Identification of pharmacogenetic predictors of lipid-lowering response to atorvastatin in Chilean subjects with hypercholesterolemia

Clinica Chimica Acta 413 (2012) 495–501

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Identification of pharmacogenetic predictors of lipid-lowering response to atorvastatin in Chilean subjects with hypercholesterolemia Alexy Rosales a, Marysol Alvear b, Alejandro Cuevas a, Nicolás Saavedra a, Tomás Zambrano a, Luis A. Salazar a,⁎ a Centro de Biología Molecular & Farmacogenética, Departamento de Ciencias Básicas, Facultad de Medicina, Universidad de La Frontera, Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile b Departamento de Ciencias Químicas & Recursos Naturales, Facultad de Ingenieria, Ciencias y Administración, Universidad de La Frontera, Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile

a r t i c l e

i n f o

Article history: Received 25 August 2011 Received in revised form 12 October 2011 Accepted 8 November 2011 Available online 19 November 2011 Keywords: Polymorphisms Hypercholesterolemia Atorvastatin CYP3A4

a b s t r a c t Background: Statins are normally the first-line therapy for hypercholesterolemia (HC); however, the lipidlowering response shows high interindividual variation. We investigated the effect of four polymorphisms in CYP3A4, CYP3A5 and ABCB1 genes on response to atorvastatin and CYP3A4 activity in Chilean subjects with HC. Methods: A total of 142 hypercholesterolemic individuals underwent atorvastatin therapy (10 mg/day/1 month). Serum lipid levels before and after treatment were measured. Genetic variants in CYP3A4 (−290A>G, rs2740574), CYP3A5 (6986A>G, rs776746) and ABCB1 (2677G>A/T, rs2032582 and 3435C>T, rs1045642) were analyzed by PCR-RFLP. CYP3A4 enzyme activity in urine samples was assessed through determination of 6β-hydroxycortisol/cortisol free ratio (6βOHC/FC). Results: After 4 weeks of therapy, a significant reduction in total cholesterol (TC) and LDL-c was observed (P b 0.001). The G allele for −290A>G polymorphism was related to higher percentage of variation in TC and LDL-c (P b 0.001). Moreover, same allele was associated with higher HDL-c variation (P = 0.017). In addition, CYP3A4 enzyme activity was lower in subjects carrying this polymorphism (P = 0.009). No differences were observed for CYP3A5 and ABCB1 variants. Conclusion: Our results suggest that presence of G allele for −290A>G polymorphism determines a better response to atorvastatin, being also associated with lower CYP3A4 activity in vivo, causing an increased atorvastatin activity. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Circulatory diseases are the leading cause of death worldwide with lipid metabolism being one of the main determinants of cardiovascular risk [1]. In our country, this situation is similar, placing cardiovascular disease as the main cause of mortality. Risk factors are similar to those elsewhere, mainly determined by age, obesity, sedentary life, hypercholesterolemia and alcoholism, increasing the number of hospitalizations and deaths [2]. This growth in cardiovascular disease (CVD) and associated mortality is a feature of western civilization, developing and industrialized countries, as a result of epidemiological and nutritional transition, increasing considerably health costs [3]. Nonetheless, an important point to consider is that more than half of all deaths from cardiovascular diseases can be avoided. It has been documented as an important piece of evidence

⁎ Corresponding author. Tel.: + 56 45 592895; fax: + 56 45 592832. E-mail address: [email protected] (L.A. Salazar). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.11.003

that prevention and treatment of CVD consist in reducing risk factors such as changes in lifestyle along with specific treatment of lipid disorders [2, 4]. According to the INTERHEART study, 52 countries worldwide suggest that therapeutic strategies which concomitantly target elevated LDL-cholesterol (LDL-c) and low HDL-cholesterol (HDL-c) levels, are likely to be the most effective in CVD care [1]. At the moment, the chief treatment for hypercholesterolemia is the use of 3-hydroxy-3methylglutaryl-CoenzimaA (HMG-CoA) reductase inhibitors, known as statins. Statins are among the most widely used drugs worldwide. They are usually very well tolerated, but they can cause myopathy as well as rhabdomyolysis, the risk of which is increased by certain interactions as a rare, plasma concentration-dependent adverse reaction [5]. The benefits of statins are well known. Experimental studies have documented a great amount of evidence about different positive effects such as an increment in the nitric oxide (NO) expression, as well as anti-inflammatory, immunomodulatory, anti-thrombotic, anti-proliferative, and anti-oxidant effects [6]. These are known as

