JPH032195A - Peptide and protease inhibitor - Google Patents

Abstract

NEW MATERIAL:Peptide having amino acid sequence selected from formula I to formula III or salt of said peptide. USE:Used as medicine such as anti-inflammatory drug, antineoplastic drug or anticarcinogenesis drug. Also used as hapten for producing antibody for immnoglobulin remedy. Having excellent protease-inhibiting activity against mammal. PREPARATION:For instance, insoluble carrier is bonded with carboxyl group of amino acid having C-terminal protecting amino group and said amino acid is successively subjected to peptide bonding with protected amino acid according to the aimed amino acid sequence, then the insoluble carrier is removed from generated peptide, thus purified with well-known means such as gel chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel peptide useful as a medicine, a hapten for producing immunoglobulin, etc., and a pathogenic virus (HIV) that causes acquired immunodeficiency syndrome, including the peptide. The present invention relates to a protease inhibitor containing an outer membrane protein-related peptide and a derivative thereof as an active ingredient.

[Prior art/issues to be solved by the invention] Protease inhibitors affect a wide range of enzyme systems in vivo;
For example 2. It is used to treat pancreatitis, shock, and to stop bleeding.

Currently used protein-based protease inhibitors have problems such as the possibility of antibody production.

Therefore, the reality is that the emergence of a protease inhibitor that is low molecular weight and has fewer side effects due to antibody production is eagerly awaited.

[Means to solve the problem]

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

That is, the amino acid sequence (Proc) is already known as an antigenic determinant in inducing neutralizing antibodies against HIV.

Natl, Acad, Set, USA, FJ4
．． 3198-3202 (1988)), Arg-
It has an amino acid sequence of Ile- and -Lys-3er-, as well as an amino acid sequence that shows high homology with the amino acid sequence of the reaction site of inter-α-trypsin inhibitor, a type of protease inhibitor present in mammalian serum. This led them to consider that a synthetic peptide containing this portion might be effective as a physiologically active substance.

Therefore, we synthesized a new peptide containing this part and examined the protease inhibitory effect of this peptide and other HIV outer membrane protein-related peptides. its pharmacologically acceptable salts;

Cys-Thr-Arg-Pro-^5n-Asn-^
5n-Thr-^yg-Lys-3er-Ile-Ar
g-Ile-Gln-Arg-Gly-Pro-Gly
-Arg-Ala-Phe-Val-Thr-Roe-G
ly-Lys-Ile-Gly-^snMet-Arg
-Gln-Ala-His-(:ys (
A) (2) A protease inhibitor containing at least a peptide having the amino acid sequence of the following formula (B) or a pharmacologically acceptable salt thereof as an active ingredient.

Asn-Asn-Thr-Arg-Lys-5er-I
le-Arg-Ile-Gln-Arg-Gly-Pr
o-Gly-Arg-Ala-Phe-Val-Thr
-Ile-Gly-Lys41e-Giy
(B) (Patent application 1986-31)
No. 0634).

Under these circumstances, the present inventors conducted further research and found that the following formula (
It has been found that having an amino acid sequence region consisting of extremely few amino acids, represented by IV), can extremely significantly inhibit the activity of proteases, especially trypsin and chymotrypsin, and the following formulas (1), (n ) and (III) were created.

The present invention was completed based on this new knowledge, and the first invention is based on the following formulas (1), (n) to (I[[
) [hereinafter referred to as Peptide (I), Peptide (n) and Peptide (I [[)] respectively] or a pharmacologically acceptable salt thereof (hereinafter also simply referred to as salt) It is.

1ie-Arg-Ile-Gln-Arg-Gly-P
ro-Gly-Arg-Ala-Phe-Val
'(1)+1e
-11is-11s-Gly-Pro-Gly-Arg
-Ala-Phe-Val ([1) Val-Phe
-Ala-Arg-Gly-Pro-Gly-Arg-
Gln-lieArg-IIs
(III) The second invention is
Protease inhibition using a peptide having at least the amino acid sequence of the following formula (IV) and not having the amino acid sequence of the following formula (V) [hereinafter, this peptide is referred to as a peptide (IV) derivative] or a salt thereof as an active ingredient It is a drug.

