Method for preparing the surface, particularly of a cardiac prosthesis - Patent application

Abstract

"Method for preparing the surface of a cardiac prosthesis in particular" The present invention discloses a method for preventing early active and passive degradation of prostheses that are in contact with biological fluids.

Description

[Technical field]

[0001] The invention is applicable in the medical field, in particular in the preparation of biological surfaces that come into contact with body fluids.

[0002] It has been reported that glutaraldehyde-fixed bioprosthetic heart valves (BHVs) are prone to dystrophic calcification when implanted in humans for a medium to long term, which is the main limiting factor affecting the life span of BHVs. Calcification is a complex and multifactorial process that is still not fully understood, including atherosclerosis-like tissue remodeling and long-term exposure to mechanical stress. Among the causes of calcific tissue dystrophy, treatment with glutaraldehyde (GLU) also deserves consideration.

[0003] GLU is often used as the preferred fixative sterilant for many commercial bioprosthetic products, especially surgical transcatheter implantable heart valves (THVs). THV implants are considered a less invasive procedure, avoiding open heart surgery, using a guide catheter for the positioning of the prosthesis. THVs may be an option for people at moderate or high risk of complications from heart valve replacement surgery. Unfortunately, GLU is chemically unstable, which is associated with the certain exposure of potential calcium binding sites (residual aldehydes, acids, Schiff bases, etc.). Interaction of GLU with amino acid residues in tissues results in negatively charged carboxylic acid groups that can bind to positively charged Ca. 2+ They interact electrostatically with ions, resulting in a tremendous attraction site for calcium. To make matters worse, even the freely reactive aldehyde groups are easily oxidized to carboxylic acid residues via air, in vivo blood and macrophage oxidation.

[0004] To reduce its impact on the mineralization process, BHV manufacturers have proposed several modifications of the GLU fixation protocol, including the addition of novel steps aimed at chemical stabilization of the reactive aldehyde and carboxylic acid groups. Detoxification of GLU with urazole, extension of the diamine spacer, treatment with 2-aminooleic acid, or incubation in ethanol are just some of the processes developed to stabilize GLU, which are expected to delay the dystrophy of mineralized tissues.

[0005] Although calcific degeneration of BHV is generally a long-term event that causes definitive failure of such biomedical devices, it should not be forgotten that there are a series of degenerative processes that begin to affect the prosthesis just a few hours after implantation, primarily compromising the structural aspects of the device.

[0006] Early structural BHV degeneration is a complex pathway with multiple active and passive mechanisms closely related to each other. It is now well established that active mechanisms of degeneration are triggered by an early host response within hours of implantation and often correlate with inflammation, subclinical leaflets thrombosis and / or bacterial infection. Passive degradation, on the other hand, is strictly related to graft fatigue, resulting in hole formation, tearing and wear of the leaflets.

[0007] Early structural alterations – passive factors As is well known, commercially available BHVs do not constitute viable tissue and, by definition, are incapable of regeneration and remodeling of the extracellular matrix, so that any alterations in the collagen meshwork (delamination, structural rearrangements and destruction) resulting from cyclic loading are considered irreversible damage. Prolonged cyclic loading during accelerated wear tests highlights a marked decrease in radial extensibility due to stiffening of the effective collagen fiber network. Stiffening of the leaflets over time causes abnormalities in the distribution of mechanical stresses, which leads, in particular, to overloading of the bending and sutured regions around the stent. Histological evaluation of implanted BHVs showed that tearing of the leaflets and destruction of collagen fiber bundles were characteristic of high strain areas, even in the absence of associated calcification.

[0008] Early structural alterations - active factors Asymptomatic valve thrombosis occurs frequently in BHV replacements and is more common in THV (13% frequency) than in surgical BHV (4%). At 1 year after implantation, 30% of BHVs are involved. Such pathology shows clinical signs within 30 days after implantation and causes a decrease in leaflet mobility. In patients with decreased leaflet mobility (HALT - hypoattenuating leaflet thickening), the leaflet thickness changes significantly and at least one valve becomes completely immobile. The occurrence of HALT indicates a mild valve dysfunction associated with leaflet thrombosis, early calcification and / or degeneration.

[0009] Anticoagulation (both the novel oral anticoagulants NOAC and warfarin) was effective in reducing HALT complications, but HALT recurred in 50% of patients who discontinued anticoagulation. It is important to note that dual antiplatelet therapy, the standard of care for transcatheter valve implantation, was not effective in preventing or treating asymptomatic valve thrombosis.

[0010] Bacterial infection is another worrying aspect that contributes to early BHV degeneration, especially in the case of THV. Infective endocarditis (IE) has a major impact on both the population and patient management. In the United States, there are 40,000-50,000 new cases per year, with average hospitalization costs exceeding $120,000 / patient. Despite improvements in diagnosis and surgical intervention, the 1-year mortality rate from IE has remained unchanged for more than 20 years. Surgical valve replacement is necessary when long-term antibiotic treatment is not sufficient.

[0011] The ability of certain bacteria to colonize BHVs is an important aspect for the future of heart valve replacement, since THVs have also shown good results in medium- and low-risk patients, significantly expanding the number of people undergoing this type of minimally invasive intervention. Replacement of degenerated surgical BHVs is often performed employing a valve-in-valve approach. In this case, a THV is placed inside the dysfunctional surgical BHV without its preliminary removal. This could potentially lead to the transfer of bacteria from the dysfunctional BHV to the new THV, resulting in bacterial growth and tissue colonization.

