Abstract: Responsible factors in progressive tubular dysfunction in chronic renal failure have not been fully identified. In the present study, we hypothesized that the mammalian target of rapamycin, mTOR, was a key molecule in the degenerative and progressive tubular damage in chronic renal failure. Everolimus, an mTOR inhibitor, was administered for 14 days in 5/6 nephrectomized (Nx) rats at 2 and 8 weeks after renal ablation. Marked activation of the mTOR pathway was found at glomeruli and proximal tubules in remnant kidneys of Nx rats. The reduced expression levels of the phosphorylated S6 indicated the satisfactory pharmacological effects of treatment with everolimus for 14 days. Everolimus suppressed the accumulation of smooth muscle alpha actin, infiltration of macrophages and expression of kidney injury molecule-1 in the proximal tubules. In addition, everolimus-treatment restored the tubular reabsorption of albumin, and had a restorative effect on the expression levels of membrane transporters in the polarized proximal tubular epithelium, when its administration was started at 8 weeks after Nx. These results indicate that the constitutively activated mTOR pathway in proximal tubules has an important role in the progressive tubular dysfunction, and that mTOR inhibitors have renoprotective effects to improve the proximal tubular functions in end-stage renal disease.
Abstract: Multidrug and toxin extrusions (MATE1/SLC47A1 and MATE2-K/SLC47A2) play important roles in the renal excretion of metformin. We have previously identified the nonsynonymous MATE variants with functional defects at low allelic frequencies. The purpose of this study was to evaluate the effects of heterozygous MATE variants on the disposition of metformin in mice and humans. Pharmacokinetic parameters of metformin in Mate1(+ or -) heterozygous mice were comparable with those in Mate1(+ or +) wild-type mice. Among 48 Japanese diabetic patients, seven patients carried heterozygous MATE variant and no patient carried homozygous MATE variant. There was no significant difference in oral clearance of metformin with or without heterozygous MATE variants. In addition, creatinine clearance, but not heterozygous MATE variants, significantly improved the model fit of metformin clearance by statistical analysis using the nonlinear mixed-effects modeling program. In conclusion, heterozygous MATE variants could not influence the disposition of metformin in diabetic patients.
Abstract: INTRODUCTION:: Although erlotinib, an orally active and selective tyrosine kinase inhibitor of epidermal growth factor receptor, is mainly metabolized in the liver, its effectiveness and safety for patients with chronic renal failure (CRF) undergoing hemodialysis (HD) has not been reported. Thus, we investigated the pharmacokinetics (PK) of erlotinib and its active metabolite OSI-420 in such patients with nonsmall cell lung cancer (NSCLC). METHOD:: We administered 150 mg erlotinib daily to two patients with NSCLC and CRF undergoing HD (HD group) and five patients with NSCLC and normal organ function (control group) and analyzed the PK of erlotinib and OSI-420. In the HD group, PK analyses were performed on day 1 (off HD), day 8 (off HD), and day 9 (on HD) after starting administration of erlotinib, and in the control group, they were performed on day 1 and day 8. RESULTS:: In the HD group, there were little differences in the PK data between day 8 and day 9. The PK data on day 1 and day 8 of the HD group were also similar to those of the control group. There were no serious adverse events in any cases, and one of the HD patients achieved partial response. CONCLUSION:: Erlotinib was hardly affected by renal function and HD, which confirms the effectiveness and safety of erlotinib treatment in patients with NSCLC and CRF undergoing HD. Erlotinib can become one treatment option for such patients.
Abstract: To clarify the specific molecular events of progressive tubular damage in chronic renal failure (CRF), we conducted microarray analyses using isolated proximal tubules from subtotally nephrectomized (Nx) rats as a model of CRF. Our results clearly demonstrated time-dependent changes in gene expression profiles localized to proximal tubules. The expression of mitosis-specific genes Cyclin B2 and Cell division cycle 2 (Cdc2) was significantly and selectively increased in the proximal tubules during the compensated period but decreased to the basal level in the end-stage period. Administration of everolimus, a potent inhibitor of mammalian target of rapamycin, markedly reduced compensatory hypertrophy and hyperplasia of epithelial cells, which was accompanied by complete abolishment of the expression of Cyclin B2 and Cdc2 enhancement; renal function was then severely decreased. Treatment with the Cdc2 inhibitor, 2-cyanoethyl alsterpaullone, clearly decreased epithelial cell hyperplasia, based on staining of phosphorylated histone H3 and Ki-67, while hypertrophy was not inhibited. In conclusion, we have demonstrated roles of Cyclin B2 and Cdc2 in the epithelial hyperplasia in response to Nx. These results advanced the knowledge of the contribution of cell cycle regulators, especially M phase, in pathophysiology of tubular restoration and/or degeneration, and these two molecules are suggested to be a marker for the proliferation of proximal tubular cells in CRF. Key words: Proximal tubule, Microarray, Cell Cycle, G2-M.
Abstract: We evaluated the stability of temozolomide, an alkylating agent, in solutions after opening the capsule. First, we established an analytical method for determination of temozolomide concentration by HPLC. The calibration curve for temozolomide was linear between 0.5-20 microg/ml (r=0.999). We then evaluated the stability of temozolomide in each buffer solution (pH 2-9) for 30 min. Temozolomide was decomposed pH-dependently between pH 7 and 9, and completely decomposed at pH 9. Temozolomide in several drinking water samples and beverages was decomposed according to their pH values. We also examined the time-dependent degradation of temozolomide in different pH solutions. Temozolomide started to decompose at 5 min in alkaline and neutral solutions, whereas 90% of temozolomide remained intact in acidic solution at 60 min. These results indicate that the stability of temozolomide after opening the capsule is affected by pH of solvents, and temozolomide is almost stable in acidic solutions.
Abstract: Multidrug and toxin extrusion 1 (MATE1/SLC47A1) is important for excretion of organic cations in the kidney and liver, where it is located on the luminal side. Although its functional and regulatory characteristics have been clarified, its pharmacokinetic roles in vivo have yet to be elucidated. In the present study, to clarify the relevance of MATE1 in vivo, targeted disruption of the murine Mate1 gene was carried out. The lack of Mate1 expression in the kidney and liver was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. The mRNA levels of other organic cation transporters such as Octs did not differ significantly between wild-type [Mate1(+/+)] and Mate1 knockout [Mate1(-/-)] mice. It is noteworthy that the Mate1(-/-) mice were viable and fertile. Pharmacokinetic characterization was carried out using metformin, a typical substrate of MATE1. After a single intravenous administration of metformin (5 mg/kg), a 2-fold increase in the area under the blood concentration-time curve for 60 min (AUC(0-60)) of metformin in Mate1(-/-) mice was observed. Urinary excretion of metformin for 60 min after the intravenous administration was significantly decreased in Mate1(-/-) mice compared with Mate1(+/+) mice. The renal clearance (CL(ren)) and renal secretory clearance (CL(sec)) of metformin in Mate1(-/-) mice were approximately 18 and 14% of those in Mate1(+/+) mice, respectively. This is the first report to demonstrate an essential role of MATE1 in systemic clearance of metformin.
Abstract: BACKGROUND: Gene polymorphisms of the UDP-glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) contribute to individual variations in adverse events among patients administered irinotecan, and the distribution of the polymorphisms shows large interethnic differences. Variation in the solute carrier organic anion-transporter family, member 1B1 (SLCO1B1) gene also has a significant effect on the disposition of irinotecan in Asian cancer patients. In the present study, we evaluated the association of genetic polymorphisms of UGT1A1 and SLCO1B1 with irinotecanrelated neutropenia in Japanese cancer patients. METHODS: One hundred and thirty-five consecutive patients treated with irinotecan were enrolled. Genotypes of UGT1A1 (*60, *28, *6, and *27) and SLCO1B1 (*1b, *5, and haplotype *15) were determined by direct sequencing. Severe neutropenia refers to events observed during the first cycle of irinotecan treatment. RESULTS: Severe neutropenia was observed in 29 patients (22%). Six patients were homozygous and 48 heterozygous for UGT1A1*6. Only 1 patient was homozygous for UGT1A1*28. Homozygosity for UGT1A1*6 was associated with a high risk of severe neutropenia (odds ratio [OR], 7.78; 95% confidence interval [CI], 1.36 to 44.51). No significant association was found between severe neutropenia and other UGT1A1 polymorphisms or SLCO1B1 polymorphisms. CONCLUSION: These findings suggest that the UGT1A1*6 polymorphism is a potential predictor of severe neutropenia caused by irinotecan in Japanese cancer patients.
