Investigating the impact of OCT transporter genotype on metformin-induced vitamin B12 deficiency

• Main Author: Green, L. A.
• Other Authors: Pirmohamed, M. ; Park, B. ; Antoine, D.
• Published: University of Liverpool 2016
• Subjects: 616.3
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706764

Metformin is the first-line therapy for the treatment of Type 2 Diabetes Mellitus (T2DM) and is the most widely prescribed anti-diabetic drug in the world. A common and potentially hazardous side-effect of metformin treatment is vitamin B12 deficiency. OCT1 (SLC22A1) and OCT2 (SLC22A2) are highly polymorphic drug transporters responsible for the hepatic and renal uptake of metformin, respectively. Studies have documented that variants in OCT1 and OCT2 can have an effect on transporter function altering the pharmacokinetic profile of metformin, which leads to inter-individual variability in metformin response. To date, there is no reported correlation between the systemic concentration of metformin, metformin dose, and vitamin B12 levels, and whether this is affected by OCT gene polymorphisms. Thus, the overall aim of the studies described in this thesis was to explore the relationship between metformin pharmacokinetic parameters, OCT genetic variants and vitamin B12. To investigate this, we used biological samples from a cohort of 75 T2DM patients receiving metformin. In order to quantify metformin in human plasma, we designed and developed a sensitive and transferable hydrophilic interaction liquid chromatography (HILIC) method for both UV and mass spectrometric detection. This was successfully used to quantify metformin plasma levels in our cohort which ranged from 49 to 4908 ng/mL with a mean of 1879 ng/mL. To genotype patients for OCT1 and OCT2 polymorphisms, the SLC22A1 AND SLC22A2 genes were sequenced using Sanger sequencing. Fifty genetic variants were identified within the 5’ untranslated region (5’UTR), 3’UTR, exon and exon-intronic boundaries across both genes. Two novel single nucleotide polymorphisms (SNPs) were identified in SLC22A1 (g.-59C > T, c.+14 A > G). The potential effect of these variants on transporter function was explored using bioinformatic algorithms and in silico 3D structural modelling and ligand docking techniques. The metformin plasma concentrations and genetic data was collated with other clinical and biological data and incorporated into a population pharmacokinetic (PopPK) model to assess the influence genetic variants on metformin pharmacokinetics. Serum urea levels were the biggest predictor of metformin clearance. Only one genetic variant, the rs113569197 insertion was shown to be a significant influence on the predicted population metformin clearance in a univariate model. The rs113569197 variant was of particular interest as it represented an 8 bp insertion across an exon-intron boundary in SLC22A1, which we predicted to result in a premature stop codon and truncated protein. However, in multivariate PopPK modelling, this variant dropped out possibly because of lack of power. Multivariate linear regression analysis was used to explore the effect of metformin clinical variables and OCT genetic variants on vitamin B12 concentrations. Metformin dose, mg/kg of body weight (P < 0.0001) and serum folate levels (P=0.048) contributed independently to explain 32% (P < 0.0001) of the variance in serum vitamin B12 concentrations, but there was no effect of metformin plasma concentration (P=0.08) or genetic variants in SLC22A1 and SLC22A2. These data show that decreased serum vitamin B12 concentrations in patients with T2DM are driven more by metformin dose than exposure indicating that the mechanism by which metformin causes vitamin B12 deficiency is due to its effects at the level of the intestine, rather than systemically. In conclusion, the work presented in this thesis demonstrates that the mechanism of metformin-induced vitamin B12 deficiency is related to the dose, but not the plasma concentration of metformin. Genetic variants in SLC22A1 and SLC22A2 did not influence vitamin B12 levels in this patient group. Further work is required to define whether metformin specific drug transporters expressed in the intestine are responsible for metformin-induced vitamin B12 deficiency in order to better understand the pathogenesis.