JPH0585530B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes human IgG with SH groups blocked.
The present invention relates to an anti-inflammatory agent containing a chain (hereinafter simply referred to as H chain) as an active ingredient. The H chain has already been reported as a constituent fragment of known IgG, for example, as reported by Fleischman et al. [Arch.Biochem.
Biophys., Supple. (1), 174, (1962)]. By the way, the present inventors have determined that the H chain is
The present invention was completed by discovering that a substance with blocked SH groups has an anti-inflammatory effect. The SH group-blocked H chain, which is the active ingredient in the present invention, is obtained from human-derived IgG by cleaving the disulfide bonds of IgG and has a molecular weight of 50,000±.
1500 in which the SH group of the polypeptide chain is blocked, and its recovery method has been established, for example, as reported by Fleischmann et al. Note that the disulfide bond may be cut not only at an intermolecular bond but also at an intramolecular bond. The outline of a typical method for recovering H chains is as follows. IgG is dissolved in 0.55M Tris-HCl buffer, pH 8.2, to a concentration of approximately 2-3%. Gently pass nitrogen gas through and add 2-mercaptoethanol to a final concentration of 0.75~
Add to 5.25M and leave at room temperature for 1 hour to perform reduction. Next, cool it in an ice water bath and add 2
- Add 0.75 to 5.25M monoiodoacetamide in the same amount as mercaptoethanol, keep the pH of the solution at 8.0 by adding trimethylamine, etc., and react for about 1 hour, then dialyze against cold saline to remove excess reagent. In this reaction, free SH groups in the H chain are blocked by -CH ₂ CONH ₂ . Next, H and L chains were dissociated by dialysis against cooled 1M propionic acid, and passed through a Sephadex G-75 column equilibrated with 1M propionic acid, resulting in two separated H and L chains. Since it is eluted as a peak, this is collected. Of course, recovery of H chains is not limited to the above method. As a reagent for cleaving intermolecular and intramolecular disulfide bonds, in addition to 2-mercaptoethanol, for example, dithiothreitol, dithioerythritol, etc. are used. After collecting the H chain fraction, it is subjected to desired treatments such as dialysis, sterilizing filtration, heat treatment, and freeze-drying in order to use it as a medicine. Although the SH group in the H chain used in the present invention is blocked [Biochemistry, 7 (5), 1950-
1958 (1968), Method in Enzymology xxv ,
185], this block is preferably a group that is not easily decomposed, such as a group that forms an S--C bond. In addition to the above-mentioned groups, examples of such groups include the following. Lower alkyl: methyl, ethyl, n-propyl, etc. N,N-dilower alkylcarbamide - lower alkyl group: N,N-diethyl-carbamidomethyl Lower alkoxycarbonyl - lower alkyl group: ethoxycarbonylmethyl, ethoxycarbonylethyl, etc. Carboxy- Lower alkyl group: Carboxymethyl, carboxyethyl, etc. Cyano-lower alkyl group: Cyanomethyl, etc. β-Amino-lower alkyl group: -
CH ₂ CH ₂ NH ₂ etc. Benzoyl - lower alkyl group: -
CH ₂ COC ₆ H ₅ etc. Carbamoyl - lower alkyl group: -
These blocked substances, such as CH ₂ CONH ₂ , can be produced by known means or similar means. The active ingredient of the anti-inflammatory agent of the present invention is an H chain in which the SH group is blocked, but it is sufficient that the IgG contains the H chain in which the SH group is blocked at a molar ratio of 60 to 100%. The remaining 40% molar ratio may contain an L chain. Next, we will show the methods and results of experiments conducted to confirm the pharmacological actions and effects, clinical trials, acute toxicity tests, dosage, administration method, etc. of H chains with blocked SH groups. () Anti-inflammatory effect Causes carrageenan edema and carrageenan granuloma,
The anti-inflammatory effect and strength of the H chain in which the SH group was blocked was investigated. Regulatory effect of SH group-blocked H chain on carrageenan edema Twelve male SD rats weighing 150-200 g were used in each group. 1.5% lambda carrageenan solution (Sigma
type Iambda−Carrageenan Lot48C−
0.094) 0.1ml was injected subcutaneously into the hind paw, and the paw volume was measured immediately after the injection and at regular intervals from the 1st hour until the 7th hour using a UGO-BASIL Volume Differential Meter.
It was measured over time. Samples (physiological saline, untreated natural IgG, SH group-blocked H chain obtained in Reference Example 1, 100 mg/Kg each) were injected with carrageenan for 30 minutes.
administrated intraperitoneally 5 minutes before administration. The increase in paw volume immediately after injection was determined as the edema rate, and the results are shown in Figure 1. Each point in FIG. 1 represents the mean±standard deviation. As is clear from the results, SH
When administering 100 mg/Kg of group-blocked H chain,
A significant effect on controlling the edema increase rate was observed compared to the native untreated IgG and saline administration groups. Carrageenin granuloma inhibition test Wistar rats weighing 150±20 g were used in groups of 10. After cutting the rat's back hair,
6 ml of air was injected subcutaneously on the back to form an air pouch. Approximately 24 hours later, 4 ml of 2% lambda carrageenan solution was injected into the same site as an inflammatory agent. Five days after administration of carrageenan, rats that had formed granulomas of the same degree were screened, and the test drugs (shown in Table 1) were administered for 7 days.
The administration was continued for 7 consecutive days. On the day after the end of administration, the rats were sacrificed and the weight of the granuloma was measured. The results are shown in Table 1. In conclusion, a significant inhibitory effect was observed in the H chain administration group in which the SH group was blocked compared to the physiological saline administration group. i.e. 100, 50, 10mg/Kg
A significant difference was observed in carrageenan granuloma formation inhibition effect between the saline administration group and the saline administration group, with a risk rate of 0.1% or less at 1 mg/Kg and a risk rate of 5% or less at 1 mg/Kg.

