WO2008055719A2

WO2008055719A2 - Medical device capable of releasing no

Info

Publication number: WO2008055719A2
Application number: PCT/EP2007/057781
Authority: WO
Inventors: Kent Høier NIELSEN; Frederik Enemark Poulsen; Christian Jensen; Lars Niklas Larsson; Steen Guldager Petersen; Finn Munk Ulrich
Original assignee: Arsenal Medical, Inc.
Priority date: 2006-11-08
Filing date: 2007-07-27
Publication date: 2008-05-15
Also published as: EP2094326A2; JP2010508925A; JP2010508924A; EP2107914A2; WO2008055718A2; WO2008055718A3; WO2008055719A3

Abstract

The invention provides a medical device for intravascular use, comprising a base material which is at least in part, coated with a coating system comprising a polymer mixture, wherein said polymer mixture comprises at least one reactive nitrogen species (RNS) adduct, wherein said adduct is capable of releasing said reactive nitrogen species under physiological conditions; and a hydrophilic polymer or hydrophilic polymer blend, wherein the reactive nitrogen species adduct and the hydrophilic polymer or polymer blend form a homogeneous phase. In a preferred embodiement, the medical device is coated with a linear poly (ethyleneimine) diazeniumdiolate (L-PEI-NONO) embedded in polyure thane (PU).

Description

COATING SYSTEM

FIELD OF THE INVENTION

The present invention relates to a coating system for controlled release of reactive nitrogen species (RNS), such as nitric oxide and/or nitroxyl, from intravascular medical devices.

BACKGROUND OF THE INVENTION

Reactive nitrogen species, such as nitric oxide, are regarded as potent mediators for many biological functions, such as acting as a vasodialator, a neurotransmitter, an inflammatory mediator, an inhibitor of platelet activation, a modulator of endothelial and leukocyte adhesion, and a modulator of macrophages and neutrophils.

However, nitric oxide and other reactive nitrogen species are highly unstable in physiological conditions, and are rapidly inactivated by oxyhemoglobin within red blood cells.

Adducts for reactive nitrogen species, such as nitroglycerin have been widely used systemically, for example in the treatment of angina. However, systemic application results in an extensive dilution of the adduct as well as potentially harmful side effects at sites remote from the site of therapeutic action.

There has therefore been a concerted effort to develop local administration technologies, which allow the direct application of nitric oxide to the required site in the body, particularly within the vascular system, where RNS can prevent or reduce thrombosis or vasospasm during vascular surgery, or prevent restenosis occurring after coronary angioplasty and stent implantation.

EP 0 752 866 discloses that polymers to which are bound nitric oxide releasing N₂O₂ ^" functional group may be used for the treatment of restenosis and related disorders. It was found that nitric oxide treated polyethylenimine was effective in triggering vaso-relaxation.

WO2005037339, which refers to expandable balloon for use in angioplasty procedures, discloses that expandable stents, used in the treatment of restenosis, may be coated with a pharmaceutical agent, such as nitric oxide (NO) and that such nitric oxide releasing matrixes may also relax or prevent arterial spasm once the medical device is in place. WO2005039664 refers to a medical device, such as a guide wire, an embolization device, or a guide shaft for a microcatheter, which comprises an outer surface layer formed by electrospun nanofibers of polymeric linear poly(ethylenimine) diazeniumdiolate. It is also disclosed that nitric oxide releasing matrixes may relax or prevent arterial spasm once the medical device is in place.

Numerous reactive nitrogen species (RNS) adducts have been developed which are capable of storing RNS in a stable form prior to use, whilst releasing the nitric oxide upon exposure to physiological media, such as blood. Such adducts have been used to coat intravascular medical devices.

Further technologies have been developed to control the release of RNS from RNS adducts in an attempt to regulate the release of RNS to provide an optimised therapeutic dosage over a defined time period :

WO 95/24908, which discloses NONOate polymers (including linear polyethylenimine diazeniumdiolate), which are capable of locally releasing nitric oxide to a site at risk of restenosis, refers to a problem in the use of polymeric NONOates in that whilst the N₂O₂ ^" groups near the surface should be available for rapid release, those deeply embedded are sterically shielded, and require more time and energy to release the stored nitric oxide.

US 6,885,366 utilises electrospinning of linear polyethylenimine diazeniumdiolate to coat medical devices in nanofibres to enable the effective surface area exposed to increased, therefore providing a high initial release rate of nitric oxide.

WOO 1/56646 refers to a coating system for a medical device for insertion into the human body where the coating system comprises an inner first layer of a biocompatible compound which provides sustained release of a biologically active agent, such as nitric oxide, and a second outer coating of the of the same biologically active agent, thereby providing an outer layer which can provide a rapid release of nitric oxide, and an inner layer which provides a longer term release of nitric oxide.

US 2003/0045865 refers to a system where upon administration of a medical article to a patient, an activator compound interacts with a nitric oxide adduct to result in the release of nitric oxide.

US 5,994,444 refers to the use of an acid-labile nitric oxide precursor (a polymer comprising inorganic nitrite), and a reducing agent which reduces the pH of the polymer, facilitating the release of nitric oxide upon use. However, none of the controlled release technologies available allow for a controlled release of a therapeutically effective amount of nitric oxide to prevent the side effects associated with the act of insertion of medical devices into the body - i.e. for the duration of the surgical procedure involved. This is because, with the exception of nanofibers technology, none of the controlled release technologies address the key issue relating to the penetration of physiological fluids into a layer of nitric oxide adduct - they merely facilitate the nitric oxide release once the physiological fluids have penetrated into the adduct layer.

Nanofibre technology allows for a very rapid penetration of physiological fluids into a polymer layer. However, the release rate is a factor of the thickness of the nanofibres, and may therefore result in a very high initial release of nitric oxide, but with insufficient release during the latter stages of a medical intervention procedure.

OBJECT OF THE INVENTION

The current invention address the problems relating to penetration of physiological fluids into the nitric oxide adduct coatings on medical devices by providing a polymer coating system, where the adduct is presented in the form of a polymer structure which comprises both a structural polymer, and a hydrophilic polymer, thereby allowing the controlled access of physiological fluids directly to the adduct, depending upon the proportion of the structural polymer, the hydrophilic polymer and the adduct present in the polymer coating system. Other reactive nitrogen species, such as nitroxyl (HNO) are also of relevance in terms of therapeutic performance, and as such the invention relates to coating systems comprising RNS adducts in general, as well as specifically nitric oxide and HNO.

SUMMARY OF THE INVENTION

The invention provides for a medical device for intravascular use, comprising a base material which is at least in part, coated with a coating system comprising a polymer mixture, wherein said polymer mixture comprises a) at least one reactive nitrogen species (RNS) adduct, wherein said adduct is capable of releasing said reactive nitrogen species under physiological conditions; and b) a hydrophilic polymer or hydrophilic polymer blend; wherein the reactive nitrogen species adduct and the hydrophilic polymer or polymer blend form a homogeneous phase.

The invention provides for a method for the manufacture of a coated medical device suitable for intravascular use, said method comprising: a) selecting a medical device suitable for use in vascular surgery, said medical device comprising a base material; b) applying a coating system comprising a polymer mixture to said external base layer, wherein said polymer mixture comprises at least one reactive nitrogen species adduct, and a hydrophilic polymer or polymer blend, wherein the reactive nitrogen adduct and the supporting polymer or polymer blend form a homogenous phase.

The invention provides a medical device for intravascular use, comprising a base material which is at least in part, coated with a coating system comprising a polymer mixture, wherein said polymer mixture comprises i) at least one RNS adduct which is capable of releasing RNS under physiological conditions and ii) a hydrophilic supporting polymer or polymer blend, and wherein the RNS adduct and the supporting polymer or polymer blend form a homogeneous phase.

The invention further provides for a method for the manufacture of a coated medical device suitable for intravascular use, said method comprising: (a) selecting a medical device suitable for use in vascular surgery, said medical device comprising a base material, (b) applying a coating system comprising a polymer mixture to said external base layer, wherein said polymer mixture comprises at least one RNS adduct capable of releasing RNS under physiological conditions, and a hydrophilic supporting polymer or polymer blend, and wherein the RNS adduct and the supporting polymer or polymer blend form a heterogeneous phase.

The invention also provides for the use of the coating system according to the invention for the manufacture of a medical device for use intravascular use, for the prevention of one or more the conditions selected from : vasospasm or vasoconstriction, prevention of cerebral vasospasm, relaxation of smooth muscle, vasodilatation, thrombosis, decreased platelet deposition or aggregation, alleviation of restenosis, increased blood pressure, oxygen free radical reperfusion injury, treatment of cardiovascular disease, preventing the adverse effects associated with the use of said medical device, preventing abnormal cell proliferation.

The invention further provides for the use of the coating system according to the invention for the application to a medical device in order to control the rate of release of the reactive nitrogen species, such as nitric oxide, from the surface of the medical device. In one embodiment, the coating system may also control the release of reactive nitrogen species, such as nitric oxide, from said inner layer into the physiological medium, and/or the diffusion of water from the physiological medium into the inner layer under physiological conditions and/or the leakage of small molecule by products from said polymer mixture into the physiological medium.

STATEMENT OF INVENTION

Related cases:

PCT/DK2006/000714, which is hereby incorporated by reference, provides a method and system for measurement of nitric oxide release from nitric oxide adducts. The method allows for the level of nitrite to be taken into account or even eliminated in order to obtain a precise measurement of NO that has hitherto not been achievable.

The embodiments of the present invention may be used in conjunction with the inventions disclosed in the following applications, which are hereby incorporated by reference: EP application No. EP06023203 and US provisional application 60/864,879, EP application No. 06023223 and US provisional 60/864,886, EP application No. 06023222 and US provisional application 60/864,893, EP application No. EP06075159 and US provisional application 60/761,359, and PCT/DK2007/000030.

The Medical Device

In one embodiment, the medical device is an intravascular medical device or a neurovascular medical device.

In one embodiment the medical device is a transient medical device which does not remain within the patient after the surgical procedure has been completed, such as an introducer sheath or assembly and components thereof, a catheter such as a coronary guiding catheter or a neuro catheter, a guide wire, a syringe needle/trocar, an angioplasty balloon, a coronary wire.

The medical device may be a peripheral medical devices such as peripheral wires, guiding catheter, peripheral balloon; stent delivery system, peripheral wires, peripheral guiding catheter, stent delivery system; introducer sheath, dialator, guide wire, syringe needle.

The medical device may be selected from the group consisting of: Neuro medical devices, such as neuro guiding catheter, neuro microcatheter, neuro microwire, neurostent delivery system, neuron balloon; coronary medical devices, such as coronary wires, coronary guiding catheter, PTCA angioplasty balloon; stent delivery system, coronary wires, coronary guiding catheter, PTA angioplasty balloon; introducer sheath, dilator, guide wire, syringe needle /trocar, introducer sheath assembly.

The medical device may for instance include an intermittent or permanent intravascular implant, such as a stent, a stent graft, a balloon, a catheter, a guiding catheter, a guidewire, an embolization device, such as wire or a coil.

In case of a balloon, the device may e.g. include an expandable coated angioplasty balloon, such as a PTA (percutaneous translumenal angioplasty) balloon, a PTCA (percutaneous translumenal coronar angioplasty) balloon or a PTNA (percutaneous translumenal neurovascular angioplasty) balloon.

In one embodiment the medical device is an implant such as a stent.

In one embodiment the medical device is a prosthetic, such as a breast implant or a penis implant.

In one embodiment, the medical device is not made from polytetrafluoroethylene (PTFE) (Teflon™) or Fluorinated polyethylene (FEP). There are two benefits in selecting an alternative substrate for manufacture of the base material of the medical device, firstly, it allows for the routine use of radiation based sterilisation techniques, and secondly it facilitates the coating of the medical device.

Therefore, preferably the medical device, such as the introducer sheath (tubing) is manufactured from a material which is capable of being sterilised using radiation based techniques, such as gamma radiation or e-beam. Nylon is a suitable based material. Other suitable materials may be selected from the group consisting of, for example, polyurethanes, aromatic polyesters, polycarbonates, polyethylenes, polystyrenes, and polysulfones.

The medical device may be made from a base material which is high density polyethylene.

Introducer Sheath

The RNS (such as nitric oxide) eluting medical device according to the present invention may be an introducer sheath, or introducer sheath assembly (kit of parts). The kit of parts comprises at least two key components: the introducer sheath including a dilator and the

RNS eluting coating applied on at least the distal portion of the sheath. The primary mode of action of this device is identical to that of the traditional introducer sheath: for use for the intravascular introduction of interventional and/or diagnostic devices. The ancillary action of the RNS elution is to prevent vessel spasm and occlusion during the introduction of interventional and/or diagnostic devices through the introducer sheath. It is envisaged that the use of the introducer sheath of the present invention will also reduce the likelihood of blood clot formation, thrombosis and related disorders.

A preferred device is an introducer sheath coated with a RNS eluting coating applied on at least the distal 10 cm of the sheath.

In one embodiment the proximal lcm of the introducer sheath is not coated.

The device is indented for radial intravascular introduction of interventional/diagnostic devices and the primary mode of action of this device is identical to that of the traditional introducer sheath.

The ancillary action of the medical device is RNS elution to enhance the clinical safety during the procedure, such as decreasing the risk of vasospasm or thrombosis.

The term an 'introducer sheath' as used herein is a device which is used to introduce medical devices into the human body, such as the vascular or neurovascular system which comprises a hollow tube, typically made of a flexible material through which other medical devices are introduced into the vascular or neurovascular system. Introducer sheaths are often coated with a lubricating surface or made from a lubricating material, such as Teflon™. The proximal end of the introducer sheath is exterior to the body, whilst the majority of the length of the introducer sheath is within the lumen of a vessel of the vascular system. The introducer sheath typically has a length of about 10 to about 30 cm in length and a diameter of 5 or 6 french. French are units which correspond to the internal diameter of the introducer sheath, and the external diameter of subsequent medical devices inserted into introducer sheath, such as catheters. 1 french is l/3^rd of a millimetre. Although wall thicknesses of the introducer sheath may vary, it is preferable to have as thin a wall as possible, whilst retaining the structural robustness of the introducer sheath.

Typically, the insertion of the introducer sheath involves a first act of making an incision into the human body using a hollow needle (a trocar) into an arterial wall (venepuncture). Subsequently a soft tipped guide wire is passed through the needle and the needle removed. An introducer sheath, comprising the dilator within is then passed over the guide wire. The dilator is removed and the medical device, typically comprising a catheter is passed over wire and wire is removed. Preferably, the external surface of the proximal end of the introducer sheath is not coated with said RNS adduct. This is because this is the region which comes into more that momentary contact with the site of injury into the vessel (insertion site), where a local vasospasm is may be desirable, both in ensuring a tight connection with the introducer sheath, thereby preventing leakage of the physiological media from the insertion site into the surrounding tissues and external to the body, and also facilitates quicker healing after removal of the introducer sheath as the blood clots at the insertion site.

Therefore, preferably, the region of the external surface of the proximal end of the introducer sheath not coated is sufficient to reduce bleeding from the entry point into the vascular or neurovascular system, as compared to an equivalent proximally coated sheath.

Typically the introducer sheath has a length of between 10 and 30cm.

Typically, the introducer sheath has an inner diameter of between 4 and 12 French. Other sizes may be appropriate depending on the size of the patient (and their arteries), and the size of the medical device to be inserted via the introducer sheath. For example, the diameter may be between 4 and 7 French, such as between 5 - 6 French, may for example be used for radial arteries. 4 French introducer sheaths may be suitable for use in small children. 8 or 9 French may be used for larger equipment to be entered via the femoral artery, and even up to 12 to 14 French for devices such as aortic stents. However, typically 5 to 6 French is sufficient for normal cardiac procedures.

In a preferred embodiment the introducer sheath is a transradial introducer sheath. The transradial introducer sheath is particularly useful in avoiding the vasospasm associated with entry via the radial arteries.

Kit of Parts

Introducer sheaths are typically manufactured as kits of parts, which in addition to the introducer sheath comprises a dilator.

The term 'dilator' as used herein, is a device which is inserted into the introducer sheath, which ensures the structural robustness of the introducer sheath upon insertion into the vascular system, and are removed prior to insertion of the medical device to be inserted to the introducer sheath. The dilator has an outer diameter allowing a close fit with the introducer sheath, whilst not impeding its removal. The dilator also has an inner diameter, through which the guide wire passes upon insertion into the patient. Dilators are also made from a flexible material, and when fully inserted into the introducer sheath they typically extends beyond the distal tip of the introducer sheath, and typically comprises a tapered end which facilitates insertion of the introducer sheath/dilator into the vessel. The dilator also may comprise a distal region of radio opaque material which, using X ray images, is used to locate the end of the introducer sheath assembly to ensure correct insertion.

The term 'guide wire for an introducer sheath' is specific term describing the guide wire which is used for guiding the insertion of the introducer sheath into the blood vessel. Preferably, the guide wire ranges from about 0.018 to about 0.038" (about 0.4572 to about 0.9652mm) in diameter and about 35 to about 80cm in length. Unlike guide wires for catheters, the guide wire for an introducer sheath does not reach the site of intervention, indeed, in one embodiment it is not necessary for the guide wire to extend beyond the distal tip of the introducer sheath or dilator.

