Characterization of the Inhibitory Effects of N-Butylpyridinium
Chloride and Structurally Related Ionic Liquids on Organic Cation
Transporters 1/2 and Human Toxic Extrusion Transporters 1/2-K
In Vitro and In Vivo
Yaofeng Cheng, Lucy J. Martinez-Guerrero, Stephen H. Wright, Robert K. Kuester,
Michelle J. Hooth, and I. Glenn Sipes
Departments of Pharmacology (Y.C., R.K.K., I.G.S.) and Physiology (L.J.M.-G., S.H.W.), College of Medicine, the University of
Arizona, Tucson, Arizona; and National Toxicology Program, National Institute of Environmental Health Sciences, Research
Triangle Park, North Carolina (M.J.H.)
Received August 13, 2010; accepted June 1, 2011
Ionic liquids (ILs) are a class of salts that are expected to be used
as a new source of solvents and for many other applications. Our
previous studies revealed that selected ILs, structurally related
organic cations, are eliminated exclusively in urine as the parent
compound, partially mediated by renal transporters. This study
investigated the inhibitory effects of N-butylpyridinium chloride
(NBuPy-Cl) and structurally related ILs on organic cation transport-
ers (OCTs) and multidrug and toxic extrusion transporters (MATEs)
in vitro and in vivo. After Chinese hamster ovary cells expressing
rat (r) OCT1, rOCT2, human (h) OCT2, hMATE1, or hMATE2-K were
constructed, the ability of NBuPy-Cl, 1-methyl-3-butylimidazolium
chloride (Bmim-Cl), N-butyl-N-methylpyrrolidinium chloride (BmPy-
Cl), and alkyl substituted pyridinium ILs to inhibit these transporters
was determined in vitro. NBuPy-Cl (0, 0.5, or 2 mg/kg per hour) was
also infused into rats to assess its effect on the pharmacokinetics of
metformin, a substrate of OCTs and MATEs. NBuPy-Cl, Bmim-Cl, and
BmPy-Cl displayed strong inhibitory effects on these transporters
(IC50? 0.2–8.5 ?M). In addition, the inhibitory effects of alkyl-substi-
tuted pyridinium ILs on OCTs increased dramatically as the length of
the alkyl chain increased. The IC50values were 0.1, 3.8, 14, and 671
?M (hexyl-, butyl-, and ethyl-pyridinium and pyridinium chloride) for
rOCT2-mediated metformin transport. Similar structurally related in-
hibitory kinetics were also observed for rOCT1 and hOCT2. The in
clearance of metformin in rats. These results demonstrate that ILs
compete with other substrates of OCTs and MATEs and could alter
the in vivo pharmacokinetics of such substrates.
Ionic liquids (ILs) are a growing class of industrial chemicals that
are being increasingly investigated for a variety of applications
(Plechkova and Seddon, 2008). They are usually composed of organic
cations and various inorganic or organic anions. They have melting
points at or less than 100°C. Because of the chemical diversity of the
cations/anions, the number of available ILs is almost unlimited (Baker
et al., 2005). ILs with specific melting points, viscosities, densities, or
ionic conductivities can be formulated for special applications (Wel-
ton, 1999). Therefore, ILs are expected to be used widely in analytical
methods, engineering processes, consumer products, and biomedical
applications (Plechkova and Seddon, 2008). Because of their ex-
tremely low vapor pressure, their capacity to pollute the air is mini-
mal. Thus, a primary application of these compounds is to replace the
classic volatile organic solvents (Rogers and Seddon, 2003).
Although the physical/chemical characteristics of ILs may mini-
mize the risk of atmospheric contamination, other sources of environ-
mental exposure are of concern. For example, Couling et al. (2005)
suggested that the increased use of ILs on a large scale in industry
could result in water pollution. Exposures from occupational and
consumer products are also likely to occur. Some studies have already
investigated the toxic effects caused by pyridinium- and imidazolium-
based ILs. On a molecular level, such ILs altered the function of some
biological enzymes, such as oxidoreductase and mushroom tyrosinase
(Pinto et al., 2008; Yang et al., 2009). Zhao et al. (2007) have
This work was supported by the National Institutes of Health National Institute
of Environmental Health Sciences [Contract N01-ES45529] (National Toxicology
Program); the National Institutes of Health National Institute of Diabetes and
Digestive and Kidney Diseases [Grant DK58251]; and the National Institutes of
Health National Institute of Environmental Health Sciences [Grant ES06694].
Article, publicationdate,and citation
informationcan be found at
ABBREVIATIONS: IL, ionic liquid; SAR, structure-activity relationship; NBuPy-Cl, N-butylpyridinium chloride; Bmim-Cl, 1-butyl-3-methylimida-
zolium chloride; BmPy-Cl, N-butyl-N-methylpyrrolidinium chloride; OCT, organic cation transporter; MATE, multidrug and toxic extrusion
transporter; Py-Cl, pyridine hydrochloride; EtPy-Cl, 1-ethylpyridinium chloride; HePy-Cl, 1-hexylpyridinium chloride; TEA, tetraethylammonium
trifluoroacetate; MPP, 1-methyl-4-phenylpyridinium; CHO, Chinese hamster ovary; r, rat; h, human; LSC, liquid scintillation counter; GFR,
glomerular filtration rate; Kt, concentration of substrate that results in half-maximal transport.
DRUG METABOLISM AND DISPOSITION
U.S. Government work not protected by U.S. copyright
DMD 39:1755–1761, 2011
Vol. 39, No. 9
Printed in U.S.A.
summarized the toxicities of these two groups of ILs to various
organisms, such as water algae, bacteria, fungi, and mammalian cell
lines. To some organisms, ILs are much more toxic than the conven-
tional organic solvents (Ranke et al., 2004).