Gly-Pro-Gly-Arg-(■)^sn-As
n-Thr-Arg-Lys-5er-Ile-Arg
-Ile-Gln8r-Gly-Pro-Gly-A
rg-Ala-(V) In the present specification, pharmacologically acceptable salts are not particularly limited as long as they have low toxicity, and include hydrochloride, acetate, phosphate, citrate, and succinate. Organic acid salts such as acid salts, alkali metal salts (sodium salts,
Examples include inorganic acid salts such as potassium salts, etc.

In the present invention, the amino acid sequence represented by formula (IV) is H
This corresponds to a part of the amino acid sequence in the coated fluorescent substance (gP120) of IV. However, the peptide (I
V) Derivatives are not particularly limited as long as they have at least the amino acid sequence region represented by the above formula (IV); These peptides can be combined with polyethylene glycol or its FaR to reduce
Peptides modified with polyethylene glycol, monomethoxypolyethylene glycol, etc., whose terminal r-
Examples include peptides (1), (II) and (I[[), HIV Examples include HIVO coat protein-related peptides, such as those derived from outer membrane proteins and modified compounds thereof.

Peptides (1), (II) and (I) in the present invention
Peptide (rV) derivatives containing [

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, a C-terminal amino acid with a protected amino group is bonded to an insoluble carrier via a carboxyl group. As the insoluble carrier, a known one can be used. After removing the amino protecting group, the amino acids with protected amino groups were sequentially peptide bonded according to the amino acid sequence and reacted step by step to synthesize the entire amino acid sequence. After that, it is manufactured by removing the insoluble carrier. At this time, it is preferable that the reactive functional group is protected by a known protecting group in peptide synthesis.

The peptide thus produced can be purified to any degree of purity by purification methods commonly used in peptide chemistry, such as using ion exchange resins, partition chromatography, gel chromatography, reversed 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 can be produced by hydrolyzing the salt by a method known per se.

In this specification, when amino acids, peptides, protecting groups, etc. are indicated by abbreviations, they are IUPAC-IUB
Co+u+1 session on Biologica
The abbreviations are based on Nomenclature or common abbreviations in the field, examples of which are given below.

Thr: Threonine ^'g: Arginine Pro
Nibroline Lys: Lysine e: Isoleucine Gln: Glutamine Gly nigericin
Ala: Alanine Phe: Phenylalanine Val: Valine Old S: Histidine t-BO
C: t-Butyloxycarbonyl [Action/Effect] Peptide (IV) derivatives including peptides (1), (II) and (Ill) and their salts can be used in mammals such as humans, dogs, cows, horses, mice and rats. It has excellent protease inhibitory activity in animals, especially trypsin inhibitory activity and chymotrypsin inhibitory activity, and is used as an anti-inflammatory agent, antitumor agent, carcinogenesis inhibitor, etc., and as a hubten for producing antibodies for immunoglobulin therapy. It is useful.

The protease inhibitors of the present invention are usually peptide (rV)
The derivative or its salt is formulated as a lyophilized product and administered orally or parenterally, and the dosage form varies depending on the route of administration.

Preferred dosage forms of the protease inhibitor of the present invention include:
Examples include injections, capsules, tablets, creams, ointments, patches, and burying agents, which are manufactured by methods known per se.

For example, when a peptide (RV) derivative or its salt is used as an anti-inflammatory therapeutic agent, the dosage is, for example, in the case of an injection, 0.1 to 1000 s+g/person per day as the peptide (IV) derivative or its salt. This is generally administered in one to several divided doses a day.

Experimental Example 1 The inhibitory effect of peptide (1) on trypsin activity was
This was demonstrated by kinetic analysis of the enzymatic reaction.

Commercially available purified trypsin tpg, 50 g + M) Lis-hydrochloride buffer pH 8, 0, 0, 15 M sodium chloride and substrate t-butyloxycarbonyl-phenylalanyl-L-seryl-L-arginine 4-methyl- In a reaction solution consisting of coumaryl-7-amide, peptide (I
), or distilled water was added as a control and 2
The enzyme activity was calculated by reacting at 5°C and measuring the fluorescence intensity at an excitation wavelength of 380 ns+ and an emission wavelength of 440 n-. The Lineleiber-Park plot pattern in the presence of 50 μH of peptide (1) is shown in FIG. Reinleiber-Park plot shows that the group [1 degree 25.33.3.50.100 μM
The enzyme activity in 1 second is 7-
The enzyme unit that produces 1 pmol of amino-4-methylcoumarin was calculated as 1 pKat, and the reciprocal of the calculated enzyme activity was plotted against the reciprocal of each substrate concentration.

