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ADME PK

Preclinical Species Hepatic Oatp Uptake Transporter Inhibition

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

Preclinical species hepatic Oatp uptake transporter inhibition 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 inhibitors 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 an inhibitor of an hepatic active uptake transporter in those species. Whilst not usually performed for a compound on the back of IC50 data indicating a positive interaction potential for inhibition of human OATP1B1, were a sponsor to consider conducting an in vivo interaction study in a preclinical species (e.g. Cynomolgus monkey)3 then it would be beneficial to understand the in vitro inhibitory potential of the molecule against that preclinical species Oatp in order to put it into context with exposure and aid interpretation of the data.
  • Cyprotex’s preclinical species hepatic Oatp transporter inhibition assay determines if your compound is an inhibitor 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).

4Chu et al., (2013) Expert Opin Drug Metab Toxicol 9(3): 237-252.

Protocol

Preclinical species hepatic Oatp uptake transporter inhibition assay protocol

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
Probe Substrate [3H]-Estradiol 17β-glucuronide
Test Article Concentrations Single concentration (for batches of 6 compounds) OR 6 concentrations plus 0 µM (final test compound concentrations dependent on customer requirements)
Time Points Dependent on transporter
Analysis Method Radiochemical detection using scintillation counting
Data Delivery Percentage inhibition (single concentration) OR
IC50

Related Services

Human SLC Transporter Inhibition

Data

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

Figure 1
Mean rat Oatp1b2-mediated estradiol 17ß-D-glucuronide (1 μM) transport in the presence of a range of concentrations of rifamycin SV expressed as a percentage of vehicle control.

The results represent the mean ± standard deviation of 3-9 replicate wells (triplicate wells per incubation condition performed on three separate occasions).
 
TransporterSubstrateInhibitorIC50 ± Standard Error (µM)
Rat Oatp1b2 Estradiol 17β-glucuronide Rifamycin SV 0.992 ± 0.188
Dog Oatp1b4 Estradiol 17β-glucuronide Rifamycin SV 0.314 ± 0.0623
Cyno Oatp1b1 Estradiol 17β-glucuronide Rifamycin SV 0.120 ± 0.0246
Table 1
Inhibition of rat Oatp1b2, dog Oatp1b4 and Cynomolgus monkey Oatp1b1-mediated transport of the prototypical substrate estradiol 17β-glucuronide.

The incubation conditions for each of the species have been fully characterised for our chosen Oatp substrate, estradiol 17ß-glucuronide, based on time linearity and uptake kinetics (Vmax and Km). The chosen substrate concentration is at least seven-times lower than the determined Km, and as such IC50 equates to Ki (assuming competitive inhibition).

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 Ufuk A et al., (2018) In Vitro-In Vivo Extrapolation of OATP1B-Mediated Drug-Drug Interactions in Cynomolgus Monkey. J Pharmacol Exp Ther 365(3): 688-699.
4 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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Please give details of the assays you are interested in. Where appropriate please specify one or more species (human, rat, mouse etc.), isoforms (CYP1A1,CYP1B1, etc) or other relevant details.

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