Aminoglycosides such as gentamicin and amikacin are the most commonly used antibiotics worldwide in the treatment of Gram-negative bacterial infections. However, serious complications like nephrotoxicity and ototoxicity are dose-limiting factors in the use of aminoglycosides. A relatively large amount of the intravenously administered dose is accumulated in the kidney (about 10% of dose), whereas little distribution of aminoglycosides to other tissues is observed. Aminoglycosides are taken up in the epithelial cells of the renal proximal tubules and stay there for a long time, resulting in nephrotoxicity. Acidic phospholipids are considered as a binding site for aminoglycosides in the brush-border membrane of the proximal tubular cells. More recently, it has been reported that megalin, a giant endocytic receptor abundantly expressed at the apical membrane of renal proximal tubules, plays an important role in binding and endocytosis of aminoglycosides in the proximal tubular cells. The elucidation of the aminoglycoside-binding receptor would help design a strategy to prevent against aminoglycoside-induced nephrotoxicity. In this review, we summarize recent advances in the understandings of the molecular mechanisms responsible for renal accumulation of aminoglycosides, especially megalin-mediated endocytosis. In addition, approaches toward prevention of aminoglycoside-induced nephrotoxicity are discussed, based on the molecular mechanisms of the renal accumulation of aminoglycosides.

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  In the last decade, many organic anion transporters have been isolated, characterized their distribution and substrates. The recently identified organic anion transporter family OATP (organic anion transporting polypeptide)/LST (liver-specific transporter) family, transport bile acids, hormones as well as eicosanoids, various compounds (BSP, HMG-CoA reductase inhibitor, angiotensin converting enzyme inhibitor, etc.). The isolation of the family revealed that not only hydrophilic compounds, drugs and hormones of lipophilic nature need a membrane transport system to penetrate cell membrane. In this family, the nomenclature becomes very complicated and the physiological role of this family is still unclear except about few organs such as the brain, liver and kidney. Even in such organs, the co-existence of the OATP/LST family and similar substrate specificity hamper the progress and clear characterization identifying the real role of the transporter family. Here, recent progress and an insight of this field are reviewed.

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  The organic cation/carnitine transporter OCTN2 transports carnitine in a sodium-dependent manner, whereas it transports organic cations sodium-independently. To elucidate the functional domain in OCTN2, we constructed chimeric proteins of human OCTN2 (hOCTN2) and mouse OCTN3 (mOCTN3) and introduced mutations at several amino acids conserved among human, rat and mouse OCTN2. We found that transmembrane domains (TMD) 1-7 are responsible for organic cation transport and for sodium dependence in carnitine transport. Within TMD1-7, Q180 and Q207 of hOCTN2 are the critical amino acids for the sodium dependence, and double mutation of Q180 and Q207 resulted in minimal change in transport activity when sodium was removed from the uptake medium. We propose that sodium-dependent affinity for carnitine is dependent on sodium recognition by these critical amino acids in hOCTN2, whereas carnitine transport by OCTN2 requires functional linkage between TMD1-7 and TMD11.

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  The objective of this study is to evaluate the effect of acute renal or hepatic failure on the intestinal absorption of tacrolimus. Simultaneous perfusion study in rat small intestine revealed that the extent of absorption into blood vessels was decreased in the jejunum and the ileum of rat of acute renal failure due to the decrease in the uptake of tacrolimus into enterocytes. In contrast, there observed no significant changes in tacrolimus absorption in rat of acute hepatic failure. Since it has been reported that tacrolimus absorption is regulated mainly by Cytochrome P-450 (CYP) mediated metabolism in the jejunum, but by P-glycoprotein (P-gp) mediated efflux in the ileum, these factors might contribute to the changes in intestinal absorption of tacrolimus in rat of acute renal failure. Enzyme inhibitor, ketoconazole, was co-perfused with tacrolimus to specify the effect of CYP and P-gp. However, since ketoconazole failed to recover the permeability in the jejunum and ileum of rat of acute renal failure, it is considered that the changes in CYP or P-gp functions might not be involved in the decreased uptake of tacrolimus. This type of kinetic study in rats should be valuable to identify the precise mechanisms of drug absorption and the effects of various diseases on it, such as acute renal or hepatic failure.

