Article Figures & Data
Figures
- Fig. 1.
Visual representation of a mechanistic two-compartmental transporter model. The movement of DCF-AG in the proposed model is described by three vectorial processes: 1) Pdif,in, passive diffusion from the buffer into the cell; 2) Kactive, active transport from the buffer into the cell; and 3) Pdif,out, passive diffusion from the cell into the buffer. Active transport for this model applies for the OATP2B1-mediated uptake of DCF-AG. Using differential equations within Berkeley Madonna software, the mass transfer of DCF-AG into and out of each compartment is modeled over time using in vitro time-course data to derive kinetic parameters such as Km, Pdif, and Vmax. Pdif reflects passive diffusion while Km and Vmax are used to characterize active uptake that adheres to Michaelis-Menten kinetics. Kinetic parameterization was performed for uptake at pH 6.0 and 7.4 to reflect the physiologic conditions of the intestine and liver, respectively.
- Fig. 2.
Concentration-versus-time profiles of DCF-AG uptake by OATP2B1. HEK-WT and HEK-OATP2B1 cells were seeded for 48 hours and incubated with increasing concentrations of DCF-AG at multiple time points in buffer titrated to either pH 6.0 or 7.4 at 37°C. Intracellular concentrations were determined by LC–MS/MS, and the concentrations were modeled in Berkeley Madonna. (A) and (B) show the uptake of DCF-AG by HEK-OATP2B1 cells at pH 6.0 and 7.4, respectively. Fitted lines from a two-compartment model represent the best fit. Data are the individual replicates from a typical study.
- Fig. 3.
Cytotoxicity of DCF and DCF-AG using HEK cells. HEK-OATP2B1 cells were incubated in the absence or presence of increasing concentrations of compound. Incubations were conducted for 3, 6, or 12 hours at 37°C. Cytotoxicity was assessed using calcein AM to determine live cells and ethidium homodimer-1 as an indicator of dead cells. The CAM and EthD-1 responses indicate concentration-dependent cell death by DCF, and the cytotoxicity was more pronounced for DCF-AG. *P < 0.05, **P < 0.01; ***P < 0.001 compound versus its time-matched vehicle incubation. RFU, relative fluorescence units.
- Fig. 4.
Generation of reactive oxygen species by in HEK cells. HEK-OATP2B1 cells were incubated in the absence or presence of increasing concentrations of compound. Incubations were conducted for 1, 2, or 3 hours at 37°C, after which ROS were detected with the use of DCFDA, which becomes oxidized by ROS to a fluorogenic form. Fluorescence was normalized to vehicle controls and expressed as percentage change from vehicle. Each bar represents the mean ± the standard deviation of n = 3 measurements per condition. *P < 0.05, **P < 0.01; ***P < 0.001 compound versus its time-matched vehicle incubation. RFU, relative fluorescence units.
- Fig. 5.
Inhibition of superoxide dismutase as an indication of oxidative stress. Copper/zinc SOD was incubated in the absence or presence of increasing concentrations of DCF (□) or DCF-AG (▪) for 30 minutes at room temperature (∼25°C). Vehicle shows assay performance in the presence of the same level of organic solvent (dimethyl sulfoxide) used in the inhibitor incubations. Each bar reflects the mean ± standard error of the mean from three separate studies (n = 2 replicates per study). **P < 0.01, ***P < 0.001 inhibitor versus its vehicle incubation. #P < 0.05, ###P < 0.001 for DCF versus DCF-AG.
- Fig. 6.
COX-1 inhibition profiles of DCF, OH-DCF, and DCF-AG. Recombinant COX-1 was incubated in the presence of inhibitors for 2 minutes at 37°C, and the formation of PGE2 from AA was monitored via LC–MS/MS. Data reflect the mean ± standard error of the mean from three separate studies (n = 2 replicates per study). Inhibition data were fit with a three-parameter model to calculate IC50. Dotted lines reflect the 95% confidence intervals of the fit.
- Fig. 7.
COX-2 inhibition profiles of DCF, OH-DCF, and DCF-AG. Recombinant COX-2 was incubated in the presence of inhibitors for 2 minutes at 37°C, and the formation of PGE2 from arachidonic acid was monitored via LC–MS/MS. Data reflect the mean ± standard error of the mean from three separate studies (n = 2 replicates per study). Inhibition data were fit with a three parameter model to calculate IC50. Dotted lines reflect the 95% confidence intervals of the fit.
- Fig. 8.
Proposed pathways on the disposition and mechanisms of toxicity for DCF-AG. DCF-AG is generated in the liver after DCF (intraperitoneal or by mouth) administration, where it undergoes excretion into bile or blood via ABC transporters. In the intestinal lumen DCF-AG is taken up by OATPs and exerts various effects that lead to intestinal injury. The absence of MRP3 exacerbates GI injury from DCF-AG owing to the loss of a clearance mechanism from enterocytes. Solid arrows indicate vectorial transport via uptake or efflux transporters, or via blood flow. Dotted arrows signify transport by passive processes. Dashed lines demonstrate possible enzymatic reactions such as inhibition of COX, generation of ROS, or cleavage by bacterial β-glucuronidases (β-gluc). The question mark denotes uncertainty regarding the efflux pathways for the indicated metabolites.
Tables
- TABLE 1
Summary of DCF-AG uptake kinetics mediated by OATP2B1
Each value represents the mean ± standard error of the mean from three studies (n = 2 replicates per study). *P < 0.05 for pH 6.0 versus pH 7.4.
Media pH Vmax Km Uptake CLint Pdif pmol/min per milligram μM μl/min per milligram μl/min per milligram 6.0 27.8 ± 4.1* 15.2 ± 0.8 1.82 ± 0.19 0.0522 ± 0.0120* 7.4 17.6 ± 1.5 14.3 ± 0.1 1.23 ± 0.11 0.0245 ± 0.0030 CLint, intrinsic uptake clearance.
- TABLE 2
Summary of in vitro COX inhibition assays
IC50 values reflect the mean ± standard error of the mean from three experiments (n = 2 replicates per experiment).
Compound COX-1 IC50 COX-2 IC50 μM μM SC-560 (COX-1 selective) 0.00166 ± 0.00022 N.D. DuP-697 (COX-2 selective) N.D. 0.00714 ± 0.0007 DCF 0.0206 ± 0.0037 0.103 ± 0.005 OH-DCF 0.375 ± 0.075 21.2 ± 0.3 DCF-AG 0.620 ± 0.105 2.91 ± 0.36 N.D., not determined.
Data Supplement
Files in this Data Supplement:
- Supplemental Figure 1 - Cytotoxicity caused by DCF-AG in HEK wild-type and OATP2B1-transfected cells.
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