(19) TZZ ¥_T

(11) EP 2 464 236 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A01N 37/36 (2006.01) A01N 61/00 (2006.01) 16.10.2019 Bulletin 2019/42 A01N 59/16 (2006.01) A01N 43/16 (2006.01) A01N 47/44 (2006.01) A01N 31/16 (2006.01) (2006.01) (2006.01) (21) Application number: 10808706.5 A01N 25/34 A61K 31/202 A61K 31/232 (2006.01) A61K 31/765 (2006.01) A61K 31/09 (2006.01) A61K 31/155 (2006.01) (22) Date of filing: 11.08.2010 A61K 31/35 (2006.01) A61K 31/28 (2006.01) A61L 29/16 (2006.01) A61L 31/16 (2006.01) A61L 15/44 (2006.01) A01P 1/00 (2006.01) A61K 33/38 (2006.01) A61K 45/06 (2006.01)

(86) International application number: PCT/US2010/045194

(87) International publication number: WO 2011/019834 (17.02.2011 Gazette 2011/07)

(54) ANTIMICROBIAL -CONTAINING BIOMATERIALS ANTIMIKROBIELLE SILBERHALTIGE BIOMATERIALIEN BIOMATÉRIAUX ANTIMICROBES CONTENANT DE L’ARGEANT

(84) Designated Contracting States: • MARTAKOS, Paul AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Pelham GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO NH 03076 (US) PL PT RO SE SI SK SM TR (74) Representative: Helbig, Christian et al (30) Priority: 11.08.2009 US 539282 Wagner + Helbig Patentanwälte (43) Date of publication of application: Pfarrstrasse 14 20.06.2012 Bulletin 2012/25 80538 München (DE)

(73) Proprietor: Atrium Medical Corporation (56) References cited: Hudson, NH 03051 (US) WO-A1-2009/091900 WO-A1-2010/042241 US-A1- 2006 110 457 US-A1- 2006 204 738 (72) Inventors: US-A1- 2006 263 330 US-A1- 2008 109 017 • FAUCHER, Keith, M. US-A1- 2009 181 937 Milford NH 03055 (US) • ZHENG ET AL: "Fatty synthesis is a target • KABIRU, Hilda for antibacterial activity of unsaturated fatty Nashua ", FEBS LETTERS, ELSEVIER, NH 03062 (US) AMSTERDAM, NL, vol. 579, no. 23, 26 September • HORTON, Anthony, Richard 2005 (2005-09-26), pages 5157-5162, Manchester XP005390460, ISSN: 0014-5793, DOI: NH 03104 (US) 10.1016/J.FEBSLET.2005.08.028 • PROWSE, Jocelyn Waltham MA 02453 (US)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 464 236 B1

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• JI-YOUNGLEE ET AL: "AntimicrobialSynergistic • MULTANEN M ET AL: "Bacterial adherence to Effect of Linolenic Acid and Monoglyceride silver coated poly-L-lactic acid urological against Bacillus cereus and Staphylococcus stents in vitro", UROLOGICAL RESEARCH, aureus", JOURNAL OF AGRICULTURAL AND SPRINGER VERLAG, BERLIN, DE, vol. 28, no. 5, FOOD CHEMISTRY, vol. 50, no. 7, 1 March 2002 1 October 2000 (2000-10-01), pages 327-331, (2002-03-01), pages 2193-2199, XP055072182, XP002451300, ISSN: 0300-5623, DOI: ISSN: 0021-8561, DOI: 10.1021/jf011175a 10.1007/S002400000133

2 1 EP 2 464 236 B1 2

Description formation of adhesions (Y.C. Cheong et al., 2001). Ab- dominal adhesions formed after hernia repair can result RELATED APPLICATION(S) in pain, bowel strangulation, infertility and in some cases death (Y.C. Cheong et al., 2001). [0001] This application claims priority to, and the ben- 5 [0005] U.S. Patent Application Publication No. US efit of, U.S. Application No. 12/539,282, filed on August 2009/0181937 A1 (Faucher et al.) discloses cross-linked 11, 2009. fatty acid-based biomaterials that may be used alone or in combination with a medical device for the release and BACKGROUND OF THE INVENTION local delivery of one or more therapeutic agents. How- 10 ever, this document does not pertain to an antimicrobial [0002] Implantable medical devices are indispensable silver hydrated fatty acid-derived biomaterial comprising in the ability to treat a variety of medical onditionsc in a fatty acid and a glyceride that is hydrated with an aque- critically and chronically ill patients. Catheters can be ous form of silver to form silver fatty acid salts within the used to deliver drugs or nutrients to a patient or to safely biomaterial. remove waste products. Stents can be used to open15 [0006] WO 2009/091900 A1 (John et al.) discloses a blocked coronary arteries and restore blood flow to the green approach in metal nanoparticle-embedded antimi- heart. Vascular grafts can also be used to restore blood crobial coatings from vegetable oils and oil-based mate- flow in addition to providing easier access and improved rials, which relates to methods of making metal nanopar- treatment of a patient with kidney failure by dialysis. Her- ticles. However, this document does not disclose the for- nia mesh devices allow for improved patient outcomes 20 mation of an antimicrobial silver hydrated fatty acid-de- in the treatment of abdominal wounds by providing ad- rived biomaterial comprising a fatty acid and a glyceride ditional strength to the surgical repair. that is hydrated with an aqueous form of silver to form [0003] Onecomplication in the use of implantable med- silver fatty acid salts within the biomaterial. ical devices is the risk of these devices becoming colo- [0007] WO 2010/042241 A1 (Faucher et al.) discloses nized with bacteria during surgical implantation (see,25 cross-linked fatty acid-based biomaterials such as per- e.g., A.M. Carbonell et al. Surg Endosc. 2005; Vol. 19, tain to fatty acid-based, pre-cure-derived biomaterials. pgs 430-435; T. Bechert et al. Nature Medicine. 2000; However, this document does not disclose the formation Vol. 6, No. 8, pgs 1053-1056; R. Kuijer et al. Biomaterials. of an antimicrobial silver hydrated fatty acid-derived bi- 2007; Vol. 28, pgs 5148-5154; C.R. Arciola et al. Bioma- omaterial comprising a fatty acid and a glyceride that is terials. 2008; Vol. 29, pgs 580-586). Once a patient30 hydrated with an aqueous form of silver to form silver shows signs of device infection, the surgeon is often re- fatty acid salts within the biomaterial. quired to perform several additional surgical procedures [0008] U.S. Patent Application Publication No. US to treat the device infection, such as drainage of the in- 2006/0263330 A1 (Emeta et al.) discloses antimicrobial fection site and the local administration of antibiotics. In polymer compositions and their uses. However, the dis- cases where the infection is not successfully resolved, 35 closed antimicrobial polymer compositions include a the surgeon will be required to remove the device from complex of an anionic polyester with an antimicrobial the implanted surgical site until the infection is resolved metal, wherein the anionic polyester has at least one car- with the treatment of oral and/or intravenous antibiotics boxylic acid group. These materials may be immersed in to the patient. Thus, an infected medical device not only a silver salt solution so as to form a complex of anionic results in increasing medical costs, but also results in 40 polyester and silver. However, these materials are sub- increased risk of morbidity and mortality to the patient stantially different from an antimicrobial silver hydrated (A.M. Carbonell et al., 2005; T. Bechert et al, 2000; R. fatty acid-derived biomaterial comprising a fatty acid and Kuijer et al., 2007). a glyceride. [0004] Another important aspect in the use of medical [0009] M. Multanen et al., Bacterial adherence to silver devices is the biological response to a medical device 45 nitrate coated poly-L-lactic acid urological stents in vitro, after surgical repair of an in-vivo injury (see,e.g., Y.C. 38 UROLOGY RESEARCH 327-31 (2000) pertains to a Cheong et al. Human Reproduction Update. 2001; Vol. studydirected to whether it is possible toprevent bacterial 7, No. 6, pgs 556-566). A typical biological response to adherence to bioabsorbable self-reinforced L-lactic acid the surgical site includes inflammation of native tissue polymer (SR-PLLA) urological stents by coating these followed by migration and proliferation of cells to mitigate 50 stents with blended epsilon-captrolac- the inflammatory response, including platelets and mac- tone/L-lactide copolymer. This document reports that the rophages, and a subsequent healing phase that includes studied silver nitrate coating prevented adherence of fibrin deposition and the formation of fibrin matrix fol- some bacteria to the SR-PLLA stents. However, the stud- lowed by tissue remodeling. In the case of hernia repair, ied materials are substantially different from an antimi- abnormal peritoneal healing can occur when there is the 55 crobial silver hydrated fatty acid-derived biomaterial expression of inflammatory cytokines from macrophages comprising a fatty acid and a glyceride. (e.g., α-TNF) that can result in an inability of the fibrin [0010] Accordingly, there remains a need for medical matrix to be properly broken down and can result in the devices that have a reduced susceptibility to colonization

3 3 EP 2 464 236 B1 4 by bacteria and other microorganisms, or other health and cresol. complications, such as adhesions. [0015] In another aspect disclosed herein, the antimi- crobial compound is an antibiotic compound. The antibi- SUMMARY OF THE INVENTION otic compound can be selected from the group consisting 5 of gentamicin , penicillin g, ephalothin, ampicillin, [0011] What is desired is a material ( e.g.,a device coat- amoxicillin, augmentin, aztreonam, imipenem, strepto- ing, gel or stand-alone film) that can be utilized to prevent mycin,gentamicin, vancomycin,clindamycin, erythromy- or diminish chronic inflammation due to the hydrolysis cin, azithromycin, polymyxin, bacitracin, amphotericin, products of the coating, as well as to reduce infection nystatin, rifampicin, tetracycline, doxycycline, chloram- resulting from surgical implantation of medical devices. 10 phenicol, nalidixic acid, ciprofloxacin, sulfanilamide, gan- Furthermore, it is desirable that the material release and trisin, trimethoprim, isoniazid, para-aminosalicylic acid, deliver therapeutic agents (e.g., an antimicrobial, e.g., and minocycline. an antibiotic agent) in a sustained and controlled fashion. [0016] In another aspect disclosed herein, of the bio- When in the form of a stand alone film, such a device material disclosed herein, the cross-linked fatty acid and can be useful in wound healing applications. 15 glyceride arederived from oil containing at leastone ome- [0012] Thus, disclosed herein is a composition com- ga-3 fatty acid, such as fish oil. prising an antimicrobial agent, a fatty acid, and a glycer- [0017] In another aspect disclosed herein, the antimi- ide, wherein the fatty acid and glyceride components are crobial-containing biomaterial contains an additional an- cross-linked. Such a composition can be referred to as timicrobial compound. The additional antimicrobial com- an "antimicrobial agent-containing biomaterial." When 20 pound can be selected from the group consisting of dia- the antimicrobial agent is a silver compound, the com- midines, and iodophors, peroxygens, , bi- position can be referred to as a "silver-containing bioma- sphenols, halophenols, and silver com- terial." This composition has both anti-inflammation and pounds. In another embodiment, the additional antimi- anti-infection properties, and can come in many forms, crobial compound is selected from the group consisting e.g., a coating for a medical device, a stand-alone film, 25 of elemental silver, silver nanoparticle, silver nitrate, sil- a gel, a particle, or an emulsion. When the composition ver , silver fluoride, silver , silver , is a coating, it can be placed on the surface of a medical , silver , , silver device in order to prevent bacterial colonization and in- tetrafluoroborate, silver , silver , silver lac- fection. The antimicrobial-containing biomaterial can al- tate, silver benzoate, silver cyclohexanebutyrate, silver so be used to prevent tissue adhesion, as well as to fa- 30 diethyldithiocarbamate, silver trifluoromethanesul- cilitate general wound healing, when, e.g., in the form of fonate, , , , hexachlo- a film. rophene, dibromopropamidine, , [0013] In one aspect, disclosed herein is a composition and cresol. comprising an antimicrobial compound, a fatty acid, and [0018] In still another aspect disclosed herein, the an- a glyceride, wherein the fatty acid and glyceride compo- 35 timicrobial-containing biomaterial contains an additional nents are cross-linked. In one aspect disclosed herein, antibiotic compound. In one embodiment, the antibiotic the antimicrobial compound is a silver compound, and compound is selected from the group consisting of gen- the silver compound is ionic silver or elemental silver. tamicin sulfate, penicillin g, ephalothin, ampicillin, amox- The silver can be elemental silver and is in the form of a icillin, augmentin, aztreonam, imipenem, streptomycin, silver nanoparticle. In another embodiment disclosed40 gentamicin, vancomycin, clindamycin, erythromycin, azi- herein, the silver is ionic silver and is selected from the thromycin, polymyxin, bacitracin, amphotericin, nystatin, group consisting of silver nitrate, , silver rifampicin, tetracycline, doxycycline, chloramphenicol, fluoride, , , silver sulfate, silver nalidixic acid, ciprofloxacin, sulfanilamide, gantrisin, tri- carbonate, silver cyanide, , silver methoprim, isoniazid, para-aminosalicylic acid, and mi- sulfide, , silver lactate, silver benzoate, sil- 45 nocycline. ver cyclohexanebutyrate, silver diethyldithiocarbamate, [0019] The antimicrobial-containing biomaterial com- silver trifluoromethanesulfonate and mixtures thereof. In position disclosed herein can be further combined with another aspect disclosed herein, the silver is ionic silver an oil containing an omega-3 fatty acid. Examples of such and is selected from the group consisting of silver nitrate, oils include, but are not limited to, fish oil, olive oil, grape silver acetate, silver oxide, and mixtures thereof. 50 oil, palm oil, or flaxseed oil. [0014] In another aspect disclosed herein, the antimi- [0020] In another aspect disclosed herein, when the crobial compound is selected from the group consisting biomaterial composition is associated with a tissue of a of diamidines, iodine and iodophors, peroxygens, phe- subject, the silver compound is released from the cross- nols, bisphenols, halophenols, biguanides and silver linked fatty acid at a controlled release rate. compounds. In still another aspect disclosed herein, the 55 [0021] In another aspect, disclosed herein is a film antimicrobial compound is selected from the group con- comprising an antimicrobial compound, a fatty acid, and sisting of triclosan, chlorhexidine, triclocarban, hexachlo- a glyceride, wherein the fatty acid and glyceride compo- rophene, dibromopropamidine, chloroxylenol, phenol nents are cross-linked.