Abstract: In human proximal tubules, organic cations are taken up from blood into cells by human organic cation transporter 2 [hOCT2/solute carrier (SLC) 22A2] and then eliminated into the lumen by apical H(+)/organic cation antiporters, human multidrug and toxin extrusion 1 (hMATE1/SLC47A1) and hMATE2-K (SLC47A2). To evaluate drug interactions of cationic drugs in the secretion process, epithelial cells engineered to express both hOCT2 and hMATE transporters are required to simultaneously evaluate drug interactions with renal basolateral and apical organic cation transporters. In the present study, therefore, we assessed the drug interaction between cimetidine and metformin with double-transfected Madin-Darby canine kidney cells stably expressing both hOCT2 and hMATE1 as an in vitro model of the proximal tubular epithelial cells. The basolateral-to-apical transport and intracellular accumulation of [(14)C]metformin by a double transfectant were markedly inhibited by 1 mM cimetidine at the basolateral side. On the other hand, 1 microM cimetidine at the basolateral side moderately decreased the basolateral-to-apical transport of [(14)C]metformin and significantly increased the intracellular accumulation of [(14)C]metformin from the basolateral side, suggesting that cimetidine at a low concentration inhibits apical hMATE1, rather than basolateral hOCT2. Actually, in concentration-dependent inhibition studies by a single transporter expression system, such as human embryonic kidney 293 stably expressing hMATE1, hMATE2-K, or hOCT2, cimetidine showed higher affinity for hMATEs than for hOCT2. These results suggest that apical hMATE1 is involved in drug interactions between cimetidine and cationic compounds in the proximal tubular epithelial cells.
Abstract: To assess the effects of intestinal cytochrome P450 2C19 on the interaction between tacrolimus and proton pump inhibitors, we examined the concentration/dose ratio [(ng/ml)/(mg/day)] of tacrolimus coadministered with omeprazole (20 mg) or lansoprazole (30 mg) to 89 adult living-donor liver transplant patients on postoperative days 22 to 28, considering the CYP2C19 genotypes of the native intestine and the graft liver, separately. The concentration/dose ratio of tacrolimus coadministered with omeprazole was significantly higher in patients with two variants (*2 or *3) for intestinal CYP2C19 (median, 6.38; range, 1.55-22.9) than intestinal wild-type homozygotes (median, 2.11; range, 1.04-2.54) and heterozygotes (median, 2.11; range, 0.52-4.33) (P = 0.010), but the extent of the increase was attenuated by carrying the wild-type allele in the graft liver even when patients were CYP3A5*1 noncarriers. Conversely, the CYP2C19 polymorphisms both in the native intestine and in the graft liver little influenced the interaction between tacrolimus and lansoprazole, but CYP3A5*1 noncarriers showed higher tacrolimus concentration/dose ratio than CYP3A5*1 carriers. Furthermore, our experiments in vitro revealed that lansoprazole had a stronger inhibitory effect on the CYP3A5-mediated metabolism of tacrolimus than omeprazole, although not significantly (IC(50) = 19.9 +/- 13.8 microM for lansoprazole, 53.7 +/- 6.1 microM for omeprazole). Our findings suggest that intestinal and graft liver CYP2C19 plays a relatively greater role in the metabolism of omeprazole than it does for lansoprazole, so that the effects of CYP3A5 on the metabolism of tacrolimus might be masked by the interaction with omeprazole associated with the CYP2C19 genotype.
Abstract: The pharmacokinetics of vancomycin was retrospectively examined based on trough concentrations obtained during routine therapeutic drug monitoring to examine possible pharmacokinetic differences between adult Japanese cancer and non-cancer patients with various degrees of renal function. A total of 231 data points from 65 cancer patients and 41 non-cancer patients were collected, and patients' background, vancomycin dose, and vancomycin clearance estimated by an empirical Bayesian method were summarized. Regarding the patients' characteristics and clinical laboratory test data, no clear differences were found between the two groups. The relationship between vancomycin clearance and creatinine clearance were similar between the groups, suggesting little effect of malignancy on vancomycin clearance. After the sub-group comparisons regarding fluid retention and cancer type, no clear differences were found in the vancomycin clearance versus creatinine clearance relationship. We conclude that the initial dose of vancomycin should not necessarily be adjusted for cancer patients. For individualized vancomycin-based therapy, dose adjustment at the appropriate time is important according to information from routine therapeutic drug monitoring and clinical laboratory tests, and to observations of the efficacy, nephrotoxicity, and other conditions in each patient.
Abstract: Although some guanidine compounds were reported as superior substrates for organic cation transporter (OCT)2 than OCT1, it was unclear whether this guanidino group was an important factor in determining the specificity of hOCT1 and hOCT2. Using HEK293 cells transfected with human (h)OCT1 or hOCT2 cDNA, we assessed the role of hOCT1 and/or hOCT2 in the transport of guanidine compounds such as uremic toxins and therapeutic agents. Guanidine, creatinine and aminoguanidine more markedly inhibited the uptake of [(14)C]tetraethylammonium (TEA) by hOCT2 than by hOCT1. [(14)C]TEA uptake by hOCT2, but not hOCT1, was trans-stimulated by unlabeled guanidine, methylguanidine, creatinine, aminoguanidine and phenylguanidine. In patients with renal failure, the impairment of hOCT2 might decrease the excretion of guanidine, methylguanidine, and creatinine as uremic toxins. The uptake of aminoguanidine, a candidate for an anti-diabetic agent, was enhanced by hOCT2 with the Michaelis constant (K(m)) of 4.10+/-0.35mM. Metformin, which was also an anti-diabetic agent, and creatinine more potently inhibited the uptake of [(14)C]aminoguanidine by hOCT2 than that by hOCT1. Aminoguanidine had little impact on the uptake of [(14)C]metformin by hOCT1, but inhibited that by hOCT2 with the IC(50) of 1.49+/-0.14mM. These results indicated that the specificity of hOCT1 and hOCT2 was not determined simply by guanidino group. Among guanidine compounds, aminoguanidine was identified as a new superior substrate for hOCT2.
Abstract: H(+)/organic cation antiporters (multidrug and toxin extrusion: MATE1 and MATE2-K) play important roles in the renal tubular secretion of cationic drugs. We have recently identified a regulatory single nucleotide polymorphism (SNP) of the MATE1 gene (-32G>A). There is no other information about SNPs of the MATE gene. In this study, we evaluated the functional significance of genetic polymorphisms in MATE1 and MATE2-K. We sequenced all exons of MATE1 and MATE2-K genes in 89 Japanese subjects and identified coding SNPs (cSNPs) encoding MATE1 (V10L, G64D, A310V, D328A and N474S) and MATE2-K (K64N and G211V). All the variants except for MATE1 V10L showed significant decrease in transport activity. In particular, MATE1 G64D and MATE2-K G211V variants completely lost transport activities. When membrane expression level was evaluated by cell surface biotinylation, those of MATE1 (G64D and D328A) and MATE2-K (K64N and G211V) were significantly decreased compared with that of wild type. These findings suggested that the loss of transport activities of the MATE1 G64D and MATE2-K G211V variants were due to the alteration of protein expression in cell surface membranes. This is the first demonstration of functional impairment of the MATE family induced by cSNPs.
Abstract: PURPOSE: This study investigated whether haplotypes in the multidrug resistance 1 (MDR1) gene had effects on mRNA expression levels of MDR1 and cytochrome P450 (CYP) 3A4, and on the pharmacokinetics of tacrolimus in living-donor liver transplant (LDLT) patients, considering the gender difference. METHODS: Haplotype analysis of MDR1 with G2677T/A and C3435T was performed in 63 de novo Japanese LDLT patients (17 to 55 years; 44.4% women). The expression levels of MDR1 and CYP3A4 mRNAs in jejunal biopsy specimens were quantified by real-time PCR. RESULTS: Intestinal CYP3A4 mRNA expression levels (amol/microg total RNA) showed significantly higher values in women carrying the 2677TT-3435TT haplotype (median, 10.7; range, 5.92-15.2) than those with 2677GG-3435CC (3.03; range 1.38-4.68) and 2677GT-3435CT (median, 4.31; range, 0.07-9.42) (P = 0.022), but not in men (P = 0.81). However, MDR1 haplotype did not influence mRNA expression levels of MDR1 nor the concentration/dose ratio [(ng/mL)/(mg/day)] of oral tacrolimus for the postoperative 7 days, irrespective of gender. CONCLUSION: MDR1 haplotype may have a minor association with the tacrolimus pharmacokinetics after LDLT, but could be a good predictor of the inter-individual variation of intestinal expression of CYP3A4 in women.
