Search

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in cholesterol biosynthesis, that are highly effective in reducing plasma low-density lipoprotein (LDL) cholesterol and decreasing the risk of cardiovascular events. In recent years, a multitude of variants in genes involved in pharmacokinetics (PK) and pharmacodynamics (PD) have been suggested to influence the cholesterol-lowering response. However, the vast majority of studies have analyzed the pharmacogenetic associations in populations in Europe and the USA, whereas data in other populations, including Brazil, are mostly lacking. This narrative review provides an update of clinical studies on statin pharmacogenomics in Brazilian cohorts exploring lipid-lowering response, adverse events and pleiotropic effects. We find that variants in drug transporter genes (SLCO1B1 and ABCB1) positively impacted atorvastatin and simvastatin response, whereas variants in genes of drug metabolizing enzymes (CYP3A5) decreased response. Furthermore, multiple associations of variants in PD genes (HMGCR, LDLR and APOB) with statin response were identified. Few studies have explored statin-related adverse events, and only ABCB1 but not SLCO1B1 variants were robustly associated with increased risk in Brazil. Statin-related pleiotropic effects were shown to be influenced by variants in PD (LDLR, NR1H2) and antioxidant enzyme (NOS3, SOD2, MTHFR, SELENOP) genes. The findings of these studies indicate that statin pharmacogenomic associations are distinctly different in Brazil compared to other populations. This review also discusses the clinical implications of pharmacogenetic studies and the rising importance of investigating rare variants to explore their association with statin response.

Anticoagulant and antiplatelet drugs effectively prevent thrombotic events in patients with cardiovascular diseases, ischemic stroke, peripheral vascular diseases, and other thromboembolic diseases. However, genetic and non-genetic factors affect the response to antithrombotic therapy and can increase the risk of adverse events. This narrative review discusses pharmacogenomic studies on antithrombotic drugs commonly prescribed in Brazil. Multiple Brazilian studies assessed the impact of pharmacokinetic (PK) and pharmacodynamic (PD) gene variants on warfarin response. The reduced function alleles CYP2C9*2 and CYP2C9*3, and VKORC1 rs9923231 (c.-1639GA) are associated with increased sensitivity to warfarin and a low dose requirement to prevent bleeding episodes, whereas CYP4F2 rs2108622 (p.Val433Met) carriers have higher dose requirements (warfarin resistance). These deleterious variants and non-genetic factors (age, gender, body weight, co-administered drugs, food interactions, and others) account for up to 63% of the warfarin dose variability. Few pharmacogenomics studies have explored antiplatelet drugs in Brazilian cohorts, finding associations between CYP2C19*2, PON1 rs662 and ABCC3 rs757421 genotypes and platelet responsiveness or clopidogrel PK in subjects with coronary artery disease (CAD) or acute coronary syndrome (ACS), whereas ITGB3 contributes to aspirin PK but not platelet responsiveness in diabetic patients. Brazilian guidelines on anticoagulants and antiplatelets recommend the use of a platelet aggregation test or genotyping only in selected cases of ACS subjects without ST-segment elevation taking clopidogrel, and also suggest CYP2C9 and VKORC1 genotyping before starting warfarin therapy to assess the risk of bleeding episodes or warfarin resistance.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in cholesterol biosynthesis, that are highly effective in reducing plasma low-density lipoprotein (LDL) cholesterol and decreasing the risk of cardiovascular events. In recent years, a multitude of variants in genes involved in pharmacokinetics (PK) and pharmacodynamics (PD) have been suggested to influence the cholesterol-lowering response. However, the vast majority of studies have analyzed the pharmacogenetic associations in populations in Europe and the USA, whereas data in other populations, including Brazil, are mostly lacking. This narrative review provides an update of clinical studies on statin pharmacogenomics in Brazilian cohorts exploring lipid-lowering response, adverse events and pleiotropic effects. We find that variants in drug transporter genes (SLCO1B1 and ABCB1) positively impacted atorvastatin and simvastatin response, whereas variants in genes of drug metabolizing enzymes (CYP3A5) decreased response. Furthermore, multiple associations of variants in PD genes (HMGCR, LDLR and APOB) with statin response were identified. Few studies have explored statin-related adverse events, and only ABCB1 but not SLCO1B1 variants were robustly associated with increased risk in Brazil. Statin-related pleiotropic effects were shown to be influenced by variants in PD (LDLR, NR1H2) and antioxidant enzyme (NOS3, SOD2, MTHFR, SELENOP) genes. The findings of these studies indicate that statin pharmacogenomic associations are distinctly different in Brazil compared to other populations. This review also discusses the clinical implications of pharmacogenetic studies and the rising importance of investigating rare variants to explore their association with statin response.

Background: The available antihypertensive drugs are effective and well tolerated agents. However, only about half of patients with treated hypertension achieve appropriate blood pressure control. Genetic and non-genetic factors contribute to the interindividual variability of the therapeutic response. Objective: This review constitutes a comprehensive update of the pharmacogenomics of antihypertensive drugs and their clinical implications in Brazil. Results: Twenty-five studies explored the influence of gene variants on drug response in patients with primary, resistant, or gestational hypertension. Variants in BDKRB2, NOS3, PRKCA, and VEGFA influenced the response to enalapril in patients with primary hypertension. AGT and MMP2 variants were associated with a high risk of resistance to antihypertensive treatment, whereas NOS2 variants were related to low risk. Moreover, NAT2 slow acetylators showed an increased response to hydralazine in patients with resistant hypertension. HMOX1, NAMPT, MMP9, NOS3, and TIMP1 variants might be markers of drug responsiveness in hypertensive or preeclamptic pregnant women. Power and replication of studies, polygenic nature of the response to therapy, and treatment with multiple drugs were important challenges to identify genetic predictors of antihypertensive response in Brazil. Conclusion: Pharmacogenomic studies in Brazilian cohorts provide some evidence of variants, mainly in pharmacodynamics genes, which influence the response to antihypertensive drugs. However, some findings are limited by cohort size or therapeutic scheme and may be influenced by interactions with other genetic and non-genetic factors. Therefore, further investigations are needed to elucidate the contribution of pharmacogenomics to the efficacy and safety of antihypertensive therapy.

