Implantable medical device

Abstract

An implantable medical device comprising a nitric oxide-generating polymeric material wherein (i) the nitric oxide-generating polymeric material comprises a polymer and: (a) particles wherein one or more single particles each contain a nitrite and a proton source; or (b) a particulate agglomerate wherein the agglomerate comprises one or more single particles comprising a nitrite, one or more single particles comprising a proton source, and optionally a binder, or the particulate agglomerate comprises one or more single particles each comprising a nitrite and a proton source, and optionally a binder; (ii) the nitric oxide generating polymeric material comprises a proton source polymer, and the nitric oxide generating polymeric material comprises a nitrite dissolved in the proton source polymer matrix; or (iii) a combination of (i) and (ii) described above.

Description

[0001] The present invention relates to an implantable medical device comprising a polymeric material that generates nitric oxide, wherein

[0002] (i) the nitric oxide generating polymeric material comprises a polymer and: (a) particles, wherein one or more individual particles each contain a nitrite and a proton source; or (b) particle agglomerates, wherein the agglomerates comprise one or more individual particles containing a nitrite, one or more individual particles containing a proton source, and optionally a binder, or the particle agglomerates comprise one or more individual particles each containing a nitrite and a proton source, and optionally a binder;

[0003] (ii) the nitric oxide generating polymeric material comprises a proton-producing polymer, and the nitric oxide generating polymeric material comprises a nitrite dissolved in the proton-producing polymer matrix; or

[0004] (iii) A combination of (i) and (ii) above. Background Art

[0005] Nitric oxide (NO) and nitric oxide precursors have been extensively studied for use in medical applications.

[0006] However, substantial problems remain in the efficient generation and delivery of nitric oxide, other nitrogen oxides, and their precursors to organisms and cells for therapeutic purposes. The widely used system for generating nitric oxide relies on acidifying nitrite with a proton source (such as an acid) to initially produce nitrous acid (HNO2), which then readily decomposes into nitric oxide and nitrate, as well as hydrogen ions and water. This decomposition can be represented by the following balanced equation (1):

[0007] 3 HNO2→2 NO+NO3 - +H + +H2O (1)

[0008] Acid and nitrite are provided as the individual components of predetermined amount usually.These individual components keep separate until when using, to minimize reaction before the moment of need.Therefore these two kinds of reactants are provided with two-piece system (two-part system), and it relates to the part containing nitrite and the individual part containing acid.In this way, these two kinds of individual components in these two individual parts can be combined or mixed when needed, to prevent releasing nitric oxide before need.

[0009] Nitric oxide (NO) has been recognized as a key regulator of tissue repair and regeneration in a variety of tissues, including but not limited to skin, tendons, and bones. In damaged skin wounds, the delivery of exogenous NO has been shown to repair and heal in experimental and clinical studies. In vivo, topical delivery of the NO prodrug glyceryl trinitrate (GTN) has been shown in several clinical studies to bring about a more rapid and sustained resolution of symptoms caused by rotator cuff tears and other tendon injuries. However, the systemic adverse side effects associated with transdermal delivery of GTN and NO production (especially migraine-like headaches) have limited its clinical application in tendon injuries.

[0010] Implantable medical devices are known for various clinical applications. Synthetic polymers widely used in medical devices such as sutures and stents include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL) or their composite materials. Over the past 40 years, these materials have been shown to have excellent biocompatibility when used in various clinical applications. However, the devices composed of these materials are mainly passive in the healing response, either holding the damaged tissue together or creating a stable environment so that the endogenous repair response can proceed without being subjected to harmful mechanical damage at the site. SUMMARY OF THE INVENTION

[0012] To address the lack of biological activity of synthetic polymers and the adverse effects of systemic exposure to NO prodrugs, the present inventors have developed a composition of synthetic biopolymers to be manufactured that delivers bioactive doses of NO directly to the site of tissue repair and regeneration. By incorporating a stable powder of an acidified nitre reaction into a synthetic medical polymer, a scaffold (device) has been made that can be implanted directly into the site of tissue damage to provide a structural scaffold that supports tissue repair, releasing nitric oxide at doses that support tissue repair and regeneration. In other words,

[0013] Most generally, the invention provides a kind of implantable medical device (implantable medical device) with the polymeric material (nitric oxide generating polymeric material) that generates nitric oxide, and the polymeric material that generates nitric oxide comprises (i) polymer and particle or the particle agglomerate that comprises nitrite and proton source, or (ii) and acid source polymer (acid source polymer), it comprises the nitrite that is dissolved in the described polymer matrix.In this way, nitrite and proton source keep close to (or tightly combined), so that the acidification of nitrite is provided when contacting with aqueous environment, but do not react substantially before needs, therefore single-component system can be provided.Comprise that the solid component of nitrite and acid source can avoid comprising moisture source (as solution or water-based gel).In this way, reactant reduces and is exposed to moisture, so that reaction is minimized before needs reaction.

[0014] In a first aspect, the present invention provides an implantable medical device comprising a nitric oxide-generating polymeric material, wherein

[0015] (i) the nitric oxide generating polymeric material comprises a polymer and: (a) particles, wherein one or more individual particles each contain a nitrite and a proton source; or (b) particle agglomerates, wherein the agglomerates comprise one or more individual particles containing a nitrite, one or more individual particles containing a proton source, and optionally a binder, and / or the particle agglomerates comprise one or more individual particles each containing a nitrite and a proton source, and optionally a binder;

[0016] (ii) the nitric oxide generating polymer material comprises a proton source polymer, and the nitric oxide generating polymer material comprises a nitrite dissolved in a matrix of the proton source polymer; or

[0017] (iii) A combination of (i) and (ii) above.

[0018] The nitric oxide generating polymeric material can have a pre-implantation water content of 10% or less, 5% or less, 2% or less, 1% or less, or the nitric oxide generating polymeric material can be substantially free of water.

[0019] The nitric oxide-generating polymer material may be the outer surface of an implantable medical device.

[0020] The nitric oxide generating polymer material may form a scaffold of an implantable medical device, the nitric oxide generating polymer material may form a coating on another component of an implantable medical device, or the nitric oxide generating polymer material may form a part of a textile of an implantable medical device.

[0021] The nitric oxide-generating polymer material may be a fiber or a coating.

[0022] An implantable medical device may be a one-part device.

[0023] The nitric oxide generating polymeric material may comprise a) particles, wherein one or more individual particles each contain a nitrite and a proton source; or (b) particle agglomerates, wherein the agglomerates comprise one or more individual particles containing a nitrite, one or more individual particles containing a proton source and an optional binder, and / or the particle agglomerates comprise one or more individual particles each containing a nitrite and a proton source and an optional binder.

[0024] The one or more individual particles or particle agglomerates may be blended or coated with excipients for influencing the rate of water ingress into the particles and / or excipients for influencing the kinetics of nitric oxide formation from the particles.

[0025] The excipient for influencing the rate of water ingress into the particles may be a polyol or a hydrophobic material such as a phospholipid, magnesium stearate or colloidal silicon dioxide, and / or the excipient for influencing the rate of water ingress into the particles may be nitric oxide or a nitric oxide precursor sequestering material such as a thiol, alcohol, amine or amide.

[0026] The particles comprising nitrite and a proton source may be formed by spray drying a mixture comprising a nitrite solution and a proton source solution.

[0027] The nitric oxide generating polymer material may include a proton-generating polymer. The proton-generating polymer may be an acidic polymer, a photoacid polymer, or an acid precursor polymer, such as a hydrolyzable ester.

[0028] The one or more particles or particle agglomerates may be embedded or partially embedded in the polymer of the nitric oxide generating polymer material. Alternatively, the one or more particles or particle agglomerates are attached to the surface of the polymer of the nitric oxide generating polymer material.

[0029] The polymeric material that generates nitric oxide can comprise proton source polymer, and the polymeric material that generates nitric oxide can comprise the nitrite that is dissolved in the proton source polymer matrix.In these embodiments, nitrite can be substantially uniformly mixed with the proton source polymer matrix.The polymeric material that generates nitric oxide can be formed by the non-aqueous solution of nitrite and proton source polymer.

[0030] The polymer of the nitric oxide generating polymer material may be a biocompatible polymer.The polymer of the nitric oxide generating polymer material may be a resorbable material.

[0031] The proton source may include an acid, an acid precursor such as an ester, or a photoacid.

[0032] The implantable medical device may include one or more additional dry components adjacent to the nitric oxide-generating polymer material. The implantable medical device may include one or more additional components adjacent to the nitric oxide-generating polymer material, provided that the water content of any component adjacent to the nitric oxide-generating polymer material is 10% or less, 5% or less, 2% or less, or 1% or less, based on the weight of the component adjacent to the nitric oxide-generating polymer material.

[0033] The implantable medical device may include an antimicrobial agent.The implantable medical device may be a one-piece medical device.

[0034] In a second aspect, the present invention provides a packaged implantable medical device comprising the implantable medical device described herein within a low moisture permeability package.

[0035] Low moisture permeability packaging includes one or more low moisture permeability materials (e.g., aluminum foil) in the walls of the package and / or can be hermetically sealed. The packaging atmosphere within the packaged implantable medical device has a low moisture content upon initial packaging, and / or the package includes a package insert that traps moisture.

[0036] In a third aspect, the present invention provides a method of implanting an implantable medical device into a subject, the method comprising implanting the implantable medical device described herein into the subject.The implantable medical device may be a one-piece implantable medical device.

[0037] In a fourth aspect, the present invention provides particles or particle agglomerates for implanting an implantable medical device as described herein into a subject, wherein (a) one or more individual particles each contain a nitrite and a proton source; or (b) the agglomerate comprises one or more individual particles containing a nitrite, one or more individual particles containing a proton source, and optionally a binder, and / or the particle agglomerate comprises one or more individual particles each containing a nitrite and a proton source, and optionally a binder. The implantable medical device may be a one-piece implantable medical device.

[0038] Optional or specific features of one aspect of the invention described herein are also applicable to other aspects of the invention, so long as the features are compatible with that aspect. In particular, optional or specific features of an implantable medical device are also applicable to a packaged implantable medical device, a method of implanting the implantable medical device, and particles or particle agglomerates for use in an implantable medical device, so long as the features are compatible with those aspects.

[0039] Details

[0040] The invention will now be described in more detail.The Examples and Figures below provide illustrations of the invention.

[0041] Figure 1 Scanning electron microscope image showing PCL fibers without nitric oxide-generating particles.

[0042] Figures 2 to 4 Scanning electron microscope images of PCL fibers loaded with 1%, 5% and 10% w / w nitric oxide generating particles are shown. The particles are visible as white spots in the fibers.

[0043] Figure 5 Scanning electron microscope image showing TPU fibers without nitric oxide generating particles.

[0044] Figures 6 to 8 Scanning electron microscope images of TPU fibers loaded with 1%, 5% and 10% w / w nitric oxide generating particles are shown, respectively. The particles are visible as white spots in the fiber.

[0045] Figure 9 The fluorescence intensity of the NO sensor (DAF-FM) of the electrospun fiber of Example 8 is shown versus time.

[0046] Figure 10 Shown are the deposition patterns of the powders of Examples 1A, 2, 3, and 4 on agarose with Hanks balanced salt solution and a pH indicator (phenol red).

[0047] Figure 11 Cumulative NO production for Examples 1A, 2, 3, and 4 is shown.

[0048] Figure 12 Shown are the sprouting intensities of HUVEC spheroids treated with Examples 1B and 6A, quantified by an image analysis system to determine the cumulative sprout length (CSL) per spheroid relative to a basal control.

[0049] Figure 13Figure 3. The morphology of VERO cells grown on NO-releasing PLGA scaffolds after 7 days of culture. A, no NO-releasing powder; B, 1% (wt / wt) NO-releasing powder; C, 5% (wt / wt) NO-releasing powder; D, 10% (wt / wt) NO-releasing powder.

[0050] FIG14 shows a scanning electron microscope image of PLGA fibers containing sodium nitrite. Figure 14A 、 14B 14A and 14C show fibers from Examples 14A, 14B, and 14C, respectively.

[0051] Figure 15A The pH of Examples 14A-D is shown over time. Figure 15B The fluorescence intensity distribution of Examples 14A-D over time is shown.

[0052] The reaction between one or more nitrites and a proton source to generate nitric oxide, optionally other nitrogen oxides and / or optionally their precursors is referred to herein as a "NOx generation reaction" or "NOx generation reaction" or similar wording, and "NOx" is used to refer to the acidification products of nitrite, particularly nitric oxide, other nitrogen oxides and their precursors, individually and in any combination collectively. It is to be understood that the various components of the NOx generated can escape as gases, or can dissolve in the reaction mixture, or can initially dissolve and subsequently escape as gases, or any combination thereof.

[0053] The term "about" is used herein to indicate that a numerical value is not strictly limiting, and that the skilled person will understand that the value may extend above or below the exact value (as the case may be) according to the skilled person's understanding of the value. The term "about" may indicate a value of up to ±10% of the value.

[0054] Unless otherwise indicated, particle sizes described herein refer to volume mean diameter (VMD).

