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SLCO2B1 c.935G>A single nucleotide polymorphism has no effect on the pharmacokinetics of montelukast and aliskiren

Tapaninen, Tuija; Karonen, Tiina; Backman, Janne T.; Neuvonen, Pertti J.; Niemi, Mikko

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Pharmacogenetics and Genomics: January 2013 - Volume 23 - Issue 1 - p 19-24
doi: 10.1097/FPC.0b013e32835bac90
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Abstract

Introduction

The organic anion-transporting polypeptide 2B1 (OATP2B1) influx transporter, encoded by the SLCO2B1 gene, belongs to the superfamily of sodium-independent solute carrier organic anion transporter proteins 1. OATP2B1 is expressed in various tissues, including the liver, intestine, pancreas, heart, brain, placenta, mammary gland, testis, skin, skeletal muscle, and platelets. In vitro, OATP2B1 transports several endogenous and xenobiotic compounds, including various drugs 1. Because of its localization in the luminal membrane of small intestinal enterocytes and in the sinusoidal membrane of hepatocytes in the liver, OATP2B1 has been suggested to participate in the active intestinal absorption and hepatic uptake of drugs 1.

In a previous study, a nonsynonymous single nucleotide polymorphism (SNP) in the SLCO2B1 gene, c.935G>A (p.R312Q; rs12422149), was associated with reduced plasma concentrations and impaired response to the leukotriene receptor antagonist montelukast in adult patients with asthma 2. In another study in adolescent and young adult patients with asthma, the area under the plasma montelukast concentration–time curve from 0 h to infinity (AUC0–∞) was about 40% lower in individuals heterozygous for the c.935 G>A SNP than in noncarriers 3. However, no homozygotes for the SNP were included in either of these studies. The c.935 G>A SNP is relatively common, with a variant allele frequency of 2.1–19% in White, 21–40% in Asian, and 6.6–13% in Sub-Saharan African or African American populations 2,4.

In addition to montelukast, the renin-inhibiting antihypertensive drug aliskiren has also been reported to be an OATP2B1 substrate 5. The pharmacokinetic properties of aliskiren make it an interesting candidate for a probe drug for studying the function of intestinal drug transporters; it has a low oral bioavailability and poor passive permeability through lipid biomembranes, it is only slightly metabolized, has an easily measurable pharmacodynamic response, and is known to interact strongly with inhibitors of intestinal influx (e.g. grapefruit juice) and efflux (e.g. itraconazole) transporters 6–8. For example, grapefruit juice, which contains OATP2B1-inhibiting constituents 9, has decreased the AUC0–∞ of aliskiren by 61% and its peak plasma concentration (Cmax) by 81% 8.

A pharmacokinetic study in individuals with SNPs associated with altered transporter function is a specific way to investigate the contribution of a transporter toward drug pharmacokinetics. To determine the functional significance of the SLCO2B1 c.935 G>A SNP, the aims of the present study were to examine the effects of this SNP on OATP2B1 function in vivo, using montelukast and aliskiren as probe substrates, in an adequately powered pharmacokinetic study in healthy volunteers.

Methods

Participants

Thirty-three healthy White Finnish volunteers participated in this study after providing written informed consent. They were recruited from a group of more than 800 individuals genotyped for the SLCO2B1 c.935G>A SNP 4. The SLCO2B1 c.935GG group included eight women and eight men, and their characteristics were (mean±SD) as follows: age 23±3 years (range, 20–30 years), height 174±10 cm (162–189 cm), and weight 67±10 kg (52–83 kg). The SLCO2B1 c.935GA group included three women and nine men, and the characteristics for this group were as follows: age 25±3 years (21–31 years), height 177±8 cm (161–192 cm), and weight 73±13 kg (54–103 kg). The SLCO2B1 c.935AA group included one woman and four men. Their characteristics were as follows: age 28±2 years (26–30 years), height 173±4 cm (167–179 cm), and weight 69±7 kg (59–76 kg). Each participant’s health was ascertained by assessment of medical history, clinical examination, and laboratory tests. None of the patients was on any continuous medication, including oral contraceptives, and none was a tobacco smoker.

