Nitric oxide generating device and method for manufacturing the same

Abstract

This invention provides a nitric oxide generating device and a method for manufacturing the same. [Solution] In an exemplary method, a solution is introduced into the lumen of a nitric oxide permeable polymer object. The solution contains a solvent and S-nitroso-1-adamantanethol dissolved in the solvent. The solution is immersed in the lumen of the nitric oxide permeable polymer object for a maximum of 12 hours. The solution is then removed from the lumen of the nitric oxide permeable polymer object.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 521,207, filed on 15 June 2023, which is incorporated herein by reference in its entirety.

[0002] Description of research or development funded by the federal government. This invention was made with government support under EB024038 and HL155100 and was recognized by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Nitric oxide (NO) is an endogenous gaseous molecule that has been shown to possess several important physiological functions, including intrinsic vasodilatory, wound healing, angiogenesis-promoting, anticancer, antiplatelet activity, and antibacterial / antiviral activity. In some cases, NO may be used to control infections, prevent biofilm formation, and minimize inflammation and fibrosis. [Brief explanation of the drawing]

[0004] The features of the examples in this disclosure will become apparent by referring to the following detailed description and drawings, where similar reference numbers correspond to similar, but sometimes not identical, components. For brevity, reference numbers or features having the aforementioned functions may or may not be described in relation to other drawings in which such reference numbers or features appear.

[0005] [Figure 1] Figure 1 is a schematic perspective view of an extracorporeal circulation circuit tube containing an example of a nitric oxide generation device, namely, S-nitroso-1-adamantanethol (SNAT) impregnated into a portion of the wall adjacent to the lumen of the tube. [Figure 2A] Figure 2A is a schematic diagram of a nitric oxide generating device, i.e., a Y-connector for an extracorporeal blood circulation circuit. [Figure 2B] Figure 2B is a schematic cross-sectional view taken along line 2B-2B in Figure 2A, showing S-nitroso-1-adamantanthiol (SNAT) impregnated into a portion of the wall adjacent to the lumen of a Y-connector, and an optional coating of a direct thrombin inhibitor attached to the surface of the lumen. [Figure 2C] Figure 2C is a schematic diagram showing an example of coating the surface of the lumen with a direct thrombin inhibitor. [Figure 3A] Figure 3A is a black and white reproduction of a photograph of a portion of the control tube. [Figure 3B] Figure 3B is a black and white reproduction of a photograph of a portion of a SNAT semi-impregnated tube. [Figure 4] Figure 4 is a graph showing the relationship between time (X axis, days) and nitric oxide (NO) flux (Y axis, x10⁻¹⁰ mol·min⁻¹·cm⁻²) for SNAT semi-impregnated tubes. [Figure 5] Figure 5 shows microscopic images (reproduced in black and white) of the performance of control tubes and SNAT semi-impregnated tubes in biofilm formation using S. aureus (Staphylococcus aureus) strains and P. aeruginosa (Pseudomonas aeruginosa) strains. [Figure 6] Figure 6 is a schematic diagram of the experimental setup for the in vivo test described herein in the Examples section. [Figure 7A] Figure 7A is a black and white reproduction of the inside of the SNAT semi-impregnated tube after a 4-hour in vivo test. [Figure 7B] Figure 7B is a black and white reproduction of the inside of the control tube after a 4-hour in vivo trial. [Figure 8] Figure 8 is a bar graph showing the platelet count (Y axis, percentage (%) relative to baseline) in SNAT semi-impregnated tubes and control tubes after exposure to the extracorporeal circulation circuit over different time periods (X axis, minutes). [Figure 9]Figure 9 shows multiple field emission scanning electron microscope images (reproduced in black and white) of the control (unmodified) polymer (row A), the SNAT-doped polymer of the example (row B), and the comparative S-nitroso-N-acetylpenicillamine (SNAP)-doped polymer (row C). [Figure 10] Figure 10 is a bar graph showing the relationship between time (X axis, days) and nitric oxide (NO) flux (Y axis, x10⁻¹⁰ mol·min⁻¹·cm⁻²) for two different SNAT fully impregnated tubes. [Figure 11] Figure 11 is a bar graph showing the one-year stability of SNAT fully impregnated tubes exposed to different storage temperatures, where stability is shown as a relationship between time (X axis, 1 year + 0 days, 1 day, or 2 days) and nitric oxide (NO) flux (Y axis, x10⁻¹⁰ mol·min⁻¹·cm⁻²). [Figure 12] Figure 12 is a bar graph showing the stability of control tubes and SNAT fully impregnated tubes exposed to different sterilization conditions, where stability is shown as a relationship between time (X axis, days) and nitric oxide (NO) flux (Y axis, x10⁻¹⁰ mol·min⁻¹·cm⁻²). [Figure 13A] Figure 13A is a bar graph showing platelet aggregation (Y axis, %) in SNAT-coated tubes and control tubes after exposure to an extracorporeal circulation circuit over different time periods (X axis, minutes). [Figure 13B] Figure 13B is a bar graph showing the thrombus area (Y axis, pixels / cm2) of the extracorporeal circulation circuit for SNAT-coated tubes and control tubes exposed to the flow of the extracorporeal circulation circuit for 4 hours. [Figure 14A] Figure 14A is a bar graph showing platelet aggregation (Y axis, %) in SNAT fully impregnated tubes and control tubes after exposure to an extracorporeal circulation circuit over different time periods (X axis, minutes). [Figure 14B] Figure 14B is a bar graph showing the thrombus area (Y axis, pixels / cm2) of the extracorporeal circulation circuit for SNAT fully impregnated tubes and control tubes exposed to the flow of the extracorporeal circulation circuit for 4 hours. [Figure 15] Figure 15 shows confocal images (reproduced in black and white) of the performance of control tubes and SNAT fully impregnated tubes in biofilm formation using S. aureus (Staphylococcus aureus) and E. coli (Escherichia coli) strains. [Figure 16] Figure 16 is a graph showing the relationship between time (X-axis, days) and nitric oxide (NO) flux (Y-axis, x10-10 mol·min-1·cm-2) for an SNAT semi-impregnated tube of another embodiment (n = 3).

