An article adapted for contact with blood, a process for the preparation thereof as well as uses thereof

_? 5 TECHNICAL FIELD

The present invention relates to the field of heparini- zation of materials for the purpose of imparting thereto a heparin layer which inhibits in contact with blood the adhesion of thrombocytβs and the adsorption of blood

10 proteins. Thus-, the article according to the invention is especially suited for applications where there are contacts with blood, e.g. medical applications. It is true that the methodology of heparinizing a material for the above-men¬ tioned purpose is previously known per se. but the present

15 invention relates to a novel, alternative method of adhering the heparin to the substrate, viz. via a special, novel type of a pre-adsorbed layer. In addition to the above-mentioned article the invention relates to a process for the prepara¬ tion thereof as well as to the use of said article for medi-

20 cal applications.

BACKGROUND OF THE INVENTION

To accomplish blood compatibility for different materials in contact with blood one of the most important

25 methods has been to heparinize the surface thereof. Thus, the heparin layer on the surface inhibits, as was mentioned above, the adhesion of thrombocytes and the adsorption of blood proteins. Furthermore, the heparin must be enzymatical- ly active in the blood coagulation process, which calls for

30 specific necessities as to molecular conformation and mobili¬ ty relative to the surface. ζ Two main principles for the heparinization have previ¬ ously been utilized. The first one is based on colloidal pre¬ cipitation, e.g. through complex-formation between a phi-

35 philic amines and heparin. The second one utilizes the possi¬ bility of covalently bonding the heparin to the surface. However, these known principles have some limitations which

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means that there is a continuous research for alternative or improved methods for the heparinization.

SUMMARY OF THE INVENTION The present invention relates to an alternative or im¬ proved technique for the heparinization of surfaces, which technique eliminates or at least reduces the limitations of the prior art while at the same time imparting thereto, at least for certain applications, additional advantageous prop- erties which have not been obtainable by the previously utilized technique. More specifically we have found that a specific protein, viz. lysozyme, possesses unexpected affin¬ ity for heparin and gives an outstanding adhesion to dif¬ ferent substrate surfaces. The unexpectedly good results which have been obtained by the protein according to the in¬ vention will be described more in detail below, but primarily it can be mentioned that a very good adhesion to metal sur¬ faces has been obtained, which is a material in connection with which previously known methods have shown deficiences. Lysozyme is a protein that is present in low concentra¬ tions in blood. Already therein there is an interesting ad¬ vantage as in this way the invention is based on the utiliza¬ tion of a substance which is non-foreign to the human organ¬ ism. In addition thereto another interesting property of lysozyme is its antimicrobial properties which, thus, impart to the novel heparinized surface an antimicrobial activity - a security factor in storage and handling.

More specifically the article according to the invention is characterized in that heparin or a heparin-based material is adhered or bonded to the substrate via a layer of lysozyme or a derivative thereof which is pre-adβorbed to said substrate.

As was indicated above the novel technique according to the invention has been shown to work especially well for metal surfaces, in connection with which previously known heparinizing methods have shown limitations. However, the invention is also applicable to other substrates which are

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chosen per ae in accordance with previously known tech¬ niques, i.e. primarily such substrates which it has previous¬ ly been desired to heparinize for the purpose of imparting to the same improved properties in applications where there is a contact with blood. Examples of such materials are polymeric materials and glass. As concerns polymeric materials it should be noted that it has turned out that the invention is especially interesting in connection with polymeric materials of the so called low energy type, which means polymeric materials that are not wetted by water but by organic solvents .

As concerns the term "lysozyme or derivatives thereof" it should be understood that of course the invention is not limited to the use of lysozyme per se. but it is also pos- sible to choose any derivative thereof which gives the corresponding or similar properties. Such a choice may for instance depend on a better solubility in the desired solvent for a derivative than for lysozyme per se. As examples of utilizable derivatives there can be mentioned salts, such as the chloride salt. Moreover, the invention is of course in¬ tended to cover such cases where the lysozyme has been modi¬ fied within the molecule at a position or site that does not have any direct connection with the effect of the invention, i.e. a modification that does not change the desired pro- perties according to the present invention.

