ABCB1 1199G > A Polymorphism Impacts Transport Ability of P-gp-Mediated Antipsychotics

Abstract

The alterations in P-glycoprotein (P-gp)-mediated transport of antipsychotics due to the ABCB1 1199G>A polymorphism were assessed in the current study. The ABCB1 _wt and ABCB1 _1199A recombinant cell models were constructed to study the sensitivity, intracellular accumulation, and transepithelial permeability of antipsychotic drugs. ABCB1 _1199A recombinant cells had more sensitivity to olanzapine (2.2-fold, p < 0.01), aripiprazole (1.8-fold, p < 0.01), amisulpride (2.3-fold, p < 0.01), and risperidone (3.1-fold, p < 0.01) than ABCB1_wt cells, while the resistance to paliperidone in both recombinant cell models was similar. In addition, the uptake quality of olanzapine, aripiprazole, amisulpride, and risperidone in ABCB1 _1199A recombinant cells was greatly decreased compared to ABCB1 _wt cells (3.2-fold, p < 0.01; 3.7-fold, p < 0.01; 3.1-fold, p < 0.01; 2.6-fold, and p < 0.01, respectively). Furthermore, apparent permeability values were greatly increased in ABCB1 _1199A recombinant cells compared with ABCB1 _wt recombinant cells for olanzapine (2.7-fold, p < 0.01), aripiprazole (2.9-fold, p < 0.01), amisulpride (3.4-fold, p < 0.01), and risperidone (4.1-fold, p < 0.01). The influence of ABCB1 1199G>A polymorphism on the transport of P-gp-mediated substrates showed up as drug-specific. Collectively, the ABCB1 1199G>A polymorphism may impact effective antipsychotics concentration in target cells via mediating the agents transport and distribution.

Introduction

The human multidrug resistance gene (ABCB1) encodes P-glycoprotein (P-gp), a transmembrane protein, which mediates ATP-dependent substrate transport. P-gp resides in plasma membrane and mediates the efflux transport for a large numbers of natural compounds and lipophilic xenobiotics (Lin and Yamazaki, 2003). Increasing evidence has demonstrated that there are more than 50 single-nucleotide polymorphisms (SNPs) in ABCB1 gene sequence were found in previous studies, suggesting that 19 SNPs locate in exon region, 18 SNPs in introns, and 11 SNPs in untranslated region (Cao et al., 2015; Ma et al., 2015; Wolking et al., 2015). The genetic mutant of ABCB1 was found to impact the expression level and functional activity of P-gp in clinical and experimental studies, and the most common SNPs of ABCB1 gene (1236C>T, 2677 G>T/A, and 3435 C>T) were also reported in published articles (Li et al., 2014; Keangpraphun et al., 2015; Zhang et al., 2015). Furthermore, a clinical study reported that there is a SNP at position 1199 in ABCB1 gene sequence (Coller et al., 2006). Recent observations have reported that ABCB1 1199G>A polymorphism (rs2229109) has an important influence on the transport of steroid drugs and anticancer agents (Woodahl et al., 2009; Peng et al., 2016), which indicates that 1199A allele can significantly increase the transport ability of P-gp-mediated substrates. An initial research has demonstrated that ABCB1 1199G>A polymorphism changed P-gp-mediated transport of a prototypic substrate, rhodamine 123, as well as HIV protease inhibitor (Green et al., 2005). A recent research has demonstrated that ABCB1 1199G>A polymorphism may be predictive of progression-free survival in patients receiving paclitaxel chemotherapy (Woodahl et al., 2009). It can be rationally hypothesized that the impact of ABCB1 1199G>A polymorphism may differentially influence the affinity and the activity of P-gp in a substrate-dependent manner.