Abstract: We attempted a switch of mammalian target of rapamycin (mTOR) inhibitors from sirolimus to everolimus, a derivative of sirolimus and now on the market in Japan, in two pancreatic islet transplant patients. Both patients were administered tacrolimus with sirolimus or everolimus. They had been administered 5 or 9 mg sirolimus once a day and had maintained a trough concentration of about 15 ng/mL as measured by high performance liquid chromatography with ultraviolet detection. After the switch from sirolimus to everolimus, they were given 10 or 12 mg/day of everolimus twice a day to maintain a trough concentration of 12-15 ng/mL as measured by a fluorescence polarization immunoassay (FPIA) method. Afterward, the blood concentrations of everolimus and sirolimus after the conversion were measured by high performance liquid chromatography with mass spectrometry and everolimus concentrations were found to be 5-10 ng/mL. These data show that a larger dosage is needed for everolimus than sirolimus to maintain the same trough blood concentration. Data obtained by the FPIA for everolimus should be carefully evaluated after switching from sirolimus to everolimus because of the cross-reactivity of the antibody with sirolimus.
Abstract: Hypertension in patients with chronic kidney disease (CKD) strongly associates with cardiovascular events. Among patients with CKD, reducing the accumulation of uremic toxins may protect against the development of hypertension and progression of renal damage, but there are no established therapies to accomplish this. Here, overexpression of human kidney-specific organic anion transporter SLCO4C1 in rat kidney reduced hypertension, cardiomegaly, and inflammation in the setting of renal failure. In addition, SLCO4C1 overexpression decreased plasma levels of the uremic toxins guanidino succinate, asymmetric dimethylarginine, and the newly identified trans-aconitate. We found that xenobiotic responsive element core motifs regulate SLCO4C1 transcription, and various statins, which act as inducers of nuclear aryl hydrocarbon receptors, upregulate SLCO4C1 transcription. Pravastatin, which is cardioprotective, increased the clearance of asymmetric dimethylarginine and trans-aconitate in renal failure. These data suggest that drugs that upregulate SLCO4C1 may have therapeutic potential for patients with CKD.
Abstract: This study assesses the effects of rabeprazole on the pharmacokinetics of tacrolimus, considering the cytochrome P450 (CYP) 2C19 and CYP3A5 genotypes of living-donor liver transplant patients (native intestine) and their corresponding donors (graft liver). We examined the concentration/dose ratio of tacrolimus in transplant patients treated with (n=17) or without (n=38) rabeprazole at 10 mg/day on postoperative days 22-28. A stratified analysis revealed no significant differences between the control and rabeprazole groups in the median (range) concentration/dose ratio of tacrolimus [(ng/mL)/(mg/day)] for CYP2C19 extensive/intermediate metabolizers [2.71 (1.00-6.15) versus 2.55 (0.96-9.25); P=0.85] and for poor metabolizers [4.92 (2.44-7.00) versus 3.82 (2.00-7.31); P=0.68], respectively. Even based on the classification of CYP2C19 genotypes of donors, no significant difference in the concentration/dose ratio of tacrolimus was found for the two groups (CYP2C19 extensive/intermediate metabolizers, P=0.52; poor metabolizers, P=0.51). The same was observed for CYP3A5(*)1 carriers (P=0.97 for native intestine; P=0.87 for graft liver) and CYP3A5(*)3/(*)3 carriers (P=0.89 for native intestine; P=0.56 for graft liver). These findings suggest a safer dosing and monitoring of tacrolimus coadministered with rabeprazole early on after liver transplantation regardless of the CYP2C19 and CYP3A5 genotypes of transplant patients and their donors.
Abstract: OBJECTIVES: The aim of this study was to evaluate whether genetic polymorphisms are responsible for individual variation in the expression levels of hepatic organic anion transporters. METHODS: The mRNA levels of multidrug resistance-associated protein 2, organic anion transporting polypeptide (OATP) 1B1, OATP1B3 and OATP2B1 were determined by real-time polymerase chain reaction using liver tissue samples from 102 Japanese. The association between the mRNA levels of these transporters and the disappearance rate of indocyanine green was examined. The promoter region of each transporter gene was sequenced and the effects of single nucleotide polymorphisms (SNPs) in the promoter region [regulatory SNPs (rSNPs)] on mRNA levels were evaluated. Western blot analysis using hepatic crude plasma membrane was carried out to assess protein levels. RESULTS: Expression levels of hepatic organic anion transporters vary considerably among individuals and possible links were observed between mRNA levels of these transporters and the disappearance rate of indocyanine green. In the OATP2B1 promoter region, we identified an rSNP (-282G > A) that significantly increased mRNA expression. Western blot analysis showed a remarkable increase in the OATP2B1 protein level in the homozygotes for the rSNP with a high mRNA level. In contrast, none of the rSNPs of multidrug resistance-associated protein 2, OATP1B1 and OATP1B3 affected the mRNA expression. CONCLUSION: OATP2B1 -282G > A is a major factor affecting expression, suggesting a contribution to inter-individual differences in the expression level of OATP2B1.
Abstract: The effect of itraconazole on the pharmacokinetics of everolimus was investigated in rats. Ten minutes after an intravenous or intraintestinal administration of itraconazole, everolimus was delivered intravenously (0.2 mg/kg) or intraintestinally (0.5 mg/kg). Blood concentrations of everolimus were measured up to 240 min, and pharmacokinetic parameters were calculated. Intraintestinally administered itraconazole (20 mg/kg) significantly increased the area under the concentration-time curve (AUC) of intraintestinally administered everolimus about 4.5-fold, but even at 50 mg/kg did not affect the AUC of intravenously administered everolimus. However, intravenously administered itraconazole (50 mg/kg) increased the AUC of both intraintestinally and intravenously administered everolimus approximately 2-fold. Using a value for hepatic blood flow from the literature (50 ml/min/kg), the apparent intestinal and hepatic extraction of everolimus without itraconazole was calculated as about 80% and 13%, respectively. Intraintestinally administered itraconazole (20 mg/kg) changed the apparent intestinal extraction by 0.26-fold from 0.829 to 0.215, but the hepatic availability of everolimus was almost unchanged after the intravenous or intraintestinal administration of itraconazole even at a dose of 50 mg/kg from 0.871 to 0.923 or 0.867, respectively. In conclusion, intraintestinally administered itraconazole dramatically increased the AUC of everolimus delivered intraintestinally by inhibiting the intestinal first-pass extraction of this drug.
Abstract: Tacrolimus is widely used to prevent acute rejection after transplantation, but achieving therapeutic blood concentrations of tacrolimus is often difficult because of large pharmacokinetic variability. In this study, the applicability of the Bayesian method to individualize tacrolimus dose was prospectively examined. Twenty adult recipients (Bayesian group) and another 20 adult patients (control group), all of whom underwent living-donor liver transplantation, were enrolled in this study. In the Bayesian group, the dose of tacrolimus during the first 3 and 4 weeks after surgery was adjusted with the Bayesian method using a population pharmacokinetic model, targeting a trough level of 5 to 12 ng/mL. The interindividual variability in tacrolimus concentrations was significantly reduced in the Bayesian group compared with the control group (average percentage coefficient of variation for all occasions, 32% vs 44% and 31% vs 39% in the first 3 and 4 weeks, respectively). In addition, more patients achieved the target concentrations in the Bayesian group than in the control group (average for all occasions, 85% vs 59% and 83% vs 70% in the first 3 and 4 weeks, respectively). These findings suggest that the Bayesian method can be used to calculate maintenance doses of tacrolimus in adult patients early after living-donor liver transplantation.
