KR20030026153A - Disulfide reduced galactosylated serum albumins and their radioisotope labeled compounds for hepatic function image thereof - Google Patents

Abstract

PURPOSE: A disulfide-reducing galactose-binding serum albumin for imaging the liver function and a radioactive isotope-labeling compound containing the same are provided, thereby the radioactive isotope can be easily labeled, the radioactive isotope-labeled compound is stable, and a large quantity of the radioactive-labeled compound can be accumulated in the liver, so that the imaging of the liver function can be improved. CONSTITUTION: The disulfide-reducing galactose-binding serum albumin is represented by formula(1):(Gal-L)n-A-(SH)m, wherein Gal is galactosyl; L is a linker or direct linkage; A is disulfide-reducing serum albumin; SH is thiol; n is an integer of 1 to 42; and m is an integer of 2 to 34. The method for preparing the disulfide-reducing galactose-binding serum albumin comprises the steps of: binding galactose to serum albumin directly or using the linker to prepare galactose-binding serum albumin; and reducing the disulfide bond of the galactose-binding serum albumin using a reducing agent containing a thiol group, wherein the reducing agent containing the thiol group is 2-mercaptoethanol, 1,4-dithiothreitol, 2-aminoethanethiol, thioglycolate, cysteine and glutathione. The radioactive isotope-labeling compound containing the disulfide-reducing galactose-binding serum albumin is provided, wherein the radioactive isotope is selected from 99mTc, 188Re, 186Re, 67Cu, 64Cu, 212Pb, 212Bi and 109Pd.

Description

Disulfide reduced galactosylated serum albumins and their radioisotope labeled compounds for hepatic function image

The present invention relates to a disulfide reduced galactose-binding serum albumin and a radioisotope labeling compound comprising the same, and more particularly to a disulfide reduced galactose-binding serum albumin and a method for preparing the same, It relates to a radioisotope labeling compound comprising the same and a kit for radiolabeling.

(Gal-L) _n -A- (SH) _m

In Chemical Formula 1,

Gal is a galactosyl group,

L is a linker or a direct bond,

A is disulfide reduced serum albumin,

SH is a thiol group,

n is an integer of 1 to 42,

m is an integer of 2-34.

Liver disease is a particularly common disease among Asians, including Koreans, and thus diagnosis of liver function is a very important field. Liver function imaging using radioisotopes in liver function diagnosis can identify the size, shape and overall liver function of the liver as well as local lesions.

Specifically, various diffuse or local diseases occur in the liver. The diffuse diseases include acute infection, chronic infection, cirrhosis of the liver, and damage caused by drugs such as alcohol (alcohol), and connective tissue damage.In particular, acute infection, chronic infection, and cirrhosis of the liver occur in Korea. Due to the deterioration of liver function, liver function imaging is useful. In addition, the local disease is liver cancer, and when the liver function imaging, as well as hepatic function as well as the location and size of the liver cancer masses can be known.

Liver cancer may be caused by metastasis of various cancers as well as primary hepatoma, and the therapeutic success of the cancer depends on the control of metastasis. Metastasis of the cancer is manifested as a local defect of liver function imaging, but similar defects are also manifested by various benign lesions, normal liver structures or artifacts. Therefore, understanding these factors is a prerequisite for reading the functional image.

Liver function imaging using conventionally used radioisotope labeling compounds results in cold lesions rather than surrounding normal tissues due to reduced concentrations in the lesions. However, the discovery of cynics is more difficult than the discovery of hot lesions in pale radioactivity, especially when finding cynics in large structures such as the liver.

Currently, liver function imaging has been enhanced by the development of radioisotope labeling compounds and single photon emission computed tomography, thereby increasing the image quality and increasing the sensitivity and specificity of the test.

On the other hand, there is a hepatic binding protein (HBP) in mammalian liver that binds to and removes glycoproteins containing sugars having galactose at their ends. Thus, in order to image the liver function, it is administered to the body by labeling the technetium to the serum albumin combined with galactose, and then it is possible to image by binding to the galactose receptor of the liver.

In the method of labeling galactose-binding serum albumin for liver function imaging with technics, an electrolysis method was initially used, and a special apparatus for performing the method was required, and the labeling operation using the apparatus was complicated. It was difficult to use widely.

