Nitric oxide generating devices and methods of making the same

Abstract

In an exemplary method, a solution is introduced into an internal opening of a nitric oxide permeable polymer object. The solution comprises a solvent and S-nitroso-1-adamantanethiol, and the S-nitroso-1-adamantanethiol is dissolved in the solvent. The solution is allowed to be soaked in the internal opening of the nitric oxide permeable polymer object for a time of up to 12 hours. The solution is then removed from the internal opening of the nitric oxide permeable polymer object.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Publication Number (43) Publication Date (21) Application Number 202480039944.1 (22) Application Date 2024.05.29 (30) Priority Data 63 / 521207 2023.06.15 US (85) PCT International Application Entering National Phase Date 2025.12.15 (86) PCT International Application Application Data PCT / US2024 / 031413 2024.05.29 (87) PCT International Application Publication Data WO2024 / 258612 EN 2024.12.19 (71) Applicant: University of Michigan Board of Trustees Address: Michigan, USA (72) Inventors: G. Lautner, O. Lautner-Solba, R.H. Bartlett, R. Gorul, M.E. Meyerhoff, S.P. Schwendman (74) Patent Agency: China Patent Agency (Hong Kong) Limited 72001 Patent Attorneys: Ma Weijun, Zhang Hua (51) Int.Cl. A61L 29 / 12 (2006.01) C01B 21 / 24 (2006.01) A61M 25 / 00 (2006.01) (54) Invention Title: Nitric Oxide Generating Apparatus and Method of Manufacturing Thereof (57) Abstract: In an exemplary method, a solution is introduced into an internal opening of a nitric oxide-permeable polymer object. The solution comprises a solvent and S-nitroso-1-adamantanethiol dissolved in the solvent. The solution is allowed to soak in the internal opening of the nitric oxide-permeable polymer object for up to 12 hours. The solution is then removed from the internal opening of the nitric oxide permeable polymer object. Claims 2 pages, Description 15 pages, Drawings 11 pages, CN 121358507 A 2026.01.16 CN 1 21 35 85 07 A 1. A method for generating a nitric oxide generator, comprising: introducing a solution into the internal opening of a nitric oxide permeable polymer object, the solution comprising: a solvent; and S-nitroso-1-adamanthiol dissolved in the solvent; allowing the solution to soak in the internal opening for up to 12 hours, whereby the S-nitroso-1-adamanthiol impregnates the wall of the nitric oxide permeable polymer object in a portion adjacent to the internal opening; and removing the solution from the internal opening. 2. The method as defined in claim 1, wherein: the nitric oxide permeable polymer object is made of poly(vinyl chloride); and the solvent is part of a solvent system further comprising a plasticizer. 3. The method as defined in claim 2,Wherein: the solvent system further comprises a second solvent; and the solvent, the plasticizer, and the second solvent are present in a predetermined volume ratio. 4. The method of claim 3, wherein the predetermined volume ratio is 0.1 to 10 of solvent: 0.1 to 10 of plasticizer: 0.1 to 10 of second solvent. 5. The method of claim 3, wherein the solvent is different from the second solvent and is independently selected from acetone, methanol, ethyl acetate, ethyl ketone, tetrahydrofuran, chloroform, and ethanol. 6. The method of any one of claims 3 to 5, wherein: the solvent is acetone; the plasticizer is trioctyl trimellitate; and the second solvent is methanol. 7. The method of any one of claims 1 to 6, wherein the solution contains up to 2000 mg / mL of S-nitroso-1-adamantanethiol. 8. The method of any one of claims 1 or 7, wherein the nitric oxide-permeable polymeric body is made of silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene. 9. The method of claim 8, wherein: the nitric oxide-permeable polymeric object is made of organosilicon; and the solvent is tetrahydrofuran. 10. The method of claim 8, wherein: the nitric oxide-permeable polymeric object is made of polyurethane; and the solvent is selected from methanol, methyl ethyl ketone, chloroform, cyclohexane, and combinations thereof. 11. The method of any one of claims 1 to 10, wherein prior to introducing the solution, the method further comprises generating the solution by dissolving S-nitroso-1-adamanthiol in a solvent. 12. The method of claim 11, wherein prior to generating the solution, the method further comprises generating S-nitroso-1-adamanthiol by exposing 1-adamanthiol to a solvent-free nitrosation reaction using tert-butyl nitrite. 13. The method as defined in any one of claims 1 to 12, further comprising: introducing a coating solution into the internal opening, the coating solution comprising a direct thrombin inhibitor linker polymer dissolved in a third solvent; and allowing the third solvent to evaporate, thereby forming a coating on the surface of the internal opening, the coating comprising the direct thrombin inhibitor linker polymer. 14. The method as defined in claim 13, wherein prior to introducing the coating solution, the method further comprises forming the coating solution by: dissolving an anchor polymer in the third solvent; introducing a linker molecule into the third solvent, thereby covalently attaching the linker molecule to the anchor polymer; and introducing a direct thrombin inhibitor into the third solvent, thereby covalently attaching the direct thrombin inhibitor to the linker molecule attached to the anchor polymer.To form the direct thrombin inhibitor conjugate polymer; precipitate the direct thrombin inhibitor conjugate polymer; and redissolve the direct thrombin inhibitor conjugate polymer in a fresh third solvent. 15. The method as defined in any one of claims 1 to 14, wherein the S-nitroso-1-adamantanethiol is a tertiary thiol having a molecular weight of about 195 Daltons to about 350 Daltons and a predicted n-octanol-water partition ratio of more than 5. 16. The method as defined in claim 1, wherein: the solvent is part of a solvent system comprising a second solvent; the solvent is different from the second solvent and is independently selected from acetone, methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, cyclohexane, and ethanol; the solvent system comprises the solvent and the second solvent in a predetermined volume ratio; and the predetermined volume ratio is 0.1:10 to 10:0.1. 17. A nitric oxide generating device comprising: a nitric oxide-permeable polymeric body; and S-nitroso-1-adamanthiol impregnated in a portion of the wall of the nitric oxide-permeable polymeric body adjacent to an internal opening of the nitric oxide-permeable polymeric body, wherein the outer surface of the wall of the nitric oxide-permeable polymeric body is substantially free of the S-nitroso-1-adamanthiol. 18. The nitric oxide generating device of claim 17, wherein the S-nitroso-1-adamanthiol extends into the wall for approximately 500 µm to approximately 1000 µm. 19. The nitric oxide generating device of claim 17 or claim 18, wherein the nitric oxide-permeable polymeric body is made of poly(vinyl chloride), silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene. 20. The nitric oxide generating device of any one of claims 17 to 19, further comprising a direct thrombin inhibitor-linked polymer attached to the surface of the internal opening. Claims 2 / 2 Page 3 CN 121358507 A Nitric Oxide Generating Apparatus and Method for Manufacturing the Same

[0001] Cross Reference to Related Applications This application claims the benefit of U.S. Provisional Application Serial No. 63 / 521,207, filed June 15, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Statement Regarding Federally Funded Research or Development This invention was made with government support under EB024038 and HL155100 granted by the National Institutes of Health. The government holds certain rights to this invention.