Mode for Carrying Out the Invention

[0006] Nitric oxide (NO) donor molecules are incorporated in a coating on a polymeric object or are impregnated in the polymeric object. Exemplary polymeric objects include tubes, catheters (e.g., hemodialysis catheters), and / or extracorporeal circuit components such as connectors, other catheters (e.g., urethral catheters, vascular catheters), pump chambers, or the like. These NO-releasing polymeric objects are used to reduce thrombosis and platelet aggregation and prevent bacterial infection and biofilm formation in a plurality of biomedical applications including extracorporeal circuits.

[0007] The inventors have found that some NO donor molecules, such as diazeniumdiolate NO donors and S-nitrosothiol-type NO donors, can be limited in terms of use and / or can lead to undesirable results. As an example, diazeniumdiolate NO donors (e.g., DBHD-N2O2) have usage limitations because they may not be impregnable in the host material or are difficult to impregnate. As another example, diazeniumdiolate NO donors coated on extracorporeal circuit tubes can be contaminated with carcinogenic nitrosamines. As yet another example, S-nitrosothiol-type NO donors (e.g., S-nitroso-N-acetylpenicillamine (SNAP)) coated on or impregnated within a polymeric object can unfavorably change the surface roughness and / or mechanical properties of the polymeric object. The solubility of S-nitrosothiol-type NO donors is also limited. Thus, with respect to impregnated S-nitrosothiol-type NO donors, the change in mechanical properties can be due to recrystallization of the material after impregnation.

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

[0009] Examples disclosed herein utilize a tertiary nitrosothiol (S-nitroso-1-adamantanethiol, i.e., SNAT) having an adamantane molecule as an NO donor molecule, and employ a method of semi-impregnating a polymer object with SNAT. "Semi-impregnation" means that a portion of the polymer object exposed to the SNAT-containing solution is impregnated, while other portions of the polymer object remain without 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) at a desired time (e.g., in the range of weeks to months) without compromising the mechanical integrity of the polymer object.

[0010] In some cases, semi-impregnated polymer objects also include direct thrombin inhibitor-linked polymer coatings on the surface of polymer objects exposed to blood or other fluids. Direct thrombin inhibitor-linked polymer coatings can aid in anticoagulation. As two examples, the direct thrombin inhibitor may be bivalirudine or argatroban.

[0011] The adamantane portion increases the lipophilicity of the drug molecule. The simplest tertiary S-nitrosothiol containing adamantane is S-nitroso-1-adamantanethiol (SNAT, thionitrite (HNOS), S-tricyclo[3.3.1.13,7]dec-1-yl ester). [ka] For example, S-nitroso-1-adamantanethiol is a tertiary thiol with a molecular weight in the range of approximately 195 daltons to approximately 350 daltons and a predicted n-octanol / water partition ratio greater than 5 (e.g., 5.9). This molecule can spontaneously release NO when the lumen of a polymer object (partially impregnated with SNAT) is exposed to a solution and / or blood under physiological conditions. In one specific example, the NO generating device exhibits antibacterial and bactericidal effects (bacterial CFU / mL decreases by at least 2 log) for at least 7 days. In another specific example, the NO generating device exhibits (0.5~4.0 x 10) for more than 1 month (e.g., approximately 35 days). -10mol·min -1 cm -2 It can continuously release a sufficient amount of NO within or exceeding the normal endothelial range.

[0012] In the examples disclosed herein, this molecule is used as a hydrophobic NO donor to generate a biocompatible surface having antithrombotic and antibacterial properties. In particular, SNAT provides desirable surface anticoagulation within the lumen of a polymer object in which the SNAT is partially impregnated. This may eliminate the need for systemic anticoagulation. SNAT may be useful in modifying all components of extracorporeal circulation circuits and is thought to be usable in applications such as hemodialysis, hemofiltration, cardiopulmonary bypass (CPB), cardiac pacing leads, urinary catheters, and artificial lung housings. Furthermore, the NO-releasing ability of the SNAT polymer provides an antithrombotic effect by inhibiting platelet activation and platelet adhesion (aggregation) to the lumen surface (as demonstrated by the in vivo model in the Examples section).

[0013] SNAT is extremely lipophilic (logD=5.9) and therefore does not easily leach from lipophilic polymers into aqueous media and / or blood (compared to other NO donors such as SNAP or diazenium diolate). Furthermore, SNAT is pH-independent, which distinguishes it from other NO donors (thiols: S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), or diamines: DBHD-N2O2). Moreover, SNAT is highly soluble in the solvents or solvent systems disclosed herein, thus simplifying the semi-impregnation process. After semi-impregnation, SNAT remains dissolved in the polymer phase, thus avoiding crystal formation and allowing the polymer object to maintain its mechanical properties. Solubilized SNAT also allows for a higher payload within the polymer object compared to crystallizing NO donors. Furthermore, SNAT provides relatively continuous NO release (i.e., no delay time when exposed to solution and / or blood under physiological conditions). Also, SNAT does not contain amine functional groups and therefore eliminates the formation of carcinogenic nitrosamines. Consequently, SNAT-impregnated polymer materials may exhibit reduced cytotoxicity compared to other polymer materials modified for NO generation.

[0014] Exemplary NO generating devices 10, 10' are shown in Figures 1 and 2A. Generally, devices 10, 10' include a polymer body 12 having a lumen 16, and a SNAT 14 partially impregnated into a portion of the polymer body 12 adjacent to the surface 17 of the lumen 16.

[0015] In each example, the nitric oxide permeable polymer object 12 is formed of a polymer capable of releasing NO, i.e., NO generated from an NO donor, in the examples disclosed herein. In one example, the nitric oxide permeable polymer object 12 is composed of poly(vinyl chloride) (PVC, medical or non-medical grade), silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene. The polymer object 12 may also include a plasticizer, e.g., tris(2-ethylhexyl) trimellitate (i.e., trioctyl trimellitate (TOTM), a phthalate-free plasticizer), or a bioplasticizer. The bioplasticizer may be any plasticizer obtained from biomass resources, e.g., vegetable oil, cardanol, vegetable fatty acids, glycerin, and / or citric acid. Such plasticizers have been extensively studied to replace petroleum-based o-phthalate plasticizers. As an example, PVC tubing containing plasticizers is commercially available under the trade name TYGON S3 (trademark) E-3603 (of Saint-Gobain Performance Plastics Corp.). Other examples include silicone, polyurethane, polycarbonate, and polypropylene, which do not necessarily have to contain plasticizers.

