Mayumi Saeki

UGT1A1 Haplotypes Associated with Reduced Glucuronidation and Increased Serum Bilirubin in Irinotecan‐administered Japanese Patients with Cancer

A comprehensive haplotype analysis of UGT1A1 in the Japanese population was conducted, and the effects of these haplotypes were investigated with respect to UGT1A1‐related phenotypic parameters in patients with cancer who received irinotecan.

Irinotecan pharmacokinetics/pharmacodynamics and UGT1A genetic polymorphisms in Japanese: roles of UGT1A1*6 and *28.

Objectives SN-38, an active metabolite of irinotecan, is detoxified by glucuronidation with UGT1A isoforms, 1A1, 1A7, 1A9, and 1A10. The pharmacogenetic information on UGT1A haplotypes covering all these isoforms is important for the individualized therapy of irinotecan. Associations between UGT1A haplotypes and pharmacokinetics/pharmacodynamics of irinotecan were investigated to identify pharmacogenetic markers. Methods Associations between UGT1A haplotypes and the area under concentration curve ratio (SN-38 glucuronide/SN-38) or toxicities were analyzed in 177 Japanese cancer patients treated with irinotecan as a single agent or in combination chemotherapy. For association analysis, diplotypes of UGT1A gene segments [(1A1, 1A7, 1A9, 1A10), and Block C (common exons 2–5)] and combinatorial haplotypes (1A9-1A7-1A1) were used. The relationship between diplotypes and toxicities was investigated in 55 patients treated with irinotecan as a single agent. Results Among diplotypes of UGT1A genes, patients with the haplotypes harboring UGT1A1*6 or *28 had significantly reduced area under concentration curve ratios, with the effects of UGT1A1*6 or *28 being of a similar scale. A gene dose effect on the area under concentration curve ratio was observed for the number of haplotypes containing *28 or *6 (5.55, 3.62, and 2.07 for 0, 1, and 2 haplotypes, respectively, P<0.0001). In multivariate analysis, the homozygotes and double heterozygotes of *6 and *28 (*6/*6, *28/*28 and *6/*28) were significantly associated with severe neutropenia in 53 patients who received irinotecan monotherapy. Conclusions The haplotypes significantly associated with reduced area under concentration curve ratios and neutropenia contained UGT1A1*6 or *28, and both of them should be genotyped before irinotecan is given to Japanese and probably other Asian patients.

mRNA expression of multiple cytochrome P450 isozymes in four types of cultured skin cells

Background: Many drugs are known to induce allergic reactions in the skin. The metabolic activation of drugs resulting in the formation of protein adducts is thought to be a first step in the induction of these allergic reactions. We postulated that dermal tissue might be a site of drug activation by cytochrome P450 (CYP) isozymes. Methods: Messenger RNA was extracted from cultured Langerhans cells, keratinocytes, fibroblasts and melanocytes from 6 individuals, and CYP mRNA expression was analyzed by RT-PCR. Results: CYP1A1, 1B1 and 2E1 were found in all four cell types. CYP2A6, 2C, 2D6, 3A5, 3A7 and 4B1 mRNA was expressed in a cell-type- and/or individual-specific manner. CYP1A2, 2A7, 2B6 and 3A4 mRNA was not detectable. Conclusions: The mRNA for a variety of CYP isozymes was expressed in all four types of skin cells examined. These CYP enzymes may be involved in the pathogenesis of drug-induced allergic reactions in the skin.

Haplotype structures of the UGT1A gene complex in a Japanese population.

Genetic polymorphisms of UDP-glucuronosyltransferases (UGTs) are involved in individual and ethnic differences in drug metabolism. To reveal co-occurrence of the UGT1A polymorphisms, we first analyzed haplotype structures of the entire UGT1A gene complex using the polymorphisms from 196 Japanese subjects. Based on strong linkage disequilibrium between UGT1A8 and 1A10, among 1A9, 1A7, and 1A6, and between 1A3 and 1A1, the complex was divided into five blocks, Block 8/10, Block 9/6, Block 4, Block 3/1, and Block C, and the haplotypes for each block were subsequently determined/inferred. Second, using pyrosequencing or direct sequencing, additional 105 subjects were genotyped for 41 functionally tagged polymorphisms. The data from 301 subjects confirmed the robustness of block partitioning, but several linkages among the haplotypes with functional changes were found across the blocks. Thus, important haplotypes and their linkages were identified among the UGT1A gene blocks (and segments), which should be considered in pharmacogenetic studies.

