KR100854985B1 - Medical products comprising a hemocompatible coating, production and use thereof - Google Patents

Abstract

The present invention relates to the use of polysaccharides, including the sugar building unit N-acylglucosamine, for the preparation of hemophilic surfaces, as well as to hemophilically coating the surfaces with these polysaccharides classified as being a common biosynthetic precursor material of heparin and heparan sulfate. It is about a method. Medical devices, in particular stents, containing paclitaxel as an antiproliferative active substance and according to the invention are described, as well as the use for the prevention of restenosis of these stents.

Description

Medical device comprising a hemocompatible coating, manufacturing method and use thereof

The present invention relates to the use of a polysaccharide (or polysaccharide) containing a sugar building block N-acylglucosamine for use in preparing a blood-compatible surface of a medical device, a method of hemophilically coating the surface of a medical device with the polysaccharide and A medical device having a blood-compatible surface.

In the human body, blood is only in contact with a surface other than the inside of a natural blood vessel when it is injured. Ultimately, when blood comes into contact with foreign surfaces, the blood coagulation system is always active, reducing bleeding and preventing life-threatening blood loss. Due to the fact that the implant is also a representative foreign surface, all patients who receive an implant that is in permanent contact with blood are treated to maintain blood contact with the drug. In other words, anticoagulants that inhibit blood coagulation have to be endowed with significant side effects.

The thrombosis risk described above also occurs as one of the risk factors in blood-bearing vessels with the use of so-called stencils called stents. For example, stents are used to expand the vessel wall, particularly in the case of vascular narrowing and closure due to atherosclerotic changes in the coronary arteries. This fixes the lime fragments in the vessels, thereby smoothing the surface of the inner space of the vessels, thereby promoting the flow of blood in the vessels. In addition, the stent is resistant to the contractile restorative force of the expanded blood vessel portion. The most commonly used material is medical stainless steel.

Stent thrombosis is an early thrombosis that occurs in less than 1% of patients in cardiac electrode laboratories or in 2% to 5% of patients in the recovery phase during hospitalization. In about 5% of patients, vascular injuries due to surgery are caused by artery occlusion, and there is also the possibility of gastric aneurysms caused by vasodilation. In addition, continued administration of heparin as an anticoagulant increases the risk of bleeding.

In addition, a very common complication is restenosis (or restenosis) in which blood vessels are reclosed. Although stents reduce the risk of closed regeneration of blood vessels, they have not been known to completely block restenosis until now. The incidence of restenosis after implantation of the stent is less than 30%, which is one of the main reasons for patients to visit the hospital periodically.

The exact concept of restenosis is not explained in the technical literature. A commonly used morphological definition for restenosis is that after successful PAT (percutaneous angioplasty) the vessel diameter is reduced to less than 50% of the diameter of a normal vessel. Although this is an empirical figure, the relationship between hemodynamic relevance and its clinical pathology is not based on a large scientific basis. Clinical deterioration of patients in practice is often observed as a sign of restenosis of previously treated vascular sites.                 

Vascular damage during the implantation of the stent causes an inflammatory response, which plays an important role in the healing process for at least seven days. The concurrent processes are included in those associated with the release of growth factors that initiate an increase in the proliferation of smooth muscle cells and with it lead to rapid restenosis, ie the closed regeneration of blood vessels due to unregulated growth. Even two weeks after the stent grows into the tissue of the blood vessel and is completely surrounded by smooth muscle, scarring (neovascular endothelial hyperplasia) may also appear and cover the stent surface as well as close the entire internal space of the stent.

Attempts have been made to solve the problem of restenosis by coating the stent with heparin (J. Whorle et al., European Heart Journal (2001)). 22, 1808-1816). Heparin treats only the first mentioned cause as an anticoagulant and furthermore its overall effect can only be exerted in solution. On the other hand, this first problem can be almost completely avoided pharmacologically by the administration of anticoagulants. An additional problem is attempted to address by inhibiting the growth of local smooth muscle cells on stents today. This is done for example by means of a stent containing a radioactive stent or a pharmacologically active agent.

In conclusion, there is a need for a non-thrombotic, hemocompatible material that does not activate the coagulation system and does not induce blood coagulation without contacting the blood without being discovered as a foreign surface, eliminating important factors of the restenosis stimulation process. The support must be added with an active agent that must inhibit the inflammatory response or regulate the healing process involving cell division.                 

There are many challenges in this area to produce stents that can reduce or completely eliminate restenosis in such a way. Other feasibility studies have been tested. The most common configuration type consists of coating a matrix, usually a biostable polymer, suitable for the stent. The matrix contains proliferative or anti-inflammatory agents that are released through temporally controlled steps to inhibit inflammatory responses and excessive cell division.

US-A-5 891 108 shows, by way of example, a stent molded in a hollow form, which contains a pharmaceutically active substance therein, which can be released through several outlets in the stent. EP-A-1 127 582 presents a trenched stent having a depth of 0.1 mm to 1 mm and a length of 7 mm to 15 mm on a surface suitable for the provision of the active material. This active substance reservoir, similar to the exit in the hollow stent, releases the pharmaceutically active substance contained in exactly high concentrations over a relatively long period of time, which may cause smooth muscle cells to no longer or only significantly slow the stent. Provide the fact that As a result, the stent is exposed to blood for longer periods of time, which in turn leads to increased vascular obstruction by thrombosis (Liistro F., Colombo A., Late acute thrombosis after paclitaxel eluting stent implantation. Heart (2001) 86 262-4) .

One solution to this problem can be addressed by phosphorylcholine coatings of biocompatible materials (WO 0101957). That is, phosphorylcholine, a component of the erythrocyte cell membrane, forms a non-thrombotic surface as a component of the non-biodegradable polymer layer coated on the stent. Depending on the molecular weight, such active agents are either absorbed by the polymer containing phosphorylcholine layer or adsorbed on the surface.

It is an object of the present invention to provide a use for hemophilically coated medical devices, hemocompatible coating methods and hematologically coated medical devices, in particular stents, for preventing or reducing side effects such as restenosis.

In particular, it is an object of the present invention, on the one hand, to inhibit cellular responses in the first few days and weeks after transplantation, with the aid of certain substances and combinations thereof, on the other hand, which may lead to complications with prolonged use with reduced effects of the active substance. It is to provide a stent that allows for continuous and controlled internal growth of the stent by providing a non-thrombotic and / or inert and / or biocompatible surface that ensures that no reaction to existing foreign surfaces occurs.

There are many attempts to produce near perfect stimulation of natural non-thrombotic states in the blood vessels located in the blood side. EP-B-0 333 730 describes a method for producing hemophilic substrates by retraction, adsorption and / or modification and fixation of polysaccharides (HSI) on non-thrombotic endothelial cell surfaces. The immobilization of specific endothelial cell surface proteoheparane sulfate HSI on biological or artificial surfaces provides the result that the coated surface is blood friendly and suitable for permanent blood contact. On the other hand, the disadvantage is that the manufacturing process of HSI presupposes the cultivation of endothelial cells, and therefore, the cultivation of endothelial cells can be obtained only when the cultivation of endothelial cells takes only a long time and a considerable cost. This is a significant constraint on.                 

The present invention solves the above object by providing a medical device showing the surface coating properties of the determined polysaccharide and paclitaxel. Simvastatin, 2-methylthiazolidine-2,4-dicarboxylic acid and corresponding sodium salts instead of paclitaxel, macrocyclic suboxides (MCS) and derivatives thereof, tyrphostin, D24851, thymosin a-1, inter Leucine-1β inhibitor, activating protein C (aPC), MSH, fumaric acid and fumaric acid esters, PETN (pentaerythritol tetranitrate), PI88, dermisidine, baccatin and derivatives thereof, further derivatives of docetaxel and paclitaxel, ta Inflammatory drug anti-inflammatory drugs, as well as other anti-inflammatory drugs that are determined, such as crawlrimus, pimecrolimus, trapidil, α- and β-estradiol, sirolimus, colchicine and melanocyte-stimulating cell stimulating hormone (α-MSH) Or combinations of these drugs with paclitaxel can be used. Methods of making these hemocompatible surfaces are described in claims 20-31. Preferred embodiments can be found in the dependent claims, the examples and the figures.

Subject of the present invention is a medical device having at least partially coated a surface with a hemophilic layer comprising at least one of the compounds of formula (1):

Formula (1)

In the above formula,

n is an integer between 4 and 1050;

Y is residues -CHO, -COCH ₃ , -COC ₂ H ₅ , -COC ₃ H ₇ , -COC ₄ H ₉ , -COC ₅ H ₁₁ , -COCH (CH ₃ ) ₂ , -COCH ₂ CH (CH ₃ ) _{2, -COCH (CH 3) C} 2 H 5, -COC (CH 3) 3, -CH 2 COO, -C 2 H 4 COO -, -C 3 H 6 COO - or -C ₄ H ₈ COO ^- is .

