The Use of Carboxyfluorescein Reveals the Transport Function of MCT6/SLC16A5 Associated with CD147 as a Chloride-Sensitive Organic Anion Transporter in Mammalian Cells.

Oral drug absorption involves drug permeation across the apical and basolateral membranes of enterocytes. Although transporters mediating the influx of anionic drugs in the apical membranes have been identified, transporters responsible for efflux in the basolateral membranes remain unclear. Monocarboxylate transporter 6 (MCT6/SLC16A5) has been reported to localize to the apical and basolateral membranes of human enterocytes and to transport organic anions such as bumetanide and nateglinide in the Xenopus oocyte expression system; however, its transport functions have not been elucidated in detail. In this study, we characterized the function of MCT6 expressed in HEK293T cells and explored fluorescent probes to more easily evaluate MCT6 function. The results illustrated that MCT6 interacts with CD147 to localize at the plasma membrane. When the uptake of various fluorescein derivatives was examined in NaCl-free uptake buffer (pH 5.5), the uptake of 5-carboxyfluorescein (5-CF) was significantly greater in MCT6 and CD147-expressing cells. MCT6-mediated 5-CF uptake was saturable with a Km of 1.07 mM and inhibited by several substrates/inhibitors of organic anion transporters and extracellular Cl ion with an IC50 of 53.7 mM. These results suggest that MCT6 is a chloride-sensitive organic anion transporter that can be characterized using 5-CF as a fluorescent probe.

A Simple and Rapid Bioluminescence-Based Functional Assay of Organic Anion Transporter 1 as a D-Luciferin Transporter.

Organic anion transporter 1 (SLC22A6/OAT1) plays a key role in renal tubular excretion of endo- and exogenous anionic substances including drugs. Since the inhibition of OAT1 function by a concomitant drug may cause pharmacokinetic drug-drug interactions (DDIs) in clinical practice, an in vitro uptake study to evaluate the inhibition potency of OAT1 is useful for the prediction and avoidance of DDIs and recommended for drug candidates in drug development. In this chapter, we describe a rapid and highly sensitive functional assay of OAT1 based on bioluminescence (BL) detection using D-luciferin as a substrate in living cells. The principle of measurement simply relies on the biochemical feature of D-luciferin to be recognized as a substrate of OAT1, and the BL intensity depending on intracellular D-luciferin level and luciferase activity, thereby allowing the quantitative analysis of OAT1-mediated D-luciferin transport. The BL measurement can be completed within 1 min without experimental procedures for removing extracellular uptake solution and washing cells, both of which involve in the conventional uptake studies using isotope-labeled or fluorescent compounds. The present method is applicable to high-throughput screening to identify and avoid potential OAT1 inhibitors in drug development.

Extracorporeal membrane oxygenation may decrease the plasma concentration of remdesivir in a patient with severe coronavirus disease 2019.

Remdesivir is an antiviral drug that results in clinical improvement after five days of treatment and accelerates recovery by 31%. No studies have discussed the pharmacokinetic analysis of remdesivir in patients with severe COVID-19 requiring extracorporeal membrane oxygenation (ECMO). A 63-year-old American man who underwent mechanical ventilation and ECMO for severe COVID-19 was administered remdesivir for ten days. The loading dosage was 200 mg at 7 PM on day 12 and 100 mg daily at 0:00 PM from day 13-21, administered within 1 h. The pharmacokinetic analysis was performed. The serum creatinine concentration was within the normal range of 0.5-0.7 mg/dL during treatment. According to the pharmacokinetic analysis, the plasma concentrations of remdesivir and GS-441524 4 h after administration (C4) were 662 ng/mL and 58 ng/mL, respectively, and the concentrations 18 h after administration (C18) were 32 ng/mL and 44 ng/mL, respectively. Therefore, the half-life of remdesivir and GS-441524 was 3.2 and 35.1 h, respectively. Monitoring the plasma concentrations of remdesivir and GS-441524 in patients undergoing ECMO may be necessary.

