DE4341274A1 - Use of annexins for coating medical materials - Google Patents

Abstract

The use of annexins for coating medical materials is new. Also claimed are: (A) a process for coating a medical material comprising immersing the material in an annexin-contg. buffer soln. to bind the annexin(s) by adsorption, and opt. washing the material in an annexin-free buffer soln.; (B) a medical material obtainable by process (A); (C) use of annexins for prodn. of coated medical materials for prevention and treatment of fibrin deposits on implants.

Description

The invention relates to the use of annexins for coating of medical devices and materials, in particular for coating Implants.

Implants are used to perform certain replacement functions in the human or animal body. When implanting Foreign materials, however, sometimes lead to severe postoperative work inflammation and bleeding that may lead to thrombus formation nen. For the implantation of artificial lenses, postoperative inflammatory ones Reactions are described that lead to a change in approximately 1.9 to 4.4% of the lens implants lead to undesirable fibrin formation on the surface of the lens material (Spitznas et al. (1990) Arch. Ophthalmol. 108, 1666; Miyake et al. (1989) Ophthalmology 96, 1228-1233; Rowland et al. (1985) Curr. Eye Res. 4, 537- 553).

Systemic administration of heparin is one way Bil prevention of fibrin or thrombi. However, it has been shown that systemic administration often with serious side effects, e.g. B. the appearance bleeding.

In recent years, to avoid this undesirable Ef We tried to implantable devices and materials with antithrombotic effects coating substances.

In US 4,526,714 a method is described by means of which the implant bare devices with a conjugate of heparin, a heparin-like material or a heparin analog and a protein. The Conjugate is by coupling using 1-ethyl-3-dimethylamino-propylcar bodiimid (EDC) formed, then the conjugate via free amino groups is covalently bonded to the surface of the implantable material.

It has also been proposed to directly apply heparin to the surface of a medical material or in its surface layer so that Heparin more or less evenly over a long period of time in that Blood is delivered. A disadvantage of this method, however, is that the control delivery of an effective amount of heparin can hardly be achieved. Also there is a risk of unwanted bleeding here (Leininger et al. (1966) Science 152, 1628, Rembaum et al. (1970) J. Macromol. Sci. Chem., A4 (3), 715; Merrill et al. (1966) TASAIO 12, 139).  

To increase the effectiveness of materials that can be implanted on surfaces The bound antithrombotic substances were still used seeks to introduce a variable distance between surface and substance. DE-OS 31 09 141 describes special spacer molecules with a length from 3 to 150 Å.

In addition to heparin, others become antithrombotic and / or anticoagu latorily acting substances for coating medical materials called. Thus, in EP-A-0470474 implants are described in which at least part of the surface with tissue plasminogen activator (t-PA) is provided. FR 2554349 proposes streptokinase, urokinase, Plasminogen activators and heparin via antibodies to implantable Tie material.

The previously known materials with anticoagulant and anti Thrombotic substances are coated, but do not develop significant effectiveness. For example, the implantation performed more artificial Eye lenses, which had covalently bound heparin, to something lower degree of postoperative inflammatory reactions, posterior Synechia and a reduction in pigment deposits. The difference between coated and uncoated lenses is minimal (Lundgren et al. (1992) J. Cataract. Refract. Surg. 18, 65-70; Fagerholm et al. (1989) J. Cataract. Refract. Surg. 15, 485-490; Spanberg et al. (1990), J. Ca. taract. Refract. Surg. 16, 170-177; Philipson et al. (1992) J. Cataract. Refract. Surg. 18, 71-78; Borgioli et al. (1992), Ophthalmology 19, 1248-1255).

Furthermore, the coating of implants is sometimes considerable Side effects associated. This was particularly evident in the case of heparin layered materials that the substance is often uncontrolled from the surface is released, which is associated with considerable side effects. also will in some cases cause the coating material to degrade rapidly Dog enzymatic processes observed.

Furthermore, the known processes for producing these are coated materials were extremely complex.