In addition, large numbers of antipsychotic drugs were found to be substrates for P-gp, such as olanzapine, aripiprazole, amisulpride, paliperidone, and risperidone (Schmitt et al., 2006; Kuzman et al., 2011; Holthoewer et al., 2013; Mi et al., 2016). Moreover, these studies have demonstrated that the common genetic polymorphism of ABCB1 was possibly involved in the transport abnormalities of antipsychotics. Therefore, this study investigates the alterations in P-gp-mediated transport of antipsychotics due to the ABCB1 1199G>A polymorphism. In the present research, the sensitivity, uptake quality, and transepithelial permeability of antipsychotic agents were assessed using recombinant cell systems transfected with ABCB1 1199G and 1199A alleles. Transepithelial transport of P-gp-mediated antipsychotics can demonstrate how ABCB1 1199G>A SNP impacts the functional activity of P-gp-mediated antipsychotics transport and guide individualized medicine.

Materials and Methods

Cell culture

LLC-PK1 control and recombinant ABCB1-trasfecting cells were cultured in complete medium consisting of DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic–antimycotic, and cultured in the presence of 5% CO₂ at 37°C.

Generation of stable recombinant systems

Plasmids expressing ABCB1 1199A allele was generated by site-directed mutagenesis using the QuickChange II XL Site-Directed Mutagenesis Kit (Strata-gene) with the mismatched primers 5′-ATTCAGAAATGTTCACTTCAATTACCCATCTCG-3′ (forward) and 5′-TTGAAGTGAAC ATTTCTGAATTCCAAATTTCCC-3′ (reverse). The sequencing of plasmids to confirm the presence of the mutation was performed by Sanger sequencer (GATC Biotech AG). LLC-PK1 cells were transfected with 10 μg of plasmid DNA. Then, cells were replated in six-well plates (1.0 × 10⁵ cells/well) after lentivirus transfection, and 2 μg/mL of puromycin were added to screen the stable recombinant cells. Then, the western blot was performed to verify the overexpression of ABCB1 1199G and 1199A in recombinant systems. Total proteins in all cell models were obtained via decomposing with RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, and 0.1% SDS [Beyotime]). Immune printing strips were incubated with P-gp (1:1000; Santa Cruz Biotechnology, Inc.) and β-actin (1:1000; Absin Bioscience, Inc., Shanghai) antibodies for overnight at 4°C. Then, bands were washed and incubated with HRP-labeled goat anti-rabbit IgG (1:3000; Google Biological Co. LTD, Wuhan) fluorescent secondary antibodies, and analyzed using Chemiluminescence Detection System (Thermo Scientific).

Antipsychotics sensitivity assay

LLC-PK1 control and recombinant cells were cultured in 96-well plates (1.0 × 10⁴ cells/well) for 12 h in the presence of 5% CO₂ at 37°C and treated with varying concentrations of olanzapine (0.05, 0.1, 0.3, 0.6, 0.8 μM), aripiprazole (0.1, 0.2, 0.4, 0.8, 1.0 μM), amisulpride (0.2, 0.4, 0.6, 1.0, 1.4 μM), paliperidone (0.02, 0.06, 0.12, 0.24, 0.48 μM), and risperidone (0.1, 0.2, 0.4, 0.6, 0.8 μM). After incubation for 48 h, the old medium was removed. Next step, we added 100 μL of complete medium containing 10 μL CCK8 reagent (Abcam) into each well and then incubated at 37°C for 1 h. Inhibitory rates were assessed using microplate reader (Molecular Devices, Biobase, Japan) via measuring the absorbance values at 450 nm. Also, 50% cell inhibition (EC₅₀) was determined to evaluate the resistance for these antipsychotics.

