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Cyclosporine (CsA)-induced nephrotoxicity can become a major obstacle to continuous use. The aim of this study was to optimize CsA dose to avoid its irreversible nephrotoxicity. Twenty-three Japanese patients with pediatric-onset systemic lupus erythematosus or idiopathic nephrotic syndrome, who were maintained in a stable condition by oral dosing of CsA microemulsion, were enrolled in this study. The patients were stratified into 3 groups; those with no, reversible, and irreversible nephrotoxicity, according to periodically performed renal pathohistological examinations. A higher concentration of CsA in blood (p=0.002-0.011) and a longer duration of CsA treatment (p=0.002) were risk factors for irreversible nephrotoxicity, and the cumulative CsA dose, the product of the maintenance dose and duration of CsA treatment, was predictive of nephrotoxicity (p=0.036). The maximum target blood concentration at 2 h post-dose, C(2), to avoid CsA-induced irreversible nephrotoxicity was 700 ng/ml, although the cumulative CsA dose of 4850 mg/kg would result in a 50% probability of nephrotoxicity.

Glucocorticoids such as prednisolone are used for their anti-inflammatory properties. But there is evidence to suggest that under certain conditions, glucocorticoids have pro-inflammatory effects, for example, enhancement of IL-1beta production. To date, it has been reported that IL-1beta production intensity was associated with single nucleotide polymorphisms at positions -1470, -511, and -31 in the promoter region and at position 3954 in exon 5 of the IL-1beta gene. In the present study, it was examined whether these IL-1beta genotypes were associated with the suppressive effect of prednisolone on IL-1beta production in human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS). A midrange concentration (10(-6) M) of prednisolone suppressed the LPS-induced increase in IL-1beta mRNA expression and protein release, while higher concentrations (10(-5) M, 10(-4) M) exhibited less suppression or had a synergistic stimulative effect on IL-1beta production in certain subjects. Under treatment with 10(-4) M prednisolone, the levels of IL-1beta protein production stimulated by LPS in PBMC extracted from the subjects with the IL-1beta TT(-31), TC(-31), and CC(-31) genotypes were suppressed to 6.0+/-3.4%, 31.4+/-57.0%, and 87.7+/-84.8%, respectively, of the level in prednisolone-untreated control cells (TT(-31) vs. CC(-31), p<0.05). Glucocorticoid-based anti-inflammatory therapy might be less effective in patients with the IL-1beta TC(-31) and CC(-31) genotypes than those with the TT(-31) genotype.

In our previous study, the MDR1/Pglycoprotein-overexpressing multidrug resistant subline, Hvr100-6, was established from the human cervical carcinoma cell line HeLa-Ohio (HeLa) by stepwise exposure to an anti-microtubule agent, vinblastine sulfate, a typical substrate of MDR1. Their gene and protein expression profiles were analyzed herein, and 148 genes were identified to be differentially expressed by cDNA microarray analysis. The up-regulation of sorcin, a soluble resistance-related calcium-binding protein of 22 kDa, was confirmed in Hvr100-6 cells by the proteome analysis. To clarify the relationship between MDR1 and sorcin, HeLa cells were treated with small interfering RNAs (siRNAs) targeted for theirs mRNAs. The siRNA for MDR1 mRNA resulted in its decrease by 86% and 61% on the days 1 and 2 after the treatment, whereas the expression level of sorcin mRNA was not changed. On the other hand, the siRNA for sorcin mRNA suppressed its expression by 80-90% on days 1-3 after the treatment. Interestingly; suppression of sorcin induced a more than 3-fold increase in the expression level for MDR1 mRNA. An efflux function of MDR1 evaluated with using rhodamine 123 as a probe showed a tendency to be increased in HeLa cells treated with siRNA for sorcin, compared with that in the cells treated with scramble siRNA. The activity and the expression of caspase-3 in the sorcin knock-down HeLa cells were relatively higher than those in the cells treated with scramble siRNA. Thus, we demonstrated that sorcin might be a partial suppressor of MDR1 expression. Furthermore, the present study suggested that sorcin repressed apoptosis via dysfunction of caspase-3.