Study design

The study protocol was approved by the Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District, and the National Agency for Medicines. In a fixed-order crossover study with two phases, following an overnight fast, the participants ingested a single 10 mg dose of montelukast (Singulair; Merck Sharp Dohme B.V., Haarlem, the Netherlands) with 150 ml water, and after a washout period of at least 1 week and following an overnight fast, a single 150 mg dose of aliskiren (Rasilez; Novartis, Horsham, UK) with 150 ml water. A standard-ized warm meal was served 4 h after drug administration and a standardized light meal after 7 and 10 h. The participants were under direct medical supervision for 12 h after drug administration. In the aliskiren phase, systolic and diastolic blood pressures were measured, as a safety assessment, from the forearm using an automatic oscillometric blood pressure monitor (Omron M5-I; Omron Healthcare Europe BV, Hoofddorp, the Netherlands), with the participant in a sitting position, before and 2, 4, 7, 9, 12, and 24 h after aliskiren ingestion. No significant differences in blood pressures were observed between the genotype groups (data not shown). The use of all drugs was prohibited during the week before drug administration, as was the use of grapefruit products in the 3 days before drug administration and during the days of blood sampling.

Timed blood samples (5 or 10 ml each) for drug concentration measurements were drawn into tubes containing ethylenediaminetetraacetic acid before and 0.5, 1, 2, 3, 4, 5, 7, 9, 12, and 24 h after montelukast administration, and before and 0.5, 1, 2, 3, 4, 5, 7, 9, 12, 24, 34, 48, and 72 h after aliskiren administration. Blood samples for the determination of plasma renin activity (5 ml each) were drawn before the administration of aliskiren and 4 and 24 h thereafter into chilled ethylenediaminetetraacetic acid tubes, which were placed on ice immediately after sampling. Plasma was separated within 30 min. Urine was collected up to 12 h after aliskiren ingestion. Urine aliquots and plasma were stored at −70°C until analysis.

Determination of drug concentrations and renin activity

Plasma concentrations of montelukast and its M6 metabolite were quantified using a Sciex API 3000 liquid chromatography–tandem mass spectrometry (LC/MS/MS) system (Sciex Division of MDS Inc., Toronto, Ontario, Canada) after protein precipitation (montelukast) or solid-phase extraction (M6) as described earlier 10. Montelukast-d6 served as an internal standard. The lower limit of quantification was 0.2 ng/ml for both montelukast and M6. The between-day coefficient of variation (CV) for montelukast was 6.8% at 0.5 ng/ml, 9.0% at 2.0 ng/ml, 4.8% at 20 ng/ml, and 7.2% at 200 ng/ml and for M6 11.5% at 0.5 ng/ml, 11.8% at 2.0 ng/ml, and 10.9% at 20 ng/ml (n=6–7).

Plasma and urine concentrations of aliskiren were quantified using an AB Sciex API 2000 Q Trap LC/MS/MS system (Sciex Division of MDS Inc.) 8. Acebutolol served as an internal standard. The lower limit of quantification of plasma aliskiren was 0.25 ng/ml, and the between-day CV was 4.5% at 2 ng/ml, 3.8% at 20 ng/ml, and 3.6% at 200 ng/ml (n=10). The lower limit of quantification of aliskiren in urine was 21 ng/ml, and the intra-day CV was 4.0% at 21 ng/ml, 2.3% at 54 ng/ml, 4.2% at 450 ng/ml, and 3.7% at 1350 ng/ml (n=6). Plasma renin activity was determined by a radioimmunoassay of generated angiotensin I using a commercially available method (Renctk; DiaSorin, Saluggia, Italy) at Medix Laboratories (Espoo, Finland).

