Treatment of Aldehyde-Fixed Tissue Behandlung Von Aldehyd-Fixierten Geweben Traitement De Tissus Fixés Par L’Aldéhyde

Europäisches Patentamt *EP000796627B1* (19) European Patent Office

Office européen des brevets (11) EP 0 796 627 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: A01N 1/02, A61L 27/36 of the grant of the patent: 14.07.2004 Bulletin 2004/29

(21) Application number: 97301843.5

(22) Date of filing: 19.03.1997

(54) Treatment of aldehyde-fixed tissue Behandlung von Aldehyd-fixierten Geweben Traitement de tissus fixés par l’aldéhyde

(84) Designated Contracting States: • Zilla, Peter DE FR NL SE Campsbay 8001, Cape Town (ZA)

(30) Priority: 19.03.1996 US 619843 (74) Representative: Golding, Louise Ann Frank B. Dehn & Co., (43) Date of publication of application: 179 Queen Victoria Street 24.09.1997 Bulletin 1997/39 London EC4V 4EL (GB)

(73) Proprietors: (56) References cited: • MEDTRONIC, INC. US-A- 4 786 287 US-A- 5 460 797 Minneapolis, Minnesota 55432-3576 (US) • Zilla, Peter • JAYAKRISHNAN A ET AL: "Glutaraldehyde as a Campsbay 8001, Cape Town (ZA) fixative in bioprostheses and drug delivery matrices" BIOMATERIALS, vol. 17, no. 5, 1 March (72) Inventors: 1996 (1996-03-01), page 471-484 XP004032769 • Trescony, Paul V. ISSN: 0142-9612 Champlin, Minnesota 55316 (US)

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 796 627 B1

Printed by Jouve, 75001 PARIS (FR) EP 0 796 627 B1

Description

[0001] This invention relates to a method of treating aldehyde-treated, particularly aldehyde-fixed biological material, such as heart valves, in order to reduce residual aldehyde levels, and to a solution in which such material may be stored. 5 [0002] The implantation of biological materials, in particular those which are collagenous, is common in a number of medical applications. These applications include implantation of heart valves, pericardium, arteries, veins, skin, tendons, ligaments etc. The biological materials may be harvested from the same individual (autologous), from a different species (heterologous), or from the same species but from a different individual (homologous). [0003] Without any prior treatment, implanted collagenous biological material that is heterologous or homologous 10 may be regarded as a foreign antigen and trigger a host immune response that destroys the biological material. Treating the biological material with aldehydes such as formaldehyde, glutaraldehyde, glyoxal, or dialdehyde starch serves to: 1) enhance the mechanical durability and resistance to proteolytic attack by crosslinking collagen within the biological material; 2) greatly reduce an immune response to the implant by combining with and masking antigenic sites within the tissue; and 3) maintain sterility prior to implantation. Such aldehyde-treated material is termed "fixed". 15 [0004] While treatment with aldehydes prevents the triggering of an immune response and subsequent rejection of the biological material, residual aldehydes are known to be slowly released from aldehyde-treated biological materials and are known to be cytotoxic. The release of cytotoxic aldehyde related products can cause a local inflammatory response and can prevent complete healing of the implant. The customary method of briefly washing the aldehyde- fixed implant with sterile saline or water just prior to implantation cannot completely remove excess aldehyde within 20 the fixed tissue because of diffusion limitations and because a substantial amount of aldehyde is released from the implant by slow hydrolysis over a long period of time. This limitation is particularly emphasized in thick aldehyde-fixed biological materials, such as stentless heart valves, vascular grafts, ligaments, and the like. Other methods are therefore necessary to further eliminate residual aldehydes, thus "detoxifying" the fixed biological material. [0005] One method of removing aldehyde residues is by using an aminodicarboxylic acid such as glutamic acid or 25 aspartic acid as disclosed in US-A-4120649 (Schechter). In addition to aminodicarboxylic acid, primary and secondary amines may be used singly or in combination to effect aldehyde removal as disclosed in US-A-4786287 (Nashef et al). The preferred amine is a primary amine having a general formula R-NH2, where R can be an aliphatic or aromatic group or a combination thereof, which may be dissolved in a rinsing solution or immobilized on a solid support. The rinsing solution is buffered to a pH of 7.0 to 7.6. Because of the high pH of the rinsing solution, elevated temperatures 30 and continuous rinsing or many rinsings are needed to promote efficient diffusion of the aldehyde from the tissue. This method, however, does not satisfactorily remove all the aldehydes within the tissue. For example, residual aldehyde remains at a level that prevents a cell lining from growing on the surface of thick-walled tissue. [0006] A method disclosed in US-A-5188834 (Grimm et al) provides improvement over the aminodicarboxylic acid and amine treatment methods described above by using a dicarboxylic acid in an acidic medium with a pH of 2.5 to 35 5.5. At this lower pH, there is an enhanced degradation of tissue-bound polymeric aldehyde species to low molecular weight diffusable species. The low molecular weight species can more readily react with the dicarboxylic acids and be removed from the tissue by diffusion. The method described in US-A-5188834 requires, however, that the aldehyde detoxified tissue be stored in a separate non-aldehyde containing storage solution. The storage solution typically con- tains two cytostatic agents, methyl and propyl paraben. 40 [0007] Thus, there remains a need for an improved method for treating aldehyde-treated material, particularly thick tissue, in order to effectively remove residual traces of aldehyde from the material, as well as a need for a storage solution having improved bacteriocidal and fungicidal activity in which such material may be stored. [0008] A method for detoxifying aldehyde-treated biological tissue prior to implantation has now been found, said method comprising the step of treating said tissue with a non-peptidyl compound having at least one amide or imide 45 group in an aqueous treatment medium having a pH of less than 10. [0009] The non-peptidyl amide-functional and/or imide-functional compounds for use in the method of the invention are conveniently used in a low pH medium for the treatment of aldehyde-treated biotissue, which is generally of a collagenous material. The method is particularly advantageous for treating thick walled (greater than 1 mm) tissue. [0010] Viewed from one aspect the invention provides a method for preparing a biological tissue for implantation, 50 said method comprising the steps of:

(a) treating said tissue with an aldehyde; and (b) treating the resulting tissue with a non-peptidyl compound having at least one amide or imide group in an aqueous treatment medium having a pH of less than 10. 55 [0011] Steps (a) and (b) are carried out sequentially. Carried out sequentially, the tissue is generally fixed (i.e. crosslinked) by the aldehyde and the residual aldehyde is subsequently detoxified. [0012] A preferred group of non-peptidyl amide-functional and/or imide-functional compounds suitable for use in the

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method of the present invention includes those compounds which can be represented generally by the formulae R-C (O) -NH-R' (I) and R-C (O) -NH-C (O) -R' (II), wherein R and R' independently represent hydrogen atoms, amino groups, or alkyl or alkenyl groups (having 1 or more carbon-carbon double bonds), e.g. C1-6, preferably C1-3 alkyl or alkenyl groups, optionally interrupted by one or more N atoms, NH or NCH3 groups and/or substituted by one or more O atoms, 5 alkyl (e.g. methyl), amino, amido and/or hydroxyl groups. Such compounds preferably contain from 1 to 3 five or six membered rings. More preferably, R and R' together with -C(O) -NH- or -C(O) -NH-C(O)- form a ring or rings. Most preferably, the amide and/or imide groups are part of a ring, as opposed to being pendant therefrom. Also, it is preferred that the compounds be water soluble. [0013] Preferred compounds of formulae (I) and (II) are those in which R and R' independently represent hydrogen 10 atoms or alkyl or alkenyl groups optionally substituted with oxygen atoms, nitrogen atoms, carbonyl groups, amino groups, amido groups, and/or hydroxyl groups. [0014] Preferably the non-peptidyl compound for use in the method of the invention is cyclic and may comprise both amide and imide groups. [0015] The cyclic non-peptidyl compound is preferably a cyclic nitrogen metabolite or a synthetic heterocycle. A 15 preferred group of such compounds includes uridine, uracil, thymidine, thymine, 5,6-dihydroxyuracil, 5,6-dihydroxythymine, inosine, hypoxanthine, xanthine, xanthosine, uric acid, allantoin, guanine, guanosine, nicotinamide, orotic acid, urazole, glycoluril, hydantoin, 5,5-dimethyl hydantoin, pyrrolid-2-one, pyrazol-3-one, imidazol-2-one, allopurinol, barbituric acid and theobromine. [0016] Suitable such compounds may be grouped into two categories of compounds. Group I comprises cyclic ni- 20 trogen metabolites including, for example, uridine, uracil, thymidine, thymine, 5,6-dihydroxyuracil, 5,6-dihydroxythymine, inosine, hypoxanthine, xanthine, xanthosine, uric acid, allantoin, guanine, guanosine, nicotinamide, and orotic acid. Group II comprises synthetic heterocyclics including, for example, urazole, glycoluril, hydantoin, 5,5-dimethyl hydantoin, pyrrolid-2-one, pyrazol-3-one, imidazol-2-one, allopurinol, barbituric acid, and theobromine.