[0012] Finally, regulatory agencies require medical devices to be tested for material-mediated pyrogenicity according to ISO 10993-11:2017 Biological evaluation of medical devices - Part 11: Systemic toxicity testing. The term pyrogen (from Greek pyros: fire) defines a substance that induces fever. Pyrogenic reactions induced by medical devices can be attributed to several causes, depending on the presence of so-called "material-mediated pyrogens". One well-known and well-characterized class of exogenous pyrogens is that of endotoxins. Endotoxins are lipopolysaccharide components present in the cell walls of gram-negative bacteria. Another broad class of exogenous pyrogens is non-endotoxic pyrogens, which include substances such as lipoteichoic acid from gram-positive bacteria and other compounds from fungi, yeasts, viruses, bacteria, and parasites. A third class of non-endotoxic pyrogens is material-mediated pyrogens. Material-borne pyrogens have no formal definition, but are believed to leach from the material or surface of the medical device. Material-borne pyrogens can also result from contaminants introduced during manufacturing or packaging, such as residues from cutting fluids, release agents, cleaning agents, and processing aids. It is therefore of fundamental importance to develop treatments that can improve the outcome of BHV without introducing chemicals or contaminants that increase the pyrogenic reaction. Summary of the Invention

[0013] The inventors of the present patent application have surprisingly found a method to prevent both active and passive early degeneration events in biological matrices (particularly medical devices, more particularly cardiac prostheses), which achieves unprecedented stabilization of GLU, resistance to both surface platelet adhesion and fibrin release, as well as surface colonization by microorganisms. As a result, such a method shows an interesting improving effect on the biomechanical properties of the treated biological matrices. [Brief description of the drawings]

[0014] [Figure 1]Percentage reduction of freely reactive aldehyde and carboxylic acid groups in polyphenol-treated GLU-fixed pericardial patches (n=16 for each type of chemical group measurement).

[0015] [Diagram 2] Percentage of thrombus accumulation in polyphenol-treated and untreated (GLU) pericardial samples (n=16 for each type of chemical group measurement).

[0016] [Diagram 3] Comparison of tensile strength between polyphenol-treated and untreated samples (GLU, n=36 for each type of chemical group measurement).

[0017] [Figure 4] Comparison of the percentage of elongation between polyphenol-treated and untreated samples (GLU, n=36 for each type of chemical group measurement).

[0018] [Diagram 5] F Young's modulus correlation between polyphenol-treated and untreated samples (GLU, n=36 for each type of chemical group measurement).

[0019] Objective of the invention In a first object, the present invention discloses a method for treating a surface in contact with a biological fluid.

[0020] In a preferred embodiment, the surface is a surface of a medical device.

[0021] In a more preferred embodiment, said surface is the surface of a biological prosthesis, which may be a cardiac prosthesis.

[0022] In a second object, the present invention discloses a surface to be contacted with a biological fluid, obtained by the method of the present invention.

[0023] In a preferred embodiment, the surface is a surface of a medical device.

[0024] In a more preferred embodiment, said surface is the surface of a biological prosthesis, which may be a cardiac prosthesis.

[0025] Medical devices, biological prostheses and cardiac prostheses comprising a surface according to the invention represent further objects of the present invention.

[0026] In a third object, the present invention discloses a method for the treatment of diseases comprising the use of the medical device, biological prosthesis or cardiac prosthesis of the present invention.

[0027] In a preferred embodiment, the disease is a heart disease.

[0028] In one embodiment, the disease is a disease in a human, and in another embodiment, the disease is a disease in an animal.

[0029] The solutions comprising a phenolic compound or a mixture of phenolic compounds used in the method of the present application represent a further object of the present invention.

[0030] In accordance with another object of the present invention, a method for preparing the inventive solution containing a phenolic compound or a mixture of phenolic compounds is disclosed.

[0031] In yet another object, the present invention discloses a kit for carrying out the method of the present invention.

[0032] Detailed Description of the Invention According to a first object, the present invention discloses a method for the treatment of a surface that is in contact with a biological fluid.

[0033] Biological fluids within the scope of the present invention are represented by blood, serum, plasma, vitreous gel, tears, urine, saliva, stool, including synovial fluid, peritoneal fluid, pericardial fluid, pleural fluid and amniotic fluid.

[0034] The biological surface may refer to a surface of human or animal origin.

[0035] In particular, the animal derived surface may be a surface of equine, porcine or bovine origin, preferably a surface of porcine or bovine origin, such a surface may be considered a biological matrix.

[0036] In particular, said surface is a surface of a medical device.

[0037] The medical device according to the invention may be indicated in heart valves, tendons, ligaments, pericardium, fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose tissue, bone tissue, pelvic tissue, abdominal tissue, breast tissue and dermal tissue.

[0038] In accordance with another embodiment of the invention, the surface is a surface of a biological prosthesis.

[0039] The biological prostheses according to the invention may be present in blood vessels, heart valves, tendons, ligaments, pericardium, fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvic tissue, abdominal tissue, breast tissue and dermal tissue.

[0040] In a preferred embodiment, the biological prosthesis according to the invention may be represented as a cardiovascular prosthesis, such as a heart valve or a pericardial tissue patch.

[0041] In a more preferred embodiment, the heart valves which can be treated according to the present invention are designated surgical heart valves.

[0042] In a further preferred embodiment, the heart valve treatable according to the present invention is depicted as a transcatheter implantable heart valve, said valve having to be implanted through a catheter and being folded to be accommodated within the catheter.

[0043] In accordance with the method of the present invention, the disclosed surface is contacted with a solution containing a phenolic compound or a mixture of phenolic compounds.

[0044] For purposes of the present invention, a phenolic compound is intended to mean a phenolic or polyphenolic compound (in some instances both of which are referred to as "phenols" or "polyphenols" and used synonymously herein) selected from the group including phenol, phenolic aldehydes, phenolic acids, phenylamines, phenol compounds, flavonoids, phenylpropanoids and tannins.

[0045] In particular, the phenolic compound is selected from the group comprising vanillin, cinnamic acid, phenylalanine, coumarin, xanthone, catechin, flavonone, flavone, chalcone, flavanonol, flavanol, leucoanthocyanidin, anthocyanidin, hydroxycinnamic acid.