4 5 EP 2 464 236 B1 6

[0022] The antimicrobial-containing biomaterial dis- taining oil are fish oil. In still another aspect disclosed closed herein can be associated with a medical device, herein, the antimicrobial compound is a silver compound, such as a bandage, a stent, a graft, a shunt, a catheter, and the silver compound is selected from the group con- a surgical mesh, or a balloon. sisting of a silver nanoparticle, silver nitrate, silver chlo- [0023] In another aspect, disclosed herein is a medical 5 ride, silver fluoride, silver bromide, silver oxide, silver sul- device at least partially coated with the antimicrobial-con- fate, , silver cyanide, silver tetrafluorob- taining biomaterial provided herein. In one embodiment, orate, , silver acetate, silver lactate, silver the medical device is a bandage, a stent, a graft, a shunt, benzoate, silver cyclohexanebutyrate, silver diethyldithi- a catheter, or a balloon. In another embodiment, the ocarbamate, silver trifluoromethanesulfonate and mix- mesh is a surgical mesh. 10 tures thereof. In another aspect of this method disclosed [0024] In another aspect, disclosed herein is a method herein, before curing the third material, the third material of forming a composition comprising an antimicrobial is associated with a medical device, such as a bandage, compound and a cross-linked fatty acid, comprising: a stent, a catheter, a surgical mesh, or a balloon. [0028] In another aspect, disclosed herein is a method curing a starting material comprising a fatty acid and 15 of forming a composition comprising an antimicrobial a glyceride according to a first curing condition to compound and a cross-linked fatty acid, comprising: form a second material; and associating the second material with an antimicrobial compound that is dis- associating a fatty acid- and glyceride-containing oil solved in a solution by immersing the second mate- with an antimicrobial compound to form a second rial in the antimicrobial compound solution or aero- 20 material; and solizing the antimicrobial compound solution onto curing the second material according to a first curing the second material; condition; such that the composition is formed. such that the antimicrobial-containing composition is formed. [0025] In one aspect of this method disclosed herein, 25 the fatty acid starting material is fish oil. In another aspect [0029] In one aspect of this method, the fatty acid-con- disclosed herein, the antimicrobial is a silver compound, taining oil is a fish oil. In still another embodiment, the and the silver compound is selected from the group con- fatty acid- and glyceride-containing oil is a mixture of fish sisting of a silver nanoparticle, silver nitrate, silver chlo- oil, and pre-cured fish oil. In another embodiment of this ride, silver fluoride, silver bromide, silver oxide, silver sul- 30 method, the antimicrobial compound is a silver com- fate, silver carbonate, silver cyanide, silver tetrafluorob- pound, and the silver compound is ionic silver in aqueous orate, silver sulfide, silver acetate, silver lactate, silver solution. In still another aspect disclosed herein, the an- benzoate, silver cyclohexanebutyrate, silver diethyldithi- timicrobial compound is a silver compound, and the silver ocarbamate, silver trifluoromethanesulfonate and mix- compound is in the form of a silver nanoparticle. tures thereof. In another disclosed aspect of the method, 35 [0030] In yet another aspect of this method disclosed before curing the fatty acid starting material, the starting herein, an additional antimicrobial compound is dis- material is associated with a medical device, such as a solved in solvent to form a third composition, and the bandage, a stent, a graft, a shunt, a catheter, or a balloon. third composition is coated onto the outer surface of the In another aspect disclosed herein, the composition is antimicrobial-containing composition. In one aspect dis- associated with a surgical mesh. 40 closed herein, the third composition is coated onto the [0026] In another aspect, disclosed herein is a method outer surface of the antimicrobial-containing composition of forming a composition comprising an antimicrobial by spraying the third composition or submerging the an- compound and a cross-linked fatty acid, comprising: timicrobial-containing composition in the third composi- tion. The additional compound can be an antimicrobial curing a starting material comprising a fatty acid and 45 compound, and the antimicrobial compound is selected a glyceride according to a first curing condition to from the group consisting of diamidines, iodine and io- form a second material; dophors, peroxygens, phenols, bisphenols, halophenols, associating the second material with a fatty acid-con- biguanides and silver compounds. In another aspect dis- taining oil and an antimicrobial compound that is dis- closed herein, the additional antimicrobial compound, solved in a solution to form a third material; and 50 and the antimicrobial compound is selected from the curing the third material according to a second curing group consisting of elemental silver, silver nanoparticle, condition; silver nitrate, silver chloride, silver fluoride, silver bro- such that the composition is formed. mide, silver oxide, silver sulfate, silver carbonate, silver cyanide, silver tetrafluoroborate, silver sulfide, silver ac- [0027] In one aspect of this method disclosed herein, 55 etate, silver lactate, silver benzoate, silver cyclohexaneb- a portion of the fatty acids of the second material are utyrate, silver diethyldithiocarbamate, silver trifluor- cross-linked. In still another embodiment disclosed here- omethanesulfonate, triclosan, chlorhexidine, triclocar- in, the fatty acid starting material and the fatty acid-con- ban, , dibromopropamidine, chloroxy-

5 7 EP 2 464 236 B1 8 lenol, phenol and cresol.In still another embodiment of ous form of silver is selected from the group consisting this method, the additional compound is an antibiotic of silver acetate and silver nitrate. compound, and wherein the antibiotic compound is se- [0040] In preferred embodiments the film is associated lected from the group consisting of gentamicin sulfate, with a medical device, and the medical device is a band- penicillin g, ephalothin, ampicillin, amoxicillin, augmen- 5 age, a stent, a graft, a shunt, a catheter, a surgical mesh, tin, aztreonam, imipenem, streptomycin, gentamicin, or a balloon. The medical device as described above can vancomycin, clindamycin, erythromycin, azithromycin, be at least partially coated with the composition as de- polymyxin, bacitracin, amphotericin, nystatin, rifampicin, scribed above, wherein the medical device is a bandage, tetracycline, doxycycline, chloramphenicol, nalidixic ac- a stent, a graft, a shunt, a catheter, a balloon, or a surgical id, ciprofloxacin, sulfanilamide, gantrisin, trimethoprim, 10 mesh. isoniazid, para-aminosalicylic acid, and minocycline. [0041] The invention is also directed to a method of [0031] In another aspect of this method disclosed here- forming a composition comprising an antimicrobial silver in, before curing the second material, the second material hydrated fatty acid-derived biomaterial, comprising: is associated with a medical device, such as a bandage, a stent, a catheter, a surgical mesh, or a balloon. 15 curing a starting material comprising a fatty acid and [0032] In another aspect, disclosed herein is a method a glyceride to form a solid bioabsorbable gel coating of treating or preventing tissue adhesion in a subject in containing thefatty acids and glycerides cross-linked need thereof, comprising administering the subject the to each other; and antimicrobial-containing biomaterial provided herein. associating the solid bioabsorbable gel coating with [0033] In another aspect, disclosed herein is a method 20 an antimicrobial compound that is dissolved in an of treating or preventing a bacterial infection in a subject aqueous solution by immersing the solid bioabsorb- in need thereof, comprising administering the subject the able gel coating in the antimicrobial compound so- antimicrobial-containing biomaterial provided herein. lution; such that the composition is formed, wherein [0034] In another aspect, disclosed herein is an emul- fatty acid starting material is fish oil, wherein the an- sion comprising an antimicrobial compound, a fatty acid, 25 timicrobial compound is a silver compound, and the and a glyceride, wherein the fatty acid and glyceride com- silver compound is selected from the group consist- ponents are cross-linked. ing of silver nitrate, silver chloride, silver fluoride, sil- [0035] In another aspect, disclosed herein is a compo- ver bromide, silver oxide, silver sulfate, silver car- sition comprising a silver compound, a fatty acid, and a bonate, silver cyanide, silver tetrafluoroborate, silver glyceride, wherein the fatty acid and glyceride compo- 30 sulfide, silver acetate, silver lactate, silver benzoate, nents are cross-linked. silver cyclohexanebutyrate, silver diethyldithiocar- [0036] The invention is directed to a composition com- bamate, silver trifluoromethanesulfonate and mix- prising an antimicrobial silver hydrated fatty acid-derived tures thereof. biomaterial comprising a fatty acid, and a glyceride, wherein the fatty acid and glyceride components are35 [0042] The composition of the invention can comprise cross-linked to each other to form a bioabsorbable gel fish oil cured to form a coating that includes the silver containing the cross-linked fatty acids and glycerides, fatty acid salt, wherein silver of the silver fatty acid wherein the biomaterial is hydrated with an aqueous form salt are characterized by an FTIR peak at about 1512 of silver to form silver fatty acid salts within the biomate- cm-1, and in said composition the coating can be formed rial, wherein the aqueous form of silver is selected from 40 on a medical device. the group consisting of silver acetate and silver nitrate. [0043] In preferred embodiments the invention is di- [0037] In preferred embodiments the composition is rected to the composition as described above, wherein derived from an oil containing an omega-3 fatty acid the cross-linked fatty acid and glyceride are derived from and/or wherein the oil is a fish oil, olive oil, grape oil, palm fish oil. oil, or flaxseed oil. 45 [0044] In preferred embodiments the invention is di- [0038] The inventionis also directed to the composition rected to the method as described above, wherein the as defined above for use in a method of treating or pre- composition is associated with a medical device, and the venting tissue adhesion in a subject in need thereof com- medical device is a bandage, a stent, a graft, a shunt, a prising administering to the subject the composition of catheter, a balloon, or a surgical mesh. claim. 50 [0045] The invention is also directed to a composition [0039] The invention is also directed to a film compris- as described above for use in a method of treating or ing an antimicrobial silver hydrated fatty acid-derived bi- preventing a bacterial infection in a subject in need there- omaterial comprising a fatty acid, and a glyceride, where- of, comprising administering to the subject the composi- in the fatty acid and glyceride components are cross- tion. linked to each other to form the film containing the cross- 55 [0046] In preferred embodiments the invention is di- linked fatty acids and glycerides, wherein the biomaterial rected to the composition as described above, wherein is hydrated with an aqueous form of silver to form silver the medical device is a bandage, a stent, a graft, a shunt, fatty acid salts within the biomaterial, wherein the aque- a catheter, a balloon, or a surgical mesh.

6 9 EP 2 464 236 B1 10

BRIEF DESCRIPTION OF THE DRAWINGS Figure 11 graphically depicts different zone sizes in mm in a zone of inhibition in-vitro study achieved [0047] The foregoing and other embodiments, objects, from fatty acid-derived biomaterial samples pre- features and advantages of the invention can be more pared with different concentrations of reduced silver fully understood from the following description in con- 5 (reference example); junction with the accompanying drawings. In the draw- Figure 12 depicts a bar graph of the log reduction ings, like reference characters generally refer to like fea- of colony forming units per cm 2 of a fatty acid-derived tures and structural elements throughout the various fig- biomaterial sample incorporated with reduced silver ures. The drawings are not necessarily to scale, empha- (reference example) together with a control fatty ac- sis instead being placed upon illustrating the principles 10 id-derived biomaterial sample in a three day in-vitro of the invention. S. aureus biofilm study; [0048] This includes various embodiments that are Figure 13 represents FTIR analysis of fatty acid-de- based on observed antimicrobial properties of medical rived biomaterial with reduced silver (reference ex- articles augmented with fatty acid antimicrobial compo- ample) and a fatty acid-derived biomaterial control sitions containing various pharmaceutical and anti-infec- 15 between the regions of 800-1800 cm-1 without the tive agents. The examples set forth in this specification presence of the silver fatty acid peak at approximate- are not intended to be limiting and merely set forth some ly 1512 cm -1, showing a variation of the samples an- of the many possible embodiments of the claimed inven- alyzed in Figures 9A and 9B; tion. Figure 14 depicts silver release data of the silver 20 nanoparticle (reference example) coated fatty acid- Figure 1 is a schematic of reactions that result in the derived biomaterial in aqueous media as described formation of ester bonds; in reference Example 3; Figure 2 shows bar graphs showing similarity of fatty Figure 15 illustrates the silver assay loading of dif- acid composition between fatty acid-derived bioma- ferent samples with different concentrations of silver terial coating and biological tissue; 25 nanoparticles in the fatty acid-derived biomaterial Figure 3 illustrates the silver release profile of the coating as discussed in reference Example 4; fatty acid-derived biomaterial coating hydrated in sil- Figure 16 depicts silver release data of the silver ver acetate over 4 hours; nanoparticle fatty acid film-derived biomaterial as Figure 4 illustrates the silver release profile of the described in reference Example 3; fatty acid-derived biomaterial coating hydrated in sil- 30 Figure 17 depicts silver release data of the silver ver nitrate over 4 hours; nanoparticle coated fatty acid-derived biomaterial Figure 5 depicts a bar graph of the total silver in with gentamicin added in-situ as described in refer- mg/cm2, assayed from Fatty Acid Biomaterial sam- ence Example 3; ples hydrated in aqueous silver nitrate and aqueous Figure 18 depicts gentamicin release data of the sil- silver acetate over a period of 6 hours; 35 ver nanoparticle coated fatty acid-derived biomate- Figures 6A-6B represent FTIR analysis of fatty acid- rial with gentamicin added in-situ as described in ref- derived biomaterial samples hydrated in silver nitrate erence Example 3; and silver acetate between the regions of 900-1850 Figure 19 provides a bar graph of zones measured cm-1 illustrating an increase in silver fatty acid salts in mm of Silver nanoparticle fatty acid-derived bio- with increased hydration time as indicated by the 40 material surface coated with gentamicin, a Silver na- peaks at 1512cm-1; noparticle fatty acid-derived biomaterial and a fatty Figures 7 is a bar graph of the recorded log reduction acid-derived biomaterial control in a three day zone of colony forming units per cm2 of silver nitrate hy- of inhibition study as described in reference Example drated fatty acid-derived biomaterial samples 5; against a control fatty acid-derived biomaterial in a 45 Figure 20 illustrates a bar graph of the colony form- three day in-vitro S. aureus biofilm study; ing units per mL enumerated by extracting samples Figure 8 illustrates the silver release profile of a fatty of a Silver nanoparticle fatty acid-derived biomaterial acid-derived biomaterial sample spray coated with surface coated with gentamicin, a Silver nanoparticle layer of silver; fatty acid-derived biomaterial and a fatty acid-de- Figure 9 depicts a bar graph of the log reduction of 50 rived biomaterial control in a bacterial adherence colony forming units per cm2 of a fatty acid-derived study as described in reference Example 5; biomaterial sample coated with a layer of silver to- Figure 21 depicts the drug release data of gen- gether with a control fatty acid-derived biomaterial tamicin in an aqueous media from gentamicin Silver sample in a three day in -vitro S. aureus biofilm study; nanoparticle fatty acid film-derived biomaterial sam- Figure 10 illustrates the silver release profile in55 ples with different gentamicin concentrations loaded mg/cm2 of a fatty acid-derived biomaterial sample onto the samples as described in reference Example incorporated with reduced silver (reference exam- 5; ple); Figure 22 depicts the gentamicin assay loading data