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[0017] A yet more preferred group of compounds suitable for use in the method of the present invention includes 50 those compounds with an imide group, preferably those with both an amide group and an imide group. Such compounds include uric acid, allantoin, urazole, uridine, thymidine, uracil, thymine, 5,6-dihydroxyuracil, 5,6-dihydroxythymine, orotic acid, xanthine, xanthosine, hydantoin, 5,5-dimethylhydantoin, and theobromine. [0018] Another preferred group of compounds includes those that are natural mammalian metabolites, e.g. purine or pyrimidine metabolites. This includes uridine, thymidine, uracil, thymine, 5,6-dihydroxyuracil, and 5,6-dihydroxythym- 55 ine, orotic acid, xanthine, xanthosine, uric acid, allantoin, and nicotinamide. A more preferred group are those compounds that are natural metabolites, particularly those having both an amide group and an imide group. A yet more particularly preferred group of compounds includes hypoxanthine, allantoin, barbituric acid, and urazole. [0019] A further compound that is useful as a detoxifying compound and is a natural mammalian metabolite, but is

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not cyclic, is urea. [0020] The present invention provides a method for detoxifying aldehyde-treated tissues, preferably aldehyde-fixed tissues. That is, the method of the present invention can be used to reduce residual aldehyde levels in bioprosthetic tissue (also referred to herein as biological tissue or biotissue) prior to surgical implantation of the tissue and is par- 5 ticularly effective for thick walled tissue, i.e. tissue greater than 1 mm thick. Furthermore, a solution containing a non- peptidyl amide/imide compound, optionally together with an aldehyde, can be used as an antimicrobial storage media. [0021] Viewed from a yet further aspect, the invention thus also provides the use of an aqueous solution comprising a non-peptidyl compound as hereinbefore defined, optionally together with an aldehyde, as an implant storage solution. [0022] The tissue to be treated in accordance with the method of the invention is first extracted from its source, e.g. 10 a donor such as an animal, and typically processed by one of many conventional procedures used to prepare biological tissue for implantation as bioprosthetic material. Treatment of extracted biological tissue with aldehyde compounds is well-known, and is described for example in US-A-4120649 (Schechtter), US-A-3988782 (Dardick et al), and US-A- 4553974 (Dewanjee). Glutaraldehyde is a preferred fixing agent, although other suitable aldehydes can be used including, for example, formaldehyde, glyoxal, and dialdehyde starch. The aldehyde is used in an amount and for a time 15 that is effective to cross-link collagen in the tissue to a degree sufficient to stabilize the tissue and to render it substan- tially non-antigenic. Such tissue is referred to as aldehyde-fixed tissue or simply "fixed" tissue. [0023] In the method of the invention, aldehyde-treated tissue is further treated with a non-peptidyl compound containing at least one amide (-C(O)NH-) group, at least one imide (-C(O)NH-C(O)-) group, or both, in an aqueous medium having a pH of no greater than 10. The method of the present invention is advantageous because it can effectively 20 detoxify aldehyde-fixed tissue in no greater than 7 days, and preferably in no greater than 4 days. [0024] The detoxification method of the present invention is preferably carried out in an aqueous medium having a pH of less than 7, and more preferably in an aqueous medium having a pH of 3-5. Preferably, for detoxification in no greater than 7 days, the method of the present invention is carried out in an aqueous medium at a temperature greater than 25°C, and more preferably at a temperature of 30-45°C, although temperatures as low as 4°C can also be used. 25 [0025] The extent of detoxification, i.e. removal of aldehyde residue from the tissue, such as a heart valve tissue, may be assessed in in vitro studies by endothelial cell seeding onto the tissue. Growth of an endothelial cell monolayer directly on top of the fixed and detoxified tissue provides a sensitive biological indicator that amides and/or imides have reduced the number of residual aldehydes in the tissue. Endothelial cell growth indicates that cytotoxic aldehydes are absent within the tissue. 30 [0026] Both the mixture of products resulting from aldehyde fixation of biological tissue and from the reaction of non- peptidyl amide and/or imide reagents with these aldehyde products are expected to be complex. It is expected that residual Schiff's bases and other hydrolytically unstable unsaturated compounds will remain in the tissue after this treatment. Thus, it may be beneficial to treat the detoxified tissue subsequently with a reducing agent, such as sodium borohydride, to convert unstable unsaturated compounds to stable saturated ones. This can generally be done to 35 aldehyde-treated tissue, such as glutaraldehyde-fixed aortic wall tissue, without significantly altering the favorable detoxification achieved with non-peptidyl amides and/or imides. [0027] An advantage of the present invention is that the amide-functional and/or imide-functional compounds, particularly those that are natural metabolites, have generally low toxicity, so that the biotissue can be implanted directly after the amide/imide treatment. After the amide/imide treatment, however, it is desirable to rinse the tissue with a 40 sterile saline solution prior to implantation. [0028] Furthermore, because products of glutaraldehyde and nitrogen-containing compounds, such as amides and imides, exhibit antimicrobial activity (see e.g. US-A-4454133 (Berke et al)), the biotissue can be stored in the amide/ imide treatment solution for long periods of time. [0029] The volume of the amide/imide treatment solution, the concentration of the amide/imide, and the number and 45 duration of rinses used in the method of the present invention can vary, depending on the type of tissue, the residual aldehyde concentration in the tissue, the temperature and pH of the treatment solution, etc. Generally, it is desirable to have a stoichiometric excess of the amide/imide groups over the releasable aldehyde groups. Preferably, a five-fold excess is used, and more preferably a 100-fold excess is used. Typically, effective amide/imide concentrations are 0.01 M to 0.1M. The amide/imide can be dissolved completely in the treatment solution, it can be dispersed therein 50 without complete dissolution, or it can also be attached to a solid support. [0030] Biological tissue that can be detoxified using the method of the invention includes any tissue that can be fixed with an aldehyde such as glutaraldehyde. This includes, for example, epithelial or fibrous connective tissue, such as pericardial tissue, dura mater, fascia lata, amnion, tendon, ligament, cartilage, arteries, veins, skin patches, bone, heart valves, reconstituted collagen, etc. The method is particularly well suited for thick and/or dense biological tissues, such 55 as heart valves, which are particularly difficult to detoxify. [0031] The invention has been described with reference to various specific and preferred embodiments and will be further described by reference to the following nonlimiting examples.

