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Description  |
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The present invention relates to a method for retarding or preventing the
calcification of a prosthesis implanted in a mammal such as a human.
BACKGROUND OF THE INVENTION
The surgical implantation of prosthetic devices (prostheses) into humans
and other mammals has been carried out in recent years with increasing
frequency. Such prostheses include, by way of illustration only, heart
valves, vascular grafts, urinary bladders, heart bladders, left
ventricular-assist devices, hip prostheses, silastic breast implants,
tendon prostheses, and the like. They may be constructed from natural
tissues, inorganic materials, synthetic polymers, or combinations thereof.
By way of illustration, mechanical heart valve prostheses typically are
composed of rigid materials, such as polymers, carbons, and metals, and
employ a poppet occluder which responds passively with changes in
intracardiac pressure or flow. Valvular bioprostheses, on the other hand,
typically are fabricated from either porcine aortic valves or bovine
pericardium; in either case, the tissue is pretreated with glutaraldehyde
and then sewn onto a flexible metallic alloy or polymeric stent which
subsequently is covered with a poly(ethylene terephthalate) cloth sewing
ring covering. The assembled prostheses (often referred to in the
literature as bioprostheses) are stored in 0.2 percent glutaraldehyde.
Examples of reference of a more general nature include Helen E. Kambic et
al., "Biomaterials In Artificial Organs," Chem. Eng. News, Apr. 14, 1986,
pp. 31-48; and Frederick J. Schoen, "Pathology of Cardiac Valve
Replacement," Chapter 8 in D. Morse et al., Editors, "Guide to Prosthetic
Cardiac Valves," Springer-Verlag, N.Y., 1985, pp. 209-238.
Prostheses derived from natural tissues are preferred over mechanical
devices because of certain significant clinical advantages. Tissue-derived
prostheses generally do not require routine anticoagulation. Moreover,
when they fail, they usually exhibit a gradual deterioration which can
extend over a period of months, or even years. Mechanical devices, on the
other hand, typically undergo catastrophic failure.
While any prosthetic device can fail because of mineralization, and
especially calcification, this cause of prosthesis degeneration is
especially significant for tissue-derived prostheses. Indeed,
calcification has been stated to account for over 60 percent of the
failures of cardiac bioprosthetic valve implants. Despite the clinical
importance of the problem, the pathogenesis of calcification is
incompletely understood. Moreover, there apparently is no effective
therapy known at the present time.
References which discuss the calcification problem and its prevention
include. among others. R. J. Levy et al., "Bioprosthetic Heart Valve
Calcification: Clinical Features, Pathobiology, and Prospect for
Prevention," CRC Review in Biocompatibility, 2, 147-187 (1986); Frederick
J. Schoen et al., "Calcification of Bovine Pericardium Used in Cardiac
Valve Bioprostheses," Am. J. Pathol., 123, 134-145 (1986); Robert J. Levy
et al., "Inhibition by Diphosphonate Compounds of Calcification of Porcine
Bioprosthetic Heart Valve Cusps Implanted Subcutaneously in Rats,"
Circulation, 71, 349-356 (1985); Gershon Golomb et al., "Inhibition of
Bioprosthetic Heart Valve Calcification by Sustained Local Delivery of Ca
and Na Diphosphonate via Controlled Release Matrices," Trans. Am. Soc.
Artif. Intern. Organs, 32, 587-590 (1986); R. J. Levy et al., "Local
Controlled-Release of Diphosphonates from Ethylenevinyllacetate Matrices
Prevents Bioprosthetic Heart Valve Calcification" Trans. Am. Soc. Artif.
Intern. Organs, 31, 459-463 (1985); and Frederick J. Schoen et al., "Onset
and Progression of Experimental Bioprosthetic Heart Valve Calcification,"
Lab. Invest., 52, 523-532 (1985).
As a reading of many of the foregoing references will show, previous
efforts at preventing the calcification of tissue-derived prostheses
include:
(a) detergent pretreatment of the prosthesis;
(b) daily injection of a diphosphonate, such as 1-hydroxyethylidene
diphosphonic acid;
(c) covalent binding of a diphosphonate, such as
1-hydroxy-3-aminopropane-1,1-diphosphonic acid, to bioprosthetic tissue
proteins via residual aldehyde groups remaining after a glutaraldehyde
pretreatment; and
(d) controlled-release, site-specific diphosphonate delivery by an osmotic
pump or a controlled-release matrix, such as an ethylene-vinyl acetate
copolymer, typically with 1-hydroxyethylidene diphosphonic acid or
1-hydroxy-3-aminopropane-1,1-diphosphonic acid as the diphosphonate.
Suitable polymers typically include those disclosed in U.S. Pat. No.
