KR100669498B1 - Serine protease inhibitor and composition for septic shock treatment containing the same - Google Patents

Abstract

The present invention relates to a serine protease inhibitor and to the treatment of sepsis containing the same.

Serine Protease Inhibitors, Sepsis Treatment

Description

Serine protease inhibitor and composition for septic shock treatment containing the same

1 is a partial amino acid sequence of a serine protease inhibitor according to the present invention.

Figure 2 is a graph showing the effect of inhibiting the blood pressure drop by the serine protease inhibitor according to the present invention.

Figure 3 is a graph showing the effect of serine protease inhibitors according to the present invention on bradykinin production.

Figure 4 is a graph showing the effect of serine protease inhibitors according to the present invention on cali Crane activity.

The present invention relates to a serine protease inhibitor and an agent for treating sepsis containing the same.

Sepsis is the leading cause of death in patients suffering from sepsis due to bacterial infection. With regard to the onset of sepsis, the activation of proteases is known to be one of the main factors for the onset of septic syndrome, so protease inhibitors were expected to be useful therapeutics for bacterial protease-induced sepsis. As an example, aprotinin, a pancreatic protease inhibitor, is widely used as a therapeutic for sepsis, pancreatitis, and other diseases with hyperfibrinolysis, and kunitz type trypsin inhibitor isolated from soybeans is evaluated for its physiological role. And studies to improve bioavailability are underway (Shin, YH et al., Immunopharmacology, 33: 369-373).

Protease inhibitors are present everywhere in various forms in plants and animals. For plants, soybeans are the most abundant source of protease inhibitors, and in 1964 Kunitz isolated kun amino acid inhibitors with 181 amino acid residues and two disulfide bonds from soybeans. In addition, it was investigated that the Bowman-Birk soybean inhibitor (BBI) isolated by Birk was a protein consisting of 71 amino acid residues containing seven disulfide bonds.

The present invention provides serine protease inhibitors.

The present invention also provides a treatment for sepsis.

The present invention relates to serine protease inhibitors.

Inhibitors according to the invention are named DI-1 or an active fragment thereof, DI-2 or an active fragment thereof, DI-3 or an active fragment thereof and a DI mixture.

In the present invention, 'DI-1' has a molecular weight of 15.9 kDa by electrophoresis under reducing conditions, shows inhibitory activity against trypsin and plasmin, has inhibitory activity against chymotrypsin, and thrombin and human plasma calilikrein (kallikrein) It does not show inhibitory activity against swine pancreatic calricin.

'DI-2' has a molecular weight of 12.1 kDa by electrophoresis under reducing conditions, shows inhibitory activity against trypsin and plasmin, inhibitory activity against elastase, thrombin, human plasma cali Crane, and pig pancreatic cali crane. Does not exhibit inhibitory activity.

'DI-3' has a molecular weight of 14.6 kDa by electrophoresis under reducing conditions and exhibits inhibitory activity against trypsin and plasmin, and does not show inhibitory activity against thrombin, human plasma cali Crane, and pig pancreatic cali Crane.

'DI mixture' is a mixture of DI-1 or an active fragment thereof, DI-2 or an active fragment thereof and DI-3 or an active fragment thereof, and is DI-1 or an active fragment thereof, DI-2 or A composition comprising an active fragment and DI-3 or an active fragment thereof. DI mixtures according to the invention include both mixtures comprising other substances in addition to DI-1, DI-2 and DI-3 or mixtures obtained by mixing purely separated DI-1, DI-2 and DI-3. .

'Active fragment' means a polypeptide having a portion of the sequence of a serine protease inhibitor according to the invention and having a serine protease inhibitory activity according to the invention.

The partial amino acid sequence of the serine protease inhibitor according to the present invention is as follows, DI-1 and DI-3 is BBI isolated from Macrotyloma axillare seed (Jouber et al., 1979), BBI isolated from Azuki bean (Ishikawa et al. , 1985), and partly similar amino acid sequences to BBI type 1 (Odani et al. 1972) isolated from soybeans, and DI-2 shows sequence similarity with soybean BBI type 3 (Odani et al. 1977) sequences.

