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Pentoxifylline and its major oxidative metabolites exhibit different pharmacological properties
European Journal of Pharmacology 535 (2006) 301 – 309 www.elsevier.com/locate/ejphar
Pentoxifylline and its major oxidative metabolites exhibit different pharmacological properties Marianna Fantin a,b , Luigi Quintieri b , Erzsébet Kúsz a , Emese Kis a , Hristos Glavinas c , Maura Floreani b , Roberto Padrini b , Ernő Duda a , Csaba Vizler a,⁎ a
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesvari krt 62, 6701 Szeged, Hungary b Department of Pharmacology and Anesthesiology, Pharmacology Section, University of Padova, Padova, Italy c SOLVO Biotechnology, Szeged, Hungary Received 26 November 2005; received in revised form 25 January 2006; accepted 10 February 2006 Available online 20 March 2006
Abstract Previous investigations indicate that some of the metabolites of the hemorheological agent pentoxifylline (PTX), namely 1-(5-hydroxyhexyl)3,7-dimethylxanthine (M1), 1-(4-carboxybutyl)-3,7-dimethylxanthine (M4) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (M5), concur to some of the biological effects of the drug. However, information on the bioactivity of the major circulating oxidative metabolites of PTX (M4 and M5) is scanty. Here, we compared the effects of M4 and M5 with that of PTX and its major reductive metabolite, M1, on TNF-α production and cytotoxicity, endothelial cell proliferation and on the ATPase activity related to some ATP-binding cassette (ABC) transporters. Unlike PTX and M1, M4 and M5 poorly inhibited lipopolysaccaride-stimulated tumor necrosis factor-α (TNF-α) release by RAW 264.7 murine macrophages, and did not affect at all cell proliferation and upregulation of TNF-α-induced vascular cell adhesion molecule-1 (VCAM-1) in H5V endothelioma cells. By contrast, M4 and M5 were more effective than PTX and M1 in protecting WC/1 murine fibrosarcoma cells from TNF-α cytotoxicity. Moreover, results from ATP hydrolase assays indicated that neither PTX nor its tested metabolites interacted significantly with the human multidrug resistance transporters p-glycoprotein/multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1), and breast cancer resistance protein (BCRP). Based on these results and literature data, M5, retaining some of the PTX effects but lacking in significant inhibition of TNF-α production, may be a promising candidate drug for certain pathologic conditions. © 2006 Elsevier B.V. All rights reserved. Keywords: Pentoxifylline; Carboxylated metabolite; TNF-α; Macrophage; Endothelium; ABC transporter
1. Introduction Pentoxifylline [1-(5-oxohexyl)-3,7-dimethylxanthine, PTX; Fig. 1] is a tri-substituted xanthine derivative known as a nonspecific inhibitor of cyclic AMP (cAMP) phosphodiesterases and broadly used clinically in the treatment of various peripheral vascular and cerebrovascular disorders characterized by an inadequate tissue perfusion (Frampton and Brodgen, 1995; Moher et al., 2000; Jull et al., 2002). The beneficial effects of PTX on microcirculation are thought to be based mainly on its ability to improve microvascular blood flow via an increase of erythrocyte and leukocyte deformability, and a
⁎ Corresponding author. Tel.: +36 62 599 649; fax: +36 62 433 506. E-mail address: [email protected] (C. Vizler). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.02.017
reduction of platelet and erythrocyte aggregability (for a review, see Frampton and Brodgen, 1995). More recently, attention has focused on the therapeutic potential of PTX as an immunomodulatory, anti-inflammatory and antitumor agent. Numerous studies have shown that PTX suppresses the production of tumor necrosis factor α (TNF-α) by murine and human macrophages and leukocytes, both at the mRNA and the protein level (Han et al., 1991; Weinberg et al., 1992). Further studies have shown that PTX interferes with the synthesis of other pro-inflammatory cytokines such as interleukin-1, interleukin-6 and interleukin-8, as well as with neutrophil superoxide anion production and degranulation, and neutrophil and lymphocyte adhesion and transendothelial migration (Currie et al., 1990; Crouch and Fletcher, 1992; D'Hellencourt et al., 1996; Dominguez-Jimenez et al., 2002). These findings may explain why the drug exhibits significant therapeutic
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above-mentioned reports represent the only available evidence of biological activity of M4 and M5. This study was therefore undertaken with the major aim of further exploring the bioactivity of M4 and M5. Namely, we compared the effects of the two carboxylated metabolites of PTX with that of the parent drug and M1 on lipopolysaccharide (LPS)-induced TNFα production in macrophages, TNF-α cytotoxic activity toward tumor cells, and endothelial cell viability and vascular cell adhesion molecule-1 (VCAM-1) expression in response to TNF-α. Moreover, we explored the possible interaction of PTX and its major circulating metabolites with some members of the ATP-binding cassette (ABC) transporter superfamily. Fig. 1. Chemical structures of pentoxifylline and its major metabolites.
2. Materials and methods 2.1. Materials and stock solutions
effects in different immune- and inflammatory-mediated diseases in both animal models and humans (Schwarz et al., 1993; Nataf et al., 1993; Zabel et al., 1993). Reports also suggest that PTX administration might increase the effectiveness of antitumor radio- and chemotherapy. In particular, by increasing tumor blood flow and oxygenation and abrogating the G2 checkpoint, it sensitizes tumors to both irradiation and alkylating agents (Song et al., 1992, Li et al., 1998; Collingridge and Rockwell, 2000). Moreover, PTX inhibits endothelial cell proliferation in vitro and tumor-driven angiogenesis in vivo (Gude et al., 2001), and reverses the classical multidrug resistance (MDR) phenotype in murine tumor cells through a downregulation of the mdr1 gene expression (Drobná et al., 2002). PTX undergoes extensive metabolism in humans, resulting in formation of at least seven metabolites (denoted metabolite M1-7), the major circulating ones being M1 [1-(5-hydroxyhexyl)-3,7-dimethylxanthine], M4 [1-(4-carboxybutyl)-3,7dimethylxanthine] and M5 [1-(3-carboxypropyl)-3,7-dimethylxanthine] (Fig. 1; Hinze et al., 1972). Notably, upon oral or intravenous administration of PTX to healthy volunteers, the plasma levels of both M1 and M5 greatly exceeded those of the parent drug, PTX; in the same subjects, circulating levels of M4 were comparable to those of PTX (Beerman et al., 1985; Nicklasson et al., 2002). The hemorheological and anticytokine properties of PTX are known to be retained by racemic (R,S)M1 and (R)-M1 (lisofylline), respectively (Ambrus et al., 1995; Rice et al., 1994; Van Furth et al., 1997); lisofylline is being under developed as a drug in its own right (Yang et al., 2005 and references therein). By contrast, information on the pharmacological properties of the major oxidative metabolites of PTX, M4 and M5, are still scanty. A study by Crouch and Fletcher (1992) demonstrated that M4 and M5 were more potent than PTX in inhibiting neutrophil superoxide anion production, degranulation (lactoferrin release) and surface expression of the β-2 integrin CD11b/CD18 (mac-1). In another study, Ambrus et al. (1995), exploring the hemorheological effects of various PTX metabolites, found that M1 and M5 were similar to PTX in their activity on erythrocyte deformability; notably, M5 was more potent than pentoxifylline in inhibiting epinephrineinduced platelet aggregation. To the best of our knowledge, the
Racemic (R,S)-M1, M4 and M5 were supplied by Aventis Pharma Deutschland GmbH (Frankfurt am Main, Germany). Unless indicated otherwise, all other reagents, including PTX, were obtained from Sigma-Aldrich Co. (Budapest, Hungary). Stock solutions of PTX and its metabolites (10 mg/ml) were prepared in Dulbecco's Modified Eagle's Medium (DMEM), filtered through Millex-GV 0.2 μm filters (Millipore, Carrigtwohill, Co Cork, Ireland) and kept at − 20 °C. Murine recombinant TNF-α (mrTNF-α) was produced and purified in our laboratory as previously described (Gyorfy et al., 1997). Membranes isolated from cells expressing individual human ATP-binding cassette (ABC) transporters were provided by SOLVO Biotechnology (Szeged, Hungary). 2.2. Cell lines and culture conditions The murine macrophage cell line RAW 264.7, the murine endothelioma cell line H5V (Vizler et al., 1998) and the highly TNF-α-sensitive WEHI-164 murine fibrosarcoma cell subline WC/1, which was established in our laboratory (Gyorfy et al., 1997), were grown in complete medium (CM), which consisted of DMEM/Ham's F12 (50:50 ratio) supplemented with 10% fetal calf serum (FCS) and 2mM glutamine. All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. 2.3. TNF-α production in cultured macrophage cells To assess the effects of PTX and its major metabolites on TNF-α production by LPS-stimulated macrophages, RAW 264.7 cells were seeded in 24-well culture plates (Orange Scientific, Braine-l'Alleud, Belgium) at a density of 3 × 105 cells/well in 1 ml of CM and left to adhere to the plastic plates for 1 h (37 °C, 5% CO2). The cells were then left untreated (control) or preincubated with PTX, M1, M4 or M5 (final concentration, 1 mg/ml) for 1h before adding LPS (final concentration, 10 or 100ng/ml) and culturing for 4 h (37 °C, 5% CO2). Thereafter, aliquots of the supernatants (500 μl) were harvested, centrifuged and stored at 4 °C until analyzed for TNF-α content.