Abstract: We propose a simple and practical modeling approach for analysis of the data for myelosuppression after cancer chemotherapy, which can be applied when pharmacokinetic data are not available and several anticancer drugs were simultaneously administered. The model equation is based on the probability density function for the Erlang distribution. The data for cell counts of leukocytes (white blood cell, WBC), platelets (PLT), and reticulocytes (RET) obtained in routine clinical laboratory tests after the ICE (ifosfamide/carboplatin/etoposide) regimen for cancer chemotherapy were retrospectively collected from 28 patients, and a population analysis was applied. The time course profiles could be well explained by the proposed model. The individual values of the time to reach the nadir were obtained by the Bayesian method, and their medians (days) were 16.8 for WBC, 12.8 for PLT, and 8.2 for RET. Such information would be useful to determine the day of visit for outpatients especially for additional treatment to prevent side effects such as infections. The model is simple and applicable to explain the time course profiles for myelosuppression irrespective of cell types, and also practical because it requires only the data from routine clinical laboratory tests without any additional burden to patients.
Abstract: Although the organic cation transporter 2 (OCT2/SLC22A2) mediate renal tubular uptake of cisplatin from the circulation, neither apical multidrug and extrusion (MATE) 1 or MATE2-K mediate tubular secretion of the agent. Therefore, the highly concentrated tubular cisplatin potentiates nephrotoxicity, and these are considered to be a critical mechanism for cisplatin-induced nephrotoxicity. In the present study, we examined the protective effect of imatinib, a cationic anticancer agent, on that nephrotoxicity. Imatinib markedly reduced cisplatin-induced cytotoxicity and platinum accumulation in OCT2-expressing HEK293 cells, but almost no change was found in the cells expressing human MATE1, MATE2-K and rat MATE1. In rats, the renal accumulation of platinum and subsequent nephrotoxicity, based on the blood urea nitrogen, plasma creatinine and creatinine clearance, were significantly decreased with the oral administration of imatinib. The orally administered imatinib significantly increased the area under the plasma concentration-time curve of intravenously administered cisplatin for 3 min by an average of 120%. In additional, the concomitant administration of imatinib clearly avoided the severe renal impairment by the histological examination. In conclusion, the concomitant administration of imatinib with cisplatin prevents cisplatin-induced nephrotoxicity inhibiting the OCT2-mediated renal accumulation of cisplatin.
Abstract: OBJECTIVE: The potential influence of the multidrug resistance 1 (MDR1) gene and the cytochrome P450 (CYP) genes, CYP3A4 and CYP3A5, on the oral clearance (CL/F) of tacrolimus in adult living-donor liver transplant patients was examined. Furthermore, the development of renal dysfunction was analyzed in relation to the CYP3A5 genotype. METHODS: Sixty de novo adult liver transplant patients receiving tacrolimus were enrolled in this study. The effects of various covariates (including intestinal and hepatic mRNA levels of MDR1 and CYP3A4, measured in each tissue taken at the time of transplantation, and the CYP3A5*3 polymorphism) on CL/F during the first 50 days after surgery were investigated with the nonlinear mixed-effects modeling program. RESULTS: CL/F increased linearly until postoperative day 14, and thereafter reached a steady state. The initial CL/F immediately after liver transplantation was significantly affected by the intestinal MDR1 mRNA level (P<0.005). Furthermore, patients carrying the CYP3A5*1 allele in the native intestine, but not in the graft liver, showed a 1.47 times higher (95% confidence interval, 1.17-1.77 times, P<0.005) recovery of CL/F with time than patients having the intestinal CYP3A5*3/*3 genotype. The cumulative incidence of renal dysfunction within 1 year after transplantation, evaluated by the Kaplan-Meier method, was significantly associated with the recipient's but not donor's CYP3A5 genotype (*1/*1 and *1/*3 vs. *3/*3: recipient, 17 vs. 46%, P<0.05; donor, 35 vs. 38%, P=0.81). CONCLUSION: These findings suggest that the CYP3A5*1 genotype as well as the MDR1 mRNA level in enterocytes contributes to interindividual variation in the CL/F of tacrolimus in adult recipients early after living-donor liver transplantation. Furthermore, CYP3A5 in the kidney may play a protective role in the development of tacrolimus-related nephrotoxicity.
Abstract: Perfluorooctanoic acid, an environmental contaminant, is found in both wild animals and human beings. There are large species and sex differences in the renal excretion of perfluorooctanoic acid. In the present study, we aimed to characterize organic anion transporters 1-3 (OAT1-3) in human beings and rats to investigate whether the species differences in the elimination kinetics of perfluorooctanoic acid from the kidneys can be attributed to differences in the affinities of these transporters for perfluorooctanoic acid. We used human (h) and rat (r) OAT transient expression cell systems and measured the [(14)C] perfluorooctanoic acid transport activities. Both human and rat OAT1 and OAT3 mediated perfluorooctanoic acid transport to similar degrees. Specifically, the kinetic parameters, K(m), were 48.0 +/- 6.4 microM for h OAT1; 51.0 +/- 12.0 microM for rOAT1; 49.1 +/- 21.4 microM for hOAT3 and 80.2 +/- 17.8 microM for rOAT3, respectively. These data indicate that both human and rat OAT1 and OAT3 have high affinities for perfluorooctanoic acid and that the species differences in its renal elimination are not attributable to affinity differences in these OATs between human beings and rats. In contrast, neither hOAT2 nor rOAT2 transported perfluorooctanoic acid. In conclusion, OAT1 and OAT3 mediated perfluorooctanoic acid transport in vitro, suggesting that these transporters also transport perfluorooctanoic acid through the basolateral membrane of proximal tubular cells in vivo in both human beings and rats. Neither human nor rat OAT2 mediated perfluorooctanoic acid transport. Collectively, the difference between the perfluorooctanoic acid half-lives in human beings and rats is not likely to be attributable to differences in the affinities of these transporters for perfluorooctanoic acid.
Abstract: Organic cation transporters (OCTs) and organic anion transporters (OATs) (SLC22A family) play crucial roles in the renal secretion of various drugs. Messengar ribonucleic acid (mRNA) expression of transporters can be a key factor regulating interindividual differences in drug pharmacokinetics. However, the source of variations in mRNA levels of transporters is unclear. In this study, we focused on single nucleotide polymorphisms (SNP) in the promoter region [regulatory SNPs (rSNPs)] as candidates for the factor regulating mRNA levels of SLC22A. We sequenced the promoter regions of OCT2 and OAT1-4 in 63 patients and investigated the effects of the identified rSNPs on transcriptional activities and mRNA expression. In the OCT2 promoter region, one deletion polymorphism (-578_-576delAAG) was identified; -578_-576delAAG significantly reduced OCT2 promoter activity (p < 0.05), and carriers of -578_-576delAAG tend to have lower OCT2 mRNA levels, but the difference is not significant. There was no rSNP in the OAT1 and OAT2 genes. The five rSNPs of OAT3 and one rSNP of OAT4 were unlikely to influence mRNA expression and promoter activity. This is the first study to investigate the influences of rSNPs on mRNA expression of SLC22A in the kidney and to identify a regulatory polymorphism affecting OCT2 promoter activity.
Abstract: During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of beta-lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis.
Abstract: We report different effects of administration of proton pump inhibitors on tacrolimus blood concentration in two living-donor liver transplant patients. In case 1, a 51-year-old man with liver cirrhosis due to hepatitis C virus underwent living-donor liver transplantation, and tacrolimus was orally administered. Omeprazole (40 mg/day) was introduced intravenously between postoperative days 5 and 6, and oral lansoprazole (30 mg/day) was introduced from day 6, leading to an increase in the concentration/dose ratio of tacrolimus from day 10. In case 2, a 41-year-old living-donor liver transplant woman received tacrolimus, and co-administered with omeprazole (40 mg/day) intravenously during 7 days immediately after surgery. During this period, trough concentration of tacrolimus was high, but the concentration/dose ratio of tacrolimus was gradually decreasing with time. Switched to rabeprazole (10 mg/day) orally on the postoperative 8th day, the concentration/dose ratio of tacrolimus remained low, indicating little drug-drug interaction between tacrolimus and rabeprazole. In both cases, the genotypes of CYP2C19 and CYP3A5 were defective both in the graft liver and in the native intestine. A drug-drug interaction between rabeprazole and tacrolimus was not observed in this case study presented, suggesting that this combination could be safely used in tacrolimus therapy after liver transplantation.