ILs based on imidazolium, pyridinium, phosphonium, and ammo-
nium have been tested on Cyclotella meneghiniana, Selenastrum
capricornutum, Daphnia magna, Pseudokirchneriella subcapitata,
and zebrafish (Latala et al., 2005; Cho et al., 2007; Pretti et al., 2009;
Yu et al., 2009). It was found that shorter alkyl-substituted chains
demonstrated lower toxicity compared with the cations with a longer
alkyl substituent. This structure-activity relationship (SAR) was also
observed for biological enzymes, such as acetylcholine esterase (Stock
et al., 2004; Stasiewicz et al., 2008). Lactic acid production by Lactoba-
cillus, an acid-producing bacterium, was decreased as the alkyl chain
length on the imidazolium cations increased (Matsumoto et al., 2004).
Ranke et al. (2007) compared the cation lipophilicity and the cytotoxicity
of 74 ILs on IPC-81 leukemia cells and suggested that the length of the
alkyl chain played a dominant role in their cytotoxicity.
Few studies have investigated the disposition and toxicity of ILs in
mammals. Because N-butylpyridinium chloride (NBuPy-Cl), 1-methyl-
3-butylimidazolium chloride (Bmim-Cl), and N-butyl-N-methylpyrroli-
dinium (BmPy-Cl) are starting materials for many other ILs, they were
selected by the National Toxicology Program for investigation of their
disposition, metabolism, and toxicity. In previous studies, we reported
that these three compounds are absorbed (40–70%) from the rodent
gastrointestinal tract and then are widely distributed in rats (Sipes et al.,
2008; Cheng et al., 2009; Knudsen et al., 2009). For the three ILs, the
portion of the dose that became systemically available was eliminated
exclusively in the urine as the parent compound. Renal elimination was
facilitated, in part, by tubular secretion, presumably mediated by organic
cation transporter 2 (OCT2).
In this study, we examined the in vitro inhibitory effects of NBuPy-
Cl, Bmim-Cl, and BmPy-Cl on rat OCT1/2 and human OCT2 and also
determined whether these ILs inhibited human MATE1/2-K. How the
length of the alkyl chain affected the degree of inhibition was further
investigated on OCTs. The ILs used were pyridine hydrochloride
(Py-Cl), 1-ethylpyridinium chloride (EtPy-Cl), and 1-hexylpyridinium
chloride (HePy-Cl), derivatives of NBuPy-Cl (Fig. 1). In addition, in
vivo studies were conducted to determine whether NBuPy-Cl can
influence the pharmacokinetics of metformin, a known substrate of
OCTs and MATEs.
Materials and Methods
Chemicals and Materials. [3H]Tetraethylammonium trifluoroacetate
(TEA) (54 Ci/mmol) was synthesized by GE Healthcare (Chalfont St. Giles,
Buckinghamshire, UK). [14C]Metformin (112 mCi/mmol) was received from
Moravek Biochemicals (Brea, CA). [3H]1-Methyl-4-phenylpyridinium (MPP)
(80 Ci/mmol) was synthesized by the Department of Chemistry and Biochem-
istry, University of Arizona (Tucson, AZ). Metformin (98% purity) was
purchased from Sigma-Aldrich (St. Louis, MO). NBuPy-Cl, Bmim-Cl, and
BmPy-Cl (all ?98% purity) (Fig. 1) were obtained from Merck KGaA (Darm-
stadt, Germany). Py-Cl, EtPy-Cl, and HePy-Cl (all ?98% purity) were
purchased from Acros Organics (Geel, Belgium). Ketamine/xylazine and pen-
tobarbital sodium salt were purchased from Sigma-Aldrich. Solvable and
Pico-Flour scintillation cocktail solution were obtained from PerkinElmer
Life and Analytical Sciences (Waltham, MA). Fetal bovine serum, peni-
cillin/streptomycin, phleomycin (Zeocin), and hygromycin B were obtained
from Invitrogen (Carlsbad, CA). Kaighn’s modification (F12K) medium
and other chemicals (the highest quality available) were purchased from
In Vitro Studies. Cell culture and transfection. Chinese hamster ovary cells
containing a single integrated Flp recombination target site (CHO Flp-In) were
acquired from Invitrogen and were used for stable expression of the rat
ortholog of OCT1 (rOCT1) and OCT2 (rOCT2) and the human ortholog of
OCT2 (hOCT2), MATE1 (hMATE1), and MATE2-K (hMATE2-K). Trans-
porter cDNAs were inserted into the pcDNA5/FRT/V5-His-TOPO plasmid
vector (Invitrogen) following the manufacturer’s instructions. The sequences
were confirmed by the DNA Sequencing Facility at the University of Arizona
(Tucson, AZ). CHO cells were maintained in F12K medium with 10% fetal
bovine serum, penicillin (100 units/ml), streptomycin (100 ?g/ml), and phleo-
mycin (100 ?g/ml) at 37°C in a humidified atmosphere with 5% CO2, and 5 ?
106cells in 400 ?l of media were electroporated (BTX ECM 630) at 260 V
(time constant of ?25 ms) with 10 ?g of salmon sperm DNA (Invitrogen), 18
?g of pOG44, and 2 ?g of the plasmid vector containing transporter cDNA.
The cells were then transferred to T75 cell culture flasks. After a 24-h
incubation, medium containing hygromycin (300 ?g/ml), instead of phleomy-
cin, was applied as selection pressure for 2 weeks.
Transport studies. The transport of [3H]TEA (12 nM) and [14C]metformin
(9 ?M) was first characterized over time in CHO_hOCT2, CHO_rOCT1, and
CHO_rOCT2 cells along with [3H]MPP (15 nM) in CHO_hMATE1 and
CHO_hMATE2-K cells. On the basis of the time-dependent transport curve,
the inhibitory effects of ILs toward OCTs were determined during the linear
phase of uptake (30 s for [3H]TEA uptake and 60 s for [14C]metformin uptake
by hOCT2; 120 s for [3H]TEA uptake by rOCT1, [14C]metformin uptake by
rOCT2, and [3H]MPP uptake by hMATE1 and hMATE2-K).
