FI104232B - Use of hirudin derivatives as thrombin inhibitors - Google Patents

Description

104232

Use of hirudin derivatives as thrombin inhibitors

The invention relates to the use of hirudin bound to dextran, Sepharose or polyethylene glycol (MP 1500-15000 dt) as a thrombin inhibitor in vitro. The invention also relates to a method of coating the surface of medical devices with said hirudins.

Hirudins are e.g. known from EP-A

142,860, EP-A 158,564, EP-A 158,986, EP-A 168,342, EP-A

171 024, EP-A 193 175, EP-A 200 655, EP-A 209 061, EP-A

227 938 DE 3 445 517 A1 DE 3 805 540.6 and Chang FEBS,

Bd. 164, 307 (1983). Chang demonstrated, inter alia, that the C-terminal 15 shortening strongly interferes with the anti-thrombotic effect of hirudin. The importance of hirudins for anti-coagulation therapy was also adequately described [e.g. P. Walsmann and F. Markwardt; Pharmazie, 36, 653 (1981)]. Thus, it specifically inhibits thrombin but is otherwise far-20 macologically ineffective, meaning that undesirable side effects have so far not been demonstrated.

However, a drawback to medical use in the prophylaxis of • • · · · · · · · · · · 25 in the body [Richter et al., Haematol. 115: 64 (1988); (Markwardt et al., 1988, Pharmazie 43, 202).

• · · • · · ·

Thus, it is an object of the invention to find novel hirudin derivatives which have a longer half-life or low elimination.

3 0 This task is unexpectedly solved by women of hirudin derivative of hirudin or its physiologically acceptable salt and carrier.

. · **. Hirudins include compounds described in, for example, the literature cited on page 11,

35, in particular EP-A 171 024, EP-A 158 986, EP-A

: 209 061 and De 3 805 540.6, such as e.g.

• «2 104232 0 1 10 Leu-Thr-Tyr-Thr-Asp-Cye-Thr-Glu-Ser-Gly-Gin-Asn-Leu-Cys-20 5 Leu-Cys-Glu-Gly-Ser-Asn-Val -Cys-Gly-Gin-Gly-Asn-Lys-Cys-30 40 lie-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gin-Cys-Val-Thr-Gly-50

Glu-Gly-Thr-Pro-Lys-Pro-Gin-Ser-Hie-Asn-Asp-Gly-Asp-Phe-10 60

Glu-Glu-lie-Pro-Glu-Glu-Tyr-Leu-Gln.

These hirudins may be prepared by methods well known in the art of peptide chemistry, cf. e.g., Houben-Weyl, Methoden derorganchen Chemie, Volume 15/2, preferably by solid phase synthesis such as B. Merifield, J. Am. Chem. Soc. 85, 2149 (1963) or R.C. Sheppard, Int. J. Peptide Protein res. 21, 118 (1983) or similar known methods 20. Alternatively, the skilled artisan may approach said hirudins by known genetic engineering techniques.

In addition, the following hirudins modified by genetic engineering techniques are at issue. Preferably standing ···. such that, for example, two of the three lysines naturally occurring in the sequence are replaced by natural • »» I. amino acid, preferably Asg, Asn. Also, the · · · "Y hirudins modified by the directional N- (preferably · · · · 1 Lys) or C-terminal (preferably Met) extension · The N-terminal extension have the sequence cost. the lysines present in the system are replaced by another natural amino acid, · · · · 1, preferably Asn, Argr.

Y: Soluble carriers are used as carriers, especially polysaccharides such as Dextrane (MW 20,000-75,000 dt) - preferably Dextrane (MW 70,000 dt) - Laevane, 35 Heparine (MW 6,000-20,000 dt) or "low molecular weight • (• 1 «· 3 104232 of those" heparin (MW <6000 dt), polyethylene glycol (MW 1500 - 15000 dt) or gelatin batch hydrolyzate, e.g. diisocyanate-moistened gelatin batch hydrolyzate (Polygeline , RHaemaccel) or insoluble carriers such as, for example, sepharoses, e.g., CH-Sepharose 4B (Fa. Pharmacia), agarose, cellulose, hydroxymethacrylate, all activated according to known methods, however, in particular, Dextrane, Heparine is used. and low molecular weight heparin-10.

The ratio of hirudin to carrier may vary widely in hirudin derivatives. It depends on the nature of the carrier and the reaction conditions.

Also described herein is a process for the preparation of a hirudin derivative 15 wherein the hirudin is transferred to the active carrier at a temperature of 0 to 25 ° C, preferably 4 ° C.

The hirudin derivatives described herein are valuable thrombin inhibitors, which are particularly good for having a longer half-life, which in turn allows widespread use, e.g., in the prevention of vascular coke.

Thus, hirudins and hirudin derivatives are used. for example, preferably in the ceramic surface layers of heart valves (e.g., active ingredient), artificial vessels, biocompatible medical devices, catheters or filters, membranes and materials such as conventional hemidialysis devices.

• · When determining and demonstrating important blood components, the problem is that the blood to be tested has to be pre-handed 1 2 3 4 5 6. This can lead to problems with the accuracy of such 2 · · · 3 * · * 'tests. Test tubes covered with hiru- 4 • · · 5 V * din open up new possibilities for the expert.

In addition, hirudin and hirudin derivatives have utility in transplants used e.g. delayed release of the active ingredient, e.g. osmotic mi- (1 I • 104232 4 bundles, biodegradable microcapsules, rods, liposome preparations.