Abstract: In the proximal tubules of rat (r) kidney, the polyspecific organic cation transporters (OCTs), rOCT1 and rOCT2, mediate the baso-lateral uptake of various organic cations, including many drugs, toxins, and endogenous compounds, and the apical type of H(+)/ organic cation antiporter, rat multidrug and toxin extrusion 1 (rMATE1), mediate the efflux of organic cations. Renal clearances of H(2) receptor antagonists, including famotidine, were reported to be decreased in patients with kidney disease. Therefore, acute kidney injury (AKI) could influence renal excretion and disposition of organic cations accompanied by the regulation of organic cation transporters. The aim of this study was to investigate the pharmacokinetic alteration of cationic drugs and the expression of tubular organic cation transporters, rOCT1, rOCT2, and rMATE1, in ischemia/reperfusion (I/R)-induced AKI rats. I/R-induced AKI increased the plasma concentration of i.v. administrated famotidine, a substrate for rOCT1 and rOCT2, or tetraethylammonium (TEA), a substrate for rOCT1, rOCT2, and rMATE1. The areas under the plasma concentration curves for famotidine and TEA were 2- and 6-fold higher in I/R rats than in sham-operated rats, respectively. The accumulation of TEA into renal slices was significantly decreased, suggesting that organic cation transport activity at the basolateral membranes was reduced in I/R rat kidney. The protein expressions of basolateral rOCT2 and luminal rMATE1 were down-regulated in I/R rat kidneys. These data suggest that the urinary secretion of cationic drugs via epithelial organic cation transporters is decreased in AKI.
Abstract: Human organic cation transporter 2 (OCT2/SLC22A2), which is specifically expressed in the kidney, plays critical roles in the renal secretion of cationic compounds. Tissue expression and membrane localization of OCT2 are closely related to the tissue distribution, pharmacological effects, and/or adverse effects of its substrate drugs. However, the molecular mechanisms underlying the kidney-specific expression of OCT2 have not been elucidated. In the present study, therefore, we examined the contribution of DNA methylation of the promoter region for the OCT2 gene to its tissue-specific expression using human tissue samples. In vivo methylation status of the proximal promoter region of OCT2 and that of OCT1, a liver-specific organic cation transporter, were investigated by bisulfite sequencing using human genomic DNA extracted from the kidney and liver. All CpG sites in the OCT2 proximal promoter were hypermethylated in the liver, while hypomethylated in the kidney. On the other hand, the promoter region of OCT1 was hypermethylated in both the kidney and liver. The level of methylation of the OCT2 promoter was especially low at the CpG site in the E-box, the binding site of the basal transcription factor upstream stimulating factor (USF) 1. In vitro methylation of the OCT2 proximal promoter dramatically reduced the transcriptional activity, and an electrophoretic mobility shift assay showed that methylation at the E-box inhibited the binding of USF1. These results indicate that kidney-specific expression of human OCT2 is regulated by methylation of the proximal promoter region, interfering with the transactivation by USF1.
Abstract: PURPOSE: In patients with thyroid disorders, abnormalities in the pharmacokinetics of various drugs including digoxin, a substrate of P-glycoprotein (Pgp) which plays a crucial role in drug absorption and disposition, have been reported. In this study, we examined the effect of 3,5,3'-L-triiodothyronine (T(3)) on the function and expression of Pgp using the human intestinal epithelial cell line Caco-2. MATERIALS AND METHODS: The effect of T(3) on the expression of Pgp and MDR1 mRNA was assessed by Western blotting and competitive polymerase chain reaction, respectively. Digoxin uptake and transport by Pgp was assessed using Caco-2 cell monolayers. RESULTS: The expression of MDR1 mRNA was increased by T(3) treatment in a concentration-dependent manner. Pgp expression was also increased by 100 nM T(3), whereas it decreased on depletion of T(3). The amount of [(3)H]digoxin accumulated in Caco-2 cell monolayers treated with T(3) was diminished significantly compared with that in control cells. In addition, the basal-to-apical transcellular transport of [(3)H]digoxin was accelerated by T(3) treatment. CONCLUSIONS: These results indicate that T(3) regulates the expression and function of Pgp. It is possible that changes in Pgp expression alter the pharmacokinetics of Pgp substrates in patients with thyroid disorders.
Abstract: It has been difficult to set an individualized therapeutic window of tacrolimus after organ transplantation, because of wide interindividual variation of responsiveness to immunosuppressive therapy. In this study, we examined the significance of multidrug resistance 1 (MDR1) in the peripheral blood cells by comparing the trough concentration of tacrolimus with the occurrence of acute cellular rejection (ACR) in retrospectively collected pediatric living-donor liver transplant patients, who were enrolled after obtaining written informed consent. No significant difference in the intraindividual variation in MDR1 mRNA expression in the peripheral blood cells was observed between postoperative days 3 and 7. The average trough concentration of tacrolimus during the 15-day postoperative period was significantly higher in the event-free patients than in those who experienced ACR (21 of 44 cases), and they had higher levels of blood MDR1 mRNA. In addition, the average trough concentration of tacrolimus significantly correlated with the logarithmically transformed MDR1 mRNA data from the blood cells in patients of both the event-free (r = 0.5406; P = 0.0077) and ACR (r = 0.4772; P = 0.0284). The cellular accumulation of [(14)C]tacrolimus in the peripheral blood mononuclear cells was 2-fold higher in mdr1a/1b-knockout mice than in wild-type mice (P = 0.0182). These results suggest that MDR1 in blood cells decreases the leukocytic concentration of tacrolimus, and it could be a useful marker to establish an individualized target concentration of tacrolimus to prevent ACR in pediatric patients after liver transplantation.
Abstract: Vectorial secretion of cationic compounds across tubular epithelial cells is an important function of the kidney. This uni-directed transport is mediated by two cooperative functions, which are membrane potential-dependent organic cation transporters at the basolateral membranes and H+/organic cation antiporters at the brush-border membranes. More than 10 years ago, the basolateral organic cation transporters (OCT1-3/SLC22A1-3) were isolated, and molecular understandings for the basolateral entry of cationic drugs have been greatly advanced. However, the molecular nature of H+/organic cation antiport systems remains unclear. Recently, mammalian orthologues of the multidrug and toxin extrusion (MATE) family of bacteria have been isolated and clarified to function as H+/organic cation antiporters. In this commentary, the molecular characteristics and pharmacokinetic roles of mammalian MATEs are critically overviewed focusing on the renal secretion of cationic drugs.
Abstract: Human organic cation transporter 1 (OCT1/SLC22A1) plays important roles in the hepatic uptake of cationic drugs. The functional characteristics of this transporter have been well evaluated, but molecular information regarding transcriptional regulation is limited. In the present study, therefore, we examined the gene regulation of OCT1 gene focusing on basal core expression. An approximately 2.5-kb fragment of the OCT1 promoter region was isolated, and promoter activity was measured by luciferase assay in the human liver cell lines Huh7 and HepG2. Deletion analysis suggested that the region spanning -141/-69 was essential for the basal core transcriptional activity and that this region contained the sequence of a cognate E-box (CACGTG). The E-box is known to be bound by the basal transcription factors, upstream stimulating factors (USFs), and the functional involvements of USF1 and USF2 were confirmed by a transactivation effect, a mutational analysis of the E-box, and an electrophoretic mobility shift assay. The transactivation effect of USFs on the OCT1 promoter was further stimulated by hepatocyte nuclear factor 4alpha, a liver-enriched transcription factor. There were no polymorphisms in the proximal promoter region (about 400 bp) of OCT1 gene (n = 109). These findings indicated that both USF1 and USF2 bind to an E-box sequence located in the OCT1 core promoter region and are required for the basal gene expression of this transporter.
Abstract: Zinc is an essential metal ion for the body, and is widely used for nutritional and clinical purposes. Previously, we showed that zinc inhibits the transport of glycylsarcosine via the intestinal peptide transporter PEPT1 in the human intestinal cell line Caco-2. In this study, we examined the effect of zinc on the activity of peptide transporters in rats using the oral beta-lactam antibiotic ceftibuten as a model drug. The plasma ceftibuten concentration after intraintestinal administration was decreased in the presence of zinc. The maximum plasma concentration (C(max)) was significantly decreased and the time required to reach C(max) (T(max)) was prolonged by zinc coadministration. The plasma ceftibuten concentration after iron coadministration or two hours after zinc administration was not affected. The in situ loop technique revealed 50% inhibition of ceftibuten absorption by zinc. In conclusion, zinc inhibits the transport activity of PEPT1 in vivo as well in vitro.