In addition, any salt of the compound of formula (1) may be used. The hemocompatible layer may preferably be applied directly onto the surface of the non-hemophilic medical device or deposited on other biostable and / or biodegradable layers. In addition, additional biostable and / or biodegradable and / or hemophilic layers may be deposited on the hemophilic layer. In addition, the active agent paclitaxel is present on, in, and / or under each of the hemophilic layer or two or more hemophilic layers. The active substance (paclitaxel) may form its active substance layer on or under the hemophilic layer and / or may be incorporated into one or more of a biostable, biodegradable and / or hemophilic layer. Preferably, compounds of formula (1) wherein Y is one of the following groups are used: -CHO, -COCH ₃ , -COC ₂ H ₅ or -COC ₃ H ₇ . Further preferred groups are -CHO, -COCH ₃ or -COC ₂ H ₅ , particularly preferred is group -COCH ₃ .

Compounds of formula (1) contain only minor amounts of free amino groups. Due to the fact that in the ninhydrin reaction the free amino group can no longer be detected by the sensitivity of the test, less than 2%, preferably less than 1%, particularly preferably less than 0.5% of all -NH-Y groups are free It may imply that it exists as an amino group. That is, the —NH—Y group where Y is hydrogen is present at such a low percentage.

Since the polysaccharide of formula (1) contains a carboxylate group and an amino group, this formula includes not only alkalis of the corresponding polysaccharides but also alkaline earth metal salts. Alkali metal salts such as sodium salts, potassium salts, lithium salts or alkaline earth metal salts such as magnesium salts or calcium salts may be mentioned. In addition, with ammonia, primary, secondary, tertiary and quaternary amines, pyridine and pyridine derivatives, ammonium salts, preferably alkylammonium salts and pyridinium salts can be formed. Among the bases that form salts with polysaccharides include inorganic and organic bases such as NaOH, KOH, LiOH, CaCO ₃ , Fe (OH) ₃ , NH ₄ OH, tetraalkylammonium hydroxides and similar compounds.

The polysaccharide of formula (1) has a molecular weight of 2 kD to 15 kD, preferably 4 kD to 13 kD, more preferably 6 kD to 12 kD, particularly preferably 8 kD to 11 kD. The variable n is an integer between 4 and 1050. Preferred n is an integer of 9 to 400, more preferably an integer of 14 to 260, particularly preferably an integer of 19 to 210.

Formula (1) shows disaccharides. This should be understood as the basic structure of the polysaccharide used, where n of the basic structures form a continuous polysaccharide. This basic structure, consisting of two sugar molecules, should not be construed that Formula (1) contains only polysaccharides of even sugar molecules. Formula (1) also includes polysaccharides in odd sugar building units. The terminal group of the polysaccharide is a hydroxyl group.

Particularly preferred are medical devices which contain a hemophilic layer of the compound of formula (1) directly on the surface of the medical device and a paclitaxel layer thereon. The paclitaxel layer can be divided in small portions into the hemophilic layer or incorporated entirely into the hemophilic layer.

It is also preferred that at least one biostable layer is present below the hemophilic layer. In addition, one or more hemophilic layers may be coated in whole and / or partially on the biostable and / or biodegradable layers. Preference is given to placing the biodegradable or hemophilic layer outward.

A further preferred embodiment is to contain a layer of paclitaxel beneath the hemophilic layer or between the biostability and hemocompatible layer, whereby paclitaxel is released slowly through the hemophilic layer. Paclitaxel may be covalently and / or adhesively bonded in and / or on a hemophilic layer and / or a biostable and / or biodegradable layer, with adhesive bonding being preferred.

As the biodegradable material for the biodegradable layer, the following may be used:

Polyvalerolactone, poly-ε-decaractone, polylactic acid, polyglycolic acid, polylactide, polyglycolide, copolymer of polylactide and polyglycolide, poly-ε-caprolactone, polyhydroxy moiety Carbonic acid, polyhydroxybutyrate, polyhydroxy valerate, polyhydroxybutyrate-co-valerate, poly (1,4-dioxane-2,3-dione), poly (1,3-dioxane-2- On), poly-para-dioxanone, polyanhydrides such as polymaleic anhydride, polyhydroxymethacrylate, fibrin, polycyanoacrylate, polycaprolactonedimethylacrylate, poly-b- Multiblock polymers such as maleic acid, polycaprolactonebutylacrylate, oligocaprolactonediol and oligodioxanonediol, polyether ester multiblock polymers such as PEG and poly (butylene terephthalate), polypy Tolactone, polyglycolic acid trimethylcarbonate, polycaprolactone-glycolide, poly (g-ethylglutamate), poly (DTH-iminocarbonate), poly (DTE-co-DT-carbonate), poly (bisphenol- A-iminocarbonate), polyorthoester, polymethyl trimethyl carbonate, polytrimethyl carbonate, polyiminocarbonate, poly (N-vinyl) pyrrolidone, polyvinyl alcohol, polyesteramide , Glycolated polyester, polyphosphoester, polyphosphazene, poly [(p-carboxyphenoxy) propane], polyhydroxypentanoic acid, polyanhydride, polyethylene oxide-propylene oxide, soft polyurethane, backbone Polyurethanes having amino acid residues in the base, polyether esters such as polyethylene oxide, polyalkenoxalates, polyorthoesters and copolymers thereof, lipids, car Jinan, fibrinogen, starch, collagen, protein-based polymer, polyamino acid, synthetic polyamino acid, zein, modified zein, polyhydroxyalkanoate, petic acid, ethic acid, modified and unmodified fibrin and casein, carboxy Methylsulfate, albumin, hyaluronic acid, chitonic acid and derivatives thereof, heparan sulfate and derivatives thereof, heparin, chondroitin sulfate, dextran, b-cyclodextrin, copolymer of PEG and polypropylene glycol, gum arabic, guar , Gelatin, collagen, collagen-N-hydroxysuccinimide, lipids, phospholipids, variants and copolymers and / or mixtures of the aforementioned substances.

As a biostable material for the biostable layer, it can be used as: polyacrylic acid, polymethyl methacrylate as polyacrylate, polybutyl methacrylate, polyacrylamide, polyacrylonitrile, polyamide, polyether Amide, polyethyleneamine, polyimide, polycarbonate, polycarbonate, polyvinyl ketone, polyvinyl halogenide, polyvinylidene halogenide, polyvinyl ether, polyisobutylene, polyvinyl aromate , Polyvinyl ester, polyvinylpyrrolidone, polyoxymethylene, polytetramethylene oxide, polyethylene, polypropylene, polytetrafluoroethylene, polyurethane, polyetherurethane, silicone-polyetherurethane, silicone-polyurethane, silicone Polycarbonate-urethane, polyolefin elastomer, polyisobutylene, EPDM gum, fluorosilicone, Carboxymethyl chitosan, polyaryl ether ether ketone, polyether ether ketone, polyethylene terephthalate, poly valerate, carboxymethyl cellulose, cellulose, rayon, rayon triacetate, cellulose nitrate, cellulose acetate, hydroxyethyl cellulose, cellulose Butyrate, cellulose acetate butyrate, ethyl vinyl acetate copolymer, polysulfone, epoxy resin, ABS resin, EPDM gum, silicone as polysiloxane, polydimethylsiloxane, polyvinylhalogen and copolymers, cellulose ether, cellulose triacetate, chitosan and these materials Copolymers and / or mixtures thereof.

Medical devices having a hemophilic surface described herein can provide any. In particular, one may provide suitable for contact with blood or blood products for short or long term. Such medical devices include, for example, prostheses, organs, blood vessels, arteries, heart valves, tubes, organ replacement parts, implants, fibers, hollow fibers, stents, hollow needles, syringes, membranes, tinned goods, Blood vessels, titration plates, pacemakers, absorption media, chromatography media, chromatography columns, dialysis machines, connecting parts, sensors, valves, centrifuge chambers, recuperators, endoscopes, filters, pump chambers. The present invention relates in particular to stents.

The polysaccharide of formula (1) may be produced from heparin and / or heparansulfate. These materials are structurally very similar compounds. Heparan sulfate has significant differences in molecular weight, degree of acetylation and degree of sulfateization, depending on the type of mammal. Heparan sulfate derived from the liver, for example, exhibits an acetylation coefficient of about 50%, while heparan sulfate of glycocalyx derived from endothelial cells may exhibit an acetylation coefficient of at least about 90%. Heparin shows only very small amounts of acetylation up to about 5%. The sulfated coefficient of heparan sulphate and heparin from the liver is less than 2 per disaccharide unit, heparan sulphate from endothelial cells is close to zero, and heparan sulphate from other cell types is 0 to 2 per disaccharide unit.

Compounds of formula (1) are characterized by an amount of sulphate groups per disaccharide unit of less than 0.05. In addition, the amount of free amino groups in these compounds is less than 1% based on all —NH—Y groups.

