JPH06172212A - Medicine composition - Google Patents

Abstract

PURPOSE:To provide a medicine composition useful as a protease-inhibiting medicine or anti-shock medicine. CONSTITUTION:The medicine composition contains a peptide compound of the following formula or its pharmaceutically acceptable salt as an active ingredient. Tn-A-B-Pro-C-D-Leu-Tc (Tn is N-acetyl; A is arginine residue or lysine residue; B is lysine residue, asparagine residue, glycine residue, isoleucine residue, histidine residue or arginine residue; Pro is proline residue; C is tryptophan residue or tyrosine residue; D is phenylalanine residue or isoleucine residue; Leu is leucine residue; Tc is hydroxyl group).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition useful as a protease inhibitor or an anti-shock drug.

[0002]

2. Description of the Related Art When blood coagulation occurs in blood vessels, the release of plasminogen activator (hereinafter also referred to as activator) from vascular endothelial cells is promoted. The released activator is adsorbed and concentrated on the plasminogen present in the fibrous fibrin formed as a result of the hemagglutination reaction. As a result, plasminogen is activated by an activator and converted into plasmin. Plasmin decomposes fibrin and so-called fibrinolysis occurs.

Such a phenomenon is caused by various causes, and is observed, for example, in septic shock. Many leukocytes are deposited on the capillary bed of the lung due to the retention of blood and the increase in viscosity that occur during the shock of sepsis. As a result, the white blood cells are stimulated to release the internal enzyme. The enzyme free radicals of the released internal enzyme inactivate α1-antitrypsin, which is a blood coagulation inhibitor, and blood coagulation in blood vessels is enhanced. Among the above-mentioned internal enzymes, in particular, the action of mediators such as platelet activating factor (PAF) causes systemic microthrombus, resulting in organ damage.
As a result, blood coagulation factors such as fibrinogen, prothrombin, factor V or factor VII, and platelets are significantly consumed, and at the same time, activation of the fibrinolytic system is enhanced, and finally, a marked bleeding tendency is exhibited. Become. This kind of disseminated intravascular coagulation
ular coagulation sydrome (DIC) has a great impact especially on multi-organ disorders.

The underlying diseases of DIC include, for example, malignant tumors (eg, infiltrates, pancreatic metastases, destruction of tumor cells, tissue damage, cell destruction), extensive burns, obstetric diseases (eg, early placental ablation). , Amniotic fluid embolism, dead fetuses), severe infectious disease (Gram-negative bacillus, septicemia due to endotoxemia, viral infections such as zoster, rubella, etc.), hemolytic reaction (ABO incompatible transfusion, hemolysis due to snake venom), intravascular Membrane changes (giant hematoma, extracorporeal circulation, shock) can be exemplified. DIC occurs in these underlying diseases due to abrupt activation of the mechanism of blood coagulation in blood vessels. And D
As described above, IC causes multiple microthrombus, ischemic lesions of various organs such as kidney and lung, and consumable blood coagulation disorders (con-sumption) due to significant consumption of platelets and blood coagulation factors. Coagulopathy) or diffuse intravascular coagulation and fibrin formation-induced activation of the fibrinolytic system (secondary fibrinolysis), resulting in various bleeding tendencies.

In the treatment of such DIC, it is usually necessary to carry out anticoagulant therapy concurrently with the treatment of the above-mentioned underlying diseases. Conventionally, heparin and synthetic protease inhibitors have been used for this anticoagulant therapy. In addition, it is also used in combination with an antifibrinolytic solvent.

[0006] In addition, the Septic Shock
ck), the bacterial infection causes the bacterial cell wall component endotoxin to increase blood coagulation, and the platelet count in the blood is significantly decreased. As a result, endotoxin-induced vascular endothelial cell damage and shock symptoms are also induced. And cause acute circulatory failure. Anticoagulants such as heparin are also used for such shock symptoms.

