Substrate-Dependent Inhibition of the Human Organic Cation Transporter OCT2: A Comparison of Metformin with Experimental Substrates

<div><p>The importance of the organic cation transporter OCT2 in the renal excretion of cationic drugs raises the possibility of drug-drug interactions (DDIs) in which an inhibitor (perpetrator) drug decreases OCT2-dependent renal clearance of a victim (substrate) drug. In fact, there are clinically significant interactions for drugs that are known substrates of OCT2 such as metformin. To identify drugs as inhibitors for OCT2, individual drugs or entire drug libraries have been investigated <i>in vitro</i> by using experimental probe substrates such as 1-methyl-4-phenylpyridinium (MPP⁺) or 4–4-dimethylaminostyryl-N-methylpyridinium (ASP⁺). It has been questioned whether the inhibition data obtained with an experimental probe substrate such as MPP⁺ or ASP⁺ might be used to predict the inhibition against other, clinical relevant substrates such as metformin. Here we compared the OCT2 inhibition profile data for the substrates metformin, MPP⁺ and ASP⁺. We used human embryonic kidney (HEK 293) cells stably overexpressing human OCT2 as the test system to screen 125 frequently prescribed drugs as inhibitors of OCT2-mediated metformin and MPP⁺ uptake. Data on inhibition of OCT2-mediated ASP⁺ uptake were obtained from previous literature. A moderate correlation between the inhibition of OCT2-mediated MPP⁺, ASP⁺, and metformin uptake was observed (pairwise <i>r</i>_s between 0.27 and 0.48, all <i>P</i> < 0.05). Of note, the correlation in the inhibition profile between structurally similar substrates such as MPP⁺ and ASP⁺ (Tanimoto similarity <i>T</i> = 0.28) was even lower (<i>r</i>_s = 0.27) than the correlation between structurally distinct substrates, such as ASP⁺ and metformin (<i>T</i> = 0.01; <i>r</i>_s = 0.48) or MPP⁺ and metformin (<i>T</i> = 0.01; <i>r</i>_<i>s</i> = 0.40). We identified selective as well as universal OCT2 inhibitors, which inhibited transport by more than 50% of one substrate only or of all substrates, respectively. Our data suggest that the predictive value for drug-drug interactions using experimental substrates rather than the specific victim drug is limited.</p></div

Inhibitors of OCT2-mediated metformin (1000 μM) and MPP⁺ (50 μM) transport identified in a screen of 125 drugs most commonly prescribed in Germany.

<p>(A) Inhibition of metformin transport. (B) Inhibition of MPP⁺ transport. Each bar represents one compound tested in HEK-OCT2 cells at a concentration of 20 μM. Bars showing a statistically significant (one-sample t test) inhibition are shaded in black. Data are presented as the mean +/- standard error (at least two experiments each on two or more separate days, i.e., <i>n</i> = 4–15). Negative inhibition values indicate enhanced OCT2-dependent substrate uptake in cells incubated with the respective test drug compared with vehicle-treated cells; ASS, acetylsalicylic acid.</p

Comparison of IC₅₀ values for inhibition of OCT2-dependent uptake of metformin and maximum therapeutic plasma concentrations in humans.

<p>C_max, maximum steady-state plasma concentration; C_max,u, C_max of unbound drug; IC₅₀, experimentally determined concentration for half maximal inhibition of OCT2-mediated metformin (1000 μM) uptake. C_max values were obtained from Regenthal <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136451#pone.0136451.ref016" target="_blank">16</a>].</p><p>Comparison of IC₅₀ values for inhibition of OCT2-dependent uptake of metformin and maximum therapeutic plasma concentrations in humans.</p

Concentration-dependent inhibition of OCT2-mediated metformin uptake into HEK-OCT2 cells.

<p>Shown is the inhibition of metformin uptake by psychoactive (A) and non-psychoactive drugs (B). Data are presented as the mean ± standard error (at least three experiments each on two separate days, i.e., <i>n</i> = 6).</p

OCT2-mediated metformin and MPP⁺ uptake.

<p>Time-dependent uptake of metformin (A, 1000 μM) and MPP⁺ (B, 50 μM) into OCT2-expressing HEK-cells and corresponding vector controls. Uptake of metformin (C) and MPP⁺ (D) in HEK-VC and HEK-OCT2 cells after three minutes. Data are presented as the mean ± standard error (4–8 experiments each on two to four separate days, i.e., <i>n</i> = 8–29).</p

Substrate-dependent differences in the inhibitory profiles and concentration-dependent inhibition of metformin uptake.

<p>(A) Correlation analysis between inhibition of OCT2-mediated metformin and inhibition of OCT2-mediated MPP⁺ uptake in HEK-OCT2 cells. Drugs were tested at 20 μM. Substrate concentrations were 1000 μM for metformin and 50 μM for MPP⁺ (data are presented as the mean ± standard error, Spearman's rank-order correlation test). (B) Bland-Altman plot (bias and limits of agreement) for the inhibition of OCT2-mediated metformin and MPP⁺ uptake. Average of % inhibition of metformin uptake and % inhibition of MPP⁺ is plotted against the difference between % inhibition of metformin uptake and % inhibition of metformin uptake, LoA, limits of agreement. Simvastatin, simvastatin lactone</p

Descriptive statistics of edits and editors of drug articles in Wikipedia.

<p>Descriptive statistics of edits and editors of drug articles in Wikipedia.</p

Study design.

<p>Pharmacological information of randomly selected curricular drugs was retrieved form two standard textbooks of general pharmacology for five different categories (indication, mechanism of action, adverse effects, pharmacokinetics and contraindications). The textbook data overlap served as point of reference for the corresponding drug articles in Wikipedia. The analysis was performed separately for the German and English language version of Wikipedia and textbooks.</p

Accuracy and completeness of drug information in Wikipedia.

<p>Analysis of (A) accuracy and (B) completeness of the German (left panel) and English (right panel) language Wikipedia in comparison to textbooks. Accuracy was defined as the degree of closeness to the pharmacological information of the textbooks overlap. Completeness was defined as percentage of pharmacological statements of the textbook overlap present in the respective Wikipedia articles. Box plots showing median, first and third quartile with whiskers depicting the 5% and 95% percentile. In (A), statistical outliers are shown as black dots. Note that some box plots appear as vertical lines due to high accuracy scores and low variability of data. IND, indication; MA, mechanism of action; AE, adverse effects; PK, pharmacokinetics; CI, contraindications.</p

<i>Alanine-glyoxylate aminotransferase 2</i> (<i>AGXT2</i>) Polymorphisms Have Considerable Impact on Methylarginine and β-aminoisobutyrate Metabolism in Healthy Volunteers

<div><p>Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common <i>AGXT2</i> single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding <i>AGXT2</i> SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the <i>AGXT2</i> SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated <i>AGXT2</i> SNP rs16899974, which was not significantly linked to the other <i>AGXT2</i> SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [²H₆]-SDMA as substrate. <i>In vitro</i>, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). <i>In silico</i> modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of <i>AGXT2</i> affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper-β-aminoisobutyric aciduria.</p></div