CA2493488A1 - A coating composition for an implantable medical device and method for coating such a device with naphthazarin and/or a naphtharazin derivative - Google Patents
A coating composition for an implantable medical device and method for coating such a device with naphthazarin and/or a naphtharazin derivative Download PDFInfo
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- CA2493488A1 CA2493488A1 CA002493488A CA2493488A CA2493488A1 CA 2493488 A1 CA2493488 A1 CA 2493488A1 CA 002493488 A CA002493488 A CA 002493488A CA 2493488 A CA2493488 A CA 2493488A CA 2493488 A1 CA2493488 A1 CA 2493488A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
- A61L33/0041—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate characterised by the choice of an antithrombatic agent other than heparin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/10—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/416—Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
Abstract
The invention relates to a coating composition for an implantable medical device, which contains at least one polymer and at least one biologically active substance, i.e. naphthazarin and/or a naphthazarin derivative, particularly Shikonin.
Description
Coating composition for an implantable medical device and method for coating such a device The present invention relates to a coating composition for an implantable medical device, where the coating includes at least one polymer and at least one bioactive agent.
Various coating compositions and methods for coating im-plantable medical devices are sufficiently well known in the art.
Coated implantable medical devices are used for example as skin, bone or cartilage substitute and are very important as prostheses especially in vascular surgery.
Prostheses of these types are implanted in a lumen of the body, for example in a blood vessel, of a patient in order to replace these vessels for the relevant fluids over a defined distance -for example in the case of a vessel prosthesis - or in order to widen them and keep them open by a so-called stmt. In such cases, the prostheses, which usually have a cylindrical shape, support the lining of the vessel and prevent the vessels from collapsing or their lining blocking the passage through the vessels. Materials conventionally used for prostheses are, for example, synthetic materials such as woven filaments of poly-ethylene terephthalate (PET) or of expanded polytetrafluoroeth-ylene (ePTFE), but various metals are also employed in addi-tion.
Tt is not necessary in every case of a vessel constriction or a vascular occlusion (stenosis) to perform an invasive surgical procedure. Even in cases of occlusion of the coronary vessels of the heart it is possible in many cases to avoid a heart operation with opened chest through a prosthesis which is in-troduced through vessels (intravascularly). For this purpose, for example, a catheter which has on its tip an inflatable balloon and an expandable mechanical support for the vessel (stmt) is introduced via a vein as far as into the vessel constriction. The vessel and stmt are simultaneously expanded through the inflation of the balloon; the flow of blood through the stenosis thus becomes possible again.
This rather forcible widening is associated with injury to the vessel. It is supported against collapse by the rigid stmt.
Even if the stent has a very open structure in the deployed state, there are many points of contact between the rigid sup-porting materials and the injured vessel wall. This gives rise to two consecutive problems:
Various coating compositions and methods for coating im-plantable medical devices are sufficiently well known in the art.
Coated implantable medical devices are used for example as skin, bone or cartilage substitute and are very important as prostheses especially in vascular surgery.
Prostheses of these types are implanted in a lumen of the body, for example in a blood vessel, of a patient in order to replace these vessels for the relevant fluids over a defined distance -for example in the case of a vessel prosthesis - or in order to widen them and keep them open by a so-called stmt. In such cases, the prostheses, which usually have a cylindrical shape, support the lining of the vessel and prevent the vessels from collapsing or their lining blocking the passage through the vessels. Materials conventionally used for prostheses are, for example, synthetic materials such as woven filaments of poly-ethylene terephthalate (PET) or of expanded polytetrafluoroeth-ylene (ePTFE), but various metals are also employed in addi-tion.
Tt is not necessary in every case of a vessel constriction or a vascular occlusion (stenosis) to perform an invasive surgical procedure. Even in cases of occlusion of the coronary vessels of the heart it is possible in many cases to avoid a heart operation with opened chest through a prosthesis which is in-troduced through vessels (intravascularly). For this purpose, for example, a catheter which has on its tip an inflatable balloon and an expandable mechanical support for the vessel (stmt) is introduced via a vein as far as into the vessel constriction. The vessel and stmt are simultaneously expanded through the inflation of the balloon; the flow of blood through the stenosis thus becomes possible again.
This rather forcible widening is associated with injury to the vessel. It is supported against collapse by the rigid stmt.
Even if the stent has a very open structure in the deployed state, there are many points of contact between the rigid sup-porting materials and the injured vessel wall. This gives rise to two consecutive problems:
All vessel injuries have thrombogenic effects. Via activation of the blood platelets by vessel material under the endothelium there is formation in the lumen of a thrombus which is intended to prevent escape of blood. Foreign surfaces such as metals or plastics from which stems are produced also have thrombogenic effects and may through the thrombus formation lead within a short time to renewed vascular occlusion (restenosis).
During healing of the vessel which has been damaged by the widening, there is moreover scar formation especially where a permanent stress is exerted on the regenerating tissue by points of pressure from the rigid stmt. Excessive prolifera-tion of scar tissue leads to restenosis with about 300 of all uncoated stems after a relatively long time.
Coating of the stmt, which is necessarily rigid as supporting material, is intended to prevent both risks of restenosis - the short-term risk of thrombi and the long-term proliferation of scar tissue.
These problems, namely the avoidance of coagulation and prolif-eration and the prevention of activation of blood platelets, are approached in different ways in the prior art. Such re-search aims for example are coatings of stem s which are in-tended to provide increased hemocompatibility. For example, anticoagulant, antimicrobial, antiinflammatory or antiprolif-erative agents, which are generally referred to as "bioactive"
substances, have been employed for a relatively long time in the coating of stem s. These substances are intended to be released from the coating material of the stent in such a way that they prevent inflammations of the surrounding tissue, excessive growth of smooth muscle cells or dotting of blood.
During healing of the vessel which has been damaged by the widening, there is moreover scar formation especially where a permanent stress is exerted on the regenerating tissue by points of pressure from the rigid stmt. Excessive prolifera-tion of scar tissue leads to restenosis with about 300 of all uncoated stems after a relatively long time.
Coating of the stmt, which is necessarily rigid as supporting material, is intended to prevent both risks of restenosis - the short-term risk of thrombi and the long-term proliferation of scar tissue.
These problems, namely the avoidance of coagulation and prolif-eration and the prevention of activation of blood platelets, are approached in different ways in the prior art. Such re-search aims for example are coatings of stem s which are in-tended to provide increased hemocompatibility. For example, anticoagulant, antimicrobial, antiinflammatory or antiprolif-erative agents, which are generally referred to as "bioactive"
substances, have been employed for a relatively long time in the coating of stem s. These substances are intended to be released from the coating material of the stent in such a way that they prevent inflammations of the surrounding tissue, excessive growth of smooth muscle cells or dotting of blood.
US 5,788,979 discloses a method for coating a biocompatible material which comes into contact with a patient's blood, the composition of the coating being intended to prevent coagula-tion of the blood by the biomaterial. In the method, initially a biodegradable material which is compatible with blood and tissue of the human body is prepared in a liquid state, and subsequently an anticoagulant composition is put into the liq-uid, biodegradable material. A liquid coating material which can be applied in a continuous manner to a biocompatible mate-rial, and subsequently dried, is produced in this way. Layer thicknesses of less than 100 ~m can be produced with this method and the materials used.
US 5,788,979 further discloses that the biodegradable material can be in particular a biodegradable, synthetic polymer such as, for example, polyglycolic acids, polylactic acids, polyhy-droxybutyrates, polyhydroxyvalerates, polydioxanones, modified starches, celluloses etc.
It is additionally proposed that, besides the anticoagulant composition, further substances may be present in the coating, such as, for example, antiinflammatory, antiproliferative, antibiotic substances. Examples thereof mentioned in the patent are dexamethasone, gentamycin and hirudin.
A disadvantage of said coatings is that on use of anticoagulant compositions the respective administration and dosage must be observed most carefully and be suited to the individual pa-tient, because these substances are always associated with a high risk of acute hemorrhages. In addition, after contact with recombinantly produced hirudin, many patients develop antibod-ies against hirudin-thrombin complexes, making the antithrom-botic effect of this substance uncontrollable. The use of glu-cocorticoids such as dexamethasone causes, especially over relatively long periods, the side effects on protein and carbo-hydrate metabolism which are normally observed with these hor-mones.
DE 195 21 642 discloses implants which consist at least partly of an absorbable material and which have in this absorbable material an antibiotic active substance, this active substance being released into the surroundings throughout the breakdown phase of the absorbable material. The antibiotic active sub-stance mentioned in this patent is in particular gentamycin.
A disadvantage of the use of gentamycin is that there are high individual variations in its therapeutic and toxic concentra-tion. Thus, side effects such as nephrotoxicity, neuronal blocks and, in particular, vestibular and cochlear impairments have been described in connection with gentamycin.
Further compounds which may be mentioned in this connection are the cytostatics Taxol (paclitaxel) and rapamycin and deriva-tives thereof. However, many side effects and complications on use as stmt coating are also known from the literature for these, e.g. the high thrombogenicity of Taxol (F. Liistro, A. Colombo, "Late acute thrombosis after paclitaxel eluting stmt implantation", Heart, 86: 262-264 (2001)).
On use of rapamycin as stmt coating (US 2001 027 340), current clinical studies have shown only a limited reduction of reste-nosis (Cordis SIRIUS study: 10% restenosis). Besides the high toxicity of these cytostatics, disadvantages are the chemical instability and the resulting difficulty of accurate dosage.
Thus, some patients in the abovementioned study have also shown symptoms of overdosage (thinning of the vessel wall).
A further risk for example with the coated stems known in the art is that the coating material is damaged during processes of adaptation of the stent to the relevant vessel - in the form of expansions or compressions. The coating and the release of the bioactive substances are thereby impaired in their respective function and effect.
There is further a great need to be able to control more simply and more accurately the delivery of the bioactive substance in a coating to the surroundings of the implant.
WO 01/64214 discloses the use of topoisomerase inhibitors such as, for example, camptothecin, anthracyclins and 1,4-naphthoquinones such as juglone, menadione and plumbagin for the treatment of inflammatory diseases.
WO 01/21326 discloses a method for producing a polymer with a support surface, where a coating composition which may include a cyclic diketone such as, for example, naphthoquinone is ap-plied.
Despite the various degradable coating materials frequently suggested in the prior art, and despite the large number of compositions having antibiotic, antimicrobial or anticoagulant activity which have been investigated in this connection, re-stenoses of vessels and rejection reactions of implants remain a great problem. Accordingly, despite all this there is a great need for devices which can keep, for example, vessels perma-nently free, and which provide for good bioresorption.
It is therefore an object of the present invention to provide a coating which includes a polymer with a novel biocompatible bioactive agent, while preferably - with an optimization of the mechanical properties of the coating - defined amounts of this bioactive substance can be incorporated and its release from the coating can be controlled in a simple manner.