[0046] Furthermore, in particular, the phenolic compound may be selected from the group comprising resveratrol, aloin, cyanarin, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperitin, quinic acid, eleonolic acid, pinoresinol, luteolin, apigenin, tangeretin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid.

[0047] For the purposes of the present invention, derivatives of the phenolic or polyphenolic compounds disclosed above are also included, for example salts or esters or isomers may also be used.

[0048] In one embodiment of the invention, the solution of the invention comprises a mixture of two or more of the above disclosed phenolic or polyphenolic compounds.

[0049] In accordance with a preferred embodiment, the solutions of the present invention may contain a mixture of two or more of the above disclosed phenylpropanoids.

[0050] Below some components and some solutions according to the invention are reported: [Table 1]

[0051] For preparation of the solutions of the present invention, the phenolic or polyphenolic compounds are solubilized in an alcoholic solvent.

[0052] When preparing a mixture of phenolic or polyphenolic compounds, solutions of each compound are prepared separately and then mixed together.

[0053] According to a preferred embodiment of the invention, said solution comprises a mixture of phenolic compounds, more preferably a mixture of phenylpropanoid compounds.

[0054] For said purpose, the first component is solubilized in an alcoholic solution (component A), preferably at 10% of the final volume of said solution.

[0055] Alcohol solvents according to the present invention may include methanol, ethanol, isopropanol, butanol, and the like, and preferably include or are represented by ethanol.

[0056] In the solutions of the invention, the second component is solubilized in an isotonic buffer solution (component B), preferably to 90% of the final volume of said solution.

[0057] In one embodiment of the invention, the final solution is a hydroalcoholic solution.

[0058] According to the present invention, in the disclosed method, the solution of the phenolic compound or mixture of phenolic compounds preferably has a pH value of 5-7.

[0059] Once prepared, the solution may optionally be filtered through a 0.22 μm filter.

[0060] In accordance with the present invention, the method disclosed involves contacting a surface with a solution of a phenolic compound or a mixture of phenolic compounds for a period of less than 2 hours.

[0061] Preferably, the contacting continues for about 1 hour.

[0062] More preferably, the contacting continues for about 30 minutes.

[0063] In an even more preferred embodiment, the contacting comprises a first step and a second step.

[0064] In a preferred embodiment, the first contacting step is carried out for 30 minutes and the second contacting step is carried out for 30 minutes.

[0065] Optionally, a rinsing step (also called a washing step) may be carried out between two contacting steps.

[0066] According to a preferred embodiment of the invention, the method is carried out in the dark, more preferably completely in the dark (i.e. avoiding exposure to light).

[0067] According to a preferred embodiment, the process is carried out while stirring the solution.

[0068] With regard to the temperature of the contacting step, it is preferably carried out at a temperature of about +20°C ± 10°C.

[0069] In a preferred embodiment of the invention, after the contacting step, the treated surface, medical device, biological prosthesis or cardiac prosthesis may be subjected to one or more washing steps.

[0070] Preferably, each of said washing steps is carried out using a suitable buffer, for example a suitable buffer may be represented by a phosphate buffer.

[0071] In one embodiment, each washing step may be carried out for about 15-30 minutes.

[0072] In another embodiment, each washing step may be carried out for about 12 to 48 hours.

[0073] According to one embodiment of the invention, where the disclosed method is carried out on a biological surface, said biological surface may have previously been subjected to a pretreatment step.

[0074] In particular, the pretreatment step may have one or more of the following effects: - protein stabilization, - lipid stabilization or removal, - stabilization or removal of cellular structures, - Reduced antigenicity.

[0075] For the purposes of the present invention, said pretreatment step may include one or more of glutaraldehyde, formaldehyde, quercetin or genipin pretreatment steps, as well as a treatment for the removal of phospholipids.

[0076] According to certain embodiments of the invention, before subjecting the biological surface to a pretreatment step, the biological surface may be treated with a mixture of glycerol, heparin, amines (i.e., alkylamines, amino alcohols, ethanolamines), amino acids (lysine, hydroxylysine, aminosulfonates, taurine, aminosulfates, dextran sulfate, chondroitin sulfate), hydrophilic multifunctional polymers (i.e., polyvinyl alcohol, polyethyleneimine), hydrophobic multifunctional polymers (i.e., alpha-dicarbonyl, methylglyoxal, 3-deoxyglucosone, , glyoxal), hydrazides (i.e., adipic hydrazide), N,N-disuccinimidyl carbonate, carbodiimides (i.e., 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride-EDC, N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide-CMC, 1,3-di-cyclohexylcarbodiimide-DCC, 2-chloro-1-methylpyridinium iodide-CMPI, 2-chloro-1-methylpyridinium iodide-CMPI), antibiotics, cell recruiting agents, hemocompatible agents, anti-inflammatory agents, anti-proliferative agents, reducing agents (i.e., sodium cyanoborohydride, sodium borohydride, sodium bisulfite + acetylacetone, formic acid-formaldehyde, mono-, di- or polyepoxyalkanes).

[0077] In the context of the present invention, the method of treating a surface that is in contact with biological fluids according to the present invention is a stabilization method.

[0078] In particular, the stabilization method deactivates available reactive groups on the pretreated surface.

[0079] More specifically, the stabilization method deactivates available aldehyde and carboxylic acid groups on the pre-treated surface.

[0080] In the context of the present invention, the methods disclosed are anti-calcification methods.

[0081] The surfaces obtainable by the method of the invention represent another object of the present application.

[0082] Biological prostheses, medical devices, in particular cardiac prostheses, comprising a surface obtainable according to the method of the present application represent a further object of the present invention.

[0083] The surfaces obtained by the pretreatment and treatment methods of the present invention represent another object of the present application.