The term ^λAn introducer sheath assembly' refers to a kit of parts which comprises an introducer sheath and at least one other medical device which is used during the insertion of the introducer sheath into the vascular system, for example by the Seldinger technique. The Introducer sheath assembly may also comprise, in its proximal end, which is not inserted into the body, a valve to facilitate the introduction of further medical devices, such as catheters. The introducer sheath may also comprise a valve to allow insertion of fluid administration such as therapeutic agents. A suture ring on the valve housing may provide secure anchoring of the sheath.

An introducer sheath assembly suitable for coating with the compound capable of releasing nitric oxide under physiological conditions is disclosed in US 5,409,463. Suitable introducer sheaths for coating using the method of the invention are available from Thomas Medical Products Inc., Malvern, Pennsylvania, and may be prepared by e.g. spray coating, such as coating with the coating system and/or coating with nanofibres, such as by electrospinning (US 6,382,526, US 6,520,425).

In one embodiment, the kit of parts comprises a dilator according to the invention wherein said dilator is capable of being inserted into an introducer sheath, such as the introducer sheath according to the invention, or an introducer sheath that is not coated with an nitric oxide adduct. The dilator may also, preferably, be capable of being inserted over an introducer sheath guide wire, such as the introducer sheath guide wire according to the invention.

Preferably, when the dilator is fully inserted into said introducer sheath, it extends beyond the distal tip of said introducer sheath, and the region of the dilator which extends beyond the distal tip of said introducer sheath comprises a tapered end which is coated with said compound capable or releasing the RNS (such as nitric oxide) under physiological conditions.

In one embodiment, the region of said dilator which does not extend beyond the distal end of the introducer sheath when fully inserted is not coated with said compound capable or releasing the RNS, such as nitric oxide under physiological conditions.

The kit of parts may also comprise an introducer sheath guide wire which is optionally coated with an RNS adduct which is capable of releasing the RNS (such as nitric oxide) under physiological conditions, such as the RNS adducts and/or coating systems as referred to herein.

The trocar needle used for making the initial insertion may also be coated with a RNS adduct (such as a nitric oxide) adduct. The trocar is hollow to allow passage of the guide wire through into the lumen of the vessel. Whilst it is considered beneficial that the distal portion of the trocar is coated with the RNS adduct such as a nitric oxide adduct and/or coating systems as referred to herein, in one embodiment, the proximal end of the trocar, i.e. the portion which after insertion into the patient, is in contact with the wound site, is not coated with the RNS adduct such as a nitric oxide adduct or coating system as referred to herein.

Method for the Manufacture

It is envisaged that medical device according to the invention may be made from or comprise a base material which comprises the RNS adduct.

However, in a preferred embodiment, the medical device is pre-manufactured, and the subsequently coated with the RNS adduct.

In order to ensure a specific target release resulting in the desired clinical impact (optimal target release is in the range from about 0.5 nmol/min/cm² to about 40 nmol/min/cm²), the RNS donor (adduct) is incorporated into a polymeric matrix.

In a preferred embodiment, the coating comprises of 3 layers: (ι) an optional primer layer, which ensures and optimizes coating adherence to the device. (ιι) The RNS ( e.g. nitric oxide) donating layer (first domain), which may for example be a mixture of polyurethanes or other polymers referred to herein (see under coating system) and L-PEI-NONO (LPN)(whιch, may for example be suitably dissolved in an alkali organic solvent, such as pyridine or pH adjusted methanol (prepared for example by addition of a suitable alkali, such as NaOH) for spray application onto the medical device). The recipe for the nitric oxide (e.g. LPN) donating layer may include a pH adjusted solvent (e.g. an alkali organic solvent such as an alcohol such as methanol, or pyridine) to ensure that the nitric oxide (e.g. LPN) is stable during processing. The alkali agent is preferably retained in the first domain to ensure that the pH of the first domain remains above 7(Ni) an optional topcoat (Outer layer/coat), which serves to ensure coating integrity, appropriate rate of water absorption and appropriate rate of RNS (e.g. NO)diffusion (and prevents systemic release of LPEI-NONO).

The term 'homogeneous' as used herein refers to a single phasic system which is uniform in structure and/or composition throughout.

Coats (i) and (iii) may be optional, but are preferred. Typically the method for preparing a coated medical device according to the invention consists of a first optional step of applying the priming layer (i), an second step of applying the nitric oxide adduct layer (ii), and a third optional step of applying the top coat (iii). Further coatings, as referred to herein, may be applied either before during or subsequent to the application of the RNS adduct layer.

When a topcoat is applied the release of the RNS, such as NO, typically deviates significantly from a l'order release. This is due to the barrier properties of the topcoat: limiting the water absorption and the diffusion of RNS, such as NO, through the coating leading to a more constant release over time.

Prior to application of the RNS, such as NO, adduct coating, a priming layer, as herein described, may be applied. The priming layer may be applied by any suitable means, such as dipping, spaying, or extrusion.

During manufacture of the device, the layers may e.g. be formed by dip-coating, spraying, painting, printing, vapor deposition, extrusion or a combination thereof. In one embodiment the layers are not formed by electro spinning techniques. Spray coating has been found to be a highly convenient way of applying the domains and layers to medical devices.

The inner layer(s) may be textured, e.g. by sanding prior to application of the outer layer, so as to obtain a textured or roughened outer surface of the inner layer providing improved bonding of the outer layer.

The RNS adduct is applied as a coating system.

The coating system comprises a polymer mixture which is applied to said external surface of the medical device (or the primer or subsequent layer such as a further layer, such as the second or third domains), wherein said polymer mixture comprises at said at least one RNS adduct, (such as the nitric oxide adduct) and a hydrophilic polymer or polymer blend, and wherein the RNS adduct, and the polymer or polymer blend form a homogeneous phase.

In one embodiment the hydrophilic polymer or polymer blend is a hydrophilic supporting polymer or polymer blend.

It is thought that the hydrophilic polymer or polymer blend typically provides support within the polymer matrix allowing transport of water and ions to the RNS adduct and transport of the RNS to the external (physiological) environment. The hydrophilic polymer or polymer blend may therefore be referred to as a hydrophilic supporting polymer or polymer blend herein.

Further layers may then be applied over the coating system layer. For example a layer of a material which is capable of affecting the pH of the coating layer system upon insertion into physiological media may be applied (see European application No. 06075159 and PCT/DK2007/000030, which are hereby incorporated by reference). The pH modifying layer may include an pH modifying agent, which in a broad embodiment may include any atom, molecule or ion, including H⁺ and OH^", capable of affecting pH by shifting the local balance between H⁺ and OH^" ions. A change in pH may arise due to direct increase or decrease of H⁺ or OH^" ions by means of ingress of acid or base, or due to ingress of molecules or ions that trigger chemical reactions that confer a change of pH. The NO release from NO adducts is typically sensitive to pH, with release of NO being favoured in acidic conditions, whereas HNO release is favoured in alkaline conditions. Therefore the use of a further layer which is capable of shifting the local balance between H⁺ and OH^" ions upon wetting (e.g. in physiological media) allows the release rate of the RNS from the RNS adduct to be controlled. The further layer of material may comprise, for example an acid, such as an acid selected from ascorbic acid, polyacrylic acid, oxylic acid, acetic acid and lactic acid. The acid may be incorporated into a polyurethane polymer for application.

In a preferred embodiment, an outer coating as herein described is also applied. The outer coating may be applied directly to the coating system layer, or to one or more further layers applied to the coating system layer.

If premature release of the therapeutic agent is to be avoided, the donor-compound containing coating is preferably applied under conditions that are unfavourable for release, e.g. conditions of low temperature, low pressure, lower water content or low humidity. In particular, premature release of the therapeutic agent is achieved by deposition of the donor- compound, i.e. therapeutic agent, under pH conditions which inhibit release of the therapeutic agent - suitably under alkaline conditions of pH greater than 7. When HNO is the RNS acidic conditions (less than pH 7 may be preferred. Neutral pH may be useful for RNS adducts where both HNO and NO are desirable RNSs.

Thus, the method of manufacture according to the fifth aspect of the invention preferably takes place at a relative humidity of at most 40%, such as at most 30%, such as at most 25%, such as at most 20%, such as at most 15% or 10%. Likewise, in the product of the second aspect of the invention, a relative humidity of at most 40% may be maintained in the package, such as at most 30%, such as at most 25%, such as at most 20%, such as at most 15% or 10%. Similarly, to prevent premature release of the therapeutic agent, the medical device may be manufactured and stored at a pH inhibiting such release, e.g. at a pH of at least 7, such as at least 8, 9, 10, 11, 12, 13 or 14, or, in the alternative, at a pH of at most 6, such as at most 5, 4, 3, 2 or 1. Combinations of humidity and pH and optionally other parameters may be applied to further inhibit premature release of the therapeutic agent.

The medical device is then packaged, and sterilised.

It is highly advantageous to include a water absorber into the inside of the packaging material to ensure low water environment within the packaging - this not only extends the shelf life of the sealed products, but also protects the top coat during sterilisation. Suitably, levels of gaseous water as low as 0.01% within the packaging can be obtained (www.mgc- a.com).

As RNS (such as NO) release from RNS adducts is sensitive to moisture, it is preferable that the packaging is performed in a confined environment where the relative humidity does not exceed about 40%, such as does not exceed about 30%, such as does not exceed about 20% or about 10%. In one embodiment the relative humidity is less than about 1%, or substantially free of water (i.e. less than 0.1% water). Preferably, the medical device or kit of parts is packaged in a sealed pouch, such as an aluminium sealed pouch with N₂ atmosphere. In one embodiment the O₂(g) is less than about 0.1%, or substantially free of O₂(g) (i.e. less than 0.1% O₂). The packaging should preferably prevent moisture, oxygen and light from entering the package.

In one embodiment, the sterilisation is performed using radiation sterilisation, such as e- beam of gamma radiation. In one embodiment, the method of manufacture involves coating the entire external surface of the medical device. However, as described herein, in one embodiment it may be advantageous to coat only part of the medical device.

When the term "about" is used herein, it is considered that the specific value provides is also explicitly disclosed, e.g. the range from about 0.01 to about 1, explicitly discloses the range from 0.01 to 1.

Reactive nitrogen species (RNS)

The term reactive nitrogen species refers to a free radical which comprises a nitrogen atom. Typically the reactive nitrogen species has a molecular weight of less that 250, such as less than 100, such as less than 50, such as less than 40, such as less than 35. Preferably the reactive nitrogen species is gas at 20°C under normal atmospheric pressure. Particularly preferred reactive nitrogen species are nitric oxide (NO), and nitroxy (HNO).

In one embodiment the RNS is nitric oxide (NO). The RNS adduct may therefore be an NO adduct.

The RNS may also be referred to as the therapeutic agent herein.

In one embodiment the RNS is (HNO). The RNS adduct may therefore be an HNO adduct. HNO is also considered as a highly effective vasodiolator, and as such is a suitable RNS for use in intravascular medical devices.

HNO can be indirectly detected N₂O by using the propensity of HNO to undergo dimerization according to the following reaction:

HNO + HNO →[HONNOH]→N₂O + H₂O

N₂O can then be detected by eg. gas chromatography.

In one embodiment the RNS comprises both nitric oxide (NO) and (HNO) - the RNS adduct may therefore be capable of releasing both HNO and NO under physiological conditions.

The RNS is applied to the medical device in the form of an RNS adduct. For the purposes of assessing the release kinetics of in physiological fluid/media, we have used phosphate buffered saline at pH 7.4 at a temperature of 37°C.

Reactive nitrogen species adducts (RNS adducts)

The RNS is typically administered indirect as a substance which gives off the RNS as a result of a chemical reaction when wetted or exposed to enzymes or other chemicals, generally these are called RNSs adducts .

The RNS adducts may be monomers of polymers, and may be selected from compounds which, for example, comprise nitrosyl, nitrite, nitrate, nitroso, nitrosothio, nitro, metal-NO complex, nitrosamine, nitrosimine, diazetine dioxide, furoxan, benzofuroxan or NONOate (- N₂O₂ ^") groups.

In one embodiment, the RNS adducts may be selected from the group consisting of nitroglycerin, sodium nitroprusside, S-nitroso-proteins, S-nitrosothiols, long carbon-chain lipophilic S-nitrosothiols, nitrosocarbonyls, S-nitroso-dithiols, iron-nitrosyl compounds, thionitrates, thionitrites, sydnonimines, furoxans, organic nitrates, and nitrosated amino acids, nitroso-acetylcysteine, S-nitroso-captopril, S-nitroso-homocysteine, S-nitroso-cysteine, S-nitroso-glutathione, and S-nitrosopenicillamine, S-nitrosothiols, S-nitrosylated polysaccharides such as S-nitrosylated cyclodexrins, hydroxylamines, cyanamide, acyloxy nitroso compounds N-hydroxysulfenamides, diazeniumdiolates (NONOates) prepared from primary and -or secondary amines NONOate polymers.

A preferred group of RNS adducts are the diazeniumdiolates. Diazeniumdiolates may comprise primary amine groups, which typically result in the release of HNO in physiological media, and secondary amine groups, which typically result in the release of NO in physiological media. Polyalkylenimine diazeniumdiolates comprise both primary and secondary amine groups, and as such as suitable adducts for both HNO and NO. Other HNO/NO donors are disclosed in US 2005/0009789 and US 2004/0038947, which are herby incorporated by reference. It is recognised that by adjusting the pH the balance between NO and HNO release from the RNS adduct may be controlled, with higher pH typically resulting in an increase in HNO release, whereas lower pH results in a high release of NO.

When referring to RNS adduct herein, it should be understood that the term refers to NO adducts, HNO adducts, and HNO/NO adducts. The compound capable of releasing the RNS under physiological conditions (RNS adduct) preferably comprises a RNS-nucleophile complexes (such as NO-nucleophile complexes). The RNS-adduct may be monomer or a polymer.

RNS adducts which are monomeric molecules may be soluble or insoluble in physiological media. Suitable monomeric RNS adducts are, for example, disclosed in US 4,954,526.

Numerous polymers which are capable of releasing the RNS in physiologic media are known in the art. For example, the polymers disclosed in US 5,405,919 and US 6,875,840 may be used.

It is preferable that the RNS adduct, such as the NO-adduct is in the form of a polymer, such as a linear polymer, a branched polymer, and/or a cross linked polymer, to which is bound a RNS releasing functional group, such as a RNS-nucleophile complexes.

In one embodiment, the RNS adduct, such as the nitric oxide adduct is selected from the group consisting of: nitroglycerin, sodium nitroprusside, S-nitroso-proteins, S-nitrosothiols, long carbon-chain lipophilic S-nitrosothiols, nitrosocarbonyls, iron-nitrosyl compounds, thionitrates, thionitrites, sydnonimines, furoxans, organic nitrates, and nitrosated amino acids, nitroso-acetylcysteine, S-nitroso-captopril, S-nitroso-homocysteine, S-nitroso-cysteine, S-nitroso-glutathione, and S-nitrosopenicillamine, S-nitrosylated polysaccharides such as S- nitrosylated cyclodexrins, hydroxylamines, cyanamide, acyloxy nitroso compounds N- hydroxysulfenamides, diazeniumdiolates (NONOates) prepared from primary and -or secondary amines.

It is recognised that some RNS adducts/NO adducts are water inducible, also refered to as proton inducible, i.e. they accept protons from ionic water, which results in the release of the RNS, such as nitric oxide, (e.g. NONOates). It is preferable that such water inducible RNS adducts are used.

However, it is also envisaged that other RNS adducts, such as NO adducts, may also be employed. For example enzymatic release of NO may also be utilised by incorporation of suitable enzymes into the nitric oxide adduct layer, the enzymes then become activated when the come into contact with water, causing the release of nitric oxide.

In one embodiment the RNS adduct is a NONOate, such as a polymeric NONOate selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoride, polyvinylchloride, derivatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes, biopolymers such as peptides, proteins, oligonucleotides, antibodies and nucleic acids, starburst dendrimers.

Polyimines represent a diverse group of polymer which may have diazeniumdiolate moieties covalently bound thereto. Polyimines include poly(alkylenimines) such as poly(ethylenimines). For example, the polymer may be a linear poly(ethylenimine) diazeniumdiolate (L-PEI-NONO) as disclosed in US 6,737,447 which is hereby incorporated by reference. The loading of the nitric oxide donor onto the linear poly(ethylenimine) (PEI) can be varied so that 5-80%, e.g. 10-50%, such as 33%, of the amine groups of the PEI carry a diazeniumdiolate moiety. Depending on the applied conditions, the (L)-PEI-NONO can liberate various fractions of the total amount of releasable RNS, such as nitric oxide.

Polyimines with diazeniumdiolate moieties (in particular linear poly(ethylenimine) diazeniumdiolate) may e.g. be used as polymers for an electrospinning process because such polymers typically have a suitable hydrophilicity and because the load of diazeniumdiolate moieties (and thereby the load of latent RNS, such as NO molecules) can be varied over a broad range, cf. the above example for PEI-NONO.

In one embodiment the RNS adduct, such as the NO adduct is a poly(alkylenimines) diazeniumdiolate, such as PEI-NONO. PEI-NONO also exists as a branched polymer, which may also be used, or a linear polymer LPEI-NONO (linear), the linear form is particularly preferred.

A most preferred RNS adduct polymer is polyethylenimine diazeniumdiolate, such as linear polyethylenimine diazeniumdiolate (LPEI-NONO). US 6,855,366 provides methods for the preparation to LPEI-NONO. The loading of the RNS donor, such as nitric oxide onto the linear poly(ethylenimine) (PEI) can be varied so that 5-80%, e.g. 10-50%, such as about 20 to about 40%, such as about 30% of the amine groups of the PEI carry a diazeniumdiolate moiety. Depending on the applied conditions, the L-PEI-NONO can liberate various fractions of the total amount of releasable RNS, such as nitric oxide.