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2.3.1. TNF-α bioassay TNF-α content of the supernatants was determined in a 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-based bioassay using WC/1 cells as TNF-α-sensitive target. Appropriate dilutions of the cell supernatants were added in quadruplicate to 96-well plates (Orange Scientific) containing 3 × 104 WC/1 cells/well in 100μl of DMEM supplemented with 5% FCS, 2 mM glutamine and 1μg/ml actinomycin D, a sensitizer of tumor cells to the cytotoxicity of TNF-α (Holtmann et al., 1988). After a 24-h incubation period, MTT was added to each well (final concentration, 1 mg/ml) and plates were incubated at 37°C for 2h to allow reduction of MTT by viable cell dehydrogenases to a formazan product. Thereafter, the supernatant containing the unreacted dye was replaced with acidified isopropanol (0.05 M HCl–isopropanol, 100μl/well), plates were vigorously shaken, and absorbance at 570 nm was measured using a microtiter plate reader. The percentage of cell survival was calculated from the absorbance (A) values as follow: Atested / Auntreated control × 100. TNF-α levels were estimated by evaluation of the supernatant dilution resulting in 50% reduction of cell survival and comparison with a concentration– cell survival curve for mrTNF-α. 2.3.2. TNF-α immunoassay In some experiments, TNF-α levels were also measured by an enzyme-linked immunosorbent assay (ELISA) developed in our laboratory using commercially available reagents. In detail, 96-well plates (Greiner Microlon, Greiner Biosciences, Frickenhausen, Germany) were coated with capture antibody (goat anti-mouse TNF-α, R&D Systems Inc., Minneapolis, MN, USA) in coating buffer (carbonate–bicarbonate buffer, pH 9.6) by overnight incubation at 4 °C. The plates were blocked with DMEM containing 10% FCS for 1 h at room temperature. After three washing with 0.1% Tween 20 in PBS (wash buffer), the samples and calibration standards (mrTNF-α) were added to the plates, and incubated 1h at 4°C. The plates were then washed three times with wash buffer and added with biotinylated detection antibody (anti-mouse TNF-α, R&D Systems Inc.). After 1h incubation at 4 °C, the plates were washed three times with wash buffer and then streptavidin conjugated to horseradish peroxidase (ExtrAvidin-peroxidase) was added at a dilution of 1:1000. After a 45-min incubation period at room temperature, the plates were washed three times with wash buffer and the substrate solution was added (0.4 mg/ml orthophenylenediamine, 0.01% hydrogen peroxide (30%) in 0.1M citrate/phosphate buffer, pH 5.2). The plates were incubated in the dark for 15 min before spectrophotometric reading at 450 nm in a microplate reader. The concentrations of TNF-α were calculated on the basis of standard curves constructed with known amounts of mrTNF-α (from 5 to 10,000 U/ml, final concentration) and were expressed as U/ml. The detection limit was 100 U/ml of mrTNF-α. 2.4. TNF-α cytotoxicity assay The impact of PTX and its major metabolites on TNF-α cytotoxic activity was studied using WC/1 murine fibrosarcoma
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cells as tumor cell target. WC/1 cells were seeded in 96-well culture plates (Orange Scientific) at a density of 3 × 104 cells/ well in DMEM supplemented with 5% FCS, 2 mM glutamine and 1 μg/ml actinomycin D, and incubated for 24h (37 °C, 5% CO2). Thereafter, the cells were exposed to rmTNF-α (final concentration, 0, 4, 8 and 16 U/ml) and 1μg/ml actinomycin D in the absence or presence of PTX, M1, M4 or M5 (final concentration, 1 mg/ml). The plates were further incubated for 24h (37 °C, 5% CO2) and then cell viability was determined by an MTT assay performed as described above. 2.5. Evaluation of cell viability and VCAM-1 expression in endothelioma cells To compare the effects of PTX, M1, M4 and M5 on endothelial cell viability, H5V cells were seeded in 24-well culture plates (Orange Scientific) at a density of 2 × 104 cells/ well in 1 ml of CM and incubated for 24h (37 °C, 5% CO2). The cells were then left untreated (control) or treated with PTX, M1, M4 or M5 (final concentrations: 0.5, 1 and 2 mg/ml) for 24 h (37 °C, 5% CO2) before evaluation of cell survival by an MTT assay performed as described above. To investigate the effects of PTX and its major metabolites on TNF-α-induced VCAM-1 expression, H5V cells were seeded in 6-well culture plates (Orange Scientific) at a density of 2 × 105/well in 3 ml of CM and incubated for 24 h (37 °C, 5% CO2). The cells were then left untreated or exposed to mrTNF-α (100 U/ml) in the absence or presence of PTX, M1, M4 or M5 (final concentration, 1mg/ml) for 24h (37 °C, 5% CO2). After trypsinization, the cells were collected, centrifuged for 5 min at 150×g, resuspended in 200 μl phosphate-buffered saline (PBS) and incubated with 1 μl rat anti-mouse VCAM-1 monoclonal antibody (R&D Systems, Minneapolis, MN) for 30min at 4 °C. The cells were washed three times, resuspended in 100μl PBS and incubated with 1 μl of fluorescein isothiocyanate (FITC)-conjugated goat anti-rat Ig for 30 min at 4 °C. Finally, upon washing, the cells were analyzed with a FACScalibur flow cytometer (BD Bioscences, Mountain View, CA, USA). 2.6. ABC transporters ATP hydrolase (ATPase) activity assay The interaction of PTX and its major metabolites with the human ABC membrane transporters p-glycoprotein/multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) was evaluated by measuring their ability to stimulate the ATPase activity of membrane vesicles isolated from baculovirusinfected Spodoptera frugiperda (Sf9) insect cells expressing human MDR1 or human MRP1, or from a mammalian cell line engineered to express human BCRP, respectively; all these three transporters exhibit a substrate-stimulated ATPase activity, which is closely related to their substrate transport function (Bakos et al., 2000 and references therein; Janvilisri et al., 2003). Membrane vesicles were prepared as described previously (Sarkadi et al., 1992) and the assays were performed as described in detail at http://www.solvo.huwww.solvo.hu. Briefly, membrane vesicles (20 μg/well) were incubated for
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control (Fig. 3 and Table 1), the ANOVA was followed by Dunnett's test. 3. Results 3.1. M4 and M5 are less effective than PTX and M1 in inhibiting LPS-stimulated TNF-α release from RAW 264.7 macrophages
Fig. 2. Effect of PTX and its major metabolites on LPS-induced TNF-α production in RAW 264.7 murine macrophages. RAW 264.7 cells were preincubated with PTX, M1, M4 or M5 (final concentration, 1mg/ml) for 1h, and then stimulated with 10 or 100ng/ml LPS for 4 h; the supernatants were collected and their TNF-α content was determined by an MTT-based bioassay as described in Section 2. Values are the mean ± S.E.M. of four independent experiments. ⁎P < 0.05 vs. control; ⁎⁎⁎P < 0.001 vs. control; aP < 0.05 vs. PTX; b P < 0.05 vs. M1.
40 min (MDR1 and BCRP) or 60 min (MRP1) at 37 °C in a medium containing 10 mM MgCl2, 40 mM MOPS–Tris (pH 7.0), 50 mM KCl, 5 mM dithiothreitol, 0.1 mM EGTA, 4 mM sodium azide, 1mM ouabain, 5 mM ATP, 2mM glutathione and various concentrations of the tested compounds in the absence or presence of 1.2 mM sodium orthovanadate. ATPase activity was determined as the difference of inorganic phosphate liberation measured in the presence or absence of sodium orthovanadate (vanadate-sensitive ATPase activity). Results were plotted as % activity, where 0% is the activity measured in the presence of solvent and 100% is the activity measured in the presence of a strong activator of the transporter, namely verapamil (final concentration, 40 μM), N-ethylmaleimide glutathione (final concentration, 10 mM) and sulfasalazine (final concentration, 10 μM) for MDR1, MRP1 and BCRP, respectively.
PTX and lysofilline, the R isomer of M1, have been shown to markedly inhibit LPS-stimulated production of TNF-α both in vitro and in vivo (Rice et al., 1994; Van Furth et al., 1997). Therefore, a first set of experiments explored the ability of M4 and M5, in comparison with that of PTX and M1, to inhibit TNF-α release from LPS-stimulated RAW 264.7 murine macrophage cells. This macrophage cell line was chosen in the present study because of its well-documented sensitivity to LPS and subsequent regulated production of large amounts of TNF-α (Lin et al., 2004). In preliminary trials, all tested compounds concentration-dependently decreased TNF-α release from macrophages in response to stimulation with 10 ng/ml LPS, reaching a plateau at 1mg/ml (not shown); thus, this concentration was selected for use in the final series of experiments. As shown in Fig. 2, M4 and M5 significantly and comparably decreased TNF-α levels in the supernatants from cells stimulated with 10ng/ml LPS (22% decrease; P < 0.05 vs. control); however, PTX in itself and M1 were remarkably more effective than the carboxylated metabolites in decreasing TNF-α production (P < 0.001 vs. both M4 and M5). Moreover, PTX and M1, but not M4 and M5, significantly decreased TNF-α production in response to cell stimulation with a 10-fold higher concentration of LPS (100 ng/ml; Fig. 2). The poor inhibitory effect of M4 and M5 on TNF-α production by murine macrophages was confirmed by further experiments based on immunoassay detection of TNF-α. As shown in Table 1, both M4 and M5 significantly inhibited TNF-α release in response to 10 ng/ml LPS (P < 0.01 vs. LPS alone), whereas they did not decrease TNF-α production when tested in macrophages treated with 100ng/ml LPS.
Table 1 Immunoassay of TNF-α released by LPS-activated RAW 264.7 cells in the absence and in the presence of M4 and M5 TNF-α levels (U/ml) +LPS 10 ng/ml
+LPS 100 ng/ml
1110 ± 15 477 ± 7 ⁎⁎ (− 56.6 ± 1.6%) 529 ± 26 ⁎⁎ (− 52.3 ± 4.9%)
1268 ± 64 1063 ± 73 a (− 13.4 ± 4.4%) 1035 ± 27.5 a (− 18.4 ± 2.6%)
2.7. Analysis of the data
– M4 M5
Results are given as means ± S.E.M. of n experiments. The Student's t-test for unpaired data was used for comparison of the mean values between two groups (Fig. 3). The one-way analysis of variance (ANOVA) followed by Tukey's test was used for multiple comparison among treatment groups (Figs. 2, 4 and 6). When treatment groups were compared to the same
TNF-α levels were measured by an enzyme-linked immunosorbent assay (ELISA) as described in Section 2. Data represent means ± S.E.M. of three experiments carried out in quadruplicate. Percent inhibition with respect to control (LPS alone) is reported in parenthesis. a Not statistically different (P > 0.05) from values obtained in LPS-only treated cells. ⁎⁎ P < 0.01 vs. values obtained in LPS-only treated cells.