[0084] Biological prostheses, medical devices, in particular cardiac prostheses, comprising surfaces obtained according to the pretreatment and treatment methods of the present application represent a further object of the present invention.

[0085] In the context of the present invention, the method of treating a surface that is in contact with biological fluids according to the present invention is a protective method.

[0086] In particular, the method of protection prevents the occurrence of asymptomatic thrombosis.

[0087] More specifically, the method of the present invention is an anti-platelet adhesion method.

[0088] More specifically, the method of the present invention prevents the synthesis of fibrin.

[0089] The synthesis of fibrin involves the activation of soluble fibrinogen into insoluble fibrin polymers. These polymers laterally aggregate to form fibers that branch into three-dimensional networks that interact with circulating platelets, leading to the formation of fibrin clots that are essential for hemostasis and wound clotting. When the clot enters the bloodstream, it is called a thrombus and can occlude small and medium-sized blood vessels, causing ischemia, stroke, and heart attack.

[0090] In particular, the protective methods of the present invention have been demonstrated to prevent and avoid the fixation of circulating platelets to biological surfaces that have been treated and / or pretreated according to the present invention.

[0091] The surfaces obtainable by the method of the invention represent another object of the present application.

[0092] Biological prostheses, medical devices, in particular cardiac prostheses, comprising a surface obtainable according to the method of the present application represent a further object of the present invention.

[0093] The surfaces obtained by the pre-treatment and treatment methods of the invention represent another object of the present application.

[0094] Biological prostheses, medical devices and cardiac prostheses comprising surfaces obtained according to the pretreatment and treatment methods of the present invention represent further objects of the present invention.

[0095] In the context of the present invention, methods of treating surfaces that come into contact with biological fluids are methods that preserve and maintain appropriate structural and biomechanical properties.

[0096] In particular, the methods of the present invention have been demonstrated to improve the elongation properties of biologically engineered tissues.

[0097] In particular, the method preserves the collagen structure of the BHV leaflets.

[0098] Thus, the method of treating surfaces that come into contact with biological fluids according to the present invention is a protective method for maintaining the proper physiological hematological and hydrodynamic properties of the treated BHV.

[0099] The surfaces obtainable by the method of the invention represent another object of the present application.

[0100] Biological prostheses, medical devices and cardiac prostheses comprising a surface obtained according to the method of the present application represent further objects of the present invention.

[0101] The surfaces obtained by the pre-treatment and treatment methods of the present invention represent another object of the present application.

[0102] Biological prostheses, medical devices and cardiac prostheses comprising surfaces obtained according to the pretreatment and treatment methods of the invention represent further objects of the invention.

[0103] The method of the present invention for treating surfaces that come into contact with biological fluids is an antimicrobial and antiviral method in that it is a method for disinfecting the treated surface.

[0104] In particular, the antimicrobial method of the present invention has a bactericidal effect.

[0105] More particularly, the antimicrobial methods of the present invention are active against microorganisms responsible for the development of endocarditis.

[0106] In one embodiment, the microorganism is a gram + Bacteria, Gram - Bacteria, yeast and mold.

[0107] In another embodiment, the microorganism is a mycobacterium, such as Mycobacterium chelonae.

[0108] Thus, the method of treating a surface that is in contact with biological fluids according to the present invention comprises administering an anti-gram + Anti-Gram - These are antibacterial, anti-yeast, anti-fungal and anti-mycobacterial.

[0109] In particular, the antiviral method of the present invention has a virucidal effect against viruses.

[0110] More particularly, said virucidal action is in accordance with the EN 14476 standard.

[0111] In particular, said viruses belong to the families Picornaviridae, Adenoviridae and Caliciviridae.

[0112] More particularly, said microorganisms are poliovirus type 1 LSc-2ab (RVB-1260), adenovirus type 5 (ATCC VR-5) and murine norovirus strain S-99 (RVB-651).

[0113] The surfaces obtainable by the method of the invention represent another object of the present application.

[0114] Biological prostheses, medical devices and cardiac prostheses comprising a surface obtained according to the method of the present application represent further objects of the present invention.

[0115] The surfaces obtained by the pre-treatment and treatment methods of the present invention represent another object of the present application.

[0116] Bioprostheses, medical devices and cardiac prostheses comprising a surface obtained according to the pretreatment and optionally treatment method of the present invention represent further objects of the present invention.

[0117] More particularly, said biological prosthesis may be designated a cardiovascular prosthesis obtained by the pretreatment and optionally treatment method of the invention.

[0118] According to a third object of the present invention, a method for treating a disease is disclosed, comprising the use of the medical device, biological prosthesis or cardiac prosthesis disclosed above.

[0119] In a preferred embodiment, the disease is a heart disease.

[0120] In one embodiment, the disease is a disease in a human, and in another embodiment, the disease is a disease in an animal.

[0121] In certain embodiments, the method of treating a disease according to the present invention comprises a valve-in-valve approach, in which a valve is placed inside a dysfunctional valve without first removing the valve.

[0122] As a further object of the present invention, a solution containing a phenolic compound or a mixture of phenolic compounds for use in the method of the present application is disclosed.

[0123] In particular, the preparation of said solution comprises a first step of solubilizing a phenolic compound in an alcoholic solvent.

[0124] For example, the phenolic compounds of List A above may be dissolved.

[0125] If necessary, the phenolic compound can further be solubilized in an isotonic buffer.

[0126] For example, the phenolic compounds of list B above may be dissolved.

[0127] According to a preferred embodiment, the solution of compound A represents 10% (volume) of the final solution and the solution of compound B represents 90% (volume) of the final solution.

[0128] According to a preferred embodiment, the preparation of the solution is carried out in the dark, preferably in complete darkness, i.e. avoiding exposure to light.

[0129] The invention is further illustrated in connection with the experimental section below.

[0130] The following experimental section shows the results of assays carried out on surfaces treated according to the invention.

