Edmund Jon Deoon Lee

Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations.

The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.

Genetic polymorphisms in MDR1 and CYP3A4 genes in Asians and the influence of MDR1 haplotypes on cyclosporin disposition in heart transplant recipients.

Intestinal cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) both play a vital role in the metabolism of oral cyclosporine (CsA). We investigated the genetic polymorphisms in CYP3A4(promoter region and exons 5, 7 and 9) and MDR1 (exons 12, 21 and 26) genes and the impact of these polymorphisms on the pharmacokinetics of oral CsA in stable heart transplant patients (n = 14). CYP3A4 polymorphisms were rare in the Asian population and transplant patients. Haplotype analysis revealed 12 haplotypes in the Chinese, eight in the Malays and 10 in the Indians. T-T-T was the most common haplotype in all ethnic groups. The frequency of the homozygous mutant genotype at all three loci (TT-TT-TT) was highest in the Indians (31%) compared to 19% and 15% in the Chinese and Malays, respectively. In heart transplant patients, CsA exposure (AUC(0-4 h), AUC(0-12 h) and C(max)) was high in patients with the T-T-T haplotypes compared to those with C-G-C haplotypes. These findings suggest that haplotypes rather than genotypes influence CsA disposition in transplant patients.

Herbal bioactivation: The good, the bad and the ugly

Abstract It has been well established that the formation of reactive metabolites of drugs is associated with drug toxicity. Similarly, there are accumulating data suggesting the role of the formation of reactive metabolites/intermediates through bioactivation in herbal toxicity and carcinogenicity. It has been hypothesized that the resultant reactive metabolites following herbal bioactivation covalently bind to cellular proteins and DNA, leading to toxicity via multiple mechanisms such as direct cytotoxicity, oncogene activation, and hypersensitivity reactions. This is exemplified by aristolochic acids present in Aristolochia spp, undergoing reduction of the nitro group by hepatic cytochrome P450 (CYP1A1/2) or peroxidases in extrahepatic tissues to reactive cyclic nitrenium ion. The latter was capable of reacting with DNA and proteins, resulting in activation of H-ras oncogene, gene mutation and finally carcinogenesis. Other examples are pulegone present in essential oils from many mint species; and teucrin A, a diterpenoid found in germander (Teuchrium chamaedrys) used as an adjuvant to slimming diets. Extensive pulegone metabolism generated p-cresol that was a glutathione depletory, and the furan ring of the diterpenoids in germander was oxidized by CYP3A4 to reactive epoxide which reacts with proteins such as CYP3A and epoxide hydrolase. On the other hand, some herbal/dietary constituents were shown to form reactive intermediates capable of irreversibly inhibiting various CYPs. The resultant metabolites lead to CYP inactivation by chemical modification of the heme, the apoprotein, or both as a result of covalent binding of modified heme to the apoprotein. Some examples include bergamottin, a furanocoumarin of grapefruit juice; capsaicin from chili peppers; glabridin, an isoflavan from licorice root; isothiocyanates found in all cruciferous vegetables; oleuropein rich in olive oil; dially sulfone found in garlic; and resveratrol, a constituent of red wine. CYPs have been known to metabolize more than 95% therapeutic drugs and activate a number of procarcinogens as well. Therefore, mechanism-based inhibition of CYPs may provide an explanation for some reported herb-drug interactions and chemopreventive activity of herbs. Due to the wide use and easy availability of herbal medicines, there is increasing concern about herbal toxicity. The safety and quality of herbal medicine should be ensured through greater research, pharmacovigilance, greater regulatory control and better communication between patients and health professionals.

Pharmacokinetic Interactions of Drugs with St John’s Wort

There is a worldwide increasing use of herbs which are often administered in combination with therapeutic drugs, raising the potential for herb-drug interactions. St John’s wort (Hypericum perforatum) is one of the most commonly used herbal antidepressants. A literature search was performed using Medline (via Pubmed), Biological Abstracts, Cochrane Library, AMED, PsycINFO and Embase (all from their inception to September 2003) to identify known drug interaction with St John’s wort. The available data indicate that St John’s wort is a potent inducer of CYP 3A4 and P-glycoprotein (PgP), although it may inhibit or induce other CYPs, depending on the dose, route and duration of administration. Data from human studies and case reports indicate that St John’s wort decreased the blood concentrations of amitriptyline, cyclosporine, digoxin, fexofenadine, indinavir, methadone, midazolam, nevirapine, phenprocoumon, simvastatin, tacrolimus, theophylline and warfarin, whereas it did not alter the pharmacokinetics of carbamazepine, dextromethorphan, mycophenolic acid and pravastatin. St John’s wort decreased the plasma concentration of the active metabolite SN-38 in cancer patients receiving irinotecan treatment. St John’s wort did not alter the pharmacokinetics of tolbutamide, but increased the incidence of hypoglycaemia. Several cases have been reported that St John’s wort decreased cyclosporine blood concentration leading to organ rejection. St John’s wort caused breakthrough bleeding and unplanned pregnancies when used concomitantly with oral contraceptives. It also caused serotonin syndrome when coadministered with selective serotonin-reuptake inhibitors (e.g. sertaline and paroxetine). Both pharmacokinetic and pharmacodynamic components may play a role in these interactions. Because the potential interaction of St John’s wort with other drugs is a major safety concern, additional systematic research on herb-drug interactions and appropriate regulation in herbal safety and efficacy is needed.

