JPS63253028A - Organism compatibility promoter - Google Patents

Abstract

PURPOSE:To obtain an organism compatibility promoter containing a compound obtained by treating a protein toxin such as toxin of Vibrio cholerae with an amino group chemically modified substance in a specific ratio as an active ingredient. CONSTITUTION:An organism compatibility promoter containing a compound [e.g. a compound shown by the formula (R1 is toxin of Vibrio cholerae; R2 is chemically modified group obtained by replacing hydrogen atom of primary amino group of protein R1; n is 1-10 with the proviso that n is 1-8 when R1 is viscotoxin)] obtained by treating 1mol. protein toxin selected from toxin of Vibrio cholerae, abrin (obtained from seed of Abrus precatorius L), ricin (protein belonging to albumin having ethanol poison factor and blood aggregation factor) and viscotoxin (peptide toxin contained in Viscum albam L to become a heart toxin) with 1-10mol. chemically modifying substance (e.g. polyethylene oxide) as an active ingredient.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a biocompatibility promoter.

[Conventional technology]

In diseases requiring transplantation of various organs, including kidney, heart, skin, and bone marrow, rejection reactions that occur between transplanted organs and transplanted tissues have become a major problem. Currently 7 crosses to suppress this rejection? phosphorus is used.

[Problem that the invention seeks to solve]

However, Cyclos? Lin must be taken for a long period of time from pre-surgery to post-surgery, and is it necessary to take cucross? Long-term administration of phosphorus suppresses the immune system, which protects the body from invasion by bacteria, leading to many problems such as opportunistic infections. Therefore, there has been a strong desire to develop agents that suppress biological rejection reactions or promote biocompatibility without suppressing the autoimmune system.

[Means to solve the problem]

In view of these circumstances, the present inventors conducted intensive research to obtain a substance that exerts a rejection suppressing effect or a biocompatibility promoting effect without suppressing the body's inherent immune ability, and as a result, we have recently discovered that protein The present invention was completed based on the discovery that a compound obtained by applying a chemical modifier to a toxin has an excellent effect of promoting biocompatibility, that is, an effect of suppressing rejection reactions.

Therefore, the present invention provides a biocompatible compound containing as an active ingredient a compound obtained by reacting 1 to 10 moles of a chemical modifier to 1 mole of Vibrio cholerae toxin, apurin, ricin, or viscotoxin. It provides an accelerator.

Among the toxins of the present invention, apurin is
Precatorius L
Ricin is a tannin substance that belongs to albumin and has a lethal poisonous factor and a blood aggregation factor. In addition, Viscotoxin is found in western mistletoe (Vi scum album L).
It is a peptide toxin contained in the heart that is cardiotoxic. Therefore, all of these proteins, including Vibrio cholerae toxin, are foreign proteins and poisons to humans. For this reason, when administered to humans, it stimulates the immune system to produce total antibodies, and repeated administration to patients may result in severe anaphylactic shock.Also, as it is a toxic substance, The drawback is that slight errors in dosage can lead to death of the patient.

However, the present invention is based on the knowledge that replacing the hydrogen atoms of the primary amino groups of such proteins with specific atomic groups has an antiimmune effect.

The chemical modifier may be any substance as long as it can modify the amine groups of proteins, but preferably ? Liethylene oxide, 3,5-bis(0-methoxy?liethylene glycol) triazone;
? Riasuf9ragic acid,? Realanin,? Copolymers of lyrizone or glutamic acid with cin; 3,5-bis(7'lurane)triazone, dextran; fatty acids such as zolopionic acid, caproic acid, caprylic acid, and lauric acid, and the like are used.

Modification of protein toxins with the chemical modifiers described above can be carried out by conventional methods employed for chemical modification of tannolytes. for example,? Chemical modification of the toxin using lyethylene glycol can be carried out according to the method of Inada et al., Chemistry, Vol. 40, pp. 650-655, 1985. Chemical modification of Tan and Q toxins using Bori amino acids has been described, for example, by Liu et al. [Bioc et al.
hemistry J Vol. 18, pp. 690-697, 1
It can be done according to the method of 978. Also,
Chemical modification of tannolytic toxins using pullulan is described, for example, in Uaui et al., J. Immunol J Vol. 122, No. 1.
1979, pp. 266-1272. Chemical modification of tannotoxic toxins using dextran is described, for example, by King et al.
ehem.

BiophiaJ Vol. 169, pp. 464-473, 1
Do you follow the method of 975? :, can become. Furthermore, the chemical modification of tannolytic toxins using fatty acids has been described, for example, by Coon et al.
10, pp. 183-190, 1973.

The chemically modified toxin of the present invention obtained as described above is represented by the following formula, for example.

