1. Y OUNG I NNOVATORS 2009 Drug transport by genetic variants of the liver specific human organic transporter OCT1 (SLC22A1). Effect of concomitantly administered drugs on metformin uptake. Gustav Ahlin 1, Ying Chen 2, Alexandra G. Ianculescu 2, Kathleen M. Giacomini 2 and Per Artursson 1 1 Department of Pharmacy, Uppsala University, Sweden 2 Department of Biopharmaceutical Sciences, University of California San Francisco, San Francisco, California, USA.

2. A BSTRACT Purpose: The highly polymorphic organic transport protein, OCT1; SLC22A1 is involved in the translocation of the hydrophilic anti-diabetic drug metformin from the blood into hepatocytes. Since many drugs are inhibitors of the OCT1 [1], this may lead to drug-drug interactions at the OCT1. The risk of drug- drug interactions may be further increased in individuals with an OCT1 protein with reduced function. Methods: We used HEK293 cells stably transfected with OCT1-reference and the common genetic variant M420del to investigate the inhibition of metformin transport at OCT1. The concentration dependent inhibition by drugs concomitantly used with metfomin was investigated using 14 C-labeled metformin as an OCT1 substrate. Results: Inhibition of metformin transport in OCT1-M420del was more pronounced than in the OCT1- reference. The IC 50 of the concomitantly administered drug verapamil on metformin uptake in OCT1- M420del was 0.21  M, in the presence of human serum albumin. This was similar to the plasma concentration of verapamil (0.15  M). Similarly, other concomitant drugs inhibited metformin uptake at their respective clinical plasma concentration. Conclusions:This study suggests that the risk for drug-drug interactions with metformin at the liver specific OCT1 may be increased in individuals carrying an OCT1 with reduced function. Further, our results suggest that several concomitantly administered drugs may lead to drug-drug interactions with metformin. Young Innovators 2009

3. I NTRODUCTION The organic transport protein, OCT1; SLC22A1 is highly expressed at the sinusoidal membrane of the liver (figure 1, [1]) and is involved in the translocation of the hydrophilic anti-diabetic drug metformin from the blood into hepatocytes. Since many drugs are inhibitors of the OCT1 [2], this may lead to drug-drug interactions between metformin and drugs that inhibit OCT1. The OCT1 is also a highly polymorphic transport protein with many variants displaying a reduced function. The reduced function in combination with concomitant drugs interacting with OCT1 may further increase the risk for drug-drug interactions in individuals carrying an OCT1 protein with reduced function. Young Innovators 2009

4. I NTRODUCTION Young Innovators 2009 Figure 1: Out of 36 transporters involved in drug transport OCT1 is the highest expressed transport protein in human liver. Only the 14 highest expressed SLC transporters are shown in this figure [1].

5. M ATERIALS AND M ETHODS HEK293 cells stably transfected with OCT1-reference and the common genetic variant M420del were used to investigate metformin transport and inhibition of metformin transport. Subcellular localization was assessed using GFP-fused OCT1 proteins (figure 2). Drugs concomitantly used with metformin was investigated as potential inhibitors of OCT1 mediated uptake of 14 C labeled metformin uptake. Concentration dependent inhibition curves were around the reported C max of these these drugs were generated and their clinical inhibitory potential was analysed. Young Innovators 2009

6. R ESULTS Figure 2 shows that both OCT1-reference and M420del is located at the cell membrane. However, the metformin uptake show a substantial reduction for M420del compared to the OCT1-reference (figure 2). Inhibition of metformin transport in OCT1-M420del was more pronounced compared to the OCT1 reference (figure 2c). In the presence of human serum albumin, the IC 50 of the concomitantly administered drug verapamil on metformin uptake in OCT1 M420del was 0.21  M (table 1). This is lower than the reported plasma concentration of verapamil (0.60  M). Similarly, other concomitant drugs inhibited metformin uptake at their respective portal vein plasma concentration (table 1). Young Innovators 2009

7. R ESULTS Young Innovators 2009 Figure 2: Confocal fluorescence images of HEK293 cells transfected with GFP only (a). HEK293 cells stable transfected with GFP tagged OCT1-reference (b) and M420del (c). The specific GFP staining is shown in the left images as green staining whereas plasma membrane was stained (red staining) using Alexa Fluor 594 conjugated to wheat germ agglutinin. The images to the right show the overlap (yellow staining) between GFP specific staining and membrane staining.

8. R ESULTS Young Innovators 2009 ab c Figure 3: (a) The 14 C-metformin uptake in OCT1- reference and M420del relative to uptake in OCT1-reference. A significant reduction in metformin uptake was seen for M420del. (b) Michaelis-Menten kinetic curves for the uptake of 14 C-metformin in OCT1-reference and M420del. (c) The concentration dependent effect of the OCT1 inhibitor verapamil on the uptake of the metformin

9. R ESULTS Young Innovators 2009 Table 1: IC 50 values and the IC 50 ratios for the OCT1-reference and M420del. IC 50 values were derived from concentration dependent inhibition curves of metformin uptake.

10. D ISCUSSION The data presented here for the first time shows that genetic variants of transporters with reduced function are more susceptible to drug inhibition compared to variants with normal function. This may help to explain the reason for drug-drug interactions found in vivo. However, further studies are needed to validate this finding for other transporters than the OCT1. The suggested drug-drug interaction between metformin and verapamil also demands further investigation in in vivo settings. Young Innovators 2009

11. C ONCLUSION This study suggests that the risk for drug-drug interactions with metformin at the liver specific OCT1 may be increased in individuals carrying an OCT1 with reduced function. Further, our results suggest that several concomitantly administered drugs, e.g. verapamil, may lead to drug-drug interactions with metformin. Young Innovators 2009

12. A CKNOWLEDGMENTS Young Innovators 2009 Uppsala University Per Artursson Christel Bergström All other cell group members UCSF Kathleen Giacomini Ying Chen Alexandra Ianculescu Kaiser permanente, Georgia Robert Davis

13. R EFERENCES [1] Hilgendorf et al. Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines. Drug Metab Dispos 2007; 35(8): 1333-1340 [2] Ahlin et al. Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1. J Med Chem 2008; 51(19): 5932-5942 Young Innovators 2009

14. BIOS/C ONTACT INFO Gustav Ahlin PhD Adress: Department of Pharmacy, Uppsala University Box 580 751 23 Uppsala Sweden Phone: +46705167258 Mail: gustav.ahlin@farmaci.uu.se Young Innovators 2009