Introduction

Ticlopidine, an anti-platelet agent, has been widely used for the secondary prevention of atherothrombosis.¹ Severe hepatotoxicity, mainly of cholestatic type, may occur with ticlopidine treatment,² and there appears to be an increased rate of hepatic adverse reactions in Japanese compared with Caucasian patients.³

There are several reports indicating that ticlopidine-induced hepatotoxicity is caused by an immune-mediated reaction^{4, 5, 6} and therefore indicates an idiosyncratic toxicity. Idiosyncratic toxicity can be caused by drug-reactive metabolite(s), of which the thiophene ring of ticlopidine is a known example.⁷ For instance, tienilic acid, a thiophene-containing diuretic drug that was withdrawn from the market, is metabolized by cytochrome CYP2C9⁸ to yield a reactive metabolite that covalently binds to several macromolecules, including CYP2C9 itself.⁹ Immune-mediated mechanisms via such covalent binding are believed to be associated with idiosyncratic drug-induced hepatotoxicity.^{10, 11} We therefore hypothesized that ticlopidine-induced hepatotoxicity might be mediated by a similar mechanism, and searched for genes encoding CYPs that catalyze the metabolic activation of ticlopidine,^{12, 13, 14, 15, 16, 17, 18} ticlopidine-reactive metabolite(s)-detoxifying enzymes such as glutathione S-transferase,¹⁹ and associated proteins focusing on polymorphisms that might affect enzyme activity and amino-acid substitution as well as those for which variants have been confirmed in Japanese (as published in the literature and online searchable databases). Furthermore, since there is evidence supporting a relationship between idiosyncratic drug-induced hepatotoxicity and human leukocyte antigen (HLA) genotype,^{20, 21, 22, 23, 24} three HLA class I loci (HLA-A, B and C) and 3 HLA class II loci (HLA-DRB1, DQB1 and DPB1) were also investigated. Herein, we report our preliminary data on HLA and other potential pharmacogenomic factors associated with ticlopidine-induced hepatotoxicity observed by performing an unmatched case–control study in Japanese patients.

Results

A strong correlation between ticlopidine-induced hepatotoxicity and five HLA alleles such as HLA-A^*3303, HLA-B^*4403, HLA-Cw^*1403, HLA-DRB1^*1302 and HLA-DQB1^*0604 was noted (corrected probability (P)-value (Pc)<0.01; Table 1). Although HLA-A^*3303 was present only in 12 (14%) of the 85 controls, it was present in 15 (68%) of the 22 cases with all ticlopidine-induced hepatotoxicity (odds ratio, 13.04; 95% confidence interval (CI), 4.40–38.59; the corrected P-value (Pc)=1.24 10^–5) and in 12 (86%) of the 14 cases with cholestatic-type hepatotoxicity (odds ratio, 36.50; 95% CI 7.25–183.82; Pc=7.32 10^–7). A specific HLA haplotype (HLA-A^*3303, -B^*4403, -Cw^*1403, -DRB1^*1302, -DQB1^*0604) was found in 12 (55%) of the 22 cases with all ticlopidine-induced hepatotoxicity, including 10 patients with cholestatic hepatotoxicity, whereas this was observed in only nine (11%) of the 85 controls (Table 1).

Table 1 - Association between haplotype HLA-A^*3303-B^*4403-Cw^*1403-DRB1^*1302-DQB1^*0604 and hepatotoxicity associated with ticlopidine use in Japanese patients.

Full table

In the case group, none of the patients without HLA-A^*3303 had HLA-B^*4403, HLA-Cw^*1403, HLA-DRB1^*1302 or HLA-DQB1^*0604 (Table 2, highlighted in bold). Among patients with hepatocellular-type damage, none exhibited HLA-A^*3303, -B^*4403, -Cw^*1403, -DRB1^*1302 or -DQB1^*0604 (Table 2).

Table 2 - HLA genotypes in 22 patients with ticlopidine-induced hepatotoxicity.

Full table

No significant (P<0.01) data on the other genotypes that were assumed to be related to metabolism, and oxidative stress pathways of ticlopidine were observed in relation to ticlopidine-induced hepatotoxicity, in marked contrast to our results that highly significant Pc-values (0.00908–7.32 10^-7) were obtained between the HLA genotypes and ticlopidine-induced hepatotoxicity.

Discussion

Severe hepatotoxicity has been reported in patients treated with ticlopidine.² The frequency of such events appears higher in Japan than in western countries.³ Our results suggest that the presence of HLA-A^*3303 is a strong risk factor for ticlopidine-induced hepatotoxicity, especially the serious cholestatic type, in Japanese patients. The present study identified a potential association between ticlopidine-induced hepatotoxicity and HLA-A^*3303 either alone or as part of a specific HLA haplotype (HLA-A^*3303, -B^*4403, -Cw^*1403, -DRB1^*1302 and -DQB1^*0604). These five HLA alleles show a mutual linkage disequilibrium and are the second most common haplotype in the Japanese population with a frequency of 4.8%.²⁵ A survey of the allele frequency and haplotype frequency for HLA class I from five ethnic groups in North America showed that the allele frequency of HLA-A^*3303 is 0.53% in Caucasians and 11.70% in Asians²⁶ (Japanese, 9.7%²⁵). The frequency of the HLA-A^*3303-B^*4403-Cw^*1403 haplotype was 2.09% in Asians (Japanese, 5.8%²⁵), but <0.6% in Caucasians.²⁶ Significant correlations among several drug-induced idiosyncratic toxicities and specific HLA alleles have also been reported.^{20, 21, 22, 23, 24} As HLA protein plays a key role in immune-related reactions, the association observed in the present study likely suggests an immune-mediated mechanism and may also explain the higher incidence of ticlopidine-induced hepatotoxicity in Japanese patients.