Aim: To analyze the expression of urinary exosome-derived miRNAs (exo-miRs) in kidney recipients on tacrolimus-based therapy. Patients & methods: Clinical and drug monitoring data were recorded from 23 kidney recipients. Expression of 93 exo-miRs was measured by quantitative PCR array and mRNA targets were explored. Results: 16 exo-miRs were differentially expressed, including marked upregulation of miR-155-5p, and downregulation of miR-223-3p and miR-1228-3p. Expression of miR-155-5p and miR-223-3p correlated with tacrolimus dose (p

BACKGROUND: Genetic and epigenetics factors have been implicated in drug response, graft function and rejection in solid organ transplantation. Differential expression of genes involved in calcineurin and mTOR signaling pathway and regulatory miRNAs was analyzed in the peripheral blood of kidney recipient cohort (n=36) under tacrolimus-based therapy. METHODS: PPP3CA, PPP3CB, MTOR, FKBP1A, FKBP1B and FKBP5 mRNA expression and polymorphisms in PPP3CA and MTOR were analyzed by qPCR. Expression of miRNAs targeting PPP3CA (miR-30a, miR-145), PPP3CB (miR-10b), MTOR (miR-99a, miR-100), and FKBP1A (miR-103a) was measured by qPCR array. RESULTS: PPP3CA and MTOR mRNA levels were reduced in the first three months of treatment compared to pre-transplant (PA (GG genotype) and MTOR c.2997C>T (TT genotype) were associated with reduced expression of PPP3CA mRNA and MTOR, respectively. FKBP1B mRNA levels were higher in patients with acute rejection (P=0.026). CONCLUSIONS: The expression of PPP3CA, MTOR and miR-99a in the peripheral blood of renal recipients is influenced by tacrolimus-based therapy and by PPP3CA and MTOR variants. These molecules can be potential biomarkers for pharmacotherapy monitoring.

Aim: The influence of variants in pharmacokinetics-related genes on long-term exposure to tacrolimus (TAC)-based therapy and clinical outcomes was investigated. Patients & methods: Brazilian kidney recipients were treated with TAC combined with everolimus (n = 178) or mycophenolate sodium (n = 97). The variants in CYP2C8, CYP2J2, CYP3A4, CYP3A5, POR, ABCB1, ABCC2, ABCG2, SLCO1B1 and SLCO2B1 were analyzed. Main results: CYP3A5*3/*3 genotype influenced increase in TAC concentration from week 1 to month 6 post-transplantation (p

BACKGROUND Polymorphisms in genes encoding transport proteins and metabolizing enzymes involved in tacrolimus (TAC) disposition may be important sources of individual variability during treatment. OBJECTIVE The aim of this study was to investigate the effect of combined CYP3A4 and CYP3A5 variants, using a CYP3A4/5 genetic score, and ABCB1 polymorphisms on therapeutic TAC monitoring and their relationship with clinical outcomes. MATERIAL AND METHODS Brazilian kidney transplant recipients (n=151), who received TAC over 3 months after transplantation, were genotyped for CYP3A4 rs2242480 (g.20230G>A), CYP3A5 rs15524 (g.31611C>T) and rs776746 (g.6986A>G), ABCB1 rs1128503 (c.1236C>T), rs1045642 (c.3435C>T), and rs2032582 (c.2677G>T/A) polymorphisms. RESULTS Frequencies of CYP3A4 g.20230A, CYP3A5 g.31611C, and g.6986A were 0.37, 0.26, and 0.28, respectively. These alleles were associated with TAC rapid metabolization and were used for CYP3A4/5 genetic score construction. A higher CYP3A4/5 genetic score was associated with higher TAC dose and lower concentrations for dose administered (Co/D, P

Monitoring of immunosuppressive drugs, such as calcineurin and mTOR inhibitors, is essential to avoid undesirable kidney transplant outcomes. Polymorphisms in pharmacokinetics-related genes have been associated with variability in blood levels of immunosuppressive drugs and adverse effects, but influence of pharmacodynamics-related genes remains to be elucidated. The influence of polymorphisms in genes of the mTOR and calcineurin signaling pathways on long-term clinical outcomes was investigated in Brazilian kidney transplant recipients within the 1-year post-transplant. Two-hundred and sixty-nine kidney transplant recipients were enrolled at a kidney transplant center in São Paulo city, Brazil, and treated with tacrolimus plus everolimus or mycophenolate sodium (clinical trial NCT01354301). Clinical and laboratory data, including renal function parameters and drug blood levels were recorded. Genomic DNA was extracted from blood samples. Polymorphisms in MTOR rs1057079 (c.4731G>A), rs1135172 (c.1437T>C), and rs1064261 (c.2997C>T); PPP3CA rs3730251 (c.249G>A); FKBP1A rs6033557 (n.259+24936T>C); FKBP2 rs2159370 (c.-2110G>T); and FOXP3 rs3761548 (c.-23+2882A>C) and rs2232365 (c.-22-902A>G) were analyzed by real-time PCR. Frequencies of gene polymorphisms did not differ among the treatment groups. Analysis of primary outcomes showed that patients carrying MTOR c.1437CC and FOXP3 c.-23+2882CC genotypes had higher serum creatinine than non-carriers (p < 0.05) at 1-year post-transplant. MTOR c.4731G allele (AG+GG genotype) was associated with increased risk for acute rejection (OR = 3.53, 95% CI = 1.09–11.48, p = 0.037). Moreover, 1-year cumulative incidence of rejection was higher in MTOR c.4731G allele carriers compared to AA genotype carriers (p = 0.027). Individually, analysis of secondary outcomes revealed that FKBP2 c.-2110GG genotype carriers had higher risk of leukopenia, FKBP1A n.259+24936C allele carriers had increased risk of constipation, and FOXP3 c.-22-902A or c.-23+2882A allele had higher risk of gastrointestinal disorders (p < 0.05). However, these results were not maintained in the multivariable analysis after p-value adjustment. In conclusion, variants in genes of mTOR and calcineurin pathways are associated with long-term impaired renal function, increased risk of acute rejection, and, individually, with adverse events in Brazilian kidney transplant recipients.