The expression level of MDR1 mRNA was evaluated in colorectal adenocarcinomas and adjacent noncancerous colorectal tissues obtained from 21 Japanese patients. It was lower in the former than in the latter (p=0.012), suggesting its down-regulation as a consequence of malignant transformation of colorectal tissues, possibly with the suppression of differentiation. Relatively lower expression was suggested in moderately-differentiated colorectal adenocarcinomas than well-differentiated ones, but there was no statistical difference (p=0.111). MDR1 mRNA up-regulation was found in a colorectal adenocarcinoma cell line, HCT-15, after treatment with two typical differentiating agents, sodium butyrate and all-trans retinoic acid, suggesting its involvement in the cellular events, resulting in differentiation without malignant transformation. MDR1 T-129C, but not G2677A,T and C3435T, was associated with the lower expression of MDR1 mRNA both in colorectal adenocarcinomas (p=0.040) and adjacent noncancerous colorectal tissues (p=0.023), possibly being an useful invasive marker predicting poorly-differentiated colorectal adenocarcinomas and thereby the poor prognosis of the patients, especially when no extra biopsy samples will be obtained. Further investigations with relatively large number of patients should be undertaken to confirm these preliminary results.

Synthetic DNA templates were compared with authentic cDNA templates as standards for the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The single-stranded DNA template used here targeted the multidrug resistant transporter P-glycoprotein/MDR1. The double-stranded DNA template, targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was synthesized using an exonuclease-free large fragment E. coli DNA polymerase I. The human colon carcinoma cell line Caco-2 and human duodenum biopsies were used to prepare the authentic cDNA templates. The standard lines were comparable for the synthetic DNA templates and authentic cDNA templates. Long-term cryopreservation at -80 degrees C resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic cDNA templates in the case of MDR1, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic cDNA templates. Even for the synthetic DNA templates, repetitive freeze-thawing resulted in destabilization, especially at lower concentrations, and degradation products might have interfered with the RT-PCR's efficiency. The synthetic DNA templates are better than the authentic cDNA templates, but more than 5 cycles of repetitive freeze-thawing should be avoided.

Recently, a silent polymorphism of C3435T of the MDR1 gene, encoding the multidrug resistant transporter MDR1/P-glycoprotein, has been found to be associated with susceptibility to ulcerative colitis (UC), but this remains controversial. This study was conducted to find a possible reason for the discrepancies, and it was suggested that the age of onset was important for the association, namely, C3435T was predictive of susceptibility to later onset UC, but not for early onset UC. Linkage disequilibrium of C3435T with T-129C, C1236T and G2677A, T was suggested to be altered in UC, but the analysis of their haplotype provided no advantage in terms of prediction over that with only C3435T. The effect of C3435T on susceptibility could not be explained by that on mRNA expression in rectal mucosa, but it was greater in the C(3435)-noncarriers in the early onset group, allowing the individualization of steroid-based pharmacotherapy.

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is a shrub widely distributed in South China and India. In this study, the antiproliferative activity of the ethanol extract of root and aqueous extract of leaves of R. nasutus, and the supposed active moiety rhinacanthin C was assessed in vitro using the human cervical carcinoma cell line HeLa, its MDR1-overexpressing subline Hvr100-6, human prostate carcinoma PC-3 cells and human bladder carcinoma T24 cells. Rhinacanthin C was chemically synthesized and its content in the R. nasutus extracts was determined by HPLC with a photodiode array detector. The antiproliferative activity of the R. nasutus extracts was also assessed in vivo using sarcoma 180-bearing mice. It was suggested that 1) the in vitro antiproliferative activity of rhinacanthin C was comparable with or slightly weaker than that of 5-FU, 2) rhinacanthin C showed antiproliferative activity for MDR1-overexpressing Hvr100-6 cells, similarly to parent HeLa cells, 3) the in vitro antiproliferative activity of the ethanol extract of root R. nasutus was due to rhinacanthin C, whereas that of the aqueous extract of leaves of R. nasutus was due to constituents other than rhinacanthin C, and 4) both of the R. nasutus extracts showed in vivo antiproliferative activity after oral administration once daily for 14 d.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild topical side effects including irritation. In this paper, the cytotoxicity of HQ was assessed in vitro using rat skin fibroblasts as the concentration with 50% survival after 24 h exposure to be 16.5 microM. The intradermal concentrations at 2 h after application of the HQ ointments was also estimated to be 358 mM and 51.7 mM in stratum corneum and viable tissue (viable epidermis+dermis), respectively, by an in vitro rat skin permeation study with rat full-thickness abdominal skin and Franz-type diffusion cells. It was demonstrated that the intradermal concentration of HQ was much higher than that eliciting cytotoxicity, suggesting that the topical side effects after application of HQ ointment were due to the cytotoxicity of HQ.

