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This study conducted fundamental research to develop a more effective BNCT targeting cancer stem cells. We constructed plasmids that induced the overexpression of L-type amino acid transporter 1 (LAT1) tagged with tdTomato on the cytoplasmic membranes of CD133 expressing cancer cells. After transfection of the plasmids into a glioblastoma cell line (T98G), several clones overexpressing LAT1-tdTomato in the hypoxic microenvironment of the spheroids formed from each clone were obtained. Confocal laser microscopic observation confirmed that signals from LAT1-tdTomato overlapped with immunofluorescence signals from the second antibody binding to CD133 in the hypoxic microenvironment of the spheroids. As CD133-positive cells in the hypoxic microenvironment of T98G spheroids have cancer stem cell characteristics, LAT1 seems to be selectively overexpressed in cancer stem cell-like cells. An RI tracer method showed that cells overexpressing LAT1-tdTomato in the hypoxic microenvironment of spheroids incorporate 14C-BPA much more than cells that do not overexpress LAT1-tdTomato. Neutron radiation experiments showed a more significant regression in spheroids formed with clones than in spheroids formed with parental cells when spheroids were treated with 10BPA. These results suggest that BNCT combined with gene therapy targeting cancer stem cells is more effective in glioblastoma therapy.

Drug metabolizing enzyme activity is affected by various factors such as drug-drug interactions, and a method to quantify drug metabolizing enzyme activity in real time is needed. In this study, we developed a novel radiopharmaceutical for quantitative imaging to estimate hepatic CYP3A4 and CYP2D6 activity. Iodine-123- and 125-labeled

Introduction We clarified the renal uptake and urinary secretion mechanism of [99mTc]dimercaptosuccinic acid ([99mTc]DMSA) via drug transporters in renal proximal tubules. Methods [99mTc]DMSA was added to human embryonic kidney 293 cells expressing human multidrug and toxin extrusion (MATE)1 and MATE2-K, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cation transporter (OCT)2; to Flp293 cells expressing human organic anion transporter (OAT)1 and OAT3; and to vesicles expressing P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)2, MRP4, or breast cancer resistance protein with and without probenecid (OAT inhibitor for both OATs and MRPs). Time activity curves of [99mTc]DMSA with and without probenecid were established using LLC-PK1 cells. Biodistribution and single photon emission computed tomography (SPECT) imaging in mice were conducted using [99mTc]DMSA with and without probenecid. Results [99mTc]DMSA uptake was significantly higher in Flp293/OAT3 than in mock cells. Uptake via OAT3 was inhibited by probenecid. [99mTc]DMSA uptake into vesicles that highly expressed MRP2 was significantly higher in adenosine triphosphate (ATP) than in adenosine monophosphate (AMP), and probenecid decreased uptake to similar levels as that in AMP. In the time activity curves for [99mTc]DMSA in LLC-PK1 cells, probenecid loading inhibited accumulation from the basolateral side into LLC-PK1 cells, whereas accumulation from the apical side into cells gradually increased. Transport of [99mTc]DMSA from both sides was low. Biodistribution and SPECT imaging studies showed that [99mTc]DMSA with probenecid loading resulted in significantly higher accumulation in blood, heart, liver, and bladder after [99mTc]DMSA injection compared with control mice. Probenecid induced significantly lower accumulation in the kidney after [99mTc]DMSA injection. Conclusions [99mTc]DMSA accumulates in renal proximal tubular epithelial cells from blood via OAT3 on the basolateral side, and then a small volume of [99mTc]DMSA will be excreted in urine via MRP2. Advances in knowledge [99mTc]DMSA accumulates via OAT3 in renal proximal tubular epithelial cells and is slightly excreted from the cells via MRP2. Implications for patient care [99mTc]DMSA may be useful for measuring renal transport function with OAT3 in patients.

The World Health Organization has cautioned that antimicrobial resistance (AMR) will be responsible for an estimated 10 million deaths annually by 2050. To facilitate prompt and accurate diagnosis and treatment of infectious disease, we investigated the potential of amino acids for use as indicators of bacterial growth activity by clarifying which amino acids are taken up by bacteria during the various growth phases. In addition, we examined the amino acid transport mechanisms that are employed by bacteria based on the accumulation of labeled amino acids, Na+ dependence, and inhibitory effects using a specific inhibitor of system A. We found that 3H-L-Ala accurately reflects the proliferative activity of Escherichia coli K-12 and pathogenic EC-14 in vitro. This accumulation in E. coli could be attributed to the amino acid transport systems being different from those found in human tumor cells. Moreover, biological distribution assessed in infection model mice with EC-14 using 3H-L-Ala showed that the ratio of 3H-L-Ala accumulated in infected muscle to that in control muscle was 1.20. By detecting the growth activity of bacteria in the body that occurs during the early stages of infection by nuclear imaging, such detection methods may result in expeditious diagnostic treatments for infectious diseases.

NTRODUCTION: (131)I-labeled m-iodobenzylguanidine ([(131)I]MIBG) has been used to treat neuroblastoma patients, but [(131)I]MIBG may be immediately excreted from the cancer cells by the adenosine triphosphate binding cassette transporters, similar to anticancer drugs. The purpose of this study was to clarify the efflux mechanism of [(131)I]MIBG in neuroblastomas and improve accumulation by inhibition of the transporter in neuroblastomas. METHODS: [(131)I]MIBG was incubated in human embryonic kidney (HEK)293 cells expressing human organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporter (OAT)1 and OAT2, organic cation transporter (OCT)1 and OCT2, and sodium taurocholate cotransporting polypeptide, and in vesicles expressing P-glycoprotein (MDR1), multidrug resistance associated protein (MRP)1-4, or breast cancer resistance protein with and without MK-571 and probenecid (MRP inhibitors). Time activity curves of [(131)I]MIBG with and without MK-571 and probenecid were established using an SK-N-SH neuroblastoma cell line, and transporter expression of multiple drug resistance was measured. Biodistribution and SPECT imaging examinations were conducted using [(123)I]MIBG with and without probenecid in SK-N-SH-bearing mice. RESULTS: [(131)I]MIBG uptake was significantly higher in OAT1, OAT2, OCT1, and OCT2 than in mock cells. Uptake via OCT1 and OCT2 was little inhibited by MK-571 and probenecid. [(131)I]MIBG uptake into vesicles that highly expressed MRP1 or MRP4 was significantly higher in ATP than in AMP, and these inhibitors restored uptake to levels similar to that in AMP. Examining the time activity curves for [(131)I]MIBG in SK-N-SH cells, higher expressions of MDR1, MRP1, MRP4, and MK-571, or probenecid loading produced significantly higher uptake than in control at most incubation times. The ratios of tumors to blood or muscle in SK-N-SH-bearing mice were significantly increased by probenecid loading in comparison with normal mice. CONCLUSIONS: [(131)I]MIBG exports via MRP1 and MRP4 in neuroblastoma. The accumulation and tumor-to-blood or muscle ratios of [(131)I]MIBG are improved by inhibition of MRPs with probenecid in neuroblastoma. ADVANCES IN KNOWLEDGE: [(131)I]MIBG, widely used for treatment of neuroendocrine tumors including neuroblastoma, is excreted via MRP1 and MRP4 in neuroblastoma. IMPLICATIONS FOR PATIENT CARE: Loading with probenecid, OAT, and MRP inhibitors improves [(131)I]MIBG accumulation.

Technetium-99m-labeled mercaptoacetyltriglycine ([99mTc]MAG3) is widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function. [99mTc]MAG3 is a substrate for organic anion transporter (OAT)1 and OAT3 on the basolateral membrane side for renal secretion. We investigated the transport mechanism and affinity of [99mTc]MAG3 on the apical membrane of renal proximal tubule cells for renal secretion. Adenosine triphosphate-binding cassette (ABC) transporters for renal secretion of [99mTc]MAG3 were examined using ABC transporter vesicles expressing multiple drug resistance 1 (MDR1), breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP)2, and MRP4. MK-571, a MRP inhibitor, was applied to measure the Km and Vmax of MRP2 and MRP4 in a vesicle transport assay. Single photon emission computed tomography (SPECT) was performed in normal rats and MRP2-deficient Eisai hyperbilirubinuria rats (EHBR) using [99mTc]MAG3 with and without MK-571. [99mTc]MAG3 uptake in adenosine triphosphate was significantly higher than that in adenosine monophosphate in vesicles that highly expressed MRP2 and MRP4. The affinity of [99mTc]MAG3 for MRP4 was higher than that for MRP2. Renal secretion via MRP2 and MRP4 was identified by comparing normal and EHBR rats with and without MK-571 on SPECT. [99mTc]MAG3 is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2. SIGNIFICANCE STATEMENT: [99mTc]MAG3, widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function, is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2.

