JPH1057052A - Modified superoxide dismutase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a superoxide dismutase (SOD) derivative capable of improving pharmacological effect through raising the blood half-life of SOD and low in active oxygen toxicity due to H2 O2 as a reaction product from SOD itself, and to provide a method for producing the SOD derivative. SOLUTION: This SOD derivative is a SOD modified with oligomerized keratin, esp. an oligomerized keratin incorporated with crosslink group through reaction with a lower aliphatic dicarboxylic acid (e.g. modified with succinylated keratin). This modified SOD can be obtained by reaction of a SOD with a crosslink group-incorporated keratin formed by reaction between a keratin hydrolyzate and an aliphatic lower dicarboxylic acid. This SOD derivative has excellent mitigating effect on paraquat toxicity, also exhibiting high efficacy on wound curing and further, exhibiting excellent erasing effect on active oxygen toxicity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a superoxide dismutase derivative having a long half-life in vivo and excellent drug efficacy. More specifically, the present invention relates to a superoxide dismutase useful as a therapeutic drug for anti-inflammatory agents, myocardial ischemia, rheumatism, Crohn's disease, ulcers, infectious diseases, autoimmune diseases and skin diseases, acute drug poisoning, organ transplantation, etc. Is modified with low molecular weight keratin to improve the drug efficacy and a method for producing the same.

[0002]

2. Description of the Related Art Superoxide dismutase (hereinafter referred to as S)
OD) is an enzyme that catalyzes the disproportionation of a superoxide anion radical, and has an action of protecting the human body from the toxicity of active oxygen generated by eliminating the superoxide anion radical. An enzyme that has already successfully produced human Cu, Zn-type recombinant SOD, and is expected to be used as a therapeutic agent for various conditions involving superoxide anion radicals such as inflammation, ischemic tissue lesions, shock, and infectious diseases. It is.

[Problems to be solved by the invention]

However, since the original Cu, Zn-SOD is a relatively small protein having a molecular weight of about 30,000, when it is administered to a living body, it undergoes renal glomerular filtration, so that the half-life in blood is extremely high. Short and has a low affinity for tissues,
It is known that the pharmacological effect cannot be sufficiently exerted. SOD is deactivated by H2O2 which is a reaction product of its own catalysis. At this time, a more active hydroxyl radical (.OH) may be generated. Reducing active oxygen toxins is also an important issue. To solve these problems,
Various drug carrier-SOD copolymers have been prepared.

[0004] The present inventors of the present invention have a long half-life in vivo, can exert a sufficient drug effect, have an excellent anti-inflammatory effect, superoxide radical removing effect, and have few problems concerning safety such as antigenicity. Also, SOD derivatives having functions such as high cell affinity have been studied for many years. On the other hand, the present inventors have reported that keratin hydrolyzate or a chemically modified product such as succisylation thereof (low molecular weight keratin) has blood stability, organ tropism, biodegradability and safety. It also finds that it functions as a new type of drug material that is economical.

Means for Solving the Invention

On the basis of such findings, the present inventors have attempted to improve the efficacy of SOD by chemical modification with keratin. As a result, the soluble fraction was purified with a hydrolyzate of keratin, which is a biological component, and the modified fraction was purified thereby. SOD (SOD chemically bound with low molecular weight keratin, hereinafter referred to as keratin-
SOD conjugate) was synthesized. The SOD modified with low molecular weight keratin which is a novel substance thus obtained, in particular, a keratin-SOD conjugate modified with low molecular weight keratin modified with lower aliphatic dicarboxylic acid, for example, succinylated keratin is used for the above object. And that the keratin-SOD conjugate can be produced efficiently and with high safety by reacting keratin such as succinylated keratin with a cross-linking group-introduced keratin and SOD. Was completed.

That is, the present invention relates to a superoxide dismutase modified with low molecular weight keratin. According to the present invention, the modified superoxide dismutase is obtained by reacting a keratin hydrolyzate with an aliphatic lower dicarboxylic acid. By reacting the cross-linking group-introduced keratin obtained by the above with superoxide dismutase.

BEST MODE FOR CARRYING OUT THE INVENTION

[0007] The present invention relates to S-modified with low molecular weight keratin.
OD, which can be modified using a keratin hydrolyzate as it is, but in particular, a low-molecular-weight keratin having a cross-linking group introduced by a reaction with a lower aliphatic dicarboxylic acid, for example, a succinylated low-molecular-weight keratin. SODs modified with molecular keratin are preferred because they result in higher purity products, are easier to purify, have higher safety, and have superior properties.

