WO2001002031A2

WO2001002031A2 - Targeted anticalcification treatment

Info

Publication number: WO2001002031A2
Application number: PCT/US2000/017606
Authority: WO
Inventors: Dan Simionescu; Agneta Simionescu; Jean-Marie Girardot; Marie-Nadia Girardot
Original assignee: Biomedical Design, Inc.
Priority date: 1999-07-01
Filing date: 2000-06-27
Publication date: 2001-01-11
Also published as: AU5639700A; WO2001002031A3

Abstract

Methods for providing biomaterials with increased resistance to calcification by treating with cell-targeted agents and/or with matrix-targeted agents. Cell-targeted agents are used which block calcium channels or which prevent oxidative damage to cells and/or inhibit enzymes. Other treatments of biomaterials may remove cell-derived calcium-binding components or target extracellular matrices, as by cleaving proteins with cyanogen bromide or reducing disulfide bonds to produce sulfhydryl groups which are thereafter alkylated. Combinations of the foregoing different treatments are also effective in increasing the calcification resistance of cardiovascular tissues that were previously subjected to chemical fixing and/or other anticalcification treatments.

Description

TARGETED ANTICALCIFICATION TREATMENT

The present invention relates generally to treating biomaterials destined for implantation in a human patient so as to render such materials resistant to calcification, and more particularly relates to methods for targeting anticalcification treatment to particular biological tissue that has been previously fixed, i.e. chemically cross-linked, so as to render it more resistant to calcification following its implantation in the human patient. Still more particularly, the invention relates to treatments of this type that are targeted to specific biomaterials of a certain character that have heretofore been difficult to effectively render resistant to calcification.

Background of the Invention Degenerative calcification of glutaraldehyde-fixed biological tissues used for bioprosthetic heart valve (BHV) fabrication is presently considered to be a major cause of long-term failure of these implants in a clinical setting. Mitigation of calcification has been investigated (a) by subsequently treating glutaraldehyde- treated tissues with a variety of compounds and (b) by employing fixation procedures which do not employ glutaraldehyde. The results obtained thus far indicate that the type of tissue and its precise composition may be important in determining its susceptibility to calcification. Collagen concentration, for example, varies from about 90% (w/w) in pericardium, to about 40% in aortic cusps, and to only about 25% in aortic wall tissue. On the other hand, elastin accounts for only 1- 5% in pericardium and about 10-15% in cusps whereas it may account for up to about 50% of aortic wall tissue. Furthermore, cell types and numbers differ significantly between these three tissues.

Anticalcification treatment of glutaraldehyde fixed tissues such as that disclosed in U.S. Patent No. 4,976,733 and the treatment of tissues not cross-linked by glutaraldehyde in Nos. 5,447,536 and 5,733,339 were shown to be quite effective in reducing calcification of collagenous tissues, such as pericardium and porcine aortic leaflets; however, elastin-containing aortic wall tissue has proven to be less susceptible to reduction of calcification by the above-mentioned treatments. As a result, the investigation has continued for other anticalcification treatments that would specifically target tissue relatively low in collagen, for example tissue having relatively greater amounts of elastin.

Although, the molecular mechanisms of BHV calcification are not well understood, additional elements, e.g. the presence of injured or devitalized cells, are now considered to be important along with the character of cross-linked extracellular matrix. For example, cell injury induced by fixation protocols can lead to impairment of normal calcium homeostasis, followed by a massive calcium influx, and such can, in turn, lead to cell death and calcification of cell remnants. Cross-linked extracellular matrix, on the other hand, can induce calcium deposition per se or, as a consequence of cell-mediated propagation, can induce calcification into the surrounding matrix. Therefore, the molecular substrates that can promote deposition of calcium salts in BHV and other implanted organs may generally be divided into two categories: (a.) cell- derived elements, such as lipid membranes which may contain calcium-transporting channels and calcium ATPases, integrins, cadherins, selectins and annexins, as well as cytoskeletal protein structures present in the close vicinity of devitalized cells, cell enzymes, calmodulin, mitochondria, the cell nucleus and other calcium-binding components, and (b.) extracellular matrix calcium-binding components, such as elastin-associated microfibrillar proteins (EAMF) , collagens, proteoglycans, proteolytic enzymes, such as etalloproteinases (MMPs) , matrix phosphatases, and other non-collagenous proteins. A common element that appears to characterize these calcifiable substrates is the ubiquitous presence of one or more high affinity calcium-binding sites or calcium- trapping pockets which, by means of carboxyl and/or hydroxyl groups, attract and immobilize calcium ions. The three-dimensional conformation of these sites is stabilized in the "correct" shape by intramolecular bridges, such as disulfide bonds, and hydrophobic interactions. [See Guidebook to the Calcium-binding Proteins , Celio et al., eds., Oxford University Press, Oxford, UK, p. 15- 21, 1996]

Summary of the Invention Anticalcification methods have now been devised that take into consideration the character of these molecular substrates that account for calcium deposition in tissues. These new methods accordingly target or challenge such substrates with specific compounds in such a way as to reduce or inhibit overall tissue calcification, without compromising any previously obtained cross-linking or calcification resistance that may have been obtained as a result of treatment with other reagents.

