WO2003027148A1 - Disulfide-reduced neogalactosyl serum albumin and use of radiolabeled derivative thereof for liver imaging - Google Patents

Disulfide-reduced neogalactosyl serum albumin and use of radiolabeled derivative thereof for liver imaging Download PDF

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WO2003027148A1
WO2003027148A1 PCT/KR2002/001787 KR0201787W WO03027148A1 WO 2003027148 A1 WO2003027148 A1 WO 2003027148A1 KR 0201787 W KR0201787 W KR 0201787W WO 03027148 A1 WO03027148 A1 WO 03027148A1
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serum albumin
disulfide
neogalactosyl
reduced
liver
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WO2003027148A8 (en
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Jae Min Jeong
Jaetae Lee
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Jae Min Jeong
Jaetae Lee
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/081—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the protein being an albumin, e.g. human serum albumin [HSA], bovine serum albumin [BSA], ovalbumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

  • the present invention relates to disulfide-reduced neogalactosyl serum albumin and use of its radiolabeled derivative for liver imaging, and more particularly, to disulfide-reduced neogalactosyl serum albumin represented by the following Formula 1 and a method of preparing the same, as well as its radiolabeled derivative. Also, the present invention is concerned with a kit for liver imaging.
  • liver imaging with radioisotopes makes it possible to evaluate the size and shape of liver and overall hepatic function, as well as local lesions.
  • liver cancer is a typical example of the local diseases.
  • location and size of tumors as well as liver function can be evaluated by using the technology of liver imaging.
  • liver cancer is induced by primary hepatoma or metastasis of various cancer, and whether successing medical treatment of cancer or not is dependant on control of metastatic carcinoma.
  • metastatic carcinoma appears as local defection, but many benign lesions and normal structures of liver as well as artifacts also appear similar to the local defection. Therefore, it is very important to interpret correctly the result of liver imaging with basic knowledge of such lesions.
  • liver imaging with the conventional radiolabeled compounds most liver lesions appear as cold lesions that have lower density than surrounding normal tissue. However, it is more difficult to find the cold lesions due to nonspecific background radioactivity than in case of hot lesions. Moreover, there is a need for careful attention when looking for cold lesion in phantom of large organs such as liver.
  • liver imaging technique has been improved in sensitivity and specificity thanks to the development of radiolabeled compounds applicable in nuclear medicine and advancement of single photon emission computed tomography (SPECT) .
  • SPECT single photon emission computed tomography
  • mammalian liver contains hepatic binding protein (HBP) which functions to bind glycoprotein including galactose at the terminus in blood and remove it in blood.
  • HBP hepatic binding protein
  • Liver imaging can be achieved through administration of technetium-labeled serum albumin to which galactose is bound, and then association of galactose receptor.
  • Gal n -protein- (NH 2 ) m + anhydrous cyclic DTPA -> (Gal) n _ protein- (DTPA) m
  • Gal is a galactosyl group
  • NH 2 is an amino group
  • n or m is an integer of 1 to 40.
  • glycoprotein o- ucoid is first treated with neuraminidase to remove sialic acid, and to expose galactose at the terminus of the glycoprotein, and then DTPA is attached to the glycoprotein.
  • DTPA is attached to the glycoprotein.
  • the resulting glycoprotein is easily radiolabeled with a radioactive isotope of technetium or rhenium.
  • such a method is problematic, as follows.
  • the step of binding technetium or rhenium to DTPA should be conducted at high temperature for a prolonged period, thus causing denaturation of protein in addition to requiring a long reaction time.
  • acidic DTPA is bound to protein in a large quantity, which may cause changes in properties of neogalactosyl serum albumin.
  • galactose and DTPA are able to bind to lysine residues and amino termini, when one of the two binds to the protein in a large quantity, the other lacks sufficient binding sites (G. Ashwell, C. J. Steer., J. Am . Med. Assoc. 246, 2358-2364, 1981; R. J. Stockert, A. G. Morell., Hepatology, 3, 750-757, 1983; D. R. Vera, R. C. Stadalnik, K. A. Krohn., J. Nucl . Med.
  • Japanese Pat. Nos. 05087190 and 61312434 disclose neogalactosyl serum albumin to which DTPA is attached to facilitate its radiolabeling with technetium, in which neogalactosyl serum albumin in a bound form of DTPA is prepared under a heated condition of 50°C for 30 min to bind a suitable amount of DTPA to serum albumin.
  • this method has several drawbacks.
  • radiolabeling of the neogalalactosyl protein is conducted at a relatively high temperature for a prolonged period to give strong binding between DTPA and technetium or rhenium.
  • the resultant protein thus binds to a large number of acidic DTPA, which may cause changes in properties of serum albumin.
  • the binding of galactose and DTPA to serum albumin is competitive, leading to limitation of binding of one of the two.
  • monoclonal antibody much research on methods of radiolabeling methods with technetium has been done, resulting in the finding that technetium metal binds stably to thiol groups in monoclonal antibodies, which are generated by reduction of disulfide bonds.
  • Korean Pat. No. 178961 discloses a method of radiolabeling with technetium or rhenium through binding of a radioisotope of the metal to thiol groups that are spatially adjacent each other.
  • a radioisotope of the metal to thiol groups that are spatially adjacent each other.
  • the present invention aims to provide disulfide-reduced neogalactosyl serum albumin containing exposed galactosyl and thiol groups and a method of preparing the same.
  • the present invention aims to provide a radiolabeled compound containing disulfide-reduced neogalactosyl serum albumin.
  • the present invention aims to provide a kit for liver imaging, comprising disulfide-reduced neogalactosyl serum albumin, a weak chelating agent and stannous chloride, additionally containing an antioxidant.
  • Fig. 1 shows a simplified structure of disulfide- reduced neogalactosyl serum albumin, in which (a) shows serum albumin with directly bound galactosyl groups, and (b) shows serum albumin with galactosyl groups indirectly bound using linkers;
  • Gal is galatosyl group
  • L means linker
  • SH is thiol group
  • Fig. 2 is a graph showing stability of 99m ⁇ c- disulfide-reduced neolactosyl serum albumin; and Fig. 3 is a graph showing biodistribution of 9m Tc- disulfide-reduced neolactosyl serum albumin in mouse.
  • disulfide-reduced neogalactosyl serum albumin represented by the following Formula 1, which can be used for liver imaging.
  • Formula 1 (Gal-L) n -A-(SH) m wherein, Gal is galactosyl group, L means linker or direct bonding, A is disulfide-reduced serum albumin, SH is thiol group, n is an integer of 1 to 42, and m is an integer of 2 to 34.
  • Fig. 1 shows a simplified structure of an embodiment of disulfide-reduced neogalactosyl serum albumin represented by Formula 1.