It is preferable that after synthesis, during storage, manufacture of the medical devices and/or storage of the medical device the RNS adduct (e.g. NO adduct), such as (L)PEI-NONO is kept in an alkaline environment during the formulation and processing of the (L)PEI-NONO and the application of such (alkali) (L)PEI-NONO formulations to medical devices.

In one embodiment, the invention provides for alkali stabilised formulations of polyethylenimine diazeniumdiolate, such as linear polyethylenimine diazeniumdiolate, and medical devices which comprise, such as are coated with such alkali stabilised formulations, such as medical devices which comprise at least said first domain as described herein.

We have discovered that despite the use of non-aqueous solvents such as methanol, as a solvent for polyethylenimine diazeniumdiolate, the release of NO from polyethylenimine diazeniumdiolate can occur prematurely, such as during the formulation, storage and processing of the polyethylenimine diazeniumdiolate, resulting in sub-optimal nitric oxide donation capacity. In the case that formulation and processing of the nitric oxide adduct occurs in the presence of oxygen (e.g. during spray coating of medical devices), formation of nitrite is an unavoidable effect of spontaneous nitric oxide release.

The MW of the RNS adduct polymer, such as NO adduct polymer, will, in one embodiment, such as when the RNS adduct is LPEI-NONO, depend upon the size of the starting materials used to prepare the RNS adduct, and in some cases the degree of branching of the polymer. Typically, larger polymers sizes are preferable in terms of reducing in vivo solubility, however, large polymers may also interfere with the ability to solubilise and coat the medical device. In the case of LPEI-NONO, the use of branched precursors results in cleavage of the molecular structure at branch points, reducing the average molecular weight of the polymer molecules present in the final NO polymer adduct Hence MW is used to define whether the LPEI-NONO is linear:

In a preferred embodiment, the RNS adduct, such as the LPEI-NONO polymer has an average molecular weight of between about 5kDa and about 20OkDa, such as between about 1OkDa and about 10OkDa, such as more preferably between about 3OkDa and about 7OkDa, such as about 55kDa with a preferred polydispersity between about 1 and 3 and more preferably about 2.

The average molecular weight is determined by size exclusion chromatography (SEC) of the polymer backbone, linear polyethyleneimine (L-PEI) using various polyethylene glycols (PEG) with known molecular weights as standards. The total amount of NO in the NO loaded polymer (L-PEI-NONO) can be determined my elemental analysis (EA). Hence, the average molecular weight of the RNS, such as NO, loaded polymer (L-PEI-NONO) can be calculated based on the results from the size exclusion chromatography (SEC) and the elemental analysis (EA).

In a preferred embodiment, the RNS, such as NO, adduct, which may form a coating (i.e. an NO eluting coating), is based on linear polyethyleneimine (L-PEI) with pendant NONO groups. Each NONO group is covalently attached to one of the N atoms in the L-PEI polymer chain thereby forming an N* - N⁺(O^")=N-O^" group in which N* is one of the atoms of the L-PEI backbone. This group is stabilized by the formation of a Zwitter-ion complex with an adjacent -NH₂ ⁺- amine group in the L-PEI backbone.

The RNS, such as NO, loaded linear polyethyleneimine (L-PEI-NONO) releases the RNS, such as NO, when it is exposed to a proton donating (H⁺) environment like water or blood. As described herein the RNS, such as NO, released by PEI-NONO may be NO and/or HNO.

To stabilize the coating the device is stored in a sealed aluminum pouch with N₂ atmosphere. The product is thereby protected against moisture, light and oxygen during storage.

Incorporating the L-PEI-NONO into a polymer matrix, allows controlled release of the RNS, such as nitric oxide. When introduced into the vascular system the RNS, such as nitric oxide is delivered to the vessel wall by diffusion.

The RNS dose delivered to the vessel wall (i.e. the target location) is determined by the release rate and the time in contact with the artery wall. The dose delivered to the target, i.e. the smooth muscle cell (SMC), depends furthermore on the thickness of the vessel wall, potential plaque, presence of blood/hemoglobin, the presence of oxygen and the diffusion constant (D).

It has been recognised that polymeric NONOates, such as those which contain diazeniumdiolate groups can generate undesirable small molecule side products such as nitrosamines (see US 6,875,840). The present invention overcomes the potential generation of undesirable small molecule side products, whilst allowing controlled release of the RNS, such as nitric oxide, under physiological conditions by the incorporation of polymeric NONOates, such as polyethyleminine diazeniumdiolate, in a hydrophilic (support) polymer or polymer blend.

Through careful analysis of NO release from Imear-polyethylenimine diazeniumdiolate, we have detected NO release from ionic solutions which have been exposed to medical devices, after removal of the medical device. This has led us to the discovery that contrary to previous teachings, Imear-polyethylenimine diazeniumdiolate is soluble in ionic aqueous solution, and as such there is a measurable systemic release of LPEI-NONO into the body upon use of medical devices coated with LPEI-NONO (see Figure 12).

The solubility of LPEI-NONO in physiological fluids presents a previously unrecognized problem relating to its use to coat medical devices, such as intravascular medical devices in that the release of LPEI-NONO from the medical device into the blood stream will result in the systemic release of LPEI-NONO and undesirable release of nitric oxide at sites remote to the site of therapy.

It is also considered that the release of LPEI-NONO into the blood stream is undesirable as it results in the release of a foreign compound into the body.

We have overcome this problem of systemic release by utilisation of an outer-coat layer external to the LPEI-NONO layer which effectively reduces the release of LPEI-NONO to an insignificant concentration upon insertion into body fluid, such as blood. In addition, by affecting the fluxes of water and RNS the outer-coat can have a beneficial controling effect upon the release kinetics of the RNS, such as nitric oxide from polyethylenimine diazeniumdiolate coated medical devices.

For example, in one aspect, upon insertion of the medical device into the body there is a controllable initial period, where the RNS, such as NO, release is relatively low. This prevents premature release of the RNS, which can cause issues of excessive bleeding from the entry wound due to the anti-thrombosis effect of the RNS. The properties of the outer-layer, and in some respects the first, second and third domains, as referred to herein, particularly the nature of the hydrophilic support polymer/polymer blend used in said domains, are important in determining (i.e. controlling) the nature of this initial period.

The RNS release mechanism

Diazeniumdiolates, as an example of an RNS adduct, can act both as NO and/or as HNO donors depending on whether the are attached to a primary (HNO donor) or an secondary (NO donor) amine. The NO -release from NONO'ates is pH-dependant and follows first order kinetics. Hence the only requirements for NO release are an aqueous environment and sufficient amounts of protons (Dutton et al, 2004). The overall reaction with H⁺ results in 2 NO and a secondary amine group on the Nitric Oxide donor. Significantly less knowledge is accumulated on HNO chemistry than NO chemistry. However, HNO release is known to occur from e.g. diazeniumdiolates linked to a primary amine by a pH dependent release mechanism expected to occur as illustrated below, (e.g. Miranda et al., 2005)

R JO R

.N-N. + H N-H + 2 NO*

R ^'N-O R ,N-N / O

H₂O ^■*^■ ,N=N HNO R N-O R

However, the simplicity of the release system also puts high demands on how to store and handle the material. Any exposure to normal atmospheric moisture can initiate the release of the RNS (such as NO). Typically the NO will react with O₂ (in aqueous and humid conditions) and form NO₂ ^" (nitrite) (Kharitonov et al., 1994), which will contribute to a decrease in pH and further release of NO.

Polymeric backbones of linear Polyethylenimine diazeniumdiolate, (LPEI) with covalently bound NONO-groups (L-PEI-NONO) may be mixed and immobilized together with other polymers, such as biocompatible polymers. This results in NO being released from a LPEI- NONO coated product (no small molecule by-products).

Incorrect handling of the material may result in the formation of significant amounts of nitrite that mistakenly can be interpreted as NO (during release measurements) if the analysis method is not specific for NO (e.g. the commonly used Griess method). In the case of primary amine NONO'ates (e.g. Angeli's salt) a release of HNO has been observed (Miranda et al., 2005), although this has not been proven for the secondary amine NONO'ates illustrating that the chemistry of NO and HNO appears to be orthogonal.

Other NO-donors such as sodium nitroprusside, molsidomine, and the nitrate esters requires redox activation in order to release the NO and the more recently introduced agents of the S- nitrosothiol family which can release NO through hemolytic cleavage, forming reactive thiyls as a by-product, or through trace transition metal (concentration dependant) catalytic breakdown, forming disulfide in the process (Singh et al., 1996). They are also prone to produce larger amounts of by-products, such as nitrite and nitrate and the small molecule donors are generally more toxic than the polymeric donors.

In one embodiment, density (i.e concentration) of application of the RNS adduct, such as polyethylenimine diazeniumdiolate onto the surface of the medical device is between about 0.05 and about lOOmg/cm², such as between about 0.1 and about 10 mg/cm², such as preferably between about 0.2 and about 3 mg/cm².

In one embodiment, density of application of the RNS adduct, such as polyethylenimine diazeniumdiolate onto the surface of the medical device is between about 0.05 and about 200 μmol NO/cm², such as between about O. land about 20 μmol NO/cm², such as preferably between about 0.5 and about 3 μmol NO/cm².

A key aspect in controlling this premature release of nitric oxide from polyethylenimine diazeniumdiolate is therefore to increase the pH of the polyethylenimine diazeniumdiolate (local environment - e.g. first domain) to above 7, such as between pH 8 and 13.

Alkaline formulations of polyethylenimine diazeniumdiolate are typically made by dissolving the polyethylenimine diazeniumdiolate in alkalinized organic solvents, such as alcohols, such as ethanol and methanol, tetrahydrofurane, pyridine, and the like.

The alkalinized solvents are typically prepared by dissolving suitable alkali compounds (bases) prior to the addition of the (L)PEI-NONO polymer. The OH^" added should be between about 1 mM to about 100 mM OH- such as between 10 mM to about 75 mM OH- such as about between 20 mM and about 50 mM,. The pH of the solvent is modulated by adjusting the amount of alkali added.

It will be recognised that although PEI-NONO may be preferred, in one embodiment other polyalkylenimine diazenumdiolates may be used.

Numerous suitable alkali agents may be used, such as inorganic alkali, such as NaOH, KOH, Ca(OH)₂, and LiOH, or organic alkali, such as lithium diisopropylamide and methylamine. The alkali may be an alkoxide such as methoxide or ethoxide. The alkali may be a organic or inorganic alkali buffer such as phosphate, ethanolamine, ADA, carbonate, ACES, PIPES , MOPSO, imidazole, BIS-TRIS propane, BES, MOPS, HEPES, TES, MOBS, DIPSO , TAPSO, triethanolamine (TEA), pyrophosphate, HEPPSO, POPSO, tricine, hydrazine, glycylglycine, Trizma (tris), EPPS, HEPPS, BICINE, HEPBS, TAPS, 2-amino-2-methyl-l,3-propanediol (AMPD), TABS, AMPSO, taurine (AES), borate, CHES, 2-amino-2-methyl-l-propanol (AMP), glycine, ammonium hydroxide, CAPSO, methylamine, piperazine, CAPS, CABS, and pipidine.

Most preferred is an alkali methanol, such as NaOH dissolved in methanol (typically at a concentration of between 0.1 and 10 μg/ml, such as between 1 and 2 μg/ml, such as about 1.67μg/ml). Coating Systems (or Donor Layer)

The medical device according to the invention comprises a RNS adduct, such as nitric oxide adduct, in the form of a coating system comprising a polymer mixture, wherein said polymer mixture comprises said RNS adduct and a hydrophilic polymer or polymer blend, and wherein the said RNS adduct and the polymer or polymer blend form a homogeneous phase.

Therefore, the RNS adduct is in the form of a coating applied to at least part of the external surface of the medical device according to the medical device according to the invention.

In one embodiment, the coating system does not include an acidic agent such as lactic acid or vitamin C.

The coating system preferably comprises an antioxidant, such as a sterically hindered phenolic antioxidant (eg. Octadecyl-3-(3,5-di-tert.butyl-4-hydroxyphenyl)-propionate, tradename Irganox 1076, Ciba specialty chemicals), such as between about 0.01 and 1%; such as between about 0. 1% and 0.5%. The use of such an antioxidant increases stability during radiation sterilization and improves the shelf life of the medical device.

The term 'hydrophilic' refers to a substance which has a higher solubility in water than in oil or other hydrophobic solvents. Hydrophilic molecules are capable of forming hydrogen bonds, which makes then soluble not only in water but other polar solvents.

It has been discovered that the use of such a polymer mixture can control the release of the RNS adduct (such as nitric oxide adduct) from said inner layer into the physiological medium. By adjusting the ratio between the RNS releasing adduct and the additional polymer blend the embodiment of the RNS releasing adduct will be improved to minimize the release of the RNS releasing adduct into the surrounding environment. This is particularly of relevance for the medical devices, such as transient medical devices, where the release of the RNS adducts may lead to a low level of undesirable systemic effects, and may therefore also require more stringent regulatory approval process. It is also envisaged that the use of the polymer matrix can prevent the leakage of small molecule by-products from said polymer mixture into the physiological medium.

The use of the polymer mixture is also a key determinant in controlling the rate and timing of the release of RNS (such as nitric oxide) from said inner layer into the physiological medium. This is controlled by adjusting the polymer or polymer blend to balance the level of support compared to hydrophilic characteristics, thereby creating the desired structure which can provide the controlled diffusion of water from the physiological medium into the inner layer under physiological conditions to release the RNS.

Parameters important for the RNS releasing adduct are, as mentioned above, good stability, a fast activation of the RNS release, a continuous release within physiological relevant concentration and ensuring no, or minimal, release of the RNS releasing polymer into the external environment.

To ensure good stability, the polymer mixture, polymer blend, coating system and/or single polymer should withstand a traction force applied to the surface of the polymer coat of about 4 to 100 newton cm^"2 such as about 8 to 15 newton cm^"2 such as preferably between 10 to 20 Newton cm^"2

To ensure a rapid activation of the RNS (e.g. NO) release, the polymer mixture should show a good water conductance performance. In one embodiment, this parameter can be measured by determining the release rate of RNS from a RNS adduct coated with the polymer and a suitable RNS adduct. This may be achieved by supplying a polymer with high water conductance and a high water conductance/swelling ratio to the RNS releasing polymer layer. The polymer mixture, polymer blend, coating system, and/or hydrophilic polymer should allow water transport into the RNS adduct layer rapidly enough to induce a increase in RNS release of about 0.01 to about 50 nmol cm^"2 min^"1 per minute such as about 0.25 to about 20 nmol cm^"2 min^"1 per minute such as preferably between 0.5 to about 10 nmol cm^"2 min^"1 per minute when measured at the maximum rate of increase of RNS release (see Figure 6). This may be determined by preparing a 30% LPEI-NONO, 70% hydrophilic polymer (or polymer mixture or polymer blend), on an inert base material such as nylon, and measuring the rate of release of RNS (e.g. NO) over time when inserted into phosphate buffered saline, pH 7.4, 37°C. Suitably the RNS (e.g. NO) adduct used to determine the RNS (e.g. NO) release may be the LPEI-NONO as prepared in the example 1.

In one embodiment, the hydrophilic polymer or polymer blend, when used in the homogeneous polymer blend, provides a water (proton) transport rapidly enough to obtain a maximum rate of increase of RNS release with a slope of about 0.01 and about 80

(nmol/min/cm²)/min when inserted into physioloigical conditions, such as when inserted into phosphate buffered saline, pH 7.4 at 37°C.

In one embodiment, the ratio of polymer mixture to said RNS (e.g. NO) adduct present in the coating system is between about 5/95 to about 95/5, such as between 40/60 and 90/10, preferably between about 50/50 and about 80/20, such as about 70/30, as measured weight/weighty

In one embodiment, the RNS (e.g. NO) donor itself may form the hydrophilic or support polymer, or polymer blend, and as such the RNS (e.g. NO) adduct is suitable for use as a coating system per se. In such an embodiment, the coating system may comprise up to 100% of the RNS (e.g. NO) adduct.

In one preferred embodiment, the polymer blend comprises a mixture of a support polymer and a hydrophilic polymer.

The hydrophilic polymer may be selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinyhdene difluoπde, polyvinylchloπde, deπvatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes, and silicones.

More specifically they may be selected from the group consisting of: aromatic polyurethanes, ethylene ocrviato polyolefins, hydrophilic aliphatic polyurethanes or silicones, all with a high ratio of water conductance towards water absorbance. These polymers can be selected from the tecophihc, estane, EMAC and EBAC polyolefins families or high water vapour conducting silicones.

The support polymer may be selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinyhdene difluoπde, polyvinylchloπde, deπvatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes with good adhesion and structural stability, fulfilling the stability criteria stated above. However, it could preferably be an aliphatic polyether- based polyurethane such as eg. Tecoflex SG 85A.

In one embodiment, the ratio of support polymer to hydrophilic polymer used in the coating system can be between about 95/5 to about 35/65. by weight depending on the characteristics of the polymers used. In one system, using an aliphatic polyether-based polyurethane together with an aromatic polyurethane, the ratio can vary from about 90/10 to about 50/50 by weight.