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Fig. 3. Effect of PTX and its major metabolites on TNF-α-induced cytotoxicity in WC/1murine fibrosarcoma cells. WC/1 cells were treated for 24 h with the indicated concentrations of TNF-α in the presence or absence of each xanthine derivative (final concentration, 1 mg/ml) and cell viability was then determined by an MTT assay as described in Section 2. Values are the mean ± S.E.M. of three independent experiments carried out in quadruplicate. ⁎⁎P < 0.01 vs. control; ⁎⁎⁎P < 0.001 vs. control.
3.2. M4 and M5, but not PTX and M1, significantly attenuate TNF-α cytotoxic activity toward WC/1 fibrosarcoma cells TNF-α exhibits cytotoxicity against a wide range of murine and human tumor cell lines, and PTX has been found to attenuate TNF-α-induced cell death in cultured L292 murine fibrosarcoma cells via induction of heme oxigenase-1 (Oh et al., 2003). Therefore, we compared the ability of PTX and its major metabolites to protect tumor cells from TNF-α-induced cytotoxicity. WC/1 murine fibrosarcoma cells were exposed to increasing concentrations of mrTNF-α (0, 4, 8 and 16U/ml) in the presence of actinomycin D and in the presence or absence of increasing concentrations (from 0.1 to 2 mg/ml) of PTX, M1, M4 or M5 for 24h, and cell survival was then evaluated. For the active compounds, the activity reached a plateau at 1 mg/ml (data not shown). As shown in Fig. 3, M4 and M5 (1 mg/ml) significantly attenuated TNF-α cytotoxicity toward WC/1 tumor cells. By contrast, at all tested concentrations of TNFα, the antagonistic effects of PTX and M1 were weak (Fig. 3).
TNF-α-induced VCAM-1 expression in H5V murine endothelioma cells. As shown in Fig. 4, a 24-h exposure to PTX and M1 (final concentration 0.5, 1 and 2 mg/ml) concentration-dependently reduced H5V cell viability as judged by an MTT assay; M4 and M5 were completely ineffective in this respect (Fig. 4). As expected, cytofluorimetric analysis of H5V cells revealed a marked increase of fluorescence associated to VCAM-1 expression upon stimulation with 100 U/ml rmTNF-α (Figs. 5 and 6); both PTX and M1 (final concentration, 1 mg/ml) were capable of completely suppressing the VCAM-1 inducing effect
3.3. Effects of PTX and its major metabolites on cell survival and TNF-α-induced VCAM-1 expression in H5V endothelioma cells PTX was shown to be cytostatic and/or cytotoxic in vitro toward both normal and transformed endothelial cells (Gude et al., 2001). Moreover, a pharmacological elevation of cAMP levels was found to inhibit TNF-α-induced VCAM-1 expression in human umbilical vein endothelial cells (Pober et al., 1993; Otsuki et al., 2001). Thus, further experiments compared the effects of PTX and its major metabolites on cell viability and
Fig. 4. Survival curves of H5V murine endothelioma cells in response to PTX and its major metabolites. H5V endothelial cells were cultured for 24h in the presence of different concentrations of each xanthine derivative and then cell viability was determined by an MTT assay, as described in Section 2. Values are the mean ± S.E.M. of three experiments carried out in triplicate. ⁎P < 0.05; ⁎⁎P < 0.01; ⁎⁎⁎P < 0.001 vs. control (no drug) values; asignificantly different vs. M4 (P < 0.05 or less); csignificantly different vs. M4 (P < 0.05, or less); b significantly different vs. M5 (P < 0.05, or less); dsignificantly different vs. M5 (P < 0.05, or less).
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M. Fantin et al. / European Journal of Pharmacology 535 (2006) 301–309 Table 2 A comprehensive summary of the pharmacological effects of PTX and its major metabolites
Fig. 5. Effect of PTX and its major metabolites on TNF-α-induced VCAM-1 expression in H5Vmurine endothelioma cells. H5V cells were left untreated or exposed for 24 h to 100U/ml mrTNF-α in the presence or absence of PTX, M1, M4 or M5 (final concentration, 1mg/ml), and then VCAM-1 expression was analyzed by flow cytometry as described in Section 2. Light gray histogram: no staining; dark gray histogram: unstimulated cells; thin line: TNF-α-stimulated cells; thick line: cells stimulated in the presence of TNF-α and PTX or its metabolites. The experiment was repeated three times with similar results.
of TNF-α, since the cells treated with the cytokine plus either PTX or M1 were undistinguishable from TNF-α-untreated cells. By contrast, neither M4 nor M5 (both at 1 mg/ml) did interfere with TNF-α-induced upregulation of VCAM-1, since the cells treated with the cytokine plus either M4 or M5 were undistinguishable from those treated with TNF-α only (Figs. 5 and 6).
Effect
PTX
M1
M4
M5
Macrophages, inhibition of LPS-induced TNF release Endothelium, growth inhibition Endothelium, inhibition of TNF-α-induced VCAM-1 expression WC/1 fibrosarcoma cells, protection from TNF-α cytotoxicity Interaction with MDR1 Interaction with BCRP Interaction with MRP1 Neutrophils, inhibition superoxide production in response to warming or formyl-methionyl-leucyl-phenylalanine (FMLP) (Crouch and Fletcher, 1992) Neutrophils, inhibition of lactoferrin release in response to warming or FMLP (Crouch and Fletcher, 1992) Neutrophils, inhibition of CD11b/CD18 expression in response to warming or FMLP (Crouch and Fletcher, 1992) Improvement of red blood cell filterability (Ambrus et al., 1995) Inhibition of ADP-induced platelet aggregation (Ambrus et al., 1995) Inhibition of epinephrine-induced platelet aggregation (Ambrus et al., 1995)
++
++
+/−
+/−
+ +
+ +
− −
− −
+/−
+/−
+
+
− + − −
− − − +
− − − +
− − − +
+/−
+/−
+
+
+
+
++
+++
+
+
−
+
+
++
−
+/−
+
+
−
++
sulfasalazine at the highest concentration tested (50 μg/ml). By contrast, PTX metabolites were devoid of any effect on the ATPase activity related to BCRP. 4. Discussion Although M4 and M5 are major circulating species of PTX in humans, so far only few and scattered literature data are available on their pharmacological properties. In this study, aimed at investigating some possible biological effects of M4
3.4. Interaction of PTX and its metabolites with human ABC membrane transporters PTX has been found to reverse the p-glycoprotein-mediated MDR phenotype and to downregulate mdr1 gene expression in cultured murine tumor cells (Drobná et al., 2002; Viladkar and Chitnis, 1994). The possible interaction of PTX and its major circulating metabolites with MDR1, MRP1 and BCRP, all known to be ATP-dependent pumps capable of conferring an MDR phenotype (Gottesman et al., 2002), has been investigated measuring drug-stimulated ATPase activity in membranes from cells engineered to express individual recombinant transporters. In our hands, none of the tested compounds (final concentration, up to 50 μg/ml) increased the ATPase activity of membrane vesicles expressing MDR1 or MRP1 (data not shown), suggesting that neither PTX nor its major metabolites interact with these ATP-dependent transporters. As shown in Fig. 7, the ATPase activity of membranes expressing BCRP was concentration-dependently stimulated by PTX, reaching approximately 50% of the ATPase activity observed in the presence of 10 μM
Fig. 6. Effect of PTX and its major metabolites on TNF-α-induced VCAM-1 expression in H5Vmurine endothelioma cells. H5V cells were left untreated or exposed for 24h to 100 U/ml mrTNF-α in the presence or absence of PTX, M1, M4 or M5 (final concentration, 1 mg/ml), and then VCAM-1 was stained by anti-VCAM-1 antibody and FITC labeled secondary antibody. Increase of mean fluorescence level (MFL) was calculated by dividing the MFL of treated cells with MFL of non-stimulated, identically stained controls. The results are mean ± S.E.M. of three independent experiments. ⁎P < 0.05 vs. TNF-treated control; ⁎⁎not significant vs. TNF-treated control.
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Fig. 7. Concentration–effect curves for pentoxifylline and its metabolites on MDR1 and BCRP ATPase activity assessed in isolated cell membrane vesicles. Vanadate sensitive ATPase activity was determined using the technological platform of SOLVO Biotechnology, as described in Section 2. The results are presented as % activity where 0% is the vanadate-sensitive ATPase activity measured in the presence of solvent alone, while 100% is the vanadate-sensitive ATPase activity measured in presence of 40μM verapamil (MDR1) or 10μM sulfasalazine (BCRP). The results are means ± S.E.M. of three experiments carried out in triplicate.
