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Research ArticleArticle

Transport of Dicationic Drugs Pentamidine and Furamidine by Human Organic Cation Transporters

Xin Ming, Wujian Ju, Huali Wu, Richard R. Tidwell, James E. Hall and Dhiren R. Thakker
Drug Metabolism and Disposition February 2009, 37 (2) 424-430; DOI: https://doi.org/10.1124/dmd.108.024083

Abstract

The antiparasitic activity of aromatic diamidine drugs, pentamidine and furamidine, depends on their entry into the pathogenic protozoa via membrane transporters. However, no such diamidine transporter has been identified in mammalian cells. The goal of this study is to investigate whether these dicationic drugs are substrates for human organic cation transporters (hOCTs, solute carrier family 22A1–3) and whether hOCTs play a role in their tissue distribution, elimination, and toxicity. Inhibitory and substrate activities of pentamidine and furamidine were studied in stably transfected Chinese hamster ovary (CHO) cells. The results of [3H]1-methyl-4-phenylpyridinium uptake study showed that pentamidine is a potent inhibitor for all three OCT isoforms (IC50 < 20 μM), whereas furamidine is a potent inhibitor for hOCT1 and hOCT3 (IC50 < 21 μM) but a less potent inhibitor for hOCT2 (IC50 = 189.2 μM). Both diamidines are good substrates for hOCT1 (Km = 36.4 and 6.1 μM, respectively), but neither is a substrate for hOCT2 or hOCT3. The cytotoxicity of pentamidine and furamidine was 4.4- and 9.3-fold greater, respectively, in CHO-hOCT1 cells compared with the mock cells. Ranitidine, an hOCT1 inhibitor, reversed this hOCT1-mediated potentiation of cytotoxicity. This is the first finding that dicationic drugs, such as pentamidine and furamidine, are substrates for hOCT1. In humans, aromatic diamidines are primarily eliminated in the bile but are distributed and cause toxicity in both liver and kidney. These transporters may play important roles in the disposition of aromatic diamidines in humans, as well as resultant drug-drug interactions and toxicity involving diamidine drugs.

Organic cation transporters (OCTs) in the solute carrier family (SLC) 22A family are mainly expressed in the major elimination organs, kidney (the basolateral membrane of tubular cells), and liver (the sinusoidal membrane of hepatocytes) (Koepsell et al., 2007). There are three distinct OCT transporters, namely, OCT1, OCT2, and OCT3, which mediate the entry of organic cations into cells (Koepsell et al., 2007). In humans, human (h) OCT1 is predominantly expressed in the liver, whereas hOCT2 is mainly expressed in the kidney (Gorboulev et al., 1997; Zhang et al., 1997). OCTs interact with many drugs, including the H2 antagonist ranitidine (Bourdet et al., 2005), the antidiabetic drug metformin (Wang et al., 2002), and the anticancer drug oxaliplatin (Yokoo et al., 2007). The majority of OCT substrates are monovalent and comparatively small cations, the so-called type I organic cations (Meijer et al., 1999; Wright, 2005) as exemplified by the prototypical substrates tetraethylammonium (TEA) and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) (Koepsell et al., 2007). In a recent study, paraquat was identified as a substrate for hOCT2 (Chen et al., 2007). This is the first and only report of a dicationic compound to be transported by a member of the SLC22A family. Chen et al. (2007) speculated that transporters in this family may accept dications as substrates. The results presented in this study confirm this prediction and show that the antifungal and anti-parasitic agent pentamidine and its analog furamidine (Fig. 1), which carry two positive charges at all the physiologic pH values, are selective substrates for hOCT1.

