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

Most commonly used definite anticholinergic drugs (i.e. ACB score 2 or 3) among the total study population.

<p><i>Left y-axis</i> Absolute number of drugs with definite anticholinergic properties received by patients. <i>Right y-axis</i> Proportion of the respective drug of all definite anticholinergics according to the ACB scale.</p

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

Cognitive impairment measured by the Mini-Mental State Examination (MMSE) and mean anticholinergic cognitive burden in patients of the GiB-DAT database.

<p>A MMSE score of 0–17 indicates severe, 18–24 moderate and 25–30 no cognitive impairment. Error bar 95% confidence interval, *** p < 0.001 for overall and all pairwise comparisons.</p

High-QT-drugs and SmPC-high-risk-QT-drugs.

<p>High-QT-drugs and SmPC-high-risk-QT-drugs.</p

Anticholinergic burden and cognitive function in a large German cohort of hospitalized geriatric patients

<div><p>Purpose</p><p>Previous studies suggest an association between use of anticholinergic drugs in elderly patients and cognitive impairment. However, there are still limited data on the association of anticholinergic drug use and cognitive impairment as well as contribution of individual drugs to anticholinergic load using large, well-documented patient cohorts treated in geriatric units from Europe.</p><p>Methods</p><p>We investigated 797,440 prescriptions to 89,579 hospitalized patients treated in geriatric units within the GiB-DAT database. Data of all patients discharged between 1 January 2013 and 30 June 2015 was included. The Anticholinergic Cognitive Burden (ACB) scale was used to classify anticholinergic drugs as definite (score 2 or 3) and possible anticholinergics (score 1). Cognitive function was determined using Mini-Mental State Examination (MMSE) and the standardized scale for dementia (4D+S).</p><p>Results</p><p>In two multivariable logistic regression models age, sex, number of drugs and ACB total scores were identified as variables independently associated with cognitive impairment as measured by MMSE (odds ratio per ACB unit 1.114, 95% CI 1.099–1.130) or the diagnosis dementia (odds ratio 1.159 per ACB unit, 95% CI 1.144–1.173, both p < 0.0001). High anticholinergic load was associated with patients with severe cognitive impairment (p < 0.05 for all pairwise comparisons). ACB score 3 anticholinergic drugs contributed 77.9% to the cumulative amount of ACB points in patients with an anticholinergic load of 3 and higher.</p><p>Conclusions</p><p>Using a cross-sectional study design, a significant positive association between anticholinergic drug load and cognitive impairment in European patients treated in specialised geriatric units was found. The most frequently used definitve anticholinergic drugs were quetiapine, amitriptyline and carbamazepine.</p></div

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