[0131] Polyphenol Solution In the following table some examples of polyphenol solutions according to the invention are reported. [Table 2]

[0132] In particular, the surface has been treated with solution 5 according to the disclosure above.

[0133] Preparation of polyphenol solution 5 Caffeic acid as component A is weighed according to the indicated concentration and solubilized in ethanol to 10% of the final volume of the polyphenol mixture. Tannic acid as component B column is weighed according to the indicated concentration and solubilized in denatured phosphate buffer to 90% of the final volume of the polyphenol mixture. Both steps are performed in the dark. Once solubilization is complete, the two solutions are mixed. The pH is adjusted to 5-7. The solution is filtered through a 0.22 μm filter. This solution is referred to as solution 5 or polyphenol solution.

[0134] Other solutions according to the present invention can be prepared similarly as disclosed above.

[0135] Stabilization of glutaraldehyde Tissue processing Several bovine pericardium were carefully selected to obtain rectangular patches (n=32). All patches were subjected to a preliminary GLU cross-linking treatment. Briefly, pericardial tissues were incubated in a buffered GLU solution for 24 h in three steps in the dark. In this example, the GLU solution was 0.6%±0.5% v / v in the first and second steps, and 0.2%±0.15% v / v in the third step. The GLU-treated pericardial patches were washed twice in phosphate buffer for 15 min each.

[0136] Sixteen patches were incubated with inventive solution 5, prepared as above, in two stages for 25±10 minutes each at room temperature (RT) in the dark with moderate but constant agitation. At the end of the incubation period, the treated patches were washed five times for 15-30 minutes with phosphate buffer. The samples are designated as TREATED.

[0137] The remaining GLU-fixed pericardial patches served as controls (GLU, n = 16).

[0138] Determination of free carboxylic acid group content The pericardial patches were placed in OCT (optimal cutting temperature) and frozen by immersion in isopentane precooled with liquid nitrogen. Cryosections of 7 μm thickness were then made on MirrIR slides, suitable for infrared reflectance studies, and analyzed by FT-IR microscopy with 64 scans in reflectance and mosaic modes for each selected area. The detector was fitted with a 4 cm -1 A high-resolution FPA was used. The treated tissue showed a higher fluorescence intensity at wavenumber 1233 cm compared to the GLU-fixed sample. -1 It was shown that the concentration of carboxylic acid groups in (corresponding to the CO bond stretching of the carboxylic acid group) is low.

[0139] Considering the sum of the free carboxylic acid groups determined in the GLU immobilized samples as 100% of the available groups, a reduction corresponding to an overall 76% was reported in the treated samples.

[0140] Measurement of free aldehyde group content Prepare 150 ml of solution A by dissolving 0.2 M citric acid, 0.5 M sodium hydroxide, and 8 mM stannic chloride in ultrapure water. Prepare 25 ml of solution B by dissolving 0.22 M ninhydrin in 25 ml of ethylene glycol monomethyl ether (Cellosolve). Combine one volume of solution A with an equal volume of solution B and mix for 45 minutes in the dark to obtain solution C. Note that 2 ml of solution C is required for each sample to be analyzed.

[0141] Tissue samples should be prepared to approximately 20 mg wet weight each. Each sample is incubated in 2 ml of solution C at 100°C in the dark for 20 minutes, cooled in water, and diluted with 15 ml of 50% isopropanol. The color developed is read at 570 nm within 30 minutes. The nmoles of aldehyde groups are determined relative to a glycine standard.

[0142] Taking the total amount of free aldehydes determined in the GLU-fixed samples as 100%, a reduction equal to 56.3% of the total was reported in the treated samples.

[0143] FIG. 1 shows the percentage reduction of freely reactive aldehyde and carboxylic acid groups in treated pericardial tissue patches (n=16 for measurement of each type of chemical group).

[0144] Assessment of platelet adhesion Tissue processing An in vitro blood flow model was employed to evaluate platelet adhesion and the tendency of fibrin release under flow conditions. Several bovine pericardium were carefully selected to obtain rectangular patches (n=12). All patches were subjected to a preliminary GLU cross-linking treatment. Briefly, in a dark room, pericardial tissues were incubated in buffered GLU solution for 24 h each in three steps. In this example, the GLU solution is 0.6% ± 0.5% v / v in the first and second steps, and 0.2% ± 0.15% v / v in the third step. The GLU-treated pericardial patches were subjected to two washings in phosphate buffer for 15 min each.

[0145] Solution 5 of the present invention, prepared as described above, was incubated with six patches in two stages for 25±10 minutes each at room temperature (RT) in the dark with moderate but constant agitation. At the end of the incubation, the treated patches were washed five times for 15-30 min in phosphate buffer. The samples are designated as TREATED.

[0146] The remaining GLU-fixed pericardial patches were employed as controls (GLU, n = 6).

[0147] Quantification of platelet adhesion Heparinized bovine blood was collected from three different animals and spiked with radioisotopes for thrombus quantification. Pericardial tissue strips were placed in 25.4 mm conduits and pumped with a peristaltic pump for 1 h at 2.5 L / min blood flow. The strips were rinsed with saline and placed in a gamma counter to quantify radioactivity (reflecting relative thrombosis). The mean radioactivity was 73.133 counts per minute (cpm) for Glu and 35.165 cpm for treated samples. In Figure 2, the results are presented as a percentage of platelet prevalence reduction in polyphenol-treated tissues, with GLU samples taken as 100%.

[0148] Mechanical properties of treated tissue Tissue processing 72 rectangular strips (approximately 10 mm long and 8 mm wide) of bovine pericardium were treated with GLU solution as disclosed above ("Tissue Treatment"). The GLU-treated pericardium stripes were subjected to two washing steps in phosphate buffer for 15 min each. At room temperature, 36 patches were incubated with solution 5 disclosed above, in the dark and under moderate but constant agitation, in two steps for 25±10 min each. At the end of the incubation, the treated patches were subjected to five washing steps in phosphate buffer for 15-30 min each. The samples are called TREATED.

