JP2782232B2 - Protease inhibitor - Google Patents

Description

The present invention relates to a pathogenic virus (HI) of acquired immunodeficiency syndrome.
The present invention relates to a protease inhibitor comprising, as an active ingredient, the outer membrane protein-related peptide V) and its derivative thereof.

[Problems to be solved by the prior art and the invention]

Protease inhibitors affect a wide range of enzyme systems in vivo, and are used, for example, in the treatment of acute pancreatitis, shock, hemostasis during bleeding, and the like.

Currently used proteinase inhibitors have problems such as the possibility of antibody production. Therefore, the fact is that the appearance of a protease inhibitor having a low molecular weight and having few side effects due to antibody production is expected.

[Means for solving the problem]

The present inventors have conducted research on HIV outer membrane protein-related peptides such as a peptide consisting of the amino acid sequence of the HIV coat protein (gp120). As a result, the peptide has an amino acid represented by the formula (IV) described below. It has been found that as long as it has a sequence site, it has an excellent protease inhibitory action.

That is, an amino acid sequence already known as an antigenic determinant in the induction of neutralizing antibodies to HIV (Proc. Natl.Acad.Sc.
i.USA, 88 , 3198-3202 (1988)], -Arg-Ile- and -Ar, which are the reaction sites of the soybean trypsin inhibitor and Bowman-Birk inhibitor, respectively, which are widely known as proteinaceous trypsin inhibitors. Lys-Ser- and an amino acid sequence showing high homology with the amino acid sequence of the reaction site of inter-α-trypsin inhibitor, which is a kind of protease inhibitor present in mammalian serum. It has been concluded that a synthetic peptide containing is effective as a physiologically active substance.

Therefore, while synthesizing a novel peptide containing this portion and examining the protease inhibitory effect of the peptide related to the outer membrane protein of HIV including the peptide, (1) a peptide having the amino acid sequence of the following formula (A) or Its pharmacologically acceptable salts.

(2) A protease inhibitor comprising, as an active ingredient, at least a peptide having an amino acid sequence of the following formula (B) or a pharmacologically acceptable salt thereof.

(Japanese Patent Application No. 63-310634).

Under such circumstances, the present inventors have further studied and found that an amino acid sequence site consisting of a very small number of amino acids represented by the following formula (IV), which is a part of the amino acid sequence of the formula (B), If present, they have found that they significantly inhibit the activity of proteases, especially trypsin and chymotrypsin, and have created peptides of formulas (I), (II) and (III) below.

The present invention has been completed based on such new findings, and the present invention relates to the following (1) and (2).

(1) A peptide having at least an amino acid sequence site of the following formula (IV) and not having an amino acid sequence site of the following formula (V) and having protease inhibitory activity [hereinafter, this peptide is referred to as a peptide (IV) derivative] Or a protease inhibitor comprising a pharmacologically acceptable salt thereof (hereinafter, also simply referred to as a salt) as an active ingredient.

(2) A peptide having protease inhibitory activity is a peptide having an amino acid sequence selected from the following formulas (I) to (III) [hereinafter, peptide (I) and peptide (I, respectively)
(1) and peptide (III)].

In the present specification, a pharmacologically acceptable salt is not particularly limited as long as it has low toxicity, and is an organic acid salt such as hydrochloride, acetate, phosphate, citrate, and succinate. ,
Inorganic acid salts such as alkali metal salts (sodium salt, potassium salt, etc.) can be mentioned.

In the present invention, the amino acid sequence represented by the formula (IV) is HI
It corresponds to a part of the amino acid sequence in V coat protein (gp120). However, the peptide (IV) derivative is not particularly limited as long as it has at least the amino acid sequence site represented by the above formula (IV), and one or more amino acids are additionally peptide-bonded to the amino acid sequence. Or a peptide obtained by modifying these peptides with polyethylene glycol or a derivative thereof (polyethylene glycol, monomethoxypolyethylene glycol, etc.) to reduce their antigenicity, and hydrogen is present at the terminal "-Gly" or / and "Arg-" site. Examples thereof include those in which each of the above-mentioned portions is a terminal amino acid, and the above-mentioned peptides (I), (II) and (III), those derived from the coat protein of HIV, and modified compounds such as HIV Are exemplified.

