Development of radioiodine-labeled mequitazine for evaluation of hepatic CYP2D activity.

Background: As drug-metabolizing enzyme activities are affected by a variety of factors, such as drug-drug interactions, a method to evaluate drug-metabolizing enzyme activities in real time is needed. In this study, we developed a novel SPECT imaging probe for evaluation of hepatic CYP2D activity. Methods: Iodine-123- and 125-labeled 4-iodobenzylmequitazine (123/125I-BMQ) was synthesized with high labeling and purity. CYP isozymes involved in the metabolism of 125I-BMQ in mouse liver microsomes were evaluated, and the utility of 123/125I-was assessed from biological distribution and SPECT imaging evaluation in normal and CYP2D-inhibited mice. Results: In vitro metabolite analysis using mouse liver microsomes showed that 125I-BMQ is specifically metabolized by CYP2D. Biological distribution and SPECT imaging of 123/125I-BMQ in normal mice showed that injection 123/125I-BMQ accumulated early in the liver and was excreted into the gallbladder and intestines. In CYP2D-inhibited mice, accumulation in the liver was increased, but accumulation in the gallbladder and intestines, the excretory organ, was delayed. Since only metabolites of 125I-BMQ are detected in bile, visualization and measuring of the accumulation of metabolites over time in the intestine, where bile is excreted, could predict the amount of metabolites produced in the body and evaluate CYP2D activity, which would be useful in determining the dosage of various drugs metabolized by CYP2D. Conclusion: 123/125I-BMQ is useful as a SPECT imaging probe for comprehensive and direct assessment of hepatic CYP2D activity in a minimally invasive and simple approach.

SPECT Imaging of P. aeruginosa Infection in Mice Using 123I-BMIPP.

Pseudomonas aeruginosa infection is an infectious disease that must be controlled because it becomes chronic and difficult to treat, owing to its unique system of toxin production/injection and elimination of other bacteria. Here, we noninvasively monitored P. aeruginosa using single-photon emission computed tomography (SPECT) imaging. Determining the amount and localization of the P. aeruginosa will enable making faster clinical diagnoses and selecting the most appropriate therapeutic agents and methods. Nonclinically, this information can be used for imaging in combination with biofilms and toxin probes and will be useful for discovering drugs targeting P. aeruginosa. To study P. aeruginosa accumulation, we conducted in vitro and in vivo studies using iodine-123 ß-methyl-p-iodophenyl-pentadecanoic acid (123I-BMIPP), which we previously reported using for Escherichia coli. In vitro, 123I-BMIPP accumulated in P. aeruginosa by being taken up into the bacteria and adsorbing to the bacterial surface. In vivo, 123I-BMIPP accumulated significantly more in infected sites than in noninfected sites and could be quantified by SPECT. These results suggest that 123I-BMIPP can be used as a probe for P. aeruginosa for SPECT. Establishing a noninvasive monitoring method using SPECT will allow further progress in studying P. aeruginosa.

Evaluation of L-Alanine Metabolism in Bacteria and Whole-Body Distribution with Bacterial Infection Model Mice.

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.

Biological Distribution after Oral Administration of Radioiodine-Labeled Acetaminophen to Estimate Gastrointestinal Absorption Function via OATPs, OATs, and/or MRPs.

We evaluated the whole-body distribution of orally-administered radioiodine-125 labeled acetaminophen (125I-AP) to estimate gastrointestinal absorption of anionic drugs. 125I-AP was added to human embryonic kidney (HEK)293 and Flp293 cells expressing human organic anion transporting polypeptide (OATP)1B1/3, OATP2B1, organic anion transporter (OAT)1/2/3, or carnitine/organic cation transporter (OCTN)2, with and without bromosulfalein (OATP and multidrug resistance-associated protein (MRP) inhibitor) and probenecid (OAT and MRP inhibitor). The biological distribution in mice was determined by oral administration of 125I-AP with and without bromosulfalein and by intravenous administration of 125I-AP. The uptake of 125I-AP was significantly higher in HEK293/OATP1B1, OATP1B3, OATP2B1, OAT1, and OAT2 cells than that in mock cells. Bromosulfalein and probenecid inhibited OATP- and OAT-mediated uptake, respectively. Moreover, 125I-AP was easily excreted in the urine when administered intravenously. The accumulation of 125I-AP was significantly lower in the blood and urinary bladder of mice receiving oral administration of both 125I-AP and bromosulfalein than those receiving only 125I-AP, but significantly higher in the small intestine due to inhibition of OATPs and/or MRPs. This study indicates that whole-body distribution after oral 125I-AP administration can be used to estimate gastrointestinal absorption in the small intestine via OATPs, OATs, and/or MRPs by measuring radioactivity in the urinary bladder.

Measurement of Hepatic CYP3A4 and 2D6 Activity Using Radioiodine-Labeled O-Desmethylvenlafaxine.

