JPH04262781A - Superoxide dismutase modified with gelatin - Google Patents

Abstract

PURPOSE:To provide a superoxide dismutase modified with gelatin, having long half-life in blood, exhibiting excellent antiinflammatory action and superoxide radical scavenging action, free from the problems of antigenicity, etc., and having high biocompatibility. CONSTITUTION:A superoxide dismutase modified with gelatin. The enzyme is preferably produced by usually reacting gelatin with a lower aliphatic dicarboxylic acid anhydride to introduce crosslinking carboxyl group to the amino group of gelatin and bonding the crosslinking carboxyl group to the amino group of the superoxide dismutase through an acid amide bond by a carbodiimide process using dicyclohexyl carbodiimide, etc., as a bonding agent. The lower aliphatic dicarboxylic acid anhydride is preferably maleic anhydride, succinic anhydride, etc.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is characterized by modified gelatin (
It is related to superoxide dismutase (chemically bound to gelatin), and in addition to being an anti-inflammatory agent, it is also expected to have effects such as attenuating tissue damage caused by the production of superoxide radicals that occur during ischemic reperfusion, and improving peripheral circulation. It is useful as a medicine.

0002

[Prior art] Superoxide dismutase (hereinafter S
It's called OD. ) is said to have the effect of preventing damage caused by superoxide radicals generated in vivo, and bovine SOD
Orgotein is used as an anti-inflammatory agent. Furthermore, it is attracting attention as a scavenger for superoxide radicals generated in blood vessels when blood flow is re-opened after blood flow is blocked. However, unmodified SOD has a very short half-life in the body and cannot exhibit sufficient medicinal efficacy depending on the application, so SOD modified with polyalkylene glycol (
Attempts have been made to lengthen the half-life and increase its effectiveness, such as in Japanese Patent Application Laid-Open No. 61-249388. On the other hand, the present inventors have demonstrated that gelatin and its hydrolysates or chemically modified products such as its succinylation, or hydrolysates of naturally insoluble collagen and its chemically modified products such as its succinylation, have the ability to improve microcirculation. are finding.

0003

[Problem to be solved by the invention] The present inventors have discovered that SOD
has a long half-life, can fully demonstrate its medicinal efficacy, and has anti-inflammatory and anti-inflammatory effects.
In order to find SOD derivatives that have excellent superoxide radical scavenging effects, fewer problems such as antigenicity, high biocompatibility, and the ability to improve peripheral circulation, we have developed various chemically modified SODs that are bonded with various synthetic polymers, etc. investigated.

0004

[Means for solving the problem] As a result, the present invention has been completed, and SOD modified with gelatin, which is a biological component,
The inventors have discovered that (SOD in which gelatin is chemically bonded to SOD) (hereinafter referred to as gelatin-SOD conjugate) achieves the above object, and have completed the present invention.

The gelatin-SOD conjugate of the present invention is usually produced as follows. (1) Introduction of a crosslinking group into gelatin A lower aliphatic dicarboxylic acid anhydride such as a dicarboxylic acid anhydride having about 2 to 8 carbon atoms, preferably maleic anhydride, succinic anhydride, citraconic anhydride, aspartic acid, Glutamic acid, glutaric anhydride, ascorbic acid, etc. are reacted directly or in the presence of a condensing agent if necessary, to introduce carboxyl groups that become crosslinking groups into the amino groups of gelatin. The advantage of introducing a carboxyl group into the crosslinking group is that it does not accumulate in specific organs, increases blood concentration, increases solubility, and is expected to reduce reactivity with living organisms such as antigenicity. It is.

(2) Formation of gelatin-SOD conjugate The bond between the carboxyl group on the gelatin side into which the crosslinking group obtained in (1) above is introduced and the amino group of SOD forms an acid amide bond. , any of the following methods commonly used in peptide chemistry can be used.

For example, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-cyclohexyl-3-(
Carbodiimide method using 2-morpholinoethyl) carbodiimide, diisopropylcarbodiimide, etc. or diphenylphosphorazidate, diethylphosphorocyanidate, N-ethoxycarbonyl-2-ethoxydihydroquinoline or N-ethyl-5-phenyl isoxa as a condensing agent. A method using zolium-3'-sulfonate, etc., and in some cases, a method using N-
Examples include a method in which hydroxysuccinimide, p-nitrophenol, pentachlorophenol, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboximide, etc. are used in combination.

The proportion of the condensing agent used is usually about 1 to 20 molar equivalents to the crosslinking group-introduced gelatin. The solvent used in this condensation is not particularly limited as long as it does not affect the reaction, but a polar solvent that dissolves the raw material compound is usually preferred, and water is most commonly used. The reaction temperature can usually be carried out in a range of about 0°C to 50°C. The obtained gelatin-SOD conjugate can be isolated by gel filtration or the like.

[0009] Gelatin used as a raw material in the present invention has a broad meaning, including not only gelatin generally obtained by hydrolyzing collagen, but also partially hydrolyzed products thereof.
Usually, gelatin with a molecular weight of about 2,000 to 500,000 is used, and more preferably gelatin with a molecular weight of 2,000 to 500, which has less antigenicity and is separated and purified by hydrolysis etc.
,000. Although there are no particular limitations on the SOD, human CuZnSOD, which is produced by genetic recombination and has no antigenicity to humans, is used.