Comprehensive UGT1A1 Genotyping in a Japanese Population by Pyrosequencing

Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), is important in the detoxification and enhanced elimination of a large number of endogenous and exogenous substrates. The human UGT1A gene complex contains at least nine variations of exon 1, common exons 2–5, and a single exon 1 splices to exons 2–5 (1). Of the UGT1A isoforms, UGT1A1 is primarily responsible for the glucuronidation of bilirubin in the human liver and can also conjugate phenols, anthraquinones, flavonoids, and a variety of therapeutic drugs and their metabolites (e.g., SN-38, an active irinotecan metabolite) (2)(3). Several functional polymorphisms in UGT1A1 are associated with reduced bilirubin glucuronidation activity and cause hyperbilirubinemia (Gilbert and Crigler–Najjar syndromes). UGT1A1 TATA box variants [A(TA)6TAA>A(TA)5/7/8TAA] are associated with enhanced [(TA)5] or reduced [(TA)7/8] UGT1A1 transcription (4). Among them, the (TA)6 and (TA)7 repeats have been reported in Asians. The variant (TA)7 is associated with reduced glucuronidation of SN-38 and bilirubin, as well as the pathogenesis of Gilbert syndrome (5). In addition, a T-to-G substitution at nucleotide −3279 (A of the translational start codon in GenBank accession no. AF297093.1 is nucleotide number 1) in the UGT1A1 phenobarbital-responsive enhancer module reduces transcriptional activity (6). The most common nonsynonymous single-nucleotide polymorphism (SNP) (211G>A) that causes an amino acid alteration (glycine to arginine at codon 71) is found in Asian populations at frequencies of 13–23% (7)(8). The 686C>A (P229Q) variation in the Taiwanese has a frequency of 2.8% (8). Also associated with Gilbert syndrome are 211G>A (G71R) and 686C>A (P229Q). Rare in Japanese and Taiwanese patients is 1456T>G (Y486D), which is associated with the more severe type II Crigler–Najjar syndrome (8)(9). Our previous study demonstrated that …

Identification of the functional vitamin D response elements in the human MDR1 gene

P-glycoprotein, encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. The expression of MDR1 is induced by a variety of compounds, of which 1alpha,25-dihydroxyvitamin D(3) is known to be an effective inducer. However, it remains unclear how 1alpha,25-dihydroxyvitamin D(3) regulates the expression of MDR1. In this study, we demonstrated that the vitamin D receptor (VDR) induces MDR1 expression in a 1alpha,25-dihydroxyvitamin D(3)-dependent manner. Luciferase assays revealed that the region between -7.9 and -7.8k bp upstream from the transcription start site of the MDR1 is responsible for the induction by 1alpha,25-dihydroxyvitamin D(3). Electrophoretic mobility shift assays revealed that several binding sites for the VDR/retinoid X receptor alpha (RXRalpha) heterodimer are located between the -7880 and -7810 bp region, to which the three molecules of VDR/RXRalpha are able to simultaneously bind with different affinities. Luciferase assays using mutated constructs revealed that the VDR-binding sites of DR3, DR4(I), MdC3, and DR4(III) contribute to the induction, indicating that these binding sites act as vitamin D response elements (VDREs). The contribution of each VDRE to the inducibility was different for each response element. An additive effect of the individual VDREs on induced luciferase activity by 1alpha,25-dihydroxyvitamin D(3) was also observed. These results indicate that the induction of MDR1 by 1alpha,25-dihydroxyvitamin D(3) is mediated by VDR/RXRalpha binding to several VDREs located between -7880 and -7810bp, in which every VDRE additively contributes to the 1alpha,25-dihydroxyvitamin D(3) response.

Genetic variations and haplotype structures of the DPYD gene encoding dihydropyrimidine dehydrogenase in Japanese and their ethnic differences

AbstractDihydropyrimidine dehydrogenase (DPD) is an inactivating and rate-limiting enzyme for 5-fluorouracil (5-FU), and its deficiency is associated with a risk for developing a severe or fatal toxicity to 5-FU. In this study, to search for genetic variations of DPYD encoding DPD in Japanese, the putative promoter region, all exons, and flanking introns of DPYD were sequenced from 341 subjects including cancer patients treated with 5-FU. Fifty-five genetic variations, including 38 novel ones, were found and consisted of 4 in the 5′-flanking region, 21 (5 synonymous and 16 nonsynonymous) in the coding exons, and 30 in the introns. Nine novel nonsynonymous SNPs, 29C>A (Ala10Glu), 325T>A (Tyr109Asn), 451A>G (Asn151Asp), 733A>T (Ile245Phe), 793G>A (Glu265Lys), 1543G>A (Val515Ile), 1572T>G (Phe524Leu), 1666A>C (Ser556Arg), and 2678A>G (Asn893Ser), were found at allele frequencies between 0.15 and 0.88%. Two known nonsynonymous variations reported only in Japanese, 1003G>T (*11, Val335Leu) and 2303C>A (Thr768Lys), were found at allele frequencies of 0.15 and 2.8%, respectively. SNP and haplotype distributions in Japanese were quite different from those reported previously in Caucasians. This study provides fundamental information for pharmacogenetic studies for evaluating the efficacy and toxicity of 5-FU in Japanese and probably East Asians.