DI-1 SG-HHQXT-- ---------- ------ SIP- ---------- ------- SCL

DI-3 SG-HHQSTDE PSESSK ---- ------ SIPP QCHCSDLRLD SCHSACKSCL

MAI DE-3 --DHHHSTDE PSESSKPCCD ECACTKSIPP QCRCTDVRLN SCHSACSSCV

AB-1B SGHHEHSTDZ PSSZSKPCCB HCACTKSIPP QCRCTDLRLD SCHSACKSCI

BBI-1 -------- DD --ESSKPCCD QCACTKSNPP QCRCSDMRLN SCHSACKSCI

DI-1 CTFSINATCV C -------- ---------- ---

DI-3 CTFSIPAQCV CADTNDFYE PCK ------- ---

MAI DE-3 CTFSIPAQCV CADTNDFYE PCK ------- ---

AB-1 CTLSIPAQCV CBBIBDFYE PCKSSHSDDD NNN

BBI-1 CALSYPAQCV CVDTTDFYE PCKPSEDDK- -EN

DI-2 ------- HHD DSSDEPSESL --ACCDLCIC TASIPPQCSC TDIRLNSCHS

BBI-3 MCILSFLKSD QSSSYDDDEY SKPCCDLCMC TRSMPPQCsc EDIRLNSCHS

DI-2 ACK ------ S VPGLCRCLDT NDFCYQP --- ---

BBI-3 DCKSCMCTRS QPGQCRCLDT NDFCYKPCKS RDD

Serine protease inhibitors according to the present invention, when used individually or in combination, showed an inhibitory effect on Pseudomonal elastase (P.E) -induced hypotension and kalcreine activity and on bradykinin production. These results indicate that the serine protease inhibitor according to the present invention does not directly act on the plasma kallikrein of the kinin-calikrein system when it is involved in the inhibition of the increase in calikrein activity and the bradykinin production by PE. It is presumed to exhibit an inhibitory effect.

In addition, the inhibitor of the present invention inhibits blood pressure drop induced by Pseudomonas elastase.

The present invention therefore relates to a treatment for sepsis. The sepsis therapeutic agent according to the present invention contains a DI mixture. That is, the therapeutic agent for sepsis according to the present invention has a molecular weight of 15.9 kDa by electrophoresis under reducing conditions, exhibits inhibitory activity against trypsin and plasmin, inhibits chymotrypsin, and thrombin and human plasma callicraine, porcine pancreas. Serine protease inhibitor, which does not show inhibitory activity against cali crane, molecular weight of 12.1 kDa by electrophoresis under reducing conditions, inhibitory activity against trypsin, chymotrypsin and plasmin, inhibitory activity against elastase , Serine protease inhibitors that do not show inhibitory activity against thrombin, human plasma cali Crane, and pig pancreatic cali Crane, and molecular weight by electrophoresis under reducing conditions and 14.6 kDa, showing inhibitory activity against trypsin, chymotrypsin and plasmin , Thrombin and human plasma callicrane, porcine pancreas cali It contains a serine protease inhibitor which does not exhibit the inhibitory activity for the lane.

The types and compositions of buffers used in this study were as follows, and protein quantification was determined by micro-lowry method (Lowry, O.H. et al. (1951) J. Biol. Chem. 193 265).

How to measure trypsin activity

Trypsin activity was determined by Erlanger spectroscopy using BAPNA (N-benzoyl-D, L-arginine- p -nitrid hydrochloride) as substrate. BAPNA is hydrolyzed by trypsin to produce p -nitroalanine with maximum absorbance at 410 nm. 100 μl of 60 μg / ml trypsin solution and 200 μl of 50 mM Tris / HCl (pH 8.2) containing 50 mM CaCl ₂ were incubated at 37 ° C. for 10 minutes to 400 μg / ml BAPNA. 600 μl of the solution was added thereto, followed by further incubation for 10 minutes at 37 ° C., 30% acetic acid was added to terminate the reaction, and the absorbance was measured at 410 nm.