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 O-desmethylvenlafaxine (123/125I-ODV) was obtained with high labeling and purity, and its metabolism was found to strongly involve CYP3A4 and CYP2D6. SPECT imaging in normal mice showed that the administered 123I-ODV accumulated early in the liver and was excreted into the gallbladder, as evaluated by time activity curves. In its biological distribution, 125I-ODV administered to mice accumulated early in the liver, and only the metabolite of 125I-ODV was quickly excreted into the bile. In CYP3A4- and CYP2D6-inhibited model mice, the accumulation in bile decreased more than in normal mice, indicating inhibition of metabolite production. These results indicated that imaging and quantifying the accumulation of radioactive metabolites in excretory organs will aid in determining the dosages of various drugs metabolized by CYP3A4 and CYP2D6 for individualized medicine. Thus, 123/125I-ODV has the potential to direct, comprehensive detection and measurement of hepatic CYP3A4 and CYP2D6 activity by a simple and less invasive approach.

123I-BMIPP, a Radiopharmaceutical for Myocardial Fatty Acid Metabolism Scintigraphy, Could Be Utilized in Bacterial Infection Imaging.

In this study, we evaluated the use of 15-(4-123I-iodophenyl)-3(R,S)-methylpentadecanoic acid (123I-BMIPP) to visualize fatty acid metabolism in bacteria for bacterial infection imaging. We found that 123I-BMIPP, which is used for fatty acid metabolism scintigraphy in Japan, accumulated markedly in Escherichia coli EC-14 similar to 18F-FDG, which has previously been studied for bacterial imaging. To elucidate the underlying mechanism, we evaluated changes in 123I-BMIPP accumulation under low-temperature conditions and in the presence of a CD36 inhibitor. The uptake of 123I-BMIPP by EC-14 was mediated via the CD36-like fatty-acid-transporting membrane protein and accumulated by fatty acid metabolism. In model mice infected with EC-14, the biological distribution and whole-body imaging were assessed using 123I-BMIPP and 18F-FDG. The 123I-BMIPP biodistribution study showed that, 8 h after infection, the ratio of 123I-BMIPP accumulated in infected muscle to that in control muscle was 1.31 at 60 min after 123I-BMIPP injection. In whole-body imaging 1.5 h after 123I-BMIPP administration and 9.5 h after infection, infected muscle exhibited a 1.33-times higher contrast than non-infected muscle. Thus, 123I-BMIPP shows potential for visualizing fatty acid metabolism of bacteria for imaging bacterial infections.

Indirect SPECT Imaging Evaluation for Possible Nose-to-Brain Drug Delivery Using a Compound with Poor Blood-Brain Barrier Permeability in Mice.

Single-photon emission computed tomography (SPECT) imaging using intravenous radioactive ligand administration to indirectly evaluate the time-dependent effect of intranasal drugs with poor blood-brain barrier permeability on brain drug distributions in mice was evaluated. The biodistribution was examined using domperidone, a dopamine D2 receptor ligand, as the model drug, with intranasal administration at 0, 15, or 30 min before intravenous [123I]IBZM administration. In the striatum, [123I]IBZM accumulation was significantly lower after intranasal (IN) domperidone administration than in controls 15 min after intravenous [125I]IBZM administration. [123I]IBZM SPECT was acquired with intravenous (IV) or IN domperidone administration 15 min before [123I]IBZM, and time-activity curves were obtained. In the striatum, [123I]IBZM accumulation was clearly lower in the IN group than in the control and IV groups. Time-activity curves showed no significant difference between the control and IV groups in the striatum, and values were significantly lowest during the first 10 min in the IN group. In the IN group, binding potential and % of receptor occupancy were significantly lower and higher, respectively, compared to the control and IV groups. Thus, brain-migrated domperidone inhibited D2R binding of [123I]IBZM. SPECT imaging is suitable for research to indirectly explore nose-to-brain drug delivery and locus-specific biological distribution.

Comparison of L- and D-Amino Acids for Bacterial Imaging in Lung Infection Mouse Model.

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.

A single high-dose irradiation changes accumulation of methotrexate and gene expression levels of SLC and ABC transporters in cancer cells.

Chemoradiotherapy is frequently used to treat cancer. Stereotactic body radiotherapy (SBRT) is a single high-dose radiotherapy used to treat a variety of cancers. The anticancer drug methotrexate (MTX) shows affinity for solute carrier (SLC) and ATP-binding cassette (ABC) transporters. This study investigated relationships between accumulation of methotrexate and gene expression levels of solute carrier and ATP-binding cassette transporters in cancer cells after a single and high-dose X-ray irradiation. Cancer cell lines were selected from lung and cervical cancer cell line that are commonly used for stereotactic body radiotherapy and effective with methotrexate. We examined expression levels of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, OATP1B7, and organic anion transporter (OAT)1 as solute carrier transporters and multidrug resistance-associated protein (MRP)1 and MRP2 as ATP-binding cassette transporters, using real-time polymerase chain reaction and accumulation of 3H-MTX in cancer cells after 10-Gy irradiation, assuming stereotactic body radiotherapy. Cells were divided into three groups: Control without irradiation; 4 h after irradiation; and 24 h after irradiation. In control, gene expression levels of OAT1 in all cells was below the limit of measurement. After irradiation, gene expression levels of OATP1B1/1B3/1B7 showed changes in each cell line. Gene expression levels of MRP1/2 tended to increase after irradiation. Gene expression levels of OATP1B1/1B3/1B7 were much lower than those of MRP1/2. Accumulation of 3H-MTX tended to decrease over time after irradiation. Irradiation of cancer cells thus alters gene expression levels of both solute carrier transporters (OATP1B1/1B3/1B7) and ABC transporters (MRP1/2) and decreases accumulation of 3H-MTX in cancer cells over time due to elevated expression of MRP1/2.

