SUPPLEMENTAL DIGITAL CONTENT METHODS
In Vitro Metformin Transport Studies
Effect of Dolutegravir on Metformin Transport by MATE1 and MATE2-K
HEK293 cells transfected with human MATE1, MATE2-K, or vector control were established by Sekisui Medical Co., Ltd. (Naka-gun, Ibaraki, Japan). Cells were cultured in 75-cm2 bottom flasks and subjected to passage every 3 to 4 days. The culture medium was exchanged every 4 days. MATE1 cells were seeded in collagen I-coated 24-well plates at a density of 2.5×105 cells/well and incubated (37°C, 5% CO2) for 2 days. For MATE2-K, HEK293 cells were seeded in collagen I-coated 12-well plates at a density of 3.5×105 cells/well and incubated (37°C, 5% CO2) for 1 day; cells were then transfected with MATE2-K cDNA and incubated for an additional 2 days.
Uptake experiments were initiated by pre-incubation (15 minutes, 37°C) with Gibco® Hank’s Balanced Salt Solution (HBSS; pH 8.5 to invert MATE transport directionality inward) containing dolutegravir or cimetidine (0-100 µM). After pre-incubation, cells were incubated (5 minutes, 37°C) with HBSS (pH 8.5) solutions containing 10 µM [14C]metformin, dolutegravir, or cimetidine (0-100 µM).
Cellular uptake of [14C]metformin was quantified by radiometric detection; protein content was determined by Pierce™ BCA Protein Assay Kit per the manufacturer’s instructions.
Effect of Dolutegravir on Metformin Transport by PMAT and OCT3
MDCK-II cells transfected with human PMAT, OCT3, or vector control were established by Optivia Biotechnology, Inc (Menlo Park, CA). MDCK-II cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose, low sodium bicarbonate, and 10% fetal bovine serum (FBS).
Cells between passage 7 and 30 were seeded at a density of 6±1×104 cells/well on 96-well transwell membrane plates 1 day before transfection; transport assays were carried out following 2-day incubation (37°C, 5% CO2) after transfection.
Uptake experiments were initiated by pre-incubation in both apical and basolateral chambers (15 minutes, 37°C, orbital shaking at 60 rpm) with HBSS containing inhibitors dolutegravir (0-100 µM), quinidine (1 mM), decynium-22 (100 µM), or verapamil (0-100 µM). After pre-incubation, cells were incubated in the basolateral chambers (5 minutes, 37°C, 60 rpm) with HBSS solutions containing
inhibitors and probe substrates: 10 µM [14C]metformin for both PMAT and OCT3; 10 µM [14C]1-methyl- 4-phenylpyridinium (MPP+) for PMAT, 2 µM [14C]MPP+ for OCT3, and 100 µM
[14C]tetraethylammonium for OCT3. Cellular uptake of probe substrate was determined by radiometric detection.
Effect of Dolutegravir on Paracellular Permeability and Cellular Uptake of Metformin
Caco-2 cells (wild type C2BBe1, ATCC catalog # CRL-2102) were received from Sigma- Aldrich, Inc (St. Louis, MO) as a high throughput assay ready plate on day 16 or 17 of culture. Plates were Millicell® 24-cell culture multi-well inserts with polycarbonate membranes, 0.4 µm pore size, and 0.7 cm2 surface area. Pre-seeded plates were unpacked and processed according to the vendor’s
instructions. Briefly, plates were left at room temperature for 24 to 48 hours then elevated to 37°C, 5%
CO2 in a cell culture incubator for 4 hours to liquefy the shipping medium and to allow a culture medium change (DMEM, 20% FBS, supplemented with 2 mM L-glutamine). Following the initial media change, plates were incubated at 37°C with 5% CO2 and culture medium was replaced every 2 to 3 days. Plates were used prior to day 25 of culture as recommended by the vendor.
Effect of dolutegravir on Lucifer yellow paracellular flux: basolateral and apical chamber incubation solutions were transport medium (DMEM supplemented with L-glutamine, 25 mM HEPES, pyridoxine HCl, but without sodium pyruvate and phenol red, pH 7.4) containing dolutegravir (0.3-100 µM) for the first 15 minutes. For the subsequent 90 minutes, the apical incubation solutions were transport medium containing dolutegravir (0.3-100 µM) and 100 µM Lucifer yellow. In a separate set of experiments, basolateral-to-apical flux of Lucifer yellow was determined in confluent MDCKII cell monolayers in the absence or presence of dolutegravir (0.3-100 µM).
