Shuji Kojima

Tumor uptake of 67Ga-carrying liposomes

The in vivo distribution, excretion, and tumor localization of liposome-encapsulated 67Ga in normal and Ehrlich tumor (solid form)-bearing mice were studied. In normal mice, multilamellar vesicles (MLVs) were taken up mainly by the liver and spleen, whereas small unilamellar vesicles (SUVs) exhibited a broader tissue distribution. When 67Ga was encapsulated in MLVs or SUVs, the excretion of the radiotracer in the urine and feces was less than that observed for free tracer at 72 h after i.v. administration. In tumor-bearing mice, SUVs were found to accumulate preferentially in tumors. The tumor uptake of neutral, positive, and negative SUVs was 10%–13% of the administered dose per gram of tumor tissue at 24 h after their injection. These values were about three times higher than those found for free 67Ga-nitrilotriacetic acid (67Ga-NTA) or 67Ga-citrate. Significant differences in tumor uptake due to different surface charges of liposomes were not observed. Enhanced tumor-to-blood and tumor-to-muscle ratios were also observed at 24 h after injection. These results suggest that 67Ga-carrying liposomes may be a useful for tumor imaging.

Elevated uptake of 67Ga and increased heparan sulfate content in liver-damaged rats

Values of percent 67Ga binding with standard acid mucopolysaccharides (AMPS) were determined. The binding percent of 67Ga with heparan sulfate (HS) was especially high. The relation between 67Ga and HS content was investigated in liver-damaged rats. Elevated uptake of 67Ga occurred in two stages, corresponding to the early and the later stages during feeding with 2-acetylaminofluorene (AAF). Uptake of 67Ga in the liver was first observed at 5–6 weeks after the start of AAF feeding. There was a decrease at about week 7 almost to the control level, followed by a sustained increase. This changing pattern of 67Ga uptake was in good accord with that of liver HS content. A similar relation between 67Ga uptake and HS content was also observed in rat liver treated with a single dose of carbon tetrachloride (CCl4). The elevated 67Ga uptake and increased HS content induced by CCl4 were both significantly inhibited by aminoacetonitrile (AAN) or papain pretreatment.Moreover, we screened cations having a high affinity with HS from the exchangeability of 45Ca with those in 45Ca-HS complex. Fe3+ and other trivalent cations such as Al3+, La3+, Ce3+, Er3+, Sm3+, Yb3+, In3+, Ru3+, and Eu3+, and Le2+ represented strong affinity to HS. These cations also effectively decreased the 67Ga tissue distribution in normal rats. These results suggest that HS may be involved in the mechanism of 67Ga uptake in tumor and inflammatory tissues.

Cholesterol enhances the delivery of liposome-encapsulated gallium-67 to tumors

The effect of incorporation of cholesterol (CH) into liposome membranes on the delivery of 67Ga encapsulated in liposomes to tumors was studied. The changes of the blood clearance of liposomes, liposome, stability in serum and liposome uptake by the liver and spleen were also examined. Liposomes were prepared from distearoylphosphatidylcholine with various amounts of CH. It became clear that large amounts of CH (above 33 mol%) dramatically enhanced liposomal delivery of 67Ga to sarcoma 180 solid tumor in mice. Large amounts of CH increased the liposome stability in serum and decreased liposome uptake by the liver and spleen after intravenous injection, thus prolonging the blood clearance of liposomes. These observations suggested that the large delivery of 67Ga by CH-rich liposomes resulted from the extended retention and increased amount of liposomes in the circulation, caused by the incorporation of large amounts of CH. Small amounts of CH decreased liposome stability and hastened blood clearance, but had little effect on 67Ga delivery to the tumor. CH-rich liposomes showed high tumor uptake, with high tumor to blood and tumor to tissue ratios, of 67Ga. It is anticipated that 67Ga-carrying liposomes will be an excellent tumor imaging agent for clinical use, provided that a correct choice of CH content is made.

Biodistribution of neoglycoproteins in mice bearing solid Ehrlich tumor.