Recently, a method using chelates having two functional groups for linking proteins to label the technetium has been proposed. The labeling method binds chelates to large molecules such as antibodies, and chelates preferably use various forms of diethylenetriamine pentaacetic acid (hereinafter referred to as "DTPA"). As an example, a labeling method using a chelate is shown in Scheme 1 below.

(Gal) _n -protein- (NH ₂ ) _m + anhydrous cyclic DTPA → (Gal) _n -protein- (DTPA) _m

(Wherein

Gal is a galactosyl group,

NH ₂ is an amino group,

n and m are each an integer of 1 to 40.)

According to the above scheme, the o-mucoid, a glycoprotein, is treated with neuraminidase to remove sialic acid, thereby allowing galactose to be exposed at the end of the glycoprotein. By combining DTPA with glycoproteins, the radiolabeled technetium or rhenium label can be easily achieved. However, since this method does not form a strong bond with the radioisotope technetium or rhenium, it is necessary to prepare DTPA by heating it at a relatively high temperature for a long time to label the radioisotope. There are problems that take place and take a long time. In addition, by combining a large amount of acidic DTPA, there is a problem in that the properties of the serum albumin to which galactose is bound are changed a lot, and both galactose and DTPA bind to the lysine residue or amino terminus of albumin. There is a disadvantage in that excessive binding does not allow other materials to bind sufficiently [G. Ashwell, CJ Steer., J. Am. Med. Assoc . 246, 2358-2364, 1981; RJ Stockert, AG Morell., Hepatology , 3, 750-757, 1983; DR Vera, RCStadalnik, KA Krohn., J. Nucl. Med. , 26, 1157-1167, 1985; G. Galli, CL Maini, P. Orlando, G. Deleide, G. Valle., J. Nucl. Med. Allied Sci ., 32, 110-116, 1988].

Recently, Japanese Patent Nos. 05087190 and 61312434 have suggested a substance combining DTPA with galactose-binding serum albumin in order to facilitate the labeling of technetium. In this patent, in order to bind an appropriate amount of DTPA to serum albumin, heating was performed at 50 ° C. for 30 minutes to produce DTPA-bound galactose-binding serum albumin. However, since the prepared DTPA-bound galactose-binding serum albumin is not strong in binding with technetium or rhenium, it was prepared by heating at a relatively high temperature for a long time, and also by binding a large amount of acidic DTPA. There is a problem that the properties of the serum albumin combined with galactose is changed a lot, and since the galactose and DTPA competitively bind to serum albumin, the binding of DTPA and galactose is limited.

On the other hand, the development of monoclonal antibodies (monoclonal antibody), accelerated the research on the technique of labeling. As a result, it was found that the monoclonal antibody was selectively and stably labeled at the site where the thiol group and the technetium were bonded. As a method using such a monoclonal antibody, Korean Patent Registration No. 0178961 proposes a method and kit for labeling technetium and rhenium using a plurality of spatially adjacent free thiol groups in an antibody and / or antibody fragment. However, specific materials for liver function imaging of the present invention and experimental results thereof have not been reported yet.

Accordingly, the present inventors prepared disulfide reduced galactose-binding serum albumin to effectively perform liver function imaging to solve the above problems, and as a result of producing a radioisotope labeling compound and a kit for radiolabeling, It was found that radiolabeling is easier than conventional radioisotope labeling compounds for liver function imaging, and that the radiolabeling compound has excellent stability and high liver accumulation rate. The present invention has been completed.

Disclosure of Invention It is an object of the present invention to provide a radioactive isotope and easy-disulfide reduced galactose-binding serum albumin and its preparation for imaging liver function.

Specifically, it is an object of the present invention to provide a disulfide reduced galactose-binding serum albumin including a galactosyl group and a thiol group at the serum albumin terminal and a method for producing the same.

It is another object of the present invention to provide a radioisotope labeling compound having excellent stability after radiolabeling and a rate of liver accumulation.

Specifically, it is an object of the present invention to provide a radioisotope labeling compound comprising the disulfide reduced galactose binding serum albumin.

In addition, another object of the present invention is to provide a kit for radioactive isotope and easy label liver imaging.

Specifically, it is an object of the present invention to provide a kit for use in liver function imaging comprising disulfide reduced galactose binding serum albumin, weak chelating agent and stannous chloride, further antioxidant.