Pentamidine (Fig. 1), an aromatic diamidine, has been used in the clinic against early stage human African trypanosomiasis since 1941 to treat Pneumocystis carinii pneumonia in AIDS patients and to treat antimony-resistant leishmaniasis (Barrett et al., 2007). Furamidine, previously called DB75, is a diamidine analog of pentamidine in which the alkoxy chain linking the phenyl rings has been replaced with a furan ring (Fig. 1) (Das and Boykin, 1977). Furamidine has shown excellent in vitro and in vivo activity in mouse and monkey models of early stage African trypanosomiasis (Barrett et al., 2007). The O-methyl amidoxime prodrug of furamidine, pafuramidine, is in phase III clinical trials as a p.o. active agent to treat human African trypanosomiasis (Barrett et al., 2007).

Carrier-mediated uptake and accumulation in target parasites is crucial for the antiparasitic activity of aromatic diamidines (Barrett et al., 2007). Over 1 mM intracellular concentration of pentamidine was achieved in trypanosomes when they were exposed to 1 μM concentration of this drug (Carter et al., 1995). The uptake of pentamidine into trypanosomes is mediated by multiple transporters, including the P2 transporter, the high-affinity pentamidine transporter 1, and the low-affinity pentamidine transporter 1 (Barrett et al., 2007), none of which share significant sequence homology with hOCTs in the SLC22A family. Mutations in these parasite-specific transporters render the target parasites resistant to pentamidine and other aromatic diamidines (Barrett et al., 2007). It is surprising that a transporter for pentamidine (and other diamidine compounds) has not been identified to date in mammalian cells despite its use in humans for fungal and parasitic infections. The results in this study clearly show that the aromatic diamidines, pentamidine and furamidine, are good substrates for hOCT1 and potent inhibitors for multiple hOCTs. These findings have important implications in understanding their disposition and organ-specific toxicity in humans and in anticipating potential drug-drug interactions when these diamidine drugs are coadministered with other cationic drugs.

Fig. 1.
Fig. 1.

Chemical structures and pKa's of pentamidine and furamidine.

Materials and Methods

Materials. F-12 Nutrient Mixture, penicillin-streptomycin-amphotericin B solution (100×), and HEPES (1 M) were obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum and trypsin-EDTA solution (1×) were obtained from Sigma-Aldrich (St. Louis, MO). Geneticin was obtained from Invitrogen. Hanks' balanced salt solution was obtained from Mediatech, Inc. (Herndon, VA). Pentamidine isethionate salt (purity >98%), TEA chloride, MPP+, and quinidine were purchased from Sigma-Aldrich. Furamidine dihydrochloride salt (purity >98%) was synthesized by Medichem (Chicago, IL) using previously described methods (Das and Boykin, 1977). [3H]MPP+ (85 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). [3H]Pentamidine (4.6 Ci/mmol, purity 97.4%) was obtained from Moravek Biochemicals (Brea, CA). [14C]Furamidine (55.3 mCi/mmol, purity 96%) was obtained from Huntingdon Life Sciences (Huntingdon, UK). Chinese hamster ovary (CHO) cells were obtained from the American Tissue Culture Collection (Manassas, VA).

hOCT1 cDNA in pcDNA3.1 vector and hOCT2 cDNA in pCMV vector were provided by Prof. Hermann Koepsell (Julius-Maximilians-University, Würzburg, Germany). hOCT3 cDNA in pSPORT1 vector was provided by Dr. Vadivel Ganapathy (Medical College of Georgia, Augusta, GA) and was subcloned into pcDNA3.1 vector (Invitrogen).

Transfection of CHO Cells with hOCT cDNAs. CHO cells were transfected with pcDNA3.1 empty vector (mock cells) or the vectors containing the full-length hOCT1, hOCT2, or hOCT3 using the Nucleofector System (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's protocol specific for CHO cells. Transfectants were selected with 500 μg/ml geneticin for 10 days. A clone with the highest [3H]MPP+ uptake activity was chosen as a stably transfected cell line for further studies. The stably transfected CHO cells were cultured in F-12 Nutrient Mixture with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B plus 500 μg/ml geneticin. All the cell lines were grown at 37°C in a humidified atmosphere with 5% CO2.