[0007]

However, it is not possible to improve the bleeding tendency by breaking the vicious cycle in which bleeding symptoms are caused by the consumable blood coagulation disorder caused as a result of the blood reaching the hypercoagulable state as described above. It's extremely difficult. For example, when heparin is administered, heparin is a protease inhibitor, antithrombin II.
Since it exerts an anticoagulant effect by binding to I (ATIII) and changing its three-dimensional structure to immediately change the protease inhibitory effect, a certain amount (70%) or more in blood is exhibited. AT-III is required. Also,
When heparin is administered in excess, it may cause an increase in bleeding tendency rather than in bleeding. In clinical practice, continuous administration by infusion is performed instead of single administration. Therefore, there is a problem that the medication management becomes complicated.

[0008] Synthetic protease inhibitors such as gabexate mesylate and nafamostat mesylate are also intravenously injected by continuous infusion. In addition, the side effect of synthetic protease inhibitors causing vasculitis has also been reported.

Furthermore, antifibrinolytic agents such as streptokinase, urokinase and tranexamic acid are secondary to the fibrin formation by blood coagulation in blood vessels as described above. Since it is understood that the living body is a protective reaction that tries to dissolve and remove thrombus, it is not preferable to inhibit only this fibrinolysis with an antifibrinolytic agent. Contraindicated.

As described above, in addition to the above-mentioned DIC, venous thrombosis, cerebral thrombosis, transient myocardial infarction, hemolytic anemia, and platelets are caused by abnormalities of biological homeostasis caused by blood coagulation in blood vessels. It is known to cause diseases such as diminished purpura.

The present invention has been made in view of the above points, and provides a pharmaceutical composition having a protease inhibitory action and an anti-shock action, which is useful for diseases caused by increased blood coagulation in blood vessels.

[0012]

The present invention provides a pharmaceutical composition containing a peptide compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. Tn-AB-Pro-CD-Leu-Tc (I)

(In the formula, Tn represents an amino terminal site, and
Indicates a D-type or L-type arginine residue or lysine residue, and B indicates a D-type or L-type lysine residue, asparagine residue, glycine residue, isoleucine residue, histidine residue or arginine residue. , Pro represents a D-type or L-type proline residue, C represents a D-type or L-type tryptophan residue or tyrosine residue, and D represents a D-type or L-type phenylalanine residue or Isoleucine residue, Leu represents a D-type or L-type leucine residue, Tc represents a carboxyl terminal site), where Tn is N-acetyl, pGlu-Gly, or pGlu-Leu-Tyr −Glu−Asn −Lys −Pro

(In the formula, pGlu represents a D-type or L-type pyroglutamic acid residue, Leu represents a D-type or L-type leucine residue, Tyr represents a D-type or L-type tyrosine residue, Glu indicates a glutamic acid residue, Asn indicates D
Type or L-type asparagine residue, and Lys is D
Type or L-type lysine residue, and Pro is a D-type or L-type proline residue).
The pharmaceutical composition of the present invention can be used as a protease inhibitor and an anti-shock drug. Hereinafter, the pharmaceutical composition of the present invention will be described in more detail.

The above-mentioned peptide compound of the present invention and a pharmaceutically acceptable salt thereof (hereinafter referred to as the present peptide compound)
Was disclosed by Caraway and Leeman in 1973 and is incorporated by Lehman in US Pat. No. 3,92.
No. 6,756, hypothalamic inducer
ed substance), which includes neurotensin. The peptide compounds also include a neurotensin-related peptide proposed as Xenopsin in Uchiyama, US Pat. No. 3,926,306. Xenopsin is disclosed to have blood pressure lowering and gastric contractile effects. Table 1 below shows an example of the above-mentioned neurotensin and neurotensin-related peptides including xenopsin.

