Transcript Young Innovators 2009

YOUNG INNOVATORS 2009
Drug 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
2 Department
of Pharmacy, Uppsala University, Sweden
of Biopharmaceutical Sciences, University of California San Francisco, San Francisco,
California, USA.
ABSTRACT
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•
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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 drugdrug 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 OCT1reference. The IC50 of the concomitantly administered drug verapamil on metformin uptake in OCT1M420del 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.
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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.
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INTRODUCTION
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].
0
OATP2B1
OATN2
OATP4C1
MCT5
OCT3
PEPT1
OATP1A2
OAT2
UST3
MCT1
OATP1B3
NTCP
OATP1B1
OCT1
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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.
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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).
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RESULTS
Figure 2: Confocal fluorescence
images of HEK293 cells
GFP only a
transfected with GFP only (a).
HEK293 cells stable transfected
with GFP tagged OCT1-reference
(b) and M420del (c). The specific
OCT1 -reference
b
GFP staining is shown in the left
images as green staining whereas
plasma membrane was stained
(red staining) using Alexa Fluor
OCT1 -M420del c
594 conjugated to wheat germ
agglutinin. The images to the right
show the overlap (yellow staining)
between GFP specific staining and
membrane staining.
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100 %
b
100
50
22.4%
9.0%
3
-1
-1
nmolmin mg protein
150
14
a
C-metformin uptake (%)
RESULTS
2
1
0
0
Reference
M420del
R61C
0
250
500
750 1000 1250 1500 1750
Metformin (M)
50
pmolmg protein
-1
c
40
30
20
10
0
-3
-2
-1
0
log C (M)
1
2
3
Figure 3: (a) The 14C-metformin uptake in OCT1reference 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
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RESULTS
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.
Frequency
Frequency
(%)11
(%)
Amitriptyline
Amitriptyline
Glibenclamide
Glibenclamide
Pioglitazone
Pioglitazone
Simvastatin
Simvastatin
Verapamil
Verapamil
3.2
3.2
37.0
Reported
2
Reported
Cmax (μM)
2
Ratio
M420del
Reference
Predicted
4
3
Predicted (μM)Reference
Ref/M420del
IC50 (M) Ratio
IC50 (M) M420del
C
max, portal 3
4
Ref/M420del
Cmax (μM)
Cmax, portal (μM)
IC50 (M)
0.72
33.0
11.3 (1.66)
12.6 (1.69)
0.90
85.8 (1.97)***
2.32
0.72
0.73
33.0
1.41
IC50 (M)
11.3 (1.66)
ns
12.6 (1.69)
ns
0.90
199 (1.47)
85.8 (1.97)***
2.32
0.73
1.41
199 (1.47)
21.3
21.3
4.49
4.49
12.3
12.3
178 (2.62)* 1.04
(1.46)
185 185
(1.46)
178 (2.62)*
1.04
20.7
20.7
0.13
0.13
9.03
9.03
26.5 (1.87)***3.36
(1.25)
89.089.0
(1.25)
26.5 (1.87)***
3.36
37.0
4.9
4.9
0.60
0.60
15.05
15.05
ns
ns
0.21 (1.46) 8.33
(1.62)
1.751.75
(1.62)
0.21 (1.46)
8.33
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.
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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.
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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.
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ACKNOWLEDGMENTS
Uppsala University
• Per Artursson
• Christel Bergström
• All other cell group members
UCSF
• Kathleen Giacomini
• Ying Chen
• Alexandra Ianculescu
Kaiser permanente, Georgia
• Robert Davis
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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
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BIOS/CONTACT INFO
Gustav Ahlin
PhD
Adress:
Department of Pharmacy, Uppsala University
Box 580
751 23 Uppsala
Sweden
Phone: +46705167258
Mail: [email protected]
Young Innovators 2009