Several classes of antiretroviral (ARV) drugs (ARDs) comprising nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) are combined to the highly active ARV therapy in human immunodeficiency virus (HIV) (Piacenti, 2006). Long-term efficacy and avoidance of toxic effects are the main therapeutic challenges. For example, severe liver toxicity has been described for PIs like indinavir, nelfinavir, and ritonavir and for NNRTIs like nevirapine and efavirenz and different NRTIs like lamivudine (Carr et al., 2001; Clark et al., 2002). Nephrotoxicity is a common side effect of the NRTI tenofovir (Callens et al., 2003), and combination therapy leads to an aggravation of toxic reactions (Zimmermann et al., 2006).

Understanding of ARD interactions with their cellular targets (i.e., metabolizing enzymes and xenobiotic transporters) is essential for the improvement of the therapy. Interactions of ARDs with enzymes have been studied in detail and have tailored therapeutic use. In particular, ritonavir, which is a potent inhibitor of cytochrome P450 isoenzymes in liver and intestinum, is clinically used to boost the plasma levels of other CYP3A4 substrates such as the PIs saquinavir and indinavir (Hsu et al., 1998).

In contrast, the role of xenobiotic transporters for the highly active ARV therapy is less clearly defined (Thomas, 2004). More than 50% of the currently used ARDs carry positively charged nitrogen moieties under physiological conditions. Hence, they may be considered as potential substrates or inhibitors of the organic cation transporters (OCTs), especially of the subtypes 1 (SLC22A1) and 2 (SLC22A2), which are predominantly expressed in liver and kidney, respectively, and thus have been suggested to be centrally involved in the elimination of cationic xenobiotics (Okuda et al., 1996; Gorboulev et al., 1997). Moreover, ARV inhibitors or substrates of OCT1 and OCT2 may also influence drug elimination of other drugs translocated by OCT1 and OCT2 or alter the physiological function of their recently discovered endogenous substrates cyclo(his-pro) or salsolinol (Taubert et al., 2007).

Apart from the predominant localization in liver and kidney, little is known about differential expression and regulation of OCT1 and OCT2 in other organ systems, especially in lymphoid tissues, which are the main sites of accumulation and replication of HIV (Haase, 1999). Hence, transporter-mediated uptake of ARDs in lymphoid cells may be important for the control of the HIV infection to avoid disease propagation (Kinman et al., 2006; Salama et al., 2006).

The purpose of the present study was to investigate the interaction of common cationic ARV and concomitantly administered cationic drugs such as pentamidine and trimethoprim with OCT1 and OCT2 using a heterologous expression system. Furthermore, we explored the expression profile of OCT1 and OCT2 in different peripheral tissues of healthy donors. In addition, we examined the expression of OCT1 and OCT2 in lymph nodes (LNs) of HIV-infected patients compared with healthy controls.

Materials and Methods

Chemicals. The pharmaceuticals were provided by the following companies: ritonavir, Abbott (Wiesbaden, Germany); nevirapine, Boehringer (Ingelheim, Germany); atazanavir, efavirenz, and stavudine, Bristol-Myers Squibb (München, Germany); isoniazid, trimethoprim, and rifampicin, Sigma-Aldrich (Taufkirchen, Germany); adefovir, cidofovir, emtricitabine, and tenofovir, Gilead (Martinsried/München, Germany); abacavir, acyclovir, amprenavir, lamivudine, pentamidine, and zidovudine, GlaxoSmithKline (Bad Oldesloe, Germany); indinavir, MSD Sharp and Dohme (Harr, Germany); and nelfinavir, saquinavir, and zalcitabine, Roche (Grenzach-Wyhlen, Germany). H³-1-Methyl-4-phenylpyridinium (MPP+) (3.0 TBq/mmol) was purchased from Amersham Biosciences (Piscataway, NJ). All the other chemicals were purchased from Sigma-Aldrich, Merck (Darmstadt, Germany), and Roth (Karlsruhe, Germany).

Tissue Preparation and Quantification of mRNA. LNs from HIV-1–infected people in different stages of disease (Centers for Disease Control and Prevention stage A, n = 5; Centers for Disease Control and Prevention stage C, n = 7) without ARV therapy and healthy controls without any infectious or inflammatory disease (n = 5) were surgically dissected either from the cervical or the inguinal region, immediately placed in saline, and mononuclear cells were dissociated mechanically. Lymphoid tissue mononuclear cells were isolated by density gradient centrifugation over Ficoll (BD, Heidelberg, Germany) and stored in fetal calf serum (PAA, Cölbe, Germany) with 10% dimethyl sulfoxide (Sigma-Aldrich) in liquid nitrogen until analysis. Total RNA was extracted by the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) accompanied by digestion of contaminating DNA by DNase I treatment. For the expression profiling, human total RNA samples were obtained from the Clontech (Mountain View, CA) collection, comprising equally pooled samples of 10 to 30 normal, 20- to 60-year-old female and male Caucasian individuals (except the gender-specific organs).