4,378,224 to Moses J. Folkman et al.; see, also, U.S. Pat, Nos. 4,164,560
and 4,391,797, both to Moses J. Folkman and Robert S. Langer, Jr., neither
of which appears to be directed to preventing calcification. For a similar
disclosure which also does not appear to be directed at preventing
calcification of implanted prostheses, see U.S. Pat. No. 4,357,312 to Dean
S. T. Hsieh and Robert S. Langer, Jr. In addition to the two
diphosphonates mentioned [see, also, Krammsch et al., Circ. Res., 42,
562-571, (1978)], other anticalcification agents which apparently are
suitable for controlled-release applications include calcium channel
blockers such as nifedipine [Henry et al., J. Clin. Invest., 68. 1366-1369
(1981)]; calcium chelating agents such as ethylenediamine-tetraacetic acid
[Wartman et al., J. Atheroscler, Res., 7, 331-341 (1967)]; ionic
antagonists such as lanthanum trichloride [Kramsch, supra]; thiophene
compounds [Krammsch, supra]; and phosphocitrate analogues such as
2-aminotricarballylate.
As a variation of method (c), above, pretreatments of fixed natural tissue
prostheses which apparently do not involve covalent binding are known.
Several representative references are described below.
U.S. Pat. No. 4,402,697 to Elisabeth M. Pollock and David J. Lent describes
a pretreatment using a solution of a water-soluble phosphate ester such as
sodium dodecyl hydrogen phosphate.
A similar pretreatment using a solution of a water-soluble quaternary
ammonium salt such as dodecyltrimethylammonium chloride is described in
U.S. Pat, No. 4,405,327 to Elisabeth M. Pollock.
Finally, U.S. Pat, No. 4,323,358 to David J. Lentz and Elisabeth M. Pollock
describes a pretreatment using a solution of a water-soluble salt of a
sulfated higher aliphatic alcohol, such as sodium dodecyl sulfate.
While such methods were capable of lowering bioprosthetic tissue
calcification, they are not free from difficulties. For example, detergent
pretreatment, while having a short-term effectiveness, does not appear to
be a viable approach for the long-term inhibition of tissue calcification.
Diphosphonate injection at effective levels is accompanied by severe
untoward effects on bone and overall somatic growth. The use of an osmotic
pump requires the subdermal surgical implantation of the pump, and the
long-term supply of the anticalcification agent administered by the pump
is an issue which must be addressed. The long-term administration of an
anticalcification agent also is an issue with controlled-release methods.
In addition, the glutaraldehyde pretreatment used with all cardiac
bioprostheses apparently facilitates prosthesis tissue calcification.
Thus, there is a pressing need for a more effective method for reducing or
preventing the calcification of prostheses.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method for retarding or
preventing the calcification of a prosthesis implanted in a mammal by
covalently coupling to the prosthesis before implantation an effective
amount of an anticalcification agent in the form of a substituted
aliphatic carboxylic acid or a derivative thereof. The acid contains from
about 8 to about 30 carbon atoms, and it may be straight-chain or
branched-chain and saturated or unsaturated. The covalent coupling is via
the substituent moiety which may be an amino, a mercapto, a carboxyl or a
hydroxyl group. Also, the invention provides prostheses suitable for
implantation in a mammal made by the aforesaid method.
In one preferred embodiment, the anticalcification agent is a substituted
aliphatic carboxylic acid which contains from about 12 to about 24 carbon
atoms and no more than about three carbon-carbon double bonds. In a
particularly preferred embodiment, the anticalcification agent is a
substituted aliphatic straight-chain carboxylic acid which contains from
about 12 to about 22 carbon atoms and one carbon-carbon double bond.
The method of the present invention is useful for retarding or preventing
the calcification of a prosthesis implanted in a mammal, such as a human,
and has particular application with respect to those prostheses which are
especially susceptible to degeneration as a result of calcification.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "prosthesis" is meant to include any device which
is implanted in a mammal. Thus, the term includes heart valves and other
heart components, vascular replacements or grafts, artificial hearts,
urinary tract and bladder replacements, bowel and tissue resections in
general, left ventricular-assist devices, hip replacements, silastic
breast implants, artificial tendons, electrodes, catheters, and the like.
However, it will be recognized by those having ordinary skill in this art
that the present invention may be of most importance in relation to
prostheses for which calcification after implantation has been a clinical
problem. Thus, while the present invention can be used with essentially
any prosthesis, it may not be as beneficial for a prosthesis which is not
likely to suffer degeneration or malfunction as a result of
mineralization.
The material from which the prosthesis is prepared is not critical. Thus,
the prosthesis can be one which is made from natural tissues, including
but not limited to bovine, ovine, porcine, and human tissue; metals;
synthetic organic materials, such as polyurethanes, polyetherurethanes;
silicones; polyesters; polycarbonates; polyacrylates and methacrylates;
polyacetates; polyolefins, such as polyethylene and polypropylene;
polyalcohols; combinations and derivatives thereof; and the like. Other
materials, well known to those having ordinary skill in the art, also can
be used.
In general, the anticalcification agent is selected from the group
consisting of aliphatic carboxylic acids and alkali metal salts and
derivatives thereof, each of which acids can contain from about 8 to about
30 carbon atoms. Preferably, the anticalcification agent will contain from
about 12 to about 22 or 24 carbon atoms and most preferably from about 15
to about 20 carbon atoms. The preferred alkali metal salts are the
potassium and sodium salts.