and M5, in comparison with those of the parent drug and its major reductive metabolite, M1, we present evidence that the pharmacological properties of both carboxylated metabolites differ quantitatively and/or qualitatively from those of PTX and M1. Our present data and those of others comparing the bioactivity of PTX with that of its major metabolites are summarized in Table 2. One of the best characterized effects of PTX is its ability to decrease TNF-α production both in vitro and in vivo (Zabel et al., 1993). The anti-TNF-α activity of PTX is known to be retained by lisofylline, the R isomer of M1 (Rice et al., 1994; Van Furth et al., 1997), which has been recently proposed as a PTX-like anti-inflammatory drug candidate on its own right. On the other hand, to the best of our knowledge, there is no data in
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the literature on the modulation of TNF-α release by M4 or M5. Here, we found that both carboxylated metabolites of PTX were remarkably less potent than the parent drug and M1 in inhibiting TNF-α production by LPS-stimulated murine macrophages (Fig. 2). This finding suggests that M4 and M5 are unlikely to contribute to the suppression of TNF-α circulating levels observed in response to PTX administration (Zabel et al., 1993). Furthermore, because suppression of TNF-α production by xanthine derivatives is thought to be mediated by inhibition of cAMP phosphodiesterase activity (Semmler et al., 1993), our data suggest that both carboxylated metabolites may be weaker inhibitors of cAMP phosphodiesterase than the parent drug and M1; experiments on partially purified preparations of cAMP phosphodiesterase aimed at confirming this hypothesis are in progress. Furthermore, information on the structure-activity relationships for PTX and its major metabolites will be obtained from docking studies on theoretical models of cAMP phosphodiesterase. M5, being devoid of major effects on TNF-α production but retaining the beneficial hemorheological activity of the parent drug (Ambrus et al., 1995), might be a safer drug than PTX for the treatment of microcirculatory disorders. Indeed, on a theoretical basis, an indiscriminate blockade of TNF-α production and/or effects might be associated with an increased susceptibility to some types of infections, as it has been observed experimentally in PTX-treated mice bearing chronic pulmonary tuberculosis (Turner et al., 2001). Moreover, since both carboxylated metabolites of PTX are more effective than the parent drug in inhibiting several functions of human neutrophils, including degranulation and superoxide anion production (Crouch and Fletcher, 1992), they might be even more effective than PTX in those conditions in which neutrophil-mediated tissue damage plays a prominent role, such as myocardial infarction, varicose ulcers and adult respiratory distress syndrome (Fantone and Ward, 1985). PTX was recently shown to decrease the susceptibility of L929 murine fibrosarcoma cells to the cytotoxic action of TNFα (Oh et al., 2003). As shown in Fig. 3, under our experimental conditions, the protective effect of PTX and M1 on TNF-αinduced toxicity in WC/1 murine fibrosarcoma cells was relatively weak (Fig. 3). Interestingly, both the carboxylated metabolites of PTX were significantly more effective than the parent drug and M1 in attenuating WC/1 tumor cell sensitivity to TNF-α (Fig. 3). Because the cytoprotection against TNF-α afforded by PTX was related to induction of the activity of heme oxygenase-1 (Oh et al., 2003), a protein capable of preventing cells from undergoing apoptosis triggered by various agents inducing oxidative damage (Fang et al., 2004 and references therein), M4 and M5 might be therapeutic tools in diseases associated with oxidative stress. Evidence that induction of heme oxigenase-1 may have therapeutic implications in these types of diseases has been provided by studies demonstrating a cytoprotective potential of the protein in models of lung injury (Otterbein et al., 1999) and ischemia/reperfusion injury (Katori et al., 2002). Further experiments demonstrated that PTX and M1, but not M4 and M5, reduced cell proliferation and/or viability and
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blocked TNF-α-induced VCAM-1 expression in endotheliumderived murine cells. In agreement, other studies have shown that a pharmacological elevation of cAMP levels inhibits both endothelial cell proliferation and VCAM-1 expression in response to TNF-α (Kim et al., 2001; Pober et al., 1993; Otsuki et al., 2001). These findings further support a possible lack of inhibition of cAMP phosphodiesterase activity by the carboxylated metabolites of PTX and possibly preclude their usefulness as inhibitors of tumor angiogenesis, as it was proposed for PTX by Gude et al. (2001). Though PTX was shown to reverse the classical MDR phenotype in cultured murine tumor cells (Drobná et al., 2002; Viladkar and Chitnis, 1994), neither PTX nor its major metabolites did stimulate the ATPase activity of human MDR1 or human MRP1 (not shown). In other words, none of the tested compounds seem to be a good substrate of these membrane transporters and, therefore, conceivably, could efficiently compete with a cytotoxic drug substrate for transport outside of the cell. However, these data do not exclude that PTX might be capable of reversing the MDR phenotype of human tumors since the drug was shown to suppress mdr1 gene expression in multidrug-resistant L1210/VCR murine leukemia cells (Drobná et al., 2002). In conclusion, based on our present and previous findings and, in spite of the lack of knowledge concerning the exact molecular targets, a therapeutic use of M4 and M5 in certain pathologic conditions is conceivable. In particular, M5, retaining the modulatory effects of PTX on erythrocyte deformability, platelet aggregation and neutrophil function, but lacking substantial inhibitory activity toward TNF-α production, deserves further preclinical evaluation for its therapeutic potential as an hemorheological and anti-inflammatory agent. Acknowledgements Pentoxifylline metabolites were kindly supplied by Dr. J. Pünter, Aventis Pharma Deutschland GmbH. The H5V cell line was kindly supplied by Dr. A. Vecchi, Laboratory of Immunopharmacology, Istituto Ricerche Farmacologiche Mario Negri (Milano, Italy). This work was supported by the Hungarian National Research and Development Program (NKFP 1A/057/04). References Ambrus, J.L., Stadler, S., Kulaylat, M., 1995. Hemorheologic effects of metabolites of pentoxifylline (Trental). J. Med. 26, 65–75. Bakos, E., Evers, R., Sinko, E., Varadi, A., Borst, P., Sarkadi, B., 2000. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol. Pharmacol. 57, 760–768. Beerman, B., Ings, R., Mansby, J., Chamberlain, J., McDonald, A., 1985. Kinetics of intravenous and oral pentoxifylline in healthy subjects. Clin. Pharmacol. Ther. 37, 25–28. Collingridge, D.R., Rockwell, S., 2000. Pentoxifylline improves the oxygenation and radiation response of BA1112 rat rabdomyosarcomas and EMT6 mouse mammary carcinomas. Int. J. Cancer 90, 256–264. Crouch, S.P., Fletcher, J., 1992. Effect of ingested pentoxifylline on neutrophil superoxide anion production. Infect. Immun. 60, 4504–4509.
Currie, M.S., Rao, K.M., Padmanabhan, J., Jones, A., Crawford, J., Cohen, H.J., 1990. Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation, and superoxide production. J. Leukoc. Biol. 47, 244–250. D'Hellencourt, C.L., Diaw, L., Cornillet, P., Guenounou, M., 1996. Differential regulation of TNF alpha, IL-1 beta, IL-6, IL-8, TNF beta, and IL-10 by pentoxifylline. Int. J. Immunopharmacol. 18, 739–748. Dominguez-Jimenez, C., Sancho, D., Nieto, M., Montova, M.C., Barreiro, O., Sanchez-Madrid, F., Gonzales-Amaro, R., 2002. Effect of pentoxifylline on polarization and migration of human leukocytes. J. Leukoc. Biol. 71, 588–596. Drobná, Z., Stein, U., Walther, W., Barancik, M., Breier, A., 2002. Pentoxifylline influences drug transport activity of P-glycoprotein and decreases mdrl gene expression in multidrug resistant mouse leukemic L1210/VCR cells. Gen. Physiol. Biophys. 21, 103–109. Fang, J., Akaike, T., Maeda, H., 2004. Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment. Apoptosis 9, 27–35. Fantone, J.C., Ward, P.A., 1985. Polymorphonuclear leukocyte-mediated cell and tissue injury: oxygen metabolites and their relations to human disease. Human Pathol. 16, 973–978. Frampton, J.E., Brodgen, R.N., 1995. Pentoxifylline (oxpentifylline). A review of its therapeutic efficacy in the management of peripheral vascular and cerebrovascular disorders. Drugs Aging 7, 480–503. Gottesman, M.M., Fojo, T., Bates, S.E., 2002. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev., Cancer 2, 48–58. Gude, R.P., Binda, M.M., Boquete, A.L., Bonfil, R.D., 2001. Inhibition of endothelial cell proliferation and tumor-induced angiogenesis by pentoxifylline. J. Cancer Res. Clin. Oncol. 127, 625–630. Gyorfy, Z., Benko, S., Kusz, E., Maresca, B., Vigh, L., Duda, E., 1997. Highly increased TNF sensitivity of tumor cells expressing the yeast delta 9desaturase gene. Biochem. Biophys. Res. Commun. 18, 465–470. Han, J., Huez, G., Beutler, B., 1991. Interactive effects of the tumor necrosis factor promoter and 3′-untranslated regions. J. Immunol. 146, 1843–1848. Hinze, H.J., Bedessem, G., Soder, A., 1972. Structure of excretion products of 3,7-dimethyl-1-(5-oxo-hemyl)-xanthine (BL 191) in man. Arzneim.-Forsch. 22, 1144–1151. Holtmann, H., Hahn, T., Wallach, D., 1988. Interrelated effects of tumor necrosis factor and interleukin 1 on cell viability. Immunobiology 177, 7–22. Janvilisri, T., Venter, H., Shahi, S., Reuter, G., Balakrishnan, L., van Veen, H.W., 2003. Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis. J. Biol. Chem. 278, 20645–20651. Jull, A., Waters, J., Arroll, B., 2002. Pentoxifylline for treatment of venous leg ulcers: a systematic review. Lancet 359, 1550–1554. Katori, M., Anselmo, D.M., Busuttil, R.W., Kupiec-Weglinski, J.W., 2002. A novel strategy against ischemia and reperfusion injury: cytoprotection with heme oxygenase system. Transpl. Immunol. 9, 227–233. Kim, T.Y., Kim, W.I., Smith, R.E., Kay, E.D., 2001. Role of p27(Kip1) in cAMP- and TGF-beta2-mediated antiproliferation in rabbit corneal endothelial cells. Invest. Ophthalmol. Visual Sci. 42, 3142–3149. Li, Y.X., Weber-Johnson, K., Sun, L.Q., Paschoud, N., Mirimanoff, R.O., Coucke, P.A., 1998. Effect of pentoxifylline on radiation-induced G2-phase delay and radiosensitivity of human colon and cervical cancer cells. Radiat. Res. 149, 338–342. Lin, H.I., Chu, S.J., Wang, D., Feng, N.H., 2004. Pharmacological modulation of TNF production in macrophages. J. Microbiol. Immunol. Infect. 37, 8–15. Moher, D., Pham, B., Ausejo, M., Saez, A., Hood, S., Barber, G., 2000. Pharmacological management of intermittent claudication. Drugs 59, 1057–1070. Nataf, S., Louboutin, J.P., Chabannes, D., Feye, J.R., Muller, J.Y., 1993. Pentoxifylline inhibits experimental allergic encephalomyelitis. Acta Neurol. Scand. 88, 97–99. Nicklasson, M., Bjorkman, S., Roth, B., Jonsson, M., Hoglund, P., 2002. Stereoselective metabolism of pentoxifylline in vitro and in vivo in humans. Chirality 14, 643–652.