The effectiveness of L- and D-amino acids for detecting the early stage of infection in bacterial imaging was compared. We evaluated the accumulation of 3H-L-methionine (Met), 3H-D-Met, 3H-L-alanine (Ala), and 3H-D-Ala in E. coli EC-14 and HaCaT cells. Biological distribution was assessed in control and lung-infection-model mice with EC-14 using 3H-L- and D-Met, and 18F-FDG. A maximum accumulation of 3H-L- and D-Met, and 3H-L- and D-Ala occurred in the growth phase of EC-14 in vitro. The accumulation of 3H-L-Met and L-Ala was greater than that of 3H-D-Met and D-Ala in both EC-14 and HaCaT cells. For all radiotracers, the accumulation was greater in EC-14 than in HaCaT cells at early time points. The accumulation was identified at 5 min after injection in EC-14, whereas the accumulation gradually increased in HaCaT cells over time. There was little difference in biodistribution between 3H-L-and D-Met except in the brain. 3H-L- and D-Met were sensitive for detecting areas of infection after the spread of bacteria throughout the body, whereas 18F-FDG mainly detected primary infection areas. Therefore, 11C-L- and D-Met, radioisotopes that differ only in terms of 3H labeling, could be superior to 18F-FDG for detecting bacterial infection in lung-infection-model mice.

We clarified the renal uptake and urinary secretion mechanism of [[[[

OBJECTIVES Drugs are mainly metabolized by hepatic enzymes, the activity of which can differ between individuals. Although it is ideal to measure the hepatic clearance of liver-targeted drugs in individualized medicine, blood enzyme tests typically measure metabolic drug clearance in the entire body, and not just in the liver. We investigated whether I-iomazenil imaging can directly assess and quantify the activity of hepatic drug-metabolizing enzymes. MATERIALS AND METHODS Hepatic enzymes that metabolize I-iomazenil were identified by thin-layer chromatography in mouse liver homogenates with bis(4-nitrophenyl) phosphate (BNPP) inhibitor for carboxylesterase enzymes and nicotinamide adenine dinucleotide phosphate (NADPH) generator for cytochrome P450 enzymes. Whole-body images of mice were acquired using I-iomazenil with and without BNPP, and the distribution was also obtained. The metabolism of I-iomazenil in the blood, liver, gall bladder, and bladder was investigated by thin-layer chromatography. RESULTS From the in-vitro metabolism of I-iomazenil using BNPP, the enzyme converting I-iomazenil to I-R-COOH was identified as carboxylesterase, and that converting I-iomazenil to M2 was identified as cytochrome P450 in experiments with and without an NADPH generator. The biological distribution and whole-body imaging showed increased accumulation in the liver of mice administered BNPP compared with normal mice, but decreased levels in the gall bladder and small intestine. The main fraction in bile and urine was I-R-COOH, with two unknown metabolites (M1 and M2), I, and I-iomazenil also being present. CONCLUSION I-iomazenil whole-body imaging has good possibility of direct measurement of hepatic carboxylesterase activity as accumulation of I-R-COOH in the gall bladder through bile and in the bladder through urine.

In boron neutron capture therapy (BNCT), 10 B-4-borono-L-phenylalanine (BPA) is commonly used as a 10 B carrier. PET using 4-borono-2-18 F-fluoro-phenylalanine (18 F-FBPA PET) has been performed to estimate boron concentration and predict the therapeutic effects of BNCT; however, the association between tumor uptake of 18 F-FBPA and boron concentration in tumors remains unclear. The present study investigated the transport mechanism of 18 F-FBPA and BPA, and evaluated the utility of 18 F-FBPA PET in predicting boron concentration in tumors. The transporter assay revealed that 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid, an inhibitor of the L-type amino acid transporter, significantly inhibited 18 F-FBPA and 14 C-4-borono-L-phenylalanine (14 C-BPA) uptake in FaDu and LN-229 human cancer cells. 18 F-FBPA uptake strongly correlated with 14 C-BPA uptake in 7 human tumor cell lines (r = .93; P < .01). PET experiments demonstrated that tumor uptake of 18 F-FBPA was independent of the administration method, and uptake of 18 F-FBPA by bolus injection correlated well with BPA uptake by continuous intravenous infusion. The results of this study revealed that evaluating tumor uptake of 18 F-FBPA by PET was useful for estimating 10 B concentration in tumors.

Introduction Due to its poor prognosis, specific imaging for early detection of malignant melanoma is strongly desired. Although radioiodine-labeled 4-hydroxyphenylcysteamine, which we previously developed, has good affinity for tyrosinase, an enzyme in the melanin metabolic pathway, image contrast of the melanoma:organ ratios is not sufficiently high for detection of primary melanoma and metastases at early injection times. In this study, we developed radioiodine-labeled acetaminophen (I-AP) for specific, high-contrast imaging of malignant melanoma. Methods Radioiodine-125-labeled AP ( 125 I–AP) was prepared using the chloramine-T method under no carrier-added conditions. Accumulation of radioactivity and the mechanism were evaluated in vitro using B16 melanoma cells incubated with 125 I–AP or 14 C(U)-labeled AP ( 14 C–AP) with and without l-tyrosine as a substrate of tyrosinase, phenylthiourea as an inhibitor of tyrosinase, and thymidine as an inhibitor of DNA polymerase. The biological distribution of radioactivity in B16 melanoma-bearing mice was evaluated to determine the accumulation of 125 I–AP. The stability of 125 I–AP over time was evaluated in mice. Results The labeling efficiency and radiochemical purity of 125 I–AP were >80% and 95%, respectively. Accumulation of 125 I–AP was higher than that of 14 C–AP at 60min of incubation in vitro. The affinity of 14 C–AP for tyrosinase and DNA polymerase was higher than that of 125 I–AP, whereas the V max of 125 I–AP was higher than that of 14 C–AP. 125 I–AP showed the highest accumulation in the gall bladder, and clearance from the blood and kidney was rapid. Melanoma:muscle and melanoma:normal skin ratios of 125 I–AP for imaging contrast were the highest at 15min after injection, whereas the melanoma:blood and melanoma:bone ratios gradually increased over time. 125 I–AP remained stable for 60min after injection in mice. Conclusions 125 I–AP has affinity for tyrosinase and high image contrast at early time points after injection. Therefore, 123 I–AP imaging has great potential for specific, high-contrast detection of malignant melanoma. Advances in Knowledge 123 I–AP will provide specific, high-contrast imaging for malignant melanoma at early injection times. Implications for patient care 123 I–AP has good potential for the diagnosis of malignant melanoma compared with 123 I–labeled 4-hydroxyphenylcysteamine, which we previously developed.

Introduction To clarify the difference between system A and L amino acid transport imaging in PET clinical imaging, we focused on the use of α-[ N -methyl- 11 C ]-methylaminoisobutyric acid ([ 11 C ]MeAIB), and compared it with [ S -methyl- 11 C ]-L-methionine ([ 11 C ]MET). The aim of this study was to assess the correlation of accumulation of these two radioactive amino acid analogs with expression of amino acid transporters and cell proliferative activity in carcinoma cells. Methods Amino acid uptake inhibitor studies were performed in four human carcinoma cells (epidermal carcinoma A431, colorectal carcinoma LS180, and lung carcinomas PC14/GL and H441/GL) using the radioisotope analogs [ 3 H]MET and [ 14 C]MeAIB. MeAIB was used to inhibit the A system and 2-amino-2-norbornane-carboxylic acid (BCH) was used to inhibit the L system. The carcinoma gene expression levels of a number of amino acid transporters were measured by microarray and quantitative polymerase chain reaction. Carcinoma proliferative activity was assessed using accumulation of [methyl- 3 H]-3'-deoxy-3'-fluorothymidine ([ 3 H]FLT). Results and conclusion [ 14 C]MeAIB uptake occurred principally via a Na + -dependent A type mechanism whereas [ 3 H]MET uptake occurred predominantly via a Na + -independent L type mechanism although other transporters were also utilized depending on cell type. There was no correlation between [ 3 H]MET uptake and total system L amino acid transporter (LAT) expression. In contrast, [ 14 C]MeAIB uptake strongly correlated with total system A amino acid transporter (SNAT) expression and proliferative activity in this preliminary study using four human carcinoma cell lines. Carcinoma proliferative activity also correlated with total SNAT expression. Advances in Knowledge and Implications for Patient Care: Because there is a significant correlation between the accumulation of [ 14 C]MeAIB and the gene expression level of total SNAT as well as the accumulation of [ 3 H]FLT, it is suggested that use of the analog [ 11 C]MeAIB in PET may provide an indication of tumor cell proliferative activity. [ 11 C]MeAIB is therefore expected to be very useful in PET imaging.