That is why today mostly normal, uncoated medicines cal materials used for implantation. Only in a few cases, e.g. B. at  Uveitis and diabetes patients, there is an occasional use of artificial eye lenses coated with heparin.

The object of the invention was to avoid the disadvantages described.

The present invention surprisingly allows the effective Use of annexes for coating medical materials, esp especially for coating implants. Coating with annexes leads to a significant improvement in anticoagulant and antithroma botical activity on the surface of the treated devices and materia lien. The coating with Annexin V shows a particularly strong Ef perfect.

Furthermore, the present invention includes a new one, with little Coating process to be carried out. This procedure allows the effective adsorption of annexins on surfaces of medical materials lien for the implantation, while the biological activity of the well which annexins are preserved without causing a significant loss Protein comes through detachment or enzymatic breakdown.

Description of the figures

Fig. 1 shows a rabbit eye on the first postoperative day.
a: lens coated with Annexin V.
b: control lens (uncoated).

Fig. 2 shows the extent of fibrin formation on the first postoperative day. The results are given as the average degree of coagulation.
AV +: lenses coated with Annexin V.
C: uncoated control lenses

Fig. 3 shows the average number of days until the brine deposits are dissolved. The results are shown for eyes in which lenses coated with nexin V (AV +) and for eyes in which uncoated control lenses were implanted (C). The difference is statistically significant (p <0.05).

Fig. 4 shows cellular precipitates and pigment deposits on the 10th postoperative day.
a: lens coated with Annexin V.
b: control lens (uncoated).

Fig. 5 light microscopic ( 5 a) and electron microscopic photographs ( 5 b₁, 5 b₂) of the corneal endothelium on the 90th day after implantation. There are no changes in the endothelium ( 5 b₁). The corneal structure is also normal. The corneal structures on the 10th postoperative day showed the same results ( 5 b₂).

If bleeding occurs during the implantation of medical materials, this triggers a cascade of enzymatic reactions, at the end of which the Formation of thrombin stands, which ultimately converts fibrinogen into fibrin. Ver different procoagulatory reactions, e.g. B. the activation of Pro thrombin by the factors Xa and Va are thereby caused by phospholipid Catalyzes surfaces to which the coagulation factors bind.

Proteins that are involved in these phospholipid surface-dependent processes and whose phospholipid binding is calcium (Ca ²⁺ ) -dependent are referred to as annexins. The Annexin protein family includes e.g. B. Li pocortin I, calpactin I, protein II, lipocortin III, p67-calelectrin, the vascular anticoagulant proteins (VAC), IBC, PAP, PAPI, PP4, endonexin II and lipocortin V. At the moment, about 10 different annexins are known ( Crumpton et al. (1990) Nature 345, 212 and Barton et al. (1991) Eur. J. Biochem. 198, 749-760 and the literature cited here). In particular, Annexin V (VACα) shows a strong anti-inflammatory and anticoagulant activity (Chollet et al. (1992) Brit. J. Ophthalmol 76 450-452; Iwasaki et al. (1987) J. Biochem. 102, 1261-1273; Reutelingsperger et al. (1985) Eur. J. Biochem. 151, 625-629).

The structural similarities of the annexins are obviously the basis for their similar Ca ²⁺ and phospholipid binding properties, whereby there is a clear individuality regarding the affinity for Ca ²⁺ and the different phospholipid types.

The physiological functions of the Annexine extend to mem industry-related processes. The exact in vivo function of the annexins is still  not finally clarified, but due to the biochemical properties properties of the annexins demonstrate two biological properties: annexins have an anti-inflammatory effect due to their ability, the phospholi pase A2 (PLA₂) to inhibit and an antithrombotic effect by Inhibition of the coagulation cascade (Wallner et al. (1986) Nature 320, 77-81; Chap et al. (1988) Biochem. Biophys. Res. Comm. 150, 972-978). This ef effects are mainly due to the affinity of the annexins for phospholipids return. Phospholipids, substrates of phospholipase A2, are described under be agreed circumstances bound by Annexine. You are then for the correspond The lipase is not accessible or can play its role as a catalytic surface no longer play in relation to the coagulation cascade. The fibrin formation is inhibited by the annexins (Chollet et al. (1992) Brit. J. Ophthalmol. 76, 450-452; Burgogne et al. (1984) Cell Calcium 10, 1-10 Klee (1988) Biochemi stry 27, 6645-6652).