These methods of treatment generally fall into two main classes: cell-targeted treatments and matrix- targeted treatments. The cell-targeted class of treatment includes three general categories; however, in some instances treatments from different classes or from different categories within one class may be used in combination and, as such, may produce an effect that is greater than the effect produced by either class or category of treatment alone. Generally, it has been found that progression from reversible, physiological calcium-binding towards the irreversible deposition of calcium salts into or onto these substrates can be effectively stopped by treatments that destabilize, modify and/or destroy the original conformation of high affinity calcium-binding sites and/or by treatments that permanently block the access and influx of counterions into otherwise unaltered sites.

In the class of treatment that targets cells, injured cells are protected from degeneration and calcium overload by treating tissue containing such cells to reduce calcium influx into the cells or prevent oxidative and/or enzymatic damage. One may use a calcium channel blocking agent, such as nifedipine (NIF) or diltiazem hydrochloride (DIL) , an antioxidant, or an agent, such as captopril (CAP) , that inhibits damaging enzymes.

Potential cell-related calcification substrates, such as the cytoskeletal proteins actin, yosin, troponin and actinin, can also be removed, extracted or inactivated by using an appropriate extractant, such as a high potassium salt/MgATP mixture (KMA) .

In the other class of treatment which targets the extracellular matrix, the structure of calcium-binding components, such as EAMF and MMPs and the like, in extracellular matrices can be appropriately modified to reduce the capacity thereof either to calcify per se or to induce calcification in adjacent components. For example, treatment with a suitable cleaving agent, such as cyanogen bromide (CB) , will effect partial cleavage of proteins at methionine residues, whereas protein disulfide bonds can be effectively broken by treatment with a suitable reducing agent, such as dithiothreitol (DTT) , and then alkylated with a suitable reactant, such as N-ethylmaleimide (NEM) .

Detailed Description of the Preferred Embodiments As mentioned above, it was known that certain tissues, such as the wall of aortic root tissue, have not heretofore been rendered as successfully resistant to calcification as a result of present anticalcification treatments or non-glutaraldehyde cross-linking, as have, for example, tissues containing significantly higher amounts of collagen. However, as indicated, it has now been found that these and other such tissues can be treated by specific cell-targeted and matrix-targeted anticalcification treatments that are indeed effective. It has been found that the irreversible deposition of calcium salts into or onto these substrates can be effectively stopped by the use of different treatments within these two classes, which may be used individually or in combination with one another. One category of treatment permanently blocks the access and influx of counterions into such sites in cells. A second category of treatment provides antioxidants which prevent oxidative damage and inhibit the action of enzymes that promote calcification of cells. A third category extracts cell-related potential calcification sites. Another category of treatment destabilizes, modifies and/or destroys the original conformation of such high affinity calcium-binding sites and is principally effective against calcification in matrices.

Tissues and biomaterials generally that can benefit from this technology may be characterized as being constituents of either biologic or synthetic origin which, once implanted in a human, are expected to directly or indirectly suffer the effects of calcification while implanted in a patient. Because these biomaterials are expected to be susceptible to concomitant calcium overload, oxidative damage and/or enzymatic hydrolysis, the incorporation of protective agents into these biomaterials provides them with reduced susceptibility towards calcification as well as increased biostability and durability.

Biological tissues, as a result of modified extracellular matrix components or injured cells or both, will frequently present calcifiable substrates to which attention needs to be given; they may be cardiovascular tissues or non-cardiovascular tissues. Examples of the former group include cardiac valves with or without associated stents, i.e. aortic, mitral, pulmonary and tricuspid valves, pericardium and blood vessels, such as (a) arterial segments of large, medium or small caliber, e.g. aortic, carotid or coronary, and (b) venous segments of large, medium or small caliber with or without accompanying venous valves, e.g. saphenous, jugular or cavae. Examples of the non-cardiovascular tissue group include tendons, ligaments, articulations, aponeuroses, cartilages, organ capsules and sheaths, membranes, such as fasciae and dura matter, conduits, such as esophagus, trachea, hepatic ducts and ureter, and cavitary organs from the digestive and urinary tracts. Such implanted tissue may be heterologous, homologous or autologous, i.e. of animal or human origin. Overall, the biomaterials which may be treated prior to implantation may be whole tissues, organs or products thereof which are composed of extracellular matrix components, both with and without cells, and they may be in solid, liquid or gel form, e.g. in the form of sheets, sponges or fibers. They may also be products of tissue engineering or of guided tissue regeneration, wherein scaffolds and scaffolds with cells are used.

The biomaterials which are to be treated by the invention may have been previously chemically processed for removal of selected components, such as antigenic determinants, cell remnants, lipids, sugars and the like. As earlier indicated, such tissues may also have been chemically fixed or cross-linked using glutaraldehyde or other procedures. These tissues may also be further processed by pre- or post-fixation treatments with various anticalcification compounds, e.g. 2-aminooleic acid, phosphonates, detergents, ions and dyes; moreover, these biomaterials may be freeze-dried or dehydrated tissues. They may also be tissues or organs that were preserved by deep-freezing in the presence of cryoprotectants , as well as tissues or organs preserved in antibiotic-containing cold solutions.