To determine the inhibitory effects of ILs, the transport of [3H]TEA (12 nM)
by rOCT1 and hOCT2 and of [14C]metformin (9 ?M) by rOCT2 and hOCT2
was measured in the presence of increasing concentrations of NBuPy-Cl,
Bmim-Cl, BmPy-Cl, Py-Cl, EtPy-Cl, or HePy-Cl. Various concentrations of
NBuPy-Cl were used to investigate its inhibitory effects on hMATE1- or
hMATE2-K-mediated [3H]MPP transport. The transport assay was performed
as described previously (Cheng et al., 2009). In general, cells were cultured in
12-well plates. Once reaching confluence, they were incubated in 0.4 ml of
Waymouth’s buffer containing [14C]metformin, [3H]TEA, or [3H]MPP and
one of the ILs for a predetermined time as described above. After solubilization
of the cells with 0.4 ml of NaOH (0.5 M) with 1% SDS and neutralization with
0.2 ml of HCl (1 M), the cell lysate solution (0.5 ml) was counted using a liquid
scintillation counter (LSC).
FIG. 1. Chemical structures of NBuPy-Cl and
other structurally related ionic liquids.
CHENG ET AL.
Animal Studies. Animal surgeries. Male Fischer-344 rats (300–350 g) were
purchased from Harlan (Indianapolis, IN). The rats were housed in the Uni-
versity of Arizona Animal Care Facility (accredited by the Association for
Assessment and Accreditation for Laboratory Animal Care) with controlled
temperature (25°C), humidity (40–60%), and light/dark cycle (12 h). After 7
to 10 days of acclimation, 1 ml/kg ketamine/xylazine solution was adminis-
tered (intraperitoneally) to induce anesthesia. Once the animals were totally
anesthetized, the jugular vein and carotid artery were cannulated with PE-50
tubing (i.d. 0.58 mm and o.d. 0.965 mm; BD, Franklin Lakes, NJ). The surgical
openings were covered with water-saturated gauze sponges. After surgery,
pentobarbital (65 ?g/kg) was administered subcutaneously to maintain anes-
thesia. The rats were then placed in a ventral position on a heating pad
maintained at 37°C. A syringe infusion pump (Harvard Apparatus, Holliston,
MA) was used for the infusion of NBuPy-Cl. All the protocols were approved
by the Institutional Animal Care and Use Committee at the University of
Dosing selection and sample collection. It was observed previously that
NBuPy-Cl (5 mg/kg) did not affect the GFR in rats after a single intravenous
dose (Cheng et al., 2009). However, when this dose (5 mg/kg per hour, 2 ml/kg
per hour) was infused over 4 h (total dose 20 mg/kg), the clearance of the
metformin (assessed over 3 h) was reduced to ?1% of that observed in the
control (saline group). Therefore, two lower doses (2 mg/kg per hour, 0.5
mg/kg per hour) of NBuPy-Cl were used in the studies described here. They
provided total doses of 8 and 2 mg/kg, respectively.
After surgery, the jugular vein was infused with saline (2 ml/kg per hour) or
NBuPy-Cl (0.5 or 2 mg/kg per hour, 2 ml/kg per hour) over 4 h. A bolus dose
of [14C]metformin (5 mg/kg, 50 ?Ci/kg) was administered through the jugular
vein after 1 h of perfusion. Blood samples (300 ?l) were then collected at 7.5,
15, 30, 45, 60, 90, 120, 150, and 180 min from the carotid artery. An equal
volume of saline was administered to replace the blood that was withdrawn. At
the end of the experiment (3 h after metformin dosing), animals were eutha-
nized by CO2inhalation. Selected tissues (adipose, heart, kidney, liver, lung,
muscle, spleen, and testes) were collected.
Sample Analysis. Blood samples were centrifuged at 750g for 10 min.
Aliquots of the plasma samples (two 50-?l samples) were mixed with 15 ml of
the Pico-Flour scintillation cocktail solution, and samples were counted using
a LSC. Tissues samples were analyzed as described by Sipes et al. (2008). In
brief, the samples were solubilized with Solvable, quenched with H2O2(30%),
and counted using a LSC.
Data Analysis. For the in vitro transport studies, estimates of the IC50
values were determined by the modified Michaelis-Menten equation (Malo and
Berteloot, 1991; Groves et al., 1994):
IC50? [I]? C
J is the transport rate of radiolabeled compound at the concentration of [T*], Jmapp
is the Jmaxfor [*T] transport times the ratio of Ki/Kt, IC50is equal to Ki(1 ?
[T*/Kt]), [I] is the concentration of unlabeled inhibitor, and C is a constant
representing the component of total uptake that is not saturable over the concen-
tration range tested.
The application of this equation, given its structure, carries the tacit as-
sumption that the inhibitory interactions observed are competitive in nature
and reflect binding of substrate and inhibitor at a common binding site.
Although the substrates/inhibitors studied here have all been shown to be
transported by OCTs, inhibitory interactions between OCT substrates have, in
some cases, been shown to reflect a “mixed-type” inhibitory profile, presum-
ably reflecting both competition for a common binding region within the
transporter and longer-range allosteric interactions (e.g., Koepsell et al., 2007).
Thus, we refer to the kinetic constants calculated through the above equation
as “IC50” values and make no claim as to their precise mechanistic basis.
The pharmacokinetics of metformin after intravenous administration in rats
was defined by WinNonlin software (version 6.1; Pharsight, Mountain View,
CA) using a noncompartment model. All individual data points are presented
as means ? S.E.M. The data were analyzed statistically by Student’s t test or
one-way analysis of variance with the Newman-Keuls post-test using Graph-
Pad Prism 4 (GraphPad Software Inc., San Diego, CA). A level of p ? 0.05
was considered significant.