Dyslipidemia, diabetes, obesity and hypertension are common metabolic diseases. In the last decades, unhealthy lifestyle and aging have leads to an increased incidence of these diseases, increasing morbidity and mortality by cardiovascular causes. The treatment of metabolic diseases includes life-style interventions as healthy diet and physical exercise, as well as pharmacological interventions. Several drugs are available for the management of metabolic diseases including among others lipid-lowering antidiabetics and antihypertensive drugs. Variability in response to these drugs is influenced by both genetic and non-genetic factors. Polymorphisms in genes related to drug pharmacokinetics and pharmacodynamics have been shown to influence drug efficacy and safety. This review is focused on pharmacogenetic studies related to the management of metabolic diseases in samples of the Brazilian population. Associations of variants in drug metabolizing enzymes and transporters, drug target and metabolism-related genes with the efficacy and safety of lipid-lowering, antidiabetic and antihypertensive drugs are described. Most pharmacogenetic studies in Brazil have focused in pharmacological response to a small group of drugs, as statins and some antihypertensives, while there are almost no studies on antidiabetic and antiobesity drugs. Some studies reported significant associations of gene polymorphisms with drug response confirming previous data from other populations, whereas other works did not replicate, which may relay on the genetic admixture of our population. In conclusion, further studies are necessary considering larger sample sizes, new unexplored drugs and more genetic variants to obtain stronger conclusions to explore clinical applications of pharmacogenetic studies in our population.

SUMMARY Introduction: Cholesterol-lowering therapy has been related with several pleiotropic effects including anti-inflammatory action in vascular endothelium; however, their influ- ence on monocyte adhesion molecules is poorly described. Aims: To investigate the effect of inhibitors of synthesis (statins) and absorption (ezetimibe) of cholesterol on expression of adhesion molecules L-selectin, PSGL-1, VLA-4, LFA-1, and Mac-1 in mononuclear cells in vivo and in vitro using THP-1 cells. Methods: The influence of simvastatin (10 mg/day), ezetimibe (10 mg/day), and their combination (10 mg each/day) on mRNA expression of adhesion molecules was analyzed in peripheral blood mononuclear cells (PBMC) from hy- percholesterolemics. The effects of atorvastatin, simvastatin, and ezetimibe on mRNA and protein expression of adhesion molecules were also evaluated in THP-1 cells. Results: Sim- vastatin/ezetimibe combination, but not the monotherapies, reduced the mRNA expression of the PSGL-1, LFA-1, and Mac-1 genes in PBMC from hypercholesterolemics. Total and LDL cholesterol in serum correlated with PSGL-1 mRNA expression, whereas HDL choles- terol negatively correlated with mRNA levels of L-selectin and VLA-4 genes (P < 0.05). Plasma hsCRP was also correlated with mRNA levels of VLA-4, LFA-1, and Mac-1 (P < 0.05). Atorvastatin and simvastatin at 10 lM reduced mRNA and protein expression of L-selectin, PSGL-1, and VLA-4 in THP-1 cells (P < 0.05). Conclusion: Cholesterol-low- ering therapy modulates gene expression of adhesion molecules in PBMC from hypercho- lesterolemics and THP-1 cells. Simvastatin/ezetimibe combination gives more benefits by reducing to a larger extent the expression of adhesion molecules in mononuclear cells.

Background Reverse cholesterol transport (RCT) has been inversely related to atherosclerosis and cardiovascular risk. The influence of menopause in the RCT process is poorly understood and the effects of cholesterol-lowering interventions, including statins and hormone therapy (HT), on genes controlling the RCT in postmenopausal women are also unknown. Methods The effects on serum lipids and expression profile of genes involved in RCT – APOA1 , ABCA1 , ABCG1 , SCARB1 and LXRA – were evaluated by TaqMan ® quantitative PCR in peripheral blood mononuclear cells (PBMC) from 87 postmenopausal hypercholesterolemic women treated with atorvastatin (AT, n = 17), estrogen or estrogen plus progestin (HT, n = 34) and estrogen or estrogen plus progestin associated with atorvastatin (HT + AT, n = 36). Results Atorvastatin and HT treatments reduced the mRNA levels of APOA1 and SCARB1 , respectively, whereas ABCA1 expression was reduced after all treatments. Although the expression of LXRA , an important transcription factor controlling the expression of genes involved in RCT, was not modified after any treatment, it was correlated with ABCA1 , APOA1 and SCARB1 RNAm values before and after treatments, however no correlation with ABCG1 was observed. In a linear regression analysis, HT was related to an increase in apoAI levels after treatment when compared to atorvastatin and, moreover, higher SCARB1 and ABCA1 basal expression were also associated with decreased apoAI levels after treatments. Conclusion ABCA1 mRNA levels are decreased by atorvastatin and HT, however these treatments have a differential effect on APOA1 and SCARB1 expression in PBMC from postmenopausal women. Basal ABCA1 and SCARB1 expression profile could be helpful markers in predicting the effect of atorvastatin and HT on RCT, according to the changes in apoAI levels in this sample population.