According to the present invention, this object is achieved with the coating composition mentioned at the outset in that the bioactive agent is naphthazarin and/or a naphthazarin de-rivative.
The object on which the invention is based is completely achieved in this way.
The inventor has realized that it is possible through the use of polymers and of particular naphthazarin derivatives in a coating to produce an implantable medical device which has excellent mechanical properties, i.e. can withstand both com-pressions and expansions, and that the incorporation of naph-thazarin derivatives has no adverse effect on the mechanical properties, rather the substance is retained as bioactive agent.
The inventor has further realized that it is possible through the use of certain naphthazarin derivatives to check visually whether, and to what extent, the coating of implantable medical devices has suceeded. This is achieved through the coloring inherent in the naphthazarin derivatives: if a coating com-prises a naphthazarin derivative, it is possible to ascertain on the basis of the coloring whether a device is uncoated, coated or only partly coated.
Naphthazarin is known in the art as a basic structure in many natural pigments which additionally also represent medicinal agents. It is possible through the use of naphthazarin and/or naphthazarin derivatives to achieve two advantageous effects with one active substance. Firstly, these natural products are colored, so that the success of the coating can be checked visually, and secondly they simultaneously have a healing ef-fect, which makes the addition of further bioactive substances in the coating unnecessary.
Fig. lb shows the basic structure of naphthazarin. Naphthazarin in turn is regarded as a derivative of 1,4-naphthoquinone, whose basic structure is shown in fig. la.
Naphthazarin derivatives in the present case include all com-pounds which have the basic structure of naphthazarin, while the radical R for example can be any aliphatic radical which may be acyclic or cyclic, unbranched or branched, or in substi-tuted (for example hydroxy-substituted) form.
In a further embodiment, the derivative is selected from the group comprising shikonin, alkannin, arnebin and derivatives thereof. Particularly preferred in this connection is shikonin.
The inventor was able to show in his own experiments that pre-vention of blood platelet and fibroblast aggregation was possi-.. CA 02493488 2005-O1-20 ble with stems coated with the naphthazarin derivative shikonin.
The range of effects of shikonin is considerably broader by comparison with the compounds previously used in connection with coatings, such as, for example, rapamycin and Taxol.
Shikonin, alkannin and derivatives thereof have been known for some time as red natural pigments and as medicinal agents.
Thus, for example, Papageorgiou et al., "Chemie and Biologie von Alkannin, Shikonin and verwandten Naphthazarin-Naturstoffen", Angew. Chemie 111: 280-311, 1999, present in their review article the biological and pharmacological proper-ties which have been known for a relatively long time for these agents, and discuss bioorganic, preparative and medical as-pects: thus, the article cites other publications in which it was shown that shikonin itself has an antiinflammatory and antimicrobial effect. The article by Papageorgiou et al. addi-tionally cites a publication in which an antithrombotic effect of a few naphthazarin derivatives is described, but this effect could not be unambiguously ascribed to the shikonin family.
Shikonin has been proved to have an antitumor effect, and is antimycotic, antimicrobial and wound-healing. The cytostatics (for example rapamycin or Taxol) used to date do not achieve the unique profile of effects of shikonin.
Hisa et al., "Shikonin, an Ingredient of Lithospermum Eryth-rorhizon, Inhibits Angiogenesis in Vivo and in Vitro", Antican-cer Res. 18: 783-790, 1998, showed that shikonin was able to inhibit the proliferation of bovine endothelial cells, leading to inhibition of angiogenesis. By contrast, this article does not describe or explain whether, and how, shikonin or sub-,. CA 02493488 2005-O1-20 stances related to shikonin are able to prevent the prolifera-tion of fibroblasts, especially of myofibroblasts, whose pro-liferation is, as previously mentioned, involved in the reste-nosis of vessels.
The results shown by the inventor were not to be expected espe-cially taking account of the article by Sakaguchi et al., "Granulomatous Tissue Formation of Shikon and Shikonin by Air Pouch Method", Biol. Pharm. Bull 24(6): 650-655, 2001, in which it is explained that Shikonin stimulates neoangiogenesis. On the contrary, the known properties of shikonin have to date been a deterrent to the use of shikonin or other naphthoquinone or naphthazarin derivatives in connection with implantable medical devices. The inventor was the first to be able to show that shikonin is able to prevent the aggregation of blood platelets and thus both thromboses and long-term restenoses.
Naphthazarins have not previously been proposed for coating implantable medical devices.
In addition, naphthazarin derivatives are suitable for better examination of the kinetics of release in development. Other colorants, for example, without any biological activity, would merely be further foreign substances in the coating and would thus increase the risk of an allergic or inflammatory reaction.
Accordingly, the use of shikonin or of other naphthazarin de-rivatives provides a final visual check of whether, and how well, an implant has been coated.
It is not precluded in this connection that the coating compo-sition also includes a plurality of naphthazarin derivatives, or else further concomitant substances are also incorporated into the coating composition, such as, for example, anticoagu-lant, antimicrobial or antiinflammatory substances.
In a further preferred embodiment, the coating composition comprises naphthazarin and/or a naphthazarin derivative in a content of from 0.01 to to by weight.
It is further preferred for naphthazarin and/or naphthazarin derivative to be comprised in a content which is selected from the group comprising 0.040 by weight, 0.05% by weight, 0.06% by weight, 0.07% by weight, 0.080 by weight, 0.09% by weight, to by weight.
The inventor has been able to show in his own experiments that the use of from 0 . 1 to 1 o by weight of a naphthazarin deriva-tive, especially shikonin, in the coating is suitable for ex-erting an adequate inhibitory effect on blood platelets and fibroblast adhesion.
The polymer may in this connection be a biocompatible polymer out of which the bioactive substance diffuses, or an absorbable polymer out of which the active substance is released through degradation of the polymer.
In a further embodiment, it is preferred for the polymer to be an absorbable polyester and to be selected in particular from the group comprising polyglycolic acid, polylactic acid, poly-caprolactone, polyhydroxyalkanoates and copolyesters thereof.
Such absorbable polyesters and copolyesters are sufficiently well known in the art and have proved to be sufficiently useful in particular in medical uses.
It is particularly preferred in this connection for the polyhy-droxyalkanoate to be selected from the group comprising polyhy-droxybutyrate, polyhydroxyvalerate and copolyesters thereof.
The inventor has been able to show in his own experiments that, in particular, copolyesters of polyhydroxybutyrate and polyhy-droxyvalerate have outstanding properties as coating material.
These polyesters ensure that the coating is both biodegradable and has excellent mechanical properties. It further emerged from his own experiments that a naphthazarin derivative incor-porated into this copolyester showed excellent biological ac-tivities.
It is known that many organic biodegradable polymers are able to release active substances within a certain time window.
However, many materials are unsuitable as coating because they are not entirely biocompatible. Thus, for example, van der Giessen et al., "Marked Inflammatory Sequelae to Implantation of Biodegradable and Nonbiodegradable Polymers in Porcine Coro-nary Arteries", Circulation 94: 1690-1697, 1996, showed that polylactides and other polymers thought to be biocompatible led to inflammatory tissue reactions on degradation in the body.
It is additionally known that many biodegradable polymers do not have appropriate mechanical properties. Thus, for example, crystalline regions may lead to sudden fissuring. For these reasons, a coating for an irnplantable medical device must have particular mechanical properties, and withstand both compres-sion and expansion - for example an inflation of the catheter.
In a further embodiment, the copolyester comprises polyhydroxy-valerate in a content of from 20 to 30 o by weight, preferably of 25°s by weight, and polyhydroxybutyrate in a content of from 70 to 80o by weight, preferably of 75o by weight.
The inventor was able to show in his own experiments that this ratio is particularly suitable for use as coating material, because good solubility is ensured with this ratio and, at the same time, the excellent mechanical properties of the polyester are attained.
It is further preferred in a further embodiment for the absorb-able polymer and the shikonin to be preferably dissolved in at least one solvent, preferably dimethylacetamide and/or tetrahy-drofuran.
It could be shown that the mechanical and biological properties of the coating are not impaired by dissolving the active compo-nents in these solvents.
The invention further relates to a method for coating an im-plantable medical device comprising the steps:
{a) applying the novel coating composition to the implantable medical device, (b) drying of the implantable medical device and (c) where appropriate repetition of steps (a) and (b).
In one embodiment it is preferred in this connection for the coating composition to be sprayed onto the implantable medical device.
Various techniques can be used for the spraying, all of which are sufficiently well known in the art.
In another preferred embodiment, the coating is applied by immersing the implantable medical device into the coating.
The coating processes are moreover repeated until the desired layer thickness for the coating on the implantable medical device is reached, for example a layer thickness of from 1 to 100 Vim.
A preferred embodiment of the method of the invention for coat-ing an implantable medical device consists of applying a coat-ing which includes a polyhydroxybutyrate-polyhydroxyvalerate copolyester in which the poly-hydroxybutyrate . polyhydroxyvalerate ratio is 3 . 1 and in which shikonin is present in a content of from 0.01 to So by weight.
The inventor has realized that it is possible with this compo-sition to produce a particularly suitable coating with which, besides the optimal mechanical properties of a coating, it is also possible to utilize effectively the dual function of shikonin - as colorant and bioactive agent. The properties of shikonin - colorant and active agent - are retained even after incorporation into the coating according to the invention.
In a further preferred embodiment, a stmt is employed as de-vice in the method for coating an implantable medical device.
It is possible in this connection to employ for example stems which include at least one metal and/or one synthetic material.
Stems of these types, and methods for coating stents of these types, are sufficiently well known in the art.
However, it is not precluded that other types of implantable medical devices can be coated with the coating composition of the invention. Thus, for example, skin implants, a cartilage ar bone substitute are also suitable, which may be flat, rectangu-lar, cylindrical or configured as valve.
If a vessel prosthesis is used as implantable medical device, the tubular design thereof can be of any shape, that is to say for example as branched or unbranched tube etc.
The invention further relates to the use of a novel coating composition as indicated above for coating implantable medical devices.
The invention further relates to the use of naphthazarin and/or naphthazarin derivatives, especially of shikonin, for producing a coating composition for an implantable medical device and to the use for coating an implantable medical device.
The invention further relates to an implantable medical device which is coated with a novel coating composition as mentioned above, and especially stents.
Further advantages are evident from the description hereinaf-ter.
It will be understood that the features mentioned above and to be explained below can be used not only in the combinations indicated in each case, but also in other combinations or on their own, without leaving the scope of the present invention.
The invention is explained in more detail below by means of use and implementation examples and by the figures. In these Fig. la shows the formula of the substance 1,4-naphtho-quinone; and Fig. lb shows the general formula of the basic structure of naphthazarin derivatives.
