JPH11147838A - New nitrogen monoxide supplying agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new nitrogen monoxide supplying agent. SOLUTION: This nitrogen monoxide supplying agent comprises S nitrosated α1 protease inhibitor as a main form and acts as a bifunctional function protein as the nitrogen monoxide supplying agent. The agent can be used as a suitable medicine for improving peripheral vascular circulatory failure, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a new use of plasma proteins. More specifically, S1 nitrosated α1
The present invention relates to a nitric oxide donor based on a protease inhibitor.

[0002]

2. Description of the Related Art α1
Protease inhibitors (hereinafter sometimes referred to as α1PI) are single-chain glycoproteins having a molecular weight of 53,000 and are synthesized mainly in the liver. The blood concentration of a healthy person is 300
At mg / dl (〜60 μM), it is present at the highest concentration among blood protease inhibitors. It has a strong inhibitory activity on neutrophil elastase and cathepsin G, and has a function as a serine protease inhibitor. α1
PI is antithrombin III, heparin cofactor I
I, amino acid sequence homology with protein C inhibitor, α1 antichymotrypsin, α2 plasmin inhibitor, plasminogen activator inhibitor, C1 inhibitor, etc. is high, and a group of serine protease inhibitors (generally referred to as serpin (SERPIN)) ) (Stein P and Ch
othia C, J. Mol. Biol., 221 , p.6
15-621, (1991)).

[0003] α1PI deficiency and abnormalities are closely related to the onset of emphysema, and the onset of emphysema cannot prevent elastase from neutrophils accumulated in the alveoli due to α1PI deficiency. It is believed that this is due to the inability to suppress fiber collapse (Kei
th B and Travis J, J. Biol. Che
m., 255 , pp. 3931-3934 (1980)). α1
Due to its broad protease inhibition spectrum, PI has the ability to inhibit proteases in the above-mentioned tissues, activates blood coagulation system Factor XI (XIa), inhibitors of fibrinolytic plasmin, inhibitors of immune complement system protease, and suppresses the progression of inflammation. (Koji Suzuki, Protein Nucleic Acid Enzyme, 34 , (8)
(1989)).

[0004] The amino acid sequence of human α1PI has been determined from both proteins and cDNAs. Consisting of 394 amino acids, three asparagine residues (Asn
46, Asn83 and Asn247) are linked to a complex type sugar chain. There is one residue cysteine in the molecule, but no SS bond. Me at C-terminal site
t358-Ser is the reaction site and is 1: 1 with the target enzyme.
To form a complex and inactivate protease (Research Monographsin Ce)
ll and Tissue Physiology, v
ol.12, Protease inhibitors,
Barrett AJ and Salvesen G,
441-456 (1986), Elisevier;
Hemostasis, thrombus, fibrinolysis, Michio Matsuda and Koji Suzuki, p.200
-208 (1994), Chugai Medical)

In 1980, Furchigott et al. Discovered an endothelial cell-derived relaxing factor (EDRF).
In 1987, interest in NO has increased worldwide since the fact that the substance was nitric oxide (hereinafter sometimes referred to as NO) was proved by Moncada et al.
ncada Pet et al., Nature 327 , p.
524, (1987)). NO has a wide variety of physiological activities,
It plays an important role in biological defense functions such as vascular smooth muscle relaxation, inhibition of vascular smooth muscle cell proliferation, inhibition of platelet and leukocyte adhesion and aggregation, inflammatory response, and immune response. NO is produced and released from three types of NO synthase (neural type, endothelial type, and inducible type). Many studies so far have revealed the effects of NO produced and released from three types of NO synthase (hereinafter sometimes referred to as NOS). That is, nervous NO
NO produced from S is involved as a neurotransmitter in higher-order structures in the central nervous system, and in gastrointestinal and cardiovascular neuroregulation in the peripheral nervous system. NO produced from endothelial NOS plays an important role in regulating vascular tonus and maintaining homeostasis of vascular functions such as antithrombotic action and antiplatelet aggregation action. It has been clarified that NO, which is produced in large quantities transiently by inducible NOS, plays a part in host defense reactions against bacteria, viruses, tumor cells, etc. (vascular and endothelial, NO: life and death Involvement, Special Issue (199
7)).

[0006] Stamler et al. Disclose μM in human plasma.
An S-nitroso albumin of the order was found, and it was reported that the cysteine residue of hemoglobin in blood was nitrosated. These results indicate that S-nitrosation of in vivo proteins is involved in NO transport and storage,
It is thought to control the physiological action of O (Sta
mler JS et al., Proc Natl Ac
ad Sci USA, 89 , p.7674-7677, (1
992); Lia J et al., Nature, 38.
0 , p. 221-226, (1996)). Recently, due to the attractive physiological activity of NO as described above, various NO donors have been developed and used as agents from a clinical standpoint. Actually, S-nitrosoglutathione (hereinafter, GSN)
O), which inhibits platelet aggregation but does not lower blood pressure, as a platelet aggregation inhibitor during percutaneous transluminal coronary angioplasty (PTCA), It has been applied to the treatment of preeclampsia in women (Langford EJ et al., Lanc.
et, 344 , pp. 1458-1460, (1994); d.
e Belder et al., Lancet, 345 ,
pp. 124-125, (1996)).

At present, NO donors such as GSNO, which are clinically used, have a short half-life in blood or have an adverse effect on by-products produced simultaneously with the release of NO.
There are still insufficient points and many problems as NO donors. On the other hand, for S-nitrosated proteins represented by hemoglobin, Stamler J.S.
Report their usefulness (PCT International Patent Publication, WO 96/30006 and WO 97
/ 10265), it is easily predicted that the introduction rate of nitroso groups is non-uniform due to the presence of a plurality of cysteine residues in the protein used. In addition, there is a concern that hemoglobin itself may impair the function of vascular endothelial cells, or may cause a decrease in renal function due to the deposition of an iron component in kidney tissue.

[0008]

Means for Solving the Problems, Constitution of the Invention The present invention relates to α, which is a physiological various protease inhibitor.
1PI is focused on its properties and the structural property of having only one cysteine in the molecule, and by introducing an S-nitroso group into it, a novel protease inhibitor and N
There is a technical feature in providing a bifunctional functional protein as an O donor and using it as an agent for improving vascular circulatory failure or the like. The present inventors have found that α1P
Focusing on the above-mentioned properties of I, S-
Nitrosylated α1PI (hereinafter sometimes referred to as SNO-α1PI) was prepared, and its potential as an agent for improving peripheral circulatory failure was pursued. That is, as a result of examining the inhibitory activity of SNO-α1PI, the stability of SNO-α1PI, and the NO-donating ability (vascular relaxation action, antiplatelet action, antibacterial action), surprisingly, SNO-α1PI was unmodified with unmodified α1PI. It retains the same protease activity, and according to comparison with nitrosated albumin, SNO-
α1PI was found to have extremely high stability, and furthermore had good NO donating ability (vascular relaxation action, antiplatelet action, ischemia / reperfusion injury inhibitory action, and antibacterial action). Based on these findings, the present invention has been completed.

The outline of the study results on SNO-α1PI of the present invention is as follows. (1) About 99% of a nitroso group was successfully introduced into unmodified α1PI. The transduction rate was much higher than the transduction rate of about 30% of albumin, one of the plasma proteins. (2) The protease inhibitory ability of SNO-α1PI was almost equivalent to that of unmodified α1PI. Also, SNO-α1PI
The dissociation of the nitroso group and the inhibitor activity were stable for more than 2 weeks in liquid storage at 4 ° C. On the other hand, the stability of the nitroso group of nitrosoalbumin was SNO-α1PI
Was very low as compared with only about 50% of the nitroso groups remained in 2 weeks. (3) SNO-α1PI showed a vasorelaxing action in a concentration-dependent manner. However, the effect was not observed for unmodified α1PI. (4) SNO-α1PI showed an antiplatelet effect in a concentration-dependent manner. On the other hand, no effect was observed for unmodified α1PI. (5) SNO-α1PI showed an inhibitory effect on ischemia / reperfusion injury. On the other hand, no effect was observed for unmodified α1PI. (6) Furthermore, SNO-α1PI showed a growth inhibitory effect on various bacteria in a concentration-dependent manner. The effect is GSNO
Was more effective. However, the effect was not recognized with unmodified α1PI. The results of (3) to (6) show that SNO-α1PI functions well as a NO donor. From the above findings found by the present inventors, the SNO of the present invention
The usefulness of -α1PI as a NO donor is clear.
In particular, it is expected to act as a suitable agent for improving peripheral circulatory insufficiency.

It should be noted that SE which is structurally similar to α1PI
Inhibitors of the RPIN genus, such as α1 chymotrypsin and protein C inhibitor, have only one residue of cysteine in the molecule and have no SS bond. Heparin cofactor II, which has three cysteines in the molecule but has no SS bond, also undergoes nitrosation to give αα
It can be estimated that 1PI has the same function as a NO donor, and the usefulness of those nitrosated compounds is expected.

Preparation method of α1PI used in the present invention
Although there is no particular limitation on, for example,
Obtained by the separation method or genetic recombination technology
There is a method of preparing using α1PI-producing cells. blood
As a method for producing α1PI derived from, for example, plasma
Ammonium sulfate fractionation, blue sepharose, DEA
E-cellulose and Sephadex G-75
Travis et al. For performing chromatographic methods
(Travis J., Method in Enzymo
loy,80pp. 754-765) or ammonium sulfate
After the analysis, Zinc chelate, DE-52 Cellulo
Ku combined with each column chromatography
The method of rekki is known (Kurekki
T., Analytical Biochemistry
99pp. 415-420 (1979)). Above all, α1
The PI-containing fraction is spread on a cation exchange resin,
Adsorb white matter and separate and collect α1PI into non-adsorbed fraction
That can efficiently prepare α1PI
Is recommended (JP-A-8-99999).

Introducing NO (nitrosation) to thiol groups of proteins and synthetic reagents can be achieved by a method of reacting with nitrite. As typified by the nitrosation of hemoglobin, there have been cases where the nitrosation of proteins has been successful, but in general, reactions are performed under severe conditions for proteins. Therefore, a method using isoamyl nitrite, which allows the introduction of NO into a thiol group under milder conditions (DeMaster EG.
et al., Biochemistry, 34 , p.
494-11499, (1995)) and a reaction with n-butyl nitrite (Meyer DJ et al.).
l., FEBS Letters, 345 , p. 177-1
80, (1994)).

The SNO-α1PI thus prepared is
It can be freeze-dried or stored in a liquid state together with a stabilizer such as human albumin, salt, sodium citrate, sugar or amino acid, and furthermore, can be frozen and stored. Further, for the purpose of inactivating infectious contaminating viruses, heat treatment at a predetermined temperature in a lyophilized state or a liquid state, for example, at 65 ° C. for 96 hours in a lyophilized state and at 60 ° C. for 10 hours in a liquid state, This is a very preferable embodiment from the viewpoint of drug safety. In the present invention, SNO-α1PI as such an active ingredient is combined with a known suitable excipient to obtain a NO donor which maintains the function as a protease inhibitor of the present invention by a known method.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention.

[0015]

EXAMPLES Preparation Example 1 ( Preparation of S-nitrosated α1PI) Corn fraction FIV-1
Was dissolved in 9.0 L of 0.1 M Tris-HCl / 0.02 M sodium chloride buffer (pH 8.8). To this solution, a 50% polyethylene glycol 4000 solution was added to a final concentration of 12%, and then adjusted to pH 5.2 with an acetate buffer at pH 4.0 to obtain a centrifuged supernatant. Next, the pH of the centrifuged supernatant was corrected to 6.5, and a powdery polyethylene glycol 4000 was added to a final concentration of 30%, and the resulting precipitate fraction was separated by centrifugation. The collected precipitate was dissolved in 25 mM phosphate buffer (pH 6.0) and then dissolved in DEAE-Sepharos.
e Developed on FF and eluted with 90 mM phosphate buffer (pH 6.0). The eluate was diluted with water so that the electric conductivity was 2.0 or less, and the pH was adjusted to 5.2.
SP-S equilibrated with phosphate / acetate buffer (pH 5.2)
The solution was developed on epharose FF, and the non-adsorbed fraction was collected. This fraction was subjected to a virus removal membrane (PLANOVA: 15).
N was filtered using Asahi Kasei Corporation and concentrated using an ultrafiltration membrane. Sodium citrate was added to the concentrated solution to a final concentration of 0.4 M, and sucrose was added to a concentration of 40%.
Liquid heating was performed at 0 ° C. for 10 hours. Add this solution to 25
mM phosphate buffer (pH 7.0).
Mixed albumin was removed using epharose. The flow-through fraction of Blue-Sepharose contains 10
A double molar amount of DTT (Dithiothreitol) was added, and the mixture was reacted at room temperature for 2 hours, followed by re-chromatography by DEAE in the same manner as above. High-purity purified α1PI was obtained in the eluted fraction. After measuring the -SH group content of the obtained highly purified purified α1PI, 100 mM was determined using an ultrafiltration membrane.
The dialysis was replaced with a phosphoric acid / 5 mM EDTA solution (pH 7.0).

The obtained α1PI and isoamylnitrite (Wako Pure Chemical Industries, Ltd.)
Co., Ltd.) at final concentrations of 0.1 mM and 1.0 mM, respectively.
1 ml of 0.1 M phosphate buffer (pH 7.8)
And reacted at 37 ° C. for 30 minutes. The reaction solution is
Seph equilibrated with 0.1 M phosphate buffer (pH 7.8)
After developing on adex-G25 to remove unreacted low-molecular-weight reaction products such as isoamyl nitrite and isoamyl alcohol, the resulting S-nitrosated α1PI (S
-NOα1PI) was concentrated by ultrafiltration. The amount of the recovered protein was 5.04 mg, and the recovery rate as a protein was 95%.

Example 1 (Analysis of properties of S-nitrosated α1PI) The α1PI amount in the present invention was assayed by the following method. The ability of α1PI to inhibit elastase was evaluated by using a chromogenic substrate for elastase. Hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide by elastase increases the absorption at 405 nm. This increase is continuously measured at 37 ° C., and the amount of hydrolysis per unit time is calculated. α1P
The linear change in absorption with and without I is compared over time. The amount of α1PI as an inhibitor was then evaluated based on the fact that elastase and α1PI react stoichiometrically at 1: 1 and the known amount of elastase (Trav
is J, Method in Enzymology, 8
0 , p. 754-765).

Similarly, the ability of S-NOα1PI to inhibit trypsin was evaluated by using a chromogenic substrate for trypsin (Travis J, Method in E).
nzymology, 80 , pp. 754-765). Table 1 shows the results. As a result, it was revealed that about 90% or more of the inhibitory activity of S-NOα1PI on elastase and trypsin remained.

[0019]

Table 1 Experiment No. α1PI protein amount Elastase inhibitory activity Trifusin inhibitory activity (mg) Remaining (%) Remaining (%) 1 4.8 95.8 97.9 2 4.7 93.6 95.7 35.7 5.1 90.2 92.2 4 5.2 92.3 94.2 5 4.9 91.8 95.9

The S-nitroso group was quantified by high performance liquid chromatography connected to a flow reactor for continuously reacting the sample with mercury chloride and the Glyce reagent (Aka).
ike T, et al., J. Biochemistry
y, in press). Table 2 shows the results. as a result,
The introduction efficiency of the S-nitroso group is about 99%, and α1PI
Was found to be almost completely S-nitrosated. Similarly,
Purified albumin was also nitrosated and the introduction rate of the nitroso group was quantified. As a result, the introduction rate was about 30%.

[0021]

Table 2 Experiment No. Nitroso group introduction rate (%) α1PI albumin 1 98 26 299 34 3 100 32 4 98 28 5 100 31

The stability of the prepared S-NOα1PI was evaluated by aseptically leaving the liquid at 4 ° C. as a result,
As shown in Table 3, the dissociation of the nitroso group of S-NOα1PI and the inhibitory activity were stable for 2 weeks or more. On the other hand, the nitroso group of nitrosated albumin attenuated by about 50% in about 2 weeks, which revealed the superiority of S-NOα1PI in stability.

[0023]

Table 3 EIC: Elastase inhibitory activity TIC: Trifusin inhibitory activity Storage period S-NOα1PI S-NO albumin (days) SNO content (%) EIC (%) TIC (%) SNO content (%) 0 100 100 100 100 1 98 97 98 96 96 99 98 97 834 100 100 99 99 71 8 97 99 100 62 12 100 98 99 51 51 16 98 97 98 98 42

Example 2 (Vascular relaxing effect of S-nitrosated α1PI) Body weight 2.5-
A 3.0 kg female New Zealand white rabbit was sacrificed under sodium pentobarbitone anesthesia and the thoracic aorta was removed. After removing fat and excess connective tissue from the excised aorta, a 5 mm wide aortic ring was prepared. The aortic ring was mounted on an organ bath filled with 20 ml Krebs solution and the isometric tension was recorded. Krebs solution at 37 ° C
And 95% O ₂ /5% CO ₂ gas was bubbled. After pre-contracting the aortic ring with 0.3 μM phenylephrine, S-NOα1PI was increased to 1, 3, 10,
Organs to be 30, 100 and 300 nM
It was added into the bath and the change in tension was recorded. The state contracted by 0.3 μM phenylephrine was defined as 0% relaxation, the tension before the addition of phenylephrine was defined as 100% relaxation, and the percentage of relaxation due to each concentration of S-NOα1PI was expressed as%. Table 4 shows the results. S-NOα1PI relaxed the aortic ring in a concentration-dependent manner, and 300 nM S-NOα1PI caused complete relaxation of the aortic ring. On the other hand, in the case of adding unmodified α1PI performed as a control, no apparent relaxation effect was observed.

[0025]

Table 4 Amount of drug added Vascular relaxation rate (%) (nM) S-NOα1PI α1PI 0.0 0.0 1 5.6 1.0 3 19.0 1.5 10 35.6 2.0 30 66 .7 2.0 100 87.3 2.4 300 1000.0 3.0

Example 3 (Antiplatelet effect of S-nitrosated α1PI) Platelet-rich plasma
(PRP), collagen, ADP (adenosine 5'-
The effect of S-NOα1PI on platelet aggregation induced by an aggregation-inducing substance such as diphosphate) or epinephrine was examined. The control was unmodified α1PI. PRP was collected according to a standard method, and the plasma was mixed with S-NOα1PI or unmodified α1PI at a ratio of 9: 1. 360 μl of the mixed plasma was added to the measurement cuvette, and left at 37 ° C. for 3 minutes. Then, 40 μl of an aggregation-inducing substance-containing solution was added.
A TRACER1 PAT-4A). The maximum aggregation rate was calculated from the recorded data. Here, the maximum aggregation rate is represented by the ratio of the maximum transmittance of PRP to the transmittance of platelet-poor plasma, and is usually 70% or more in ADP aggregation. That is, a high maximum aggregation rate means a high platelet aggregation rate, and the maximum aggregation rate decreases due to the action of the inhibitor. S-NOα of the present invention for the maximum aggregation rate
The effect of 1PI was compared to a control unmodified α1PI. Table 5
As shown in Table 2, platelet aggregation induced by the three aggregation-inducing substances was clearly suppressed by the treatment with S-NOα1PI.

[0027]

[Table 5] Drug Maximum aggregation rate (%) Collagen ADP Epinephrine S-NOα1PI 830 15 (5 μM) Unmodified α1PI 75 80 75 (5 μM)

Example 4 (Suppression of ischemia / reperfusion injury by S-nitrosated α1PI)
Wistar rat (male, 200)
２５０250 g) was anesthetized under ether and the abdomen was opened in the midline. The portal vein and hepatic artery were nodulated and blood circulation was blocked for 30 minutes.
Immediately after dissolving the nodules and reperfusion, the rats of each group were given 0. 1 S-NOα1PI and 0.
3 mg and 0.3 ml of physiological saline were administered as a negative control. GOT, GPT and LDH were used as indices and 3
Hepatic damage after hours was evaluated. Table 6 shows the results. From the numerical value of each index, the S-value of the present invention is compared with that of the unmodified α1PI.
It was revealed that NOα1PI has a significant effect on suppressing ischemia / reperfusion injury.

[0029]

Table 6 Drugs to be administered Indicator GOT GPT LDH (IU / L) S-NOα1PI 900 900 6000 (0.3 mg) Unmodified α1PI 1450 1550 18000 (0.3 mg) Physiological saline 1800 2050 15000 (0.3 ml)

Example 5 (Antibacterial effect of S-nitrosated α1PI) Staphylo
coccus aureus ATCC 25923 (hereinafter referred to as S. aureus ) .
aureus ), Staphylococcus
s pyrogenes S121 (hereinafter, S. pyogene)
s ), Escherichia coli AT
CC25922 (hereinafter referred to as E. coli ), Salmon
ella typhi GIFU10751 (hereinafter, S.typ)
hi ) and Salmonella typhi
murium LT2 (hereinafter S. typhimurium)
The antibacterial effect of S-NOα1PI against GSNO and unmodified α1PI was evaluated using the increase in turbidity accompanying bacterial growth as an index. S. aureus , S. pyog
enes , E. coli , S. typhi and S. ty
phymurium (1 × 10 ⁶ colony forming units / ml each) was added to Krebs-Ringer-Linn buffer pH 7.4.
(Hereinafter referred to as KRP) in S-NOα1PI (0.0
13, 0.04, 0.12 mM), GSNO (10, 10
0, 1000 mM) and unmodified α1PI (0.013,
0.04, 0.12 mM) at 37 ° C for 1 hour.
In addition, what did not add a chemical | medical agent was set as the control. The reaction solution was diluted 10-fold with a Brain Heart Infusion liquid medium, and cultured at 37 ° C. to grow the bacteria. S-NOα1PI, GSNO and unmodified α1PI
The growth rate of each bacterium in the presence was evaluated based on the increase in turbidity by culturing for 6 hours. Table 7 shows the results. S-NO
α1PI and GSNO inhibited the growth of each bacterium, and comparing their activities, the former was approximately 100 times more active than the latter. On the other hand, unmodified α1PI did not show any inhibitory effect on bacterial growth.

[0031]

Table 7 Drug Bacterial Species (Growth Rate (%)) Addition Concentration (mM) ABCDE Control 100 100 100 100 100 GSNO 0.196 150 116 116 106 106 186 100 109 109 136 106 10 0 43 173 3 S-NOα1PI 0.013 76 144 122 99 100 0.04 839 23 3 3 0.12 66 61 2 1 α1PI 0.013 102 214 114 114 131 92 0.04 104 269 106 139 105 0.12 109 184 116 120 120 116 A: S. aureus , S. aureus pyogenes , C: E. coli , D: S. typhi , E: S. typhimurium

Continuation of the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/00 643 A61K 31/00 643D (72) Inventor Takayoshi Hamamoto 1999-8 Asoda, Shimizu-cho, Kumamoto-shi, Kumamoto (72) Inventor Kazuhiko Tomoyoshi 70-1 Yuge, Tatsuta-cho, Kumamoto-shi, Kumamoto (72) Inventor Tomohiro Nakagaki 2527-311, Toyooka, Koshimachi, Kikuchi-gun, Kumamoto (72) Inventor Seiji Miyamoto 2066-8, Suya, Nishi-Goshi-cho, Kikuchi-gun, Kumamoto

Claims (5)

[Claims] 1. A nitric oxide donor based on S-nitrosated α1 protease inhibitor. 2. A vascular circulatory insufficiency improving agent comprising the nitric oxide donor according to claim 1 as a main component. 3. An antiplatelet agent comprising the nitric oxide donor according to claim 1 as a main component. 4. An ischemic / reperfusion injury inhibitor comprising the nitric oxide donor according to claim 1 as a main component. 5. An antibacterial agent comprising the nitric oxide donor according to claim 1 as a main component.