JPS63313581A - Modified superoxide dismutase - Google Patents

Abstract

PURPOSE:To obtain a modified SOD by bonding an SOD useful for the remedy of tissue disorder caused by O2<-> generated from oxygen molecule in a living body with a compound having pharmacological activity and useful as a drug. CONSTITUTION:A modified SOD (superoxide dismutase) is produced by bonding a carboxyl group (or amino group) of a compound having physiological activity to an amino group (or carboxyl group) of SOD using a carbodiimide and separating and purifying the objective substance e.g. by gel-filtration. The physiologically active compound is those having a molecular weight of 150-400, exhibiting anti-inflammatory action and having functional group such as amino group, carboxyl group, etc., e.g. indomethacin or ketoprofen. The SOD used as a starting material is human SOD produced e.g. by genetic engineering technique. The amount of the physiologically active compound to be bonded to SOD is preferably 1-10mol. per 1mol. of SOD.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to modified superoxide dismutase (hereinafter abbreviated as SOD) useful for the treatment of tissue damage caused by superoxide (0;) generated from oxygen molecules in living bodies. ).

[Description of prior art]

Superoxide dismutase (SOD) is an enzyme that has the effect of eliminating superoxide (0;) through a dismutation reaction shown in the following formula.

SOD 20; + 2 H" -m-→0□+Hz O'z
Therefore, SOD is generated from oxygen molecules in the body;
Tissue damage caused by, for example, osteoarthritis, rheumatoid arthritis, damage caused by radiation exposure, damage caused by ultraviolet rays, oxygen retinopathy of prematurity, cataracts, side effects of anticancer drugs such as adriamycin, problems associated with resumption of blood flow to ischemic areas, etc. It is attracting attention as an effective treatment for.

Although SOD is thus promising as a medicine, its pharmacological activity is often not fully demonstrated because its half-life in the bloodstream is very short (about 5 minutes).

The reason why SOD has a very short half-life in the bloodstream is that its molecular weight (32,000) is smaller than the filtration limit of the renal glomerulus (approximately 50,000 in molecular weight), so it disappears quickly from the blood. , is thought to be excreted in the urine.

Therefore, in order to fully demonstrate the pharmacological activity of SOD,
Polyethylene glycol (Pyatok.

P, S, et al, ;Re5earch C.

mmunications in Chemica
l Pathology and Pharma
cology, 29, 113 (1980)), albumin (Wong, et at; Agent
, and Actions, 10, 231 (1980
)), Ficoll (McCord, J.

M, et al; Proceedings.

f National Academic 5c
ience of American, Engineering 1, 11
59 (1980)), polyalkylene glycol (JP-A-58-32826) and inulin (JP-A-6L-24).
Attempts have been made to increase the half-life of SOD in the blood by turning SOD into a macromolecule using compounds such as 9388) and blocking its filtration in renal glomeruli.

In addition, when SOD is modified with styrene maleic acid (average molecular weight 1,600) and is injected intravenously, this modified SO
An attempt to prevent SOD from being filtered through renal glomeruli by utilizing the property that D binds non-covalently to serum proteins (e.g., albumin, globulin, etc.) and becomes a macromolecule (Masayasu Inoue et al.; No. 37) A collection of lecture abstracts from the Protein Structure Conference, page 61 (1986)) has also been published.

However, these modified SODs such as macromolecular SODs and styrene-maleic acid-bonded SODs have problems such as decreased activity of SOD due to SOD modification and non-uniformity of molecular weight due to wide molecular weight distribution of the obtained modified SODs. There is.

[Problem that the invention seeks to solve]

The purpose of the present invention is to obtain modified SOD (SOD) which is useful for the treatment of tissue damage caused by oxygen generated from oxygen molecules in the living body and a compound that has pharmacological activity useful as a medicine.
(hereinafter abbreviated as T-modified 5ODJ).