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pleiotropic effects. The statins' mechanism of action is to inhibit in a competitive and reversible way the HMG-CoA reductase enzyme, which catalyzes the biosynthesis of cholesterol in the liver. Once inhibited, it stops the formation of mevalonic acid and reduces intracellular cholesterol synthesis, resulting in a compensatory increase in expression of LDL receptors in liver cells, reducing LDL-c levels of circulating total cholesterol (about 20%–50%, respectively). They also give rise to a modest decrease in triglycerides (TG, 10% to 40%) and a small increase in high density lipoprotein (HDL) cholesterol (5% to 15%) levels [7]. Important studies have shown that for every 1% reduction in LDL-c level, there is an associated 1% reduction in risk of clinical cardiovascular events [8]. Statins are metabolized in the intestine and in the liver by cytochrome P450-system (CYP450), mainly by CYP3A sub-family, including CYP3A4 and CYP3A5 isoforms. The CYP3A plays a role in their metabolism in humans, participating in the metabolism of bile acids and steroids (such as aldosterone, testosterone, and estrogens), but also in the biotransformation of xenobiotics such as immunosuppressive drugs [9, 10]. Simvastatin, lovastatin, and atorvastatin are metabolized by cytochrome P450 (CYP) 3A4 [11, 12]. A drug may be conditioned by its own factors (dose, via of administration, interactions, pharmacokinetics and pharmacodynamics), by individual characteristics (ethnicity, age, sex, genetic factors), or by conditions attached (associated diseases, smoking or ingestion of alcohol) such as a slow metabolizer, fast metabolizer or nonresponder individual. To date, more than 40 genes that could affect response to statins have been investigated, related to both, pharmacokinetics (metabolizing enzymes and transport proteins) and pharmacodynamics (receptors and signal transduction pathways), although with contradictory results [12, 13]. Large interindividual pharmacokinetic differences observed among humans are partially due to genetic polymorphisms in drug-metabolizing enzymes such as cytochrome P450, or transporter proteins such a ABCB1 [14]. These variations include genes involved in intestinal absorption of cholesterol and apolipoprotein E (APOE), ATP transporter (ATP binding cassette) the production of cholesterol, including HMG-CoA reductase, metabolism of lipoproteins and apolipoprotein B and LDL receptor, and also of those who act in the way of the cytochrome P450 [15–19]. In the case of atorvastatin, variation from identifiable genetic polymorphisms has been shown [20]. For example in the case of CYP3A sub-family, variations in genes involved in uptake, distribution, and metabolism of statins may also significantly modulate clinical response. In the same way, P-glycoprotein, the gene product of ABCB1 (MDR1), belonging to the adenosine triphosphate-binding cassette superfamily of membrane transporters, gives multidrug resistance, playing an important role in the bioavailability (absorption, distribution and elimination) of common drugs in medical care, including atorvastatin. Human ABCB1 (MDR1) is located on chromosomal region 7q21, and provides genetic variants of interest in exons 21 and 26 [21]. These genetic variants of ABCB1 can naturally affect interindividual variability in pharmacokinetics and pharmacodynamics of many drugs and account for differences in the bioavailability of various P-gp substrates. Some studies analyzed single nucleotide polymorphisms (SNPs) in candidate genes, including genes relevant to metabolizing enzymes such as CYP3A4 and CYP3A5 genes [22], transport protein such an ABCB1 gene [23], and cholesterol biosynthesis (HMGCR). Common variants were identified, among these −290A>G (CYP3A4) [24], 6986A>G (CYP3A5) [25], 3435C>T and 2677G>A>T (ABCB1) [26] which were associated to a possible response and efficacy of lipidlowering therapy, elevating controversial results [27]. The −290A>G (rs2740574) polymorphism is located in the promoter region, altering transcription efficiency, and thus the overall enzymatic activity of CYP3A4. The G allele has enhanced CYP3A4 expression due to reduced binding of a transcriptional repressor. This

effect is discussed elsewhere [28]. CYP3A5*1 (6986A>G, rs776746) is one of the key alleles responsible for drug response variation. This sequence variant in CYP3A5 gene, was found to cause alternative splicing and protein truncation (incorrect splicing of pre-mRNA forming an aberrant mRNA which is degraded), resulting in the absence of functional CYP3A5 from liver tissue [29]. Similarly, polymorphisms in the ABCB1 gene (2677G>A>T, rs2032582 and 3435C>T, rs1045642) are associated with an impaired efflux pump of the ABCB1 transporter, resulting in increased drug levels [26, 27]. Finally, because this drug is metabolized primarily by CYP3A4 enzyme, variations could interfere with the effectiveness of treatment, such as CYP3A4*1B, CYP3A5*3 and polymorphisms in the ABCB1 gene. Determination of the metabolic activity is also important, being studied via a urinary excretion ratio of 6β-hydroxycortisol to free cortisol. Based on these data, the aims of this study were to determine the allelic frequency and evaluate the role of four common SNPs on therapeutic response to atorvastatin, and their possible relation between genetic variation and the activity of CYP3A4 enzyme in Chilean individuals with hypercholesterolemia. 2. Materials and methods 2.1. Subjects A total of 142 non related individuals, with a mean age of 56.4 ± 10.7 years, diagnosed with hypercholesterolemia, according to NCEP criteria [30], treated with atorvastatin 10 mg/day for one month, were selected from the Federico Thieme Health Center (Región de La Araucanía, Chile). This drug is delivered to all hypercholesterolemic patients attended in the public hospitals of our country. None of the subjects had diabetes, hepatic disease, kidney disease, endocrinological disorders, and malignant disease or was receiving concomitant lipid-lowering therapy. Patients under treatment with medications that could affect the lipid profile like beta-blockers and diuretics were not included in this study. Patients with clinical diagnosis of familial hypercholesterolemia (FH) were also excluded. All the participants voluntarily signed an informed consent. The study protocol was approved by the local Scientific Ethics Committee. Blood samples were obtained by venipuncture following a 10 to 12-h overnight fast. Urine samples were collected, separated in aliquots, and frozen at −20° until analysis. Biochemical measurements were determined by enzymatic methods already described [31] and low-density lipoprotein cholesterol was calculated using Friedewald's formula, when triglycerides did not exceed 400 mg/dL (4.8 mmol/L). The accuracy of the biochemical determinations was controlled using normal and pathological commercial serums (Human, Germany). 2.2. Molecular analysis Genomic DNA was extracted from blood leukocytes by a salting out procedure optimized by Salazar et al. [32]. The integrity of the genomic DNA was visualized by electrophoresis in 1.0% agarose gel. The −290A>G (CYP3A4), 6986A>G (CYP3A5), 2677G>A>T and 3535C>T (ABCB1) gene polymorphisms were detected using polymerase chain reaction (PCR) followed by enzymatic restriction according to conditions described by Cavalli et al. [33], Kim et al. [25], Rodrigues et al. [26], and Cascorbi et al. [20], respectively. PCR amplification was performed using a Mycycler™ System (Bio-Rad Laboratories Inc, CA, USA). The correct assessment of genotypes for − 290A>G (CYP3A4), 6986A>G (CYP3A5), 2677G>A>T and 3535C>T (ABCB1) polymorphisms was evaluated using a homozygous sample for restriction site as a positive control. In addition, all gels were reread blindly by two persons without any change, and 20% of the analyses were repeated randomly. The digestion fragments were identified by electrophoresis (3.0% agarose gel), stained with ethidium bromide and