[0003] Background of the Invention Nitric oxide (NO) is an endogenous gas molecule that has been shown to have several important physiological functions,This includes its unique vasodilatory properties, wound-healing properties, pro-angiogenic properties, anticancer efficacy, antiplatelet activity, and antimicrobial / antiviral activity. In some cases, NO can be used to control infection, prevent biofilm formation, and minimize inflammation and fibrosis.

[0004] Brief Description of the Drawings Features of examples of this disclosure will become apparent from the following detailed description and the accompanying drawings, wherein the same reference numerals correspond to similar but may not be the same components. For the sake of brevity, reference numerals or features having the functions previously described may or may not be described in conjunction with other drawings in which they appear.

[0005] Figure 1 is a schematic and perspective view of an example of a nitric oxide generator, namely an extracorporeal circuit tube comprising S-nitroso-1-adamantanethiol (SNAT) impregnated in the portion of the wall adjacent to the internal opening of the tube; Figure 2A is a schematic diagram of a nitric oxide generator, namely a Y-connector for an extracorporeal blood circuit; Figure 2B is a cross-sectional view taken along line 2B-2B of Figure 2A, schematically depicting S-nitroso-1-adamantanethiol (SNAT) impregnated in the portion of the wall adjacent to the internal opening of the Y-connector and an optional coating of direct thrombin inhibitor attached to the surface of the internal opening; Figure 2C schematically depicts an example of a direct thrombin inhibitor coating on the surface of the internal opening; Figure 3A is a black and white reproduction of a photograph of a portion of a control tube; Figure 3B is a black and white reproduction of a photograph of a portion of an SNAT semi-impregnated tube; Figure 4 depicts the nitric oxide (NO) flux (Y-axis, × 10⁻¹⁰ mol min⁻¹) of the SNAT semi-impregnated tube. Figure 5 shows microscopic images (reproduced in black and white) of the performance of control tubes and SNAT semi-impregnated tubes on biofilm formation using Staphylococcus aureus and Pseudomonas aeruginosa strains; Figure 6 is a schematic diagram of the experimental setup for the in vivo test described in the Examples section; Figure 7A is a black and white photograph of the interior of the SNAT semi-impregnated tube after 4 hours of in vivo testing; Figure 7B is a black and white photograph of the interior of the control tube after 4 hours of in vivo testing; Figure 8 is a bar graph depicting platelet counts (Y-axis, %) after exposure of the SNAT semi-impregnated tubes and control tubes in an in vitro circuit for different time periods (X-axis, minutes); Figure 9 shows multiple field emission scanning electron microscope images (reproduced in black and white) of the control (unmodified) polymer (row A), the example SNAT-doped polymer (row B), and the comparative S-nitroso-N-acetylpenicillamine (SNAP)-doped polymer (row C). Figure 10 depicts the nitric oxide (NO) flux (Y-axis) of two different SNAT fully impregnated tubes.Figure 11 is a bar graph depicting the 1-year stability of SNAT fully impregnated tubes exposed to different storage temperatures, where stability is shown as the relationship between nitric oxide (NO) flux (Y-axis, ×10⁻¹⁰ mol min⁻¹ cm⁻²) and time (X-axis, 1 year + 0, 1, or 2 days); Figure 12 is a bar graph depicting the stability of control tubes and SNAT fully impregnated tubes exposed to different sterilization conditions, where stability is shown as the relationship between nitric oxide (NO) flux (Y-axis, ×10⁻¹⁰ mol min⁻¹ cm⁻²) and time (X-axis, days); Figure 13A is a bar graph depicting platelet aggregation (Y-axis, %) of SNAT-coated tubes and control tubes after exposure to different time periods (X-axis, minutes) in an in vitro circuit. Figure 13B is a bar graph depicting the extracorporeal circuit thrombus area (Y-axis, pixels / cm2) of SNAT-coated tubes and control tubes exposed to extracorporeal circuit flow for 4 hours; Figure 14A is a bar graph depicting platelet aggregation (Y-axis, %) of SNAT-completely impregnated tubes and control tubes after exposure to extracorporeal circuit flow for different time periods (X-axis, minutes); Figure 14B is a bar graph depicting the extracorporeal circuit thrombus area (Y-axis, pixels / cm2) of SNAT-completely impregnated tubes and control tubes exposed to extracorporeal circuit flow for 4 hours; Figure 15 depicts a confocal image (reproduced in black and white) of the performance of control tubes and SNAT-completely impregnated tubes on biofilm formation using Staphylococcus aureus and Escherichia coli strains; and Figure 16 is a graph depicting the nitric oxide (NO) flux (Y-axis, ×10⁻¹⁰ mol min⁻¹ cm⁻²) vs time (X-axis, days) of another example of SNAT semi-impregnated tubes.

[0006] The invention details that nitric oxide (NO) donor molecules have been incorporated into coatings on polymer objects or have been impregnated into polymer objects. Exemplary polymer objects include extracorporeal circuit components such as tubes, catheters (e.g., hemodialysis catheters) and / or connectors, other catheters (e.g., urinary catheters, vascular catheters), pump chambers, etc. NO-releasing polymer objects have been used in a variety of biomedical applications, including extracorporeal circuits, to reduce thrombosis, platelet aggregation, and prevent bacterial infection and biofilm formation.

[0007] The inventors have found that some NO donor molecules, such as diazenium diolate NO donors and S-nitrosothiol type NO donors, may be limited in use and / or lead to undesirable results. As one example, diazenium diolate NO donors (e.g., DBHD-N2O2) involve limitations in use because they cannot be impregnated or are difficult to impregnate into the host material. In another instance, the azodium glycol NO donor coated on the external circuit tubing may be contaminated with carcinogenic nitrosamines. In yet another instance,S-nitrosothiol type NO donors (e.g., S-nitroso-N-acetylpenicillamine (SNAP)) coated onto or impregnated within polymer objects may adversely alter the surface roughness and / or mechanical properties of the polymer object. The solubility of S-nitrosothiol type NO donors is also limited. Therefore, in the case of impregnated S-nitrosothiol type NO donors, changes in mechanical properties may be due to recrystallization of the impregnated material.

[0008] The inventors have also discovered that some NO donor introduction techniques, such as coating techniques (e.g., dip coating, spin coating, etc.) or complete impregnation, may also lead to undesirable results. Coating techniques may result in undesirable increases in surface roughness, tube (or other component) thickness, and / or peeling. Complete impregnation can impair the mechanical integrity of the tube (or other component) and may require a large number of NO donor molecules.

[0009] The examples disclosed herein employ a tertiary nitrosothiol (S-nitroso-1-adamantane thiosulfate, specification 2 / 15 page 5 CN 121358507 A alcohol or SNAT) containing adamantane molecules as the NO donor molecule, and also employ a method of semi-impregnating the polymer object with SNAT. "Semi-impregnation" means that a portion of the polymer object exposed to a solution containing SNAT is impregnated, while the other portion of the polymer object remains free of SNAT. The resulting NO-generating object provides a desired level of NO (e.g., a minimum of 0.5 flux units to 4 flux units) for a desired time (e.g., weeks to months) without compromising the mechanical integrity of the polymer object.

[0010] In some examples, the semi-impregnated polymer object further includes a direct thrombin inhibitor-linked polymer coating on the surface of the polymer object to be exposed to blood or other fluids. The direct thrombin inhibitor-linked polymer coating can contribute to anticoagulation. As two examples, the direct thrombin inhibitor may be bivalirudin or argatroban.