1 is a schematic diagram of the disulfide reduced galactose-binding serum albumin of the present invention,

(a) is serum albumin directly bound to galactosyl groups,

(b) is blood serum albumin in which galactosyl groups are bound via a linker.

In this case, Gal represents a galactosyl group, L represents a linker, and SH represents a thiol group.

2 is a graph showing the stability test of ^99m Tc-disulfide reduced lactosyl serum albumin of the present invention,

Figure 3 is a graph showing the distribution results of the mouse body of ^99m Tc-disulfide reduced lactosyl serum albumin of the present invention.

In order to achieve the above object,

The present invention provides disulfide reduced galactose-binding serum albumin represented by the following formula (1) and a method for preparing the same for liver function imaging.

The present invention provides a radioisotope labeling compound comprising the disulfide reduced galactose binding serum albumin.

The present invention also provides a kit for use in liver function imaging comprising the disulfide reduced galactose-binding serum albumin, weak chelating agent and stannous chloride, and additionally an antioxidant.

Hereinafter, the present invention will be described in more detail.

The present invention includes disulfide reduced galactose binding serum albumin represented by the following formula (1) for liver function imaging.

Formula 1

(Gal-L) _n -A- (SH) _m

(Wherein

Gal is a galactosyl group,

L is a linker or a direct bond,

A is disulfide reduced serum albumin,

SH is a thiol group,

n is an integer of 1 to 42,

m is an integer of 2 to 34.)

1 is a diagram illustrating an example of the disulfide reduced galactose-binding serum albumin represented by Chemical Formula 1. FIG.

As shown in Formula 1 and Figure 1 , disulfide reduced galactose-binding serum albumin is a galactosyl group directly bonded to the terminal of the serum albumin ( Figure 1 (a)) or using a linker ( Figure 1 (b ), And contains a thiol group by reducing disulfide in serum albumin.

Human serum albumin is a protein with a molecular weight of 66,462, a major axis of 8 nm, a short axis of 6 nm, an isoelectric point (IEP) of 4.8, and constitute 50% (4 g / dl) of plasma protein. It consists of a single polypeptide chain.

The serum albumin has 17 disulfide bonds so that 2 to 34 thiol groups bind to serum albumin upon reduction using a reducing agent containing a specific thiol group. The thiol group is a moiety that binds to the radioisotope and has two or more adjacent thiol groups in order to act as a chelate for the radioisotope.

Thiol groups bound to the serum albumin are stable under oxygen-free conditions, and stability increases at low pH below pH 6. In addition, rapid cooling to liquid nitrogen temperature allows for long-term storage without contamination or loss of thiol groups.

The galactose binding serum albumin of the present invention is bound either directly or by using a linker when galactose and serum albumin (HSA) are bound. The linker may be a C ₁ to C ₁₀ alkyl group, C _{4 to} C ₁₀ aryl group, monopeptide, dipeptide, oligopeptide, C _{4 to} C ₁₀ cycloalkyl group, benzyl group, thioether, ether, amine, hydra And one or more complexes of the group consisting of jides, pentose sugars, hexose sugars and alcohols.

The galactosyl group including the galactosyl and the linker is combined with the amino group (-NH ₂ ) of human serum albumin (HSA). For example, serum albumin to which galactose is bound directly or by using a linker is represented by the following Chemical Formulas 2 to 4. Formula 2 below shows galactosyl serum albumin in which galactose and serum albumin are directly bonded. Formula 3 shows lactosyl serum albumin in which galactose is bound using glucose as a linker, and formula 4 below shows a phenyl group as a linker. Phenylgalactosyl serum albumin used. If necessary, the galactose-binding serum albumin may include a galactosyl group including 1 to 42 galactosyls or linkers.

Galactose-binding serum albumin represented by the above formula forms a disulfide-reduced galactose-binding serum albumin in which disulfide bonds in serum albumin are reduced by thiol groups using a reducing agent containing a thiol group.

The present invention includes a method for preparing a disulfide reduced galactose-binding serum albumin represented by Formula 1 used for liver function imaging.

Specifically, galactose is combined with serum albumin directly or using a linker to produce galactose-binding serum albumin (step 1); And,

Reducing the disulfide bond of the galactose binding serum albumin using a reducing agent containing a thiol group (step 2).