Example 1. Coating compositions used The following polymer compositions were tested as coating mate-rial:
- a composite coating of the non-absorbable polyurethane Elastollan, with and without shikonin;
- a biodegradable copolyester of poly((3-hydroxybutyrate-co-(3-hydroxyvalerate) (P(HB-HV) hereinafter) with a content of 12o by weight polyhydroxyvalerate;
- a biodegradable copolyester of P(HB-HV) with a content of 25o by weight polyhydroxyvalerate, with and without shikonin.
The solvents tested with dimethylacetamide (DMAA hereinafter) and tetrahydrofuran (THF hereinafter).
It emerged from this that a composition of 25 o by weight poly-hydroxyvalerate (PHV hereinafter) in relation to 75o polyhy-droxybutyrate (PHB hereinafter) is most suitable. In addition, a composition with this ratio in both solvents with a maximum concentration of up to 1.5o by weight could be dissolved in both solvents by heating.
In this connection, a working solution (I) comprising P(HB-HV) with 25% by weight PHV with the following composition was used for the coating composition:
- P(HB-HV): 1.5 g - DMAA or THF: 100 ml Besides this pure copolyester, coating with another composi-tion, namely P (HB-HV) with a content of 25% by weight PHV, and with shikonin, was tested. The working solution (II) used for this had the following composition:
- P(HB-HV): 1.5 g - shikonin: from 15 to 75 mg - DMMA or THF: 100 ml.
The tests were carried out using firstly stents (made of stain-less steel with electropolished surface, Translumina GmbH, Hechingen, Germany) coated with working solutions I and II, and secondly polymer films from indicated working solutions I and II cast in Petri dishes.
2. Cell adhesion and cell proliferation studies a) Elastollan films Materials and methods Elastollan films containing 0.1 and 0.5o by weight shikonin, and Elastollan films without shikonin, were tested.
Although Elastollan is a non-absorbable polymer, nevertheless the results obtained with this polymer show the activity of shikonin as inhibitor of cell adhesion and proliferation. This means that in this case the active agent is released by leach-ing and not by degradation of the Elastollan matrix.
Human embryonic fibroblasts were employed in these experiments.
The cells were cultivated in Eagle's medium with the addition of 10% fetal calf serum and passaged twice a week using a tryp-sin-EDTA solution. The cells present after the 14th passage were used for the tests. Mitomycin C dissolved in culture me-dium in a concentration of 20 ~g/ml was used as negative con-trol.
Extracts from Elastollan films without shikonin, Elastollan films with 0.1% by weight, 0.5% by weight, 1% by weight and 5%
by weight shikonin (based on polyurethane), and shikonin alone, were tested.
Extracts were obtained by incubating dishes covered by the polymers to be tested (i.e. Elastollan films without or with shikonin) with Eagle's medium at 37°C for 3 h. Undissolved solids were removed by filtering. The control with shikonin only (in the culture medium) was cultivated under the same conditions.
The respective filtrates were employed as test extracts, with the pH of the shikonin containing Elastollan extracts having been adjusted previously with 0.1 N HCl, and with the same amount of phosphate buffer as was necessary for adjusting the pH of the Elastollan/shikonin extracts having been added to the control extract (Elastollan without shikonin) and to the ex-tract with shikonin alone.
In parallel, 1 ml portions of the cell suspension (fibroblasts, see above) with 4 x 104 cells/ml were applied to 24-well plates.
After a cultivation time of 24 hours, the medium was removed and the extracts to be tested were added (positive control:
fresh medium without extracts; negative controls; addition of mitomycin C for 2 h).
The cells were then washed with Hank's solution, and fresh medium was put into the wells. The cells were then incubated for 72 h. After this incubation time, the cultures were washed twice with phosphate buffer and incubated with 2.5o glutaralde-hyde at 4 °C for 30 min. They were then washed again twice and stained with Giemsa at 37°C in humid atmosphere for 3 h.
The stain retained by the cells was eluted with a phosphate buffer/alcohol mixture (1:1) at room temperature for 15 min.
The optical density of the resulting solutions was determined by a spectrophotometer with a wavelength of 620 nm.
n.-,......~ a-..
In the positive control (tested extract: only culture medium), the fibroblasts covered almost the entire surface of the well and showed an elongate shape and a typical growth pattern.
In the negative control (tested extract: culture medium + mito-mycin C), no cell growth was observed. The cell density was low and corresponded to that on the second day of the experiment.
The cells had an elongate shape but were not in contact with one another. Some cells were rounded or were lyzed.
Cell growth on extract from Elastollan alone was similar to that in the positive control. On use of shikonin-containing Elastollan extracts it was possible to observe an inhibitory and even cytotoxic effect: a few cells were adherent, most were rounded.
This test showed that shikonin and extracts from shikonin-containing Elastollan films have an inhibitory effect on human cells in vitro.
b) Tn vitro adhesion of human embryonic lung fibroblasts to Elastollan- and shikonin-containing Elastollan films Petri dishes coated with films of Elastollan- and shikonin-containing Elastollan-DMAA solutions were used for this test.
The concentration of shikonin in the polyurethane samples was 0.01% by weight, 0.05a by weight and 0.5% by weight (based on polyurethane). A plastic Petri dish served as positive control.
The cells were seeded on the surface of the Petri dishes coated with the polymer compositions in a concentration of 4 x 104 cells/ml in Eagle's medium with 10o fetal calf serum.
The number of adherent fibroblasts was counted after incubation at 37°C for 0.5 and 2 h.
Results The counts obtained are shown in table I below.
As is evident from the table through comparison with the posi-tive control, a concentration of 0.5o by weight shikonin in the Elastollan films inhibits the adhesion of fibroblasts virtually completely:
Type of surface Number of counted fibroblasts (after an incubation time of) 0.5 h 2 h Control (plastic Petri ca. 60 ca. 95 dish) Elastollan 10 - 40 none Elastollan + shikonin < 20 none (O.Olo by weight) Elastollan + shikonin ca. 30 none (0.050 by weight) Elastollan + shikonin 0 - 5 none (0.5s by weight) c) Adhesion of blood platelets onto copolyester P(HB-HV) films and onto shikonin-containing copolyester P(HB-HV) films !PlHB-HV)-Sh) and onto stents coated with these films It is known that the adhesion of blood platelets to biomateri-als reflects the compatibility of medical devices with blood in relation to blood and tissue cell activation.
It is presumed that adhesion proceeds in a plurality of steps:
initially the blood platelets bind to the surface, are then activated and develop pseudopods, and then they spread out and form aggregates. The subsequent release of intracellular compo-nents, including blood clotting factors, stimulates adhesion and aggregation of further blood platelets.
Besides, infiltration of actively proliferating myocytes from the media into the intima, accompanied by the production of abundant extracellular matrix components (collagen, proteo-aminoglycans), is presumed to be the fundamental mechanism of restenosis. Immediate deposition of blood platelets at the point where the vessel was injured, and the subsequent release of myoproliferative substances (for example the growth factor (PDGF), ~-transforming factor (~-TGF), endothelium-derived growth factor (EDGF), etc.} probably represent the stimulating factors.
Thus, adhesion of blood platelets during stmt implantation plays a key role both in relation to thromboses and to resteno-ses.
The activation status of adherent blood platelets can be esti-mated from their morphology. A larger effect of the material on blood platelets results in more adherent cells being distrib-uted or aggregated on the material.
Both uncoated stents and stems coated with P(HB-HV)-Sh were tested for this test. The average layer thickness of the coat-ing was approximately 15 to 20 Vim.
The following solutions were used for coating the stents:
- 0.75% P(HB-HV) with 25% HV in DMAA or THF;
0.75% P(HB-HV) with 25% HV + 0.5% by weight shikonin (based on the weight of P(HB-HV)) in DMAA or THF;
- 0.75% P(HB-HV) with 25% HV + to by weight shikonin (based on the weight of P(HB-HV}) in DMAA or THF;
The stem s were coated by several times being immersed into the various solutions or sprayed. The drying time between the indi-vidual spraying steps was between 2 and 15 min. Finally, the stents were dried at 45 to 50°C for 30 min. The total amount of shikonin on the sprayed P(HB-HV)-l.OSh stems was 2 ~g to 4 fig.
The shikonin coating of the stems proved to be stable in the expanded state over a period of 28 days.
In addition, polymer films from the indicated solutions (P(HB-HV) with or without shikonin) were tested on stainless steel plates from the same solutions with a layer thickness of 20 to 30 ~,m. The surface of an uncoated stmt served as con-trol.
Preparation of the blood Whole blood from healthy adult donors was put into siliconized glass vessels. Blood clotting of 10 ml of blood was prevented by adding sodium citrate in a ratio of (blood: sodium citrate) 9:1. Blood platelet-enriched plasma was obtained by centrifug-ing the whole blood at 100 x g for 20 minutes at room tempera-ture. The blood platelet-enriched plasma fraction was removed with a plastic pipette and immediately employed in the experi-ments.
50 ~l drops of this blood platelet-enriched plasma fraction were put on the surfaces of the plate samples and incubated for 15 min. To test the coated stem s, they were put in a vessel with blood platelet-enriched plasma and incubated for 30 minutes. The number of blood platelets which adhere to the surface during this period was sufficient for a qualitative analysis, because the platelets formed no large thrombus-like structures. The samples were washed with physiological saline solution in order to wash off unadsorbed plasma proteins and weakly adhering blood platelets. The samples were then fixed in 2.5o glutaraldehyde, and subsequently dehydrated by standard techniques with an increasing ethanol content.
Adhesion of the blood platelets was investigated by scanning electron microscopy (SEM) (JSM T330, JEOL, Japan). All the samples were made conductive by coating of copper with 1.2 kV, 10 mA for 7 minutes (JEOL JFC-1100, Japan). The microscopic investigations were carried out with a voltage of 5 kV. 25 sections each 400 ~m2 in size were selected at random for each sample. The qualitative total blood platelet number Ntot and the number of blood platelets NI of the following two categories of cells which explicitly reflect the activation of the blood platelets were then evaluated. Each category comprises two morphological classes of adherent blood platelets:
Ia) single, non-activated or only slightly activated deformed cells;
Ib) pseudopod-forming cells or cells in an early stage of spreading.
IIa) fully spread blood platelets;
IIb) aggregates (of two or more blood platelets).
With reference to this classification, blood platelets from category I interact only weakly with the surface; in contrast thereto, a strong interaction takes place between the surface and the blood platelets of category II.
Results a) Coating with a 0.75% by weight solution of P(HB-HV) and P(HB-HV)+shikonin in DM.AA
The number of cells on the uncoated stems proved to be very low, and where adherent blood platelets were present virtually all the cells were completely spread on the uncoated stmt. The total number on the P(HB-HV) film was higher than that for the control and all morphological classes of the cells were present on this surface, but the number of aggregates proved to be very low.