Surprisingly, it now turned out that the Annexine was extremely good suitable proteins are to medical materials, especially implantable Materials to coat. The coating of annexins is avoided largely the negative observed when coating with heparin Effects without the anticoagulant and antithrombotic activity of the Annexine is lost.

Methods for the preparation of annexins are known to the person skilled in the art and are described, for example, by Crompton et al. (1988) Cell 35 1-3; Tait et al. (1988) Biochem. 27 6268-6276, Zaetzel et al. (1989) J. Biol. Chem. 284, 14463-14470; Fukanoshi et al. (1987) Biochem 26 8087-8092, Geisow et al. (1986) FEBS Lett. 203, 99-103; Geisow et al. (1987) Biochem. Soc. Trans. 25, 800-802, US 4,937,324, US 4,736,018, US 4,507,229, WO 88/05659, EP 0181465, EP 0293567, EP 0318703 and EP 0279459.

The medical materials that can be used for a coating are primarily implants. Medical materials therefore refer to here Substances and parts that are used to fulfill certain replacement functions for a for a limited period of time or for life become. Support and control come as replacement functions or the partial replacement of organ functions, the support of Heilpro eating (e.g. immobilizing a fracture using osteosynthesis), the transfer development of forces (e.g. exercise-stable and exact reduction of fracture ends by means of osteosynthesis), the regulation of deformities or the plastic  Space filling, e.g. B. mammoplasty using plastic implants) in Be dress. In this context, pacemakers, heart valves, Ge vascular implants, contact lenses, artificial eye lenses, catheters, membranes, etc. are called.

Material with a high adsorption capacity is particularly suitable for loading layering of implants with annexes. Above all, there is material made of plastic (plastic), e.g. B. polymethyl methacrylate (PMMA), and silicones in Question. PMMA and silicones can be used, for example, for artificial eye lenses be used. Of course, others, like the above me medical materials, for example contact lenses, for coating to be pulled.

The specialist is responsible for the coating of medical material there are many known methods available. So the annexins can be covalent, e.g. B. according to U.S. Patent No. 4,526,714 with EDC (1-ethyl-3-dime thylaminopropylcarbodiimide) as a coupling reagent or with spacer molecules variable length are bound (DE-OS-31 09 141). The specialist are beyond known other methods such. B. binding via antibody con jugate according to FR 2554349. However, these procedures are quite complex and do not allow reproducible coating and no controlled clearance setting the bound annexins.

Surprisingly, however, it was found that an adsorptive Be Layering medical materials with annexes all the advantages of the above mentioned procedures united. The invention is particularly suitable Process for coating plastic implants, such as. B. Polymethyl methacrylate, or from silicones. In contrast to the known methods he this method allows the production of a uniform coating at ge little expenditure on equipment. The adsorptive process with annexins leads surprisingly to implants with a substantially stable Be layering. There is only a very slight release of the respective Annexins. On the one hand, the stable coating of the material leads to a long-term antithrombotic and anticoagulant activity in the Be rich on the surface of the implant, on the other hand this is very low release of the anticoagulant and antithrombotic substance between the implant and the surrounding tissue, so that a total optimal effect is achieved.  

The coating can ver with a single or with several different annexes take place; optionally with the addition of pharmaceuticals suitable additives, for example heparin, streptokinase, urokinase, t-PA, etc. According to the invention, the coating is advantageously carried out with An nexin V, which is in native form, e.g. B. from highly perfused tissues like that Placenta or in recombinant form can be isolated by known methods can (EP-A-0181465, EP-A-0293567).