Step 1 is modified to have a bondable functional group such as thiocyanate, ester, aldehyde, iminomethoxyalkyl, or the like by using a conventional chemical reaction to bind the terminal portion of the galactose or linker with serum albumin. To prepare galactose binding serum albumin through.

Specifically, Scheme 2 below is 2-imino-2-methoxyethyl-1-thio-β-D-galactose represented by Chemical Formula 2 (2-imino-2-methoxyethyl-1-thio-β-D -galactose (hereinafter referred to as "IME-thiogalactose") to show the preparation of galactosyl serum albumin.

As shown in Scheme 2, D-galactose is acetylated and brominated, thiourea is bound, and chloroacetonitrile is reacted to react cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-. β-D-galactopyranoside is synthesized. This was reacted with sodium methoxide in methanol solution to synthesize IME-thiogalactose, and the amino group (NH _2- ) and thiocarbamyl of human serum albumin (HSA) were synthesized under the conditions of pH 8-9. It combines in the form to prepare galactosyl serum albumin represented by the formula (2).

Scheme 3 below shows a process of preparing lactosyl serum albumin represented by Formula 3 by combining serum albumin and lactose directly.

Since lactose is a disaccharide obtained by combining galactose with glucose, as shown in Scheme 3, the aldehyde of the glucose moiety and the amino acid of albumin are combined to form a Schiff base (Schiff'sbase) and then reduced to NaCNBH ₃ , NaBH _4, and the like. To form a stable bond to prepare lactosyl serum albumin represented by the formula (3). In the case of lactosyl serum albumin, by linkage of galactose including a linker, the linker used glucose to connect the serum protein and galactose.

In addition, the following Scheme 4 shows a process for preparing phenylgalactosyl serum albumin represented by Formula 4 by directly combining phenylgalactose with serum albumin.

As shown in Scheme 4, phenylgalactose produces stable phenylgalactosyl serum albumin represented by Chemical Formula 4 by combining the SCN portion of the phenyl group and the amino group of serum albumin.

Step 2 is a thiol group is bonded to the disulfide bond in the galactose binding serum albumin using a reducing agent to prepare a disulfide reduced galactose binding serum albumin of the formula (1). The reducing agent is a reducing agent containing a thiol group in the structural formula, for example, 2-mercaptoethanol, dithiothreitol, thioglycolate, cysteine and glutathione are used, in addition to containing a thiol group used in this field Reducing agents may be used.

In the reduction reaction of step 2, an additional chelating agent may be added to remove the remaining metal ions, thereby increasing the stability of the resulting compound and increasing the labeling efficiency of subsequent radioisotopes. The chelating agent may be used conventional chelate, preferably using EDTA.

The present invention includes a radioisotope labeling compound containing a disulfide reduced galactose binding serum albumin of formula (1).

The radioisotope is preferably ^99m Tc, ¹⁸⁸ Re, ¹⁸⁶ Re, ⁶⁷ Cu, ⁶⁴ Cu, ²¹² Pb, ²¹² Bi or ¹⁰⁹ Pd, more preferably ^99m Tc or ¹⁸⁸ Re.

The radioisotope can be stably labeled by binding to the thiol group of the disulfide reduced galactose binding serum albumin.

The radioisotope labeling compound is prepared by adding a disulfide reduced galactose-binding serum albumin of Formula 1 and a radioisotope. These labeling reactions can be injected into the human body immediately after labeling in an sterile, pyrogen-free aqueous solution for administration to the human body.

The labeled radioisotope labeling compound can be parenterally injected into the body, accumulated in the liver, and used for imaging liver function through conventional nuclear medicine equipment.

In addition, the present invention is 0.1 to 100 mg / unit dose of disulfide reduced galactose-binding serum albumin represented by the following formula (1), 0.1 to 500 mg / unit dose of the weak chelating agent and 0.01 μg / unit dose of stannous chloride Kits for liver function imaging in a pharmaceutically acceptable nonpyrogenic sterile form comprising a dose of 100 mg / unit, and additionally an antioxidant of 0-500 mg / unit.