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Examples

Harvesting and Fixation of Porcine Aortic Wall Tissue

5 [0032] Aortas from large pigs were roughly dissected at the abattoir and transported to the laboratory in Hank's Balanced Salt Solution (HBSS) containing the following antibiotics: Amphotericine B, 25 µg/ml; Clindamycine Phos- phate, 120 µg/ml; Vancomycine, 50 µg/ml; and [0033] Polymyxine B, 130 µg/ml. After dissecting fatty and connective tissue from the aortas in the laboratory, 1.2 cm diameter discs were punched from the aortic wall and stored in fresh Hank's Balanced Salt Solution (HBSS) with 10 antibiotics at 4°C for up to 2 days. [0034] The aortic wall discs were fixed by placing in 0.2% glutaraldehyde (GA) in Phosphate Buffered Saline (PBS) at 4°C for 7 days with 10 ml of fixative per disc. The discs were then placed in PBS containing 0.2% GA and 1.0% isopropanol at 4°C for 32 hours with 10 ml of solution per disc. The discs were then placed in sterile PBS alone at 37°C for 24 hours with 10 ml of PBS per disc. 15 Preparation of Detoxifying Compounds

[0035] The following detoxifying compounds were prepared as described below at ambient temperature (25-30°C) unless otherwise noted and then sterilized by 0.2 µm filtration. 20 [0036] 0.1 M allantoin (ALAN) : 16.3 g of ALAN were added to 800 ml of 0.625 M acetic acid heated to 40°C and titrated to pH 4.5 with 1 N NaOH and made up to 1 litre total volume with water. [0037] 0.02 M hypoxanthine (HYPX) : 2.7 g of HYPX were added to 640 ml of 0.625 M acetic acid, heated to 40°C, titrated to pH 4.5 with 1N NaOH and made up to 1 litre total volume with water. [0038] 0.1 M urazole (URAZ): 10.1 g of URAZ were added to 800 ml of 0.625 M acetic acid, titrated to pH 4.5 with 25 1.0 N NaOH, and made up to 1 litre total volume with water. [0039] 0.1 M barbituric acid (BBA) : 12.8 g of BBA were added to 640 ml of 0.625 M acetic acid, titrated to pH 4.5 with 0.1 N NaOH, and made up to 1 litre total volume with water. [0040] 0.1 M histidine (HIST) : 15.5 g of HIST were added to 800 ml of 0.625 M acetic acid, titrated to pH 4.5 with 1.0 M NaOH, and made up to 1 litre total volume with water. 30 [0041] 0.005 M 1-lysine (LYS) : 0.73 g of LYS were added to 1000 ml of 0.625 M acetic acid. The solution pH was 5.75. [0042] 0.8% 1-glutamic acid (GLTA) : 8.0 g of GLTA were added to 1000 ml of distilled water and stirred at 37°Cto dissolve.