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Table 1 Clinical and demographic characteristics of the study group. Parameters

N = 142

Age (years) Male/Female, n BMI, kg/m2 SBP, mm Hg DBP, mm Hg Total cholesterol, mg/dL HDL-cholesterol, mg/dL LDL-cholesterol, mg/dL Triglycerides, mg/dL ASAT/GOT, UI/L ALAT/GOT, UI/L

56.4 ± 10.7 89/53 25.6 ± 2.7 106.8 ± 12.3 72.7 ± 9.2 274.4 ± 18.3 46.4 ± 8.8 185.4 ± 17.5 212.8 ± 50.5 24.4 ± 7.2 22.7 ± 8.3

visualized on a UV transilluminator (E-Box 1000, Vilber Lourmant, France). 2.3. Determination of CYP3A4 activity in urine samples The assessment of CYP3A4 activity was performed by determining the 6β-hidroxycortisol/free cortisol ratio in first morning urine samples by reversed-phase high-performance liquid chromatography (RPLC), using conditions previously described by Hong et al. [34], after extraction of urine sample in a solid phase using a SPE C-18 cartridge (UCT, USA), followed by a liquid/liquid phase and RPLC, in order to eliminate the interference maximum. The standards of Cortisol (FC), 6β-hidroxycortisol (6β-OHC) and dexamethasone (IS, internal standard) were purchased from Sigma (St. Louis, Missouri, USA) and were of at least 98% purity. All other chemical and solvents were of liquid chromatographic and analytical-reagent grade. Briefly, 2.0 mL of urine was added to solutions containing known amounts of internal standard (2000 mg/mL) and applied to SPE C-18 extraction cartridges pretreated with methanol and water. The cartridges were washed and then eluted with a solution of ethyl acetate-diethyl ether. Then, 1 M NaOH saturated and 1% acetic acid saturated solutions were added. The organic extract was evaporated to dryness by a stream of nitrogen. The residue was reconstituted in a solution of acetonitrilo-water (1:4, v/v). A 20 μL aliquot of the solution was injected to HPLC. Peaks areas of FC, 6β-OHF and IS were measured. Chromatography was performed with a Merck Hitachi Pump L-7100 HPLC systems and Merck Hitachi L-4250 UV–vis detector. Chromatographic separation of the analytes was achieved on C18 column, using mobile phase composed Acetonitrile (A) ammonium sulfate solution in lineal gradient. The flow-rate was 1.0 mL/min, the detection wavelength was 240 nm (λ), and the column temperature was 45 °C. 2.4. Statistical analysis The analysis of the collected data was done using SigmaStat program, version 2.0 (Jandel Sci., San Rafael, USA). Association between the different analyzed variables was verified using Student t test or one-way ANOVA. For comparison of proportions and to evaluate

Fig. 1. Individual therapeutic response to atorvastatin 10 mg/day/4 weeks, considering the LDL-C serum values as efficacy variable. Each bar represents the percent change in LDL-cholesterol from baseline for one study subject; these data are arranged in rank order to show the range of responses.

the achievement of the Hardy–Weinberg equilibrium we used the Chi-square test (χ 2). Statistical significance was considered as P b 0.05. 3. Results 3.1. Clinical variables and lipid-lowering response to atorvastatin The clinical and demographic characteristics of the study group are summarized in Table 1. Serum lipids levels at baseline and after treatment with atorvastatin at a dose of 10 mg daily for 4 weeks are summarized in Table 2. The reduction percentage of total cholesterol, LDL-cholesterol and triglycerides was significant at the end of treatment (P b 0.001). In addition, the drug produced a significant increase in HDL-cholesterol levels (P b 0.001). Figure 1 shows the inter-individual therapeutic response to atorvastatin in the population studied, using as endpoint the reduction of LDL-cholesterol. No adverse reactions were observed. 3.2. Genotypes and lipid-lowering response to atorvastatin Table 3 summarizes genotype distribution and the relative allele frequencies for all genetic variants investigated. The genotype distribution for all polymorphisms investigated was consistent with Hardy–Weinberg equilibrium. When comparing lipid-lowering response to atorvastatin according to the four polymorphisms investigated (Tables 4 and 5), we see that only the common variant

Table 3 Genotype distribution and relative allele frequencies of four common genetic polymorphisms of CYP3A4, CYP3A5 and ABCB1 in Chilean hypercholesterolemic individuals. Polymorphism Genotypes − 290A>G (CYP3A4)

Table 2 Serum lipids levels at baseline and after four weeks of treatment with atorvastatin (10 mg daily).

Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides

Baseline (mg/dL)

Treatment (mg/dL)

Change (mg/dL)

P-value

274.4 ± 18.3 46.4 ± 8.8 185.4 ± 17.5 212.8 ± 50.5

224.5 ± 26.2 54.1 ± 6.7 137.3 ± 26.1 165.9 ± 48.4

− 49.8 ± 30.0 7.6 ± 6.7 − 48.1 ± 31.6 − 47.1 ± 43.2

b 0.001 b 0.001 b 0.001 b 0.001

Results are expressed as mean ± SD; P-values from Student's t-test.