SNMMI2018

Introduction A specific diagnosis for melanoma is strongly desired because malignant melanoma has poor prognosis. In a previous study, although radioiodine-125-labeled 4-hydroxyphenyl-L-cysteine ( 125 I-L-PC) was found to have good substrate affinity for tyrosinase enzyme in the melanin metabolic pathway, 123/131 I-L-PC had insufficient substrate affinity for tyrosinase to diagnose melanoma. In this study, we synthesized 4-hydroxyphenylcysteamine (4-PCA) and developed a novel radioiodine-125-labeled 4-hydroxyphenylcysteamine ( 125 I-PCA) to increase affinity for the melanin biosynthesis pathway. Methods 4-PCA was separated with 2-hydroxyphenylcysteamine (2-PCA), which is an isomer of 4-PCA, and was examined using melting point, proton nuclear magnetic resonance, mass spectrometry and elemental analysis. 125 I-PCA was prepared using the chloramine-T method under no-carrier added conditions. We performed biodistribution experiments using B16 melanoma-bearing mice using 125 I-PCA, 125 I-L-PC, 125 I-α-methyl-L-tyrosine, 123 I- m -iodobenzylguanidine and 67 Ga-citrate. In vitro assay was performed with B16 melanoma cells, and affinity for tyrosinase, DNA polymerase and amino acid transport was evaluated using phenylthiourea, thymidine, ouabine and L-tyrosine inhibitor. In addition, partition coefficients of 125 I-PCA were evaluated. Results In the synthesis of 4-PCA, analysis values did not differ between calculated and reported values, and 4-PCA was separated from 2-PCA at high purity. In biodistribution experiments, 125 I-PCA was accumulated and retained in B16 melanoma cells when compared with 125 I-L-PC. 125 I-PCA showed the highest values at 60min after radiotracer injection in melanoma-to-muscle ratios, melanoma-to-blood ratios and melanoma-to-skin ratios. Accumulation of 125 I-PCA was significantly inhibited by phenylthiourea and thymidine. Partition coefficients of 125 I-PCA were lower than those of N -isopropyl- p -[ 123 I]iodoamphetamine and were not significantly different from 125 I-L-PC. Conclusions 125 I-PCA is a better substrate for tyrosinase and DNA polymerase and has higher uptake and longer retention in B16 melanoma cells when compared with 125 I-L-PC. Therefore, 123/131 I-PCA has good potential for diagnosis for malignant melanoma. Advance in Knowledge 125 I-PCA will be a specific diagnosis tool for malignant melanoma. Implications for Patient Care 123/131 I-PCA has good potential for the diagnosis of malignant melanoma when compared with other SPECT tracers, as well as anti-melanoma chemotherapeutic drugs.

In boron neutron capture therapy (BNCT), 10B‐4‐borono‐L‐phenylalanine (BPA) is commonly used as a 10B carrier. PET using 4‐borono‐2‐18F‐fluoro‐phenylalanine (18F‐FBPA PET) has been performed to estimate boron concentration and predict the therapeutic effects of BNCT; however, the association between tumor uptake of 18F‐FBPA and boron concentration in tumors remains unclear. The present study investigated the transport mechanism of 18F‐FBPA and BPA, and evaluated the utility of 18F‐FBPA PET in predicting boron concentration in tumors. The transporter assay revealed that 2‐aminobicyclo‐(2.2.1)‐heptane‐2‐carboxylic acid, an inhibitor of the L‐type amino acid transporter, significantly inhibited 18F‐FBPA and 14C‐4‐borono‐L‐phenylalanine (14C‐BPA) uptake in FaDu and LN‐229 human cancer cells. 18F‐FBPA uptake strongly correlated with 14C‐BPA uptake in 7 human tumor cell lines (r = .93; P < .01). PET experiments demonstrated that tumor uptake of 18F‐FBPA was independent of the administration method, and uptake of 18F‐FBPA by bolus injection correlated well with BPA uptake by continuous intravenous infusion. The results of this study revealed that evaluating tumor uptake of 18F‐FBPA by PET was useful for estimating 10B concentration in tumors.

To clarify the difference between system A and L amino acid transport imaging in PET clinical imaging, we focused on the use of α-[N-methyl-Amino acid uptake inhibitor studies were performed in four human carcinoma cells (epidermal carcinoma A431, colorectal carcinoma LS180, and lung carcinomas PC14/GL and H441/GL) using the radioisotope analogs [[

Introduction Although [S-methyl-11C]-labeled L-methionine and D-methionine ( 11 C-L-MET and 11 C-D-MET) are useful radiotracers for positron emission tomography imaging of brain tumors, it is not known whether the accumulation and transport mechanisms underlying uptake of 11 C-D-MET and 11 C-L-MET are the same. 11 C-L-MET is mainly taken up by the amino acid transport system L. We evaluated accumulation and the transport mechanism of D-MET in high- and low-grade human glioma cells in vitro. Methods The expression of transport system genes in high- (A172 and T98G) and low-grade (SW1088 and Hs683) glioma cells was quantitatively analyzed. Accumulation of [S-methyl- 3 H]-L-MET ( 3 H-L-MET) and [S-methyl- 3 H]-D-MET ( 3 H-D-MET) in these cells was compared during 60min of incubation. The transport mechanism of 3 H-L-MET and 3 H-D-MET was investigated by incubating the cells with these compounds and examining the effect of the inhibitors 2-amino-2-norbornane-carboxylic acid or α-(methylamino) isobutyric acid. Results Absolute expression levels of system L and system alanine–serine–cysteine (ASC) in high-grade glioma cells were higher than in low-grade cells. In high-grade glioma cells, expression of system ASC genes was higher than that of system L genes. 3 H-D-MET, which is transported by systems L and ASC, accumulated at higher levels than 3 H-L-MET at all incubation times because 3 H-D-MET is more sensitive to system ASC than 3 H-L-MET. Conversely, in low-grade glioma cells with lower expression of system L and ASC, 3 H-D-MET accumulated at higher levels than 3 H-L-MET in early incubation times because 3 H-D-MET may be more sensitive to system ASC than system L. Conclusion 3 H-D-MET was mainly transported by systems L and ASC and sensitive to system ASC, whereas 3 H-L-MET was transported by system L in human glioma cells. In vitro, the accumulation of 3 H-D-MET was significantly higher than that of 3 H-L-MET during the entire incubation time in high-grade glioma cells, and in early incubation times in low-grade glioma cells.

Introduction S -methyl- 11 C-labeled l- and d-methionine ( 11 C-l- and d-MET) are useful as radiotracers for tumor imaging. However, it is not known whether the transport mechanism of 11 C-d-MET is the same as that for 11 C-l-MET, which is transported by the amino acid transport system L. In this study, we investigated the transport mechanism of 11 C-l- and d-MET by analyzing the expression of transport system genes in human-derived tumor cells. Methods The expression of transport system genes in human-derived tumor cells was quantitatively analyzed. The mechanism of MET transport in these cells was investigated by incubating the cells with [ S -methyl- 3 H]-l-MET ( 3 H-l-MET) or [ S -methyl- 3 H]-d-MET ( 3 H-d-MET) and the effect of 2-amino-2- norbornane-carboxylic acid, a system L transport inhibitor, or α-(methylamino)isobutyric acid, a system A transport inhibitor, on their transport was measured. The transport and metabolic stability of [ S -methyl- 14 C]-l-MET ( 14 C-l-MET) and 3 H-d-MET was also analyzed using bearing mice with H441 or PC14 tumor cells. Results 3 H-d-MET was mainly transported by both systems L and alanine–serine–cysteine (ASC), while system L was involved in 3 H-l-MET transport. There was a high correlation between both 3 H-l-MET and 3 H-d-MET uptake and the expression of amino acid transport system genes. In the in vivo study, H441-cell accumulation of 3 H-d-MET was higher than that of 14 C-l-MET. Hepatic and renal accumulation of 3 H-d-MET was lower than that of 14 C-l-MET. Conclusion The transport mechanism of 3 H-d-MET was different from that of 3 H-l-MET. Since 3 H-d-MET has high metabolic stability, its accumulation reflects the transporter function of system L and ASC.

Trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) is an amino acid PET tracer that has shown promise for visualizing prostate cancer. Therefore, we aimed to clarify the anti-(18)F-FACBC transport mechanism in prostate cancer cells. We also studied the fate of anti-(18)F-FACBC after it is transported into cells.For convenience, because of their longer half-lives, (14)C compounds were used instead of (18)F-labeled tracers. Trans-1-amino-3-fluoro-1-(14)C-cyclobutanecarboxylic acid ((14)C-FACBC) uptake was examined in human prostate cancer DU145 cells with the following substrates of amino acid transporters: α-(methylamino) isobutyric acid (a system A-specific substrate) and 2-amino-2-norbornanecarboxylic acid (a system L-specific substrate). The messenger RNA expression of amino acid transporters in human prostate cancer specimens was analyzed by complementary DNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression in DU145 cells was analyzed by qRT-PCR. We also examined the knockdown effect of the amino acid transporters system ASC transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter 2 (SNAT2) on (14)C-FACBC uptake. In addition, the possibility of (14)C-FACBC incorporation into proteins was examined.(14)C-FACBC uptake by DU145 cells was markedly decreased to approximately 20% in the absence of Na(+), compared with that in its presence, indicating that Na(+)-dependent transporters are mainly responsible for the uptake of this tracer. Moreover, 2-amino-2-norbornanecarboxylic acid inhibited the transport of (14)C-FACBC to the basal level in Na(+)-free buffer. In contrast, α-(methylamino) isobutyric acid did not inhibit (14)C-FACBC accumulation in DU145 cells. Human prostate tumor specimens and DU145 cells had similar messenger RNA expression patterns of amino acid transporter genes. Although SNAT2 and ASCT2 are 2 major amino acid transporters expressed in prostate tumor tissues and DU145 cells, ASCT2 knockdown using small interfering RNA was more effective in lowering (14)C-FACBC transport than SNAT2. Almost all intracellular (14)C-FACBC was recovered from the nonprotein fraction.ASCT2, which is a Na(+)-dependent amino acid transporter, and to a lesser extent Na(+)-independent transporters play a role in the uptake of (14)C-FACBC by DU145 cells. Among the Na(+)-independent transporters, system L transporters are also involved in the transport of (14)C-FACBC. Moreover, (14)C-FACBC is not incorporated into proteins in cells. These findings suggest a possible mechanism of anti-(18)F-FACBC PET for prostate cancer.

We examined the protective effects of N-acetylcysteine (NAC) on the death of glia-free neurons in culture. Under normoxic conditions, the protection by NAC was observed only in cystine-free but not complete medium. When the cells were cultured under hypoxic conditions, NAC much elongated their survival even in the presence of cystine. H2O2 was found to be generated to considerable concentration in the presence of both NAC and cystine, and the administration of catalase prevented the cell death. These results suggest that the harmful effect of NAC is because of H2O2 generated by autoxidation of cysteine, which derives from the reaction between NAC and cystine. The present results raise the possibility that NAC can act as either antioxidant or prooxidant depending on the milieu.

Introduction We examined 3-[ 123 I]iodo-α-methyl- l -tyrosine ([ 123 I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure. Methods Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [ 125 I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [ 125 I]IMT uptake were examined. Results Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B 0 AT was not detected. [ 125 I]IMT uptake in DLD-1 cells involved Na + -independent system L primarily and Na + -dependent system(s). Uptake of [ 125 I]IMT in Na + -free buffer followed Michaelis–Menten kinetics, with a K m of 78 μM and V max of 333 pmol/10 6 cells per minute. Neutral d - and l -amino acids with branched or aromatic large side chains inhibited [ 125 I]IMT uptake. Tyrosine analogues, tryptophan analogues, l -phenylalanine and p -halogeno- l -phenylalanines, and gamma amino acids [including 3,4-dihydroxy- l -phenylalanine ( l -DOPA), dl - threo -β-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]- l -phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [ 125 I]IMT uptake, but l -tyrosine methyl ester and R(+)/S(−)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [ 125 I]IMT uptake except l -serine and d / l -cysteine. Conclusions [ 125 I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1.

The alpha(v)beta(3) integrin plays a pivotal role in angiogenesis and tumor metastasis. Angiogenic blood vessels overexpress alpha(v)beta(3) integrin, as in tumor neovascularization, and alpha(v)beta(3) integrin expression in other microvascular beds and organs is limited. Therefore, alpha(v)beta(3) integrin is a suitable receptor for tumor-targeting imaging and therapy. Recently, tetrameric and dimeric RGD peptides have been developed to enhance specificity to alpha(v)beta(3) integrin. In comparison to the corresponding monomeric peptide, however, these peptides show high levels of accumulation in kidney and liver. The purpose of this study is to evaluate tumor-targeting properties and the therapeutic potential of 111In- and 90Y-labeled monomeric RGD peptides in BALB/c nude mice with SKOV-3 human ovarian carcinoma tumors. DOTA-c(RGDfK) was labeled with 111In or 90Y and purified by HPLC. A biodistribution study and scintigraphic images revealed the specific uptake to alpha(v)beta(3) integrin and the rapid clearance from normal tissues. These peptides were renally excreted. At 10 min after injection of tracers, 111In-DOTA-c(RGDfK) and 90Y-DOTA-c(RGDfK) showed high uptake in tumors (7.3 +/- 0.6% ID/g and 4.6 +/- 0.8% ID/g, respectively) and gradually decreased over time (2.3 +/- 0.4% ID/g and 1.5 +/- 0.5% ID/g at 24 hr, respectively). High tumor-to-blood and -muscle ratios were obtained from these peptides. In radionuclide therapeutic study, multiple-dose administration of 90Y-DOTA-c(RGDfK) (3 x 11.1 MBq) suppressed tumor growth in comparison to the control group and a single-dose administration (11.1 MBq). Monomeric RGD peptides, 111In-DOTA-c(RGDfK) and (90)Y-DOTA-c(RGDfK), could be promising tracers for alpha(v)beta(3) integrin-targeting imaging and radiotherapy.

Introduction Early detection and/or prediction of metastasis provide more prognostic relevance than local recurrence. Direct spread into the peritoneum is frequently found in pancreatic cancer patients, but positron emission tomography (PET) with 2-deoxy-2-fluoro-d-glucose (FDG) is not useful for identifying such metastasis. We investigated a method to enhance FDG accumulation using AsPC-1 human ascites tumor cells. Methods 14 C-FDG accumulation was assessed under the following conditions: 1) characteristics of 14 C-FDG transport were examined using phloridzin, a Na + -free buffer, and various hexoses, and 2) accumulation of 14 C-FDG was measured in cells that were pretreated with hexose for various time periods, and activity of 6-phosphofructo-1-kinase (PFK-1) was assayed. Results 14 C-FDG transport into AsPC-1 cells was mediated primarily by a Na + -independent transport mechanism. Aldohexoses such as d-glucose, d-mannose, and d-galactose inhibited 14 C-FDG transport. Cells pretreated with d-glucose, d-mannose, or d-fructose exhibited augmented 14 C-FDG accumulation. Pretreatment with higher concentrations of d-glucose or d-fructose tended to increase PFK-1 activity. Conclusions Very little information has been published about the association between PFK-1 and FDG accumulation, and we confirmed the impacts of various hexoses on the activity of PFK-1 and FDG accumulation in AsPC-1 cells. Clarifying the relevance of PFK-1 in FDG accumulation will contribute to developing new features of FDG-PET, because PFK-1 is the main regulator of glycolysis.

Activation of the PI3K-Akt pathway is known to induce tumor radioresistance. In the current study, we examined the ability of 17AAG, which decreases the levels of Hsp90 client proteins including components of the PI3K-Akt pathway, to sensitize radioresistant human squamous cell carcinoma cells to X-irradiation. Human squamous cell carcinoma cell lines (SQ20B, SCC61 and SCC13) were incubated for 16 h at 37 degrees C in medium containing 17AAG. Radiation sensitivity was determined by clonogenic assays, and protein levels were examined by western blotting. Apoptosis was determined in monolayer cells by AO/EB double staining and in spheroids using the TdT-mediated dUTP nick end labeling assay. 17AAG (0.2 microM) enhanced the radiosensitivity more effectively in radioresistant SQ20B and SCC13 cells than in radiosensitive SCC61 cells. However, in all three cell lines, 17AAG increased radiation-induced apoptosis by reducing the expression of EGFR and ErbB-2 and inhibiting the phosphorylation of Akt. Furthermore, 17AAG (1 microM) sensitized SQ20B spheroids to radiation, and inhibition of Akt activation by 17AAG increased radiation-induced apoptosis in spheroids. The findings suggest that 17AAG effectively sensitizes radioresistant cells to radiation by inhibiting the PI3K-Akt pathway. Targeting the PI3K-Akt pathway with 17AAG could be a useful strategy for radiosensitization of carcinomas.

