Effect of genetic variation in the organic cation transporter 2 on the renal elimination of metformin

Abstract

Objective: The goal of this study was to determine the effect of a genetic variant in the organic cation transporter 2 (OCT2), OCT2-808G/T, which results in an amino acid change, A270S, on the pharmacokinetics of the antidiabetic drug, metformin.

Methods: The uptake of metformin was performed in stably transfected HEK-293 cells expressing the empty vector (MOCK), the reference OCT2-808G, and the variant OCT2-808T. Healthy individuals with known OCT2 genotypes [14 homozygous for the OCT2 reference allele (808G/G) and nine heterozygous for the variant allele (808G/T, *3D)] were recruited to this study. Metformin concentrations in plasma and urine were measured by liquid chromatography-tandem mass spectrometry method. Creatinine levels were also measured in plasma and urine. Pharmacokinetic parameters were evaluated for both the groups.

Results: We observed that in HEK-293 stably transfected cells, OCT2-808T had a greater capacity to transport metformin than did the reference OCT2. Metformin pharmacokinetics was characterized in 23 healthy volunteers of Caucasian and African-American ancestries. We observed that the renal clearance (CL(R)) and the net secretion (SrCL(R)) of metformin were significantly different between the volunteers heterozygous for the variant allele (808G/T), and the volunteers homozygous for the reference allele (808G/G) (P<0.005). Multivariate analysis revealed that OCT2 genotype was a significant predictor of CL(R) and SrCL(R) of metformin (P<0.01).

Conclusion: We conclude that genetic variation in OCT2 plays an important role in the CL(R) and SrCL(R) of metformin in healthy volunteers.

Figure 1. Transport of metformin by reference OCT2 (OCT2-808G) and the variant (OCT2-808T)

(A) HEK-293 cells stably transfected with empty vector, OCT2-808G or OCT2-808T were incubated with 9.25 μM [¹⁴C] metformin for 30 seconds and uptake of metformin in these cells was determined. (B) These cells were incubated with 9.25 μM [¹⁴C] metformin as well as different concentrations of unlabeled metformin for 30 seconds. Final uptake values were obtained by subtracting the uptake in MOCK cells from that in the cells expressing OCT2 reference or the variant at each corresponding substrate concentration. Studies were performed in triplicate in each individual experiment. Data represent three separate experiments.

Figure 2. Expression level of OCT2 protein in cells stably transfect with OCT2-808G and OCT2-808T

A. Cells stably transfected with empty vector, OCT2-808G and OCT2-808T were harvested and analyzed by Western blot for levels of OCT2. Anti-β actin was used as the loading control. B. OCT2 levels were quantified by scanning densitometry and expressed as mean ± SD (n=3), with OCT2 reference value set to 1. * indicates that OCT2 level in OCT2-808T was statistically significant from that of OCT2-808G (p < 0.05, Student's t-test).

Figure 3. Mean metformin plasma concentration-time curves in individuals with OCT2 genetic variants

A single oral dose of 850 mg metformin HCl tablet was administered to subjects homozygous for the reference allele (808G/G, n=14, triangles) or those heterozygous for the variant allele (808G/T, n=9, squares). Blood was drawn at the indicated time points. Significant differences in metformin plasma concentrations between two genotypes are indicated by asterisks. Data represent mean ± SD.

Figure 4. Effect of OCT2 genotype on renal clearance (CL_R) or net tubular secretion (SrCL_R) of metformin

The metformin CL_R or SrCL_R of each individual is shown graphically. Note that there were significant differences in CL_R (p= 0.005) and SrCL_R (p= 0.002) between genotype groups.