[0055] As used herein, the terms "one-part," "single-part," and "two-part" are used to refer to the number of pieces of an implantable medical device prior to the time of need (e.g., implantation into a subject). For example, a one-piece implantable medical device is provided as a single piece prior to the time of need. A one-piece implantable medical device is typically implanted into a subject as a single piece. In contrast, a two-piece implantable medical device is provided as two pieces prior to the time of need and is typically combined into a single-piece implantable medical device prior to implantation into a subject. It should also be noted that the one-piece implantable medical devices described herein can be formed from a nitric oxide-generating polymeric material and one or more other components.

[0056] Implantable medical devices

[0057] As used herein, an "implantable medical device" is a device intended to be implanted in a subject (e.g., a human or animal). Implantable medical devices are typically manufactured to replace missing biological structures, support damaged biological structures, or enhance existing biological structures. Implantable medical devices are of a wide variety and are known per se.

[0058] Examples of implantable medical devices include, but are not limited to, sensory and neural implants (e.g., intraocular lenses, intrastromal corneal ring segments, cochlear implants, tympanostomy tubes, and neurostimulators), cardiovascular implants (e.g., artificial hearts, artificial heart valves, implantable cardioverter-defibrillators, artificial cardiac pacemakers, and coronary stents), orthopedic implants (e.g., pins, rods, screws, plates, and combinations thereof, used to anchor broken bones while they heal), electrical implants, contraceptive implants (e.g., copper- and hormone-based intrauterine devices), and implants for the treatment of cervical spondylosis. devices), cosmetic implants, and other organs and systems (e.g., LINX, implantable gastric stimulators, diaphragmatic / phrenic nerve stimulators, neurostimulators, surgical meshes, artificial urinary sphincters, and penile implants).

[0059] The polymeric material that generates nitric oxide can replace the material in known implantable medical device configurations. For example, the polymeric material that generates nitric oxide can form the framework in, for example, a cardiovascular stent, or can form the coating of, for example, an electrical implant. Alternatively, the polymeric material that generates nitric oxide can be included in known implantable medical device configurations as any additional material. For example, the polymeric material that generates nitric oxide can form an additional coating, for example, an additional coating on the outer surface of an artificial joint.

[0060] Polymer materials that generate nitric oxide

[0061] Implantable medical device of the present invention comprises the polymeric material that generates nitric oxide to generate nitric oxide with the acidification by nitrite, and wherein the material that generates nitric oxide comprises polymer, nitrite component and proton source component.The arrangement of nitrite in polymer can be as particle or particle agglomerate, and wherein this particle or agglomerate contain nitrite component and proton source component.Perhaps, nitrite can be dissolved in the proton source polymer.

[0062] In this way, the nitrite and the proton source can be in close proximity to fully react when exposed to an aqueous environment. The nitrite and the proton source are present in the polymer material of the implantable medical device. In this way, these components do not need to be combined at the time of use (e.g., as part of a two-part system).

[0063] Typically, the polymeric material is a solid polymeric material. Typically, the particles or particle agglomerates are solid particles. In this way, nitric oxide generation before use is reduced because the water content can be minimized.

[0064] In some embodiments, the polymer material of nitric oxide is a mixture of nitrite and acid component.Usually, all components of the polymer material that generates nitric oxide are dry components.In this way, before use, the reaction of nitrite and acid component is minimized.Based on the weight meter of the polymer material that generates nitric oxide, the water content of the polymer material that generates nitric oxide can be 10% or less, 5% or less, 2% or less or 1% or less.In this way, before use, the reaction between nitrite and the proton source reactant is minimized.Water content can be measured by standard laboratory method, as by sample weighing, removes moisture (for example, by drying in a baking oven exceeding 100 ℃), and then again by sample weighing.

[0065] The nitric oxide generating polymeric material can include one or more fibers. The nitric oxide generating polymeric material can be a woven or non-woven fiber material. The nitric oxide generating polymeric material can be a coating on a substrate. The nitric oxide generating polymeric material can be formed into a medical device stent.

[0066] The polymer of the polymeric material that generates nitric oxide can be an adsorbent. The polymeric material can be composed of woven or non-woven fibers or solid foam. The polymeric material can be composed of cotton fibers, rayon, polyester (such as PLGA) and / or gelling fibers, such as carboxymethyl cellulose and its salts. Additionally or alternatively, the polymeric material can be a solid foam of a hydrophilic material (such as silicone).

[0067] In certain embodiments, the nitric oxide generating polymeric material is on or forms the outer surface of the implantable medical device when in use. In this way, the nitric oxide generating layer is exposed to the implantation site to directly provide nitric oxide to the implantation site.

[0068] Alternatively, the implantable medical device may have one or more permeable layers or components outside the polymer layer that generates nitric oxide. The implantable medical device may include one or more permeable layers adjacent to the polymer material that generates nitric oxide, and may be configured so that the one or more permeable layers contact the implantation site in use. The one or more permeable layers may be made of any permeable material, typically any gas and / or liquid permeable material. In this way, nitric oxide may enter these layers and / or liquid may enter the polymer material that generates nitric oxide through these layers.

[0069] The polymeric material that generates nitric oxide can further include one or more active pharmaceutical ingredients (API). In this way, the polymeric material that generates nitric oxide can deliver one or more active pharmaceutical ingredients directly to the implantation site. There are no particular restrictions on the API. Specific API can include one or more analgesics, one or more anti-inflammatory drugs, one or more additional antimicrobials and / or one or more anticoagulants.

[0070] The acidification of the nitrite component and the proton source component generally has antimicrobial activity. In some instances, the implantable medical device includes an additional antimicrobial agent. Antimicrobial agents are known per se. In some instances, the implantable medical device includes AgNO2 as both an antimicrobial agent and a nitrite.

[0071] Polymers of nitric oxide-generating polymer materials

[0072] The polymer material that generates nitric oxide includes at least one polymer. The at least one polymer can be a natural polymer or a synthetic polymer. In a specific embodiment, the at least one polymer is a synthetic polymer.

[0073] Synthetic polymers are widely used in implantable medical devices. Examples of synthetic polymers that may be included in the nitric oxide polymer material include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), thermoplastic polyurethane (TPU), and blends thereof.

[0074] Additionally or alternatively, at least one natural polymer may be included in the polymeric material generating nitric oxide. Such natural polymer may serve as a cell binding motif to promote cell adhesion and / or proliferation. Examples of natural polymers that may be included in the nitric oxide polymeric material include, but are not limited to, gelatin, chitin, or collagen.

[0075] In some embodiments, the nitric oxide generating polymeric material comprises only synthetic polymers or a blend of synthetic polymers. In some embodiments, the nitric oxide generating polymeric material may comprise a blend of at least one natural polymer and at least one synthetic polymer.

[0076] The at least one polymer can be a biocompatible polymer. In other words, the at least one polymer can be compatible with living cells. In certain embodiments, the polymer present in the polymeric material that generates nitric oxide is biocompatible. Where more than one polymer is present, each polymer present in the polymeric material that generates nitric oxide can be biocompatible.

[0077] In some embodiments, the at least one polymer is a biodegradable or bioresorbable polymer. In other words, the at least one polymer degrades when exposed to a biological environment, such as a cell or tissue of a subject, and can be absorbed by the body over time. In a specific embodiment, the polymer present in the polymeric material that generates nitric oxide is biodegradable or bioresorbable. In the presence of more than one polymer, each polymer present in the polymeric material that generates nitric oxide can be biodegradable or bioresorbable.

[0078] The polymer may be an electrospinning polymer for forming one or more electrospun fibers. Polymers suitable for electrospinning are known per se. Examples include, but are not limited to, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), thermoplastic polyurethane (TPU), polymers that can serve as cell binding motifs (such as, but not limited to, gelatin or collagen), and blends thereof.

[0079] In some embodiments, the polymer is a proton source polymer. The proton source can be an acid polymer or an acid precursor polymer. An "acid precursor polymer" is a polymer species that can undergo a chemical reaction to provide an acid species. For example, an acid precursor polymer can be a polymer species that can undergo hydrolysis to provide an acid species. In other words, the acid precursor polymer can be a hydrolyzable acid precursor polymer for releasing an acid when hydrolyzed. For example, the acid precursor polymer can be a polyester. The acid precursor polymer can be a photoacid. In other words, the acid precursor polymer can be a species that becomes more acidic when absorbing light. For the avoidance of doubt, the term "photoacid" as used herein includes species that undergo reversible proton photodissociation and species that undergo irreversible proton photodissociation.

[0080] Such proton-generating polymers include, but are not limited to, polyacrylic acid (particularly partially neutralized polyacrylic acid) and polyesters (particularly PLGA).

[0081] Particles or particle agglomerates comprising a nitrite component and a proton source component

[0082] In some embodiments, the implantable medical device of the present invention includes particles or particle agglomerates comprising a nitrite component and a solid proton source component in a single nitric oxide-generating polymeric material. In this manner, the single nitric oxide-generating polymeric material can release nitric oxide by acidification of the nitrite when exposed to moisture in an aqueous environment or the atmosphere.

[0083] Solid nitrite component

[0084] The solid nitrite component includes nitrite. There is no particular restriction on the selection of nitrite. Nitrite can be selected from one or more alkali metal nitrites or alkaline metal nitrites. For example, the one or more nitrites can be selected from LiNO2, NaNO2, KNO2, RbNO2, CsNO2, FrNO2, AgNO2, Be(NO2)2, Mg(NO2)2, Ca(NO2)2, Sr(NO2)2, Mn(NO2)2, Ba(NO2)2, Ra(NO2)2 and any mixture thereof. Nitrite can be NaNO2 or KNO2. Nitrite can be NaNO2.

[0085] The nitrite can be a pharmaceutical grade nitrite. In other words, the nitrite can follow one or more valid pharmacopoeial monographs on nitrite. For example, the nitrite can follow one or more nitrite monographs in the United States Pharmacopoeia (USP), the European Pharmacopoeia or the Japanese Pharmacopoeia.

[0086] In particular, the nitrite used may have one or more of the characteristics provided in Table 1 in paragraphs

[0032] to

[0060] and / or paragraph

[0204] of WO 2010 / 093746, the disclosures of which are incorporated herein by reference in their entirety.

[0087] Solid proton source components

[0088] The proton source component includes a proton source. The proton source can be any species that can serve as a proton source for acidification of nitrite. There is no particular limitation on the choice of the proton source. The proton source can be, for example, an acid.

[0089] The solid proton-producing component may be provided as a solid proton-producing component. Additionally or alternatively, the solid proton-producing component may be provided as part of the polymer (eg as a proton-producing polymer).

[0090] The acid may be selected from one or more organic carboxylic acids or organic non-carboxylic reducing acids.

[0091] The expression "organic carboxylic acid" herein refers to any organic acid containing one or more -COOH groups in the molecule. The organic carboxylic acid may be linear or branched. The carboxylic acid may be saturated or unsaturated. The carboxylic acid may be aliphatic or aromatic. The carboxylic acid may be acyclic or cyclic. The carboxylic acid may be a vinylogous carboxylic acid.

[0092] The organic carboxylic acid may carry one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic carboxylic acids useful in the present disclosure include α-hydroxy-carboxylic acids, β-hydroxy-carboxylic acids, and γ-hydroxy-carboxylic acids.

[0093] The expression "organic non-carboxylic acid reducing acid" herein refers to any organic reducing acid that does not contain a -COOH group in the molecule. The organic non-carboxylic acid reducing acid may be linear or branched. The non-carboxylic acid reducing acid may be saturated or unsaturated. The non-carboxylic acid reducing acid may be aliphatic or aromatic. The non-carboxylic acid reducing acid may be acyclic or cyclic. The non-carboxylic acid reducing acid may be vinylogous.

[0094] The organic non-carboxylic acid reducing acid may have one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic non-carboxylic acid reducing acids useful in the present disclosure include acidic reductones, such as reductic acid (2,3-dihydroxy-2-cyclopentanone).

[0095] The one or more organic carboxylic acids or non-carboxylic acid reducing acids may have a pKa1 of less than about 7.

[0096] The one or more organic carboxylic acids may comprise, consist of, or be one or more reducing carboxylic acids. The organic carboxylic acids may be, for example, selected from salicylic acid, acetylsalicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, α-hydroxypropionic acid, β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamoic acid (emboic acid), stearic acid, malonic acid, succinic acid, fumaric acid, glucoheptonic acid, glucuronic acid, lactobionic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, salts thereof, and combinations thereof.

[0097] The organic carboxylic acid may be citric acid or a salt thereof.

[0098] The carboxylic acid may be or comprise a polymeric or polymeric carboxylic acid, such as polyacrylic acid, polymethacrylic acid, a copolymer of acrylic acid and methacrylic acid, polylactic acid, polyglycolic acid or a copolymer of lactic acid and glycolic acid. The term "organic carboxylic acid" as used herein also encompasses partial or complete esters of organic carboxylic acids or partial or complete salts thereof, provided that these can serve as a proton source for use according to the present invention.

[0099] The organic non-carboxylic acid reducing acid can be, for example, selected from ascorbic acid; ascorbatepalmitic acid (ascorbyl palmitate); ascorbic acid derivatives such as 3-O-ethyl ascorbic acid, other 3-alkyl ascorbic acids, 6-O-octanoyl ascorbic acid, 6-O-dodecanoyl ascorbic acid, 6-O-tetradecanoyl ascorbic acid, 6-O-octadecanoyl ascorbic acid and 6-O-dodecandioyl ascorbic acid; acidic reductones such as reductic acid; isoascorbic acid; salts thereof; and combinations thereof.

