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ADME PK

Human SLC Uptake Transporter Substrate Identification (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, OATP1A2, OATP2B1, OAT2, OAT4, OCTN2, PEPT1, PEPT2, NTCP) for Screening or Regulatory Reporting Purposes

Understand if your compound is a substrate for the main human SLC uptake transporters, OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, OATP1A2, OATP2B1, OAT2, OAT4, OCTN2, PEPT1, PEPT2 or NTCP.

SLC 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 the SLC uptake transporters in vitro

  • The SLC (solute carrier) family transport a wide range of different solutes across biological membranes using diverse energy coupling mechanisms1.
  • Members of the SLC transporters include the OATP, OAT, OCT, MATE, OCTN and the PEPT transporters. These transporters are based predominantly in the intestine, the blood brain barrier, the kidneys and the liver where they influence the absorption, distribution, metabolism and excretion of drugs within the body.
  • The draft FDA guidance2 and the EMA guidance3 recommend investigating for potential OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1 and MATE2-K substrate identification due to the role of these transporters in clinical drug-drug interactions and the impact of genetic polymorphism of some of these transporters on therapy outcome and toxicity.
  • The EMA3 and International Transporter Consortium (ITC)4 also suggests that potential interactions with OCT1 should be considered.
  • It is only necessary to evaluate potential OAT1, OAT3, OCT2, MATE1 and MATE2-K substrates when renal active secretion of the investigational drug is significant (e.g., active secretion by the kidney is more than or equal to 25% of total clearance) and it is only necessary to evaluate potential OATP1B1, OATP1B3 and OCT1 substrates when hepatic or biliary secretion is more than or equal to 25% of total clearance.2,3,4
  • Cyprotex’s SLC transporter substrate identification assay determines if your compound is a substrate of the key transporters recommended in the regulatory guidelines.
Membrane transporters can have clinically relevant effects on the pharmacokinetics and pharmacodynamics of a drug in various organs and tissues by controllong its absorption, distribution and elimination.

2Draft FDA Guidance for Industry– In Vitro Metabolism- and Transporter-Mediated Drug-Drug Interaction Studies, October 2017

Protocol

SLC uptake transporter substrate identification assay protocol for screening (1 or 2 concentrations) or regulatory type studies (4 concentrations & single concentration plus inhibitor)

Test System Mammalian HEK293 cells transiently overexpressing a single transporter (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, OATP1A2, OATP2B1, OAT2, OAT4, OCTN2, PEPT1, PEPT2 or NTCP – other transporters available on request)

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

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

Screening study - two concentrations (typically, 1 µM and 10µM), two timepoints for a single compound

Regulatory study - typically 1, 10, 50 and 100 µM (depending on customer requirements) plus inhibition at a single substrate concentration (two timepoints)
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
Written report available on request

Related Services

P-gp
BCRP
BSEP
MRPs

Data

Data from Cyprotex's SLC uptake transporter substrate identification assay

Figure 1
Uptake of 3H-estradiol 17β-glucuronide (1 µM) in OATP1B1-transfected HEK293 cells and control HEK293 cells over 20 min in the presence and absence of the inhibitor rifamycin (100 µM).

To confirm transporter involvement in the uptake of estradiol 17β-glucuronide in the OATP1B1 transfected cells, the inhibitor rifamycin was included in the incubations. This reduced the uptake to similar levels (< 2 fold) as observed in the control cells.
Figure 2
Uptake of 3H-estrone 3-sulfate (1 µM) in OAT3-transfected HEK293 cells and control HEK293 cells over 20 min in the presence and absence of the inhibitor probenecid (100 µM).

To confirm transporter involvement in the uptake of estrone 3-sulfate in the OAT3-transfected cells, the inhibitor probenecid was included in the incubations. This reduced the uptake to similar levels (< 2 fold) 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 Draft FDA Guidance for Industry– In Vitro Metabolism- and Transporter-Mediated Drug-Drug Interaction Studies, October 2017.
3 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012).
4Zamek-Gliszczynski M et al., (2018) Transporters in Drug Development: 2018 ITC Recommendations for Transporters of Emerging Clinical Importance. Clin Pharmacol Ther 104(5): 890-899.

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