Transport Studies in hOCT-Expressing CHO Cells. Stably transfected CHO cells were grown as monolayers in 24-well plates. Medium was changed every other day. The cells were used 5 to 7 days postseeding. Cells were preincubated for 30 min at 37°C in transport buffer (Hanks' balanced salt solution with 25 mM d-glucose and 10 mM HEPES, pH 7.2). Experiments were initiated by replacement of the transport buffer with 0.4 ml of radiolabeled dose solutions in transport buffer. Uptake was determined within the linear uptake region, 4 min for [3H]MPP+ and 10 min for [3H]pentamidine and [14C]furamidine, after which the dose solution was aspirated and cells were washed three times with 4°C transport buffer and dissolved in 500 μl of 0.1 N NaOH/0.1% SDS for 4 h with shaking. Radioactivity was determined by scintillation counting. Protein content was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) with bovine serum albumin as a standard.

For kinetic studies, varying amounts of unlabeled diamidines were added to the uptake solutions to give increasing total (radiolabeled plus unlabeled) substrate concentrations. Uptake was determined in the mock cells or CHO-hOCT1 cells over a 10-min period. Nonspecific cell-associated radioactivity was determined by measuring substrate uptake in the mock cells at each substrate concentration, and these values were then subtracted from the results in transfected cells to obtain uptake values and the final kinetic data. The Km and Vmax values were obtained by fitting the Michaelis-Menten equation V = Vmax × [S]/(Km + [S]) to the data using WinNonlin (Pharsight, Mountain View, CA), where V refers to the rate of substrate transport, Vmax refers to the maximum rate of substrate transport, [S] refers to the concentration of substrate, and Km is defined as the concentration of substrate at the half-maximal transport rate.

For inhibition studies, different concentrations of unlabeled inhibitors were added to uptake solution containing 1 μM[3H]MPP+. Uptake was determined over a 4-min period in the absence or presence of increasing concentrations of inhibitors. Nonspecific cell-associated radioactivity was determined by measuring [3H]MPP+ uptake in the mock cells at each inhibitor concentration, and these values were then subtracted from the values in hOCT-transfected cells to give corrected data that were used for generation of the inhibition curves. The IC50 value was estimated by fitting the data to the equation V = Vo/[1 + (I/IC50)n] using WinNonlin (Pharsight), where V is the uptake rate of [3H]MPP+ in the presence of inhibitor, Vo is the uptake rate of [3H]MPP+ in the absence of inhibitor, I is the concentration of inhibitor, and n is the Hill coefficient.

Cytotoxicity Assay. The cytotoxicity of diamidines was measured by the Alamar Blue assay (Schoonen et al., 2005). In brief, cells were seeded in 96-well plates at 3000 cells/well. After 24 h, cells were exposed to different concentrations of diamidines in the presence or absence of 1 mM ranitidine, an OCT1 inhibitor (Bourdet et al., 2005), for 24 h. Drug-containing medium was replaced with fresh medium, and cells were incubated for an additional 24 h. Alamar Blue reagent was added and incubated for 4 h. The samples were read in a microplate reader set at 544-nm excitation wavelength and 584-nm emission wavelength. The IC50 values were obtained by fitting F, the percentage of the maximal cell growth at different drug concentrations, to the equation F = 100/[1 + (C/IC50)n] using WinNonlin (Pharsight); the maximal cell growth was the cell growth in the medium without diamidine; C is the concentration of diamidine, and n is the slope factor.

Statistical Analysis. Data are expressed as mean ± S.D. from three measurements unless otherwise noted. Statistical significance was evaluated using analysis of variance followed by Dunnett's test for multiple comparisons. The data were analyzed with SigmaStat 2.0 (Systat Software, Inc., San Jose, CA).