6986A>G (CYP3A5) 3435C>T (ABCB1) 2677G>A>T (ABCB1)

AA AG 78.9% (112) 21.1% (30) HWE: χ2 = 1.980, P = 0.159 AA AG 51.4% (73) 43.6% (62) 2 HWE: χ = 1.840, P = 0.174 CC CT 42.2% (60) 47.8% (68) HWE: χ2 = 0.696, P = 0.403 GG GT TT GA 34.6 % 31.5% 6.9 % 17.7 % (45) (41) (9) (23) HWE: χ2 = 1.980, P = 0.159

HWE, Hardy–Weinberg Equilibrium.

Allele frequencies GG 0% (0)

A 0.89

G 0.11

GG 4.9% (7)

A 0.27

G 0.73

TT 9.8% (14)

C 0.66

T 0.34

TA 7.7 % (10)

G A T 0.59 0.14 0.27

AA 0.77 % (1)

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Table 4 Response to atorvastatin treatment (10 mg/day/1 month) according to genotypes for − 290A>G (CYP3A4) and 6986A>G (CYP3A5) gene polymorphism in Chilean subjects with hypercholesterolemia. − 290A>G (CYP3A4) AA (112)

6986A>G (CYP3A5) AG (30)

P-value

AA (73)

AG (62)

GG (7)

P-value

Baseline (mg/dL) Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides

272.7 ± 17.8 46.9 ± 9.2 183.7 ± 17.8 210.5 ± 41.4

280.5 ± 19.1 44.5 ± 7.1 191.6 ± 14.8 221.4 ± 66.6

0.004 0.184 0.027 0.295

277.8 ± 8.4 49.4 ± 8.1 184.1 ± 14.8 221.7 ± 35.5

273.3 ± 19.8 48.7 ± 11.2 182.2 ± 20.1 211.6 ± 45.1

275.0 ± 17.8 44.3 ± 5.4 188.1 ± 14.8 212.9 ± 56.2

0.763 0.088 0.160 0.096

Treatment (mg/dL) Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides

228.1 ± 24.0 53.2 ± 6.8 141.8 ± 23.4 165.9 ± 47.5

211.1 ± 29.9 57.9 ± 5.1 129.3 ± 29.2 164.8 ± 52.2

0.001 b 0.001 b 0.001 0.914

224.8 ± 39.1 55.7 ± 6.8 136.2 ± 41.1 162.5 ± 48.8

228.3 ± 26.6 54.7 ± 7.8 139.2 ± 25.4 173.5 ± 52.3

221.2 ± 24.3 53.5 ± 5.6 135.7 ± 24.5 159.3 ± 44.3

0.291 0.517 0.743 0.232

− 16.1 ± 9.1 14.9 ± 13.0 − 22.2 ± 13.5 − 20.3 ± 19.5

− 24.4 ± 11.8 31.8 ± 16.1 − 36.4 ± 17.8 − 23.9 ± 18.2

b 0.001 b 0.001 b 0.001 0.356

− 18.9 ± 14.3 14.6 ± 19.0 − 25.2 ± 23.3 − 27.7 ± 11.4

− 16.1 ± 10.2 14.7 ± 14.5 − 22.9 ± 15.1 − 17.2 ± 20.3

− 19.3 ± 9.7 22.1 ± 14.9 − 27.2 ± 15.2 − 23.7 ± 18.4

0.186 0.017 0.275 0.096

Change (%) Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides

Number of samples in parenthesis; results are expressed as mean ± SD; p values from Student's t-test or ANOVA; Multiple comparisons by Holm–Sidak test.

−290A>G affects drug response (Table 4). Individuals with the mutated AG genotype exhibited a greater reduction in serum total cholesterol and LDL-c. They also showed an increase in HDL-c levels (P b 0.001). In addition, a significant difference was observed in basal and post-treatment levels of total cholesterol and LDL-c by genotype (P b 0.05). 3.3. Effect of − 290A>G variant of CYP3A4 gene on CYP3A4 activity To evaluate the effect of −290A>G polymorphism in CYP3A4 gene over CYP3A4 activity, we used the 6β-hidroxycortisol/free cortisol ratio. A total of 28 first morning urine samples were analyzed (20 samples from subjects with AA homozygous genotype and 8 samples from AG carriers genotype for −290A>G variant of CYP3A4 gene). The CYP3A4 enzyme activity in urine was lower in subjects carrying the mutated G allele for the −290A>G variant of the CYP3A4 gene (AA = 2.5 ± 1.7 vs. AG = 0.79 ± 0.4, P = 0.009, Fig. 2). 4. Discussion Cardiovascular disease is considered one of the biggest problems of our decade, causing more deaths each year than any other

pathology in the world. Some authors have suggested a slowdown in mortality rates from these causes, contradictory to the increasing prevalence of cardiovascular risk factors [35]. Currently there is great interest in the relationship between the development of a pathology and pharmacology response, in this particular case, the lipid-lowering drug response and its relationship with the presence of genetic variants. Thus, in this study we investigated the frequency of four common variants and their association with lipid-lowering response to atorvastatin in Chilean hypercholesterolemic subjects. Biochemical analysis, showed a 25.3% reduction in LDL-cholesterol levels when atorvastatin medication was fulfilled (primary efficacy variable), similar to the one reported in the ASCOT-LLA study (29.1%), using the same drug and dose. In our study we also observed a significant reduction in plasma concentrations of total cholesterol (P b 0.001), triglycerides (P b 0.001), and an increase in HDL-cholesterol (P b 0.001). Nonetheless, a wide variability in therapeutic response was observed. Similarly, other groups have reported a significant variability in individual response in the same conditions described in our study [36, 37]. When we analyzed the allele frequencies for the polymorphisms investigated, our data demonstrate that the mutated G allele for