Nor concerning heparin the requisite is that heparin per se has to be utilized to obtain the desired effect. Thus, the expression "heparin-based material" is intended to cover those heparin compounds which give a corresponding or similar effect, reference in this context being made to the prior art which discloses numeral examples of heparin compounds for the purpose referred to. Thus, in this connection the invention does not differ from the prior art.

Those applications for which the article claimed is especially well suited are also selected in accordance with the prior art, which means that this need not be described more here. However, through the fact that certain improve-

ments of the properties or additional advantageous properties are obtained by the invention medical uses or applications will become even more interesting in connection with the in¬ vention than according to the prior art. The process according to the invention is characterized by firstly contacting the substrate with a solution of the lysozyme or the derivative thereof to the formation of a lysozyme layer and then exposing the substrate with its lysozyme layer to a heparin or heparin-based solution so as to adhere or bond the heparin or the heparin-based material to said lysozyme layer.

As is often the case for surfaces which are to be coated, such surfaces have to be comparatively clean to obtain the desired result. This is true also in connection with the present invention, especially in the case where the substrate is a metal. In such a case the surface should be very clean, i.e. be comprised of the metal or the metal oxide. In the ideal case this means that the surface should be cleaned or purified in a so called plasma cleaner and immediately thereafter transferred into distilled water.

Alternatively, a consecutive washing in lye, acid and distil¬ led water can be accepted. For a plastic surface, especially a low energetic one, the cleaning preferably means that the material is cleaned in water with a detergent and then an organic solvent. As concerns other substrates in principle those cleaning methods which have previously been utilized in connection therewith are applicable.

After said cleaning of the substrate surface, if re¬ quired, the substrate is contacted with the lysozyme solu- tion, which is commonly a water solution or an aqueous solu¬ tion, and distilled water is often preferred relative to a buffer solution. In order to obtain a lysozyme layer the solution should have a concentration of at least 0.1 percent by weight. The upper limit is not especially critical as concerns the desired effect, but generally the concentration should not exceed 10 percent by weight, since otherwise viscosity effects will interfere with the process. An

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especially preferable range as concerns the concentration of lysozyme or derivative thereof is 0.1-2 percent by weight.

The residence time of said stage of the treatment should be at least 15 minutes, e.g. about 20 minutes, as such a period is normally required to attain a plateau value for the adsorption of lysozyme. Once said plateau or maximum value has been attained there is normally no reason to further extend the residence time, which generally means that said residence or treatment time is within the range of 15-30 minutes.

After said treatment with lysozyme solution the sub¬ strate should be rapidly rinsed in water and then directly exposed to a heparin solution or heparin based solution. Thus, it has turned out, especially in connection with metals, that drying should not or must not be performed between the two coating stages, in order to obtain the optimum effect .

Also the solution of heparin or heparin-based compound is preferably water-based. For adsorption reasons its con- centration should be above 0.05 percent by weight, especially above 0.1 percent by weight. Nor is in this case the upper limit especially critical, and any additional effect is hard¬ ly obtained at a concentration value exceeding about 5 per¬ cent by weight. Therefore, a generic range is 0.05-5, especially 0.1-5, percent by weight. However, in many cases said concentration should not even exceed about 2 percent by weight, as otherwise the viscosity will cause interferences. Thus, the specially preferred range is 0.1-2 percent by weight. However, as concerns the heparin treatment in princi- pie all experiences from the prior art can be utilized, i.e. said stage is principally performed per se in accordance with the guide-lines of the prior art in this field.

The exposure time as concerns the heparin solution or the heparin based solution is generally at least 20 minutes, e.g. about 30 minutes, such as 20-45 minutes.

After said exposure to the heparin solution the sub¬ strate is suitably rinsed in distilled water, whereupon it is

allowed to dry or is dryed after drainage of the excess of solution. By the rinsing in distilled water before said drying the amount of heparin can be reduced to a monomole- cular layer. However, for most applications a certain surface excess of dissolved adsorbed heparin is preferred.