How Amides Activity Is Measured

Amidate activity measurement methods are used to investigate the substrate specificity of a serine protease inhibitor. Boc-Gln-Ala-Arg-MCA, a synthetic substrate for trypsin (0.03 μg), chymotrypsin (0.015 units), thrombin, plasmin (0.02 units) and elastays (0.02 units), aspartin proteases, Suc-Leu-Leu-Val-Tyr-MCA, Boc-Val-Pro-Arg- MCA, Boc-Val-Leu-Lys-MCA, Suc-Ala-Pro-Ala-MCA as substrates 500 μl of 50 mM Tris / HCl (pH 8.2) buffer containing protease and 50 mM CaCl ₂ was added and reacted at 37 ° C. for 5 minutes, followed by adding 2 μl of 10 mM MCA (4-methyl-7-coumaryl amide) substrate. The reaction was completed for 5 minutes at 37 ° C. and 17% acetic acid was added to terminate the reaction. The amount of AMC (7-amino-4-methyl coumarin) produced was measured at 380 and 460 nm, respectively. Ki values of inhibitors for each protease are calculated by the Dixon plot method (Dixon, M. (1953), Biochem. J. 55: 170-171). IC ₅₀ measures the protease activity by varying the concentration of the inhibitor, and then sets the concentration of the inhibitor that inhibits each protease activity by 50%.

Example 1.

2 L of hexane was added to 500 g of the powdered white bean, and stirred at room temperature for 12 hours, followed by filtration with 0.8 μm filter paper. After sufficiently drying it was extracted with 3 L of extraction buffer (10 mM NaH ₂ PO ₄ .H ₂ O, 10 mM Na ₂ HPO ₄ , 25 mM sodium chloride, 1 mM thiourea; pH 6.0) with stirring at 37 ° C. for 3 hours. After removing the residue, the pH of the extract was adjusted to 4.1 by adding c-HCl. The precipitated material was centrifuged at 32.000 × g for 15 minutes at 4 ° C., and the supernatant was heat treated at 100 ° C. for 10 minutes to remove precipitated protein. After centrifugation, the supernatant was concentrated using an Amicon ultrafiltration (3,000 cut membrane). It was loaded into a Sephadex G-50 column (Φ3.5 x 120 cm) equilibrated with 10 mM sodium phosphate (pH 7.2) buffer containing 0.1 M NaCl and eluted at 0.2 ml / min with the same buffer. The obtained fraction was measured for absorbance at 280 nm, and trypsin activity was measured using BAPNA to obtain an active fraction.

The obtained trypsin active fraction was loaded into a C ₁₈ reverse phase open column (Φ 1.0 × 20.5 cm) equilibrated with H ₂ O and collected at a rate of 1 ml per minute. The pass-through fraction was washed with 5% acetonitrile containing 0.05% TFA until absorbance disappeared at 280 nm, followed by 5% acetonitrile containing 0.05% TFA and 60% acetonitrile each containing 0.05% TFA 200 Elution was carried out in linear gradient conditions. Pass-through fractions and elution fractions were collected separately and trypsin activity was measured for each fraction. No activity was observed in the passage fraction and the activity was confirmed from the elution fraction. In the initial fraction of the elution fraction, a fraction containing DI-2 and DI-3 was obtained, followed by a fraction containing DI-1. They were subjected to electrophoresis to collect only trypsin inhibitor fraction without pigment, which was named DI mixture ( Dolichos inhibitor). The DI mixture contained DI-1, DI-2, DI-3 in a ratio of 5: 2: 1.