Functional Expression of OCTs and MATEs in CHO Cells. The
accumulation of 12 nM [3H]TEA by CHO cells stably expressing
OCTs was time-dependent, i.e., nearly linear for at least 60 s and
approaching steady state by 10 min (Fig. 2A). In all three cell lines,
the presence of 5 mM unlabeled TEA reduced the 10-min accumula-
tion of [3H]TEA by 90% or more. The uptake of [3H]TEA displayed
kinetic characteristics of carrier-mediated transport minimally influ-
enced by surface binding (Suhre et al., 2005). The profiles of hOCT2-
and rOCT2-mediated [14C]metformin transport were similar to that of
[3H]TEA, but the transport rate of [14C]metformin by rOCT1 was too
low to conduct an inhibition study (data not shown). Therefore, uptake
of [3H]TEA was used to determine the inhibitory effects of ILs on
The function of CHO cells expressing hMATE1 or hMATE2-K
was also determined, and the results are shown in Fig. 2B. As with the
OCTs, MATE1- or MATE2-K-mediated transport of radiolabeled
substrate, in this case, [3H]MPP, was blocked more than 90% by
unradiolabeled 1 mM MPP. These results demonstrate that OCTs and
MATEs were expressed in the CHO cells and were functionally
Inhibitory Effect of NBuPy-Cl, Bmim-Cl, and BmPy-Cl on
OCTs. In the presence of NBuPy-Cl, the intracellular uptake of
[3H]TEA by rOCT1 was inhibited in a concentration-dependent man-
ner (Fig. 3A). Similar inhibitory kinetics of NBuPy-Cl on [14C]met-
formin transport were observed for rOCT2 and hOCT2 (Fig. 3, B and
C). The IC50values, which are the concentrations of inhibitors (ILs)
A uptake (fmol
05 10 20
FIG. 2. Characterization of the function of OCTs and MATEs in transfected CHO
cells. A, [3H]TEA uptake by rOCT1 (f), rOCT2 (Œ), or hOCT2 (F) over time in
the absence (solid symbols) or presence (open symbols) of 5 mM TEA. B, [3H]MPP
uptake by hMATE1 (f) or hMATE2-K (F) over time in the absence (solid symbols)
or presence (open symbols) of 1 mM MPP. n ? 3; mean ? S.E.M.
INHIBITORY EFFECTS OF IONIC LIQUIDS ON OCTs AND MATEs
that result in half-maximum transport of TEA or metformin by OCTs,
are presented in Table 1. The three ILs were potent inhibitors of
rOCT1/2 and hOCT2, with IC50values ranging from 0.15 to 7.53 ?M.
As shown in Table 1, IC50values were significantly lower for hOCT2
when metformin was used as the probe substrate.
Inhibition of OCTs by Alkyl-Substituted Pyridiniums. To de-
termine the relationship between the structure of ILs and their inhib-
itory effects (structure-activity relationship) on OCTs, pyridinium-
based ILs with different alkyl chain lengths were investigated. Uptake
of TEA or metformin by OCTs was measured in the presence of
increasing concentrations of Py-Cl, EtPy-Cl, NBuPy-Cl, and HePy-
Cl. As shown in Fig. 4A, all the pyridinium compounds tested
inhibited rOCT2-mediated [14C]metformin transport. Py-Cl was the
weakest inhibitor with an IC50of 671 ?M (Table 1). The inhibition
curve shifted left gradually, and the IC50values decreased approxi-
mately 10-fold for every 2 carbons added to the alkyl chain. HePy-Cl
showed the strongest inhibitory activity (IC50? 0.1 ?M). The struc-
in uptake (
0 01 0.01
FIG. 4. Influence of alkyl chain length on the inhibitory effects of pyridinium-based
ILs on OCTs. A, intracellular uptake of [14C]metformin (120 s) by CHO_rOCT2 in
the presence of increasing concentrations of RPy-Cl (RPy-Cl: Py-Cl, EtPy-Cl,
NBuPy-Cl, and HePy-Cl; n ? 3–5, mean ? S.E.M.). B, relationship between
inhibitory effects (IC50) and alkyl chain length (number of carbons) of RPy-Cl (n ?
3–5; mean ? S.E.M.). The statistical analysis compared the log(IC50) using one-way
analysis of variance with the Newman-Keuls post-test.
ol min-1 cm
0 2040 60 80 100
in uptake (
0 20 406080 100
in uptake (
0 20406080 100
FIG. 3. Inhibitory effects of NBuPy-Cl on OCTs. Intracellular uptake of [3H]TEA
by CHO_rOCT1 (A, 120 s) and [14C]metformin by CHO_rOCT2 (B, 120 s) and
CHO_hOCT2 (C, 60 s) in the presence of increasing concentrations of NBuPy-Cl
(n ? 3–5; mean ? S.E.M.).
Inhibitory effects (IC50) of NBuPy-Cl and structurally related ILs on
OCT-mediated TEA or metformin transport
Data are means ? S.E.M., n ? 3 to 5.
rOCT1: TEA rOCT2: Metformin
0.48 ? 0.05**
1.50 ? 0.43
790 ? 51**
36.7 ? 3.7*
2.29 ? 0.64
0.35 ? 0.03**
0.15 ? 0.01*,#
0.44 ? 0.04
555 ? 40**,#
20.1 ? 2.2*,#
0.69 ? 0.08#
0.10 ? 0.01*,#
4.70 ? 1.37
7.53 ? 0.97
762 ? 254*
72.1 ? 13.3*
4.74 ? 0.66
2.21 ? 0.23*
1.80 ? 0.26**
1.81 ? 0.08**
671 ? 92**
14.4 ? 0.9**
3.78 ? 0.23
0.10 ? 0.01**
* P ? 0.05 and ** P ? 0.01, compared with NBuPy-Cl.
# P ? 0.05, compared with the probe substrate of TEA for hOCT2.
CHENG ET AL.
turally related inhibitory effects of these pyridinium ILs on rOCT1
and hOCT2 were similar to those of rOCT2 (Fig. 4B). The IC50values
are presented in Table 1 for both probe substrates, TEA and
Inhibitory Effects of NBuPy-Cl on hMATE1 and hMATE2-K.
The inhibitory effects of NBuPy-Cl on the apical membrane transporters,
hMATE1 and hMATE2-K, were also determined. Figure 5 shows the
inhibitory kinetic curves of NBuPy-Cl on intracellular uptake of
[3H]MPP by CHO cells expressing hMATE1 or hMATE2-K. The IC50
values were 8.5 ? 2.6 and 1.6 ? 0.2 ?M, respectively.
Intravenous Coadministration of NBuPy-Cl and Metformin.