Peptides (I), (II) and (II) of the present invention
The peptide (IV) derivative containing I) is produced by degradation of gp120, a peptide synthesis method used in ordinary peptide chemistry, a modification method and the like.

The peptide synthesis method may be either a solid phase method or a liquid phase method. The solid phase method is performed, for example, as follows.
That is, first, the C-terminal amino acid whose amino group is protected is bound to an insoluble carrier by a carboxyl group. Any known insoluble carrier may be used. Next, after removing the amino protecting group, the amino acid-protected amino acid is sequentially peptide-bonded in accordance with the amino acid sequence, and reacted step by step to synthesize the entire amino acid sequence, followed by removing the insoluble carrier. At this time, the reactive functional group is preferably protected by a known protecting group in peptide synthesis.

The peptide thus produced can be purified to any purity by a purification method commonly used in peptide chemistry such as ion exchange resin, partition chromatography, gel chromatography, reverse phase chromatography and the like.

The peptide produced as described above can be converted into a salt by a method known per se, and a free peptide is produced by hydrolyzing the salt by a method known per se.

In the present specification, when abbreviations are used for amino acids, peptides, protecting groups, and the like, they are IUPACIUB Commiss
This is based on the abbreviation by ion on Biological Nomenclature or the abbreviation commonly used in the art, and examples thereof are as follows.

Thr: Threonine Arg: Arginine Pro: Proline Lys: Lysine Ile: Isoleucine Gln: Glutamine Gly: Glycine Ala: Alanine Phe: Phenylalanine Val: Valine His: Histidine t-BOC: t-butyloxycarbonyl [Action / Effect] Peptide (I ), (II) and (III), including peptide (IV) derivatives and salts thereof, human, dog, bovine,
It has excellent protease inhibitory activity, especially trypsin inhibitory activity and chymotrypsin inhibitory activity for mammals such as horses, mice and rats, and as an anti-inflammatory agent, an antitumor agent, a carcinogenesis inhibitor, etc., and immunoglobulin therapy It is useful as a hapten or the like for preparing antibodies for use.

The protease inhibitor of the present invention usually comprises a peptide (IV)
A derivative or a salt thereof is formulated as a freeze-dried product and administered orally or parenterally, and the dosage form varies depending on the administration route and the like.

Preferred dosage forms of the protease inhibitor of the present invention include, for example, injections, capsules, tablets, creams, ointments, patches, emollients, and the like, which are produced by methods known per se.

For example, when a peptide (IV) derivative or a salt thereof is used as a therapeutic agent for anti-inflammation, for example, in the case of an injection, the dose is generally 0.1 to 1000 mg / day as a peptide (IV) derivative or a salt thereof. And this one day a day
It is administered in several divided doses.

Experimental Example 1 The inhibitory effect of peptide (I) on trypsin activity was shown by kinetic analysis of an enzyme reaction.

Commercially available 1 pg of purified trypsin, 50 mM Tris-hydrochloride buffer pH 8.0, 0.15 sodium chloride and the substrate t-butyloxycarbonyl-L-phenylalanyl-L-seryl-L-arginine 4-methyl-coumaryl-7 By adding peptide (I) or distilled water as a control to a reaction solution comprising amide, reacting in a fluorescent cell at 25 ° C., and measuring the fluorescence intensity at an excitation wavelength of 380 nm and an emission wavelength of 440 nm. The enzyme activity was calculated. 50 μM peptide (I)
The pattern of the Lineweaver-Burk plot in the presence of is shown in FIG. Lineweaver-Burk plots show substrate concentrations of 25, 33.3, 50, and 100 in the above reaction mixture.
The enzyme activity at μM was calculated as 1 pKat, which is the enzyme unit that causes the enzyme to react with the substrate in one second to produce 1 picomole of 7-amino-4-methylcoumarin, and the enzyme activity calculated for each reciprocal of the substrate concentration. By plotting the reciprocal of.

This result indicates that the inhibition of trypsin activity by peptide (I) is of an antagonistic type. In FIG. 1, .largecircle. Indicates a control, and .circle-solid. Indicates a result in the presence of 50 .mu.M of peptide (I).

