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99mTc-Neolactosylated
Human Serum Albumin for Imaging the Hepatic Asialoglycoprotein Receptor Jae Min Jeong,§,† Mee Kyoung Hong,§,† Jaetae Lee,‡ Miwon Son,| Young So,§,† Dong Soo Lee,§ June-Key Chung,*,§,† and Myung Chul Lee§ Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea, Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, Korea, and Research Laboratories, Dong-A Pharmaceutical Co. Ltd., Yongin, Kyunggi, Korea. Received November 10, 2003
99m
Tc-Labeled diethylenetriaminepentaacetic acid (DTPA)-coupled neogalactosyl human serum albumin (GSA) is used as an imaging agent for asialoglycoprotein receptor of the liver. However, its labeling is inconvenient because it should be incubated for 30 min at 50 °C. In addition, the conjugated DTPAs can cause decrease of pI and denaturation of protein. Therefore, we developed an improved agent 99mTc-neolactosyl human serum albumin (LSA) which contains a terminal galactose. LSA was synthesized by conjugating lactose to human serum albumin by the formation of a Schiff’s base and successive reduction with sodium cyanoborohydride. The number of conjugated lactose molecules per LSA was 40.7 ( 12.3. To simplify the labeling procedure, we used a direct labeling method that adopts a high affinity 99mTc binding site concept in antibody labeling. The produced LSA was reduced by β-mercaptoethanol to generate sulfhydryl groups and purified by PD-10 size-exclusion column. The number of generated sulfhydryl groups per LSA was 21.9 ( 3.0. Medronate and stannous chloride were added to the reduced LSA and freeze-dried. Finally, 99mTc-pertechnetate (37 MBq, 1 mL) was added to the vial and incubated for 10 min at room temperature. The labeling efficiency of 99mTc-LSA was higher than 98%, and the stability in human serum at 37 °C for 24 h was over 90%. Biodistribution study using balb/c mice and imaging study using SD rats showed high initial liver uptake and slow increase in the intestine due to hepatobiliary excretion after metabolism in the hepatocytes. Negligible spleen uptake was found while 99mTc-tin colloid showed significant amount of spleen uptake due to reticuloendothelial uptake. In conclusion, an improved agent, 99mTc-LSA, for imaging asialoglycoprotein receptor of the liver was successfully developed which showed a simple labeling procedure, high labeling efficiency, high stability, and high initial liver uptake.
INTRODUCTION
Traditionally, radiocolloids, e.g. 99mTc-tin colloid, 99mTcsulfur colloid, 99mTc-phytate colloid, etc., have been used for imaging liver function (Stern et al., 1966; Arzoumanian et al., 1977). However, the images obtained by radiocolloids do not represent the function of hepatocytes which comprise 85% of the liver cells playing a key role in liver function. Rather, the images represent the function of the reticuloendothelial system which comprises only 15% of the liver cells playing a defensive role. Although, the function of hepatocytes could be indirectly estimated by measuring serum level of liver enzymes such as ALT and AST, direct estimation of liver function using functional imaging can provide more useful information. Asialoglycoprotein receptors are known to exist on the mammalian liver (Ashwell and Morell, 1974) and play an important role in the hepatic metabolism of serum * Corresponding author: Department of Nuclear Medicine, Seoul National University Hospital, 28 Yungun-dong Chongroku, Seoul 110-744 Korea;
[email protected]; tel: +822-760-3376; fax: +82-2-745-7690. § Department of Nuclear Medicine, Seoul National University College of Medicine. † Cancer Research Institute, Seoul National University College of Medicine. ‡ Kyungpook National University Hospital. | Dong-A Pharmaceutical Co. Ltd.