[0016] The polymer object 12 may be any medical device having a lumen 16 that comes into contact with another bodily fluid, such as blood or urine, during use. For example, the polymer object 12 may be any extracorporeal circulation circuit component, e.g., a tube, catheter, and / or a connector (as schematically shown in Figure 1), another catheter, a pump chamber, or any other medical device that benefits from nitric oxide generation in its lumen 16 during use. The lumen 16 may be the lumen of a tube, catheter, or connector, the interior of a pump chamber, or similar.

[0017] An example of an NO generating device 10 is shown in Figure 1. This example is an extracorporeal circulation circuit tube. As shown in Figure 1, the NO generating device 10 comprises a nitric oxide permeable polymer body 12 and S-nitroso-1-adamantanethol (SNAT) 14 impregnated in a portion of the wall W of the nitric oxide permeable polymer body 12 adjacent to the lumen 16 of the nitric oxide permeable polymer body 12, and the outer surface 18 of the wall W of the nitric oxide permeable polymer body 12 is substantially free of S-nitroso-1-adamantanethol 14. "Substantially free" means that less than 1% (partially impregnated) SNAT 14 is present on the outer surface 18 of the polymer body 12. In some examples, 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 "on the outer surface 18" and "within a portion of the wall W of the polymer object 12 adjacent to the outer surface 18" include both the outer surface 18 and the distance measured from the outer surface 18 that extends into the wall thickness. In one example, approximately 25% to approximately 80% of the wall W thickness (measured from the outer surface 18) substantially does not contain SNAT 14. Therefore, the portion of wall W that substantially does not contain SNAT 14 depends on the wall thickness. In one specific example, if the wall thickness is 1 / 16 inch (1,587.5 μm), SNAT 14 is not present in wall W within 500 μm of the outer surface 18. In this particular example, SNAT 14 is not present in approximately 31% of wall W (measured inward from the outer surface 18).

[0018] The SNAT 14 is semi-impregnated using the method disclosed herein, and therefore the SNAT 14 is located within a portion of the wall W of the nitric oxide permeable polymer body 12 adjacent to the lumen 16 of the nitric oxide permeable polymer body 12, and not on the outer surface 18 of the wall W (as described above). The SNAT 14 is located near the surface 17 adjacent to the lumen 16. The phrase "near the surface 17" means that the SNAT 14 is located at a distance measured from the inner surface 17 and the surface 17 extending within the thickness of the wall. As described above, this distance is less than the total thickness of the wall W, since the portion of the outer surface 18 does not substantially include the SNAT 14. In one example, about 20% to about 75% of the wall W thickness (measured from the inner surface 17) includes 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 to a depth of approximately 500 μm to 1000 μm. The degree to which the SNAT 14 is partially impregnated may depend on the thickness of the wall W. Thinner walls may have a smaller impregnation distance, while thicker walls may have a larger impregnation distance (measured from the inner surface 17).

[0019] As stated, S-nitroso-1-adamantanethol remains dissolved in the polymer phase, and therefore, the NO generating device 10 does not contain solid S-nitroso-1-adamantanethol.

[0020] Furthermore, as simulated in Figure 1, the inner surface 17 may be coated with a direct thrombin inhibitor-linked polymer 20. The direct thrombin inhibitor-linked polymer 20 is further described with reference to Figure 2C.

[0021] Another example of an NO generating device 10' is shown in Figure 2A. This example is an extracorporeal circulation circuit Y connector. As shown in the cross-sections of Figures 2A and 2B, the NO generating device 10' comprises a nitric oxide permeable polymer body 12 and S-nitroso-1-adamantanethol (SNAT) 14 impregnated into a portion of the wall W of the nitric oxide permeable polymer body 12 adjacent to the lumen 16 of the nitric oxide permeable polymer body 12. Similar to Figure 1, the outer surface 18 of the wall W of the nitric oxide permeable polymer body 12 is substantially free of S-nitroso-1-adamantanethol 14.

[0022] Furthermore, as simulated in Figure 2B, the inner surface 17 can be directly coated with a thrombin inhibitor-linked polymer 20.

[0023] Here, with reference to Figure 2C, an example of a direct thrombin inhibitor-linked polymer 20 is schematically shown. The direct thrombin inhibitor-linked polymer 20 comprises an anchor polymer 22, a linking molecule 24 bonded to the anchor polymer 22, and a direct thrombin inhibitor molecule 26 bonded to the linking molecule 24. A coating can be formed on the inner surface 17 of the base polymer (i.e., polymer body 12), and any anchor polymer 22 containing a face functional group that bonds to the linking molecule 24 can be used. Similarly, any linking molecule 24 containing a functional group that bonds to the anchor polymer 22 and the direct thrombin inhibitor molecule 26 can be used.

[0024] In one example, the anchor polymer 22 is polyurethane or polyurethane copolymer (e.g., CARBOSIL®, a silicone and polycarbonate-urethane copolymer owned by DSM IP Assets), and the linking molecule is 4,4′-methylene-bis(cyclohexyl isocyanate) (HMDI) or polyethylene glycol (PEG). In another example, the direct thrombin inhibitor molecule 26 is bivalirudin having the following structure: [ka] The guanidine group at the N-(amino) terminus can be used as an anchor point for the HMDI linking molecule via the isocyanate group of the linking group. The direct thrombin inhibitor molecule 26 may be 100% pure bivalirudine or bivalirudine trifluoroacetate (bivalirudine TFA). Other examples of direct thrombin inhibitors may be pyridine, decilidine, and argatroban. The other anchor polymer 22 includes poly(vinyl chloride), silicone, or polyurethane, or any combination of the listed polymers. The linking molecule 24 is then selected to bond to a particular anchor polymer 22.

[0025] The semi-impregnation method disclosed herein comprises introducing a solution into the lumen 16 of a nitric oxide permeable polymer object 12, wherein the solution comprises a solvent and S-nitroso-1-adamantanethol 14 dissolved in the solvent; immersing the solution in the lumen 16 for a maximum of 12 hours; and removing the solution from the lumen 16 of the nitric oxide permeable polymer object 12.