Pharmacokinetic analysis using a noncompartment model revealed
that the elimination half-life of [14C]metformin (5 mg/kg i.v.) from
plasma of male F-344 rats was 2.3 h (Fig. 6; Table 2). The volume of
distribution was 484 ml, and the systemic clearance was 3.4 ml/min.
Because the liver metabolism/excretion of metformin is negligible, its
systemic clearance reflects its renal clearance (Scheen, 1996). Infu-
sion of NBuPy-Cl increased the plasma level of metformin in a
dose-dependent manner (Fig. 6). Thus, the plasma area under the
curve from 0 to 180 min for metformin was significantly increased
because of the reduced renal clearance (Table 3). NBuPy-Cl had a
minimal effect on the volume of distribution of metformin. This
observation was further substantiated by the findings that tissue levels
of metformin were not significantly elevated when they were har-
vested at the 3-h time point (Table 3). The small increases that were
observed may reflect the presence of blood, which, in the NBuPy-Cl-
infused rats, had elevated concentrations of metformin. The only
tissue that showed a small increase in the tissue/plasma ratio, indic-
ative of actual increases in tissue levels, was the kidney, but this value
was not significant because of the large interanimal variations.
OCTs are widely expressed on the basolateral membrane of prox-
imal tubule cells, where they function to transport a wide array of
organic cations from plasma to the intracellular space of these cells
(Choi and Song, 2008). OCT2 is richly expressed in human and rat
kidneys, but OCT1 is weakly detected in human kidneys (Okuda et al.,
1996; Motohashi et al., 2002). On the apical membrane of these
tubular cells, organic cations are most likely exported to the urinary
lumen by MATEs (Nies et al., 2011). There are two mammalian
MATEs, MATE1 and MATE2-K. Human kidneys express both ho-
mologs, whereas rodent kidneys express only MATE1 (Terada et al.,
2006). In several studies, we observed that three ILs, NBuPy-Cl,
Bmim-Cl, and BmPy-Cl, were excreted rapidly in the urine of rats as
the parent compounds (Sipes et al., 2008; Cheng et al., 2009; Knudsen
et al., 2009). Because the rate of clearance from plasma exceeded the
glomerular filtration rate, it was proposed that these three ILs, which
are organic cations, undergo transport-mediated secretion.
NBuPy-Cl and BmPy-Cl were significantly accumulated in CHO
cells expressing hOCT2 after a 10-min incubation, compared with
FIG. 5. Inhibitory effects of NBuPy-Cl on hMATE1 and hMATE2-K. Intracellular
uptake of [3H]MPP by CHO_hMATE1 (A) or CHO_hMATE2-K (B) in 2 min with
the presence of increasing concentrations of NBuPy-Cl (n ? 3–6; mean ? S.E.M.).
0 3060 90 120150180
FIG. 6. Effects of NBuPy-Cl on the pharmacokinetics of metformin. Plasma con-
centrations of [14C]metformin (n ? 3–4; mean ? S.E.M.) after a single intravenous
administration (5 mg/kg) to male F344 rats with saline (Œ, 2 ml/h) or NBuPy-Cl (f,
0.5 mg/kg per hour; F, 2 mg/kg per hour) infusion through the jugular vein.
Kinetic parameters of metformin in male F-344 rats after intravenous
administration (5 mg/kg) with NBuPy-Cl or saline infusion
Data are means ? S.E.M.; n ? 3 to 4. Predicted parameters were calculated using
noncompartment model analysis.
min ? ?g/ml
358 ? 47
391 ? 37
687 ? 86*
3.4 ? 1.0
2.3 ? 0.2
1.2 ? 0.4*
0.5 mg/kg per hour
2 mg/kg per hour
2.3 ? 0.6
2.8 ? 0.4
4.5 ? 1.2
484 ? 47
473 ? 22
334 ? 22
AUC0_180, area under the curve from 0 to 180 min; t1/2, elimination half-life; CL, systemic
clearance; Vss, volume of distribution.
* P ? 0.05, compared with saline group.
INHIBITORY EFFECTS OF IONIC LIQUIDS ON OCTs AND MATEs
naive CHO cells (Cheng et al., 2009; Knudsen et al., 2009). The
transport kinetics of these two ILs, as assessed by Ktvalues (Kt? 18
?M for NBuPy-Cl and 37 ?M for BmPy-Cl), were similar to that of
the model substrate of hOCT2, TEA (Kt? 40 ?M). NBuPy-Cl and
BmPy-Cl were also well transported in CHO cells expressing
rOCT1 or rOCT2 (unpublished data). Results of subsequent exper-
iments performed after the disposition studies on Bmim-Cl were
published revealed that Bmim-Cl was also transported by hOCT2,
rOCT1, and rOCT2 (data not presented). These in vitro results
suggest that OCTs could contribute to the rapid renal elimination
of these ILs.
The results presented in this article demonstrate that, in addition to
serving as substrates, NBuPy-Cl, BmPy-Cl, and Bmim-Cl are also
potent inhibitors of rOCT1/2 and hOCT2. The IC50values for inhi-
bition of hOCT2-mediated TEA transport by these three ILs ranged
from 0.5 to 2.3 ?M. These inhibitory values are similar to those
observed for MPP (2 ?M) and tetrapentylammonium (10 ?M), com-
pounds considered to be potent inhibitors of hOCT2 (Suhre et al.,
2005). When metformin, the widely prescribed type 2 antidiabetic
drug, was used as the probe substrate for hOCT2, these ILs demon-
strated even stronger inhibitory effects. This observation was also
noted when tetraalkylammonium compounds were used to inhibit
hOCT2-mediated MPP and metformin transport; i.e., transport of
metformin was inhibited to a greater degree (Dresser et al., 2002; Choi
et al., 2007). The reason for the different inhibitory effects observed
for the two probe substrates is still not clear. The transport character-
istic of NBuPy-Cl by hOCT2 (Kt? 18 ?M) is also significantly
different from its inhibitory potency on hOCT2-mediated metformin
uptake (IC50? 0.7 ?M). These results might be related to different
binding sites of two chemicals within the large binding surface of
OCTs and/or the influence of noncompetitive or other allosteric
interactions (Koepsell et al., 2007).