The following figure shows the glycoside units of heparin or heparansulfate, with the typical orientation of heparin, having a random orientation of sulfate groups and a sulfated coefficient of 2 per disaccharide unit:

All heparan sulfates have a consensus sequence in biosynthesis with heparin. First of all, key proteins with xylose-containing binding regions are formed. It consists of xylose and two galactose residues linked thereto. At the end of the two galactose units, glucuronic acid and galactosamine are alternately connected until an appropriate chain length is formed. Finally, several levels of enzymatic modification of all common heparan sulfate and heparin such common polysaccharide precursors are by means of sulfotransferase and epimerase, and these enzymes undergo different conversions in different spectra of different hepases. Forms lansulfate and ultimate heparin.

Heparin is an alternate linkage between D-glucosamine and D-glucuronic acid or L-iduronic acid, with an amount of L-iduronic acid of 75% or less. D-glucosamine and D-glucuronic acid are in β-1,4-glycosidic manner, and L-iduronic acid is linked to disaccharides by α-1,4-glycosidic bonds to form heparin subunits. These subunits are in turn connected to each other in a β-1,4-glycosidic manner to form heparin. The location of sulfonyl groups varies. On average one glycoside unit contains 4 to 5 sulfuric acid groups. Heparan sulfate, also called heparin sulfate, contains less N- and O-linked sulfonyl groups than heparin, except heparan sulfate, which is derived from the liver, and on the contrary, contains more N-acetyl groups. The amount of L-iduronic acid is less than heparin.

As is apparent from FIG. 1, the compound of formula (1) (eg, for example FIG. 1B) is structurally similar to the natural heparan sulphate of endothelial cells but overcomes the drawbacks first mentioned with the use of endothelial heparan sulphate. .

In the commercial heparin preparations, certain pentose units, which can be found about every third molecule, are responsible for antithrombotic activity. Other antithrombotic heparin preparations can be produced by specific separation techniques. In highly active, for example preparations obtained by antithrombin-III-affinity chromatography (high affinity heparin) such active sequences are found in all heparin molecules, while in the case of affinity preparations the characteristic saccharide sequence Not present and thus no coagulation inhibitory activity can be detected. Through these interactions with the saccharide, the activity of antithrombin III, an inhibitor of the coagulation core factor thrombin, is essentially exponential (binding affinity is increased by 2 × 10 ³ fold) [Stiekema JCJ; Clin Nephrology 26, Suppl. Nr 1, S3-S8, 1986].

Most amino groups of heparin are either N-sulfated or N-acetylated. The most important O-sulfated sites are C2 in idouronic acid as well as C6 and C3 in glucosamine. The activity of the saccharide in plasma coagulation is basically the sulfate group on C6 and the other functional groups in a small proportion.

The surface of the medical graft coated with heparin or heparan sulfate is only limited by the coating and remains in such a state. Heparin or heparan sulphate added on the artificial surface significantly loses antithrombotic activity associated with limited interactions with antithrombin III due to the steric hindrance of the pentose units mentioned. Due to the immobilization of these polyanionic substances, the strong adsorption of plasma proteins on the heparinized surface eliminates the coagulation inhibitory effect of heparin on the one hand, separate from heparan sulfate on the one hand, and on the other hand, thereby allowing tertiary structure to be formed. Changing plasma proteins (eg, albumin, fibrinogen, thrombin) and platelets adsorbed thereto are observed in all cases of initiating a specific coagulation process.

Thus, a correlation exists on the one hand between the limited interaction of antithrombin III with the pentose units by immobilization and on the other hand, precipitation of plasma proteins occurs on the heparin layer versus the heparan sulfate layer of the medical implant, Loss of antithrombogenic properties and even reverse, because plasma protein adsorption that occurs over a few seconds leads to loss of anticoagulant surface and that the adsorbed plasma proteins alter their tertiary structure, resulting in surface antithrombogenicity. This is due to the change and the formation of a thrombotic surface. Surprisingly, it can be detected that the compound of formula (1) still shows heparin's blood affinity properties despite the structural difference of heparin to heparan sulfate, and further represented as the first step in the activation of the coagulation process after immobilization of the compound. It could not be observed to the extent that the coagulation of plasma protein was revealed. The compound blood affinity properties according to the invention still remain after being fixed to these artificial surfaces.

In addition, it is assumed that the sulfate group of heparin and heparan sulfate is required for interaction with antithrombin III, thereby providing the anticoagulant effect of heparin and heparan sulfate. The compounds of the present invention are not active coagulation inhibitors, ie anticoagulants, due to the almost complete desulphationation in which the sulfate groups are removed up to a small amount of less than 0.2 sulfate groups per disaccharide unit.

Compounds of formula (1) of the present invention may be produced from heparin or heparansulfate by the first near complete desulfation of the polysaccharide followed by almost complete N-acylation. The term “nearly complete desulfation” refers to the degree of desulfation of at least 90%, preferably at least 95%, particularly preferably at least 98%. The desulfation coefficient can be determined according to the so-called ninhydrin test, which indicates a free amino group. Desulfation takes place in such a way that no color reaction is obtained using DMMB (dimethylmethylene blue). This color test is suitable for the display of sulfated polysaccharides but the limit of detection is unknown in the literature. Desulfation can be carried out, for example, by pyrolysis of pyridinium salts in a solvent mixture. In particular, a mixture of DMSO, 1,4-dioxane and methanol has proven to be suitable.

Heparin as well as heparan sulfate were desulphated through total hydrolysis and subsequently reacylated. The number of sulfate groups per disaccharide unit (S / D) was then determined by ¹³ C-NMR. Table 1 below shows their results for examples of heparin and desulfated and reacetylated heparin (Ac-heparin).

Table 1: Distribution of functional groups per disaccharide unit of heparin and Ac-heparin example determined by ¹³ C-NMR measurement

2-S 6-S 3-S NS N-Ac NH ₂ S / D Heparin 0.63 0.88 0.05 0.90 0.08 0.02 2.47 Ac-heparin 0.03 0 0 0 1.00 - 0.03

2-S, 3-S, 6-S: sulfate groups in positions 2, 3 and 6, respectively

NS: Sulfate group on amino group

N-Ac: Acetyl group on amino group

NH ₂ : free amino group

S / D: Sulfate group per disaccharide unit.

A sulfate content of about 0.03 sulfate group / disaccharide unit (S / D) in Ac-heparin was regenerated as compared to about 2.5 sulfate group / disaccharide units in heparin.

As mentioned above, the difference in sulfate content of heparin and heparan sulfate has a significant effect on the anti-thrombin III counter activity and on the coagulation effect of these compounds. These compounds have a content of sulfate group per dimer unit of less than 0.2, preferably less than 0.07, more preferably less than 0.05 and particularly preferably less than 0.03.

By removing the sulfate group of heparin, which is responsible for the active coagulation inhibitory action mechanism, hemophilic, coagulant inactive oligosaccharides and polysaccharides suitable for surface refining are obtained. These on the one hand exhibit no activity in the coagulation process and on the other hand are not detected by the coagulation system as a foreign surface. Thus, these coatings successfully mimic the highest standards of blood affinity and persistence for the coagulation active component of blood. Examples 3 and 4 illustrate such surfaces, and these surfaces are coated with the compounds of the invention, in particular covalently, to provide a continuous, non-thrombotic and hemophilic coating. This is clearly demonstrated by the example of Ac-heparin.

Almost completely N-acylated refers to a degree of N-acylation of at least 94%, preferably at least 97%, particularly preferably at least 98%. Acylation is carried out in such a way that the color reaction is no longer obtained with the nihydrin reaction for the detection of free amino groups. Preferably used as the acylating agent is carboxylic acid chloride, -bromide or anhydride. Acetic anhydride, propionic anhydride, butyric acid anhydride, acetic acid chloride, propionic acid chloride or butyric acid chloride are for example suitable for synthesizing the compounds of the present invention. Particularly suitable as acylating agents are carboxylic acid anhydrides.

In particular, as a solvent for carboxylic acid anhydride, deionized water is used in particular together with a cosolvent, and the cosolvent is added in an amount of 10 to 30% by volume. Suitable as cosolvents are methanol, ethanol, DMSO, DMF, acetone, dioxane, THF, ethyl acetate and other polar solvents. When using carboxylic acid halogenides, preferably polar anhydrous solvents such as DMSO or DMF are used.

Compounds of the formula according to the invention comprise a carboxylate group in half the sugar molecule and an N-acyl group in the other half.                 

The present invention relates to the use of the compounds of formula (1) as well as salts of these compounds for the coating of natural and / or artificial surfaces, in particular hemophilic coatings. A characteristic of a compound according to the invention in the context of "hemophilicity" means that it does not interact with the compound or platelet of the blood coagulation system and thereby does not initiate the blood coagulation process.