Reverse transcription was performed as described before (Taubert et al., 2007). Briefly, the integrity of the RNA was assessed by denaturing gel electrophoresis; RNA was reversed-transcribed into cDNA; and the cDNA was quantified by spot test. Expression of OCT1 and OCT2 mRNAs was determined from 10-ng samples of cDNA by quantitative real-time polymerase chain reaction (PCR) (LightCycler; Roche Diagnostics) relative to the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using TaqMan gene expression assays (Applied Biosystems, Foster City, CA). Assay IDs were as follows: Hs00427550_m1 (OCT1), Hs00533907_m1 (OCT2), and Hs99999905_m1 (GAPDH). Relative quantifications were performed by pair-wise fixed reallocation randomization test (Pfaffl, 2001) and corrected for amplification efficiency as evaluated from serial dilutions of cDNA. The expression values presented show the copy number of transporter transcript relative to the copy number of GAPDH in the same cDNA. Failing expression (expression value = 0) was considered after more than 45 cycles of amplification without increase in fluorescence intensity.

Cloning and Cell Culture. The cDNA encoding OCT1 (GenBank accession number NM_003057) and OCT2 (NM_003058) was amplified by reverse transcription-PCR from human liver and kidney total RNA and subcloned into the eukaryotic expression vector pcDNA3. Human embryonic kidney (HEK)-293 cells lacking endogenous OCT1 and OCT2 mRNA expression were stably transfected with the respective and the empty vectors and selected with geneticin as described (Ey et al., 2007). Experiments were performed on cell clones exhibiting an equal mRNA expression level of the transporters, as determined by quantitative real-time PCR. HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum.

Transport Experiments. For uptake experiments to identify substrates, cells were grown on polystyrene dishes of 60-mm diameter to at least 80% confluence. Before addition of test compounds, culture medium was replaced by 3 ml of HEPES-modified Krebs buffer (140.0 mM NaCl, 5.0 mM KCl, 2.0 mM CaCl₂, 1.0 mM MgCl₂, 10.0 mM HEPES, and 5.0 mM d-glucose, adjusted to pH 7.40), and cells were left to equilibrate for 30 min at 37°C. Uptake was terminated after indicated time points by washing cells three times with 3 ml of ice-cold buffer. Cells were lysed with 1 ml of 4 mM perchloric acid and subjected to electrospray ionization–liquid chromatography/tandem mass spectrometry quantification. Cellular protein content was determined after solubilization with 1 ml of 0.1 mM NaOH using the bicinchoninic assay (BCA Protein Assay Kit; Pierce, Rockford, IL).

For uptake experiments to identify inhibitors, test compounds were added to the preincubation buffer for 30 min and then coincubated with the radiolabeled model substrate ³H-MPP+ (100 nM). Uptake was stopped after 1 min by washing with ice-cold buffer. Subsequently cells were solubilized with 0.1% v/v Triton X-100 in 5 mM Tris-HCl, pH 7.4. Radioactivity (disintegrations per minute) was determined by liquid scintillation counting. Cellular protein content was determined as described above.

Liquid Chromatography/Tandem Mass Spectrometry Quantification of Transport Substrates. The lysates of uptake experiments were analyzed by a triple-quadrupole tandem mass spectrometer (TSQ Quantum; Thermo Electron, Dreieich, Germany) equipped with a thermostated (10°C) Surveyor autosampler and thermostated (30°C) Surveyor high-performance liquid chromatography system (Thermo Electron) operating in positive electrospray ionization mode as described (Taubert et al., 2007). Spray voltage was set at 4000 V, and capillary temperature was kept at 350°C. Nitrogen sheath gas and auxiliary gas pressure were 40 and 4 psi, respectively. Argon collision gas pressure was 1.5 mTorr. Twenty-microliter aliquots of the samples were injected onto an Aquasil C18 column (3 μm, 100 × 4.6 mm; Thermo Electron) and eluted isocratically at a flow rate of 300 μl/min. The mobile phase consisted of 0.1% formic acid in deionized water and 0.1% formic acid in acetonitrile (30:70% v/v). Quantification was performed by using the single reaction monitoring mode to study the specific precursor ion [M + H]⁺ →product ion transitions (collision energy) for lamivudine, m/z 230→112 (25 eV); zalcitabine, m/z 212→112 (25 eV); stavudine, m/z 225→127 (25 eV); nevirapine, m/z 267→226 (25 eV); efavirenz, m/z 314→244 (20 eV); zidovudine, m/z 268→127 (25 eV); emtricitabine, m/z 248→130 (25 eV); abacavir, m/z 248→152 (25 eV); tenofovir, m/z 288→176 (25 eV); acyclovir, m/z 226→152 (17 eV); adefovir, m/z 274→162 (25 eV); nelfinavir, m/z 568→330 (25 eV); indinavir, m/z 615→421 (10 eV); ritonavir, m/z 721→296 (25 eV); saquinavir, m/z 671→570 (25 eV); amprenavir, m/z 506→245 (25 eV); atazanavir, m/z 705→168 (30 eV); pentamidine, m/z 341→324 (22 eV); trimethoprim, m/z 291→230 (25 eV); isoniazid, m/z 138→121 (22 eV); rifampicin, m/z 823→791 (25 eV); and MPP+, m/z 170→128 (36 eV, internal standard, 50 ng/ml). Detection limit was at least 0.001 nmol/mg protein.

