Drug-Drug Interactions & Transporters

Membrane drug transporters, located in many barrier tissues in the body, may interact with drugs to affect absorption, distribution and elimination in vivo, and are the cause of clinically relevant drug-drug interactions (DDIs). Identification of the test article as a substrate or an inhibitor of transporters can explain clinically relevant altered exposures and toxicities of concomitantly administered drugs.

Intestinal permeability of a drug is an important factor driving the fraction absorbed and can be evaluated using the Caco-2 monolayer transport assay.

Regulatory considerations for transporter interactions

These studies are recommended by both FDA and EMA drug-drug interaction (DDI) guidelines to evaluate transporter interactions before going into first-in-human trials. Transporter studies highlight potential issues with achieving efficacious plasma concentrations of concomitantly administered medications due to a drug's impact on absorption or distribution.

Several transporters interact with drugs in clinical use. Those recommended for testing include:

  • ATP binding cassette (ABC) efflux transporters P-glycprotein (P-gp), Breast Cancer Resistance Protein (BCRP), and Bile Salt Export Pump (BSEP).
  • Solute carrier (SLC) uptake transporters Organic Anion Transporter (OAT) 1 and OAT3, Organic Cation Transporter (OCT) 2, Organic Anion Transporting Polypeptide (OATP) 1B1, OATP1B3, and Multidrug and Toxin Extrusion (MATE) 1 and MATE2 K.

Methods for Transporter Interactions:

Uptake Transporter Assay
  • Test system: Transfected cell lines expressing a single uptake (SLC) transporter or vector control, grown in monolayers in a humidified incubator at 37°C in 5% CO2.
  • Assay buffer: HBSS with HEPES, pH 7.4
  • For MATE transporters only, cells are pretreated with NH4Cl to introduce a pH gradient across the cell membrane, required for MATE uptake activity
  • Test article concentrations: 2 for substrate assays, 2 for inhibition assays
  • Radioalabeled probe substrates specific for each transporter
  • Positive control inhibitors for each transporter

Transporter-expressing cells in culture are preincubated with an assay buffer for 30 minutes in a humidified incubator at 37°C in 5% CO2. MATE-expressing cells are treated with NH4CI to create a pH gradient across cell membrane to facilitate uptake activity. A probe substrate or test article is added to triplicate wells, and inhibitors are added where applicable. Plates are incubated for one time point from 2 - 10 minutes depending on the transporter. The buffer is aspirated; and cells are washed, then lysed, collected and analyzed for presence of the test article using LC-MS.

Substrate assessment: Uptake of the test article by each transporter alone or in the presence of selective inhibitor will be compared to uptake by the vector control after incubation for 2-5 minutes. Uptake of a probe substrate by each transporter alone or in the presence of selective inhibitor and by the vector control will be performed as controls.

Inhibitor assessment: Uptake of a probe substrate by each transporter will be conducted alone or in the presence of a selective inhibitor or the test article. Uptake of the probe substrate by the vector control will also be performed as a control. If inhibition is observed, the IC50 may be determined.

Efflux Transporter Assay
  • Test system: Caco-2 human intestinal endothelial cells, cultured on Transwell plates in a humidified incubator at 37°C in 5% CO2 for 21- 24 days
  • Assay buffer: HBSS with HEPES, pH 7.4 in both apical and basolateral chambers
  • Transendothelial  electrical resistance (TEER) is measured prior to assay to confirm the formation of tight junctions
  • Test article concentrations: 2 for substrate assays, 2 for inhibition assays
  • Radiolabeled permeability markers mannitol and caffeine are used to verify tight junction formation
  • Radiolabeled probe substrates are used, specific for each transporter
  • Positive control selective inhibitors for each transporter with negative inhibitor controls

Apparent permeability of test article and probe substrates is determined in both the apical to basolateral (A-B) and basolateral to apical (B-A) directions on transwell plates. After 30 minutes of preincubation in an assay buffer in a humidified incubator at 37°C in 5% CO2, the probe or test article is added to one chamber (donor) with a blank buffer in the opposite chamber (receiver). Inhibitors are then added to both chambers where applicable. Plates are incubated at 37°C in 5% CO2 for 2 hours. Donor and receiver chamber samples are collected and analyzed for presence of the test article using LC-MS. Then test article permeability and efflux ratio are determined.

Substrate assessment: The transport of the test article alone or in the presence of selective inhibitors will be determined in both directions. Transport of a probe substrate for each transporter alone or in the presence of selective inhibitors will be performed as controls.

Inhibitor assessment:  Transport of a probe substrate will be determined in both directions after incubation for 2 hours alone or in the presence of selective inhibitor or the test article. If inhibition of efflux transport is observed, the IC50 may be determined.

Vesicular Membrane Uptake Assay
  • Test system: Membrane vesicles (50 µg/well) expressing efflux (ABC) transporter BSEP.
  • Test article concentrations: 2 for substrate assays; 2 for inhibition assays.
  • Adenosine triphosphate (ATP)-dependent activity measured, with adenosine monophosphate (AMP) used as control.
  • Radiolabeled probe substrate specific for BSEP
  • Positive control inhibitors for each transporter

Membrane vesicles expressing BSEP and the test article or probe substrate will be incubated in 96 well plates. Uptake will be initiated by the addition of adenosine triphosphate (ATP), followed by incubation for 30 minutes at 37°C. Adenosine monophosphate (AMP) will be used in parallel as a negative control. The uptake will be terminated by vacuum aspiration and rinsing the filters twice with excess ice‑cold transport buffer. Vesicles are then extracted from filter plates for LC‑MS quantitation and calculation of ATP-dependent activity.

Substrate assessment: Uptake of the test article alone and with selective inhibitor in the presence of ATP will be compared to uptake in the presence of AMP.  Uptake of a probe substrate by BSEP alone and in the presence of selective inhibitor will be performed as controls.

Inhibitor assessment: Uptake of a probe substrate will be conducted alone and in the presence of selective inhibitor or the test article; uptake in the presence of ATP will be compared to uptake in the presence of AMP.  If inhibition of ATP-dependent uptake of the test article is observed, the IC50 may be determined.

 

Deliverables

These assays will identify and profile substrate or inhibition interactions with membrane drug transporters. Data will describe the extent of uptake and/or efflux, test article permeability, transporter inhibition and IC50 values as applicable.

These data may also be used to assess DDI risks based on likely interactions with concomitant therapeutics and help to rank new drug candidates. If interactions are observed, pharmacometrics may allow additional guidance when assessing the need for a clinical trial.