[0011] The adamantane moiety enhances the lipophilicity of the drug molecule. The simplest tertiary S-nitrosothiol having adamantane is S-nitroso-1-adamantanethiol (SNAT, thionitrous acid (HNOS), S-tricyclic [3.3.1.13,7]dec-1-yl ester): .

[0012] In one example, the S-nitroso-1-adamantanethiol is a tertiary thiol having a molecular weight of about 195 Daltons to about 350 Daltons and a predicted n-octanol-water partition ratio of more than 5 (e.g., 5.9). This molecule is capable of spontaneously releasing NO when the internal opening of the polymer object (in which SNAT is semi-impregnated) is exposed to solution and / or blood under physiological conditions. In one specific example, the NO generating device exhibits antimicrobial and bactericidal effects (wherein bacterial CFU / mL is reduced by at least 2 logarithmic orders) for at least 7 days. In another specific example,The NO generating device is capable of continuously releasing a sufficient amount of NO within or above the normal endothelial range (0.5–4.0 × 10⁻¹⁰ mol min⁻¹ cm⁻²) for more than a month (e.g., approximately 35 days).

[0013] In the examples disclosed herein, the molecule is used as a hydrophobic NO donor to produce a biocompatible surface with antithrombotic and antimicrobial properties. In particular, SNAT provides desirable surface anticoagulation within the internal openings of its semi-impregnated polymeric object. This eliminates the need for systemic anticoagulation. SNAT is believed to be suitable for modifying all extracorporeal circuit components and for applications such as hemodialysis, hemofiltration, cardiopulmonary bypass (CPB), cardiac pacing leads, urinary catheters, and artificial lung shells. Furthermore, the NO-releasing capacity of the SNAT polymer provides antithrombotic effects (as demonstrated in the in vivo models in the Examples section) by inhibiting platelet activation and platelet adhesion (aggregation) to the surface of the internal openings.

[0014] SNAT is highly lipophilic (logD = 5.9), and therefore less readily (e.g., compared to other NO donors such as SNAP or azomonium glycol salts) leach from lipophilic polymers into aqueous media and / or blood. SNAT is also pH-independent, unlike other NO donors (thiols: S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), or diamines: DBHD-N2O2). Furthermore, SNAT is highly soluble in the solvents or solvent systems disclosed herein, thereby simplifying the semi-impregnation process. After semi-impregnation, SNAT remains dissolved within the polymer phase, thereby preventing crystal formation and allowing the polymer object to retain its mechanical properties. Dissolved SNAT also enables a higher effective loading within the polymer object compared to crystalline NO donors. Moreover, SNAT provides a relatively continuous release of NO (i.e., no lag time when exposed to solution and / or blood under physiological conditions). SNAT also does not contain amine functional groups, thereby eliminating the formation of carcinogenic nitrosamines. Therefore, compared to other polymeric objects modified for NO generation, SNAT semi-impregnated polymeric objects can exhibit reduced cytotoxicity.

[0015] Exemplary NO generating devices 10, 10' are shown in Figures 1 and 2A. Devices 10, 10' generally include a polymeric object 12 having an internal opening 16 and an SNAT 14 semi-impregnated in the portion of the polymeric object 12 adjacent to the surface 17 of the internal opening 16.

[0016] In each example, the nitric oxide-permeable polymeric object 12 is formed of a polymer capable of releasing NO generated by the SNAT (i.e., the NO donor in the examples disclosed herein). In one example,The nitric oxide-permeable polymeric object 12 is made of poly(vinyl chloride) (PVC, medical or non-medical grade), silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene. The polymeric object 12 may also include plasticizers such as tris(2-ethylhexyl) trimellitate (i.e., trioctyl trimellitate (TOTM), a phthalate-free plasticizer) or bio-based plasticizers. Bio-based plasticizers can be any plasticizer derived from biomass resources, such as vegetable oils, cashew nut shells, vegetable fatty acids, glycerol, and / or citric acid. These plasticizers have been extensively studied as alternatives to petroleum-based phthalate plasticizers. As an example, PVC pipes containing the plasticizer are commercially available under the trade name TYGON S3™ E-3603 (from Saint-Gobain Performance Plastics Corp.). As other examples, silicone, polyurethane, polycarbonate, and polypropylene may not contain the plasticizer.

[0017] The polymeric object 12 can be any medical device having an internal opening 16 that will come into contact with blood or another bodily fluid (such as urine) during use. As an example, the polymeric object 12 can be any external circuit assembly, such as a tube (schematically shown in FIG. 1), a catheter and / or connector (schematically shown in FIG. 2A), other catheters, a pump chamber, or any other medical device that will benefit from the generation of nitric oxide at its internal opening 16 during its use. The internal opening 16 can be the lumen of the tube or catheter or connector, the interior of a pump chamber, etc.

[0018] An example of a NO generating device 10 is shown in FIG. 1. This example is an external circuit tube. As shown in Figure 1, the NO generating device 10 includes a nitric oxide-permeable polymer body 12 and S-nitroso-1-adamanthiol (SNAT) 14 impregnated in the portion of the wall W of the nitric oxide-permeable polymer body 12 adjacent to an internal opening 16 of the nitric oxide-permeable polymer body 12, wherein the outer surface 18 of the wall W of the nitric oxide-permeable polymer body 12 is substantially free of S-nitroso-1-adamanthiol 14. "Substantially free" means that less than 1% of SNAT 14 is present at the outer surface 18 of the polymer body 12 (i.e., partially impregnated). In some instances, SNAT 14 is not present in the portion of the wall W of the polymer body 12 adjacent to the outer surface 18 of the wall W. The phrases "at the outer surface 18" and "in the portion of the wall W of the polymer body 12 adjacent to the outer surface 18" include the outer surface 18 and the distance extending from the outer surface 18 into the wall thickness. In one instance, approximately 25% to approximately 80% of the wall W thickness (as measured from the outer surface 18) is substantially free of SNAT 14. Thus,The portion of the wall W that is substantially free of SNAT 14 will depend on the wall thickness. In one specific example, when the wall thickness is 1 / 16 inch (1,587.5 μm), SNAT 14 is absent in the wall W within 500 μm of the outer surface 18. In this particular example, approximately 31% of the wall W (measured inward from the outer surface 18) is free of SNAT 14.

[0019] SNAT 14 is semi-impregnated using the method disclosed herein, and thus SNAT 14 is located within the portion of the wall W of the nitric oxide permeable polymer body 12 adjacent to its internal opening 16, and not at the outer surface 18 of the wall W (as described above). SNAT 14 is located near the surface 17 adjacent to the internal opening 16. The phrase "near the surface 17" means that SNAT 14 is present on the inner surface 17 and at a distance extending into the wall thickness as measured from the surface 17. As described above, this distance is less than the total thickness of the wall W because a portion of the outer surface 18 is substantially free of SNAT 14. In one example, approximately 20% to approximately 75% of the wall W thickness (as measured from the inner surface 17) comprises the SNAT 14. In one example, the wall W has a thickness of approximately 3 / 32 inch (approximately 2,381 μm), and the SNAT 14 extends from the inner surface 17 into the wall W by approximately 500 μm to approximately 1000 μm. The degree of semi-impregnation of the SNAT 14 can depend on the thickness of the wall W. Thinner walls can have a smaller impregnation distance, while thicker walls can have a larger impregnation distance (measured from the inner surface 17).