  • disulfide-reduced neogalactosyl serum albumin contains galactosyl groups directly bound to the terminus of serum albumin (see, Fig. 1(a)) or indirectly bound thereto through mediation by linkers (see, Fig. 1(b)), and has thiol groups through reduced disulfide bond of serum albumin.
  • Human serum albumin has a molecular weight of 66,462 Da, and is 8 nm in long axis and 6 nm in short axis, with an isoelectric point (IEP) of 4.8.
  • the protein occupies 50 % (4g/dl) of proteins present in blood plasma and comprises of a single bond.
  • HSA human serum albumin
  • 2-34 thiol groups would be generated on the human serum albumin.
  • the newly generated thiol groups serve as sites for binding of radioactive isotopes, where over two thiol groups are preferably located to be adjacent each other in order to act as a chelate for radioactive isotopes.
  • the thiol groups on the reduced neogalactosyl serum albumin are stable in the absence of oxygen, and their stability is increased in pH values less than pH 6.
  • the thiol groups-containing serum albumin is flash-frozen in liquid nitrogen, there is no contamination as well as no loss of reduced thiol groups, thus making its storage for a prolonged period possible.
  • galactose is directly or indirectly bound to human serum albumin, where indirect binding is achieved using a linker.
  • linker useful in the present invention is selected by one or more than one from the group consisting of alkyl groups of C 1 -C 10 , aryl groups of C 4 -C ⁇ o, monopeptides, dipeptides, oligopeptides, cycloalkyl groups of C-C ⁇ o, benzyl groups, thioethers, ethers, amines, hydrazides, pentoses, hexoses and alcohols.
  • the galactosyl group and the linker-containing galactosyl group is bound to amino group (-NH 2 ) of human serum albumin.
  • human serum albumin bound to galactose directly or indirectly by linker is represented by the following Formulas 2 to 4.
  • Formula 2 shows neogalactosyl serum albumin to which galactose directly binds
  • Formula 3 shows neolactosyl serum albumin to which galactose indirectly binds using glucose as a linker
  • Formula 4 shows phenylgalactosylated serum albumin to which galactose indirectly binds using phenyl group as a linker.
  • the neogalactosyl serum albumin of the present invention may include 1 to 42 galactosyl groups or linker- containing galactosyl groups according to intended use.
  • HSA human serum albumin
  • the method comprises steps of preparing neogalactosyl serum albumin by binding directly or using a linker to bind galactose to human serum albumin (Step 1), and reducing disulfide bonds in the neogalactosyl serum albumin using a thiol groups-containing reducing agent (Step 2) .
  • neogalactosyl serum albumin is produced by binding galactose to human serum albumin directly or using a linker, where the terminal end of galactose or the linker is modified using a chemical reaction common in the art to generate a functional group available for a binding, such as thiocyanate, ester, aldehyde, iminomethoxyalkyl groups, and the like, thus facilitating its binding to human serum albumin.
  • neogalactosyl serum albumin is prepared using 2-imino-2-methoxyethyl-l-thio- ⁇ -D-galactose (hereinafter referred to as "I E-thiogalactose”) represented by Formula 2 according to Reaction Scheme 2, below.
  • I E-thiogalactose 2-imino-2-methoxyethyl-l-thio- ⁇ -D-galactose
  • D-galactose is acetylated and brominated, and thiourea binds to the modified D-galactose.
  • Cyanomethyl 2, 3, 4, 6-tetra-0-acetyl- 1-thio- ⁇ -D-galatose is formed through reaction with chloroacetonitrile, and then reacts with sodium methoxide in a methanol solution, producing I E-thiogalactose.
  • IME- thiogalactose binds to an amino group (-NH 2 ) in human serum albumin (HSA) at pH 8-9, forming a thiocarbamyl unit, thereby producing neogalactosyl serum albumin, represented by Formula 2.
  • Neolactosyl serum albumin represented by Formula 3 is prepared by directly binding lactose to human serum albumin, as in the following Reaction Scheme 3. [Reaction Scheme 3]
  • Lactose is a disaccaride consisting of galactose and glucose. As shown in Reaction Scheme 3, lactose binds to human serum albumin through reaction between an aldehyde in glucose component and an amino group in serum albumin, forming a Schiff s base, and is then stabilized through reduction using NaCNBH 3 or NaBH, thereby producing neolactosyl serum albumin represented by Formula 3. In case of neolactosyl serum albumin, galactose binds to human serum albumin using glucose as a linker.
  • Reaction Scheme 4 shows a process for preparing phenylgalactosyiated serum albumin represented by Formula 4, which consists of phenylgalactose and human serum albumin.
  • phenylgalactosyiated serum albumin represented by Formula 4 is prepared by binding SCN unit at phenyl group' s terminus of phenylgalatose to an amino group in human serum albumin.
  • Step 2 disulfide bonds present in the neogalacosyl serum albumin prepared in Step 1 are reduced using a reducing agent, thereby producing disulfide- reduced neogalactosyl serum albumin represented by Formula 1.
  • the reducing agent contains thiol groups. Examples of the reducing agent useful in the present invention include 2-mercaptoethanol, dithiothreitol, thioglycholate, cysteine, glutathione, and those common in the art.
  • a chelating agent can be added to remove residual metal ions, thus improving stability of a product and increase labeling efficiency of radioisotopes.
  • the chelating agent is selected from those common in the art, and preferably, EDT .
  • a radiolabeled compound containing disulfide- reduced neogalactosyl serum albumin represented by Formula 1.
  • radioisotopes for use in the present invention are 99m Tc, 188 Re, 186 Re, 67 Cu, 6 Cu, 212 Pb, 212 Bi or 109 Pd, and more preferably, 9m Tc or 188 Re.
  • the radioisotope binds stably to thiol groups in disulfide-reduced neogalactosyl serum albumin.
  • the radiolabeled compound is prepared by reacting disulfide-reduced neogalactosyl serum albumin represented by Formula 1 with the radioisotope, and the preparation is performed in a sterile liquid solution not containing pyrogen, thus allowing its application to a human body immediately after preparation.
  • the radiolabeled compound When being administered to a human patient intravenously, the radiolabeled compound is accumulated in the liver, thereby making it possible to image hepatic function using apparatuses common in nuclear medicine.
  • the present invention provides a kit for liver imaging, which is in a pharmaceutically acceptable and non-pyrogenic sterile form, comprising a unit dose of disulfide-reduced neogalactosyl serum albumin represented by Formula 1 from 0.1 to 100 mg, a unit dose of a weak chelating agent from 0.1 to 500 mg, a unit dose of stannous chloride from 0.01 ⁇ g to 100 mg, and a unit dose of an antioxidant from 0 to 500 mg.