M. Fantin et al. / European Journal of Pharmacology 535 (2006) 301–309 Oh, G.S., Pae, H.O., Moon, M.K., Choi, B.M., Yun, Y.G., Rim, J.S., Chung, H. T., 2003. Pentoxifylline protects L929 fibroblasts from TNF-alpha toxicity via the induction of heme oxygenase-1. Biochem. Biophys. Res. Commun. 302, 109–113. Otsuki, M., Saito, H., Xu, X., Sumitani, S., Kouhara, H., Kurabayashi, M., Kasayama, S., 2001. Cilostazol represses vascular cell adhesion molecule-1 gene transcription via inhibiting NF-kappaB binding to its recognition sequence. Atherosclerosis 158, 121–128. Otterbein, L.E., Kolls, J.K., Mantell, L.L., Cook, J.L., Alam, J., Choi, A.M., 1999. Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Clin. Invest. 103, 1047–1054. Pober, J.S., Slowik, M.R., De Luca, L.G., Ritchie, A.J., 1993. Elevated cyclic AMP inhibits endothelial cell synthesis and expression of TNF-induced endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1, but not intercellular adhesion molecule-1. J. Immunol. 150, 5114–5123. Rice, G.C., Brown, P.A., Nelson, R.J., Bianco, J.A., Singer, J.W., Bursten, S., 1994. Protection from endotoxic shock in mice by pharmacologic inhibition of phosphatidic acid. Proc. Natl. Acad. Sci. U. S. A. 91, 3857–3861. Sarkadi, B., Price, E.M., Boucher, R.C., Germann, U.A., Scarborough, G.A., 1992. Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J. Biol. Chem. 267, 4854–4858. Schwarz, A., Krone, C., Trautinger, F., Aragane, Y., Neuner, P., Luger, T.A., Schwarz, T., 1993. Pentoxifylline suppresses irritant and contact hypersensitivity reactions. J. Invest. Dermatol. 101, 549–552. Semmler, J., Gebert, U., Eisenhut, T., Moeller, J., Schonharting, M.M., Allera, A., Endres, S., 1993. Xanthine derivatives: comparison between suppression of tumour necrosis factor-alpha production and inhibition of cAMP phosphodiesterase activity. Immunology 78, 520–525.
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Song, C.W., Hasegawa, T., Kwon, H.C., Lyons, J.C., Levitt, S.H., 1992. Increase in tumor oxygenation and radiosensitivity caused by pentoxifylline. Radiat. Res. 130, 205–210. Turner, J., Frank, A.A., Brooks, J.V., Marietta, P.M., Orme, I.M., 2001. Pentoxifylline treatment of mice with chronic pulmonary tuberculosis accelerates the development of destructive pathology. Immunology 102, 248–253. van Furth, A.M., Verhard-Seijmonsbergen, E.M., van Furth, R., Langermans, J. A., 1997. Effect of lisofylline and pentoxifylline on the bacterial-stimulated production of TNF-alpha, IL-1 beta IL-10 by human leucocytes. Immunology 91, 193–196. Viladkar, A., Chitnis, M., 1994. In vitro effects of pentoxifylline and doxorubicin on cell survival and DNA damage in sensitive and MDRP388 leukemia cells. Cancer Biother. 9, 143–151. Vizler, C., Rosato, A., Calderazzo, F., Quintieri, L., Fruscella, P., Wainstok de Calmanovici, R., Mantovani, A., Vecchi, A., Zanovello, P., Collavo, D., 1998. Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity. Br. J. Cancer 77, 656–662. Weinberg, J.B., Mason, S.N., Wortham, T.S., 1992. Inhibition of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) messenger RNA (mRNA) expression in HL-60 leukemia cells bypentoxifylline and dexamethasone: dissociation of acivicin-induced TNF-alpha andIL-1 beta mRNA expression from acivicin-induced monocytoid differentiation. Blood 179, 3337–3343. Yang, Z., Chen, M., Nadler, J.L., 2005. Lisofylline: a potential lead for the treatment of diabetes. Biochem. Pharmacol. 69, 1–5. Zabel, P., Schade, F.U., Schlaak, M., 1993. Inhibition of endogenous TNF formation by pentoxifylline. Immunobiology 187, 447–463.
Pentoxifylline and its major oxidative metabolites exhibit different pharmacological properties Marianna Fantin a,b , Luigi Quintieri b , Erzsébet Kúsz a , Emese Kis a , Hristos Glavinas c , Maura Floreani b , Roberto Padrini b , Ernő Duda a , Csaba Vizler a,⁎ a
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesvari krt 62, 6701 Szeged, Hungary b Department of Pharmacology and Anesthesiology, Pharmacology Section, University of Padova, Padova, Italy c SOLVO Biotechnology, Szeged, Hungary Received 26 November 2005; received in revised form 25 January 2006; accepted 10 February 2006 Available online 20 March 2006
Abstract Previous investigations indicate that some of the metabolites of the hemorheological agent pentoxifylline (PTX), namely 1-(5-hydroxyhexyl)3,7-dimethylxanthine (M1), 1-(4-carboxybutyl)-3,7-dimethylxanthine (M4) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (M5), concur to some of the biological effects of the drug. However, information on the bioactivity of the major circulating oxidative metabolites of PTX (M4 and M5) is scanty. Here, we compared the effects of M4 and M5 with that of PTX and its major reductive metabolite, M1, on TNF-α production and cytotoxicity, endothelial cell proliferation and on the ATPase activity related to some ATP-binding cassette (ABC) transporters. Unlike PTX and M1, M4 and M5 poorly inhibited lipopolysaccaride-stimulated tumor necrosis factor-α (TNF-α) release by RAW 264.7 murine macrophages, and did not affect at all cell proliferation and upregulation of TNF-α-induced vascular cell adhesion molecule-1 (VCAM-1) in H5V endothelioma cells. By contrast, M4 and M5 were more effective than PTX and M1 in protecting WC/1 murine fibrosarcoma cells from TNF-α cytotoxicity. Moreover, results from ATP hydrolase assays indicated that neither PTX nor its tested metabolites interacted significantly with the human multidrug resistance transporters p-glycoprotein/multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1), and breast cancer resistance protein (BCRP). Based on these results and literature data, M5, retaining some of the PTX effects but lacking in significant inhibition of TNF-α production, may be a promising candidate drug for certain pathologic conditions. © 2006 Elsevier B.V. All rights reserved. Keywords: Pentoxifylline; Carboxylated metabolite; TNF-α; Macrophage; Endothelium; ABC transporter
1. Introduction Pentoxifylline [1-(5-oxohexyl)-3,7-dimethylxanthine, PTX; Fig. 1] is a tri-substituted xanthine derivative known as a nonspecific inhibitor of cyclic AMP (cAMP) phosphodiesterases and broadly used clinically in the treatment of various peripheral vascular and cerebrovascular disorders characterized by an inadequate tissue perfusion (Frampton and Brodgen, 1995; Moher et al., 2000; Jull et al., 2002). The beneficial effects of PTX on microcirculation are thought to be based mainly on its ability to improve microvascular blood flow via an increase of erythrocyte and leukocyte deformability, and a
⁎ Corresponding author. Tel.: +36 62 599 649; fax: +36 62 433 506. E-mail address: [email protected] (C. Vizler). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.02.017
reduction of platelet and erythrocyte aggregability (for a review, see Frampton and Brodgen, 1995). More recently, attention has focused on the therapeutic potential of PTX as an immunomodulatory, anti-inflammatory and antitumor agent. Numerous studies have shown that PTX suppresses the production of tumor necrosis factor α (TNF-α) by murine and human macrophages and leukocytes, both at the mRNA and the protein level (Han et al., 1991; Weinberg et al., 1992). Further studies have shown that PTX interferes with the synthesis of other pro-inflammatory cytokines such as interleukin-1, interleukin-6 and interleukin-8, as well as with neutrophil superoxide anion production and degranulation, and neutrophil and lymphocyte adhesion and transendothelial migration (Currie et al., 1990; Crouch and Fletcher, 1992; D'Hellencourt et al., 1996; Dominguez-Jimenez et al., 2002). These findings may explain why the drug exhibits significant therapeutic
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above-mentioned reports represent the only available evidence of biological activity of M4 and M5. This study was therefore undertaken with the major aim of further exploring the bioactivity of M4 and M5. Namely, we compared the effects of the two carboxylated metabolites of PTX with that of the parent drug and M1 on lipopolysaccharide (LPS)-induced TNFα production in macrophages, TNF-α cytotoxic activity toward tumor cells, and endothelial cell viability and vascular cell adhesion molecule-1 (VCAM-1) expression in response to TNF-α. Moreover, we explored the possible interaction of PTX and its major circulating metabolites with some members of the ATP-binding cassette (ABC) transporter superfamily. Fig. 1. Chemical structures of pentoxifylline and its major metabolites.