[0020] As described, S-nitroso-1-adamanthiol remains dissolved in the polymer phase, and thus the NO generating device 10 does not include solid S-nitroso-1-adamanthiol.

[0021] As shown in the virtual image in FIG1, the inner surface 17 may also be coated with a direct thrombin inhibitor linked polymer 20. The direct thrombin inhibitor connecting polymer 20 will be further described with reference to FIG2C.

[0022] Another example of the NO generating device 10' is shown in FIG2A. This example is an extracorporeal circuit Y-connector. As shown in the cross-sectional views of FIG2A and FIG2B, the NO generating device 10' includes a nitric oxide permeable polymer body 12; and S-nitroso-1-adamanthiol (SNAT) 14 impregnated in the portion of the wall W of the nitric oxide permeable polymer body 12 adjacent to the internal opening 16 of the nitric oxide permeable polymer body 12. Similar to FIG1, the outer surface 18 of the wall W of the nitric oxide permeable polymer body 12 is substantially free of S-nitroso-1-adamanthiol 14.

[0023] As shown in the virtual image in FIG2B,The inner surface 17 may also be coated with a direct thrombin inhibitor linker polymer 20.

[0024] Referring now to FIG. 2C, an example of a direct thrombin inhibitor linker polymer 20 is schematically depicted. The direct thrombin inhibitor linker polymer 20 includes an anchor polymer 22, a linker molecule 24 attached to the anchor polymer 22, and a direct thrombin inhibitor molecule 26 attached to the linker molecule 24. Any anchor polymer 22 capable of forming a coating on the inner surface 17 of the base polymer (i.e., polymer body 12) and containing surface functional groups to attach to the linker molecule 24 can be used. Similarly, any linker molecule 24 containing functional groups to attach to the anchor polymer 22 and to the direct thrombin inhibitor molecule 26 can be used.

[0025] In one example, the anchor polymer 22 is a polyurethane or a polyurethane copolymer (e.g., CARBOSIL®—a copolymer of silicone and polycarbonate-urethane—owned by DSM IP Assets), and the linking molecule is 4,4'-methylene-bis(cyclohexyl isocyanate) (HMDI) or polyethylene glycol (PEG). In one example, the direct thrombin inhibitor molecule 26 is bivalirudin, having the following structure: , and the guanidine group at the N-(amino) terminus can serve as an anchoring point for the isocyanate group to the HMDI linking molecule via a linker. The direct thrombin inhibitor molecule 26 can be 100% pure bivalirudin or its trifluoroacetate salt (bivalirudin TFA). As other examples, the direct thrombin inhibitor can be pirudin, desirudin, and argatroban. Other anchor polymers 22 include poly(vinyl chloride), silicone, or polyurethane, or any combination of the listed polymers. Subsequently, linker molecule 24 is selected to attach to a specific anchor polymer 22.

[0026] The semi-impregnation method disclosed herein includes introducing a solution into an internal opening 16 of a nitric oxide permeable polymer body 12, the solution comprising a solvent and S-nitroso-1-adamanthiol 14 dissolved in the solvent; allowing the solution to soak in the internal opening 16 for up to 12 hours; and removing the solution from the internal opening 16 of the nitric oxide permeable polymer body 12.

[0027] SNAT 14 is nonpolar and therefore hydrophobic. The selected solvent or solvent system should dissolve SNAT (see page 5 / 15 of specification 8 CN 121358507 A 14). In some cases, the selected solvent may also be nonpolar. In some instances, the solvent is used alone (i.e., without any other solvent or plasticizer). In other instances, the solvent is part of a solvent system containing one or more other solvents. When a combination of solvents is used, one of the selected solvents is more nonpolar than some of the other solvents. For example,In the case of any of methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, or cyclohexane, acetone may be selected. In some other examples, the solvent is part of a solvent system containing a plasticizer. In a further example, the solvent is used in combination with another solvent and a plasticizer.

[0028] The solvent used in the semi-impregnation method can dissolve SNAT but not the polymer body 12. However, the solvent can penetrate into the polymer body 12. The solvent is selected from acetone, methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, cyclohexane, and ethanol.

[0029] In one example of a single solvent system, the nitric oxide-permeable polymer body 12 is made of organosilicon, and the solvent is tetrahydrofuran.

[0030] When multiple solvents are used, the hydrophobicity and lipophilicity of the selected solvents will be related to the volume ratio used. In one example, first and second solvents that are different from each other are used. The solvent is different from the second solvent and is independently selected from acetone, methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, cyclohexane, and ethanol. In this example, the solvent system includes a solvent and a second solvent in a predetermined volume ratio, and the predetermined volume ratio is 0.1:10 to 10:0.1. In a specific example, when using two solvents, the volume ratio of the two solvents can be 2:1 to 1:2. It should be understood that any volume ratio within the widest range can be used, such as 1:1, 1.5:1, 1:1.5, etc.

[0031] In one example of a dual-solvent system, the nitric oxide-permeable polymer body 12 is made of PVC, and the two solvents are acetone and methanol.

[0032] In another example of a dual-solvent system, the nitric oxide-permeable polymer body 12 is made of polyurethane, and the two solvents are methanol and chloroform. In the case of polyurethane, the solvent can be selected from methanol, methyl ethyl ketone, chloroform, cyclohexane, and combinations thereof.

[0033] In some instances, the solvent system further comprises a plasticizer. The plasticizer is added when the NO-permeable polymer body 12 contains a plasticizer as part of its composition (e.g., some PVC polymer bodies). The additional plasticizer in the solvent system can replenish the plasticizer leached from the NO-permeable polymer body 12 during the semi-impregnation process. The minimum predetermined volume ratio of the plasticizer is ≥0.1 to ensure that the overall mechanical properties of the NO-permeable polymer body 12 remain substantially unchanged after the semi-impregnation process. When the plasticizer is added to the solvent, the predetermined volume ratio is the volume ratio of the first solvent:plasticizer:second solvent. As an example, this predetermined volume ratio is 0.1 to 10 for the first solvent: 0.1 to 10 for the plasticizer: 0.1 to 10 for the second solvent. As a specific example,The volume ratio can be 1:3:1, 1.5:2:1.5, 1:2:2, 2:1:2, or 2:2:1. Ratios between the widest possible volume ratios can also be used. Other volume ratios can also be used, as long as they can dissolve the selected amount of SNAT. While volume ratios are described herein, it should be understood that the given ratios also apply to weight ratios.

[0034] Partly due to the very high lipophilicity of SNAT, the amount of SNAT contained in the solution depends on the application. Typically, the solution contains up to 2000 mg / mL of S-nitroso-1-adamantanethiol. For a polymer object 12 with a wall W of 1 / 16” (1,587.5 μm) thickness, the concentration of S-nitroso-1-adamanthiol in the solution may be 1000 mg / mL. For a thinner wall W, the concentration of S-nitroso-1-adamanthiol may be less than 1000 mg / mL (e.g., 50 mg / mL to 900 mg / mL). For a thicker wall W, the concentration of S-nitroso-1-adamanthiol may be greater than 1000 mg / mL (e.g., from greater than 1000 mg / mL to at most 2000 mg / mL).