In accordance with the first treatment category mentioned above, lipid membranes from injured cells which may contain naturally occurring calcium channels and calcium ATPases, integrins, cadherins, selectins and annexins may be protected from calcium overload and/or degeneration by treating such tissue with a calcium channel blocking agent and/or an antihypertensive agent that is capable of preventing oxidative damage and inhibiting the action of enzymes which have been reported to cause calcification. Examples of one group of suitable calcium-channel blocking agents include nifedipine (NIF), i.e., 1, 4-Dihydro-2 , 6-dimethyl-4-(2- nitrophenyl) -3 , 5-pyridinedicarboxylic acid dimethyl ester, nimodipine, nisoldipine, nitredipine, nicardipine, nilvadipine, amlodipine, lacidipine, verapamil, diltiazem hydrochloride (DIL), i.e., 1, 5-Benzothiazepin-4 ( 5H) one, 3- (acetyloxy) -5- [ 2- (dimethylamino) ethyl ] -2 , 3-dihydro-2- (4-methoxyphenyl) -monohydrochloride, trifluoperazine, bepridil, cinnarizine, fendiline, flunarizine, lidoflazine, phenylamine, pryanodine, ruthenium red and veratridine.

Useful in the second category of treatment are agents capable of preventing oxidative damage, which may also have antihypertensive properties; such agents include captopril (CAP), i.e., 1- (3-Mercapto-2-methyl-l- oxopropyl) -L-proline, quinalapril, enalapril, lisinopril and zofenopril. Other examples include allopurinol, nicotinamide, ebselen, resveratrol, xanthine, diphenyl phenylene diamine, chlorpromazine, manitol, catalase, peroxidase, desferroxamine, polyphenols, N-acetyl cysteine, ubiquinol, butylated hydroxytoluene, probucol, alpha-tocopherol, trolox, superoxid dismutase, thiourea, taurine, propyl galate, histidine, vitamin C, beta- carotene, beta-mercaptoethanol, reduced glutathion, reduced glutathion monoisopropyl esther, reducible dyes (phenazine methosulfate, nitroblue tetrazolium chloride, tiazolyl blue, methylene blue, toluidine blue) , N-tert butyl phenyl nitrone, antioxidant peptides (anserine, carnosine, carcinine) , Val-Phe-aldehyde, PMSF, leupeptin benzamidine, soybean trypsin inhibitor. Some compounds, such as CAP, are both antioxidants and inhibitors of metal-containing enzymes. Combinations of agents from the foregoing two groups may advantageously be employed.

Either as an alternative to, or in addition to, the above treatments, it may be desirable and feasible as a third treatment category, to remove potential cell- related calcification substrates, e.g. cytoskeletal proteins such as actin, yosin, troponin and actinin, as well as cell enzymes, calmodulin, mitochondria, cell nuclei and other calcium-binding or calcium-trapping components. Such tissue is treated using a compound which functions as a suitable cell extractant, e.g. a high concentration potassium salt/MgATP mixture (KMA) , to remove, extract and/or inactivate such substrates that are prone to calcium salt accumulation or inducing same, and such treatment of walls with KMA showed 52-56% reduction in wall calcification following 8 weeks implantation in the rat subder al model of calcification. Other suitable cell extractants may also be used. Such extraction treatments may be advantageously used in combination with treatment by proteolytic enzyme inhibitors, such as PMSF, leupeptin, benzamidine and soybean trypsin inhibitor.

