Jun-ichi Sawada

Population pharmacokinetics of gemcitabine and its metabolite in Japanese cancer patients: impact of genetic polymorphisms.

Background and ObjectiveGemcitabine (2′,2’-difluorodeoxycytidine) is an anticancer drug, which is effective against solid tumours, including non-small-cell lung cancer and pancreatic cancer. After gemcitabine is transported into cells by equilibrative and concentrative nucleoside transporters, it is phosphorylated by deoxycytidine kinase (DCK) and further phosphorylated to its active diphosphorylated and triphosphorylated forms. Gemcitabine is rapidly metabolized by cytidine deaminase (CDA) to an inactive metabolite, 2′,2′-difluorodeoxyuridine (dFdU), which is excreted into the urine. Toxicities of gemcitabine are generally mild, but unpredictable severe toxicities such as myelosuppression and interstitial pneumonia are occasionally encountered. The aim of this study was to determine the factors, including genetic polymorphisms of CDA, DCK and solute carrier family 29A1 (SLC29A1 [hENT1]), that alter the pharmacokinetics of gemcitabine in Japanese cancer patients.Patients and Methods250 Japanese cancer patients who received 30-minute intravenous infusions of gemcitabine at 800 or 1000mg/m2 in the period between September 2002 and July 2004 were recruited for this study. However, four patients were excluded from the final model built in this study because they showed bimodal concentration-time curves. Two patients who experienced gemcitabine-derived life-threatening toxicities in October 2006 and January 2008 were added to this analysis. One of these patients received 30-minute intravenous infusions of gemcitabine at 454 mg/m2 instead of the usual dose (1000 mg/m2).Plasma concentrations of gemcitabine and dFdU were measured by high-performance liquid chromatography-photodiode array/mass spectrometry. In total, 1973 and 1975 plasma concentrations of gemcitabine and dFdU, respectively, were used to build population pharmacokinetic models using nonlinear mixed-effects modelling software (NONMEM® version V level 1.1).Results and DiscussionTwo-compartment models fitted well to plasma concentration-time curves for both gemcitabine and dFdU. Major contributing factors for gemcitabine clearance were genetic polymorphisms of CDA, including homozygous CDA*3 [208G>A (Ala70Thr)] (64% decrease), heterozygous *3 (17% decrease) and CDA -31delC (an approximate 7% increase per deletion), which has a strong association with CDA*2 [79A>C (Lys27Gln)], and coadministered S-1, an oral, multicomponent anti-cancer drug mixture consisting of tegafur, gimeracil and oteracil (an approximate 19% increase). The estimated contribution of homozygous CDA*3 to gemcitabine clearance provides an explanation for the life-threatening severe adverse reactions, including grade 4 neutropenia observed in three Japanese patients with homozygous CDA*3. Genetic polymorphisms of DCK and SLC29A1 (hENT1) had no significant correlation with gemcitabine pharmacokinetic parameters. Aging and increased serum creatinine levels correlated with decreased dFdU clearance.ConclusionA population pharmacokinetic model that included CDAgenotypes as a covariate for gemcitabine and dFdU in Japanese cancer patients was successfully constructed. The model confirms the clinical importance of the CDA*3 genotype.

Association of carboxylesterase 1A genotypes with irinotecan pharmacokinetics in Japanese cancer patients

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * Association of UDP-glucuronosyltransferase 1A1 (UGT1A1) genetic polymorphisms *6 and *28 with reduced clearance of SN-38 and severe neutropenia in irinotecan therapy was demonstrated in Japanese cancer patients. * The detailed gene structure of CES1 has been characterized. * Possible functional SNPs in the promoter region have been reported. WHAT THIS STUDY ADDS * Association of functional CES1 gene number with AUC ratio [(SN-38 + SN-38G)/irinotecan], an in vivo index of CES activity, was observed in patients with irinotecan monotherapy. * No significant effects of major CES1 SNPs on irinotecan PK were detected. AIMS Human carboxylesterase 1 (CES1) hydrolyzes irinotecan to produce an active metabolite SN-38 in the liver. The human CES1 gene family consists of two functional genes, CES1A1 (1A1) and CES1A2 (1A2), which are located tail-to-tail on chromosome 16q13-q22.1 (CES1A2-1A1). The pseudogene CES1A3 (1A3) and a chimeric CES1A1 variant (var1A1) are also found as polymorphic isoforms of 1A2 and 1A1, respectively. In this study, roles of CES1 genotypes and major SNPs in irinotecan pharmacokinetics were investigated in Japanese cancer patients. METHODS CES1A diplotypes [combinations of haplotypes A (1A3-1A1), B (1A2-1A1), C (1A3-var1A1) and D (1A2-var1A1)] and the major SNPs (-75T>G and -30G>A in 1A1, and -816A>C in 1A2 and 1A3) were determined in 177 Japanese cancer patients. Associations of CES1 genotypes, number of functional CES1 genes (1A1, 1A2 and var1A1) and major SNPs, with the AUC ratio of (SN-38 + SN-38G)/irinotecan, a parameter of in vivo CES activity, were analyzed for 58 patients treated with irinotecan monotherapy. RESULTS The median AUC ratio of patients having three or four functional CES1 genes (diplotypes A/B, A/D or B/C, C/D, B/B and B/D; n= 35) was 1.24-fold of that in patients with two functional CES1 genes (diplotypes A/A, A/C and C/C; n= 23) [median (25th-75th percentiles): 0.31 (0.25-0.38) vs. 0.25 (0.20-0.32), P= 0.0134]. No significant effects of var1A1 and the major SNPs examined were observed. CONCLUSION This study suggests a gene-dose effect of functional CES1A genes on SN-38 formation in irinotecan-treated Japanese cancer patients.