7 11 EP 2 464 236 B1 12

in mg/cm2 in gentamicin Silver nanoparticle fatty acid may occur during surgery, nosocomial infections are also film-derived biomaterial samples containing different associated with medical device infections which neces- gentamicin concentrations as described in reference sitate the use of antimicrobial compositions within or on Example 5; the surface of these medical devices in order to prevent Figure 23 shows a bar graph of zones measured in 5 bacterial colonization and infection. mm from a three day bacterial zone of inhibition study [0050] Provided herein is a biomaterial containing an of Silver nanoparticle fatty acid-derived reference bi- antimicrobial silver hydrated fatty acid-derived biomate- omaterial surface coated with chlorhexidine diace- rial comprising a fatty acid, and a glyceride, wherein the tate and a Silver nanoparticle fatty acid-derived ref- fatty acid and glyceride components are cross-linked to erence biomaterial control; 10 each other, wherein the biomaterial is hydrated with an Figure 24 is a bar graph of a bacterial adherence aqueous form of silver to form silver fatty acid salts within assay of silver nanoparticle fatty acid reference bio- the biomaterial. material surface coated with chlorhexidine[0051] The biomaterial can be further combined with diacetate , chlorhexidine-silver fatty acid biomaterial other antimicrobial agents such as, but not limited to, surface coated with chlorhexidine diacetate together 15 triclosan, chlorhexidine, gentamicin, gentamicin sulfate, with fatty acid biomaterial and silver nanoparticle fat- and other antibiotics. One advantage of the present an- ty acid biomaterial controls; timicrobial-containing biomaterial is the anti-inflammato- Figure 25 shows triclosan release data from a Silver ry properties derived from the fatty acids, while providing nanoparticle fatty acid-derived reference biomaterial a suitable vehicle for sustained controlled release of the sample surface coated with Triclosan; 20 antimicrobial (silver ions and optionally: triclosan, chlo- Figure 26 illustrates the zones measured from a Sil- rhexidine, gentamicin, gentamicin sulfate, etc.). The an- ver nanoparticle fatty acid-derived biomaterial sam- timicrobial-containing biomaterial furthermore provides ple surface coated with triclosan over approximately a favorable surface for adding topical layers of additional three weeks; antimicrobial agents that provide different sustained re- Figure 27a is a S. aureus bacterial suspension as- 25 lease and efficacy properties. say plot of Silver nanoparticle fatty acid-derived ref- [0052] When associated with a medical device, the an- erence biomaterial sample surface coated with Tri- timicrobial-containing biomaterialprovided herein can re- closan; duce the incidence of inflammatory and foreign body re- Figure 27b is a S. aureus bacterial adherence assay sponses after implantation of that medical device. The plot of Silver nanoparticle fatty acid-derived refer- 30 antimicrobial-containing biomaterial can also provide the ence biomaterial sample surface coated with Tri- release of one or more therapeutic agents (silver-based closan; agents, triclosan, chlorhexidine, gentamicin, gentamicin Figure 28 portrays a S. aureus zone of inhibition plot sulfate, etc.) over a period of time in order to reduce the showing the zone sizes measured from a Triclosan- ability of bacteria to colonize during implantation and to Silver nanoparticle fatty acid-derived reference bio- 35 prevent a device-related infection. The antimicrobial- material triclosan surface coated sample over two containing biomaterial can also be used to prevent tissue weeks; adhesion, as well as to facilitate general wound healing. Figure 29 is a S. aureus bacterial adherence plot of When in the form of a stand-alone film, or when associ- Silver nanoparticle fatty acid-derived reference bio- ated with a bandage, the antimicrobial-containing bioma- material sample surface coated with Triclosan; 40 terial can be used in wound healing applications. The Figure 30 portrays a S. aureus zone of inhibition plot antimicrobial-containing biomaterial can also be metab- showing the zone sizes measured from a Triclosan- olized via a bioabsorption mechanism. Silver nanoparticle fatty acid-derived reference bio- [0053] The antimicrobial-containing biomaterial can be material sample over two and a half weeks; and utilized alone or optionally in combination with a medical Figure 31 illustrates the efficacy of different refer- 45 device for the release and local delivery of one or more ence antimicrobial formulations of fatty acid-based antimicrobial agents to prevent colonization of bacteria biomaterials containing different doses of triclosan onto the device during surgical implantation. Methods of with and without silver nanoparticles as evaluated in forming and tailoring the properties of said biomaterials an in-vivo infectability model with a clinical strain of are also provided. Additionally, due to the unique prop- MRSA. 50 erties of the underlying chemistry of the biomaterial, the biomaterial (e.g., coating or stand-alone film) contains DETAILED DESCRIPTION specific chemical components that assist in reducing a foreign body response and inflammation at the site of [0049] Implantable medical devices are designed to tissue injury during implantation that improves its in-vivo improve the quality of life of patients with varying medical 55 performance. conditions. These devices, however, are highly suscep- tible to infection and can affect patient mortality and mor- bidity leading to high medical costs. While such infection

8 13 EP 2 464 236 B1 14

Antimicrobial-Containing Fatty Acid-Derived Biomateri- rent with the formation of hydroperoxides is the isomer- als ization of (C=C) double bonds from cis to trans in addition to double bond conjugation. Continued heating of the oil [0054] In various aspects, the antimicrobial-containing results in the solidifying of the coating through the forma- biomaterial can be, but is not limited to, a coating for a 5 tion of cross-linking and by the further reaction of the medical device, a stand-alone film, or a gel. The bioma- hydroperoxides and the cleavage of C=C double bonds, terial can be a hydrophobic biomaterial comprising cross- which convert them into secondary oxidation byproducts linked fatty acids and glycerides. In certain instances, as including , ketones, , fatty acids, es- described herein, the source of the fatty acid and glyc- ters, lactones,ethers, and hydrocarbons whichcan either eride is an oil, e.g., a fish oil. The oil can also be olive oil, 10 remain within the coating and/or are volatilized during grape oil, palm oil, or flaxseed oil. In such an instance, the process. the antimicrobial-containing biomaterial can also be re- [0059] The type and amount of cross-links formed dur- ferred to as an "oil-derived antimicrobial-containing bio- ing oil oxidation can be tailored depending on the condi- material" (e.g., an "oil-derived silver-containing biomate- tions selected ( e.g., coating thickness, temperature, met- rial"). 15 al composition, etc.). For instance, heating of the oil al- [0055] Various methods can be used to prepare the lows for cross-linking between the fish oil unsaturated antimicrobial-containing biomaterial described herein. chains using a combination of peroxide (C-O-O-C), ether The fatty-acid and glyceride (e.g., oil, e.g., fish oil) can (CO-C), and hydrocarbon (C-C) bridges (see, e.g., F.D. be cured (using heat, UV, etc.) to induce fatty-acid oxi- Gunstone, "Fatty Acid and Lipid Chemistry." 1999.). dation and crosslinking of a portion, majority, or all of the 20 Heating at lower temperatures (below 150°C) results in fatty acids and glycerides to form a gel. The aqueous the formation of predominantly peroxide cross-links, form of silver is added to the fatty acid after the curing where heating at higher temperatures ( i.e. above 150°C) process. results in the thermal degradation of peroxides and C=C [0056] Several methods are available to cure the oil and ether cross-links dominate (F.D. Gunstone, 1999). starting material containing one or more therapeutic25 [0060] In addition to thermal curing processes, oxida- agents to produce a fatty acid-derived biomaterial for a tion of oils can also be induced by light ( e.g., photo-oxy- drug release and delivery coating or stand-alone film in genation). Photo-oxygenation is limited to C=C carbon accordance with the present invention (for example, as atoms and results in a conversion from cis to trans C=C described in US Patent Application Publicationsisomers during curing (as occurs with heat initiated cur- 2008/0118550, 2007/0202149, 2007/0071798,30 ing). However, photo-oxygenation using UV is a relatively 2006/0110457, 2006/0078586, 2006/0067983,quicker reaction than autoxidation from heat curing, in 2006/0067976, 2006/0067975,). the realm of about 1000-1500 times faster. The quicker [0057] Preferred methods for curing the starting mate- reaction especially holds true for methylene interrupted rial to produce an antimicrobial agent-containing, fatty polyunsaturated fatty acids, such as EPA and DHA, acid-derived biomaterial of the present invention include, 35 which are found in the fish oil based embodiments of the but are not limited to, heating (e.g., employing an oven, present invention. a broadband infrared (IR) light source, a coherent IR light [0061] An important aspect of UV curing when com- source (e.g., laser), and combinations thereof) and ultra- pared to heat curing is that although the byproducts ob- violet (UV) irradiation. The starting material may be tained by both curing methods are similar, they are not cross-linked through auto-oxidation ( i.e., oxidative cross- 40 necessarily identical in amount or chemical structure. linking). One reason for this is due to the ability of photo-oxygen- [0058] In accordance with various embodiments de- ation to create hydroperoxides at more possible C=C scribedherein, thedrug releasecoatings and stand alone sites. films of the present invention are formed of a fatty acid- [0062] Photo-oxygenation, such as that which results derived biomaterial, which can be derived from saturated 45 from UV curing, due to its enhanced ability to create inner and unsaturated fatty acid compounds e.g.,( free fatty hydroperoxides, also results in the ability to form rela- acids, fatty acid ester, monoglycerides, diglycerides, trig- tively greater amounts of cyclic byproducts, which also lycerides, metal salts, etc.). Preferably, the source of fatty relates to peroxide cross-linking between fish oil hydro- acids described herein is saturated and unsaturated fatty carbon chains. For example, photo-oxygenation of lino- acids such as those readily available in triglyceride form 50 lenate results in 6 different types of hydroperoxides to be in various oils (e.g., fish oils). One method of the forma- formed, whereas autoxidation results in only 4. The great- tion of a fatty acid-derived biomaterial is accomplished er amount of hydroperoxides created using photo-oxy- through autoxidation of the oil. As a liquid oil containing genation results in a similar, but slightly different, struc- unsaturated fatty acid is heated, autoxidation occurs with ture and amount of secondary byproducts to be formed the absorption of oxygen into the oil to create hydroper- 55 relative to autoxidation from heat curing. Specifically, in an amount dependent upon the amount of un- these byproducts are aldehydes, ketones, alcohols, fatty saturated (C=C) sites in the oil. However, the (C=C) acids, esters, lactones, ethers, and hydrocarbons. bonds are not consumed in this initial reaction. Concur- [0063] Depending on the oil curing conditions and the

9 15 EP 2 464 236 B1 16 fatty acid composition of the starting oil, a fatty acid-de- compound and the biomaterial itself. The antimicrobial- rived biomaterial can be produced by curing the oil so as containing biomaterials described herein are created by to oxidize the double bonds of the unsaturated fatty acid thermal oxidation of the unsaturated double bonds in an chains while predominantly preserving triglyceride ester oil, initially resulting in the formation of hydroperoxides, functional groups. The oxidation of the unsaturated fatty 5 which are then subsequently further oxidized into other acid chains results in the formation of hydroperoxides, byproducts including fatty acids. Carboxyl and hydroxyl which, with continued curing, are converted into and al- groups formed during the oxidation of the oil i.e.,( fatty dehydes, ketones, alcohols, fatty acids, esters, lactones, acids and glycerides) can react together upon further ethers, and hydrocarbons. With continued heating of the heating by esterification, which results in the formation oxidized oil, the byproducts are volatilized, resulting in 10 of lactone and ester cross-links to gel the coating. Thus, an increase in the coating in addition to the for- different antimicrobial silver containing coatings can mation of ester cross-links. The formation of ester and be produceddue to thefinal resultant chemistry produced lactone cross-links can occur via different types of mech- by the interaction of the antimicrobial compound with the anisms (i.e., esterification, alcoholysis, acidolysis, inter- different molecular species present in the oil-derived esterification as described in F.D. Gunstone,1999, 15 coating. Additionally, unlike traditional hydrophobic med- Chapter 8, incoprated herein by reference) between the ical device materials such as PTFE, the antimicrobial- hydroxyl and carboxyl functional components in the coat- containing biomaterial becomes readily hydrated in an ing formed from the oxidation process ( i.e., glyceride and aqueous environment, which results in the release of an- fatty acid). The cross-linking reaction can form different timicrobial compounds comprising silver ions to be ob- types of ester linkages such as ester, anhydride, aliphatic 20 served. peroxide, and lactones. One possible mechanism for the [0067] As discussed above, the fatty-acid and glycer- formation of the fish-oil derived biomaterial used herein ide (e.g., oil, e.g., fish oil) is cured to induce fatty-acid can be provided as follows: fish oil (triglycerides) absorbs oxidation and crosslinking of a portion, majority, or all of oxygen; oxidation of the C=C bonds in the oil occurs; the fatty acids and glycerides to form a gel. The antimi- fatty acid and glycerides (a mixture of mono, di- and tri) 25 crobial agent is added to the fatty acid after the curing are formed; continued curing results in dehydration of process. neighboringcarboxyl and hydroxyl functional groups;lac- [0068] In the embodiment, a method to load antimicro- tone/ester cross-links are formed between fatty acids and bial compounds into a silver-containing biomaterial is to glycerides; and the oil solidifies into a bioabsorbable gel first create the coating using a thermal cure process and containing cross-linked fatty acids and glycerides. The 30 then soaking the coating in a water soluble form of silver reaction chemistry of this formation is as follows: isomer- selected from silver nitrate or silver acetate. This process ization and oxidation of C=C bonds; volatilization of wa- creates silver fatty acid salts within the silver-containing ter,hydrocarbons andaldehydes, resulting inan increase biomaterial. Additionally, the amount of silver fatty acid in coating viscosity; and ester and lactone cross-links salts formed within the biomaterial can be altered as a formed results in solidifying the coating into a gel. 35 function of pH, silver compound selected and soak time. [0064] Figure 1 provides a schematic of different meth- Using this process, the amount of silver ions produced ods to form esters from oils for illustrative purposes, but within the biomaterial are released more rapidly, typically is not meant to be limiting in its scope to the invention. in less than a week as measured using in-vitro dissolution [0065] In various aspects, provided herein is a means testing methodology. Accordingly, this process can be to load aqueous form of silver into fatty acid-derived bi- 40 used with any other water soluble antimicrobial agent. omaterials. In one embodiment, these silver-containing [0069] It will be appreciated that the present invention biomaterials can be combined with a medical device in can be implemented as a coating, as well as a stand- order to enhance the device’s short term antimicrobial alone material, or other forms as described herein ( e.g., activity. For example, the release of silver ions from the a gel). As would be understood by those of ordinary skill oil silver-containing biomaterials (e.g., coating or stand- 45 in the art these biomaterials will have an outer surface alone film) can be altered depending on the silver com- that interacts with its environment upon implantation or pound selected ( e.g., whether it is an insoluble or soluble application. form of silver) in addition to the method of loading silver [0070] Antimicrobial-containing biomaterial coatings into the coating and/or formulation e.g.,( sprayed onto and stand-alone films of the present invention are formed cured coating, or soaked into cured coating). Depending 50 from an oil component. The term "oil component" is also on the method of incorporation of the silver compounds referred to herein as the "oil acid-containing starting ma- into the biomaterial, the silver ions release in water can terial" or "fatty acid-containing starting material." The "fat- bealtered, which can alterthe efficacy of the antimicrobial ty acid-containing starting material" can be natural or de- -containing biomaterial. rived from synthetic sources. Preferably, the "oil contain- [0066] In one embodiment, the antimicrobial behavior 55 ing starting material" comprises unsaturated fatty acids. of the compounds incorporated into the antimicrobial- The oil component can be either an oil, or an oil compo- containing biomaterials e.g., ( coatings or stand-alone sition. The oil component can be a naturally occurring films) can be altered by the interaction between the silver oil, such as fish oil, flax seed oil, grape seed oil, or palm

