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ADME PK

Human MRP Efflux Transporter Substrate Identification (MRP2, MRP3, MRP4) for Screening or Regulatory Reporting Purposes

Understand if your compound and/or metabolite is a substrate for the human efflux transporters, MRP2, MRP3 or MRP4.

MRP efflux 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 MRP efflux transporters in vitro

  • MRP2 (multidrug resistance associated protein 2; ABCC2), MRP3 (ABCC3) and MRP4 (ABCC4) are ATP binding cassette (ABC) efflux transporters which are located on the brush border membrane of enterocytes (MRP2), the canalicular membrane (MRP2) or sinusoidal membrane (MRP3, MRP4) of hepatocytes, the brush border membrane of renal proximal tubule epithelial cells (MRP2, MRP4) and at the blood-brain barrier (MRP4)1.
  • Consequently, these efflux transporters influence the absorption, distribution, metabolism and excretion of drugs/and or metabolites within the body.
  • The International Transporter Consortium (ITC)1 indicate that because MRP2, MRP3 and MRP4 are important determinants of hepatobiliary disposition of polar drug metabolites, for example glucuronide conjugates, then being a substrate of these transporters may contribute to the overall victim and perpetrator DDI potential of the parent drug. Furthermore, the draft FDA guidances2,3 indicate that the DDI potential of metabolites versus the major drug transporters, and other emerging transporters such as MRPs when appropriate, be assessed on a case by case basis.
  • Cyprotex’s MRP efflux transporter substrate identification assay determines if your compound is a substrate of these key hepatobiliary transporters.
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

MRP transporter substrate identification assay protocol for screening (1 or 2 concentrations) or regulatory type studies (4 concentrations & two concentrations plus inhibitor)

Test System Sf9 insect cell-derived or mammalian (HEK293) cell-derived inside-out membrane vesicles overexpressing a single transporter (MRP2, MRP3 or MRP4) incubated in the presence of ATP and AMP (absence of ATP)
Test Article Concentrations Screening study – single concentration (typically 1 µM), single timepoint for 7 compounds

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

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

Regulatory study - typically 1, 10, 50 and 100 µM (depending on customer requirements) plus inhibition at two substrate concentrations (two timepoints)
Time Points Typically, 10 min or 10 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
Human SLC Transporters

Data

Data from Cyprotex's MRP efflux transporter substrate identification assay

Figure 1
Uptake of [3H]-estradiol 17β-glucuronide (30 µM) in MRP2 membrane vesicles with ATP or AMP in the presence and absence of the inhibitor MK-571 (300 µM).

To confirm ATP-dependent transporter involvement in the uptake of estradiol 17β-glucuronide in the MRP2 membrane vesicles, the inhibitor MK-571 was included in the incubations. This reduced the uptake to similar levels (< 2 fold) as observed in the plus AMP background condition.
Figure 2
Uptake of [3H]-estradiol 17β-glucuronide (1 µM) in MRP3 membrane vesicles with ATP or AMP in the presence and absence of the inhibitor terfenadine (300 µM).

To confirm ATP-dependent transporter involvement in the uptake of estradiol 17β-glucuronide in the MRP3 membrane vesicles, the inhibitor terfenadine was included in the incubations. This reduced the uptake to similar levels (< 2 fold) as observed in the plus AMP background condition.
Figure 3
Uptake of [3H]-estradiol 17β-glucuronide (1 µM) in MRP4 membrane vesicles with ATP or AMP in the presence and absence of the inhibitor MK-571 (10 µM).

To confirm ATP-dependent transporter involvement in the uptake of estradiol 17β-glucuronide in the MRP4 membrane vesicles, the inhibitor MK-571 was included in the incubations. This reduced the uptake to similar levels (< 2 fold) as observed in the plus AMP background condition.

References

1 Zamek-Gliszczynski MJ et al., (2018) Transporters in Drug Development: 2018 ITC Recommendations for Transporters of Emerging Clinical Importance. Clin Pharmacol Ther 104(5): 890-899
2 Draft FDA Guidance for Industry – Drug Interaction Studies – Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations, February 2012
3 Draft FDA Guidance for Industry – In Vitro Metabolism- and Transporter- Mediated Drug-Drug Interaction Studies, October 2017

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