With respect primarily to sites in extracellular matrices where there also are calcium-binding components, e.g. fibrillin, other EAMF proteins, collagens, proteoglycans, proteolytic enzymes, such as MMPs, phosphatases, and non-collagenous proteins, such as laminin, fibronectin, thrombospondin, tenascin, osteonectin, osteopontin and matrix Gla-protein, the other main class of matrix-targeted treatment is used. It has been found that such protein components can be effectively modified in such way as to reduce their capacity to calcify per se or to induce calcification in adjacent or related components. One preferred treatment is to use an appropriate cleaving agent, such as cyanogen bromide (CB) , to cleave such proteins at methionine (Met) residues; alternative cleaving agents are well known in the art and include hydroxylamine, N-bromosuccinimide, N-chlorosuccinimide, thiocyanobenzoic acid, ortho-iodosobenzoic acid and trifuoroperazine. By use of such a modification regimen, the configuration of methionine-containing proteins is changed, and their ability to thereafter bind calcium salts or to induce or promote such binding is very significantly reduced. It has also been found that effective modification to the same end can be effected by breaking intramolecular bridges within proteins, e.g., by altering hydrophobic interactions and/or breaking disulfide bonds. Such modifications can be effected by reduction with dithiothreitol (DTT) or similar well known reducing agents, such as ammonium sulfite, dithioerythritol , sodium sulfite, tri-n-butylphosphine and beta- mercaptoethanol, and then preventing the reversal of such reduction by reacting with a reagent that will bond with a sulfhydryl group. Examples of suitable blocking reagents include alkylating reactants, such as N- ethylmaleimide (NEM) , dithiobis-(2-nitrobenzoic acid), iodoacetamide, iodoacetate, p-hydroxymercuri-benzoate and the methanethiosulfonates. Because there may be more proteins with Met residues than there are proteins with disulfide bonds in such extracellular matrices, treatment with CB or an equivalent cleaving agent may be preferred. However, both these modifications have advantageous effects in reducing calcification resulting from the presence of EAMF proteins, MMPs and the like, and the combination of these two treatments may produce the most desirable effect. Treatments are carried out using the cell-targeted agents at appropriate concentrations, temperatures, pH and durations as generally known in this art for use of such reagents. CAP or similar agents may be used at a concentration of from 1-200 πiM (preferably 25-75 mM) , at about 15-40°C, and at about pH 6-8 for about 2-72 hours. NIF, DIL and related calcium channel blocking agents would be used at concentrations of about 0.1-50 mM (preferably about 5-25 M) under otherwise similar conditions. Cell extractants are used at a similar pH and for a similar duration at temperatures in the range of about 0-20°C. KMA may be used at a KCl concentration between about 0.4 M and about 1.5 M and usually between 0.5-0.8 M and MgATP at concentrations between 0.01-10 mM and usually between about 0.05-8 mM. Although any sequence of treatments with agents from the three categories of cell-targeted agents may generally be used, when such a combination of treatments are employed, the following sequences are most often used: Category 1 followed by Category 2; Category 2 followed by Category 1; Category 3 followed by Category 2; Category 3 followed by Category 1; and Category 3 followed by Category 1 and Category 2. A cleaving agent, such as CB, is used at a concentration of about 1-200 M (preferably about 10-50 mM) , at a similar pH and temperature as for CAP, but for a shorter duration of about 1-24 hours, e.g., about 3 hours. A reducing agent, such as DTT, might be employed at a concentration of about 1-200 mM (preferably about 25-75 mM) and at other conditions as for CAP, and treatment with such an agent is preferably followed by treatment with a blocking agent, such as NEM, at a similar concentration and about the same temperature and pH as for CAP, but for a duration of about 12-48 hours, e.g., 24 hours. When combinations of the two classes of treatment are employed, it is preferred that the matrix- targeted class of treatment precede the cell-targeted treatment. For example, treatment with CB, or treatment with DTT preferably followed by reaction with an alkylating agent, would usually be carried out prior to treatment with a Category 2 agent, with optional treatment thereafter with a Category 1 agent.

The aforementioned treatments are usually carried out in a buffered aqueous solution, e.g. using a borate buffer or HEPES, PIPES, MOPSO or the like. Washing is carried out following the anticalcification treatment and prior to sterilization. Normal saline or a buffered aqueous solution, as described above, may be used at about 0-40°C for 15 minutes to 4 hours, with the optional inclusion of up to about 25% isopropanol or another lower alkanol. When a combination of treatment steps is used, washing or rinsing between steps is desirable but not always necessary so long as there is washing prior to sterilization.

The anticalcification treatment may be applied to tissue that is not cross-linked, such as cryopreserved homografts, or to tissue that is cross-linked. When the treatment is applied to cross-linked tissues, it is generally performed after fixation treatment of the biological tissue, although it may be performed previous thereto, or even both before and after. However, when a cell extractant or a reducing agent is used, treatment is preferably carried out prior to fixation. When the present treatment is carried out in combination with another type of anticalcification treatment, such as treating with 2-aminooleic acid as described in the '733 patent, the present treatment is preferably carried out subsequent thereto, except for treatment with a cell extractant or a reducing agent, which is preferably carried out prior to such other type of anticalcification treatment. It is also important that such targeted methods of anticalcification treatment, when carried out following fixing, do not adversely affect the desirable cross- linking of tissue that has been previously effected, and tests to date show this be true. These treatments also do not appear to have any adverse effect upon the anticalcification properties of the overall tissue, which properties may have been the result of a previously administered anticalcification treatment or non- glutaraldehyde cross-linking, such as described in the aforementioned three U.S. patents that have been proven to provide excellent calcification resistance for porcine valves leaflets. The foregoing is felt to be important because calcification may concomitantly occur, both in BHV and in other biomaterials, in a variety of different substrates, e.g. in cells and in extracellular matrix. As a result, sequential treatment using several targeted treatment methods that are compatible with one another may very well be important in achieving the overall desired effect. For example, it may be desirable to treat cell- containing BHV tissue to block calcium channels and/or reduce oxidizing and/or enzymatic damage, and/or extract or inactivate certain proteins, such as actin and myosin, in combination with modifying proteins in the extracellular matrix that may have a propensity to bind calcium per se or to induce calcification (as by partially cleaving such proteins and/or reducing cyclizing S-S bonds followed by alkylating) ; as a result of such treatment, overall calcification of BHV tissue is found to be very effectively reduced. Moreover, testing has shown that treatment with two agents in categories one and two, i.e. a combination of a calcium-channel- blocking agent and an antioxidant and/or enzyme inhibitor, may be more effective than either treatment alone. Furthermore, certain of the treatments described with respect to one class may also have some beneficial effects upon targets from the other class. For example, treatment with CAP, in addition to protecting targeted cells, also inhibits the action of MMPs and phosphatases; similarly, treatment with CB or by DTT/NEM may also have an anticalcification effect upon certain cell-derived substrates.