For both of the above-mentioned surface treatments it should be noted that they are preferably performed at room temperature. A somewhat raised temperature can be utilized if desired, but generally the temperature should not exceed about 50°C, as otherwise structural changes may appear in the lysozyme.

Finally the invention relates to the use of the above- defined article or of an article prepared by the process defined above, for medical applications where there are contacts with blood. In this connection it should be noted that of course the term "medical applications" should be interpreted in a broad meaning, i.e. the use is not speci¬ fically limited to therapeutica.l treatments only.

EXAMPLES

The invention will now be further described by means of the following non-limiting examples. The percentages used therein relate to percentages by weight unless otherwise specifically stated.

EXAMPLE 1

A commercially available lysozyme from poultry egg white is checked by means of gel electrophoresis to be free from other egg white proteins. The lysozyme is then de-salted by means of dialysis. A solution of 0,5 percent by weight of lysozyme in distilled water is then prepared. Metal cannulae are submersed in a bath of said solution for 20 minutes. Said metal cannulae have been pre-cleaned for 5 minutes in a so called plasma cleaner at an air pressure of 5 torr. They are picked up from the bath, given a shower of distilled water and immediately transferred to a bath consisting of a 0.1 % heparin solution in distilled water. After 30 minutes the

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cannulae are picked up, rapidly given a shower of distilled water and allowed to dry in a sterile chamber at 30°C. In this way metal cannulae having a heparin coating adhered via a pre-adsorbed layer of lysozyme are obtained.

EXAMPLE 2

Catheters of polyethylene are washed in a one percent Triton X100 solution and then in ethanol (96 X). Said catheters are submersed in a 0.1 X lysozyme solution in dis- tilled water. After about 20 minutes they are passed through a bath of distilled water, whereupon they are transferred to a 0.1 % heparin solution in distilled water. After 30 minutes the catheters are washed and are then allowed to dry so as to form articles according to the invention.

CLINICAL INVESTIGATION OF HEPARINIZED STEEL TUBES ACCORDING TO THE INVENTION

Several methods have been utilized in order to deter¬ mine thrombogenicity for artificial materials. A previously utilized method means that steel tubes are inserted into blood vessels and that said steel tubes are incubated in the vessel. During said incubation the coagulation system is incubated, an adsorption of proteins on the extraneous surface as well as an adhesion of thrombocyteβ and possible thrombosification of the inserted tube being obtained. From an animal experimental point of view said methodology was found to be a good method especially to study the formation of thrombosis in arteries as well as in veins.

Lately essentially such methods which utilize labeled radio isotopes have been used for studies of thrombogenicity. However, to estimate the thrombogenicity of steel tubes the previously used technique with an intravascular insertion of the steel tube and a determination of the weight differences before and after incubation is the best one for an optimum determination of the thrombogenicity of the material. Steel tubes having a diameter of 4 mm, a length of 25 mm and a thickness of 0.1 mm were heparinized in accordance with Example 1 above.

MATERIALS AND METHODS

Animals: 3 sheep, about 40 kg. Anesthesia: Penta- barbital initially 30 mg/kg, then a continuous infusion with ^* 7.5 mg/ in. The sheep intubated, respirator ventilated with 40 X of O2 , respirator frequency 20/min, volume 10 1/min. Exploration of both carotides, which are opened by a small longitudinal incision, and the 25 mm long steel tube, tapered and polished, is inserted. In carotis on one side there is inserted a heparinized tube and in the other side a πon-heparinized tube. Between the different incubations the sides are changed. After pilots tests the incubation time was selected to 15 minutes.

^' RESULTS

25 incubation periods were performed. In all these the thrombus weights were considerably much less on the heparini¬ zed tube than on the non-heparinized one (32 + 4 mg as compa¬ red to 210 + 10 mg) . In addition thereto there were addi- tional thrombus masses in the vessel in seven cases when the steel tube was removed. All these thrombus masses were in non-heparinized tubes (weights 96, 201, 143, 369, 374, 216 and 199 mg) .

The statistical calculation when using student's paired t-teβt gives a t-value of t=9.20, df 25, i.e. a considerably significant reduction of the thrombogenicity.

ORIGINAL