[0026] SNAT14 is nonpolar and therefore hydrophobic. The selected solvent or solvent system should dissolve SNAT14. In some examples, the selected solvent may also be nonpolar. In some examples, the solvent is used alone (i.e., without any other solvents or plasticizers). In other examples, 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 the others. For example, acetone may be selected with methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, or cyclohexane. In yet another example, the solvent is part of a solvent system containing a plasticizer. In yet another example, the solvent is used in combination with another solvent and a plasticizer.

[0027] The solvent used in the semi-impregnation method can dissolve SNAT but cannot dissolve the polymer object 12. However, the solvent can penetrate into the polymer object 12. The solvent is independently selected from the group consisting of acetone, methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, cyclohexane, and ethanol.

[0028] In one example of a single solvent system, the nitric oxide-permeable polymer object 12 is composed of silicone, and the solvent is tetrahydrofuran.

[0029] When multiple solvents are used, the hydrophobicity and lipophilicity of the selected solvents contribute to the volume ratio used. In one example, two different solvents, a first and a second, are used. The first and second solvents are different and independently selected from the group consisting of acetone, methanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, chloroform, cyclohexane, and ethanol. In this example, the solvent system includes the first and second solvents in a predetermined volume ratio, with the predetermined volume ratio range being 0.1:10 to 10:0.1. In one specific example, when two solvents are used, the volume ratio of the two solvents may be in the range of 2:1 to 1:2. It should be understood that any volume ratio within the widest range, such as 1:1, 1.5:1, 1:1.5, etc., can be used.

[0030] In one example of two solvent systems, the nitric oxide-permeable polymer object 12 is composed of PVC, and the two solvents are acetone and methanol.

[0031] In another example of two solvent systems, the nitric oxide-permeable polymer object 12 is composed of polyurethane, and the two solvents are methanol and chloroform. With respect to polyurethane, the solvent can be selected from the group consisting of methanol, methyl ethyl ketone, chloroform, cyclohexane, and combinations thereof.

[0032] In some examples, the solvent system further includes a plasticizer. A plasticizer is added when the NO-permeable polymer object 12 contains a plasticizer as part of its composition (e.g., some PVC polymer objects). The additional plasticizer in the solvent system may compensate for the plasticizer leaching from the NO-permeable polymer object 12 during the semi-impregnation method. The minimum value of plasticizer in a given volume ratio is ≥0.1 to ensure that the overall mechanical properties of the NO-permeable polymer object 12 do not change substantially after the semi-impregnation process is performed. When a plasticizer is added to the solvent, the given volume ratio is the volume ratio of the first solvent:plasticizer:second solvent. As an example, this given volume ratio is 0.1–10 of the first solvent:0.1–10 of the plasticizer:0.1–10 of the second solvent. For example, this volume ratio may be 1:3:1, 1.5:2:1.5, 1:2:2, 2:1:2, or 2:2:1. Ratios between the widest range of volume ratios may also be used. Further volume ratios may be used as long as they allow the selected amount of SNAT to dissolve. While volume ratios are described herein, it should be understood that these ratios are equally applicable as weight ratios.

[0033] Due in part to SNAT's extremely high lipophilicity, the amount of SNAT present in a solution depends on the application. Generally, a solution contains up to 2000 mg / mL of S-nitroso-1-adamantanethol. For a polymer object 12 with walls W that are 1 / 16 inch (1,587.5 μm) thick, the concentration of S-nitroso-1-adamantanethol in the solution may be 1000 mg / mL. For thinner walls W, the concentration of S-nitroso-1-adamantanethol may be less than 1000 mg / mL (e.g., 50 mg / mL to 900 mg / mL). For thicker walls W, the concentration of S-nitroso-1-adamantanethol may be greater than 1000 mg / mL (e.g., greater than 1000 mg / mL and up to 2000 mg / mL).

[0034] After introducing the solution into the lumen 16 of the nitric oxide permeable polymer object 12, the lumen 16 can be sealed for an incubation time. The incubation time is up to 12 hours. In some cases, the incubation time is less than 12 hours. In one example, the incubation time ranges from 1 hour to about 8 hours. Specifically, when the wall thickness W is about 0.6 mm (600 μm), the incubation time ranges from about 30 minutes to about 2.5 hours. In another example, the incubation time is 4 hours (this is particularly desirable for maintaining the mechanical properties of a polymer object with a wall thickness W of about 1.5875 mm (1,587.5 μm)).

[0035] After the incubation period has expired, the solution is removed from the lumen 16. When the polymer object 12 is transparent or translucent, the SNAT-impregnated portion of the polymer object 12 appears green.

[0036] Prior to introducing the solution into the lumen 16, the method may further include generating the solution by dissolving S-nitroso-1-adamantanethol in a solvent system. Prior to generating the solution, the method further includes generating S-nitroso-1-adamantanethol by nitrosating 1-adamantanethol: [ka] In the examples disclosed herein, the nitrosation process is carried out as solvent-free nitrosation using t-butyl nitrite (i.e., tertiary butyl nitrite (tBuNO2)). t-butyl nitrite functions as both a solvent and a reactant in the nitrosation process and is therefore considered solvent-free. The solvent-free process avoids solvents that may otherwise adversely affect the stability of SNAT. During the process, 1-adamantanethol is mixed with a desired amount (e.g., 1.1 molar equivalents) of tertiary butyl nitrite and reacted with it. First, stirring may be performed, and then the reaction vessel is kept on ice for a predetermined time (e.g., 30 minutes) as the reaction is exothermic. The reaction may then be carried out at room temperature (e.g., 18°C ​​to 21°C) for a desired time up to 12 hours. After the reaction, excess tertiary butyl nitrite and the by-product tertiary butanol (t-BuOH) are removed, for example, by turning at room temperature. After this, the product may be vacuum-dried overnight.

[0037] It should be understood that solvent-free nitrosation used to form SNAT can be carried out in conjunction with a semi-impregnation method or as a standalone method to produce SNAT. In the latter case, solvent-free nitrosation is not carried out in conjunction with the semi-impregnation method disclosed herein.

