DE2808396C2 - Protease inhibitors - Google Patents

Description

3. Pharmaceutical agent characterized by a content of eglin B and / or eglin C in one customary carrier, optionally with customary additives.

The application relates to protease inhibitors Leech extracts.

It is known that extracts from leeches (Hirudo medicinalis) contain protease inhibitors. From Methods Enzymol. 19, pages 924 to 932 is e.g. B. known that a specific inhibitor against thrombin can be obtained from leeches. This Inhibitor is from the authors of the above literature labeled hirudin.

From Proc. Int Res. Conf. on Proteinase Inhibitors, 1970, pages 271 to 280 it is known that the extracts leeches also contain trypsin-plasmin-specific inhibitors.

When examining the extracts of leeches (Hirudo medicinalis), two new ones were found Protease inhibitors of the protein type found with the scientific names Eglin B and Eglin C were designated. The invention therefore relates to protease inhibitors Eglin B and Eglin C from Leech extracts, where

jo a) Eglin B have a calculated molecular weight of 8,073 and Eglin C a molecular weight of 8,099
b) eglin B per molecule

7 Asp
5 th

3 Ser
7 GIu
6 per
5 GIy

1 AIa

0 Cys

(after performic acid oxidation)

11 VaI

0 Met

(after performic acid oxidation)

0 Ue

5 leu

4-5 Tyr

5 phe

so 4 His

2 Lys

4 arg
0-1 trp

(determined spectrophotomevrically) and

Eglin C has the following amino acid sequence:

10

NHj-Thr-Glu-Phe-Gly-Ser-Glu-Leu-Lys-Ser-Phe-

20 Pro-Glu-Val-Val-Gly-Lys-Thr-Val-Asp-Gln-

30 Ala-Arg-Glu-Tyr-Phe-Thr-Leu-His-Tyr-Pro-

40 Gln-Tyr-Asp-Val-Tyr-Phe-Leu-Pro-Glu-Gly-

50 Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-Asn-

60 Arg-Val-Arg-Val-Phe-Tyr-Asn-Pro-Gly-Thr-

70 Asn-Val-Val-Asn-His-Val-Pro-His-Val-Gly-COOH

c) Eglin B.

Chymotrypsin complex with a dissociation constant of K, ■ = 3 ■ 10- ' ⁰ M, (substrate: 3-carboxypropionyl-L-phenyl-alanine-pnitroanilide) or / C, = 1.8 · 10- "M, (substrate: NK benzoyl-L-tyrosine ethyl ester), subtilisin complex with a dissociation constant of Ki = 2 · 10- ^{: o} M, (substrate: azocasein),

Granulocytic elastase complex with a dissociation constant of Kj = 2.3 · 10- ' ⁰ M, (substrate: N ^ -Succinyl-falanyljs-p-nitroanilide), granulocytic cathepsin G complex with a dissociation constant of

Ki = 2.5 x 10- ¹⁰ M, (substrate: Nh-benzoyl-L-arginine-p-nitroanilide), and
Eglin C.

Chymotrypsin complex with a dissociation constant of Κ, =! - 10- ' ⁰ M, (substrate: 3-carboxypropionyl-L-phenyl-aIanine-pnitroanilide) or K ₁ = 2.6 · 10- "M, (substrate: NK-benzoyl- L-tyrosine ethyl ester), Subtilisinkomplex with a dissociation constant of K ₁ = 1.2 ■ 10 ^-10 M, (substrate: azo-casein),

Granuiocytic elastase complex with a dissociation constant of K, ■ = 2 ■ 10 "' ⁰ M, (substrate: N ^ -Succinyl-falanylJs-p-nitroanilide) Granulocytic cathepsin G complex with a dissociation constant of