[0016]

[Table 1] The symbols in Table 1 have the following meanings. pGlu; Pyroglutamyl-glutamic acid Gly; Glycine Leu; Leucine Tyr; Tyrosine Glu; Glutamic acid Asn; Asparagine Lys; Lysine Pro; Proline Arg; Arginine Ile; Isoleucine Phe; Phenylalanine His; Histidine Ala; Alanine Indicates an amino acid residue. An amino acid residue refers to an amino acid having both an α-amino group and a carboxyl group in a free state.

The present inventors have newly found that the present peptide compound containing the above-mentioned neurotensin and its related peptide has a protease inhibitory action. Here, the protease is, for example, a serine protease such as thrombin, plasmin, trypsin and the like.
Among these proteases, thrombin is a fibrinolytic enzyme and has an action of promoting blood coagulation. Further, plasmin is a fibrinolytic enzyme and has an action of promoting the dissolution of fibrin (fibrin). Therefore, the peptide compound of the present invention has a combined effect of inhibiting thrombin and plasmin, respectively, and therefore exerts a remarkable effect in improving the symptoms of DIC. In addition, the peptide compound has an action of reducing the number of platelets in blood and acts to improve the hypercoagulable state.

Further, the present peptide compound can suppress shock symptoms due to endotoxin, for example. This is because the enhancement of blood coagulation in blood vessels caused by endotoxin can be suppressed by the peptide compound.

The peptide compounds of the present invention can form pharmaceutically acceptable salts with organic or inorganic acids. Suitable acids to form suitable pharmaceutically acceptable salts include, but are not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, benzoic acid, citric acid, salicylic acid, malic acid, malonic acid. Examples thereof include fumaric acid, succinic acid, tartaric acid, lactic acid, gluconic acid, ascorbic acid, maleic acid, benzenesulfonic acid, methane or ethanesulfonic acid, and human roxylmethane or hydroxyl ethanoic acid.

Further, the peptide compound of the present invention can form a base addition salt formed by adding a pharmaceutically acceptable organic base. The organic base used here is not particularly limited, and examples thereof include mono-, di-, and trialkylamines such as methylamine, dimethylamine, or triethylamine; mono-, di-, and triethanolamine; arginine. Or amino acids such as lysine; guanidine; N-methylglucosamine; N-
Methylglucamine; L-glutamine; N-methylpiperidine; morpholine; ethylenediamine; N-benzylphenethylamine or tris (hydroxyl) aminomethane. Examples of these organic bases are "Pharmaceutica
l Salts "[J. Pharm. Sci. 66 (1): 1-19 (1977)].

The peptide compounds of the present invention can be synthesized according to various chemical methods. Preferably developed by RB Merrifield, JM Steward and JD
It is synthesized by the manual or fully automatic solid phase synthesis method disclosed in Young's "Solid Phase Peptide Synthesis i" (1984) (hereinafter referred to as Reference 1).

In the solid phase synthesis method, amino acids corresponding to the peptide compound to be synthesized are sequentially bound from the C-terminal side. First, a protected α-amino acid corresponding to the C-terminal of a peptide compound (hereinafter referred to as the C-terminal α-amino acid) is bound to a suitable insoluble resin support. The α-amino group of amino acids must be protected until it forms a peptide bond with the resin support or previously bound amino acid residues. After the binding reaction to the carboxyl terminal is completed, the protecting group that protects the α-amino group (hereinafter referred to as the α-amino group protecting group) is removed, and the next amino acid residue is added. Protecting groups protecting various α-amino groups and acids for removing them are disclosed in the above cited reference 1. A typical protecting group for the α-amino group is a urethane-based tertiary butyloxycarbonyl (Boc), which is acid labile. Other protecting groups and related chemistries are also available, such as base stable 9-fluorenylmethyloxycarbonyl (FMOC).

Similarly, active amino acid side chain functional groups need to be blocked until the end of the synthesis. The group for blocking this functional group (hereinafter referred to as a blocking group) is described in Reference 1
And M. Bodanski, "Peptide Synthesis" (1976).

The solid phase synthesis method begins with the attachment of the C-terminal α-amino acid residue described above to a resin support. An activator is required for this binding. As the activator, for example, dichlorohexycarbodiimide (DCC), alone,
Or 1-hydroxybenzotriazole (HOBT),
Used with diisopropylcarbodiimide (DIIPC) or ethyldimethylaminopropylcarbodiimide (EDC).

After linking the C-terminal α-amino acid residue,
The α-amino protecting group is removed. When the protecting group is BOC, it is eliminated with a dichloromethane solution of trifluoroacetic acid (25% or more). The reaction solution is neutralized with triethylamine dichloromethane solution (10%) and the free amine is recovered as a salt.

Then, the amino acids corresponding to the desired peptide are sequentially added to the reaction solution containing the resin support,
By repeating the steps of binding, deprotection and neutralization, the peptide of interest is extended on the resin support. A cleaning process is performed between each step. Each amino acid is used by dissolving it in a suitable solvent together with an equivalent amount of coupling agent.
After this, the peptide on the resin support is eliminated from the resin support, and the side chain blocking group is eliminated. Anhydrous hydrogen fluoride cleaves BOC and can be used for elimination of blocking groups. Nucleophilic substitution scavengers such as dimethyl sulfide and anisole can be combined to specifically avoid reactions at side chain functional groups. The method for synthesizing the peptide compound of the present invention is not limited to the solid phase synthesis method, and other synthesis methods such as a liquid phase method that are commonly used by those skilled in the art can be applied.

The peptide compound can be administered orally or parenterally in a therapeutically effective amount. The effective amount of the peptide compound is within the range of 1 to 100 μg / human. The dose of the peptide compound can be determined within this range according to various factors such as the degree of disease, the weight of the patient, and the age.

The peptide compound can be administered in combination with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier is, for example, a liquid, gel or solid diluent or excipient containing physiological saline, phosphate buffer and the like. In addition, the drug of the present invention may contain additives which can be generally used in drug compositions, such as common preservatives and antioxidants, if necessary.

The pharmaceutical composition of the present invention can be administered as a preparation for oral administration or a preparation for parenteral administration. The preparation for oral administration is, for example, a usual tablet, capsule, powder, solution or suspension. Further, the preparation for parenteral administration is, for example, an ordinary solution injection or suspension injection, suppository or nasal mucosal spray. The drug of the present invention is preferably administered by intravenous injection or subcutaneous injection. The dosage form of the pharmaceutical composition of the present invention includes the patient's condition, age,
It can be selected according to the degree of the disease. The pharmaceutical composition of the present invention is particularly preferably administered as an amino acid infusion, a carbohydrate infusion or an electrolyte infusion.

The pharmaceutical composition of the present invention contains the above peptide compound as an active ingredient and has a protease inhibitory action. Thereby, it can be used as a protease inhibitor. For example, it has an action of inhibiting thrombin, which is an enzyme of the blood coagulation system, and is therefore expected as a blood coagulation inhibitor. In particular, it has an action of inhibiting plasmin, which is an enzyme of the fibrinolytic system, so that bleeding tendency which is a result of secondary fibrinolysis caused by the significant consumption of blood coagulation factors and platelets due to blood coagulation in blood vessels, that is, DIC Is extremely effective in the treatment of

Furthermore, since the pharmaceutical composition of the present invention has an action of inhibiting various proteases, not limited to thrombin and plasmin, it is expected to be useful for treating various diseases caused by abnormal protease action. To be done.

Further, the pharmaceutical composition of the present invention can be used as an anti-shock drug. The peptide compound has an action of reducing the number of platelets in blood. As a result, it is possible to suppress acute circulatory insufficiency that presents with a shock symptom, which is caused by endotoxin which causes a hypercoagulable state and the number of platelets in blood is significantly reduced.