A series of clinical studies on the cytochrome P450 2C19 (CYP2C19) genotype and the pharmacokinetics and pharmacodynamics of three proton pump inhibitors (PPIs), omeprazole, lansoprazole and rabeprazole, have been conducted to establish the individualized pharmacotherapy based on the CYP2C19 genotyping, and in the present study, the CYP2C19 genotype-dependency was more pronounced for omeprazole than the other two. Herein, to validate further the difference among 3 PPIs in CYP2C19 genotype-dependency on the phenotype, a comparative in vitro study was conducted using the human liver microsomes and newly developed anti-human CYP antibodies. The residual concentrations of omeprazole and lansoprazole in 5 lots of human liver microsomes were dependent on the CYP2C19 activities, however, for rabeprazole, there was no correlation. The hydroxylation of omeprazole was more inhibited by anti-CYP2C19 antibody than lansoprazole, whereas anti-CYP3A4 antibody showed similar inhibition. In conclusion, the relative contribution of CYP2C19 on total metabolism of 3 PPIs elucidated herein coincided with the CYP2C19 genotype-dependent pharmacokinetics.

In this paper, the effects of H2O2, a typical reactive oxygen species (ROS), on cell proliferation or death were examined using the human cervical carcinoma cell line HeLa and its MDR1-overexpressing subline, Hvr100-6, which was established by stepwise exposure to vinblastine. It was confirmed that the growth of HeLa cells was enhanced by H2O2 at relatively low concentrations in a concentration-dependent manner, and the growth enhancement was suppressed by antioxidants. Doxorubicin and daunorubicin also enhanced the growth of HeLa cells at concentrations lower than IC50 values, and the antioxidants suppressed this effect, being consistent with the fact that both anticancer drugs generate ROS. The growth enhancement by H2O2 or doxorubicin and daunorubicin was not observed in Hvr100-6 cells. In addition, it was suggested that antioxidants had no effect on MDR1 mRNA expression in HeLa and Hvr100-6 cells, and thereby hardly reverse multidrug resistance in tumor cells.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild side effects. Chromatic aberration is expected to induce non-compliance, and this may be the reason for the relatively large variability in efficacy. In this paper, the effects of various storage conditions on the chromatic aberration and HQ content of HQ ointments were evaluated, and it was suggested that the chromatic aberration was accelerated by exposure to high temperature, air and light, although these had no effect on the HQ content. In addition, various types of HQ ointments were prepared to find a formulation to minimize chromatic aberration, and it was found that the concentrations of antioxidants, Na(2)SO(3) and L(+)-ascorbic acid (AsA), seemed to be too high, and that the protective effect of AsA on chromatic aberration was mainly due to its acidifying effect.

The multidrug transporter, MDR1-mediated interaction of digoxin with antiarrhythmic or antianginal drugs was examined in vitro by using the MDR1-overexpressing LLC-GA5-COL150 cells, which were established by transfection with human MDR1 cDNA into porcine kidney epithelial LLC-PK(1) cells. Amiodarone, its active metabolite monodesethyl-amiodarone (DEA), and quinidine markedly inhibited the basal-to-apical transport (renal secretion) of [(3)H]digoxin and increased the apical-to-basal transport (reabsorption), but cibenzoline and lidocaine showed slight inhibition of the transport, and disopyramide and mexiletin had no such effects. The IC(50) values for amiodarone, DEA and quinidine on [(3)H]digoxin transport in LLC-GA5-COL150 cells were 5.48 microM, 1.27 microM and 9.52 microM, respectively. These were comparable to, or only several times the achievable concentration in clinical use, suggesting that MDR1 could be responsible for the drug interaction between digoxin and amiodarone found in clinical reports and that DEA contributes the elevation of digoxin serum concentration. Similarly, dipyridamole altered the transport, but isosorbide showed only slight modification of the transport. The IC(50) value for dipyridamole was 40.0 microM, also only several times the achievable concentration in clinical use, indicating a risk of interaction.