Intracellular accumulation

LLC-PK1 control and recombinant cells were cultured in six-well plates (1.0 × 10⁵ cells/well) in the presence of 5% CO₂ at 37°C for 48 h, then olanzapine (0.5 μM), aripiprazole (0.7 μM), amisulpride (0.8 μM), paliperidone (0.24 μM), and risperidone (0.4 μM) were added in serum-free medium after the old medium was removed for 4 h, respectively. After incubation with these drugs, the supernatant was removed by splashing the plate. The cells were collected and lysed in 200 μL of RIPA lysis buffer. The total protein concentration in each well was measured by BCA assay. Then, cellular antipsychotics were extracted by lipid–lipid extraction assay and ethyl acetate (500 μL) as the organic phase. The supernatant was collected completely and was evaporated to dryness using nitrogen concentration (Ember, China). The dry residue was redissolved with ethanol. Cellular antipsychotics concentrations were determined by using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The UPLC-MS/MS equipment consisted of liquid phase system (Ekspert ultraLC 100-XL) and mass spectrometry system (AB SCIEX 4500 QTRAP). In brief, chromatographic separation was carried out on ACQUITY UPLC BEH Shield RP18 (1.7 μm, 2.1 × 50 mm) analytic column (Waters) maintained at 40°C temperature. The mobile phase consisted of (A) 5 mM ammonium formate (pH = 4.0) and (B) a mixture of methanol and acetonitrile (50:50, v/v). The flow rate of the mobile phase under gradient elution conditions (0–1 min: 20% B; 1–3 min: 20–50% B; 3–4 min: 50–90% B; 4–5 min: 90–20% B; 5–6 min: 20% B) was kept at 0.25 mL/min. The autosampler temperature was set at 5°C, and the injection volume was 20 μL. The relative contents were normalized by measuring concentrations of total protein in each well.

Transepithelial permeability assay

LLC-PK1 control and recombinant cells (2 × 10⁴/well) were grown in 24 mm well on permeable supports (Transwell^™, 3.0 μm membrane pore size; Corning) in the presence of 5% CO₂ at 37°C for 72 h. The transepithelial electrical resistance (TEER) values were evaluated to verify the integrity of monolayer cells. To investigate the transepithelial transport of apical-to-basal (A→B), 200 mL of antipsychotics were added in the apical (A) chamber, and only 200 mL of HBSS-GH were added in basal (B) chamber. In basal-to-apical (B→A) efflux, the olanzapine (0.5 μM), aripiprazole (0.7 μM), amisulpride (0.8 μM), paliperidone (0.24 μM), and risperidone (0.4 μM) solutions were added in opposite chamber compared with A→B. After LLC-PK1 cells were treated with antipsychotics in the presence of 5% CO₂ at 37°C for 2 h, 200 mL aliquots were covered from the blank chamber. Inhibition of P-gp transport activity was carried out by addition of 10 nM GF120918 in the HBSS-GH medium to both chambers. Apparent permeability values (P_app) were calculated based on a previous study (Peng et al., 2016). In brief, P_app = (ΔQ/Δt)/(C₀ × A), where ΔQ/Δt is the rate of transport of agent into the receiver chamber, C₀ is the initial level of the test drug, A is the surface area of permeable support. The directional efflux of antipsychotics was assessed by taking the ratio of the P_app(B→A) over the P_app(A→B) values. Samples were run in triplicate.

Statistical analysis

Student's two-sided t-test was performed to evaluate the differences among means. p < 0.05 was regarded as statistically significant.

Results

Overexpression of P-gp in recombinant systems

The expression levels of P-gp were detected by western blot analysis in all cell systems. As displayed in Figure 1, the results showed that the expression levels of P-gp in recombinant ABCB1 1199G-trasfecting cells and 1199A-trasfecting cells were similar and significantly higher than control cells (p < 0.01). As shown in the histogram, the expression levels of P-gp transfected with ABCB1 1199G and 1199A cells were approximately 3.2 times higher than control cells.

FIG. 1.

Observation of P-gp expression in recombinant LLC-PK1 cells. **p < 0.05, compared with control group. P-gp, P-glycoprotein.