proteins (Ashwell and Steer, 1981). It has been reported that the asialoglycoprotein receptors exist on the surface of hepatocytes (Stokert and Morell, 1983), and it has also been suggested that quantitative imaging of asialoglycoprotein receptors estimates the function of the liver (Krohn et al., 1982; Lee, 1998; Miki et al., 2001). A novel neogalactosylalbumin has been developed and labeled with 99mTc for imaging the liver (Krohn et al., 1980). Receptor-mediated binding to hepatocytes was also investigated both in vitro and in vivo (Vera et al., 1984a,b). Later preclinical studies proved the stability and safety of 99mTc-neogalactosylalbumin (Vera et al., 1985). However, it was necessary to develop a simpler and more convenient method, because the electrolytic labeling method used could not be performed practically in most clinical fields. To make the labeling procedure simple, diethylenetriaminepentaacetic acid (DTPA)-conjugated neogalactosylalbumin (GSA) has been developed as an instant kit (Kubota et al., 1986). The GSA kit can be labeled with 99m Tc more easily and consequently they are used widely for imaging liver function especially in Japan. The development and application of 99mTc-GSA has been published in an informative review (Stadalnik and Vera, 2001). However, it is predicted that 99mTc would make less stable complexes with hard bases such as carboxyl or tertiary amine groups of DTPA, because it is a soft acid (Huheey, 1983). In fact, it has been reported that a
10.1021/bc0342074 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/03/2004
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significant amount of 99mTc nonspecifically binds to antibodies instead of the DTPA residue when attempting to label DTPA-conjugated antibodies (Childs and Hnatowich, 1985). In addition, conjugation of DTPA to human serum albumin can lead to denaturation, polymerization, and decrease of isoelectric point. Conjugation of DTPA and galactose to the same protein molecule would make them compete with each other because both bind to the amino group. In this paper, we introduced a direct labeling concept instead of conjugation of DTPA to human serum albumin. Thiol-containing antibodies, after reduction of disulfide bonds using a reducing agent such as β-mercaptoethanol, can be labeled directly with 99mTc (Schwarz and Steinstra¨sser, 1987; Mather and Ellison, 1990). Similarly, human serum albumin containing 17 intramolecular disulfide bonds should also be labeled with 99mTc directly after reduction with β-mercaptoethanol. Another important aspect of this experiment is that the conjugated sugar is not a galactose but a lactose. Lactose is a disaccharide of galactose and glucose: 4-Oβ-D-galactopyranosyl-D-glucose. The aldehyde group of the glucose residue and the amino group of human serum albumin form a reversible Schiff’s base and are subsequently reduced into an secondary amine to form an irreversible covalent bond. Thus the galactose residues of neolactosylated human serum albumin (LSA) are intact even after conjugation and act as ligands of asialoglycoprotein receptors.
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Figure 1. Conjugation of lactose to human serum albumin by reductive amination. The aldehyde group of glucose residue makes a Schiff’s base with the amino group of human serum albumin, and then it is reduced to a stable amine by sodium cyanoborohydride.
EXPERIMENTAL PROCEDURES
Materials and Instruments. Human serum albumin was purchased from Green Cross Corp. (Seoul, Korea) as a 20% solution in normal saline. For purification of neolactosylated human serum albumin (LSA), readymade Sephadex G-25 columns were purchased as PD-10 columns from Pharmacia (Uppsala, Sweden). Disposable aseptic 0.22 µm filters were purchased from Millipore Company. 99Mo/99mTc-generators were purchased from Du Pont Company. ITLC plates were purchased from Gelman Company. Tin colloid kits (0.15 mg of stannous fluoride and 1 mg of sodium fluoride) and human serum albumin (HSA) kits (50 mg of HSA and 0.5 mg of stannous chloride hydrate) for 99mTc-labeling were purchased from Daiichi Co. The particle size of 99mTc-tin colloid has been reported as 0.1-0.5 µm (McClelland et al. 2003). All the other reagents and solvents, if not specified, were purchased from Aldrich, Fluka, or Sigma and were used without further purification. Radio-TLC chromatograms were obtained using a BioScan System 200 imaging scanner. A gamma scintillation counter Cobra II model from Packard Canberra Company was used for counting radioactivity of organs in the biodistribution study. A gamma camera Sigma 410 model from Ohio-Nuclear equipped with a low-energy collimator was used for imaging rats. Preparation of LSA. LSA was prepared using minor modification of the previously reported reductive lactosamination method (Lim et al., 1996; Wilson, 1978). The overall reaction procedure is described in Figure 1. In 50 mL of 0.2 M potassium phosphate buffer (pH ) 8.0), 680 mg of human serum albumin was dissolved completely and 1 g of R-lactose was dissolved successively. One gram of sodium cyanoborohydride was added, and the solution was filtered using a 0.22 µm filter. The filtered solution was incubated at 37 °C with slow stirring for 14 days. The resulting reactant was centrifuged at 3000 rpm for 5 min to remove any precipitate. The supernatant was recovered and stored at -70 °C until used.