Table 5 Response to atorvastatin treatment (10 mg/day/1 month) according to genotypes for 2677G>A>T and 3435C>T polymorphisms of ABCB1 gene in Chilean subjects with hypercholesterolemia. 2677G>A>T(ABCB1) Non GG (84)

3435C>T (ABCB1) GG (45)

P-value

CC (60)

CT (68)

TT (14)

P-value

Baseline (mg/dL) Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides

274.6 ± 15.8 47.1 ± 9.4 184.7 ± 18.6 214.5 ± 49.8

273.0 ± 14.9 43.7 ± 6.8 185.2 ± 10.4 214.5 ± 56.3

0.708 0.122 0.892 0.999

272.2 ± 17.8 46.0 ± 8.4 184.2 ± 16.8 209.7 ± 53.5

275.6 ± 17.8 47.1 ± 9.3 185.8 ± 17.3 212.6 ± 48.8

278.3 ± 22.5 44.8 ± 8.4 188.1 ± 21.9 226.7 ± 47.0

0.394 0.595 0.720 0.528

Treatment (mg/dL) Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides

226.6 ± 25.5 56.6 ± 6.8 138.7 ± 24.9 166.9 ± 48.9

231.8 ± 23.3 51.2 ± 6.5 143.4 ± 26.9 165.8 ± 52.1

0.399 0.038 0.444 0.925

222.9 ± 26.2 54.1 ± 6.6 139.9 ± 25.5 164.2 ± 49.0

226.2 ± 26.2 54.2 ± 7.0 139.0 ± 26.2 166.6 ± 48.4

227.2 ± 27.1 54.1 ± 6.1 134.5 ± 29.4 166.9 ± 48.5

0.750 0.992 0.742 0.923

− 17.2 ± 10.0 18.1 ± 16.0 − 24.1 ± 15.2 − 21.2 ± 18.8

− 16.3 ± 10.0 18.0 ± 10.6 − 22.4 ± 14.2 − 23.1 ± 23.0

0.705 0.963 0.647 0.690

− 17.8 ± 10.1 19.2 ± 14.6 − 25.6 ± 14.5 − 20.6 ± 18.1

− 17.6 ± 10.1 16.9 ± 15.6 − 24.5 ± 15.7 − 20.5 ± 20.9

− 19.3 ± 12.5 22.7 ± 15.4 − 27.0 ± 19.8 − 25.5 ± 15.6

0.854 0.384 0.831 0.665

Change (%) Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides

Number of samples in parenthesis; results are expressed as mean ± SD; p values from Student's t-test or ANOVA.

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6β-Hydroxicortisol/ Free Cortisol .

8

6

4

2

0

CYP3A4*1B

Wild Type

Fig. 2. Distribution of the urinary 6β-hydroxycortisol/free cortisol ratios of individuals carrying the mutated − 290G allele (CYP3A4*1B, n = 8) and Wild type allele (n = 20) for the − 290A>G polymorphism of CYP3A4 gene (P = 0.009).

−290A>G polymorphism of CYP3A4 gene was found in 10.6% of the studied individuals. Comparing this frequency with those reported by other studies, we see a similarity with Brazilian (0.13) [33], Asian (0.16) [38] and Hispanic (0.09) population [39], and a significant difference with American population, both White (0.04) and Black (0.54) [39], also German (0.05) [40], Africans (0.82) [41] and AfroAmerican (0.67) individuals [40]. In relation to the 6986A>G variant of CYP3A5 gene, the G allele was observed at a frequency of 0.73 in the studied subjects. The results are very similar to the respective frequencies observed in some Asian populations such as Chinese [42] and Japanese [43] (0.77 and 0.76, respectively) and very different as in the reported frequencies in Americans (0.93) [27], Africans (0.15) [44], European Caucasians (0.93) [45], and Afro- American populations (0.36) [46]. Our data also indicates that polymorphism 3435C>T in exon 21 of ABCB1 gene, has a frequency for the T allele of 0.34. These results are very similar to the respective frequencies observed by Wielandt et al. [47] in Mapuche and Mestizo ethnicities of our country, having allele frequencies of 0.35 and 0.33, respectively. In addition, the frequency found in our study, for the T allele for the variant 3435C>T is similar to that observed in Asian population such as Koreans (0.37) [48], slightly lower in Rapanui Chilean populations (0.25) [47], but differs significantly from that observed in Brazilian (0.47) [26], AfroAmericans (0.21) [46], Africans (0.17) [49], European Caucasian (0.52) [45] and Oceania (0.53) [50] populations. With respect to G allele for the 2677G>A>T polymorphism of the ABCB1 gene, the frequency found was 0.59, slightly lower than reported in ethnic populations in Chile, Mestiza (0.65) and Mapuche (0.69) [47]. Comparing our result with other populations, we can see a similarity with those reported by Cascorbi et al. [20] and Rodrigues et al. [26] who observed frequencies of 0.56 (Caucasian) and 0.58 (Brazilian), respectively. In contrast, Yi et al. [48] reported in Korean individuals, an allele frequency of 0.44 for this allele. In relation to the effectiveness of lipid-lowering treatment and its influence with the presence of the SNPs investigated, an important finding in our study was the relation observed between the presence of G allele in polymorphism −290A>G (CYP3A4 gene) and increased therapeutic response to atorvastatin in subjects carrying this variant.