10 17 EP 2 464 236 B1 18 oil, or other oils having desired characteristics. The oil acid. There can be both monounsaturated and polyun- can also be a synthetic oil. One embodiment of the saturated omega fatty acids. present invention makes use of a fish oil in part because [0075] Omega-3 and omega-6 fatty acids are also of the high content of omega-3 fatty acids, which can known as essential fatty acids because they are impor- provide healing support for damaged tissue, as dis-5 tant for maintaining good health, despite the fact that the cussed herein. The fish oil can also serve as an anti- human body cannot make them on its own. As such, ome- adhesion agent. In addition, the fish oil maintains anti- ga-3 and omega-6 fatty acids must be obtained from ex- inflammatory or non-inflammatoryproperties as well. The ternal sources, such as food. Omega-3 fatty acids can present invention is not limited to formation of the fatty be further characterized as containing eicosapentaenoic acid-derived biomaterial with fish oil as the oil. However, 10 acid (EPA), docosahexanoic acid (DHA), and alpha-lino- the present application makes reference to the use of lenic acid (ALA). Both EPA and DHA are known to have fish oil as one example embodiment. Other naturally oc- anti-inflammatory effects and wound healing effects with- curring oils or synthetic oils can be utilized in accordance in the human body. with the present invention as described herein. [0071] It should be noted that as utilized herein, the 15 Antimcrobial Agents term "fish oil" includes but is not limited to omega-3 fatty acid, unsaturated fatty acid, polyunsaturated fatty acid, [0076] As discussed above, provided herein are com- fish oil fatty acid, free fatty acid, monoglycerides, di-glyc- positions comprising an antimicrobial silver hydrated fat- erides, or triglycerides, esters of fatty acids, or a combi- ty acid-derived biomaterial comprising a fatty acid, and nation thereof. The fish oil may include one or more of 20 a glyceride, wherein the fatty acid and glyceride compo- arachidic acid, gadoleic acid, arachidonic acid, eicosap- nents are cross-linked to each other, wherein the bioma- entaenoic acid, docosahexaenoic acid or derivatives, terial is hydrated with an aqueous form of silver to form analogs and pharmaceutically acceptable salts thereof. silver fatty acid salts within the biomaterial. [0072] Furthermore, as utilized herein, the term free [0077] As used herein the term "antimicrobial," "anti- fatty acid includes, but is not limited to, one or more of 25 microbial agent" or "antimicrobial composition" refers to butyric acid, caproic acid, caprylic acid, capric acid, lauric a composition that has the effect of inhibiting the growth acid, myristic acid, palmitic acid, palmitoleic acid, stearic of bacteria, fungi, yeast, algae, etc., or killing these mi- acid, oleic acid, vaccenic acid, linoleic acid, alpha-lino- croorganisms. The aqueous form of silver is selected lenic acid, gamma-linolenic acid, behenic acid, erucic ac- from the group consisting of silver acetate and silver ni- id, lignoceric acid, analogs and pharmaceutically accept- 30 trate. Optional further antimicrobial compounds include, able salts thereof. The naturally occurring oils, including but are not limited to, diamidines, iodine and iodophors, fish oil, are cured as described herein to form a hydro- peroxygens, phenols, bisphenols, halophenols, bigua- phobic cross-linked fatty acid-derived biomaterial, creat- nides and silver compounds. ing the coating. [0078] An antibiotic is another example of an antimi- [0073] With regard to the aforementioned oils, it is gen- 35 crobial agent. The term "antibiotic" as used herein refers erally known that the greater the degree of unsaturation to any compound known to one of ordinary skill in the art in the fatty acids the lower the of a fat, and that will inhibit the growth of, or kill, bacteria. Non-limiting the longer the hydrocarbon chain the higher the melting examples of antimicrobial agents that can be used with point of the fat. An unsaturated fat, thus, has a lower the biomaterials provided herein include gentamicin sul- melting point, and a saturated fat has a higher melting 40 fate, penicillin g, ephalothin, ampicillin, amoxicillin, aug- point. Those fats having a lower melting point are more mentin, aztreonam, imipenem, streptomycin, gen- often oilsat roomtemperature. Thosefats havinga higher tamicin, vancomycin, clindamycin, erythromycin, azithro- melting point are more often waxes or solids at room mycin, polymyxin, bacitracin, amphotericin, nystatin, ri- temperature. Therefore, a fat having the physical state fampicin, tetracycline, doxycycline, chloramphenicol, na- of a liquid at room temperature is an oil. In general, un- 45 lidixic acid, ciprofloxacin, sulfanilamide, gantrisin, tri- saturated fats are liquid oils at room temperature, and methoprim, isoniazid, para-aminosalicylic acid, and mi- saturated fats are waxes or solids at room temperature. nocycline. [0074] Polyunsaturated fats are one of four basic types [0079] These anti-infective agents can be used either of fat derivedby the bodyfrom food. The other fats include alone or in combination with one another to create an saturated fat, as well as monounsaturated fat and cho- 50 infection-resistant fatty acid based biomaterial. lesterol. Polyunsaturated fats can be further composed [0080] Silver exists in two forms, ionic (Ag+) and ele- of omega-3 fatty acids and omega-6 fatty acids. Under mental (Ag0), but only the ionic form of silver has antimi- the convention of naming, the unsaturated fatty acid ac- crobial properties (S. Pal et al. 2007, "Applied and Envi- cording to the position of its first double bond of carbons, ronmental Microbiology" Vol. 73 (6), 1712-1720; A. B. G. those fatty acids having their first double bond at the third 55 Lansdown. 2006,"Curr. Probl. Dermatol." Vol 33, 17-34; carbon atom from the methyl end of the molecule are R.O. Darouiche, 1999, "Clinical Infectious Diseases" Vol. referred toas omega-3 fattyacids. Likewise, afirst double 29, 1371-1377; V. Sambhy et al., 2006, "J. American bond at the sixth carbon atom is called an omega-6 fatty Chem. Society. Vol. 128, 9798-9808; Matsumura, K et

11 19 EP 2 464 236 B1 20 al., 2003, "Applied & Environmental Microbiology" Vol. in the coating or stand alone film can be altered as a 69, No. 7, 4278-4281). The antimicrobial activity of silver function of pH, silver compound selected (silver acetate depends on the interaction of silver ions (Ag+) with the or silver nitrate) and soak time. Using this process, silver bacteria’s cellular membrane, which results in preventing ions are released from the coating more rapidly, typically DNA replication and subsequent proliferation of bacteria. 5 in less than a week in a water dissolution test using an All forms of silver produce silver ions, but the rate of silver ICP silver assay. ionization and/or release in a hydrated environment is [0084] An optional antimicrobial used with the bioma- dependent on the silver compound selected (A. B. G. terials provided herein can be triclosan. Triclosan (2,4,4’- Lansdown. 2006,"Curr. Probl. Dermatol." Vol 33, 17-34; trichloro-2’-hydroxydiphenyl ether) is a broad spectrum R.O. Darouiche, 1999, "Clinical 10 Infectiousantimicrobial agent that is classified as a chlorinated bi- Diseases" Vol. 29, 1371-1377; V. Sambhy et al.,sphenol that has long been used as a biocide in various 2006, "J. American Chem. Society. Vol. 128, 9798-9808; products including soap products, oral care products and Matsumura, K et al., 2003, "Applied & Environmental cosmetics. Triclosan activity covers a broad range of Microbiology" Vol. 69, No. 7, 4278-4281). For instance, gram positive bacteria such as Bacillus subtilis, Myco- silver nitrate rapidly forms silver ions in an aqueous en- 15 bacterium smegmatis, Staphylococcal aureus, gram vironment whereas silver metal slowly forms silver ions. negative bacteria including E. coli, Salmonella typhimu- The amount of silver needed to produce an antimicrobial rium, Shigella flexneri, yeasts and some fungi (M. J. effect has been previously reported to be in the range of Stewart et. al, "J. Mol. Biol., 1999, Vol 290, pgs 859-865). only 50 ppb (K.C. Chaw et al. 2005). Thus, only small At low concentrations, triclosan is bacteriostatic and bac- amounts of silver need to be loaded on the device to be 20 tericidal at higher concentrations and has been found to effective. The incorporation of the silver antimicrobial into have a low toxicity profile making it ideal for use in per- the coating is designed to protect the device from bacte- sonal hygiene products and various medical devices (A. rial colonization. D. Russell, Journal of Antimicrobial Chemotherapy, 2004 [0081] The release of silver ions from the silver-con- Vol 53, pgs693-695). Triclosan’s mechanism of action taining biomaterial can be altered depending on the silver 25 targets a specific enzyme, enoyl-acyl carrier re- compound selected (e.g., whether it is an insoluble or ductase(ENR), involved in the lipid biosynthesispathway soluble form of silver) in addition to the method of loading by acting as competitive inhibitor. ENR catalyzes the ter- silver into the coating and/or formulation ( e.g., silver com- minal reaction in the fatty acid elongation cycle during pound sprayed onto cured coating, or soaked into cured fatty acid biosynthesis. Enzyme inhibition studies using coating). Depending on the chemical form of the silver 30 E. coli have demonstrated that the enzyme-inhibitor com- compound and the method of incorporation of the silver plex formed between triclosan and ENR are strong, slow compounds into the biomaterial, the silver ion’s release to dissociate and relatively irreversible (M. in water and can be altered, which can alter the bioma- Kapoor, "Biochem. J." 2004, Vol 381, 719-724; J. Stew- terial’s efficacy. art et. al, "J. Mol. Biol., 1999, Vol 290, 859-865). The [0082] As discussed above, the antimicrobial behavior 35 lipophilic nature of triclosan makes it an ideal antimicro- of the silver compounds incorporated into the silver-con- bial agent to incorporate into the fatty acid biomaterial taining biomaterials can be altered by not only the form coating as described in the present invention. of silver compounds that are incorporated into a bioma- [0085] Another optional antimicrobial used with the bi- terial, but also by the chemical interaction between the omaterials provided herein can be chlorhexidine. Chlo- silver compound and the biomaterial itself. Depending 40 rhexidine is a biocide belonging to the biguanides group. on the type of silver compound and the stage of incorpo- It is widely used as a preservative and as an ration of the silver compound into the coating, different agent in hand washing products, oral products and coat- silver-containing biomaterials can be produced by not ed in combination with silver-sulfadiazine on some med- only the type of silver compound selected, but also due ical devices such as urinary catheters to prevent biofilm to the final resultant chemistry produced by the interac- 45 formation. Uptake of chlorhexidine is pH dependent and tion of the silver compound with the different molecular its antibacterial action occurs by diffusing into and attack- species present in the oil-derived coating. ing thebacterial cytoplasm after damaging bacterial outer [0083] The method to load silver antimicrobial com- cell membranes (G. McDonnell et. al., "Clinical Biological pounds into silver-containing biomaterial is to first create Reviews." 1997, Vol 12(1), 147-179; S. Bassetti, 2001, a coating or stand alone film using a thermal cure process 50 Vol 45(4), 1535-1538). and then soak or spray a water soluble form of silver [0086] A further optional antimicrobial used with the (e.g., silver nitrate or silver acetate) onto the coating. This biomaterials provided herein can be gentamicin. Gen- is performed by soaking the oil-derived gel into a solution tamicin is a water soluble aminoglycoside antibiotic that of either silver nitrate or silver acetate for a fixed period is active against a range of gram positive and gram neg- of time to load silver into the coating. This process pre- 55 ative bacteria. The mechanism of transport of gentamicin dominantly creates silver fatty acid salts within the coat- across cell membranes is not well known but it is hypoth- ing, which was confirmed using FTIR spectroscopy. Ad- esized that gentamicin interacts with some components ditionally, the amountof silver fatty acidsalts formedwith- of the cell membrane by the interaction of gentamicin

12 21 EP 2 464 236 B1 22 amino groups and free fatty aldehydes via a Schiff-base groups are typically broken down by either chemical reaction (D. E. Auslander et. al., "J Pharm Sci." 1975, and/or enzymatic hydrolysis mechanisms (K. Park et al., 64(3), 516-519). "Biodegradable Hydrogels for Drug Delivery." 1993; J. [0087] In one embodiment, the antimicrobial-contain- M. Andersen, "Perspectives on the In-Vivo Responses ing biomaterial contains more than one antimicrobial5 of Biodegradable Polymers." in Biomedical Applications compound. For example, the antimicrobial agent can be of Synthetic Biodegradable Polymers, edited by Jeffrey combined with the fatty acid/glyceride starting material O. Hollinger.1995, pgs 223-233). Chemical hydrolysis of to form a composition, such that the composition is cross- an antimicrobial -containing biomaterial occurs when the linked so the antimicrobial-containing biomaterial is functional group present in the material is cleaved by formed, and then an additional antimicrobial agent is add- 10 water. Enzymatic hydrolysis is the cleavage of functional ed to the surface of the biomaterial (by spraying, sub- groups in an antimicrobial-containing biomaterial caused mersing, etc.). The additional agent can be the same as by the reaction with a specific enzyme ( e.g., triglycerides the antimicrobial agent incorporated in the cross-linked are broken down by lipases (enzymes) that result in free matrix, or different. In another embodiment, two or more fatty acids that can then be transported across cell mem- different antimicrobial agents are combined with the fatty 15 branes). The length of time a biodegradable antimicro- acid/glyceride starting material to form a composition, bial-containing biomaterial takes to be hydrolyzed is de- such that the composition is cross-linked so an antimi- pendent on several factors such as the cross-linking den- crobial-containing biomaterial is formed that that con- sity of the material, the thickness, the hydration ability of tains two or more antimicrobial agents. the coating, the crystallinity of the fatty acid-derived bio- [0088] In another embodiment, provided herein is the 20 material, and the ability for the hydrolysis products to be silver-containing biomaterial comprising fish oil, wherein metabolized by the body (K. Park et al., 1993 and J. M. the fatty acids and glycerides of the fish oil are cross- Andersen, 1995). linked, and wherein the silver-containing biomaterial fur- [0093] It should be noted that a bioabsorbable sub- ther comprises an additional therapeutic agent, such as stance is different from a biodegradable substance. Bi- an antimicrobial agent. In a particular embodiment, the 25 odegradable is generally defined as capable of being de- silver-containing biomaterial further comprises triclosan, composed by biological agents, or capable of being bro- chlorhexidine or gentamicin. ken down by microorganisms or biological processes. [0089] In another embodiment, the additional thera- Biodegradable substances can cause an inflammatory peutic agent that is loaded into the antimicrobial-contain- response due to either the parent substance or those ing biomaterial (the silver-containing biomaterial) is an 30 formed during hydrolysis, and they may or may not be anti-adhesive agent. As used herein, the term "anti-ad- absorbed by tissues. Some biodegradable substances hesion agent" refers to any compound that prevents ad- are limited to a bulk erosion mechanism for hydrolysis. hesions or accretions of body tissues formed in response For example, a commonly used biodegradable polymer, to injury of various kinds, e.g., surgery, infection, chem- PLGA (poly (lactic-co-glycolic acid)) undergoes chemical otherapy, radiation. Anti-adhesion agents include, but 35 hydrolysis in-vivo to form two alpha-hydroxy acids, spe- are not limited to, hyaluronic acid, human plasma derived cifically glycolic and lactic acids. Thus, in one embodi- surgical sealants, and agents comprised of hyaluronate ment, the antimicrobial-containing biomaterial provided and carboxymethylcellulose that are combined with herein is non-polymeric. Although glycolic and lactic ac- dimethylaminopropyl, ethylcarbodimide, hydrochloride, ids are byproducts of various metabolic pathways in the PLA, and/or PLGA. 40 body, it has been demonstrated in previous medical im- [0090] In various aspects, the additional antimicrobial plant and local drug delivery applications that a local con- agent is sprayed onto the outside surface of the antimi- centration of these products results in an acidic environ- crobial-containing biomaterial e.g., ( coating or stand- ment to be produced, which can lead to inflammation and alone film). In one embodiment, the antimicrobial agent damage to local tissue (S. Dumitriu, "Polymeric Bioma- is triclosan. Alternatively, the triclosan can be sprayed in 45 terials." 2002). Clinically, this can lead to impaired clinical combination with the soluble silver compound onto the outcomes such as restenosis (D.E. Drachman and D.I. outside surface of the silver-containing biomaterial ( e.g., Simon. Current Atherosclerosis Reports. 2005, Vol 7, pgs coating or stand-alone film). 44-49; S.E. Goldblum et al. Infection and Immunity. 1989, [0091] In another embodiment, the second antimicro- Vol. 57, No. 4, pgs 1218-1226) and impaired healing in bial agent can be loaded by incorporating the agent into 50 a coronary stent application which can lead to late-stent the biomaterial during the thermal cure process onto a thrombosis or adhesion formation in an abdominal hernia finished device. repair (Y.C. Cheong et al. Human Reproduction Update. 2001; Vol. 7, No. 6: pgs 556-566). Thus, an ideal antimi- Coating Hydrolysis and Bioabsorption Chemistry of Fatty crobial -containing biomaterial should not only demon- Acid-Derived Biomaterials 55 strate excellent biocompatibility upon implantation, but should also maintain that biocompatibility during the life [0092] Biodegradable and bioabsorbable implantable of its implantation with its hydrolysis byproducts being materials with ester, lactone, and anhydride functional absorbable by local tissue.