The present invention also provides polysaccharides for hemophilic coating of the surface. Preferred are polysaccharides having a range of molecular weight limits mentioned above. The polysaccharides used are that they characteristically contain significant amounts of the sugar building unit N-acylglucosamine. This means that 40 to 60% of the sugar building units are N-acylglucosamine and substantially the remaining sugar building units contain each carboxyl group. Polysaccharides generally consist of only two sugar building units of at least 95%, preferably at least 98%, whereas one sugar building unit contains a carboxyl group and the other contains N-acyl groups.

One sugar building unit of the polysaccharide is N-acylglucosamine, preferably N-acetylglucosamine, and the other case is uronic acid glucuronic acid and iduronic acid. Preferred are polysaccharides that substantially mimic sugar glucosamine, whereas substantially half of the sugar building units have N-acyl groups, preferably N-acetyl, while the other half of the glucosamine building units are by amino groups or one or more It has one carboxyl group bonded directly by a methylenyl group. In the case of these carboxylic acid groups bonded to amino groups, carboxymethyl- or carboxyethyl groups are preferred. Also preferred are polysaccharides that substantially simulate half of N-acylglucosamine, preferably half of N-acetylglucosamine and the other half substantially mimic those of uronic acid glucuronic acid and iduronic acid. Especially preferred are polysaccharides which show a substantially alternating sequence of N-acylglucosamine with one of the two uronic acids.

Surprisingly, desulfated and substantially N-acylated heparin is particularly suitable in the use of the present invention. In particular, N-acetylated heparin is suitable for hemocompatible coatings.

The term "substantially" refers to statistical variables (deviations). One substantial alternating sequence of sugar building units generally implies that two identical sugar building units are not linked to one another but do not completely rule out such defective linkages. "Substantially half" means almost 50% but allows a few variables. This is because, in particular for biosynthesized macromolecules, the ideal case is never achieved and some variables must always be considered, enzymes do not work completely and some error is always expected in catalytic degradation. For natural heparin there is a significant alternating sequence of N-acetylglucosamine and uronic acid.

Also described is a method of blood affinity coating of surfaces. In particular, cases where direct blood contact is required are described. For these methods, natural and / or artificial surfaces are provided and the polysaccharides described above are immobilized on these surfaces.

Fixation of polysaccharides on these surfaces can be achieved by hydrophobic interactions, van der Waals forces, electrostatic interactions, hydrogen bonding, ionic interactions, crosslinking of polysaccharides and / or covalent bonds on the surface. Preferred are covalent linkages (side chain bonds) of the polysaccharide, more preferred are covalent single point linkages (side chain bonds), and particularly preferred are covalent end point linkages (terminal bonds).

Hereinafter, the coating method of the present invention is described. The biological and / or artificial surface of the medical device can be treated with a blood affinity coating using the following methods:

a) providing a surface of the medical device;

b) depositing at least one of the compounds of formula (1) according to claim 1 as a hemophilic layer on this surface; And / or

b ') A biostable and / or biodegradable layer is deposited on the surface of the medical device or hemophilic layer.

"Deposition" means coating at least a portion of a surface with a suitable compound, wherein the compound is located and / or fixed on or in contact with the surface abuted or fixed by any means.

“Substantially the remaining sugar building units” should be understood as 93%, preferably 96%, particularly preferably 98% of the remaining 60% to 40% of the remaining sugar building units have carboxyl groups. .

Preferably an uncoated and / or non-hemophilic surface is provided. A "non-blood affinity" surface refers to a surface that is capable of activating a blood coagulation system and is therefore thrombogenic.

Further aspects include the following steps:

a) providing a surface of the medical device;

b) depositing at least one of the polysaccharides of formula (1) according to the invention;                 

b ') depositing a biostable layer on the surface of the medical device;

d ') deposits an additional hemophilic layer of at least one of the polysaccharides of formula (1) according to the invention.

The last mentioned embodiment ensures that the surface coating does not lose blood affinity characteristics, for example even in the case of mechanical damage of the polymer layer and in parallel with mechanical damage of the hemophilic outer layer.

A "biological or artificial" surface is an example in which an artificial part is incorporated into an artificial medical device, such as a pig heart having an artificial heart valve.

The single layer is preferably deposited by dipping or spraying methods. On the other hand, paclitaxel can be deposited simultaneously on one layer of the medical device and then on individual layers covalently and / or bonded. In this way it is possible to deposit the active substance paclitaxel while simultaneously depositing a hemophilic layer on the medical device. Materials for biostable or biodegradable layers have been mentioned above.

It is then possible to deposit the active material layer of paclitaxel as an additional optional step c) on the first biostable and / or biodegradable or hemophilic layer. In a preferred embodiment paclitaxel is covalently bonded to the underlying layer. In addition, paclitaxel is preferably deposited on and / or in the hemophilic layer or biostable layer by dipping or spraying methods.

After step b) or c), further step d) may be carried out by depositing at least one biodegradable layer and / or at least one biostable layer on the hemophilic layer and / or the paclitaxel layer.

In another embodiment, after step b ') or step c), the step d') of depositing at least one of the compounds of formula (1) as a hemophilic layer on the biostable and / or biodegradable layer and / or the paclitaxel symptoms may be carried out. Can be.

Deposition of paclitaxel after step d) and / or d ') may occur in and / or on one or more biodegradable and / or biostable layers or hemophilic layers. The paclitaxel as well as the monolayer is preferably deposited and / or executed on and / or in the underlying layer by dipping and / or spraying methods.

In a preferred embodiment, the biostable layer is deposited on the surface of the medical device and on which the hemophilic layer is deposited, preferably covalently bonded.

Preferably, the hemophilic layer is characterized by a different degree of sulfateization (sulfation degree) and acylation coefficient (degree of acylation) in the molecular weight range of the saccharide that is responsible for antithrombotic activity, ie, up to the commercially available heparin standard molecular weight of 13 kD. Naturally heparin, heparansulfate and derivatives thereof of the site-selectively synthesized derivatives, oligo- and polysaccharides of erythrocyte glycocalyx, desulphated and N-reacetylated heparin, N-carboxymethylated and / or partially N- Acetylated chitosan and / or mixtures of these materials.

Subjects of the invention are also medical devices that are hemophilically coated according to one of the methods mentioned herein. For medical devices, stents are preferred.

Conventional stents that can be coated according to the method of the present invention consist of stainless steel, nitinol or other metals and alloys or consist of synthetic polymers.                 

The stent of the present invention is coated with the compound of formula (1), preferably the blood affinity layer is coated with a covalent bond. The second layer completely or incompletely covers this first hemophilic layer. This second layer preferably conspires paclitaxel. The hemophilic layer of the stent, on the one hand, preferably provides the necessary blood affinity and thus reduces the risk of thrombosis and the containment of the inflammatory response due to invasion and absence of non-endogenous surfaces and is evenly distributed over the entire surface of the stent. Paclitaxel occurs in a controlled manner when the surface of the stent is covered with cells, especially smooth muscle and endothelial cells, resulting in the interaction of the thrombosis and inflammatory reactions, the release of growth factors, and the proliferation and migration of cells during the recovery process. To provide for the formation of a newly "recovered" cell layer.

Thus, the use of paclitaxel covalently and / or adhesively bonded to the underlying layer and / or provided covalently and / or adhesively to one or more layers results in the release of this active material continuously and in small amounts, As a result, the clustering of cells on the surface of the stent is suppressed, but it is ensured that excessive clustering and internal growth of cells into the vascular cavity are prevented. The combination of the two affects the ability of the stent according to the invention to grow rapidly into the vessel wall and reduce both the risk of restenosis and the risk of thrombus. The release of paclitaxel occurs over a period of 1 to 12 months, preferably 1 to 3 months after transplantation.

Paclitaxel is preferably contained at a pharmacologically active concentration of 0.001-10 mg, preferably 0.01-5 mg, particularly preferably 0.1-1.0 mg per cm ² of stent surface. Additional active substances may be contained in the same or similar concentrations in the hemophilic layer.

The amount of polymer applied per layer is 0.01 mg to 3 mg, preferably 0.20 mg to 1 mg, particularly preferably 0.2 mg to 0.5 mg. Such coated stents release the continuously controlled active substance paclitaxel and are therefore well suited for the prevention and reduction of restenosis.

These stents with a hemocompatible coating are formed by providing the stent and preferably covalently depositing one hemophilic layer of formula (1). The hemophilic layer permanently shields the surface of the implant after the release of the active material and thus after the influence of the active material is dissipated.

Preferred embodiments of the stent according to the invention show a coating consisting of two or more layers. This layer thus deposited on the first layer is called the second layer. According to the design of the two layers, the first layer simulates a blood affinity layer, which layer is substantially completely covered by covalent bonding and / or adhesion by a second layer of paclitaxel.

