TABLE 1.
Reaction constants and their derivations for use during in silico modeling
| Reaction | Kinetic Parameters of Reaction | Source of Data |
|---|---|---|
| r1 | Hydrolysis of CDFDA (s14) to CDF (s5) within the cell | Zamek-Gliszczynski et al., 2003 |
| Sandwich culture of primary rat hepatocytes: all CDFDA was converted to CDF within 10 s. Presume this is 20 half-lives; therefore, t1/2 = 0.5 s. | ||
| For a first-order reaction, t1/2 = ln2/k. Therefore, k = 0.693/0.5 = 1.39 s-1. | ||
| r2 | Passive diffusion of CDFDA (s13) into the cell. | Zamek-Gliszczynski et al., 2003 |
| Uptake clearance of 10, 100, and 500 μM CDFDA into rat hepatocytes = 4.9, 687, and 3374 μl/min/mg protein, respectively. | ||
| Assume to be a reversible first-order process, so rate = k[s]. Therefore, k ≈ 6.8 min-1. | ||
| r3 | Efflux of CDF (s12) via Abcc2 into the bile canaliculi. | Pratt et al., 2006 and experimentally derived |
| Abcc2 overexpression in human embryonic kidney cells: KM = 18.9 ± 2 μM and Vmax = 95.5 ± 14.8 pmol/min/mg protein. | ||
| Sandwich culture of primary rat hepatocytes: MK571 IC50 value at 10 μM CDFDA = 1.9 ± 3.7 μM. | ||
| r4 | Uptake of CDFDA (s13) into the cell via active transport. | Zamek-Gliszczynski et al., 2003; Wu and Benet, 2005 |
| Because CDFDA is a very lipid-soluble molecule, the presence of active transport into the cell will be minimal and transport is unaltered by temperature. Discounted in the model. | ||
| r8 | Binding of CDFDA (s13) to extracellular proteins. | Zamek-Gliszczynski et al., 2003 |
| In vivo CDFDA is 22% protein-bound. The model simulates the in vitro assay and because hepatocytes were dosed with CDFDA in HBSS, containing no extracellular protein, this figure will be zero. | ||
| Discounted in the model. | ||
| r10 | Conversion of CDFDA (s13) to CDF (s26) outside of the cell. | Zamek-Gliszczynski et al., 2003 |
| In phosphate-buffered saline, t1/2 = 7.6 ± 0.1 h-1. For a first-order reaction, t1/2 = ln2/k. Therefore, k = 0.693/456 = 0.00152 min-1 | ||
| r11 | Uptake of CDF (s26) into the cell via active transport. | Zamek-Gliszczynski et al., 2003 |
| Sandwich culture of primary rat hepatocytes: uptake clearance of CDF into rat hepatocytes = 1.9 ± 0.1 μl/min/mg protein and was inhibited by low temperature (10 μM dose). | ||
| Uptake was saturable with and inhibited by substrates of organic anion-transporting polypeptides, e.g., taurocholate and rifampicin. | ||
| KM of 22 ± 10 μM and Vmax = 97 ± 9 pmol/min/mg protein. | ||
| r12 | Passive diffusion of CDF (s26) into the cell. | Zamek-Gliszczynski et al., 2003 and experimentally derived |
| Sandwich culture of primary rat hepatocytes: uptake inhibited by low temperature so mainly transporter-mediated. Octanol/water coefficient demonstrates no evidence for partition into lipid membranes. | ||
| Discounted in the model. | ||
| r13 | Binding of CDF (s26) to extracellular proteins. | Zamek-Gliszczynski et al., 2003 |
| In vivo CDF is 20% protein-bound. The model simulates the in vitro assay and as hepatocytes were cultured in HBSS, containing no extracellular protein, this figure will be zero. | ||
| Discounted in the model. | ||
| r14 | Efflux of CDF (26) across the sinusoidal membrane via Abcc3. | Zamek-Gliszczynski et al., 2003 and experimentally derived |
| Sandwich culture of primary rat hepatocytes: rate of efflux is approximately a quarter of efflux via Abcc2. | ||
| Vmax ≈ 24 pmol/min/mg protein and KM ≈ 20 μM. | ||
| Sandwich culture of primary rat hepatocytes: MK571 IC50 value at 10 μM CDFDA = 1.9 ± 3.7 μM. |