[0008] The SOD used as a raw material in the present invention is not particularly limited.
n SOD is preferred, and it is particularly preferred to use human Cu Zu SOD which has no antigenicity to humans produced by genetic engineering. Other SOs derived from bovine and porcine erythrocytes
D can also be used.

[0009] Low molecular weight keratin is obtained by hydrolyzing keratin and converting it into low molecular weight. It uses a hoof or horn (hoof angle) of a cow or calf as a raw material, steam-heat-treats it, and then produces a protease (for example, KAKEN). "Pronase"), for example, by treating with protease / raw material = 1/100.
Those of about 00 to 50000 are used.

The low molecular weight keratin-SOD conjugate of the present invention is usually produced as follows. (1) Introduction of a cross-linking group into low molecular weight keratin Various keratin hydrolysates (average molecular weight 5,000 to 50,000) and lower aliphatic dicarboxylic acids, especially anhydrides thereof, for example, dicarboxylic anhydrides having about 2 to 8 carbon atoms Preferably, maleic anhydride, succinic anhydride, citracoleic anhydride, aspartic acid, glutamic acid and the like are reacted with keratin molecules directly or, if necessary, in the presence of a crosslinking agent.
As a result, the carboxyl group of the dicarboxylic acid reacts with the amino group of the keratin molecule to cause cross-linking, thereby suppressing the formation of a reaction conjugate between keratins (the amino group of keratin and the carboxyl group of keratin), and Carboxyl group easily reacts with the amino group of SOD as shown in the next section. In addition, by introducing a carboxylic acid group that crosslinks the amino group of keratin using dicarboxylic acid, it does not accumulate in specific organs, increases blood concentration, increases solubility, and increases antigenic immunogenicity. Etc.
It has the effect of reducing the reactivity with the living body.

(2) Formation of a low molecular weight keratin-SOD conjugate An acid amide bond (carbamide bond) is formed by the bond between the carboxyl group on the keratin side into which the crosslinking group obtained in the above (1) is introduced and the amino group of SOD. ) Can be formed by any of the following methods commonly used in peptide chemistry.

For example, dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) as a binder
Carbodiimide, carbodiimide method using 1-cyclohexyl-3- (monofoliethyl) carbodiimide or the like or a method using diphenylphosphodiazide, diethylphosphorocyanide, N-ethoxycarbonyl-2-ethoxydihydroquinoline as a binder, or depending on the case And a method in which N-hydroxybenzotrimazole or the like is used in combination with these binders.

The proportion of the binder used is usually about 1 molar equivalent to 100 molar equivalents relative to the keratin into which the crosslinking group has been introduced. The solvent used for the condensation is not particularly limited as long as it does not affect the reaction. The reaction temperature is in the range of about 10 to 45 ° C,
The reaction pH can be in the range of about 3.0 to 8.0. The obtained low molecular weight keratin-SOD conjugate can be isolated by a gel filtration method or the like.

The ratio of low molecular weight keratin to SOD in the obtained low molecular weight keratin-SOD conjugate varies depending on reaction conditions and the like.
0, preferably about 2 to 8 low molecular weight keratin binds. Therefore, the average molecular weight of the keratin-SOD conjugate is about 40
It is about 000 to 500,000.

The obtained low-molecular-weight keratin-SOD conjugate can be used as it is, or can be formulated as a drug for anti-inflammatory and other purposes by freeze-drying or the like with a commonly used pharmaceutical carrier such as maltose. . The ratio of the low molecular weight keratin-SOD conjugate to the preparation material is not particularly limited, but the low molecular weight keratin-SOD, 1
Can be used in a ratio of 0 to 10.

[0016]

Next, the present invention will be described specifically with reference to examples and test examples. [Example 1] Synthesis of low molecular weight keratin-SOD conjugate (1) Preparation of succinylated keratin 500 mg of low molecular weight keratin (average molecular weight: 20,000) was dissolved in 50 ml of 0.5 M aqueous sodium hydrogen carbonate solution, and 2.5 g of succinic anhydride was obtained.
Were added sequentially. During the reaction, the pH was kept between 8 and 8.3 by adding sodium carbonate powder. After the addition,
Stirred at 25 ° C. for 60 minutes. After the completion of the reaction, the solution was desalted and freeze-dried to obtain 412 mg of a succinylated keratin preparation. The modification rate of the amino group of the obtained succinylated keratin was 93.4% as measured by the TNBS method.