Shikonin by contrast was able distinctly to reduce the amount of adherent blood platelets compared with films coated only with P (HB-HV) . In this case, only two morphological classes of cells of category I were observable: slightly activated blood platelets and pseudopods-forming cells.
It is evident on the basis of these results that P(HB-HV)-Sh films are more suitable than surfaces with pure P(HB-HV) be-cause the shikonin-containing surfaces showed a lower affinity for cell binding.
In order to determine the connection between blood platelet activation and shikonin concentration, the blood platelet adhe-sion to films of pure P(HB-HV), of P(HB-HV) with O.lo by weight shikonin (P(HB-HV)-O.lSh) and of P(HB-HV) with 0.5o by weight shikonin (P(HB-HV)-0.5Sh) was determined for an incubation time of 15 minutes and 30 minutes. It was possible to demonstrate thereby that addition of shikonin was able to reduce the number and the degree of activation of the blood platelets.
a) Coating with a 0.750 by weight solution of P(HB-HV) and P(HB-HV)+shikonin in THF
The coatings were applied as multilayer films to expanded and unexpanded stems. The stents were coated with the polymer by immersing it in the diluted working polymer solution (about 2 to 4 times). Each layer was then dried with hot air.
A comparative analysis of the adhesion of blood platelets to the uncoated stmt, to a stmt coated with diamond-like carbon and to a stmt coated with P(HB-HV)-0.5Sh was carried out. The incubation time of the samples in plasma enriched with blood platelets was from 15 to about 30 minutes.
No blood platelets were observed on the stems coated with P(HB-HV)-0.5Sh. However, in the same time it was possible to find blood platelets on the uncoated and on the carbon-coated stems. This means that the properties of shikonin in relation to the activation of blood platelets are also retained on use of THF as solvent.
c) Coating with 1% by weight shikonin (P(HB-HV)-l.OSh) com-pared with stems with a diamond-like coating (DLC) In the comparative analysis of the stem s coated with to by weight shikonin with DLC-coated stems it emerged that virtu-ally no platelets adhered to the coated stems in the case of the stem s coated with P(HB-HV)-l.OSh in vitro. On the other hand, a few spread-out platelets were to be found on DLC-coated stents.
3. Determination of the biolorqical properties of the films produced These investigations were carried out in accordance with the international standard for biological investigations on medical devices ISO 10993 and in accordance with the national standard GOST R ISO 10993.
a) Determination of the hemolytic properties The hemolysis was determined by preparing extracts of the mate-rials, in particular of P(HB-HV) and of P(HB-HV)-0.5Sh, in each case in physiological saline solution. Whole blood was added to these extracts, followed by incubation at 37°C for one hour.
The extracts were removed, the mixture was centrifuged (50 minutes at 400 x g) and the cell-free supernatant was care-fully removed. The hemoglobin concentration for this super-natant was determined by photometry, and the hemolytic index (%) was calculated from the ratio of liberated hemoglobin to hemoglobin present. A pure saline solution was used as control in this case.
Under the given conditions, both extracts, that is to say the extract of P (HB-HV) and that of P (HB-HV) -Sh, proved to be non-hemolytic (hemolytic index: 0%).
b) Determination of the complement system activation The compatibility of the polymer films with blood was tested by determining the hemolytic activity of the complement system of human blood plasma before and after its incubation with a film sample or extract. The tested samples were films of P(HB-HV), P(HB-HV)-Sh and of cuprophane (control).
The concentration of the hemoglobin liberated by the lytic effect of the complement system from sheep erythrocytes coated with antibodies reflects the complement activity in the serum sample. The parameter for the assay was the time required for 100 ~1 of the tested serum to lyze (i1~2) 50 0 of 5 ml of sensi-tized sheep erythrocytes (5 x 108 cells/ml). The temperature of the reaction mixture was kept constant at 37°C by means of thermostats.
Human serum was first diluted with a buffer (comprising 0.15 mM
CaCl2 and 0.5 mM MgCl2) and then incubated with or without polymer samples at 37°C for 60 min. The values for the remain-ing complement activity ((CH50)R%) were determined as criterion of the activating properties of the polymers in the following way:
(CH50) Ri ' (Z1/2) 0 / (Z1~2) (t) i(c1 1OO o where:
(ii/z) o = time for 50% lysis of sheep erythrocytes by the serum complement before incubation with the sample;
(tl/2) (t) i = time for 50% lysis of sheep erythrocytes by the serum complement after incubation time t with the polymer sam-ple;
(il/2) (t) c~~ - time for 50% lysis of sheep erythrocytes by the serum complement after incubation time t in the control (with-out polymer sample).
The results of these tests are summari2ed in table II below:
Tested samples CHS~ (n = 3) Cuprophane (control material) 89 5 P(HB-HV) extract 85 5 / 80 5 P(HB-HV)-Sh extract 90 5 / 85 5 Serum after 1 h incubation (control serum) As is evident from the table, the complement activity of serum incubated with P(HB-HV)-Sh extract proved to be lower than that of serum incubated with pure P(HB-HV) extract. In addition, it showed approximately the same CHso as cuprophane.
c) ATP release tests In a further comparative analysis, DLC-coated stems were in-vestigated with P(HB-HV)-l.OSh-coated stems. Fresh human platelet-rich plasma (PRP) was employed for this purpose. The stems were incubated with 450 ~,1 of PRP at 37°C for 5 min. The platelet aggregation and the secretion ability was measured by means of bioluminescence using the highly sensitive LUMI-AGREGOMETER (CHRONO-LOG Corp,, USA).
It was shown that the shikonin-coated stents did not change the functional status of the platelets (in relation to ATP re-lease), whereas the DLC-coated stents inhibited ATP release.
d) Intramuscular implantation test 1. Films of P(HB-HV) only and of P(HB-HV)-Sh were implanted in accordance with ISO 10993 Part 6 into the muscles of guinea pigs. The size of the film samples was 0.5 x 1 cm with a thickness of 20 to 30 Vim. For each type of sample, two guinea pigs with the implant were observed for 7 days.
It was shown after 7 days that fibroblast monolayers formed around the P(HB-HV) and P(HB-HV)-Sh films. Infil-tration of macrophages and lymphocytes around these im-plants proved to be a typical tissue response after im-plantation for 7 days. It was nevertheless possible to show that the degree of the inflammatory response to the biodegradable polymer with shikonin was less than with the pure P(HB-HV) film.
2. Adult male Wistar rats (230 - 250 g) were divided into two groups: animals of group A (n=6) were implanted with P(HB-HV)-l.OSh-coated stems and those of group B (n=4) with stems with a diamond-like coating (DLC stems) as control. The stems remained subcutaneously implanted in the backs of the rats over a period of 2 and 6 weeks for subsequent macroscopic and microscopic determination of subchronic (short-term) effects (for each of the individ-ual period n=3 for group A and n=2 for group B). The ani-mals were anesthetized with sodium thiopental and, after completion of the experiments, euthanized with an overdose of this substance.
For the histological investigations, the implants with surrounding tissue were fixed in 12s formaldehyde, embed-ded in gelatin and divided into sections with a diamond blade.
Histological and macroscopic investigations showed that no chronic inflammatory foreign-body reactions could be ob-served in both groups, but with the DLC stems a consider-able number of macrophages and some carbon particles were detected in the surrounding tissue. The fibrous capsule which formed around the stems was significantly thinner with the P(HB-HV)-l.OSh-coated stems than with the DLC
stems .
Overall, the stability and biocompatibility of the P(HB-HV)-l.OSh-coated stents was higher than that of the DLC stems .
3. In further experiments, the stents were implanted into the aorta of cats. The experimental animals were for this pur-pose divided into two groups: group A (n=2) received P(HB-HV)-l.OSh-coated stems, group B (n=2) received DLC
stems. Adult female cats (2.5 to 3.5 kg) were anesthe-tized intramuscularly with a 5 percent ketamine solution (0.65 mg/kg) and a 0.25 percent droperidol solution (0.1 mg/kg) . The stems were implanted into the abdominal part of the aorta. No additional anticoagulants were ad-ministered.
18 to 24 days after the implantation, the animals were anesthetized, and the aorta segments were removed and fixed in 10% formalin and dehydrated. The stmt-containing segments were then embedded in methyl methacrylate and di-vided into sections using a diamond blade.
No acute thrombotic occlusion or growth of fibrous connec-tive tissue into the lumen was observed in both test groups. The histological investigations revealed that for the DLC stems some carbon particles and a relatively large number of macrophages were observed in the aorta wall surrounding the DLC stems. 24 days after implanta-tion, neointima formation was observed with the P(HB-HV)-l.OSh-coated stems. It had a more regular arrangement than with the DLC stems. No indications of acute or chronic inflammation were observed.
Summary With the results it could be shown that firstly the composition of 25% by weight polyhydroxyvalerate and 75% by weight polyhy-droxybutyrate proved to be an optimal composition for the coat-ing, and that this mixture together with a particular shikonin content showed high adhesion to metallic surfaces. In addition, it could be shown that the properties of the polymer coating were not affected by repeated expansion of the coated stmt.
Further, it cold be shown that a concentration of shikonin of up to 5% by weight inhibited virtually completely the adhesion of human blood platelets to the polymer surface in vitro. Thus, shikonin can be used in suitable concentrations for preventing excessive cell proliferation.
A comparative study on the biological properties of a film of pure P(HB-HV) or of P(HB-HV)-Sh in THF in vitro showed that shikonin had good compatible properties in relation to comple-ment activation and blood platelet adhesion.
With the results it could further be shown that a smooth, thin, fibrocellular layer is formed around the stmt through a coat-ing with P(HB-HV)-Sh, leading to reendothelialization with an adequate diameter for normal blood flow.
US 5,788,979 further discloses that the biodegradable material can be in particular a biodegradable, synthetic polymer such as, for example, polyglycolic acids, polylactic acids, polyhy-droxybutyrates, polyhydroxyvalerates, polydioxanones, modified starches, celluloses etc.
It is additionally proposed that, besides the anticoagulant composition, further substances may be present in the coating, such as, for example, antiinflammatory, antiproliferative, antibiotic substances. Examples thereof mentioned in the patent are dexamethasone, gentamycin and hirudin.
A disadvantage of said coatings is that on use of anticoagulant compositions the respective administration and dosage must be observed most carefully and be suited to the individual pa-tient, because these substances are always associated with a high risk of acute hemorrhages. In addition, after contact with recombinantly produced hirudin, many patients develop antibod-ies against hirudin-thrombin complexes, making the antithrom-botic effect of this substance uncontrollable. The use of glu-cocorticoids such as dexamethasone causes, especially over relatively long periods, the side effects on protein and carbo-hydrate metabolism which are normally observed with these hor-mones.
DE 195 21 642 discloses implants which consist at least partly of an absorbable material and which have in this absorbable material an antibiotic active substance, this active substance being released into the surroundings throughout the breakdown phase of the absorbable material. The antibiotic active sub-stance mentioned in this patent is in particular gentamycin.