Genetic Variations and Haplotype Structures of the ABCB1 Gene in a Japanese Population: An Expanded Haplotype Block Covering the Distal Promoter Region, and Associated Ethnic Differences

As functional ABCB1 haplotypes were recently reported in the promoter region of the gene, we resequenced the ABCB1 distal promoter region, along with other regions (the enhancer and proximal promoter regions, and all 28 exons), in a total of 533 Japanese subjects. Linkage disequilibrium (LD) analysis based on 92 genetic variations revealed 4 LD blocks with the same make up as previously described (Blocks −1, 1, 2 and 3), except that Block 1 was expanded to include the distal promoter region, and that a new linkage between polymorphisms −1789G>A in the distal promoter region and IVS5 + 123A>G in intron 5 was identified. We re‐assigned Block 1 haplotypes, and added novel haplotypes to the other 3 blocks. The reported promoter haplotypes were further classified into several types according to tagging variations within Block 1 coding or intronic regions. Our current data reconfirm the haplotype profiles of the other three blocks, add more detailed information on functionally‐important haplotypes in Block 1 and 2 in the Japanese population, and identified differences in haplotype profiles between ethnic groups. Our updated analysis of ABCB1 haplotype blocks will assist pharmacogenetic and disease‐association studies carried out using Asian subjects.

Genetic Polymorphisms and Haplotypes of the Human Cardiac Sodium Channel α Subunit Gene (SCN5A) in Japanese and their Association with Arrhythmia

Genetic variations in cardiac ion channels have been implicated not only as the causes of inherited arrhythmic syndromes, but also as genetic risk factors for some acquired arrhythmias. To elucidate the potential roles of genetic polymorphisms of the α subunit of the voltage‐gated sodium channel type V (SCN5A) in cardiac rhythm disturbance, the entire SCN5A coding exons and their flanking introns were sequenced in 166 Japanese arrhythmic patients and 232 healthy controls. We detected 69 genetic variations, including 54 novel ones. Out of the 12 novel nonsynonymous single nucleotide polymorphisms (SNPs), p.Leu1988Arg was found at a frequency of 0.015. The other 11 SNPs were rare (0.001), with 6 found in arrhythmic patients and 5 in healthy controls. The frequency of a novel intronic SNP, c.703+130G>A, was significantly higher in the patients than in the controls, suggesting this SNP is associated with an unknown risk factor for arrhythmia. Following linkage disequilibrium analysis, the haplotype structure of SCN5A was inferred using high‐frequency SNPs. The frequency of the haplotype harbouring both p.Leu1988Arg and the common SNP p.His558Arg (haplotype GG) was significantly lower in the patients than in the controls. This finding suggests that this haplotype (GG) might have been positively selected in the controls because of its protective effect against arrhythmias. This study provides fundamental information necessary to elucidate the effect of genetic variations in SCN5A on channel function and cardiac rhythm in Japanese, and probably in the Asian population.

Considerations for non-clinical safety studies of therapeutic peptide vaccines

Guidelines for non-clinical studies of prophylactic vaccines against infectious diseases have been published widely, but similar guidelines for therapeutic vaccines, and especially therapeutic peptide vaccines, have yet to be established. The approach to non-clinical safety studies required for therapeutic vaccines differs from that for prophylactic vaccines due to differences in the risk-benefit balance and the mechanisms of action. We propose the following guidelines for non-clinical safety studies for therapeutic peptide vaccines. (i) Since the main safety concern is related to the immune response that might occur at normal sites that express a target antigen, identification of these possible target sites using in silico human expression data is important. (ii) Due to the strong dependence on HLA, it is not feasible to replicate immune responses in animals. Thus, the required non-clinical safety studies are characterized as those detecting off-target toxicity rather than on-target toxicity.