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  To investigate the regulation of drug absorption from the small intestine by the enteric nervous system (ENS), the vascular-luminal perfusion study and the in-vitro transport study were performed by employing phenol red as a poorly absorbable model compound. The effect of ENS on the intestinal absorption of phenol red was examined by adding epinephrine, an adrenergic agonist, or bethanechol, a cholinergic agonist into the vascular perfusate in the vascular-luminal perfused rat small-intestine preparation. The viability of the perfused intestine was checked by the recovery of the vascular perfusate, net water flux and absorbability of antipyrine, a well absorbable drug, and it was confirmed that the function of the perfused small-intestine preparation was maintained for at least 1 hr. The effect of epinephrine or bethanechol on the function of the small intestine was recognized as the increase in net water absorption, or the promotion of the water secretion, respectively. These phenomena are ones that are typically observed when adrenergic or cholinergic neuron is stimulated. Then, we investigated the small-intestinal absorption of phenol red in the vascular-luminal perfused preparation. Absorption clearance (CL_abs) of phenol red was gradually increasing during the perfusion for 1 hr, but the 20-min vascular perfusion with the perfusate containing epinephrine made CL_abs of phenol red constant and significantly lower than those for control study. Furthermore, after the perfusate was changed with the one without any agonist, again, CL_abs of phenol red started to increase. These results clearly indicate that the stimulation of adrenergic neuron by epinephrine leads to the decrease in the small-intestinal absorption of phenol red. On the other hand, the vascular perfusion of bethanechol resulted in the increase in CL_abs of phenol red comparing to the control study. Removing bethanechol from the vascular perfusate decreased CL_abs of phenol red, again. The in-vitro transport study using the isolated jejunum sheet also showed that epinephrine in the serosal solution significantly decreased the transport of phenol red, which can be ascribed to the paracellular pathway tightened by the action of epinephrine because of the increase in transmucosal electrical resistance (TER). On the other hand, although the effect of bethanechol on both the transport of phenol red and TER was not statistically significant, the transport of phenol red tended to increase and the values of TER are smaller than those of control study.

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  TAS-102, a new oral drug, is composed of an antitumor drug, α,α,α-trifluorothymidine (FTD), and its metabolic inhibitor, 5-chloro-6-(2-iminopyrrolidine-1-yl)methyl-2,4(1H,3H)-pyrimidinedione hydrochloride (TPI). It has been reported that the oral administration of TAS-102 increases the AUC of FTD in rodents and monkeys in different manners. In this study, a pharmacokinetic model was developed, in an attempt to evaluate the bioavailability of FTD in these animals after the co-administration of TPI. Since TPI inhibits FTD metabolism competitively, a time-dependent as well as concentration-dependent model for the hepatic intrinsic clearance of FTD was developed including the time courses of both FTD and TPI. Based on this modeling, we were able to quantitatively explain the TPI dose-dependent enhancement of AUC of FTD in monkeys, while little increase was observed in rats. These results are consistent to observations that thymidine phosphorylase (TPase) is predominantly expressed in monkeys; while uridine phosphorylase (UPase) is superior to TPase in rats. Since TPase is also predominantly expressed in humans, the pharmacokinetic model developed in this study can be used to explain the bioavailability of TAS-102 in humans.

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  Regioselective sulfation of the phytoestrogens daidzein (DZ, 7,4′-dihydroxyisoflavone) and genistein (GS, 5,7,4′-trihydroxyisoflavone) was investigated using human liver cytosol and purified recombinant human sulfotransferase (SULT) isoforms, SULT1A1, SULT1A3, SULT2A1, and SULT1E1. 7-Position-preferential sulfation of DZ and GS was observed in human hepatic cytosols from 3 male and 3 female subjects. Average ratios for 7- to 4′-sulfate formation were 4.5:1 from DZ and 8.4:1 from GS in these human liver cytosols. Apparent K_m values for the 7- and 4′-sulfation of DZ and GS by these cytosols were similar and in a range from 0.46 to 0.66 μM. All recombinant human SULTs had activity for 7- and 4′-sulfation of these phytoestrogens except for 7-sulfating activity of SULT1A3. SULT1A1 and SULT1E1 exhibited much higher catalytic efficiency, k_cat/K_m, for 7- and 4′-sulfation of these substrates than did the other two, SULT1A3 and SULT2A1. SULT1A1 showed K_m values of 0.47 and 0.52 μM for the mono-sulfation of DZ and GS, respectively, which were very similar to those of human cytosol. The observed k_cat/K_m indicated that SULT1A1 catalyzed 7-sulfation of DZ and GS at rates 4.4- and 8.8-fold higher, respectively, than such 4′-sulfation. However, with SULT1E1, catalytic efficiency was very similar for the sulfation of both positions. These data strongly suggest that SULT1A1 plays a major role in monosulfation of the phytoestrogens and determines the regioselectivity of sulfation in human hepatic cytosol. A kinetic study for 7,4′-disulfate formation of DZ and GS from their 7- and 4′-monosulfates indicated that SULT1E1 most efficiently catalyzed both reactions among human SULTs.

