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ADME PK

Human SLC Uptake Transporter Inhibition (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 inhibits the 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 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.

The effect of SLC uptake transporter inhibition and its measurement 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 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 inhibition 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 the International Transporter Consortium (ITC)4 also suggests that potential interactions with OCT1 should be considered.
  • Cyprotex’s SLC transporter inhibition assay determines if your compound is an inhibitor of the key transporters recommended in the regulatory guidelines. Additionally since 2016 and ahead of guidance recommendations, for IC50 determination, the assay has incorporated a 15 min preincubation step with test compound for OATP1B1, OATP1B3 and all other SLC transporters. Inclusion of this 15 min inhibitor preincubation step produces an identical IC50 value for the OATP inhibitor cyclosporin A when compared to that determined with inclusion of a 30 min preincubation step recommended in the 2017 draft FDA guidance2, indicating that the preincubation methods are equivalent for DDI risk assessment (see Cyprotex Inhibitor Preincubation poster). Furthermore, the use of a shorter 15 min preincubation time might lessen any impact of potential cytotoxic effects during cell incubations.
Membrane transporters can have clinically relevant effects on the pharmacokinetics and pharmacodynamics of a drug in various organs and tissues by controlling 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 inhibition assay protocol

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
Probe Substrate [3H]-Estradiol 17β-glucuronide (OATP1B1, OATP1B3)
[3H]-PAH (OAT1)
[3H]-Estrone 3-sulfate (OAT3, OAT4, OATP1A2, OATP2B1)
[14C]-Metformin (OCT2, MATE1) (upon request for MATE2-K)
[14C]-TEA (OCT1, MATE2-K) (upon request for MATE1)
[3H]-cGMP (OAT2)
[3H]-L-Carnitine (OCTN2)
[3H]-Glycyl sarcosine (PEPT1, PEPT2)
[3H]-Taurocholic acid (NTCP)
Test Article Concentration Single concentration (for batches of 6 compounds) OR
6 concentrations plus 0 µM (final test compound concentrations dependent on customer requirements)
Time Point Dependent on transporter
Analysis Method Radiochemical detection using scintillation counting
Data Delivery Percentage inhibition (single concentration) OR
IC50
Written report available on request

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Data

Data from Cyprotex's SLC Uptake Transporter Inhibition assay

Figure 1
Representative graph showing inhibition of OAT3-mediated transport of estrone 3-sulfate by the OAT3 inhibitor, probenecid. Data shown represents the mean of 3 replicates ± standard deviation.
TransporterSubstrateInhibitorIC50 ± Standard Error (µM)
OATP1B1 Estradiol 17β-glucuronide Rifamycin 0.67 ± 0.18
Cyclosporin A 1.53 ± 0.22
OATP1B3 Estradiol 17β-glucuronide Rifampicin 0.79 ± 0.11
Cyclosporin A 0.96 ± 0.24
OAT1 PAH Probenecid 16.6 ± 11.7
Diclofenac 1.00 ± 0.36
OAT3 Estrone 3-sulfate Probenecid 11.5 ± 2.64
Diclofenac 18.7 ± 3.91
OCT1 TEA Verapamil 7.59 ± 2.42
Quinidine 30.5 ± 4.20
OCT2 Metformin Verapamil 26.3 ± 2.42
Quinidine 35.6 ± 2.03
MATE1 Metformin Cimetidine 1.22 ± 0.09
Trimethoprim 2.64 ± 0.27
MATE2-K Metformin Cimetidine 3.34 ± 1.02
Trimethoprim 0.35 ± 0.06
MATE1 TEA Cimetidine 0.92 ± 0.10
Verapamil 17.9 ± 3.88
MATE2-K TEA Cimetidine 7.02 ± 5.27
Verapamil 21.6 ± 1.79
OATP1A2 Estrone 3-sulfate Rifamycin SV 3.44 ± 0.78
Ritonavir 1.41 ± 0.77
OATP2B1 Estrone 3-sulfate Rifamycin SV 2.72 ± 0.33
Ritonavir 4.88 ± 0.85
Atorvastatin 0.23 ± 0.08
OAT2 cGMP Indomethacin 8.31 ± 0.62
Cimetidine 239 ± 35.80
OAT4 Estrone 3-sulfate Losartan 46.4 ± 3.12
Furosemide 159 ± 25.70
OCTN2 L-Carnitine Meldonium 32.3 ± 6.15
Verapamil 111 ± 42.0
PEPT1 Glycyl sarcosine Losartan 399 ± 62.55
Cefadroxil 629 ± 157.50
PEPT2 Glycyl sarcosine Losartan 34.9 ± 5.92
Cefadroxil 22.6 ± 7.43
NTCP Taurocholic acid Pioglitazone 10.2 ± 0.98
Cyclosporin A 7.21 ± 1.75
Table 1
Inhibition of OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, OATP1A2, OATP2B1, OAT2, OAT4, OCTN2, PEPT1, PEPT2 and NTCP-mediated transport of prototypical substrates.

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).
4 Zamek-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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