The paclitaxel layer slowly dissolves, thus releasing the active material at the rate of dissolution process. The first hemophilic layer ensures the necessary blood miscibility of the stent to the extent that the active substance is removed. By the release of the active substance, the adsorption of cells is significantly reduced for only a certain period of time and the desired controlled adsorption is possible, with the outer layer already widespread. Finally, the hemophilic layer remains a non-thrombotic surface and shields the foreign surface in such a way that a life-threatening reaction may no longer occur.                 

Such stents can be produced by blood affinity coating of the stent in the following manner:

a. Providing the stent

b. Preferably deposits a covalently bound hemophilic layer

c. The affinity layer is substantially completely covered with the antiproliferative active substance paclitaxel by a dipping or spraying method.

The stent of the present invention solves both the problem of acute thrombus and the problem of neointimal hyperplasia after stent implantation. The stents of the invention are also particularly suitable because they are coatings for the continuous release of one or more antiproliferative, immunosuppressive active substances. The coated stent of the present invention almost completely prevents the risk of restenosis since it is capable of continuously releasing the active substance in the required amount as desired.

The natural and / or artificial surface coated with the hemophilic layer of the polysaccharide according to the method described above is in the form of a stent in combination with an antiproliferative active substance, preferably paclitaxel, for the prevention of restenosis, blood vessels and blood It is particularly suitable as an implant and / or organ replacement in direct contact with it.

Medical devices coated according to the present invention not only are particularly suitable for direct and permanent blood contact, but also surprisingly show features that reduce or even prevent the adsorption of proteins on similarly coated surfaces. Adsorption of plasma proteins onto foreign surfaces in contact with blood is an essential first step for further progress in the recognition and transitional actions of the blood system.                 

This is important, for example, for in vitro diagnosis from body fluids. Thus, the deposition of the coatings according to the invention generally results in nonspecific adsorption of proteins onto microtiter plates or other supporting media used in diagnostic detection methods, which can interfere with sensitive test reactions and lead to errors in assay results. Prevent or at least reduce it.

By using the coatings according to the invention on an adsorption medium or on a chromatography medium, nonspecific adsorption of proteins is also prevented or reduced, so that better separation can be achieved and products of higher purity can be produced.

FIG. 1 shows the glycoside units of heparin or heparansulfate, with the sulfated coefficient of 2 per disaccharide and the statistical distribution of sulfate groups as a typical case of heparin (FIG. 1A). 1B shows an example of a compound of the formula described herein to compare structural similarity.

2 shows the effect of PVC-tubular expanded, surface modified stainless steel tubular stents on platelet loss (PLT-loss). Uncoated stainless steel tubular stents were measured by reference. As a value of zero, the level of platelet loss was set in the case of PVC-tubes without a stainless steel tubular stent. As such, SH1 is a heparin covalently coated stent, SH2 is a chondroitinsulfate coated stent, SH3 is a stent coated with a polysaccharide derived from red blood cell glycocalyx, and SH4 is an ac-heparin covalently coated Stainless steel tubular stent.                 

Figure 3 shows the restenosis rate of oligosaccharides and polysaccharide coated stents of fully desulfated and N-reacetylated heparin (Ac-heparin) covalently coated stents and erythrocyte glycocalyx uncoated stents and polyacrylic acid (PAS) Coated stents were compared 4 weeks after transplantation into pigs.

FIG. 4 is a quantitative inertial prototype: image (a) of a cross section through a stent containing vessel segment with an Ac-heparin coated stent and a corresponding image (b) of an uncoated stent as a comparison. Obvious differences were observed in the thickness of neointimal formation formed after 4 weeks in animal experiments (pigs).

5 is an elution plot of paclitaxel from a stent (without support medium).

Example 1

Synthesis of Desulfated and Reacetylated Heparin:

100 ml Amberlite IR-122 cation exchange resin was added to the 2 cm diameter column with 400 ml 3M HCl in the converted H ⁺ -form and washed with distilled water until the eluate was free of chloride and the pH was neutral. 1 g sodium-heparin was dissolved in 10 ml water and added on a cation exchange column, then eluted with 400 ml water. The eluate was added dropwise to a vessel containing 0.7 g pyridine and then titrated with pyridine to make pH 6 and lyophilized.

0.9 g heparin-pyridinium-salt was added to a round flask equipped with a reflux condenser (condenser) and containing 90 ml of a 6/3/1 mixture of DMSO / 1,4-dioxane / methanol (v / v / v). Next, it heated to 90 degreeC for 24 hours. 823 mg pyridinium chloride was then added and heated to 90 ° C. for another 70 hours. Then, diluted with 100 ml water and titrated with dilute sodium hydroxide to make pH 9. Desulfated heparin was dialyzed against water and lyophilized.

100 mg of desulphated heparin was dissolved in 10 ml of water, cooled to 0 ° C. and added under stirring with 1.5 ml methanol. The solution to the OH ^{- -} 4 ml of the type also Wexford 1x4 was added to an anion exchange resin was added to 150 μl of acetic anhydride was stirred for 2 hours at 4 ℃. The resin was then filtered off and the solution dialyzed against water and then lyophilized.

Example 2

Synthesis of Desulfated N-propionylated Heparin:

100 ml Amberlite IR-122 cation exchange resin was added to the 2 cm diameter column with 400 ml 3M HCl in the converted H ⁺ -form and washed with distilled water until the eluate was free of chloride and the pH was neutral. 1 g sodium-heparin was dissolved in 10 ml water and added on a cation exchange column, then eluted with 400 ml water. The eluate was added dropwise to a vessel containing 0.7 g pyridine and then titrated with pyridine to make pH 6 and lyophilized.

0.9 g heparin-pyridinium-salt is added to a round flask equipped with a reflux condenser and containing 90 ml of a 6/3/1 mixture of DMSO / 1,4-dioxane / methanol (v / v / v), and then 90 Heated to ° C. for 24 h. 823 mg pyridinium chloride was then added and heated to 90 ° C. for another 70 hours. Then, diluted with 100 ml water and titrated with dilute sodium hydroxide to make pH 9. Desulfated heparin was dialyzed against water and lyophilized.

100 mg of desulphated heparin was dissolved in 10 ml of water, cooled to 0 ° C. and added under stirring with 1.5 ml methanol. To this solution was added 4 ml Dow's 1 × 4 anion exchange resin in the form of OH ⁻ −, then 192 μl of propionic anhydride was added and stirred at 4 ° C. for 2 hours. The resin was then filtered off and the solution dialyzed against water and then lyophilized.

Example 3

Determination of blood affinity of compounds of formula (1) according to ISO 10933-4 (in vitro measurement):

Covalent coating of the compound of formula (1) on cellulose membranes, silicone tubes and stainless steel stents to determine the blood affinity of the compound of formula (1) and the corresponding done on heparin as well as uncoated material surfaces used in a single test Was tested.

3.1 Cellulose membrane coated with desulphated and reacetylated heparin (Ac-heparin) (cupropane)

An open perfusion system of Sakariassen-modified Baumgartner-chambers is used for the examination of the coagulating physiological interactions between citrated whole blood and Ac-heparin- and / or heparin-coated cupropane membranes [Sakariassen K.S. et al .; J. Lab. Clin. Med. 102: 522-535 (1983). The chamber consists of four building parts, conical faucets and connected fittings, made of polymethylmethacrylate, and includes a statistical coverage at every run, allowing equilibrium investigation of two modified membranes. The structure of this chamber allows for quasi-lamina reflux conditions.

After permeation at 37 ° C. for 5 minutes, the membrane is extracted, the platelets are occupied after fixation of adjacent platelets. Individual results were established with respect to the highly thrombotic endothelial matrix as a negative standard with 100% platelet occupancy. Adsorption of platelets occurs secondary before the formation of a plasma protein layer on foreign material. Plasma protein fibrinogen acts as a cofactor of platelet aggregation. Such induced activation of platelets results in the binding of several kinds of aggregated associated plasma proteins, such as Vitronectin, Fibronectin and von Villebrand-Factor, to the platelet surface. These effects finally lead to irreversible aggregation of platelets.

Platelet occupancy provides an acceptable power of the surface thrombogenicity in the case of foreign surface contact of blood because of the described interactions. From this fact the following conclusions are drawn: The lower the platelet occupancy on the perfused surface, the higher the affinity of the tested surface. The results of the tested heparin-coated and Ac-heparin-coated membranes show a marked improvement in the affinity of the foreign surface through coating with Ac-heparin. Heparin-coated membranes show 45-65% platelet occupancy, while Ac-heparin-coated surfaces show values of 0-5% (see subcutaneous matrix with 100% platelet occupancy).