[0035] After the solution is introduced into the internal opening 16 of the nitric oxide-permeable polymer object 12, the internal opening 16 may be sealed for an incubation period. The incubation period is at most 12 hours. In some cases, the incubation period is less than the specified period. 12 hours. In one example, the incubation period is from 1 hour to about 8 hours. As a specific example, when the wall W thickness is about 0.6 mm (600 μm), the incubation period is from about 30 minutes to about 2.5 hours. In another example, the incubation period is 4 hours (which is particularly desirable for maintaining the mechanical properties of polymer objects having a wall W thickness of about 1.5875 mm (1,587.5 μm).

[0036] After the incubation period has ended, the solution is removed from the internal opening 16. When the polymer object 12 is transparent or translucent, the SNAT-impregnated portion of the polymer object 12 will appear green.

[0037] Before introducing the solution into the internal opening 16, the method may further include generating the solution by dissolving S-nitroso-1-adamanthiol in a solvent system. Prior to generating the solution, the method further includes generating S-nitroso-1-adamanthiol by exposing 1-adamanthiol to nitrosation: In the examples disclosed herein, the nitrosation process is carried out as a solventless nitrosation using tert-butyl nitrite (i.e., tert-butylnitrite (tBuNO2)). The tert-butyl nitrite acts as both a solvent and a reagent in the nitrosation process.And thus it is considered solvent-free. The solvent-free method avoids solvents that could otherwise adversely affect the stability of SNAT. During this method, 1-adamanthiol is mixed with the desired amount (e.g., 1.1 molar equivalents) of tert-butyl nitrite and allowed to react. Initial stirring can be performed, and the reaction vessel is then placed on ice for a predetermined time (e.g., 30 minutes) because the reaction is exothermic. The reaction can then be allowed to proceed at room temperature (e.g., 18°C ​​to 21°C) for a desired time of up to 12 hours. After the reaction, excess tert-butyl nitrite and the byproduct tert-butanol (t-BuOH) are removed, for example, by rotation at room temperature. The product can then be vacuum dried overnight.

[0038] It should be understood that the solvent-free nitrosation for the formation of SNAT can be carried out in conjunction with the semi-impregnation method or as a separate method to produce SNAT. In the latter case, the solvent-free nitrosation is not carried out in conjunction with the semi-impregnation method disclosed herein.

[0039] As a result of the semi-impregnation method, S-nitroso-1-adamantanethiol 14 extends from the inner surface 17 into the wall W as described herein. In one example, SNAT 14 is present at the inner surface 17 and subsequently extends into the wall W by approximately 500 μm to approximately 1000 μm. In other words, S-nitroso-1-adamantanethiol is present at the inner surface 17 of the internal opening 16 and extends a distance into the wall thickness, wherein, as described herein, this distance is less than the total thickness. The remainder of the wall thickness extending to the outer surface 18 is substantially free of S-nitroso-1-adamantanethiol.

[0040] Some examples of the method further include incorporating a direct thrombin inhibitor linker polymer 20 as a coating on the inner surface 17. The direct thrombin inhibitor linker polymer 20 may be formed prior to its application to the inner surface 17.

[0041] In one exemplary method, the direct thrombin inhibitor linker polymer 20 can be formed by: dissolving an anchor polymer 22 in a solvent; introducing a linker molecule 24 into the solvent, whereby the linker molecule 24 is covalently attached to the anchor polymer 22; introducing a direct thrombin inhibitor 26 into the solvent, whereby the direct thrombin inhibitor 26 is covalently attached to the linker molecule 24 attached to the anchor polymer 22, to form the direct thrombin inhibitor linker polymer 20. Any instance of the anchor polymer 22 and linker molecule 24 set forth herein can be used, and the selected solvent is capable of dissolving each of components 22, 24, and 26. In the exemplary method, precipitation, drying, and redissolution can be performed between reactions. For example, after the linker molecule 24 is attached to the anchor polymer 22, precipitation can be performed with hexane, and the solid product (e.g., pellets) can be dried and redissolved in fresh solvent before the introduction of the direct thrombin inhibitor 26. Subsequently,After the introduction of the direct thrombin inhibitor 26, the reaction can be allowed to proceed for a predetermined amount of time (e.g., 20 to 40 minutes), and then precipitation can be carried out with water. The solid product (e.g., pellets of the direct thrombin inhibitor-linked polymer 20) can be dried and redissolved in fresh solvent. In one example, the final solution contains approximately 10 µM of the direct thrombin inhibitor-linked polymer 20.

[0042] In one example of the method, the anchor polymer 22 is in the form of small polymer beads. In another example of the method, the solvent is tetrahydrofuran (THF).

[0043] Once the direct thrombin inhibitor-linked polymer solution is formed, it is introduced into the internal opening 16. After a few seconds, the solution drains from the internal opening 16. The solvent is allowed to evaporate, which occurs relatively quickly, leaving a coating of the direct thrombin inhibitor-linked polymer 20 on the surface 17 of the internal opening 16 (of the base polymer 12). It should be understood that the direct thrombin inhibitor-linked polymer 20 is NO-permeable and therefore does not inhibit the action of the semi-impregnated SNAT 14.

[0044] Various examples are given herein to further illustrate the present disclosure. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the present disclosure. Examples

[0045] Example 1 Initially, several examples of NO-generating polymer tubes disclosed herein were produced using medical-grade PVC TYGON™ ND-100-65 tubes (without topcoat and inner diameter 3 / 8") and SNAT. Two different solutions and two different half-immersion times (4 hours or 12 hours) were tested. The solvent systems of the solutions comprised acetone:plasticizer:methanol in ratios of 1:3:1 and 2:1:2. Each solution contained 1000 mg / mL of SNAT, and the plasticizer was tri(2-ethylhexyl) trimellitate (TOTM).

[0046] The example tubes were filled with the corresponding solutions, sealed, and allowed to incubate for 4 hours or 12 hours.

[0047] To compare mechanical properties, various control polymer tubes were prepared according to Table 1. These controls were filled with different solvent systems (excluding SNAT) for a sustained half-immersion time (in Table 1, "n" indicates the number of tests for each type). Young's modulus was measured, and the average results for the control polymer tubes are also shown in Table 1 (where SEM is the average standard error).p < 0.05). The first naïve control value was provided by the manufacturer. The second naïve control value represents the measured value of the as-received tube without any solvent exposure.

[0048] Table 1 Specification 8 / 15 pages 11 CN 121358507 A.

[0049] These results confirm the effect of swelling time and solvent volume ratio on the mechanical properties of tubes without added SNAT. Longer incubation time has a greater effect on Young's modulus and may be more desirable when using thicker polymer objects.

[0050] The Young's modulus of a polymer tube of one example (containing a 1:3:1 solvent system and a solution of SNAT, incubated for 4 hours) was also measured, and the average results are also shown in Table 2 (where SEM is the standard error mean, p < 0.05). For ease of comparison, some controls in Table 1 are also repeated in Table 2.

[0051] Table 2.

[0052] These results indicate that SNAT semi-impregnation does not harmfully affect the mechanical properties of the polymer tube.