Kinetic and Statistical Analysis. Uptake velocity versus substrate concentration data were fitted by the Michaelis-Menten equation: V₀ = V_max · [S]/(K_m + [S]), where V₀ and V_max represent initial and maximal transport velocity, respectively (nmol/mg protein/min), [S] initial substrate concentration (μM), K_m substrate concentration at half-maximal transport velocity (μM), and V_max/K_m efficacy of transport. For IC₅₀ studies, data were fit to the equation V = V₀/[1+(I/IC₅₀)ⁿ] where V is the uptake of [³H] MPP+ (1 min) in the presence of inhibitor (nmol/mg protein/min), V₀ is the uptake of [³H] MPP+ in the absence of inhibitor, I is the inhibitor concentration (μM), and n is the Hill coefficient. Statistical significance of differences between two treatments was evaluated by the unpaired two-tailed t test, differences between more than two treatments by one-way analysis of variance or Kruskal-Willis test, as applicable; p < 0.05 was considered statistically significant.

Results

Screening of OCT1 and OCT2 Inhibitors. As shown in Fig. 1, A–F, of the 22 cationic drugs tested, the PIs nelfinavir, ritonavir, saquinavir, and indinavir and the anti-infective drugs pentamidine and trimethoprim inhibited the uptake of radiolabeled MPP⁺ by OCT1 and OCT2. The IC₅₀ values ranged from 0.4 μM for pentamidine (OCT1) up to 275 μM for indinavir (OCT2). All the drugs inhibited OCT1 2 to 10 times more effectively than OCT2. Nelfinavir was the strongest inhibitor among the PIs. Pentamidine showed the strongest inhibitory potential [IC₅₀ 0.4 μM (OCT1); 3.8 μM (OCT2)] exceeding that of the other drugs by a factor of 100 (Table 1; Fig. 2). The IC₅₀ values are in the range of the C_max values of the drugs, which have been determined in human pharmacokinetic studies.

Identification of Lamivudine and Zalcitabine as Substrates of OCT1 and OCT2. A significant specific uptake by OCT1 and OCT2 was observed for lamivudine, zalcitabine, pentamidine, and trimethoprim. However, pentamidine and trimethoprim exhibited a high nonspecific uptake, which excluded a relevant contribution of OCT1 and OCT2 to the transport of these substances. Nelfinavir showed an equal accumulation in OCT1, OCT2, and control cells (unspecific OCT1 and OCT2 independent transport) (Fig. 3A). Drugs without detectable uptake in OCT1, OCT2, or control cells were abacavir, acyclovir, adefovir, amprenavir, atazanavir, efavirenz, emtricitabine, indinavir, isoniazid, nevirapine, rifampicin, ritonavir, saquinavir, stavudine, tenofovir, and zidovudine (data not shown).

Assessment of the transport kinetics revealed V_max and K_m values of lamivudine and zalcitabine as shown in Fig. 3B. Transport efficacy V_max/K_m was higher for lamivudine compared with zalcitabine and higher in OCT1 compared with OCT2 cells for both drugs. The inhibitors of the MPP+ uptake in OCT1 and OCT2 cells (Fig. 1, A–F) also inhibited lamivudine and zalcitabine uptake, with pentamidine showing the strongest efficacy followed by nelfinavir, ritonavir, trimethoprim, and saquinavir (Fig. 3, C and D).

