Junya Nagai

Interaction with P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells

The effect of cytochrome P-450 3A (CYP3A) substrates (erythromycin, midazolam) and an inhibitor (ketoconazole) on P-glycoprotein-mediated transport was studied in Caco-2, the human colon adenocarcinoma cell line expressing various functions of differentiated intestinal epithelial cells. The involvement of P-glycoprotein in the transport of these drugs was also examined. The basal-to-apical transport of rhodamine 123, a P-glycoprotein substrate, was inhibited by erythromycin, midazolam and ketoconazole, as well as by P-glycoprotein inhibitors such as verapamil. The apical-to-basal transport of rhodamine 123 was increased by these drugs. The transepithelial transport of erythromycin and midazolam, but not of ketoconazole, was much greater from the basal to apical side than from the apical to basal side. The inhibitory effect of verapamil was observed on the basal to apical transport of erythromycin, but not on midazolam and ketoconazole transport. In conclusion, erythromycin, midazolam and ketoconazole could interact with P-glycoprotein-mediated transport, and P-glycoprotein could be, at least in part, involved in the transport of erythromycin, but not of midazolam and ketoconazole, in the intestinal epithelia.

Novel Polymyxin Derivatives Carrying Only Three Positive Charges Are Effective Antibacterial Agents

ABSTRACT The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-d-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.

Expression and function of P-glycoprotein in rats with glycerol-induced acute renal failure

The effect of glycerol-induced acute renal failure on P-glycoprotein expression and function was evaluated in rats. The in vivo function of P-glycoprotein was evaluated by measuring renal secretory and biliary clearance and brain distribution of rhodamine 123 (Rho-123), a P-glycoprotein substrate, under a steady-state plasma concentration. In acute renal failure rats, the P-glycoprotein level increased 2.5-fold in the kidney, but not in the liver and brain. In contrast, P-glycoprotein function in these tissues was suppressed. Interestingly, not only the renal but also the biliary clearance of Rho-123 was correlated with the glomerular filtration rate. In Caco-2 cells, plasma from renal failure rats exhibited a greater inhibitory effect on P-glycoprotein-mediated transport of Rho-123 than did plasma from control rats. In conclusion, P-glycoprotein function was systemically suppressed in acute renal failure, even though the level of P-glycoprotein remained unchanged or rather increased. This may be due to the accumulation of some endogenous P-glycoprotein substrates/modulators in the plasma in disease states.

Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells

AIMS The purpose of this study was to characterize the internalization mechanism of human IgG into the epithelial cells of human small intestine, employing human intestinal epithelial cell line Caco-2 as an in vitro model system. MAIN METHODS Real-time PCR analysis and uptake studies of fluorescein isothiocyanate-labeled IgG (FITC-IgG) from human serum were performed using Caco-2 cells. KEY FINDINGS Real-time PCR analysis showed that mRNA level of the neonatal Fc receptor (FcRn) was increased during the differentiation process in Caco-2 cells. The binding of FITC-labeled human IgG to the membrane surface of Caco-2 cells increased with a decrease in pH of incubation buffer. The uptake of FITC-IgG was also stimulated at acidic pH and was time-dependent. The binding and uptake of FITC-IgG at pH 6.0 was partially, but significantly, decreased by human gamma-globulin in a concentration-dependent manner. A mixture of metabolic inhibitors (sodium azide and 2-deoxyglucose) significantly inhibited the uptake, but not the binding, of FITC-IgG. In addition, endosomal acidification inhibitors such as bafilomycin A(1) and chloroquine significantly increased the accumulation of FITC-IgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) and caveolin-dependent endocytosis inhibitors (nystatin and indomethacin) did not decrease the uptake of FITC-IgG at pH 6.0. In contrast, macropinocytosis inhibitors such as cytochalasin B and 5-(N-ethyl-N-isopropyl) amiloride significantly decreased the uptake of FITC-IgG at pH 6.0. SIGNIFICANCE The internalization of human IgG in human intestine might be, at least in part, due to FcRn-mediated endocytosis, which could occur by a process other than clathrin- and caveolin-dependent mechanisms.