Effect of dolutegravir absorptive (apical-to-basolateral) flux of [14C]metformin: basolateral and apical chamber incubation solutions were transport medium containing dolutegravir (0.3-100 µM) for the first 15 minutes. For the subsequent 90 minutes, the apical incubation solutions were transport medium containing dolutegravir (0.3-100 µM) and [14C]metformin (0.05-5 mM).
Effect of dolutegravir on the cellular uptake of [14C]metformin: basolateral and apical chamber incubation solutions were transport medium containing dolutegravir (0.3-100 µM) for the first 15 minutes. Subsequently, the apical incubation solutions were transport medium containing dolutegravir (0.3-100 µM) and [14C]metformin (0.05-5 mM). Cell monolayers were incubated at 37°C with shaking in the time-linear range for metformin uptake (10 minutes at 0.05 mM, 2.5 minutes at 0.5 and 5 mM).
Lucifer yellow concentrations were measured by fluorescence spectrophotometry. [14C]metformin was quantified by radiometric detection; protein content was determined by Thermo Scientific™ Pierce™
BCA Protein Assay Kit per the manufacturer’s instructions.
SUPPLEMENTAL DIGITAL CONTENT RESULTS
In Vitro Metformin Transport Studies
Effect of Dolutegravir on Metformin Transport by MATE1 and MATE2-K
Dolutegravir (GSK1349572) was not a clinically relevant inhibitor of [14C]metformin transport by MATE1 (IC50 = 6.3±1.1 μM) and MATE2-K (IC50 = 24.8±4.7 μM) (Supplemental Figure 1). The positive control MATE inhibitor, cimetidine, inhibited MATE1- and MATE2-K-mediated [14C]metformin
transport with IC50 values of 2.8±0.5 and 3.6±0.6 μM, respectively (Supplemental Figure 2). Cell viability in control, MATE1, and MATE2-K-expressing HEK293 cells in the presence of dolutegravir ranged from 99% to 111% of vehicle control for up to 100 μM dolutegravir (data not shown).
Effect of Dolutegravir on Metformin Transport by PMAT and OCT3
Dolutegravir (≤100 μM) did not inhibit [14C]MPP+ uptake by PMAT or OCT3 (Supplemental Figure 3). Likewise, dolutegravir (≤100 μM) did not inhibit [14C]metformin uptake by PMAT, and it did not inhibit OCT3 at concentrations ≤10 μM, while 10% to 25% inhibition of [14C]metformin transport by OCT3 was noted at 30 to 100 μM dolutegravir concentrations (Supplemental Figure 4). Positive control inhibitors, 100 μM decynium-22 for PMAT and 1 mM quinidine for OCT3, inhibited PMAT and OCT3 transport activities by 78% to 88% and 92% to 95%, respectively. In addition, the positive control OCT3 inhibitor, verapamil, inhibited transport of 100 μM [14C]tetraethylammonium via OCT3, with an IC50 of 2.94 µM, and 95.8% inhibition at 100 µM verapamil (data not shown).
Effect of Dolutegravir on Paracellular Permeability and Cellular Uptake of Metformin
Dolutegravir (0.3-100 µM) did not increase the paracellular permeability of Lucifer yellow in a concentration-dependent manner in Caco-2 cell monolayers, with mean absorptive (apical-to-basolateral) permeability of 7.5 nm/sec and 14.2 nm/sec at 0.3 µM and 1 to 100 µM dolutegravir, respectively.
Likewise, in MDCKII cell monolayers Lucifer yellow basolateral-to-apical Lucifer yellow permeability was comparable in the absence and presence of (0.3-100 µM) dolutegravir (11.1±5.0 vs 9.4±4.2 nm/sec, respectively). Finally, dolutegravir (≤100 µM) did not increase the absorptive (apical-to-basolateral) flux
of metformin in Caco-2 cell monolayers (Supplemental Table 1). Dolutegravir (0.3-100 µM) did not affect the cellular uptake of metformin (0.05, 0.5, 5 mM) into Caco-2 monolayers (Supplemental Table 2).