Objective: 3-[123I]iodo-α-methyl-L-tyrosine ([123I]IMT) is an imaging agent for amino acid transport. In order to obtain fundamental data related to tumor imaging with [123I]IMT and renal physiological accumulation of [123I]IMT, we investigated the transport characteristics of [123I]IMT in porcine kidney epithelial cell line LLC-PKi using cell monolayers grown on microporous membrane filters.Methods: LLC-PKi monolayers were created on a collagen-coated microporous (3μm) membrane (4.7 cm2). To examine transcellular transport (secretion and reabsorption) and accumulation, the monolayers were incubated for up to 90 min at 37°C with 18.5 kBq [125I]IMT in Dulbecco’s phosphate-buffered saline (pH 7.4) as an uptake solution. After incubation, transcellular transport was assessed by quantifying the radioactivity of the solutions on each side of the monolayer. For the accumulation experiment, the cells were solubilized in NaOH solution, and the radioactivity was quantified. For the inhibition experiment, the inhibitor was added at a final concentration of 1 mM. For the pH dependence experiment, the pH of the apical-side uptake solution was varied from pH 5 to pH 8. Transport of [14C]Tyr was examined for comparison.Results: Bi-directional transcellular transport of [125I]IMT was observed, corresponding to secretion and reabsorption in proximal tubule. Accumulation of [125I]IMT from the basolateral side (1.62 ± 0.15%) and the apical side (2.62 ± 0.35%) was observed at 90 min. 2-Amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L), L-Tyr (mother compound of [125I]IMT) and 2-aminoisobutyric acid (an inhibitor of system L and A) inhibited both directional transport (p < 0.01) and accumulation (p < 0.01). 2-(Methylamino)isobutyric acid (a specific inhibitor of system A) appeared to inhibit transport and accumulation, but the results were not significant. Decreasing apical pH significantly enhanced accumulation of [125I]IMT from both sides (p < 0.001), whereas accumulation of mother L-Tyr was significantly suppressed.Conclusions: The inhibition experiments suggest that the main contributor to [125I]IMT transport is system L, rather than Na+-dependent transport, in both apical and basolateral membrane. [125I]IMT was transported by the system that transported L-Tyr, but the observed pH dependence of transport suggests that different mechanisms are involved in accumulation of [125I]IMT and [14C]Tyr.

We examined transport of 3-[(125)I]iodo-alpha-methyl-L-tyrosine ([(125)I]IMT) in Xenopus laevis oocytes co-expressing human L-type amino acid transporter 1 (a component of system L) and human 4F2hc. Human LAT1 mediated transport of [(125)I]IMT. [(125)I]IMT uptake was decreased by the presence of L-isomers of Cys, Leu, Ileu, Phe, Met, Tyr, His, Trp and Val and D-isomers of Leu, Phe and Met. Human LAT1-mediated [(125)I]IMT uptake was highly stereoselective for the L-isomers of Tyr, His, Trp, Val and Ileu. To examine the effects of 3-iodination and alpha-methylation on IMT transport, kinetic parameters of IMT were compared with those of mother Tyr and 3-[(125)I]iodo-L-tyrosine (3-I-Tyr). Uptake of Tyr, 3-I-Tyr and [(125)I]IMT followed Michaelis-Menten kinetics, with K(m) values of 29.0 +/- 5.1, 12.6 +/- 6.1 and 22.6 +/- 4.1 microM, respectively. Neither the alpha-methyl group nor the size of the 3-iodinated Tyr residue was an obstacle to transport via hLAT1. Furthermore, affinity of IMT for hLAT1 is higher than that of the natural parent tyrosine. The level of efflux mediated by hLAT1 was highly stimulated by extracellularly applied L-Leu, suggesting exchange of [(125)I]IMT and L-Leu via hLAT1.

Introduction: 123I-N-isopropyl-p-iodoamphetamine (123I-IMP) is metabolized and converted to 123I-p-iodoamphetamine (123I-PIA) by CYP2C19 in humans. Since variations in 123I-PIA levels reflect variations in CYP2C19 activity, CYP2C19 activity can be estimated by quantitative analysis of 123I-PIA levels. Thus, 123I-IMP administration can provide diagnostic information not only regarding cerebral blood flow (rCBF) but also regarding metabolic function. The aim of the present study was to detect variations in CYP activity in mice using metabolite analysis. Methods: Metabolism of 125I-IMP in pooled homogenates of mouse liver (MLH) was analyzed by high-performance liquid chromatography (HPLC) in the presence or absence of NADPH. The amount of 125I-PIA generated was calculated as the normalized peak area of the chromatogram. Inhibition of 125I-IMP metabolism was evaluated using the inhibitor SKF-525A. A biodistribution study of 125I-IMP was performed to determine the organ distribution of 125I-IMP/125I-IMP metabolites and the effect of SKF-525A. Variations in CYP activity in vivo were detected by administration of 123I-IMP and/or SKF-525A to mice. The liver and the kidney were then excised, homogenized and analyzed using HPLC. Results: 125I-IMP was metabolized by MLH in the presence of NADPH, and the production of 125I-PIA was inhibited by SKF-525A. SKF-525A did not greatly affect the biodistribution of 125I-IMP/125I-IMP metabolites in vivo. Both 123I-IMP and 123I-PIA were detected in organs of control mice. However, 123I-PIA was not detected in the livers or kidneys of mice treated with SKF-525A. Conclusions: CYP activity in vivo was inhibited by SKF-525A treatment. Variations in CYP activity could be detected in the blood, liver and kidney as changes in the peak area of 123I-PIA. Advances in knowledge and implications for patient care: 123I-IMP metabolite analysis has the potential to provide beneficial information for prediction of the effect of medicines, in addition to its contribution to more accurate rCBF diagnosis that reflects individual CYP activity., Nuclear Medicine and Biology, 42(2), pp.171-176; 2015

The Patlak plot method of measuring cerebral blood flow (CBF) to improve the repeatability and quantitative capability, by using technetium-99m ethyl cysteinate dimer (99mTc-ECD). We calculated CBF and then statistically analyzed its relationships with various hematological and biochemical parameters. There were significant statistical correlations between these clinical parameters and the measured values of mean CBF (mCBF), also between these biochemical parameters and post-acetazolamide (p-ACZ) mCBF, in terms of estimated glomerular filtration rate (eGFR), serum albumin level, red blood cell count, blood urea nitrogen level, and random blood glucose level. In addition, statistically significant correlations were found between these parameters and increased mCBF. Another significant correlation was found between cerebrovascular reserve capacity (CVR) and platelet count. Values of p-ACZ mCBF and CVR were lower in a group with HbA(1C)7% and high blood glucose levels than in healthy subjects. In addition, values of resting mCBF and p-ACZ mCBF were lower in a group with kidney dysfunction (eGFR30 ml/min/1.73 m2) than in subjects with normal renal function or mild dysfunction. A multiple linear regression analysis showed a correlation between resting mCBF value and eGFR. Therefore, there were correlations between CBF and the levels of these parameters of diabetes or chronic kidney disease. These results suggest that our Patlak plot modified method may be a potentially useful tool for analyzing the relationships between CBF and underlying diseases and/or the pathophysiology of CBF dysfunction. The post-ACZ ECD Patlak resting and vascular reserve (p-ACZ ECD Patlak RVR) test provides a way of detecting minor changes in CBF, which is difficult to reveal by only resting Patlak plot method, in patients with lifestyle diseases such as diabetes or chronic kidney disease. In addition, we believe that a new modified method contribute to predict risk of cerebral vascular disorders along with clinical parameters.

BPA used in BNCT has a similar structure to some essential amino acids and is transported into tumor cells by amino acid transport systems. Previous study groups have tried various techniques of loading BPA to increase intracellular boron concentration. CHO-K1 cells demonstrate system L (LAT1) activity and are suitable for specifying the transport system of a neutral amino acid. In this study, we examined the intracellular accumulation of boron in CHO-K1 cells by amino acid transport control, which involves co-loading with L-type amino acid esters. Intracellular boron accumulation in CHO-K1 cells showed the greatest increased upon co-loading 1.0 mM BPA, with 1.0 mM l-Tyr-O-Et and incubating for 60 min. This increase is caused by activation of a system L amino acid exchanger between BPA and l-Tyr. The amino acid esters are metabolized to amino acids by intracellular hydrolytic enzymes that increase the concentrations of intracellular amino acids and stimulate exchange transportation. We expect that this amino acid transport control will be useful for enhancing intracellular boron accumulation.