In one embodiment the ration of support to hydrophilic polymer used in the coating system is about 70/30 by weight. The ratio of support polymer to hydrophilic polymer is a ratio which excludes the RNS (e.g. NO) adduct, which is also present in the coating system.

It is envisaged that in one embodiment the RNS (e.g. NO) adduct may itself be part of either of the hydrophilic and/or supporting polymer. However, in a preferred embodiment, the RNS (e.g. NO) adduct is not the supporting polymer or hydrophilic polymer referred to in the coating system, although may comprise support and/or hydrophilic properties. Suitable methods for the preparation of hydrophilic and/or supporting polymer nitric oxide adducts are disclosed in US 5,405,919 or other references referring to synthesis of RNS (e.g. NO) polymers, such as those referred to herein.

In one embodiment, the thickness of the coating system is between about 1 to about 100 μm, such as between 2 and about 20 μm, such as between 5 and about 20 μm in dry state.

In one aspect, the coating or coating system comprising the RNS (e.g. NO) adduct is applied to the exterior surface of said medical device by one or more of the following methods: spray coating, painting, dipping.

The First Domain

The RNS (e.g. NO) adduct may be in the form of a first domain in the form of the coating system referred to herein. Suitably, the first domain is a discrete domain which does not form a homogenous phase with the second domain, if present(See figure 7, for examples. In a preferable embodiment, the first domain may be in the form of a uniform layer of approximately uniform thickness (Figure 7A-E). However, it will be apparent to the skilled person that the first domain may be in many other forms (see Figure 7, F to I for example).

The first domain may comprise the hydrophilic polymer or polymer blend combined with one or more alkali (base) agents or basic side groups. The hydrophilic polymer or polymer blend suitable for use in the first domain is described in EP application No. 06023223 and US provisional 60/864,886, and PCT/DK2007/00030.

In one embodiment, the first domain may form discrete particles, such as nanoparticles, which are embedded into a further domain, such as the second domain. Alternatively the second domain may form discrete particles which are embedded in a further domain, e.g. the outer-layer and/or the first domain (see Figure 7, F to I for example).

It is however preferred the first domain forms a homogeneous layer. The first domain or coating system is not in the form of, or does not comprise, LPEI-NONO fibres, such as electrospun fibres or nanofibres.

The first domain or coating system forms a homogeneous phase.

The first domain or coating system is not heterogeneous.

The pH of the first domain or coating system may be controlled in numerous ways known in the art. For application to a medical device, (L)PEI-NONO is typically dissolved in a nonaqueous solvent,

Typιcally,when the RNS is NO, the first domain or coating system is an alkaline domain, i.e. when it comes into contact with a suitable solvent, for example water, the pH of the local domain is above pH 7, such as above about pH 7.5, such as above about pH 8, such as above about pH 9, such as above about pH 10, such as above about pH 11, such as above about pH 12, such as above about pH 13, such as about pH 14, or such as between above pH 7 and about pH 14. In a preferred embodiment the pH of first domain is between about pH 9 and about pH 12, most preferred between about pH 8 and about pH 11, such as between about pH 9 to about pH 10. We have noted that high pH, e.g. 12 and above, the polymer systems, such as the hydrophilic support polymer/polymer blend, that the high pH can alter the physical properties of the polymer, which in some cases may be detrimental, for example high pH may affect the stability of the coating systems.

The first domain, when the RNS is NO, therefore typically comprises an alkaline compound which may be organic or inorganic. Suitable inorganic alkali compounds include by way of example NaOH, KOH, Ca(OH)₂, and LiOH. Suitable alkali organic compound include, by way of example lithium dnsopropylamide, methylamine or methoxide. The alkali compound may be a alkaline buffer, such as a buffer selected from the group consisting of: phosphate, ethanolamine, ADA, carbonate, ACES, PIPES , MOPSO, imidazole, BIS-TRIS propane, BES, MOPS, HEPES, TES, MOBS, DIPSO , TAPSO, tπethanolamine (TEA), pyrophosphate, HEPPSO, POPSO, tπcine, hydrazine, glycylglycine ,Tπzma (tπs), EPPS, HEPPS, BICINE, HEPBS, TAPS, 2-amιno-2-methyl-l,3-propanedιol (AMPD), TABS, AMPSO, taurine (AES), borate, CHES, 2- amιno-2-methyl-l-propanol (AMP), glycine,, ammonium hydroxide, CAPSO, methylamine, CAPS, CABS, and pipidine.

The alkaline compound may also be an alkaline polymer, such as a polymer containing a inorganic or organic base, such as an alkaline side group. The alkaline compound, including alkali polymers, preferably has a pKb of less than 6, more preferable a pKb of less than 5

The alkali compound or group may be selected from the group consisting of a primary amine, a secondary amine and a tertiary amine.

The alkali compound or group may be selected from the group consisting of lithium dnsopropylamide, methylamιne,and chloroquine.

In addition to (L)PEI-NONO the first domain typically comprises a further polymer, such as a polyurethane, or a hydrophilic polymer or polymer blend as referred to herein (see under coating systems).

In one embodiment, the first domain may comprise a polymer selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoπde, polyvinylchloπde, deπvatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes, silicones.

In one embodiment, the first domain may comprise a polymer selected from the group consisting of: aromatic polyurethanes, ethylene acrylate polyolefins, hydrophilic aliphatic polyurethanes or silicones, all with a high ratio of water conductance towards water absorbance. These polymers can be selected from the tecophihc, estane, EMAC and EBAC polyolefins families or high water vapour conducting silicones.

In one embodiment, the first domain may comprise a polymer selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoπde, polyvinylchloπde, deπvatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes with good adhesion and structural stability, fulfilling the stability criteria stated above. However, it could preferably be an aliphatic polyether-based polyurethane such as eg. Tecoflex SG 85A.

PCT/DK2007/000030 provides further examples of the different domains, including further domains. The Second Domain

The second domain is a discrete domain which does not form a homogenous phase with the first domain (See Figure 7 for example). In a preferable embodiment, the second domain may be in the form of a uniform layer of approximately uniform thickness- see Figure 7 A and B for examples.. However, it will be apparent to the skilled person that the second domain may be in many other forms.

The second domain may comprise the hydrophilic supporting polymer or polymer blend combined with one or more acidic agents or acidic side groups. The hydrophilic supporting polymer or polymer blend suitable for use in the second domain is described in EP application No. 06023223 and US provisional 60/864,886.

The second domain may form discrete particles, such as nanoparticles, which are embedded into a further domain, such as the first domain. Alternatively the second domain may form discrete particles which are embedded in a further domain, e.g. the outer-layer and/or the first domain (see e.g. figure 7).

In one embodiment the second domain may also comprise (L)PEI-NONO. However, in one preferred embodiment, the second domain does not comprise (L)PEI-NONO.

In one embodiment, the second layer is not in the form of, or does not comprise, LPEI-NONO fibres, such as electrospun fibers or nanofibers.

It is preferable that the second domain forms a homogeneous phase.

The pH of the second domain may be controlled in numerous ways known in the art. For example, the second domain may e.g. comprise ascorbic acid, polyacrylic acid, lactic acid, acetic acid and/or oxylic acid as H⁺-releasing agents for affecting release of the therapeutic agent from the first domain. The second domain may comprise a polymer which is acidic, or comprises acidic side groups, which are capable of releasing protons upon contact with water.

Further acidic agents or side groups, which may be used in the second domain include lactic acid or vitamin C, and/or an acid agent selected from the group consisting of: ascorbic acid, polyacrylic acid, oxylic acid, acetic acid and lactic acid.

The acidic agent may have a pKa of less than 6, more preferable any organic acid with a pKa of less than 5. In one embodiment the acidic agent is an inorganic acid, such as hydrochloric acid, sulphuric acid, nitric acid or hydrobromic acid.

The acidic agent may be, or comprise an acidic buffer, such as a buffer selected form the group consisting of: maleate, phosphate, glycine, citrate, glycylglycine, malate, formate, , succinate, acetate, propionate, pyridine, piperazine, cacodylate, MES, histidine, bis-tπs, ethanolamine, ADA and carbonate.

The acidic compound may be, in one embodiment, a polymer containing an inorganic or organic acid such as a side group. In a preferable embodiment, the acidic compound, such as the acidic polymer comprises carboxylic groups.

The acidic compound may therefore be a fruit acid, or an equivalent, for example a hydroxy acid, or an acidic derivative thereof.

Therefore, it is considered that the second domain may be either internal or external to the first domain.

The second domain is capable of affecting the pH of the first domain by shifting the local balance between H⁺ and OH^" ions upon wetting of at least a portion of the medical device, i.e. the second domain is capable of reducing the pH of the first domain upon wetting of at least a portion of the medical device. Therefore release rate of the nitric oxide from the first domain is dependent on the pH of at least one of the first domain and in one embodiment the second domain.

Typically, when the RNS is NO, the second domain is an acidic domain, i.e. when it comes into contact with a suitable solvent, for example water, the pH of the local domain is below pH 7, such as below about pH 6.5, such as below about pH 6, such as below about pH 5, such as below about pH 4, such as below about pH 3, such as below about pH 2, such as about pH 1, or such as between below pH 7 and about pH 1. The pH of the second domain may, for example be between about pH 2 and about pH 6. or between about pH 3 to about pH 5.

We have also noted that at very low acidity, such as below pH 2, the acidity can effect the physical properties of the polymers used in the second coat, and this may effect the stability of the coat detrimentally. First and second domains

In one embodiment of the invention, the medical device is at least partially coated with both a first and a second domain.

In one embodiment, the first domain comprises a first coating layer of the device which is applied to, or is immediately adjacent to, either the base material of the medical device or the primer layer. The second domain may therefore comprise a second coating layer of the device which is applied to, or is immediately adjacent to said first coating

Suitably, the release rate of the RNS (e.g. NO) from the first domain is dependent on the pH of at least one of the first domain and/or the second domain.

The first and second domains may have any form or shape, however, layered structures are preferred, such that the first domain forms one coating layer and that the second domain forms another coating layer. For example, the medical device may be coated with one material forming the second domain as an initial surface coating, which subsequently is being covered with another material forming the first domain as a further coating layer onto of the initial surface coating. For some applications it may be preferable that the donor-compound containing layer (i.e. first domain) is applied prior to the pH modifying layer (i.e. second domain), whereas it may be preferable for other applications that the pH modifying layer (i.e. second domain) is applied prior to the donor layer (i.e. first domain). It is also envisaged that the two layers may be applied simultaneously, for example by extrusion of two independent layers, or by coating of a layer which comprises both the first and second domains, e.g. for example when the first and/or second domains are in the form of particles, one domain may form a 'matrix', whist the other domain forms discrete (i.e. non-homogeneous) particles, such as nano-particles within the 'matrix' domain, or both first and second domains may be in the form of discrete particles within a suitable matrix composition, such as the hydrophilic supporting polymer or polymer blend referred to herein.

In the embodiment where there is at least a first domain and a second domain, the first domain is capable of releasing one or more therapeutic agents, the release rate being e.g. dependent on the pH of the domain. As the second domain is capable of affecting the pH of the first domain by release of H⁺ ions, upon wetting of at least a portion of the medical device, the pH of the first domain decreases upon wetting of a portion of the device, and accordingly the release of the therapeutic agent is triggered or enhanced.

Hence, in one preferred embodiment, the present invention provides for the controlled release of the therapeutic agent (typically nitric oxide or HNO) in dependency of the humidity or water content of a portion of the medical device, such as the water content of the second domain. In a typical application of the medical device, the second domain is wetted by blood upon entry into the vascular system of a human or animal body. The timing of the release of RNS (e.g. NO) may be further influenced by the arrangement of the first and second domains, and by the addition of further layers, such as the outer-coat.

In a preferred embodiment, the second domain is applied prior to, i.e. is interior to, the first domain. This ensures that as the protons diffuse out of the second domain to the external environment they must pass through the first domain, thereby ensuring the maximum RNS (e.g. NO) release.

Herein, the first domain is also referred to as the bioactive domain, and the second domain is also referred to as the pH active domain. However, it should be recognised that the first domain is also pH active, but in contrary to the second domain the pH (or potential pH upon wetting) of the first domain is alkaline, where as the pH (or potential pH upon wetting) of the second domain is acidic.

The Third Domain

The medical device may comprise a third domain.

The third domain may be an additional domain which is positioned between the first domain and the second domain.

In one embodiment, the medical device may comprise a first domain and a third domain.

The third domain may be a neutral layer, which controls the rate of influx of water (and protons) from the body fluid (optionally via the second domain) into the first domain. The third domain may, in one embodiment, comprise a buffer, which limits the influx of protons into the first domain, thereby providing for 'long and low' RNS (e.g. NO) release kinetics.

By using a third domain of about neutral pH (7) between the fist and second domains, the premature degradation of the (L)PEI-NONO (NO release) can be avoided or reduced even further, this particularly relevant during the processing steps (manufacture) involved in preparation of the medical device. In one embodiment the third domain comprises or consists of the hydrophilic supporting polymer or polymer blend as referred to herein, such as that disclosed in EP application No. 06023223 and US provisional application 60/864/886.

In one embodiment, the third domain consists or comprises a hydrophilic polymer.

In one embodiment, the third domain consists or comprises a hygroscopic polymer.

The third domain may comprise a buffer, such as a buffer of pH around 7, when it comes into contact with water. As such the third domain can control the rate at which the protons diffuse from the second domain into the first domain by acting as a proton quencher. This is useful when the medical device may come into contact with water or water vapour prior to the time at which the therapeutic release is required or optimal. Likewise the third domain can act as a -OH quencher, preventing the undesirable alkalisation of the acidic layer.

The thickness of the third domain, may, for example be between about O. lμm and about lOμm, such as between lμm and about 5μm.

In one embodiment the third domain does not comprise a buffer. In one embodiment the pH of the third domain is about 7.

It is recognised that the composition of the outer-coat and the third layer may, in one embodiment be identical.

Inner Priming Layer

A primer is typically a first coating formulated to seal raw surfaces and hold succeeding finish coats.

In a preferred embodiment, the base material of the medical device (i.e. external surface and/or the surface which comes into contact with the physiological media, is coated with an inner priming layer between the base layer of the medical device and said coating system or nitric oxide adduct, or said first or second domains.

In one embodiment the inner priming layer may be selected from the group consisting of polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoπde, polyvinylchloride, deπvatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes. In one embodiment the inner priming layer may be polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoride, polyvinylchloride, derivatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes.

Suitably the inner priming layer has good adhesion and structural stability, withstanding a traction force applied to the surface of the polymer coat of about 4 to 100 Newton cm^"2 such as about 8 to 50 newton cm^"2 such as preferably between 10 tp 20 Newton cm^"2.

A preferred primer polymer is an aliphatic polyether-based polyurethane such as eg. Tecoflex SG 85A.

In one embodiment, the thickness of the priming layer is between 0.2 and 5μm. The priming layer need not be uniform in thickness, over the surface as its role is to secure the coating and/or NO adduct to the medical device. It is therefore required to provide sufficient anchor points to provide a robust platform on which to apply the addition coating(s). Preferably, the priming layer has effectively complete coverage over the base material to be coated as this ensures maximum structural integrity of the subsequent layers.

Further layers

As described above with respect to the method for the manufacture of a sterilised medical device, a further layer or layers may be applied over the RNS (e.g. NO) adduct, such as the coating system or fibre layers. Such further layers include the second domain and third domains as well as the outer-coating (outer-layer) as herein disclosed.

In one embodiment, at least one of the first, second, third domains comprises polyurethane, such as both the first and second domains, such as the first and third domains.

Therefore in one embodiment, the medical device according to the invention comprises a RNS (e.g. NO) adduct (first domain) capable of releasing RNS (e.g. NO) under physiological conditions as herein described, and at least one further layer (second domain) of a material which is capable of affecting the pH of the RNS (e.g. NO) adduct (the first domain), such as the RNS (e.g. NO) adduct coating system or fibres, upon insertion into physiological media, (see European application No. 06075159 and PCT/DK2007/000030 which are hereby incorporated by reference).

In one embodiment, it is preferred that the further layers (including the outer layer referred to below) are sufficiently flexible to allow movement and flexing of the medical device without risking the integrity of the further layers. Suitably, the further layers may be hydroscopic and swell with water when in use. The routine submersion of introducer sheath apparatus in isotonic solution prior to use therefore allows the further layers to absorb sufficient water to ensure sufficient flexibility, and reduced friction upon insertion. Some polymers, for example the tecophillic polymers used for the outer coating, may swell to about 60% of their volume when inserted in aqueous media. A further advantage of using further coats of polymers which swell is that the swelling will mask any pin-hole defects or imperfections in the further coats, therefore ensuring the integrity of the coating system when in use.

Whilst it is, in one embodiment, preferable that the further layer or layers are applied externally to said RNS (e.g. NO) adduct (i.e. located between the RNS (e.g. NO) adduct and the physiological media when in use), it is also recognised that the further layer may be applied as a layer between the base material of the medical device and the RNS (e.g. NO) adduct. It is also envisaged that the RNS (e.g. NO) adduct coating, such as coating system may be formulated with a coating component that regulates the pH, and therefore NO release from the nitric oxide adduct when it comes into contact with physiological media.

In one embodiment, an outer coating as herein described is also applied. The outer coating may be applied directly to the coating system layer, or to one or more further layers applied to the coating system layer.