In a special embodiment of the invention can be used for the coating for example proceed as follows: the medical material, preferably made of plastic, such as. B. polymethyl methacrylate, or silicone placed in an annexin-containing buffer solution. The concentration of annexin or the annexin mixture results for the person skilled in the art from the amount of adsorbent material and the residence time of the medical material in the Annexin solution. For Annexin V z. B. a concentration of 10 µg / ml is sufficient accordingly, this concentration being increased or decreased depending on the residence time can be. The annexin-containing solution should have a stable pH sen, which does not impair the biological activity of the annexin used and enables good adsorption. For Annexin V, the pH of the Solution between pH 6.0 and 9.0, preferably at pH 8.0. Depending on the However, smaller and larger pH values are also possible with annexin. When Buffer solution can e.g. B. a Tris-HCl, CaCl₂ solution can be used. On other solutions that have a buffering effect in this area are natural can also be used. For the preparation of coated eye lenses PMMA or silicone has a 20 mM Tris-HCl, 1 mM CaCl₂ solution, pH 8.0, proven to be particularly advantageous.

The medical material is in the Annexin V solution for approximately 1 Incubated for one hour at room temperature, advantageously with shaking. To Washing with annexin-free buffer can the coated material to Im plantation in a suitable rinsing solution (e.g. "Balanced Salt Solution"; BSS (Alcon Pharma GmbH, Freiburg, Germany)) who are kept under cooling the.

The extent and stability of the coating can be compared with known Ver driving are checked, e.g. B. with immunological methods such as ELISA test. The production of annexin-specific antibodies is a specialist man known (DE 38 10 331 and Harlow and Lane: "Antibodies. A Labora tory Manual "; Cold Spring Harbor, 1988, USA).  

All native annexins come with anti for coating the lenses coagulant and antithrombotic properties in question. As mentioned Processes for the production of the native annexins are known. It is next to it Of course, it is also possible to use modified annexins, such as the one in of the mutants described in EP-A-0293567.

The annexins can also be produced using recombinant DNA methods become. As described in European patent application EP-A-0293567, can the corresponding annexin, for example from strongly vascular Tissue according to EP-A-0181465 can be obtained, N-terminal se be quoted. With the help of this information, according to known off Selection criteria synthetic DNA probes are made for screening appropriate cDNA banks can be used. After isolation and The annexin can characterize a corresponding cDNA clone in an egg a suitable host system (e.g. in bacteria, yeast or mammalian cells) to be expressed.

In the following, the invention is explained using several examples.  

Examples 1. Coating intracapsular lenses with Annexin V

For this experiment, normal polymethyl methacrylate (PMMA) lin used for the anterior chamber. The coating of these lenses was carried out with recombinantly produced Annexin V (VACα).

15 lenses were separated in 1 ml each of Annexin V solution (10 µg An nexin V / ml 20 mM Tris-HCl buffer, pH 8.0, 1 mM CaCl₂) for one hour coated on a shaker at room temperature. As a control, 15 Lenses in the same way with 20 mM Tris-HCl buffer, pH 8.0, 1 mM CaCl₂ (without addition of Annexin V). Then the lenses were with deionized water and washed in 1 ml of a 20 mM Tris-HCl solution transferred. After shaking for 30 minutes, the lenses were placed in "Balanced Salt Solu tion "(Alcon BSS®, Alcon-Pharma GmbH, Freiburg, Germany) at 4 ° C kept. BSS was also the irrigation solution used for the surgical procedure was used (Example 2).

15 pairs of PMMA lenses (coated with Annexin V and the Kon troll lenses) were made in this way. Of these, 13 were lens pairs used as implants. Another two pairs of lenses were used for the determination the coating efficiency:

To determine the bound Annexin V, these lenses were coated with egg a monoclonal biotin conjugate antibody against Annexin V (VAA-27) (Diluted 1: 1000) in PBS / BSA for one hour and ge with water to wash. The lenses were then in streptavidin-HRPO ("horse radish peroxidase ") (1: 50000 in PBS / BSA). After further washing the lenses were incubated in TMB substrate solution and the reaction after 30 min stopped by adding 500 µl 2 N H₂SO₄. Then the UV absorption determined photometrically at 450 nm.