Singapore Genome Variation Project: A haplotype map of three Southeast Asian populations

The Singapore Genome Variation Project (SGVP) provides a publicly available resource of 1.6 million single nucleotide polymorphisms (SNPs) genotyped in 268 individuals from the Chinese, Malay, and Indian population groups in Southeast Asia. This online database catalogs information and summaries on genotype and phased haplotype data, including allele frequencies, assessment of linkage disequilibrium (LD), and recombination rates in a format similar to the International HapMap Project. Here, we introduce this resource and describe the analysis of human genomic variation upon agglomerating data from the HapMap and the Human Genome Diversity Project, providing useful insights into the population structure of the three major population groups in Asia. In addition, this resource also surveyed across the genome for variation in regional patterns of LD between the HapMap and SGVP populations, and for signatures of positive natural selection using two well-established metrics: iHS and XP-EHH. The raw and processed genetic data, together with all population genetic summaries, are publicly available for download and browsing through a web browser modeled with the Generic Genome Browser.

An interethnic comparison of polymorphisms of the genes encoding drug-metabolizing enzymes and drug transporters : Experience in Singapore

Much of the interindividual variability in drug response is attributable to the presence of single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolizing enzymes and drug transporters. In recent years, we have investigated the polymorphisms in a number of genes encoding phase I and II drug-metabolizing enzymes including CYP1A1, CYP3A4, CYP3A5, GSTM1, NAT2, UGT1A1, and TPMT and drug transporter (MDR1) in three distinct Asian populations in Singapore, namely the Chinese, Malays, and Indians. Significant differences in the frequencies of common alleles encoding these proteins have been observed among these three ethnic groups. For example, the frequency of the variant A2455G polymorphism of CYP1A1 was 28% in Chinese and 31% in Malays, but only 18% in Indians. CYP3A4*4 was detected in two of 110 Chinese subjects, but absent in Indians and Malays. Many Chinese and Malays (61–63%) were homozygous for the GSTM1*0 null genotype compared with 33% of Indians. The frequency of the UGT1A1*28 allele was highest in the Indian population (35%) compared to similar frequencies that were found in the Chinese (16%) and Malay (19%) populations. More importantly, our experience over the years has shown that the pharmacogenetics of these drug-metabolizing enzymes and MDR1 in the Asian populations are different from these in the Caucasian and African populations. For example, the CYP3A4*1B allele, which contains an A-290G substitution in the promoter region of CYP3A4, is absent in all three Asian populations of Singapore studied, but occurs in more than 54% of Africans and 5% of Caucasians. There were no difference in genotype and allelic variant frequencies in exon 12 of MDR1 between the Chinese, Malay, and Indian populations. When compared with other ethnic groups, the distribution of the wild-type C allele in exon 12 in the Malays (34.2%) and Indians (32.8%) was relatively high and similar to the Japanese (38.5%) and Caucasians (41%) but different from African–Americans (15%). The frequency of wild-type TT genotype in Asians (43.5% to 52.1%) andJapanese (61.5%) was much higher than those found in Caucasians (13.3%) All the proteins we studied represent the primary hepatic or extrahepatic enzymes, and their polymorphic expression may be implicated in disease risk and the disposition of drugs or endogenous substances. As such, dose requirements of certain drugs may not be optimal for Asian populations, and a second look at the factors responsible for this difference is necessary.

Chirality Clinical Pharmacokinetic and Pharmacodynamic Considerations

The implications of the effects of chirality on the pharmacokinetics and pharmacodynamics of drugs are far-reaching. The pharmacokinetics of a racemic drug cannot be adequately described unless the enantiomers are assayed specifically

Influence of ABCB1 and ABCG2 polymorphisms on doxorubicin disposition in Asian breast cancer patients.