Abstract: BACKGROUND: Little is known about long-term outcome of tacrolimus therapy for ulcerative colitis. Aim To evaluate long-term efficacy and safety of tacrolimus in Japanese patients with refractory ulcerative colitis. METHODS: Twenty-seven patients with UC refractory to conventional therapy were administered tacrolimus with trough whole-blood levels of 10-15 ng/mL to induce remission and 5-10 ng/mL to maintain remission. Median treatment duration was 11 months (1-39 months) and median follow-up duration was 17 months (2-65 months). Evaluation of the clinical response was based on a modified Truelove-Witts severity index (MTWSI). RESULTS: Tacrolimus produced a clinical response in 21 patients (77.8%), and remission was achieved in 19 of these 21 (70.4%) within 30 days. Overall cumulative colectomy-free survival was estimated as 62.3% at 65 months. In 18 of 19 patients treated with corticosteroids at the initiation of tacrolimus therapy, corticosteroids were discontinued or tapered. Adverse events were tremor (25.9%), renal function impairment (18.5%), infectious disease (14.8%), hot flashes (11.1%), hyperkalaemia (7.4%), headache (7.4%), epigastralgia (7.4%) and nausea (3.7%). No mortality occurred. CONCLUSION: Long-term administration of tacrolimus appears to be an effective and well-tolerated treatment for Japanese patients with refractory ulcerative colitis.
Abstract: The aim of this study was to quantitatively evaluate the effects of intestinal and hepatic extraction on the pharmacokinetics of everolimus in rats. Everolimus was administered intravenously, intraportally or intraintestinally in order to separately evaluate the intestinal and hepatic first-pass extraction. Cyclosporine or tacrolimus was administered into rat intestines, and after 10 min everolimus pharmacokinetics were evaluated. The blood concentrations of everolimus were measured by the high-performance liquid chromatography with tandem mass spectrometry. Total body clearance of everolimus was constant in the dosage from 0.2 to 1.0 mg/kg. The bioavailability after intraportal and intraintestinal administration were 48.0% and 21.2%, respectively. Concomitantly administered cyclosporine (5 mg/kg) , but not tacrolimus (1 mg/kg), significantly decreased the total body clearance of everolimus compared with the control, and also increased the bioavailabilty of everolimus after intraintestinal administration 1.75-fold. Cyclosporine significantly increased the area under the blood concentration-time curve of everolimus after the intraintestinal constant infusion 3-fold, and increased that after the intraportal constant infusion only 1.35-fold. In conclusion, the intestine as well as liver contributes to the first-pass extraction for everolimus in rats. Intestinally administered cyclosporine inhibited the intestinal extraction of everolimus more than its hepatic extraction.
Abstract: The effect of oxaliplatin against colorectal cancer is superior to that of cisplatin, but the molecular mechanism(s) involved is not clear. We found previously that oxaliplatin, but not cisplatin, was transported by human (h) and rat organic cation transporter 3 (OCT3)/SLC22A3. In the present study, we examined whether hOCT3 was significantly involved in the oxaliplatin-induced cytotoxicity and accumulation of platinum in colorectal cancer. The level of hOCT3 mRNA in the colon was 9.7-fold higher in cancerous than in normal tissues in six Japanese patients (P = 0.0247). In human colorectal cancer-derived cell lines, the mRNA of hOCT3 was highly expressed compared with that of other organic cation transporters. The release of lactate dehydrogenase (LDH) and accumulation of platinum with oxaliplatin treatment were increased in SW480 cells transfected with hOCT3 cDNA compared with empty vector-transfected cells. T84 and SW837 cells, with high levels of hOCT3, released more LDH and accumulated more platinum after oxaliplatin treatment than low hOCT3-expressing cells such as SW480, HCT116, HT29, and Lovo. However, the amount of platinum accumulated after cisplatin treatment did not differ among these six cell lines. The levels of hOCT3 expression in colon and rectum were also higher in cancerous than in normal tissues in Caucasian patients as determined by dot blotting. In conclusion, the hOCT3-mediated uptake of oxaliplatin into the cancers was suggested to be important for its cytotoxicity, and hOCT3 expression may be a marker for cancer chemotherapy including oxaliplatin.
Abstract: The profiles of tacrolimus metabolites in the whole blood and bile were examined in two living-donor liver transplant patients, who transiently required higher doses of tacrolimus. Even when the 16 mg/day or oral 10 mg/day and intravenous infusion of 0.5 mg/day of tacrolimus were administered, its trough level in each patient did not reach over 15 ng/mL. By use of liquid chromatography-tandem mass spectrometry/mass spectrometry methods, a minor metabolite M-II was found to be a major metabolite both in blood and bile in these cases. However, a primary metabolite M-I was confirmed as the majority in the bile of other 8 control cases. Each graft liver and native intestine carried CYP3A5*1/*3 or *3/*3 and *1/*3 or *1/*3, respectively. Therefore, the CYP3A5 genotype could not explain the present phenomena. After removing the bile drainage tube to allow the bile flow into intestine, the required doses of tacrolimus were decreased to around 20% compared to each maximum dosage. In conclusion, a minor metabolite M-II was first found in the human bile, suggesting that the appearance of M-II in bile could associate with the extensive metabolism of tacrolimus and/or the requirement of larger oral dosage.
Abstract: The intestinal H(+)/peptide cotransporter 1 (PEPT1) plays important roles as a nutrient and drug transporter. Previously, we reported that rat intestinal PEPT1 showed a diurnal rhythm and that this rhythm is closely related to the feeding schedule. Furthermore, we also demonstrated that transcription factors, Sp1, Cdx2, and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) contribute to the basal, intestine-specific, and fasting-induced expression of PEPT1, respectively. In this study, to clarify the molecular mechanism governing the diurnal rhythm of PEPT1 expression, we compared expression profiles of these transcription factors under two kinds of feeding schedules. The intestinal Sp1 and Cdx2 did not show a circadian accumulation of mRNA or response to the daytime feeding regimen. Plasma free fatty acids, endogenous PPAR-alpha ligands, exhibited a robust circadian fluctuation in phase with that of PEPT1. However, subsequent experiments using PPAR-alpha-null mice revealed the absence of any association between the circadian rhythm of PEPT1 and PPAR-alpha. We then focused on the clock genes (Clock, Bmal1, Per1-2, and Cry1) and clock-controlled gene, albumin D site-binding protein (DBP). A robust and coordinated circadian expression of the clock genes was observed, and daytime feeding entirely inverted the phase except for Clock. The expression of DBP was in phase with that of PEPT1 in both groups. Electrophoretic mobility shift assays and reporter assays revealed that DBP has the ability to bind the DBP binding site located in the distal promoter region of the rat PEPT1 gene and induce the transcriptional activity. These findings indicate that DBP plays pivotal roles in the circadian oscillation of PEPT1.
Abstract: To clarify the transcellular transport of organic cations via basolateral and apical transporters, we established double-transfected Madin-Darby canine kidney (MDCK) cells expressing both human organic cation transporter hOCT1 and hMATE1 (MDCK-hOCT1/hMATE1), and hOCT2 and hMATE1 (MDCK-hOCT2/hMATE1) as models of human hepatocytes and renal epithelial cells, respectively. Using the specific antibodies, hOCT1 and hMATE1 or hOCT2 and hMATE1 were found to be localized in the basolateral and apical membranes of MDCK-hOCT1/hMATE1 or MDCK-hOCT2/hMATE1 cells, respectively. A representative substrate, [14C]tetraethylammonium, was transported unidirectionally from the basolateral to apical side in these double transfectants. The optimal pH was showed to be 6.5 for the transcellular transport of [14C]tetraethylammonium, when the pH of the incubation medium on the apical side was varied from 5.5 to 8.5. The basolateral-to-apical transport also decreased in the presence of 10 mM 1-methyl-4-phenylpyridinium or 1 mM levofloxacin on the basolateral side of both double transfectants. In MDCK-hOCT2/hMATE1 cell monolayers, but not in MDCK-hOCT1/hMATE1 cell monolayers, the accumulation of [14C]tetraethylammonium was decreased in the presence of 10 mM 1-methyl-4-phenylpyridinium, but significantly increased in the presence of 1 mM levofloxacin. The uptake of [14C]tetraethylammonium, [3H]1-methyl-4-phenylpyridinium, [14C]metformin and [3H]cimetidine, but not of [14C]procainamide and [3H]quinidine, by HEK293 cells was stimulated by expression of the hOCT1, hOCT2 or hMATE1 compared to control cells. However, transcellular transport of [14C]procainamide and [3H]quinidine was clearly observed in both double-transfectants. These cells could be useful for examining the routes by which compounds are eliminated, or predicting transporter-mediated drug interaction.