[0053] The additional example NO-generating polymer tubes were generated using medical-grade PVC TYGON™ ND-100-65 tubes (uncoated and 3 / 8" inner diameter) and SNAT. Two different solutions (acetone:TOTM:methanol, volume ratios of 1:3:1 and 2:1:2) were tested at a 4-hour half-immersion time. As above, each solution contained 1000 mg / mL of SNAT. The tubes were filled with the corresponding solutions, sealed, and allowed to incubate for 4 hours.

[0054] After the incubation period, the solutions were emptied. A portion of one of the original control sample and the additional example sample was photographed, and the black and white reproductions are shown in Figures 3A and 3B, respectively. The original control sample remained transparent, while the example sample included a green portion (depicted as a darker black portion in Figure 2B). The green in the example tubes is evidence of SNAT half-immersion.

[0055] Compared with the original control (ECC) (medical-grade PVC TYGON™) Compared to ND-100-65 tubes (no solution exposure, no outer coating, and 3 / 8” inner diameter, as per specification page 9 / 15, CN 121358507 A), the NO donor loading, NO release curve (measured using ozonoluminescence, see Figure 4), and tensile strength (measured using a texture analyzer) of the tubes were tested in vitro. The antibacterial properties of the NO-generating polymer tubes of the examples were also evaluated for 7 days using Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacterial strains (see Figure 5). The tubes were filled with the corresponding bacteria and allowed to incubate for 7 days, after which the bacteria were removed.Samples were tested using CDC closed containers. All these results are summarized in Table 3.

[0056] Table 3.

[0057] The semi-impregnation method, compared with full impregnation, estimated a 50% reduction in NO donor consumption, making the method disclosed herein more economical. The 2:1:2 solvent combination produced a significantly higher loading when compared with the 1:3:1 solvent combination (p ≤ 0.05, Table 3, day 35).

[0058] The tensile strength of the Example 1:3:1 group was closest to the original control (p ≥ 0.05, Table 1) and maintained better original properties than the Example 2:1:2 group (Table 3). When comparing NO release values ​​on day 35, the Example 2:1:2 group had a higher overall NO release capacity, but the Example 1:3:1 group still showed sufficient NO flux (Table 3, Figure 4).

[0059] Biofilm studies also showed sufficient antimicrobial properties in both groups, with a reduction in bacterial colonies of approximately 2 logarithmic orders (Table 3, Figure 5). The confocal images in Figure 5 clearly show more biofilm formation in the control group compared to the Example 1:3:1 group.

[0060] The anticoagulant properties of the Example 1:3:1 group (n=3) were also tested in vivo in an acute rabbit model and compared with the original control (n=9). The in vivo study was a 4-hour test. The experimental setup is shown in Figure 6. An arteriovenous shunt was used in the rabbits. An extracorporeal circuit (ECC) loop with an indwelling needle (angiocath) (16G) was introduced into the lower common carotid artery, and an extracorporeal circuit (ECC) loop with an indwelling needle (14G) was introduced into the right jugular vein, and flow was allowed from the lower common carotid artery through the thrombus formation chamber and into the right jugular vein. In this experiment, smaller thrombus area was observed at the end of the test: SNAT 0.5±0.4 cm2 vs control 9.8±0.8 cm2; and higher platelet retention: 108% vs 74% of baseline (Figure 8). Figures 7A and 7B show images (reproduced in black and white) of the example and control tubes after 4 hours of blood exposure. The SNAT semi-impregnated tube is green with a small amount of thrombus. In contrast, the control tube is red due to being entirely thrombus. Clearly, the SNAT semi-impregnated tube exhibits better anticoagulant properties.

[0061] The data in this example clearly demonstrate that SNAT semi-impregnation provides a more cost-effective surface modification method with suitable NO flux and retains the original mechanical properties of the polymer used. It also has excellent antithrombotic properties without requiring systemic anticoagulation in vivo.

[0062] Example 2 Specification 10 / 15 pages 13 CN 121358507 A Initially, medical grade PVC TYGON™ ND-100-65 tubes (without outer coating) were used.And an example of NO-generating polymer tubes with an inner diameter of 3 / 8” and SNAT was produced. A solution was tested. The solvent system of the solution consisted of acetone:plasticizer:methanol in a volume ratio of 1:2:2. The solution contained 1000 mg / mL of SNAT and the plasticizer was tri(2-ethylhexyl) trimellitate (TOTM).

[0063] The example tubes were filled with the solution, sealed, and allowed to incubate for 4 hours.

[0064] The Young's modulus of the control and example tubes was measured, and the average results of the four control polymer tubes and the four example tubes are shown in Table 4 (where SEM is the standard error mean, p<0.05).

[0065] Table 4.

[0066] Similar to the results in Example 1, these results confirm that SNAT semi-impregnation does not adversely affect the mechanical properties of the polymer tubes.

[0067] Compared with the original control (ECC) (medical grade PVC TYGON™) NO release curves of the example tubes were tested in vitro (measured using ozonoluminescence, see Figure 16) compared to ND-100-65 tubes (no solution exposure, no outer coating, and 3 / 8” inner diameter). The 1:2:2 solvent combination produced the desired NO flux (>0.5 × 10⁻¹⁰ mol / min / cm² flux) for more than 14 days and was at the threshold on day 21.

[0068] One of the example tubes was exposed to a bivalirudin-linked polymer to form a coating on the inner surface. The bivalirudin-linked polymer was prepared as described herein by dissolving CARBOSIL® beads in tetrahydrofuran and subsequently adding HMDI. The reaction product was precipitated using hexane and the precipitate was dried. The precipitate was dissolved in fresh THF and then bivalirudin trifluoroacetate or bivalirudin (100% pure) was added. The reaction was allowed to proceed for approximately 20 minutes and the reaction product was subsequently precipitated with water. The precipitate was dried and then dissolved in fresh THF. A solution containing a bivalirudin-linked polymer was added to the interior of the SNAT semi-impregnated example tube, and the solvent was evaporated. The bivalirudin-linked polymer coated the inner surface of the example tube.

[0069] As described in Example 1 and Figure 6, the example tube and a control tube were tested in vivo in rabbits. After removal from the rabbits, no clots were observed in the SNAT semi-impregnated tube coated with the bivalirudin-linked polymer. In contrast, it was 10.97 ± 0.95 cm2 (data ± standard deviation, n = 7) in the original untreated tube.

[0070] These results demonstrate the additional effects of the bivalirudin-linked polymer with semi-impregnated SNAT.

[0071] Example 3 An example of using a polyurethane vascular catheter and an SNAT-generating NO-generating polymer vascular catheter. Two solutions were tested. The solvent system for each solution consisted of methanol:chloroform in a volume ratio of 1:1. One solution contained 1000 mg / mL of SNAT,Another solution contained 600 mg / mL SNAT.

[0072] The vascular catheters of the examples were filled with the solution, sealed, and allowed to incubate for 4 hours.

[0073] The NO release curves of these vascular catheters of the examples were tested in vitro (measured using ozonoluminescence). The average results of three for each example are shown in Table 5.

[0074] Table 5 Specification 11 / 15 pages 14 CN 121358507 A.