ABCB1 1199G>A polymorphism showed different resistance to antipsychotics

As is known to all, olanzapine, aripiprazole, amisulpride, paliperidone, and risperidone were found to be the P-gp-mediated substrates, yet, the impact of ABCB1 polymorphisms on the efficacy of antipsychotics remains to be poorly understood. Therefore, this study assessed the influence of ABCB1 1199G>A mutant on the cytotoxicity of three types of LLC-PK1 cell modes after treatment with different concentrations of antipsychotic drugs, and EC₅₀ values in all types of cells, which were calculated according to a concentration-inhibition ratio response curve (Table 1). These findings indicated that the genotypic effect of ABCB1 1199G>A polymorphism in those three types of LLC-PK1 systems on the resistance to antipsychotics was dissimilar and showed as antipsychotics specific. Our results suggested that both ABCB1 _wt and ABCB1 _1199A recombinant cells are resistance to antipsychotics (all p < 0.05). ABCB1 _1199A recombinant cells were more sensitivity to olanzapine (2.2-fold, p < 0.01), aripiprazole (1.8-fold, p < 0.01), amisulpride (2.3-fold, p < 0.01), and risperidone (3.1-fold, p < 0.01) compared with ABCB1 _wt cells. However, the resistance to paliperidone in both recombinant cell models was found to be similar. These results suggested that ABCB1 1199A mutant can increase the transport activity of P-gp-mediated olanzapine, aripiprazole, amisulpride, and risperidone.

Table 1.

ABCB1 1199G>A Polymorphisms Impact the Resistance to Antipsychotics

	EC₅₀ (nM) ± SD
LLC-PK1 cells	Control	ABCB1_wt	ABCB1_1199A
Olanzapine	52.3 ± 3.9	136.9 ± 6.4^*	298.2 ± 13.4^**##
Aripiprazole	79.5 ± 5.1	159.7 ± 7.6^*	290.6 ± 14.7^**#
Amisulpride	126.3 ± 7.9	276.5 ± 8.9^*	634.5 ± 25.8^**##
Paliperidone	26.8 ± 2.5	89.5 ± 3.7^*	91.4 ± 3.6^*
Risperidone	63.4 ± 4.9	114.2 ± 7.3^*	352.4 ± 12.5^**##

Significant difference among control cells and ABCB1 overexpression cells (either wt or 1199A), ^* p < 0.05, ^** p < 0.01; Significant difference among ABCB1 _wt cells and ABCB1 _1199A cells, ^# p < 0.05, ^## p < 0.01.

ABCB1 1199G>A polymorphism impacted intracellular accumulation of antipsychotics

Intracellular accumulation of antipsychotics in recombinant cell systems was determined to investigate the effects of ABCB1 1199G>A polymorphism on uptake accumulation of these drugs. These findings demonstrated that the accumulation of all antipsychotics in both ABCB1 _wt and ABCB1 _1199A recombinant cell models were significantly decreased compared with control cells (all p < 0.05, Table 2). The uptake quality of olanzapine, aripiprazole, amisulpride, and risperidone in ABCB1 _1199A recombinant cells were significantly decreased compared with ABCB1 _wt cells (3.2-fold, p < 0.01; 3.7-fold, p < 0.01; 3.1-fold, p < 0.01; 2.6-fold, p < 0.01, respectively), suggesting that the transport ability of P-gp, encoded by ABCB1 1199A allele, is greatly increased compared with 1199G allele. However, the intracellular accumulations of paliperidone between ABCB1 _wt and ABCB1 _1199A recombinant cell models were found to be similar.

Table 2.

ABCB1 1199G>A Polymorphisms Affect the Cellular Accumulation of Antipsychotics in Recombinant Cell Models

	Concentration (nM) ± SD
LLC-PK1 cells	Control	ABCB1_wt	ABCB1_1199A
Olanzapine	320.6 ± 12.7	104.5 ± 8.9^*	32.3 ± 3.7^**,##
Aripiprazole	542.6 ± 15.4	235.2 ± 12.6^*	63.5 ± 6.2^**,#
Amisulpride	786.3 ± 21.9	324.5 ± 11.8^*	105.9 ± 8.4^**,##
Paliperidone	185.4 ± 8.7	76.9 ± 4.7^*	81.2 ± 4.3^*
Risperidone	289.6 ± 9.5	123.5 ± 5.3^*	47.6 ± 3.2^**,##

Significant difference among control cells and ABCB1 overexpression cells (either wt or 1199A), ^* p < 0.05, ^** p < 0.01; significant difference among ABCB1 _wt cells and ABCB1 _1199A cells, ^# p < 0.05, ^## p < 0.01.