Experimental Example 2 The inhibitory effect of peptide (I) on α-chymotrypsin activity was shown by kinetic analysis of an enzyme reaction.

Commercially available purified α-chymotrypsin 20 ng, 50 mM Tris-hydrochloride pH 8.0, 10 mM sodium chloride and the substrate succinyl-L-alanyl-L-alanyl-L-prolyl-
Peptide (I) or, in contrast, distilled water was added to a reaction solution containing L-phenylalanyl 4-methyl-comaryl-7-amide, and a Lineweaver-Burk plot was obtained in the same manner as in Experimental Example 1. Obtained. The pattern of the Lineweaver-Burk plot in the presence of 50 μM peptide (I) is shown in FIG.

This result indicates that the inhibition of α-chymotrypsin activity by peptide (I) is of an antagonistic type. In FIG. 2, .largecircle. Indicates a control, and .circle-solid. Indicates the result in the presence of 50 .mu.M of peptide (I).

Experimental Example 3 Inhibition constants against trypsin and α-chymotrypsin were determined for peptide (I), a peptide consisting of a part of the amino acid sequence of peptide (I), and a peptide consisting of a sequence different from the amino acid sequence of peptide (I).

The inhibition constant was calculated from a Dickson plot for trypsin showing antagonistic inhibition and a Lineweaver-Burk plot for α-chymotrypsin showing non-antagonistic inhibition. The Dickson plot calculates the enzyme activity when a peptide having a concentration of 0, 20, 40, or 60 μM is added in the same reaction solution as in Experimental Example 2, and plots the reciprocal of the enzyme activity for each peptide concentration. Was obtained.

Inhibition constants for trypsin and α-chymotrypsin are shown in Table 1. The results showed that only peptide (I) had a significant inhibitory effect on both trypsin and α-chymotrypsin. In addition, it can be seen that peptide (I) specifically inhibits trypsin while α-chymotrypsin is inhibited depending on the number of arginine and lysine.

I: Isoleucine R: Arginine Q: Glutamine G: Glycine P: Proline A: Alanine F: Phenylalanine V: Valine Experimental Example 4 Peptide (I), Peptide (II), Peptide (III)
Was examined for its inhibitory effect on chymotrypsin activity.

The chymotrypsin activity when the substrate concentration was 100 μM in the same manner as in Experimental Example 1 was expressed as a relative activity, where the activity of the control containing no peptide was 100%. FIG. 3 shows the concentration dependence of the peptides (I), (II) and (III).

These results show that the peptides (II) and (III), which are derivatives of the peptide (IV), exhibit a trypsin inhibitory effect similar to that of the peptide (I). In FIG. 3, ● represents peptide (I), ▲ represents peptide (II), Δ represents peptide (II).
Results in the presence of I).

Experimental Example 5 α of peptide (I), peptide (II) and (III)
-The inhibitory effect on chymotrypsin activity was investigated.

The α-chymotrypsin activity when the substrate concentration was 100 μM in the same manner as in Experimental Example 2 was expressed as a relative activity when the activity of the control without the peptide was set to 100%. FIG. 4 shows the concentration dependence of the peptides (I), (II) and (III).

These results indicate that peptides (II) and (III) exhibit a similar chymotrypsin inhibitory effect as peptide (I). In FIG. 4, ● represents the results in the presence of peptide (I), ▲ represents the results in the presence of peptide (III), and Δ represents the results in the presence of peptide (III).

Comparative Example 1 The inhibitory effect of peptide (I) was compared with that of L-1-tosylamido-2-phenylethylchloromethylketone (TPCK), a commercially available protease synthesis inhibitor that specifically inhibits α-chymotrypsin. .

In the same manner as in Experimental Example 2, the substrate concentration was set to 50 μM,
The relative activity was compared with that to which TPCK was added instead of peptide (I).

FIG. 5 shows the concentration dependency of peptide (I) and TPCK.

These results show that peptide (I) has a greater inhibitory effect than TPCK. In FIG. 5, ● indicates the results in the presence of peptide (I), and ○ indicates the results in the presence of TPCK.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention at all.