[0038] As a result of the semi-impregnation method, S-nitroso-1-adamantanethol 14 extends partially from the inner surface 17 into the wall W, as described herein. In one example, SNAT 14 is present on the inner surface 17 and then extends into the wall W to a depth of approximately 500 μm to approximately 1000 μm. In other words, S-nitroso-1-adamantanethol is present on the inner surface 17 of the lumen 16, for a certain distance within the wall thickness, where this distance is less than the total thickness as described herein. The remainder of the wall thickness extending to the outer surface 18 is substantially free of S-nitroso-1-adamantanethol.

[0039] Some examples of the method further include the incorporation of a direct thrombin inhibitor-linked polymer 20 as a coating to the inner surface 17. The direct thrombin inhibitor-linked polymer 20 may be formed before it is applied to the inner surface 17.

[0040] In one exemplary method, the direct thrombin inhibitor-linked polymer 20 may be formed by dissolving the anchor polymer 22 in a solvent, introducing the linking molecule 24 into the solvent to covalently bond the linking molecule 24 to the anchor polymer 22, and introducing the direct thrombin inhibitor 26 into the solvent to covalently bond the direct thrombin inhibitor 26 to the linking molecule 24 bonded to the anchor polymer 22, thereby forming the direct thrombin inhibitor-linked polymer 20. Any example of the anchor polymer 22 and linking molecule 24 described herein may be used, and the selected solvent may be capable of dissolving each of the components 22, 24, and 26. In the exemplary method, precipitation, drying, and redissolution may be carried out between reactions. For example, after the linking molecule 24 has bonded to the anchor polymer 22, precipitation may be carried out using hexane, and the solid product (e.g., pellet) may be dried and redissolved in a fresh solvent before the direct thrombin inhibitor 26 is introduced. Then, after the direct thrombin inhibitor 26 is introduced, the reaction may be allowed to proceed for a predetermined amount of time (e.g., 20 to 40 minutes), and then precipitation may be carried out using water. This solid product (e.g., a pellet of direct thrombin inhibitor-linked polymer 20) can be dried and redissolved in a fresh solvent. In one example, the final solution contains approximately 10 μM of direct thrombin inhibitor-linked polymer 20.

[0041] In one example, the anchor polymer 22 is in the form of small polymer beads. In another example, the solvent is tetrahydrofuran (THF).

[0042] Once the direct thrombin inhibitor-linked polymer solution is formed, the direct thrombin inhibitor-linked polymer solution is introduced into the lumen 16. After a few seconds, the solution is discharged from the lumen 16. The solvent evaporates, which occurs relatively quickly, leaving a coating of the direct thrombin inhibitor-linked polymer 20 on the surface 17 of the lumen 16 (of the base polymer 12). It should be understood that the direct thrombin inhibitor-linked polymer 20 is permeable to NO and therefore does not interfere with the effect of the semi-impregnated SNAT 14.

[0043] To further illustrate this disclosure, various examples are given herein. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of this disclosure.

[0044] Examples Example 1

[0045] First, several examples of NO-generating polymer tubes disclosed herein were fabricated using medical-grade PVC Tygon™ ND-100-65 tubing (without topcoat, 3 / 8 inch (approximately 9.525 mm) inner diameter) and SNAT. Two different solutions were tested with two different semi-impregnation times (4 hours or 12 hours). The solvent systems of the solutions contained 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 tris(2-ethylhexyl) trimellitate (TOTM).

[0046] The tubes in the examples were filled with the respective solutions, sealed, and incubated for 4 or 12 hours.

[0047] For comparison of mechanical properties, several control polymer tubes were prepared according to Table 1. These controls were filled with different solvent systems (without SNAT) for a half-impregnation time. The number of tests of each type is represented by "n" in Table 1). Young's modulus was measured, and the average results for the control polymer tubes are also shown in Table 1 (where SEM is the standard error, and *p<0.05). The first untreated control value was supplied by the manufacturer. The second untreated control value represents the measurement of the tube upon receipt, which had not been exposed to any solvent. [Table 1]

[0048] These results demonstrate the effects of swelling time and solvent volume ratio on the mechanical properties of the tube without additional SNAT. Longer incubation times have a greater effect on Young's modulus and may be more preferable when thicker polymer materials are used.

[0049] The Young's modulus of one example polymer tube (solution containing a 1:3:1 solvent system and SNAT, incubated for 4 hours) was also measured, and the average results are shown in Table 2 (where SEM is the standard error, *p<0.05). Table 2 also repeats some of the controls from Table 1 for ease of comparison. [Table 2]

[0050] These results indicate that SNAT semi-impregnation did not adversely affect the mechanical properties of the polymer tubes.

[0051] Additional Examples: NO-generating polymer tubes were fabricated using medical-grade PVC Tygon® ND-100-65 tubing (without topcoat, inner diameter 3 / 8 inch (approximately 9.525 mm)) and SNAT. Two different solutions (acetone:TOTM:methanol in volume ratios of 1:3:1 and 2:1:2) were tested with a half-impregnation time of 4 hours. As described above, each solution contained 1000 mg / mL of SNAT. The tubes were filled and sealed with the respective solutions and incubated for 4 hours.

[0052] After the incubation time, the solution was emptied. Parts of one untreated control sample and one additional example sample were photographed, and black and white reproductions are shown in Figures 3A and 3B, respectively. The untreated control sample remained clear, while the example sample contained a green area (shown as a darker black area in Figure 2B). The green color in the example tube was evidence of partial impregnation with SNAT.

[0053] The tubes were tested in vitro and compared to an untreated control (ECC) (medical-grade PVC Tygon® ND-100-65 tube (no solution exposure, no topcoat, inner diameter 3 / 8 inch (approximately 9.525 mm))) for NO donor addition, NO emission profile (measured using ozone chemiluminescence (see Figure 4)), and tensile strength (measured using a texture analyzer). The antibacterial properties of the example NO-generating polymer tubes 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 respective bacteria, incubated for 7 days, then the bacteria were removed, and the samples were tested using CDC sealed containers. All of these results are summarized in Table 3. [Table 3]

[0054] The semi-impregnation method consumes an estimated 50% less NO donor compared to full impregnation, making the method disclosed herein more economical. The 2:1:2 solvent combination resulted in significantly higher additions compared to the 1:3:1 solvent combination (p ≤ 0.05, Table 3, 35 days).