The multidrug resistant transporter MDR1/P-glycoprotein, the gene product of MDR1, is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily of membrane transporters. MDR1 acts as an energy-dependent efflux pump that exports its substrates out of cells. MDR1 was originally isolated from resistant tumor cells as part of the mechanism of multidrug resistance, but over the last decade, it has been elucidated that human MDR1 is also expressed throughout the body to confer intrinsic resistance to the tissues by exporting unnecessary or toxic exogeneous substances or metabolites. A number of structurally unrelated drugs are substrates for MDR1, and MDR1 and other transporters are recognized as an important class of proteins for regulating pharmacokinetics and pharmacodynamics. In 2000, Hoffmeyer et al. performed a systemic screening for MDR1 polymorphisms and detected 15 single nucleotide polymorphisms (SNPs). They also indicated that a polymorphism in exon 26 at position 3435 (C3435T), a silent mutation, affected the expression level of MDR1 protein in duodenum, and thereby the intestinal absorption of digoxin. To date, the genotype frequencies of C3435T have been investigated extensively using a larger population and interethnic difference has been elucidated, and a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical studies on MDR1 genotype-related MDR1 expression and pharmacokinetics have also been performed around the world; however, results were not always consistent with Hoffmeyer's report. In this review, published reports are summarized for the future individualization of pharmacotherapy based on MDR1 genotyping. In addition, recent investigations have raised the possibility that MDR1 and related transporters play a fundamental role in regulating apoptosis and immunology, and in fact, there are reports of MDR1-related susceptibility to inflammatory bowel disease, HIV infection and renal cell carcinoma. Herein, these issues are also summarized, and the current status of the knowledge in the area of pharmacogenomics of other transporters is briefly introduced.

In the present study, we examined whether polymorphisms in the ATP-binding cassette (ABC) transporter genes, MDR1, MRP1 and MRP2, were associated with their respective mRNA expression levels in duodenal enterocytes of 13 healthy Japanese volunteers. MDR1 genotypes of T-129C, G2677(A,T) and C3435T, MRP1 genotypes of G128C, C218T, G2168A and G3173A, and MRP2 genotypes of C-24T, G1249A, C2302T, C2366T and G4348A were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or direct sequencing. Mutations T-129C, G2677(A,T) and C3435T of MDRI gene were found at allele frequencies of 2/26, 16/26 and 12/26, respectively. Mutations G2168A of the MRPI gene and C-24T of the MRP2 gene were also found at allele frequencies of 1/26 and 6/26, respectively, whereas other mutations were not detected in MRP1 and MRP2 genes. The relative concentrations (mean +/- S.E.) of MDR1 mRNA to villin mRNA were 0.38 +/- 0.15, 0.56 +/- 0.14 and 1.13 +/- 0.42 in the subjects with C/C3435, C/T(3435) and T/T(3435), respectively, which supported the lower serum concentrations of digoxin after single oral administration in the subjects with the mutant T-allele at position 3435. Genetic collaboration between positions 3435 and 2677 was suggested, and those in G/G2677, G/(A,T)(2677) and T/(A,T)(2677) were 0.16 +/- 0.05, 1.10 +/- 0.40, and 0.63 +/- 0.16, respectively (p = 0.107). However, there was no remarkable effect of the G2168A of the MRP1 gene or of C-24T of the MRP2 gene on the relative MRP1 or MRP2 mRNA concentrations, respectively.

Sulfapyridine (SP), one of the metabolites of sulfasalazine (SASP), is further metabolized into N-acetylsulfapyridine (AcSP) by polymorphic N-acetyltransferase 2 (NAT2). NAT2 activity has been diagnosed by phenotyping, that is, evaluating plasma concentrations or urinary excretions of tentatively administered test drugs for dose individualization and avoidance of serious adverse events. Herein, we investigated the relationship between NAT2 genotypes and the pharmacokinetics of SP in healthy Japanese subjects, as well as the adverse events of SASP in patients with inflammatory bowel disease (IBD). Eight healthy subjects and 13 IBD patients were classified into three groups by NAT2 genotyping; the homozygote for the wild-type allele (Rapid Types), the compound heterozygote for the wild-type and mutant alleles (Intermediate Types), and the homozygote for mutant alleles (Slow Types). A single oral dose of 40 mg/kg SASP was administered to each healthy subject, and plasma and urine samples were taken until 51 and 72 h after administration, respectively. Both the SP and AcSP concentrations in each sample were determined by the HPLC method. The NAT2 genotypes were well-correlated with the plasma concentrations or urinary excretions of SP and AcSP in 8 healthy subjects, except for one Slow Type. In patients with IBD, skin rash was seen in 3 of 6 Rapid Types and 1 of 6 Intermediate Types, consistent with the concept that hypersensitive reactions are independent of serum SP concentrations. In contrast, SASP dosing-related acute pancreatitis was found in the Slow Type patient. In this case, the NAT2 activity was diagnosed by genotyping in advance, and the medical staff could pay scrupulous attention, resulting in no serious subjective symptoms such as abdominal pain, anorexia or fever. Further investigations on the relationship between the NAT2 genotype and adverse events are required, although genotyping appeared to be a promising method to avoid such serious adverse events.