SummarySynthetic neoglycoproteins were employed for the specific detection of their corresponding cellular sugar receptors, such as endogenous lectins, by specific protein-carbohydrate interaction. A panel of 16 radioiodinated probes with defined carbohydrate content, attached to the carrier protein bovine serum albumin, disclosed marked differences in the expression of corresponding sugar receptors on Ehrlich ascites tumor cells in vitro as an exemplary tumor model. To quantify tumor uptake in the more complex in vivo situation and to assess binding to individual organs attributable to the various types of carbohydrates we determined the biodistribution of the radiolabelled neoglycoproteins 48 h after injection into tumor-bearing mice. The individual pattern of retention of radioactivity demonstrated distinct properties of the different organs that need to be accounted for in drug-targeting and tumor-imaging studies, based on protein-carbohydrate interactions. Combined tumor-to-blood and tumor-to-muscle ratios of neoglycoprotein accumulation were well above 1 after covalent attachment of xylose or glucuronic acid, respectively, to the carrier protein. These data constitute the basis for further refinements of the carbohydrate part of suitable neoglycoproteins to allow a potentially rational application of neoglycoproteins as drug-targeting vehicles and tumor-imaging radiopharmaceuticals.

67Ga accumulation and heparan sulfate metabolism in lysosomes

Abstract67Ga incorporation in the mitochondrial-lysosomal (Mt-Ly) fraction of rat liver increased sharply from 1 to 24 h after 67Ga IV injection, whereas that in the 105,000 g supernatant fraction decreased sharply from 6 to 48 h. Further, when 14C-labeled heparan sulfate (HS: biosynthesized by injection of [1-14C] glucosamine into a rat) was injected intravenously into a rat treated with Triton WR-1339, and the liver was isolated and fractionated, the radioactivity of 14C was concentrated in the lysosomal fraction.The effect of HS on 67Ga uptake in normal rat liver was also studied. On administration of 67Ga-HS complex, the peak uptake of administered radioactivity of 67Ga in rat liver occurred sooner than after the injection of 67Ga alone. The change was greatest in the Mt-Ly fraction among the subcellular fractions. Moreover, the 67Ga uptake in rat liver increased with increasing doses of HS.These results suggest that administered 67Ga may be bound to HS and that the concentration of 67Ga in lysosomes may be related to the in vivo metabolism of HS.

Uptake of67Ga in the heart of rats treated with isoproterenol

Gallium-67 citrate (67Ga) accumulation and various enzyme activities during the repair of rat heart with infarct-like lesions induced by isoproterenol (ISP) treatment were measured for 10 days after treatment. Serum creatine phosphokinase (CPK) and glutamic oxalacetic transaminase (GOT) activities were increased immediately after ISP treatment, reaching maximum levels of activity of 545±64 U/ml and 542±94 KU/ml, respectively, within 12 h. Uptake of67Ga in the rat heart was elevated 12 h after ISP treatment, reaching a maximum on day 1 (0.473±0.015% dose/g heart). This pattern was essentially similar to the pattern of uronic acid content in the 1.2 M NaCl fraction, which contained mainly heparan sulfate (HS). The activity of glucose-6-phosphate dehydrogenase (G-6-PDH), a marker enzyme for fibrogenesis of damaged tissues, was also elevated 12 h after the ISP treatment, reaching a maximum of approximately 2.47 times that of the control heart on day 1. On the other hand, there were no significant changes in the67Ga uptake and uronic acid content in any of the fractions of the liver and kidneys. These findings suggested that HS might be an acceptor for67Ga accumulation during the repair of rat heart with infarct-like lesions, in accord with our previous results on CCl4-damaged rat liver.

Application of lectins to tumor imaging radiopharmaceuticals

We investigated the in vitro binding of 125I-lectins to Ehrlich ascites tumor (EAT) cells and in vivo uptake of 125I-lectins in Ehrlich solid tumor (EST) bearing mice. In in vitro binding assays, phaseolus vulgaris agglutinin (PHA), pisum sativum agglutinin (PSA), and concanavalia agglutinin (Con A) showed a high affinity for EAT cells. The in vivo biodistribution of 125I-lectins showed 125I-PSA to be significantly taken up into EST tissues 24 h postinjection. After IV injection of 125I-PSA, uptake of the radioactivity into the tumor tissues reached a maximum at 6 h, and thereafter decreased. Rapid clearance of the radioactivity from blood and its exretion into kidney soon after injection of 125I-PSA were observed. When compared with the biodistribution of 67Ga-citrate in EST bearing mice 24 h postinjection, tumor to liver (T/B), tumor to muscle (T/M), and tumor to blood (T/B) ratios were superior for 125I-PSA. At 6 h postinjection, the T/B-ratio of 125I-PSA was 2.5, and this value may be sufficient to enable discernable diagnostic images. Our results suggest that PSA might be a useful tumor imaging radiopharmaceutical.