Detoxification of Glutaraldehyde Fixed Tissue Discs 35 [0043] Glutaraldehyde fixed aortic wall discs were incubated in a sterile aqueous solution of a detoxifying compound at 37°C for 7 days with 40 ml of solution per disc. The discs were then transferred to PBS and incubated at 4°C for 3 days with 10 ml of PBS per disc. The discs were then attached to TEFLON framing rings in a laminar flow hood and placed in HBSS with antibiotics as described above and incubated at 37°C for 2 days with 2 ml of solution per disc 40 prior to endothelial cell seeding.

Post-detoxification Treatment with Sodium Borohydride

[0044] Immediately after detoxifying glutaraldehyde fixed aortic wall discs, selected discs were placed under aseptic 45 conditions in sterile filtered 0.1 M NaBH4 in PBS and incubated at 37°C for 3 days with 40 ml of solution per disc. The discs were then attached to TEFLON framing rings in a laminar flow hood and placed in HBSS with antibiotics as described above and incubated at 37°C for 2 days with 2 ml of solution per disc prior to endothelial cell seeding.

Combined Glutaraldehyde - Amide/Imide Treatment(outside scope of claims) 50 [0045] Aortic wall discs were transferred under aseptic conditions from HBSS with antibiotic to 0.625 M acetic acid with 0.2% GA and 0.1 M detoxifying compound and incubated at 4°C for 7 days with 10 ml of solution per disc. The discs were then placed in PBS containing 0.2% GA and 1.0% isopropanol and incubated at 4°C for 32 hours and then stored in sterile PBS alone at 4°C for 1-2 weeks. 55 Endothelial Cell Seeding Experiments

[0046] Aortic wall discs (1.2 cm diameter) were attached to TEFLON framing rings and placed in 24 tissue culture

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plate wells along with supplemented M199 media. Second passage human veinous endothelial cells or primary porcine aortic endothelial cells were seeded onto the tissue samples. Replicate samples were collected at several time points and processed for SEM analysis. An initial qualitative visual assessment of cell coverage and morphology was made from SEM photographs. Subsequent experiments included determination of cell number from SEM photos (magnifi- 5 cation 1500x) where 15 areas on the seeded tissue surface were counted by 2-3 persons. The mean ±1SD was de- termined from 30-45 counted fields per sample. [0047] Table 1 presents the results of in vitro seeding studies using primary cultures of porcine aortic endothelial cells or second passage human endothelial cells seeded onto glutaraldehyde-fixed tissue with or without additional detoxification. When several of the fixed tissue post-fixation treated with cyclic amide and/or imide reagents including 10 allantoin, hypoxanthine, urazole, and barbituric acid for 7 days at 37°C followed by three days of incubation in PBS at 4°C, cell growth was maintained over 10 days at levels comparable with treatment with amino acids including L-glutamic acid. Table 1 also shows that subsequent reduction of the fixed and detoxified tissue with sodium borohydride may in some cases be beneficial.

15 Table 1 PORCINE AORTIC ENDOTHELIAL CELL SEEDING ONTO GLUTARALDEHYDE FIXED AORTIC WALL TISSUE ±DETOXIFICATION ±NaBH4 REDUCTION Experiment 1@ 20 Detox Reagent Cell Number x 1000 at Day 10 Cell Coverage at Day 10

No NaBH4 Red. + NaBH4 Red. No NaBH4 Red. + NaBH4 Red. 12.3±7.3 17.0±6.5 C- C- allantoin 10.5±4.5 17.5±6.8 >50% C- 25 urazole 20.4±6.5 21.5±7.1 PC C- barbituric acid 5.1±4.3 15.6±3.4 >50% PC 1-glutamic acid 15.0±7.3 28.3±6.9 PC PC to C- 30 1-histidine 9.9±6.5 19.8±6.2 >50% C- 1-lysine 14.5±7.0 14.6±5.2 C- C- combined GTA fix + 19.1±6.5* N.D. C N.D. urazole 35 No Detoxification spherical/ dead 7.8±6.0 spherical/ dead 50% No fixation 5.5±3.4* N.D. C* N.D.

* = day 7, day 10 sample not available 2 @ = Experiments without additional NaBH4 reduction were seeded with 2nd passage human venous endothelial cells at 125,000 cells/cm of sample 2 40 area; Experiments with additional NaBH4 reduction were seeded with primary culture porcine aortic endothelial cells at 97,000 cells/cm of sample area. C = Confluent cell layer PC = Preconfluent cell layer with isolated gaps between cells

[0048] Table 2 shows the results of three separate additional porcine aortic endothelial cell seeding experiments 45 comparing the detoxification effect of both amide/imide compounds with and without subsequent reduction with sodium borohydride. As with all cell seeding experiments, cell growth is a function of seeding density and the vigour of individual cell isolates. The results presented in Table 2 again show the ability of various amide/imide compounds to detoxify glutaraldehyde fixed tissue with or without additional reduction with sodium borohydride.