The hirudin derivatives described herein show that it is possible to modify hirudin with a high molecular weight carrier material, thus surprisingly maintaining hirudin activity. The pharmacokinetic behavior of hirudin is advantageously altered. Injection of hirudin and Dextran-hirudin in a rat and plasma concentration measurement using a thrombin inhibition test results in a 10-fold reduction in Dextran-hirudin inhibition over an hour. Surprisingly, an additional advantage is the extension of the half-life. It increases with Dextran-hirudin to 6-7 hours, whereas with hirudin-1 it increases to 1-2 hours (Markwardt et al., Pharmazie 43 15 (1988) 202).

The following examples, using the isohirudin described in German Patent Application 3,805,540.6 ("Leu-Thr-Hirudin"), should illustrate, but are not limited to. The skilled artisan will appreciate that all known hirudins can be converted to the equivalent of the used hirudin by the carrier and also the hirudins described on page 2. In this case: conditions for individual reactions may vary.

: ***: Example 1 • «· 25 Preparation of Hirudin • · t:. Synthesis of hirudin occurs, for example, in yeast cells which, according to German patent application P 3 805 540.6 * (HOE 88 / F 043), direct hirudin out. Its residue hirudin is enriched in HP20 column chromatography 30 (German Patent Application P 3 738 541.0; HOE 87 / F 337).

· .1 'After dialysis and subsequent affinity chromatography on Trombin-Seharose (Walsmann, P.:

Pharmazie 36: 860-861 (1981)).

1 I

Elution in reverse phase HPLC.

4 4 4 4 4 4 4 «· · 5 104232

The synthesis may also be carried out by other known methods of genetic engineering or peptide chemistry.

Example 2

Use of hirudin followed by thrombin-5 inhibition test

Female and male Wistar rats weighing approximately 300 g are stunned with ethyl urethane (1.5 g / kg i.p.). The rats are placed i.v. 1000 AT-U / kg hirudin derivative or authentic "Leu-Thr hirudin" dissolved in physiological saline. For blood collection, animals are implanted with a catheter in the carotid artery. To determine anti-thrombin inactivity, animals are bled and treated with citrate. 0.1 ml of the thus treated blood is mixed with 0.2 ml of Tris / HCl buffer (0.1 15 mol / L; pH 7.4) and preincubated at 37 ° C. After adding 0.1 ml of thrombin solution (10 NIH-U / ml), the coagulation time is determined by a coagulation meter according to Schnitger and Gross. A calibration curve with reference plasma is made in parallel with the assay and the respective plasma hirudin or plasma hirudin derivative concentration is read from the curve.

Example 3

Dextran-hirudin

:: Combination of "Leu - Thr hirudin" with dextran (MW

25,000,000 dt) occurs in Parikh, J. et al. by method, • Ϊ Meth. Enzymol 34 (1974) 77. For this, M »· is activated.

Dextran is treated with meta-periodate at 4 ° C followed by dialysis and lyophilization. 2 g activated • · ·

Dextran is then dissolved in 285 ml of 0.1 mol / l sodium, To the phosphate buffer (pH 8.8) at room temperature, cool the solution to 4 ° C, add 20 mg of hirudin and incubate for 14 hours at 4 ° C. 75 ml are then separated and lyophilized. After removal of the small molar buffer salts by gel filtration (RSephadex G-35 25, 1 cm x 100 cm), the solution is lyophilized.

I «1« · · · · · «« · «% • · 6 104232

The reaction occurs between at least one of the three lysines and activated Dextran.

To reduce the formed Schiffschen base, 75 mg of NaBH 4 is added to 75 ml of incubated solution, the mixture is stirred for 40 minutes at 4 ° C and then the solution is dialyzed against water for 24 hours at 4 ° C (membrane: Servapor Dialysis Lubing). 10 mm diameter, Fa. Serva Feinbiochemica GmbH Heidelberg). Then gel filtration on RSephadex G-25 and lyophilization. Dextran-hirudin is purified by Thrombin-Sepharose affinity chromatography similar to Example 1. Dextran-hirudin activity is determined by the method of Griesbach et al. (Thromb. Res. 37, 347-350 (1985)). The inhibition of thrombin-catalyzed chromozym TH is then measured. When fitted to a molar base, the activity measured corresponds to the activity of hirudin. The Dextran hirudin thus purified is placed in a Wistar rot i.v. After one hour, a detectable concentration in the plasma of rats is found. This can be described in reference 20 only with a dose of hirudin about 10 times higher. If plasma hirudin concentration is further monitored, a elimination half-life of 6-7 hours is observed. This is a clear prolongation compared to,, ···, hirudins (1-3 hours). The experimental animals used do not show any behavioral disturbance.

* · 9 I. Example 4 • 1 L · 1 / Sepharose Hirudin • · · · · 18 mg of hirudin is dissolved in 2 mL of 0.1 M NaHCO 3 · · V · and 0.5 M NaCl pH = 8. at 0 ° C. 400 mg of activated CH-Sepharose 4 B (Pharmacia) is swollen according to the manufacturer's instructions ::. The protein solution is then added to the support and allowed to stand for 1.5 hours at 23 ° C. Subsequently, the immobilisate is aspirated and allowed to inactivate residual binding groups for 1 hour in 0.1 M TRIS, pH 35.0. The carrier is then washed according to the manufacturer's instructions.

«·« «♦ · · · • · 7 104232

The protein-coupling yield is 5%. The biological activity is determined in nitro as in Example 2. Sepharose hirudin is isolated from the treated plasma, washed with 1-1.5 M NaCl, and retested. 5 Measured activity is within the error limits of 1st measurement.

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Claims (2)

Use of hirudin bound to dextran, Sepharose or polyethylene glycol (MW 1,500 - 15,000 5 dt) as an in vitro thrombin inhibitor. 2. Method of coating the surface of medical agents, characterized in that they are coated with hirudin bound to dextran, Sepharose or polyethylene glycol (MW 1,500 - 15,000 dt). • · · • · · · · · · · · ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · *