JPH0360806B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anti-inflammatory agent containing a human IgG L chain (hereinafter simply referred to as L chain) as an active ingredient. The L chain has already been reported as a component 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 completed the present invention by discovering that the L chain has an anti-inflammatory effect. The L chain, which is the active ingredient in the present invention, is derived from human
It is a polypeptide chain with a molecular weight of 23,000±1,000 obtained from IgG by cleaving the disulfide bonds of IgG, and its recovery method has been established, for example, in the above-mentioned report by Fleischman et al. The outline of a typical method for recovering L chain is as follows. IgG is dissolved in 0.55M Tris-HCl buffer, pH 8.2, to a concentration of approximately 2%. After gently blowing nitrogen gas, 2-mercaptoethanol is added to the final concentration of 0.75M, and the mixture is left at room temperature for 1 hour to perform reduction. Next, the mixture was cooled in an ice water bath, 0.75M monoiodoacetamide in the same amount as 2-mercaptoethanol was added, and the solution was reacted for 1 hour while maintaining the pH of the solution at 8.0 by adding trimethylamine, and then dialyzed against cold saline. Remove excess reagent. In this reaction, free SH groups are blocked by alkylation.
Next, H and L chains were dissociated by dialysis against cooled IM propionic acid and equilibrated with IM propionic acid.
When passed through a Sephadex G-75 column, the H chain and L chain are welded as two separate peaks, which are collected. Of course, recovery of the L chain is not limited to the above method. After collecting the L 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. The L chain also includes derivatives in which the SH group is modified (alkylated, sulfonated, etc.) in order to improve the stability of the SH group. Next, the methods and results of experiments conducted to confirm the anti-inflammatory effect of L chain, clinical tests, acute toxicity tests, dosage and administration method will be shown. () Anti-inflammatory effect Causes carrageenan edema and carrageenan granuloma,
The anti-inflammatory effect and strength of the L chain was investigated. Inhibitory effect of L chain on carrageenan edema Twelve male SD rats weighing 200 to 250 g were used in each group. 1.5% lambda carrageenan solution (Sigma
type IV Iambda−Car−rageenan Lot 48C−
0.094) 0.1ml was injected subcutaneously into the hind leg, and the paw volume was measured immediately after the injection and at regular intervals from the 1st hour until the 7th hour.
Measured over time at er. Samples (physiological saline, terminally treated natural IgG, L chain obtained by iodoacetic acid treatment in reference example, each 100 mg/Kg) were injected into carrageenan at 30 mg/kg.
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, the L chain
At 100 mg/Kg administration, a significantly suppressive effect on the edema increase rate was observed compared to the native untreated IgG and saline administration groups. Carrageenin granuloma inhibition test Wistar rats weighing 100 to 120 g were used in groups of 5 rats. 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. Rats that had formed granulomas of the same degree on the 5th day after administration of carrageenan were screened, and the test drug (shown in Table 1; the L chain is a reference example treated with iodoacetic acid) was continuously administered by the 7th day. It was administered for 3 days. On the day after the end of administration, the rats were sacrificed and the weight of the granuloma was measured. The results are in Table 1
It was shown to. In conclusion, compared to the physiological saline administration group, L
A significant inhibitory effect was observed in the chain administration group. 100
mg/Kg and 50mg/Kg with a risk rate of less than 1%,
In addition, at 10 mg/Kg, an inhibitory effect on carrageenan granuloma formation was observed with a risk rate of less than 5% compared to the saline administration group.

[Table] () Mechanism of anti-inflammatory action We investigated the mechanism of anti-inflammatory action of L chain. The method of heating hemolysis of L chain was investigated by Glenn et al. (EMGlenn, B.J., Bowman and J.C.
Koslowske, Biochem.Pharmacol.Supple−
ment, 27 , 1968). I followed the instructions. 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. Add 3.8ml of this erythrocyte suspension
Add 0.2ml of 400mM sodium dodecyl sulfate (SDS) and adjust the absorbance of the supernatant at 540nm to 0.4-0.5 during total hemolysis.
The concentration was adjusted so that red blood cell suspension
Add 0.2 ml of the sample (as shown in Figure 2, the amount of L chain added was 10 to 200 μg/ml; L chain is the one treated with iodoacetamide in the reference example) to 3.8 ml, and incubate at 53°C. Incubated for 20 minutes. Thereafter, the suspension was rapidly cooled and centrifuged (3000 rpm x 15 minutes), and 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, a 62% hemolysis inhibition effect was observed when 100 mg or more of L chain was added. This is thought to be because the L chain increases the stability of the red blood cell membrane. Therefore, it is surmised that the reason for the series of anti-inflammatory effects caused by the L chain is its biomembrane stability. () Toxicity Since the L chain-containing preparation according to the present invention is made from human IgG, it is guaranteed to be as safe as human IgG in terms of toxicity. () Subject, dose and method of administration L chain is used as an anti-inflammatory prophylactic or therapeutic agent for mammals (e.g. humans, dogs, mice, rats, horses, cows), and the dose is usually 1.
100mg/Kg per day. The L chain is usually administered in the form of injections, oral preparations, etc. Examples of injections include those that are dissolved in distilled water for injection before use. The method of administration is intravenous or intramuscular. Examples of oral preparations include capsules, tablets, powders, 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 L chain as its main component, has extremely low toxicity and exhibits remarkable pharmacological effects, making it extremely useful as a drug for treating and preventing inflammation. Next, reference examples and formulation examples of the present invention will be explained. Reference Example 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 disuride bonds. Next, 0.75M iodoacetic acid or 0.2 iodoacetamide was added, the pH was maintained at 8.0, and the reaction was allowed to proceed for 1 hour, after which excess reagent was removed using a Sephadex G-25 column. Next, SephadexG200 in the presence of SDS
Column (4.0 x 120cm) [Solvent: 0.04MSDS-0.05M
phosphate buffer (PH8.0)], the absorbance was measured at 280 nm, and the L chain fraction was collected. SDS was removed from the L chain fraction, it was dialyzed against physiological saline, and after sterilization filtration was performed, it was made into a lyophilized product. Formulation example 1 (Oral preparation) (1) Carboxymethylated L 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) Carboxymethyl cellulose/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 gastroinsoluble film coating agent (eg, polyvinyl acetal diethylamino acetate) or an edible coloring agent, if necessary. Formulation example 2 (intravenous injection) (1) Carbamoylmethylated L chain 50mg (2) Glucose 10mg (3) Physiological saline 10mg Add (1) and (2) to (3) in the above weight ratio. Stir,
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 membrane 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. Formulation Example 3 (Capsule) (1) Carboxymethylated L chain 50g (2) Lactose 935g (3) Magnesium stearate 15g Weigh each of the above ingredients, mix 1000g uniformly, and mix the mixed powder with hard gelatin. into a capsule
Fill 200mg each.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are graphs showing the effects of the formulation of the present invention, respectively.

Claims (1)

[Claims] 1. An agent for treating and preventing inflammation containing the L chain of human IgG as an active ingredient. 2. The agent for treating and preventing inflammation according to claim 1, wherein the L chain is an alkylated L 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.