Adsorption of platelets on Ac-heparinized surfaces becomes quite difficult due to their absence, for activation of platelet essential plasma proteins. In contrast, heparin-coated surfaces with immediate initial plasma protein adsorption provide optimal prerequisites for activation, deposition and aggregation of platelets, and ultimately the blood reacts with the corresponding defense mechanisms on the inserted foreign surface. . Ac-heparin satisfies the requirements for foreign affinity of the foreign surface much better than heparin.

The interaction of plasma protein adsorption and platelet occupancy as a direct driver for surface thrombusability, depending on the blood provided coating, is particularly well demonstrated by this in vitro test. Thus, the use of covalently bound heparin as an antithrombogenic surface is only quite limited or not at all possible. The interaction of immobilized heparin with blood leads to unwanted effects-heparin-coated surfaces induce thrombus formation.

Obviously, the significant importance of heparin as an antithrombotic agent does not apply to covalently immobilized heparin. When administered systemically in dissolved form, their properties can be fully revealed. However, if heparin is not covalently immobilized, the antithrombotic properties are only short-lived. Ac-heparin, in fact, completely loses the antithrombogenic properties of the initial molecule due to desulfation and N-reacetylation, but differs by reacquiring distinct nonthrombotic properties. This property is demonstrated in the passiveness to antithrombin III and the loss affinity for the initial aggregation process and is maintained after covalent binding.

As such, Ac-heparin and thus the compound of formula (1) are optimal as gastrointestinal means of foreign surfaces in contact with the aggregation system.

3.2. Fixation on silicone

100 ml of a mixture of ethanol / water 1/1 (v / v) were pumped through a 1 mm long silicone tube with a 3 mm inner diameter at 40 ° C. for 30 minutes. Then 2 ml of 3- (triethoxysilyl) -propylamine are added and pumped for an additional 15 hours at 40 ° C. by circulation. It is then washed with 100 ml ethanol / water and 100 ml water for 2 hours each.

3 mg of deacetylated and reacetylated heparin (Ac-heparin) was dissolved in 30 ml 0.1 M MES-buffer pH 4.75 at 4 ° C. and 30 mg CME-CDI (N-cyclohexyl-N ′-(2- Morpholinoethyl) carbodiimidemethyl-p-toluenesulfonate). This solution is pumped through the tube in circular motion at 4 ° C. for 15 hours. Then rinse with water, 4M NaCl solution and water for 2 hours each.

3.3. Measurement of platelet count (EN30993-4)

Two 2 cm long glass tubes made for this shape were placed in a 2 m long silicone tube with an internal diameter of 3 mm. The tube was then closed with a shrinking round tube and filled with 0.154 M NaCl solution through a syringe under the exclusion of air. In doing so, one syringe was used to fill the solution and the other syringe was used to remove air. The solution was exchanged under the exclusion of air with two syringes for citrated whole blood of healthy test subjects. The concave hole of the syringe was then closed by pushing the glass tube over them and the tube tightly inserted into the dialysis pump. Blood was pumped at a flow rate of 150 ml / min for 10 minutes. Platelet content of blood was measured before and after perfusion with a Coulter counter. Platelet loss was 10% for uncoated silicone tubes. In contrast, the silicon tube coated according to Example 5.2 had an average loss of 0% (number of experiments: n = 3).

In this dynamic test system it is also shown that the activation of platelets on the Ac-heparin coated surface is reduced. At the same time, the fixation of heparin can be recorded to achieve a negative effect on the affinity of the surface used. In contrast, Ac-heparin shows no effect on contact with platelets according to its passive nature.

3.4 Whole Blood Testing on 316 LVM Stainless Steel Coronary Stents

In addition to biocompatibility experiments, a 31 mm long 316 LVM stainless steel stent was covalently coated with Ac-heparin. For the occupancy factor of the entire surface of 2 cm ^{2 and} the surface of about 20 pm / cm ² stents, the charge of this stent is about 0.35 μg Ac-heparin. In comparison, for the prevention of thrombosis, the daily application rate of heparin is usually 20-30 mg, which is therefore at least 60,000 times.

These experiments were performed with a set hemodynamic Chandler loop-system [A. Henseler, B. Oedekoven, C. Andersson, K. Mottaghy; KARDIOTECHNIK 3 (1999)]. The coated and uncoated stents were expanded and tested in PVC tubes (medical grade PVC) with a length of 600 mm and an internal diameter of 4 mm. The results of these experiments verify that of the experiments discussed in the silicon tube. Initially, 50% platelet loss in the perfusion due to stents is reduced to over 80% by refining the stent surface with Ac-heparin.

The effect on platelet loss of tubular expanded and surface modified coronary stents is assessed in an additional Chandler test during 45 minutes of whole blood perfusion. To this end, a stent-glass PVC tube is first analyzed, with a result of zero. The empty tube shows an average platelet loss of 27.4% for donor blood at a standard deviation of only 3.6%. This baseline was the basis that different surface modified stents were expanded under PVC tubes and analyzed under similar conditions for platelet loss caused by them. Even in this case, the stent coated surface, which accounts for only about 0.84% of the total test surface, appears to have a repetitive effect corresponding to the platelet content. According to the empty tube (default), a smooth, chemically uncoated stent results in an additional 22.7% platelet loss on average. Together this results in nearly similar platelet loss with measurable foreign surfaces of less than 1% when compared to PVC hollow tubes. The direct result is that even though this test surface only accounts for 0.84% of the total surface, medical stainless steel 316 LVM used as a stent induces about 100 times more potent platelet damage compared to medical grade PVC surfaces.

Analyzed surface coatings of stainless steel coronary stents show that it is possible to very clearly reduce large sized stent induced platelet damage (see FIG. 2). The most effective is 81.5% Ac-heparin (SH4).

If one considers the effect of the Ac-heparin-coated stent on platelet loss, a good match is obtained. The correlation of platelet loss in perfusion versus platelet adsorption on a given surface shows the reliability of the measurements.

3.4.1 Covalent Hemophilic Coating of Stents

The unexpanded stent of medical stainless steel LVM 316 was deoiled with acetone and ethanol for 15 minutes in an ultrasonic bath and dried at 100 ° C. in a dryer. They were then dipped in a 2% solution of 3-aminopropyltriethoxysilane in a mixture of ethanol / water mixture (50/50: (v / v) for 5 minutes and then dried at 100 ° C. for 5 minutes. The stent was then washed overnight with demineralized water.

3 mg of desulphated and reacetylated heparin was dissolved in 30 ml 0.1M MES-buffer (2- (N-morpholino) ethanesulfonic acid) at pH 4.75 at 4 ° C. and 30 mg of N-cyclohexyl Mix with -N '-(2-morpholinoethyl) carbodiimide-methyl-p-toluenesulfonate. Ten stents were stirred in this solution at 4 ° C. for 15 hours. They were then washed with water, 4M NaCl solution and water for 2 hours each.

3.4.2 Determination by HPLC of Glucosamine Content in Coated Stents

Hydrolysis: The coated stent is placed in a small hydrolysis tube and treated with 3 ml of 3M HCl for exactly 1 minute at room temperature. Remove the metal probe and seal the tube in a dryer at 100 ° C for 16 hours before incubating. Then allow them to cool, evaporate three times until dry, then immerse in 1 ml of degassed and filtered water, and measure against hydrolyzed standards by HPLC:

Stent Sample area Desulfate + Reacetylated Heparin [g / sample] Area [cm ² ] Desulfate + Reacetylated Heparin [g / cm ² ] Desulfate + Reacetylated Heparin [pmol / cm ² ] One 129.021 2.70647E-07 0.74 3.65739E-07 41.92 2 125.615 2.63502E-07 0.74 3.56084E-07 40.82 3 98.244 1.93072E-07 0.74 2.60908E-07 29.91 4 105.455 2.07243E-07 0.74 2.80058E-07 32.10 5 119.061 2.33982E-07 0.74 3.16192E-07 36.24 6 129.202 2.53911E-07 0.74 3.43124E-07 39.33 7 125.766 2.53957E-07 0.74 3.43185E-07 39.34

Example 4

In vivo examination of the coated coronary stent (FIG. 5)                 

4.1. In Vivo Testing of Coronary Stents Coated with Ac-heparin

Due to the data on the affinity that Ac-heparin provided in the in vitro experiments, the suitability of Ac-heparin surface as a non-thrombotic coating of metal stents was discussed in vivo (animal experiments).

The aim of the experiment was primarily to assess the effect of Ac-heparin coating on stent induced vascular responses. In addition to recording possible thrombus processes, variables associated with the restenosis process, such as neointimal zone, vascular lumen and stenosis, were recorded. Six to nine month old breeding pigs were used for testing and one was for the validity of the stent for long-term established and approved animal models.

As expected in these experiments, none of the acute, subacute or subacute thrombus processes were recorded. This can be assessed as evidence of the nonthrombotic properties of Ac-heparin.