This result shows that the inhibition of trypsin activity by peptide (1) is antagonistic. In FIG. 1, O is the control, and * is the result in the presence of 50 μM peptide (+).

Experimental Example 2 The inhibitory effect of peptide (1) on α-chymotrypsin activity was demonstrated by kinetic analysis of the enzymatic reaction.

20 μg of commercially available purified α-chymotrypsin, 50 mM Tris-hydrochloride pH 8, 0, 10 mM sodium chloride and the substrate succinyl-L-alanyl-shi-alanyl-shi-prolyl-shi-phenylalanyl 4-methyl-coumaryl-7. - Peptide (I) in the reaction solution consisting of amide
Or, as a control, Rheinliver bark pronto was obtained in the same manner as in Experimental Example 1 except that distilled water was added. 5
0 μM pef.

The Rheinleiber-Park plot pattern in the presence of Tido(1) is shown in FIG.

This result shows that the inhibition of α-chymotrypsin activity by peptide (1) is non-competitive. In Figure 2,
○ indicates the control, and . indicates the result in the presence of 50 μM peptide (1).

Experimental Example 3 Inhibition constants for trypsin and α-chymotrypsin were determined for peptide (I), a peptide consisting of a part of the amino acid sequence of peptide (1), and a peptide consisting of a sequence different from the amino acid sequence of peptide (I).

Inhibition constants were calculated from the Dixon Pronto plot for trypsin showing competitive inhibition, and from the Reinleiber-Park plot for α-chymotrypsin showing non-competitive inhibition. The Dixon plot shows that in the same reaction solution as in Experimental Example 2, the concentration was 0.20.40.60I! The enzyme activity when peptide M was added was calculated and obtained by plotting the reciprocal of the enzyme activity for each peptide concentration.

The inhibition constants for trypsin and α-chymotrypsin are shown in Table 1. This result showed that only peptide (1) had a significant inhibitory effect on both trypsin and α-chymotrypsin. Furthermore, it can be seen that the inhibition of trypsin depends on the number of arginine and lysine, whereas the peptide (1) specifically inhibits α-chymotrypsin.

Table 1 Summer: Isoleucine R: Arginine 0: Glutamine G Nigericin P Nibroline
A: Alanine F: Phenylalanine
V: Valine Experimental Example 4 Peptide (■), Peptide (■), Peptide (III)
The inhibitory effect of chymotrypsin on chymotrypsin activity was investigated.

The chymotrypsin activity when the substrate concentration was set to 100 μM in the same manner as in Experimental Example 1 was expressed as a relative activity, with the activity of the control to which no peptide was added as 100%.

The concentration dependence of peptides (1), (II) and (III) is shown in FIG.

From this result, peptide (TV) derivative, peptide (
II) and (II[) are likely to exhibit similar trypsin inhibitory effects to peptide (I). In Figure 3, . is peptide (I), mu is peptide (n), and peptide ([
[I) These are the results in the presence of

Experimental Example 5 The inhibitory effects of peptide (■), peptides (II) and peptides (III) on α-chymotrypsin activity were investigated.

The α-chymotrypsin activity when the substrate concentration was set to 100 μM in the same manner as in Experimental Example 2 was expressed as a relative activity with the activity of the control to which no peptide was added as 100%. The concentration dependence of peptides ([), (U) and (I[l) is shown in FIG. 4.

This result shows that peptides (U) and (III) exhibit similar chymotrypsin inhibitory effects as peptide (+). In FIG. 4, * is the result in the presence of peptide (■), mu is the peptide (■), and ■ is the result in the presence of peptide (III).

Comparative Example 1 The inhibitory effect of peptide (r) was suppressed by L, a commercially available protease synthesis inhibitor that specifically inhibits α-chymotrypsin.
The effect was compared with that of -1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK).

In the same manner as in Experimental Example 2, the substrate concentration was set to 50 μM,
The relative activity was compared with that in which TPCK was added instead of peptide (1).

The concentration dependence of peptide (1) and TPCK is shown in FIG.

