JP3522798B2 - Method for producing sugar-modified protein - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an artificially synthesized sugar-modified protein, and more particularly to a sugar-modified bioactive protein.

[0002]

2. Description of the Related Art In recent years, attempts have been made to utilize a biologically active protein or glycoprotein having biological activity as a drug or a diagnostic agent. However, in order to use it effectively and specifically, it enhances the stability in vivo, enhances the signal action in metabolism, the signal action in the intracellular region, and the signal action as recognition for receptors and target cells. It has become essential to do it.
As the method, by chemically modifying the protein, the blood stability of the protein is enhanced, the signal action is enhanced, the uptake into target cells or target organs is promoted, and the physiological activity of the protein is increased. that it further has such to impart new physiological activity is expected.

The affinity of galactose with the liver has been reported in relation to its accumulation in the liver as a target organ (Kawasaki.T & Ashwell.G., J. Biol. Chem., 251,129.
6,1976 and Lee.Y C et al., J. Biol. Chem., 258, 199, 198.
3). Based on this finding, a protein having a physiological activity effective for liver diseases such as liver cancer, cirrhosis, and hepatitis is linked to a sugar having a galactose at the end, thereby efficiently increasing the uptake of the protein into the liver. , Can enhance the therapeutic effect.

For example, a bioactive protein modified with β-D-galactopyranosyl polyethylene glycol is known (JP-A-63-152393). There is also known a technique for producing a glycoprotein containing a large amount of sugar chains having a galactose-galactose bond at its end by genetic engineering (JP-A-1-102099). However, the conventional techniques require a complicated production process, reaction conditions that may denature a physiologically active protein, and require expression in a glycoprotein using a special eukaryotic cell, which is sufficiently satisfactory. Results have not been obtained, nor has it been put to practical use.

[0005]

An object of the present invention is to provide a sugar-modified protein by a simple and versatile chemical method, and in particular, by biomodifying a bioactive protein with a sugar, liver accumulation properties are improved. And to provide a sugar-modified protein having enhanced physiological activity.

[0006]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors were able to obtain a sugar-modified protein in which galactose was introduced into the protein by the following means, and The present invention has been completed by finding that it is possible to prevent a decrease in the activity of a physiologically active protein due to modification, and that the obtained glycosylated protein has an extremely high liver accumulation property.

Accordingly, the present invention provides a lactose lactone and protein in an aqueous solvent, is reacted at 0 to 45 ° C., lacto
Exists in galactose derivatives formed by ring-opening of lactones
Existing carboxyl groups and ligation present in protein molecules
Between the ε-amino groups of amino acid residues or the N-terminal amino groups of proteins
The method for producing a sugar-modified protein is characterized in that the amide bond reaction is carried out. Further, the present invention is represented by the following formula (a):
Protein represented by lactose lactone with the following formula (b) and (excluding interferon-.alpha.) by the above process that
A sugar-modified protein represented by the formula (c) obtained by amide bond reaction .

[Chemical 2] (In the formula, m is the number of primary amino groups of the protein, and n is an amino group.
The number of bonds, R, is a protein skeleton, and m ≧ n. ) The present invention will be described in detail below.

Lactose lactone is a known substance and can be produced by the following method. Lactose is oxidized with an oxidizing agent such as iodine in a lower alcohol (eg, methanol) to open the reducing terminal glucopyranose to a lactanate, and then a strong acid cation exchange resin (eg, Dowex). ) 50 (H ⁺ )) and the like under acidic conditions for dehydration ring closure to obtain lactose lactone (Polymer Journal, 17,
(4), 567-575 (1985)).

The protein to be reacted with lactose lactone is not particularly limited as long as it has at least one functional group capable of binding with lactose lactone. The structure of the protein used in the reaction may be a protein derivative having a sugar chain or other modifying group in addition to the amino acid. In the present invention, as the protein used in the reaction, a protein having physiological activity is particularly useful.

The functional group is not particularly limited as long as it reacts with the carboxyl group present in the galactose derivative formed by ring-opening of lactose lactone, but a primary amino group or the like can be generally exemplified. . By the reaction of the present invention, lactose lactone reacts with the functional group, whereby a sugar-modified protein having a galactose residue in the protein molecule and / or at the terminal of the protein molecule can be extremely efficiently produced.