13 23 EP 2 464 236 B1 24

[0094] The bio-absorbable nature of the antimicrobial- Med. 2003; Vol. 81, pgs 613-626). Additionally, omega- containing biomaterial (derived, e.g., from fish oil con- 3 fatty acids are known to be important for human health taining triglycerides and fatty acids) used as a stand- and specifically EPA and DHA are known to have anti- alone film, a coating for a medical device, or in drug de- inflammatory properties in-vivo. However, EPA and DHA livery applications results in the biomaterial being ab- 5 are not anti-inflammatory themselves, but it is the oxida- sorbed over time by the cells of the body tissue. In various tivebyproducts they arebiochemically converted into that embodiments, there are substantially no substances in produce anti-inflammatory effects in-vivo (V.N. Bochkov the biomaterial, or in vivo conversion by-products of the and N. Leitinger, 2003; L.J. Roberts II et al. The Journal biomaterial, which induce an inflammatory response, of Biological Chemistry. 1998; Vol. 273, No. 22, pgs e.g., the antimicrobial-containing biomaterial converts in 10 13605-13612.). Therefore, by selecting the appropriate vivo into non-inflammatory components. For example, in process conditions, an antimicrobial-containing bioma- various embodiments, the antimicrobial-containing bio- terial (derived from, e.g., fish oil) can be created and con- material of the present invention upon absorption and trolled using oil oxidation chemistry with a final chemical hydrolysis do not produce lactic acid and glycolic acid profile that will have a favorable biological performance break-down products in measurable amounts. The15 in-vivo. chemistry of the antimicrobial-containing biomaterial de- [0097] The process of making an antimicrobial-con- scribed herein consists of predominantly fatty acid and taining biomaterial as described herein leads to a final glyceride components that can either be hydrolyzed in- chemical profile that is biocompatible, minimizes adhe- vivo by chemical and/or enzymatic means which results sion formation, acts as a tissue separating barrier, and in the release of fatty acid and glyceride components that 20 is non-inflammatory with respect to the material chemis- can be transported across cell membranes. Subsequent- try and the products produced upon hydrolysis and ab- ly, the fatty acid and glyceride components eluted from sorption by the body in-vivo. The reason for these prop- the antimicrobial-containing biomaterials are directly me- erties is due to several unique characteristics of the an- tabolized by cells ( i.e., they are bio-absorbable). The bio- timicrobial-containing biomaterials e.g., ( coatings or absorbable nature of the coating and stand-alone film of 25 stand-alone films). the present invention results in the biomaterial being ab- [0098] In some embodiments, no toxic, short-chained sorbed over time, leaving only an underlying delivery or cross-linking agents (such as ) are used other medical device structure that is biocompatible. to form the antimicrobial-containing biomaterials e.g., ( There is substantially no foreign body inflammatory re- coatings or stand-alone films) of the invention. Short sponse to the biomaterial or its hydrolysis products in the 30 chain cross-linking agents can elute during hydrolysis of preferred embodiments of the present invention. biodegradable polymers and cause local tissue inflam- [0095] Because the materials of the invention (derived, mation. The process of creating antimicrobial-containing e.g., from fish oil containing triglycerides and fatty acids) biomaterials does not involve adding external cross-link- are biocompatible, and they hydrolyze into non-inflam- ing agents because the oil is solely cured into a coating matory components, and are subsequently bio-absorbed 35 using oil autoxidation or photo-oxidation chemistry. The by surrounding tissue, they are referred to as "biomate- oxidation process results in the formation of carboxyl and rials." hydroxyl functional groups that allow for the fatty acid- derived biomaterial to become hydrated and become Fatty Acid-Derived Biomaterial Biocompatibility and In- slippery, which allows for frictional injury during and after Vivo Performance 40 implantation to be significantly reduced and/or eliminat- ed. The methods of making the antimicrobial-containing [0096] The process of making the antimicrobial-con- biomaterials described herein allow the alkyl chains of taining biomaterials (e.g., coating or stand-alone film) as the fatty acid, glyceride and other lipid byproducts described herein led to some unexpected chemical proc- present in the antimicrobial-containing biomaterial to be esses and characteristics in view of traditional scientific 45 disordered, which creates a biomaterial that is flexible reports in the literature about the oxidation of oils. Oil andaids in handlingof the material whilebeing implanted. oxidation has traditionally been of concern for oil curing Thus, in one embodiment, the antimicrobial-containing procedures due to the formation of reactive byproducts biomaterials (derived, e.g., from fish oil) do not contain such as hydroperoxides and alpha-beta unsaturated al- any cross-linking agents. dehydes that are not considered to be biocompatible50 [0099] There are several individual chemical compo- (H.C. Yeo et al. Methods in Enzymology. 1999, Vol. 300, nents of the antimicrobial-containing biomaterial that aid pgs 70-78.; S-S. Kim et al. Lipids. 1999, Vol. 34, No. 5, in its biocompatibility and its low to non-inflammatory re- pgs 489-496.). However, the oxidation of fatty acids from sponse observed in-vivo. The processes of creating a oils and fats are normal and important in the control of fatty acid-derived biomaterial described herein result in biochemical processes in-vivo. For example, the regula- 55 low to non-detectable amounts of oxidized lipid byprod- tion of certain biochemical pathways, such as to promote ucts of biocompatibility concern, such as aldehydes. or reduce inflammation, is controlled by different lipid ox- These products are either almost completely reacted or idation products (V.N. Bochkov and N. Leitinger. J. Mol. volatilized during the curing process as described in this

14 25 EP 2 464 236 B1 26 invention. The process of creating an antimicrobial-con- nents, free fatty acids and glycerides that are derived taining biomaterial largely preserves the esters of the na- from, e.g., fish oil. The source of fatty acids in this inven- tive oil triglycerides and forms ester and/or lactone cross- tion may consist of but are not limited to other oils includ- links, which are biocompatible. ing flaxseed oil, grapeseed oil, olive oil, corn oil, peanut [0100] In addition to general chemical properties of an 5 oil, safflower oil and soybean oil. antimicrobial-containing biomaterialthat assists in its bio- [0104] Generally, gram positive bacteria have been compatibility, there are also specific chemical compo- shown to be more sensitive to the bactericidal effects of nents that have positive biological properties. Another fatty acids with gram negative bacteria being less sensi- aspect is that the fatty acid chemistry produced upon tive. Longer chain unsaturated fatty acids have greater creation of a fatty acid-derived biomaterial is similar to 10 bactericidal activity than shorter chain saturated fatty ac- the fatty acid chemistry of tissue, as presented in Figure ids, particularly against gram positive bacteria. This dif- 2. Thus, as fatty acids are eluting from the biomaterial ference in bacterial sensitivity towards long chain unsatu- they are not viewed as being "foreign" by the body and rated fatty acids and short chain saturated fatty acids are cause an inflammatory response. In fact, C14 (myristic) likely a result of the differences in bacterial surface struc- and C16 (palmitic) fatty acids present in the coating have 15 ture and the mechanism of antimicrobial effects that oc- been shown in the literature to reduce production of α- cur between the fatty acid and bacteria. Longer chain TNF, an inflammatory cytokine. The expression of α-TNF fatty acids may be able to better insert the hydrocarbon has been identified as one of the key cytokines respon- chain into the bacterial phospholipid bilayer thereby con- sible for "turning on" inflammation in the peritoneal after ferring membrane destabilization and subsequent bac- hernia repair, which can then lead to abnormal healing 20 tericidal effects. Monoglycerides have also been identi- and adhesion formation. α-TNF is also an important cy- fied to act as nonionic surfactants that alter membrane tokine in vascular injury and inflammation, such as vas- permeability by penetrating the bacterial cell membrane cular injury caused during a stent deployment. In addition causing plasma membrane disintegration. Likewise, to the fatty acids just specified, there have also been short chain and medium chain fatty acids can enter bac- additional oxidized fatty acids identified that have anti- 25 terial cells by diffusion into bacterial cells in an undisso- inflammatory properties. Other components identified ciated form and cause intracellular acidification by dis- from the fatty acid-derived coatings as described in this sociating within the bacterial protoplasm. The decreased invention are delta-lactones ( i.e., 6-membered ring cyclic intracellular pHcan in turnresult inenzymatic inactivation esters). Delta-lactones have been identified as having and interrupt amino acid transport which is important in anti-tumor properties. 30 protein synthesis. An advantage of the antibacterial prop- [0101] These components identified are not meant to erties exhibited by fatty acids and their respective be limiting in scope to this invention as changes in starting monoglycerides is the decreased potential to develop oil composition and/or process conditions can invariably bacterial resistance due to the mechanisms by which alter the fatty acid and/or oxidative byproduct profiles and bacteria are killed. can be tailored as needed depending on the intended 35 purpose and site of application of the fatty acid-derived Uses biomaterial. [0102] In summary, the biocompatibility and observed [0105] The antimicrobial-containing biomaterials pro- in in-vivo performance of antimicrobial-containing bioma- vided herein can be used, for example, for wound healing terialsdescribed herein is not only beneficialas to prevent 40 in a subject, e.g., a human or non-human animal. The a foreign body response in-vivo due to the similarity of process of wound healing involves tissue repair in re- the fatty acid composition of the material to native tissue sponse to injury and it encompasses many different bi- (i.e., a biological "stealth" coating), but the specific fatty ologic processes, including epithelial growth and differ- acids and/or other lipid oxidation components eluting entiation, fibrous tissue production and function, angio- from the coating aid in preventing foreign body reactions 45 genesis, and inflammation. Accordingly, the antimicrobi- and reducing or eliminating inflammation, which leads to al-containing material provides an excellent material suit- improved patient outcomes. Additionally, the fatty acid able for wound healing applications. and glyceride components eluted from the antimicrobial- [0106] The antimicrobial-containing biomaterials pro- containing biomaterials are able to be absorbed by local vided herein can be used, for example, to prevent tissue tissue and metabolized by cells. Hence, the antimicrobi- 50 adhesion. The tissue adhesion can be a result of blunt al-containing biomaterial (e.g., coating or stand-alone dissection. Blunt dissection can be generally described film) described in this invention is also bioabsorbable. as dissection accomplished by separating tissues along natural cleavage lines without cutting. Blunt dissection is Antimicrobial Properties of Antimicrobial-Containing Bi- executed using a number of different blunt surgical tools, omaterials 55 as is understood by those of ordinary skill in the art. Blunt dissection is often performed in cardiovascular, colorec- [0103] In the present invention, the antimicrobial-con- tal, urology, gynecology, upper GI, and plastic surgery taining biomaterial is composed of various lipid compo- applications, among others.

15 27 EP 2 464 236 B1 28

[0107] After the blunt dissection separates the desired a dura patch, buttressing material, internal wound care tissues into separate areas, there is often a need to main- (such as a graft anastomotic site), and internal drug de- tain the separation of those tissues. In fact, post surgical livery system. The antimicrobial-containing biomaterial adhesions can occur following almost any type of sur- (e.g., coating or stand-alone film) may also be used in gery, resulting in serious postoperative complications. 5 applications in transdermal, wound healing, and non-sur- The formation of surgical adhesions involves complex gical fields. The antimicrobial-containing biomaterial may inflammatory processes in which tissues that normally be used in external wound care, such as a treatment for remain separated in the body come into physical contact burns or skin ulcers. The antimicrobial-containing bioma- with one another and attach to each other as a result of terial may be used without any therapeutic agent as a surgical trauma. 10 clean, non-permeable, non-adhesive, non-inflammatory, [0108] It is believed that abdominal adhesions are anti-inflammatory dressing, or the antimicrobial-contain- formed when bleeding and leakage of plasma ing biomaterial may be used with one or more therapeutic from damaged tissue deposit in the abdominal cavity and agents for additional beneficial effects. In another em- form what is called a fibrinous exudate. Fibrin, which re- bodiment, the antimicrobial-containing biomaterial is as- stores injured tissues, is sticky, so the fibrinous exudate 15 sociated with bandage or dressing for a wound. When may attach to adjacent anatomical structures in the ab- used in any of the aforementioned methods, the fatty domen. Post-traumatic or continuous inflammation ex- acid-based material (in particle form or particles pressed aggerates this process, as fibrin deposition is a uniform into a film) may or may not be associated with a thera- host response to local inflammation. This attachment peutic agent. seems to be reversible during the first few days after in- 20 [0111] In another embodiment, the antimicrobial-con- jury because the fibrinous exudates go through enzymat- taining biomaterials provided herein can be used as a ic degradation caused by the release of fibrinolytic fac- coating on a medical device to minimize the risk of these tors, most notably tissue-type plasminogen activator (t- devices becoming colonized with bacteria during surgical PA). There is constant play between t-PA and plasmino- implantation. The formulations described herein can also gen-activator inhibitors. Surgical trauma usually de-25 be spread onto a medical device surface and allowed to creases t-PA activity and increases plasminogen-activa- cure for a period of time, thereby providing a medical tor inhibitors. When this happens, the fibrin in the fibri- device coating comprising an antimicrobial-containing bi- nous exudate is replaced by collagen. Blood vessels be- omaterial. Examples of such devices include, but are not gin to form, which leads to the development of an adhe- limited to, a graft, a catheter balloon, a stent or surgical sion. Once this has occurred, the adhesion is believed 30 mesh. to be irreversible. Therefore, the balance between fibrin [0112] In still another embodiment, the antimicrobial- deposition and degradation during the first few days post- containing biomaterials provided herein ( e.g., in the form trauma is critical to the development of adhesions (Hol- of a stand-alone film) can be used for wound healing mdahl L. Lancet 1999; 353: 1456-57). If normal fibrino- applications. The antimicrobial-containing biomaterials lytic activity can be maintained or quickly restored, fibrous 35 provided herein can also be used incombination with a deposits are lysed and permanent adhesions can be bandage or dressing for wound healing applications. The avoided. Adhesions can appear as thin sheets of tissue term "wound" refers broadly to injuries to the skin and or as thick fibrous bands. subcutaneous tissue initiated in any one of a variety of [0109] Often, the inflammatory response is also trig- ways (e.g., pressure sores from extended bed rest, gered by a foreign substance in vivo, such as an implant- 40 wounds induced by trauma, cuts, ulcers, burns and the ed medical device. The body sees this implant as a for- like) and with varying characteristics. Wounds are typi- eign substance, and the inflammatory response is a cel- cally classified into one of four grades depending on the lular reaction to wall off the foreign material. This inflam- depth of the wound: (i) grade I: wounds limited to the mation can lead to adhesion formation to the implanted epithelium; (ii) grade II: wounds extending into the der- device; therefore a material that causes little to no inflam- 45 mis; (iii) grade III: wounds extending into the subcutane- matory response is desired. ous tissue; and (iv) grade IV (or full-thickness wounds): [0110] Thus, the antimicrobial-containing biomaterial wounds wherein bones are exposed (e.g., a bony pres- (e.g., coating or stand-alone film) can be used as a barrier sure point such as the greater trochanter or the sacrum). to keep tissues separated to avoid the formation of ad- The antimicrobial-containing biomaterials described hesions, e.g., surgical adhesions. Application examples 50 herein can be used for treatment of any of the aforemen- for adhesion prevention include abdominal surgeries, tioned wounds. spinal repair, orthopedic surgeries, tendon and ligament [0113] The term "treat," "treated," "treating" or "treat- repairs, gynecological and pelvic surgeries, and nerve ment" includes the diminishment or alleviation of at least repair applications. The antimicrobial-containing bioma- one symptom associated or caused by the state, disorder terial may be applied over the trauma site or wrapped 55 or disease being treated. For example, treatment can be around the tissue or organ to limit adhesion formation. diminishment of one or several symptoms of a disorder Other surgical applications of the antimicrobial-contain- or complete eradication of a disorder (e.g., bacterial in- ing biomaterial-based film may include using the film as fection, tissue adhesion, and/or external wounds).