[Table] () Mechanism of anti-inflammatory action We investigated the mechanism of anti-inflammatory action of H chains whose SH groups are blocked. The suppressive effect of H chains with blocked SH groups on the heating plate was investigated using the method of Glenn et al. (EMGlenn, B.
J.Bowmanand JCKoslowske.Biochem.
Pharmacol. Supplement, 27, 1968). Blood was collected from male Wistar rats, fibrin was removed using a glass rod, and then 0.15M phosphate buffer (PH
7.4) was added and centrifuged at 1500 rpm for 15 minutes to obtain red blood cells as a precipitate. The supernatant was washed with the same buffer until no hemoglobin was observed. 400mM in 3.8ml of this erythrocyte suspension
0.2 ml of sodium dodecyl sulfate (SDS) was added to adjust the concentration so that the absorbance at 540 nm of the supernatant during total hemolysis was 0.4-0.5. To 3.8 ml of red blood cell suspension, add the sample (as shown in Figure 2, add the SH group-blocked H chain obtained in Reference Example 1 in an amount of 10 to 200 μg/ml).
0.2 ml of ) was added and incubated at 53°C for 20 minutes. Then rapid cooling and centrifugation (3000rpm×15)
The absorbance of the suspension was measured at 540 nm. The amount of hemolysis was calculated and shown in FIG. 2 as the heating hemolysis inhibition rate. In conclusion, an effect of inhibiting hemolysis of 60% or more was observed when 75 mg or more of H chain with blocked SH groups was added. This is thought to be because the H chain in which the SH group is blocked increases the stability of the red blood cell membrane. Therefore SH
It is surmised that the reason for the series of anti-inflammatory effects caused by the group-blocked H chain is the biomembrane stability it possesses. () Toxicity Since the preparation containing an H chain with blocked SH groups according to the present invention is made from human IgG, it is guaranteed to be as safe as human IgG in terms of toxicity. By the way, IgG and reference example 1 were obtained.
The LD ₅₀ value was determined for the H chain in which the SH group was blocked, and the results are shown in Table 2.

[Table] () Target of administration, dosage, and method of administration H chains with blocked SH groups are used in mammals (e.g., humans, dogs, mice, rats, horses, and cows).
It is used as an anti-inflammatory preventive and therapeutic agent for
The dosage is usually 100 to 500 per adult human day.
mg/Kg. H chains with blocked SH groups are usually used for injections,
It is administered in the form of an oral preparation. As an injection,
For example, it may be dissolved in distilled water for injection before use. The method of administration is intravenous or intramuscular. Examples of oral preparations include capsules, tablets, powders, ribosome preparations, and oral liquid preparations. These preparations are prepared according to known methods such as those described in the Japanese Pharmacopoeia. The anti-inflammatory therapeutic and preventive agent of the present invention, which has an H chain with blocked SH groups as its main component, has extremely low toxicity and exhibits remarkable pharmacological effects, making it extremely useful as a therapeutic and preventive drug for inflammation. Next, reference examples and formulation examples of the present invention will be explained. Reference Example 1 IgG was dissolved in 0.05M Tris-HCl buffer, pH 8.2, to a concentration of about 2%, and 2-mercaptoethanol was added to a final concentration of 0.75M to cleave disulfide bonds. Then add 0.75M iodoacetic acid and adjust the pH
After keeping the temperature at 8.0 and reacting for 1 hour, SephadexG−
Excess reagent was removed with 25 columns. Sephadex G200 column (4.0 x 120 cm) in the presence of SDS (sodium dodecyl sulfate)
[Solvent: 0.04MSDS−0.05M phosphate buffer (PH
8.0)] and the absorbance was measured at 280 nm, and the H chain fraction was collected. After removing SDS from the H chain fraction, dialysis against physiological saline, and further sterilization filtration, a freeze-dried product was obtained. Reference example 2 IgG in 0.5M Tris-HC1 containing 2mMEDTA
Dissolve in buffer solution (PH8.2) to a concentration of approximately 2%. After passing nitrogen gas for about 20 minutes, dithiothreitol was added to a final concentration of 0.01 to 0.068 M, and the mixture was reacted at room temperature for 2 hours. Next, iodoacetamide was reacted to a final concentration of 0.04 to 0.272M for 1 hour under ice cooling, and then dialyzed against 0.1M acetate buffer (PH5.5) and distilled water at 4°C. Note that all operations up to now were performed under light-shielding conditions. Next, propionic acid was added to the solution to a final concentration of 1M, and gel filtration was immediately performed using a Sephadex G-100 column equilibrated with 1M propionic acid. Measurement was carried out at OD280 nm, the H chain fraction was collected, concentrated by ultrafiltration, dialyzed against distilled water, further filtered for sterilization, and then freeze-dried. The yields obtained when the above operations were carried out were as shown in Table 3.