The sugar modification rate of a protein, that is, the galactose residue binding rate varies depending on the reaction conditions such as the amount of lactose lactone used with respect to the protein and the reaction time, but the sugar modified protein of the present invention is at least contained in the protein molecule. It suffices that one or more sugars be modified, and various selections can be made according to the purpose of use. For example, when a sugar-modified protein is used as a physiologically active substance, it is generally desirable that 70% or less, preferably 10 to 50%, of the primary amino group in the protein molecule is modified with the sugar. .

The primary amino group is ε of a lysine residue.
An amino group or an amino group at the N-terminal of the protein. For example, the reaction formula when lactose lactone and a protein having a primary amino group produce a sugar-modified protein only by an amide bond reaction is as follows.

[0013]

[Chemical 1]

In the formula, (a) shows a structure of lactose lactone, (b) shows a general formula of a protein, and (c) shows a general formula of a produced sugar-modified protein. Further, m is the number of primary amino groups of the protein, n is the number of amide bonds, R is the protein skeleton,
Further, m ≧ n. 100n / m shows a sugar modification rate.
In the synthesis of the sugar-modified protein of the present invention, it is preferable to carry out the reaction between lactose lactone and the protein in an aqueous solvent at 0 to 45 ° C. As the reaction solvent, an aqueous solvent such as water or a buffer solution (eg, borate buffer solution, phosphate buffer solution, phosphate buffered saline) is used. The reaction temperature may be a temperature at which the protein is not denatured or inactivated, but is usually about 0 to 45.
It is preferably in the range of ° C, especially around room temperature. PH of reaction is about 3
A wide range of 10 to 10 can be used, but a neutral range is preferable.
The reaction time is about 0.5 to 100 hours, preferably 20.
It may be up to 50 hours. In the reaction, the amounts of lactose lactone and protein to be used are appropriately selected depending on the desired sugar modification rate, but when the protein is a physiologically active substance, it is determined by preliminary experiments using the physiological activity of the resulting sugar modified protein as an index. be able to. Usually, it is about 0.5 to 50 times by mole with respect to the primary amino group in the physiologically active protein.

After the above reaction, the reaction solution is dialyzed, salted out, ultrafiltered, ion-exchange chromatography, gel filtered, HPL.
The desired sugar-modified protein can be obtained by purification by a conventional protein purification method such as C or electrophoresis. In the glycosylated protein of the present invention, since a galactose residue is introduced into the protein, the properties of hepatic parenchymal cells having an affinity for galactose are utilized to selectively and efficiently provide a physiologically active protein to liver tissue. It is particularly effective in treating or preventing liver diseases such as liver cancer, cirrhosis and hepatitis. Furthermore, the sugar-modified protein of the present invention has an extended half-life in vivo and is durable.

The glycosylated protein of the present invention is used as a suitable pharmaceutical composition (eg, injection, tablet, capsule) using a carrier, a diluent and the like known per se in a parenterally or orally human form. It can be administered to mammals. For example,
To use a sugar-modified SOD using superoxide dismutase (hereinafter abbreviated as “SOD”) as a protein of the sugar-modified protein according to the present invention as an anti-inflammatory agent, for example, in the form of an injection, a tablet or the like, the amount of SOD is about 0.1-5m
It can be administered to the patient at a dose of g / kg / day.

Other proteins that can be used as the glycosylated protein of the present invention include those derived from various animals including humans (including those derived from cell culture), those derived from microorganisms, those derived from plants, genetically engineered products, and synthetic products. Any of the items may be used.
For example, cytokine {eg, interferon-β, interferon-γ, interleukin 2 etc.}, hormone {eg, growth hormone, insulin}, enzyme {eg, asparaginase, various proteases, various peptidases etc.}, immunoglobulin, protease inhibitor, Examples include various cytochromes. Particularly preferred bioactive proteins include S derived from various animals including humans, derived from microorganisms, derived from plants, or produced by gene recombination technology.
OD (human Cu, Zn type-SOD, Mn type-SOD, F
e-type-SOD etc.) and the like.

[0018]

EXAMPLES Hereinafter, specific examples of the present invention will be described.
The present invention is not limited to this. In the examples, galactose sugar chains were analyzed by the hydrazine decomposition method (Zokusei Kagaku Kenkyu Koza, Vol. 4, p. 142).
The sugar modification rate is TNBS (2,4,6-trinitrobenzenesulfonic acid) method (AFSAHABEEB., Anal.Biochem., 14,3).
28 (1966)).