Comparisons of labeling efficiency, biological activity and biodistribution among 125I-, 67Ga-DTPA- and 67Ga-DFO-lectins

The labeling efficiency, biological activity and biodistribution of 125I labeled and 67Ga chelating agent conjugated lectins were investigated. Pisum sativum agglutinin (PSA) and Lens culinaris agglutinin (LCH) were efficiently labeled with 67Ga using bifunctional chelating agents such as diethylenetriaminepentaacetic acid (DTPA) and deferoxamine (DFO), whereas labeling with 125I was significantly less efficient. The agglutinating activity of these lectins towards Ehrlich ascites tumor (EAT) cells was retained on conjugation with DFO, but not with DTPA. The in vitro binding ratio of 67Ga-DFO-lectins for EAT cells was almost the same as that of 125I-lectins. However, the value was significantly decreased in the case of 67Ga-DTPA-lectins. In the biodistribution study of radiolabeled lectins in Ehrlich solid tumor (EST) bearing mice, the accumulation of radioactivity in tumor tissue was very much less with 67Ga-DTPA-lectins than with 125I-lectins. However, the concentration was significantly elevated in the case of 67Ga-DFO-lectins. While, these lectins accumulated in liver, spleen, lung, and kidney to a greater extent than 67Ga citrate, the tumor to organ ratios became very low. These low tumor to organ ratios, in contrast to 67Ga citrate, will certainly inhibit the tumor delineation, and therefore it seems that in spite of a high accumulation ratio of 67Ga-DFO-lectins in tumor tissue, these agents are not useful in tumor detection.

Enhancement of clearance of plant lectins as radiopharmaceuticals by chemically glycosylated antilectin antibody

The influence of chemical glycosylation on clearance of plant lectins, employed as radiopharmaceuticals, was assessed. Controlled chemical modification of the antibody to concanavalia ensiformis agglutinin did not affect its immunologic activity, but led to rapid clearance from circulation. In tumor-bearing mice, notable increases of selected tumor/non tumor ratios even after only 1 h of presence of the galactosylated antibody to the lectin in circulation were observed. Similarly, galactosylated antibody to pisum sativum agglutinin caused significant increases of tumor/non tumor ratios.

Glucose-6-phosphate dehydrogenase and γ-glutamyltranspeptidase activities in the liver during chemically induced hepatocarcinogenesis in rats and mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) and γ-glutamyltranspeptidase (γ-GTP) were studied in the liver of rats and mice receiving a diet which contained 3′-methyl-4-(dimethylamino)azobenzene (3′-Me-DAB), diethylnitrosamine (DEN), or o-aminoazotoluene (o-AT). Elevated G-6-PDH activity was first observed within 2 weeks after the start of 3′-Me-DAB feeding, reaching a maximum at 4 weeks. There was a decrease from the 4-week level at about 6 weeks, followed by a sustained increase. The maintained activity after 8 weeks was about 6 times higher than in control rat liver. γ-GTP activity in rat liver increased immediately after the start of 3′-Me-DAB feeding; it was about 12 times the control value after 3–4 weeks. At 5–6 weeks, the activity decreased somewhat from the 3 to 4-week level, but this was followed by a further sustained increase. In rats receiving the DEN-containing water, the liver G-6-PDH and γ-GTP activities changed in the same way as in the 3′-Me-DAB-fed rats. Both activities increased in the early stage, reaching a maximum by 6 weeks, subsided at 7 weeks, and rose again from 9 weeks. In mice receiving o-AT-containing diet, hepatic G-6-PDH activity also changed in a biphasic pattern and closely related with that of liver γ-GTP activity.