Ki = 2.8 · 10- ' ⁰ M, (substrate: form N ^ -Benzoyl-L-arginine-p-nitroanilide,

d) Eglin B in the discontinuous gel electrophoresis at a current strength of 5 mA, a running time of 1.5 hours and a pH value of 4.3 an Rp value of 0.61 and at a pH value of 8.9 a Rf value of 0.53, and Eglin C in the discontinuous gel electrophoresis at a current strength of 5 mA, a running time of 1.5 hours and a pH value of 4.3 an Rf value of 0.54 and at a pH 8.9 has an R _F value of 0.59 and in SDS gel electrophoresis at a current of 2.5 mA and a running time of 3 hours has an RF value of 0.85 and 0.86, respectively and

e) Eglin B and C in neutral and weakly acidic solution and against unspecific proteolysis are stable, obtainable by

Table 1

A) Leech extracts over a chromatography column are made with a three-dimensional cross-linked polysacchride (Sephadex ® G-75) is filled, the column with a 0.05 m TRA · HCl / 0.4 M sodium chloride solution with a pH of 7.8 equilibrated and eluted (TRA = triethanolamine),

B) the desalted and lyophilized fractions with a molecular weight of approx. 6000

ίο (determined by means of SDS gel electrophoresis)

(Fraction II) in the equilibration buffer solution mentioned below dissolves and then on a chromatography column with diethylaminoethyl groups as functional groups

i-j send cellulose is filled, that gives that with a

0.03 M ammonium acetate buffer solution with a pH 6.5 equilibrated column first eluted with this buffer and then with a buffer solution with a linear salt gradient, produced by continuous

Mix each 300 ml of the 0.03 M ammonium acetate buffer solution with a pH 6.5 and a 0.4 M sodium chloride / 0.06 M ammonium acetate solution with a pH of 6.5 and eluted

C) the eluted fractions (fraction II b, c) after desalting and lyophilizing in the equilibration buffer solution below dissolves and puts it on a chromatography column,

ω those with a three-dimensional network,

Sulfopropyl groups as polysaccharide containing functional groups (SP-Sephadex ®) is filled, the column with a 0.05 M ammonium acetate solution with a pH of 5.0

J5 equilibrated and with a buffer solution with

linear salt gradient produced by continuously mixing each 300 ml of the 0.05 M ammonium acetate buffer solution with a pH of 5.0 and a 0.5 m sodium chloride / 0.05 m ammonium ace

tat solution at pH 5.0 eluted, with Eglin B and Eglin C in separate Fractions are obtained.

The inhibitors according to the invention form complex compounds with chymotrypsin, subtilisin, elastase and cathepsin-G, which have very small dissociation constants and a more labile complex with trypsin. The inhibition is of particular importance here

for example the proteases chymotrypsin, subtilisin, granulocytic elastase and granulocytic cathepsin G. The dissociation constants K for eglin B and eglin C with respect to the above proteases are summarized in Table 1 below

Protease

Chymotrysin (Bovine)

Subtilisin

human granulocytic elastase

human granuliocytic cathepsin G.

*) Substrate: 3-carboxypropionyl-L-phenylalanine-p-nitroanilide (Merck AG, ArL No. 10 754). **) Substrate: N "-Benzoyl-L-tyrosine ethyl ester (Fluka AG, Art. No. 13 110).

K ₁ [M] Eglin C. Eglin B. 7x 10 "'" *) 3 x 10 "'" *) 2.6 x 10 "" **) 1.8 x 10 "" **) 1.2 x _{] O} -io 2 x 10 "'" 2x _lo -io 2.3 x ΙΟ " ¹⁰ 2.8 x J ₀ -IO 2.5 χ 10 "'"

The inhibitor according to the invention Eglin B has opposite to the system consisting of the enzyme chymotrypsin and the substrate 3-carboxypropionyl-L-phenylalanine-p-nitroanilide, a specific inhibitory activity of 115mIU / mg. The specific inhibitory activity of the Eglins C according to the invention compared to the system consisting of the enzyme chymotrypsin and the Substrate 3-carboxypropionyl-L-phenylalanine-p-nitroanilide, is 120mlU / mg. The inhibition of the proteinases or proteases by the inventive Compounds were determined according to known methods (see Methods of enzymatic analysis, 3rd edition, VoLI, pages 1105 to 1121 and Anal. Biochem. 54, Pages 262 to 265). An inhibitor unit (IU) reduces the one catalyzed by the respective enzyme Hydrolysis of the 1 μΜοΙ per minute at 25 ° C. It will the complex consisting of the enzyme and the inhibitor by adding the two compounds to it a suitable buffer solution at 25 ° C. The enzyme and the inhibitor are about 5 minutes before added to the addition of the substrate to the buffer solution.