[0149] The remaining GLU-fixed pericardial stripes were employed as controls (GLU, n = 36).

[0150] Mechanical property evaluation Each stripe was attached to a tensile apparatus and clamped with rubber grips (Thumler Z3-X500 equipped with 100N / 500N load cells) for uniaxial tensile testing. The initial length of the sample (distance between the grips) was 50 mm. Tensile tests were performed at 50 mm / min.

[0151] Each stripe was dimensionally characterized in terms of length (useful length 50 mm), width and thickness (average of the measurements) and the cross-sectional area was calculated (at thickness x). The following parameters were obtained from each tensile curve: · Breaking strength [N]: in terms of maximum strength before breakage; Tensile strength [MPa]: in terms of ultimate strength divided by cross-sectional area (UTS); · Failure strain [%]: in terms of strain at maximum strength; · Young's modulus [MPa]: in terms of the slope of the linear region (elastic phase) of the stress-strain curve; For each parameter, one value was obtained from each patch and the average was calculated from the samples. The patches were then averaged again to give one value for each test group.

[0152] Figure 3 shows a comparison of the tensile strength of treated and untreated samples (GLU). Tensile strength (UTS), sometimes abbreviated as tensile strength (TS), is the maximum stress that a material can withstand while being stretched before breaking. Polyphenol treatments reported no statistically significant differences from the control sample (GLU).

[0153] FIG. 4 compares the percentage of elongation of treated and untreated samples (GLU).

[0154] Figure 5 shows the correlation of Young's modulus between treated and untreated samples (GLU). The percentage of elongation is strictly related to Young's modulus. Young's modulus or elastic modulus is a characteristic of a material, describing the relationship between tension and deformation under uniaxial loading conditions, and the elastic (reversible) behavior of the material. Young's modulus is defined as the ratio between the applied stress and the resulting deformation. As Young's modulus increases, the stiffness of the material also increases. The increased level of elasticity of the treated tissue results in a decrease in Young's modulus compared to the untreated pericardium (GLU).

[0155] The increased elasticity allows for better distribution of mechanical loads, which is particularly beneficial in the BHV region where high pressures are present, thus avoiding the formation of tears and preserving the collagen structure of the valve.

[0156] Bactericidal potential of polyphenol solutions The bactericidal activity (BA) of the polyphenol solution of the present invention was evaluated against various microorganisms: Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 19111, Salmonella enterica typhimurium ATCC 14028, Streptococcus viridans ATCC 6249, the nontuberculous mycobacterium Mycobacterium chelonae ATCC 35752, the yeast Candida albicans ATCC 35752, and the yeast Staphylococcus faecalis ATCC 29212. albicans ATCC 10231 and the fungus Aspergillus brasiliensis ATCC 16404.

[0157] The BA assay consists of a suspension method involving a single incubation of bacteria with a known concentration of solution 5 of the invention (inoculum) for 24 hours. At the end of the incubation period, the contents of each test tube are diluted with tryptone-salt broth (MRD broth) and then plated onto 90 mm sterile Petri dishes in a specific agar medium, either by pour plate method or spread plate method, depending on the microorganism being tested. The resulting plates are then incubated under specific conditions and temperatures according to the growth requirements of each microorganism.

[0158] Inoculum preparation For each type of microorganism, the microbial suspension in MRD broth was quantified at a wavelength of 620 nm via a spectrophotometer in a disposable 10 mm path length cuvette. The absorbance of an aliquot portion of the suspension was measured: a range of 0.150-0.460 corresponds to a concentration of cells between 1x10^8 CFU / ml and 3x10^8 CFU / ml (with Candida albicans between 1x10^7 CFU / ml and 3x10^7 CFU / ml). For Streptococcus oralis, there was no correlation between absorbance measurements and bacterial concentration, so quantification was performed with cell counts under a microscope.

[0159] Data were presented as a percentage of bactericidal activity, comparing the bacterial concentration at time t0 with the growth on the samples to determine the effect of growth promotion or inhibition, and the results were compared to a control sample (antibiotic and ethanol solution).

[0160] In the table below the percentage of bactericidal activity compared to standard antibody and ethanol solutions is reported.

[0161] [Table 3]

[0162] Virucidal potential of polyphenol solutions The virucidal activity of polyphenol solution 5 was evaluated according to the guideline: Test methods and requirements European Standard EN 14476:2013+A2:2019 / UNI EN 14476:2019 - Chemical disinfectants and antiseptics. Quantitative suspension test for the evaluation of virucidal activity in the medical field. Test methods and requirements (2 phase / 1 step). The virucidal assays were carried out for the following virus strains: poliovirus type 1 LSc-2ab (RVB-1260), adenovirus type 5 (ATCC VR-5) and murine norovirus S99 (RVB-651). In the table below, the percentage of virucidal activity of polyphenol solution 5 diluted to 80% is reported (corresponding to the highest concentration assessable according to the method). [Table 4]

[0163] Anti-adhesive effect of polyphenol solutions against various strains of microorganisms The anti-adhesive activity of the treated surfaces was evaluated against various microorganisms: Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 19111, Salmonella enterica typhimurium ATCC 14028, Streptococcus viridans ATCC 6249, the nontuberculous mycobacterium Mycobacterium chelonae ATCC 35752, the yeast Candida albicans, and the microbial strains Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 19111, Salmonella enterica typhimurium ATCC 14028, Streptococcus viridans ATCC 6249, the nontuberculous mycobacterium Mycobacterium chelonae ATCC 35752, the yeast Candida albicans, and the microbial strains Staphylococcus faecalis ATCC 29212. albicans ATCC 10231 and the fungus Aspergillus brasiliensis ATCC 16404.