The anticalcification agent may be a straight-chain or a branched-chain
compound which is appropriately substituted. Except for the substituent
through which covalent linking is achieved, the nature and number of the
substituents are not critical, provided that they do not significantly
adversely effect the anticalcification properties of the compound and also
that such substituents do not have a significant adverse physiological
effect upon implantation of the prosthesis treated with such agent. In
general, the number and nature of substituents present should be such that
a very rigid molecule is avoided. Examples of substituents which can be
present include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, hydroxy, alkoxy aryloxy, carbonyl, halo, amino,
alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino,
mercapto, alkylthio, arylthio, and the like. As explained in more detail
hereinafter, there should be at least one substituent selected from amino,
hydroxy, mercapto and carboxyl to create the desired covalent linkage.
The acids which comprise the anticalcification agents of the present
invention can be saturated or unsaturated. Such unsaturation can be
monounsaturation or polyunsaturation resulting from the presence in the
compound of one or more ethylenic or acetylenic groups, i.e., one or more
carbon-carbon double bonds, one or more carbon-carbon triple bonds, or a
combination of one or more carbon-carbon double bonds and one or more
carbon-carbon triple bonds. Moreover, such double or triple bonds can be
present anywhere in the molecule. In addition, carbon-nitrogen double
and/or triple bonds also can be present, provided such bonds do not
adversely affect either the anticalcification properties or physiological
compatibility of the compound. Carbon-carbon double bonds are the
preferred unsaturation, with one or two of such bonds being more
preferred. Most preferably, the anticalcification agent will have a single
carbon-carbon double bond in the main carboxylic acid chain, independent
of the presence or absence of unsaturation in any substituents which may
be present.
It may be noted at this point that the present invention contemplates the
use of a single anticalcification agent as well as a mixture of two or
more different anticalcification agents. If a mixture is employed, such
mixture may include a free acid and a salt thereof, or more than one free
acid or salts of different acids, as well as derivatives of any of the
foregoing. Thus, the use of the term "anticalcification agent" throughout
this specification and the appended claims is meant to include a single
anticalcification agent and any mixture of two or more anticalcification
agents, including such mixtures as those set forth above.
As used herein, the term "derivative" is meant to include any compound
having at least one portion or moiety which is substantially an aliphatic
carboxylic acid as defined hereinabove. By way of clarification, such
aliphatic carboxylic acid may be represented by the general formula,
##STR1##
with R representing the aliphatic chain with the carboxyl group preferably
located at one end thereof. For a moiety to consist substantially of such
aliphatic carboxylic acid, it should contain at least the portion,
##STR2##
the aliphatic carboxylic acid. Preferably, such moiety will contain the
portion,
##STR3##
of the aliphatic carboxylic acid. As will become evident later, if a
derivative is employed, it should still be covalently coupled to the
prosthesis through the aliphatic carboxylic acid portion of the molecule,
lest the aliphatic carboxylic acid portion be removed from the prosthesis
as a result of hydrolysis or other reaction of the derivative.
As a practical matter, derivatives of the aliphatic carboxylic acids which
comprise the anticalcification agents of the present invention typically
will be either esters or amides, with esters being preferred. By way of
illustration, examples of suitable classes of esters are given below.
(1) aliphatic, cycloaliphatic, and aromatic esters of the aliphatic
carboxylic acids;
(2) phosphoric acid esters of the aliphatic carboxylic acids; and
(3) mono- and polyesters of one or more aliphatic carboxylic acids, which
may be the same or different, with di- or polyhydric aliphatic or aromatic
alcohols, optionally via a phosphoric acid ester group, examples of such
mono- and polyesters including, by way of illustration only, 1-
monoacylglycerols, 2-monoacylglycerols, 1,2-diacylglycerols,
triacylglycerols, alkyl ether acylglycerols, glycosyldiacyl-glycerols,
phosphoglycerides, plasmalogens, sphingolipids, waxes, and the like.
Examples of aliphatic carboxylic acids coming within the foregoing
definition include, among others, octanoic acid, 2-aminooctanoic acid,
oct-3-enoic acid, 2-methyl-nonanoic acid, 10-hydroxydec-5-ynoic acid,
undecanoic acid, phenyltetradecanoic,
3-chloro-8-(2-hydroxylethoxy)pentadec-4-en-9-ynoic acid, hexadecanoic
acid, hexadec-9-enoic acid, 3,7-dimethylhexadec-9-enoic acid,
octadec-9-enoic acid (oleic acid), 2-aminooctadec-9-enoic acid,
octadec-9,12-dienoic acid, octadec-9,12,15-trienoic acid, eicosanoic acid,
eicos-5,8,11,14-tetraenoic acid, tetracosanoic acid, and the like. These
would be used in suitably substituted form, such as 2-aminooctanoic acid,
10-hydroxydec-5-ynoic acid, 12-amino dodecanoic acid,
2-aminooctadec-9-enoic acid (2-amino oleic acid) and 2,2-dicarboxyl
octadec-9-enoic acid.