[0100] The organic non-carboxylic acid reducing acid may be ascorbic acid or a salt thereof.

[0101] The one or more organic carboxylic acids or organic non-carboxylic acid reducing acids of the proton source can suitably exist together with their conjugate bases. When contacting or being exposed to an aqueous environment, the acid and its conjugate base can suitably form a buffer. The acid and its conjugate base can be provided in a ratio that realizes the required pH when exposed to an aqueous environment.

[0102] Can select buffer system, to realize required pH and keep required pH when NOx generation reaction is carried out when being exposed to aqueous environment.Can select buffer system, so that the pH of this reaction can be in the scope of about 3 to 9, for example about 4 to 8.For physiological contact or contact with living cell and organism, the pH of this reaction can be in the scope of about 5 to about 8.When existing, conjugate base can add separately, or can by using acid and / or alkali, for example mineral acid and / or inorganic base regulate pH and be generated by proton source original position.

[0103] The proton source may be a citric acid / citrate buffer system, such as a citric acid / trisodium citrate buffer system.

[0104] The proton source can be or can comprise an acid precursor. An "acid precursor" is a species that can undergo a chemical reaction to provide an acid species. For example, the acid precursor can be a species that can undergo hydrolysis to provide an acid species. In other words, the acid precursor can be a hydrolyzable acid precursor that releases an acid upon hydrolysis. For example, the acid precursor can be an ester. The acid precursor can be a photoacid. In other words, the acid precursor can be a species that becomes more acidic upon absorption of light. For the avoidance of doubt, the term "photoacid" as used herein includes species that undergo reversible proton photodissociation and species that undergo irreversible proton photodissociation.

[0105] The solid proton source component can be provided as a part of a polymer of a polymeric material (e.g., as a proton source polymer). In some embodiments, the solid proton source assembly includes a proton source fiber. In other words, the solid proton source component includes a fiber that can provide protons. Such proton source fibers include, but are not limited to, polyacrylic acid fibers (particularly partially neutralized polyacrylic acid fibers) and polyester fibers (particularly PLGA fibers).

[0106] In certain embodiments, the solid proton source component may include a combination of a solid proton source component and proton source fibers.

[0107] In certain embodiments, the solid proton source component comprises a solid proton source component.

[0108] The skilled person will appreciate that the choice of acid component / proton source can be selected according to the desired application. Combination of solid nitrite component and solid proton source component

[0109] Solid nitrite component and solid proton source component can be provided by one or more particles that contain nitrite and proton source separately.It should be understood that, when particle contained proton source and nitrite, particle can contain nitrite and proton source in same particle.The particle that contains nitrite and proton source separately can be used as the single particle in the polymeric material that generates nitric oxide and / or as particle agglomerate (wherein one or more particles in the agglomerate contain nitrite and proton source separately) and provide.

[0110] Additionally or alternatively, solid nitrite component and solid proton source component can be provided as one or more particles containing nitrite without proton source and one or more particles containing proton source without nitrite.It is to be understood that particle can contain nitrite or proton source, rather than containing nitrite and proton source in the same particle.The one or more particles containing nitrite or proton source can be blended to provide substantially uniform particle mixture.

[0111] The expressions "agglomerate", "agglomerate" and "agglomerated together" herein refer to a collection or aggregation of primary (single) particles that exhibit a recognizable collective behaviour.

[0112] In the present invention, the agglomerates of single particles may comprise (i) single particles containing nitrite and single particles containing a proton source, (ii) single particles containing nitrite and a proton source, or (iii) a combination thereof, and optionally, a binder.

[0113] In the present invention, an identifiable collective behavior may be the resistance to mechanical separation, ie the particles adhere to each other.

[0114] The particles or agglomerates of the solid nitrite component and the solid proton source component can be of a particle size suitable for their desired use or application. For example, the particles or agglomerates of the solid composition can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.

[0115] Alternatively, the particles or agglomerates of the solid nitrite component and the solid proton source component may have a particle size greater than 5 μm. For example, the particles or agglomerates of the solid composition may have a particle size greater than 50 μm, greater than 100 μm, greater than 250 μm, greater than 500 μm, greater than 750 μm, greater than 1000 μm.

[0116] The weight ratio of nitrite to proton source in the mixture of the solid nitrite component and the solid proton source component can be in the range of about 1:1 to about 1:99, such as about 1:4 to about 1:49 or about 1:7 to about 1:24.

[0117] The mixture of the solid nitrite component and the solid proton source component may comprise further optional additives, such as a binder (as mentioned above) or an organic polyol.

[0118] Adhesives

[0119] The mixture of the solid nitrite salt component and the solid proton source component may be substantially free of one or more binders. Alternatively, the mixture of the solid nitrite salt component and the solid proton source component may further comprise one or more binders.

[0120] As used herein, "binder" refers to an agent that promotes the adhesion of particles, ie, promotes the formation of particle agglomerates.

[0121] Suitable adhesive can comprise sugar, natural adhesive or synthetic or semisynthetic polymer adhesive.Sugar class can comprise for example sucrose or liquid glucose.Natural adhesive can comprise for example gum arabic, tragacanth, gelatin, starch paste, pregelatinized starch, alginic acid or cellulose.Synthetic or semisynthetic polymer adhesive can comprise for example methylcellulose, ethylcellulose, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol, polymethacrylate.Adhesive can be the copolymer (copolyvidone (copovidone)) of 1-vinyl-2-pyrrolidone and vinyl acetate.Adhesive can be microcrystalline cellulose.

[0122] The binder can be incorporated into the mixture of the solid nitrite salt component and the solid proton source component at a % w / w of about 5% w / w to about 30% w / w. For example, the binder can be incorporated into the mixture of the solid nitrite salt component and the solid proton source component at a % w / w of about 10% w / w to about 25% w / w.

[0123] Organic polyols

[0124] The mixture of solid nitrite component and solid proton source component can be substantially free of one or more organic polyols.Perhaps, the mixture of solid nitrite component and solid proton source component can further include one or more organic polyols.When the mixture of solid nitrite component and solid proton source component comprises one or more organic polyols, preferably after relating to any processing of removing solvent (for example after spray drying or freeze drying step), organic polyol is added in the mixture of solid nitrite component and solid proton source component.In other words, polyol can be added in the composition comprising one or more particles that contain nitrite and proton source; Or add in the mixture comprising one or more particles that contain nitrite and / or one or more particles that contain proton source (before or after forming the agglomerate of these particles).

[0125] The expression "organic polyol" herein refers to an organic molecule having two or more hydroxyl groups, which is not a proton source, in particular for the nitrite reaction, and which is not a sugar or polysaccharide (the terms "sugar" and "polysaccharide" include oligosaccharides, polysaccharides and glycosaminoglycans). The organic polyol thus has a pKa1 of about 7 or higher.

[0126] The expression "organic polyol" herein preferably does not include reducing agents. Examples of reducing agents that are organic molecules having two or more hydroxyl groups and are not sugars or polysaccharides are thioglycerol (e.g. 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid, ascorbic acid esters, isoascorbic acid and isoascorbic acid esters. Thioglycerol (e.g. 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid esters and isoascorbic acid esters are therefore preferably excluded from the expression "organic polyol" because they are reducing agents. In any case, ascorbic acid and isoascorbic acid are excluded from this expression because they are proton sources, in particular for the nitrite reaction.

[0127] The organic polyol may be cyclic or acyclic, or may be a mixture of one or more cyclic organic polyols and one or more acyclic organic polyols. For example, the one or more organic polyols may be selected from one or more alkane hydrocarbons substituted with two or more OH groups, one or more cycloalkanes substituted with two or more OH groups, one or more cycloalkylalkane hydrocarbons substituted with two or more OH groups, and any combination thereof. The organic polyol may not have any substituents other than OH.

[0128] The one or more organic polyols can be one or more acyclic organic polyols. The one or more acyclic organic polyols can be selected from sugar alcohols with 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The one or more acyclic organic polyols can be selected from alditols, for example, alditols with 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The one or more organic polyols can not include saponin, sapogenin, steroid or steroidal glycoside.

[0129] Alternatively, the one or more organic polyols can be one or more cyclic organic polyols. The one or more cyclic organic polyols can be cyclic sugar alcohols or cyclic alditols. For example, the one or more cyclic polyols can be cyclic sugar alcohols with 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms or cyclic alditols with 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. A specific example of a cyclic polyol is inositol.

[0130] The one or more organic polyols may have 7 or more hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 7 or more hydroxyl groups. The one or more organic polyols may have 9 or more hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 9 or more hydroxyl groups. The one or more organic polyols may have 20 or fewer hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 20 or fewer hydroxyl groups. The one or more organic polyols may have 15 or fewer hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 15 or fewer hydroxyl groups. The one or more organic polyols may have a hydroxyl number in the range of 7 to 20, for example in the range of 9 to 15. The one or more organic polyols may include 9, 12, 15 or 18 hydroxyl groups.

[0131] The one or more organic polyols may be a sugar alcohol compound comprising one or more monosaccharide units and one or more acyclic sugar alcohol units, for example, a sugar alcohol compound consisting of one or more monosaccharide units and one or more acyclic sugar alcohol units. The one or more organic polyols may be a sugar alcohol compound comprising a linear chain of one or more monosaccharide units and one or more acyclic sugar alcohol units or a branched chain of one or more monosaccharide units and one or more acyclic sugar alcohol units, for example, a sugar alcohol compound consisting thereof.

[0132] As used herein, "monosaccharide unit" refers to a monosaccharide that is covalently linked to at least one other unit in a compound (whether another monosaccharide unit or an acyclic sugar alcohol unit). As used herein, "acyclic sugar alcohol unit" refers to an acyclic sugar alcohol that is covalently linked to at least one other unit in a compound (whether a monosaccharide unit or another acyclic sugar alcohol unit). The units in the compound can be linked via ether bonds. One or more monosaccharide units can be covalently linked to other units of the compound via glycosidic bonds. Each monosaccharide unit can be covalently linked to other units of the compound via glycosidic bonds. The sugar alcohol compound can be a glycoside with a monosaccharide or oligosaccharide glycone and an acyclic sugar alcohol aglycone.

[0133] The acyclic sugar alcohol unit may be a sugar alcohol unit having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The acyclic sugar alcohol unit may be selected from the group consisting of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol and volemitol.

[0134] One or more monosaccharide units may be C5 or C6 monosaccharide units, i.e. pentose or hexose units. Each monosaccharide unit may be a C5 or C6 monosaccharide unit. One or more sugar alcohol units may be C5 or C6 sugar alcohol units. Each sugar alcohol unit may be a C5 or C6 sugar alcohol unit.

[0135] Sugar alcohol compound can comprise n monosaccharide units and m acyclic sugar alcohol units, for example, can be made up of n monosaccharide units and m acyclic sugar alcohol units, wherein n is an integer and is at least 1, m is an integer and is at least 1, and (n+m) is not more than 10.Sugar alcohol compound can comprise the chain of n monosaccharide units with an acyclic sugar alcohol unit end-blocking, for example, can be made up of it, wherein n is an integer between 1 and 9.The chain of monosaccharide units can be covalently linked by glycosidic bond.Each monosaccharide unit can be covalently linked to another monosaccharide unit or acyclic sugar alcohol unit by glycosidic bond.Sugar alcohol compound can comprise the chain of 1,2 or 3 monosaccharide units with an acyclic alcohol unit end-blocking, for example, can be made up of it.1,2,3 or each monosaccharide unit can be C5 or C6 monosaccharide unit.Acyclic alcohol unit can be C5 or C6 sugar alcohol unit. Examples of sugar alcohol compounds include, but are not limited to, isomalt, maltitol, and lactitol (n=1); maltotriitol (n=2); and maltotetraitol (n=3).

[0136] Such sugar alcohol compounds can be described as sugar alcohols derived from disaccharides or oligosaccharides. As used herein, "oligosaccharide" refers to a sugar consisting of 3 to 10 monosaccharide units. The sugar alcohol derived from disaccharides or oligosaccharides can be synthesized (e.g., by hydrogenation) by disaccharides, oligosaccharides or polysaccharides (e.g., from hydrolysis and hydrogenation), but is not limited to compounds synthesized by disaccharides, oligosaccharides or polysaccharides. For example, the sugar alcohol derived from disaccharides can be formed by the dehydration reaction of monosaccharides and sugar alcohols. The one or more organic polyols can be sugar alcohols derived from disaccharides, trisaccharides or tetrasaccharides. The example of the sugar alcohol derived from disaccharides includes but is not limited to isomalt, maltitol and lactitol. The example of the sugar alcohol derived from trisaccharides includes but is not limited to maltotriitol. The example of the sugar alcohol derived from tetrasaccharides includes but is not limited to maltotetraitol.

[0137] Organic polyol can be selected from erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fuctol, iditol, inositol, heptaheptol, isomalt, maltitol, lactitol, maltotriose alcohol, malt tetraose alcohol, polyglycitol (polyglycitol) and any combination thereof.Glycerol can be used, and when present, preferably be combined with one or more other organic polyols, for example erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fuctol, iditol, inositol, heptaheptol, isomalt, maltitol, lactitol, maltotriose alcohol, malt tetraose alcohol, polyglycitol or its any combination.