[Means for solving problems]

As a result of intensive research to solve the above-mentioned problems, the present inventors found that the modified 5OD (f-modified 5ODJ) consisting of SOD and a pharmacologically active compound of the present invention is easy to synthesize, separate and purify. There was almost no molecular weight heterogeneity due to a decrease in SOD activity due to SOD modification or a wide molecular weight distribution of the obtained r-modified 5ODJ.
.

It was discovered that the blood half-life of "D" was also significantly increased, and the present invention was completed.

That is, the present invention provides superoxide dismutase (S
Modified S obtained from OD) and a compound with pharmacological activity
The present invention relates to a modified SOD, which is characterized in that the compound has a functional group that exhibits reactivity with SOD.

The present invention will be explained in detail below.

The compound having pharmacological activity used for modifying SOD in the present invention is preferably insoluble in water or hardly soluble in water.

Such a compound is a compound that does not inhibit SOD activity and has a functional group (
For example, it may be a substance having a hydrophobic group (for example, an amino group or a carboxyl group) or a salt thereof, but it is preferable that it has a hydrophobic group even after the reaction with SOD, so that the blood concentration of serum proteins (e.g., albumin, globulin, etc.) Compounds that can non-covalently bind to the fluorescent substance (highly fluorescent matter) are preferred.

Further, it is more preferable that the compound having pharmacological activity is a low-molecular compound in order to further increase the uniformity of the molecular weight of the "r-modified SOD" and use it as a highly pure drug.

Such compounds include compounds with anti-inflammatory properties having a molecular weight in the range of 150 to 400 (e.g., indomethacin, ketoprofen, naproxen, mefenamic acid, ibuprofen, flurbiprofen, alloclofenac, tinoridine, flufenamic acid). , fenbufen, aspirin, acemethacin, tolfenamic acid, tiaprofenic acid, fenoprofen, pranoprofen, etc.) and salts of compounds (e.g., diclozenaf sodium, tolmetin sodium, etc.) are preferred. can be mentioned.

As the SOD of the present invention, those derived from animals such as cows and humans, plants such as spinach, and microorganisms such as Serratia can be used. , human SOD
It is preferable to use

As such human SOD, SOD of human red blood cells, placenta, etc. can be used, but in recent years, human SOD produced by applying genetic engineering technology (for example,
1-111690, etc.) is more preferable because a stable sample can be obtained in large quantities.

The binding ratio of SOD and a compound having pharmacological activity in the production of modified SO and DJ of the present invention is preferably 1 to 10 molecules of the compound per 1 molecule of SOD.

The bonding method is carbodiimide (for example, 1-
ethyl-3-dimethylaminopropylcarbodiimide hydrochloride, 1-benzyl-3-dimethylaminopropylcarbodiimide tosylic acid, etc.) to convert the carboxyl group (or amino group) of a pharmacologically active compound to the amino group (or amino group) of SOD. carboxyl group) [e.g., Marnet, 'D, et al.; Nature
(London), 292, 301-306).

After binding a pharmacologically active compound and SOD in this way, the r-modified 5ODJ is processed through gel filtration, ion-exchange resin-filled columns, chromatography, and high-pressure liquid chromatography, which are used as methods for the immediate separation and purification of proteins. It can be obtained by chemistries, dialysis, etc.

The purity can be examined by gel electrophoresis or absorption spectroscopy.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 A solution of ketoprofen, a low-molecular hydrophobic compound with anti-inflammatory effects (10 mg suspended in ethylene glycol with a molecular weight of 254.1 mf), 1 mffi and N-ethyl-N
'-(3-dimethylaminopropyl)carbodiimide hydrochloric acid (hereinafter abbreviated as EDC) solution (5% in 1 m2 of distilled water)
(dissolved mg) in 1 m2, adjust the pH to about 5,
The reaction was allowed to take place at room temperature for about 1 hour.

In this reaction solution, human Cu, Zn-3OD producing bacteria E and c shown in JP-A No. 61-111690 were added.

1 i A solution prepared using human Cu, Zn-3OD produced and purified using W3110 (pUBE2) (6 mg SOD, 1 ml of 150% ethylene glycol solution)
! , adjusted to pH 5-6) 1 mJl! was added and reacted overnight at room temperature.