Four weeks later the animals were euthanized and stent provided coronary sections were extracted and histologically analyzed. No signs of possible acute or subacute toxicity, allergic reactions or stimulation behind as a result of transplantation of Ac-heparin coated stents are observed during the completed experimental phase, especially on histological examination. Subsequent coronary-contrast data sets were validated during stent implantation, which allows interpretation of the vascular response to stent implantation.

The difference between the uncoated control stent and the Ac-heparin coated stent is evident. The production of distinct neointimal layers is very well observed in the case of uncoated control stents. Even after 4 weeks, the proliferation promoting effect of the uncoated stent surface on the surrounding tissue ultimately occurs to the extent that there is a risk of vascular closure in the stent area. In contrast, for Ac-heparin coated stents, a clearly thinner neointimal layer is observed, indicating well-controlled internal growth of the stent under the maintenance of a wide and free vascular cavity.

Detailed histomorphologic and coronary angiographic data consistently showed that Ac-heparin coating (SH4) reduced neoplastic hyperplasia (restenosis) by about 17-20% compared to uncoated control stents. Prove that conclusion as you can. This result is unexpected and significant at the same time. Certainly, in addition to the preconditions of hemophilic character, it affects the process of inducing neointimal hyperplasia, i.e., non-thrombotic surfaces are not required to prevent restenosis.

On the one hand the dense and permanent occupancy of the stent surface by Ac-heparin prevents direct cellular contact with the metal surface. Leakage of certain metal ions into the implant suture tissue could be found as one of the coating induced prevention of direct metal contact, as in the literature discussed as one possible reason for restenosis.

On the other hand, such positive side effects are convincing. The reason is that on the passive non-thrombotic stent surface in the absence of platelet aggregation, the proliferative effect of the released growth factor will be lost. Thus, significant stimulation of neointimal proliferation starting on the vascular cavity side is excluded.

Example 5

Coating of stolo stents by spray method                 

The unexpanded stents prepared in Examples 1 and 2 are weighed and suspended horizontally on a thin metal rod (d = 0.2 mm) fixed to the rotating shaft of the rotating and feeding device and rotated at 28 r / min. The stent is fixed in this manner and the interior of the stent does not contact the rod. Spray the stent with a specific spray at a feed width of 2.2 cm, a feed rate of 4 cm / s, and a 6 cm distance between the stent and the spray valve. After drying at room temperature for about 15 minutes, it is dried overnight in a steam hood and reweighed.

Composition of Spray: 44 mg taxol was dissolved in 6 g chloroform.

Stent number Before coating After coating Coating mass One 0.0194 g 0.0197 g 0.30 mg

Example 6

Determination of Elution Status in PBS-Buffer

2 ml PBS-buffer per stent in a sufficiently small flask is added, sealed with para-film and incubated in a dryer at 37 ° C. After each time interval has ended, the excess solution is pipetted out and the UV absorption at 306 nm is measured.

Claims (37)

At least one portion of the surface of the medical device is directly coated with a hemophilic layer comprising a compound of formula (1) and at least one of salts of these compounds or further coated on one or more interposed biostable or biodegradable layers, Medical devices in which the active substance paclitaxel is present on, in, below or in combination thereof: (Formula 1)