Genetic polymorphisms of FCGR2A encoding Fcγ receptor IIa in a Japanese population and functional analysis of the L273P variant

Fcγ receptor IIa (FcγRIIa) plays an important role in antibody-dependent cellular cytotoxicity and inflammation. Changes in FcγRIIa expression levels or activity caused by genetic polymorphisms in FCGR2A, the gene encoding FcγRIIa, may lead to differences in disease progression as well as efficacy of antibody therapeutics between individuals. In this study, we sequenced the 5′-flanking region along with all exons and their flanking regions of FCGR2A from 111 Japanese subjects. Forty-eight genetic variations were found including 12 novel ones. Beside the well-known functional 497Au2009>u2009G (H166R) polymorphism, we detected 818Tu2009>u2009C (L273P) at 0.005 frequency. Since the functional significance of this polymorphism has not been revealed, we next assessed the functions of the L273P substitution by expressing wild-type and the variant proteins in human Jurkat cells. The L273P variant protein showed similar cell surface expression and IgG-binding properties as the wild-type, but it exhibited a stronger signal transduction activity based on the approximately 2-fold enhancement of tyrosine phosphorylation of FcγRIIa itself. The current results suggest that L273P could have functional significance in the antibody-dependent clinical responses through FcγRIIa.

Investigating toxicity specific to adjuvanted vaccines

ABSTRACT In an attempt to understand the unique toxicity of adjuvanted vaccines, we studied how toxicity develops over time following vaccine administration. In addition to on‐ and off‐target toxicity typically observed with general pharmaceuticals, we observed toxicity associated with both the generation and the broad action of effectors (antibodies and/or cytotoxic T lymphocytes, CTLs). The impact on effector generation appears to be related to local tolerance specific to the adjuvant. The vaccine immune response by effectors serves to demonstrate species relevance as outlined in the recent WHO guideline on the nonclinical evaluation of adjuvanted vaccines. When regarded as pharmaceuticals that function at sites of local administration, adjuvants have inherent on‐ and off‐target toxicity. On‐target toxicity of the adjuvant is typically associated with effector generation, and could vary depending on animal species. Therefore, the use of species with sensitivity to adjuvants described in the WHO guidelines is required to evaluate the toxicity of the vaccine associated with effector generation. Changes in safety pharmacology endpoints would be considered off‐target and further studies are conducted only if changes in these endpoints are observed in nonclinical or clinical studies. Thus our decision tree does not recommend the routine conduct of stand‐alone safety pharmacology studies. Highlights“Time–toxicity profiles” of adjuvanted vaccines were elucidated.Unique profiles other than ordinary on‐vs off‐target toxicity were observed.Adjuvants are also found to have their own on‐vs off‐target toxicity profiles.These profiles account for animal species specificity of vaccines and adjuvants.The profiles were also useful for understanding safety pharmacology of vaccines.

Note on Regulatory Toxicology Requirements for Adjuvants and Vaccines; in View of the Newly Established WHO Guidelines

1 Review Division, Pharmaceuticals and Medical Devices Agency (PMDA), Kasumigaseki 3-3-2, Chiyoda-ku, Tokyo 100-0013, Japan 2 Research and Development Department, POC Clinical Research Inc. Taishidou 4-11, Setagaya-ku, Tokyo 154-0004, Japan 3 Pathology Department, The Chemo-SeroTherapeutic Research Institute, Kyokushi Kawabe 1314-1, Kikuchi-shi, Kumamoto 869-1298, Japan 4 Vaccine Research Laboratories, Kitasato Daiichi Sankyo Vaccine Co. Ltd., Kita-Kasai 1-16-13, Edogawa-ku, Tokyo 134-8630, Japan 5 Research Division, Chugai Pharmaceutical Co. Ltd., Komakado, 1-135, Gotemba, Shizuoka 412-8513, Japan

Impact of UDP-Glucuronosyltransferase 1A Haplotypes on Irinotecan Treatment

Irinotecan, an antineoplastic-prodrug, is widely used for the treatment of colorectal, lung and other cancers, and is one of model pharmaceuticals for personalized medicine. The active metabolite, SN-38, is a topoisomerase I inhibitor generated by hydrolysis of irinotecan by carboxylesterases. SN-38 is subsequently glucuronidated by uridine diphosphate glucuronosyltransferase 1As (UGT1As) to form an inactive metabolite, SN-38 G. A reduction in SN-38 G formation is closely related to severe irinotecan toxicities (diarrhea and neutropenia). Therefore, the association of UGT1A1 polymorphisms with irinotecan toxicities has been intensively studied. A number of recent irinotecan-pharmacogenetic studies have revealed significant associations between UGT1A1*28 and severe irinotecan toxicities, leading to the introduction of the clinical application of *28 genetic testing in the United States. This paper provides an overview of recent progress in irinotecan pharmacogenetics, focusing on updated findings on the haplotype structures of UGT1As and addressing the clinical significance of UGT1A1 genotypes/haplotypes to severe irinotecan toxicities. Issues that must be addressed for improvement of personalized irinotecan therapy are also discussed.