Drug Metabolism: Renal Excretion of Rhodamine 123, a P‐Glycoprotein Substrate, in Rats with Glycerol‐induced Acute Renal Failure

To clarify renal handling of rhodamine 123, a substrate for P‐glycoprotein, in normal and diseased states, in‐vivo clearance studies were performed with normal rats and rats with glycerol‐induced acute renal failure.

Transport of prostaglandin E1 across the blood-brain barrier in rats

The transport of prostaglandin E1 (PGE1) across the blood‐brain barrier (BBB) was characterized using an in‐situ rat brain perfusion technique. The uptake of [3H]PGE1 was not affected by short‐chain monocarboxylic acids (butyric acid and valeric acid). On the other hand, uptake of [3H]PGE1 was significantly inhibited by medium‐chain monocarboxylic acids such as hexanoic acid, enanthic acid and octanoic acid. These medium‐chain monocarboxylic acids showed a more potent inhibitory effect on [3H]PGE1 uptake with increasing number of carbon atoms. In contrast, there was no decrease in [3H]PGE1 transport by any dicarboxylic acids with 5–8 carbon atoms. Valproic acid decreased [3H]PGE1 uptake, whereas p‐aminohippuric acid, a substrate for the organic anion transporter family, did not inhibit [3H]PGE1 transport. Bromocresol green, an inhibitor of prostaglandin transporter (PGT), strongly decreased [3H]PGE1 transport across the BBB. In addition, digoxin and taurocholate, substrates for organic anion transporting polypeptide subtype 2 (Oatp2), significantly inhibited [3H]PGE1 uptake. RT‐PCR analysis revealed that PGT mRNA and Oatp2 mRNA are expressed in a capillary‐rich fraction from rat brain. Thus, it is suggested that PGE1 transport across the BBB is mediated by some specific transport systems, possibly by the members of the Oatp family.

Expression and function of P-glycoprotein in rats with carbon tetrachloride-induced acute hepatic failure

Acute hepatic failure was induced experimentally in rats by intraperitoneal injection of 2.5 mL kg−1 carbon tetrachloride (CCl4), and the effects on the expression and function of P‐glycoprotein in the liver, kidney and brain were evaluated. The CCl4 injection significantly increased the indicators of hepatic function (glutamate oxaloacetate transaminase, glutamate pyruvate transaminase), but not of renal function (blood urea nitrogen, glomerular filtration rate). In rats with acute hepatic failure, the hepatic P‐glycoprotein concentration increased 1.5‐fold and the ATP concentration decreased to approximately 40% that in control rats. In contrast, P‐glycoprotein concentrations in the kidney and brain and ATP concentrations in the kidney remained unchanged. The in‐vivo P‐glycoprotein function in these tissues was suppressed as evaluated by biliary and renal secretory clearances and brain distribution of rhodamine 123, a P‐glycoprotein substrate. These findings suggest that factors other than P‐glycoprotein concentration are involved in the systemic suppression of P‐glycoprotein function in diseased rats. In Caco‐2 cells, plasma collected from CCl4‐treated rats exhibited a greater inhibitory effect on P‐glycoprotein‐mediated transport of rhodamine 123 than that from control rats, suggesting the accumulation of an endogenous P‐glycoprotein substrate/inhibitor in the plasma of diseased rats. In fact, the plasma concentration of corticosterone, an endogenous P‐glycoprotein substrate, increased 2‐fold in CCl4‐treated rats compared with control rats. It was demonstrated that P‐glycoprotein function is systemically suppressed in rats with CCl4‐induced acute hepatic failure, not only in the target organ (liver), but also in other organs (kidney and brain), although the P‐glycoprotein concentration remained unchanged in the kidney and brain, and increased in the liver. In the systemic suppression of the P‐glycoprotein function in the diseased state, the alteration of plasma concentrations or components of endogenous P‐glycoprotein‐related compounds, such as corticosterone, would likely be involved.