OBJECTIVE: The purpose of this study was to evaluate the appropriate parameters of a filter and of subsets (S) and iterations (I) of the ordered-subset expectation maximization (OSEM) algorithm in 11C-acetate PET. METHODS: A Hanning filter (HF) and a Gaussian filter (GF) were selected for filtered back-projection (FBP) and the OSEM algorithm, respectively. After evaluation of the optimal HF size, the GF size was optimized using healthy volunteers (HV). Myocardial blood flow (MBF) and oxygen consumption (k(mono)) values were calculated by combining 4S, 16S, or 28S with 2I, 4I, 6I, or 8I of the OSEM (MBF(OSEM) and k(monoOSEM), respectively) in eight HV and eight coronary artery disease (CAD) patients. These MBF(OSEM) and k(monoOSEM) values were compared with those obtained using FBP (MBF(FBP) and k(monoFBP), respectively). RESULTS: Optimal HF and GF (10.0GF) sizes for the FBP and OSEM algorithms, respectively, were 10.0 mm full-width resolution at half-maximum. MBF(OSEM) was changed by modifying the parameters of the OSEM algorithm. The best correlations were between MBF(FBP) and MBF(OSEM), with 28S6I and 10.0GF for HV patients and 28S8I for CAD patients. However, the MBF(OSEM) with 28S8I was significantly different from MBF(FBP) at the global myocardium in HV. The k(monoOSEM) with 28S6I was not significantly different from k(monoFBP) in HV or CAD patients. CONCLUSION: Appropriate parameters are 28S6I with a 10.0GF on the MBF(OSEM) and k(monoOSEM) measurement using 11C-acetate. Diagnostic performance will improve using noiseless, artifact-reduction images, and accurate quantitative values that are provided by the OSEM algorithm with the appropriate parameters.

We examined the biodistribution and urinary excretion of 125I-4-iodo-L-meta-tyrosine (4-125I-L-mTyr) in mice, in order to compare the results with that of 3-[125I]iodo-alpha-methyl-L-tyrosine (125I-L-AMT). Three hours after administration, more than 70% of the excretion of 4-125I-L-mTyr was in the urine, and less than 5% was found in the feces. The kidney homogenate and urine analysis on its metabolites revealed that most of the accumulated radioactivity belonged to intact 4-125I-L-mTyr (>95%). These results indicated its high metabolic stability, which was comparable to 125I-L-AMT stability. However, the blood clearance of 4-125I-L-mTyr was faster than that of 125I-L-AMT. 4-125I-L-mTyr accumulation in mouse kidney cortex was approximately 80% lower than that of 125I-L-AMT. Probenecid reduced the 4-125I-L-mTyr accumulation in the kidney and urinary excretion, similar to 125I-L-AMT.

The first step of technetium-99m-mercaptoacetylglycylglycylglycine (99mTc-MAG3) secretion by the proximal tubule cells is the extraction of 99mTc-MAG3 from the peritubular plasma by proximal tubule cells through the basolateral membrane. The evidence for 99mTc-MAG3 being excreted from renal proximal tubules via rat organic anion transporter 1 (OAT1) by molecular biological experiments with expressed OAT1 in Xenopus laevis oocytes was discussed. Inhibition studies of 99mTc-MAG3 revealed that OAT1 mediated Na+-independent 99mTc-MAG3 uptake, and that this transport was energy-independent. Accumulated dicarboxylate, such as glutarate, stimulated 99mTc-MAG3 uptake. Tc-99m-MAG3, as well as paminohippurate (PAH), and o-iodohippurate (OIH), was confirmed to be a transport substrate of OAT1 by the uptake of expressed oocytes.

We have clarified the membrane transport characteristics of 3-[123I]Iodo-α-methyl-L-tyrosine by molecular biological methods with cDNAs of an amino acid transporter. Uptake and inhibition studies of this compound were performed with human L-type amino acid transporter-1 and a heavy chain of human 4F2 cell surface antigen co-expressed Xenopus laevis oocytes. 3-[125I]Iodo-α-methyl-L-tyrosine uptake was decreased by the presence of Ala, Ser, Thr, Cys, Leu, IIeu, Phe, Met, Tyr, His, Trp, Val, Asn, and Gln. The uptake was Na+-independent, and was activated by a heavy chain of human 4F2 cell surface antigen.

Introduction High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of 14 C(U)-l-tyrosine ( 14 C-Tyr), 125 I-4-iodo-l- meta -tyrosine (4- 125 I- m Tyr), 125 I-6-iodo-l- meta -tyrosine (6- 125 I- m Tyr), 125 I-3-iodo-α-methyl-l-tyrosine ( 125 I-IMT) and 125 I-3-iodo-l-tyrosine (3- 125 I-Tyr) using Chinese hamster ovary cells (CHO-K1). Methods Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na + at 37°C or 4°C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na + -containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na + -free uptake solution); sodium azide and 2,4-dinitrophenol (NaN 3 and DNP, inhibitors of the generation of adenosine triphosphate); p -aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and l- and d-isomers of natural amino acids. Results 14 C-Tyr exhibited affinity for systems L, A and ASC. 4- 125 I- m Tyr and 3- 125 I-Tyr exhibited high specificity for system L, whereas 6- 125 I- m Tyr and 125 I-IMT exhibited affinity for both systems L and ASC. Uptake of 4- 125 I- m Tyr was markedly reduced by incubation at 4 °C, and was not significantly inhibited by NaN 3 , DNP, PAH or TEA. The inhibition profiles of the l- and d-isomers of natural amino acids indicated that system L mediates the transport of 4- 125 I- m Tyr. Conclusions 4- 125 I- m Tyr exhibited the greatest system L specificity (93.46±0.13%) of all of the tested amino acids.

Almost all D-glucose (GLU) filtered through the glomerulus is reabsorbed by the renal proximal tubules, whereas a high portion of 2-[18F]fluoro-2-deoxy-D-glucose [(18F)FDG] is excreted through the urine. However, [18F]FDG is not entirely excreted in the urine suggesting that it may be partially reabsorbed by the proximal tubules. The purpose of this study was to compare the time course of transcellular transport of administered [14C] labeled FDG ([14C]FDG) with that of [14C] labeled GLU ([14C]GLU) using the kidney epithelial cell line, LLC-PK1.Transcellular transport of [14C]FDG and [14C]GLU by LLC-PK1 cells was measured in Na+-containing or Na+-free Dulbecco's phosphate-buffered saline [PBS(+) and PBS(-), respectively] in the presence or absence of phlorizin, phloretin, probenecid, or tetraethylammonium bromide inhibitors that predominantly inhibit sodium-dependent glucose transporters (SGLTs), sodium-independent glucose transporters, organic anion transporters, and organic cation transporters, respectively.When assayed in PBS(+), less [14C]FDG than [14C]GLU was reabsorbed by the proximal tubular cells over the entire incubation time. Reabsorption of [14C]FDG was mediated mainly by SGLT at early time points in the incubation, whereas high reabsorption of [14C]GLU was mediated by both SGLT and glucose transporter over 90 min of incubation. Secretion of [14C]FDG also tended to be slightly higher than that of [14C]GLU over 90 min of incubation.Transcellular transport of [14C]FDG over time by LLC-PK1 cells was clarified. The polarized distribution of transcellular transporters of [14C]FDG and [14C]GLU in LLC-PK1 cells differs.

When a therapeutic drug is competitively displaced at the binding sites of serum proteins, the free fraction of the drug will be increased, with an increase in the manifestation of pharmacologic properties. In the case of molecular imaging probes, total clearance and tissue distribution are increased in such circumstances. The aim of this study was to observe the increase in cerebral accumulation of N-isopropyl-p-(123)I-iodoamphetamine ((123)I-IMP) using the protein-binding displacement method with amino acid infusion.(123)I-IMP binding to human serum was investigated and identified. In addition, protein-binding sites and the specific binding sites of human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) were examined by ultrafiltration. Then, serum-binding sites and the displacement effects of amino acid infusion, including Proteamin 12X Injection and Kidomin, were confirmed in vitro. Subsequently, displacement of (123)I-IMP serum protein binding with Proteamin amino acid infusion was tested in monkeys. A scintigraphic study of (123)I-IMP in monkeys loaded with or without Proteamin was performed, and time-activity-curves of (123)I-IMP brain accumulation in monkeys were evaluated.(123)I-IMP was bound to HSA site II and AGP to nearly equal extents. Compared with control conditions, loading with Proteamin and Kidomin markedly increased free fractions of binding site markers for HSA site II ((14)C-diazepam: 0.95% +/- 0.04% for control, 1.40% +/- 0.06% for Proteamin, 1.62% +/- 0.05% for Kidomin) and AGP ((3)H-propranolol: 10.60% +/- 0.32% for control, 13.18% +/- 0.14% for Proteamin, 13.82% +/- 0.72% for Kidomin). Amino acid infusions were thus suitable for use as displacers for binding site II and AGP. With use of Proteamin amino acid infusion to displace protein binding, the free fraction of (125)I-IMP (14.95% +/- 0.74%) was significantly increased in serum (19.24% +/- 0.87%). In a (123)I-IMP scintigraphic study of monkeys, average cerebral uptake in 2 monkeys increased by 1.34-fold with Proteamin. Our findings suggested that Proteamin treatment increased the free fraction of (123)I-IMP, yielding rapid and pronounced cerebral accumulation in vivo.Amino acid infusion can improve brain accumulation by competitive displacement of serum protein binding in vivo. Further similar studies are needed with other radiopharmaceuticals.

