Transporter activity assays
For URAT1 and OAT4, DNA constructs pCMV/neo-URAT1 and pCMV/neo-OAT4 were purchased from Origene Technologies. In addition, a glycine codon was introduced just prior to the stop codon using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s instructions, producing pCMV/neo-URAT1-553G and pCMV/neo-OAT4-551G. The added glycine has no effect on the activity of the proteins (data not shown). Stably transfected HEK293-OAT4-551G cells were used for OAT4 transport activity assays, which were grown for several weeks in media (DMEM with 10 % fetal bovine serum and 1 mM sodium pyruvate) containing 500 μg/mL geneticin sulfate. An isolated clone with high OAT4 activity was selected for activity assays. One day prior to the assay, cells were plated at 200,000 cells/well into each well of white, clear bottom poly-D-lysine-coated assay plates (BD Biosciences). URAT1-553G-expressing cells were prepared by transient reverse transfection of pCMV/neo-URAT1-553G into HEK-293 T cells using DreamFect Gold (Boca Scientific) according to the manufacturer’s instructions, and plated directly into assay plates in the same way as the OAT4-551G cells.
The cells were assayed on the following day. For measuring urate transport, cells were washed once with wash buffer (25 mM MES pH 5.5, 125 mM sodium gluconate), and then incubated in assay buffer (25 mM MES pH 5.5, 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM calcium gluconate, and 5.6 mM glucose) containing different amounts of lesinurad, for 5 minutes: 14C-urate (American Radiolabeled Chemicals, Inc.) at a final concentration of 100 μM in assay buffer was then added and incubated with the cells for 10 minutes. The free urate was removed by aspiration and the cells rinsed three times in 150 μL wash buffer. The cells were then subjected to liquid scintillation counting to measure transported urate. Lesinurad dose-response curves and half-maximal inhibition constants (IC50 values) were generated from the urate transport results using GraphPad Prism software (GraphPad Software, Inc.), variable slope (four parameter) model. Each point represents the mean and standard error of the mean (SEM) from triplicates.
For OAT1, HEK-293 T cells were transfected as described above using pSPORT6-hOAT1 (Thermo Scientific) and assayed for transport of 5 μM 6-carboxyfluorescein (CF) for 2 minutes with or without lesinurad, in assay buffer containing 125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES, pH 7.3. The cells were washed three times in a buffer containing 125 mM NaCl and 25 mM HEPES, pH 7.3, and then lysed in 1 M sodium hydroxide prior to fluorescence measurement.
For ABCG2, monolayers of Caco-2 cells expressing ABCG2 were grown on a permeable support (1 μM PET membranes) in 24-transwell plates. Cells were washed and incubated in Hank’s balanced salt solution, with or without different concentrations of lesinurad or with 100 μM chrysin for 30 minutes at 37 °C in both apical (A) and basolateral (B) compartments. Each treatment was assayed in triplicate: [
3H]-genistein substrate at 2 nM was then added for an additional 120 minutes at 37 °C. For basolateral-to-apical (B → A) flux measurements, genistein was placed in the basolateral compartment only (donor well) and a sample was taken after the 120-minute incubation period from the apical compartment (receiver well). For apical-to-basolateral (A → B) flux measurements, genistein was placed in the apical compartment only (donor well) and a sample was taken after the 120-minute incubation period from the basolateral compartment (receiver well). Samples were then subjected to scintillation counting. The inhibition of BCRP/ABCG2 was then calculated by the net B → A flux method [
39]. The substrate flux (A → B or B → A) is determined by the apparent permeability (P
app) using the following equation:
$$ {\mathrm{P}}_{\mathrm{app}} = \left[1/\left(\mathrm{Area}\ *\ {\mathrm{C}}_{\mathrm{D}}(0)\right)\right]\ *\ {\mathrm{dM}}_{\mathrm{r}}/\mathrm{d}\mathrm{t}. $$
where Area is the area of the filter in the transwell, CD(0) is the initial dosing concentration of genistein, and dMr/dt is the slope of the flux divided by the incubation time.
