JPS61134316A - Complement activity inhibiting agent - Google Patents

Abstract

PURPOSE:To provide a complement activity inhibiting agent containing zinc ion as an active component, inhibiting the activation of the complement first path based on the antigen and antibody composite and the complement second path occurring in the absence of antibody, effective for the remedy of autoimmune diseases, and having low toxicity. CONSTITUTION:The objective complement activity inhibiting agent is produced by using zinc ion as an active component. It has been found that zinc ion (Zn<2+>) has excellent complement activity inhibiting effect, and is useful as a complement activity inhibiting agent. The zinc ion formed by the administration of a zinc compound (e.g. zinc chloride, zinc phosphate, zinc acetate, glycine zinc, etc.) inhibits the formation of both C3 convertase (C142 and C3b, Bp, P) playing the most important role in the activation of a complement. The complement activity inhibiting effect of zinc ion takes place through the complement controlling mechanism inherent to a living body. It is useful for the remedy of autoimmune diseases such as glomerulonephritis, etc., and drug allergy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to complement activity inhibitors effective in the treatment of autoimmune diseases.

In more detail, c. Activation of the classical complement pathway and alter-n.
The present invention relates to a complement activity inhibitor containing zinc ions as an active ingredient, which inhibits the activation of the active pathway.

Complement originally plays a role as one of the biological defense mechanisms against foreign substances such as bacteria. In other words, when bacteria invade the body, antibodies bind to the bacteria (complement 1
pathway) or bacterial cells) (complement 2
Pathway) Complement is activated and bacteria are destroyed by activated complement.

However, in many autoimmune diseases11 such as glomerular gastritis and autoimmune hemolytic anemia, antibodies against self tissues (autoantibodies) are constantly present, and then complement activation occurs continuously. Anaphylatoxin 03ts produced by activation of complement, which continues to destroy self-tissues
, C5a is a neutrophil,! Activation of clophages further promotes tissue destruction by their phagocytes.

Therefore, in the treatment of such autoimmune diseases, a substance that inhibits the activation of complement (complement activation inhibitor*) is required. Complement activity inhibitors are also required for the treatment of drug allergies for similar reasons.

Several complement activity inhibitors have been discovered for the above purpose and have been confirmed to be effective against experimental autoimmune diseases, but none of them are highly toxic and have not yet become effective therapeutic agents for autoimmune diseases. do not have.

The present inventors conducted intensive research to obtain a drug with low toxicity and excellent complement activation inhibitory effect, and found that zinc ion is superior to 7? ,
The present invention was completed based on the discovery that it has a complement activity inhibiting effect.

Divalent metal ion Oa is used for complement activation reaction.

It is already known that Mg2+ is required.

That is, C~2+ is involved in the expression of complement component C1 activity, and Mg'' is involved in C3 convertase (0142) of the first complement pathway.
and 03 convertase of the alternative complement pathway (
It is essential for the formation of 03b, Eb, P).

On the other hand, human serum contains zinc ions (Zn) or
The present inventors focused on zinc ions and studied their effects on complement activity, and found that
We found that complement activity is regulated by serum zinc concentration.

That is, zinc ion (Zn) inhibits the complement activity of human P and guinea pig serum in a concentration-dependent manner, with 50%
Inhibition was achieved at 8-12 μM.

This value is below the serum zinc concentration.

The fact that serum complement activity is regulated by zinc ions is demonstrated by the use of a chelating agent selective for zinc ions, 0-phenan.
It is also clear from the fact that when human serum is treated with throline, zinc ions in the serum are chelated to increase complement activity, and that this increasing effect is suppressed by adding an equivalent amount of zinc ions.

The site of inhibition of complement activity by zinc ions is one of the rate-limiting reactions in the complement 1 and 2 pathways.
Based on inhibition of formation of ase (0142 and 03b, Bb, P').

Based on the new findings revealed by the inventors,
The use of zinc ions as a complement activity inhibitor has the following advantages.

1) The complement activation inhibitory effect of zinc ions is due to the zinc ions (Zn'') of zinc compounds.

2) Zinc ions (Zn) produced by administration of zinc compounds
2+), unlike other complement activity inhibitors, is not a foreign substance to living organisms and can exist in relatively large amounts in serum.

3) Zinc ions (Zn) produced by administration of zinc compounds
) is carried out through the complement control mechanism by zinc ions, which the body naturally possesses.

4) Zinc ions (Zn) produced by administration of zinc compounds
The inhibition site of the complement activation inhibitory effect by 03 con-vertase is the most important site for complement activation.
(C142 and 03b, Bb, P) formation is inhibited, making it an optimal site for inhibition.

5) Zinc compounds generally have low toxicity, although this varies depending on the administration method.

There is no particular limitation on the zinc compound that is produced in the body by the zinc ion contained in the active ingredient of the complement activity inhibitor of the present invention, as long as it does not have any adverse effects on other organisms in the body. For example, zinc-containing inorganic Examples include salts, zinc-containing organic compounds, and zinc-containing proteins.

Examples of zinc-containing inorganic salts include zinc chloride, zinc bromide, zinc iodide, zinc fluoride, zinc hydroxide, zinc sulfate, zinc carbide, zinc carbonate, zinc silicate, zinc phosphate, zinc oxide, zinc nitrate, and nitrite. It can contain zinc, zinc pyrophosphate, zinc selenide, etc.