Transepithelial permeability

The transepithelial bidirectional permeability of antipsychotic drugs across LLC-PK1 monolayers were also investigated in transwell insert-plate unit. As shown in Table 3, the bidirectional transport of all antipsychotics in both ABCB1 _wt and ABCB1 _1199A recombinant cell systems were greatly increased compared with control cells (all p < 0.05), indicating that P-gp overexpression in both recombinant cell models increased antipsychotics efflux. The apparent permeability values (P_app) of antipsychotics were next evaluated in both ABCB1_wt and ABCB1 _1199A recombinant cell systems. Our data suggested that the efflux ratios of paliperidone were unchanged between ABCB1 _wt and ABCB1 _1199A recombinant cells, indicating that 1199A allele dose not impact the efflux ability of paliperidone. However, the P_app values were greatly increased in ABCB1 _1199A recombinant cells compared with ABCB1 _wt recombinant cells for olanzapine (2.7-fold, p < 0.01), aripiprazole (2.9-fold, p < 0.01), amisulpride (3.4-fold, p < 0.01), and risperidone (4.1-fold, p < 0.01), suggesting that genetic mutant of ABCB1 gene can influence the transport ability of certain antipsychotics.

Table 3.

Apparent Permeability Ratios for Antipsychotics in Three Types of LLC-PK1 Cells

	P_appB→A/P_appA→B ± SD
LLC-PK1 cells	Control	ABCB1_wt	ABCB1_1199A
Olanzapine	0.84 ± 0.13	3.42 ± 1.05^*	9.34 ± 1.94^**,##
Olanzapine+GF120918	0.91 ± 0.14	1.04 ± 0.16⁺	0.94 ± 0.11⁺
Aripiprazole	1.34 ± 0.17	6.04 ± 0.24^*	17.56 ± 0.47^**,##
Aripiprazole+GF120918	1.32 ± 0.16	1.52 ± 0.19⁺	1.30 ± 0.17⁺
Amisulpride	1.05 ± 0.11	5.06 ± 0.19^*	17.24 ± 0.51^**,##
Amisulpride+GF120918	0.99 ± 0.10	1.09 ± 0.08⁺	1.17 ± 0.11⁺
Paliperidone	0.67 ± 0.08	2.49 ± 0.16^*	2.68 ± 0.14^*
Paliperidone+GF120918	0.71 ± 0.07	0.84 ± 0.11⁺	0.92 ± 0.13⁺
Risperidone	1.09 ± 0.13	5.27 ± 0.21^*	21.8 ± 0.54^**,##
Risperidone+GF120918	0.94 ± 0.07	1.13 ± 0.14⁺	1.25 ± 0.16⁺

The efflux ratios of antipsychotics in all cell models after treatment with P-gp inhibitor, GF120918 (10 nM), were then investigated. The P_app values of olanzapine, aripiprazole, amisulpride, paliperidone, and risperidone in the presence of P-gp inhibitor were greatly decreased in both recombinant cells. Our results displayed that ABCB1 1199A allele increases the transport ability of olanzapine, aripiprazole, amisulpride, and risperidone, but not paliperidone, by increasing P-gp-mediated activity.

Discussion

To assess the functional role of ABCB1 1199G>A polymorphism in the transport activity of P-gp-mediated antipsychotics, ABCB1 _wt and ABCB1 _1199A recombinant cell systems were constructed based on previous report (Peng et al., 2016). Large numbers of antipsychotic drugs were found to be substrates for P-gp, such as olanzapine, aripiprazole, amisulpride, paliperidone, and risperidone (Schmitt et al., 2006; Kuzman et al., 2011; Holthoewer et al., 2013; Mi et al., 2016), and whether ABCB1 1199G>A polymorphism can have an impact in the transport activity of these antipsychotics, which needs to be further observed. Clinical study has demonstrated that a possible effect of ABCB1 2677G>T and 3435C>T genetic polymorphism based on the development of metabolic abnormalities among female patients supplemented with olanzapine/risperidone was found (Holthoewer et al., 2013). The ABCB1 2677TT/3435TT genotypes were found to have a statistically significant increase of aripiprazole transport compared to patients with other ABCB1 genotypes (Rafaniello et al., 2017), while another study indicated that ABCB1 genotypes seemed unlikely to have an effect (Suzuki et al., 2014). A link between ABCB1 genetic polymorphism and efficacy and adverse reaction to risperidone or paliperidone was found in Han Chinese schizophrenic subjects (Mi et al., 2016). However, the associations between ABCB1 1199G>A polymorphism and the transport activity of these drugs in vitro remain to be poorly understood. Therefore, we used ABCB1 _wt and ABCB1 _1199A recombinant cell models to assess the functional impacts of 1199A allele on sensitivity, cellular accumulation, and net efflux ratios of antipsychotics.