Introduction Transport of the amino acid analog 123I-3-iodo-α-methyl- l -tyrosine, which is used in clinical SPECT imaging, occurs mainly via l -type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of 125I-3-iodo-α-methyl- l -tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. Methods Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. l -Gly, l -Ser, l -Leu, l -Phe, l -Met, l -Tyr, d -Tyr, l -Val and l -Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of l -Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of l - and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of l - and d -Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37°C or under ice-cold conditions. Results Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of l -Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. Conclusions The l -Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and l -Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.

Introduction The binding of radiopharmaceutical to serum proteins is thought to be an important factor that restricts its excretion and accumulation in tissue. We calculated the effect of inhibitors of serum protein binding using a hypothetical radiopharmaceutical. In vitro experiments and protein binding inhibitor-loaded monkey scintigraphy were then conducted using 123 I- N -isopropyl- p -iodoamphetamine (IMP) as the radiopharmaceutical. Methods Free fraction ratios of radiopharmaceutical were calculated with one radiopharmaceutical, two serum proteins and two specific inhibitors in the steady state at various serum protein concentrations. In vitro protein binding inhibition studies using human, rat and monkey sera were performed with site-selective displacers of specific binding sites: 400 μM 6-methoxy-2-naphthylacetic acid (6MNA; a major nabumeton metabolite) as a serum albumin Site II inhibitor and 400 μM erythromycin (ETC) as an α 1 -acid glycoprotein (AGP) site inhibitor. Scintigraphy with or without 6MNA loading of monkeys was performed. Results The theoretical findings roughly corresponded to the experimental results. Approximately 75% of IMP bound to serum albumin Site II and AGP in the species examined. The free fraction of IMP (25.0±0.6% for human, 22.8±0.4% for monkey, 23.7±0.3% for rat) increased with loading of specific protein binding inhibitors (6MNA: 28.0±0.3% for human, 24.5±0.7% for monkey, 24.3±0.2% for rat; ETC: 26.3±0.4% for human, 29.5±1.1% for monkey, 26.0±0.7% for rat) and was serum protein concentration dependant based on the results of calculations. Simultaneous administration of 6MNA and ETC produced a higher free fraction ratio of IMP (31.9±1.0% for human, 34.6±0.4% for monkey, 27.0±0.3% for rat) than summation of the single administrations of 6MNA and ETC (domino effect) in human, rat and monkey sera. Rapid cerebral accumulation was observed with 6MNA loading in monkey scintigraphy. Conclusions 6MNA appears to change the pharmacokinetics and brain accumulation of IMP in monkeys. Further studies in human are required.

The alpha(v)beta(3) integrin plays a pivotal role in angiogenesis and tumor metastasis. Angiogenic blood vessels overexpress alpha(v)beta(3) integrin, as in tumor neovascularization, and alpha(v)beta(3) integrin expression in other microvascular beds and organs is limited. Therefore, alpha(v)beta(3) integrin is a suitable receptor for tumor-targeting imaging and therapy. Recently, tetrameric and dimeric RGD peptides have been developed to enhance specificity to alpha(v)beta(3) integrin. In comparison to the corresponding monomeric peptide, however, these peptides show high levels of accumulation in kidney and liver. The purpose of this study is to evaluate tumor-targeting properties and the therapeutic potential of 111In- and 90Y-labeled monomeric RGD peptides in BALB/c nude mice with SKOV-3 human ovarian carcinoma tumors. DOTA-c(RGDfK) was labeled with 111In or 90Y and purified by HPLC. A biodistribution study and scintigraphic images revealed the specific uptake to alpha(v)beta(3) integrin and the rapid clearance from normal tissues. These peptides were renally excreted. At 10 min after injection of tracers, 111In-DOTA-c(RGDfK) and 90Y-DOTA-c(RGDfK) showed high uptake in tumors (7.3 +/- 0.6% ID/g and 4.6 +/- 0.8% ID/g, respectively) and gradually decreased over time (2.3 +/- 0.4% ID/g and 1.5 +/- 0.5% ID/g at 24 hr, respectively). High tumor-to-blood and -muscle ratios were obtained from these peptides. In radionuclide therapeutic study, multiple-dose administration of 90Y-DOTA-c(RGDfK) (3 x 11.1 MBq) suppressed tumor growth in comparison to the control group and a single-dose administration (11.1 MBq). Monomeric RGD peptides, 111In-DOTA-c(RGDfK) and (90)Y-DOTA-c(RGDfK), could be promising tracers for alpha(v)beta(3) integrin-targeting imaging and radiotherapy.

Introduction In order to improve tumor imaging, changes in the pharmacokinetics of 3-[ 12 3 I]iodo-α-methyl-l-tyrosine ([ 12 3 I]IMT), an artificial amino acid that exhibits high tumor accumulation, after probenecid (PBC) loading was studied in mice implanted with colon cancer DLD-1 cells using 12 5 I-labeled IMT ([ 12 5 I]IMT). Methods DLD-1-implanted KSN-slc nude male mice received 740 kBq of [ 12 5 I]IMT via the tail vein at 5 min after 50 mg/kg body weight PBC loading, and autoradiography was performed at 5, 15 and 30 min after injection. Male ddY mice then received 670 kBq of [ 12 5 I]IMT and 50 mg/kg 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or p -aminohippurate (PAH) via the tail vein, and kidney autoradiography was performed at 5 min after injection. In vitro inhibition study was then performed based on the accumulation mechanisms of [ 12 5 I]IMT in DLD-1, using 1 mM l-tyrosine, BCH, α-(methylamino)-isobutyric acid, N -benzoyl-β-alanine, PBC, PAH, 2,4-dinitrophenol and sodium azide. Both Na + -dependent and Na + -independent uptake were investigated. Results Higher tumor accumulation in PBC-loaded DLD-1-implanted mice was seen when compared to control mice. PAH and BCH, respectively, reduced renal accumulation in the tubule segment-2 (S2)-like and S1-like regions. We confirmed that [ 12 5 I]IMT transport is predominantly mediated by l-type amino acid transporter-1 in DLD-1 cells. Conclusions [ 12 5 I]IMT uptake is mediated by organic anion and amino acid transporters in the kidney. Organic anion transporter inhibitors may yield better tumor images with good tumor/normal tissue radioactivity ratios if adequate administration plans are developed.

Introduction The fact that d-amino acids have been found in various tissues and are involved in various functions is a clue to how to develop new imaging agents. We examined d-amino acid transport mechanisms in Chinese hamster ovary (CHO-K1) cells because CHO-K1 cells are widely used in biomedical studies and are thought to be useful for expression of genes involved in metabolism of d-amino acids. Methods Uptake experiments were performed. CHO-K1 cells cultured in 60-mm plastic culture dishes under ordinary culture conditions were incubated with 18.5 kBq of radiolabeled amino acid in 2 ml of phosphate-buffered-saline-based uptake solution at 37°C. The following radiolabeled amino acid tracers were used: d-[1- 14 C]-alanine, l-[1- 14 C]-alanine, d-[1- 14 C]-serine, l-[1- 14 C]-serine, d-[1- 14 C]-methionine, l-[1- 14 C]-methionine, d-[1- 14 C]-phenylalanine, l-[1- 14 C]-phenylalanine, d-[1- 14 C]-leucine, l-[1- 14 C]-leucine, d-[1- 14 C]-valine, l-[1- 14 C]-valine, d-[1- 14 C]-tyrosine, l-[1- 14 C]-tyrosine, d-[1- 14 C]-glutamic acid, l-[1- 14 C]-glutamic acid, d-[1- 14 C]-lysine, l-[1- 14 C]-lysine, d-[1- 14 C]-arginine and l-[1- 14 C]-arginine. We tested the inhibitory effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na + -containing uptake solution) and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na + -free uptake solution). Results d-[1- 14 C]-methionine, d-[1- 14 C]-phenylalanine and d-[1- 14 C]-tyrosine accumulated mainly via system L. d-[1- 14 C]-alanine and d-[1- 14 C]-serine accumulated primarily via system ASC. High uptake of d-[1- 14 C]-alanine, d-[1- 14 C]-methionine, d-[1- 14 C]-phenylalanine and d-[1- 14 C]-leucine was observed. The uptake of radiolabeled serine, valine, tyrosine, glutamic acid and arginine into CHO-K1 was highly stereoselective for l-isomers. Conclusions We observed high uptake of d-[1- 14 C]-alanine via system ASC (most likely alanine–serine–cysteine-selective amino acid transporter-1) and high uptake of d-[1- 14 C]-methionine and d-[1- 14 C]-phenylalanine via system L (most likely l-type amino acid transporter-1).