To determine the amino acid composition of the inhibitors according to the invention, samples of Eglin B and Eglin C hydrolyzed in 6 η HCl for 20., 40 and 72 hours. The amino acid analysis was carried out with a Durrum analyzer carried out. The content of cysteine and methionine was determined according to the in Methods Enzymol. 11, pages 197 to 199 described performic acid oxidation method certainly. It was found that the compounds according to the invention do not contain any cysteine and methionine residues.

The tryptophan content was measured photometrically according to the method described in Biochem. 6, pages 1948 to 1954 The method described was determined for the determination of the end groups in Eur. J. Biochem. 7, pages 463 to 470 used the DANSYL method.

The compounds according to the invention contain an unusually high content of hydrophobes Amino acids, but no methionine, no isoleucine and especially no cysteine or disulfide bridges. The compounds according to the invention are thus among the first protease inhibitors of the protein type which have no disulfide bridges. This structural feature is of particular biochemical interest because It was previously assumed that protein protease inhibitors in the vicinity of the reactive center a Disulfide bridge included. It is believed that the compounds of the invention by hydrophobic Interactions of neighboring amino acid residues in the molecule are stabilized by the zwkrhpn the complex forming the compounds according to the invention and the abovementioned proteases is over stable for a long time, even in the presence of an excess of the protease to be complexed.

The addition of equimolar amounts is sufficient for the complete inhibition of the abovementioned proteases of the compounds according to the invention. Tests were carried out in each of which a constant Amount of the protease to be inhibited with increasing amounts of the compounds according to the invention in one suitable buffer solution at a pH of 7.8 and a temperature of 25 ° C was put together and 5 minutes later the corresponding Substrate added in order to determine the residual activity or the activity of the residual free protease. The amounts of inhibitor according to the invention used in each case were on the abscissa of the following 1 to 6 and the measured free enzyme activity as a function of the added The amount of inhibitor was determined on the ordinate of the following Fig. 1 to 6 applied.

The F i g. 1 shows the reduction in the hydrolyzing effect of trypsin compared to the substrate N-Benzoyl-L-arginine-p-nitroanilide. The Eglin B and Eglin C were used in amounts between 0 to 30 μg used. The amounts of inhibitor used show that the inhibiting effect of the invention Compounds against trypsin is relatively weak.

The F i g. Figure 2 shows the increasing inhibition of the hydrolyzing effects of chymotrypsin versus the substrate 3-carboxypropionyl-L-phenyl-ala-

! 5 nin-n-nitroanilide by adding increasing amounts (0 to 8 μg) of Eglin B and Eglin C.

The F i g. 3 shows the increasing inhibition of the hydrolyzing effects of chymotrypsin against the substrate N * -Benzoyl-L-tyrosine ethyl ester by adding increasing amounts (0 to 0.7 μg) of Eglin B and Eglin C.

The F i g. 4 shows the inhibition of the hydrolyzing Effect of subtilisin through the addition of the compounds according to the invention to the substrate Azo casein.

The F i g. 5 shows the inhibition of elastase by the addition of increasing amounts of the invention Compounds towards the substrate N * -Succinyl- [alanyl] 3-p-nitroanilide.

The F i g. 6 shows the inhibition of the hydrolyzing action of kethepsin G by the addition of increasing amounts of the compounds according to the invention compared with the substrate N * -benzoyl-L-tyrosine ethyl ester.
The compounds according to the invention are prepared from leech extracts. In the first purification stage, the raw leech extract (1.5 g) is placed on a gel chromatography column ^{which is filled with a three-dimensionally crosslinked polysaccharide (Sephadex β} G-75), the extract in an equilibration buffer solution consisting of 0.05 molar TRA.HCl / 0.4 moairem NaCl with a pH = 7.8 is dissolved and the column material is equilibrated with the same buffer solution. The column is eluted with the said buffer solution in amounts of 52 ml per hour. Fractions of 9.7 ml are collected.