[0164] Each microbial strain was grown overnight in its own broth at 37° C. At the end of incubation, colony forming units (UFC) were counted to determine the effective concentration of the microorganism.

[0165] Tissue processing Approx. 2cm 2 Ninety bovine pericardium samples each were treated with GLU solution as disclosed above ("Tissue Treatment"). The GLU-treated pericardium samples were subjected to two washes in phosphate buffer for 15 minutes each. At the end of the incubation, 45 patches were treated with solution 5 of the invention according to the disclosed method and then subjected to five washes in phosphate buffer for 15-30 minutes each. These samples are called TREATED.

[0166] The remaining GLU-fixed pericardial specimen was used as a control (GLU).

[0167] Evaluation of anti-adhesion effect Polyphenol-treated and untreated patches were washed with PBS and incubated overnight at room temperature in PBS + antibiotics (300 μg / mL). Different types of antibiotics specific for each type of microbial strain were used (neomycin, penicillin, cephalosporin, polymyxin, rifamycin, lipiarmycin, quinolones, sulfonamides, macrolides, lincosamides, tetracyclines, aminoglycosides, doxycycline, minocycline, ampicillin, amoxicillin / clavulanic acid, azithromycin, carbapenems, piperacillin / tazobactam, quinolones, chloramphenicol, ticarcillin, trimethoprim / sulfamethoxazole). After overnight incubation, tissue patches were washed extensively with PBS to remove traces of unbound antibiotics. The treated and untreated samples were then incubated with various strains of microorganisms (microbial load 1 × 10) for 90 min at room temperature under moderate but constant agitation. 7 CFU / mL) were singly exposed to 100% microbial strains of bacteria. At the end of the incubation, tissue samples were subjected to three moderate vortexing passages to facilitate detachment of loosely bound bacteria. Finally, samples were homogenized in a Stomacher® 400 and serial dilutions of the resulting homogenates were plated onto Petri dishes containing the appropriate selective growth medium. After 24 h of incubation at 37°C, colony forming units were counted for each sample type.

[0168] Results are presented as the percentage reduction of adherent microorganisms assessed by comparing treated pericardial patches with untreated GLU-fixed pericardial patches (n=5 for each microbial strain).

[0169] The following table reports the percentage reduction of adherent microorganisms assessed on treated pericardial patches. Percentage values ​​were determined comparing treated and untreated GLU-fixed pericardial patches (n=5 for each microbial strain).

[0170] [Table 5]

[0171] Non-pyrogenicity of polyphenol-treated tissues The Monocyte Activation Test (MAT) was qualified and validated for pyrogen detection by the European Center for the Validation of Alternative Methods (ECVAM) in 2005 and by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) in 2008. ECVAM has been included in the European Pharmacopoeia's governing method for pyrogen detection since 2010 (Chapter 2.6.30) and is described in the FDA's "Guidance For Industry - Pyrogen and Endotoxins testing: Questions and Answers". The Monocyte Activation Test (MAT) is an alternative in vitro human test to the Rabbit Pyrogen Test (RPT) and is capable of detecting all pyrogens, including endotoxins and non-endotoxin pyrogens (NEPs).

[0172] Tissue processing Approx. 2cm 2Eighteen bovine pericardium samples, each of which was treated with GLU solution as disclosed above ("tissue treatment"). The GLU-treated pericardium samples were subjected to two washes in phosphate buffer for 15 min each. Nine patches were incubated with solution 5 in the dark for 25±10 min each in two steps. At the end of the incubation, the treated patches were subjected to five washes in phosphate buffer for 15-30 min each. The samples are called TREATED.

[0173] The remaining nine GLU-fixed pericardial specimens were employed as controls (GLU, n = 9).

[0174] Heat generation evaluation The samples were placed in 40 ml of endotoxin-free water at 37°C with moderate shaking for 1 hour. The water was analyzed with the MAT test. Briefly, the water was brought into contact with human monocyte cells, mimicking what happens in the human body, where in the presence of pyrogens, the monocytes become activated and produce several cytokines, including interleukin-6 (IL6). The cytokines are then detected using an immunological assay (ELISA) involving specific antibodies and an enzymatic color-developing reaction.

[0175] The following table reports the endotoxin unit assessment in treated and untreated (GLU) pericardial patches: Criteria for being considered pyrogenic: NMT 20 EU / device. [Table 6]

[0176] In view of the above disclosure, the advantages of the methods of the present invention will be apparent.

[0177] In particular, the method of the present invention has been shown not to modify other properties of surfaces that have been treated, and optionally pretreated, according to the disclosure above, and of medical devices, biological prostheses, in particular cardiac prostheses, that contain said surfaces.

[0178] As another advantage, the disclosed method has been shown to deactivate available aldehyde and carboxylic acid reactive groups on treated or pretreated surfaces.

[0179] Furthermore, the method of the present invention prevents dysfunction of the BHV, since platelet deposition on the fibrin network contributes to the development of subclinical leaflets thrombosis (SLT), which causes alteration of valve mobility.

[0180] It has been found that the protective action of the method of the invention contributes to a better distribution of mechanical loads, which avoids tears, abrasions and the formation of holes.

[0181] As an additional advantage, the methods of the present invention prevent microbial adhesion and biofilm formation on the treated and optionally pre-treated surfaces.

[0182] Again, the disclosed method avoids bacterial and viral contamination of surfaces that have been treated, and optionally pre-treated, according to the present invention.

Claims

1. An antimicrobial and antiviral method for treating a surface that comes into contact with biological fluids, comprising the step of bringing the surface into contact with a solution containing a phenolic compound or a mixture of phenolic compounds.

2. The method according to claim 1, wherein the surface is the surface of a medical device.

3. The method according to claim 1, wherein the surface is the surface of a biological prosthesis.

4. The method according to claim 1, wherein the surface is the surface of a cardiac prosthesis, a heart valve, a pericardial tissue patch, or a surgical heart valve.

