Association of polymorphisms in four bilirubin metabolism genes with serum bilirubin in three Asian populations

Numerous studies have shown that the (TA)n repeat polymorphism in the uridine diphosphate glycosyltransferase 1 (UGT1A1) gene promoter is associated with hyperbilirubinemia. Several studies also indicated that single nucleotide polymorphism (SNP) rs4148323:G>A at Exon 1 of UGT1A1 is associated with hyperbilirubinemia. However, it remains unclear what role the polymorphisms play in influencing serum total bilirubin (TBIL) levels in general populations, and whether polymorphisms in other genes involved in the bilirubin metabolism pathway are associated with TBIL levels. The present study addressed these questions by investigating the association of four bilirubin metabolism genes with TBIL levels in three Asian populations: 11 genetic polymorphisms in heme oxygenase‐1 (HMOX1); biliverdin reductase A (BLVRA); solute carrier organic anion transporter family member 1B1 (SLCO1B1); and UGT1A1. The populations consisted of 502 Kazak herdsmen, 769 Uyghur farmers, and 789 Han farmers, with distinct genetic backgrounds. UGT1A1 was found to be associated with the (TA)₇ allele of the (TA)n repeat polymorphism. We also showed that the A allele of SNP rs4148323:G>A was strongly associated with high TBIL levels in all three populations (each P<0.005). Among polymorphisms in other genes, only the (GT)n repeat polymorphism in the HMOX1 promoter region showed association with TBIL levels in the Uyghur population, but not in the Han and Kazak populations. We also assessed the contributions of (TA)n polymorphism and rs4148323:G>A to phenotypic variations in all three populations. Finally, we observed that significant differences of TBIL levels existed among the three populations; however, this could not be completely explained by the differences at the (TA)n repeat polymorphism and SNP rs4148323:G>A. Hum Mutat 0, 1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Biotransformation and in vitro assessment of metabolism-associated drug–drug interaction for CRx-102, a novel combination drug candidate

CRx-102 is an oral synergistic combination drug which contains the cardiovascular agent, dipyridamole (DP) and a very low dose of the glucocorticoid, prednisolone (PRED). CRx-102 works through a novel mechanism of action in which DP selectively amplifies the anti-inflammatory activity of PRED without replicating its side effects. CRx-102 is in clinical trials for the treatment of osteoarthritis. Here we delineate the in vitro metabolism and explore the potential for a drug–drug interaction between the active agents in CRx-102. Our study using human hepatocyte suspensions showed that both DP and PRED were metabolized by CYP3A4 isozymes, resulting in the formation of diverse arrays of both oxidative and oxidative-reduced metabolites. Within phase 1 biotransformation, CYP3A4 was one of the pathways responsible for the metabolism of PRED, while phase 2 biotransformation played a significant role in the metabolism of DP. Glucuronidation of DP was substantial and was catalyzed by many UGT members, specifically those in the UGT1A subfamily. Based on the tandem mass (MS/MS) product ion spectra (PIS) acquired, the major metabolites of both agents, namely, monooxygenated, mono-N-deethanolaminated, dehydrogenated and O-glucuronidated metabolites of DP and the monooxygenated (e.g., 6-hydroxyl), dehydrogenated (prednisone) and reduced (20-hydroxyl) metabolites of PRED, were identified and elucidated. The affinities for DP biotransformation, including CYP3A4-mediated oxidative pathways and UGT-mediated O-glucuronidation, appeared high (K_m < 10 μM), as compared with the modest affinities of PRED biotransformation catalyzed by CYP3A4 (K_m ∼ 40–170 μM). DP, but not PRED, exerted a minimal inhibitory effect on the drug-metabolizing CYP isoforms, including CYP3A4, which was determined using a panel of CYP isoform-preferred substrate activities in pooled human liver microsomal (HLM) preparations and microsomal preparations containing the recombinant enzymes (K_i ∼ 2–12 μM). Using the DP maximal plasma concentration (C_max) observed in the clinic and a predictive mathematical model for metabolism-associated drug–drug interaction (DDI), we have demonstrated that there is little likelihood of a pharmacokinetic interaction between the two active agents in CRx-102.     