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Significant differences in the percentages of reduction of total cholesterol and LDL-c were observed at the end of pharmacological therapy in subjects carrying the mutant allele compared to homozygous wild type genotype. In addition, these individuals reported a greater increase in HDL-c levels after treatment. This result is similar to that reported by Gao et al. [51] who observed an association between the presence of G allele in hypercholesterolemic individuals, who had a greater reduction in serum total cholesterol levels, when treated with atorvastatin 20 mg/day for 4 weeks. However, no association was found regarding LDL-c reduction. Furthermore, our data differ with those obtained by Kajinami et al. [24] who associated the presence of this variant (allele G) to higher levels of LDL cholesterol after treatment, in 340 HC individuals treated with atorvastatin 10 mg/day for 52 weeks. Similarly, Poduri et al. [52] found no association between this variant and the reduction in lipid levels by analyzing a population of 265 subjects treated with atorvastatin (20 mg/day) during a period of 6 weeks. In relation to the variant 6986A>G of CYP3A5 gene, we only observed a significant difference in the increased percentage of variation in HDL-c levels after 1 month of treatment with atorvastatin. Other investigations reported controversial results between this variant and the percentage variation in response to statins. Kivisto et al. [53] suggest that this variant affects the pharmacological response observing a greater reduction in LDL-c levels in a group of 69 individuals after one year treatment with statins. More recently, Kim et al. [25] described an association between this variant and response to simvastatin in a group of 22 subjects, suggesting that the presence of this polymorphism provides a possible explanation for interindividual variability. In addition, for the polymorphisms 3435C>T and 2677G>A>T of ABCB1 gene, our study showed a significant difference in HDL-c concentrations post-treatment by comparing the subjects with the wild type allele (2677G) versus the mutated allele (P = 0.038), however, no such association was observed when comparing the percentages of variations in HDL-c levels (P = 0.963). Similarly, Rodrigues et al. [26] in Brazilian hypercholesterolemic subjects, found no relationship between the individual presence of this polymorphism and response to atorvastatin (10 mg/day/1 month). Moreover, Kajinami et al. [46] have reported that hypercholesterolemic white women carrying at least one 2677nonG (2677T or 2677A) allele showed higher reduction in LDL cholesterol levels than non-carriers, but this was not seen in men. They also showed that there was a relation between basal and variation of HDL cholesterol in subjects with the mutated allele (T) for the variant 3435C>T of the ABCB1 gene. Finally, considering that metabolism of atorvastatin is mainly achieved by CYP3A4, the enzyme activity was analyzed in morning spot urine samples for −290A>G variant. We used the 6β-hydroxicortisol/ free cortisol ratio, and a non-invasive assay to determine CYP3A4 activity [54]. CYP3A4 catalyzes the cortisol to form 6β-hydroxicortisol, and this metabolite is then excreted as an unconjugated form in urine. Although its use has been debated by some investigators, this ratio is considered as the simplest and most practical assay in the assessment of CYP3A4 activity [10]. Inhibition of drug metabolizing enzymes leads to increased plasma levels of the drug, prolonging its pharmacological effects and increasing the possibility of adverse reactions or toxicity. Individuals with higher CYP3A4 activity rates should receive a different dosage of the drug than for subjects with rates closer to zero [34]. Our data indicates that individuals carrying the mutated allele for the variant −290A>G of the CYP3A4 gene, have a 6βOHC/FC ratio lower than subjects homozygous for the normal allele (AA genotype, P = 0.009). These results are similar to those reported by Wang et al. [18] which analyzed the Ile118Val variant in 211 hypercholesterolemic subjects of Asian origin treated with simvastatin (20 mg/day) for 4 weeks. They found a significant difference in reducing total cholesterol and triglycerides, however, no significant difference in reducing LDL-c