[0138] Many organic polyols contain one or more chiral centers and therefore exist in stereoisomeric forms. All stereoisomeric forms and optical isomers and isomer mixtures of organic polyols are intended to be included within the scope of the present invention. In particular, D and / or L forms of all chiral organic polyols and all mixtures thereof can be used.

[0139] Particle agglomeration

[0140] Agglomeration of the particles may be achieved by any suitable means known to those skilled in the art.

[0141] Agglomeration of the particles can be achieved by mechanical means, for example by mechanically pressing the particles together. Agglomeration by mechanical means can be achieved by micronizing the nitrite particles and the proton source particles. Alternatively, agglomeration by mechanical means can be achieved by providing particles that are substantially free of static electricity.

[0142] The agglomeration of particle can be realized by chemical means, for example the adhesion or chemical coating of chemical promotion.The agglomeration by chemical means can be realized by adhesion promoter (for example moisture).Perhaps, the agglomeration by chemical means can be realized by the coating material that the primary particle of nitrite and the primary particle of proton source are bonded together.Previously discussed suitable adhesive, suitable coating material is discussed in " coating particle " part below.

[0143] Coated particles

[0144] The one or more particles of the mixture of solid nitrite component and solid proton source component may be coated with an excipient (also referred to herein as coated particles).

[0145] The coated particles may include a single particle containing a nitrite and a proton source and coated with an excipient.

[0146] Alternatively, the coated particles may be particle agglomerates coated with an excipient, and the particle agglomerates include (a) particles comprising nitrite and a proton source and / or (b) a mixture of one or more nitrite particles comprising nitrite and one or more proton source particles comprising a proton source.

[0147] In this way, the coated particles include the nitrite and the proton source within the same coating.

[0148] Excipient can be hydrophobic. Excipient can be any material that can coat particle or agglomerate so that particle or agglomerate are coated with hydrophobic layer. This hydrophobic material can be polymeric material, for example organic polymer material, as polyol. This hydrophobic material can be amphiphilic species, for example surfactant type species, as nonionic, anionic, cationic or amphoteric surfactant type species. This hydrophobic material can be for example inorganic mineral material and the inorganic mineral material that forms 3D skeleton. This hydrophobic material can be biocompatible. This hydrophobic material can comprise poly (lactic acid-co-glycolic acid) (poly (lactic-co-glycolic acid), PLGA), phospholipid such as dipalmitoylphosphatidylcholine (dipalmitoylphosphatidylcholine, DPPC), magnesium stearate and mesoporous silica (mesoporoussilica) one or more. The hydrophobic material may comprise a polymeric material poly(lactic-co-glycolic acid) (PLGA) without acid end groups, or may comprise a polymeric material poly(lactic-co-glycolic acid) (PLGA) with acid end groups. Excipients may include polyols, magnesium stearate, colloidal silicon dioxide.

[0149] As used herein, "surfactant" refers to a surfactant that can reduce the surface tension of a species in a medium or the interfacial tension between media. Surfactant species generally have a hydrophilic head and a hydrophobic tail.

[0150] The hydrophobic material may be attached to the particles or agglomerates by chemical bonding or by electrostatic or intermolecular forces.

[0151] The coating of the coated particles or coated particle agglomerates can affect the reaction dynamics, such as reaction kinetics, of the acidification of nitrite when the coated particles or coated agglomerates are exposed to an aqueous environment.

[0152] The excipient may be a species capable of trapping or sequestering nitric oxide or a nitric oxide precursor. For example, the excipient may include a thiol, an alcohol, an amine, or an amide.

[0153] The coated particles or coated particle agglomerates of the mixture of the solid nitrite component and the solid proton source component can be a particle size suitable for the desired use or application. The coated particles or coated particle agglomerates of the mixture of the solid nitrite component and the solid proton source component can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less. Alternatively, the coated particles or coated agglomerates of the mixture of the solid nitrite component and the solid proton source component can have a particle size greater than about 5 μm. For example, the particles or agglomerates of the mixture of the solid nitrite component and the solid proton source component can have a particle size greater than about 50 μm, greater than about 100 μm, greater than about 250 μm, greater than about 500 μm, greater than about 750 μm, greater than about 1000 μm.

[0154] Particles are formed from a mixture containing a nitrite solution and a proton source solution

[0155] The mixture of a solid nitrite component and a solid proton source component may be formed by spray drying or freeze drying of a mixture containing a nitrite solution and a proton source solution.

[0156] The particle of the mixture of solid nitrite component and solid proton source component can be formed by the mixture that contains nitrite solution and proton source solution.The particle formed in this way should be by after mixing nitrite solution and proton source solution at short notice (for example 30 seconds or shorter) desolvating, and / or after mixing nitrite solution and proton source solution and in order to desolvate, mixture is placed under reaction delay condition (for example under the temperature lower than solvent freezing point) and forms.In this way, from this mixture, desolvate, minimize the acidification of nitrite simultaneously.Therefore in gained powder composition, can there be nitrite and the proton source of effective dose.

[0157] When after mixing nitrite solution and proton source solution, desolvate at short notice, can after mixing nitrite solution and proton source solution, desolvate in 30 seconds or less time.In some instances, after mixing nitrite solution and proton source solution, desolvate in 10 seconds or less time, 5 seconds or less time, 2 seconds or less time or 1 second or less time.In some instances, after mixing nitrite solution and proton source solution, desolvate in 500 milliseconds or less time, 100 milliseconds or less time, 50 milliseconds or less time or 10 milliseconds or less time.

[0158] In one example, particles can be formed by spray drying a mixture containing a nitrite solution and a proton source solution. Spray drying of the mixture can enable removal of the solvent within 30 seconds or less after mixing the nitrite solution and the proton source solution. Spray drying of materials is known per se.

[0159] In some embodiments, the mixture is a mixture of an aqueous nitrite solution and a proton source aqueous solution. When using the aqueous solution, the time between mixing these two aqueous solutions is minimized to suppress the acidification of nitrite. Before spray drying occurs, the aqueous nitrite solution and the aqueous solution of acid can be mixed online for approximately 1 to approximately 10 milliseconds, for example, approximately 3 to approximately 5 milliseconds. Spray drying can be carried out immediately after nitrite and acid solution mix. It should be understood that, as described, mixing and spray drying the mixture containing nitrite solution and proton source solution limits the potential reaction time between proton source and the nitrite component.

[0160] Particles formed by spray drying a mixture containing a nitrite solution and an acid solution can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.

[0161] Spray drying the mixture comprising the nitrite solution and the acid solution as described can produce a mixture of a solid powder nitrite component and a solid powder proton source component, wherein each particle contains both the nitrite and the proton source components.

[0162] The particles formed by spray drying a mixture containing a nitrite solution and a proton source solution can be in any suitable form. For example, the particles formed by spray drying a mixture containing a nitrite solution and a proton source solution can be in a crystalline form or an amorphous form. The particles formed by spray drying a mixture containing a nitrite solution and a proton source solution can be in an amorphous form.

[0163] Additionally or alternatively, before, during or immediately after mixing nitrite solution and proton source solution, the mixture of nitrite solution and proton source solution is placed under reaction delay conditions (e.g., at a temperature below the solvent freezing point) and used to remove the solvent. In this way, the acidification of nitrite is delayed until the solvent is removed. In particular, the solvent can be an aqueous solvent.

[0164] A particular example of reaction delay condition is that the temperature of mixture is lower than the freezing point of solvent.In this way, the acidifying reaction rate of nitrite can be slowed down when removing solvent.In the case that the temperature of this mixture is lower than the freezing point of solvent, nitrite solution and proton source solution mix at a temperature higher than the freezing point of solvent usually, and then the temperature of this mixture is reduced to the freezing point lower than solvent.In this way, the good mixing of solution can be realized.

[0165] In some examples, solvent removal can be performed under reduced gas pressure. In particular, solvent removal can be performed under reduced gas pressure and at a temperature below the freezing point of the solvent to be removed.

[0166] A particularly useful technique for removing solvents under conditions of delayed reaction time is freeze drying (also known as "lyophilization").

[0167] It should be noted that the terms "removing solvent" and / or "drying" as used herein are intended to provide a solid powder composition. These terms include, but are not limited to, complete removal of solvent. In some instances, the solid powder composition may include trace amounts of residual solvent. For example, the powder composition may contain up to about 10% residual solvent, such as up to about 5% residual solvent, up to about 3% residual solvent, or up to about 1% residual solvent. Additional drying techniques, such as vacuum drying, may be employed after the initial removal of solvent to provide a solid powder composition.

[0168] Combining solids to form particle agglomerates

[0169] The mixture of a solid nitrite component and a solid proton source component may be formed by combining a nitrite-containing solid with a proton source-containing solid to form particle agglomerates, wherein the particle agglomerates include one or more nitrite-containing particles and one or more proton source-containing particles.

[0170] Combining a nitrite-containing solid with a proton source-containing solid to form particle agglomerates can be achieved, for example, by: (a) blending one or more nitrite particles and one or more proton source particles, wherein the nitrite particles are formed by spray drying a nitrite solution and the proton source particles are formed by spray drying a proton source solution; or (b) forming one or more particles by micronizing a nitrite solid and a proton source solid together.

[0171] Blended spray-dried nitrite particles and spray-dried acid particles

[0172] The mixture of a solid nitrite component and a solid proton source component can be formed as follows:

[0173] (i) spray drying or freeze drying a solution containing nitrite,

[0174] (ii) spray drying or freeze drying a solution containing a proton source,

[0175] (iii) Blending the species of (i) and (ii).

[0176] The mixture of solid nitrite component and solid proton source component can be a blend of nitrite particles and proton source particles, wherein the nitrite particles are formed by spray drying a nitrite solution, and the proton source particles are formed by spray drying a proton source solution. The spray-dried nitrite particles and the spray-dried proton source particles can be blended to provide a blended solid composition by standard means known to those skilled in the art.

[0177] The spray-dried nitrite particles and the spray-dried proton source particles can be blended in a nitrite / proton source weight ratio ranging from about 1:1 to about 1:99, such as from about 1:4 to about 1:49 or from about 1:7 to about 1:24.

[0178] The spray-dried nitrite particles and the spray-dried proton source particles can be blended for a period of about 5 to about 60 minutes, such as a period of about 10 to about 40 minutes, or a period of about 15 to about 30 minutes. The spray-dried nitrite particles and the spray-dried proton source particles can be blended for a period of about 20 minutes.

[0179] The particles formed by spray drying the nitrite solution and the acid solution and blending these components can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.

[0180] The spray-dried nitrite solution and the spray-dried proton source solution and blending these components can produce a mixture of a solid nitrite component and a solid proton source component comprising particle agglomerates, wherein the agglomerates include one or more particles containing nitrite and one or more particles containing a proton source.

[0181] The particles formed by spray drying nitrite solution and spray drying proton source solution and blending these components can be any suitable form. For example, the particles formed by spray drying nitrite solution and spray drying proton source solution and blending these components can be crystalline form or amorphous form. The particles formed by spray drying a mixture containing nitrite solution and proton source solution can be amorphous form.

[0182] Particles formed by micronizing nitrite solid and acid solid

[0183] The particles may be formed by micronizing the nitrite solid together with the proton source solid.

[0184] As used herein, the expression "micronization" refers to a process for reducing the average particle size of a solid composition to typically within the micrometer range. Micronization can be achieved by standard methods known to those skilled in the art. For example, micronization can be performed by milling or grinding the particles or by utilizing a supercritical fluid.

[0185] In the case where the proton source is a buffered acid system, the proton source solid can be two components - a solid acid component and a solid conjugate base component. The nitrite solid and the proton source solid can be micronized in a ratio of about 1:1 to about 1:99, such as in the range of about 1:4 to about 1:49 or about 1:7 to about 1:24, for example 1:9 w / w nitrite:proton source.

[0186] Particles formed by micronizing the nitrite solid and the proton source solid together can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.

[0187] Micronization of the nitrite solution and the proton source solution together can produce a solid powder composition comprising particles containing nitrite and particles containing a proton source. Micronization of the nitrite solution and the proton source solution together can produce a solid powder composition comprising agglomerates comprising particles containing nitrite and particles containing a proton source.

[0188] The particles formed by micronizing a nitrite solution with a proton source solution can be in any suitable form. For example, the particles formed by micronizing a nitrite solution with a proton source solution can be in a crystalline form or an amorphous form. The particles formed by micronizing a nitrite solution with a proton source solution can be in a crystalline form.

[0189] The particles formed by micronization can include one or more optional additives as described above (in addition to the proton source and nitrite). In particular, the particles formed by micronization can include a binder as described above. The binder can be micronized together with the nitrite solid and the proton source solid.

[0190] When the polymeric material that generates nitric oxide comprises particle or particle agglomerate, particle or particle agglomerate can be incorporated to or be encapsulated in the polymer of the polymeric material that generates nitric oxide.In this way, particle or particle agglomerate can be remained in this material by polymer, until being exposed to moisture or aqueous environment.Particle or particle agglomerate can be exposed or partly exposed on the surface of the polymer of the polymeric material that generates nitric oxide, or can be encapsulated in the polymer of the polymeric material that generates nitric oxide fully.

[0191] The nitric oxide generating polymeric material can be a fibrous material comprising fibers of the polymer and particles or particle agglomerates incorporated or encapsulated into the fibrous material. The particles or particle agglomerates can be exposed or partially exposed on the surface of the substrate fibers, or can be completely encapsulated in the fiber network and fiber cross-section.