2. Materials and methods 2.1. Materials and stock solutions
effects in different immune- and inflammatory-mediated diseases in both animal models and humans (Schwarz et al., 1993; Nataf et al., 1993; Zabel et al., 1993). Reports also suggest that PTX administration might increase the effectiveness of antitumor radio- and chemotherapy. In particular, by increasing tumor blood flow and oxygenation and abrogating the G2 checkpoint, it sensitizes tumors to both irradiation and alkylating agents (Song et al., 1992, Li et al., 1998; Collingridge and Rockwell, 2000). Moreover, PTX inhibits endothelial cell proliferation in vitro and tumor-driven angiogenesis in vivo (Gude et al., 2001), and reverses the classical multidrug resistance (MDR) phenotype in murine tumor cells through a downregulation of the mdr1 gene expression (Drobná et al., 2002). PTX undergoes extensive metabolism in humans, resulting in formation of at least seven metabolites (denoted metabolite M1-7), the major circulating ones being M1 [1-(5-hydroxyhexyl)-3,7-dimethylxanthine], M4 [1-(4-carboxybutyl)-3,7dimethylxanthine] and M5 [1-(3-carboxypropyl)-3,7-dimethylxanthine] (Fig. 1; Hinze et al., 1972). Notably, upon oral or intravenous administration of PTX to healthy volunteers, the plasma levels of both M1 and M5 greatly exceeded those of the parent drug, PTX; in the same subjects, circulating levels of M4 were comparable to those of PTX (Beerman et al., 1985; Nicklasson et al., 2002). The hemorheological and anticytokine properties of PTX are known to be retained by racemic (R,S)M1 and (R)-M1 (lisofylline), respectively (Ambrus et al., 1995; Rice et al., 1994; Van Furth et al., 1997); lisofylline is being under developed as a drug in its own right (Yang et al., 2005 and references therein). By contrast, information on the pharmacological properties of the major oxidative metabolites of PTX, M4 and M5, are still scanty. A study by Crouch and Fletcher (1992) demonstrated that M4 and M5 were more potent than PTX in inhibiting neutrophil superoxide anion production, degranulation (lactoferrin release) and surface expression of the β-2 integrin CD11b/CD18 (mac-1). In another study, Ambrus et al. (1995), exploring the hemorheological effects of various PTX metabolites, found that M1 and M5 were similar to PTX in their activity on erythrocyte deformability; notably, M5 was more potent than pentoxifylline in inhibiting epinephrineinduced platelet aggregation. To the best of our knowledge, the
Racemic (R,S)-M1, M4 and M5 were supplied by Aventis Pharma Deutschland GmbH (Frankfurt am Main, Germany). Unless indicated otherwise, all other reagents, including PTX, were obtained from Sigma-Aldrich Co. (Budapest, Hungary). Stock solutions of PTX and its metabolites (10 mg/ml) were prepared in Dulbecco's Modified Eagle's Medium (DMEM), filtered through Millex-GV 0.2 μm filters (Millipore, Carrigtwohill, Co Cork, Ireland) and kept at − 20 °C. Murine recombinant TNF-α (mrTNF-α) was produced and purified in our laboratory as previously described (Gyorfy et al., 1997). Membranes isolated from cells expressing individual human ATP-binding cassette (ABC) transporters were provided by SOLVO Biotechnology (Szeged, Hungary). 2.2. Cell lines and culture conditions The murine macrophage cell line RAW 264.7, the murine endothelioma cell line H5V (Vizler et al., 1998) and the highly TNF-α-sensitive WEHI-164 murine fibrosarcoma cell subline WC/1, which was established in our laboratory (Gyorfy et al., 1997), were grown in complete medium (CM), which consisted of DMEM/Ham's F12 (50:50 ratio) supplemented with 10% fetal calf serum (FCS) and 2mM glutamine. All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. 2.3. TNF-α production in cultured macrophage cells To assess the effects of PTX and its major metabolites on TNF-α production by LPS-stimulated macrophages, RAW 264.7 cells were seeded in 24-well culture plates (Orange Scientific, Braine-l'Alleud, Belgium) at a density of 3 × 105 cells/well in 1 ml of CM and left to adhere to the plastic plates for 1 h (37 °C, 5% CO2). The cells were then left untreated (control) or preincubated with PTX, M1, M4 or M5 (final concentration, 1 mg/ml) for 1h before adding LPS (final concentration, 10 or 100ng/ml) and culturing for 4 h (37 °C, 5% CO2). Thereafter, aliquots of the supernatants (500 μl) were harvested, centrifuged and stored at 4 °C until analyzed for TNF-α content.
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2.3.1. TNF-α bioassay TNF-α content of the supernatants was determined in a 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-based bioassay using WC/1 cells as TNF-α-sensitive target. Appropriate dilutions of the cell supernatants were added in quadruplicate to 96-well plates (Orange Scientific) containing 3 × 104 WC/1 cells/well in 100μl of DMEM supplemented with 5% FCS, 2 mM glutamine and 1μg/ml actinomycin D, a sensitizer of tumor cells to the cytotoxicity of TNF-α (Holtmann et al., 1988). After a 24-h incubation period, MTT was added to each well (final concentration, 1 mg/ml) and plates were incubated at 37°C for 2h to allow reduction of MTT by viable cell dehydrogenases to a formazan product. Thereafter, the supernatant containing the unreacted dye was replaced with acidified isopropanol (0.05 M HCl–isopropanol, 100μl/well), plates were vigorously shaken, and absorbance at 570 nm was measured using a microtiter plate reader. The percentage of cell survival was calculated from the absorbance (A) values as follow: Atested / Auntreated control × 100. TNF-α levels were estimated by evaluation of the supernatant dilution resulting in 50% reduction of cell survival and comparison with a concentration– cell survival curve for mrTNF-α. 2.3.2. TNF-α immunoassay In some experiments, TNF-α levels were also measured by an enzyme-linked immunosorbent assay (ELISA) developed in our laboratory using commercially available reagents. In detail, 96-well plates (Greiner Microlon, Greiner Biosciences, Frickenhausen, Germany) were coated with capture antibody (goat anti-mouse TNF-α, R&D Systems Inc., Minneapolis, MN, USA) in coating buffer (carbonate–bicarbonate buffer, pH 9.6) by overnight incubation at 4 °C. The plates were blocked with DMEM containing 10% FCS for 1 h at room temperature. After three washing with 0.1% Tween 20 in PBS (wash buffer), the samples and calibration standards (mrTNF-α) were added to the plates, and incubated 1h at 4°C. The plates were then washed three times with wash buffer and added with biotinylated detection antibody (anti-mouse TNF-α, R&D Systems Inc.). After 1h incubation at 4 °C, the plates were washed three times with wash buffer and then streptavidin conjugated to horseradish peroxidase (ExtrAvidin-peroxidase) was added at a dilution of 1:1000. After a 45-min incubation period at room temperature, the plates were washed three times with wash buffer and the substrate solution was added (0.4 mg/ml orthophenylenediamine, 0.01% hydrogen peroxide (30%) in 0.1M citrate/phosphate buffer, pH 5.2). The plates were incubated in the dark for 15 min before spectrophotometric reading at 450 nm in a microplate reader. The concentrations of TNF-α were calculated on the basis of standard curves constructed with known amounts of mrTNF-α (from 5 to 10,000 U/ml, final concentration) and were expressed as U/ml. The detection limit was 100 U/ml of mrTNF-α. 2.4. TNF-α cytotoxicity assay The impact of PTX and its major metabolites on TNF-α cytotoxic activity was studied using WC/1 murine fibrosarcoma
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cells as tumor cell target. WC/1 cells were seeded in 96-well culture plates (Orange Scientific) at a density of 3 × 104 cells/ well in DMEM supplemented with 5% FCS, 2 mM glutamine and 1 μg/ml actinomycin D, and incubated for 24h (37 °C, 5% CO2). Thereafter, the cells were exposed to rmTNF-α (final concentration, 0, 4, 8 and 16 U/ml) and 1μg/ml actinomycin D in the absence or presence of PTX, M1, M4 or M5 (final concentration, 1 mg/ml). The plates were further incubated for 24h (37 °C, 5% CO2) and then cell viability was determined by an MTT assay performed as described above. 2.5. Evaluation of cell viability and VCAM-1 expression in endothelioma cells To compare the effects of PTX, M1, M4 and M5 on endothelial cell viability, H5V cells were seeded in 24-well culture plates (Orange Scientific) at a density of 2 × 104 cells/ well in 1 ml of CM and incubated for 24h (37 °C, 5% CO2). The cells were then left untreated (control) or treated with PTX, M1, M4 or M5 (final concentrations: 0.5, 1 and 2 mg/ml) for 24 h (37 °C, 5% CO2) before evaluation of cell survival by an MTT assay performed as described above. To investigate the effects of PTX and its major metabolites on TNF-α-induced VCAM-1 expression, H5V cells were seeded in 6-well culture plates (Orange Scientific) at a density of 2 × 105/well in 3 ml of CM and incubated for 24 h (37 °C, 5% CO2). The cells were then left untreated or exposed to mrTNF-α (100 U/ml) in the absence or presence of PTX, M1, M4 or M5 (final concentration, 1mg/ml) for 24h (37 °C, 5% CO2). After trypsinization, the cells were collected, centrifuged for 5 min at 150×g, resuspended in 200 μl phosphate-buffered saline (PBS) and incubated with 1 μl rat anti-mouse VCAM-1 monoclonal antibody (R&D Systems, Minneapolis, MN) for 30min at 4 °C. The cells were washed three times, resuspended in 100μl PBS and incubated with 1 μl of fluorescein isothiocyanate (FITC)-conjugated goat anti-rat Ig for 30 min at 4 °C. Finally, upon washing, the cells were analyzed with a FACScalibur flow cytometer (BD Bioscences, Mountain View, CA, USA). 2.6. ABC transporters ATP hydrolase (ATPase) activity assay The interaction of PTX and its major metabolites with the human ABC membrane transporters p-glycoprotein/multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) was evaluated by measuring their ability to stimulate the ATPase activity of membrane vesicles isolated from baculovirusinfected Spodoptera frugiperda (Sf9) insect cells expressing human MDR1 or human MRP1, or from a mammalian cell line engineered to express human BCRP, respectively; all these three transporters exhibit a substrate-stimulated ATPase activity, which is closely related to their substrate transport function (Bakos et al., 2000 and references therein; Janvilisri et al., 2003). Membrane vesicles were prepared as described previously (Sarkadi et al., 1992) and the assays were performed as described in detail at http://www.solvo.huwww.solvo.hu. Briefly, membrane vesicles (20 μg/well) were incubated for
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control (Fig. 3 and Table 1), the ANOVA was followed by Dunnett's test. 3. Results 3.1. M4 and M5 are less effective than PTX and M1 in inhibiting LPS-stimulated TNF-α release from RAW 264.7 macrophages
Fig. 2. Effect of PTX and its major metabolites on LPS-induced TNF-α production in RAW 264.7 murine macrophages. RAW 264.7 cells were preincubated with PTX, M1, M4 or M5 (final concentration, 1mg/ml) for 1h, and then stimulated with 10 or 100ng/ml LPS for 4 h; the supernatants were collected and their TNF-α content was determined by an MTT-based bioassay as described in Section 2. Values are the mean ± S.E.M. of four independent experiments. ⁎P < 0.05 vs. control; ⁎⁎⁎P < 0.001 vs. control; aP < 0.05 vs. PTX; b P < 0.05 vs. M1.
