DE2154556A1 - Crystalline kallikreins a and b - from crude kallikrein - Google Patents

Abstract

Crystalline kallikrein component A (K-A) or B (K-B) is produced by (a) eluting the precipitate obtained from organs by known methods of salt-precipitation, (b) desalting the eluate and chromatographing on suitable basically substd. macroporous ion-exchangers, then treating with a suitable acidically substd. ion-exchanger, (c) sepg. the resulting soln. of highly purified kallikrein into K-A and K-B by known procedures and (d) crystallising the separated components from their soln. by addn. of a suitable precipitant. The new procedure enables K-A and K-B (which have vasodilating activity) to be obtained in very good yields and in high purity.

Description

Process for the purification and crystallization of the elidinogenasez allikrein) components A and B The present invention relates to a method for purification and crystallization the kallikrein components A and B as well as the use of the crystalline kallikrein component A and / or B as a drug.

When acting on the body's own kininogen, kallikrein sets the circulatory effects Kinine free. Kallikrein preparations are therefore used to treat various circulatory disorders used therapeutically (E.K. Frey, K. Kraut, E. Werle, Das Kallikrein-Kinin-System and his inhibitors, F. Enke, Stuttgart 1968, p. 150 f).

So far, partially purified preparations of kallikrein have been used for this purpose from the pancreas, submaxillary or urine.

It is already known that preparations of kallikrein can be used from the pancreas, submaxillary or urine.

There are various ways of obtaining partially purified kallikrein Process has become known, e.g. according to DBP 890 857 kallikreinh @ ltige glands thermolyzed in aqueous suspension at temperatures between 78 and 650C and the Aqueous phases containing kallikrein are separated from insoluble fractions. To the Extraction of partially purified kallikrein can also be done according to DBP 910 580 urine as well as autolysates of the pancreas or submaxillary with aqueous solutions of lead or zinc salts are precipitated, the precipitates separated, in phosphate buffer solutions, primarily in diammonium hydrogen phosphate solutions, dissolved, the solutions dialyzed, the dialysates concentrated and the active ingredient from the concentrates with organic solvents, e.g. with ethanol or acetone. The still impure kallikrein preparations can e.g. 3. according to DBP 1 102 973 so further purified that kallikrein at pH values from 3 to 10, mainly at pH G.0 to 7, on basic-substituted celluloses is adsorbed, the basic substituted cellulose separated and ineffective Accompanying substances are washed out with a very diluted buffer solution. The active substance can be made from basic substituted celluloses with 1 to 5% electrolyte solutions be desorbed. Preparations with approximately 50 to 100 KE / mg protein (KE = Kallikrein unit) * obtained. Even very impure kallikrein preparations can according to DBP 1 124 187 for further purification using electrodialysis are subjected to exchange membranes. In this way, preparations with 50 units / rng won. Kallikrein preparations cleaned in this way are only conditionally suitable for parenteral use Use. On the one hand, the content of foreign proteins can be used to raise immunological sensitivity and with more- * Determination of the kallikrein unit by measuring the decrease in blood pressure on dogs (E. Frey, H. Kraut, E. Nterle, The kallikrein-kinin system and its inhibitors, F. Enke-Verlag Stuttgart 1968, 5.11).

single use then for life-threatening anaphylactic shock to lead. On the other hand, it was difficult with the previous settlement procedures to obtain preparations that were sufficiently free of pyrogens. It wasn't either possible to remove pyrogens by filtration through clarifying and sterile filter sheets, because kallikrein is highly self-adsorbed by these materials.

It has also become known that smaller amounts can be used of highly purified kallikrein can be obtained by various processes. To H. Fritz et al. (Hoppe Seyler's Z. Physiol. Chem. 348, 1120-1132 (1967)) a preparation with a specific activity of 1516 KE / mg protein by a product which has been pre-cleaned according to DBP 910 580 is applied 1 to 3 times on hydroxylapatite cellulose chromatographed.

However, it is necessary to use the optimal concentration of the elution buffer for each kallikrein sample and each new batch of hydroxyapatite cellulose in preliminary tests to redefine. This method is therefore not process-technically feasible.

According to another proposal (H, Moriya et al., Biochem.