Examples of zinc-containing organic compounds include zinc formate, zinc acetate, zinc glycine, zinc acetate, zinc caproate, zinc caprylate, zinc lafricate, zinc stearate, zinc oleate, zinc oxalate, zinc lactate, zinc tartrate, and citric acid. Zinc organic acids such as zinc acid, zinc gluconate, zinc benzoate, zinc salicylate, zinc picrate, zinc amino acids such as zinc amide, zinc cyanide, zinc hydrogen sulfide, zinc thiocyanate, zinc glycine, etc. I can do it.

Examples of zinc-containing proteins include those that non-specifically adsorb zinc ions and zinc-containing proteins that coordinate zinc ions.

The complement activity inhibitor of the present invention can be administered orally2 or parenterally such as by intravenous injection.

The dosage varies depending on the age, medical condition, and weight of the patient. Generally, for adults, the active ingredient zinc ion (Zn) can be administered in doses of about 10 to 3001n9 per day, and can be divided into several doses per day if necessary.

Although the zinc compound itself can be administered as the complement activity inhibitor of the present invention, it is usually administered as various pharmaceutical compositions. Examples of dosage forms of such pharmaceutical compositions include capsules, granules, powders, tablets, tablets, syrups, injections, and the like.

None, the toxicity of zinc compounds e.g.
D5o is 2.46 p/d after oral administration, and LD of zinc chloride in rats is 60-901 p/d when administered intravenously.

In addition, the complement activity inhibitory effect of the complement activity inhibitor of the present invention is demonstrated by the effect of human serum complement and purified complement components on sensitive red plate EA (
sheep erythrocytes sensitized with rabbit anti-sheep erythrocyte antibodies) and complement reaction intermediate l1iAC14.

This was confirmed by measuring the hemolytic reaction of KA (:!431).

Hereinafter, the present invention will be explained in more detail with reference to Examples.
It is not limited to these.

Example 1 Human serum containing Oa HMg + glucose (
) heel/< ツ77- (D G ”I
B”) 1/1o.

Add zinc io y (Zn
(J2)? :Including DGVB” 0.1 +d 3
The reaction was carried out at 0°C for 15 minutes. NextIrlX1G' 1il
t7) EAf! 0.1 d of DCkVB was added thereto, and the mixture was further reacted at 3F'C for 60 minutes.

Centrifuge the reaction solution and measure the amount of hemoglobin in the supernatant using absorbance A41.
Determine the degree of hemolysis (y)'It by measuring at 4 mμ,
Furthermore, complement activity Z (Z--jn(1-7)) was calculated. The complement activity obtained in the above reaction without the addition of ZnCl2 is set as 20, and the inhibitory activity is expressed as Z/ZO, which is shown in Figure 1.
Shown below.

From FIG. 1, ZnCl2 inhibits human complement activity in a concentration-dependent manner, and its SOq & inhibitory concentration is approximately 8 μM. 4
ks Zn C72 At 35 μM, complement activity was reduced to approximately 1.
I don't understand that it inhibits oo96.

Example 2 0.1-DGVB containing 4M human complement component C2
K Zn (DGVB including 42ft"0.1a?'
4 and reacted at 30°C for 15 minutes. Then lX10
Add DGVB2+Q and IJlj containing 1% ZnCl4 (an intermediate formed by the reaction of KA Ic Cu2 and C4) and react at 37°C for 10 minutes. The generated 1IiA (14
Activity of 2 (Z)? :Measurement next. The activity of MAC142 was measured by adding 03-09 and allowing the hemolytic reaction to proceed.

The activity without the addition of ZnO12 was set as 20, and the inhibitory activity was expressed as Z/zo.

The results are shown in Figure 2.

From U2, ZnO12 is 03 convertase
[EA]42 formation reaction could be inhibited in a ttyav-dependent manner, and its 50% inhibition degree was about 6 μM.

Example 3 DG containing human alternative complement pathway components B, D and P'4
DGVB containing VE” 0.1 ml K ZnCl2
(1,1,m) were added and reacted at 30°C for 15 minutes.

Then I X 1G'a(D KAO43b(Fi
A ni04 (!: aA (combined thing) including DG
VB" 0.1 agwoiJD, t, reacted at 37°C for 30 minutes. KA produced 0431), Bt),
The activity of P (Z) 'a' was measured. ! , the activity without the addition of n012 was expressed as zO, and the inhibitory activity was expressed as Z/zo.

The results are shown in Figure 3.

From Figure 3, Zn (J2 is the alternative complement pathway 03 con
verea as.

K A O431), Bl), can inhibit the formation reaction of P in a once-dependent manner, and its so96m harmful concentration is about 1
btsuno at 0 μM.

Examples of formulations are shown below.

Formulation example 1. Tablet zinc chloride 30 damil, ii
95 corn flour
24,150 da The above formulation was made into tablets according to a conventional method.

Formulation Example L Injection Zinc chloride was dissolved in 5 to 20 degrees of distilled water adjusted to pH 5, and then sealed in a 2aat ampoule and sterilized according to a conventional method to prepare an injection.

[Brief explanation of the drawing]

FIG. 1 shows the inhibitory activity of Zn 012 on human serum complement activity. Figure 2 shows C 3 convert by znct2.
Figure 3 shows the inhibitory activity of the formation reaction of taseEAO142.
tase KAO431), Bl), shows the inhibitory activity of P formation reaction. In each figure, the vertical axis shows the inhibitory activity, and the horizontal axis shows the inhibitory activity of Zn012.

Claims (1)

[Claims] A complement activity inhibitor containing zinc ions as an active ingredient.