A disadvantage of the use of gentamycin is that there are high individual variations in its therapeutic and toxic concentra-tion. Thus, side effects such as nephrotoxicity, neuronal blocks and, in particular, vestibular and cochlear impairments have been described in connection with gentamycin.
Further compounds which may be mentioned in this connection are the cytostatics Taxol (paclitaxel) and rapamycin and deriva-tives thereof. However, many side effects and complications on use as stmt coating are also known from the literature for these, e.g. the high thrombogenicity of Taxol (F. Liistro, A. Colombo, "Late acute thrombosis after paclitaxel eluting stmt implantation", Heart, 86: 262-264 (2001)).
On use of rapamycin as stmt coating (US 2001 027 340), current clinical studies have shown only a limited reduction of reste-nosis (Cordis SIRIUS study: 10% restenosis). Besides the high toxicity of these cytostatics, disadvantages are the chemical instability and the resulting difficulty of accurate dosage.
Thus, some patients in the abovementioned study have also shown symptoms of overdosage (thinning of the vessel wall).
A further risk for example with the coated stems known in the art is that the coating material is damaged during processes of adaptation of the stent to the relevant vessel - in the form of expansions or compressions. The coating and the release of the bioactive substances are thereby impaired in their respective function and effect.
There is further a great need to be able to control more simply and more accurately the delivery of the bioactive substance in a coating to the surroundings of the implant.
WO 01/64214 discloses the use of topoisomerase inhibitors such as, for example, camptothecin, anthracyclins and 1,4-naphthoquinones such as juglone, menadione and plumbagin for the treatment of inflammatory diseases.
WO 01/21326 discloses a method for producing a polymer with a support surface, where a coating composition which may include a cyclic diketone such as, for example, naphthoquinone is ap-plied.
Despite the various degradable coating materials frequently suggested in the prior art, and despite the large number of compositions having antibiotic, antimicrobial or anticoagulant activity which have been investigated in this connection, re-stenoses of vessels and rejection reactions of implants remain a great problem. Accordingly, despite all this there is a great need for devices which can keep, for example, vessels perma-nently free, and which provide for good bioresorption.
It is therefore an object of the present invention to provide a coating which includes a polymer with a novel biocompatible bioactive agent, while preferably - with an optimization of the mechanical properties of the coating - defined amounts of this bioactive substance can be incorporated and its release from the coating can be controlled in a simple manner.
According to the present invention, this object is achieved with the coating composition mentioned at the outset in that the bioactive agent is naphthazarin and/or a naphthazarin de-rivative.
The object on which the invention is based is completely achieved in this way.
The inventor has realized that it is possible through the use of polymers and of particular naphthazarin derivatives in a coating to produce an implantable medical device which has excellent mechanical properties, i.e. can withstand both com-pressions and expansions, and that the incorporation of naph-thazarin derivatives has no adverse effect on the mechanical properties, rather the substance is retained as bioactive agent.
The inventor has further realized that it is possible through the use of certain naphthazarin derivatives to check visually whether, and to what extent, the coating of implantable medical devices has suceeded. This is achieved through the coloring inherent in the naphthazarin derivatives: if a coating com-prises a naphthazarin derivative, it is possible to ascertain on the basis of the coloring whether a device is uncoated, coated or only partly coated.
Naphthazarin is known in the art as a basic structure in many natural pigments which additionally also represent medicinal agents. It is possible through the use of naphthazarin and/or naphthazarin derivatives to achieve two advantageous effects with one active substance. Firstly, these natural products are colored, so that the success of the coating can be checked visually, and secondly they simultaneously have a healing ef-fect, which makes the addition of further bioactive substances in the coating unnecessary.
Fig. lb shows the basic structure of naphthazarin. Naphthazarin in turn is regarded as a derivative of 1,4-naphthoquinone, whose basic structure is shown in fig. la.
Naphthazarin derivatives in the present case include all com-pounds which have the basic structure of naphthazarin, while the radical R for example can be any aliphatic radical which may be acyclic or cyclic, unbranched or branched, or in substi-tuted (for example hydroxy-substituted) form.
In a further embodiment, the derivative is selected from the group comprising shikonin, alkannin, arnebin and derivatives thereof. Particularly preferred in this connection is shikonin.
The inventor was able to show in his own experiments that pre-vention of blood platelet and fibroblast aggregation was possi-.. CA 02493488 2005-O1-20 ble with stems coated with the naphthazarin derivative shikonin.
The range of effects of shikonin is considerably broader by comparison with the compounds previously used in connection with coatings, such as, for example, rapamycin and Taxol.
Shikonin, alkannin and derivatives thereof have been known for some time as red natural pigments and as medicinal agents.
Thus, for example, Papageorgiou et al., "Chemie and Biologie von Alkannin, Shikonin and verwandten Naphthazarin-Naturstoffen", Angew. Chemie 111: 280-311, 1999, present in their review article the biological and pharmacological proper-ties which have been known for a relatively long time for these agents, and discuss bioorganic, preparative and medical as-pects: thus, the article cites other publications in which it was shown that shikonin itself has an antiinflammatory and antimicrobial effect. The article by Papageorgiou et al. addi-tionally cites a publication in which an antithrombotic effect of a few naphthazarin derivatives is described, but this effect could not be unambiguously ascribed to the shikonin family.
Shikonin has been proved to have an antitumor effect, and is antimycotic, antimicrobial and wound-healing. The cytostatics (for example rapamycin or Taxol) used to date do not achieve the unique profile of effects of shikonin.
Hisa et al., "Shikonin, an Ingredient of Lithospermum Eryth-rorhizon, Inhibits Angiogenesis in Vivo and in Vitro", Antican-cer Res. 18: 783-790, 1998, showed that shikonin was able to inhibit the proliferation of bovine endothelial cells, leading to inhibition of angiogenesis. By contrast, this article does not describe or explain whether, and how, shikonin or sub-,. CA 02493488 2005-O1-20 stances related to shikonin are able to prevent the prolifera-tion of fibroblasts, especially of myofibroblasts, whose pro-liferation is, as previously mentioned, involved in the reste-nosis of vessels.
The results shown by the inventor were not to be expected espe-cially taking account of the article by Sakaguchi et al., "Granulomatous Tissue Formation of Shikon and Shikonin by Air Pouch Method", Biol. Pharm. Bull 24(6): 650-655, 2001, in which it is explained that Shikonin stimulates neoangiogenesis. On the contrary, the known properties of shikonin have to date been a deterrent to the use of shikonin or other naphthoquinone or naphthazarin derivatives in connection with implantable medical devices. The inventor was the first to be able to show that shikonin is able to prevent the aggregation of blood platelets and thus both thromboses and long-term restenoses.
Naphthazarins have not previously been proposed for coating implantable medical devices.
In addition, naphthazarin derivatives are suitable for better examination of the kinetics of release in development. Other colorants, for example, without any biological activity, would merely be further foreign substances in the coating and would thus increase the risk of an allergic or inflammatory reaction.
Accordingly, the use of shikonin or of other naphthazarin de-rivatives provides a final visual check of whether, and how well, an implant has been coated.
It is not precluded in this connection that the coating compo-sition also includes a plurality of naphthazarin derivatives, or else further concomitant substances are also incorporated into the coating composition, such as, for example, anticoagu-lant, antimicrobial or antiinflammatory substances.
In a further preferred embodiment, the coating composition comprises naphthazarin and/or a naphthazarin derivative in a content of from 0.01 to to by weight.
It is further preferred for naphthazarin and/or naphthazarin derivative to be comprised in a content which is selected from the group comprising 0.040 by weight, 0.05% by weight, 0.06% by weight, 0.07% by weight, 0.080 by weight, 0.09% by weight, to by weight.
The inventor has been able to show in his own experiments that the use of from 0 . 1 to 1 o by weight of a naphthazarin deriva-tive, especially shikonin, in the coating is suitable for ex-erting an adequate inhibitory effect on blood platelets and fibroblast adhesion.
The polymer may in this connection be a biocompatible polymer out of which the bioactive substance diffuses, or an absorbable polymer out of which the active substance is released through degradation of the polymer.
In a further embodiment, it is preferred for the polymer to be an absorbable polyester and to be selected in particular from the group comprising polyglycolic acid, polylactic acid, poly-caprolactone, polyhydroxyalkanoates and copolyesters thereof.
Such absorbable polyesters and copolyesters are sufficiently well known in the art and have proved to be sufficiently useful in particular in medical uses.
It is particularly preferred in this connection for the polyhy-droxyalkanoate to be selected from the group comprising polyhy-droxybutyrate, polyhydroxyvalerate and copolyesters thereof.
The inventor has been able to show in his own experiments that, in particular, copolyesters of polyhydroxybutyrate and polyhy-droxyvalerate have outstanding properties as coating material.
These polyesters ensure that the coating is both biodegradable and has excellent mechanical properties. It further emerged from his own experiments that a naphthazarin derivative incor-porated into this copolyester showed excellent biological ac-tivities.
It is known that many organic biodegradable polymers are able to release active substances within a certain time window.
However, many materials are unsuitable as coating because they are not entirely biocompatible. Thus, for example, van der Giessen et al., "Marked Inflammatory Sequelae to Implantation of Biodegradable and Nonbiodegradable Polymers in Porcine Coro-nary Arteries", Circulation 94: 1690-1697, 1996, showed that polylactides and other polymers thought to be biocompatible led to inflammatory tissue reactions on degradation in the body.
It is additionally known that many biodegradable polymers do not have appropriate mechanical properties. Thus, for example, crystalline regions may lead to sudden fissuring. For these reasons, a coating for an irnplantable medical device must have particular mechanical properties, and withstand both compres-sion and expansion - for example an inflation of the catheter.
In a further embodiment, the copolyester comprises polyhydroxy-valerate in a content of from 20 to 30 o by weight, preferably of 25°s by weight, and polyhydroxybutyrate in a content of from 70 to 80o by weight, preferably of 75o by weight.
The inventor was able to show in his own experiments that this ratio is particularly suitable for use as coating material, because good solubility is ensured with this ratio and, at the same time, the excellent mechanical properties of the polyester are attained.
It is further preferred in a further embodiment for the absorb-able polymer and the shikonin to be preferably dissolved in at least one solvent, preferably dimethylacetamide and/or tetrahy-drofuran.
It could be shown that the mechanical and biological properties of the coating are not impaired by dissolving the active compo-nents in these solvents.
The invention further relates to a method for coating an im-plantable medical device comprising the steps:
{a) applying the novel coating composition to the implantable medical device, (b) drying of the implantable medical device and (c) where appropriate repetition of steps (a) and (b).
In one embodiment it is preferred in this connection for the coating composition to be sprayed onto the implantable medical device.