In anti-Helicobacter pylori therapy using omeprazole and antimicrobials, the efficacy can be related to the CYP2C19 genotype groups; the eradication rates were 83% in extensive metabolizers and 100% in poor metabolizers. The present study was undertaken to help predict the optimal dosage of omeprazole for extensive metabolizers in this therapy. Seven healthy Japanese subjects, classified based on the CYP2C19 genotype into extensive metabolizers (n=4) and poor metabolizers (n=3), participated in this study. Each subject received a single oral dose of omeprazole 20, 40, and 80 mg, with at least a 1-week washout period between each dose. Plasma concentrations of omeprazole and its two metabolites were monitored for 12 h after each dose of medication. After each dose was administered, the pharmacokinetic profiles of omeprazole and its two metabolites were significantly different between extensive metabolizers and poor metabolizers. The area under the plasma concentration-time curve (AUC) of omeprazole in extensive metabolizers was disproportionally increased 3.2- or 19.2-fold with dose escalation from 20 to 40 or 80 mg omeprazole, respectively. In contrast, the AUC of omeprazole was proportionally increased with the higher dose in poor metabolizers. The AUC of omeprazole after 20 mg administration to poor metabolizers was almost equal to the AUC in extensive metabolizers after 80 mg administration. In anti-H. pylori therapy, the recommended dose of omeprazole for extensive metabolizers is suggested to be a maximum of 80 mg x 2/d based on pharmacokinetic considerations.

The cytotoxic effects of 27 anticancer drugs including amrubicin, vinorelbine, paclitaxel, docetaxel, gemcitabine, and irinotecan were evaluated in human cervical carcinoma HeLa cells, and drug-resistant HeLa-derived Hvrl-1, HvrlO-6, and Hvr100-6 cells, which were newly established by stepwise exposure to vinblastine. FACS and RT-PCR analysis indicated that MDR1 (P-glycoprotein) was induced without any alterations in expression of its related transporters. Hvrl00-6 cells showed 2- to 200-fold higher resistance to anthracyclines than HeLa cells, and unexpectedly showed slight resistance to idarubicin and amrubicin. The relative resistance to vinca-alkaloids was 300- to 600,000-fold, and HvrlOO-6 cells showed the highest relative resistance to vinorelbine. HvrlOO-6 cells also showed 4000- and 60000-fold resistance to the taxanes paclitaxel and docetaxel, respectively. Hvr100-6 cells were also resistant to 6-mercaptopurine, actinomycin D, etoposide, and mitomycin C, with relative resistance of 8-, 45000-, 12-, and 9-fold, respectively. In contrast, HvrlOO-6 cells showed no or slight resistance to platinum derivatives, pyrimidine analogues, and alkylating agents or to irinotecan and its active form, or tamoxifen. The cytotoxicity of anthracyclines, vinca-alkaloids, taxanes, actinomycin D, and etoposide was extensively reversed by cyclosporin A. Cyclosporin A had no effect on the cytotoxicity of 6-mercaptopurine or mitomycin C, suggesting that resistance to these drugs was not mediated via MDR1. The alterations in cytotoxicity by overexpression of MDR1 and effects of cyclosporin A could be also qualitatively explained by [3H]vinblastine uptake experiments. The 27 anticancer drugs analyzed here could be classified into substrates and nonsubstrates for MDR1. This will be useful for designing effective regimens for chemotherapy.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in Japan by imitating skin lightening creams commercially available in the U.S.A. and European Union. In our hospital, HQ ointments consisting of 5 or 10% HQ, 1.6% L(+)-ascorbic acid (AsA), 0.5% (w/w) Na2SO3, 10% (v/w) glycerin and hydrophilic ointment have been prepared. However, various problems have been observed including chromatic aberration of HQ ointments, relatively large variability of efficacy, and undesirable side effects although they were mild. Herein, the pharmaceutical and clinical properties of the HQ ointments were evaluated. HQ ointments were highly effective for treatment of various types of skin pigmentation. Chromatic aberration occurred during 3 months of storage, but this could be suppressed by storage at 4 degrees C. Chromatic aberration was independent of prescribed HQ content, and was not explained by alterations of HQ or p-benzoquinone (p-BQ) contents. Unexpectedly, removal of both antioxidants resulted in suppression of chromatic aberration, but an increase in p-BQ content. Acidification by removal of Na2SO3 only was further effective for the suppression of chromatic aberration, but with a decrease of p-BQ content except in the initial period. Chromatic aberration was due to water soluble material and insoluble material both formed by co-existence of HQ and p-BQ at a molecular ratio of 5:3 to 1:1. 1H-NMR analysis elucidated that the water soluble material was not HQ or p-BQ, and the insoluble material was a complex of HQ and p-BQ with non-covalent binding.