Preparation of Disulfide-Reduced LSA (rLSA). Reduction of LSA was performed in a similar way with that of monoclonal antibody for labeling with 99mTc (Mather and Ellison, 1990). To 1 mL of the above prepared LSA, 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 added successively and incubated at 37 °C for 1 h. After purification of the rLSA using Sephadex G-25 column/phosphate-buffered saline (pH ) 6.0), 0.3 mL of sodium medronate (1.25 mg/mL, pH 7.6, containing 68 µg SnF2) was added. The solution was dispensed into vials as an aliquot containing 3 mg of protein and stored in a refrigerator after freeze-drying. Determination of the Conjugation Number of Lactose. The conjugated number of lactose per molecule of human serum albumin was calculated by measuring galactose concentration from the absorbance at 485 nm using the phenol/sulfuric acid method (Dubios et al., 1956); since the secondary amine formed by reductive amination is acid-stable, only galactose is produced during the hydrolysis (Wilson, 1978). Determination of the Number of Thiols. The number of thiol groups on an rLSA molecule was determined before addition of sodium medronate containing SnF2. It was determined by measuring absorbance at 410 nm after 400 µL of sample was reacted with 2 mL of 0.1 M Ellman’s reagent (5,5′-dithio-bis(2-nitrobenzoic acid). Serially diluted cysteine solution was used as a standard. Labeling with 99mTc. To the above freeze-dried vial, 2-5 mL aliquot of generator-eluted 99mTc-pertechnetate was added and incubated at room temperature for 1-30 min. Unreduced LSA was also labeled using the same method for comparison. The labeling efficiencies were checked using ITLC/Umezawa (ethanol:10% ammonium acetate ) 1:1) and 5% bovine serum albumin (BSA) impregnated paper chromatography/saline. Unbound 99m Tc moved to the solvent-front, and LSA-bound 99mTc stayed at the origin on ITLC/Umezawa. All 99mTc activity
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Figure 2. Number of lactose molecules conjugated to human serum albumin by incubation time. The conjugated number becomes maximized after incubation for 8 days at 37 °C.
except reduced hydrolyzed 99mTc moved to the solventfront on 5% BSA-impregnated paper chromatography/ saline. Stability Test. Stability of the labeled LSA was checked at room temperature. Stability in human serum at 37 °C was also checked. The bound and free form of 99m Tc was determined using ITLC/Umezawa and 5% BSA-impregnated paper chromatography/saline as described above. The pH of the reaction mixture during stability test was measured by pH paper. Biodistribution Study in Mice. For biodistribution, 37 kBq of 99mTc-LSA was injected into each mouse (male, ICR, ∼20 g, n ) 4) through the tail vein. The mice were sacrificed at 10 and 60 min. The weight and radioactivity of the blood, muscle, fat tissue, heart, lung, liver, spleen, stomach, intestine, kidney, and bone from the sacrificed mice were measured using an electronic balance and gamma scintillation counter. The percentage of injected dose per gram tissue was calculated. Imaging Study in Rats. For imaging, 7.4 MBq of 99m Tc-LSA was injected into each rat (male, SpragueDawley, ∼200 g) through the tail vein. Planar images of the rats in prone position after 15 and 30 min were obtained using a gamma camera equipped with a lowenergy collimator. Imaging with 99mTc-tin colloid by the same procedure was performed for comparison. RESULTS
Preparation and Characterization of rLSA. The entire procedure was performed in an aseptic manner to prevent bacterial growth. Excess lactose (lactose:HSA ) 285:1) was added to make the reaction close to complete. The conjugated number of lactose to human serum albumin reached almost maximum after incubation at 37 °C for 8 days (Figure 2). The maximum number of conjugated lactose per human serum albumin was about 43. The average conjugated number after 11 days incubation was 40.7 ( 12.3. For labeling with 99mTc, the LSA was reduced by β-mercaptoethanol. The remaining β-mercaptoethanol was removed by PD-10 size-exclusion column, and rLSA was obtained in phosphate-buffered saline (pH 6.0). The average number of sulfhydryl groups per rLSA measured by Ellman’s reagent was 21.9 ( 3.0. For 99mTc labeling and stabilization, 0.3 mL of sodium medronate (1.25 mg/ mL, pH 7.6, containing 68 µg SnF2) was added, then dispensed into 3-mg protein aliquots and freeze-dried. Labeling with 99mTc and Radiochemical Study. After labeling of rLSA with 99mTc, free 99mTc was determined by ITLC/Umezawa and colloid was determined by 5% BSA-impregnated Whatman No.1/saline (Figure 3).