The following examples illustrate the effectiveness of treatments carried out using some of the preferred embodiments of the invention. EXAMPLE 1 - Cell Targeted Treatment Multiple samples of porcine aortic roots were fixed and treated as follows: (a) Glutaraldehyde - Samples were treated with 0.2% glutaraldehyde in phosphate-buffered saline, pH 7.4, for 12 days at room temperature. Tissues were then sterilized for 24 hours at 37°C using 1% glutaraldehyde, 20% isopropanol in phosphate- buffered saline, pH 7.4, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in phosphate-buffered saline, pH 7.4, and then stored in the same solution; (b) Glutaraldehyde plus 2-aminooleic acid - additional samples were treated with 0.2% glutaraldehyde in phosphate-buffered saline, pH 7.4, for 12 days at room temperature followed by incubation in an aqueous buffered solution of 2-aminooleic acid according to the '733 patent, and then rinsed. Tissues were then sterilized for 24 hours at 37°C in 1% glutaraldehyde, 20% isopropanol in borate- buffered saline, pH 7.4, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in borate- buffered saline, pH 7.4, and stored in the same solution; (c) Glutaraldehyde plus 2-aminooleic acid plus CAP - additional samples were treated with 0.2% glutaraldehyde in phosphate-buffered saline, pH 7.4, for 12 days at room temperature, followed by treatment with aminooleic acid (as above) , followed by incubation in 50 mM CAP in borate-buffered saline, pH 7.4 , in 10% isopropanol for 24 hours at 37°C and then rinsed. Tissues were then sterilized for 24 hours at room temperature in 1% glutaraldehyde, 20% isopropanol in borate-buffered saline, pH 7.4, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in borate-buffered saline, pH 7.4, and stored in the same solution; (d) Glutaraldehyde/AOA plus NIF - additional tissues were treated as in Example 1 group (b) (in which tissues were glutaraldehyde-fixed, then treated with 2-aminooleic acid) . Tissues were then rinsed and incubated in 5 mM NIF in borate buffer saline, pH 7.4, containing 20% isopropanol for 24 hours at 37°C. After rinsing in borate buffer saline, pH 7.4, containing 20% isopropanol, tissues were sterilized in 1% glutaraldehyde 20% isopropanol in borate buffer saline, pH 7.4 for 24 hours at 37°C, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in borate buffer saline, pH 7.4, and then stored in the same solution.

(e) EDC(sulfo-NHS) -type fixation - Additional tissues first fixed according to the teaching of the '339 patent using EDC plus sulfo-NHS and hexanediamine and/or suberic acid in HEPES buffer, and then sterilized according to U.S. patent number 5,911,951, e.g., by incubation for 24 hours at 40°C in 25 mM EDC, 20% isopropanol in 10 mM HEPES, pH 6.5, and stored in the same solution;

(f) EDC (sulfo-NHS) -type fixation plus CAP - Additional tissue was fixed with EDC (sulfo-NHS) as indicated above and then further incubated in 50 mM CAP in 10 mM HEPES, pH 6.5, in 10% isopropanol for 24 hours at 37°C and then rinsed. Tissues were then sterilized according to the '951 patent and stored in the same solution; and

(g) EDC/sulfo-NHS-fixed plus NIF - additional tissues were fixed as in Example 1 group (e) (in which tissues were fixed using the EDC/sulfo-NHS process) . Tissues were then rinsed and incubated in 5 mM NIF in HEPES buffered saline, pH 6.5, containing 20% isopropanol, for 24 hours at 37°C. After rinsing in HEPES buffer saline, pH 6.5, containing 20% isopropanol, tissues were sterilized according to the "951 patent and stored in the same solution.

Following undergoing the foregoing treatments, the sterilized roots were washed in normal saline, and cusps were dissected away from the aortic walls. For calcification studies, 20 wall coupons and 20 cusp halves were randomly selected from each experimental condition; they were implanted subdermally in three-week old, male /02031

Wistar rats. Samples were explanted at 4 and 8 weeks, and calcium was quantitated by Atomic Absorbtion Spectrophotometry (AAS) . Selected samples from each experimental condition were processed for histology and stained with hematoxylin and eosin (H&E) for cells, and with von Kossa reagent for calcium deposits. The results are set forth in the table that follows.

RESULTS

Table 1

Mean ± SEM (milligrams calcium/errant dry tissue)

Walls Cusps

Treatment 4-week 8-week 4-week 8-week

Group a 64.0 ± 5.0 114.0 ± 16 53.0 1 4.0 155 1 30 Group b 50.1 ± 6.0 99.6 ± 5.0 5.2 1 1.0 5.6 1 1.0 Group c 5.2 ± 2.0 21.5 ± 3.0 3.7 1 1.0 3.5 1 1.0 Group d 7.8 1 4.0 18.7 1 7.0 2.7 1 1.3 2.3 1 0.6 Group e 63.2 1 6.0 100.0 1 7.0 2.8 1 0.9 2.9 ± 1.0 Group f 2.6 1 1.1 8.0 1 3.0 1.7 1 0.3 1.9 ± 0.6 Group g 0.3 1 0.1 8.9 1 3.0 0.5 1 0.3 0.7 1 0.2