[0033]

EXAMPLES Examples of the present invention will be described in detail below. Example 1 Protease Inhibitory Action (1) Thrombin

Solutions obtained by diluting the peptide compounds 1 to 3 shown in Table 2 below with various concentrations of 0.1 M Tris buffer (pH 8.4) were mixed with thrombin (final concentration 10 U / ml). This mixed solution was incubated at 37 ° C. for 5 minutes. Next, in the mixed solution, the synthetic color development substrate test team S-
0.125 mg of 2238 (registered trademark; manufactured by Daiichi Pure Chemicals Co., Ltd.) was mixed and reacted at 37 ° C. for 15 minutes. After that, 2% citric acid was added to the reaction solution to stop the reaction, and p-nitroaniline hydrolyzed by the remaining thrombin and released from the synthetic chromogenic substrate was colorimetrically determined at a wavelength of 405 nm to obtain a control solution. The inhibitory ability was determined by comparison with [0.1 M Tris buffer (pH 8.4)]. Based on the determined inhibitory potency, each peptide compound 1 to 3 has thrombin activity of 5
The concentration at which 0% inhibition (IC ₅₀ ) was calculated. The results are shown in Table 3.

[0035]

[Table 2]

[0036]

[Table 3]

From the above results, it was confirmed that the peptide compound which is the active ingredient of the pharmaceutical composition of the present invention has an inhibitory effect on thrombin and can exert a protease inhibitory effect. Example 2 Protease Inhibitory Action (2) Plasmin

Solutions obtained by diluting the peptide compounds 1 to 3 shown in Table 2 below with various concentrations of 0.1 M Tris buffer (pH 7.4) were mixed with plasmin (final concentration 10 U / ml). This mixed solution was incubated at 37 ° C. for 5 minutes. Next, in the mixed solution, the synthetic color development substrate test team S-
2251 (registered trademark; manufactured by Daiichi Pure Chemicals Co., Ltd.) (0.2 mg) was mixed and reacted at 37 ° C. for 15 minutes. After this, 2% citric acid was added to the reaction solution to stop the reaction, and the p-nitroaniline hydrolyzed by residual plasmin and liberated from the synthetic chromogenic substrate was colorimetrically determined at a wavelength of 405 nm to obtain a control solution. The inhibitory ability was determined by comparison with [0.1 M Tris buffer (pH 7.4)]. Based on the obtained inhibitory potency, each peptide compound 1 to 50 has a plasmin activity of 50
The% inhibitory concentration (IC50) was calculated. The results are shown in Table 4.
Shown in.

[0039]

[Table 4]

From the above results, it was confirmed that the peptide compound which is the active ingredient of the pharmaceutical composition of the present invention has an inhibitory effect on plasmin and can exert a protease inhibitory effect. Example 3 Anti-shock action

In this example, mixed-breed dogs (6 dogs per group) were used as samples. Each sample was anesthetized by intravenous injection of pentobarbital at a dose of 30 mg / kg. Next, the peptide compounds 1 to 3 shown in Table 2 were intravenously injected at a dose of 32 nmol / kg. After 30 minutes from the administration, endotoxin derived from Escherichia coli was intravenously injected at a dose of 3 mg / kg while performing artificial respiration with air. Immediately after the injection of the peptide compound and 120 minutes after the intravenous injection of endotoxin, blood was collected from the sample, and the average number of platelets in the blood was measured. As a control, the experiment was conducted according to the same procedure as described above except that the peptide compound was not administered. The results are shown in Table 5. Here, the platelet count is 1 mm ³ of blood The number per (1 μl) was used.

[0042]

[Table 5]

From the above results, it was confirmed that the peptide compound, which is the active ingredient of the pharmaceutical composition of the present invention, has an action of reducing the hypercoagulable state caused by endotoxin and suppressing the tendency of the platelet count to decrease. . This allows
It was found that the pharmaceutical composition of the present invention is effective in improving the shock symptoms of acute circulatory failure caused by endotoxin. This endotoxin-induced shock symptom is partly due to the increase in vascular permeability due to vascular endothelial cell damage in addition to the decrease in the number of platelets, but the pharmaceutical composition of the present invention is shocked by improving the hypercoagulable state as described above. It suppresses the symptoms.