The weak chelating agent plays a role in preventing the phenomenon of labeling at the weak binding site of galactose binding albumin and colloid formation in the preparation of the radioisotope labeling compound using the liver function imaging kit. , Phosphonate, glucoheptonate, gluconate, glucarate, tartarate, succinate and citric acid, consisting of one or two or more, and the kit of the present invention is preferably 0.1 to 500 mg per unit dose. .

As for stannous chloride, 0.01 microgram-100 mg are preferable.

In addition, the kit for imaging liver function of the present invention further comprises an antioxidant. The antioxidant is to prevent the formation of a polymer that can be produced by the oxidation of the thiol group of the disulfide reduced galactose-binding serum albumin is bonded to each other, preferably vitamin C or gentic acid, etc., in the kit of the present invention 0 500 mg is preferable.

The liver function imaging kit is frozen or lyophilized in a sterile container and in an inert gas atmosphere, preferably cooled in liquid nitrogen and slowly dissolved immediately before use. The kit may supplement buffer sterile vials, saline, syringes, filters, columns, and other auxiliary devices to prepare injection preparations for use by clinical pathologists or technicians. It is well known to those of ordinary skill in the art that the kits can be changed and modified in accordance with the patient's individual needs or diet, as well as in the form in which radioisotopes can be provided or obtained. .

The kit for liver function imaging is prepared by radioactive isotope labeling compound by adding 0.1-500 mCi of radioisotope per mg of disulfide reduced galactose-binding serum albumin at room temperature immediately before use. .

In one embodiment, the acid salt and tekeune syum made of a generator ^(99m TcO ₄ ^-) was added to the liver image Purification Kit was reacted for 1~30 minutes at room temperature to prepare a tekeune syum labeled compound in 98-100% efficiency.

According to an embodiment of the present invention, when the labeling efficiency of the radioisotope labeling compound was measured, ^99m Tc-lactosyl serum albumin was able to obtain a labeling efficiency of 98% or more, and the stability of the radioisotope labeling compound was measured. As shown in FIG. 2 , the radiochemical purity was stable for 24 hours. Also, as a result of measuring the accumulation rate of the radioisotope labeled compound in the liver, as shown in FIG. It was accumulated, and the accumulation rate of 77.4% ID / g was observed in the liver, indicating that the labeling efficiency, post-labeling stability, and liver accumulation rate were excellent.

An embodiment of the present invention will be described below.

However, the present invention is not limited by the examples.

Example 1 Preparation of Disulfide Reduced Galactosyl Serum Albumin

Step 1: Preparation of Galactosyl Serum Albumin

As in Scheme 1, 100 g of D-galactose was added to 400 ml of acetic anhydride and 3 ml of perchloric acid while stirring slowly over 30 minutes while maintaining the temperature at 30 to 40 ° C. After 30 g of amorphous phosphorus was added to the mixed solution containing D-galactose, the reaction vessel was cooled with ice. 180 g of bromine was slowly added while maintaining the temperature of the liquid mixture at 20 ° C. or lower, and 36 ml (0.9 equivalent) of distilled water was slowly added over 30 minutes while preventing the temperature from rising. After the reaction vessel was blocked and reacted for another 2 hours at room temperature, 300 ml of chloroform was added, and the mixture was poured into a separatory funnel containing 800 ml of ice water. The chloroform layer was collected, filtered to remove phosphorus, and then washed twice with the same amount of ice water. The solution was washed once more with an aqueous sodium bicarbonate solution to remove residual acid, dehydrated with calcium chloride, dried under reduced pressure, dissolved in diethyl ether and recrystallized to obtain a crystal having a melting point of 87 ° C. 9.74 g (20 mmol) of 2-S- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl) -2-thioshudourea hydrobromide was added to 40 ml of water and acetone 1 Dissolve in the mixed solution and add 5 ml (79 mmol) of chloroacetonitrile to dissolve completely. Add 3.2 g (23.2 mmol) of potassium carbonate and 4.0 g (40.4 mmol) of sodium bisulfite, stir well at room temperature for 30 minutes, then add the reaction mixture to 160 ml of ice water and stir for another 2 hours. The resulting precipitate is collected by filtration and washed with cold water. The precipitate, dried in air, is dissolved in boiling methanol, filtered to remove impurities and stored in a refrigerator to obtain crystals: 72% recovery, melting point 95-97 ° C. 40 mg of the recovered crystals are dissolved in 1.5 ml of anhydrous methanol. At this time, the mixture is heated to about 40 ° C. to be dissolved well. Here, 0.8 mg of sodium methoxide is added thereto, mixed well, and reacted at room temperature for 48 hours to synthesize IME-thiogalactose: 22 mg, yield 55%. Next, 100 mg of serum albumin was dissolved in 1 ml of 0.2 M boric acid buffer solution (pH = 8.5), followed by vigorous mixing with 22 mg IME-thiogalactose. The reaction was carried out at 37 ° C. for 1.5 hours and frozen at −70 ° C.