The results indicate that post-fixation treatment of wall tissue with CAP, i.e., groups (c) and (f) , significantly reduces the calcification compared to wall tissue of groups (b) and (e) . In (b) , wall tissue samples had been fixed and then treated with 2-aminooleic acid, and in group (e) , wall tissue samples had been fixed according to the '339 patent. Moreover, the treatment does not compromise the earlier established calcification resistance of the cusps created by such prior treatment, but instead it may actually slightly improve the resistance of the cusps. Similar results were obtained in the case of post-fixation treatments with 5 mM NIF, i.e. groups (d) and (g) , and are also obtained when 5 mM DIL is used. In all samples screened from each experimental condition, histology revealed the absence of inflammatory reactions (H&E) , while von Kossa reagent staining essentially confirmed the calcium analysis results. Alternatively, a pre-fixation treatment to accomplish actin and myosin extraction using KMA was shown to reduce the number of such cells in calcification-prone areas in the wall tissue, but it proved less effective than those treatments detailed in the above-reported tests in increasing calcification resistance.

The foregoing results suggest that targeting cell calcification by treating with calcium-blocking agents or oxidative damage-preventing agents or with other modifying or extracting agents can significantly reduce aortic wall calcification.

EXAMPLE 2 - Extracellular Matrix Targeted Treatments

Multiple samples of porcine aortic roots were fixed and treated as follows.

(a) Glutaraldehyde - Samples as treated as in case of group (a) for Example 1.

(b) Glutaraldehyde plus aminooleic acid - Samples as treated as in case of group (b) for Example 1.

(c) Glutaraldehyde plus 2-aminooleic acid plus CB - Samples were treated with 0.2% glutaraldehyde in phosphate-buffered saline, pH 7.4, for 12 days at room temperature, followed by treatment with aminooleic acid (as above) , followed by incubation in 18.8 mM CB in borate-buffered saline, pH 7.4, for 3 hours at 37°C, and then rinsed. Tissues were sterilized for 24 hours at room temperature in 1% glutaraldehyde, 20% isopropanol in borate-buffered saline, pH 7.4, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in borate-buffered saline, pH 7.4, and stored in the same solution. (d) EDC (sulfo-NHS) -type fixation - Samples were treated as in case of group (e) for Example 1. (e) EDC (sulfo-NHS) -type fixation plus CB - Additional tissue was fixed using EDC and sulfo-NHS as indicated above and then further incubated in 18.8 mM CB in 10 mM HEPES, pH 6.5, for 3 hours at 37°C. Tissues were then sterilized according to the '951 patent and stored in the same solution. Following the foregoing treatments, the sterilized roots were washed in normal saline, and cusps were dissected away from the aortic walls. For calcification studies, 20 wall coupons and 20 cusp halves were randomly selected from each experimental condition and were implanted subdermally in three-week old, male Wistar rats. Samples were explanted at 4 and 8 weeks, and calcium was quantitated by AAS. Selected samples from each experimental condition were processed for histology and stained as in Example 1. The results are tabulated in the table which follows.

RESULTS Table 2

Mean 1 SEM (milligrams calcium/gram dry tissue)

Walls Cusps

Treatment 4-week 8-week 4-week 8-week

Group a 65.5 1 6 0 111.0 1 14 58.0 1 5.0 165 1 28

Group b 58.3 ± 5 0 109.5 1 5.0 5.2 1 1.0 5.1 1 1.2

Group c 7.9 ± 3 0 22.6 1 3.0 3.5 1 0.8 3.8 1 1.0

Group d 60.0 1 6 0 102.0 1 8.0 2.5 1 0.8 5.0 1 1.0

Group e 8.9 1 1 1 20.2 1 3.0 4.2 1 1.0 3.3 1 0.8

The results indicate that post-fixation treatment with CB, i.e., groups (c) and (e) , reduces the calcification of wall tissue that has been either treated with glutaraldehyde and 2-aminooleic acid or fixed according to the '339 patent, without increasing calcification of the corresponding cusps. Studies on calcification of purified elastic fibers obtained from aortic walls confirmed the efficacy of CB treatment as an anticalcification treatment. Moreover, shrinkage temperature for the fixed cusps was not significantly affected by the CB treatment, indicating that cross-link density was not significantly reduced by treatment with CB. Histology revealed little, if any, inflammatory reactions in all samples screened from each experimental condition, and von Kossa staining confirmed the calcium quantitation results. Alternative testing showed that similar post-fixation modification of aortic wall tissue with DTT/NEM was also effective in reducing calcification; however, it was not as effective in one particular test as treatment with CB, which is presently preferred.