Various techniques can be used for the spraying, all of which are sufficiently well known in the art.
In another preferred embodiment, the coating is applied by immersing the implantable medical device into the coating.
The coating processes are moreover repeated until the desired layer thickness for the coating on the implantable medical device is reached, for example a layer thickness of from 1 to 100 Vim.
A preferred embodiment of the method of the invention for coat-ing an implantable medical device consists of applying a coat-ing which includes a polyhydroxybutyrate-polyhydroxyvalerate copolyester in which the poly-hydroxybutyrate . polyhydroxyvalerate ratio is 3 . 1 and in which shikonin is present in a content of from 0.01 to So by weight.
The inventor has realized that it is possible with this compo-sition to produce a particularly suitable coating with which, besides the optimal mechanical properties of a coating, it is also possible to utilize effectively the dual function of shikonin - as colorant and bioactive agent. The properties of shikonin - colorant and active agent - are retained even after incorporation into the coating according to the invention.
In a further preferred embodiment, a stmt is employed as de-vice in the method for coating an implantable medical device.
It is possible in this connection to employ for example stems which include at least one metal and/or one synthetic material.
Stems of these types, and methods for coating stents of these types, are sufficiently well known in the art.
However, it is not precluded that other types of implantable medical devices can be coated with the coating composition of the invention. Thus, for example, skin implants, a cartilage ar bone substitute are also suitable, which may be flat, rectangu-lar, cylindrical or configured as valve.
If a vessel prosthesis is used as implantable medical device, the tubular design thereof can be of any shape, that is to say for example as branched or unbranched tube etc.
The invention further relates to the use of a novel coating composition as indicated above for coating implantable medical devices.
The invention further relates to the use of naphthazarin and/or naphthazarin derivatives, especially of shikonin, for producing a coating composition for an implantable medical device and to the use for coating an implantable medical device.
The invention further relates to an implantable medical device which is coated with a novel coating composition as mentioned above, and especially stents.
Further advantages are evident from the description hereinaf-ter.
It will be understood that the features mentioned above and to be explained below can be used not only in the combinations indicated in each case, but also in other combinations or on their own, without leaving the scope of the present invention.
The invention is explained in more detail below by means of use and implementation examples and by the figures. In these Fig. la shows the formula of the substance 1,4-naphtho-quinone; and Fig. lb shows the general formula of the basic structure of naphthazarin derivatives.
Example 1. Coating compositions used The following polymer compositions were tested as coating mate-rial:
- a composite coating of the non-absorbable polyurethane Elastollan, with and without shikonin;
- a biodegradable copolyester of poly((3-hydroxybutyrate-co-(3-hydroxyvalerate) (P(HB-HV) hereinafter) with a content of 12o by weight polyhydroxyvalerate;
- a biodegradable copolyester of P(HB-HV) with a content of 25o by weight polyhydroxyvalerate, with and without shikonin.
The solvents tested with dimethylacetamide (DMAA hereinafter) and tetrahydrofuran (THF hereinafter).
It emerged from this that a composition of 25 o by weight poly-hydroxyvalerate (PHV hereinafter) in relation to 75o polyhy-droxybutyrate (PHB hereinafter) is most suitable. In addition, a composition with this ratio in both solvents with a maximum concentration of up to 1.5o by weight could be dissolved in both solvents by heating.
In this connection, a working solution (I) comprising P(HB-HV) with 25% by weight PHV with the following composition was used for the coating composition:
- P(HB-HV): 1.5 g - DMAA or THF: 100 ml Besides this pure copolyester, coating with another composi-tion, namely P (HB-HV) with a content of 25% by weight PHV, and with shikonin, was tested. The working solution (II) used for this had the following composition:
- P(HB-HV): 1.5 g - shikonin: from 15 to 75 mg - DMMA or THF: 100 ml.
The tests were carried out using firstly stents (made of stain-less steel with electropolished surface, Translumina GmbH, Hechingen, Germany) coated with working solutions I and II, and secondly polymer films from indicated working solutions I and II cast in Petri dishes.
2. Cell adhesion and cell proliferation studies a) Elastollan films Materials and methods Elastollan films containing 0.1 and 0.5o by weight shikonin, and Elastollan films without shikonin, were tested.
Although Elastollan is a non-absorbable polymer, nevertheless the results obtained with this polymer show the activity of shikonin as inhibitor of cell adhesion and proliferation. This means that in this case the active agent is released by leach-ing and not by degradation of the Elastollan matrix.
Human embryonic fibroblasts were employed in these experiments.
The cells were cultivated in Eagle's medium with the addition of 10% fetal calf serum and passaged twice a week using a tryp-sin-EDTA solution. The cells present after the 14th passage were used for the tests. Mitomycin C dissolved in culture me-dium in a concentration of 20 ~g/ml was used as negative con-trol.
Extracts from Elastollan films without shikonin, Elastollan films with 0.1% by weight, 0.5% by weight, 1% by weight and 5%
by weight shikonin (based on polyurethane), and shikonin alone, were tested.
Extracts were obtained by incubating dishes covered by the polymers to be tested (i.e. Elastollan films without or with shikonin) with Eagle's medium at 37°C for 3 h. Undissolved solids were removed by filtering. The control with shikonin only (in the culture medium) was cultivated under the same conditions.
The respective filtrates were employed as test extracts, with the pH of the shikonin containing Elastollan extracts having been adjusted previously with 0.1 N HCl, and with the same amount of phosphate buffer as was necessary for adjusting the pH of the Elastollan/shikonin extracts having been added to the control extract (Elastollan without shikonin) and to the ex-tract with shikonin alone.
In parallel, 1 ml portions of the cell suspension (fibroblasts, see above) with 4 x 104 cells/ml were applied to 24-well plates.
After a cultivation time of 24 hours, the medium was removed and the extracts to be tested were added (positive control:
fresh medium without extracts; negative controls; addition of mitomycin C for 2 h).
The cells were then washed with Hank's solution, and fresh medium was put into the wells. The cells were then incubated for 72 h. After this incubation time, the cultures were washed twice with phosphate buffer and incubated with 2.5o glutaralde-hyde at 4 °C for 30 min. They were then washed again twice and stained with Giemsa at 37°C in humid atmosphere for 3 h.
The stain retained by the cells was eluted with a phosphate buffer/alcohol mixture (1:1) at room temperature for 15 min.
The optical density of the resulting solutions was determined by a spectrophotometer with a wavelength of 620 nm.
n.-,......~ a-..
In the positive control (tested extract: only culture medium), the fibroblasts covered almost the entire surface of the well and showed an elongate shape and a typical growth pattern.
In the negative control (tested extract: culture medium + mito-mycin C), no cell growth was observed. The cell density was low and corresponded to that on the second day of the experiment.
The cells had an elongate shape but were not in contact with one another. Some cells were rounded or were lyzed.
Cell growth on extract from Elastollan alone was similar to that in the positive control. On use of shikonin-containing Elastollan extracts it was possible to observe an inhibitory and even cytotoxic effect: a few cells were adherent, most were rounded.
This test showed that shikonin and extracts from shikonin-containing Elastollan films have an inhibitory effect on human cells in vitro.
b) Tn vitro adhesion of human embryonic lung fibroblasts to Elastollan- and shikonin-containing Elastollan films Petri dishes coated with films of Elastollan- and shikonin-containing Elastollan-DMAA solutions were used for this test.
The concentration of shikonin in the polyurethane samples was 0.01% by weight, 0.05a by weight and 0.5% by weight (based on polyurethane). A plastic Petri dish served as positive control.
The cells were seeded on the surface of the Petri dishes coated with the polymer compositions in a concentration of 4 x 104 cells/ml in Eagle's medium with 10o fetal calf serum.
The number of adherent fibroblasts was counted after incubation at 37°C for 0.5 and 2 h.
Results The counts obtained are shown in table I below.
As is evident from the table through comparison with the posi-tive control, a concentration of 0.5o by weight shikonin in the Elastollan films inhibits the adhesion of fibroblasts virtually completely:
Type of surface Number of counted fibroblasts (after an incubation time of) 0.5 h 2 h Control (plastic Petri ca. 60 ca. 95 dish) Elastollan 10 - 40 none Elastollan + shikonin < 20 none (O.Olo by weight) Elastollan + shikonin ca. 30 none (0.050 by weight) Elastollan + shikonin 0 - 5 none (0.5s by weight) c) Adhesion of blood platelets onto copolyester P(HB-HV) films and onto shikonin-containing copolyester P(HB-HV) films !PlHB-HV)-Sh) and onto stents coated with these films It is known that the adhesion of blood platelets to biomateri-als reflects the compatibility of medical devices with blood in relation to blood and tissue cell activation.
It is presumed that adhesion proceeds in a plurality of steps:
initially the blood platelets bind to the surface, are then activated and develop pseudopods, and then they spread out and form aggregates. The subsequent release of intracellular compo-nents, including blood clotting factors, stimulates adhesion and aggregation of further blood platelets.
Besides, infiltration of actively proliferating myocytes from the media into the intima, accompanied by the production of abundant extracellular matrix components (collagen, proteo-aminoglycans), is presumed to be the fundamental mechanism of restenosis. Immediate deposition of blood platelets at the point where the vessel was injured, and the subsequent release of myoproliferative substances (for example the growth factor (PDGF), ~-transforming factor (~-TGF), endothelium-derived growth factor (EDGF), etc.} probably represent the stimulating factors.
Thus, adhesion of blood platelets during stmt implantation plays a key role both in relation to thromboses and to resteno-ses.
The activation status of adherent blood platelets can be esti-mated from their morphology. A larger effect of the material on blood platelets results in more adherent cells being distrib-uted or aggregated on the material.
Both uncoated stents and stems coated with P(HB-HV)-Sh were tested for this test. The average layer thickness of the coat-ing was approximately 15 to 20 Vim.
The following solutions were used for coating the stents:
- 0.75% P(HB-HV) with 25% HV in DMAA or THF;
0.75% P(HB-HV) with 25% HV + 0.5% by weight shikonin (based on the weight of P(HB-HV)) in DMAA or THF;
- 0.75% P(HB-HV) with 25% HV + to by weight shikonin (based on the weight of P(HB-HV}) in DMAA or THF;
The stem s were coated by several times being immersed into the various solutions or sprayed. The drying time between the indi-vidual spraying steps was between 2 and 15 min. Finally, the stents were dried at 45 to 50°C for 30 min. The total amount of shikonin on the sprayed P(HB-HV)-l.OSh stems was 2 ~g to 4 fig.
The shikonin coating of the stems proved to be stable in the expanded state over a period of 28 days.
In addition, polymer films from the indicated solutions (P(HB-HV) with or without shikonin) were tested on stainless steel plates from the same solutions with a layer thickness of 20 to 30 ~,m. The surface of an uncoated stmt served as con-trol.