Essential Contribution of CD4+ T Cells to Antigen-Induced Nasal Hyperresponsiveness in Experimental Allergic Rhinitis.

Nasal hyperresponsiveness (NHR) is a characteristic feature of allergic rhinitis (AR); however, the pathogenesis of NHR is not fully understood. In this study, during the establishment of an experimental AR model using ovalbumin-immunized and -challenged mice, augmentation of the sneezing reaction in response to nonspecific proteins as well as a chemical stimulant was detected. Whether NHR is independent of mast cells and eosinophils was determined by using mast cell- and eosinophil-deficient mice. NHR was suppressed by treatment with anti-CD4 antibody, suggesting the pivotal contribution of CD4+ T cells. Furthermore, antigen challenge to mice to which in vitro-differentiated Th1, Th2, and Th17 cells but not naïve CD4+ T cells had been adoptively transferred led to the development of equivalent NHR. Since antigen-specific IgE and IgG were not produced in these mice and since antigen-specific IgE-transgenic mice did not develop NHR even upon antigen challenge, humoral immunity would be dispensable for NHR. CD4+ T cells play a crucial role in the pathogenesis of AR via induction of NHR, independent of IgE-, mast cell-, and eosinophil-mediated responses.

Identification of biomarker sets for predicting the efficacy of sublingual immunotherapy against pollen-induced allergic rhinitis

Sublingual immunotherapy (SLIT) is effective against allergic rhinitis, although a substantial proportion of individuals is refractory. Herein, we describe a predictive modality to reliably identify SLIT non-responders (NRs). We conducted a 2-year clinical study in 193 adult patients with Japanese cedar pollinosis, with biweekly administration of 2000 Japanese allergy units of cedar pollen extract as the maintenance dose. After identifying high-responder (HR) patients with improved severity scores and NR patients with unchanged or exacerbated symptoms, differences in 33 HR and 34 NR patients were evaluated in terms of peripheral blood cellular profiles by flow cytometry and serum factors by ELISA and cytokine bead array, both pre- and post-SLIT. Improved clinical responses were seen in 72% of the treated patients. Pre-therapy IL-12p70 and post-therapy IgG1 serum levels were significantly different between HR and NR patients, although these parameters alone failed to distinguish NR from HR patients. However, the analysis of serum parameters in the pre-therapy samples with the Adaptive Boosting (AdaBoost) algorithm distinguished NR patients with high probability within the training data set. Cluster analysis revealed a positive correlation between serum Th1/Th2 cytokines and other cytokines/chemokines in HR patients after SLIT. Thus, processing of pre-therapy serum parameters with AdaBoost and cluster analysis can be reliably used to develop a prediction method for HR/NR patients.

Selective Suppression of Th2 Cell-Mediated Lung Eosinophilic Inflammation by Anti-Major Facilitator Super Family Domain Containing 10 Monoclonal Antibody

BACKGROUND The eosinophil is deeply associated with the pathogenesis of bronchial asthma and other allergic diseases. We recently identified a novel eosinophil-specific cell surface molecule, major facilitator super family domain containing 10 (Mfsd10). A monoclonal antibody (mAb) against Mfsd10 (M2) showed selective binding and neutralizing activities for eosinophils. However, the relative potency of the blockage of Mfsd10 and other eosinophil-specific molecules for the treatment of allergic diseases has not been evaluated. Therefore, in this study, the effects of M2 and an anti-Siglec-F mAb on antigen-immunized and antigen-specific Th2 cell-transferred murine eosinophilic inflammation models were comparatively investigated. METHODS Ovalbumin (OVA)-specific Th2 cells were differentiated from naïve CD4+ T cells of DO11.10/RAG-2-/- mice in vitro and cytokine producing activity of the Th2 cells was examined. OVA-immunized and Th2 cell-transferred BALB/c mice were treated with M2 or anti-Siglec-F and challenged with OVA. Then the number of inflammatory cells and the concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) were determined. RESULTS Antigen-specific Th2 cells produced large amounts of IL-4, IL-5 and IL-13 but not IL-17A or IFN-γ. Administration of M2 significantly suppressed antigen-induced lung eosinophil infiltration both in OVA-immunized and Th2 cell-transferred mice. The potency as well as selectivity of M2 for down-regulating eosinophils was quite similar to that of anti-Siglec-F. Both mAbs did not affect antigen-induced IL-5 production in the lungs. CONCLUSIONS Mfsd10 as well as Siglec-F could be an effective target to treat eosinophil-related disorders including bronchial asthma.