Figure 3. Determination of labeling efficiency. Labeled and colloidal 99mTc remains at the origin, and all the other activities move to the solvent-front by ITLC/Umezawa and all activities except colloidal 99mTc move to the solvent-front of the paper chromatography/saline.
Figure 4. Stability test of 99mTc-LSA at room temperature and at 37 °C in human serum. Table 1. Labeling Efficiency of 99mTc-LSA Determined by ITLC and Paper Chromatography added volume of 99mTc-pertechnetatea incubation time (min)
2 mL
5 mL
1 3 7 15 30
98.5 98.2 98.9 98.9 100
98.9 99.0 100 99.3 100
a
The values denote labeling efficiency (%).
Labeling was finished in 1 min with >98% efficiency at room temperature (Table 1). The volume of 99mTcpertechnetate, either 2 mL or 5 mL, did not affect the labeling efficiency. The preparation of the 99mTc-LSA was stable for at least 24 h when stored at room temperature, and it was also stable in serum at 37 °C for 24 h (Figure 4). Biodistribution Study in the Mice. 99mTc-HSA showed high blood pool activity. High activities in the liver, spleen, and kidney seem to be caused by high blood content in those tissues (Figure 5A). 99mTc-LSA ac-
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little or no metabolism in the liver (Figure 5C). Unlike Tc-HSA, both 99mTc-LSA and 99mTc-tin colloid showed very low blood activity due to rapid uptake by the liver. Imaging Study in Rats. 99mTc-LSA was taken up by the liver quickly at 15 min, and some activity was excreted into the intestine at 30 min (Figure 6A). No spleen uptake was found. 99mTc-Tin colloid accumulated mainly in the liver, although some activity was found in the spleen. The activity did not change significantly with time (Figure 6B). These results were consistent with the biodistribution results in the mice. 99m
DISCUSSION
Figure 5. Biodistribution of 99mTc-HSA (A), 99mTc-LSA (B), and 99mTc-tin colloid (C) in mice (male, ICR).
Figure 6. Imaging after injection of 99mTc-LSA (A) and 99mTctin colloid (B) in rats (male, Sprague-Dawley).
cumulated mainly in the liver and was excreted into the intestine slowly, which is evidence of specific uptake by the asialoglycoprotein receptor and successive metabolism (Figure 5B). 99mTc-Tin colloid, known to be taken up by the reticuloendothelial system, showed high accumulation in the liver, and the radioactivity was not excreted as expected (Figure 5C). It also showed high accumulation in the spleen. The radioactivity in the lung is due to capillary blockage by aggregated 99mTc-tin colloid. Very low radioactivity in the intestine indicates
Previously reported galactosylation method has a problem because it requires long preliminary steps for synthesis of precursor of galactosylation (Kudo et al., 1994). In addition the precursor, 2-imino-2-methoxyethyl1-thio-β-D-galactose, is not suitable for long-term storage due to instability. So the precursor of the precursor, 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactose, is generally synthesized and used for synthesis of 2-imino-2-methoxyethyl-1-thio-β-D-galactose to conjugate to human serum albumin. On the other hand, lactose, the precursor of lactosylation, is commercially available at an economical price. After reductive lactosaminylation, glucose residue of lactose is opened and reduced to a glucitol type linker which is neutral and hydrophilic. Generally, neutral and hydrophilic linkers are ideal for conjugation of haptens to proteins because they may not have much of an effect on the tertiary structure of the conjugated proteins. Major advances in the labeling of proteins with 99mTc have been developed with monoclonal antibodies. Many bifunctional chelating agents for linking 99mTc and antibody have been used. Although DTPA was the first introduced bifunctional chelating agent for labeling antibodies with 99mTc, it was not used widely because of the problems previously discussed in the Introduction section. Another extensively studied bifunctional chelating agent was N3S type compound (Kasina et al., 1998). Although this bifunctional chelating agent showed excellent radiochemical and biological properties, the labeling procedure was complicated because of prelabeling and postconjugating procedure. A preconjugation and postlabeling type bifunctional chelating agent, hydrazinonicotinic acid (HYNIC), has been developed to facilitate labeling (Abrams et al., 1990) and has been adapted by many researchers due to excellent labeling efficiency and simple