The coating and washing solutions containing Annexin V were washed with egg Modified plasma ELISA test for Annexin V measured. To do this 10 µg / ml plates of monoclonal antibody specific for Annexin V (VAA-33) coated. The VAA-27 / biotin conjugate and streptavidin-HRPO were used in dilutions of 1: 1000 and 1: 50000. For determination The reference values became standard values starting with 10 ng annexin V / ml used. PBS / BSA was used as the dilution buffer. Which he mean values are shown in Table 1.  

Table 1

Annexin V concentration in buffer and wash solutions

In addition, 8 lenses coated with Annexin V and 8 control lenses for the determination of the Annexin V concentration in the postoperative cam merwasser used.

In addition, two silicone lenses were described as An as described above nexin V coated to determine if Annexin V is similar on silicone material effective as on PMMA. The binding efficiency was determined as you can read above.  

The determined values show that lenses made of PMMA or silicone be can be layered without the coating being removed by washing can be. This also shows the immunochemical analysis as well as the Be adjustment of the buffer solutions using ELISA.

2. Implantation of the lenses coated with Annexin V. a) Surgical procedure

All animals were grown according to the recommendations of the Association for Research in Vision and Ophthalmology "for the use of animals in research treated. There were 13 white New Zealand rabbits weighing 2.0 to 2.5 kg used. The rabbits were intramuscularly injected injection of 50 mg ketamine anesthetized.

The surgery was performed by a surgeon and was identical for every eye. A 7 mm wide corneal incision was made and the viscoelastic material in the anterior chamber without previous lens extraction introduced. The Lin. Coated with Annexin V. and the control lenses were arbitrarily placed in the right or left Eye inserted so that each rabbit has a lens coated with Annexin V. and received an uncoated control lens. An iridotomy was performed in the 12th Clock position performed and the wound interrupted with five 10-0 mono filamentous nylon seams closed. The viscoelastic material was manually aspirated at the end of the procedure and washed with BSS. Five minutes later the following day was used to produce fibrin deposits Cyclocryotherapy performed. A cryoprobe was placed on the limbus in the three, six, nine and placed in twelve o'clock position. Freezing took place for 45 Seconds at -80 ° C.

No antibiotic or anti-inflammatory treatment was given postoperatively performed. The rabbits were examined daily: after instillation of a topical anesthetic, the anterior chamber became microscopic examined and estimated the presence or absence of fibrin. All subs Research was carried out in a double-blind trial by two doctors.

b) Prevention of postoperative fibrin formation by Annexin V coated lenses

Fibrin formation was similar to that of Johnson et al. described me method (Johnson et al. (1987) Ophthalmology 94, 597-601). The fol The following parameters were determined:  

1. The fibrin deposition area on the first postoperative day

The surface of the lenses was divided into four quadrants. In each quadrant, the fibrin deposition was classified on a scale between 0+ and 2+: 0+ (no fibrin); 1+ (moderate exudate); 2+ (significant exudate). The results for the four quadrants were then added to give a measurement for each eye. The individual values are shown in Table 2. Fig. 2 shows the average fibrin formation on the first post-operative day.

2. Time to dissolve the fibrin deposit

The day was recorded on which no more fibrin could be detected on the surface of the lenses. The determined individual values are shown in Table 3. Fig. 3 shows a graphical representation of the average values.

Table 2

Fibrin formation

Table 3

Fibrin breakdown

3. Cellular precipitates and pigment deposits on the lenses tenth postoperative day

The extent of cellular precipitates and pigment deposits on the Surface of the lenses was the third parameter for the assessment of the Annexin V coated lenses. All lenses were released on the tenth post operational day explanted. The corneal wound was reopened and the viscoelastic material implanted in the anterior chamber visibly removed. The lenses were then fixed in formaldehyde solution and stained with hematoxylin eosin. Photographs of the lenses were on finally with the "Biocom image analyis system" software and the Wilcoxon test evaluated.