  • a unit dose of disulfide-reduced neogalactosyl serum albumin represented by Formula 1 from 0.1 to 100 mg
  • a unit dose of a weak chelating agent from 0.1 to 500 mg
  • stannous chloride from 0.01 ⁇ g to 100 mg
  • an antioxidant from 0 to 500 mg.
  • the weak chelating agent functions to prevent binding of the radioisotope to a weak-binding region in neogalactosyl serum albumin or forming colloids.
  • the weak chelating agent is selected from the group consisting of phosphonate, glucoheptonate, gluconate, glucarate, tartrate, succinate, citrate, and mixtures thereof, and preferably contained in a unit dose of 0.1 to 500 mg in the kit.
  • Stannous chloride is preferably contained in a unit dose of 0.01 ⁇ g to 500 mg in the kit.
  • the kit for liver imaging according to the present invention further comprises an antioxidant.
  • the antioxidant which serves to prevent formation of complexes caused by oxidation of thiol groups in disulfide-reduced neogalactosyl serum albumin,- is preferably vitamin C or gentisic acid, and is preferably ' contained in a unit dose of 0 to 500 mg in the kit.
  • the kit for liver imaging is frozen or lyophilized in a sterile container under inert gas atmosphere, and preferably, stored in liquid nitrogen in a frozen state and slowly thawed right before use.
  • the kit may be provided along with a vial containing sterile buffer solution, saline, syringes, filters, columns, and other auxiliary apparatuses, thus facilitating preparation for injection, which may be performed by clinical pathologists or technicians ' . It is well known to those of ordinary skill in the art that the kit may be modified and transformed according to individual needs or dietary requirement of patients as well as changed in the form in which a radioisotope is supplied or acquird.
  • the radiolabeled compound is prepared as follows. Right before application, a radioisotope is added to the kit at room temperature in an amount of 0.1 to 500 mCi per mg disulfide-reduced neogalactosyl serum albumin, followed by reaction for 0.1 to 30 minutes.
  • a technetium-labeled compound can be prepared by adding 99 Tc0 ⁇ prepared in a generator to the kit for liver imaging and reacting for 1 to 30 minutes at room temperature, giving labeling efficiency of 98 to 100 %.
  • STEP 1 Preparation of neogalactosyl human serum albumin According to Reaction Scheme 1, above, 100 g of D- galactose was slowly added to a mixture of 400 ml acetic anhydride and 3 ml perchloric acid, which was maintained at 30-40 °C, over 30 minutes with gentle agitation, and the D- galactose-containing mixture was then supplemented with 30 g of amorphous phosphorus and cooled on ice. 180 g of bromine was slowly added to the mixture maintained below 20 °C, along with slow addition of 36 ml (0.9 equivalent) distilled water over 30 minutes to prevent increase in temperature of the mixture. The container containing the mixture was covered and allowed to react for 2 more hours at room temperature.
  • the produced precipitate was collected through filtration and washed with cold water. After being dried in air, the precipitate was dissolved in boiled methanol and filtered to remove impurities, and then stored in a refrigerator to obtain crystals, where recovery rate is 72 %, and the recovered crystal has a melting point of 95-97 °C. 40 mg of the recovered crystal was dissolved in 1.5 ml anhydrous methanol heated to about 40 °C, and 0.8 mg of sodium methoxide was then added thereto and well mixed, followed by reaction for 48 hours at room temperature, resulting in synthesis of IME-thiogalactose (22 mg; a yield: 55 %).
  • neogalactosyl human serum albumin (13.6 mg/ml) prepared in Step 1, above, was mixed with 40 ⁇ l of 0.3 M EDTA (pH 8.0), 40 ⁇ l of 1 M sodium bicarbonate and 50 ⁇ l of 1.5 M ⁇ -mercaptoethanol, and allowed to react for 1 hour at 37 °C. Thereafter, disulfide-reduced neogalactosyl human serum albumin was obtained using a Sephadex G-25 column.
  • NaCNBH 3 sodium cyanoborohydride
  • reaction mixture was centrifuged at 3,000 rpm for 5 minutes, and supernatant was stored at -70°C in a frozen state.
  • neolactosyl human serum albumin (13.6 mg/ml) prepared in Step 1, above, was mixed with 40 ⁇ l of 0.3 M EDTA (pH 8.0), 40 ⁇ l of 1 M sodium bicarbonate and 50 ⁇ l of 1.5 M ⁇ -mercaptoethanol, and allowed to react for 1 hour at 37°C.
  • disulfide-reduced neolactosyl human serum albumin was separated using a Sephadex G-25 column, mixed with 0.3 ml of a glucarate solution (2 mg/ml, pH 7.0) containing 6 ⁇ g of SnCl 2 ⁇ 2 0, and then aliquotted in a protein amount of 3 mg and stored at -70°C in a frozen or lyophilized state.
  • EXAMPLE 3 Preparation of disulfide-reduced neophenylgalactosyl human serum albumin
  • STEP 1 Preparation of neophenylgalactosyl human serum albumin
  • neophenylgalactosyl human serum albumin (13.6 mg/ml) prepared in Step 1, above, was mixed with 40 ⁇ l of 0.3 M EDTA (pH 8.0), 40 ⁇ l of 1 M sodium bicarbonate and 50 ⁇ l of 1.5 M. ⁇ -mercaptoethanol, and allowed to react for 1 hour at 37°C.
  • COMPARATIVE EXAMPLE 1 DTPA-bound neogalactosyl serum albumin( 99 ⁇ Tc-DTPA-GSA-Gal)
  • DTPA-bound enogalactosyl serum albumin (GSA, Nihon Medi-Physics, Japan) was dissolved in 1.2 ml of a physiological saline solution, and then mixed with 99 Tc0 " , followed by reaction for 30 minutes at 50 °C , resulting in production of 99m Tc- labeled DTPA-bound neogalactosyl serum albumin ( 9 a 9 a m m n TC-DTPA-GSA-Gal)
  • labeling efficiency according to reaction time and added amount of the technetium- containing solution shows that 99 ⁇ n Tc-disulfide-reduced neolactosyl human serum albumin is radiolabeled with a labeling efficiency of over 98 % after 1 minute of reaction at the room temperature, while labeling efficiency of 99m ⁇ c- DTPA-GSA-Gal is over 95 % (data not shown), indicating that labeling efficiency of 99m Tc-disulfide-reduced neolactosyl human serum albumin according to the present invention is over 3 % higher than that of 99m Tc-DTPA-GSA-Gal .
  • Table 3 and Fig. 3 show radioactivity distributed in each organ of mouse 10 and 60 minutes after injection, which is given as a percentage, respectively. After a lapse of 10 minutes, radioactivity in liver was found to be 77.4 % ID/g, while other tissues showed very low radioactivities. These results indicate that when being labeled with 99m, Tc- disulfide-reduced neogalactosyl serum albumin according to the present invention is greatly useful for liver imaging.