ILs are called “designable chemicals” because they can be custom-
ized structurally to fit specific applications. These alterations in struc-
ture not only will change the chemical and physical properties of ILs
but also may alter their disposition, transport, and toxicity. As dem-
onstrated by the SAR results presented here, the inhibitory effects of
the pyridinium-based ILs changed dramatically as the length of the
alkyl side chain was altered. The IC50values decreased significantly
with an increase in the number of carbons on the alkyl chain. This
SAR has also been reported for other chemical classes (Ullrich, 1997;
Bednarczyk et al., 2003; Suhre et al., 2005; Choi et al., 2007). It was
suggested that the hydrophobicity of the ILs increased with increasing
alkyl chain length (Ranke et al., 2007). This increased hydrophobicity
could facilitate their interaction on the pharmacophore of OCTs and
thus contribute to the enhanced inhibitory activity (Bednarczyk et al.,
Results presented here show that NBuPy-Cl is also a potent inhib-
itor of hMATE1 and hMATE2-K. Because both OCTs and MATEs
play critical roles in the renal secretion of organic cations, disruption
of any of these transporters may alter the renal elimination profile of
certain chemicals. For example, renal secretion of TEA was reduced
in both rats and mice with impeded OCT1/2 function (Jonker et al.,
2003; Matsuzaki et al., 2008). Likewise, plasma concentrations of
metformin were increased in MATE1 knockout mice, and its urinary
excretion was significantly reduced (Tsuda et al., 2009). Thus, it is not
surprising that infusion of NBuPy-Cl markedly altered the plasma
pharmacokinetics of metformin. It has the capacity to inhibit the
transport processes related to both the uptake of metformin into
proximal tubules and its extrusion from these cells into the tubular
Metformin is a comparatively low-affinity substrate for hOCT1,
hOCT2, hMATE1, and hMATE2-K, with Ktvalues of 1.47, 0.99,
0.78, and 1.98 mM, respectively (Koepsell et al., 2007; Tanihara et al.,
2007). As determined here, NBuPy-Cl blocks both OCTs and MATEs
with IC50values in the low micromolar range. Based on the equation
for calculating the steady-state plasma concentration after intravenous
infusion (Rowland and Tozer, 1995), the estimated steady-state con-
centration of NBuPy-Cl in plasma is 0.8 ?g/ml (4.7 ?M), with an
infusion dose of 2 mg/kg per hour. Therefore, it was not surprising
that renal clearance of metformin was reduced in the presence of this
compound. Indeed, the NBuPy-Cl inhibitory profile suggests that a
plasma concentration of ?5 ?M would block approximately half of
the rOCT1 (IC50? 4.7 ?M) and rOCT2 (IC50? 3.8 ?M) activity.
Because of the negative intracellular potential (?70 mV) of renal
proximal tubule cells, NBuPy-Cl, the positively charged moiety, could
reach substantially higher levels intracellularly and inhibit MATEs
more extensively. Thus, exit of metformin from renal proximal tubule
cells may have been rate-limiting under these conditions. In fact, the
exit step was shown to be rate-limiting in renal secretion of organic
cations (Scha ¨li et al., 1983), which is consistent with the accumulation
of metformin in kidney tissue associated with the highest dose of
Because metformin is primarily eliminated in the urine as the parent
compound and at a clearance rate that exceeds the GFR (Scheen,
1996), the increased plasma area under the curve of metformin in
NBuPy-Cl-infused animals relates, at least in part, to the inhibitory
effects of NBuPy-Cl on OCTs and/or MATEs. In our previous pub-
lication, we reported that NBuPy-Cl, at a single intravenous dose of 5
mg/kg, did not alter the plasma clearance of inulin in unanesthetized
animals. This result indicated that NBuPy-Cl at this dose did not affect
rat GFR. However, it should be cautioned that the GFR could have
been decreased in animals coadministered NBuPy-Cl and the anes-
thetics that were used. For example, pentobarbital has been reported to
decrease GFR (Walker et al., 1986). On the basis of the clearance of
metformin in unanesthetized rats (1.4 ml/min per 100 g) reported by
Choi et al. (2010) and that of the control rats (1.0 ml/min per 100 g)
reported here, anesthesia could account for up to 30% of the reduced
metformin clearance. Another factor that could reduce GFR is im-
pairment of renal function because of renal toxicity. However, in
studies with Bmim-Cl, a closely related ionic liquid, no evidence of
renal toxicity was observed (as assessed by both serum chemistry and
histopathology) at oral doses up to 50 mg/kg. This dose, which was
?50% bioavailable, maintained blood levels of Bmim-Cl at ?2 ?g/ml
for several hours, close to the estimated steady-state blood levels of
NBuPy-Cl reported here. Thus, the in vivo data presented here support
the hypothesis that ionic liquids have the potential to inhibit renal
Metformin concentration in selected tissues of F-344 rats after intravenous
administration of metformin (5 mg/kg) to male F-344 rats infused with saline
Data are presented as means ? S.E.M. ?tissue (g)/plasma (ml) metformin concentration
0 (Saline) (n ? 4) 0.5 mg/kg per hour (n ? 4) 2 mg/kg hour (n ? 4)
1.8 ? 0.4 ?1.5?
31.6 ? 2.5 ?25?
151 ? 47 ?121?
13.7 ? 0.3 ?11?
15.0 ? 0.8 ?12?
5.8 ? 0.4 ?4.7?
8.5 ? 0.5 ?6.8?
4.6 ? 0.4 ?3.7?
4.2 ? 2.3 ?4.6?
41.3 ? 4.4 ?56?
204 ? 73 ?213?
16.9 ? 2.9 ?21?
14.5 ? 2.7 ?19?
7.1 ? 2.0 ?8.8?
8.8 ? 1.6 ?11?
3.9 ? 0.3 ?5.3?
7.3 ? 0.9 ?3?
45.8 ? 8.4 ?19?
756 ? 192 ?266?
44.9 ? 11.1 ?16?
28.7 ? 4.8 ?11?