Abstract: Absorption of riboflavin is mediated by transporter(s). However, a mammalian riboflavin transporter has yet to be identified. In the present study, the novel human and rat riboflavin transporters hRFT1 and rRFT1 were identified on the basis of our rat kidney mRNA expression database (Horiba N, Masuda S, Takeuchi A, Saito H, Okuda M, Inui K. Kidney Int 66: 29-45, 2004). hRFT1 and rRFT1 cDNAs have an open reading frame encoding 448- and 450-amino acid proteins, respectively, that exhibit 81.1% identity and 96.4% similarity to one another. In addition, an inactive splice variant of hRFT1, hRFT1sv, was also cloned. The hRFT1sv cDNA, which encodes a 167-amino acid protein, retains an intron between exons 2 and 3 of hRFT1. Real-time PCR revealed that the sum of hRFT1 and hRFT1sv mRNAs was expressed strongly in the placenta and small intestine and was detected in all tissues examined. In addition, hRFT1 and hRFT1sv were expressed in human embryonic kidney (HEK)-293 and Caco-2 cells. HEK-293 cells transfected with green fluorescent protein-tagged hRFT1 and rRFT1 exhibited a fluorescent signal in the plasma membrane. Overexpression of hRFT1 and rRFT1, but not hRFT1sv, increased the cellular accumulation of [(3)H]riboflavin. The transfection of small interfering RNA targeting both hRFT1 and hRFT1sv significantly decreased the uptake of [(3)H]riboflavin by HEK-293 and Caco-2 cells. Riboflavin transport is Na(+), potential, and pH independent. Kinetic analyses demonstrated that the Michaelis-Menten constants for the uptake by HEK-293 and Caco-2 cells were 28.1 and 63.7 nM, respectively. We propose that hRFT1 and rRFT1 are novel mammalian riboflavin transporters, which belong to a new mammalian riboflavin transporter family.
Abstract: Irinotecan is widely used in the treatment of colorectal, gastric, and lung cancers. However, adverse drug reactions such as severe diarrhea and neutropenia limit the dose of this drug. Irinotecan is metabolized by carboxylesterase to form an active metabolite, 7-ethyl-10-hydroxycamptothecin(SN-38), which in turn is subsequently conjugated by UGT-glucuronosyltransferase 1A1(UGT1A1)to yield an inactive form, SN-38 glucuronide(SN-38 G). The UGT1A1 gene polymorphisms contribute to the individual variation in adverse events among patients administered irinotecan. However, the distribution of polymorphisms shows large interethnic differences. The distribution of UGT1A1*28 greatly differs between Caucasians and Japanese; the frequency of UGT1A1*28 is high in Caucasians, whereas it is low in Asians including Japanese. Recently, it has been demonstrated that genetic variants of UGT1A1*6 in addition to UGT1A1*28 are associated with the occurrence of adverse events in irinotecan chemotherapy in Asians. This review summarizes recent studies to outline the role of UGT1A1*28 and UGT1A1*6 for irinotecan-induced adverse drug reaction in Japanese cancer patients.
Abstract: The substrate specificities of human (h) multidrug and toxin extrusion (MATE) 1 and hMATE2-K were examined to find functional differences between these two transporters by the transfection of the cDNA of hMATE1 and hMATE2-K into HEK293 cells. Western blotting revealed specific signals for hMATE1 and hMATE2-K consistent with a size of 50 and 40kDa, respectively, in the transfectants as well as human renal brush-border membranes under reducing conditions. In the presence of oppositely directed H(+)-gradient, the transport activities of various compounds such as tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, creatinine, guanidine, procainamide, and topotecan were stimulated in hMATE1- and hMATE2-K-expressing cells. In addition to cationic compounds, anionic estrone sulfate, acyclovir, and ganciclovir were also recognized as substrates of these transporters. Kinetic analyses demonstrated the Michaelis-Menten constants for the hMATE1-mediated transport of tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, guanidine, procainamide, topotecan, estrone sulfate, acycrovir, and ganciclovir to be (in mM) 0.38, 0.10, 0.17, 0.78, 2.10, 1.23, 0.07, 0.47, 2.64, and 5.12, respectively. Those for hMATE2-K were 0.76, 0.11, 0.12, 1.98, 4.20, 1.58, 0.06, 0.85, 4.32, and 4.28, respectively. Although their affinity for hMATE1 and hMATE2-K was similar, the zwitterionic cephalexin and cephradine were revealed to be specific substrates of hMATE1, but not of hMATE2-K. Levofloxacin and ciprofloxacin were not transported, but were demonstrated to be potent inhibitors of these transporters. These results suggest that hMATE1 and hMATE2-K function together as a detoxication system, by mediating the tubular secretion of intracellular ionic compounds across the brush-border membranes of the kidney.
Abstract: The immunosuppressant mycophenolate mofetil (MMF) is frequently administered with calcineurin inhibitors and corticosteroids to recipients of organ transplantations. However, the renal handling of the active metabolite mycophenolic acid (MPA) and 7-O-MPA-glucuronide (MPAG) has been unclear. The purpose of the present study was to assess the interaction of MPA and MPAG with the human renal organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8), by conducting uptake experiments using HEK293 cells stably expressing these transporters. MPA and MPAG inhibited the time-dependent uptake of p-[(14)C]aminohippurate by hOAT1 and that of [(3)H]estrone sulfate by hOAT3. The apparent 50% inhibitory concentration (IC(50)) of MPA for hOAT1 and hOAT3 was estimated at 10.7 and 1.5 microM, respectively. In the case of MPAG, the IC(50) values were calculated at 512.3 microM for hOAT1 and 69.1 microM for hOAT3. Eadie-Hofstee plot analyses showed that they inhibited hOAT1 noncompetitively and hOAT3 competitively. No inhibitory effects of tacrolimus, cyclosporin A and azathioprine on transport of p-[(14)C]aminohippurate by hOAT1 and of [(3)H]estrone sulfate by hOAT3 were observed. No transport of MPA by these transporters was observed. On the other hand, the uptake of MPAG into cells was stimulated by the expression of hOAT3, but not hOAT1. These findings propose the possibility that the administration of MMF decreases the renal clearance of drugs which are substrates of hOAT1 and hOAT3. Present data suggest that hOAT3 contributes to the renal tubular secretion of MPAG.
Abstract: The mechanism of severe nephrotoxicity caused by cisplatin, but not carboplatin, oxaliplatin, and nedaplatin, is not fully understood. The renal accumulation and subsequent nephrotoxicity of platinum agents were examined in rats. Among these four drugs, only cisplatin induced nephrotoxicity at 2 days after its intraperitoneal administration. The urinary activity of N-acetyl-beta-D-glucosaminidase and expression of kidney injury molecule-1 mRNA and osteopontin were markedly enhanced in the cisplatin-treated rats. Although some markers were affected in the rats administered nedaplatin, only minor histological change was observed. The renal accumulation of cisplatin was much greater than that of the other drugs. In the in vitro study, the cellular accumulation of cisplatin and oxaliplatin was stimulated by the expression of rat (r) OCT2. Oxaliplatin was also transported by rOCT3. A luminal H(+)/organic cation antiporter, rMATE1 (multidrug and toxin extrusion) as well as human (h) MATE1 and hMATE2-K, stimulated the H(+)-gradient-dependent antiport of oxaliplatin, but not of cisplatin. Carboplatin and nedaplatin were not transported by these transporters. In conclusion, the nephrotoxicity of platinum agents was closely associated with their renal accumulation, which is determined by the substrate specificity of the OCT and MATE families.