Supplemental Table 1. Effect of Dolutegravir (GSK1349572) on [
14C]Metformin Flux in Caco-2 Cell Monolayers
Metformin Concentration
GSK1349572 Concentration (μM)
Rate A→B (pmoles/min/cm2)
A→B Mass Balance (%)
0.05 mM [14C]metformin 0 1.3±0.21 98±0.041
0.3 35±27 105±0.060
1 2.1±0.87 98±0.010
3 5.9±5.8 99±0.027
10 34±30 103±0.059
30 16±15 101±0.036
100 13±9.6 97±0.015
0.5 mM [14C]metformin 0 17±3.6 102±0.036
0.3 15±0.34 101±0.0060
1 20±2.2 101±0.022
3 353±365 107±0.042
10 18±0.83 102±0.028
30 17.6±2.96 99.3±0.024
100 27.9±19.8 96.2±0.025
5 mM [14C]metformin 0 136±11.3 99.7±0.018
0.3 150±42.0 98.3±0.015
1 142±16.1 98.4±0.019
3 211±120 99.1±0.017
10 150±24.2 99.2±0.032
30 469±504 97.8±0.050
100 1709±1265 99.6±0.025
Supplemental Table 2. Effect of Dolutegravir (GSK1349572) on [
14C]Metformin Cellular Uptake in Caco-2 Cell Monolayers.
GSK1349572 (μM)
0.05 mM [14C]Metformin
0.5 mM [14C]Metformin
5 mM [14C]Metformin (pmol/μg
protein) SD
(pmol/μg
protein) SD
(pmol/μg
protein) SD
0 17 5.2 14 11 11 4.3
0.3 11 5.1 10 2.3 6.9 1.4
1 9.6 4.9 9.9 3.1 8.5 3.4
3 11 3.2 6.7 1.9 8.7 0.27
10 21 0.94 14 1.1 6.7 1.7
30 17 8.2 25 15 6.7 1.7
100 16 7.8 22 19 8.2 1.3
SD, standard deviation.
Data are average of samples from triplicate wells.
Each bar represents the mean ± SD of three samples.
0 5 10 15 20 25 30
0 0.1 0.3 1 3 10 30 50 100
Concentration of GSK1349572 (µM) Control cells
MATE1-expressing cells
Cleared volume (µL/mg protein)
Inhibitory effect of GSK1349572 on MATE1-mediated transport of metformin IC50value for the inhibition by GSK1349572 on MATE1-mediated transport of metformin
Concentration of GSK1349572 (µM) Slope factor: 0.830
[14C]Metformin GSK1349572 0 1.98 ± 0.24 24.6 ± 1.4 100.0 6.34 ± 1.11
(10 µM) 0.1 1.85 ± 0.15 24.4 ± 0.9 99.7
0.3 2.12 ± 0.48 24.4 ± 1.4 98.5
1 1.73 ± 0.16 20.5 ± 0.1 83.0
3 1.70 ± 0.30 17.8 ± 0.8 71.2
10 1.35 ± 0.15 8.91 ± 0.53 33.4
30 1.10 ± 0.08 6.67 ± 0.41 24.6
50 0.874 ± 0.049 5.21 ± 0.38 19.2
100 0.981 ± 0.084 3.50 ± 0.18 11.1
Each value represents the mean ± SD of three samples.
The IC50 value represents the mean ± SE.
Substrate
(concentration) Test compound Concentration (µM)
Cleared volume (µL/mg protein)
% of control IC50
Control cells MATE1-expressing cells
Supplemental Figure 1. Effect of dolutegravir (GSK1349572) on metformin transport by MATE1 and MATE2-K.
Each bar represents the mean ± SD of three samples.
0 5 10 15
0 0.1 0.3 1 3 10 30 50 100
Concentration of GSK1349572 (µM) Control cells
MATE2-K-expressing cells
Cleared volume (µL/mg protein)
Inhibitory effect of GSK1349572 on MATE2-K-mediated transport of metformin IC50value for the inhibition by GSK1349572 on MATE2-K-mediated transport of metformin
Concentration of GSK1349572 (µM) Slope factor: 0.917
[14C]Metformin GSK1349572 0 1.30 ± 0.11 9.82 ± 0.94 100.0 24.8 ± 4.7
(10 µM) 0.1 1.19 ± 0.11 10.2 ± 0.2 105.8
0.3 1.05 ± 0.06 10.0 ± 0.5 105.0
1 0.952 ± 0.083 10.2 ± 0.7 108.5
3 1.15 ± 0.06 8.89 ± 0.44 90.8
10 0.941 ± 0.030 7.00 ± 1.00 71.1
30 0.862 ± 0.055 5.46 ± 0.19 54.0
50 0.683 ± 0.009 3.03 ± 0.08 27.5
100 0.683 ± 0.006 3.19 ± 0.36 29.4
Each value represents the mean ± SD of three samples.
The IC50 value represents the mean ± SE.
Substrate
(concentration) Test compound Concentration (µM)
Cleared volume (µL/mg protein)
% of control IC50
Control cells MATE2-K-expressing cells
Each bar represents the mean ± SD of three samples.