In addition, all amino acids used in the following examples are L-forms unless otherwise specified.

Synthesis Example 1 Peptide (I) was synthesized.

Peptide synthesis was performed by a solid phase method using a peptide synthesizer 430A manufactured by Applied Biosystems. That is, 0.5 mmol of t-Boc-L-valine-bonded phenylacetamidomethyl resin (styrene-1% divinylbenzene copolymer) was combined with 2 mmol of t-Boc amino acid whose N-terminal was protected with t-Boc, by N, N Condensation was carried out by a symmetric amino acid anhydride method using 1 mmol N, N-dicyclohexylcarbodiimide in dimethylformamide and a sequential extension method in which peptide bonds were extended one by one. However, regarding the condensation of Gln and Arg, the condensation was performed twice in N, N-dimethylformamide by the active ester method using 1 mmol of hydroxybenzotriazole. Examples of the protective group for the side chain of the t-Boc amino acid include a 2,4,6-trimethylbenzenesulfonyl group for Arg, an Nt-butoxycarbonyl-N-2-chlorobenzyloxycarbonyl group for Lys, and a benzyl group for Ser and Thr. Each group was used.

Standard peptide synthesis program uses 60% deprotection
1 minute or 15 minutes with trifluoroacetic acid / dichloromethane, 3 times 1 minute with dichloromethane for washing, 2 times 1 minute with 10% N, N-diisopropylethylamine for neutralization, and 1 minute with N, N-dimethylformamide for washing. N, N-dimethylformamide as condensation
Symmetric amino acid anhydride method using dicyclohexylcarbodiimide for 20 minutes, washing twice with N, N-dimethylformamide for 1 minute and with dichloromethane four times. In these standard synthesis programs, the reaction temperature, reaction time and the like for each amino acid are controlled in detail by a microcomputer.

The peptide resin obtained by the automatic peptide synthesizer was cleaved with trifluoromethanesulfonic acid to cleave the protecting group of the support resin and the amino acid side chain. That is, 1 g of the synthetic peptide resin, 1 ml of thioanisole and 500 μ of ethanedithiol are stirred at room temperature for 10 minutes, 10 ml of trifluoroacetic acid is added while cooling the flask with ice water, and the mixture is stirred for 10 minutes. Then add 1 ml of trifluoromethanesulfonic acid,
Remove the flask from the ice water and stir at room temperature for 30 minutes. Cold diethyl ether was added to the flask until no precipitation of peptide was observed, and the mixture was stirred for 1 minute, and the crystals were filtered with a Teflon filter. The crystals were further dissolved in a small amount of trifluoroacetic acid, transferred into cold diethyl ether, and again filtered with a Teflon filter. This was dissolved in 2N acetic acid or the like to obtain a crude purified peptide.

The crude peptide is a reverse phase column (diameter 2 cm, length 25 cm) made of silica gel packing with octadecyl group attached.
m) high-performance liquid chromatography (column: YMC
R-ODS-5) was used for purification. The elution was carried out by using 0.1% trifluoroacetic acid as a mobile phase and performing a linear gradient of 90% acetonitrile from 30% to 70% (flow rate: 5 ml / min.). The chromatogram of the purified sample is as shown in FIG. 6 (detected by ultraviolet absorption at 220 nm).

1 nmole of the purified peptide was analyzed by a gas phase sequencer. As a result, the amino acid residues detected in analysis cycles 1 to 12 are, in order, Ile, Arg, Ile, Gln, Arg, Gl
y, Pro, Gly, Arg, Ala, Phe, and Val, and it was confirmed that the desired peptide (I) was obtained. The peptide yield after purification was 110 mg.

Synthesis Example 2 Peptide (II) was synthesized.

Peptide synthesis was performed in the same manner as in Synthesis Example 1.
In addition, a dinitrophenyl group was used as the protecting group for the t-Boc amino acid side chain in His.