Pheramcol. 18, 549-552 (1968)) electrophoretically pure is obtained Kallikrein in 6-9% yield, starting from autolyzed pancreatic pulp by successive Application of DEAE cellulose, precipitation with 2-ethoxy-6,9-diamino-acridinium-lactate, Extraction of the precipitate with ammonium sulfate solution, precipitation with acetone, chromatography on hydroxyapatite and chromatography on Sephadex G 100. This process is also possible technically unusable because of the low yield and the large number of steps.

Kallikrein obtained by this method on a laboratory scale is a mixture of two isoenzymes. By electrophoresis or by chromatography on anion exchangers, kallikrein can be broken down into two components, Separate kallikrein component A and kallikrein component B (E. Habermann, Hoppe-Seyler t s Z.Physiol. Chemistry 328, 15-23; F. Fiedler and E. Werle, ibid. 348, 1097-1089 (1967)). It is not yet known whether the small structural difference in the amino acid sequence or in the carbohydrate portion of the glycoprotein kallikrein. The two components cannot be distinguished in their biological effectiveness.

The present invention deals with a simple method of recovery of the pure, crystalline kallikrein components A and B in high yield.

It was found that pure kallikrein components A and B were obtained can be obtained by a according to DP 910 580 from the precipitation of the lead or The eluate obtained from zinc salt precipitation is desalinated, and the desalinated solution is transferred to a suitable one macroporous anion exchanger, e.g. DEAE-Cellulose or DEAE-Sephadex # chromatographed, the solution of the partially purified kallikrein obtained in this way with a suitable one macroporous cation exchanger, such as CM-Sephadex or SE-Sephadex treated, the Kallikrein separates into components A and B by known processes and separates them crystallized by adding ammonium sulfate. The kallikrein components crystallized in this way surpass all previously known preparations in terms of specific activity and are definitely free from antigenic foreign proteins and pyrogens.

The enzymatic activity of the kallikrein is determined by measuring the hydrolysis of N-benzoyl-L-arginine ethyl ester (E. Werle and B. Kaufmann-Bretsch, Natural Sciences 46, 559 (1959)) in the form of the F.I.P. (Federation international Pharmaceutique) standardized embodiment as a titrimetric test (publication 1972 in J.mond.

Pharm.). For the conversion of the enzymatic units into the by Measurement of lower blood pressure in dogs using specific biological units of kallikrein (KE) (E. Frey, H. Kraut, E. Werle, The kallikrein-kinin system and side inhibitors, F. Enke, Stuttgart 1968, p. 11) the factor 1 KE = 6.37 F.I.P.-units is used. The protein content of solutions of the pure kallikrein is determined from the absorbance 280 mm, with an extinction coefficient of E180 ° = = 280 - 19.3 (= extinction a 1% solution of kallikrein at pH 7.0) is used.

It was found that the desalination of the obtained according to DBP 910 580 Eluate can be done by dialysis or by gel filtration. For the following According to the invention, chromatography on macroporous anion exchangers is particularly useful basic substituted celluloses or basic substituted gels with sufficient Pore size used. This cleaning step is preferred on DEAE-Sephadex columns carried out. Adsorption takes place at a pH of 4.0-8.0, preferably at pH 4.5-5.0. Elution takes place by increasing the salt concentration at a constant pH or lowering the pH value with constant salt concentration or a combination both procedures. The change in pH and salt concentration can be continuous take place as gradient elution or in stages. All known buffer salts can be used be used; volatile buffer salts, e.g. ammonium formate, are preferred Ammonium acetate, ammonium propionate or the corresponding alkyl ammonium salts, e.g.

Triethylammonium formate. In a preferred embodiment of the invention Procedure, kallikrein is made from 0.2 M ammonium acetate pH 5.0 on DEAE-Sephadex in the form adsorbed on a column, inactive protein washed out with 0.25 M ammonium acetate pH 5.0 and then enriched kallikrein eluted with 0.3 M ammonium acetate pH 4.5.

The chromatography on macroporous anion exchangers represents one substantial improvement of the process according to DBP 1 102 973, the yield and simplicity of implementation on an industrial scale will.

The thus enriched kallikrein is treated according to the invention further purified with macroporous cation exchangers. This makes preparations obtained of about 90% purity. As macroporous cation exchangers, in particular acid-substituted celluloses or acid-substituted gels, e.g. from cross-linked Dextrans or? Olyacrylamide, with sufficient pore size, can be used. The treatment the solution of the enriched kallikrein with the cation exchanger can take place, by stirring the solution with the exchanger or by stirring the solution through Layers of the exchanger filtered. If columns are used, it is advisable to concentrate the solution of the enriched kallikrein from the previous stage and desalinate. During the stirring or during the filtration process Accompanying substances are retained by the gels, while kallikrein passes through unhindered the filter layer runs.

