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ADME PK

Single and Double Transporter Knockout Cell-Based Assays

  • Functional single and double knockouts of the intestinal efflux transporters, MDR1, BCRP, MRP2, MDR1/MRP2, MDR1/BCRP and MRP2/BCRP are currently available.
  • Cells created by Sigma® Life Science using CompoZr® Zinc Finger Nuclease (ZFN) technology.
  • The Caco-2-derived cell line (C2BBe1) is utilized which is more physiologically relevant than most other cell line systems because:
    • Caco-2 cells express multiple transporters
    • C2BBe1 cells are a human-derived cell line with no interference from animal transporters
    • By comparing parental (wild type) cells against the knockout transporter cells, the transporter(s) responsible for drug efflux can be elucidated without the reliance on specific substrates and/or inhibitors.
    • Double knockout cell lines provide greater scrutiny of drug efflux when multiple transporters are involved.
Zinc-finger nucleases (ZFNs) are targetable DNA cleavage reagents that have been adopted as gene-targeting tools. ZFN-induced double strand breaks are subject to cellular DNA repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies.

1Carroll D (2011) Genetics 188; 773-782

Protocol

Single and Double Transporter Knockout Cell-Based assay protocol

Test Article Concentration 10 μM
Transporter Systems Available Parental Caco-2 derived cell line (C2BBe1) and MDR1, BCRP and MRP2 single and double knockout cell lines
Period of Cell Culture 20-22 days
Number of Replicates 2
Incubation Time 2 hr
Integrity Marker Lucifer Yellow
Analysis Method LC-MS/MS
Data Delivery Papp
Efflux ratio
Experimental recovery

Data

Data from Cyprotex's Single and Double Transporter Knockout Cell-Based assays

 
Functional Knockouts of Efflux Transporters
Figure 1
Functional Knockouts of Efflux Transporters created by Sigma® Life Science using CompoZr® Zinc Finger Nuclease Technology.
Proposed Screening Strategy for Determining Role of MDR1, BCRP and MRP2 in Transporter Activity
Figure 2
Proposed Screening Strategy for Determining Role of MDR1, BCRP and MRP2 in Transporter Activity.

The screening strategy described above can be used to identify if a test compound is a substrate for MDR1, BCRP or MRP2. Initially this involves investigating the efflux ratio in the parental C2BBe1 cell line. If the efflux ratio is less than 2 then it is assumed that the test compound is not a substrate for MDR1, BCRP or MRP2. If the efflux ratio is greater than 2 then the test compound can be screened in the MDR1, BCRP and MRP2 knockout cell lines to determine if the test compound is a substrate for these transporters.
Functional Knockouts of Efflux Transporters
Figure 3
Efflux Ratios for Colchicine (MDR1 Substrate), Nitrofurantoin (BCRP Substrate), CDCFDA (MRP2 Substrate) and Cimetidine (MDR1 and BCRP Substrate) in C2BBe1 Wild Type and Single and Double Knockout Transporter Cell Lines.

The single knockout cells are effective at identifying compounds which are effluxed by a single intestinal transporter. For more complex interactions where multiple transporters are involved (e.g., in the case of cimetidine), the double knockouts are able to provide confirmation of the role of more than one transporter and which transporters are responsible for the efflux.

References

1 Carroll D (2011) Genetics 188; 773-782

Sigma and CompoZr are registered trademarks of Sigma-Aldrich Co. LLC.

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