Step 2: Preparation of Disulfide Reduced Galactosyl Serum Albumin

40 μl of 0.3 M EDTA (pH 8.0), 40 μl of 1 M sodium bicarbonate, and 50 μl of 1.5 M β-mercaptoethanol were prepared in 1 ml galactosyl serum albumin prepared in step 1 above (37). The reaction was carried out at 1 ° C. for 1 hour. After the reaction, disulfide reduced galactosyl serum albumin was prepared using a Sephadex G-25 column.

<Example 2> Preparation of disulfide reduced lactosyl serum albumin.

Step 1: preparing lactosyl serum albumin

As in Scheme 2, 680 mg of serum albumin was completely dissolved in 50 ml of 0.2 M potassium phosphate buffer (pH = 8.0), followed by further dissolving 1 g α-lactose. After dissolving, 1 g sodium cyanoborohydride (NaCNBH ₃ ) was added to dissolve and filtered using a 0.22 μm filter. After filtration, the mixture was reacted for 11 days while stirring slowly at 37 ° C. The reaction solution was centrifuged at 3,000 rpm for 5 minutes to remove precipitates, and the supernatant was taken and stored frozen at -70 ° C.

Step 2: preparation of disulfide reduced lactosyl serum albumin

To 40 ml of 0.3 M EDTA (pH 8.0), 40 µl of 1 M sodium bicarbonate and 50 µl of 1.5 M β-mercaptoethanol were added to 1 ml lactosyl serum albumin (13.6 mg / ml) prepared in Step 1, at 37 ° C. The reaction was carried out for 1 hour. Disulfide reduced lactosyl serum albumin was isolated using a Sephadex G-25 column, followed by addition of 0.3 ml of glucarate solution (2 mg / ml, pH 7.0, including 6 μg SnCl ₂ · H ₂ O), and protein. Aliquots were dispensed into vials at an amount corresponding to 3 mg and then stored frozen or lyophilized at -70 ° C.

Example 3 Disulfide Reduced Phenylgalactosyl Serum Albumin

Step 1: Preparation of Phenylgalactosyl Serum Albumin

As in Scheme 3, 268 mg serum albumin was dissolved in 25 ml of 0.1 M carbonic acid buffer (pH 9.5), followed by addition of 25 mg α-L-galactopyranosylphenylisothiocyanate. The reaction was stirred for 20 hours while stirring. The reaction solution was stored frozen at -70 ° C.

Step 2: Preparation of Disulfide Reduced Phenylgalactosyl Serum Albumin

To 40 ml of 0.3 M EDTA (pH 8.0), 40 µl of 1 M sodium bicarbonate and 50 µl of 1.5 M β-mercaptoethanol were added to 1 ml phenylgalactosyl serum albumin (13.6 mg / ml) prepared in Step 1 above. It was made to react at 1 degreeC. After the reaction, disulfide-reduced phenylgalactosyl serum albumin was isolated using a Sephadex G-25 column, and then 0.3 ml of glucrate solution (2 mg / ml, pH 7.0, containing 6 μg SnCl ₂ · H ₂ O) was added. The solution was added to the vial in an amount corresponding to 3 mg of protein, and then stored at -70 ° C by freezing or freeze-drying.

Example 4 Preparation of Liver Function Imaging Kit

1 ml disulfide reduced lactosyl serum albumin (13.6 mg / ml), 0.3 ml glucarate solution (2 mg / ml, pH 7.0, 6 μg SnCl ₂ · H ₂ O) were added, followed by 3 mg of protein The aliquots were dispensed into vials and then frozen or lyophilized at -70 ° C and stored.