40 min (MDR1 and BCRP) or 60 min (MRP1) at 37 °C in a medium containing 10 mM MgCl2, 40 mM MOPS–Tris (pH 7.0), 50 mM KCl, 5 mM dithiothreitol, 0.1 mM EGTA, 4 mM sodium azide, 1mM ouabain, 5 mM ATP, 2mM glutathione and various concentrations of the tested compounds in the absence or presence of 1.2 mM sodium orthovanadate. ATPase activity was determined as the difference of inorganic phosphate liberation measured in the presence or absence of sodium orthovanadate (vanadate-sensitive ATPase activity). Results were plotted as % activity, where 0% is the activity measured in the presence of solvent and 100% is the activity measured in the presence of a strong activator of the transporter, namely verapamil (final concentration, 40 μM), N-ethylmaleimide glutathione (final concentration, 10 mM) and sulfasalazine (final concentration, 10 μM) for MDR1, MRP1 and BCRP, respectively.
PTX and lysofilline, the R isomer of M1, have been shown to markedly inhibit LPS-stimulated production of TNF-α both in vitro and in vivo (Rice et al., 1994; Van Furth et al., 1997). Therefore, a first set of experiments explored the ability of M4 and M5, in comparison with that of PTX and M1, to inhibit TNF-α release from LPS-stimulated RAW 264.7 murine macrophage cells. This macrophage cell line was chosen in the present study because of its well-documented sensitivity to LPS and subsequent regulated production of large amounts of TNF-α (Lin et al., 2004). In preliminary trials, all tested compounds concentration-dependently decreased TNF-α release from macrophages in response to stimulation with 10 ng/ml LPS, reaching a plateau at 1mg/ml (not shown); thus, this concentration was selected for use in the final series of experiments. As shown in Fig. 2, M4 and M5 significantly and comparably decreased TNF-α levels in the supernatants from cells stimulated with 10ng/ml LPS (22% decrease; P < 0.05 vs. control); however, PTX in itself and M1 were remarkably more effective than the carboxylated metabolites in decreasing TNF-α production (P < 0.001 vs. both M4 and M5). Moreover, PTX and M1, but not M4 and M5, significantly decreased TNF-α production in response to cell stimulation with a 10-fold higher concentration of LPS (100 ng/ml; Fig. 2). The poor inhibitory effect of M4 and M5 on TNF-α production by murine macrophages was confirmed by further experiments based on immunoassay detection of TNF-α. As shown in Table 1, both M4 and M5 significantly inhibited TNF-α release in response to 10 ng/ml LPS (P < 0.01 vs. LPS alone), whereas they did not decrease TNF-α production when tested in macrophages treated with 100ng/ml LPS.
Table 1 Immunoassay of TNF-α released by LPS-activated RAW 264.7 cells in the absence and in the presence of M4 and M5 TNF-α levels (U/ml) +LPS 10 ng/ml
+LPS 100 ng/ml
1110 ± 15 477 ± 7 ⁎⁎ (− 56.6 ± 1.6%) 529 ± 26 ⁎⁎ (− 52.3 ± 4.9%)
1268 ± 64 1063 ± 73 a (− 13.4 ± 4.4%) 1035 ± 27.5 a (− 18.4 ± 2.6%)
2.7. Analysis of the data
– M4 M5
Results are given as means ± S.E.M. of n experiments. The Student's t-test for unpaired data was used for comparison of the mean values between two groups (Fig. 3). The one-way analysis of variance (ANOVA) followed by Tukey's test was used for multiple comparison among treatment groups (Figs. 2, 4 and 6). When treatment groups were compared to the same
TNF-α levels were measured by an enzyme-linked immunosorbent assay (ELISA) as described in Section 2. Data represent means ± S.E.M. of three experiments carried out in quadruplicate. Percent inhibition with respect to control (LPS alone) is reported in parenthesis. a Not statistically different (P > 0.05) from values obtained in LPS-only treated cells. ⁎⁎ P < 0.01 vs. values obtained in LPS-only treated cells.
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Fig. 3. Effect of PTX and its major metabolites on TNF-α-induced cytotoxicity in WC/1murine fibrosarcoma cells. WC/1 cells were treated for 24 h with the indicated concentrations of TNF-α in the presence or absence of each xanthine derivative (final concentration, 1 mg/ml) and cell viability was then determined by an MTT assay as described in Section 2. Values are the mean ± S.E.M. of three independent experiments carried out in quadruplicate. ⁎⁎P < 0.01 vs. control; ⁎⁎⁎P < 0.001 vs. control.
3.2. M4 and M5, but not PTX and M1, significantly attenuate TNF-α cytotoxic activity toward WC/1 fibrosarcoma cells TNF-α exhibits cytotoxicity against a wide range of murine and human tumor cell lines, and PTX has been found to attenuate TNF-α-induced cell death in cultured L292 murine fibrosarcoma cells via induction of heme oxigenase-1 (Oh et al., 2003). Therefore, we compared the ability of PTX and its major metabolites to protect tumor cells from TNF-α-induced cytotoxicity. WC/1 murine fibrosarcoma cells were exposed to increasing concentrations of mrTNF-α (0, 4, 8 and 16U/ml) in the presence of actinomycin D and in the presence or absence of increasing concentrations (from 0.1 to 2 mg/ml) of PTX, M1, M4 or M5 for 24h, and cell survival was then evaluated. For the active compounds, the activity reached a plateau at 1 mg/ml (data not shown). As shown in Fig. 3, M4 and M5 (1 mg/ml) significantly attenuated TNF-α cytotoxicity toward WC/1 tumor cells. By contrast, at all tested concentrations of TNFα, the antagonistic effects of PTX and M1 were weak (Fig. 3).
TNF-α-induced VCAM-1 expression in H5V murine endothelioma cells. As shown in Fig. 4, a 24-h exposure to PTX and M1 (final concentration 0.5, 1 and 2 mg/ml) concentration-dependently reduced H5V cell viability as judged by an MTT assay; M4 and M5 were completely ineffective in this respect (Fig. 4). As expected, cytofluorimetric analysis of H5V cells revealed a marked increase of fluorescence associated to VCAM-1 expression upon stimulation with 100 U/ml rmTNF-α (Figs. 5 and 6); both PTX and M1 (final concentration, 1 mg/ml) were capable of completely suppressing the VCAM-1 inducing effect
3.3. Effects of PTX and its major metabolites on cell survival and TNF-α-induced VCAM-1 expression in H5V endothelioma cells PTX was shown to be cytostatic and/or cytotoxic in vitro toward both normal and transformed endothelial cells (Gude et al., 2001). Moreover, a pharmacological elevation of cAMP levels was found to inhibit TNF-α-induced VCAM-1 expression in human umbilical vein endothelial cells (Pober et al., 1993; Otsuki et al., 2001). Thus, further experiments compared the effects of PTX and its major metabolites on cell viability and
Fig. 4. Survival curves of H5V murine endothelioma cells in response to PTX and its major metabolites. H5V endothelial cells were cultured for 24h in the presence of different concentrations of each xanthine derivative and then cell viability was determined by an MTT assay, as described in Section 2. Values are the mean ± S.E.M. of three experiments carried out in triplicate. ⁎P < 0.05; ⁎⁎P < 0.01; ⁎⁎⁎P < 0.001 vs. control (no drug) values; asignificantly different vs. M4 (P < 0.05 or less); csignificantly different vs. M4 (P < 0.05, or less); b significantly different vs. M5 (P < 0.05, or less); dsignificantly different vs. M5 (P < 0.05, or less).
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M. Fantin et al. / European Journal of Pharmacology 535 (2006) 301–309 Table 2 A comprehensive summary of the pharmacological effects of PTX and its major metabolites
Fig. 5. Effect of PTX and its major metabolites on TNF-α-induced VCAM-1 expression in H5Vmurine endothelioma cells. H5V cells were left untreated or exposed for 24 h to 100U/ml mrTNF-α in the presence or absence of PTX, M1, M4 or M5 (final concentration, 1mg/ml), and then VCAM-1 expression was analyzed by flow cytometry as described in Section 2. Light gray histogram: no staining; dark gray histogram: unstimulated cells; thin line: TNF-α-stimulated cells; thick line: cells stimulated in the presence of TNF-α and PTX or its metabolites. The experiment was repeated three times with similar results.
of TNF-α, since the cells treated with the cytokine plus either PTX or M1 were undistinguishable from TNF-α-untreated cells. By contrast, neither M4 nor M5 (both at 1 mg/ml) did interfere with TNF-α-induced upregulation of VCAM-1, since the cells treated with the cytokine plus either M4 or M5 were undistinguishable from those treated with TNF-α only (Figs. 5 and 6).