(2) Preparation of succinylated keratin-SOD conjugate 5 mg of SOD and 150 mg of succinylated keratin were added to 5 ml of 0.1 M
Dissolve in sodium phosphate buffer (pH 6.0) and add 100 mg of 1-
Ethyl-3- [3- (dimethylamino) propyl] -carbodiimide was added. This is at 25 ° C for 1 hour and at 4 ° C
Stirred for 20 hours. After completion of the reaction, excess succinylated keratin was removed by an ultrafiltration apparatus (YM-30, molecular weight cut off: 30000), and 50 mM potassium phosphate buffer (pH 7.0) was added by Sephacryl S-200 (3.5 × 70 cm). Elution was performed using eluent. Fractions of the conjugate (282 to 498 ml) were collected, desalted with an ultrafiltration apparatus, and lyophilized to obtain 105 mg of a succinylated keratin-SOD conjugate preparation.

(3) Succinylated keratin-human CuZn-
Properties of SOD Conjugate To determine the number of succinylated keratin bound to SOD, unmodified SOD and succinylated keratin-
Amino acid analysis and atomic absorption analysis of the SOD conjugate were performed. Also, unmodified SOD and succinylated keratin-SO
The Cu and Zn contents in the D-complex were measured and the results
The OD content and the number of keratin bonds were determined. Also, SO
The amino acid group of D or succinylated keratin-SOD conjugate was quantified by the TNBS method to determine the SOD content. Table 1 shows the results.

[0019]

Table 1 Properties of succinylated keratin-human CuZn-SOD conjugate

[Test Example 1] Succinylated keratin-S
Paraquat toxicity reduction effect of OD-conjugated paraquat dissolved in physiological saline at a dose of 50 mg / 10 ml / Kg was intraperitoneally administered to male ICR mice weighing 25 g. The succinylated keratin-SOD conjugate of the invention was administered through the tail vein at a dose of 50000 units / 10 ml / Kg. For comparison, physiological saline and unmodified S were used instead of the succinylated keratin-SOD conjugate.
The same test was carried out for the same amount of OD and polystyrene maleic acid (SMA) -SOD conjugate, and the efficacy was compared. After the administration, the solid sample and water were freely taken, and the survival rate was observed up to 4 days later. As a result, as shown in FIG. 1, the physiological saline administration group and the unmodified SOD administration group all died within 4 days, whereas the succinylated keratin-
Even after 4 days, the SOD conjugate administration group (─ ● ─)
The succinylated keratin-SOD conjugate of the present invention exhibited a significantly high survival rate of 5.9%, indicating that paraquat toxicity reduction effect was very high. On the other hand, used as a control
The paraquat toxicity reducing effect of the SMA-SOD derivative was high only on the first day, and thereafter the survival rate sharply decreased.

[Test Example 2] Succinylated keratin-S
Wound healing effect of OD conjugate An 8-week-old Wistar rat was anesthetized with ether, the back was shaved with an electric razor, the back skin was pulled, and a circular (inner diameter)
A 12 mm) chisel was applied and punched out with a mallet to form a circular skin defect. Thereafter, the succinylated keratin-SOD conjugate of the present invention (approximately 100 mg / bo
dy) and the same amount of unmodified SOD was applied twice a day for 5 days for comparison. Using a caliper, the major axis and minor axis of the injured part were measured, the area was determined, and the area change according to the following equation was used as an index of the wound healing effect. Table 2 shows the results. As is clear from Table 2, a clear healing area reduction promoting effect was observed in the succinylated keratin-SOD conjugate of the present invention.

[0022]

TABLE 2 Healing effect of SOD and succinylated keratin-SOD conjugate on defective wounds

Test Example 3 Succinylated keratin-SO
Reducing effect of D-conjugate on toxicity of active oxygen: the present SOD
The anti-radical activity test of the unmodified SOD is expected to not only be inactivated by its own reaction product, H2O2, but also to generate more active hydroxyl radical (.OH). H2
Exposing the active center copper to the outside is considered to be an important process in order to express the toxicity of SOD by reaction with O2. The degree of exposure of the active center copper to the outside is determined by detecting the hydroxyl radical resulting from the reaction between H2O2 and copper (Fenton reaction), generally by converting phenylhydrazine to phenyl radical by contact with phenylhydrazine. Therefore, the effect of reducing the toxicity of active oxygen was evaluated by measuring the degree of external exposure of copper as described below.

