Is N 1-Methylnicotinamide a Good Organic Cation Transporter 2 (OCT2) Biomarker?

Abstract

Background/Objectives: The impact of potential precipitant drugs on plasma or urinary exposure of endogenous biomarkers is emerging as an alternative approach to evaluating drug-drug interaction (DDI) liability. N1-Methylnicotinamide (NMN) has been proposed as a potential biomarker for renal organic cation transporter 2 (OCT2). NMN is synthesized in the liver from nicotinamide by nicotinamide N-methyltransferase (NNMT) and is subsequently metabolized by aldehyde oxidase (AO). Multiple clinical studies have shown a reduction in NMN plasma concentration following the administration of OCT inhibitors such as cimetidine, trimethoprim, and pyrimethamine, which contrasts with their inhibition of NMN renal clearance by OCT2. We hypothesized that OCT1-mediated NMN release from hepatocytes is inhibited by the administration of OCT inhibitors. Methods: Re-analysis of the reported NMN pharmacokinetics with and without OCT inhibitor exposure was performed. We assessed the effect of cimetidine on NMN uptake in OCT1-HEK293 cells and evaluated the potential confounding effects of cimetidine on enzymes involved in NMN formation and metabolism. Results: A re-analysis of previous NMN pharmacokinetic DDI data suggests that NMN plasma systemic exposure decreased by 17-41% during the first 4 h following different OCT inhibitor administration except dolutegravir. Our findings indicate that NMN uptake was significantly higher (by 2.5-fold) in OCT1-HEK293 cells compared to mock cells, suggesting that NMN is a substrate of OCT1. Additionally, our results revealed that cimetidine does not inhibit NNMT and AO activity. Conclusions: Our findings emphasize the limitations of using NMN as an OCT2 biomarker and reveal potential mechanisms behind the reduction in NMN plasma levels associated with OCT inhibitors. Instead, our data suggest that NMN could be tested further as a potential biomarker for OCT1 activity.

Keywords: drug–drug interactions; endogenous biomarkers; hepatic transport; organic cation transport; pharmacokinetic; renal transport.

Figure 1

Schematic presentation of the formation, metabolism, and release of N1-methylnicotinamide (NMN) in the liver. NMN is synthesized in the liver from nicotinamide through the enzymatic action of nicotinamide N-methyltransferase (NNMT). NMN undergoes further metabolism catalyzed by aldehyde oxidase (AO), forming N1-methyl-2-pyridone-5-carboxamide (2PYR) and N1-methyl-4-pyridone-3-carboxamide (4PYR). NMN is then released into the systemic circulation through hepatic organic cation transporter 1 (OCT1). NMN is eliminated in the urine primarily through active tubulure secretion mediated by OCT2 and multidrug and toxin extrusion 1 (MATE1) and MATE2K transporters. The figure was created using BioRender.

Figure 2

Effect of cimetidine, pyrimethamine, trimethoprim, and dolutegravir (OCT inhibitors) on N1-methyl nicotinamide (NMN) plasma concentrations in healthy adults. Plasma concentration–time profiles of NMN with and without cimetidine exposure (Data from Ailabouni et al., 2024 [13] (A), Data from Müller et al., 2023 [11] (B), and Data from Koishikawa et al., 2024 [14] (C)); pyrimethamine exposure (Data from Ito et al., 2012 [9] (D)); trimethoprim exposure (Data from Müller et al., 2015 [10] (E)); and dolutegravir exposure (Data from Koishikawa et al., 2024 [14] (F)). NMN plasma concentrations in absence of OCT inhibitor (baseline) are indicated with green open circles, while NMN plasma concentrations in presence of OCT inhibitors are indicated with blue solid circles.

Figure 3

Inhibition of OCT1-mediated N1-ethylnicotinamide (NMN) transport by cimetidine in HEK293-OCT1 cell lines. Uptake of 50 µM NMN by HEK293 cells overexpressing OCT1 and mock HEK293 cells after 5 min incubation (A). Uptake of 50 µM NMN by HEK293 cells overexpressing OCT1 alone and with three different concentrations of cimetidine (1,10, 100, and 200 µM) (B). Uptake of 100 µM metformin (positive control) by HEK293 cells overexpressing OCT1 alone and with three different concentrations of cimetidine (1,10, 100, and 200 µM) (C). Each point represents mean ± SE (n = 4). Two-tailed unpaired t-test; p-value < 0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

Figure 4

Effect of cimetidine on aldehyde oxidase (AO) activity. The 4-Oxo-carbazeran MRM signal intensity and retention time (A). Mass fragmentation pattern of carbazeran (CBZ) and its metabolite oxo-carbazeran (4-oxo-CBZ) in ESI positive mode. The exact masses of fragment ions (in red text) are depicted on the structure, while neutral losses are shown in the HR–MS/MS line spectra (green dotted lines) (B). Carbazeran metabolism to 4-oxo-carbazeran by AO in human liver cytosol alone and with three different concentrations of cimetidine (1, 10, and 100 µM) (C). In (C), each point represents mean ± SE (n = 3). Two-tailed unpaired t-test; p-value > 0.05 (ns, not significant).

Figure 5

Effect of cimetidine on nicotinamide N-methyltransferase (NMNT) activity for N1-methylnicotinamide (NMN) formation. NMN MRM signal intensity and retention time (A). Mass fragmentation pattern of nicotinamide and its metabolite NMN in ESI positive mode. The exact masses of fragment ions (in red text) are depicted on the structure, while neutral losses are shown in the HR–MS/MS line spectra (green dotted lines) (B). Nicotinamide metabolism to NMN by using recombinant human NNMT alone and with three different concentrations of cimetidine (1, 10, and 100 µM) (C). In (C), each point represents mean ± SE (n = 3). Two-tailed unpaired t-test; p-value > 0.05 (ns, not significant).