[0075] The examples prepared with 1000 mg / mL SNAT showed the desired NO flux (>0.5 × 10⁻¹⁰ mol / min / cm² flux) for more than 31 days and were at the threshold on day 35. The examples prepared with 600 mg / mL SNAT showed the desired NO flux (>0.5 × 10⁻¹⁰ mol / min / cm² flux) for more than 14 days and were below the threshold on day 21. These results demonstrate the effectiveness of the semi-impregnation method in polyurethane objects.

[0076] Example 4: A NO-generating polymer tube was produced using silicone tubes and SNAT. A solution containing 70 mg / mL SNAT in tetrahydrofuran was tested.

[0077] The silicone tubes of the example were filled with the solution, sealed, and allowed to incubate for 4 hours.

[0078] The NO release curves of the tubes of the example were tested in vitro (measured using ozonoluminescence). The results are shown in Table 6.

[0079] Table 6: Specification, 12 / 15 pages, 15 CN 121358507 A.

[0080] The example prepared with 70 mg / mL SNAT in silicone exhibited near-desirable NO flux (>0.5 × 10⁻¹⁰ mol / min / cm² flux) for 4 days. It is believed that these results will improve with higher SNAT loadings. These results demonstrate the effectiveness of the semi-impregnation method in silicone objects.

[0081] Comparative Examples: SNAT was used in the case of a full impregnation technique or a coating technique. SNAT was synthesized as described herein, and the in vitro circuit (PVC tubing (ND 100-65 TYGONT™, 3 / 8” ID) and connectors, as well as sleeves) were modified with the NO donor via a complete impregnation or coating technique. The complete impregnation technique involves completely immersing the in vitro circuit in a solution of SNAT (1000 mg / mL) in a combination of an organic solvent and a plasticizer (acetone:plasticizer:methanol, 1:3:1) until the circuit swells in the solution. The circuit is then dried. The coating technique involves dissolving SNAT in a polymer solution and applying the solution to the inner surface of the ECC assembly and drying. In other complete impregnation techniques,SNAP was used as the NO donor. The control examples were identical to the original control examples.

[0082] To evaluate the surface roughness of the NO donor-doped polymers (SNAP, SNAT) and the unmodified control polymers, field emission scanning electron microscopy (JEOL JSM-7800F) was used. These images are shown in Figure 9, where row A depicts the original control examples, row B depicts PVC tubes fully impregnated with SNAT (1000 mg / ml), and row C depicts PVC tubes fully impregnated with SNAP (250 mg / ml, maximum load limit). The PVC surface loaded with SNAT looks similar to the original control, but is more exaggerated than the original control. The PVC surface loaded with SNAP clearly shows the difference.

[0083] In vitro NO release curves of PVC loop tubes fully impregnated with SNAT (600 mg / ml and 1000 mg / ml) were measured by a nitric oxide analyzer (NOA). As shown in Figure 10, the PVC tube impregnated with 1000 mg / ml SNAT exhibited endothelial level NO release capacity for up to 49 days (Figure 10).

[0084] The storage stability of PVC tubes impregnated with 1000 mg / ml SNAT was tested. Storage conditions were as follows: dry condition; in air; different temperatures of 21°C, 4°C, and -20°C; and a 1-year period—where d0 marks the 1-year storage date, and d1 and d2 are subsequent dates after 1 year. The 1-year results (Figure 11) show that fairly stable storage stability was achieved for PVC tubes impregnated with 1000 mg / ml SNAT at 4°C and -20°C.

[0085] The NO release capacity of the polymer examples and the original control impregnated with 1000 mg / ml SNAT was also tested after exposure to different sterilization techniques. Sterilization was performed using ethylene oxide (EtO), hydrogen peroxide (H2O2), and OPS liquid chemicals (shown as PVC liquid). The results are shown in Figure 12. These results show that OPS liquid chemicals are the optimal sterilization liquid because it did not alter NO release until day 14. The results also indicate that widely used H2O2 could be another option for sterilizing the tubes of the examples while maintaining the original NO release capacity.

[0086] To evaluate the antithrombotic activity and platelet preservation capacity of the novel NO donor (SNAT) coated or impregnated extracorporeal loop tubes, and to determine whether the modified polymer tubes have a lower thrombogenic tendency compared to unmodified polymer materials.A rabbit arteriovenous (A-V) thrombosis model without systemic heparinization was used (similar to the apparatus shown in Figure 6). Determined endpoint parameters included thrombus area in the tube, coagulation, plasma fibrinogen levels, platelet count, and aggregation via aggregation assay. A total of 13 white male New Zealand rabbits were evaluated (n=7 controls, n=3 NO-600 mg / ml coated, n=3 NO-1000 mg / ml impregnated). 1000 mg / ml SNAT coated tubes (data not shown) were used, but the high NO concentration in the thin coating made its production technically difficult: 1) the surface became uneven, “bumpy” due to the formation of gaseous NO; and 2) flow readings could not be taken via the Transonic System due to the uneven surface and the very dark color of the coating.

[0087] In the NO-coated group, blood pressure and blood flow velocity remained steady during the 4-hour test period compared to baseline. Platelet function (77.9+ / 1.4% SNATi-1000 vs. 56.0+ / 4.2% control, compared to baseline 78.1+ / 5.9%) and platelet count (Fig. 13A), as measured by aggregation, were maintained throughout the study, and no significant clot formation was observed in this group (Fig. 13B).

[0088] However, the control group showed higher variance in the above parameters. Platelet counts dropped below 50%, and all circuits were severely clogged (Figs. 13A and 13B, 1.2+ / -1.2 cm2 SNAT-600, n=3 vs. 11.0+ / -0.4 cm2 control, n=7). Both control circuits were clogged within the first hour of the experiment.

[0089] As described, the impregnation technique (1000 mg / ml) was also used to prepare the NO-releasing surface. This technique treats the entire polymer on both the inner and outer surfaces.

[0090] The NO-impregnated circuit showed even better antithrombotic properties than the coated group (0.001+ / -0.0 cm² (SNATi 1000, n=3) vs. 10.28+ / -0.20 cm² control, n=7), and no clot formation was observed throughout the circuit (Figs. 14A and 14B). Aggregation assays in the NO-impregnated group (SNATi) were not significantly different from baseline (79.8+ / 0.3% SNATi-1000 vs. 56.0+ / 4.2% control, compared to baseline 74.1+ / 1.9%). SNAT-impregnated PVC also showed good antimicrobial activity (reduction of 3 log units after 5 days). Confocal images are shown in Fig. 15. Tables 7A and 7B show the high antimicrobial efficacy (reduced bacterial colonies) of the SNAT-impregnated polymer against Gram-positive and Gram-negative strains after a 5-day biofilm study (Table 7A).And the bacterial kill effect (7 days, a reduction of 7 log units (Table 7B)) compared to the control.

[0091] Table 7A – Average bacterial colony count (CFU / ml) in biofilm test. Specification 14 / 15 pages 17 CN 121358507 A.

[0092] Table 7B – Average bacterial colony count (CFU / ml) in bacterial kill test.

[0093] All these results indicate that SNAT-treated (coated or impregnated) circuits exhibit superior antithrombotic and antibacterial activity compared to untreated control ECC, and do indeed help maintain platelet counts during the study. For the SNAT semi-impregnated examples, the antithrombotic and antibacterial activities may be similar. However, these results also confirm that these techniques may have detrimental effects on the surface roughness and potential mechanical properties of the coated or fully impregnated polymers, unlike the semi-impregnated examples disclosed herein.