[0055] The tensile strength of the 1:3:1 group was closest to that of the untreated control (p≧0.05, Table 1), maintaining superior inherent properties compared to the 2:1:2 group (Table 3). While the 2:1:2 group had a generally higher NO-releasing capacity when comparing the NO-releasing values ​​at 35 days, the 1:3:1 group still showed sufficient NO flux (Table 3, Figure 4).

[0056] Furthermore, biofilm testing showed sufficient antimicrobial properties in both groups, with an approximately 2-log reduction in bacterial colonies (Table 3, Figure 5). The confocal image in Figure 5 clearly shows more biofilm formation in the control group than in the Example 1:3:1 group.

[0057] Furthermore, the anticoagulant properties of the 1:3:1 group (n=3) were tested in vivo in an acute rabbit model and compared with an untreated control (n=9). The in vivo test was a 4-hour long-term test. The experimental setup is shown in Figure 6. Arteriovenous shunts were used in rabbits. An extracorporeal circulation circuit (ECC) loop with a vascular catheter (16G) was introduced into the inferior common carotid artery, and an extracorporeal circulation circuit (ECC) loop with a vascular catheter (14G) was introduced into the right jugular vein, with flow from the inferior common carotid artery through the thrombus formation chamber to the right jugular vein. In this experiment, a reduction in thrombus area (SNAT 0.5±0.4 cm) was observed. 2 Comparison: 9.8 ± 0.8 cm 2)Improved platelet preservation rates (108% vs. 74% relative to baseline, respectively) were observed at the end of the study (Figure 8). Images (reproduced in black and white) of the example tubes and control tubes after 4 hours of blood exposure are shown in Figures 7A and 7B, respectively. The SNAT semi-impregnated tubes were green and contained only a small amount of thrombus. In contrast, the control tubes were red due to all thrombus. Clearly, the SNAT semi-impregnated tubes exhibit superior anticoagulant properties.

[0058] The data in this embodiment clearly demonstrate that SNAT semi-impregnation provides a more cost-effective surface modification method with appropriate NO flux and while preserving the original mechanical properties of the polymer used. This SNAT semi-impregnation also has excellent antithrombotic properties in vivo, eliminating the need for systemic anticoagulation.

[0059] Example 2

[0060] First, the examples of NO-generating polymer tubes disclosed herein were prepared using medical-grade PVC Tygon™ ND-100-65 tubing (without topcoat, inner diameter 3 / 8 inch (approximately 9.525 mm)) and SNAT. One solution was tested. The solvent system of the solution contained acetone:plasticizer:methanol in a volume ratio of 1:2:2. The solution contained 1000 mg / mL of SNAT, and the plasticizer was tris(2-ethylhexyl) trimellitate (TOTM).

[0061] The example tubes were filled with the solution, sealed, and incubated for 4 hours.

[0062] The Young's modulus of the control and example tubes was measured, and the average results for the four control polymer tubes and four example tubes are shown in Table 4 (where SEM is the standard error, and *p<0.05). [Table 4]

[0063] Similar to the results in Example 1, these results indicate that SNAT semi-impregnation did not adversely affect the mechanical properties of the polymer tube.

[0064] The example tubes were tested in vitro and compared to an untreated control (ECC) (medical-grade PVC Tygon® ND-100-65 tube (no solution exposure, no topcoat, inner diameter 3 / 8 inch (approximately 9.525 mm)) for NO emission profiles (measured using ozone chemiluminescence (see Figure 16)). The 1:2:2 solvent combination yielded the desired NO flux (>0.5x10) over 14 days. -10 mol / min / cm 2 This resulted in a flux (which reached the threshold in 21 days).

[0065] One of the example tubes was exposed to bivalirudine-linked polymer to form a coating on its inner surface. The bivalirudine-linked polymer was prepared as described herein by dissolving CARBOSIL® beads in tetrahydrofuran and then adding HMDI. The reaction product was precipitated using hexane, and the precipitate was dried. The precipitate was dissolved in fresh THF, and then bivalirudine trifluoroacetate or bivalirudine (100% purity) was added. The reaction was allowed to proceed for about 20 minutes, and then the reaction product was precipitated using water. The precipitate was dried and redissolved in fresh THF. The solution containing the bivalirudine-linked polymer was added inside the SNAT semi-impregnated example tube, and the solvent was evaporated. The bivalirudine-linked polymer coated the inner surface of the example tube.

[0066] The example tubes and one control tube were tested in vivo in rabbits as described in Example 1 and Figure 6. After removal from the rabbits, no thrombi were observed in the SNAT semi-impregnated tubes coated with bivalirudin-linked polymer. In contrast, the untreated tubes showed a thrombus of 10.97 ± 0.95 cm². 2(Data ± standard deviation, n = 7).

[0067] These results show the additional effect of the vivalidine-linked polymer with semi-impregnated SNAT.

[0068] Example 3

[0069] Examples of the NO-generating polymer vascular catheter were prepared using a polyurethane vascular catheter and SNAT. Two solutions were tested. The solvent system of each solution contained methanol:chloroform in a volume ratio of 1:1. One solution contained 1000 mg / mL of SNAT and the other solution contained 600 mg / mL of SNAT.

[0070] The example vascular catheter was filled with the solution, sealed, and the vascular catheter was incubated for 4 hours.

[0071] The example vascular catheter was tested in vitro for the NO release profile of the vascular catheter (measured using the ozone chemiluminescence method). The average results for three of each example are shown in Table 5.

Table 5

[0072] The example prepared with 1000 mg / mL of SNAT exhibited a desired NO flux (>0.5x10 -10 mol / min / cm 2 flux) for more than 31 days and reached the threshold at 35 days. The example prepared with 600 mg / mL of SNAT exhibited a desired NO flux (>0.5x10 -10 mol / min / cm 2 flux) for more than 14 days and dropped below the threshold at 21 days. These results show the effectiveness of the semi-impregnation method in polyurethane objects.

[0073] Example 4

[0074] One NO-generating polymer tube was fabricated using a silicone tube and SNAT. One solution containing 70 mg / mL of SNAT in tetrahydrofuran was tested.

