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Thiamine, which consists of an aminopyrimidine and a thiazolium ring linked by a methylene bridge, is positively charged at pH 7.4. THTR‐1 transports thiamine with a K _m value of approximately 7 μM, whereas THTR‐2 has a K _m of approximately 4 μM. Both transporters are considered high‐affinity, low‐capacity thiamine transporters, whereas other human transporters that take up thiamine (i.e., OCT1, OCT2, MATE1) are of low affinity, high capacity [18]. Thiamine transport by both THTR‐1 and THTR‐2 is pH sensitive, optimal at neutral pH, and decreases as extracellular pH becomes acidic [55]. In November 2013, a clinical trial of fedratinib, a JAK2 kinase inhibitor, was halted due to the observation of Wernicke’s encephalopathy, a severe neurological disease induced by thiamine deficiency, in several subjects [56]. Subsequent studies revealed fedratinib as a potent inhibitor of THTR‐2, with the hypothesis that the aminopyrimidine structure present in fedratinib is responsible for the THTR‐2 interaction [57]. Fedratinib is also a substrate of THTR‐2 with a K _m ~ 0.44 μM, but is not a substrate of THTR‐1. Another JAK2 kinase inhibitor, AZD1480, inhibits both THTR‐1 and THTR‐2, with IC₅₀ values of 22 μM and 15 μM, respectively. A pharmacophore model created to identify features associated with potent inhibition of THTR‐1 and THTR‐2 validated the importance of the 2,4 diaminopyrimidine structure for substrate and inhibitor [56].

Additional prescription drugs have been identified as substrates of THTR‐1 and THTR‐2 using in vitro assays and computational models. For example, metformin has been identified as a substrate and inhibitor of THTR‐2 [55]. Additionally, MPP⁺, a neurotoxin associated with Parkinson’s disease, and famotidine, an over‐the‐counter histamine receptor antagonist used to treat acid reflux disease, have been identified as substrates of THTR‐2. Verapamil and chloroquine are inhibitors of THTR‐2‐mediated uptake of metformin at 1 mM. Substrate and inhibitor specificity differences exist between species, as exemplified by metformin, which is a substrate for human, but not mouse THTR‐2, and fedratinib, which demonstrates weaker inhibition of mTHTR‐2 compared with hTHTR‐2. Trimethoprim, an antibiotic used for a wide variety of infections, is not only an inhibitor but also a substrate of both THTR‐1 and THTR‐2, with K _m values of 22 μM and 1 μM, respectively.

A high‐throughput THTR‐2 drug inhibitor screen evaluating over 1,300 compounds, including many prescription and over‐the‐counter medications, identified several prescription drugs as novel inhibitors of THTR‐2 at low‐ or mid‐micromolar concentrations including hydroxychloroquine sulfate (IC₅₀ ~ 17 μM), sertraline hydrochloride (IC₅₀ ~ 1 μM), amitriptyline hydrochloride (IC₅₀ ~ 11 μM), amoxapine (IC₅₀ ~ 46 μM), quinapril (IC₅₀ ~ 34 μM). Based on the current FDA guidelines, all of these drugs are predicted to reach the threshold required for a clinical drug–drug interaction study [58].

The increasing number of approved drugs identified as potent THTR‐1 and/or THTR‐2 inhibitors initiates concerns regarding the potential for these common medications to cause clinical adverse events related to thiamine deficiency. Many of these drugs are predicted to reach concentrations in the intestine sufficiently high to significantly inhibit absorption of thiamine by the primary intestinal thiamine transporter, THTR‐2 [58]. The overwhelming majority of these drugs are taken chronically, and thus prolonged inhibition of thiamine absorption is possible. Individuals already at risk for thiamine deficiency (i.e., individuals with alcoholism, malnourished individuals, and the elderly) who take such THTR‐2 inhibitor drugs may be at risk for thiamine deficiency. This risk may be increased for individuals on multiple medications, several of which may inhibit the transporter.

PMAT substrates have been identified using an in vitro screen of a variety of radiolabeled compounds. As noted, despite its sequence similarity to the ENT transporters, PMAT does not transport most nucleosides or nucleoside analogs; instead, PMAT transports neurotransmitters such as serotonin, dopamine, epinephrine, and norepinephrine, as well as the prototypical OCT substrate MPP⁺. The K _m and V _max values for these substrates suggest that PMAT is a low‐affinity, high‐capacity neurotransmitter transporter. PMAT substrate uptake is pH‐dependent (i.e., decreased pH, increased transport). Metformin and atenolol, a prescription medication used for hypertension, are substrates of PMAT, with greatest uptake at pH ~ 6 [59, 60]). There is significant overlap in exogenous drugs that are substrates and inhibitors of PMAT and OCT transporters (e.g., HIV protease inhibitors). Lopinavir is a PMAT‐selective inhibitor that does not inhibit OCT transporters and can be used to differentiate PMAT‐mediated monoamine and organic cation transport from OCT1‐3 mediated transport [61].