  • disulfide-reduced neogalactosyl serum albumin use of its radiolabeled derivative and a kit for radiolabeling according to the present invention can be radiolabeled more effectively than the conventional compounds used in liver imaging, have excellent stability as well as a high accumulation rate in liver, thereby allow its application in liver imaging, using thiol groups obtained from reduction of disulfide bonds.

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Abstract

Disclosed are disulfide-reduced neogalactosyl serum albumin and use of its radiolabeled derivative for liver imaging. More particularly, the present invention relates to disulfide-reduced neogalactosyl serum albumin represented by the following Formula 1 and a method of preparing the same, as well as its radiolabeled derivative. Also, the present invention is concerned with a kit for liver imaging, which is capable of radiolabeling neogalactosyl human serum albumin. The radiolabeled compound according to the present invention has excellent stability as well as a high accumulation rate in liver, thereby allowing its application in liver imaging Formula (I), wherein, Ga1, L, A, n, and m are described in detail in the specification.

Description

DISULFIDE-REDUCED NEOGALACTOSYL SERUM ALBUMIN AND USE OF RADIOLABELED DERIVATIVE THEREOF FOR LIVER IMAGING
TECHNICAL FIELD The present invention relates to disulfide-reduced neogalactosyl serum albumin and use of its radiolabeled derivative for liver imaging, and more particularly, to disulfide-reduced neogalactosyl serum albumin represented by the following Formula 1 and a method of preparing the same, as well as its radiolabeled derivative. Also, the present invention is concerned with a kit for liver imaging. [Formula 1] (Gal-L)„-A-(SH)m wherein, Gal is a galactosyl group, L means linker or direct bonding, A is disulfide-reduced serum albumin, SH is a thiol group, n is an integer of 1 to 42, and m is an integer of 2 to 34.
BACKGROUND ART Liver-related diseases occur frequently among Orientals including Koreans, and accordingly, it is considered to be very important to diagnose the hepatic function. Among diagnostic methods of hepatic function, liver imaging with radioisotopes makes it possible to evaluate the size and shape of liver and overall hepatic function, as well as local lesions.
It is known that various diffuse diseases or local diseases develop in the liver. Examples of the diffuse diseases include acute infection, chronic infection, hepatic cirrhosis, injury caused by drugs including alcohol, and injury of connective tissue, and acute infection, chronic infection and hepatic cirrhosis are especially common among Koreans. In case of diffuse disease, which results in decrease of hepatic function, liver imaging is usefully applied. Also, liver cancer is a typical example of the local diseases. In case of liver cancer, location and size of tumors as well as liver function can be evaluated by using the technology of liver imaging.
Liver cancer is induced by primary hepatoma or metastasis of various cancer, and whether successing medical treatment of cancer or not is dependant on control of metastatic carcinoma. In a liver image, metastatic carcinoma appears as local defection, but many benign lesions and normal structures of liver as well as artifacts also appear similar to the local defection. Therefore, it is very important to interpret correctly the result of liver imaging with basic knowledge of such lesions.
In liver imaging with the conventional radiolabeled compounds, most liver lesions appear as cold lesions that have lower density than surrounding normal tissue. However, it is more difficult to find the cold lesions due to nonspecific background radioactivity than in case of hot lesions. Moreover, there is a need for careful attention when looking for cold lesion in phantom of large organs such as liver.
At present, liver imaging technique has been improved in sensitivity and specificity thanks to the development of radiolabeled compounds applicable in nuclear medicine and advancement of single photon emission computed tomography (SPECT) .
On the other hand, mammalian liver contains hepatic binding protein (HBP) which functions to bind glycoprotein including galactose at the terminus in blood and remove it in blood. Liver imaging can be achieved through administration of technetium-labeled serum albumin to which galactose is bound, and then association of galactose receptor.
Initially, such a neogalactosyl serum albumin was radiolabeled with technetium using an electrolysis method, which requires specific apparatuses and comprises complicated procedures, resulting in limitation in universal use thereof.
Recently, there has been a proposal for a method of radiolabeling proteins with technetium using chelates with two functional groups capable of connecting proteins, in which chelates bind to macromolecules such as antibodies. As a chelate, preferably, diethylenetriamine pentaacetic acid (hereinafter referred to simply as "DTPA") of various forms can be used. For example, radiolabeling using chelates can be achieved in the manner represented by the following Reaction Scheme 1 : [Reaction Scheme 1]
(Gal)n-protein- (NH2)m + anhydrous cyclic DTPA -> (Gal)n _ protein- (DTPA)m wherein, Gal is a galactosyl group, NH2 is an amino group, and n or m is an integer of 1 to 40.
According to the Reaction Scheme 1, glycoprotein o- ucoid is first treated with neuraminidase to remove sialic acid, and to expose galactose at the terminus of the glycoprotein, and then DTPA is attached to the glycoprotein. Thus, the resulting glycoprotein is easily radiolabeled with a radioactive isotope of technetium or rhenium. However, such a method is problematic, as follows. In order to ensure strong binding between DTPA and technetium or rhenium, the step of binding technetium or rhenium to DTPA should be conducted at high temperature for a prolonged period, thus causing denaturation of protein in addition to requiring a long reaction time. In addition, acidic DTPA is bound to protein in a large quantity, which may cause changes in properties of neogalactosyl serum albumin. Further, since both galactose and DTPA are able to bind to lysine residues and amino termini, when one of the two binds to the protein in a large quantity, the other lacks sufficient binding sites (G. Ashwell, C. J. Steer., J. Am . Med. Assoc. 246, 2358-2364, 1981; R. J. Stockert, A. G. Morell., Hepatology, 3, 750-757, 1983; D. R. Vera, R. C. Stadalnik, K. A. Krohn., J. Nucl . Med. , 26, 1157-1167, 1985; G. Galli, C. L. Maini, P. Orlando, G. Deleide, G. Valle., J. Nucl . Med. Allied Sci . , 32, 110-116, 1988). Japanese Pat. Nos. 05087190 and 61312434 disclose neogalactosyl serum albumin to which DTPA is attached to facilitate its radiolabeling with technetium, in which neogalactosyl serum albumin in a bound form of DTPA is prepared under a heated condition of 50°C for 30 min to bind a suitable amount of DTPA to serum albumin. However, this method has several drawbacks. That is, radiolabeling of the neogalalactosyl protein is conducted at a relatively high temperature for a prolonged period to give strong binding between DTPA and technetium or rhenium. The resultant protein thus binds to a large number of acidic DTPA, which may cause changes in properties of serum albumin. In addition, the binding of galactose and DTPA to serum albumin is competitive, leading to limitation of binding of one of the two. On the other hand, with the development of monoclonal antibody, much research on methods of radiolabeling methods with technetium has been done, resulting in the finding that technetium metal binds stably to thiol groups in monoclonal antibodies, which are generated by reduction of disulfide bonds. Korean Pat. No. 178961 discloses a method of radiolabeling with technetium or rhenium through binding of a radioisotope of the metal to thiol groups that are spatially adjacent each other. However, there is still no report of specific compounds and their experimental results useful for liver imaging.