The net transport in the B → A direction was determined by subtracting the substrate flux in the A → B direction from that in the B → A direction:
$$ \mathrm{N}\mathrm{e}\mathrm{t}\ \mathrm{B}\ \to\ \mathrm{A}\ \mathrm{flux} = {\mathrm{P}}_{\mathrm{app}}\left(\mathrm{B}\ \to\ \mathrm{A}\right)\hbox{-} {\mathrm{P}}_{\mathrm{app}}\left(\mathrm{A}\to \mathrm{B}\right). $$
Percent inhibition is calculated by dividing the Net (B → A) flux in the presence of the inhibitor by the Net (B → A) flux in the absence of the inhibitor (inh):
$$ \mathrm{Percent}\ \mathrm{Inhibition} = 100\ \hbox{-} \left[100*\mathrm{N}\mathrm{e}\mathrm{t}{\left(\mathrm{B}\to \mathrm{A}\right)}_{\mathrm{inh}}/\mathrm{mean}\ \mathrm{N}\mathrm{e}\mathrm{t}\left(\mathrm{B}\to \mathrm{A}\right)\right]. $$
For GLUT9, SLC2A9v2/GLUT9ΔN in pCMV6/neo was linearized with XmaI and NotI and cRNA transcribed using the mMessage mMachine T7 kit (Ambion). Oocytes were injected with either water or 25 ng cRNA and incubated for 3 to 5 days in ND96 buffer. For each point, 10 oocytes were incubated with 100 μM 14C-uric acid in ND96 buffer with and without test compounds at room temperature for 60 minutes. The oocytes were then washed three to four times in ice-cold ND96 buffer and lysed prior to scintillation counting.
Mitochondrial toxicity assay
HepG2 cells were counted via hemocytometer and diluted into equal volumes of serum-free Opti-MEM (Life Technologies) and JC-1 Staining Solution (Sigma catalog number CS0390) at 300,000 cells/mL in the presence of 2.5 μg/mL JC-1. Cells were incubated at 37 °C and 5 % CO2 for 20 minutes, rinsed once with Hank's Balanced Salt Solution, and resuspended to the same concentration in serum-free Opti-MEM. Cells were then seeded into 96-well black-sided clear-bottom tissue culture plates at 30,000 cells/100 μL/well. Three replicates per experimental condition group were used. Compound was added with the final dimethyl sulfoxide (DMSO) concentration at 0.5 %. After 2 hours at 37 °C and 5 % CO2, medium was aspirated and plates were read on a Gemini EM spectrofluorometer (Molecular Devices). Fluorescence of JC-1 monomers was monitored by excitation at 490 nm and emission at 530 nm (green) and of aggregates by excitation at 525 nm and emission at 590 nm (red). The ratio of J-aggregates to monomers was calculated, with a lower ratio indicating increased cell toxicity. The percent change in mitochondrial membrane potential (Ψm) was calculated as follows:
[Em590/Em530 (vehicle only) – Em590/Em530 (test compound)]/[Em590/Em530 (vehicle only) – Em590/Em530 (100 μM menadione)].
Clinical studies
Lesinurad effects on FEUA
From a phase 1 clinical trial, RDEA594-109, eight healthy male volunteers were administered single oral doses of 200, 400, or 600 mg lesinurad tablets (with food). In each treatment period, plasma samples were collected within 30 minutes before dosing (pre-dose) and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 30, 36, and 48 hours post-dose. Urine (total catch) was collected pretreatment at hours –24 to –18, –18 to –12, and –12 to 0, and post-dose at hours 0 to 6, 6 to 12, 12 to 24, 24 to 30, 30 to 36, and 36 to 48.
After protein precipitation of the samples, lesinurad levels in plasma and urine were measured at Ardea Biosciences by quantitative high performance liquid chromatography with tandem mass spectrometry. The lesinurad plasma PK parameters maximum plasma concentration (Cmax) and area under the curve (AUC) were derived using the software WinNonlin Professional, Version 5.2 (Pharsight Corporation). The parameters from individual profiles of lesinurad were determined using noncompartmental methods. Urine samples were analyzed for uric acid and creatinine using enzymatic methods by Covance Clinical Research Unit (Dallas, TX, USA), and pharmacodynamic (PD) parameters were determined by Covance Clinical Research Unit using SAS, Version 8.2 (SAS Institute Inc). Calculated PD parameters for uric acid included urinary excretion, renal clearance, and FEUA. FEUA was calculated by dividing the uric acid clearance by the creatinine clearance. For analyzing the relationship between plasma lesinurad concentrations and FEUA, the weighted average plasma lesinurad concentration was calculated for plasma samples from each time period that corresponded to the associated urine samples.
Lesinurad effects on sUA
Serum was collected within 30 minutes prior to drug administration, and at 6, 12, 16, 24, 30, 36, and 48 hours after dosing. Serum urate levels and PD parameters were assayed and calculated by Covance Clinical Research Unit as described above.