C ₁₈ reverse phase HPLC was performed to purely separate DI-1, DI-2, DI-3. Using A buffer (H ₂ O with 0.05% TFA) and B buffer (CH ₃ CN with 0.05% TFA), the concentration of B buffer to 40% CH ₃ CN at 45 minutes at 1 ml per minute flow rate Elution with increasing linear gradient conditions yielded three peaks showing serine protease inhibitor activity. According to the obtained amount, each peak was named DI-1, DI-2, DI-3. DI-1, DI-2, and DI-3 were shown as single bands on SDS-PAGE, respectively, and were 15.9, 12.1, and 14.6 kDa, respectively.

Example 2. Amino Acid Sequencing                     

Purified and separated into a single substance Partial amino acid sequences were determined for fragments obtained by pretreatment with DI-1, DI-2, and DI-3 with trypsin. In the trypsin treatment method, 7 μg of protein was dissolved in 100 μl of 0.4M NH ₄ HCO ₃ buffer containing 8M urea, 5 μl of 45 mM Dithiothreitol was added thereto, and the protein was reduced by incubating at 50 ° C. for 15 minutes. 20 μl of 100 mM iodoacetide was added and allowed to react for 15 minutes at room temperature to alkylate. 2 μg of trypsin was added to the alkylated protein and allowed to react for 12 hours at 37 ° C. for fragmentation. The fragmented protein was linearly gradient using C ₁₈ reversed phase HPLC (Chemosorb 5-ODS-H 2.1 × 150W 11660) [A: 0% 0.06% TFA in CH ₃ CN, B: 0.052% TFA in 80% C _H3 CN] Fragmented protein was fractionated at a flow rate of 0.2 ml per minute using a program of 90% increase in B% at 90 minutes, and the amino acid sequence was determined by Edman digestion. The results are shown in FIG.

Example 3. Investigation of Substrate Specificity and Activity for Enzymes

Using the known substrates of serine protease and aspartin protease, the IC ₅₀ value was determined by measuring the amidase activity of DI-1, DI-2, DI-3, and DI mixtures thereof. As a control, BBI and SBTI (Kunitz) were used. This is shown in Table 1.

DI-1, DI-2, and DI-3 showed similar activity to trypsin and the same activity against plasmin. DI-1 has high activity against chymotrypsin and DI-2 is specific for elastase. DI-1, DI-2, and DI-3 showed no activity for thrombin, human plasma caliquene, and pig pancreatic calicheine. BBI inhibited trypsin and chymotrypsin, and SBTI showed activity against trypsin and plasma cali Crane (IC ₅₀ = 0.064 μg for 0.01 unit of cali Crane). However, neither BBI nor SBTI showed activity in porcine pancreatic elastase. The IC ₅₀ values thus obtained correspond to the dissociation constants (Ki) of these inhibitors, and Ki values are shown in Table 2.

Protease IC ₅₀ (μg) DI-1 DI-2 DI-3 DI mixture BBI SBTI Trypsin 0.168 0.117 0.165 0.062 0.259 0.155 Chymotrypsin 0.027 1.323 0.937 0.027 1.313 - Ella Stays - 0.066 - - - - Thrombin - - - - - - Plasmin 0.197 0.556 0.200 0.240 - - Kallikrein - - - - - 0.064

Protease Ki (M)  temperament Inhibitor DI-1 DI-2 DI-3 Trypsin 2.5x10 ^-8 3.1 x 10 ^-8 2.4 x 10 ^-8 Boc-Gln-Ala-Arg-MCA Chymotrypsin 3.6 x 10 ^-9 7.1 x 10 ^-7 2.1 x 10 ^-8 Suc-Leu-Leu-Val-Tyr-MCA Thrombin -a - - Boc-Val-Pro-Arg-MCA Ella Stays - 6.2 x 10 ^-8 - Suc-Ala-Pro-Ala-MCA Plasmin 5.2 x 10 ^-8 1.7 x 10 ^-7 5.7 x 10 ^-8 Boc-Val-Leu-Lys-MCA Kallikrein - - - Z-Phe-Arg-MCA