[0044]

Industrial Applicability As described above, the pharmaceutical composition of the present invention contains a peptide compound having a protease inhibitory action and an antishock action and a pharmaceutically acceptable salt thereof as an active ingredient. This allows, for example, a DIC
It is extremely useful for the treatment of various diseases caused by the abnormal action of protease such as that described above, and for the suppression of shock symptoms caused by the hypercoagulable state. [Sequence Listing] SEQ ID NO: 1 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics 1 EX is pyroglutamic acid. Sequence Xaa Gly Lys Arg Pro Trp Ile Leu 15 Sequence number: 2 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics 1 EX is pyroglutamic acid. Sequence Xaa Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 5 10 SEQ ID NO: 3 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Arg Arg Pro Tyr Ile Leu 1 5 SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics 1 E The α-amino group is acetylated. Sequence Arg Arg Pro Tyr Ile Leu 1 5 Sequence length: 6 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Lys Ile Pro Tyr Ile Leu 1 5 Sequence number: 6 Sequence length Length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ile Ala Arg Arg His Pro Tyr Phe Leu 1 5 SEQ ID NO: 7 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence Type: Peptide Sequence Lys Asn Pro Tyr Ile Leu 15

Claims (4)

[Claims] 1. A protease inhibitor comprising a peptide compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. Tn-A-B-Pro-C-D-Leu-Tc (I) (In the formula, Tn represents an amino terminal site, A represents a D-type or L-type arginine residue or a lysine residue, B
Is a D-type or L-type lysine residue, asparagine residue, glycine residue, isoleucine residue, histidine residue or arginine residue, and Pro is a D-type or L-type residue.
C is a D-type or L-type tryptophan residue or a tyrosine residue, and D is a D-type proline residue.
Type or L type phenylalanine residue or isoleucine residue, Leu indicates D type or L type leucine residue, and Tc indicates a carboxyl terminal site) 2. Tn is N-acetyl, pGlu-Gly, or pGlu-Leu-Tyr-Glu-Asn-Lys-Pro (in the formula, pGlu represents a D-type or L-type pyroglutamic acid residue, Leu indicates a D-type or L-type leucine residue, Tyr indicates a D-type or L-type tyrosine residue, and Gl
u represents a glutamic acid residue, Asn represents D type or L
Type asparagine residue, Lys is D type or L
Type is a lysine residue, and Pro is a D-type or L-type proline residue.) The protease inhibitor according to claim 1. 3. An anti-shock drug comprising a peptide compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. Tn-A-B-Pro-C-D-Leu-Tc (I) (In the formula, Tn represents an amino terminal site, A represents a D-type or L-type arginine residue or a lysine residue, B
Is a D-type or L-type lysine residue, asparagine residue, glycine residue, isoleucine residue, histidine residue or arginine residue, and Pro is a D-type or L-type residue.
C is a D-type or L-type tryptophan residue or a tyrosine residue, and D is a D-type proline residue.
Type or L type phenylalanine residue or isoleucine residue, Leu indicates D type or L type leucine residue, and Tc indicates a carboxyl terminal site) 4. Tn is N-acetyl, pGlu-Gly, or pGlu-Leu-Tyr-Glu-Asn-Lys-Pro (wherein, pGlu represents a D-type or L-type pyroglutamic acid residue, Leu indicates a D-type or L-type leucine residue, Tyr indicates a D-type or L-type tyrosine residue, and Gl
u represents a glutamic acid residue, Asn represents D type or L
Type asparagine residue, Lys is D type or L
Is a type lysine residue, and Pro is a D-type or L-type proline residue).