【table】

[Table] Example 1 (Oral preparation) (1) Carbamoylmethylated H chain 5.0mg (2) Fine particles for direct injection No. 209 (manufactured by Fuji Chemical) 46.6mg Magnesium aluminate metasilicate 20% Corn starch 30% Lactose 50% (3) Crystalline cellulose 24.0mg (4) Carboxylmethylcellulose/calcium
4.0mg (5) Magnesium stearate 0.4mg Pass all of (1), (3) and (4) through a 100 mesh sieve in advance. These (1), (3), (4), and (2) are each dried to reduce the moisture content to a certain level, and then mixed using a mixer in the above weight ratio. Add (5) to the uniformly mixed powder, mix for a short time (30 seconds), and tablet the mixed powder (punch: 6.3mmφ, 6.0mmR) to make one tablet.
It was made into an 80mg tablet. The tablets may be coated with a commonly used gastric soluble film coating agent (eg, polyvinyl acetal diethylamino acetate) or an edible coloring agent, if necessary. Example 2 (Intravenous injection) (1) Carbamoylmethylated H chain 50mg (2) Glucose 100mg (3) Physiological saline 10ml (3) Add (1) and (2) in the above weight ratio and stir death,
Dissolve completely. After this solution is filtered using a membrane filter with a pore size of 0.45 μm, sterilization filtration is performed again using a main blank filter with a pore size of 0.20 μm. Aseptically dispense 10 ml of the filtrate into vials, fill with nitrogen gas, and seal to prepare an intravenous injection. Example 3 (Capsule) (1) Carboxymethylated heavy chain 50g (2) Lactose 935g (3) Magnesium stearate 15g Weigh each of the above ingredients, mix the total of 1000g uniformly, and put the mixed powder into hard gelatin capsules. to
Fill 200mg each. Example 4 (Liposomal formulation) Carbamoylmethylated H chain derived from human IgG
Dissolve in 0.01 phosphate buffer (PH7.2) containing 0.125M NaCl to a concentration of approximately 0.5%. On the other hand, 0, 5, 10, 20
% (w/w) of phosphatidic acid (PA) was dissolved in 10 ml of chloroform, and a phospholipid film was formed using a rotary evaporator. By adding 1 ml of the above H chain solution and shaking, a closed fat body was formed and the H chain was incorporated. This fat body was fractionated by centrifugation (10,000 rpm, 30 minutes), and the amount of protein incorporated therein was measured. The results are shown in Table 4.

【table】 [Brief explanation of the drawing]

FIG. 1 and FIG. 2 are graphs showing the effects of the formulation of the present invention, respectively. Black circles: saline administration, white circles: untreated natural
IgG (100mg/Kg) administration, white triangle...H chain IgG with SH group blocked (100mg/Kg) administration, black triangle...
...H chain IgG with blocked SH group (10 mg/Kg) administered, *...R<0.01, **...R<0.001.

Claims (1)

[Claims] 1. An anti-inflammation agent containing as an active ingredient the H chain of human IgG whose SH phase is blocked. 2. The agent for treating and preventing inflammation according to claim 1, wherein the H chain is an alkylated H chain. 3. The anti-inflammatory agent according to claim 1, which is in the form of a solid preparation. 4. The anti-inflammatory agent according to claim 1, which is in the form of a liquid preparation.