Example 4 Investigation of Inhibitory Effect on Blood Pressure Drop

Using a sepsis model of guinea pigs induced with pseudomonal elastase (PE), according to Khan's method (Khan et al., 1993a: Khan et al., 1993b), the inhibitor according to the present invention was applied to a protease secreted from bacteria. Inhibition of blood pressure drop induced by When male guinea pigs pre-anesthetized with pentobarbital were injected intravenously with a half-treatment of 0.3 mg / kg of PE, guinea pig blood pressure of 64.0 ± 6.4 mmHg was 14.7 ± It dropped to 9.4 mmHg (n = 10). This drop was peaked 3 minutes after P.E injection and recovered 30 minutes later. Experiments were carried out using DI-1, DI-2, DI-3, and DI mixtures as inhibitors. Intravenous injection of 33 mg / kg of DI-1, DI-2, or DI-3 3 minutes prior to PE injection did not inhibit blood pressure drop, but intravenous injection of 33 mg / kg of DI mixture prevented the drop of blood pressure. n = 5, 63.0 ± 6.4 to 56.8 ± 6.7 mmHg). PE-induced blood pressure drop was also inhibited even when pretreatment with 11 mg / kg of DI-1, DI-2, and DI-3 (n = 2, 61.0 ± 8.5 mmHg to 55.3 ± 4.2 mmHg) . The results are shown in FIG. Injecting only the DI mixture without P.E did not significantly affect basal blood pressure (n = 5, from 63.4 ± 6.4 mmHg to 64.2 ± 9.5 mmHg).

Example 5 Effect on the production of calikrein and bradykinin

The effects of the inhibitor according to the present invention on the production of bradykinin and cali Crane activity in the plasma involved in blood pressure drop were investigated. Blood was collected from venous blood vessels three minutes before physiological saline or inhibitor injection, three minutes after PE injection, and after a drop in blood pressure, and 100% trichloroacetic acid (TCA) was added to the collected blood to precipitate the protein. 6,300 × g, 5 min centrifugation and TCA-soluble fractions were recovered and used for the determination of bradykinin and cali Crane activity. When measuring bradykinin, enzyme immunization was used according to the test method of the bradykinin kit manufacturer.

  The amount of bradykinin in the plasma of normal guinea pigs was 122.3 ± 18.8 pg / ml (n = 3). After 3 minutes of PE injection, the plasma level of bradykinin was markedly increased when blood pressure dropped (n = 4, 1182.5 ± 665.6 pg / ml plasma), and after 30 minutes, the level of bradykinin was almost normal. (N = 3, 261.0 ± 41.1 pg / ml plasma). P.E. Pretreatment of 33 mg / ml DI mixture prior to administration significantly inhibited the increase in bradykinin concentration (n = 4, 224.25 ± 45.33 pg / ml plasma). The results are shown in FIG.

Intravenous injection of 0.3 mg / kg of PE increased calcinine activity of guinea pigs after 3 minutes (n = 3, from 80.8 ± 33.1 pg / ml to 238.7 ± 34.90 pg / ml), but with DI mixture 33 mg / kg. The increase in cali Crane activity was inhibited by the pretreatment of kg (n = 3, 105.5 ± 22.2 pg / ml plasma). The results are shown in FIG.

According to the present invention, it is possible to provide an economical serine protease inhibitor, and also to provide an agent for treating sepsis.