5. The method according to claim 1, wherein the biological fluid is selected from the group comprising blood, serum, plasma, vitreous gel, tears, urine, saliva, feces; synovial fluid, peritoneal fluid, pericardial fluid, pleural fluid, and amniotic fluid.

6. The method according to claim 5, wherein the phenolic compound or mixture of the phenolic compounds is selected from the group comprising phenol, phenolaldehyde, phenolic acid, phenylamine, phenolic compound, flavonoid, phenylpropanoid, and tannin.

7. The method according to claim 6, wherein the phenolic compound or a mixture of the phenolic compounds is selected from the group comprising vanillin, cinnamic acid, phenylalanine, coumarin, xanthones, catechins, flavonones, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidins, anthocyanidins, and hydroxycinnamic acid.

8. The method according to claim 7, wherein the phenolic compound or a mixture of the phenolic compounds is selected from the group comprising resveratrol, aloin, cynarin, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperitin, quinic acid, elenolic acid, pinoresinol, luteolin, apigenin, tangeretin, isorhamnetin, kaempferol, myricetin, eriodictiol, hesperetin, naringenin, theaflavin, thearubidin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidine, peonidine, chicoriic acid, ferulic acid, and salicylic acid.

9. The method according to claim 1, further comprising the step of bringing the surface into contact with a solution of the phenolic compound or a mixture of the phenolic compounds for a period of less than two hours.

10. The method according to claim 1, comprising a first contact step and a second contact step, wherein a rinsing step is performed between the first contact step and the second contact step.

11. The method according to claim 1, comprising a pretreatment step with one or more compounds selected from the group comprising glutaraldehyde, formaldehyde, quercetin, or genipin.

12. The method according to claim 1, wherein the pretreatment step further comprises the removal of phospholipids.

13. The method according to claim 1, wherein the pretreatment step further includes a preparation step including the use of a capping agent.

14. A surface that comes into contact with biological fluids, obtained by the method described in claim 1.

15. A medical device or biological prosthesis comprising the surface described in claim 14.

16. A medical device according to claim 15, selected from the group including heart valves, tendons, ligaments, pericardium, fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose tissue and bone tissue, pelvic tissue, abdominal tissue, breast tissue and dermal tissue.

17. A medical device or biological prosthesis according to claim 15 for use in a method of treating a disease.

18. The medical device or biological prosthesis according to claim 17, wherein the disease is a heart disease.

19. The medical device or biological prosthesis according to claim 17, wherein the disease is a disease in humans or animals.

20. The medical device or biological prosthesis according to claim 18, wherein the disease is a disease in humans or animals.

21. A medical device or biological prosthesis according to any one of claims 17 to 20, wherein the method includes a valve-in-valve approach.

22. A method for sterilizing a surface that comes into contact with biological fluids, comprising the step of bringing the surface into contact with a phenolic compound or a mixture of phenolic compounds.

23. The sterilization method according to claim 22, wherein the surface is the surface of a medical device.

24. The sterilization method according to claim 22, wherein the surface is the surface of a biological prosthesis.

25. The sterilization method according to claim 22, wherein the surface is the surface of a cardiac prosthesis, a heart valve, a pericardial tissue patch, or a surgical heart valve.

26. The sterilization method according to claim 22, wherein the biological fluid is selected from the group comprising blood, serum, plasma, vitreous gel, tears, urine, saliva, feces; synovial fluid, peritoneal fluid, pericardial fluid, pleural fluid, and amniotic fluid.

27. The sterilization method according to claim 26, wherein the phenolic compound or mixture of the phenolic compounds is selected from phenol, phenolaldehyde, phenolic acid, phenylamine, phenolic compound, flavonoid, phenylpropanoid, and tannin.

28. The sterilization method according to claim 27, wherein the phenolic compound or a mixture of the phenolic compounds is selected from the group comprising vanillin, cinnamic acid, phenylalanine, coumarin, xanthones, catechins, flavonones, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidins, anthocyanidins, and hydroxycinnamic acid.

29. The sterilization method according to claim 28, wherein the phenolic compound or a mixture of the phenolic compounds is selected from the group comprising resveratrol, aloin, cynarin, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperitin, quinic acid, elenolic acid, pinoresinol, luteolin, apigenin, tangeretin, isorhamnetin, kaempferol, myricetin, eriodictiol, hesperetin, naringenin, theaflavin, thearubidin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidine, peonidine, chicoriic acid, ferulic acid, and salicylic acid.

30. The sterilization method according to claim 22, further comprising the step of bringing the surface into contact with a solution of the phenolic compound or a mixture of the phenolic compounds for a period of less than two hours.

31. The sterilization method according to claim 22, comprising a first contact step and a second contact step, wherein a rinsing step is performed between the first contact step and the second contact step.

32. The sterilization method according to claim 22, comprising a pretreatment step with one or more compounds selected from the group comprising glutaraldehyde, formaldehyde, quercetin, or genipin.

33. The sterilization method according to claim 22, wherein the pretreatment step further comprises the removal of phospholipids.

34. The sterilization method according to claim 22, wherein the pretreatment step further includes a preparation step including the use of a capping agent.

35. The sterilization method according to claim 22, wherein the bactericidal activity is effective against microorganisms that cause endocarditis.

36. The aforementioned microorganism, Gram + bacteria, Gram - The sterilization method according to claim 22, wherein the bacteria are bacteria, yeast, mold or virus.

37. The sterilization method according to claim 22, wherein the microorganism is mycobacteria.

38. The sterilization method according to claim 22, wherein the virus belongs to the family Picornaviridae, Adenoviridae, and Caliciviridae.

39. A surface obtained by the sterilization method described in claim 22.

40. A biological prosthesis, medical device, and cardiac prosthesis comprising the surface described in claim 39.

41. A biological prosthesis according to claim 40, represented by a cardiovascular prosthesis.

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