Fanconi syndrome is a renal dysfunction characterized by various combinations of renal tubular transport dysfunction involving amino acids, glucose, protein and other substances. Most reabsorption of amino acids occurs in proximal renal tubule segment 1 (S1). The present study evaluated the possibility of early detection of drug-induced Fanconi syndrome, based on decreased renal accumulation of 125I-3-iodo-alpha-methyl-L-tyrosine (125I-IMT), an amino acid transport marker, in the S1 region of renal cortex. The present experimental model used maleate (MAL)-induced Fanconi syndrome in mice. Results were compared between 125I-IMT and 3 other clinical renal radiopharmaceuticals: 99mTc-2,3-dimercaptosuccinic acid (99mTc-DMSA); 99mTc-mercaptoacetylglycylglycylglycine (99mTc-MAG3); and 99mTc-diethylenetriaminepentaacetic acid (99mTc-DTPA).Male ddY mice (age, 6 weeks; body weight, 25 g) were used to create a Fanconi model of renal dysfunction. A single dose of maleate disodium salt was administered by intraperitoneal injection (6 mmol/kg). Hematoxylin and eosin (HE) staining of the renal cortex, renal autoradiography and measurement of renal radioactivity of labeled compounds were performed at 30, 60, 90 and 120 min after MAL injection. At 5 min after injection of labeled compounds (18.5 kBq for accumulation experiment, 670 kBq for autoradiography), animals were sacrificed by ether overdose and kidneys were removed. For the accumulation experiment, radioactivity was measured using a well-type scintillation counter. For autoradiography, 20-microm sections of frozen kidney were used with Bio-Imaging Analyzer.At 30 min after MAL injection, HE staining showed pyknosis in some proximal tubule cells. At that time, accumulations of 125I-IMT and 99mTc-DMSA in the S1 region were approximately 67% and 55% of control levels (p0.005). MAL increased accumulation of 99mTc-DTPA in the S1 region, but had no effect on accumulation of 99mTc-MAG3 in the S 1 region.Decreased accumulation of 123I-IMT in the S1 region appears to represent a useful marker for detection of MAL-induced Fanconi syndrome. In future, we plan to assess the efficacy of using 125I-IMT to monitor renal dysfunction induced by nephrotoxic clinical drugs.

Objective: We investigated mechanisms of renal accumulation of radioiodinated 3-iodo-α-methyl-L-tyrosine (IMT), which has been used clinically for tumor imaging and as an amino acid transport marker in studies of brain and pancreas function.Methods: In this study, we used125I- or123I-labeled IMT ([125I]IMT or [123I]IMT) as the transport marker. Partition coefficients of [125I]IMT were determined for hypothetic urine at pH ranging from 5 to 8. The examination of uptake and inhibition of [125I]IMT was performed using normal human renal proximal tubule epithelial cells (RPTEC), which are characteristic of the proximal convoluted tubule. The plasma protein binding ratio of [125I]IMT was determined using rats. In thein vivo experiments using mice, we examined biodistribution and excretion inhibition, and performed whole body autoradiography. Also, renal SPECT using [123I]IMT was performed using a normal canine.Results: Very low lipophilicity of [125I]IMT in hypothetic urine suggests that a carrier-mediated pathway contributes to its marked kidney accumulation. [125I]IMT uptake into RPTEC was significantly inhibited by 2-amino-bicyclo[2,2,l]heptane-2-carboxylic acid (BCH) in a sodium-dependent manner, suggesting reab-sorption mainly via system B° in apical membrane of proximal tubule. Plasma protein binding ratio of IMT was 45.4 ± 5.6%. At 6 hr after administration of IMT to mice, excretion via urinary tract was 77.51% of injected dose, and excretion into feces was 0.25%. Furosemide, ethacrynic acid and probenecid inhibited tubular secretion of [125I]IMT in mice. We obtained very clear autoradio-graphs of mouse renal cortex and a canine renal SPECT image (S2-like region).Conclusions: We believe that [123I]IMT is useful for kidney imaging. In future studies, we plan to examine the use of [123I]IMT in diagnosis of disease.

Recently, complementary DNA (cDNA) encoding a p-aminohippurate (PAH) transporter designated rat organic anion transporter 1 (OAT1) was isolated. OAT1, a multispecific organic anion transporter at the basolateral membrane, is exclusively expressed in the middle segment of the proximal tubule in the rat kidney. It has been proposed that OAT1 is indirectly involved in PAH uptake via the Na(+) dicarboxylate cotransporter. In this study, in molecular biologic experiments using OAT1-expressing Xenopus laevis oocytes, we obtained evidence that (99m)Tc-mercaptoacetylglycylglycylglycine (MAG3) is transported via OAT1.Capped OAT1 complementary RNA (cRNA) was synthesized from library plasmid cDNA linearized with BamHI using in vitro transcription. Defolliculated oocytes were injected with 10 ng of OAT1 cRNA. Two to 3 d after injection, uptake of (99m)Tc-MAG3 was measured using ND96 solution containing 18.5 kBq of (99m)Tc-MAG3. Before the uptake experiments, OAT1-expressing oocytes were preincubated for 2 h with 1 mmol/L glutarate (a dicarboxylate), to generate an outwardly directed glutarate gradient. Then, after incubation for 60 min at room temperature, radioactivity of oocytes was determined. For the inhibition experiments, uptake was assessed in the absence or presence of inhibitor: 2 mmol/L of PAH, o-iodohippurate (OIH), probenecid, 3,5-diiodo-4-pyridone-N-acetate (iodopyracet), furosemide, ethacrynic acid, glucoheptonate, maleic acid, L-Tyr, or tetraethylammonium (TEA) or 0.1 mmol/L of 2,4-dinitrophenol (DNP).Na(+) had a significant effect on (99m)Tc-MAG3 uptake (P0.05). Accumulated glutarate stimulated simultaneous (99m)Tc-MAG3 uptake and glutarate excretion (P0.001). The following compounds significantly inhibited (99m)Tc-MAG3 uptake: PAH, 8.5% +/- 16.2% of (99m)Tc-MAG3 uptake in the absence of an inhibitor; OIH, 26.4% +/- 21.7%; probenecid, 29.1% +/- 12.4%; iodopyracet, 15.8% +/- 7.9%; furosemide, 30.5% +/- 15.7%; ethacrynic acid, 21.6% +/- 10.6%; glucoheptonate, 35.6% +/- 22.6%; and maleic acid, 60.1% +/- 18.7%. (99m)Tc-MAG3 accumulation in Xenopus laevis oocytes was not significantly inhibited by TEA, L-Tyr, or DNP.The following substances had a cis-inhibitory effect on (99m)Tc-MAG3 transport: PAH, OIH, probenecid, iodopyracet, furosemide, ethacrynic acid, and glucoheptonate. Glutarate had a trans-stimulative effect on (99m)Tc-MAG3 transport. (99m)Tc-MAG3 acts as a substrate of OAT1, an organic anion/dicarboxylate exchanger.

The substance 4-[(125)I]iodo-L-meta-tyrosin (4-[(125)I]mTyr) is a radioiodinated amino acid that exhibits high in vivo stability and rapid renal elimination in vivo. We investigated transport of 4-[(125)I]mTyr in LLC-PK(1) (porcine kidney epithelial cell line) monolayers grown on collagen-coated, micro-porous membrane filters. We found that 4-[(125)I]mTyr transport in LLC-PK(1) cells was carrier-mediated and sodium-independent, and that 4-[(125)I]mTyr transport was similar to that of L-Tyr and 3-iodo-alpha-methyl-L-tyrosine. The results of the inhibition experiments suggest that 4-[(125)I]mTyr transport is predominantly mediated by a L-type amino acid transporter 1-like porcine homologue (a component of system L) in both basolateral and apical membrane.