Results

Expression of Functional hOCT1, hOCT2, and hOCT3 in Stably Transfected CHO Cells. CHO cells that were stably transfected with hOCT cDNAs were evaluated for the expression of functional hOCT1, hOCT2, and hOCT3 genes. Uptake of 1 μM[3H]MPP+, a model substrate for OCTs, as a function of time was determined in CHO cells transfected with hOCT1 (CHO-hOCT1), hOCT2 (CHO-hOCT2), or hOCT3 (CHO-hOCT3) and compared with its uptake in the mock cells. The concentration of [3H]MPP+ used is significantly lower than the reported Km values of MPP+ for hOCT1 (15–32 μM), hOCT2 (19–78 μM), and hOCT3 (47 μM) (Koepsell et al., 2007). Uptake of [3H]MPP+ in each transfected cell line was linear up to 5 min and was severalfold (p < 0.001) greater than in the mock cells (Fig. 2), providing evidence for expression of functional hOCT transporters in these cell lines.

Fig. 2.
Fig. 2.

Functional expression of hOCT1, hOCT2, and hOCT3 in stably transfected CHO cells. CHO cells, stably transfected with hOCT1, hOCT2, hOCT3 cDNA (•), or empty vector (mock cells, ○) were incubated with [3H]MPP+ (1 μM) at 37°C for the indicated time. Data represent mean ± S.D. from a representative experiment performed in triplicate.

Inhibition of hOCT1, hOCT2, and hOCT3 by Aromatic Diamidines in CHO Cells. As expected, uptake of 1 μM[3H]MPP+ into CHO-hOCT1, CHO-hOCT2, and CHO-hOCT3 cells was inhibited in a concentration-dependent manner by the prototypical OCT inhibitors TEA and quinidine (Fig. 3), with quinidine showing over an order of magnitude greater potency than TEA (Table 1). The two aromatic diamidine compounds tested, pentamidine and furamidine (Fig. 1), proved to be potent inhibitors of all three hOCTs. The results in Table 1 show that pentamidine is a potent inhibitor of hOCTs with IC50 values of <20 μM for all three transporters. Thus, its potency toward all the hOCTs is considerably greater than that of TEA and similar to that of quinidine, which is considered to be one of the most potent inhibitors of these transporters. Furamidine, a structural analog of pentamidine, is also a potent inhibitor of hOCT1 and hOCT3 (IC50 of 7.4 ± 0.9 and 20.4 ± 2.8 μM, respectively) but a surprisingly weak inhibitor of hOCT2 (IC50 of 182.0 ± 30.3 μM).

View this table:
TABLE 1

Inhibition of hOCT1-, hOCT2-, or hOCT3-mediated [3H]MPP+ uptake by pentamidine and furamidine in the respective hOCT-expressing CHO cells

Fig. 3.
Fig. 3.

Concentration-dependent inhibition of hOCT1, hOCT2, and hOCT3 by diamidines. Uptake of [3H]MPP+ (1 μM) in CHO-hOCT1, CHO-hOCT2, CHO-hOCT3, and the mock cells was determined in the absence or presence of increasing concentrations of pentamidine (•), furamidine (▾), quinidine (○), and TEA (□) for 4 min. Nonspecific cell-associated radioactivity was determined by measuring substrate uptake in the mock cells at each inhibitor concentration, and these values were then subtracted from the values in hOCT1-transfected cells to give corrected data that were used for generation of the inhibition curves. Thus, the data represent inhibition of the transporter-mediated portion of [3H]MPP+ uptake. Data are expressed as mean ± S.D. of experiments in triplicate.

Uptake of Pentamidine and Furamidine into hOCT1-, hOCT2-, and hOCT3-Expressing CHO Cells. As shown in Fig. 4A, the uptake of 0.1 μM[3H]pentamidine into CHO-hOCT1 cells over a 10-min period (linear range, see Figs. 4B and 5B) was 5.1-fold (p < 0.001) greater than that into the mock cells. In contrast, [3H]pentamidine was taken up into CHO-hOCT2 cells at only a marginally greater rate (1.2-fold, p < 0.05) than into the mock cells, and no transporter-mediated uptake into CHO-hOCT3 cells could be shown. The uptake of 1 μM[14C]furamidine was 4.6-fold (p < 0.001) and 1.6-fold (p < 0.01) greater into CHO-hOCT1 and CHO-hOCT2 cells, respectively, compared with the mock cells, whereas the uptake into CHO-hOCT3 cells was not above the control value (Fig. 5A). These data suggest that pentamidine and furamidine are selective substrates of hOCT1, showing statistically significant but nonetheless extremely weak substrate activity toward hOCT2 and none toward hOCT3. In CHO-hOCT1 cells, accumulation of pentamidine (Fig. 4B) and furamidine (Fig. 5B) was linear over 10 min. The uptake, measured as a function of concentration, was saturable (Figs. 4C and 5C) with Km values of 36.4 ± 8.3 and 6.1 ± 1.1 μM, respectively, and Vmax values of 156.0 ± 3.7 and 27.2 ± 1.3 pmol/mg/min, respectively.