tudy Objective To investigate the influence of single nucleotide polymorphisms (SNPs) in genes encoding metabolizing enzymes (CYP2C8, CYP2J2, and UGT2B7) and transporters (ABCC2 and ABCG2) on dose and dose-adjusted trough blood concentrations (C:D ratio), clinical outcomes, and occurrence of adverse events of tacrolimus and mycophenolate sodium in Brazilian kidney transplant recipients. Design Pharmacogenetic analysis of patients enrolled in a previously published study. Patients One hundred forty-eight adult kidney transplant recipients treated with tacrolimus, enteric-coated mycophenolate sodium, and prednisone for 90 days posttransplantation. Measurements and Main Results ABCC2 c.−24C>T and c.3972C>T, ABCG2 c.421C>A, CYP2C8*3, CYP2J2 c.−76G>T, and UGT2B7 c.372A>G SNPs were determined by real-time polymerase chain reaction. The CYP3A5*3C SNP data were used to eliminate the confounding effect of this variant on the results. ABCC2 c.3972T allele carriers showed higher tacrolimus C:D values than did carriers of the c.3972CC genotype. The CYP2C8*3 variant was also associated with slightly higher tacrolimus C:D values and higher estimated glomerular filtration rate but only in CYP3A5-nonexpressing patients (CYP3A5*3C/*3C carriers). None of the SNPs were associated with mycophenolate sodium dose or episodes of biopsy-confirmed acute rejection or delayed graft function. The CYP2J2 c.−76T allele was associated with increased risk for treatment-induced nausea and/or vomiting (OR: 5.30, 95% confidence interval 1.49-18.79, pT polymorphism affected tacrolimus C:D in Brazilian kidney transplant recipients. Further, CYP2C8*3 and CYP2J2 c.−76G>T SNPs influenced the renal function of these patients and the occurrence of adverse events during treatment with tacrolimus and mycophenolate sodium.

BACKGROUND Polymorphisms in genes encoding for drug-metabolizing enzymes and drug transporters are among multiple factors that modulate the pharmacokinetic variability of tacrolimus (TAC) and sirolimus (SRL). This study aimed to evaluate the influence of single nucleotide polymorphisms (SNPs) on TAC and SRL dose-adjusted concentrations (C0/D) in stable kidney transplant recipients. METHODS This is an exploratory and prospective study, which includes 46 stable kidney transplant recipients. These patients were monitored from the 3rd to the 24th month after transplantation. The SRL group consisted of 25 patients receiving TAC, prednisone (PRED), and mycophenolate sodium (MPS), which were converted from TAC to SRL at 3rd month after transplantation. The TAC group consisted of 21 patients who underwent treatment with TAC, PRED, and MPS. Both groups were genotyped for CYP3A4 rs2242480 (g.20230G>A), CYP3A5 rs15524 (g.31611C>T), CYP2C8 rs10509681 (c.1196A>G) and ABCB1 rs1045642 (c.3435C>T), rs1128503 (c.1236C>T), and rs2032582 (c.2677G>T/A) polymorphisms. RESULTS In the TAC group, CYP3A4 rs2242480 A allele carriers were associated with lower TAC C0/D. For CYP3A5 rs15524 SNP, C0/D was higher among patients carrying TT genotype when compared with CT and CC genotype carriers in the SRL and, more consistently, in the TAC groups. For ABCB1 rs1045642 SNP, TT genotype was associated with reduced SRL C0/D, but only at month 15. CONCLUSIONS CYP3A4 rs2242480 and CYP3A5 rs15524 SNPs resulted in significant changes in SRL and TAC C0/D at different times after transplantation.

Menopause is associated with changes in lipid levels resulting in increased risk of atherosclerosis and cardiovascular events. Hormone therapy (HT) and atorvastatin have been used to improve lipid profile in postmenopausal women.Effects of HT, atorvastatin and APOE polymorphisms on serum lipids and APOE and LXRA expression were evaluated in 87 hypercholesterolemic postmenopausal women, randomly selected for treatment with atorvastatin (AT, n=17), estrogen or estrogen plus progestagen (HT, n=34) and estrogen or estrogen plus progestagen associated with atorvastatin (HT+AT, n=36). RNA was extracted from peripheral blood mononuclear cells (PBMC) and mRNA expression was measured by TaqMan® PCR. APOE ɛ2/ɛ3/ɛ4 genotyping was performed using PCR-RFLP.Total cholesterol (TC), LDL-c and apoB were reduced after each treatment (p

Aims: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated. Material and Methods: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot® and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (−71T>C) gene polymorphisms were identified by TaqMan® Real-time PCR. Results: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%:1.3–8.0, p < 0.05). Conclusion: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy. This work was supported by grants from FAPESP (2008/06667-9). A.C. Rodrigues, F.D.V. Genvigir and M.A.V. Willrich are recipients of fellowships from FAPESP, Sao Paulo, Brazil. M.H. Hirata and R.D.C. Hirata are recipients of fellowships from CNPq, Brasilia, Brazil. This work was partially supported by Life Technologies, Sao Paulo, SP, Brazil SI

The SR-BI is a key component on the cholesterol metabolism. Polymorphisms in the SR-BI gene (SCARB1) were related with variations on plasma lipoprotein profile and other risk factors for cardiovascular disease. We tested the relationship of 3 SCARB1 single nucleotide polymorphisms (SNPs) with hypercholesterolemia in a Brazilian population and whether these variants can influence lipid-lowering response to atorvastatin.c.4GA, c.726+54CT and c.1050CT SNPs and serum concentrations of lipid and apolipoproteins were evaluated in 147 hypercholesterolemic (HC) and 185 normolipidemic (NL) unrelated Brazilian subjects. HC patients were treated with atorvastatin (10 mg/day/4 weeks).Frequencies of SCARB1 polymorphisms were similar between the HC and NL groups (p0.05). The T allele for c.726+54CT was associated with higher LDL-c in NL and with higher apoB and apoB/apoAI in HC (p0.05). HC individuals carrying c.1050C allele carriers (CC and CT genotypes) had lower change of total cholesterol, LDL-c, apoB and apoB/apoAI ratio (p0.05) than the TT genotype carriers in response to atorvastatin.The SCARB1 polymorphisms are related with variations in serum lipids in the Brazilian population and c.1050CT SNP is associated with lipid-lowering atorvastatin response.

Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and efflux/ uptake transporters. There is little information on the regulation of these proteins in human cells after statin therapy. In this study, the effects of atorvastatin and simvastatin on mRNA expression of efflux (ABCB1, ABCG2 and ABCC2) and uptake (SLCO1B1, SLCO2B1 and SLC22A1) drug transporters in Caco-2 and HepG2 cells were investigated.Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins.Differences in mRNA basal levels of the transporters were as follows: ABCC2ABCG2ABCB1SLCO1B1SLC22A1SLC O2B1 for HepG2 cells, and SLCO2B1ABCC2ABCB1ABCG2SLC22A1 for Caco-2 cells. While for HepG2 cells, ABCC2, ABCG2 and SLCO2B1 mRNA levels were significantly up-regulated at 1, 10 and 20 micromol/L after 12 or 24 h treatment, in Caco-2 cells, only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin. Interestingly, whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells, in Caco-2 cells the statin significantly down-regulated ABCB1, ABCC2, SLC22A1, and SLCO2B1 mRNA levels after 12 or 24 h treatment.These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters, and this effect depends on the cell type. Furthermore, alterations in the expression levels of drug transporters in the liver and/or intestine may contribute to the variability in oral disposition of statins.Acta Pharmacologica Sinica (2009) 30: 956-964; doi: 10.1038/aps.2009.85; published online 22 June 2009.

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. One hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p=0.039). Basal ABCB1 mRNA in the lower quartile (0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4+/-12.4%) and apolipoprotein B (apoB) (17.0+/-31.3%) when compared with the higher quartile (0.085: LDL-c=40.3+/-14.3%; apoB=32.5+/-10.7%; p0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin.

The transcription factors SREBP1 and SCAP are involved in intracellular cholesterol homeostasis. Polymorphisms of these genes have been associated with variations on serum lipid levels and response to statins that are potent cholesterol-lowering drugs. We evaluated the effects of atorvastatin on SREBF1a and SCAP mRNA expression in peripheral blood mononuclear cells (PBMC) and a possible association with gene polymorphisms and lowering-cholesterol response.Fifty-nine hypercholesterolemic patients were treated with atorvastatin (10 mg/day for 4 weeks). Serum lipid profile and mRNA expression in PBMC were assessed before and after the treatment. Gene expression was quantified by real-time PCR using GAPD as endogenous reference and mRNA expression in HepG2 cells as calibrator. SREBF1 -36delG and SCAP A2386G polymorphisms were detected by PCR-RFLP.Our results showed that transcription of SREBF1a and SCAP was coordinately regulated by atorvastatin (r=0.595, p0.001), and that reduction in SCAP transcription was associated with the 2386AA genotype (p=0.019). Individuals who responded to atorvastatin with a downregulation of SCAP had also a lower triglyceride compared to those who responded to atorvastatin with an upregulation of SCAP.Atorvastatin has differential effects on SREBF1a and SCAP mRNA expression in PBMC that are associated with baseline transcription levels, triglycerides response to atorvastatin and SCAP A2386G polymorphism.