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levels. In relation to the CYP3A4 activity observed 6βOHC/FC ratios were significantly lower in individuals with the variant studied (CYP3A4*4), reaching up to 50 fold between genotype. It has been well known that an interindividual difference in metabolic profile of many drugs is in part due to sequence variants in gene encoding drug metabolizing enzymes. Therefore, knowledge of the prevalence of SNPs in a population is essential for the estimation of the likelihood of interindividual differences of drug efficacy or side effects. Recently, Lagos et al. [36] showed that the ethnicity of the Chilean population could be a cause in the inadequacy of atorvastatin, since individuals characterized as haplogroup B by mitochondrial DNA (mtDNA) have higher lipid levels post-treatment. Thus, the influence of ethnicity on pharmacogenetic responses must be taken into account by performing and comparing clinical trials in various ethnic groups. In summary, our results show that atorvastatin treatment (10 mg/day/1 month) is effective in reducing serum LDL-c levels. Nonetheless, wide variability in therapeutic response was observed. Our results suggest that presence of G allele for − 290A>G polymorphism of CYP3A4 gene determines a better response to atorvastatin, being also associated with lower CYP3A4 activity in vivo, causing an increased atorvastatin activity. This finding can explain, in part, the observed variability for this therapy and the presence of adverse/ side effects in some individuals. However, this study is restricted by its sample size. Pharmacogenetic testing has an increasing impact in the individualization of drug treatment and could contribute significantly to enhanced drug safety and efficacy. Therefore, it would be interesting to complement the present study investigating whether −290A>G polymorphism may influence the response to other doses of atorvastatin, as well as to other statins available in our country. Acknowledgments This study was supported by grants from Dirección de Investigación y Desarrollo, Universidad de La Frontera, Temuco, Chile (DIUFRO DI09-1007). References [1] Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case–control study. Lancet 2004;364(9438):937–52. [2] Medina E, Kaempffer A. Epidemiology of cardiovascular disease in Chile. Rev Chil Cardiol 2007;26:219–26. [3] Luque C, Cisternas FA, Araya M. Changes in the patterns of disease after the epidemiological transition in health in Chile, 1950–2003. Rev Med Chil 2006;134(6): 703–12. [4] Lanas F, Avezum A, Bautista LE, et al. Risk factors for acute myocardial infarction in Latin America: the INTERHEART Latin American study. Circulation 2007;115(9): 1067–74. [5] Niemi M. Transporter pharmacogenetics and statin toxicity. Clin Pharmacol Ther 2010;87(1):130–3. [6] Kirmizis D, Chatzidimitriou D. Pleiotropic vasoprotective effects of statins: the chicken or the egg? Drug Des Devel Ther 2009;3:191–204. [7] Vaughan CJ, Gotto Jr AM, Basson CT. The evolving role of statins in the management of atherosclerosis. J Am Coll Cardiol 2000;35(1):1–10. [8] LaRosa JC. Low-density lipoprotein cholesterol reduction: the end is more important than the means. Am J Cardiol 2007;100(2):240–2. [9] Igel M, Sudhop T, Von Bergmann K. Metabolism and drug interactions of 3hydroxy-3-methylglutaryl coenzyme A-reductase inhibitors (statins). Eur J Clin Pharmacol 2001;57(5):357–64. [10] Galteau MM, Shamsa F. Urinary 6ß-hydroxycortisol: a validated test for evaluating drug induction or drug inhibition mediated through CYP3A in humans and in animals. Eur J Clin Pharmacol 2003;59(10):713–33. [11] Malinowski JM. Atorvastatin: a hydroxymethylglutaryl-coenzyme A reductase inhibitor. Am J Health Syst Pharm 1998;55(21):2253–67. [12] Neuvonen PJ, Niemi M, Backman JT. Drug interactions with lipid-lowering drugs: mechanisms and clinical relevance. Clin Pharmacol Ther 2006;80(6): 565–81. [13] Voora D, Shah SH, Reed CR, et al. Pharmacogenetic predictors of statin-mediated low-density lipoprotein cholesterol reduction and dose response. Circ Cardiovasc Genet 2008;1(2):100–6.

[14] Evans WE, Johnson JA. Pharmacogenomics: the inherited basis for interindividual differences in drug response. Annu Rev Genomics Hum Genet 2001;2:9–39. [15] Evans WE. Pharmacogenomics: marshalling the human genome to individualise drug therapy. Gut 2003; 52 (Suppl 2): ii10-18. [16] Salazar LA, Hirata MH, Quintao EC, Hirata RD. Lipid-lowering response of the HMG-CoA reductase inhibitor fluvastatin is influenced by polymorphisms in the low-density lipoprotein receptor gene in Brazilian patients with primary hypercholesterolemia. J Clin Lab Anal 2000;14(3):125–31. [17] Kajinami K, Akao H, Polisecki E, Schaefer EJ. Pharmacogenomics of statin responsiveness. Am J Cardiol 2005; 96(9A); 65K-70K. [18] Wang A, Yu BN, Luo CH, et al. Ile118Val genetic polymorphism of CYP3A4 and its effects on lipid-lowering efficacy of simvastatin in Chinese hyperlipidemic patients. Eur J Clin Pharmacol 2005;60(12):843–8. [19] Kerola T, Lehtimäki T, Kähönen M, Nieminen T. Statin pharmacogenomics: lipid response and cardiovascular outcomes. Current Cardiovascular Risk Reports 2010;4(2):150–8. [20] Cascorbi I, Gerloff T, Johne A, et al. Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther 2001;69(3):169–74. [21] Bodor M, Kelly EJ, Ho RJ. Characterization of the human MDR1 gene. AAPS J 2005;7(1):E1–5. [22] Lee CR, Goldstein JA, Pieper JA. Cytochrome P450 2C9 polymorphisms: a comprehensive review of the in-vitro and human data. Pharmacogenetics 2002;12(3): 251–63. [23] Ayrton A, Morgan P. Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 2001;31(8–9):469–97. [24] Kajinami K, Brousseau ME, Ordovas JM, Schaefer EJ. CYP3A4 genotypes and plasma lipoprotein levels before and after treatment with atorvastatin in primary hypercholesterolemia. Am J Cardiol 2004;93(1):104–7. [25] Kim KA, Park PW, Lee OJ, Kang DK, Park JY. Effect of polymorphic CYP3A5 genotype on the single-dose simvastatin pharmacokinetics in healthy subjects. J Clin Pharmacol 2007;47(1):87–93. [26] Rodrigues AC, Rebecchi IM, Bertolami MC, Faludi AA, Hirata MH, Hirata RD. High baseline serum total and LDL cholesterol levels are associated with MDR1 haplotypes in Brazilian hypercholesterolemic individuals of European descent. Braz J Med Biol Res 2005;38(9):1389–97. [27] Mega JL, Morrow DA, Brown A, Cannon CP, Sabatine MS. Identification of genetic variants associated with response to statin therapy. Arterioscler Thromb Vasc Biol 2009;29(9):1310–5. [28] Amirimani B, Ning B, Deitz AC, Weber BL, Kadlubar FF, Rebbeck TR. Increased transcriptional activity of the CYP3A4*1B promoter variant. Environ Mol Mutagen 2003;42(4):299–305. [29] Kuehl P, Zhang J, Lin Y, et al. Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet 2001;27(4):383–91. [30] Grundy SM, Cleeman JI, Merz CN, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 2004;110(2):227–39. [31] Jaramillo PC, Muñoz A, Lanas C, Lanas F, Salazar LA. Endothelial nitric oxide synthase G894T gene polymorphism in Chilean subjects with coronary artery disease and controls. Clin Chim Acta 2006;371(1–2):102–6. [32] Salazar LA, Hirata MH, Cavalli SA, Machado MO, Hirata RDC. Optimized procedure for DNA isolation from fresh and cryopreserved clotted human blood useful in clinical molecular testing. Clin Chem 1998;44(8):1748–50. [33] Cavalli SA, Hirata MH, Hirata RDC. Detection of MboII polymorphism at the 5′ promoter region of CYP3A4. Clin Chem 2001;47(2):348–51. [34] Hong Z, Yu F, Ying L, Aibin L. A highly sensitive SPE- liquid/liquid extraction–RPLC analytical method for the determination of 6[beta]-hydroxycortisol and cortisol in cancer patients' urine. Journal of Medical Colleges of PLA 2010;25(2):75–83. [35] Romero T, Romero CX. Stagnant in cardiovascular prevention: alarming trends and persistent socioeconomic obstacles. Rev Esp Cardiol 2010;63(11):1340–8. [36] Lagos J, Lemus J, Sierra F, et al. Association of serum lipid levels and mitochondrial DNA Amerindian haplogroups in hypercholesterolemic subjects receiving atorvastatin. Rev Chil Cardiol 2010;29(2):208–13. [37] Cerda A, Genvigir FD, Arazi SS, et al. Influence of SCARB1 polymorphisms on serum lipids of hypercholesterolemic individuals treated with atorvastatin. Clin Chim Acta 2010;411(9–10):631–7. [38] Thompson JF, Man M, Johnson KJ, et al. An association study of 43 SNPs in 16 candidate genes with atorvastatin response. Pharmacogenomics J 2005;5(6):352–8. [39] Ball SE, Scatina J, Kao J, et al. Population distribution and effects on drug metabolism of a genetic variant in the 5′ promoter region of CYP3A4. Clin Pharmacol Ther 1999;66(3):288–94. [40] Sata F, Sapone A, Elizondo G, et al. CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity. Clin Pharmacol Ther 2000;67(1):48–56. [41] Zanger UM, Turpeinen M, Klein K, Schwab M. Functional pharmacogenetics/ genomics of human cytochromes P450 involved in drug biotransformation. Anal Bioanal Chem 2008;392(6):1093–108. [42] Hu YF, He J, Chen GL, et al. CYP3A5*3 and CYP3A4*18 single nucleotide polymorphisms in a Chinese population. Clin Chim Acta 2005;353(1–2):187–92. [43] Horinouchi M, Sakaeda T, Nakamura T, et al. Significant genetic linkage of MDR1 polymorphisms at positions 3435 and 2677: functional relevance to pharmacokinetics of digoxin. Pharm Res 2002;19(10):1581–5. [44] Kudzi W, Dodoo AN, Mills JJ. Genetic polymorphisms in MDR1, CYP3A4 and CYP3A5 genes in a Ghanaian population: a plausible explanation for altered metabolism of ivermectin in humans? BMC Med Genet 2010;11:111.