SUMMARY OF THE INVENTION
Leading to the present invention, the intensive and thorough research into effective liver imaging using disulfide-reduced neogalactosyl serum albumin, a radiolabeled compound using the same and a kit for radiolabeling, with the aim to solve the above problems, resulted in the finding that the compound of the present invention is more easily radiolabeled than the conventional compounds for radiolabeling, and is highly stable when being radiolabeled, as well as shows a high accumulation rate in the liver, thus being capable of providing excellent liver images .
It is therefore an object of the present invention to provide disulfide-reduced neogalactosyl serum albumin for liver imaging, which is easily radiolabeled, and a method of preparing the same.
In detail, the present invention aims to provide disulfide-reduced neogalactosyl serum albumin containing exposed galactosyl and thiol groups and a method of preparing the same.
It is another object of the present invention to provide a radiolabeled compound having high stability as well as an excellent accumulation rate in the liver. In detail, the present invention aims to provide a radiolabeled compound containing disulfide-reduced neogalactosyl serum albumin.
It is a further object of the present invention to provide a kit for liver imaging, comprising an easily radiolabelable compound.
In detail, the present invention aims to provide a kit for liver imaging, comprising disulfide-reduced neogalactosyl serum albumin, a weak chelating agent and stannous chloride, additionally containing an antioxidant.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 shows a simplified structure of disulfide- reduced neogalactosyl serum albumin, in which (a) shows serum albumin with directly bound galactosyl groups, and (b) shows serum albumin with galactosyl groups indirectly bound using linkers;
Wherein Gal is galatosyl group, L means linker and SH is thiol group.
Fig. 2 is a graph showing stability of 99mτc- disulfide-reduced neolactosyl serum albumin; and Fig. 3 is a graph showing biodistribution of 9mTc- disulfide-reduced neolactosyl serum albumin in mouse.
DISCLOSURE OF THE INVENTION
In accordance with the present invention, there is provided disulfide-reduced neogalactosyl serum albumin represented by the following Formula 1, which can be used for liver imaging. [Formula 1] (Gal-L)n-A-(SH)m wherein, Gal is galactosyl group, L means linker or direct bonding, A is disulfide-reduced serum albumin, SH is thiol group, n is an integer of 1 to 42, and m is an integer of 2 to 34.
Fig. 1 shows a simplified structure of an embodiment of disulfide-reduced neogalactosyl serum albumin represented by Formula 1.
As shown in Formula 1 and Fig. 1, disulfide-reduced neogalactosyl serum albumin contains galactosyl groups directly bound to the terminus of serum albumin (see, Fig. 1(a)) or indirectly bound thereto through mediation by linkers (see, Fig. 1(b)), and has thiol groups through reduced disulfide bond of serum albumin.
Human serum albumin has a molecular weight of 66,462 Da, and is 8 nm in long axis and 6 nm in short axis, with an isoelectric point (IEP) of 4.8. In addition, the protein occupies 50 % (4g/dl) of proteins present in blood plasma and comprises of a single bond.
When human serum albumin (HSA) , having seventeen disulfide bonds, is reduced by a reducing agent, 2-34 thiol groups would be generated on the human serum albumin. The newly generated thiol groups serve as sites for binding of radioactive isotopes, where over two thiol groups are preferably located to be adjacent each other in order to act as a chelate for radioactive isotopes. The thiol groups on the reduced neogalactosyl serum albumin are stable in the absence of oxygen, and their stability is increased in pH values less than pH 6. In addition, when the thiol groups-containing serum albumin is flash-frozen in liquid nitrogen, there is no contamination as well as no loss of reduced thiol groups, thus making its storage for a prolonged period possible.
In accordance with the present invention, galactose is directly or indirectly bound to human serum albumin, where indirect binding is achieved using a linker. Examples of the linker useful in the present invention is selected by one or more than one from the group consisting of alkyl groups of C1-C10, aryl groups of C4-Cχo, monopeptides, dipeptides, oligopeptides, cycloalkyl groups of C-Cιo, benzyl groups, thioethers, ethers, amines, hydrazides, pentoses, hexoses and alcohols.
The galactosyl group and the linker-containing galactosyl group is bound to amino group (-NH2) of human serum albumin. For example, human serum albumin bound to galactose directly or indirectly by linker is represented by the following Formulas 2 to 4. Formula 2, below, shows neogalactosyl serum albumin to which galactose directly binds, Formula 3 shows neolactosyl serum albumin to which galactose indirectly binds using glucose as a linker, and Formula 4 shows phenylgalactosylated serum albumin to which galactose indirectly binds using phenyl group as a linker. The neogalactosyl serum albumin of the present invention may include 1 to 42 galactosyl groups or linker- containing galactosyl groups according to intended use.
[Formula 2]
Figure imgf000013_0001
;Formula 3]
Figure imgf000013_0002
[Formula 4]
Figure imgf000013_0003
As represented by Formulas 2 to 4, disulfide bonds in human serum albumin (HSA) are reduced by a reducing agent containing thiol groups to form disulfide-reduced neogalactosyl serum albumin.
In accordance with the present invention, there is provided a method of preparing disulfide-reduced neogalactosyl serum albumin for liver imaging, which is represented by Formula 1, above.
The method comprises steps of preparing neogalactosyl serum albumin by binding directly or using a linker to bind galactose to human serum albumin (Step 1), and reducing disulfide bonds in the neogalactosyl serum albumin using a thiol groups-containing reducing agent (Step 2) . In Step 1, neogalactosyl serum albumin is produced by binding galactose to human serum albumin directly or using a linker, where the terminal end of galactose or the linker is modified using a chemical reaction common in the art to generate a functional group available for a binding, such as thiocyanate, ester, aldehyde, iminomethoxyalkyl groups, and the like, thus facilitating its binding to human serum albumin.
In more detail, neogalactosyl serum albumin is prepared using 2-imino-2-methoxyethyl-l-thio-β-D-galactose (hereinafter referred to as "I E-thiogalactose") represented by Formula 2 according to Reaction Scheme 2, below.
[Reaction Scheme 2]
Figure imgf000015_0001
As shown in Reaction Scheme 2, D-galactose is acetylated and brominated, and thiourea binds to the modified D-galactose. Cyanomethyl 2, 3, 4, 6-tetra-0-acetyl- 1-thio-β-D-galatose is formed through reaction with chloroacetonitrile, and then reacts with sodium methoxide in a methanol solution, producing I E-thiogalactose. IME- thiogalactose binds to an amino group (-NH2) in human serum albumin (HSA) at pH 8-9, forming a thiocarbamyl unit, thereby producing neogalactosyl serum albumin, represented by Formula 2.