Example 5 Preparation of Technicium Labeling Compound Using Liver Function Imaging Kit

To the kit of Example 4, 2 ml or 5 ml of a physiological saline solution ( ^99m TcO ₄ ⁻ ) of pertechnic acid salt ( ^99m TcO ₄ ⁻ ) prepared with ⁹⁹ Mo / ^99m Tc-generator (Du Pont) was added for 1 to 30 minutes at room temperature. Reaction.

<Comparative Example 1> Galactose-binding serum albumin (including DTPA) ^99m Tc-DTPA-GSA-Gal)

Of 3 mg of DTPA is bonded galactosyl federated serum albumin (Galactosylated serum albumin, GSA, Nihon Medi-Physics, Japan) were dissolved in physiological saline for 1.2 ㎖, ^99m TcO ₄ ^- was mixed for 30 minutes at 50 ℃ culture ^Was labeled ( ^99m Tc-DTPA-GSA-Gal).

Experimental Example 1 Measurement of Radioisotope Labeling Efficiency

The radioisotope labeling efficiency of Technicium bound to lactosyl serum albumin ( ^99m Tc-DTPA-GSA-Gal) and lactosyl serum albumin was developed by physiological saline after taking a small amount of reactant in instant thin layer chromatography (ITLC). -Was determined by measuring the radioactivity distribution with a scanner. Technicium bound to lactosyl serum albumin remained at the origin, all other technics moved along the solvent shear. The results are shown in Table 1 below.

Labeling Efficiency of Lactosyl Serum Albumin Reaction time (minutes) Technetium amount (ml) 2 5 One 98.45 98.86 3 98.16 99.04 7 98.87 100 15 98.88 99.26 30 100 100

As shown in Table 1 , as a result of measuring the labeling efficiency according to the reaction time and the added volume of technetium, it was confirmed that the labeling was performed at an efficiency of 98% or more in 1 minute at room temperature, and Comparative Example 1 ( ^99m Tc- The radioisotope labeling efficiency of serum albumin of DTPA-GSA-Gal) is 95% or more, and the radioisotope labeling efficiency of serum albumin of the present invention is increased by 3% or more.

Experimental Example 2 Stability Experiment of Radioisotope Labeling Compound

Stability was measured through radiochemical purity when the labeled technic labeled substance in the above example was kept at room temperature and incubated with phosphorous serum at 37 ° C. The results are shown in Table 2 below.

Radiochemical Purity over Time (%) time 0.125 0.25 0.5 One 2 3 5 20 24 25 ℃ (room temperature) 100 100 99.5 99.5 99.1 98.3 98.0 98.1 99.0 37 degrees Celsius (serum) 100 99.9 99.5 97.6 99.1 97.0 88.1 92.3 88.5

As shown in Table 2 and Figure 2 , when left at room temperature showed the purity of 100% for 24 hours the same as the initial prepared purity, when incubated with serum at 37 ℃ slightly decreased after 4 hours , It was confirmed that it is stable by maintaining a purity of 88% or more for 24 hours.

Experimental Example 3 Confirmation of Liver Intake by In Vivo Distribution Experiment

Mice (weight average 20 g, male, ICR) were used to determine liver accumulation rates by in vivo distribution experiments.

Into the tail vein of the mouse was administered 10 μCi each of the technetium labeling material prepared in Example 4 and Comparative Example 1. The mice were sacrificed and dissected, each organ was removed, and the radioactivity distributed in each tissue was measured with a gamma counter (CobraIII, Packard, USA), weighed using a scale, and a percentage of the injection per unit g was obtained. In vivo distribution was measured by standard methods. The results are shown in Tables 3 and 4 below.

Percent Radioactivity According to Biological Tissues Following Administration of Example 4 (% ID / g) blood muscle Fat Heart lungs liver spleen Camouflage Intestine kidney bone 10 minutes 0.99 0.27 0.15 0.62 0.81 77.42 7.69 0.60 0.20 2.66 2.88 60 mins 0.68 0.10 0.29 0.38 0.68 60.97 7.10 5.72 12.04 4.49 2.88

Table 3 and Figure 3 is a graph showing the percentage of radioactivity distributed in each tissue after 10 minutes and 60 minutes, the radioactivity percentage after 10 minutes was 77.4% ID / g, the amount of other tissues You can see that this is a very small amount. Through this, it can be seen that the technetium-labeled compound including the disulfide reduced galactose-binding serum albumin of the present invention can be sufficiently used for liver function imaging.