A eficácia das estatinas em reduzir o risco de eventos coronarianos não é completamente explicada por seus efeitos em diminuir colesterol de lipoproteína de baixa densidade (LDL-C). Um dos seus efeitos adicionais pode ser decorrente da modificação na concentração de lipoproteína de alta densidade (HDL), reconhecida como ateroprotetora, principalmente por seu papel no transporte reverso do colesterol (TRC). Os transportadores de membrana do tipo ATP-binding cassette, ABCA1 e ABCG1, e o scavenger receptor BI (SRBI) são proteínas importantes envolvidas no TRC e seus genes são regulados por vários fatores de transcrição, entre eles os liver-x-receptors (LXRs). Com a finalidade de avaliarmos os efeitos dos hipolipemiantes sobre expressão dos transportadores ABC e do receptor SRBI, a expressão de RNAm do ABCA1, ABCG1, SCARB1, NR1H3 (LXRα) e NR1H2 (LRXβ) foi avaliada por PCR em tempo real em células das linhagens HepG2 (origem hepática) e Caco-2 (origem intestinal) tratadas com atorvastatina ou sinvastatina (10 µM) e/ou ezetimiba (até 5 µM) por até 24 horas. Além disso, a expressão desses genes também foi avaliada em células mononucleares do sangue periférico (CMSP) de 50 pacientes normolipidêmicos (NL) e 71 hipercolesterolêmicos (HC) tratados com atorvastatina (10mg/dia/4semanas, n=48) ou sinvastatina e/ou ezetimiba (10mg/dia/4 ou 8 semanas, n=23). A possível associação entre os polimorfismos ABCA1 C-14T e R219K e a expressão de RNAm em CMSP também foi avaliada por PCR-RFLP. O SCARB1 foi o gene mais expresso nas células HepG2 e Caco-2, seguido por NR1H2, NR1H3, ABCG1 e ABCA1 em HepG2 ou por ABCA1 e ABCG1 em Caco-2. O tratamento com estatinas (1 ou 10 µM) ou ezetimiba (5 µM), por 12 ou 24 horas, aumentou a expressão de RNAm do ABCG1, mas não de ABCA1 e SCARB1, em células HepG2. Ainda nesta linhagem, o aumento na transcrição dos genes NR1H2 e NR1H3 foi observado somente com a maior concentração de atorvastatina (10 µM) e, ao contrário, o tratamento com ezetimiba causou redução na transcrição de NR1H2, sem alteração de NR1H3. Em células Caco-2, o tratamento com atorvastatina ou sinvastatina por 12 ou 24 horas reduziu a quantidade do transcrito ABCA1 e não alterou a expressão do SCARB1 e do ABCG1, embora, para este último, tenha havido uma tendência à diminuição da expressão após tratamento com sinvastatina (p=0,07). Após tratamento com ezetimiba isolada (até 5 µM) nenhuma alteração de expressão de RNAm foi observada em células Caco-2; no entanto, após 24 horas de tratamento com sinvastatina e ezetimiba, foi reduzida a taxa de transcrição de ABCA1 e ABCG1, mas não de SCARB1. Ao contrário das linhagens celulares, em CMSP os genes NR1H2 e ABCG1 foram os mais expressos, seguidos pelos genes SCARB1 e ABCA1 e, finalmente, pelo NR1H3. Indivíduos HC tiveram maior expressão basal de NR1H2 e NR1H3, mas não de outros genes, quando comparados aos NL (pT quando comparados aos portadores do genótipo -14CC (p=0,034). O tratamento com estatinas, com ezetimiba ou com a terapia combinada diminuiu a transcrição de ABCA1 e ABCG1. Para o SCARB1, NR1H2 e NR1H3, nenhuma alteração de expressão de RNAm em CMSP foi detectada após os tratamentos in vivo. Após todas as fases de tratamento, ABCA1 e ABCG1 e também NR1H2 e NR1H3 foram significativamente correlacionados entre si, mas nenhuma correlação com perfil lipídico sérico foi relevante. Coletivamente, esses resultados dão indícios de que os hipolipemiantes analisados (estatinas e ezetimiba) têm um importante papel na regulação da expressão de genes envolvidos no transporte reverso do colesterol e sugerem a existência de regulação tecido-específica para os dois transportadores ABC. Além disso, o efeito das estatinas ou da ezetimiba sobre a expressão do ABCA1, do ABCG1 ou do SCARB1 não sofreu influencia de alterações diretas da transcrição dos LXRs. The efficacy of statins in reducing the risk of coronary events is not completely explained by their effects in decreasing cholesterol low-density lipoprotein (LDL-C). One of their additional effects may result from the change in concentration of high-density lipoprotein (HDL), recognized as atheroprotective, mainly for the role in reverse cholesterol transport (RCT). The membrane transporters, as ATP-binding cassette, ABCA1 and ABCG1, and scavenger receptor BI (SRBI) are important proteins involved in the RCT and their genes are regulated by various transcription factors, including the liver-X-receptors (LXRs) . In order to evaluate the effects of lipid lowering on expression of ABC transporters and SRBI receptor, the mRNA expression of ABCA1, ABCG1, SCARB1, NR1H3 (LXRα) and NR1H2 (LRXβ) was assessed by real time PCR in HepG2 (hepatic origin) and Caco-2 (intestinal origin) cells treated with atorvastatin or simvastatin (10 µM) and/or ezetimibe (up to 5 µM) for 24 hours. Furthermore, the expression of these genes was evaluated in peripheral blood mononuclear cells (PBMC) of 50 normolipidemic (NL) and 71 hypercholesterolemic (HC) patients treated with atorvastatin (10mg/d/4 weeks, n = 48) or simvastatin and/or ezetimibe (10mg/d/4 or 8 weeks, n = 23). The possible association between ABCA1 C-14T and R219K polymorphisms and mRNA expression in PBMC was also evaluated by PCR-RFLP. SCARB1 was the most expressed in HepG2 and Caco-2 cells, followed by NR1H2, NR1H3, ABCG1 and ABCA1 in HepG2 or by ABCG1 and ABCA1 in Caco-2. The treatment with statins (1 or 10 µM) or ezetimibe (5 µM) for 12 or 24 hours, increased mRNA expression of ABCG1 but not ABCA1 and SCARB1 in HepG2 cells. Moreover, in HepG2 cells, atorvastatin also upregulated NR1H2 and NR1H3 only at 10.0 µM, meanwhile ezetimibe downregulated NR1H2 but did not change NR1H3 expression. In Caco-2 cells, atorvastatin or simvastatin treatment for 12 or 24 hours reduced the amount of ABCA1 transcript and did not alter the ABCG1 and SCARB1 expressions, despite the tendency to decrease ABCG1 mRNA expression after simvastatin treatment (p = 0.07). After treatment with ezetimibe alone (up to 5 µM) no change in mRNA expression was observed in Caco-2 cells; however, after 24 hours- simvastatin and ezetimibe treatments decreased the transcription of ABCA1 and ABCG1, but not of SCARB1. Unlike cell lines, in PBMC, NR1H2 and ABCG1 were the most expressed, followed by SCARB1 and ABCA1 and finally by the NR1H3. HC patients showed higher NR1H2 and NR1H3 basal expressions, but not of other genes, compared to NL (p T polymorphism when compared with -14CC carriers (p = 0.034). Treatment with statins, ezetimibe, or combined therapy downregulated ABCA1 and ABCG1 expression. For SCARB1, NR1H2 and NR1H3, no change in mRNA expression in PBMC was detected after treatments. After all phases of treatment, ABCA1 and ABCG1 as well as NR1H2 and NR1H3 were significantly correlated, but no correlation with serum lipid profile was relevant. Collectively, these results provide evidences that the lipid lowering (statins and ezetimibe) have an important role in mRNA expression regulation of genes involved in reverse cholesterol transport and suggest the existence of tissue-specific regulation for the ABC transporters. Furthermore, the effect of statins or ezetimibe on ABCA1, ABCG1 or SCARB1 expression was not directly influenced by changes of LXR transcription.