R+ (-Rz n yu (Formula R1: Vibrio cholerae toxin, apurine, ricin, or biscotoxin residue; R1: Chemical modification group that substitutes a hydrogen atom for the primary amino group of the protein.

n: If R1 is a Vibrio cholerae toxin or a ricin residue, n=1
~10, when R1 is an apurine residue, n=1~10; when R1 is biscotoxin, n=1~8. ) Among these, preferable examples of the group R2 include the following.

(1) 3,5 bis(
0-methoxy? lyethylene glycol)-triazole group.

(2) Average molecular weight 500-5,000,000 g riasunoqraginic acid, ? Realanin,? Residue of lylizone, a copolymer of glutamic acid and cin.

(3) 3.5 bis(pullulan)-triazole group with an average molecular weight of 20,000 to 5oooooo.

(4) Dextran residue having an average molecular weight of 70,000 to 200,000.

(5) Residues of propionic acid, casillonic acid, casorylic acid, and lauric acid.

By modifying the chemically modified Tan/Q toxin of the present invention with various polymers, its original toxic effect is almost completely eliminated. For example, the toxicity of this product is LD and o value of 3t/KP or more when administered intraperitoneally to rats, and 1 when administered orally.
It is more than 0 and is highly safe.

The biocompatibility promoter of the present invention is preferably orally administered in an amount of about 300 Q as a chemically modified tanno-male toxin per adult male per day for 1 to 3 days before surgery.

[Effect]

It has not yet been elucidated by what mechanism the biocompatibility promoting effect, that is, the rejection suppressing effect, of the chemically modified protein of the present invention is expressed.

However, although the Vibrio cholerae toxin, apurin, etc. used as raw materials in the present invention are all poisonous, by replacing the hydrogen atoms of the primary amine groups of such proteins with specific atomic groups, The fact that it has an anti-immune effect is thought to be due to the poisonous effect of Tanno9 toxin suppressing the immune response.

〔Effect of the invention〕

As shown above, the chemically modified protein of the present invention exhibits an excellent anti-immune effect, exhibits an extremely excellent biocompatibility promoting effect with low toxicity and few side effects, and is useful as a biocompatibility promoter.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 (1) Vibrio cholerae toxin (molecules [84,000; Funakoshi #
Add 0.4M boric acid packarz to make the ratio 5z9/-.
Dissolve in 5- and add 100 q to this? Lieethylene glycol (average molecular weight s, ooo ~ 20.000; trade name Activated? Lieethylene glycol, manufactured by Seikagaku Corporation) was added, and after reacting for 1 hour with slow stirring under a nitrogen stream, 50mM acetate/ba'/ 77- (pH5,5) 1
00d't- was added to terminate the reaction. This solution was purified by ultrafiltration using Amicon-Diaflow (PM-30), and this product was freeze-dried. 201 g of lyethylene glycol-modified Vibrio cholerae toxin was obtained.

The average molecular weight of this product was 159,000.

(The polyethylene glycol-modified Vibrio cholerae toxin obtained in step 1 (+) was subjected to dull filtration using Sephadex G-200 (manufactured by Pharmacia). The column size was Z5 x 55 m, and the eluate was 0.05 M vinegar rRd. 77
- pH 5.5f was used.

The results are shown in FIG. ? The one modified with lyethylene glycol was eluted at A in FIG. 1, whereas the unmodified one was eluted at B in FIG. Furthermore, the results of amino acid analysis revealed that PEG was introduced into an average of 7.5 amino acid residues.

Example 2 (1) 1w1t of Vibrio cholerae toxin 20IIg at 0.01g
Dissolved in M phosphate buffer (pH 7), added 50 μj of a 25 g/-trimethylformamide solution of m-maleimidobenzoyl-N-hydroxysuccinimide ester (manufactured by Sigma), and reacted at room temperature for 30 minutes. I let it happen. After the reaction was completed, the mixture was desalted by a glue method using Sephadex G-25t and lyophilized to obtain m-maleimidobenzoyl-conjugated Vibrio cholerae toxin.

(it) glue with an average molecular weight of 110,000? Rimmer 2
0.125M Phosphate Phosphate in which 0.00g is dissolved (
pH 7,2) 1- to 1519/fId of S-acetylmercazotosuccinic anhydride (manufactured by Sigma)
20μjfic of zomethylformamide solution was added, and the mixture was allowed to react at room temperature for 30 minutes. Sephadex G-2 after completion of reaction
After desalting by Dull filtration method using 5, it is freeze-dried to obtain acetylmercazotosacnyl-amino acid? A remer was prepared.