Identification of Human UDP-Glucuronosyltransferase Responsible for the Glucuronidation of Niflumic Acid in Human Liver

Purpose To assess the uridine diphosphate (UDP)-glucuronosyltransferase (UGT) isozymes involved in the glucuronidation of niflumic acid in human liver. Methods The glucuronidation activity of niflumic acid was determined in liver microsomes and recombinant UGT isozymes by incubation of niflumic acid with UDP-glucuronic acid (UDPGA). Results Incubation of niflumic acid with liver microsomes and UDPGA produced one peak, which was identified as a glucuronide from mass spectrometric analysis. A study involving a panel of recombinant human UGT isozymes showed that glucuronidation activity was highest in UGT1A1 among the isozymes investigated. The glucuronidation in human liver microsomes (HLMs) followed Michaelis-Menten kinetics with a K_m value of 16 μM, which is similar to that found with recombinant UGT1A1. The glucuronidation activity of niflumic acid in microsomes from eight human livers significantly correlated with UGT1A1-catalyzed estradiol 3β-glucuronidation activity (r=0.78, p<0.05). β-Estradiol inhibited niflumic acid glucuronidation with an IC₅₀ of 25 μM in HLMs, comparable to that for UGT1A1. Conclusions These findings indicate that UGT1A1 is the main isozyme involved in the glucuronidation of niflumic acid in the human liver.     

Crigler-Najjar综合征Ⅱ型患者及其家系UGT1A1基因突变的分析

目的:分析1例遗传性高非结合胆红素血症Crigler—Najjar综合征Ⅱ型(CN—Ⅱ)患者及其家系尿苷二磷酸葡萄糖醛酸转移酶基因(UGT1A1)的突变特征,研究其遗传特点和诊断。方法:采用肝功能试验、低热卡试验、肝脏病理组织学和苯巴比妥诱导试验治疗等方法确诊1例CN—Ⅱ,追踪并抽取该先证者及其家系11例成员的外周静脉血,提取基因组DNA,采用聚合酶链反应(PCR)方法扩增UGT1A1基因第1～5外显子基因片段,用直接DNA测序法筛查UGT1A1基因突变。结果:该先证者UGT1A1基因的第5外显子第486密码子发生了T→G碱基的错义突变,导致了酪氨酸变成了天门冬氨酸(Tyr486Asp)。先证者为该突变的纯合子,其父母、妹妹及三位祖父为该突变的杂合子。其余4例未患病成员均未发现UGT1A1基因突变。结论:Tyr486Asp突变是本例CN—Ⅱ家系的致病性基因,该突变不影响UGT1A1基因启动子区苯巴比妥酸的应答元件,其遗传规律符合常染色体隐性遗传。经查询人类基因文库证实,本例CN—Ⅱ基因突变类型在我国是首例,国外尚未见到相同的家系分析报告。     

A homozygous mutation in a Chinese man with Crigler-Najjar syndrome type II and a family genetic analysis

OBJECTIVE: To investigate the family genetic background of a 22-year-old man with Crigler-Najjar syndrome type II (CN-II). METHODS: After the proband (patient) with CN-II was diagnosed by liver function tests, a low calorie intake test and an oral phenobarbital enzyme-induction trial, blood samples were collected from 11 family members for identifying DNA gene groups. Exons 1-5 of the UGT1A1 gene were amplified by polymerase chain reaction (PCR) and mutations of the UGT1A1 gene were screened by a direct DNA sequencing. RESULTS: The serum unconjugated bilirubin increased in the proband from 156.4 micromol/L to 243.5 micromol/L after he started a low calorie intake, and it decreased to 51.8 micromol/L within a month of taking oral phenobarbital daily. Both functional tests and ultrasonographic images of the liver were normal except for the unconjugated hyperbilirubinemia. A missense mutation of Tyr486Asp at exon 5 in the UGT1A1 gene and a homozygous mutation were confirmed in the proband. Heterozygous mutations were found in his parents, his younger sister and three great-uncles, while no mutation of the UGT1A1 gene was detected in the remaining four family members. CONCLUSION: A missense mutation of Tyr486Asp is considered to be the cause of the CN-II in this patient. It is a recessive trait that is autosomally inherited in this family. No influence of the mutation was found on the response elements for phenobarbital in the promoter region.     

Phase II study of pharmacogenetic-tailored therapy in elderly colorectal cancer patients

Background

Retrospective analyses suggested that a pharmacogenetic approach may allow a tailored selection of chemotherapy for metastatic colorectal cancer.

