Product and method for the treatment of bioprosthetic tissues

Abstract

The invention concerns the treatment of bioprosthetic tissues a Cyclodextrin, preferably in association with Ethanol.

Claims

1. A method of treating bioprosthetic tissues used for cardiovascular prostheses, the method comprising the step of: removing phospholipids from the bioprosthetic tissues by using a Cyclodextrin that is able by itself to remove phospholipids from the bioprosthetic tissues wherein the Cyclodextrin is a β-Cyclodextrin.

2. The method according to claim 1, wherein the bioprosthetic tissue is further treated with Polyethylene Glycol for achieving a tissue dehydration and with Ethylene Oxide for a sterilization of the bioprosthetic tissues.

3. The method according to claim 1, wherein the Cyclodextrin includes an element selected from the group consisting of: 2-hydroxypropyl β-Cyclodextrin, Sulfobutyl Ether β-Cyclodextrin, and Maltosyl β-Cyclodextrin.

4. The method according to claim 1, wherein the bioprosthetic tissues are further treated with Ethanol for removing phospholipids from the bioprosthetic tissues.

5. The method according to claim 4, wherein the bioprosthetic tissue is treated with Ethanol and Cyclodextrin simultaneously.

6. The method according to claim 4, wherein the bioprosthetic tissue is treated with Ethanol followed by the treatment with Cyclodextrin.

7. The method according to claim 4, wherein, the bioprosthetic tissue is treated with Cyclodextrin followed by the treatment with Ethanol.

8. The method according to claim 1, further comprising a step of cross-linking the bioprosthetic tissue.

9. The method according to claim 8, wherein the step of cross-linking occurs after removing phospholipids from the bioprosthetic tissue by using Cyclodextrin.

10. The method according to claim 8, further comprising a second treating of the bioprosthetic tissue with the Cyclodextrin, said second treating with Cyclodextrin being performed after the step of cross-linking.

11. The method according to claim 8, wherein the step of cross-linking occurs before the step of removing phospholipids from the bioprosthetic tissue by using Cyclodextrin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows schematically different cyclodextrins.

(2) FIG. 2 shows schematically the structure of p-cyclodextrin.

(3) FIG. 3 shows different isomers of cyclodextrins.

(4) FIG. 4 shows different cyclodextrins and their characteristics.

(5) FIG. 5 shows a direct action of cyclodextrins on cells.

(6) FIG. 6 shows the extraction of cholesterol/phospholipids as a function of ethanol content.

(7) FIG. 7 shows a flow chart of a combined delipidation treatment.

(8) FIG. 8 shows a flowchart of a phospholipid extraction in two phases.

(9) FIG. 9 shows a flowchart of an alternative phospholipid extraction in two phases.

(10) FIG. 10 shows a flowchart of a method including a further detoxification process.

(11) FIG. 11 shows a flowchart of a method including a tissue dehydration procedure.

DESCRIPTION OF THE INVENTION

(12) The present invention generally relates to a novel and original use of Cyclodextrin in the treatment of bioprosthetic tissues.

(13) Using a Cyclodextrin in this treatment provides bioprosthetic tissues with a long-term mechanical and biological durability, after implant. Such properties are especially critical for heart valve bioprostheses.

(14) Cyclodextrins belong to a large family of molecules but for the present invention the most accredited are those of the β family. The functionalized β-Cyclodextrins and in particular the HP β-Cyclodextrin and the SBE β-Cyclodextrins have been approved for parenteral use. They therefore are expressing all desired chemical action without any damages for the excretory organs even if they are present in traces.

(15) Advantageously, the Cyclodextrin is used in combination with Ethanol.

(16) The tissue treatment based on Ethanol in aqueous concentration, higher than 50%, has demonstrated to be highly effective in extracting the phospholipids. The Ethanol effectiveness is closely related to the Glutaraldehyde cross-linking of bioprosthetic tissues. The mechanism is unclear but the Ethanol treatment appears to be more effective when applied after a Glutaraldehyde based cross-linking process.

(17) The Cyclodextrin action can be expressed as direct, with primary extraction of lipidic molecules, and indirect with complexation of the lipidic molecules already extracted. It is in this second action mode that Cyclodextrins can complex phospholipids already solubilized by Ethanol.