(lit) 30m17 of the 9m-maleimidobenzoyl-conjugated V. cholerae toxin obtained in (1) was dissolved in 0.1M phosphate buffer (pH 6,0), and this was dissolved in the S
-acetylmercazotosuccinyl amino acid? vinegar? -40
11i1e dissolved 0,01 M EDTA-containing P
Added to BS solution 4- and mixed. After degassing, 70 μl of 0.5M hydroxylamine was added to perform deacetylation, followed by reaction at 25° C. for 20 minutes. Sephadex G-
Desalting and purification were performed by the glue filtration method using 100i to obtain ricin-modified Vibrio cholerae toxin 120 Nfl. The average molecular weight of this product was 684,000. Furthermore, as a result of amino acid analysis, an increase in lysine was observed, confirming that it had been chemically modified.

Example λ (1) Zorlan (manufactured by Sigma; average molecular weight 1
After dissolving 50 g of 250,000), it was cooled on ice, and dimethylformamide 1 in which 7 anuric chloride (manufactured by Sigma) was dissolved at zoq/ml was added thereto, and the mixture was reacted for 2 to 3 hours under water cooling.

During the reaction, 10% Na2CO1f was used to maintain the system pH'i7.
I kept it.

(j) After the reaction in (1) was completed, Vibrio cholerae toxin 10Q was added to the reaction solution, and the mixture was allowed to react at 37°C for 3 hours, and then left at 4°C overnight. Next, purification was performed by the gel filtration method using Sephadex G-200, which also served as desalting.
06119 was obtained. The average molecular weight of this product is 1.584
It was 000. The results of amino acid analysis revealed that an average of 6 amino acid residues were introduced into the target product. Furthermore, when sugar coloring was performed using anisaldehyde, sugar was confirmed.

Example t (+) Dextran (manufactured by Sigma; average molecular weight 120,000
) was dissolved in 25 ml, and 50 ml of sodium periodate was added to this solution.
89 was added and reacted at room temperature for 1 to 3 hours. After the reaction was completed, dialysis and desalination were performed to obtain active dextran.

(ii) 0.025M borate buffer (pH
10 Jj9 of Vibrio cholerae toxin and 50 kg of activated dextran obtained in (1) above were added to the mixture (9,05) and left for 30 minutes. Add IM NaBH4100ttl to this and
The reaction was carried out at 5°C for 30 minutes. Sephadex G-100
Desalting and purification are carried out by the gel filtration method using t-
Dextran-modified Vibrio cholerae toxin 3 was obtained. The average molecular weight of this product was 804.000, and as a result of amino acid analysis, dextran was introduced into an average of 6 amine groups.

Example 5 30q of Vibrio cholerae toxin was dissolved in 0.5M carbonate buffer (pH 9.5). Anhydrous Laughlin 11 for this! ! <
A 30% t-butyl alcohol solution (5111t'e) of molecular weight 382 (manufactured by Sigma) was added, and the mixture was reacted at 37°C for 4 hours. After the reaction was completed, the product was desalted by dialysis and then freeze-dried to obtain 22 g of lauric acid-modified Vibrio cholerae toxin. The average molecular weight of this product was 85,600, and when all free amines were quantified, it was found that 58% of the amine 7 groups had reacted, confirming that lysine was modified.

Example 6 Biocompatibility-promoting effect: The biocompatibility-promoting effect of the present interstitial compound was investigated using 4- to 8-week-old mice (!sex). In the test, mice receiving skin grafts (acceptors) were exposed to 50j19/0 the day before the surgery.
Group in which KP's modified Vibrio cholerae toxin was injected intraperitoneally, ■Cyclos? Rin (manufactured by Sandoz) for 2 weeks before and after surgery.
Groups administered orally at 0q/~/day and ■What.

The animals were divided into 3 groups, including a non-treated group and 20 animals per group, and the number of days the grafted skin survived was examined and compared. The results are shown in Table 1.

As shown in Table 1, the modified V. cholerae toxin administration group had more than 10 times the concentration of NCR toxin compared to the control group. Survival days were prolonged by more than 8 times compared to the phosphorus-administered group, confirming the biocompatibility-promoting effect of the present invention.

[Brief Description of the Drawings] Figure 1 was obtained with the present invention? FIG. 2 is a diagram comparing nine turns of dull filtration of lyethylene-modified Vibrio cholerae toxin (6) and unmodified Vibrio cholerae toxin (13). that's all

Claims (1)

[Claims] 1. 1 to 10 per mole of protein toxin selected from Vibrio cholerae toxin, apurin, ricin, or viscotoxin
A biocompatibility enhancer containing as an active ingredient a compound obtained by the action of a molar amount of an amino group chemical modifier.