The fractions obtained are either purified by multiple ultrafiltration (Amicon cell with UM 05 Membranes) or by gel filtration through a

So ion exchange column (Sephadex · G-25 or Fracto- <Tf »l 70nn \ unH Fliiii» riin <T with 0.1 M acetic acid

desalinated. The salt-free solution which contains the protease inhibitors according to the invention is lyophilized.
The fractions which contain the substances with molecular weights of approx. 38,000 and approx. 8500 (determined by means of SDS gel electrophoresis, SDS = Na dodecyl sulfate) are not processed further.

The fractions which contain the substances with molecular weights of approx. 6000 (fraction II) become one subjected to further cleaning. Fraction II (70 mg) is added to the equilibration buffer solution below dissolved and then placed on a chromatography column containing cellulose, the diethylaminoethyl groups as having functional groups, is filled.

The column material is washed with a 0.03 M ammonium acetate buffer solution equilibrated with a pH of 6.5 The column is eluted with this buffer solution in proportions of 17 ml per hour until only

Fractions are obtained which have no appreciable inhibitory effect on chymotrypsin. Thereafter, the column is filled with a buffer solution with a linear salt gradient prepared by continuous Mix 300 ml each of the above-described equilibration buffer solution and one 0.4 M sodium chloride / 0.06 M ammonium acetate solution of pH 6.5 eluted. There are fractions of 3.7 ml collected.

The fractions obtained (II b, c) are desalted as indicated above and then lyophilized.

The fraction II b, c (50 mg) is in a 0.05 M ammonium acetate buffer solution with a pH of 5.0 dissolved and placed on a chromatography column for further purification, which is marked with a three-dimensional cross-linked polysaccharide, the sulfopropyl groups as functional groups (SP-Sephadex) has, is filled. The column material is previously with a 0.05 M ammonium acetate solution with a pH equilibrated to 5.0. The column is prepared with a buffer solution with a linear salt gradient by continuously mixing each 300 ml of the equilibration buffer solution described above and a 0.5 M sodium chloride / 0.05 M ammonium acetate solution, pH 5.0, in amounts of 17 ml per hour eluted, collecting two different fractions. Fraction I contains the Eglin B and the Fraction II the EgHnC. By desalting and lyophilizing the eluates, the Compounds obtained in the pure state.

It was the specific inhibitory activity of the fractions of the individual cleaning mares compared to the System consisting of the enzyme chymotrypsin and the substrate 3-carboxypropionyl-L-phenylalanine-p-nitroanilide certainly. The results are summarized in Table 2 below.

Table 2

fraction

Specific Hcmma'itivity
(mIU / mg)

Starting material 5.5

II 15.5

Hb, c 74

Eglin b 115

Eglin C 120

In the following table 3 is the specific Inhibitory activity of the invention. Substances in the individual purification stages against chymotrypsin (Substrate: N ^ -Succinyl-L-phenylalanine-p-nitroanilide) summarized in inhibitor units per mg. Table 3 shows that the specific inhibitory activity with increasing cleaning increases.

Table 3

40

fraction Specific inhibitory activity versus chymotrypsin (Substrate: N "-Succinyl- L-phenylalanine-p-nitroanilide) imIU / mg] Raw hirudin extract 5.5 II 15.5 Hb, c 74 Eglin B. 115 Eglin C 120

mlU = milliinhibitor units.