Expression Profile of OCT1 and OCT2. In keeping with previous studies, the highest RNA expression of OCT1 and OCT2 was detected in liver and kidney, respectively. Additionally we found significant levels of OCT1 in kidney, prostate, ovary, colon, heart, lung, mammary gland, pancreas, skeletal muscle, small intestine, spleen, and testis and of OCT2 in testis, colon, heart, skeletal muscle, and small intestine (Fig. 4, A and B).

Increased Expression of OCT1 and OCT2 in LNs of HIV-Infected Patients with Severe Disease. Because of their crucial role in the pathogenesis of HIV infection, we focused attention on the expression of the transporters in LNs. Whereas OCT1 and OCT2 levels in LN of healthy controls were negligible, we determined much higher expression in HIV-infected persons in correlation with the disease stage (Fig. 5, A and B). In comparison, LN expression of the phylogenetically related extraneuronal monoamine transporter SLC22A3 or carnitine transporter SLC22A5 (OCTN2) was not different between HIV-infected and noninfected individuals (data not shown).

Discussion

The main findings of the present study were as follows: 1) we could identify and characterize several inhibitors of OCT1 and OCT2 among different PIs and anti-infective drugs; 2) we could identify and characterize lamivudine and zalcitabine as substrates of OCT1 and OCT2 with high transport efficacy; 3) we could identify new localizations of OCT1 in ovary, prostate, and testis and of OCT2 in colon, heart, skeletal muscle, and testis; and 4) we could detect an increased expression of OCT1 and OCT2 in LNs of HIV-infected persons with disease stage C.

The PIs nelfinavir, ritonavir, saquinavir, and indinavir and the anti-infective drugs pentamidine and trimethoprim could be identified as inhibitors of OCT1 and OCT2 with IC₅₀ values ranging from 0.4 μM for pentamidine (OCT1) up to 275 μM for indinavir (OCT2). These concentrations are in the range of therapeutically relevant plasma levels after p.o. dosing (Table 1). Our data of the inhibition of the OCT1-mediated transport by the PIs nelfinavir, ritonavir, saquinavir, and indinavir are supported by Zhang et al. (2000), who examined the inhibition of the uptake of C¹⁴-tetraethylammonium into transiently transfected HeLa cells and obtained similar IC₅₀ values.

Applying the liquid chromatography/tandem mass spectrometry technology, we could identify two NRTIs, lamivudine and zalcitabine, as substrates of OCT1 and OCT2 with high transport efficacies. Takubo et al. (2002) and Leung and Bendayan (2001) support our data by the detection of the uptake of lamivudine in brush-border membrane vesicles from rat renal cortex and in a porcine renal epithelial cell line (LLC-PK), respectively. They proposed the existence of a specific transporter system, which we could identify (Leung and Bendayan, 2001; Takubo et al., 2002). In contrast to our results, Takeda et al. (2002) identified acyclovir as a substrate of OCT1 in stably transfected S2 cells, but the transport efficacy was very low (V_max/K_m = 0.7 μl/mg/min), namely, 5 to 10 times lower than the efficacies we determined for lamivudine and zalcitabine (Takeda et al., 2002) (Fig. 2B).

Lamivudine is a broadly used drug in HIV therapy as it belongs to one of the alternative components recommended by the Food and Drug Administration for the first choice in the combined treatment of therapy-naive patients. Lamivudine is often combined with ritonavir to boost plasma levels of a second PI. This could lead to an accumulation of the substrate lamivudine by reduced renal or hepatic elimination or enhanced excretion by reduced cellular uptake caused by the inhibition of OCT1 and OCT2 by ritonavir.

Furthermore, trimethoprim and pentamidine are frequently administered with lamivudine in HIV patients as these anti-infective drugs are used for primary and secondary Pneumocystis jirovecii prophylaxis. Interactions of lamivudine with trimethoprim have been described both in in vitro experiments and in clinical studies: the coadministration of trimethoprim led to a decrease of 35% in the renal clearance of lamivudine (Moore et al., 1996; Takubo et al., 2000). This decrease may be caused by the inhibition of the renal transport of lamivudine via OCT2 by trimethoprim.

The combination of zalcitabine with lamivudine for the ARV therapy is not recommended and should even be avoided as competition for intracellular phosphorylation may occur (Barry et al., 1999). A possible mechanism could be the competition of zalcitabine and lamivudine, which we could characterize as substrates of OCT1 and OCT2, for the transport into the cells, leading to decreased drug levels intracellularly, where phosphorylation takes place.

Because OCT2 is highly expressed in the kidney and both zalcitabine and lamivudine are mainly eliminated by the kidney (70%), pharmacokinetic effects may occur, but unfortunately no pharmacokinetic studies have been performed (Barry et al., 1999).