The Outer Layer

In one embodiment, the medical device comprises an outer layer situated externally to the RNS (e.g. NO) adduct, and if relevant any further coating layers, and said outer layer comprises a polymer which controls one or more of the following: ι) the release of the RNS (e.g. NO) adduct from said inner layer into the physiological medium either by forming a diffusion barrier between the RNS (e.g. NO) releasing adduct and the physiological medium, or by applying a diffusion resistance to the diffusing molecule and thereby significantly slowing down the diffusion process . ιι) the release of RNS (e.g. NO) from said inner layer into the physiological medium by presenting a low conductance to the RNS (e.g. NO) molecule. ιιι) the diffusion of water from the physiological medium into the inner layer under physiological conditions by either facilitating or restricting the water flow through the top layer depending on the needs in the given application and/or ιv) the leakage of small molecule by products from said polymer mixture into the physiological medium. To ensure a controllable release of RNS (e.g. NO), the outer layer should be able to restrict the activation, and/or the continuous release, of the nitric oxide release by a factor of about 1 to 20 such as about 1.5 to 10 such as preferably between 2 to 5 compared to the activation of RNS (e.g. NO) release from the RNS (e.g. NO) coating layer itself.

Suitable outer-coatings and methods of manufacture of medical devices coated with out- coats, and the medical devices and uses thereof are disclosed in European application No 06023222 and US provisional application 60/864,893.

The outer layer may also provide structural stability, ensuring the RNS (e.g. NO) adduct and/or coating system remains in place during manufacture, storage, preparation and use of the medical device.

As described above, the outer coat may consist or comprises of a polymer which has the ability to swell upon insertion into aqueous media. An advantage of using polymers which swell is that the swelling will mask any pin-hole defects or imperfections in the further coats, therefore ensuring a high integrity of the coating system when in use.

The ability to swell may be determined by a simple experiment where a known volume of polymer, such as in a granular form, is added to an excess of pure water and allowed to reach an equilibrium in term of water absorption. The swollen granules are then removed from the aqueous media, excess water removed, and the change in volume is assessed. Typically, polymers suitable for use in the outer coat have an ability to swell of at least 1% volume, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%. Such polymers are referred to as hydroscopic polymers. It will however be recognised that the use of hydroscopic polymers should be used carefully so as to not interfere with the functionality of the medical device or removal of the medical device from the patient.

Whilst disclosures in the prior art refer to the insolubility of LPEI-NONO (see US 6,855,366 for example), we have surprisingly found that (linear) polyethylenimine diazeniumdiolate is soluble in physiological media despite it previously being considered insoluble in aqueous media. It appears that the solubility is due to the presence of ions within the physiological media, which are absent in pure water in which polyethylenimine diazeniumdiolate is stated insoluble. The use of the coating system and/or outer layer reduces or prevents the inappropriate release of the LPEI-NONO from the coated medical device.

In one embodiment, the outer layer comprises a hydrophilic polymer, such as a hydroscopic polymer. Polymers which may be used in the outer coating may be selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoride, polyvinylchloride, derivatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes, silicones, or celluloses. More specifically they can be aromatic polyurethanes, ethylene acrylate polyolefins, hydrophilic aliphatic polyurethanes or silicones.

High water vapour conducting polymers, such as EMAC and EBAC polyolefins families or high water vapour conducting silicones may also be used. Water vapour conducting polymers can be identified using the ASTM Method E96B - 50%RH, 23°F, preferable water vapour conducting polymers range from moderately breathable, about 350g/m²/24hrs MVT to a highly breathable, about 760g/m²/24hrs HMVT. Such polymers may, in one embodiment also be useful as hydrophilic polymers for use in the polymer blend/coating systems disclosed herein.

In one preferred embodiment, the outer layer polymer forms a hydroscopic gel upon insertion into physiological media.

It is preferred that the outer coating is selected for its ability to facilitate the insertion and withdrawal of the medical device from the vessel. In this respect hydroscopic or hydrophilic polymers may be preferred in some applications as they form a slippery surface upon insertion into aquatic environments, such as physiological media. It may therefore be preferred that the medical device is contacted with a suitable aqueous solution prior to use, such as isotonic water, for a brief period prior to use, such as for between 1 and 3 minutes.

In one preferred embodiment, the outer layer polymer comprises a hydrophilic polyurethane.

It has been discovered that the use of such a polymer can control the release of the RNS (e.g. NO) adduct from said inner layer into the physiological medium. This is particularly of relevance for intravascular medical devices, such as transient intravascular medical devices (i.e. not implants), where the release of the RNS (e.g. NO) adducts may lead to a low level of undesirable systemic effects, and may therefore also require more stringent regulatory approval process.

Hence it is preferred that both the coating system (as first domain) as herein disclosed and the outer layer are used, (optionally with the second and/or third or domains) as both these can control the undesirable release of the RNS (e.g. NO) adduct into the physiological media. The outer layer can also be used to control the rate and timing of the release of RNS (e.g. NO) from the RNS (e.g. NO) adduct, such as the coating system as disclosed herein, into the physiological medium. In this respect the outer layer may be only partially or unevenly applied, thereby allowing part of the RNS (e.g. NO) adduct to rapidly come into contact with physiological media, giving an initial release of RNS (e.g. NO), whilst the adduct which has an outer coating provides a more sustained release for longer duration.

In one embodiment, the outer-coat provides a further protection against premature release of RNS (e.g. NO) by preventing the premature hydration of the first and/or second domains. This may be achieved by utilising an outer-coat which is, for example, only partially permeable to water, or only permeable to water vapour, for example an outer-coat made of a hydrophobic polymer - such outer-coats are particularly useful for delayed release, for example in the case of implants such as stents. Suitable hydrophobic polymers are known in the art and include various polymers such as silicones (which may for example be permeable to water vapour but not water) and polyvinyl chloride (PVC) polyurethanes (PU), polyacrylates or other polymers with restricted water conductance or mixtures hereof.

In one embodiment, the outer layer (coat) does not comprise an acidic agent. Such an outer-layer may be used in the coatings of medical devices where a further layer or layers are applied which comprise an agent which modulates the pH as herein referred to (i.e. a pH modifying layer).

The pH of the outer-layer may suitably be about physiological pH, such as between about pH 7 and about pH 8, such as about pH 7.4. The outer layer may comprise a buffer which maintains such a pH, such as a phosphate buffer. By maintaining a pH of about physiological pH the outer-layer can protect the cells which come into contact with the medical device from the pH of the first and/or second domains.

Nitric Oxide Release

One of the dominant processes in the blood vessel is the scavenging of nitric oxide by haemoglobin. Most nitric oxide combines with oxyhaemoglobin in a 60-100% oxygen saturated environment to form methaemoglobin and subsequently nitrate. In low oxygen saturated environment, nitric oxide combines with deoxyhaemoglobin to form nitrosylhaemoglobin, which in the presence of oxygen forms nitrogen oxides and methaemoglobin. The end products of nitric oxide that enter the systemic circulation are methaemoglobin and subsequently nitrate. The nitrate is then transferred to the serum, and the greater part of the nitrate is excreted into the urine through the kidney. The typical half life of nitric oxide in blood is milliseconds. The half-life of nitric oxide is several hundred times longer in tissue than in blood. The biological half-life of haemoglobin- bound nitric oxide is about 15 mm.

Physiological concentration range of nitric oxide activity: The concentration in the smooth muscle cells resulting in relaxation is, based on the literature findings, expected to be larger than 200 nM but significantly below 1 mM. The lower limit (200 nM) is determined by the concentration required to activate soluble guanylyl cyclase, which acts as the enzyme that generates the second messenger cyclic GMP resulting in smooth muscles relaxation. The upper limit (1 mM) is determined by the concentration leading to significant oxidative stress and mutations.

For comparison the physiological level of nitric oxide concentration in tissue stated in the literature ranges form 100 to 500 nM. The physiological concentration in healthy endothelial cell is about 100 μM.

Dose - nitric oxide release: According to Vaughn et al., Am J Physiol. 1998 Jun;274(6 Pt T)₁ 0.32 nmol/min/cm² is one of the highest reported release rates from the natural endothelium. In order to obtain the desired biological response values higher may therefore required. For example, considering the short contact with the vessel wall and the thickness of tissue that the nitric oxide needs to pass before reaching the media of the vessel the desired release rate should preferably be higher to obtain the optimal vasorelaxing response.

To support the determination of an appropriate release rate ex-vivo studies on human as well as rat arteries were carried out. The aim of the study was to investigate the effect of nitric oxide released from test tubes coated with the nitric oxide eluting polymer. It was shown that the nitric oxide eluting polymer induced forceful relaxation of the arteries. The applied release rates were in the magnitude of 0.5 and up to 3 nmol/min/cm². Testing has been performed in PBS as well as in blood.

According to the literature findings there is about a factor of 5000 between the concentration required to activate soluble guanylyl cyclase (200 nM) and the concentration level leading to significant oxidative stress and mutations (1 mM).

The effective device release rate has according to the preclinical studies and literature findings a broad range. To minimize the risk of toxicity the upper limit is preferably set to 40 nmol/min/cm². The lower limit is preferably set to 0.5 nmol/min/cm². Lower release rates than 0.5 nmol/min/cm² probably also induce relaxation, however they may be less effective. Suitably the (maximum) rate of NO release from the medical device according to the invention may be greater than about 0.1 nmol/min/cm², such as greater thanθ.25 nmol/min/cm², preferably greater than about 0.32 nmol/min/ cm², such as greater than 0.5 nmol/min/cm², such as greater than lnmol/min/ cm².

Suitably the maximum rate of NO release from the medical device according to the invention is no more than about 40 nmol/min/cm², or no greater than about 60 nmol/min/cm², or no greater than about 80 nmol/min/cm².

In one embodiment, (maximum) release rate from the medical device according to the invention is between about 0.5 and up to about 3 nmol/min/cm².

When a topcoat (outer layer/coating) is applied the release of nitric oxide deviates significantly from a l'order release. This is due to the barrier properties of the topcoat: limiting the water absorption and the diffusion of nitric oxide through the coating leading to a more constant release over time.

Preferably, the peak release of nitric oxide, and/or the release after 5 minutes after insertion into an isotonic solution, from the outer surface of the medical device is (e.g. between about 0.5 and about 40 nmol/min/cm²) measured by using a dynamic headspace chamber connected to a chemoluminescence NO detector: The object coated with the described nitric oxide releasing coating system is placed in a head space chamber containing pbs buffer (pH7.4) with 0.00004% Tween 20, kept at 37°C. The solution is continuously flushed with 250 ml_ N₂ gas ensuring oxygen free conditions. The nitric oxide released from the coat into the oxygen free environment is stripped off from the solution and carried to the chemoluminescence NO detector by the NO gas. The examples provide a NO assay which is used to determine the release rate of nitric oxide.

In one embodiment the peak release rate is obtained within the first fifteen minutes after wetting or inserting the device, such as within the first ten minutes such as within the first five minutes, such as within the first three minutes after wetting or inserting the device.

In order to determine whether a potential nitric oxide adduct is capable of releasing nitric oxide 'under physiological conditions' as used herein, the assay as described above may be used.

Preferably, the release of nitric oxide from the outer surface of the medical device has a half life in physiological media of at least 30 minutes, such as at least 60 minutes, such as at least 90 minutes or at least 2 hours such as at least 4 hours, at least 6 hours or at least 12 hours.

In one embodiment the maximum rate of decrease of NO release after the point of maximum rate of NO release has been obtained is less than about -0.015 nmol/min/cm²/min, such as less than about -0.03nmol/min/cm²/min, such as less than about -0.06 nmol/min/cm²/min.

Further Therapeutic Agents

The medical device may be capable of releasing one or more further therapeutic agents. These further therapeutic agents may be provided in the first, second and/or third domains, and or the outer-coating. Alternatively the therapeutic device may provide other means for delivery of the further therapeutic agents. Like the release of NO or HNO from polyethylenimine diazeniumdiolate, the release of the further therapeutic agents may also be pH dependant, and as such the acidification of the first domain may cause the release of the therapeutic agent, or the alkalinisation of the second or third domain or outer-layer from the alkali first domain, may trigger the release of the further therapeutic agents.

In addition to (or in one embodiment as an alternative to) the polyethylenimine diazeniumdiolate, the medical device may be coated in one or more further therapeutic agents, such as a human growth factor, an anti coagulant, such as heparin, an antibiotic agent, such as an antibiotic, a chemotherapeutical agent, a further smooth muscle cell proliferation reducing agents, such as nitric oxide (NO) or a nitric oxide donor, and/or a vasodilation agents, such as NO or an NO donor. Ascorbic acid (vitamin C) may be provided as an antioxidant or as a catalyst for release of nitric oxide (i.e. within the second domain). In one embodiment, in case the release rate of the further therapeutic agent is not per se dependent on pH, the therapeutic agent may be bonded to or encapsulated in a carrier compound, which is characterised by a pH-dependent release rate or a pH-dependent degradation rate of a carrier material encapsulating the therapeutic agent.

The further therapeutic agent may be immobilised in a hydrogel, e.g. a hydrogel. Certain hydrogels swell under acidic conditions. One possible way to produce such a hydrogel which swells in blood but not in pure water is to co-deposit the therapeutic agent with glucoseoxidase (GOD). When glucose diffuses into the hydrogel from the blood, GOD will transform the glucose to cluconic acid and hydrogen peroxide.

The further therapeutic agent may e.g. comprise at least one of: heparin or another thrombin inhibitor, hirudin, hirulog, argatroban, D-phenylalanyl-L-poly-L-arginyl chloromethyl ketone or another antithrombogenic agent, or mixtures thereof; streptokinase, urokinase, a tissue plasminogen activator, or another thrombolytic agent, or mixtures thereof; paclitaxel; estrogen or estrogen derivatives; a fibrinolytic agent; a vasospasm inhibitor; a calcium channel blocker, a nitrate, nitrite, nitric oxide, a nitric oxide promoter, such as ascorbic acid, or another vasodilator; an antimicrobial agent or antibiotic; aspirin, ticlopdine or another antiplatelet agent; colchicine or another antimitotic, or another microtubule inhibitor; cytochalasin or another actin inhibitor; a remodelling inhibitor; GPIIb/Illa, GPIb-IX or another inhibitor or surface glycoprotein receptor; deoxyribonucleic acid, an antisense nucleotide or another agent for molecular genetic intervention; methotrexate or another antimetabolite or antiproliferative agent; an anti-cancer chemotherapeutic agent; dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate or another dexamethasone derivative, or another anti-inflammatory steroid; dopamine, bromocriptine mesylate, pergolide mesylate or another dopamine agonist; ⁶⁰Co (having a half life of 5.3 years), ¹⁹²Ir (half life of 73.8 days), ³²P (half life of 14.3 days), ¹¹¹In (68 hours), ⁹⁰Y (64 hours), ^99mTc (6 hours) or another radiotherapeutic agent; iodine-containing compounds, barium-containing compounds, gold, tantalum, platinum, tungsten or another heavy metal functioning as a radiopaque agent; a peptide, a protein, an enzyme, an extracellular matrix component, a cellular component or another biologic agent ; captopril, enalapril or another angiotensin converting enzyme (ACE) inhibitor; ascorbic acid, alphatocopherol, superoxide dismutase, deferoxyamine, a 21-aminosteroid (lasaroid) or another free radical scavenger, iron chelator or antioxidant; angiopeptin; a ¹⁴C-, ³H-, ¹³¹I-, ³²P or ³⁶S-radiolabelled form or other radiolabeled form of any of the foregoing. Mixtures of any of these are also envisaged.

It is recognised that one or more of the therapeutic agents listed above, may in one embodiment, be used either in the absence of (L)PEI-NONO, or in addition to (L)PEI-NONO. They may be present in the first domain, second domain, third domain or outer coat and/or further layers as referred to herein.

Method of Treatment

The medical devices/kits of parts according to the invention may be used for performing intravascular or neurovascular surgery.

The site of entry of the medical device may, in one embodiment, be selected form the group consisting of: the femoral artery, the radial artery, the carotid artery, the brachial artery, the auxiliary artery.

In one embodiment, the site of entry of the medical device is selected form the group consisting of: the radial artery, the brachial artery & the auxiliary artery. In one aspect the invention provides for a method for performing intravascular or neurovascular surgery said method comprising introducing the medical device according to the invention into the vascular or neurovascular system of a subject (patient).

In one aspect the invention provides for a method of preventing or reducing vasospasm associated with the introduction of a medical device into the vascular or neurovascular system of a subject, said method comprising introducing the medical device according to the invention into the vascular or neurovascular system of a subject (patient).

In one aspect the invention provides for the use of a coating system according to the invention in the manufacture of a medical device for the prevention or reduction of vasospasm associated with the introduction of the medical device into the vascular system of a subject.

FURTHER EMBODIMENTS

The following embodiments may be combined with the other features of the invention as referred to herein.

1. A medical device for intravascular use, comprising a base material which is at least in part, coated with a coating system comprising a polymer mixture, wherein said polymer mixture comprises i) at least one nitric oxide adduct which is capable of releasing nitric oxide under physiological conditions, such as a water inducible NO adduct, and ii) a hydrophilic supporting polymer or polymer blend, and wherein the nitric oxide adduct and the supporting polymer or polymer blend form a homogeneous phase.