Abstract: Chloride ion has a stimulatory effect on the transport of organic anions across renal basolateral membranes. However, the exact mechanisms at molecular levels have been unclear as of yet. Human organic anion transporters hOAT1 and hOAT3 play important roles in renal basolateral membranes. In this study, the effects of Cl(-) on the activities of these transporters were evaluated by using HEK293 cells stably expressing hOAT1 or hOAT3 (HEK-hOAT1 or HEK-hOAT3). The uptake of p-[(14)C]aminohippurate by HEK-hOAT1 and [(3)H]estrone sulfate by HEK-hOAT3 was greater in the presence of Cl(-) than in the presence of SO(4)(2-) or gluconate. Additionally, the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3 was significantly higher in the Cl(-)-containing medium than the gluconate-containing medium, suggesting that the influences of Cl(-) are not dependent on substrate and that Cl(-) directly stimulates the functions of hOAT1 and hOAT3. The substitution of gluconate with Cl(-) did not change the K(m) value for the uptake of p-[(14)C]aminohippurate by HEK-hOAT1 but caused an approximately threefold increase in the maximal uptake rate (V(max)) value. On the other hand, replacement of gluconate with Cl(-) decreased the K(m) value for the uptake of [(3)H]estrone sulfate and cefotiam by HEK-hOAT3 to about one-third, while it did not change the V(max) value. In summary, Cl(-) upregulates the activities of both hOAT1 and hOAT3, but its effects on transport kinetics differ between these transporters. It was suggested that Cl(-) participates in the trans-location process for hOAT1, and the substrate recognition process for hOAT3.
Abstract: Proton-pump inhibitors (PPIs, e.g. omeprazole and rabeprazole) are often administered to transplant patients as a treatment or prophylaxis for ulcers after surgery. Since tacrolimus and PPIs share the CYP3A4 system for metabolism, pharmacokinetic interactions are anticipated when they are administered simultaneously. We present a Japanese male patient who underwent a living-donor kidney transplantation having received tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression. The concentration/dose (C/D) ratio for tacrolimus was markedly higher during the period of treatment with omeprazole than ranitidine or rabeprazole. The results of liver functional tests were within the normal range during the use of these three antacid drugs. Since the higher C/D ratio for tacrolimus when omeprazole was being administered did not result from a decrease in the elimination of tacrolimus due to hepatic dysfunction, drug interaction between omeprazole and tacrolimus was strongly suspected. The present case indicates that rabeprazole can be used safely in place of omeprazole in kidney transplant recipients receiving tacrolimus.
Abstract: We previously reported that MEEK, a generic product of vancomycin hydrochloride (VCM), was less nephrotoxic than a conventional preparation (S-VCM) in normal rats at a nephrotoxic dose (400 mg/kg) of VCM.(1)) To infer the clinical significance of this finding, we compared the risk of nephrotoxicity of these two formulations in rats with chronic renal failure in this study. MEEK or S-VCM was given intravenously to two weeks post-5/6 nephrectomy rats, and the pharmacokinetic profile of VCM and pathological evaluation were compared. There were no differences at the daily clinical dose (40 mg/kg), but at the twice the daily clinical dose (80 mg/kg), the mean plasma concentration of VCM was higher after S-VCM administration than after MEEK and the CL(tot) and CL(r) decreased to approximately 60% of those after MEEK. The renal tissue concentration of VCM was 1.5-fold higher at 24hr after S-VCM administration than after MEEK. Pathologically, no marked differences between the findings were observed at 24hr after administration of each formulation. These findings suggest that MEEK reduces renal damage caused by VCM and prevents the iatrogenic aggravation of nephrotoxicity. These results hold out hope that MEEK will permit high-dose administration of VCM, while revealing clinical significance of the nephrotoxicity-reduction by MEEK.
Abstract: A H+/organic cation antiporter (multidrug and toxin extrusion 1: MATE1/SLC47A1) plays important roles in the tubular secretion of various clinically important cationic drugs such as cimetidine. We have recently found that the regulation of this transporter greatly affects the pharmacokinetic properties of cationic drugs in vivo. No information is available about the regulatory mechanisms for the MATE1 gene. In the present study, therefore, we examined the gene regulation of human (h) and rat (r) MATE1, focusing on basal expression. A deletion analysis suggested that the regions spanning -65/-25 and -146/-38 were essential for the basal transcriptional activity of the hMATE1 and rMATE1 promoter, respectively, and that both regions contained putative Sp1-binding sites. Functional involvement of Sp1 was confirmed by Sp1 overexpression, a mutational analysis of Sp1-binding sites, mithramycin A treatment, and an electrophoretic mobility shift assay. Furthermore, we found a single nucleotide polymorphism (SNP) in the promoter region of hMATE1 (G-32A), which belongs to a Sp1-binding site. The allelic frequency of this rSNP was 3.7%, and Sp1-binding and promoter activity were significantly decreased. This is the first study to clarify the transcriptional mechanisms of the MATE1 gene and to identify a SNP affecting the promoter activity of hMATE1.
Abstract: BACKGROUND: We investigated the pharmacokinetics and pharmacodynamics of paclitaxel with carboplatin or gemcitabine in patients with urogenital cancer to clarify the significance of monitoring of the serum concentration of paclitaxel. METHODS: Paclitaxel was administered at 175 mg/m(2) or 150 mg/m(2) to patients with hormone-refractory prostate cancer (n = 10) or advanced transitional cell carcinoma (n = 6) along with carboplatin or gemcitabine, respectively. The relationships between pharmacokinetic parameters and hematological adverse effects, as well as pharmacological effects, were examined. The effects of patient characteristics, including single-nucleotide polymorphisms of MDR1(ABCB1), CYP2C8, CYP3A4, and CYP3A5, on the total body clearance of paclitaxel were evaluated. RESULTS: Total body clearance and volume of distribution at a steady-state after the intravenous infusion of paclitaxel were not significantly different between patients with carboplatin or gemcitabine. The percent decreases in neutrophils and platelets for the regimen with gemcitabine were significantly greater than those with carboplatin, and showed a significant positive relationship with the observed concentration at the end of infusion or time above 0.1-microM concentration of paclitaxel. Post-therapy decreases in prostate-specific antigen were not positively correlated with the extent of paclitaxel exposure in the prostate cancer patients. Neither the polymorphisms at exon 26 (C3435T) and at exon 21 (G2677A/T) in MDR1 nor the CYP3A5*1 allele significantly affected the total body clearance of paclitaxel. CONCLUSION: The hematological side effects of paclitaxel were intensified by gemcitabine, and were correlated with paclitaxel pharmacokinetics. Monitoring of the serum concentration of paclitaxel will facilitate the therapy, with less myelosuppression and without any loss of therapeutic efficacy.
Abstract: BACKGROUND: Limb ischemia remains a challenge. To overcome shortcomings or limitations of gene therapy or cell transplantation, a sustained release system of basic fibroblast growth factor (bFGF) using biodegradable gelatin hydrogel has been developed. METHODS AND RESULTS: A phase I-IIa study was performed, in which 7 patients had critical limb ischemia. They were intramuscularly injected with 200 microg of bFGF-incorporated gelatin hydrogel microspheres into the gastrocnemius of the ischemic limb. End-points were safety and feasibility of treatment after 4 and 24 weeks. One patient was excluded from the study for social reasons, but only after symptomatic improvements. In the evaluation of the other 6 patients, significant improvements were observed in the distance walked in 6 min (295+/-42 m vs 491+/-85 m for pretreatment vs after 24 weeks, p=0.023) and in transcutaneous oxygen pressure (53.5+/-5.2 mmHg vs 65.5+/-4.0 mmHg, p=0.03). The rest pain scale also improved (3.5+/-0.2 vs 1.0+/-0.6, p=0.022). The ankle-brachial pressure index improved at 4 weeks but not at 24 weeks. Among 5 patients who had a non-healing foot ulcer, the ulcer was completely healed in 3 patients, reduced in 1, and there was no change in 1 patient at 24 weeks. The blood levels of bFGF were undetected or within the normal level in all patients. CONCLUSIONS: The sustained release of bFGF from gelatin hydrogel might be simple, safe, and effective to achieve therapeutic angiogenesis because it did not need genetic materials or collection of implanted cells, and because it did not have any general effects, which was supported by there being no elevation of the bFGF serum level.