As already noted, included within the definition of anticalcification
agents are the alkali metal salts of the aliphatic carboxylic acids. Also
included are esters of such acids with monohydric alcohols, as well as
mono- and polyesters with polyhydric alcohols, examples of which alcohols
include, by way of illustration only, methanol, ethanol,
2-(4-ethoxyphenyl)ethanol, 2-chloropropanol, 1-butanol, 2-butanol,
hex-3-en-1-ol, octanol, eicosanol, phenol, 3-methylphenol, 1,2-ethanediol,
1,4-butanediol, glycerol, L-glycerol 3-phosphoric acid, phosphoric acid,
sphingosine, dihydrosphingosine, cholesterol, lanosterol, cholic acid,
aldosterone, esterone, testosterone, and the like.
As already noted, the method of the present invention involves covalently
coupling an anticalcification agent to a prosthesis. As a consequence of
such covalent coupling, the anticalcification agent will certainly be
associated with the surfaces of the prosthesis. However, such agent also
may migrate into, and become associated with, part or all of the bulk
materials from which the prosthesis is constructed, depending upon a
number of factors such as the porosity of the materials and the
permeability of such materials to the anticalcification agent. For the
purposes of this invention, no distinction is made between the location of
the agent on the surfaces only or with partial or complete penetration
into the bulk materials. That is, the term "covalent coupling" and
variations thereof include the coupling of the anticalcification agent to
the prosthesis surfaces only and also coupling of the anticalcification
agent to part or all of the bulk materials of which the prosthesis is
composed.
Reaction conditions for the covalent coupling of the anticalcification
agent to the prosthesis may vary, depending upon the presence or absence
of biomaterials, the type of functional groups present on the prosthesis,
the nature of the anticalcification agent, and the coupling chemistry to
be employed. In general, the coupling reaction typically will be carried
out in an aqueous solution which may be buffered at a suitable pH. The
nature of the buffer is not believed to be critical. Examples of suitable
buffers include phosphate, tartrate, bicarbonate, borate, citrate,
formate, acetate, N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid
(HEPES), tris(hydroxymethyl) aminomethane (TRIS),
3-(N-morpholino)propanesulfonic acid (MOPS), and the like. For additional
details on buffers, see, e.g., Gerald D. Fasman, Editor, "CRC Handbook of
Biochemistry and Molecular Biology," 3rd Edition, Physical and Chemical
Data Volume I, CRC Press, Inc., Boca Raton, Fla., 1976, pp.354-377. In
addition, the buffer concentration is not critical and can vary widely.
The pH of the buffer also can vary over a wide range, typically from about
6 to about 14. Thus, the pH of the buffer preferably will be in the range
of from about 7 to 11, most preferably from about 9 to about 11, and in
such a buffer the carboxylic acid anticalcification agent will usually be
in the form of its alkali metal salt, e.g., the sodium salt.
The concentration of the anticalcification agent in the aqueous solution
can vary, for example, from about 0.1 percent by weight or less to about
20 percent by weight or more. As a practical matter, less concentrated
solutions are preferred, with concentrations in the range of from about
0.1 percent to about 5 percent perhaps being typical.
The coupling reaction may be carried out at any temperature which does not
adversely affect the prosthesis, e.g., from about 0.degree. C. to about
100.degree. C., unless the prosthesis is composed in whole or in part of
biomaterials, i.e., tissue, in which case the temperature typically will
not exceed about 40.degree. C. As a practical matter, however, such
aqueous solution will be at ambient temperature. Exposure time can vary
over a wide range, e.g., from about 5 minutes to about a week, and it will
be dependent upon the particular coupling reaction being utilized and the
overall conditions. In general, very long exposure times, e.g., more than
a week, do not appear to be necessary. Thus, the exposure time may be in
the range of from about 10 minutes to about 24 hours; however, for tissue
materials, as opposed to synthetic polymeric materials, coupling reactions
may be carried out at room temperature for periods of about five days or
even longer to assure that an adequate amount of the agent is covalently
bonded to the prosthesis.
After a sufficient reaction time (incubation), the prosthesis is rinsed or
washed to remove excess reaction solution. The rinse or wash solution can
be distilled water, deionized water, a buffer, or any other suitable
liquid. If the prosthesis contains biomaterial, it is not allowed to dry;
such biomaterial prosthesis is preferably stored in, for example, 0.2
percent, buffered aqueous glutaraldehyde solution.
By way of illustration only, when 2-aminooleic acid or a salt thereof is to
be covalently coupled to a bioprosthesis, the treating solution typically
will be a buffer, such as borate buffer, having a pH in the range of from
about 8 to about 11. Incubation can vary from about 2 hours or less to
about 24 hours or more, and incubation periods of from about 48 hours to
about 120 hours are preferred for tissue prostheses. While the incubation
temperature can range from about 0.degree. C. to about 40.degree. C.,
temperatures from about 20.degree. C. to about 35.degree. C. appear to be
preferred.
In general, covalent coupling of the anticalcification agent to a
prosthesis may be accomplished by any of a variety of methods known to
those having ordinary skill in the art. Such methods typically involve
such reactive substituent groups as amino, carboxy, hydroxy, and mercapto.