Neolactosyl serum albumin represented by Formula 3 is prepared by directly binding lactose to human serum albumin, as in the following Reaction Scheme 3. [Reaction Scheme 3]
Figure imgf000016_0001
Lactose is a disaccaride consisting of galactose and glucose. As shown in Reaction Scheme 3, lactose binds to human serum albumin through reaction between an aldehyde in glucose component and an amino group in serum albumin, forming a Schiff s base, and is then stabilized through reduction using NaCNBH3 or NaBH, thereby producing neolactosyl serum albumin represented by Formula 3. In case of neolactosyl serum albumin, galactose binds to human serum albumin using glucose as a linker.
In addition, the following Reaction Scheme 4 shows a process for preparing phenylgalactosyiated serum albumin represented by Formula 4, which consists of phenylgalactose and human serum albumin. [Reaction Scheme 4 ]
Figure imgf000017_0001
As shown in Reaction Scheme 4, phenylgalactosyiated serum albumin, represented by Formula 4 is prepared by binding SCN unit at phenyl group' s terminus of phenylgalatose to an amino group in human serum albumin.
In Step 2, disulfide bonds present in the neogalacosyl serum albumin prepared in Step 1 are reduced using a reducing agent, thereby producing disulfide- reduced neogalactosyl serum albumin represented by Formula 1. Wherein, the reducing agent contains thiol groups. Examples of the reducing agent useful in the present invention include 2-mercaptoethanol, dithiothreitol, thioglycholate, cysteine, glutathione, and those common in the art.
In the reduction reaction of Step 2, a chelating agent can be added to remove residual metal ions, thus improving stability of a product and increase labeling efficiency of radioisotopes. The chelating agent is selected from those common in the art, and preferably, EDT . In accordance with the present invention, there is provided a radiolabeled compound containing disulfide- reduced neogalactosyl serum albumin represented by Formula 1.
Preferred radioisotopes for use in the present invention are 99mTc, 188Re, 186Re, 67Cu, 6Cu, 212Pb, 212Bi or 109Pd, and more preferably, 9mTc or 188Re. The radioisotope binds stably to thiol groups in disulfide-reduced neogalactosyl serum albumin.
The radiolabeled compound is prepared by reacting disulfide-reduced neogalactosyl serum albumin represented by Formula 1 with the radioisotope, and the preparation is performed in a sterile liquid solution not containing pyrogen, thus allowing its application to a human body immediately after preparation. When being administered to a human patient intravenously, the radiolabeled compound is accumulated in the liver, thereby making it possible to image hepatic function using apparatuses common in nuclear medicine.
In addition, the present invention provides a kit for liver imaging, which is in a pharmaceutically acceptable and non-pyrogenic sterile form, comprising a unit dose of disulfide-reduced neogalactosyl serum albumin represented by Formula 1 from 0.1 to 100 mg, a unit dose of a weak chelating agent from 0.1 to 500 mg, a unit dose of stannous chloride from 0.01 μg to 100 mg, and a unit dose of an antioxidant from 0 to 500 mg.
When a radiolabeled compound, using a kit for liver imaging, is prepared, the weak chelating agent functions to prevent binding of the radioisotope to a weak-binding region in neogalactosyl serum albumin or forming colloids. The weak chelating agent is selected from the group consisting of phosphonate, glucoheptonate, gluconate, glucarate, tartrate, succinate, citrate, and mixtures thereof, and preferably contained in a unit dose of 0.1 to 500 mg in the kit.
Stannous chloride is preferably contained in a unit dose of 0.01 μg to 500 mg in the kit.
In addition, the kit for liver imaging according to the present invention further comprises an antioxidant. The antioxidant, which serves to prevent formation of complexes caused by oxidation of thiol groups in disulfide-reduced neogalactosyl serum albumin,- is preferably vitamin C or gentisic acid, and is preferably' contained in a unit dose of 0 to 500 mg in the kit. The kit for liver imaging is frozen or lyophilized in a sterile container under inert gas atmosphere, and preferably, stored in liquid nitrogen in a frozen state and slowly thawed right before use. The kit may be provided along with a vial containing sterile buffer solution, saline, syringes, filters, columns, and other auxiliary apparatuses, thus facilitating preparation for injection, which may be performed by clinical pathologists or technicians'. It is well known to those of ordinary skill in the art that the kit may be modified and transformed according to individual needs or dietary requirement of patients as well as changed in the form in which a radioisotope is supplied or acquird.
The radiolabeled compound is prepared as follows. Right before application, a radioisotope is added to the kit at room temperature in an amount of 0.1 to 500 mCi per mg disulfide-reduced neogalactosyl serum albumin, followed by reaction for 0.1 to 30 minutes.
For example, a technetium-labeled compound can be prepared by adding 99Tc0~ prepared in a generator to the kit for liver imaging and reacting for 1 to 30 minutes at room temperature, giving labeling efficiency of 98 to 100 %.
In accordance with example of the present invention, upon preparing a radiolabeled compound using the kit of the present invention, there is produced 99mTc-disulfide- reduced neolactosyl serum albumin having labeling efficiencies of over 98 % as well as maintaining stability of its radiochemical purity for 24 hours, as shown in Fig. 2. In addition, 99Tc-disulfide-reduced neolactosyl serum albumin is accumulated in the liver in 77.4 % ID/g, while accumulating in other organs in a very small amount, as shown in Fig. 3, thereby labeling efficiency, stability, and accumulation rate in the liver, after radiolabeling, are excellent. The present invention will be explained in more detail with reference to the following examples in conjunction with the accompanying drawings. However, the following examples are provided only to illustrate the present invention, and the present invention is not limited to them.