Cytotoxicity of Aromatic Diamidines in CHO-hOCT1 Cells. In light of the known renal and hepatic toxicity of pentamidine (O'Brien et al., 1997), the question that was addressed next was whether the hOCT1-mediated cellular uptake of the diamidines and resultant high intracellular concentrations could render these diamidines toxic toward CHO-hOCT1 cells. The toxicity of pentamidine toward CHO-hOCT1 cells was 4.4-fold (p < 0.001) greater than toward the mock cells (IC50 of 46.2 ± 5.3 and 202 ± 16.4 μM, respectively), indicating that hOCT1 enhances pentamidine-induced cytotoxicity (Fig. 6A; Table 2). Although furamidine was significantly more cytotoxic than pentamidine toward the mock cells (IC50 of 10.2 ± 0.7 μM), its cytotoxicity was enhanced by nearly 9.3-fold (p < 0.001) when hOCT1 mediated its cellular uptake (IC50 of 1.1 ± 0.05 μM in CHO-hOCT1 cells) (Fig. 6B; Table 2). To confirm that the enhancement of cytotoxicity of the diamidines in CHO-hOCT1 cells was caused by the increased accumulation of drugs mediated by hOCT1, diamidine toxicity to the mock and CHO-hOCT1 cells was evaluated in the presence of an hOCT1 inhibitor. Ranitidine, a known inhibitor of hOCTs (Bourdet et al., 2005), was used for these experiments because it could completely abolish the hOCT1 activity at a concentration (1 mM) that showed no cytotoxicity toward the CHO-hOCT1 cells and that did not affect the toxicity of diamidines to the mock cells (because of hOCT1-unrelated mechanisms). As expected, ranitidine treatment almost completely eliminated the hOCT1-mediated enhancement of the diamidine cytotoxicity (Figs. 6, A and B).

View this table:
TABLE 2

Cytotoxicity of pentamidine and furamidine in CHO-hOCT1 cells and the mock CHO cells

The IC50 values of pentamidine and furamidine in CHO-hOCT2 and CHO-hOCT3 cells were not significantly different from those in the mock cells (Tables 3 and 4), consistent with the results reported here that the diamidines are not substrates of hOCT2 or hOCT3.

View this table:
TABLE 3

Cytotoxicity of pentamidine and furamidine in CHO-hOCT2 cells and the mock CHO cells

View this table:
TABLE 4

Cytotoxicity of pentamidine and furamidine in CHO-hOCT3 cells and the mock CHO cells

Fig. 4.
Fig. 4.

Uptake of [3H]pentamidine by CHO-hOCT1, CHO-hOCT2, and CHO-hOCT3 cells. A, uptake of [3H]pentamidine (0.1 μM) was determined in CHO-hOCT1, CHO-hOCT2, CHO-hOCT3, and the mock cells over 10 min. B, uptake of [3H]pentamidine (0.1 μM) was determined in the mock cells or CHO-hOCT1 cells for the indicated time periods. C, uptake of [3H]pentamidine (indicated concentration) was determined in the mock cells or CHO-hOCT1 cells for 10 min. Nonspecific cell-associated radioactivity was determined by measuring the compound uptake in the mock cells at each substrate concentration, and these values were then subtracted from the values in hOCT1-transfected cells to obtain the final kinetic curves. Data represent mean ± S.D. of a representative experiment in triplicate for both the mock and the transfected cells. ***, p < 0.001, **, p < 0.01, and *, p < 0.05 compared with uptake in the mock cells.