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Claims

1. A method for preparing a biological tissue for implantation, said method comprising the steps of:

5 (a) treating said tissue with an aldehyde; and (b) treating the resulting tissue with a non-peptidyl compound having at least one amide or imide group in an aqueous treatment medium having a pH of less than 10; wherein steps (a) and (b) are carried out sequentially.

2. A method as claimed in claim 1 wherein said non-peptidyl compound comprises a compound of formula (I) or (II) : 10

R-C(O)-NH-R' (I)

R-C(O)-NH-C(O)-R' (II) 15

(wherein R and R' independently represent hydrogen atoms, amino groups, or alkyl or alkenyl groups optionally interrupted by one or more N atoms, NH or NCH3 groups and/or substituted by one or more O atoms, alkyl, amino, amido and/or hydroxyl groups; and wherein R and R' may optionally link together to form one or more 5-or 6-mem- 20 bered heterocyclic rings).

3. A method as claimed in claim 1 or claim 2 wherein said non-peptidyl compound is cyclic.

4. A method as claimed in claim 3 wherein said non-peptidyl compound is a cyclic nitrogen metabolite or a synthetic 25 heterocycle.

5. A method as claimed in claim 4 wherein said non-peptidyl compound is uridine, uracil, thymidine, thymine, 5,6-dihydroxyuracil, 5,6-dihydroxythymine, inosine, hypoxanthine, xanthine, xanthosine, uric acid, allantoin, guanine, guanosine, nicotinamide, orotic acid, urazole, glycoluril, hydantoin, 5,5-dimethyl hydantoin, pyrrolid-2-one, pyrazol- 30 3-one, imidazol-2-one, allopurinol, barbituric acid or theobromine.

6. A method as claimed in any one of claims 1 to 3 wherein said non-peptidyl compound comprises both amide and imide groups.

35 7. A method as claimed in claim 6 wherein said non-peptidyl compound is uric acid, allantoin, urazole, uridine, thymidine, uracil, thymine, 5,6-dihydroxyuracil, 5,6-dihydroxythymine, orotic acid, xanthine, xanthosine, hydantoin, 5,5-dimethyl hydantoin or theobromine.

8. A method as claimed in any one of claims 1 to 3 wherein said non-peptidyl compound is a natural mammalian 40 metabolite.

9. A method as claimed in claim 8 wherein said non-peptidyl compound is uridine, thymidine, uracil, thymine, 5,6-dihydroxyuracil, 5,6-dihydroxythymine, orotic acid, xanthine, xanthosine, uric acid, allantoin or nicotinamide.

45 10. A method as claimed in claim 8 wherein said non-peptidyl compound comprises both amide and imide groups.

11. A method as claimed in claim 10 wherein said non-peptidyl compound is hypoxanthine, allantoin, barbituric acid or urazole.

50 12. A method as claimed in claim 1 or claim 2 wherein said non-peptidyl compound is urea.

13. A method as claimed in any preceding claim wherein the aqueous treatment medium has a pH of less than 7.

14. A method as claimed in any preceding claim carried out at a temperature of greater than 25°C. 55 15. A method as claimed in claim 14 carried out at a temperature of from 30 to 45°C.

16. A method as claimed in any preceding claim wherein said biological tissue is greater than lmm thick.

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17. A method as claimed in any preceding claim wherein said biological tissue is a heart valve.

18. A method as claimed in any preceding claim further comprising the step of treating the biological tissue with a reducing agent. 5 19. A method as claimed in claim 18 wherein said reducing agent is sodium borohydride.

20. Use of an aqueous solution comprising a non-peptidyl compound as defined in any one of claims 1 to 12, optionally together with an aldehyde, as an implant storage solution. 10

Patentansprüche

1. Verfahren zur Vorbehandlung eines biologischen Gewebes für die Implantation, mit den Schritten 15 (a) Behandlung des Gewebes mit einem Aldehyd; und (b) Behandlung des resultierenden Gewebes mit einer nicht-peptidischen Verbindung, die wenigstens eine Amid- oder Imidgruppe aufweist, in einem wässrigen Behandlungsmedium mit einem pH-Wert von weniger als 10; 20 wobei die Schritte (a) und (b) nacheinander ausgeführt werden.