Representative coupling methods involve, by way of illustration only,
carboxylic acid azide groups, carboxylic acid chlorides, carbodiimides and
Woodward's reagent, diazotization, isothiocyanates, cyanuric chloride,
cyanogen bromide, titanium chloride activation, the Ugi reaction, the use
of intermediate coupling agents, such as organofunctional silanes, peptide
bond formation, the formation of a Schiff's base, as well as the formation
of an ester linkage between a carboxyl group substituent and a free
hydroxy group on the prosthesis, to name but a few. See, e.g., Howard H.
Weetall, "Immobilization by Covalent Attachment and by Entrapment,"
Chapter 6 in Ralph A. Messing, Editor, "Immobilized Enzymes for Industrial
Reactors," Academic Press, New York, 1975, pp. 99-123; and William H.
Scouten, Editor, "Solid Phase Biochemistry. Analytical and Synthetic
Aspects," John Wiley & Sons, Inc., New York, 1983. Moreover, further
chemical modification can be accomplished after coupling the
anticalcification agent to the prosthesis. For example, the
carbon-nitrogen double bond of the Schiff's base can be reduced, e.g., by
reaction with sodium borohydride under mild conditions, to increase the
stability of the coupled material, e.g., against hydrolysis. It will be
appreciated by those having ordinary skill in the art, however, that not
all coupling methods necessarily can be used with any given prosthesis.
That is, the coupling chemistry to be employed is in part dependent upon
the nature of the functional groups present on the prosthesis.
The present invention is further described by examples which follow. Such
examples however, are not to be construed as limiting in any way either
the spirit or the scope of the present invention. In the examples, all
percentages are percents by weight and all temperatures are in degrees
Celsius, unless otherwise specified.
EXAMPLE 1
The method of the present invention is illustrated by the covalent coupling
of an amino-substituted oleic acid anticalcification agent to bovine
pericardium tissue.
I. Formation of Oleic Acid Agent
A substituted oleic acid compound is synthesized in a form which is well
suited for covalently coupling to glutaraldehyde-treated bovine
pericardium tissue or other natural tissue by a linkage which takes
advantage of the glutaraldehyde pretreatment of the tissue. To prepare the
compound, 2-aminooleic acid, the procedure of F. Amat Guerri, Grasas
Aceites (Seville), 26. 90 (1975), was generally followed. The series of
reactions described below was repeated three times, with similar results,
in order to accumulate a supply of intermediates and the final compound.
Only one reaction sequence is described, however.
A. Preparation of 2-(Hexadec-7-enyl)propane Dicarboxylic Acid
A 250-ml., three-necked, round-bottomed flask was fitted with a 50ml.
pressure-equalized side arm addition funnel, condenser, and rubber septum.
The addition funnel and condenser also were fitted with rubber septa. The
flask was purged continuously with dry nitrogen (Matheson extra dry grade)
which was introduced via a syringe needle inserted through the rubber
septum fitted on one of the three necks of the flask; the nitrogen exited
via another syringe needle inserted through the condenser-mounted rubber
septum. Using a syringe, the flask was charged with 21.5 ml. (0.155 mole)
of diisopropylamine (99 percent, Aldrich chemical Company, Inc.,
Milwaukee, Wis.) and 50 ml. of dry tetrahydrofuran (THF) (Gold Label, 99.9
percent, Aldrich Chemical Company, Inc.). The resulting solution was
cooled in an ice-salt bath while being stirred with a magnetic stirrer. To
the cold solution (of the order of -20 to -10 degrees) was added slowly 60
ml. of n-butyllithium in hexane (Aldrich Chemical Company, Inc.) which had
been added via a syringe to the addition funnel. The resulting mixture was
stirred for two hours, after which time 20.0 g. (0.071 mole) of oleic acid
(Fisher Scientific, Pittsburgh, Pa.) was added dropwise by means of the
addition funnel (charged by syringe injection), over a one-hour period;
during the addition, the reaction solution turned dark red, indicating the
formation of the dianion. To the reaction mixture then was added dropwise
over an approximately 30-minute period a mixture of 51 g. of
hexamethylphosphoramide (Aldrich Chemical Company, Inc., 99 percent, which
has been dried over calcium hydride and then stored over a molecular
sieve) and 50 ml. of THF, the two liquids having been added previously to
the addition funnel by syringe injection. The resulting mixture was
stirred for one hour and poured into a 600ml. beaker containing 100 g. of
solid carbon dioxide (the red color disappeared immediately upon
contacting the dry ice). The mixture was allowed to stir overnight. The
reaction mixture, now a pale yellow solution at ambient temperature, was
acidified with approximately 40 ml. of concentrated hydrochloric acid, an
amount sufficient to turn blue litmus paper red. The acidified solution
was extracted three times with 100-ml. portions of diethyl ether (Fisher
Scientific). The ether extracts were combined, washed once with distilled
water, and dried over anhydrous magnesium sulfate (reagent grade, J. T.