[0075] In the example, the silicone tubes were filled with a solution, sealed, and incubated for 4 hours.

[0076] The tube in this example was tested in vitro for its NO emission profile (measured using ozone chemiluminescence). The results are shown in Table 6. [Table 6]

[0077] Examples prepared with 70 mg / mL SNAT in silicone showed nearly the desired NO flux (>0.5 x 10) over 4 days. -10 mol / min / cm 2 The flux was observed. These results may be improved by adding a higher amount of SNAT. These results demonstrate the effectiveness of the semi-impregnation method in silicone materials.

[0078] Comparative Example

[0079] Comparative examples used SNAT with either a full impregnation technique or a coating technique. SNAT was synthesized as described herein, and the extracorporeal circulation circuit (PVC tubing (ND100-65 TYGONT®, 3 / 8 inch inner diameter (approximately 9.525 mm)) and connectors and cannulas) was modified with the NO donor via full impregnation or coating techniques. The full impregnation technique involved completely immersing the extracorporeal circulation circuit in a solution of SNAT (1000 mg / mL) in a combination of organic solvent and plasticizer (1:3:1 acetone:plasticizer:methanol) until the circuit swelled with the solution. The circuit was then dried. The coating technique involved dissolving SNAT in a polymer solution, applying the solution to the inner surface of the ECC components, and drying. SNAP was used as the NO donor with other full impregnation techniques. The control example was the same as the untreated control in the examples.

[0080] An electro-emission scanning electron microscope (JEOL JSM-7800F) was used to evaluate the surface roughness of NO donor-doped polymers (SNAP, SNAT) and unmodified control polymers. These images are shown in Figure 9, where row A shows the untreated control example, row B shows the SNAT-fully impregnated PVC tube (1000 mg / ml), and row C shows the SNAP-fully impregnated PVC tube (maximum dosing limit of 250 mg / ml). The SNAT-added PVC surface was similar to the untreated control, but more significantly so. The SNAP-added PVC surface showed a clear difference.

[0081] The in vitro NO release profiles of SNAT (600 mg / ml and 1000 mg / ml) fully impregnated PVC circuit tubing were measured by a nitric oxide analyzer (NOA). As shown in Figure 10, the 1000 mg / ml SNAT-impregnated PVC tubing exhibited endothelial-level NO release capacity for up to 49 days (Figure 10).

[0082] The storage stability of SNAT-impregnated PVC tubes containing 1000 mg / ml was tested. The storage conditions were as follows: dry, under air, at different temperatures of 21°C, 4°C, and -20°C, and for a period of one year, where d0 is the date one year after storage, and d1 and d2 are subsequent dates after one year). The results after one year (Figure 11) show that relatively stable storage is possible for SNAT-impregnated PVC tubes containing 1000 mg / ml at 4°C and -20°C.

[0083] Furthermore, the NO-releasing capacity of 1000 mg / ml impregnated SNAT polymer examples and untreated controls was tested after undergoing different sterilization techniques. Sterilization was performed using ethylene oxide (EtO), hydrogen peroxide (H2O2), and OPS liquid chemical (shown as PVC liquid). The results are shown in Figure 12. Since the OPS liquid chemical did not alter NO-releasing capacity up to day 14, these results indicate that the OPS liquid chemical is the best sterilization liquid. The results also indicate that the widely used H2O2 may be a further option for sterilizing example tubes while maintaining their original NO-releasing capacity.

[0084] To evaluate the antithrombotic activity and platelet preservation capacity of extracorporeal circulation circuit tubing coated or impregnated with a novel NO donor (SNAT), and to determine whether the modified polymer tubing was less thrombotic compared to unmodified polymer materials, an arteriovenous (AV) rabbit thrombosis model without systemic heparinization (similar to the configuration shown in Figure 6) was used. The determined endpoint parameters included thrombus area within the tubing, coagulation, plasma fibrinogen levels, platelet count by aggregateometry, and platelet aggregation. A total of 13 male New Zealand white rabbits (n=7 controls, n=3 coated with NO-600 mg / ml, n=3 impregnated with NO-1000 mg / ml) were evaluated. While 1000 mg / ml SNAT coated the tubing (data not shown), the NO concentration was very high in the thin coating layer, leading to the following technical difficulties. Specifically, 1) the formation of gaseous NO resulted in an uneven and "bumpy" surface, and 2) the uneven surface and the very dark color of the coating made it impossible to read the flow through the transonic system.

[0085] In the coated NO group, blood pressure and blood flow remained stable compared to baseline throughout the 4-hour study period. Platelet function (SNATi-1000: 77.9±1.4% vs. control: 56.0±4.2%, compared to baseline 78.1±5.9%) and platelet count (Figure 13A), as measured by aggregateometry, were maintained throughout the study period, and no significant thrombus formation (Figure 13B) was observed in this group.

[0086] However, the control group showed higher variance in the parameters mentioned above. Platelet counts decreased to less than 50%, and significant thrombus formation was observed in all circuits (Figures 13A and 13B, SNAT-600: 1.2±1.2cm). 2 n=3 pairs vs. control: 11.0±0.4cm 2 (n=7). Two of the control circuits formed thrombi within the first hour of the experiment.

[0087] As described, the impregnation technique (1000 mg / ml) was also used to prepare the NO-releasing surface. This technique treated the entire polymer, both on the inner and outer surfaces.

[0088] The impregnated NO circuit exhibits even superior antithrombotic properties compared to the coated group (SNATi1000: 0.001 ± 0.0 cm). 2 n=3 vs. control: 10.28±0.20cm 2 The control group (n=7) showed no thrombus formation throughout the circuit (Figures 14A and 14B). Aggregometry of the NO-impregnated group (SNATi) did not show a significant difference compared to baseline values ​​(SNATi-1000: 79.8±0.3% vs. control: 56.0±4.2%, compared to baseline 74.1±1.9%). SNAT-impregnated PVC also showed excellent antibacterial activity (5-day, 3-log reduction). Confocal images are shown in Figure 15. Tables 7A and 7B show the high antibacterial effect (reduction in bacterial colonies) (Table 7A) and bacterial killing effect (7-day, 7-log reduction (Table 7B)) of SNAT-impregnated polymers against Gram-positive and Gram-negative bacterial strains after a 5-day biofilm test compared to the control group. [Table 7A] [Table 7B]

[0089] All of these results suggest that the SNAT treatment (both coating and impregnation) circuit exhibits superior antithrombotic and antimicrobial activity compared to untreated control ECC, contributing to the maintenance of platelet counts throughout the test period. The antithrombotic and antimicrobial activity may be similar to that of the SNAT semi-impregnation examples. However, these results also indicate that these techniques may have adverse effects on the surface roughness and, possibly, the mechanical properties of the coated or fully impregnated polymer, which differs from the semi-impregnation examples disclosed herein.