(18) In general the action of Cyclodextrins, when applied to biologic tissues, can be explained as a steric interaction or a weak covalent bound between its hydrophobic cavity and the lipidic molecules. In other terms it is about a weak covalent bound of Cyclodextrins and lipidic molecules without occurring any chemical reaction.

(19) The use of a specific derivative of Cyclodextrin for the treatment of biologic tissues is disclosed in patent application US2002/137024. This prior art teaches that sulfonated and sulfated_polyanions are able to block the calcium nucleation sites in biologic tissues used for prosthetic devices. The mechanism of action of these chemicals is not described but one can unambiguously deduct from the teaching of this document that the sulfonated and/or sulfated functional groups are responsible for the blocking of the calcium nucleation sites. As a matter of fact, the examples reported in US2002/137024 refer to completely different molecules, having in common the sulfonate/sulfate groups only. A few examples of the polyanions mentioned in US2002/137024 are sulfated Cyclodextrins being just one of them. It is possible to infer that the sulfonate/sulfate groups are able to block the calcium nucleation sites, presumably because of the affinity between calcium and sulfate anions. In other words, a sort of competitive action of sulfate/sulfonate anions against the phosphate groups of the phospholipids, which are clearly identified in US2002/137024 as calcium nucleation sites despite already well known in the prior art.

(20) In general, Cyclodextrins do not contain functional sulfonate or sulfate groups. It should be underlined that Cyclodextrins are usually neutral molecules and not ionic compounds. Sulfated Cyclodextrin is just one derivative of the large class of Cyclodextrins and the functionalization is aimed at obtaining a more soluble or tolerable molecules for i.v. injection. Therefore there is nothing in US2002/137024 suggesting the use of Cyclodextrin itself as blocking agents for calcium nucleation sites. In fact, US 2002/137024 is even teaching away the use of Cyclodextrin by itself as blocking agent for calcium nucleation sites.

(21) In the present invention, the selected Cyclodextrin is acting in a completely different way in respect to what described in US2002/137024. The Cyclodextrin by itself is removing phospholipids from the tissue thanks to its hydrophobic cavity, which would sequestrate the phospholipid fat chains.

(22) Ethanol and Cyclodextrin can be used simultaneously or separately, in any order.

(23) The bioprosthetic tissues (native valve cusps, bovine or porcine pericardial tissues) are selected for absence of defects and thickness. The selected patches or cusps are submitted to a cross-link process aimed at stabilizing the collagen in order to avoid any immunologic or foreign body tissue response. The cross-link process can be conducted with different molecules, but typically Glutaraldehyde at a concentration ranging between 0.1% to 1% for a period of 12 h to 48 h or more is used.

(24) Preferably, the combined delipidation treatment (FIG. 7) is performed combining Ethanol at a concentration between 35% and 80% solubilized in a buffered solution at PH 7.4 with 10 mM to 200 mM of β-Cyclodextrin for 2 h to 24 h at a temperature ranging between 25° C. and 40° C.

(25) After the delipidation treatment the patches are assembled in semi-finished or finished assemblies and then chemically sterilized with an aldehyde based solution eventually added with short chain alcohol molecules.

(26) Finished devices are then stored in a solution composed by aldehyde at concentration of 0.1% to 1% and eventually added with short chain alcohol molecules in concentration of 10% to 50%.

(27) In order to add a tissue detoxification process, aimed at removing aldehyde free molecules from the bioprosthesis before the implant, a pre-implantation rinsing procedure is performed.

(28) This pre-implantation rinsing is performed with three aliquots of 500 ml of a solution of p-Cyclodextrins at a concentration of 10 mM to 200 mM at a temperature of 15° C. to 30° C.

(29) In another embodiment the phospholipid extraction can be performed, after the tissue cross-link, in a disjoined manner in two phases (FIG. 8). First the Ethanol treatment is performed followed by the β-Cyclodextrin. Both treatments performed at the same concentrations and conditions described in FIG. 7.

(30) The phospholipid extraction, as described in FIG. 8, can be conducted in inverted way anticipating the exposition to β-Cyclodextrin followed by Ethanol treatment at the same concentrations and conditions.

(31) The combined treatment of Ethanol and β-Cyclodextrin can be performed anticipating the p-Cyclodextrin treatment directly on the bioprosthetic tissue before the cross-link procedure (FIG. 9) followed by a treatment with an Ethanol solution. The concentration of Ethanol and β-Cyclodextrin can be the same as described in FIG. 7. This disjoined treatment can be applied in inverted order if needed.