Comparative Example 1 (% ID / g, ± is the standard deviation) according to the biological tissue after administration of ^99m Tc-DTPA-GSA-Gal time 10 minutes 30 minutes 2 hours 6 hours blood 0.47 ± 0.10 0.35 ± 0.60 0.25 ± 0.05 0.18 ± 0.20 liver 73.7 ± 1.31 54.7 ± 2.82 38.8 ± 5.21 24.7 ± 1.85 Intestine 0.40 ± 0.10 4.6 ± 1.50 9.2 ± 2.2 17.4 ± 5.01

As shown in Table 4 , the radioactivity percentage for Comparative Example 1 ( ^99m Tc-DTPA-GSA-Gal) was about 74% after 10 minutes, similar to Example 4 of the present invention, but after about 30 minutes It can be seen that it is reduced to 55% and released in the liver faster than the radioisotope labeling compound of the present invention.

As described above, the disulfide reduced galactose-binding serum albumin of the present invention and the radioisotope labeling compound comprising the same and a kit for radiolabeling reduce the disulfide in serum albumin than the radioisotope labeling compound for conventional liver function imaging. It is easy to label radioisotopes using the generated thiol group, and has excellent characteristics for imaging liver function due to high stability and high liver accumulation rate.

Claims (14)

Disulfide reduced galactose-binding serum albumin represented by the following formula (1). Formula 1 (Gal-L) _n -A- (SH) _m (Wherein Gal is a galactosyl group, L is a linker or a direct bond, A is disulfide reduced serum albumin, SH is a thiol group, n is an integer of 1 to 42, m is an integer of 2 to 34.) The method according to claim 1, wherein the linker (L) is C _{1 ~} C ₁₀ alkyl group, C _{4 ~} C ₁₀ aryl group, monopeptide, dipeptide, oligopeptide, C ₄ ~ C ₁₀ cycloalkyl group, benzyl group, Disulfide reduced galactose-binding serum albumin, characterized in that it is a complex of one or more of the group consisting of thioether, ether, amine, hydrazide, pentose, hexose and alcohol. The disulfide reduced galactose binding serum albumin according to claim 1, wherein the Gal-L is a galactosyl group, a lactosyl group, or a phenylgalactosyl group. Binding galactose to serum albumin directly or using a linker to produce galactose binding serum albumin (step 1); And, A method for producing the disulfide reduced galactose-binding serum albumin of claim 1, comprising reducing the disulfide bond of the galactose-binding serum albumin using a reducing agent containing a thiol group (step 2). The method according to claim 4, wherein the reducing agent containing a thiol group is 2-mercaptoethanol, 1,4-dithiodreitol, 2-aminoethanethiol, thioglycolate, cysteine and glutathione. The method of claim 4, wherein the reduction further comprises a chelating agent. 7. A process according to claim 6, wherein said chelating agent is EDTA. Radioisotope labeling compound for imaging liver function, including the disulfide reduced galactose binding serum albumin of claim 1. The radioisotope labeling compound for liver function imaging according to claim 8, wherein the metallic radioisotope is ^99m Tc, ¹⁸⁸ Re, ¹⁸⁶ Re, ⁶⁷ Cu, ⁶⁴ Cu, ²¹² Pb, ²¹² Bi or ¹⁰⁹ Pd. A kit for imaging liver function in a pharmaceutically acceptable nonpyrogenic sterile form comprising the disulfide reduced galactose binding serum albumin, a weak chelating agent and stannous chloride. 11. The method according to claim 10, wherein the disulfide reduced galactose-binding serum albumin is 0.1-100 mg / unit dose, the weak chelating agent is 0.1-500 mg / unit dose, and the stannous chloride 0.1 μg / t. Liver function imaging kit, characterized in that the unit dose-100 mg / unit dose. 11. The method according to claim 10, wherein the weak chelating agent comprises one or more of a group consisting of phosphonate, glucoheptonate, gluconate, glucarate, tartarate, succinate and citric acid. Kit. The kit for liver function imaging according to claim 10, further comprising 0 to 500 mg / unit dose of antioxidant. The kit for imaging liver function according to claim 13, wherein the antioxidant is vitamin C or gentic acid.