P-gp is highly expressed in the capillary endothelial cells of the blood–brain barrier (BBB), and it suppresses the brain penetration of many agents, including antipsychotics treatment for schizophrenia (Emmert et al., 2014). Extensive observations have demonstrated that many antipsychotics show limited distribution into the central nervous system (CNS), partly due to poor penetration across BBB and excessive removal by P-gp-dependent transport (O'Sullivan et al., 2014; Iseger and Bossong, 2015). ABCB1 genetic polymorphism can change P-gp activity and further impact antipsychotics distribution into the CNS as well as resistance to drugs. Therefore, our study was carried out to assess the influence of ABCB1 1199G>A polymorphism on the efflux ability of P-gp-mediated antipsychotics.

The recombinant cell models were used to assess the genetic differences in cell sensitivity to olanzapine, aripiprazole, amisulpride, paliperidone, and risperidone in LLC-PK1 cells, which transfected with either ABCB1 _wt or ABCB1 _1199A. ABCB1 _1199A recombinant cells have more sensitivity to olanzapine, aripiprazole, amisulpride, and risperidone compared with ABCB1 _wt cells. However, the resistance to paliperidone in both recombinant cell models was found to be similar. These results suggested that ABCB1 1199A mutant can increase the transport activity of P-gp-mediated olanzapine, aripiprazole, amisulpride, and risperidone. The resistance may be due to reduced intracellular accumulation and further decreased cytotoxicity. Therefore, the intracellular accumulations of these antipsychotics were also observed in the present study. The uptake quality of olanzapine, aripiprazole, amisulpride, and risperidone in ABCB1 _1199A recombinant cells were significantly decreased compared with ABCB1 _wt cells, while the intracellular accumulations of paliperidone between ABCB1 _wt and ABCB1 _1199A recombinant cell models were found to be similar.

We then evaluated the effect of ABCB1 1199G>A polymorphism on P-gp-mediated directional transepithelial transport in our recombinant cell models and indicated that variations in transepithelial transport correspond to the changes investigated in above studies. The efflux ratios of paliperidone were unchanged between ABCB1 _wt and ABCB1 _1199A recombinant cells, while the P_app values were greatly increased in ABCB1 _1199A recombinant cells compared with ABCB1 _wt recombinant cells for olanzapine, aripiprazole, amisulpride, and risperidone, suggesting that genetic mutant of ABCB1 gene can influence the transport ability of certain antipsychotics. ABCB11199A variant allele greatly increased the activity of P-gp to drive efflux of these antipsychotics across cellular membranes. The cells transfected with ABCB11199A variant allele greatly increased resistance to olanzapine, aripiprazole, amisulpride, and risperidone, while decreased intracellular content of these antipsychotics in both cell models.

Conclusions

We found that ABCB1 1199G>A polymorphism changes cellular sensitivity, intracellular accumulation, and transepithelial transport of antipsychotics in our recombinant cell models. However, the occurrence of 1199A allele appears to be drug specific. Further phenotype-genotype correlation studies are needed to verify our findings. Collectively, our results may help to optimize the drug dosage of antipsychotics in clinical.

Footnotes

Acknowledgments

The authors thank the Central Laboratory of Renmin Hospital for providing all laboratory equipment.

Disclosure Statement

No competing financial interests exist.

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