A. Rosales et al. / Clinica Chimica Acta 413 (2012) 495–501 [45] Dally H, Bartsch H, Jager B, et al. Genotype relationships in the CYP3A locus in Caucasians. Cancer Lett 2004;207(1):95–9. [46] Kajinami K, Brousseau ME, Ordovas JM, Schaefer EJ. Polymorphisms in the multidrug resistance-1 (MDR1) gene influence the response to atorvastatin treatment in a gender-specific manner. Am J Cardiol 2004;93(8):1046–50. [47] Wielandt AM, Vollrath V, Chianale J. Polymorphisms of the multiple drug resistance gene (MDR1) in Mapuche, Mestizo and Maori populations in Chile. Rev Med Chil 2004;132(9):1061–8. [48] Yi SY, Hong KS, Lim HS, et al. A variant 2677A allele of the MDR1 gene affects fexofenadine disposition. Clin Pharmacol Ther 2004;76(5):418–27. [49] Ameyaw MM, Regateiro F, Li T, et al. MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity. Pharmacogenetics 2001;11(3):217–21. [50] Roberts RL, Joyce PR, Mulder RT, Begg EJ, Kennedy MA. A common P-glycoprotein polymorphism is associated with nortriptyline-induced postural hypotension

[51]

[52]

[53] [54]

501

in patients treated for major depression. Pharmacogenomics J 2002;2(3): 191–6. Gao Y, Zhang LR, Fu Q. CYP3A4*1G polymorphism is associated with lipidlowering efficacy of atorvastatin but not of simvastatin. Eur J Clin Pharmacol 2008;64(9):877–82. Poduri A, Khullar M, Bahl A, Sehrawat BS, Sharma Y, Talwar KK. Common variants of HMGCR, CETP, APOAI, ABCB1, CYP3A4, and CYP7A1 genes as predictors of lipid-lowering response to atorvastatin therapy. DNA Cell Biol 2010;29(10): 629–37. Kivisto KT, Niemi M, Schaeffeler E, et al. Lipid-lowering response to statins is affected by CYP3A5 polymorphism. Pharmacogenetics 2004;14(8):523–5. Lykkesfeldt J, Loft S, Poulsen HE. Simultaneous determination of urinary free cortisol and 6 beta-hydroxycortisol by high-performance liquid chromatography to measure human CYP3A activity. J Chromatogr B Biomed Appl 1994;660(1): 23–9.