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ADME PK

Preclinical Species Hepatic Oatp Uptake Transporter Substrate Identification

Understand if your compound is an in vitro substrate for the main hepatic Oatp uptake transporter of preclinical species, namely rat Oatp1b2, dog Oatp1b4 or Cynomolgus monkey Oatp1b1.

Preclinical species hepatic Oatp uptake transporter substrate identification is within our portfolio of in vitro drug transporter services. Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Identifying potential substrates of preclinical species hepatic uptake transporters in vitro

  • The SLC (solute carrier) family transport a wide range of different solutes across biological membranes using diverse energy coupling mechanisms1.
  • One of the most important human SLC transporters expressed in human liver is OATP1B1 which is responsible for the hepatic uptake and rate-determining elimination of a wide range of endogenous compounds and drugs that are substrates2.
  • Species differences in drug transporters with regard to their tissue distribution, expression levels and substrate specificity can be problematic for preclinical cross-species extrapolation of drug disposition (clearance) and DDI potential to human.
  • The use of in vitro cell test systems that each overexpress the major hepatic Oatp transporter of preclinical species (Oatp1b2, Oatp1b4 or Oatp1b1 for rat, dog or Cynomolgus monkey, respectively) may be useful towards understanding whether a molecule is a substrate of an hepatic active uptake transporter in those species. Such knowledge may assist in the interpretation of any observed liver accumulation in vivo, or help towards qualitatively understanding an underprediction of in vivo hepatic clearance from in vitro microsomal clearance data for that preclinical species.
  • Cyprotex’s preclinical species hepatic Oatp transporter substrate identification assay determines if your compound is a substrate of key preclinical species transporters.
It has become increasingly clear that there are significant differences between rodents, dog, monkey, and human in the substrate specificity, tissue distribution, and relative abundance of transporters. These differences complicate cross-species extrapolations, which is important when attempting to predict human pharmacokinetics (PK) of drug candidates and assess risk for drug–drug interactions (DDIs).

3Chu X et al., (2013) Expert Opin Drug Metab Toxicol 9(3): 237-252.

Protocol

Preclinical species hepatic Oatp uptake transporter substrate identification screening assay protocol (1 or 2 concentrations)

Test System Mammalian HEK293 cells transiently overexpressing a single preclinical species transporter (rat Oatp1b2, dog Oatp1b4 or Cynomolgus monkey Oatp1b1)

Control vector-transfected HEK293 cells
Test Article Concentrations Options:
- single concentration (typically, 1 µM), single timepoint for 7 compounds

- two concentrations (typically, 1 µM and 10µM), single timepoint for 3 compounds

- two concentrations (typically, 1 µM and 10µM), two timepoints for a single compound
Time Points Typically, 2 min or 2 min and 20 min (depending on customer requirements)
Analysis Method MicroBeta® scintillation counter (radiolabelled substrates)
LC-MS/MS analysis (non-radiolabelled substrates)
Data Delivery Cellular uptake and fold accumulation

Related Services

Human SLC Transporter Substrate Identification

Data

Data from Cyprotex's preclinical species hepatic Oatp uptake transporter substrate identification assay

Figure 1
Uptake of 3H-estradiol 17β-glucuronide (1 µM) in rat Oatp1b2-transfected HEK293 cells and control HEK293 cells over 1.5 min in the presence and absence of the inhibitor rifamycin SV (30 µM).

To confirm transporter involvement in the uptake of estradiol 17β-glucuronide in the rat Oatp1b2-transfected cells, the inhibitor rifamycin SV was included in the incubations. This reduced the uptake ratio by well over 50% with the transporter uptake giving similar levels (~2-fold uptake ratio) as observed in the control cells.
Figure 2
Uptake of 3H-estradiol 17β-glucuronide (1 µM) in dog Oatp1b4-transfected HEK293 cells and control HEK293 cells over 3 min in the presence and absence of the inhibitor rifamycin SV (10 µM).

To confirm transporter involvement in the uptake of estradiol 17β-glucuronide in the dog Oatp1b4-transfected cells, the inhibitor rifamycin SV was included in the incubations. This reduced the uptake ratio by well over 50% with the transporter uptake giving similar levels (~ 2 fold uptake ratio) as observed in the control cells.
Figure 3
Uptake of 3H-estradiol 17β-glucuronide (1 µM) in Cynomolgus monkey Oatp1b1-transfected HEK293 cells and control HEK293 cells over 2 min in the presence and absence of the inhibitor rifamycin SV (3 µM).

To confirm transporter involvement in the uptake of estradiol 17β-glucuronide in the Cynomolgus monkey Oatp1b1-transfected cells, the inhibitor rifamycin SV was included in the incubations. This reduced the uptake ratio by well over 50% with the transporter uptake giving similar levels (< 2 fold uptake ratio) as observed in the control cells.

References

1 Schlessinger A et al., (2013) Molecular modeling and ligand docking for solute carrier (SLC) transporters. Curr Top Med Chem 13(7): 843-856.
2 Shitara Y et al., (2013) Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption. Biopharm Drug Dispos 34: 45-78.
3 Chu X et al., (2013) Species differences in drug transporters and implications for translating preclinical findings to humans. Expert Opin Drug Metab Toxicol 9(3): 237-252.

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