Inhibition of the Hepatic Uptake of 99mTc-Tetrofosmin Using an Organic Cation Transporter Blocker.

The accumulation of high levels of 99mTc-tetrofosmin (99mTc-TF) in the hepatobiliary system can lead to imaging artifacts and interference with diagnosis. The present study investigated the transport mechanisms of 99mTc-TF and attempted to apply competitive inhibition using a specific inhibitor to reduce 99mTc-TF hepatic accumulation. In this in vitro study, 99mTc-TF was incubated in HEK293 cells expressing human organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, organic anion transporter 2 (OAT2), organic cation transporter 1 (OCT1), OCT2, and Na+-taurocholate cotransporting polypeptide with or without each specific inhibitor to evaluate the contribution of each transporter to 99mTc-TF transportation. In vivo studies, dynamic planar imaging, and single photon emission computed tomography (SPECT) experiments with rats were performed to observe alterations to 99mTc-TF pharmacokinetics using cimetidine (CMT) as an OCT1 inhibitor. Time-activity curves in the liver and heart were acquired from dynamic data, and the 99mTc-TF uptake ratio was calculated from SPECT. From the in vitro study, 99mTc-TF was found to be transported by OCT1 and OCT2. When CMT-preloaded rats and control rats were compared, the hepatic accumulation of the 99mTc-TF was reduced, and the time to peak heart count shifted to an earlier stage. The hepatic accumulation of 99mTc-TF was markedly suppressed, and the heart-to-liver ratio increased 1.6-fold. The pharmacokinetics of 99mTc-TF were greatly changed by OCT1 inhibitor. Even in humans, the administration of OCT1 inhibitor before cardiac SPECT examination may reduce 99mTc-TF hepatic accumulation and contribute to the suppression of artifacts and the improvement of SPECT image quality.

Assessment of drug transporters involved in the urinary secretion of [99mTc]dimercaptosuccinic acid.

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.

Biological Distribution of Orally Administered [123I]MIBG for Estimating Gastrointestinal Tract Absorption.

Gastrointestinal tract absorption of cationic anticancer drugs and medicines was estimated using whole-body imaging following oral [123I]MIBG administration. [123I]MIBG was added to cultures of human embryonic kidney (HEK)293 cells expressing human organic anion transporting polypeptide (OATP)2B1, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cation transporter (OCT)1, OCT2, and OCT3 with and without cimetidine (an OCTN and OCT inhibitor) and L-carnitine (an OCTN inhibitor). Biodistribution analyses and single-photon emission computed tomography (SPECT) imaging in normal and dextran sodium sulfate (DSS)-induced experimental colitis mice were conducted using [123I]MIBG with and without cimetidine. [123I]MIBG uptake was significantly higher in HEK293/OCTN1, 2, and OCT1-3 cells than in mock cells. Uptake via OCTN was inhibited by L-carnitine, whereas OCT-mediated uptake was inhibited by cimetidine. Biodistribution analyses and SPECT imaging studies showed significantly lower accumulation of [123I]MIBG in the blood, heart, liver, and bladder in DSS-induced experimental colitis mice and mice with cimetidine loading compared with normal mice, whereas significantly higher accumulation in the stomach and kidney was observed after [123I]MIBG injection. [123I]MIBG imaging after oral administration can be used to estimate gastrointestinal absorption in the small intestine via OCTN and/or OCT by measuring radioactivity in the heart, liver, and bladder.

[131I]MIBG exports via MRP transporters and inhibition of the MRP transporters improves accumulation of [131I]MIBG in neuroblastoma.

INTRODUCTION: 131I-labeled m-iodobenzylguanidine ([131I]MIBG) has been used to treat neuroblastoma patients, but [131I]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 [131I]MIBG in neuroblastomas and improve accumulation by inhibition of the transporter in neuroblastomas. METHODS: [131I]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 [131I]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 [123I]MIBG with and without probenecid in SK-N-SH-bearing mice. RESULTS: [131I]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. [131I]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 [131I]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: [131I]MIBG exports via MRP1 and MRP4 in neuroblastoma. The accumulation and tumor-to-blood or muscle ratios of [131I]MIBG are improved by inhibition of MRPs with probenecid in neuroblastoma. ADVANCES IN KNOWLEDGE: [131I]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 [131I]MIBG accumulation.