2. The medical device according to embodiment 1, wherein the at least one nitric oxide adduct is selected from the group consisting of: nitroglycerin, sodium nitroprusside, S-nitroso-proteins, S-nitrosothiols, long carbon-chain lipophilic S-nitrosothiols, S- nitroso-dithiols, iron-nitrosyl compounds, thionitrates, thionitrites, sydnonimines, furoxans, organic nitrates, and nitrosated amino acids, nitroso-acetylcysteine, S- nitroso-captopril, S-nitroso-homocysteine, S-nitroso-cysteine, S-nitroso-glutathione, and S-nitrosopenicillamine, S-nitrosothiols, S-nitrosylated polysaccharides such as S- nitrosylated cyclodexrins, NONOate compounds (i.e. compounds which comprise the anionic NONOate functional group (N₂O₂ ^")), NONOate polymers.

3. The medical device according to embodiment 2, wherein the at least one nitric oxide adduct is a NONOate polymer. 4. The medical device according to embodiment 3 wherein the NONOate polymer backbone is selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoride, polyvinylchloride, derivatized polyolefins such as polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes, biopolymers such as peptides, proteins, oligonucleotides, antibodies and nucleic acids, starburst dendrimers.

5. The medical device according to embodiment 4 wherein the polymer which comprise the NONOate functional group (N₂O₂ ^") is polyethylenimine diazeniumdiolate, such as linear polyethylenimine diazeniumdiolate (LPEI-NONO).

6. The medical device according to any one of embodiments 2 to 5, wherein the nitric oxide adduct has a molecule weight of between about 5kDa and about 20OkDa, such as between about 2OkDa and about 10OkDa, such as between about 5OkDa and about 6OkDa, such as about 55kDa.

7. mixture to said nitric oxide adduct present in the coating system is between about 5% to about 80%, such as between 10% and 50%, preferably between about 20% and about 50%, such as about 30%,

8. The medical device according to any one of the preceding embodiments wherein said polymer blend comprises a mixture of a support polymer and a hydrophilic polymer.

9. The medical device according to embodiment 8, wherein the hydrophilic polymer or polymer blend, when used in the homogeneous polymer blend, provides a release rate of about 0.5 and about 80 nmol/min/cm² as measured by using a dynamic headspace chamber connected to a chemoluminescence NO detector at the point of maximum rate of increase of release of nitric oxide when inserted into phosphate buffered saline solution, pH 7.4 at 37°C.

10. The medical device according to embodiment 9, wherein the support polymer and/or polymer blend and/or coating system can withstand a traction force applied to the surface of the polymer coat of about 4 to 100 newton cm^"2 such as about 8 to 50 newton cm^"2 such as preferably between 10 to 20 Newton cm^"2.

11. The medical device according to anyone of embodiments 8 to 10, wherein the ratio of support polymer to hydrophilic polymer used in the in coating system is between about 95/5 to about 35/65 by weight. 12. The medical device according to any one of the preceding embodiments, wherein the thickness of the coating system is about 1 to about 100 μm, such as between 5 and about 20μm in dry state.

13. The medical device according to anyone of the preceding embodiments wherein the base material is coated with an inner priming layer prior to application of the coating system.

14. The medical device according to embodiment 13, wherein the inner priming layer is a polyurethane primer

15. The medical device according to embodiments 13 or 14, wherein the thickness of the priming layer is between about 0.2 and about 5μm.

16. The medical device according to any one of the preceding embodiments which comprises at least one further layer which may be either internal or external to the coating system, wherein said further layer is capable of affecting the pH of the coating system by shifting the local balance between H⁺ and OH^" ions upon wetting of at least a portion of the medical device.

17. The medical device according to any one of the preceding embodiments which comprises an outer layer situated externally to the nitric oxide adduct, and said outer layer comprises a polymer which controls one or more of the following: i) the release of polyethylenimine diazeniumdiolate from said inner layer into the physiological medium and ii) the release of nitric oxide from said inner layer into the physiological medium and iii) the diffusion of water from the physiological medium into the inner layer under physiological conditions and iv) the leakage of small molecule by products from said polymer mixture into the physiological medium.

18. The medical device according to embodiment 17, wherein said outer layer comprises a hydrophilic polymer

19. The medical device according to embodiment 17 or 18, wherein said hydrophilic polymer forms a hydroscopic gel upon insertion into physiological media.

20. The medical device according to any one of embodiments 17 to 19, wherein said outer layer comprised a hydrophilic polyurethane. 21. The medical device according to any one of embodiments 17 to 19, wherein the thickness of said outer layer is between about 0.3 and about 5 μm.

22. The medical device according to any one of the preceding embodiments wherein the release of nitric oxide from the outer surface of the medical device is between about 0.5 and about 80 nmol/min/cm2 as measured by using a dynamic headspace chamber connected to a chemoluminescence NO detector.

23. The medical device according to any one of the preceding embodiments wherein the release of nitric oxide from the outer surface of the medical device has a half life under physiological conditions as measured by using a dynamic headspace chamber connected to a chemoluminescence NO detector, of at least 30 minutes, such as at least 60 minutes, such as at least 90 minutes.

24. The medical device according to anyone of the preceding embodiments wherein the release of nitric oxide from the outer surface of the medical device has a half life under physiological conditions, of no greater than 6 hours, such as no greater than 4 hours, such as no greater than 3 hours.

25. The medical device according to anyone of the preceding embodiments wherein the medical device is selected from the group consisting of: Neuro medical devices, such as neuro guiding catheter, neuro microcatheter, neuro microwire, neurostent delivery system, neuron ballon; coronary medical devices, such as coronary wires, coronary guiding catheter, PTCA angioplasty balloon; stent delivery system, coronary wires, coronary guiding catheter, PTA angioplasty balloon, stent delivery system; introducer sheath, dialator, guide wire, syringe needle.

26. The medical device according to anyone of the preceding embodiments wherein the base material consists or comprises of one or more of the compounds selected from the group consisting of: a metal, such as stainless steel, titanium, gold, plantinum; a plastic, such as PVC, PA, PS, Epoxy Resins, Silicone Rubber, Natural Rubber, Polyurethane, PE, PP, Polyester, Nylon, PET, PMMA, Polysulphones, Polyphosphazenes, Thermoplastic Elastomers Polydimethylsiloxane (PDMS).

27. A medical device according to any one of the preceding embodiments wherein the density of the nitric oxide adduct, such as polyethylenimine diazeniumdiolate, on the surface of the medical device is between about 0.05 and about 100mg/cm2, such as between about 0.1 and about 10 mg/cm2, such as preferably between about 0.2 and about 5 mg/cm2. 28. The medical device according to any one of the preceding embodiments, wherein the support polymer and/or polymer blend and/or coating system can withstand a traction force applied to the surface of the polymer coat of about 4 to 100 newton cm- 2 such as about 8 to 50 newton cm-2 such as preferably between 10 to 20 Newton cm-2.

29. A method for the manufacture of a coated medical device suitable for intravascular use, said method comprising :

a. selecting a medical device suitable for use in vascular surgery, said medical device comprising a base material.

b. applying a coating system comprising a polymer mixture to said external base layer, wherein said polymer mixture comprises at least one nitric oxide adduct capable of releasing nitric oxide under physiological conditions, and a hydrophilic supporting polymer or polymer blend, and wherein the nitric oxide adduct and the supporting polymer or polymer blend form a heterogeneous phase.

30. The method according to embodiment 29, wherein the coating system is applied by spray coating, dipping, painting or extrusion.

31. The method according to embodiment 29 or 30, wherein prior to applying the coating system, said base material is at least partially coated with a primer layer, such a primer according to any one of embodiments 13 to 15.

32. The method according to any one of embodiments 29-31, which comprises applying at least one further layer which may be applied either internal (prior to) or external (subsequent) to the coating system layer such as the coating system, wherein said further layer is capable of affecting the pH of the nitric oxide adduct or coating system by shifting the local balance between H⁺ and OH^" ions upon wetting of at least a portion of the medical device.

33. The method according to any one of embodiments 29 to 32, wherein subsequent to the application of the coating system and any further layers, at least part of the coating system surface is coated with an outer layer, such as the outer layer according to any one of embodiments 17 to 20. 34. A method of performing intravascular surgery comprising inserting the medical device according to any one of embodiments 1 to 28, or the medical device prepared by the methods according to any one of embodiments 29 to 33, into the vascular system of a patient.

35. The use of the coating system as defined in any one of embodiments 1 to 34 for the manufacture of a medical device for use intravascular use, for the prevention of one or more the conditions selected from: vasospasm or vasoconstriction, prevention of cerebral vasospasm, relaxation of smooth muscle, vasodilatation, thrombosis, decreased platelet deposition or aggregation, alleviation of restenosis, increased blood pressure, oxygen free radical reperfusion injury, treatment of cardiovascular disease, preventing the adverse effects associated with the use of said medical device, preventing abnormal cell proliferation.

36. Use of a coating system which controls the rate of release of nitric oxide from the surface of a medical device which comprises a nitric oxide adduct when said medical device is inserted in vivo, said coating system comprises a polymer mixture, wherein said polymer mixture comprises i) at least one nitric oxide adduct and ii) a hydrophilic supporting polymer or polymer blend, and wherein the nitric oxide adduct and the supporting polymer or polymer blend form a heterogeneous phase.

FIGURE LEGENDS

FIGURE 1: Schematic presentation for the synthesis of L-PEI and further processing to L-PEI-NONO'ate. The poly(2-ethyl-2-oxazoline) used was produced by Polymer Chemistry Innovations Inc. (AQUAZOL^® 500) and has an average molecular weight of 500.000 g/mol.

FIGURE 2. Diagram of the introducer sheath purchased from Thomas Medical Products, US.

FIGURE 3: Principal of the spray pattern analyzer. The spray plume is illuminated by a laser sheet and captured by a high speed camera.

FIGURE 4: Simple illustration of the concept: The release of nitric oxide is impacted by the water absorption, the pH and the diffusion rate of nitric oxide through the coating layers.

FIGURE 5: Diazeniumdiolates and their suggested the mechanism of release. FIGURE 6: Nitric oxide release dynamics using the primer/ NO adduct coating system/outer layer.

FIGURE 7: illustrates various coated medical devices according to the invention as described herein. OL refers to outer layer, 1^st refers to the first domain, 2^nd refers to the second domain, 3^rd refers to the third domain, BM refers to the base material of the medical device - typically the external surface prior to coating. All embodiments show may also comprise a primer layer on top of the base material. The pH of the 1^st domain is preferably alkaline in embodiments A, B, C, D, F, G, H, and I, and may also be alkaline in E. The pH of the second domain is acidic in all embodiments where it is shown. The pH of the 3^rd domain is typically about neutral. The pH of the outer-coat is typically around physiological pH. Diagrams F, G, H and I represent particles of the first and/or second domains, optionally coated with the third domain, which are embedded in either the first, second or third domains as illustrated.

FIGURE 8: A balloon coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI-NONO) as disclosed in US 6,737,447 embedded in polyurethane and provided with a top coat of pure polyurethane was immersed in the head space chamber. Nitric oxide release started essentially immediately upon immersion of the inflated balloon in the head space chamber and increased asymptotically to a level of approximately 1.5 nmol/min/cm² in approximately 50 minutes. Due to the high load of NO in the LPEI-NONO, the release of NO maintained essentially constant for the remaining measurement period (approximately 140 minutes).

FIGURE 9: A balloon coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI-NONO) as disclosed in US 6,737,447 embedded in polyurethane and supported by a coating layer of 90% polyacrylic acid and 10% polyurethane was immersed in the headspace chamber. Nitric oxide release started essentially immediately upon immersion of the inflated balloon in the head space chamber at a level above the threshould measurement maximum of the nitric oxide analysis apparatus (10 nmol/min/cm²). An asymptotic decrease to a level of approximately 1.2 nmol/min/cm² was observed until the measurement was interrupted after approximately 82 minutes.

FIGURE 10: A balloon coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI- NONO) as disclosed in US 6,737,447 embedded in polyurethane and supported by a coating layer of 80% polyacrylic acid and 20% polyurethane, and further comprising a top coat of polyurethane was immersed in the headspace chamber. Nitric oxide release started essentially immediately upon immersion of the inflated balloon in the head space chamber at a level above the threshold measurement maximum of the nitric oxide analysis apparatus (10 nmol/min/cm²). An asymptotic decrease to a level of approximately 0.4 nmol/min/cm² was observed until the measurement was interrupted after approximately 105 minutes.

FIGURE 11: A and B represent balloons coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI-NONO) as disclosed in US 6,737,447 and embedded in polyurethane without top coat. Formulation and processing is equal for both (A) and (B) except from the pH of the methanol used for dissolving the LPEI-NONO. The two balloons were dissolved in respectively alkaline methanol (A) and neutral methanol (B). The Balloons were immersed in the head space chamber. The peaks at approximately 15-17 minutes and at the end of the measurement, represent nitrite measurements (samples are indexed for comparison). 2005 1102 SGP E1-B2 #710 pH adjusted, 20051102 SGP El-Bl #719 not pH adjusted.

FIGURE 12: A and B represent nitric oxide release from a modified nylon (pebax™) tube coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI-NONO) as disclosed in US 6,737,447 and embedded in polyurethane. Formulation and processing is equal for both (A) and (B) except for the presence of a top coat in A and the absence of top coat in B. The arrows illustrate where the coated tube has been removed and re-entered the measuring chamber. The nitric oxide signal between the arrow is equal to the nitric oxide release from the LPEI-NONO leaked out of the coat (samples are indexed for comparison).

FIGURE 13: Graphical summary of the visual angiographic analysis.

EXAMPLES

EXAMPLE 1: Manufacturing of the nitric oxide donor (L-PEI-NONO'ate)

In this example, the nitric oxide eluting coating applied to the medical device is based on linear polyethyleneimine (L-PEI) with pendant NONO groups. Each NONO group is covalently attached to one of the N atoms in the L-PEI polymer chain thereby forming an N* - N⁺(O^")=N-O^" group in which N* is one of the atoms of the L-PEI backbone. This group is stabilized by the formation of a Zwitter-ion complex with an adjacent -NH₂ ⁺- amine group in the L-PEI backbone. the manufacturing of L-PEI-NONO'ate may be divided into the following basic steps:

1. Synthesis of L-PEI (synthesized from poly (2-ethyl-oxazoline - see Warakomski and

Thill, J. Polymer Science 1990;28:3551-63 for methodology)

2. Preparation of L-PEI powder.

3. Loading of L-PEI powder with Nitric Oxide. Loading occured a reaction chamber under Nitric Oxide pressure where the LPEI was suspended in acetonitrile. The Nitric Oxide forms covalent bonds with the nitrogen atoms on the L-PEI, thereby forming the L-

PEI-NONO'ate. This reaction was continued until the reaction reached equilibrium and no more NO was consumed (4-6 days depending on batch size). Subsequently, the L- PEI-NONO'ate was evacuated of any solvents and then grinded to a very fine powder.

The overall chemical reaction is illustrated in Figure 1.

Nitric oxide (NO) is a colorless and odorless lipophilic gas that with a aiπwater partition coefficient of 20: 1 and a maximum solubility of 1.7xlO^"3 M at 1 atm 25 °C, making it easily diffusible across membranes. NO has an unpaired electron and is thus characterized as a free radical, allowing it to react with other species with unpaired electrons such as O₂ ^" and other radical species. Additionally, NO possesses high capacity to ligate with hemeproteins such as hemoglobin.

The NO used in the manufacture of the NO Eluting Introducer Sheath contains at least 99% V/V of NO. The impurities for the NO include the following gasses: carbon dioxide, nitrogen, nitrogen dioxide, nitrous oxide and water.

The Nitric Oxide was purchased from Linde Gas. The purity is larger than 99% weight.

Impurities are specified as non specific NOX < 0.5% by weight (N₂O, N₂O₃, N₂O₄, NO₂, N₂O₅) and Nitrogen < 0.5% by weight (N₂). Poly(2-ethyl-oxazolιne) was boiled for 24 hours in sulphuric acid. Boiling in a sulfuric acid solution was needed to reach the desired > 90% level of hydrolysis of poly(2-ethyl 2- oxazohne). In the isolation procedure of the formed product, polyethyleneimine, the formed propionic acid was distilled off and the sulfuric acid was neutralized with sodium hydroxide and then recrystallised several times in water to remove salt impurities, i.e. sodium sulphate and propionic acid residuals.

The production of LPEI from Poly(2-ethyl-oxazolιne) eliminates the risk of residual ethyleneimine monomers.

The average molecular weight of the L-PEI-NONO'ate was found to be between 25 and 3OkDa, such as about 28kDa, with a relatively narrow distribution.

EXAMPLE 2: The Introducer Sheath

The introducer sheaths to be coated were purchased bulk from Thomas Medical Products, US. The (percutaneous) introducer sheath is used for intravascular introduction of interventional/diagnostic devices (See Figure 2).

Table 1: Materials used in the coating of the introducer sheath

Tradenames of polymers used: Tecoflex SG85A, Estane 58237, Tecophillic SP-93A.

EXAMPLE 3: 100 % Tecoflex in Pyridine (Primer layer)

Solution : 2 % solid, (w/w), Tecoflex in Pyridine (in total 80 g).

1. 2 g Tecoflex SG85A was dried for 4 hours at 75 ⁰C 2. After drying the vessel was sealed and left at room temperature.

3. 1.6 g dried Tecoflex SG 85A was mixed with 78.4 g Pyridine and incubated overnight at 75°C. "Dried" Pyridine, 34945 from Riedel-de Haen was used.

