JPS6255079A - Modified uricase - Google Patents

Abstract

PURPOSE:To obtain a modified uricase having remarkably decreased or eliminated antigenicity while keeping the enzymatic activity, by substituting the amino acid hydrogen atom in the uricase molecule partially with a specific substituent group. CONSTITUTION:A triazine derivative having double-stranded water-soluble polymer residue is made to react with uricase to obtain a modified uricase wherein the amino acid hydrogen in the uricase molecule is substituted partially with the group of formula (R is O-methoxypolyethylene glycol residue having a molecular weight of >=2,000).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a modified uricase with reduced or eliminated antigenicity, which has improved safety as a medicine.

Uricase is an enzyme protein with a molecular weight of approximately 120,000, consisting of four subunits, and is a protein that catalyzes the reaction that decomposes uric acid into allantoin, hydrogen peroxide, and carbon dioxide gas. Uricase activity is low in humans and primates, and uric acid is thought to be the main end product of purine metabolism;
Patients with hyperuric acid plasma accumulate large amounts of uric acid in their tissues and blood, causing extreme pain.

Although treatment is currently strongly desired, no fundamental treatment has been established.

Currently, uricase has been isolated and purified from liver and yeast, and is used for clinical testing to quantify uric acid. The idea of treating gout by administering this enzyme directly into the blood and decomposing uric acid in the blood is because uricase was isolated from a non-human animal, so it is foreign to humans. Since it is a protein, antibodies are produced, and repeated administration of uricase causes anaphylactic shock, leading to death. The reason for this is that there is an antigen-determining site on the surface of the uricase molecule that humans recognize as non-self, and this site is thought to be composed of two to three amino acid residues. The reduction or disappearance of the antigenicity of uricase converts non-self to self for humans and allows readministration to gout patients (hyperuricemia).

The present inventor previously identified amino acid residues present in the uricase molecule as a group (active PE
We discovered that antigenicity could be significantly reduced or eliminated while retaining enzymatic activity by partially substituting with
189).

These previous inventions are characterized by blocking antigen-antibody reactions by covering the surface of uricase with pleated polymeric residues, and eliminating antigenicity while retaining enzymatic activity. There is. However, there was a drawback that the enzyme activity was also considerably reduced in order to eliminate antigenicity, so further improvements were needed.

As a result of research, the present inventor found that by reacting a triazine with a double-stranded water-soluble polymer residue with uricase, it was possible to react a triazine with a single-stranded water-soluble polymer residue with the same enzyme. We were able to reduce the number of modifications of amino acid residues in the same enzyme, increase the effectiveness of covering the enzyme surface with water-soluble polymers, and succeeded in suppressing the decrease in enzyme activity.

That is, the present invention provides that the amino group in the uricase molecule is a group (active PE
G2) is a modified uricase with reduced or eliminated antigenicity.

An example of the method for producing the modified uricase of the present invention is shown.

By reacting O-methoxy-polyethylene glycol (I) with a molecular weight of 2000 or more with 2.4.6-
His(0-methoxypolyethylene glycol)-6-
Chlor-8-triazine (m) is produced.

The reaction is carried out at reflux temperature in the presence of a base.

Next, 2,4-
His(0-methoxypolyethylene glycol)-6-chloro-8-)riazine (I[I) is added in an amount of 1.5 to 15 times (molar ratio) per amino group present in the uricase molecule to avoid protein denaturation. By reacting under these conditions, the modified cricase (IV) of the present invention in which the amino group in the uricase molecule is partially substituted can be obtained.

(III) (IV) When the molecular weight of the modified uricase of the present invention is measured,
This is confirmed by the fact that the molecular weight of uricase, 120,000, coincides with the sum of the molecular weights of bound compound (I[).

Regarding the modified uricase of the present invention, the compound (I[I
) The addition rate was determined by a method of measuring unreacted amino groups by m-trinitrobenzenesulfonic acid, and the number of bound amino groups in the uricase molecule was measured. Enzyme activity was determined by measuring the decrease in absorbance in 293 dishes due to uric acid oxidation. The molecular extinction coefficient of uric acid at 293 nm is 1.26X10'! J liter was 1 mol/cm. The antigenicity was measured using an antiserum obtained by immunizing a rabbit with uricase by a method of measuring the amount of precipitate produced by the antigen-antibody reaction, and the binding ability with the antibody was measured.

The enzyme activity and antibody binding ability of the modified uricase of the present invention produced by the method of the example were measured and the results are shown in the following table.

-OO100100 tt Zoo 25
70 49tt 150
63 6tt 2
From the 50 36 45 0 table, uricase in which 36 out of 98 total amino groups were modified with active PEG2 had completely lost its binding ability, but still had 45 enzyme activities remaining. On the other hand, the enzyme modified with active PEG had only 15 enzyme activities remaining.

lQQmA containing anhydrous carbonated soda of Reference Example 1011! After dissolving 20 Iit of monomethoxypolyethylene glycol having a molecular weight of 5000 in anhydrous benzene and refluxing at 80°C for 30 minutes, 2,4.6-)dichloro-S-triazine 365
Add da, 1. The reaction was carried out under reflux at 80°C day and night. The reaction residue was removed, 300 ml of petroleum ether was added to form a precipitate, the precipitate was washed several times with petroleum ether, and 2,4-
Bis(0-methoxypolyethylene glycol)-6-chloro-8-)riazine was produced.

Example 5 0.1 M borate buffer (pH 1) containing uricase 5
0), 2,4-bis(0-methoxypolyethylene glycol)-6-chloro-8-triazine prepared above was separated at a molar ratio of 10 times the amino group in the uricase molecule, and heated at 37°C for 1 hour. Made it react. Purified by conventional methods,
A white powder of modified uricase was obtained. This thing has a molecular weight of 450,000, and as a result of the analysis of amino groups, 30 were bonded, so the molecular weight of the added part is 30 x 5000 x 2 = 30
0000 and the molecular weight of uricase of 120,000 (420,000) were in y agreement.

Claims (1)

[Claims] 1. A group in which the amino group in the uricase molecule has the formula ▲ Numerical formula, chemical formula, table, etc. ▼ (Here, R represents an O-methoxypolyethylene glycol residue having a molecular weight of 2000 or more.) A modified uricase with reduced or eliminated antigenicity that is partially substituted with