Since the genotype distribution did not significantly deviate from the Hardy–Weinberg equilibrium, we believe that the genotyping methods used in the present study are reliable. However, in this unmatched study, potential confounders such as previous hepatic illness, adverse events such as drug-induced hepatitis, and alcohol consumption were not assessed among the cases and controls. Therefore, our preliminary findings should be confirmed by further investigation with a larger number of cases and matched controls. Such studies may provide us with a better understanding of the associations we observed in a small sample size of Japanese patients. Whether our findings can be extrapolated to other ethnic groups remains unknown.

Subjects and methods

Subjects

We identified 22 Japanese patients with ticlopidine-induced hepatotoxicity (cases) and 85 patients who experienced no adverse reactions during ticlopidine treatment (controls). The diagnosis of ticlopidine-induced hepatotoxicity was based on clinical history and laboratory findings (serum bilirubin > 1.5 mg/dL or serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) at least twice the upper limit of normal). Cases were divided into three drug-induced liver injury categories according to Danan's²⁷ criteria for hepatotoxicity cholestatic (n=14), mixed (n=5), and hepatocellular damage (n=3). There were no differences of age, gender or ticlopidine dosage between the two groups. According to the anthropological genetic classification of the Japanese population based on HLA allele frequencies or haplotype frequencies, all patients included in this study were Hondo Japanese (the majority of the population of contemporary Japan); consequently, the case and control groups of this study can be anthropologically considered as one ethnic subgroup of Japanese from the genetic point of view. All patients provided written informed consent to participate, and blood samples were anonymous. The study was approved by the ethics committees of Daiichi Pharmaceutical Co., Ltd and of each participating institution. The protocol conforms to the Ethical Guidelines for Human Genome/Gene Research in Japan and ethical guidelines of the 1975 Declaration of Helsinki.

Genotyping

Selected for analysis were the following 12 genes known to be involved in drug metabolism and oxidative stress pathways: CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, glutathione synthetase (GSS), glutathione S-transferase P1 (GSTP1), nuclear factor-erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (KEAP1) and heme-oxygenase 1 (HMOX1). Ninety-six polymorphic loci of these genes were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), PCR single-strand conformational polymorphism (PCR-SSCP) or direct sequencing. Furthermore, three HLA class I loci (HLA-A, B and C) and three HLA class II loci (HLA-DRB1, DQB1 and DPB1) were genotyped by PCR-sequence-specific primers (SSP) method. All laboratory personnel were blinded as to whether samples came from cases or controls.

Statistical analysis

Fisher's exact test was used to evaluate the significance of differences between the cases and controls regarding the frequencies of genetic polymorphisms related to ticlopidine metabolism. Significance was accepted at a P-value of <0.05. Odds ratios and 95% CI were also calculated to estimate the strength of the association between mutations at different loci and the occurrence of ticlopidine-induced hepatotoxicity. The significance of the association between HLA alleles and ticlopidine-induced hepatotoxicity was assessed by ² contingency table analysis with Yates' correction and by t-test. Odds ratios were calculated from the cross-product ratio of entries in the ² 2 2 table. Pc was also calculated as the product of the P-value and the number of alleles tested at each locus. Pc<0.05 was accepted as statistically significant.

Notes

Duality of Interest

This study was supported by Daiichi Pharmaceutical Co., Ltd.

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Acknowledgements

We thank all of the patients who agreed to take part in this study. We also thank Dr Kingo Fujimura (Professor, Hiroshima University, Faculty of Medicine), Dr Takaaki Isshiki (Professor, Teikyo University, Department of Medicine), Dr Tsutomu Imaizumi (Professor, Kurume University School of Medicine), Dr Hideo Kusuoka (Vice Director, Osaka National Hospital), Dr Takahito Sone (Director, Ogaki Municipal Hospital), Dr Genki Mizukoshi (Nippon Medical School), Dr Masayuki Taniguchi (Associate Professor, Jikei University School of Medicine Daisan Hospital), Dr Satoshi Hirose (Assistant Professor, University of Fukui Hospital) and Dr Hiroyuki Takashima (Teaching Assistant, Shiga University of Medical Science) for patient recruitment and data collection, as well as Dr Masayuki Yamamoto (Professor, Center for Tsukuba Advanced Research Alliance), Dr Noritaka Ariyoshi (Associate Professor, Chiba University School of Medicine), ADME/TOX Research Institute of Daiichi Pure Chemicals Co., Ltd, and Mitsubishi Kagaku Bio-Clinical Laboratory Inc. for genotyping analyses.