EXAMPLE 1: Preparation of disulfide-reduced neogalactosyl serum albumin
STEP 1 : Preparation of neogalactosyl human serum albumin According to Reaction Scheme 1, above, 100 g of D- galactose was slowly added to a mixture of 400 ml acetic anhydride and 3 ml perchloric acid, which was maintained at 30-40 °C, over 30 minutes with gentle agitation, and the D- galactose-containing mixture was then supplemented with 30 g of amorphous phosphorus and cooled on ice. 180 g of bromine was slowly added to the mixture maintained below 20 °C, along with slow addition of 36 ml (0.9 equivalent) distilled water over 30 minutes to prevent increase in temperature of the mixture. The container containing the mixture was covered and allowed to react for 2 more hours at room temperature. Thereafter, 300 ml chloroform was added thereto and the mixture was then poured into a separatory funnel containing 800 ml ice water. The chloroform phase was collected and filtered to remove phosphorus, and then washed twice with an equivalent volume of ice water. After one more washing with a solution of sodium bicarbonate to eliminate residual acid, the chloroform phase was dehydrated with calcium chloride and dried under reduced pressure, and then dissolved in diethylether, recrystallized to lead to formation of crystals having a melting point of 87 °C. 9.47 g (20 mmol) of 2-S-(2,3,4, 6-tetra-0-acethyl-β-D- galactopyranosyl)-2-thiopseudourea hydrobromide was dissolved in 40 ml of a mixture of water and acetone in a ratio of 1:1 and completely dissolved with addition of 5 ml (79 mmole) of chloroacetonitrile. 3.2 g (23.2 mmol) of potassium carbonate and 4.0 g (40.4 mmol) of sodium bisulfite were then added to the mixture and well mixed with stirring for 30 minutes at room temperature, and the reaction mixture was added to 160 ml ice water and well stirred for 2 hours. The produced precipitate was collected through filtration and washed with cold water. After being dried in air, the precipitate was dissolved in boiled methanol and filtered to remove impurities, and then stored in a refrigerator to obtain crystals, where recovery rate is 72 %, and the recovered crystal has a melting point of 95-97 °C. 40 mg of the recovered crystal was dissolved in 1.5 ml anhydrous methanol heated to about 40 °C, and 0.8 mg of sodium methoxide was then added thereto and well mixed, followed by reaction for 48 hours at room temperature, resulting in synthesis of IME-thiogalactose (22 mg; a yield: 55 %). Thereafter, 100 mg human serum albumin dissolved in 1 ml of 0;2 M boron-buffered solution (pH 8.5) was vigorously mixed with 22 mg of IME-thiogalactose for 1.5 hours at 37 °C, and then stored at -70°C.
STEP 2 : Preparation of disulfide-reduced neogalactosyl human serum albumin
1 ml of neogalactosyl human serum albumin (13.6 mg/ml) prepared in Step 1, above, was mixed with 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, and allowed to react for 1 hour at 37 °C. Thereafter, disulfide-reduced neogalactosyl human serum albumin was obtained using a Sephadex G-25 column.
EXAMPLE 2: Preparation of disulfide-reduced neolactosyl human serum albumin
STEP 1 : Preparation of neolactosyl human serum albumin
According to Reaction Scheme 2, above, 680 mg of human serum albumin was completely dissolved in 50 ml of 0.2 M potassium phosphate-buffered solution (pH 8.0), and 1 g of α-lactose was then added thereto and completely dissolved.
1 g of sodium cyanoborohydride (NaCNBH3) was added to the mixture and completely dissolved. The mixture was then filtered using a 0.22 μm membrane. The filtered mixture was reacted for 11 days with gentle agitation at 37 °C.
Thereafter, the reaction mixture was centrifuged at 3,000 rpm for 5 minutes, and supernatant was stored at -70°C in a frozen state.
STEP 2 : Preparation of disulfide-reduced neolactosyl human serum albumin
1 ml of neolactosyl human serum albumin (13.6 mg/ml) prepared in Step 1, above, was mixed with 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, and allowed to react for 1 hour at 37°C. Thereafter, disulfide-reduced neolactosyl human serum albumin was separated using a Sephadex G-25 column, mixed with 0.3 ml of a glucarate solution (2 mg/ml, pH 7.0) containing 6 μg of SnCl2Η20, and then aliquotted in a protein amount of 3 mg and stored at -70°C in a frozen or lyophilized state.
EXAMPLE 3 : Preparation of disulfide-reduced neophenylgalactosyl human serum albumin STEP 1 : Preparation of neophenylgalactosyl human serum albumin
According to Reaction Scheme 3, above, 268 mg of human serum albumin was completely dissolved in 25 ml of 0.1 M
carbonate-buffered solution (pH 9.5), and 25 mg of α-L- galactopyranosylphenylisothiocyanate was then added thereto, followed by reaction for 20 hours with stirring. The reaction mixture was stored at -70°C.
STEP 2 : Preparation of disulfide-reduced neophenylgalactosyl human serum albumin
1 ml of neophenylgalactosyl human serum albumin (13.6 mg/ml) prepared in Step 1, above, was mixed with 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, and allowed to react for 1 hour at 37°C. Thereafter, disulfide-reduced neophenylgalactosyl human serum albumin was separated using sephadex G-25 column, mixed with 0.3 ml of a glucarate solution (2 mg/ml, pH 7.0) containing 6 μg of SnCl2Η20, and then aliquotted in a protein amount of 3 mg and stored at - 70 °C in a frozen or lyophilized state. EXAMPLE 4: Preparation of a kit for liver imaging
1 ml of disulfide-reduced neolactosyl huaman serum albumin (13.6 mg/ml) was added to 0.3 ml of a glucarate solution (2 mg/ml, pH 7.0) containing 6 μg of SnCl2-H20, and then aliquotted in a protein amount of 3 mg and stored at -
70°C in a frozen or lyophilized state.
EXAMPLE 5 : Preparation of technetium-labeled compound using the kit for liver imaging
2 ml and 5 ml of a physiological saline solution containing 99mTc04 ", which was prepared in a 99Mo/99mTc- generator (Du Pont), were added to the kit prepared in Example 4, respectively, and the mixtures were reacted for 1 to 30 minutes at room temperature, thus producing 99mTc- disulfide-reduced neolactosyl serum albumin.
COMPARATIVE EXAMPLE 1: DTPA-bound neogalactosyl serum albumin(99πιTc-DTPA-GSA-Gal)
3 mg of DTPA-bound enogalactosyl serum albumin (GSA, Nihon Medi-Physics, Japan) was dissolved in 1.2 ml of a physiological saline solution, and then mixed with 99Tc0", followed by reaction for 30 minutes at 50 °C , resulting in production of 99mTc- labeled DTPA-bound neogalactosyl serum albumin ( 9a9amm nTC-DTPA-GSA-Gal)
EXPERIMENTAL EXAMPLE 1: Assay for radiolabeling efficiency
Labeling efficiencies in 99mTc-DTPA-GSA-Gal prepared in Comparative Example 1 and 99mTc-disulfide-reduced neolactosyl human serum albumin were evaluated by performing ITLC (instant thin layer chromatography) , where a small amount of the reactant was spotted on a ITLC strip and then developed with a physiological saline solution, and analyzing distribution of radioisotope using a TLC-scanner. As a result, technetium bound to disulfide-reduced neolactosyl human serum albumin remained at the origin, while unbound technetium migrated with development of the solvent. The result for 99mTc-disulfide-reduced neolactosyl human serum albumin is given in Table 1, below.