Baker Chemical Co.). The ether solution was decanted from the drying agent
and evaporated under reduced pressure by means of a rotating evaporator
(Buchi Rotovap, Model RE 120). The residue was a pale yellow semisolid
weighing 20.2 g. (87 percent yield).
B. Preparation of Diethyl 2-(Hexadec-7-enyl) Propane Dicarboxylate
A solution of 5 g. (15.3 mmole) of the product from step A, above, four
drops of concentrated hydrochloric acid as catalyst, and 100 ml. of 95
percent ethanol (Mallinckrodt, Inc., St. Louis, Mo.) was heated at reflux
temperature for two hours in a 250-ml. single-necked round-bottomed flask
fitted with a condenser. The solvent was removed by distillation at
atmospheric pressure. The light yellow residue then was distilled at about
0.1 mm Hg to yield 5.1 g. (87 percent) of a clear, colorless oil, b.p.
140-3 at 0.1 mm Hg, which was shown to be diethyl 2-(hexadec-7-enyl)
propanedicarboxylic acid by infrared analysis (absorption maxima were
observed at 1735, 1710, and 1190 cm.sup.-1).
C. Preparation of Ethyl 2-Hydroxyiminooctadec-9-eneoate
The method of D. J. Drinkwater and P. W. G. Smith, J. Chem. Soc., 1971,
1305, was followed. A 50-ml., three-necked, round-bottomed flask was
fitted with a pressure-equalized addition funnel and charged with 1.0 g.
(2.62 mmole) of diethyl 2-(hexadec-7-enyl) propanedicarboxylate. The
material was cooled to about -10 degrees while being stirred magnetically.
The flask then was charged with 0.27 g. (2.62 mmole) of n-butylnitrite
(Aldrich Chemical Company, Inc.), followed by the addition over a period
of one hour of a sodium ethoxide solution prepared by reacting 0.06 g. of
sodium spheres (Aldrich Chemical Company, Inc.) with 1.8 ml. of absolute
ethanol. The reaction mixture then was stirred overnight at -10 degrees.
Ethanol was removed under reduced pressure and the residue was mixed with
an equal volume of ice water. The resulting solution was washed with
diethyl ether and the pH was adjusted to 5 by the addition of hydrochloric
acid while being cooled in an ice bath. The acidified solution was
extracted twice with 50-ml. portions of diethyl ether. The ether extracts
were combined, dried over anhydrous magnesium sulfate, and decanted from
the drying agent. The ether was removed under reduced pressure to give
0.79 g. (85 percent) of the desired product as shown by infrared analysis
(absorption maxima were observed at 3260 and 1734 cm.sup.-1).
D. Preparation of 2-Hydroxyiminooctadec-9-enoic Acid
A mixture of 0.6 g. (1.77 mmole) of ethyl 2-hydroxy-iminooctadec-9-eneoate
and 20 ml. of 1 N aqueous sodium hydroxide was heated at reflux
temperature for 10 minutes. The reaction mixture was cooled, acidified
with concentrated hydrochloric acid, and extracted twice with 50-ml.
portions of diethyl ether. The extracts were combined, dried over
anhydrous magnesium sulfate, and decanted from the drying agent. The ether
was removed to give a light yellow oil which was recrystallized from
ether-light petroleum (b.p. 40-60 degrees, Fisher Scientific) to yield 0.5
g. (91 percent) of 2-hydroxyiminooctadec-9-eneoic acid. Infrared analysis
of the material showed absorption maxima at 3220, 3080, and 1695
cm.sup.-1.
E. Preparation of 2-Aminooleic Acid
A mixture of 0.7 g. of 2-hydroxyiminooctadec-9-enoic acid, 0.77 g. of zinc
powder (Fluka Chemical Corporation, Ronkonkoma, N.Y.), and 12.3 ml. of a
2:1 glacial acetic acid: water mixture was heated at reflux temperature
for 90 minutes. The reaction mixture was filtered through a fine scintered
glass funnel while still hot, and the filtrate was allowed to cool
overnight in a freezer. A solid had precipitated which was filtered on a
Buchner funnel to yield 0.61 g. (91 percent) of white, powdery
2-aminooleic acid, m.p. 216-7 degrees (dec.). The material was analyzed by
infrared spectroscopy in a potassium bromide pellet; absorption maxima
were seen at 3200-3500 and 1650 cm.sup.-1.
II. Covalent Coupling of 2-Aminooleic Acid to Bovine Pericardium Cusps
2-Aminooleic acid, as the sodium salt, was coupled to two groups of ten
glutaraldehyde-pretreated bovine pericardium cusps (the cusps were
obtained from a local slaughterhouse and stored in 0.2 percent aqueous
glutaraldehyde solution) by simply incubating the cusps overnight at
ambient temperature in a 1.0 percent solution of the anticalcification
agent in 0.05 M sodium borate buffer at pH 11. Coupling occurred through
residual aldehyde groups via Schiff base formation. After incubation, the
cusps were rinsed ten times with 10-ml. portions of physiological saline
solution (0.9 percent aqueous sodium chloride).