Interaction of valproic acid and carbapenem antibiotics with multidrug resistance-associated proteins in rat erythrocyte membranes

We recently reported that the decrease in plasma valproic acid (VPA) level by carbapenem antibiotics (CPs) may partly be due to the increased erythrocyte distribution of VPA. In order to clarify the mechanisms underlying altered VPA distribution in erythrocytes, we examined the role of multidrug resistance-associated proteins (Mrps). The uptake of 2,4-dinitrophenyl-S-glutathione (DNP-SG), a substrate of Mrps, by inside-out vesicles (IOVs) prepared from rat erythrocytes was an ATP-dependent, active process. DNP-SG uptake was mediated by high- and low-affinity transport systems, and was inhibited by various Mrp inhibitors such as probenecid and indomethacin. Glutathione stimulated only the high-affinity transport system. VPA inhibited the low-affinity transport of DNP-SG, while panipenem, a CP, inhibited both high- and low-affinity transport. ATP-dependent, Mrp-mediated transport of methotrexate, another Mrp substrate, in IOVs was also observed, and VPA and various CPs inhibited the transport. The uptake of [(3)H]VPA was examined, and found to be ATP-dependent. ATP-dependent uptake of [(3)H]VPA was inhibited by Mrp inhibitors and panipenem, while the inhibition was not observed in the absence of ATP. These results indicate that VPA and CPs interact with Mrp-mediated transport in erythrocyte membranes, and VPA itself is transported by Mrps, which is inhibited by panipenem. Thus, the increased erythrocyte distribution of VPA by CPs observed under in vivo conditions may partly be explained by their interaction with Mrps in erythrocyte membranes.

Role of intestinal efflux transporters in the intestinal absorption of methotrexate in rats

The role of intestinal efflux transporters such as P‐glycoprotein (P‐gp), breast cancer resistance protein (BCRP) and multidrug resistance‐associated proteins (MRPs) in intestinal absorption of methotrexate was examined in rats. In everted intestine, the mucosal efflux of methotrexate after application to serosal side was higher in jejunum than ileum, and the efflux in jejunum was suppressed by pantoprazole, a BCRP inhibitor, and probenecid, an MRP inhibitor, but not by verapamil, a P‐gp inhibitor. The mucosal methotrexate efflux in ileum was suppressed by pantoprazole, but not by other inhibitors. On the other hand, the serosal efflux of methotrexate after application to mucosal side was greater in ileum than jejunum, and was suppressed by probenecid. In in‐vivo rat studies, the intestinal absorption of methotrexate was significantly higher when methotrexate was administered to ileum than jejunum. Pantoprazole increased methotrexate absorption from jejunum and ileum. Probenecid increased the absorption of methotrexate from jejunum but decreased the absorption from ileum, as evaluated by peak plasma methotrexate levels. In conclusion, BCRP and MRPs are involved in the regional difference in absorption of methotrexate along the intestine, depending on their expression sites.

Inhibition of PAH transport by parathyroid hormone in OK cells: involvement of protein kinase C pathway

We have previously shown that the p-aminohippurate (PAH) transport system in OK kidney epithelial cell line is under the regulatory control of protein kinase C. Parathyroid hormone (PTH) could activate protein kinase C, as well as protein kinase A, in OK cells. In the present study, the effect of PTH on PAH transport was studied in OK cells. PTH inhibited the transcellular transport of PAH from the basal to the apical side, as well as the accumulation of PAH in OK cells. Basolateral PAH uptake was inhibited by PTH in a dose- and time-dependent manner. Protein kinase A activators did not affect the transcellular transport or the accumulation of PAH. The PTH-induced inhibition of the accumulation of PAH was blocked by a protein kinase C inhibitor staurosporine. These results suggest that PTH inhibits the PAH transport in OK cells and that the messenger system mediated by protein kinase C, not protein kinase A, plays an important role in the regulation of PAH transport by PTH.We have previously shown that the p-aminohippurate (PAH) transport system in OK kidney epithelial cell line is under the regulatory control of protein kinase C. Parathyroid hormone (PTH) could activate protein kinase C, as well as protein kinase A, in OK cells. In the present study, the effect of PTH on PAH transport was studied in OK cells. PTH inhibited the transcellular transport of PAH from the basal to the apical side, as well as the accumulation of PAH in OK cells. Basolateral PAH uptake was inhibited by PTH in a dose- and time-dependent manner. Protein kinase A activators did not affect the transcellular transport or the accumulation of PAH. The PTH-induced inhibition of the accumulation of PAH was blocked by a protein kinase C inhibitor staurosporine. These results suggest that PTH inhibits the PAH transport in OK cells and that the messenger system mediated by protein kinase C, not protein kinase A, plays an important role in the regulation of PAH transport by PTH.