Eleni Aklillu

A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants

Many drugs, including proton pump inhibitors and certain antidepressants, are metabolized by the polymorphic cytochrome P450 (CYP) 2C19 enzyme. A significant portion of extensive metabolizers do not reach appropriate drug levels, and our objective was to investigate any genetic background.

Comparisons of CYP1A2 genetic polymorphisms, enzyme activity and the genotype-phenotype relationship in Swedes and Koreans.

ObjectivesTo investigate the CYP1A2 genotype-phenotype relationship and to compare CYP1A2 genetic polymorphisms and enzyme activity in terms of the effect of smoking and oral contraceptive (OC) use in Swedes and Koreans.MethodsCYP1A2 enzyme activity was determined in 194 and 150 healthy Swedish and Korean subjects, respectively, on the basis of the 4-h plasma paraxanthine/caffeine (17X/137X) ratio determined using high-performance liquid chromatography. Genotyping for the −3860G>A, −2467delT, −739 T>G, −729 C>T, −163C>A and −3113A>G polymorphisms was performed by PCR-restriction fragment length polymorphism analysis.ResultsThe mean 17X/137X ratio was 1.54-fold higher in Swedes than in Koreans (mean difference: 0.16; 95% CI of the mean difference: 0.12, 0.20; pu2009<u20090.0001). Smokers had a significantly higher 17X/137X ratio (higher CYP1A2 activity) than non-smokers, while Swedish OC users had a significantly lower 17X/137X ratio than non-users (mean difference: 0.31, 95% CI of the mean difference: 0.23, 0.39; pu2009<u20090.0001). No effect of gender differences on enzyme activity was observed. Four known (CYP1A2*1A, *1D, *1F, and *1L) and two novel haplotypes (CYP1A2*1V and CYP1A2*1W) were found. CYP1A2*1K was rare in Swedes and absent in Koreans. No significant genotype-phenotype relationship was observed, with the exception of CYP1A2*1F in Swedish smokers, where it was associated with higher enzyme inducibility (pu2009=u20090.02). Koreans displayed a significantly lower mean 17X/137X ratio than Swedes having the same CYP1A2 genotype, smoking habit and OC use.ConclusionsWe found significant differences in CYP1A2 enzyme activity between Swedes and Koreans that could not be explained by environmental factors or the CYP1A2 haplotypes examined, despite differences in allele frequencies. None of the investigated CYP1A2 haplotypes are critical in inducing variations in enzyme activity, with the exception of CYP1A2*1F.

4??-Hydroxycholesterol is a new endogenous CYP3A marker: relationship to CYP3A5 genotype, quinine 3-hydroxylation and sex in Koreans, Swedes and Tanzanians

Objectives To study the potential endogenous marker of CYP3A activity, 4&bgr;-hydroxycholesterol, and its relation to sex and the CYP3A5 geno/haplotypes and compare with CYP3A4/5 catalyzed 3-hydroxylation of quinine in the three major races. Methods The plasma concentration of 4&bgr;-hydroxycholesterol was measured in healthy Tanzanians (n=138), Swedes (n=161) and Koreans (n=149) by gas chromatography–mass spectrometry. The metabolic ratio of quinine/3-hydroxyquinine in plasma 16-h post dose was determined by high performance liquid chromatography, previously reported in Tanzanians and Swedes, and now also in Koreans. The participants were genotyped for relevant alleles of CYP3A5. Results The mean plasma concentrations of 4&bgr;-hydroxycholesterol in Koreans, Swedes and Tanzanians were 29.3, 26.8 and 21.9u2009ng/ml, respectively (P<0.01 between all three populations). Within all three populations there were significant differences in 4&bgr;-hydroxycholesterol levels between the CYP3A5 genotypes. Women had higher concentrations than men, but the difference was only significant in Tanzanians (P<0.001) and Koreans (P<0.00001). The quinine/3-hydroxyquinine metabolic ratio was significantly different in all three populations with the highest CYP3A activity in Koreans and the lowest in Tanzanians. Korean women had a lower metabolic ratio than men (P<0.00001). Significant correlations between 4&bgr;-hydroxycholesterol and quinine 3-hydroxylation were found in Tanzanians and Koreans. Conclusion Clear differences in the activity of both CYP3A4 and CYP3A5 were shown in the three major human races. Both 4&bgr;-hydroxycholesterol and quinine/3-hydroxyquinine metabolic ratio showed a higher CYP3A activity in women than in men. The results give strong evidence that the plasma concentration of 4&bgr;-hydroxycholesterol may be used as an endogenous marker of CYP3A activity (CYP3A4+5).