labeling procedure (Schwartz et al., 1991; Blankenberg et al., 1999). The most up-to-date method of labeling proteins might be using tricarbonyl intermediate complex (Alberto et al., 1998; Egli et al., 1999). This method is currently widely studied by many researchers due to high labeling efficiency and high stability despite its two-step labeling procedure. We applied a direct labeling method to label LSA with 99m Tc, which has been studied for a long time. Direct labeling can be applied only to proteins containing disulfide bonds or sulfhydryl groups. Direct 99mTc-labeling method of HSA using stannous ion was first reported by Eckelman et al. in 1971. It has been found that preliminary reduction of proteins with stannous tartrate allows for labeling small amounts of proteins (Pettit et al., 1980). A specific labeling method to the high affinity sites of antibodies with 99mTc to acquire high stability after labeling has been reported (Paik et al., 1985). A practical method with higher than 90% labeling efficiency which employs preliminary reduction with β-mercaptoethanol
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and using a weak chelator has also been reported (Mather and Ellison, 1990). The role of the weak chelator was the prevention of colloid formation and weak labeling to the antibodies. In this experiment, we used medronate as a weak chelator and found that colloid formation was completely suppressed, and labeled LSA was stable. Labeling efficiency was also very high (>98%) even after 1 min incubation at room temperature. It is a significant improvement compared to a commercialized GSA kit (neogalactosyl-DTPA-human serum albumin) which requires 30 min incubation at 50 °C for labeling. Dosage of LSA was formulated as 3 mg/vial in this research. It has been reported that administration of 3 mg of 99mTc-GSA would be less extensively affected by the physiological asialoglycoprotein (Ha-Kawa et al., 1997). The number of total asialoglycoprotein receptors on the normal liver is 2.5-10 × 1016. About 2.4 × 1016 molecules are in 3 mg of LSA. To evaluate the function of the liver, it is necessary to administer close to the saturation number of ligands. If the number of ligands is low, then the image will be normal even though the number of asialoglycoprotein receptors decreases. Slow increase of radioactivity in the intestines after initial uptake in the liver is due to metabolism in the hepatocytes and successive hepatobiliary excretion. 99mTcTin colloid which is not metabolized showed constant biodistribution as expected. A blood pool imaging agent, 99mTc-HSA, showed high blood activity. Biodistribution and imaging study show well-correlated results in this experiment. CONCLUSION
A new imaging agent for asialoglycoprotein receptors of the liver, 99mTc-LSA, was successfully developed. The new agent showed promising practicability with easy labeling, high labeling efficiency, high stability, and high liver uptake. ACKNOWLEDGMENT
We would like to express our gratitude to Mr. Keagan H. Lee for his assistance in the English language for this manuscript. LITERATURE CITED (1) Abrams, M. J., Juweid, J., tenKate, C. I., Schwartz, D. A., Hauser, M. M., Gaul, F. E., Fuccello, A. J., Rubin, R. H., Strauss, H. W., and Fischman, A. J. (1990) Technetium-99mhuman polyclonal IgG radiolabeled via the hydrazine nicotinamide derivative for imaging focal sites of infection in rats. J. Nucl. Med. 31, 2022-2026. (2) Alberto, R., Schibli, R., Egli, A., and Schubiger, A. P. (1998) A novel organometallic aqua complex of technetium for the labeling of biomolecules: synthesis of [99mTc(OH2)3(CO)3]+ form [99mTcO4]- in aqueous solution and its reaction with a bifunctional ligand. J. Am. Chem. Soc. 120, 7987-7988. (3) Arzoumanian, A., Rosenthall, L., and Seto, H. (1977) Clinical comparison of 99mTc-labeled preformed phytate colloid and sulfur colloid: concise communication. J. Nucl. Med. 18, 118. (4) Ashwell, G., and Morell, A. G. (1974) The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. 41, 99-128. (5) Ashwell, G., and Steer, C. J. (1981) Hepatic recognition and catabolism of serum glycoprotein. J. Am. Med. Assoc. 246, 2358-2364. (6) Blankenberg, F. G., Katsikis, P. D., Tait, J. F., Davis, E., Naumovski, L., Ohtsuki, K., Kopiwoda, S., Abrams, M. J., and Strauss, H. W. (1999) Imaging of apoptosis (programmed cell death) with 99mTc annexin V. J. Nucl. Med. 40, 184-191.
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