0 5 10 15 20 25 30
0 0.1 0.3 1 3 10 30 50 100
Concentration of cimetidine (µM) Control cells
MATE1-expressing cells
Cleared volume (µL/mg protein)
Inhibitory effect of cimetidine on MATE1-mediated transport of metformin IC50value for the inhibition by cimetidine on MATE1-mediated transport of metformin
Concentration of cimetidine (µM) Slope factor: 1.08
[14C]Metformin Cimetidine 0 1.98 ± 0.24 24.6 ± 1.4 100.0 2.82 ± 0.46
(10 µM) 0.1 1.99 ± 0.28 25.6 ± 3.3 104.4
0.3 1.70 ± 0.19 25.8 ± 1.1 106.5
1 1.67 ± 0.03 18.4 ± 2.3 74.0
3 1.19 ± 0.09 12.9 ± 0.3 51.8
10 1.09 ± 0.06 5.81 ± 0.27 20.9
30 0.953 ± 0.038 3.33 ± 1.01 10.5
50 0.975 ± 0.150 2.15 ± 0.06 5.2
100 1.10 ± 0.07 2.25 ± 0.09 5.1
Each value represents the mean ± SD of three samples.
The IC50 value represents the mean ± SE.
Substrate
(concentration) Test compound Concentration (µM)
Cleared volume (µL/mg protein)
% of control IC50
Control cells MATE1-expressing cells
Supplemental Figure 2. Effect of cimetidine on metformin transport by MATE1 and MATE2-K.
Each bar represents the mean ± SD of three samples.
0 5 10 15
0 0.1 0.3 1 3 10 30 50 100
Concentration of cimetidine (µM) Control cells
MATE2-K-expressing cells
Cleared volume (µL/mg protein)
Inhibitory effect of cimetidine on MATE2-K-mediated transport of metformin IC50value for the inhibition by cimetidine on MATE2-K-mediated transport of metformin
Concentration of cimetidine (µM) Slope factor: 0.689
[14C]Metformin Cimetidine 0 1.30 ± 0.11 9.82 ± 0.94 100.0 3.65 ± 0.59
(10 µM) 0.1 1.08 ± 0.05 8.77 ± 0.86 90.3
0.3 1.13 ± 0.06 8.15 ± 0.38 82.4
1 0.866 ± 0.039 7.31 ± 0.41 75.6
3 0.818 ± 0.078 5.29 ± 0.54 52.5
10 0.921 ± 0.083 3.35 ± 0.22 28.5
30 0.714 ± 0.117 2.33 ± 0.57 19.0
50 0.681 ± 0.036 1.96 ± 0.16 15.0
100 0.653 ± 0.071 1.77 ± 0.15 13.1
Each value represents the mean ± SD of three samples.
The IC50 value represents the mean ± SE.
Substrate
(concentration) Test compound Concentration (µM)
Cleared volume (µL/mg protein)
% of control IC50
Control cells MATE2-K-expressing cells
GSK1349572 (µM)
Percent of Control (%)
0.1 1 10 100
0 20 40 60 80 100 120
Inhibition of 2M MPP+ uptake mediated by OCT3
2 µM MPP+ uptake by OCT3:
GSK 1349572 (µM)
Cellular Accumulation
(transporter) (pmol/min/cm2)
Cellular Accumulation (control) (pmol/min/cm2)
Net Transporter Mediated Cellular
Accumulation (pmol/min/cm2)
Inhibition (%)
0 8.50 ± 1.20 0.375 0.0590 8.13 1.20 0.00 14.8 0.1 uM 8.14 0.950 0.343 0.0804 7.79 0.950 4.14 11.7 0.3 uM 7.86 0.986 0.311 0.0335 7.55 0.986 7.13 12.1 0.5 uM 7.47 0.918 0.288 0.0255 7.19 0.918 11.6 11.3 1 uM 7.39 0.211 0.281 0.0442 7.11 0.211 12.5 2.60 3 uM 7.37 0.782 0.347 0.0585 7.03 0.782 13.6 9.62 5 uM 8.19 0.857 0.384 0.0274 7.81 0.857 3.96 10.5 10 uM 8.46 0.781 0.396 0.0374 8.06 0.781 0.849 9.61 30 uM 8.56 0.620 0.330 0.0274 8.23 0.620 -1.20 7.63 50 uM 7.95 0.783 0.349 0.0601 7.60 0.783 6.56 9.63 100 uM 7.87 0.276 0.329 0.0731 7.54 0.276 7.27 3.40 1mM Quinidine 0.745 0.0304 0.342 0.0551 0.403 0.0304 95.0 0.374