(32) The rationale for anticipating the β-Cyclodextrin treatment, before the cross-link procedure (FIG. 9), is based on the direct active mode of Cyclodextrins to directly delipidate the bioprosthetic tissues. This is the same active extraction capacity expressed by Cyclodextrins observed in experiments where these molecules were able to extract cholesterol and other lipids from the atherosclerotic plaques in arterial vessels (FDA approved a Cyclodextrins as orphan drug treatment in a rare pediatric disease where infants show abnormally high plasma concentration of cholesterol with atherosclerotic plaques at 2-3 years age).

(33) In another embodiment after the phospholipids extraction as described in the previous treatments with Ethanol and Cyclodextrins the process could include a further detoxification process, based on Cyclodextrins, aimed at removing, in effective way, the residual aldehyde molecules (FIG. 10). The aim of this chemical treatment variation is required in order to store the bioprosthesis in a bacteriostatic aldehyde-free storage solution. As previously described it is quite important the removal of aldehydes from the storage solution since it has been demonstrated that storing the bioprosthesis in Glutaraldehyde could partially override the positive anticalcification effect given by the Ethanol treatment. This is the reason why in the previous embodiments the storage media, described in FIGS. 7 to 9 where based on Glutaraldehyde solution added with a certain amount of short-chain alcohols.

(34) An important step forward in the bioprosthetic tissue treatment is represented by the tissue dehydration in association with a ethylene oxide sterilization. This is done in order to more easily store the bioprostheses avoiding chemical sterilization and their handling especially when they must be collapsed and used in transcatheter procedures.

(35) The previous treatment embodiments previously presented, as possible variations, can be associated to the tissue dehydration procedure. As for example in FIG. 11, after the delipidation, a detoxification process is performed with β-Cyclodextrin is completed with the aim to remove aldehyde molecules from the tissue. When the detoxification is completed a tissue dehydration procedure can be started. This treatment is based on a progressive removal of water from the bioprosthetic tissue obtained with Polyethylene Glycol (e.g. MW 100 to 800) in aqueous solution ranging from 80% to 90%. The treatment is performed at a temperature between 20° C. to 50° C. for 12 h to 48 h. In order to obtain a more effective dehydration short chain alcohols can be added at a concentration of 10% to 20%.

(36) The dehydration process is completed with a tissue drying for several hours in a clean environment. It allows a final storage of the bioprostheses in a dry packaging that is submitted to sterilization by means of Ethylene Oxide.

(37) All the treatment processes, above described, can be performed on semi-finished assemblies or directly on the final assembled bioprostheses. In this case the processes can be applied as an individual prosthetic treatment.

BIBLIOGRAPHY

(38) 1. Schoen F J, Levy R J. Calcification of tissue heart valve substitutes: progress toward understanding and prevention. Ann Thorac Surg 2005; 79:1072-80. 2. Konakci K Z, Bohle B, Blumer R, Hoetzenecker W, Roth G, Moser B, Boltz-Nitulescu G, Gorlitzer M, Klepetko W, Wolner E, Ankersmit H J. Alpha-GAL on bioprostheses: xenograft immune response in cardiac surgery. Eur J Clin Invest 2005; 35:17-23. 3. Manji R A, Zhu L F, Nijjar N K, Rayner D C, Korbutt G S, Churchill T A, Rajotte R V, Koshal A, Ross D B. Glutaraldehyde-fixed bioprosthetic heart valve conduits calcify and fail from xenograft rejection. Circulation 2006; 114:318-27. 4. Vyavahare N, Hirsch D, Lerner E, Baskin J Z, Schoen F J, Bianco R, Kruth H S, Zand R, Levy R J. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms. Circulation 1997; 95:479-88. 5. Jorge-Herrero E, Ferna'ndez P, Escudero C, Garci'a-Pa'ez J M, Castilo-Olivares J L. Calcification of pericardial tissue pretreated with different amino acids. Biomaterials 1996; 17:571-5. 6. Bina Gidwani, Amber Vyas. A comprehensive review on cyclodextrin-based carriers for celivery of chemotherapeutic cytotoxic anticancer drugs. BioMed Research International Volume 2015, Article ID 198268, 15 pages http://dx.doi.org/10.1155/2015/198268. 7. Ankitkumar S. Jain, Abhijit A. Date, Raghuvir R. S. Pissurlenkar, Evans C. Coutinho, Mangal S. Nagarsenker. Sulfobutyl Ether.sub.7 β-Cyclodextrin (SBE.sub.7 β-CD) Carbamazepine complex: preparation, characterization, molecular modeling, and evaluation of in vivo anti-epileptic activity. AAPS PharmSciTech, Vol. 12, No. 4, December 2011; 1163-75. 8. E. M. Martin Del Valle. Cyclodextrins and their uses: a review. Process Biochemistry 39 (2004) 1033-1046. 9. Caroline Coisne, Sebastien Tilloy, Eric Monflier, Daniel Wils, Laurence Fenart, Fabien Gosselet. Cyclodextrins as emerging therapeutic tools in the treatment of cholesterol-associated vascular and neurodegenerative diseases. Molecules 2016, 21, 1748. 10. Narendra R. Vyavahare, Danielle Hirsch, Eyal Lerner, Jonathan Z. Baskin, Robert Zand, Frederick J.