Pharmacokinetics and pharmacodynamics

Peak plasma concentration (Cmax), time to Cmax (tmax), elimination half-life (t½), and area under the plasma concentration–time curve (AUC) values from 0 to 24 h (AUC0–24) or 0 to 72 h (AUC0–72), as well as from 0 h to infinity (AUC0–∞), were calculated for montelukast and aliskiren by noncompartmental methods using MK-Model, version 5.0 (Biosoft, Cambridge, UK). The terminal log-linear part of each concentration–time curve was identified visually, and the elimination rate constant (ke) was determined from the logarithmically transformed data using linear regression analysis. The t½ was calculated using the equation t½=ln2/ke. The AUC values were calculated using a combination of the linear and log-linear trapezoidal rules with extrapolation to infinity, when appropriate, by division of the last measured concentration by ke. The renal clearance (CLR) of aliskiren was calculated from the amount of aliskiren excreted into urine from 0 to 12 h (Ae) divided by the AUC0–12. Aliskiren pharmacodynamics were characterized by plasma renin activity at 4 and 24 h after aliskiren administration.

Statistical analysis

Results are expressed as geometric means and CV and geometric mean ratios and 90% confidence intervals (CIs), or median with range (tmax). The data were analyzed using the statistical program IBM SPSS Statistics 19 (SPSS Inc., Chicago, Illinois, USA). The Cmax, AUC, and Ae values were adjusted for a body weight of 70 kg. All variables, except tmax, were transformed logarithmically before statistical analysis. Statistical comparisons were made between the SLCO2B1 c.935GG, c.935GA, and c.935AA genotypes using analysis of variance (ANOVA) with a-priori pair-wise comparisons with the Fisher’s least significant difference method. The tmax data were analyzed using the Kruskal–Wallis test with a-priori pair-wise comparisons with the Mann–Whitney U-test. Possible correlations between the AUC0–∞ of montelukast and aliskiren were investigated using the Pearson correlation coefficient. Differences were considered statistically significant when P-value was less than 0.05. It was estimated that the number of patients in each genotype group was sufficient to detect a 50% higher or 33% lower mean AUC0–∞ of aliskiren or montelukast in the SLCO2B1 c.935GA group than in the c.935GG group and a 100% higher or 50% lower AUC0–∞ in the c.935AA group than in the c.935GG group, with a power of at least 80% (α level 5%) 7,10.

Results

The SLCO2B1 c.935G>A genotype had no significant effect on the pharmacokinetics of montelukast, its primary metabolite M6, or aliskiren (Tables 1 and 2, Figs 1 and 2). The geometric mean ratios (90% CI) of montelukast Cmax in participants with the c.935GA or the c.935AA genotype to those with the c.935GG genotype were 1.06 (0.92, 1.23) and 0.92 (0.76, 1.13), respectively (ANOVA P=0.501), and the corresponding geometric mean ratios (90% CI) of montelukast AUC0–∞ were 1.02 (0.87, 1.21) and 0.88 (0.71, 1.10), respectively (ANOVA P=0.557). The weight-adjusted Cmax and AUC0–∞ of montelukast varied 2.6-fold and 3.0-fold, respectively, between individual participants. The geometric mean ratios (90% CI) of the M6 metabolite Cmax in participants with the c.935GA or the c.935AA genotype to those with the c.935GG genotype were 0.99 (0.70, 1.40) and 0.84 (0.53, 1.33), respectively (ANOVA P=0.795), and the corresponding geometric mean ratios (90% CI) of M6 AUC0–∞ were 1.02 (0.71, 1.47) and 0.85 (0.52, 1.40), respectively (ANOVA P=0.830).