[0192] In some instances, the polymer material that generates nitric oxide is porous, and the particle of solid nitrite component and / or solid proton source component or particle agglomerate at least some are in the hole of this material.In other words, this material can be porous and be impregnated with the particle or particle agglomerate of solid nitrite component and / or solid proton source component.In some instances, make this material be porous by comprising hole in the surface of this material.In other instances, this material can be the porous net of material element (as polymer fiber), and particle or particle agglomerate are in the space between material element.As a particular instance, the particle of solid nitrite component and / or solid proton source component or particle agglomerate can be impregnated in the space of polymer fiber net.

[0193] The particles or particle agglomerates of the solid nitrite component and / or the solid proton source component can be of a particle size suitable for dispersion in the gelling fibers. The particles or particle agglomerates of the solid nitrite component and / or the solid proton source component can have a particle size greater than about 5 μm. For example, the particles or particle agglomerates of the solid nitrite component and / or the solid proton source component can have a particle size greater than about 50 μm, greater than about 100 μm, greater than about 250 μm, greater than about 500 μm, greater than about 750 μm, or greater than about 1000 μm.

[0194] In order to achieve a larger particle size, particles or particle agglomerates can be granulated. "Granulation" refers to a process in which particulate matter is combined to form larger particles known as granules. Granulation can be performed, for example, by compressing particles or agglomerates to provide tablets, which can then be broken into particles. Particles or agglomerates can be compressed at approximately 1 to approximately 10 MT (metric tons), for example, at approximately 3 to approximately 7 MT. Particles or agglomerates can be compressed at approximately 3.8 MT. Particles or agglomerates can be compressed at approximately 6.5 MT. A sieve, for example, a 1 mm sieve, can be used to break tablets into particles.

[0195] In order to promote compression, adhesive can be added in particle or agglomerate.Suitable adhesive can comprise sugar, natural adhesive or synthetic or semisynthetic polymer adhesive.Sugar class can comprise for example sucrose or liquid glucose.Natural adhesive can comprise for example gum arabic, tragacanth, gelatin, starch paste, pregelatinized starch, alginic acid or cellulose.Synthetic or semisynthetic polymer adhesive can comprise for example methylcellulose, ethylcellulose, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol, polymethacrylate.Adhesive can be the copolymer (copolyvidone) of 1-vinyl-2-pyrrolidone and vinyl acetate.Adhesive can be microcrystalline cellulose.

[0196] The binder may be incorporated into the composition at a % w / w of about 5% w / w to about 30% w / w. For example, the binder may be incorporated into the composition at a % w / w of about 10% w / w to about 25% w / w.

[0197] Alternatively, the composition may be substantially free of binder.

[0198] The particle size may be increased in this way to ensure that the particles or agglomerated particles remain trapped (incorporated or encapsulated) between the fibers.

[0199] A method for incorporating or encapsulating particles or agglomerates of solid nitrite and / or solid proton source components into a nitric oxide generating polymeric material comprises the steps of: (i) mixing particles or agglomerates of solid nitrite and / or solid proton source components with a non-polar liquid containing a polymer to form a liquid-particle mixture, and (ii) solidifying the liquid-particle mixture to form a material incorporating or encapsulating particles or agglomerates of solid nitrite and / or solid proton source components.

[0200] The liquid-particle mixture can be solidified by spinning the mixture into fibers. Techniques known to those skilled in the art for fiber spinning can be used. For example, the liquid-particle mixture can be solidified by dry spinning, wet spinning, gel spinning, or electrospinning. The liquid-particle mixture can be solidified by electrospinning. "Electrospinning" refers to a fiber production method in which a charged wire of a polymer solution or polymer melt is pulled to a fiber diameter using electricity. The liquid-particle mixture can be solidified by gel spinning. "Gel spinning" refers to a fiber production method that relies on temperature-induced physical gelation for solidification.

[0201] Alternatively, the particles or particle agglomerates of solid nitrite component and / or solid proton source component can be incorporated into the polymer material after the solid polymer has been formed. For example, the particles or particle agglomerates of solid nitrite component and / or solid proton source component can be impregnated into a porous polymer material, such as a fiber mesh substrate. In these examples, the solid polymer has been formed, and the particles or particle agglomerates of solid nitrite component and / or solid proton source component are added thereto. A particular example of the method in which the solid powder composition is impregnated into the porous polymer material comprises those (and other technology available from Fibroline France) described in EP2331309. The nitrite dissolved in the polymer matrix

[0202] In some embodiments, the nitrite component is dissolved in the polymer matrix of the proton source polymer. In certain embodiments, the nitrite component is substantially uniformly dissolved in the polymer matrix.

[0203] The nitrite component and the proton source polymer are as previously described herein. The method for dissolving the nitrite component in the polymer matrix can include dissolving the nitrite component in a polar solvent such as methanol and mixing with a polar solution of the polymer (such as dimethyl sulfoxide, DMSO). In a specific embodiment, the mixing provides a substantially uniform mixture of the nitrite component and the polymer.

[0204] The solvent used to dissolve the nitrite component and the polymer is generally selected so that the solution containing the nitrite source and the polymer is miscible and the solutes remain dissolved and uniformly mixed.

[0205] The solvent system can contain a kind of solvent or multiple solvents to make co-solvent solution.Suitable solvent can be a single solvent or a mixture of solvents, which is preferably polar or polar aprotic for the polymer, and polar or polar protic for the nitrite component.Water is not necessarily excluded as the solvent selected for sodium nitrite, as long as once mixed with the polymer solution, a uniform solution of nitrite source and polymer can still be obtained.In some embodiments, the solvent for the nitrite component is non-aqueous, and / or the solvent for the polymer is non-aqueous.

[0206] In certain embodiments, the solvent used may be suitable for electrospinning or thermal spinning processes and are known to those skilled in the art.

[0207] The mixture of the nitrite component and the polymer can then be formed into a nitric oxide-generating polymer material by known methods. For example, the mixture can be electrospun or thermally spun into fibers. Alternatively, the mixture can be coated onto a substrate. The mixture can be cast into a film. The solvent of the mixture can be removed at this stage.

[0208] Other characteristics of implantable medical devices

[0209] The implantable medical device may include one or more additional dry components or layers adjacent to the nitric oxide generating polymer material. The water content of any component or layer adjacent to the nitric oxide generating polymer material may be 10% or less, 5% or less, 2% or less, or 1% or less, based on the weight of the component or layer adjacent to the nitric oxide generating polymer material.

[0210] Packaged implantable medical devices

[0211] The present invention also provides a packaged implantable medical device comprising the implantable medical device described herein in a low moisture permeability package.

[0212] The low moisture permeability package can include one or more low moisture permeability materials (e.g., aluminum foil) in the wall of the package. In a specific embodiment, the low moisture permeability package includes one or more low moisture permeability materials (e.g., aluminum foil) in the wall of the package and the implantable medical device and is hermetically sealed. The low moisture permeability package can include one or more low moisture permeability materials (e.g., aluminum foil) in at least a portion of all outer walls of the package.

[0213] The packaging atmosphere within the packaged implantable medical device can have a low moisture content upon initial packaging. The packaging atmosphere can have a relative humidity of 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. Relative humidity can be measured using a hygrometer.

[0214] The packaging atmosphere may comprise an inert packaging gas such as nitrogen, argon, helium or CO 2 . The packaging atmosphere comprises 10% or less, 8% or less, 5% or less, 2% or less, 1% or less oxygen. In some embodiments, the packaging atmosphere is substantially free of oxygen.

[0215] Additionally or alternatively, the package may include one or more package inserts that seal in moisture. Such package inserts may be desiccant packets, such as silica gel packets.

[0216] Methods of implanting implantable medical devices

[0217] The present invention provides a method of implanting an implantable medical device as described herein into a subject. The implantable medical device may be a one-piece implantable medical device. In other words, the implantable medical device may be provided as a single piece until needed.

[0218] The present invention also provides particles or particle agglomerates for implanting an implantable medical device as herein described into a subject, wherein (a) one or more single particles each contain a nitrite and a proton source; or (b) agglomerates include one or more single particles containing nitrite, one or more single particles containing a proton source and optional adhesives, and / or particle agglomerates include one or more single particles each containing a nitrite and a proton source and optional adhesives. The implantable medical device can be a one-piece implantable medical device. In other words, the implantable medical device can be provided as a single piece before the moment of need.

[0219] In some embodiments, the method includes adding water (including aqueous solutions, suspensions, gels or other forms comprising water) to the polymeric material that generates nitric oxide before the implantable medical device is implanted into the subject. The addition of water can be directly added to the polymeric material that generates nitric oxide, or can be indirectly added to the polymeric material that generates nitric oxide (e.g., through one or more permeable components or layers adjacent to the polymeric material that generates nitric oxide). The water added can be a sterile aqueous solution. The aqueous environment can be a sterile saline solution.

[0220] Alternatively, the implantable medical device is implanted in a subject without adding water. In this manner, aqueous fluids from the subject (e.g., blood and / or exudate) can be absorbed by the nitric oxide-generating polymer material of the implantable medical device and activate nitric oxide production.

[0221] The subject can be a human or an animal subject. The subject can be a human or a domesticated animal.

[0222] Method for producing solid powder components

[0223] Method for producing a solid powder composition by removing a solvent

[0224] The method of making a mixture of a solid powdered nitrite component and a solid powdered proton source component may include removing solvent from the mixture of nitrite solution and proton source solution to minimize acidification prior to formation of the powder composition.

[0225] In one example, the method includes the step of removing the solvent (eg, by spray drying) in less than 30 seconds after mixing the nitrite solution and the proton source solution to form a solid.

[0226] In another example, the method includes providing reaction delay conditions (eg, freeze drying) during solvent removal and immediately before, during, and / or after mixing the nitrite solution and the proton source solution.

[0227] In one example, the method may include the step of removing the solvent from the aqueous mixture containing the nitrite solution and the proton source solution to form a solid powder.

[0228] The aqueous solution of nitrite can have a concentration in the range of about 0.1 M to about 5 M. The aqueous solution of nitrite can have a concentration of at least about 0.1 M, at least about 0.2 M, at least about 0.5 M, at least about 0.75 M, or at least about 1 M. The aqueous solution of nitrite can have a concentration of at most about 5 M, at most about 4 M, at most about 3 M, or at most about 2 M. For example, the aqueous solution of nitrite can have a concentration in the range of about 1 M to about 2 M, such as about 1.5 M. The aqueous solution of nitrite can have a pH of about 6.5 to about 9, for example, about 7 to about 8.

[0229] The aqueous solution of the proton source can have a concentration in the range of about 0.1M to about 5M. The aqueous solution of the nitrite can have a concentration of at least about 0.1M, at least about 0.2M, at least about 0.5M, at least about 0.75M, or at least about 1M. The aqueous solution of the nitrite can have a concentration of at most about 5M, at most about 4M, at most about 3M, or at most about 2M. For example, the aqueous solution of the nitrite can have a concentration in the range of about 0.5M to about 1.5M, such as about 1M. The aqueous solution of citric acid can have a pH of about 4 to 6. The pH of the aqueous solution of the proton source can be adjusted using, for example, an inorganic base such as sodium hydroxide.

[0230] In some instances, the step of removing the solvent takes 20 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, or 1 second or less after mixing the nitrite solution and the proton source solution. In some instances, the solvent is removed within 500 milliseconds or less, 100 milliseconds or less, 50 milliseconds or less, or 10 milliseconds or less after mixing the nitrite solution and the proton source solution.

[0231] spray drying

[0232] The mixture of a solid powdered nitrite component and a solid powdered proton source component can be produced by spray drying a nitrite solution and a proton source solution.

[0233] The aqueous solution of nitrite and the aqueous solution of acid can be mixed online for about 1 to about 10 milliseconds, for example about 3 to about 5 milliseconds before spray drying occurs. Spray drying can be carried out immediately after nitrite and proton source solution mix. It will be appreciated that, as described, mixing and spray drying the mixture containing nitrite solution and proton source solution greatly limits the potential reaction time between proton source and the nitrite component, and stops the reaction completely when removing moisture quickly.

[0234] Spray drying can be carried out at an outlet temperature in the range of about 60 to about 80° C., such as about 65 to about 75° C. or about 68 to about 70° C. Spray drying can be carried out at an atomization pressure of about 1 to 6 bar. Spray drying can be carried out at a liquid feed rate of about 1 to about 5 g / min, such as about 2 g / min to about 4 g / min, or about 3 g / min.

[0235] Reaction delay conditions

[0236] Alternatively, the method may include providing reaction delay conditions (eg, freeze drying) during solvent removal and immediately before, during, and / or after mixing the nitrite solution and the proton source solution.

[0237] A particular example of reaction delay condition is that the temperature of mixture is lower than the freezing point of solvent.In this way, the acidifying reaction rate of nitrite can be slowed down when removing solvent.In the case that the temperature of this mixture is lower than the freezing point of solvent, nitrite solution and proton source solution mix at a temperature higher than the freezing point of solvent usually, and then the temperature of this mixture is reduced to the freezing point lower than solvent.In this way, the good mixing of solution can be realized.

[0238] In some examples, solvent removal can be performed under reduced gas pressure. In particular, solvent removal can be performed under reduced gas pressure and at a temperature below the freezing point of the solvent to be removed.