2. Verfahren nach Anspruch 1, wobei die nicht-peptidische Verbindung eine Verbindung der Formel (I) oder (II):

25 R-C(O)-NH-R' I)

R-C(O)-NH-C(O)-R' (II)

30 umfasst (worin R und R' unabhängig voneinander für Wasserstoffatome, Aminogruppen oder Alkyl- und Alkenyl- gruppen, die gegebenenfalls durch eine oder mehrere N-Atome, NH- oder NCH3-Gruppen unterbrochen und/oder durch ein oder mehrere O-Atome, Alkyl-, Amino-, Amido- und/oder Hydroxylgruppen substituiert sind; und worin R und R' gegebenenfalls unter Bildung eines oder mehrerer 5- oder 6-gliedriger heterocyclischer Ringe verknüpft 35 sind).

3. Verfahren nach Anspruch 1 oder 2, wobei die nicht-peptidische Verbindung cyclisch ist.

4. Verfahren nach Anspruch 3, wobei es sich bei der nicht-peptidischen Verbindung um einen cyclischen Stickstoff- 40 metaboliten oder einen synthetischen Heterocyclus handelt.

5. Verfahren nach Anspruch 4, wobei es sich bei der nicht-peptidischen Verbindung um Uridin, Uracil, Thymidin, Thymin, 5,6-Dihydroxyuracil, 5,6-Dihydroxythymin, Inosin, Hypoxanthin, Xanthin, Xanthosin, Harnsäure, Allantoin, Guanin, Guanosin, Nicotinamid, Orotsäure, Urazol, Glycoluril, Hydantoin, 5,5-Dimethylhydantoin, Pyrrolid-2-on, 45 Pyrazol-3-on, Imidazol-2-on, Allopurinol, Barbitursäure oder Theobromin handelt.

6. Verfahren nach einem der Ansprüche 1 bis 3, wobei die nicht-peptidische Verbindung sowohl Amid- als auch Imidgruppen umfasst.

50 7. Verfahren nach Anspruch 6, wobei es sich bei der nicht-peptidischen Verbindung um Harnsäure, Allantoin, Urazol, Uridin, Thymidin, Uracil, Thymin, 5,6-Dihydroxyuracil, 5,6-Dihydroxythymin, Orotsäure, Xanthin, Xanthosin, Hy- dantoin, 5,5-Dimethylhydantoin oder Theobromin handelt.

8. Verfahren nach einem der Ansprüche 1 bis 3, wobei die nicht-peptidische Verbindung ein natürliches Stoffwech- 55 selprodukt eines Säugers ist.

9. Verfahren nach Anspruch 8, wobei es sich bei der nicht-peptidischen Verbindung um Uridin, Thymidin, Uracil, Thymin, 5,6-Dihydroxyuracil, 5,6-Dihydroxythymin, Orotsäure, Xanthin, Xanthosin, Harnsäure, Allantoin oder Ni-

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cotinamid handelt.

10. Verfahren nach Anspruch 8, wobei die nicht-peptidische Verbindung sowohl Amid- als auch Imidgruppen umfasst.

5 11. Verfahren nach Anspruch 10, wobei es sich bei der nicht-peptidischen Verbindung um Hypoxanthin, Allantoin, Barbitursäure oder Urazol handelt.

12. Verfahren nach Anspruch 1 oder 2, wobei es sich bei der nicht-peptidischen Verbindung um Harnstoff handelt.

10 13. Verfahren nach einem der vorhergehenden Ansprüche, wobei das wässrige Behandlungsmedium einen pH-Wert von weniger als 7 aufweist.

14. Verfahren nach einem der vorhergehenden Ansprüche, das bei einer Temperaturoberhalb 25 °C durchgeführt wird.

15 15. Verfahren nach Anspruch 14, das in einem Temperaturbereich von 30 bis 45 °C durchgeführt wird.

16. Verfahren nach einem der vorhergehenden Ansprüche, wobei das biologische Gewebe eine Dicke von mehr als 1 mm aufweist.

20 17. Verfahren nach einem der vorhergehenden Ansprüche, wobei das biologische Gewebe eine Herzklappe ist.

18. Verfahren nach einem der vorhergehenden Ansprüche, welches als weiteren Schritt die Behandlung des biologischen Gewebes mit einem Reduktionsmittel umfasst.

25 19. Verfahren nach Anspruch 18, wobei es sich bei dem Reduktionsmittel um Natriumborhydrid handelt.

20. Verwendung einer eine nicht-peptidische Verbindung umfassenden wässrigen Lösung wie in einem der Ansprüche 1 bis 12 definiert, gegebenenfalls zusammen mit einem Aldehyd, als Implantat-Aufbewahrungslösung.

