Atorvastatin (ATV) was purchased from Pfizer (New York, NY) and the d5-ATV calcium salt from Alsachim (CAS number: 222412-82-0). Gentamicin (G418), hygromycin B, dimethyl sulfoxide (DMSO), ampicillin and kanamycin were purchased from Sigma-Aldrich (St. Louis, MO).
Human Embryonic Kidney cells (HEK 293, ATCC N°CRL-1573) control and transfected cells were grown in Dulbecco’s Modified Eagle Medium with high glucose, L-glutamine and sodium pyruvate (DMEM GIBCO®) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) of penicillin-streptomycin purchased from ThermoFisher Scientific (Waltham, MA). Cells were cultured at 37°C with 5% CO2. For re-culturing, cells were detached with enzyme-free cell dissociation buffer (ThermoFisher Scientific).
Generation of SLCO2B1935G and SLCO2B1935A plasmids
The expression vector pCMV-V-OFPSpark® coding for the wild-type OATP2B1 protein (SLCO2B1935G) and a fluorescent red tag (OFPSpark®) was purchased from Bio-Connect life sciences (cat. HG18756-ACR, Huissen, The Netherlands). The construct is resistant to bacterial (kanamycin) and eukaryotic (hygromycin B) antibiotics, allowing further bacterial and cell selection. The variant plasmid expressing SLCO2B1935A (rs12422149), was generated by site-directed mutagenesis directed by PCR (Icycler IQ - BioRad, Hercules, CA) using Phusion Site-Directed Mutagenesis kit (Waltham, MA) following the manufacturer’s protocol. The back-to-tail primers phosphorylated in 5’-OH that include the point mutation were 5’-CAG TTT CGG CAA AAG GTC TTA G-3’ (forward) and 5’-AAG CTC ACG TTT TTC CTT G-3’ (reverse) and were purchased from Eurofins Scientific SE (Ebersberg, Germany). The plasmid transformation and amplification were performed in dH5α Escherichia Coli strains through Heat-Shock. The product was then purified by maxiprep using the PureLink™ HiPure Plamid Filter Maxiprep (ThermoFisher Scientific, Waltham, MA). The amplicon was finally sequenced using Sanger method (Eurofins, Germany) to confirm the introduction of the point mutation c.935G>A in the coding nucleotide sequence of SLCO2B1 (Fig. 6a, b).
Generation of ABCC12012G and ABCC12012T plasmids
The expression vector pcDNA3.1-eGFP was kindly offered by Dr. Susan Cole (PMID: 24080162), and codes for the wild-type MRP1 protein (ABCC12012G) with a green fluorescent tag (GFP). The construct is resistant to bacterial (ampicillin) and eukaryotic (gentamicin) antibiotics, allowing further bacterial and cell selection. As for SLCO2B1, the variant plasmid expressing ABCC12012T (rs45511401) was generated by site-directed mutagenesis directed by PCR. The back-to-tail primers phosphorylated in 5’-OH that include the point mutation were 5’-ATC CCC GAA GTT GCT TTG GTG-3’ (forward) and 5’-GGA GAA GGT GAT GCC ATT C-3’ (reverse). The product was amplified and purified in the same conditions as previously described and the variant was successfully introduced in the coding sequence of ABCC1. (Fig. 6c, d)
Generation of stable recombinant cell lines
For single transfectants overexpressing either the wild-type (WT) or the variant (var) OATP2B1 protein, i.e. HEKOATP2B1WT and HEKOATP2B1var, 5 x 105 HEK293 cells/well were seeded in 12-well plates the day before the transfection with lipofectamine 3000 Transfection Kit (Invitrogen, Carlsbad, CA) with 1.25µg of plasmid DNA (either SLCO2B1935G or SLCO2B1935A plasmids). The same method was used to obtain HEKMRP1WT and HEKMRP1var single transfectants. For the double transfectants, 1 x 106 cells already overexpressing one of the two proteins (either HEKOATP2B1WT or HEKOATP2B1var) were cultured in 6-well plates one day before the lipofection. Transfection was then achieved using 2.5µg of plasmid DNA per well (either ABCC12012G or ABCC12012T plasmids). The antibiotic selection was performed 72h post-lipofection with hygromycin B and G418 at a final concentration of 0.5 mg/ml and 1mg/ml to select cells that stably expressed OATP2B1 and/or MRP1 proteins, respectively. Resistant strains were reseeded after 14 days for future development and sorted by flow cytometry.
Double transfectant models will be renamed thereafter with the model overexpressing both WT transporters being HEKOATP2B1WT-MRP1WT, the model overexpressing SLCO2B1 WT and ABCC1 variant; HEKOATP2B1WT-MRP1var, and the model overexpressing SLCO2B1 variant and ABCC1 WT; HEKOATP2B1var-MRP1WT. According to the relatively low frequency of the polymorphisms in the population, the impact of both mutations in the double transfectant model HEKOATP2B1var-MRP1var was not investigated (theorical frequency of the combination is 0.66%).