10 µM MPP+ uptake by PMAT:
GSK 1349572 (µM)
Cellular Accumulation
(transporter) (pmol/min/cm2)
Cellular Accumulation (control) (pmol/min/cm2)
Net Transporter Mediated Cellular
Accumulation (pmol/min/cm2)
Inhibition (%)
0 12.9 ± 0.173 0.789 0.0972 12.2 0.173 0.00 1.42 0.1 13.3 0.402 0.712 0.0590 12.6 0.402 -3.65 3.30 0.3 12.5 1.49 0.763 0.0972 11.8 1.49 3.12 12.2 0.5 13.8 1.01 0.680 0.102 13.2 1.01 -8.24 8.27 1 14.0 0.381 0.974 0.0675 13.0 0.381 -7.12 3.14 3 14.2 0.492 0.842 0.111 13.4 0.492 -10.0 4.04 5 13.2 1.25 0.923 0.0359 12.3 1.25 -1.18 10.3 10 13.6 0.946 0.876 0.0903 12.7 0.946 -4.75 7.78 30 13.7 0.332 0.756 0.104 13.0 0.332 -6.78 2.73 50 12.9 0.488 0.685 0.110 12.2 0.488 -0.475 4.01 100 13.2 0.686 0.712 0.0866 12.5 0.686 -2.91 5.65 100 µM Decynium-22 3.06 0.310 0.449 0.105 2.61 0.310 78.5 2.55
Supplemental Figure 3. Effect of dolutegravir (GSK1349572) on MPP+ transport by PMAT and OCT3.
GSK1349572 (µM)
Percent of Control (%)
0.1 1 10 100
0 20 40 60 80 100 120
Inhibition of 10 µM Metformin uptake mediated by OCT3
10 µM Metformin uptake by PMAT:
GSK 1349572 (µM)
Cellular Accumulation
(transporter) (pmol/min/cm2)
Cellular Accumulation (control) (pmol/min/cm2)
Net Transporter Mediated Cellular
Accumulation (pmol/min/cm2)
Inhibition (%)
0 2.87 ± 0.0976 0.265 0.0357 2.61 0.0976 0.00 3.74 0.1 2.73 0.00689 0.293 0.0655 2.44 0.00689 6.45 0.264 0.3 3.04 0.183 0.317 0.0855 2.72 0.183 -4.35 7.03 0.5 3.20 0.107 0.234 0.0371 2.97 0.107 -13.9 4.12 1 3.25 0.225 0.199 0.0371 3.05 0.225 -17.1 8.61 3 3.27 0.225 0.214 0.00852 3.05 0.225 -17.1 8.62 5 2.75 0.286 0.374 0.158 2.38 0.286 8.87 11.0 10 2.68 0.231 0.402 0.101 2.28 0.231 12.6 8.85 30 2.75 0.195 0.376 0.0100 2.37 0.195 8.93 7.47 50 2.72 0.281 0.239 0.0746 2.49 0.281 4.67 10.8 100 2.57 0.321 0.220 0.0385 2.35 0.321 9.81 12.3 100 µM Decynium-22 0.529 0.0264 0.229 0.0588 0.299 0.0264 88.5 1.01 10 µM Metformin uptake by OCT3:
GSK 1349572 (µM)
Cellular Accumulation (transporter) (pmol/min/cm2)
Cellular Accumulation (control) (pmol/min/cm2)
Net Transporter Mediated Cellular
Accumulation (pmol/min/cm2)
Inhibition (%)
0 1.17 ± 0.0591 0.250 0.0295 0.923 0.0591 0.00 6.41 0.1 1.10 0.166 0.241 0.0142 0.858 0.166 7.03 18.0 0.3 1.20 0.0810 0.269 0.0246 0.932 0.0810 -1.00 8.78 0.5 1.10 0.0148 0.283 0.0590 0.822 0.0148 11.0 1.60
1 1.09 0.262 0.283 0.0147 0.805 0.262 12.8 28.4 3 1.01 0.0718 0.273 0.0614 0.740 0.0718 19.8 7.78 5 1.11 0.0466 0.198 0.0142 0.914 0.0466 1.01 5.05 10 1.16 0.0931 0.303 0.0456 0.861 0.0931 6.72 10.1 30 1.12 0.0536 0.284 0.0375 0.833 0.0536 9.73 5.80 50 1.04 0.0317 0.336 0.0666 0.705 0.0317 23.6 3.43 100 1.05 0.0997 0.360 0.0420 0.690 0.0997 25.2 10.8 1mM Quinidine 1.70 0.0760 1.63 0.0436 0.0733 0.0760 92.1 8.24