Contamination of lenalidomide on blister packages after administration and its exposure countermeasures.

BACKGROUND: Reliable exposure control measures are needed to avoid occupational exposures from hazardous drugs. However, there is little information on blister packages concerning exposure. We investigated the contamination and exposure control methods of lenalidomide. MATERIALS AND METHODS: Nine facilities involved with the RevMate program (the Japanese REMS program) participated in this study. Blister packages (10 capsules/ sheet, no cuts) were collected from each institution after the administration of 5-mg Revlimid capsules. Additionally, the safety performance of different gloves was tested. RESULTS: A total of 18 samples were analyzed and the results revealed that all samples were contaminated with lenalidomide. Our questionnaire revealed that all pharmacists handled the blister packages with their bare hands when they were checking the remaining capsules of lenalidomide. We analyzed gloves made from four different materials (nitrile, polyvinyl chloride, latex, and polyethylene) and found no permeability in any glove type. CONCLUSION: We conclude that the spent blister package is a potential source of exposure to lenalidomide. All medical staff and caregivers should wear gloves when they handle lenalidomide.

Influene of Pharmaceutical Excipients on the Membrane Transport of a P-glycoprotein Substrate in the Rat Small Intestine.

BACKGROUND AND OBJECTIVES: Generic drugs are generally used worldwide because of affordability compared to brand-name drugs. One of the main differences between brand-name and generic drugs is pharmaceutical excipients. We previously reported the effects of pharmaceutical excipients on the membrane permeation of drugs via the paracellular and transcellular routes, which are passive transport routes. P-glycoprotein (P-gp) is a typical ATP-binding cassette transporter and is mostly responsible for drug-drug interactions involving transporters. In the present study, rhodamine 123 (Rho123) was selected as the P-gp substrate, and the effects of pharmaceutical excipients on its membrane transport in the rat jejunum and ileum were examined. METHODS: Twenty major pharmaceutical excipients widely used in the pharmaceutical industry were selected. The in vitro diffusion chamber method using the rat jejunum and ileum was employed to investigate the effects of pharmaceutical excipients on the membrane permeation of Rho123. RESULTS: The results obtained showed that the membrane permeability of Rho123 significantly (P < 0.05) changed under certain dosage conditions of pharmaceutical excipients such as sodium carboxymethyl starch, pullulan, glyceryl monostearate and so on. Furthermore, the effects of pharmaceutical excipients were site specific in the small intestine. CONCLUSION: The present results demonstrated that some pharmaceutical excipients altered the membrane permeability of Rho123 in the rat small intestine.

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23 µM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36 µM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.

Effects of pharmaceutical excipients on membrane permeability in rat small intestine.

Pharmaceutical excipients should not disturb the effects of drug therapy. In recent years, however, it has been reported that excipients induce some changes to the tight junction (TJ) and P-glycoprotein (P-gp), which can affect drug disposition. In this study, we examined the effects of 20 common pharmaceutical excipients from different classes on mucosal membrane and the differences of such effects among regions of the small intestine. We used the in vitro sac method in rat jejunum and ileum to study the effects of excipients on the membrane permeation of 5(6)-carboxyfluorescein (5-CF). 5-CF was used as a model of water-soluble compounds. In some dosage conditions of methyl-ß-cyclodextrin, the membrane permeability of 5-CF was significantly increased in the jejunum, but such change was not observed in the ileum. Similarly, in the cases of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and croscarmellose sodium, the membrane permeability of 5-CF was significantly increased in the jejunum, but no change was observed in the ileum. On the other hand, in both the jejunum and the ileum, the membrane permeation of 5-CF was decreased with 0.02% (w/v) hydroxypropyl cellulose, but significantly increased with it at 0.20% (w/v). It was shown that excipients affected the membrane permeability of water-soluble compounds via the paracellular route, and these effects on absorption differed among regions of the small intestine. Moreover, in the case of 20 excipients, not only an increase in membrane permeability but also a decrease was observed. Therefore, it was suggested that a more effective formulation could be designed by changing the combination of excipients.