Characterization of OATP2B1 and MRP1 protein expression
Cells were detached, counted and centrifuged at 400g for 5 minutes at 4°C on the day of the experiment. The supernatant was discarded and the cell pellet resuspended in 2mL of sterile FACS-Buffer (Dulbecco’s PBS, 1% (v/v) decomplemented FBS, 1mM EDTA [ThermoFischer Scientific]). Cells were washed twice then resuspended either in FACS-buffer for analysis with BD-FACS Verse or sorting with BD FACSAria® III (BD Biosciences, New Jersey). OFPSpark® (red OATP2B1) and eGFP (green MRP1) fluorescence were excited with the blue laser (488nm) with bandpass filters that were BP 586/42 and 527/32 respectively. Raw data were analyzed using the FlowJo software (Ashland, OR). Since fluorescent OFPSpark® and eGFP emission spectra partially overlap, data were compensated through FlowJo.
All generated recombinant models were sorted with fluorescence parameters gated on the same level of intensity to ensure similar protein expression. Sorted cells were reseeded in 12-well plates with the adequate selective antibiotic concentrations to allow growth expansion.
Inverted fluorescence microscope
Cells were seeded in Ibidi 4 chambers pH+ µ-slide (Gräfelfing, Germany) 48h prior to microscopy analysis using Zeiss Observer Z1 (Zeiss, Germany). Fluorescent signal was obtained using Zeiss Colibri 7 LED light source. The red fluorescence (OFPSpark®) emitted by the tag attached to OATP2B1 was observed with the yellow LED at an excitation of 555nm and an emission QBP 582/30. The green fluorescence (eGFP) emitted by the tag attached to MRP1 was observed with the blue LED at an excitation of 475nm and an emission QBP 514/30. Images were taken with Zeiss Axiocam 506 mono and processed with Zeiss Zen 2 lite software.
Characterization of OATP2B1 and MRP1 activity
Atorvastatin intracellular accumulation and efflux kinetics
The day before the accumulation, cells (3 x 105) were seeded in 24-well plates coated with poly-L-lysine (Sigma-Aldrich), reaching a confluence of ~90% the next day. The day of the experiment, 50µL of each well were removed before the addition of 50µL of freshly diluted ATV solution. For drug accumulation, cells were incubated for 120 minutes with increasing ATV concentrations (0, 50, 75, 100, 150 and 500nM) whereas a concentration of 75nM was selected for the efflux kinetics. All experiments were performed at least twice (N=2) and in triplicate (n=3). After ATV incubation at 37°C with 5% of CO2 saturation, plates were centrifuged (Eppendorf 417R) at 400g for 5 minutes at 4°C and the supernatant was discarded. Cells were washed three times with 500µL of ice-cold D-PBS and either recovered for accumulation experiments or allowed to efflux in a drug-free medium for 30, 60, 120, 300 minutes. Cells were finally resuspended in 500µL of D-PBS supplemented with 1mM EDTA, transferred to a 1.5ml Eppendorf® (Hamburg, Germany) and centrifuged at 400g for 5 minutes at 4°C. The supernatant was finally discarded and the cell pellet stored at -20°C for further extraction. For efflux kinetics, aliquots of media (500µl) were collected at each time point to quantify the fraction of drug effluxed as well as the cell pellet collected with the same method previously described. The efflux ratio was calculated by normalizing the amount of effluxed ATV in the medium at the time points (30, 60, 120 or 300 min) on the intracellular amount accumulated at the start of the efflux period (i.e. after 2h of accumulation).
Atorvastatin extraction and quantification
Thawed cell pellets were resuspended in 100µL of extraction solvent constituted of a mix of methanol-water (60:40, v/v) and d5-ATV at a final concentration of 5nM. Samples were vortexed then sonicated for 10 minutes and centrifuged during 5 minutes at 10,600g at room temperature. The cell pellets were stored at -20°C for protein assay and supernatants were transferred to an injection vial and dried under nitrogen flow. The residue was resuspended in 30µL of methanol before injection in the HPLC-MS/MS system. For ATV media quantification, 500µl of media were added to 1.2mL of ice-cold acetone supplemented with d5-ATV at a final concentration of 5nM. The mix was vigorously vortexed and stored at -20°C for 2h and then centrifuged at 10,600g for 10 minutes. The supernatant was subsequently evaporated and resuspended in MeOH. 5µL of each extract was injected in the HPLC-MS/MS system (Xevo TQ-S, Waters). Analyte separation was obtained using a gradient between mobile phase A (MeOH-H2O (75:25) with 0.1% (v/v) of acetic acid) and mobile phase B (MeOH with 0.1% (v/v) of acetic acid) and a UPLC column (Ascentis Express C18 column (2.7µm, 150x4mm)) equipped with a pre-column. For MS/MS detection, ionized compounds were generated using an electrospray source operated in positive mode. Detection was performed by Multiple Reaction Monitorinig (MRM) mode using the quantification transitions 564.3 à 440.3 for d5-ATV and 559.2 à 440.2 for ATV. Calibration curves were established between 0.5 and 1000 fmol of ATV on column. Data analysis was performed on MassLynx software (Waters). ATV concentrations were normalized on the total protein content.
Protein normalization and statistical analysis
Protein assay was performed in triplicate for each sample with thawed cell pellet using DC™ Protein Assay (BioRad, Hercules, CA).
Graphs and statistical analysis were realized with GraphPad Prism 8.4.2. To assess transporters impact on ATV intracellular accumulation, ANOVA-2 ways was used to evaluate the impact of 2 independent factors, i.e. ATV incubation concentrations (accumulation experiment) or efflux time (efflux kinetics) and the cell lines. Bonferroni post-hoc tests were performed for multiple comparisons in order to determine which mean differed from the others. In all cases, results were considered significant when p-value was < 0.05.