Table 1
Table 1:
Pharmacokinetic variables of a single 10 mg oral dose of montelukast in healthy participants with the SLCO2B1 c.935GG (n=16), c.935GA (n=12), or c.935AA (n=5) genotype
Table 2
Table 2:
Pharmacokinetic variables of and plasma renin activity response to a single 150 mg oral dose of aliskiren in healthy participants with the SLCO2B1 c.935GG (n=16), c.935GA (n=12), or c.935AA (n=5) genotype
Fig. 1
Fig. 1:
Geometric mean (90% confidence interval) weight-adjusted plasma concentrations of montelukast after a single 10 mg oral dose of montelukast in healthy participants with the SLCO2B1 c.935GG (n=16, open circles), c.935GA (n=12, solid circles), or c.935AA (n=5, triangles) genotype. The inset shows the same data on a semilogarithmic scale. Some error bars have been omitted for clarity.
Fig. 2
Fig. 2:
Geometric mean (90% confidence interval) weight-adjusted plasma concentrations of aliskiren after a single 150 mg oral dose of aliskiren in healthy participants with the SLCO2B1 c.935GG (n=16, open circles), c.935GA (n=12, solid circles), or c.935AA (n=5, triangles) genotype. The inset shows the same data on a semilogarithmic scale. Some error bars have been omitted for clarity.

The geometric mean ratios (90% CI) of aliskiren Cmax in participants with the c.935GA or c.935AA genotype to those with the c.935GG genotype were 1.11 (0.78, 1.60) and 1.47 (0.91, 2.39), respectively (ANOVA P=0.408), and the corresponding geometric mean ratios (90% CI) of aliskiren AUC0–∞ were 0.98 (0.74, 1.30) and 1.24 (0.85, 1.80), respectively (ANOVA P=0.576). The weight-adjusted Cmax, AUC0–∞, and Ae of aliskiren, and its CLR varied 12.5-fold, 5.6-fold, 9.6-fold, and 4.2-fold, respectively, between individual participants. No differences were found in the renin activity response to aliskiren between the genotypes. The AUC0–∞ values of montelukast and aliskiren showed no significant correlation (r=−0.153, P=0.394; Fig. 3).

Fig. 3
Fig. 3:
Relationship between the AUC0–∞ values of montelukast and aliskiren in healthy participants with the SLCO2B1 c.935GG (n=16, open circles), c.935GA (n=12, solid circles), or c.935AA (n=5, triangles) genotype.

Discussion

In this study, the SLCO2B1 c.935G>A SNP had no effect on the single-dose pharmacokinetics of the OATP2B1 substrates montelukast or aliskiren in healthy volunteers. This is in contrast to two earlier studies, which suggested that the pharmacokinetics of montelukast could be affected by this SNP 2,3. The first of these earlier studies was carried out as part of a clinical trial comparing the efficacy of low-dose theophylline, montelukast, and placebo 2. In that study, the morning plasma concentrations of montelukast following an evening dose (measured to evaluate compliance) were 70–80% lower in 20 patients with the c.935GA genotype than in 60 patients with the c.935GG genotype 2. The compliance of these patients and the timing of the montelukast dose before blood sampling were likely to be variable, because the morning concentrations reported in this study were lower than those observed 12 h after a single 10 mg dose of montelukast in other studies 10. The second study was an interaction study carried out in patients with asthma, with a primary aim to examine the effects of orange juice and grapefruit juice on the pharmacokinetics of montelukast, with Gatorade as a comparator 3. In that study, the AUC0–∞ of montelukast was on average 46% lower in five patients with the c.935GA genotype than in 21 patients with the c.935GG genotype when taken together with Gatorade 3. In both of these studies, genotyping was carried out retrospectively and the studies were not designed to examine the effect of the SLCO2B1 genotype on montelukast pharmacokinetics as a primary aim. Moreover, both studies included patients of different ethnicities (study 1 : 20 African-American, 55 White, 5 Hispanic 2; study 2 : 12 African-American, 2 Asian, 11 White, 1 Hispanic 3), and it cannot be ruled out that population stratification could have yielded false-positive results. Therefore, we found it important to try to replicate the earlier findings in a prospective study. Even though our study also included c.935AA homozygotes, we did not observe any trend for altered montelukast or aliskiren pharmacokinetics in carriers of this SNP.