Preparation of the blood Whole blood from healthy adult donors was put into siliconized glass vessels. Blood clotting of 10 ml of blood was prevented by adding sodium citrate in a ratio of (blood: sodium citrate) 9:1. Blood platelet-enriched plasma was obtained by centrifug-ing the whole blood at 100 x g for 20 minutes at room tempera-ture. The blood platelet-enriched plasma fraction was removed with a plastic pipette and immediately employed in the experi-ments.
50 ~l drops of this blood platelet-enriched plasma fraction were put on the surfaces of the plate samples and incubated for 15 min. To test the coated stem s, they were put in a vessel with blood platelet-enriched plasma and incubated for 30 minutes. The number of blood platelets which adhere to the surface during this period was sufficient for a qualitative analysis, because the platelets formed no large thrombus-like structures. The samples were washed with physiological saline solution in order to wash off unadsorbed plasma proteins and weakly adhering blood platelets. The samples were then fixed in 2.5o glutaraldehyde, and subsequently dehydrated by standard techniques with an increasing ethanol content.
Adhesion of the blood platelets was investigated by scanning electron microscopy (SEM) (JSM T330, JEOL, Japan). All the samples were made conductive by coating of copper with 1.2 kV, 10 mA for 7 minutes (JEOL JFC-1100, Japan). The microscopic investigations were carried out with a voltage of 5 kV. 25 sections each 400 ~m2 in size were selected at random for each sample. The qualitative total blood platelet number Ntot and the number of blood platelets NI of the following two categories of cells which explicitly reflect the activation of the blood platelets were then evaluated. Each category comprises two morphological classes of adherent blood platelets:
Ia) single, non-activated or only slightly activated deformed cells;
Ib) pseudopod-forming cells or cells in an early stage of spreading.
IIa) fully spread blood platelets;
IIb) aggregates (of two or more blood platelets).
With reference to this classification, blood platelets from category I interact only weakly with the surface; in contrast thereto, a strong interaction takes place between the surface and the blood platelets of category II.
Results a) Coating with a 0.75% by weight solution of P(HB-HV) and P(HB-HV)+shikonin in DM.AA
The number of cells on the uncoated stems proved to be very low, and where adherent blood platelets were present virtually all the cells were completely spread on the uncoated stmt. The total number on the P(HB-HV) film was higher than that for the control and all morphological classes of the cells were present on this surface, but the number of aggregates proved to be very low.
Shikonin by contrast was able distinctly to reduce the amount of adherent blood platelets compared with films coated only with P (HB-HV) . In this case, only two morphological classes of cells of category I were observable: slightly activated blood platelets and pseudopods-forming cells.
It is evident on the basis of these results that P(HB-HV)-Sh films are more suitable than surfaces with pure P(HB-HV) be-cause the shikonin-containing surfaces showed a lower affinity for cell binding.
In order to determine the connection between blood platelet activation and shikonin concentration, the blood platelet adhe-sion to films of pure P(HB-HV), of P(HB-HV) with O.lo by weight shikonin (P(HB-HV)-O.lSh) and of P(HB-HV) with 0.5o by weight shikonin (P(HB-HV)-0.5Sh) was determined for an incubation time of 15 minutes and 30 minutes. It was possible to demonstrate thereby that addition of shikonin was able to reduce the number and the degree of activation of the blood platelets.
a) Coating with a 0.750 by weight solution of P(HB-HV) and P(HB-HV)+shikonin in THF
The coatings were applied as multilayer films to expanded and unexpanded stems. The stents were coated with the polymer by immersing it in the diluted working polymer solution (about 2 to 4 times). Each layer was then dried with hot air.
A comparative analysis of the adhesion of blood platelets to the uncoated stmt, to a stmt coated with diamond-like carbon and to a stmt coated with P(HB-HV)-0.5Sh was carried out. The incubation time of the samples in plasma enriched with blood platelets was from 15 to about 30 minutes.
No blood platelets were observed on the stems coated with P(HB-HV)-0.5Sh. However, in the same time it was possible to find blood platelets on the uncoated and on the carbon-coated stems. This means that the properties of shikonin in relation to the activation of blood platelets are also retained on use of THF as solvent.
c) Coating with 1% by weight shikonin (P(HB-HV)-l.OSh) com-pared with stems with a diamond-like coating (DLC) In the comparative analysis of the stem s coated with to by weight shikonin with DLC-coated stems it emerged that virtu-ally no platelets adhered to the coated stems in the case of the stem s coated with P(HB-HV)-l.OSh in vitro. On the other hand, a few spread-out platelets were to be found on DLC-coated stents.
3. Determination of the biolorqical properties of the films produced These investigations were carried out in accordance with the international standard for biological investigations on medical devices ISO 10993 and in accordance with the national standard GOST R ISO 10993.
a) Determination of the hemolytic properties The hemolysis was determined by preparing extracts of the mate-rials, in particular of P(HB-HV) and of P(HB-HV)-0.5Sh, in each case in physiological saline solution. Whole blood was added to these extracts, followed by incubation at 37°C for one hour.
The extracts were removed, the mixture was centrifuged (50 minutes at 400 x g) and the cell-free supernatant was care-fully removed. The hemoglobin concentration for this super-natant was determined by photometry, and the hemolytic index (%) was calculated from the ratio of liberated hemoglobin to hemoglobin present. A pure saline solution was used as control in this case.
Under the given conditions, both extracts, that is to say the extract of P (HB-HV) and that of P (HB-HV) -Sh, proved to be non-hemolytic (hemolytic index: 0%).
b) Determination of the complement system activation The compatibility of the polymer films with blood was tested by determining the hemolytic activity of the complement system of human blood plasma before and after its incubation with a film sample or extract. The tested samples were films of P(HB-HV), P(HB-HV)-Sh and of cuprophane (control).
The concentration of the hemoglobin liberated by the lytic effect of the complement system from sheep erythrocytes coated with antibodies reflects the complement activity in the serum sample. The parameter for the assay was the time required for 100 ~1 of the tested serum to lyze (i1~2) 50 0 of 5 ml of sensi-tized sheep erythrocytes (5 x 108 cells/ml). The temperature of the reaction mixture was kept constant at 37°C by means of thermostats.
Human serum was first diluted with a buffer (comprising 0.15 mM
CaCl2 and 0.5 mM MgCl2) and then incubated with or without polymer samples at 37°C for 60 min. The values for the remain-ing complement activity ((CH50)R%) were determined as criterion of the activating properties of the polymers in the following way:
(CH50) Ri ' (Z1/2) 0 / (Z1~2) (t) i(c1 1OO o where:
(ii/z) o = time for 50% lysis of sheep erythrocytes by the serum complement before incubation with the sample;
(tl/2) (t) i = time for 50% lysis of sheep erythrocytes by the serum complement after incubation time t with the polymer sam-ple;
(il/2) (t) c~~ - time for 50% lysis of sheep erythrocytes by the serum complement after incubation time t in the control (with-out polymer sample).
The results of these tests are summari2ed in table II below:
Tested samples CHS~ (n = 3) Cuprophane (control material) 89 5 P(HB-HV) extract 85 5 / 80 5 P(HB-HV)-Sh extract 90 5 / 85 5 Serum after 1 h incubation (control serum) As is evident from the table, the complement activity of serum incubated with P(HB-HV)-Sh extract proved to be lower than that of serum incubated with pure P(HB-HV) extract. In addition, it showed approximately the same CHso as cuprophane.
c) ATP release tests In a further comparative analysis, DLC-coated stems were in-vestigated with P(HB-HV)-l.OSh-coated stems. Fresh human platelet-rich plasma (PRP) was employed for this purpose. The stems were incubated with 450 ~,1 of PRP at 37°C for 5 min. The platelet aggregation and the secretion ability was measured by means of bioluminescence using the highly sensitive LUMI-AGREGOMETER (CHRONO-LOG Corp,, USA).
It was shown that the shikonin-coated stents did not change the functional status of the platelets (in relation to ATP re-lease), whereas the DLC-coated stents inhibited ATP release.
d) Intramuscular implantation test 1. Films of P(HB-HV) only and of P(HB-HV)-Sh were implanted in accordance with ISO 10993 Part 6 into the muscles of guinea pigs. The size of the film samples was 0.5 x 1 cm with a thickness of 20 to 30 Vim. For each type of sample, two guinea pigs with the implant were observed for 7 days.
It was shown after 7 days that fibroblast monolayers formed around the P(HB-HV) and P(HB-HV)-Sh films. Infil-tration of macrophages and lymphocytes around these im-plants proved to be a typical tissue response after im-plantation for 7 days. It was nevertheless possible to show that the degree of the inflammatory response to the biodegradable polymer with shikonin was less than with the pure P(HB-HV) film.
2. Adult male Wistar rats (230 - 250 g) were divided into two groups: animals of group A (n=6) were implanted with P(HB-HV)-l.OSh-coated stems and those of group B (n=4) with stems with a diamond-like coating (DLC stems) as control. The stems remained subcutaneously implanted in the backs of the rats over a period of 2 and 6 weeks for subsequent macroscopic and microscopic determination of subchronic (short-term) effects (for each of the individ-ual period n=3 for group A and n=2 for group B). The ani-mals were anesthetized with sodium thiopental and, after completion of the experiments, euthanized with an overdose of this substance.
For the histological investigations, the implants with surrounding tissue were fixed in 12s formaldehyde, embed-ded in gelatin and divided into sections with a diamond blade.
Histological and macroscopic investigations showed that no chronic inflammatory foreign-body reactions could be ob-served in both groups, but with the DLC stems a consider-able number of macrophages and some carbon particles were detected in the surrounding tissue. The fibrous capsule which formed around the stems was significantly thinner with the P(HB-HV)-l.OSh-coated stems than with the DLC
stems .
Overall, the stability and biocompatibility of the P(HB-HV)-l.OSh-coated stents was higher than that of the DLC stems .
3. In further experiments, the stents were implanted into the aorta of cats. The experimental animals were for this pur-pose divided into two groups: group A (n=2) received P(HB-HV)-l.OSh-coated stems, group B (n=2) received DLC
stems. Adult female cats (2.5 to 3.5 kg) were anesthe-tized intramuscularly with a 5 percent ketamine solution (0.65 mg/kg) and a 0.25 percent droperidol solution (0.1 mg/kg) . The stems were implanted into the abdominal part of the aorta. No additional anticoagulants were ad-ministered.
18 to 24 days after the implantation, the animals were anesthetized, and the aorta segments were removed and fixed in 10% formalin and dehydrated. The stmt-containing segments were then embedded in methyl methacrylate and di-vided into sections using a diamond blade.
No acute thrombotic occlusion or growth of fibrous connec-tive tissue into the lumen was observed in both test groups. The histological investigations revealed that for the DLC stems some carbon particles and a relatively large number of macrophages were observed in the aorta wall surrounding the DLC stems. 24 days after implanta-tion, neointima formation was observed with the P(HB-HV)-l.OSh-coated stems. It had a more regular arrangement than with the DLC stems. No indications of acute or chronic inflammation were observed.