OBJECTIVE: The aim of the study was to investigate whether adiposity and metabolic markers, such as leptin, glucose, and lipids, are influenced by leptin (LEP) and leptin receptor (LEPR) gene polymorphisms in a sample of our population. SUBJECTS AND METHODS: A group of 326 individuals of Caucasian-European descent, aged 30 to 80 years, 87 men and 239 women, 148 obese and 178 non-obese, was randomly selected at two clinical hospitals in the city of Sao Paulo, Brazil. All individuals declared their ethnic group as white during the initial interview. Anthropometric measurements, body mass index (BMI), and fat mass were evaluated. Blood samples were drawn for DNA extraction and measurements of leptin, soluble leptin receptor, glucose, and lipids. LEP -2548G>A and LEPR Lys109Arg (c.326A>G), Gln233Arg (c.668A>G) and Lys656Asn (c.1968G>C) polymorphisms were detected by PCR-RFLP. RESULTS: Increased leptin and serum lipids, and LEPR Arg223Arg (GG genotype) were associated with higher risk for obesity (p < 0.05), while reduced risk was found in LEPR Arg109Arg (GG genotype) carriers (OR: 0.38, 95%CI: 0.19-0.77, p = 0.007). Multiple linear regression analysis showed a relationship between LEPR 223Arg, increased waist circumference, and leptinemia (p < 0.05), while LEPR 109Arg was associated with high total cholesterol and triglycerides (p < 0.05). LEPR haplotype 3 (AGG: 109Lys/233Arg/656Lys) carriers have increased risk for obesity (OR: 2.56, 95% CI: 1.19-5.49, p = 0.017). Moreover, this haplotype was associated with increased BMI, waist circumference, and leptinemia (p < 0.05). CONCLUSIONS: LEPR polymorphisms are associated with obesity, hyperleptinemia, and atherogenic lipid profile, suggesting their potential role for leptin resistance and cardiovascular risk. Moreover, LEPR haplotype 3 confers susceptibility to adiposity and hyperleptinemia in our population.

Objective Using candidate gene approach, we have investigated the effect of single nucleotide polymorphism (SNP) in genes related to lipid metabolism and atherosclerosis on dyslipidemia and atorvastatin response. Methods The study included 157 patients treated with atorvastatin and 145 controls. Genomic DNA was isolated and genotyped using SNPlex technology. Results Allele and genotype disease association test revealed that APOB rs693 (OR: 2.2 [1.5–3.2], p = 0.0001) and CD36 rs1984112 (OR: 3.7 [1.9–7.0], p = 0.0002) SNPs were independent risk factors for hypercholesterolemia. Only APOB rs693 T variant allele was associated with increased LDL cholesterol levels (> 160 mg/dL). After atorvastatin treatment (10 mg/day/4 weeks), LIPC − 514T allele was positively associated with LDL cholesterol reduction. Conclusion The current study reinforces the current knowledge that carrying APOB rs693 is an independent risk factor for dyslipidemia and higher LDL levels. Furthermore, we found that a variant of CD36 was associated with dyslipidemia as a risk (rs1984112) factor. Finally, atorvastatin response could be predicted by LIPC − 514C > T SNP and physical activity. In conclusion, our data evidences the contribution of genetic markers and their interaction with environmental factor in the variability of statin response.

Background Variability of response to statins has been related to polymorphisms in genes involved in cholesterol homeostasis and statin metabolism, such as CYP3A4 and CYP3A5. We investigated the effects of atorvastatin on CYP3A4 and CYP3A5 mRNA expression in mononuclear cells and on CYP3A activity and their interactions with common variants. Methods Unrelated individuals (n = 121) with hypercholesterolemia (HC) were treated with atorvastatin (10 mg/day/4 weeks). Ninety-two normolipidemic (NL) subjects were selected as a control group. Genotype analysis of CYP3A4*1B (rs2740574), CYP3A4*22 (rs35599367), CYP3A5*3C (rs776746), and CYP3A5*1D (rs15524) and mRNA levels in peripheral blood mononuclear cells (PBMCs) were estimated. CYP3A activity was phenotyped by the urinary cortisol to 6-beta-hydroxy-cortisol ratio. Results LDL cholesterol reduction in response to atorvastatin was positively correlated with change in CYP3A4 (R2 = 0.039, p = 0.037) and CYP3A5 (R2 = 0.047, p = 0.019) mRNA levels and negatively correlated with CYP3A activity (R2 = 0.071, p = 0.022). CYP3A5*3C (AGT haplotype) was associated to lower basal CYP3A5 mRNA expression in HC (p

Background: Balancing the subject composition of case and control groups to create homogenous ancestries between each group is essential for medical association studies. Methods: We explored the applicability of single-tube 34-plex ancestry informative markers (AIM) single nucleotide polymorphisms (SNPs) to estimate the African Component of Ancestry (ACA) to design a future case–control association study of a Brazilian urban sample. Results: One hundred eighty individuals (107 case group; 73 control group) self-described as white, brown-intermediate or black were selected. The proportions of the relative contribution of a variable number of ancestral population components were similar between case and control groups. Moreover, the case and control groups demonstrated similar distributions for ACA 0.50 categories. Notably a high number of outlier values (23 samples) were observed among individuals with ACA

Enterocytes play a crucial role in high-density lipoprotein (HDL) biogenesis. Statins and ezetimibe are potent lowering-cholesterol drugs, which can also influence HDL plasma concentrations. We hypothesized that these drugs could modulate the expression of intestinal ABCA1 and ABCG1, two genes involved in HDL metabolism.Caco-2 cells were used as a model of the human intestinal cells and were treated with statins (0.01-1 μmol/L) and/or ezetimibe (0.5-5.0 μmol/L) for 12 h or 24 h. Gene expression was examined using real-time PCR.ABCA1 level was more abundant than ABCG1 in Caco-2 cells. ABCA1 was downregulated after 12-h and 24-h treatment with atorvastatin (0.1 and 1.0 μmol/L) or simvastatin (0.01, 0.1 and 1 μmol/L) (p0.05). In statin-treated cells, ABCG1 levels remained unaltered. Ezetimibe alone did not induce change of ABCA1 or ABCG1 mRNA levels (p0.05) but 24-h ezetimibe (2.5 or 5.0 μmol/L) plus simvastatin (1 μmol/L) treatment decreased the transcription of ABCA1 and ABCG1 (p0.05).Our findings reveal that, at the concentrations studied, statins isolated or combined with ezetimibe, but not ezetimibe alone, downregulate ABCA1 mRNA expression in Caco-2 cells. Moreover, simvastatin combined with ezetimibe treatment also decrease the ABCG1 levels in these cells.