12.8 ? 1.2 ?5.4?
18.3 ? 3.4 ?7?
7.4 ? 1.2 ?2.9?
CHENG ET AL.
transporters and alter the pharmacokinetics of substrates of these
It should be noted that the doses of NBuPy-Cl infused into rats were
high. It is unlikely that such blood levels would be achieved and
maintained in humans exposed orally and/or dermally to environmen-
tal/occupational levels of NBuPy-Cl or other ILs. In rats, dermal
absorption of these three ILs was less than 35% of the applied dose (5
mg/kg, 125 ?g/cm2), and the absorbed dose was readily eliminated.
Dermal absorption in humans is not expected to exceed that observed
for rats. It is clear that additional studies are needed to focus on how
alterations in structure affect absorption of ILs after oral dosing or
dermal application. More lipophilic ILs may achieve higher internal
concentrations. This action, coupled with the greater inhibitory effects
on OCTs, could influence their pharmacokinetic parameters as well as
In summary, in in vitro studies, NBuPy-Cl strongly inhibited OCTs
and MATEs. In in vivo studies with rats, this inhibition resulted in a
reduction in the plasma clearance of metformin. The structurally
related ILs, Bmim-Cl and BmPy-Cl, and pyridinium-based ILs with
increasing alkyl chain length, were also inhibitors of OCTs. The
inhibitory effect increased as the length to the alkyl chain increased.
We thank Dr. Michael Cunningham (National Toxicology Program and
National Center for Toxicogenomics, National Institute of Environmental
Health Sciences) for his advice and support. We also thank Xiaohong Zhang
(Department of Physiology, University of Arizona) for her assistance in cell
transfection work, as well as Dr. Helen Cunny and Dr. Matt Stout (National
Toxicology Program, National Institute of Environmental Health Sciences) for
their critical review of this manuscript and Dr. Binfeng Xia (University of
South Carolina) for his support in the pharmacokinetic modeling.
Participated in research design: Cheng, Wright, Kuester, and Sipes.
Conducted experiments: Cheng and Martinez-Guerrero.
Contributed new reagents or analytic tools: Hooth.
Performed data analysis: Cheng, Martinez-Guerrero, and Sipes.
Wrote or contributed to the writing of the manuscript: Cheng, Wright,
Hooth, and Sipes.
Baker GA, Baker SN, Pandey S, and Bright FV (2005) An analytical view of ionic liquids.
Bednarczyk D, Ekins S, Wikel JH, and Wright SH (2003) Influence of molecular structure on
substrate binding to the human organic cation transporter, hOCT1. Mol Pharmacol 63:489–
Cheng Y, Wright SH, Hooth MJ, and Sipes IG (2009) Characterization of the disposition and
toxicokinetics of N-butylpyridinium chloride in male F-344 rats and female B6C3F1 mice and
its transport by organic cation transporter 2. Drug Metab Dispos 37:909–916.
Cho CW, Pham TP, Jeon YC, Vijayaraghavan K, Choe WS, and Yun YS (2007) Toxicity of
imidazolium salt with anion bromide to a phytoplankton Selenastrum capricornutum: effect of
alkyl-chain length. Chemosphere 69:1003–1007.
Choi MK, Jin QR, Jin HE, Shim CK, Cho DY, Shin JG, and Song IS (2007) Effects of
tetraalkylammonium compounds with different affinities for organic cation transporters on the
pharmacokinetics of metformin. Biopharm Drug Dispos 28:501–510.
Choi MK and Song IS (2008) Organic cation transporters and their pharmacokinetic and
pharmacodynamic consequences. Drug Metab Pharmacokinet 23:243–253.
Choi YH, Lee U, Lee BK, and Lee MG (2010) Pharmacokinetic interaction between itraconazole
and metformin in rats: competitive inhibition of metabolism of each drug by each other via
hepatic and intestinal CYP3A1/2. Br J Pharmacol 161:815–829.
Couling DJ, Bernot RJ, Docherty KM, Dixon JK, and Maginn EJ (2005) Assessing the factors
responsible for ionic liquid toxicity to aquatic organism via quantitative structure-property
relationship modeling. Green Chem 8:82–90.
Dresser MJ, Xiao G, Leabman MK, Gray AT, and Giacomini KM (2002) Interactions of
n-tetraalkylammonium compounds and biguanides with a human renal organic cation trans-
porter (hOCT2). Pharm Res 19:1244–1247.
Groves CE, Evans KK, Dantzler WH, and Wright SH (1994) Peritubular organic cation transport
in isolated rabbit proximal tubules. Am J Physiol 266:F450–F458.
Jonker JW, Wagenaar E, Van Eijl S, and Schinkel AH (2003) Deficiency in the organic cation
transporters 1 and 2 (Oct1/Oct2 [Slc22a1/Slc22a2]) in mice abolishes renal secretion of
organic cations. Mol Cell Biol 23:7902–7908.
Knudsen GA, Cheng Y, Kuester RK, Hooth MJ, and Sipes IG (2009) Effects of dose and route
on the disposition and kinetics of 1-butyl-1-methylpyrrolidinium chloride in male F-344 rats.
Drug Metab Dispos 37:2171–2177.
Koepsell H, Lips K, and Volk C (2007) Polyspecific organic cation transporters: structure,
function, physiological roles, and biopharmaceutical implications. Pharm Res 24:1227–1251.
Latała A, Stepnowski P, Nedzi M, and Mrozik W (2005) Marine toxicity assessment of
imidazolium ionic liquids: acute effects on the Baltic algae Oocystis submarina and Cyclotella
meneghiniana. Aquat Toxicol 73:91–98.
Malo C and Berteloot A (1991) Analysis of kinetic data in transport studies: new insights from
kinetic studies of Na?-D-glucose cotransport in human intestinal brush-border membrane
vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 122:127–141.
Matsumoto M, Mochiduki K, and Kondo K (2004) Toxicity of ionic liquids and organic solvents
to lactic acid-producing bacteria. J Biosci Bioeng 98:344–347.