Only one study with lamivudine and zalcitabine, which evaluated the efficacy, has been conducted and could show that triple therapy of lamivudine, zalcitabine, and zidovudine was slightly more effective than lamivudine and zidovudine, and both regimens exceeded the combined therapy of zalcitabine and zidovudine (Ruiz et al., 1996). However, a potential contribution of other transporters [e.g., the organic anion transporter (OAT) 1] should also be considered as Jin and Han (2006) described uptake of zalcitabine in Chinese hamster ovary cells transfected with OAT1 and Wada et al. (2000) detected uptake of zalcitabine and lamivudine in Xenopus laevis oocytes injected with rat OAT1.

Interactions of the ARV drugs with the native substrates of the transporters may also be of clinical relevance. Of particular interest could be the inhibition of cyclo(his-pro), the native substrate of OCT2, because lowering the uptake into the dopaminergic neurons entails the risk of neurotoxic side effects of the ARV drugs (Taubert et al., 2007).

Our RNA expression studies revealed a broad distribution of OCT1 and OCT2 in different organs. A reliable detection of the protein expression was not possible because the commercially available antibodies we tested showed no sufficient specificity against OCT1 or OCT2. However, we have previously shown a strong correlation between the RNA expression of OCT1 and OCT2 and the intracellular accumulation of the native substrate cyclo(his-pro) (Taubert et al., 2007), indicating that the mRNA expression level correlates with transporter function and hence with the protein expression level.

Drug penetration in different organs is especially important for the effectivity of the ARV drugs because HIV hides and replicates in different tissues. For example, in testis, where we could detect a high expression of OCT2 and a basal expression of OCT1, HIV supports productive infection, and this represents a potential source of virus in semen (Zhang et al., 1998; Roulet et al., 2006). Furthermore, the adverse effects of the ARV therapy affect different organs. Therefore, increased drug transport via OCT1 and OCT2 could contribute to an aggravation of the toxicity especially in colon, heart, and muscle, leading to a disruption of the intestinal barrier, cardiomyopathy, or degeneration and myonecrosis of the skeletal muscles (Bode et al., 2005; Hofman and Nelson, 2006). Of particular relevance is our finding of the increased expression (up to 1000-fold) of OCT1 and OCT2 in the mononuclear cells of the LNs of HIV-infected people with Centers for Disease Control and Prevention stage C compared with HIV-negative controls.

This difference should not be because of different cell types as the LNs of the HIV-infected and the healthy persons were dissected from similar locations; the mononuclear cells were prepared according to the same procedure; and microscopic control showed no differences in morphology and cell count. However, the possibility that different cell types account for the observed differences in transporter expression cannot be completely excluded. Evidence exists that HIV infection leads to a hyperplasia of the lymphoid tissue and a perturbation of cytokine expression, redistribution, and sequestration of lymphocyte subpopulations in the LNs (Orenstein et al., 1999; Biancotto et al., 2007). Nevertheless, the higher mRNA levels of the OCTs in the LNs of HIV-infected persons indicate a strongly increased potential for the accumulation of OCT substrates in the LNs.

The activation of the immune system with the complex alteration of cytokine expression induced by the HIV infection could lead to the up-regulation of the expression of OCT1 and OCT2 (Grossman et al., 2006). Minuesa et al. (2008) could already show that an in vitro T-cell stimulation of peripheral blood mononuclear cells (PBMCs) could lead to an up-regulation of OCT1 but not of OCT2. The divergent results of the OCT2 expression could be because of the differences in the experimental settings as Minuesa et al. (2008) used PBMCs instead of lymphoid tissue mononuclear cells, stimulated the cells with unspecific agents for T cell activation compared with the immune activation by HIV, and chose an in vitro setting in contrast to our in vivo studies with HIV-infected patients. The increased expression cannot be a consequence of therapy because all the patients included were therapy-naive.

It appears that the HIV-induced up-regulation is confined to specific transporters because genetically related or unrelated carriers were not modulated. This also indicates special involvement of OCT1 and OCT2 in the pathophysiology of HIV.

The up-regulation of OCT1 and OCT2 in progressive HIV infection can lead to a better uptake of the substrate lamivudine into the cells. This could provide an explanation of the results of Anderson et al. (2007), who described increased levels of lamivudine in PBMCs with decreased CD4 cells in advanced HIV infection.

Our data provide the experimental basis to further elucidate HIV drug interactions in vivo. Additional studies are needed to determine the expression of OCT1 and OCT2 in further targets of HIV as T-helper cells or monocytes and to explore the regulatory mechanisms of OCT1 and OCT2 up-regulation in progressive HIV infection and the potential pathophysiological role of their endogenous substrates.