The inhibitors according to the invention obtained in the purification of leech extracts are like was determined by discontinuous gel electrophoresis (10% gel) - chromatographically pure (cf. H. R. Maurer, Disc. Electrophoresis, 1971, Walter de Gruyter, Berlin). Also based on SDS gel electrophoresis (15% Gel) it could be demonstrated that the inventive prepared by the above process Compounds are present in pure form (cf. SDS gel electrophoresis K. Weber, M. Osborn, The Proteins (Neurath, H, Hill R. L, eds.) Vol. I, pages 179-223 Academic Press, New York).

In discontinuous electrophoresis (disc electrophoresis) at a running time of 1.5 hours, a current of 5 mA and a pH of 8.9, Eglin B has an R _F value of 0.53 and at a pH value of 4.3 has an R _F value of 0.61.

_{In discontinuous electrophoresis, Eglin C has an R F} value of 0.59 at a running time of 1.5 hours, a current intensity of 5 mA and a pH value of 8.9 and an R F value of 0.59 at a pH value of 4.3 R _F value of 0.54 and in SDS gel electrophoresis with a running time of 3 hours and a current strength of 2.5 mA an Rt> value of 0.85 and 0.86, respectively.

In addition 6 sheets of drawings

Claims (2)

Patent claims: 1. Protease inhibitors (Eglin B and Eglin C) from leech extracts, where a) Eglin B has a calculated molecular weight of and Eglin C has a molecular weight of exhibit b) eglin B per molecule ι ο 7 Asp 5 th 3 Ser 7 GIu 6 per 5 GIy 1 AIa 0 Cys (after performic acid oxidation) 11 VaI 0 met (after performic acid oxidation) 0 Ue 5 Leu 4-5 Tyr 5 phe 4 His 2 Lys 4 Arg 0-1 Trp (determined spectrophotometrically) has and Eglin C has the following amino acid sequence: 10 NHrThr-Glu-Phe-Gly-Ser-Glu-Leu-Lys-Ser-Phe- 20 Pro-Glu-Val-Val-Gly-Lys-Thr-Val-Asp-Gln- 40 30 Ala-Arg-Glu-Tyr-Phe-Thr-Leu-His-Tyr-Pro- 40 Gln-Tyr-Asp-Val-Tyr-Phe-Leu-Pro-Glu-Gly- 50 Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-Asn- 60 so Arg-Val-Arg-Val-Phe-Tyr-Asn-Pro-Gly-Thr- 70 Asn-Val-Val-Asn-His-Val-Pro-His-Val-Gly-COOH 55 c) Eglin B. Chymotrypsinkomplex with a dissociation constant of K, - = 3-10- ¹⁰ M (substrate: 3-carboxypropionyl-L-phenylalanine-p-nitroanilide) or to Ki = 1.8 x 10 "M (substrate: N <* - benzoyl-L-tyrosine ethyl ester), Subtilisinkomplex with a dissociation constant of K = 2 ■ 10 ^-10 M (substrate: azo-casein) & 5 Granulocytic elastase complex with a dissociation constant of K, = 2.3 · 10- '° M (substrate: N ^ -Succinyl- [alanyl] 3-p-nitroanilide), granulocytic cathepsin G complex with a dissociation constant of K ₁ r - 24 · ΙΟ - · ⁰ M, (substrate: NKBenzoyl-L-arginine-p-nitroanilide), and eglin C one Chymotrypsinkoniplex with a dissociation constant of Ki = 10 T ¹⁰ M, (substrate: 3-carboxypropionyl-L-phenylalanine-p-nitroanilide) or K ₁ = 2.6 · 10- "M (substrate: N * -Benzoyl-L-tyrosine ethyl ester), Subtilisin complex with a dissociation constant of Ki = 1.2 · 10- ' ⁰ M, (substrate: azo-casein), Granulozy'ary elastase complex with a dissociation constant of K ₁ · = 2 · 10- ' ⁰ M, (substrate: N «-Succinyl- [alanyl] 3-p-nitroanilide),
granulocytic cathepsin G complex with a dissociation constant of Ki = 2.8 x 10- ¹⁰ M, (substrate: * N-benzoyl-L-arginine-p-nitroanilide), which form, d) Eglin B in the discontinuous gel electrophoresis at a current strength of 5 mA, a running time of 1.5 hours and a pH value of 4.3 an Rf value of 0.61 and at a pH value of 8.9 a R _F value of 0.53, and Eglin C in the discontinuous gel electrophoresis at a current strength of 5 mA, a running time of 1.5 hours and a pH value of 4.3 a Rh value of 0.54 and at a pH value of 8.9 an R _F -Value of 0.59 and in SDS gel electrophoresis at a current strength of 2.5 mA and a running time of hours has an Rk value of 0.85 or 0.86 and e) Eglin B and C in neutral and weakly acidic solution and against unspecific proteolysis are stable, obtainable by A) Leech extracts over a chromatography column are made with a three-dimensional cross-linked polysaccharide Sephadex ® G-75) is filled, the column with a 0.05 m TRA · HCl / 0.4 M sodium chloride solution with a pH of 7.8 and equilibrated eluted (TRA = triethanolamine), B) the desalted and lyophilized fractions with a molecular weight of approx. 