[0010] The ratio of gelatin to SOD in the obtained gelatin-SOD conjugate varies depending on the reaction conditions, etc., so it cannot be stated unconditionally, but it is usually 1- to 1 molecule of SOD.
20, preferably about 2 to 10 gelatin molecules are bound. Therefore, the average molecular weight of the gelatin-SOD conjugate is approximately 40,000 to 240,000.

The obtained gelatin-SOD conjugate can be used as it is or by lyophilization or the like with a commonly used pharmaceutical carrier such as lactose, and used as a medicine for anti-inflammatory and other purposes. Gelatin-S
The ratio of the OD conjugate to the pharmaceutical carrier is not particularly limited, but it can be used in a ratio of 0 to 10 to 1 part of the gelatin-SOD conjugate.

Next, the present invention will be explained in detail with reference to Examples and Test Examples. Example 1. Synthesis of gelatin-SOD conjugate (1) Preparation of succinylated gelatin Dissolve 1 g of gelatin [Bromoids (registered trademark) W52, average molecular weight 10,000, Seiwa Kasei Co., Ltd.] in 40 ml of 0.5 molar sodium bicarbonate aqueous solution. Then, 100 mg of succinic anhydride was added five times every 10 minutes. This was left stirring at room temperature for 60 minutes. During the reaction, the pH was adjusted to 8 by adding a 0.5M aqueous sodium carbonate solution at any time.
I kept it. After the reaction is completed, ultrafiltration method [Amicon (Ami
con) (registered trademark) YW5, molecular weight cut off 5,000]
After removing excess reagents, salts, etc., the mixture was freeze-dried to obtain 850 mg of a succinylated gelatin sample. Free amino groups remaining in the obtained succinylated gelatin were removed with TNB.
When measured by the S (sodium 2,4,6-trinitrobenzene-1-sulfonate) method, the residual rate was 5% or less.

(2) Preparation of gelatin-SOD conjugate SO
50 mg of D (human recombinant CuZnSOD) and 160 mg of succinylated gelatin were dissolved in 5 ml of 0.1 mol phosphate buffer (pH 6), and water-soluble carbodiimide [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] was prepared. 100 mg was added. This was left stirring at room temperature for 1 hour and then at 4°C for 16 hours. After removing excess succinylated gelatin by ultrafiltration, it was desalted by gel filtration and freeze-dried to obtain 64.5 mg of gelatin-SOD conjugate specimen.

Properties of gelatin-SOD conjugate In order to clarify the number of gelatin bound to SOD, unmodified SO
D and the gelatin-SOD conjugate were subjected to amino acid analysis and atomic absorption spectrometry. In the amino acid analysis, the gelatin content and SOD content in the gelatin-SOD conjugate were determined using hydroxyproline (Hyp), which is a constituent amino acid of collagen, as an index. Furthermore, Cu and Z in unmodified SOD and gelatin-SOD conjugates were determined by atomic absorption spectrometry.
The n content was measured, and the SOD content and the number of gelatin bonds were determined from the results. Further, SOD residual activity was determined by the cytochrome C reduction method. The results are shown in Table 1.

Table 1. Properties of gelatin-SOD conjugate S
OD content SOD residual Gelatin
(W/W%) Activity (%) Bond number Amino acid analysis (Hyp) 32.5
100.0 6.6 Atomic absorption spectrometry (Cu) 33.3 97
．． 9 6.4 〃
(Zn) 31.7 102.
8 6.9 From the results in the table above, the average molecular weight is about 96,000 to 110,000.

Test Example 1 Half-life in blood of gelatin-SOD conjugate Unmodified SOD and gelatin-S were administered to ddy male mice (20-30 g).
The OD conjugate was administered at 15,000 units/kg via the tail vein. Blood was collected at each time, and the SOD activity in the plasma was measured by the cytochrome C reduction method. The obtained blood half-life was approximately 5 minutes for unmodified SOD and approximately 30 minutes for the gelatin-SOD conjugate. Furthermore, when looking for the improvement in in-vivo activity by looking at the area under the curve from a graph plotting changes in activity over time, it was found to be about 4 times that of unmodified SOD.

Test Example 2 Pharmacological effect of gelatin-SOD conjugate Suppression rate on the formation of ischemic paw edema in mice DDY male mice (30 to 35 g) were placed in a transparent plastic fixture, and only one paw was exposed through the incision. After wrapping an office rubber band around this leg 10 times, it was removed from the fixture and placed in a cage. After 20 minutes, the mouse was transferred to the fixture again, the rubber was cut with scissors, and the thickness of the paw was immediately measured using a Mitutoyo model N11025 micrometer. The mouse was then returned to the gauge, and the thickness of the paw was measured after a certain period of time, and the difference between the thickness and the value immediately after cutting the rubber was defined as swelling. The drug was administered intravenously 30 minutes before the start of ischemia. In the control group, 0.2 ml of physiological saline alone was administered, and unmodified SO
D and gelatin-SOD conjugate were both administered at 20,000 units/kg/0.2 ml as SOD activity. The results are shown in Table 2.

[0018]

[0019]

[0020]

Claims (1)

[Claims] [Claim 1] Superoxide dismutase modified with gelatin