The ATP-binding cassette transporter A1 (ABCA1) has an essential role in the formation of nascent high-density lipoprotein particles and also participates in the cholesterol efflux from macrophages in the artery wall. Several substances, such as statins, or even gene variants are able to modulate ABCA1 expression. There is strong evidence that statin treatment downregulates the ABCA1 expression in nonloaded macrophages. Interestingly, in cholesterol-loaded macrophages, which are more relevant to atherogenesis, this effect is lost. We observed an inhibitory effect of atorvastatin in peripheral blood mononuclear cells of hypercholesterolemic individuals. Moreover, in these individuals, the ABCA1 -14C>T polymorphism was associated with high baseline gene-expression levels. Other studies are needed to evaluate how relevant these findings are to the formation of arterial foam cells in vivo.

The cytochrome P450 isoenzyme 3A5 (CYP3A5) has an important role on biotransformation of xenobiotics. CYP3A5 SNPs have been associated with variations on enzyme activity that can modify the metabolism of several drugs.In order to evaluate the influence of CYP3A5 variants on response to lowering-cholesterol drugs, 139 individuals with hypercholesterolemia were selected. After a wash-out period of 4 weeks, individuals were treated with atorvastatin (10 mg/day/4 weeks). Genomic DNA was extracted by a salting-out procedure. CYP3A5*3C, CYP3A5*6 and CYP3A5*1D were analyzed by PCR-RFLP and DNA sequencing.Frequencies of the CYP3A5*3C and CYP3A5*1D alleles were lower in individuals of African descent (*3C: 47.8% and *1D: 55.2%) than in non-Africans (*3C: 84.9% and *1D 84.8%, p0.01). Non-Africans carrying *3A allele (*3C and *1D combined alleles) had lower total and LDL-cholesterol response to atorvastatin than non-*3A allele carriers (p0.05).CYP3A5*3A allele is associated with reduced cholesterol-lowering response to atorvastatin in non-African individuals.

ABCA1 plays an important role in HDL metabolism. Single nucleotide polymorphisms (SNPs) in ABCA1 gene were associated with variation in plasma HDL-c.The effect of the ABCA1 SNPs C-14T, R219K and of a novel variant C-105T on serum lipids was investigated in 367 unrelated Brazilian individuals (224 hypercholesterolemic and 143 normolipidemic). The relation between ABCA1 SNPs and the lipid-lowering response to atorvastatin (10 mg/day/4 weeks) was also evaluated in 141 hypercholesterolemic (HC) individuals. The polymorphisms were detected by PCR-RFLP and confirmed by DNA sequencing.Linkage disequilibrium was found between the SNPs C-105T and C-14T in the HC group. HC individuals carrying -105CT/TT genotypes had higher serum HDL-c and lower triglyceride and VLDL-c concentrations as well as lower TG/HDL-c ratio compared to the -105CC carriers (p0.05). The R219K SNP was associated with reduced serum triglyceride, VLDL-c and TG/HDL-c ratio in the HC group (p0.05), and with an increased serum apoAI in NL individuals. The effects of ABCA1 SNPs on basal serum lipids of HC individuals were not modified by atorvastatin treatment.The ABCA1 SNPs R219K and C-105T were associated with a less atherogenic lipid profile but not with the lowering-cholesterol response to atorvastatin in a Brazilian population.

Background Apolipoprotein E (apoE) is a key component of the lipid metabolism. Polymorphisms at the apoE gene (APOE) have been associated with cardiovascular disease, lipid levels and lipid-lowering response to statins. We evaluated the effects on APOE expression of hypercholesterolemia, APOE ε2/ε3/ε4 genotypes and atorvastatin treatment in Brazilian individuals. The relationship of APOE genotypes and plasma lipids and atorvastatin response was also tested in this population. Methods APOE ε2/ε3/ε4 and plasma lipids were evaluated in 181 normolipidemic (NL) and 181 hypercholesterolemic (HC) subjects. HC individuals with indication for lowering-cholesterol treatment (n = 141) were treated with atorvastatin (10 mg/day/4-weeks). APOE genotypes and APOE mRNA in peripheral blood mononuclear cells (PBMC) were analyzed by TaqMan real time PCR. Results HC had lower APOE expression than NL group (p < 0.05) and individuals with low APOE expression showed higher plasma total and LDL cholesterol and apoB, as well as higher apoAI (p < 0.05). Individuals carrying ε2 allele have reduced risk for hypercholesterolemia (OR: 0.27, 95% I.C.: 0.08-0.85, p < 0.05) and NL ε2 carriers had lower total and LDL cholesterol and apoB levels, and higher HDL cholesterol than non-carriers (p < 0.05). APOE genotypes did not affect APOE expression and atorvastatin response. Atorvastatin treatment do not modify APOE expression, however those individuals without LDL cholesterol goal achievement after atorvastatin treatment according to the IV Brazilian Guidelines for Dyslipidemia and Atherosclerosis Prevention had lower APOE expression than patients with desirable response after the treatment (p < 0.05). Conclusions APOE expression in PBMC is modulated by hypercholesterolemia and the APOE mRNA level regulates the plasma lipid profile. Moreover the expression profile is not modulated neither by atorvastatin nor APOE genotypes. In our population, APOE ε2 allele confers protection against hypercholesterolemia and a less atherogenic lipid profile. Moreover, low APOE expression after treatment of patients with poor response suggests a possible role of APOE level in atorvastatin response.