First, the succinylated keratin-SO of the present invention is used.
D-conjugate and original, unmodified CuZn-type SOD for comparison
H2O2 was added to each of the polyethylene glycol-SOD conjugates, and the generation of the generated phenyl radical was measured using an electron spin resonance apparatus (ESR). In this system, the phenyl radical was detected by adding Nt-butyl-α-phenylnitrone (PBN) as a spin trap and trapping it with PBN. 5000
units / ml SOD derivative in 10 mM H2O2 and 25 mM phosphate buffer
(pH 7.4) at 37 ° C. After a certain period of time, catalase (250 units / ml) was added, and the reaction was stopped by shaking at 37 ° C for 30 minutes. In the presence of PBN (final concentration, 5 mM), phenylhydrazine (final concentration: 2 mM) was added and reacted at 25 ° C. for 3 minutes. 10 mM in unmodified SOD, succinylated keratin-SOD and polyethylene glycol-SOD (5000 units / ml)
FIG. 2 shows the change over time of the PBN-phenyl radical after the reaction with H2O2 in terms of relative intensity to MnO 2. As is apparent from FIG. 2, the succinylated keratin-S of the present invention is obtained.
The increase in PBN-phenyl radical of the OD conjugate (─ ● ─) was found to be much smaller than unmodified SOD and PEG-SOD.

Further, the degree of external exposure of copper to 1 mM H2O2 reaction for up to 1 hour was determined for various other derivatives and compared with the succinylated keratin-SOD conjugate. The result is shown in FIG. As can be seen from FIG. 3, the degree of exposure of the succinylated keratin-SOD conjugate of the present invention to the outside of the copper was determined based on unmodified SOD, polyvinyl alcohol-SOD, polyethylene glycol-SOD, polystyrene maleic acid-SOD, succinylated It is much smaller than the gelatin-SOD and pyran-SOD conjugates, indicating that it is the most resistant and stable derivative to H2O2 and hydroxyl radicals.

The above results indicate that succinylated keratin-S
OD suppresses the release of copper at the active center, thereby suppressing the generation of hydroxy radicals and suggesting that the toxicity of CuZn-SOD was reduced. SOD modified with molecular keratin
Is superior to unmodified SOD and other SOD conjugates.

[0027]

According to the present invention, by modifying SOD with low molecular weight keratin, the half-life in vivo is prolonged, thereby improving the drug efficacy, suppressing the release of the active center copper, and improving the SOD itself. It has the effect of suppressing the generation of hydroxyl radicals by the reaction product H2O2 and reducing the toxicity of hydroxyl radicals derived from CuZn-SOD.
Besides being used as an anti-inflammatory agent, it is useful as a therapeutic drug for myocardial ischemia, rheumatism, Crohn's disease, ulcer, infectious disease, autoimmune disease and skin disease, acute drug poisoning such as paraquat, and organ transplantation.

[Brief description of the drawings]

FIG. 1 is a graph showing the paraquat toxicity reducing effect of low molecular weight keratin-SOD according to the present invention.

FIG. 2 is a graph showing the change over time of the amount of phenyl radicals generated by the generation of hydroxyl radicals generated by copper at the active center after H2O2 treatment in SOD, low molecular weight keratin-SOD conjugate and PEG-SOD conjugate. is there.

FIG. 3 is a diagram comparing the amounts of phenyl radicals generated by the generation of hydroxyl radicals generated by copper at the active center in various SOD derivatives after H2O2 treatment.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication A61K 38/44 ACJ A61K 37/50 ACJ ACL ACL ADA ADA ADQ ADQ ADS ADS ADW ADW ADW (72) Inventor Hiroshi Maeda 3-21-19 Hodakubo, Kumamoto City, Kumamoto Prefecture (72) Inventor Takaaki Akaike 2041, Nagaminecho, Kumamoto City, Kumamoto Prefecture

Claims (3)

[Claims] 1. Superoxide dismutase modified with low molecular weight keratin. 2. The modified superoxide dismutase according to claim 1, wherein the modified superoxide dismutase is modified with low-molecular-weight keratin having a cross-linking group introduced by reaction with a lower aliphatic dicarboxylic acid. 3. The modified keratin according to claim 2, wherein the keratin hydrolyzate is reacted with a lower aliphatic dicarboxylic acid to react a low-molecular-weight keratin with a cross-linking group obtained therefrom with superoxide dismutase. A method for producing a superoxide dismuter.