16 29 EP 2 464 236 B1 30

[0114] Various aspects and embodiments are further [0119] Silver assay of fatty acid-derived biomaterial described by way of the following Examples. The Exam- samples hydrated in silver acetate and silver nitrate over ples are offered by way of illustration and not by way of a six hour period was performed by ICP. Figure 5 illus- limitation. trates that the silver content in a fatty acid-derived bio- 5 material sample increases as a function of hydration time EXAMPLES using aqueous silver nitrate or aqueous silver acetate as the hydration media. A greater amount of silver was able [0115] The following examples are for demonstration to be loaded using silver acetate than silver nitrate within purposes and are not meant to be limiting. the same hydration time as result of the additional pen- 10 etration of the silver acetate into the coating due to the EXAMPLE 1 higher pH and the similarity of the acetate group to the fatty acid components within the coating. Analysis of the Silver fatty acid salts within the Fatty Acid-Derived silver hydrated fatty acid-biomaterial samples by FTIR, Biomaterial shows evidence of the formation of fatty acid represented 15 bythe peak at1512 cm -1, which increases with increasing [0116] In accordance with the present invention, silver hydration time as shown in Figures 6A-6B for silver ni- in its aqueous form was used to hydrate a fatty acid- trate and silver acetate loading solutions, respectively. derived biomaterial sample. One possible mechanism for [0120] The efficacy of the silver nitrate hydrated fatty the formation of the antimicrobial-containing biomaterial acid-derived biomaterial was tested in a three day in-vitro hydrated in aqueous silver is as follows: fish oil (triglyc- 20 S. aureus biofilm study. A fatty acid-derived biomaterial erides) absorbs oxygen into the oil; oxidation of the C=C sample, without silver present, was used as a control and bonds in the oil occurs; fatty acid and glycerides (a mix- the results obtained are shown in Figure 7. These results ture of mono, di- and tri) are formed; continued curing show the silver hydrated sample had an approximate 5 results in dehydration of neighboring carboxyl and hy- log reduction in colony forming units (CFU’s) per cm2 droxyl functional groups; lactone/ester cross-links are 25 after one day and no CFU’s were present on days 2 and formed between fatty acids and glycerides; the oil solid- 3. The fatty acid-derived biomaterial control sample had ifies into a bioabsorbable gel containing cross-linked fatty an approximate 3 log reduction in CFU’s after day one acids and glycerides; the cross-linked fatty acid bioma- as a result of the antibacterial properties of short-chain terial is immersed in an aqueous Ag solution; and the fatty acids (T. Kitahara, "Biol Pharm. Bull.: Second Edi- hydrated silver coated fatty acid biomaterial is dried to 30 tion." 2004, Vol 27(9), pgs 1321-1326) but the CFU’s evaporate the solvent. gradually increased on day 2 and 3. Figure 7 demon- [0117] The silver hydrated fatty acid-derived biomate- strates that adding silver to the fatty acid- derived bioma- rial was prepared as follows. terial coating through hydration in aqueous silver media [0118] Aqueous silver acetate was massed and dis- enhances the coating’s ability to form a biofilm within an solved to a final concentration of 0.06M. In addition, a 35 in-vitro S. aureus infected environment. fatty acid biomaterial was prepared by curing native fish oil, at a of approximately 100 mg/inch 2, onto poly- EXAMPLE 2 propylene mesh at a temperature of 200 °F for 24 hours to form a solid coating. To incorporate silver by hydration, Fatty Acid-Derived Biomaterial surface coated with a 1 inch2 sample of the fatty acid biomaterial was then 40 silver immersed in the 0.06M aqueous silver acetate solution for four hours. By immersing the sample in aqueous sil- [0121] In a different embodiment of the present inven- ver, the fatty acid biomaterial becomes hydrated in an tion, silver was coated onto the surface of a fatty acid- aqueous environment over an extended period of time derived biomaterial. Sample preparation was done as allowing for the formation of silver fatty acid salts. Deter- 45 follows. mination of free silver content released in mg/cm2 from [0122] Silver nitrate was massed and dissolved in the silver hydrated fatty acid biomaterial sample was car- methanol to a final concentration of 0.02M. In addition, ried out in water and analyzed by Inductively Coupled a fatty acid biomaterial was prepared by encapsulating Plasma (ICP). The silver release profile obtained is polypropylene mesh with approximately 100 mg of fish shown in Figure 3. Theresults indicate thatan immediate 50 oil per inch2 then thermally curing the fish oil cast onto burst of silver is released from the hydrated fatty acid- the polypropylene mesh at a temperature of 200 °F for derived coating. Similarly, Figure 4 shows the silver re- 24 hours to form a solid fatty acid- derived coating. To lease profile of silver from a 1 inch2 fatty acid-derived incorporate silver on the surface, a 1 inch 2 sample of the biomaterial sample hydrated in 0.07M silver nitrate. The fatty acid biomaterial was then sprayed onto the surface free silver released from the silver nitrate hydrated sam- 55 with the 0.02M silver nitrate solution and allowed to dry ple also occurs as a burst indicating that high levels of to evaporate off the methanol solvent. One possible silver would be released rapidly in an aqueous environ- mechanism for the formation of the antimicrobial-contain- ment. ing biomaterial coated with aqueous silver is as follows:

17 31 EP 2 464 236 B1 32 fish oil (triglycerides) absorbs oxygen into the oil; oxida- and thoroughly mixing these components with intermit- tion of the C=C bonds in the oil occurs; fatty acid and tent heating. The emulsified silver formulation is then cast glycerides (a mixture of mono, di- and tri) are formed; onto a medical device and thermally cured at 200 °F for continued curing results in dehydration of neighboring approximately 24 hours to form a solid reduced silver carboxyl and hydroxyl functional groups; lactone/ester 5 fatty acid-derived biomaterial coating. Thermal curing is cross-links are formed between fatty acids and glycer- hypothesized to convert the aqueous silver present in ides; the biomaterial solidifies into a bioabsorbable gel the coating to an insoluble form of silver within the fatty containing cross-linked fatty acids and glycerides; the acid-derived coating. This is accomplished by the inter- cross-linked fatty acid biomaterial is sprayed with an action of the silver ions in the silver nitrate solution with aqueous Ag solution; and the sprayed silver coated fatty 10 the lipid hydroperoxides formed during the oxidation of acid biomaterial is dried to evaporate the solvent. the oil-derived cross-linked gel, which are then reduced [0123] The silver release properties of a fatty acid-de- to silver metal (A. Kumar et al., "Nature Materials". 2008 rived monofilament coated biomaterial sprayed with sil- Vol 7, pgs 236-241). One possible mechanism for the ver nitrate was evaluated in water. The silver content in formation of the biomaterial coating containing reduced the dissolution media was analyzed over 30 days by ICP. 15 silver is as follows: emulsified fish oil, pre-cured fish oil Figure 8shows thesilver amounts released into aqueous and silver (Ag) absorb oxygen; oxidation of the C=C media depicting a dose dump of silver in the dissolution bonds in the oil occurs; fatty acid and glycerides (mixture media. The silver release profile of the sprayed fatty acid- of mono, di- and tri) and reduced silver are formed; con- derived biomaterial is similar to the silver hydrated fatty tinued curing results in dehydration of neighboring car- acid-derived biomaterial in Figures 3-4. 20 boxyl and hydroxyl functional groups; lactone/ester [0124] Results illustrating the efficacy of the fatty acid- cross-links are formed between fatty acids and glycer- derived biomaterial sprayed with a layer of silver in a ides; and the material solidifies into a bioabsorbable gel three day in-vitro S. aureus biofilm study are shown in containing cross-linked fatty acids, glycerides and re- Figure 9. A 5 log reduction in colony forming units duced Ag. (CFU’s) per cm2 was noted after one day and no CFU’s 25 [0126] Thesilver release profile ofa reducedsilver fatty were present on days 2 and 3. The fatty acid-derived acid-derived coating was evaluated in water as the dis- biomaterial control sample had an approximate 3 log re- solution media. ICP was used to analyze the free silver duction in CFU’s after day one but the CFU’s per cm2 content in the dissolution media for a period of approxi- gradually increased on day 2 and 3 indicating that the mately two months. Silver released in mg/cm2 over time addition of the surface silver layer on the fatty acid-de- 30 is depicted in Figure 10. Silver is released in steady grad- rived biomaterial helped reduce the formation of a biofilm ual manner over an extended amount of time in water in this 3 day in-vitro study. indicating that the aqueous silver that was cured with the fatty acid-derived biomaterial is converted to an insoluble REFERENCE EXAMPLE 3 form. In an aqueous environment, a reduced silver fatty 35 acid-derived biomaterial sample would release low levels Formation of reduced silver within the fatty acid-de- of silver unlike Examples 1 and 2 where a rapid burst of rived biomaterial silver is released in an aqueous environment. [0127] The reduced silver fatty acid-derived biomate- [0125] In this example, a different method of forming rial was tested for its bactericidal properties in a 2 day S. a silver coated fatty acid-derived material is described. 40 aureus zone of inhibition assay using samples prepared The method of preparation is as follows: using increasing concentrations of aqueous silver nitrate. Three components are used to make up a silver-contain- Figure 11 shows the graphical results of the zone of in- ing fatty acid emulsion formulation which is then thermally hibition assay. Zones measured on day 1 were found to cured onto polypropylene mesh to form a solid reduced be larger in samples prepared from a higher concentra- silver fatty acid-derived biomaterial coating. The three 45 tion of silver. This demonstrates that the silver added to components that make up the emulsion include an aque- the fatty acid-derived biomaterial is responsible for the ous silver solution, native fish oil and partially cured fish growth inhibiting effects observed. Day 2 of the zone as- oil that has a viscous consistency. An aqueous silver so- say had a comparable size in all three samples tested. lution was prepared by massing silver nitrate or silver Similarly, a biofilm assay was performed on a reduced acetate and dissolving it in water to give final concentra- 50 silver fatty acid-derived biomaterial sample and found to tions of 0.07M and 0.06M respectively. Separately, par- have no bacteria on the surface of the device after three tially cured fish oil was prepared by exposing native fish days as shown inFigure 12. The overall reduction in oil totemperatures of approximately 90°C in the presence CFU’s was approximately 5 log. of oxygen for 16 hours which results in a partially cured [0128] FTIR analysis of the reduced silver coating is fish oil component with a viscous consistency. The silver 55 shown in Figure 13. The spectra of the fatty acid bioma- containing emulsion is then prepared by massing out terial control and the reduced silver fatty acid sample (w/w) 45% of the native fish oil, 45% of the partially cured have similar spectral profiles and do not show the pres- fish oil and 10% of the aqueous silver solution in one vial ence of fatty acid salts as seen in Figures 6A-6B indi-

18 33 EP 2 464 236 B1 34 cating that the aqueous silver in these samples interacts pressure; and the fatty acid silver nanoparticle film is cast differently after the thermal curing process. onto a polymeric device. [0133] The silver release profile of this embodiment is REFERENCE EXAMPLE 4 shown in Figure 16, which also shows a sustained re- 5 lease profile in an aqueous environment. Formation of silver nanoparticle fatty acid-derived biomaterial REFERENCE EXAMPLE 5