The macroporous cation exchangers used for filtration or stirring are pretreated in a known manner.

For filtration, they are mixed with dilute buffer solutions and, for example, embedded in columns. The column length / column diameter ratio is of secondary importance for the filtration process. Preferably used Columns with a large column diameter. For the production of the buffer solutions the known buffer salts, especially volatile buffer salts, e.g. ammonium formate, Ammonium acetate ammonium propylene or the corresponding alkylammonium salts z0B. Triäthylarnmoiuinformiat used. The pH of the buffer solutions can be between 4.5 and s.5, mainly between 5.0 and 7.0 can be chosen That The highly purified kallikrein thus obtained is chromatographed according to known methods separated into components A and B on DEAE cellulose or DEA-Sephadex.

One can also separate the components by this method already carry out at an earlier stage of the cleaning path and the separate Then continue cleaning the components separately.

The highly purified kallikrein components are according to the invention separated by the addition of ammonium sulfate in solid form or as a concentrated solution crystallized at a pH of 4.0 - 8.0. The crystallization starts with one Ammonium sulfate saturation of about 45- O / o within a few days, preferably at a temperature of 15 - 250 C. The crystals have the shape of thin needles, which -tangle together in clusters. The crystals can pass through from the mother liquor Centrifugation or filtration separated, with 50 o / o saturated ammonium sulfate solution washed and dissolved in dilute saline solutions or water.

The crystals consist of pure electrophoretically uniform Kallikrein A and Kallikrein B. FIG. 2 shows the result of an electrophoresis experiment. Work was carried out with veronal Na buffer (pH 8.6). The ionic strength was 0.075; Tension: 100 V; Running time: 20 minutes. At time 0 it was level with the dashed line applied.

At 1 was kallikrein, at 2 the kallikrein component A and for 3 the kallikrein component B was used. The specific activity of about 1600 KE / mg Rrotein cannot be obtained even by repeated recrystallization further increase.

Surprisingly, this method according to the invention succeeds partially purified kallikrein from its accompanying substances and foreign enzymes extremely easy to separate without the use of cumbersome elution methods. It was not foreseeable that the accompanying substances and the foreign enzymes, but not Kallikrein are retained by the macroporous cation exchangers and that Kallikrein remaining in solution by the process according to the invention is very good Yields and is obtained in high purity. Through the crystallization of the two Kallikrein components A and B also become difficult-to-remove pyrogenic substances almost completely separated. The simplicity of the method according to the invention allows the implementation on a technical scale. The method according to the invention provides thus an enrichment of the technology.

The solution of the crystals is characterized by a UV spectrum at pH 7 with a maximum at 282 nm, a minimum at 251 nm, 2 shoulders at 277 and 290 nm and a ratio of the absorbances at 280 and 260 nm of 1.78.

In 0.1 n NaOH the UV spectrum shows two maxima at 280 and 289 nm (Fig. 1).

The crystalline kallikreinw components A and B according to the invention can be used as effective components of medicines, as a result of them kallikrein component A or kallikrein component can have a vasodilating effect B-containing drugs, especially for peripheral circulatory disorders, E.g. endangiitis obliterans, arteriosclerosis obliterans, Raynaud's disease, in the case of disturbed Fracture and wound healing, e.g. Sudeck's syndrome, burns, and for circulatory disorders of age can be used with success.

The application can be orally in the form of tablets or dragees, which the crystalline kallikrein component A or kallikrein component B in a mixture with contain inert, non-toxic pharmaceutically acceptable carriers or parenterally as an intramuscular or intravenous injection of an aqueous solution of the crystalline Kallikre in comp ons be A or B. Especially for parenteral use The crystalline kallikrein components offer better than a few pure preparations the advantage of the far lower risk of undesirable side effects and allergic reactions Reactions.