Each group of foregoing treated cusps was implanted subcutaneously in the
ventral mid-abdominal area of a group of five three-week old
Sprague-Dawley rats (Charles River Breeding Laboratories, Inc.,
Wilmington, Mass.), two cusps per rat (one on each side). The implantation
procedure was essentially the same as that described in U.S. Pat, No.
4,402,697. One group of ten untreated cusps was implanted as a first
control. As a second control, a group of ten cusps was incubated in 0.05
M, pH 11 sodium borate buffer which did not contain an anticalcification
agent.
Two separate studies were conducted. After 21 days, the implanted cusps
were removed from the rats and analyzed for calcium by atomic absorption
spectrometry as described by Frederick J. Schoen et al., Am. J. Pathol.
123, 134-145 (1986), supra. The animals were sacrificed and autopsied; no
abnormalities were observed. A group of ten untreated cusps which had not
been implanted also was analyzed for calcium. The results are summarized
in Table 1.
TABLE 1
______________________________________
Effect of Covalent Coupling of 2-Aminooleic Acid
on the Calcification of Bovine Pericardium Cusps
Implanted Subcutaneously in Rats
Group Tissue Ca Concentration
______________________________________
Untreated, unimplanted
2.3 .+-. 0.5
Implanted, untreated
142.6 .+-. 6.8
Implanted, untreated.sup.a
141.7 .+-. 12.8
Implanted, treated
1.5 .+-. 0.2
Implanted, untreated.sup.a
144.0 .+-. 7.9
Implanted, treated
0.8 .+-. 0.4
______________________________________
.sup.a Incubated overnight at ambient temperature in 0.05 M, pH 11 HEPES
buffer.
The effectiveness of the covalently coupled 2-aminooleic acid in inhibiting
mineralization is readily apparent from Table 1. Calcium concentrations in
the treated tissues were at levels equivalent, or perhaps even lower, than
the level of calcium normally present in the tissue.
The significance of the method of the present invention is made more
evident upon studying the effect of a generally similar, but
non-covalently linked, anticalcification agent on the calcification
process. Such a study is detailed in Examples 2 and 3.
EXAMPLE 2
Several process variables were studied with regard to pretreatment of
tissue prostheses before carrying out implantation experiments with
respect to a non-covalently bound anticalcification agent.
Incubation Time
Glutaraldehyde-treated bovine pericardium cusps were incubated at ambient
temperature in 1 percent by weight aqueous solution of sodium oleate which
contained .sup.14 C-labeled sodium oleate for one and seven days,
respectively. The solution pH was 10.6. The cusps were washed ten times
with 10-ml. portions of 0.05 M HEPES buffer at pH 7.4. The cusps then were
analyzed for sodium oleate uptake. The results are summarized in Table 2.
TABLE 2
______________________________________
Sodium Oleate Concentration in Tissue
after One and Seven Days
Incubation Period
Tissue NaOleate Concn.
______________________________________
One day 119.3 nmoles/mg. tissue
Seven days 124.2 nmoles/mg. tissue
______________________________________
The data in Table 2 show essentially no difference in tissue uptake of
sodium oleate, indicating that incubation times of longer than one day,
with non-covalently bound sodium oleate, do not appear to substantially
increase uptake.
Effect of Wash pH
The above described procedure was repeated, using a concentration of sodium
oleate of 10 percent instead of 1 percent and an incubation period of one
day. Three groups of cusps were employed. One group of cusps were not
washed; they were blotted to remove excess fluid. The second group was
washed with distilled water, and the third group was washed with pH 7.4
HEPES buffer as described above. Table 3 summarizes the results.
TABLE 3
______________________________________
Sodium Oleate Concentration in Tissue
under Varying Wash Conditions
Wash Medium Tissue Na Oleate Concn.
______________________________________
None (blotted)
845.2 nmoles/mg. tissue
Distilled water
263.9 nmoles/mg. tissue
Buffer, pH 7.4
631.9 nmoles/mg. tissue
______________________________________
It may be noted that the buffer wash resulted in a lower amount of sodium
oleate being removed from the tissue as a consequence of the washing step.
In addition, it appears that the use of a 10 percent sodium oleate
significantly increases tissue pickup of sodium oleate, compared with a 1
percent sodium oleate solution.
In order to evaluate the effect of an extended wash cycle, the above
experiment was repeated. At the end of the incubation period, cusps were
immersed in 10 cc of either distilled water or pH 7.4 HEPES buffer. The
cusps were agitated on a shaking platform at 37 degrees for 16 days. The
cusps then were analyzed for tissue sodium oleate. The results are
summarized in Table 4.
TABLE 4
______________________________________
Sodium Oleate Concentration in Tissue
under Varying Wash Conditions
Wash Condition
Tissue NaOleate Concn.
______________________________________
Distilled water
15.5 nmoles/mg. tissue
Buffer, pH 7.4
218.2 nmoles/mg. tissue
______________________________________
The data in Table 4 again demonstrate the advantages to a pH 7.4 wash
compared with a distilled water wash.