Since the synthesized peptide resin contained His, the following pretreatment was performed before cleavage with trifluoromethanesulfonic acid as in Synthesis Example 1. N, N-dimethylformamide 24ml, thiophenol 1ml to the peptide resin,
After stirring at room temperature for 1 hour, the mixture was suction-filtered with a Teflon filter, washed with 10 ml of dichloromethane three times, and dried.
Further, while keeping this resin at 0 ° C, m-cresol
1 ml, dimethyl sulfide 3 ml, trifluoroacetic acid 5 ml,
The reaction was carried out in 1 ml of trifluoromethanesulfonic acid for 3 hours, washed with cold diethyl ether on a Teflon filter, dried, and cut in the same manner as in Synthesis Example 1.

Purified by high performance liquid chromatography in the same manner as in Synthesis Example 1. The chromatogram of the purified sample is as shown in FIG. 7 (detected by ultraviolet absorption at 220 nm).

Analysis cycle 1 analyzed by the same method as in Synthesis Example 1
The amino acid residues detected in ~ 11 are Ile, His, Ile, Gly, Pro,
Gly, Arg, Ala, Phe, Val, and Thr, and it was confirmed that the desired peptide (II) was obtained. The peptide yield after purification was 180 mg.

Synthesis Example 3 Peptide (III) was synthesized.

 Peptide synthesis was performed in the same manner as in Synthesis Example 1.

Purified by high performance liquid chromatography in the same manner as in Synthesis Example 1. The chromatogram is shown in FIG.
m, detected by ultraviolet absorption).

As a result of analysis in the same manner as in Synthesis Example 1, amino acid residues detected in analysis cycles 1 to 12 were Val, Phe, Ala, Arg,
Gly, Pro, Gly, Arg, Gln, Ile, Arg, and Ile, and it was confirmed that the desired peptide (III) was obtained.

Formulation Example 1 After the peptide (I) was purified, it was injected into an ampoule, freeze-dried and sealed to obtain a 100 mg / amble injectable formulation.

Formulation Example 2 1 g of a freeze-dried peptide (I) was mixed with Macrogol ointment (the 8th revision Japanese Pharmacopoeia) (a mixture of Macrogol 4000 50 g and Macrogol 400 50 g) to obtain a 1% ointment formulation. Was.

Formulation Example 3 Injection formulation was obtained by using Peptide (II) instead of Peptide (I) in Formulation Example 1.

Formulation Example 4 In Formulation Example 2, an ointment was obtained using peptide (II) instead of peptide (I).

Formulation Example 4 In Formulation Example 2, an ointment was obtained using peptide (III) instead of peptide (I).

[Brief description of the drawings]

Fig. 1 is a graph showing the inhibitory effect of the protease inhibitor of the present invention (peptide (I)) on trypsin, using a Lineweaver-Burk plot.
Α- of the protease inhibitor of the present invention [peptide (I)]
It is a graph which shows the inhibitory effect with respect to chymotrypsin. FIG. 3 is a graph showing the concentration-dependent inhibitory effect of peptides (I), (III) and (IV) on trypsin. FIG. 4 shows the α of peptides (I), (III) and (IV).
-It is a graph which shows the concentration-dependent inhibitory effect with respect to chymotrypsin. FIG. 5 is a graph comparing the inhibitory effects on α-chymotrypsin of the protease inhibitor of the present invention [peptide (I)] and a commercially available synthetic protease inhibitor (TPCK). FIG. 6 is a chart of high performance liquid chromatography of the purified peptide (I). FIG. 7 is a chart of high performance liquid chromatography of the purified peptide (II). FIG. 8 is a chart of high performance liquid chromatography of the purified peptide (III).

────────────────────────────────────────────────── ⁶ Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12N 9/99 A61K 37/64 ADU (56) References JP-A-2-69194 (JP, A) International Publication 88/9181 (WO , A1) European publication 311219 (EP, A1) European publication 339504 (EP, A2) Acta Biochem. Pol. , 28 (3-4) (1981), pp. 241-251 (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14 / 155,7 / 06,7 / 08 A61K 37/64 C12N 9 / 99 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] (1) at least a formula Having an amino acid sequence site of A protease inhibitor comprising, as an active ingredient, a peptide having a protease inhibitory activity or a pharmacologically acceptable salt thereof, having no amino acid sequence site. 2. The protease inhibitor according to claim 1, wherein the peptide having protease inhibitory activity is a peptide having an amino acid sequence selected from the following formulas (I) to (III).