In the above formula, n is an integer between 4 and 1050; Y is residues -CHO, -COCH ₃ , -COC ₂ H ₅ , -COC ₃ H ₇ , -COC ₄ H ₉ , -COC ₅ H ₁₁ , -COCH (CH ₃ ) ₂ , -COCH ₂ CH (CH ₃ ) _{2, -COCH (CH 3) C} 2 H 5, -COC (CH 3) 3, -CH 2 COO -, -C 2 H 4 COO -, -C 3 H 6 COO - or -C ₄ H ₈ COO ^- to be. The medical device of claim 1, wherein Y is residues —CHO, —COCH ₃ , —COC ₂ H ₅ , —COC ₃ H ₇ . The medical device of claim 2, wherein Y is —COCH ₃ . The medical device according to claim 1, wherein a blood affinity layer is directly deposited on the surface of the medical device, and paclitaxel is deposited on the blood affinity layer. The medical device according to any one of claims 1 to 3, wherein at least one biostable layer, or biodegradable layer or blended layer thereof is present below or between the two hemocompatible layers. The medical device according to any one of claims 1 to 3, wherein at least one additional biostable layer, or biodegradable layer or compounding layer thereof, is completely or incompletely coated over the hemophilic layer. The medical device according to any one of claims 1 to 3, wherein at least one active substance paclitaxel layer is present between the biostable and hemophilic layers. 4. The method of claim 1, wherein the paclitaxel is covalently attached or adhered to or adhered to, in combination with, in, or in a biocompatible layer, or in, or in a biodegradable layer. Combined medical device. The method according to any one of claims 1 to 3, wherein the biodegradable material for the biodegradable layer is polyvalerolactone, poly-ε-decaractone, polylactic acid, polyglycolic acid, polylactide, polyglycolide , Copolymers of polylactide and polyglycolide, poly-ε-caprolactone, polyhydroxybutanoic acid, polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxybutyrate-co-valerate, Polyanhydrides such as poly (1,4-dioxane-2,3-dione), poly (1,3-dioxan-2-one), poly-para-dioxanone, polymaleic anhydride From polyhydroxymethacrylate, fibrin, polycyanoacrylate, polycaprolactonedimethylacrylate, poly-b-maleic acid, polycaprolactonebutylacrylate, oligocaprolactonediol and oligodioxanonediol As far as being Block polymers, polyether ester multiblock polymers such as PEG and poly (butylene terephthalate), polyfibolactone, polyglycolic acid trimethylcarbonate, polycaprolactone-glycolide, poly (g-ethylglutamate), poly (DTH-iminocarbonate), poly (DTE-co-DT-carbonate), poly (bisphenol-A-iminocarbonate), polyorthoesters, polyglycolic acid trimethyl-carbonate, polytrimethylcarbonate, Polyiminocarbonate, poly (N-vinyl) pyrrolidone, polyvinyl alcohol, polyesteramide, glycolated polyester, polyphosphoester, polyphosphazene, poly [(p-carboxyphenoxy) propane] , Polyhydroxypentanoic acid, polyanhydrides, polyethylene oxide-propylene oxide, soft polyurethanes, polyurethanes with amino acid rests in the backbone, polyethylene jade Polyether esters such as cation, polyalkenoxalates, polyorthoesters and copolymers thereof, lipids, carrageenan, fibrinogen, starch, collagen, protein-based polymers, polyamino acids, synthetic polyamino acids, zeins, modified zeins, Polyhydroxyalkanoates, pectic acid, actinic acid, modified and unmodified fibrin and casein, carboxymethylsulfate, albumin, hyaluronic acid, chitonic acid and derivatives thereof, heparan sulfate and Derivatives thereof, heparin, chondroitin sulfate, dextran, b-cyclodextrin, copolymers of PEG and polypropylene glycol, gum arabic, guar, gelatin, collagen, collagen-N-hydroxysuccinimide, lipids, phospholipids, Medical devices characterized in that variants and copolymers, mixtures or combinations thereof of the abovementioned materials are used. The polyacrylic acid as a biostable material for the biostable layer, polyacrylate as polymethyl methacrylate, polybutyl methacrylate, polyacrylamide, polyacrylonitrile according to claim 1. , Polyamide, polyetheramide, polyethyleneamine, polyimide, polycarbonate, polycarbonate, polyvinyl ketone, polyvinyl halogenide, polyvinylidene halogenide, polyvinyl ether, polyisobutyl Ethylene, polyvinyl aromate, polyvinyl ester, polyvinylpyrrolidone, polyoxymethylene, polytetramethylene oxide, polyethylene, polypropylene, polytetrafluoroethylene, polyurethane, polyetherurethane, silicone-polyetherurethane, Silicone-polyurethane, silicone-polycarbonate-urethane, polyolefin elastomer, polyisobutylene, EPDM gum, fluoro Silicone, carboxymethyl chitosan, polyaryl ether ether ketone, polyether ether ketone, polyethylene terephthalate, poly valerate, carboxymethyl cellulose, cellulose, rayon, rayon triacetate, cellulose nitrate, cellulose acetate, hydroxyethyl cellulose , Cellulose butyrate, cellulose acetate butyrate, ethylvinylacetate copolymer, polysulfone, epoxy resin, ABS resin, EPDM gum, silicone as polysiloxane, polydimethylsiloxane, polyvinylhalogen and copolymer, cellulose ether, cellulose triacetate, chitosan and A medical device characterized in that copolymers, mixtures or combinations thereof of these substances are used. The medical device according to any one of claims 1 to 3, wherein one of the following active substances is used instead of the active substance paclitaxel: simvastatin, 2-methylthiazolidine-2,4-dicarboxylic acid and the corresponding sodium Salts, macrocyclic suboxides (MCS) and derivatives thereof, activating protein C (aPC), PETN (pentaerythritol tetranitrate), trapidyl, β-estradiol as well as mixtures of these active substances or A mixture of one of these active substances with paclitaxel. A medical device according to any one of claims 1 to 3, wherein the medical device is a prosthesis, organ, blood vessel, artery, heart valve, tube, organ replacement part, implant, fiber, hollow fiber, stent, hollow needle, syringe , Membranes, tinned goods, blood vessels, titration plates, pacemakers, absorption media, chromatography media, chromatography columns, dialysis machines, connecting parts, sensors, valves, centrifuge chambers, recuperators, endoscopes And a filter or a pump chamber. The medical device of claim 12, wherein the medical device is a stent. The medical device according to claim 13, wherein the polymer of formula (1) as defined in claim 1 is deposited on the stent surface in an amount of 0.01 mg to 3 mg / layer. The medical device according to claim 13, wherein the active substance is used at a pharmaceutically active concentration of 0.001 to 10 mg per cm ² of stent surface and per layer. A medical device according to claim 13 used for the prevention or reduction of restenosis. Paclitaxel, simvastatin, 2-methylthiazolidine-2,4-dicarboxylic acid sodium salt, macrocyclic suboxide (MCS), derivatives of MCS, activated protein C (aPC), PETN (pentaerythritol tetra Nitrate), trapidyl, β-estradiol or the medical device according to claim 13 used for the continuous release thereof. The medical device according to any one of claims 1 to 3, which is used for direct contact with blood. The medical device according to any one of claims 1 to 3, which is used for the prevention or reduction of nonspecific adsorption or deposition of proteins on the coated surface of the medical device or both. 19. The medical device of claim 18, wherein the blood-compatible coated surface of the device is used as the surface of micro-titer plates or other carrier media for the detection process. The medical device of claim 19, wherein the hemophilically coated surface of the medical device is used as the surface of the microtiter plate or other carrier medium for the detection process. 19. The medical device of claim 18, wherein the blood affinity coated surface of the medical device is used as the surface of the adsorption medium or the chromatography medium. 20. The medical device of claim 19, wherein the blood affinity coated surface of the medical device is used as the surface of the adsorption medium or the chromatography medium. a) providing a surface of the medical device; b) depositing at least one of the compounds of formula (1) as defined in claim 1 as a hemophilic layer on the surface, thereby providing a blood-friendly coating of the biological or artificial surface of the medical device. Way. The method of claim 24, wherein the hemophilic layer is coated by a dipping or spraying method with at least one biodegradable layer or biostable layer comprising paclitaxel bonded in a covalently bonded or bonded manner. The method of claim 24, further comprising c) depositing paclitaxel into the hemophilic layer, on the hemophilic layer, or in a combination thereof. 27. The method of claim 26, wherein paclitaxel is provided, deposited or deposited on a hemophilic layer or in a hemophilic layer or in a compounding layer thereof by a dipping or spraying method to covalently or adhesively bond or combine with the hemophilic layer How they are combined in a way. 27. The method of claim 26, further comprising: d) depositing one or more biodegradable layers, one or more biostable layers or biodegradable layers or blended layers thereof on a hemophilic layer or paclitaxel layer, respectively, d ') further comprising depositing at least one compound of formula (1) as defined in claim 1 on the paclitaxel layer as a hemophilic layer. The method of claim 28, further comprising e) depositing paclitaxel on at least one or more biodegradable layers, or on a biostable or hemophilic layer. The method of claim 29, wherein paclitaxel is deposited on at least one or more biodegradable layers, or on a biostable layer or on a hemophilic layer, or by at least one biodegradable, biostable or hemophilic layer by a dipping or spraying method. By covalent or adhesive bonding or combinations thereof. delete 31. The method according to any one of claims 24 to 30, wherein the hemophilic layer has a different sulfated and acylation coefficient within the molecular weight range of the pentose sugar responsible for antithrombotic activity, i. Natural heparin, heparan sulfate and derivatives thereof, oligo- and polysaccharides of erythrocyte glycocalix, desulphated and N-reacetylated heparin, N-carboxymethylated and partially N-acetyl Oxidized chitosan, or partially N-acetylated chitosan or N-carboxymethylated chitosan or mixtures of these materials. 31. The method according to any one of claims 28 to 30, wherein the biodegradable material for the biodegradable layer is polyvalerolactone, poly-ε-decaractone, polylactic acid, polyglycolic acid, polylactide, polyglycolide , Copolymers of polylactide with polyglycolide, poly-ε-caprolactone, polyhydroxybutanoic acid, polyhydroxybutyrate, polyhydroxy valerate, polyhydroxybutyrate-co-valerate, poly (1, Polyanhydrides such as 4-dioxane-2,3-dione), poly (1,3-dioxan-2-one), poly-para-dioxanone, polymaleic anhydride, polyhydroxy Multi, such as formed from methacrylate, fibrin, polycyanoacrylate, polycaprolactone dimethyl acrylate, poly-b-maleic acid, polycaprolactone butyl acrylate, oligocaprolactonediol and oligodioxanonediol Block Polymer, PEG Polyether ester multiblock polymers such as poly (butylene terephthalate), polyfibotolactone, polyglycolic acid trimethylcarbonate, polycaprolactone-glycolide, poly (g-ethylglutamate), poly (DTH-imino Carbonates), poly (DTE-co-DT-carbonate), poly (bisphenol-A-iminocarbonate), polyorthoesters, polyglycolic acid trimethyl-carbonate, polytrimethylcarbonate, polyiminocarbonate , Poly (N-vinyl) pyrrolidone, polyvinyl alcohol, polyesteramide, glycolated polyester, polyphosphoester, polyphosphazene, poly [(p-carboxyphenoxy) propane], polyhydroxyphene Polycarbonates such as carbonic acid, polyanhydrides, polyethylene oxide-propylene oxide, soft polyurethanes, polyurethanes having amino acid residues in the backbone, and polyethylene oxides. Le esters, polyalkenoxalates, polyorthoesters and copolymers thereof, lipids, carrageenans, fibrinogen, starch, collagen, protein-based polymers, polyamino acids, synthetic polyamino acids, zein, modified zein, polyhydroxyal Decanoate, petroleum acid, ethic acid, modified and unmodified fibrin and casein, carboxymethylsulfate, albumin, hyaluronic acid, chitonic acid and derivatives thereof, heparan sulfate and derivatives thereof, heparin, chondroitin sulfate, dextran, b Cyclodextrins, copolymers of PEG and polypropylene glycol, gum arabic, guar, gelatin, collagen, collagen-N-hydroxysuccinimide, lipids, phospholipids, variants, copolymers, mixtures of the aforementioned substances or Characterized in that a combination thereof is used. 31. The method according to any one of claims 28 to 30, wherein polyacrylic acid as a biostable material for the biostable layer, polyacrylate as a polymethylmethacrylate, polybutylmethacrylate, polyacrylamide, polyacrylonitrile , Polyamide, polyetheramide, polyethyleneamine, polyimide, polycarbonate, polycarbonate, polyvinyl ketone, polyvinyl halogenide, polyvinylidene halogenide, polyvinyl ether, polyisobutyl Ethylene, polyvinyl aromate, polyvinyl ester, polyvinylpyrrolidone, polyoxymethylene, polytetramethylene oxide, polyethylene, polypropylene, polytetrafluoroethylene, polyurethane, polyetherurethane, silicone-polyetherurethane, Silicone-polyurethane, silicone-polycarbonate-urethane, polyolefin elastomer, polyisobutylene, EPDM gum, fluorine Silicone, carboxymethyl chitosan, polyaryl ether ether ketone, polyether ether ketone, polyethylene terephthalate, poly valerate, carboxymethyl cellulose, cellulose, rayon, rayon triacetate, cellulose nitrate, cellulose acetate, hydroxyethyl cellulose , Cellulose butyrate, cellulose acetate butyrate, ethyl vinyl acetate copolymer, polysulfone, epoxy resin, ABS resin, EPDM gum, silicone as polysiloxane, polydimethylsiloxane, polyvinylhalogen and copolymer, cellulose ether, cellulose triacetate, chitosan, Copolymers, mixtures or combinations thereof of these materials are used. 31. The method according to any one of claims 24 to 30, wherein the deposition of the polysaccharide of formula (1) as defined in claim 1 comprises hydrophobic interactions, van der Waals forces, electrostatic interactions, hydrogen bonding, ionic interactions. , By means of crosslinking or covalent bonding or a combination thereof. 31. The method according to any one of claims 25 to 30, wherein one of the following active substances is used instead of the active substance paclitaxel: simvastatin, 2-methylthiazolidine-2,4-dicarboxylic acid sodium salt, macro Cyclic suboxides (MCS), derivatives of MCS, activated protein C (aPC), PETN, trapidyl, β-estradiol as well as mixtures of these active substances or mixtures of paclitaxel with one of these active substances. A medical device available by one method according to any one of claims 24 to 30.

Images