This result shows that peptide (1) has a greater inhibitory effect than TPCK. In Figure 5, . is a peptide (
1), O is the result in the presence of TPCK.

〔Example〕

EXAMPLES The present invention will be described in more detail with reference to Examples below, but these are not intended to limit the present invention in any way.

Note that all amino acids used in the following examples are L-units unless otherwise specified.

Synthesis example 1 Peptide (1) was synthesized.

Peptide synthesis was performed by a solid-phase method using Peptide Synthesizer 43OA manufactured by Applied Biosystems. That is, 0.5 mmol of L-Boc-L-valine-bonded phenylacetamidomethyl resin (styrene-1
% divinylbenzene copolymer), the N-terminus was
2 mmol of t-Boc amino acid protected with N,N-
Condensation was carried out by a symmetrical amino acid anhydride method using 1 mmol N,N-dicyclohexylcarbodiimide in dimethylformamide, followed by a sequential extension method in which peptide bonds were extended one by one. However, regarding the condensation of Gln and Arg, condensation was performed twice by an active ester method using 1 mmol of hydroxybenzotriazole in N,N-dimethylformamide.

t-BO (, as a protecting group for the side chain of an amino acid, Arg
2,4.6-trimethylbenzenesulfonyl group, L
ys is N-t-butoxycarbonyl-N-2-chlorobenzyloxycarbonyl group, Ser and Thr
In each case, a benzyl group was used.

The standard synthesis program for peptides is 60% deprotection.
trifluoroacetic acid/dichloromethane for 1 min or 15 min, 3 x 1 min with dichloromethane as a wash, 2 x 1 min with 10% N, N-diisopropylethylamine as a neutralization, and 1 x 1 min with N, N-dimethylformamide as a wash. N in N,N-dimethylformamide as condensation for 6 min.

Symmetrical amino acid anhydride method with N-dicyclohexycarbodiimide for 20 min, two 1 min washes with N,N-dimethylformamide and 4x 1 min with dichloromethane.
In these standard synthesis programs, which are performed multiple times, the reaction temperature, reaction time, etc. for each amino acid are controlled in detail by a microcomputer.

The peptide resin obtained by the automatic peptide synthesizer is
The support resin and the protecting groups on the amino acid side chains were cleaved with trifluoromethanesulfonic acid.

That is, 1 g of synthetic peptide resin and thioanisole II
d. Stir 500 µl of ethanedithiol at room temperature for 10 minutes, add 10% of trifluoroacetic acid while cooling the flask with ice water, and stir for 10 minutes.

Next, 1 ml of trifluoromethanesulfonic acid is added, and the flask is taken out of the ice water and stirred at room temperature for 30 minutes. Add cold diethyl ether to the flask until no more peptide precipitates appear, stir for 1 minute, filter the crystals with a Teflon filter, further dissolve this in a small amount of trifluoroacetic acid, transfer to cold diethyl ether, and pass through the Teflon filter again. The crude peptide was filtered and dissolved in 2N acetic acid or the like to obtain a crudely purified peptide.

The crudely purified peptide was purified using a reverse phase column (diameter 21, length 25C) consisting of a silica gel packing with octadecyl groups bonded to it.
11) high performance liquid chromatography (column:
Purification was performed using YMCR-ODS-5). Elution was performed using O, 1% trifluoroacetic acid as the mobile phase and a linear concentration gradient of 90% acetonitrile from 30% to 70% (flow rate 5 d/sin). The chromatogram of the purified sample is shown in Figure 6 ( (detected by ultraviolet absorption at 220 nm).

One nanomole of the purified peptide was analyzed using a gas phase sequencer. As a result, the amino acid residues detected in analysis cycles 1 to 12 are, in order, IIe+Arg, rl
e, Gln+Arg+ Gly+ Pro+ GIy
+Arg, Ala.

Phe and Vat, and it was confirmed that the target peptide (1) was obtained. The yield of peptide after purification was 110 μ.

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 contains Hls, the following pretreatment was performed before the cleavage with trifluoromethanesulfonic acid as in Synthesis Example 1. N, N- on peptide resin
Dimethylformamide 24tel, thiophenol Id
After stirring at room temperature for 1 hour, the mixture was suction filtered using a Teflon filter, washed three times with 10-dichloromethane, and dried.

