation of the reaction mixture gave a 70% yield
of the dialkyl acetyl phosphonate, specifically diethyl acetylphosphonate
(b.p. 63.degree.-65.degree. C.).
Diethyl hydrogen phosphite (1.0 mole) and diethyl amine (1.0 mole) were
combined in a round bottom flask. Diethyl acetylphosphonate (1.0 mole) was
added dropwise, with stirring. The reaction mixture was maintained at a
temperature of about 75.degree. C. for an additional 2 hours after all of
the diethyl acetyl phosphonate was added. The resulting mixture was
distilled under reduced pressure to yield tetraethyl 1-hydroxyethylidene
bisphosphonic acid as a pale yellowish liquid which crystallized at
-15.degree. C. into small needle-shaped crystals.
ii.) Tetraalkyl 1-hydroxyethylidene bisphosphonic acid can also be formed
by the esterification of a polyphosphonate anticalcificatio | | |
