Y OUNG I NNOVATORS 2009 Drug transport by genetic variants of the liver specific human organic transporter OCT1 (SLC22A1). Effect of concomitantly administered

YOUNG INNOVATORS 2009Drug transport by genetic variants of the liver

specific human organic transporter OCT1 (SLC22A1). Effect of concomitantly administered

drugs on metformin uptake.

Gustav Ahlin1, Ying Chen2, Alexandra G. Ianculescu2, Kathleen M. Giacomini2 and Per Artursson1

1 Department of Pharmacy, Uppsala University, Sweden 2 Department of Biopharmaceutical Sciences, University of California San Francisco, San Francisco,

California, USA.

ABSTRACT

• 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 14C-labeled metformin as an OCT1 substrate.

• Results: Inhibition of metformin transport in OCT1-M420del was more pronounced than in the OCT1-reference. The IC50 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

INTRODUCTION

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.


INTRODUCTION


OATP2B1

OATN2

OATP4C1

MCT5

OCT3

PEPT1

OATP1A2

OAT2

UST3

MCT1

OATP1B3

NTCP

OATP1B1

OCT1

0 1 2 3 6 9

relative gene expression level

OATP2B1

OATN2

OATP4C1

MCT5

OCT3

PEPT1

OATP1A2

OAT2

UST3

MCT1

OATP1B3

NTCP

OATP1B1

OCT1

0 1 2 3 6 9

relative gene expression level

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].

MATERIALS AND METHODS

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 14C labeled metformin uptake. Concentration dependent inhibition

curves were around the reported Cmax of these these drugs were generated and

their clinical inhibitory potential was analysed.


RESULTS

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 IC50 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).


RESULTS


OCT1 -reference

GFP only

OCT1 -M420del

a

b

c

OCT1 -reference

GFP only

OCT1 -M420del

a

b

c

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.

0 250 500 750 1000 1250 1500 17500

1

2

3

Metformin (M)

nm

ol

min

-1 m

g p

rote

in-1

-3 -2 -1 0 1 2 30

10

20

30

40

50

log C (M)

pm

ol

mg

pro

tein

-1100 %

22.4%9.0%

0

50

100

150

Reference M420del R61C

14C-

met

form

in u

ptak

e (%

)RESULTS


a b

c Figure 3: (a) The 14C-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 14C-metformin in OCT1-reference and M420del. (c) The concentration dependent effect of the OCT1 inhibitor verapamil on the uptake of the metformin

RESULTS


1. The treatment frequency of the drugs were derived from prescription data for 11319 US patients treated with the type 2 diabetes drug metformin.2. The reported total Cmax. Obtained from Goodman and Gillman´s and Clarke´s isolation and identification of drugs.3. Predicted portal vein concentration as described by Ito et. al. 1998.4. IC50 ratios between OCT1-reference and M420del.

Amitriptyline 3.2 0.72 33.0

Glibenclamide 37.0 0.73 1.41

Pioglitazone 21.3 4.49 12.3

Simvastatin 20.7 0.13 9.03

Verapamil 4.9 0.60 15.05

Frequency Reported Predicted Reference M420del Ratio(%)1

Cmax (μM)2 Cmax, portal (μM)3 IC50 (M) IC50 (M) Ref/M420del4

Amitriptyline 3.2 0.72 33.0 11.3 (1.66) 12.6 (1.69)ns0.90

Glibenclamide 37.0 0.73 1.41 199 (1.47) 85.8 (1.97)*** 2.32

Pioglitazone 21.3 4.49 12.3 185 (1.46) 178 (2.62)* 1.04

Simvastatin 20.7 0.13 9.03 89.0 (1.25) 26.5 (1.87)*** 3.36

Verapamil 4.9 0.60 15.05 1.75 (1.62) 0.21 (1.46)ns8.33

Table 1: IC50 values and the IC50 ratios for the OCT1-reference and M420del. IC50 values were derived from concentration dependent inhibition curves of metformin uptake.

DISCUSSION

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.


CONCLUSION

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.


ACKNOWLEDGMENTS


Uppsala University • Per Artursson• Christel Bergström• All other cell group members

UCSF• Kathleen Giacomini• Ying Chen• Alexandra Ianculescu

Kaiser permanente, Georgia • Robert Davis

REFERENCES

[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


BIOS/CONTACT INFO

Gustav Ahlin

PhD

Adress:

Department of Pharmacy, Uppsala University

Box 580

751 23 Uppsala

Sweden

Phone: +46705167258

Mail: [email protected]


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Y OUNG I NNOVATORS 2009 Drug transport by genetic variants of the liver specific human organic transporter OCT1 (SLC22A1). Effect of concomitantly administered