The results of this testing indicate that matrix components, such as EAMF proteins, MMPs, and other calcium-binding components can be effectively chemically modified in a manner so as to reduce aortic wall calcification. EXAMPLE III - Combination of Targeted Treatments

Multiple samples of porcine aortic wall segments were fixed and treated as follows. (a) Glutaraldehyde/AOA - additional tissue was treated as in Example 1 group (b) (in which the tissues were glutaraldehyde-fixed, then treated with aminooleic acid, followed by sterilization) . (b) Glutaraldehyde/AOA plus CAP plus NIF - additional tissue was treated as in Example 1 group (c) (in which tissues were glutaraldehyde-fixed, then treated with aminooleic acid, followed by treatment with the CAP) . Tissues were then rinsed and incubated for 24 hours at 37°C in 5 mM NIF in borate buffer saline, pH 7.4, containing 20% isopropanol. After rinsing in borate buffer saline, pH 7.4, containing 20% isopropanol, tissues were sterilized for 24 hours at 37°C in 1% glutaraldehyde and 20% isopropanol in borate buffer saline, pH 7.4, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in borate buffer saline, pH 7.4, and stored in the same solution. (c) Glutaraldehyde/AOA plus CB plus CAP - additional tissue was treated as in Example 2 group (c) - (in which tissues were glutaraldehyde-fixed, then treated with aminooleic acid, and then treated with CB) . Tissues were further rinsed and incubated for 24 hours at 37°C in 50 mM CAP in borate buffer saline, pH 7.4, containing 10% isopropanol. After rinsing, the tissues were sterilized for 24 hours at 37°C in 1% glutaraldehyde and 20% isopropanol in borate buffer saline, pH 7.4, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in borate buffer saline, pH 7.4, and stored in the same solution.

(d) Glutaraldehyde/AOA plus CB plus NIF - additional tissue was treated as in Example 2 group (c) - (in which tissues were glutaraldehyde-fixed, then treated with aminooleic acid, and then treated with CB) . Tissues were further rinsed and incubated for 24 hours at 37°C in 5 M NIF in borate buffer saline, pH 7.4, containing 20% isopropanol. After rinsing, the tissues were sterilized for 24 hours at 37°C in 1% glutaraldehyde and 20% isopropanol in borate buffer saline, pH 7.4, followed by 24 hours incubation at 40°C in 0.2% glutaraldehyde in borate buffer saline, pH 7.4, and stored in the same solution.

(e) EDC/sulfo-NHS - additional tissue was treated as in Example 1 group (e) (in which tissues were fixed with the EDC sulfo-NHS process, followed by sterilization) .

(f) EDC/sulfo-NHS plus CAP plus NIF - additional tissue was treated as in Example 1 group (f) (in which tissues were fixed with the EDC sulfo-NHS process, then treated with CAP) . Tissues were then rinsed and incubated for 24 hours at 37°C in 5 mM NIF in HEPES buffered saline, pH 6.5, containing 20% isopropanol. After rinsing in HEPES buffer saline, pH 6.5, containing 20% isopropanol, tissues were sterilized according to the "951 patent and stored in the same solution. (g) EDC/sulfo-NHS plus CB plus CAP - additional tissue was treated as in Example 2 group (e) (in which tissues were fixed with the EDC sulfo-NHS process, then treated with CB) . Tissues were then rinsed and incubated for 24 hours at 37°C in 50 mM CAP in HEPES buffered saline, pH 6.5, containing 10% isopropanol.

After rinsing in HEPES buffer saline, pH 6.5, containing 10% isopropanol, tissues were sterilized according to the ^"951 patent and stored in the same solution, (h) EDC/sulfo-NHS plus CB plus NIF - additional tissue was treated as in Example 2 group (e) (in which tissues were fixed with the EDC sulfo-NHS process, then treated with CB) . Tissues were then rinsed and incubated for 24 hours at 37°C in 5 mM NIF in HEPES buffered saline, pH 6.5, containing 20% isopropanol.

After rinsing in HEPES buffer saline, pH 6.5, containing 20% isopropanol, tissues were sterilized according to the "951 patent and stored in the same solution.

Following the above-mentioned treatments, the sterilized wall segments were washed in normal saline, and 20 wall coupons were randomly selected from each experimental group and implanted subdermally in rats; they were analyzed for calcification after 4 and 8 weeks as described in Example 1. The results are set forth in the table that follows: RESULTS Table 3

Mean ± SEM (milligrams calcium/gram dry tissue)

Walls

Treatment 4 -week 8 -week

Group a 49.9 1 8.0 114.9 1 12.0 Group b 4.4 1 3.5 4.2 1 2.1 Group c 3.9 i 2.6 12.0 1 8.8 Group d 1.4 1 0.9 3.8 1 2.9 Group e 65.4 1 7.0 103.0 1 9.0 Group £ 1.8 1 1.2 7.1 1 3.1 Group g 2.3 1 0.7 6.7 1 3.0 Group h 3.4 1 1.5 4.9 1 3.7

The results indicate that post-fixation treatments with CAP followed by NIF, as well as treatments which employ CB followed by CAP or NIF, significantly reduce calcification of wall tissue that has earlier been either treated with glutaraldehyde and aminooleic acid or fixed according to the "399 patent. Histology performed on samples randomly selected from each experimental group revealed the absence of inflammatory reactions, and von Kossa staining confirmed the calcium quantitation results.