Summary With the results it could be shown that firstly the composition of 25% by weight polyhydroxyvalerate and 75% by weight polyhy-droxybutyrate proved to be an optimal composition for the coat-ing, and that this mixture together with a particular shikonin content showed high adhesion to metallic surfaces. In addition, it could be shown that the properties of the polymer coating were not affected by repeated expansion of the coated stmt.
Further, it cold be shown that a concentration of shikonin of up to 5% by weight inhibited virtually completely the adhesion of human blood platelets to the polymer surface in vitro. Thus, shikonin can be used in suitable concentrations for preventing excessive cell proliferation.
A comparative study on the biological properties of a film of pure P(HB-HV) or of P(HB-HV)-Sh in THF in vitro showed that shikonin had good compatible properties in relation to comple-ment activation and blood platelet adhesion.
With the results it could further be shown that a smooth, thin, fibrocellular layer is formed around the stmt through a coat-ing with P(HB-HV)-Sh, leading to reendothelialization with an adequate diameter for normal blood flow.
Claims (23)
1. A coating composition for an implantable medical device, the coating composition comprising at least one polymer and at least one bioactive agent, characterized in that the bioactive agent is naphthazarin and/or a naphthazarin derivative.
2. The coating composition as claimed in claim 1, character-ized in that the derivative is selected from the group comprising shikonin, alkannin, arnebin.
3. The coating composition as claimed in claim 1 or 2, char-acterized in that the derivative is shikonin.
4. The coating composition as claimed in any of claims 1 to 3, characterized in that naphthazarin and/or naphthazarin derivatives are present in the content of from 0.01 to 5%
by weight.
by weight.
5. The coating composition as claimed in any of claims 1 to 3, characterized in that the naphthazarin and/or naph-thazarin derivative is present in a content which is se-lected from the group comprising 0.04% by weight, 0.05% by weight, 0.06% by weight, 0.07% by weight, 0.08% by weight, 0.09% by weight, 1% by weight.
6. The coating composition as claimed in any of claims 1 to 5, characterized in that the polymer is selected from the group: biocompatible polymer, absorbable polymer and ab-sorbable polyester.
7. The coating composition as claimed in claim 6, character-ized in that the absorbable polyester is selected from the group comprising polyglycolic acid, polylactic acid, poly-caprolactone, polyhydroxyalkanoate and copolyesters thereof.
8. The coating composition as claimed in claim 7, character-ized in that the polyhydroxyalkanoate is selected from the group comprising polyhydroxybutyrate, polyhydroxyvalerate and copolyesters thereof.
9. The coating composition as claimed in claim 8, character-ized in that the copolyester is a polyhydroxybutyrate-polyhydroxyvalerate copolyester.
10. The coating composition as claimed in claim 9, character-ized in that in the copolyester polyhydroxyvalerate is present in a content of from 20 to 30% by weight, prefera-bly of 25% by weight, and polyhydroxybutyrate is present in a content of from 70 to 80% by weight, preferably of 75% by weight.
11. The coating composition as claimed in any of claims 1 to 10, characterized in that the absorbable polymer and naph-thazarin and/or naphthazarin derivative are present dis-solved in at least one solvent.
12. The coating composition as claimed in claim 11, character-ized in that the solvent is dimethylacetamide and/or tet-rahydrofuran.
13. A method for coating an implantable medical device com-prising the steps:
(a) applying the coating composition as claimed in any of claims 1 to 12 onto the implantable medical device, (b) drying of the implantable medical device.
(a) applying the coating composition as claimed in any of claims 1 to 12 onto the implantable medical device, (b) drying of the implantable medical device.
14. A method for coating an implantable medical device com-prising the steps:
(a) applying the coating composition as claimed in any of claims 1 to 12 onto the implantable medical device, (b) drying of the implantable medical device and (c) repeating steps (a) and (b) .
(a) applying the coating composition as claimed in any of claims 1 to 12 onto the implantable medical device, (b) drying of the implantable medical device and (c) repeating steps (a) and (b) .
15. The method as claimed in claim 13 or 14, characterized in that the coating composition is sprayed on.
16. The method as claimed in claim 13 or 14, characterized in that the coating composition is applied by immersing the implantable medical device into the coating composition.
17. The method for coating an implantable medical device as claimed in any of claims 13 to 16, characterized in that a coating composition which includes a copolyester of poly-hydroxybutyrate-polyhydroxyvalerate in a ratio of 3:1 and shikonin in a content of from 0.01 to 5% by weight is ap-plied.
18. The method for coating an implantable medical device as claimed in any of claims 13 to 17, characterized in that a stmt is employed as implantable device.
19. Use of a coating composition as claimed in any of claims 1 to 12 for coating implantable medical devices.
20. Use of naphthazarin and/or of a naphthazarin derivative, in particular shikonin, for producing a coating composi-tion for an implantable medical device.
21. Use of naphthazarin and/or of a naphthazarin derivative, in particular shikonin, for coating an implantable medical device.
22. An implantable medical device coated with a coating compo-sition as claimed in any of claims 1 to 12.
23. A stent coated with a coating composition as claimed in any of claims 1 to 12.
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| Application Number | Priority Date | Filing Date | Title |
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| DE10234398A DE10234398B4 (en) | 2002-07-23 | 2002-07-23 | An implantable medical device coating composition and method of coating such device and its use |
| DE10234398.5 | 2002-07-23 | ||
| PCT/EP2003/007913 WO2004009148A1 (en) | 2002-07-23 | 2003-07-19 | Coating composition for an implantable medical device and method for coating such a device |
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| CA002493488A Abandoned CA2493488A1 (en) | 2002-07-23 | 2003-07-19 | A coating composition for an implantable medical device and method for coating such a device with naphthazarin and/or a naphtharazin derivative |
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| EP (1) | EP1523346B1 (en) |
| JP (1) | JP4851086B2 (en) |
| AT (1) | ATE318624T1 (en) |
| AU (1) | AU2003254547B2 (en) |
| CA (1) | CA2493488A1 (en) |
| DE (2) | DE10234398B4 (en) |
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| US20150140117A1 (en) * | 2012-07-14 | 2015-05-21 | Nobel Biocare Services Ag | Bioactivated bone substitute material |
| US20150224224A1 (en) * | 2012-07-14 | 2015-08-13 | Nobel Biocare Services Ag | Bioactivated material |
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| FR2866239B1 (en) * | 2004-02-12 | 2007-01-12 | Perouse Laboratoires | IMPLANTABLE TUBULAR PROSTHESIS. |
| RU2447902C2 (en) * | 2010-07-21 | 2012-04-20 | Федеральное Государственное Автономное Образовательное Учреждение Высшего Профессионального Образования "Сибирский Федеральный Университет" | Biologically active polymeric medical composition (version) |
| RU2555502C2 (en) * | 2013-10-08 | 2015-07-10 | Федеральное государственное бюджетное учреждение "Научно-исследовательский институт комплексных проблем сердечно-сосудистых заболеваний" Сибирского отделения Российской академии медицинских наук (ФГБУ "НИИ КПССЗ" СО РАМН) | Suture material with antithrombotic coating |
| RU2709091C1 (en) * | 2018-12-26 | 2019-12-13 | Федеральное государственное автономное образовательное учреждение высшего образования "Национальный исследовательский университет "Московский институт электронной техники" | Method of producing a coating with high hydrophilicity based on a biodegradable polymer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61143334A (en) * | 1984-12-18 | 1986-07-01 | Mitsui Petrochem Ind Ltd | 5,8-dehydroxy-1,4-naphthoquinone derivative |
| JPH04108804A (en) * | 1990-08-28 | 1992-04-09 | Shin Etsu Chem Co Ltd | Agent for preventing deposition of polymer scale and method for preventing deposition thereof |
| JPH04108805A (en) * | 1990-08-28 | 1992-04-09 | Shin Etsu Chem Co Ltd | Agent for preventing deposition of polymer scale and method for preventing deposition thereof |
| CA2059742A1 (en) * | 1991-01-22 | 1992-07-23 | Mikio Watanabe | Coating solution for preventing adhesion of polymer scale and method for preventing scale adhesion during preparation of polymers |
| US5464650A (en) * | 1993-04-26 | 1995-11-07 | Medtronic, Inc. | Intravascular stent and method |
| WO2001021326A1 (en) * | 1999-09-22 | 2001-03-29 | Surmodics, Inc. | Water-soluble coating agents bearing initiator groups and coating process |
| WO2001064214A2 (en) * | 2000-02-28 | 2001-09-07 | The University Of British Columbia | Compositions and methods for the treatment of inflammatory diseases using topoisomerase inhibitors |
-
2002
- 2002-07-23 DE DE10234398A patent/DE10234398B4/en not_active Expired - Fee Related
-
2003
- 2003-07-19 RU RU2005104943/15A patent/RU2308295C2/en not_active IP Right Cessation
- 2003-07-19 JP JP2004522524A patent/JP4851086B2/en not_active Expired - Fee Related
- 2003-07-19 EP EP03765062A patent/EP1523346B1/en not_active Expired - Lifetime
- 2003-07-19 AT AT03765062T patent/ATE318624T1/en not_active IP Right Cessation
- 2003-07-19 CA CA002493488A patent/CA2493488A1/en not_active Abandoned
- 2003-07-19 DE DE50302545T patent/DE50302545D1/en not_active Expired - Lifetime
- 2003-07-19 AU AU2003254547A patent/AU2003254547B2/en not_active Ceased
- 2003-07-19 WO PCT/EP2003/007913 patent/WO2004009148A1/en active IP Right Grant
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150140117A1 (en) * | 2012-07-14 | 2015-05-21 | Nobel Biocare Services Ag | Bioactivated bone substitute material |
| US20150224224A1 (en) * | 2012-07-14 | 2015-08-13 | Nobel Biocare Services Ag | Bioactivated material |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2004009148A8 (en) | 2004-07-22 |
| RU2005104943A (en) | 2005-07-10 |
| AU2003254547A1 (en) | 2004-02-09 |
| EP1523346B1 (en) | 2006-03-01 |
| JP4851086B2 (en) | 2012-01-11 |
| DE50302545D1 (en) | 2006-04-27 |
| RU2308295C2 (en) | 2007-10-20 |
| DE10234398B4 (en) | 2006-07-27 |
| DE10234398A1 (en) | 2004-02-12 |
| WO2004009148A1 (en) | 2004-01-29 |
| ATE318624T1 (en) | 2006-03-15 |
| EP1523346A1 (en) | 2005-04-20 |
| JP2006502753A (en) | 2006-01-26 |
| AU2003254547B2 (en) | 2009-01-08 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |
Effective date: 20130719 |