Discussion

OCTs had previously been found to interact mainly with monovalent organic cations (type I cations) (Wright, 2005). In a recent study, one divalent organic cation, paraquat, has been identified to be an hOCT2 substrate (Chen et al., 2007). The two amidine groups in pentamidine exhibit successive pKa's of 11.5 and 12.9, and those in furamidine have successive pKa's of 10.4 and 11.8 (Saulter, 2005). Thus, both diamidines behave as divalent cations in the body because their pKa's are more than 3 units above any physiologic pH encountered by them in the blood circulation and other tissue compartments. Our finding that the divalent cations, aromatic diamidines, are substrates of hOCT1 not only confirms what Chen et al. (2007) characterized as a speculative suggestion about the functional transport activity of OCTs for dications but also extends their observation by showing that hOCT1, like hOCT2, also transports divalent cations. This is also the first report in which a drug with divalent cation functionality is shown to be an OCT substrate because paraquat is an herbicide without clinical applications.

Fig. 5.
Fig. 5.

Uptake of [14C]furamidine by CHO-hOCT1, CHO-hOCT2, and CHO-hOCT3 cells. A, uptake of [14C]furamidine (1 μM) was determined in CHO-hOCT1, CHO-hOCT2, CHO-hOCT3, and the mock cells over 10 min. B, uptake of [14C]furamidine (1 μM) was determined in the mock cells or CHO-hOCT1 cells for the indicated time periods. C, uptake of [14C]furamidine (indicated concentration) was determined in the mock cells or CHO-hOCT1 cells for 10 min. Nonspecific cell-associated radioactivity was determined by measuring the compound uptake in the mock cells at each substrate concentration, and these values were then subtracted from the values in hOCT1-transfected cells to obtain the final kinetic curves. Data represent mean ± S.D. of a representative experiment in triplicate for both the mock and the transfected cells. ***, p < 0.001, **, p < 0.01, and *, p < 0.05 compared with uptake in the mock cells.

These results also provide the first evidence that the clinically used antifungal and antiparasitic agent pentamidine and its analog furamidine, which is also a clinical candidate, are substrates for human transporters. The results further show that the three hOCT isoforms exhibit differential substrate and inhibition profiles toward the divalent cations. For example, aromatic diamidines are hOCT1 substrates but show no substrate activity for hOCT2 or hOCT3. In contrast, paraquat has been reported as an hOCT2 substrate with poor or no substrate activity for hOCT1 and hOCT3 (Chen et al., 2007). It is important to note that the aromatic diamidines are potent inhibitors of hOCT3, although they are not transported by it. While this article was in preparation, Jung et al. (2008) reported that pentamidine was a potent inhibitor of hOCT1 and hOCT2, which is consistent with the results in this study. However, they did not show the contribution of hOCT1 and hOCT2 to the transport of pentamidine because of a high nonspecific uptake in human embryonic kidney cells, in which hOCTs were transfected (Jung et al., 2008).

Fig. 6.
Fig. 6.

Effect of hOCT1-mediated transport on pentamidine and furamidine cytotoxicity. After 1-day culture in 96-well plates, CHO-hOCT1 (circle) or the mock cells (square) were incubated with different concentrations of pentamidine (A) or furamidine (B) in the presence (solid) or absence (open) of 1 mM ranitidine, an OCT1 inhibitor, for 24 h followed by another 24-h culture in fresh media. Alamar Blue assays were performed afterward, and the IC50 values or cell growth inhibition were calculated. Studies were performed in triplicate in each individual experiment.