Pharmacogenetic & Pharmacokinetic Biomarker for Efavirenz Based ARV and Rifampicin Based Anti-TB Drug Induced Liver Injury in TB-HIV Infected Patients

Background Implication of pharmacogenetic variations and efavirenz pharmacokinetics in concomitant efavirenz based antiviral therapy and anti-tubercular drug induced liver injury (DILI) has not been yet studied. We performed a prospective case-control association study to identify the incidence, pharmacogenetic, pharmacokinetic and biochemical predictors for anti-tubercular and antiretroviral drugs induced liver injury (DILI) in HIV and tuberculosis (TB) co-infected patients. Methods and Findings Newly diagnosed treatment naïve TB-HIV co-infected patients (nu200a=u200a353) were enrolled to receive efavirenz based ART and rifampicin based anti-TB therapy, and assessed clinically and biochemically for DILI up to 56 weeks. Quantification of plasma efavirenz and 8-hydroxyefaviernz levels and genotyping for NAT2, CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 genes were done. The incidence of DILI and identification of predictors was evaluated using survival analysis and the Cox Proportional Hazards Model. The incidence of DILI was 30.0%, or 14.5 per 1000 person-week, and that of severe was 18.4%, or 7.49 per 1000 person-week. A statistically significant association of DILI with being of the female sex (pu200a=u200a0.001), higher plasma efavirenz level (pu200a=u200a0.009), efavirenz/8-hydroxyefavirenz ratio (pu200a=u200a0.036), baseline AST (pu200a=u200a0.022), ALT (pu200a=u200a0.014), lower hemoglobin (pu200a=u200a0.008), and serum albumin (pu200a=u200a0.007), NAT2 slow-acetylator genotype (pu200a=u200a0.039) and ABCB1 3435TT genotype (pu200a=u200a0.001). Conclusion We report high incidence of anti-tubercular and antiretroviral DILI in Ethiopian patients. Between patient variability in systemic efavirenz exposure and pharmacogenetic variations in NAT2, CYP2B6 and ABCB1 genes determines susceptibility to DILI in TB-HIV co-infected patients. Close monitoring of plasma efavirenz level and liver enzymes during early therapy and/or genotyping practice in HIV clinics is recommended for early identification of patients at risk of DILI.

S-mephenytoin hydroxylation phenotype and CYP2C19 genotype among Ethiopians

The polymorphic metabolism of S-mephenytoin and the distribution of two known deleterious mutant CYP2C19 alleles was determined among 114 healthy unrelated black Ethiopians. Six subjects (5.2%) were poor metabolizers (PMs) of S-mephenytoin. The frequencies of the defective CYP2C19*2 (CYP2C19m1) and CYP2C19*3 (CYP2C19m2) alleles were 0.14 and 0.02, respectively. Three of the PMs were homozygous for the CYP2C19*2 allele and the remaining three PMs were heterozygous for both the CYP2C19*2 and CYP2C19*3 mutant alleles. It is concluded that the frequency of PMs for S-mephenytoin is similar in Ethiopians, Zimbabweans and Caucasians and that the CYP2C19*3 allele, for the first time identified in a black population, together with the CYP2C19*2 allele account for all of the defective CYP2C19 alleles among the Ethiopian PMs.

Evidence for environmental influence on CYP2D6-catalysed debrisoquine hydroxylation as demonstrated by phenotyping and genotyping of Ethiopians living in Ethiopia or in Sweden

Black Africans show lower rates of CYP2D6- and CYP2C19-dependent drug metabolism compared to Caucasians of the same apparent genotype. To determine if environmental factors are responsible for this difference, the genotypes and phenotypes of CYP2D6 and CYP2C19 among Ethiopians living in Sweden (n = 70) were assessed and compared to our previously published data from Ethiopians living in Ethiopia (n = 114) and Swedish Caucasians (n = 134). There was no significant difference in CYP2C19 genotype or phenotype as assessed by mephenytoin between Ethiopians in Sweden or in Ethiopia. However, Swedes were significantly more rapid for CYP2C19 activity than both Ethiopian groups (P < 0.01). A comparison of the debrisoquine MR among individuals of the same CYP2D6 genotype revealed that Swedes exhibited the highest rate of debrisoquine metabolism, followed by Ethiopians in Sweden and Ethiopians in Ethiopia. The difference between the Ethiopian groups was significant (P < 0.02 using a univariate test ANOVA) and amounted to approximately 50% of the magnitude of the MR difference between Swedes and Ethiopians in Ethiopia. It is tempting to speculate that inhibitory dietary factors may explain the differences seen between the two Ethiopian groups and that these components in the past might have contributed to dietary stress-mediated selection of duplicated and multiduplicated active CYP2D6 genes, as frequently seen in Ethiopians. In conclusion, the results indicate a significant influence of environmental factors as an explanation for the difference in capacity for CYP2D6, but not CYP2C19 metabolism between Caucasians and Black Africans. Additional factors remain to be elucidated to fully explain the interethnic differences in CYP2D6 activity.

Importance of ethnicity, CYP2B6 and ABCB1 genotype for efavirenz pharmacokinetics and treatment outcomes: a parallel-group prospective cohort study in two sub-Saharan Africa populations.