Reference Example: Production of Lactose-lactone (1) Production of lactanate 26 g of lactose was dissolved in 450 ml of water, 35 ml of methanol was added, 600 ml of methanol containing 37.45 g of iodine was added at 40 ° C., and then 4% water was added. Potassium oxide solution (35.2 g / 875 ml) was added and the temperature was 40 ° C.
And reacted for 60 minutes. When the color of iodine disappears, cool with ice,
1000 ml of methanol was added, the precipitate was collected by filtration, washed with cold methanol and ether, dissolved in 150 ml of water and added with methanol, and the precipitate was collected by filtration to open the glucose of lactose, and the 1-position thereof had a carboxyl group. 18 g of a compound (lactanate) which is a potassium salt of. (2) Preparation of lactose lactone 10 g of the lactanate prepared in (1) was dissolved in 200 ml of water and passed through a Dowex 50 (H ⁺ ) column to give a free form, which was concentrated and methanol was added. Concentrated. Methanol was distilled off, ethanol was added to the formed precipitate, and the precipitate was collected by filtration to obtain lactose lactone.

Example 1: Modification of SOD with lacto-lactone (production) SOD derived from bovine erythrocytes (molecular weight 32000; 22 amino groups; 3000 units / mg protein) 5 mg (0.156 μmol;
Dissolve 3.44 μmol) as a primary amino group in 1 ml of water, and add 6 mg (16.76 μmol) and 15 mg (41.9 μmol) lactose lactone.
And 30 mg (83.8 μmol) each were added, and the mixture was reacted at room temperature for 48 hours. The reaction solution was dialyzed against water and freeze-dried.

Each lot of sugar-modified SOD produced in the order of the amount of lacto-slactone used was LL-SOD-1,
2 and 3. Yield, sugar modification rate (first modified with sugar
The number of primary amino groups is 4, 8 and 12 respectively),
Table 1 shows the elution time by high performance liquid chromatography (HPLC) (DEAE 5PW) using active and ion exchange resins.
In addition, acetyl cellulose electrophoresis {solvent: 0.1 M pyridine / formic acid (pH 3.0), migration: 30 minutes (0.5 mA / cm), Coomassie-blue staining} is shown in FIG.

[0022]

[Table 1]

(Pharmacokinetics) Lot LL-SOD-3 and SOD were treated with [2,3- ³ H] succinimidyl propionate (succinim).
idyl propionate) and 2500 kBq / mg of ³ H-LL-SOD-
3 and 2500 kBq / mg of ³ H-SOD was prepared. Male C ₃ H / HeN 6
10 μg of protein equivalent was administered from the tail vein of week-old mice, the animals were sacrificed over time after the administration, and radioactivity of blood and each organ was measured to examine the pharmacokinetics. The results are shown in Table 2.

[0024]

[Table 2]

[0025]

EFFECTS OF THE INVENTION The sugar-modified protein of the present invention has a very high accumulation property in the liver and an extended half-life in vivo as compared with the unmodified protein. Therefore, when the protein is used for treating or preventing liver diseases such as liver cancer, cirrhosis, and hepatitis,
An extremely high therapeutic effect or preventive effect can be achieved as compared with the case where an unmodified protein is used.

In the method for producing a sugar-modified protein of the present invention, since the protein is not exposed to harsh reaction conditions, it is possible to prevent a decrease in physiological activity due to chemical modification and obtain a sugar-modified protein having high physiological activity. This is an extremely excellent method. Further, in the present invention, since proteins having various functions can be sugar-modified proteins having various sugar-modification rates, various functional proteins can be produced according to the purpose.

[Brief description of drawings]

FIG. 1 shows electrophoretic diagrams of a sugar-modified SOD according to the present invention and an untreated SOD by acetylcellulose electrophoresis.

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Claims (2)

(57) [Claims] 1. Lactose lactone is allowed to react with protein in an aqueous solvent at 0 to 45 ° C. to release the lactose lactone.
Carboxy present in cyclic galactose derivatives
Group and the ε-amido of the lysine residue present in the protein molecule.
Amide bond between the amino group or the amino group at the N-terminal of the protein
A method for producing a sugar-modified protein, which is characterized in that 2. A lactose lactone represented by the following formula (a) and a protein represented by the following formula (b) (interferon-
and (except α) by the production method according to claim 1.
A sugar-modified protein represented by the formula (c) obtained by the reaction . [Chemical 1] (In the formula, m is the number of primary amino groups of the protein, and n is an amino group.
The number of bonds, R, is a protein skeleton, and m ≧ n. )