Claims (8)

As a composition for the treatment or prevention of sepsis comprising a serine protease inhibitor extracted from the white migraine as an active ingredient, The serine protease inhibitor 1) obtained by extracting white bean head with non-polar organic solvent and fractional eluting with phosphate buffer 2) molecular weight by electrophoresis under reduced conditions is 15.9 kDa, 3) shows inhibitory activity against trypsin and plasmin, has inhibitory activity against chymotrypsin, and does not show inhibitory activity against thrombin, human plasma kallikrein and porcine pancreatic calichein, 4) A composition for treating or preventing sepsis, wherein the 14-amino acid sequence corresponding to the amino acid sequence of the 38th to 51st amino acids of Bowman-Birk inhibitor type 1 (BBI-1), a protease inhibitor isolated from soybean, is SCLCTFSINATCVC . As a composition for the treatment or prevention of sepsis comprising a serine protease inhibitor extracted from the white migraine as an active ingredient, The serine protease inhibitor 1) obtained by extracting white bean head with non-polar organic solvent and fractional eluting with phosphate buffer 2) the molecular weight by electrophoresis under reducing conditions is 12.1 kDa, 3) shows inhibitory activity against trypsin, chymotrypsin and plasmin, has inhibitory activity against elastase, and does not show inhibitory activity against thrombin, human plasma kallikrein and porcine pancreatic calikrein, 4) The 13 amino acid sequence corresponding to the amino acid sequence of the 8th to 20th amino acids of Bowman-Birk inhibitor type 3 (BBI-3), a protease inhibitor isolated from soybean, is HHDDSSDEPSESL, and the 23rd amino acid to 53 A composition for treating or preventing sepsis, wherein the 31 amino acid sequence corresponding to the amino acid sequence up to the first amino acid is ACCDLCICTASIPPQCSCTDIRLNSCHSACK, and the 18 amino acid sequence corresponding to the amino acid sequence from the 60th amino acid to the 77th amino acid is SVPGLCRCLDTNDFCYQP. As a composition for the treatment or prevention of sepsis comprising a serine protease inhibitor extracted from the white migraine as an active ingredient, The serine protease inhibitor 1) obtained by extracting white bean head with non-polar organic solvent and fractional eluting with phosphate buffer 2) molecular weight by electrophoresis under reduced conditions is 14.6 kDa, 3) shows inhibitory activity against trypsin, chymotrypsin, and plasmin, and does not show inhibitory activity against thrombin and human plasma kallikrein and porcine pancreatic calikrein; 4) A composition for treating or preventing sepsis, wherein the 46-amino acid sequence corresponding to the amino acid sequence of the 17th to 63rd amino acids of Bowman-Birk inhibitor type 1 (BBI-1), a protease inhibitor isolated from soybean, is SIPPQCHCSDLRLDSCHSACKSCLCTFSIPAQCVCADTNDFYEPCK . A serine protease inhibitor of claim 1, a serine protease inhibitor of claim 2 and a serine protease inhibitor of claim 3 comprising the composition for the treatment or prevention of sepsis. Extract the white bean head with a non-polar organic solvent to obtain a primary extract, The primary extract was extracted again with an extraction buffer comprising Na ₂ H ₂ PO ₄ .H ₂ O, Na ₂ HPO ₄ , sodium chloride, and thiourea, to obtain a secondary extract, The secondary extract is added to the acid solution to obtain a precipitate, sepsis treatment or prevention composition comprising a white migraine extract prepared as a supernatant obtained by centrifuging the precipitate as an active ingredient. The white migraine extract is a composition for treating or preventing sepsis, which is a fraction extract of the supernatant fractions eluted with a phosphate buffer in a Sephadex column. The method of claim 2, The white migraine extract is a composition for treating or preventing sepsis, which is an elution fraction obtained by eluting the trypsin active fraction from reversed phase High Performance Liquid Chromatography (HPLC). The method of claim 2, The white migraine extract comprises 5% acetonitrile and 0.05% trifluroacetic acid, including fractions fractionated by passage using 5% acetonitrile containing 0.05% trifluoroacetic acid (TFA) and 0.05% trifluoroacetic acid. A composition for treating or preventing sepsis, which is an elution fraction obtained by eluting under linear gradient conditions using 60% acetonitrile.

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