[0129] In a different embodiment of this invention, el- Formation of silver nano-particle fatty acid-derived emental silver is used in the form of silver nanoparticles 10 biomaterial in combination with gentamicin sulfate and added to a fatty acid biomaterial to in order to en- hance antibacterial properties of the fatty acid biomaterial [0134] In this embodiment, the silver nanoparticle fatty coating. The sample preparation was done as follows: acid-derived biomaterial coating is combined with a phar- Silver nanopowder surface coated with oleic acid was maceutical agent such as gentamicin to enhance micro- purchased from American Elements, 99.9% silver metal. 15 bial stasis. A fish oil and silver nanopowder formulation was then [0135] Silver nanopowder surface coated with oleic ac- prepared by mixing 1% silver nano powder in 99% fish id was purchased from American Elements (Product oil (w/w). The fish oil and silver nanopowder was thor- Number AG-M-03M-NPC), 99.9% silver metal. gen- oughly mixed in a sonication bath for an hour then cast tamicin sulfate was purchased from Spectrum Chemicals onto a medical device and thermally cured at 200 °F for 20 and Laboratory Products, part number G1174. The fish 24 hours to form a solid coating. One possible mecha- oil and silver nanopowder formulation was then prepared nism for the formation of the nano-silver coated bioma- by mixing 0.16 % silver nano powder and 2.57% gen- terial coating is as follows: fish oil and silver nanoparticles tamicin sulfate powder in 97% fish oil (w/w). The gen- absorb oxygen; oxidation of the C=C bonds in the oil tamicin sulfate, silver nanopowder and fish oil was then occurs; fatty acid and glycerides (mixture of mono, di- 25 thoroughly mixed using a cryo-grinding apparatus under and tri) are formed; continued curing results in dehydra- cryogenic temperatures forming a cryoground formula- tion of neighboring carboxyl and hydroxyl functional tion containing fish oil gentamicin and silver. Cryogenic groups;lactone/ester cross-links areformed between fat- grinding (cryogrinding) is a process carried out at ex- ty acids and glycerides; and the material solidifies into a tremely low temperatures enabled by using liquid nitro- bioabsorbable gel containing cross-linked fatty acids,30 gen (-196 °C), to finely pulverize and mix sample com- glycerides and Ag nano particles ponents by means of a magnetic grinding mechanism, [0130] The silver release properties of the silver nan- making the sample brittle under these low temperatures, oparticle fatty acid biomaterial samples in water are thereby yielding a thoroughly mixed homogenous formu- shown in Figure 14. The release profiles indicate that lation. A Cryogenic impact grinder (6750 Freezer Mill) more silver is released as a function of the concentration 35 was used for homogenizing a fish oil, gentamicin and of silver nanoparticles added to the fatty acid biomaterial silver formulation. The cryogenic grinding apparatus’ tub coating. The silver nanoparticles in the fatty acid bioma- was filled with 4-5L of liquid nitrogen and set to grind for terial coating also produces a steady controlled release eight cycles. Each cycle consists of 2 minutes of grinding of silver in aqueous media over a period of approximately with a 2 minute cooling period at a rate of 15 impacts per 75 days. The release profiles in Figure 20 are an improve- 40 second during the grinding cycle. ment to the quick burst of silver released when silver is [0136] Fish oil, gentamicin powder and silver nanopar- sprayed onto the fatty acid-derived biomaterial or hydra- ticles were appropriately massed into a cryogenic grind- tion method is used to incorporate silver fatty acid salts ing polycarbonate center cylinder vial capped on one end into the coating as described in Example 1 and 2. with a stainless steel end plug. A stainless steel magnetic [0131] The amount of silver loaded into the fatty acid- 45 bar was added into the polycarbonate vial containing the derived biomaterial was determined by ICP. Theformulation components and the open end was capped amounts of silver loaded within the fatty acid biomaterial with a stainless steel end plug. The vial was then placed can be controlled by altering the concentration of the sil- into the cryogenic grinding apparatus programmed to ver nanoparticles in the coating as shown in Figure 15. produce an alternating magnetic field that causes the [0132] In a related method of this embodiment, a silver 50 stainless steel bar to impact the stainless steel end plugs nanoparticle fatty acid-derived film can be prepared sep- of the polycarbonate vial containing the formulation com- arately and cast onto a medical device to produce a var- ponents, resulting in a finely ground and homogenized iation of the mechanism described inFigure 13. One formulation. The cryoground formulation was allowed to possible mechanism for the formation of the silver nan- thaw, cast onto a medical device and thermally cured at oparticle-containing biomaterial coating is as follows: fat- 55 200 °F for 24 hours to form a solid coating. One possible ty acid film particles and silver nanoparticles are thor- mechanism for the formation of the gentamicin and nano- oughly mixed together; the blended particles and Ag na- silver oil-derived biomaterial coating is as follows: fish noparticles are pressed into a thin film under heat and oil, gentamicin and silver nanoparticles absorb oxygen;

19 35 EP 2 464 236 B1 36 oxidation of the C=C bonds in the oil occurs; fatty acid layer. The bacterial adherence results in Figure 20 sim- and glycerides (mixture of mono, di- and tri) are formed; ilarly show that significantly fewer bacteria adhered to continued curing results in dehydration of neighboring the surface of the gentamicin sprayed silver nanoparticle carboxyl and hydroxyl functional groups; lactone/ester fatty acid-derived biomaterial. Addition of gentamicin to cross-links are formed between fatty acids and glycer- 5 the silver nanoparticle fatty acid-derived biomaterial ides; and the coating solidifies into a bioabsorbable gel therefore improved the antibacterial properties of the bi- containing cross-linked fatty acids, glycerides, gen- omaterial coating. tamicin and Ag nano particles [0140] In another embodiment of this invention, a fatty [0137] The release of gentamicin and silver ions in acid-derived film containing silver nanoparticles and gen- mg/cm2 was tested in aqueous conditions. Figure 17 il- 10 tamicin is prepared and cast onto a polymeric medical lustrates a plot of the silver ions released over approxi- device. The mechanism used to form the gentamicin and mately three weeks whereas Figure 18 shows the re- silver nanoparticle fatty acid-derived biomaterial coating lease profile of gentamicin in water. The silver release is as follows: fatty acid film particles, genatmicin and sil- profile was gradual but the gentamicin produced a burst vernanoparticles thoroughly mixed together;the blended of the drug dose in less than 48 hours. This indicates that 15 particles, gentamicin and Ag nanoparticles are pressed in this particular embodiment, silver would be slow re- into a thin film under heat and pressure; and the gen- leasing over extended time periods but the gentamicin tamicin fatty acid silver nanoparticle film is cast onto a would be fast releasing within a relatively short amount polymeric device. of time in an aqueous environment. gentamicin release in aqueous media occurred as a burst [0138] Another method of incorporating gentamicin in- 20 over 250 hours but the total amount released was able to the silver nanoparticle fatty acid biomaterial coating is to be modified by varying the total amount of gentamicin accomplished by spraying a solution of gentamicin sul- loaded onto the sample as shown in Figure 21. The total fate on a cured silver nanoparticle fatty acid-derived bi- amount of gentamicin loaded per sample can also be omaterial sample. The antibiotic solution is prepared by controlled by altering the amount of gentamicin added to dissolving gentamicin in water; the concentration of the 25 the initial formulation. Assay values of gentamicin in this gentamicin can range between 1 and 50 mg/ml depend- example were determined to be between 180 and 900 ing on how much is drug to be loaded onto the surface mg/cm2 for concentrations of gentamicin ranging be- of the coating and how many sprayed layers are to be tween 2 and 6% (Figure 22). applied. The material can be prepared as follows: fish oil and silver nanoparticles absorb oxygen; oxidation of the 30 REFERENCE EXAMPLE 6 C=C bonds in the oil occurs; fatty acid and glycerides (mixture of mono, di- and tri) are formed; continued curing Formation of silver nano-particle fatty acid-derived results in dehydration of neighboring carboxyl and hy- biomaterial in combination with Chlorhexidine diac- droxyl functional groups; lactone/ester cross-links are etate formed between fatty acids and glycerides; the material 35 solidifies into a bioabsorbable gel containing cross-linked [0141] In this example, chlorhexidine diacetate is used fatty acids, glycerides, and Ag nano particles; and gen- as an additional antimicrobial agent to augment the silver tamicin is sprayed onto the surface of the cross-linked nanoparticle fatty acid-derived biomaterial. One possible silver nanoparticle biomaterial and allowed to dry. mechanism for the formation of the silver nanoparticle [0139] The efficacy of the gentamicin sprayed silver 40 fatty acid biomaterial surface coated with chlorhexidine nanoparticle fatty acid-derived biomaterial coating was diacetate is as follows: fish oil and silver nanoparticles evaluated by zone of inhibition (ZOI) and bacterial ad- absorb oxygen: oxidation of the C=C bonds in the oil herence tests shown in Figures 19 and 20. In the ZOI occurs; fatty acid and glycerides (mixture of mono, di- test, silver nanoparticle fatty acid biomaterial coating and and tri) are formed; continued curing results in dehydra- gentamicin sprayed silver nanoparticle fatty acid bioma- 45 tion of neighboring carboxyl and hydroxyl functional terial coating sample discs with a 0.5 inch diameter were groups; lactone/ester cross-links are formed between fat- placed on a bed of S. aureus bacteria in an agar plate ty acids and glycerides; the coating solidifies into a bio- and incubated for 24 hours before transferring the disc absorbable gel containing cross-linked fatty acids, glyc- to a fresh bacterial agar plate. In this particular test, the erides, and Ag nano particles; and chlorhexidine is ZOI was done for 3 days comparing the silver nanopar- 50 sprayed onto the surface of the cross-linked silver nan- ticle fatty acid biomaterial coating with and without the oparticle biomaterial and allowed to dry. gentamicin sprayed layer. The plot in Figure 19 shows [0142] The antimicrobial solution was prepared by dis- that both silver nanoparticle fatty acid biomaterial coating solving chlorhexidine diacetate in water to a final con- with and without the gentamicin sprayed layer were effi- centration of 10 mg/ml and was evenly sprayed onto the cacious in reducing the number of bacteria growing55 surface of the silver nanoparticle fatty acid-derived bio- around the surface of the device but the sample with the material created using the methodology described in Ex- added gentamicin layer was more efficacious in this re- ample 4. gard when compared to the sample without a gentamicin [0143] Bacterial adherence and ZOI tests were per-

20 37 EP 2 464 236 B1 38 formed on the chlorhexidine sprayed silver nanoparticle biomaterial coating. Figure 27a further depicts the fattyacid-derived biomaterial samples. The ZOI testsam- number of colony forming units enumerated per ml of ples had measurable zones for three days with the chlo- media after performing a S. aureus bacterial suspension rhexidine sprayed silver nanoparticle fatty acid-derived assay using various fatty acid biomaterial samples with- biomaterial samples having larger zones compared to 5 out silver, with silver and with both silver and triclosan the control silver nanoparticle fatty acid biomaterial sam- present. This assay monitors the ability of the antimicro- ples without chlorhexidine as show in Figure 23. Bacte- bial to elute from the device to the surrounding media or rial adherence test results of the chlorhexidine sprayed environment. Figure 27a showed that the lowest number silver nanoparticlefatty acid-derived biomaterialsamples of colony forming units within the suspension media was showed that adding a chlorhexidine layer significantly re- 10 obtained from the fatty acid biomaterial coated with both duced the number of bacteria adhered to the surface of triclosan and silver. Figure 27b shows the results ob- the when compared to a silver nanoparticle fatty acid- tained from a S. aureus bacterial adherence assay of the derived biomaterial control sample without chlorhexidine fatty acid biomaterial samples without silver, with silver as shown in Figure 24. and with both silver and triclosan. This assay is a meas- 15 ure of the ability to prevent bacterial colonization and REFERENCE EXAMPLE 7 adherence onto the surface of the device. The results in Figure 27b showed reduced bacterial colony forming Formation of silver nano-particle fatty acid-derived units extracted from the surface of the silver nanoparticle biomaterial in combination with Triclosan fatty acid biomaterial silver compared to the control but 20 the most significant reduction was noted in the silver na- [0144] This example describes the use of triclosan to noparticle fatty acid biomaterial with triclosan which had augment the antimicrobial properties of the silver nano- no bacteria adhered to the surface. particle fatty acid-derived biomaterial coating. One pos- [0146] In a related embodiment, triclosan can be incor- sible mechanism for the formation of the silver nanopar- porated into the silver nanoparticle fatty acid-derived ticle fatty acid-derived biomaterial surface coated with 25 coating and additional sprayed with a layer of triclosan triclosan is as follows: fish oil and silver nanoparticles to further improve upon antibacterial properties. The absorb oxygen; oxidation of the C=C bonds in the oil mechanism used to form this triclosan silver nanoparticle occurs; fatty acid and glycerides (mixture of mono, di- fatty acid-derived biomaterial coating surface coated with and tri) are formed; continued curing results in dehydra- triclosan is as follows: fish oil, triclosan and silver nano- tion of neighboring carboxyl and hydroxyl functional30 particles absorb oxygen; oxidation of the C=C bonds in groups;lactone/ester cross-links areformed between fat- the oil occurs; fatty acid and glycerides (mixture of mono, ty acids and glycerides; the coating solidifies into a bio- di- and tri) are formed; continued curing results in dehy- absorbable gel containing cross-linked fatty acids, glyc- dration of neighboring carboxyl and hydroxyl functional erides, and Ag nano particles; and triclosan is applied to groups; lactone/ester cross-links are formed between fat- the surface of the solidified coating containing cross-35 ty acids and glycerides; the coating solidifies into a bio- linked fatty acids, glycerides, and Ag nano particles absorbable gel containing cross-linked fatty acids, glyc- [0145] A triclosan solution was prepared by dissolving erides, gentamicin and Ag nano particles; solidified coat- triclosan in to a concentration ranging between ing containing fatty acids, triclosan, and Ag nanoparticles 1 and 20 mg/ml and the solution was then sprayed onto is surface coated with an additional layer of triclosan. the silver fatty acid coating surface created using the40 [0147] Sample preparation involves combining 1% tri- process described in Example 4. Multiple layers were closan powder, 1% silver nanoparticles and fish oil, thor- sprayed on the surface if lower triclosan solutions were oughly mixing the formulation which is then cast onto a used and fewer triclosan layers were sprayed on the coat- medical device and thermally cured to solidify the coat- ing surface if higher triclosan solutions were used. An ing. The surface of the solid coating containing triclosan, illustration of triclosan dissolution in a buffer solution is 45 silver nanoparticles and fatty acids is then sprayed with shown in Figure 25 showing low level amounts of tri- a 1-20 mg/ml triclosan solution. Figure 28 depicts a plot closan are gradually released in this medium. The effi- of ZOI results obtained from triclosan silver nanoparticle cacy of the triclosan sprayed silver nanoparticle fatty ac- fatty acid-derived samples surface coated with triclosan. id-derived biomaterial coating was evaluated by zone of Significant zones were measured through day 13 dem- inhibition (ZOI) and bacterial adherence tests shown in 50 onstrating a high antimicrobial efficacy of these samples. Figures 26 27a and 27b. The plot in Figure 26 showed The results of the bacterial adherence test illustrated in improved zones measured that were relatively consistent Figure 29 were acquired using bacterial adherence over a period of approximately 3 weeks. Previous ZOI counts extracted from the surface of the device after 24 tests of the silver nanoparticle fatty acid biomaterial coat- hours. The extraction bacterial counts obtained after 24 ing without surface sprayed Triclosan, exhibited zones 55 hours showed that the silver fatty acid biomaterial with for approximately 3 days. Addition of triclosan to the coat- triclosan had the lowest number of colony forming units ing surface therefore significantly improved the bacteri- extracted from the sample’s surface. ostatic capabilities of the silver nanoparticle fatty acid [0148] In another related embodiment, triclosan is in-

21 39 EP 2 464 236 B1 40 corporated into the silver nanoparticle fatty acid-derived thesurface coated triclosanmay be attributed tothe lower biomaterial before the coating formulation is thermally bacterial counts in samples with both triclosan and silver cured (i.e., triclosan not sprayed on the surface of the nanoparticles. finished device). One possible mechanism for the forma- [0151] The section headings used herein are for or- tion of the triclosan and nano-silver hydrophobic oil-de- 5 ganizational purposes only and are not to be construed rived cross-linked biomaterial coating is as follows: fish as limiting the subject matter described in any way. oil, triclosan and silver nanoparticles absorb oxygen; ox- idation of the C=C bonds in the oil occurs; fatty acid and glycerides (mixture of mono, di- and tri) are formed; con- Claims tinued curing results in dehydration of neighboring car- 10 boxyl and hydroxyl functional groups; lactone/ester 1. A composition comprising an antimicrobial silver hy- cross-links are formed between fatty acids and glycer- drated fatty acid-derived biomaterial comprising a ides; the coating solidifies into a bioabsorbable gel con- fatty acid, and a glyceride, wherein the fatty acid and taining cross-linked fatty acids, glycerides, triclosan and glyceride components are cross-linked to each other Ag nano particles. 15 to form a bioabsorbable gel containing the cross- [0149] ZOI test results showing antimicrobial proper- linked fatty acids and glycerides, wherein the bioma- ties of this coating are displayed in Figure 30 which il- terial is hydrated with an aqueous form of silver to lustrates zones measured through day 19. This demon- form silver fatty acid salts within the biomaterial, strates that the coating has sufficient antimicrobial effi- wherein the aqueous form of silver is selected from cacy to prevent bacterial growth on the device in a S. 20 the group consisting of silver acetate and silver ni- aureus bacterial environment. trate.