By comparing the 8-week calcium results from Example 3 with the comparable results in Examples 1 and 2, the data indicate that the effect of certain combinations of targeted treatments can reduce wall calcification to a higher extent than either treatment alone. For example, calcification of glutaraldehyde- and aminooleic acid- treated walls at 8-week post-implantation was reduced to 22.6±3 milligrams of calcium/gram dry tissue by treatment with CB alone (Example 2, group c) and to 18.7±7.0 milligrams of calcium/gram dry tissue by treatment with NIF alone (Example 1, group d) , while treatment with CB, followed by treatment with NIF, reduced calcium levels to 3.812.9 milligrams of calcium/gram dry tissue (Example 3, group d) . These data suggest that the major determinants of wall calcification are related to both cells and components of the extracellular matrix. As a result, it now appears that multiple targeting of relevant calcifying substrates can still further significantly reduce aortic wall calcification.

Although the invention has been described with regard to certain preferred embodiments which constitute the best mode presently known to the inventors for carrying out the invention, it should be understood that various changes and modifications that would be obvious to one having the ordinary skill in this art may be made without deviating from the scope of the invention as set forth in the claims appended hereto. For example, although various sequences of treatment are set forth, in many instances, the steps can be carried out in different sequences and still obtain the advantageous anticalcification characteristics in the ultimate products. Although washing between steps is considered desirable to avoid any potential interaction between reagents, if there is no such interaction expected, such washing step may be omitted. Moreover, some reagents may be fully compatible with each other, in which case a combination of treatments may be performed simultaneously, and as such, simultaneous treatment is often considered to be the equivalent of sequential treatment with agents from certain categories. Likewise, although the working examples show treatment of cardiovascular tissues, i.e. porcine aortic roots with walls and cusps or porcine aortic wall segments alone, such was done for purposes of allowing reasonable comparison, and it should be understood, as set forth in the description, that the invention is considered to be applicable to a wide variety of biomaterials destined for implantation in mammals, particularly humans, where calcification is considered to be a distinct problem because of its adverse effect on ultimate lifetime.

The disclosures of the previously enumerated U.S. patents are expressly incorporated by reference.

Particular features of the invention are set forth in the claims that follow.

Claims

CLAIMS :

1. A method for the treatment of biomaterials destined for implantation in mammals, including humans, which method comprises:

(a) treating said biomaterial with an effective amount of a cell-targeted agent, which agent (i) decreases calcification by blocking calcium channels, (ii) prevents oxidative or selective enzymatic damage and/or (iii) removes cell-derived calcium-binding components; followed by washing said treated biomaterial; or

(b) treating said biomaterial with an effective amount of a matrix-targeted agent which chemically modifies proteins in matrix-derived components thereof that bind calcium or that induce calcification in adjacent components, followed by washing said treated biomaterial; or

(c) sequentially treating said biomaterial with a combination of (a) followed by (b) or of (b) followed by (a) with an intermediate washing step being optional, whereby said treated and washed biomaterial thereafter resists in vivo calcification.

2. The method according to claim 1 wherein treating is carried out according to step (a) (ii) using a concentration of between about 1 and about 200 mM of an antihypertensive agent which prevents oxidative and/or enzymatic damage.

3. The method according to claim 2 wherein a concentration of captopril between about 25 and about 75 mM is employed.

4. The method according to any one of claims 1-3 wherein treating is carried out in accordance with step (a) (i) using a concentration of between about 0.1 and about 50 mM of a calcium channel-blocking agent.

5. The method according to claim 4 wherein a concentration of between about 5 and about 25 mM of nifedipine or diltiazem hydrochloride is used.

6. The method of any one of claims 1-5 wherein treating is carried out according to step (b) using a protein cleaving agent at a concentration of between about 1 and about 200 mM.

7. The method according to claim 6 wherein a concentration of about 10 and about 50 mM of cyanogen bromide is used.

8. The method according to claim 6 wherein, following treatment according to step (b) , said biomaterial is treated according to step (a) (i) .

9. The method according to claim 6 wherein, following treatment in accordance with step (b) , said biomaterial is treated in accordance with step (a) (ii) , and optionally then treated according to step (a) (i) .

10. The method according to any one of claims 1-9 wherein treating is carried out according to step (a) (iii) using KMA at a KCl concentration of between about 0.5-0.8 M and MgATP at a concentration of between about 0.05 and about 8 mM.

11. The method according to any one of claims 1-10 wherein step (b) is carried out using a reducing agent at a concentration of between about 1 and about 200 mM.

12. The method according to claim 11 wherein step (b) is carried out using a concentration of between about 25 and about 75 mM of DTT and is followed by treatment with an alkylating agent.

13. A method for the treatment of chemically cross- linked cardiovascular tissues prior to implantation in the human body, which method comprises:

(a) treating said tissue with an effective amount of a cell-targeted agent which decreases calcification by blocking calcium channels, and then washing said treated tissue; or

(b) treating said tissue with an effective amount of a cell-targeted agent which decreases calcification by preventing oxidative and enzymatic damage, and then washing said treated tissue; or I to σ>

1

20. The method according to claim 13 wherein there is sequential treatment of said tissue in the form of one of the following combinations: (a) followed by (b) ; (b) followed by (a) ; (c) followed by (b) ; (c) followed by (a) ; or (c) followed by (a) and (b) .