Furthermore, while keeping this resin at 0°C, m-Fresol 1rId11 Dimethyl sulfide 3ffii! , trifluoroacetic acid 5d, trifluoromethanesulfonic acid 1
The reaction mixture was reacted for 3 hours in a Teflon filter, washed with cold diethyl ether on a Teflon filter, dried, and then cut in the same manner as in Synthesis Example 1.

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

Results of analysis using the same method as Synthesis Example 1 Analysis cycle 1~
The amino acid residue detected in No. 11 is He.

His, lie, Gly, Pro, Gly, A
rg, Ala, Phe, Vat.

It was confirmed that the target peptide (n) was obtained. The yield of peptide after purification was 1801g.
Met.

Synthesis example 3 Peptide (Ill) was synthesized.

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

It was purified by high performance liquid chromatography in the same manner as in Synthesis Example 1. The current chromatogram is shown in Figure 8 (22
(detected by ultraviolet absorption at 0 nm).

As a result of analysis in the same manner as in Synthesis Example 1, analysis cycle l
The amino acid residues detected at ~12 are Val.

Phe, Ala, Arg, Gly, Pro
+ GIy+ Arg, Gin, Ile.

It was confirmed that the target peptide (1) was obtained.

Formulation Example 1 After the peptide (1) was purified, it was injected into ampoules, freeze-dried and sealed to obtain an injection preparation of 100 μ/ampule.

Formulation Example 2 Macrogol ointment (8th edition Japanese Pharmacopoeia) (mixture of 50 g of Macrogol 4000 and 50 g of Macrogol 4005) was mixed with 1 g of lyophilized peptide (1) and kneaded thoroughly to obtain a 1% ointment preparation. .

Formulation Example 3 In Formulation Example 1, peptide (I) was replaced with peptide (I).
II) to obtain an injectable preparation.

Formulation Example 4 In Formulation Example 2, peptide (1) was replaced with peptide (
If) was used to obtain an ointment.

Formulation Example 4 In Formulation Example 2, peptide (1) was replaced with peptide (
III) to obtain an ointment.

[Brief explanation of drawings]

FIG. 1 is a Reinleiber-Park plot graph showing the inhibitory effect of the protease inhibitor of the present invention [peptide (■)] on totricin. Figure 2 shows the Rheinleiber-Park plot.
α of the protease inhibitor [peptide (■)] of the present invention
- is a graph showing the inhibitory effect on chymotrypsis. Figure 3 shows peptides (1), (I[I) and (TV)
1 is a graph showing the concentration-dependent inhibitory effect on trypsin. Figure 4 shows peptides (1), (III) and (IV)
2 is a graph showing the concentration-dependent inhibitory effect of α-chymotrypsin on α-chymotrypsin. FIG. 5 shows the protease inhibitor [peptide (I) of the present invention.
)] and a commercially available synthetic protease inhibitor (TPCK) are graphs comparing the inhibitory effects on α-chymotrypsin. The sixth view is a high performance liquid chromatography chart of purified peptide (1). FIG. 7 is a chart of high performance liquid chromatography of purified peptide (II). FIG. 8 is a chart of high performance liquid chromatography of purified peptide (II). Fig. 8 1/(51(rnM)-1 1/(S) (r!IM)-1 Fig. 3 Ao704 Toi/l Ifi M+ To Fig. 4〇7° child doi/blindness (μM) Figure 6 Figure 7

Claims (2)

[Claims] (1) A peptide having an amino acid sequence selected from the following formulas (I) to (III) or a pharmacologically acceptable salt thereof. He-Arg-Ile-Gln-Arg-Gly-P
ro-Gly-Arg-Ala-Phe-Val(I
) Ile-His-Ile-Gly-Pro-Gly-A
rg-Ala-Phe-Val(II) Val-Phe-
Ala-Arg-Gly-Pro-Gly-Arg-G
ln-Ile-Arg-Ile(III) (2) has at least an amino acid sequence site of the formula -Gly-Pro-Gly-Arg-(IV), and has the formula -Asn-Asn-Thr-Arg-Lys-Ser-
He-Arg-Ile-Gln-Arg-Gly-P
A protease inhibitor containing as an active ingredient a peptide that does not have the amino acid sequence ro-Gly-Arg-Ala-(V) or a pharmacologically acceptable salt thereof.