EXAMPLE 3
Two groups, each containing ten glutaraldehyde-treated bovine pericardium
cusps per group, were incubated for two hours at ambient temperature in
solutions containing 1 percent sodium oleate (Fisher Scientific). The
first solution was obtained by dissolving the sodium oleate in 0.05 M, pH
7.4 HEPES buffer. The second solution was obtained by dissolving the
sodium oleate in distilled water; the solution pH was 10.6.
Each group of cusps was implanted subcutaneously in rats as described in
Example 1. Two control groups of the same size also were established by
cusps which had not been pretreated with sodium oleate.
The first control group of rats received daily sterile saline injections
subcutaneously, one injection of 500 microliters per animal per day. The
saline was 0.9 percent aqueous sodium chloride solution having a pH of
7.4. The second control group received equivalent daily injections of a
sodium oleate solution at a level of 100 mg. sodium oleate per kg. of body
weight per 24-hour period.
After 14 days, the cusps were retrieved from all four groups. The animals
were sacrificed and autopsied, with the sodium oleate-injected animals
showing lower blood calcium levels. The cusps were analyzed for calcium by
atomic absorption spectrometry as described in Example 1. The results are
summarized in Table 5.
TABLE 5
______________________________________
Effect of Sodium Oleate Treatment
on the Calcification of Bovine Pericardium Cusps
Implanted Subcutaneously in Rats
Group Tissue Ca Concentration.sup.a
______________________________________
1st Control (saline injection)
70.4 .+-. 12.8
2nd Control (Na oleate injection)
62.3 .+-. 8.3
Na Oleate pretreatment, pH 7.4
51.4 .+-. 6.2
Na Oleate pretreatment, pH 10.6
34.7 .+-. 7.9
______________________________________
.sup.a Reported as the mean of micrograms calcium per mg. of cusp tissue
.+-. standard error.
The data in the above table show that pretreatment of a prosthesis with
sodium oleate reduces calcification. While the reduction in calcification
was of the order of 30 to 50 percent, based on the first control, such
reduction is not believed to be sufficient for the long-term retardation
or prevention of calcification. It is likely that the sodium oleate is
associated with the prosthesis tissue only on the basis of hydrogen
bonding and Van der Waals forces. Such association may be readily
disrupted with the probability that the sodium oleate is removed from the
locus of the implant, thus rendering it less effective than a covalently
coupled anticalcification agent.
EXAMPLE 4
Treatment of Glutaraldehyde-Preserved Porcine Tissue With 2-Amino Oleic
Acid Sodium Salt (2-AOASS) and With 12-Amino Dodecanoic Acid Sodium Salt
(12-ADASS)
The cusps or leaflets from glutaraldehyde-preserved porcine valves were
dissected, rinsed for 5 min. with sterile physiological saline (0.9% NaCl,
10 cusps in 50 ml.) and placed in 10 mM sodium borate, pH 11.0. This
allows the excess glutaraldehyde to be washed out of the tissue and also
to adjust the pH of the leaflets.
The cusps were placed in 1% (w/v) solution (1 cusp per b 2 ml.) of either
2-AOASS or 12-ADASS and incubated without shaking for the varying times
and at the varying temperatures indicated in Experiments A-D hereinafter.
The control leaflets were incubated under similar conditions but in the
absence of active anticalcification agents. After incubation, the cusps
were rinsed twice with 50 ml. of 10 mM sodium borate buffer, pH 11.0, to
remove unbound material and twice with 50 ml. of distilled water. The
cusps were then stored in 0.2% buffered glutaraldehyde solution. It is
possible that some of the anticalcification agent that permeates into the
tissue during incubation but associates with it without being covalently
bonded may become cross-linked therewithin during storage in the 0.2%
glutaraldehyde solution and add to anticalcification protection.
Experiments to date have not proved that cross-linking in fact occurs.
Immediately prior to implantation, the cusps were rinsed with sterile
physiological saline to remove the excess glutaraldehyde.
One control and one or two treated cusps were implanted subcutaneously in
three-week old male rats (30 to 50 g.) as set forth with regard to Example
1.
After 21, 60, 90 or 120 days the implants were retrieved, cleaned of
adherent tissue and rinsed 5 times with sterile physiological solution and
5 times with distilled water. The explanted tissue was lyophilized,
weighted and hydrolyzed in an oxygen-free atmosphere in 6 N HCl for 24 hr.
at 110.degree. C. The hydrolysates were dried and resolubilized in dilute
HCl, and the calcium level was determined by the Inductively Coupled
Plasma Analysis method.
Experiment A
The treated samples were glutaraldehyde-preserved cusps incubated at room
temperature (about 22.degree. C.) for 24 hours with either 2-AOASS or
12-ADASS at a concentration of 1% (w/v). The control samples were
glutaraldehyde-preserved cusps incubated in the absence of active agents.
Both treated and control cusps were implanted for 21, 60, 90 and 120 days.
The results are presented in Table A below:
TABLE A
______________________________________
Ca++ .mu.g./mg. dry tissue
Implant time
Mean .+-. SEM
(days) n Control n Treated
______________________________________
2-AOASS
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