The influence of three high frequency ABCB1 polymorphisms (c.1236C>T, c.2677G>A/T, and c.3435C>T) and the ABCG2 c.421C>A polymorphism on the disposition of doxorubicin in Asian breast cancer patients receiving adjuvant chemotherapy was investigated in the present study. The allelic frequency of the ABCB1 c.1236T, c.2677T, c.2677A, and c.3435T variants were 60%, 38%, 7%, and 22%, respectively, and the frequency of the ABCG2 c.421A allele was 23%. Pairwise analysis showed increased exposure levels to doxorubicin in patients harboring at least one ABCB1 c.1236T allele (P = 0.03). Patients homozygous for the CC‐GG‐CC genotype had significantly lower doxorubicin exposure levels compared to the patients who had CT‐GT‐CT (P = 0.02) and TT‐TT‐TT genotypes (P = 0.03). Significantly increased clearance of doxorubicin was also observed in patients harboring CC‐GG‐CC genotypes when compared to patients harboring the CT‐GT‐CT genotype (P = 0.01). Patients harboring the CC‐GG‐CC genotypes had significantly lower peak plasma concentrations of doxorubicinol compared to patients who had TT‐TT‐TT genotypes (P = 0.03). No significant influences on doxorubicin pharmacokinetic parameters were observed in relation to the ABCG2 c.421C>A polymorphism. In conclusion, the present exploratory study suggests that the three high frequency linked polymorphisms in the ABCB1 gene might be functionally important with regards to the altered pharmacokinetics of doxorubicin in Asian breast cancer patients, resulting in significantly increased exposure levels and reduced clearance. (Cancer Sci 2008; 99: 816–823)

Impact of CYP2D6, CYP3A5, CYP2C9 and CYP2C19 polymorphisms on tamoxifen pharmacokinetics in Asian breast cancer patients

AIM To investigate the impact of genetic polymorphisms in CYP2D6, CYP3A5, CYP2C9 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Asian breast cancer patients. METHODS A total of 165 Asian breast cancer patients receiving 20 mg tamoxifen daily and 228 healthy Asian subjects (Chinese, Malay and Indian; n= 76 each) were recruited. The steady-state plasma concentrations of tamoxifen and its metabolites were quantified using high-performance liquid chromatography. The CYP2D6 polymorphisms were genotyped using the INFINITI™ CYP450 2D6I assay, while the polymorphisms in CYP3A5, CYP2C9 and CYP2C19 were determined via direct sequencing. RESULTS The polymorphisms, CYP2D6*5 and *10, were significantly associated with lower endoxifen and higher N-desmethyltamoxifen (NDM) concentrations. Patients who were *1/*1 carriers exhibited 2.4- to 2.6-fold higher endoxifen concentrations and 1.9- to 2.1-fold lower NDM concentrations than either *10/*10 or *5/*10 carriers (P < 0.001). Similarly, the endoxifen concentrations were found to be 1.8- to 2.6-times higher in *1/*5 or *1/*10 carriers compared with *10/*10 and *5/*10 carriers (P≤ 0.001). Similar relationships were observed between the CYP2D6 polymorphisms and metabolic ratios of tamoxifen and its metabolites. No significant associations were observed with regards to the polymorphisms in CYP3A5, CYP2C9 and CYP2C19. CONCLUSIONS The present study in Asian breast cancer patients showed that CYP2D6*5/*10 and *10/*10 genotypes are associated with significantly lower concentrations of the active metabolite of tamoxifen, endoxifen. Identifying such patients before the start of treatment may be useful in optimizing therapy with tamoxifen. The role of CYP3A5, CYP2C9 and CYP2C19 seem to be minor.

MDR1, the blood–brain barrier transporter, is associated with Parkinson’s disease in ethnic Chinese

Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease. It is characterised by bradykinesia, rigidity, resting tremor, and postural instability.1 It is a genetically heterogeneous disorder. Pathogenic mutations in several genes—including α-synuclein , Parkin , UCH-L1 (ubiquitin-C terminal hydrolase-L1) and DJ -1—have previously been identified in rare monogenic forms of this disease showing autosomal dominant, autosomal recessive, or maternal transmission, with or without genetic anticipation.2,3 The more common, sporadic form of Parkinson’s disease appears to result from an interaction between genetic and environmental factors.4 Polymorphisms in several genes, including those implicated in familial forms of the disease such as α-synuclein 5 and Parkin ,6,7 are also reported to be associated with the sporadic form.8 Genetic susceptibility to sporadic Parkinson’s disease was also found to be modulated by genes involved in xenobiotic management. A meta-analysis of 84 association studies of 14 genes showed that polymorphisms in four genes are significantly associated with the disease.9 These genes are either responsible for xenobiotic metabolism, such as NAT 210,11 and GST T1,12 or may interact with environmental agents, such as monoamine oxidase ( MAO B).13 Poor metaboliser alleles of the cytochrome P450 xenobiotic metabolism enzyme, CYP2D6, may also be associated with increased risk of Parkinson’s disease.14–20 Furthermore, there may be sex effects in the association of CYP 2D6 mutant alleles with Parkinson’s disease.21 These genetic association studies corroborate epidemiological studies, which have long suggested that Parkinson’s disease is associated with exposure to certain environmental xenobiotics. Although most of the specific agents remain to be identified, rural living, well water consumption, industrialisation, and herbicide/pesticide exposure have been implicated as potential risk factors.1,22,23 Another category of genes that may influence susceptibility to Parkinson’s disease is the …