TABLE 1
Labeling efficiency of neolactosyl human serum albumin
Figure imgf000027_0001
Figure imgf000028_0001
As apparent in Table 1, labeling efficiency according to reaction time and added amount of the technetium- containing solution shows that 99ιnTc-disulfide-reduced neolactosyl human serum albumin is radiolabeled with a labeling efficiency of over 98 % after 1 minute of reaction at the room temperature, while labeling efficiency of 99mτc- DTPA-GSA-Gal is over 95 % (data not shown), indicating that labeling efficiency of 99mTc-disulfide-reduced neolactosyl human serum albumin according to the present invention is over 3 % higher than that of 99mTc-DTPA-GSA-Gal .
EXPERIMENTAL EXAMPLE 2: Test for stability of radiolabeled compound
Stability of 99mTc-disulfide-reduced neolactosyl human serum albumin prepared in Example was evaluated by investigating its radiochemical purity when held at room temperature or incubated in serum at 37 °C. The results are given in Table 2, below.
TABLE 2 Radiochemical purity (%) according to time
Figure imgf000029_0001
As shown in Table 2 and Fig. 2, when being placed at room temperature, 99ltlTc-disulfide-reduced neolactosyl human serum albumin was found to retain radiochemical purity of nearly 100 % for 24 hours. Also, when being incubated in serum at 37 °C, radiochemical purity of the radiolabeled compound was slightly reduced after 4 hours, but it was maintained at over 88 % for 24 hours, demonstrating that the radiolabeled compound has high stability.
EXPERIMENTAL EXAMPLE 3: Identification of accumulating rate in the liver by biodistribution
Accumulating rate in the liver through biodistribution of the two 99mTc-labeled compounds was investigated in male mice (ICR) having average body weight of 20g.
10 μCi of 99mTc-labeled compounds prepared in Example 5 and Comparative Example 1 were injected into mice via tail veins. After the mice were sacrificed and anatomized, organs were excised. Radioactivity distributed in the isolated organs was analyzed using a gamma ray counter (Cobralll, Packard, USA), and the weight of the organs was measured using a electric balance. Biodistribution of the two radiolabeled compounds were determined according to a standard method to find injected dose per gram tissue (% ID/g), which is given as a percentage. The results are given in Tables 3 and 4, below.
TABLE 3 The percentage of radioactivity according to organs after injection of the radiolabeled compound prepared by example 4(%ID/g)
Figure imgf000030_0001
Table 3 and Fig. 3 show radioactivity distributed in each organ of mouse 10 and 60 minutes after injection, which is given as a percentage, respectively. After a lapse of 10 minutes, radioactivity in liver was found to be 77.4 % ID/g, while other tissues showed very low radioactivities. These results indicate that when being labeled with 99m, Tc- disulfide-reduced neogalactosyl serum albumin according to the present invention is greatly useful for liver imaging. TABLE 4
The percentage of radioactivity according to organ after injection of the compound(99mTc-DTPA-GSA-Gal) prepared by
comparative example 1(% ID/g, ± means SD)
Figure imgf000031_0001
As shown in Table 4, after a lapse of 10 minutes, 99mTc-DTPA-GSA-Gal in liver showed radioactivity of about 74 % ID/g, which is similar to that of the radiolabeled compound prepared in Example 4. However, after 30 minutes, radioactivity of 99mTc-DTPA-GSA-Gal in liver was reduced to about 55 %, indicating that 99mTc-DTPA-GSA-Gal is eliminated in a faster speed than 99mTc-disulfide-reduced neolactosyl serum albumin according to the present invention.
INDUSTRIAL APPLICABILITY
As described hereinbefore, disulfide-reduced neogalactosyl serum albumin, use of its radiolabeled derivative and a kit for radiolabeling according to the present invention can be radiolabeled more effectively than the conventional compounds used in liver imaging, have excellent stability as well as a high accumulation rate in liver, thereby allow its application in liver imaging, using thiol groups obtained from reduction of disulfide bonds.

Claims

WHAT IS CLAIMED IS;
1. A disulfide-reduced neogalactosyl serum albumin, represented by the following Formula 1 : [Formula 1] (Gal-L)n-A-(SH)m wherein, Gal is a galactosyl group, L means linker or direct bonding, A is disulfide-reduced serum albumin, SH is a thiol group, n is an integer of 1 to 42, and m is an integer of 2 to 34.
2. The disulfide-reduced neogalactosy serum albumin according to claim 1, wherein the linker (L) is selected from the group consisting of alkyl groups of Ci-Cio, aryl groups of C-Cι0, monopeptides, dipeptides, oligopeptides, cycloalkyl groups of C-Cιo, benzyl groups, thioethers, ethers, amines, ' hydrazides, pentoses, hexoses, alcohols and mixtures thereof.
3. The disulfide-reduced neogalactosyl serum albumin according to claim 1, wherein the Gal-L is galactosyl, lactosyl, or phenylgalactosyl.
4. A method of preparing disulfide-reduced neogalactosyl serum albumin, comprising the steps of: preparing neogalactosyl serum albumin by binding,' directly or using a linker, galactose to human serum albumin (Step 1); and reducing disulfide bonds in the neogalactosyl serum albumin using a thiol groups-containing reducing agent (Step 2) .
5. The method according to claim 4, wherein the thiol groups-containing reducing agent is selected from the group consisting of 2-mercaptoethanol, 1, 4-dithiothreitol, 2-aminoethanethiol, thioglycholate, cysteine, and glutathione.
6. The method according to claim 4, wherein a chelating agent is added in the Step 2.
7. The method according to claim 6, wherein the chelating agent is EDTA.
8. A radiolabeled compound containing the disulfide- reduced neogalactosyl serum albumin of claim 1 for liver imaging.
9 . The radiolabeled compound according to claim 8 , wherein the compound is labeled with a radioisotope selected from 99mTc, 188Re, 186Re, 67Cu, 6 Cu, 212Pb, 212Bi or 109 Pd .
10. A kit for liver imaging comprising disulfide- reduced neogalactosyl serum albumin of claim 1, a weak chelating agent and stannous chloride, which is in a pharmaceutically acceptable and non-pyrogenic sterile form.
11. The kit according to claim 10, wherein the disulfide-reduced neogalactosyl serum albumin is contained in a unit dose of 1 to 100 mg, the weak chelating agent is contained in a unit dose of 0.1 to 500 mg, and the stannous chloride is contained in a unit dose of 0.1 μg to 100 mg.
12. The kit according to claim 10, wherein the weak chelating agent is selected from the group consisting of phosphonate, glucoheptonate, gluconate, glucarate, tartrate, succinate, citrate, and mixtures thereof.
13. The kit according to claim 10, wherein further an antioxidant is contained in a unit dose of 0 to 500 mg.
14. The kit according to claim 13, wherein the antioxidant is vitamin C or gentisic acid.
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