6000 (determined by means of SDS gel electrophoresis) (fraction II) in the below mentioned Dissolve equilibration buffer solution and then transfer it to a chromatography column containing Diethylaminoethyl groups are filled as cellulose containing functional groups, there, the column equilibrated with a 0.03 M ammonium acetate buffer solution with a pH of 6.5 initially with this Buffer eluted and then with a linear salt gradient buffer solution prepared by continuous mixing of each 300 ml of the 0.03 M ammonium acetate buffer solution with a pH 6.5 and a 0.4 M sodium chloride / 0.06 M ammonium acetate solution with a pH value of 6.5 eluted and C) the eluted fractions (fraction II b, c) after desalting and lyophilization in dissolves the equilibration buffer solution below and applies it to a chromatography column there, those with a three-dimensionally cross-linked, sulfopropyl groups as functional Polysaccharide containing groups (SP-Sephadex ®) is filled, the column equilibrated with a 0.05 M ammonium acetate solution with a pH of 5.0 and with a buffer solution with a linear salt gradient prepared by continuous Mix 300 ml each of the 0.05 zN ammonium acetate buffer solution with a pH of 5.0 and a 0.5 m N atrium chloride / 0.05 ammonium acetate solution eluted at pH 5.0, with Eglin B and Eglin C in separate Fractions are obtained. 2. Procedure for obtaining the protease inhibitors according to claim 1, characterized in that one A) Leech extracts over a chromatography column are made with a three-dimensional cross-linked polysaccharide Sephadex ® G-75) is filled, the column with a 0.05 m TRA · HCl / 0.4 M sodium chloride solution with a pH of 7.8 equilibrated and eluted (TRA = triethanolamine), B) the desalted and lyophilized fractions with a molecular weight of approx. 6000 (determined by means of SDS gel electrophoresis) (fraction II) in the equilibration buffer solution mentioned below dissolves and then on a chromatography column with diethylaminoethyl groups is filled as cellulose containing functional groups, there that with a 0.03 M ammonium acetate buffer solution with a pH value of 6.5, the column equilibrated first with this buffer and then eluted with a buffer solution with a linear salt gradient, prepared by continuous mixing of 300 ml each of the 0.03 M ammonium acetate buffer solution with a pH of 6.5 and a 0.4 M sodium chloride / 0.06 m Ammonium acetate solution with a pH of 6.5 and C) the eluted fractions (fraction II b, c) after desalting and lyophilization in the Dissolve the following equilibration buffer solution and apply it to a chromatography column, those with a three-dimensional "network", sulfopropyl groups as functional groups containing polysaccharide (SP-Sephadex ®) is filled, the column with a 0.05 M ammonium acetate solution equiübriert with a pH value of 5.0 and with a buffer solution with linear salt gradient, produced by continuously mixing each 300 ml of the 0.05 M ammonium acetate buffer solution with a pH of 5.0 and a 0.5 m Sodium chloride / 0.05 M ammonium acetate solution with a pH of 5.0 eluted, whereby Eglin B and Eglin C can be obtained in separate fractions.