There appear to be no in-vitro studies on the effects of the SLCO2B1 c.935G>A SNP on OATP2B1 function. All OATP transporters contain 12-transmembrane domains 1, and the p.R312Q amino acid substitution resulting from the c.935G>A SNP is located in the predicted third and largest intracellular loop of OATP2B1, suggesting that it should not affect substrate recognition or binding. Moreover, predictions by four protein function-predicting algorithms (SIFT, Human Protein DB tool, PolyPhen-2, PMut) suggest that the amino acid substitution does not have a deleterious effect on protein function (http://sift.jcvi.org, http://genetics.bwh.harvard.edu/pph2, http://mmb.pcb.ub.es/PMut, http://www.snps3d.org). These predictions are well in agreement with the present in-vivo data and further support the lack of effect of this SNP on OATP2B1 activity.

Other sequence variants have also been described in the SLCO2B1 gene, but their clinical relevance is still uncertain 4,11–13. The SLCO2B1 c.43C>T (p.P15S), c.601G>A (p.V201M), and c.1175C>T (p.T392I) SNPs, as well as a 3-amino acid (26–28) deletion variant (Glu-Asn-Thr) have been associated with a reduced transport activity, and the SLCO2B1 c.1457C>T (p.S486F) SNP with either a reduced or an increased transport activity of the protein in vitro11,12. In addition, the SLCO2B1 g.-282G>A (rs2712807) SNP in the promoter region has been associated with increased OATP2B1 protein expression level in the human liver 13. In vivo in humans, the SLCO2B1 c.1457C>T SNP has been associated with a reduced AUC of fexofenadine and celiprolol 14,15. However, in the previous study of montelukast pharmacogenetics in patients with asthma, the SLCO2B1 c.1457C>T SNP had no effect on the plasma concentrations or effects of montelukast 2. Therefore, further studies with adequate numbers of participants are required to replicate the associations between the c.1457C>T SNP and fexofenadine and celiprolol pharmacokinetics, as well as to examine the effects of other sequence variants on OATP2B1 activity and function in vivo.

The studies identifying montelukast and aliskiren as OATP2B1 substrates have been challenged recently 16,17. In the original study by Mougey et al. 2, the translocation of [3H]montelukast from the basal to the apical side of OATP2B1-expressing MDCKII cell monolayers was shown to be increased by 1.4-fold. However, in studies carried out by researchers from Merck, polarized transport of montelukast could not be found in MDCKII-OATP2B1 monolayers 16. Moreover, no accumulation of montelukast into MDCKII-OATP2B1 cells could be found compared with control MDCKII cells 16. It is therefore possible that the transcellular transport of montelukast in MDCKII-OATP2B1 cell monolayers, observed in the original study 2, was because of transport by an unknown endogenous transporter expressed in MDCKII cells. However, [14C]aliskiren was found to be taken up into OATP2B1-expressing HEK293 cells with a Km of 72 µmol/l 5. In a more recently published study, no uptake of [14C]aliskiren into HEK293-OATP2B1 cells could be found, but this may be because of the low (2.5 or 10 µmol/l) aliskiren concentrations used 17. Taken together, the balance of evidence suggests that montelukast may not be a substrate of OATP2B1 after all, whereas aliskiren seems to be a low-affinity OATP2B1 substrate.

Conclusion

The results of this prospective pharmacokinetic study show that the SLCO2B1 c.935G>A SNP has no clinically significant effect on the pharmacokinetics of montelukast and aliskiren. These findings suggest that the SNP exerts no meaningful effect on OATP2B1 activity in vivo or that OATP2B1 has no, or only a minor, importance in the pharmacokinetics of montelukast and aliskiren.

Acknowledgements

The authors thank Jouko Laitila for montelukast concentration measurements, Kaisa Kurkinen (MSc) for aliskiren concentration measurements, and Eija Mäkinen-Pulli and Lisbet Partanen for skilled assistance.

This study was supported by grants from the Sigrid Jusélius Foundation (Helsinki, Finland).

Conflicts of interest

There are no conflicts of interest.

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Keywords:

aliskiren; montelukast; OATP2B1; pharmacogenetics; pharmacokinetics; SLCO2B1

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