Objectives We evaluated the importance of ethnicity and pharmacogenetic variations in determining efavirenz pharmacokinetics, auto-induction and immunological outcomes in two African populations. Methods ART naïve HIV patients from Ethiopia (nu200a=u200a285) and Tanzania (nu200a=u200a209) were prospectively enrolled in parallel to start efavirenz based HAART. CD4+ cell counts were determined at baseline, 12, 24 and 48 weeks. Plasma and intracellular efavirenz and 8-hydroxyefvairenz concentrations were determined at week 4 and 16. Genotyping for common functional CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 variant alleles were done. Result Patient country, CYP2B6*6 and ABCB1 c.4036A>G (rs3842A>G) genotype were significant predictors of plasma and intracellular efavirenz concentration. CYP2B6*6 and ABCB1 c.4036A>G (rs3842) genotype were significantly associated with higher plasma efavirenz concentration and their allele frequencies were significantly higher in Tanzanians than Ethiopians. Tanzanians displayed significantly higher efavirenz plasma concentration at week 4 (p<0.0002) and week 16 (pu200a=u200a0.006) compared to Ethiopians. Efavirenz plasma concentrations remained significantly higher in Tanzanians even after controlling for the effect of CYP2B6*6 and ABCB1 c.4036A>G genotype. Within country analyses indicated a significant decrease in the mean plasma efavirenz concentration by week 16 compared to week 4 in Tanzanians (pu200a=u200a0.006), whereas no significant differences in plasma concentration over time was observed in Ethiopians (pu200a=u200a0.84). Intracellular efavirenz concentration and patient country were significant predictors of CD4 gain during HAART. Conclusion We report substantial differences in efavirenz pharmacokinetics, extent of auto-induction and immunologic recovery between Ethiopian and Tanzanian HIV patients, partly but not solely, due to pharmacogenetic variations. The observed inter-ethnic variations in efavirenz plasma exposure may possibly result in varying clinical treatment outcome or adverse event profiles between populations.

Analysis of CYP2C9*5 in Caucasian, Oriental and Black-African populations

AbstractnObjective.CYP2C9 is a polymorphic gene with at least six known allelic variants (CYP2C9*1 to *6). CYP2C9*5 has been recently described in African-Americans. The lower activity of CYP2C9*5 encoded enzyme than *1 has been reported for the S-warfarin 7-hydroxylation in vitro. The aim of the present study was to develop an assay for the analysis of this variant and to determine the frequency of this polymorphism in different ethnic populations.nMaterials and methods. A PCR-based endonuclease digestion method, using a mismatched forward primer that introduced a recognition site for AvaII in all the CYP2C9 genotypes except CYP2C9*5, is described. DNA samples from 150 Ethiopians, 183 Tanzanians, 200 Caucasians from Sweden and 150 Orientals from Korea were screened for this variant allele.nResults and conclusion. The CYP2C9*5 allele was analysed using a polymerase chain reaction-based endonuclease method, and it was found in three Tanzanians (allele frequency, 0.0082) but not in Ethiopians, Caucasians or Orientals.

Anti-tuberculosis therapy-induced hepatotoxicity among Ethiopian HIV-positive and negative patients.

Background To assess and compare the prevalence, severity and prognosis of anti-TB drug induced hepatotoxicity (DIH) in HIV positive and HIV negative tuberculosis (TB) patients in Ethiopia. Methodology/Principal Findings In this study, 103 HIV positive and 94 HIV negative TB patients were enrolled. All patients were evaluated for different risk factors and monitored biochemically and clinically for development of DIH. Sub-clinical hepatotoxicity was observed in 17.3% of the patients and 8 out of the 197 (4.1%) developed clinical hepatotoxicity. Seven of the 8 were HIV positive and 2 were positive for HBsAg. Conclusions/Significance Sub-clinical hepatotoxicity was significantly associated with HIV co-infection (pu200a=u200a0.002), concomitant drug intake (pu200a=u200a0.008), and decrease in CD4 count (pu200a=u200a0.001). Stepwise restarting of anti TB treatment was also successful in almost all the patients who developed clinical DIH. We therefore conclude that anti-TB DIH is a major problem in HIV-associated TB with a decline in immune status and that there is a need for a regular biochemical and clinical follow up for those patients who are at risk.

Allele-specific expression and gene methylation in the control of CYP1A2 mRNA level in human livers

The basis for interindividual variation in the CYP1A2 gene expression is not fully understood and the known genetic polymorphisms in the gene provide no explanation. We investigated whether the CYP1A2 gene expression is regulated by DNA methylation and displays allele-specific expression (ASE) using 65 human livers. Forty-eight percent of the livers displayed ASE not associated to the CYP1A2 mRNA levels. The extent of DNA methylation of a CpG island including 17 CpG sites, close to the translation start site, inversely correlated with hepatic CYP1A2 mRNA levels (P=0.018). The methylation of two separate core CpG sites was strongly associated with the CYP1A2 mRNA levels (P=0.005) and ASE phenotype (P=0.01), respectively. The CYP1A2 expression in hepatoma B16A2 cells was strongly induced by treatment with 5-aza-2′-deoxycytidine. In conclusion, the CYP1A2 gene expression is influenced by the extent of DNA methylation and displays ASE, mechanisms contributing to the large interindividual differences in CYP1A2 gene expression.