[0094] Throughout the specification, references to “an example,” “another example,” “an instance,” etc., mean that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the embodiment is included in at least one example described herein and may or may not be present in other examples. Furthermore, it should be understood that, unless the context explicitly specifies otherwise, the elements described in any example may be combined in any suitable manner across a variety of examples.

[0095] It should be understood that the ranges provided herein include the range and any values ​​or subranges within the range. For example, a molecular weight of approximately 195 Daltons to approximately 350 Daltons should be interpreted as including not only the explicitly listed limit of approximately 195 Daltons to approximately 350 Daltons, but also individual molecular weights (e.g., 197 Daltons, 250 Daltons, 275.5 Daltons, etc.) and subranges of molecular weights (approximately 250 Daltons to approximately 350 Daltons, approximately 197 Daltons to approximately 297 Daltons, etc.). Furthermore, when “approximately” is used to describe a value, this means that minute variations (at most + / - 10%) by the value are covered.

[0096] In describing and claiming protection for the embodiments disclosed herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise.

[0097] While several embodiments have been described in detail, it should be understood that the disclosed embodiments can be modified. Therefore,The aforementioned instructions are considered non-restrictive. Instruction Manual Page 15 / 15 18 CN 121358507 A Figure 1 Figure 2A Instruction Manual Appendix 1 / 11 Page 19 CN 121358507 A Figure 2B Figure 2C Figure 3A Instruction Manual Appendix 2 / 11 Page 20 CN 121358507 A Figure 3B Figure 4 Instruction Manual Appendix 3 / 11 Page 21 CN 121358507 A Figure 5 Figure 6 Figure 7A Instruction Manual Appendix 4 / 11 Page 22 CN 121358507 A Figure 7B Figure 8 Instruction Manual Appendix 5 / 11 Page 23 CN 121358507 A Figure 9 Figure 10 Instruction Manual Appendix 6 / 11 Page 24 CN 121358507 A Figure 11 Instruction Manual Appendix 7 / 11 Page 25 CN 121358507 A Figure 12 Instruction Manual Appendix 8 / 11 Page 26 CN 121358507 A Figure 13A Figure 13B, Appendix to the Specification, Page 9 / 11, 27 CN 121358507 A; Figure 14A, Figure 14B, Appendix to the Specification, Page 10 / 11, 28 CN 121358507 A; Figure 15, Figure 16, Appendix to the Specification, Page 11 / 11, 29 CN 121358507 A.

Claims

1. A method for producing a nitric oxide generating device, comprising: introducing a solution into an interior opening of a nitric oxide permeable polymeric object, the solution comprising: a solvent; and S-nitroso-l-adamantane thiol dissolved in the solvent; allowing the solution to soak in the interior opening for a period of up to 12 hours, whereby the S-nitroso-l-adamantane thiol impregnates into portions of the nitric oxide permeable polymeric object adjacent to the interior opening; and removing the solution from the interior opening.

2. The method as defined in claim 1, wherein: the nitric oxide permeable polymeric object is made of poly(vinyl chloride); and the solvent is part of a solvent system further comprising a plasticizer.

3. The method as defined in claim 2, wherein: the solvent system further comprises a second solvent; and the solvent, the plasticizer, and the second solvent are present in a predetermined volume ratio.

4. The method as defined in claim 3, wherein the predetermined volume ratio is 0.1 to 10 of the solvent: 0.1 to 10 of the plasticizer: 0.1 to 10 of the second solvent.

5. The method as defined in claim 3, wherein the solvent and the second solvent are different and independently selected from the group consisting of acetone, methanol, ethyl acetate, ethyl ketone, tetrahydrofuran, chloroform, and ethanol.

6. The method as defined in any one of claims 3 to 5, wherein: the solvent is acetone; the plasticizer is trioctyl trimellitate; and the second solvent is methanol.

7. The method as defined in any one of claims 1 to 6, wherein solution comprises up to 2000 mg / mL of S-nitroso-l-adamantane thiol.

8. The method as defined in any one of claims 1 or 7, wherein the nitric oxide permeable polymeric object is made of silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene.

9. The method as defined in claim 8, wherein: the nitric oxide permeable polymeric object is made of silicone; and the solvent is tetrahydrofuran.

10. The method as defined in claim 8, wherein: the nitric oxide permeable polymeric object is made of polyurethane; and the solvent is selected from the group consisting of methanol, methyl ethyl ketone, chloroform, cyclohexane, and combinations thereof.

11. The method as defined in any one of claims 1 to 10, wherein prior to introducing the solution, the method further comprises producing the solution by dissolving S-nitroso-l-adamantane thiol in a solvent.

12. The method as defined in claim 11, wherein prior to producing the solution, the method further comprises producing S-nitroso-l-adamantane thiol by exposing l-adamantane thiol to solvent-free nitrosation using tert-butyl nitrite.

13. The method as defined in any one of claims 1 to 12, further comprising: introducing a coating solution into the interior opening, the coating solution comprising a direct thrombin inhibitor-linked polymer dissolved in a third solvent; and allowing the third solvent to evaporate, thereby forming a coating on the surface of the interior opening, the coating comprising the direct thrombin inhibitor attachment polymer.

14. The method as defined in claim 13, wherein prior to introducing the coating solution, the method further comprises forming the coating solution by: dissolving an anchor polymer in the third solvent; introducing a linker molecule into the third solvent, thereby covalently attaching linker molecules to the anchor polymer; introducing a direct thrombin inhibitor into the third solvent, thereby covalently attaching the direct thrombin inhibitor to the linker molecules attached to the anchor polymer to form the direct thrombin inhibitor attachment polymer; precipitating the direct thrombin inhibitor attachment polymer; and resolubilizing the direct thrombin inhibitor attachment polymer in fresh third solvent.

15. The method as defined in any one of claims 1 to 14, wherein the S-nitroso-1- adamantane thiol is a tertiary thiol having a molecular weight of about 195 daltons to about 350 daltons and a predicted n-octanol-water partition ratio of more than 5.

16. The method as defined in claim 1, wherein: the solvent is part of a solvent system comprising a second solvent; the solvent is different from the second solvent and is independently selected from the group consisting of acetone, methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, cyclohexane, and ethanol; the solvent system comprises a predetermined volume ratio of the solvent and the second solvent; and the predetermined volume ratio is 0.1 : 10 to 10 : 0.

1.

17. A nitric oxide generation device comprising: a nitric oxide permeable polymeric object; and S-nitroso-1-adamantane thiol impregnated in a portion of a wall of the nitric oxide permeable polymeric object adjacent to an interior opening of the nitric oxide permeable polymeric object, wherein an exterior surface of the wall of the nitric oxide permeable polymeric object is substantially free of the S-nitroso-1-adamantane thiol.

18. The nitric oxide generation device as defined in claim 17, wherein the S-nitroso-1- adamantane thiol extends into the wall by about 500 pm to about 1000 pm.

19. The nitric oxide generation device as defined in any one of claim 17 or claim 18, wherein the nitric oxide permeable polymeric object is made of poly(vinyl chloride), silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene.

20. The nitric oxide generation device as defined in any one of claims 17 to 19, further comprising a direct thrombin inhibitor attachment polymer attached to a surface of the interior opening.

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