By adding high molecular colloids according to DBP 941 585, e.g. Polyvinylpyrrolidone or dextran, or by forming the complex with the kallikrein inhibitor Trasylol # according to DBP 1 000 566 can be made from the crystalline kallikrein components A or B delayed-acting injection preparations are produced. Kallikrein for injection purposes can be prepared as a ready-to-use isotonic solution as well as in solid form by freeze-drying a mixture of crystalline KalliLrein components A or B with suitable carriers, e.g. dextran, mannitol, Lactose, gelatin, from which by dissolving in a suitable solvent the ready-to-use Solution is prepared immediately before use.

Example: a) 200 1 of one obtained from pork pancr @ as according to DP 910 580 and dialysis eluate from a lead salt precipitation containing 10.8 million KE concentrated in a thin film evaporator to 15 1 and then by gel filtration through a Desalted column of Sephadex G-25. The desalted solution was made up with acetic acid Adjusted to pH 5.0 and a slight precipitate formed in the process was filtered off. Then Solid ammonium acetate was added up to a specific conductivity of 12 mS. This solution was passed through a 20 x 65 cm column (20 l) of DEAE-Sephadex A-50, equilibrated with 0.2 M ammonium acetate pH 5.0, given, whereby kallikrein adsorbs will. The column was then first with approx. 40 liters of 0.25 M ammonium acetate pH 5.0 and then eluted with 0.1 M ammonium acetate pH 4.5. Kallikrein appears in the eluate shortly after the pH has dropped to e-twa 4-5 (Fig. 3), the active fractions contained 9.35 million

KE (86.5%). The solution was concentrated in vacuo and over a Desalted column of Sephadex G-25; a lyophilized sample had the specific Activity 414 CU / mg weight.

b) The desalted solution with a volume of 300 ml was via a 10x130 cm column (10 1) from CM-Sephadex C-50, equilibrated with 0.01 M ammonium acetate pH 5.0, filtered. Kallikrein appeared first in the eluate, followed by inactive impurities (Figure 4). The kallikrein fraction (930 ml) contained 5.5 million KE (59%). A lyophilized The sample had the specific activity of 1060 KE / mg weight or 1220 KE / mg protein.

c) 4.42 million KE of a solution of highly purified kallikrein obtained according to b), were on a column 5 x 100 cm of DEAE-Sephadex A-50, equilibrated with 0.2 I M ammonium acetate, pH 6.7, adsorbed. The elution took place with a convex gradient of 0.2 to 0.55 M ammonium acetate pH 6.7 with a 2-1 container. Two slightly overlapping activity peaks appeared in the eluate of kallikrein, the first of which is electrophoretically called kallikrein component B, the second identified as kallikrein component A. The pure kallikrein Fractions containing A and B, respectively, were combined. Was received: 2.1 million KE Kallikrein component B (47.5%), 1.25 million KE kallikrein component A (28%) and 0.32 million KE of a mixture of kallikrein components A and D from the overlap area the summit (7.2%), together 83%.

d) Part of the Lö solution containing the kallikrein component B was concentrated to a concentration of approx. 20,000 KE / ml and saturated with Ammonium sulfate solution added up to 45% saturation. After 6 days at 20 to 250C continued crystallization. After 4 weeks, the crystals were centrifuged off, with 50% saturated ammonium sulfate solution and washed in 0.05 M phosphate buffer pH 7.0 solved. The specific activity was 16.50 KE / mg protein, the yield was 64 %.

Kallikrein component A was also crystallized in the same way. There is no difference in the microscopic appearance of the crystals.

Claims (5)

Patent claims 1. Process for the preparation of a crystalline kallidinogenase (kallikrein) component A or 3, characterized in that the organs by known methods Ketallsalzfällung obtained precipitate is eluted, the eluate is desalted on suitable basic substituted macroporous ion exchangers ohromatographed, then with treated a suitable acid-substituted ion exchanger, the thus obtained Solution of the highly purified kallidinogenase (kallikrein) according to known methods in X the components A and B are separated, and these separated components by Addition of a suitable precipitation means to be crystallized from their solution. 2. Crystalline kallidinogenase (kallikrein) component A. 7. Crystalline kallidinogenase (kallikrein) component B. 4. Medicines, characterized by a content of a crystalline Kallidinogenase (kallikrein) component according to claim 2 and / or 3. 5. A method of making a chemotherapeutic agent, thereby characterized in that crystalline kallidinogenase (kallikrein) components according to Claims 2 and 3 with inert, non-toxic pharmaceutically suitable carriers mixed.