30 Revendications

1. Procédé de préparation d'un tissu biologique pour implantation, ledit procédé comprenant les étapes de:

35 (a) traitement dudit tissu avec un aldéhyde ; et (b) traitement du tissu résultant avec un composé non peptidique possédant au moins un groupe amide ou imide dans un milieu de traitement aqueux possédant un pH de moins de 10 ;

dans lequel les étapes (a) et (b) sont effectuées de manière séquentielle. 40 2. Procédé tel que revendiqué dans la revendication 1 dans lequel ledit composé non peptidique comprend un com- posé de formule (I) ou (II):

45 R-C(O)-NH-R' (I)

R-C(O)-NH-C(O)-R' (II)

50 (dans lesquelles R et R' représentent indépendamment des atomes d'hydrogène, des groupes amino, ou des groupes alkyle ou alcényle éventuellement interrompus par un ou plusieurs atomes N, groupes NH ou NCH3 et/ ou substitués par un ou plusieurs atomes O, groupes alkyle, amino, amido et/ou hydroxy ; et dans lesquelles R et R' peuvent éventuellement se lier ensemble pour former un ou plusieurs noyaux hétérocycliques possédant 5 ou 6 chaînons). 55 3. Procédé tel que revendiqué dans la revendication 1 ou la revendication 2 dans lequel ledit composé non peptidique est cyclique.

13 EP 0 796 627 B1

4. Procédé tel que revendiqué dans la revendication 3 dans lequel ledit composé non peptidique est un métabolite azoté cyclique ou un hétérocycle synthétique.

5. Procédé tel que revendiqué dans la revendication 4 dans lequel ledit composé non peptidique est l'uridine, l'uracile, 5 la thymidine, la thymine, la 5,6-dihydroxyuracile, la 5,6-dihydroxythymine, l'inosine, l'hypoxanthine, la xanthine, la xanthosine, l'acide urique, l'allantoïne, la guanine, la guanosine, la nicotinamide, l'acide orotique, l'urazole, la gly- colurile, l'hydantoïne, la 5,5-diméthylhydantoïne, la pyrrolid-2-one, la pyrazol-3-one, l'imidazol-2-one, l'allopurinol, l'acide barbiturique ou la théobromine.

10 6. Procédé tel que revendiqué dans l'une quelconque des revendications 1 à 3 dans lequel ledit composé non peptidique comprend à la fois des groupes amide et imide.

7. Procédé tel que revendiqué dans la revendication 6 dans lequel ledit composé non peptidique est l'acide urique, l'allantoïne, l'urazole, l'uridine, la thymidine, l'uracile, la thymine, la 5,6-dihydroxyuracile, la 5,6-dihydroxythymine, 15 l'acide orotique, la xanthine, la xanthosine, l'hydantoïne, la 5,5-diméthylhydantoïne, ou la théobromine.

8. Procédé tel que revendiqué dans l'une quelconque des revendications 1 à 3 dans lequel ledit composé non peptidique est un métabolite mammifère naturel.

20 9. Procédé tel que revendiqué dans la revendication 8 dans lequel le composé non peptidique est l'uridine, la thymidine, l'uracile, la thymine, la 5,6-dihydroxyuracile, la 5,6-dihydroxythymine, l'acide orotique, la xanthine, la xanthosine, l'acide urique, l'allantoïne, ou la nicotinamide.

10. Procédé tel que revendiqué dans la revendication 8 dans lequel ledit composé non peptidique comprend à la fois 25 des groupes amide et imide.

11. Procédé tel que revendiqué dans la revendication 10 dans lequel ledit composé non peptidique est l'hypoxanthine, l'allantoïne, l'acide barbiturique ou l'urazole.

30 12. Procédé tel que revendiqué dans la revendication 1 ou la revendication 2 dans lequel ledit composé non peptidique est l'urée.

13. Procédé tel que revendiqué dans l'une quelconque des revendications précédentes dans lequel le milieu de traitement aqueux possède un pH de moins de 7. 35 14. Procédé tel que revendiqué dans l'une quelconque des revendications précédentes effectué à une température supérieure à 25°C.

15. Procédé tel que revendiqué dans la revendication 14 effectué à une température comprise entre 30 et 45°C. 40 16. Procédé tel que revendiqué dans l'une quelconque des revendications précédentes dans lequel ledit tissu biologique possède une épaisseur supérieure à 1 mm.

17. Procédé tel que revendiqué dans l'une quelconque des revendications précédentes dans lequel ledit tissu biolo- 45 gique est une valve de coeur.

18. Procédé tel que revendiqué dans l'une quelconque des revendications précédentes comprenant de plus l'étape de traitement du tissu biologique avec un agent réducteur.

50 19. Procédé tel que revendiqué dans la revendication 18 dans lequel ledit agent réducteur est le borohydrure de sodium.

20. Utilisation d'une solution aqueuse comprenant un composé non peptidique comme défini dans l'une quelconque des revendications 1 à 12, éventuellement ensemble avec un aldéhyde, comme solution de stockage d'implant. 55