4. The solution was homogeneous prior to use.

EXAMPLE 4 : Tecoflex/ Estane 70/30 to LPN formulation in amount 70/30

1. 2 g Tecoflex and SG85A 1 g Estane 58237 were dried for 4 hours at 75 ⁰C

2. After drying the vessel was sealed and left at room temperature.

3. Preparation of mixture A: 0.48 g dried Tecoflex SG85A, 0.12 g dried Estane 58237 and 29.4 g dried Pyridine were mixed. The mixture was sealed and incubated at 75 °C overnight. The vessel was shaken vigorously several times during the incubation.

4. To prepare pH adjusted methanol, 50 mg NaOH was added to 30 ml water free methanol.

5. Preparation of mixture B: 0.3 g LPN and 14.7 g (18.6 ml) pH adjusted methanol were mixed in a vial. The mixture was shaken briefly. The vessel was sealed and protected against light, and incubated at room temperature with stirring for 1 hours. The mixture was filtered through a 0.45 μm filter into a new vial.

6. Preparation of mixture C: Mixtures A and B were mixed in ratio 7:3 (e.g. 7 ml + 3 ml) into a new vial and sealed in a light proof vessel. 7. Mixture C was used for down stream processing. The maximum time from final preparation of mixture C and until termination of the down stream processes was 9 hours. The solution was shaken just prior to down stream processing.

Example 5: 100 % Tecophillic SP-93A-100 in Pyridine

Solution: 2 % solid, (w/w) Tecophillic in Pyridine (in total 80 g).

1. 2 g Tecophillic SP-93A-100 was dried for 4 hours at 75 ⁰C

2. After drying the Tecophillic SP-93A-100 was sealed and stored at room temperature.

3. 1.6 g Tecophillic and 78.4 g Pyridine were mixed and incubated overnight at 75°C. The vessel was shaken vigorously several times during the incubation. 4. The solution was homogeneous prior to down stream applications.

EXAMPLE 6: Coating The coating was applied in a three step spray coating process. The first step was to apply a USP class VI tested primer onto the sheath. For spray coating onto the sheath, the primer is dissolved in pyridine.

The second step is to spray a mixture of polyurethanes and L-PEI-NONO dissolved in methanol and pyridine onto the introducer sheath.

The third step is to apply a top coat.

The three different spray coating mixtures are described in detail in below.

The spray coating is performed with conventional air spraying equipment designed for a small fluid flow and low air pressure. The spray coating process is monitored and controlled to ensure that the spray process delivers a smooth uniform layer, and that the layer thicknesses are not subject to deviations.

The most critical source of variation in the spray coating setup is variation in the spray plume. Variations can include particle size variations and variations in spray plume geometry, e.g. that the spray plume will not be concentrated on the center of the rotating sheath during the spray-coating process.

Variations in the spray coating process are typically due to spray fluid drying up on the spray nozzle orifice thus altering the airflow. One way to manage this problem is to optimize the design of the spray nozzle and thereby avoid spray residue build-up. In addition, formulation of the spray mixture is optimized to prevent drying of the spray solution on the spray nozzle.

The spray process may be examined and optimised using advanced laser technique and highspeed camera. The equipment enables measuring the angle and orientation of the spray plume and analyzing variations. Furthermore, the equipment enables analysis of drop sizes and the impact of different parameters such as air flow and distance to the device surface. The working principal of this spray coating analysis equipment is illustrated by FIGURE 3.

The described technique is similar to the method required to validate the spray plume geometry for oral and nasal sprays.

In order to ensure a specific target release resulting in the desired clinical impact (target release is in the range from 0.5 nmol/min/cm² - 40 nmol/min/cm²), the nitric oxide donoi was incorporated into a polymeric matrix (FIGURE 4). The coating consists of 3 layers: • Primer

To ensure and optimize coating adherence to the device.

• Nitric oxide donating layer

The nitric oxide donating layer is a mixture of polyurethanes and L-PEI-NONO (LPN) dissolved in pyridine and methanol onto the medical device.

The recipe for the nitric oxide donating layer includes pH adjusted solvent (methanol) to ensure that the LPN is stable during processing.

• Topcoat

The polymeric topcoat serves to ensure coating integrity, a barrier solubilisation of the LPN polymer, appropriate rate of water absorption and appropriate rate of NO diffusion.

When a topcoat is applied the release of nitric oxide deviates significantly from a l'order release. This is due to the barrier properties of the topcoat: limiting the water absorption and the diffusion of nitric oxide through the coating.

EXAMPLE 7: Layer thickness

When using confocal fluorescence microscopy the LPN layer has a relatively strong autofluorescence (blue/green). The other layers cannot be detected by this method, probably because they have no or very weak autofluorescence and because the top layer and the priming layers are too thin (less than 0.6-0.7 μm). By this method the thickness of the LPN layer, when dry, is determined to be in the magnitude of 4-5 μm, when applying 44 cycles of LPN (normal spray-coating parameters). When using only 22 cycles of LPN applied on the devices and when proportionality is assumed the LPN coating is in the magnitude of 2 μm.

The total coat is considered to be in the magnitude of 3 μm when using 22 cycles. However, coats of up to 40 μm may be appropriate and can be achieved by increasing the number of coating, such as spraying cycles.

When merged into an isotonic solution the coating swells as a result of water absorption. We have determined experimentally that the swelling increase the coating thickness by approximately 0.03 mm (30 μm).

EXAMPLE 8: Nitric oxide measurement

Nitric oxide measurements were carried out by using a chemiluminescence NO analyzer

(Sievers NO Analyser NOA 280i-2). The NO analyzer detects the total amount of NO(g) that passes the detector after a sample injection. The detection is based on the reaction : NO + O₃ + O₂

NO₂* -> NO₂ + hv

The emitted light (hv) is detected in a photomultiplier and is directly correlated to the amount Of NO.

The principle is that NO eluting samples are placed in acid wash bottle (named head space chamber) containing PBS buffer (pH 7.4) added 0.0004%o Tween 20 covering the sample, which is continuously flushed with N₂ gas which carries the released NO gas to the NO analyzer. Due to the continuous flow of N₂, The presence of oxygen is thereby avoided and so is the formation of nitrite (NO₂ ^"). This method ensures that all NO and only NO is measured.

EXAMPLE 9: PACKING & STERILIZATION

Products are then packaged and sealed in an aluminum pouch are appropriate for sterilization by electron beam irradiation.

Coated medical devices may be sterilized by electron beam sterilization through a certified sub-contractor, such as Steπgenics, Espergade, DK.

The validation and routine sterilization is performed in accordance with the requirements of EN 552 (Sterilization of medical devices - Validation and routine control of sterilization by irradiation) and ISO 11137 (Sterilization of health care products - Radiation) and the products are sterile in accordance with EN 556 (Sterilization of Medical Devices) (SAL 10^"6).

EXAMPLE 10:

The release of nitric oxide (NO) from medical devices according to the present invention is illustrated in the below examples with reference to Figs. 8 - 10 Coated balloons with various coatings were immersed in a head space chamber containing phosphate-buffered saline (PBS) maintained at pH = 7.4 and body temperature, i.e. 37°C. The head space chamber was continuously flushed with N₂ gas, which carried the released NO to a nitric oxide analysis apparatus. The nitric oxide analysis apparatus comprised a so-called high-sensitivity detector for measuring nitric oxide based on a gas-phase chemiluminescent reaction between nitric oxide and ozone:

NO + O₃ -> NO₂* + O₂ NO₂* -> NO₂ + hv As emission from electronically excited nitrogen dioxide is in the red and near-infrared region of the spectrum, it could be detected by a thermoelectrically cooled, red-sensitive photomultiplier tube. The analysis apparatus used was a Nitric Oxide Analyzer NOA™ 28Oi provided by Sievers^®, Boulder, Colorado, USA.

In Figs. .8 - 10 the irregular discontinuities in the NO output signal derives from a discontinuity in the flow of NO to the analysis apparatus.

Example IQa. (Fig. 8) A balloon coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI-NONO) as disclosed in US 6,737,447 embedded in polyurethane and provided with a top coat of pure polyurethane was immersed in the head space chamber. Nitric oxide release started essentially immediately upon immersion of the inflated balloon in the head space chamber and increased asymptotically to a level of approximately 1.5 nmol/min/cm² in approximately 50 minutes. Due to the high load of NO in the LPEI-NONO, the release of NO maintained essentially constant for the remaining measurement period (approximately 140 minutes).

Example IQb (Fig. 9): A balloon coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI-NONO) as disclosed in US 6,737,447 embedded in polyurethane and supported by a coating layer of 90% polyacrylic acid and 10% polyurethane was immersed in the headspace chamber. Nitric oxide release started essentially immediately upon immersion of the inflated balloon in the head space chamber at a level above the threshould measurement maximum of the nitric oxide analysis apparatus (10 nmol/min/cm²). An asymptotic decrease to a level of approximately 1.2 nmol/min/cm² was observed until the measurement was interrupted after approximately 82 minutes.

Example IQc, (Fig. 10): A balloon coated with a linear poly(ethylenimine) diazeniumdiolate (LPEI-NONO) as disclosed in US 6,737,447 embedded in polyurethane and supported by a coating layer of 80% polyacrylic acid and 20% polyurethane, and further comprising a top coat of polyurethane was immersed in the headspace chamber. Nitric oxide release started essentially immediately upon immersion of the inflated balloon in the head space chamber at a level above the threshould measurement maximum of the nitric oxide analysis apparatus (10 nmol/min/cm²). An asymptotic decrease to a level of approximately 0.4 nmol/min/cm² was observed until the measurement was interrupted after approximately 105 minutes.

EXAMPLE 11:

An animal study was performed in accordance with Good Laboratory Practices. The control device was a non-coated introducer sheath (without nitric oxide, Terumo, Radiofocus® Introducer II). In this study, a total of 22 animals were assigned into two groups; an acute group and a 7 days follow-up group. A total of 20 animals (10 in each group) were successfully included in the trial. The purpose of this porcine animal study was to evaluate the safety, performance and efficacy of the Introducer Sheath prepared according to the previous examples.

The primary endpoint of the study was the safety histological analysis that was performed on the femoral arteries of both the acute animals and the 7 day animals, determining potential negative cellular changes and changes in lumen diameter around where the sheath has been placed.

Efficacy was evaluated as any vasospasm-related changes in the vessel lumen diameter during placement and use of a NO-eluting sheath compared to a control sheath, through angiographic analysis.

Animal preparation

Twenty-two domestic pigs (weight 30-35 kg, fed on a standard natural diet without lipid or cholesterol supplementation throughout the study) were included in the study. For practical reasons the animals were not randomized into the two groups (acute and 1-week follow up) since the trial was executed over two consecutive weekends. Hence, all the animals for the 1- week follow up was included during the first weekend.

One day before the experiment, a loading dose of 250 mg Aspirin and 300 mg Clopidogrel per os were administered.

The pigs were fasted overnight and sedated with 12 mg/kg ketamin-hydrochloπd + 1 mg/kg xylazin and 0.04 mg/kg atropin. After premedication the anaesthesia were deepened with isofluran and oxygen by using a mask. When the pharyngeal reflex was eliminated the pigs were intubated intratracheally and the anaesthesia was maintained with 1.5-2.5 vol% isofluran, 1.6-1.8 vol% oxygen and 0.5 vol% N₂O. During anaesthesia the following parameters were measured: puls/min, breath/mm, SpO₂, and ECG.

Procedure

Access to the right or left femoral artery was performed through direct puncture of the artery under sterile conditions, and a 6F introducer sheath (either a test device or a control device) was inserted in the artery. Either a test device or a control device (depending on the first sheath inserted) was inserted in the contralateral artery using the same access procedure. Heart rate, arterial blood pressure and temperature were monitored. The procedure was not blinded for the physician since the test device and the control device have obvious differences in packaging and appearance.

After administration of 60 IU/kg of heparin sodium, a 6Fguιdιng catheter was advanced into the ascending aorta. Furthermore, heparin (400 I.U./h) was given as a slow continuous infusion during the procedure. A coronary angiography was performed using regular contrast agent, followed by a sham stenting procedure (balloon dilation of the artery with low pressure and short time not to expose the animals for an unexpected coronary event). After a coronary complication in the pig No 6, which induced the death of the animal, the sham stenting procedure was modified and the balloon was dilated inside the guiding catheter.

After the sham stenting, the guiding catheter was removed. Angiography of the femoral artery was repeated.

The sheaths were left in place in order to reach an inserted time of at least Ih in total. Angiography of the femoral artery was repeated.

Approximately 50% of the sheaths length was retracted from the artery and the angiographic procedure was repeated in order to document the vessel diameter at the site where the sheaths were previously located.

Following, the sheaths were pulled out completely.

In the survival animals, an attempt was made to close the puncture site with an Angio-Seal™ (St. Jude Medical) closure device. The 7 day animals were allowed to recover from the anaesthesia and received metamizol for pain relief. The acute animals were euthanized with 10 ml saturated potassium chloride.

Follow-up examinations

After 7 days, control angiography of the femoral arteries was performed through carotid artery access. After insertion of a 6F sheath into the surgically prepared right or left carotid artery, a guide wire (0.035") was navigated into the descending aorta. A 6F diagnostic right coronary catheter was pushed to the aorto-iliaca bifurcation. Angiography of the left and right sided arteria iliaca communis, and femoralis communis was performed. The right or left arteria femoralis communis was then selectively cannulated, and a selective arteriography of the arteria femoralis superficialis was also performed. Subsequently, the animals were euthanized with 10 ml saturated potassium chloride.

All baseline and follow-up procedures lasted approximately 60 to 90 minutes. The stress for the animals and the angiographic procedure were comparable to coronary interventions in humans.

Assessment of femoral angiography

Peri-procedure and follow-up quantitative angiographic parameters were measured by means of a computer-assisted quantitative coronary arteriographic edge detection algorithm (ACOMPC, Siemens, Germany). The minimal lumen, reference diameters and percent diameter of potential lumen narrowing were assessed. The measurements were done at least four times during the procedure: before sheath placement (baseline), after sheath placement, after sham stenting, after pull back of the sheath with a length of 50%, and after Ih (final).

Histopathology and histomorphometrv of the femoral arteries

An experienced observer, experienced in vascular pathology has analysed all slides, without knowledge of groups.

Occurrence of intravascular trauma induced by placement of the sheath in the vessel was recorded. Gross findings from the pathological investigation were reported if any arterial external alterations related to the sheath placement were observed. The explanted vessels were evaluated for outer diameter enlargement, lumen narrowing, filling defects, and medial thinning, if present.

The following sections of the femoral arteries were sectioned:

1. reference segment within 5 mm distally to the puncture site 2. sections per one centimetre where the sheath has been placed

3. reference segment within 5 mm proximally to where the distal end of the sheath was placed

Sections of each arterial segment were stained with both hematoxylin-eosin and Verhoeff-van Gieson-elastin to determine the location and extent of injury.

The quantitative analysis included: • lumen area (area of the vessel lumen, mm²)

• intimal or neointima area (area of the intimal or neointimal tissue, mm²)

• internal elastic lamina area (area of the internal elastic lamina, mm²)

• media area (area of the media, mm²) • external elastic lamina area (area of the elastic external lamina, mm²)

• adventitial area (area of the adventitia, mm²)

For each arterial segment, inflammation description (cell types and locations), haemorrhage and necrosis and vessel wall and luminal thrombosis were described.

RESULTS

Safety and performance

Available for angiographic evaluation :

Acute experiment: 10 NO sheath

9 control sheath

1-week survival: 9 NO-sheath

9 control sheath

Available for histological evaluation :

Acute experiment: 10 NO sheath

9 control sheath

1-week survival: 9 NO-sheath

10 control sheath

1. Weight gain at follow-up time (weight day 7 - weight day 0): 32.5±1.0 at day 7, and 32.0±1.2 kg at day 0. Animals in the non-survival group weighed 34±1.2 kg. No fever, weight loss or any general symptoms were observed. 2. General well-being up to, and at follow-up time (episodes of fever etc): There was 1 unexpected death (MIS-6) 2 h after closure of both femoral arteries, which was coronary artery related (ST-elevation after coronary procedure). No sheath-related events occurred 3. The Angio-Seal™ did not function in all cases. In the cases where it was necessary to close the vessel for the 1-week follow-up, surgical closure of the arteries had to be done (3 control sheaths and 2 NO-sheaths)

4. ECG changes (ECG was recorded before, during, and after the procedure and at the follow-up time in order to exclude any effects on the myocardium by NO). No sheath- related ECG-changes were observed

5. Unexpected death during the follow-up period: MIS-6 died as a consequence of the coronary procedure. Autopsy was performed, which confirmed, that the death was not related to the sheath placements

6. The baseline laboratory blood parameters were in porcine normal ranges. After angiography, PT was unchanged, but due to the application of the unfractionated heparin, the aPTT increased in both groups, without difference between the groups

7. Autopsy of the animals revealed that all of the animals (except MIS-6) were healthy, and no NO-related alterations were recorded.

Visual angiographic analysis Definitions:

Open, no spasm : open artery without compromisation of the arterial lumen Open, mild spasm : open artery with mild stenosis distal to the sheath placement Open, moderate spasm : open artery with appr. 50% lumen stenosis Open, severe spasm: open artery with TIMI flow 1 at the distal part Occluded: artery completely occluded distal to the puncture site

Table 3. Summarized results of the visual angiographic analysis

loccluded 10 11 0 4 0 3 0 3 I

Quantitative angiographic analysis

The overall results are summarized in the table below.