[0239] A particularly useful technique for removing solvents under conditions of delayed reaction time is freeze drying (also known as "lyophilization").

[0240] In some embodiments, the method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent. The method of the present invention relates to a method for removing the solvent in the presence of a solvent.

[0241] It should be noted that the terms "removing solvent" and / or "drying" as used herein are intended to provide a solid powder composition. These terms include, but are not limited to, complete removal of solvent. In some instances, the solid powder composition may include trace amounts of residual solvent. For example, the powder composition may contain up to about 10% residual solvent, such as up to about 5% residual solvent, up to about 3% residual solvent, or up to about 1% residual solvent. Additional drying techniques, such as vacuum drying, may be employed after the initial removal of solvent to provide a solid powder composition.

[0242] Method of combining particles to form particle agglomerates

[0243] The formation of particle agglomerates comprising nitrite-containing particles and proton source-containing particles can be achieved in a variety of ways.

[0244] In one example, the method may include the steps of:

[0245] (i) spray drying or freeze drying a nitrite solution to form nitrite particles;

[0246] (ii) spray drying or freeze drying the proton source solution to form proton source particles; and

[0247] (iii) blending the nitrite particles and the proton source particles.

[0248] The aqueous solution of nitrite can have a concentration in the range of about 0.1 M to about 5 M. The aqueous solution of nitrite can have a concentration of at least about 0.1 M, at least about 0.2 M, at least about 0.5 M, at least about 0.75 M, or at least about 1 M. The aqueous solution of nitrite can have a concentration of at most about 5 M, at most about 4 M, at most about 3 M, or at most about 2 M. For example, the aqueous solution of nitrite can have a concentration in the range of about 1 M to about 2 M, such as about 1.5 M. The aqueous solution of nitrite can have a pH of about 6.5 to about 9, for example, about 7 to about 8.

[0249] The aqueous solution of the proton source can have a concentration in the range of about 0.1M to about 5M. The aqueous solution of the nitrite can have a concentration of at least about 0.1M, at least about 0.2M, at least about 0.5M, at least about 0.75M, or at least about 1M. The aqueous solution of the nitrite can have a concentration of at most about 5M, at most about 4M, at most about 3M, or at most about 2M. For example, the aqueous solution of the nitrite can have a concentration in the range of about 0.5M to about 1.5M, such as about 1M. The aqueous solution of citric acid can have a pH of about 4 to 6. The pH of the aqueous solution of the proton source can be adjusted using, for example, an inorganic base such as sodium hydroxide.

[0250] Spray drying can be carried out at an outlet temperature of about 60 to about 80° C., such as about 65 to about 75° C. or about 68 to about 70° C. Spray drying can be carried out at an atomization pressure of about 1 to 6 bar. Spray drying can be carried out at a liquid feed rate of about 1 to about 5 g / min, such as about 2 g / min to about 4 g / min, or about 3 g / min.

[0251] In some examples, the spray-dried particles are further dried, such as by vacuum drying.

[0252] The spray-dried or freeze-dried nitrite particles and the spray-dried or freeze-dried proton source particles may be blended by standard means known to those skilled in the art to provide a blended solid powder composition.

[0253] The spray-dried or freeze-dried nitrite particles and the spray-dried or freeze-dried proton source particles can be blended in a nitrite / proton source weight ratio in the range of about 1:1 to about 1:99, such as in the range of about 1:4 to about 1:49 or about 1:7 to about 1:24.

[0254] The spray-dried nitrite particles and the spray-dried proton source particles can be blended for a period of about 5 to about 60 minutes, such as a period of about 10 to about 40 minutes, or a period of about 15 to about 30 minutes. The spray-dried nitrite particles and the spray-dried proton source particles can be blended for a period of about 20 minutes.

[0255] Method for producing a mixture of a solid powder nitrite component and a solid powder proton source component by micronization

[0256] The method for producing a mixture of a solid powder nitrite component and a solid powder proton source component may include the step of micronizing the nitrite solid and the proton source solid to produce a solid powder composition. Micronization itself is known. Micronization can be achieved by standard methods known to those skilled in the art. For example, micronization can be performed by grinding or milling particles or by utilizing a supercritical fluid.

[0257] The nitrite solid can be micronized with the proton source solid for a time of about 5 to about 30 minutes, such as about 5 to about 20 minutes, or about 5 to about 15 minutes. The nitrite solid can be micronized with the proton source solid for a time of about 10 minutes.

[0258] The nitrite solid can be micronized together with the proton source solid at a venturi pressure of 8 bar and a grinding pressure of 2 bar.

[0259] The present inventors have discovered that micronizing a nitrite solid together with (i.e., simultaneously with) a proton source solid can produce a solid powder composition that releases nitric oxide better when exposed to an aqueous environment than a solid powder composition formed by blending a separately micronized nitrite powder and a separately micronized proton source powder.

[0260] Method for producing a solid powder composition having coated particles

[0261] A mixture of a solid powder nitrite component and a solid powder proton source component comprising particles coated in a hydrophobic material may be produced. The method may include any of the following steps:

[0262] (i) coating particles containing nitrite and a proton source with a hydrophobic material; or

[0263] (ii) combining one or more nitrite particles containing nitrite and one or more proton source particles containing a proton source, and then coating the mixture.

[0264] The hydrophobic material may be the same hydrophobic material as described above.

[0265] The particles or particle agglomerates may be coated in any suitable manner known to those skilled in the art.

[0266] Particles or particle agglomerates can be coated by dispersing the particles or agglomerates in a solution containing a hydrophobic material and drying the solution to provide particles or particle agglomerates coated with a hydrophobic material layer. In some instances, the solution comprises a non-polar solvent. In a particular instance, the solution does not contain a polar solvent (e.g., methanol). Such a polar solvent can dissolve at least a portion of the particles. In particular, the solution can be anhydrous.

[0267] The hydrophobic material may be, for example, PLGA. The particles or particle agglomerates may be dried with the hydrophobic material in a 1:1 w / w ratio. The solution in which the particles or particle agglomerates are dispersed or suspended may be a solution of DCM and the hydrophobic material.

[0268] In certain embodiments, the suspension of particles in the hydrophobic material solution is dried by spray drying. The solution containing the hydrophobic material in which the particles or particle agglomerates are dispersed can be spray dried at an outlet temperature of about 28 to 30°C. The solution containing the hydrophobic material in which the particles or agglomerates are dispersed can be spray dried at an atomization pressure of about 1 bar. The solution containing the hydrophobic material in which the particles or agglomerates are dispersed can be spray dried at a liquid feed rate of about 2 g / min.

[0269] The coated particles or coated particle agglomerates can have a particle size of less than about 10 μm, such as less than about 9 μm, eg, less than about 8 μm, less than about 7 μm, less than about 6 μm, or less than about 5 μm.

[0270] Particles or particle agglomerates can be coated with particles or particle agglomerates by blending them with a hydrophobic material to provide particles or agglomerates coated with a hydrophobic material layer. The hydrophobic material can be, for example, DPPC, magnesium stearate, mesoporous silica, or a combination thereof. The particles or agglomerates can be blended with the hydrophobic material at a ratio of 1:1 w / w. The hydrophobic material can be sieved before blending. Alternatively, the hydrophobic material can be not sieved before blending.

[0271] The particles or particle agglomerates may be blended with the hydrophobic material for a period of about 10 to about 40 minutes, or about 15 to about 30 minutes.The spray-dried nitrite particles and the spray-dried proton source particles may be blended for a period of about 20 minutes.

[0272] Water-based environment

[0273] The nitric oxide generating layer of the present invention generally releases NOx when in contact with an aqueous environment. There is no particular limitation on the aqueous environment.

[0274] The aqueous environment can be an aqueous biological fluid, such as a body fluid. Such body fluids can include wound exudate or exudate, and / or blood (such as plasma, serum).

[0275] Alternatively, the aqueous environment can be a sterile aqueous solution.The aqueous environment can be a saline solution.

[0276] In some embodiments, the solid powder composition may be sufficiently hygroscopic to absorb moisture from the air, which is sufficient to initiate the release of NOx. Example

[0277] Preparation of solid powder compositions

[0278] Materials and analytical methods

[0279] The following materials were obtained from commercial sources: sodium nitrite from Honeywell, citric acid from Sigma Aldrich, trisodium citrate from Merck, sodium hydroxide from Fisher, PLGA RG502H from Sigma Aldrich, mesoporous silica (Syloid 244FP) from Grace, dipalmitoylphosphatidylcholine (DPPC) from Avanti, Kollidon VA64 Fine from BASF, microcrystalline cellulose from JRS Pharma, and dichloromethane (DCM) from Sigma Aldrich. Deionized (DI) water (18.2 MΩ) was prepared using an ELGA water purification system.

[0280] Unless otherwise stated, the following analytical methods were used.

[0281] Particle size distribution (PSD) of dry powder measured by Sympatec

[0282] Laser particle size analysis of the spray-dried powder was performed using a Sympatec HELOS particle size analyzer equipped with an R3 lens (0.5-175.0 μm range) / R5 lens (0.5-875.0 μm range) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a reduced pressure of 60 mbar. ASPIROS glass tubes were filled with powder in a reduced humidity environment (<25% RH) and sealed with parafilm until measurement was performed. Unless otherwise stated, measurements were performed in triplicate and the average data were reported.

[0283] Example 1: Spray drying a mixture containing a nitrite solution and a proton source solution to form a solid powder composition

[0284] A feed solution of 1.5 M sodium nitrite was prepared by dissolving the desired mass of sodium nitrite in deionized water (feed solution 1). A feed solution of 1 M citric acid adjusted to pH 4 was prepared by dissolving the desired mass of citric acid in deionized water and adjusting the pH to 4 using 10 M aqueous sodium hydroxide solution (feed solution 2). The pH of the solutions was measured using a Mettler Toledo SevenCompact pH meter.

[0285] Feed solutions 1 and 2 were spray dried using a Buchi B290 spray dryer equipped with a Buchi two-fluid nozzle. The two feed solutions were pumped simultaneously using separate feed lines (platinum-cured silicone L / S14 tubing) connected using a Y-piece fitting and a single Masterflex peristaltic pump that combined the feed solutions just before atomization. A standard Buchi cyclone separator and collection tank were installed for product collection.

[0286] The feed solution was spray dried in two batches under the following conditions:

[0287] Example Outlet temperature (℃) Atomization pressure (bar) Liquid feed rate (g / min) 1A 68-70 5.5 3.06 1B 68-70 1.5 3.05

[0288] Both batches were then vacuum dried using an Edwards Super Modulyo freeze dryer set to 25°C for 24 hours.

[0289] The particle size distribution of the two batches was then measured using a Sympatec HELOS particle size analyzer equipped with an R3 lens (0.5-175.0 μm range) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a reduced pressure of 60 mbar. The measurements were performed in triplicate.

[0290] The resulting particle size distribution was measured as follows:

[0291]

[0292] VMD = Volume Mean Diameter

[0293] Example 2: Spray drying nitrite and proton source separately and then blending to produce a solid composition

[0294] Prepare a 1.5 M sodium nitrite solution by dissolving the required mass of sodium nitrite in deionized water.

[0295] A 1 M citric acid solution adjusted to pH 5.6 was prepared by dissolving the required mass of citric acid in deionized water and adjusting the pH to 5.6 using 10 M aqueous sodium hydroxide solution.The pH of the solution was measured using a Mettler Toledo Seven Compact pH meter.

[0296] These feed solutions were spray dried using a Buchi B290 spray dryer under the following conditions:

[0297]

[0298] All batches were then dried under vacuum using an Edwards Super Modulyo freeze dryer set to 25°C for 24 hours.

[0299] The particle size distribution of the three batches was then measured using a Sympatec HELOS particle size analyzer equipped with an R3 lens (0.5-175.0 μm range) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a depression of 60 mbar. The measurements were performed in triplicate.

[0300]

[0301] The spray-dried nitrite solids (Component 2A) and the spray-dried citric acid solids at pH 5.6 (Component 2C) were then blended at a ratio of 9:1 w / w citrate solids:nitrite solids using a Turbula T2F mixer at 46 rpm for 20 minutes to give the powder composition of Example 2.

[0302] Example 3: Micronizing a nitrite solid with a proton source solid to produce a solid powder composition

[0303] Sodium nitrite, citric acid and trisodium citrate were combined in the following weight proportions: 10.79%, 14.74% and 74.47%, respectively.The mixture was blended using a Turbula T2F mixer at 47 rpm for 10 minutes.

[0304] The blend was micronized using an Atritor M3 fluid energy mill at a venturi pressure of 8 bar and a grinding pressure of 2 bar. The blend was fed directly into the hopper at a target feed rate of ˜2 g / min. The resulting powder (Example 3) was collected in a single collection jar at reduced humidity (20% RH).

[0305] The particle size distribution was then measured using a Sympatec HELOS particle size analyzer equipped with an R3 lens (0.5-175.0 μm range) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a depression of 60 bar. The measurements were performed in triplicate.

[0306] The resulting particle size distribution was measured as follows:

[0307]

[0308] VMD = Volume Mean Diameter

[0309] Reference Example 4: Nitrite and proton source are micronized separately and then blended to produce a solid composition

[0310] Sodium nitrite is micronized using an Atritor M3 fluid energy mill at a venturi pressure of 8 bar and a grinding pressure of 2 bar. Sodium nitrite is directly fed into the hopper at a target feed rate of ~2 g / min. The powder (component 4A) produced is collected in a single collection jar at a reduced humidity (20% RH).