Effect
PTX
M1
M4
M5
Macrophages, inhibition of LPS-induced TNF release Endothelium, growth inhibition Endothelium, inhibition of TNF-α-induced VCAM-1 expression WC/1 fibrosarcoma cells, protection from TNF-α cytotoxicity Interaction with MDR1 Interaction with BCRP Interaction with MRP1 Neutrophils, inhibition superoxide production in response to warming or formyl-methionyl-leucyl-phenylalanine (FMLP) (Crouch and Fletcher, 1992) Neutrophils, inhibition of lactoferrin release in response to warming or FMLP (Crouch and Fletcher, 1992) Neutrophils, inhibition of CD11b/CD18 expression in response to warming or FMLP (Crouch and Fletcher, 1992) Improvement of red blood cell filterability (Ambrus et al., 1995) Inhibition of ADP-induced platelet aggregation (Ambrus et al., 1995) Inhibition of epinephrine-induced platelet aggregation (Ambrus et al., 1995)
++
++
+/−
+/−
+ +
+ +
− −
− −
+/−
+/−
+
+
− + − −
− − − +
− − − +
− − − +
+/−
+/−
+
+
+
+
++
+++
+
+
−
+
+
++
−
+/−
+
+
−
++
sulfasalazine at the highest concentration tested (50 μg/ml). By contrast, PTX metabolites were devoid of any effect on the ATPase activity related to BCRP. 4. Discussion Although M4 and M5 are major circulating species of PTX in humans, so far only few and scattered literature data are available on their pharmacological properties. In this study, aimed at investigating some possible biological effects of M4
3.4. Interaction of PTX and its metabolites with human ABC membrane transporters PTX has been found to reverse the p-glycoprotein-mediated MDR phenotype and to downregulate mdr1 gene expression in cultured murine tumor cells (Drobná et al., 2002; Viladkar and Chitnis, 1994). The possible interaction of PTX and its major circulating metabolites with MDR1, MRP1 and BCRP, all known to be ATP-dependent pumps capable of conferring an MDR phenotype (Gottesman et al., 2002), has been investigated measuring drug-stimulated ATPase activity in membranes from cells engineered to express individual recombinant transporters. In our hands, none of the tested compounds (final concentration, up to 50 μg/ml) increased the ATPase activity of membrane vesicles expressing MDR1 or MRP1 (data not shown), suggesting that neither PTX nor its major metabolites interact with these ATP-dependent transporters. As shown in Fig. 7, the ATPase activity of membranes expressing BCRP was concentration-dependently stimulated by PTX, reaching approximately 50% of the ATPase activity observed in the presence of 10 μM
Fig. 6. Effect of PTX and its major metabolites on TNF-α-induced VCAM-1 expression in H5Vmurine endothelioma cells. H5V cells were left untreated or exposed for 24h to 100 U/ml mrTNF-α in the presence or absence of PTX, M1, M4 or M5 (final concentration, 1 mg/ml), and then VCAM-1 was stained by anti-VCAM-1 antibody and FITC labeled secondary antibody. Increase of mean fluorescence level (MFL) was calculated by dividing the MFL of treated cells with MFL of non-stimulated, identically stained controls. The results are mean ± S.E.M. of three independent experiments. ⁎P < 0.05 vs. TNF-treated control; ⁎⁎not significant vs. TNF-treated control.
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Fig. 7. Concentration–effect curves for pentoxifylline and its metabolites on MDR1 and BCRP ATPase activity assessed in isolated cell membrane vesicles. Vanadate sensitive ATPase activity was determined using the technological platform of SOLVO Biotechnology, as described in Section 2. The results are presented as % activity where 0% is the vanadate-sensitive ATPase activity measured in the presence of solvent alone, while 100% is the vanadate-sensitive ATPase activity measured in presence of 40μM verapamil (MDR1) or 10μM sulfasalazine (BCRP). The results are means ± S.E.M. of three experiments carried out in triplicate.
and M5, in comparison with those of the parent drug and its major reductive metabolite, M1, we present evidence that the pharmacological properties of both carboxylated metabolites differ quantitatively and/or qualitatively from those of PTX and M1. Our present data and those of others comparing the bioactivity of PTX with that of its major metabolites are summarized in Table 2. One of the best characterized effects of PTX is its ability to decrease TNF-α production both in vitro and in vivo (Zabel et al., 1993). The anti-TNF-α activity of PTX is known to be retained by lisofylline, the R isomer of M1 (Rice et al., 1994; Van Furth et al., 1997), which has been recently proposed as a PTX-like anti-inflammatory drug candidate on its own right. On the other hand, to the best of our knowledge, there is no data in
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the literature on the modulation of TNF-α release by M4 or M5. Here, we found that both carboxylated metabolites of PTX were remarkably less potent than the parent drug and M1 in inhibiting TNF-α production by LPS-stimulated murine macrophages (Fig. 2). This finding suggests that M4 and M5 are unlikely to contribute to the suppression of TNF-α circulating levels observed in response to PTX administration (Zabel et al., 1993). Furthermore, because suppression of TNF-α production by xanthine derivatives is thought to be mediated by inhibition of cAMP phosphodiesterase activity (Semmler et al., 1993), our data suggest that both carboxylated metabolites may be weaker inhibitors of cAMP phosphodiesterase than the parent drug and M1; experiments on partially purified preparations of cAMP phosphodiesterase aimed at confirming this hypothesis are in progress. Furthermore, information on the structure-activity relationships for PTX and its major metabolites will be obtained from docking studies on theoretical models of cAMP phosphodiesterase. M5, being devoid of major effects on TNF-α production but retaining the beneficial hemorheological activity of the parent drug (Ambrus et al., 1995), might be a safer drug than PTX for the treatment of microcirculatory disorders. Indeed, on a theoretical basis, an indiscriminate blockade of TNF-α production and/or effects might be associated with an increased susceptibility to some types of infections, as it has been observed experimentally in PTX-treated mice bearing chronic pulmonary tuberculosis (Turner et al., 2001). Moreover, since both carboxylated metabolites of PTX are more effective than the parent drug in inhibiting several functions of human neutrophils, including degranulation and superoxide anion production (Crouch and Fletcher, 1992), they might be even more effective than PTX in those conditions in which neutrophil-mediated tissue damage plays a prominent role, such as myocardial infarction, varicose ulcers and adult respiratory distress syndrome (Fantone and Ward, 1985). PTX was recently shown to decrease the susceptibility of L929 murine fibrosarcoma cells to the cytotoxic action of TNFα (Oh et al., 2003). As shown in Fig. 3, under our experimental conditions, the protective effect of PTX and M1 on TNF-αinduced toxicity in WC/1 murine fibrosarcoma cells was relatively weak (Fig. 3). Interestingly, both the carboxylated metabolites of PTX were significantly more effective than the parent drug and M1 in attenuating WC/1 tumor cell sensitivity to TNF-α (Fig. 3). Because the cytoprotection against TNF-α afforded by PTX was related to induction of the activity of heme oxygenase-1 (Oh et al., 2003), a protein capable of preventing cells from undergoing apoptosis triggered by various agents inducing oxidative damage (Fang et al., 2004 and references therein), M4 and M5 might be therapeutic tools in diseases associated with oxidative stress. Evidence that induction of heme oxigenase-1 may have therapeutic implications in these types of diseases has been provided by studies demonstrating a cytoprotective potential of the protein in models of lung injury (Otterbein et al., 1999) and ischemia/reperfusion injury (Katori et al., 2002). Further experiments demonstrated that PTX and M1, but not M4 and M5, reduced cell proliferation and/or viability and
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blocked TNF-α-induced VCAM-1 expression in endotheliumderived murine cells. In agreement, other studies have shown that a pharmacological elevation of cAMP levels inhibits both endothelial cell proliferation and VCAM-1 expression in response to TNF-α (Kim et al., 2001; Pober et al., 1993; Otsuki et al., 2001). These findings further support a possible lack of inhibition of cAMP phosphodiesterase activity by the carboxylated metabolites of PTX and possibly preclude their usefulness as inhibitors of tumor angiogenesis, as it was proposed for PTX by Gude et al. (2001). Though PTX was shown to reverse the classical MDR phenotype in cultured murine tumor cells (Drobná et al., 2002; Viladkar and Chitnis, 1994), neither PTX nor its major metabolites did stimulate the ATPase activity of human MDR1 or human MRP1 (not shown). In other words, none of the tested compounds seem to be a good substrate of these membrane transporters and, therefore, conceivably, could efficiently compete with a cytotoxic drug substrate for transport outside of the cell. However, these data do not exclude that PTX might be capable of reversing the MDR phenotype of human tumors since the drug was shown to suppress mdr1 gene expression in multidrug-resistant L1210/VCR murine leukemia cells (Drobná et al., 2002). In conclusion, based on our present and previous findings and, in spite of the lack of knowledge concerning the exact molecular targets, a therapeutic use of M4 and M5 in certain pathologic conditions is conceivable. In particular, M5, retaining the modulatory effects of PTX on erythrocyte deformability, platelet aggregation and neutrophil function, but lacking substantial inhibitory activity toward TNF-α production, deserves further preclinical evaluation for its therapeutic potential as an hemorheological and anti-inflammatory agent. Acknowledgements Pentoxifylline metabolites were kindly supplied by Dr. J. Pünter, Aventis Pharma Deutschland GmbH. The H5V cell line was kindly supplied by Dr. A. Vecchi, Laboratory of Immunopharmacology, Istituto Ricerche Farmacologiche Mario Negri (Milano, Italy). This work was supported by the Hungarian National Research and Development Program (NKFP 1A/057/04). References Ambrus, J.L., Stadler, S., Kulaylat, M., 1995. Hemorheologic effects of metabolites of pentoxifylline (Trental). J. Med. 26, 65–75. Bakos, E., Evers, R., Sinko, E., Varadi, A., Borst, P., Sarkadi, B., 2000. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol. Pharmacol. 57, 760–768. Beerman, B., Ings, R., Mansby, J., Chamberlain, J., McDonald, A., 1985. Kinetics of intravenous and oral pentoxifylline in healthy subjects. Clin. Pharmacol. Ther. 37, 25–28. Collingridge, D.R., Rockwell, S., 2000. Pentoxifylline improves the oxygenation and radiation response of BA1112 rat rabdomyosarcomas and EMT6 mouse mammary carcinomas. Int. J. Cancer 90, 256–264. Crouch, S.P., Fletcher, J., 1992. Effect of ingested pentoxifylline on neutrophil superoxide anion production. Infect. Immun. 60, 4504–4509.
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