The examinations showed that the surgical procedure in any case caused fibrin to be deposited on the surface of the lenses. With the exception of 2 rabbits, all other rabbits in which a lens coated with Annexin V was implanted showed a statistically less severe first inflammatory reaction (Table 2, Fig. 1a and 1b) and a shorter time until the fibrin deposit was broken down (Table 3 ). FIG. 2 shows the mean of the extent of the respective fibrin deposition on day 1. From FIG. 2 it clearly follows that there is a smaller value for the lenses coated with Annexin V (3, 15 versus 5 of the control lenses; p <0, 05).

Table 4

Cellular precipitates and pigment deposits

Fig. 3 shows the average number of days until the Fi brine deposit is broken down. The mean was 6.75 days for the control lenses (from a range of 3 to 10 days) and 4.91 days for the lenses coated with Annexin V (from a range of 2 to 8 days).

The cellular precipitates and deposits were determined by morphometri analysis carried out on 13 lenses. Eight pairs of lenses showed a diff in favor of Annexin V (Table 4). When analyzing the cellular pre zipitate and pigment deposits were less deposits with the Annexin V treated lenses were found. Regarding secondary effects, no opposite effects were observed after the procedure. In particular  in particular, there was no bleeding from the lenses coated with Annexin V. caused and no endothelial toxicity 10 and 90 days after implantation observed.

3. Annexin V concentration in aqueous humor after implantation

To determine the Annexin V release in aqueous humor after the Implantation was carried out as follows: Eight lenses were as described wrote coated with Annexin V and implanted; eight more uncoated lenses were used as control lenses. In four rabbits on 3rd postoperative day, with another four rabbits on the 7th postoperative day a puncture of the anterior chamber was performed and the concentration determined by Annexin V as described (ELISA test).

There were Annexin V concentrations that were below the detection limit of the test (0.28 ng / ml). Consequently, the coated lens material has an ne essentially permanent and stable Annexin V coating. The An nexin V coating and the low release of Annexin V in the reverse Exercise fluid is sufficient for the development of anti-inflammatory and antithrombotic activity on the surface of the lenses and in the Au gene chamber fluid. Of course, this ensures fewer secondary effects, that could be caused by the coating.

4. Short and long term toxicity

In four rabbit eyes, Annexin V was injected into the anterior chamber. Two of them were removed on the 7th postoperative day and two on the 60th postoperative day. After fixation with formaldehyde solution, the corea and retina were examined using light and electron microscopy. No toxicity caused by Annexin V was shown. In particular, the corneal endothelial cells showed a normal morphology and a normal density distribution in comparison with corresponding control cells ( FIGS. 4 and 5).

Claims (14)

1. Use of annexins for coating medical materia lien. 2. Use according to claim 1, characterized in that it is the Annexin is Annexin V (VACα). 3. Use of annexins according to one of claims 1 or 2, since characterized in that the medical material is artificial eye lenses. 4. Use of annexins according to claim 3, characterized in that that the lens material made of polymethyl methacrylate (PMMA) or silicone consists. 5. Process for coating medical material, characterized records that the material is placed in an annexin-containing buffer solution the Annexin or the Anhexine are bound adsorptively and that medical material if necessary in an annexin-free buffer solution is washed. 6. The method according to claim 5, characterized in that for coating tion Annexin V is used. 7. The method according to claim 5, characterized in that artificial Lens material is coated. 8. The method according to claim 7, characterized in that PMMA or Silicone lenses are coated. 9. The method according to any one of claims 5-8, characterized in that

• a) artificial lens material made of PMMA or silicone is added to an Annexin V-containing 20 mM Tris-HCl solution, 1 mM CaCl₂, pH 6 to 9,
• b) the Annexin V is adsorptively bound to the surface and
• c) Excess Annexin V is removed by washing with an Annexin V-free Tris-HCl buffer solution, pH 6 to 9.

10. Medical material, producible according to one of claims 5 to 9. 11. Medical material, characterized in that it has an annexin or several annexins, optionally with the addition of a pharmazeu table suitable additive has been coated. 12. Medical material according to claim 11, characterized in that they are artificial eye lenses. 13. Medical material according to one of claims 11 or 12, characterized characterized that it is coated with Annexin V. 14. Use of annexins for the production of coated medical Materials for the prevention and treatment of fibrin deposits Implants.