Table 4. Summarized quantitative angiographic results

Results are summarized by means and standard deviations. Differences between the groups were tested by using two-sided t-test for identification of statistical significance. The same types of statistics were used throughout the report.

Histological results

Histopatholoqical results

The following score system was used :

0 = no abnormality

1 = mild 2 = moderate

3 = severe changes (eg inflammation, necrosis, haemorrhagia)

The pathological changes were observed in most cases in the adventitia. In rare cases, if the changes were observed also in the intima or media, a comment was made.

The thrombotisation was scored as 0 = no thrombus formation, otherwise, in case of in vivo thrombosis, the proportion of the thrombus in relation to the lumen was given (in which degree was the lumen occupied by the thrombus).

Consecutive numbering of the histological samples: The most distal part of the artery contacted with the sheath (site of the puncture) received the number "1". The most proximal part (at the tip of the previously placed sheath) received the number "11". R means right side, L means left side. Distal or proximal references represent arterial segments 1 cm within distal (distal to the puncture site) or proximal (proximal to the tip of the previously placed sheath) to the previously placed sheath, respectively. The summarizing results are listed in table 5.

No difference was observed between the sheaths in the acute experiment. After 1 week, a trend towards less inflammation and thrombus formation was observed using NO-sheaths. No vessel wall necrosis was observed. The histopathological changes were focused on the adventitia, which might be partially related to the mechanical injury (puncture of the artery). Foreign body reactions with giant cells were recorded at the vessel closure sites.

Table 5. Summary of the histopathological results

Table 6. Summarized histomorphometry results. Abbr.: IEL: internal elastic lamina, EEL: external elastic lamina, %AS: percent area stenosis

Minimal incipient intimal proliferation starts during the 1st hour, while the sheath is placed in the artery. In the acute experiment, the incipient neointimal proliferation was less in the NO- sheath group. Consequently, the %area stenosis was less in the NO-sheath group. Note that the amount of the neoinitma and the measured %area stenosis is clinically negligible.

After 1 week, probably also due to the closure of the puncture site of the arteries, a small neointimal proliferation can be observed in both sheath-groups. The cellular proliferation was less in the NO-sheath group (probably due to less lumen thrombosis), resulting in a significantly larger lumen area and less %area stenosis.

The amount of neointima and a mildly higher %area stenosis in the control sheath group remains in the acceptable and clinically not relevant ranges. CONCLUSIONS

• Safety histological analysis: passed

• Blood pressure changes during the procedure: passed

• Episodes of fever: passed • ECG changes: passed

• Unexpected death : passed (one product unrelated event)

• Weight gain : passed

• Vasospasm-related changes in the vessel lumen diameter during placement and use of a NO-eluting sheath compared to a control sheath, through angiographic analysis: passed (significant changes (less vessel constriction) was observed during, and at the end of the procedure)

The insertion of the NO-eluting sheath proved to be safe and effective to prevent acute vasospasm during catheterization. Insertion of NO-eluting sheaths resulted in a trend towards less vessel wall and luminal thrombosis and significantly less cellular proliferation, initiated by the local vascular wall injury. Additionally, the mechanical properties of the NO- eluting sheath were similarly excellent as compared to that of the conventional sheath. The use of the NO-eluting sheath did not cause any systemic response related to the local NO- release from the sheath, and did not influence the systemic circulation and heart function.

In detail:

1. The NO-eluting sheath placement was safe; no sheath- or NO-elution-related complication was observed.

2. The performance parameters were excellent.

3. The baseline vessel size was similar in the 2 groups. 4. After sheath placement a trend towards a larger vessel diameter at the distal part was observed in NO-sheath group. 5. Angiography after coronary artery procedure revealed a significantly less vessel constriction expressed as %DS and a wider distal vessel diameter after placement of

NO-sheaths as compared with the control sheaths. 6. After 50% withdrawal of the sheaths (triggered spasm); NO-sheath placement led to a significantly higher vessel size both proximal and distal to the sheath placement.

7. Final angiography showed a significantly larger vessel diameter at the sheath placement and at the proximal and distal reference sites.

8. At 1-week FUP a trend towards a higher proximal vessel site was observed after NO- sheath placement. 9. The histopathological changes were focused on the adventitia, which might be partially related to the mechanical injury (puncture of the artery).

10. No histological difference was observed between the sheaths in the acute experiment.

11. After 1 week a trend towards less inflammation and thrombus formation was observed for the NO-sheaths.

12. No vessel wall necrosis was observed in neither the acute nor the 1-week survival experiment.

13. Foreign body reaction with giant cells was recorded at the vessel closure sites independently from the type of the inserted sheath. 14. Histomorphometry revealed that minimal incipient intimal proliferation starts during the 1 hour sheath placement period. In the acute experiment, the incipient neointimal proliferation was less in the NO-sheath group. Consequently, the %area stenosis was also less in the NO-sheath group. To be noted is that the amount of the neoinitma and the measured %area stenosis is clinically negligible. 15. After 1 week small neointimal proliferation can be observed in both sheath-groups, probably also due to the closure of the puncture site of the arteries. The cellular proliferation was less in the NO-sheath group.

16. In accordance with the less cellular proliferation, and mildly less lumen thrombosis, a significantly larger lumen area and less %area stenosis was measured in the NO- sheath group.

17. The amount of neointima and a mildly higher %area stenosis in the control sheath group remains in the acceptable and clinically still not relevant ranges.

18. No differences between the groups were observed as regards to the media thickness, neither in the acute nor in the 1-week groups.

REFERENCES

[1] Iganarro et al.

Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: Comparison with enzymatically formed nitric oxide from L-arginine Pharmacology, Vol. 90, pp. 8103-8107, September 1999

[2] L. K. Keefer et al.

Chemistry of the Diazeniumdiolates. 2. Kinetics and Mechanism of Dissociation to Nitric

Oxide in Aqueous Solution.

J. Am. Chem. Soc. 2001, 123, 5473-5481 [3] Keith M. Davies,*,t David A. Wink,Φ Joseph E. Saavedra,§ and Larry K. Keefer.

Chemistry of the Diazeniumdiolates. 2. Kinetics and Mechanism of Dissociation to Nitric Oxide in Aqueous Solution. J. Am. Chem. Soc. 2001, 123, 5473-5481

[4] Dennis K. Taylor, Ian Bytheway, Derek H . R. Barton, Craig A. Bayse, and Michael B.

Hall. Toward the Generation of NO in Biological Systems. Theoretical Studies of the N 2 0 2 Grouping. J. Org. Chem. 1996,60, 435-444

[5] Andrew S. Dutton, Jon M. Fukuto and K. N. Houk. The Mechanism of NO Formation from the Decomposition of Dialkylamino Diazeniumdiolates: Density Functional Theory and CBS-QB3 Predictions. Inorganic Chemistry, Vol. 43, No. 3, 2004

[6] Katrina M. Miranda, Herbert T. Nagasawa and John P Toscano. Donors of HNO. Current topics in Medicinal Chemistry 2005, 5, 649-664

Claims

1. A medical device for intravascular use, comprising a base material which is at least in part, coated with a coating system comprising a polymer mixture, wherein said polymer mixture comprises:

a. at least one reactive nitrogen species (RNS) adduct, wherein said adduct is capable of releasing said reactive nitrogen species under physiological conditions; and

b. a hydrophilic polymer or hydrophilic polymer blend,

wherein the reactive nitrogen species adduct and the hydrophillic polymer or polymer blend form a homogeneous phase.

2. The medical device according to claim 1, wherein the hydrophilic polymer or hydrophilic polymer blend is a hydrophilic support polymer or support polymer blend.

3. The medical device according to claims 1 or 2, wherein the reactive nitrogen species adduct is proton inducible.

4. The medical device according to any one of claims 1 - 3, wherein the at least one reactive nitrogen species is selected from the group consisting of nitric oxide (NO) and nitroxyl (HNO).

5. The medical device according to any one of claims 1 - 4, wherein the at least one reactive nitrogen species adduct is at least one nitric oxide adduct.

6. The medical device according to any one of claims 1 - 5, wherein the at least one reactive nitrogen species adduct is selected from the group consisting of: nitroglycerin, sodium nitroprusside, S-nitroso-proteins, S-nitrosothiols, long carbon- chain lipophilic S-nitrosothiols, nitrosocarbonyls, iron-nitrosyl compounds, thionitrates, thionitrites, sydnonimines, furoxans, organic nitrates, and nitrosated amino acids, nitroso-acetylcysteine, S-nitroso-captopril, S-nitroso-homocysteine, S- nitroso-cysteine, S-nitroso-glutathione, and S-nitrosopenicillamine, S-nitrosylated polysaccharides such as S-nitrosylated cyclodexrins, hydroxylamines, cyanamide, acyloxy nitroso compounds N-hydroxysulfenamides, diazeniumdiolates (NONOates) prepared from primary and -or secondary amines NONOate polymers.

7. The medical device according to claim 6, wherein at least one RNS adduct is a NONOate polymer.

8. The medical device according to claim 7 wherein the NONOate polymer backbone is selected from the group consisting of: polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene difluoride, polyvinylchloride, derivatized polyolefins such as polyalkylenimine, polyethylenimine, polyethers, polyesters, polyamides such as nylon, polyurethanes, biopolymers such as peptides, proteins, oligonucleotides, antibodies and nucleic acids, starburst dendrimers.

9. The medical device according to claim 8 wherein the polymer which comprise the

NONOate functional group (N₂O₂ ^") is polyethylenimine diazeniumdiolate, such as linear polyethylenimine diazeniumdiolate (LPEI-NONO).

10. The medical device according to any one of claims 5 to 9, wherein the RNS adduct has a molecule weight of between about 5kDa and about 20OkDa, such as between about 2OkDa and about 10OkDa, such as between about 5OkDa and about 6OkDa, such as about 55kDa.

11. The medical device according to any one of claims 5 to 10, wherein the mixture of said RNS adduct present in the coating system is between about 5% to about 80%, such as between 10% and 50%, preferably between about 20% and about 50%, such as about 30% (wt%).

12. The medical device according to any one of claims 1 - 11, wherein said polymer blend comprises a mixture of a support polymer and a hydrophilic polymer.

13. The medical device according to claim 12, wherein the hydrophilic polymer or polymer blend, when used in the homogeneous polymer blend, provides a maximum rate of increase of RNS release with a slope of about 0.01 and about 80 (nmol/min/cm²)/min when inserted into phosphate buffered saline at pH 7.4 at a temperature of 37°C.

14. The medical device according to claim 12 or 13, wherein the support polymer and/or polymer blend and/or coating system can withstand a traction force applied to the surface of the polymer coat of about 4 to 100 newton cm^"2 such as about 8 to 50 newton cm^"2 such as preferably between 10 to 20 Newton cm^"2.

15. The medical device according to anyone of claims 12 to 14, wherein the ratio of support polymer to hydrophilic polymer used in the in coating system is between about 95/5 to about 35/65 by weight.

16. The medical device according to any one of claims 1 - 15, wherein the thickness of the coating system is about 1 to about 100 μm, such as between 2 and about 20μm in dry state.

17. The medical device according to any one of claims 1 - 16 wherein the base material is coated with an inner priming layer prior to application of the coating system.

18. The medical device according to claim 17, wherein the inner priming layer is a polyurethane primer

19. The medical device according to claims 17 or 18, wherein the thickness of the priming layer is between about 0.2 and about 5μm.

20. The medical device according to any one of claims 1 to 19 which comprises at least one further layer which may be either internal or external to the coating system, wherein said further layer is capable of affecting the pH of the coating system by shifting the local balance between H⁺ and OH^" ions upon wetting of at least a portion of the medical device.

21. The medical device according to any one of claims 1 - 20 which comprises an outer layer situated externally to the nitric oxide adduct.

22. The medical device according to claim 21, wherein the outer layer controls or prevents the release of the reactive nitrogen species adduct from said inner layer into the physiological medium.

23. The medical device according to claim 22, wherein the reactive nitrogen species adduct is polyethylenimine diazeniumdiolate.

24. The medical device according to any one of claims 21 - 23, wherein the outer layer controls the release of the reactive nitrogen species, from said coating system (inner layer) into the physiological medium and/or the diffusion of water from the physiological medium into the inner layer under physiological conditions and/or the leakage of small molecule by products from said polymer mixture into the physiological medium.

25. The medical device according to any one of claims 21 - 24, wherein said outer layer comprises a hydrophilic polymer.

26. The medical device according to claim 25, wherein said hydrophilic polymer forms a hydroscopic gel upon insertion into physiological media.

27. The medical device according to any one of claims 21 - 26, wherein said outer layer comprised a hydrophilic polyurethane.

28. The medical device according to any one of claims 21 to 27, wherein the thickness of said outer layer is between about 0.3 and about 5 μm in dry state.

29. The medical device according to any one claims 1 - 28 wherein the release of the reactive nitrogen species, such as nitric oxide and/or nitroxyl from the outer surface of the medical device is between about 0.5 and about 80 nmol/min/cm2.

30. The medical device according to any one of claims 1 - 29, wherein the release of the reactive nitrogen species, such as nitric oxide and/ or nitroxyl, from the outer surface of the medical device has a half life under physiological conditions of at least 30 minutes, such as at least 1 hour.

31. The medical device according to any one of claims 1 - 30, wherein the release of the reactive oxygen species, such as nitric oxide, from the outer surface of the medical device has a half life under physiological conditions, of no greater than 6 hours, such as no greater than 4 hours, such as no greater than 3 hours.

32. The medical device according to any one of the claims 1 - 31 wherein the medical device is selected from the group consisting of: Neuro medical devices, such as neuro guiding catheter, neuro microcatheter, neuro microwire, neurostent delivery system, neuron ballon; coronary medical devices, such as coronary wires, coronary guiding catheter, PTCA angioplasty balloon; stent delivery system, coronary wires, coronary guiding catheter, PTA angioplasty balloon, stent delivery system; introducer sheath, dialator, guide wire, syringe needle; peripheral medical devices such as peripheral wires, guiding catheter, peripheral balloon; stent delivery system, peripheral wires, peripheral guiding catheter, stent delivery system; introducer sheath, dialator, guide wire, and syringe needle.

33. The medical device according to any one of the claims 1 - 32, wherein the base material consists or comprises of one or more of the compounds selected from the group consisting of: a metal, such as stainless steel, titanium, gold, plantinum; a plastic, such as PVC, PA, PS, Epoxy Resins, Silicone Rubber, Natural Rubber, Polyurethane, PE, PP, Polyester, Nylon, Polyester, PET, PMMA, Polysulphones,

Polyphosphazenes, Thermoplastic Elastomers Polydimethylsiloxane (PDMS)..

34. A medical device according to any one of claims 1 - 33 wherein the concentration of the reactive nitrogen species adduct, such as nitric oxide and -or nitroxyl adduct, such as polyethylenimine diazeniumdiolate, on the surface of the medical device is between about 0.05 and about 100mg/cm2, such as between about 0.1 and about 10 mg/cm2, such as preferably between about 0.2 and about 0.5 mg/cm2.

35. The medical device according to any one of claims 1 - 34, wherein the hydrophillic polymer and/or polymer blend and/or coating system can withstand a traction force applied to the surface of the polymer coat of about 4 to 100 newton cm-2 such as about 8 to 50 newton cm-2 such as preferably between 10 to 20 Newton cm-2.

36. A method for the manufacture of a coated medical device suitable for intravascular use, said method comprising :

b. applying a coating system comprising a polymer mixture to said external base layer, wherein said polymer mixture comprises at least one reactive nitrogen species adduct as according to any one of claims 1 - 35, and a hydrophilic polymer or polymer blend, as according to any one of the preceding claims, and wherein the reactive nitrogen adduct and the supporting polymer or polymer blend form a homogenous phase.

37. The method according to claim 36, wherein the coating system is applied by spray coating, dipping, painting or extrusion.

38. The method according to claim 36 or 37, wherein prior to applying the coating system, said base material is at least partially coated with a primer layer, such a primer according to any one of claims 17 to 19.

39. The method according to any one of claims 36-38, which comprises applying at least one further layer which may be applied either internal (prior to) or external (subsequent) to the coating system layer such as the coating system, wherein said further layer is capable of affecting the pH of the reactive nitrogen adduct or coating system by shifting the local balance between H⁺ and OH^" ions upon wetting of at least a portion of the medical device.

40. The method according to any one of claims 36 to 39, wherein subsequent to the application of the coating system and any further layers, at least part of the coating system surface is coated with an outer layer, such as the outer layer according to any one of claims 21 to 28.

41. A method of performing intravascular or neurovascular surgery comprising inserting the medical device according to any one of claims 1 to 35, or the medical device prepared by the methods according to any one of claims 36 to 40, into the vascular or neurovascular system of a patient.

42. The use of the coating system as defined in any one of claims 1 to 35 for the manufacture of a medical device for use intravascular use, for the prevention of one or more the conditions selected from: vasospasm or vasoconstriction, prevention of cerebral vasospasm, relaxation of smooth muscle, vasodilatation, thrombosis, decreased platelet deposition or aggregation, alleviation of restenosis, increased blood pressure, oxygen free radical reperfusion injury, treatment of cardiovascular disease, preventing the adverse effects associated with the use of said medical device, preventing abnormal cell proliferation.

43. Use of a coating system according to any of the preceding claims for the coating of a medical device for intravascular or neurovascular surgery.

44. Use of a coating system according to any of the preceding claims for the manufacture of a medical device for intravascular or neurovascular surgery for the control of vasospasm associated with the use of said medical device.