In humans, pentamidine is mainly distributed into the liver (Thomas et al., 1997) and eliminated via biliary secretion (Conte, 1991). In contrast, small hydrophilic cationic drugs, such as metformin (Pentikainen et al., 1979) and cimetidine (Weiner and Roth, 1981), are mainly excreted via the kidney. Selective distribution of certain OCTs in the kidney probably contributes to the preferential renal elimination of these compounds because both metformin (Kimura et al., 2005) and cimetidine (Tahara et al., 2005) are superior substrates for hOCT2, predominantly expressed in the kidney (Gorboulev et al., 1997), than for hOCT1 that is predominantly expressed in the liver (Gorboulev et al., 1997). The results reported in this study show that pentamidine and furamidine are good substrates for hOCT1 but have practically no substrate activity toward hOCT2. These results are consistent with the existing literature on the disposition of pentamidine in humans. AIDS patients treated with 4 mg of pentamidine/kg by 2 h i.v. infusion had peak plasma concentrations of 1.80 ± 1.09 μM (Food and Drug Administration label of pentamidine). Taking into consideration ∼70% plasma protein binding of pentamidine in humans (Benet and Hoener, 2002), the free drug concentrations of pentamidine are likely to be much lower than the Km value of pentamidine for hOCT1 (36.4 μM), suggesting that hOCT1 would not be saturated at typical therapeutic doses and should influence pentamidine disposition in the patients treated with it. Furthermore, coadministered drugs that are hOCT1 inhibitors are likely to affect pentamidine disposition. In rats, Oct1 is expressed in the liver and the kidney; in fact, highest Oct1 expression occurs in the kidney (Grundemann et al., 1994). This may explain why similar amounts of pentamidine were detected in urine and feces after i.v. administration to rats (Bronner et al., 1995). Hepatic elimination via biliary secretion of hydrophilic drugs involves entry into the hepatocytes across the sinusoidal membrane from the blood, followed by intracellular disposition, and secretion from the cells across the canalicular membrane. The cation-selective sinusoidal uptake transporters, including hOCT1, mediate the first step of hepatic uptake and thus play a critical role in determining the tissue distribution and hepatic elimination of cationic drugs (Urban and Giacomini, 2007). Likewise, canalicular efflux transporters, including P-glycoprotein and multidrug and toxin extrusion H+/drug antiporters, also play a role in hepatic elimination of organic cations by mediating the second step of egress to the bile (Koepsell et al., 2007).

Parenteral pentamidine therapy caused adverse drug reactions including nephrotoxicity and hepatotoxicity (O'Brien et al., 1997). Pentamidine and furamidine are DNA minor groove binders (Barrett et al., 2007), and furamidine selectively accumulates in nuclei of tumor cells (Lansiaux et al., 2002). The rapid entry of these charged compounds into cells could not be accounted for by passive diffusion process (Lansiaux et al., 2002). It was speculated that transporters, analogous to those responsible for the entry of aromatic diamidines into parasites (Barrett et al., 2007), must exist to promote intracellular accumulation of these diamidines. These results showed that overexpression of hOCT1 not only increased the uptake of pentamidine and furamidine into CHO cells but also enhanced the potency of these compounds as cytotoxic agents. OCT-mediated transport has been reported to enhance the cytotoxicity of cisplatin (Ciarimboli et al., 2005), oxaliplatin (Zhang et al., 2006), and paraquat (Chen et al., 2007). Transport of organic cations across cell membrane via OCTs is driven by membrane potential and the substrate concentration gradient, and cellular uptake of positively charged molecules is thermodynamically favorable because of the electrical potential that exists across the cell membrane with the inside of the cell more negative than the outside (Koepsell et al., 2007). The hOCT1-assisted cell entry of organic cations can produce over 10-fold higher intracellular concentrations compared with the dose concentration (Koepsell et al., 2007). With two positive charges in each molecule, aromatic diamidines may achieve even higher intracellular concentrations, driven by membrane potential. These results suggest that hOCT1 could promote the accumulation of aromatic diamidines in human tissues and may play an important role in diamidine-induced cytotoxicity in the organs that express hOCT1, such as liver. The contribution of the transporter in vivo to the hepatic clearance and hepatotoxicity of aromatic diamidines needs to be examined in animal and clinical studies. In human kidney, hOCT2 is highly expressed but not hOCT1 (Gorboulev et al., 1997). Aromatic diamidines are practically not substrates toward hOCT2; therefore, transport by renal OCTs may not account for the severe nephrotoxicity of pentamidine (O'Brien et al., 1997). However, in a recent study that used quantitative real-time polymerase chain reaction technique, expression of hOCT1 mRNA was shown to be only a few-fold lower than that of hOCT2 in the kidney (Hilgendorf et al., 2007), suggesting that hOCT1 may contribute to basolateral uptake of cations, including diamidines, in the kidney. Therefore, whether hOCTs modulate nephrotoxicity of diamidines needs further investigation.

