Search

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.

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.

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.

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.

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.

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.

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 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.

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

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.

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.

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).

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.