[0311] Citric acid and trisodium citrate were combined in the following weight proportions: 16.51% and 83.49%, respectively.The mixture was blended using a Turbula T2F mixer at 47 rpm for 10 minutes.

[0312] The blend was micronized using an Atritor M3 fluid energy mill at a venturi pressure of 8 bar and a grinding pressure of 2 bar. The blend was fed directly into the hopper at a target feed rate of ˜2 g / min. The resulting powder (component 4B) was collected in a single collection jar at reduced humidity (20% RH).

[0313] The micronized nitrite solid (component 4A) and the micronized citric acid solid (component 4B) were then blended at a ratio of 9:1 w / w citrate solids:nitrite solids using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Reference Example 4.

[0314] NOx precipitation

[0315] Examples 1A, 2, 3, and 4 were loaded into an APTAR Unidose nasal sprayer (https: / / www.aptar.com / products / pharmaceutical / uds / ), which was supported in a rig 30 cm above a petri dish (9.8 cm diameter) containing agarose with Hanks balanced salt solution and a pH indicator (phenol red). Figure 1 Shows the powder deposition pattern due to local pH adjustments where the particles land.

[0316] After applying, plate is transferred to a sealed chamber immediately, and nitrogen oxides (NOx) are measured through 15 minutes by Selected Ion Flow Tube Mass Spectrometry (SIFT-MS). Regardless of its preparation method, all powders separate out nitrogen monoxide. However, between four kinds of powders, the difference of the total amount of NOx separated out is observed in the process of 15 minutes.

[0317] It should be noted that agarose is buffered at a neutral to slightly alkaline pH, which should inhibit the reaction, but the particles are able to overcome this buffering effect in a short time and counteract the buffering in local areas. Figure 2 Cumulative NO generation is shown for Examples 1A, 2, 3, and 4. Cumulative NO / nmols per mg nitrite. Experimental results were normalized to the % nitrite in the powder.

[0318] Example Accumulated NO / nmols <![CDATA[Cumulative NO / nmols.mg nitrite -1 > Example 1A 2410 526 Example 3 1571 322 Example 4 562 120 Example 2 2393 552

[0319] Coated solid powder composition

[0320] Example 5: Particles coated with hydrophobic material DPPC or mesoporous silica

[0321] Example 1B was blended with mesoporous silica at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to give the powder composition of Example 5A.

[0322] Example 1B was blended with DPPC at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to give the powder composition of Example 5B.

[0323] Example 3 was blended with mesoporous silica at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 5C.

[0324] Example 3 was blended with DPPC at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to give the powder composition of Example 5D.

[0325] Example 6: Particles coated with PLGA

[0326] A PLGARG 502H solution was prepared by dissolving 1.5 grams of PLGA in approximately 30 milliliters of DCM to form a clear and colorless solution. 1.5 grams of Example 1B was added to this solution with stirring to form a 1:1 w / w ratio feed suspension 6A as a visually homogeneous white suspension.

[0327] Similarly, a separate PLGA RG 502H solution was prepared by dissolving 1.5 grams of PLGA in approximately 30 milliliters of DCM to form a clear and colorless solution. 1.5 grams of Example 3 was added to this solution with stirring to form a 1:1 w / w ratio feed solution 6B as a visually homogeneous white suspension.

[0328] These feed suspensions were spray dried using a Buchi B290 spray dryer according to the method detailed above. The spray drying parameters are summarized below.

[0329]

[0330] In the environment (28%RH) of humidity reduction, the sample bottle is placed horizontally in a single weighing dish. Remove the lid and cover the opening with a foil with a hole (pierced with a needle). The sample is transferred to an Edwards Super Modulyo freeze dryer set to 25°C and vacuum dried for 24 hours (the maximum vacuum pressure observed is ~ 0.1 mbar). After vacuum drying, the sample is transferred to a low humidity (~ 24%RH) environment and covered with nitrogen. The bottle is then sealed with parafilm (Parafilm) and sealed in a foil bag with a desiccant to store at 2-8°C.

[0331] The particle size distribution was then measured using a Sympatec HELOS particle size analyzer equipped with an R3 lens (0.5-175.0 μm range) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a depression of 60 bar. The measurements were performed in triplicate.

[0332] The resulting particle size distribution was measured as follows:

[0333]

[0334] VMD = Volume Mean Diameter

[0335] Example 7: NOx precipitation of coated particles

[0336] The aliquots (30 mg) of the powder sample are deposited in 60 mm petri dishes. Cellulose filter paper (50 mm diameter) is placed on the sample and slight pressure is applied. Sodium phosphate solution (10 mM, 250 μ l) is dispensed onto the cellulose filter paper. The sample is immediately placed in a 650 ml chamber, which is sealed, and moist air is then drawn through the chamber at 650 ml / min for 30 minutes. The air stream from the discharging is analyzed by Single Ion Flow Tube Mass Spectrometry (SIFT-MS).

[0337]

[0338] Preparation of electrospun fibers containing nitrite / acid particles

[0339] Particles containing nitrite and acid formed by spray drying as described above (according to Example 1A) and having a particle size <10 μm were used to form electrospun fibers incorporating the particles.

[0340] Electrospun fibers are prepared using methods known to those skilled in the art by dispersing powder particles containing a nitrite source and a proton source in a solution of a polymer polycaprolactone ("PCL") or thermoplastic polyurethane ("TPU") and electrospinning the resulting mixture to form electrospun fibers containing powder particles comprising a nitrite source and a proton source.

[0341] Example 8: PCL-based biodegradable fibers

[0342] The following examples show PCL-based biodegradable fibers with and without particles comprising a nitrite source and a proton source.

[0343]

[0344] *Reference fiber

[0345] Using microscopy techniques such as scanning electron microscopy it can be observed that the particles are dispersed within the fibers. Figure 1 is a control sample without powder particles. Figures 2 to 4 Shown are electrospun fibers having particles containing a nitrite source and a proton source on the surface of the fiber and within the fiber body.

[0346] Example 9: TPU-based non-biodegradable fibers

[0347] The following examples show non-biodegradable fibers based on TPU:

[0348]

[0349] *refer to

[0350] Using microscopy techniques such as scanning electron microscopy it can be observed that the particles are dispersed within the fibers. Figure 5 is a control sample without powder particles. Figures 6 to 8 Shown are electrospun fibers having particles containing a nitrite source and a proton source on the surface of the fiber and within the fiber body.

[0351] Example 10: Nitric Oxide Generation of the Fiber of Example 8

[0352] The generation of nitric oxide and its precursor was assessed using an established fluorometric method. In brief, the NO probe (NO-sensor) diaminofluorescein-FM (DAF-FM) is known to be converted into a fluorescent triazole in the presence of nitric oxide (and oxygen) and its precursor. When excited with a wavelength of approximately 490 nm, the resulting triazole (DAF-T) emits 520 nm of light.

[0353] Where applicable, the cast liner was removed from the sample prior to testing. Electrospun fiber discs containing powders comprising a nitrite source and a proton source (Examples 8B-D) and a control without powder (Example 8A) were placed individually in 5 mL polyethylene tubes with screw caps, and the weight was recorded (the discs needed to be folded into quarters to fit in the container).

[0354]

[0355] *refer to

[0356] An aliquot of DAF-FM (1 mM in DMSO) was diluted with deionized water to produce a 1 μM DAF-FM aqueous solution ("DAF solution"). Example 10A was loaded with DAF solution (1 μM, 5 mL). Examples 10B-D were loaded with DAF-FM solution (1 μM, 4 mL). The volume was set to ensure that the electrospun disc was completely immersed in the solution. A blank sample containing only DAF-FM solution was also prepared as a control.

[0357] An initial fluorescence reading was taken of the DAF-FM solution from each sample as follows: After mixing, an aliquot of the DAF solution in each experiment was transferred individually to a clean cuvette (approximately 3 mL volume) and the fluorescence intensity was measured. The pH of the solution was also measured before returning the solution to the same respective sample vial. Samples were incubated at 30°C between time points.

[0358] Repeat fluorescence and pH measurements at 0, 4, 24 and 96 hours (the control showed an increased fluorescence signal over a longer time frame). At each time point, the vial was removed from the incubator and stirred to ensure that the DAF solution was thoroughly mixed. After recording any visual observations (such as the physical state of the electrospun disc), the volume required for the test (approximately 3 mL) was removed from the container and transferred to the same cuvette used to obtain the 0 hour sample. After recording the fluorescence measurement, the pH was also measured. Using a pipette, the sample for testing was withdrawn from the cuvette and returned to the original sample container with the same disc, which was then resealed with the same lid and returned to the incubator. The same method was used for all samples and controls.

[0359] The fluorescence intensity of the blank sample at each time point was subtracted from the fluorescence intensity of the test sample. The corrected intensity is shown in Figure 9 The sample containing 10% powder had the greatest fluorescence intensity, followed by the sample containing 5% powder.

[0360] Biological evaluation of solid powder compositions

[0361] Example 11: Evaluation of the efficacy of four formulations against Pseudomonas aeruginosa

[0362] Petri dishes containing nutrient agar (NA, available from AcuMedia) were prepared and allowed to solidify. An inoculum of Pseudomonas aeruginosa (ATCC 9027) was prepared in phosphate buffered saline (PBS, Sigma-Aldrich) and serially diluted to 1×10 5 CFU mL -1 100 mL of the inoculum was pipetted onto a NA plate, spread, and allowed to dry at room temperature for 15 minutes. The lid was removed from the inoculated agar plate and the open plate was placed in an Aptar Unidose nasal sprayer.

[0363] An Aptar delivery device containing the powder of Example 1A, Example 3, Reference Example 4, or Example 2 was attached to an Aptar nasal spray device and the powder was aerosolized (approximately 50 mg dose) onto an agar plate. The following table shows examples for each formulation.

[0364] Formulation 1 Example 1A Formulation 2 Example 3 Formulation 3 Reference Example 4 Formulation 4 Example 2

[0365] After 5 seconds, the agar plate cover was replaced and the agar plate was incubated at 37°C ± 2°C for 16 hours. After incubation, the plate was photographed. For all plates, three biopsy punches were taken from a 2x2 cm area in the center of the agar plate. Sterile swabs moistened with PBS were used to remove bacteria from each biopsy, and any cells were suspended in 10 mL of PBS, then ultrasonically treated for 5 minutes, serially diluted, and plated onto NA.

[0366] A negative control plate that was not exposed to the aerosolized powder and a positive control plate to which 1 mL of bleach had been added were also tested simultaneously. All tests were performed in quintuplicate.

[0367] For each test item, three replicates were randomly selected and DNA was extracted from 400 μL of each biopsy using the DN easy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions. The samples were eluted in AE buffer in a final volume of 100 μL.

[0368] For each extraction, qPCR was performed in triplicate using the QuantiNova Pathogen and IC kit (Qiagen) according to the manufacturer's instructions. Each reaction tube contained a final concentration of 16 μM of each primer and 5 μM of the labeled probe.

[0369] The cycling conditions were as follows: 50°C for 10 min, 95°C for 2 min, 35 cycles of 95°C for 5 sec, 55°C for 30 sec, and 72°C for 1 min. Each assay run was validated by positive (Pseudomonas aeruginosa) and negative (RNase-free water) controls. Data were analyzed using Q-Rex software (Qiagen) to obtain Cq values from predetermined thresholds. For each sample, the average Cq value was compared with a 1×10 2 Up to 1x10 8 CFU mL -1 The determined range of the standard curve was compared to calculate the Log 10 CFUmL -1 The final sample concentration was calculated.

[0370] Table 1: The effect of treatment with formulations 1, 2, 3, 4 and bleach on the growth of 1 x 10 5 CFU mL -1 Average recovery and reduction of P. aeruginosa from three biopsy punches obtained from the center of nutrient agar

[0371]

[0372] SD = standard deviation, CFU = colony forming unit, N / A = not applicable, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

[0373] 7.44 ± 0.17 Log was observed in biopsies taken from negative control plates. 10 CFU mL -1 The average recovery of P. aeruginosa was observed from biopsies taken from formulations 2 and 3. 3.52 ± 3.12 and 1.36 ± 2.13 Log 10 CFU mL -1 No viable P. aeruginosa was recovered from biopsies taken from Formulations 1 and 4 or the positive control plates.

[0374] Table 2: The results of the experiments on the samples from the culture medium inoculated with 1 x 10 5 CFU mL -1 Molecular Quantification of Pseudomonas aeruginosa in Nutrient Agar Biopsy Punches

[0375]

[0376] SD = standard deviation, CFU = colony forming unit. # = Quantitation below the detection limit. ～ = Quantification of positive control samples was performed up to N = 1, therefore standard deviation could not be calculated. N / A = not applicable, ** = p < 0.01, *** = p < 0.001.

[0377] Compared to the untreated negative control, after treatment with Formulation 1 and Formulation 4 powders, 5 CFU mL -1 A significant decrease in the recovery of viable P. aeruginosa was observed in biopsies of nutrient agar plates of the inoculum, as no viable P. aeruginosa was recovered. Molecular quantification reflected recovery from colony counts.

