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.

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.

Discovery of Carbono(di)thioates as Indoleamine 2,3-Dioxygenase 1 Inhibitors.

A structure-activity relationship study unexpectedly showed that carbonothioates 4a and 4b, obtained by a unique alkaline hydrolysis of 2-alkylthio-oxazolines 3a and 3b, respectively, are a novel scaffold for indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors. Derivatization of the carbonothioates enhanced inhibitory activity against IDO1 and cellular kynurenine production without cytotoxicity and led to the discovery of the related scaffolds carbonodithioates 5 and cyanocarbonimidodithioates 6 as IDO1 inhibitors. Incorporation of an OH group provided the most potent analogue 5i. UV-visible absorption spectroscopy of the Soret band, as well as docking and peptide mapping studies, suggested that these molecules bind to the heme in the active site of IDO1. Our unique IDO1 inhibitors are potential leads for future development.

Hydrostatic pressure promotes endothelial tube formation through aquaporin 1 and Ras-ERK signaling.

Vascular tubulogenesis is tightly linked with physiological and pathological events in the living body. Endothelial cells (ECs), which are constantly exposed to hemodynamic forces, play a key role in tubulogenesis. Hydrostatic pressure in particular has been shown to elicit biophysical and biochemical responses leading to EC-mediated tubulogenesis. However, the relationship between tubulogenesis and hydrostatic pressure remains to be elucidated. Here, we propose a specific mechanism through which hydrostatic pressure promotes tubulogenesis. We show that pressure exposure transiently activates the Ras/extracellular signal-regulated kinase (ERK) pathway in ECs, inducing endothelial tubulogenic responses. Water efflux through aquaporin 1 and activation of protein kinase C via specific G protein-coupled receptors are essential to the pressure-induced transient activation of the Ras/ERK pathway. Our approach could provide a basis for elucidating the mechanopathology of tubulogenesis-related diseases and the development of mechanotherapies for improving human health.