Drug-drug interactions via OCTs have been well documented (Li et al., 2006). For example, cimetidine is a potent hOCT2 inhibitor, and coadministration with cimetidine reduced renal clearance of other cationic drugs (Ayrton and Morgan, 2001). Although pentamidine and furamidine are selectively transported by hOCT1, they are potent inhibitors of multiple hOCTs. In fact, their inhibitory potency is similar to that of quinidine, which is one of the most potent OCT inhibitors known among the marketed drugs (Urakami et al., 2002; Bednarczyk et al., 2003; Bourdet et al., 2005). The free plasma concentrations of pentamidine are likely to be lower than its IC50 values for hOCTs. However, it is important to note that pentamidine is accumulated in the liver, and to a lesser extent in the kidney, in humans (Thomas et al., 1997), and furamidine is mainly accumulated in the liver after p.o. administration of its prodrug to rats (Midgley et al., 2007). Therefore, it is reasonable to conclude that their intracellular concentrations in hepatocytes, and perhaps in renal tubular cells, should be much higher than that in the plasma. Because the OCTs are bidirectional transporters (Koepsell et al., 2007), the diamidines may influence their activity (inhibit or activate) from the intracellular side. If so, pentamidine may affect the renal clearance of other cations that are hOCT2 substrates, and both diamidines may interfere with hepatic uptake of other hOCT1 substrates, such as metformin (Shu et al., 2008), and change their pharmacokinetic and pharmacodynamic behaviors.

The results presented in this study make a strong case that divalent organic cations are likely to be transported across cell membranes via hOCT1, and that this class of compounds may have potent inhibitory activity toward multiple hOCTs. This study has shown for the first time that the antifungal and antiparasitic drug pentamidine and a structurally similar diamidine drug, furamidine, are substrates for a human transporter (hOCT1) and inhibitors for multiple hOCTs. Although further studies are warranted, these findings provide a rationale for the observed disposition and excretory organ toxicity of pentamidine and raise the specter of potential drug-drug interactions when an aromatic diamidine is coadministered with monovalent and divalent organic cations.

Acknowledgments

We thank Prof. Hermann Koepsell (Julius-Maximilians-University) for providing the plasmids of pcDNA3.1/hOCT1 and pCMV/hOCT2 and Dr. Vadivel Ganapathy (Medical College of Georgia) for providing the plasmid of pSPORT1/hOCT3.

Footnotes

  • This study was partially supported by the Bill and Melinda Gates Foundation [Grant 38920].

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.108.024083.

  • ABBREVIATIONS: OCT, organic cation transporter; SLC, solute carrier family; h, human; TEA, tetraethylammonium; MPP+, 1-methyl-4-phenylpyridinium; CHO, Chinese hamster ovary.

    • Received August 28, 2008.
    • Accepted October 28, 2008.

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Transport of Dicationic Drugs Pentamidine and Furamidine by Human Organic Cation Transporters

Xin Ming, Wujian Ju, Huali Wu, Richard R. Tidwell, James E. Hall and Dhiren R. Thakker
Drug Metabolism and Disposition February 1, 2009, 37 (2) 424-430; DOI: https://doi.org/10.1124/dmd.108.024083
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