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  Fast disintegrating lansoprazole tablet (LFDT) has been developed as a multiple unit formulation and evaluated using human subjects as compared to the conventional lansoprazole (LPZ) capsule containing enteric coated granules. Twelve healthy male volunteers, who were confirmed as extensive metabolizers (EMs) based on the plasma levels of LPZ sulphone metabolite, were enrolled into the study and genotype of CYP2C19 was confirmed. They kept 30 mg LFDT in their mouths for 2 min and the saliva was recovered without swallow. Eight subjects did not show LPZ in their serum after intake. Although LPZ was detected in 4 subjects' serum, their concentrations were less than 5 ng/mL. LPZ was thought to be not absorbed from the oral cavity. LFDT was orally administered to 12 healthy male EMs at two doses, 15 mg and 30 mg, and serum LPZ concentrations were measured. The mean C_max and AUC_0-24 were 474.1±254.0 ng/mL and 1105.3±1101.4 ng·h/mL (15 mg) and 992.8±384.3 ng/mL and 2216.5±1270.1 ng·h/mL (30 mg). By comparing to that obtained after oral administration of the same doses of LPZ capsule, serum LPZ concentration vs. time curve was almost the same level, i.e., C_max and AUC_0-24 did not have significant differences. From these results, LFDT has been shown to be equivalent to LPZ capsule and will show the same acid suppressing effects in the clinical situation.

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  We sequenced all nine exons and exon-intron junctions of the cytochrome P450 2C19 (CYP2C19) gene from a Japanese subject with a lowered capacity of CYP2C19-mediated 4′-hydroxylation after an oral administration of mephobarbital. We found a novel single nucleotide polymorphism (SNP) of CYP2C19 gene as follows: SNP, 040110MoritaJ001; GENENAME: CYP2C19; ACCESSION NUMBER: NT_030059.8; LENGTH; 25 bases; 5′-GAGGGCCTGGCCC/TGCATGGAGCTGT-3′. The SNP (168946C>T) induced an amino acid alteration (Arg442Cys) located in exon 9 close to the heme-binding region of CYP2C19, which may result in the decrease in the catalytic properties of CYP2C19. A new allele having this SNP was designated as CYP2C19*16.

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  Thirty-three genetic variations including fourteen novel ones were found in the SLC22A2 gene from 116 Japanese individuals. The novel variations were as follows: 596C>T (MPJ6_OC2003), 602C>T (MPJ6_OC2004), IVS5+20A>G (MPJ6_OC2010), IVS5-84_-83insG (MPJ6_OC2013), IVS6+30T>C (MPJ6_OC2014), IVS6+146G>T (MPJ6_OC2016), IVS6+179G>T (MPJ6_OC2017), IVS6-16delT (MPJ6_OC2018), 1920G>A (MPJ6_OC2022), 2153G>A (MPJ6_OC2026), 2157C>T (MPJ6_OC2028), 2306T>C (MPJ6_OC2031), 2342+5T>C (the last nucleotide number of mRNA+the position in the 3′-flanking region; MPJ6_OC2032) and 2342+127T>C (MPJ6_OC2033). Six variations were located in the exons, four of which were in the 3′-untranslated region (3′-UTR) of exon 11; six were in the introns; and two were in the 3′-flanking region. The frequencies were 0.802 for IVS5-84_-83insG, 0.013 for 602C>T, 0.009 for 596C>T, and 0.004 for the other 11 variations. Among them, 596C>T and 602C>T resulted in amino acid substitutions (Thr199Ile and Thr201Met, respectively).

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