[0090] References throughout the specification to "one example," "another example," "example," etc., mean that certain elements (e.g., features, structures, and / or properties) described in relation to the examples are included in at least one example described herein and may or may not be present in other examples. In addition, it should be understood that elements described for any example may be combined in any preferred manner in various examples unless otherwise clearly defined in the context.

[0091] It should be understood that the ranges provided herein include the stated range and any values ​​or subranges within that range. For example, molecular weights in the range of about 195 daltons to about 350 daltons should be interpreted to include not only the explicitly enumerated limits of about 195 daltons to about 350 daltons, but also individual molecular weights (e.g., 197 daltons, 250 daltons, 275.5 daltons, etc.) and subranges of molecular weights (e.g., about 250 daltons to about 350 daltons, about 197 daltons to about 297 daltons, etc.). Furthermore, when "about" is used to state a value, it means that a small variation (up to ±10%) from the stated value is included.

[0092] In describing and claiming the examples disclosed herein, the singular forms "a," "an," and "the" shall include multiple subjects unless the context clearly indicates otherwise.

[0093] While several examples are described in detail, it should be understood that the disclosed examples are subject to modification. Therefore, the foregoing explanation should be considered non-limiting.

Claims

1. A method for producing a nitric oxide generating device, including the following steps: Introducing a solution into the lumen of a nitric oxide permeable polymer object, where the solution is: Solvent, and S-nitroso-1-adamantanthiol dissolved in the aforementioned solvent Including; Immerse the lumen in the solution for a maximum of 12 hours, thereby impregnating a portion of the wall of the nitric oxide-permeable polymer object adjacent to the lumen with the S-nitroso-1-adamantanthiol; and To remove the solution from the lumen.

2. The nitric oxide permeable polymer material is composed of poly(vinyl chloride), and The aforementioned solvent is part of a solvent system that further contains a plasticizer. The method according to claim 1.

3. The solvent system further comprises a second solvent, and The solvent, the plasticizer, and the second solvent are present in a predetermined volume ratio. The method according to claim 2.

4. The method according to claim 3, wherein the predetermined volume ratio is 0.1 to 10 parts of the solvent, 0.1 to 10 parts of the plasticizer, and 0.1 to 10 parts of the second solvent.

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

6. The aforementioned solvent is acetone. The aforementioned plasticizer is trioctyl trimelite, and The second solvent is methanol. The method according to any one of claims 3 to 5.

7. The method according to any one of claims 1 to 6, wherein the solution contains up to 2000 mg / mL of S-nitroso-1-adamantanthiol.

8. The method according to any one of claims 1 or 7, wherein the nitric oxide permeable polymer material is composed of silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene.

9. The nitric oxide permeable polymer material is composed of silicone, and The method according to claim 8, wherein the solvent is tetrahydrofuran.

10. The nitric oxide permeable polymer object is composed of polyurethane, and The solvent is selected from the group consisting of methanol, methyl ethyl ketone, chloroform, cyclohexane, and combinations thereof. The method according to claim 8.

11. The method according to any one of claims 1 to 10, further comprising, before introducing the solution, dissolving the S-nitroso-1-adamantanthiol in the solvent to produce the solution.

12. The method according to claim 11, further comprising generating the S-nitroso-1-adamantanthiol by solvent-free nitrosation of 1-adamantanthiol with t-butyl nitrite before generating the solution.

13. The method according to any one of claims 1 to 12, further comprising the following steps: Introducing a coating solution into the lumen, wherein the coating solution comprises a direct thrombin inhibitor-linked polymer dissolved in a third solvent; and The third solvent is evaporated, thereby forming a coating on the surface of the lumen, wherein the coating comprises the direct thrombin inhibitor-linked polymer.

14. The method according to claim 13, further comprising forming the coating solution by the following steps before introducing the coating solution: Dissolving the anchor polymer in the third solvent; The linking molecule is introduced into the third solvent, thereby covalently bonding the linking molecule to the anchor polymer; The process involves introducing a direct thrombin inhibitor into the third solvent, thereby covalently bonding the direct thrombin inhibitor to the linking molecule bonded to the anchor polymer, thereby forming the direct thrombin inhibitor linking polymer; Precipitating the direct thrombin inhibitor linked polymer; and The direct thrombin inhibitor-linked polymer is redissolved in a new third solvent.

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

16. The aforementioned solvent is part of a solvent system that includes the second solvent. The aforementioned solvent and the second solvent are different from each other and are independently selected from the group consisting of 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 in the range of 0.1:10 to 10:0.

1. The method according to claim 1.

17. Nitric oxide permeable polymer material, and S-nitroso-1-adamantanethol impregnated in a portion of the wall of the nitric oxide permeable polymer object adjacent to the lumen of the nitric oxide permeable polymer object Includes, The outer surface of the wall of the nitric oxide permeable polymer material is substantially free of the S-nitroso-1-adamantanthiol. Nitric oxide generating device.

18. The nitric oxide generating device according to claim 17, wherein the S-nitroso-1-adamantanthiol extends within the wall to a depth of approximately 500 μm to approximately 1000 μm.

19. The nitric oxide generating device according to any one of claims 17 or 18, wherein the nitric oxide permeable polymer material is composed of poly(vinyl chloride), silicone, polyurethane, a combination of silicone and polyurethane, polycarbonate, polypropylene, or polytetrafluoroethylene.

20. The nitric oxide generating device according to any one of claims 17 to 19, further comprising a direct thrombin inhibitor-linked polymer attached to the surface of the lumen.

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