(39) Schoen, Robert J. Levy. Prevention of calcification of glutaraldehyde-crosslinked porcine aortic cusps by ethanol preincubation: Mechanistic studies of protein structure and water-biomaterial relationships. J Biomed Mater Res, 40, 577-585, 1998. 11. W. K. Ramp and D. N. Demaree, “Inhibition of net calcium efflux from bone by ethanol in vitro,” Am. J. Physiol., 246, C30-C36 (1984). 12. K. E. Friday and G. A. Howard, “Ethanol inhibits human bone cell proliferation and function in vitro,” Metabolism, 40, 562-565 (1991). 13. E. Rubin and R. Hagai, “Ethanol-induced injury and adaptation in biological membranes,” Fed. Proc., 41, 2465-2471 (1982). 14. M. S. Tung and T. J. O'Farrell. The effect of ethanol on the solubility of dicalcium phosphate dihydrate in the system Ca(OH)2-H3PO4-H2O at 37° C. J. Mol. Liq., 56, 237-243 (1993). 15. Narendra Vyavahare, Danielle Hirsch, Eyal Lerner, Jonathan Z. Baskin, Frederick J. Schoen, Richard Bianco, Howard S. Kruth, Robert Zand, Robert J. Levy. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Circulation. 1997; 95:479-488. 16. Anil Madhav Patwardhan, M.Ch., Pradeep Vaideeswar. Stress strain characteristics of glutaraldehyde treated porcine aortic valve tissue following ethanol treatment. IJTCVS 2004; 20: 67-71. 17. Vyavahare N R, Jones P L, Hirsch D, Levy R J. Prevention of glutaraldehyde-fixed bioprosthetic heart valve calcification by alcohol pretreatment: further mechanistic studies. J Heart Valve Dis. 2000 July; 9(4):561-6. 18. Ming Shen, Ali Kara-Mostefa, Lin Chen, Michel Daudon, Marc Thevenin, Bernard Lacour, and Alain Carpentier. Effect of ethanol and ether in the prevention of calcification of bioprostheses. Ann Thorac Surg 2001; 71:5413-6. 19. Connolly J M, Alferiev I, Kronsteiner A, Lu Z, Levy R J. Ethanol inhibition of porcine bioprosthetic heart valve cusp calcification is enhanced by reduction with sodium borohydride. J Heart Valve Dis. 2004 May; 13(3):487-93. 20. Hyoung Woo Changa, Soo Hwan Kimb, Kyung-Hwan Kima, Yong Jin Kima. Combined anti-calcification treatment of bovine pericardium with amino compounds and solvents. Interactive CardioVascular and Thoracic Surgery 12 (2011) 903-907. 21. Kwan-Chang Kim, Soo-Hwan Kim, Yong-Jin Kim. Detoxification of glutaraldehyde treated porcine pericardium using L-Arginine & NABH.sub.4. Korean J Thorac Cardiovasc Surg 2011; 44:99-107. 22. Evandro Antonio Sardeto, Francisco Diniz Affonso da Costa, Iseu do Santo Elias Affonso da COSTA, João Gabriel Roderjan, Eduardo Discher, Ricardo Alexandre Schneider, Carlos Henrique Gori Gomes, Claudinei Colattusso, Daniel Precoma, Andrea Dumsch, Sergio Veiga Lopes, Jairo Leal. Efficacy of AlCl.sub.3 and ethanol in the prevention of calcification of fragments of porcine aortic wall fixed in GDA.