In vitro validation of an in vivo phenotyping drug cocktail for major drug transporters in humans

Eur J Pharm Sci. 2023 Jul 1:186:106459. doi: 10.1016/j.ejps.2023.106459. Epub 2023 May 3.


Purpose: Cocktails of transporter probe drugs are used in vivo to assess transporter activity and respective drug-drug interactions. An inhibitory effect of components on transporter activities should be ruled out. Here, for a clinically tested cocktail consisting of adefovir, digoxin, metformin, sitagliptin, and pitavastatin, inhibition of major transporters by individual probe substrates was investigated in vitro.

Methods: Transporter transfected HEK293 cells were used in all evaluations. Cell-based assays were applied for uptake by human organic cation transporters 1/2 (hOCT1/2), organic anion transporters 1/3 (hOAT1/3), multidrug and toxin extrusion proteins 1/2K (hMATE1/2K), and organic anion transporter polypeptide 1B1/3 (hOATP1B1/3). For P-glycoprotein (hMDR1) a cell-based efflux assay was used whereas an inside-out vesicle-based assay was used for the bile salt export pump (hBSEP). All assays used standard substrates and established inhibitors (as positive controls). Inhibition experiments using clinically achievable concentrations of potential perpetrators at the relevant transporter expression site were carried out initially. If there was a significant effect, the inhibition potency (Ki) was studied in detail.

Results: In the inhibition tests, only sitagliptin had an effect and reduced hOCT1- and hOCT2- mediated metformin uptake and hMATE2K mediated MPP+ uptake by more than 70%, 80%, and 30%, respectively. The ratios of unbound Cmax (observed clinically) to Ki of sitagliptin were low with 0.009, 0.03, and 0.001 for hOCT1, hOCT2, and hMATE2K, respectively.

Conclusion: The inhibition of hOCT2 in vitro by sitagliptin is in agreement with the borderline inhibition of renal metformin elimination observed clinically, supporting a dose reduction of sitagliptin in the cocktail.

Keywords: ABC; Membrane transporters; Membrane vesicles; SLC; Transporter-mediated drug-drug interactions.

MeSH terms

  • Biological Transport
  • Drug Interactions
  • HEK293 Cells
  • Humans
  • Metformin* / metabolism
  • Organic Cation Transport Proteins* / genetics
  • Organic Cation Transport Proteins* / metabolism
  • Organic Cation Transporter 2 / metabolism
  • Sitagliptin Phosphate / pharmacology


  • Organic Cation Transport Proteins
  • Sitagliptin Phosphate
  • Metformin
  • Organic Cation Transporter 2