Matsuzaki T, Morisaki T, Sugimoto W, Yokoo K, Sato D, Nonoguchi H, Tomita K, Terada T,
Inui K, Hamada A, et al. (2008) Altered pharmacokinetics of cationic drugs caused by
down-regulation of renal rat organic cation transporter 2 (Slc22a2) and rat multidrug and toxin
extrusion 1 (Slc47a1) in ischemia/reperfusion-induced acute kidney injury. Drug Metab
Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, and Inui
K (2002) Gene expression levels and immunolocalization of organic ion transporters in the
human kidney. J Am Soc Nephrol 13:866–874.
Nies AT, Koepsell H, Damme K, and Schwab M (2011) Organic cation transporters (OCTs,
MATEs), in vitro and in vivo evidence for the importance in drug therapy. Handb Exp
Okuda M, Saito H, Urakami Y, Takano M, and Inui K (1996) cDNA cloning and functional
expression of a novel rat kidney organic cation transporter, OCT2. Biochem Biophys Res
Pinto PC, Saraiva ML, and Lima JL (2008) Oxidoreductase behavior in ionic liquids: a review.
Anal Sci 24:1231–1238.
Plechkova NV and Seddon KR (2008) Applications of ionic liquids in the chemical industry.
Chem Soc Rev 37:123–150.
Pretti C, Chiappe C, Baldetti I, Brunini S, Monni G, and Intorre L (2009) Acute toxicity of ionic
liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and
Danio rerio. Ecotoxicol Environ Saf 72:1170–1176.
Ranke J, Mo ¨lter K, Stock F, Bottin-Weber U, Poczobutt J, Hoffmann J, Ondruschka B, Filser J,
and Jastorff B (2004) Biological effects of imidazolium ionic liquids with varying chain
lengths in acute Vibrio fischeri and WST-1 cell viability assays. Ecotoxicol Environ Saf
Ranke J, Mu ¨ller A, Bottin-Weber U, Stock F, Stolte S, Arning J, Sto ¨rmann R, and Jastorff B
(2007) Lipophilicity parameters for ionic liquid cations and their correlation to in vitro
cytotoxicity. Ecotoxicol Environ Saf 67:430–438.
Rogers RD and Seddon KR (2003) Chemistry ionic liquids—solvents for the future? Science
Rowland M and Tozer NH (1995) Clinical Pharmacokinetics, Concepts and Applications, 3rd ed.
Lippincott Williams & Wilkins, Philadelphia.
Scha ¨li C, Schild L, Overney J, and Roch-Ramel F (1983) Secretion of tetraethylammonium by
proximal tubules of rabbit kidneys. Am J Physiol 245:F238–F246.
Scheen AJ (1996) Clinical pharmacokinetics of metformin. Clin Pharmacokinet 30:359–371.
Sipes IG, Knudsen GA, and Kuester RK (2008) The effects of dose and route on the toxicoki-
netics and disposition of 1-butyl-3-methylimidazolium chloride in male F-344 rats and female
B6C3F1 mice. Drug Metab Dispos 36:284–293.
Stasiewicz M, Mulkiewicz E, Tomczak-Wandzel R, Kumirska J, Siedlecka EM, Gołebiowski M,
Gajdus J, Czerwicka M, and Stepnowski P (2008) Assessing toxicity and biodegradation of
novel, environmentally benign ionic liquids (1-alkoxymethyl-3-hydroxypyridinium chloride,
saccharinate and acesulfamates) on cellular and molecular level. Ecotoxicol Environ Saf
Stock F, Hoffmann J, Ranke J, Stormann R, Ondruschka B, and Jastorff B (2004) Effects of ionic
liquids on the acetylcholinesterase—a structure-activity relationship consideration. Green
Suhre WM, Ekins S, Chang C, Swaan PW, and Wright SH (2005) Molecular determinants of
substrate/inhibitor binding to the human and rabbit renal organic cation transporters hOCT2
and rbOCT2. Mol Pharmacol 67:1067–1077.
Tanihara Y, Masuda S, Sato T, Katsura T, Ogawa O, and Inui K (2007) Substrate specificity of
MATE1 and MATE2-K, human multidrug and toxin extrusions/H?-organic cation antiporters.
Biochem Pharmacol 74:359–371.
Terada T, Masuda S, Asaka J, Tsuda M, Katsura T, and Inui K (2006) Molecular cloning,
functional characterization and tissue distribution of rat H?/organic cation antiporter MATE1.
Pharm Res 23:1696–1701.
Tsuda M, Terada T, Mizuno T, Katsura T, Shimakura J, and Inui K (2009) Targeted disruption
of the multidrug and toxin extrusion 1 (mate1) gene in mice reduces renal secretion of
metformin. Mol Pharmacol 75:1280–1286.
Ullrich KJ (1997) Renal transporters for organic anions and organic cations. Structural require-
ments for substrates. J Membr Biol 158:95–107.
Walker LA, Gellai M, and Valtin H (1986) Renal response to pentobarbital anesthesia in rats:
effect of interrupting the renin-angiotensin system. J Pharmacol Exp Ther 236:721–728.
Welton T (1999) Room-temperature ionic liquids. Solvent for synthesis and catalysis. Chem Rev
Yang Z, Yue YJ, Huang WC, Zhuang XM, Chen ZT, and Xing M (2009) Importance of the ionic
nature of ionic liquids in affecting enzyme performance. J Biochem 145:355–364.
Yu M, Wang SH, Luo YR, Han YW, Li XY, Zhang BJ, and Wang JJ (2009) Effects of the
1-alkyl-3-methylimidazolium bromide ionic liquids on the antioxidant defense system of
Daphnia magna. Ecotoxicol Environ Saf 72:1798–1804.
Zhao D, Liao Y, and Zhang Z (2007) Toxicity of ionic liquids. Clean Soil Air Water 35:42–48.
Address correspondence to: Dr. I. Glenn Sipes, Department of Pharmacol-
ogy, College of Medicine, The University of Arizona, P.O. Box 245050, Tucson, AZ
85724-5050. E-mail: firstname.lastname@example.org
INHIBITORY EFFECTS OF IONIC LIQUIDS ON OCTs AND MATEs