Aim

We conducted a phase II study of pharmacogenetic-selected first-line chemotherapy in elderly patients with advanced colorectal cancer, with the aim to improve efficacy and to reduce toxicity in this group of patients.

Methods

24 patients were enrolled in this study. Chemotherapy regimen was prospectively assigned based on TS, DPD, ERCC-1 and UGT1A1 genotyping results. Twelve patients (50%) were treated with modified FOLFIRI, 11 patients (46%) with modified FOLFOX6 and 1 (4%) with De Gramont regimen.

Results

A partial remission was obtained in 4 cases (17%), stable disease in 8 cases (33%) and progressive disease in 12 cases (50%). Grade 3-4 neutropenia was observed in 7 patients (29%) and diarrhoea in 3 cases (12%). The trial was then interrupted according to study design requiring 13 partial remissions out of the first 24 patients enrolled as the necessary response rate level in order to continue.

Conclusion

Prospective selection of chemotherapy based on TS, DPD, ERCC-1 and UGT1A1 expression in elderly advanced colorectal cancer patients failed to confirm previous results. A more accurate validation of retrospective findings is warranted before these molecular markers can be used for treatment selection in the clinical practice.     

Role of UGT1A1 mutation in fasting hyperbilirubinemia

BACKGROUND AND AIM: Low-grade fasting hyperbilirubinemia is a common observation in healthy subjects (HS), whereas high-grade fasting hyperbilirubinemia is believed to be a characteristic finding of Gilbert's syndrome. This study was undertaken to assess the role of mutation in bilirubin UDP- glycosyltransferase gene (UGT1A1) on fasting hyperbilirubinemia. METHODS: Analysis of UGT1A1 and a caloric restriction test (400 kcal for 24 h) were performed in 56 healthy subjects (25 males, 31 females), and 28 patients with Gilbert's syndrome (18 males, 10 females). There were 29 healthy subjects with no mutation in UGT1A1, and 27 healthy subjects and 26 Gilbert's syndrome patients with mutations in the coding and/or promoter (TATA box) regions of UGT1A1. RESULTS: The mean increment of serum bilirubin (DeltaSB) was 7.6 micromol/L [corrected] (males) and 4.1 micromol/L (females) in subjects with no UGT1A1 mutation. Subjects with mutation in UGT1A1 showed higher levels of DeltaSB than individuals without mutation. Among healthy subjects, gender difference in DeltaSB values was observed only in individuals with the wild type of UGT1A1, but not in those with mutations in this gene. CONCLUSION: The results of the present study suggest that UGT1A1 mutation has a role in the development of high-grade fasting hyperbilirubinemia after caloric restriction.     

Tolerance and Efficacy of Regorafenib according to UGT Pharmacogenetical Status in the Treatment of Metastatic Refractory Colorectal Cancer

Introduction: Regorafenib is a multi tyrosin-kinase inhibitor prescribed in metastatic refractory colorectal cancer treatment. Its toxicity is significant but inconstant. The metabolism of regorafenib includes oxydation via cytochrome P3A4, then glucuroconjugation. A pharmacogenetical approach of mutational status of Uridine-Diphospho-Glucuronosyltransfersase (UDP-glucuronosyl-transferase, UGT) could be a strategy to optimise the use of regorafenib. Patients and Method: This is a restrospective, unicentric study. All adult patients treated with regorafenib for a metastatic colorectal cancer in our center between 2013 and 2017 were analysed. UGT1A1 polypmorphism was previously researched in the laboratory after written informed consent. Results: Thirty-five patients received regorafenib during the study period. A TA repetition in UGT1A1 gene was present for 16 patients including two Gilbert syndrome. There were no more adverse events on patients with a heterozygous TA repetition or with a Gilbert syndrome compared with patients without UGT polymorphism, whatever the dosage at initiation of regorafenib. Adverse events grade 2 or superior, and grade 3 or superior tended to be more noticeable when the starting dose was 120 or 160 mg per day compared to 80 mg per day, but not statistically significant. No difference was observed on progression-free survival neither depending on UGT status nor depending on initating dose of regorafenib. Conclusion: This is a preliminary study evaluating safety of regorafenib according to UGT1A1 polymorphism. Larger and prospective studies are needed to evaluate dose-escalation strategy in patients with variable activity of glucuroconidation, especially Gilbert syndrome, or with abnormalities in other UGT enzymes such as UGT1A9.