[0378] Example 12: Cell Binding and Proliferation

[0379] method

[0380] Four PLGA nonwoven fiber scaffolds containing 0, 1, 5, and 10% (wt / wt) NO-generating powder (described above) were prepared by electrospinning. After preliminary testing to confirm low bioburden and optimal cell loading density, 8 mm diameter discs were aseptically cut from the as-received electrospun fiber sheets and placed in separate sterile plates. The VERO suspension was diluted to 5 × 10 4A density of 10 cells / disc was applied to each disc. As a positive control, the same number of cells was seeded into a cell culture plate of the same size. As a negative control, we incubated the discs in culture medium alone (no cells) and in culture medium alone on cell culture plastic. At 24 hours and 7 days, the discs were harvested, rinsed in sterile PBS to remove non-adherent cells, and then:

[0381] 1. Apply cell lysis buffer and quantify cell content by total double-stranded DNA (dsDNA)

[0382] 2. Preparation for Cell Morphology Analysis by Electron Microscopy

[0383]

[0384] result

[0385] Morphological evaluation of cells bound to scaffolds at day 7

[0386] Figure 12 showed that cells bound to the PLGA matrix alone maintained a rounded shape with minimal extension along the fibers ( Figure 12 A). In contrast, cell cultures were observed to spread along the fibers and bridge between them ( Figure 12 BD). Similar spreading was observed at all doses of NO generating powder. Incorporating nitric NO releasing powder into the PLGA nonwoven scaffold did not adversely affect cell binding or proliferation and improved cell spreading and bridging between fibers within the scaffold.

[0387] Example 13: Effect of powder composition on sprouting of human umbilical vein endothelial cells (HUVEC) in a spheroid-based cell angiogenesis assay

[0388] 10x concentrated stock solutions / suspensions of Examples 1B and 6A were prepared in basal medium (without supplements and FCS) by vortexing and pipetting. Subsequently, semi-log dilution series were prepared in the same medium.

[0389] # Example 1B [mg / mL] Example 6A [mg / mL] 1 5.000 10.000 2 1.500 3.000 3 0.500 1.000 4 0.150 0.300 5 0.050 0.100 6 0.015 0.030 7 0.005 0.010

[0390] endothelial cells

[0391] Cells: HUVEC, primary human umbilical vein endothelial cells (PromoCell, Heidelberg, Germany), passage 3 to 4.

[0392] Morphology: adherent, cobblestone-like growth in a single layer

[0393] Culture medium: Endothelial cell growth and basal medium (ECGM / ECBM, PromoCell)

[0394] Subculture: Split 1:3; every 3-5 days, at approximately 1x 10 4 cells / cm 2 Inoculation and cultivation: 37°C, 5% CO2

[0395] Doubling time: 24-48 hours

[0396] Storage: Use 70% culture medium, 20% FCS, 10% DMSO to store approximately 1x 10 6 Cells / ampoule frozen

[0397] Source: Human umbilical vein, pooled donor

[0398] Test Method

[0399] Experiments were performed in a modification of the originally published protocol (Korff and Augustin: J Cell Sci 112:3249-58, 1999). Briefly, spheroids were prepared as described (Korff and Augustin: J Cell Biol 143:1341-52, 1998) by pipetting 400 HUVEC in a hanging drop onto a plastic dish to allow the spheroids to aggregate overnight. 50 HUVEC spheroids were then seeded in 0.9 ml of collagen gel and pipetted into each well of a 24-well plate to allow polymerization. After 30 minutes, pre-incubated test samples were added by pipetting 100 μl of a 10-fold concentrated working solution onto the polymerized gel (see Table 1 for final assay concentration). The plates were incubated at 37° C. for 24 hours and fixed by adding 4% PFA (Roth, Karlsruhe, Germany).

[0400] Quantitative

[0401] The germination intensity of HUVEC spheroids treated with the test samples was quantitatively measured by an image analysis system that measures the cumulative germination length (CSL) of each spheroid. Photos of individual spheroids were taken using an inverted microscope and digital imaging software NIS-Elements BR 3.0 (Nikon). Subsequently, the spheroid photos were uploaded to the homepage of Wimasis for image analysis. The cumulative germination length of each spheroid was measured using the imaging analysis tool WimSprout. The average value of the cumulative germination length of 10 randomly selected spheroids was analyzed as a single data point. The mean and SD values of each triplicate experiment were converted to % of the basic control.

[0402] result

[0403] Figure 13 The CSL of Examples 1B and 6A relative to a basal control is shown. The effect of Example 1B (spray-dried particles without coating) was less than that of the basal control. In contrast, the PLGA-coated particles of Example 6A exhibited a significant dose-dependent effect compared to the basal control. This suggests that despite being in a largely neutral environment, the coated particles provide a local environment capable of acidifying nitrite.

[0404] Example 14: Nitrite dissolved in polymeric material

[0405] Preparation of PLGA fibers

[0406] Poly(lactic-co-glycolic acid) (PLGA) of varying lactic acid:glycolic acid ratios was dissolved in dimethyl sulfoxide (DMSO). Sodium nitrite was dissolved in methanol. After the sodium nitrite / methanol solution was added to the DMSO-PLGA solution, a slight precipitation was observed, which readily redissolved under stirring to form a clear, homogeneous solution. The solid content of the PLGA polymer in the DMSO was varied in the initial DMSO-PLGA solution to ensure that the final solution comprising sodium nitrite and methanol was sufficiently viscous to achieve stable electrospinning. The sodium nitrite concentration in the resulting electrospun fibers was at most the limit that allowed stable electrospinning, which could be, but was not necessarily limited to, 0.15% in the final fibers.

[0407] The following examples show PLGA-based biodegradable fibers with various concentrations of sodium nitrite.

[0408]

[0409] *Reference sample

[0410] Figure 14A -C shows scanning electron microscopy images of samples A to C (Examples 14A-C).

[0411] Nitric oxide release

[0412] The generation of nitric oxide and its precursor was assessed using established fluorometric methods. In brief, the NO probe (NO-sensor) diaminofluorescein-FM (DAF-FM) is known to be converted into a fluorescent triazole in the presence of nitric oxide (and oxygen) and its precursor. When excited with a wavelength of approximately 490nm, the resulting triazole (DAF-T) emits 520nm of light.

[0413] Where applicable, the cast liner was removed from the sample prior to testing. Electrospun fiber discs containing sodium nitrite (Examples 14B-D) and blanks without sodium nitrite (Example 14A) were placed individually in 5 mL polyethylene tubes with screw caps, and the weights were recorded (the discs needed to be folded into quarters to fit in the containers).

[0414]

[0415]

[0416] An aliquot of DAF-FM (1 mM in DMSO) was diluted with deionized water to make a 1 μM DAF-FM aqueous solution ("DAF solution"). All samples were loaded with DAF solution (1 μM, 5 mL). The volume was set to ensure that the electrospun disc was completely immersed in the solution. A blank sample with only DAF solution was also prepared as a control.

[0417] An initial fluorescence reading was taken on the DAF solution from each sample as follows: After mixing, an aliquot of the DAF solution in each experiment was transferred individually to a clean cuvette (approximately 3 mL volume) and the fluorescence intensity was measured. The pH of the solution was also measured before returning the solution to the same respective sample vial. Samples were incubated at 30°C between time points.

[0418] Repeat fluorescence and pH measurements at 0, 24, 48, and 120 hours. At each time point, the vial was removed from the incubator and stirred to ensure that the DAF solution was fully mixed. After recording any visual observations (such as the physical state of the electrospun disc), the volume required for the test (approximately 3 mL) was removed from the container and transferred to the same cuvette used to obtain the 0 hour sample. After recording the fluorescence measurement, pH was also measured. Using a pipette, the sample for testing was withdrawn from the cuvette and returned to the original sample container with the same disc, which was then resealed with the same lid and returned to the incubator. The same method was used for all samples and controls.

[0419] The pH of the sample is displayed on Figure 15A As PLGA fibers hydrolyze, the pH decreases:

[0420] The fluorescence intensity from diaminofluorescein-type compounds is known to decrease with increasing acidity [Angew. Chem. Int. Ed. 1999, 38, No. 21]. Using the data given in the literature [Angew. Chem. Int. Ed. 1999, 38, No. 21] and the pH of the sample, a proportional adjustment was applied to the intensity results to account for the loss of fluorescence intensity as the pH decreased in the experiment, thereby normalizing all pH results and thus enabling them to be directly compared with each other.

[0421] In addition, the fluorescence intensity of the control sample without any PLGA material was subtracted from the fluorescence intensity of the test sample at each time point. The corrected intensity distribution is shown in Figure 15B middle.

[0422] The results showed that, especially in the sample containing 0.15% sodium nitrite, there was a fluorescent signal, thus the material produced nitric oxide and its precursors.

Claims

1. An implantable medical device comprising a polymeric material that generates nitric oxide, wherein (i) the nitric oxide generating polymeric material comprises a polymer and: (a) particles, wherein one or more individual particles each contain a nitrite and a proton source; or (b) particle agglomerates, wherein the agglomerates comprise one or more individual particles containing a nitrite, one or more individual particles containing a proton source, and optionally a binder, and / or the particle agglomerates comprise one or more individual particles each containing a nitrite and a proton source, and optionally a binder; (ii) the nitric oxide generating polymeric material comprises a proton-producing polymer, and the nitric oxide generating polymeric material comprises a nitrite dissolved in the proton-producing polymer matrix; or (iii) A combination of (i) and (ii) above.

2. The implantable medical device of claim 1 , wherein the nitric oxide generating polymer material has a water content of 10% or less, 5% or less, 2% or less, 1% or less prior to implantation, or the nitric oxide generating polymer material is substantially free of water.

3. An implantable medical device according to claim 1 or claim 2, wherein the nitric oxide generating polymer material forms a scaffold of the implantable medical device, the nitric oxide generating polymer material forms a coating on another component of the implantable medical device, or the nitric oxide generating polymer material forms part of a textile of the implantable medical device.

4. The implantable medical device according to any one of claims 1 to 3, wherein the nitric oxide generating polymer material is a fiber or a coating.

5. The implantable medical device according to any one of claims 1 to 4, wherein one or more of the single particles or particle agglomerates are blended with or coated with an excipient for influencing the rate of water ingress into the particles and / or an excipient for influencing the kinetics of nitric oxide formation by the particles.

6. The implantable medical device according to claim 5, wherein the excipient for influencing the rate of water ingress into particles is a polyol or a hydrophobic material, such as a phospholipid, magnesium stearate or colloidal silicon dioxide, and / or the excipient for influencing the rate of water ingress into particles is nitric oxide or a nitric oxide precursor chelating material, such as a thiol, an alcohol, an amine or an amide.

7. An implantable medical device according to any one of claims 5 or 6, wherein the particles containing both nitrite and a proton source are formed by spray drying a mixture containing a nitrite solution and a proton source solution.

8. The implantable medical device according to any one of claims 1 to 7, wherein the proton-generating polymer is an acidic polymer, a photoacidic polymer or an acid precursor polymer, such as a hydrolyzable ester.

9. An implantable medical device according to any one of claims 5 to 8, wherein one or more of the particles or particle agglomerates are embedded or partially embedded in a polymer of the nitric oxide generating polymer material, or One or more of the particles or particle agglomerates are attached to the surface of a polymer of the nitric oxide-generating polymeric material.

10. The implantable medical device of claim 1, wherein in option (ii) the nitrite is substantially uniformly mixed with the proton-producing polymer matrix.

11. The implantable medical device of claim 10, wherein the nitric oxide generating polymer material is formed from a non-aqueous solution of a nitrite and a proton-generating polymer.

12. The implantable medical device according to any one of claims 1 to 11, wherein the polymer of the nitric oxide generating polymer material is a biocompatible polymer, optionally wherein the polymer of the nitric oxide generating polymer material is resorbable.

13. The implantable medical device of any one of claims 1 to 12, wherein the implantable medical device comprises one or more additional dry components adjacent to the nitric oxide generating polymer material.

14. The implantable medical device of any one of claims 1 to 13, further comprising one or more additional components adjacent to the nitric oxide generating polymer material, provided that the water content of any component adjacent to the nitric oxide generating polymer material is 10% or less, 5% or less, 2% or less, or 1% or less, based on the weight of the component adjacent to the nitric oxide generating polymer material.

15. The implantable medical device of any one of claims 1 to 14, wherein the implantable medical device comprises an antimicrobial agent.

16. The implantable medical device according to any one of claims 1 to 15, wherein the implantable medical device is a one-piece medical device.

17. The implantable medical device of any one of claims 1 to 16, wherein the nitric oxide generating polymer material is on an outer surface of the implantable medical device.

18. A packaged implantable medical device comprising the implantable medical device according to any one of claims 1 to 17 in a low moisture permeability package.

19. The packaged implantable medical device according to claim 18, wherein the low moisture permeability package (i) including one or more low moisture permeability materials (e.g., aluminum foil) in the walls of the package; (ii) is hermetically sealed; or (iii) Includes a package insert to trap moisture.

20. Particles or particle agglomerates for implanting an implantable medical device according to any one of claims 1 to 19 into a subject, wherein (a) one or more individual particles each contain a nitrite and a proton source; or (b) the agglomerate comprises one or more individual particles containing a nitrite, one or more individual particles containing a proton source and optionally a binder, and / or the particle agglomerate comprises one or more individual particles each containing a nitrite and a proton source and optionally a binder, optionally wherein the implantable medical device is a one-piece implantable medical device.

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