REFERENCE EXAMPLE 8 2. The composition of claim 1 wherein the composition is derived from an oil containing an omega-3 fatty Formationof fatty acid-derived biomaterial and silver 25 acid. nano-particle fatty acid-derived biomaterial in com- bination with Triclosan 3. The composition of claim 2, wherein the oil is a fish oil, olive oil, grape oil, palm oil, or flaxseed oil. [0150] The efficacy of triclosan fatty acid-derived bio- materials were evaluated in an in-vivo infectability model 30 4. The composition as defined in claim 1 for use in a using a clinical strain of MRSA. An additional embodi- method of treating or preventing tissue adhesion in ment of this invention uses triclosan coated onto the sur- a subject in need thereof comprising administering face of a fatty acid-derived biomaterial sample without to the subject the composition of claim 1. silver present within the coating matrix. Further yet, silver nanoparticles may be encapsulated within the lipid coat- 35 5. A film comprising an antimicrobial silver hydrated fat- ing matrix as described in Example 4 and then surface ty acid-derived biomaterial comprising a fatty acid, coated with triclosan. The combination of triclosan and and a glyceride, wherein the fatty acid and glyceride silver in the fatty acid-derived biomaterial coating may components are cross-linked to each other to form be serve to enhance the antimicrobial properties of the the film containing the cross-linked fatty acids and fatty acid-derived biomaterial coating as seen in Figure 40 glycerides, wherein the biomaterial is hydrated with 31. The plot displays bacterial counts scrapped from the an aqueous form of silver to form silver fatty acid samples’ surface that represent 7 day explant samples salts within the biomaterial, wherein the aqueous obtained from an in vivo infectability study. The sample form of silver is selected from the group consisting sets evaluated include fatty acid-derived biomaterial of silver acetate and silver nitrate. samples surface coated with 20ug/cm2 of triclosan and 45 60ug/cm2 silver nanoparticles in the fatty acid-derived 6. The film of claim 5, wherein the film is associated biomaterial samples surface coated with 20ug/cm2 and with a medical device, and the medical device is a 40ug/cm2 triclosan. While some efficacy was evident in bandage, a stent, a graft, a shunt, a catheter, a sur- all sample sets tested in this study,Figure 31 demon- gical mesh, or a balloon. strates that the lowest bacterial counts were recorded in 50 the sample sets which had both silver nanoparticles and 7. A medical device at least partially coated with the triclosan. Adding triclosan to the surface of a fatty acid- composition of claim 1, wherein the medical device derived biomaterial confers a benefit in reducing bacterial is a bandage, a stent, a graft, a shunt, a catheter, a adherence onto the surface of the device but having both balloon, or a surgical mesh. silver nanoparticles and triclosan present in the coating 55 results in a more significant reduction of bacterial adher- 8. A method of forming a composition comprising an ence as shown in Figure 31. A possible synergistic effect antimicrobial silver hydrated fatty acid-derived bio- between the silver encapsulated in the lipid coating and material, comprising:

22 41 EP 2 464 236 B1 42

curing a starting material comprising a fatty acid Silberfettsäuresalze innerhalb des Biomaterials zu and a glyceride to form a solid bioabsorbable bilden, wobei die wässrige Form von Silber aus der gel coating containing the fatty acids and glyc- Gruppe ausgewählt ist, die aus Silberacetat und Sil- erides cross-linked to each other; and bernitrat besteht. associating the solid bioabsorbable gel coating 5 with an antimicrobial compound that is dissolved 2. Zusammensetzung nach Anspruch 1, wobei die Zu- in an aqueous solution by immersing the solid sammensetzung von einem Öl abgeleitet ist, das ei- bioabsorbable gel coating in the antimicrobial ne Omega-3-Fettsäure enthält. compound solution; such that the composition is formed, wherein fatty acid starting material is 10 3. Zusammensetzung nach Anspruch 2, wobei das Öl fish oil, wherein the antimicrobial compound is ein Fischöl, Olivenöl, Traubenöl, Palmöl oder Lein- a silver compound, and the silver compound is samenöl ist. selected from the group consisting of silver ni- trate, silver chloride, silver fluoride, silver bro- 4. Zusammensetzung nach Anspruch 1 zur Verwen- mide, silver oxide, silver sulfate, silver carbon- 15 dung in einem Verfahren zum Behandeln oder Ver- ate, silver cyanide, silver tetrafluoroborate, sil- hindern von Gewebeanhaftung bei einem Patienten, ver sulfide, silver acetate, silver lactate, silver derdessen bedarf, umfassend das Verabreichen der benzoate, silver cyclohexanebutyrate, silver di- Zusammensetzung nach Anspruch 1 an den Patien- ethyldithiocarbamate, silver trifluorometh- ten. anesulfonate and mixtures thereof. 20 5. Film, umfassend ein antimikrobielles silberhydrati- 9. The composition of claim 1, comprising fish oil cured siertes, von Fettsäure abgeleitetes Biomaterial, das to form a coating that includes the silver fatty acid eine Fettsäure und ein Glycerid umfasst, wobei die salt, wherein silver ions of the silver fatty acid salt Fettsäure- und Glycerid-Komponenten miteinander are characterized by an FTIR peak at about 1512 25 vernetzt sind, um den Film zu bilden, der die ver- cm-1. netzten Fettsäuren und Glyceride enthält, wobei das Biomaterial mit einer wässrigen Form von Silber hy- 10. The composition of claim 9, wherein the coating is dratisiert ist, um Silberfettsäuresalze innerhalb des formed on a medical device. Biomaterials zu bilden, wobei die wässrige Form von 30 Silber aus der Gruppe ausgewählt ist, die aus Silbe- 11. The composition of claim 1, wherein the cross-linked racetat und Silbernitrat besteht. fatty acid and glyceride are derived from fish oil. 6. Film nach Anspruch 5, wobei der Film mit einem Me- 12. The method of claim 8, wherein the composition is dizinprodukt verbunden ist und das Medizinprodukt associated with a medical device, and the medical 35 eine Bandage, ein Stent, ein Transplantat, ein Shunt, device is a bandage, a stent, a graft, a shunt, a cath- ein Katheter, ein chirurgisches Netz oder ein Ballon eter, a balloon, or a surgical mesh. ist.

13. The composition as defined in claim 1 for use in a 7. Medizinprodukt, das zumindest teilweise mit der Zu- method of treating or preventing a bacterial infection 40 sammensetzung nach Anspruch 1 beschichtet ist, in a subject in need thereof, comprising administer- wobei das Medizinprodukt eine Bandage, ein Stent, ing to the subject the composition. ein Transplantat, ein Shunt, ein Katheter, ein Ballon oder ein chirurgisches Netz ist. 14. The composition of claim 10, wherein the medical device is a bandage, a stent, a graft, a shunt, a cath- 45 8. Verfahren zum Bilden einer Zusammensetzung, die eter, a balloon, or a surgical mesh. ein antimikrobielles silberhydratisiertes, von Fett- säure abgeleitetes Biomaterial umfasst, umfassend:

Patentansprüche Härten eines Ausgangsmaterials, das eine Fett- 50 säure und ein Glycerid umfasst, um eine feste 1. Zusammensetzung, umfassend ein antimikrobielles bioabsorbierbare Gelbeschichtung zu bilden, silberhydratisiertes, von Fettsäure abgeleitetes Bio- die die miteinander vernetzten Fettsäuren und material, das eine Fettsäure und ein Glycerid um- Glyceride enthält; und fasst, wobei die Fettsäure- und Glycerid-Komponen- Verbinden der festen bioabsorbierbaren Gelbe- ten miteinander vernetzt sind, um ein bioabsorbier- 55 schichtung mit einer antimikrobiellen Verbin- bares Gel zu bilden, das die vernetzten Fettsäuren dung, die in einer wässrigen Lösung aufgelöst und Glyceride enthält, wobei das Biomaterial mit ei- ist, indem die feste bioabsorbierbare Gelbe- ner wässrigen Form von Silber hydratisiert ist, um schichtung in die antimikrobielle Verbindungs-

23 43 EP 2 464 236 B1 44

lösung eingetaucht wird; sodass die Zusam- 2. Composition selon la revendication 1, dans laquelle mensetzung gebildet wird, wobei Fettsäureaus- la composition est dérivée d’une huile contenant un gangsmaterial Fischöl ist, wobei die antimikro- acide gras oméga-3. bielle Verbindung eine Silberverbindung ist und die Silberverbindung aus der Gruppe ausge- 5 3. Composition selon la revendication 2, dans laquelle wählt ist, die aus Silbernitrat, Silberchlorid, Sil- l’huile est une huile de poisson, une huile d’olive, berfluorid, Silberbromid, Silberoxid, Silbersulfat, une huile de raisin, une huile de palme, ou une huile Silbercarbonat, Silbercyanid, Silbertetrafluor- de lin. borat, Silbersulfid, Silberacetat, Silberlactat, Sil- berbenzoat, Silbercyclohexanbutyrat, Silber- 10 4. Composition selon la revendication 1 pour une utili- diethyldithiocarbamat, Silbertrifluormethansul- sation dans un procédé de traitement ou de préven- fonat und Mischungen davon besteht. tion d’une adhérence tissulaire chez un patient en ayant besoin comprenant l’administration au patient 9. Zusammensetzung nach Anspruch 1, umfassend de la composition selon la revendication 1. Fischöl, das gehärtet ist, um eine Beschichtung zu 15 bilden, die das Silberfettsäuresalz enthält, wobei Sil- 5. Film comprenant un biomatériau antimicrobien dé- berionen des Silberfettsäuresalzes durch eine FTIR- rivé d’un acide gras hydraté contenant de l’argent Spitze bei ungefähr 1512 cm -1 gekennzeichnet sind. comprenant un acide gras, et un glycéride, dans le- quel les composants acide gras et glycéride sont ré- 10. Zusammensetzung nach Anspruch 9, wobei die Be- 20 ticulés l’un à l’autre pour former le film contenant les schichtung auf einem Medizinprodukt gebildet ist. acides gras et les glycérides réticulés, dans lequel le biomatériau est hydraté avec une forme aqueuse 11. Zusammensetzung nach Anspruch 1, wobei die/das d’argent pour former des sels d’acide gras d’argent vernetzte Fettsäure und Glycerid von Fischöl abge- au sein du biomatériau, dans lequel la forme aqueu- leitet sind. 25 se d’argent est sélectionnée dans le groupe consti- tué de l’acétate d’argent et du nitrate d’argent. 12. Verfahren nach Anspruch 8, wobei die Zusammen- setzung mit einem Medizinprodukt verbunden ist 6. Film selon la revendication 5, dans lequel le film est und das Medizinprodukt eine Bandage, ein Stent, associé à un dispositif médical, et le dispositif médi- ein Transplantat, ein Shunt, ein Katheter, ein Ballon 30 cal est un bandage, un stent, un greffon, un shunt, oder ein chirurgisches Netz ist. un cathéter, une maille chirurgicale, ou un ballonnet.

13. Zusammensetzung nach Anspruch 1 zur Verwen- 7. Dispositif médical au moins partiellement revêtu de dung in einem Verfahren zum Behandeln oder Ver- la composition selon la revendication 1, dans lequel hindern einer bakteriellen Infektion bei einem Pati- 35 le dispositif médical est un bandage, un stent, un enten, der dessen bedarf, umfassend das Verabrei- greffon, un shunt, un cathéter, un ballonnet, ou une chen der Zusammensetzung an den Patienten. maille chirurgicale.

14. Zusammensetzung nach Anspruch 10, wobei das 8. Procédé de formation d’une composition compre- Medizinprodukt eine Bandage, ein Stent, ein Trans- 40 nant un biomatériau antimicrobien dérivé d’un acide plantat, ein Shunt, ein Katheter, ein Ballon oder ein gras hydraté contenant de l’argent, comprenant : chirurgisches Netz ist. le durcissement d’un matériau de départ com- prenant un acide gras et un glycéride pour for- Revendications 45 mer un revêtement gélifié bioabsorbable solide contenant les acides gras et les glycérides réti- 1. Composition comprenant un biomatériau antimicro- culés les uns aux autres ; et bien dérivé d’un acide gras hydraté contenant de l’association du revêtement gélifié bioabsorba- l’argent comprenant un acide gras, et un glycéride, ble solide avec un composé antimicrobien qui dans laquelle les composants acide gras et glycéride 50 est dissous dans une solution aqueuse par l’im- sont réticulés l’un à l’autre pour former un gel bioab- mersion du revêtementgélifié bioabsorbable so- sorbable contenant les acides gras et les glycérides lide dans la solution de composé antimicrobien ; réticulés, dans laquelle le biomatériau est hydraté de telle sorte que la composition est formée, avec une forme aqueuse d’argent pour former des dans lequel le matériau de départ de type acide sels d’acide gras d’argent au sein du biomatériau, 55 gras est une huile de poisson, dans lequel le dans laquelle la forme aqueuse d’argent est sélec- composé antimicrobien est un composé d’ar- tionnée dans le groupe constitué de l’acétate d’ar- gent, et le composé d’argent est sélectionné gent et du nitrate d’argent. dans le groupe constitué du nitrate d’argent, du

24 45 EP 2 464 236 B1 46

chlorure d’argent, du fluorure d’argent, du bro- mure d’argent, de l’oxyde d’argent, du sulfate d’argent, du carbonate d’argent, du cyanure d’argent, du tétrafluoroborate d’argent, du sul- fure d’argent, de l’acétate d’argent, du lactate 5 d’argent, du benzoate d’argent, du cyclohexa- nebutyrate d’argent, du diéthyldithiocarbamate d’argent, du trifluorométhane-sulfonate d’argent et des mélanges de ceux-ci. 10 9. Composition selon la revendication 1, comprenant une huile de poisson durcie pour former un revête- ment qui inclut le sel d’acide gras d’argent, dans la- quelle les ions d’argent du sel d’acide gras d’argent sont caractérisés par un pic FTIR à environ 1 512 15 cm-1.

10. Composition selon la revendication 9, dans laquelle le revêtement est formé sur un dispositif médical. 20 11. Composition selon la revendication 1, dans laquelle l’acide gras et le glycéride réticulés sont dérivés d’une huile de poisson.

12. Procédé selon la revendication 8, dans lequel la25 composition est associée à un dispositif médical, et le dispositif médical est un bandage, un stent, un greffon, un shunt, un cathéter, un ballonnet, ou une maille chirurgicale. 30 13. Composition selon la revendication 1 pour une utili- sation dans un procédé de traitement ou de préven- tion d’une infection bactérienne chez un patient en ayant besoin, comprenant l’administration au patient de la composition. 35

14. Compositionselon la revendication 10, danslaquelle le dispositif médical est un bandage, un stent, un greffon, un shunt, un cathéter, un ballonnet, ou une maille chirurgicale. 40

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

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