Add 0.5M Tris-HCl buffer (pH 7,
The reaction solution was neutralized by adding several drops of 0) to stop the reaction.

This reaction stop solution was centrifuged (10,00 Orpm,
The supernatant obtained (for 5 minutes) was transferred to a C15C15x50 column packed with Sephadex G-10 (manufactured by Pharmacia) equilibrated with physiological saline for 1.5 m per purification.
1 to separate and purify the modified SOD fraction.
I got ODJ.

This r-modified 5ODJ was concentrated using an ultrafiltration membrane through which substances with a molecular weight of 10,000 or less pass.

“The average molecular weight of modified 5OD1 is approximately 34,000 (the average number of ketoprofen bonds per enzyme molecule is 2).
As calculated from the difference absorption spectrum between r-modified 5ODJ and unmodified SOD at 60 nm, it was about 8 molecules), indicating a nearly uniform molecular weight distribution.

The activity retention rate of r-modified 5ODJ was determined by a method [Oy
Anagui, Y; Analytical Bio
chemistry, 142°290-296 (19
84)), it was 90% or more.

“The half-life of modified 5ODJ in the bloodstream was measured as follows.

Wistar rat (female, weight 250±30g) 2
The rats were given 1 ml of r-modified 5OD1 solution dissolved in physiological saline.
0 and 6 mg of SOD protein were injected into the common jugular vein per animal.

2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes after injection
0, 2 ml after 50 minutes, 60 minutes, 150 minutes
Collect blood and measure the amount of SOD in the plasma using the SO of the large gutter.
D activity measurement method and SOD concentration measurement method using monoclonal antibodies against human Cu and Zn-3OD (Japanese Patent Application
-201697).

“To determine the half-life of modified 5ODJ in the bloodstream, we plotted the relative activity and relative concentration of these SODs in plasma against time, and found that in both cases of relative activity and relative concentration, the half-life is It also took about 2.5 hours.

Example 2 The method for producing r-modified 5ODJ was the same as Example 1, except that indomethacin (molecular weight: 358), a low-molecular hydrophobic compound with anti-inflammatory effect, was used instead of ketoprofen.
is the same as

The average molecular weight of r-modified 5ODJ (7) is approximately 33,000 (r-modified SOD with an average number of indomethacin bonds per enzyme molecule of 260 nm).

(calculated from the difference absorption spectrum between SOD and unmodified SOD, it was about 3 molecules), indicating a nearly uniform molecular weight distribution.

The activity retention rate of r-modified 5ODJ was 95% or more as a result of measuring the SOD specific activity before and after modification using indomethacin in the same manner as in Example 1.

The half-life of r-modified 5ODJ in the bloodstream was measured in the same manner as in Example 1 using two Wistar rats (female, weight 250±30 g), and was found to be approximately 40 minutes.

Comparative Example 1 In order to determine the half-life of unmodified SOD in the bloodstream, the relative activity of SOD in plasma was determined using Wistar rats (male, body weight 250 ± 30 g) in the same manner as in Example 1. The half-life was approximately 5 minutes in both cases of relative activity and relative concentration.

〔Effect of the invention〕

The r-modified 5ODJ of the present invention has a much improved half-life in the bloodstream than unmodified SOD, and has not only the effect of SOD as an anti-inflammatory drug, but also the pharmacology used to modify this SOD. The active compound can also be expected to have a medicinal effect.

Claims (3)

[Claims] (1) Modified S obtained from superoxide dismutase (hereinafter abbreviated as SOD) and a compound with pharmacological activity
A modified SOD, which is characterized in that the compound has a functional group that exhibits reactivity with SOD. (2) Claim 1 in which the pharmacological activity is an anti-inflammatory effect
Modified SOD as described in Section. (3) Modified S according to claim 1 or 2, wherein the functional group of the compound is an amino group or a carboxyl group.
O.D.